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Signaling pathways of isoproterenol-induced ERK1/2 phosphorylation in primary cultures of astrocytes are concentration-dependent
Journal of Neurochemistry
Abstract
J. Neurochem. (2010) 115, 1007–1023.
Stimulation of β-adrenoceptors activates the canonical adenylate cyclase pathway (via Gs protein) but can also evoke phosphorylation of extracellular-regulated kinases 1 and 2 (ERK1/2) via Gs/Gi switching or β-arrestin-mediated recruitment of Src. In primary cultures of mouse astrocytes, activation of the former of these pathways required micromolar concentrations of the β1/β2-adrenergic agonist isoproterenol, that acted on β1-adrenoceptors, whereas the latter was activated already by nanomolar concentrations, acting on β2 receptors. Protein kinase A activity was required for Gs/Gi switching, which was followed by Ca2+ release from intracellular stores and Giα- and metalloproteinase-dependent transactivation of the epidermal growth factor receptor (EGFR; at its Y1173 phophorylation site), via its receptor-tyrosine kinase, β-arrestin 1/2 recruitment, and MAPK/ERK kinase-dependent ERK1/2 phosphorylation. ERK1/2 phosphorylation by Src activation depended on β-arrestin 2, but not β-arrestin 1, was accompanied by Src/EGFR co-precipitation and phosphorylation of the EGFR at the Src-phosphorylated Y845 site and the Y1045 autophosphorylation site; it was independent of transactivation but dependent on MAPK/ERK kinase activity, suggesting EGFR phosphorylation independently of the receptor-tyrosine kinase or activation of Ras or Raf directly from Src. Most astrocytic consequences of activating either pathway (or both) are unknown, but morphological differentiation and increase in glial fibrillary acidic protein in response to dibutyryl cAMP-mediated increase in cAMP depend on Gs/Gi switching and transactivation.
... This promising neuroprotective action is further discussed and reviewed in [74]. The pathway activated by b 1 -adrenergic receptors in mature astrocytes [75] reminds of that opened by [K ? ] o C10 mM. ...
... ] o C10 mM. That is because a protein kinase A-mediated G s /G i switch leads to an increase in [Ca 2? ] i , activation of a metalloproteinase (not ADAM17) and release of a growth factor activating the EGF receptor and causing ERK phosphorylation; however Src is not involved [75] and NKCC1 is not activated. The b 1 -adrenergic signaling pathway is inhibited by the inhibitors H89, PTX, GM6001, AG1478 and U0126, blockers of protein kinase A (and thus G s /G i switching), G i , metalloproteinases, EGF receptor phosphorylation and ERK phosphorylation. ...
... However, the situation is probably more complex, with stimulation of b 2 -adrenergic receptors causing glycogenolysis in chicken astrocytes, which express no b 1 -adrenergic receptors [80] and probably also in white matter astrocytes in mammalian brain [81]. The pathway for b 2 -adrenergic stimulation includes no Ca 2? -releasing processes [75], but extracellular Ca 2? might be accumulated. However, besides mediating signaling in a G protein-independent fashion as shown in Fig. 5b, b-arrestins also desensitize b-adrenergic receptors [82] and additional b 2 -adrenergic pathways in other cell types cannot be excluded. ...
This paper describes the roles of the astrocytic Na+, K+-ATPase for K+ homeostasis in brain. After neuronal excitation it alone mediates initial cellular re-accumulation of moderately increased extracellular K+. At higher K+ concentrations it is assisted by the Na+, K+, 2Cl− transporter NKCC1, which is Na+, K+-ATPase-dependent, since it is driven by Na+, K+-ATPase-created ion gradients. Besides stimulation by high K+, NKCC1 is activated by extracellular hypertonicity. Intense excitation is followed by extracellular K+ undershoot which is decreased by furosemide, an NKCC1 inhibitor. The powerful astrocytic Na+, K+-ATPase accumulates excess extracellular K+, since it is stimulated by above-normal extracellular K+ concentrations. Subsequently K+ is released via Kir4.1 channels (with no concomitant Na+ transport) for re-uptake by the neuronal Na+, K+-ATPase which is in-sensitive to increased extracellular K+, but stimulated by intracellular Na+ increase. Operation of the astrocytic Na+, K+-ATPase depends upon Na+, K+-ATPase/ouabain-mediated signaling and K+-stimulated glycogenolysis, needed in these non-excitable cells for passive uptake of extracellular Na+, co-stimulating the intracellular Na+-sensitive site. A gradual, spatially dispersed release of astrocytically accumulated K+ will therefore not re-activate the astrocytic Na+, K+-ATPase. The extracellular K+ undershoot is probably due to extracellular hypertonicity, created by a 3:2 ratio between Na+, K+-ATPase-mediated Na+ efflux and K+ influx and subsequent NKCC1-mediated volume regulation. The astrocytic Na+, K+-ATPase is also stimulated by β1-adrenergic signaling, which further stimulates hypertonicity-activation of NKCC1. Brain ischemia leads to massive extracellular K+ increase and Ca2+ decrease. A requirement of Na+, K+-ATPase signaling for extracellular Ca2+ makes K+ uptake (and brain edema) selectively dependent upon β1-adrenergic signaling and inhibitable by its antagonists.
... The signaling pathways for both 1 -and 2 -adrenergic signaling H89 have been determined in cultured astrocytes with the aid of specific inhibitors [39]. As shown in Figure 1, phosphorylation of extracellular regulated kinase 1 and kinase 2 (ERK 1/2 ) by isoproterenol is inhibited by H89, an inhibitor of protein-kinase A (PKA) and thus of G s -mediated signaling by isoproterenol. ...
... As shown in Figure 1, phosphorylation of extracellular regulated kinase 1 and kinase 2 (ERK 1/2 ) by isoproterenol is inhibited by H89, an inhibitor of protein-kinase A (PKA) and thus of G s -mediated signaling by isoproterenol. Pertussis toxin (PTX), an inhibitor of the G s -G i switch occurring in the astrocytic signaling pathway of 1 -but not 2 -adrenergic signaling [39], has a similar effect. In this pathway G s activation is followed by an increase in [Ca 2+ ] i , which in astrocytes leads to metalloproteinasemediated release of a growth factor that transactivates the epidermal growth factor receptor (EGFR). ...
... As can be seen from Table 2 each of these inhibitors also prevents 1 -adrenergic edema formation in vivo after ischemia/reperfusion. In contrast, the Src inhibitor PP1 that is active in the 2 -but not 1 -adrenergic signaling [39] had no effect. The consistency between results obtained in cultured astrocytes and the present in vivo results further supports the validity of the used cultures as models of their in situ counterparts. ...
Infarct size and brain edema following ischemia/reperfusion are reduced by inhibitors of the Na+, K+, 2Cl−, and water cotransporter NKCC1 and by β
1-adrenoceptor antagonists. NKCC1 is a secondary active transporter, mainly localized in astrocytes, driven by transmembrane Na+/K+ gradients generated by the Na+,K+-ATPase. The astrocytic Na+,K+-ATPase is stimulated by small increases in extracellular K+ concentration and by the β-adrenergic agonist isoproterenol. Larger K+ increases, as occurring during ischemia, also stimulate NKCC1, creating cell swelling. This study showed no edema after 3 hr medial cerebral artery occlusion but pronounced edema after 8 hr reperfusion. The edema was abolished by inhibitors of specifically β
1-adrenergic pathways, indicating failure of K+-mediated, but not β
1-adrenoceptor-mediated, stimulation of Na+,K+-ATPase/NKCC1 transport during reoxygenation. Ninety percent reduction of extracellular Ca2+ concentration occurs in ischemia. Ca2+ omission abolished K+ uptake in normoxic cultures of astrocytes after addition of 5 mM KCl. A large decrease in ouabain potency on K+ uptake in cultured astrocytes was also demonstrated in Ca2+-depleted media, and endogenous ouabains are needed for astrocytic K+ uptake. Thus, among the ionic changes induced by ischemia, the decrease in extracellular Ca2+ causes failure of the high-K+-stimulated Na+,K+-ATPase/NKCC1 ion/water uptake, making β
1-adrenergic activation the only stimulus and its inhibition effective against edema.
... concentrations. From previous studies by Du et al. [10] it is known that isoproterenol concentrations C1 lM stimulate b 1 -adrenergic receptors and via a G s -mediated pathway activate cAMP and protein kinase A (PKA). However, PKA induces a G s /G i switch [10] followed by an increase in [Ca 2? ] i , a release of an agonist of the epidermal growth factor (EGF) receptor, stimulation of this receptor, and eventually phosphorylation of extracellular regulated kinases 1 and 2 (ERK 1/2 ). ...
... From previous studies by Du et al. [10] it is known that isoproterenol concentrations C1 lM stimulate b 1 -adrenergic receptors and via a G s -mediated pathway activate cAMP and protein kinase A (PKA). However, PKA induces a G s /G i switch [10] followed by an increase in [Ca 2? ] i , a release of an agonist of the epidermal growth factor (EGF) receptor, stimulation of this receptor, and eventually phosphorylation of extracellular regulated kinases 1 and 2 (ERK 1/2 ). In the present study we confirmed the involvement of a b 1 -adrenergic effect, whereas inhibition of b 2 -adrenergic signaling was of no consequence, and we tested the influence on stimulated glycogenolysis by the PKA inhibitor H-89, the G i inhibitor PTX, GM6001 an inhibitor of the release of EGF receptor agonist, and AG 1478, an EGF receptor antagonist, as well as of administration of the EGFR agonist EGF. ...
... Under control conditions the glycogen content averaged from all experiment was 90 ± 8.1 nmol/mg protein, with only minor differences between different experiments. One lM isoproterenol stimulates both the higher affinity b 2adrenergic and the lower affinity b 1 -adrenergic receptors [10]. However, Fig. 1a shows that the b 1 -adrenergic antagonist betaxolol inhibited glycogenolysis in response to isoproterenol, whereas the b 2 -adrenergic antagonist ICI118551 had no effect. ...
Glycogenolysis, in brain parenchyma an astrocyte-specific process, has changed from being envisaged as an emergency procedure to playing central roles during brain response to whisker stimulation, memory formation, astrocytic K(+) uptake and stimulated release of ATP. It is activated by several transmitters and by even very small increases in extracellular K(+) concentration, and to be critically dependent upon an increase in free cytosolic Ca(2+) concentration ([Ca(2+)]i), whereas cAMP plays only a facilitatory role together with increased [Ca(2+)]i. Detailed knowledge about the signaling pathways eliciting glycogenolysis is therefore of interest and was investigated in the present study in well differentiated cultures of mouse astrocytes. The β-adrenergic agonist isoproterenol stimulated glycogenolysis by a β1-adrenergic effect, which initiated a pathway in which cAMP/protein kinase A activated a Gi/Gs shift, leading to Ca(2+)-activated glycogenolysis. Inhibition of this pathway downstream of cAMP but upstream of the Gi/Gs shift abolished the glycogenolysis. However, inhibitors operating downstream of the Ca(2+)-sensitive step, but preventing transactivation-mediated epidermal growth factor (EGF) receptor stimulation, a later step in the activated pathway, also caused inhibition of glycogenolysis. For this reason the effect of EGF was investigated and it was found to be glycogenolytic. Large increases in extracellular K(+) activated glycogenolysis by a nifedipine-inhibited L-channel opening allowing influx of Ca(2+), known to be glycogenolysis-dependent. Small increases (addition of 5 mM KCl) caused a smaller effect by a similarly glycogenolysis-reliant opening of an IP3 receptor-dependent ouabain signaling pathway. The same pathway could be activated by GABA (also in brain slices) due to its depolarizing effect in astrocytes.
... Song, J. Xu, L. Hertz, W. Walz and L. Peng, in press, BioMed Research International). The pathway for both β 1 -and β 2 -mediated signaling is known in astrocytes [73]. In well-differentiated astrocyte cultures this pathway reminds of that shown in Fig. 2a for the effect of highly elevated K + concentrations. ...
... In well-differentiated astrocyte cultures this pathway reminds of that shown in Fig. 2a for the effect of highly elevated K + concentrations. Although β-adrenergic receptors are G s -linked, a G s to G i switch mediated by protein kinase A (PKA) leads to an increase in [Ca 2+ ] i and a subsequent growth factor release, which transactivates the EGF receptor and stimulates ERK 1/2 phosphorylation [73]. Two important differences between the two signaling pathways are that Src and the metallopropteinase ADAM 17 are not involved in the pathway activated by stimulation of β 1 -adrenergic receptors, although Src constitutes part of the β 2 -adrenoceptor-mediated pathway. ...
Brain edema is a serious complication in ischemic stroke because even relatively small changes in brain volume can compromise cerebral blood flow or result in compression of vital brain structures on account of the fixed volume of the rigid skull. Literature data indicate that administration of either antagonists of the V1 vasopressin (AVP) receptor or the β1-adrenergic receptor are able to reduce edema or infarct size when administered after the onset of ischemia, a key advantage for possible clinical use. The present review discusses possible mechanisms, focusing on the role of NKCC1, an astrocytic cotransporter of Na+, K+, 2Cl- and water and its activation by highly increased extracellular K+ concentrations in the development of cytotoxic cell swelling. However, it also mentions that due to a 3/2 ratio between Na+ release and K+ uptake by the Na+,K+-ATPase driving NKCC1 brain extracellular fluid can become hypertonic, which may facilitate water entry across the blood-brain barrier, essential for development of edema. It shows that brain edema does not develop until during reperfusion, which can be explained by lack of metabolic energy during ischemia. V1 antagonists are likely to protect against cytotoxic edema formation by inhibiting AVP enhancement of NKCC1-mediated uptake of ions and water, whereas β1-adrenergic antagonists prevent edema formation because β1-adrenergic stimulation alone is responsible for stimulation of the Na+,K+-ATPase driving NKCC1, first and foremost due to decrease in extracellular Ca2+ concentration. Inhibition of NKCC1 also has adverse effects, e.g. on memory and the treatment should probably be of shortest possible duration.
... Astrocytes express numbers of receptors for neurotransmitters. In particular, activation of a 2or b 1 -adrenoceptors stimulates intracellular Ca 2? release [10,11]. Dexmedetomidine is a potent and highly specific a 2adrenergic agonist [12]. ...
... Dexmedetomidine is a potent and highly specific a 2adrenergic agonist [12]. Isoproterenol is a non-specific agonist of b-adrenergic receptors, which stimulates b 1 receptors at concentrations higher than 1 lM [10]. Regulated release of Ca 2? from the endoplasmic reticulum (ER) through intracellular Ca 2? release channels, such as the InsP 3 receptors (InsP 3 R) and (to a lesser extend) ryanodine receptors (RyRs) (for review, see [8,13] ) assumes the leading role in glial Ca 2? signalling. ...
AimThe primary aim of this study was to identify the effects of hyperammonemia on functional expression of Cav1.2 L-type Ca2+ channels in astroglia.Methods
Primary cultures of mouse astrocytes were used to study effects of chronic treatment (1-5 days) with ammonium chloride, at 1, 3 and 5 mM on depolarisation-induced increases in free cytosolic Ca2+ concentration ([Ca2+]i, measured with fura-2 based microfluorimetry) in control conditions and following treatment with the L-type Ca2+ channel inhibitor nifedipine or with ryanodine receptors inhibitor ryanodine. Expression of Cav1.2 mRNA was identified with RT-PCR, whereas protein content was determined by Western blotting. Sustained hyperammonemia in vivo was induced by daily injections of urease (33 units/kg body weight, i.p.) for 3 days.ResultsDepolarisation-induced [Ca2+]i transients sensitive to nifedipine (peak of the response) and to ryanodine (plateau phase) were significantly increased in astrocytes chronically exposed to ammonium. The ammonium-induced increase in Ca2+ influx in astrocytes resulted from an up-regulation of Cav1.2 channels expression detected at mRNA and protein levels. Increase in Cav1.2 expression was prevented by ouabain antagonist canrenone. Similar up-regulation of Cav1.2 gene expression was found in the brains of adult mice subjected to intraperitoneal injection of urease. In transgenic mice tagged with an astrocyte-specific or neurone-specific markers and treated with intraperitoneal injections of ureas, the fluorescence-activated cell sorting of neurones and astrocytes demonstrated that Cav1.2 mRNA expression was up-regulated in astrocytes, but not in neurones.Conclusions
Ammonium-induced deregulation of astroglial Ca2+ signalling, is, in part, associated with up-regulation of Cav1.2 L-type calcium channels.This article is protected by copyright. All rights reserved.
... ERK 1/2 and AKT stayed maximally phosphorylated for at least 4 h. This is much longer than EGFR transactivation by stimulation of G protein-coupled receptors (Li et al., 2008a,b;Du et al., 2010) and by elevation of the extracellular K + concentration (Cai et al., 2011) in astrocytes, and in the present study several EGFR sites were phosphorylated already after 30 min. Nevertheless, swelling did not become apparent until after 8 h of exposure. ...
... b 1 -adrenergic receptors, 5-HT 2B receptors or high extracellular potassium ion (K + ) concentrations. In all these situations metalloproteinase-mediated shedding of growth factor(s) was a necessary component of the signal pathway, demonstrated by the inhibitory effects of GM6001(Li et al., 2008aDu et al., 2010;Cai et al., 2011). In contrast, no shedding of growth factor(Ammonia-induced EGFR transactivation requires association of the a 1 isoform of Na/K-ATPase and Src in lipid raft, but not of the a 2 isoform of Na/K-ATPase. ...
Ammonia toxicity is clinically important and biologically poorly understood. We reported previously that 3 mM ammonia chloride (ammonia), a relevant concentration for hepatic encephalopathy studies, increases production of endogenous ouabain and activity of Na,K-ATPase in astrocytes. In addition, ammonia-induced upregulation of gene expression of α2 isoform of Na,K-ATPase in astrocytes could be inhibited by AG1478, an inhibitor of the EGF receptor (EGFR), and by PP1, an inhibitor of Src, but not by GM6001, an inhibitor of metalloproteinase and shedding of growth factor, suggesting the involvement of endogenous ouabain-induced EGF receptor transactivation. In the present study, we investigated ammonia effects on phosphorylation of EGF receptor and its intracellular signal pathway towards MAPK/ERK1/2 and PI3K/AKT; interaction between EGF receptor, α1, and α2 isoforms of Na,K-ATPase, Src, ERK1/2, AKT and caveolin-1; and relevance of these signal pathways for ammonia-induced cell swelling, leading to brain edema, an often fatal complication of ammonia toxicity. We found that i) ammonia increases EGF receptor phosphorylation at EGFR(845) and EGFR(1068); ii) ammonia-induced ERK1/2 and AKT phosphorylation depends on the activity of EGF receptor and Src, but not on metalloproteinase; iii) AKT phosphorylation occurs upstream of ERK1/2 phosphorylation; iv) ammonia stimulates association between the α1 Na,K-ATPase isoform, Src, EGF receptor, ERK1/2, AKT and caveolin-1; v) ammonia-induced ROS production might occur later than EGFR transactivation; vii) both ammonia induced ERK phosphorylation and ROS production can be abolished by canrenone, an inhibitor of ouabain, and vi) ammonia-induced cell swelling depends on signaling via the Na,K-ATPase/ouabain/Src/EGF receptor/PI3K-AKT/ERK1/2, but in response to 3 mM ammonia it does not appear until after 12 hr. Based on literature data it is suggested that the delayed appearance of the ammonia-induced swelling at this concentration reflects required ouabain-induced oxidative damage of the ion and water cotransporter NKCC1. This information may provide new therapeutic targets for treatment of hyperammonic brain disorders.
... As discussed later in the text, the IP 3 receptor and the increase in [Ca 2+ ] i by its stimulation constitute part of the astrocytic K + /Na + ,K + -ATPase/ouabain pathway assumed to be essential for normal Na + ,K + -ATPase function and thus K + reaccumulation. It is also an intermediate in the β 1 -adrenergic pathway (Du et al., 2010) accelerating the operation of the astrocytic Na + /K + -ATPase/NKCC1 ion/water transport system that provides regulatory volume increase during hypertonicity, and probably contributes to the undershoot. ...
... The interpretation of these findings are that isoproterenol-mediated signaling stimulating the Na + ,K + -ATPase required for NKCC1 function caused regulatory volume increase and in the process at the very least may contribute to the establishment of the undershoot in intact brain or brain slices (see Table 2 and later text). This conclusion is consistent with two previously discussed observations in intact brain tissue: inhibition of the undershoot by furosemide (Figure 4), and dependence of the undershoot on intact IP 3 receptor function (Figure 7), a receptor operating within the β 1 -adrenergic signaling pathway in astrocytes (Du et al., 2010). It cannot be excluded that α 2 -adrenergic stimulation of the neuronal Na + ,K + -ATPase also contributes to the undershoot, since another co-transporter, KCC2 which is expressed in neurons, is also blocked by furosemide (Russell, 2000;Blaesse et al., 2009). ...
Brain excitation increases neuronal Na(+) concentration by 2 major mechanisms: (i) Na(+) influx caused by glutamatergic synaptic activity; and (ii) action-potential-mediated depolarization by Na(+) influx followed by repolarizating K(+) efflux, increasing extracellular K(+) concentration. This review deals mainly with the latter and it concludes that clearance of extracellular K(+) is initially mainly effectuated by Na(+),K(+)-ATPase-mediated K(+) uptake into astrocytes, at K(+) concentrations above ~10 mM aided by uptake of Na(+),K(+) and 2 Cl(-) by the cotransporter NKCC1. Since operation of the astrocytic Na(+),K(+)-ATPase requires K(+)-dependent glycogenolysis for stimulation of the intracellular ATPase site, it ceases after normalization of extracellular K(+) concentration. This allows K(+) release via the inward rectifying K(+) channel Kir4.1, perhaps after trans-astrocytic connexin- and/or pannexin-mediated K(+) transfer, which would be a key candidate for determination by synchronization-based computational analysis and may have signaling effects. Spatially dispersed K(+) release would have little effect on extracellular K(+) concentration and allow K(+) accumulation by the less powerful neuronal Na(+),K(+)-ATPase, which is not stimulated by increases in extracellular K(+). Since the Na(+),K(+)-ATPase exchanges 3 Na(+) with 2 K(+), it creates extracellular hypertonicity and cell shrinkage. Hypertonicity stimulates NKCC1, which, aided by β-adrenergic stimulation of the Na(+),K(+)-ATPase, causes regulatory volume increase, furosemide-inhibited undershoot in [K(+)]e and perhaps facilitation of the termination of slow neuronal hyperpolarization (sAHP), with behavioral consequences. The ion transport processes involved minimize ionic disequilibria caused by the asymmetric Na(+),K(+)-ATPase fluxes.
... [15][16][17][18][19][20]. The G-protein-coupled receptors reported on include the adrenergic receptors that are relevant for brown fat [21][22][23][24][25][26]. ...
... Concerning the β-adrenoceptors, the picture is more complex, especially since also an intracellular route to EGF receptor activation has been suggested. The suggestion is that for β 1 -and β 2 -adrenoceptors, the activation process may result in protein kinase A-mediated phosphorylation of the receptor at serine/ threonine residues in the C-terminal tail, which leads to recruitment of β-arrestin, and to a G s -to G i -switch [17] which results in Src activation, leading to EGF-receptor transactivation [23,26,30,48,49] possibly without ectodomain shedding, and through this to Erk1/2 activation. However, in COS-7 cells with transfected β 1 adrenoceptors, Erk1/2 activation is mediated via Gαs, but with no involvement of the EGF receptor [50]. ...
... We did not accumulate evidence for a role of ADRB2 in cell cycle progression but these effects seem to be highly cell type specific (29,30). It is nevertheless conceivable that HIC1 could inhibit cell cycle progression by two means: directly by repress-ing Cyclin D1 (8) but also indirectly by decreasing ADRB2 levels. ...
... In our experiments, we used isoproterenol, an adrenaline/ noradrenaline mimetic, to activate ADRB2 as previously described (16,20,28,29). Isoproterenol is also able to target ADRB1 and ADRB3. ...
The transcriptional repressor HIC1 (Hypermethylated in Cancer 1) is a tumor suppressor gene inactivated in many human cancers including breast carcinomas. In
this study, we show that HIC1 is a direct transcriptional repressor of β-2 adrenergic receptor (ADRB2). Through promoter luciferase activity, chromatin immunoprecipitation (ChIP) and sequential ChIP experiments, we demonstrate
that ADRB2 is a direct target gene of HIC1, endogenously in WI-38 cells and following HIC1 re-expression in breast cancer cells. Agonist-mediated
stimulation of ADRB2 increases the migration and invasion of highly malignant MDA-MB-231 breast cancer cells but these effects
are abolished following HIC1 re-expression or specific down-regulation of ADRB2 by siRNA treatment. Our results suggest that early inactivation of HIC1 in breast carcinomas could predispose to stress-induced metastasis through up-regulation of the β-2 adrenergic receptor.
... Three images were acquired and averaged together every 4 min for 20 min in control aCSF to establish a baseline and then for an additional 40 min with either NE (20 µM; A0937, Sigma-Aldrich), ISO (5 µM; I5627, Sigma-Aldrich), or control aCSF. Five µM ISO elicits astrocyte morphological changes (Vardjan et al., 2014;Sherpa et al., 2016), recruiting both β 1 -and β 2 -ARs to increase cAMP in astrocytes (Du et al., 2010). Such β-AR activation is observed during sustained NE elevation during vigilance tasks (Wahis and Holt, 2021) and is crucial for astrocyte participation in memory consolidation (Gao et al., 2016). ...
Glutamate spillover from the synapse is tightly regulated by astrocytes, limiting the activation of extrasynaptically located NMDA receptors (NMDAR). The processes of astrocytes are dynamic and can modulate synaptic physiology. Though norepinephrine (NE) and β-adrenergic receptor (β-AR) activity can modify astrocyte volume, this has yet to be confirmed outside of sensory cortical areas, nor has the effect of noradrenergic signaling on glutamate spillover and neuronal NMDAR activity been explored. We monitored changes to astrocyte process volume in response to noradrenergic agonists in the medial prefrontal cortex of male and female mice. Both NE and the β-AR agonist isoproterenol (ISO) increased process volume by ∼20%, significantly higher than changes seen when astrocytes had G-protein signaling blocked by GDPβS. We measured the effect of β-AR signaling on evoked NMDAR currents. While ISO did not affect single stimulus excitatory currents of Layer 5 pyramidal neurons, ISO reduced NMDAR currents evoked by 10 stimuli at 50 Hz, which elicits glutamate spillover, by 18%. After isolating extrasynaptic NMDARs by blocking synaptic NMDARs with the activity-dependent NMDAR blocker MK-801, ISO similarly reduced extrasynaptic NMDAR currents in response to 10 stimuli by 18%. Finally, blocking β-AR signaling in the astrocyte network by loading them with GDPβS reversed the ISO effect on 10 stimuli-evoked NMDAR currents. These results demonstrate that astrocyte β-AR activity reduces extrasynaptic NMDAR recruitment, suggesting that glutamate spillover is reduced.
... This may be an important observation that warrants further investigation. In addition, there are a number of drugs that can modulate ERK1/2 activity, including serotonin , amphetamines (Choe and Wang, 2002), isoproterenol (Du et al., 2010), and cocaine (Hoffmann et al., 2012). Whether these treatments alter the levels of sulfotransferases in the brain remains to be determined. ...
SULT4A1 is a brain-selective sulfotransferase-like protein that has recently been shown to be essential for normal neuronal development in mice. In the present study, SULT4A1 was found to co-localize with SULT1A1/3 in human brain neurons. Using immunoprecipitation, SULT4A1 was shown to interact with both SULT1A1 and 1A3 when expressed in human cells. Mutation of the conserved dimerization motif located in the C-terminus of the sulfotransferases prevented this interaction. Both ectopically expressed and endogenous SULT4A1 decreased SULT1A1/3 protein levels in neuronal cells, which was also prevented by mutation of the dimerization motif. During differentiation of neuronal SH-SY5Y cells, there was a loss in SULT1A1/3 protein but an increase in SULT4A1 protein. This resulted in an increase in the toxicity of dopamine, a substrate for SULT1A3. Inhibition of SULT4A1 using siRNA abrogated the loss in SULT1A1/3 and reversed dopamine toxicity. These results show a reciprocal relationship between SULT4A1 and the other sulfotransferases suggesting that it may act as a chaperone to control the expression of SULT1A1/3 in neuronal cells. SIGNIFICANCE STATEMENT: The catalytically inactive sulfotransferase SULT4A1 may regulate the function of other SULTs by interacting with them via a conserved dimerization motif. In neuron-like cells, SULT4A1 is able to modulate dopamine toxicity by interacting with SULT1A3, potentially decreasing the metabolism of dopamine.
... ATP, by itself, induced stellation by breaking down into adenosine, which stimulated P1 purinoceptors (Abe & Saito, 1999). Using primary cultures of mouse astrocytes, it has been demonstrated that at low concentrations, ISP acts on β 2 receptors to activate protein kinase A (PKA) activity which is required for Gs/Gi switching (Du et al., 2010). This is followed by Ca 2+ release from intracellular stores and G iα -and metalloproteinase-dependent transactivation of the EGFR, resulting in β-arrestin 1/2 recruitment and MAPK/ERK kinase-dependent ERK1/2 phosphorylation. ...
Thyroid hormones (THs) have important contributions to the development of the mammalian brain, targeting its actions on both neurons and glial cells. Astrocytes, which constitute about half of the glial cells, characteristically undergo dramatic changes in their morphology during development and such changes become necessary for the proper development of the brain. Interestingly, a large number of studies have suggested that THs play a profound role in such morphological maturation of the astrocytes. This review discusses the present knowledge on the mechanisms by which THs elicit progressive differentiation and maturation of the astrocytes. As a prelude, information on astrocyte morphology during development and its regulations, the role of THs in the various functions of astrocyte shall be dealt with for a thorough understanding of the subject of this review.
... The surplus of intracellular calcium binds to calmodulin (Cam) and phosphorylates eNOS to promote the synthesis of NO [19]. NO is released into smooth muscle cells where it binds to soluble guanylyl cyclase receptors (sGC) [7,4]. The activation of sGC increases cGMP production, activating protein kinase G (PKG) that phosphorylates myosin light chain kinase. ...
Peripheral arterial occlusive disease (PAOD) occurs due to the build up of atherosclerotic plaque and reduces blood flow to cause chronic ischemia. Patients with PAOD may experience intermittent claudication, or the pain in limb skeletal muscles due to a decease in blood flow. Collateral arteries can act as a natural bypass and improve blood flow to hypoxic tissue by creating an alternate route for blood to flow, but not all patients with PAOD have pre-existing collateral networks. Animal studies indicate that tissues without pre-existing collateral networks can form de novo collaterals from capillaries following occlusion of a feed artery. Unfortunately, theses de novo collaterals, termed arterialized collateral capillaries (ACCs) lack functional vasodilation at day-7 following feed artery occlusion. To induce the formation of ACCs, we ligated the lateral feed artery in the spinotrapzeius muscle in Balb/c mice. We evaluated the potential mechanism of impaired functional vasodilation in immature arterialized collateral capillaries (7 days following occlusion) by measuring endothelial-dependent vasodilation to bradykinin and endothelial-independent vasodilation to isoproterenol and sodium nitroprusside. Vasodilation to both the endothelial-dependent and endothelial-independent vasodilators was impaired in the immature ACCs as compared to the terminal arterioles on the unoperated sham side. Similar responses to the endothelial cell and smooth muscle cell-dependent vasodilations suggest that impaired functional vasodialtion is due to impaired vascular smooth muscle cell function, which is consistent with our preivous research. We speculate that the SMCs of the ACCs are immature and may still be remodeling, rearranging, or modulating phenotype in the newly formed collaterals. Determining factors to induce mature arterialized collateral capillaries in patients with PAOD lacking pre-existing collateral netoworks could reduce ischemia and improve prognosis.
... At a low isoproterenol concentration (B100 nM) b 2 -adrenergic (green arrows) stimulation activates Src via the function of b-arrestin 2. Src stimulates ERK 1/2 phosphorylation and also phosphorylates the EGF receptor without involvement of the receptor-tyrosine kinase. This ERK 1/2 phosphorylation is secondary to MEK activation, which probably is induced by direct activation of Raf by Src, although an effect on Ras cannot be excluded From [106] Neurochem Res (2017) 42:254-271 263 according to the revised method (including use of dBcAMP during the culturing) was measured by Hertz and Hertz [30] in total, intact cultures by the aid of an oxygen electrode inserted directly into the culture flask. The initial respiratory rate was intense (300 lmol per hr per 100 mg protein or with *200 mg protein per g wet wt. ...
Based on differences in gene expression between cultured astrocytes and freshly isolated brain astrocytes it has been claimed that cultured astrocytes poorly reflect the characteristics of their in vivo counterparts. This paper shows that this is not the case with the cultures of mouse astrocytes we have used since 1978. The culture is prepared following guidelines provided by Drs. Monique Sensenbrenner and John Booher, with the difference that dibutyryl cyclic AMP is added to the culture medium from the beginning of the third week. This addition has only minor effects on glucose and glutamate metabolism, but it is crucial for effects by elevated K(+) concentrations and for Ca(2+) homeostasis, important aspects of astrocyte function. Work by Liang Peng and her colleagues has shown identity between not only gene expression but also drug-induced gene upregulations and editings in astrocytes cultured by this method and astrocytes freshly isolated from brains of drug-treated animals. Dr. Norenberg's laboratory has demonstrated identical upregulation of the cotransporter NKCC1 in ammonia-exposed astrocytes and rats with liver failure. Similarity between cultured and freshly isolated astrocytes has also been shown in metabolism, K(+) uptake and several aspects of signaling. However, others have shown that the gene for the glutamate transporter GLT1 is not expressed, and rat cultures show some abnormalities in K(+) effects. Nevertheless, the overall reliability of the cultured cells is important because immunohistochemistry and in situ hybridization poorly demonstrate many astrocytic genes, e.g., those of nucleoside transporters, and even microarray analysis of isolated cells can be misleading.
... In clinical settings, PD-associated depression is treated with antidepressants, including selective serotonin reuptake inhibitors (SSRIs). Astrocytes express multiple neurotransmitter receptors, that include 5-HT 2 receptors to serotonin, and αand β-adrenergic receptors (Li et al., 2008a,b;Du et al., 2010;Ding et al., 2013). Previously, we reported that a SSRI fluoxetine acts as an agonist at 5-HT 2B receptors and induces transactivation of epidermal growth factor receptor (EGFR) in astrocytes (Li et al., 2008b). ...
Astrocytes contribute to pathogenesis of neuropsychiatric disorders, including major
depression. Stimulation of astroglial 5-HT2B receptors transactivates epidermal growth
factor receptors (EGFRs) and regulates gene expression. Previously we reported that
expression of 5-HT2B receptors in cortical astrocytes is down-regulated in animals, which
developed anhedonia in response to chronic stress; moreover this down-regulation
as well as anhedonia, are reversed by chronic treatment with fluoxetine. In this study
we have investigated whether astrocytic 5-HT2B receptor is involved in anhedonia in
C57BL/6 mice model of Parkinson’ disease (PD) induced by intraperitoneal injection of
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 7 days. The MPTP treatment
induced anhendonia in 66.7% of animals. The appearance of depressive behavior
was accompanied with motor deficiency and decrease of tyrosine hydroxylase (TH)
expression. Expression of mRNA and protein of 5-HT2B receptor in animals that became
anhedonic decreased to 77.3 and 79.3% of control groups, respectively; in animals that
received MPTP but did not develop anhedonia the expression of 5-HT2B receptor did
not change. Experiments with FACS-sorted isolated cells demonstrated that decrease
in 5-HT2B receptor expression was confined to astrocytes, and did not occur in
neurons. Fluoxetine corrected MPTP-induced decrease of 5-HT2B receptor expression
and depressive behavior. Our findings indicate that regulation of gene expression of 5-
HT2B receptors in astroglia may be associated with pathophysiological evolution of PDinduced
depression.
... Endogenous GPCR activation not only initiates signaling via heterotrimeric G proteins, but also recruits proteins of the arrestin family, which act as scaffolding proteins and promote G protein-independent signaling (Pierce et al., 2002;Rajagopal et al., 2010;Shukla et al., 2011). Research has shown that arrestin 3 (β-arrestin 2) is expressed in astrocytes ex vivo (Bruchas et al., 2006;McLennan et al., 2008), and it is involved in kappa opioid receptor (KOR)-induced proliferation (McLennan et al., 2008;Miyatake et al., 2009), reduction of chemical-induced apoptosis (Zhu and Reiser, 2014), CXCR7 mediated inflammatory response (Odemis et al., 2012;Lipfert et al., 2013) and beta 2-adrenergic receptor (β2AR)-mediated glycogenolysis (Dong et al., 2001;Du et al., 2010) in astrocytes. As a scaffolding protein, β-arrestins also mediate internalization and ubiquitylation for many ion channels and transporters expressed in astrocytes (Shukla et al., 2011). ...
Astrocytes are the predominant glial type in the central nervous system and play important roles in assisting neuronal function and network activity. Astrocytes exhibit complex signaling systems that are essential for their normal function and the homeostasis of the neural network. Altered signaling in astrocytes is closely associated with neurological and psychiatric diseases, suggesting tremendous therapeutic potential of these cells. To further understand astrocyte function in health and disease, it is important to study astrocytic signaling in vivo. In this review, we discuss molecular tools that enable the selective manipulation of astrocytic signaling, including the tools to selectively activate and inactivate astrocyte signaling in vivo. Lastly, we highlight a few tools in development that present strong potential for advancing our understanding of the role of astrocytes in physiology, behavior, and pathology.
... Reproduced fromDu T, Li B, Li H, Li M, Hertz L, Peng L. 2010. Signaling pathways of isoproterenol-induced ERK 1/2 phosphorylation in primary cultures of astrocytes are concentration-dependent. ...
The cotransporter of Na+, K+, 2Cl–, and water, NKKC1, is activated under two conditions in the brain, exposure to highly elevated extracellular K+ concentrations, causing astrocytic swelling, and regulatory volume increase in cells shrunk in response to exposure to hypertonic medium. NKCC1-mediated transport occurs as secondary active transport driven by Na+/K+-ATPase activity, which establishes a favorable ratio for NKCC1 operation between extracellular and intracellular products of the concentrations of Na+, K+, and Cl– × Cl–. In the adult brain, astrocytes are the main target for NKCC1 stimulation, and their Na+/K+-ATPase activity is stimulated by elevated K+ or the β-adrenergic agonist isoproterenol. Extracellular K+ concentration is normal during regulatory volume increase, so this study investigated whether the volume increase occurred faster in the presence of isoproterenol. Measurement of cell volume via live cell microscopic imaging fluorescence to record fluorescence intensity of calcein showed that this was the case at isoproterenol concentrations of ≥1 µM in well-differentiated mouse astrocyte cultures incubated in isotonic medium with 100 mM sucrose added. This stimulation was abolished by the β1-adrenergic antagonist betaxolol, but not by ICI118551, a β2-adrenergic antagonist. A large part of the β1-adrenergic signaling pathway in astrocytes is known. Inhibitors of this pathway as well as the glycogenolysis inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol hydrochloride and the NKCC1 inhibitors bumetanide and furosemide abolished stimulation by isoproterenol, and it was weakened by the Na+/K+-ATPase inhibitor ouabain. These observations are of physiological relevance because extracellular hypertonicity occurs during intense neuronal activity. This might trigger a regulatory volume increase, associated with the post-excitatory undershoot. © 2014 Wiley Periodicals, Inc.
... Previous reports showed that isoprenaline activation of β-ARs elicits an increase of p-ERK1/2 in different cell types (for review see [3]), such as HEK293 cells (present results and [29]), primary astrocytes [31] and cardiac [10] and dermal fibroblasts [32]. However, ERK1/2 phosphorylation was decreased in keratinocytes [33], corneal epithelial cells [33,34] and detrusor smooth muscle cells [35]. ...
β-adrenoceptors (β-ARs) modulate ERK1/2 and p38 in different cells, but little is known about the contribution of these signaling pathways to the function of β-ARs in vascular tissue. Immunoblotting analysis of rat aortic rings, primary endothelial (ECs) and smooth muscle cells (SMCs) isolated from aorta showed that β-AR stimulation with isoprenaline activated p38 in aortic rings and in both cultured cell types, whereas it had a dual effect on ERK1/2 phosphorylation, decreasing it in ECs while increasing it in SMCs. These effects were reversed by propranolol, which by itself increased p-ERK1/2 in ECs. Isoprenaline β-AR mediated vasodilation of aortic rings was potentiated by the ERK1/2 inhibitor, U0126, in the presence or absence of endothelium or L-NAME; whereas inhibition of p38 had no impact. Isoprenaline moderately decreased sprouting from aorta rings in the Matrigel angiogenesis assay; conversely propranolol not only prevented isoprenaline inhibition, but stimulated angiogenesis. ERK1/2 inhibition decreased angiogenesis, while a dramatic stimulation was observed by p38 blockade. Our results suggest that ERK1/2 activation after β-ARs stimulation in the smooth muscle hinders the vasodilator effect of isoprenaline, but in the endothelium β-ARs decreases ERK1/2 and increases p38 activity reducing therefore angiogenesis.
... Effects of transmitters operating via the protein kinase C (PKC) and the phosphatidylinositide signaling system might therefore become reduced primarily in astrocytes but possibly also in neurons by antibipolar drug treatment. Effects exerted via protein kinase A (PKA), like those of adrenaline acting on -adrenergic receptors, may also be affected in astrocytes on account of a G s /G i shift in their pathway [109]. This might include -adrenergic glycogenolysis, which is dependent on the increase in [Ca 2+ ] occurring after this shift. ...
Chronic treatment with fluoxetine or other so-called serotonin-specific reuptake inhibitor antidepressants (SSRIs) or with a lithium salt "lithium", carbamazepine, or valproic acid, the three classical antibipolar drugs, exerts a multitude of effects on astrocytes, which in turn modulate astrocyte-neuronal interactions and brain function. In the case of the SSRIs, they are to a large extent due to 5-HT2B-mediated upregulation and editing of genes. These alterations induce alteration in effects of cPLA2, GluK2, and the 5-HT2B receptor, probably including increases in both glucose metabolism and glycogen turnover, which in combination have therapeutic effect on major depression. The ability of increased levels of extracellular K(+) to increase [Ca(2+)] i is increased as a sign of increased K(+)-induced excitability in astrocytes. Acute anxiolytic drug treatment with benzodiazepines or GABAA receptor stimulation has similar glycogenolysis-enhancing effects. The antibipolar drugs induce intracellular alkalinization in astrocytes with lithium acting on one acid extruder and carbamazepine and valproic acid on a different acid extruder. They inhibit K(+)-induced and transmitter-induced increase of astrocytic [Ca(2+)] i and thereby probably excitability. In several cases, they exert different changes in gene expression than SSRIs, determined both in cultured astrocytes and in freshly isolated astrocytes from drug-treated animals.
... This transactivation was first reported by Daub et al. in 1996 [79]; following this initial report, other RTKs (receptor tyrosine kinases), such as PDGFR (platelet-derived growth factor receptor) and IGF-1R (insulin-like growth factor-1 receptor), have been shown to be transactivated. Importantly, this crosstalk mechanism has been found in a wide variety of cell types, including human keratinocytes, primary mouse astrocytes, PC-12 cells, human gastric cancer cells, ovarian cancer cell lines, VSMCs (vascular smooth muscle cells) and others [74,[80][81][82][83][84][85]. ...
GPCRs (G-protein-coupled receptors) are among the most important targets for drug discovery due to their ubiquitous expression and participation in cellular events under both healthy and disease conditions. These receptors can be activated by a plethora of ligands, such as ions, odorants, small ligands and peptides, including angiotensins and kinins, which are vasoactive peptides that are classically involved in the pathophysiology of cardiovascular events. These peptides and their corresponding GPCRs have been reported to play roles in other systems and under pathophysiological conditions, such as cancer, central nervous system disorders, metabolic dysfunction and bone resorption. More recently, new mechanisms have been described for the functional regulation of GPCRs, including the transactivation of other signal transduction receptors and the activation of G-protein-independent pathways. The existence of such alternative mechanisms for signal transduction and the discovery of agonists that can preferentially trigger one signalling pathway over other pathways (called biased agonists) have opened new perspectives for the discovery and development of drugs with a higher specificity of action and, therefore, fewer side effects. The present review summarizes the current knowledge on the non-canonical signalling and roles of angiotensins and kinins.
... In this particular case of transactivation, the priming of the EGFR activity via either the ligand stimulation or the overexpression of the receptor protein is also necessary [112]. Isoproterenol-induced transactivation of EGFR in astrocytes was also shown to involve Src-dependent Y845 phosphorylation; in this case, nanomolar concentrations, but not micromolar concentrations, of isoproterenol activation of β2-adrenergic receptor signaling are the trigger for this functional interaction, the astrocytic consequences of which are morphological differentiation and an increase in glial fibrillary acidic protein [113]. Transactivation of EGFR by α2-adrenergic receptor signaling in astrocytes in mature brain also involves Y845 phosphorylation [114]. ...
The Src gene product (Src) and the epidermal growth factor receptor (EGFR) are prototypes of oncogene products and function primarily as a cytoplasmic non-receptor tyrosine kinase and a transmembrane receptor tyrosine kinase, respectively. The identification of Src and EGFR, and the subsequent extensive investigations of these proteins have long provided cutting edge research in cancer and other molecular and cellular biological studies. In 1995, we reported that the human epidermoid carcinoma cells, A431, contain a small fraction of Src and EGFR in which these two kinase were in physical association with each other, and that Src phosphorylates EGFR on tyrosine 845 (Y845) in the Src-EGFR complex. Y845 of EGFR is located in the activation segment of the kinase domain, where many protein kinases contain kinase-activating autophosphorylation sites (e.g., cAMP-dependent protein kinase, Src family kinases, transmembrane receptor type tyrosine kinases) or trans-phosphorylation sites (e.g., cyclin-dependent protein kinase, mitogen-activated protein kinase, Akt protein kinase). A number of studies have demonstrated that Y845 phosphorylation serves an important role in cancer as well as normal cells. Here we compile the experimental facts involving Src phosphorylation of EGFR on Y845, by which cell proliferation, cell cycle control, mitochondrial regulation of cell metabolism, gamete activation and other cellular functions are regulated. We also discuss the physiological relevance, as well as structural insights of the Y845 phosphorylation.
... In mature cultured astrocytes stimulation of β 1 -adrenoceptors activates protein kinase A (via G s ) and causes an in increase in free cytosolic calcium concentration ([Ca 2+ ] i ) after a G s -G i switch Frontiers in Integrative Neuroscience www.frontiersin.org April 2013 | Volume 7 | Article 20 | 3 (Du et al., 2010), with the latter probably responsible for most of the glycogenolytic effect. Addition of 5 mM extracellular K + concentrations also increases [Ca 2+ ] i in astrocytes, secondary to an Na + ,K + -ATPase effect (Xu et al., 2013). ...
The involvement of glycogenolysis, occurring in astrocytes but not in neurons, in learning is undisputed (Duran et al., 2013). According to one school of thought the role of astrocytes for learning is restricted to supply of substrate for neuronal oxidative metabolism. The present "perspective" suggests a more comprehensive and complex role, made possible by lack of glycogen degradation, unless specifically induced by either (1) activation of astrocytic receptors, perhaps especially β-adrenergic or (2) even small increases in extracellular K(+) concentration above its normal resting level. It discusses (1) the known importance of glycogenolysis for glutamate formation, requiring pyruvate carboxylation; (2) the established role of K(+)-stimulated glycogenolysis for K(+) uptake in cultured astrocytes, which probably indicates that astrocytes are an integral part of cellular K(+) homeostasis in the brain in vivo; and (3) the plausible role of transmitter-induced glycogenolysis, stimulating Na(+),K(+)-ATPase/NKCC1 activity and thereby contributing both to the post-excitatory undershoot in extracellular K(+) concentration and the memory-enhancing effect of transmitter-mediated reduction of slow neuronal afterhyperpolarization (sAHP).
... Isoproterenol (ISO) is a synthetic catecholamine that is widely used for stimulation of all subtypes of βAR in cell [3] and animal model [4]. In the cultured cells, ISO-induced βAR stimulation activated ERK in cardiomyocytes [5] and astrocytes via PKA pathway [6]. In the rat aorta, 7 days of ISO treatment induced endothelial dysfunction and increased vasoconstriction [7]. ...
Beta adrenergic overstimulation may increase the vascular damage and stroke. However, the underlying mechanisms of beta adrenergic overstimulation in cerebrovascular dysfunctions are not well known. We investigated the possible cerebrovascular dysfunction response to isoproterenol induced beta-adrenergic overstimulation (ISO) in rabbit cerebral arteries (CAs).
ISO was induced in six weeks aged male New Zealand white rabbit (0.8-1.0 kg) by 7-days isoproterenol injection (300 μg/kg/day). We investigated the alteration of protein expression in ISO treated CAs using 2DE proteomics and western blot analysis. Systemic properties of 2DE proteomics result were analyzed using bioinformatics software. ROS generation and following DNA damage were assessed to evaluate deteriorative effect of ISO on CAs. Intracellular Ca(2+) level change and vascular contractile response to vasoactive drug, angiotensin II (Ang II), were assessed to evaluate functional alteration of ISO treated CAs. Ang II-induced ROS generation was assessed to evaluated involvement of ROS generation in CA contractility.
Proteomic analysis revealed remarkably decreased expression of cytoskeleton organizing proteins (e.g. actin related protein 1A and 2, α-actin, capping protein Z beta, and vimentin) and anti-oxidative stress proteins (e.g. heat shock protein 9A and stress-induced-phosphoprotein 1) in ISO-CAs. As a cause of dysregulation of actin-cytoskeleton organization, we found decreased level of RhoA and ROCK1, which are major regulators of actin-cytoskeleton organization. As functional consequences of proteomic alteration, we found the decreased transient Ca(2+) efflux and constriction response to angiotensin II and high K(+) in ISO-CAs. ISO also increased basal ROS generation and induced oxidative damage in CA; however, it decreased the Ang II-induced ROS generation rate. These results indicate that ISO disrupted actin cytoskeleton proteome network through down-regulation of RhoA/ROCK1 proteins and increased oxidative damage, which consequently led to contractile dysfunction in CA.
... In particular, at low b-agonist (ISO) concentrations , b2-adrenoceptors are activated resulting in b-arrestin-2-dependent activation of Src, which in turn activates the EGFR. However, at high agonist concentrations, b1-adrenoceptors transactivate EGFR via a Gi-proteinmediated pathway involving b-arrestin, MMP and HB-EGF in astrocytes (Du et al., 2010). In addition, b2-adrenoceptors can transactivate EGFR through a pathway similar to that of b1-adrenoceptors, as it involves Src, MMP and HB-EGF in cardiac fibroblasts (Kim et al., 2002), but is independent of MMP/HB-EGF in COS7 cells (Drube et al., 2006). ...
The MMPs and their inhibitors [tissue inhibitor of MMPs (TIMPs) ] form the mainstay of extracellular matrix homeostasis. They are expressed in response to numerous stimuli including cytokines and GPCR activation. This review highlights the importance of adrenoceptors and phosphoprotein phosphatases (PPP) in regulating MMPs in the cardiovascular system, which may help explain some of the beneficial effects of targeting the adrenoceptor system in tissue remodelling and will establish emerging crosstalk between these three systems. Although - and β-adrenoceptor activation increases MMP but decreases TIMP expression, MMPs are implicated in the growth stimulatory effects of adrenoceptor activation through transactivation of epidermal growth factor receptor. Furthermore, they have recently been found to catalyse the proteolysis of β-adrenoceptors and modulate vascular tone. While the mechanisms underpinning these effects are not well defined, reversible protein phosphorylation by kinases and phosphatases may be key. In particular, PPP (Ser/Thr phosphatases) are not only critical in resensitization and internalization of adrenoceptors but also modulate MMP expression. The interrelationship is complex as isoprenaline (ISO) inhibits okadaic acid [phosphoprotein phosphatase type 1/phosphoprotein phosphatase type 2A (PP2A) inhibitor]-mediated MMP expression. While this may be simply due to its ability to transiently increase PP2A activity, there is evidence for MMP-9 that ISO prevents okadaic acid-mediated expression of MMP-9 through a β-arrestin, NF-κB-dependent pathway, which is abolished by knock-down of PP2A. It is essential that crosstalk between MMPs, adrenoceptors and PPP are investigated further as it will provide important insight into how adrenoceptors modulate cardiovascular remodelling, and may identify new targets for pharmacological manipulation of the MMP system.
... Cyclic nucleotides produced by astrocytes play critical roles as second messengers in a variety of astroglial functions. For instance, increased levels of either cGMP or cAMP affect astrocyte morphology (e.g., soma retraction , process elongation, and branching) and their communication with neurons and blood vessel cells (Boran and Garcia, 2007; Pollenz and McCarty, 1986 ). Astrocytes are targets of several physiologically important agents that can elevate cyclic nucleotide levels, including nitric oxide (NO) and natriuretic peptides, which increase cGMP levels (Baltrons and Garc ıa, 2001; Baltrons et al., 2008; Prado et al., 2010; Zielinska et al., 2007 ), and b-adrenoreceptor agonists, pituitary adenylate cyclase-activating polypeptide, vasoactive intestinal peptide, adenosine, and ATP, which increase cAMP concentrations (Dar e et al., 2007; Du et al., 2010; Duffy and MacVicar, 1995; Jozwiak-Bebenista et al., 2007; Masmoudi-Kouki et al., 2007). Many of the effects exerted by these modulators have been linked to the activation of protein kinases, but we hypothesized that CNG channels represent an additional target for cyclic nucleotides in astrocytes, as they do in neurons. ...
Cyclic nucleotide-gated (CNG) channels are nonselective cation channels activated by cyclic AMP (cAMP) or cyclic GMP (cGMP). They were originally identified in retinal and olfactory receptors, but evidence has also emerged for their expression in several mammalian brain areas. Because cGMP and cAMP control important aspects of glial cell physiology, we wondered whether CNG channels are expressed in astrocytes, the most functionally relevant glial cells in the CNS. Immunoblot and immunofluorescence experiments demonstrated expression of the CNG channel olfactory-type A subunit, CNGA2, in cultured rat cortical astrocytes. In patch-clamp experiments, currents elicited in these cells by voltage ramps from -100 to +100 mV in the presence of the cGMP analogue, dB-cGMP, were significantly reduced by the CNG channel blockers, L-cis-diltiazem (LCD) and Cd(2+) . The reversal potentials of the LCD- and Cd(2+) -sensitive currents were more positive than that of K(+) , as expected for a mixed cation current. Noninactivating, voltage-independent currents were also elicited by extracellular application of the membrane permeant cGMP analogue, 8-Br-cGMP. These effects were blocked by LCD and were mimicked by natriuretic peptide receptor activation and inhibition of phosphodiesterase activity. Voltage-independent, LCD-sensitive currents were also elicited by 8-Br-cGMP in astrocytes of hippocampal and neocortical brain slices. Immunohistochemistry confirmed a broad distribution of CNG channels in astrocytes of the rat forebrain, midbrain, and hindbrain. These findings suggest that CNG channels are downstream targets of cyclic nucleotides in astrocytes, and they may be involved in the glial-mediated regulation of CNS functions under physiological and pathological conditions.
... The induced expression of the EGF receptor (EGFR) in progenitor cells during the neurogenic period resulted in an increase of cells that expressed the astrocyte markers S100b or GFAP prematurely [87]. Recently, Du et al. [88] suggested that EGFR phosphorylation and activation of MAPK/ERK pathways have consequences that may include morphological differentiation and increase in GFAP in primary cultures of mouse astrocytes. In fact, during normal embryonic cortical development, when progenitor cells begin to generate astrocytes in response to IL-6 and BMP families, there is an increase of EGFR, which regulates the competence of progenitors to interpret LIF as an astrocyte-inducing signal [89]. ...
Neuron-astroglia interactions play a key role in several events of brain development, such as neuronal generation, migration, survival, and differentiation; axonal growth; and synapse formation and function. While there is compelling evidence of the effects of astrocyte factors on neurons, their effects on astrocytes have not been fully determined. In this review, we will focus on the role of neurons in astrocyte generation and maturation. Further, we highlight the great heterogeneity and diversity of astroglial and neural progenitors such as radial glia cells, and discuss the importance of the variety of cellular interactions in controlling the structural and functional organization of the brain. Finally, we present recent data on a new role of astrocytes in neuronal maturation, as mediators of the action of biolipids in the cerebral cortex. We will argue that the functional architecture of the brain depends on an intimate neuron-glia partnership, by briefly discussing the emerging view of how neuron-astrocyte dysfunctions might be associated with neurodegenerative diseases and neurological disorders.
Spinal cord injury (SCI) is a serious central nervous system (CNS) trauma that results in permanent and severe disability. The extracellular matrix (ECM) can affect the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) by interacting with the ERK integrin subunits. In this study, we built a model of SCI with glial fibrillary acidic protein-green fluorescent protein (GFAP-GFP) and thymus cell antigen 1-yellow fluorescent protein-H (Thy1-YFPH) in mice that express specific transgenes in their astrocytes or neurons. Then, we collected spinal cord neurons or astrocytes by fluorescence-activated cell sorting (FACS). In this way, we investigated the SCI-induced phosphorylation of ERK1/2 and epidermal growth factor receptor (EGFR) in neurons and astrocytes, and we discovered that the SCI-induced EGFR signaling pathways differed between neurons and astrocytes. In the present study, we found that the Src-dependent phosphorylation of EGFR induced by SCI occurred only in neurons, not in astrocytes. This phenomenon may be due to the involvement of Thy-1, which promoted the binding between Src and EGFR in neurons after SCI. In addition, the expression of the integrin subunits after SCI differed between neurons and astrocytes. Our present study shows that the EGFR signaling pathway triggered by SCI in neurons differed from the EGFR signaling pathway triggered in astrocytes, a finding that may help to pave the way for clinical trials of therapies that inhibit EGFR signaling pathways after SCI.
This paper deals exclusively with gray matter astrocytes because the equally important white matter astrocytes are discussed elsewhere. α- and β-adrenergic receptors have repeatedly been demonstrated immunologically and autoradiographically, by determination of their mRNA or by effects on intracellular Ca²⁺ response. The predominant subtypes are α1A, α2A, and β1. Signaling by the two latter subtypes is complex and involves transactivation of the epidermal growth factor receptor and phosphorylation of extracellular regulated kinases 1/2 as well as Gs/Gi switch during β1-adrenergic signaling. Adrenergic signaling stimulates glucose metabolism at many different points as well as Na⁺, K⁺-ATPase activity. Intermittent stimulation of glycogen synthesis by α2-adrenergic activation and of glycogenolysis by β-adrenergic activity is essential for learning. Reduction of α-adrenergic activity is probably therapeutic in bipolar disorder, and impairment of adrenergic signaling may be causatively involved in Alzheimer’s disease. Compensation for missing adrenergic stimulation during growth of astrocytic cultures is important.
Astroglia express β1- and β2-adrenergic receptors. Stimulation of astrocytic β1-adreneergic receptors induces extracellular regulated kinase 1 and 2 (ERK1/2) phosphorylation via protein kinase A, Gs/Gi switching, Ca²⁺ release from intracellular stores, metalloproteinase-catalyzed release of growth factor, and transactivation of epidermal growth factor receptor; while stimulation of β2-adrenergic receptors induces ERK1/2 phosphorylation by β-arrestin-mediated Src activation, without the involvement of epidermal growth factor receptor activation. Brain edema after 3 h of focal ischemia followed by 8 h reperfusion can be prevented by antagonists of β1-adrenergic receptor and inhibitors of the associated signaling pathway, whereas inhibition of β2-adrenergic cascade has no effect. In astrocytes in primary cultures, stimulation of β1-adrenergic receptor increases the activity of both Na,K-ATPase and Na–K–Cl cotransporter NKCC1. Here we discuss mechanisms underlying the effects of β1-adrenergic receptor activation on brain edema, with particular emphasis on the signaling pathway of β1-adrenergic receptor, extracellular ions and mitogen-activated protein kinase/ERK1/2 cascade during ischemia and reperfusion periods.
In brain glycogen, formed from glucose, is degraded (glycogenolysis) in astrocytes but not in neurons. Although most of the degradation follows the same pathway as glucose, its breakdown product, L-lactate, is released from astrocytes in larger amounts than glucose when glycogenolysis is activated by noradrenaline. However, this is not the case when glycogenolysis is activated by high K⁺ concentrations – possibly because noradrenaline in contrast to high K⁺ stimulates glycogenolysis by an increase not only in free cytosolic Ca²⁺ concentration ([Ca²⁺]i) but also in cyclic AMP (c-AMP), which may increase the expression of the monocarboxylate transporter through which it is released. Several transmitters activate glycogenolysis in astrocytes and do so at different time points after training. This stimulation is essential for memory consolidation because glycogenolysis is necessary for uptake of K⁺ and stimulates formation of glutamate from glucose, and therefore is needed both for removal of increased extracellular K⁺ following neuronal excitation (which initially occurs into astrocytes) and for formation of transmitter glutamate and GABA. In addition the released L-lactate has effects on neurons which are essential for learning and for learning-related long-term potentiation (LTP), induction of the neuronal gene Arc/Arg3.1 and activation of gene cascades which are mediated by CREB and cofilin. Inhibition of glycogenolysis blocks learning and all related molecular events, but all changes can be reversed by injection of L-lactate. The effect of extracellular L-lactate is due to both astrocyte-mediated signaling which activates noradrenergic activity on all brain cells and to a minor uptake, possibly into dendritic spines.
The astrocytic Na+,K+-ATPase is important because increasing evidence indicates that increased extracellular K+ in brain following neuronal excitation initially is accumulated into astrocytes. This is due to higher Na+,K+-ATPase activity in astrocytes than in neurons and because the extracellular K+-sensitive site of the astrocytic Na+,K+-ATPase, in contrast to that of the neuronal enzyme, has low enough affinity for K+ to be further activated by increased K+ concentrations. However, K+ must eventually be re-accumulated into neurons in order to prevent neuronal K+ depletion. Accumulated astrocytic K+ is released through Kir4.1 channels, but a presently unsolved problem is how renewed astrocytic uptake is prevented. Experiments in well-differentiated cultured astrocytes providing a solution of this problem are discussed. At the same time subunit composition of the astrocytic Na+,K+-ATPase and its influence on the enzyme’s kinetic parameters is reviewed together with stimulation of the enzyme by noradrenaline and its functional importance. So are details of Na+,K+-ATPase signaling in response to submicromolar concentrations of ouabain and/or low mM K+ concentrations without which the catalytic activity of the astrocytic enzyme is abolished. Two pathophysiological conditions are discussed, cerebral ischemia/reperfusion and hepatic encephalopathy. In the former ouabain signaling dependence on extracellular Ca2+ is crucial and provides therapeutic possibilities. In the latter the ability of NH4+ to mimic K+ in both catalytic and signaling effects of the Na+,K+-ATPase is essential. In both conditions it is important that operation of the Na+, K+, Cl− and water cotransporter NKCC1 is dependent upon ion gradients created by the Na+,K+-ATPase.
β-arrestins, important soluble proteins mediating receptor desensitization, have diverse biological functions, such as regulating cell proliferation, cell survival, apoptosis and gene transcription. β-arrestins regulate inflammatory and immune reactions by inhibiting the basal activity of pro-inflammatory transcription factor NF-kB and participating in Toll-like receptors (TLR)/NF-kB signal pathway-mediated NF-kB activation. β-arrestins are involved in the pathogenesis of various inflammatory diseases, such as inflammatory bowel disease, rheumatoid arthritis, ankylosing spondylitis, asthma and so on. The study on β-arrestins will reveal the mechanisms of inflammatory diseases and provide new strategy for clinical treatment. Here we reviewed the recent progress on the role of β-arrestins in regulating the inflammatory diseases.
The Lund project (1992) recommended treatment with clonidine (α2-adrenergic agonist) and metoprolol (β1-adrenergic antagonist) to improve recovery after brain trauma, and discouraged use of the V1 agonist vasopressin (ADH). Brain effects of these drugs and the ability of a post-traumatic elevation of extracellular K+ concentrations ([K+]o) to activate mechanism(s) leading to secondary cytotoxic (cellular) edema were then virtually unknown. Now, it is established that interactions occur between effects on astrocytes by high [K+]o and vasopressin or α2 and β1-adrenergic agonists and antagonists, and that the effects modify edema and thus intracranial pressure. In mouse astrocytes in primary cultures, reliably expressing characteristics of their in vivo counterparts, high [K+]o and each of the transmitters agonists activate a signal mechanism, transactivation, in which Ca2+ entry through depolarization-mediated channel opening or stimulation of Gq-or Gi/o protein-coupled receptors via PKC-, Ca2+-and metalloproteinase- mediated signaling leads to release of an epidermal growth factor (EGF) receptor agonist. Minor, but important, differences exist between individual pathways. The agonist released by dexmedetomidine decreases neuronal vulnerability to oxidative damage by a paracrine effect, and in all cases the released EGF receptor agonist has autocrine effects. These include mitogen-activated protein (MAP) kinase-mediated phosphorylation of astrocytic extracellular-regulated kinase (ERK), and with high [K+]o also the cotransporter NKCC1, accumulating Na+ and K+ together with 2 Cl-and water, causing edema. This effect, exerted specifically on astrocytes, is enhanced by β1-adrenergic or vasopressinergic V1 signaling, explaining the beneficial effect of β1-adrenergic antagonists and why vasopressin should be omitted in edema treatment after brain trauma.
Both ATP and glutamate are on one hand essential metabolites in brain and on the other serve a signaling function as transmitters. However, there is the major difference that the flux in the pathway producing transmitter glutamate is comparable to the rate of glucose metabolism in brain, whereas that producing transmitter ATP is orders of magnitude smaller than the metabolic turnover between ATP and ADP. Moreover, de novo glutamate production occurs exclusively in astrocytes, whereas transmitter ATP is produced both in neurons and astrocytes. This chapter deals only with ATP and exclusively with its formation and release in astrocytes, and it focuses on potential associations with glycogenolysis, which is known to be indispensable for the synthesis of glutamate. Glycogenolysis is dependent upon an increase in free intracellular Ca(2+) concentration (Ca(2+)]i). It can be further stimulated by cAMP, but in contrast to widespread beliefs, cAMP can on its own not induce glycogenolysis. Astrocytes generate ATP from accumulated adenosine, and this process does not seem to require glycogenolysis. A minor amount of the generated ATP is utilized as a transmitter, and its synthesis requires the presence of the mainly intracellular nucleoside transporter ENT3. Many transmitters as well as extracellular K(+) concentrations high enough to open the voltage-sensitive L-channels for Ca(2+) cause a release of transmitter ATP from astrocytes. Adenosine and ATP induce release of ATP by action at several different purinergic receptors. The release evoked by transmitters or elevated K(+) concentrations is abolished by DAB, an inhibitor of glycogenolysis.
Until the demonstration little more than 20 years ago that glycogenolysis occurs during normal whisker stimulation glycogenolysis was regarded as a relatively uninteresting emergency procedure. Since then, a series of important astrocytic functions has been shown to be critically dependent on glycogenolytic activity to support the signaling mechanisms necessary for these functions to operate. This applies to glutamate formation and uptake and to release of ATP as a transmitter, stimulated by other transmitters or elevated K(+) concentrations and affecting not only other astrocytes but also most other brain cells. It is also relevant for astrocytic K(+) uptake both during the period when the extracellular K(+) concentration is still elevated after neuronal excitation, and capable of stimulating glycogenolytic activity, and during the subsequent undershoot after intense neuronal activity, when glycogenolysis may be stimulated by noradrenaline. Both elevated K(+) concentrations and several transmitters, including the β-adrenergic agonist isoproterenol and vasopressin increase free cytosolic Ca(2+) concentration in astrocytes, which stimulates phosphorylase kinase so that it activates the transformation of the inactive glycogen phosphorylase a to the active phosphorylase b. Contrary to common belief cyclic AMP plays at most a facilitatory role, and only when free cytosolic Ca(2+) concentration is also increased. Cyclic AMP is not increased during activation of glycogenolysis by either elevated K(+) concentrations or the stimulation of the serotonergic 5-HT2B receptor. Not all agents that stimulate glycogenolysis do so by directly activating phophorylase kinase-some do so by activating processes requiring glycogenolysis, e.g. for synthesis of glutamate.
Chronic treatment with anti-bipolar drugs (lithium, carbamazepine, and valproic acid) down-regulates mRNA and protein expression of kainate receptor GluK2 in mouse brain and cultured astrocytes. It also abolishes glutamate-mediated, Ca(2+)-dependent ERK(1/2) phosphorylation in the astrocytes. Chronic treatment with the SSRI fluoxetine enhances astrocytic GluK2 expression, but increases mRNA editing, abolishing glutamate-mediated ERK(1/2) phosphorylation and [Ca(2+)](i) increase, which are shown to be GluK2-mediated. Neither drug group affects Glu4/Glu5 expression necessary for GluK2's ionotropic effect. Consistent with a metabotropic effect, the PKC inhibitor GF 109203X and the IP(3) inhibitor xestospongin C abolish glutamate stimulation in cultured astrocytes. In CA1/CA3 pyramidal cells in hippocampal slices, activation of extrasynaptic GluK2 receptors, presumably including astrocytic, metabotropic GluK2 receptors, causes long-lasting inhibition of slow neuronal afterhyperpolarization mediated by Ca(2+)-dependent K(+) flux. This may be secondary to the induced astrocytic [Ca(2+)](i) increase, causing release of 'gliotransmitter' glutamate. Neuronal NMDA receptors respond to astrocytic glutamate release with enhancement of excitatory glutamatergic activity. Since reduction of NMDA receptor activity is known to have antidepressant effect in bipolar depression and major depression, these observations suggest that the inactivation of astrocytic GluK2 activity by antidepressant/anti-bipolar therapy ameliorates depression by inhibiting astrocytic glutamate release. A resultant strengthening of neuronal afterhyperpolarization may cause reduced NMDA-mediated activity.
The betagamma subunits of heterotrimeric G proteins play important roles in regulating receptor-stimulated signal transduction processes. Recently appreciated among these is their role in the signaling events that lead to the phosphorylation and subsequent desensitization of muscarinic cholinergic (Haga, K., and Haga, T. (1992) J. Biol. Chem. 267, 2222-2227) and beta-adrenergic (Pitcher, J. A., Inglese, J., Higgins, J. B., Arriza, J. L., Casey, P. J., Kim, C., Benovic, J. L., Kwatra, M. M., Caron, M. G., and Lefkowitz, R. J. (1992) Science 257, 1264-1267) receptors. Betagamma mediates the membrane targeting of the beta-adrenergic receptor kinase (betaARK), in response to receptor activation, through a specific betaARK-betagamma interaction. This process utilizes the membrane-anchoring properties of the isoprenylated gamma subunit of betagamma. In the present study, we have employed three distinct approaches to identify the region within the carboxyl terminus of betaARK which binds betagamma and thereby results in membrane translocation. We studied the ability of betagamma to enhance the enzymatic activity of a series of truncated mutants of bovine betaARK1, the ability of glutathione S-transferase fusion proteins containing various lengths of the carboxyl terminus of betaARK to bind betagamma subunits, and the ability of synthetic peptides comprised of betaARK sequences to inhibit betagamma activation of betaARK1. We find that the minimal betagamma binding domain of betaARK is localized to a 125-amino acid residue stretch, the distal end of which is located 19 residues from the carboxyl terminus. A single 28-mer peptide (Trp643 to Ser670) derived from this sequence effectively inhibited betagamma activation of betaARK1, with an IC50 of 76 muM. The identification of this ''betagamma binding domain'' on betaARK and the development of peptide inhibitors provide important tools for the study of G protein-coupled receptor desensitization, as well as for the investigation of betagamma activation of other G protein-effector systems.
Upon their discovery, β-arrestins 1 and 2 were named for their capacity to sterically hinder the G protein coupling of agonist-activated seven-transmembrane receptors, ultimately resulting in receptor desensitization. Surprisingly, recent evidence shows that β-arrestins can also function to activate signaling cascades independently of G protein activation. By serving as multiprotein scaffolds, the β-arrestins bring elements of specific signaling pathways into close proximity. β-Arrestin regulation has been demonstrated for an ever-increasing number of signaling molecules, including the mitogen-activated protein kinases ERK, JNK, and p38 as well as Akt, PI3 kinase, and RhoA. In addition, investigators are discovering new roles for β-arrestins in nuclear functions. Here, we review the signaling capacities of these versatile adapter molecules and discuss the possible implications for cellular processes such as chemotaxis and apoptosis.
Activation of β-adrenoreceptors induces cardiomyocyte hypertrophy. In the present study, we examined isoproterenol-evoked
intracellular signal transduction pathways leading to activation of extracellular signal-regulated kinases (ERKs) and cardiomyocyte
hypertrophy. Inhibitors for cAMP and protein kinase A (PKA) abolished isoproterenol-evoked ERK activation, suggesting that
Gs protein is involved in the activation. Inhibition of Gi protein by pertussis toxin, however, also suppressed isoproterenol-induced ERK activation. Overexpression of the Gβγ subunit binding domain of the β-adrenoreceptor kinase 1 and of COOH-terminal Src kinase, which inhibit functions of Gβγ and the Src family tyrosine kinases, respectively, also inhibited isoproterenol-induced ERK activation. Overexpression of
dominant-negative mutants of Ras and Raf-1 kinase and of the β-adrenoreceptor mutant that lacks phosphorylation sites by PKA
abolished isoproterenol-stimulated ERK activation. The isoproterenol-induced increase in protein synthesis was also suppressed
by inhibitors for PKA, Gi, tyrosine kinases, or Ras. These results suggest that isoproterenol induces ERK activation and cardiomyocyte hypertrophy
through two different G proteins, Gs and Gi. cAMP-dependent PKA activation through Gs may phosphorylate the β-adrenoreceptor, leading to coupling of the receptor from Gs to Gi. Activation of Giactivates ERKs through Gβγ, Src family tyrosine kinases, Ras, and Raf-1 kinase.
Insulin resistance, a hallmark of type 2 diabetes, is a defect of insulin in stimulating insulin receptor signalling, which has become one of the most serious public health threats. Upon stimulation by insulin, insulin receptor recruits and phosphorylates insulin receptor substrate proteins, leading to activation of the phosphatidylinositol-3-OH kinase (PI(3)K)-Akt pathway. Activated Akt phosphorylates downstream kinases and transcription factors, thus mediating most of the metabolic actions of insulin. Beta-arrestins mediate biological functions of G-protein-coupled receptors by linking activated receptors with distinct sets of accessory and effecter proteins, thereby determining the specificity, efficiency and capacity of signals. Here we show that in diabetic mouse models, beta-arrestin-2 is severely downregulated. Knockdown of beta-arrestin-2 exacerbates insulin resistance, whereas administration of beta-arrestin-2 restores insulin sensitivity in mice. Further investigation reveals that insulin stimulates the formation of a new beta-arrestin-2 signal complex, in which beta-arrestin-2 scaffolds Akt and Src to insulin receptor. Loss or dysfunction of beta-arrestin-2 results in deficiency of this signal complex and disturbance of insulin signalling in vivo, thereby contributing to the development of insulin resistance and progression of type 2 diabetes. Our findings provide new insight into the molecular pathogenesis of insulin resistance, and implicate new preventive and therapeutic strategies against insulin resistance and type 2 diabetes.
The role of epidermal growth factor (EGF) receptor autophosphorylation sites in the regulation of receptor functions has been studied using cells transfected with mutant EGF receptors. Simultaneous point mutation of 4 tyrosines (Y1068, Y1086, Y1148, Y1173) to phenylalanine, as well as removal of these sites by truncation of the carboxyl-terminal 123 amino acid residues, resulted in reduced receptor phosphorylation of an in vivo specific substrate phospholipase C-gamma 1 to less than 50% compared to the wild-type receptor. The internalization rate constant Ke was also significantly lower in these mutants (0.15/min) compared to cells transfected with wild-type receptor (0.27/min). Additional mutation of tyrosine 992 to phenylalanine in the truncated receptor mutant (Dc-123F) further decreased the receptor internalization rate to a minimal level (ke = 0.07-0.10/min), equivalent to the ke measured for cells expressing kinase-negative receptor (A721). Moreover, tyrosine kinase activity of the Dc-123F receptor toward phospholipase C-gamma 1, compared to wild-type receptor, was reduced by 90%. Taken together, these results show that EGF receptor lacking five autophosphorylation sites functions similar to a kinase-negative receptor. Mutation of tyrosine residue Y992 alone in the context of full length EGF receptor, however, did not affect receptor internalization or kinase activity toward phospholipase C-gamma 1. These data indicate that tyrosine 992 is critical for substrate phosphorylation and internalization only in the context of the truncated receptor, and that minor autophosphorylation sites, such as Y992, may act as compensatory regulatory sties in the absence of the major EGF receptor autophosphorylation sites.
Autophagic sequestration of endogenous lactate dehydrogenase or electroinjected [3H]raffinose in isolated rat hepatocytes was strongly suppressed by the Ca2+ chelator EGTA, unless the cells had previously been electroloaded in the presence of high concentrations of Ca2+ (1.2 mM). The extracellular Ca2+ chelator bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) and the intracellular Ca2+ chelator BAPTA/tetra(acetoxymethyl)-ester (BAPTA/AM) both inhibited autophagy to the same extent as did EGTA. Inhibitors of Ca(2+)-activated protein kinases (KN-62, H-7, W-7) had little or no effect on autophagy, indicating that the Ca2+ requirement of autophagy was not mediated by such kinases. Agents that elevate cytosolic Ca2+ by releasing Ca2+ from intracellular stores, like thapsigargin, 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) and the ionophores A23187 and ionomycin, inhibited autophagy strongly, implicating depletion of sequestered rather than of cytosolic intracellular Ca2+ as a common mechanism of inhibition. Lysosomal (propylamine-sensitive) protein degradation, known to be largely autophagy-dependent, was inhibited by thapsigargin and tBuBHQ. Thapsigargin had no effect on cellular ATP levels, but all agents tested (thapsigargin, tBuBHQ, ionophores) inhibited protein synthesis. Our results suggest that autophagy, like protein synthesis, is dependent on the presence of Ca2+ in some intracellular storage compartment.
Stimulation of human neutrophils by the receptor agonist N-formylmethionyl-leucyl-phenylalanine (fMLP) results in a respiratory burst, catalysed by an NADPH oxidase. Concomitantly, phospholipase D (PLD) is activated. To investigate the role of protein kinase C (PKC) in these neutrophil responses, we have compared the effects of staurosporine and a structural analogue of staurosporine (cgp41251), that reflects a higher selectivity towards PKC. Both staurosporine and cgp41251 dose-dependently inhibited the production of superoxide induced by phorbol 12-myristate 13-acetate (PMA). Both compounds also caused inhibition of the fMLP-induced respiratory burst, but with a lower efficacy during the initiation phase of this response. This latter observation cannot be taken as evidence against PKC involvement in the activation of the respiratory burst, because pretreatment of neutrophils with ionomycin before PMA stimulation also results in a lower efficacy of inhibition. Activation of PLD by fMLP was enhanced in the presence of staurosporine, but not in the presence of cgp41251. Enhancement of PLD activation was also observed in the presence of H-89, an inhibitor of cyclic-AMP-dependent protein kinase (PKA). Both staurosporine and H-89 reversed the dibutyryl-cyclic-AMP-induced inhibition of PLD activation whereas cgp41251 was without effect. These results indicate that the potentiating effect of staurosporine on PLD activation induced by fMLP does not reflect a feedback inhibition by PKC activation, but instead a feedback inhibition by PKA activation. Taken together, our results indicate that in human neutrophils: (i) PKC activity is not essential for fMLP-induced activation of PLD; (ii) PKC activity does play an essential role in the activation of the respiratory burst by fMLP, other than mediating or modulating PLD activation; (iii) there exists a negative-feedback mechanism on fMLP-induced PLD activation by concomitant activation of PKA.
The βγ subunits of heterotrimeric G proteins play important roles in regulating receptor-stimulated signal transduction processes. Recently appreciated among these is their role in the signaling events that lead to the phosphorylation and subsequent desensitization of muscarinic cholinergic (Haga, K., and Haga, T. (1992) J. Biol. Chem. 267, 2222-2227) and β-adrenergic (Pitcher, J. A., Inglese, J., Higgins, J. B., Arriza, J. L., Casey, P. J., Kim, C., Benovic, J. L., Kwatra, M. M., Caron, M. G., and Lefkowitz, R. J. (1992) Science 257, 1264-1267) receptors, βγ mediates the membrane targeting of the β-adrenergic receptor kinase (βARK), in response to receptor activation, through a specific βARK-βγ interaction. This process utilizes the membrane-anchoring properties of the isoprenylated γ subunit of βγ. In the present study, we have employed three distinct approaches to identify the region within the carboxyl terminus of βARK which binds βγ and thereby results in membrane translocation. We studied the ability of βγ to enhance the enzymatic activity of a series of truncated mutants of bovine βARK1, the ability of glutathione S-transferase fusion proteins containing various lengths of the carboxyl terminus of βARK to bind βγ subunits, and the ability of synthetic peptides comprised of βARK sequences to inhibit βγ activation of βARK1. We find that the minimal βγ binding domain of βARK is localized to a 125-amino acid residue stretch, the distal end of which is located 19 residues from the carboxyl terminus. A single 28-mer peptide (Trp643 to Ser670) derived from this sequence effectively inhibited βγ activation of βARK1, with an IC50 of 76 μM. The identification of this "βγ binding domain" on βARK and the development of peptide inhibitors provide important tools for the study of G protein-coupled receptor desensitization, as well as for the investigation of βγ activation of other G protein-effector systems.
Multipin peptide synthesis has been employed to produce biotinylated 11-mer phosphopeptides that account for every tyrosine residue in insulin receptor substrate-1 (IRS-1) and the cytoplasmic domains of the insulin-, epidermal growth factor-, platelet-derived growth factor- and basic fibroblast growth factor receptors. These phosphopeptides have been screened for their capacity to bind to the SH2 domains of Shc and Grb in a solution phase enzyme-linked immunosorbent assay. The data revealed new potential Grb2 binding sites at Tyr-1114 (epidermal growth factor receptor (EGFR) C-tail); Tyr-743 (platelet-derived growth factor receptor (PDGFR) insert region), Tyr-1110 from the E-helix of the catalytic domain of insulin receptor (IR), and Tyr-47, Tyr-939, and Tyr-727 in IRS-1. None of the phosphopeptides from the juxtamembrane or C-tail regions of IR bound Grb2 significantly, and only one phosphopeptide from the basic fibroblast growth factor receptor (Tyr-556) bound Grb2 but with medium strength. Tyr-1068 and -1086 from the C-tail of EGFR, Tyr-684 from the kinase insert region of PDGFR, and Tyr-895 from IRS-1 were confirmed as major binding sites for the Grb2 SH2 domain. With regard to Shc binding, the data revealed new potential binding sites at Tyr-703 and Tyr-789 from the catalytic domain of EGFR and at Tyr-557 in the juxtamembrane region of PDGFR. It also identified new potential Shc binding sites at Tyr-764, in the C-tail of basic fibroblast growth factor receptor, and Tyr-960, in the juxtamembrane of IR, a residue previously known to be required for Shc phosphorylation in response to insulin. The study confirmed the previous identification of Tyr-992 and Tyr-1173 in the C-tail of EGFR and several phosphopeptides from the PDGFR as medium strength binding sites for the SH2 domain of Shc. None of the 34 phosphopeptides from IRS-1 bound Shc strongly, although Tyr-690 showed medium strength binding. The specificity characteristics of the SH2 domains of Grb2 and Shc are discussed. This systematic peptide mapping strategy provides a way of rapidly scanning candidate proteins for potential SH2 binding sites as a first step to establishing their involvement in kinase-mediated signaling pathways.
Epidermal growth factor receptor (EGFR) signaling was analyzed in mammalian cells conditionally defective for receptor-mediated
endocytosis. EGF-dependent cell proliferation was enhanced in endocytosis-defective cells. However, early EGF-dependent signaling
events were not uniformly up-regulated. A subset of signal transducers required the normal endocytic trafficking of EGFR for
full activation. Thus, endocytic trafficking of activated EGFR plays a critical role not only in attenuating EGFR signaling
but also in establishing and controlling specific signaling pathways.
PC12 cells respond to a variety of external stimuli such as growth factors, neurotransmitters, and membrane depolarization
by activating the Ras/mitogen-activated protein kinase pathway. Here we demonstrate that both depolarization-induced calcium
influx and treatment with bradykinin stimulate tyrosine phosphorylation of the epidermal growth factor receptor (EGFR). Using
a tetracycline-controlled expression system in conjunction with a dominant-negative EGFR mutant, we demonstrate that depolarization
and bradykinin triggered signals involve EGFR function upstream of SHC and MAP kinase. Furthermore, bradykinin-stimulated
EGFR transactivation is critically dependent on the presence of extracellular calcium, and when triggered by ionophore treatment,
calcium influx is already sufficient to induce EGFR tyrosine phosphorylation. Taken together, our results establish calcium-dependent
EGFR transactivation as a signaling mechanism mediating activation of the Ras/mitogen-activated protein kinase pathway in
neuronal cell types.
Rap1 is a small, Ras-like GTPase that was first identified as a protein that could suppress the oncogenic transformation of cells by Ras. Rap1 is activated by several extracellular stimuli and may be involved in cellular processes such as cell proliferation, cell differentiation, T-cell anergy and platelet activation. At least three different second messengers, namely diacylglycerol, calcium and cyclic AMP, are able to activate Rap1 by promoting its release of the guanine nucleotide GDP and its binding to GTP. Here we report that activation of Rap1 by forskolin and cAMP occurs independently of protein kinase A (also known as cAMP-activated protein kinase). We have cloned the gene encoding a guanine-nucleotide-exchange factor (GEF) which we have named Epac (exchange protein directly activated by cAMP). This protein contains a cAMP-binding site and a domain that is homologous to domains of known GEFs for Ras and Rap1. Epac binds cAMP in vitro and exhibits in vivo and in vitro GEF activity towards Rap1. cAMP strongly induces the GEF activity of Epac towards Rap1 both in vivo and in vitro. We conclude that Epac is a GEF for Rap1 that is regulated directly by cAMP and that Epac is a new target protein for cAMP.
Ligand binding to the EGF receptor initiates both the activation of mitogenic signal transduction pathways plus trafficking events that relocalize the receptor on the cell surface and within intracellular compartments. The trafficking compartments include caveolae, clathrin-coated pits, and various endosome populations prior to receptor degradation in lysosomes. Evidence is presented that distinct signaling pathways are initiated from these different compartments. These include the Ras/MAP kinase cascade and the PLC-dependent hydrolysis of PI-4,5 P2. Multiple tyrosine kinase substrates that facilitate EGF receptor trafficking between these various compartments, as well as the participation of phosphoinositides and Ras-like G proteins in the trafficking pathway are also described. BioEssays 22:697–707, 2000.
Many of the G-grotein-coupled receptors for hormones that bind to the cell surface can signal to the interior of the cell through several different classes of G protein(1-4). Far example, although most of the actions of the prototype beta(2)-adrenergic receptor are mediated through G(s) proteins and thr cyclic-AMP-dependent protein kinase (PKA) system(5,6), beta-adrenergis receptors can also couple to G(i) proteins(1,2). Here we investigate the mechanism that controls the specificity of this coupling. We show that in HEK293 cells, stimulation of mitogen-activated protein (MAP) kinase by the beta(2)-adrenergic receptor is mediated by the beta gamma subunits of pertussis-toxin-sensitive G proteins through a pathway involving the non-receptor tyrosine kinase c-Src and the G protein Ras, Activation of this pathway by the beta(2)-adrenergic receptor requires that the receptor be phosphorylated by PKA because it is blocked by H-89, an inhibitor of PKA. Additionally, a mutant of the receptor, which lacks the sites normally phosphorylated by PKA, can activate adenylyl cyclase(5), the enzyme that generates cAMP, but not MAP kinase. Our results demonstrate that a mechanism previously shown to mediate uncoupling of the beta(2)-adrenergic receptor from G(s) and thus heterologous desensitization(7) (PKA-mediated receptor phosphorylation), also serves to 'switch' coupling of this receptor from G(s) to G(i) and initiate a new set of signalling events.
Homeostasis of multicellular organisms is critically dependent on the correct interpretation of the plethora of signals which cells are exposed to during their lifespan. Various soluble factors regulate the activation state of cellular receptors which are coupled to a complex signal transduction network that ultimately generates signals defining the required biological response. The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases represents both key regulators of normal cellular development as well as critical players in a variety of pathophysiological phenomena. The aim of this review is to give a broad overview of signal transduction networks that are controlled by the EGFR superfamily of receptors in health and disease and its application for target-selective therapeutic intervention. Since the EGFR and HER2 were recently identified as critical players in the transduction of signals by a variety of cell surface receptors, such as G-protein-coupled receptors and integrins, our special focus is the mechanisms and significance of the interconnectivity between heterologous signalling systems.
Astroglial β-adrenergic receptors (β-ARs) are functionally linked to regulate cellular morphology. In primary cultures, the β-AR agonist isoproterenol (ISP) can transform flat polygonal astrocytes into process-bearing, mature stellate cells by 48 h, an effect that can be blocked by the β-AR antagonist, propranolol. ISP induced immediate activation of protein kinase A (PKA) which persisted up to 2 h, with no visible change in cell morphology. However, activation of PKA was sufficient to drive the process of transformation to completion, suggesting the involvement of downstream regulators of PKA. In addition to PKA inhibitors, the mitogen-activated protein kinase (MAPK) kinase inhibitor PD098059 also blocked ISP-induced morphological transformation. ISP treatment resulted in a biphasic response of cellular phosphorylated MAPK (phosphorylated extracellular signal-regulated kinase; p-ERK) level: an initial decline in p-ERK level followed by a sustained induction at 12–24 h, both of which were blocked by PKA inhibitor. The induction in pERK level coincided with initiation of morphological differentiation of the astrocytes and nuclear translocation of p-ERK. A long-lasting activation of p-ERK activity by ISP, at a later stage, appears to be critical for the transformation of astrocytes.
: Secondary microcultures of newborn rat cerebrum astroglial (AG) cells, maintained in a serum-free, chemically defined medium, were treated with various agents known to elevate intracellular cyclic AMP (cAMP) levels. Earlier studies had shown these drugs to induce a process-bearing (stellate) morphology in the AG cells, a response that was antagonized by the presence of gangliosides. One millimolar dibutyryl cyclic AMP (dBcAMP), 10 μM forskolin, 12 nM cholera toxin, and 30 μM isoproterenol all raised intracellular cAMP levels, from basal values of 3 pmol/106 cells to 30–30,000 pmol/106 cells, depending on the agent tested. dBcAMP caused the greatest elevation, and forskolin the least. The timing and/or the level of the AMP response did not precisely correlate with those of the stella-tion response. Values of ED50 with the four agents, as determined for the cAMP response, were always higher than stellation ED50 values in all treatments, and ED50 did not correlate with the maximal levels of cyclic AMP induced by the four agents. The capacity of ganglioside GMl to block the stellation response to the four agents was not accompanied by a similar capacity to block the cAMP responses. Lysophosphatidylserine (lysoPS) had the capacity to induce AG cell stellation as well, without altering the basal level of cAMP. Both lysoPS and gangliosides, therefore, may act directly on the cellular machinery underlying the stellation response without involving changes in intracellular AMP.
The developing cerebral cortex is likely to exhibit synaptic circuitries differing from those in adulthood, due to the asynchronous maturation of the various neurotransmitter systems. Two antisera directed against mammalian β-adrenergic receptors (βAR), βAR248 and βAR404, were used to characterize the laminar, cellular, and subcellular distributions of βAR in postnatally developing visual cortex of rats. The antigenic sites were the receptor's third intracellular loop for βAR248 and the C-terminus for βAR404. During week 1, most of the βAR404- and βAR248-immunoreactive sites were dendritic. Morphologically identifiable synapses were rare, even in layer 1: yet, semiquantitative analysis revealed that βAR404-immunoreactive synapses comprise half of those in layer 1. During week 2, the two antisera began to diverge in their immunoreactivity patterns. With βAR248, there was an overall decline in immunoreactivity, while with βAR404, there was an increase in immunoreactive sites, primarily due to labeled astrocytic processes that increased 200-fold in areal density by week 3. In contrast, the areal density of synaptic labeling by βAR404 barely doubled, in spite of the 30-fold increase in areal density of synapses. These results suggest that βAR undergo conformational changes during early postnatal periods, causing alterations in their relative antigenicity to the two antisera. Furthermore, the first 2 weeks appear to be characterized by modulation of earliest-formed synapses, and the subsequent phase is marked by addition of astrocytic responses that would be more diffuse temporally and spatially. Activation of βAR is recognized to increase visually evoked activity relative to spontaneous activity. Moreover, astrocytic βAR are documented to regulate extracellular concentrations of glutamate, ATP, and neurotrophic factors important for the formation of binocular connections. Thus, neuronal and astrocytic responses may, together and in tandem, facilitate strengthening of intracortical synaptic circuitry during early life.
This chapter describes transactivation as a process, in which signaling to a G-protein coupled receptor (GPCR) leads to the release of a growth factor that in turn stimulates the receptor tyrosine-kinases (RTK) on the same or adjacent cells and evokes a response that is indistinguishable from that which can be directly obtained by direct RTK stimulation. It discusses that transactivation plays a major role in not only astrocytic functions but also in interactions between astrocytes and neurons, including astrocyte-mediated neuroprotection. The chapter reviews that transactivation may be specific for the epithelial growth factor receptor EGFR and not a phenomenon common to many growth factor receptors. However, different GPCRs can lead to EGFR transactivation, albeit probably utilizing different transduction pathways in the donor cells, as exemplified by similar effects of the group I metabotropic glutamate receptor agonist, [RS ]-3,5-dihydroxyphenylglycine, and the α2A-adrenergic agonist dexmedetomidine. Both drugs were found to act on astrocytes and these cells are likely to be a major source of soluble heparin-binding EGF-like growth factor (HB-EGF), since GPCRs are related to EGF activation and HB-EGF are expressed at high level in astrocytes in primary cultures and in the brain in vivo, and since expression of the HB-EGF gene may mainly occur in astrocytes in neurodegenerative diseases.
During recent years it has become increasingly clear that classical neurotransmitters such as monoamines and the amino acids glutamate and GABA affect the development and subsequent functional differentiation of neurons and astrocytes. The present review focuses on an outline of effects of these substances on cell morphology, enzyme activities and receptor expression. Moreover, the mechanisms for these effects are discussed and it is concluded that receptor activation is likely to be coupled to second messenger systems and regulation of intracellular Ca2+ levels.
Src-related kinases have been recently implicated in signaling from Gi-coupled receptors to MAP kinase. Whether Src-like kinases participate in MAP kinase activation by the large family of receptors coupled to G proteins of the Gq family is still unclear. Here, we show that a specific inhibitor for Src-like kinases, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1), and dominant negative mutants of Src suppress MAP kinase activation in COS-7 cells when elicited by either m1 and m2 muscarinic receptors, which are typical Gq and Gi-coupled receptors, respectively. Furthermore, activation of MAP kinase by overexpression of beta gamma subunits, but not by stimulation with phorbol esters was also inhibited by the dominant-negative Src. In contrast, a dominant negative Pyk2 had only mild effects on m1 and m2 mediated-MAP kinase activation. We concluded that Src like kinase(s), acting downstream from beta gamma dimers, play an important role relaying signals from both Gq and Gi-coupled receptors to MAP kinase.
EGF receptor transactivation has been known for more than ten years. It is a signal pathway in which a G-protein-coupled receptor (GPCR) signal leads to release of a growth factor, which in turn activates the EGF receptor-tyrosine kinase in the same or adjacent cells. Astrocytes express a number of GPCRs and play key roles in brain function. Astrocytic transactivation is of special interest, since its autocrine effect may regulate gene expression and alter cell functions in the cells themselves and its paracrine effect may provide additional opportunities for cross-talk between astrocytes and their neighbors, such as neurons. The signal pathways of EGF transactivation are complicated. This does not only apply to the pathways leading to shedding of growth factor(s), but also to the downstream signal pathways of the EGF receptor, i.e., MAPK and PI3K. The latter may vary according to the type of growth factor released, the sites of tyrosine phosphorylation on the EGF receptor, and the duration of the phosphorylation. Using primary cell cultures we have found that dexmedetomidine, a specific alpha(2)-adrenergic receptor, induced shedding of HB-EGF from astrocytes, which in turn transactivated EGF receptors and stimulated astrocytic c-Fos and FosB expression. At the same time released HB-EGF protected neurons from injury caused by H(2)O(2). We have also confirmed dexmedetomidine transactivation in the brain in vivo. EGF transactivation by 5-HT(2B) receptor stimulation was responsible for up-regulation of cPLA(2) in astrocytes by fluoxetine, an antidepressant and inhibitor of the serotonin transporter, which also is a specific 5-HT(2B) agonist.
Recent in vivo studies have established astrocytes as a major target for locus coeruleus activation (Bekar et al., 2008), renewing interest in cell culture studies on noradrenergic effects on astrocytes in primary cultures and calling for additional information about the expression of adrenoceptor subtypes on different types of brain cells. In the present communication, mRNA expression of alpha(1)-, alpha(2)- and beta-adrenergic receptors and their subtypes was determined in freshly isolated, cell marker-defined populations of astrocytes, NG2-positive cells, microglia, endothelial cells, and Thy1-positive neurons (mainly glutamatergic projection neurons) in murine cerebral cortex. Immediately after dissection of frontal, parietal and occipital cortex of 10-12-week-old transgenic mice, which combined each cell-type marker with a specific fluorescent signal, the tissue was digested, triturated and centrifuged, yielding a solution of dissociated cells of all types, which were separated by fluorescence-activated cell sorting (FACS). mRNA expression in each cell fraction was determined by microarray analysis. alpha(1A)-Receptors were unequivocally expressed in astrocytes and NG2-positive cells, but absent in other cell types, and alpha(1B)-receptors were not expressed in any cell population. Among alpha(2)-receptors only alpha(2A)-receptors were expressed, unequivocally in astrocytes and NG-positive cells, tentatively in microglia and questionably in Thy1-positive neurons and endothelial cells. beta(1)-Receptors were unequivocally expressed in astrocytes, tentatively in microglia, and questionably in neurons and endothelial cells, whereas beta(2)-adrenergic receptors showed tentative expression in neurons and astrocytes and unequivocal expression in other cell types. This distribution was supported by immunochemical data and its relevance established by previous studies in well-differentiated primary cultures of mouse astrocytes, showing that stimulation of alpha(2)-adrenoceptors increases glycogen formation and oxidative metabolism, the latter by a mechanism depending on intramitochondrial Ca(2+), whereas alpha(1)-adrenoceptor stimulation enhances glutamate uptake, and beta-adrenoceptor activation causes glycogenolysis and increased Na(+), K(+)-ATPase activity. The Ca(2+)- and cAMP-mediated association between energy-consuming and energy-yielding processes is emphasized.
Evidence accumulates for a key role of the beta(2)-adrenergic receptors in the many homeostatic and neuroprotective functions of astrocytes, including glycogen metabolism, regulation of immune responses, release of neurotrophic factors, and the astrogliosis that occurs in response to neuronal injury. A dysregulation of the astrocytic beta(2)-adrenergic-pathway is suspected to contribute to the physiopathology of a number of prevalent and devastating neurological conditions such as multiple sclerosis, Alzheimer's disease, human immunodeficiency virus encephalitis, stroke and hepatic encephalopathy. In this review we focus on the physiological functions of astrocytic beta(2)-adrenergic receptors, and their possible impact in disease states.
Our previous work demonstrated dexmedetomidine-activated phosphorylation of extracellular regulated kinases 1 and 2 (ERK(1/2)) in primary cultures of mouse astrocytes and showed that it is evoked by alpha(2)-adrenoceptor-mediated transactivation of epidermal growth factor (EGF) receptors, a known response to activation of G(i/o)- or G(q)-coupled receptors [Li, B., Du, T., Li, H., Gu, L., Zhang, H., Huang, J., Hertz, L., Peng, L., 2008a. Signaling pathways for transactivation by dexmedetomidine of epidermal growth factor receptors in astrocytes and its paracrine effect on neurons. Br. J. Pharmacol. 154, 191-203]. Like most studies of transactivation, that study used cultured cells, raising the question whether a similar effect can be demonstrated in intact brain tissue and the brain in vivo. In the present study we have shown that (i) dexmedetomidine-mediated ERK(1/2) phosphorylation occurs in mouse brain slices with a similar concentration dependence as in cultured astrocytes (near-maximum effect at 50nM); (ii) intraperitoneal injection of dexmedetomidine (3microg/kg) in adult mice causes rapid phosphorylation of the EGF receptor (at Y845 and Y992) and of ERK(1/2) in the brain; (iii) both EGF receptor and ERK(1/2) phosphorylation are inhibited by intraventricular administration of (a) AG 1478, a specific inhibitor of the receptor-tyrosine kinase of the EGF receptor; (b) GM 6001, an inhibitor of metalloproteinase(s) required for release of EGF receptor agonists from membrane-bound precursors; or (c) heparin, neutralizing heparin-binding EGF (HB-EGF). Thus, in intact brain HB-EGF, known to be expressed in brain, may be the major EGF agonist released in response to stimulation of alpha(2)-adrenoceptors, the released agonist(s) activate(s) EGF receptors, and ERK(1/2) is phosphorylated as a conventional response to EGF receptor activation. Our previous paper (see above) showed that dexmedetomidine evokes no ERK(1/2) phosphorylation in cultured neurons, but neurons respond to astrocyte-conditioned medium (and to EGF) with ERK(1/2) phosphorylation. The present findings therefore suggest that EGF receptor transactivation in astrocytes in the mature brain in vivo is an important process in response to alpha(2)-adrenoceptor stimulation and may lead to phosphorylation of ERK(1/2) both in astrocytes themselves and in adjacent neurons.
beta-Adrenergic receptors can activate extracellular signal-regulated kinases (ERKs) via different mechanisms. In this study, we investigated the molecular mechanism of beta1-adrenergic receptor (beta1AR)-mediated ERK activation in African green monkey kidney COS-7 cells. Treatment of cells with isoproterenol (ISO), a beta1AR selective agonist, induced phosphorylation of ERK1/2 in a dose-dependent manner. ISO-stimulated ERK phosphorylation was not influenced by the Gbetagamma inhibitor, betaAR kinase carboxyl terminal (betaARKct) or by the Gi inhibitor, pertussis toxin (PTX), but it was clearly abolished via inhibition of protein kinase A (PKA) with H89, or of mitogen-activated protein kinase kinase (MEK1) with PD98059, revealing that the Galphas subunit is involved in ERK regulation through the PKA/MEK1 pathway. We also tested the effect of the adenylate cyclase activator forskolin on ERK activation, and the result was identical to that of ISO stimulation. Moreover, pretreatment with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478 or with the Src tyrosine kinase inhibitor PP2 did not affect ERK activation. These observations suggest a mechanism of beta1AR-mediated ERK activity that involves the Galphas subunit, but not EGFR or Src tyrosine kinase.
Fluoxetine has relatively high affinity for Gq/11 protein-coupled 5-HT(2) receptors. Part of these receptors in brain are on astrocytes, where fluoxetine causes an increase in free cytosolic calcium concentration ([Ca(2+)](i)) and phosphorylation of extracellular regulated kinase 1 and 2 (ERK(1/2)).
The objectives of the study are to identify subtype of the 5-HT(2) receptor involved, to establish whether ERK(1/2) phosphorylation is a result of 5-HT(2)-mediated transactivation of epidermal growth factor (EGF) receptors (EGFRs), and to determine signaling pathways up- and downstream of ERK(1/2).
Primary cultures of mouse astrocytes, which express all three subtypes of the 5-HT(2) receptor but no 5-HT(2) transporter, were used. ERK(1/2) phosphorylation and c-Fos and FosB protein expression were determined with Western blotting, and c-fos and fosB mRNA expression with reverse transcription polymerase chain reaction. Receptor subtype was investigated with subtype-specific 5-HT antagonists and 5-HT(2B) receptor depletion and signaling pathways by EGFR phosphorylation, using immunoprecipitation and Western blotting, inhibition of protein kinase C (PKC), and [Ca(2+)](i) chelation by BAPTA/AM.
ERK(1/2) phosphorylation was abolished by SB204741, a universal 5-HT(2) receptor antagonist, and in 5-HT(2B) receptor-depleted cells, but unaffected by 5-HT(2A) or 5-HT(2C) receptor antagonists (M100907 and SB242084). Phosphorylation of ERK(1/2) and EGFRs was abolished by AG 1478, an inhibitor of EGFR tyrosine kinases, and GM 6001, an inhibitor of Zn-dependent metalloproteinases, suggesting growth factor "shedding" and transactivation of EGFRs. Chelation of [Ca(2+)](i) or PKC inhibition with GF 109203X abrogated ERK(1/2) phosphorylation. Up-regulated mRNA and protein expression of c-fos and fosB was abolished by SB204741, AG1478, and by U0126, an inhibitor of ERK phosphorylation by MAP kinase/ERK kinase.
The development of (Na⁺+K⁺) ATPase, carbonic anhydrase and HCO3⁻-stimulated ATPase activity was studied in developing rat brain in vivo, and in primary astrocyte cultures from 1–3-day-old rat brain as a function of increasing cell growth. The primary cultures showed an increase in all the above enzyme activities during cell growth, with time courses which were qualitatively similar to their development in vivo. Cell cultures grown separately from the cerebellum plus brain stem regions showed greater carbonic anhydrase activity than cerebral cultures over the entire 4-week growth period, correspondincg to development of this activity in these same regions in vivo. HCO3⁻-stimulated ATPase activity was slightly greater in cerebellar cultures and (Na⁺+K⁺) ATPase activity was greater in cerebral cultures up to the second week of growth, resembling development of the same enzyme activities in vivo. C6 glioma and neuroblastoma cells showed no and 10-fold lower carbonic anhydrase activities respectively, compared to the primary astrocyte cultures.
[3H]-Dihydroalprenolol ([3H]-DHA) binds to cerebral membranes of the frog, chick, rat, mouse, rabbit and human with a dissociation equilibrium constant (KD) of about 1 nM and displays binding characteristics indicative of an interaction with beta-adrenoceptors. However, the maximum number of specific binding sites labelled by this beta-adrenoceptor ligand varies substantially between the species with the chick and mouse having the highest, and the frog the lowest density. The structure--activity relationships of adrenergic agents to inhibit specific [3H]-DHA binding suggests that whereas the membrane sites from all the species had similar affinities for non-selective beta-adrenergic agents, several drugs that have been reported to show selectivity for beta1-adrenoceptors demonstrated considerably higher affinities for mammalian rather than avian or amphibian membrane sites. By this pharmacological criteria it is likely that all the beta-adrenoceptor binding sites in frog and chick cerebral tissue have properties resembling beta2-receptors. However, in mammalian cerebral cortex, evidence is presented that beta1- and beta2-adrenoceptors coexist in a ratio of 70%/30% respectively.
—Addition of norepinephrine or isoproterenol to primary cultures started from the brains of 1-3 day old rats caused up to 200-fold increases in cAMP levels, which reached a maximum by 5-10 min and then declined. This effect was studied in detail for norepinephrine. The rise in cAMP levels was followed by morphological changes, in which up to 65% of the cells exhibited an astrocyte-like morphology, and 2-3 fold increases in carbonic anhydrase and (Na+-K+) ATPase activities. However, morphological transformation also occurred after much smaller increases in total cAMP levels. These effects on cell morphology and enzyme activities reached a maximum 1-2 h after addition of norepinephrine and then declined. Carbonic anhydrase activity was found both in the particulate and post 100,000 g supernatant fractions from homogenates of these cultured cells, and in the latter case the activity was activated 3-fold by addition of cAMP. The significance of these obscrvations on the cellular localization of, and functional role for similar increases in cAMP in brain tissue is discussed.
Kinetics for uptake and release of glutamate were measured in normal, i.e., nontransformed, astrocytes in cultures obtained from the dissociated, cortexenriched superficial parts of the brain hemispheres of newborn DBA mice. The uptake kinetics indicated a minor, unsaturable component together with an intense uptake following Michaelis-Menten kinetics. TheK
m
(50 μM) was reasonably comparable to the corresponding values in brain slices and in other glial preparations. TheV
max (58.8 nmol min−1 mg−1 protein) was, however, much higher than that observed in glial cell lines or peripheral satellite cells, and also considerably higher than that generally reported for brain slices. The release of glutamate was much smaller than the uptake, and only little affected by an increase of the external glutamate concentration, suggesting a net accumulation of glutamate rather than a homoexchange. Such an intense accumulation of glutamate into normal astrocytes may play a major role in brain metabolism and may help keep the extracellular glutamate cohcentration below excitatory levels.
In recent years evidence has accumulated indicating the presence of functional receptors for most neurotransmitters on astrocytes. In particular, receptors coupled to adenylate cyclase have been demonstrated, in primary astrocyte cultures, for vasoactive intestinal peptide (VIP), noradrenaline (NA) and adenosine. Here we provide, in primary cultures of cerebral cortical astrocytes prepared from neonatal mice, a detailed characterization of a cAMP-dependent process elicited by VIP, NA and adenosine, i.e. the hydrolysis of glycogen. The EC50s for the glycogenolytic effect of VIP, NA and adenosine are 3, 20 and 800 nM, respectively. The initial rate of glycogen hydrolysis is, in nmol/mg prot/min, 9.1 for VIP and 7.5 for NA. The effect of NA is predominantly mediated by beta-adrenoceptors, although an alpha 1-adrenergic component, acting most likely through protein kinase C activation, is also present. The action of VIP is mimicked by peptides sharing sequence homologies such as PHI and secretin. Glutamate, GABA, carbachol and the peptides NPY and somatostatin do not influence glycogen levels. The glycogen content of the cultures can be markedly increased by anabolic factors present in fetal calf serum, by high (e.g. 25 mM) glucose in the medium and by 48-h pretreatment of the cultures with dibutyryl cAMP. These results indicate that the glycogen content of astrocytes is under the dynamic control of various factors, including certain neurotransmitters. They also further stress the notion of a functional interaction between neurons and glial cells aimed at maintaining local energy metabolism homeostasis.
Elevation of the extracellular potassium concentration above its "resting" level of 5.4 mM stimulated uptake of 45Ca2+ in primary cultures of astrocytes. This effect was only observed when cells were exposed to excess potassium shortly after their exposure to 45Ca2+ and was potently inhibited (IC50 congruent to 3 nM) by the calcium channel blocker nimodipine. In contrast, nimodipine exerted little effect on unstimulated basal uptake of 45Ca2+. These findings suggest that the therapeutic benefit of calcium channel blockers in epilepsy may result in part from the ability of these drugs to prevent calcium entry into astrocytes during seizures when the extracellular potassium is elevated four- to fivefold above normal.
The adenylate cyclase system consists of stimulatory and inhibitory hormone and drug receptors coupled through different GTP-binding proteins to a catalytic unit, responsible for the synthesis of cAMP from ATP. Pertussis toxin blocks the effect of inhibitory agonists on the catalytic unit by enzymatically inactivating the inhibitory GTP-binding protein (Gi). Study of the inhibitory arm of the cyclase system has been facilitated by the dissection of the overall process of hormonal inhibition of cAMP formation into a series of reactions characteristic of the individual protein components of this complex system; pertussis toxin has proven to be a useful tool with which to study these individual reactions. Exposure of cells or membranes to pertussis toxin in the presence of NAD results in ADP-ribosylation of a 41,000 Da subunit of Gi. ADP-ribosylation of Gi has a number of effects on the overall and partial reactions of the cyclase system, including a loss of a) hormonal inhibition of cAMP formation, b) hormonal stimulation of GTPase and c) agonist-induced release of membrane-bound guanyl nucleotides. In addition, in toxin-treated membranes, the affinity of inhibitory receptors for agonist but not antagonist is decreased with no significant change in receptor number.
The agonist specificity pattern of the beta-adrenergic adenylate cyclase in glial primary cultures was not typical of either beta 1- or beta 2-adrenergic receptors. The dose-response curves for adrenaline did not correspond to simple mass action kinetics and their computer analysis suggests the presence of both beta 1- and beta 2-adrenergic-sensitive adenylate cyclase (58 plus or minus 17% and 42 plus or minus 17% respectively). Similar properties of beta 1- and beta 2-adrenergic-sensitive adenylate cyclases were found by computer analysis of the dose-response curves for isoprenaline in the presence of a constant concentration of practolol (a selective beta 1 antagonist) (55 plus or minus 10% and 45 plus or minus 10% of beta 1- and beta 2-sensitive adenylate cyclase respectively). The curves for displacement of [3H]dihydroalprenolol by practolol confirm these results. For purpose of comparison, the beta-adrenergic receptors of meningeal cells in cultures were subjected to similar analysis. The results clearly showed that these cells exclusively contained beta 2-adrenergic receptors.
Beta-Adrenergic receptors were studied in intact cells of chick, rat and mouse embryo brain in primary cultures, by the specific binding of [3H]dihydro-L-alprenolol ([3H]DHA). The results were compared to the receptor binding of broken cell preparations derived from the cell cultures or from the forebrain tissues used for the preparation of the cultures. Detailed analysis of [3H]DHA binding to living chick brain cells revealed a high-affinity, stereoselective, beta-adrenergic-type binding site. Equilibrium measurements indicated the apparent positive cooperativity of the binding reaction. By direct fitting of the Hill equation to the measured data, values of Bmax = 12.01 fmol/10(6) cells (7200 sites/cell), Kd = 60.23 pM and the Hill coefficient n = 2.78 were found. The apparent cooperative character of the binding was confirmed by the kinetics of competition with L-alprenolol, resulting in maximum curves at low ligand concentrations. The rate constants of the binding reaction were estimated as k+ = 8.31 X 10(7) M-1 X min-1 and k- = 0.28 min-1 from the association results, and k- = 0.24 min-1 from the dissociation data. The association kinetics supported the cooperativity of the binding, providing a Hill coefficient n = 1.76; Kd, as (k-/k+)1/n was found to be 101 pM. Analysis of the equilibrium binding of [3H]DHA to rat and mouse living brain cells resulted in values of Bmax = 13.04 fmol/10(6) cells (7800 sites/cell), Kd = 43.85 pM and n = 2.52, and Bmax = 8.08 fmol/10(6) cells (4800 sites/cell), Kd = 46.70 pM and n = 1.63, respectively, confirming the apparent cooperativity of the beta-receptor in mammalian objects, too. The [3H]DHA equilibrium binding to broken cell preparations of either chick, rat or mouse brain cultures or forebrain tissues was found to be non-cooperative, with a Hill coefficient n = 1, Kd in the range 1-2 nM, and a Bmax of 10(3) - 10(4) sites/cell. Our findings demonstrate that cell disruption causes marked changes in the kinetics of the beta-receptor binding and in the affinity of the binding site, although the number of receptors remains unchanged.
The beta-receptors of intact neuronal and glial cells of chick embryonic brain were studied via the specific binding of the beta-antagonist [3H]dihydro-L-alprenolol ( [3H]DHA). Cells were cultivated in either highly homogeneous or mixed populations; the neuronal cells were also grown under the influence of glial conditioned medium (GCM) or 10(-11)-10(-10) M L-norepinephrine or L-isoproterenol. The beta-receptors of both neuronal and glial cells proved to be positively cooperative (n = 2.5) and of high affinity, with a Kdapp of 98 and 44 pM, respectively. The Kdapp value was influenced only slightly by the different culture conditions. The receptor concentration was relatively low in the homogeneous neuronal and glial cultures (Bmax = 6.4 and 3.3 fmol/10(6) cells, respectively). It increased by a factor of 2-3 if development of the neuron-glia contacts in the culture was possible (mixed cultures). GCM and beta-agonists elevated the number of beta-receptors of the neuronal cells approximately 4-fold, even in the absence of glial cells. This receptor-number change was preceded by a well observable morphological differentiation. Both the morphological and the beta-receptor effects of L-norepinephrine were antagonized by L-propranolol. The beta-receptor number increased about 2-fold during a 10-day in vitro development, even in neuron-glia mixed cultures.
The effects of the β 1 ‐ and β 2 ‐adrenoceptor selective antagonists, CGP 20712A and ICI 118551 respectively, on responses to isoprenaline‐induced relaxation of rat distal colon were investigated in order to determine the contributions of these subtypes to relaxation. In addition, the properties of ICI D7114, a novel putative stimulant of atypical β‐adrenoceptors, were investigated. Our preliminary experiments with ICI D7114 showed that this compound lacked agonist activity in rat distal colon and in fact antagonized responses to isoprenaline. We therefore studied the antagonism of isoprenaline by ICI D7114 in more detail.
Longitudinal segments of rat distal colon were suspended in Krebs solution at 37°C for isometric recording. The Krebs solution contained EDTA (23 μ m ) and prazosin (0.1 μ m ) and was gassed with 95/5% O 2 /CO 2 . After an initial equilibration period, reproducible contractions to a submaximal concentration of methacholine (1 μ m ) were obtained before carrying out a concentration‐response curve (CRC) to isoprenaline in a non‐cumulative manner. Four consecutive CRCs to isoprenaline were carried out in each tissue with a 1 h interval between each curve. Antagonists were present in increasing concentrations during the intervals between CRCs. Control tissues received no antagonists to allow estimation of the magnitude of time‐dependent changes.
Isoprenaline produced a concentration‐dependent inhibition of methacholine‐induced contractions. CRCs to isoprenaline were reproducible with no significant time‐dependent changes. Propranolol produced no shift of the isoprenaline CRC at 0.01 μ m and a 5 fold shift at 0.1 μ m . No further shift was observed with 1 μ m . CGP 20712A had no effect on the CRC to isoprenaline at 0.1, 1 and 3 μ m . ICI 118551 produced little or no shift at 0.1 μ m and a six fold shift with 1 μ m . No further shift was observed with 3 μ m . ICI D7114 produced a concentration‐dependent parallel rightward shift of the CRC to isoprenaline. Schild analysis gave a slope close to unity and a mean pA 2 value of 7.29 for ICI D7114.
The results with propranolol and β 1 ‐ and β 2 ‐adrenoceptor antagonists confirm the mainly atypical nature of β‐adrenoceptors in rat distal colon. There may also be a small contribution from β 2 ‐adrenoceptors in the response to isoprenaline but β 1 ‐adrenoceptors are absent. ICI D7114 has no agonist activity and behaves as a relatively high affinity reversible competitive antagonist of atypical β‐adrenoceptors in this preparation.
The reverse transcription/polymerase chain reaction was used to demonstrate beta 3-adrenoceptor mRNA in rat brain regions. Levels were highest in hippocampus, cerebral cortex and striatum and lower in hypothalamus, brainstem and cerebellum.
The benzodiazepines diazepam and midazolam at submicromolar concentrations potentiated the increase in free cytosolic calcium concentration in astrocytes in primary cultures evoked by an elevation of the extracellular potassium concentration ([K+]0), but they had little stimulatory effect at normal [K+]0 and none at maximally elevated [K+]0. Nifedipine, an inhibitor of the L-channel, counteracted both the effect of the elevated [K+]0 as such and the benzodiazepine modulation. PK 11195, an antagonist of the peripheral-type benzodiazepine receptor, counteracted the effect of the benzodiazepines, but had no effect on the increase in free cytosolic calcium evoked by the elevated [K+]0.
The c-Raf-1 protein kinase plays a critical role in intracellular signaling downstream from many tyrosine kinase and G-protein-linked receptors. c-Raf-1 binds to the proto-oncogene Ras in a GTP-dependent manner, but the exact mechanism of activation of c-Raf-1 by Ras is still unclear. We have established a system to study the activation of c-Raf-1 in vitro. This involves mixing membranes from cells expressing oncogenic H-RasG12V, with cytosol from cells expressing epitope-tagged full-length wild-type c-Raf-1. This results in a fraction of the c-Raf-1 binding to the membranes and a concomitant 10- to 20-fold increase in specific activity. Ras was the only component in these membranes required for activation, as purified recombinant farnesylated K-Ras.GTP, but not non-farnesylated K-Ras.GTP or farnesylated K-Ras.GDP, was able to activate c-Raf-1 to the same degree as intact H-RasG12V membranes. The most potent activation occurred under conditions in which phosphorylation was prohibited. Under phosphorylation-permissive conditions, activation of c-Raf-1 by Ras was substantially inhibited. Consistent with the results from other groups, we find that the activation of c-Raf-1 by Src in vivo occurs concomitant with tyrosine phosphorylation on c-Raf-1, and in vitro, activation of c-Raf-1 by Src requires the presence of ATP. Therefore we propose that activation of c-Raf-1 by Ras or by Src occurs through different mechanisms.
Many of the G-protein-coupled receptors for hormones that bind to the cell surface can signal to the interior of the cell through several different classes of G protein. For example, although most of the actions of the prototype beta2-adrenergic receptor are mediated through Gs proteins and the cyclic-AMP-dependent protein kinase (PKA) system, beta-adrenergic receptors can also couple to Gi proteins. Here we investigate the mechanism that controls the specificity of this coupling. We show that in HEK293 cells, stimulation of mitogen-activated protein (MAP) kinase by the beta2-adrenergic receptor is mediated by the betagamma subunits of pertussis-toxin-sensitive G proteins through a pathway involving the non-receptor tyrosine kinase c-Src and the G protein Ras. Activation of this pathway by the beta2-adrenergic receptor requires that the receptor be phosphorylated by PKA because it is blocked by H-89, an inhibitor of PKA. Additionally, a mutant of the receptor, which lacks the sites normally phosphorylated by PKA, can activate adenylyl cyclase, the enzyme that generates cAMP, but not MAP kinase. Our results demonstrate that a mechanism previously shown to mediate uncoupling of the beta2-adrenergic receptor from Gs and thus heterologous desensitization (PKA-mediated receptor phosphorylation), also serves to 'switch' coupling of this receptor from Gs to Gi and initiate a new set of signalling events.