[Show abstract][Hide abstract] ABSTRACT: Most cells express more than one receptor plus degrading enzymes for adenine nucleotides or nucleosides, and cellular responses to purines are rarely compatible with the actions of single receptors. Therefore, these receptors are viewed as components of a combinatorial receptor web rather than self-dependent entities, but it remained unclear to what extent they can associate with each other to form signalling units. P2Y(1), P2Y(2), P2Y(12), P2Y(13), P2X(2), A(1), A(2A) receptors and NTPDase1 and -2 were expressed as fluorescent fusion proteins which were targeted to membranes and signalled like the unlabelled counterparts. When tested by FRET microscopy, all the G protein-coupled receptors proved able to form heterooligomers with each other, and P2Y(1), P2Y(12), P2Y(13), A(1), A(2A), and P2X(2) receptors also formed homooligomers. P2Y receptors did not associate with P2X, but G protein-coupled receptors formed heterooligomers with NTPDase1, but not NTPDase2. The specificity of prototypic interactions (P2Y(1)/P2Y(1), A(2A)/P2Y(1), A(2A)/P2Y(12)) was corroborated by FRET competition or co-immunoprecipitation. These results demonstrate that G protein-coupled purine receptors associate with each other and with NTPDase1 in a highly promiscuous manner. Thus, purinergic signalling is not only determined by the expression of receptors and enzymes but also by their direct interaction within a previously unrecognized multifarious membrane network.
Full-text · Article · Nov 2008 · Biochimica et Biophysica Acta
[Show abstract][Hide abstract] ABSTRACT: Neurons communicate through the exocytotic release of transmitters from presynaptic axon terminals and the ensuing activation of postsynaptic receptors. Instantaneous responses of postsynaptic cells to released neurotransmitters are mediated by ligand-gated ion channels, whereas G protein-coupled receptors mediate rather delayed effects. Moreover, the actions of ionotropic receptors are transient (milliseconds to seconds) and those of G protein-coupled receptors are more long lasting (seconds to minutes). Accordingly, neuronal signalling via ligand-gated ion channels is termed neurotransmission, whereas signalling via G protein-coupled receptors is termed neuromodulation. Exocytotic transmitter release is modulated by a variety of mechanisms such as previous activity at the synapse and the presence of extracellular neurotransmitters. Like the postsynaptic responses, presynaptic modulation is not only mediated by slowly acting G protein-coupled receptors, but also by fast acting ligand-gated ion channels. Accordingly, members of all known families of ligand-gated ion channels (cys-loop receptors, such as GABA(A), glycine, nicotinic acetylcholine, and 5-HT(3) receptors, ionotropic glutamate receptors, P2X receptors, and vanilloid receptors) are known to control transmitter release. All these ligand-gated ion channels display heterogeneous structures and functions. Therefore, activation of such presynaptic receptors can control transmitter release in different ways and through a multitude of mechanisms. This review provides a summary of the functions of the different presynaptic ligand-gated ion channels and presents prototypic examples for the physiological and pharmacological relevance of these presynaptic receptors.
Preview · Article · Jun 2008 · Current Molecular Pharmacology
[Show abstract][Hide abstract] ABSTRACT: The release of transmitters through vesicle exocytosis from nerve terminals is not constant but is subject to modulation by various mechanisms, including prior activity at the synapse and the presence of neurotransmitters or neuromodulators in the synapse. Instantaneous responses of postsynaptic cells to released transmitters are mediated by ionotropic receptors. In contrast to metabotropic receptors, ionotropic receptors mediate the actions of agonists in a transient manner within milliseconds to seconds. Nevertheless, transmitters can control vesicle exocytosis not only via slowly acting metabotropic, but also via fast acting ionotropic receptors located at the presynaptic nerve terminals. In fact, members of the following subfamilies of ionotropic receptors have been found to control transmitter release: ATP P2X, nicotinic acetylcholine, GABAA
, ionotropic glutamate, glycine, 5-HT3, andvanilloid receptors. As these receptors display greatly diverging structural and functional features, a variety of different mechanisms are involved in the regulation of transmitter release via presynaptic ionotropic receptors. This text gives an overview of presynaptic ionotropic receptors and briefly summarizes the events involved in transmitter release to finally delineate the most important signaling mechanisms that mediate the effects of presynaptic ionotropic receptor activation. Finally, a few examples are presented to exemplify the physiological and pharmacological relevance of presynaptic ionotropic receptors.
No preview · Article · Feb 2008 · Handbook of experimental pharmacology
[Show abstract][Hide abstract] ABSTRACT: Nucleotides are released not only from neurons, but also from various other types of cells including fibroblasts, epithelial, endothelial and glial cells. While ATP release from non-neural cells is frequently Ca(2+) independent and mostly non-vesicular, neuronal ATP release is generally believed to occur via exocytosis. To evaluate whether nucleotide release from neuroendocrine cells might involve a non-vesicular component, the autocrine/paracrine activation of P2Y(12) receptors was used as a biosensor for nucleotide release from PC12 cells. Expression of a plasmid coding for the botulinum toxin C1 light chain led to a decrease in syntaxin 1 detected in immunoblots of PC12 membranes. In parallel, spontaneous as well as depolarization-evoked release of previously incorporated [(3)H]noradrenaline from transfected cells was significantly reduced in comparison with the release from untransfected cells, thus indicating that exocytosis was impaired. In PC12 cells expressing the botulinum toxin C1 light chain, ADP reduced cyclic AMP synthesis to the same extent as in non-transfected cells. Likewise, the enhancement of cyclic AMP synthesis either due to the blockade of P2Y(12) receptors or due to the degradation of extracellular neucleotides by apyrase was not different between non-transfected and botulinum toxin C1 light chain expressing cells. However, the inhibition of cyclic AMP synthesis caused by depolarization-evoked release of endogenous nucleotides was either abolished or greatly reduced in cells expressing the botulinum toxin C1 light chain. Together, these results show that spontaneous nucleotide release from neuroendocrine cells may occur independently of vesicle exocytosis, whereas depolarization-evoked nucleotide release relies predominantly on exocytotic mechanisms.
Preview · Article · Oct 2007 · Purinergic Signalling
[Show abstract][Hide abstract] ABSTRACT: The inhibitor cystine-knot motif identified in the structure of CSTX-1 from Cupiennius salei venom suggests that this toxin may act as a blocker of ion channels. Whole-cell patch-clamp experiments performed on cockroach neurons revealed that CSTX-1 produced a slow voltage-independent block of both mid/low- (M-LVA) and high-voltage-activated (HVA) insect Ca(v) channels. Since C. salei venom affects both insect as well as rodent species, we investigated whether Ca(v) channel currents of rat neurons are also inhibited by CSTX-1. CSTX-1 blocked rat neuronal L-type, but no other types of HVA Ca(v) channels, and failed to modulate LVA Ca(v) channel currents. Using neuroendocrine GH3 and GH4 cells, CSTX-1 produced a rapid voltage-independent block of L-type Ca(v) channel currents. The concentration-response curve was biphasic in GH4 neurons and the subnanomolar IC(50) values were at least 1000-fold lower than in GH3 cells. L-type Ca(v) channel currents of skeletal muscle myoballs and other voltage-gated ion currents of rat neurons, such as I(Na(v)) or I(K(v)) were not affected by CSTX-1. The high potency and selectivity of CSTX-1 for a subset of L-type channels in mammalian neurons may enable the toxin to be used as a molecular tool for the investigation of this family of Ca(v) channels.
[Show abstract][Hide abstract] ABSTRACT: 5-HT(3) (serotonin type 3) receptors are targets of antiemetics, antipsychotics, and antidepressants and are believed to play a role in cognition. Nevertheless, contrasting results have been obtained with respect to their functions in the CNS and in the control of transmitter release. We used rat hippocampal neurons in single-neuron microcultures to identify the roles of presynaptic 5-HT(3) receptors at central synapses. 5-HT (10 microm) caused a transient > 10-fold increase in the frequency of miniature inhibitory postsynaptic currents without affecting amplitudes or kinetics. This effect was abolished by tropisetron (30 nm) and when Ca(2+) channels were blocked by 100 microm Cd(2+) it was mimicked and occluded when neurons were depolarized by 20 mm, but not 10 mm, K(+). Thus, activation of presynaptic 5-HT(3) receptors increased spontaneous GABA release by causing depolarization and opening of voltage-gated Ca(2+) channels. In microculture neurons, 5-HT transiently reduced action potential-evoked inhibitory autaptic currents by > 50%; this effect was blocked by tropisetron and mimicked by 20 mm, but not 10 mm, K(+). Miniature excitatory postsynaptic currents were not altered by 5-HT. Excitatory autaptic currents were tonically reduced, an effect attenuated by 5-HT(1A) antagonists. Thus, presynaptic 5-HT(3) receptors control GABA, but not glutamate, release and mediate opposite effects on spontaneous and action potential-dependent release.
Preview · Article · Jan 2007 · Journal of Neurochemistry
[Show abstract][Hide abstract] ABSTRACT: The release of noradrenaline from nerve terminals is modulated by a variety of presynaptic receptors. These receptors belong to one of the following three receptor superfamilies: transmitter-gated ion channels, G protein-coupled receptors (GPCR), and membrane receptors with intracellular enzymatic activities. For representatives of each of these three superfamilies, receptor activation has been reported to cause either an enhancement or a reduction of noradrenaline release. As these receptor classes display greatly diverging structures and functions, a multitude of different molecular mechanisms are involved in the regulation of noradrenaline release via presynaptic receptors. This review gives a short overview of the presynaptic receptors on noradrenergic nerve terminals and summarizes the events involved in vesicle exocytosis in order to finally delineate the most important signaling cascades that mediate the modulation via presynaptic receptors. In addition, the interactions between the various presynaptic receptors are described and the underlying molecular mechanisms are elucidated. Together, these presynaptic signaling mechanisms form a sophisticated network that precisely adapts the amount of noradrenaline being released to a given situation.
No preview · Article · Nov 2006 · Pharmacology [?] Therapeutics
[Show abstract][Hide abstract] ABSTRACT: KT-362 is an antiarrhythmic and antihypertensive agent with vasodilating activity. Since it carries a homoveratryl group in the side chain, an obvious relation exists to the verapamil-type calcium antagonists. Replacement of the fused aromatic moiety in KT-362 with thiophene provided 8-[N-[2-(3,4-dimethoxyphenyl)ethyl]-β-analyl]-5,6,7,8-tetrahydrothieno[3,2-b][1,4] thiazepine (1). Compound 1 shows a negative chronotropic activity in spontaneously beating right atria (IC50 = 23 μM, n = 7), and a negative inotropic effect in papillary muscles (IC50 = 2.7 μM, n = 7) and left atria (IC50 = 4 μM, n = 6) of the guinea-pig heart. The decrease of contractility in papillary muscles could be antagonized by increasing the extracellular calcium concentration. Compound 1 was found to affect high (IC50: 70 ± 5 μM) and low (IC50: 129 ± 34 μM) voltage-activated calcium channel currents as well as voltage-activated sodium channel currents (IC50: 80 ± 13 μM) in chick dorsal root ganglion neurons. In addition, nicotine-induced currents were potently inhibited (IC50: 6 ± 0.7 μM) in bovine chromaffin cells.
No preview · Article · Sep 2006 · Archiv der Pharmazie
[Show abstract][Hide abstract] ABSTRACT: Within the last 15 years, at least eight different G protein-coupled nucleotide receptors, i.e., P2Y receptors, have been characterized by molecular means. While ionotropic P2X receptors are mainly involved in fast synaptic neurotransmission, P2Y receptors rather mediate slower neuromodulatory effects. This P2Y receptor-dependent neuromodulation relies on changes in synaptic transmission via either pre- or postsynaptic sites of action. At both sites, the regulation of voltage-gated or transmitter-gated ion channels via G protein-linked signaling cascades has been identified as the predominant underlying mechanisms. In addition, neuronal P2Y receptors have been found to be involved in neurotoxic and neurotrophic effects of extracellular adenosine 5-triphosphate. This review provides an overview of the most prominent actions mediated by neuronal P2Y receptors and describes the signaling cascades involved.
No preview · Article · Sep 2006 · Pflügers Archiv - European Journal of Physiology
[Show abstract][Hide abstract] ABSTRACT: Many membrane proteins incur a folding problem during biosynthesis; only a fraction thereof is exported from the endoplasmic reticulum (ER), because quality control is stringent. This is also true for G protein-coupled receptors. Here, we identify the deubiquitinating enzyme Usp4 as an interaction partner of the A2a adenosine receptor, a Gs-coupled receptor. Usp4 binds to the carboxyl terminus of the A2A receptor and allows for its accumulation as deubiquinated protein. This relaxes ER quality control and enhances cell surface expression of functionally active receptor. The effect of Usp4 on the A2A receptor was specific because 1) it was not seen in C-terminally truncated versions of the receptor; 2) it was not mimicked by Usp14, another member of the ubiquitin-specific protease family; and 3) it was not seen with the metabotropic glutamate receptor-5, another G protein-coupled receptor with a high propensity for intracellular retention. These observations show that deubiquinating enzymes can regulate quality control in the ER.
Full-text · Article · May 2006 · Molecular Pharmacology
[Show abstract][Hide abstract] ABSTRACT: Mammalian Staufen2 (Stau2) is a member of the double-stranded RNA-binding protein family. Its expression is largely restricted to the brain. It is thought to play a role in the delivery of RNA to dendrites of polarized neurons. To investigate the function of Stau2 in mature neurons, we interfered with Stau2 expression by RNA interference (RNAi). Mature neurons lacking Stau2 displayed a significant reduction in the number of dendritic spines and an increase in filopodia-like structures. The number of PSD95-positive synapses and miniature excitatory postsynaptic currents were markedly reduced in Stau2 down-regulated neurons. Akin effects were caused by overexpression of dominant-negative Stau2. The observed phenotype could be rescued by overexpression of two RNAi cleavage-resistant Stau2 isoforms. In situ hybridization revealed reduced expression levels of beta-actin mRNA and fewer dendritic beta-actin mRNPs in Stau2 down-regulated neurons. Thus, our data suggest an important role for Stau2 in the formation and maintenance of dendritic spines of hippocampal neurons.
Full-text · Article · Feb 2006 · The Journal of Cell Biology
[Show abstract][Hide abstract] ABSTRACT: The physiological function of neurotransmitter transporter proteins like the serotonin transporter (SERT) is reuptake of neurotransmitter that terminates synaptic serotoninergic transmission. SERT can operate in reverse direction and be induced by SERT substrates including 5-HT, tyramine and the positively charged methyl-phenylpyridinium (MPP(+)), as well as the amphetamine derivatives para-chloroamphetamine (pCA) and methylene-dioxy-methamphetamine (MDMA). These substrates also induce inwardly directed sodium currents that are predominantly carried by sodium ions. Efflux via SERT depends on this sodium flux that is believed to be a prerequisite for outward transport. However, in recent studies, it has been suggested that substrates may be distinct in their properties to induce efflux. Therefore, the aim of the present study was a pharmacological characterization of different SERT substrates in uptake experiments, their abilities to induce transporter-mediated efflux and currents. In conclusion, the rank order of affinities in uptake and electrophysiological experiments correlate well, while the potencies of the amphetamine derivatives for the induction of efflux are clearly higher than those of the other substrates. These discrepancies can be only explained by mechanisms that can be induced by amphetamines. Therefore, based on our pharmacological observations, we conclude that amphetamines distinctly differ from non-amphetamine SERT substrates.
Full-text · Article · Dec 2005 · Neuropharmacology
[Show abstract][Hide abstract] ABSTRACT: Presynaptic inhibition of transmitter release is commonly mediated by a direct interaction between G protein betagamma subunits and voltage-activated Ca2+ channels. To search for an alternative pathway, the mechanisms by which presynaptic bradykinin receptors mediate an inhibition of noradrenaline release from rat superior cervical ganglion neurons were investigated. The peptide reduced noradrenaline release triggered by K+-depolarization but not that evoked by ATP, with Ca2+ channels being blocked by Cd2+. Bradykinin also reduced Ca2+ current amplitudes measured at neuronal somata, and this effect was pertussis toxin-insensitive, voltage-independent, and developed slowly within 1 min. The inhibition of Ca2+ currents was abolished by a phospholipase C inhibitor, but it was not altered by a phospholipase A2 inhibitor, by the depletion of intracellular Ca2+ stores, or by the inactivation of protein kinase C or Rho proteins. In whole-cell recordings, the reduction of Ca2+ currents was irreversible but became reversible when 4 mM ATP or 0.2 mM dioctanoyl phosphatidylinositol-4,5-bisphosphate was included in the pipette solution. In contrast, the effect of bradykinin was entirely reversible in perforated-patch recordings but became irreversible when the resynthesis of phosphatidylinositol-4,5-bisphosphate was blocked. Thus, the inhibition of Ca2+ currents by bradykinin involved a consumption of phosphatidylinositol-4,5-bisphosphate by phospholipase C but no downstream effectors of this enzyme. The reduction of noradrenaline release by bradykinin was also abolished by the inhibition of phospholipase C or of the resynthesis of phosphatidylinositol-4,5-bisphosphate. These results show that the presynaptic inhibition was mediated by a closure of voltage-gated Ca2+ channels through depletion of membrane phosphatidylinositol bisphosphates via phospholipase C.
Preview · Article · Dec 2005 · Molecular Pharmacology
[Show abstract][Hide abstract] ABSTRACT: Bradykinin is known to stimulate neurons in rat sympathetic ganglia and to enhance transmitter release from their axons by interfering with the autoinhibitory feedback, actions that involve protein kinase C. Here, bradykinin caused a transient increase in the release of previously incorporated [3H] noradrenaline from primary cultures of dissociated rat sympathetic neurons. When this effect was abolished by tetrodotoxin, bradykinin caused an inhibition of tritium overflow triggered by depolarizing K+ concentrations. This inhibition was additive to that caused by the alpha2-adrenergic agonist UK 14304, desensitized within 12 min, was insensitive to pertussis toxin, and was enhanced when protein kinase C was inactivated. The effect was half maximal at 4 nm and antagonized competitively by the B2 receptor antagonist Hoe 140. The cyclooxygenase inhibitor indomethacin and the angiotensin converting enzyme inhibitor captopril did not alter the inhibition by bradykinin. The M-type K+ channel opener retigabine attenuated the secretagogue action of bradykinin, but left its inhibitory action unaltered. In whole-cell patch-clamp recordings, bradykinin reduced voltage-activated Ca2+ currents in a pertussis toxin-insensitive manner, and this action was additive to the inhibition by UK 14304. These results demonstrate that bradykinin inhibits noradrenaline release from rat sympathetic neurons via presynaptic B2 receptors. This effect does not involve cyclooxygenase products, M-type K+ channels, or protein kinase C, but rather an inhibition of voltage-gated Ca2+ channels.
Preview · Article · Jul 2005 · Journal of Neurochemistry
[Show abstract][Hide abstract] ABSTRACT: Within the last 15 years, at least 8 different G protein-coupled P2Y receptors have been characterized. These mediate slow metabotropic effects of nucleotides in neurons as well as non-neural cells, as opposed to the fast ionotropic effects which are mediated by P2X receptors. One class of effector systems regulated by various G protein-coupled receptors are voltage-gated and ligand-gated ion channels. This review summarizes the current knowledge about the modulation of such neuronal ion channels via P2Y receptors. The regulated proteins include voltage-gated Ca(2+) and K(+) channels, as well as N-methyl-D: -aspartate, vanilloid, and P2X receptors, and the regulating entities include most of the known P2Y receptor subtypes. The functional consequences of the modulation of ion channels by nucleotides acting at pre- or postsynaptic P2Y receptors are changes in the strength of synaptic transmission. Accordingly, ATP and related nucleotides may act not only as fast transmitters (via P2X receptors) in the nervous system, but also as neuromodulators (via P2Y receptors). Hence, nucleotides are as universal transmitters as, for instance, acetylcholine, glutamate, or gamma-aminobutyric acid.
Preview · Article · Jan 2005 · Purinergic Signalling
[Show abstract][Hide abstract] ABSTRACT: Although feedback inhibition of noradrenaline release by coreleased nucleotides is a well known phenomenon, it remained unclear which P2 receptor subtypes and associated signalling cascades may be involved. In the rat pheochromocytoma cell line PC12, 2-methylthio-ADP reduced noradrenaline release triggered by K+ depolarization more potently than ADP and ATP, whereas UDP or UTP failed to do so. The inhibition by ADP was abolished by pertussis toxin and antagonized by reactive blue 2, 2-methylthio-AMP, and AR-C69931MX, but not by suramin. AR-C69931MX acted as a competitive antagonist with an apparent affinity of 2 nm, but did not alter noradrenaline release, when PC12 cells were continuously superfused. However, when the superfusion was halted during K+ depolarization, release was significantly reduced and this inhibition was attenuated by AR-C69931MX, thus revealing ongoing autoinhibition. Rises in cellular cyclic AMP did not alter depolarization-evoked release nor its reduction by ADP, even though the nucleotide did inhibit cyclic AMP accumulation. ADP and the direct Ca2+ channel blocker Cd2+ inhibited voltage-activated Ca2+ currents, but not ATP-induced currents, and both agents reduced K+-evoked, but not ATP-evoked, release. Hence, if voltage-gated Ca2+ channels do not contribute to stimulation-evoked release, ADP fails to exert its inhibitory action. In primary cultures of rat sympathetic neurons, ADP also reduced Ca2+ currents and K+-evoked noradrenaline release, and AR-C69931MX acted again as competitive antagonist with an apparent affinity of 3 nm. These results show that P2Y12 receptors mediate an autoinhibition of transmitter release from PC12 cells and sympathetic neurons through an inhibition of voltage-gated Ca2+ channels.
No preview · Article · Jan 2005 · European Journal of Neuroscience
[Show abstract][Hide abstract] ABSTRACT: The core complex, formed by the SNARE proteins synaptobrevin 2, syntaxin 1 and SNAP-25, is an important component of the synaptic fusion machinery and shows remarkable in vitro stability, as exemplified by its SDS-resistance. In western blots, antibodies against one of these SNARE proteins reveal the existence of not only an SDS-resistant ternary complex but also as many as five bands between 60 and >200 kDa. Structural conformation as well as possible functions of these various complexes remained elusive. In western blots of protein extracts from PC12 cell membranes, an antibody against SNAP-25 detected two heat-sensitive SDS-resistant bands with apparent molecular weights of 100 and 230 kDa. A syntaxin antibody recognized only the 230 kDa band and required heat-treatment of the blotting membrane to detect the 100 kDa band. Various antibodies against synaptobrevin failed to detect SNARE complexes in conventional western blots and detected either the 100 kDa band or the 230 kDa band on heat-treated blotting membranes. When PC12 cells were exposed to various extracellular K(+)-concentrations (to evoke depolarization-induced Ca(2+) influx) or permeabilized in the presence of basal or elevated free Ca(2+), levels of these SNARE complexes were altered differentially: moderate Ca(2+) rises (</=1 microM) caused an increase, whereas Ca(2+) elevations of more than 1 microM led to a decrease in the 230 kDa band. Under both conditions the 100 kDa band was either increased or remained unchanged. Our data show that various SDS-resistant complexes occur in living cells and indicate that they represent SNARE complexes with different structures and diverging functions. The distinct behavior of these complexes under release-promoting conditions indicates that these SNARE structures have different roles in exocytosis.
Preview · Article · Feb 2004 · Journal of Cell Science
[Show abstract][Hide abstract] ABSTRACT: Acetylcholine has long been known to excite sympathetic neurons via M1 muscarinic receptors through an inhibition of M-currents. Nevertheless, it remained controversial whether activation of muscarinic receptors is also sufficient to trigger noradrenaline release from sympathetic neurons. In primary cultures of rat superior cervical ganglia, the muscarinic agonist oxotremorine M inhibited M-currents with half-maximal effects at 1 microM and induced the release of previously incorporated [3H]noradrenaline with half-maximal effects at 10 microM. This latter action was not affected by the nicotinic antagonist mecamylamine which, however, abolished currents through nicotinic receptors elicited by high oxotremorine M concentrations. Ablation of the signalling cascades linked to inhibitory G proteins by pertussis toxin potentiated the release stimulating effect of oxotremorine M, and the half-maximal concentration required to stimulate noradrenaline release was decreased to 3 microM. Pirenzepine antagonized the inhibition of M-currents and the induction of release by oxotremorine M with identical apparent affinity, and both effects were abolished by the muscarinic toxin 7. These results indicate that one muscarinic receptor subtype, namely M1, mediates these two effects. Retigabine, which enhances M-currents, abolished the release induced by oxotremorine M, but left electrically induced release unaltered. Moreover, retigabine shifted the voltage-dependent activation of M-currents by about 20 mV to more negative potentials and caused 20 mV hyperpolarisations of the membrane potential. In the absence of retigabine, oxotremorine M depolarised the neurons and elicited action potential discharges in 8 of 23 neurons; in its presence, oxotremorine M still caused equal depolarisations, but always failed to trigger action potentials. Action potential waveforms caused by current injection were not affected by retigabine. These results indicate that the inhibition of M-currents is the basis for the stimulation of transmitter release from sympathetic neurons via M1 muscarinic receptors.
Preview · Article · Jan 2004 · The Journal of Physiology
[Show abstract][Hide abstract] ABSTRACT: Activation of P2Y receptors by released nucleotides subserves important autocrine-paracrine functions in various non-neural tissues. To investigate how P2Y receptors are activated in a neuronal environment, we used PC12 cells in which nucleotides were found to elicit increases in inositol phosphates via P2Y2 and decreases in cAMP via P2Y12 receptors. Depolarization of PC12 cells raised inositol phosphates, and blockade of voltage-gated Ca2+ channels by Cd2+ or degradation of extracellular nucleotides by apyrase prevented this effect. In nondepolarized cells, apyrase did not affect inositol phosphates. Depolarization of PC12 cells also reduced the A2A receptor-mediated synthesis of cAMP. This effect was again prevented by Cd2+ or apyrase, but apyrase enhanced the synthesis of cAMP even in nondepolarized cells. Overexpression of rat P2Y2 receptors increased the nucleotide-dependent inositol phosphate accumulation and enhanced the effect of K+ depolarization. Nevertheless, apyrase still failed to alter spontaneous inositol phosphate accumulation. Expression of rat P2Y1 receptors, in contrast, led to huge increases in spontaneous inositol phosphate accumulation, which was reduced by a receptor antagonist or by apyrase. This increased synthesis of inositol phosphates could not be further enhanced by depolarization or receptor agonists, but when endogenous nucleotides were removed by superfusion, recombinant P2Y1 receptors could be activated to mediate an inhibition of M-type K+ channels. These results indicate that nucleoside diphosphate-sensitive (P2Y12 and P2Y1) receptors are activated by spontaneous nucleotide release, whereas triphosphate-sensitive (P2Y2) receptors require an excess of depolarization-evoked release to become activated.
No preview · Article · Sep 2003 · The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
[Show abstract][Hide abstract] ABSTRACT: The adenosine A(2A) receptor and the dopamine D(2) receptor are prototypically coupled to G(s) and G(i)/G(o), respectively. In striatal intermediate spiny neurons, these receptors are colocalized in dendritic spines and act as mutual antagonists. This antagonism has been proposed to occur at the level of the receptors or of receptor-G protein coupling. We tested this model in PC12 cells which endogenously express A(2A) receptors. The human D(2) receptor was introduced into PC12 cells by stable transfection. A(2A)-agonist-mediated inhibition of D(2) agonist binding was absent in PC12 cell membranes but present in HEK293 cells transfected as a control. However, in the resulting PC12 cell lines, the action of the D(2) agonist quinpirole depended on the expression level of the D(2) receptor: at low and high receptor levels, the A(2A)-agonist-induced elevation of cAMP was enhanced and inhibited, respectively. Forskolin-stimulated cAMP formation was invariably inhibited by quinpirole. The effects of quinpirole were abolished by pretreatment with pertussis toxin. A(2A)-receptor-mediated cAMP formation was inhibited by other G(i)/G(o)-coupled receptors that were either endogenously present (P(2y12)-like receptor for ADP) or stably expressed after transfection (A(1) adenosine, metabotropic glutamate receptor-7A). Similarly, voltage activated Ca(2+) channels were inhibited by the endogenous P(2Y) receptor and by the heterologously expressed A(1) receptor but not by the D(2) receptor. These data indicate functional segregation of signaling components. Our observations are thus compatible with the proposed model that D(2) and A(2A) receptors are closely associated, but they highlight the fact that this interaction can also support synergism.
Preview · Article · Aug 2003 · Neuropsychopharmacology