[Show abstract][Hide abstract] ABSTRACT: We have examined the potential roles of intracellular Ca2+ regulation and of multiple cytoskeletal elements in control of the directed migration of cultured oligodendrocyte progenitor cells (OPs). OPs were found to migrate in response to platelet-derived growth factor (PDGF) or to a lesser extent to basic fibroblast growth factor (FGF) in a non-additive manner. This response was inhibited by chelation of intracellular Ca2+ by using BAPTA-AM. OP migration was not evoked by the neurotransmitter agonists phenylephrine or methacholine, which elevate OP Ca2+ levels. Inhibition of the MAP kinase pathway with PD 098059 did not affect OP migration to PDGF. Within growth cone-like leading edges of migratory OP processes, monomeric and filamentous actin were found to be colocalized with myosin and filamentous actin was prominent in filopodia extending beyond the leading edge. Tubulin was distributed throughout OP processes and cell bodies. Inhibition of actin or tubulin polymerization, by using cytochalasin B or nocodazole, respectively, altered OP morphology and markedly impaired migration. Inhibition of the myosin ATPase by BDM, which prevents force-generating actin/myosin interactions, greatly inhibited the chemotaxic response at concentrations that did not disrupt cell morphology. These results indicate that growth factors stimulate OP migration by activating pathways which include intracellular Ca2+ regulation, and characterize the distribution of multiple cytoskeletal elements involved in the generation of directed OP movement.
[Show abstract][Hide abstract] ABSTRACT: This study was undertaken to examine the expression and role of the endoplasmic reticulum (ER) proteins calreticulin and ryanodine receptors, and mitochondria, in cultured astrocytes. Using several lines of investigation, we have identified a key role for mitochondria in astrocyte Ca2+ signalling: (1) a significant correlation was found between sites of regenerative Ca2+ wave amplification (possessing high amplitude ER Ca2+ release) and the location of mitochondria in the cell; (2) norepinephrine (2 microM) caused a rapid-onset increase in rhod 2 fluorescence in 34% of astrocyte mitochondria, indicating that cytosolic Ca2+ responses result in mitochondrial Ca2+ elevation; and (3) pretreatment with the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone to inhibit mitochondrial activity markedly reduced the amplitude of subsequent norepinephrine-evoked cytosolic Ca2+ responses. We then investigated the roles of several ER proteins in Ca2+ signalling by immunocytochemistry. Ryanodine receptors and calreticulin were found to be expressed in heterogeneous patterns in astrocytes. The expression pattern of calreticulin corresponded closely with the distribution of mitochondria, whereas the expression of ryanodine receptors was not similar to that of either of these cellular factors. We measured Ca2+ wave kinetics in a single astrocyte, then assessed protein distribution by immunocytochemistry in the same cell. Cross-correlation between norepinephrine-evoked Ca2+ wave amplitude and calreticulin distribution indicated a close spatial relationship between this Ca2+-binding protein and sites of regenerative wave amplification. These results demonstrate that amplification sites for Ca2+ waves in astrocytes are identifiable by accumulations of calreticulin (and type 2 InsP3Rs), and by the presence of mitochondria, which may regulate the ER Ca2+ release process.
Journal of Neuroscience Research 07/1998; 52(6):672-83. DOI:10.1002/(SICI)1097-4547(19980615)52:63.0.CO;2-5 · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this study we have investigated the expression of ryanodine receptors (RyRs), and the ability of caffeine to evoke RyR-mediated elevation of intracellular Ca2+ levels ([Ca2+]i) in glial cells of the oligodendrocyte/type 2 astrocyte lineage. Immunocytochemistry with specific antibodies identified ryanodine receptors in cultured oligodendrocytes, type 2 astrocytes, and O-2A progenitor cells, at high levels in the perinuclear region and in a variegated pattern along processes. Glia acutely isolated from rat brain and in aldehydefixed sections of cortex were similarly found to express RyRs. Caffeine (5-50 mM) caused an increase in [Ca2+]i in most cultured type 2 astrocytes and in 50% of oligodendrocytes. Responses elicited by caffeine were inhibited by pretreatment with ryanodine (10 microM) or thapsigargin (1 microM), and the peak response was unaffected by removal of [Ca2+]o. O-2A progenitor cells, in contrast, were largely unresponsive to caffeine treatment. Pretreatment with kainate (200 microM) to activate Ca2+ entry increased the magnitude of caffeine-evoked [Ca2+]i elevations in type 2 astrocytes and oligodendrocytes, and caused caffeine to activate responses in a significant proportion of previously non-responding O-2A progenitors. In both type 2 astrocytes and oligodendrocytes, caffeine evoked Ca2+ changes which propagated as wavefronts from several initiation sites. These wave amplification sites were characterized by significantly higher local Ca2+ release kinetics. Our results indicate that several glial cell types express RyRs, and that their functionality differs within different cell types of the oligodendrocyte lineage. In addition, ionotropic glutamate receptor activation fills the caffeine-sensitive Ca2+ stores in these cells.
Journal of Neuroscience Research 06/1998; 52(4):468-82. DOI:10.1002/(SICI)1097-4547(19980515)52:43.0.CO;2-# · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this study, we have developed a mathematical method to derive the Ca2+ fluxes underlying agonist-evoked Ca2+ waves in cultured rat cortical astrocytes. Astrocytes were stimulated with norepinephrine (100 nM) to evoke Ca2+ waves, which were recorded by measuring FIuo-3 fluorescence changes with high spatial and temporal resolution. Normalized fluorescence (ΔF/F) was analyzed in discrete cellular spaces in a series of successive slices along the length of the cell. From these data, Ca2+ flux was then calculated using a one dimensional reaction-diffusion equation which utilizes the temporal and spatial derivatives of the fluorescence data and the diffusion coefficient of Ca2+ in the cytosol. This method identified distinct sites of positive flux (Ca2+ release into the cytosol) and of negative flux (Ca2+ removal from cytosol) and showed that in astrocytes, sites of Ca2+ release from stores regularly alternate with sites of Ca2+ removal from the cytosol. Cross correlation analysis of the two distribution patterns gave positive correlation at 2 μm out of phase and a negative correlation in phase. Thapsigargin-induced Ca2+ waves were analyzed to determine if the negative flux was due to Ca2+ uptake via thapsigargin-sensitive Ca2+ pumps. Negative flux sites were still found under these conditions, suggesting that multiple mechanisms of Ca2+ removal from the cytosol may contribute to negative flux sites. This method of calculation of flux may serve as a means to describe the distribution of functional ion channels and pumps participating in cellular Ca2+ signalling.
[Show abstract][Hide abstract] ABSTRACT: Many physiologically important activities of oligodendrocyte progenitor cells (O-2A cells), including proliferation, migration and differentiation, are regulated by cytosolic Ca2+ signals. However, little is known concerning the mechanisms of Ca2+ signalling in this cell type. We have studied the interactions between Ca2+ entry, Ca2+ release from endoplasmic reticulum and Ca2+ regulation by mitochondria in influencing cytosolic Ca2+ responses in O-2A cells.Methacholine (MCh; 100 μM) activated Ca2+ waves that propagated from several initiation sites along O-2A processes.During a Ca2+ wave evoked by MCh, mitochondrial membrane potential was often either depolarized (21% of mitochondria) or hyperpolarized (20% of mitochondria), as measured by changes in the fluorescence of 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazole carbocyanine iodide (JC-1).Stimulation with kainate (100 μM) evoked a slowly rising, sustained cytosolic Ca2+ elevation in O-2A cells. This also, in some cases, resulted in either a depolarization (15% of mitochondria) or hyperpolarization (12% of mitochondria) of mitochondrial membrane potential.Simultaneous measurement of cytosolic (fluo-3 AM) and mitochondrial (rhod-2 AM) Ca2+ responses revealed that Ca2+ elevations in the cytosol evoked by either MCh or kainate were translated into long-lasting Ca2+ elevations in subpopulations of mitochondria. In some mitochondria, Ca2+ signals appeared to activate Ca2+ release into the cytosol.Inhibition of the mitochondrial Na+-Ca2+ exchanger by CGP-37157 (25 μM) decreased kainate Ca2+ response amplitude and increased the rate of return of the response to basal Ca2+ levels.Thus, both ionotropic and metabotropic stimulation evoke changes in mitochondrial membrane potential and Ca2+ levels in O-2A cells. Ca2+ uptake into some mitochondria is activated by Ca2+ entry into cells or release from stores. Mitochondrial Ca2+ release appears to play a key role in shaping kainate-evoked Ca2+ responses.
The Journal of Physiology 04/1998; 508(2). DOI:10.1111/j.1469-7793.1998.413bq.x · 5.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: It is becoming increasingly clear that mitochondrial Ca2+ uptake from and release into the cytosol has important consequences for neuronal and glial activity. Ca2+ regulates mitochondrial metabolism, and mitochondrial Ca2+ uptake and release modulate physiological and pathophysiological cytosolic responses. In glial cells, inositol 1,4,5-trisphosphate-dependent Ca2+ responses are faithfully translated into elevations in mitochondrial Ca2+ levels, which modifies cytosolic Ca2+ wave propagation and may activate mitochondrial enzymes. The location of mitochondria within neurones may partially determine their role in Ca2+ signalling. Neuronal death due to NMDA-evoked Ca2+ entry can be delayed by an inhibitor of the mitochondrial permeability transition pore, and mitochondrial dysfunction is being increasingly implicated in a number of neurodegenerative conditions. These findings are illustrative of an emerging realization by neuroscientists of the importance of mitochondrial Ca2+ regulation as a modulator of cellular energetics, endoplasmic reticulum Ca2+ release and neurotoxicity.
Brain Research Reviews 04/1998; 26(1-26):72-81. DOI:10.1016/S0165-0173(97)00056-8 · 5.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In oligodendrocyte processes, methacholine-evoked Ca2+ waves propagate via regions of specialized Ca2+ release kinetics (wave amplification sites) at which the amplitude and rate of rise of local Ca2+ signals are markedly higher than in surrounding areas (Simpson, P. B., and Russell, J. T. (1996) J. Biol. Chem. 271, 33493-33501). In the present study we have examined the effects of other phosphoinositide-coupled agonists on Ca2+ in these cells, and the structural specializations underlying regenerative wave amplification sites. Both bradykinin and norepinephrine evoke Ca2+ waves, which initiate at the same loci and propagate through the cell body and multiple processes via identical wave amplification sites. Antibodies against type 2 inositol 1,4,5-trisphosphate receptors (InsP3R2) and calreticulin identify expression of these proteins in oligodendrocyte membranes in Western blots. Immunocytochemistry followed by high resolution fluorescence microscopy revealed that both InsP3R2 and calreticulin are expressed in high intensity patches along processes. Cross-correlation analysis of the profiles of local Ca2+ release kinetics during a Ca2+ wave and immunofluorescence for these proteins along cellular processes showed that the domains of high endoplasmic reticulum protein expression correspond closely to wave amplification sites. Staining cells with the mitochondrial dye, MitoTracker(R), showed that mitochondria are only found in intimate association with these sites possessing high density endoplasmic reticulum proteins, and they remain in the same locations over relatively long periods of time. It appears, therefore, that multiple specializations are found at domains of elevated Ca2+ release in oligodendrocyte processes, including high levels of calreticulin, InsP3R2 Ca2+ release channels, and mitochondria.
[Show abstract][Hide abstract] ABSTRACT: We have characterized the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA) pumps in cultured rat cortical type-1 astrocytes, type-2 astrocytes and oligodendrocytes. Perfusion with 10 microM cyclopiazonic acid (CPA) or 1 microM thapsigargin evoked a large and persistent elevation in cytosolic [Ca2+] in normal Ca2+-containing medium and a small and transient increase in nominally Ca2+-free medium. Subtraction of the response in Ca2+-free medium from that in the control revealed a slow-onset Ca2+-entry response to SERCA inhibition, which began after most of the store depletion had occurred. Thapsigargin- and CPA-induced responses propagated as Ca2+ waves, which began in several distinct cellular sites and travelled throughout the cell and through nearby cells, in confluent cultures. Propagation was supported by specialized Ca2+-release sites where the amplitude of the response was significantly higher and the rate of rise steeper. Such higher Ca2+-release kinetics were observed at these sites during Ins(1,4,5)P3-mediated Ca2+ waves in the same cells. Fluorescently tagged thapsigargin labelled SERCA pumps throughout glial cell bodies and processes. In oligodendrocyte processes, multiple domains with elevated SERCA staining were always associated with mitochondria. Our results are consistent with a model in which only a single Ca2+ store, expressing Ins(1,4,5)P3 receptors and SERCAs sensitive to both thapsigargin and CPA, is present in rat cortical glia, and indicate that inhibition of SERCA activates both Ca2+ release as a wavefront and Ca2+ entry via store-operated channels. The spatial relationship between SERCAs and mitochondria is likely to be important for regulating microdomains of elevated Ca2+-release kinetics.
[Show abstract][Hide abstract] ABSTRACT: We have examined the mechanisms that underlie Ca2+ wave propagation in cultured cortical astrocytes. Norepinephrine evoked Ca2+ waves in astrocytes that began at discrete initiation loci and propagated throughout the cell by regenerative amplification at a number of cellular sites, as shown by very high Ca2+ release rates at these regions. We have hypothesized previously that domains displaying elevated Ca2+ release kinetics in astrocytes may correspond to sites of high inositol 1,4,5-trisphosphate receptor (InsP3R) density. To examine this possibility, we compared the distribution pattern of endoplasmic reticulum (ER) and InsP3Rs with Ca2+ release kinetics in subcellular regions during propagation of norepinephrine-evoked waves. 3,3'-Dihexyloxacarbocyanine iodide staining revealed that the ER in astrocytes exists as a meshwork of membranes extending throughout the cells, including fine processes. A specific antibody directed against type 2 InsP3Rs (InsP3R2) detected a 260-kDa band in western blotting of astrocyte membranes. Immunocytochemistry using this antibody stained the entire ER system in a punctate, variegated manner. When Ca2+ responses and InsP3R2 immunofluorescence were compared in the same cell, domains of elevated Ca2+ response kinetics (high amplitude and rapid rate of rise) showed significant positive correlation with high local intensity of InsP3R2 staining. It appears, therefore, that specializations in the ER responsible for discrete local Ca2+ release sites that support regenerative wave propagation include increased levels of InsP3R2 expression.
Journal of Neurochemistry 07/1997; 68(6):2317-27. · 4.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have examined the mechanisms that underlie Ca2+ wave propagation in cultured cortical astrocytes. Norepinephrine evoked Ca2+ waves in astrocytes that began at discrete initiation loci and propagated throughout the cell by regenerative amplification at a number of cellular sites, as shown by very high Ca2+ release rates at these regions. We have hypothesized previously that domains displaying elevated Ca2+ release kinetics in astrocytes may correspond to sites of high inositol 1,4,5-trisphosphate receptor (InsP3R) density. To examine this possibility, we compared the distribution pattern of endoplasmic reticulum (ER) and InsP3Rs with Ca2+ release kinetics in subcellular regions during propagation of norepinephrine-evoked waves. 3,3′-Dihexyloxacarbocyanine iodide staining revealed that the ER in astrocytes exists as a meshwork of membranes extending throughout the cells, including fine processes. A specific antibody directed against type 2 InsP3Rs (InsP3R2) detected a 260-kDa band in western blotting of astrocyte membranes. Immunocytochemistry using this antibody stained the entire ER system in a punctate, variegated manner. When Ca2+ responses and InsP3R2 immunofluorescence were compared in the same cell, domains of elevated Ca2+ response kinetics (high amplitude and rapid rate of rise) showed significant positive correlation with high local intensity of InsP3R2 staining. It appears, therefore, that specializations in the ER responsible for discrete local Ca2+ release sites that support regenerative wave propagation include increased levels of InsP3R2 expression.
[Show abstract][Hide abstract] ABSTRACT: To understand how extracellular signals may produce long-term effects in neural cells, we have analyzed the mechanism by which neurotransmitters and growth factors induce phosphorylation of the transcription factor cAMP response element binding protein (CREB) in cortical oligodendrocyte progenitor (OP) cells. Activation of glutamate receptor channels by kainate, as well as stimulation of G-protein-coupled cholinergic receptors by carbachol and tyrosine kinase receptors by basic fibroblast growth factor (bFGF), rapidly leads to mitogen-activated protein kinase (MAPK) phosphorylation and ribosomal S6 kinase (RSK) activation. Kainate and carbachol activation of the MAPK pathway requires extracellular calcium influx and is accompanied by protein kinase C (PKC) induction, with no significant increase in GTP binding to Ras. Conversely, growth factor-stimulated MAPK phosphorylation is independent of extracellular calcium and is accompanied by Ras activation. Both basal and stimulated MAPK activity in OP cells are influenced by cytoplasmic calcium levels, as shown by their sensitivity to the calcium chelator bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid. The kinetics of CREB phosphorylation in response to the various agonists corresponds to that of MAPK activation. Moreover, CREB phosphorylation and MAPK activation are similarly affected by calcium ions. The MEK inhibitor PD 098059, which selectively prevents activation of the MAPK pathway, strongly reduces induction of CREB phosphorylation by kainate, carbachol, bFGF, and the phorbol ester TPA. We propose that in OPs the MAPK/RSK pathway mediates CREB phosphorylation in response to calcium influx, PKC activation, and growth factor stimulation.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 03/1997; 17(4):1291-301. · 6.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have examined the spatial and temporal nature of Ca2+ signals activated via the phosphoinositide pathway in oligodendrocytes and the cellular specializations underlying oligodendrocyte Ca2+ response characteristics. Cultured cortical oligodendrocytes were incubated with fluo 3 or fura 2, and digital video fluorescence microscopy was used to study the effect of methacholine on [Ca2+]i. Single peaks, oscillations, and steady-state plateau [Ca2+]i elevations were evoked by increasing agonist concentration. The peaks and oscillations were found to be Ca2+ wave fronts, which propagate via distinct amplification regions in the cell where the kinetics of Ca2+ release (amplitude and rate of rise of response) are elevated. Staining with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolecarbocyanine++ + iodide (JC-1) and 3,3'-dihexyloxacarbocyanine iodide revealed that mitochondria are found in groups of three or more in oligodendrocyte processes and that the groups are distributed with considerable distance separating them. Cross-correlation analysis showed a high degree of correlation between sites where mitochondria are present and peaks in the amplitude and rate of rise of the Ca2+ response. Intramitochondrial Ca2+ concentration, measured using rhod 2, increased upon treatment with methacholine. Methacholine also evoked a rapid change in mitochondrial membrane potential as measured by the J-aggregate fluorescence of JC-1. Pretreatment with the mitochondrial inhibitors carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (1 microM, 2 min) or antimycin (2 microg/ml, 2 min) altered the methacholine-evoked Ca2+ response in most cells studied, responses being either markedly potentiated or inhibited. The results of this study demonstrate that stimulation of phosphoinositide-coupled muscarinic acetylcholinoceptors activates propagating Ca2+ wave fronts in oligodendrocytes and that the characteristics of these waves are dependent on mitochondrial location and function.