Modification of human 5-HT(2C) receptor function by Cys23Ser, an abundant, naturally occurring amino-acid substitution.

A human serotonin (5-HT)(2C) receptor gene polymorphism leads to the substitution of cysteine for serine at codon 23 (Cys23Ser); the frequency of the Ser23 allele in unrelated Caucasians is approximately 0.13. In the present study, we assessed whether Cys23Ser could affect receptor function. The two alleles were functionally compared following expression in COS-7 cells. The constitutive activity of the receptor in an in situ reconstitution system was also evaluated following expression of each allele in Sf9 cells. Using radioligands, Ser23-expressed membranes showed reduced high-affinity binding to meta-chlorophenylpiperazine (m-CPP) and 5-HT. Although the amplitude of the 5-HT-induced intracellular Ca(2+) peak did not differ between the alleles, Ser23 required higher 5-HT concentrations to elicit the same response. These differences might be due to more extensive desensitization in the Ser23 form. In the in situ reconstitution system, the 5-HT(2C) receptor displayed considerable constitutive activity, with the Ser23 allele being significantly higher in this regard than the Cys23 form. After prolonged serum deprivation in order to resensitize the receptor, four of the 15 cells expressing Ser23 showed abnormally higher m-CPP-induced sensitivity of the Ca(2+) response. These results indicate that the Ser23 allele may be constitutively more active than Cys23. Thus, Ser23 appears to be an abundant candidate allele capable of directly influencing inter-individual variation in behavior, susceptibility to mental disorder, and response to drugs including atypical antipsychotic and some antidepressant drugs that are potent 5-HT(2C) inverse agonists or antagonists.

Ca(2+) binding protein frequenin mediates GDNF-induced potentiation of Ca(2+) channels and transmitter release.

Molecular mechanisms underlying long-term neurotrophic regulation of synaptic transmission and plasticity are unknown. We report here that long-term treatment of neuromuscular synapses with glial cell line-derived neurotrophic factor (GDNF) potentiates spontaneous and evoked transmitter release, in ways very similar to presynaptic expression of the Ca(2+) binding protein frequenin. GDNF enhances the expression of frequenin in motoneurons, and inhibition of frequenin expression or activity prevents the synaptic action of GDNF. GDNF also facilitates Ca(2+) influx into the nerve terminals during evoked transmission by enhancing Ca(2+) currents. The effect of GDNF on Ca(2+) currents is blocked by inhibition of frequenin expression, occluded by overexpression of frequenin, and is selective to N-type Ca(2+) channels. These results identify an important molecular target that mediates the long-term, synaptic action of a neurotrophic factor.

Bax translocation to mitochondria subsequent to a rapid loss of mitochondrial membrane potential.

Bax, a pro-apoptotic member of the Bcl-2 family, is a cytosolic protein that inserts into mitochondrial membranes upon induction of cell death. Using the green fluorescent protein fused to Bax (GFP-Bax) to quantitate mitochondrial binding in living cells we have investigated the cause of Bax association with mitochondria and the time course relative to endogenous and induced changes in mitochondrial membrane potential (DeltaPsi(m)). We have found that staurosporine (STS) induces a loss in DeltaPsi(m) before GFP-Bax translocation can be measured. The onset of the DeltaPsi(m) loss is followed by a rapid and complete collapse of DeltaPsi(m) which is followed by Bax association with mitochondria. The mitochondria uncoupler FCCP, in the presence of the F(1)-F(0) ATPase inhibitor oligomycin, can trigger Bax translocation to mitochondria suggesting that when ATP levels are maintained a collapse of DeltaPsi(m) induces Bax translocation. Neither FCCP nor oligomycin alone alters Bax location. Bax association with mitochondria is also triggered by inhibitors of the electron transport chain, antimycin and rotenone, compounds that collapse DeltaPsi(m) without inducing rapid ATP hydrolysis that typically occurs with uncouplers such as FCCP. Taken together, our results suggest that alterations in mitochondrial energization associated with apoptosis can initiate Bax docking to mitochondria.

Sparks and puffs in oligodendrocyte progenitors: cross talk between ryanodine receptors and inositol trisphosphate receptors.

Investigating how calcium release from the endoplasmic reticulum (ER) is triggered and coordinated is crucial to our understanding of how oligodendrocyte progenitor cells (OPs) develop into myelinating cells. Sparks and puffs represent highly localized Ca(2+) release from the ER through ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP(3)Rs), respectively. To study whether sparks or puffs trigger Ca(2+) waves in OPs, we performed rapid high-resolution line scan recordings in fluo-4-loaded OP processes. We found spontaneous and evoked sparks and puffs, and we have identified functional cross talk between IP(3)Rs and RyRs. Local events evoked using the IP(3)-linked agonist methacholine (MeCh) showed significantly different morphology compared with events evoked using the caffeine analog 3,7-dimethyl-1-propargylxanthine (DMPX). Pretreatment with MeCh potentiated DMPX-evoked events, whereas inhibition of RyRs potentiated events evoked by low concentrations of MeCh. Furthermore, activation of IP(3)Rs but not RyRs was critical for Ca(2+) wave initiation. Using immunocytochemistry, we show OPs express the specific Ca(2+) release channel subtypes RyR3 and IP(3)R2 in patches along OP processes. RyRs are coexpressed with IP(3)Rs in some patches, but IP(3)Rs are also found alone. This differential distribution pattern may underlie the differences in local and global Ca(2+) signals mediated by these two receptors. Thus, in OPs, interactions between IP(3)Rs and RyRs determine the spatial and temporal characteristics of calcium signaling, from microdomains to intracellular waves.

Müller cell Ca2+ waves evoked by purinergic receptor agonists in slices of rat retina.

We have measured agonist evoked Ca2+ waves in Müller cells in situ within freshly isolated retinal slices. Using an eye cup dye loading procedure we were able to preferentially fill Müller glial cells in retinal slices with calcium green. Fluorescence microscopy revealed that bath perfusion of slices with purinergic agonists elicits Ca2+ waves in Müller cells, which propagate along their processes. These Ca2+ signals were insensitive to tetrodotoxin (TTX, 1.0 microM) pretreatment. Cells were readily identified as Müller cells by their unique morphology and by subsequent immunocytochemical labeling with glial fibrillary acidic protein antibodies. While cells never exhibited spontaneous Ca2+ oscillations, purinoreceptor agonists, ATP, 2 MeSATP, ADP, 2 MeSADP, and adenosine readily elicited Ca2+ waves. These waves persisted in the absence of [Ca2+]o but were abolished by thapsigargin pretreatment, suggesting that the purinergic agonists tested act by releasing Ca2+ from intracellular Ca2+ stores. The rank order of potency of different purines and pyrimidines for inducing Ca2+ signals was 2 MeSATP = 2MeSADP > ADP > ATP >> alphabetameATP = uridine triphosphate (UTP) > uridine diphosphate (UDP). The Ca2+ signals evoked by ATP, ADP, and 2 MeSATP were inhibited by reactive blue (100 microM) and suramin (200 microM), and the adenosine induced signals were abolished only by 3,7-dimethyl-1-propargylxanthine (200 microM) and not by 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine) or 8-cyclopentyl-1,3-dipropylxanthine at the same concentration. Based on these pharmacological characteristics and the dose-response relationships for ATP, 2 MeSATP, 2 MeSADP, ADP, and adenosine, we concluded that Müller cells express the P1A2 and P2Y1 subtypes of purinoceptors. Analysis of Ca2+ responses showed that, similar to glial cells in culture, wave propagation occurred by regenerative amplification at specialized Ca2+ release sites (wave amplification sites), where the rate of Ca2+ release was significantly enhanced. These data suggest that Müller cells in the retina may participate in signaling, and this may serve as an extra-neuronal signaling pathway.

Elevation of resting mitochondrial membrane potential of neural cells by cyclosporin A, BAPTA-AM, and bcl-2.

This study tested the hypothesis that the activity of the mitochondrial membrane permeability transition pore (PTP) affects the resting mitochondrial membrane potential (DeltaPsi) of normal, healthy cells and that the anti-apoptotic gene product Bcl-2 inhibits the basal activity of the PTP. DeltaPsi was measured by both fluorometric and nonfluorometric methods with SY5Y human neuroblastoma cells and with GT1-7 hypothalamic cells and PC12 pheochromocytoma cells in the absence and presence of Bcl-2 gene overexpression. The resting DeltaPsi of Bcl-2 nonexpressing PC12 and wild-type SY5Y cells was increased significantly by the presence of the PTP inhibitor cyclosporin A (CsA) or by intracellular Ca(2+) chelation through exposure to the acetoxymethyl ester of 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM). The DeltaPsi of Bcl-2-overexpressing PC12 cells was larger than that of Bcl-2-negative cells and not significantly increased by CsA or by Ca(2+) chelation. CsA did not present a significant effect on the DeltaPsi monitored in unstressed GT1-7 cells but did inhibit the decrease in DeltaPsi elicited by the addition of t-butyl hydroperoxide, an oxidative inducer of the mitochondrial permeability transition. These results support the hypothesis that an endogenous PTP activity can contribute to lowering the basal DeltaPsi of some cells and that Bcl-2 can regulate the endogenous activity of the mitochondrial PTP.

Frequency-dependent regulation of rat hippocampal somato-dendritic excitability by the K+ channel subunit Kv2.1.

The voltage-dependent potassium channel subunit Kv2.1 is widely expressed throughout the mammalian CNS and is clustered primarily on the somata and proximal dendrites, but not axons, of both principal neurones and inhibitory interneurones of the cortex and hippocampus. This expression pattern suggests that Kv2.1-containing channels may play a role in the regulation of pyramidal neurone excitability. To test this hypothesis and to determine the functional role of Kv2. 1-containing channels, cultured hippocampal slices were incubated with antisense oligonucleotides directed against Kv2.1 mRNA. Western blot analysis demonstrated that Kv2.1 protein content of cultured slices decreased > 90 % following 2 weeks of treatment with antisense oligonucleotides, when compared with either control missense-treated or untreated cultures. Similarly, Kv2.1 immunostaining was selectively decreased in antisense-treated cultures. Sustained outward potassium currents, recorded in both whole-cell and outside-out patch configurations, demonstrated a selective reduction of amplitude only in antisense-treated CA1 pyramidal neurones. Under current-clamp conditions, action potential durations were identical in antisense-treated, control missense-treated and untreated slices when initiated by low frequency stimulation (0.2 Hz). In contrast, spike repolarization was progressively prolonged during higher frequencies of stimulation (1 Hz) only in cells from antisense-treated slices. Similarly, action potentials recorded during electrographic interictal activity in the 'high [K+]o' model of epilepsy demonstrated pronounced broadening of their late phase only in cells from antisense-treated slices. Consistent with the frequency-dependent spike broadening, calcium imaging experiments from single CA1 pyramidal neurones revealed that high frequency Schaffer collateral stimulation resulted in a prolonged elevation of dendritic [Ca2+]i transients only in antisense-treated neurones. These studies demonstrate that channels containing Kv2.1 play a role in regulating pyramidal neurone somato-dendritic excitability primarily during episodes of high frequency synaptic transmission.

Mitochondria in Ca2+ signaling and apoptosis.

Cellular Ca2+ signals are crucial in the control of most physiological processes, cell injury and programmed cell death; mitochondria play a pivotal role in the regulation of such cytosolic Ca2+ ([Ca2+]c) signals. Mitochondria are endowed with multiple Ca2+ transport mechanisms by which they take up and release Ca2+ across their inner membrane. These transport processes function to regulate local and global [Ca2+]c, thereby regulating a number of Ca2+-sensitive cellular mechanisms. The permeability transition pore (PTP) forms the major Ca2+ efflux pathway from mitochondria. In addition, Ca2+ efflux from the mitochondrial matrix occurs by the reversal of the uniporter and through the inner membrane Na+/Ca2+ exchanger. During cellular Ca2+ overload, mitochondria take up [Ca2+]c, which, in turn, induces opening of PTP, disruption of mitochondrial membrane potential (delta(psi)m) and cell death. In apoptosis signaling, collapse of delta(psi)m and cytochrome c release from mitochondria occur followed by activation of caspases, DNA fragmentation, and cell death. Translocation of Bax, an apoptotic signaling protein from the cytosol to the mitochondrial membrane, is another step during this apoptosis-signaling pathway. The role of permeability transition in the context of cell death in relation to Bcl-2 family of proteins is discussed.

Permeability transition pore regulates both mitochondrial membrane potential and agonist-evoked Ca2+ signals in oligodendrocyte progenitors.

In this study, we investigated the importance of mitochondrial permeability transition pore (PTP) in agonist-evoked cytosolic Ca2+ ([Ca2+]c) signals in oligodendrocyte progenitor cells (OP cells). We measured transmembrane potential across the mitochondrial inner membrane (delta psi m) and [Ca2+]c in the immediate vicinity simultaneously using tetramethylrhodamine ethyl ester (TMRE) and calcium green respectively. Stimulation of OP cells with methacholine evoked robust [Ca2+]c signals in approximately 80% of cells which were either oscillatory or showed a peak followed by a plateau. Elevations in [Ca2+]c induced by supramaximal concentrations of the agonist (> 200 microM) were accompanied by changes in delta psi m in 33-42% of the total mitochondria investigated. The mitochondria that responded either depolarized (26-29%), hyperpolarized (7-13%) or showed no change (58-67%). Thus, of the responsive mitochondria, most (70%) depolarized during agonist-evoked [Ca2+]c signals. Blockade of PTP with cyclosporin A (CSA) reduced the number of mitochondria that depolarized with a corresponding increase in the number that hyperpolarized. In addition, CSA or its analogue methyl valine-4- CSA (MeVal-CSA), reduced the frequency of agonist-evoked global [Ca2+]c oscillations. In resting cells, CSA (63%) and MeVal-CSA (77%) hyperpolarized a majority of the mitochondria suggesting that PTP is constitutively active and may show flickering openings. Such hyperpolarizations were not mimicked by either cyclosporine H or verapamil and were inhibited by Ru360, which blocks the mitochondrial uniporter. This observation suggested that in resting cells, Ca2+ ions might redistribute between cytosol and mitochondrial matrix through the uniporter and the PTP. Taken together, these data suggest that PTP may play an important role in regulating delta psi m and local [Ca2+]c signals during agonist stimulation in OP cells.

Differential cellular expression of isoforms of inositol 1,4,5-triphosphate receptors in neurons and glia in brain.

Inositol 1,4,5-trisphosphate receptors (IP3R) are mediators of second messenger-induced intracellular calcium release. Three isoforms are known to be expressed in brain, but their regional distributions and cellular localizations are little known. In order to better understand the roles of IP3 receptor isoforms in brain function, a first step is to define their distributions. We have used affinity-purified antibodies directed against peptides unique to each isoform to determine their sites of expression in rat brain. Type 1 IP3R (IP3R1) is dramatically enriched in Purkinje neurons in cerebellum and neurons in other regions, consistent with previous studies. By contrast, IP3R2 is only detected in glia, whereas IP3R3 is predominantly neuronal, with little detected in glia. IP3R3 is enriched in neuropil, especially in neuronal terminals (which often contain large dense core vesicles) in limbic and basal forebrain regions including olfactory tubercle, central nucleus of the amygdala, and bed nucleus of the stria terminalis. In addition, IP3R1 and IP3R3 have clearly distinct time courses of expression in developing brains. These data suggest separate roles for inositol 1,4,5-trisphosphate receptor isoforms in development, and for glial and neuronal function. The IP3R3 may be involved in regulation of neurotransmitter or neuropeptide release in terminals within specific nuclei of the basal forebrain and limbic system.

Specialized distributions of mitochondria and endoplasmic reticulum proteins define Ca2+ wave amplification sites in cultured astrocytes.

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.

Mitochondrial Ca2+ uptake and release influence metabotropic and ionotropic cytosolic Ca2+ responses in rat oligodendrocyte progenitors.

1. 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. 2. Methacholine (MCh; 100 microM) activated Ca2+ waves that propagated from several initiation sites along O-2A processes. 3. 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). 4. Stimulation with kainate (100 microM) 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. 5. 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. 6. Inhibition of the mitochondrial Na+-Ca2+ exchanger by CGP-37157 (25 microM) decreased kainate Ca2+ response amplitude and increased the rate of return of the response to basal Ca2+ levels. 7. 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.

Role of mitochondrial Ca2+ regulation in neuronal and glial cell signalling.

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.

Characterization of ryanodine receptors in oligodendrocytes, type 2 astrocytes, and O-2A progenitors.

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.

Neurotransmitter-induced novel modulation of a nonselective cation channel by a cAMP-dependent mechanism in rat pineal cells.

In the rat, circadian rhythm in melatonin is regulated by noradrenergic and neuropeptide inputs to the pineal via adenosine 3',5'-cyclic monophosphate (cAMP)- and Ca2+-dependent mechanisms. We have identified a large conductance (170 pS), voltage-dependent, nonselective cation channel on rat pineal cells in culture that shows a novel mode of modulation by cAMP. Pituitary adenylate cyclase activating peptide (PACAP), norepinephrine, or 8-Br-cAMP increase channel open probability (Po) with a hyperpolarizing shift in voltage dependence such that the channel becomes active at resting membrane potentials. The increase in Po was accompanied by a change in current rectification properties such that the channel was transformed from being inactive at rest to an inwardly rectifying cation conductance in the presence of agonist, which depolarizes the cell. This channel is calcium insensitive, is blocked by Cs+, and shows a permeability sequence: K+ > Na+ >/= NH+4 > Li+. The data suggest that PACAP and norepinephrine acting through a cAMP-dependent mechanism modulate this nonselective cation channel, resulting in a slow onset depolarization that may be important in regulation of pineal cell excitability.

Images of Ca2+ flux in astrocytes: evidence for spatially distinct sites of Ca2+ release and uptake.

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 Fluo-3 fluorescence changes with high spatial and temporal resolution. Normalized fluorescence (delta F/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 microns 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.

High density distribution of endoplasmic reticulum proteins and mitochondria at specialized Ca2+ release sites in oligodendrocyte processes.

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.

Role of sarcoplasmic/endoplasmic-reticulum Ca2+-ATPases in mediating Ca2+ waves and local Ca2+-release microdomains in cultured glia.

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.

Comparison of type 2 inositol 1,4,5-trisphosphate receptor distribution and subcellular Ca2+ release sites that support Ca2+ waves in cultured astrocytes.

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.