Ca mobilization and capacitative Ca entry regulate DNA synthesis in cultured chick retinal neuroepithelial cells

Release of Ca²⁺ from intracellular Ca²⁺ stores (Ca²⁺ mobilization) and capacitative Ca²⁺ entry have been shown to be inducible in neuroepithelial cells of the early embryonic chick retina. Both types of Ca²⁺ responses decline parallel with retinal progenitor cell proliferation. To investigate their potential role in the regulation of neuroepithelial cell proliferation, we studied the effects of 2,5-di-tert-butylhydroquinone (DBHQ), an inhibitor of the Ca²⁺ pump of intracellular Ca²⁺ stores, and of SK&F 96365, an inhibitor of capacitative Ca²⁺ entry, on DNA synthesis in retinal organ cultures from embryonic day 3 (E3) chicks and in dissociated cultures from E7 and E9 chick retinae. We demonstrate that both antagonists inhibit [³H]-thymidine incorporation in a dose-dependent manner without affecting cell viability or morphology. The inhibition of [³H]-thymidine incorporation by SK&F 96365 occurred in the same concentration range (IC₅₀: 4 μM) as the blockade of capacitative Ca²⁺ entry in the E3 retinal organ culture. At a concentration of 5 μM SK&F 96365, DNA synthesis was reduced by 71, 40 and 32% in the E3, E7 and E9 cultures, respectively. Application of DBHQ at concentrations which led to depletion of intracellular Ca²⁺ stores also inhibited [³H]-thymidine incorporation with IC₅₀ values of 20–30 μM in the different cultures. Our results suggest the involvement of Ca²⁺ mobilization and capacitative Ca²⁺ entry in the regulation of DNA synthesis in the developing neural retina.     

Involvement of P2 purinoceptors in the regulation ofdna synthesis in the neural retina of chick embryo

The activation of P2 purinoceptors induces Ca2+ mobilization in the early embryonic chick neural retina. This purinergic Ca2+ response declines parallel with the decrease in mitotic activity during retinal development. To investigate the role of P2 purinoceptors in the regulation of retinal cell proliferation, we studied the effects of the P2 purinoceptor antagonists suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), and of the agonist ATP on DNA synthesis in retinal organ cultures from embryonic day 3 (E3) chick. Suramin inhibited [3H]-thymidine incorporation in a dose-dependent manner (IC50: approximately 70 microM). PPADS also reduced [3H]-thymidine incorporation with maximum inhibition of 46% at 100 microM. Exogenous ATP enhanced [3H]-thymidine incorporation in a dose-dependent manner to maximally 200% of control (EC50: approximately 70 microM). In dissociated retinal cultures from E7 chick, both antagonists showed similar inhibitory effects on [3H]-thymidine incorporation without affecting cell viability. In line with these observations, the presence of extracellular ATP was demonstrated both in vitro and in vivo. In the medium of E3 retinal organ cultures, the concentration of ATP increased 25-fold within 1 h of incubation and this concentration was kept for at least 24 h. In the chick amniotic fluid, the ATP concentration was nearly 3 microM at E3 and declined to 0.15 microM at E7. The results indicate that P2 purinoceptors activated by autocrine or paracrine release of ATP are involved in the regulation of DNA synthesis in the neural retina at early embryonic stages.     

A store-operated Ca2+ influx activated in response to the depletion of thapsigargin-sensitive Ca2+ stores is developmentally regulated in embryonic cortical neurons from mice

Store-operated channels (SOCs) are recruited in response to the release of Ca2+ from intracellular stores. They allow a voltage-independent entry of Ca2+ into the cytoplasm also termed capacitative Ca2+ entry (CCE). In neurons, the functional significance of this Ca2+ route remains elusive. Several reports indicate that SOCs could be developmentally regulated. We verified the presence of a CCE in freshly dissociated cortical cells from E13, E14, E16, E18 fetuses and from 1-day-old mice. Intracellular Ca2+ stores were depleted by means of the SERCA pump inhibitor thapsigargin. At E13, the release of Ca2+ from thapsigargin-sensitive compartments gave rise to an entry of Ca2+ in a minority of cells. This Ca2+ route, insensitive to voltage-gated Ca2+ channel antagonists like Cd2+ and Ni2+, was blocked by the SOC inhibitor SKF-96365. After E13 and on E13 cells kept in culture, there is a marked increase in the percentage of cells with functional SOCs. The lanthanide La3+ fully inhibited the CCE from neonatal mice whereas it weakly blocked the thapsigargin-dependent Ca2+ entry at E13. This suggests that the subunit composition of the cortical SOCs is developmentally regulated with La3+-insensitive channels being expressed in the embryonic cortex whereas La3+-sensitive SOCs are found at birth. Our data argue for the presence of SOCs in embryonic cortical neurons. Their expression and pharmacological properties are developmentally regulated.     

Zinc ions are endogenous modulators of neurotransmitter-stimulated capacitative Ca2+ entry in both cultured and in situ mouse astrocytes

Astrocytes express a variety of metabotropic receptors and their activation leads to a biphasic Ca2+ response due to Ca2+ release from intracellular stores and subsequent capacitative Ca2+ entry. We performed Ca2+ imaging with Fura-2 on cultured mouse astrocytes and showed that extracellular zinc reversibly blocks the capacitative Ca2+ entry following application of the metabotropic ligands ATP, glutamate and endothelin-1. Zinc blocked the plateau phase of the ligand-triggered Ca2+ responses. When ligands were repetitively applied in the presence of zinc the calcium responses progressively decayed and even disappeared, indicating that capacitative Ca2+ entry is required to refill the stores. Zinc inhibited the capacitative Ca2+ entry with a K(i) of approximately 6 microM, which is well within the physiological concentration range of zinc found in the brain. Application of the reducing agent DTT prevented the blocking effect by zinc ions but not the inhibition elicited by the nonphysiological metal ions Gd3+ and La3+, indicating that zinc has a distinct binding site. To monitor the capacitative Ca2+ entry in astrocytes in situ and to determine the effect of zinc on this pathway we utilized X-rhod-1 imaging in hippocampal slices of a transgenic mouse line with green fluorescent astrocytes. Zinc affected the repetitive metabotropic Ca2+ response in the following fashion: (i) after depleting stores in Ca(2+)-free solution, re-addition of Ca2+ led to an influx of Ca2+ via a zinc-sensitive Ca2+ entry route; (ii) with repetitive application of metabotropic ligands, Ca2+ responses became smaller and even disappeared in the presence of zinc. We conclude that zinc, which is co-released from glutamatergic synaptic vesicles upon neuronal activity, has a major impact on shaping the astrocytic calcium responses.     

Intracellular alkalinization augments capacitative Ca2+ entry in platelets

In order to elucidate the significance of intracellular alkalinization in signal transduction of platelets, we investigated the effects on capacitative Ca(2+) entry (CCE) of intracellular alkalinization that was induced by NH(4)Cl. Addition of NH(4)Cl (10 mM) to the medium resulted in an elevation of intracellular pH by about 0.35, which was eliminated by simultaneous addition of propionate (20 mM), an inducer of intracellular acidification, to the medium. CCE was induced by an extracellular addition of Ca(2+) to platelets in which Ca(2+) stores had been depleted by stimulation with thapsigargin in nominally Ca(2+)-free medium. NH(4)Cl markedly augmented CCE and subsequent platelet aggregation, both of which were abolished in the presence of SKF-96365, an inhibitor of capacitative Ca(2+) entry in non-excitable cells such as platelets. The augmentation of CCE and subsequent aggregation by NH(4)Cl was not observed in the presence of propionate or SKF-96365. Extracellular alkalosis induced by Tris also markedly augmented CCE and subsequent aggregation. These augmenting effects of extracellular alkalosis by Tris were significantly but incompletely inhibited by simultaneous addition of propionate (20 mM), which completely eliminated elevation of intracellular pH elicited by Tris. Thus, the augmenting effect of extracellular alkalosis on CCE was in part mediated by intracellular alkalosis. These findings suggest that intracellular alkalinization is a potent signal that augments CCE in platelets.     

Hypoxic regulation of Ca2+ signaling in cultured rat astrocytes

Acute hypoxia modulates various cell processes, such as cell excitability, through the regulation of ion channel activity. Given the central role of Ca2+ signaling in the physiological functioning of astrocytes, we have investigated how acute hypoxia regulates such signaling, and compared results with those evoked by bradykinin (BK), an agonist whose ability to liberate Ca2+ from intracellular stores is well documented. In Ca2+-free perfusate, BK evoked rises of [Ca2+]i in all cells examined. Hypoxia produced smaller rises of [Ca2+]i in most cells, but always suppressed subsequent rises of [Ca2+]i induced by BK. Thapsigargin pre-treatment of cells prevented any rise of [Ca2+]i evoked by either BK or hypoxia. Restoration of Ca2+ to the perfusate following a period of acute hypoxia always evoked capacitative Ca2+ entry. During mitochondrial inhibition (due to exposure to carbonyl cyanide p-trifluromethoxyphenyl hydrazone (FCCP) and oligomycin), rises in [Ca2+]i (observed in Ca2+-free perfusate) evoked by hypoxia or by BK, were significantly enhanced, and hypoxia always evoked responses. Our data indicate that hypoxia triggers Ca2+ release from endoplasmic reticulum stores, efficiently buffered by mitochondria. Such liberation of Ca2+ is sufficient to trigger capacitative Ca2+ entry. These findings indicate that the local O2 level is a key determinant of astrocyte Ca2+ signaling, likely modulating Ca2+-dependent astrocyte functions in the central nervous system.     

The beta-amyloid precursor protein controls a store-operated Ca2+ entry in cortical neurons

A polyclonal antibody (APP-Ab) raised against the extracellular domain of the beta-amyloid precursor protein (APP) triggers a marked neuronal cell death preceded by activation of Ca(2+)-dependent enzymes, neurite degeneration, oxidative stress and nuclear condensation [Mbebi et al. (2002) J. Biol. Chem., 277, 20979-20990]. We have investigated whether activation of APP by this antibody could promote cell death through cellular Ca2+ homeostasis alteration. We carried out time-lapse recordings of intracellular Ca2+ signals in cultured mice cortical neurons by means of a scanning confocal microscope. When applied in the presence of 2 mm external Ca2+, APP-Ab elicited a long-lasting elevation of the intracellular concentration of Ca2+ ([Ca2+]i). Experiments performed in the absence of external Ca2+ showed that APP-Ab triggers the release of Ca2+ from intracellular stores. The re-admission of external Ca2+ provides an additional rise of Ca2+ most likely through store-operated channels. A pretreatment of the cells with pertussis toxin, to inhibit the activity of Gi/Go proteins, or with the phospholipase C inhibitor, 3-nitrocoumarin, prevented both the APP-dependent elevation of Ca2+ as well as the APP-Ab-mediated cell death. Similarly, the store-operated channel inhibitors, 2-APB or SKF-96365 block both the APP-Ab-dependent Ca2+ entry and the APP-Ab-mediated cell death. Altogether, our data provide functional evidence that APP can perturb intracellular Ca2+ homeostasis by emptying intracellular Ca2+ stores and triggering Ca2+ entry through store-operated channels. In response to APP activation, the long-lasting elevation of [Ca2+]i due to an entry of Ca2+ via store-operated channels appears as a major event that leads to neuronal cell death.     

Caffeine-sensitive Ca2+ stores in carp retinal bipolar cells

High K+- or caffeine-induced Ca2+ signal was studied in freshly dissociated carp retinal ON-type bipolar cells using a confocal laser-scanning microscope. In response to 35 mM K+ exposure, a rise in [Ca2+]i appeared in both the terminal and soma, but was absent after removal of external Ca2+ or in the presence of 100 microM nifedipine. It is indicated that, for high K+-induced increase of [Ca2+]i, Ca2+ influx through voltage-gated L-type Ca2+ channels is essential and Ca2+ entry through reversed Na+/Ca2+ exchange may be negligible. Interestingly, caffeine-induced elevation of [Ca2+]i was restricted to the soma, and could be abolished by 50 microM ryanodine, suggesting that caffeine-sensitive Ca2+ stores gated by ryanodine receptors were present in the soma but not in the terminal of bipolar cells. After treatment with 50 microM ryanodine for 20 min, the peak of the Ca2+ transients evoked by 35 mM K+ in the soma decreased to 48.2+/-5.7% of the control. The results suggest that depolarization-evoked Ca2+ influx can cause Ca2+ release from caffeine-sensitive Ca2+ stores, and in turn amplify Ca2+ signal in the soma of retinal bipolar cells.     

Reduction of Ca2+ stores and capacitative Ca2+ entry is associated with the familial Alzheimer's disease presenilin-2 T122R mutation and anticipates the onset of dementia

Mutations in the presenilin genes PS1 and PS2, the major cause of familial Alzheimer's disease (FAD), are associated with alterations in Ca2+ signalling. In contrast to the majority of FAD-linked PS1 mutations, which cause an overload of intracellular Ca2+ pools, the FAD-linked PS2 mutation M239I reduces Ca2+ release from intracellular stores [Zatti, G., Ghidoni, R., Barbiero, L., Binetti, G., Pozzan, T., Fasolato, C., Pizzo, P., 2004. The presenilin 2 M239I mutation associated with Familial Alzheimer's Disease reduces Ca2+ release from intracellular stores. Neurobiol. Dis. 15/2, 269-278]. We here show that in human FAD fibroblasts another PS2 mutation (T122R) reduces both Ca2+ release and capacitative Ca2+ entry. The observation, done in two monozygotic twins, is of note since only one of the subjects showed overt signs of disease at the time of biopsy whereas the other one developed the disease 3 years later. This finding indicates that Ca2+ dysregulation anticipates the onset of dementia. A similar Ca2+ alteration occurred in HeLa and HEK293 cells transiently expressing PS2-T122R. Based on these data, the "Ca2+ overload" hypothesis in AD pathogenesis is here discussed and reformulated.     

Capacitative Ca2+ influx in glial cells is inhibited by glycolytic inhibitors

In non-excitable cells, stimulation of phosphoinositide (PI) turnover and inhibition of the endoplasmic reticulum (ER) Ca²⁺-ATPase are methods commonly used to deplete calcium stores, resulting in a capacitative Ca²⁺ influx (i.e., Ca²⁺ release-activated Ca²⁺ influx). Since this Ca²⁺ influx in glial cells has not been thoroughly investigated, we have used C₆ glioma cells as a glial cell model to study this phenomenon. On adding cyclopiazonic acid (CPA) or thapsigargin (TG) (two ER Ca²⁺-ATPase inhibitors) in Ca²⁺-free medium, only a small transient increase in intracellular Ca²⁺ was seen. After depletion of the stored Ca²⁺, a marked Ca²⁺ influx, followed by a prolonged plateau, was seen on re-addition of extracellular Ca²⁺ ions (2 mM), i.e., capacitative Ca²⁺ influx. A similar effect was seen on adding ATP, known to deplete the inositol triphosphate (IP₃)-sensitive Ca²⁺ store in C₆ cells. After various degrees of store depletion, the amplitude of the capacitative Ca²⁺ influx was found to be highly dependent on the amount of Ca²⁺ remaining in the store. This Ca²⁺ influx was markedly inhibited by (1) La³⁺ and Ni²⁺, (2) SK&F 96365, econazole, and miconazole, and (3) membrane depolarization, clearly showing that this Ca²⁺ influx after store depletion in C₆ cells is a capacitative mechanism. Interestingly, the capacitative Ca²⁺ influx can be inhibited by a reduction in intracellular ATP (ATP_i) levels in glial cells. The role of ATP_i in the capacitative Ca²⁺ influx is discussed. GLIA 21:315–326, 1997. © 1997 Wiley-Liss, Inc.     

Effects of calmodulin and protein kinase C modulators on transient Ca2+ increase and capacitative Ca2+ entry in human platelets: relevant to pathophysiology of bipolar disorder

Disturbed intracellular calcium (Ca(2+)) homeostasis has been implicated in bipolar disorder, which mechanisms may be involved in the dysregulation of protein kinase C (PKC) and calmodulin systems. In this study, we investigated a transient intracellular Ca(2+) increase induced by thapsigargin, an inhibitor of sarco/endoplasmic reticulum Ca(2+)-ATPase pump (SERCA), and a capacitative Ca(2+) entry followed by addition of extracellular Ca(2+), in the presence or absence of PKC/calmodulin modulators in the platelets of healthy subjects in order to elucidate the role of SERCA in Ca(2+) homeostasis and to assess how both PKC and calmodulin systems regulate the two Ca(2+) responses. Moreover, we also examined the thapsigargin-elicited transient Ca(2+) increase and capacitative Ca(2+) entry in patients with mood disorders. PKC and calmodulin systems have opposite regulatory effects on the transient Ca(2+) increase and capacitative Ca(2+) entry in the platelets of normal subjects. The inhibitory effect of PKC activation on capacitative Ca(2+) entry is significantly increased and the stimulatory effect of PKC inhibition is significantly decreased in bipolar disorder compared to major depressive disorder and normal controls. These results suggest the possibility that increased PKC activity may activate the inhibitory effect of capacitative Ca(2+) entry in bipolar disorder. However, this is a preliminary study using a small sample, thus further studies are needed to examine the PKC and calmodulin modulators on the capacitative Ca(2+) entry in a larger sample.     

Blockade of NMDA receptor-mediated mobilization of intracellular Ca2+ prevents neurotoxicity.

NMDA receptor activation leads to elevated Ca2+ in cultured rat cortical and retinal ganglion cell neurons. If excessive, this Ca2+ response is associated with delayed neurotoxicity. We used dantrolene and ionomycin to test if the Ca2+ response to NMDA was due to mobilization of intracellular Ca2+ stores rather than merely to Ca2+ influx. In the presence of EGTA, ionomycin resulted in release and subsequent depletion of intracellular Ca2+ stores. Henceforth, despite normal extracellular Ca2+, NMDA elicited only about half of its former Ca2+ response. Similarly, when dantrolene was used to block Ca2+ release from intracellular stores, we observed > 50% smaller NMDA-evoked Ca2+ responses. These results quite surprisingly indicate that at least half the Ca2+ response to NMDA is due to release of intracellular Ca2+, a process triggered by influx of extracellular Ca2+. Dantrolene also protected neurons from NMDA receptor-mediated neurotoxicity. Release of intracellular Ca2+ may therefore be a necessary step in the cascade leading to neuronal damage induced by excessive NMDA receptor stimulation and may be amenable to pharmacological intervention.     

Pharmacologic characterization of refilling inositol 1,4,5-trisphosphate-sensitive Ca stores in NG108-15 cells

Following mobilization with the inositol 1,4,5-trisphosphate (IP₃)-generating agonist bradykinin, Ca²⁺ stores in neuroblastoma × glioma hybrid, NG108-15 cells require extracellular Ca²⁺ to refill. The process by which this store refills with Ca²⁺ was characterized by recording bradykinin-induced intracellular free Ca²⁺ concentration transients as an index of the degree of refilling of the store. Cyclopiazonic acid, a microsomal Ca²⁺ ATPase inhibitor, reversibly depleted intracellular Ca²⁺ stores in these cells, but did not recruit detectable Ca²⁺ influx, suggesting that these cells lack substantial capacitative Ca²⁺ entry. The paucity of voltage-sensitive Ca²⁺ channels in undifferentiated NG108-15 cells, suggested that a channel analogous to that proposed to mediate capacitative Ca²⁺ entry in nonexcitable cells might assist refilling IP₃-sensitive Ca²⁺ stores in these cells. The possibility that compounds shown previously to inhibit capacitative Ca²⁺ entry in nonexcitable cells might inhibit the refilling of the IP₃-sensitive store in NG108-15 cells was explored. The IP₃-sensitive store was depleted by exposure to bradykinin, allowed to refill briefly in the presence of the test compound and then challenged again with bradykinin to evaluate the degree of refilling of the store. The imidazole derivatives, econazole (10 μM), L-651582 (10 μM)and SKF 96365 (20 μM), all completely blocked the bradykinin-induced Ca²⁺ response. Calmodulin antagonists, W-7 (100 μM)and trifluoperazine (10 μM), were also effective, although at concentrations well above those required to inhibit calmodulin. Because of the high concentrations required to inhibit bradykinin responses, the possibility that these agents might have additional effects was explored. Compounds were tested in a paradigm in which the store was preloaded with Ca²⁺ before treatment. All of these agents depleted, at least partially, the preloaded store. Econazole was the least effective of the compounds tested for releasing stores, although it was comparable to the other compounds for inhibition of refilling. Although NG108-15 cells refill intracellular Ca²⁺ stores by a plasmalemmal Ca²⁺ leak, this leak shares a pharmacology similar to the capacitative Ca²⁺ entry pathway described for nonexcitable cells.     

ATP induces proliferation of retinal cells in culture via activation of PKC and extracellular signal-regulated kinase cascade

Both ATP and acetylcholine can induce the mobilization of intracellular calcium in the early developing chick embryo retina, a response that decreases during retinal development. In this study, the effects of these transmitters on the turnover of phosphoinositides and proliferation of developing retinal cells in culture were characterized. While ATP, UTP or carbachol were able to induce a >400% accumulation of phosphoinositides in retinal cell cultures, only ATP promoted a dose-dependent increase in [(3)H]-thymidine incorporation in cultured cells (EC(50)=8.6 microM), a response that was inhibited by the P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (0.1 or 0.25 mM). ADP, but not UTP or adenosine, also stimulated the proliferation of retinal cells (EC(50)=5.8 microM), indicating that activation of P2Y1 receptors mediates the proliferative response of retinal cells to ATP. The mitogenic effect of ATP was completely prevented by the PKC inhibitor chelerythrine chloride (0.5 microM) and the phospholipase C (PLC) inhibitor U73122 (0.5 microM). PD 98059 (25 or 50 microM), an inhibitor of the activation of extracellular signal-regulated kinases (ERKs) also blocked the increase in [(3)H]-thymidine incorporation induced by ATP. Moreover, the effect of ATP was pronounced in cultures obtained from retinas at embryonic days 6-8, but not at day 9. Since Müller and bipolar cells are the predominant cell types that proliferate at these embryonic stages, our data suggest that ATP, through activation of P2Y1 receptors coupled to phospholipase C, PKC and MAP kinases, affects DNA synthesis in one or both of these cell types in culture.     

The presenilin 2 M239I mutation associated with familial Alzheimer's disease reduces Ca2+ release from intracellular stores

Mutations in presenilin (PS) genes account for the majority of the cases of the familial form of Alzheimer's disease (FAD). PS mutations have been correlated with both over-production of the amyloid-beta-42 (Abeta42) peptide and alterations of cellular Ca(2+) homeostasis. We here show, for the first time, the effect of the recently described PS2 FAD-associated M239I mutation on two major parameters of intracellular Ca(2+) homeostasis: the Ca(2+) storing capacity of the endoplasmic reticulum (ER) and the activation level of capacitative Ca(2+) entry (CCE), the Ca(2+) influx pathway activated by depletion of intracellular stores. Ca(2+) release from intracellular stores was significantly reduced in fibroblasts from FAD patients, compared to that found in cells from healthy individuals or patients affected by sporadic forms of Alzheimer's Disease (AD). No significant difference was however found in CCE between FAD and control fibroblasts. Similar results were obtained in two cell lines (HEK293 and HeLa) stably or transiently expressing the PS2 M239I mutation.     

Differential thapsigargin-sensitivities and interaction of Ca2+ stores in human SH-SY5Y neuroblastoma cells

In human SH-SY5Y neuroblastoma cells, two distinct intracellular Ca2+ stores, a KCl-/caffeine-sensitive and a carbachol-/IP3-sensitive store, were demonstrated previously. In this study, responses of these two intracellular Ca2+ stores to thapsigargin were characterized. Ca2+-release from these stores was evoked either by high K+ (100 mM KCl) or by 1 mM carbachol, and changes in the intracellular Ca2+ level were monitored using Fura-2 fluorimetry. A sequential stimulation protocol (KCl-->carbachol or vice versa) allowed evaluation of the individual contribution of different Ca2+ stores to the evoked intracellular Ca2+ ([Ca2+]i)-transients and the dynamic interaction between them. Thapsigargin (0.05 nM - 20 microM) alone induced a [Ca2+]i-transient. Both the carbachol- and the KCl-evoked [Ca2+]i-transients were inhibited by thapsigargin, but with very different sensitivities. Thapsigargin inhibited the carbachol-evoked [Ca2+]i-transients with (IC50 = 0.353 nM) or without (IC50 = 0.448 nM) a KCl-prestimulation, but an additional small component, with a much lower sensitivity (IC50=4814 nM), was observed in the absence of a KCl-prestimulation. In contrast, the KCl-evoked [Ca2+]i-transients displayed only one component with a very low sensitivity to thapsigargin in both absence (IC50=3343 nM) and presence (IC50=6858 nM) of a carbachol-prestimulation. These findings suggest that the sarco-/endoplasmic reticular Ca2+ ATPases associated with the KCl-/caffeine- and carbachol-/IP3-sensitive intracellular Ca2+ stores differ from each other, either in types or in their post-translational modification. Such difference might play important role in the regulation of neuronal Ca2+ homeostasis.     

Complex interactions between mGluRs, intracellular Ca2+ stores and ion channels in neurons

Metabotropic glutamate receptors (mGluRs) can increase intracellular Ca2+ concentration via Ins(1,4,5)P3- and ryanodine-sensitive Ca2+ stores in neurons. Both types of store are coupled functionally to Ca2+-permeable channels found in the plasma membrane. The mGluR-mediated increase in intracellular Ca2+ concentration can activate Ca2+-sensitive K+ channels and Ca2+-dependent nonselective cationic channels. These mGluR-mediated effects often result from mobilization of Ca2+ from ryanodine-sensitive, rather than Ins(1,4, 5)P3-sensitive, Ca2+ stores, suggesting that close functional interactions exist between mGluRs, intracellular Ca2+ stores and Ca2+-sensitive ion channels in the membrane.     

Ca(2+) stores and capacitative Ca(2+) entry in human neuroblastoma (SH-SY5Y) cells expressing a familial Alzheimer's disease presenilin-1 mutation

Presenilins are involved in the proteolytic production of Alzheimer's amyloid peptides, but are also known to regulate Ca(2+) homeostasis in various cells types. In the present study, we examined intracellular Ca(2+) stores coupled to muscarinic receptors and capacitative Ca(2+) entry (CCE) in the human neuroblastoma SH-SY5Y cell line, and how these were modulated by over-expression of either wild-type presenilin 1 (PS1wt) or a mutant form of presenilin 1 (PS1 deltaE9) which predisposes to early-onset Alzheimer's disease. Ca(2+) stores discharged by application of 100 microM muscarine (in Ca(2+)-free perfusate) in PS1wt and PS1 DeltaE9 cells were significantly larger than those in control cells, as determined using Fura-2 microfluorimetry. Subsequent CCE, observed in the absence of muscarine when Ca(2+) was re-admitted to the perfusate, was unaffected in PS1wt cells, but significantly suppressed in PS1 deltaE9 cells. However, when Ca(2+) stores were fully depleted with thapsigargin, CCE was similar in all three cell groups. Western blots confirmed increased levels of PS1 in the transfected cells, but also demonstrated that the proportion of intact PS1 in the PS1 deltaE9 cells was far greater than in the other two cell groups. This study represents the first report of modulation of both Ca(2+) stores and CCE in a human, neurone-derived cell line, and indicates a distinct effect of the PS1 mutation deltaE9 over wild-type PS1.     

Extracellular pH affects platelet aggregation associated with modulation of store-operated Ca(2+) entry.

The pH dependence of store-operated Ca(2+) influx (SOCI) into human platelets, as well as its physiological consequence, aggregation, was studied. In Ca(2+)-free medium, thapsigargin (1 microM) induced a small increase in intracellular free-Ca(2+) ([Ca(2+)](i)), which was not affected by changes in extracellular pH. The addition of Ca(2+) (0.5-3 mM) after Ca(2+) store depletion caused by thapsigargin resulted in concentration-dependent increases in [Ca(2+)](i) (SOCI), which were strongly inhibited by SKF-96365 (100 microM), an inhibitor of receptor-mediated Ca(2+) entry. SOCI was inhibited by acidosis (pH 6.9) and augmented by alkalosis (pH 7.9). The addition of Ca(2+) (0.5-3 mM) to platelets, which were kept in Ca(2+)-free medium, slightly but significantly increased [Ca(2+)](i). This Ca(2+) leak entry was also decreased and increased by extracellular acidosis (pH 6.9) and alkalosis (pH 7.9), respectively, but not affected by SKF-96365. Neither thapsigargin (1 microM) stimulation in Ca(2+)-free solution nor elevation of extracellular Ca(2+) alone was sufficient to induce platelet aggregation. In contrast, the addition of Ca(2+) (1 mM) to platelets activated by thapsigargin resulted in aggregation, which was markedly inhibited by SKF-96365 (100 microM). Platelet aggregation associated with SOCI was also inhibited by extracellular acidosis (pH 6.9) and augmented by extracellular alkalosis (pH 7.9). These results suggest that acidosis-induced inhibition, as well as alkalosis-induced promotion of platelet aggregation, involve pH effects on SOCI.     

Modifications of intracellular Ca2+ signalling during nerve growth factor-induced neuronal differentiation of rat adrenal chromaffin cells

Postnatal sympathetic neurons (SNs) and chromaffin cells (CCs) derive from neural crest precursors. CCs can differentiate in vitro into SN-like cells after nerve growth factor (NGF) exposure. This study examines changes of intracellular Ca2+ homeostasis and dynamics of CCs under conditions that promote a neuronal phenotype. Spontaneous Ca2+ fluctuations, a frequent observation in early cultures of CCs, diminished after > 10 days in vitro in control cells and ceased in NGF-treated ones. At the same time, Ca2+ rises resulting from entry upon membrane depolarization, gradually increased both their size and peak d[Ca2+]i/dt, resembling those recorded in SNs. Concomitantly, caffeine-induced Ca2+ rises, resulting from Ca2+ release from intracellular stores, increased their size and their peak d[Ca2+]i/dt by > 1000%, and developed transient and sustained release components, similar to those of SNs. The transient component, linked to regenerative Ca2+ release, appeared after > 10 days of NGF treatment, suggesting a delayed steep enhancement of Ca2+-induced Ca2+ release (CICR). Immunostaining showed that proteins coded by the three known isoforms of ryanodine receptors (RyRs) are present in CCs, but that only RyR2 increased significantly after NGF treatment. Since the transient release component increased more steeply than RyR2 immunostaining, we suggest that the development of robust CICR requires both an increased expression of RyRs and more efficient functional coupling among them. NGF-induced transdifferentiation of chromaffin cells involves the enhancement of both voltage-gated Ca2+ influx and Ca2+ release from intracellular stores. These modifications are likely to complement the extensive morphological and functional reorganization required for the replacement of the endocrine phenotype with the neuronal one.