Differential Stimulation of Noradrenaline Release by Reversal of the Na+/Ca2+ Exchanger and Depolarization in Chromaffin Cells

We compared the effectiveness of Ca²⁺ entering by Na⁺/Ca²⁺ exchange with that of Ca²⁺ entering by channels produced by membrane depolarization with K⁺ in inducing catecholamine release from bovine adrenal chromaffin cells. The Ca²⁺ influx through the Na⁺/Ca²⁺ exchanger was promoted by reversing the normal inward gradient of Na⁺ by preincubating the cells with ouabain to increase the intracellular Na⁺ and then removing Na⁺ from the external medium. In this way we were able to increase the cytosolic free Ca²⁺ concentration ([Ca²⁺]_c) by Na⁺/Ca²⁺ exchange to 325 ± 14 nM, which was similar to the rise in [Ca²⁺]_c observed upon depolarization with 35 mM K⁺ of cells not treated with ouabain. After incubating the cells with ouabain, K⁺ depolarization raised the [Ca²⁺]_c to 398 ± 31 nM, and the recovery of [Ca²⁺]_c to resting levels was significantly slower. Reversal of the Na⁺ gradient caused an −6-fold increase in the release of noradrenaline or adrenaline, whereas K⁺ depolarization induced a 12-fold increase in noradrenaline release but only a 9-fold increase in adrenaline release. The ratio of noradrenaline to adrenaline release was 1.24 ± 0.23 upon reversal of the Na⁺/Ca²⁺ exchange, whereas it was 1.83 ± 0.19 for K⁺ depolarization. Reversal of the Na⁺/Ca²⁺ exchange appeared to be as efficient as membrane depolarization in inducing adrenaline release, in that the relation of [Ca²⁺]_c to adrenaline release was the same in both cases. In contrast, we found that for the same average [Ca²⁺]_c, the Ca²⁺ influx through voltage-gated channels was much more efficient than the Ca²⁺ entering through the Na⁺/Ca²⁺ exchanger in inducing noradrenaline release from chromaffin ceils. This greater effectiveness of membrane depolarization in stimulating noradrenaline release suggests that there is a pool of noradrenaline vesicles which is more accessible to Ca²⁺ entering through voltage-gated Ca²⁺ channels than to Ca²⁺ entering through the Na⁺/Ca²⁺ exchanger, whereas the adrenaline vesicles do not distinguish between the source of Ca²⁺.     

Divalent Cation Entry in Cultured Rat Cerebellar Granule Cells Measured Using Mn2+ Quench of Fura 2 Fluorescence

In this study the rate of Mn²⁺ quench of fura-2 fluorescence evoked by glutamatergic and cholinergic agonists, depolarization and Ca²⁺ store modulators was measured in cultured cerebellar granule cells, in order to study their effects on Ca²⁺ entry in isolation from effects on Ca²⁺ store release. The rate of fluorescence quench by 0.1 mM Mn²⁺ was markedly increased by 25 mM K⁺- evoked depolarization or by 200 μM N-methyl-D-aspartate (NMDA), with a significantly greater increase occurring during the rapid-onset peak phase compared to the plateau phase of the K⁺- or NMDA-evoked [Ca²⁺]_i response. The stimulatory effect of NMDA on Mn²⁺ quench was abolished by dizocilpine (10 μM), but nitrendipine (2 μM), while decreasing the rate of basal quench, did not affect NMDA-stimulated Mn²⁺ entry. This suggests that nitrendipine may not act on NMDA channels in granule cells, at least under these conditions, and that voltage-operated Ca²⁺ channels are involved in control quench whereas the NMDA-evoked quench is dependent on entry through the receptor channel. The t_1/2 of quench was unaffected by α-amino-hydroxyisoxazole propionic acid (200 μM) and carbamyl choline (1 mM). Neither thapsigargin (10 μM) nor dantrolene (30 μM) significantly affected the rate of quench under control or NMDA- or K⁺-stimulated conditions, which confirms that the previously reported inhibitory effects on [Ca²⁺]_i elevations evoked by these agents are due to actions on Ca²⁺ stores. However, thapsigargin elevated [Ca²⁺Ii in the presence of normal [Ca²⁺]_i, but not in nominally Ca²⁺-free medium, indicating that it evokes Ca²⁺ entry in cerebellar granule cells, probably subsequent to store depletion, which appears to be either too small to be detected by Mn²⁺ quench or to occur via Mn²⁺-impermeant channels.     

Modulation of neurotransmitter release by dihydropyridine-sensitive calcium channels involves tyrosine phosphorylation

Cultured rat cerebellar granule cells depolarized by high KCl, display a large component of Ca²⁺ influx through L-type voltage-dependent Ca²⁺ channels as defined by a sensitivity to 1 μm nifedipine. This Ca²⁺ influx is not coupled to neurotransmitter exocytosis but has implications for neuronal development. KCl stimulation in the absence of external Ca²⁺ followed by the re-addition of Ca²⁺ allows the coupling of a class of L-type Ca²⁺ channels to neurotransmitter exocytosis as assessed by loading of glutamatergic pools with [³H]-d -aspartate. KCl stimulation in the absence of external Ca²⁺ (‘predepolarization’) enhances tyrosine phosphorylation of several cellular proteins, and inhibitors of tyrosine kinases block both phosphorylation and the neurotransmitter release coupled to the L-type Ca²⁺ channel. More specifically, an inhibitor of src family tyrosine kinases, PP1, blocks the effects of predepolarization suggesting a role for a src family kinase in the process. Furthermore, L-type Ca²⁺ channel recruitment and modulation of release could be activated with the tyrosine phosphatase inhibitor sodium orthovanadate. The phosphoproteins enhanced by predepolarization, which include the cytoskeletal proteins focal adhesion kinase (FAK) and vinculin, are also highly phosphorylated early on in culture when neurite outgrowth occurs. As the neurons develop a network of neurites, both tyrosine phosphorylation and L-type Ca²⁺ channel activity decrease. These results show a novel mechanism for the recruitment of L-type Ca²⁺ channels and their coupling to neurotransmitter release which involves tyrosine phosphorylation. This phenomenon has a role in cerebellar granule cell development.     

Sodium-dependent glutamate transport in Müller glial cells: regulation by phorbol esters

The regulation of the Na⁺-dependent high affinity glutamate/aspartate transporter system expressed in cultured Müller glia cells from chick retina was studied. Treatment of the cells with the Ca²⁺/diacylglycerol dependent protein kinase C (PKC) activator, phorbol 12-tetradecanoil-13-acetate (TPA) produced a decrease in [³H] -aspartate uptake which was reversed by staurosporine and partially by H7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochoride], two PKC inhibitors. Long-term treatment with TPA resulted in a drastic decrease in the uptake activity, correlated with a substantial fall in the expression of the transporter protein. These findings suggest that PKC is involved in transport modulation at two different levels: phosphorylation and transporter expression in retinal Müller glial cells.     

Transduction of a protein kinase C-generated signal into the long-lasting facilitation of glutamate release

The present study investigated the delayed and persistent effects of 4β-phorbol 12, 13-dibutyrate (PDBu) on the K⁺ -evoked release of endogenous glutamate and dynorphin B-like immunoreactivity from a subcellular fraction (P₃) that is enriched in hippocampal mossy fiber synaptosomes. It is demonstrated that the alpha, beta, gamma, epsilon, and zeta isoforms of protein kinase C (PKC) are present in the P₃ fraction obtained using the guinea pig hippocampus as starting tissue. The K⁺ -evoked release of glutamate was found to be selectively enhanced when mossy fiber-enriched synaptosomes were preincubated with PDBu for 15 minutes and extensively washed with a PDBu-free medium. The persistent enhancement of glutamate release observed under this condition was not reversed by the protein kinase inhibitor staurosporine and was desensitized to the potentiating effects of an acute reexposure to PDBu. The overall content and activity of PKC was not substantially altered during the initial 15 minutes of treatment with PDBu (10 μM). More prolonged pretreatments with PDBu altered the substrate specificity of PKC and decreased the content of all PKC isoforms, but did not reverse the facilitation of glutamate release that followed preincubation in the presence of PDBu. It is concluded that the persistent activation of PKC enhances K⁺ -evoked glutamate release from hippocampal mossy fiber-enriched synaptosomes and that, once established, this presynaptic facilitation is sustained by a process that is no longer directly dependent on continued PKC phosphotransferase activity.     

Partial characterization of K-induced increase in [Ca]cyt and GnRH release in GT1-7 neurons

Secretion of pituitary gonadotropins is regulated centrally by the hypothalamic decapeptide gonadotropin releasing hormone (GnRH). Using the immortalized hypothalamic GT1-7 neuron, we characterized pharmacologically the dynamics of cytosolic Ca²⁺ and GnRH release in response to K⁺-induced depolarization of GT1-7 neurons. Our results showed that K⁺ concentrations from 7.5 to 60 mM increased [Ca²⁺]_cyt in a concentration-dependent manner. Resting [Ca²⁺]_cyt in GT1-7 cells was determined to be 69.7 ± 4.0 nM (mean ± S.E.M.; N = 69). K⁺-induced increases in [Ca²⁺]_cyt ranged from 58.2 nM at 7.5 mM [K⁺] to 347 nM at 60 mM [K⁺]. K⁺-induced GnRH release ranged from about 10 pg/ml at 7.5 mM [K⁺] to about 60 pg/ml at 45 mM [K⁺]. K⁺-induced increases in [Ca²⁺]_cyt and GnRH release were enhanced by 1 μM BayK 8644, an L-type Ca²⁺ channel agonist. The BayK enhancement was completely inhibited by 1 μM nimodipine, an L-type Ca²⁺ channel antagonist. Nimodipine (1 μM) alone partially inhibited K⁺-induced increases in [Ca²⁺]_cyt and GnRH release. Conotoxin (1 μM) alone had no effect on K⁺-induced GnRH release or [Ca²⁺]_cyt, but the combination of conotoxin (1 μM) and nimodipine (1 μM) inhibited K⁺-induced increase in [Ca²⁺]_cyt significantly more (p < 0.02) than nimodipine alone, suggesting that N-type Ca²⁺ channels exist in GT1-7 neurons and may be part of the response to K⁺. The response of [Ca²⁺]_cyt to K⁺ was linear with increasing [K⁺] whereas the response of GnRH release to increasing [K⁺] appeared to be saturable. K⁺-induced increase in [Ca²⁺]_cyt and GnRH release required extracellular [Ca²⁺]. These experiments suggest that voltage dependent N- and L-type Ca²⁺ channels are present in immortalized GT1-7 neurons and that GnRH release is, at least in part, dependent on these channels for release of GnRH.     

Ca2+ changes and 86Rb efflux activated by hyposmolarity in cerebellar granule neurons

Hyposmotic swelling increased ⁸⁶Rb release in cultured cerebellar granule neurons (1 day in vitro [DIV]) with a magnitude related to the change in osmolarity. ⁸⁶Rb release was partially blocked by quinidine, Ba²⁺, and Cs⁺ but not by TEA, 4-AP, or Gd³⁺. ⁸⁶Rb efflux decreased in Cl⁻-depleted cells or cells treated with DDF or DIDS, suggesting an interconnection between Cl⁻ and K⁺ fluxes. Swelling induced a substantial increase in [Ca²⁺]_i to which both external and internal sources contribute. However, ⁸⁶Rb efflux was independent of [Ca²⁺]_o, unaffected by depleting the endoplasmic reticulum (ER) by ionomycin or thapsigargin and insensitive to charybdotoxin, iberiotoxin, and apamin. Swelling-activated ⁸⁶Rb efflux in differentiated granule neurons after 8 DIV, which express Ca²⁺-sensitive K⁺ channels, was not different from that in 1 DIV neurons, nor in time course, net release, Ca²⁺-dependence, or pharmacological sensitivity. We conclude that the swelling-activated K⁺ efflux in cerebellar granule neurons is not mediated by Ca²⁺-sensitive large conductance K⁺ channels (BK) as in many cell types but resembles that in lymphocytes where it is possibly carried by voltage-gated K⁺ channels. J. Neurosci. Res. 53:626–635, 1998. © 1998 Wiley-Liss, Inc.     

4-Aminopyridine stimulates B-50 (GAP-43) phosphorylation in rat synaptosomes

Recently, we have shown that stimulation of [³H]-noradrenaline release from hippocampal slices by 4-aminopyridine (4-AP) is accompanied by an enhancement of the phosphorylation of B-50, a major presynaptic substrate of protein kinase C (PKC). PKC has been implicated in the regulation of transmitter release. In this study, we investigated the effects of 4-AP on B-50 phosphorylation in synaptosomes from rat brain and compared the effects of 4-AP with those of depolarization with K⁺, in order to gain more insight into the mechanism of action of 4-AP. B-50 phosphorylation was stimulated by incubation with 4-AP for 2 minutes at concentrations ranging from 10 μM to 5 mM. 4-AP (100 μM) stimulated B-50 phosphorylation already within 15 seconds; longer incubations revealed a sustained increase in the presence of 4-AP. B-50 phosphorylation was also stimulated by depolarization with 30 mM K⁺ for 15 seconds. The effects of both 4-AP or K⁺ depolarization on B-50 phosphorylation were abolished at low extracellular Ca²⁺ concentrations. The increase in B-50 phosphorylation induced by 4-AP seemed to be dependent on the state of depolarization, since the effect of 4-AP was largest under nondepolarizing conditions. Comparing the effects of 4-AP and K⁺ depolarization on B-50 phosphorylation suggests that a different mechanism of action is involved. These results indicate that the stimulation of B-50 phosphorylation by 4-AP in hippocampal slices can be attributed to a direct action of 4-AP on presynaptic terminals. In addition, our results support the hypothesis that B-50 phosphorylation by PKC is involved in Ca²⁺-dependent transmitter release evoked by 4-AP. This research was supported by CLEO-TNO grant A66 of the Dutch Epilepsy Foundation.     

Opposing effects of thapsigargin on the survival of developing cerebellar granule neurons in culture

Elevated levels of potassium (K⁺) promote maturation and survival of cerebellar granule neurons in culture. When switched from a culture medium containing high K⁺ (25 mM) to one with low K⁺ (5 mM) mature granule neurons undergo death by apoptosis. The mechanism by which high K⁺ promotes neuronal survival and conversely inhibits apoptosis) is unclear. Several pieces of evidence indicate that an increase in intracellular calcium (Ca²⁺) resulting from depolarization mediated-influx of extracellular Ca²⁺ is necessary. We examined the effect of thapsigargin on granule neuron cultures. Thapsigargin is an inhibitor of the endoplasmic reticular Ca²⁺ ATPase causing a depletion of Ca²⁺ from internal stores. this treatment would therefore be expected to raise intracellular cytosolic Ca²⁺ without membrane depolarization. We find that treatment of mature neurons with thapsigargin at doses 5 nM inhibits death resulting from the lowering of extracellular K⁺. The survival effect of thapsigargin was not affected by inhibitors of extracellular Ca²⁺ influx including nifedipine, verapamil, methoxyverapamil, Mg²⁺, and Ni²⁺, nor was it inhibited by the NMDA receptor antagonist, MK801. We have further examined whether thapsigargin could substitute for elevated K⁺ during the maturation of granule cells. Unexpectedly, treatment of younger (immature) neuronal cultures with the same dose of thapsigargin (5 nM) induced cell death. DNA fragmentation analysis suggested that death was due to apoptosis and not toxicity. As observed with the survival effect on mature neurons, the lethal effect of thapsigargin on immature granule cells was not prevented by inhibitors of Ca²⁺ influx.     

Glutamate increases the [Ca]i but stimulates Ca-independent release of [H]GABA in cultured chick retina cells

The effect of glutamate of [Ca²⁺]_i and on [³H]γ-aminobutyric acid (GABA) release was studied on cultured chick embryonic retina cells. It was observed that glutamate (100 μM) increases the [Ca²⁺]_i by Ca²⁺ influx through Ca²⁺ channels sensitive to nitrendipine, but not to ω-conotoxin GVIA (ω-Cg Tx) (50%), and by other channels insensitive to either Ca²⁺ channel blocker. Mobilization of Ca²⁺ by glutamate required the presence of external Na⁺, suggesting that Na⁺ mobilization through the ionotropic glutamate receptors is necessary for the Ca²⁺ channels to open. The increase in [Ca²⁺]_i was not related to the release of [³H]GABA induced by glutamate, suggesting that the pathway for the entry of Ca²⁺ triggered by glutamate does not lead to exocytosis. In fact, the glutamate-induced release of [³H]GABA was significantly depressed by Ca_o²⁺, but it was dependent on Na_o⁺, just as was observed for the [³H]GABA release induced by veratridine (50 μM). The veratridine-induced release could be fully inhibited by TTX, but this toxin had no effect on the glutamate-induced [³H]GABA release. Both veratridine- and glutamate-induced [³H]GABA release were inhibited by 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid (NNC-711), a blocker of the GABA carrier. Blockade of the NMDA and non-NMDA glutamate receptors with MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively, almost completely blocked the release of [³H]GABA evoked by glutamate. Continuous depolarization with 50 mM K⁺ induced maximal release of [³H]GABA of about 1.5%, which is much smaller than the release evoked by glutamate under the same conditions (6.0–6.5%). Glycine (3 μM) stimulated [³H]GABA release induced by 50 mM K⁺, and this effect was blocked by MK-801, suggesting that the effect of K⁺ on [³H]GABA release was partially mediated through the NMDA receptor which probably was stimulated by glutamate released by K⁺ depolarization. We conclude that glutamate induces Ca²⁺-independent release of [³H]GABA through reversal of the GABA carrier due to Na⁺ entry through the NMDA and non-NMDA, TTX-insensitive, channels. Furthermore the GABA carrier seems to be inhibited by Ca²⁺ entering by the pathways open by glutamate. This Ca²⁺ does not lead to exocytosis, probably because the Ca²⁺ channels used are located at sites far from the active zones.     

The muscarinic modulation of [H]

In goldfish, gonadotropin-releasing hormone (GnRH) stimulation of growth hormone (GH) release has been shown to involve extracellular Ca²⁺ entry through voltage-sensitive Ca²⁺ channels and the activation of protein kinase C (PKC). In this study, the possible involvement of extracellular Na⁺ in mediating the GH response to GnRH was examined using dispersed pituitary cells. Perifusion with Na⁺-depleted medium reversibly reduced the acute GH response to 5-min pulses of either 10 nM salmon (s)GnRH or 10 nM chicken (c)GnRH-II. Similarly, replacement of normal medium with Na⁺-depleted medium attenuated the long-term GH release response to sGnRH and cGnRH-II under static incubation conditions. These results suggest that GnRH-induced GH release requires the presence of extracellular Na⁺. Treatment with 5-min pulses of the Na⁺-channel agonist veratridine (10 μM) increased GH release in an extracellular Ca²⁺-dependent manner, presumably due to activation of voltage-sensitive Ca²⁺ channels resulting from the depolarizing effect of increased Na⁺ influx. On the other hand, Na⁺ entry through tetrodotoxin (TTX)-sensitive, voltage-dependent Na⁺ channels is not involved in GnRH-induced GH release. Application of 250 nM TTX, which abolished the voltage-sensitive Na⁺ currents in identified goldfish somatotropes, did not affect the acute GH responses to 5-min pulses of sGnRH and cGnRH-II. The possible participation of Na⁺/H⁺ antiport in mediating the extracellular Na⁺-dependent GnRH action on GH release was then examined. In static incubation experiments, sGnRH- and cGnRH-II-induced GH secretion were reduced by inhibitors of the Na⁺/H⁺ antiport, amiloride and dimethylamiloride (DMA). Likewise, the GH response to the PKC activator, tetradecanoyl phorbol acetate, was attenuated by treatment with Na⁺-depleted medium, amiloride, and DMA. The inhibitory actions of amiloride and DMA were selective as these drugs did not affect the GH response elicited by the Ca²⁺ ionophore ionomycin and the voltage-sensitive Ca²⁺ channel agonist, Bay K 8644. Taken together, these results indicate that extracellular Na⁺ and the Na⁺/H⁺ exchanger are involved in the mediation of GnRH-stimulated GH release in goldfish. Furthermore, this dependence on Na⁺ and Na⁺/H⁺ antiport probably occurs distal to the activation of PKC by GnRH.     

Extracellular Sodium Dependence of GnRH-Stimulated Growth Hormone Release in Goldfish Pituitary Cells

In goldfish, gonadotropin-releasing hormone (GnRH) stimulation of growth hormone (GH) release has been shown to involve extracellular Ca²⁺ entry through voltage-sensitive Ca²⁺ channels and the activation of protein kinase C (PKC). In this study, the possible involvement of extracellular Na⁺ in mediating the GH response to GnRH was examined using dispersed pituitary cells. Perifusion with Na⁺-depleted medium reversibly reduced the acute GH response to 5-min pulses of either 10 nM salmon (s)GnRH or 10 nM chicken (c)GnRH-II. Similarly, replacement of normal medium with Na⁺-depleted medium attenuated the long-term GH release response to sGnRH and cGnRH-II under static incubation conditions. These results suggest that GnRH-induced GH release requires the presence of extracellular Na⁺. Treatment with 5-min pulses of the Na⁺-channel agonist veratridine (10 μM) increased GH release in an extracellular Ca²⁺-dependent manner, presumably due to activation of voltage-sensitive Ca²⁺ channels resulting from the depolarizing effect of increased Na⁺ influx. On the other hand, Na⁺ entry through tetrodotoxin (TTX)-sensitive, voltage-dependent Na⁺ channels is not involved in GnRH-induced GH release. Application of 250 nM TTX, which abolished the voltage-sensitive Na⁺ currents in identified goldfish somatotropes, did not affect the acute GH responses to 5-min pulses of sGnRH and cGnRH-II. The possible participation of Na⁺/H⁺ antiport in mediating the extracellular Na⁺-dependent GnRH action on GH release was then examined. In static incubation experiments, sGnRH- and cGnRH-II-induced GH secretion were reduced by inhibitors of the Na⁺/H⁺ antiport, amiloride and dimethylamiloride (DMA). Likewise, the GH response to the PKC activator, tetradecanoyl phorbol acetate, was attenuated by treatment with Na⁺-depleted medium, amiloride, and DMA. The inhibitory actions of amiloride and DMA were selective as these drugs did not affect the GH response elicited by the Ca²⁺ ionophore ionomycin and the voltage-sensitive Ca²⁺ channel agonist, Bay K 8644. Taken together, these results indicate that extracellular Na⁺ and the Na⁺/H⁺ exchanger are involved in the mediation of GnRH-stimulated GH release in goldfish. Furthermore, this dependence on Na⁺ and Na⁺/H⁺ antiport probably occurs distal to the activation of PKC by GnRH.     

Modulation of big K+ channel activity by ryanodine receptors and L‐type Ca2+ channels in neurons

As metabotropic glutamate receptor type 1 (mGluR1) is known to couple L-type Ca²⁺ channels and ryanodine receptors (RyR, 1 ) in cerebellar granule cells, we examined if such a coupling could activate a Ca²⁺-sensitive K⁺ channel, the big K⁺ (BK) channel, in cultured cerebellar granule cells. We observed that (±)-1-amino-cyclopentane-trans-1,3-dicarboxylic acid (t-ACPD) and quisqualate (QA) stimulated the activity of BK channels. On the other hand, (2S, 3S, 4S)-α-carboxycyclopropyl-glycine (L-CCG-I) and l -(+)-2-amino-4-phosphonobutyrate (L-AP4) had no effect on BK channels, indicating a specific activation by group I mGluRs. Group I mGluRs stimulation of the basal BK channel activity was mimicked by caffeine and both effects were blocked by ryanodine and nifedipine. Interestingly, carbachol stimulated BK channel activity but through a pertussis toxin (PTX)-sensitive pathway that was independent of L-type Ca²⁺ channel activity. Our report indicates that unlike the muscarinic receptors, group I mGluRs activate BK channels by mobilizing an additional pathway involving RyR and L-type Ca²⁺ channels.     

Trophic effect of cholera toxin B subunit in cultured cerebellar granule neurons: Modulation of intracellular calcium by GM1 ganglioside

Survival of cerebellar granule cells (CGC) in culture was significantly improved in the presence of cholera toxin B subunit (Ctx B), a ligand which binds to GM1 with specificity and high affinity. This trophic effect was linked to elevation of intracellular calcium ([Ca²⁺]_i), and was additive to that of high K⁺. Survival was optimized when Ctx B was present for several days during the early culture period. ⁴⁵Ca²⁺ and cell survival studies indicated the mechanism to involve enhanced influx of Ca²⁺ through L-type voltage-sensitive channels, since the trophic effect was blocked by antagonists specific for that channel type. Inhibitors of N-methyl-D-aspartate receptor/channels were without effect. During the early stage of culture Ctx B, together with 25 mM K⁺, caused [Ca²⁺]_i to rise to 0.2–0.7 μM in a higher proportion of cells than 25 mM K⁺ alone. A significant change in the nature of GM1 modulation of Ca²⁺ flux occurred after 7 days in culture, at which time Ctx B ceased to elevate and instead reduced [Ca²⁺]_i below the level attained with 25 mM K⁺. GM1 thus appears to serve as intrinsic inhibitor of one or more L-type Ca²⁺ channels during the first 7 days in vitro, and then as intrinsic activator of (possibly other) L-type channels after that period. This is the first demonstration of a modulatory role for GM1 ganglioside affecting Ca²⁺ homeostasis in cultured neurons of the CNS. © 1996 Wiley-Liss, Inc.     

Stimulus-coupled release of amino acids from cerebellar granule cells in culture

Cerebellar cultures greatly enriched in excitatory granule neurones were depolarized by exposure to either elevated K⁺ or veratrine. Stimulation of the release of not only Glu, but also of certain amino acids, including Gly, Ala and Ser, was observed. The effect was specific, as depolarization did not induced the release of all the estimated amino acids or of lactate dehydrogenase. In comparison with the characteristics of the evoked release of Glu, those of the responsive neutral amino acids were similar in terms of Ca²⁺-dependence, but differences were also noted. Thus, upon stimulation, the relative rise was smaller than for Glu and the degree of depolarization causing maximal release was lower. The questions of whether stimulus-coupled release of the non-transmitter amino acids from granule cells may play a neuromodulatory role in the cerebellum is discussed.     

Release of γ-[H]aminobutyric acid from synaptosomes: effect of external cations and of ouabain

In the present study we have investigated the effect of cations and ouabain on Ca²⁺-independent and Ca²⁺-dependent release of γ-[³H]aminobutyric acid ([³H]GABA) from sheep brain synaptosomes. The presence of Na⁺ in the external medium is essential for the Ca²⁺-independent release induced by K⁺ or ouabain. Thus, in the absence of Ca²⁺, ouabain of K⁺ causes the release of [³H]GABA provided that Na⁺ is present in the external medium. Under K⁺-depolarizing conditions, in a Na⁺ medium, either ouabain or Ca²⁺ further increases the [³H]GABA release induced by depolarization, but their effects are not additive. The presence of external Na⁺ is not required for the Ca²⁺-dependent release of [³H]GABA due to K⁺ depolarization, and this release, which occurs in a choline medium, is not modified by ouabain. Under these conditions (choline medium) K⁺-depolarization dependent release is absolutely dependent on external Ca²⁺, which suggests that this release of [³H]GABA occurs only by exocytosis, without the carrier-mediated efflux which normally co-exists with exocytosis due to K⁺-depolarization in a Na⁺ medium. It is likely that the release induced by ouabain or K⁺ involves the membrane carrier which responds to changes in membrane potential.     

The survival of cultured mouse cerebellar granule cells is not dependent on elevated potassium-ion concentration

The effects of K⁺-induced membrane depolarization were studied on the survival and biochemical parameters in mouse and rat cerebellar granule cells grown in micro-well cultures. Cell numbers were determined by estimating DNA content using the Hoechst 33258 fluorochrome binding assay. DNA from degenerated cells was removed by prior DNAase treatment. These DNA estimates of cell numbers were comparable with values obtained by direct counting of fluorescein diacetate-stained viable cells. In agreement with previous studies, the survival of rat granule cells was promoted by increasing the concentration of K⁺ in the medium from 5 to 25 mM throughout a 7-day culture period. In contrast, mouse granule cells survived in culture containing ‘low’ K⁺ (5 or 10 mM), as well as in the presence of ‘high’ K⁺ (25 mM). On the other hand, several biochemical parameters in mouse granule cells were markedly increased by cultivation in ‘high’ as compared with ‘low’ K⁺-containing media, demonstrated by increased fluorescein diacetate esterase activity, enhanced rate of NADPH-dependent tetrazolium reduction, augmented 2-deoxy-d-glucose accumulation and increased N-methyl-d-aspartate-evoked ⁴⁵Ca²⁺ influx. It was concluded that although cultivation in ‘high’ K⁺ promotes biochemical differentiation in mouse cerebellar granule cells, these cells differ from their rat counterparts in that they do not develop a survival requirement for K⁺-induced membrane depolarization.     

Potassium depolarization of mammalian vestibular sensory cells increases [Ca2+]i through voltage‐sensitive calcium channels

The existence of voltage-sensitive Ca²⁺ channels in type I vestibular hair cells of mammals has not been conclusively proven. Furthermore, Ca²⁺ channels present in type II vestibular hair cells of mammals have not been pharmacologically identified. Fura-2 fluorescence was used to estimate, in both cell types, intracellular Ca²⁺ concentration ([Ca²⁺]_i) variations induced by K⁺ depolarization and modified by specific Ca²⁺ channel agonists and antagonists. At rest, [Ca²⁺]_i was 90 ± 20 nm in both cell types. Microperifusion of high-K⁺ solution (50 mm ) for 1 s increased [Ca²⁺]_i to 290 ± 50 nm in type I (n = 20) and to 440 ± 50 nm in type II cells (n = 10). In Ca²⁺-free medium, K⁺ did not alter [Ca²⁺]_i. The specific L-type Ca²⁺ channel agonist, Bay K, and antagonist, nitrendipine, modified in a dose-dependent manner the K⁺-induced [Ca²⁺]_i increase in both cell types with maximum effect at 2 μm and 400 nm , respectively. Ni²⁺, a T-type Ca²⁺ channel blocker, reduced K⁺-evoked Ca²⁺ responses in a dose-dependent manner. For elevated Ni²⁺ concentrations, the response was differently affected by Ni²⁺ alone, or combined to nitrendipine (500 nm ). In optimal conditions, nitrendipine and Ni²⁺ strongly depressed by 95% the [Ca²⁺]_i increases. By contrast, neither ω-agatoxin IVA (1 μm ), a specific P- and Q-type blocker, nor ω-conotoxin GVIA (1 μm ), a specific N-type blocker, affected K⁺-evoked Ca²⁺_i responses. These results provide the first direct evidence that L- and probably T-type channels control the K⁺-induced Ca²⁺ influx in both types of sensory cells.     

Facilitation of glutamate release from rat cerebrocortical glutamatergic nerve terminals (synaptosomes) by phosphatidylserine and phosphatidylcholine

Phosphatidylserine (PS) and phosphatidylcholine (PC) have been shown to enhance cognitive function. Considering that brain glutamatergic system is thought to participate in cognitive processing, our objective was to determine the effect of PS and PC on glutamate release from the nerve terminal (synaptosome) freshly isolated from rat cerebral cortex. Data showed that both PS and PC potently facilitate 4‐aminopyridine (4‐AP)‐evoked Ca²⁺‐dependent and Ca²⁺‐independent glutamate release. Facilitation of glutamate release by PS or PC was associated with an increase of 4‐AP‐evoked depolarization and downstream elevation of cytoplasmic free calcium concentration ([Ca²⁺]_c). In addition, glutamate release elicited by direct Ca²⁺‐entry with Ca²⁺‐ionophore (ionomycin) was also facilitated by PS or PC. Furthermore, PS‐ or PC‐mediated facilitation of 4‐AP‐evoked glutamate release was superseded or suppressed by protein kinase C (PKC) activator and inhibitor, respectively. Together, these results suggest that PS or PC effects a facilitation of glutamate exocytosis by increasing nerve terminal excitability and Ca²⁺ influx into cerebrocortical nerve terminals through a signaling cascade involving PKC. Synapse 63:215–223, 2009. © 2008 Wiley‐Liss, Inc.     

Intracellular acidification induced by membrane depolarization in rat hippocampal slices: roles of intracellular Ca and glycolysis

To elucidate the mechanism of pH_i changes induced by membrane depolarization, the variations in pH_i and [Ca²⁺]_i induced by a number of depolarizing agents, including high K⁺, veratridine, N-methyl-

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-aspartate (NMDA) and ouabain, were investigated in rat hippocampal slices by the fluorophotometrical technique using BCECF or fura-2. All of these depolarizing agents elicited a decrease in pH_i and an elevation of intracellular calcium ([Ca²⁺]_i) in the CA1 pyramidal cell layer. The increases in [Ca²⁺]_i caused by the depolarizing agents almost completely disappeared in the absence of Ca²⁺ (0 mM Ca²⁺ with 1 mM EGTA). In Ca²⁺ free media, pH_i acid shifts produced by high K⁺, veratridine or NMDA were attenuated by 10–25%, and those produced by ouabain decreased by 50%. Glucose-substitution with equimolar amounts of pyruvate suppressed by two-thirds the pH_i acid shifts induced by both high K⁺ and NMDA. Furthermore, lactate contents were significantly increased in hippocampal slices by exposure to high K⁺, veratridine or NMDA but not by ouabain. These results suggest that the intracellular acidification produced by these depolarizing agents, with the exception of ouabain, is mainly due to lactate accumulation which may occur as a result of accelerated glycolysis mediated by increased Na⁺–K⁺ ATPase activity. A Ca²⁺-dependent process may also contribute to the intracellular acidification induced by membrane depolarization. Since an increase in H⁺ concentration can attenuate neuronal activity, glycolytic acid production induced by membrane depolarization may contribute to the mechanism that prevents excessive neuronal excitation.