Effects of the N-methyl-D-aspartate antagonists on the rise in [Ca2+]i following depolarization in aged rat brain synaptosomes.

The effects of non-competitive NMDA antagonists, MK-801 and dextrorphan in relation to the rise in intracellular Ca2+ concentrations ([Ca2+]i) after stimulation with 15 mM K+ in whole brain synaptosomes from young (3 months old) and aged (24 months old) Fisher344 rats were examined. A fluorescent chelating agent, Rhod-2, was employed to monitor any alterations of K(+)-evoked [Ca2+]i. In young rats, the rise in [Ca2+]i following depolarization was affected by neither dextrorphan (1, 10, 100 microM) nor MK-801 (0.1, 1, 10 microM), while in aged rats, 1 microM dextrorphan and 0.1 microM MK-801 brought about a significant increase in [Ca2+]i following depolarization. In low Mg2+ medium, 10 microM MK-801 and 100 microM dextrorphan significantly inhibited the rise in [Ca2+]i after stimulation with 15 mM K+ in young rats, while neither dextrorphan nor MK-801 could affect the rise in [Ca2+]i significantly in aged rats. When 100 microM NMDA was applied in a medium containing 1.2 mM Mg2+, the rise in [Ca2+]i following depolarization was slightly inhibited by 1 microM MK-801 in young rats, but it was not inhibited significantly by dextrorphan. In aged rats, both 100 microM dextrorphan and 10 microM MK-801 strongly inhibited the rise in [Ca2+]i following depolarization in the presence of 100 microM NMDA. Instead of NMDA, when 100 microM alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), a non-NMDA receptor agonist, was applied, dextrorphan did not inhibit the rise in [Ca2+]i. In low Mg2+ medium, 100 microM NMDA potentiated the inhibitory effect of 10 microM dextrorphan in young rats, while 100 microM dextrorphan or MK-801 did not show any further inhibition by adding 100 microM NMDA. The addition of 100 microM AMPA did not affect the effect of dextrorphan in a low Mg2+ medium in young rats. These results suggest that NMDA antagonist-mediated [Ca2+]i homeostatic system may alter through aging. In addition, the findings that NMDA potentiated the inhibitory effect of NMDA antagonist, which being further potentiated by aging or lowered extrasynaptosomal Mg2+, indicate the possibility that the Mg2+ block to NMDA receptors might be attenuated through aging.     

Modulation of N-methyl-D-aspartate receptor-mediated increases in cytosolic calcium in cultured rat cerebellar granule cells.

The concentration of intracellular free Ca2+ ([Ca2+]i) was measured in rat cerebellar granule cells using the fluorescent indicator fura-2. Culturing the cells as monolayers on plastic squares which could be placed into cuvettes allowed measurements of [Ca2+]i to be performed on large and homogeneous populations of CNS neurons. Granule cells so cultured maintained low levels of [Ca2+]i (around 90 nM) which increased promptly upon the addition of various excitatory amino acids including N-methyl-D-aspartate (NMDA). Increases in [Ca2+]i elicited by NMDA were inhibited by Mg2+ (1 mM) and often potentiated by glycine (1 microM). The addition of TTX or strychnine (5 microM each) did not alter responses to NMDA or NMDA plus glycine. Cytosolic Ca2+ responses to NMDA/glycine were dependent on the presence of extracellular Ca2+ and were unaffected by concentrations of nifedipine or verapamil that blocked increases in [Ca2+]i elicited by K+ depolarization. Responses elicited by NMDA/glycine were inhibited competitively by 2-amino-5-phosphonovalerate or 3-((+-)-2-carboxypiperazin-4-yl)-propyl-1- phosphonic acid and non-competitively by MK-801 or Mg2+. HA-966 and 7-chlorokynurenate inhibited responses to NMDA alone and blocked competitively the potentiating effects of glycine. The results demonstrate NMDA-mediated increases in [Ca2+]i in cerebellar granule cells that arise solely from influx of extracellular Ca2+ through dihydropyridine-insensitive channels. The strict dependence of the NMDA-evoked response on extracellular Ca2+ provides little evidence for a coupling of NMDA receptors to inositol phosphate metabolism and mobilization of intracellular Ca2+. The effect of various agents on NMDA/glycine-induced increases in [Ca2+]i parallels their effects on ligand binding to or current flow through the NMDA receptor-channel complex. The measurement of cytosolic Ca2+ in this preparation of neuronal cells thus appears especially well suited for assessing, on a functional level, the regulation of NMDA receptors in the CNS.     

Process extension and intracellular Ca2+ in cultured murine oligodendrocytes.

Previous investigations have shown that phorbol esters stimulate process extension in oligodendrocytes (OL), likely by the activation of protein kinase C (PKC). In this report, we demonstrate that treatment of OL with 4beta-phorbol-12, 13-dibutyrate (PDB; 0.1-1 microM) resulted in an increase in intracellular Ca2+ concentration ([Ca2+]i) from 94+/-2 nM (mean+/-S.E.M.) to 244+/-10 nM. This increase was produced by Ca2+ influx through a La3+-insensitive pathway. Changes in [Ca2+]i were also produced by modifying the extracellular Ca2+ concentration ([Ca2+]o) where [Ca2+]i was increased by elevations in [Ca2+]o. In parallel experiments we found that increased [Ca2+]o alone, without concurrent phorbol ester application, resulted in increased OL process extension as determined by the percent of OL with long processes (greater than 3 times the cell body diameter). These results demonstrate that increasing [Ca2+]o stimulates OL process outgrowth. Furthermore, both elevations in [Ca2+]o and PDB exposure increase [Ca2+]i, suggesting that some of the effects of phorbol esters on OL process extension are likely mediated by changes in [Ca2+]i.     

NMDA-induced increase in [Ca2+]i and 45Ca2+ uptake in acutely dissociated brain cells derived from adult rats.

A preparation of acutely dissociated brain cells derived from adult (3-month-old) rat has been developed under conditions preserving the metabolic integrity of the cells and the function of N-methyl-D-aspartate (NMDA) receptors. The effects of glutamate and NMDA on [Ca2+]i measured with fluo3 and 45Ca2+ uptake have been studied on preparations derived from hippocampus and cerebral cortex. Glutamate (100 microM) and N-methyl-DL-aspartate (200 microM) increased [Ca2+]i by 26-12 nM and 23-9 nM after 90 s in cerebral cortex and hippocampus, and stimulated 45Ca2+ uptake about 16-10% in the same regions. The increases in [Ca2+]i and 45Ca2+ uptake were inhibited by 40% in the presence of 1 mM MgCl2 and by 90-50% in the presence of MK-801. The results indicate (a) that a large fraction of the [Ca2+]i response to glutamate in freshly dissociated brain cells from the adult rat involves NMDA receptors, (b) when compared with results in newborn rats, there is a substantial blunting of the [Ca2+]i increase in adult age.     

Effect of lead on intracellular free calcium ion concentration in a presynaptic neuronal model: 19F-NMR study of NG108-15 cells

The intracellular free calcium ion concentration ([Ca2+]i) of the neuroblastoma x glioma hybrid cell line, NG108-15, was measured using the 19F-nuclear magnetic resonance divalent cation indicator, 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetra-acetic acid (5F-BAPTA). The basal [Ca2+]i was measured to be 106 +/- 14 nM. Treatment with 5 microM lead (Pb) for 2 h produced a 2-fold increase in [Ca2+]i to 200 +/- 24 nM and a measurable intracellular free Pb2+ concentration ([Pb2+]i) of 30 +/- 10 pM. Intracellular free Zn2+ concentrations ([Zn2+]i) were also observed in the presence of Pb. This represents the first direct demonstration that Pb elevates the [Ca2+]i in neurons, thus providing evidence for a role of [Ca2+]i in mediating the neurotoxicity of Pb.     

Excitatory amino acid regulation of intracellular Ca2+ in isolated catfish cone horizontal cells measured under voltage- and concentration-clamp conditions.

[Ca2+]i was measured using fura-2-loaded isolated catfish horizontal cells in the presence of L-glutamate and the glutamate analogs kainate (KA), quisqualate (QA), and NMDA. Caffeine was used to release Ca2+ from intracellular stores. Cell membrane potential was controlled with a voltage clamp to prevent activation of voltage-dependent Ca2+ channels in the presence of agonist. All excitatory amino acid agonists produced a rapid and sustained rise in [Ca2+]i with the order of potency being QA greater than Glu greater than KA greater than NMDA. The agonist-induced [Ca2+]i increase was blocked in reduced [Ca2+]o and by 6-cyano-7-nitroquinoxaline-2,3-dione and 2-amino-5-phosphonopentanoate, which are specific blockers for QA/KA and NMDA receptors, respectively. The metabotropic receptor agonist trans-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD; 10-200 microM) had no effect on [Ca2+]i. Hill coefficients from curves fitted to concentration-response data suggested an amplification of the Ca2+ signal that was interpreted as calcium-induced calcium release (CICR) from intracellular Ca2+ stores. Caffeine (10 mM) produced a rapid transient rise in [Ca2+]i, confirming the existence of a Ca(2+)-sensitive store. Following caffeine-induced depletion of Ca2+ from intracellular stores, agonists were still able to produce increases in [Ca2+]i, confirming Ca2+ influx through the agonist-gated channel. The agonist-induced increase in [Ca2+]i was decreased following caffeine-induced depletion, confirming a process of CICR. These results are consistent with the hypothesis that excitatory amino acids can produce direct modulation of [Ca2+]i by influx through the agonist-gated channel and by CICR from intracellular stores.     

Propylene glycol increases cytosolic free calcium in rat cerebrocortical synaptosomes

In these studies, the authors investigated the effect of propylene glycol (PG) on the cytosolic free Ca2+ concentration ([Ca2+]i) in rat cerebrocortical synaptosomes using the fluorescent Ca2+ indicator fura-2. PG (0.5-5% v/v) increased [Ca2+]i in a concentration-dependent manner. The PG-induced increase in [Ca2+]i was inhibited approximately 50% by the omission of extracellular Ca2+ or the addition of Ni2+ (100 microM). Decrease of extracellular Na+ (6.2 mM) or addition of tetrodotoxin (1 microM), verapamil (10 microM), nifedipine (10 microM), omega-agatoxin IVA (200 nM), omega-conotoxin GVIA (1 microM), or omega-conotoxin MVIIC (1 microM) had no effect on the increase in [Ca2+]i. Also, addition of TMB-8 (100 microM), ryanodine (50 microM) or thapsigargin (1 microM) did not modify the increase in [Ca2+]i in the absence of extracellular Ca2+. These results suggest that PG increases [Ca2+]i in rat cerebrocortical synaptosomes by both stimulating Ca2+ entry through a Ni2+-sensitive pathway and releasing Ca2+ from TMB-8-, ryanodine- and thapsigargin-insensitive Ca2+ stores.     

The effects of excitatory amino acids on intracellular calcium in single mouse striatal neurons in vitro

Using microspectrofluorimetry and the calcium-sensitive dye fura-2, we examined the effect of excitatory amino acids on [Ca2+]i in single striatal neurons in vitro. N-methyl-D-aspartic acid (NMDA) produced rapid increases in [Ca2+]i. These were blocked by DL-2-amino-5-phosphonovaleric acid (AP5), by Mg2+, by phencyclidine, and by MK801. The block produced by Mg2+ and MK801 could be relieved by depolarizing cells with veratridine. When external Ca2+ was removed, NMDA no longer increased [Ca2+]i. Furthermore, the effects of NMDA were not blocked by concentrations of La3+ that blocked depolarization induced rises in [Ca2+]i. Substitution of Na+o by Li+ did not block the effects of NMDA. Concentrations of L-glutamate greater than or equal to 10(-6) M also increased [Ca2+]i. The effects of moderate concentrations of glutamate were blocked by AP5 but not by La3+ or by substitution of Na+ by Li+. The effects of glutamate were blocked by removal of external Ca2+ but were not blocked by concentrations of Mg2+ or MK801 that completely blocked the effects of NMDA. The glutamate analogs kainic acid (KA) and quisqualic acid also increased [Ca2+]i. The effects of KA were blocked by removal of external Ca2+ but not by La3+, Mg2+, MK801, or replacement of Na+ by Li+. Although AP5 was able to block the effects of KA partially, very high concentrations were required. These results may be explained by considering the properties of glutamate-receptor-linked ionophores. Excitatory amino acid induced increases in [Ca2+]i are consistent with the possibility that Ca2+ mediates excitatory amino acid induced neuronal degeneration.     

Effect of the removal of extracellular Ca2+ on the response of cytosolic concentrations of Ca2+ to ouabain in carotid body glomus cells of adult rabbits.

Effect of the removal of extracellular Ca2+ on the response of cytosolic concentrations of Ca2+ ([Ca2+]i) to ouabain, an Na+/K+ exchanger antagonist, was examined in clusters of cultured carotid body glomus cells of adult rabbits using fura-2AM and microfluorometry. Application of ouabain (10 mM) induced a sustained increase in [Ca2+]i (mean+/-S.E.M.; 38+/-5% increase, n=16) in 55% of tested cells (n=29). The ouabain-induced [Ca2+]i increase was abolished by the removal of extracellular Na+. D600 (50 microM), an L-type voltage-gated Ca2+ channel antagonist, inhibited the [Ca2+]i increase by 57+/-7% (n=4). Removal of extracellular Ca2+ eliminated the [Ca2+]i increase, but subsequent washing out of ouabain in Ca2+-free solution produced a rise in [Ca2+]i (62+/-8% increase, n=6, P<0.05), referred to as a [Ca2+]i rise after Ca2+-free/ouabain. The magnitude of the [Ca2+]i rise was larger than that of ouabain-induced [Ca2+]i increase. D600 (5 microM) inhibited the [Ca2+]i rise after Ca2+-free/ouabain by 83+/-10% (n=4). These results suggest that ouabain-induced [Ca2+]i increase was due to Ca2+ entry involving L-type Ca2+ channels which could be activated by cytosolic Na+ accumulation. Ca2+ removal might modify the [Ca2+]i response, resulting in the occurrence of a rise in [Ca2+]i after Ca2+-free/ouabain which mostly involved L-type Ca2+ channels.     

Metabotropic purinoreceptors in rat dorsal horn neurones: predominant dendritic location.

Elevations of cytosolic free Ca2+ concentration ([Ca2+]i) induced by addition of ATP have been compared in rat dorsal horn neurones in slices and after their isolation. ATP application induced in neurones in situ a rise of [Ca2+]i by 201 +/- 12 nM. In Ca2+-free external solution the rise was 156 +/- 14 nM (n = 45 of 76), indicating the presence of active purinergic metabotropic receptors in about 59% of neurones. [Ca2+]i transients induced by 2MeSATP in Ca2+-free external solution were completely abolished by 10 microM PPADS, indicating that some of the corresponding receptors are of the P2Y1 type. In acutely isolated neurones which lost their dendrites, there were no metabotropic response. The results confirm the presence of metabotropic postsynaptic purinoreceptors located in the dendritic tree of dorsal horn neurones.     

In vitro status epilepticus causes sustained elevation of intracellular calcium levels in hippocampal neurons

Calcium ions and calcium-dependent systems have been implicated in the pathophysiology of status epilepticus (SE). However, the dynamics of intracellular calcium ([Ca2+]i) levels during SE has not yet been studied. We have employed the hippocampal neuronal culture (HNC) model of in vitro SE that produces continuous epileptiform discharges to study spatial and dynamic changes in [Ca2+]i levels utilizing confocal laser scanning microscopy and the calcium binding dye, indo-1. During SE, the average [Ca2+]i levels increased from control levels of 150-200 nM to levels of 450-600 nM. This increased [Ca2+]i was maintained for the duration of SE. Following SE, [Ca2+]i levels gradually returned to basal values. The duration of SE was shown to affect the ability of the neuron to restore resting [Ca2+]i levels. Both N-methyl-D-aspartate (NMDA) receptor-gated and voltage-gated Ca2+ channels (VGCCs) contributed to the increased calcium entry during SE. Moreover, this elevation in [Ca2+]i occurred in both the nucleus and cytosol. These results provide the first dynamic measurement of [Ca2+]i during prolonged electrographic seizure discharges in an in vitro SE model and suggest that prolonged epileptiform discharges give rise to abnormal sustained increases in [Ca2+]i levels that may play a role in the neuronal cell damage and long-term plasticity changes associated with SE.     

Spontaneous and beta-adrenergic receptor-mediated taurine release from astroglial cells are independent of manipulations of intracellular calcium

Stimulation of beta-adrenergic receptors on LRM55 astroglial cells results in cAMP-dependent release of taurine. We have previously demonstrated that extracellular Ca2+ is not required for either spontaneous or receptor-mediated taurine release (Martin et al., 1988b). In the present series of experiments we investigated the relationship between changes in intracellular free Ca2+ ([Ca2+]i) and taurine release. [Ca2+]i was measured using the fluorescent probe fura-2 and was manipulated by changing the concentration of Ca2+ in the incubation medium and by using the Ca2+ ionophore ionomycin. [Ca2+]i was reduced from 150 +/- 95 nM (n = 46) in control medium (containing 1.1 mM CaCl2) to 46 +/- 10 nM (n = 43) in saline containing no CaCl2 and 10 microM EGTA. [Ca2+]i was rapidly elevated to greater than or equal to 1 microM in medium containing 100 microM CaCl2 and 10 microM ionomycin. Taurine release, either spontaneous or stimulated by isoproterenol, was not significantly affected by these manipulations of [Ca2+]i. [Ca2+]i did not change when cells were stimulated with 100 nM isoproterenol in either control saline containing 1.1 mM CaCl2 or in CaCl2-free saline containing 10 microM EGTA. Other secretogogs (serotonin and ethanol) did not cause changes in [Ca2+]i. These data indicate that neither spontaneous or receptor-mediated taurine release from astroglial cells is Ca2+ dependent. However, when cells were preloaded with Ca2+, allowed to recover briefly, and then stimulated with isoproterenol, it was possible to demonstrate transient increases in Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

L-glutamate increases internal free calcium levels in synaptoneurosomes from immature rat brain via quisqualate receptors

Internal free calcium concentrations ([Ca2+]i) have been monitored in synaptoneurosomes from 8-d-old rat whole brain previously loaded with the calcium-sensitive fluorescent probe Fura 2. Under basal conditions, [Ca2+]i was around 200 nM, this concentration increasing only slowly during storage of the synaptoneurosomes at room temperature (40% increase 2 hr after loading). Opening of sodium channels with veratridine- (10 microM) or KCl- (30 mM) induced depolarization caused rapid increases in synaptoneurosomal [Ca2+]i. [Ca2+]i was also markedly increased by addition of the Ca2+ ionophore A23187 (10-100 nM). The effect of veratridine, but not of KCl was prevented by previous addition of TTX (1 microM). KCl-induced [Ca2+]i increase was dependent on external Ca2+ and was partially blocked by the dihydropyridine derivative PN 200-110 (IC50 0.15 microM, maximal inhibition 55% at 3 microM). L-Glutamate elicited a concentration-dependent fast increase in synaptoneurosomal [Ca2+]i in the 8-d-old (but not in the adult) rat brain (EC50 = 2 microM). The effect of glutamate was stereospecific, the EC50 of the D-isomer being 47 times higher than that of L-isomer. The magnitude of the L-glutamate response differed in several brain regions, being highest in the cerebral cortex and lowest in the cerebellum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitric oxide applications prior and simultaneous to potentially excitotoxic NMDA-evoked calcium transients: cell death or survival

Nitric oxide (NO) is a molecule that plays a prominent role in neurotoxic as well as neuroprotective pathways. Here, we investigated the effects of NO on potentially excitotoxic glutamate-induced intracellular calcium ([Ca2+]i) dynamics. Our hypothesis was that pre- and coexposure to NO in conjunction with glutamate receptor stimulation modulates [Ca2+]i responses differentially. [Ca2+]i transients, assessed by the fluorescent cytosolic Ca2+ indicator dye fluo-4, were elicited in mouse striatal neurons by consecutive NMDA applications (200 microM for 100 s each). Subgroups of neuronal cultures were additionally exposed to a NO donor (S-nitroso-N-acetyl-d,l-penicillamine, SNAP, 50-500 microM), either by pre- (for 6 h prior to NMDA) or cotreatment (for 30 min during NMDA). Pretreatment with NO led to dramatically decreased NMDA-evoked [Ca2+]i rises in comparison to controls (NMDA alone). Annexin V/propidium iodide staining showed consistently that NO pretreatment is protective against NMDA-induced cell death. In contrast, NO/NMDA cotreatment caused a potentiation of [Ca2+]i rises, whereby the duration of [Ca2+]i transients following NMDA application was prolonged and remained at an increased plateau level. Simultaneous application of the mitochondrial permeability transition pore (mtPTP) blocker cyclosporin A (2 microM) during the NO/NMDA cotreatment prevented the deregulation of [Ca2+]i. The observed [Ca2+]i deregulation was accompanied by a decrease in the mitochondrial membrane potential as indicated by tetramethylrhodamine methylester (TMRM) fluorescence. These findings suggest that NO can act in a protective way due to preconditioning or can have a possibly detrimental impact in case of acute release. They provide a possible explanation for the ambivalence of NO in neurodegenerative processes where glutamate receptor stimulation and mitochondrial [Ca2+]i sequestration are causally involved.     

Neuropeptide-induced cytosolic Ca2+ transients and phosphatidylinositol turnover in cultured human retinal pigment epithelial cells.

Neuropeptide-induced mobilization of cytosolic free Ca2+ concentration ([Ca2+]i) and phosphatidylinositol (PI) turnover in cultured human retinal pigment epithelial (RPE) cells were studied and their temporal relationship was compared. After RPE cells were loaded with fura-2/AM, [Ca2+]i was analyzed using a digital imaging microscopy system. Bombesin-related peptides which include bombesin, neuromedin B, and neuromedin C induced significant [Ca2+]i transients in RPE cells, whereas other neuropeptides, neuropeptide Y, vasoactive intestinal polypeptide (VIP), and substance P were not effective to produce [Ca2+]i transients. The percentage of reactive cells which showed positive [Ca2+]i transients induced by bombesin-related peptides was around 50%. Bombesin (1 microM) showed a peak concentration of 663 +/- 27.0 nM (mean +/- S.E.M., n = 61), neuromedin B (1 microM), 327 +/- 28.7 nM (mean +/- S.E.M., n = 38), and neuromedin C (1 microM), 357 +/- 22.7 nM (mean +/- S.E.M., n = 32). Ca2+ transients occurred within 30 s and lasted less than 5 min after the application of the neuropeptides. Chelation of the extracellular Ca2+ by EGTA significantly shortened the total time of [Ca2+]i transients induced by the above. The measurements of phosphoinositides in RPE cells revealed that neuropeptide-induced PI turnover was as quick as [Ca2+]i transients. Inositol biphosphate (IP2) and inositol triphosphate (IP3) in RPE cells showed transient increases at 15 s after the stimulation by bombesin-related peptides. These data show that changes in [Ca2+]i and PI turnover are directly linked and both are important in the signal transduction system of bombesin-related peptides in RPE cells. The data also suggest that bombesin-related peptides may play some possible roles in RPE cells.     

The bradykinin receptor--a putative receptor-operated channel in PC12 cells: studies of neurotransmitter release and inositol phosphate accumulation.

Bradykinin (BK) induced [3H]norepinephrine [( 3H]NE) release and phosphatidylinositol turnover were investigated in PC12 cells. Induction of [3H]NE release by BK is mediated by activation of BK-B2-receptors, as determined using type specific BK receptor antagonists. BK induces [3H]NE release with a half maximal effective concentration of 30 +/- 0.5 nM, and reaches maximal net fractional release of 9.0 +/- 1% with 200 nM BK. The BK-induced release is Ca2+ dependent, reaching maximal release at 1.0 mM Ca2+, is pertussis toxin insensitive (1 microgram/ml), slightly increased by a dibutyryl cAMP (1 mM) and not affected by inhibitors of the cyclooxygenase or lipoxygenase pathways. Voltage-sensitive Ca2+ channel blockers, verapamil (10 microM), nifedipine (10 microM), and omega-conotoxin (CgTx 10 nM), do not block the BK-induced release. However, a considerable inhibitory effect was obtained by divalent cations Co2+ (ED50 = 0.2 mM) and Ni2+ (ED50(2)+ = 1 mM). These results indicate the involvement of a Ca2+ channel in the BK-mediated release which is different from the L- or N-type voltage sensitive calcium channels. Whereas [Ca2+]ex is essential for the BK-induction of catecholamine release, the rise in level of InsP's induced by BK in the presence or in the absence of [Ca2+]ex is similar up to concentration of 1 microM. This indicates that the rise in InsP's induced by BK is not sufficient to cause neurotransmitter release. Moreover, subsequent addition of Ca2+ to BK-stimulated cells in Ca(2+)-free medium yields no release. Hence, no activity triggered by BK alone could be further stimulated by Ca2+ for induction of release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of CA2+ on K+ efflux during regulatory volume decrease in cultured astrocytes.

The calcium (Ca2+) dependence of potassium (K+) efflux activated by hyposmolarity in cultured cerebellar astrocytes was investigated, measuring in parallel experiments (86)Rb release and changes in cytosolic Ca2+ ([Ca2+]i). Hyposmotic (50%) medium increased [Ca2+]i from 117 to 386 nM, with contributions of extracellular Ca2+ and Ca2+ from the endoplasmic reticulum. Hyposmotic medium increased (86)Rb efflux rate from 0.015 min(-1) to a maximal of 0. 049 min(-1) and a net release of 30%. This osmosensitive efflux was inhibited by Ba(2+) (0.028 min(-1)), quinidine (0.024 min(-1)), and charybdotoxin (0.040 min(-1)), but was unaffected by TEA, 4-AP, or apamin. Removal of external Ca2+ from the hyposmotic medium increased (86)Rb efflux to a maximal rate constant of 0.056 min(-1) and a net release of 38% and caused a delay of inactivation. These changes were due to the overlaping of an efflux activated by Ca2+ removal in isosmotic medium. This isosmotic 86Rb efflux was unaffected by TEA or 4-AP, reduced by verapamil, and abolished by Ba2+, nitrendipine, and Mg2+. With the swelling-induced [Ca2+]i rise suppressed by ethyleneglycoltetraacetic acid-acetoxy-methyl ester (EGTA-AM), hyposmotic (86)Rb was 30% reduced. The Ca2+ entry blockers Cd2+, Ni2+, La3+, and Gd3+ did not affect (86)Rb efflux. A 40% decrease observed with verapamil and nitrendipine was found unrelated to Ca2+, because these agents did not affect the [Ca2+]i rise and the inhibition persisted in the absence of external Ca2+. The phospholipase C blocker U-73122 did not affect [Ca2+]i nor (86)Rb efflux. Blockers of Ca2+/calmodulin W7 and KN-93 decreased (86)Rb efflux to the same extent as EGTA-AM. Ionomycin markedly potentiated (86)Rb release in hyposmotic conditions only when [Ca2+]i was raised to about 1 microM, suggesting the implication of maxi-K+ channels at this [Ca2+]i threshold, which nonetheless, was not attained during hyposmotic swelling. It is concluded that (86)Rb efflux in cerebellar astrocytes is largely (70%) Ca2+-independent and the Ca2+-dependent fraction is sustained essentially by Ca2+ released from the endoplasmic reticulum and mediated by a mechanism involving Ca2+/calmodulin.     

A significant increase in intracellular Ca2+ concentration induced by (2S,3R,4S)-2-(carboxycyclopropyl)glycine, a new potent NMDA agonist, in cultured rat hippocampal neurons.

The increase in intracellular Ca2+ concentration, [Ca2+]i, induced by isomers of 2-(carboxycyclopropyl)glycine (CCG) was examined in cultured rat hippocampal neurons. Some CCG isomers and N-methyl-D-aspartate (NMDA) increased [Ca2+]i in a concentration dependent manner. The 2S,3R,4S isomer of CCG (L-CCG-IV) was the most potent in elevating [Ca2+]i, and its activity was more than 100 times higher than that of NMDA and about 10 times higher than that of L-glutamate. The increase in [Ca2+]i was effectively blocked by NMDA blockers and Mg2+, and was markedly augmented by the addition of a low concentration of glycine. L-CCG-IV would be a useful tool for elucidation of functions of NMDA receptors.     

Low concentrations of ethanol deplete type-2 astrocytes of intracellular free magnesium

The acute effects of low concentrations of ethanol on intracellular free magnesium ions ([Mg2+]i) in cultured type-2 astrocytes were studied by digital imaging microscopy using the Mg2+ fluorescent probe, mag-fura-2. In 0-mM ethanol, the basal level of [Mg+]i was 124.7+/-2.56 microM with a heterogeneous distribution within the cells. Treatment of the cells with 10 and 25 mM ethanol (10 min) resulted in rapid concentration-dependent reduction in [Mg2+]i; the greater the concentration of alcohol, the greater the depletion of [Mg2+]i. Exposure of cells to 10 and 25 mM resulted in approximately 27 and 50% reductions in [Mg2+]i, respectively. Reincubation in normal Mg2+-physiological buffer solution restored [Mg2+]i levels. These observations may suggest that acute "binge drinking" of ethanol, which often results in cerebral ischemia and stroke, may do so as a result of depletion of astrocytic [Mg2+]i, possibly producing disruption of the blood-brain barrier.     

Relationship of calcium and adenylate cyclase messenger systems in rat brain synaptosomes

The effects of cyclic AMP on the rise in cytosolic free calcium concentration, [Ca2+]i, after stimulation with 15 mM K+ in rat brain synaptosomes were investigated. The fluorescent chelating agent Quin-2 was employed to monitor alterations of K+-evoked [Ca2+]i. Under normoxic conditions, clonidine (1, 10 microM), an alpha 2-adrenoceptor agonist, decreased the 15 mM K+-evoked [Ca2+]i. Although yohimbine (1, 10 microM), an alpha 2-adrenoceptor antagonist, had little or no effect on K+-evoked [Ca2+]i, the inhibitory effects of clonidine were blocked by yohimbine. 8-Bromo cyclic AMP, a cyclic AMP analogue, (50-500 microM), increased K+-evoked [Ca2+]i in a dose-dependent manner. The addition of cyclic AMP analogues subsequent to clonidine treatment reversed the clonidine-induced suppression of K+-evoked [Ca2+]i. On the other hand, under hypoxic conditions, K+-evoked [Ca2+]i was reduced by about 50-60%. 8-Bromo cyclic AMP and the adenylate cyclase activators, yohimbine (1-10 microM) and isoproterenol, a beta-adrenoceptor agonist, (0.1-10 microM), transiently reversed the reduction of the K+-evoked [Ca2+]i caused by hypoxia. These results indicate that the activation of alpha 2-adrenoceptor produces a rapid, sustained decrease in [Ca2+]i which may be due to a decrease in the levels of intracellular cyclic AMP. In addition, the increase in cellular levels of cyclic AMP reversed the reduction of the Ca2+ response to high K+ stimulation caused by hypoxia. If this is so, there is the possibility that increased cyclic AMP might improve the hypoxic damage.