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.     

Release from intracellular stores is responsible for calcium signaling with von Willebrand factor in human platelets

Interaction of von Willebrand factor (VWF) with the platelet promotes hemostasis upon vascular injury. Such interaction raises intracellular free calcium concentration ([Ca2+]i) and induces platelet activation. The platelet [Ca2+]i increase is generally attributed to influx across the plasma membrane. The present study defined the contribution of intracellular calcium stores. Platelet [Ca2+]i was monitored with Fura-PE3. Ristocetin-mediated binding of VWF transiently elevated [Ca2+]i after a lag phase. Studies with 63 healthy donors consistently revealed a VWF-induced platelet [Ca2+]i signal in the absence of extracellular calcium; there was only modest enhancement with extracellular calcium. Blockade of plasma membrane calcium channels did not diminish the signal, whereas depletion or blockade of the intracellular calcium stores abolished it. These findings imply that release from intracellular stores is responsible for the VWF-induced platelet [Ca2+]i increase. Influx across the plasma membrane plays no more than a minor role, probably representing "capacitative entry" to refill the intracellular stores.     

Calcimycin potentiates responses of rat hippocampal neurons to N-methyl-D-aspartate

We examined the effect of elevating intracellular calcium ([Ca2+]i) on responses to iontophoretically applied N-methyl-D-aspartate (NMDA), and quisqualate in CA1 neurons of the hippocampal slice. Topical application of calcimycin (A23187), a calcium ionophore, potentiated responses to NMDA but not to quisqualate. This potentiation was prevented by loading cells with the calcium chelator, BAPTA, suggesting that the action of calcimycin on NMDA receptors was mediated by an elevation of [Ca2+]i in the recorded cell. The potentiation was also recorded in voltage-clamped and in cesium-loaded cells, suggesting that it was not mediated by non-specific changes in voltage or input resistance of the cell that may have resulted from the rise in [Ca2+]i. We propose that intracellular calcium plays a crucial role in regulating the activity of the NMDA subtype of L-glutamate receptor.     

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.     

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.     

Involvement of intracellular calcium stores during oxygen/glucose deprivation in striatal large aspiny interneurons.

Striatal large aspiny interneurons were recorded from a slice preparation using a combined electrophysiologic and microfluorometric approach. The role of intracellular Ca2+ stores was analyzed during combined oxygen/glucose deprivation (OGD). Before addressing the role of the stores during energy deprivation, the authors investigated their function under physiologic conditions. Trains of depolarizing current pulses caused bursts of action potentials coupled to transient increases in intracellular calcium concentration ([Ca2+]i). In the presence of cyclopiazonic acid (30 micromol/L), a selective inhibitor of the sarcoendoplasmic reticulum Ca2+ pumps, or when ryanodine receptors were directly blocked with ryanodine (20 [micromol/L), the [Ca2+]i transients were progressively smaller in amplitude, suggesting that [Ca2+]i released from intracellular stores helps to maintain a critical level of [Ca2+]i during physiologic firing activity. As the authors have recently reported, brief exposure to combined OGD induced a membrane hyperpolarization coupled to an increase in [Ca2+]i. In the presence of cyclopiazonic acid or ryanodine, the hyperpolarization and the rise in [Ca2+]i induced by OGD were consistently reduced. These data support the hypothesis that Ca2+ release from ryanodine-sensitive Ca2+ pools is involved not only in the potentiation of the Ca2+ signals resulting from cell depolarization, but also in the amplification of the [Ca2+]i rise and of the concurrent membrane hyperpolarization observed in course of OGD in striatal large aspiny interneurons.     

HIV-1 envelope protein evokes intracellular calcium oscillations in rat hippocampal neurons

Treatment of single rat hippocampal neurons with 200 pM recombinant HIV-1 envelope glycoprotein, gp120, resulted in large increases in the intracellular free calcium concentration ([Ca2+]i) as measured with indo-1-based microfluorimetry. Three patterns of [Ca2+]i increases were observed: in one pattern the [Ca2+]i rose rapidly and transiently as a single peak, in a second pattern gp120 induced [Ca2+]i oscillations that subsided when the protein was removed, and in a third pattern the oscillations continued long after washout of gp120. Both single peak and oscillatory [Ca2+]i increases were completely blocked by the Ca2+ channel blocker nitrendipine (1 microM). The sustained oscillatory responses were also blocked completely and reversibly by the N-methyl-D-aspartate (NMDA) receptor antagonist CGS19755 (10 microM) and the Na+ channel blocker tetrodotoxin (1 microM). Complete block by antagonists of Ca2+, Na+, and NMDA-gated ion channels suggests that at least two cells are required to maintain the [Ca2+]i oscillations. We hypothesize that gp120 acts as an excitotoxin by increasing synaptic activity in the network of neurons established in primary culture.     

Mode of action of taurine as a neuroprotector

Previously, it has been shown that taurine exerts its protective function against glutamate-induced neuronal excitotoxicity through its action in reducing glutamate-induced elevation of intracellular free calcium, [Ca2+]i. Here, we report the mechanism underlying the effect of taurine in reducing [Ca2+]i. We found that taurine inhibited glutamate-induced calcium influx through L-, P/Q-, N-type voltage-gated calcium channels (VGCCs) and NMDA receptor calcium channel. Surprisingly, taurine had no effect on calcium influx through NMDA receptor calcium channel when cultured neurons were treated with NMDA in Mg2+-free medium. Since taurine was found to prevent glutamate-induced membrane depolarization, we propose that taurine protects neurons against glutamate excitotoxicity by preventing glutamate-induced membrane depolarization, probably through its effect in opening of chloride channels and, therefore, preventing the glutamate-induced increase in calcium influx and other downstream events.     

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.     

Activation of P-type calcium channel regulates a unique thapsigargin-sensitive calcium pool in embryonic motoneurons

By regulating voltage-dependent Ca2+ influx and intracellular Ca2+ homeostasis, electrical activity plays a central role in motoneuron development. Dissociated cultures of purified embryonic rat motoneurons were used to explore the molecular mechanisms by which Ca2+ influx control [Ca2+]i transients in these neurons. Thapsigargin (250 nm) and cyclopiazonic acid (10 micro m), which deplete Ca2+ stores in the endoplasmic reticulum, decrease by 30% the depolarization-induced [Ca2+]i transients in motoneurons without affecting voltage-activated calcium currents. This thapsigargin-sensitive intracellular Ca2+ pool differs from other previous described Ca2+ stores that are sensitive to ryanodine or caffeine, inositol triphosphate, insulin and from mitochondrial Ca2+ pools. Thapsigargin affected the Cav2.1 P-type Ca2+ channel component of the depolarization-induced [Ca2+]i transient in motoneurons but spared [Ca2+]i transient induced by Cav1 L-type and Cav2.2 N-type Ca2+ channel components, suggesting a close functional relationship between Cav2.1 subunit and this unique thapsigargin-sensitive Ca2+ store. Altogether the present results demonstrate a new pathway, used by embryonic motoneurons, to regulate Ca2+ signalling through voltage-activated (Cav2.1) Ca2+ channels.     

L-type voltage-gated calcium channels modulate kainic acid neurotoxicity in cerebellar granule cells

This study reports on the regulation of kainate neurotoxicity in cerebellar granule cells by calcium entry through voltage-gated calcium channels and by calcium release from internal cellular stores. Kainate neurotoxicity was prevented by the AMPA selective antagonist LY 303070 (10 microM). Kainate neurotoxicity was potentiated by cadmium, a general voltage-gated calcium channel blocker, and the L-type voltage-gated calcium channel blocker nifedipine. The antagonists of intracellular Ca2+ ([Ca2+]i) release, thapsigargin and ryanodine, were also able to potentiate kainate neurotoxicity. Kainate treatment elevated [Ca2+]i concentration with a rapid initial increase that peaked at 1543 nM and then declined to plateau at approximately 400 nM. Nifedipine lowered the peak response to 764 nM and the plateau response to approximately 90 nM. Thapsigargin also lowered the kainate-induced increase in [Ca2+]i (640 nM peak, 125 nM plateau). The ryanodine receptor agonist caffeine eliminated the kainate-induced increase in [Ca2+]i, and reduced kainate neurotoxicity. Kainate neurotoxicity potentiated by nifedipine was not prevented by RNA or protein synthesis inhibitors, nor by the caspase inhibitors YVAD-CHO and DEVD-CHO. Neither DNA laddering nor the number of apoptotic nuclei were increased following treatment with kainate and nifedipine. Increased nuclear staining with the membrane impermeable dye propidium iodide was observed immediately following kainate treatment, indicating a loss of plasma membrane integrity. Thus, kainate neurotoxicity is prevented by calcium entry through L-type calcium channels.     

Nitric oxide modulates NMDA-induced increases in intracellular Ca2+ in cultured rat forebrain neurons.

We studied the effects of nitric oxide (NO) and the NO-releasing agents sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP) and isosorbide dinitrate (ISDN) on N-methyl-D-aspartate (NMDA)-induced increases in intracellular Ca2+ ([Ca2+]i), whole-cell patch-clamp currents and on glutamate-stimulated [3H]dizocilpine binding. NO and agents that release NO partially inhibit increases in [Ca2+]i at concentrations between 1 microM and 1 mM. These agents also decrease [Ca2+]i changes produced by kainate and potassium, but to a smaller extent. As the effects of NO are still present following alkylation of the redox modulatory site on the NMDA receptor this action of NO is probably not a consequence of oxidation of the redox site. In contrast to SNP, ISDN does not inhibit NMDA-induced whole cell patch-clamp currents suggesting that NO modulates [Ca2+]i via perturbation of a Ca2+ homeostatic process. Furthermore, SNP may have a direct action on the NMDA receptor complex in addition to the generation of NO. 8-Bromo-cGMP does not mimic the inhibitory effect of NO suggesting that this effect is not the result of NO stimulation of neuronal cGMP production. As the production of NO in neurons is dependent on increases in [Ca2+]i associated with NMDA receptor activation, these data suggest that NO-mediated decreases in [Ca2+]i may represent a novel feedback inhibitory mechanism for NO production in the brain.     

Sodium nitroprusside blocks NMDA receptors via formation of ferrocyanide ions.

The effects of a nitric oxide (NO) donor, sodium nitroprusside (SNP), on N-methyl-D-aspartate (NMDA) receptors were assessed by optical measurements of intracellular calcium concentration ([Ca2+]i) and patch-clamp techniques in cultured central neurons. SNP selectively blocked NMDA-mediated currents and increases in [Ca2+]i. SNP inhibited the binding of [3H]-CGS 19755. The blockade of NMDA responses by SNP was prevented by CPP or APV which are selective competitive NMDA receptor antagonists. These effects were not necessarily mediated by NO, since they were mimicked by ferrocyanide ions, the NO companion photolysis product of SNP.     

Role of Ca2+ in induction of neurotransmitter-related gene expression by butyrate

We examined the effect of butyrate on neurotransmitter-related gene expression and calcium homeostasis in PC12 cells. Pretreatment with Ca2+ chelators (EGTA or BAPTA-AM) attenuated the butyrate-triggered accumulation of TH and ppEnk mRNA indicating that Ca2+ plays a role in butyrate-induced regulation of neuronal genes. Butyrate alone did not alter intracellular Ca2+ levels as determined by Fura-PE3 fluorescence; however, pretreatment with butyrate (18-24 h) reduced the first Ca2+ peak and prevented the second sustained rise in [Ca2+]i as induced by nicotine or ryanodine. In contrast, butyrate had no effect on Ca2+ transients when added shortly before or during nicotine or ryanodine stimulation. These results suggest that chronic butyrate exposure can modulate cell responses by affecting intracellular Ca2+ signaling.     

Secreted phospholipase A2 potentiates glutamate-induced calcium increase and cell death in primary neuronal cultures

Secreted phospholipases A2 (sPLA2s) modulate neuronal survival and neurotransmitter release. Here we show that sPLA2 (group III) synergistically increases glutamate-induced cell death and intracellular calcium ([Ca2+]i) in cultured primary cortical and hippocampal neurons. Whereas 1 microM glutamate elicited transient [Ca2+]i increases in all neurons that recovered 66% to baseline, 25 ng/ml sPLA2 pretreatment resulted in sustained [Ca2+]i increases, with only 5% recovery. At 250 nM glutamate, 25% of neurons failed to respond, and the average recovery time was 101 +/- 12 sec; sPLA2 increased recovery time to 158 +/- 6 sec, and only 2% of cells failed to respond. Both the noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 and the calcium-channel blocker cobalt inhibited this effect. Experiments with the glutamate uptake inhibitor L-trans-pyrollidine-2,4-dicarboxylic acid (2.5 microM) indicated that glutamate uptake sites are not a likely modulation point by sPLA2, whereas arachidonic acid (AA) potentiated calcium responses to glutamate. Thus the enhancement of glutamate-induced [Ca2+]i increases by sPLA2 may be due to modulation at NMDA receptors and/or calcium channels by AA. These results indicate that sPLA2 affects neuronal responses to both nontoxic (0.1-10 microM) and toxic (=25 microM) concentrations of glutamate, implicating this enzyme in neuronal functions in pathology.     

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.     

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.     

Fructose-1,6-bisphosphate stabilizes brain intracellular calcium during hypoxia in rats.

BACKGROUND AND PURPOSE: Exogenously administered fructose-1,6-bisphosphate reduces neuronal injury from hypoxic or ischemic brain insults. To test the hypothesis that fructose-1,6-bisphosphate prevents changes in intracellular calcium ([Ca2+]i) and high-energy phosphate levels, we measured [Ca2+]i, intracellular pH (pHi), and adenosine triphosphate in cultured rat cortical astrocytes and cortical brain slices during hypoxia. METHODS: The fluorescent indicators fura-2 and bis-carboxyethyl-carboxyfluorescein were used to simultaneously measure [Ca2+]i and pHi with a fluorometer. RESULTS: Exposure to hypoxia (95% N2, 5% CO2) or 100 microM sodium cyanide produced transient increases in [Ca2+]i in astrocytes and sustained increases in [Ca2+]i in brain slices. Adenosine triphosphate levels fell in slices exposed to hypoxia or cyanide. Fructose-1,6-bisphosphate (3.5 mM) blocked increases in [Ca2+]i and prevented depletion of adenosine triphosphate. Fructose-1,6-bisphosphate also partially prevented adenosine triphosphate depletion in brain slices incubated in glucose-free medium. Iodoacetate (a specific inhibitor of glycolysis) elevated [Ca2+]i and partially prevented these actions of fructose-1,6-bisphosphate. Changes in pHi during hypoxia were not affected by fructose-1,6-bisphosphate. CONCLUSIONS: Fructose-1,6-bisphosphate supports adenosine triphosphate production via stimulation of glycolysis and results in the maintenance of normal [Ca2+]i during hypoxia or hypoglycemia.     

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.