Changes In Intracellular Free Ca2 Research Articles

NMDA receptor characterization and subunit expression in rat cultured mesencephalic neurones.

1. NMDA-induced changes in free intracellular Ca2+ concentration ([Ca2+]i) were determined in individual cultured rat mesencephalic neurones by the fura-2 method. mRNA expression encoding NMDA receptor subunits (NR1, NR2A-D) was examined by RT-PCR. 2. NMDA (1-100 microM, plus 10 microM glycine) induced a concentration-dependent increase in [Ca2+]i (EC50 = 5.7 microM). The effect of NMDA was virtually insensitive to tetrodotoxin (0.3 microM) and nitrendipine (1 microM), but dependent on extracellular Ca2+. 5,7-Dichlorokynurenic acid (10 microM), a specific antagonist at the glycine binding site on the NMDA receptor, abolished the NMDA response. 3. Memantine, an open-channel blocker, and ifenprodil, a preferential non-competitive NR1/NR2B receptor antagonist diminished the NMDA effect with an IC50 value of 0.17 and 1 microM, respectively. Ethanol at 50 and 100 mM caused about 25 and 45%-inhibition, respectively. 4. Agarose gel analysis of the PCR products followed by ethidium bromide fluorescence or CSPD chemiluminescence detection revealed an almost exclusive expression of the NR1 splice variants lacking exon (E) 5 and E22. The 3' splice form without both E21 and E22 exceeded that containing E21 by approximately 4 fold. The relative amounts of NR2A, NR2B, NR2C corresponded to approximately 1:2:1. NR2D mRNA was also detectable. 5. In conclusion, mesencephalic neurones bear ethanol-sensitive NMDA receptors which might be involved in the development of ethanol dependence and withdrawal. The high affinity of NMDA to this receptor, its sensitivity to ifenprodil and memantine may suggest that the mesencephalic NMDA receptor comprises the NR1 splice variant lacking E5, NR2B, and NR2C, respectively.

Effects of taurine on the motility and intracellular free Ca2+ concentration of fowl spermatozoa in vitro.

The effects of taurine on the motility and intracellular free Ca2+ concentration of fowl spermatozoa were investigated in vitro. The addition of taurine, within the range of 0-5 mmol l(-1), did not appreciably affect the motility of intact fowl spermatozoa. Motility remained almost negligible at 40 degrees C, while vigorous movement was observed at 25 degrees C. Even with the addition of Ca2+ before the addition of taurine, neither stimulation nor inhibition of motility was observed compared with the control (no addition of taurine). Similar results were obtained by the addition of taurine and calyculin A, a specific inhibitor of protein phosphatases. There were no changes in intracellular free Ca2+ concentrations, measured by a fluorescent Ca2+ indicator, fura-2, in taurine-treated spermatozoa. These results suggest that taurine is not involved in the regulation of fowl sperm motility and metabolism by intracellular Ca2+ mobilization in vitro.

Increase of intracellular Ca2+ and relocation of E-cadherin during experimental decompaction of mouse embryos.

To determine the role of intracellular Ca2+ in compaction, the first morphogenetic event in embryogenesis, we analyzed preimplantation mouse embryos under several decompacting conditions, including depletion of extracellular Ca2+, blocking of Ca2+ channels, and inhibition of microfilaments, calmodulin, and intracellular Ca2+ release. Those treatments induced decompaction of mouse morulae and simultaneously induced changes in cytosolic free Ca2+ concentration and deregionalization of E-cadherin and fodrin. When morulae were allowed to recompact, the location of both proteins recovered. In contrast, actin did not change its cortical location with compaction nor with decompaction-recompaction. Calmodulin localized in areas opposite to cell-cell contacts in eight-cell stage embryos before and after compaction. Inhibition of calmodulin with trifluoperazine induced its delocalization while morulae decompacted. A nonspecific rise of intracellular free Ca2+ provoked by ionomycin did not affect the compacted shape. Moreover, the same decompacting treatments when applied to uncompacted embryos did not produce any change in intracellular Ca2+. Our results demonstrate that in preimplantation mouse embryos experimentally induced stage-specific changes of cell shape are accompanied by changes of intracellular free Ca2+ and redistribution of the cytoskeleton-related proteins E-cadherin, fodrin, and calmodulin. We conclude that intracellular Ca2+ specifically is involved in compaction and probably regulates the function and localization of cytoskeleton elements.

Calcium channel subtypes responsible for voltage-gated intracellular calcium elevations in embryonic rat motoneurons

The central role of electrical activity and Ca 2+ influx in motoneuron development raises important questions about the regulation of Ca 2+ signalling induced by voltage-dependent Ca 2+ influx. In the purified embryonic rat motoneuron preparation, we recorded barium currents through voltage-activated Ca 2+ channels using the whole-cell configuration of the patch-clamp technique. We found that motoneurons express at least four types of high-voltage-activated Ca 2+ channels, based on their kinetics, voltage-dependences and pharmacological properties. Of the sustained Ca 2+ current activated at 0 mV from a holding potential of −100 mV, approximatively 45% was omega-conotoxin-GVIA (1 μM) sensitive, 25% was omega-agatoxin-IVA (30 nM) sensitive and 20% was nitrendipine (250 nM) sensitive. The residual current, after applying these three antagonists, was an inactivating current that differs from classical T-type Ca 2+ currents. Based on this pharmacology, changes in intracellular free Ca 2+ concentrations were then monitored by Fura 2 digital imaging microspectrofluorimetry. Upon K + depolarization, the intracellular Ca 2+ transient induced by the activation of each type of Ca 2+ channel appeared to be quantitatively proportional to their Ca 2+ influx. The existence of a calcium-induced calcium release mechanism through activation of caffeine-, ryanodine-sensitive intracellular stores was then investigated. High doses of caffeine and low doses of ryanodine failed to increase intracellular free calcium concentrations and low concentrations of caffeine and high concentrations of ryanodine did not affect K +-induced intracellular free calcium concentration transients indicating both the absence of Ca 2+-gated Ca 2+-release channels and of a Ca 2+-induced Ca 2+ release mechanism. Together, these data provide evidence that embryonic motoneurons express multiple Ca 2+ channels that function as important regulators of intracellular Ca 2+ signalling and may be involved in their development.

Human autoantibodies specific for the alpha1A calcium channel subunit reduce both P-type and Q-type calcium currents in cerebellar neurons.

The pharmacological properties of voltage-dependent calcium channel (VDCC) subtypes appear mainly to be determined by the alpha1 pore-forming subunit but, whether P-and Q-type VDCCs are encoded by the same alpha1 gene presently is unresolved. To investigate this, we used IgG antibodies to presynaptic VDCCs at motor nerve terminals that underlie muscle weakness in the autoimmune Lambert-Eaton myasthenic syndrome (LEMS). We first studied their action on changes in intracellular free Ca2+ concentration [Ca2+]i in human embryonic kidney (HEK293) cell lines expressing different combinations of human recombinant VDCC subunits. Incubation for 18 h with LEMS IgG (2 mg/ml) caused a significant dose-dependent reduction in the K+-stimulated [Ca2+]i increase in the alpha1A cell line but not in the alpha1B, alpha1C, alpha1D, and alpha1E cell lines, establishing the alpha1A subunit as the target for these autoantibodies. Exploiting this specificity, we incubated cultured rat cerebellar neurones with LEMS IgG and observed a reduction in P-type current in Purkinje cells and both P- and Q-type currents in granule cells. These data are consistent with the hypothesis that the alpha1A gene encodes for the pore-forming subunit of both P-type and Q-type VDCCs.

V7 (CD101) Ligation Inhibits TCR/CD3-Induced IL-2 Production by Blocking Ca2+ Flux and Nuclear Factor of Activated T Cell Nuclear Translocation

Ligation of the V7 (CD101) molecule on T cells with anti-V7 mAb blocks TCR/CD3-induced proliferation by inhibiting IL-2 transcription. To explore the basis for this observation, we analyzed the effects of V7 ligation on CD3/TCR-induced changes in intracellular free Ca2+ and Ca2+-dependent nuclear factor of activated T cells (NF-AT) translocation to the nucleus, which is required for IL-2 transcription. T cells exposed to anti-V7 mAb fluxed Ca2+ transiently, but did not flux Ca2+ in response to subsequent treatment with anti-CD3; however, they recovered the capacity to flux Ca2+ after treatment with pervanadate, indicating that tyrosine dephosphorylation of a critical V7-related substrate is required in the desensitization process. One such substrate, phospholipase C (PLC)-gamma1, becomes tyrosine phosphorylated on CD3/TCR activation and mediates inositol triphosphate-dependent Ca2+ flux. Co-cross-linking of T cells with anti-CD3 and anti-V7 resulted in selective inhibition of PLC-gamma1 tyrosine phosphorylation, which may explain V7-mediated blockade of anti-CD3-induced Ca2+ flux. Moreover, anti-CD3-induced binding of transcription factors to a consensus NF-AT-binding oligonucleotide, which is dependent on Ca2+, was blocked completely by treatment of the cells with anti-V7, whereas binding to a consensus-activating protein-1 oligonucleotide was unaffected. Western blot analysis of cytoplasmic and nuclear extracts confirmed that anti-V7 prevented nuclear translocation of NF-ATc induced by anti-CD3. We conclude that V7 ligation interferes with T cell activation and IL-2 secretion through a Ca2+ and tyrosine kinase-dependent pathway that inhibits PLC-gamma1 phosphorylation and prevents NF-AT translocation to the nucleus.

Suppression of Sprouting: An Early Function of NMDA Receptors in the Absence of AMPA/Kainate Receptor Activity

Electrophysiological studies have documented the existence of synapses showing only NMDA ionotropic glutamate receptor function that are therefore presumably "silent" at resting membrane potentials. Silent synapses are more prevalent in young than in older neurons, and NMDA receptor activity at such contacts may facilitate the appearance of functional AMPA receptors. However, it is uncertain whether such silent synapses actually have a function in young neurons independent of AMPA receptor induction. Using a newly characterized culture system for neurons from larval Xenopus tecta, we show that blocking NMDA receptors or preventing changes in intracellular free Ca2+ concentration with BAPTA AM significantly increases neurite sprouting and elongation in contacted but not in isolated neurons. Blocking AMPA/KA receptors or Na+-dependent action potentials does not mimic this effect. Moreover, in these young neurons, NMDA receptor-dependent Ca2+ responses to glutamate measured with confocal fluo-3 imaging are retained during AMPA/KA receptor blockade. The data suggest that many of the young contacts in these cultures are active even though they use only NMDA ionotropic glutamate receptors. Calcium influx through the NMDA receptor at these contacts seems to reduce neurite motility. This effect should lead to the accumulation of glutamatergic inputs on NMDA receptor-expressing dendrites, which could facilitate the onset of AMPA/KA receptor function and the action potential-dependent phase of synaptogenesis.

Eosinophil chemotactic activity in bronchoalveolar lavage from idiopathic pulmonary fibrosis is dependent on cytokine priming of eosinophils.

Increased numbers of eosinophils have been found in bronchoalveolar lavage (BAL) fluid obtained from patients with idiopathic pulmonary fibrosis (IPF). This suggests the presence of one or more cytokines in the lung tissue of patients with IPF, which are involved in the induction of migration of eosinophils towards the pulmonary compartment. To evaluate this hypothesis, we studied migratory responses of blood eosinophils towards BAL fluid. Migratory responses were tested by means of a modified Boyden chamber assay in 21 patients with IPF and 14 healthy controls. Experiments were performed with unprimed eosinophils and in vitro primed eosinophils (preincubated with 10(-11) M granulocyte macrophage-colony-stimulating factor). Changes in intracellular free Ca2+ ([Ca2+]i) in eosinophils in response to BAL fluid were also investigated, to characterize putative chemotaxins further. Chemotactic responses of eosinophils were observed towards BAL fluid from both patients with IPF and controls, provided that the eosinophils were primed. No changes in [Ca2+]i in eosinophils were detected in response to BAL fluid. Furthermore, neither a blocking antibody against interleukin-8 nor one against regulated on activation, normal T-cell, expressed and secreted (RANTES) influenced the response. Since a chemotactic response of in vitro primed eosinophils was also observed towards bronchoalveolar lavage fluid from normals, it was concluded, that in idiopathic pulmonary fibrosis, apart from the presence of a chemotactic factor in the lung tissue, other mechanisms such as priming of eosinophils in the peripheral blood are responsible for the extravasation of eosinophils into the pulmonary compartment. As no changes in [Ca2+]i were observed in the eosinophils after incubation with bronchoalveolar lavage fluid, the chemotaxin responsible for the migratory responses is probably not one of the known eosinophil-activating chemokines.

Protective effect of GV150526A on the glutamate-induced changes in basal and electrically-stimulated cytosolic Ca ++ in primary cultured cerebral cortical cells

Glutamate-induced changes in intracellular free Ca ++ concentration ([Ca ++] i) were recorded in resting and electrically-stimulated primary cultures of rat cerebral cortical cells, employing the Ca ++ indicator Fura 2. A brief (10 min) exposure to glutamate led to a concentration-dependent basal [Ca ++] i increase, measured 30 min after glutamate removal. In order to unmask more subtle modifications in [Ca ++] i movements associated with neurosecretion, the glutamate effect was also studied in electrically-stimulated cells. The application of trains (10 s) of electrical pulses (intensity 30 mA, duration 1 ms) induced frequency-related Na +- and Ca ++-dependent [Ca ++] i transients. A 5 min treatment with 50 μM glutamate reduced to 48% the electrically-evoked [Ca ++] i transients, evaluated 30 min after glutamate challenge. The neuroprotective effect of sodium 4-6-dichloro-3-[(E)]-3-( N-phenyl)propenamide]indole-2-carboxylate (GV150526A), a new indole derivative with high affinity and selectivity for the glycine site of the NMDA receptor-channel complex, was compared with that of dl-2-amino-5-phosphonopentanoic acid (AP5), infenprodil, 7-chlotokynurenic acid and 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)-quinoxaline (NBOX) on glutamate-induced [Ca ++] i changes in resting and electrically-stimulated cells. In both experimental conditions, GV150526A showed to be the most potent compound. Moreover, GV150526A and 7-chlorokynurenic acid were 2–3 times more active in stimulated neurons than in resting neurons, indicating a major involvement of the glycine site in the protection of the cells kept in an active state.

Neutrophil Elastase Promotes Rapid Exocytosis in Human Airway Gland Cells by Producing Cytosolic Ca2 +Oscillations

The molecular and ionic mechanisms responsible for the regulation of mucus exocytosis in human airway gland cells remain poorly defined. To determine whether dynamic changes of intracellular free Ca2+ concentration [Ca2+]i can promote different exocytotic responses, we monitored dynamic changes in [Ca2+]i and secretory granule (SG) exocytosis in individual human tracheal submucosal serous gland (HTG) cells. These changes were in response to exposure of the cells to three different secretagogues associated with airway inflammation and disease: human neutrophil elastase (HNE), histamine, and ATP. Dynamic changes in [Ca2+]i from single cells were determined with Indo-1/AM using quantitative UV laser microspectrofluorometry. The rate of SG exocytosis was measured in single cells by fluorescence videomicroscopy of SG degranulation and by the ELISA method. Exposure of HTG cells to a low concentration of HNE (1.0 microM) caused a high rate of SG exocytosis (52% decrease in the initial quinacrine fluorescence) during the first 8-min stimulation period compared with that observed following exposure of the cells to 100 microM histamine (10% decrease) or 100 microM ATP (6% decrease). In contrast to a rapid and transient rise in [Ca2+]i induced by histamine (1.0-100 microM) and ATP (10-100 microM), HNE (0.01-1 microM) generated asynchronous oscillations in [Ca2+]i over the first 8-min period. Depletion of internal Ca2+ stores with thapsigargin (500 nM) induced a significant reduction (P < 0.01) in the observed increases in [Ca2+]i upon addition of each of the secretagogues, but did not greatly affect the SG exocytotic responses. Interestingly, the removal of extracellular Ca2+ (+5 mM EGTA) significantly reduced (P < 0.01) both the [Ca2+]i increases and the rate of SG exocytosis following exposure to the secretagogues. We also demonstrate that the influx of extracellular Ca2+ and [Ca2+]i oscillations rather than the absolute level of [Ca2+]i regulate the rapid onset and extent of exocytotic responses to HNE in airway gland cells. Taken together, these results provide strong evidence that [Ca2+]i is a critical intracellular messenger in the regulation of exocytosis process in human airway gland cells.

Mitogens and Calcium Channels

Mitogens and, in particular, growth factors can induce changes in free intracellular Ca2+ concentration by opening voltage-independent cationic channels, with different degrees of Ca2+ selectivity. Recent work has provided preliminary insights into the nature of these channels and their role in the control of cell cycle progression.

Cytosolic Calcium Transient and Light Response in Limulus Ventral Nerve Photoreceptors: Supralinearity in Response-Intensity Curves

Abstract Light-evoked intracellular electrical responses were measured simultaneously with the changes in intracellular free Ca2+ or Sr2+ concentration by means of arsenazo III. The response-versus-stimulus intensity characteristics was measured in two states of relative light adaptation in strontium-containing saline (10 mᴍ Sr2+; 0.25 mᴍ Ca2+). Supralinear parts of the curves with slopes &gt; 1 were found for the receptor current amplitude and the current­-time integral as well as for the amplitude of the arsenazo signal within at least 0.3 log units of identical stimulus intensity range. Maximum slopes of response-versus-intensity curves are on average approximately 1.7 for the receptor current and the arsenazo signal in both adaptational states. The rise in time of the arsenazo signal evoked by a light flash shows two phases. In strontium saline we observe a fast increase at the onset of the arsenazo signal which is detectable within the first 20-100 ms of the signal rise and a slow further increase to the maximum, with a duration of 0.7-6 s. In the detailed analysis of both phases we observed that the amplitude of each phase increases supralinear, i.e. disproportionate to the stimulus intensity. The fast rising phase of the arsenazo signal is not visibly voltage-dependent. This fast rising phase might be a monitor of an IP3-sensitive calcium or strontium release. Since the amplitude of the fast rising phase increases supralinear with the stimulus intensity, we propose the interpretation that more than one IP3-molecule must bind to the IP3-receptor to open this channel of the intracellular calcium store. Only the slow rising phase is voltage-dependent. The kinetic of this slow rising phase was accelerated with more negative membrane potentials. This predominant slow phase correlates with the receptor current-time integral. Therefore the slow rising phase of the arsenazo signal might be a monitor for influx of calcium or strontium through light activated ion channels in the plasma membrane. The supralinearity of the amplitude of the slow rising phase might indicate the supralinear increase in calcium or strontium, which is carried by the receptor current. Additionally, we calculated the fraction of strontium and calcium ions of the total receptor current. When calcium was replaced by strontium in the superfusate about 3-30% of the receptor current are carried by strontium and calcium ions. In physiological saline calcium contributes about 1-1.5% to the receptor current.

Norepinephrine-mediated calcium signaling is altered in vascular smooth muscle of diabetic rat

We studied the influence of diabetes on norepinephrine (NE)-induced changes in intracellular free Ca 2+ levels (receptor-mediated Ca 2+ signaling) in single tail artery vascular smooth muscle (VSM) cells. VSM cells from 12–16 week streptozotocin-induced diabetic (SID) rats showed an increase in sensitivity to NE when compared to control VSM cells in that the concentration of NE needed to elicit half maximal response of the initial Ca 2+ transient was reduced more than 4-fold though the maximal response attained was apparently reduced. In addition, the slope factor (steepness) of the dose-response relation was lowered 4-fold. Moreover, VSM cells of diabetic animals had a higher incidence of NE-induced Ca 2+ oscillatory responses. The shift of the dose-response curve to the left, coupled with a higher incidence of oscillations, indicate that the noradrenergic receptor-mediated Ca 2+ signaling pathways in tail artery VSM of diabetic rat may be altered.

Glutamate stimulates neurogranin phosphorylation in cultured rat hippocampal neurons

Neurogranin is a calmodulin-binding and a protein kinase C substrate, that is expressed in telencephalic regions of the rat brain and has been associated with signal transduction and long-term potentiation (LTP). We here report that neurogranin is present in cultured hippocampal neurones, although in amounts lower than those present in the adult hippocampus, and that is also phosphorylated `in vivo'. Glutamate receptor activation rapidly and significantly increases neurogranin phosphorylation, which achieves maximal phosphate labeling after ionotropic receptor stimulation (kainate and N-methyl- d-aspartate) and more moderate one after metabotropic receptor activation. It is proposed that neurogranin phosphorylation responds to changes in intracellular free Ca 2+ and, also, that an increase in neurogranin phosphorylation contributes to enhance and extend calmodulin action, and therefore participate in post-synaptic signal transduction and LTP.

The relationship between mitochondrial state, ATP hydrolysis, [Mg2+]i and [Ca2+]i studied in isolated rat cardiomyocytes.

1. As ATP has a higher affinity for Mg2+ than ADP, the cytosolic magnesium concentration rises upon ATP hydrolysis. We have therefore used the Mg(2+)-sensitive fluorescent indicator Magnesium Green (MgG) to provide an index of changing ATP concentration in single rat cardiomyocytes in response to altered mitochondrial state. 2. In response to FCCP, [Mg2+]i rose towards a plateau coincident with the progression to rigor, which signals ATP depletion. Contamination of the MgG signal by changes in intracellular free Ca2+ concentration (the KD of MgG for Ca2+ is 4.7 microM) was excluded by simultaneous measurement of [Ca2+]i and [Mg2+]i in cells dual loaded with fura-2 and MgG. The response to FCCP was independent of external Mg2+, confirming an intracellular source for the rise in [Mg2+]i. 3. Simultaneous measurements of mitochondrial NAD(P)H autofluorescence and mitochondrial potential (delta psi m; .-1 fluorescence) and of autofluorescence and MgG allowed closer study of the relationship between [Mg2+]i and mitochondrial state. Oligomycin abolished the FCCP-induced rise in [Mg2+]i without altering the change in autofluorescence. Thus, the rise in [Mg2+]i in response to FCCP is consistent with the release of intracellular Mg2+ following ATP hydrolysis by the mitochondrial F1F0-ATPase. 4. The rise in [Mg2+]i was correlated with cell-attached recordings of ATP-sensitive K+ channel (KATP) activity. In response to FCCP, an increase in KATP channel activity was seen only as [Mg2+]i reached a plateau. In response to blockade of mitochondrial respiration and glycolysis with cyanide (CN-) and 2-deoxyglucose (DOG), [Mg2+]i rose more slowly but again KATP channel opening increased only when [Mg2+]i reached a plateau and the cells shortened. 5. Oligomycin decreased the rate of rise of [Mg2+]i delayed the onset of rigor and increased the rate of mitochondrial depolarization in response to CN-_DOG. Thus, with blockade of mitochondrial respiration delta psi m is maintained by the mitochondrial F1F0-ATPase at the expense of ATP reserves. 6. In response to CN-_DOG, the initial rise in [Mg2+]i was accompanied by a small rise in [Ca2+]i. After [Mg2+]i reached a plateau and rigor developed, [Ca2+]i rose progressively. On reperfusion, in hypercontracted cells, [Ca2+]i recovered before [Mg2+]i and [ca2+]i oscillations were sustained while [Mg2+]i decreased. Thus on reperfusion, full recovery of [ATP]i is slow, but the activation of contractile elements and the restoration of [Ca2+]i does not require the re-establishment of millimolar concentrations of ATP.

Calcium homeostasis in cultured embryonic rat septohippocampal neurons is altered by ethanol and nerve growth factor before and during depolarization

Ethanol and nerve growth factor (NGF) affect the survival of septohippocampal (SH) neurons. The effect of ethanol and NGF on calcium (Ca 2+ ) homeostasis in these neurons was investigated in this study. Changes in intracellular-free Ca 2+ concentration ([Ca 2+ ] i ) were measured using indo-1 in cultured embryonic (E21) SH neurons before stimulation (basal) and during stimulation with 30 mM potassium cloride (KCl + ). SH neurons were treated with 0, 100, 200, 400, or 800 mg% ethanol with NGF (+NGF) or without NGF (−NGF). NGF treatment decreased, while ethanol did not affect basal [Ca 2+ ] i . The combination of ethanol and NGF treatment led to increases in basal [Ca 2+ ] i . While [Ca 2+ ] i was lower during stimulation with KCl + following ethanol or NGF treatment, ethanol and NGF treatment together led to significantly greater increases or decreases in [Ca 2+ ] i compared to similarly treated NGF neurons. Responses of SH neurons were compared to those of medial septal (MS) neurons. Changes in [Ca 2+ ] i during treatment with ethanol and/or NGF were reduced in SH neurons compared with MS neurons. We conclude that changes in Ca 2+ homeostasis can occur in SH neurons in the presence of ethanol and/or NGF. The changes following ethanol treatment are enhanced by NGF. By altering Ca 2+ homeostasis, NGF may enhance the survival of SH neurons during ethanol-induced neurotoxicity.

Calcium homeostasis in cultured embryonic rat septohippocampal neurons is altered by ethanol and nerve growth factor before and during depolarization

Ethanol and nerve growth factor (NGF) affect the survival of septohippocampal (SH) neurons. The effect of ethanol and NGF on calcium (Ca 2+) homeostasis in these neurons was investigated in this study. Changes in intracellular-free Ca 2+ concentration ([Ca 2+] i) were measured using indo-1 in cultured embryonic (E21) SH neurons before stimulation (basal) and during stimulation with 30 mM potassium cloride (KCl +). SH neurons were treated with 0, 100, 200, 400, or 800 mg% ethanol with NGF (+NGF) or without NGF (−NGF). NGF treatment decreased, while ethanol did not affect basal [Ca 2+] i. The combination of ethanol and NGF treatment led to increases in basal [Ca 2+] i. While [Ca 2+] i was lower during stimulation with KCl + following ethanol or NGF treatment, ethanol and NGF treatment together led to significantly greater increases or decreases in [Ca 2+] i compared to similarly treated NGF neurons. Responses of SH neurons were compared to those of medial septal (MS) neurons. Changes in [Ca 2+] i during treatment with ethanol and/or NGF were reduced in SH neurons compared with MS neurons. We conclude that changes in Ca 2+ homeostasis can occur in SH neurons in the presence of ethanol and/or NGF. The changes following ethanol treatment are enhanced by NGF. By altering Ca 2+ homeostasis, NGF may enhance the survival of SH neurons during ethanol-induced neurotoxicity.

A unique combination of plasma membrane Ca2+-ATPase isoforms is expressed in islets of Langerhans and pancreatic β-cell lines

Changes in free intracellular Ca2+ concentration regulate insulin secretion from pancreatic beta-cells. The existence of steep Ca2+ gradients within the beta-cell requires the presence of specialized Ca2+ exclusion systems. In this study we have characterized the plasma membrane Ca2+-ATPases (PMCAs) which extrude Ca2+ from the cytoplasm. PMCA isoform- and subtype-specific mRNA expression was investigated in rodent pancreatic alpha- and beta-cell lines, and in human and rat islets of Langerhans using reverse-transcription PCR with primers flanking the calmodulin-binding region of rat PMCA. The expression pattern of PMCA 1 and 2 was conserved in different species and islet-cell types since both rat and human islets of Langerhans and all cell lines tested contained the 1b and 2b forms. PMCA 4 isoform subtypes, however, were expressed in a cell-type-specific manner since beta-cells expressed PMCA 4b only, whereas in islets of Langerhans, which contain alpha, beta, delta and polypeptide-secreting cells, PMCA 4a and 4b were simultaneously present. No evidence was obtained for the expression of PMCA 3. Characterization of the beta-cell Ca2+-pump protein showed that it shared several similarities with the erythrocyte PMCA. It is a P-type ATPase; its phosphorylated intermediate was stabilized by La3+; it reacted with a PMCA-specific antibody; and it was not N-glycosylate. However, the beta-cell PMCA had a higher molecular mass than that of the erythrocyte; this difference could be explained by either predominant translation of the PMCA2 form, which has a molecular mass 3-8 kDa higher than the erythrocyte PMCA 1 and 4 proteins, or by a possible sequence insertion. Thus a unique combination of functionally distinct PMCA isoforms (1b, 2b, 4b) participates in Ca2+ homoeostasis in the beta-cell.

2′-Phospho-Cyclic ADP-ribose, a Calcium-mobilizing Agent Derived from NADP

Cyclic adenosine diphosphoribose (cADPR), a metabolite of NAD, appears to modulate changes in intracellular free Ca2+ levels by activation of ryanodine-sensitive Ca2+ channels. We report here that an ADPR cyclase purified from Aplysia californica readily catalyzes the conversion of NADP to 2'-phospho-cyclic adenosine diphosphoribose (2'-P-cADPR), cyclized at N-1 of the adenine moiety. An enzyme from canine spleen previously shown to contain NAD glycohydrolase, ADPR cyclase, and cADPR hydrolase activities also utilized NADP and 2'-P-cADPR as substrates. The apparent Km value for NADP was 1.6 microM compared with 9.9 microM for NAD, and the Vmax with NADP was twice that with NAD, indicating that 2'-P-cADPR is a likely metabolite in mammalian cells. 2'-P-cADPR was as active as cADPR in eliciting Ca2+ release from rat brain microsomes, but was unable to elicit Ca2+ release following conversion to 2'-P-ADPR by the action of canine spleen NAD glycohydrolase. 2'-P-cADPR and 1-D-myo-inositol 1,4,5-trisphosphate (IP3) appear to act by distinct mechanisms as microsomes desensitized to IP3 still released Ca2+ in response to 2'-P-cADPR and vice versa. Also, inhibition of IP3-induced Ca2+ release by heparin had no effect on release by 2'-P-cADPR. Both 2'-P-cADPR and cADPR appear to act by a similar mechanism based on similar kinetics of Ca2+ release, similar dose-response curves, cross-desensitization, and partial inhibition of release by procaine. The results of this study suggest that 2'-P-cADPR may function as a new component of Ca2+ signaling and a possible link between NADP metabolism and Ca2+ homeostasis.

Effect of Ca2+ influx on intracellular free Ca2+ responses in antigen-stimulated RBL-2H3 cells.

We undertake a quantitative investigation of changes in intracellular free Ca2+ concentration ([Ca2+]i) in antigen-stimulated rat basophilic leukemia (RBL-2H3) cells, which include contributions of both Ca2+ store release and Ca2+ influx from the medium. Following Keizer and De Young (J. Keizer and G. De Young. Biophys. J. 61: 649-660, 1992), we develop a highly constrained mathematical model for [Ca2+]i oscillations in RBL-2H3 cells, which includes activation of the inositol trisphosphate receptor (IP3R) by inositol 1,4,5-trisphospate, indirect Ca2+ activation of the IP3R via Ca2+ -dependent activity of phospholipase C-gamma, slow inhibition of the IP3R by cytosolic Ca2+, refilling of Ca2+ stores by a Ca2+ -ATPase (SERCA)-type pump, and a simple representation of the dependence of plasma membrane (PM) fluxes on experimental conditions. Using this full (open cell) model, we simulate [Ca2+]i responses for protocols in which antigen concentration and external Ca2+ are manipulated and compare out calculations with experimental data. In protocol A, cells are stimulated in the presence of external Ca2+, in protocols B and C, cells are stimulated in the absence of external Ca2+, with external Ca2+ later reapplied in protocol C. We are able to reproduce quantitatively the important features of all three protocols, including the dose response of protocol B, the [Ca2+]i response to thapsigargin, and lag time results, and we provide qualitative explanations for the responses derived from our calculations. We also develop a simplified (closed cell) version of the model in which PM fluxes are neglected and total free Ca2+ concentration ([Ca2+]T) is a slowly varying parameter. This permits us to explain in a simple graphical fashion how PM fluxes may influence [Ca2+]i responses in RBH-2H3 cells through modulation of [Ca2+]T.