Intraterminal Ca(2+) and spontaneous transmitter release at the frog neuromuscular junction

We investigated the relationship between intraterminal Ca(2+) concentration ([Ca(2+)](i)) and the frequency of miniature end plate potentials (MEPPs) at the frog neuromuscular junction by use of ratiometric imaging of fura-2-loaded nerve terminals and intracellular recording of MEPPs. Elevation of extracellular [KCl] over the range of 2-20 mM resulted in increases in [Ca(2+)](i) and MEPP frequency. Loading terminals with the fast and slow Ca(2+)-buffers bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-acetoxymethyl (BAPTA-AM) and EGTA-AM resulted in equivalent reductions in the KCl-dependent increases in MEPP frequency. The [Ca(2+)](i) dependence of MEPP frequency determined by elevation of [Ca(2+)](i) due to application of 0.1-10 microM ionomycin was similar to that determined when [Ca(2+)](i) was raised by increasing extracellular KCl. Measurements in 10 mM extracellular KCl revealed that application of the phorbol ester phorbol 12 myristate 13-acetetate (PMA) caused an increase in MEPP frequency while the inactive analogue, 4 alpha-PMA, did not. PMA application also caused an increase in [Ca(2+)](i). The relationship between [Ca(2+)](i) and MEPP frequency in PMA was the same as was determined by the other methods of raising [Ca(2+)](i). Under all conditions tested, our data revealed a low [Ca(2+)](i) threshold for activation of transmitter release and are consistent with a K(d) for [Ca(2+)](i) on the order of 1 microM.     

Ca(2+)- and metabolism-related changes of mitochondrial potential in voltage-clamped CA1 pyramidal neurons in situ

In hippocampal slices from rats, dialysis with rhodamine-123 (Rh-123) and/or fura-2 via the patch electrode allowed monitoring of mitochondrial potential (DeltaPsi) changes and intracellular Ca(2+) ([Ca(2+)](i)) of CA1 pyramidal neurons. Plasmalemmal depolarization to 0 mV caused a mean [Ca(2+)](i) rise of 300 nM and increased Rh-123 fluorescence signal (RFS) by 相似文献   

NPC-14686, a novel anti-inflammatory agent, increased intracellular Ca(2+) concentrations in MDCK renal tubular cells

The effect of NPC-14686 (Fmoc-L-homophenylalanine), a novel anti-inflammatory agent on intracellular free Ca(2+) concentrations ([Ca(2+)](i)) in Madin Darby canine kidney (MDCK) renal tubular cells, was investigated, using fura-2 as a Ca(2+) dye. At concentrations between 10 and 200 microM NPC-14686 increased [Ca(2+)](i) concentration dependently. The [Ca(2+)](i) signal comprised an initial rise and a sustained phase. Ca(2+) removal inhibited the Ca(2+) signals by 90%. In Ca(2+)-free medium, pretreatment with 100 microM NPC-14686 nearly abolished the [Ca(2+)](i) increase induced by 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) and abolished the [Ca(2+)](i) increase induced by 2 microM carbonylcyanide m-chlorophenylhydrazone (CCCP) (a mitochondrial uncoupler). NPC-14686 (100 microM) induced a slight [Ca(2+)](i) increase after pretreatment with 2 microM CCCP and 1 microM thapsigargin. Addition of 3 mM Ca(2+) elicited a [Ca(2+)](i) increase in cells pretreated with 100 microM NPC-14686 in Ca(2+)-free medium. Inhibition of inositol-1,4,5-trisphosphate (IP(3)) production by suppressing phospholipase C with 2 microM U73122 did not alter NPC-14686-induced Ca(2+) release. Trypan blue exclusion revealed that incubation with 10 or 200 microM NPC-14686 for 1-30 min decreased cell viability by 10-20% concentration dependently. Collectively, the results demonstrate that, in MDCK tubular cells, NPC-14686 induced Ca(2+) release followed by Ca(2+) entry, with the latter playing a major role. NPC-14686 appears to release intracellular Ca(2+) in an IP(3)-uncoupled manner. NPC-14686 may be of mild cytotoxicity.     

Na(+)-Ca(2+) exchanger controls the gain of the Ca(2+) amplifier in the dendrites of amacrine cells

We have previously shown that disabling forward-mode Na(+)-Ca(2+) exchange in amacrine cells greatly prolongs the depolarization-induced release of transmitter. To investigate the mechanism for this, we imaged [Ca(2+)](i) in segments of dendrites during depolarization. Removal of [Na(+)](o) produced no immediate effect on resting [Ca(2+)](i) but did prolong [Ca(2+)](i) transients induced by brief depolarization in both voltage-clamped and unclamped cells. In some cells, depolarization gave rise to stable patterns of higher and lower [Ca(2+)] over micrometer-length scales that collapsed once [Na(+)](o) was restored. Prolongation of [Ca(2+)](i) transients by removal of [Na(+)](o) is not due to reverse mode operation of Na(+)-Ca(2+) exchange but is instead a consequence of Ca(2+) release from endoplasmic reticulum (ER) stores over which Na(+)-Ca(2+) exchange normally exercises control. Even in normal [Na(+)](o), hotspots for [Ca(2+)] could be seen following depolarization, that are attributable to local Ca(2+)-induced Ca(2+) release. Hotspots were seen to be labile, probably reflecting the state of local stores or their Ca(2+) release channels. When ER stores were emptied of Ca(2+) by thapsigargin, [Ca(2+)] transients in dendrites were greatly reduced and unaffected by the removal of [Na(+)](o) implying that even when Na(+)-Ca(2+) exchange is working normally, the majority of the [Ca(2+)](i) increase by depolarization is due to internal release rather than influx across the plasma membrane. Na(+)-Ca(2+) exchange has an important role in controlling [Ca(2+)] dynamics in amacrine cell dendrites chiefly by moderating the positive feedback of the Ca(2+) amplifier.     

Intracellular [Ca(2+)] transients in Ca(2+) wave in single rat ventricular myocyte

Ca(2+) release from the sarcoplasmic reticulum (SR) in heart muscle grades depending on Ca(2+) influx in the physiological twitch; Ca(2+( wave results from regenerative Ca(2+) release from the SR. To examine if the Ca(2+) release from the SR in the Ca(2+) wave takes a duration similar to the physiological one, a transient rise of intracellular [Ca(2+)] ([Ca(2+)](i) transient) was recorded during both a propagating Ca(2+) wave and an electrically evoked twitch with single rat ventricular myocytes, using a laser scanning confocal microscope. Care was taken to record the fluo-3 fluorescence from a segmental region with little lateral movement, especially during a propagating Ca(2+) wave. During a typical Ca(2+) wave, the time-to-peak (TP) and the half-width (HD) of the averaged [Ca(2+)](i) transient were 161 and 253 ms respectively, but they were 76 and 145 ms during an electrically evoked twitch. The difference in the duration between the two types of [Ca(2+)](i) transients could not be accounted for by modification of duration of [Ca(2+)](i) transient by possible asynchronous Ca(2+) release from the SR during a Ca(2+) wave, suggesting that the regenerative Ca2+) release from the SR in the Ca2+) wave occurs more slowly than the physiological one in rat ventricular myocytes.     

Ca2+-dependent inactivation of Ca2+-induced Ca2+ release in bullfrog sympathetic neurons

We studied inactivation of Ca(2+)-induced Ca(2+) release (CICR) via ryanodine receptors (RyRs) in bullfrog sympathetic neurons. The rate of rise in [Ca(2+)](i) due to CICR evoked by a depolarizing pulse decreased markedly within 10-20 ms to a much slower rate despite persistent Ca(2+) entry and little depletion of Ca(2+) stores. The Ca(2+) entry elicited by the subsequent pulse within 50 ms, during which the [Ca(2+)](i) level remained unchanged, did not generate a distinct [Ca(2+)](i) rise. This mode of [Ca(2+)](i) rise was unaffected by a mitochondrial uncoupler, carbonyl cyanide p-trifluromethoxy-phenylhydrazone (FCCP, 1 microm). Paired pulses of varying interval and duration revealed that recovery from inactivation became distinct >or= 50 ms after depolarization and depended on [Ca(2+)](i). The inactivation was prevented by BAPTA (>or= 100 microm) but not by EGTA (相似文献   

Effect of extracellular [Ca(2+)] on Ca(2+) release from sarcoplasmic reticulum in rat ventricular myocytes

The effect of external [Ca(2+)] ([Ca(2+)](o)) on Ca(2+) release from the sarcoplasmic reticulum (SR) was examined with rested-state twitches in rat ventricular myocytes. The magnitude of transient rise of intracellular [Ca(2+)] ([Ca(2+)](i)) relative to the resting one, F/F(o), as measured with fluo-3, was 1.75+/-0.07 (mean+/-SEM, n=9) and 1.86+/-0.13 (n=9) at 0.3 and 1.8 mM [Ca(2+)](o), respectively; the difference was insignificant. The time from onset to peak and the rate of rise of the [Ca(2+)](i) transient were 0.107+/-0.017 s (n=9) and 18.8+/-3.38 F/F(o)/s, respectively, at 0.3 mM [Ca(2+)](o), they were 0.064+/-0.005 s (n=9) and 31.1+/-0.03 F/F(o)/s, respectively, at 1.8 mM [Ca(2+)](o). The difference in the corresponding values at the two [Ca(2+)](o) was significant (t-test, p<0.05). The half decay time of the [Ca(2+)](i) transient was 0.217+/-0.016 s (n=8) at 0.3 mM [Ca(2+)](o) and was similar to the value of 0.230+/-0.022 s (n=8) at 1.8 mM [Ca(2+)](o), indicating that the rate of decrease of [Ca(2+)](i) is independent of the [Ca(2+)](o). The duration of action potential was similar at 0.3 and 1.8 mM [Ca(2+)](o) as examined with papillary muscle. The results suggest that a lowering of [Ca(2+)](o), i.e., reducing the Ca(2+) influx, slows the rate of Ca(2+) release from the SR fully loaded with Ca(2+) with little effect on the total amount of the Ca(2+) release. An instantaneous relationship between the [Ca(2+)](i) and the myocyte shortening at 0.3 and 1.8 mM [Ca(2+)](o) suggested that the time course of unloaded contraction is related not only to the magnitude, but also to the rate of rise of [Ca(2+)](i).     

Contribution of Ca(2+)-permeable AMPA/KA receptors to glutamate-induced Ca(2+) rise in embryonic lumbar motoneurons in situ

Intracellular Ca(2+) ([Ca(2+)](i)) was fluorometrically measured with fura-2 in lumbar motoneurons of acutely isolated spinal cord slices from embryonic rats. In ester-loaded cells, bath-applied glutamate (3 microM to 1 mM) evoked a [Ca(2+)](i) increase by up to 250 nM that was abolished by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) plus 2-amino-5-phosphonovalerate (APV). CNQX or APV alone reduced the response by 82 and 25%, respectively. The glutamatergic agonists kainate (KA), quisqualate (QUI), and S-alpha-amino-3-hydroxy-5-methyl-4-isoxalone (S-AMPA) evoked a similar [Ca(2+)](i) transient as glutamate. N-methyl-D-aspartate (NMDA) was only effective to increase [Ca(2+)](i) in Mg(2+)-free saline, whereas [1S,3R]-1-aminocyclopentane-1,3-dicarboxylic acid ([1S,3R]-ACPD) had no effect. The glutamate-induced [Ca(2+)](i) rise was suppressed in Ca(2+)-free superfusate. Depletion of Ca(2+) stores with cyclopiazonic acid (CPA) did not affect the response. Thirty-six percent of the [Ca(2+)](i) increase in response to membrane depolarization induced by a 50 mM K(+) solution persisted on combined application of the voltage-gated Ca(2+) channel blockers nifedipine, omega-conotoxin-GVIA and omega-agatoxin-IVA. In fura-2 dialyzed motoneurons, the glutamate-induced [Ca(2+)](i) increase was attenuated by approximately 70% after changing from current to voltage clamp. Forty percent of the remaining [Ca(2+)](i) transient and 20% of the concomitant inward current of 0.3 nA were blocked by Joro spider toxin-3 (JSTX). The results show that voltage-gated Ca(2+) channels, including a major portion of R-type channels, constitute the predominant component of glutamate-induced [Ca(2+)](i) rises. NMDA and Ca(2+)-permeable KA/AMPA receptors contribute about equally to the remaining component of the Ca(2+) rise. The results substantiate previous assumptions that Ca(2+) influx through JSTX-sensitive KA/AMPA receptors is involved in (trophic) signaling in developing motoneurons.     

Efficient Ca2+ buffering in fast-spiking basket cells of rat hippocampus

Fast-spiking parvalbumin-expressing basket cells (BCs) represent a major type of inhibitory interneuron in the hippocampus. These cells inhibit principal cells in a temporally precise manner and are involved in the generation of network oscillations. Although BCs show a unique expression profile of Ca(2+)-permeable receptors, Ca(2+)-binding proteins and Ca(2+)-dependent signalling molecules, physiological Ca(2+) signalling in these interneurons has not been investigated. To study action potential (AP)-induced dendritic Ca(2+) influx and buffering, we combined whole-cell patch-clamp recordings with ratiometric Ca(2+) imaging from the proximal apical dendrites of rigorously identified BCs in acute slices, using the high-affinity Ca(2+) indicator fura-2 or the low-affinity dye fura-FF. Single APs evoked dendritic Ca(2+) transients with small amplitude. Bursts of APs evoked Ca(2+) transients with amplitudes that increased linearly with AP number. Analysis of Ca(2+) transients under steady-state conditions with different fura-2 concentrations and during loading with 200 microm fura-2 indicated that the endogenous Ca(2+)-binding ratio was approximately 200 (kappa(S) = 202 +/- 26 for the loading experiments). The peak amplitude of the Ca(2+) transients measured directly with 100 microm fura-FF was 39 nm AP(-1). At approximately 23 degrees C, the decay time constant of the Ca(2+) transients was 390 ms, corresponding to an extrusion rate of approximately 600 s(-1). At 34 degrees C, the decay time constant was 203 ms and the corresponding extrusion rate was approximately 1100 s(-1). At both temperatures, continuous theta-burst activity with three to five APs per theta cycle, as occurs in vivo during exploration, led to a moderate increase in the global Ca(2+) concentration that was proportional to AP number, whereas more intense stimulation was required to reach micromolar Ca(2+) concentrations and to shift Ca(2+) signalling into a non-linear regime. In conclusion, dentate gyrus BCs show a high endogenous Ca(2+)-binding ratio, a small AP-induced dendritic Ca(2+) influx, and a relatively slow Ca(2+) extrusion. These specific buffering properties of BCs will sharpen the time course of local Ca(2+) signals, while prolonging the decay of global Ca(2+) signals.     

Ca2+ dynamics at the frog motor nerve terminal

Rises in free [Ca2+]i in response to various tetanic stimuli (Ca2+ transient) in frog motor nerve terminals were measured by recording fluorescence changes of Ca2+ indicators and analyzed in relation to short-term synaptic plasticity. Ca2+ transients reached a plateau after 10-20 impulses at 100 Hz and decayed in a three-exponential manner, in which the fast component was predominant. The plateau and fast component of the Ca2+ transient were elevated infralinearly with an increase in tetanus frequency. Computer simulation showed that the Ca2+ transients estimated from fluorescence changes faithfully reflect the true changes in [Ca2+]i except for the initial 20 ms. A slow Ca2+ chelator, EGTA, loaded into the nerve terminal, decreased the magnitude of both the fast and slow components of facilitation of transmitter release and the time constant of the former. A fast Ca2+ chelator, BAPTA, decreased the magnitude of fast facilitation but slightly increased its time constant. These results suggest that Ca2+ transients in the frog motor nerve terminals are primarily caused by Ca2+ entry and are dissipated by three components, in which the rate of the fast component is equivalent to that of free Ca2+ diffusion. The residual Ca2+ in the nerve terminals after stimulation accounts for the fast component of facilitation.     

Distinct intracellular calcium profiles following influx through N- versus L-type calcium channels: role of Ca2+-induced Ca2+ release

Selective activation of neuronal functions by Ca(2+) is determined by the kinetic profile of the intracellular calcium ([Ca(2+)](i)) signal in addition to its amplitude. Concurrent electrophysiology and ratiometric calcium imaging were used to measure transmembrane Ca(2+) current and the resulting rise and decay of [Ca(2+)](i) in differentiated pheochromocytoma (PC12) cells. We show that equal amounts of Ca(2+) entering through N-type and L-type voltage-gated Ca(2+) channels result in significantly different [Ca(2+)](i) temporal profiles. When the contribution of N-type channels was reduced by omega-conotoxin MVIIA treatment, a faster [Ca(2+)](i) decay was observed. Conversely, when the contribution of L-type channels was reduced by nifedipine treatment, [Ca(2+)](i) decay was slower. Potentiating L-type current with BayK8644, or inactivating N-type channels by shifting the holding potential to -40 mV, both resulted in a more rapid decay of [Ca(2+)](i). Channel-specific differences in [Ca(2+)](i) decay rates were abolished by depleting intracellular Ca(2+) stores with thapsigargin or by blocking ryanodine receptors with ryanodine, suggesting the involvement of Ca(2+)-induced Ca(2+) release (CICR). Further support for involvement of CICR is provided by the demonstration that caffeine slowed [Ca(2+)](i) decay while ryanodine at high concentrations increased the rate of [Ca(2+)](i) decay. We conclude that Ca(2+) entering through N-type channels is amplified by ryanodine receptor mediated CICR. Channel-specific activation of CICR provides a mechanism whereby the kinetics of intracellular Ca(2+) leaves a fingerprint of the route of entry, potentially encoding the selective activation of a subset of Ca(2+)-sensitive processes within the neuron.     

Fibronectin induces pseudopod formation and cell migration by mobilizing internal Ca(2+) in blastoderm cells from medaka embryos

During vertebrate embryogenesis, blastoderm cells at the gastrula stage migrate to new locations for subsequent development. The cellular mechanism of migration was studied in medaka (Oryzias latipes) embryos at the early gastrula stage. When fibronectin was applied iontophoretically or by the puff method, cell surface protrusion known as pseudopods and a local [Ca(2+)](i) rise at the site of application were observed in approximately half of the isolated blastoderm cells. When the pseudopod adhered to the substrate, the cell body moved toward the direction of the pseudopod as [Ca(2+)](i) declined and the pseudopod was withdrawn. Local puff application of ionomycin, a Ca(2+) ionophore, in the presence of external Ca(2+) induced protrusions of the plasma membrane similar to pseudopods, suggesting that the [Ca(2+)](i) rise itself is causing pseudopod formation. On the other hand, fibronectin induced pseudopods even in the absence of external Ca(2+), suggesting the mobilization of Ca(2+) from internal stores. In accordance with this interpretation, fibronectin failed to induce [Ca(2+)](i) rises after pretreatment with thapsigargin, a blocker of Ca(2+)-ATPase in the endoplasmic reticulum. Furthermore, chelating internal Ca(2+) with BAPTA prevented fibronectin from inducing pseudopods. U-73122, a blocker of phospholipase C, completely suppressed both the [Ca(2+)](i) rise and morphological changes accompanied with fibronectin application, suggesting involvement of the inositol phosphate pathway. On the other hand, caffeine evoked a [Ca(2+)](i) rise in a great majority of the fibronectin-responsive cells and the percentage of fibronectin-responsive cells was greatly reduced by a blocking dose of ryanodine. These results suggest that fibronectin activates phospholipase C and the initial [Ca(2+)](i) rise through IP(3) receptors further activates ryanodine receptors, achieving the local [Ca(2+)](i) rise. The decay time course of [Ca(2+)](i) after fibronectin application was prolonged in the absence of external Na(+). DCB, an inhibitor of Na(+)/Ca(2+) exchangers, also prolonged the time course of the [Ca(2+)](i) decay, suggesting the contribution of Na(+)/Ca(2+) exchangers. Cytochalasin D, an inhibitor of actin polymerization by binding to the barbed end of F-actin, induced swelling in fibronectin-responsive cells and prevented fibronectin from inducing pseudopod formation without suppressing the [Ca(2+)](i) rise. These results support the hypothesis that fibronectin facilitates cell migration via pseudopod formation during gastrulation.     

Somatic Ca(2+) dynamics in response to choline-mediated excitation in histaminergic tuberomammillary neurons

Histaminergic tuberomammillary (TM) neurons of the posterior hypothalamus have been implicated in cognition, alertness and sleep-wakefulness cycles. Spontaneous firing of TM neurons has been associated with histamine release and wakefulness. The expression of alpha7 nicotinic acetylcholine receptors (nAChRs) in TM neurons suggests a role for endogenous choline and for nicotinic drugs in the regulation of intracellular Ca(2+) metabolism, normal TM neuronal activity and histamine release. First, we established the link between TM neuronal spontaneous firing frequency and cytosolic free Ca(2+) concentration ([Ca(2+)](i)). A strong correlation was observed: an onset of spontaneous firing (3-4Hz) was accompanied by a 20-fold increase in [Ca(2+)](i) from 56+/-18 nM to 1.0+/-0.6 microM. The same range of firing frequencies has been observed in TM neurons in vivo and is associated with wakefulness. Secondly, choline-induced activation of alpha7 nAChRs did not elevate [Ca(2+)](i) directly, i.e. in the absence of high-threshold voltage-gated Ca(2+) channel (HVGCC) activation. Cd(2+) (200 microM) completely blocked all Ca(2+) signals, but inhibited only 37+/-16% of alpha7 nAChR-mediated currents. Thirdly, the responsiveness of [Ca(2+)](i) to choline-mediated excitation was inhibited by hyperpolarization and enhanced by depolarization, sensitizing [Ca(2+)](i) at membrane voltages associated with normal TM neuronal activity. These properties of [Ca(2+)](i) define the ability of TM neurons to translate cholinergic stimuli of identical strengths into different cytosolic Ca(2+) effects, providing the physiological substrate for state-specific modulation of incoming cholinergic information and would be expected to play a very important role in determining activity profiles of TM neurons exposed to elevated concentrations of cholinergic agents, such as choline and nicotine.     

Polycyclic aromatic hydrocarbon diol epoxides increase cytosolic Ca(2+) of airway epithelial cells

Polycyclic aromatic hydrocarbons (PAHs) increase cytosolic Ca(2+) concentration ([Ca(2+)](i)) in lymphocytes and mammary epithelial cells, but little is known regarding their effects on [Ca(2+)](i) in airway epithelium. We hypothesized that benzo[a]pyrene (BP) and/or anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), a carcinogenic BP metabolite, increases [Ca(2+)](i) in untransformed human small airway epithelial (SAE) cells and that their effects on [Ca(2+)](i) are directly proportional to carcinogenicity. SAE [Ca(2+)](i) was determined by a ratiometric digital Ca(2+) imaging system. BPDE increased SAE [Ca(2+)](i) within 20 s in media with high (1 mM) and low (10 nM) Ca(2+) at a threshold concentration of 0.2 nM. Elevation of [Ca(2+)](i) persisted longer with high Ca(2+). Neither BP nor solvent altered [Ca(2+)](i). Thapsigargin and inositol 1,4,5- phosphate receptor (InsP(3)R) antagonists inhibited this BPDE action with low Ca(2+). We conclude that BPDE but not BP increases [Ca(2+)](i) partly by mobilizing Ca(2+) from cytosolic stores through an InsP(3)R. The most potent carcinogenic PAH diol epoxide increased in SAE [Ca(2+)](i) at the lowest threshold concentration, suggesting that carcinogenicity is directly proportional to the action of PAHs on SAE [Ca(2+)](i). Short-term exposure to BPDE 36 to 48 h before the study rendered SAE cells less sensitive to BPDE, suggesting that BPDE may also induce persistent changes in Ca(2+) signaling pathways.     

Haloperidol prolongs diastolic phase of Ca(2+) transient in cardiac myocytes

Haloperidol (HPL), a widely used antipsychotic drug, is known to induce serious ventricular arrhythmias. However, the mechanism underlying their induction is not clear. We therefore examined the effects of HPL on the intracellular Ca(2+) ([Ca(2+)](i)) transient and on cell motion in cultured cardiac myocytes, as well as the pathways involving the HPL-induced abnormality of Ca(2+) homeostasis. HPL prolonged the diastolic phase of the Ca(2+) transient, with a mid-diastolic re-elevation of [Ca(2+)](i). The re-elevation of [Ca(2+)](i) was shown to be provoked by Ca(2+) release from sarcoplasmic reticulum (SR), which can trigger delayed afterdepolarization, the major arrhythmogenic factor. The re-elevation of [Ca(2+)](i) coincided with cell re-contraction during diastole. The induction of this abnormality by HPL appears to be independent of the mechanisms of the antipsychotic action.     

Different effects of toosendanin on perineurially recorded Ca(2+) currents in mouse and frog motor nerve terminals.

By perineurial recording, the effects of toosendanin (TSN), a presynaptic blocker, on nerve terminal calcium currents (I(Ca)) were observed in innervated triangularis sterni of the mouse and cutaneous pectoris of the frog. It was found that TSN blocked the slow component of I(Ca) insensitive to nifedipine and omega-conotoxin-GVIA, and increasing the extracellular Ca(2+) concentration partially antagonized the inhibitory effect in mouse motor nerve terminals. However, in the frog, TSN increased the slow component of I(Ca) and this effect disappeared in the presence of nifedipine in perfusion solution. Based on previous data showing that the slow component of I(Ca) were mediated by different subtypes of calcium channels in mouse and frog motor nerve terminals, we presume that TSN could exercise different effects on various subtypes of calcium channels.     

Na(+) and Ca(2+) homeostasis pathways, cell death and protection after oxygen-glucose-deprivation in organotypic hippocampal slice cultures

Intracellular ATP supply and ion homeostasis determine neuronal survival and degeneration after ischemic stroke. The present study provides a systematic investigation in organotypic hippocampal slice cultures of the influence of experimental ischemia, induced by oxygen-glucose-deprivation (OGD). The pathways controlling intracellular Na(+) and Ca(2+) concentration ([Na(+)](i) and [Ca(2+)](i)) and their inhibition were correlated with delayed cell death or protection. OGD induced a marked decrease in the ATP level and a transient elevation of [Ca(2+)](i) and [Na(+)](i) in cell soma of pyramidal neurons. ATP level, [Na(+)](i) and [Ca(2+)](i) rapidly recovered after reintroduction of oxygen and glucose. Pharmacological analysis showed that the OGD-induced [Ca(2+)](i) elevation in neuronal cell soma resulted from activation of both N-methyl-d-aspartate (NMDA)-glutamate receptors and Na(+)/Ca(2+) exchangers, while the abnormal [Na(+)](i) elevation during OGD was due to Na(+) influx through voltage-dependent Na(+) channels. In hippocampal slices, cellular degeneration occurring 24 h after OGD, selectively affected the pyramidal cell population through apoptotic and non-apoptotic cell death. OGD-induced cell loss was mediated by activation of ionotropic glutamate receptors, voltage-dependent Na(+) channels, and both plasma membrane and mitochondrial Na(+)/Ca(2+) exchangers. Thus, we show that neuroprotection induced by blockade of NMDA receptors and plasma membrane Na(+)/Ca(2+) exchangers is mediated by reduction of Ca(2+) entry into neuronal soma, whereas neuroprotection induced by blockade of AMPA/kainate receptors and mitochondrial Na(+)/Ca(2+) exchangers might result from reduced Na(+) entry at dendrites level.     

Inhibition of Ca(2+) entry caused by depolarization in acetylcholine-stimulated antral mucous cells of guinea pig: G protein regulation of Ca(2+) permeable channels

The effects of depolarizing conditions resulting from increasing extracellular K(+) concentration or nystatin treatment on intracellular Ca(2+) concentration ([Ca(2+)](i)) were studied in guinea pig antral mucous cells following acetylcholine (ACh) stimulation. ACh stimulation evoked a biphasic increase in [Ca(2+)](i), that is, an initial transient increase followed by a plateau. Depolarizing conditions reduced the [Ca(2+)](i) in the plateau phase during ACh stimulation. However, pertussis toxin (PTX, a G protein inhibitor) treatment caused [Ca(2+)](i) in the ACh-evoked plateau phase to increase under depolarizing conditions, while it had no effect on [Ca(2+)](i) under hyperpolarized conditions. Based on these observations, Ca(2+) permeable channels are regulated by a G protein which is activated by depolarized conditions and inhibited by hyperpolarized conditions and PTX; activation of the G protein (depolarization) causes Ca(2+) permeable channels to inhibit, and in turn, inhibition of the G protein (hyperpolarization) causes them to activate.     

Effects of hyperosmotic shrinking on ventricular myocyte shortening and intracellular Ca(2+) in streptozotocin-induced diabetic rats

Evidence exists for a specific diabetic cardiomyopathy independent of concurrent vascular disease. We tested the hypothesis that chronic hyperglycaemia found in streptozotocin- (STZ) induced diabetic rats leads to an altered response to and contractile effects of hyperosmotic shrinkage in ventricular myocytes. Analysis confirmed significant hyperglycaemia and revealed significant blood hyperosmolarity in STZ-treated rats. Myocyte volume changes, shortening and intracellular Ca(2+) ([Ca(2+)](i)) transients were measured in cells superfused with normal Tyrode (NT, 300 mmol/kg) and then hyperosmotic Tyrode (HT, 440 mmol/kg) at 35-36 degrees C. Shrinking significantly reduced the amplitude of shortening, whilst the [Ca(2+)](i) transient was significantly increased. The time course of both shortening and the [Ca(2+)](i) transient were prolonged in myocytes from STZ-treated compared to control rats. Time to peak shortening was 130.3 ms in STZ compared to 100.2 ms in control myocytes. Time to peak [Ca(2+)](i) transient was 70.8 ms in STZ compared to 44.6 ms in control myocytes and the time from peak to half recovery was 191.0 ms in STZ compared to 169.1 ms in control myocytes. Fractional SR Ca(2+) release, assessed by the application of caffeine, was increased by shrinking. However, the effects of raised extracellular osmolarity on volume changes, contractility and [Ca(2+)](i) were not altered by the chronic hyperglycaemia found in STZ-treated rats.