Region-specific activity of the plasma membrane Ca2+ pump and delayed activation of Ca2+ entry characterize the polarized, agonist-evoked Ca2+ signals in exocrine cells

The initial release of Ca2+ from the intracellular Ca2+ stores is followed by a second phase during which the agonist-dependent Ca2+ response becomes sensitive to the extracellular Ca2+, indicating the involvement of the plasma membrane (PM) Ca2+ transport systems. The time course of activation of these transport systems, which consist of both Ca2+ extrusion and Ca2+ entry pathways, is not well established. To investigate the participation of these processes during the agonist-evoked Ca2+ response, isolated pancreatic acinar cells were exposed to maximal concentrations of an inositol 1,4,5-trisphosphate-mobilizing agonist (acetylcholine, 10 microM) in different experimental conditions. Following the increase of [Ca2+]i, there was an almost immediate activation of the PM Ca2+ extrusion system, and maximal activity was reached within less than 2s. The rate of Ca2+ extrusion was dependent on the level of [Ca2+]i, with a steep activation at values just above the resting [Ca2+]i and reached a plateau value at 700 nM Ca2+. In contrast, the PM Ca2+ entry pathway was activated with a much slower time course. There was also a delay of 3-4 s between the maximal effective depletion of the intracellular Ca2+ stores and the activation of this entry pathway. By use of digital imaging data, the PM Ca2+ transport systems were also analyzed independently in two regions of the cells, the lumenal and the basal poles. With respect to the activation of the Ca2+ entry pathways, no significant difference existed between these two regions. In contrast, the PM Ca2+ pump displayed a different pattern of activity in these regions. In the basal pole, the pump activity was more sensitive to changes of [Ca2+]i and had a higher maximal activity. Also, in the lumenal pole, the pump became saturated at values of [Ca2+]i around 700 nM, whereas at the basal pole [Ca2+]i had a biphasic effect on the pump activity, and higher [Ca2+]i inhibited the pump. It is argued that these differences in sensitivity to the levels of [Ca2+]i and the different relationship between [Ca2+]i and the rate of extrusion at the two functional poles of the pancreatic acinar cells indicate that the plasma membrane Ca2+ ATPase might play an important role in the polarization of the Ca2+ response.     

Propofol and ketamine only inhibit intracellular Ca2+ transients and contraction in rat ventricular myocytes at supraclinical concentrations

BACKGROUND: The cellular mechanisms that mediate the cardiodepressant effects of intravenous anesthetic agents remain undefined. The objective of this study was to elucidate the direct effects of propofol and ketamine on cardiac excitation-contraction coupling by simultaneously measuring intracellular calcium concentration ([Ca2+]i) and shortening in individual, field-stimulated ventricular myocytes. METHODS: Freshly isolated rat ventricular myocytes were loaded with the Ca2+ indicator, fura-2, and placed on the stage of an inverted fluorescence microscope in a temperature-regulated bath. [Ca2+]i and myocyte shortening (video edge detection) were monitored simultaneously in individual cells that were field-stimulated at 0.3 Hz. RESULTS: Baseline [Ca2+]i (mean +/- SEM) was 80 +/- 12 nM, and resting cell length was 112 +/- 2 microm. Field stimulation increased [Ca2+]i to 350 +/- 23 nM, and the myocytes shortened by 10% of diastolic cell length. Both intravenous anesthetic agents caused dose-dependent decreases in peak [Ca2+]i and shortening. At 300 microM, propofol prolonged time to peak concentration and time to 50% recovery for [Ca2+]i and shortening. In contrast, changes in time to peak concentration and time to 50% recovery in response to ketamine were observed only at the highest concentrations. Neither agent altered the amount of Ca2+ released from intracellular stores in response to caffeine. Propofol but not ketamine, however, caused a leftward shift in the dose-response curve to extracellular Ca2+ for shortening, with no concomitant effect on peak [Ca2+]i. CONCLUSIONS: These results indicate that both intravenous anesthetic agents have a direct negative inotropic effect, which is mediated by a decrease in the availability of [Ca2+]i. Propofol but not ketamine may also alter sarcoplasmic reticulum Ca2+ handling and increase myofilament Ca2+ sensitivity. The effects of propofol and ketamine are primarily apparent at supraclinical concentrations, however.     

Depletion of ryanodine-sensitive Ca2+ store activates Ca2+ entry in rat submandibular gland acinar cells

The existence of ryanodine-sensitive Ca2+ stores and their role in the Ca2+ entry mechanism were examined in the rat submandibular gland acinar cells, using the microfluorimetry of intracellular Ca2+ concentration ([Ca2+]i). In the presence of thapsigargin, a Ca(2+)-ATPase inhibitor of inositol (1, 4, 5) triphosphate (InsP3)-sensitive Ca2+ stores, caffeine caused an increase in [Ca2+]i, which was inhibited by treatment with ryanodine (a ligand to the Ca(2+)-induced Ca2+ release channels). In the cells treated with ryanodine, 1 mM Ca2+ addition to a Ca(2+)-free solution caused a marked increase in [Ca2+]i, which was eliminated by application of Ni2+ or SK & F 96365, suggesting a Ca2+ entry triggered by ryanodine. The maximal change in the net increase in [Ca2+]i caused by the ryanodine-coupled Ca2+ entry, was 104.0 +/- 16.0 nM, which intense was caused by 10 microM ryanodine. Emptying the InsP3-sensitive stores by treatment with thapsigargin also caused Ca2+ entry, which maximally changed [Ca2+]i by 349.6 +/- 15.1 nM. Ten mumol/liter ryanodine was confirmed to cause a release of 45Ca2+ from the parotidic microsomal fraction enriched in endopalsmic reticulum. We propose that ryanodine-sensitive Ca2+ stores are present in rat submandibular gland acinar cells. We further propose that release of Ca2+ from the ryanodine-sensitive stores, which means eventually depletion of the ryanodine-sensitive Ca2+ stores, can activate the Ca2+ entry. The ability for Ca2+ entry coupled with the ryanodine-sensitive Ca2+ stores seems to be about 30% of the ability for Ca2+ entry coupled with the thapsigargin-sensitive Ca2+ stores.     

Isoproterenol evokes extracellular Ca2+ spikes due to secretory events in salivary gland cells

Secretory cells should in principle export substantial amounts of calcium via exocytosis since Ca2+ is sequestered in secretory granules. Based on a new technique for measurements of the extracellular calcium concentration in the vicinity of the cell membrane and on the droplet technique, we have monitored the rate of calcium extrusion from salivary gland acinar cells. Isoproterenol (ISP), a beta-adrenergic agonist and powerful secretogogue, evoked no change in the cytosolic free Ca2+ concentration ([Ca2+]i) but induced vigorous extracellular Ca2+ concentration ([Ca2+]i) spiking. The absence of [Ca2+]i elevation and the pulsatile nature of the changes in [Ca2+]i indicate that these spikes are most likely due to calcium release from secretory granules. The cholinergic agonist acetylcholine (ACh), which induces moderate secretion, evoked a marked rise in [Ca2+]i and a smooth rise in [Ca2+]i, most likely induced by plasma membrane calcium pumps, on which shortlasting [Ca2+]i spikes were superimposed. The rate of ISP-induced calcium efflux was very substantial. The calculated calcium loss during the first 100 s of supramaximal stimulation corresponded to a reduction of the total cellular calcium concentration of approximately 0.4 mM. We conclude that in salivary glands, calcium release via exocytosis is one of the main mechanisms extruding calcium from cells to the extracellular milieu.     

Spatial heterogeneity of caffeine- and inositol 1,4,5-trisphosphate-induced Ca2+ transients in isolated snail neurons

Inositol 1,4,5-trisphosphate- and caffeine-induced Ca2+ release was examined in neurons isolated from the mollusc Helix pomatia using Ca2+ indicator fura-2 and fluorescent digital-imaging microscopy technique. Extracellular application of caffeine caused a fast and pronounced augmentation of [Ca2+]i whose amplitude and kinetics differ in the centre of the cell and near its membrane. Mean values of caffeine-induced increase of [Ca2+]i were 0.97 +/- 0.11 microM at the periphery and 0.53 +/- 0.13 microM in the centre. The rates of rise and relaxation of caffeine-evoked [Ca2+]i transients were faster near the membrane. Pressure injection of inositol, 1,4,5-trisphosphate into the same neurons produced an abrupt and significant increase of [Ca2+]i in the centre (mean value of inositol 1,4,5-trisphosphate-induced elevation = 0.55 +/- 0.11 microM) while the response was smaller or even absent near the cellular membrane. Inositol 1,4,5-trisphosphate- and caffeine-induced Ca2+ transients did not affect each other. The data obtained indicate that in snail neurons these two calcium pools are not overlapping and at least some part of the caffeine-sensitive store is located close to the cellular membrane and that the inositol 1,4,5-trisphosphate-sensitive one is located in the centre of the cell.     

Na+/Ca2+ exchanger modulates kainate-triggered Ca2+ signaling in Bergmann glial cells in situ

The role of sodium-calcium exchanger in calcium homeostasis in Bergmann glial cells in situ was investigated by monitoring cytoplasmic calcium ([Ca2+]i) and sodium ([Na+]i) concentrations. The [Ca2+]i and [Na+]i transients were measured either separately by using fluorescent indicators fura-2 and SBFI, respectively, or simultaneously using the indicators fluo-3 and SBFI. Since the removal of extracellular Na+ induced a relatively small (approximately 50 nM) elevation of [Ca2+]i, the Na+/Ca2+ exchanger seems to play a minor role in regulation of resting [Ca2+]i. In contrast, kainate-triggered [Ca2+]i increase was significantly suppressed by lowering of the extracellular Na+ concentration ([Na+]o). In addition, manipulations with [Na+]o dramatically affected the recovery of the kainate-induced [Ca2+]i transients. Simultaneous recordings of [Ca2+]i and [Na+]i revealed that kainate-evoked [Ca2+]i transients were accompanied with an increase in [Na+]i. Moreover, kainate induced significantly larger [Ca2+]i and smaller [Na+]i transients under current-clamp conditions as compared to those recorded when the membrane voltage was clamped at -70 mV. The above results demonstrate that the Na(+)-Ca2+ exchanger is operative in Bergmann glial cells in situ and is able to modulate dynamically the amplitude and kinetics of [Ca2+]i signals associated with an activation of ionotropic glutamate receptors.     

Blockade by ifenprodil of high voltage-activated Ca2+ channels in rat and mouse cultured hippocampal pyramidal neurones: comparison with N-methyl-D-aspartate receptor antagonist actions

1. The block by ifenprodil of voltage-activated Ca2+ channels was investigated in intracellular free calcium concentration ([Ca2+]i) evoked by 50 mM K+ (high-[K+]o) in Fura-2-loaded rat hippocampal pyramidal neurones in culture and on currents carried by Ba2+ ions (IBa) through Ca2+ channels in mouse cultured hippocampal neurones under whole-cell voltage-clamp. The effects of ifenprodil on voltage-activated Ca2+ channels were compared with its antagonist actions on N-methyl-D-aspartate- (NMDA) evoked responses in the same neuronal preparations. 2. Rises in [Ca2+]i evoked by transient exposure to high-[K+]o in our preparation of rat cultured hippocampal pyramidal neurones are mediated predominantly by Ca2+ flux through nifedipine-sensitive Ca2+ channels, with smaller contributions from nifedipine-resistant, omega-conotoxin GVIA-sensitive Ca2+ channels and Ca2+ channels sensitive to crude funnel-web spider venom (Church et al., 1994). Ifenprodil (0.1-200 microM) reversibly attenuated high-[K+]o-evoked rises in [Ca2+]i with an IC50 value of 17 +/- 3 microM, compared with an IC50 value of 0.7 +/- 0.1 microM for the reduction of rises in [Ca2+]i evoked by 20 microM NMDA. Tested in the presence of nifedipine 10 microM, ifenprodil (1-50 microM) produced a concentration-dependent reduction of the dihydropyridine-resistant high-[K+]o-evoked rise in [Ca2+]i with an IC50 value of 13 +/- 4 microM. The results suggest that ifenprodil blocks Ca2+ flux through multiple subtypes of high voltage-activated Ca2+ channels. 3. Application of the polyamine, spermine (0.25-5 mM), produced a concentration-dependent reduction of rises in [Ca2+]i evoked by high-[K+]o. The antagonist effects of ifenprodil 20 micro M on high-[K+]0-evoked rises in [Ca2+]. were attenuated by spermine 0.25 mM but not by putrescine 1 or 5 mM. In contrast,spermine 0.1 mM increased rises in [Ca2+]i evoked by NMDA and enhanced the ifenprodil (5 micro M) block of NMDA-evoked rises in [Ca2+]i.4. Similar results were obtained in mouse cultured hippocampal pyramidal neurones under whole-cell voltage-clamp. Ifenprodil attenuated both the peak and delayed whole-cell IB. with an IC% value of 18 +/- 2 micro M, whilst it attenuated steady-state NMDA-evoked currents with an IC50 of 0.8 +/- 0.2 micro M. Block of IBa by ifenprodil 10 JaM was rapid in onset, fully reversible and occurred without change in thecurrent-voltage characteristics of Ba. The ifenprodil block of IBa was enhanced on membrane depolarization and was weakly dependent on the frequency of current activation. Spermine 0.1 mM potentiated control NMDA-evoked currents but attenuated IB,. In agreement with the microspectrofluorimetric studies, co-application of spermine produced a small enhancement of the inhibitory effect of ifenprodil 10 micro M on NMDA-evoked responses whereas the reduction of I4 by ifenprodil 10 micro M in the presence of spermine was less than expected if the inhibitory effects of ifenprodil and spermine on IBa were simply additive.5. The results indicate that ifenprodil blocks high voltage-activated Ca2+ channels in rat and mouse cultured hippocampal pyramidal neurones. Although the Ca2+ channel blocking actions of ifenprodil are observed at higher concentrations than those associated with NMDA antagonist activity, Ca2+ channel blockade may contribute, at least in part, to the established neuroprotective and anticonvulsant properties of the compound.     

Membrane voltage initiates Ca2+ waves and potentiates Ca2+ increases with abscisic acid in stomatal guard cells

In higher plants changes and oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) are central to hormonal physiology, including that of abscisic acid (ABA), which signals conditions of water stress and alters ion channel activities in guard cells of higher-plant leaves. Such changes in [Ca2+]i are thought to encode for cellular responses to different stimuli, but their origins and functions are poorly understood. Because transients and oscillations in membrane voltage also occur in guard cells and are elicited by hormones, including ABA, we suspected a coupling of [Ca2+]i to voltage and its interaction with ABA. We recorded [Ca2+]i by Fura2 fluorescence ratio imaging and photometry while bringing membrane voltage under experimental control with a two-electrode voltage clamp in intact Vicia guard cells. Free-running oscillations between voltages near -50 mV and -200 mV were associated with oscillations in [Ca2+]i, and, under voltage clamp, equivalent membrane hyperpolarizations caused [Ca2+]i to increase, often in excess of 1 microM, from resting values near 100 nM. Image analysis showed that the voltage stimulus evoked a wave of high [Ca2+]i that spread centripetally from the peripheral cytoplasm within 5-10 s and relaxed over 40-60 s thereafter. The [Ca2+]i increases showed a voltage threshold near -120 mV and were sensitive to external Ca2+ concentration. Substituting Mn2+ for Ca2+ to quench Fura2 fluorescence showed that membrane hyperpolarization triggered a divalent influx. ABA affected the voltage threshold for the [Ca2+]i rise, its amplitude, and its duration. In turn, membrane voltage determined the ability of ABA to raise [Ca2+]i. These results demonstrate a capacity for voltage to evoke [Ca2+]i increases, they point to a dual interaction with ABA in triggering and propagating [Ca2+]i increases, and they implicate a role for voltage in "conditioning" [Ca2+]i signals that regulate ion channels for stomatal function.     

Effect of caffeine on mucus secretion and agonist-dependent Ca2+ mobilization in human gastric mucus secreting cells

Caffeine is known to stimulate gastric acid secretion, but, the effects of caffeine on gastric mucus secretion have not been clarified. To elucidate the action of caffeine on gastric mucin-producing cells and its underlying mechanism, the effects of caffeine on mucus glycoprotein secretion and agonist-induced [Ca2+]i mobilization were examined in human gastric mucin secreting cells (JR-I cells). The measurement of [Ca2+]i using Indo-1 and the whole cell voltage clamp technique were applied. Mucus glycoprotein secretion was assessed by release of [3H]glucosamine. Caffeine by itself failed to increase [Ca2+]i and affect membrane currents, while it dose-dependently inhibited agonist (acetylcholine (ACh) or histamine)-induced [Ca2+]i rise, resulting in inhibiting activation of Ca2+-dependent K+ current (I(K.Ca)) evoked by agonists. The effect of caffeine was reversible, and the half maximal inhibitory concentration was about 0.5 mM. But, caffeine did not suppress [Ca2+]i rise and activation of I(K.Ca) induced by A23187 or inositol trisphosphate (IP3). Theophylline or 3-isobutyl-1-methyl-xanthine (IBMX) did not mimic the effect of caffeine. Caffeine failed to stimulate mucus secretion, while it significantly decreased ACh-induced mucus secretion. These results indicate that caffeine selectively inhibits agonist-mediated [Ca2+]i rise in human gastric epithelial cells, probably through the blockade of receptor-IP3 signaling pathway, which may affect the mucin secretion.     

Warm cells revealed by microfluorimetry of Ca2+ in cultured dorsal root ganglion neurons

Warm cells were identified by Fura-PE3-based microfluorimetry of Ca2+ in cultured dorsal root ganglion (DRG) neurons. In response to a physiologically relevant stimulus temperature (43 degrees C), a subpopulation of small DRG neurons from new born rats increased the intracellular Ca2+ concentration ([Ca2+]i). Seven percent of the cells responded to the warm stimulus. The stimulus evoked elevation in [Ca2+]i from 52.5 +/- 9.5 nM (mean +/- S.D., n = 18) to 171.0 +/- 15.6 nM in cells between 15 and 25 microns in diameter. The depletion of extracellular Ca2+ diminished the Ca2+ elevation. The Na(+)-free condition also diminished the response. We concluded that the heat stimulation opens nonselective cation channels in putative warm cells from DRG neurons.     

Relation of exocytotic release of gamma-aminobutyric acid to Ca2+ entry through Ca2+ channels or by reversal of the Na+/Ca2+ exchanger in synaptosomes

The specific inhibitor of the gamma-aminobutyric acid (GABA) carrier, NNC-711, (1-[(2-diphenylmethylene)amino]oxyethyl)- 1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride, blocks the Ca(2+)-independent release of [3H]GABA from rat brain synaptosomes induced by 50 mM K+ depolarization. Thus, in the presence of this inhibitor, it was possible to study the Ca(2+)-dependent release of [3H]GABA in the total absence of carrier-mediated release. Reversal of the Na+/Ca2+ exchanger was used to increase the intracellular free Ca2+ concentration ([Ca2+]i) to test whether an increase in [Ca2+]i alone is sufficient to induce exocytosis in the absence of depolarization. We found that the [Ca2+]i may rise to values above 400 nM, as a result of Na+/Ca2+ exchange, without inducing release of [3H]GABA, but subsequent K+ depolarization immediately induced [3H]GABA release. Thus, a rise of only a few nanomolar Ca2+ in the cytoplasm induced by 50 mM K+ depolarization, after loading the synaptosomes with Ca2+ by Na+/Ca2+ exchange, induced exocytotic [3H]GABA release, whereas the rise in cytoplasmic [Ca2+] caused by reversal of the Na+/Ca2+ exchanger was insufficient to induce exocytosis, although the value for [Ca2+]i attained was higher than that required for exocytosis induced by K+ depolarization. The voltage-dependent Ca2+ entry due to K+ depolarization, after maximal Ca2+ loading of the synaptosomes by Na+/Ca2+ exchange, and the consequent [3H]GABA release could be blocked by 50 microM verapamil. Although preloading the synaptosomes with Ca2+ by Na+/Ca2+ exchange did not cause [3H]GABA release under any conditions studied, the rise in cytoplasmic [Ca2+] due to Na+/Ca2+ exchange increased the sensitivity to external Ca2+ of the exocytotic release of [3H]GABA induced by subsequent K+ depolarization. Thus, our results show that the vesicular release of [3H]GABA is rather insensitive to bulk cytoplasmic [Ca2+] and are compatible with the view that GABA exocytosis is triggered very effectively by Ca2+ entry through Ca2+ channels near the active zones.     

High PCO does not alter pHi, but raises [Ca2+]i in cultured rat carotid body glomus cells in the absence and presence of CdC12

We measured the effect of high PCO (500-550 Torr) on the pHi and [Ca2+]i in cultured glomus cells of adult rat carotid body (CB) as a test of the two models currently proposed for the mechanism of CB chemoreception. The metabolic model postulates that the rise in glomus cell [Ca2+]i, the initiating reaction in the signalling pathway leading to chemosensory neural discharge, is due to [Ca2+] release from intracellular Ca2+ stores. The membrane potential model postulates that the rise in [Ca2+]i comes from influx of extracellular Ca2+ through voltage-dependent Ca2+ channels (VDCC) of the L-type. High PCO did not change pHi at PO2 of 120-135 Torr, showing that CO-induced changes in [Ca2+]i are not due to changes in pHi. High PCO caused a highly significant rise in [Ca2+]i from 90+/-12 nM to 675+/-65 nM, both in the absence and in the presence of 200 microM CdCl2, a potent blocker of L-type VDCCs. This result is fully consistent with release of Ca2+ from glomus cell intracellular stores according to metabolic model, but inconsistent with influx of extracellular Ca2+ through VDCCs according to the membrane potential model.     

Dissociation of intracellular Ca2+ release and Ca2+ entry response to 5-hydroxytryptamine in cultured canine tracheal smooth muscle cells

The relationship between the agonist-sensitive Ca2+ pool and those discharged by the Ca2+ -ATPase inhibitor thapsigargin (TG) were investigated in canine tracheal smooth muscle cells (TSMCs). In fura-2-loaded TSMCs, 5-hydroxytryptamine (5-HT) stimulated a rapid increase in intracellular Ca2+ ([Ca2+]i), followed by a sustained plateau phase that was dependent on extracellular Ca2+. In such cells, TG produced a concentration-dependent increase in [Ca2+]i, which remained elevated over basal level for several minutes and was substantially attenuated in the absence of extracellular Ca2+. Application of 5-HT after TG demonstrated that the TG-sensitive compartment partly overlapped the 5-HT-sensitive stores. Pre-treatment of TSMCs with TG significantly inhibited the increase in [Ca2+]i induced by 5-HT in a time-dependent manner. Similar results were obtained with two other Ca2+ -ATPase inhibitors, cyclopiazonic acid and 2,5-di-t-butylhydroquinone. Although these inhibitors had no effect on phosphoinositide hydrolysis, Ca2+ -influx was stimulated by these agents. These results suggest that depletion of the agonist-sensitive Ca2+ stores is sufficient for activation of Ca2+ influx. Some characteristics of the Ca2+ -influx activated by depletion of internal Ca2+ stores were compared with those of the agonist-activated pathway. 5-HT-stimulated Ca2+ influx was inhibited by La3+, membrane depolarisation, and the novel Ca2+ -influx blocker 1-?beta-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl?-1H-imidazole hydrochloride (SKF96365). Likewise, activation of Ca2+ influx by TG also was blocked by La3+, membrane depolarisation, and SKF96365. These results suggest that (1) in the absence of PI hydrolysis, depletion of the agonist-sensitive internal Ca2+ stores in TSMCs is sufficient for activation of Ca2+ influx, and (2) the agonist-activated Ca2+ influx pathway and the influx pathway activated by depletion of the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool are indistinguishable.     

Cytoplasmic sodium, calcium and free energy change of the Na+/Ca2+-exchanger in rat ventricular myocytes

The relationship between changing driving force of the Na+/Ca2+-exchanger (deltaG(exch)) and associated cytosolic calcium fluxes was studied in rat ventricular myocytes. DeltaG(exch) was abruptly reversed by the reduction of extracellular sodium ([Na+]o) with or without sustained depolarization by the elevation of potassium ([K+]o). Cytosolic sodium ([Na+]i) and calcium ([Ca2+]i) were measured with SBFI and indo-1 respectively and the time course of recovery of deltaG(exch) was calculated. Following abrupt reversal of deltaG(exch) from +4.1 to -9.2 kJ/mol [Na+]i exponentially decreased from 9.6-2.5 mmol/l (t(1/2) about 30 s) and [Ca2+]i transiently increased to a peak value after about 30 s. Negative values of deltaG(exch) were associated with an increase and positive values with a decrease of [Ca2+]i. Equilibrium (deltaG(exch) = 0) was reached after about 30 s coinciding with the time to peak [Ca2+]i. After 180 s deltaG(exch) reached a new steady state at +3.5 kJ/mol. Inhibition of SR with ryanodine or thapsigargin reduced the amplitude of the [Ca2+]i transient and shifted its peak to 80 s, but did not affect the time course of [Na+]i changes. In the presence of ryanodine or thapsigargin the time required for deltaG(exch) to recover to equilibrium was also shifted to 80 s. When we changed the deltaG(exch) to the same extent by the reduction of [Na+]o in combination with a sustained depolarization, [Na+]i decreased less and the amplitude of [Ca2+]i transient was much enhanced. This increase of [Ca2+]i was completely abolished by verapamil. DeltaG(exch) only recovered to a little above equilibrium (+1 kJ/mol). Inhibition of the Na+/K+-ATPase with ouabain entirely prevented the decrease of [Na+]i and caused a much larger increase of [Ca2+]i, which remained elevated; deltaG(exch) recovered to equilibrium and never returned to positive values. The rate of change of total cytosolic calcium was related to deltaG(exch), despite the fact that the calcium flux associated with the exchanger itself contributed only about 10%; SR related flux contributed by about 90% to the rate of change of total cytosolic calcium. In summary, reduction of [Na+]o causes reversal of the Na+/Ca2+-exchanger and its driving force deltaG(exch), a transient increase of [Ca2+]i and a decrease of [Na+]i. The influx of calcium associated with reversed deltaG(exch) triggers the release of calcium from SR. Both the decrease of [Na+]i and the increase of [Ca2+]i contribute to the recovery of deltaG(exch) to equilibrium. The time at which deltaG(exch) reaches equilibrium always coincides with the time to peak of [Ca2+]i transient. Activation of the Na+/K+-ATPase is required to reduce [Na+]i and recover deltaG(exch) to positive values in order to reduce [Ca2+]i. We conclude that deltaG(exch) is a major regulator of cytosolic calcium by interaction with SR.     

Alkalinization prolongs recovery from glutamate-induced increases in intracellular Ca2+ concentration by enhancing Ca2+ efflux through the mitochondrial Na+/Ca2+ exchanger in cultured rat forebrain neurons

Increasing extracellular pH from 7.4 to 8.5 caused a dramatic increase in the time required to recover from a glutamate (3 microM, for 15 s)-induced increase in intracellular Ca2+ concentration ([Ca2+]i) in indo-1-loaded cultured cortical neurons. Recovery time in pH 7.4 HEPES-buffered saline solution (HBSS) was 126 +/- 30 s, whereas recovery time was 216 +/- 19 s when the pH was increased to 8.5. Removal of extracellular Ca2+ did not inhibit the prolongation of recovery caused by increasing pH. Extracellular alkalinization caused rapid intracellular alkalinization following glutamate exposure, suggesting that pH 8.5 HBSS may delay Ca2+ recovery by affecting intraneuronal Ca2+ buffering mechanisms, rather than an exclusively extracellular effect. The effect of pH 8.5 HBSS on Ca2+ recovery was similar to the effect of the mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxyphenyl)hydrazone (FCCP; 750 nM). However, pH 8.5 HBSS did not have a quantitative effect on mitochondrial membrane potential comparable to that of FCCP in neurons loaded with a potential-sensitive fluorescent indicator, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine++ + iodide (JC-1). We found that the effect of pH 8.5 HBSS on Ca2+ recovery was completely inhibited by the mitochondrial Na+/Ca2+ exchange inhibitor CGP-37157 (25 microM). This suggests that increased mitochondrial Ca2+ efflux via the mitochondrial Na+/Ca2+ exchanger is responsible for the prolongation of [Ca2+]i recovery caused by alkaline pH following glutamate exposure.     

Lysophosphatidylcholine induces Ca2+-independent cellular injury attenuated by d-propranolol in rat cardiomyocytes

In isolated rat cardiomyocytes, exogenous lysophosphatidylcholine (LPC) (15 microM) increased the intracellular Ca2+ concentration (Ca2+]i) from 72 +/- 5 to 3042 +/- 431 nM accompanied by cell injury as indicated by the hypercontracture of the cells and the increase in creatine phosphokinase (CPK) release. In order to understand whether the cell injury induced by LPC was a consequence of the elevation of [Ca2+]i, the effect of LPC was examined in the Ca2+-free solution containing EGTA. Under the Ca2+ -free conditions, LPC did not increase [Ca2+]i, whereas it still inflicted injury on the cells in terms of cell-shape change and CPK release to the same degree as that under the Ca2+-present condition. Addition of ryanodine (10 microM) failed to prevent the changes in cell-shape and CPK release induced by LPC under both Ca2+-free and Ca2+-present conditions. Preincubation of the myocytes with d-propranolol (50 microM) inhibited the LPC-induced changes in cell-shape and CPK release under both Ca2+ -free and Ca2+ -present conditions (p < 0.05). Our study provides clear evidence that the cellular injury induced by LPC could be independent of the increase in [Ca2+]i, and the Ca2+-independent cellular injury induced by LPC could be attenuated by d-propranolol, although the mechanism remains unknown.     

Mechanisms underlying changes of tetanic [Ca2+]i and force in skeletal muscle

Force development in skeletal muscle is driven by an increase in myoplasmic free [Ca2+]i ([Ca2+]i) due to Ca2+ release from the sarcoplasmic reticulum (SR). The magnitude of [Ca2+]i elevation during stimulation depends on: (a) the rate of Ca2+ release from the SR; (b) the rate of Ca2+ uptake by the SR; and (c) the myoplasmic Ca2+ buffering. We have used fluorescent Ca2+ indicators to measure [Ca2+]i in intact, single fibres from mouse and Xenopus muscles under conditions where one or more of the above factors are changed. The following interventions resulted in increased tetanic [Ca2+]i: beta-adrenergic stimulation, which potentiates the SR Ca2+ release; application of 2.5-di(tert-butyl)-1,4-benzohydroquinone, which inhibits SR Ca2+ pumps; application of caffeine, which facilitates SR Ca2+ release and inhibits SR Ca2+ uptake; early fatigue, where the rate of SR Ca2+ uptake is reduced; acidosis, which reduces both the myoplasmic Ca2+ buffering and the rate of SR Ca2+ uptake. Reduced tetanic [Ca2+]i was observed in late fatigue, due to reduced SR Ca2+ release, and in alkalosis, due to increased myoplasmic Ca2+ buffering. Force is monotonically related to [Ca2+]i but depends also on the myofibrillar Ca2+ sensitivity and the maximum force cross-bridges can produce. This is clearly illustrated by changes of intracellular pH where, despite a lower tetanic [Ca2+]i, tetanic force is higher in alkalosis than acidosis due to increases of myofibrillar Ca2+ sensitivity and maximum cross-bridge force.     

Osmotic swelling-induced changes in cytosolic calcium do not affect regulatory volume decrease in rat cultured suspended cerebellar astrocytes

Hyposmotic swelling-induced changes in intracellular Ca2+ concentration ([Ca2+]i) and their influence on regulatory volume decrease (RVD) were examined in rat cultured suspended cerebellar astrocytes. Hyposmotic media (50 or 30%) evoked an immediate rise in [Ca2+]i from 117 nM to a mean peak increase of 386 (50%) and 220 nM (30%), followed by a maintained plateau phase. Ca2+ influx through the plasmalemma as well as release from internal stores contributed to this osmosensitive [Ca2+]i elevation. Omission of external Ca2+ or addition of Cd2+, Mn2+, or Gd3+ did not reduce RVD, although it was decreased by La3+ (0.1-1 mM). Verapamil did not affect either the swelling-evoked [Ca2+]i or RVD. Maneuvers that deplete endoplasmic reticulum (ER) Ca2+ stores, such as treatment (in Ca2+-free medium) with 0.2 microM thapsigargin (Tg), 10 microM 2,5-di-tert-butylhydroquinone, 1 microM ionomycin, or 100 microM ATP abolished the increase in [Ca2+]i but did not affect RVD. However, prolonged exposure to 1 microM Tg blocked RVD regardless of ER Ca2+ content or cytosolic Ca2+ levels. Ryanodine (up to 100 microM) and caffeine (10 mM) did not modify [Ca2+]i or RVD. BAPTA-acetoxymethyl ester (20 microM) abolished [Ca2+]i elevation without affecting RVD, but at higher concentrations BAPTA prevented cell swelling and blocked RVD. We conclude that the osmosensitive [Ca2+]i rise occurs as a consequence of increased Ca2+ permeability of plasma and organelle membranes, but it appears not relevant as a transduction signal for RVD in rat cultured cerebellar astrocytes.     

Oscillatory Cl- current induced by angiotensin II in rat pulmonary arterial myocytes: Ca2+ dependence and physiological implication

We have previously reported that angiotensin II (ANG II) induces oscillations in the cytoplasmic calcium concentration ([Ca2+]i) of pulmonary vascular myocytes. The present work was undertaken to investigate the effect of ANG II in comparison with ATP and caffeine on membrane currents and to explore the relation between these membrane currents and [Ca2+]i. In cells clamped at -60 mV, ANG II (10 microM) or ATP (100 microM) induced an oscillatory inward current. Caffeine (5 mM) induced only one transient inward current. In control conditions, the reversal potential (Erev) of these currents was close to the equilibrium potential for Cl- ions (Ecl = -2.1 mV) and was shifted towards more positive values in low-Cl- solutions. Niflumic acid (10-50 microM) and DIDS (0.25-1 mM) inhibited this inward current. Combined recordings of membrane current and [Ca2+]i by indo-1 microspectrofluorimetry revealed that ANG II- and ATP-induced currents occurred simultaneously with oscillations in [Ca2+]i whereas the caffeine-induced current was accompanied by only one transient increase in [Ca2+]i. Niflumic acid (25 microM) had no effect on agonist-induced [Ca2+]i responses, whereas thapsigargin (1 microM) abolished both membrane current and the [Ca2+]i response. Heparin (5 mg/ml in the pipette solution) inhibited both [Ca2+]i responses and membrane currents induced by ANG II and ATP, but not by caffeine. In pulmonary arterial strips, ANG II-induced contraction was inhibited by niflumic acid (25 microM) or nifedipine (1 microM) to the same extent and the two substances did not have an additive effect. This study demonstrates that, in pulmonary vascular smooth muscle, ANG II, as well as ATP, activate an oscillatory calcium dependent chloride current which is triggered by cyclic increases in [Ca2+]i and that both oscillatory phenomena are primarily IP3-mediated. It is suggested that ANG II-induced oscillatory chloride current could depolarise the cell membrane leading to activation of voltage-operated Ca2+ channels. The resulting Ca2+ influx contributes to the component of ANG II-induced contraction that is equally sensitive to chloride or calcium channel blockade.     

Modulation of pancreatic acinar cell to cell coupling during ACh-evoked changes in cytosolic Ca2+

The temporal changes in cytosolic free Ca2+ ([Ca2+]i), Ca2+-dependent membrane currents (Im), and gap junctional current (Ij) elicited by acetylcholine (ACh) were measured in rat pancreatic acinar cells using digital imaging and dual perforated patch-clamp recording. ACh (50 nM-5 microM) increased [Ca2+]i and evoked Im currents without altering Ij in 19 of 37 acinar cell pairs. Although [Ca2+]i rose asynchronously in cells comprising a cluster, the delay of the [Ca2+]i responses decreased with increasing ACh concentrations. Perfusion of inositol 1,4,5-trisphosphate (IP3) into one cell of a cluster resulted in [Ca2+]i responses in neighboring cells that were not necessarily in direct contact with the stimulated one. This suggests that extensive coupling between acinar cells provides a pathway for cell-to-cell diffusion of Ca2+-releasing signals. Strikingly, maximal (1-5 microM) ACh concentrations reduced Ij by 69 +/- 15% (n = 9) in 25% of the cell pairs subjected to dual patch-clamping. This decrease occurred shortly after the Im peak and was prevented by incubating acinar cells in a Ca2+-free medium, suggesting that uncoupling was subsequent to the initiation of the Ca2+-mobilizing responses. Depletion of Ca2+-sequestering stores by thapsigargin resulted in a reduction of intercellular communication similar to that observed with ACh. In addition, ACh-induced uncoupling was prevented by blocking nitric oxide production with L-nitro-arginine and restored by exposing acinar cells to dibutyryl cGMP. The results suggest that ACh-induced uncoupling and capacitative Ca2+ entry are regulated concurrently. Closure of gap junction channels may occur to functionally isolate nearby cells differing in their intrinsic sensitivity to ACh and thereby to allow for sustained activity of groups of secreting cells.