Ethanol acutely decreases calcium transients in cultured human myotubes

Ethanol consumption frequently leads to a number of skeletal muscle disorders, including acute and chronic alcoholic myopathy. Ethanol has been found to interfere with signal transduction mechanisms in cardiac and smooth muscle cells. We studied the effects of ethanol on the intracellular calcium ([Ca2+]i) transients responsible for excitation-contraction coupling in human myotubes from chronic alcoholic patients and healthy controls. Cultured myotubes were loaded with the fluorescent Ca2+ indicator fura-2 and evaluated on a single-cell basis. Following electrical stimulation, ethanol caused a significant reversible dose-dependent reduction in [Ca2+]i transient amplitude, achieving a mean decrease of 36+/-5% at 300 mM ethanol (p < 0.01), without modifying the basal [Ca2+]i. This acute effect of ethanol was similar in myotubes obtained from chronic alcoholics and controls. Similarly, ethanol caused a dose-dependent reduction of [Ca2+]i transient amplitude in control samples when depolarization was elicited by 100 mM KCl (p < 0.01). Several potential mechanisms of ethanol action were studied in control muscle samples. Sarcolemmal Ca2+ entry was measured indirectly by monitoring Mn2+-quenching of intracellular fura-2 via the nitrendipine-sensitive Ca2+ channels during electrical pacing. Ethanol at doses of 100 mM and greater caused a dose-dependent reduction in the rate of quench (p < 0.01). In addition, the intracellular pool of Ca2+ releasable by caffeine was found to be reduced at 300 mM ethanol (p < 0.05). We conclude that ethanol reduces the [Ca2+]i transients underlying excitation-contraction coupling in human myotubes, and that this occurs to a similar extent in cells obtained from chronic alcoholics and controls. This acute effect of ethanol was primarily due to an inhibitory effect of ethanol on sarcolemmal Ca2+ influx via voltage-operated Ca2+ channels, although there may also be an effect on the Ca2+ sarcoplasmic reticulum loading state.     

The proliferative effect of vascular endothelial growth factor requires protein kinase C-alpha and protein kinase C-zeta

The heparin-binding protein vascular endothelial growth factor (VEGF) is a highly specific growth factor for endothelial cells. VEGF binds to specific tyrosine kinase receptors, which mediate intracellular signaling. We investigated 2 hypotheses: (1) VEGF affects intracellular calcium [Ca2+]i regulation and [Ca2+]i-dependent messenger systems; and (2) these mechanisms are important for VEGF's proliferative effects. [Ca2+]i was measured in human umbilical vein endothelial cells using fura-2 and fluo-3. Protein kinase C (PKC) activity was measured by histone-like pseudosubstrate phosphorylation. PKC isoform distribution was observed with confocal microscopy and Western blot. Inhibition of PKC isoforms was assessed by specific antisense oligonucleotides (ODN) for the PKC isoforms. VEGF (10 ng/mL) induced a transient increase in [Ca2+]i followed by a sustained elevation. The sustained [Ca2+]i plateau was abolished by EGTA. Pertussis toxin also abolished the plateau phase, whereas the initial peak was not affected. The PKC isoforms alpha, delta, epsilon, and zeta were identified in endothelial cells. VEGF induced a translocation of PKC-alpha and PKC-zeta toward the nucleus and the perinuclear area, whereas cellular distribution of PKC-delta and PKC-epsilon was not influenced. Cell exposure to TPA led to a down-regulation of PKC-alpha and reduced the proliferative effect of VEGF. VEGF-induced endothelial cell proliferation also was reduced by the PKC inhibitors staurosporine and calphostin C. Specific down-regulation of PKC-alpha and PKC-zeta with antisense ODN reduced the proliferative effect of VEGF significantly. Our data show that VEGF induces initial and sustained Ca2+ influx. VEGF leads to the translocation of the [Ca2+]i-sensitive PKC isoform alpha and the atypical PKC isoform zeta. Antisense ODN for these PKC isoforms block VEGF-induced proliferation. These findings suggest that PKC isoforms alpha and zeta are important for VEGF's angiogenic effects.     

Heat shock proteins come of age: primitive functions acquire new roles in an adaptive world

We measured [Ca2+]i and [Na+]i in isolated transgenic (TG) mouse myocytes overexpressing the Na+-Ca2+ exchanger and in wild-type (WT) myocytes. In TG myocytes, the peak systolic level and amplitude of electrically stimulated (ES) [Ca2+]i transients (0.25 Hz) were not significantly different from those in WT myocytes, but the time to peak [Ca2+]i was significantly prolonged. The decline of ES [Ca2+]i transients was significantly accelerated in TG myocytes. The decline of a long-duration (4-s) caffeine-induced [Ca2+]i transient was markedly faster in TG myocytes, and [Na+]i was identical in TG and WT myocytes, indicating that the overexpressed Na+-Ca2+ exchanger is functionally active. The decline of a short-duration (100-ms) caffeine-induced [Ca2+]i transient in 0 Na+/0 Ca2+ solution did not differ between the two groups, suggesting that the sarcoplasmic reticulum (SR) Ca2+-ATPase function is not altered by overexpression of the Na+-Ca2+ exchanger. There was no difference in L-type Ca2+ current density in WT and TG myocytes. However, the sensitivity of ES [Ca2+]i transients to nifedipine was reduced in TG myocytes. This maintenance of [Ca2+]i transients in nifedipine was inhibited by Ni2+ and required SR Ca2+ content, consistent with enhanced Ca2+ influx by reverse Na+-Ca2+ exchange, and the resulting Ca2+-induced Ca2+ release from SR. The rate of rise of [Ca2+]i transients in nifedipine in TG myocytes was much slower than when both the L-type Ca2+ current and the Na+-Ca2+ exchange current function together. In TG myocytes, action potential amplitude and action potential duration at 50% repolarization were reduced, and action potential duration at 90% repolarization was increased, relative to WT myocytes. These data suggest that under these conditions, overexpression of the Na+-Ca2+ exchanger in TG myocytes accelerates the decline of [Ca2+]i during relaxation, indicating enhanced forward Na+-Ca2+ exchanger function. Increased Ca2+ influx also appears to occur, consistent with enhanced reverse function. These findings provide support for the physiological importance of both these modes of Na+-Ca2+ exchange.     

Modulations of early and late secretory processes by activation of protein kinases in the rat adrenal medulla

Modulatory effects of the activation of either protein kinase C (PKC) by phorbol 12,13-dibutyrate (PDBu) or protein kinase A (PKA) by forskolin on stimulant-evoked secretory processes in the perfused rat adrenal medulla were studied. PDBu or forskolin was applied during repetitive stimulation (30 s each at 10-min intervals) with nicotine, bradykinin, muscarine or histamine, and changes in [Ca2+]i (fura-2 microfluorometry) and catecholamine secretions (electrochemical detection) were simultaneously measured. PDBu markedly potentiated the nicotine-evoked secretion without altering the [Ca2+]i response. PDBu partially inhibited the muscarine-evoked secretion and almost completely blocked the histamine-evoked secretion, concomitantly with extensive suppressions of the [Ca2+]i responses to these stimulants. The bradykinin-evoked secretion was enhanced by PDBu despite a slight attenuation of the [Ca2+]i response. PDBu reduced bradykinin-induced intracellular Ca2+ release in a Ca2+-free medium but enhanced the secretion associated with the released Ca2+. These results suggest that PDBu-activated PKC modulates secretory processes at, at least, two different stages. An early-stage modulation may downregulate receptor/G protein systems, which accounts for the inhibitory effect of PDBu on the muscarine- and histamine-evoked responses. A late-stage modulation may generally promote Ca2+-triggered exocytosis after elevation of [Ca2+]i, which explains the potentiation of the nicotine-evoked secretion by PDBu. The late-stage modulation may counteract the early-stage modulation in bradykinin-stimulated cells. Forskolin potentiated the secretory responses to the four secretagogues without increasing the [Ca2+]i responses. PKA may modulate secretory process at a step(s) distal to the rise in [Ca2+]i as is the case with the late-stage modulation by PKC.     

Mechanism of nitric oxide-induced vasodilatation: refilling of intracellular stores by sarcoplasmic reticulum Ca2+ ATPase and inhibition of store-operated Ca2+ influx

The precise mechanisms by which nitric oxide (NO) decreases free [Ca2+]i, inhibits Ca2+ influx, and relaxes vascular smooth muscle are poorly understood. In rabbit and mouse aorta, agonist-induced contractions and increases in [Ca2+]i were resistant to nifedipine, suggesting Ca2+ entry through non-L-type Ca2+ channels. Relaxations to NO were inhibited by thapsigargin (TG) or cyclopiazonic acid (CPA) indicating the involvement of sarcoplasmic reticulum ATPase (SERCA). Studies of the effect of NO on [Ca2+]i and the rate of Mn2+ influx with fura-2 fluorometry in rabbit aortic smooth muscle cells in primary culture were designed to test how SERCA is involved in mediating the response to NO. When cells were stimulated with angiotensin II (AII), NO accelerated the removal of Ca2+ from the cytoplasm, decreased [Ca2+]i, and inhibited Ca2+ and Mn2+ influx. Inhibition of SERCA abolished all the effects of NO. In contrast, inhibition of the Na+/Ca2+exchanger or the plasma membrane Ca2+ ATPase had no influence on the ability of NO to decrease [Ca2+]i. NO maximally decreased [Ca2+]i within 5 s, whereas significant inhibition of AII-induced Ca2+ and Mn2+ influx required more than 15 s. The inhibition of cation influx strictly depended on [Ca2+]o and functional SERCA, suggesting that during the delay before NO inhibits Ca2+ influx, the influx of Ca2+ and the uptake into intracellular stores are required. In the absence of [Ca2+]o, NO diminished the AII-induced [Ca2+]i transient by a SERCA-dependent mechanism and increased the amount of Ca2+ in the stores subsequently released by ionomycin. The present study indicates that the initial rapid decrease in [Ca2+]i caused by NO in vascular smooth muscle is accounted for by the uptake of Ca2+ by SERCA into intracellular stores. It is proposed that the refilling of the stores inhibits store-operated Ca2+ influx through non-L-type Ca2+ conducting ion channels and that this maintains the decrease in [Ca2+]i and NO-induced relaxation.     

Ca2+ transients in cardiac myocytes measured with high and low affinity Ca2+ indicators

Intracellular calcium ion ([Ca2+]i) transients were measured in voltage-clamped rat cardiac myocytes with fura-2 or furaptra to quantitate rapid changes in [Ca2+]i. Patch electrode solutions contained the K+ salt of fura-2 (50 microM) or furaptra (300 microM). With identical experimental conditions, peak amplitude of stimulated [Ca2+]i transients in furaptra-loaded myocytes was 4- to 6-fold greater than that in fura-2-loaded cells. To determine the reason for this discrepancy, intracellular fura-2 Ca2+ buffering, kinetics of Ca2+ binding, and optical properties were examined. Decreasing cellular fura-2 concentration by lowering electrode fura-2 concentration 5-fold, decreased the difference between the amplitudes of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes by twofold. Thus, fura-2 buffers [Ca2+]i under these conditions; however, Ca2+ buffering is not the only factor that explains the different amplitudes of the [Ca2+]i transients measured with these indicators. From the temporal comparison of the [Ca2+]i transients measured with fura-2 and furaptra, the apparent reverse rate constant for Ca2+ binding of fura-2 was at least 65s-1, much faster than previously reported in skeletal muscle fibers. These binding kinetics do not explain the difference in the size of the [Ca2+]i transients reported by fura-2 and furaptra. Parameters for fura-2 calibration, Rmin, Rmax, and beta, were obtained in salt solutions (in vitro) and in myocytes exposed to the Ca2+ ionophore, 4-Br A23187, in EGTA-buffered solutions (in situ). Calibration of fura-2 fluorescence signals with these in situ parameters yielded [Ca2+]i transients whose peak amplitude was 50-100% larger than those calculated with in vitro parameters. Thus, in vitro calibration of fura-2 fluorescence significantly underestimates the amplitude of the [Ca2+]i transient. These data suggest that the difference in amplitude of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes is due, in part, to Ca2+ buffering by fura-2 and use of in vitro calibration parameters.     

Pituitary adenylate cyclase-activating polypeptide causes rapid Ca2+ release from intracellular stores and long lasting Ca2+ influx mediated by Na+ influx-dependent membrane depolarization in bovine adrenal chromaffin cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been reported to increase intracellular Ca2+ concentrations ([Ca2+]i) and catecholamine release in adrenal chromaffin cells. We measured [Ca2+]i with fura-2 and recorded ion currents and membrane potentials with the whole cell configuration of the patch-clamp technique to elucidate the mechanism of PACAP-induced [Ca2+]i increase in bovine adrenal chromaffin cells. PACAP caused [Ca2+]i to increase due to Ca2+ release and Ca2+ influx, and this was accompanied by membrane depolarization and inward currents. The Ca2+ release was suppressed by ryanodine, an inhibitor of caffeine-sensitive Ca2+ stores, but was unaffected by cinnarizine, an inhibitor of inositol trisphosphate-induced Ca2+ release. Ca2+ influx and inward currents were both inhibited by replacement of extracellular Na+, and Ca2+ influx was inhibited by nicardipine, an L-type Ca2+ channel blocker, or by staurosporine, a protein kinase C (PKC) inhibitor, but was unaffected by a combination of omega- conotoxin-GVIA, omega-agatoxin-IVA, and omega-conotoxin- MVIIC, blockers of N-, P-, and Q-type Ca2+ channels. Moreover, 1-oleoyl-2-acetyl-sn-glycerol, a PKC activator, induced inward currents and Ca2+ influx. These results indicate that PACAP causes both Ca2+ release, mainly from caffeine-sensitive Ca2+ stores, and Ca2+ influx via L-type Ca2+ channels activated by membrane depolarization that depends on PKC-mediated Na+ influx.     

Protective ability of acetylsalicylic acid (aspirin) to scavenge radiation induced free radicals in J774A.1 macrophage cells

Indirect studies suggested that protein kinase C (PKC) has a role in sperm motility and the acrosome reaction. Physiological inducers of the sperm acrosome reaction include progesterone, which can increase intracellular calcium ([Ca2+]i), tyrosine phosphorylation of proteins and chloride efflux in human spermatozoa. PKC may be involved in progesterone-stimulated acrosome reaction, although controversial results have been obtained concerning the effect of PKC inhibition on progesterone-stimulated [Ca2+]i increase. In the present study, we investigated the direct effect of progesterone on the activity of PKC, as well as the effect of a panel of PKC inhibitors on progesterone-stimulated [Ca2+]i increase and tyrosine phosphorylation of proteins. We found that progesterone stimulates sperm PKC activity and that PKC inhibition with staurosporine and bisindolylmaleimide partially reversed the effect of progesterone on acrosome reaction, indicating an involvement of the enzyme in the effect of the steroid. We next evaluated the effect of three different PKC inhibitors (sangivamycin, staurosporine and bisindolylmaleimide) on progesterone-stimulated [Ca2+]i increase. Neither short-term (15 min) nor long-term (90 min) preincubation with any of the three compounds had a substantial effect on the stimulatory effect of progesterone on sperm [Ca2+]i. Nor was responsiveness to progesterone affected by either short-term (determining activation of PKC) or long-term (determining down-regulation of PKC) incubation with the tumour promoter phorbol myristate acetate (PMA), a known non-physiological stimulator of PKC. These results indicate that progesterone-stimulated calcium influx is independent of PKC activation. In addition, we found that preincubation with PKC inhibitors had a stimulatory effect per se on tyrosine phosphorylation of sperm proteins. When compared with the appropriate control, the effect of progesterone on tyrosine phosphorylation was slightly (but not significantly) reduced by the inhibitors, sangivamycin, staurosporine and bisindolylmaleimide, but was significantly inhibited by calphostin C. These results do not permit a final conclusion on the involvement of PKC in progesterone-stimulated tyrosine phosphorylation of sperm proteins. However, the lack of effect of PMA on tyrosine phosphorylation indicates that PKC stimulation is not sufficient to induce this effect. In conclusion, our results indicate that PKC plays a role in progesterone-induced acrosome reaction and that progesterone-stimulated PKC activation is downstream to stimulation of calcium influx by the steroid.     

New advances in sex preselection

The effects of peroxynitrite (ONOO-) on cultured cardiac myocytes were examined by simultaneous measurements of intracellular Ca2+ ([Ca2+]i) and contractile function. On exposure to 0.2 mM ONOO-, [Ca2+]i increased to beyond the systolic level within 5 min with a concomitant decrease in spontaneous contraction of myocytes followed by complete arrest. Addition of a L-type Ca2+ channel blocker or removal of extracellular Ca2+ prevented the ONOO(-)-induced increase in [Ca2+]i, indicating that the increase in [Ca2+]i was caused by the enhanced influx of Ca2+ through the plasma membrane and not by the enhanced release from sarcoplasmic reticulum (SR). Plasma membrane fluidity and concentration of the thiobarbiturate acid-reactive substance (TBARS) in the cells remained unchanged by the ONOO- treatment. The complete cessation of contraction of myocytes persisted even under the massive increase in [Ca2+]i, which was induced by an additional saponin (5 microM) treatment. In conclusion, ONOO- increases [Ca2+]i in myocytes through disturbance of Ca2+ transport systems in the plasma membrane and impairs contractile protein.     

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.     

Increased calcium buffering in basal forebrain neurons during aging

Increased calcium buffering in basal forebrain neurons during aging. J. Neurophysiol. 80: 350-364, 1998. Alterations of neuronal calcium (Ca2+) homeostasis are thought to underlie many age-related changes in the nervous system. Basal forebrain neurons are susceptible to changes associated with aging and to related dysfunctions such as Alzheimer's disease. It recently was shown that neurons from the medial septum and nucleus of the diagonal band (MS/nDB) of aged (24-27 mo) F344 rats have an increased current influx through voltage-gated Ca2+ channels (VGCCs) relative to those of young (1-4. 5 mo) rats. Possible age-related changes in Ca2+ buffering in these neurons have been investigated using conventional whole cell and perforated-patch voltage clamp combined with fura-2 microfluorimetric techniques. Basal intracellular Ca2+ concentrations ([Ca2+]i), Ca2+ influx, Ca2+ transients (Delta[Ca2+]i), and time course of Delta[Ca2+]i were quantitated, and rapid Ca2+ buffering values were calculated in MS/nDB neurons from young and aged rats. The involvement of the smooth endoplasmic reticulum (SER) was examined with the SER Ca2+ uptake blocker, thapsigargin. An age-related increase in rapid Ca2+ buffering and Delta[Ca2+]i time course was observed, although basal [Ca2+]i was unchanged with age. The SER and endogenous diffusible buffering mechanisms were found to have roles in Ca2+ buffering, but they did not mediate the age-related changes. These findings suggest a model in which some aging central neurons could compensate for increased Ca2+ influx with greater Ca2+ buffering.     

The role of immune complex in otitis media with effusion

Lead characteristically perturbs processes linked to the calcium messenger system. This study was undertaken to determine the role of PKC in the Pb2+ induced rise of [Ca2+]i. [Ca2+]i was measured using the divalent cation indicator, 1,2-bis(2-amino-5-fluorophenoxy) ethane N, N,N',N'-tetraacetic acid (5F-BAPTA) and 19F-NMR in the osteoblast cell line, ROS 17/2.8. Treatment of cells with Pb2+ at 1 and 5 microM produced a rise in [Ca2+]i from a basal level of 125 nM to 170 nM and 230 nM, respectively, while treatment with phorbol 12-myristate 13-acetate (PMA) (10 microM), an activator of PKC, produced a rise in [Ca2+]i to 210 nM. Pretreatment with calphostin C, a potent and highly selective inhibitor of PKC activation failed to produce a change in basal [Ca2+]i and prevented any rise in [Ca2+]i in response to Pb2+. To determine whether Pb2+ acts directly on PKC, we measured the Pb2(+)-dependent activation of phosphatidylserine/diolein-dependent incorporation of 32P from ATP into histone and endogenous TCA precipitable proteins in the 100,000 X g supernatant from homogenized ROS 17/2.8 cells. The free concentrations of Pb2+ and Ca2+ were set using 5F-BAPTA; and [Ca2+] and [Pb2+] in the PKC reaction mixtures were confirmed by 19F-NMR. We found that Pb2+ activates PKC in the range of 10(-11)-10(-7) M, with an activation constant of 1.1 X 10(-10) M, whereas Ca2+ activates PKC in the range from 10(-8) to 10(-3) M, with an activation constant of 3.6 X 10(-7) M. These data suggest that Pb2+ activates PKC in ROS 17/2.8 cells and that Pb2+ activation of PKC mediates the documented rise in [Ca2+]i and, perhaps, other toxic effects of Pb2+.     

Effect of 5-hydroxytryptamine on intracellular calcium dynamics in cultured rat vascular smooth muscle cells

The present study elucidated the precise mechanism of 5-hydroxytryptamine (5-HT)-induced increase of intracellular Ca2+ concentration ([Ca2+]i) in cultured vascular smooth muscle cells isolated from rat aortic media. [Ca2+]i was measured using fluorescent Ca2+ indicator, fura-2. 5-HT caused a dose-dependent increase in [Ca2+]i, which was completely inhibited by ketanserin. alpha-Methyl-5-HT had an equipotent effect to 5-HT. Diltiazem at 10 microM partially suppressed the 5-HT-induced increase in [Ca2+]i. 5-HT also augmented Mn2+ influx, when monitored by Mn2+ quenching of fura-2 fluorescence. When extracellular Ca2+ (1.3 mM) was removed, a decrease in resting level and a small, transient increase in [Ca2+]i were observed. 5-HT stimulation also induced an increase in the production of inositol triphosphate. 5-HT-induced increase in [Ca2+]i was significantly, but partially inhibited by staurosporin and H-7. Phorbol 12-myristate 13-acetate induced an increase in [Ca2+]i, which was abolished by removal of extracellular Ca2+. 5-HT-induced increase in [Ca2+]i was not affected by the pretreatment with pertussis toxin (PTX), and was not accompanied by a change in cyclic AMP content. These results suggest that, in cultured rat aortic smooth muscle cells, 5-HT increases [Ca2+]i via 5-HT2 receptor subtype by inducing influx of extracellular Ca2+ partially through L-type voltage-dependent Ca2+ channel, as well as by mobilizing Ca2+ from its intracellular stores. Activation of protein kinase C may be positively involved in the regulatory mechanism of Ca2+ influx, but PTX-sensitive G protein and cyclic AMP seem to be not involved.     

JTT-501, a novel oral antidiabetic agent, improves insulin resistance in genetic and non-genetic insulin-resistant models

The effects of histamine on the intracellular Ca2+ concentration ([Ca2+]i), action potential and membrane currents were assessed in single atrial myocytes prepared from guinea-pigs. Histamine caused a concentration-dependent increase in the [Ca2+]i transient in indol/AM loaded myocytes when stimulated electrically at 0.5 Hz. However, the maximum increase in [Ca2+]i transient produced by histamine was less than 50% of that elicited by isoprenaline. The histamine-induced increase in [Ca2+]i transient was significantly inhibited by chlorpheniramine, but not by cimetidine. Pretreatment with nifedipine nearly completely suppressed the histamine-induced increase in [Ca2+]i transient. Cyclopiazonic acid did not affect the histamine response. In the whole-cell current-clamp mode of the patch-clamp method, both histamine and isoprenaline prolonged action potential duration (APD) in atrial myocytes. In the presence of Co2+ or nifedipine, the isoprenaline-induced APD prolongation was abolished and an APD shortening effect was manifested, while histamine still increased APD. The APD prolongation elicited by histamine was reversed by chlorpheniramine. In the voltage-clamp mode, the histamine-sensitive membrane current was inwardly rectifying and reversed close to the calculated value of the K+ equilibrium potential. Histamine had no apparent effect on L-type Ca2+ current, in contrast to the pronounced effect of isoprenaline. These results indicate that in guinea-pig atrial myocytes stimulation of H1-receptors with histamine does not directly activate Ca2+ channels but causes an elevation of [Ca2+]i transient by increasing Ca2+ influx through the channels during the prolonged repolarization of action potentials resulting from inhibition of the outward K+ current.     

Rilmenidine elevates cytosolic free calcium concentration in suspended cerebral astrocytes

Rilmenidine, a ligand for imidazoline and alpha2-adrenergic receptors, is neuroprotective following focal cerebral ischemia. We investigated the effects of rilmenidine on cytosolic free Ca2+ concentration ([Ca2+]i) in rat astrocytes. Rilmenidine caused concentration-dependent elevation of [Ca2+]i, consisting of a transient increase (1-100 microM rilmenidine) or a transient increase followed by sustained elevation above basal levels (1-10 mM rilmenidine). A similar elevation in [Ca2+]i was induced by the imidazoline ligand cirazoline. The transient response to rilmenidine was observed in Ca2+-free medium, indicating that rilmenidine evokes release of Ca2+ from intracellular stores. However, the sustained elevation of Ca2+ was completely dependent on extracellular Ca2+, consistent with rilmenidine activating Ca2+ influx. Pretreatment with thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, abolished the response to rilmenidine, confirming the involvement of intracellular stores and suggesting that rilmenidine and thapsigargin activate a common Ca2+ influx pathway. The alpha2-adrenergic antagonist rauwolscine attenuated the increase in [Ca2+]i induced by clonidine (a selective alpha2 agonist), but not the response to rilmenidine. These results indicate that rilmenidine stimulates both Ca2+ release from intracellular stores and Ca2+ influx by a mechanism independent of alpha2-adrenergic receptors. In vivo, rilmenidine may enhance uptake of Ca2+ from the extracellular fluid by astrocytes, a process that may contribute to the neuroprotective effects of this agent.     

Endothelin: receptor subtypes, signal transduction, regulation of Ca2+ transients and contractility in rabbit ventricular myocardium

Endothelin (ET) isopeptides, ET-1, ET-2 and ET-3, elicit a positive inotropic effect (PIE) in association with a negative lusitropic effect, essentially with identical efficacies and potencies in the isolated rabbit papillary muscle, but with different concentration-dependent properties. Pharmacological analysis indicates that the PIE of ET-1 is mediated by an ETA2 subtype that is less sensitive to BQ-123 and FR139317, whereas the PIE of ET-3 is mediated by an ETA1 subtype that is highly sensitive to these ETA antagonists. ETs increased the amplitude of intracellular Ca2+ transient (CaT) in indo-1 loaded rabbit ventricular myocytes, but the increase was much smaller than that produced by elevation of [Ca2+]o or isoproterenol for a given extent of PIE, an indication of increased myofibrillar Ca2+ sensitivity. ETs stimulate phosphoinositide (PI) hydrolysis, which leads to production of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Evidence for the role of IP3-induced Ca2+ release in cardiac E-C coupling is tenuous. Generation of IP3 induced by ET-1 was transient and returned to the baseline level when the PIE reached an elevated steady level. Protein kinase C (PKC) that is activated by DAG and also via other pathways triggered by ETs stimulates Na+-H+ exchanger to lead to an increased [Na+]i and alkalinization. The former may contribute to an increase in the amplitude of CaT through Na+-Ca2+ exchanger, and the latter, to an increase in myofibrillar Ca2+ sensitivity. A number of PKC inhibitors, such as staurosporine, H-7, calphostin C and chelerythrine, consistently and selectively inhibited the PIE of ET-3 without affecting the PIE of isoproterenol and Bay k 8644. The maximum inhibition was 20-30% of the total response. A Na+-H+ exchange inhibitor, [5-(N-ethyl-N-isopropyl) amiloride (EIPA)] or a Ca2+ antagonist, verapamil, could not completely inhibit the PIE of ET-3, but the combination of both inhibitors totally abolished the PIE of ET-3. These findings indicate that activation of PKC and subsequent activation of Na+-H+ exchanger and/or L-type Ca2+ channels may play a crucial role in the cardiac action of ET isopeptides in the rabbit ventricular myocardium.     

Sarcoplasmic reticulum-sarcolemma interactions and vascular smooth muscle tone

A characteristic of vascular smooth muscle cell morphology is a close apposition of its peripheral sarcoplasmic reticulum (SR) with the sarcolomma; this arrangement gives rise to important functional interactions whereby the peripheral SR regulates Ca2+ influx and vascular tone. We review here the key evidence supporting the following aspects of SR-sarcolemma interactions while establishing a conceptual framework encompassing (i) the SR ultrastructure and functions, (ii) the integration of the sarcolemmal Na+-Ca2+ exchanger and the peripheral SR in the mediation of a bidirectional Ca2+ exchange between the peripheral SR and the extracellular space, (iii) the existence of a higher myoplasmic free Ca2+ concentration [Ca2+]myo in the subsarcolemmal space formed between the sarcolemma and the peripheral SR relative to the [Ca2+]myo of the inner myoplasm in the resting smooth muscle cell, (iv) the division of the subsarcolemmal space into functional microdomains, (v) the existence of spontaneous localized bursts of Ca2+ release from the peripheral SR (Ca2+ sparks) towards the sarcolemma, (vi) the physiological triggering of nonlocalized Ca2+ release from the peripheral SR by Ca2+ influx (Ca2+-induced Ca2+ release), and (vii) capacitative Ca2+ entry in vascular smooth muscle. We present an overview of the physiological and pathological implications of these interactions.     

Sensitivity of G-protein involved in endothelin-1-induced Ca2+ influx to pertussis toxin in porcine endothelial cells in situ

1. We designed a new method to determine quantitatively the intracellular Ca2+ concentration ([Ca2+]i) in endothelial cells in situ, using front-surface fluorometry and fura-2-loaded porcine aortic valvular strips. Using this method, we investigated the characteristics of the G-protein involved in endothelin-1 (ET-1)-induced changes in [Ca2+]i of endothelial cells in situ. 2. Endothelial cells were identified by specific uptake of acetylated-low density lipoprotein labelled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI-Ac-LDL). Double staining with DiI-Ac-LDL and fura-2 showed that the valvular strip was covered with a monolayer of endothelial cells and that the cellular component which contributed to the fura-2 fluorescence, [Ca2+]i signal, was exclusively endothelial cells. 3. ET-1 (10(-7) M) induced an elevation of [Ca2+]i consisting of two components: the first was a rapid and transient elevation to reach a peak, followed by a second, sustained elevation (the second phase). The first phase was composed of extracellular Ca(2+)-independent and -dependent components, while the second phase was exclusively extracellular Ca(2+)-dependent. The extracellular Ca(2+)-independent component of the first phase was due to the release of Ca2+ from intracellular storage sites. The second phase and part of the first phase of [Ca2+]i elevation were attributed to the influx of extracellular Ca2+. The Ca2+ influx component was completely inhibited by 10(-3) M Ni2+ but was not affected by 10(-5) M diltiazem. 4. Pertussis toxin (IAP) markedly inhibited the extracellular Ca2+-dependent elevation of [Ca2+]j, but had no effect on the extracellular Ca2+-independent elevation of [Ca2+], caused by ET-1 (10-7M).5. Bradykinin (10-7 M) or ATP (10- 5M) elevated [Ca2+]i and these responses also consisted of extracellular Ca2+-independent and extracellular Ca2+-dependent components. IAP had no effect on either component of the [Ca2+]i elevation induced by bradykinin or ATP.6. From these findings we conclude that, in porcine endotheliel cells in situ, ET-1 elevates [Ca2+]i as are result of a Ca2+ influx component from the extracellular space and release of intracelluarly stored Ca2+ .The Ca2+ influx is regulated by an IAP-sensitive G-protein, while the release of Ca2+ from the intracellular store is not.     

Mechanisms through which high glucose concentration raises [Ca2+]i in renal proximal tubular cells

BACKGROUND: The basal levels of cytosolic calcium ([Ca2+]i) of renal proximal tubular cells of rats with streptozotocin-induced diabetes are elevated. It is possible that this phenomenon is mediated by the hyperglycemia, which may cause both increased calcium influx into and/or decreased calcium efflux out of these cells. METHODS: We examined whether high glucose concentration in vitro causes acute rise in [Ca2+]i of freshly isolated renal proximal tubular cells and explored the pathways that are involved in such an event. RESULTS: There were dose and time dependent increments in [Ca2+]i of renal proximal tubular cells exposed to high concentrations of glucose. A similar effect was observed with equimolar concentrations of mannitol or choline chloride but not urea. A substantial part of the rise in [Ca2+]i was inhibited when the media contained verapamil, nifedipine, amlodipine or ryanodine and when the cells were placed in a calcium free media. Inhibitors of G protein(s) (GDPbetaS or pertussis toxin), inhibitors of cAMP-protein kinase A pathway (RpcAMP or H-89), inhibitors of protein kinase C (staurosporine or calphostin) and inhibitor of Na+-H+ exchanger (HOE 694) blocked the rise in a dose dependent manner. High glucose concentration also caused a decrease in ATP content of these cells and a reduction in the Vmax of their Ca2+ATPase. CONCLUSIONS: The results are consistent with the formulation that the osmotic activity (cell shrinkage) of the high glucose concentration may activate a stretch receptor with subsequent stimulation of various cellular pathways including G protein(s), cAMP-protein kinase A and phospholipase C systems and calcium channels. Activation of these cellular pathways permits both calcium influx into renal tubular cells and mobilization of calcium from their intracellular stores. Further, a decrease in calcium efflux secondary to the reduction in the Vmax of Ca2+ ATPase may occur. It is possible that the rise in [Ca2+]i is critical for the stimulation of the events that lead to restoration of cell volume to normal.     

Integrin alphavbeta5 participates in the binding of photoreceptor rod outer segments during phagocytosis by cultured human retinal pigment epithelium

Because glycolysis is thought to be important for maintenance of cellular ion homeostasis, the aim of the present study was to examine the role of glycolysis in the control of cytosolic calcium ([Ca2+]i) and cell shortening during conditions of increased calcium influx. Thus, [Ca2+]i and unloaded cell shortening were measured in fura-2/AM loaded rat ventricular myocytes. All cells were superfused with Tyrode's solution containing glucose and pyruvate (to preserve oxidative metabolism), and glycolysis was inhibited by iodoacetate (IAA, 100 microM). Calcium influx was increased, secondary to an increase in intracellular sodium, by addition of veratrine (1 microgram/ml), or directly by either elevating [Ca2+]o from 2 to 5 mM or by exposing the cells to isoproterenol (1 to 100 nm). Veratrine exposure caused a time-dependent increase in both diastolic and systolic [Ca2+]i that resulted in cellular calcium overload and hypercontraction. The rate of increase in [Ca2+]i was more rapid in IAA-treated than in untreated myocytes, leading to a 13+/-3 v 5+/-2% increase (P<0.05) in diastolic [Ca2+]i after 5 min of exposure. The corresponding increases in systolic [Ca2+]i were 43+/-6 and 24+/-5% (P<0.05). Elevated [Ca2+]o resulted in increased [Ca2+]i transient amplitudes and cell shortening. These responses were each attenuated by inhibiting glycolysis, so that the increase was 38+/-5 v 68+/-9% ([Ca2+]i transient amplitude, P<0.05) and 41+/-11 v 91+/-18% (cell shortening, P<0.05). Inhibition of glycolysis did not, however, affect the increase in calcium transient or cell shortening during addition of isoproterenol. We conclude that glycolysis plays an essential role in the maintenance of intracellular calcium homeostasis during severe calcium overload. Glycolysis was also essential for signalling the inotropic effect that accompanied elevation in extracellular calcium, while the changes in intracellular calcium following administration of isoproterenol were not influenced by glycolysis in the present model.