Dose-dependent effects of chloroquine on secretory granule formation in the melanotroph

Intracellular calcium ([Ca2+]i) and hydrogen ion concentrations (pHi) are important regulators of cell function. Those ions also may interact and it is important, therefore, to measure their concentrations simultaneously. In the present studies we used a system developed for that purpose, a fluorescent emission ratio technique for simultaneous analysis of calcium (Indo-1) and pH (SNARF-1) in single cells at video rates, and determined if arginine vasopressin (AVP, 12.5 mumol/l) evoked [Ca2+]i and pHi signals interact in MDCK cells. We also employed a simple system for analysing the side specific (basolateral or apical) application of agonist to polarized cell layers on permeable membranes. AVP is found to evoke simultaneous changes in both pHi and [Ca2+]i. Basolateral application induced transient acidification, followed by partial recovery, and a [Ca2+]i transient with kinetic pattern similar to that of the pHi. Apical application also caused a mirror image pHi and [Ca2+]i pattern but of smaller magnitude (no peak). Selective removal of extracellular calcium ([Ca2+]e) or sodium ([Na+]e) dissociated the pHi and [Ca2+]i responses in both cases. Na+e removal abolished the pHi changes, but not the [Ca2+]i transients. [Ca2+]e removal abolished the [Ca2+]i changes and reduced, but did not abolish, the pHi responses. Thus, AVP induces pHi changes which are modified by calcium while calcium signalling is not modified by changes in pHi.     

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.     

Catecholamine secretion from bovine adrenal chromaffin cells: the role of the Na+/Ca2+ exchanger and the intracellular Ca2+ pool

The role of the Na+/Ca2+ exchanger and intracellular nonmitochondrial Ca2+ pool in the regulation of cytosolic free calcium concentration ([Ca2+]i) during catecholamine secretion was investigated. Catecholamine secretion and [Ca2+]i were simultaneously monitored in a single chromaffin cell. After high-K+ stimulation, control cells and cells in which the Na+/Ca2+ exchange activity was inhibited showed similar rates of [Ca2+]i elevation. However, the recovery of [Ca2+]i to resting levels was slower in the inhibited cells. Inhibition of the exchanger increased the total catecholamine secretion by prolonging the secretion. Inhibition of the Ca2+ pump of the intracellular Ca2+ pool with thapsigargin caused a significant delay in the recovery of [Ca2+]i and greatly enhanced the secretory events. These data suggest that both the Na+/Ca2+ exchanger and the thapsigargin-sensitive Ca2+ pool are important in the regulation of [Ca2+]i and, by modulating the time course of secretion, are important in determining the extent of secretion.     

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.     

Intracellular sodium concentration in cultured cerebellar granule cells challenged with glutamate

We monitored simultaneously the changes in the intracellular sodium concentration ([Na+]i) and intracellular calcium concentration ([Ca2+]i) in individual neurons from primary cultures of cerebellar granule cells loaded with sodium-binding benzofuran isophthalate and fluo-3. An application of glutamate (50 microM) in Mg(2+)-free medium containing 10 microM glycine evoked [Na+]i and [Ca2+]i increases that exceeded 60 mM and 1 microM, respectively. The kinetics of [Na+]i and [Ca2+]i decreases after the termination of the glutamate pulse were different. [Na+]i failed to decrease immediately after glutamate withdrawal and the delay in the onset of [Na+]i decrease after the glutamate pulse termination was proportional to the glutamate dose, the glutamate pulse duration, and the extent of [Ca2+]i elevation elicited by glutamate. The kinetics of [Ca2+]i decrease were biphasic, with the first phase occurring immediately after glutamate withdrawal and the second phase being correlated in time with a [Na+]i value lower than 15-20 mM. These results were interpreted to indicate that the glutamate-evoked calcium influx may lead to sodium homeostasis destabilization. The delay in the restoration of the sodium gradient may in turn prolong the neuronal exposure to toxic [Ca2+]i values, due to the decrease in the efficiency of the Na+/Ca2+ exchanger to extrude calcium. The glutamate effects on [Na+]i and [Ca2+]i were potentiated by glycine. Glycine (10 microM) added alone also evoked [Na+]i and [Ca2+]i increases; this effect was inhibited by a competitive inhibitor of the N-methyl-D-aspartate receptor, 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid, indicating an involvement of endogenous glutamate.     

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.     

Different mechanisms for [Ca2+]i oscillations induced by carbachol and high concentrations of [Ca2+]o in the rat ventricular myocyte

1. The purpose of the present study was to explore the different mechanisms of [Ca2+]i oscillations induced by high concentrations of either carbachol (CCh) or extracellular Ca2+ ([Ca2+]o). First, we compared the oscillations induced by CCh at concentrations of 100-300 micromol/L and [Ca2+]o (5 mmol/L) in the single rat ventricular myocyte. Second, we studied CCh- and [Ca2+]o-induced [Ca2+]i oscillations following either interference with the production of inositol trisphosphate (IP3), reductions in cytosolic Ca2+ ([Ca2+]i), inhibition of Ca2+ influx and Na+-Ca2+ exchange or depletion of Ca2+ from its intracellular store. 2. The [Ca2+]i oscillations induced by CCh were frequent and were superimposed on [Ca2+]i transients in electrically stimulated cells, whereas those induced by high [Ca2+]o were occasional and occurred in quiescent cells and between [Ca2+]i transients in electrically stimulated cells. In both cases, [Ca2+]i oscillations were preceded by an increase in resting levels of [Ca2+]i. 3. Carbachol-induced [Ca2+]i oscillations were accompanied by an increase in amplitude and prolongation of the time of decline to 80% of the peak of the [Ca2+]i transient, while high [Ca2+]o-induced [Ca2+]i oscillations were the opposite. 4. A reduction of [Ca2+]o to 0.1 mmol/L and treatment with Ni2+ or ryanodine or 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid AM (BAPTA-AM) abolished the [Ca2+]i oscillations induced by both CCh and high [Ca2+]o. 5. The calcium channel blockers verapamil and nifedipine and inhibitors of phospholipase C (neomycin and U-73122) abolished the [Ca2+]i oscillations induced by CCh; Li+ accelerated the onset of the [Ca2+]i oscillations induced by CCh. 6. These observations suggest that the mechanisms responsible for the [Ca2+]i oscillations induced by CCh and high [Ca2+]o are different from each other. Other than an increase in extracellular Ca2+ influx as a mechanism common for both CCh- and high [Ca2+]o-induced [Ca2+]i oscillations, the CCh-induced [Ca2+]i oscillations involve influx of Ca2+ via L-type Ca2+ channels, Na+-Ca2+ exchange, mobilization of intracellular Ca2+ and IP3 production.     

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.     

Changes in pattern of heat loss at high ambient temperature caused by water deprivation in a large flightless bird, the emu

We investigated the mechanism by which H2O2 mediates an increase in [Na+]i in L929 cells and the relevance of this Na+ load for H2O2-induced cell injury. [Na+]i increased early after exposure to H2O2 as monitored by fluorescence spectrophotometry of cells loaded with SBFI. The omission of Na+ from the incubation buffer significantly reduced H2O2-cytotoxicity. This protection could not be mimicked by inhibition of either the Na+/H+-antiporter, the Na+/HCO3- -cotransporter, or the Na+/K+/2Cl- -cotransporter by using Hoechst 694 (0.02 mM) or 4-acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid (SITS) (0.02 mM) or furosemide (1 mM) and bumetanide (0.5 mM). Only the blocker of the Na+/Ca2+-exchanger bepridil (0.2 mM) significantly reduced H2O2-cytotoxicity but without interfering with the increase in [Na+]i. H2O2 caused a rapid and sustained increase in [Ca2+]i, which was significantly reduced in bepridil pretreated cells and after replacing extracellular Na+ by choline. H2O2 was found to initiate a cellular uptake of unphysiological Ni2+ by using Newport Green diacetate as fluorescent dye. Our data suggest that H2O2 mediates Na+-influx across the plasma membrane rather unspecifically than through specific transporters. The protective effect of bepridil against H2O2-cytotoxicity occurs as a consequence of a reduced cellular Ca2+-uptake. We conclude that H2O2-mediated unspecific accumulation of Na+ seems to favor a Ca2+-influx into the cells, which takes place on the Na+/Ca2+-exchanger operating in reverse mode in exchange for Na+-efflux. Therefore, H2O2-induced cellular Na+ accumulation appears to play a permissive rather than a triggering role in H2O2-mediated cell injury.     

The protective action of tetrodotoxin and (+/-)-kavain on anaerobic glycolysis, ATP content and intracellular Na+ and Ca2+ of anoxic brain vesicles

Because recent reports point to Na+ channel blockers as protective agents directed against anoxia-induced neuronal damage including protection of anaerobic glycolysis, the influences of tetrodotoxin (TTX) and (+/-)-kavain on anoxic rat brain vesicles were investigated with respect to lactate synthesis, vesicular ATP content and cytosolic free Na+ and Ca2+ ([Na+]i, [Ca2+]i), both of the latter determined fluorometrically employing SBFI and FURA-2, respectively. After anoxia, basal lactate production was increased from 2.9 to 9.8 nmol lactate/min/mg protein. Although lactate synthesis seemed to be stable for at least 45 min of anoxia, as deduced from the linearity of lactate production, the ATP content declined continuously with a half life (tau 1/2) of 14.5 min, indicating that anaerobic glycolysis was insufficient to cover the energy demand of anoxic vesicles. Correspondingly, [Na+]i and [Ca2+]i increased persistently after anoxia by 22.1 mmol/l Na+ and 274.9 nmol/l Ca2+, determined 6.3 min after onset. An additional stimulation of vesicles with veratridine accelerated the drop of ATP (tau 1/2 = 5.1 min) and provoked a massive Na+ overload, which levelled off to 119 mmol/l Na+ within a few minutes. Concomitantly, [Ca2+]i increased linearly with a rate of 355 nmol Ca2+/l/min. Despite the massive perturbation of ion homeostasis, lactate production was unaffected during the first 8 min of veratridine stimulation. However, complete inhibition of lactate synthesis took place 30 min after veratridine was added. The Na+ channel blockers TTX and (+/-)-kavain, if applied before anoxia, preserved vesicular ATP content, diminished anoxia-induced increases in [Na+]i and [Ca2+]i and prevented both the veratridine-induced increases of [Na+]i and [Ca2+]i and the inhibition of lactate production. The data indicate a considerable Na+ influx via voltage-dependent Na+ channels during anoxia, which speeds up the decline in ATP and provokes an increase in [Ca2+]i. A massive Na+ and Ca2+ overload induced by veratridine failed to influence lactate synthesis directly, but initiated its inhibition.     

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.     

P2 receptor-mediated signal transduction in Ehrlich ascites tumor cells

The mechanisms, by which the P2 receptor agonists adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP) evoke an increase in the free cytosolic calcium concentration ([Ca2+]i) and in intracellular pH (pHi), have been investigated in Ehrlich ascites tumor cells. The increase in [Ca2+]i evoked by ATP or UTP is abolished after depletion of intracellular Ca2+ stores with thapsigargin in Ca2+-free medium, and is inhibited by U73122, an inhibitor of phospholipase C (PLC), indicating that the increase in [Ca2+]i is primarily due to release from intracellular, Ins(1,4,5)P3-sensitive Ca2+ stores. ATP also activates a capacitative Ca2+-entry pathway. ATP as well as UTP evokes a biphasic change in pHi, consisting of an initial acidification followed by alkalinization. Suramin and 4,4'-diisothiocyano-2,2'-stilbene-disulfonic acid (DIDS) inhibit the biphasic change in pHi, apparently by acting as antagonists at P2 receptors. The alkalinization evoked by the P2 receptor agonists is found to be due to activation of a 5'-(N-ethyl-N-isopropyl)amiloride (EIPA)-sensitive Na+/H+ exchanger. ATP and UTP elicit rapid cell shrinkage, presumably due to activation of Ca2+ sensitive K+ and Cl- efflux pathways. Preventing cell shrinkage, either by incubating the cells at high extracellular K+ concentration, or by adding the K+-channel blocker, charybdotoxin, does not affect the increase in [Ca2+]i, but abolishes the activation of the Na+/H+ exchanger, indicating that activation of the Na+/H+ exchanger is secondary to the Ca2+-induced cell shrinkage.     

Parameters of lymphocyte Na+-Ca2+ regulation and blood pressure: the gender effect

Alterations in cellular Ca2+ and Na+ regulation play a role in the pathogenesis of essential hypertension. Using peripheral lymphocytes from 68 normal persons, we observed the following relationships for major cellular Ca2+ regulatory parameters. Among men and women, Na+-Ca2+ exchanger activity was positively correlated with the resting cytosolic free Ca2+ ([Ca2+]c) (r=0.43, P=0.0003), and the resting [Ca2+]c was positively correlated with cytosolic Na+ ([Na+]c) (r=0.50, P=0.0001). For men only, store-operated Ca2+ entry was positively correlated with Na+-Ca2+ exchanger activity (r=0.63, P=0.0001). In addition, systolic and diastolic blood pressures were positively correlated with [Na+]c in men (r=0.53, P=0.001, and r=0. 41, P=0.017, respectively) but not in women (r=0.30, P=0.088, and r=0.24, P=0.17, respectively). Some of the relationships between cellular and blood pressure parameters were confounded by serum triglycerides. These observations indicate a gender effect on cellular Ca2+-Na+ regulation and its relationship with blood pressure.     

Calcium homeostasis in rat septal neurons in tissue culture

Septal neurons from embryonic rats were grown in tissue culture. Microfluorimetric and electrophysiological techniques were used to study Ca2+ homeostasis in these neurons. The estimated basal intracellular free ionized calcium concentration ([Ca2+]i) in the neurons was low (50-100 nM). Depolarization of the neurons with 50 mM K+ resulted in rapid elevation of [Ca2+]i to 500-1,000 nM showing recovery to baseline [Ca2+]i over several minutes. The increases in [Ca2+]i caused by K+ depolarization were completely abolished by the removal of extracellular Ca2+, and were reduced by approximately 80% by the 'L-type' Ca2+ channel blocker, nimodipine (1 microM). [Ca2+]i was also increased by the excitatory amino acid L-glutamate, quisqualate, AMPA and kainate. Responses to AMPA and kainate were blocked by CNQX and DNQX. In the absence of extracellular Mg2+, large fluctuations in [Ca2+]i were observed that were blocked by removal of extracellular Ca2+, by tetrodotoxin (TTX), or by antagonists of N-methyl D-aspartate (NMDA) such as 2-amino 5-phosphonovalerate (APV). In zero Mg2+ and TTX, NMDA caused dose-dependent increases in [Ca2+]i that were blocked by APV. Caffeine (10 mM) caused transient increases in [Ca2+]i in the absence of extracellular Ca2+, which were prevented by thapsigargin, suggesting the existence of caffeine-sensitive ATP-dependent intracellular Ca2+ stores. Thapsigargin (2 microM) had little effect on [Ca2+]i, or on the recovery from K+ depolarization. Removal of extracellular Na+ had little effect on basal [Ca2+]i or on responses to high K+, suggesting that Na+/Ca2+ exchange mechanisms do not play a significant role in the short-term control of [Ca2+]i in septal neurons. The mitochondrial uncoupler, CCCP, caused a slowly developing increase in basal [Ca2+]i; however, [Ca2+]i recovered as normal from high K+ stimulation in the presence of CCCP, which suggests that the mitochondria are not involved in the rapid buffering of moderate increases in [Ca2+]i. In simultaneous electrophysiological and microfluorimetric recordings, the increase in [Ca2+]i associated with action potential activity was measured. The amplitude of the [Ca2+]i increase induced by a train of action potentials increased with the duration of the train, and with the frequency of firing, over a range of frequencies between 5 and 200 Hz. Recovery of [Ca2+]i from the modest Ca2+ loads imposed on the neuron by action potential trains follows a simple exponential decay (tau = 3-5 s).     

Effects of chronic run training on Na+-dependent Ca2+ efflux from rat left ventricular myocytes

The effects of endurance run training on Na+-dependent Ca2+ regulation in rat left ventricular myocytes were examined. Myocytes were isolated from sedentary and trained rats and loaded with fura 2. Contractile dynamics and fluorescence ratio transients were recorded during electrical pacing at 0.5 Hz, 2 mM extracellular Ca2+ concentration, and 29 degreesC. Resting and peak cytosolic Ca2+ concentration ([Ca2+]c) did not change with exercise training. However, resting and peak [Ca2+]c increased significantly in both groups during 5 min of continuous pacing, although diastolic [Ca2+]c in the trained group was less susceptible to this elevation of intracellular Ca2+. Run training also significantly reduced the rate of [Ca2+]c decay during relaxation. Myocytes were then exposed to 10 mM caffeine in the absence of external Na+ or Ca2+ to trigger sarcoplasmic reticular Ca2+ release and to suppress cellular Ca2+ efflux. This maneuver elicited an elevated steady-state [Ca2+]c. External Na+ was then added, and the rate of [Ca2+]c clearance was determined. Run training significantly reduced the rate of Na+-dependent clearance of [Ca2+]c during the caffeine-induced contractures. These data demonstrate that the removal of cytosolic Ca2+ was depressed with exercise training under these experimental conditions and may be specifically reflective of a training-induced decrease in the rate of cytosolic Ca2+ removal via Na+/Ca2+ exchange and/or in the amount of Ca2+ moved across the sarcolemma during a contraction.     

Effect of human recombinant erythropoietin (Epo) on cellular Ca2+, Na+, and K+ regulation of vascular smooth muscle cells grown in vitro--a new insight into the pathogenesis of Epo induced hypertension

To gain an insight into the effect of erythropoietin (Epo) upon cation transporters and cytosolic free Ca2+ concentration ([Ca2+]i) of vascular smooth muscle cells (VSMC), we studied whether 1) Epo, per se, alters Ca2+ Na+, K+ fluxes and [Ca2+]i of VSMC, and 2) Epo may modify the effect of endothelin (ET-1). Using serially passaged quiescent cultured VSMC, the following results were obtained. 1) Epo had no direct effect on steady state Na(+)-K+ transporters (Na(+)-K+ pump, Na(+)-K+ cotransport and Na(+)-H+ antiport). 2) ET-1 alone substantially stimulated Na(+)-K+ pump, Na(+)-H+ antiport and 45Ca uptake, although these effects were not potentiated in the presence of Epo. 3) Epo alone substantially stimulated 45Ca uptake, leading to an increase in [Ca2+]i, which effect was not seen in Ca2+ deficient medium, and was partially inhibited with diltiazem but not with TMB-8. 4) Even in the presence of Epo, ET-1 and angiotensin II (A II) had substantial stimulatory effect on [Ca2+]i of cultured VSMC. The present data indicate that Epo, per se, elicits an increase in [Ca2+]i of VSMC through the stimulation of inward Ca2+ flux without affecting Na(+)-K+ transporters. In contrast, Epo did not potentiate ET-1's stimulatory effect on the transporters. Although the effect of Epo was subtle compared to ET-1 and A II, it may alter an overall characteristic of vascular smooth muscle cell contractility, possibly leading to blood pressure elevation in patients on maintenance dialysis.     

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.     

Spontaneous electrical and calcium oscillations in unstimulated pituitary gonadotrophs

Single pituitary cells often fire spontaneous action potentials (APs), which are believed to underlie spiking fluctuations in cytosolic calcium concentration ([Ca2+]i). To address how these basal [Ca2+]i fluctuations depend on changes in plasma membrane voltage (V), simultaneous measurements of V and [Ca2+]i were performed in rat pituitary gonadotrophs. The data show that each [Ca2+]i spike is produced by the Ca2+ entry during a single AP. Using these and previously obtained patch-clamp data, we develop a quantitative mathematical model of this plasma membrane oscillator and the accompanying spatiotemporal [Ca2+]i oscillations. The model demonstrates that AP-induced [Ca2+]i spiking is prominent only in a thin shell layer neighboring the cell surface. This localized [Ca2+]i spike transiently activates the Ca2(+)- dependent K+ current resulting in a sharp afterhyperpolarization following each voltage spike. In accord with experimental observations, the model shows that the frequency and amplitude of the voltage spikes are highly sensitive to current injection and to the blocking of the Ca(2+)-sensitive current. Computations also predict that leaving the membrane channels intact, the firing rate can be modified by changing the Ca2+ handling parameters: the Ca2+ diffusion rate, the Ca2+ buffering capacity, and the plasma membrane Ca2+ pump rate. Finally, the model suggests reasons that spontaneous APs were seen in some gonadotrophs but not in others. This model provides a basis for further exploring how plasma membrane electrical activity is involved in the control of cytosolic calcium level in unstimulated as well as agonist-stimulated gonadotrophs.     

Long-term activation of capacitative Ca2+ entry in mouse microglial cells

The cytoplasmic free calcium concentration ([Ca2+]i) was measured in cultured microglial cells with the Ca2+-sensitive fluorescent dye Fura-2 using a digital imaging system. Stimulation of P2 purinergic receptors by ATP or UTP always evoked a [Ca2+]i elevation. The ATP-induced Ca2+ response involved both Ca2+ influx through ionotropic receptors and Ca2+ release from intracellular pools, whereas UTP selectively stimulated intracellular Ca2+ release. When intracellular Ca2+ release was stimulated in the absence of extracellular Ca2+, the readmission of extracellular Ca2+ caused a large rebound [Ca2+]i increase. Following this rebound, [Ca2+]i did not return to the initial resting level, but remained for long periods of time (up to 20 min), at a new, higher steady-state level. Both the amplitude of the rebound Ca2+ transient and the new plateau level strongly correlated with the degree of intracellular Ca2+ depletion, indicating the activation of a store-operated Ca2+ entry pathway. The elevated steady-state [Ca2+]i level was associated with a significant increase in the plasma membrane permeability to Ca2+, as changes in extracellular Ca2+ were reflected in almost immediate changes of [Ca2+]i. Similarly, blocking plasma-lemmal Ca2+ channels with the non-specific agonist La3+ (50 microM) caused a decrease in [Ca2+]i, despite the continuous presence of Ca2+ ions in the extracellular medium. After the establishment of the new, elevated steady-state [Ca2+]i level, stimulation of P2U metabotropic purinoreceptors did not induce a [Ca2+]i response. In addition, application of either thapsigargin (1 microM) or carbonyl cyanide chlorophenyl hydrazone (10 microM) failed to affect [Ca2+]i. We conclude that the maximal depletion of intracellular Ca2+ stores in mouse brain microglia determines the long-term activation of a plasma membrane Ca2+ entry pathway. This activation appears to be associated with a significant decrease in the capability of the intracellular Ca2+ stores to take up cytosolic Ca2+ once they have been maximally depleted.     

A prototypic intracellular calcium antagonist, TMB-8, protects cultured cerebellar granule cells against the delayed, calcium-dependent component of glutamate neurotoxicity

The effect(s) of a prototypic intracellular Ca2+ antagonist, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), on glutamate-induced neurotoxicity was investigated in primary cultures of mouse cerebellar granule cells. Glutamate evoked an increase in cytosolic free-Ca2+ levels ([Ca2+]i) that was dependent on the extracellular concentration of Ca2+ ([Ca2+]o). In addition, this increase in [Ca2+]i correlated with a decrease in cell viability that was also dependent on [Ca2+]o. Glutamate-induced toxicity, quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining, was shown to comprise two distinct components, an "early" Na+/Cl(-)-dependent component observed within minutes of glutamate exposure, and a "delayed" Ca(2+)-dependent component (ED50 approximately 50 microM) that coincided with progressive degeneration of granule cells 4-24 h after a brief (5-15 min) exposure to 100 microM glutamate. Quantitative analysis of cell viability and morphological observations identify a "window" in which TMB-8 (at > 100 microM) protects granule cells from the Ca(2+)-dependent, but not the Na+/Cl(-) -dependent, component of glutamate-induced neurotoxic damage, and furthermore, where TMB-8 inhibits glutamate-evoked increases in [Ca2+]i. These findings suggest that Ca2+ release from a TMB-8-sensitive intracellular store may be a necessary step in the onset of glutamate-induced excitotoxicity in granule cells. However, these conclusions are compromised by additional observations that show that TMB-8 (1) exhibits intrinsic toxicity and (2) is able to reverse its initial inhibitory action on glutamate-evoked increases in [Ca2+]i and subsequently effect a pronounced time-dependent potentiation of glutamate responses. Dantrolene, another putative intracellular Ca2+ antagonist, was completely without effect in this system with regard to both glutamate-evoked increases in [Ca2+]i and glutamate-induced neurotoxicity.