Regulation of the plasma membrane Ca2+ pump by cyclic nucleotides in cultured vascular smooth muscle cells

We investigated the mechanisms of Ca2+ extrusion from cultured rat aortic smooth muscle cells while monitoring changes in the cytosolic Ca2+ concentration ([Ca2+]i) using fura 2 fluorescence. 45Ca2+ efflux from these cells consisted of two major mechanisms; one was dependent on the extracellular sodium concentration (Na+o) and the other was independent of Na+o. Na+o-dependent efflux increased monotonically with increasing [Ca2+]i between 0.1 and 1.0 microM, whereas Na+o-independent efflux reached a plateau at 0.6-1 microM [Ca2+]i with a half-maximum obtained at about 0.16 microM. At [Ca2+]i below 1 microM, the latter was significantly greater than the former. Unlike the Na+o-dependent mechanism, Na+o-independent 45Ca2+ efflux was inhibited almost entirely by extracellularly added La3+ or a combination of high extracellular pH (pH 8.8) and 20 mM Mg2+. It was also inhibited, although not completely, by compound 48/80, a calmodulin antagonist, and vanadate. These results strongly suggest that Na+o-dependent and Na+o-independent 45Ca2+ effluxes occur via the Na+/Ca2+ exchanger and the ATP-dependent Ca2+ pump, respectively. Sodium nitroprusside and atrial natriuretic factor, which are agents that stimulate intracellular production of cGMP, and 8-BrcGMP significantly accelerated the Na+o-independent 45Ca2+ efflux especially at low [Ca2+]i. Forskolin, dibutyryl cAMP, and 8-Br-cAMP, however, showed no stimulation. These results suggest that the plasma membrane Ca2+ pump is regulated by cGMP but not by cAMP in intact vascular smooth muscle cells.     

Cyclic GMP stimulates Na+/Ca2+ exchange in vascular smooth muscle cells in primary culture.

We examined the effect of cGMP on Na+/Ca2+ exchange in rat aortic smooth muscle cells (VSMCs) in primary culture. The intracellular Ca2+ concentration [( Ca2+]i) was raised by adding ionomycin to VSMCs incubated at high extracellular pH (pH0) (pH0 = 8.8) and high extracellular Mg2+ (Mg2+0) (Mg2+0 = 20 mM), conditions that inhibit activity of the sarcolemmal Ca2+ pump. 45Ca2+ efflux observed under these conditions was mostly extracellular Na+ (Na+0)-dependent and thus presumably catalyzed by the Na+/Ca2+ exchanger. Brief treatment of VSMCs with 8-bromo-cGMP or atrial natriuretic peptide increased this Na+0-dependent 45Ca2+ efflux by about 50%. The 8-bromo-cGMP treatment did not significantly influence total cell Na+, membrane potential, and cell pH. Conversely, when VSMCs were loaded with Na+ and then exposed to a Na+0-free medium, the rate of 45Ca2+ uptake into VSMCs increased as cell Na+ increased. Prior treatment of VSMCs with 8-bromo-cGMP accelerated 45Ca2+ uptake by up to 60% without influencing Na+ loading itself. Treatment of VSMCs with 25 microM 2,5-di-(tert-butyl)-1,4-benzohydroquinone, an inhibitor of the sarcoplasmic reticulum Ca(2+)-ATPase, induced a transient elevation of [Ca2+]i. 8-Bromo-cGMP stimulated the rate of recovery phase of this Ca2+ transient measured in the high pHo/high Mg2+o medium. All these results indicate that cGMP stimulates Na+/Ca2+ exchange in VSMCs.     

Modulation of plasma membrane Ca2+ pump by membrane potential in cultured vascular smooth muscle cells

We examined the effect of membrane potential (Em) on the activity of the plasma membrane Ca2+ pump in cultured rat aortic smooth muscle cells (VSMCs). Inside-negative K+ diffusion potential higher or lower than the resting Em (-46 mV) was artificially imposed on VSMCs with various concentrations of extracellular K+ (K+o) and 1 microM valinomycin. We found that the recovery phase of the intracellular Ca2+ transient elicited with 1 microM ionomycin was accelerated by depolarizing Em, whereas it was retarded by hyperpolarizing Em. The rate of extracellular Na+ (Na+o)-independent 45Ca2+ efflux from VSMCs stimulated with 1 microM ionomycin increased almost linearly with a change in Em from -98 to -3 mV. This effect of Em was abolished by extracellularly added LaCl3 or a combination of high pH (pH 8.8) and high Mg2+ (20 mM), conditions that presumably inhibit the plasma membrane Ca2+ pump (Furukawa, K.-I., Tawada, Y., & Shigekawa, M. (1988) J. Biol. Chem. 263, 8058-8065). Intracellular contents of Na+ and K+ and intracellular pH, on the other hand, were not influenced by the change in Em under the conditions used. These results indicate that alteration in Em can modulate the intracellular Ca2+ concentration in intact VSMCs by changing the rate of Ca2+ extrusion by the plasma membrane Ca2+ pump. The data strongly suggest that the plasma membrane Ca2+ pump in VSMCs is electrogenic.     

Kinetics and stoichiometry of coupled Na efflux and Ca influx (Na/Ca exchange) in barnacle muscle cells

Coupled Na+ exit/Ca2+ entry (Na/Ca exchange operating in the Ca2+ influx mode) was studied in giant barnacle muscle cells by measuring 22Na+ efflux and 45Ca2+ influx in internally perfused, ATP-fueled cells in which the Na+ pump was poisoned by 0.1 mM ouabain. Internal free Ca2+, [Ca2+]i, was controlled with a Ca-EGTA buffering system containing 8 mM EGTA and varying amounts of Ca2+. Ca2+ sequestration in internal stores was inhibited with caffeine and a mitochondrial uncoupler (FCCP). To maximize conditions for Ca2+ influx mode Na/Ca exchange, and to eliminate tracer Na/Na exchange, all of the external Na+ in the standard Na+ sea water (NaSW) was replaced by Tris or Li+ (Tris-SW or LiSW, respectively). In both Na-free solutions an external Ca2+ (Cao)-dependent Na+ efflux was observed when [Ca2+]i was increased above 10(-8) M; this efflux was half-maximally activated by [Ca2+]i = 0.3 microM (LiSW) to 0.7 microM (Tris-SW). The Cao-dependent Na+ efflux was half-maximally activated by [Ca2+]o = 2.0 mM in LiSW and 7.2 mM in Tris-SW; at saturating [Ca2+]o, [Ca2+]i, and [Na+]i the maximal (calculated) Cao-dependent Na+ efflux was approximately 75 pmol#cm2.s. This efflux was inhibited by external Na+ and La3+ with IC50's of approximately 125 and 0.4 mM, respectively. A Nai-dependent Ca2+ influx was also observed in Tris-SW. This Ca2+ influx also required [Ca2+]i greater than 10(-8) M. Internal Ca2+ activated a Nai-independent Ca2+ influx from LiSW (tracer Ca/Ca exchange), but in Tris-SW virtually all of the Cai-activated Ca2+ influx was Nai-dependent (Na/Ca exchange). Half-maximal activation was observed with [Na+]i = 30 mM. The fact that internal Ca2+ activates both a Cao-dependent Na+ efflux and a Nai-dependent Ca2+ influx in Tris-SW implies that these two fluxes are coupled; the activating (intracellular) Ca2+ does not appear to be transported by the exchanger. The maximal (calculated) Nai-dependent Ca2+ influx was -25 pmol/cm2.s. At various [Na+]i between 6 and 106 mM, the ratio of the Cao-dependent Na+ efflux to the Nai-dependent Ca2+ influx was 2.8-3.2:1 (mean = 3.1:1); this directly demonstrates that the stoichiometry (coupling ratio) of the Na/Ca exchange is 3:1. These observations on the coupling ratio and kinetics of the Na/Ca exchanger imply that in resting cells the exchanger turns over at a low rate because of the low [Ca2+]i; much of the Ca2+ extrusion at rest (approximately 1 pmol/cm2.s) is thus mediated by an ATP-driven Ca2+ pump.(ABSTRACT TRUNCATED AT 400 WORDS)     

Acetylcholine-evoked increase in the cytoplasmic Ca2+ concentration and Ca2+ extrusion measured simultaneously in single mouse pancreatic acinar cells.

The intracellular free Ca2+ concentration ([free Ca2+]i) was measured simultaneously with the Ca2+ extrusion from single isolated mouse pancreatic acinar cells placed in a microdroplet of extracellular solution using the fluorescent probes fura-2 and fluo-3. The extracellular solution had a low total calcium concentration (15-35 microM), and acetylcholine (ACh), applied by microionophoresis, therefore only evoked a transient elevation of [free Ca2+]i lasting about 2-5 min. The initial sharp rise in [free Ca2+]i from about 100 nM toward 0.5-1 microM was followed within seconds by an increase in the total calcium concentration in the microdroplet solution ([Ca]o). The rate of this rise of [Ca]o was dependent on the [free Ca2+]i elevation, and as [free Ca2+]i gradually decreased Ca2+ extrusion declined with the same time course. Ca2+ extrusion following ACh stimulation was not influenced by removal of all Na+ in the microdroplet solution indicating that the Ca2+ extrusion is not mediated by Na(+)-Ca2+ exchange but by the Ca2+ pump. The amount of Ca2+ extruded during the ACh-evoked transient rise in [free Ca2+]i corresponded to a decrease in the total intracellular Ca concentration of about 0.7 mM which is close to previously reported values (0.5-1 mM) for the total concentration of mobilizable calcium in these cells. Our results therefore demonstrate directly the ability of the Ca2+ pump to rapidly remove the large amount of Ca2+ released from the intracellular pools during receptor activation.     

Effect of thapsigargin on cytosolic Ca2+ level and amylase release in rat parotid acinar cells.

At concentrations greater than 0.01 microM, thapsigargin (ThG) dose-dependently caused an increase in cytosolic free Ca2+ concentration ([Ca2+]i) in rat parotid acinar cells, as measured by the fluorescent Ca(2+)-indicator fura-2. In the absence of extracellular Ca2+, a transient increase in [Ca2+]i by ThG was observed, and subsequent addition of carbachol (CCh) did not produce a further [Ca2+]i response, suggesting that ThG released Ca2+ from the CCh-sensitive intracellular Ca2+ pool. Since ThG did not stimulate formation of inositol phosphates, the ThG-induced Ca2+ mobilization is independent of phosphoinositide breakdown. High concentrations (greater than 0.1 microM) of ThG induced amylase release from rat parotide acini, but the effect was very poor as compared with that of CCh or the protein kinase C activator, PMA (phorbol 12-myristate 13-acetate). Combined addition of ThG and PMA modestly potentiated amylase release induced by PMA alone. These results support the view that amylase release by muscarinic stimulation is mediated mainly by activation of protein kinase C rather than a rise in [Ca2+]i, although Ca2+ may modulate the secretory response.     

Interaction between beta-adrenergic signaling and protein kinase C increases cytoplasmic Ca2+ in alveolar type II cells

The interaction between beta-adrenergic signaling and the activation of protein kinase C in alveolar type II cell plays an important role in the regulation of surfactant secretion because the combined application of beta-adrenergic agonist with protein kinase C activator to the cells stimulates the secretion synergistically. However, the mechanisms underlying the interaction are not clear. In the present study, we examined the combined effect of terbutaline with phorbol 12-myristate 13-acetate (PMA) on cytoplasmic free Ca2+ concentration ([Ca2+]i) in rat alveolar type II cells. The combined application of terbutaline with PMA to the cells rapidly increased [Ca2+]i, although neither of them affected it by itself. Similar increases of [Ca2+]i were observed in other combinations, such as terbutaline with 1-oleoyl-2-acetyl-sn-glycerol, and forskolin with PMA. Either the removal of extracellular Ca2+ or the addition of Co2+ remarkably suppressed the increase of [Ca2+]i induced by the combination of terbutaline with PMA. In addition, Co2+ inhibited the phosphatidylcholine secretion induced by the combination of terbutaline and PMA. These results suggested that the [Ca2+]i increased as a result of the interaction between formation of cyclic AMP and activation of protein kinase C in alveolar type II cells, and that the increase in [Ca2+]i was mediated by the Ca2+ influx through the plasma membrane. This mechanism to modulate [Ca2+]i may play a role in the regulation of surfactant secretion by alveolar type II cells.     

Effects of calcium, calmodulin, protein kinase C and protein tyrosine kinases on volume-activated taurine efflux in human erythroleukemia cells.

The effects of calcium, calmodulin, protein kinase C (PKC) and protein tyrosine kinase (PTK) modulators were examined on the volume-activated taurine efflux in the erythroleukemia cell line K562. Exposure to hypoosmotic solution significantly increased taurine efflux and intracellular calcium concentration ([Ca2+]i). The Ca2+ channel blockers La3+ (1 mM), verapamil (200 microM) and nifedipine (100 microM) inhibited the hypoosmotically-induced [Ca2+]i increase by more than 90%, while the volume-activated taurine efflux was inhibited by 61.3 +/- 9.5, 74.1 +/- 9.3 and 38.0 +/- 1.5%, respectively. Furthermore, the calmodulin inhibitors W7 (50 microM) and trifluoperazine (10 microM) and the Ca2+/calmodulin-dependent protein kinase II inhibitor KN-62 (2 microM) significantly blocked the volume-activated taurine efflux by 93.4 +/- 2.7, 77.9 +/- 3.5 and 61.3 +/- 15.8%, respectively. In contrast, the PKC inhibitor staurosporine (200 nM) or the PKC activator phorbol 12-myristate 13-acetate (100 nM) did not have significant effects on the volume-activated taurine efflux. However, pretreatment with PTK inhibitors genistein, tyrphostin A25, and tyrphostin A47 blocked the volume-activated taurine efflux. These results suggest that the volume-activated taurine efflux in K562 cells may not directly involve Ca2+, but may require the presence of calmodulin and/or PTK.     

Inhibition of bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilisation by phorbol ester in rat cultured vascular smooth muscle cells

Regulation of the increase in inositol phosphate (IP) production and intracellular Ca2+ concentration ([Ca2+]i by protein kinase C (PKC) was investigated in cultured rat vascular smooth muscle cells (VSMCs). Pretreatment of VSMCs with phorbol 12-myristate 14-acetate (PMA, 1 microM) for 30 min almost abolished the BK-induced IP formation and Ca2+ mobilisation. This inhibition was reduced after incubating the cells with PMA for 4 h, and within 24 h the BK-induced responses were greater than those of control cells. The concentrations of PMA giving a half-maximal (pEC50) and maximal inhibition of BK induced an increase in [Ca2+]i, were 7.8 +/- 0.3 M and 1 microM, n = 8, respectively. Prior treatment of VSMCs with staurosporine (1 microM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Paralleling the effect of PMA on the BK-induced IP formation and Ca2+ mobilisation, the translocation and downregulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of the cells with PMA for various times, translocation of PKC-alpha, betaI, betaII, delta, epsilon, and zeta isozymes from the cytosol to the membrane were seen after 5 min, 30 min, 2 h, and 4 h of treatment. However, 24-h treatment caused a partial downregulation of these PKC isozymes in both fractions. Treatment of VSMCs with 1 microM PMA for either 1 or 24 h did not significantly change the K(D) and Bmax of the BK receptor for binding (control: K(D) = 1.7 +/- 0.2 nM; Bmax = 47.3 +/- 4.4 fmol/mg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. In conclusion, these results demonstrate that translocation of PKC-alpha, betaI, betaII, delta, epsilon, and zeta induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca2+ mobilisation in VSMCs.     

Mechanisms of lysophosphatidic acid-induced increase in intracellular calcium in vascular smooth muscle cells

Although lysophosphatidic acid (LPA) is known to cause an increase in intracellular Ca2+ concentration ([Ca2+]i) in vascular smooth muscle cells (VSMCs), the mechanisms of [Ca2+]i mobilization by LPA are not fully understood. In the present study, the effect of LPA on [Ca2+]i mobilization in cultured A10 VSMCs was examined by Fura-2 fluorescence technique. The expression of LPA receptors was studied by immunostaining. LPA was observed to increase [Ca2+]i in a concentration-dependent manner; this increase was dependent on the concentration of extracellular Ca2+. Both sarcolemmal (SL) Na(+)-Ca2+ exchange inhibitors (amiloride, Ni2+ and KB-R7943) and Na(+)-H+ exchange inhibitor (MIA) as well as SL store-operated Ca2+ channel (SOC) antagonists (SK&F 96365, tyrphostin A9 and gadolinium), unlike SL Ca2+ channel antagonists (verapamil and diltiazem), inhibited the LPA-induced increase in [Ca2+]i. In addition, sarcoplasmic reticulum (SR) Ca2+ channel blocker (ryanodine), SR Ca2+ channel opener (caffeine), SR Ca2+ pump ATPase inhibitor (thapsigargin) and inositol 1,4,5-trisphosphate (InsP3) receptor antagonists (xestospongin and 2-aminoethoxydiphenyl borate) were found to inhibit the LPA-induced Ca2+ mobilization. Furthermore, phospholipase C (PLC) inhibitor (U 73122) and protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate) attenuated the LPA-induced increase in [Ca2+]i. These results indicate that Ca2+ mobilization by LPA involves extracellular Ca2+ entry through SL Na(+)-Ca2+ exchanger, Na(+)-H+ exchanger and SL SOCs. In addition, ryanodine-sensitive and InsP(3)-sensitive intracellular Ca2+ pools may be associated with the LPA-induced increase in [Ca2+]i. Furthermore, the LPA-induced [Ca2+]i mobilization in VSMCs seems to be due to the activation of both PLC and PKC.     

Phorbol ester induces both gene expression and phosphorylation of the plasma membrane Ca2+ pump.

Regulation of the plasma membrane Ca2+ pump in the cell is of critical importance in maintaining calcium homeostasis. Since protein kinase C is known to regulate functions of cellular proteins by direct phosphorylation or by inducing their gene expression, we investigated the possible involvement of protein kinase C in the regulation of the plasma membrane Ca2+ pump. The Ca2+ pump was isolated by immunoprecipitation from [32P]orthophosphate-labeled cultured rat aortic endothelial cells grown in the absence or presence of phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C. PMA treatment of cells led to a rapid increase in the phosphorylation level (1.3-fold) within 5 min and a further increase to 2.9-fold after 3 h. Prolonged PMA treatment also induced the accumulation of the Ca2+ pump mRNA, followed by increased levels of the pump protein. The peak level of the pump mRNA induction occurred at 4 h and was 8-20-fold higher than the control culture without PMA. The rate of the Ca2+ pump protein accumulation was slower, reaching a maximum of 3.5-fold after 6 h. Induction of the pump mRNA was suppressed by the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and by down-regulation of protein kinase C. Inactive phorbol ester 4 alpha-phorbol didecanoate also failed to mimic the PMA effect. These results suggest that the induction of Ca2+ pump expression is mediated by a protein kinase C-dependent mechanism. Furthermore, since the induction of the Ca2+ pump mRNA was blocked when cycloheximide and PMA were added together, this suggests that newly synthesized protein factor is needed to produce the mRNA induction. Our results suggest that protein kinase C is involved in the regulation of the Ca2+ pump in endothelial cells. At the protein level, it modifies the Ca2+ pump by phosphorylation, and at the gene level, it stimulates the expression of its mRNA and thereby increases the amount of the pump protein.     

Mercury-induced Ca2+ increase and cytotoxicity in renal tubular cells

The effect of mercury (Hg2+), a known nephrotoxicant, on intracellular free Ca2+ levels ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells was explored. [Ca2+]i was measured by using the Ca2+ -sensitive dye fura-2. Hg2+ increased [Ca2+]i in a concentration-dependent manner with an EC50 of 6 microM. The Ca2+ signal comprised a gradual increase. Removal of extracellular Ca2+ decreased the Hg2+ -induced [Ca2+]i increase by 27%, suggesting that the Ca2+ signal was due to both extracellular Ca2+ influx and store Ca2+ release. In Ca2+ -free medium, the Hg2+ -induced [Ca2+]i increase was nearly abolished by pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), and conversely, pretreatment with Hg2+ abolished thapsigargin-induced Ca2+ increase. Hg2+ -induced Ca2+ release was not altered by inhibition of phospholipase C but was potentiated by activation of protein kinase C. Overnight treatment with 1 microM Hg2+ did not alter cell proliferation rate and mitochondrial activity, but 10 microM Hg2+ killed all cells. Collectively, this study shows that Hg2+ induced protein kinase C-regulated [Ca2+]i increases in renal tubular cells via releasing store Ca2+ from the endoplasmic reticulum in a manner independent of phospholipase C activity. Hg2+ also caused cytotoxicity at higher concentrations.     

Muscarinic receptor-mediated increase in cytoplasmic free Ca2+ in isolated bovine adrenal medullary cells. Effects of TMB-8 and phorbol ester TPA

The change in cytoplasmic free calcium, [Ca2+]i in isolated bovine adrenal medullary cells during stimulation by acetylcholine (ACh) in Ca2+-free incubation medium was measured using the fluorescent Ca2+ indicator quin2. ACh (1-100 microM) caused an increase in [Ca2+]i by mobilization of Ca2+ from the intracellular pool. Nicotine (10 microM) did not increase [Ca2+]i in the absence of extracellular Ca2+. Pretreatment of the cells with atropine (10 microM) completely inhibited ACh-induced increase in [Ca2+]i, whereas pretreatment with hexamethonium (100 microM) did not. The intracellular Ca2+ antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), inhibited ACh-induced increase in [Ca2+]i. The activator of protein kinase C 12-O-tetradecanoylphorbol-13-acetate (TPA), but not its 'inactive' analog 4 alpha-phorbol-12,13-didecanoate (PDD), also inhibited ACh-induced increase in [Ca2+]i. These findings suggest that in bovine adrenal medullary cells, stimulation of muscarinic ACh receptor causes an increase in [Ca2+]i by mobilizing Ca2+ from the intracellular pool and that protein kinase C is involved in 'termination' or 'down regulation' of this response.     

Calcium stimulates luteinizing-hormone (lutropin) exocytosis by a mechanism independent of protein kinase C.

Using permeabilized gonadotropes, we examined whether Ca2(+)-stimulated luteinizing-hormone (LH) exocytosis is mediated by the Ca2(+)-activated phospholipid-dependent protein kinase (protein kinase C). In the presence of high [Ca2+]free (pCa 5), alpha-toxin-permeabilized sheep gonadotropes secrete a burst of LH and then become refractory to maintained high [Ca2+]free. The protein kinase C activator phorbol myristate acetate (PMA) is able to stimulate further LH release from cells made refractory to high [Ca2+]free, suggesting that Ca2+ does not stimulate LH release by activating protein kinase C. Staurosporine, a protein kinase C inhibitor, inhibited PMA-stimulated (50% inhibition at 20 nM), but not Ca2(+)-stimulated, LH exocytosis. In cells desensitized to PMA by prolonged exposure to a high PMA concentration, Ca2(+)-stimulated LH exocytosis (when corrected for depletion of total cellular LH) was not inhibited. Ba2+ was able to stimulate LH exocytosis to a maximal extent similar to Ca2+, although higher Ba2+ concentrations were necessary. Ba2+ and Ca2+ stimulated LH exocytosis with a similar time course, and both were inhibitory at high concentrations. Furthermore, cells made refractory to Ca2+ were also refractory to Ba2+. These data strongly suggest that Ba2+ and Ca2+ act through the same mechanism. Since Ba2+ is a poor activator of protein kinase C, these findings are additional evidence against a major role for protein kinase C in mediating Ca2(+)-stimulated LH exocytosis.     

Presence of Na+/Ca2+ exchange activity and its role in regulation of intracellular calcium concentration in bovine adrenal chromaffin cells.

The presence of a Na+/Ca2+ exchanger in bovine adrenal chromaffin cells was demonstrated by measuring the efflux of 45Ca2+ which had been preloaded into cells by a brief depolarization. The efflux of 45Ca2+ was dependent on extracellular Na+ (Na+o); 45Ca2+ efflux was significantly decreased by replacing Na+o with N-methylglucamine (NMG), or Li+. Replacement of Na+o by NMG increased the resting intracellular Ca2+ concentration ([Ca2+]i) of freshly isolated chromaffin cells. This could be reversed by adding Na+, suggesting that Na+/Ca2+ exchanger activity was involved in maintaining [Ca2+]i at its resting level. The initial rate of Na(+)-dependent [Ca2+]i recovery after Ca2+ loading by depolarization was dependent on the level of [Ca2+]i. There was an apparent linear relationship between the activity of the Na+/Ca2+ exchanger and [Ca2+]i both in the presence and absence of Na+o. When cells were treated with other stimuli, including 10 microM DMPP or 40 mM caffeine, the ability of the stimulated cells to decrease [Ca2+]i was significantly reduced upon replacing Na+o with NMG. Our data show that the Na+/Ca2+ exchanger is one of the major pathways for regulating [Ca2+]i in chromaffin cells in both resting and stimulated states.     

Ca2+ dependence of transverse tubule-mediated calcium release in skinned skeletal muscle fibers

Isometric force and 45Ca efflux from the sarcoplasmic reticulum were measured at 19 degrees C in frog skeletal muscle fibers skinned by microdissection. After Ca2+ loading, application of the ionophores monensin, an Na+(K+)/H+ exchanger, or gramicidin D, an H+ greater than K+ greater than Na+ channel-former, evoked rapid force development and stimulated release of approximately 30% of the accumulated 45Ca within 1 min, whereas CCCP (carbonyl cyanide pyruvate p-trichloromethoxyphenylhydrazone), a protonophore, and valinomycin, a neutral, K+-specific ionophore, did not. When monensin was present in all bathing solutions, i.e., before and during Ca2+ loading, subsequent application failed to elicit force development and to stimulate 45Ca efflux. 5 min pretreatment of the skinned fibers with 50 microM digitoxin, a permeant glycoside that specifically inhibits the Na+,K+ pump, inhibited monensin and gramicidin D stimulation of 45Ca efflux; similar pretreatment with 100 microM ouabain, an impermeant glycoside, was ineffective. Monensin stimulation of 45Ca efflux was abolished by brief pretreatment with 5 mM EGTA, which chelates myofilament-space calcium. These results suggest that: monensin and gramicidin D stimulate Ca2+ release from the sarcoplasmic reticulum that is mediated by depolarization of the transverse tubules, which seal off after sarcolemma removal and form closed compartments; a transverse tubule membrane potential (myofilament space-negative) is maintained and/or established by the operation of the Na+,K+ pump in the transverse tubule membranes and is sensitive to the permeant inhibitor digitoxin; the transverse tubule-mediated stimulation of 45Ca efflux appears to be entirely Ca2+ dependent.     

Effect of nortriptyline on Ca2+ handling in SIRC rabbit corneal epithelial cells

To explore the effect of nortriptyline, a tricyclic antidepressant, on cytosolic free Ca2+ concentrations ([Ca2+]i) in corneal epithelial cells, [Ca2+]i levels in suspended SIRC rabbit corneal epithelial cells were measured by using fura-2 as a Ca2+-sensitive fluorescent dye. Nortriptyline at concentrations between 20-200 microM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. Nortriptyline-induced Ca2+ influx was inhibited by the store-operated Ca2+ channel blockers econazole and SK&F96365, the phospholipase A2 inhibitor aristolochic acid, and alteration of activity of protein kinase C. In Ca2+-free medium, 200 microM nortriptyline pretreatment greatly inhibited the rise of [Ca2+]i induced by the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin. Conversely, pretreatment with thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ; another endoplasmic reticulum Ca2+ pump inhibitor) nearly abolished nortriptyline-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 decreased nortriptyline-induced [Ca2+]i rise by 75%. Taken together, nortriptyline induced [Ca2+]i rises in SIRC cells by causing phospholipase C-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via store-operated Ca2+ channels.     

Differential effects of protein kinase C activators on carbamylcholine- and high K+-induced rises in intracellular free calcium concentration in cultured adrenal chromaffin cells

Pretreatment of adrenal chromaffin cells with protein kinase C activators, i.e. 12-O-tetradecanoyl phorbol-13-acetate (TPA) and 1-oleoyl 2-acetyl glycerol (OAG), partially inhibited carbamylcholine (CCh)-induced rise in intracellular free Ca2+ concentration ([Ca2+]i). The apparent IC50 values of TPA and OAG were 3 nM and 25 microM, respectively. The effect of TPA on the CCh-induced rise in [Ca2+]i was overcome by pretreatment of the cells with a protein kinase C inhibitor, 1-(5-isoquinidinesulfonyl)-2-methylpiperazine hydrochloride (H-7). In contrast, KCl-induced rise in [Ca2+]i was not affected by pretreating the cells with TPA or OAG. An inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate failed to affect the CCh-induced rise in [Ca2+]i. CCh-induced 45Ca2+ uptake was also partially inhibited by pretreatment of the cells with TPA or OAG, but KCl-induced 45Ca2+ uptake was not affected by these pretreatments. These results indicate that protein kinase C activation causes an uncoupling of signal transduction between the nicotinic receptors and Ca2+ channels.     

Protein kinase C stimulates dense tubular Ca2+ uptake in the intact human platelet by increasing the Vm of the Ca(2+)-ATPase pump: stimulation by phorbol ester, inhibition by calphostin C.

The effects of protein kinase C (PKC) on Ca2+ transport were investigated in human intact platelets. The indicator quin2 was used to measure the free cytoplasmic Ca2+ concentration ([Ca2+]cyt) and to search for possible PKC effects on the Ca(2+)-ATPase extrusion pump located in the plasma membrane. The Ca2+ indicator chlorotetracycline (CTC) was used to study PKC effects on the dense tubular Ca(2+)-ATPase uptake pump. The activity of PKC was stimulated by phorbol 12-myristate 13-acetate (PMA) and was inhibited with calphostin C. Neither PKC activation nor inhibition had any effect on [Ca2+]cyt or the Ca2+ extrusion pump. Substantial activation of the dense tubular pump was observed with PMA. In resting platelets bathed in 2 mM external Ca2+ giving [Ca2+]cyt = 102-106 nM, activation of PKC by PMA (100 nM) increases the rate and extent of dense tubular Ca2+ uptake to 1.62 +/- 0.35 and 1.25 +/- 0.3 times control value (respectively). The Vm of the dense tubular pump was measured by using ionomycin to manipulate [Ca2+]cyt. It is shown that PMA increases the Vm by a factor of 1.7 +/- 0.4 but has no effect on the Km value (= 180 nM). An unexpected finding was that PKC activity supports a portion of the basal activity of the dense tubular Ca2+ pump in resting platelets. Preincubation with the inhibitor calphostin C (100 nM) decreases the rate and extent of dense tubular Ca2+ uptake in resting platelets by 38 +/- 5% and 29 +/- 21% (respectively). This is due to a 28 +/- 9% decrease in the Vm of the dense tubular pump. This suggests that there is a low level of stimulation of dense tubular Ca2+ pump mediated by PKC in resting platelets.     

Ionomycin releases calcium from the sarcoplasmic reticulum and activates Na+/Ca2+ exchange in vascular smooth muscle cells

Ionomycin (1 microM) produced a large spike in cytosolic free Ca2+ [( Ca2+]i). The ionophore had no effect on [Ca2+]i if the sarcoplasmic reticulum had previously been Ca2+ depleted by stimulating neurohormone receptors. Ionomycin markedly increased 45Ca2+ efflux and decreased total cell Ca2+ by 60 to 70% in 1 min. Replacing extracellular Na+ [( Na+]o) with choline or N-methyl-D-glucamine strongly inhibited the effects of ionomycin on 45Ca2+ efflux and total Ca2+. Ionomycin caused similar peak increases in [Ca2+]i in the presence and absence of [Na+]o, but the exponential fall from the peak was faster in the presence of [Na+]o. Dimethylbenzamil, a potent blocker of Na+/Ca2+ exchange in these cells, strongly inhibited the effects of ionomycin on 45Ca2+ efflux and total cell Ca2+. We conclude that the increase in cytosolic free Ca2+ produced by ionomycin may be sufficient to activate the plasma membrane Na+/Ca2+ exchanger which removes Ca2+ from the cytosol and helps restore basal [Ca2+]i.