Regulation of phosphorylase A formation and calcium content in aortic smooth muscle and smooth muscle cells: effects of atrial natriuretic peptide II

Atrial natriuretic peptide II (ANP II) raises cyclic GMP and relaxes vascular smooth muscle in vitro. The manner in which ANP II relaxes vascular smooth muscle is unknown but may involve alterations in the concentration of free intracellular Ca++. To examine this possibility, changes in intracellular Ca++ were monitored in rat aortic strips using the Ca++-dependent conversion of phosphorylase b to a, while Ca++ levels and phosphorylase were measured in cultured rat aortic smooth muscle cells. ANP II produced time- and concentration-dependent decreases in phosphorylase a and tension in norepinephrine-contracted aortic strips. The decrease in the formation of phosphorylase a was accompanied by an increase in cyclic GMP content. ANP II also decreased phosphorylase a formation in K+-depolarized tissues but to a lesser extent. Agonists such as angiotensin II and arginine vasopressin, and depolarizing concentrations of K+ elevated Ca++ levels in cultured aortic cells. ANP II inhibited Ca++ accumulation to either agonists or K+, but was more effective against agonists. Phosphorylase a formation which was increased by agonists and K+ in cultured cells was also inhibited by ANP II. We conclude that phosphorylase a formation can be a useful indicator of intracellular Ca++ concentrations in smooth muscle preparations and that ANP II regulates Ca++ levels in agonist and depolarized smooth muscle, suggesting that ANP II affects mainly Ca++ removal from the cytoplasm.     

Ca++ inhibition of isoproterenol responses in mammalian skeletal muscle

In noncontracting mouse hemidiaphragms incubated in modified Krebs-Ringer--bicarbonate buffer with 10 mM Ca++, isoproterenol-stimulated phosphorylase a formation, conversion of phosphorylase kinase to the activated form, elevation of cyclic AMP-dependent protein kinase activity ratios and increase in cyclic AMP concentrations were reduced 35 to 50% over the responses in buffer with 2.5 mM Ca++. In buffer with 10 mM Ca++, the initial rate of isoproterenol-stimulated cyclic AMP accumulation was 59% of that in buffer with 2.5 mM Ca++. The inhibitory action of Ca++ on cyclic AMP accumulation was antagonized by verapamil, but not by inhibitors of cyclic nucleotide phosphodiesterase activity. In buffer with 2.5 mM Ca++, isoproterenol-stimulated cyclic AMP accumulation was inhibited by A23187 and caffeine, agents that can increase intracellular Ca++ concentrations. In addition to Ca++, high concentrations of Co++, Ni++, Mn++ and, to a lesser extent, Sr++ inhibited the isoproterenol response. The results of these studies indicate that high buffer Ca++ concentrations inhibit the response of the glycogenolytic pathway to isoproterenol by an action on cyclic AMP formation. We propose that the site of the inhibitory action of Ca++ is the divalent metal activator site associated with hormone-stimulated adenylate cyclase activity.     

Muscarine-stimulated neurotransmitter release from PC12 cells

The effect of muscarine on neurosecretion was studied in the rat pheochromocytoma cell line, PC12. When PC12 cells were exposed to muscarine the cells responded rapidly with elevation of cellular inositol trisphosphate levels, elevation of intracellular free Ca++ and release of stored transmitter. These three phenomena were totally inhibited by the muscarinic antagonist, atropine, but were unaffected by the nicotinic antagonist, d-tubocurarine. Muscarine did not stimulate the production of cyclic GMP in these cells. The muscarine-stimulated increases in inositol trisphosphate, intracellular free Ca++ and neurotransmitter release displayed similar time courses and concentration dependencies suggesting that the secretion observed may be associated with the formation of inositol trisphosphate and elevation of intracellular free Ca++. The increase in intracellular free Ca++ appeared to be due to a mobilization of Ca++ from intracellular stores inasmuch as the increase in intracellular free Ca++ was not inhibited by the voltage-dependent Ca++ channel antagonist, nifedipine, at concentrations demonstrated to block K+-induced Ca++ influx into the cells, and little or no uptake of 45Ca++ was noted when cells were stimulated with muscarine. Elevation of inositol trisphosphate, intercellular free Ca++ and stimulation of transmitter release were, however, inhibited by the absence of extracellular Ca++. The results suggest that muscarine-stimulated release of neurotransmitter may be associated with an inositol trisphosphate-induced mobilization of intracellular Ca++.     

Comparative effects of divalent cations on the methylmercury-induced alterations of acetylcholine release

Bath application of methylmercury (MeHg) at the murine neuromuscular junction blocks synchronous evoked release of acetylcholine (ACh) and then increases spontaneous release of ACh effects observed electrophysiologically as cessation of EPPS, and increased MEPP frequency (MEPPf), respectively. The objectives of the present study were to test whether the effect of MeHg on spontaneous release was Ca++-specific by substituting Sr++ or Ba++ for Ca++, whether the time course of MeHg-induced block of synchronous evoked release was altered by varying Ca++ concentrations or substituting Sr++ and whether the processes involved in the decay of elevated MEPPf after repetitive stimulation (asynchronous evoked release) were altered by MeHg. MEPPf was recorded continuously from the rat hemidiaphragm using conventional methods during pretreatment with 2 mM Ca++, 2 mM Sr++ or 0.5 mM Ba++ and subsequently with the cation plus 100 microM MeHg. The time to peak MEPPf in MeHg was not different under any condition; however, peak MEPPf was lower in Sr++ solutions than in Ca++ or Ba++ solutions. EPPs were recorded from the rat hemidiaphragm cut muscle preparation during pretreatment with either 2, 4 or 8 mM Ca++ or 2 or 4 mM Sr++ and subsequently with the cation plus 100 microM MeHg. The latency to block of the EPP in 4 and 8 mM Ca++ was not significantly different from the latency in 2 mM Ca++. The latency to block in 2 or 4 mM Sr++ was also not different from that in Ca++. In addition, under all conditions EPP amplitude remained virtually unchanged from pretreatment values until block occurred after 8 to 9 min exposure to MeHg.(ABSTRACT TRUNCATED AT 250 WORDS)     

An adenosine triphosphate-dependent calcium uptake pump in human neutrophil lysosomes.

Regulation of the cytosolic free calcium concentration is important to neutrophil function. In these studies, an ATP-dependent calcium uptake pump has been identified in human neutrophil lysosomes. This energy-dependent Ca++ uptake pump has a high affinity for Ca++ (Michaelis constant [Km] Ca++ = 107 nM) and a maximum velocity (Vmax) of 5.3 pmol/mg of protein per min. ATP was the only nucleotide that supported Ca++ uptake by lysosomes. The Km for ATP was 177 microM. ATP-dependent Ca++ uptake by neutrophil lysosomes was temperature- and pH-sensitive with optimal Ca++ pump activity at 37 degrees C and pH 7.0-7.5. Mg++ was also essential for ATP-dependent Ca++ uptake by lysosomes. Azide and antimycin A had no effect on the energy-dependent uptake of Ca++ by neutrophil lysosomes. The chemotactic peptide formyl-methionyl-leucyl-phenylalanine inhibited ATP-dependent Ca++ accumulation by isolated lysosomes. Butoxycarbonyl-phenylalanine-leucine-phenylalanine-leucine-phenylalanine , a competitive antagonist of the chemotactic peptide, blocked this inhibitory effect. These studies demonstrate the presence of an ATP-dependent Ca++ uptake pump in human neutrophil lysosomes that functions at physiologic intracellular concentrations of Ca++, ATP, and H+ and may be important to regulating neutrophil function by modulating cytosolic Ca++.     

Modulation by intracellular calcium pool of arginine vasopressin-induced cellular cyclic AMP production in rat renal papillary collecting tubule cells in culture.

The present study was undertaken to determine whether endoplasmic reticulum is involved in the cellular action of arginine vasopressin (AVP) in rat renal papillary collecting tubule cells in culture, using three dissimilar agents. 1 x 10(-7) M AVP increased cytosolic free Ca++ concentration ([Ca++]i) from 93.2 to 188.6 nM (P < .01). Exposure to 1 x 10(-4) M 3, 4, 5-trimethoxybenzoic acid, 8-(diethylamino) octylester hydrochloride (TMB-8), which inhibits intracellular Ca++ mobilization and blocks the function of endoplasmic reticulum, attenuated the [Ca++]i response to AVP. A significant increase in [Ca++]i in response to 1 x 10(-7) M AVP was obtained with Ca(++)-free medium containing 1 x 10(-4) M ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA) (52.3 to 98.3 nM). However, when cells were preincubated with Ca(++)-free medium containing a mixture of 1 x 10(-4) M EGTA and 1 x 10(-4) M TMB-8, the 1 x 10(-7) M AVP-mobilized [Ca++]i was completely abolished. In the presence of 5 x 10(-4) M 3-isobutyl-1-methylxanthine, AVP increased cellular cyclic AMP (cAMP) production in a dose-dependent manner. Such an AVP-induced cAMP production was significantly reduced in cells exposed to Ca(++)-free medium containing 1 x 10(-4) M EGTA. After exposure to Ca(++)-free medium containing a mixture of 1 x 10(-4) M EGTA and 1 x 10(-4) M TMB-8, the cAMP response to AVP was markedly reduced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of cellular Ca2+ depletion on phospholipid turnover and glycogen phosphorylase a in rat hepatocytes

The influences of changes in cellular Ca2+ level on membrane phospholipid turnover and cellular function (monitored by glycogen phosphorylase a activity) were investigated in vasopressin- and ionophore A23187-stimulated rat hepatocytes. Addition of vasopressin or A23187 to rat hepatocytes in the presence of extracellular Ca2+ enhanced the phosphorylase a activity by 3 to 4-fold within 1 min, returning to initial activity with further incubation. There was the marked generation of 1,2-diacylglycerol resulted from phospholipase C activation, which followed the transient activation of phosphorylase a. When the incorporation of [32P]phosphate into phospholipids was examined, phosphatidylinositol (PI) labeling due to vasopressin-stimulation remained rather unchanged up to 5 min but then rose gradually. On the other hand, A23187 had little effect on the incorporation into phosphatidylinositol although marked phosphatidic acid (PA) labeling was consequently produced, showing inhibitory effect on the conversion of PA to PI. Deprivation of extracellular Ca2+, which also reduced slightly the intracellular Ca2+ from 3.33 micrograms to 1.38 micrograms/10(7) cells, suppressed but not abolished stimuli-induced phosphorylase a activation without affecting the enhancement of phospholipid metabolism. Hepatocytes depleted of intracellular Ca2+ (0.50 microgram/10(7) cells) no longer showed both phosphorylase a activation and the enhancement of phospholipid metabolism. These findings seem to indicate that phosphorylase a activity is more sensitive than membrane phospholipid turnover to changes of intracellular Ca2+ concentration. The results demonstrate that marked and selective changes in membrane phospholipids depending on the type of stimulants occur upon stimulation of hepatocytes and provide the possibility that these reactions do not trigger glycogen phosphorylase a activation through Ca2+ mobilization.     

Ionic and secretory response of pancreatic islet cells to minoxidil sulfate

Minoxidil sulfate is an antihypertensive agent belonging to the new class of vasodilators, the "K+ channel openers." The present study was undertaken to characterize the effects of minoxidil sulfate on ionic and secretory events in rat pancreatic islets. The drug unexpectedly provoked a concentration-dependent decrease in 86Rb outflow. This inhibitory effect was reduced in a concentration-dependent manner by glucose and tolbutamide. Minoxidil sulfate did not affect 45Ca outflow from islets perfused in the presence of extracellular Ca++ and absence or presence of glucose. However, in islets exposed to a medium deprived of extracellular Ca++, the drug provoked a rise in 45Ca outflow. Whether in the absence or presence of extracellular Ca++, minoxidil sulfate increased the cytosolic free Ca++ concentration of islet cells. Lastly, minoxidil sulfate increased the release of insulin from glucose-stimulated pancreatic islets. These results suggest that minoxidil sulfate reduces the activity of the ATP-sensitive K+ channels and promotes an intracellular translocation of Ca++. The latter change might account for the effect of the drug on the insulin-releasing process. However, the secretory response to minoxidil sulfate could also be mediated, at least in part, by a modest Ca++ entry.     

Inositol 1,4,5-trisphosphate may regulate rat brain Cai++ by inhibiting membrane bound Na(+)-Ca++ exchanger.

The role of inositol 1,4,5-trisphosphate (1,4,5-IP3) in regulating cytosolic Ca++ by stimulating Ca++ release from intracellular organelles is well established. However, other modes of intracellular Ca++ regulation by 1,4,5-IP3 have not been determined. To determine if 1,4,5-IP3 may regulate cell cytosolic Ca++ by acting on plasma membrane bound Na(+)-Ca++ exchanger, we investigated Ca++ transport in synaptosomes using 45Ca++ as tracer. In the presence of either an inhibitor of voltage gated Na+ channels (tetrodotoxin) or the K+ ionophore (valinomycin), Ca++ uptake was significantly inhibited (P less than 0.05) by 1,4,5-IP3 in a concentration dependent manner, with half-maximal inhibition occurring at submicromolar concentrations between 10(-9) M and 10(-10) M 1,4,5-IP3. Similarly, Ca++ efflux by the exchanger was significantly inhibited 40% by 1,4,5-IP3. The inhibitory effect of 1,4,5-IP3 on the Na(+)-Ca++ exchanger was observed in the presence of Ca++ channel blockers, and in vesicles pretreated with caffeine to deplete the 1,4,5-IP3-sensitive stores of Ca++. These results suggest that during signal transduction in brain, 1,4,5-IP3 may increase cytosolic [Ca++] in part by inhibiting the Na(+)-Ca++ exchanger and thus, Ca++ efflux from cell.     

Effects of ethanol on neurotransmitter release and intracellular free calcium in PC12 cells

The effect of ethanol on muscarine-stimulated release of l-[3H]norepinephrine ([3H]NE) was studied using the rat pheochromocytoma cell line, PC12. At concentrations of 25 mM and above, ethanol produced a dose-dependent inhibition of muscarine-stimulated release of [3H]NE. The inhibition of muscarine-stimulated transmitter release occurred in the absence of any detectable effect of ethanol on [3H]NE uptake or on muscarinic binding to the cells. However, ethanol produced an inhibition of muscarine-stimulated elevation of intracellular free Ca++ which corresponded with the inhibition of transmitter release. At concentrations greater than 100 mM, ethanol produced an increase in the basal release of [3H]NE. Intracellular free Ca++ also was increased by ethanol concentrations greater than 100 mM. The elevation of basal transmitter release and intracellular free Ca++ by concentrations of ethanol greater than 100 mM occurred independently of the inhibition by ethanol of muscarine-stimulated elevation of intracellular free Ca++ and transmitter secretion. These results suggest that the effects of ethanol on neurotransmitter release are associated with the effects of ethanol on intracellular free Ca++.     

Effects of nicorandil on cytosolic calcium concentrations in quin2-loaded rat aortic vascular smooth muscle cells in primary culture

We made use of quin2 microfluorometry to observe the effects of nicorandil (2-nicotinamidoethyl nitrate) on cytosolic Ca++ concentrations [( Ca++]i) in rat aortic vascular smooth muscle cells in primary culture. Regardless of whether cells were at rest, in a state of Ca++-depletion or at K+-depolarization, nicorandil rapidly and dose-dependently decreased [Ca++]i to the lower steady-state level. Nicorandil dose-dependently inhibited norepinephrine-induced Ca++ transients in physiological salt solution containing 1 mM Ca++. Nicorandil accelerated the reduction of [Ca++]i observed when the cells were exposed to Ca++-free solution. When the cells were treated with nicorandil in Ca++-free solution, Ca++ transients induced by the first application of caffeine were little affected, but those induced by subsequent repetitive caffeine applications were reduced strongly and progressively. In contrast, pretreatment with nicorandil markedly inhibited Ca++ transients induced by the first application of norepinephrine, in Ca++-free solution. These effects of nicorandil on [Ca++]i and Ca++ transients were similar to those seen with nitroglycerin. The denitrated compound of nicorandil, N-(2-hydroxyethyl)nicotinamide, had no such effect. Thus, it is apparently the nitrate moiety of the chemical structure by which nicorandil actively and strongly reduces [Ca++]i in vascular smooth muscle cells. The reduction of [Ca++]i by nicorandil may result in a decrease in Ca++ in the norepinephrine-sensitive store; hence, the reduction of [Ca++]i elevation by norepinephrine.     

Release of Ca2+ from the endoplasmic reticulum is not the mechanism for bile acid-induced cholestasis and hepatotoxicity in the intact rat liver.

The hypothesis that monohydroxy bile acids exert their cholestatic and hepatotoxic effects via a sustained elevation of cytosolic [Ca2+] was tested in the isolated perfused rat liver. Infusion of the specific inhibitor of microsomal Ca2+ sequestration, 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) (25 microM for 10 min) produced efflux of Ca2+ from the liver and a sustained (20 min) increase in cytosolic [Ca2+] as indicated by the threefold increase in hepatic glucose output. Release of the endoplasmic reticular Ca2+ pool was demonstrated by the complete abolition of vasopressin- and phenylephrine-induced Ca2+ exchange between the liver and perfusate. Despite the profound perturbation of intracellular Ca2+ homeostasis produced by tBuBHQ, there was no decrease in bile flow and no evidence of hepatocellular injury (for 60 min), as indicated by lactate dehydrogenase release. In contrast, lithocholic acid (25 microM for 10 or 30 min) or taurolithocholic acid (5 microM for 10 or 30 min) produced an 80-90% inhibition of bile flow and a progressive increase in perfusate lactate dehydrogenase activity. During and after bile acid infusion, there was no change in Ca2+ fluxes between liver and perfusate, no stimulation of glucose output from the liver, and hormone-stimulated Ca2+ responses were preserved. It is concluded that the mechanisms for bile acid-induced cholestasis and hepatotoxicity in the intact liver are not attributable to changes in intracellular Ca2+ homeostasis, and especially not to prolonged release or depletion of Ca2+ sequestered in the endoplasmic reticulum.     

Relationship between phosphatidylinositol turnover and Ca++ mobilization induced by alpha-1 adrenoceptor stimulation in the rat aorta

In this study the causal relationship between alpha-1 adrenoceptor activation mediating contraction in rat aorta and the mediatory responses, such as phosphatidylinositol turnover and intracellular Ca++ release has been evaluated. Norepinephrine (1 X 10(-5) M) increased maximally the accumulation of [3H]inositol-1-PO4. In the presence of LiCl (10 mM) the norepinephrine-induced accumulation of [3H]inositol-1-PO4 occurred in a time-dependent, linear fashion (0-60 min), achieving a 13-fold increase over the unstimulated control at 60 min of exposure. This stimulation could be inhibited by prazosin (1 X 10(-7) M) but not by yohimbine (1 X 10(-7) M), whereas it was also unaffected by nifedipine (3 X 10(-7) M). Potassium depolarization did not invoke [3H]inositol-1-PO4 production nor did Sgd 101/75 in concentrations of up to 3 X 10(-5) M, although both have been found effective in stimulating a large influx of Ca++ for their contraction. However, the effect of norepinephrine on the formation of [3H] inositol-1-PO4 was antagonized by Sgd 101/75. A positive correlation (correlation coefficient 0.966) between intracellular Ca++ release and phosphatidylinositol turnover induced by a series of alpha-1 adrenoceptor agonists was demonstrated. These data support the hypothesis that stimulation of alpha-1 adrenoceptors in rat aorta can elicit two distinct processes of Ca++ utilization for contraction. One facilitates exclusively an influx of extracellular Ca++ which is independent of phosphatidylinositol turnover, whereas the other activates the release of intracellularly bound Ca++ that may be mediated primarily by phosphatidylinositol metabolism.     

Chronic ethanol administration to rats decreases receptor-operated mobilization of intracellular ionic calcium in cultured hepatocytes and inhibits 1,4,5-inositol trisphosphate production: relevance to impaired liver regeneration.

We tested the hypothesis that ethanol impairs liver regeneration by abrogating receptor-mediated elevation of cytosolic free calcium ([Ca2+]i). In rats fed for 16 weeks with ethanol, hepatocellular proliferation induced by partial hepatectomy was greatly impaired. Similarly, EGF-induced DNA synthesis was reduced in cultured hepatocytes from ethanol-fed rats. There was no change in the number or affinity of EGF receptors on hepatocytes from ethanol-fed rats. Despite this, EGF-mediated production of inositol 1,4,5-trisphosphate (Ins[1,4,5]P3) was lower in hepatocytes from ethanol-fed rats, and the EGF-induced [Ca2+]i transient appeared to be abrogated. When vasopressin or phenylephrine were used as cell surface receptor ligands, hepatocytes cultured from ethanol-fed rats exhibited major reductions in Ins(1,4,5)P3 synthesis. This was associated with greatly truncated [Ca2+]i transients. These changes were not due to an effect on the Ins(1,4,5)P3 receptor on the endoplasmic reticulum or to a decrease in the size of the Ins(1,4,5)P3-mobilizable intracellular Ca+2 store. Further, mobilization of the same Ca2+ store by 2,5-di-tert-butylhydroquinone or thapsigargin restored the ability of hepatocytes from ethanol-fed rats to proliferate when exposed to EGF. It is concluded that chronic ethanol consumption inhibits liver regeneration by a mechanism that is, at least partly, the result of impaired receptor-operated [Ca2+]i signaling due to reduced generation of Ins(1,4,5)P3.     

Effects of ionophore A23187 on base-line and vasopressin-stimulated sodium transport in the toad bladder.

The cation specific ionophore A23187 (Io) is a useful tool for studying the role of intracellular Ca++ (Ca++)i in physiologic processes. The present studies explore the role of (Ca++)i on Na transport in the toad bladder. Scraped bladder cells exposed to 1 muM Io for 60 min took up 100% more 45Ca than control cells. Io, 1 muM, added to the serosal side of bladders incubated in standard Ringers containing 2.5 mM Ca++ inhibited short circuit current (SCC) values by a mean of 30% at 60 min and 50% at 90 min. Io did not inhibit SCC significantly in bladders incubated in Ringers containing 0.2 mM Ca++. These data indicate that the effects of Io on SCC depend on the levels of external Ca++ and suggest that entry of Ca++ into cells mediates the inhibition of base-line SCC. PReincubation of the bladders with either lanthanum chloride or pentobarbital prevented the increased 45Ca uptake produced by ionophore as well as theinhibition of SCC caused by the antibiotic. Vasopressin, antidiuretic hormone (ADH). 10 MU/ml, increased peak SCC by 247% in bladders preincubated for 1 h in Ringers with 2.5 mM Ca++ and 1 muM Io and by 318% in control bladders (P less than 0.01). Bladders exposed to 1 muM Io in Ringers with 0.2 mM Ca++ had an increase in SCC after ADH comparable to that observed in controls. Since the effects of ADH on SCC are mediated by cyclic AMP, we tested the effects of Io on cAMP production by scraped toad bladder cells. ADH increased cAMP from 8 to 30 pmol/mg protein in controls but it did not increase cAMP over base-line values in the presence of Io when the Ringers contained 2.5 mM Ca++. Io did not inhibit cAMP production in response to ADH when the Ca++ in the Ringers was 0.2 mM. The results indicate that Io inhibits baseline and ADH stimulated SCC by increasing (Ca++)i or Ca++ bound to the cell membrane. It is suggested that: ()( (Ca++)i or membrane-bound Ca++ plays a key role in base-line and ADH stimulated Na transport in the toad bladder; (2) inhibition of ADH stimulated SCC may be due inpart to decreased cAMP generation in response to ADH when (Ca++)i or membrane-bound Ca++ levels are increased.     

Dependency of cyclic AMP-induced insulin release on intra- and extracellular calcium in rat islets of Langerhans.

Calcium and cyclic AMP are important in the stimulation of insulin release. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) raises islet cAMP levels and causes insulin release at nonstimulatory glucose concentrations. In isolated rat pancreatic islets maintained for 2 d in tissue culture, the effects of IBMX on insulin release and 45Ca++ fluxes were compared with those of glucose. During perifusion at 1 mM Ca++, 16.7 mM glucose elicited a biphasic insulin release, whereas 1 mM IBMX in the presence of 2.8 mM glucose caused a monophasic release. Decreasing extracellular Ca++ a monophasic release. Decreasing extracellular Ca++ to 0.1 mM during stimulation reduced the glucose effect by 80% but did not alter IBMX-induced release. Both glucose and IBMX stimulated 45Ca++ uptake (5 min). 45Ca++ efflux from islets loaded to isotopic equilibrium (46 h) was increased by both substances. IBMX stimulation of insulin release, of 45Ca++ uptake, and of efflux were not inhibited by blockade of Ca++ uptake with verapamil, whereas glucose-induced changes are known to be inhibited. Because IBMX-induced insulin release remained unaltered at 0.1 mM calcium, it appears that cAMP-stimulated insulin release is controlled by intracellular calcium. This is supported by perifusion experiments at 0 Ca++ when IBMX stimulated net Ca++ efflux. In addition, glucose-stimulated insulin release was potentiated by IBMX. These results suggest that cAMP induced insulin release is mediated by increases in cytosolic Ca++ and that cAMP causes dislocation of Ca++ from intracellular stores.     

Ca(2+) signaling via sigma(1)-receptors: novel regulatory mechanism affecting intracellular Ca(2+) concentration

The sigma(1)-receptor is a one-transmembrane endoplasmic reticulum protein that binds neurosteroids and dextrorotatory benzomorphans. The roles of sigma(1)-receptors in regulating intracellular Ca(2+) in NG108 cells were examined in this study. sigma(1)-Ligands pregnenolone sulfate, (+)-pentazocine, and 2-(4-morpholino)ethyl-1-phenylcyclohexane-1-carboxylate hydrochloride modulate Ca(2+) signaling in NG108 cells via two modes of action. First, nanomolar concentrations of the ligands, without effect by themselves, potentiated the bradykinin-induced increase of the cytosolic free Ca(2+) concentration in a bell-shaped manner. This effect of sigma(1)-ligands was unaffected by depletion of Ca(2+) from perfusion buffer and was blocked by a 21-mer antisense oligodeoxynucleotide against the cloned sigma(1)-receptors. Second, after the cells were depleted of the endoplasmic reticulum Ca(2+) stores, the depolarization (75 mM KCl)-induced increase in cytosolic free Ca(2+) was potentiated by 2-(4-morpholino)ethyl-1-phenylcyclohexane-1-carboxylate hydrochloride, whereas it was inhibited by pregnenolone sulfate and (+)-pentazocine. These effects, albeit opposite in direction, were blocked by both the 21-mer antisense oligodeoxynucleotide and pertussis toxin. Western blotting indicates that sigma(1)-receptors are increased on the plasma membrane and the nuclear membrane in the presence of sigma(1)-ligand. These results suggest that Ca(2+) signaling via sigma(1)-receptors may represent a novel mechanism that affects intracellular Ca(2+) concentrations.     

Effect of calcium on the availability of platelet von Willebrand factor

The binding of a polyclonal immune-purified rabbit anti-human von Willebrand factor (VWF) antibody to activated and nonactivated normal human platelets was studied in the presence and absence of extracellular calcium. Extracellular Ca++ in the incubation buffer results in no increase in the amount of antibody bound to the surface of unstimulated platelets when compared with that bound in the absence of Ca++. Alpha thrombin stimulation of the platelets increases antibody binding to the platelets in both the presence and absence of extracellular Ca++; however, the increase is approximately four times as great in the presence of extracellular Ca++. The amount of VWF detected in the incubation supernatants of unstimulated platelets is similar in either the presence or absence of Ca++, and this amount is increased without demonstrating a Ca++ dependency after thrombin stimulation. Measurement of thrombin-induced beta thromboglobulin (beta TG) release reveals some parallel between anti-VWF antibody binding and beta TG release. However, the amount of beta TG release after thrombin stimulation is independent of extracellular Ca++, whereas the amount of bound anti-VWF is markedly increased in the presence of Ca++. These studies demonstrate that thrombin induces an increase in the surface exposure of platelet VWF even in the absence of extracellular Ca++. The data also suggest that there may be a quantity of platelet VWF that can become exposed on the platelet surface by an action of thrombin that is independent of the Ca++-dependent granule release reaction induced by thrombin.     

Calcium-dependent stimulation of renal medullary prostaglandin synthesis by furosemide

The present study examined the actions of furosemide and other "loop" diuretics on immunoreactive prostaglandin E (iPGE) and [14C]arachidonate (AA) release in vitro in incubates of slices from rat and dog outer or inner medulla. The loop diuretics furosemide, ethacrynic acid, bumetanide and 3-benzylamino-4-phenylthio-5-sulfamoylbenzoic acid all significantly increased [14C]AA and iPGE release (1.5- to 4-fold) into the media of rat outer and inner medulla and dog outer medullary slice incubates. By contrast, equimolar concentrations of chlorothiazide and hydrochlorothiazide were without effects on these parameters. Stimulation of [14C]AA or iPGE by furosemide was abolished by exclusion of Ca++ from the incubation media or by addition of verapamil to complete media, but was not altered by exclusion of Na+. Ca++-free media or verapamil also abolished the increases in [14C]AA and iPGE induced by ionophore A23187. By contrast, these incubation conditions did not influence the iPGE responses to hypertonic mannitol or exogenous AA. The presence of Ca++-responsive acyl hydrolase activity was demonstrated in the microsomal fraction from rat outer medulla. However, this activity was not altered by addition of furosemide to the subcellular fraction in the presence or absence of Ca++. Thus, furosemide and other loop diuretics stimulate renal medullary iPGE synthesis in vitro, and may do so through Ca++-mediated or dependent enhancement of the release of AA.     

Deficient activity of dephosphophosphorylase kinase and accumulation of glycogen in the liver

Low activity of phosphorylase and increased concentration of glycogen were found in liver tissue from five children with asymptomatic hepatomegaly. In vitro activation of liver phosphorylase in these patients occurred at the rate of 10% or less of normal. Elimination of the defect by the addition of kinase that activates phosphorylase demonstrated the integrity of the phosphorylase enzyme and the deficient activity of dephophophosphorylase kinase.On the average, 60% of the phosphorylase enzyme of normal human liver was in the active form. Phosphorylase kinase of rabbit muscle activated phosphorylase of normal human liver to a final value that was significantly higher than the one obtained in the absence of muscle phosphorylase kinase.The ultrastructural examination of hepatic tissue from the five patients revealed increased amounts of glycogen. There was scarcity of endoplasmic reticulum. There was intercellular glycogen in continuity with the glycogen of the hepatocytes through breaks in their circumference. Lipid droplets with lucid areas in the form of needles and plates contained aggregates of glycogen. There were numerous lysosomes, some containing glycogen. Large vacuoles filled with glycogen and surrounded by a membrane were seen occasionally. The vacuoles might reflect the lysosomal pathway of glycogen degradation, since there was apparent fusion of such autophagic vacuoles with small vesicles resembling primary lysosomes.