Agonist induced release of intracellular Ca2+ in the rabbit aorta

Summary The effects of hormonal agonists (norepinephrine, angiotensin, and histamine) on⁴⁵Ca efflux from the rabbit aorta were studied using a Ca-EGTA buffered efflux medium. Each caused a transient stimulation of efflux rate which probably reflected the release of an intracellular⁴⁵Ca store. The size of the stimulation of efflux correlated with the size of the initial rapid phase of contraction. The norepinephrine-sensitive intracellular Ca fraction was estimated to be greater than 21 moles/Kg wet tissue weight. This fraction is separate from intracellular Ca which is accumulated during relaxation. Evidence is presented for the lack of cyclic nucleotide involvement in the release of Ca²⁺, and possible alternative modes of coupling are discussed.     

Hormone-evoked calcium release from intracellular stores is a quantal process

Ca2+ mobilization by hormones, ionomycin, and inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) were studied to determine whether Ca2+ release is a continuous or a quantal process. Hormone-mediated Ca2+ release occurs only during the first 2-4 s of stimulation. Stimulation of acini with a maximal hormone concentration following stimulation with a submaximal concentration resulted in free cytosolic Ca2+ concentration ([Ca2+]i) increase and 45Ca efflux. The peak [Ca2+]i increase induced by a maximal concentration of agonist was nearly constant when cells were prestimulated with a submaximal dose for 1-15 min. Submaximal hormone concentrations release only a fraction of intracellular 45Ca2+, after which intracellular Ca2+ content remains constant. The partially released stores remain depleted until cell stimulation is terminated, at which time the stores reload with Ca2+. For comparison, increasing concentrations of ionomycin resulted in increasing rates of Ca2+ release. Each ionomycin concentration released all the Ca2+ from intracellular stores. We therefore conclude that hormone-evoked Ca2+ release is a quantal rather than a continuous process. In permeabilized cells, increasing concentrations of Ins-1,4,5-P3 resulted in an increased fraction of Ca2+ release. No submaximal Ins-1,4,5-P3 concentration was capable of releasing all the Ins-1,4,5-P3-mobilizable Ca2+. Therefore, it appears that the quantal properties of hormone-evoked Ca2+ release reflect the quantal properties of Ins-1,4,5-P3-mediated Ca2+ release from intracellular stores.     

Glucose-, calcium- and concentration-dependence of acetylcholine stimulation of insulin release and ionic fluxes in mouse islets.

Mouse islets were used to define the glucose-dependence and extracellular Ca2+ requirement of muscarinic stimulation of pancreatic beta-cells. In the presence of a stimulatory concentration of glucose (10 mM) and of Ca2+, acetylcholine (0.1-100 microM) accelerated 3H efflux from islets preloaded with myo-[3H]inositol. It also stimulated 45Ca2+ influx and efflux, 86Rb+ efflux and insulin release. In the absence of Ca2+, only 10-100 microM-acetylcholine mobilized enough intracellular Ca2+ to trigger an early but brief peak of insulin release. At a non-stimulatory concentration of glucose (3 mM), 1 microM- and 100 microM-acetylcholine increased 45Ca2+ and 86Rb+ efflux in the presence and absence of extracellular Ca2+. However, only 100 microM-acetylcholine marginally increased 45Ca2+ influx and caused a small, delayed, stimulation of insulin release, which was abolished by omission of Ca2+. At a maximally effective concentration of glucose (30 mM), 1 microM- and 100 microM-acetylcholine increased 45Ca2+ influx and efflux only slightly, but markedly amplified insulin release. Again, only 100 microM-acetylcholine mobilized enough Ca2+ to trigger a peak of insulin release in the absence of Ca2+. The results thus show that only high concentrations of acetylcholine (greater than or equal to 10 microM) can induce release at low glucose or in a Ca2+-free medium. beta-Cells exhibit their highest sensitivity to acetylcholine in the presence of Ca2+ and stimulatory glucose. Under these physiological conditions, the large amplification of insulin release appears to be the result of combined effects of the neurotransmitter on Ca2+ influx, on intracellular Ca2+ stores and on the efficiency with which Ca2+ activates the releasing machinery.     

Insulin release and metabolism of calcium, adenine and adenosine 3',5'-cyclic monophosphate.

In order to assess further the mechanisms involved in insulin release, we prelabeled rat pancreatic islets of Langerhans by incubating either 45Ca or [2-3H]adenine. When prelabeled islets were perfused with a glucose-free medium (the experiment with 45Ca) and a medium containing 2.8 mM glucose (the experiment with [2-3H]adenine) respectively, a constant rate of efflux of the radioactivity was established by 30 min in each case. D-Glucose at 16.7 mM concentration elicited a rapid efflux of 45Ca and [2-3H]adenine derivatives ([3H]Ad) within 4 to 6 min after commencing the step-wise stimulation by glucose, concomitantly with insulin release. However, L-glucose and D-galactose littel stimulated both 45Ca and [3H]Ad release. Lanthanum chloride caused a burst peak of 45Ca release in the absence of glucose. A rapid efflux of 45Ca was caused by beta-D-glucose and D-glyceraldehyde to much lesser extent than by alpha-D-glucose. The slowly rising concentration of glucose at 0.1 mM/min of gradient level failed to elicit any rapid efflux of 45Ca or [3H]Ad, although insulin release occurred in accordance with an increase in glucose concentration. Even when the gradient of glucose concentration was raised to 0.7 mM/min, glucose failed to stimulate an efflux of [3H]Ad but the subsequent stimulation by 16.7 mM glucose caused a rapid efflux of [3H]Ad concomitantly with the release of insulin. No rapid efflux of 45Ca was observed under a slow-rise glucose stimulation until the gradient level of the glucose concentration was raised to 6.7 mM. Analysis of distribution of the radioactive adenine derivatives after incubation showed that the adenosine fraction had the highest radioactivity in the medium followed by the ATP, adenine and cAMP fraction in that order, and the ATP fraction had the highest radioactivity in the islet. The ratio of radioactivity in the cAMP fraction in the medium to the total count was the highest among all. On the basis of these results, it was suggested that the discharge of [3H]Ad and 45Ca might occur with the alteration of the membrane permeability induced by a rapid change of the glucose concentration, and that their discharge might perhaps link to the glucoreceptor mechanism directly controlling insulin release.     

The influence of high potassium depolarization and acetylcholine on calcium exchange in the rat uterus

Net and radioactive calcium movements were studied in the rat uterus during stimulation with acetylcholine and high potassium solutions. High potassium did not affect the efflux of intracellular Ca⁴⁵, but was able to release Ca⁴⁵ from a small parallel Ca fraction which was believed to be located in the cell membranes. High potassium did markedly slow the influx of Ca⁴⁵ and caused a net calcium efflux. Acetylcholine had no effect on calcium movements in polarized myometrium, but it increased the Ca⁴⁵ influx in depolarized uteri. Ca⁴⁵ taken up during contraction exchanged more slowly during subsequent efflux than Ca⁴⁵ taken up at rest. The results were interpreted as supporting the hypothesis that myometrial contraction is induced by a release of calcium from the inside of the cell membrane and the endoplasmic reticulum, and relaxation follows the removal of ionic cytoplasmic calcium by these same structures.     

Calcium mobilization by cadmium or decreasing extracellular Na+ or pH in coronary endothelial cells

Replacing extracellular Na+ with choline transiently increased cytoplasmic free Ca2+ ([Ca2+]i) more than 5-fold in coronary endothelial cells. Removing external Na+ stimulated 45Ca2+ efflux approximately 4-fold and influx approximately 1.7-fold. The stimulation of efflux was independent of extracellular Ca2+ and the osmotic Na+ substitute. The release of stored Ca2+, rather than Ca2+ influx via Na(+)-Ca2+ exchange, probably causes the increase in [Ca2+]i and 45Ca2+ efflux. Cadmium or decreasing external, not intracellular, pH transiently increased [Ca2+]i. Cd2+ and some other divalent metals also stimulated 45Ca2+ efflux. The potency order of the metals that stimulated efflux was Cd2+ greater than CO2+ greater than Ni2+ greater than Fe2+ greater than Mn2+. Incubating the cells with Zn2+ prior to assaying efflux in the absence of Zn2+ strongly inhibited the stimulation of 45Ca2+ efflux by Cd2+, pH 6, and the removal of external Na+ without affecting the stimulation of efflux by ATP. These findings support the hypothesis that certain trace metals or decreasing external Na+ or pH trigger the release of stored Ca2+ by stimulating a cell surface "receptor."     

Dissociation between intracellular calcium mobilization and insulin secretion in isolated rat islets of Langerhans

The neuropeptide bombesin provoked a dose-dependent stimulation of 45Ca2+ efflux from pre-loaded islets of Langerhans. This response occurred rapidly, was not sustained and did not depend on the presence of extracellular calcium, suggesting that it resulted from the mobilization of intracellular calcium stores. Under conditions when large increases in 45Ca2+ efflux were observed, bombesin completely failed to stimulate the rate of insulin secretion. Similar results were also obtained with the muscarinic cholinergic agonist, carbachol. The data suggest that the release of calcium from intracellular pools is not sufficient to induce an increase in insulin secretion in normal islet cells.     

Alpha 1-adrenergic receptor activation mobilizes cellular Ca2+ in a muscle cell line

Regulation of cellular Ca2+ movements by alpha 1-adrenergic receptors has been studied using 45Ca2+ flux techniques in monolayer cultures of intact BC3H-1 cells. Unidirectional 45Ca2+ efflux from BC3H-1 cells reveals multiphasic kinetics, with a major fraction of cellular Ca2+ residing in a slowly exchanging intracellular compartment. Stimulation of alpha 1-adrenergic receptors by the agonist phenylephrine substantially increases 45Ca2+ unidirectional efflux, accompanied by a far smaller increase in 45Ca2+ influx. The selective enhancement of 45Ca2+ unidirectional efflux upon alpha 1-adrenergic receptor activation results in a net 30-40% decline in total cell Ca2+ content, measured either by radioisotopic equilibrium techniques or by atomic absorption spectroscopy. The relatively large pool of Ca2+ responsive to alpha-adrenergic stimulation is not displaced by La3+ but can be depleted with the Ca2+ ionophore A-23187. These results indicate that alpha 1-adrenergic receptor activation predominantly mobilizes Ca2+ from intracellular stores, together with a much smaller increase in transmembrane Ca2+ permeability. This interpretation is supported by comparative 45Ca2+ flux studies using a sister clone of BC3H-1 cells possessing surface nicotinic acetylcholine receptors but no alpha 1-adrenergic receptors. Agonist stimulation of the cholinergic receptor opens a well characterized transmembrane ion permeability gate. Cholinergic receptor activation greatly enhances the observed 45Ca2+ unidirectional influx relative to efflux, leading to net elevation of cellular Ca2+ content as Ca2+ moves down its inwardly directed concentration gradient.     

Rapid calcium mobilization by vasopressin and prostaglandin F2 alpha is independent of sodium influx in quiescent 3T3 cells

Vasopressin (VP) rapidly increased 45Ca2+ efflux. A VP antagonist prevented VP from mobilizing Ca2+ and stimulating DNA synthesis. Prostaglandin F2 alpha (PGF2 alpha) also stimulated rapid 45Ca2+ release. The effectiveness of different prostaglandins corresponded to their effectiveness as mitogens. The removal of external Na+ or Ca2+ had no effect on VP-or PGF2 alpha-induced 45Ca2+ release. The present results indicate that the mobilization of intracellular Ca2+ by these hormones is independent of Na+ or Ca2+ influx and that Ca2+ mobilization is important for growth stimulation.     

5,6-Epoxyeicosatrienoic acid mobilizes Ca2+ in anterior pituitary cells

Luteinizing hormone releasing hormone stimulates the concomitant release of luteinizing hormone and 45Ca2+ from prelabeled anterior pituitary cells. Indomethacin (10 microM) and nordihydroguaiaretic acid (10 microM) had no effect on the luteinizing hormone releasing hormone-stimulated release of either luteinizing hormone or 45Ca2+. Eicosatetraynoic acid (10 microM) blocked both luteinizing hormone releasing hormone-stimulated luteinizing hormone secretion and luteinizing hormone releasing hormone-stimulated 45Ca2+ efflux. 5,6-Epoxyeicosatrienoic acid stimulated both luteinizing hormone secretion and 45Ca2+ efflux from anterior pituitary cells. Additionally, 5,6-epoxyeicosatrienoic acid closely mimics the ability of luteinizing hormone releasing hormone to increase intracellular free calcium. These results are consistent with the hypothesis that 5,6-EET alters calcium homeostasis in a manner similar to that observed during luteinizing hormone releasing hormone stimulation of luteinizing hormone release.     

Serum rapidly mobilizes calcium from an intracellular pool in quiescent fibroblastic cells

Addition of dialysed fetal bovine serum to quiescent cultures of Swiss 3T3 cells loaded with 45Ca2+ causes a very rapid increase in the rate of 45Ca2+ efflux from an intracellular pool. Exposure to serum for 2 min leads to a fall of 0.59 nmol Ca2+/mg protein in the intracellular Ca2+ content of the cells. Inhibitors of mitochondrial function prevent the stimulation of 45Ca2+ efflux by serum. The stimulation of 45Ca2+ efflux by serum is also observed in quiescent cultures of Rat-1, Swiss 3T6 and BHK cells and in secondary cultures of whole mouse embryo fibroblasts.     

Feedback inhibition of Ca2+ release by Ca2+ is the underlying mechanism of agonist-evoked intracellular Ca2+ oscillations in pancreatic acinar cells.

Oscillations of free intracellular Ca2+ concentration ([Ca2+]i) are known to occur in many cell types during physiological cell signaling. To identify the basis for the oscillations, we measured both [Ca2+]i and extracellular Ca2+ concentration ([Ca2+]o) to follow the fate of Ca2+ during stimulation of [Ca2+]i oscillations in pancreatic acinar cells. [Ca2+]i oscillations were initiated by either t-butyloxycarbonyl-Tyr(SO3)-Nle-Gly-Tyr-Nle-Asp-2-phenylethyl ester (CCK-J), which mobilized Ca2+ from the inositol 1,4,5-trisphosphate (IP3)-insensitive pool, or low concentration of cholecystokinin octapeptide (CCK-OP), which mobilized Ca2+ from the IP3-sensitive internal pool. Little Ca2+ efflux occurred during the oscillations triggered by CCK-J or CCK-OP in spite of a large average increase in [Ca2+]i. When internal store Ca2+ pumps were inhibited with thapsigargin (Tg) during [Ca2+]i oscillations, a rapid Ca2+ efflux occurred similar to that measured in intensely stimulated, nonoscillatory cells. Tg also stimulated 45Ca efflux from internal pools of cells stimulated with CCK-J or a low concentration of CCK-OP. Hence, a large fraction of the Ca2+ released during each spike is reincorporated by the internal store Ca2+ pumps. Surprisingly, when the increase in [Ca2+]i during stimulation of oscillations was prevented by loading the cells with 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid, a persistent activation of Ca2+ release and Ca2+ efflux occurred. This was reflected as a persistent increase in [Ca2+]o in cells suspended at low [Ca2+]o or persistent efflux of 45Ca from internal stores of cells maintained at high [Ca2+]o. Since agonist-stimulated Ca2+ release evidently remains activated when [Ca2+]i is highly buffered, the primary mechanism determining Ca2+ oscillations must include an inhibition of Ca2+ release by [Ca2+]i. Loading the cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid had no apparent effect on the levels or kinetics of IP3 formation in agonist-stimulated cells. This suggests that [Ca2+]i regulated the oscillation by inhibition of Ca2+ release independent of its possible effects on cellular levels of IP3.     

Involvement of Ca2+ entry and inositol trisphosphate-induced internal Ca2+ mobilization in muscarinic receptor-mediated catecholamine release in dog adrenal chromaffin cells.

Catecholamine (CA) release from adrenal medulla evoked by muscarinic receptor stimulation has been studied using isolated perfused adrenal gland and cultured chromaffin cells from dogs. Muscarine and oxotremorine (1-100 microM), and bethanechol (0.1-1 mM) dose-dependently stimulated CA release. Muscarine-evoked CA release was antagonized with M1-antagonist, pirenzepine and, to a lesser extent, with atropine; and was reduced either by removal of extracellular Ca2+ or treatment with Ca2+ channel blockers. Muscarine caused an increase of 45Ca uptake and 22Na uptake. Tetrodotoxin (TTX) did not affect muscarine-evoked increase of 22Na uptake and CA release. Under the absence of extracellular Ca2+, muscarine stimulated a 45Ca efflux. Muscarine-induced CA release was attenuated by treating the cells with 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate-HCl (TMB-8) which blocks Ca2+ release from the intracellular store. A phospholipase C inhibitor, neomycin, markedly reduced muscarine-induced CA release but not nicotine- and high K(+)-evoked release. Cinnarizine, a Ca2+ channel blocker, attenuated muscarine-evoked but not caffeine-induced CA release and 45Ca efflux in the absence of extracellular Ca2+. Muscarine caused an increase in intracellular free Ca2+ concentration ([Ca2+]i) in the presence of extracellular Ca2+. It caused a similar increase, but to a lesser extent, in the absence of extracellular Ca2+. The increase of [Ca2+]i induced by muscarine without extracellular Ca2+ was reduced by neomycin and cinnarizine. Polymixin B and retinal, which reduced 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced CA release, had little effect on muscarine-induced CA release. Muscarine increased cellular Ins(1,4,5)P3 production, and atropine inhibited this increase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of platelet-activating-factor-induced calcium influx and intracellular calcium release in platelets by phorbol esters.

1. The rate of 45Ca2+ efflux from prelabelled rat islets of Langerhans was stimulated by carbachol in a dose-dependent manner. 2. Significant stimulation occurred in the presence of 0.2 microM-carbachol; the response was half-maximal at 3-5 microM and was maximal at 20 microM. 3. Stimulation of 45Ca2+ efflux by carbachol was not dependent on the presence of extracellular Ca2+ and was enhanced in Ca2+-depleted medium. 4. Stimulation of 45Ca2+ efflux by 5 microM-carbachol occurred independently of any change in [3H]arachidonic acid release in prelabelled islets, and probably reflected generation of inositol trisphosphate in the cells. 5. The amphipathic peptide melittin failed to increase islet-cell 45Ca2+ efflux at a concentration of 1 microgram/ml, and caused only a modest increase at 10 micrograms/ml. 6. Despite its failure to increase 45Ca2+ efflux, melittin at 1 microgram/ml caused a marked enhancement of 3H release from islets that had been prelabelled with [3H]arachidonic acid. 7. The stimulation of 3H efflux caused by melittin correlated with a dose-dependent increase in the unesterified [3H]arachidonic acid content of prelabelled islets and with a corresponding decrease in the extent of labelling of islet phospholipids. 8. Combined addition of melittin (1 microgram/ml) and 5 microM-carbachol to perifused islets failed to augment 45Ca2+ efflux relative to that elicited by carbachol alone. 9. The data indicate that melittin promotes an increase in arachidonic acid availability in intact rat islets. They do not, however, support the proposal that this can either directly reproduce or subsequently modify the extent of intracellular Ca2+ mobilization induced by agents that cause an increase in inositol trisphosphate.     

Effects of platelet-derived growth factor on Ca2+ in 3T3 cells

The effect of platelet-derived growth factor (PDGF) on cellular Ca2+ was examined in BALB/c-3T3 cells. PDGF induced: A decrease in cell 45Ca2+ content. An apparent increased rate of efflux of preloaded 45Ca2+. A decrease in residual intracellular 45Ca2+ remaining after rapid efflux. When added after the rapid phase of efflux of 45Ca2+ had occurred, an immediate decrease in post-efflux residual intracellular 45Ca2+. All of the observed changes in 45Ca2+ induced by PDGF are consistent with a rapid release of Ca2+ from an intracellular Ca2+ pool that has the slowest efflux and is relatively inaccessible to extracellular EDTA. When incubated with chlortetracycline (CTC), a fluorescent Ca2+ probe, 3T3 cell mitochondria became intensely fluorescent. Addition of PDGF resulted in a rapid decrease in CTC fluorescence intensity in both adherent and suspended 3T3 cells. The effects of PDGF on 3T3 cell Ca2+ stores and CTC fluorescence intensity were identical with the effects of the Ca2+ ionophore A23187 and of the proton ionophore carbonyl cyanide m-chlorophenyl hydrazone. Serum, which contains PDGF, also altered intracellular Ca2+ stores, but platelet-poor plasma, which does not contain PDGF, had no effect. EGF, insulin, and tetradecanoyl phorbol acetate (TPA), other factors which stimulate 3T3 cell growth, did not alter 3T3 cell Ca2+ stores. Release of Ca2+ from intracellular sequestration sites may be a mechanism by which PDGF stimulates cell growth.     

Regulation of calcium fluxes in rat pancreatic islets: Quinine mimics the dual effect of glucose on calcium movements

The effects of quinine and 9-aminoacridine, two blockers of potassium conductance in islet cells, on ⁴⁵Ca efflux and insulin release from perifused islets were investigated in order to elucidate the mechanisms by which glucose initially reduces ⁴⁵Ca efflux and later stimulates calcium inflow in islet cells. In the absence of glucose, 100 μM quinine stimulated ⁴⁵Ca net uptake, ⁴⁵Ca outflow rate and insulin release. Quinine also dramatically enhanced the cationic and the secretory response to intermediate concentrations of glucose, but had little effect on ⁴⁵Ca net uptake, ⁴⁵Ca fractional outflow rate and insulin release at a high glucose concentration (16.7 mM). The ability of quinine to stimulate ⁴⁵Ca efflux depended on the presence of extracellular calcium, suggesting that it reflects a stimulation of calcium entry in the islet cells. In the absence of extracellular calcium, quinine provoked a sustained decrease in ⁴⁵Ca efflux. Such an inhibitory effect was not additive to that of glucose, and was reduced at low extracellular Na⁺ concentration. At a low concentration (5 μM), quinine, although reducing ⁸⁶Rb efflux from the islets to the same extent as a non-insulinotropic glucose concentration (4.4 mM), failed to inhibit ⁴⁵Ca efflux. In the presence of extracellular calcium, 9-aminoacridine produced an important but transient increase in ⁴⁵Ca outflow rate and insulin release from islets perifused in the absence of glucose. In the absence of extracellular calcium, 9-aminoacridine, however, failed to reduced ⁴⁵Ca efflux from perifused islets. It is concluded that quinine, by reducing K⁺ conductance, reproduces the effect of glucose to activate voltage-sensitive calcium channels and to stimulate the entry of calcium into the B-cell. However, the glucose-induced inhibition of calcium outflow rate, which may also participate in the intracellular accumulation of calcium, does not appear to be mediated by changes in K⁺ conductance.     

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.     

Mathematical modeling of stimulus-secretion coupling in the pancreatic beta-cell. III. Glucose-induced inhibition of calcium efflux.

The inhibitory effect of glucose upon 45Ca efflux from prelabeled pancreatic islets was simulated in a mathematical model for Ca2+-cyclic AMP interaction in the process of glucose-induced insulin release. At variance with a previous interpretation, it was postulated that glucose inhibits 45Ca efflux by facilitating the uptake of the cation by the vacuolar system. The latter facilitation did not hinder glucose from provoking a rapid accumulation of cytosolic Ca2+ and, hence, insulin release. The postulated facilitation was also suitable in simulating the effect of glucose upon 45Ca efflux, uptake, and intracellular distribution in the pancreatic islets.     

The effect of caffeine on calcium efflux and calcium translocation in skeletal and visceral muscle.

1. KCl-induced depolarization resulted in a large stimulation of the 45Ca efflux from both cockroach skeletal muscle and rat ileal smooth muscle. 2. Caffeine (10 mM) induced a large stimulation of 45Ca efflux from skeletal muscle, but a fall in the efflux from ileal muscle, especially if the efflux was previously stimulated by KCl depolarization. 3. Caffeine inhibited calcium uptake by skeletal muscle mitochondria and sarcoplasmic reticulum, was without effect on ileal muscle mitochondria, but significantly increased caclium binding by ileal muscle membrane vesicular preparations. 4. The induction of contractures and stimulation of 45Ca efflux in skeletal muscle by caffeine are clearly related to inhibition of intracellular calcium binding by the sarcoplasmic reticulum and mitochondria. 5. The relaxation of ileal muscle by caffeine and the inhibition of fibre calcium efflux correlate well with caffeine enhancement of intracellular calcium binding. These experiments suggest that the membrane vesicular compartment may be the main agency centrally involved in fibre calcium regulation in this muscle during the contraction-relaxation cycle.     

Excitation of skinned muscle fibers by imposed ion gradients. II. Influence of quercetin and ATP removal on the Ca2+-insensitive component of stimulated 45Ca efflux

Ionic gradients imposed by choline Cl replacement of K methanesulfonate (Mes) at constant [K][Cl] product stimulate 45Ca efflux from skinned muscle fibers; a small, sustained Ca2+-insensitive efflux component, observed in EGTA, appears to grade a much larger Ca2+-dependent component responsible for contractile activation and is likely to reflect intermediate steps in excitation-contraction coupling. The present studies examined ATP-related effects on the Ca2+-insensitive stimulation. 45Ca efflux was measured on segments of frog semitendinosus muscle skinned by microdissection, with isometric force monitored continuously. The Ca2+-insensitive component was potentiated by quercetin, a flavonoid thought to inhibit the sarcoplasmic reticulum (SR) Ca pump by stabilizing a phosphorylated intermediate. Quercetin increased the stimulated net 45Ca release in the absence of EGTA, as expected from inhibition of reaccumulation, but its effectiveness in EGTA indicated potentiation of unidirectional efflux as such. Quercetin also increased unstimulated (control) 45Ca efflux in EGTA, to a smaller extent; potentiation appeared to be a function of efflux, with stimulation above control loss increased approximately 2.6-fold. ATP removal before stimulation, which led to rigor force and increased stiffness, prevented all quercetin effects in EGTA. ATP removal by itself inhibited ionic stimulation of the Ca2+-insensitive component, with little residual increase above the parallel control loss. Addition of the nonhydrolyzable ATP analogue AMP-PCP ([adenylyl-beta,gamma-methylene]diphosphate) (0.8 mM) after ATP removal gave similar results to ATP-free solution, which suggests that adenine nucleotide binding alone does not support stimulation by choline Cl. These results imply a fundamental role for ATP in the excitation of skinned fibers by imposed diffusion potentials; they also suggest that ATP regulates the SR Ca efflux channel, in a manner that could provide the positive feedback in Ca2+-dependent Ca release.