Calcium binding in cardiac sarcolemma.

The relationship between calcium binding and ATPase activity has been investigated for guinea pig cardiac sarcolemma. The concentrations of calcium ions and of ATP were the main factors in determining the amount of calcium bound. With a saturating concentration of ATP, at low calcium concentrations (0.1 mM) more than 50% of the total calcium bound was ATP dependent while at high concentrations of calcium (10 mM) only 20% of the calcium bound was ATP dependent. The ATP-dependent calcium binding process involves one class of calcium binding sites while the non-ATP-dependent calcium binding process involves two classes of binding sites. Inhibitor studies of Ca2+-stimulated MgATPase, MgATPase, and CaATPase activities suggest Ca2+ and Mg2+ are either interacting with different sites on the same enzyme or with different enzymes.     

ATP-driven proton fluxes across membranes of secretory organelles

The ATP-dependent proton uptake by chromaffin granule membranes, lysosomes, and synaptosomes was examined. In synaptosomes the reaction was absolutely dependent on the presence of chloride, while in chromaffin granules chloride had a profound effect and in lysosomes only a minor effect. The presence of chloride markedly increases the rate of collapse of delta pH by carbonyl cyanide p-trifluoromethoxyphenylhydrazone in all three organelles. Ascorbate with phenazine methosulfate uncoupled the ATP-dependent proton uptake by chromaffin granules, but had no effect on lysosomes and synaptosomes. Proton uptake by submitochondrial particles was about 50-fold more sensitive to dicyclohexylcarbodiimide than the proton uptake by chromaffin granule membranes. Chromaffin granule membranes were treated with 2 M sodium bromide to inactivate the mitochondrial ATPase. The treatment caused a complete inhibition of the ATP-dependent proton uptake. Solubilization of these membranes by sodium cholate, followed by reconstitution by cholate dilution revealed the ATP-dependent proton uptake of the system. It is concluded that the genuine ATPase enzyme of chromaffin granules is a proton translocator.     

Inhibition of the sarcoplasmic reticulum Ca2+ transport ATPase by thapsigargin at subnanomolar concentrations.

ATP-dependent calcium uptake by isolated sarcoplasmic reticulum vesicles is inhibited by concentrations of free thapsigargin as low as 10(-10) M. This effect is due to primary inhibition of the Ca(2+)-dependent ATPase which is coupled to active transport. When binding of calcium to the activating sites of the enzyme is measured under equilibrium conditions in the absence of ATP, addition of thapsigargin produces strong inhibition. On the other hand, if [tau-32P]ATP is added to ATPase preincubated with Ca2+ under favorable conditions, significant levels of 32P-phosphorylated intermediate are still formed transiently, even in the presence of thapsigargin. The phosphoenzyme, however, decays rapidly as the calcium-enzyme complex is destabilized as a consequence of ATP utilization, and formation of the thapsigargin-enzyme complex is favored. Formation of the thapsigargin-enzyme complex is also favored by Ca2+ chelation with EGTA, with consequent inhibition of the enzyme reactivity to Pi (i.e. reverse of the ATPase hydrolytic reaction). Neither the Ca(2+)- and ATP-induced Ca2+ release from junctional sarcoplasmic reticulum nor the Ca(2+)- and calmodulin-dependent ATPase of plasma membranes (erythrocyte ghosts) were found to be altered by thapsigargin at such low concentrations.     

The metal sites on sarcoplasmic reticulum membranes that bind lanthanide ions with the highest affinity are not the ATPase Ca2+ transport sites.

We attempted to establish whether lanthanide ions, when added to sarcoplasmic reticulum (SR) membranes in the absence of nucleotide, compete with Ca2+ for binding to the transport sites of the Ca(2+)-ATPase in these membranes, or whether they bind to different sites. Equilibrium measurements of the effect of lanthanide ions on the intrinsic fluorescence of SR ATPase and on 45Ca2+ binding to it were performed either at neutral pH (pH 6.8), i.e. when endogenous or contaminating Ca2+ was sufficient to nearly saturate the ATPase transport sites, or at acid pH (pH 5.5), which greatly reduced the affinity of calcium for its sites on the ATPase. These measurements did reveal apparent competition between Ca2+ and the lanthanide ions La3+, Gd3+, Pr3+, and Tb3+, which all behaved similarly, but this competition displayed unexpected features: lanthanide ions displaced Ca2+ with a moderate affinity and in a noncooperative way, and the pH dependence of this displacement was smaller than that of the Ca2+ binding to its own sites. Simultaneously, we directly measured the amount of Tb3+ bound to the ATPase relative to the amount of Ca2+ and found that Tb3+ ions only reduced significantly the amount of Ca2+ bound after a considerable number of Tb3+ ions had bound. Furthermore, when we tested the effect of Ca2+ on the amount of Tb3+ bound to the SR membranes, we found that the Tb3+ ions which bound at low Tb3+ concentrations were not displaced when Ca2+ was added at concentrations which saturated the Ca2+ transport sites. We conclude that the sites on SR ATPase to which lanthanide ions bind with the highest affinity are not the high affinity Ca2+ binding and transport sites. At higher concentrations, lanthanide ions did not appear to be able to replace Ca2+ ions and preserve the native structure of their binding pocket, as evaluated in rapid filtration measurements from the effect of moderate concentrations of lanthanide ions on the kinetics of Ca2+ dissociation. Thus, the presence of lanthanide ions slowed down the dissociation from its binding site of the first, superficially bound 45Ca2+ ion, instead of specifically preventing the dissociation of the deeply bound 45Ca2+ ion. These results highlight the need for caution when interpreting, in terms of calcium sites, experimental data collected using lanthanide ions as spectroscopic probes on SR membrane ATPase.     

Inhibition of the SERCA Ca2+ pumps by curcumin. Curcumin putatively stabilizes the interaction between the nucleotide-binding and phosphorylation domains in the absence of ATP.

Curcumin is a compound derived from the spice, tumeric. It is a potent inhibitor of the SERCA Ca2+ pumps (all isoforms), inhibiting Ca2+-dependent ATPase activity with IC50 values of between 7 and 15 microm. It also inhibits ATP-dependent Ca2+-uptake in a variety of microsomal membranes, although for cerebellar and platelet microsomes, a stimulation in Ca2+ uptake is observed at low curcumin concentrations (<10 microm). For the skeletal muscle isoform of the Ca2+ pump (SERCA1), the inhibition of curcumin is noncompetitive with respect to Ca2+, and competitive with respect to ATP at high curcumin concentrations ( approximately 10-25 microm). This was confirmed by ATP binding studies that showed inhibition in the presence of curcumin: ATP-dependent phosphorylation was also reduced. Experiments with fluorescein 5'-isothiocyanate (FITC)-labelled ATPase also suggest that curcumin stabilizes the E1 conformational state. The fact that FITC labels the nucleotide binding site of the ATPase (precluding ATP from binding), and the fact that curcumin affects FITC fluorescence indicate that curcumin must be binding to another site within the ATPase that induces a conformational change to prevent ATP from binding. This observation is interpreted, with the aid of recent structural information, as curcumin stabilizing the interaction between the nucleotide-binding and phosphorylation domains, precluding ATP binding.     

Caclium uptake and associated adenosine triphosphatase activity in fragmented sarcoplasmic reticulum. Requirement for potassium ions.

The effects of monovalent cations on calcium uptake by fragmented sarcoplasmic reticulum have been clarified. Homogenization of muscle tissue in salt-containing solutions leads to contamination of this subcellular fraction with actomyosin and mitochondrial membranes. When, in addition, inorganic cations are contributed by the microsomal suspension and in association with nucleotide triphosphate substrates there is an apparent inhibition of the calcium transport system by potassium and other cations. However, when purified preparations were obtained after homogenization in sucrose medium followed by centrifugation on a sucrose density gradient in a zonal rotor, calcium uptake and the associated adenosine triphosphatase activity were considerably activated by potassium and other univalent cations. When plotted against the log of the free calcium concentration there was only a slight increase in calcium uptake and ATPase activity in the absence of potassium ions but sigmoid-shaped curves were obtained in 100 mM K+ with half-maximal stimulation occurring at 2 muM Ca2+ for both calcium uptake and ATPase activity. The augmentation in calcium uptake was not due to an ionic strength effect as Tris cation at pH 6.6 was shown to be inactive in this respect. Other monovalent cations were effective in the order K+ greater than Na+ greater than NH4+=Rb+=Cs+ greater than Li+ with half-maximal stimulation in 11 mM K+, 16 mM Na+, 25 mM NH4+, Rb+, and Cs+ and in 50 mM Li+. There was nos synergistic action between K+ AND Na+ ions and both calcium uptak and associated ATPase were insensitive to ouabain. Thallous ions stimulate many K+-requiring enzymes and at one-tenth the concentration were nearly as effective as K+ ions in promoting calcium uptake. The ratio of Ca2+ ions transported to P1 released remained unchanged at 2 after addition of K+ ions indicating an effect on the rate of calcium uptake rather than an increased efficiency of uptake. In support of this it was found that during the stimulation of calcium uptake by Na+ ions there was a reduction in the steady state concentration of phosphorylated intermediate formed from [gamma-32P]ATP. It is considered that there is a physiological requirement for potassium ions in the relaxation process.     

Stimulation of calcium accumulation in cardiac sarcolemma by protein kinase.

Sarcolemmal membranes isolated from guinea pig heart ventricles contained an ATP-dependent calcium-sequestering activity. Sarcolemmal calcium accumulation but not binding was enhanced by preincubation of membranes with exogenous protein kinase, with cyclic AMP, or with isoproterenol. Protein kinase (EC 2.7.1.37) increased the V of Ca2+ accumulation by sarcolemma without any significant effect on the affinity for Ca2+. The endogenous protein kinase activity present in isolated sarcolemma affected membrane phosphorylation. Cyclic AMP increased the endogenous kinase activity modestly, whereas histone increased it significantly. Exogenous protein kinase also catalyzed phosphorylation of these membranes. Endogenous and exogenous kinase-catalyzed phosphorylation of sarcolemma was hydroxylamine-insensitive. Ca2+-dependent ATPase (EC 3.6.1.3) (extra ATPase) activity of sarcolemma was also increased by protein kinase.     

Calmodulin abolishes the changes in Ca2+ binding and transport by heart sarcolemmal membranes of spontaneously hypertensive rats

Both Ca2+ transport and binding properties of heart sarcolemmal membranes are altered in spontaneously hypertensive rats (SHR) when compared to their normotensive controls (WKY). The effects of calmodulin on these two processes were studied at free calcium concentrations presumed to be the physiological levels in the cytosol. At a calcium concentration of 2.10(-8)M, calmodulin did not significantly modify either binding or ATP-dependent accumulation of calcium by membranes of both origins. In contrast, at a free calcium concentration of 4.10(-7)M, calmodulin enhanced the calcium binding to SHR membranes and the ATP-dependent calcium transport by SHR and WKY membranes. Differences in calcium binding and ATP-dependent accumulation between the two substrains were suppressed in presence of calmodulin. These data demonstrate that modifications in calcium handling by SHR cardiac plasma membranes might be due to altered intracellular content or function of calmodulin in SHR.     

Inactivation of Na+, K+ -ATPase from cattle brain by sodium fluoride

The influence of the physiological ligands and modifiers on the plasma membrane Na+, K+ -ATPase from calf brain inactivation by sodium fluoride (NaF) is studied. ATP-hydrolyzing activity of the enzyme was found to be more stable as to NaF inhibition than its K+ -pNPPase activity. The activatory ions of Na+, K+ -ATPase have different effects on the process of the enzyme inhibition by NaF. K+ intensifies inhibition, but Na+ does not affect it. An increase of [Mg2+free] in the incubation medium (from 0.5 to 3.0 mM) rises the sensitivity of Na+, K+ -ATPase to NaF inhibition. But an increase of [ATP] from 0.3 to 1.5 mM has no effect on this process. Ca and Mg ions modify Na+, K+ -ATPase inhibition by fluoride differently. Ca2+free levels this process, and Mg2+free on the contrary increases it. In the presence of Ca ions and in the neutral-alkaline medium (pH 7.0-8.5) the recovery of activity of the transport ATPase inhibited by-NaF takes place. Sodium citrate also protects both ATP-hydrolizing and K-pNPPase activity of the Na+, K+ -ATPase from NaF inhibition. Under the modifing membranous effects (the treatment of plasma membranes by Ds-Na and digitonin) the partial loss of Na+, K+ -ATPase sensitivity to NaF inhibition is observed. It is concluded that Na+, K+ -ATPase inactivation by NaF depends on the influence of the physiological ligands and modifiers as well as on the integrity of membrane structure.     

The effects of lubrol WX on brain membrane Ca2+/Mg2+ ATPase and ATP-dependent Ca2+ uptake activity following acute and chronic ethanol

Acute administration of ethanol (2.5 gm/kg, i.p.) to rats inhibits the cytosolic buffering of Ca2+ in nerve terminals. Ca2+ ATPase and ATP-dependent Ca2+ uptake are both inhibited 30 min after a single dose of ethanol. Chronic ethanol administration (6%, 14 days) did not inhibit Ca2+ ATPase but significantly stimulated ATP-dependent Ca2+ uptake. Lubrol WX treatment of acute ethanolic membranes reverses the inhibition of Ca2+ ATPase seen following ethanol. Lubrol WX treatment of chronic ethanolic membranes prevents the increase in ATP-dependent Ca2+ uptake seen in ethanolic membranes. Both acute and chronic ethanol-induced changes in Ca2+ transport within nerve terminals may involve lipid-dependent parameters of the membrane which may underlie neuronal adaptation.     

Stimulation of polymorphonuclear leukocytes by dicyclohexylcarbodiimide: calcium homeostasis

The involvement of calcium in N,N'-dicyclohexylcarbodiimide (DCCD)-mediated stimulation of guinea pig neutrophils was investigated. Exposure to DCCD resulted in a fast though moderate elevation of cytosolic calcium concentration. Exchange experiments indicated that DCCD enhanced 45Ca2+ efflux without affecting uptake of the radioisotope from the medium. Plasma membranes isolated from DCCD-stimulated cells failed to support ATP-dependent 45Ca2+ uptake indicating inhibition of their Ca-ATPase. The finding that the enhanced efflux of 45Ca2+ depended on the presence of Na+ ions in the medium implicated a Na+/Ca2+ exchanger in efflux of the ion observed in DCCD-stimulated neutrophils. This is the first indication for the participation of this carrier in calcium homeostasis in stimulated neutrophils. Experiments carried out with 14C-DCCD indicated covalent binding of the reagent to 20 and 150 Kd membrane proteins.     

Characterization of calcium transport by basal plasma membranes from human placental syncytiotrophoblast.

We have studied the mechanisms involved in calcium (Ca2+) transport through the basal plasma membranes (BPM) of the syncytiotrophoblast cells from full-term human placenta. These purified membranes were enriched 25-fold in Na+/K(+)-adenosine triphosphate (ATPase), 37-fold in [3H] dihydroalprenolol binding sites, and fivefold in alkaline phosphatase activity compared with the placenta homogenates. In the absence of ATP and Mg2+, a basal Ca2+ uptake was observed, which followed Michaelis-Menten kinetics, with a Km Ca2+ of 0.18 +/- 0.05 microM and Vmax of 0.93 +/- 0.11 nmol/mg/min. The addition of Mg2+ to the incubation medium significantly decreased this uptake in a concentration-dependent manner, with a maximal inhibition at 3 mM Mg2+ and above. The Lineweaver-Burk plots of Ca2+ uptake in the absence and in the presence of 1 mM Mg2+ suggest a noncompetitive type of inhibition. Preloading the BPM vesicles with 5 mM Mg2+ had no significant effect on Ca2+ uptake, eliminating the hypothesis of a Ca2+/Mg2+ exchange mechanism. This ATP-independent Ca2+ uptake was not sensitive to 10(-6) M nitrendipine nor to 10(-4) M verapamil. An ATP-dependent Ca2+ transport was also detected in these BPM, whose Km Ca2+ was 0.09 +/- 0.02 microM and Vmax 3.4 +/- 0.2 nmoles/mg/3 min. This Ca2+ transport requires Mg2+, the optimal concentration of Mg2+ being approximately 1 mM. Preincubation of the membrane with 10(-6) M calmodulin strongly enhanced the initial ATP-dependent Ca2+ uptake. Finally, no Na+/Ca2+ exchange process could be demonstrated.     

Relation between phosphorylation and adenosine triphosphate-dependent Ca2+ binding of swine and bovine erythrocyte membranes

The correlation between the ATP-dependent Ca2+ binding and the phosphorylation of the membranes from swine and bovine erythrocytes was studied. The Ca2+ binding was measured by using 45CaCl2, and the phosphorylation by [gamma-32P]ATP was studied with the technique of SDS polyacrylamide gel electrophoresis. 200 mM NaCl and KCl markedly repressed the Ca2+ binding of swine erythrocyte membranes. The radioactivity of 32P-labelled membranes was revealed mainly in 250,000 dalton protein and a lipid fraction. NaCl and KCl also repressed the phosphorylation of the lipid which was identified as triphosphoinositide by paper chromatography. The membranes prepared from trypsin-digested erythrocytes completely retained the Ca2+-binding activity, and lost 30% of (Ca2+ + Mg2+)-ATPase activity. The Ca2+-binding and ATPase activity of isolated membranes decreased to 55% and to 0%, respectively, by tryptic digestion. Neither the Ca2+ binding nor the phosphorylation of polyphosphoinositides were detected in bovine erythrocyte membranes. These results suggest that the formation of triphosphoinositide rather than the (C2+ + Mg2+)-ATPase of membranes is linked to the ATP-dependent Ca2+ binding of erythrocyte membranes.     

Effects of Taurine on Calcium Ion Uptake and Protein Phosphorylation in Rat Retinal Membrane Preparations

The effects of taurine on ATP-dependent calcium ion uptake and protein phosphorylation of rat retinal membrane preparations were investigated. Taurine (20 mM) stimulates ATP-dependent calcium ion uptake by twofold in crude retinal homogenates. In contrast, it inhibits the phosphorylation of specific membrane proteins as shown by acrylamide gel electrophoresis and autoradiography. The close structural analogue of taurine, 2-aminoethylhydrogen sulfate, demonstrates similar effects in both systems, i.e., stimulation of ATP-dependent calcium ion uptake and inhibition of protein phosphorylation, whereas isethionic acid and guanidinoethanesulfonate have no effect on either system. A P1 subcellular fraction of the retinal membrane preparation that contains photoreceptor cell synaptosomes has a higher specific activity for the uptake of calcium ions. Phosphorylation of specific proteins in the P1 fraction is also inhibited by the addition of 20 mM taurine. Taurine has no effect on retinal ATPase activities or on phosphatase activity, thus suggesting that it directly affects a kinase system.     

Calmodulin Stimulation of Ca2+-Dependent ATP Hydrolysis and ATP-Dependent Ca2+ Transport in Synaptic Membranes

We report here characterization of calmodulin-stimulated Ca2+ transport activities in synaptic plasma membranes (SPM). The calcium transport activity consists of a Ca2+-stimulated, Mg2+-dependent ATP hydrolysis coupled with ATP-dependent Ca2+ uptake into membraneous sacs on the cytosolic face of the synaptosomal membrane. These transport activities have been found in synaptosomal subfractions to be located primarily in SPM-1 and SPM-2. Both Ca2+-ATPase and ATP-dependent Ca2+ uptake require calmodulin for maximal activity (KCm for ATPase = 60 nM; KCm for uptake = 50 nM). In the reconstituted membrane system, KCa was found to be 0.8 microM for Ca2+-ATPase and 0.4 microM for Ca2+ uptake. These results demonstrate for the first time the calmodulin requirements for the Ca2+ pump in SPM when Ca2+ ATPase and Ca2+ uptake are assayed under functionally coupled conditions. They suggest that calmodulin association with the membrane calcium pump is regulated by the level of free Ca2+ in the cytoplasm. The activation by calmodulin, in turn, regulates the cytosolic Ca2+ levels in a feedback process. These studies expand the calmodulin hypothesis of synaptic transmission to include activation of a high-affinity Ca2+ + Mg2+ ATPase as a regulator for cytosolic Ca2+.     

Mechanisms of petroleum hydrocarbon toxicity: destruction of liver microsomal and mitochondrial calcium pump activities by a Prudhoe Bay crude oil

Administration of Prudhoe Bay crude oil (PBCO) to rats resulted in an abrupt drop in liver mitochondrial and microsomal ATP-dependent calcium uptake activity. Also, in vitro incubations of either mitochondria or microsomes in the presence of a dimethyl sulfoxide (DMSO) extract of PBCO resulted in a dose-dependent inhibition of calcium influx. The release of calcium from calcium-loaded mitochondria and microsomes was also observed in the presence of the PBCO extract. At concentrations which effect calcium sequestration, the PBCO extract produced swelling of mitochondria. Microsomal ATPase activity in the presence or absence of calcium was unaffected by PBCO. The results indicate that increased permeability of the membranes to calcium is a contributory factor in the inhibition of calcium uptake by PBCO.     

Ouabain induces acetylcholine release from pure cholinergic synaptosomes independently of extracellular calcium concentration

We have studied the correlation between [³H]ouabain binding sites, (Na⁺+K⁺)ATPase (EC 3.6.1.3) activity and acetylcholine (ACh) release in different subcellular fractions ofTorpedo marmorata electric organ (homogenate, synaptosomes, presynaptic plasma membranes). Presynaptic plasma membranes contained the greater number of [³H]ouabain binding sites in good agreement with the high (Na⁺+K⁺)ATPase activity found in this fraction. Blockade of this enzymatic activity by ouabain dose-dependently induced ACh release from pure cholinergic synaptosomes, either in the presence or absence of extracellular calcium ions. We suggest that one of the mechanisms involved in the ouabain-induced ACh release in the absence of Ca²⁺ _o may be an increase in Na⁺ _i that could (a) evoke Ca²⁺ release from internal stores and (b) inhibit ATP-dependent Ca²⁺ uptake by synaptic vesicles.     

The effect of ionomycin on calcium fluxes in sarcoplasmic reticulum vesicles and liposomes.

Ionomycin, a recently discovered calcium ionophore, inhibits the ATP-dependent active Ca2+ transport of rabbit sarcoplasmic reticulum vesicles at concentrations as low as 10(-8) to 10(-6) M. The effect is due to an increase in the Ca2+ permeability of the membrane which is also observed on liposomes. The inhibition of Ca2+ uptake is accompanied by an increase in the Ca2+-sensitive ATPase activity of sarcoplasmic reticulum vesicles.     

The Effect of Calcium Ionophores on Fragmented Sarcoplasmic Reticulum

X-537 A and A 23187, two antibiotics which form liphophilic complexes with divalent cations, function as ionophores in vesicular fragments of sarcoplasmic reticulum (SR). Addition of either ionophore to SR preloaded with calcium in the presence of adenosine triphosphate (ATP), causes rapid release of calcium. Furthermore, net calcium accumulation by SR is prevented, when the ionophores are added to the reaction mixture before ATP. On the contrary, ATP-independent calcium binding to SR is not inhibited. This effect is specific for the two antibiotics and could not be reproduced, either by inactive derivatives, or by other known ionophores. Neither ionophore produces alterations of the electron microscopic appearance of SR membranes or inhibition of the calcium-dependent ATPase. In fact, the burst of ATP hydrolysis obtained on addition of calcium, is prolonged in the presence of the ionophores. Lanthanum inhibits ATP-independent calcium binding to SR, ATP-dependent calcium accumulation and calcium-dependent ATPase. However, addition of lanthanum to SR preloaded in the presence of ATP, does not cause calcium release. The reported experiments indicated that: (a) ATP-dependent calcium accumulation by SR results in primary formation of calcium ion gradients across the membrane. (b) Most of the accumulated calcium is not available for displacement by lanthanum on the outer surface of the membrane. (c) Calcium ionophores induce rapid equilibration of the gradients, by facilitating cation diffusion across the membrane.     

Comparison of the properties of active Ca2+ transport by the islet-cell endoplasmic reticulum and plasma membrane

The properties of active or ATP-dependent calcium transport by islet-cell endoplasmic reticulum and plasma membrane-enriched subcellular fractions were directly compared. These studies indicate that the active calcium transport systems of the two membranes are fundamentally distinct. In contrast to calcium uptake by the endoplasmic reticulum-enriched fraction, calcium uptake by islet-cell plasma membrane-enriched vesicles exhibited a different pH optimum, was not sustained by oxalate, and showed an approximate 30-fold greater affinity for ionized calcium. A similar difference in affinity for calcium was exhibited by the Ca²⁺-stimulated ATPase activities which are associated with these islet-cell subcellular fractions. Consistent with the effects of calmodulin on calcium transport, calmodulin stimulated Ca²⁺-ATPase in the plasma membranes, but did not increase calcium-stimulated ATPase activity in the endoplasmic reticulum membranes. The physiological significance of the differences observed in calcium transport by the endoplasmic reticulum and plasma membrane fractions relative to the regulation of insulin secretion by the islets of Langerhans is discussed.