Extent Of Ca2 Research Articles

Receptor for myo-inositol trisphosphate from the microsomal fraction of Vigna radiata.

The microsomal fraction from mung-bean (Vigna radiata) hypocotyl was found to contain Ins (1,4,5)P3- and Ins(2,4,5)P3-binding activity. Preincubation of the microsomal fraction with thiol-containing reagents reduced specific InsP3 binding. A single class of binding site with a Kd value of 1.5 nM and Bmax. of 1.1 pmol/mg of protein was detected. Other myo-inositol phosphates exhibited little affinity for this protein. The binding protein was purified to homogeneity and the molecular mass of the native form recorded as 400 kDa. However, under denaturing conditions the molecular mass was 110 kDa, suggesting that the protein is a homotetramer. That this protein is associated with Ca2+ release was confirmed by including it in proteoliposomes and adding Ins(1,4,5)P3 or Ins(2,4,5)P3. The affinity of Ins(1,4,5)P3 is 3-fold higher than that of Ins(2,4,5)P3. The binding affinity of InsP3 is also reflected in the extent of Ca2+ released from the microsomal fraction. Heparin inhibits binding of InsP3 to the protein, the K1/2 being 0.26 microM. It is also shown that the protein acts as a receptor for InsP3 with characteristics of high affinity and low density.

Endothelin-3 Decreases Ca2+ Uptake in Platelet Membrane Vesicles

Summary: To analyze the mechanisms by which endothelin-3 (ET-3) attenuates agonist-mediated Ca2+ mobilization from an internal pool, we investigated ET-3 effects on 45Ca2+ uptake in platelet membrane vesicles. They were compared to those of thapsigargin (Tg), a specific inhibitor of the dense tubule Ca2+ pumps. In the absence of ATP, ET-3 up to 1 μM did not affect the amount of Ca2+ bound to membrane sites. In the presence of ATP, ET-3 dose-dependently reduced the initial rate and the extent of Ca2+ uptake (p < 0.001). In comparison, Tg dose-dependently inhibited both the ATP-independent Ca2+ binding (p < 0.001) and the ATP-dependent Ca2+ accumulation (p < 0.001), with half-maximal effects at 7 nM. Pretreatment with 1 μM ET-3 decreased the inhibitory effect of 10 nM Tg, but only on the initial rate of ATP-dependent Ca2+ uptake (p = 0.04; n = 6). These results indicate that ET-3 is functionally coupled to Ca2+ -ATPases of the dense tubules. Its inhibitory effects are probably due to inhibition of the catalytic cycle of the Ca2+ pumps. Such inhibition could lead to a depletion of Ca2+ stores and therefore to reduced Ca2+ release in response to agonists.

Endothelin-3 Decreases Ca2+ Uptake in Platelet Membrane Vesicles

To analyze the mechanisms by which endothelin-3 (ET-3) attenuates agonist-mediated Ca2+ mobilization from an internal pool, we investigated ET-3 effects on 45Ca2+ uptake in platelet membrane vesicles. They were compared to those of thapsigargin (Tg), a specific inhibitor of the dense tubule Ca2+ pumps. In the absence of ATP, ET-3 up to 1 microM did not affect the amount of Ca2+ bound to membrane sites. In the presence of ATP, ET-3 dose-dependently reduced the initial rate and the extent of Ca2+ uptake (p < 0.001). In comparison, Tg dose-dependently inhibited both the ATP-independent Ca2+ binding (p < 0.001) and the ATP-dependent Ca2+ accumulation (p < 0.001), with half-maximal effects at 7 nM. Pretreatment with 1 microM ET-3 decreased the inhibitory effect of 10 nM Tg, but only on the initial rate of ATP-dependent Ca2+ uptake (p = 0.04; n = 6). These results indicate that ET-3 is functionally coupled to Ca(2+)-ATPases of the dense tubules. Its inhibitory effects are probably due to inhibition of the catalytic cycle of the Ca2+ pumps. Such inhibition could lead to a depletion of Ca2+ stores and therefore to reduced Ca2+ release in response to agonists.

Gonadotropin releasing hormone stimulation of pituitary cell 3′–5′ cyclic AMP in a carp(Cyprinus carpio) is dependent on extracellular calcium

Pituitaries were collected from a common carp,yprinss carpi, belonging to vitellogenic phase and cells were disaggregated by using 0.3% collagenase and 0.05% tsypsin. Enzymatically dispersed cells were incubatedin vitro in Ca2+-free medium to observe the effect ofCanna punctatus GnRH (cGnRH) and Ca2+ on pituitary cell cAMP accumulation. Addition of cGnRH (20 Big) to pituitary cell incubation (6 × 104 cells/well) containing 4 mM theophylline, a phosphodiesterase inhibitor, caused two-fold increase of cAMP accumulation in comparison to control, Addition of Ca2+ (2 mM) to cGnRH further augmented cAMP accumulation, i.e., four-fold as compared to control. Increasing concentrations of cGnRH in the presence of Ca2+ resulted in a dose-dependent increase in cAMP accumulation. To examine the specificity of Ca2+ augmentory effect on cGnRH-stimulated pituitary cell cAMP accumulation, a specific Ca2+-channel blocker, verapamil was used, At 3 µM dose verapamil completely waived Ca2+-augmentation of cGnRH stimulatory effect on cAMP. Interestingly, verapamil also significantly inhibited cGnRH stimulation of cAMP in the Ca2+-free medium. Extent of Ca2+ plus cGnRH stimulatory effect on cAMP was further increased by the addition of 25 pmol of calmodulin, a Ca2+-carrier protein, Addition of verapamil to this system strongly inhibited Ca2+ and ealmodulin augnientory effect on cGnRH. Reduced level of cAMP in the pituitary cell due to verapamil was even lower than that of cGnRH plus ealmodulin incubation. Data indicates a contamination of Ca2+ in an apparently Ca2+-free medium, Results suggest that in lower vertebrate, i.e., fish, GnRH stimulation of pituitary cell cAMP is dependent on extracellulnr Ca2+ and incubation of pituitary cell in Ca2+-free medium is truly not free of Ca2+.

Modulation of the mitochondrial cyclosporin A-sensitive permeability transition pore by matrix pH. Evidence that the pore open-closed probability is regulated by reversible histidine protonation

Energized mitochondria in sucrose medium take up a Ca2+ pulse but do not show opening of the permeability transition pore (MTP) upon membrane depolarization by uncoupler. This is due to locking of the pore in the closed conformation by matrix acidification and fast Ca2+ efflux following membrane depolarization (Petronilli, V., Cola, C., & Bernardi P. (1993) J. Biol. Chem. 268, 1011-1016). Here we show that addition of diethyl pyrocarbonate (DPC) prior to membrane depolarization restores the ability of uncoupler to induce MTP opening. Since DPC does not modify the rate and extent of matrix acidification and the rate and extent of Ca2+ release following addition of uncoupler, its effects on pore opening appear to be due to modification of histidyl residues regulating the pore open-closed probability. This hypothesis was confirmed in studies with deenergized mitochondria incubated in potassium thiocyanate medium. While at acidic pH values pore opening is otherwise prevented, DPC allows Ca2(+)-dependent pore opening at pH 6.5 in a process that maintains full sensitivity to cyclosporin A. Pore induction by DPC can be completely prevented and partially reversed by hydroxylamine, indicating that the effect of DPC can be specifically traced to carbethoxylation of histidyl residue(s) rather than to reaction with tyrosyl or sulfhydryl groups, while the possible involvement of lysyl residues cannot be excluded. Since DPC increases the pore open probability even at matrix pH values between 7.0 and 7.7, we propose that reversible protonation of one or more histidyl residues on the matrix side of the MTP plays a role in the physiological modulation of pore opening.

IP3 receptor-mediated spatial and temporal Ca2+ signaling of the cell.

It is now revealed that "intracellular Ca2+ excitation" exists for the generation of spatial and temporal Ca2+ signals based on Ca2+ release from stores. It could be understood in analogy with electrical excitation in the cell surface, the plasma membrane. A local Ca2+ response is generated by the second messenger IP3 following agonist/receptor binding and PI turnover, corresponsing to the receptor potential or excitatory synaptic potential in neurons. The local Ca2+ response triggers a regenerative "intracellular Ca2+ spike" corresponding to the action potential. The regenerative process is the inherent property of the IP3R/Ca2+ release channel which is under positive and negative feedback controls by cytosolic and luminal Ca2+, like activation and inactivation of the voltage-gated Na+ channel. An intracellular Ca2+ spike can be induced artificially by local application of Ca2+, like induction of the action potential by a depolarizing pulse. The Ca2+ spike propagates as a spatial Ca2+ signal throughout the cell, as the action potential does. The excitation is followed by a refractory period. Repeated Ca2+ spikes are generated, depending on the magnitude of stimulus. Interval between Ca2+ spikes depends on the readiness of Ca2+ stores in terms of the sensitivity of the IP3R and extent of Ca2+ accumulation. The cycle of Ca2+ mobilization includes extracellular medium. The intracellular Ca2+ signals are thus frequency-encoded. The temporal pattern of transient Ca2+ pulses may be favorable for accomplishing cellular functions without the side effect of cell toxicity.

Platelet Aggregation Induced by Plasma from Type IIB von Willebrand’s Disease Patients Is Associated with an Increase in Cytosolic Ca2+ Concentration

The effect of type IIB von Willebrand's factor (vWF) on platelet cytosolic Ca2+ ion concentration, measured by means of the probe fura 2, was investigated. Seven patients with type IIB von Willebrand disease (vWD) were studied. Addition of type IIB vWD plasma to platelet suspensions induced a cytosolic calcium increase accompanied by platelet aggregation. Both processes were completely abolished by addition of the calcium-chelating agent EGTA, indomethacin, peptide RGDS, and monoclonal antibodies blocking the vWF binding site on GPIb-IX (LJIB1) or the cytoadhesive receptor on GPIIb-IIIa (LJCP8). The ADP-scavenger apyrase and the protein kinase C-inhibitor staurosporine partially inhibited the rate of the cytosolic calcium increase. No direct correlation between the extent of Ca2+ rise and the phenotypic expression of IIB vWD, such as the degree of spontaneous platelet aggregation or thrombocytopenia was apparent. It is suggested that aggregation and cytosolic Ca2+ increase in platelets exposed to plasma from type IIB vWD patients is mediated by a self-potentiating mechanism involving both GPIb and GPIIb-IIIa receptors as well as the thromboxane biosynthetic pathway.

Dihydropyridine-sensitive calcium channels from skeletal muscle. II. Functional effects of differential phosphorylation of channel subunits.

Dihydropyridine-sensitive Ca2+ channels from skeletal muscle are multisubunit proteins and are regulated by protein phosphorylation. The purpose of this study was to determine: 1) which subunits are the preferential targets of various protein kinases when the channels are phosphorylated in vitro in their native membrane-bound state and 2) the consequences of these phosphorylations in functional assays. Using as substrates channels present in purified transverse (T) tubule membranes, cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and a multifunctional Ca2+/calmodulin-dependent protein kinase (CaM protein kinase) preferentially phosphorylated the 165-kDa alpha 1 subunit to an extent that was 2-5-fold greater than the 52-kDa beta subunit. A protein kinase endogenous to the skeletal muscle membranes preferentially phosphorylated the beta peptide and showed little activity toward the alpha 1 subunit; however, the extent of phosphorylation was low. Reconstitution of partially purified channels into liposomes was used to determine the functional consequences of phosphorylation by these kinases. Phosphorylation of channels by PKA or PKC resulted in an activation of the channels that was observed as increases in both the rate and extent of Ca2+ influx. However, phosphorylation of channels by either the CaM protein kinase or the endogenous kinase in T-tubule membranes was without effect. Phosphorylation did not affect the sensitivities of the channels toward the dihydropyridines. Taken together, the results demonstrate that the alpha 1 subunit is the preferred substrate of PKA, PKC, and CaM protein kinase when the channels are phosphorylated in the membrane-bound state and that phosphorylation of the channels by PKA and PKC, but not by CaM protein kinase or an endogenous T-tubule membrane protein kinase, results in activation of the dihydropyridine-sensitive Ca2+ channels from skeletal muscle.

Demonstration of the phosphorylation of dihydropyridine-sensitive calcium channels in chick skeletal muscle and the resultant activation of the channels after reconstitution

We have examined the effects of cAMP elevating agents on the phosphorylation of dihydropyridine-sensitive Ca2+ channels in intact newborn chick skeletal muscle. In situ treatment with the beta-adrenergic receptor agonist isoproterenol resulted in the phosphorylation of the 170-kDa alpha 1 subunit in the intact cells, as evidenced by a marked decrease in the ability of the alpha 1 peptide to serve as a substrate in in vitro back phosphorylation reactions with [gamma-32P]ATP and the purified catalytic subunit of cAMP-dependent protein kinase. The phosphorylation of the 52-kDa beta subunit was not affected. The effects of isoproterenol were time- and concentration-dependent and were mimicked by other cAMP elevating agents but not by the Ca2+ ionophore A23187 or a protein kinase C activator. To test for functional effects of the observed phosphorylation, purified channels were reconstituted into liposomes containing entrapped fluo-3, and depolarization-sensitive and dihydropyridine-sensitive Ca2+ influx was measured. Channels from isoproterenol-treated muscle exhibited an increased rate and extent of Ca2+ influx compared to control preparations. The effects of isoproterenol pretreatment could be mimicked by phosphorylating the channels with cAMP-dependent protein kinase in vitro. These results demonstrate that the alpha 1 subunit of the dihydropyridine-sensitive Ca2(+)-channels is the primary target of cAMP-dependent phosphorylation in intact muscle and that the phosphorylation of this protein leads to activation of channel activity.

ATP-dependent and ATP-independent pathways of exocytosis revealed by interchanging glutamate and chloride as the major anion in permeabilized mast cells.

Most investigations of the mechanism of regulated exocytosis have involved the use of secretory cells permeabilized in glutamate-based electrolyte solutions. In our previous work we have used NaCl-based electrolyte solutions. For secretion to occur from rat mast cells under these latter conditions, a dual effector system comprising Ca2+ and a guanine nucleotide are required; together they are sufficient. Here we compare the secretion from mast cells permeabilized in solutions of different electrolytes. Replacement of Na+ by K+ had little effect. Replacement of Cl- by Br-, SO4-, gluconate, isethionate, acetate, tartrate, succinate, etc. affected the maximal extent of secretion elicited by the dual effectors Ca2+ and guanosine-5'-O-(3-thiotriphosphate) (Ca2(+)-plus-GTP-gamma-S) but had little influence on the effective affinity for Ca2+. The dicarboxylic amino acids (L- and D-glutamate, and L-aspartate) permitted exocytosis to be elicited by Ca2+ or GTP-gamma-S alone. Secretion stimulated by GTP-gamma-S is strongly inhibited by Cl- (50% inhibition by 20 mM Cl-), whereas the extent of Ca2(+)-induced secretion is proportional to the concentration of glutamate in mixed electrolyte buffers. Unlike dual-effector stimulation, secretion due to the single effectors requires adenosine triphosphate (ATP) and is prevented by inhibitors of protein kinase C. These results point to the existence of two parallel pathways for control of exocytosis in permeabilized cells, one ATP dependent, the other ATP independent.

Glutamine and Aspartate Loading of Synaptosomes: A Reevaluation of Effects on Calcium‐Dependent Excitatory Amino Acid Release

Guinea-pig cerebral cortical synaptosomes were preincubated for 60 min with 100 microM D-aspartate, L-aspartate, or L-glutamate. The total D- plus L-aspartate content of the synaptosomal fraction increased to 235%, 195%, or 164%, respectively, of the control. Despite this no increase was seen in the very low KCl evoked, Ca2+-dependent release of aspartate. Preincubation with the three amino acids changed the synaptosomal glutamate content to 78% (D-aspartate), 149% (L-aspartate), or 168% (L-glutamate) of control. However there was no statistically significant effect of these preincubations on the extent of Ca2+-dependent glutamate release. Thus the Ca2+-dependent release of aspartate and glutamate is not determined by the total synaptosomal content of these amino acids. The addition of 0.1-0.5 mM glutamine to the incubation caused a massive appearance of glutamate in the extrasynaptosomal medium. Analysis of specific activities showed that glutamine was hydrolysed directly by an extrasynaptosomal glutaminase, and that intrasynaptosomal glutamate was predominantly labelled by uptake of this glutaminase-derived glutamate. No increase was seen in the extent of Ca2+-dependent release of glutamate (by fluorimetry) either after preincubation with glutamine or in the continued presence of glutamine. Thus we are unable to confirm reports that glutamine expands the transmitter pool of glutamate. The extrasynaptosomal glutaminase activity in the synaptosomal preparation was inhibited by Ca2+ and activated by phosphate. Identical kinetics were obtained with "free" brain mitochondria, confirming the origin of the glutamine-derived glutamate.

Modulation of agonist-activated calcium influx by extracellular pH in rat pancreatic acini.

The biochemical and Ca2+ transport pathways involved in generating the hormone-evoked Ca2+ signal are reported to be influenced by pH. The present study was designed to determine the effect of extracellular pH (pHo) and intracellular pH (pHi) on hormone-stimulated Ca2+ transport. We used rat pancreatic acini and measured free cytosolic Ca2+ concentration ([Ca2+]i) with fura-2, pHi with 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), and Ca2+ fluxes with 45Ca2+. In the presence of external Ca2+, increasing pHo increased steady-state [Ca2+]i during sustained agonist stimulation; in the absence of external Ca2+, this increase in [Ca2+]i did not occur. The addition of an antagonist or blocking plasma membrane Ca2+ influx with La3+ in stimulated cells suspended at pHo 8.2 resulted in a reduction in [Ca2+]i. Increasing pHo increased the rate and extent of 45Ca2+ uptake into stimulated cells and the rate and extent of Ca2+ reloading of intracellular stores. The increased Ca2+ content of the intracellular stores with increased pHo indicated that at physiological pHo and pHi the agonist-mobilizable internal stores are not saturated with Ca2+. Changes in pHo affected pHi. However, changes in pHi at constant pHo had no effect on hormone-evoked [Ca2+]i increase, reduction in [Ca2+]i after hormone stimulation, or reloading of intracellular stores. We conclude that the hormone-activated plasma membrane Ca2+ entry pathway responsible for Ca2+ reloading is directly modulated by external H+.

Relationship between hormonal, GTP and Ins(1,4,5)P3-stimulated Ca2+ uptake and release in pancreatic acinar cells.

Electrically permeabilized rat pancreatic acini were used to evaluate the contributions of GTP and Ins(1,4,5) P3 to hormone-stimulated Ca2+ uptake and release from intracellular pools. Treatment of permeabilized acini with Ca2+-mobilizing hormones, GTP or GTP[S] resulted in stimulation of an ATP-dependent, VO4(2-)-sensitive Ca2+ uptake into a non-mitochondrial intracellular pool. GTP and GTP[S] also augmented the hormone-mediated stimulation of Ca2+ uptake. Including oxalate in the uptake medium increased Ca2+ uptake into this pool but did not modify the stimulation of Ca2+ uptake induced by hormones or GTP. Ins(1,4,5)P3 released all the extra Ca2+ accumulated as a result of hormone, GTP or GTP[S] stimulation. Hence, these stimuli activated the Ca2+ pump localized in the membrane of the hormone and Ins(1,4,5)P3-sensitive Ca2+ pool. Including 2,3-diphosphoglyceric acid (PGA) [an inhibitor of Ins(1,4,5)P3 hydrolysis] in the incubation medium blunted the GTP and GTP[S]-stimulated Ca2+ uptake. In the presence of PGA, the hormones inhibited Ca2+ accumulation, and GTP and GTP[S] augmented this effect. Accordingly, PGA stabilized the Ins(1,4,5)P3-evoked Ca2+ release from intracellular pools. Only in the presence of PGA was it possible to demonstrate hormonally-evoked Ca2+ release from permeabilized cells. GTP, and more importantly GTP[S], augmented the hormone-evoked Ca2+ release. Hormones and Ins(1,4,5)P3 in the presence or absence of GTP or GTP[S] released Ca2+ from the same intracellular pool. The extent of Ca2+ release caused by the combination of hormones and GTP or GTP[S] was similar to that evoked by Ins(1,4,5)P3 alone. Taken together, these results suggest that GTP or GTP[S] facilitates stimulation of phospholipase C by hormones. Such stimulation results in stimulation of protein kinase C and increased levels of Ins(1,4,5)P3 and is sufficient to explain the effects of GTP and GTP[S] on Ca2+ uptake and release from pancreatic acinar cells.

Characterization of the tetraphenylboron‐induced calcium release from skeletal sarcoplasmic reticulum

Release of Ca2+ from skeletal sarcoplasmic reticulum vesicles was studied by the spectrophotometric stopped-flow technique using tetraphenylboron as a releasing agent. The extent of Ca2+ release shows a sigmoidal response, with respect to the tetraphenylboron concentration, being dependent on Ca2+ preloading and Ca2+-ATPase activity, since these experiments were performed on actively loaded vesicles. The release process has a rapid component with an apparent rate constant of 6-8 s-1, showing a linear relationship between the rapid rate of Ca2+ release and the Ca2+ content of the vesicles. The release is not mediated by the reversal of the Ca2+ pump. Since the amphipathic anion tetraphenylboron was unable to elicit a Ca2+-release response when added to a preparation of sarcoplasmic reticulum phospholipid vesicles, it is suggested that there may be an interaction with some membrane protein(s) at the hydrophobic/hydrophilic interface leading to the opening of some specific Ca2+-release pathway.

GTP- and inositol 1,4,5-trisphosphate-activated intracellular calcium movements in neuronal and smooth muscle cell lines.

Recent evidence has revealed that a highly sensitive and specific guanine nucleotide regulatory process controls intracellular Ca2+ release within N1E-115 neuroblastoma cells (Gill, D. L., Ueda, T., Chueh, S. H., and Noel, M. W. (1986) Nature 320, 461-464). The present report documents GTP-induced Ca2+ release within quite distinct cell types, including the DDT1MF-2 smooth muscle cell line. GTP-induced Ca2+ release has similar GTP sensitivity and specificity among cells and rapidly mobilizes up to 70% of Ca2+ specifically accumulated within a nonmitochondrial Ca2+-pumping organelle within permeabilized DDT2MF-2 cells. Maximal GTP-induced release of Ca2+ is observed to be greater than inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release (the latter being approximately 30% of total releasable Ca2+). After maximal IP3-induced release, further IP3 addition is ineffective, whereas subsequent addition of GTP further releases Ca2+ to equal exactly the extent of Ca2+ release observed by addition of GTP in the absence of IP3. This suggests that IP3 releases Ca2+ from the same pool as GTP, whereas GTP also releases from an additional pool. The effects of GTP appear to be reversible since simple washing of GTP-treated cells restores their previous Ca2+ uptake properties. Electron microscopic analysis of GTP-treated membrane vesicles reveals their morphology to be unchanged, whereas treatment of vesicles with 3% polyethylene glycol, known to enhance GTP-mediated Ca2+ release, clearly induces close coalescence of membranes. In the presence of 4 mM oxalate, GTP induces a rapid and profound uptake, as opposed to release, of Ca2+. The findings suggest that GTP-activated Ca2+ movement is a widespread phenomenon among cells, which can function on the same Ca2+ pool mobilized by IP3, and although activating Ca2+ movement by a mechanism distinct from IP3, does so via a process that does not appear to involve fusion between membranes.

The effects of quinine on the calcium and magnesium content of the sarcoplasmic reticulum and the temperature-dependence of quinine contractures

A significant decrease in the Ca2+ and increase in the Mg2+ content of the terminal cisternae (TC) of the sarcoplasmic reticulum (SR) during quinine contraction was demonstrated by electron probe analysis of rapidly frozen frog muscles. The extent of Ca2+ release (71% of total) from the TC and the absence of an increase in total cell Ca2+ support the conclusion that quinine contractures are caused by passive efflux of Ca2+ from the SR when the latter is uncompensated due to inhibition of the SR Ca2+ pump by quinine. A rapid warming contraction (RWC) was observed, in the presence of quinine, when the temperature of intact and skinned muscles was increased from about 5 degrees C to 18-23 degrees C. The duration of the latency of quinine contracture, in intact muscle bundles, was approximately 31 s at 3 degrees C and 2 s at 23 degrees C. The results suggest a significant temperature sensitivity of the passive Ca2+ channels of the SR membrane, although an effect of temperature on the lipid partition coefficient of quinine into the SR has not been ruled out.

Mitochondrial changes and associated alterations induced in mice by streptozotocin administered in vivo and in vitro

Isolated mouse liver mitochondria incubated with streptozotocin showed decreased rate and extent of Ca2+ uptake, and, dependent on the concentration of streptozotocin and the addition of alpha-ketoglutarate, glutamate, fluorocitrate or guanosine 5'-triphosphate, the retention of Ca2+ was either increased or decreased. Similar observations were made in liver mitochondria incubated with succinyl-CoA. In mitochondria isolated from the kidneys and islets of mice injected with streptozotocin, with and without additional injections of glucose and/or glucagon, the rate and extent of Ca2+ uptake were reduced and the release of accumulated Ca2+ was stimulated. Electron microscopy and X-ray microanalysis showed dislocation of Ca2+-containing precipitates from the mitochondria to the cytosol, and stereology disclosed increased mitochondrial volume in the B cells of streptozotocin-treated mice. State 3 and state 4 respiration with NAD-linked substrates was inhibited, but succinate oxidation was unaffected, in mitochondria isolated from the kidneys of mice treated with streptozotocin. In the kidneys of streptozotocin-injected mice, the concentration of succinyl-CoA was increased, that of citrate and guanosine 5'-triphosphate was decreased, that of glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-diphosphate was unaffected, and the metabolite concentration ratios suggested increased mitochondrial [NAD+]/[NADH] ratio and decreased cytoplasmic [NAD+]/[NADH] ratio. It is suggested as a new hypothesis that the cytotoxicity and the diabetogenicity of streptozotocin are dependent on inhibited citric acid cycle enzyme activity (primarily that of succinyl-CoA synthetase and citrate synthetase) with altered metabolite concentrations, leading to impairment of the mitochondrial uptake of Ca2+ and the activation of the pyruvate, isocitrate and alpha-ketoglutarate dehydrogenases.

Modulation of the .GAMMA.-aminobutyric acid-gated chloride current by intracellular calcium in frog sensory neurones.

The effects of the voltage-dependent Ca2+ inward current (ICa) on gamma-aminobutyric acid (GABA)-induced Cl- current (ICl) in isolated frog sensory neurones were examined using the "concentration clamp" technique, an approach which combines internal perfusion by a suction pipette with a rapid external solution change method. A preceding ICa suppressed the GABA-activated ICl. While the inhibition was not influenced by changes of membrane potential between -40 and -100 mV, it was reduced when the internal EGTA concentration was increased. A preceding ICa shifted the GABA dose-response curve to the right without affecting the maximum current or the Hill coefficient (n = 2). Inhibition of the GABA-activated ICl by ICa showed a recovery in a single exponential manner, the time constant of which depended upon the extent of Ca2+ influx. At a low temperature (10 degrees C), however, the recovery from Ca2+-mediated inhibition was only slight. We conclude that, as a result of the voltage-dependent Ca2+ influx, a subsequent increase of intracellular Ca2+ concentration modulates the GABA-gated ICl in the soma membrane. Possible mechanisms are discussed.

Platelet Ca2+ Homeostasis: Na+-Ca2+ Exchange in Plasma Membrane Vesicles

A vesicular plasma membrane-enriched fraction obtained from human platelets exhibited 45Ca2+ uptake in exchange for intravesicular Na+. The rate of Ca2+ uptake was linear up to 4 sec. The apparent Km for Ca2+ was 22 microM and the Vmax 280 pmol/mg/sec. Ca2+ efflux from Ca2+ loaded vesicles was obtained upon dilution into a NaCl but not a KCl medium. The extent of Ca2+ uptake was monotonically increased as the pH increased from 6 to 9. Na+-Ca2+ exchange was shown to be electrogenic. Ca2+ uptake was distinguished from binding by the induction of Ca2+ release after A23187 addition. These findings support a role for Na+-Ca2+ exchange in human platelet Ca2+ transport.

Silver ions trigger Ca2+ release by acting at the apparent physiological release site in sarcoplasmic reticulum.

Rapid Ca2+ release from Ca2+ -loaded sarcoplasmic reticulum vesicles (SR) was previously shown to occur upon the addition of micromolar concentrations of heavy metals, and the extent of Ca2+ release was dependent on the binding affinity of the metal to sulfhydryl group(s) on an SR protein (Abramson, J.J., Weden, L., Trimm, J.L., and Salama, G. (1982) Biophys. J. 37, 134a; Abramson, J.J., Trimm, J.L., Weden, L., and Salama, G. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1526). The nature of this Ca2+ release site was examined further and found to be predominantly distributed in heavy SR (HSR) rather than light SR fractions. Ag+ -induced Ca2+ release from heavy SR was blocked by local anesthetics and ruthenium red which are known to inhibit Ca2+ release in skeletal fibers and in heavy SR, respectively. The rate of Ca2+ efflux from SR triggered by Ag+ was dependent on pH, Mg2+, and ionic strength of the medium. Efflux rates increased by a factor of 4 from pH 6.0 to 7.0 and then decreased in more alkaline reaction mixtures. Efflux rates from actively or passively loaded SR increased by a factor of 2.5 with increasing Mg2+ from 0 to 1 mM and then decreased in the range of 1 to 10 mM Mg2+. ATP-dependent Ca2+ uptake by SR was similar in 100 mM KCl and in 200 mM sucrose solutions, but the extent and rate of Ca2+ efflux induced by Ag+ were dramatically reduced with decreasing ionic strength of the medium. In solutions containing 5 mM Mg2+, the rate of Ca2+ efflux from heavy SR averaged over the first 1.5 s after the addition of Ag+ was 58 nmol of Ca2+/mg of SR/s, a value comparable to the fast initial rate of ATP-dependent Ca2+ uptake. The maximum initial rate of Ag+ -induced Ca2+ efflux from heavy SR in 1 mM Mg2+ may be comparable to the rate of Ca2+ release and tension development in muscle fibers. Our data indicate that Ag+ reacts with a protein or proteins in the SR, probably not the (Ca2+, Mg2+)-ATPase, to induce a rapid release of Ca2+, possibly from the physiological Ca2+ release site.