Increase in Ca2+ permeability of intracellular Ca2+ store membrane of saponin-treated guinea pig peritoneal macrophages by inositol 1,4,5-trisphosphate

Inositol 1,4,5-trisphosphate (InsP3) releases Ca2+ from the non-mitochondrial Ca2+ store site of various types of cells. To study the mechanisms of the Ca2+ release from the store site, the effect of InsP3 on the passive Ca2+ release and influx, and the active Ca2+ uptake in the presence of oxalate, was examined using saponin-treated guinea pig peritoneal macrophages. InsP3 stimulated the passive Ca2+ release and influx. Although InsP3 slightly inhibited the active Ca2+ uptake in the presence of oxalate, it seems unlikely that the Ca2+ release by this agent is caused by the inhibition of the Ca2+ uptake, because the addition of apyrase or hexokinase (which removes ATP within 30 s, so that no more Ca2+ can be accumulated) or vanadate (which inhibits the Ca2+ uptake) resulted in very slow release of Ca2+. These results suggest that the Ca2+ permeability of the Ca2+ store membrane is increased by InsP3. InsP3 did not cause an increase in the Ca2+ permeability of phospholipid vesicles (liposomes), indicating that this agent may bring about Ca2+ release by a specific effect on the physiologically relevant Ca2+ channels or carriers in the non-mitochondrial Ca2+ store site. The passive Ca2+ release by InsP3 was enhanced by ATP and an unhydrolyzable ATP analogue, 5'-adenylyimidodiphosphate, but not by ADP or AMP. The passive Ca2+ release by InsP3 was observed even at 0 degree C.     

Biphasic Ca2+ dependence of inositol 1,4,5-trisphosphate-induced Ca release in smooth muscle cells of the guinea pig taenia caeci

Ca2+ dependence of the inositol 1,4,5-trisphosphate (IP3)-induced Ca release was studied in saponin-skinned smooth muscle fiber bundles of the guinea pig taenia caeci at 20-22 degrees C. Ca release from the skinned fiber bundles was monitored by microfluorometry of fura-2. Fiber bundles were first treated with 30 microM ryanodine for 120 s in the presence of 45 mM caffeine to lock open the Ca-induced Ca release channels which are present in approximately 40% of the Ca store of the smooth muscle cells of the taenia. The Ca store with the Ca-induced Ca release mechanism was functionally removed by this treatment, but the rest of the store, which was devoid of the ryanodine-sensitive Ca release mechanism, remained intact. The Ca2+ dependence of the IP3-induced Ca release mechanism was, therefore, studied independently of the Ca-induced Ca release. The rate of IP3-induced Ca release was enhanced by Ca2+ between 0 and 300 nM, but further increase in the Ca2+ concentration also exerted an inhibitory effect. Thus, the rate of IP3-induced Ca release was about the same in the absence of Ca2+ and at 3 microM Ca2+, and was about six times faster at 300 nM Ca2+. Hydrolysis of IP3 within the skinned fiber bundles was not responsible for these effects, because essentially the same effects were observed with or without Mg2+, an absolute requirement of the IP3 phosphatase activity. Ca2+, therefore, is likely to affect the gating mechanism and/or affinity for the ligand of the IP3-induced Ca release mechanism. The biphasic effect of Ca2+ on the IP3-induced Ca release is expected to form a positive feedback loop in the IP3-induced Ca mobilization below 300 nM Ca2+, and a negative feedback loop above 300 nM Ca2+.     

Characterization of the inositol 1,4,5-trisphosphate-induced Ca2+ release in pancreatic beta-cells.

Pancreatic beta-cells isolated from obese-hyperglycaemic mice released intracellular Ca2+ in response to carbamoylcholine, an effect dependent on the presence of glucose. The effective Ca2+ concentration reached was sufficient to evoke a transient release of insulin. When the cells were deficient in Ca2+, the Ca2+ pool sensitive to carbamoylcholine stimulation was equivalent to that released by ionomycin. Unlike intact cells, cells permeabilized by high-voltage discharges failed to generate either inositol 1,4,5-triphosphate (InsP3) or to release Ca2+ after exposure to carbamoylcholine. However, the permeabilized cells released insulin sigmoidally in response to increasing concentrations of Ca2+. Also in the absence of functional mitochondria these cells exhibited a large ATP-dependent buffering of Ca2+, enabling the maintenance of an ambient Ca2+ concentration corresponding to about 150 nM even after several additional pulses of Ca2+. InsP3, maximally effective at 6 microM, promoted a rapid and pronounced release of Ca2+. The InsP3-sensitive Ca2+ pool was rapidly filled and lost its Ca2+ late after ATP depletion. The transient nature of the Ca2+ signal was not overcome by repetitive additions of InsP3. It was possible to restore the response to InsP3 after a delay of approx. 20 min, an effect which had less latency after the addition of Ca2+. These latter findings argue against degradation and/or desensitization as factors responsible for the transiency in InsP3 response. It is suggested that Ca2+ released by InsP3 is taken up by a part of the endoplasmic reticulum (ER) not sensitive to InsP3. On metabolism of InsP3, Ca2+ recycles to the InsP3-sensitive pool, implying that this pool indeed has a very high affinity for the ion. The presence of functional mitochondria did not interfere with the recycling process. The ER in pancreatic beta-cells is of major importance in buffering Ca2+, but InsP3 only modulates Ca2+ transport for a restricted period of time following immediately upon its formation. Thereafter the non-sensitive part of the ER takes over the continuous regulation of Ca2+ cycling.     

Distinct occurrence of phosphatidylinositol 4,5-bisphosphate-induced Ca2+ release and inositol 1,4,5-triphosphate-induced release in ATP-dependent Ca2+-transporting platelet microsomes

The effects of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) and inositol 1,4,5-triphosphate(InsP3) on the Ca2+ release from ATP-dependent Ca2+-transporting microsomes prepared from ox platelets were investigated. Under optimal conditions, both PtdInsP2 and InsP3 released Ca2+ from the microsomes in a similar dose-dependent manner. However, the maximal amount of Ca2+ released by InsP3 was almost one-fourth of that released by PtdInsP2. Neither PtdInsP2 nor InsP3 appeared to act as a Ca2+ ionophore since they showed no effect on the Ca2+ content of liposomes prepared from platelet microsomal lipids. InsP3-induced but not PtdInsP2-induced Ca2+ release was decreased with increasing extravesicular Ca2+ from 0.1 microM to 10 microM and it was completely inhibited by 10 microM Ca2+. PtdInsP2-induced but not InsP3-induced Ca2+ release was markedly inhibited by Mg2+, ruthenium red and neomycin. In addition, InsP3 could induce no additional Ca2+ release after the accumulated Ca2+ had been maximally released by PtdInsP2. These results indicate that PtdInsP2 releases Ca2+ from platelet microsomes more effectively than InsP3 by a mechanism distinct from that of InsP3-induced release, and further that InsP3-sensitive microsomes are included within the population of PtdInsP2-sensitive microsomes.     

Internal Ca2+ mobilization and secretion in bovine adrenal chromaffin cells

Since secretion from intact bovine adrenal chromaffin cells in response to depolarization by nicotine is triggered by a rise in the concentration of intracellular Ca2+ ([Ca2+]i) to about 200-300 nM above basal, it has been assumed that the failure of the inositol 1,4,5-trisphosphate (InsP3)-mobilizing muscarinic agonists to induce secretion reflects the fact that the 50 nM rise in [Ca2+]i they elicit is insufficient to trigger the exocytotic machinery. A recent report, however, has demonstrated that some of the nicotine-induced rise in [Ca2+]i could originate from the InsP3-releasable Ca2+ store. The role of this Ca2+ store in secretion from bovine adrenal chromaffin cells is therefore unclear. In order to investigate in more detail the role of the InsP3-sensitive Ca2+ store in secretion from these cells, we have used a combination of an InsP3-mobilizing muscarinic agonist and the sesquiterpene lactone thapsigargin (TG), which releases internal Ca2+ without concomitant breakdown of inositol lipids or protein kinase C activation, to examine the events which follow depletion of the releasable Ca2+ store in these cells. Monitoring [Ca2+]i using Fura-2 demonstrated that TG released Ca2+ from the InsP3-sensitive store and, additionally, that the Ca2+ response to TG was composed of two distinct, temporally separated, components: a) a slow (1 min) increase in [Ca2+]i to approximately 50 nM above basal that was independent of extracellular Ca2+ and b) the maintenance of this level at a new steady-state that was dependent on the continual entry of extracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibody to the inositol trisphosphate receptor blocks thimerosal-enhanced Ca(2+)-induced Ca2+ release and Ca2+ oscillations in hamster eggs.

The sulfhydryl reagent thimerosal enhanced the sensitivity of hamster eggs to injected inositol 1,4,5-trisphosphate (InsP3) or Ca2+ to generate regenerative Ca2+ release from intracellular pools. A monoclonal antibody (mAb) to the InsP3 receptor blocked both the InsP3-induced Ca2+ release (IICR) and Ca(2+)-induced Ca2+ release (CICR). The mAb also blocked Ca2+ oscillations induced by thimerosal. The results indicate that thimerosal enhances IICR sensitized by cytosolic Ca2+, but not CICR from InsP3-insensitive pools, and causes repetitive Ca2+ releases from InsP3-sensitive pools.     

Effects of Ca2+ and Mg2+ on ATP-dependent 45Ca2+ influx in A-431 human epidermoidal carcinoma cells

Upon stimulation with 10(-6) -10(-3) M ATP, A-431 human epidermoidal carcinoma cells incorporated radioactive calcium from their medium in a temperature-dependent manner. The rate of incorporation of 45Ca2+ was rapid for the initial 5 min, but decreased immediately thereafter. The preincubation of cells for 2 h in medium depleted of both Ca2+ and Mg2+ abolished the ATP-dependent 45Ca2+ incorporation, irrespective of whether or not the subsequent incubation medium contained Mg2+ ions. ATP-dependent 45Ca2+ incorporation could be restored by a second preincubation (1 h) in medium containing 1 mM Mg2+, but no Ca2+. The Mg2+ ions in the second preincubation medium could be replaced by Ca2+, Co2+, or Cu2+ for restoration of such activity. Elevation of inositol trisphosphate (InsP3) was observed in cells depleted of either Ca2+ or Mg2+, but not in cells depleted of both ions. A parallel effect was observed in changes in [Ca2+]i. Since the concentration of cytosolic calcium ions does not change by incubation of cells in medium depleted of and (or) restored with calcium ions, we conclude that either calcium or magnesium ions associated with some cellular component(s) are responsible for production of InsP3, which then supposedly mobilizes Ca2+ and provokes 45Ca2+ influx.     

Inositol 1,4,5-trisphosphate and intracellular Ca2+ homeostasis in clonal pituitary cells (GH3). Translocation of Ca2+ into mitochondria from a functionally discrete portion of the nonmitochondrial store

Ca2+-specific minielectrodes were used to monitor changes in the ambient free Ca2+ concentration [( Ca2+]a) maintained by the intracellular organelles of permeabilized GH3 cells. Mitochondria maintained a [Ca2+]a steady state of around 500 nM and displayed a very high capacity for Ca2+ uptake. A nonmitochondrial pool, tentatively identified as the endoplasmic reticulum (ER), displayed higher affinity for Ca2+ by maintaining a steady state of approximately 170 nM. The capacity of this pool was around 10 nmol/mg cell protein. Inositol 1,4,5-trisphosphate (InsP3) released Ca2+ specifically from the ER, with an EC50 of approximately 2 microM, and gave maximal release of around 4 nmol Ca2+/mg of cell protein. Repeated InsR3 additions under conditions allowing for functional mitochondrial transport resulted in successively attenuated peaks, leading eventually to the depletion of the InsP3 sensitive portion of the ER. However, Ca2+ could still be released from the total ER pool with the ATPase inhibitor, vanadate. This InsP3-insensitive store did not reaccumulate InsP3 releasable Ca2+ nor could it directly refill the sensitive pool. However, the attenuation of the InsP3 responses could be overcome by repleting the sensitive pool with exogenous Ca2+ or by inhibiting Ca2+ uptake into the mitochondria. The results suggest: 1) the ER is the major intracellular organelle buffering Ca2+ in nonstimulated GH3 cells; 2) InsP3 releases Ca2+ from only a portion of the ER; 3) the InsP3-sensitive and -insensitive ER pools are functionally distinct; 4) InsP3 addition results in a transfer of Ca2+ from the ER to the mitochondria.     

Regulation by Ca2+ and inositol 1,4,5-trisphosphate (InsP3) of single recombinant type 3 InsP3 receptor channels. Ca2+ activation uniquely distinguishes types 1 and 3 insp3 receptors

The inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP3R) is an endoplasmic reticulum-localized Ca2+ -release channel that controls complex cytoplasmic Ca(2+) signaling in many cell types. At least three InsP3Rs encoded by different genes have been identified in mammalian cells, with different primary sequences, subcellular locations, variable ratios of expression, and heteromultimer formation. To examine regulation of channel gating of the type 3 isoform, recombinant rat type 3 InsP3R (r-InsP3R-3) was expressed in Xenopus oocytes, and single-channel recordings were obtained by patch-clamp electrophysiology of the outer nuclear membrane. Gating of the r-InsP3R-3 exhibited a biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). In the presence of 0.5 mM cytoplasmic free ATP, r-InsP3R-3 gating was inhibited by high [Ca2+]i with features similar to those of the endogenous Xenopus type 1 Ins3R (X-InsP3R-1). Ca2+ inhibition of channel gating had an inhibitory Hill coefficient of approximately 3 and half-maximal inhibiting [Ca2+]i (Kinh) = 39 microM under saturating (10 microM) cytoplasmic InsP3 concentrations ([InsP3]). At [InsP3] < 100 nM, the r-InsP3R-3 became more sensitive to Ca2+ inhibition, with the InsP(3) concentration dependence of Kinh described by a half-maximal [InsP3] of 55 nM and a Hill coefficient of approximately 4. InsP(3) activated the type 3 channel by tuning the efficacy of Ca2+ to inhibit it, by a mechanism similar to that observed for the type 1 isoform. In contrast, the r-InsP3R-3 channel was uniquely distinguished from the X-InsP3R-1 channel by its enhanced Ca2+ sensitivity of activation (half-maximal activating [Ca2+]i of 77 nM instead of 190 nM) and lack of cooperativity between Ca2+ activation sites (activating Hill coefficient of 1 instead of 2). These differences endow the InsP3R-3 with high gain InsP3-induced Ca2+ release and low gain Ca2+ -induced Ca2+ release properties complementary to those of InsP3R-1. Thus, distinct Ca2+ signals may be conferred by complementary Ca2+ activation properties of different InsP3R isoforms.     

Chronic muscarinic stimulation of SH-SY5Y neuroblastoma cells suppresses inositol 1,4,5-trisphosphate action. Parallel inhibition of inositol 1,4,5-trisphosphate-induced Ca2+ mobilization and inositol 1,4,5-trisphosphate binding.

The possibility that chronic activation of the phosphoinositide-mediated signaling pathway modifies the Ca(2+)-mobilizing action of inositol 1,4,5-trisphosphate (InsP3) was examined. SH-SY5Y human neuroblastoma cells were exposed to carbachol, permeabilized electrically, loaded with 45Ca2+, and 45Ca2+ mobilization in response to exogenous InsP3 was assessed. In control permeabilized cells, InsP3 released 65 +/- 2% of sequestered 45Ca2+ (EC50 = 0.32 +/- 0.05 microM). Pre-treatment with carbachol reduced both maximal InsP3-induced 45Ca2+ release (to 34 +/- 3%, with half-maximal and maximal inhibition at approximately 3 and 6 h, respectively) and the potency of InsP3 (EC50 = 0.92 +/- 0.13 microM). This inhibitory effect of carbachol was half-maximal at approximately 5 microM, was mediated by muscarinic receptors, and was reversible following withdrawal of agonist. Pretreatment with phorbol 12,13-dibutyrate did not alter the maximal effect of InsP3 but doubled its EC50. Evidence suggesting that the inhibitory effects of carbachol pretreatment resulted from altered Ca2+ homeostasis was not forthcoming; both 45Ca2+ uptake and release induced by ionomycin and thapsigargin were identical in control and pretreated permeabilized cells, as were the characteristics of reuptake of released Ca2+. In contrast, carbachol pretreatment, without altering the affinity of InsP3 (Kd = 64 +/- 7 nM), reduced the density of [32P]InsP3-binding sites from 2.0 +/- 0.1 to 1.0 +/- 0.1 pmol/mg protein with a time course essentially identical to that for the reduction in responsiveness to InsP3. This effect was not mimicked by pretreatment of cells with phorbol 12,13-dibutyrate. These data indicate that chronic activation of phosphoinositide hydrolysis can reduce the abundance of InsP3 receptors and that this causes a reduction in size of the InsP3-sensitive Ca2+ store. This modification, possibly in conjunction with a protein kinase C-mediated event, appears to account for the carbachol-induced suppression of InsP3 action. As intracellular InsP3 mass remained elevated above basal for at least 24 h after addition of carbachol, suppression of the Ca(2+)-mobilizing activity of InsP3 represents an important adaptive response to cell stimulation that can limit the extent to which intracellular Ca2+ is mobilized.     

Effects of sulfhydryl reagents and other inhibitors on Ca2+ transport and inositol trisphosphate-induced Ca2+ release from human platelet membranes

The effects of modifiers of Ca2+ uptake and release in sarcoplasmic reticulum were studied in human platelet membranes. AgNO3,p-chloromercuribenzoate (pClHgBzO), N-ethylmaleimide (MalNEt), quercetin, vanadate, A23187, and caffeine all had the same effects on Ca2+ uptake in platelet membranes as had been observed for sarcoplasmic reticulum. These results strengthen our earlier conclusion that the Ca2+-pump proteins from internal human platelet membranes and muscle sarcoplasmic reticulum are very similar in functional properties. The sulfhydryl reagents Ag+ and pClHgBzO elicited rapid release of Ca2+ from platelet membranes in the presence of ATP, whereas MalNEt induced slow release. Quercetin also caused slow release of Ca2+ from platelet membranes in the presence of ATP. The effects of all three sulfhydryl reagents could be reversed by dithiothreitol, and Ag+-induced release was also reversed by ruthenium red. These effects are similar to those observed in sarcoplasmic reticulum, but in contrast caffeine did not induce Ca2+ release. In the absence of ATP, passively loaded platelet membranes did not release Ca2+ when exposed to sulfhydryl reagents. However, AgCl and pClHgBzO inhibited inositol trisphosphate (InsP3)-induced Ca2+ release from platelet membranes and this effect was reversed by dithiothreitol. Ruthenium red also inhibited InsP3-induced release, but ATP was found not to be required for InsP3-mediated release. LiCl enhanced Ca2+ release from platelet membranes. These results demonstrate that the InsP3-gated Ca2+ release channel is a separate entity from the Ca2+-pump and that essential protein sulfhydryls are involved in the release process.     

A high-affinity plasma membrane Ca2+-ATPase in Dictyostelium discoideum: its relation to cAMP-induced Ca2+ fluxes

Chemotactic stimulation of Dictyostelium discoideum induces an uptake of Ca2+ by the cells followed by a release of Ca2+. In this study we investigated the mechanism of Ca2+ release and found that it was inhibited by La3+, Cd2+ and azide. Ca2+ release occurred in the absence of external Na+, indicating that an Na+/Ca2+ exchange was not involved. Plasma membranes contained high- and low-affinity ATPase activities. Apparent K0.5 values were 8 microM for the major Mg2+-ATPase and 1.1 microM for the high-affinity Ca2+-ATPase, respectively. The Mg2+-ATPase activity was inhibited by elevated concentrations of Ca2+, whereas both Ca2+-ATPases were active in the absence of added Mg2+. The activities of the Ca2+-ATPases were not modified by calmodulin. The high-affinity Ca2+-ATPase was competitively inhibited by La3+ and Cd2+; we suggest that this high-affinity enzyme mediates the release of Ca2+ from D. discoideum cells.     

The inositol 1,4,5-trisphosphate receptor of cerebellum. Mn2+ permeability and regulation by cytosolic Mn2+

The inositol 1,4,5-trisphosphate receptor (InsP3R), an intracellular calcium release channel, is found in virtually all cells and is abundant in the cerebellum. We used Mn2+ as a tool to study two aspects of the cerebellar InsP3R. First, to investigate the structure of the ion pore, Mn2+ permeation through the channel was determined. We found that Mn2+ can pass through the InsP3R; the selectivity sequence for divalent cations is Ba2+ > Sr2+ > Ca2+ > Mg2+ > Mn2+. Second, to begin characterization of the cytosolic regulatory sites responsible for the Ca(2+)-dependent modulation of InsP3R function, the ability of Mn2+ to replace Ca2+ was investigated. We show that Mn2+, as Ca2+, modulates InsP3R activity with a bell-shaped dependence where the affinity of the activation site of the InsP3R is similar for both ions, but higher concentrations of Mn2+ were necessary to inhibit the channel. These results suggest that the two regulatory sites are structurally distinct. Our findings are also important for the understanding of cellular responses when Mn2+ is used to quench the intracellular fluorescence of Ca2+ indicator dyes.     

Regulation of inositol trisphosphate receptors by luminal Ca2+ contributes to quantal Ca2+ mobilization.

The quantal behaviour of inositol trisphosphate (InsP3) receptors allows rapid graded release of Ca2+ from intracellular stores, but the mechanisms are unknown. In Ca2+-depleted stores loaded with Fura 2, InsP3 caused concentration dependent increases in the rates of fluorescence quench by Mn2+ that were unaffected by prior incubation with InsP3, indicating that InsP3 binding did not cause desensitization. When Fura 2 was used to report the luminal free [Ca2+] after inhibition of further Ca2+ uptake, submaximal concentrations of InsP3 caused rapid, partial decreases in fluorescence ratios. Subsequent addition of a maximal InsP3 concentration caused the fluorescence to fall to within 5% of that recorded after ionomycin. Addition of all but the lowest concentrations of InsP3 to stores loaded with the lower affinity indicator, Calcium Green-5N, caused almost complete emptying of the stores at rates that increased with InsP3 concentration. The lowest concentration of InsP3 (10 nM) slowly emptied approximately 80% of the stores, but within 3 min the rate of Ca2+ release slowed leaving approximately 7 microM Ca2+ within the stores, which was then rapidly released by a maximal InsP3 concentration. In stores co-loaded with both indicators, InsP3-evoked Ca2+ release appeared quantal with Fura 2 and largely non-quantal with Calcium Green-5N; the discrepancy is not, therefore, a direct effect of the indicators. The fall in luminal [Ca2+] after activation of InsP3 receptors may, therefore, cause their inactivation, but only after the Ca2+ content of the stores has fallen by approximately 95% to < or = 10 microM.     

Kinetic properties of the purified Ca2+-translocating ATPase from human erythrocyte plasma membrane

The basic kinetic properties of the solubilized and purified Ca2+-translocating ATPase from human erythrocyte membranes were studied. A complex interaction between the major ligands (i.e., Ca2+, Mg2+, H+, calmodulin and ATP) and the enzyme was found. The apparent affinity of the enzyme for Ca2+ was inversely proportional to the concentration of free Mg2+ and H+, both in the presence or absence of calmodulin. In addition, the apparent affinity of the enzyme for Ca2+ was significantly increased by the presence of calmodulin at high concentrations of MgCl2 (5 mM), while it was hardly affected at low concentrations of MgCl2 (2 mM or less). In addition, the ATPase activity was inhibited by free Mg2+ in the millimolar concentration range. Evidence for a high degree of positive cooperativity for Ca2+ activation of the enzyme (Hill coefficient near to 4) was found in the presence of calmodulin in the slightly alkaline pH range. The degree of cooperativity induced by Ca2+ in the presence of calmodulin was decreased strongly as the pH decreased to acid values (Hill coefficient below 2). In the absence of calmodulin, the Hill coefficient was 2 or slightly below over the whole pH range tested. Two binding affinities of the enzyme for ATP were found. The apparent affinity of the enzyme for calmodulin was around 6 nM and independent of the Mg2+ concentration. The degree of stimulation of the ATPase activity by calmodulin was dependent on the concentrations of both Ca2+ and Mg2+ in the assay system.     

Competitive, reversible, and potent antagonism of inositol 1,4,5-trisphosphate-activated calcium release by heparin

The action of inositol 1,4,5-trisphosphate (InsP3) in releasing intracellular Ca2+ is shown to be competitively and potently antagonized by the glycosaminoglycan, heparin. Using either permeabilized cells of the DDT1MF-2 smooth muscle cell line, or an isolated microsomal membrane fraction derived from intact cells, heparin (4-6 kDa) at 10 micrograms/ml was observed to completely block the action of InsP3 in releasing Ca2+ accumulated via the ATP-dependent Ca2+ pump. In permeabilized cells, heparin had no effect on Ca2+ pump activity or on passive Ca2+ fluxes contributing to equilibrium Ca2+ accumulation. Heparin up to 100 micrograms/ml had no effect on the GTP-activated Ca2+ translocation process previously characterized in this cell line. Half-maximal inhibition of Ca2+ release activated by 10 microM InsP3 occurred with heparin at approximately 0.6 and 0.2 microgram/ml in permeabilized cells and isolated microsomes, respectively. Using microsomes, InsP3 dose-response curves in the presence and absence of 0.2 microgram/ml heparin (approximately 40 nM) revealed a 10-fold increase in apparent Km for InsP3 (0.31 microM in the absence of heparin) with no change in Vmax, indicating a competitive action of heparin. The results revealed a very high apparent affinity of heparin for the InsP3 active site, with a calculated Ki value of 2.7 nM. Heparin was shown to rapidly (within 20 s) reverse prior full activation of InsP3-mediated Ca2+ release returning the Ca2+ equilibrium back to that observed without InsP3. This reversal occurs even after prolonged (6 min) InsP3 activation. These results indicate a specific, high affinity, and competitive antagonism of the InsP3 active site by heparin. The rapidly induced reversal of InsP3-activated Ca2+ release by heparin strongly suggests that InsP3 directly activates a channel which remains open only while InsP3 is associated and closes immediately upon InsP3 dissociation.     

Ca2+-stimulated, Mg2+-dependent ATPase activity in neutrophil plasma membrane vesicles. Coupling to Ca2+ transport

Low concentrations of free Ca2+ stimulated the hydrolysis of ATP by plasma membrane vesicles purified from guinea pig neutrophils and incubated in 100 mM HEPES/triethanolamine, pH 7.25. In the absence of exogenous magnesium, apparent values obtained were 320 nM (EC50 for free Ca2+), 17.7 nmol of Pi/mg X min (Vmax), and 26 microM (Km for total ATP). Studies using trans- 1,2-diaminocyclohexane- N,N,N',N',-tetraacetic acid as a chelator showed this activity was dependent on 13 microM magnesium, endogenous to the medium plus membranes. Without added Mg2+, Ca2+ stimulated the hydrolysis of several other nucleotides: ATP congruent to GTP congruent to CTP congruent to ITP greater than UTP, but Ca2+-stimulated ATPase was not coupled to uptake of Ca2+, even in the presence of 5 mM oxalate. When 1 mM MgCl2 was added, the vesicles demonstrated oxalate and ATP-dependent calcium uptake at approximately 8 nmol of Ca2+/mg X min (based on total membrane protein). Ca2+ uptake increased to a maximum of approximately 17-20 nmol of Ca2+/mg X min when KCl replaced HEPES/triethanolamine in the buffer. In the presence of both KCl and MgCl2, Ca2+ stimulated the hydrolysis of ATP selectively over other nucleotides. Apparent values obtained for the Ca2+-stimulated ATPase were 440 nM (EC50 for free Ca2+), 17.5 nmol Pi/mg X min (Vmax) and 100 microM (Km for total ATP). Similar values were found for Ca2+ uptake which was coupled efficiently to Ca2+-stimulated ATPase with a molar ratio of 2.1 +/- 0.1. Exogenous calmodulin had no effect on the Vmax or EC50 for free Ca2+ of the Ca2+-stimulated ATPase, either in the presence or absence of added Mg2+, with or without an ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid pretreatment of the vesicles. The data demonstrate that calcium stimulates ATP hydrolysis by neutrophil plasma membranes that is coupled optimally to transport of Ca2+ in the presence of concentrations of K+ and Mg2+ that appear to mimic intracellular levels.     

Glucagon and vasopressin interactions on Ca2+ movements in isolated hepatocytes.

The effects of glucagon and vasopressin, singly or together, on cytosolic free Ca2+ concentration [( Ca2+]i) and on the 45Ca2+ efflux were studied in isolated rat liver cells. In the presence of 1 mM external Ca2+, glucagon and vasopressin added singly induced sustained increases in [Ca2+]i. The rate of the initial fast phase of the [Ca2+]i increase and the magnitude of the final plateau were dependent on the concentrations (50 pm-0.1 microM) of glucagon and vasopressin. Preincubating the cells with a low concentration of glucagon (0.1 nM) for 2 min markedly accelerated the fast phase and elevated the plateau of the [Ca2+]i increase caused by vasopressin. In the absence of external free Ca2+, glucagon and vasopressin transiently increased [Ca2+]i and stimulated the 45Ca2+ efflux from the cells, indicating mobilization of Ca2+ from internal store(s). Preincubating the cells with 0.1 nM-glucagon accelerated the rate of the fast phase of the [Ca2+]i rise caused by the subsequent addition of vasopressin. However, unlike what was observed in the presence of 1 mM-Ca2+, glucagon no longer enhanced the maximal [Ca2+]i response to vasopressin. In the absence of external free Ca2+, higher concentrations (1 nM-0.1 microM) of glucagon, which initiated larger increases in [Ca2+]i, drastically decreased the subsequent Ca2+ response to vasopressin (10 nM). At these concentrations, glucagon also decreased the vasopressin-stimulated 45Ca2+ efflux from the cells. It is suggested that, in the liver, glucagon accelerates the fast phase and elevates the plateau of the vasopressin-mediated [Ca2+]i increase respectively by releasing Ca2+ from the same internal store as that permeabilized by vasopressin, probably the endoplasmic reticulum, and potentiating the influx of extracellular Ca2+ caused by this hormone.     

Influence of Mg ions and spermine on ATP-dependent Ca2+ transport in myometrial intracellular structures. II. Comparative study of spermine, Mg ions and cyclosporin A effects on Ca2+ transport in mitochondria

In experiments carried out with the use of the radioactive label (45Ca2+) on suspension of the rat uterus myocytes processed by digitonin solution (0.1 mg/ml), influence of spermine and cyclosporin A on Mg2+, ATP-dependent Ca2+ transport in mitochondria at different Mg2+ concentration were investigated. Ca2+ accumulation in mitochondria was tested as such which was not sensitive to thapsigargin (100 nM) and was blocked by ruthenium red (10 microM). It has been shown, that spermine (1 mM) stimulates Mg2+, ATP-dependent Ca2+ accumulation in mitochondria irrespective of Mg2+ concentration (3 or 7 mM) in the incubation medium. At the same time cyclosporin A (5 microM) effects on Ca2+ accumulation in mitochondria depend on Mg2+ concentration in the incubation medium: at 3 mM Mg2+ the stimulating effect was observed, and at 7 mM Mg2+ - the inhibitory one. In conditions which led to the increase of nonspecific mitochondrial permeability and, accordingly, to dissipation of electrochemical potential (it was reached by 5 min. preincubation of myocytes suspension in the medium that contained 10 microM Ca2+, 2 mM phosphate and 3 or 7 mM Mg2+, but not ATP) significant inhibition of Mg2+, ATP-dependent Ca2+ accumulation in mitochondria was observed. The inhibition to the greater degree was observed when medium ATP and Mg2+ were absent simultaneously in the preincubation. Thus the quality of spermine effects on Ca2+ accumulation was kept: stimulation in the presence both of 3 mM and 7 mM Mg2+. Ca2+ accumulation did not reach the control level when 3 mM Mg2+ and 1 mM spermine was present and ATP absent in the preincubation medium. However, in the presence of 7 mM Mg2+ and 1 mM spermine practically full restoration (up to a control level) of Ca2+ accumulation was observed. At the same time with other things being equal such restoration was not observed at simultaneous absence of ATP and Mg2+ in the preincubation medium. The quality of cyclosporin A effects on Ca2+ accumulation in mitochondria was also kept: stimulation - in the presence of 3 mM Mg2+, inhibition - in the presence of 7 mM Mg2+ in the preincubation medium. And, at last, in the presence of cyclosporin A irrespective of the fact which preincubation medium was used, Ca2+ accumulation level practically did not depend on Mg2+ concentration.     

Granulocyte-macrophage colony-stimulating factor can stimulate macrophage proliferation via persistent activation of Na+/H+ antiport. Evidence for two distinct roles for Na+/H+ antiport activation.

Thapsigargin stimulates an increase of cytosolic free Ca2+ concentration [( Ca2+]c) in, and 45Ca2+ efflux from, a clone of GH4C1 pituitary cells. This increase in [Ca2+]c was followed by a lower sustained elevation of [Ca2+]c, which required the presence of extracellular Ca2+, and was not inhibited by a Ca2(+)-channel blocker, nimodipine. Thapsigargin had no effect on inositol phosphate generation. We used thyrotropin-releasing hormone (TRH) to mobilize Ca2+ from an InsP3-sensitive store. Pretreatment with thapsigargin blocked the ability of TRH to cause a transient increase in both [Ca2+]c and 45Ca2+ efflux. The block of TRH-induced Ca2+ mobilization was not caused by a block at the receptor level, because TRH stimulation of InsP3 was not affected by thapsigargin. Rundown of the TRH-releasable store by Ca2(+)-induced Ca2+ release does not appear to account for the action of thapsigargin on the TRH-induced spike in [Ca2+]c, because BAY K 8644, which causes a sustained rise in [Ca2+]c, did not block Ca2+ release caused by TRH. In addition, caffeine, which releases Ca2+ from intracellular stores in other cell types, caused an increase in [Ca2+]c in GH4C1 cells, but had no effect on a subsequent spike in [Ca2+]c induced by TRH or thapsigargin. TRH caused a substantial decrease in the amount of intracellular Ca2+ released by thapsigargin. We conclude that in GH4C1 cells thapsigargin actively discharges an InsP3-releasable pool of Ca2+ and that this mechanism alone causes the block of the TRH-induced increase in [Ca2+]c.