Potentiation by Ca2+ ionophores and inhibition by extracellular KCl of endothelin-induced phosphoinositide turnover in C6 glioma cells.

Interactions between endothelin-1 (ET)-induced phosphoinositide (PI) hydrolysis and agents that increase Ca2+ influx (i.e. A23187 and ionomycin) or induce depolarization (i.e. KCl) were investigated using C6 glioma. A23187 dose-dependently potentiated ET (30 nM)- and ATP (100 microM)-induced [3H]inositol phosphate (IP) accumulation. This potentiation was associated with an increase in the maximal stimulation elicited by both ET and ATP but their EC50 values were unchanged. This effect of A23187 occurred at concentrations that did not affect basal PI turnover; i.e. 10 nM-3 microM. Ionomycin within the range of 1 nM-1 microM also significantly enhanced ET-induced PI breakdown and this effect was associated with an increase of [Ca2+]i. KCl in a concentration-dependent manner (14.7-54.7 mM) markedly inhibited PI breakdown elicited by ET and ATP, but had much less inhibition on basal activity and no effect on A23187- and ionomycin-induced responses. In parallel, KCl added before or after ET, sharply attenuated the increase of ET-induced [Ca2+]i but did not affect basal level or ionomycin-induced [Ca2+]i response. Neither the potentiation by A23187 nor the inhibition by KCl of ET-induced PI turnover was observed in cultured cerebellar astrocytes. Our results suggest that the cell type-specific regulation by Ca2+ ionophores and KCl on ET-induced PI metabolism is closely related to perturbation of [Ca2+]i.     

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

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

Galanin Reduces Carbachol Stimulation of Phosphoinositide Turnover in Rat Ventral Hippocampus by Lowering Ca2+ Influx Through Voltage-Sensitive Ca2+ Channels

The 29-amino-acid peptide galanin (GAL) caused concentration-dependent inhibition of the accumulation of 3H-inositol phosphates (3H-InsPs) induced by the muscarinic agonist carbachol (CARB; 10(-3)-10(-5) M) in the presence of 5 mM lithium, specifically in tissue miniprisms from rat ventral hippocampus. The inhibitory effect of GAL involved the mono-, bis-, tris-, and tetrakisphosphates formed during activation for 2 min of phospholipase C by CARB (1 mM) in the absence of lithium. GAL (1 microM) did not affect alpha-adrenergic or serotonergic type 2 receptor-mediated phosphoinositide (PI) breakdown in the same tissue. GAL by itself neither acted on basal levels of 3H-InsPs nor affected muscarinic receptors in binding studies. Blockade of the T-, N-, and L-types of voltage-sensitive calcium channel (VSCC) with 200 microM Cd2+ reduced muscarinic receptor-mediated PI breakdown by 50% and abolished the inhibitory effect of GAL (1 microM). Reduction of the extracellular Ca2+ concentration from 1.3 mM to 0.49 microM abolished the GAL inhibition of CARB-stimulated PI hydrolysis. Ca2+ influx promoted by 18 mM K+ depolarization or by 1 microM Bay K 8644, a selective agonist of the L-type VSCC, prevented the inhibitory effect of GAL. Blockade of the L-type VSCC with nifedipine (1 microM) potentiated the inhibitory effects of GAL without affecting muscarinic stimulation of PI breakdown.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+-stimulated phospholipid phosphoesterase activities in rabbit erythrocyte membranes

The properties of the enzymes involved in Ca2+-stimulated breakdown of phosphatidylinositol 4'-phosphate (PIP), phosphatidylinositol 4',5'-bisphosphate (PIP2), and phosphatidic acid (PA) in rabbit erythrocyte ghosts were studied. At 25 degrees C, 1 to 180 microM Ca2+ rapidly stimulated the breakdown of PIP and PIP2, and maximal breakdown occurred within 10 minutes at all Ca2+ concentrations. The rate and the total amount of breakdown of PA, PIP, and PIP2 increased with Ca2+ concentration. MgCl2 inhibited the rate of Ca2+-stimulated breakdown of PIP and PIP2 at Ca2+ concentrations less than 10 microM, but did not have any appreciable effects at higher Ca2+ concentrations. MgCl2 also protected against Ca2+-stimulated breakdown of PA. In the presence and absence of 5 mM MgCl2, Ca2+ stimulated half-maximal breakdown of PIP and PIP2 at 2-3 microM under hypotonic and isotonic conditions. In the presence of 5 mM MgCl2, Ca2+-stimulated breakdown of PIP and PIP2 was associated with the release of Pi and inositol bisphosphate. In the absence of MgCl2, Ca2+ stimulated the release of 32P-labeled Pi, inositol bisphosphate, and inositol trisphosphate from labeled PIP, PIP2, and PA. Ca2+ increased phosphatidylinositol content and decreased PIP and PIP2 content in these membranes. The results of this investigation suggest that Ca2+ stimulates the breakdown of polyphosphoinositides by stimulating polyphosphoinositide phosphomonoesterase and phosphodiesterase activities in rabbit erythrocyte ghosts. These activities were activated by less than 3 microM Ca2+ in the presence of MgCl2 under hypotonic or isotonic conditions. These Ca2+-stimulated polyphosphoinositide phosphoesterase activities could therefore be active under physiological conditions in normal rabbit erythrocytes.     

Concerted stimulation of PI-turnover, Ca2+-influx and histamine release in antigen-activated rat mast cells

Phospholipid metabolism in rat mast cells activated by antigen was examined with reference to phosphatidylinositol (PI) turnover. Upon antigen stimulation, histamine release from passively sensitized mast cells with IgE was potentiated by adding phosphatidylserine (PS). The addition of antigen to [3H]glycerol-prelabeled and sensitized mast cells induced a marked loss of radioactivity of PI and a concurrent accumulation of 1,2-diacylglycerol (DG) and phosphatidic acid (PA) within 5 to 60 sec. Furthermore, this antigen-induced PI breakdown was enhanced in the presence of Mg2+. Histamine release occurred in parallel with PI breakdown. On the other hand, the transient Ca2+ influx into mast cells, as measured by uptake of 45Ca2+, was found to occur quickly after cells were activated by antigen, which was concerted with PI breakdown. These results suggest that enhanced PI turnover may be an important step in the biochemical sequence of events leading to release of histamine, and that not only Ca2+ but also Mg2+ appears to take a part in stimulus-response coupling in rat mast cells.     

The rise in concentration of free Ca2+ and of pH provides sequential, synergistic signals for secretion in antigen-stimulated rat basophilic leukemia (RBL-2H3) cells

Ag stimulation of rat basophilic leukemia (RBL-2H3) cells results in hydrolysis of inositol phospholipids, a transient increase in concentration of cytosol Ca2+ [( Ca2+]i), a gradual increase in cytosolic pH (pHi) and the activation of protein kinase C. To determine whether all these changes serve as signals for secretion, studies were conducted with cells permeabilized with streptolysin O in which pHi and [Ca2+]i could be varied independently of each other and enzyme activities could be manipulated. At resting pHi (approximately 7.0) and [Ca2+]i (0.1 microM), the permeabilized cells showed little secretory response to Ag. At resting pHi, elevated levels of Ca2+ (0.33 microM) were required for maximal secretory response to Ag. At a pHi of 7.4, however, 0.1 microM [Ca2+]i was sufficient to sustain near maximal responses to Ag. Therefore, a small increase of [Ca2+]i to 0.33 microM was required to initiate secretion, but once the pHi was elevated secretion could be sustained at near basal levels of [Ca2+]i. Since elevating the [Ca2+]i and pHi, by themselves promoted little secretion, another potentiating signal must have been generated by antigen stimulation. This signal was possibly transduced via hydrolysis of inositol phospholipids and protein kinase C. Even with an elevated [Ca2+]i (0.33 microM) the hydrolysis of the phospholipids and secretion stimulated by Ag were inhibited by guanosine 5'(2-O-thio)diphosphate and neomycin. Furthermore, both protein-kinase C and the secretory response to Ag were lost after permeabilized cells were washed but both were retained if cells were exposed to PMA before permeabilization.     

Effects of Na+, Ca2+, and Acetylcholine on Phosphoinositide- and ATP-Phosphate Turnover in 32P-Labeled Rabbit Iris Smooth Muscle

Parallel studies were carried out in the rabbit iris on (a) the effects of Na+ and/or Ca2+ on the acetylcholine-stimulated 32P labeling of phosphatidic acid (PA) and phosphatidylinositol (PI) and the breakdown of polyphosphoinositides (poly PI), and (b) the effects of these cations on the specific radioactivity of [gamma-32P]ATP. Incorporation of 32P1 into ATP and phosphoinositides is time-dependent, and it is remarkably dependent upon Na+ concentration in the incubation medium. The Na+ effect is reversible. Calcium ion, in the absence of Na+, had no effect on the specific radioactivity of ATP in 32P-labeled iris muscle; however, it moderately stimulated the 32P labeling of PA and PI and the breakdown of poly PI. In contrast, the addition of Na+, in the presence or absence of Ca2+, significantly reduced the specific radioactivity of ATP and 32P labeling of phospholipids in the 32P-labeled iris muscle. Acetylcholine had no measurable effect on the specific radioactivity of ATP. Furthermore, the neurotransmitter stimulated the 32P labeling of PA and PI and the breakdown of poly PI in the 32P-labeled muscle only in the presence of both Na+ and Ca2+. These data provide additional support for the concept that in the rabbit iris receptor-activated Ca2+ fluxes mediate or precede the effects of alpha-adrenergic and cholinergic muscarinic agents on phosphoinositide breakdown into 1,2-diacylglycerol and inositol phosphates and that restoration of the polar head groups to the 1,2-diacylglycerol (i.e., the recovery stage) is probably associated with Na+ outflux, via the Na+ -pump mechanism.     

The relationship of calcium to receptor-controlled stimulation of phosphatidylinositol turnover. Effects of acetylcholine, adrenaline, calcium ions, cinchocaine and a bivalent cation ionophore on rat parotid-gland fragments.

The possibility that Ca2+ ions are involved in the control of the increased phosphatidylinositol turnover which is provoked by alpha-adrenergic or muscarinic cholinergic stimulation of rat parotid-gland fragments has been investigated. Both types of stimulation provoked phosphatidylinositol breakdown, which was detected either chemically or radiochemically, and provoked a compensatory synthesis of the lipid, detected as an increased rate of incorporation of 32Pi into phosphatidylinositol. Acetylcholine had little effect on the incorporation of labelled glycerol, whereas adrenaline stimulated it significantly, but to a much lower extent than 32P incorporation: this suggests that the response to acetylcholine was entirely accounted for by renewal of the phosphorylinositol head-group of the lipid, but that some synthesis de novo was involved in the response to adrenaline. The responses to both types of stimulation, whether measured as phosphatidylinositol breakdown or as phosphatidylinositol labelling, occurred equally well in incubation media containing 2.5 mm-Ca2+ or 0.2 mm-EGTA [ethanedioxybis(ethylamine)-tetra-acetic acid]. Incubation with a bivalent cation ionophore (A23187) led to a small and more variable increase in phosphatidylinositol labelling with 32Pi, which occurred whether or not Ca2+ was available in the extracellular medium: this was not accompanied by significant phosphatidylinositol breakdown. Cinchocaine, a local anaesthetic, produced parallel increases in the incorporation of Pi and glycerol into phosphatidylinositol. This is compatible with its known ability to inhibit phosphatidate phosphohydrolase (EC 3.1.3.4) and increase phosphatidylinositol synthesis de novo in other cells. These results indicate that the phosphatidylinositol turnover evoked by alpha-adrenergic or muscarinic cholinergic stimuli in rat parotid gland probably does not depend on an influx of Ca2+ into the cells in response to stimulation. This is in marked contrast with the K+ efflux from this tissue, which is controlled by the same receptors, but is strictly dependent on the presence of extracellular Ca2+. The Ca2+-independence of stimulated phosphatidylinositol metabolism may mean that it is controlled through a mode of receptor function different from that which controls other cell responses. Alternatively, it can be interpreted as indicating that stimulated phosphatidylinositol breakdown is intimately involved in the mechanisms of action of alpha-adrenergic and muscarinic cholinergic receptor systems.     

U73122 inhibits Ca2+ oscillations in response to cholecystokinin and carbachol but not to JMV-180 in rat pancreatic acinar cells.

Stimulation of rat pancreatic acinar cells with low concentrations of phosphatidylinositol (PI)-linked secretagogues induces [Ca2+]i oscillations, without measurable changes in the formation of inositol 1,4,5-trisphosphate. Therefore, we tested U73122 a new phospholipase C inhibitor to determine if PI turnover is necessary for the generation of [Ca2+]i oscillations. In acini prelabeled with [3H]inositol, PI hydrolysis on stimulation with either cholecystokinin or carbachol was inhibited dose-dependently by U73122, with a maximal effect seen at 10 microM; the formation of inositol 1,4,5-trisphosphate, measured using a radioreceptor assay, was also similarly inhibited. By contrast secretin- or vasoactive intestinal peptide-stimulated production of cAMP was unaffected by 10 microM U73122. These studies indicate that U73122 is a relatively specific inhibitor of G-protein-mediated phospholipase C activation in pancreatic acini. In fura-2-loaded acini, U73122 inhibited the increases in [Ca2+]i stimulated by these high concentrations of secretagogues which can be demonstrated to elicit PI turnover. The [Ca2+]i signal generated by directly stimulating G-proteins with sodium fluoride was also inhibited by U73122; however, the [Ca2+]i rise induced by thapsigargin was unaffected. These data indicate that the mechanism of inhibition was distal to the occupation of cell surface receptors but did not involve an interference of Ca2+ metabolism in general. When [Ca2+]i oscillations were elicited by low concentrations of cholecystokinin or carbachol, U73122 rapidly inhibited the oscillating [Ca2+]i signal. In contrast, oscillations induced by an analogue of cholecystokinin, JMV-180, which does not stimulate changes in PI metabolism at any concentration, were unaffected. This indicates that cholecystokinin- and carbachol-induced oscillations are probably initiated by small, localized changes in PI metabolism, which are not readily detectable. However, the inability of U73122 to inhibit JMV-180-induced oscillations indicates that PI metabolism may not necessarily be a prerequisite for the generation of [Ca2+]i oscillations.     

The effect of heart and skeletal muscle troponin complexes and calmodulin on the Ca2+-dependent reactions of phosphorylase kinase isoenzymes

The dephosphorylated form of phosphorylase kinase was purified 700-fold from rabbit heart extract. The purified enzyme had a pH 6.8/pH 8.2 activity ratio of 0.04-0.08 and was completely dependent on Ca2+ with an apparent Ka value for Ca2+ of 2.59 microM at pH 6.8. At free Ca2+ concentrations between 0.057 microM and 400 microM, 1.5 microM rabbit heart troponin complex had no significant effect on the reaction. However, 1.5 microM rabbit skeletal muscle troponin complex stimulated the reaction 1.5-2-fold with a concomitant decrease in the Ka value for Ca2+ to 1.40 microM. No differences in the effects of these troponin complexes were observed when heart-type and skeletal muscle-type phosphorylase b isoenzymes from either rabbit or pig were used as substrate. Similar effects of heart and skeletal muscle troponin complexes were observed on the Ca2+-dependent reaction of the dephosphorylated form of phosphorylase kinase partially purified from rabbit skeletal muscle. A saturating concentration (1.36 microM) of bovine brain calmodulin stimulated 2-5-fold the Ca2+-dependent reaction of skeletal muscle phosphorylase kinase, but not the reaction of heart phosphorylase kinase. Heart troponin complex (12 microM) suppressed 80-100% the stimulatory effect of skeletal muscle troponin complex on the reactions of phosphorylase kinase isoenzymes, but had no significant effect on the stimulation by calmodulin of skeletal muscle phosphorylase kinase reaction.     

Palmitic acid stimulates glucose incorporation in the adipocyte by a mechanism likely involving intracellular calcium

The effect of palmitic acid on basal and insulin-stimulated incorporation of glucose into rat adipocytes was studied. Palmitic acid (2.40 mM) stimulated basal as well as insulin-stimulated glucose incorporation in rat adipocytes three and twofold, respectively. Similar degrees of stimulation of basal glucose oxidation by palmitate were also observed. The ability of palmitic acid to stimulate glucose uptake was additive with respect to the stimulation induced by insulin and was proportional to the palmitic acid concentration between 0.15 mM and 2.40 mM. Stimulation of glucose incorporation by palmitic acid was inhibited by preincubating the cells with quin2-AM, which accumulates intracellularly yielding the trapped chelator form. quin2, which binds intracellular Ca2+.The concentration of quin2-AM required for half-maximal inhibition of palmitic acid stimulated glucose incorporation was 3.8 +/- 1.2 microM (mean +/- SEM). The inhibition of palmitic acid-stimulated glucose incorporation by quin2-AM (10 microM) was overcome by incubating cells with the Ca2+ ionophore, A23187, in the presence of extracellular Ca2+ (2.6 mM). Chelation of extracellular Ca2+ with EGTA did not significantly affect the magnitude of palmitic acid-stimulated glucose incorporation. Dantrolene (12.5-100 microM) failed to affect basal or palmitic acid-stimulated glucose incorporation. These findings suggest that palmitic acid stimulates incorporation of glucose in the adipocyte by a mechanism dependent upon intracellular but not extracellular Ca2+.     

Agonist-mediated Ca2+ release in permeabilized UMR-106-01 cells. Transport properties and generation of inositol 1,4,5-trisphosphate

Permeabilized and intact UMR-106-01 cells attached to culture plates or coverslips were used to evaluate compartmentalized generation and the effective concentration of inositol 1,4,5-trisphosphate (In-1,4,5-P3) during agonist-mediated Ca2+ release. In permeabilized cells, Ca2+ release had the following characteristics. In-1,4,5-P3 released approximately 65% of the Ca2+ incorporated into intracellular stores. Prostaglandin F2 alpha (PGF2 alpha), endothelin, or GTP(gamma S) alone released a small amount or no Ca2+. However, the agonists together with GTP(gamma S) were as effective as In-1,4,5-P3 in releasing Ca2+. Both agonist- and In-1,4,5-P3-mediated Ca2+ release required the presence of permeable ion. Agonists, like In-1,4,5-P3, stimulated 45Ca uptake from low Ca2+ medium devoid of permeable ions into Ca2(+)-loaded intracellular stores. The permeabilized cell system was then used to evaluate compartmentalized generation and action of In-1,4,5-P3 during agonist stimulation. Mass measurement shows that in intact resting cells In-1,4,5-P3 concentration was 1.4 microM and was reduced to 0.05 microM following permeabilization. Stimulation with agonists increases In-1,4,5-P3 concentration from 0.05 to 0.34 microM. Ca2+ release by this concentration of In-1,4,5-P3 evenly distributed in the cytosol can account for only part of the agonist-mediated Ca2+ release. However, the effects of saturating In-1,4,5-P3 concentration and agonists were blocked by the specific inhibitor heparin. Measurement of heparin dependency of In-1,4,5-P3-mediated Ca2+ release was used to calculate an affinity for In-1,4,5-P3 of 0.39 microM. Similar measurements with agonists show that In-1,4,5-P3 concentration at the site of Ca2+ release during agonist stimulation is 11.2 microM. Hence, the total increase in In-1,4,5-P3 is reflected in considerably higher localized concentrations. This is interpreted to suggest compartmentalized generation and action of In-1,4,5-P3 during agonist stimulation.     

Cyclic AMP stimulates luteinizing-hormone (lutropin) exocytosis in permeabilized sheep anterior-pituitary cells. Synergism with protein kinase C and calcium.

Sheep anterior-pituitary cells permeabilized with Staphylococcus aureus alpha-toxin were used to investigate the role of cyclic AMP (cAMP) in exocytosis of luteinizing hormone (lutropin, LH) under conditions where the intracellular free Ca2+ concentration ([Ca2+]free) is clamped by Ca2+ buffers. At resting [Ca2+]free (pCa 7), cAMP rapidly stimulated LH exocytosis (within 5 min) and continued to stimulate exocytosis for at least 30 min. When cAMP breakdown was inhibited by 3-isobutyl-1-methylxanthine (IBMX), the concentration giving half-maximal response (EC50) for cAMP-stimulated exocytosis was 10 microM. cAMP-stimulated exocytosis required millimolar concentrations of MgATP, as has been found with Ca2(+)- and phorbol-ester-stimulated LH exocytosis. cAMP caused a modest enhancement of Ca2(+)-stimulated LH exocytosis by decreasing in the EC50 for Ca2+ from pCa 5.6 to pCa 5.9, but had little effect on the maximal LH response to Ca2+. Activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) dramatically enhanced cAMP-stimulated LH exocytosis by both increasing the maximal effect 5-7-fold and decreasing the EC50 for cAMP to 3 microM. This synergism between cAMP and PMA was further augmented by increasing the [Ca2+]free. Gonadotropin-releasing hormone (gonadoliberin, GnRH) stimulated cAMP production in intact pituitary cells. Since GnRH stimulation is reported to activate PKC and increase the intracellular [Ca2+]free, our results suggest that a synergistic interaction of the cAMP, PKC and Ca2+ second-messenger systems is of importance in the mechanism of GnRH-stimulated LH exocytosis.     

Muscarinic Cholinergic Stimulation of Exogenous Phosphatidylinositol Hydrolysis Is Regulated by Guanine Nucleotides in Rabbit Brain Cortical Membranes

Rabbit brain cortical membranes, which have been extracted with 2 M KCl, hydrolyze exogenously added [3H]phosphatidylinositol [( 3H]PI) in a guanine nucleotide- and carbachol-dependent manner. Both oxotremorine-M and carbachol are full agonists with EC50 values of 8 and 73 microM, respectively. Pirenzepine and atropine inhibit carbachol-stimulated [3H]PI hydrolysis. The hydrolysis-resistant guanine nucleotide analog guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) is the most potent in supporting carbachol-stimulated hydrolysis of PI. There is no effect of carbachol in the absence of guanine nucleotides or in the presence of 100 microM adenosine 5'-O-(3-thiotriphosphate), adenosine-5'-(beta, gamma-imido)triphosphate, or sodium pyrophosphate. Guanylyl-5'-(beta,gamma-imido)triphosphate [Gpp(NH)p] in the presence of carbachol also stimulates PI hydrolysis although much less than that seen with GTP gamma S. GDP and Gpp(NH)p are potent antagonists of the GTP gamma S-dependent carbachol response. Optimal stimulation by carbachol and GTP gamma S was observed at 0.3-1 microM free Ca2+ and 6 mM MgCl2. Limited trypsinization resulted in loss of receptor-regulated PI breakdown and a slight decrease in basal activity. These results demonstrate that phospholipase C hydrolysis of exogenous PI by rabbit cortical membranes may be stimulated by carbachol in a guanine nucleotide-dependent manner.     

Ca2+ dependence of stimulated 45Ca efflux in skinned muscle fibers

Stimulation of sarcoplasmic reticulum Ca release by Mg reduction of caffeine was studied in situ, to characterize further the Ca2+ dependence observed previously with stimulation by Cl ion. 45Ca efflux and isometric force were measured simultaneously at 19 degrees C in frog skeletal muscle fibers skinned by microdissection; EGTA was added to chelate myofilament space Ca either before or after the stimulus. Both Mg2+ reduction (20 or 110 microM to 4 microM) and caffeine (5 mM) induced large force responses and 45Ca release, which were inhibited by pretreatment with 5 mM EGTA. In the case of Mg reduction, residual efflux stimulation was undetectable, and 45Ca efflux in EGTA at 4 microM Mg2+ was not significantly increased. Residual caffeine stimulation at 20 microM Mg2+ was substantial and was reduced further in increased EGTA (10 mM); at 600 microM Mg2+, residual stimulation in 5 mM EGTA was undetectable. Caffeine appears to initiate a small Ca2+-insensitive efflux that produces a large Ca2+-dependent efflux. Additional experiments suggested that caffeine also inhibited influx. The results suggest that stimulated efflux is mediated mainly or entirely by a channel controlled by an intrinsic Ca2+ receptor, which responds to local [Ca2+] in or near the channel. Receptor affinity for Ca2+ probably is influenced by Mg2+, but inhibition is weak unless local [Ca2+] is very low.     

Inositol Phospholipid Hydrolysis in Rat Cerebral Cortical Slices: II. Calcium Requirement

The calcium requirement for agonist-dependent breakdown of phosphatidylinositol and polyphosphoinositides has been examined in rat cerebral cortex. The omission of added Ca2+ from the incubation medium abolished [3H]inositol phosphate accumulation from prelabelled phospholipid induced by histamine, reduced that due to noradrenaline and 5-hydroxytryptamine, but did not affect carbachol-stimulated breakdown. EC50 values for agonists were unaltered in the absence of Ca2+. Removal of Ca2+ by preincubation with EGTA (0.5 mM) abolished all responses, but complete restoration was achieved by replacement of Ca2+. The EC50 for Ca2+ for histamine-stimulated [3H]inositol phosphate accumulation was 80 microM. Noradrenaline-stimulated breakdown was antagonised by manganese (IC50 1.7 mM), but not by the calcium channel blockers nitrendipine or nimodipine (30 microM). The calcium ionophore A23187 stimulated phosphatidylinositol/polyphosphoinositide hydrolysis with an EC50 of 2 microM, and this response was blocked by EGTA. Omission of Ca2+ or preincubation with EGTA or Mn2+ (EC50 = 230 microM) greatly enhanced the incorporation of [3H]inositol into phospholipids. The IC50 for Ca2+ in inhibiting incorporation was 25 microM. The results show that different receptors mediating phosphatidylinositol/polyphosphoinositide breakdown in rat cortex have quantitatively different Ca2+ requirements, and it is suggested that rigid opinions regarding phosphatidylinositol/polyphosphoinositide breakdown as either cause or effect of calcium mobilisation in rat cortex are inappropriate.     

Cadmium-induced insulin release does not involve changes in intracellular handling of calcium

A possible interaction between Cd2+ and Ca2+ as a component in Cd2+-induced insulin release was investigated in beta cells isolated from obese hyperglycemic mice. The glucose stimulated Cd2+ uptake was dependent on the concentration of sugar. This uptake was sigmoidal with a Km for glucose of about 5 mM and was suppressed by both 50 microM of the voltage-activated Ca2+ channel blocker D-600 and 12 mM Mg2+. In the presence of 8 mM glucose 5 microM Cd2+ evoked a prompt and sustained stimulatory response, corresponding to about 3-fold of the insulin release obtained in the absence of the ion. Whereas 5 microM Cd2+ was without effect on the glucose-stimulated 45Ca efflux in the presence of extracellular Ca2+, 40 microM inhibited it. At a concentration of 5 microM, Cd2+ had no effect on the resting membrane potential or the depolarization evoked by either glucose or K+. In the absence of extracellular Ca2+ there was only a modest stimulation of 45Ca efflux by 5 microM Cd2+. Studies of the ambient free Ca2+ concentration maintained by permeabilized cells also indicate that 5 microM Cd2+ do not mobilize intracellularly bound Ca2+ to any great extent. On the contrary, at this concentration, Cd2+ even suppressed inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release. The present study suggests that Cd2+ stimulates insulin release by a direct mechanism which does not involve an increase in cytoplasmic free Ca2+ concentration.     

Modulation of carbachol-stimulated inositol phospholipid breakdown in rat cerebral cortical miniprisms by excitatory amino acids and by BAY K-8644 is dependent upon the assay calcium and potassium concentrations used.

The calcium and potassium ion dependency of the inositol phospholipid breakdown response to stimulatory agents has been investigated in rat cerebral cortical miniprisms. The calcium channel agonist BAY K-8644 (10 microM) potentiated the response to carbachol at 6 mM K+ when Ca2(+)-free, but not when 2.52 mM Ca2+ assay buffer was used. In Ca2(+)-free buffer, verapamil (10 microM) inhibited the response to carbachol at both 6 and 18 mM K+ but higher concentrations (30-300 microM) were needed when 2.52 mM Ca2+ was used. At these higher concentrations, however, verapamil inhibited the binding of 2 nM [3H]pirenzepine to muscarinic recognition sites. N-Methyl-D-Aspartate (NMDA, 100 microM) significantly reduced the basal phosphoinositide breakdown rate at 18 mM K+ at 1.3 mM Ca2+, but was without effect on the basal rate at other K+ and Ca2+ concentrations. In the presence of NMDA (100 microM) or quisqualate (100 microM), the responses to carbachol were reduced, the degree of reduction showing a complex dependency upon the assay K+ and Ca2+ concentrations used. These results indicate that the inositol phospholipid breakdown response to carbachol in cerebral cortical miniprisms can be modulated in a manner dependent upon the extracellular calcium and potassium concentrations used.     

Dose-dependency in spatial dynamics of [Ca2+]c in adrenal chromaffin cells

The spatial dynamics of cytosolic Ca2+ concentration, [Ca2+]c, in guinea pig adrenal chromaffin cells was monitored by a digital image analysing technique using Fura-2. When a freshly isolated cluster of cells was stimulated with lower concentrations of carbachol (CCh; 0.3-1 microM), the [Ca2+]c began to increase in the region beneath the plasma membrane facing the extracellular environment. The [Ca2+]c increase depended on the presence of extracellular Ca2+ ([Ca2+]o). CCh at a higher concentration (100 microM), however, caused [Ca2+]c increase even in the absence of [Ca2+]o. These results are compatible with the view that the receptor activation with a physiological concentration of secretagogue accelerates Ca2+ entry, and that stimulation with a higher concentration of the secretagogue induces small transient Ca2+ release from intracellular stores and predominant continuous Ca2+ entry.     

Demonstration of Na+-dependent Ca2+ efflux using low concentrations of fluorometric Ca2+ probes

The effects of different concentrations of the fluorometric Ca2+ probes, fura-2 and indo-1, on Ca2+ transients in cultured rat aortic smooth muscle cells were examined. When stimulated with the agonists, angiotensin II and arginine vasopressin, cells incubated with low concentrations of fura-2 or indo-1 (less than 1 microM) produced Ca2+ transients characterized by a small increase followed by a dramatic decrease in fluorescence below the original baseline. This effect of agonists was concentration-dependent, reversible, and blocked by receptor antagonists. In contrast to the agonists, stimulation of Ca2+ transients with depolarizing concentrations of K+ or with caffeine did not produce decreases in fluorescence and Ca2+ levels at any loading concentration of probe. The decrease in Ca2+ observed with agonists was dependent on the presence of extracellular Na+. These data suggest that under certain loading conditions, fluorescent Ca2+ indicators measure agonist-stimulated Ca2+ efflux mediated by a Na+/Ca2+ exchange mechanism.