Increase In 45Ca Efflux Research Articles

Possible mechanisms of inhibitory action of protamine on contractile activity of rat aorta.

Experiments were performed to determine possible mechanisms of inhibitory action of protamine chloride on noradrenaline (10 microM)-, KCl (40 mM)-, BaCl2 (1 mM)- and CaCl2 (10 mM)-induced contractions in rat aorta. Protamine, La3+ and gallopamil (D600), inhibited the K+-induced contractions more effectively than the noradrenaline-induced responses on the basis of the concentrations giving 40% inhibition. Lanthanum (1-5 mM) reduced tissue Ca content in both normal and Ca2+-depleted Tris-buffered solutions and produced an increase in 45Ca efflux from the aortic strip into the Ca2+-depleted Tris solution. Protamine (1-5 mg ml-1) reduced tissue Ca content in normal Tris solution, but to a lesser extent than La3+ in the Ca2+-depleted solution. Furthermore, protamine (3 mg ml-1) produced no increase in 45Ca efflux from aorta. These results suggest that protamine chloride may preferentially inhibit the Ca2+ influx stimulated by K+ depolarization and that its inhibitory action on rat aorta may be due to non-specific displacement of the superficially located bound Ca2+ of the cell membrane, which can also be readily removed by treatment with Ca2+-depleted solution.

Effects of chlorbutol on 45Ca movements and contractile responses of rat aorta and its relevance to the actions of Syntocinon.

The effects of chlorbutol (0.7, 1.4 and 2.8 mM) on the contractile responses induced by KCl and noradrenaline (NA) and on 45Ca movements have been studied on rat isolated thoracic aorta. Chlorbutol decreased, in a dose-dependent manner, contractions induced by KCl and NA and this effect was observed whether it was added before or after the induced contractions. Preincubation with chlorbutol inhibited the contractile responses elicited by addition of Ca (1-5 mM) to Ca-free high-potassium solution. It also inhibited in a dose-dependent manner the 45Ca influx but increased 45Ca efflux in rat aortic strips. These results suggest that chlorbutol decreases peripheral resistance by reducing the availability of intracellular Ca to the contractile machinery in vascular smooth muscle cells. The effects of synthetic oxytocin (Syntocinon) at concentrations containing the same chlorbutol concentration were quantitatively similar from those produced by chlorbutol alone. Therefore, the inhibitory cardiovascular effects ascribed previously to synthetic oxytocin may be attributed to its preservative, chlorbutol, and not to oxytocin itself.

ATP pulse and calcium homeostasis in cells from hepatopancreas of Dilocarcinus pagei, a freshwater crab

Calcium (Ca) is critical for crustaceans due to their molting cycle and its presence in the carapace as calcium carbonate, apart from the usual functions of Ca, such as cell signalling. Ca transport in Dilocarcinus pagei, a freshwater crab, was studied in isolated cells from hepatopancreas to further characterize Ca transport mechanisms in these crabs. Cells were isolated and loaded with Fluo-3, a calcium fluorescent dye. Three different cell treatments were performed: Group 1 cells were Ca free during cell dissociation, and calcium was present (at 1mM) for fluorescence cell loading and transport experiments (FC); Group 2 cells were calcium free during cell dissociation and for transport experiments, but not during cell loading (LC); and Group 3 cells were Ca free during cell dissociation, cell loading and transport experiments (WC). Intracellular Ca was recorded through time after ATP was added to the cells and ATP caused an increase in Ca efflux within 30s in all cells. WC cells showed the smallest Ca efflux compared to the other cells, probably because it was intracellularly Ca “depleted”. Vanadate and amiloride decreased the Ca efflux when ATP was added to the cells, while verapamil did not cause any effect in Ca efflux, confirming the presence of a Ca2+-ATPase sensitive to vanadate in hepatopancreas of D. pagei. In a different set of experiments, cells were also exposed to a Ca pulse of 1 and 10mM during 180s. 10mM Ca increased intracellular Ca compared to 1mM, and the increase was not recovered during the experimental time. Additionally, Ca influx was reduced by verapamil and amiloride, but not completely. The results suggest that Ca influx probably occurs through an undefined exchanger, apart from Ca channels (verapamil sensitive) and electrogenic 1Na+(1H+)/1 Ca2+ exchanger (amiloride-sensitive). Similarities between freshwater and seawater crabs, lobsters and crayfish in relation to plasma membrane Ca transporters, although the environment where they live is quite diverse, suggest that universal mechanisms for Ca homeostasis are widespread among crustaceans.

Anomeric Specificity of the Stimulatory Effect ofd-Glucose on d-Fructose Phosphorylation by Human Liver Glucokinase

D-Glucose was recently reported to stimulate d-fructose phosphorylation by human B-cell glucokinase. The present study aims at investigating the anomeric specificity of such a positive cooperativity. The alpha-anomer of D-glucose was found to increase much more markedly than beta-D-glucose the phosphorylation of D-fructose by human liver glucokinase. Such an anomeric preference diminished at high concentrations of the D-glucose anomers, i.e. when the effect of the aldohexose upon d-fructose phosphorylation became progressively less marked. A comparison between the effects of the two anomers of D-glucose and those of equilibrated D-glucose upon D-fructose phosphorylation by human liver glucokinase indicated that the results obtained with the equilibrated aldohexose were not significantly different from those expected from the combined effects of each anomers of D-glucose. In isolated rat islets incubated for 60 min at 4 degrees C, alpha-D-glucose (5.6 mm), but not beta-D-glucose (also 5.6 mm), augmented significantly the conversion of D-[U-(14)C]fructose (5.0 mm) to acidic radioactive metabolites. Likewise, in islets prelabeled with (45)Ca and perifused at 37 degrees C, D-fructose (20.0 mm) augmented (45)Ca efflux and provoked a biphasic stimulation of insulin release from islets exposed to alpha-D-glucose (5.6 mm), while inhibiting (45)Ca efflux and causing only a sluggish and modest increase in insulin output from islets exposed to beta-D-glucose (also 5.6 mm). The enhancing action of D-glucose upon D-fructose phosphorylation by glucokinase thus displays an obvious anomeric preference for alpha-D-glucose, and such an anomeric specificity remains operative in intact pancreatic islets.

Tetracaine stimulates insulin secretion through the mobilization of Ca2+from thapsigargin- and IP3-insensitive Ca2+reservoir in pancreatic β-cells

The effect of tetracaine on45Ca efflux, cytoplasmic Ca2+concentration [Ca2+]i, and insulin secretion in isolated pancreatic islets and β-cells was studied. In the absence of external Ca2+, tetracaine (0.1-2.0 mM) increased the45Ca efflux from isolated islets in a dose-dependant manner. Tetracaine did not affect the increase in45Ca efflux caused by 50 mM K+or by the association of carbachol (0.2 mM) and 50 mM K+. Tetracaine permanently increased the [Ca2+]iin isolated β-cells in Ca2+-free medium enriched with 2.8 mM glucose and 25 µM D-600 (methoxiverapamil). This effect was also observed in the presence of 10 mM caffeine or 1 µM thapsigargin. In the presence of 16.7 mM glucose, tetracaine transiently increased the insulin secretion from islets perfused in the absence and presence of external Ca2+. These data indicate that tetracaine mobilises Ca2+from a thapsigargin-insensitive store and stimulates insulin secretion in the absence of extracellular Ca2+. The increase in45Ca efflux caused by high concentrations of K+and by carbachol indicates that tetracaine did not interfere with a cation or inositol triphosphate sensitive Ca2+pool in β-cells.

Tetracaine stimulates insulin secretion through the mobilization of Ca<sup>2+</sup> from thapsigargin- and IP<sub>3</sub>-insensitive Ca<sup>2+</sup> reservoir in pancreatic β-cells

The effect of tetracaine on 45Ca efflux, cytoplasmic Ca2+ concentration [Ca2+]i, and insulin secretion in isolated pancreatic islets and beta-cells was studied. In the absence of external Ca2+, tetracaine (0.1-2.0 mM) increased the 45Ca efflux from isolated islets in a dose-dependentOFF efflux caused by 50 mM K+ or by the association of carbachol (0.2 mM) and 50 mM K+. Tetracaine permanently increased the [Ca2+]i in isolated beta-cells in Ca2+-free medium enriched with 2.8 mM glucose and 25 microM D-600 (methoxiverapamil). This effect was also observed in the presence of 10 mM caffeine or 1 microM thapsigargin. In the presence of 16.7 mM glucose, tetracaine transiently increased the insulin secretion from islets perfused in the absence and presence of external Ca2+. These data indicate that tetracaine mobilises Ca2+ from a thapsigargin-insensitive store and stimulates insulin secretion in the absence of extracellular Ca2+. The increase in 45Ca efflux caused by high concentrations of K+ and by carbachol indicates that tetracaine did not interfere with a cation or inositol triphosphate sensitive Ca2+ pool in beta-cells.

Protein kinase C activation blocks calcium receptor signaling in Xenopus laevis oocytes

We examined whether calcium receptor (CaR) signaling is affected by protein kinase C (PKC) activation by assessing the effects of phorbol-12-myristate-13-acetate (PMA) on 45Ca 2+ efflux from Xenopus laevis oocytes expressing wild-type (WT) and mutant bovine parathyroid CaRs. Raising extracellular [Ca 2+] ([Ca 2+] 0) from 0.5 to 5.5 mM increased 45Ca efflux (26±3-fold) in oocytes expressing full-length and C-terminally truncated receptor (amino acid 1–895). These increases in 45Ca efflux were blocked by 88±3% after PMA treatment for 20 min. Three consensus PKC phosphorylation sites (Thr-647, Ser-795, and Thr-889) were mutated in the context of the full-length and truncated CaR. PMA treatment inhibited high [Ca 2+] 0-induced responses in oocytes expressing the Ser795Ala CaR (1–895), Thr889Ala CaR (1–895), and Ser795Ala/Thr889Ala CaR (1–895) by 30–40% compared with untreated controls ( P<0.05). A triple mutant of the full-length CaR demonstrated similarly reduced susceptibility to inhibition of 45Ca efflux by PMA. Thus, these sites are important in mediating the effects of PKC activation on CaRs, but other residues and effector molecules are likely to participate in the effects of PKC on CaR-induced signal transduction in target cells.

Effect of D-mannoheptulose upon the cationic and secretory responses to alpha-D-glucose pentaacetate in perifused rat pancreatic islets.

The metabolism of alpha-D-glucose pentaacetate and its positive insulinotropic action in isolated rat pancreatic islets are both unexpectedly resistant to D-mannoheptulose, as judged from experiments conducted over 90-120 min incubation. In the present study, the possible effects of the heptose upon the immediate cationic and secretory response to the ester were investigated in perifused islets prelabeled with either 86Rb or 45Ca. At a 10 mM concentration, sufficient to abolish the inhibitory action of unesterified D-glucose upon 86Rb outflow, D-mannoheptulose failed to suppress the decrease in 86Rb outflow and increase in 45Ca efflux caused by alpha-D-glucose pentaacetate at normal extracellular Ca2+ concentration and also failed to prevent the decrease in both 45Ca and insulin release provoked by the ester in the absence of extracellular Ca2+. The sole obvious effect of the heptose was to change the early peak-shaped positive secretory response to alpha-D-glucose pentaacetate to a transient inhibition of insulin release. This change was observed in islets either deprived of any other exogenous nutrient or exposed to L-leucine throughout the experiments. These findings support the view that the islet functional response to alpha-D-glucose pentaacetate is largely resistant to D-mannoheptulose. They also reinforce the concept that the insulinotropic action of this and other monosaccharide esters involves a dual modality of B-cell activation, linked to both the catabolism of their carbohydrate moieties and a direct effect of the esters themselves upon a specific receptor system.

Coupling of calcium receptors to inositol phosphate and cyclic AMP generation in mammalian cells and Xenopus laevis oocytes and immunodetection of receptor protein by region-specific antipeptide antisera.

Ca2+ and other divalent cations modulate parathyroid hormone secretion by interacting with cell-surface Ca2+-sensing receptors (CaRs). We assessed the ability of these receptors to couple to Ca2+ mobilization, inositol phosphate (InsP) accumulation, and cyclic AMP production in different expression systems. In Xenopus laevis oocytes injected with bovine parathyroid CaR cRNA, the addition of extracellular cations to 1.5 mM Ca2+, 5.5 mM Mg2+, or 10 microM Gd3+ significantly increased 45Ca efflux (p < 0.01). InsP accumulation also increased dramatically when adding these cations to human embryonic kidney (HEK) 293 cells stably transfected with wild-type bovine parathyroid CaR cDNA. Raising the extracellular [Ca2+] ([Ca2+]o) from 0.1 to > 1.4 mM in oocytes and to > 1.0 mM in HEK 293 cells stimulated significant increments in 45Ca efflux and InsP accumulation, respectively (p < 0.05). In contrast, Ca2+ and Mg2+ increased InsPs to a lesser extent in COS 7 cells transiently transfected with CaR cDNA. In HEK 293 cells stably expressing CaR cDNA, there were significant reductions in cAMP content when adding high Ca2+, Mg2+, Gd3+, or the CaR modulator NPS R-467. Three region-specific anti-CaR peptide antisera immunoblotted bands of approximately 140 and 155 kDa in membranes from CaR-transfected HEK 293 cells and bovine parathyroid tissue. Immunocytochemistry demonstrated strong cell-surface staining in CaR-transfected HEK 293 cells and parathyroid tissue, which was absent when antisera were preabsorbed with CaR peptides. These results indicate that the activation of the recombinant CaR by extracellular Ca2+ can couple negatively to adenylate cyclase but positively to phospholipase C (PLC), the latter at physiological [Ca2+]o.

Tetracaine stimulates extracellular Ca 2+-independent insulin release

The effect of the local anesthetic, tetracaine, on 45Ca efflux, cytoplasmic Ca 2+ concentration [Ca 2+] i and insulin secretion in pancreatic B-cells was studied. At a physiological level of [Ca 2+] o, tetracaine (0.1–5 mM) dose-dependently inhibited insulin secretion induced by 22 mM glucose. Paradoxically, at the same glucose concentration but in the absence of external Ca 2+, tetracaine dose-dependently increased insulin secretion. At a low glucose level (2.8 mM) tetracaine failed to affect secretion, either in the presence or absence of external Ca 2+. At high (22 mM) or low (2.8 mM) glucose, [Ca 2+] i was increased by tetracaine in a dose-dependent manner. Tetracaine (2 mM) also increased the 45Ca efflux from isolated islets. This effect was of the same magnitude at both low and high glucose concentrations, and was independent of the presence of extracellular Ca 2+. Finally, tetracaine increased 45Ca efflux from islets perifused in the presence of thapsigargin. In conclusion, our data indicate that tetracaine releases Ca 2+ from a thapsigargin-insensitive store in pancreatic B-cells. Under suitable experimental conditions, insulin release can be elicited by a [Ca 2+] o-independent pathway. The existence of a ryanodine-like Ca 2+ channel in pancreatic B-cells is proposed.

N-methyl-D-aspartate-induced 45Ca2+ release from pre-labelled adult rat hippocampus in vivo.

We report the results of microdialysis experiments investigating the NMDA-induced release of intracellular Ca2+ in different brain regions. Microdialysis probes were implanted stereotaxically into the striatum, thalamus and hippocampus dentate gyrus (DG) of adult rats. Dialysates were analysed for alterations in the concentration of ionized Ca2+ in an initially calcium-free medium and for changes in 45Ca efflux from the pre-labelled endogenous Ca2+ pools. The application of 5 mM of NMDA to the dialysis medium for 20 min in the striatum, resulted in increases in Ca2+ and 45Ca concentrations by 25% and 35% respectively. After NMDA perfusion in the hippocampus DG and in the thalamus, decreases in the Ca2+ concentration to 65.6% and 38.6% of the basal level respectively, were accompanied by increases in 45Ca efflux, exceeding 1,500% of the basal level in the hippocampus. Cell swelling, and the corresponding reduction of the extracellular space volume was insufficient to explain the huge increase in 45Ca efflux. Thus, our experiments demonstrated that in vivo in the rat hippocampus DG, NMDA induces the release of 45Ca to the extracellular space from unidentified intracellular calcium stores.

Differential effects of N-methyl-d-aspartate on Ca2+ homeostasis in developing and adult rat striatum: in vivo microdialysis approach

This in vivo study concerns developmental differences in the sensitivity of striatal neurons to N-methyl-d-aspartate (NMDA). Changes in calcium homeostatis in adult vs immature rats at postnatal days 8–10, evoked by NMDA, were evaluated by measurements of 45Ca efflux and of Ca2+ taurine and phosphoethanolamine concentrations in striatal microdialysates. The efflux of [14C]sucrose was employed in order to measure changes in extracellular space volume. In adult rats the addition of 5 mM NMDA for 20 min to the perfusion medium resulted in a 30–40% increase in 45Ca efflux, and in a 15% increase in [14C]sucrose efflux. Ten minutes after NMDA perfusion, 45Ca and [14C]sucrose efflux returned to the baseline. No significant changes in Ca2+ or amino acid concentrations were observed in the dialysate of the adult rat striatum. NMDA perfusion in the striatum of immature rats initially induced a transient (5 min) increase in the efflux of 45Ca (by 13%) and [14C]sucrose (by 9%). This was followed by a prolonged (lasting 45–50 min) 45% decrease in 45Ca efflux, an 80% reduction of Ca2+ concentration, and increases in taurine and phosphoethanolamine concentrations in the dialysate, whereas [14C]sucrose efflux recovered within 10 min.These data illustrate differences in the NMDA response between developing and adult rat striatum. Only in developing rats did NMDA induce a large and prolonged influx of extracellular calcium to neurons that may explain the enhanced NMDA neurotoxicity in immature rats.

Effect of N-methyl-D,L-aspartate on isolated rat somatotrophs

The effect of excitatory amino acid receptor agonists on GH secretion was tested in isolated male rat somatotrophs. N-Methyl-D,L-Aspartate (NMDA) had a dose-dependent stimulatory effect on GH secretion in perifused somatotrophs. The effect was observed already during the first minute after exposure to NMDA and was reversible after its omission. The effect of 1 microM NMDA was inhibited by the NMDA receptor antagonists 10 microM AP7 and 5 microM MK801, and by 5 microM dextromethorphan. L-Glutamate, 100 microM, and 100 microM kainic acid also stimulated GH secretion. The stimulatory effect of NMDA on GH release was paralleled by an increase in 45Ca efflux and an increase in somatotroph intracellular calcium concentration. Efflux of 86Rb (tracer for potassium) was not affected by NMDA. It is concluded that excitatory amino acids can stimulate GH secretion in rats through a direct effect on the somatotrophs.

Effect of N-methyl-D,L-aspartate on isolated rat somatotrophs.

The effect of excitatory amino acid receptor agonists on GH secretion was tested in isolated male rat somatotrophs. N-Methyl-D,L-Aspartate (NMDA) had a dose-dependent stimulatory effect on GH secretion in perifused somatotrophs. The effect was observed already during the first minute after exposure to NMDA and was reversible after its omission. The effect of 1 microM NMDA was inhibited by the NMDA receptor antagonists 10 microM AP7 and 5 microM MK801, and by 5 microM dextromethorphan. L-Glutamate, 100 microM, and 100 microM kainic acid also stimulated GH secretion. The stimulatory effect of NMDA on GH release was paralleled by an increase in 45Ca efflux and an increase in somatotroph intracellular calcium concentration. Efflux of 86Rb (tracer for potassium) was not affected by NMDA. It is concluded that excitatory amino acids can stimulate GH secretion in rats through a direct effect on the somatotrophs.

Ionic, electrical, and secretory effects of inhibitors of arachidonic acid metabolism in mouse pancreatic beta-cells.

Mouse islets were used to study the effects of inhibitors of cyclooxygenase and lipoxygenase pathways on insulin release, ionic fluxes, and beta-cell membrane potential. The cyclooxygenase inhibitors, Na-salicylate and Na-acetylsalicylate, potentiated glucose-induced insulin release, despite a decrease in Ca influx evidenced by inhibition of the Ca-dependent electrical activity in beta-cells and 45Ca efflux from islets perifused with a medium containing Ca. This paradox can probably be explained by a mobilization of intracellular Ca (acceleration of 45Ca efflux in the absence of Ca) with subsequent activation of K+ channels (acceleration of 86Rb efflux) and repolarization of the membrane. These effects of salicylate could not be ascribed to a change in intracellular pH because they were not mimicked by 2-Cl-benzoate, which has a similar pK as salicylate but increased insulin release by stimulating Ca influx in beta-cells. Among the other cyclooxygenase inhibitors tested, indomethacin caused a slight potentiation of insulin release accompanied by marginal increases in 45Ca efflux and electrical activity, whereas flurbiprofen and ibuprofen were ineffective. Among the lipoxygenase inhibitors, compound BW 755c reversibly decreased glucose-induced insulin release by inhibiting Ca influx in beta-cells, but nordihydroguaiaretic acid had no effect. Inhibitors of arachidonic acid metabolism have effects on ionic fluxes and beta-cell membrane potential, which may explain some of the changes in insulin release they produce.

Cationic and Secretory Effects of Glimepiride and Glibenclamide in Perifused Rat Islets

The effects of glimepiride and glibenclamide upon 86Rb outflow, 45Ca outflow and insulin release were examined in rat islets perifused at low (zero to 2.8 mM) or close-to-normal (8.3 mM) D-glucose concentrations. At the low hexose concentrations, a marked and not reversible decrease in 86Rb outflow contrasted with a rapid and reversible increase in 45Ca outflow. The latter increase was abolished in the absence of extracellular Ca2+, and was not associated with any pronounced stimulation of insulin release. Inversely, in the presence of 8.3 mM D-glucose, a comparable increase in 45Ca efflux now coincided with an increase in 86Rb efflux and a marked and not reversible stimulation of insulin release. Whether in terms of the time course or glucose dependency of the cationic and secretory responses, a coupled increase in both 40Ca inflow and 45Ca outflow thus coincided with either negative or positive changes in 86Rb outflow and either minimal or marked changes in insulin output. Such dissociated behaviours suggest that the insulinotropic action of hypoglycemic sulfonylureas is not necessarily attributable solely to a primary decrease in K+ conductance.

Prolactin Increases Cytosolic Free Calcium Concentration in Hepatocytes of Lactating Rats*

PRL at a physiological concentration (10(-8) M) produced a very rapid and transient increase in 45Ca efflux in freshly isolated hepatocytes, which reached the highest value within 5 min and returned to baseline level after 20 min. PRL-induced 45Ca2+ efflux resulted in a loss of 15% of total cell calcium, which was similar to that found in vasopressin-treated cells. However, in contrast with the PRL effect, 45Ca2+ efflux induced by vasopressin was sustained. We demonstrate by using two different approaches, glycogen phosphorylase-a activation and direct cytosolic calcium concentration [( Ca2+]i) measurements, that PRL elicits a [Ca2+]i increase. The treatment of hepatic cells with PRL caused a 4-fold stimulation in glycogen phosphorylase-alpha activity after 2 min of PRL addition. Direct [Ca2+]i determination in fluo-3-loaded hepatocytes showed a 11% increase after 5 min of PRL addition. Similar data were observed in hepatocytes stimulated either with vasopressin (10(-7) M) or calcium ionophore A23187 (200 nM). The increase in [Ca2+]i promoted by PRL was independent of extracellular calcium or voltage-operated calcium channels. The data demonstrate that calcium is involved in the intracellular signaling of PRL in liver cells and that PRL initiates its action by a Ca2+ mobilization from the intracellular stores.

Effect of membrane potential on H1-receptor-mediated 45Ca influx in coronary artery.

These studies were undertaken to determine whether agonist-induced 45Ca influx in the coronary artery is modified under conditions in which Na+ pump activity (and consequently membrane potential) is altered. 45Ca influx and contraction were measured in the rabbit coronary artery with the H1-receptor agonist 2-(2-aminoethyl)pyridine (AEP, 10(-4) M) after prolonged Na+ pump block (leading to profound depolarization) and after enhanced Na+ pump activity (leading to hyperpolarization). AEP contracted vessels, enhanced 45Ca influx into the smooth muscle, and increased 45Ca efflux. The AEP-induced contraction was reduced but not abolished with either nifedipine (10(-6) M) or Ca(2+)-free solution. In tissues subjected to prolonged Na+ pump block AEP caused contraction but 45Ca influx was not increased over the unstimulated tissue. Ca(2+)-free solution and nifedipine reduced but did not abolish these contractions. In tissues with enhanced Na+ pump activity AEP caused contraction but 45Ca influx was not increased. Ca(2+)-free solution reduced these contractions but nifedipine did not. We conclude that the measurable 45Ca influx changes in a manner that is compatible with the characteristics of voltage-gated Ca2+ channels that have a low open probability during hyperpolarization and are inactivated by a period of long slow depolarization.

Calcium-dependent and independent mechanisms of contraction in canine lingual artery to U-46619.

The thromboxane A2/prostaglandin H2 (TXA2/PGH2) mimetic, U-46619, is a potent platelet aggregating agent and constrictor of vascular smooth muscle. Aggregation and contraction to U-46619, and other TXA2/PGH2 mimetics, such as U-44069 and carbocyclic TXA2, are associated with both an influx of extracellular Ca2+ and a release of Ca2+ from binding and sequestration sites within the platelet and arterial smooth muscle (Greenberg, 1981; Loutzenhiser and van Breemen, 1981; Dorn et al., 1987; Santoian et al., 1987). These mechanisms were shown by the ability of both calcium channel blocking agents and intracellular calcium antagonists, such as dantrolene sodium and TMB-8, to inhibit contraction of vascular smooth muscle to TXA2/PGH2 receptor mimetics and by the ability of these mimetics to increase 45Ca efflux without affecting 45Ca uptake (Ally et al., 1978; Greenberg, 1981; Loutzenhiser and Van Breemen, 1981; Angerio et al., 1982; Dorn et al., 1987; Santoian et al., 1987). The pool of Ca2+ utilized for the physiologic and pharmacologic actions of U-46619 appears to depend on the site and species from which the arteries and platelets are obtained (Greenberg, 1981; Loutzenhiser and Van Breemen, 1981; Angerio et al., 1982; Dorn et al, 1987; Santoian et al., 1987; Verheyen et al., 1989).

Intracellular alkalinization leads to Ca2+ mobilization from agonist-sensitive pools in bovine aortic endothelial cells.

Receptor-stimulated phosphoinositide turnover leads to activation of Na+/H+ exchange and subsequent intracellular alkalinization. To probe the effect of increased intracellular pH (pHi) on Ca2+ homeostasis in cultured bovine aortic endothelial cells (BAEC), we studied the effect of weak bases, ammonium chloride (NH4Cl) and methylamine (agents which increase pHi by direct passive diffusion), on resting and ATP (purinergic receptor agonist)-induced Ca2+ fluxes. Changes in cytosolic free Ca2+ ([Ca2+]i) or pHi were monitored in BAEC monolayers using the fluorescent dyes, fura-2 or 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, respectively. NH4Cl-induced, dose-dependent (5-20 mM) increases in [Ca2+]i (maximum change = 195 +/- 26 nM) which were temporally similar to the NH4Cl-induced pHi increases. Methylamine (20 mM) induced a more sustained pHi increase and also stimulated a prolonged [Ca2+]i increase. When BAEC were bathed in HCO3- buffer, removal of extracellular CO2/bicarbonate caused pHi to increase and also induced [Ca2+]i to increase transiently. Extracellular Ca2+ removal did not abolish the rapid NH4Cl-induced rise in [Ca2+]i, although the response was blunted and more transient. NH4Cl addition to BAEC cultures resulted in an increase in 45Ca efflux and decrease in total cell 45Ca content. BAEC treatment with ATP (100 microM) to deplete inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pools completely blocked the NH4Cl (20 mM)-induced rise in [Ca2+]i. Likewise, prior NH4Cl addition partially inhibited ATP-induced increases in [Ca2+]i, as well as slowed the frequency of repetitive [Ca2+]i spikes in single endothelial cells due to agonist. NH4Cl augmented the rate of [Ca2+]i increase that occurs in response to the depletion of agonist-sensitive intracellular Ca2+ pools. However, the internal Ca2+ store remained depleted during the continued presence of NH4Cl, as indicated by a decreased [Ca2+]i response to ATP in Ca2(+)-free medium. Finally, NH4Cl exerted these actions without affecting basal or ATP-stimulated IP3 formation. These observations provide direct evidence that increased pHi leads to Ca2+ mobilization from an agonist-sensitive pool and impairs Ca2+ pool(s) refilling mechanisms without altering cellular IP3 levels.