Comparison of effects of a potassium channel opener BRL34915, a specific potassium ionophore valinomycin and calcium channel blockers on endothelin-induced vascular contraction

The effects of a potassium (K+) channel opener BRL34915 and a specific K+ ionophore valinomycin on vasoconstriction induced by endothelin (ET) were compared with those of calcium (Ca2+) channel blockers, nicardipine and verapamil, using helical strips from rat thoracic aorta. ET induced potent and persistent contraction in control solution and similar but smaller contraction in Ca2+-free solution. BRL34915 and valinomycin inhibited the ET-induced contraction dose-dependently in control solution, but not in Ca2+-free solution. The ET-induced contraction was also inhibited by nicardipine and verapamil, though less strongly. On the other hand, high K+ (35 mM)-induced vasoconstriction was strongly inhibited by nicardipine and verapamil, but not by BRL34915 or valinomycin. These results support the idea that the extracellular Ca2+-dependent component of the ET-induced contraction may be mediated by Ca2+ influx by a route other than voltage-dependent Ca2+-channels.     

Specific receptors for endothelin-3 in cultured bovine endothelial cells and its cellular mechanism of action

Among three endothelin (ET) isopeptides, ET-3 shows the most potent initial depressor response through the endothelium-dependent mechanism. We studied the presence of specific binding sites for ET-3 in cultured bovine endothelial cells (EC) and its cellular mechanism of action. Binding studies revealed the presence of two distinct subclasses of ET-3 receptors with high and low affinities. ET-3 dose-dependently (10(-10)-10(-7) M) increased both intracellular Ca2+ levels ([Ca2+]i) and inositol trisphosphate (IP3) formation. The ET-3-induced increase in [Ca2+]i was not affected by either removal of extracellular Ca2+ or Ca2(+)-channel blockers. These data suggest that ET-3 induces phosphoinositide breakdown and increase in [Ca2+]i in ECs, possibly resulting from intracellular Ca2+ mobilization, thereby leading to vasodilatation.     

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.     

Phorbol Myristate Acetate Inhibition of Phospholipase C Activation by Carbachol in Slices of Rat Brain Cortex Is a Delayed and Indirect Effect

We examined the effect of phorbol esters on phospholipase C activation in rat brain cortical slices and membranes. There was little effect of concurrent addition of phorbol 12-myristate 13-acetate (PMA) with carbachol on phosphoinositide breakdown due to carbachol over a 1-h incubation of brain slices. However, if slices were preincubated for 3 h with 1 microM PMA or 200 microM sphingosine before addition of carbachol, there was a 35-50% inhibition of phosphoinositide breakdown. There was also a marked loss of protein kinase C (PKC) activity from both cytosol and membranes after a 3-h exposure to PMA. The loss in responsiveness to the muscarinic agonists in slices was not reflected in carbachol-stimulated phospholipase C activation using isolated membranes. However, the decrease in carbachol-induced phosphoinositide breakdown seen in slices after a 3-h exposure to PMA was abolished if the extracellular K+ concentration was elevated from 5.9 to 55mM. Because elevation of the K+ level induces depolarization and increases Ca2+ entry, we examined the effect of ionomycin, a Ca2+ ionophore. Ionomycin potentiated the effects of carbachol on phosphoinositide breakdown but was unable to reverse the effects of a 3-h incubation with PMA. Because apamin, an inhibitor of Ca2(+)-dependent K+ channels, mimicked the effects of exposure to PMA for 3 h, it is possible that these channels are involved in muscarinic cholinergic regulation of phosphoinositide breakdown in rat brain slices. These results support the hypothesis that prolonged PMA treatment in rat brain cortex has no direct effect on phospholipase C activation by muscarinic cholinergic stimulation.     

Impaired endothelin-induced vasoconstriction in coronary arteries of Zucker obese rats is associated with uncoupling of [Ca2+]i signaling

Although insulin resistance (IR) is a major risk factor for coronary artery disease, little is known about the regulation of coronary vascular tone in IR by endothelin-1 (ET-1). We examined ET-1 and PGF(2alpha)-induced vasoconstriction in isolated small coronary arteries (SCAs; approximately 250 microM) of Zucker obese (ZO) rats and control Zucker lean (ZL) rats. ET-1 response was assessed in the absence and presence of endothelin type A (ET(A); BQ-123), type B (ET(B); BQ-788), or both receptor inhibitors. ZO arteries displayed reduced contraction to ET-1 compared with ZL arteries. In contrast, PGF(2alpha) elicited similar vasoconstriction in both groups. ET(A) inhibition diminished the ET-1 response in both groups. ET(B) inhibition alone or in combination with ET(A) blockade, however, restored the ET-1 response in ZO arteries to the level of ZL arteries. Similarly, inhibition of endothelial nitric oxide (NO) synthase with N(omega)-nitro-l-arginine methyl ester (l-NAME) enhanced the contraction to ET-1 and abolished the difference between ZO and ZL arteries. In vascular smooth muscle cells from ZO, ET-1-induced elevation of myoplasmic intracellular free calcium concentration ([Ca2+]i) (measured by fluo-4 AM fluorescence), and maximal contractions were diminished compared with ZL, both in the presence and absence of l-NAME. However, increases in [Ca2+]i elicited similar contractions of the vascular smooth muscle cells in both groups. Analysis of protein and total RNA from SCA of ZO and ZL revealed equal expression of ET-1 and the ET(A) and ET(B) receptors. Thus coronary arteries from ZO rats exhibit reduced ET-1-induced vasoconstriction resulting from increased ET(B)-mediated generation of NO and diminished elevation of myoplasmic [Ca2+]i.     

Endothelin increases single-channel calcium currents in coronary arterial smooth muscle cells

Endothelin (ET), a newly identified vasoconstrictor peptide produced by endothelial cells, depends on extracellular calcium for its action [(1988) Nature 332, 411-415]. It is not yet known whether the increase in calcium influx induced by ET results from a direct effect on the Ca2+ channels or is secondary to a reduction in membrane potential. To address this question, we studied the effects of ET on single-channel calcium currents of freshly dissociated porcine coronary artery smooth muscle cells using the cell-attached mode of the patch-clamp technique. We show that ET increases Ca2+-channel activity with no effect on channel open time or conductance. The ability of bath-applied ET to increase single-channel calcium currents in the cell-attached mode is evidence that the peptide acts via a second messenger system.     

Magnesium-Dependent Inhibition of Agonist-Stimulated Phosphoinositide Breakdown in Rat Cortical Slices by Excitatory Amino Acids

The excitatory amino acid agonists kainate, N-methyl-D-aspartate (NMDA), and quisqualate inhibited ligand-stimulated phosphoinositide hydrolysis in rat cortical slices. The NMDA channel blocker MK-801 antagonized the inhibition by NMDA but had no effect on the inhibition due to kainate or quisqualate. The antagonist 6-cyano-7-nitroquinoxaline-2,3-dione blocked the effects of quisqualate and kainate but not the effect of NMDA. These data indicate that activation of the NMDA, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, and kainate types of ionotropic receptors has the same effect. In membranes prepared from cortical slices, there was no inhibition of carbachol-stimulated phosphoinositidase C activity by excitatory amino acids, suggesting that excitatory amino acids indirectly affect carbachol-stimulated phosphoinositide hydrolysis. The inhibition by excitatory amino acids of carbachol-stimulated phosphoinositide breakdown was dependent on extracellular Mg2+ and was abolished by procedures that increase intracellular Ca2+. Veratridine inhibition of carbachol-stimulated phosphoinositide hydrolysis was reversed by ouabain but not by other procedures that increase intracellular Ca2+. In contrast to excitatory amino acids, veratridine potentiated carbachol-stimulated phosphoinositide breakdown in the presence of 10 mM extracellular Mg2+. These data suggest that excitatory amino acids inhibit carbachol-stimulated phosphoinositide breakdown in rat cortex by lowering intracellular Ca2+ through a mechanism dependent on extracellular Mg2+.     

Isoform-specific protein kinase C activity at variable Ca2+ entry during coronary artery contraction by vasoactive eicosanoids.

Vasoactive eicosanoids have been implicated in the pathogenesis of coronary vasospasms. The signaling mechanisms of eicosanoid-induced coronary vasoconstriction are unclear, and a role for protein kinase C (PKC) has been suggested. Activated PKC undergoes translocation to the surface membrane in the vicinity of Ca2+ channels; however, the effect of Ca2+ entry on the activity of the specific PKC isoforms in coronary smooth muscle is unknown. In the present study, 45Ca2+ influx and isometric contraction were measured in porcine coronary artery strips incubated at increasing extracellular calcium concentrations ([Ca2+]e) and stimulated with prostaglandin F2alpha (PGF2alpha) or the stable thromboxane A2 analog U46619, while in parallel, the cytosolic (C) and particulate (P) fractions were examined for PKC activity and reactivity with anti-PKC antibodies using Western blot analysis. At 0-300 microM [Ca2+]e, both PGF2alpha and U46619 (10(-5) M) significantly increased PKC activity and contraction in the absence of a significant increase in 45Ca2+ influx. At 600 microM [Ca2+]e, PGF2alpha and U46619 increased P/C PKC activity ratio to a peak of 9.52 and 14.58, respectively, with a significant increase in 45Ca2+ influx and contraction. The 45Ca2+ influx--PKC activity--contraction relationship showed a 45Ca2+-influx threshold of approximately 7 micromol x kg(-1) x min(-1) for maximal PKC activation by PGF2alpha and U46619. 45Ca2+ influx > 10 micromol x kg(-1) x min(-1) was associated with further increases in contraction despite a significant decrease in PKC activity. Western blotting analysis revealed alpha-, delta-, epsilon-, and zeta-PKC in porcine coronary artery. In unstimulated tissues, alpha- and epsilon-PKC were mostly distributed in the cytosolic fraction. Significant eicosanoid-induced translocation of epsilon-PKC from the cytosolic to the particulate fraction was observed at 0 [Ca2+]e, while translocation of alpha-PKC was observed at 600 microM [Ca2+]e. Thus, a significant component of eicosanoid-induced coronary contraction is associated with significant PKC activity in the absence of significant increase in Ca2+ entry and may involve activation and translocation of the Ca2+-independent epsilon-PKC. An additional Ca2+-dependent component of eicosanoid-induced coronary contraction is associated with a peak PKC activity at submaximal Ca2+ entry and may involve activation and translocation of the Ca2+-dependent alpha-PKC. The results also suggest that a smaller PKC activity at supramaximal Ca2+ entry may be sufficient during eicosanoid-induced contraction of coronary smooth muscle.     

Effect of endothelin-1(1-31) on intracellular free calcium in cultured human mesangial cells

We found that human chymase selectively produces 31-amino-acid length endothelins (1-31) (ETs(1-31)). We investigated the effect of synthetic ET-1(1-31) on intracellular free Ca2+ concentration ([Ca2+]i) in cultured human mesangial cells. ET-1(1-31) increased [Ca2+]i in a concentration-dependent manner to a similar extent as ET-1. The ET-1 (1-31)-induced [Ca2+]i increase was not influenced by removal of extracellular Ca2+ but was inhibited by thapsigargin. ET-1(1-31)-induced [Ca2+]i increase was not affected by phosphoramidon. It was inhibited by BQ123, but not by BQ788. These results suggest that ET-1(1-31) by itself exhibits bioactive properties probably through endothelin ET(A) or ET(A)-like receptors. Since human chymase has been reported to exist in the kidney, ET-1(1-31) may be a candidate substance for mesangium-relevant diseases.     

Mode of action of ergonovine on isolated porcine coronary artery: the role of Ca2+

Coronary vasoconstrictor responses to ergonovine were examined in helical coronary arterial strips of young swine. Both ergonovine and serotonin (5-hydroxytryptamine) produced dose-dependent contractions of the strips. The distal region (less than 1.00 mm outer diameter) of the circumflex coronary artery was most sensitive to the responses of serotonin and ergonovine. Methysergide and nifedipine significantly depressed the contractions induced by ergonovine and serotonin. Atropine, propranolol, and the alpha 1 blocker, prazosin, did not antagonize ergonovine-induced contractions. The ergonovine response may depend entirely upon extracellular Ca2+ while the effect of serotonin may be mediated in part through the mobilization of intracellular Ca2+ stores. Increases in 45Ca2+ cellular contents occurred after ergonovine or serotonin and these increases were blocked by methysergide or nifedipine at concentrations which blocked mechanical responses to the agonist. It is concluded that the contractions of the porcine coronary artery produced by ergonovine and serotonin are as follows: (i) regionally sensitive; (ii) blocked by Ca2+ antagonists and therefore may utilize Ca2+ channels similar to those described in other excitable tissues; (iii) blocked by methysergide. These studies indicate that the major mechanism of ergonovine's action in the porcine coronary artery is through the activation of serotonin receptors on coronary arteries which are, in turn, linked to Ca2+ channels. However, this mechanism of action may be different in an intact animal.     

Production of NAADP and its role in Ca2+ mobilization associated with lysosomes in coronary arterial myocytes

The present study was designed to determine the production of nicotinic acid adenine dinucleotide phosphate (NAADP) and its role associated with lysosomes in mediating endothelin-1 (ET-1)-induced vasoconstriction in coronary arteries. HPLC assay showed that NAADP was produced in coronary arterial smooth muscle cells (CASMCs) via endogenous ADP-ribosyl cyclase. Fluorescence microscopic analysis of intracellular Ca2+ concentration ([Ca2+]i) in CASMCs revealed that exogenous 100 nM NAADP increased [Ca2+]i by 711 +/- 47 nM. Lipid bilayer experiments, however, demonstrated that NAADP did not directly activate ryanodine (Rya) receptor Ca2+ release channels on the sarcoplasmic reticulum. In CASMCs pretreated with 100 nM bafilomycin A1 (Baf), an inhibitor of lysosomal Ca2+ release and vacuolar proton pump function, NAADP-induced [Ca2+]i increase was significantly abolished. Moreover, ET-1 significantly increased NAADP formation in CASMCs and resulted in the rise of [Ca2+]i in these cells with a large increase in global Ca2+ level of 1,815 +/- 84 nM. Interestingly, before this large Ca2+ increase, a small Ca2+ spike with an increase in [Ca2+]i of 529 +/- 32 nM was observed. In the presence of Baf (100 nM), this ET-1-induced two-phase [Ca2+]i response was completely abolished, whereas Rya (50 microM) only markedly blocked the ET-1-induced large global Ca2+ increase. Functional studies showed that 100 nM Baf significantly attenuated ET-1-induced maximal constriction from 82.26 +/- 4.42% to 51.80 +/- 4.36%. Our results suggest that a lysosome-mediated Ca2+ regulatory mechanism via NAADP contributes to ET-1-induced Ca2+ mobilization in CASMCs and consequent vasoconstriction of coronary arteries.     

Endothelin-1 stimulates phosphoinositide hydrolysis in the rat pineal gland

The presence of functional endothelin receptors and their signal transduction mechanism has not been determined so far in the pineal gland. We examined the effect of endothelin-1 (ET-1) on phosphoinositide turnover in whole pineal gland. Endothelin-1 increased monophosphate accumulation in a dose-dependent manner. The phosphoinositide (PI) response elicited by ET-1 was dependent on the presence of extracellular Ca (++) since its chelation resulted in a marked decrease in ET-1-stimulated InsP(1) accumulation. On the contrary, phosphoinositide hydrolysis was not changed by the calcium blocker amlodipine. ET-1 induced PI breakdown was inhibited by neomycin, an inhibitor of phospholipase C. However, mastoparan 7, a G protein activator via Gi/Go s timulation, did not alter ET-1-induced InsP(1) accumulation. Our data indicate that stimulation of PI turnover constitutes one of the signaling pathways of ET in rat pineal gland through the stimulation of a receptor-coupled phospholipase C. And they demonstrate, for the first time, the presence of functional binding sites for endothelin in the pineal gland.     

Biological profiles of highly potent novel endothelin antagonists selective for the ETA receptor.

We describe novel potent endothelin (ET) antagonists that are highly potent and selective for the ETA receptor (selective to ET-1). Of the synthetic analogs based on ETA antagonist BE-18257A isolated from Streptomyces misakiensis (IC50 value for ETA receptor on porcine aortic smooth muscle cells (VSMCs); 1.4 microM), the compounds BQ-123 and BQ-153 greatly improved the binding affinity of [125I]ET-1 for ETA receptors on VSMCs (IC50; 7.3 and 8.6 nM, respectively), whereas they barely inhibited [125I]ET-1 binding to ETB receptors (nonselective with respect to isopeptides of ET family) in the cerebellar membranes (IC50; 18 and 54 microM, respectively). Associated with the increased affinity for ETA receptors, these peptides antagonized ET-1-induced constriction of isolated porcine coronary artery. However, there was a small amount of ET-1-induced vasoconstriction resistant to these antagonists, which paralleled the incomplete inhibition of [125I]ET-1 binding in the membrane of the aortic smooth muscle layer. These data suggest that the artery has both ETA and ETB receptors responsible for ET-1-induced vasoconstriction. The antagonists shifted the concentration-response curve to the right for ET-1 in the coronary artery, and increased the apparent dissociation constant in the Scatchard analysis of [125I]ET-1 binding on the VSMCs without affecting the binding capacity, indicative of the competitive antagonism for ETA receptor. In conscious rats, pretreatment with the antagonists markedly antagonized ET-1-induced sustained pressor responses in dose-dependent fashion without affecting ET-1-induced transient depressor action, suggesting that the pressor action is mediated by ETA receptors, while the depressor action is mediated by ETB receptors. In addition, pretreatment with the potent antagonists prevented ET-1-induced sudden death in mice. Thus, these potent ETA antagonists should provide a powerful tool for exploring the therapeutic uses of ETA antagonists in putative ET-1-related disorders.     

Coronary microvascular dysfunction in a porcine model of early atherosclerosis and diabetes

Detailed evaluation of coronary function early in diabetes mellitus (DM)-associated coronary artery disease (CAD) development is difficult in patients. Therefore, we investigated coronary conduit and small artery function in a preatherosclerotic DM porcine model with type 2 characteristics. Streptozotocin-induced DM pigs on a saturated fat/cholesterol (SFC) diet (SFC + DM) were compared with control pigs on SFC and standard (control) diets. SFC + DM pigs showed DM-associated metabolic alterations and early atherosclerosis development in the aorta. Endothelium-dependent vasodilation to bradykinin (BK), with or without blockade of nitric oxide (NO) synthase, endothelium-independent vasodilation to an exogenous NO-donor (S-nitroso-N-acetylpenicillamine), and vasoconstriction to endothelin (ET)-1 with blockade of receptor subtypes, were assessed in vitro. Small coronary arteries, but not conduit vessels, showed functional alterations including impaired BK-induced vasodilatation due to loss of NO (P < 0.01 vs. SFC and control) and reduced vasoconstriction to ET-1 (P < 0.01 vs. SFC and control), due to a decreased ET(A) receptor dominance. Other vasomotor responses were unaltered. In conclusion, this model demonstrates specific coronary microvascular alterations with regard to NO and ET-1 systems in the process of early atherosclerosis in DM. In particular, the altered ET-1 system correlated with hyperglycemia in atherogenic conditions, emphasizing the importance of this system in DM-associated CAD development.     

Functional endothelin/sarafotoxin receptors in rat heart myocytes: structure-activity relationships and receptor subtypes

Functional receptors for the peptides of the endothelin (ET) and sarafotoxin (SRTX) family were characterized in newborn rat heart myocytes using human and rat endothelins (ET-1 and ET-3, respectively), SRTX-b and SRTX-c. Binding studies in intact cells and homogenates revealed significantly higher affinities of ET-1 and SRTX-b than of ET-3 and SRTX-c towards these receptors. This binding profile of ET/SRTX peptides points to their interaction with the receptor subtype designated E-S alpha. All four peptides induced time- and dose-dependent phosphoinositide hydrolysis with the following rank order of potency: ET-1 greater than SRTX-b greater than SRTX-c greater than ET-3. Thus, ET-3 which possesses an intermediate affinity toward the receptor was the least effective with regard to this response. These results confirm and extend our earlier report that the ET/SRTX peptides interact with a newly characterized receptor(s) associated with phosphoinositide metabolism and Ca2+ mobilization. The initiation of inositol phosphate formation is largely independent of extracellular Ca2+, verapamil and nifedipine, indicating that the ET/SRTX peptides are not agonists for the voltage-dependent Ca2+-channels.     

Nicorandil is a potent dilator of endothelin-induced vascular contraction

Nicorandil, an antianginal drug, is known to open K+ channel and to increase cGMP production. The effects of nicorandil on vascular contraction induced by endothelin (ET), a potent newly discovered vasoconstrictor peptide, were investigated using helical strips from rat thoracic aorta. ET at a concentration of 5 x 10(-9) M induced strong and persistent contraction in the presence of extracellular Ca2+ and similar persistent but smaller contraction in the absence of extracellular Ca2+. Nicorandil at concentrations greater than 10(-7) M, strongly and dose-dependently inhibited ET-induced contraction in the presence of extracellular Ca2+. Nicorandil also suppressed ET-induced contraction in the presence of 10(-4) M methylene blue, an inhibitor of cGMP production, in the presence of extracellular Ca2+ but not in the absence of extracellular Ca2+. ET-induced contraction was also inhibited to lesser extents by the Ca2+ channel blockers nicardipine and verapamil. Nicorandil also strongly suppressed ET-induced increase in cytosolic free Ca2+ concentration in cultured vascular smooth muscle cells. These results suggest that nicorandil is a potent dilator of ET-induced vasoconstriction.     

L-type Ca(2+) channels, resting [Ca(2+)](i), and ET-1-induced responses in chronically hypoxic pulmonary myocytes

In the lung, chronic hypoxia (CH) causes pulmonary arterial smooth muscle cell (PASMC) depolarization, elevated endothelin-1 (ET-1), and vasoconstriction. We determined whether, during CH, depolarization-driven activation of L-type Ca(2+) channels contributes to 1) maintenance of resting intracellular Ca(2+) concentration ([Ca(2+)](i)), 2) increased [Ca(2+)](i) in response to ET-1 (10(-8) M), and 3) ET-1-induced contraction. Using indo 1 microfluorescence, we determined that resting [Ca(2+)](i) in PASMCs from intrapulmonary arteries of rats exposed to 10% O(2) for 21 days was 293.9 +/- 25.2 nM (vs. 153.6 +/- 28.7 nM in normoxia). Resting [Ca(2+)](i) was decreased after extracellular Ca(2+) removal but not with nifedipine (10(-6) M), an L-type Ca(2+) channel antagonist. After CH, the ET-1-induced increase in [Ca(2+)](i) was reduced and was abolished after extracellular Ca(2+) removal or nifedipine. Removal of extracellular Ca(2+) reduced ET-1-induced tension; however, nifedipine had only a slight effect. These data indicate that maintenance of resting [Ca(2+)](i) in PASMCs from chronically hypoxic rats does not require activation of L-type Ca(2+) channels and suggest that ET-1-induced contraction occurs by a mechanism primarily independent of changes in [Ca(2+)](i).     

Inhibition by nicardipine of endothelin-mediated inositol phosphate formation and Ca2+ mobilization in smooth muscle cell

We have investigated the effects of endothelin on phosphoinositide metabolism and Ca2+ mobilization in cultured A10 cells. Endothelin stimulated a significant increase in inositol phosphate formation in a time- and dose-dependent manner. IP3 was significantly elevated by 30 sec and reached a 2.0-fold above control at 1 min. The EC50 for endothelin was 0.5 nM. The initiation of inositol phosphate formation was independent of extracellular Ca2+, and the Ca2+ ionophore, A23187, did not stimulate IP3 formation. However, the sustained elevation of inositol phosphates was partially inhibited by incubating cells in buffer lacking Ca2+ or in buffer containing nicardipine. Endothelin mobilized both intracellular and extracellular Ca2+ reaching a peak intracellular concentration of 350 +/- 11 nM by 1 min when cells were bathed with Ca2+-complete buffer. Intracellular Ca2+ remained 2-fold above baseline for at least 15 min. In contrast, when cells were exposed to endothelin in Ca2+-free buffer, the peak value of [Ca2+]i was 195 +/- 20 nM and returned to baseline by 2 min. Nicardipine completely blocked the influx of extracellular Ca2+ but did not interfere with the mobilization of intracellular stores. We conclude that endothelin produces a rapid and sustained elevation in inositol phosphate formation. The rapid production of IP3 is consistent with the time course for mobilization of intracellular Ca2+. Elevated cytosolic Ca2+ levels are maintained by the influx of extracellular Ca2+ through a nicardipine-sensitive Ca2+ channel and are involved in the sustained formation of inositol phosphates. These data provide an explanation for the sustained, nicardipine-inhibitable contraction of coronary artery strips induced by endothelin.     

Inhibition of prostaglandin E2-induced phosphoinositide metabolism by phorbol ester in bovine adrenal chromaffin cells.

In bovine adrenal chromaffin cells, prostaglandin E2 (PGE2) stimulates the formation of inositol phosphates and Ca2+ mobilization through its specific receptor [Yokohama, Tanaka, Ito, Negishi, Hayashi & Hayaishi (1988) J. Biol. Chem. 263, 1119-1122]. Here we show that PGE2-induced phosphoinositide metabolism was blocked by pretreatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). Using intact cells, we also examined the inhibitory effect of TPA on the individual steps of the activation process of phosphoinositide metabolism. The inhibition was observed within 1 min and complete by 10 min after addition of 1 microM-TPA, and half-maximal inhibition by TPA occurred at 20 nM. TPA prevented Ca2+ mobilization induced by PGE2, but not by the Ca2+ ionophore ionomycin. The inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate did not inhibit the formation of inositol phosphates and Ca2+ mobilization by PGE2. TPA treatment affected neither the high-affinity binding of [3H]PGE2 to intact cells and membrane fractions nor the ability of guanosine 5'-[gamma-thio]triphosphate to decrease the binding in membrane fractions. TPA also abolished phosphoinositide metabolism induced by muscarinic-receptor activation. NaF plus AlCl3 and ionomycin caused the accumulation of inositol phosphates, probably by directly activating a GTP-binding protein(s) and phospholipase C respectively; neither accumulation was inhibited by TPA treatment. These results suggest that protein kinase C serves as a feedback regulator for PGE2-induced phosphoinositide metabolism. The site of action of TPA appears to be distal to the coupling of the receptor to GTP-binding protein, but on a component(s) specific to the agonist-induced phosphoinositide metabolism.