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Protein kinase C activation alters the sensitivity of agonist-stimulated endothelial-cell prostacyclin production to intracellular Ca2+.

ABSTRACT: Agonist-stimulated release of prostacyclin (PGI2) from endothelial cells requires elevation of the concentration of intracellular ionized calcium ([Ca2+]i) above a threshold value, and raised [Ca2+]i provides a sufficient transduction signal to account for the extent of PGI2 production. However, chronic activation of protein kinase C has been reported separately to potentiate PGI2 release, but to depress agonist-induced elevations of [Ca2+]i. We show here that pretreatment with phorbol 12-myristate 13-acetate (PMA) dose-dependently induces PGI2 release over many minutes after a significant lag period without any change in [Ca2+]i. In addition, PMA potentiates the transient release of PGI2 in response to agonists in a complex manner depending on the time of pre-incubation and the concentrations of both PMA and agonist. Concomitant measurement of [Ca2+]i and PGI2 release demonstrates that PMA pretreatment dose-dependently inhibits both the peak [Ca2+]i transient and the subsequent steady-state elevation of [Ca2+]i in response to agonists. Determination of the quantitative [Ca2+]i/PGI2 dose/response relationship, when PGI2 release is driven purely by elevating [Ca2+]i with ionomycin, demonstrates that PMA also enhances the Ca2+-sensitivity of PGI2 release. The observed effects of PMA on PGI2 release can be explained quantitatively by its abilities to lower the threshold [Ca2+]i required for PGI2 synthesis and to depress the peak [Ca2+]i evoked by agonist. We propose that these effects are due respectively to actions of PMA on phospholipase A2 and on a G-protein (Gp) that couples activated receptors to phospholipase C.

SUBMITTER: Carter TD

PROVIDER: S-EPMC1133286 | biostudies-other | 1989 Sep

REPOSITORIES: biostudies-other

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Project description:Endothelial cells are known to release prostacyclin (PGI2) in response to agonists, and this has generally been assumed to be caused, at least in part, by activation of a phospholipase A2 by elevated concentrations of cytoplasmic free calcium ([Ca2+]i). However, it has been shown in the blood platelet that agonists can cause arachidonate release without elevating [Ca2+]i. In the present study, rigorous analysis is made of the [Ca2+]i-dependence of PGI2 production in the human umbilical-vein endothelial cell. Thrombin caused a rapid increase in [Ca2+]i from the resting basal value of 0.1 microM to a peak, within 10-15 s, of approx. 2 microM. In the absence of extracellular Ca2+, [Ca2+]i then declined back to the resting value within 2-3 min. In the presence of extracellular Ca2+, [Ca2+]i partly decreased to a new steady-state value of approx. 1 microM. The elevated [Ca2+]i was maintained while the stimulus and the source of extracellular Ca2+ were present, suggesting that it was dependent on influx of Ca2+ across the plasma membrane. Thrombin stimulated the production of PGI2 in the presence or in the absence of extracellular Ca2+. However, the production of PGI2 was more prolonged in the presence of extracellular Ca2+. Total accumulated amounts of 6-oxo-prostaglandin F1 alpha on stimulation with thrombin without extracellular Ca2+ were only 65% of those accumulated with extracellular Ca2+ present. Cells depleted of extracellular and intracellular sources of Ca2+ by incubation with 1 mM extracellular EGTA and exposing them to ionomycin to discharge intracellular stores produced no elevation of [Ca2+]i on stimulation with thrombin or production of PGI2. The threshold [Ca2+]i required to support the production of PGI2 was measured to be 0.8-1.0 microM by using different doses of ionomycin selectively to increase [Ca2+]i. This relationship between [Ca2+]i and PGI2 production was similar to that produced by using different doses of thrombin. Our results show that the major and probably exclusive intracellular stimulus for the production of PGI2 by the vascular endothelial cell in response to thrombin is the elevation of [Ca2+]i.

Project description:The rapid synthesis and release of prostacyclin (PGI2) and the exocytotic secretion of von Willebrand Factor (vWF) elicited by activation of G-protein-coupled receptors on endothelium occur via signaling mechanisms which are incompletely defined. Activation of protein tyrosine kinases (PTKs) and modulation of the tyrosine-phosphorylation state of endogenous proteins have been implicated in several cellular processes including arachidonate release and exocytosis. In the present study we have examined the regulatory role of PTKs in agonist-stimulated release of PGI2 and vWF from human umbilical vein endothelial cells (HUVECs) using two chemically and mechanistically dissimilar PTK inhibitors (genistein and ST271). Genistein, but not the less active analogue daidzein, dose-dependently attenuated PGI2 release in response to thrombin and histamine (IC50 approx. 20 microM), and to the thrombin-receptor-activating peptide. A more potent inhibition of thrombin- and histamine-induced PGI2 synthesis was observed in cells exposed to ST271. In contrast, neither genistein nor ST271 modulated agonist-drive vWF secretion. At concentrations that abolished PGI2 release, genistein blocked thrombin- or histamine-evoked tyrosine phosphorylation of a 42 kDa protein. Ca2+ ionophore-induced PGI2 generation, but not vWF secretion, was also inhibited by both genistein and ST271, suggesting that these agents modulate PGI2 synthesis by acting at, or distal to, agonist-induced changes in intracellular CA2+ ([Ca2+]i). In fura-2-loaded HUVECs genistein partially reduced the histamine-induced peak [Ca2+]i but had no effect on the thrombin response. Ca(2+)-induced PGI2 release from electrically permeabilized HUVECs was abolished in the presence of ST271 or genistein, but not diadzein. The generation of PGI2 in response to exogenous arachidonic acid was not modulated by genistein or ST271, suggesting that PTK inhibitors do not directly inhibit cyclo-oxygenase activity. Taken together, these results suggest that PTKs regulate PGI2 synthesis and release in HUVECs by modulating, directly or indirectly, a CA(2+)-sensitive step upstream of cyclo-oxygenase.

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Protein kinase C activation alters the sensitivity of agonist-stimulated endothelial-cell prostacyclin production to intracellular Ca2+.

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