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Synergism between thrombin and adrenaline (epinephrine) in human platelets. Marked potentiation of inositol phospholipid metabolism.

ABSTRACT: We have studied synergism between adrenaline (epinephrine) and low concentrations of thrombin in gel-filtered human platelets prelabelled with [32P]Pi. Suspensions of platelets, which did not contain added fibrinogen, were incubated at 37 degrees C to measure changes in the levels of 32P-labelled phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP) and phosphatidate (PA), aggregation and dense-granule secretion after stimulation. Adrenaline alone (3.5-4.0 microM) did not cause a change in any parameter (phosphoinositide metabolism, aggregation and dense-granule secretion), but markedly enhanced the thrombin-induced responses over a narrow range of thrombin concentrations (0.03-0.08 units/ml). The thrombin-induced hydrolysis of inositol phospholipids by phospholipase C, which was measured as the formation of [32P]PA, was potentiated by adrenaline, as was the increase in the levels of [32P]PIP2 and [32P]PIP. The presence of adrenaline caused a shift to the left for the thrombin-induced changes in the phosphoinositide metabolism, without affecting the maximal levels of 32P-labelled compounds obtained. A similar shift by adrenaline in the dose-response relationship was previously demonstrated for thrombin-induced aggregation and dense-granule secretion. Also, the narrow range of concentrations of thrombin over which adrenaline potentiates thrombin-induced platelet responses is the same for changes in phosphoinositide metabolism and physiological responses (aggregation and dense-granule secretion). Our observations clearly indicate that adrenaline directly or indirectly influences thrombin-induced changes in phosphoinositide metabolism.

SUBMITTER: Steen VM

PROVIDER: S-EPMC1149337 | biostudies-other | 1988 Jul

REPOSITORIES: biostudies-other

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Project description:Inositol phospholipids in cultured GH3 cells, a prolactin secreting, thyrotropin-releasing hormone (TRH) sensitive rat pituitary cell line, exhibit a preferential selectivity for incorporating arachidonic acid. Fatty acid composition data show that all inositol phospholipids are enriched in stearic and arachidonic acids to a much greater degree than other cellular phospholipids. Incubation of GH3 cells with radioactive stearate, oleate, arachidonate, eicosapentaenoate or docosahexaenoate also showed that much more stearate and arachidonate were incorporated into inositol phospholipids. In short term incubations with tracer amounts of radioactive arachidonate, incorporation was initially into phosphatidylinositol (PtdIns), with phosphatidylinositol 4-phosphate (PtdIns4P), and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] being labelled at later times. During longer incubations, all of the inositol phospholipids reach equilibrium at about 10 h, and the resulting specific activities of the three fractions were similar. These findings suggest that arachidonate is incorporated initially into PtdIns and that PtdIns is then phosphorylated. There was no release of either arachidonate or eicosanoid products when GH3 cells were incubated with TRH. However, TRH stimulation of 32P-labelled GH3 cells resulted in rapid breakdown of PtdIns(4,5)P2 and PtdIns4P, with concomitant increases in [32P]phosphatidic acid and [32P]PtdIns. When the [32P]PtdIns was further analysed by argentation chromatography to separate PtdIns molecular species, it was found that tetraenoic (stearate/arachidonate) species accounted for 80% of the stimulated labelling. The selectivity for arachidonate incorporation into inositol phospholipids coupled with turnover of the arachidonate-containing molecular species suggests that inositol phospholipids containing arachidonic acid or the diacylglycerol resulting therefrom may play a vital cellular role in GH3 cells. This role may involve the operation of the PtdIns cycle itself rather than a stimulated release of arachidonate for eicosanoid formation.

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Synergism between thrombin and adrenaline (epinephrine) in human platelets. Marked potentiation of inositol phospholipid metabolism.

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