This study investigated the cellular mechanisms underlying the endothelin-1 (ET-1)-induced contraction of rat aorta with focus on the involvement of phospholipase D (PLD). Preincubating rat aorta in Ca(2+)-free solution reduced the contraction by 80%, whereas diltiazem (10 microM), a voltage-operated Ca2+ channel blocker, caused only a small reduction (27%, P less than 0.05) of the contraction. In myo-[3H]inositol-labeled aorta, ET-1 stimulated the formation of [3H]inositol bisphosphate and [3H]inositol trisphosphate, indicating the activation of phospholipase C (PLC). In aorta labeled with 32PO4, [3H] myristic acid or [32P]lyso-platelet-activating factor followed by exposure to ethanol (0.5%), ET-1 stimulated phosphatidylethanol (PEt) production, suggesting that ET-1 activates PLD. The PEt response was not attenuated by staurosporine (ST, 0.1 microM), an inhibitor of protein kinase C (PKC) but was inhibited by removal of Ca2+. The ET-1-induced PEt response was at least additive to that induced by phorbol 12-myristate 13-acetate (1 microM). ET-1 also stimulated the release of 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) into the tissue medium. Unlike the PEt responses, the 6-keto-PGF1 alpha response could be inhibited by ST. Removal of Ca2+ abolished the response. These results suggest that 1) ET-1 activates multiple cellular mechanisms including PLC, PLD, and the arachidonate cascade; 2) PKC activation may not be essential for the ET-1 activation of PLD but may play an important role in the ET-1 stimulation of 6-keto-PGF1 alpha release; and 3) Ca2+ is an important factor in the ET-1-induced PLD activity and 6-keto-PGF1 alpha release.

We sought to relate norepinephrine (NE) stimulation of phosphatidic acid (PA) production to functional responses of rat aorta and pathways for PA production. The time course for changes in PA was closely related to Ca-dependent tonic responses in 42K efflux and contraction. NE (30 microM for 1 min) increased PA and reduced phosphatidylcholine (PC) and phosphatidylinositol (PI) based on Pi analyses and 32P labeling of phospholipids. The 32P-to-Pi ratio in PA (0.8 +/- 0.2, n = 13) was similar to PC (0.8 +/- 0.1, n = 14) but was significantly lower (P < 0.001) than PI (4.6 +/- 0.5, n = 14). The 32P-to-Pi ratio in PA was also lower (P < 0.02) than phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. NE also increased [3H]PA twofold (P < 0.05) when PC was selectively labeled with [3H]myristic acid. These observations are more consistent with PA being formed from the hydrolysis of PC by phospholipase D (PLD) than by the phosphorylation of diacylglycerol produced by the action of phospholipase C. PLD was assayed by the formation of phosphatidylethanol (PEt) via a transphosphatidylation reaction with ethanol (half-maximal stimulation at 0.4-0.5% vol/vol). The time course for PLD stimulation by NE was similar to PA, with significant increases (P < 0.002) during 10 s to 30 min exposure. Once formed, PEt was degraded slowly, with a half time > 3 h. It is concluded that NE stimulates PLD in rat aorta, which forms a significant amount of PA from the hydrolysis of PC.(ABSTRACT TRUNCATED AT 250 WORDS)

SUMMARY Factors that lead to supersensitivity of vascular smooth muscle to norepinephrine during aldosterone-salt-induced hypertension in rats appear to reside beyond ligand-a-adrenergic receptor binding, which we have shown previously to be normal. The objective of this study was to determine whether significant shifts occur in the coupling between receptors and the production of putative second messengers. Measures of [ 3 H]mjo-inositol phosphates in aorta (endothelium removed) exhibited a concentration-dependent increase to norepinephrine, with the 50% response shifted significantly to the left in the hypertensive group (7.0 ± 0.9 x 10~7 M in 8 control rats vs 1.1 ± 0.2 x 10" 7 M in 8 hypertensive rats; p<0.001). The production of [ 32 P]phosphatidic acid was also shifted (6.5 ± 2.5 x 10" 7 M in 16 control vs 1.9 ± 0.8 x 10" 7 M in 12 hypertensive rats; p<0.05). The functional responses of 42 K efflux and contraction to norepinephrine were also significantly shifted threefold to 15-fold in the hypertensive group (p<0.001), but the 50% response typically occurred at a 10 to 100 times lower concentration than that for the production of mvo-inositol phosphates and phosphatidic acid. The amplification between receptor occupancy and functional responses apparently occurs beyond the production of phosphoinositide metabolites. The fivefold shift in the 50% response of biochemical end points for the hypertensive group accounted for most of the shift (sixfold) in the functional end points. It is concluded that the increased efficacy in the hypertensive group resulted more from shifts in the relation between receptor occupancy and production of phosphoinositide metabolites than from shifts in the action of these metabolites on functions that control to catecholamines has been associated with several models of hypertension. 1 * 3 Moreover, it precedes the elevation of blood pressure in the mineralocorticoid-salt hypertensive rat, thus implicating supersensitivity as a pathogenic factor. '• 4 -5 A systematic characterization of a-adrenergic receptors revealed no significant alteration in receptor type (a,), equilibrium dissociation constants, or maximum binding (receptor concentration) of aortic smooth muscle from the aldosterone-salt hypertensive rat (AHR).6 7 Analyses of the ligand binding and dose-response curves revealed that the agonist dissociation constant (A" A ) for norepinephrine (NE) was not changed in AHR, while the efficacy was 4.4 times higher. These findings support the conclusion that postreceptor events underlie the supersensitivity in AHR.There has been an explosion of information relating phosphoinositide metabolites to cellular regulation in many tissues, including smooth muscle.8 "' 2 The regulation of phospholipase C (PLC) by a-adrenergic receptor occupancy is a potential site for the development of supersensitivity. PLC acts on phosphatidylinositol 4,5-bisphosphate to form mjo-inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol (DAG), which are thought to be important regulators of calcium rele...

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