PHOSPHATIDIC-ACID INCREASES IN RESPONSE TO NORADRENALINE AND ENDOTHELIN-1 IN ADULT-RABBIT VENTRICULAR MYOCYTES

Objective: The aim was to assess whether noradrenaline and endothelin- 1 can stimulate endogenous production of phosphatidic acid in adult ve ntricular myocytes. Methods: After stimulation of rabbit ventricular m yocytes with noradrenaline and endothelin-1, total lipids were extract ed using the Bligh and Dyer procedure and separated by thin layer chro matography, and phosphatidic acid was quantified using photodensitomet ric analysis of visualised lipids with CuSO4/H3PO4. Results: Noradrena line (10(-5) M) elicited a rapid increase in phosphatidic acid at 2 mi n, followed by a decrease at 5 min. A second delayed and sustained inc rease in phosphatidic acid occurred at 10 min. The response to noradre naline (10(-9) to 10(-5) M) was concentration dependent with a half ma ximum response (EC(50)) of 3.1 x 10(-8) M and the maximum effect at 10 (-6) M. The increase in phosphatidic acid production in response to no radrenaline was abolished by an alpha(1) adrenergic receptor blocking agent beta-(4-hydroxyphenyl)-ethylaminomethyl]tetralone) but unaffecte d by the beta adrenergic blocking agent L-propranolol. An increase in phosphatidic acid was also elicited in rabbit ventricular myocytes in response to endothelin-1. The response was time and concentration depe ndent with the maximal increase at 12 min, EC(50) 5.3 X 10(-9) M, and maximum effect at 10(-6) M. Both noradrenaline and endothelin-1 stimul ated phosphatidylbutanol production in the presence of butanol (100 mM ), indicating that both agonists activate phospholipase D. Conclusions : Noradrenaline at physiological concentrations elicits both a rapid a nd a delayed increase in phosphatidic acid in adult rabbit ventricular myocytes. Endothelial-1, at physiological concentrations, also stimul ates an increase in the mass of phosphatidic acid in myocytes, but the increase induced by endothelin-1 is monophasic, in contrast to the bi phasic response seen during stimulation with noradrenaline. Activation of phospholipase D contributes to the increase in phosphatidic acid s een during stimulation of myocytes with either noradrenaline or endoth elin-1. These are the first data to characterise endogenous production of phosphatidic acid in isolated adult ventricular myocytes.