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 ventricular myocytes. Methods: After stimulation of rabbit ventricular myocytes with noradrenaline and endothelin-1, total lipids were extracted using the Bligh and Dyer procedure and separated by thin layer chromatography, and phosphatidic acid was quantified using photodensitometric analysis of visualised lipids with CuSO4/H3PO4. Results: Noradrenaline (10−5 M) elicited a rapid increase in phosphatidic acid at 2 min, followed by a decrease at 5 min. A second delayed and sustained increase in phosphatidic acid occurred at 10 min. The response to noradrenaline (10−9 to 10−5 M) was concentration dependent with a half maximum response (EC50) of 3.1 × 10−8 M and the maximum effect at 10−6 M. The increase in phosphatidic acid production in response to noradrenaline was abolished by an α1 adrenergic receptor blocking agent (2-[β-(4-hydroxyphenyl)-ethylaminomethyl]tetralone) but unaffected by the β 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 dependent with the maximal increase at 12 min, EC50 5.3 × 10−9 M, and maximum effect at 10−6 M. Both noradrenaline and endothelin-1 stimulated phosphatidyl- butanol production in the presence of butanol (100 mM), indicating that both agonists activate phospholipase D. Conclusions: Noradrenaline at physiological concentrations elicits both a rapid and a delayed increase in phosphatidic acid in adult rabbit ventricular myocytes. Endothelial-1, at physiological concentrations, also stimulates an increase in the mass of phosphatidic acid in myocytes, but the increase induced by endothelin-1 is monophasic, in contrast to the biphasic response seen during stimulation with noradrenaline. Activation of phospholipase D contributes to the increase in phosphatidic acid seen during stimulation of myocytes with either noradrenaline or endothelin-1. These are the first data to characterise endogenous production of phosphatidic acid in isolated adult ventricular myocytes. Cardiovascular Research 1994;28:1828-1834