Diguanosine pentaphosphate: an endogenous activator of Rho-kinase possibly involved in blood pressure regulation

Objective Rho-kinase activity is increased in cardiovascular disease and in the pathophysiology of hypertension. Few endogenous factors are known that activate the Rho-kinase pathway. Stimulation of P2Y receptors activates the Rho-kinase pathway. Recently identified diguanosine pentaphosphate (Gp(5)G) possibly activates P2Y receptors. In this study, Gp(5)G was identified and quantified in human plasma. The influence of Gp(5)G on vascular tone was studied. Methods Gp(5)G in human plasma was purified to homogeneity by several steps. Gp(5)G was quantified and identified by matrix-assisted laser desorption/ ionization mass spectrometry and enzymatic analysis. The vasoactive effects of Gp(5)G were studied in the isolated perfused rat kidney and after intra-aortic application. Activation of Rho-kinase was measured using western blot analysis. Results The plasma level of Gp(5)G in healthy donors is 9.47 +/- 4.97 nmol/l. Gp(5)G increases contractile responses induced by angiotensin II in a dose-dependent way [ED50 (-log mol) angiotensin II: 10.9 +/- 0.1; angiotensin II plus Gp(5)G (100 nmol/l): 11.5 +/- 0.1]. P-2 receptor antagonists inhibited the Gp(5)G-induced increase in angiotensin II vasoconstriction. MRS2179, a selective P-2Y1 receptor antagonist, had no effect on Gp5G-mediated angiotensin II potentiation. Rho-kinase inhibition by Y27632 abolished the Gp(5)G-induced increase of contractile responses to angiotensin II. Concentrations of 10 nmol/l Gp(5)G activated the translocation of RhoA from the cytosolic to the membranous fraction indicating the activation of Rho-kinase. The intra-aortic application of 100 pmol Gp(5)G significantly increased mean arterial blood pressure by 13.5 +/- 4.2 mmHg. Conclusion Gp(5)G is an endogenous activator of Rho-kinase, which might affect vascular tone control by Rho-kinase at physiological levels. Gp(5)G activates P-2Y4&6 receptors, and might play a role in physiological and pathophysiological vascular tone control.