Thromboxane A 2 Mediates the Stimulation of Inositol 1,4,5-Trisphosphate Production and Intracellular Calcium Mobilization by Bradykinin in Neonatal Rat Ventricular Cardiomyocytes

Abstract

Bradykinin is a mediator of the protection of myocardium by angiotensin I–converting enzyme/kininase II inhibitors. We reported that the activation of B 2 bradykinin receptors in neonatal rat cardiac myocytes in primary culture was followed by hydrolysis of phosphatidylinositol 4,5-bisphosphate and formation of inositol 1,4,5-trisphosphate (IP 3 ). Here we examine the regulation of IP 3 formation stimulated by bradykinin. Activation of myocytes with 1 μmol/L bradykinin increased IP 3 production from 117±8.3 to 1011±48.6 pmol/mg protein. Treatment of the cells with 10 μmol/L indomethacin or 1 μmol/L dexamethasone partially blocked this bradykinin-induced response. Moreover, either U73122, a phospholipase C inhibitor, or ( p -amylcinnamoyl) anthranilic acid, a phospholipase A 2 inhibitor, blunted the IP 3 response to bradykinin. Because thromboxane A 2 stimulates inositol bisphosphate metabolism in guinea pig atria, we also investigated the effect of the thromboxane A 2 receptor antagonist BM 13177 (1 μmol/L), which strongly attenuated the stimulated IP 3 production. Since thromboxane A 2 appears to partly mediate the IP 3 response to bradykinin, we examined the effect of the stable thromboxane A 2 mimetic U46619. Control cultures were stimulated more by U46619 than by bradykinin (1629±14.5 versus 1011±48.6 pmol IP 3 /mg protein). This property of U46619 was selectively antagonized by BM 13177. Inhibition of either phospholipase C or phospholipase A 2 blunted the IP 3 response to U46619. Short-term (30 minutes) activation of protein kinase C with phorbol 12-myristate 13-acetate (10 pmol/L to 1 μmol/L) attenuated the IP 3 accumulation in response to bradykinin; the effect of phorbol 12-myristate 13-acetate was reversed with 1 μmol/L staurosporine, a protein kinase C inhibitor. Treatment with 1 μg/mL cholera toxin or pertussis toxin for 4 hours amplified the IP 3 response to 10 nmol/L bradykinin from 570±20.0 to 1150±51.3 and to 1016.7±21.9 pmol/mg protein. Bradykinin mobilized 9.4% of intracellular calcium stores in cardiomyocytes as assessed by chlortetracycline-based fluorometry, and this effect of bradykinin was blocked by BM 13177 or the B 2 bradykinin receptor blocker Hoe 140 by more than 70%. In functional studies, bradykinin (1 μmol/L) increased by 12% the twitch contractile force of neonatal rat ventricular strips paced at threshold intensity, but this was unaffected by BM 13177. In conclusion, in cardiomyocytes, bradykinin enhances IP 3 production mostly via phospholipase A 2 stimulation and thromboxane A 2 formation. This prostanoid in turn stimulates its receptor and activates phospholipase C, which then splits phosphatidylinositol 4,5-bisphosphate into IP 3 and diacylglycerol. The effect of bradykinin on phospholipase C, via thromboxane A 2 , is negatively regulated by protein kinase C activation.