title: Thromboxane A2 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 B2 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 (IP3). Here we examine the regulation of IP3 formation stimulated by bradykinin. Activation of myocytes with 1 mu/L bradykinin increased IP3 production from 117 +/- 8.3 to 1011 +/- 48.6 pmol/mg protein. Treatment of the cells with 10 mu/L indomethacin or 1 mu/L dexamethasone partially blocked this bradykinin-induced response. Moreover, either U73122, a phospholipase C inhibitor, or (p-amylcinnamoyl) anthranilic acid, a phospholipase A2 inhibitor, blunted the IP3 response to bradykinin. Because thromboxane A2 stimulates inositol bisphosphate metabolism in guinea pig atria, we also investigated the effect of the thromboxane A2 receptor antagonist BM 13177 (1 mu/L), which strongly attenuated the stimulated IP3 production. Since thromboxane A2 appears to partly mediate the IP3 response to bradykinin, we examined the effect of the stable thromboxane A2 mimetic U46619. Control cultures were stimulated more by U46619 than by bradykinin (1629 +/- 14.5 versus 1011 +/- 48.6 pmol IP3/mg protein). This property of U46619 was selectively antagonized by BM 13177. Inhibition of either phospholipase C or phospholipase A2 blunted the IP3 response to U46619. Short-term (30 minutes) activation of protein kinase C with phorbol 12-myristate 13-acetate (10 pmol/L to 1 mu/L) attenuated the IP3 accumulation in response to bradykinin; the effect of phorbol 12-myristate 13-acetate was reversed with 1 mu/L staurosporine, a protein kinase C inhibitor. Treatment with 1 microgram/mL cholera toxin or pertussis toxin for 4 hours amplified the IP3 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 B2 bradykinin receptor blocker Hoe 140 by more than 70%. In functional studies, bradykinin (1 mu/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 IP3 production mostly via phospholipase A2 stimulation and thromboxane A2 formation. This prostanoid in turn stimulates its receptor and activates phospholipase C, which then splits phosphatidylinositol 4,5-bisphosphate into IP3 and diacylglycerol. The effect of bradykinin on phospholipase C, via thromboxane A2, is negatively regulated by protein kinase C activation.
authors.affiliation: Department of Anesthesiology and Pain Management, Cook County Hospital, Chicago, IL 60612, USA.
authors.forename: F
authors.initials: F
authors.lastname: Nakamura
authors.forename: R D
authors.initials: RD
authors.lastname: Minshall
authors.forename: G C
authors.initials: GC
authors.lastname: Le Breton
authors.forename: S F
authors.initials: SF
authors.lastname: Rabito
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001692:Biological Transport / Q000187:drug effects; D001920:Bradykinin / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D006352:Heart Ventricles; D015544:Inositol 1,4,5-Trisphosphate / Q000096:biosynthesis*; D007425:Intracellular Membranes / Q000378:metabolism*; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D010741:Phospholipases A / Q000502:physiology; D054467:Phospholipases A2; D011493:Protein Kinase C / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013928:Thromboxane A2 / Q000502:physiology*; D010738:Type C Phospholipases / Q000502:physiology
chemical_list: D013928:Thromboxane A2; D015544:Inositol 1,4,5-Trisphosphate; D011493:Protein Kinase C; D010741:Phospholipases A; D054467:Phospholipases A2; D010738:Type C Phospholipases; D001920:Bradykinin; D002118:Calcium