BRADYKININ-EVOKED CA2-DARBY CANINE KIDNEY-CELLS( MOBILIZATION IN MADIN)

We studied the mechanisms underlying the bradykinin-evoked changes in intracellular calcium concentration ([Ca2+](i)) in Madin Darby canine kidney (MDCK) cells. Bradykinin evoked a [Ca2+](i) transient in a dose -dependent manner, measured by fura-2 fluorimetry and digital video im aging. The transient consisted of a rise and a decay and [Ca2+], retur ned to baseline without oscillations. External Ca2+ influx occurred, a s demonstrated by Mn2+ quench and external Ca2+ removal measurements. Bradykinin acted by stimulating bradykinin B-2 receptors as evidenced by blockade by l-D-1,2,3,4-tetrahydro-3-isoquinolinecarbonyl-L-(2 alph a,3 beta,7a beta)-octahydro-1H-indole-2-carbonyl-L-arginine (HOE 140) but not by l-D-1,2,3,4-tetrahydro-3-isoquinolinecarbonyl-L-(2 alpha,3 beta,7a beta)-octahydro-1H-indole-2-carbonyl ([Des-Arg]HOE 140). The [ Ca2+](i) signal was abolished by 1-(6-((17 (10)-trien-17-yl)amino)hexy l)-1H-pyrrole-2,5-dione (U73122) and partially inhibited by neomycin, implying mediation by phospholipase C. The transient was initiated by a release of Ca2+ from internal stores since it was abolished by pretr eatment with thapsigargin or cyclopiazonic acid. The mobilization of t he internal Ca2+ store subsequently triggered a xyphenyl)propoxy]-4-me thoxyphenethyl]-1H-imidazole hydrochloride (SKF 96365)-insensitive Ca2 + entry. Pretreatment with carbonylcyanide m-chlorophynylhydrozone and gly-phe-beta-naphthylamide did not alter the transient, thus excludin g the participation of mitochondria and lysosomes. Efflux via Ca2+ pum ps contributed to the decay of the transient. Efflux via Na+/Ca2+ exch ange or sequestration by mitochondria and lysosomes was insignificant. The transient was blunted by the protein kinase C activator phorbol 1 2-myristate 13-acetate, and was enhanced by the protein kinase C inhib itors sphingosine and chelerythrine, the protein kinase A inhibitor 2, 5-di-(t-butyl)-1,4-hydroquinone, bromocinnamylamino)ethyl]5-isoquinoli nesulfonamide (H-89), the agent 8-(diethylamino)octyl 3,4,5-trimethoxy benzoate (TMB-8), and agents that elevated levels of 3',5'-cyclic guan osine monophosphate. The transient did not heterologously desensitize with that evoked by ATP, ADP or UTP. (C) 1998 Elsevier Science B.V. Al l rights reserved.