European Journal of Pharma~oh)gy - Molecular Pharma,,'oh~w &'cumin. 225 ( 1992~, 43-49 43 k, 1992 Elsevier Science Publishers B.V. All righls rcserxed 0922-41116/92/$05.(10 EJPMOL 90253 Is the bradykinin-induced Ca z + influx and the formation of endothelium-derived relaxing factor mediated by a G protein? Wolfgang F. Graier, Kurt Schmidt and Walter R. Kukovetz htstitut fi~r Pharmakodynamik und To.tikohgie, Univer~itiit (;ru:, A-80IO (;ra:. Austria Received 24 July 199!. revb, ed MS received 24 September t991. accepted 8 October t991 In cultured porcine aortic endothelial cclis bradykinin produced a tong-lasting Ca 2 + influx. In contrast to Ihc G protein-inde- pendent Ca 2+ entry evoked by ionomycin or digitonin, bradykinin-induced Ca 2~ influx was antagonized by Ni 2. with an ICs, value of about 50 .aM. Since identical IC~. values for Ni 2" were tound when Ca 2~ entry was induced by sodium fluoride or GTPyS, we suggest that stimulmion of G protein(s) results in the activation of the same Ca 2° channels as stimuiatkm by bradykinin. This conclusion is supported by our findings that inhibition ef GTPasc by mcpacrinc amplified bradykinin-stimutatcd Ca 2 ' influx, but did not interfere wi{l~ the effect of the Ca 2 ' ionophorc A23i87. Similar to its effect on Ca z" influx, mepacrinc also potentiated endothelium-derived relaxing factor (EDRF) formation by bradykinin and sodium fluoride, but did not affect A23187-induced EDRF biosynthesis. W,: therefore suggest that in endothelial cells the bradykininqnduced ('a ~' influx and the resulting formation of EDRF are regulat~,'d by a G protein. Bradykinin: Endothelial cells; Ca 2" channels: G protein: Endothclium-derivcd relaxing factor (EDRF): cGMP I. Introduction Endothelial cells play an important role in tim regu- lation of vascular tone. Upon stimulation with various agonists they respond with the formation and the re- lease of a so-called endothelium-derived relaxing factor (EDRF; Furchgott and Zawadzki, 198(1) which relaxes vascular smooth muscle by activation of soluble guany- late cyclase (Busse et al., 1985; Ignarro et al., 1986; Miilsch et al., 1987). The biosynthesis of EDRF is strictly correlated to the intracellular free Ca 2" con- centration ([Ca2+]i; Johns et al., 1988; Liickhoff et al., 1988; Schmidt et ai., 1989; Graier et al., t99(I). Stimula- tion of endothelial cells with bradykinln resul~ i, a~_.' increase in [Ca2+]i (Colden-Stanfieid et al., t987; Schilling et al., 1988, 1989; Myers and t~arkins, 1989; Graier et al., 1990) and leads to an enhanced form> tion of EDRF (Gruetter and Lemke, 1986; Graier et al., 19901 which can be easily monitored by measuring endothelial cGMP levels (Martin et al,, 1988; Schmidt et al., 1989; Boulanger et al., 199(I). The bradykinin-induced increase in [Ca2 ~]i is regu- lated (1) by the release of intracellularly stored Ca 2-, ('orrespondence lo: Dr. Wolfgang F. Graicr, lnslilu~ flit Pharmako- dynamik und Toxikotogie, Universitfitsplatz 2, A-8010 Graz. Austria. Tel. 316-38(!-5569: Fa× 316-33541. and (2) by a long-lasting influx of Ca 2" (Schilling et aL, 1988, 1989), which prolonged and amplified the forma- tion of EDRF (Graier et al., I991a). While i~ is widely accepted that the release of intracellulariy stored Ca:" is due to a stimulation of phospholipase C and the resulting formation of inositol t,4,5-trisphosphate (Freay et al., t9891, the mechanism of the regulation of bradykinin-induced Ca 2" influx is still unclear, but it is probably not regulated by voltage-dependent Ca 2+ channels (Colden-Stanfield et al., 19871. Na-/Ca 2+ exchange can be observed in endothelial cells (Bocynaems et at.. 19891 but plays apparently only a minor role in bradykinin-induced Ca-'* influx (Schilling ct at., 19881 and also the involvement of {he formation of inositot phosphates, such as inositot 1,4,5-tris- pho:-phate or inositol 1,3,4,5-tetrakisphosphate seems to be uMikely (Graier et al. 1991b). It appears to be more likely that Ca-'* enters the cytoplasm through ion channels, which are regulated by a ligand/receptor interaction (Hallam and Pearson, 1986: John,, et al., 1987). This so-called 'receptor-medi- ated Ca 2~ emry" is certainly not voltage-regulated (Colden-Stanfield et al., 19891 and has already been observed in many tissues (Rink, 1990) but the exact mechanism of ~he regulation of this Ca 2+ ently is unclear. Although it is possible that the membrane receptor directly regulates the Ca 2' channel (Benham and Tsien, 1987), the involvement of a G protein in the