THE NEUROPEPTIDE BRADYKININ STIMULATES PHOSPHOINOSITIDE TURNOVER IN HSDM1C1 CELLS - B2-ANTAGONIST-SENSITIVE RESPONSES AND RECEPTOR-BINDING STUDIES

Bradykinin (BK) and its analogs (1 nM-100 muM) stimulated phosphoinosi tide (PI) turnover in murine fibrosarcoma (HSDM1C1) cells in a concent ration-dependent manner. The relative potencies (EC50) were: BK = 48 /- 4 nM; Lys-BK = 39 +/- 3 nM; Met-Lys-BK = 158 +/- 33 nM; Des-Arg9-BK = 2617 +/- 598 nM (means +/- SEM, n = 3-14). All these analogs were f ull agonists and they produced up to 5.4 +/- 0.4-fold stimulation of P I turnover at the highest concentration (10-100 muM) of the peptides. In contrast, the analogs [D-Arg0-HYP3-Thienyl5,8-D-Phe7]-BK (HYP3-anta gonist), [D-Arg0-HYP3-Thienyl5,8-D-Phe7]-BK (Thienyl antagonist) and D es-Arg9-Leu8-BK were inactive, as agonists, at 0.1 nM-1 muM in this sy stem. These data suggested that BK-induced PI turnover in these cells was mediated via B2-type of BK receptors. This was confirmed further b y the fact that both the B2-selective Hyp3- and Thienyl-antagonists in hibited BK-induced PI turnover with K(B)S of 369 +/- 51 nM and 368 +/- 118 nM respectively while the B1-selective antagonist, Des-Arg9-Leu8- BK, was inactive at 1 muM. [H-3]BK receptor binding studies revealed t wo binding sites, one with high affinity (K(d) = 0.24 +/- 0.06 nM; B(m ax) = 1.4 +/- 0.4 pmol/g tissue) and the other with low affinity (K(d) = 18.5 +/- 0.95 nM; B(max) = 25.1 +/- 0.52 pmol/g tissue), on HSDM1C1 cell homogenates. The rank order of affinity of BK analogs at inhibit ing specific [H-3]BK binding was similar to that found for PI turnover . Taken together, these data have provided evidence for the presence o f two B2-type BK binding sites on the HSDM1C1 cells. Based on the affi nity parameters, the low-affinity component of [H-3]BK binding in HSDM 1C1 cells appears to be coupled to the phospholipase C-induced PI turn over mechanism. The high-affinity component has been previously shown to mediate the production of prostaglandins by activation of phospholi pase A2.