Bradykinin induces formation of inositol phosphates and causes an increase in cytoplasmic Ca2+ in the osteoblastic cell line MC3T3-E1.

Abstract

Recordings of fura-2 fluorescence from single osteoblastic MC3T3-E1 cells showed that bradykinin (BK, 1 microM) induced a rapid increase in cytoplasmic free Ca2+ (Cai2+, from 114 +/- 13 to 239 +/- 17 nM, mean +/- SEM). Following this initial transient (less than 1 minute) increase there was a second slow increase in Cai2+ (from 117 +/- 11 to 151 +/- 12 nM). Incubation in buffer with no Ca2+ did not affect the first rapid BK-induced increase in Cai2+ but eliminated the second slow increase. Addition of indomethacin or hydrocortisone to the incubation buffer did not inhibit the effect of BK on Cai2+. BK caused a dose-dependent initial rapid increase in Cai2+ with threshold at 1 nM and a maximal effect (241 +/- 30% of basal Cai2+ concentration) at 0.1 microM. The B1 BK receptor agonist des-Arg9-BK (1 microM) caused only a small increase in Cai2+ in MC3T3-E1 cells (from 101 +/- 20 to 140 +/- 4 nM). BK dose and time dependently stimulated the formation of inositol phosphates in MC3T3-E1 cells with EC50 at 2.4 nM and a significant increase in inositol trisphosphate already seen after 15 s. The Ca2+ ionophore ionomycin induced a rapid increase in Cai2+ and prostaglandin E2 (PGE2) formation in MC3T3-E1 cells. Forskolin (10-30 microM) increased cyclic AMP accumulation but did not affect Cai2+ or PGE2 formation. Depletion of extracellular Ca2+ significantly reduced (but did not abolish) BK-induced PGE2 formation. The initial action of BK on Cai2+ is probably due to an inositol-(1,4,5)-trisphosphate-mediated rapid release of Ca2+ from intracellular stores in osteoblasts and is followed by an influx of extracellular Ca2+. The effect is due to B2 BK receptor occupancy and is not secondary to the prostaglandin synthesis. The BK-induced breakdown of phosphatidylinositol-(4,5)-bisphosphate with a subsequent increase in Cai2+ may be involved in BK-induced prostaglandin formation in osteoblasts.