Effect of cell culture on rabbit colonic smooth muscle bradykinin receptors

Abstract The aim of this study was to assess the effect of cell culture on the bradykinin receptor of rabbit colon myocytes. In longitudinal muscle strips prepared from distal colon, bradykinin stimulated dose-dependent contraction that was 62% of the maximal response to bethanecol. At 4 °C, [ 3 H]bradykinin binding to fresh muscle homogenates from the distal colon was time dependent, saturable, and linearly related to tissue concentration. Specific binding of 0.6 nmol/L [ 3 H]bradykinin was 80% ± 2% of total binding. In competitive binding studies, Hill coefficients approached unity, suggesting the presence of a single class of receptors. The order of potency was bradykinin > [d-Phe 7 ]bradykinin ⪢ desArg 9 , [Leu 8 ]bradykinin, which is consistent with results of a B 2 receptor subclass. Colon myocytes from the longitudinal muscle layer achieved confluence and were harvested for studies after 12–14 days in culture. Bradykinin receptors were of high affinity [disassociation constant (K d ) = 672 pmol/L] and numbered 10,217 ± 2567/cell. To show that the receptors on cultured myocytes were functional, the effect of bradykinin was measured (a) on intracellular calcium concentration using Fura 2 and (b) on prostaglandin E 2 concentration in the culture media using radioimmunoassay. In cells grown to confluence on cover slips and preloaded with Fura 2, bradykinin stimulated the threshold response at 1 nmol/L and maximal response (increased intracellular calcium concentration from 229 to 633 nmol/L) at 1 μmol/L. Bradykinin, 100 nmol/L, increased Prostaglandin E 2 in the culture media threefold. In summary, colon myocytes express functioning bradykinin receptors, which, unlike muscarinic receptors, persist in culture. Bradykinin appears to be a suitable agonist for studies of receptor-mediated intracellular events in cultured colon myocytes.