REMOVAL AND RESTORATION OF EPITHELIAL CHLORIDE SECRETORY ACTIVITY OF KININS BY GENE MANIPULATION

Kinins are known to stimulate electrogenic chloride secretion in many mammalian epithelia, including those of the airways and the alimentary tract. In this study the chloride secretory activity of lysylbradykin in (LBK) on murine colonic epithelium has been examined, specifically to discover the primary and final effector mechanisms in this process, i.e. which kinin receptors are involved and which chloride channels a re responsible for chloride secretion. The approach used was to modify the mice genetically and assess the effects on kinin mediated chlorid e secretion using voltage clamping at zero potential. Briefly, LBK inc reased SCC in mouse colon by similar to 150 mu A cm(-2) with an EC(50) of similar to 5 nM. In null CF mice LBK, 1 mu M had no effect on chlo ride secretion, but reduced SCC due to K+ secretion. This effect is no rmally masked in wild-type tissues by dominant chloride secretion, but can be shown to occur to the same extent by measuring K+ secretion wi th radioisotopes. Null CF mice produce no cftr, but CFTR was introduce d into CF mice by injecting a YAC containing the human CF gene into th e pronucleus of CF zygotes. Colonic epithelia from mice with the incor porated YAC showed the same sensitivity to LBK as wild-type tissues an d achieved the same maximal chloride secretory response. Colonic epith elia from mice in which the B(2)r gene had been disrupted showed no re sponse to LBK at normally supramaximally effective concentrations, alt hough responses to other secretagogues were normal. Similarly des-Arg- BK caused no acute chloride secretory response in colonic epithelia fr om B-2 knockout mice, however small responses appeared if tissues were incubated in vitro for 3-6 h. It is concluded that cftr chloride chan nels and B(2)rs are required for electrogenic chloride secretion. Furt her CFTR can replace cftr with no effect on either the sensitivity or extent of chloride secretion. In vitro, colonic epithelia may generate B(1)rs which, upon activation, have a minor effect on chloride secret ory activity.