BOMBESIN-STIMULATED CERAMIDE PRODUCTION AND MAP KINASE ACTIVATION IN RABBIT RECTOSIGMOID SMOOTH-MUSCLE CELLS

We have investigated the hypotheses that 1) bombesin activation of pro tein kinase C (PKC) results in the hydrolysis of sphingolipids and the production of ceramide and that 2) ceramide produced on activation by bombesin mediates sustained contraction of smooth muscle cells by act ivation of PKC and mitogen-activated protein (MAP) kinase. Ceramide pr oduction was assessed using a technique that involved benzoylation of purified ceramide extracts, followed by reverse-phase high-performance liquid chromatography. Contraction of smooth muscle cells isolated fr om the rabbit rectosigmoid and stimulated with bombesin gave a signifi cant increase in newly formed ceramide (38 +/- 3.5%). 12-O-tetradecano ylphorbol-13-acetate also induced production of ceramide, which was bl ocked by calphostin C. The short-chain permeable C-2 ceramide induced a sustained contraction and activation of MAP kinase, which was blocke d by calphostin C. The increase in MAP kinase activity was maximal at 30 s and declined at 2 min. The data suggest that stimulation of smoot h muscle cells by bombesin results in a functional coupling between sn -1,2-diacylglycerol (DAG)/PKC and a sphingomyelinase, whereby DAG acti vates the hydrolysis of sphingomyelin to produce ceramide. Ceramide in turn activates PKC, which then activates MAP kinase. This could be th e basis for the sustained contraction observed with bombesin.