ORIGIN OF MOLECULAR-SPECIES OF DIACYLGLYCEROL INDUCED BY BOMBESIN IN SMOOTH-MUSCLE CELLS FROM RABBIT RECTOSIGMOID

The source of early production of sn-1,2-diacylglycerol (DAG) has for a long time been exclusively linked to hydrolysis of phosphatidylinosi tol 4,5-diphosphate, which on receptor activation is hydrolyzed into D AG and inositol 1,4,5-trisphosphate. We have investigated the origin o f lipid sources of DAG production in smooth muscle cells, in response to contraction induced by peptide agonists. We have performed a quanti tative analysis of the molecular species of DAG formed in relation to the known molecular composition of parent phospholipids. The molecular species of phospholipids are sufficiently unique that the phospholipi d origin of DAGs and its quantitative contribution to their formation can be measured by HPLC. Cell suspensions (10-15 x 10(6) cells/ml) fro m the circular muscle of rabbit rectosigmoid were incubated in the pre sence of the contractile peptide agonist bombesin (BB) at 10(-6) M. Re actions were stopped at different time intervals from 30 s to 4 min. D AGs were extracted, purified by TLC, and benzoylated with benzoic anhy dride. The benzoylated DAGs were first purified by TLC and then by nor mal phase HPLC before they were injected onto a reverse-phase column a nd eluted isocratically. Furthermore, phospholipids in the lipid extra ct [phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidyls erine (PS), and phosphatidylethanolamine (PE)] were purified by TLC an d similarly analyzed after hydrolysis to DAGs with phospholipase C (PL C). The DAG molecular species profiles for PI, PC, PS, and PE were all unique. Contraction of cells with BB gave noticeable increases (17-55 %) in newly formed DAGs. The major phospholipid source of the newly fo rmed DAGs at 30 s was only similar to 30% from PI, and the remainder w as from PC. In contrast, after 4 min of BB stimulation, a decrease was seen in newly formed DAGs in the peak specific for PI hydrolysis. The data suggest that BB-induced contraction by activation of PLCs result s in hydrolysis of different phospholipids. The DAGs formed as a resul t are qualitatively and quantitatively distinct. This could be the bas is for the kinetically different pattern of sustained contraction obse rved with BB.