D. Sbrissa et al., ORIGIN OF MOLECULAR-SPECIES OF DIACYLGLYCEROL INDUCED BY BOMBESIN IN SMOOTH-MUSCLE CELLS FROM RABBIT RECTOSIGMOID, American journal of physiology: Gastrointestinal and liver physiology, 38(1), 1998, pp. 138-150
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.