EFFECT OF CARBACHOL ON PHOSPHOLIPASE C-MEDIATED PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE HYDROLYSIS, AND ITS MODULATION BY ISOPROTERENOL IN RABBIT CORNEAL EPITHELIAL-CELLS
Y. Zhang et al., EFFECT OF CARBACHOL ON PHOSPHOLIPASE C-MEDIATED PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE HYDROLYSIS, AND ITS MODULATION BY ISOPROTERENOL IN RABBIT CORNEAL EPITHELIAL-CELLS, Current eye research, 14(7), 1995, pp. 563-571
The effects of carbachol (CCh) on phospholipase C(PLC)-mediated phosph
atidylinositol 4,5-bisphosphate (PIP2) hydrolysis and its modulation b
y isoproterenol were investigated in SV40-adenovirus transformed rabbi
t corneal epithelial cells (RCEC). When examined under light microscop
e, these cells exhibited a cobblestone-like appearance typical of the
corneal epithelial cells grown in primary culture. Addition of CCh (0.
1 mM) for 30 min to RCEC, prelabeled with (32)Pi, decreased the radioa
ctivity in phosphatidylinositol LC-phosphate and PIP2 by 15 and 27%, r
espectively, and concomitantly increased the radioactivity in phosphat
idylinositol and phosphatidic acid by 14 and 38%, respectively. When t
he concentration of CCh was increased to 1 mM, the changes in radioact
ivity were even more pronounced. Addition of CCh (0.1 mM) to the cells
, prelabeled with myo[H-3]inositol, increased the accumulation of [H-3
]inositol 1,4,5-trisphosphate ([H-3]InsP(3)) by 115%, indicating stimu
lation of PLC-mediated PIP2 hydrolysis. Similar increases were also ob
served in [H-3]InsP(1) and [H-3]InsP(2). The effects of CCh on inosito
l phosphate accumulation were time- and dose-dependent, and were inhib
ited by atropine (10 mu M), suggesting that the observed effects of CC
h were mediated by activation of muscarinic cholinergic receptors. The
effects of CCh were antagonized more potently by 4-diphenylacetoxy N-
methyl-piperidine than by pirenzepine, indicating that the muscarinic
receptors involved in PLC activation are probably of M(3) type BY West
ern immunoblotting analysis with various anti-PLC antibodies, the RCEC
were shown to contain PLC gamma 1 and PLC delta 1 in the soluble frac
tion and PLC beta 1 in the microsomal fraction. Addition of isoprotere
nol to RCEC, increased cAMP both in a time- and dose-dependent manner.
Pre-treatment of the cells with isoproterenol or other cAMP elevating
agents resulted in a significant inhibition of the CCh-induced InsP(3
) accumulation. It can be concluded from these data that activation of
M(3) muscarinic receptors in RCEC results in stimulation of PLC beta
1-mediated PIP2 hydrolysis, and that activation of beta-adrenergic rec
eptors causes inhibition of the CCh-stimulated PIP2 hydrolysis.