The hydrolysis of phosphoinositides (PI) in peripheral tissues can be stimulated by a number of putative neurotransmitters and this stimulation can be blocked by specific antagonists. Epithelial cells derived from the nonpigmented layer of the ocular ciliary epithelium were transfected by simian virus 40 and grown in culture to semiconfluency. The cells were incubated in 3 μCi/ml of (3H)-myoinositol for 2 days. The accumulation of inositol phosphates in response to several agonists (carbachol, 1 mM; ATP, 100 μM; arginine vasopressin, 1 μM; and phenylephrine, 100 μM) was determined for times ranging from 5 sec to 15 min. In the presence of 10 mM LiCl, the maximum net production of the (3H)-inositol phosphates (expressed as a percent of conversion of (3H)-phospholipids) was approximately 7.5% for inositol-1 phosphate, 0.5% for inositol-1,4 bisphosphate, and 1% for inositol-1,4,5 trisphosphate. Carbachol elicited PI hydrolysis with an EC50 value of 39 ± 9 μM. The EC50 values obtained for arginine vasopressin and ATP-initiated PI breakdown were 32 ± 10 nM and 11.9 ± 1 μM, respectively. Phenylephrine alone failed to stimulate the production of (3H)-inositol phosphates in these cells. The production of all (3H)-inositol phosphates in response to carbachol (1 mM) was inhibited by atropine (K(i) = 0.3 nM) and the selective muscarinic antagonists 4-DAMP (K(i) = 4.2 nM), pirenzepine (K(i) = 102 nM) and AFDX-116 (K(i) = 1.49 μM). Thus the muscarinic receptors that are coupled to PI hydrolysis in these cells have the pharmacologic characteristics of the M3 subtype. Phorbol 12,13 dibutyrate (1 μM) rapidly desensitized the production of (3H)-inositol phosphates by carbachol to 25% of control values. Pertussis toxin treatment reduced the PI hydrolysis elicited by carbachol to approximately 40% of control values. We have therefore demonstrated a role for G protein regulation in receptor-mediated, agonist-induced PI hydrolysis in these cells.
|Original language||English (US)|
|Number of pages||5|
|Journal||Investigative Ophthalmology and Visual Science|
|State||Published - Jan 1 1989|
ASJC Scopus subject areas
- Sensory Systems
- Cellular and Molecular Neuroscience