Kinetic analysis of the formation of inositol 1:2-cyclic phosphate in carbachol-stimulated pancreatic minilobules. Half is formed by direct phosphodiesteratic cleavage of phosphatidylinositol.
The issue as to whether there is direct phosphodiesteratic cleavage of phosphatidylinositol (PI), in addition to that of phosphatidylinositol 4,5-bisphosphate (PIP2), on agonist stimulation of cells has been controversial. In an attempt to resolve this issue, we have studied the kinetics of the formation and breakdown of the cyclic inositol phosphates. This approach is fairly straightforward, since the turnover of the cyclic inositol phosphates is very slow as compared to that of the noncyclic inositol phosphates and proceeds from inositol 1:2-cyclic 4,5-trisphosphate to inositol 1:2-cyclic phosphate (I(c1:2)P) directly by dephosphorylation without any branching pathways, in contrast to the multiple branchpoints of the noncyclic inositol phosphate pathway. Mouse pancreatic minilobules were prelabeled with [3H]inositol for 30 min, followed by washing to remove free inositol. They were then stimulated with carbachol for 30 min. The inositol cyclic polyphosphates reached steady state at 10-15 min, and I(c1:2)P reached steady state at 25 min. We blocked the action of carbachol by addition of an excess of atropine at 30 min, and the rate of disappearance of the three cyclic inositol phosphates was measured. From these data, the contribution of the inositol cyclic polyphosphate pathway to I(c1:2)P was calculated, which was 40-50% of total I(c1:2)P formation. Thus, 40-50% of the I(c1:2)P formed must have been derived from direct phosphodiesteratic cleavage of PI. This approach should prove useful in measuring the relative contributions of PI hydrolysis and PI phosphorylation (phosphatidylinositol 4,5-bisphosphate hydrolysis) in the overall PI cascade.
Dixon
J F
JF
Department of Pharmacology, University of Wisconsin Medical School, Madison 53706.
Hokin
L E
LE
D000818:Animals; D001285:Atropine / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology*; D046508:Culture Techniques; D006868:Hydrolysis; D007295:Inositol Phosphates / Q000096:biosynthesis*; D007700:Kinetics; D051379:Mice; D010179:Pancreas / Q000187:drug effects* / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D013403:Sugar Phosphates / Q000096:biosynthesis*
D007295:Inositol Phosphates; D010716:Phosphatidylinositols; D013403:Sugar Phosphates; C010199:inositol cyclic phosphate; D001285:Atropine; D002217:Carbachol; D010727:Phosphoric Diester Hydrolases