Source of 3H-labeled inositol bis- and monophosphates in agonist-activated rat parotid acinar cells.
The kinetics of [3H]inositol phosphate metabolism in agonist-activated rat parotid acinar cells were characterized in order to determine the sources of [3H]inositol monophosphates and [3H]inositol bisphosphates. The turnover rates of D-myo-inositol 1,4,5-trisphosphate and its metabolites, D-myo-inositol 1,4-bisphosphate and D-myo-inositol 1,3,4-trisphosphate, were examined following the addition of the muscarinic receptor antagonist, atropine, to cholinergically stimulated parotid cells. D-myo-Inositol 1,4,5-trisphosphate declined with a t1/2 of 7.6 +/- 0.7 s, D-myo-inositol 1,3,4-trisphosphate declined with a t1/2 of 8.6 +/- 1.2 min, and D-myo-inositol 1,4-bisphosphate was metabolized with a t1/2 of 6.0 +/- 0.7 min. The sum of the rates of flux through D-myo-inositol 1,4-bisphosphate and D-myo-inositol 1,3,4-trisphosphate (2.54% phosphatidylinositol/min) did not exceed the calculated rate of breakdown of D-myo-inositol 1,4,5-trisphosphate (2.76% phosphatidylinositol/min). Thus, there is no evidence for the direct hydrolysis of phosphatidylinositol 4-phosphate in intact cells since D-myo-inositol 1,4-bisphosphate formation can be attributed to the dephosphorylation of D-myo-inositol 1,4,5-trisphosphate. The source of the [3H]inositol monophosphates also was examined in cholinergically stimulated parotid cells. When parotid cells were stimulated with methacholine, D-myo-inositol 1,4,5-trisphosphate, D-myo-inositol 1,3,4,5-tetrakisphosphate, D-myo-inositol 1,4-bisphosphate, and D-myo-inositol 4-monophosphate levels increased within 2 s, whereas D-myo-inositol 1-monophosphate accumulation was delayed by several seconds. Rates of [3H]inositol monophosphate accumulation also were examined by the addition of LiCl to cells stimulated to steady state levels of [3H]inositol phosphates. The sum of the rates of accumulation of D-myo-inositol 1-monophosphate and D-myo-inositol 4-monophosphate did not exceed the rate of breakdown of D-myo-inositol 1,4,5-trisphosphate or the sum of the rates of flux through D-myo-inositol 1,4-bisphosphate and D-myo-inositol 1,3,4-trisphosphate. These kinetic analyses suggest that agonist-stimulated [3H]inositol bis- and monophosphate formation in intact rat parotid acinar cells can be accounted for by the metabolism of D-myo-[3H]inositol 1,4,5-trisphosphate rather than by phospholipase C-catalyzed hydrolysis of phosphatidylinositol or phosphatidylinositol 4-phosphate.
Hughes
A R
AR
Calcium Regulation Section, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
Putney
J W
JW
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D006868:Hydrolysis; D007295:Inositol Phosphates / Q000096:biosynthesis / Q000378:metabolism*; D007700:Kinetics; D010306:Parotid Gland / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D011976:Receptors, Muscarinic / Q000187:drug effects* / Q000502:physiology; D013403:Sugar Phosphates / Q000378:metabolism*; D014316:Tritium; D010738:Type C Phospholipases / Q000378:metabolism
D007295:Inositol Phosphates; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D011976:Receptors, Muscarinic; D013403:Sugar Phosphates; D014316:Tritium; C047872:inositol 2-monophosphate; D010738:Type C Phospholipases