Kinetics of inositol 1,4,5-trisphosphate and inositol cyclic 1:2,4,5-trisphosphate metabolism in intact rat parotid acinar cells. Relationship to calcium signalling.

A R Hughes,H Takemura,J W Putney

doi:10.1016/s0021-9258(19)81517-x

Abstract

Stimulation of rat parotid acinar cells by the muscarinic cholinergic receptor agonist methacholine results in the formation of inositol 1,4,5-trisphosphate [1,4,5)IP3) and inositol cyclic 1:2,4,5-trisphosphate [c1:2,4,5)IP3) which, after 40 min, accumulate to a ratio of 1:0.57. The turnover rates of these inositol trisphosphates have been determined in cholinergically stimulated rat parotid cells by measuring the degradation of the 3H-labeled compounds following receptor blockade. (1,4,5)IP3 is rapidly metabolized, with a half-time of 7.6 s; (c1:2,4,5)IP3 declines much more slowly with a half-time of almost 10 min. Because the formation and metabolism of (c1:2,4,5)IP3 are so slow, (c1:2,4,5)IP3 gradually accumulates upon prolonged receptor activation. Inositol trisphosphate turnover was compared to the receptor-mediated changes in cytoplasmic Ca2+ concentration, as measured by the fluorescent Ca2+ indicator, fura-2. The Ca2+ signal decays upon termination of inositol phosphate formation and returns to base line within 30 s. Thus, while (c1:2,4,5)IP3 may have some yet unknown biological effects on Ca2+ homeostasis, its metabolism seems far too slow to be the primary regulator of cytosolic Ca2+ levels under long term stimulatory conditions. The rate at which the Ca2+ signal decays is, however, somewhat slowed after prolonged agonist stimulation. Furthermore, the capacity of the cells to mobilize intracellular Ca2+ in response to a second agonist stimulation is slightly delayed when the duration of the first stimulus is prolonged. The results suggest that the regulation of cytoplasmic Ca2+ levels may be more complicated than initially realized and could depend on the combined actions of more than one inositol polyphosphate.

Highlights

From the Calcium Regulation Section, Laboratory of Cellular and Molecular Pharmacology, National Institute of Environmental Health Sciences-National Institutes of Health, Research Triangle Park, North Carolina 27709
The results suggest that the regulatoifon cytoplasmic Ca2+levels maybe more complicatedthan initially realized and could depend on the combined actions of more than oneinositol polyphosphate
Inositol Phosphate Accumulation-Rat parotid acinar cells were prepared by sequential trypsin and collagenase digestion of minced parotid glands as described previously (16).The cells were vigorously shaken (300rpm) with [3H]inositol(specificactivity 15 Ci/mmol, 250 pCi/ml) in Krebs-Ringer bicarbonate solution containing 1%bovine serum albumin (BSA-KRB) (16) for 90 min at 37 "C, under 5% COZ in Oz

Summary

RELATIONSHIPTO CALCIUM SIGNALLING*

Radioactivity was monitored on-line, and peaks of radioactivity standardandsupernatant from parotid cells (terminated by the addition of boiling water) were each divided and treated with an equal volume of deionized water or 10% trichloroacetic acid for 10 min at room temperature (19). Muscarinic receptor stimulation of rat parotid acinar cells results in the formation of twowellcharacterized inositol inositol containing lipids were extracted from representative aliquots trisphosphate isomers, (1,4,5)IP3and (1,3,4)IP3,as well as a of cells for each experiment and phosphoinositide-associated radio- thirdcompound,whichappears to be (cl:2,4,5)IP3(Fig. 1). The samples were large peak of [3H]inositol 1,3,4-trisphosphate([3H](1,3,4)IP3)formed centrifuged and the resulting supernatant divided into two equal upon muscarinic receptor stimulation of rat parotid cells. The results are trast, [3H](cl:2,4,5)IP3decays very slowly.Rate constantsfor the mean +- S.E. from eight analyses performed in four independent the metabolism of (1,4,5)IP3, (cl:2,4,5)IP3, and (1,3,4,5)1P4 experiments

Inositol phosphate tu

Findings

DISCUSSION