ORGANIZATION OF THE RECEPTOR-MEDIATED PHOSPHOINOSITIDE CYCLE - RELATIONSHIP BETWEEN RECEPTOR OCCUPANCY AND ACCESSION OF PHOSPHATIDYLINOSITOL

We have previously reported the existence of separate hormone-responsi ve and -unresponsive pools of inositol phospholipids in WRK-1 cells. I n order to further explore this concept we have performed experiments to examine the relationship between the plasma membrane receptor and t he pool of phosphatidyl inositol (PtdIns) that is metabolized in respo nse to hormonal stimulation. The results support the following conclus ions. 1) The amount of PtdIns metabolized in WRK-1 cells in response t o vasopressin is proportional to the number of receptors occupied; nei ther prolonged activation with nor readdition of a submaximal concentr ation of vasopressin induced the same degree of PtdIns metabolism as a maximal concentration of vasopressin. 2) Dissociation of cytoskeletal structures by incubation with cytochalasin D did not alter the amount of PtdIns accessed during hormonal stimulation. 3) Accession of PtdIn s from internal membranes does not depend on internalization and recyc ling of the receptor; cells incubated in potassium-free medium failed to internalize receptor-ligand complexes, yet they accessed the same a mount of PtdIns in response to vasopressin as did control cells. 4) Go lgi-mediated phosphatidylinositol transport is not involved in hormone -stimulated phosphoinositide turnover since brefeldin A, which interfe res with Golgi-mediated transport processes, had no effect on the amou nt of PtdIns accessed during vasopressin stimulation. 5) Phosphoinosit ide breakdown and compensatory resynthesis is not a closed process; ne wly synthesized PtdIns is not preferentially localized to a hormone-re sponsive pool but is generally redistributed between responsive and un responsive pools. (C) 1997 Wiley-Liss, Inc.dagger