Organization of the receptor-mediated phosphoinositide cycle: Relationship between receptor occupancy and accession of phosphatidylinositol

Abstract

We have previously reported the existence of separate hormone-responsive and -unresponsive pools of inositol phospholipids in WRK-1 cells. In order to further explore this concept, we have performed experiments to examine the relationship between the plasma membrane receptor and the pool of phosphatidylinositol (Ptdlns) that is metabolized in response to hormonal stimulation. The results support the following conclusions. 1) The amount of Ptdlns metabolized in WRK-1 cells in response to vasopressin is proportional to the number of receptors occupied; neither prolonged activation with nor readdition of submaximal concentration of vasopressin induced the same degree of Ptdlns metabolism as maximal concentration of vasopressin. 2) Dissociation of cytoskeletal structures by incubation with cytochalasin D did not alter the amount of Ptdlns accessed during hormonal stimulation. 3) Accession of Ptdlns from internal membranes does not depend on internalization and recycling of the receptor; cells incubated in potassium-free medium failed to internalize receptor-ligand complexes, yet they accessed the same amount of Ptdlns in response to vasopressin as did control cells. 4) Golgi-mediated phosphatidylinositol transport is not involved in hormone-stimulated phosphoinositide turnover, since brefeldin A, which interferes with Golgi-mediated transport processes, had no effect on the amount of Ptdlns accessed during vasopressin stimulation. 5) Phosphoinositide breakdown and compensatory resynthesis is not a closed process; newly synthesized Ptdlns is not preferentially localized to a hormone-responsive pool but is generally redistributed between responsive and unresponsive pools.