Effect of dual agonists on phosphoinositide pools in WRK-1 cells.

Both vasopressin and bradykinin activate the phosphoinositide cycle in WRK-1 rat mammary tumour cells. When the two agonists are added simultaneously, partial additivity is observed with respect to disappearance of prelabelled phosphoinositides and accumulation of inositol phosphates; no additivity is observed with respect to resynthesis of phosphatidylinositol as assessed by monitoring [32P]Pi incorporation. Lack of complete additivity can be explained, at least in part, by heterologous desensitization. In order to determine whether the two agonists were accessing a common or individual hormone-sensitive phosphoinositide pools, cells were incubated with [32P]Pi in the presence of either vasopressin or bradykinin and subsequently restimulated with the alternative agonist. The lipid pool labelled in the presence of either agonist was sensitive to subsequent treatment by the other ligand, suggesting a common phosphoinositide pool. However, when cells were incubated with [32P]Pi in the absence of agonists, the time course of labelling of the hormone-sensitive pool was different for bradykinin and vasopressin, with that for bradykinin becoming labelled within a much shorter time. Thus although there is a significant overlap between the phosphoinositide pools responding to vasopressin and bradykinin, there is a small fraction of the hormone-sensitive lipid which responds only to bradykinin.

The relationship of hormone-sensitive and hormone-insensitive phosphatidylinositol to phosphatidylinositol 4,5-bisphosphate in the WRK-1 cell.

We have previously characterized two distinct pools of phosphatidylinositol (PI) in the WRK-1 rat mammary tumor cell, one whose metabolism is enhanced in response to vasopressin and another which is insensitive to hormonal manipulation. The purpose of the present study was to examine the relationship between cellular phosphatidylinositol 4,5-bisphosphate (PIP2) and each of the two PI pools. We have found that in WRK-1 cells, vasopressin induces the rapid loss of PIP2 and the accumulation of inositol phosphates. By making use of kinetic differences in 32Pi uptake into the two pools of PI and assessing radioactivity levels in the 1-phosphate of PIP2, we have determined that hormone-sensitive PI is the precursor of approximately 60% of the cellular PIP2; the remainder is synthesized from the hormone-insensitive pool. Additional data indicate that PIP2 derived from hormone-sensitive PI is likewise hormone-sensitive, while that synthesized from hormone-insensitive PI remains stable over a long period of time and is not affected by the presence of vasopressin.

The effect of dihydroergotoxine on lipid peroxidation in vitro.

Dihydroergotoxine mesylate (DHET), an ergot alkaloid derivative, is widely used to treat senile cerebral vascular insufficiency. Aspects of this age-related phenomenon may be due to deterioration by lipid oxidation of cellular membranes. DHET stabilizes EEG alpha frequencies, increases cerebral blood flow and oxygen uptake and accumulates in lipid-rich structures of the brain. The effect of DHET was studied on iron-catalyzed peroxidation of liposomes as measured by the thiobarbituric acid assay. It was found that DHET inhibits peroxidation in vitro in a dose-dependent manner. These results suggest that DHET acts in part as a lipid antioxidant when used to treat senile cerebral vascular insufficiency.