Measurement of arachidonic acid release from permeabilised myometrial cells of guinea pig uterus.

Abstract

A technique has been developed for prelabelling and permeabilisation of guinea pig uterine myocytes to enable measurement of arachidonic acid release/phospholipase A2 activity in cells with intact membranes. Intact cells were prelabelled with [3H]inositol or [3H]arachidonic acid for measurement of phospholipase C and A2 respectively. In intact cells 10 nM endothelin-1 or 1 microM bradykinin stimulated both inositol polyphosphate and arachidonic acid release, whilst 1 microM oxytocin, arginine vasopressin or histamine were without effect. In Streptolysin-O permeabilised myometrial cells calcium-stimulation of inositol polyphosphate and arachidonic acid release was detected between 10 microM and 1 mM free calcium. The patterns of inositol polyphosphate and arachidonic acid release were broadly similar. Responses to 1 mM calcium were not detected in intact cells not treated with Streptolysin-O. For arachidonic acid release the K0.5 for calcium activation was about 7 microM, a level above that normally likely to be found in the uterine myocyte. Hence it is concluded that unless there are high local concentrations of calcium close to the plasma membrane, calcium is unlikely alone to be the primary regulator of arachidonic acid release and phospholipase A2.