MEASUREMENT OF ARACHIDONIC-ACID RELEASE FROM PERMEABILIZED MYOMETRIALCELLS OF GUINEA-PIG UTERUS

A technique has been developed for prelabelling and permeabilisation o f guinea pig uterine myocytes to enable measurement of arachidonic aci d release/phospholipase A2 activity in cells with intact membranes. In tact cells were prelabelled with [H-3]inositol or [H-3]arachidonic aci d for measurement of phospholipase C and A2 respectively. In intact ce lls 10 nM endothelin-1 or 1 muM bradykinin stimulated both inositol po lyphosphate and arachidonic acid release, whilst 1 muM oxytocin, argin ine vasopressin or histamine were without effect. In Streptolysin-0 pe rmeabilised myometrial cells calcium-stimulation of inositol polyphosp hate and arachidonic acid release was detected between 10 muM 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-0. For arachido nic acid release the K0.5 for calcium activation was about 7 muM, a le vel above that normally likely to be found in the uterine myocyte. Hen ce 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 .