Experimental Study Of Hyperpolarization Of 11,12-EET On Porcine Coronary Smooth Muscle Cell

Background In the coronary circulation, endothelialcells derive three endothelium-derived relaxing factors(EDRF), namely endothelium-derived nitric oxide(EDNO ), endothelium-derived hyperpolarizing factor(EDHF) and prostacyclin (PGI,)contributing to themaintainance of coronary blood flow. EDNO and EDHFare the two major factors and the mechanism of whichis throught to be through the regulation of smoothmuscle cells. Dysfunction of EDNO mechanism ismainly due to ischaemia-reperfusion injury. Themechanism and clinical application of NO have beenwell accepted. In contrast to EDNO, EDHF is suscept-ible to cardioplegia with hyperkalaemic components.The damage of endothelial cells may directly affect thefunction of coronary relaxation,decrease coronary flowduring the reperfusion period and impair the cardiaccontractility. Recently, the nature and mechanism ofEDHF are still obscure. Much of recent studies in thisfield fOcused on Epoxyeico-satrienoic acid (EETs) andAnandamide. EETs have fOur isoformers which were5,6-EET' 8,9-EET' l l, l2-EET' l4, l5-EET. They notonly share similar bio-chemical properties, but alsopocess identical bio-activities with known EDHF .Theaim of the study was to clarify the relationship betweenEETs and EDHF by the investigation of changes in thetransmembrane potential in coronary blood vesselsmooth muscle cells in the use of l l, l2-EET in vitrounder con focal microscope.Methods The coronary arteries were obtained from 4mini-pigs and the smooth cells were isolated enzyma-tically using collagenaseII and pancreatin(3: l ). Cellswere passaged one time and identified by immuno-histochemistry and then devided into three groups andcultured on 96-well plates. Cells with normal mor-phology and active state were selected from each welland examed by the con focal. The three groups are:EETgroup, GLB group and TEA group. Based membranepotential were measured 5 times before the addition ofl l,l2-EET (1microM/L [3] ) (but after the addition ofGLB 0. 5mi croM/L [3] and TEA l0microM/L [4] in thelatter twe groups). After the addition of l l, 12-EET forseveral seconds, thirty-five times scans document thechanges in membrane potential.Results: T-test were analyzed in the membranepotential between the baseline and the post-drug. Thelevel of the post-drug membrane potential of EETgroup (n=l l) and GLB group (n=8) were significenthigher than those of the baseline menbrane potential oftwo groups (P<0.00l ); while,no significent differcenesin TEA group (n=23,P>0. 05).Conclusion8: l l, l2-EET hyperpolarized the smoothmuscle cell cultured from porcine coronary vessels.The hyperpolarization of the membrane potential beblocked by TEA (Kca channel blockers), but not beblocked by GLB(K.., channel blockers). The resultssuggest that l l, l2-EET is most likely EDHF.