PHORBOL ESTER STIMULATES ACETYLCHOLINE SYNTHESIS IN CULTURED ENDOTHELIAL-CELLS ISOLATED FROM PORCINE CEREBRAL MICROVESSELS

Acetylcholine (ACh) is one of the factor which induces vasodilation th rough the release of endothelium-derived relaxing factor. The aim of t his study was to clarify whether endothelial cells can synthesize ACh and the types of substance which regulate the synthesis of ACh in endo thelial cells. We determined the ACh content of endothelial cells isol ated from porcine cerebral microvessels and of the culture medium. ACh was detected in the medium after 12 h incubation in the presence of d iisopropylfluorophosphate, a non-specific cholinesterase inhibitor, an d increased linearly up to 24 h. Phorbol 12-myristate 13-acetate (PMA, 10(-7) M) increased the ACh content of the medium in a dose-dependent manner. The effect of PMA was most apparent between 12 and 24 h after treatment, and was inhibited by cycloheximide. Calphostin C, a specif ic inhibitor of protein kinase C (PKC), did not inhibit the effect of PMA. Dioctanoyl glycerol, a specific activator of PKC, did not increas e the intracellular ACh content or the amount released into the cultur e medium. ACh synthesis was not inhibited by bromoacetyl-choline, a sp ecific inhibitor of choline acetyltransferase (ChAT). PMA treatment di d not affect the specific activity of ACh synthesis in endothelial cel ls. These data show that endothelial cells are able to synthesize ACh, and that ACh synthesis is up-regulated by PMA through the PKC indepen dent mechanism via protein induction. The enzyme which synthesizes ACh in endothelial cells is not ChAT. The increase in ACh synthesis induc ed by PMA may not be due to induction of the ACh synthetic enzyme.