The enzyme cyclooxygenase is found in high concentrations in vascular tissues where it synthesized the various prostaglandins involved in vascular homeostasis. Prostaglandin synthesis is down-regulated by autoinactivation of cyclooxygenase. Aspirin induces an analogous refractory state by irreversibly acetylating the enzyme. This project will investigate the molecular mechanisms regulating synthesis of the enzyme in cultured human cells of vascular origin following autoinactivation or treatment with aspirin. Following complete inactivation by aspirin the enzyme in vascular smooth muscle cells recovers within 2 hours by a cycloheximide sensitive process which has an absolute requirement for epidermal growth factor (EGF). In contrast, recovery of cultured human vascular endothelial cells takes 8-24 hours, does not require EGF, is suppressed by endothelial cell growth factor (ECGF) and is stimulated by inhibitors of DNA synthesis. In order to investigate these mechanisms in more depth, the cDNA for human cyclooxygenase will be cloned using antisera to purified cyclooxygenase to screen a human vascular endothelial cell cDNA library. This will provide probes to compare transcription and expression of the mRNA for cyclooxygenase in smooth muscle vs endothelial cells and to examine the mechanisms underlying the EGF requirement in the former cell type. Preliminary experiments have established the feasibility of these studies. Cultures prelabelled with 14C arachiodonic acid and RIA analysis will be used to characterize prostacyclin releasing factors of physiological significance. In particular the functional integration of newly synthesized cyclooxygenase with thrombin and leukotriene receptors will be studied to determine the reason for the permanent inactivation of integrated function by aspirin in non-dividing vascular cells. These experiments will provide information on the interactions of the vasculature with antiinflammatory analgesic drugs, and the recovery of prostacyclin synthesis in both the endothelial and smooth muscle components following exposure to these agents. In addition, the work represents a new approach to understanding the basic cellular functions of the prostaglandins and to defining the molecular biology of the cyclooxygenases.
