Molecular Mechanisms in Vascular Endothelin B Receptor Up-Regulation

Cardiovascular diseases are characterized by changes in morphology and function of the blood vessels. Different signalling molecules are involved in these changes. Endothelin-1 (ET-1) is one of the most potent signalling molecules found in man. Its actions are mediated by two receptors, the ETA and the ETB receptors. The ETA receptor is located on the smooth muscle cells and mediates potent contractile effects throughout the vascular system. The ETB receptor, when located on the endothelial cell mediates vasodilatation. When located on the smooth muscle cell the ETB receptor mediates vasoconstriction. In cardiovascular diseases there are changes in the smooth muscle phenotype and the contractile ETB receptor is up-regulated. In this thesis an organ culture model was used to examine the variation, the time-course and the intracellular pathways responsible for the ETB receptor up-regulation. Following organ culture, the ETB receptor was up-regulated in rat mesenteric arteries, the femoral artery and in the distal part of the caudal artery, but not in the aorta or the proximal caudal artery. The up-regulation was stronger in smaller arteries, in the mesenteric system and in veins. The up-regulation process in the mesenteric artery was preceded by mRNA transcription. The maximum pharmacological efficacy was reached after 24 h and the up-regulation was complete after 48 h. In acute experiments, non-specific inhibitors of protein kinase C (PKC) decreased the ETB receptor mediated contraction, but a specific PKC inhibitor, Ro31-7549, was ineffective at blocking contraction. In long-term experiments, PKC inhibitors abolished ETB receptor upregulation after 24 h of organ culture. Thus, PKC has an important role in ETB receptor upregulation and contraction. The ERK1/2 MAP kinase was activated following 3 h of organ culture. After blocking the ERK1/2 pathway with inhibitors of raf and MEK1/2, the ETB receptor up-regulation was inhibited, showing that this pathway is crucial for the ETB receptor induction. Cytokines are important regulators of inflammation and acute phase responses. After organ culture with the cytokines IL-1β and TNF-α the ETB receptor mediated contractions were increased. There was no increase in potency and the ETB receptor mRNA was not increased beyond that of incubated control arteries. These findings suggest an effect of IL-1β and TNF-α on the contractile machinery per se. To summarize, organ culture can be used for the study of the ETB receptor upregulation. This up-regulation is pronounced in small arteries and veins. PKC and the ERK1/2 and MAP kinase system are important for the induction of this up-regulation.