Type 1 angiotensin II receptors in bovine adrenocortical cells

• Main Author: Dell, Ghislaine C.
• Published: University of Edinburgh 1997
• Subjects: 572
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.649470

This thesis presents modifications to improve the established methods for the isolation and culture of zg cells, and characterises responses to steroidogenic agonists during short-term primary culture. The modified zg culture was used as a tool to pharmacologically characterise the type 1 AII receptor using Schild analysis in order to obtain pA<SUB>2</SUB> values for a selective AT<SUB>1</SUB> receptor antagonist, losartan (DuP753). The pA<SUB>2</SUB> value is a measure of antagonist affinity which should be constant for the antagonist at a particular receptor subtype. The pA<SUB>2</SUB> values obtained were compared to both pA<SUB>2</SUB> values previously reported for boving zfr cell at AT<SUB>1</SUB> receptors, and pA<SUB>2</SUB> values calculated from cultured rat mesenteric artery smooth muscle cells. No significant difference was found in the pA<SUB>2</SUB> values (zg : 7.02 +/- 0.20; vsmc : 7.45 +/- 0.31), indicating that pharmacologically the receptors are indistinguishable. The bovine AT<SUB>1</SUB> receptor was then cloned, from bovine spleen, using the published bovine adrenal AT<SUB>1</SUB> receptor sequence, by PCR. The resulting clones were sequenced and confirmed as AT<SUB>1</SUB> receptors. The DNA was then used to probe for AT<SUB>1</SUB> receptor mRNA expression in primary cultures of bovine zfr cells using Northern blot analysis. Specific agonists for the adrenal cortex, and second messenger analogues, were employed in these experiments. AII caused homologous downregulation (86%) of its receptor mRNA, an action found to be mimicked by stimulation of protein kinase C and calcium influx (91%). A cyclic AMP analogue (8-bromo cyclic AMP) also caused downregulation of AT<SUB>1</SUB> receptor mRNA. In contrast, AT<SUB>1</SUB> receptor mRNA was upregulated by incubation of the cells with insulin-like growth factor 1 (IGF-1) and potassium. Finally, the possibility that adrenal cortiscosteroids could exert a short-loop feedback effect on AT<SUB>1</SUB> receptor mRNA was analysed. Cortisol, but no aldosterone, significantly downregulated (41%) the level of AT<SUB>1</SUB> mRNA, which indicated a possible zone-specific effect on the regulation of AT<SUB>1</SUB> receptors in the adrenal cortex. The results of these studies suggest that the physiological regulation of AII receptors within the adrenal cortex is more complex than has been previously considered.