PHORBOL ESTER AND CALCIUM REGULATION OF CORTICOTROPIN-RELEASING FACTOR-RECEPTOR-1 EXPRESSION IN A NEURONAL CELL-LINE

We have previously demonstrated that corticotrophin-releasing factor r eceptor I (CRF-RI) mRNA levels can be down-regulated via activation of the cyclic AMP pathway in CATH.a cells, a neuronal cell line. In this study, we show evidence for down-regulation of CRF-RI mRNA levels via activation of the protein kinase C (PKC) and calcium second messenger pathways. Incubation of CATH.a cells with phorbol 12-myristate 13-ace tate (PMA), an activator of PKC, resulted in a time-and concentration- dependent down-regulation of CRF-R1 mRNA levels. Pretreatment with the inactive phorbol ester 4 alpha-phorbol failed to influence significan tly CRF-R1 mRNA levels, Incubation with carbachol, a cholinergic agoni st known to activate PKC and increase intracellular calcium levels via phosphatidylinositol breakdown, also downregulated CRF-RI mRNA levels . Intracellular calcium levels were directly increased using A23187, a calcium ionophore, and thapsigargin, a calcium-ATPase inhibitor, Elev ation of intracellular calcium content using either A23187 or thapsiga rgin significantly down-regulated levels of CRF-R1 mRNA. Furthermore, chelation of calcium with EGTA or blockade of voltage-dependent calciu m channels with nifedipine inhibited agonist-mediated downregulation o f CRF-R1 mRNA levels. These results indicate that activation of PKC or calcium signal transduction pathways is sufficient to cause down-regu lation of CRF-R1 mRNA levels and that calcium is required for agonist- mediated down-regulation of this receptor.