TRANSCRIPTIONAL DOWN-REGULATION OF M2 MUSCARINIC RECEPTOR GENE-EXPRESSION IN HUMAN EMBRYONIC LUNG (HEL-299) CELLS BY PROTEIN-KINASE-C

m2 muscarinic receptor gene expression was investigated following stim ulation of protein kinase C (PKC) with the phorbol ester 4 beta-phorbo l dibutyrate (PDBu) in HEL 299 cells. PDBu (100 nM) caused a time depe ndent decrease in the steady-state levels of m2 receptor mRNA and in s pecific [H-3]N-methyl scopolamine binding. Preincubation with the PKC inhibitor GF-109203X inhibited the reduction in M(2) receptor and mRNA levels induced by PDBu, confirming the involvement of PKC, Chronic PD Bu treatment also caused desensitization of the receptor as forskolin- stimulated cAMP accumulation, inhibited by carbachol in control cells, was lost upon treatment with PDBu for 24 h. Co-incubation with PDBu a nd the protein synthesis inhibitor cycloheximide, inhibited PDBu-media ted reduction of m2 receptor mRNA, indicating new protein synthesis is required for down-regulation. Half-life studies using the transcripti onal inhibitor actinomycin D suggested that the stability of the m2 re ceptor mRNA was not altered by PDBu treatment (t(1/2) = 2 h) Nuclear r un on assays showed a 50% reduction in the rate of m2 receptor gene tr anscription after treatment with PDBu for 12 h. In conclusion we have provided evidence for heterologous regulation of m2 receptor gene expr ession through changes in gene transcription resulting in uncoupling o f M(2) receptors. Furthermore, the synthesis of an unidentified factor is required for the down-regulation process.