PROLONGED ACTIVATION OF ALPHA(1) ADRENOCEPTORS INDUCES DOWN-REGULATION OF PROTEIN-KINASE-C IN VASCULAR SMOOTH-MUSCLE

Sustained exposure of vascular smooth muscle to catecholamines results in desensitization of alpha1-adrenoreceptor-mediated vascular smooth muscle contraction. The present study was designed to determine the effects of prolonged exposure of blood vessels to catecholamines on protein kinase C (PKC) activity. Incubation of rat aortic smooth muscle with 10 muM norepinephrine (NE) for 4 h resulted in a threefold decrease in sensitivity of the contractile response of rat aortic smooth muscle to the phorbol ester 4beta-phorbol 12,13-dibutyrate (PDBu); this loss in sensitivity was dependent on the presence of endothelium. NE induced a 45% decrease in enzymatic activity of the soluble and particulate forms of PKC. With [H-3]PDBu used to label phorbol ester receptor binding sites in the aorta, there was a 34% decrease in [H-3]PDBu binding sites in NE-treated blood vessels without change in binding affinity for the ligand. To determine whether this loss in enzymatic activity and [H-3]PDBu binding resulted from a decrease in the quantity of the enzyme, Western blot analyses were performed using a monoclonal antibody (MoAb) against PKC. This approach confirmed the presence of an 80-Kd immunoreactive PKC in the soluble fraction of rat aortic smooth muscle and demonstrated a 44% decrease in the amount of PKC in blood vessels after sustained exposure to catecholamines. Our results demonstrate that prolonged activation of alpha-adrenoceptors in blood vessels leads to down-regulation of PKC which may contribute to desensitization of contraction mediated by vasoconstrictors.