Guanine nucleotide regulatory protein alterations in the Milan hypertensive rat strain

Abstract

To examine whether the altered regulation of adenylyl cyclase that has been reported in vascular tissues from spontaneously hypertensive rats is also evident in the Milan hypertensive (MHS) rat strain. The plasma membranes of vascular smooth muscle cells derived from thoracic aortae from adult (60-day-old) MHS and Milan normotensive (MNS) strain rats were studied. Guanine nucleotide regulatory protein (G-protein) function was inferred from adenylyl cyclase activity studies, and levels of G-protein subunits were assessed by immunoblotting. beta-Adrenergic receptor number and affinity were measured from the binding of the antagonist [125I]-cyanopindolol. Basal adenylyl cyclase activity was increased significantly in MHS rat cell membranes, and stimulation by 0.1 mmol/l isoproterenol and 0.01 mmol/l prostaglandin E1 was significantly greater in MHS than in MNS rat cell membranes. Forskolin (at 0.1 mmol/l) resulted in a significantly greater stimulatory response in MHS membranes, which was eliminated by 0.01 mol/l NaF. Biphasic effects of GTP on isoproterenol-stimulated membranes demonstrated similar Gi function in MHS and MNS rat cell membranes, although a greater stimulatory GTP response was observed in MHS rat cell membranes. The levels of Gs alpha (both forms), Gi3 alpha and the beta-subunit were reduced in MHS rat cell membranes, whereas the levels of Gi2 alpha and Gq alpha and G11 alpha were unchanged. The number of beta-adrenoceptors was increased significantly in MHS rat cell membranes, whereas receptor affinity for the antagonist was unaltered. There are differences in adenylyl cyclase stimulatory responses in MHS rat vascular smooth muscle cell membranes. We have found evidence of reduced levels of particular G-protein subunits, altered beta-adrenoceptor-Gs coupling and increased beta-adrenoceptor number.