Guanine nucleotide regulatory protein alterations in the Milan hypertensive rat strain

OBJECTIVE: 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. DESIGN: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.