Reduced adenosine A1 receptor and G alpha protein coupling in rat ventricular myocardium during aging.
Adenosine A1 receptor (A1-AdoR) function in rat ventricles has previously been shown to decrease with age. In the present study, using the ligand [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX) and coimmunoprecipitation of A1-AdoRs with their associated G proteins, we determined the specific binding of A1-AdoR and A1-AdoR/G protein coupling in ventricular myocardium of 6- to 24-month-old Fischer 344 rats. The densities (Bmax) of A1-AdoRs were 5.8 +/- 0.8 fmol/mg protein in 6-month-old rats and 6.1 +/- 1.4 fmol/mg protein in 24-month-old rats, and the dissociation constants (Kd) were 0.32 +/- 0.04 nmol/L in 6-month-old rats and 0.34 +/- 0.05 nmol/L in 24-month-old rats (P > .05). Analysis of the dose-dependent displacement of [3H]DPCPX binding by the selective A1-receptor agonist, N6-p-sulfophenyladenosine (SPA), yielded two affinity binding sites in both 6- and 24-month-old rats. However, the proportion of high-affinity A1-AdoRs was significantly lower in 24-month-old rats (23.5%) compared with 6-month-old rats (54.9%) (P < .05). In solubilized ventricular membranes, specific [3H]DPCPX binding sites were detected in immunoprecipitates of G alpha i3 and G alpha o antisera but not with antibodies for other G alpha proteins. The basal coimmunoprecipitation of A1-AdoR with G alpha i3 and G alpha o proteins decreased by 22% and 21%, respectively, in ventricular membranes of 24-month-old rats compared with that in 6-month-old animals. A1-AdoR stimulation with SPA increased the coprecipitation of A1-AdoR with G alpha i3 and G alpha o proteins by 287% and 245%, respectively, in 6-month-old rats but only by 129% and 140%, respectively, in 24-month-old rats (P < .01). In the absence of changes in A1-AdoR density and G alpha protein levels, an age-related decline in high-affinity A1-AdoR binding sites and a reduction in the association of A1-AdoR with G alpha proteins suggest that the age-related decrease in ventricular A1-AdoR-mediated response is related to a reduction in the coupling between A1-AdoR and their G proteins.
Cai
G
G
Department of Pharmacology, Allegheny University of the Health Sciences, Philadelphia, Pa 19129, USA.
Wang
H Y
HY
Gao
E
E
Horwitz
J
J
Snyder
D L
DL
Pelleg
A
A
Roberts
J
J
Friedman
E
E
D000067956:Adenylyl Cyclase Inhibitors; D000375:Aging / Q000378:metabolism*; D000818:Animals; D019204:GTP-Binding Proteins / Q000502:physiology*; D008297:Male; D009206:Myocardium / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344; D018047:Receptors, Purinergic P1 / Q000032:analysis* / Q000502:physiology; D014970:Xanthines / Q000378:metabolism
D000067956:Adenylyl Cyclase Inhibitors; D018047:Receptors, Purinergic P1; D014970:Xanthines; C051360:1,3-dipropyl-8-cyclopentylxanthine; D019204:GTP-Binding Proteins