Prolonged treatment of rat adipocytes with the A1-adenosine receptor agonist [-]N(6)-phenylisopropyl adenosine (PIA) or prostaglandin E1 down-regulates Gi and induces heterologous desensitization. alpha 2-Adrenergic receptors also inhibit adenylyl cyclase through Gi, but whether alpha 2-receptors are present on rat adipocytes has been controversial. We have investigated the effects of the highly specific alpha 2-adrenergic agonist UK 14304 (5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline) on rat adipocytes. In previous studies on young rats, we were unable to demonstrate an effect of the alpha 2-agonist. We now report that, in cells isolated from older, more obese rats (300-400 g), UK 14304 inhibited lipolysis (measured as the rate of glycerol release) by approximately 40% (EC50 approximately 40 nM). To determine whether UK 14304 would induce heterologous desensitization, we incubated adipocytes with or without 1 microM UK 14304 for 4 days in primary culture. The cells were then washed, and the rate of lipolysis was determined during a 30-min incubation in the presence of various concentrations of PIA. The concentration-response curve for PIA-induced inhibition of lipolysis was shifted to the right, with the EC50 for UK 14304-treated cells about 2-fold higher than in the control cells. This finding demonstrates that the alpha 2-agonist can desensitize the response to PIA and indicates heterologous desensitization. To investigate the mechanism of this phenomenon, we isolated crude membrane fractions from the cells and analyzed them on Western blots using antibodies against Gi alpha 1, 2 and 3. In cells treated with UK 14304 for 4 days, Gi1 alpha and Gi2 alpha were down-regulated to about 15% of the control level, and Gi3 alpha was decreased to 30% of control. We conclude that prolonged treatment of adipocytes with the alpha 2-agonist induces heterologous desensitization of lipolysis and causes down-regulation of Gi. The findings suggest that G-protein down-regulation is a mechanism for heterologous desensitization.