ATTENUATION OF G(S)ALPHA COUPLING EFFICIENCY IN BROWN-ADIPOSE-TISSUE PLASMA-MEMBRANES FROM COLD-ACCLIMATED HAMSTERS

In order to localize site(s) of beta-adrenergic desensitization found in brown adipocytes from cold-acclimated animals, total brown-adipose-tissue homogenates (postnuclear supernatant) were obtained from control or cold-acclimated hamsters and were fractionated on discontinuous sucrose gradients. A low-density band (cytosolic proteins) and a high-density band (mitochondria) were obtained; in the middle fractions only low levels of protein were recovered. However, these fractions displayed a high level of specific [H-3]ouabain binding, indicating that they represented fractions enriched in plasma membranes. The level of [H-3]ouabain binding was significantly higher in plasma membranes from cold-acclimated animals, indicating an increased density of Na,K-ATPase units. The maximal activity of adenylate cyclase, as estimated with forskolin, was not changed by cold acclimation. However, the levels of cyclase activity observed after G(s)-protein-mediated activation (with guanosine 5'-[gamma-thio]triphosphate, isoprenaline, both of these, or fluoride) were decreased, indicating a decreased coupling efficiency. Notably, a significant decrease was observed in the functional activity of the G(s) protein, as directly measured by estimation of the ability of cholate extracts of brown-fat plasma membranes to reconstitute G(s)-protein-mediated stimulation of adenylate cyclase in cyc- membranes. Further, a functionally significant decrease (to 72%) was observed in the ratio between the amount of functional G(s) proteins and adenylate cyclase units. The total content of G(s)alpha protein was decreased to the same extent as the coupling efficiency of the membranes, indicating that a lower content of functionally equivalent G(s)alpha molecules could explain the decreased coupling. It could therefore be concluded that a decrease in G(s)-protein-mediated coupling efficiency, owing to a decrease in the amount of G(s)alpha, is at least one site of beta-adrenergic desensitization in cold-acclimated animals. This may, at least in part, explain that desensitization takes place despite the fact that the beta3-adrenoceptor itself apparently lacks some of the sites known to be involved in the desensitization process in other beta-adrenergic receptors.