Tonic activity of the rat adipocyte A1-adenosine receptor

Adipocyte A1-adenosine receptors (A1 AdoR) tonically inhibit adenylyl cyclase and lipolysis. Three potential explanations for tonic activity of A1AdoR of rat epididymal adipocytes were investigated: high affinity of adenosine for the receptor, efficient coupling of receptor activation to response, and spontaneous activity of the receptor in the absence of agonist. The affinity of adenosine for the adipocyte A1AdoR was determined as 4.6 μM by analysis of effects of an irreversible receptor antagonist on agonist concentration-response relationships. In contrast, the potency of adenosine to decrease cyclic AMP in isolated adipocytes was 1.4 nM. Occupancy by agonist of the A1AdoR was efficiently coupled to functional response (decrease of adipocyte cyclic AMP content). Activation by adenosine of less than 1% of A1AdoRs caused a near-maximal decrease of cyclic AMP in adipocytes. Thus the receptor reserve for adenosine to decrease cyclic AMP content of adipocytes was greater than 99%. Affinities and receptor reserves for other A1AdoR agonists were determined. Agonists appeared to differ more in their affinity for the receptor than in their intrinsic efficacy to activate it. A1AdoRs were inactive in the absence of agonist. It is concluded that adipocyte A1AdoR are tonically activated by endogenous adenosine at nanomolar concentrations. The expression of a high density of A1AdoR that are efficiently coupled to a functional response enables the adipocyte to respond with high sensitivity to the low-affinity agonist, adenosine. Adipocytes may be a model for cells whose functions are tonically modulated by adenosine present in the interstitium of well-oxygenated tissues. Keywords: Adipocyte, adenosine, receptor reserve, cyclic AMP, A1-adenosine receptor, CVT-2759, FSCPX Introduction Activation of the A1-adenosine receptor (A1AdoR) in adipocytes reduces adenylyl cyclase activity, cyclic AMP content, and rate of lipolysis, and enhances the actions of insulin (Fain et al., 1972; Schwabe et al., 1973; 1974). Increases of cyclic AMP accumulation and lipolysis in white adipose tissue have been observed when adenosine deaminase is added to degrade endogenous adenosine (Schwabe & Ebert, 1974; Fain & Wieser, 1975; Honnor et al., 1985) and after treatments with either pertussis toxin (Moreno et al., 1983; Olansky et al., 1983; Vannucci et al., 1989) or adenosine receptor antagonists (Vannucci et al., 1989; Lanoue & Martin, 1994; Coates et al., 1994). These findings can be interpreted to indicate that adipocyte A1AdoRs are always active and mediate a tonic inhibition of adenylyl cyclase activity and lipolysis. Tonic inhibition of adenylyl cyclase and lipolysis by the adipocyte A1AdoR could be the result of several factors. A high concentration of endogenous adenosine in adipose tissue or in preparations of isolated adipocytes may cause adenosine receptors to be tonically activated. Alternatively, a high affinity of the adipocyte A1AdoR for adenosine could explain tonic activity of the receptor at unusually low adenosine concentrations. Tonic inhibitions of adenylyl cyclase and lipolysis could also be caused by agonist-independent (i.e., spontaneous) activity of adipocyte A1AdoR. We recently demonstrated the presence of spontaneous activity of human adenosine receptors expressed at a density of 8000 fmol mg protein−1 in Chinese hamster ovary cells (Shryock et al., 1998a). Because adenosine deaminase has been reported to decrease the concentration of endogenous adenosine to undetectable levels (Honnor et al., 1985; Lohse et al., 1986), increases of adenylyl cyclase activity and lipolysis caused by adenosine receptor antagonists in the presence of ⩾1 u ml−1 of adenosine deaminase (Vannucci et al., 1989; Lanoue & Martin, 1994; Parsons et al., 1988; Coates et al., 1994) may be indicative of an action of these antagonists to reduce the spontaneous activity of adipocyte adenosine receptors. However, it is also possible that adenosine deaminase may not be effective to reduce the concentration of adenosine in the receptor compartment. Lastly, tonic inhibition of adenylyl cyclase and lipolysis may be the result of binding of adenosine to a small number of receptors among a large population of A1AdoR that are efficiently coupled to these responses. The range of reported densities of A1AdoRs in membranes prepared from adipose tissue is high (500 – 2000 fmol mg protein−1 in rat). A small fraction of this large population of receptors would be occupied at the low adenosine concentrations presumably present endogenously in vivo and in vitro. However, activation of this small fraction of the receptor population could be sufficient to cause functional responses in adipocytes, if the receptor reserve (e.g., a measure of the efficiency of coupling of receptor activation to functional response) was high. It has been estimated that R-(−)-N6-(2-phenylisopropyl)adenosine (R-PIA) caused a half-maximal reduction of adipocyte cyclic AMP content by occupancy of only 10% of the population of adenosine receptors (Lohse et al., 1986). The purpose of the present investigation was to quantify three factors that may contribute to an understanding of the tonic inhibition of adenylate cyclase activity by rat adipocyte A1AdoR. A fourth factor, the concentration of adenosine in the receptor compartment, was not investigated. We have estimated the affinity of the adipocyte A1AdoR for adenosine, the efficiency of coupling (receptor reserve) of A1AdoR activation to reduction of cyclic AMP accumulation, and the contribution of spontaneous activity of the receptor to an inhibition of cyclic AMP accumulation in adipocytes. Results indicated that receptor reserve for adenosine to decrease the accumulation of cyclic AMP in adipocytes is very great and tonic activity of the adipocyte A1AdoR is expected when the concentration of endogenous adenosine is 1 – 2 nM.