Abstract
Abstract Adenosine and 2'-deoxyadenosine at concentrations in the range of 5 to 50 µm inhibited the activation of adenylate cyclase activity in fat cell ghosts by norepinephrine. 2',5'-Dideoxyadenosine was more potent than adenosine while arabinosyl adenosine, 3'-deoxyadenosine, and 2-fluoroadenosine were equi-potent to adenosine as inhibitors of adenylate cyclase. Adenine was inactive as an inhibitor of adenylate cyclase along with nucleosides such as phenylisopropyl adenosine, inosine, 2'-deoxyinosine, guanosine, and 2'-deoxyguanosine. Adenosine, 2'-deoxyadenosine, and 2-fluoroadenosine were also inhibitors of the low Km phosphodiesterase found in the supernatant fraction obtained after centrifugation of fat cell homogenates. The large accumulation of cyclic adenosine 3',5'-monophosphate (cyclic AMP) seen in fat cells incubated for 5 min with catecholamines plus theophylline was markedly inhibited by 2',5'-dideoxyadenosine, adenosine, and 2-fluoroadenosine. The small increase in cyclic AMP due to norepinephrine or theophylline alone was also blocked by adenosine and 2',5'-dideoxyadenosine. However, there was no correlation between the effects of the nucleosides on cyclic AMP accumulation and lipolysis since 2',5'-dideoxyadenosine did not inhibit lipolysis. Adenosine inhibited the lipolytic action of theophylline or low concentrations of norepinephrine but did not inhibit the lipolytic action of higher concentrations of norepinephrine under conditions in which it virtually abolished the increase in cyclic AMP accumulation due to norepinephrine. These data indicate that marked inhibition by nucleosides of the increase in total cyclic AMP accumulation seen 5 min after the addition of theophylline, norepinephrine, or norepinephrine plus theophylline is not necessarily associated with any decrease in lipolysis. Apparently most of the increased cyclic AMP accumulation seen in the presence of lipolytic agents is unrelated to the regulation of lipolysis. The effect of adenosine and related nucleosides on cyclic AMP accumulation suggest that they may play a physiological role as feedback regulators of fat cell adenylate cyclase.
Highlights
Fluoroadenosine were inhibitors of the low K, phosphodiesterase found in the supernatant fraction obtained after centrifugation of fat cell homogenates
The large accumulation of cyclic adenosine 3’,5’-monophosphate seen in fat cells incubated for 5 min with catecholamines plus theophylline was markedly inhibited by 2’,5’-dideoxyadenosine, adenosine, and Z-fluoroadenosine
Increasing the concentration of the 2’-deoxynucleotide to 55 pM did not result in any greater inhibition of cyclase. These results suggested that there was a direct inhibition of adenylate cyclase by deoxyadenosine compounds which did not require conversion to nucleotides
Summary
Free white fat cells were obtained from 120- to 160-g female Sprague-Dawley rats (Charles River CD strain) fed laboratory chow ad libitum. White fat cells were isolated by a modification of the procedure of Rodbell [13] from the pooled parametrial adipose tissue of three or more rats. The ghosts were used immediately and incubated at 37” for 20 min in a total volume of 100 ~1 containing 40 mM Tris-HCl (pH 8.0), 5 mM MgCI,, 30 mM KCl, 1 mM cyclic AMP, 8 mM phosphoenolpyruvate, 10 pg of pyruvate kinase, 1 mM ATP, and 0.2 /&i of tritiated ATP (New England Nuclear Corporation). At the end of the incubation period the tubes were boiled for 2 min after the addition of 50 ~1 of 5 mM cyclic AMP. The fraction containing cyclic AMP was collected, and 0.2 ml of 5% zinc sulfate and 0.1 ml of saturated barium hydroxide were added. Under the conditions of our assay the formation of labeled cyclic AMP in the presence of norepinephrine or fluoride was linear over the 20-min assay period
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