Abstract
Forskolin activated adenylate cyclase of purified rat adipocyte membranes in the absence of exogenous guanine nucleotides. Guanyl-5'-yl imidodiphosphate (Gpp(NH)p) inhibited the forskolin-activated cyclase immediately upon addition of the nucleotide at concentrations too low to activate adenylate cyclase (10(-9) to 10(-7) M). Inhibition seen with a very high concentration of Gpp(NH)p (10(-4) M) lasted for 3-4 min and was followed by an increase in the synthetic rate which remained constant for at least 15 min. The length of the transient inhibition did not vary with forskolin concentrations above 0.05 microM but low Gpp(NH)p (10(-8) M) exhibited a lengthened (6-7 min) inhibitory phase. The transient inhibitory effects of Gpp(NH)p were eliminated by 10(-7) M isoproterenol, high (40 mM) Mg2+, or preincubation with Gpp(NH)p in the absence of forskolin. While forskolin stimulated fat cell cyclase in the presence of Mn2+, this ion blocked the inhibitory effects of Gpp(NH)p. The well documented inhibitory effects of GTP on the fat cell adenylate cyclase system were also observed in the presence of forskolin. However, the inhibition by GTP is not transitory. These findings indicate that Gpp(NH)p regulation of forskolin-stimulated cyclase has at least two components: 1) an inhibitory component which acts through an undetermined mechanism and which acts immediately to decrease cyclase activity; and 2) an activating component which modulates the inhibited cyclase activity through the guanine nucleotide regulatory protein.
Highlights
Forskolin activated adenylate cyclase of purified rat curs in membranes from a variety of tissues [2, 3], including adipocyte membranes in the absence of exogenous rat adipocytes [4],but only after a lag
While a normal G-protein is not required for forskolin activation of adenylate cyclase in the cyc- mutant of S49 mouse lymphoma [7], guanine nucleotides modify forskolin-activated cyclase from the cyc- mutant [8] as well as from platelet [9] and rat cerebral cortical membranes [10]
The present studies demonstrate that forskolin-activated effects of GTPon the fat cell adenylate cyclase system fat cell adenylate cyclase is inhibited by Gpp(NH)pimmediwere alsoobserved in the presence of forskolin
Summary
Adipocyte Isolation-Adipocytes were isolated by a modification of the method of Rodbell [11]from parametrial, omental, and epididymal adipose tissue of 175-250 g male or female Sprague-Dawleyrats (Charles River, CD strain) fed ad libitum on laboratory chow. The adipocytes were washed free of collagenase by suspension in at least 2 volumes of buffer, centrifugation, and removal of the infranatant. The plasma membranes (a white band at the sucrose interface) were collected, diluted with 8 volumesof homogenization buffer, and centrifuged in a Sorvall SS-34 rotor a t 16,000rpm. The pellet was suspended in homogenization buffer to a membrane protein concentration of approximately 1 mg/ml. Adenylate Cyclase Assay-Unless otherwise cited, adenylate cyclase was assayed in a mixture containing 0.2 mM LY-[~’P]AT(P20-50 cpm/ pmol), 0.1 mM [3H]cyclicAMP (0.1 cpm/pmol, as standard), 4 mM MgC120,.2% bovine serum albumin fraction V, 10 mM creatine phosphate, 10 units/ml of creatine phosphokinase, 30 mM Tris-HC1, pH 7.5, in a final volume of 100 pl of reaction mix. The stock was diluted so that reaction mixtures contained less than 1%ethanol.Theseethanol concentrations had no effect on adenylate cyclase activity.
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