287

Biochimica et Biophysica Acta, 583 (1979) 287--294 © Elsevier/North-Holland Biomedical Press

BBA 28829

CATECHOLAMINE AND GUANINE NUCLEOTIDE ACTIVATION OF SKELETAL MUSCLE ADENYLATE CYCLASE

PONNAL NAMBI and G.I. DRUMMOND

Biochemistry Group, Department of Chemistry, University of Calgary, Calgary, T2N 1N4 (Canada) (Received July 4th, 1978)

Key words: Adenylate cyclase activation; Guanine nucleotide; Catecholamine; (Skeletal muscle)

Summary Activation of adenylate cyclase by guanine nucleotide and catecholamines was examined in plasma membranes prepared from rabbit skeletal muscle. The GTP analog, 5'-guanylyl imidodiphosphate caused a time and temperaturedependent activation of the enzyme which was persistent, the K a was 0.05 pM. 5'-Guanylyl imidodiphosphate binding to the membranes was time and temperature dependent, K D 0.07 pM. Beta adrenergic amines accelerated the rate of 5'-guanylyl imidodiphosphate activation of the enzyme with an order of potency isoproterenol ~ soterenol ~ salbutamol > epinephrine > > norepinephfine. Catecholamine activation was antagonized by propranolol and the ~2 antagonist butoxamine; the ~1 antagonist practolol was inactive. [3H]Dihydroalprenolol bound to the membranes and binding was antagonized by fl adrenergic agonists with an order of potency similar to the activation of adenylate cyclase and was antagonized by butoxamine but not by practolol. The data are consistent with the idea that adenylate cyclase in skeletal muscle plasma membranes is coupled to adrenergic receptors of the/~2 type. Introduction The action of hormones on adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) has been extensively studied in a variety of eukaryotic tissues. Following the findings of Rodbell et al. [1] that glucagon stimulation of the liver enzyme was enhanced by GTP, it has become recognized that a Abbreviations: Gpp(NH)p, 5t-guanylyl imidodiphosPhate; cyclic AMP, adenosine 3~,51-cyclicphosphate;

INE, isopropylnorepinephrine (isoproterenol); [3H]DHA, (--)-[propyl-2,3-3H]dihydroalprenoloL

288 primary event in hormonal activation of adenylate cyclase is binding of guanine nucleotide to a regulatory site. Most studies on the role of guanine nucleotides have employed the more stable GTP analog, 5'-guanylyl imidodiphosphate (Gpp(NH)p) [2]. Guanine nucleotide converts the enzyme to an activated state; the hormone, following interaction with its specific receptor, increases the rate of active state formation [3--7]. Adenylate cyclase has not been extensively studied in skeletal muscle. Several years ago [8] we described the preparation of plasma membranes from rabbit skeletal muscle which contained a good yield of the enzyme. The present report constitutes a study of the effects of Gpp(NH)p and a variety of adrenergic agonists and antagonists on the enzyme. Materials and Methods [a.32p] ATP (tetraethylammonitim salt, 15--20 Ci/mmol), [ G-3H] cyclic AMP (ammonium salt, 30 Ci/mmol), (--)[propyl-2,3-3H]dihydroalprenolol ([3H]DHA) (50 Ci/mmol), Aquasol and Omnifluor were purchased from New England Nuclear, Montreal; 5'-[guanyl-8-3H]guanylyl imidodiphosphate (tetrasodium salt, 5--15 Ci/mmol) was obtained from I.C.N., CA, or Amersham Searle, Arlington, Heights, IL. Samples of the unlabeled nucleotide were purchased from I.C.N. or P.L. Biochemicals, Milwaukee; prostaglandins were obtained from the latter company. Phosphoenolpyruvate, pyruvate kinase (rabbit skeletal muscle, Type II), cyclic AMP, ATP, GTP, (--)-isoproterenol hydrochloride (INE), 5-hydroxytryptamine (serotonin), L-phenylephrine hydrochloride, DLmetanephrine hydrochloride, D L ~ - m e t h y l dopa, glucagon and insulin were from Sigma Chemical Co., St. Louis. Other agents used were obtained from the following sources: dobutamine, Eli Lilly; soterenol and (--)-sotalol, Mead Johnson; salbutamol, Allen Hanburys; (+)- and (--)-propranolol, Ayerst; (--)-alprenolol, AB H~sle; and butoxamine, Burrows Wellcome. (±)-Practolol was a gift from Dr. David Godin, Dept. of Pharmacology, University of British Columbia, Canada. Plasma membranes were prepared from rabbit leg (fast) muscle by the procedure of Severson et al. [8] with the following modifications: minced muscle was homogenized for 10 s; following homogenization the fibers were washed four times with 10 vol. of 10 mM Tris-HC1, pH 8.0 [9]. After extracting the washed particles with 0.4 M LiBr for 3 h, the suspension was diluted with an equal volume of 10 mM Tris-HC1, pH 8.0, and centrifuged at 38 000 × g for 20 min. The pellet was thoroughly suspended and stirred for 5 min in 3 vols. (based on original tissue weight) of 12.5% KBr in 10 mM Tris-HC1, pH 8.0, and then sedimented at 38 000 × g for 20 min. The pellets were washed twice in 2 vols. (based on original tissue weight) of 0.25 M sucrose, 20 mM Tris-HC1, pH 7.5, 2 mM dithiothreitol, 1 mM ethylenediamine tetraacetic acid (hereafter referred to as buffer A) by sedimenting at 25 000 × g for 5 min. The membranes were homogenized in 0.5 vol. (based on original tissue) of buffer A to provide a suspension of 6--8 mg protein/ml; the preparation was stored at --80°C. Adenylate cyclase activity in such preparations ranged from 30 to 60 pmol • min -1 • mg -1 (assay temperature 30°C) and represented a 10- to 20-fold enrichment over the starting homogenate (prepared in buffer A) with a yield of 30--40%.

289 Adenylate cyclase was assayed by the procedure of Salomon et al. [10] with the modifications we have issued previously [11]. Assays in triplicate were conducted (unless otherwise indicated) at 30°C for 10 rain; MgC12 was usually 25 mM. Activity is expressed as pmol cyclic AMP formed/min per mg. Protein was determined by the method of Lowry et al. [12]. Gpp(NH)p binding was measured by the micropore filtration technique [ 13 ]. Membranes (100--150 pg protein) were incubated in buffer A containing (where indicated) 5 mM MgC12 and 0.5 uM [3H]Gpp(NH)p (1 • 104 d p m / p m o l ) for 10 min at 30 or 37°C; final volume 150 ~1. Nonspecific binding was measured in the presence of 1000-fold excess of unlabeled ligand. The reaction was terminated by adding 1.85 ml of 50 mM Tris-HC1 (pH 7 . 5 ) a n d the mixtures were filtered immediately on glass fiber filters (Gelman type A/E). The filters were washed twice with 5 ml of buffer, and dried. Radioactivity was determined in 7 ml of cocktail (4 g Omnifluor/1 of toluene). All determinations were performed in triplicate. [3H]Dihydroalprenolol binding was quantitated by the method of Mukherjee et al. [14]. Membranes were incubated in buffer A with 0.01 pM [3H]dihydroalprenolol (1 • 10 s dpm/pmol) at 30 or 37°C, final volume 150 pl. To measure nonspecific binding, the assay included 1000-fold excess of unlabeled (--)-propranolol. The reaction was terminated by diluting with 1.85 ml of cold 50 mM Tris-HC1, pH 7.5, and filtering on glass filters (Gelman type A/E). The filters were washed with 20 ml of cold buffer, dried, and radioactivity determined b y scintillation spectrometry in 7 ml of cocktail consisting of 4 g/1 of Omnifluor in toluene. All assays were performed in triplicate. Results Studies with a variety of tissues have shown that Gpp(NH)p activation of adenylate cyclase is a time and temperature-dependent process [5--7] and is irreversible [ 2,4--7]. In a series of experiments skeletal muscle membranes were incubated in buffer A containing 5 mM MgC12 for various time intervals in the presence and absence of Gpp(NH)p and Gpp(NH)p plus INE at 30 and 37°C. U n b o u n d ligand was removed by sedimentation and washing before assay. The results in Fig. 1 show that Gpp(NH)p activation increased with time at both temperatures. When INE was present with Gpp(NH)p, activation was greatly accelerated reaching maximal levels in 10 min. Like activation of adenylate cyclase, binding of Gpp(NH)p to the membranes was time and temperature dependent (data not shown). Binding of the nucleotide was proportional to membrane protein. Activation of adenylate cyclase b y Gpp(NH)p was concentration dependent (Fig. 2A); half-maximal activation occurred at approximately 0.05 pM. Concentration dependence of Gpp(NH)p binding is presented as a Scatchard plot in Fig. 2B. A single class of binding sites with a K D in the presence of 5 mM MgC12 of 0.07 pM was obtained, a value close to the K a for enzyme activation. Mg 2÷ significantly decreased maximal Gpp(NH)p binding (compare zx with A) and altered K D somewhat. In Fig. 1 it was demonstrated that INE caused a three-fold increase in rate of Gpp(NH)p activation at 30°C over that compared with membranes incubated with the nucleotide alone. Experiments were performed to examine the effec-

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tiveness of a variety of adrenergic agonists to accelerate Gpp(NH)p activation; the effect of adrenergic antagonists on INE activation was also explored. Thus membranes were incubated with Gpp(NH)p w i t h o u t and with various concentrations of adrenergic agonists or agonist plus antagonist for 10 min at 30°C followed by dilution and washing by sedimentation before resuspension and assay. Fig. 3A shows dose vs. response curves for several adrenergic agents. (--)-INE was the most p o t e n t stimulator with Ka of 0.4 pM; salbutamol and soterenol (both ~2 agonists) were almost as potent, K a values of 0.75 and 0.80 gM, respectively. Epinephrine, Ka 2 pM, was less potent, norepinephrine was much less active, Ka 100 pM. Dobutamine was much less p o t e n t than INE and seemed to act as a partial agonist. The ~-adrenergic agonist methoxamine did not affect activation at concentrations as high as 1 mM. The stimulatory action of INE

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was strongly inhibited by propranolol (Fig. 3B). Phenylephrine and dopamine were weak stimulators and appeared to act as partial agonists; their action was antagonized by propranolol (Fig. 3B). The stimulatory action of INE was antagonized in a dose-dependent manner by (--)-propranolol which was more potent than (_+)-propranolol (Fig. 3C) reflecting stereospecificity of antagonist action. Butoxamine and sotalol were active; the ~, antagonist practolol did not prevent INE stimulation. The a antagonist phentolamine did not alter the stimulatory action of 10 #M INE or 50 #M epinephrine and norepinephrine at

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Catecholamine and guanine nucleotide activation of skeletal muscle adenylate cyclase.

287 Biochimica et Biophysica Acta, 583 (1979) 287--294 © Elsevier/North-Holland Biomedical Press BBA 28829 CATECHOLAMINE AND GUANINE NUCLEOTIDE ACT...
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