The hamster adipocyte adenylate cyclase system I. Regulation of enzyme stimulation and inhibition by manganese and magnesium ions

51

Biochimica et Biophysica Acta, 676 (1981) 51-58

Elsevier/North-HollandBiomedicalPress BBA 29672 THE HAMSTER ADIPOCYTE ADENYLATE CYCLASE SYSTEM I. REGULATION OF ENZYME STIMULATION AND INHIBITION BY MANGANESE AND MAGNESIUM IONS KARL H. JAKOBSand KLAUSAKTORIES Pharmakologisches Institut der Universita'tHeidelberg, Im Neuenheimer Feld 366, D-6900 Heidelberg, (ER. G.)

(Received February 10th, 1981)

Key words: Adenylate cyclase; Enzyme stimulation; Enzyme inhibition; Mn2+;Mg2+; (Hamster adipocyte)

In hamster adipocyte ghosts, ACTH stimulates adenylate cyclase by a GTP-dependent process, whereas prostaglandin El, a-adrenergic agonists and nicotinic acid inhibit the enzyme by a mechanism which is both GTP- and sodium-dependent. The influence of the divalent cations Mn2+ and Mg2+, was studied on these two different, apparently receptor-mediated effects on the adipocyte adenylate cyclase. At low Mn2+ concentrations, GTP (1 gM) decreased enzyme activity by about 80%. Under this condition, ACTH (0.1 gM) stimulated the cyclase by 6- to 8-fold, and NaCI (100 mM) caused a similar activation. In the presence of both GTP and NaCI, prostaglandin E 1 (1 or 10 haM) and nicotinic acid (30/aM) inhibited the enzyme by about 70-80% and epinephrine (300 gM, added in combination with a ~-adrenergic blocking agent) by 40-50%. With increasing concentrations of Mn2+, the GTP-induced decrease and the NaCl-induced increase in activity diminished, with a concomitant decrease in prostaglandin E 1-, nicotinic acid- and epinephrine-induced inhibitions as well as in ACTH-induced stimulation. At 1 mM Mn2+, inhibition of the enzyme was almost abolished and stimulation by ACTH was largely reduced, whereas activation of the enzyme by KF (10 mM) was only partially impaired. The uncoupling action of Mn2+ on hormone-induced inhibition was half-maximal at 100-200 #M and appeared not to he due to increased formation of the enzyme substrate, Mn • ATP. It occurred without apparent lag phase and could not be overcome by increasing the concentration of GTP. Similar but not identical findings with regard to adenylate cyclase stimulation and inhibition by hormonal factors were obtained with Mg2+, although about 100-fold higher concentrations of Mg2+ than of Mn2+ were required. The data indicate that Mn2+ at low concentrations functionally uncouples inhibitory and stimulatory hormone receptors from adenylate adenylate cyclase in membrane preparations of hamster adipocytes, and they suggest that the mechanism leading to uncoupling involves an action of Mn2+ on the functions of the guanine nucleotide site(s) in the system.

Introduction

In hamster adipocytes, ACTH and /3-adrenergic agonists stimulate adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) by a GTP-dependent process, whereas a-adrenergic agonists and pros-

Abbreviation: ACTH,corticotropin.

taglandin E1 and E2, which are potent antilipolytic agents, inhibit the enzyme [1,2]. The effective inhibitory coupling of these hormone receptors to the adenylate cyclase in cell-free systems requires the presence of both GTP and Na ÷, with maximal effects at about 1/AVl and 100 mM, respectively. The antilipolytic agent, nicotinic acid, inhibits the enzyme in rat and hamster adipocytes by a similar GTP- and Na+-dependent process in a hormone-like manner

0304-4165/81/0000-0000/$02.50 © Elsevier/North-HollandBiomedicalPress

52 [2,3]. In studies on other hormone- or neurotransmitterhnduced adenylate cyclase inhibitions, an obligatory role of GTP and an amplifying effect of Na ÷ have also been reported, e.g., for muscarinic cholinergic agonists in rabbit myocardium [4], for opiates, a.adrenergic and muscarinic cholinergic agonists in neuroblastoma×glioma hybrid cells [5-7] and for a-adrenergic agonists and angiotensin II in rat liver [8]. As shown in the hamster adipocyte system [ 1 - 3 ] , the hormone4nduced inhibition of the adenylate cyclase is revealed by the concurrent presence of GTP, which decreases basal enzyme activity, and of Na ÷, which antagonizes the GTP-induced decrease in activity. It appeared that the inhibitory hormonal factors inhibit the enzyme by counteracting the Na÷-induced activation. In studies on hormone receptors, which are coupled in an inhibitory manner to the adenylate cyclase, it has been shown that both guanine nucleotides and Na ÷ decrease agonist receptor binding [ 9 14], although by an apparently different mechanism [11]. Recently, it has been described that divalent cations can antagonize the GTP-and Na*4nduced reductions of agonist binding at a2-noradrenergic binding sites in bovine brain [15] and at opiate receptors in rat brain [16]. The most potent divalent cation in this regard was Mn 2÷, while Mg2÷ and Ca 2+ were far less effective. The counteracting effect of Mn 2÷ on the influence of GTP on opiate agonist binding has been attributed to a Mn2+-induced breakdown of GTP to GMP and guanosine [16]. In studies on the rat adipocyte adenylate cyclase, it has been reported that Mn 2÷ can eliminate the inhibitory phase of GTP with a concomitant loss of the adenosine-induced enzyme inhibition [ 17]. In the present study, we describe the influences of Mn 2÷ and Mg2+ upon the GTP- and Na+-dependent inhibitions of adenylate cyclase in hamster adipocyte ghosts by the antilipolytic agents, prostaglandin El, nicotinic acid and epinephrine, and on the GTPdependent stimulation of the enzyme by ACTH. We report here selective interactions between the divalent cations, GTP and sodium in regulating receptormediated inhibition and stimulation of the enzyme. Materials and Methods

Materials. Bovine serum albumin (essentially fatty acid-free) and collagenase (type II; lot 20 F-6837)

were obtained from Sigma, Mtinchen. ACTH l 24 was a gift of CIBA-Geigy, Basel. Essentially GTP-free ATP was donated by Dr. K. Miihlegger, Boehringer Mannheim. All other reagents were obtained as previously described [1-3]. [a-32p]ATP and [a-32p]GTP were prepared according to Walseth and Johnson [ 18]. Preparation of hamster fat cell ghosts. Male golden hamsters, 8 0 - 1 2 0 g , were fed ad libitum. After decapitation, epididymal fat pads were removed, and isolated fat cells were prepared following the method of Hittelman et al. [19] with the modifications described previously [2]. After lysis of the isolated cells in an ice-cold medium containing 2.5 mM MgC12, 1 mM ATP, 1 mM KHCO3 and 2 mM Tris-HC1, pH 7.6, the fat cell ghosts were centrifuged for 15 rain at 1 000Xg at 4°C, washed once with 1 mM KHCO3 and again centrifuged for 10 min at 10000×g. Finally, the ghosts were suspended in 1 mM KHCO3, frozen in liquid nitrogen and stored at -85°C. Adenylate cyclase assay. Adenylate cyclase activity was determined, if not otherwise mentioned, with 0.05 mM [a-32p]ATP (0.4-0.8 #Ci/tube), 0.1 mM cyclic AMP, 1 mM 3-isobutyl-l-methylxanthine, 1 mM dithiothreitol, 5 mM creatine phosphate used as its Tris salt, 0.4 mg/ml of creatine kinase and 0.2% (w/v) bovine serum albumin in 50 mM triethanolamine-HC1, pH 7.4, in a total volume of 100 ~l. The divalent cations, Mn ~+ and Mg2+, were added as the acetate and chloride salts, respectively, to the assay medium at 0.05 mM concentration, corresponding to that of ATP. Addition of the cations at concentrations higher than that of ATP is expressed as Mn2+exeess or Mg2+excess. By this calculation, the complex formation between the divalent cations and ingredients of the assay medium other than ATP, e.g., creatine phosphate, has been neglected. Therefore, the numbers given of excess metal ions must not correspond to the actual free divalent cation concentrations in the assay medium, especially at the low Mn2+ concentrations used. The enzyme reactions were initiated by the addition of hamster adipocyte ghosts ( 5 - 2 0 /.tg protein) to the prewarmed reaction mixtures and conducted for 10 min or as indicated at 25°C. Cyclic AMP formed was isolated by co-precipitation of related 5'-nucleotides with ZnCO3 and subsequent column chromatography on neutral alumina as previously described [20]. Standard deviations of triplicate tubes were generally less than 5% of the

53 means. All experiments were repeated at least twice with similar results, and means of representative experiments are shown in the figures and tables. Protein was determined by the method of Lowry et al. [21], with human serum albumin as standard.

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Results

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With ATP and Mn 2+ (0.05 mM each) as substrate of the hamster adipocyte adenylate cyclase, GTP (1 /aM) decreased enzyme activity by 70-80% (Table I). NaC1 (100 raM) increased the activity by about 2-fold in the absence of GTP and antagonized the decrease in activity induced by GRIP, which resulted in a 6- to 7.fold activation by NaC1. The inhibitory factor, nicotinic acid (30 /aM), had no effect in the absence of GTP without or with NaC1 and caused a slight inhibition in the presence of GTP alone. However, in the presence of both GTP and NaC1, nicotinic acid reduced the enzyme activity by 70%. Addition of 1 mM excess Mn 2+ completely changed the above described characteristics of the enzyme. While NaC1 still activated the enzyme by about 1.4-fold, the GTP-induced reduction of enzyme activity and the nicotinic acid-induced inhibition of the enzyme were almost completely abolished. Addition of excess Mn 2+ up to 1 mM had only a small activating effect on con trol activity and on the TABLE I INFLUENCE OF MANGANESE ON NICOTINIC ACIDINDUCED INHIBITION OF ADIPOCYTE ADENYLATE CYCLASE In hamster adipocyte ghosts, adenylate cyclase activity was determined with 0.05 mM M n - A T P as substrate in the absence and presence of nicotinic acid (NA; 30 ~,M). GTP (1 uM), NaC1 (100 raM) and excess Mn2+ (1 mM) were present as indicated. Values are pmol cyclic AMP/mg per rain and numbers in parentheses indicate inhibition (%) by nicotinic acid. Mn2+ (mM)

NA (uM)

Control

GTP

NaC1

GTP + NaC1

0 0

0 30

103 100 (3)

24.8 22.4 (10)

197 206 (0)

162 48.7 (70)

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125 127 (0)

120 108 (10)

171 173 (0)

162 154 (5)

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Fig. 1. Influence of Mn 2+ on nicotinic acid-induced inhibition of adipocyte adenylate cyclase. Adenytate cyclase activity

was determined with Mn. ATP (0.05 raM) as substrate at increasing concentrations of excess Mn2+ in the absence (control) and presence of GTP (1 ~tM), GTP (1 /aM) plus NaCI (100 raM) or GTP (1 ~tM),NaC1(100 mM) plus nicotinic acid (NA; 30 ~tM) as indicated. The inset shows the inhibition (%) by nicotinic acid (NA) at increasing concentrations of excess Mn2+. activity measured in the presence of GTP plus NaC1 (Fig. 1). The increase in activity at low excess Mn ~+ concentrations, which varied somewhat in repeated experiments, appeared to be due to the increase in the concentration of the complex, M. ATP (M is metal), which is thought to be the actual substrate of the adenylate cyclase [22]. In the presence of GTP and in the concurrent presence of GTP, NaC1 and nciotinic acid, the enzyme response to excess Mn 2+ was completely different. Under these two conditions, Mn 2+ increased the activity by about 3- to 5-fold at 1 mM, at which concentration a plateau was obtained. Thus, at increasing concentrations of excess Mn 2÷, both the GTP-induced reduction and the nicotinic acid.induced inhibition were diminished by about the same extent and were minimal at 1 mM

54 excess Mn 2*. The reversal o f nicotinic acid-induced inhibition was half-maximal at about 200 /AVl Mn 2+ 150

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Mn2%xcess(raM) Fig. 2. Influence of Mn 2+ on prostaglandin E 14nduced inhibition of adipocyte adenylate cyclase. Adenylate cyclase activity was determined with Mn • ATP (0.05 raM) as substrate at increasing concentrations of excess Mn ~+ in the absence (control) and presence of GTP (1 ~tM), GTP (1 ;tM) plus NaC1 (100 raM) or GTP (1 ~M), NaC1 (100 mM) plus prostaglandin E 1 (PGEI; 10 ~zM) as indicated. The inset shows the inhibition (%) by prostaglandin E 1 (PGE1) at increasing concentrations of excess Mn 2÷.

(see inset o f Fig. 1). Taking the c o m p l e x f o r m a t i o n b e t w e e n Mn 2÷ and creatine phosphate (5 raM) in the assay m e d i u m into a c c o u n t [23], this c o n c e n t r a t i o n o f Mn 2+ corresponds to an actual free Mn 2+ concentration o f a b o u t 130/zlVl. Similar data as shown with nicotinic acid were obtained with prostaglandin E1 as inhibitory agent (Fig. 2). Mn z* at increasing concentrations reversed the prostaglandin-induced inhibition with half-maximal and m a x i m a l effects at about 200 and 1 raM, respectively. Table II shows that the same reversal occurred with epinephrine as inhibitory agent, which via a-adrenoceptors inhibits hamster adipocyte adenylate cyclase m a x i m a l l y 50% [2]. With increasing concentrations o f excess Mn 2+ at a f'Lxed c o n c e n t r a t i o n o f total ATP, not only the concentration o f free Mn 2÷ but also that of the c o m p l e x , Mn • ATP, rises. Therefore, it was studied whether the nicotinic acid-induced inhibition is affected by the M n . ATP c o n c e n t r a t i o n . As shown in Fig. 3, the

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TABLE II INFLUENCE OF MANGANESE ON PGEI-, NICOTINIC ACID- AND EPINEPHRINE-INDUCED INHIBITIONS OF ADIPOCYTE ADENYLATE CYCLASE

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In hamster adipocyte ghosts, adenylate cyclase activity was determined with 0.05 mM Mn. ATP as substrate in the absence and presence of prostaglandin E 1 (PGE1; 10 /~M), nicotinic acid (NA; 30 uM) and epinephrine (epi; 300 uM) at the indicated concentrations of excess Mn 2÷. GTP (1 uM), NaC1 (100 mM) and (±)-propranolol (30 uM) were present under each condition. Values are pmol cyclic AMP/mg per min and numbers in parentheses indicate inhibitions (%) compared to respective control activities.

.__u_ 100

Mn2+ (mM)

Control

PGE l

NA

epi

0 0.1 1.0

170 230 180

45 (74) 102 (56) 183 (0)

50 (71) 113 (51) 182 (0)

94 (45) 184 (20) 181 (0)

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MnATP (uM) Fig. 3. Influence of Mn. ATP on nicotinic acid-induced inhibition of adipocyte adenylate cyclase. Adenylate cyclase activity was determined at increasing concentrations of Mn • ATP without excess Mn 2+ in the absence (control) and presence of GTP (1 ~M), GTP (1 /zM) plus NaC1 (100 raM) or GTP (1 ~ I ) , NaC1 (100 mM) plus nicotinic acid (NA; 30 ~M) as indicated. The inset shows the inhibition (%) by nicotinic acid (NA) at increasing concentrations of Mn • ATP.

55 inhibition was largely independent of the M n - A T P concentration used. The observed slight decrease in inhibition appeared to be due to a concomitant rise in the free Mn 2÷ concentration, which has been calculated [24] to increase under the experimental conditions from about 5 to 40 p.M. Since with increasing concentrations of excess Mn 2÷, not only the inhibition of the adipocyte adenylate cyclase by hormonal factors but also the GTP-induced reduction of enzyme activity decreased, the influence of Mn 2+ was studied on the effect of the lipolytic hormone, ACTH, which stimulates the enzyme by a GTP-dependent process [25]. In the absence of excess Mn 2+, ACTH (0.1 /AVl)stimulated the enzyme about 7-fold, when GTP (1 p.lVl) was present (Fig. 4). At increasing concentrations of excess Mn 2+, the GTP-induced reduction of activity

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Mn2+excess(mM) Fig. 4. Influence of Mn2+ on ACTH-induced stimulation of adipocyte adenylate cyclase. Adenylate cyclase activity was determined with Mn • ATP (0.05 mM) as substrate at increasing concentrations of excess Mn2+ in the absence (control) and presence of GTP (1 ~tM) or GTP (1 /zM) plus ACTH (0.1 /.tM) as indicated.

TABLE III INFLUENCE OF MANGANESE ON FLUORIDE-INDUCED ACTIVATION OF ADIPOCYTE ADENYLATE CYCLASE In hamster adipocyte ghosts, adenylate cyclase activity was determined with Mn. ATP (0.05 mM) as substrate at increasing concentrations of excess Mn2+ without and with GTP (1 ~M) or KF (10 mM) as indicated. Values are adenylate cyclase activity, pmol cyclic AMP/mg per min. Mn2+excess (mM)

Control

GTP

KF

KF + GTP

none 0.05 0.1 0.5 1.0 5.0

207 230 237 188 167 164

44.7 77.8 96.6 142 153 159

719 710 632 421 399 407

241 329 418 440 416 413

diminished with a concomitant decrease in ACTHinduced stimulation. At 1 mM excess Mn 2÷, ACTH caused only about 1 A-fold stimulation and at 5 mM Mn 2÷, ACTH-induced stimulation became undetectable. In the absence of GTP, the adipocyte adenylate cyclase activity was stimulated by KF (10 mM) about 3- to 4-fold, and in the presence of GTP (1 /aM), which reduced basal and fluoride-stimulated activities, an activation of about 6-fold by KF was observed (Table III). At increasing concentrations of excess Mn 2÷, the KF-stimutated activity was reduced when no GTP was present and was increased in the presence of GTP. From about 0.5 mM excess Mn 2÷, the fluoride-stimulated enzyme exhibited identical activities whether GTP was present or not. Whereas the stimulation of the enzyme by ACTH was abolished at 5 mM Mn 2÷, at this Mn 2. concentration KF still activated the enzyme about 2.5-fold. Similar experiments as shown with Mn 2. were performed with Mg2÷ as divalent cation (Fig. 5). At 2 mM Mg 2÷, GTP (1/aM) reduced the enzyme activity by about 90%, and ACTH (1 p.M)stimulated the GTP-decreased activity about 8-fold; NaC1 (100 mM) caused a similar activation which was almost completely antagonized by nicotinic acid (30 t.~1). Mg2÷ at increasing concentrations diminished both the GTP-induced reduction (%) and the NaCl-induced increase (-fold) in activity with a concomitant decrease in nicotinic acid-induced inhibition (%). In

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Fig. 5. Influence of Mg2+ on inhibitory and stimulatory regulation of adipocyte adenylate cyclase. Adenylate cyclase activity was determined with Mg. ATP (0.05 mM) as substrate at increasing concentrations of excess Mg2+ in the absence (control) and presence of GTP (1 /~M), GTP (1 ~M) plus ACTH (1 ,uM), GTP (1 ~zM)plus NaCI (100 mM) or GTP (1 /2M), NaCI (100 mM) plus nicotinic acid (NA; 30 ~tM) as indicated.

terms o f abolute values, the increase in activity due to NaC1 and the decreases in activity induced by GTP and nicotinic acid rose at Mg 2÷ concentrations o f up to 20 mM with a decline thereafter. At 100 mM Mg 2÷, the NaCl-induced activation and the nicotinic acidinduced inhibition were abolished, although GTP reduced the enzyme activity still by about 15%. Similar to the inhibitory agent, nicotinic acid, the increase in enzyme activity due to the stimulatory hormone, ACTH, was enlarged at Mg 2+ concentrations up to 50 mM with a decline thereafter, whereas stimulation (-fold) b y ACTH was decreased b y Mg2+ in a concentration-dependent manner, from about 8-fold at 2 mM to about 1.4-fold at 100 mM excess Mg2÷. Mn :+ has been described as inducing a breakdown of GTP to GMP and guanosine by stimulation of phosphatases in rat brain membranes [16]. Therefore, the early time course o f the effect o f Mn 2÷ on inhibi-

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,ncubotion time ( • i n ) Fig. 6. Time course of the effect of Mn2+ on nicotinic acidinduced inhibition of adenylate cyclase. Cyclic AMP accumulation was studied up to 10 min with Mn • ATP (0.05 raM) as substrate in the absence (squares) and presence (circles) of nicotinic acid (30 ~zM). GTP (1 ~M) and NaCI (100 mM) were present under each condition. Excess Mn2+ (1 mM) was added to the control and nicotinic acid-inhibited enzyme at the 5 • i n time point as indicated by the arrows.

tion o f the adipocyte adenylate cyclase by nicotinic acid was studied (Fig. 6). In the absence o f excess Mn 2÷, nicotinic acid (30 ~Vl) reduced cyclic AMP formation by about 80%, and addition of 1 mM Mn 2÷ reversed this inhibition without an apparent lag phase. We have also studied the influence o f Mn 2÷ on the breakdown of GTP by hamster adipocyte ghosts under the conditions used for determination of adenylate cyclase activity. The assay system included 1/.aM [ ~ - n P ] G T P instead of the labelled ATP. After an incubation period o f 10 min at 25°C, the reaction mixture was applied to polyethyleneimine cellulose plates, which were developed with 1.2 M LiC1 as solvent [26]. Addition of adipocyte ghosts caused about 15% breakdown of GTP to GMP, which relation was not changed by the presence o f nicotinic acid (30 /aM) or Mn 2÷ (1 mM). Additionally, the effect o f Mn 2÷ on adenylate cyclase activity was not

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GTP (~M) Fig. 7. Influence of GTP on Mn2+-induced uncoupling of adipocyte adenylate cyclase. Adenylate cyclase activity was determined with 0.05 mM Mn • ATP as substrate and 5 mM excess Mn 2+ at increasing concentrations of GTP in the absence (squares) and presence (circles) of nicotinic acid (30 ~tM) without (open symbols) and with (closed symbols) NaC1 (100 raM).

reversed by increase in the GTP concentration (Fig. 7). In the presence of 5 mM Mn2÷, addition of GTP up to 100 ~M did not restore the GTP-induced reduction of basal activity and also not the nicotinic acidinduced inhibition, measured in the absence and presence of NaC1 (100 mM). Discussion

The data shown in this study demonstrate that in hamster adipocyte ghosts the divalent cations Mn 2÷ and Mg2÷ can functionally uncouple both stimulatory and inhibitory hormone receptors from adenylate cyclase, with Mn 2+ being about two orders of magnitude more potent than Mg2÷. Since the studies were pursued in detail with Mn 2÷, the discussion will focus primarily on its action. It has previously been described in the case of frog erythrocytes that Mn 2+ functionally uncouples stimulatory ~-adrenergic and prostaglandin E1 receptors from adenylate cyclase activation with maximal effects at about 20 mM Mn 2÷ [27]. In these membranes, Mn 2+ appears to uncouple

the functional response without affecting the total binding of the stimulatory hormones to their receptors and without altering the/3-adrenoceptor-guanine nucleotide interactions. In hamster adipocyte ghosts, Mn 2+ potently prevented the hormonal response of the adenylate cyclase with a half-maximal and maximal effect at about 200/AVI and 1 mM, respectively. The ACTH-induced stimulation appeared to be slightly less sensitive to Mn 2+ than the hormoneinduced inhibition. However, it is difficult to compare exactly these two different, opposite hormonal responses, with regard to changes in absolute enzyme activity was well as with regard to changes in -fold stimulation or % inhibition. Although a major difference in the uncoupling action of Mn 2+ has not been observed, from our experiments it cannot be excluded that such a difference between hormonal stimulation and inhibition of the adipocyte enzyme exists [17]. As found for stimulatory hormone receptor-binding [27], in studies on prostaglandin E2 binding to hamster adipocyte ghosts we observed that Mn 2÷ (1 mM), which uncouples the functional response, did not decrease total binding and also not the influence of GTP on the receptor affinity (Grandt, R., Aktories, K. and Jakobs, K.H., unpublished observations). With regard to the possible site and mechanism of action of Mn2+ in the adipocyte system, several possibilities have to be considered. The results shown in Figs. 6 and 7 make it unlikely that Mn 2÷ exerts its uncoupling action on the adipocyte'adenylate cyclase by a breakdown of total GTP to GMP and guanosine, as has been suggested by data obtained in opiate receptor binding studies in rat brain [16]. Concomitantly with or prior to the loss of the hormonal response, the influence of GTP on the adipocyte enzyme activity was reduced. Since the action of GTP in the adenylate cyclase system appears to be mediated by a guanine nucleotide-binding regulatory site distinct from the hormone receptors and the catalytic moiety [28,29], it may be speculated that Mn 2÷ functionally uncouples hormone receptors from adenylate cyclase by perturbation of cyclase-nucleotide site interactions [27]. However, the activation of the adipocyte enzyme by fluoride, the action of which on the adenylate cyclase appears also to be mediated by the guanine nucleotide site [30,31], was only partially impaired by Mn 2+, as also observed in frog

58 erythrocyte membranes [27]. Therefore, it appears to be unlikely that Mn 2+ completely disrupts functional communication between the guanine nucleotide site and the adenylate cyclase. On the other hand, it has been suggested that there exist two functionally distinct guanine nucleotide sites, one (Ns) responsible for adenylate cyclase stimulation by hormones and the other one (Ni) mediating the hormone-induced inhibition [32]. Mn 2÷ at low concentrations may specifically affect the inhibitory site.cyclase interaction, leaving the stimulatory site interaction relatively intact [17]. At high concentrations, Mn 2+ may disturb the interactions of both the inhibitory and the stimulatory site with the adenylate cyclase. It has previously been shown in several tissues that full activation of the adenylate cyclase at a putative metal ion site, which may be located on the catalytic or on the regulatory site [33,30], renders the enzyme relatively insensitive to regulation by GTP and stimulatory hormones [ 3 4 - 3 6 ] . However, since Mn 2+ uncoupled both the inhibitory and stimulatory hormone response in hamster adipocytes over a very similar concentration range, a concentration-dependent action of Mn 2+ first (at low concentrations) on the inhibitory site and then (at high concentrations) on the stimulatory site appears too unlikely in the system studied. Whatever is the precise uncoupling mechanism of Mn 2+, the data presented indicate additionally that in order to study hormone-induced inhibition o f the adenylate cyclase in membrane preparations it is extremely important to control the use of this cation concentration, as is known from studies on hormonal stimulation of the enzyme.

Acknowledgements We are indebted to Mrs. Rita Bitsch and Ms. Gabriele Gabel for excellent technical assistance. This work was supported by a grant from the Deutsche Forschungsgemeinschaft.

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