Fasting increases fat cell adenylate cyclase sensitivity to stimulatory agonists through enhanced ability of the stimulatory regulatory component Ns to dissociate

In rat fat cell membranes, a 72-hour fasting fails to alter the adenylate cyclase stimulatory responses to Mn2+, forskolin and cholera toxin and the cholera toxin catalyzed [alpha-32P] ADP ribose incorporation into the Mr = 42,000 and 46,000/48,000 alpha s peptides of Ns. In contrast, dose-response curves for GTP-stimulation of basal and isoproterenol-stimulated adenylate cyclase display higher maximal responses in fasted rats under conditions restraining (2 mM Mg2+) but not promoting (10 mM Mg2+) the dissociation of Ns. Moreover, at 10 mM Mg2+, the sensitivity of isoproterenol-stimulated adenylate cyclase to GTP is clearly increased in fasted rats. Finally, fasting reduces by 40% the lag-phase of adenylate cyclase activation by Gpp(NH)p. Taken together, these results are consistent with the hypothesis that the permissive effect of fasting on the fat cell adenylate cyclase response to stimulatory agonists is related to increased ability of Ns and the ternary H.R.Ns. complex to dissociate which is likely due to enhanced Ns affinity for guanine nucleotides.     

Adenylate cyclase stimulation by VIP in rat and human parotid membranes

Adenylate cyclase activity was stimulated by vasoactive intestinal peptide (VIP) in rat parotid membranes, in the presence of 100 microM guanosine triphosphate (GTP). The threshold concentration of VIP was 300 nM and the activity doubled at the maximal VIP concentration tested (30 microM). The relative potency of peptides of the VIP family was: VIP greater than peptide histidine isoleucinamide (PHI) greater than secretin. The beta-adrenergic agent isoproterenol was a more efficient activator of rat parotid adenylate cyclase and its stimulatory effect, like that of VIP, depended on the presence of GTP. The effects of VIP and isoproterenol were both potentiated by 10 microM forskolin. By comparison with rat parotid preparations, membranes from a human parotid gland responded similarly to the VIP family of peptides (VIP greater than PHI greater than secretin). In both rat and human parotid membranes, two proteins (Mr 44 kDa and 53 kDa) of the alpha-subunit of Ns (the guanyl nucleotide-binding stimulatory protein) were labelled by ADP-ribosylation, in the presence of cholera toxin. Taken together, these results indicate that VIP receptors, when coupled to Ns, were able to activate the adenylate cyclase system in rat and human parotid membranes.     

3H]GDP release from rat and hamster adipocyte membranes independently linked to receptors involved in activation or inhibition of adenylate cyclase. Differential susceptibility to two bacterial toxins

When rat adipocyte membranes had been labeled with [3H]GTP in the presence of a beta-adrenergic agonist, the subsequent [3H]GDP release was stimulated by beta-agonists or agonists (e.g. glucagon and secretin) of other "activatory" receptors involved in activation of adenylate cyclase, but was not stimulated by agonists (e.g. prostaglandin E1 and adenosine) of "inhibitory" receptors involved in cyclase inhibition. On the contrary, agonists of inhibitory receptors were effective in stimulating GDP release from hamster adipocyte membranes that had been labeled via inhibitory alpha 2-adrenergic receptors, but an activatory receptor agonist such as isoproterenol was not. Thus, the guanine nucleotide regulatory protein (Ni) involved in adenylate cyclase inhibition is an entity distinct from the regulatory protein (Ns) involved in cyclase activation, and multiple activatory or inhibitory receptors are coupled to a respective common pool of Ns or Ni. Preactivated cholera toxin added together with NAD enhanced GDP release from rat adipocyte membranes prelabeled with isoproterenol but was without effect on the release from hamster adipocyte membranes that had been labeled with an alpha-agonist. In sharp contrast, the active subunit of islet-activating protein, pertussis toxin, failed to alter GDP release from the former membrane but completely abolished inhibitory agonist-induced stimulation of GDP release from the latter membrane preparation in the presence of NAD. Thus, the site of action of cholera toxin is Ns, while that of islet-activating protein is Ni. The function of Ni to communicate between inhibitory receptors and adenylate cyclase was lost when it was ADP-ribosylated by islet-activating protein.     

N-Chloroacetylhydrazone of oxo-GTP irreversibly inhibits the activating function of GTP-binding protein coupled with adenylate cyclase

Guanine nucleotides are successfully used in the studies of regulatory N-proteins coupled with adenylate cyclase. In the present work N-chloroacetylhydrazones of oxo-GTP and oxo-GDP are described. After 4 hr preincubation of these nucleotides with plasma membranes from bovine brain caudate nucleus, the ability of adenylate cyclase to be activated by guanylyl-5'-methylene-diphosphonate is blocked. The degree of inhibition depends on preincubation time and increases in the presence of Mg2+. Guanylyl-5'-methylenediphosphonate protects adenylate cyclase from the action of N-chloroacetylhydrazone of oxo-GTP. These findings suggest that adenylate cyclase activation is diminished as a result of covalent modification of the Ns. N-chloroacetyl-hydrazone of oxo-GDP also causes a loss of the adenylate cyclase sensitivity to the fluoride ion and cholera toxin.     

The mechanism of action of cholera toxin in pigeon erythrocyte lysates.

The adenylate cyclase activity of intact pigeon erythrocytes begins to rise after about 20 min of exposure to cholera toxin. The maximum rate at which the cyclase activity increases appears to be limited by the number of toxin molecules which can reach an intracellular target. If the erythrocytes are made permeable to the toxin by a bacterial hemolysin, no such limit exists, and adenylate cyclase activity starts to rise immediately upon the addition of toxin, and continues to rise to a maximum at an initially constant rate which is dependent upon the concentration of toxin. On lysed erythrocytes, the addition of cholera antitoxin immediately prevents any further rise in adenylate cyclase activity, but does not reverse any activation already achieved. Erythrocyte lysates may also be activated by isolated peptide A1 of cholera toxin, although activation of adenylate cyclase of intact erythrocytes requires the complete toxin molecule. In the intact cells, toxin first attaches by its Component B to surface receptors of which there are about 30 per erythrocyte. Subsequently, peptide A1 but not Component B is inserted into the erythrocyte. It takes only about 1 min at 37 degrees for peptide A1 to be sufficiently deep within the cell membrane to be inaccessible to extracellular antitoxin, but its complete transit through the membrane appears to take longer. The surface receptors are used only once, for they remain blocked by Component B. The number of receptors available on the surface may be increased by soaking cells in ganglioside GM1. Cholera toxin also decreases the rate of apparently spontaneous loss of adenylate cyclase activity and increases the response to epinephrine. Theophylline inhibits the action of cholera toxin.     

Effects of cholera toxin and 5'-guanylylimidodiphosphate on human spermatozoal adenylate cyclase activity

The effects of cholera toxin and 5′-guanylylimidodiphosphate (Gpp(NH)p) on human spermatozoal adenylate cyclase activity were tested. Cholera toxin had no demonstrable effect on adenylate cyclase activity in human spermatozoa at concentrations between 5 and 20 μg/ml, whether the toxin was preincubated with intact spermatozoa between 5 min and 5 h prior the adenylate cyclase assay, or was added to lysed spermatozoa, where the adenylate cyclase would be accessible to the toxin. In contrast, Gpp(NH)p at concentrations between 10 and 100 μM was effective in activating human spermatozoal adenylate cyclase activity.     

Limulus ventral photoreceptors contain a homologue of the alpha-subunit of mammalian Ns

Membranes from ventral photoreceptors of Limulus were incubated with cholera toxin and [32P]NAD+. Cholera toxin catalyzes a specific ADP-ribosylation of a 43-kDa peptide from Limulus ventral photoreceptors. Possible homologies between the 43-kDa peptide of Limulus and the alpha-subunits of mammalian stimulatory, guanine nucleotide-binding regulatory component of adenylate cyclase (Ns) were investigated by comparing the electrophoretic patterns of proteolytic fragments derived from each of these peptides that are radiolabeled by [32P]NAD+ and cholera toxin. Evidence is provided for structural homology between this invertebrate peptide and mammalian Ns.     

The hysteretic effect of Gpp(NH)p on adenylate cyclase is not altered by Mg2+ in adipocyte membranes of ob/ob mice

We have established previously that the regulation of adenylate cyclase is abnormal in adipose tissue membranes of ob/ob mice. To help establish the nature of the defect, we studied the time course of guanine nucleotide activation and inhibition of adenylate cyclase. The activation of adenylate cyclase by Gpp(NH)p in adipocyte membranes of normal (+/+) and ob/ob mice proceeds with a lag phase. In +/+ membranes, this lag could be shortened by increasing the concentration of Mg2+ in the incubation medium or by pretreatment of the membranes with cholera toxin, and it could be abolished by isoproterenol in combination with 4 mM MgCl2. In contrast, in the ob/ob membranes, only pretreatment with cholera toxin was effective in shortening the lag phase. These results indicate an impediment in the activation of adenylate cyclase in ob/ob membranes. In the +/+ membranes, Gpp(NH)p inhibited foreskolin-stimulated adenylate cyclase, following a short lag phase, producing lower steady-state velocities than those seen with forskolin alone. The inhibitory effect of Gpp(NH)p on forskolin-stimulated activity was abolished by pertussis but not by cholera toxin treatment. In the ob/ob membranes, neither Gpp(NH)p nor pertussis treatment had any effect on the steady-state velocity of the forskolin-stimulated activity. These data have been interpreted as meaning that an anomaly in Ni rather than in Ns is likely to be responsible for the impairment of adenylate cyclase activity in the membranes of the ob/ob mouse.     

Monoclonal antibodies against rat kidney gamma-glutamyl transpeptidase show species and tissue specificity.

The existence of a GTP-binding protein of the Ns type in Trypanosoma cruzi was explored. Epimastigote membranes were labelled by cholera toxin in the presence of [adenine-14C]NAD+. After SDS/polyacrylamide-gel electrophoresis of extracted membrane proteins, a single labelled polypeptide band of apparent Mr approx. 45,000 was detected. Epimastigote cells were treated with N-ethylmaleimide and electrofused to lymphoma S49 cells lacking the Ns protein. Evidence indicates that in such electrofusion-generated cell hybrids a heterologous adenylate cyclase system was reconstituted with the Ns protein provided by T. cruzi epimastigotes.     

ADP-ribosylation of membrane proteins and activation of adenylate cyclase by cholera toxin in fat cell ghosts from euthyroid and hypothyroid rats.

Incubation of fat cell ghosts with activated cholera toxin, nucleoside triphosphate, cytosol, and NAD results in increased adenylate cyclase activity and the transfer of ADP-ribose to membrane proteins. The major ADP-ribose protein comigrates on sodium dodecyl sulfate-polyacrylamide gels with the putative GTP-binding protein of pigeon erythrocyte membranes (Mr 42 000), which is also ADP-ribosylated by cholera toxin. The treatment with cholera toxin enhances the stimulation of the fat cell membrane adenylate cyclase by GTP, but the stimulation by guanyl-5'-yl imidodiphosphate is unaltered. Subsequent stimulation of fat cell adenylate cyclase by 10 micrometers epinephrine is not particularly affected. These changes were qualititatively the same for membranes isolated from fat cells of hypothyroid rats. Although the cyclase of these membranes has a reduced response to epinephrine, guanyl-5'-yl imidodiphosphate or GTP, as compared to euthyroid rat fat cell membranes, the defect is not rectified by toxin treatment and cannot be explained by a deficiency in the cholera toxin target.     

Stimulation of adenylate cyclase in washed pigeon erythrocyte membrane with cholera toxin and its subunits.

Cholera toxin was found to stimulate adenylate cyclase activity in washed membrane of pigeon erythrocytes in the presence of dithiothreitol and NAD. When tested with isolated cholera toxin components, the stimulatory activity was found with subunit A or polypeptide A1 derived from this subunit, but not with A2 or subunit B. On a molar basis, polypeptide A1 was approximately four times more active than cholera toxin. Dithiothreitol was not required in the action of polypeptide A1, suggesting that the reagent was needed only to release A1 from subunit A or the holotoxin for their action on adenylate cyclase. The single SH group in polypeptide A1 was not involved in the activity of the peptide, since chemical modification of the thiol group did not alter the stimulatory activity of the peptide. The presence of NAD was, however, essential for the activation of adenylate cyclase with cholera toxin, subunit A, or polypeptide A1. Elevation of the adenylate cyclase activity was also observed when the intact pigeon erythrocytes were incubated with polypeptide A1, although a 30-fold molar excess of A1 over that of holotoxin was required for the same level of activation.     

Activation of pigeon erythrocyte adenylate cyclase by cholera toxin partial purification of an essential macromolecular factor from horse erythrocyte cytosol

A cytosolic, macromolecular factor required for the cholera toxin-dependent activation of pigeon erythrocyte adenylate cyclase and cholera toxin-dependent ADP-ribosylation of a membrane-bound 43 000 dalton polypeptide has been purified 1100-fold from horse erythrocyte cytosol using organic solvent precipitation and heat treatment. This factor, 13 000 daltons, does not absorb to anionic or cationic exchange resins, is sensitive to trypsin or 10% trichloroacetic acid and is not extractable by diethyl ether. Activation of adenylate cyclase by cholera toxin requires the simultaneous presence of ATP (including possible trace GTP), NAD⁺, dithiothreitol, cholera toxin, membranes and the cytosolic macromolecular factor. Reversal of cholera toxin activation of adenylate cyclase, and of the toxin-dependent ADP-ribosylation, requires the presence of the cytosolic factor. The ability of the purified cytosolic factor to influence the hormonal sensitivity of liver membrane adenylate cyclase may provide clues to its physiological functions.     

Interaction of GTP-binding regulatory proteins with chemosensory receptors

GTP-binding regulatory proteins (G-proteins) were identified in chemosensory membranes from the channel catfish, Ictalurus punctatus. The common G-protein beta-subunit was identified by immunoblotting in both isolated olfactory cilia and purified taste plasma membranes. A cholera toxin substrate (Mr 45,000), corresponding to the G-protein that stimulates adenylate cyclase, was identified in both membranes. Both membranes also contained a single pertussis toxin substrate. In taste membranes, this component co-migrated with the alpha-subunit of the G-protein that inhibits adenylate cyclase. In olfactory cilia, the Mr 40,000 pertussis toxin substrate cross-reacted with antiserum to the common amino acid sequence of G-protein alpha-subunits, but did not cross-react with antiserum to the alpha-subunit of the G-protein from brain of unknown function. The interaction of G-proteins with chemosensory receptors was determined by monitoring receptor binding affinity in the presence of exogenous guanine nucleotides. L-Alanine and L-arginine bind with similar affinity to separate receptors in both olfactory and gustatory membranes from the catfish. GTP and a nonhydrolyzable analogue decreased the affinity of olfactory L-alanine and L-arginine receptors by about 1 order of magnitude. In contrast, the binding affinities of the corresponding taste receptors were unaffected. These results suggest that olfactory receptors are functionally coupled to G-proteins in a manner similar to some hormone and neurotransmitter receptors.     

Studies on the time course and rate-limiting steps in the activation of adenylate cyclase in rat liver by cholera toxin.

Cholera toxin stimulates adenylate cyclase in rat liver after intravenous injection. The stimulation follows a short latent period of 10min, and maximum stimulation was attained at 120min. Half-maximal stimulation was achieved at 35min. In contrast with this lengthy time course in the intact cell, adenylate cyclase in broken-cell preparations of rat liver in vitro were maximally stimulated by cholera toxin (in the presence of NAD+) in 20min with half-maximal stimulation in 8min. Binding of cholera toxin to cell membranes by the B subunits is followed by translocation of the A subunit into the cell or cell membrane, and separation of the A1 polypeptide chain from the A2 chain by disulphide-bond reduction, and finally activation of adenylate cyclase by the A1 chain and NAD+. As the binding of cholera toxin is rapid, two possible rate-limiting steps could be the determinants of the long time course of action. These are translocation of the A1 chain from the outside of the cell membrane to its site of action (this includes the time required for separation from the whole toxin) or the availability of NAD+ for activation. When NAD+ concentrations in rat liver were elevated 4-fold, by the administration of nicotinamide, no change in the rate of activation of adenylate cyclase by cholera toxin was observed. Thus the intracellular concentration of NAD+ is not rate-limiting and the major rate-limiting determinant in intact cells must be between the time of toxin binding to the cell membrane and the appearance of subunit A1 at the enzyme site.     

Properties of cholera toxin- and NaF-stimulated adenylate cyclase from mouse thymocytes.

Kinetic parameters of mouse thymocyte adenylate cyclase activity were determined. NaF and cholera toxin stimulated adenylate cyclase. Stimulation by either agent did not change the pH or Mg2+ optima relative to control (unstimulated cyclase). The Km value for ATP of adenylate cyclase stimulated by NaF was significantly reduced from control. By contrast, cholera toxin treatment did not change the Km relative to control. Adenylate cyclase, when stimulated by NaF, had an optimum for Mn2+ alone, or Mn2+ in combination with Mg2+, at least twice that of control. In contrast, cyclase activity prepared from cells treated with cholera toxin remained unchanged with regard to these divalent cations when compared to control. Addition of NaF to adenylate cyclase prepared from cells treated with cholera toxin resulted in a significant reduction (30%) in activity suggesting that both NaF and cholera toxin were acting on the same cyclase. NaF inhibition of cholera toxin-stimulated activity was shown to be a direct interaction of fluoride on the stimulated cyclase enzyme. This inhibition appeared to be immediate and independent on pH, Mg2+ or ATP concentrations. Although NaF inhibition was lost when Mn2+ was present in the reaction mixture, the activity expressed by addition of NaF to cyclase prepared from cholera toxin-treated cells was much less than by addition of NaF to control. As observed with cholera toxin stimulation alone, activity expressed by the inhibited enzyme (cholera toxin treated + NaF) exhibited a Km for ATP and an optimum for Mn2+ alone or in combination with Mg2+ similar to control.     

Mechanisms for inhibition of the catalytic activity of adenylate cyclase by the guanine nucleotide-binding proteins serving as the substrate of islet-activating protein, pertussis toxin

Two GTP-binding trimeric proteins (referred to as alpha 41 beta gamma and alpha 39 beta gamma based on the kilodalton molecular weights of their alpha-subunits) were purified from rat brain as the specific substrates of the ADP-ribosylation reaction catalyzed by islet-activating protein, pertussis toxin, and resolved irreversibly into alpha- and beta gamma-subunits by incubation with guanosine 5'-O-(thiotriphosphate) (GTP gamma S). Some of these resolved subunits interacted directly with the adenylate cyclase catalyst partially purified from rat brain in a detergent-containing solution, resulting in inhibition of the cyclase activity as follows. 1) GTP gamma S-bound alpha 41 inhibited the catalyst, but GTP gamma S-bound alpha 39 did not; the inhibition was competitive with GTP gamma S-bound alpha-subunit of Ns, the GTP-binding protein involved in activation of adenylate cyclase. 2) beta gamma from either alpha 41 beta gamma or alpha 39 beta gamma inhibited the catalyst in a manner not competitive with the activator such as forskolin or the alpha-subunit of Ns. 3) The ADP-ribosylation of alpha 41 beta gamma by islet-activating protein did not exert any influence on the subsequent GTP gamma S-induced resolution and the ability of the resolved GTP gamma S-bound alpha 41 to inhibit the catalyst. 4) The beta gamma-induced inhibition of the catalyst was additive to the inhibition caused by GTP gamma S-bound alpha 41. Thus, the direct inhibition of the catalyst by beta gamma or GTP gamma S-bound alpha 41 is a likely mechanism involved in receptor-mediated inhibition of adenylate cyclase, in addition to the previously proposed indirect inhibition due to the reduction of the concentration of the active alpha-subunit of Ns by reassociation with beta gamma.     

Modification of the function of pertussis toxin substrate GTP-binding protein by cholera toxin-catalyzed ADP-ribosylation.

The alpha-subunit of Gi-2, in addition to that of Gs (GTP-binding proteins involved in adenylate cyclase inhibition and stimulation, respectively) was ADP-ribosylated by cholera toxin in HL-60 cell membranes when a chemotactic receptor was stimulated by formyl-Met-Leu-Phe (fMLP), and the sites modified by cholera and pertussis toxins on the alpha-subunit of Gi-2 were different (Iiri, T., Tohkin, M., Morishima, N., Ohoka, Y., Ui, M., and Katada, T. (1989) J. Biol. Chem. 264, 21394-21400). In order to investigate how the functions of Gi-2 were modified by cholera toxin, the ADP-ribosylated and unmodified proteins were purified from HL-60 cell membranes that had been incubated in the presence and absence of cholera toxin, respectively. The modified Gi-2 displayed unique properties as follows. 1) The ADP-ribosylated alpha-subunit had a more acidic pI than the unmodified one, leading to a partial resolution of the modified Gir2 trimer from the unmodified protein by an anion column chromatography. 2) When the purified proteins were incubated with [gamma-32P]GTP, the radioactivity was more greatly retained in the modified Gi-2 than in the unmodified protein. 3) The actual catalytic rate (kcat) of GTP hydrolysis was, indeed, markedly inhibited by cholera toxin-induced modification. 4) There was an increase in the apparent affinity of Gi-2 for GDP by cholera toxin-induced modification. 5) The modified Gi-2 exhibited a low substrate activity for pertussis toxin-catalyzed ADP-ribosylation. 6) A high-affinity fMLP binding to HL-60 cell membranes was more effectively reconstituted with the ADP-ribosylated Gi-2 than with the unmodified protein. These results suggested that the agonist-fMLP receptor complex was effectively coupled with the ADP-ribosylated Gi-2, resulting in the GTP-bound form, and that the hydrolysis of GTP on the modified alpha-subunit was selectively attenuated. Thus, cholera toxin ADP-ribosylated Gi-2 appeared to be not only a less sensitive pertussis toxin substrate but also an efficient signal transducer between receptors and effectors.     

The influence of EDTA on adenylate cyclase activity in membranes from rat and mouse myocardium

Inclusion of EDTA in the homogenization buffer of both mouse and rat myocardium profoundly alters the properties of the adenylate cyclase complex. EDTA leads to an increase in the Vmax for adenylate cyclase activity due to all of the following agents: isoproterenol, Gpp[NH]p, forskolin and Mg2+. For forskolin and Mg2+, the EDTA-associated increase in Vmax is not accompanied by a change in sensitivity to activation. However, EDTA is associated with enhanced sensitivity to activation by isoproterenol and increased sensitivity to the effect of Mg2+ on isoproterenol-dependent adenylate cyclase activity. A result of greater isoproterenol-dependent adenylate cyclase activity, due to the presence of EDTA, is an attenuated synergistic contribution of Gpp[NH]p. Changes in stimulatable adenylate cyclase activity as a result of EDTA occurs in concert with effects of cholera toxin upon ADPribosylation of the guanine nucleotide regulatory protein, Ns. Substantial auto-ADP-ribosylation occurs in a cholera toxin-sensitive 42 kDa band in membranes prepared in the presence of EDTA. In addition, cofactor and substrate requirements in the cholera toxin-dependent ADP-ribosylation reaction depend on the method of membrane preparation. The results suggest that the integrity of the adenylate cyclase complex depends in part on the attention given to proteolysis that may be activated during the course of homogenization.     

Effects of Bordetella pertussis toxin on catecholamine inhibition of insulin release from intact and electrically permeabilized rat islets.

Noradrenaline- and clonidine-induced inhibition of insulin release from intact and electrically permeabilized rat islets was markedly relieved by prior exposure to 100 ng of Bordetella pertussis toxin/ml. The reversal of catecholamine inhibition of insulin secretion by this toxin was not associated with a decrease in specific binding of the alpha 2-adrenergic ligand [3H]yohimbine, and could not be fully explained by an increase in intracellular cyclic AMP. Exposure of intact islets to 1 microgram of pertussis toxin/ml for 2 h, followed by electrical permeabilization and incubation with 5 microCi of [alpha-32P]NAD+, resulted in the ADP-ribosylation in situ of a protein of molecular mass approx. 41 kDa. These results suggest that pertussis toxin alleviates catecholamine inhibition of beta-cell secretory responses by ADP-ribosylating at least one protein of molecular mass 41 kDa. In analogous systems the 41 kDa substrate of pertussis toxin has been shown to be the alpha subunit of Gi, but catecholamine-activated G proteins linked to effector systems other than adenylate cyclase might also be modified by this toxin in pancreatic beta-cells.     

Adenylate cyclase in membrane fractions of RIN-A2-cells: studies with forskolin, NaF, GppNHp and NEM

In crude membrane fractions of rat pancreatic islets and of RIN-A2-cells, forskolin and NaF stimulated adenylate cyclase activity. Basal and stimulated enzyme activity was approximately 3 to 6 fold higher in membranes of RIN-A2-cells than in membranes of islet cells. In RIN-A2-cells GppNHp and NEM inhibited forskolin-stimulated enzyme activity. The inhibitory effect of GppNHp could be reduced by NEM. It is suggested that the adenylate cyclase system of RIN-A2-cells contains inhibitory and stimulatory N-proteins and that there are critical thiols related to Ni, Ns and/or the catalytic unit. Thus, membrane fractions of RIN-A2-cells may be an appropriate model for studies on the adenylate cyclase system of insulin-producing cells.