Absence of the inhibitory effect of guanine nucleotides on adenylate cyclase activity in white adipocyte membranes of the ob/ob mouse. Effect of the ob gene

In mice homozygous for the ob gene (ob/ob), the response of adipose tissue adenylate cyclase to stimulation by lipolytic hormones is abnormally low in comparison to that in lean mice (+/+). Studies on the kinetics of adenylate cyclase activation in white adipocyte membranes under a variety of conditions show the following differences between +/+ and ob/ob mice. 1) The inhibitory effects of GTP and guanyl-5'-yl imidodiphosphate, which were clearly seen in +/+ membranes, were absent in the ob/ob membranes. 2) Half-maximal activation by GTP (in the presence of isoproterenol) required at least 10 times more GTP in ob/ob than in +/+ membranes. 3) Increasing the magnesium concentration (up to 10 mM) of the assay medium facilitated the activation of cyclase by modulatory ligands proportionately more in ob/ob than in +/+ membranes; in the +/+ membranes, 10 mM Mg2+ abolished the inhibitory effects of GTP. 4) Treatment with pertussis toxin attenuated the inhibitory effects of guanine nucleotides in +/+ membranes; no effect of the treatment was seen in the ob/ob membranes. 5) Pretreatment of membranes with cholera toxin facilitated cyclase activation proportionately more in ob/ob than in +/+ membranes; in addition, this treatment led to a shift to the left of the GTP dose-response curve in the ob/ob membranes. Cholera and pertussis toxins catalyzed the incorporation of ADP-ribose into their respective substrates in both the +/+ and the ob/ob membranes, showing that the alpha subunits of the stimulatory and inhibitory proteins of the regulatory component Ns and Ni, respectively are present in both types of membranes. Taken together, the results are consistent with the hypothesis that an excess of beta subunit (either primary or secondary to an altered interaction between beta and Ni alpha or Ns alpha) is responsible for the altered sensitivity to activating ligands of the adipocyte adenylate cyclase of the ob/ob mouse. In addition to these findings, we report an effect of the ob gene on the expression of adenylate cyclase activity, since adipose tissue cyclase from heterozygous lean mice (+/ob) showed characteristics which were intermediate between those of +/+ and ob/ob membranes.     

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.     

A novel site of action of a high affinity A1 adenosine receptor antagonist

XAC, a high affinity antagonist of the A1 adenosine receptor, enhances adenylate cyclase activity by 1.3-2 fold with an EC50 of approximately 47 nM in adipocyte membranes pretreated with adenosine deaminase to eliminate adenosine and in the presence of total phosphodiesterase inhibition by 100 microM papaverine. This effect of XAC is observed only at concentrations of GTP sufficient to activate Gi (approximately 5 x 10(-6) M GTP) and is not evident in the absence or presence of lower GTP concentrations. ADP ribosylation of Gi by pertussis toxin treatment also abolishes this stimulatory action of XAC. Furthermore, in the presence of GTP activation of inhibitory prostaglandin E1 receptors diminishes the stimulatory effect of XAC on adenylate cyclase. In addition, XAC interferes with GTP-mediated inhibition of forskolin-stimulated adenylate cyclase activity in a noncompetitive manner. Finally, XAC is only a weak inhibitor of the low Km cyclic AMP phosphodiesterase, producing approximately 40% inhibition of phosphodiesterase activity at a concentration of 100 microM. These data suggest that XAC increases adenylate cyclase activity in absence of endogenous adenosine by inhibiting tonic Gi activity in a reversible manner.     

Alterations in G-protein expression, Gi function and stimulatory receptor-mediated regulation of adipocyte adenylyl cyclase in a model of insulin-resistant diabetes with obesity.

The stimulatory effect of Mn2+ (1.5-fold), forskolin (1.6-fold) and low (1 microM) concentrations of GTP (1.9-fold) on the adenylyl cyclase of adipocyte membranes from obese, diabetic CBA/Ca mice was markedly enhanced compared to that seen using membranes prepared from their lean littermates. In contrast, receptor-mediated stimulation, achieved with either isoprenaline or secretin was reduced and that by glucagon abolished in membranes from diabetic animals. The levels of expression of alpha-subunits of Gi-1, Gi-2 and Gi-3 were reduced to some 49, 76 and 54%, respectively, in membranes from diabetic animals compared with those from normal animals. Levels of G-protein beta-subunits and Gs alpha-subunits were similar. Receptor-mediated inhibition of adenylate activity elicited by either nicotinic acid or prostaglandin E1 (PGE1) was of a similar magnitude in membranes from normal and diabetic animals but the inhibitory action of N6-(L-2-phenylisopropyl)adenosine (PIA) was greater in membranes from diabetic animals by about 30%. Gi function was similarly evident in membranes from both lean and diabetic animals, as assessed using low concentrations of guanylyl 5'-imidodiphosphate to inhibit forskolin-stimulated adenylyl cyclase activity. However, assessing Gi function using GTP showed marked dissimilarities in that the elevated GTP concentrations expected to occur physiologically were incapable of reversing the stimulation achieved at low concentrations of GTP in membranes from diabetic but not normal animals. The adipocytes of CBA/Ca mice, as do other animal models of insulin resistance, show lesions in adenylyl cyclase regulation, Gi function and G-protein expression.     

Alterations in mRNA levels, expression, and function of GTP-binding regulatory proteins in adipocytes from obese mice (C57BL/6J-ob/ob)

Messenger RNA levels for the alpha subunit of G-proteins expressed in adipocytes of lean and obese (ob/ob) mice were compared with relative levels of the encoded proteins. Using both toxin labeling and Western blots, expression of Gs alpha, Gi alpha-1, and Gi alpha-3 was decreased by approximately 2-fold in adipocytes of obese mice, while levels of Gi alpha-2 did not differ between the phenotypes. The decreases in Gi alpha-1 and Gs alpha in the obese mouse were attributed to decreased mRNA levels for these proteins. Similar mRNA levels for Gi alpha-3 were noted in both phenotypes, but Gi alpha-2 message was increased 2-fold in the obese mouse. Inhibitory regulation of adipocyte adenylylcyclase through G-proteins was evaluated by comparing the ability of R-PIA to inhibit isoproterenol-stimulated responses between the phenotypes. In spite of the decrease in Gi alpha-1 and Gi alpha-3 in adipocytes from obese mice, R-PIA inhibited adenylylcyclase, cAMP-dependent protein kinase, and lipolysis in similar fashion in both phenotypes. The GTP analog, Gpp(NH)p also inhibited forskolin-stimulated adenylylcyclase in a comparable manner, but the magnitude of the inhibition was slightly less in adipocyte membranes from obese mice. In contrast, the decrease in expression of Gs alpha was translated into substantially poorer activation of isoproterenol-stimulated responses in the obese mouse. The concentration of isoproterenol producing half-maximal activation of adenylylcyclase, protein kinase, and lipolysis did not differ between the phenotypes, but the maximal responses were much lower in cells from obese mice. Similar lipolytic potential in isolated adipocytes from each phenotype and similar total forskolin-stimulated cyclase activity in adipocyte membranes from each phenotype suggest that decreased expression of Gs alpha may contribute to the characteristic alteration in mobilization of triglycerides noted in adipocytes from obese mice.     

Specific epidermal growth factor receptors on porcine thyroid cell membranes

The influence of islet-activating protein (IAP), a Bordetella pertussis toxin, was studied on adenylate cyclase and GTPase activities in rat adipocyte membranes. Pretreatment of rats or intact rat adipocytes with IAP did not affect adenylate cyclase inhibition by the stable GTP analog, GTP gamma S, whereas inhibition by GTP was abolished. Concomitantly, activation of the adipocyte enzyme by sodium and its inhibition by nicotinic acid were prevented. Furthermore, IAP treatment of adipocyte membranes prevented nicotinic acid-induced stimulation of a high affinity GTPase. The data suggest that a GTP-hydrolyzing system involved in the inhibitory regulation of adenylate cyclase is the target of IAP's action.     

Pertussis toxin reversal of the antilipolytic action of insulin in rat adipocytes in the presence of forskolin does not involve cyclic AMP

Insulin inhibition of lipolysis in the presence of forskolin was reversed by a four hour exposure of adipocytes to pertussis toxin. In contrast, the antilipolytic action of insulin against lipolysis due to theophylline was unaffected by pertussis toxin as was the ability of insulin to lower cyclic AMP in the presence of either forskolin or theophylline. The stimulation of adenylate cyclase by norepinephrine in crude plasma membranes obtained from rat adipocytes was inhibited by N6-(Phenylisopropyl)adenosine (PIA) and abolished by pretreating rat adipocytes with pertussis toxin. The stimulation of glucose metabolism by insulin was not altered by pertussis toxin pretreatment of rat adipocytes. These findings suggest that pertussis toxin selectively abolishes the antilipolytic effect of insulin in the presence of forskolin through a cyclic AMP independent mechanism.     

Pertussis toxin reverses Gpp(NH)p inhibition of basal and forskolin activated adipocyte adenylate cyclase

Inhibition of basal adenylate cyclase by GTP or guanyl-5'-yl imidodiphosphate was abolished in membranes isolated from rat adipocytes previously incubated with pertussis toxin. Forskolin (0.1 microM) stimulated adenylate cyclase about 4-fold and inhibition of cyclase by GTP or guanyl-5'-yl imidodiphosphate was also abolished by pertussis toxin treatment of rat adipocytes. Forskolin (1 microM) increased adenylate cyclase activity at least ten-fold and the inhibitory effect of GppNHp was reduced but not abolished by pertussis toxin. In rabbit adipocytes, pertussis toxin reversed the inhibition of adenylate cyclase activity by GppNHp to the same extent as that by GTP in the presence of 1 microM forskolin. The present results indicate that pertussis toxin can reverse the inhibition of adipocyte adenylate cyclase by nonhydrolyzable GTP analogs as well as that by GTP.     

Does glucocorticoid deprivation promote the expression of adenosine receptor-sites stimulating adenylate cyclase in rat adipocyte membranes?

The influence of N⁶-phenylisopropyladenosine (PIA) on adenylate cyclase was compared in adipocyte membranes from adrenalectomized and sham operated rats. In the presence of 100 mM sodium, 10 μM GTP and adenosine deaminase, PIA inhibited basal adenylate cyclase activity in sham rats, but elicited biphasic effects in adrenalectomized rats: at concentrations up to 10 nM, PIA first stimulated the enzyme, after which higher concentrations produced inhibition. In the presence of theophylline, these biphasic effects could not be observed. When isoproterenol maximally-stimulated adenylate cyclase was studied, the same biphasic effects of PIA were also observed in adrenalectomized rats, provided that no sodium was added in the assay, since with 100 mM sodium, only inhibition was seen. Finally, the stimulatory but not the inhibitory effect of PIA was prevented by glucocorticoid administration, a phenomenon which suggests that glucocorticoid deprivation may promote the expression of adenosine receptorsites which activate adenylate cyclase and which are normally absent, cryptic or unfunctional in normal adipocytes.     

Quantification of the alpha and beta subunits of the transducing elements (Gs and Gi) of adenylate cyclase in adipocyte membranes from lean and obese (ob/ob) mice.

The abundance of the alpha and beta subunits of the GTP-binding proteins (G-proteins) that transduce hormonal messages to adenylate cyclase was assessed in adipocyte membranes from lean (+/+) and obese (ob/ob) mice, using ADP-ribosylation with bacterial toxin and immunodetection. Both methods revealed two Gs alpha species (48 and 42 kDa) in the membranes. Compared with those of lean mice, the membranes from obese mice contained substantially less of the 48 kDa species of Gs alpha, as assessed by both methods. ADP-ribosylation by pertussis toxin showed that only half as much ADP-ribose was incorporated into Gi alpha in the membranes from obese as compared with lean mice. Immunodetection revealed two separate Gi alpha peptides (39 and 40 kDa) and showed that the 40 kDa species was less abundant in the membranes from obese mice, whereas the amount of the 39 kDa species was similar in membranes from both lean and obese animals. Based on ADP-ribosylation assays, in membranes from lean mice the ratio Gs alpha/Gi alpha was 1:16, whereas in the membranes from obese mice it was 1:10. Similar amounts of immunodetectable beta peptide were found in both types of membranes. On the basis of the currently accepted dissociation model of adenylate cyclase activation, the decrease in the abundance of the Gi alpha subunit in adipocyte membranes from obese mice could account for the abnormal kinetics of the enzyme in these membranes.     

Adenosine receptor-mediated stimulation of GTP hydrolysis in adipocyte membranes

The adenosine derivative, N⁶-phenylisopropyladenosine (PIA), which inhibits adenylate cyclase in adipocyte membranes by a GTP-dependent and sodium-amplified process, was studied on GTPase activity in hamster adipocyte ghosts. PIA stimulated a high affinity GTPase without apparent lag phase. Both unstimulated and PIA-stimulated GTPases exhibited very similar K_m values of about 0.2 μM GTP. PIA-induced low K_m GTPase stimulation was amplified by sodium ions and was half-maximal and maximal at about 0.02 and 0.1 μM PIA, respectively. Stimulations of the low K_m GTPase by PIA and PGE₁, both inhibiting adipocyte adenylate cyclase, were not additive. Similar to PIA-induced adenylate cyclase inhibition, stimulation of the GTPase by PIA but not by PGE₁ was prevented by the adenosine receptor antagonist, 3-isobutyl-1-methylxanthine. The data suggest that PIA-induced stimulation of a high affinity GTPase is an essential mechanism of adenosine receptor-mediated adipocyte adenylate cyclase inhibition.     

Increased sensitivity of fat cell adenylate cyclase to stimulatory agonists during fasting is not related to impaired inhibitory coupling system

In rat adipocytes, inhibition of the forskolin-stimulated cyclic AMP response by nicotinic acid and N6-phenylisopropyladenosine was unaltered by a 72 h fasting. Under assay conditions favouring inhibition, basal and forskolin-stimulated adenylate cyclase responses to inhibition by GTP and nicotinic acid were also unimpaired by fasting. Under the same conditions, however, low GTP concentrations elicited a clear activatory effect in membranes from fasted but not from fed rats. Fasting failed to alter the incorporation of [32P]ADP ribose into the alpha i-subunit of Ni and the attenuation of nicotinic acid inhibitory action that are both induced by pertussis toxin. These results, demonstrating unimpaired inhibitory control of adenylate cyclase in starved rat adipocytes, suggest that the permissive effect of fasting on the action of stimulatory receptor agonists in fat cells reflects a specific increase in the activity of the adenylate cyclase stimulatory coupling system.     

Arrhenius plot characteristics of adipocyte-plasma-membrane adenylate cyclase activity in lean and genetically obese (ob/ob) mice

Arrhenius plots of fluoride- and guanine-nucleotide-stimulated adenylate cyclase activity were linear in adipocyte plasma membranes from lean and obese (ob/ob) mice . Arrhenius plots of isoprenaline-stimulated adenylate cyclase activity in hepatic plasma membranes biphasic in both groups. The results were biphasic in membranes from Jean mice but linear in membranes from obese mice. In contrast, Arrhenius plots of glucagon-stimulated adenylate cyclase activity in hepatic plasma membranes were biphasic in both groups. The results suggest that the coupling between the -receptor and the regulatory unit of adenylate cyclase, which has been observed to be defective in adipocyte plasma membranes from obese mice, is influenced by a different lipid environment in membranes from obese animals.     

Inhibition of rat and human adipocyte adenylate cyclase in the antilipolytic action of insulin, clofibrate, and nicotinic acid.

Clofibrate (Atromid-S), nicotinic acid, and insulin are known to be potent hypolipidemic and antilipolytic agents. The present study was undertaken to define the mechanism of action of this latter effect on isolated rat and human fat cells. Sodium clofibrate (0.42 mM), nicotinic acid (0.42 mM), and insulin (100 microU/mL) were shown to inhibit norepinephrine-stimulated lipolysis in rat and human adipose cells and this inhibition was associated with a reduction in intracellular 3',5'-cyclic AMP levels. A similar cyclic AMP lowering effect was demonstrated with insulin in the presence of procaine-HCL, which uncouples the adenylate cyclase system from lipolysis. This insulin effect was attributed to inhibition of adenylate cyclase. A direct and significant inhibition of adenylate cyclase in membrane fractions obtained from isolated human adipocytes was demonstrated for all three antilipolytic agents. The common membrane site of action of these agents whereby adenylate cyclase activity is depressed, thus decreasing cyclic AMP production and free fatty acid (FFA) mobilization from adipose stores, implies a central role for the adenylate cyclase system. These findings are consistent with the view that the hypotriglyceridemic effects of clofibrate, nicotinic acid, and insulin may be partly explained by deprivation of FFA substrate for hepatic very low density lipoprotein synthesis.     

Regulation of Adenosine-Sensitive Adenylate Cyclase from Rat Brain Striatum

An adenosine-sensitive adenylate cyclase has been characterized from rat brain striatum. In whole homogenates as well as in particulate fractions, N⁶-phenylisopropyl adenosine (PIA), 2-chloroadenosine, and adenosine N′-oxide were equipotent in stimulating adenylate cyclase. Although GTP inhibited basal as well as PIA-stimulated activity of whole homogenates, the enzyme showed an absolute dependency on GTP for stimulation by PIA, dopamine, epinephrine, and norepinephrine in a particulate fraction derived from discontinuous sucrose gradient centrifugation. Adenosine exerts two effects on this adenylate cyclase, stimulation at low concentrations and inhibition at high concentrations, suggesting the presence of two adenosine binding sites. The stimulation of adenylate cyclase by PIA was dependent on the concentration of Mg^2-. The degree of stimulation by PIA was greater at a low concentration of Mg²⁺, which suggests that stimulation by PIA was accompanied by increasing the apparent affinity for Mg²⁺. Activation of adenylate cyclase by PIA was blocked by theophylline or 3-isobutyl- 1-methylxanthine (IBMX). The pH optimum for basal or (PIA + GTP)-stimulated activities was broad, with a peak between 8.5 and 9.5. In the presence of GTP, stimulation by an optimal concentration of PIA was additive, with maximal stimulation by the catecholamines. Phospholipase A₂ treatment at a concentration of 1 U/ml for 5 min completely abolished the stimulatory effect of dopamine, whereas PIA-stimulated activity remained unaltered. These data suggest that rat brain striatum either has a single adenylate cyclase, which is stimulated by catecholamines and adenosine by distinct mechanisms, or has different cyclase populations, stimulated by either adenosine or catecholamines.     

Prostaglandin-sensitive adenylyl cyclase of cultured preadipocytes and mature adipocytes of the rat: probable role of Gi in determination of stimulatory or inhibitory action

Preadipocytes of rats were obtained from the stromal-vascular fraction of collagenase-digested perirenal fat pads and grown in serum-containing medium. By day 8 of culture the cells reached confluence and by 12 days were lipid-laden. The adenylyl cyclase of the plasma membranes was compared to that of mature fat cells. Unlike the membranes from adipocytes, the preadipocytes showed adenylyl cyclase activity that was stimulated by GTP. Stimulation of preadipocyte membranes by Gpp(NH)p, NaF, and forskolin was comparable to that of membranes from adipocytes, but the response to epinephrine and isoproterenol was minimal (approximately 1.5-fold for preadipocytes vs. 4-5-fold for adipocytes). In contrast, GTP-dependent stimulation of adenylyl cyclase of preadipocytes by PGE1 was nearly 8-fold. Stimulation occurred even in the presence of both GTP and 140 mM NaCl, a condition that leads to inhibition by PGE1 of adenylyl cyclase in membranes of adipocytes. Other characteristics of the adenylyl cyclase of preadipocyte membranes that differ from those of adipocytes include lack of inhibition by GTP of forskolin-activated activity, and, following treatment with pertussis toxin, enhanced stimulation by PGE1. ADP-ribosylation of Gi and Gs with pertussis and cholera toxins, respectively, indicated that the membranes of preadipocytes contained only 5-11% of the Gi of adipocytes and a much lower ratio of Gi:Gs. These findings suggest that cultured preadipocytes have an incompletely developed Gi pathway that may account for the stimulatory effect of prostaglandins on the adenylyl cyclase of these cells as opposed to the inhibitory action of PG in mature fat cells.     

Islet-activating protein discriminates the antilipolytic mechanism of insulin from that of other antilipolytic compounds

In vivo administration of islet-activating protein to rats resulted in an increase in fat cell lipolysis in vitro, which was associated with almost complete resistance of adipocytes towards the antilipolytic effects of N6-phenylisopropyladenosine, prostaglandin E2 and nicotinic acid. Concomitantly, the inhibitory effects of these compounds on adenylate cyclase activity in membranes were impaired. In contrast, the antilipolytic action of insulin was not only preserved, but even augmented in cells from rats treated with islet-activating protein. The data suggest that insulin exerts its antilipolytic effects via mechanisms which are different from those involved in the effects of prostaglandin E2, N6-phenylisopropyladenosine and nicotinic acid.     

The inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. Subunit dissociation and guanine nucleotide-dependent hormonal inhibition

The inhibitory and stimulatory guanine nucleotide-binding regulatory components (Gi and Gs) of adenylate cyclase both have an alpha X beta subunit structure, and the beta (35,000 Da) subunits are functionally indistinguishable. Gi and Gs both dissociate in the presence of guanine nucleotide analogs or Al3+, Mg2+, and F- in detergent-containing solutions. Several characteristics of Gi- and Gs-mediated regulation of adenylate cyclase activity have been studied in human platelet membranes. The nonhydrolyzable analog of GTP, guanosine-5'-(3-O-thio)triphosphate (GTP gamma S) mimics GTP-dependent hormonal inhibition or stimulation of adenylate cyclase under appropriate conditions. This inhibition or stimulation follows a lag period. The combined addition of epinephrine or prostaglandin E1 with GTP gamma S results in the immediate onset of steady state inhibition or activation. The effects of the GTP analog are essentially irreversible. Fluoride is also an effective inhibitor of prostaglandin E1-stimulated adenylate cyclase, while it markedly stimulates the basal activity of the enzyme. The addition of the resolved 35,000-Da subunit of Gi to membranes results in inhibition of adenylate cyclase, and the resolved 41,000-Da subunit has a stimulatory effect on enzymatic activity. The inhibitory action of the 35,000-Da subunit is almost completely abolished in membranes that have been irreversibly inhibited by GTP gamma S plus epinephrine; this irreversible inhibition is almost completely relieved by the 41,000-Da subunit. Detergent extracts of membranes that have been treated with GTP gamma S plus epinephrine contain free 35,000-Da subunit. The 41,000-Da subunit of Gi contained in such extracts has a reduced ability to be ADP-ribosylated by islet-activating protein (IAP), which implies that this subunit is in the GTP gamma S-bound form. The irreversible inhibition of adenylate cyclase caused by GTP gamma S (plus epinephrine) in membranes is highly correlated with the liberation of free 35,000-Da subunit activity and is inversely related to the 41,000-Da IAP substrate activity in detergent extracts prepared therefrom. The increase in free 35,000-Da subunit activity in extracts and the inhibition of adenylate cyclase activity in GTP gamma S (plus epinephrine)-treated membranes are both markedly inhibited by treatment with IAP.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Adenosine A1 Receptors Are Associated with Cerebellar Granule Cells

The cerebellum of mouse appears to have only the adenosine A1 receptor, which decreases adenylate cyclase activity, and not the A2 receptor, which increases adenylate cyclase activity. The adenosine analog N6-(L-phenylisopropyl)adenosine (PIA), stimulates the A1 receptor in a membrane preparation and decreases basal adenylate cyclase activity by 40%. The EC50 for PIA is approximately 50 nM. To associate the A1 receptor with a cerebellar cell type, three different neurological mutant mouse strains were studied: staggerer (Purkinje and granule cell defect), nervous (Purkinje cell defect), and weaver (granule cell defect). PIA was unable to effect a maximal decrease in adenylate cyclase activity of membranes prepared from cerebella of the staggerer and weaver mice in comparison with the respective littermate control mice. In contrast, membranes from nervous mice and their littermates showed similar PIA dose-response curves. Moreover, the diminished PIA response observed in the weaver cerebellum, when compared with the control littermate, was not detected in the striatum. This suggests no overall brain defect in the adenosine A1 receptors coupled to adenylate cyclase of the weaver mouse. We conclude that a loss of granule cells coincides with an attenuated response to PIA, implying that the A1 receptors are associated with the granule cells of the cerebellum.