Exchange of Guanine Nucleotides Between Tubulin and GTP-Binding Proteins That Regulate Adenylate Cyclase: Cytoskeletal Modification of Neuronal Signal Transduction

Tubulin, the primary constituent of microtubules, is a GTP-binding proteins with structural similarities to other GTP-binding proteins. Whereas microtubules have been implicated as modulators of the adenylate cyclase system, the mechanism of this regulation has been elusive. Tubulin, polymerized with the hydrolysis-resistant GTP analog, 5'-guanylylimidodiphosphate [Gpp(NH)p], can promote inhibition of synaptic membrane adenylate cyclase which persists subsequent to washing. Tubulin with Gpp(NH)p bound was slightly less potent than free Gpp(NH)p in the inhibition of adenylate cyclase, but tubulin without nucleotide bound had no effect on the enzyme. A GTP-binding protein from the rod outer segment (transducin), with Gpp(NH)p bound, was also without effect on adenylate cyclase. Tubulin (regardless of the nucleotide bound to it) did not alter the activity of the adenylate cyclase catalytic unit directly. When tubulin was polymerized with the hydrolysis-resistant photoaffinity GTP analog, [32P]P3(4-azidoanilido)-P1-5'-GTP ([32P]AAGTP), and this protein was added to synaptic membranes, AAGTP was transferred from tubulin to the inhibitory GTP-binding protein, Gi. This transfer was blocked by prior incubation of the membranes with Gpp(NH)p or covalent binding of AAGTP to tubulin prior to exposure of that tubulin to membranes. Incubation of membranes with Gpp(NH)p subsequent to incubation with tubulin-AAGTP results in a decrease in AAGTP bound to Gi and a compensatory increase in AAGTP bound to the stimulatory GTP-binding protein, Gs. Likewise, persistent inhibition of adenylate cyclase by tubulin-Gpp(NH)p could be overridden by the inclusion of 100 microM Gpp(NH)p in the assay inhibition. Whereas Gpp(NH)p promotes persistent inhibition of synaptic membrane adenylate cyclase without incubation at elevated temperatures, tubulin [with AAGTP or Gpp(NH)p bound] requires 30 s incubation at 23 degrees C to effect adenylate cyclase inhibition. Photoaffinity experiments yield parallel results. These data are consistent with synaptic membrane tubulin regulating neuronal adenylate cyclase by transferring GTP to Gi and, subsequently, to Gs.     

Forskolin-resistant Y1 mutants harbor defects associated with the guanyl nucleotide-binding regulatory protein, Gs

The properties of the adenylate cyclase from forskolin-resistant mutants of Y1 adrenocortical tumor cells was compared with the properties of the enzyme from parental Y1 cells in order to localize the site of mutation. In parental Y1 cells, forskolin stimulated adenylate cyclase activity with kinetics suggestive of an interaction at two sites; in mutant cells, forskolin resistance was characterized by a decrease in enzymatic activity at both sites. Forskolin potentiated the enzyme's responses to NaF and guanyl-5'-yl imidodiphosphate (Gpp(NH)p) in parent and mutant clones, and the mutant enzyme showed the same requirements for Mg2+ and Mn2+ as did the parent enzyme. The adenylate cyclase associated with forskolin-resistant mutants was insensitive to ACTH and was less responsive to Gpp(NH)p than was the parent enzyme. In parental Y1 cells and in the forskolin-resistant mutants, cholera toxin catalyzed the transfer of [32P]ADP-ribose from [32P]NAD+ into three membrane proteins associated with the alpha subunit of Gs; however, the amount of labeled ADP-ribose incorporated into mutant membranes was reduced by as much as 70%. Both parent and mutant membranes were labeled by pertussis toxin to the same extent. The insensitivity of the mutant adenylate cyclase to ACTH and Gpp(NH)p and the selective resistance of the mutant membranes to cholera toxin-catalyzed ADP-ribosylation suggest that a specific defect associated with Gs is involved in the mutation to forskolin resistance in Y1 cells.     

Chronic Electroconvulsive Treatment Augments Coupling of the GTP-Binding Protein Gs to the Catalytic Moiety of Adenylyl Cyclase in a Manner Similar to That Seen with Chronic Antidepressant Drugs

A significant increase of guanylylimidodiphosphate (GppNHp)-, fluoride-, and forskolin-stimulated adenylyl cyclase was observed in synaptic membrane preparations from rat cerebral cortex subsequent to chronic electroconvulsive shock (ECS) treatment. This effect required at least five treatments over a course of 10 days. The inhibition of adenylyl cyclase induced by GppNHp was not affected by these treatments. The dissociation constant (KD) and maximal binding for the photoaffinity GTP analog, [32P]P3-(4-azidoanilido)-P1-5'-GTP [( 32P]AAGTP), to each of the synaptic membrane G proteins also were unchanged after ECS treatment. Nonetheless, the transfer of [32P]AAGTP from Gi to Gs, which we suggest is indicative of the coupling between Gs and the adenylyl cyclase catalytic moiety, was accelerated by chronic ECS treatment but not by acute or sham treatment. Furthermore, chemical uncoupling of Gs from adenylyl cyclase rendered membranes from treated animals indistinguishable from controls. Finally, in all cases tested, membranes prepared from animals subjected to chronic treatment with amitriptyline or iprindole showed similar changes in the Gs-mediated activation of adenylyl cyclase. Acute treatments produced effects similar to controls, and liver and kidney membranes from animals receiving chronic treatment showed no changes in adenylyl cyclase despite the marked changes seen in brain. These results suggest that chronic administration of ECS enhances coupling between Gs and adenylyl cyclase enzyme and modifies interactions between Gs and Gi.     

Multiple defects occur in the guanine nucleotide regulatory protein system in liver plasma membranes of obese (fa/fa) but not lean (Fa/Fa) Zucker rats: loss of functional Gi and abnormal Gs function

Hepatocyte membranes from both lean and obese Zucker rats exhibited adenylate cyclase activity that could be stimulated by glucagon, forskolin, NaF and elevated concentrations of p[NH]ppG. In membranes from lean animals, functional Gi was detected by the ability of low concentrations of p[NH]ppG to inhibit forskolin-activated adenylate cyclase. This activity was abolished by treatment of hepatocytes with either pertussis toxin or the phorbol ester TPA, prior to making membranes for assay of adenylate cyclase activity. In hepatocyte membranes from obese animals no functional Gi activity was detected. Quantitative immunoblotting, using an antibody able to detect the alpha subunit of Gi, showed that hepatocyte plasma membranes from both lean and obese Zucker rats had similar amounts of Gi-alpha subunit. This was 6.2 pmol/mg plasma membrane for lean and 6.5 pmol/mg plasma membrane for obese animals. Using thiol pre-activated pertussis toxin and [32P]-NAD+, similar degrees of labelling of the 40 kDa alpha subunit of Gi were found using plasma membranes of both lean and obese Zucker rats. We suggest that liver plasma membranes from obese Zucker rats express an inactive Gi alpha subunit. Thus lesions in liver Gi functioning are seen in insulin-resistant obese rats and in alloxan- and streptozotocin-induced diabetic rats which also show resistance as regards the acute actions of insulin. Liver plasma membranes of obese animals also showed an impairment in the coupling of glucagon receptors to Gs-controlled adenylate cyclase, with the Kd values for activation by glucagon being 17.3 and 126 nM for lean and obese animals respectively. Membranes from obese animals also showed a reduced ability for high concentration of p[NH]ppG to activate adenylate cyclase. The use of [32P]-NAD+ and thiol-preactivated cholera toxin to label the 43 kDa and 52 kDa forms of the alpha-subunit of Gs showed that a reduced labelling occurred using liver plasma membranes from obese animals. It is suggested that abnormalities in the levels of expression of primarily the 52 kDa form of alpha-Gs may give rise to the abnormal coupling between glucagon receptors and adenylate cyclase in liver membranes from obese (fa/fa) Zucker rats.     

Gs alpha mediates epidermal growth factor-elicited stimulation of rat cardiac adenylate cyclase

In an earlier study we demonstrated that epidermal growth factor (EGF) increases the cellular accumulation of cAMP in perfused rat hearts by stimulating the cardiac adenylate cyclase via a stimulatory GTP-binding protein (Nair, B. G., Rashed, H. M., and Patel, T. B. (1989) Biochem. J. 264, 563-571). Employing antiserum, CS1, generated against a synthetic decapeptide RMHLRQYELL representing the carboxyl terminus of Gs alpha, the involvement of Gs in mediating the effects of EGF on cardiac adenylate cyclase was further investigated. The CS1 antiserum specifically recognized two forms, (52 and 40 kDa) of Gs alpha in rat cardiac membranes; the 52 kDa being the predominant species. In functional assays of adenylate cyclase activity, the CS1 antiserum did not alter either aluminum fluoride- or forskolin-stimulated adenylate cyclase activity. Similarly, basal adenylate cyclase activity in the absence of guanyl-5'-yl imidodiphosphate (Gpp(NH)p) was also not altered by the CS1 antiserum. However, as compared with controls performed in the presence of non-immune serum, preincubation of cardiac membranes with the CS1 antiserum resulted in a concentration-dependent inhibition of Gpp(NH)p-, isoproterenol-, and EGF-stimulated activities. In experiments which monitored Gi function as the ability of different G(pp)NHp, (-)N6-(R-phenylisopropyl)adenosine and carbachol to inhibit forskolin-stimulated adenylate cyclase, CS1 antiserum by inhibiting Gs, increased the apparent activity of Gi. Overall, our data demonstrate that the CS1 antiserum can specifically inhibit Gs function and therefore the stimulation of adenylate cyclase by agonists whose actions are mediated by Gs. In this respect, the data presented here demonstrate that Gs is the G-protein involved in mediating EGF-elicited stimulation of cardiac adenylate cyclase. Additionally, the finding that CS1 antiserum can overcome the effects of Gpp(NH)p on Gs, but not Gi, suggests that the carboxyl-terminal region of Gs alpha is important in the interactions with GTP or its analogs.     

Supersensitivity of beta-adrenoceptor coupled adenylate cyclase in pulmonary tissue of the spontaneously hypertensive rat

Basal adenylate cyclase activity was similar in plasma membranes prepared from the lungs of 12 week old spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). However, sensitivity to Gpp[NH]p, isoproterenol plus GTP or Gpp[NH]p was significantly greater in the SHR. Beta-receptor density measured by [3H]DHA binding was unaltered. The dissociation constant, Kd, revealed a significantly greater binding affinity of the radioligand in the SHR (6.23 +/- 0.45 nM) compared with the WKY (8.53 +/- 0.82 nM). Activity of Gs was assessed by complementing S49 cyc- acceptor membranes with lung cholate extract. Basal activity of the reconstituted system was decreased 43% in the SHR. However, sensitivity to NaF, Gpp[NH]p, and isoproterenol plus Gpp[NH]p was significantly elevated. These data suggest that desensitization of the adenylate cyclase complex is not a generalized response to chronic hypertension. A tissue specific increase in sympathetic drive appears to be responsible for the lowered concentration of cardiac beta-adrenoceptors in the SHR. In contrast, both indirect and direct evidence indicate an enhanced functional sensitivity of pulmonary Gs in the hypertensive rats.     

Determination of G-protein levels, ADP-ribosylation by cholera and pertussis toxins and the regulation of adenylyl cyclase activity in liver plasma membranes from lean and genetically diabetic (db/db) mice

Liver plasma membranes prepared from genetically diabetic (db/db) mice expressed levels of Gi alpha-2, Gi alpha-3 and G-protein beta-subunits that were reduced by some 75, 63 and 73% compared with levels seen in membranes from lean animals. In contrast, there were no significant differences in the expression of the 42 and 45 kDa forms of Gs alpha-subunits. Pertussis toxin-catalysed ADP-ribosylation of membranes from lean animals identified a single 41 kDa band whose labelling was reduced by some 86% in membranes from diabetic animals. Cholera toxin-catalysed ADP-ribosylation identified two forms of Gs alpha-subunits whose labelling was about 4-fold greater in membranes from diabetic animals compared with those from lean animals. Maximal stimulations of adenylyl cyclase activity by forskolin (100 microM), GTP (100 microM), p[NH]ppG (100 microM), NaF (10 mM) and glucagon (10 microM) were similar in membranes from lean and diabetic animals, whereas stimulation by isoprenaline (100 microM) was lower by about 22%. Lower concentrations (EC50-60 nM) of p[NH]ppG were needed to activate adenylyl cyclase in membranes from diabetic animals compared to those from lean animals (EC50-158 nM). As well as causing activation, p[NH]ppG was capable of eliciting a pertussis toxin-sensitive inhibitory effect upon forskolin-stimulated adenylyl cyclase activity in membranes from both lean and diabetic animals. However, maximal inhibition of adenylyl cyclase activity in membranes from diabetic animals was reduced to around 60% of that found using membranes from lean animals. Pertussis toxin-treatment in vivo enhanced maximal stimulation of adenylyl cyclase by glucagon, isoprenaline and p[NH]ppG through a process suggested to be mediated by the abolition of functional Gi activity. The lower levels of expression of G-protein beta-subunits, in membranes from diabetic compared with lean animals, is suggested to perturb the equilibria between holomeric and dissociated G-protein subunits. We suggest that this may explain both the enhanced sensitivity of adenylyl cyclase to stimulation by p[NH]ppG in membranes from diabetic animals and the altered ability of pertussis and cholera toxins to catalyse the ADP-ribosylation of G-proteins in membranes from these two animals.     

Coupling of adrenal medullary opioid receptors to islet-activating protein-sensitive GTP-binding proteins

Possible coupling of bovine adrenal medullary opioid receptors to islet-activating protein (IAP, pertussis toxin)-sensitive GTP-binding proteins was investigated by studying effects of guanyl-5'-yl imidodiphosphate (Gpp(NH)p) and IAP treatment of membranes on opioid binding. Gpp(NH)p inhibited [3H]D-Ala2-D-Leu5-enkephalin ([3H]DADLE) binding by increasing the dissociation constant of [3H]DADLE and membranes, and enhanced slightly [3H]diprenorphine binding. IAP treatment of membranes reduced [3H]DADLE binding and abolished almost completely the Gpp(NH)p inhibition of [3H]DADLE binding. Treatment of membranes with IAP and [32P]NAD resulted in radio-labeling of membrane proteins of approximately 39,000 dalton. DADLE inhibited adenylate cyclase activity in rat brain caudate nucleus. However, DADLE, beta-endorphin, levorphanol and dynorphin A(1-13) did not show any significant inhibitory action on bovine adrenal medullary adenylate cyclase activity. These results suggest that bovine adrenal medullary opioid (DADLE) receptors are linked to IAP-sensitive GTP-binding proteins which are not directly coupled to adenylate cyclase.     

Opiate-Dependent Changes in the Sensitivity of Adenylate Cyclase to Stimulatory Agonists and 5''-Guanylylimidodiphosphate Are Independent of G Protein Abundance and Eukaryotic ADP-Ribosyltransferase Activity in NG108-15 Cells

NG108-15 cells were exposed in culture to 1 microM [D-Ala2,D-Leu5]enkaphalin (DADLE) for 17 h. This treatment increased the maximum iloprost- and 5'-(N-ethylcarboxamido)adenosine-dependent activation of adenylate cyclase, as well as basal enzyme activity. In addition, there was an increase in the capacity of 5'-guanylylimidodiphosphate [Gpp(NH)p] to inhibit adenylate cyclase activity by direct interaction with the alpha-subunit of the Gi regulatory protein. A similar effect was observed if the cells were exposed to 10 microM carbachol. These treatments of NG108-15 cells did not alter the capacity of NaF to activate adenylate cyclase by direct interaction with Gs alpha. Exposure of NG108-15 cells to DADLE alone or DADLE plus carbachol had no effect on the capacity of pertussis toxin to ADP-ribosylate membrane proteins in these cells; neither was there any change in the activity of eukaryotic ADP-ribosyltransferase expressed in these cells. Under these conditions, the endogenous enzyme did not label any protein with a molecular mass similar to Gi alpha, 41 kDa. Treatment of the cells with DADLE or carbachol had no effect on the abundance of Gs alpha, Gi alpha, or G beta. The underlying mechanism for the changes in agonist-dependent stimulatory responses or Gpp(NH)p-dependent inhibition of adenylate cyclase remains obscure, but appears not to be mediated by eukaryotic ADP-ribosyltransferase activity or a change in the abundance of G proteins known to regulate adenylate cyclase.     

In situ binding of a photo-affinity GTP analog to synaptic membrane G-proteins. Distribution of bound GTP analog reflects the status of adenylate cyclase

Regulation of synaptic membrane adenylate cyclase is likely to involve interaction between neurotransmitter receptors, G-proteins and the adenylate cyclase catalytic unit as well as several other membrane proteins and lipids. Despite intensive study of this system, regulation of guanine nucleotide binding by the G-proteins which stimulate [Gs] or inhibit [Gi] adenylate cyclase has been examined only when those proteins have been purified and removed from the influence of the membrane environment. The hydrolysis-resistant photoaffinity GTP-analog, P3-(4-azidoanilido)-P1 5'-GTP (AAGTP) is able to bind specifically to the G-proteins in rat cerebral cortex synaptic membranes and, in this study, we have used this probe to examine the specificity and selectivity of guanine nucleotide binding to each G-protein without removing those proteins from the synaptic membrane. Marked differences were noted between guanine nucleotide binding data obtained with detergent-soluble G-proteins and data from this in situ approach. In these studies it was found that the affinity of the G-proteins binding AAGTP correlated well with the expression of adenylate cyclase activity, the affinity of both forms of Gs increasing under conditions favoring the stimulation of that enzyme.     

Photoaffinity Identification of Colchicine-Solubilized Regulatory Subunit from Rat Brain Adenylate Cyclase

Five GTP binding proteins in rat cerebral cortex synaptic membranes were identified by photoaffinity labelling with [3H] or [32P](P3-azido-anilido)-P1-5' GTP (AAGTP). When AAGTP-treated membranes were incubated with colchicine or vinblastine and subsequently washed, a single AAGTP-labelled protein of 42 kD was released into the supernatant. About 30% of the total labelled 42-kD protein was released into supernatants from membranes pretreated with colchicine or vinblastine compared with 15% released from control membranes. The amount of adenylate cyclase regulatory subunit (G unit) remaining in these membranes was assessed with reconstitution studies after inactivating the adenylate cyclase catalytic moiety with N-ethylmaleimide (NEM). Forty to fifty percent of functional G units were lost from membranes treated with colchicine prior to washing. This 40-50% loss of functional G unit after colchicine treatment corresponds to the previously observed 42% loss of NaF and guanylyl-5'-imidodiphosphate [Gpp(NH)p]-activated adenylate cyclase. Release of the AAGTP-labelled 42-kD protein from colchicine-treated synaptic membranes is double that from lumicolchicine-treated membranes. This colchicine-mediated release of 42-kD protein correlates with a doubling of functional G unit released from synaptic membranes after colchicine treatment. These findings suggest multiple populations of the G unit within the synaptic plasma membrane, some of which may interact with cytoskeletal components.     

Effects of pertussis toxin-catalyzed ADP-ribosylation on interactions of transducin and the inhibitory GTP-binding protein of adenylate cyclase with guanyl nucleotides

Release of bound [3H]Gpp(NH)p from NG108-15 cell membranes was induced by carbamylcholine, enkephalinamide, and norepinephrine, all of which inhibit adenylate cyclase. Release was blocked by antagonist, was greater with multiple agonists than with one, and required guanyl nucleotides. With membranes from pertussis toxin-treated cells, both total [3H] Gpp(NH)p binding and agonist-induced [3H]Gpp(NH)p release was decreased. ADP-ribosylation by toxin of transducin, the retinal GTP-binding protein which is similar in structure and function to that in cyclase, decreased [3H]Gpp(NH)p binding. Thus, the inability to demonstrate agonist-induced [3H]Gpp(NH)p release from toxin-treated NG108-15 membranes may result in part from absence of bound [3H]Gpp(NH)p.     

Adenylate cyclase of platelets from spontaneously hypertensive rats: reduction of the inhibition by GTP

We have compared the effects of Gpp[NH]p on adenylate cyclase activity of platelet membranes in SHR and WKY rats. In the presence of 50 microM forskolin, low concentrations of Gpp[NH]p (0.01 to 0.3 microM) inhibited the enzyme activity in both strains, but the maximal level of inhibition was significantly lower in SHR (- 20%). In the absence of forskolin, 0.1 microM Gpp[NH]p was inhibitory only in WKY and the adenylate cyclase activity was greater in hypertensive rats at this nucleotide concentration. Increasing Gpp[NH]p from 0.1 to 3 microM induced the same increase of enzyme activity in both strains. In SHR, GTP itself induced a lower inhibition of the enzyme stimulated by 50 microM forskolin or 0.1 microM prostaglandin E1. These results suggest that the modulatory effect of the guanine nucleotide inhibitory protein on adenylate cyclase may be reduced in platelets from SHR.     

The alpha 2B adrenergic receptor of undifferentiated neuroblastoma x glioma hybrid NG108-15 cells, interacts directly with the guanine nucleotide binding protein, Gi2

In membranes of undifferentiated neuroblastoma x glioma hybrid cell line NG108-15, the apparent specific binding of [3H]yohimbine measured in the presence of 1 microM noradrenaline, was increased substantially by the presence of the poorly hydrolysed analogue of GTP, guanylyl-imidodiphosphate (Gpp[NH]p) or by preincubation of membranes with antibodies against the C-terminal decapeptide of the alpha subunit of the G-protein Gi2. Such an effect was not produced by antibodies against the equivalent region of Go alpha Gi3 alpha or Gs alpha or from non-immune serum. By contrast, total specific binding of [3H]yohimbine was not modified by co-incubation with Gpp[NH]p or by preincubation with the antibodies from any of the anti-G protein antisera. These results demonstrate a direct interaction of the alpha 2B adrenergic receptor of NG108-15 cells with Gi2.     

Interaction of the Inhibitory GTP Regulatory Component with Soluble Cerebral Cortical Adenylate Cyclase

Functional interaction of the inhibitory GTP regulatory component (Ni) with the adenylate cyclase catalytic subunit has not previously been demonstrated after detergent solubilization. The present report describes a sodium cholate-solubilized preparation of rat cerebral cortical membrane adenylate cyclase that retains guanine nucleotide-mediated inhibition of activity. Methods of membrane preparation, cholate extraction, and assay conditions were manipulated such that guanosine-5'-(beta-gamma-imido)triphosphate [Gpp(NH)p] inhibited basal activity 40-60%. The rank order of potency among various GTP analogs was similar in cholate extracts and in membranes: guanosine-5'-0-(3-thiotriphosphate) greater than Gpp(NH)p greater than GTP. Inclusion of 0.1 mM EGTA reduced basal activity 70-90% and abolished Gpp(NH)p inhibition of basal activity in both membranes and cholate extracts. Forskolin-stimulated activity was also inhibited by Gpp(NH)p. Treatment of either membranes or cholate extracts with N-ethylmaleimide abolished Gpp(NH)p inhibition. Gel filtration of the cholate extract over a Sepharose 6B column in 0.1% Lubrol PX partially resolved the adenylate cyclase components. However, Gpp(NH)p inhibition of basal activity (60% of the control) was maintained in select column fractions. Sucrose gradient centrifugation totally resolved the catalytic subunit from both functional Ni and stimulatory GTP regulatory component (Ns) activities. The sedimentation of functional Ni activity was detected by assaying the ability of sucrose gradient fractions to confer Gpp(NH)p inhibition of the resolved catalytic activity. Labeling of gradient or column fractions with pertussis toxin and [32P]NAD revealed that both the 39,000- and 41,000-dalton substrates comigrated with the functional Ni activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lymphocyte adenylate cyclase and human aging

Adenylate cyclase activity was determined by enzymatic conversion of [32P]ATP to [32P]cAMP using peripheral lymphocytes freshly isolated from human subjects. The lymphocyte enzyme was stimulated by the potent beta-adrenergic catecholamine agonist isoproterenol and by the nonhydrolyzable GTP-analog Gpp[NH]p. The two activators had a synergistic effect, and agonist-dependent enzyme activity followed simple Michaelis-Menten kinetics with respect to isoproterenol in the presence but not in the absence of Gpp[NH]p. Cyclic AMP production by intact lymphocytes, determined by protein binding assay, also followed simple Michaelis-Menten kinetics with respect to isoproterenol. Kact of isoproterenol was the same in intact cells and the broken cell assay in the presence of Gpp[NH]p, suggesting the indispensable role the GTP-binding coupling factors play in the intact lymphocyte. In 31 human subjects between the age of 21 and 103, adenylate cyclase activity in the presence of isoproterenol, Gpp[NH]p, or isoproterenol in the presence of Gpp[NH]p decreased with the increasing age of the subject. The sensitivity of the enzyme to stimulation by isoproterenol, defined as the Kact and determined in the presence of Gpp[NH]p, was the same in lymphocytes from young (less than 45 years) or elderly (greater than 75 years) subjects. These results suggest a deficiency in the lymphocyte adenylate cyclase system distal to the beta-adrenergic catecholamine receptor could account for deterioration of cAMP-mediated components of the immune response which occur with age.     

Binding of [3H]forskolin to human platelet membranes. Regulation by guanyl-5'-yl imidodiphosphate, NaF, and prostaglandins E1 and D2

[3H]Forskolin binds to human platelet membranes in the presence of 5 mM MgCl2 with a Bmax of 125 fmol/mg of protein and a Kd of 20 nM. The Bmax for [3H]forskolin binding is increased to 455 and 425 fmol/mg of protein in the presence of 100 microM guanyl-5'-yl imidodiphosphate (Gpp(NH)p) and 10 mM NaF, respectively. The increase in the Bmax for [3H]forskolin in the presence of Gpp(NH)p or NaF is not observed in the absence of MgCl2. The EC50 values for the increase in the number of binding sites for [3H]forskolin by Gpp(NH)p and NaF are 600 nM and 4 mM, respectively. The EC50 value for Gpp(NH)p to increase the number of [3H]forskolin binding sites is reduced to 35 mM and 150 nM in the presence of 50 microM PGE1 or PGD2, respectively. The increase in the number of [3H]forskolin binding sites observed in the presence of NaF is unaffected by prostaglandins. The binding of [3H]forskolin to membranes that are preincubated with Gpp(NH)p for 120 min or assayed in the presence of PGE1 reaches equilibrium within 15 min. In contrast, a slow linear increase in [3H]forskolin binding is observed over a period of 60 min when Gpp(NH)p and [3H]forskolin are added simultaneously to membranes. A slow linear increase in adenylate cyclase activity is also observed as a result of preincubating membranes with Gpp(NH)p. In human platelet membranes, agents that activate adenylate cyclase via the guanine nucleotide stimulatory protein (Ns) increase the number of binding sites for [3H]forskolin in a magnesium-dependent manner. This is consistent with the high affinity binding sites for [3H]forskolin being associated with the formation of an activated complex of the Ns protein and adenylate cyclase. This state of the adenylate cyclase may be representative of that formed by a synergistic combination of hormones and forskolin.     

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.     

Possible Involvement of Pertussis Toxin Substrates (Gi, Go) in Desipramine-Induced Refractoriness of Adenylate Cyclase in Cerebral Cortices of Rats

To evaluate the efficiency of coupling between beta-receptor and adenylate cyclase catalyst via a GTP-binding protein, Gs, in the brain membrane two parameters were employed: a beta-agonist-induced increase in the membrane GTP-dependent adenylate cyclase activity and a beta-agonist-induced shortening of the lag time preceding the onset of the steady-state activation by guanyl-5'-yl-beta-gamma-imidodiphosphate [Gpp(NH)p] of the membrane cyclase. Both parameters showed lower values in membranes from desipramine-treated rats compared with untreated rats. Thus, coupling of beta-adrenergic receptors to adenylate cyclase in the brain membrane was impaired by the desipramine treatment. Rats once injected intraventricularly with islet-activating protein (IAP), pertussis toxin, were subjected to desipramine treatment, for the purpose of studying effects of another kind of the GTP-binding protein (Gi), which loses its function as a signal transducer on being ADP-ribosylated selectively by the toxin. IAP treatment did not impair the beta-receptor coupling by itself, since neither of the above two parameters for the coupling were reduced by IAP treatment. Moreover, the first parameter was normalized, though the second one was not, by superimposition of the IAP treatment upon the desipramine-treated rats. It seems likely, therefore, that Gi interacts with a Gs-adenylate cyclase coupling in an inhibitory fashion in brain membranes. The desensitization might be overcome when the inhibitory interaction of Gi on the subsequent process is attenuated by IAP treatment.     

GTP-binding proteins and adenylate cyclase activity in v-Ki-ras transformed NIH/3T3 fibroblast cells

To identify the role of ras oncogene and p21 in the coupling mechanism of GTP-binding proteins to adenylate cyclase, we used v-Ki-ras transformed NIH/3T3 fibroblast cells. In the previous study, we investigated that NaF, cholera toxin and forskolin remarkably enhanced the adenylate cyclase activity in transformed cells compared to normal NIH/3T3 cells. In the present study, adenylate cyclase was more enhanced by GTP gamma S in transformed cells than in normal cells. It was considered that p21 plays enhancing role in coupling of GTP-binding proteins to adenylate cyclase. Further, as measured by the degree of [32P] ADP-ribosylation of GTP-binding proteins by cholera toxin and pertussis toxin respectively, the amount of Gs (46 kDa) was almost equal in both cells, while the amount of Gi (41 kDa) in transformant was about one third of that in normal cells. This difference seems to be reflected in either the biological situations or the quantities of Gi. Our data suggest that v-Ki-ras transformation resulted in the decrease of Gi protein so that the inhibitory regulation on adenylate cyclase relatively becomes low and then stimulatory influence of Gs seems to be enhanced.