The activation of adenylate cyclase from small intestinal epithelium by cholera toxin

ADP-ribosylation of membrane proteins from rabbit small intestinal epithelium was investigated following incubation of membranes with [32P]NAD and cholera toxin. Cholera toxin catalyzes incorporation of 32P into three proteins of 40 kDA, 45 kDa and 47 kDa located in the brush-border membrane. In contrast, basal lateral membranes do not contain any protein which becomes labeled in a toxin-dependent manner when incubated with cholera toxin and [32P]NAD. The modification of membrane proteins from brush border occurred in spite of the virtual absence in these membranes of adenylate cyclase activatable either by cholera toxin, vasoactive intestinal peptide (VIP) or fluoride. The three agents activated adenylate cyclase when crude plasma membrane were used. Cholera toxin activated fivefold at 10 micrograms/ml. Vasoactive intestinal peptide activated at concentrations from 10-300 nM, the maximal stimulation being sixfold. Fluoride activated 10-fold at 10 mM. When basal lateral membranes were assayed for adenylate cyclase it was found that, with respect to the crude membranes, the specific activity of fluoride-activated enzyme was 3.3-fold higher, VIP stimulated enzyme was maintained while cholera-toxin-stimulated enzyme showed half specific activity. Moreover, while fluoride stimulated ninefold and VIP stimulated fivefold, cholera toxin only stimulated twofold at the highest concentration. The results suggest that the activation by cholera toxin of adenylate cyclase located at the basal lateral membrane requires ADPribosylation of proteins in the brush border membrane.     

Some comparative properties and localization of porcine jejunal adenylate cyclase.

Cholera toxin is thought to cause intestinal secretion by activating adenylate cyclase and increasing intracellular 3',5'-cyclic AMP concentrations in intestinal mucosa. Cholera toxin causes profuse secretion of fluid into ligated intestinal loops of both pigs and rabbits, but cholera toxin-induced increases in 3',5'-cyclic AMP concentration are much lower in the pig than in the rabbit. Porcine jejunal adenylate cyclase was examined for unusual properties which might account for a lack of 3'-5'-cyclic AMP accumulation after treatment with cholera toxin. The divalent cation requirements, the pH optimum, and the stimulation by fluoride ion were unremarkable. The Km for ATP was 0.11 mM with negative cooperativity indicated by a Hill coefficient of 0.83. Triton X-100 was inhibitory and guanosine diphosphate methylenephosphate stimulated enzyme activity. Adenylate cyclase activity was highest in the basal and lateral membrane fractions of jejunal mucosa and relatively low in brush-border preparations. Pretreatment of pig jejunum with cholera toxin caused a 30-40% activation of the crude and of the partly purified enzyme. A relatively low activation of adenylase cyclase in pig jejunal mucosa, compared with rabbit, may account for the absence of 3',5'-cyclic AMP accumulation after cholera-toxin treatment in the pig.     

Biphasic regulation of adenylate cyclase by cholera toxin in neuroblastoma x glioma hybrid cells is due to the activation and subsequent loss of the alpha subunit of the stimulatory GTP binding protein (GS)

Exposure of neuroblastoma x glioma hybrid (NG108-15) cells to low concentrations of cholera toxin produced a stimulation of both basal and forskolin-amplified adenylate cyclase activity in membranes prepared from these cells. Higher concentrations of cholera-toxin reversed this effect. Mn2+ activation of adenylate cyclase indicated that this effect was not due to a modification of the intrinsic activity of this enzyme. Cholera toxin was demonstrated to produce a concentration and time-dependent loss of GS alpha from membranes of these cells. Loss of GS alpha from membranes of these cells was preceded by its ADP-ribosylation. The effects of cholera toxin were specific for GS alpha, as no alterations in levels of the pertussis toxin-sensitive G-proteins Gi2, Gi3 and Go, were noted in parallel. Equally, no alteration in levels of G-protein beta-subunit were produced by the cholera toxin treatment. These experiments demonstrate that cholera toxin-catalysed ADP-ribosylation does not simply maintain an activated population of GS at the plasma membrane and that alterations in levels of GS at the plasma membrane can modify adenylate cyclase activity.     

Modulation of adenylate cyclase in human keratinocytes by protein kinase C

Adenylate cyclase (ATP-pyrophosphate lyase (cyclizing); EC 4.6.1.1) in the human keratinocyte cell line SCC 12F was potentiated by 12-O-tetradecanoyl-phorbol-13-acetate (TPA), phorbol-12,13-diacetate, and 1,2-dioctanoylglycerol. Keratinocytes exposed to TPA showed a 2-fold enhancement of adenylate cyclase activity when assayed in the presence of isoproterenol or GTP. The half-maximal effective concentration (EC50) for both isoproterenol and GTP were unaltered by TPA treatment of the cells. Basal adenylate cyclase activity in membranes from TPA-treated cultures was also increased 2-fold relative to activity in control membranes. Potentiation of adenylate cyclase activity was dependent on the concentration of TPA to which the keratinocytes were exposed (EC50 for TPA = 3 nM). TPA actions on adenylate cyclase were maximal after 15 min of incubation of the cells with the compound, correlating well with the time course of translocation of protein kinase C (Ca2+/phospholipid-dependent enzyme) from cytosol to membrane. The action of cholera toxin on adenylate cyclase was additive with TPA. In contrast, pertussis toxin actions on adenylate cyclase were not additive with TPA. Treatment of control cells with pertussis toxin activated adenylate cyclase 1.5-fold, whereas cells exposed to pertussis toxin for 6 h followed by TPA for 15 min showed the same 2-fold increase in adenylate cyclase activity as observed in membranes from cells exposed to TPA without prior exposure to pertussis toxin. Pertussis toxin catalyzed ADP-ribosylation was increased 2-fold in membranes from SCC 12F cells exposed to TPA, indicating an increase in the alpha beta gamma form of Gi. These data suggest that exposure of human keratinocytes to phorbol esters increases adenylate cyclase activity by a protein kinase C-mediated increase in the heterotrimeric alpha beta gamma form of Gi resulting in decreased inhibition of basal adenylate cyclase activity.     

Cholera toxin and adenylate cyclase: properties of the activated enzyme in liver plasma membranes.

Adenylate cyclase (EC 4.6.1.1) activity in mouse liver plasma membranes is increased fivefold when animals are pretreated with cholera toxin. The increase in activity is detectable within 20 min of an intravenous injection of the toxin. The response of the control and cholera-toxin-activated adenylate cyclase to hormones, GTP, and NaF is complex. GTP causes the same fold stimulation of control and toxin-activated cyclase, but glucagon and NaF remain the most potent activators of liver adenylate cyclase irrespective of whether the enzyme is activated by cholera toxin. Determination of kinetic parameters of adenylate cyclase indicates that cholera toxin, hormones, and NaF do not change the affinity of the enzyme for ATP-Mg nor do they alter the Ka for free Mg2+. High concentrations of Mg2+ inhibit adenylate cyclase that is stimulated by either cholera toxin, glucagon, or NaF. These same Mg2+ concentrations have no effect on the basal activity of the enzyme or its activity in the presence of GTP.     

Regulation of ciliary adenylate cyclase by Ca2+ in Paramecium.

In the ciliated protozoan Paramecium, Ca2+ and cyclic nucleotides are believed to act as second messengers in the regulation of the ciliary beat. Ciliary adenylate cyclase was activated 20-30-fold (half-maximal at 0.8 microM) and inhibited by higher concentrations (10-20 microM) of free Ca2+ ion. Ca2+ activation was the result of an increase in Vmax., not a change in Km for ATP. The activation by Ca2+ was seen only with Mg2+ATP as substrate; with Mn2+ATP the basal adenylate cyclase activity was 10-20-fold above that with Mg2+ATP, and there was no further activation by Ca2+. The stimulation by Ca2+ of the enzyme in cilia and ciliary membranes was blocked by the calmodulin antagonists calmidazolium (half-inhibition at 5 microM), trifluoperazine (70 microM) and W-7 (50-100 microM). When ciliary membranes (which contained most of the ciliary adenylate cyclase) were prepared in the presence of Ca2+, their adenylate cyclase was insensitive to Ca2+ in the assay. However, the inclusion of EGTA in buffers used for fractionation of cilia resulted in full retention of Ca2+-sensitivity by the ciliary membrane adenylate cyclase. The membrane-active agent saponin specifically suppressed the Ca2+-dependent adenylate cyclase without inhibiting basal activity with Mg2+ATP or Mn2+ATP. The ciliary adenylate cyclase was shown to be distinct from the Ca2+-dependent guanylate cyclase; the two activities had different kinetic parameters and different responses to added calmodulin and calmodulin antagonists. Our results suggest that Ca2+ influx through the voltage-sensitive Ca2+ channels in the ciliary membrane may influence intraciliary cyclic AMP concentrations by regulating adenylate cyclase.     

Alterations in components of adenylate cyclase associated with transformation of chicken embryo fibroblasts by Rous sarcoma virus

Regulation of adenylate cyclase coincident with transformation of chicken embryo fibroblasts by Rous sarcoma virus is manifest as a 10-50% decrease in basal, Mg2+-, and forskolin-stimulated activities; activities elicited by fluoride and guanosine 5'-O-(3-thiotriphosphate) are unaltered. The level of the catalytic component of adenylate cyclase, assessed with activated stimulatory guanine nucleotide-binding protein (Gs), increases approximately 1.5-fold. The level of the beta subunit common to Gs and the inhibitory regulatory protein assessed by enzyme-linked immunotransfer blotting, increases 2.7-fold. The isoelectric behavior of the beta subunit is unaltered. The amount of radiolabel incorporated into the alpha subunit of Gs (Mr = 45,000) upon incubation of membranes with 32P-labeled NAD and cholera toxin increases 3-fold upon transformation. Detergent extracts prepared from membranes of untransformed and transformed fibroblasts nevertheless exhibit equivalent abilities to reconstitute fluoride-stimulated activities to membranes of the cyc-variant of mouse S49 lymphoma cells. Islet-activating protein catalyzes incorporation of radiolabel from 32P-labeled NAD into 39,000- and 41,000-dalton proteins; the extent of radiolabel incorporation does not change upon transformation. Modest alterations in the isoelectric behaviors of substrates for cholera toxin and islet-activating protein occur.     

Involvement of calmodulin in the regulation of adenylate cyclase activity in guinea-pig enterocytes

The involvement of calmodulin as an activator of adenylate cyclase activity was examined in isolated guinea-pig enterocytes and in a membrane preparation. In enterocytes, which responded to prostaglandin E1, vasoactive intestinal peptide and cholera toxin with a significant increase in the rate of cAMP formation trifluoperazine, a calmodulin antagonist, completely inhibited cAMP formation. In a membrane preparation adenylate cyclase activity was stimulated 10-20-fold by the GTP analog, guanosine 5'-[beta-imido]5'-triphosphate (Gpp[NH]p). Prostaglandin E1 and vasoactive intestinal peptide enhanced cAMP formation in this system by 2-3- and 1.2-1.6-fold. respectively. Addition of 200 nM calmodulin to membranes, in which endogenous calmodulin was decreased from 1.4 microgram/mg protein to 0.5 microgram/mg protein by washing with buffer containing EGTA and EDTA, resulted in a 3-4-fold increase of adenylate cyclase activity. The absolute increment in adenylate cyclase activity caused by calmodulin (10-15 pmol cAMP/min per mg protein) was approximately the same in the absence or presence of Gpp[NH]p. The apparent Ka for Gpp[NH]p (6 . 10-7 M) was not significantly changed by the addition of calmodulin. Although endogenous calcium (approx. 10 microM) in the enzyme assay was adequate to affect stimulation by calmodulin, a maximal effect was observed at a calcium concentration of 100 microM. These findings indicate that a calmodulin-sensitive form of adenylate cyclase is present in guinea-pig enterocytes, and that stimulation of cAMP formation in the intestinal mucosa may involve a calmodulin-mediated mechanism.     

Secretion-stimulating and secretion-inhibiting hormones stimulate high-affinity pertussis-toxin-sensitive GTPases in membranes of a pituitary cell line

Different peptide hormones influence hormone secretion in pituitary cells by diverse second messenger systems. Recent data indicate that luteinizing-hormone-releasing hormone (LHRH) stimulates and somatostatin inhibits voltage-dependent Ca2+ channels of GH3 cells via pertussis-toxin-sensitive mechanisms [Rosenthal et al. (1988) EMBO J. 7, 1627-1633]. In other pituitary cell lines, somatostatin has been shown to cause a pertussis-toxin-sensitive decrease in adenylate cyclase activity, and LHRH and thyrotropin-releasing hormone (TRH) stimulate phosphoinositol lipid hydrolysis in a pertussis-toxin-independent manner. Whether stimulation of Ca2+ influx by TRH is affected by pertussis toxin is not known. In order to elucidate which of the hormone receptors interact with pertussis-toxin-sensitive and -insensitive G-proteins, we measured the effects of LHRH, somatostatin and TRH on high-affinity GTPases in membranes of GH3 cells. In control membranes, both LHRH and TRH stimulated the high-affinity GTPase by 20%, somatostatin by 25%. Maximal hormone effects were observed at a concentration of about 1 microM. Pretreatment of cells with pertussis toxin abolished pertussis-toxin-catalyzed [32P]ADP-ribosylation of 39-40-kDa proteins in subsequently prepared membranes and reduced basal GTPase activity. The toxin also reduced by more than half the increases in GTPase activity induced by LHRH and TRH; stimulation of GTPase by somatostatin was completely suppressed. Stimulation of adenylate cyclase by vasoactive intestinal peptide (VIP) was not impaired by pretreatment of cells with pertussis toxin. Somatostatin but not LHRH and TRH decreased forskolin-stimulated adenylate cyclase activity. The results suggest that the activated receptors for LHRH and TRH act via pertussis-toxin-sensitive and -insensitive G-proteins, whereas effects of somatostatin are exclusively mediated by pertussis-toxin-sensitive G-proteins.     

Influence of dietary omega-3 fatty acids on transmembrane signalling in rat submandibular salivary gland

We have previously shown the incorporation of dietary omega-3 and omega-6 fatty acids from menhaden oil and corn oil, respectively, into membrane phospholipids of submandibular salivary gland (SMSG) of rat [Alam S. Q. and Alam B. S. (1988) Arch. Oral Biol. 33, 295-299]. We now demonstrate the influence of such incorporation on the regulation of G proteins and adenylate cyclase activity. Cholera toxin ribosylated three protein peptides (Mr 42,000, 44,000 and 46,000) to different extents in the two groups. We found 4.9- and 2.6-fold higher and 0.4-fold lower ribosylation of Mr 42,000, 44,000 and 46,000 peptides, respectively, in SMSG membranes of rats fed a diet containing 10% menhaden oil (group II) compared to those fed 10% corn oil (group I). Functional distinctions between different forms of these peptides are not known. Cholera toxin also exhibited radiolabelling of three peptides in the SMSG membranes from normal or fasting rats. In these membranes inhibitory G proteins were not detected by pertussis toxin dependent ADP ribosylation or by a low concentration of guanylyl 5-imidodiphosphate (10(-8) M), which selectively activates inhibitory G proteins which inhibit forskolin stimulated activity of adenylate cyclase. In group II membranes both basal and fluoride stimulated activities of adenylate cyclase were found to be significantly higher than the corresponding values in group I (P less than 0.02). In cholera toxin dependent ribosylated membranes of group I, basal and fluoride stimulated activities of adenylate cyclase were significantly higher than those obtained in the absence of cholera toxin (P less than 0.02). Surprisingly, corresponding values were found to be lower in ribosylated membranes of group II. This could be due either to conformational changes in heavily ribosylated G proteins, which alters coupling with the catalytic subunit of adenylate cyclase, or due to dissociation of excessive inhibitory beta gamma complex from alpha beta gamma complex upon the activation of G proteins.     

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.     

Mechanism of activation of adenylate cyclase by Vibrio cholerae enterotoxin. Relations to the mode of activation by hormones.

The influence of Vibrio cholerae enterotoxin (choleragen) on the response of adenylate cyclase to hormones and GTP, and on the binding of 125I-labeled glucagon to membranes, has been examined primarily in rat adipocytes, but also in guinea pig ileal mucosa and rat liver. Incubation of fat cells with choleragen converts adenylate cyclase to a GTP-responsive state; (-)-isoproterenol has a similar effect when added directly to membranes. Choleragen also increases by two- to fivefold the apparent affinity of (-)-isoproterenol, ACTH, glucagon, and vasoactive intestinal polypeptide for the activation of adenylate cyclase. This effect on vasoactive intestinal polypeptide action is also seen with the enzyme of guinea pig ileal mucosa; the toxin-induced sensitivity to VIP may be relevant in the pathogenesis of cholera diarrhea. The apparent affinity of binding of 125I-labeled glucagon is increased about 1.5- to twofold in choleragen-treated liver and fat cell membranes. The effects of choleragen on the response of adenylate cyclase to hormones are independent of protein synthesis, and they are not simply a consequence to protracted stimulation of the enzyme in vivo or during preparation of the membranes. Activation of cyclase in rat erythrocytes by choleragen is not impaired by agents which disrupt microtubules or microfilaments, and it is still observed in cultured fibroblasts after completely suppressing protein synthesis with diphtheria toxin. Choleragen does not interact directly with hormone receptor sites. Simple occupation of the choleragen binding sites with the analog, choleragenoid, does not lead to any of the biological effects of the toxin.     

Potent and cooperative feedback inhibition of adenylate cyclase activity by calcium in pituitary-derived GH3 cells

Calcium (Ca2+) ion concentrations that are achieved intracellularly upon membrane depolarization or activation of phospholipase C stimulate adenylate cyclase via calmodulin (CaM) in brain tissue. In the present study, this range of Ca2+ concentrations produced unanticipated inhibitory effects on the plasma membrane adenylate cyclase activity of GH3 cells. Ca2+ concentrations ranging from 0.1 to 0.8 microM exerted an increasing inhibition on enzyme activity, which reached a plateau (35-45% inhibition) at around 1 microM. This inhibitory effect was highly cooperative for Ca2+ ions, but was neither enhanced nor dependent upon the addition of CaM (1 microM) to EGTA-washed membranes. The inhibition was greatly enhanced upon stimulation of the enzyme by vasoactive intestinal peptide (VIP) and/or GTP. Prior exposure of cultured cells to pertussis toxin did not affect the inhibition of plasma membrane adenylate cyclase activity by Ca2+, although in these membranes, hormonal (somatostatin) inhibition was significantly attenuated. Maximally effective concentrations of Ca2+ and somatostatin produced additive inhibitory effects on adenylate cyclase. The addition of phosphodiesterase inhibitors demonstrated that inhibitory effects of Ca2+ were not mediated by Ca2(+)-dependent stimulation of a phosphodiesterase activity. These observations provide a mechanism for the feedback inhibition by elevated intracellular Ca2+ levels on cAMP-facilitated Ca2+ entry into GH3 cells, as well as inhibitory crosstalk between Ca2(+)-mobilizing signals and adenylate cyclase activity.     

Inhibition of ADP-ribosyltransferase activity of cholera toxin by MDL 12330A and chlorpromazine

ADP-ribosylation by cholera toxin of the guanine nucleotide binding regulatory protein (Gs) of rat liver membrane adenylate cyclase was inhibited by 0.1-1 mM MDL 12330A or 0.1-1 mM chlorpromazine. Basal as well as cholera toxin activated adenylate cyclase activity in liver membranes was also inhibited by the two drugs. NAD glycohydrolase activity and self-ADP-ribosylation of cholera toxin were also inhibited by MDL 12330A and chlorpromazine. These effects of MDL 12330A and chlorpromazine may be related to their effects on cholera toxin-induced fluid secretion in vivo.     

Stimulation of intestinal adenylate cyclase by cholera toxin in malnourished rats

The stimulation of intestinal adenylate cyclase by cholera toxin (CT) was studied in normal and malnourished rats 4 to 24 hr after a 30-min incubation of intestinal loops with the toxin. Whereas in control rats the enzyme activity returned to basal levels after 12 hr of incubation, in malnourished rats the activity of the enzyme remained significantly elevated even after 24 hr of the initial incubation. Malnourished animals had a reduced turnover rate of intestinal cells as determined by thymidine kinase activity. The delayed turnover of intoxicated cells may account for continuous activation of mucosal adenylate cyclase and possibly for prolongation of diarrhea in malnutrition.     

Opiate-Inhibited Adenylate Cyclase in Rat Brain Membranes Depleted of Gs-Stimulated Adenylate Cyclase

Opiate agonists inhibit adenylate cyclase in brain membranes, but under normal conditions the maximal inhibition is small (10-15%). When rat brain membranes were preincubated at pH 4.5, washed, and then assayed for adenylate cyclase at pH 7.4, stimulation of activity by agents (fluoride, guanylyl-5'-imidodiphosphate, cholera toxin) that act through the stimulatory GTP-binding coupling protein (Gs) protein was lost. At the same time, inhibition of basal adenylate cyclase by opiate agonists was increased to a maximum of 30-40%. Opiate inhibition was maximal at low magnesium concentrations (less than 5 mM), required guanine nucleotides, and decreased the Vmax, not Km, of the enzyme. Incubation of membranes with pertussis toxin lowered the apparent affinity for agonists in inhibiting activity. The delta opioid agonists were more potent than mu agonists, and the Ke values for naloxone in blocking agonist inhibition were similar for both mu and delta agonists (50-90 nM). These results suggest that inhibition of adenylate cyclase in brain is not mediated by mu opiate receptors, but whether classic high-affinity delta and kappa receptors are involved with this enzyme cannot be confirmed by these experiments.     

The adenylate cyclase system and calcitonin secretion from perfused dog thyroid lobes

The aim of the study was to assess the involvement of the adenylate cyclase system in calcitonin (CT) secretion from thyroidal C-cells. The cAMP analogues Br-cAMP (10(-6) and 10(-4) mol/l) and DB-cAMP (10(-4) mol/l) and the activators of adenylate cyclase cholera toxin (0.1 microgram/ml and 5 micrograms/ml) and forskolin (10(-7) mol/l and 10(-5) mol/l) were infused for 6 min periods in perfused dog thyroid lobes. CT was measured in thyroid effluent by radioimmunoassay. Br-cAMP and cholera toxin did not alter basal CT secretion. DB-cAMP had a minimal stimulatory effect and forskolin 10(-5) mol/l a moderate stimulatory effect. This was much less than the effect of increasing perfusate Ca++ from 1.5 to 2.0 mmol/l. 10(-4) mol/l Br-cAMP increased the response to Ca++ with approximately 50 per cent. These results suggest that the activity of the adenylate cyclase system of the C-cells by itself is of little importance for CT secretion, but that it may have a role as modulator of the response to Ca++.     

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.     

Opioids, Noradrenaline and GTP Analogs Inhibit Cholera Toxin Activated Adenylate Cyclase in Neuroblastoma × Glioma Hybrid Cells

D-Ala2-Met5-enkephalin, morphine, and noradrenaline inhibit the adenylate cyclase in homogenates of neuroblastoma x glioma hybrid cells in a dose-dependent manner even after the enzyme has been preactivated by cholera toxin. Half-maximal inhibition and extent of inhibition are the same with native or cholera toxin-activated enzyme. The inhibition caused by opioids or noradrenaline are antagonized by naloxone or phentolamine, respectively. The effect of D-Ala2-Met5-enkephalin on cholera toxin-activated enzyme is immediate in onset and rapidly reversed by the addition of naloxone. Guanyl-5'-yl-imidodiphosphate stimulates basal activity but inhibits the enzyme activated by cholera toxin or prostaglandin E1. Stimulation occurs at a concentration of 100 microM or above, inhibition even at 0.1 microM. The inhibitory effect of the non-hydrolysable GTP analog is antagonized by GTP. Guanyl-5'-yl-methylenediphosphonate, another nonhydrolysable GTP analog, inhibits basal as well as cholera toxin-stimulated or prostaglandin E1-stimulated adenylate cyclase. Other guanine derivatives such as GDP, GMP, cyclic GMP, guanyl-5'-yl-phosphoric acid amide and guanosine have no effect under the same conditions. The results may be taken as a piece of evidence for two separate guanyl nucleotide-binding sites accompanying the adenylate cyclase in the hybrid cells and mediating, respectively, stimulation and inhibition of the enzyme by hormones.     

Inhibition of hepatic adenylate cyclase by NADH

Adenylate cyclase activity in isolated rat liver plasma membranes was inhibited by NADH in a concentration-dependent manner. Half-maximal inhibition of adenylate cyclase was observed at 120 microM concentration of NADH. The effect of NADH was specific since adenylate cyclase activity was not altered by NAD+, NADP+, NADPH, and nicotinic acid. The ability of NADH to inhibit adenylate cyclase was not altered when the enzyme was stimulated by activating the cyclase was not altered when the enzyme was stimulated by activating the Gs regulatory element with either glucagon or cholera toxin. Similarly, inhibition of Gi function by pertussis toxin treatment of membranes did not attenuate the ability of NADH to inhibit adenylate cyclase activity. Inhibition of adenylate cyclase activity to the same extent in the presence and absence of the Gpp (NH) p suggested that NADH directly affects the catalytic subunit. This notion was confirmed by the finding that NADH also inhibited solubilized adenylate cyclase in the absence of Gpp (NH)p. Kinetic analysis of the NADH-mediated inhibition suggested that NADH competes with ATP to inhibit adenylate cyclase; in the presence of NADH (1 mM) the Km for ATP was increased from 0.24 +/- 0.02 mM to 0.44 +/- 0.08 mM with no change in Vmax. This observation and the inability of high NADH concentrations to completely inhibit the enzyme suggest that NADH interacts at a site(s) on the enzyme to increase the Km for ATP by 2-fold and this inhibitory effect is overcome at high ATP concentrations.