Sweet tastants stimulate adenylate cyclase coupled to GTP-binding protein in rat tongue membranes.
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ABSTRACT:
SUBMITTER: Striem BJ
PROVIDER: S-EPMC1138634 | biostudies-other | 1989 May
REPOSITORIES: biostudies-other
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Project description:In isolated perfused rat hearts, epidermal growth factor (EGF; 15 nM) increased cellular cyclic AMP (cAMP) content by 9.5-fold. In rat cardiac membranes, EGF also stimulated adenylate cyclase activity in a dose-dependent manner, with maximal stimulation (35% above control) being observed at 10 nM-EGF. Half-maximal stimulation of adenylate cyclase was observed at 40 pM-EGF. Although the beta-adrenergic-receptor antagonist propranolol markedly attenuated the isoprenaline-mediated increase in cAMP content of perfused hearts and stimulation of adenylate cyclase activity, it did not alter the ability of EGF to elevate tissue cAMP content and stimulate adenylate cyclase. The involvement of a guanine-nucleotide-binding protein (G-protein) in the activation of adenylate cyclase by EGF was indicated by the following evidence. First, the EGF-mediated stimulation of adenylate cyclase required the presence of the non-hydrolysable GTP analogue, guanyl-5'-yl-imidodiphosphate (p[NH]ppG). Maximal stimulation was observed in the presence of 10 microM-p[NH]ppG. Secondly, in the presence of 10 microM-p[NH]ppG, the stable GDP analogue guanosine 5'-[beta-thio]diphosphate at a concentration of 10 microM blocked the stimulation of the adenylate cyclase by 1 nM- and 10 nM-EGF. Third, NaF + AlCl3-stimulated adenylate cyclase activity was not altered by EGF. The ability of EGF to stimulate adenylate cyclase was not affected by pertussis-toxin treatment of cardiac membranes. However, in cholera-toxin-treated cardiac membranes, when the adenylate cyclase activity was stimulated by 2-fold, EGF was ineffective. Finally, PMA by itself did not alter the activity of cardiac adenylate cyclase, but abolished the EGF-mediated stimulation of this enzyme activity. The experimental evidence in the present paper demonstrates, for the first time, that EGF stimulates adenylate cyclase in rat cardiac membranes through a stimulatory GTP-binding regulatory protein, and this effect is manifested in elevated cellular cAMP levels in perfused hearts exposed to EGF.
| S-EPMC1133616 | biostudies-other
Project description:1. Synaptosomal membranes were isolated from rats made hypothyroid by treatment with propylthiouracil and a low iodine diet. 2. When assayed in the presence of 100 mM-Na+, inhibition of forskolin-stimulated adenylate cyclase by GTP was enhanced in membranes from hypothyroid animals. 3. Hypothyroidism also enhanced inhibition of adenylate cyclase by phenylisopropyladenosine (with 100 mM-Na+ and 10 microM-GTP present). 4. Hypothyroidism did not increase binding of the A1 adenosine receptor agonist phenylisopropyladenosine to synaptosomal membranes; rather, the maximum binding was slightly decreased without any change in the KD. 5. The effect of GTP in modifying the displacement of the antagonist [3H]diethylphenylxanthine from synaptosomal membranes by unlabelled phenylisopropyladenosine was more pronounced in the hypothyroid state. 6. These findings are consistent with hypothyroidism causing modification of the brain adenylate cyclase system at the level of the coupling protein Gi.
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Project description:1. Renal tubular membranes from rat kidneys were prepared, and adenylate cyclase activity was measured under basal conditions, after stimulation by NaF or salmon calcitonin. Apparent Km value of the enzyme for hormone-linked receptor was close to 1 x 10(-8) M. 2. The system was sensitive to temperature and pH. pH was found to act both on affinity for salmon calcitonin-linked receptor and maximum stimulation, suggesting an effect of pH on hormone-receptor binding and on a subsequent step. 3. KCl was without effect areas whereas CoCl and CaCl2 above 100 muM and MnCl2 above 1 muM inhibited F- -and salmon calcitonin-sensitive adenylate cyclase activities. The Ca2+ inhibition of the response reflected a fall in maximum stimulation and not a loss of affinity of salmon calcitonin-linked receptor for the enzyme. 4. The measurement of salmon calcitonin-sensitive adenylate cyclase activity as a function of ATP concentration showed that the hormone increases the maximum velocity of the adenylate cyclase. GTP, ITP and XTP at 200 muM did not modify basal, salmon calcitonin- and parathyroid hormone-sensitive adenylate cyclase activities. 5. Basal, salmon calcitonin- and F- -sensitive adenylate cyclase activities decreased at Mg2+ concentrations below 10 mM. High concentrations of Mg2+ (100 mM) led to an inhibition of the F- -stimulated enzyme. 6. Salmon calcitonin-linked receptor had a greater affinity for adenylate cyclase than human or porcine calcitonin-linked receptors. There was no additive effect of these three calcitonin peptides whereas parathyroid hormone added to salmon calcitonin increased adenylate cyclase activity, thus showing that both hormones bound to different membrane receptors. Human calcitonin fragments had no effect on adenylate cyclase activity. 7. Salmon calcitonin-stimulated adenylate cyclase activity decreased with the preincubation time. This was due to progressive degradation of the hormone and not to the rate of binding to membrane receptors.
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Project description:Adenylate cyclase activity in platelet membrane preparations was measured in the presence of prostaglandin E1 (PGE1), GTP and a non-hydrolysable analogue of GDP, guanosine 5'-[beta-thio]diphosphate (GDP[beta S]). A dose-dependent inhibition of adenylate cyclase by GDP[beta S] was observed that could be reversed either by adding increased amounts of GTP or of PGE1.
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Project description:Adenylate cyclase toxin (ACT) is one of the principal virulence factors secreted by the whooping cough causative bacterium Bordetella pertussis, and it has a critical role in colonization of the respiratory tract and establishment of the disease. ACT targets phagocytes via binding to the CD11b/CD18 integrin and delivers its N-terminal adenylate cyclase (AC) domain directly to the cell cytosol, where it catalyzes unregulated conversion of cytosolic ATP into cAMP upon activation by binding to cellular calmodulin. High cAMP levels disrupt bactericidal functions of the immune cells, ultimately leading to cell death. In spite of its relevance in the ACT biology, the mechanism by which its ?400 amino acid-long AC domain is transported through the target plasma membrane, and is released into the target cytosol, remains enigmatic. This article is devoted to refresh our knowledge on the mechanism of AC translocation across biological membranes. Two models, the so-called "two-step model" and the recently-proposed "toroidal pore model", will be considered.
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Project description:Binding assays and assays of activation of adenylate cyclase with the agonists 5'-N-ethylcarboxyamidoadenosine (NECA) and CGS21680 have been used to compare adenosine receptors in rat pheochromocytoma PC12 cells and in rat striatum. The [3H]NECA binding showed two components, whereas [3H]CGS21680 bound to one component in both tissues. The Kd value for the high affinity site labeled with [3H]NECA in PC12 cell membranes (2.3 nM) was lower than that in striatum (6.5 nM). The [3H]CGS21680 binding site showed a Kd value of 6.7 nM and 11.3 nM in PC12 cells and striatum, respectively. In the presence of GTP the KD values of [3H]NECA and [3H]CGS21680 for the high affinity site were increased severalfold, whereas the low affinity sites for [3H]NECA were no longer detected with filtration assays. A comparison of the ability of a series of agonists and antagonists to inhibit high affinity binding of [3H]NECA to A2 receptors in PC12 cell and striatal membranes indicated that agonists had higher affinities and antagonists had lower affinities in PC12 cells, compared with affinities in striatal membranes. Analysis of activation of adenylate cyclase in PC12 cell membranes suggested that the dose-dependent stimulation by NECA involved two components, whereas CGS21680 stimulated via one component. The maximal stimulation by NECA significantly exceeded that caused by CGS21680. In intact PC12 cells, NECA caused a greater accumulation of AMP than did CGS21680, as was the case in membranes. In striatal membranes, NECA and CGS21680 showed similar maximal stimulations of adenylate cyclase. Both NECA and CGS21680 were more potent in PC12 cell membranes than in striatal membranes, in agreement with binding data. However, in contrast to binding data, antagonists were not less potent versus stimulation of adenylate cyclase by NECA or CGS21680 in PC12 cell membranes, compared with striatal membranes. In toto, the results suggest that A2A receptors in striatum are virtually identical to the A2A receptors in PC12 cells. But, in addition to an A2A receptor, it appears that a lower affinity functional receptor, probably an A2B receptor, is present in PC12 cells and PC12 cell membranes, whereas such a functional low affinity receptor is not detectable in striatal membrane.
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Project description:The direct effects of pituitary adenylate cyclase-activating polypeptides (PACAP) on sympathetic neurons were investigated using rat superior cervical ganglion neurons. Electrophysiological and pharmacological analyses were used to evaluate PACAP modulation of sympathetic neuron membrane potentials and to investigate potential ionic and intracellular signaling mechanisms mediating the responses. More than 90% of the sympathetic neurons were depolarized by the PACAP peptides even when stimulated release was blocked, indicating that the PACAP peptides elicited primary responses in the postganglionic neurons. The response profile was consistent for activation of PACAP-selective PAC(1) receptors: nanomolar concentrations of PACAP27 and PACAP38 were required to stimulate depolarization, whereas vasoactive intestinal peptide failed to evoke any response. Furthermore, depolarizations elicited by PACAP27 were reduced by the PAC(1) receptor antagonist PACAP(6-38). Both sodium influx and inhibition of a potassium current contributed to the peptide-induced depolarizations. Activation of neither pertussis toxin- nor cholera toxin-sensitive G-proteins was required for generation of the depolarizations. cAMP and diacylglycerol production and activation of protein kinase A or protein kinase C also were not requisite for the responses. By contrast, phospholipase C (PLC)-dependent inositol 1,4,5-triphosphate (IP(3)) synthesis was crucial to the PACAP-mediated depolarizations. Although calcium release from IP(3)-sensitive stores was not required for the PACAP-induced responses, inhibition of IP(3) receptors reduced the depolarizations. Thus, among the many signal transduction pathways coupled to the PAC(1) receptor, the PACAP-induced depolarization of sympathetic neurons appears to require activation of PLC and subsequent generation of IP(3).
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Project description:Desensitization of adenylate cyclase-coupled beta-adrenergic receptors in avian erythrocytes results in a 40-65% decrease in agonist-stimulated adenylate cyclase activity and correlates with increased phosphorylation of beta-adrenergic receptors. To assess the role of phosphorylation in desensitization, membranes from isoprenaline- and dibutyryl cyclic AMP-desensitized turkey erythrocytes were incubated with alkaline phosphatase for 30 min at 37 degrees C, pH 8.0. In both preparations alkaline phosphatase treatment significantly decreased desensitization of agonist-stimulated adenylate cyclase activity by 40-75% (P less than 0.05). Similar results were obtained after alkaline phosphatase treatment of membranes from isoprenaline- and dibutyryl cyclic AMP-desensitized duck erythrocytes. Moreover, alkaline phosphatase treatment of membranes from duck erythrocytes desensitized with 12-O-tetradecanoylphorbol 13-acetate returned agonist-stimulated adenylate cyclase activity to near control values. In all experiments, inclusion of 20 mM-sodium phosphate to inhibit alkaline phosphatase during treatment of membranes attenuated the enzyme's effect on agonist-stimulated adenylate cyclase activity. In addition, alkaline phosphatase treatment of membranes from control and isoprenaline-desensitized turkey erythrocytes increased the mobility of beta-adrenergic-receptor proteins, specifically photoaffinity-labelled with [125I]iodocyanopindolol-diazirine, on SDS/polyacrylamide-gel electrophoresis. The increased mobility of the beta-adrenergic-receptor proteins after alkaline phosphatase treatment of membranes was again inhibited by 20 mM-phosphate. These results provide additional evidence for a direct role for phosphorylation in desensitization of adenylate cyclase-coupled beta-adrenergic receptors in avian erythrocytes.
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Project description:Incubation of rat liver plasma membranes with liposomes of dioleoyl phosphatidic acid (dioleoyl-PA) led to an inhibition of adenylate cyclase activity which was more pronounced when fluoride-stimulated activity was followed than when glucagon-stimulated activity was followed. If Mn2+ (5 mM) replaced low (5 mM) [Mg2+] in adenylate cyclase assays, or if high (20 mM) [Mg2+] were employed, then the perceived inhibitory effect of phosphatidic acid was markedly reduced when the fluoride-stimulated activity was followed but was enhanced for the glucagon-stimulated activity. The inhibition of adenylate cyclase activity observed correlated with the association of dioleoyl-PA with the plasma membranes. Adenylate cyclase activity in dioleoyl-PA-treated membranes, however, responded differently to changes in [Mg2+] than did the enzyme in native liver plasma membranes. Benzyl alcohol, which increases membrane fluidity, had similar stimulatory effects on the fluoride- and glucagon-stimulated adenylate cyclase activities in both native and dioleoyl-PA-treated membranes. Incubation of the plasma membranes with phosphatidylserine also led to similar inhibitory effects on adenylate cyclase and responses to Mg2+. Arrhenius plots of both glucagon- and fluoride-stimulated adenylate cyclase activity were different in dioleoyl-PA-treated plasma membranes, compared with native membranes, with a new 'break' occurring at around 16 degrees C, indicating that dioleoyl-PA had become incorporated into the bilayer. E.s.r. analysis of dioleoyl-PA-treated plasma membranes with a nitroxide-labelled fatty acid spin probe identified a new lipid phase separation occurring at around 16 degrees C with also a lipid phase separation occurring at around 28 degrees C as in native liver plasma membranes. It is suggested that acidic phospholipids inhibit adenylate cyclase by virtue of a direct headgroup specific interaction and that this perturbation may be centred at the level of regulation of this enzyme by the stimulatory guanine nucleotide regulatory protein NS.
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Project description:Serotonin (5-HT) is involved in the control of various behaviors in Aplysia californica, including reproduction, feeding, locomotion, circadian rhythm, synaptic plasticity, and synaptic growth. The large variety of functions of 5-HT is mediated by different receptor subtypes that are coupled to different second-messenger systems. Here, we report the cloning of a cDNA coding for an Aplysia G-protein-coupled 5-HT receptor (5-HTap1). Its deduced amino acid sequence resembles those of the 5-HT1 receptor subfamily. When expressed in stable cell lines, 5-HTap1 exhibits high-affinity binding for the serotonergic radioligand [N-methyl-3H]lysergic acid diethylamide. This binding is competed by several 5-HT agonists and antagonists, and the pharmacological profile of inhibition has some similarities with those of 5-HT1 and 5-HT7 receptors. Application of 5-HT or its agonists 5-carboxamidotryptamine maleate and (+/-)-8-hydroxy-2-(di-n-propyl-amino) tetralin hydrobromide on cells transformed with 5-HTap1 produced a dose-dependent inhibition of forskolin-stimulated cAMP accumulation. 5-HTap1 is thus negatively coupled to adenylate cyclase. The production of antiserum against the 5-HTap1 receptor allowed us to examine its expression in animal tissues. The receptor protein is detected in every tissue examined, although it seems only weakly expressed in some samples. The receptor is also found in every ganglia of the nervous system, both in the sheath and in the neurons. 5-HTap1 mRNA is absent from the sheath, indicating that the protein observed there is probably located on the nerve terminals.
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