The role of GTP in prostaglandin E1 stimulation of adenylate cyclase in platelet membranes.
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ABSTRACT:
SUBMITTER: Stein JM
PROVIDER: S-EPMC1152231 | biostudies-other | 1983 Jul
REPOSITORIES: biostudies-other
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Project description:Sucrose and other saccharides, which produce an appealing taste in rats, were found to significantly stimulate the activity of adenylate cyclase in membranes derived from the anterior-dorsal region of rat tongue. In control membranes derived from either tongue muscle or tongue non-sensory epithelium, the effect of sugars on adenylate cyclase activity was either much smaller or absent. Sucrose enhanced adenylate cyclase activity in a dose-related manner, and this activation was dependent on the presence of guanine nucleotides, suggesting the involvement of a GTP-binding protein ('G-protein'). The activation of adenylate cyclase by various mono- and di-saccharides correlated with their electrophysiological potency. Among non-sugar sweeteners, sodium saccharin activated the enzyme, whereas aspartame and neohesperidin dihydrochalcone did not, in correlation with their sweet-taste effectiveness in the rat. Sucrose activation of the enzyme was partly inhibited by Cu2+ and Zn2+, in agreement with their effect on electrophysiological sweet-taste responses. Our results are consistent with a sweet-taste transduction mechanism involving specific receptors, a guanine-nucleotide-binding protein and the cyclic AMP-generating enzyme adenylate cyclase.
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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: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:Treatment of human platelets with concentrations of benzyl alcohol up to 50 mM augmented adenylate cyclase activity when it was assayed in the basal state and when stimulated by prostaglandin E1 (PGE1), isoprenaline or NaF. Benzyl alcohol antagonized the stimulatory effect exerted on the catalytic unit of adenylate cyclase by the diterpene forskolin. Benzyl alcohol did not modify the magnitude of the inhibitory response when the catalytic unit of adenylate cyclase was inhibited by using either low concentrations of guanosine 5'-[beta gamma-imido]triphosphate, which acts selectively on the inhibitory guanine nucleotide-regulatory protein Gi, or during alpha 2-adrenoceptor occupancy, by using adrenaline (+ propranolol). Some 34% of the potent inhibitory action of adrenaline on PGE1-stimulated adenylate cyclase was obliterated in a dose-dependent fashion (concn. giving 50% inhibition = 12.5 mM) by benzyl alcohol, with the residual inhibitory action being apparently resistant to the action of benzyl alcohol at concentrations up to 50 mM. Treatment of membranes with benzyl alcohol did not lead to the release of either the alpha-subunit of Gi or G-protein subunits. The alpha 2-adrenoceptor-mediated inhibition of adenylate cyclase was abolished when assays were performed in the presence of Mn2+ rather than Mg2+ and, under such conditions, dose-effect curves for the action of benzyl alcohol on PGE1-stimulated adenylate cyclase activity were similar whether or not adrenaline (+propranolol) was present. We suggest that (i) alpha 2-adrenoceptor- and Gi-mediated inhibition of PGE1-stimulated adenylate cyclase may have two components, one of which is sensitive to inhibition by benzyl alcohol, and (ii) the Gi-mediated inhibition of forskolin-stimulated adenylate cyclase exhibits predominantly the benzyl alcohol-insensitive component.
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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:Progenitors in human vasculature expanded in-vitro were differentiated with adipogenic cocktail for 12 days, following which they were stimulated with forskolin for 7 days Microarrays were used to detail the program of differentiation of thermogenic human adipose cells
2018-01-22 | GSE65190 | GEO
Project description:Diabetes mellitus (DM) is associated with increased platelet activation and reduced platelet inhibition by clopidogrel. Prostaglandin E1 (PGE1) stimulates adenyl cyclase activity in platelets and increases cyclic AMP concentrations, which inhibit Ca(2+)release and platelet aggregation induced by P2Y1 receptor activation. PGE1 is included in the VerifyNow P2Y12 assay to suppress P2Y1 induced platelet aggregation. We hypothesized that diabetes mellitus may be associated with altered response to PGE1 in subjects treated with clopidogrel. Subjects with established coronary artery disease who were taking clopidogrel 75?mg daily and aspirin for >14 days were enrolled (n?=?96). Diabetic (n?=?34) were compared with non-diabetic subjects (n?=?62). VerifyNow P2Y12 assay and light transmittance aggregometry (LTA) were performed using ADP as agonist with and without addition of PGE1. Genomic DNA was genotyped for common cytochrome P450 (CYP) 2C19 variants using Taqman assays. Residual on-treatment platelet aggregation induced by 20?µM ADP was not significantly different between subjects with and without DM. Addition of 22?nM and 88?nM PGE1 to 20?µM ADP resulted in a significant reduction of maximal platelet aggregation (MPA). Residual LTA platelet aggregation with PGE1 and VerifyNow P2Y12 platelet reactivity were significantly higher in subjects with DM than those without DM and in carriers of CYP 2C19*2 polymorphism. We conclude that an impaired inhibitory response to PGE1 may contribute to the high platelet reactivity phenotype in subjects with DM treated with clopidogrel. Addition of PGE1 to ADP agonist platelet assays may identify subjects with blunted inhibitory response to prostaglandins and result in a higher proportion of subjects with DM being classified as non-responders.
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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.
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Project description:Pituitary adenylate cyclase-activating polypeptide (PACAP, ADCYAP1) dysregulation has been associated with multiple stress-related psychopathologies that may be related to altered hippocampal function. In coherence, PACAP- and PAC1 receptor (ADCYAP1R1)-null mice demonstrate changes in hippocampal-dependent behavioral responses, implicating the PACAPergic system function in this structure. Within the hippocampus, the dentate gyrus (DG) may play an important role in discerning the differences between similar contexts, and DG granule cells appear to both highly express PAC1 receptors and receive inputs from PACAP-expressing terminals. Here, we review the evidence from our laboratories and others that PACAP is an important regulator of activity within hippocampal circuits, particularly within the DG. These data are consistent with an increasing literature implicating PACAP circuits in stress-related pathologies such as post-traumatic stress disorder (PTSD) and implicate the hippocampus, and in particular the DG, as a critical site in which PACAP dysregulation can alter stress-related behaviors.
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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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