Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.6.1.1 (adenylate cyclase)
19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Heterologous desensitization is a term that indicates that exposure of a cell to an agonist attenuates the response of that cell to other agonists. We examined heterologous desensitization of muscarinic cholinergic receptors of pancreatic acini and characterized mechanisms that might be responsible for desensitization. Muscarinic cholinergic receptor binding was measured by using N-[3H]methscopolamine bromide ([3H]NMS). N-Methscopolamine bromide (NMS), a receptor antagonist, bound to a single class of receptors with an affinity of 0.22 +/- 0.04 nM and a capacity of 61.5 +/- 5.1 fmol/mg of protein. These parameters of NMS binding sites were not altered by an addition of cholecystokinin (CCK) octapeptide, CCK-JMV-180, vasoactive intestinal peptide, 8-bromo-cAMP, 4-bromo-A23187, thapsigargin, or 12-O-tetradecanoylphorbol-13-acetate (TPA). Analysis of competitive inhibition curve of [3H] NMS binding by carbachol showed apparently two classes of carbachol binding sites with high affinity (38.6%) and low affinity (61.4%). Simultaneous incubation of carbachol with CCK or TPA increased the relative affinity of [3H]NMS binding, and the competitive inhibition curves showed a single class of carbachol binding site. L-364,718 blocked the effect of CCK, and staurosporine blocked the effects of TPA and partially blocked the effect of CCK. CCK-JMV-180, vasoactive intestinal peptide, 8-bromo-cAMP, 4-bromo-A23187, and thapsigargin had no effects on the competitive binding. Second, the carbachol-induced sequestration of the receptors was examined. Incubation of acini with carbachol resulted in a decrease of [3H] NMS binding sites, and the addition of CCK or TPA caused an inhibition of the carbachol-induced disappearance of [3H]NMS binding sites. Finally, studies that examined the biological response of the acinar cells showed that biphasic amylase release in response to carbachol was completely suppressed by 10 nM CCK for entire range of carbachol. Taken together, these results suggest that the effect of CCK on carbachol-induced sequestration is important for the alteration of the apparent affinity of carbachol binding sites and the biological response of acinar cells to carbachol. Further, the results suggest that another factor that induces uncoupling of receptor from effector might be involved in agonist-regulated desensitization. The results, that CCK-JMV-180 or other agonists that activate the adenylate cyclase pathway did not exert these effects of CCK, suggest that protein kinase C may be one of the key factors involved in heterologous desensitization by CCK on the carbachol binding sites and the suppression of carbachol-induced amylase release.
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PMID:Agonist-regulated alteration of the affinity of pancreatic muscarinic cholinergic receptors. 769 70

In cultured vascular smooth muscle cells, interferon gamma (IFN-gamma) induced the accumulation of nitrite, a stable metabolite of nitric oxide, in a dose- and time-dependent manner. In parallel with this reaction, this cytokine increased the mRNA and protein levels of an inducible macrophage-type of nitric oxide synthase (iNOS). Forskolin, a direct activator of adenylate cyclase, or dibutyryl cAMP alone caused small increases in nitrite accumulation and iNOS mRNA and protein levels and synergistically enhanced the IFN-gamma-stimulated reactions. 8-Bromo-cGMP neither increased by itself nor synergized with IFN-gamma to increase the same reactions. Prostaglandin E1 and beraprost, a stable analogue of prostaglandin I2, which by themselves showed only marginal effects on these reactions, also synergized with IFN-gamma to stimulate the reactions. Interleukin 1 beta or tumor necrosis factor alpha stimulated the same reactions which were similarly enhanced by forskolin. These results indicate that an elevation of intracellular cAMP, particularly in combination with inflammatory cytokines, positively regulates nitric oxide production at the level of iNOS mRNA expression in vascular smooth muscle cells.
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PMID:Cyclic AMP-elevating agents induce an inducible type of nitric oxide synthase in cultured vascular smooth muscle cells. Synergism with the induction elicited by inflammatory cytokines. 769 10

Both mu and delta opioid receptors are expressed in undifferentiated human neuroblastoma SHSY5Y cells and are negatively coupled to adenylate cyclase. The ability of various mu opioid, delta opioid and alpha-2 adrenergic agonists to inhibit acutely forskolin-stimulated adenylate cyclase activity in undifferentiated SHSY5Y cells after chronic administration with the selective mu opioid agonist [N-MePhe3,D-Pro4]morphiceptin (PLO17) or delta opioid agonist, [D-Pen2,D-Pen5]enkephalin (DPDPE) was assessed. In control cells, both PLO17 and DPDPE inhibited cyclic AMP (cAMP) formation with equal maximal inhibition, i.e., 60 +/- 3 and 66 +/- 2%, having IC50 values of 51.1 +/- 1.3 and 3.7 +/- 1.0 nM, respectively. The inhibition of intracellular cAMP formation by both agonists could be blocked by pertussis toxin pretreatment. After 24 hr of chronic administration of PLO17 (50 nM to 10 microM), a concentration-dependent loss of the ability of mu opioid agonists PLO17 and DAMGO, but not the delta opioid agonists DPDPE, nor alpha-2 adrenergic agonist UK-14304 (5-Bromo-N-(4,5,-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine) to inhibit adenylate cyclase activity was observed. In contrast, chronic administration of DPDPE (0.1 nM to 0.3 microM) resulted in a concentration-dependent reduction in the inhibition of cAMP formation produced by delta opioid agonists DPDPE and DSLET, but not mu opioid, nor alpha-2 adrenergic agonists tested. The observed homologous desensitization was also time-dependent. In addition, antagonist-induced increases in adenylate cyclase activity were observed only after chronic PLO17 administration.2+ Finally, chronic pretreatment of cells with PLO17 (10 microM) resulted in a significant decrease in mu opioid, but not delta opioid receptor, binding, whereas treatment with DPDPE (0.3 microM) resulted in a significant decrease in delta opioid, but not mu opioid receptor binding. Therefore, undifferentiated SHSY5Y cells may provide an excellent model system to study not only the signal transduction mechanisms of mu and/or delta opioid receptors, but also the cellular adaptations of specific opioid receptors.
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PMID:Mu and delta opioid receptor desensitization in undifferentiated human neuroblastoma SHSY5Y cells. 803 14

Glucocorticoid receptor levels within a given cell determine the glucocorticoid effect in the target tissue. Glucocorticoid receptors are present in adrenal medullary cells in culture where they are involved in the regulation of catecholamine biosynthesis. Modulation of glucocorticoid receptor protein and/or messenger RNA levels in response to cyclic nucleotides has been found in various cell types. In this study, we have investigated the effects of cyclic AMP and cyclic GMP on glucocorticoid receptor binding and glucocorticoid receptor-mediated function in Percoll-isolated bovine adrenal medullary cells in culture. Four-day treatment of cells with 8-bromo-cyclic AMP (10(-3) M) an analogue of cAMP, or forskolin (10(-5) M), an activator of adenylate cyclase, decreased soluble [3H]dexamethasone binding by 55 and 54%, respectively. 8-Bromo-cyclic GMP treatment decreased [3H]dexamethasone binding by 31 and 34% at 10(-5) and 10(-4) M, respectively. Treatment with 8-bromo-cyclic AMP or forskolin, but not 8-bromo-cyclic GMP, elevated cortisol levels in the medium of treated cells, presumably by elevating steroidogenesis in contaminating cortical cells. Cultures further purified to produce chromaffin-enriched cell cultures, also showed a loss (41%) in soluble [3H]dexamethasone binding when treated with 8-bromo-cyclic AMP (10(-3) M). Four-day treatment of standard Percoll-isolated cells with low concentrations of cortisol (10(-9) to 2 x 10(-7) M) similar to that found in the medium of 8-bromo-cyclic AMP-treated cells, did not decrease soluble [3H]dexamethasone binding, whereas higher cortisol concentrations (10(-6) M) produced a 62% loss in soluble binding. Adsorption of cortisol with bovine serum albumin (5 mg/ml) prevented a cortisol (10(-6) M)-induced loss in soluble [3H]dexamethasone binding with no effect on the 8-bromo-cyclic AMP-induced loss in binding, suggesting that the decrease in binding observed following 8-bromo-cyclic AMP treatment is not due to the release of cortisol from contaminating cortical cells. Finally, we report a loss in the ability of 8-bromo-cyclic AMP- or 8-bromo-cyclic GMP-treated cells to fully induce the activity of phenylethanolamine N-methyltransferase in response to cortisol, indicating that decreases in soluble [3H]dexamethasone binding translate into a decrease in the functional consequence of glucocorticoid receptor binding in adrenal medullary cells. In conclusion, these results indicate that long-term increases in cyclic nucleotide second messengers are able to decrease glucocorticoid receptor binding in bovine adrenal medullary cells, via a mechanism independent of released cortisol.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Glucocorticoid receptors in bovine adrenal medullary cells in culture: regulation by cyclic nucleotides. 839 Jun 25

The polymerase chain reaction (PCR) was used to amplify a 522-bp region of the adenylate cyclase toxin (cyaA) gene of Bordetella pertussis. As few as 100 cfu from a suspension of B. pertussis could be detected by this procedure when the amplified PCR product was detected by ethidium bromide staining of agarose gels. However, simulated clinical specimens, prepared from swabs impregnated with known numbers of B. pertussis cells, only yielded a positive reaction with > or = 10(4) cfu. Hybridisation of a Southern blot of the PCR products from the swab samples with a cya-specific probe gave a positive reaction with as few as 8 cfu, but the hybridisation signal was uniformly weak with fewer than 10(4) cfu. Nevertheless, three of 13 nasopharyngeal swabs, taken from suspected clinically defined cases of whooping cough and stored frozen for up to 18 months, gave a positive PCR reaction.
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PMID:Identification of Bordetella pertussis in nasopharyngeal swabs by PCR amplification of a region of the adenylate cyclase gene. 842 39

1. Intracellular recordings were used to study the role of metabotropic glutamate receptors (mGluRs) in modulating GABA-mediated giant depolarizing potentials (GDPs) in immature rat hippocampal CA3 neurones. 2. The mGluR antagonist (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG, 1 mM) reduced the frequency of GDPs. The broad-spectrum ionotropic glutamate receptor antagonist kynurenic acid (1 mM) blocked GDPs. 3. In the presence of kynurenic acid, both tetanic stimulation of the hilus or bath application of quisqualic acid (1 microM) and trans-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD, 20 microM) induced the appearance of GDPs. These effects were antagonized by MCPG (1 mM) or L(+)-2-amino-3-phosphonopropionic acid (L-AP3) and blocked by bicuculline (10 microM). 4. 8-Bromo-cAMP (8-Br-cAMP, 0.3 mM), 3-isobutyl-1-methylxanthine (IBMX, 200 microM) or forskolin (30 microM) mimicked the effects of mGluR agonists on GDPs. The forskolin analogue 1,9-dideoxyforskolin (30 microM), which does not activate adenylate cyclase, was ineffective. 5. Incubation of slices in the presence of the protein kinase A inhibitor Rp-adenosine 3',5'-cyclic monophosphothioate triethylamine (Rp-cAMPS) (500 microM) or superfusion of Rp-cAMPS (20 microM) prevented the effects of forskolin or t-ACPD on GDPs. In the presence of kynurenic acid, the protein kinase C activator, phorbol 12,13-diacetate (2 microM) induced the appearance of GDPs. This effect was prevented by staurosporine (1 microM). However, staurosporine (1-3 microM) did not modify the effects of t-ACPD on GDPs. 6. It is suggested that, during development, mGluRs enhance the synchronous release of GABA, responsible for GDPs, through cAMP-dependent protein kinase.
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PMID:Cyclic AMP-dependent modulation of giant depolarizing potentials by metabotropic glutamate receptors in the rat hippocampus. 858 96

Platelet responses induced by ADP are mediated by a unique P21-purinergic receptor. Although a variety of ADP analogs, substituted at C2, have been used to delineate pharmacological properties of the ADP-binding site(s), the identity of the receptor protein has not been firmly established. 2-(4-Bromo-2,3-dioxobutylthio)- ADP [2-BrCH2(CO)2CH2-S-ADP], a well-characterized ADP analog, has been previously used as an affinity label to examine the structure/function relationship of ADP-requiring enzymes [Kapetanovic, E., Bailey, J.B. & Colman, R.F. (1985) Biochemistry 24, 7586-7593]. We found that it induced platelet shape change, aggregation, exposure of fibrinogen binding sites, secretion and mobilization of intracellular calcium, but was less potent than ADP. Under non-stirring conditions, incubation of platelets with this analog for longer time periods blocked ADP-induced shape change, aggregation, and the ability to ADP to antagonize the rise in intracellular levels of cAMP induced by iloprost (a prostaglandin I2 analog). Of a variety of agonists examined, only ADP-induced aggregation was almost completely inhibited in platelets irreversibly modified by the analog. An autoradiogram of the gel obtained by SDS/PAGE of solubilized platelets modified by the ADP analog followed by reduction of the dioxo group by NaB[3H], showed the presence of a single radiolabeled protein band at 100 kDa. Platelets incubated first with either ADP, ATP, or 2-methylthio-ADP were not labeled by 2-BrCH2(CO)2CH2S-ADP and NaB[3H]4-8-BrCH2(CO)2CH2-S-ADP was previously shown by us to irreversibly antagonize ADP-induced platelet responses by selectively modifying aggregin. Incubation of platelets with 2-BrCH2(CO)2CH2S-ADP completely blocked labeling of aggregin in platelets by 8-BrCH2(CO)2CH2S-[32P]ADP. These results show that 2-BrCH2(CO)2CH2S-ADP initially interacts reversibly with aggregin (100kDa), a putative ADP receptor, and induces platelet shape change and aggregation, and at longer periods of incubation reacts irreversibly to block the ability of ADP to antagonize stimulated adenylate cyclase activity. In contrast, 6-BrCH2(CO)2CH2S-ADP was found to be a weak and reversible inhibitor of ADP-induced platelet aggregation. Prior incubation of platelets with the latter analog reduced labeling of aggregin by 8-BrCH2(CO)2CH2S-[32P]ADP. Taken together, the results further show that substitution by the BrCH2(CO)2CH2 group at the C2 and C8 positions is tolerated, while the presence of a free amino function at the C6 position is essential for its interaction with aggregin.
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PMID:Platelet activation by 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-diphosphate is mediated by its binding to a putative ADP receptor, aggregin. 866 7

We investigated the influence of the Ca(2+)-ATPase inhibitor thapsigargin (TG) on the vasorelaxant response to different endothelium-dependent and endothelium-independent relaxing agents in an isolated thoracic aorta preparation of the rabbit, precontracted by norepinephrine (NE). Pretreatment with 100 microM L-arginine methyl ester (L-NAME) an inhibitor of nitric oxide (NO) synthesis, completely prevented acetylcholine (ACh)-induced relaxation; the inactive stereoisomer D-NAME did not modify the effect of ACh. The exposure of the preparations to 1 microM TG induced a slowly developing slight increase in the basal tension during 30-min contact. The same concentration of TG also slightly reduced the response to the subsequent administration of NE. The antagonist effect of TG on the ACh response was concentration dependent in the range between 0.1 and 10 microM. A 30-min pretreatment with 1 microM TG appeared to be sufficient to induce a consistent antagonism of the ACh (0.01-10 microM) concentration-relaxant effect curve, since an increase to 60 min did not produce a further significant increment in the degree of the antagonist effect. The concentration-dependent relaxation induced by substance P (SP 0.1-3 nM) was also significantly antagonized by 1 microM TG. The effect of the calcium ionophore A23187 (0.01-1 microM) was reduced by the Ca(2+)-ATPase inhibitor only at the higher concentrations tested (0.3-1 microM). However, a 30-min contact time with 1 microM TG was completely ineffective in antagonizing the concentration-relaxant response curves to the two nitrovasodilators sodium nitroprusside (SNP 0.1-100 microM) and nitroglycerin (NTG 1-300 nM) and to the cyclic GMP analogue 8-Bromo-cyclic GMP (3-100 microM). The effects of the beta-adrenoceptor agonist isoprenaline (ISO 0.1-10 microM) and of the direct adenylate cyclase activator forskolin (FK 0.01-10 microM) were also completely unaffected by 1 microM TG. These results demonstrate that TG affects the response to agents that induce an endothelium-dependent relaxation through receptor-dependent calcium mobilization. However, they do not support the hypothesis that sarcoplasmic pump activity is essential for the development of a vasorelaxant response to endothelium-independent agents.
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PMID:Thapsigargin inhibits the response to acetylcholine and substance P but does not interfere with the responses to endothelium-independent agents. 879 40

The migration of coronary artery medial smooth muscle cells (SMCs) into the intima is proposed to be an important process of intimal thickening in coronary atherosclerotic lesions. In the current study, we examined the possible interaction of adrenomedullin, a novel vasorelaxant peptide, and angiotensin II (Ang II) on human coronary artery SMC migration using Boyden's chamber method. Ang II stimulated SMC migration in a concentration-dependent manner between 10(6) and 10(8) mol/L. This stimulation was clearly blocked by the Ang II type 1 receptor antagonist losartan but not by the type 2 receptor antagonist PD 123319. The migration stimulatory effect of Ang II was chemotactic in nature for cultured human coronary artery SMCs but was not chemokinetic. Human adrenomedullin clearly inhibited Ang II-induced migration in a concentration-dependent manner. Human adrenomedullin stimulated cAMP formation in these cells. Inhibition by adrenomedullin of Ang II-induced SMC migration was paralleled by an increase in the cellular level of cAMP. 8-Bromo-cAMP, a cAMP analogue, and forskolin, an activator of adenylate cyclase, inhibited the Ang II-induced SMC migration. These results suggest that Ang II stimulates SMC migration via type 1 receptors in human coronary artery and adrenomedullin inhibits Ang II-induced migration at least partly through a cAMP-dependent mechanism. Taken together with the finding that adrenomedullin is synthesized in and secreted from vascular endothelial cells, this peptide may play a role as a local antimigration factor in certain pathological conditions.
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PMID:Adrenomedullin is a potent inhibitor of angiotensin II-induced migration of human coronary artery smooth muscle cells. 918 Jun 34

The calcitonin receptor is a seven-transmembrane G-protein coupled receptor which is located on osteoclasts, in kidney, and in brain. The receptor signals through multiple pathways, including activation of adenylate cyclase, leading to inhibition of bone resorption. In the present study, we used antibodies raised against the C-terminus of the human calcitonin (CT) receptor to study receptor phosphorylation. In baby hamster kidney cells transfected with the human CT receptor, phosphorylation of the receptor increased approximately 2.5-fold after cells were treated with calcitonin, phorbol ester, forskolin, or calcitonin plus phorbol ester. Phosphorylation reached a maximum 20 minutes after treatment with sCT and half-maximal phosphorylation was observed at 0.1 nM sCT, a hormone concentration related to receptor occupancy. Digestion of the immunoprecipitated receptor with cyanogen bromide (CNBr) yielded a single 32P-labeled fragment which migrates at Mr 14 kD on gel electrophoresis. This corresponds to the predicted size of the CNBr fragment containing the C-terminal domain of the receptor. No 32P-labeled bands were observed for CNBr fragments predicted to contain the first, second, or third intracellular loops. An identical labeling pattern was seen with cells expressing an alternatively spliced isoform of the human receptor (insert-positive isoform). Phosphorylation of the receptor by phorbol ester and forskolin was further localized to a Mr 6 kD proteolytic fragment within the C-terminus. The protein kinase A and C inhibitors staurosporine, chelerythrine, and H-89 had little effect on CT-induced phosphorylation, suggesting that nonsecond messenger-activated kinases are involved in hormone-dependent CT receptor phosphorylation.
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PMID:Phosphorylation of the human calcitonin receptor by multiple kinases is localized to the C-terminus. 933 29


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