Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The guanylnucleotide specificity of muscarinic acetylcholine receptor (MR) inhibitory coupling to cardiac adenylate cyclase (AC) was investigated under low MgCl2 (i.e., 0.5 mM) conditions. In purified cardiac sarcolemma, carbachol maximally inhibited AC activity 60% in the presence of GTP. Carbachol-dependent inhibition in the presence of guanosine 5'-O-(3-thiotriphosphate (GTP gamma S) or guanylylimidodiphosphate [Gpp(NH)p] was of lesser magnitude (i.e., 30%) and was evident only during short incubation periods. Of greater interest, carbachol maximally inhibited AC activity in the presence of GDP and guanosine 5'-O-(2-thiodiphosphate (GDP beta S) by 35 and 60%, respectively. Control studies ruled out transphosphorylation of GDP and GDP beta S by nucleoside diphosphate kinase or guanylnucleoside triphosphate contamination as reasons for the inhibitory effects of GDP and GDP beta S. Furthermore, isoproterenol stimulated AC in the presence of GTP, GTP gamma S, and Gpp(NH)p but not in the presence of GDP or GDP beta S. Therefore, GDP and GDP beta S may serve as agonists on MR-activated Gi but not on beta-adrenergic receptor-activated Gs in these membranes. Time course studies revealed that carbachol-dependent inhibition of AC in the presence of either GTP or GDP occurred without a detectable lag period, and this inhibition was rapidly reversed by atropine. In contrast, a 1-2-min lag time was required for carbachol- and GDP beta S-dependent inhibition of AC to occur, and inhibition, once developed, was only partially and slowly reversed by atropine. Preincubation of sarcolemma with carbachol and GDP beta S, in the absence of ATP or under nonphosphorylating conditions, eliminated the lag time for inhibition of AC activity. Although it is unlikely that GDP and GDP beta S have physiological relevance of MR-Gi-AC coupling, these studies provide unique insights into this coupling mechanism in cardiac membranes.
Mol Pharmacol 1992 Jan
PMID:Guanylnucleotide specificity for muscarinic receptor inhibitory coupling to cardiac adenylate cyclase. 131 Jan 41

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.
Mol Pharmacol 1992 Feb
PMID:A2A adenosine receptors from rat striatum and rat pheochromocytoma PC12 cells: characterization with radioligand binding and by activation of adenylate cyclase. 131 11

The factors which regulate the expression of the granin family of secretory proteins have yet to be completely described. The present study investigated the effects of forskolin (FSK), an activator of adenylate cyclase, on the regulation of chromogranin B/secretogranin I (CgB) and secretogranin II (SgII) mRNA levels in rat PC12 cells. PC12 cells were treated with 10 microM FSK for time points up to 48 h and were harvested for cAMP determination, RNA isolation and Northern blot analysis, or fixed in 4% paraformaldehyde for immunocytochemistry. Cellular cAMP levels peaked after two h of FSK treatment and remained elevated for 48 h. Chromogranin B mRNA increased with FSK treatment, reaching a maximum of 7-fold above control after 24 h, while the level of SgII mRNA decreased to a level of 65 +/- 10% of control after 48 h. The effects of FSK on CgB mRNA appear to be mediated by cAMP, as 8-bromo-cAMP (500 microM) resulted in a 2.8-fold increase in CgB mRNA, and H-89 (30 microM), a selective inhibitor of cAMP-dependent protein kinase, reduced the FSK-mediated response. The level of CgB was also increased in FSK-treated cells, as evidenced by immunofluorescent analysis which showed a more intense staining in PC12 cells treated with FSK for 48 h than in untreated cells. The intensity of SgII staining was diminished by FSK treatment, most likely a result of a decreased rate of synthesis as well as an increase in the release of SgII. This study demonstrated that the mRNA and protein levels of CgB and SgII are differentially regulated by cAMP in PC12 cells.
Brain Res Mol Brain Res 1992 Jan
PMID:Differential regulation of chromogranin B/secretogranin I and secretogranin II by forskolin in PC12 cells. 131 1

In order to produce significant quantities of the human thyrotrophin (TSH) receptor we have investigated the use of two eukaryotic high expression systems. DNA encoding the receptor was obtained by the polymerase chain reaction (PCR) applied to thyroid cDNA. Receptor DNA was inserted into the baculovirus system; despite high mRNA levels there was little or no demonstrable protein production. However, using a novel amplifiable glutamine synthetase system, clones of transfected Chinese hamster ovary (CHO) cells expressed a high affinity TSH receptor (KD 0.225 +/- 0.046 nM, Bmax 20,000-45,000 sites/cell for individual clones). This was coupled to adenylate cyclase as measured by a TSH-stimulatable increase in extracellular cyclic AMP (cAMP), a detectable response being noted at 1 microU/ml TSH with half-maximal at around 25-50 microU/ml. The high expression allowed detection of both TSH binding inhibition and adenylate cyclase stimulation by autoantibodies in unfractionated sera from patients with Graves' disease.
Mol Cell Endocrinol 1992 Feb
PMID:The use of the amplifiable high-expression vector pEE14 to study the interactions of autoantibodies with recombinant human thyrotrophin receptor. 131 88

Rat ovarian membrane luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor was reconstituted into proteoliposomes. The ability of sodium cholate to extract and reconstitute hCG binding activity was dependent on the protein/detergent ratio. Trypsinization of the LH/hCG receptor containing proteoliposomes indicated that approximately 57% of hCG binding sites were oriented extravesicularly. The presence of 20% glycerol or other osmolytes during reconstitution increased the accessibility of LH/hCG receptors but not the activity of adenylate cyclase in proteoliposomes. This beneficial effect was independent of any specific detergent or its presence during detergent solubilization of proteins. Dynamic properties of membranes were monitored by electron spin resonance of 16-, 12-, and 5-doxyl stearic acid. Reconstituted proteoliposomes contain less ordered membrane lipids than do native membranes. Addition of glycerol before reconstitution increased the order of lipid bilayer and shifted it to the physical state of the native membrane. These findings are consistent with the hypothesis that a rise in membrane ordering increases the accessibility of membrane-bound LH/hCG receptors.
Mol Cell Endocrinol 1992 Feb
PMID:Osmolytes improve the reconstitution of luteinizing hormone/human chorionic gonadotropin receptors into proteoliposomes. 131 90

alpha-Thrombin (thrombin) stimulates phospholipase C and modulates the activity of adenylate cyclase in a number of cell types via G protein-coupled receptors. It is also a potent growth factor, notably for a line of hamster fibroblasts (CCL39 cells). Recently, predicted amino acid sequences for human and hamster thrombin receptors have been reported that display a putative thrombin cleavage site in the N-terminal extracellular domain. Synthetic peptides corresponding to 14 residues carboxyl to the presumed thrombin cleavage site of the human receptor have been shown to activate platelets as well as the thrombin receptor expressed in Xenopus oocytes. In the present study we have examined the effects of synthetic peptides corresponding to the same region of the hamster receptor (S-42-L-55) and shorter peptides (2-7 residues) on signal transducing systems in CCL39 cells. Our results indicate that hamster receptor peptides of greater than or equal to 5 residues effectively stimulate phospholipase C in CCL39 cells via the thrombin receptor and induce rapid desensitization of the response. The same peptides also inhibit adenylate cyclase in a pertussis toxin-sensitive manner. Although the peptides are potent agonists of serotonin release in platelets, unlike thrombin, by themselves they are not mitogenic. However, they potentiate DNA synthesis in cooperation with growth factors possessing tyrosine kinase receptors. Hence, we conclude that the potent mitogenic action of thrombin cannot be accounted for solely by the activation of the cloned receptor. We postulate the existence of an additional receptor activated by thrombin, which is required for its full mitogenic potential.
Mol Biol Cell 1992 Jan
PMID:Synthetic alpha-thrombin receptor peptides activate G protein-coupled signaling pathways but are unable to induce mitogenesis. 131 81

The Bio 14.6 cardiomyopathic Syrian hamster is an animal model of human idiopathic cardiomyopathy. The pathogenesis of the disease in this animal has not yet been clearly elucidated. It is well known that alpha- and beta-adrenergic receptors are increased in the myocardium of this animal, but that isoprenaline does not produce an augmented response. We examined the activity of cardiac stimulatory GTP-binding protein (Gs), which couple with beta-adrenergic receptors to stimulate adenylate cyclase, in Bio 14.6 cardiomyopathic hamsters at 90 and 160 days of age. The cardiac norepinephrine concentration was significantly increased in Bio 14.6 hamsters compared with control hamsters (F1B) at 90 days of age (1,739 +/- 120 vs 1,470 +/- 161 ng/g wet tissue weight, p less than 0.05). Cardiac forskolin-stimulated adenylate cyclase activities at 90 and 160 days of age were lower in the cardiomyopathic hamsters than in the F1B controls (90 days old: 98 +/- 24 vs 122 +/- 29 pmol/min/mg protein, p less than 0.05; 160 days old: 74 +/- 13 vs 124 +/- 28 pmol/min/mg protein, p less than 0.01). Cardiac Gs activities at 90 and 160 days of age were significantly lower in Bio 14.6 hamsters than those in F1B hamsters (90 days old: 204 +/- 42 vs 259 +/- 49 pmol/min/mg protein, p less than 0.05; 160 days old: 156 +/- 39 vs 211 +/- 60 pmol/min/mg protein, p less than 0.05). We thus demonstrated functional defects in cardiac Gs protein and adenylate cyclase activity in the Bio 14.6 cardiomyopathic hamsters at 90 to 160 days of age (the hypertrophic stage of cardiomyopathy).(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Biochem 1992 Mar 04
PMID:Stimulatory guanine nucleotide-binding protein and adenylate cyclase activities in Bio 14.6 cardiomyopathic hamsters at the hypertrophic stage. 131 29

Cytochrome P-450scc (P-450scc) catalyzes the cholesterol side-chain cleavage reaction, a rate-limiting enzymatic step for progesterone synthesis in trophoblastic and other steroidogenic cells. Adrenodoxin is the iron/sulfur protein donating electrons to P-450scc during this reaction. We examined the effects of cholera toxin (CT), an activator of adenylate cyclase, and 12-O-tetradecanoylphorbol acetate (TPA), a phorbol ester protein kinase C activator, on the levels of mRNAs encoding P-450scc and adrenodoxin in JEG-3 choriocarcinoma cells. CT induced in a concentration- and time-dependent manner P-450scc and adrenodoxin mRNA levels to 8-fold and 1.5-fold above that of control, respectively. TPA also increased P-450scc and adrenodoxin mRNA levels about 3-fold and 1.5-fold above that of control, respectively. Epidermal growth factor (EGF) was found to weakly induce P-450scc mRNA accumulation with a maximal 20% stimulation above basal levels. The effects of CT and TPA were apparently additive on both mRNAs. The protein synthesis inhibitor cycloheximide diminished basal, CT-, TPA-, and EGF-stimulated P-450scc mRNA accumulation whereas the opposite was observed for the adrenodoxin mRNA. Insulin-like growth factor I (IGF-I) appeared to have no effect on either mRNA. These data indicate that: (1) the accumulation of P-450scc and adrenodoxin mRNAs is mainly controlled by the cyclic adenosine 3',5'-monophosphate (cAMP)-dependent pathway but their stimulation by TPA- and EGF-induced signals may also play a weaker synergistic role; (2) the protein synthesis inhibitor cycloheximide inhibits basal, CT-, TPA- and EGF-stimulated P-450scc mRNA levels while it increases the expression of adrenodoxin mRNA suggesting that in the malignant trophoblasts these two enzyme mRNAs are differentially controlled.
Mol Cell Endocrinol 1992 Apr
PMID:Regulation of the cholesterol side-chain cleavage cytochrome P-450 and adrenodoxin mRNAs in cultured choriocarcinoma cells. 131 54

Five separate guanine nucleotide-binding proteins (G proteins) were immunologically identified in membranes from neuroblastoma x glioma NG108-15 hybrid cells. These alpha subunit proteins were Gi2 alpha, two isoforms of Gi3 alpha, and two isoforms of Go alpha. The G proteins that interacted with delta-opioid receptors in these membranes were identified using cholera toxin (CTX)-induced ADP-ribosylation and antisera selective for various G protein alpha subunits. In the presence of delta-opioid agonists, CTX induced the incorporation of [32P]ADP-ribose into three pertussis toxin substrates. Using antisera generated against peptide sequences from G alpha subunits, these three pertussis toxin substrates were identified as Gi2 alpha, Go2 alpha, and one isoform of Gi3 alpha, which has yet to be identified. This CTX-induced labeling was demonstrated to be mediated via the delta-opioid receptor in these hybrid cells by the observation that delta agonists D-Ala2-D-Leu5-enkephalin (DA-DLE) and D-Pen2-D-Pen5-enkephalin, as well as the nonselective agonists etorphine and bremazocine, were active, but the mu agonist PL017 and the kappa agonist U-50-488H did not show this activity. This incorporation into all three substrates induced by DADLE was dose dependent, with EC50 (95% confidence interval) values ranging from 12 (3-52) to 183 (65-520) nM, which compared with the Kd value of 10 +/- 1.5 nM for this agonist, a dose that produces maximal inhibition of adenylate cyclase activity. Furthermore, pretreatment of the cells with pertussis toxin or treatment of the membranes with the antagonist naloxone blocked the incorporation induced by DADLE. Incorporation of [32P]ADP-ribose into all three substrates decreased 35-83% in membranes in which the receptors had been down-regulated by chronic treatment of the cells with DADLE. Thus, a single opioid receptor type can interact with three separate G proteins.
Mol Pharmacol 1992 May
PMID:Identification of three separate guanine nucleotide-binding proteins that interact with the delta-opioid receptor in NG108-15 neuroblastoma x glioma hybrid cells. 131

Intracellular collagen degradation in normal rat hepatocytes was exponentially stimulated by db-cAMP (10-100 microM). The effect was manifested as a decrease (p less than 0.01) in net collagen production. The extent of degradation directly co-related with the intracellular cAMP levels, only up to a threshold concentration (16.2 +/- 1.3 p moles/10(6) cells) elicited by 100 microM of db-cAMP. Higher concentrations induced no further increment. Forskolin adenylate cyclase activator (10-50 microM), produced similar effects demonstrating cAMP dependence of the phenomenon. Both db-cAMP as well as Forskolin stimulated collagen degradation (p less than 0.05) in hepatocytes from rats administered CCL4. However, the extent of stimulation was significantly (p less than 0.01) less compared to that observed in normal hepatocytes. Our data demonstrates that elevated cAMP levels regulate net collagen content by signalling intracellular collagen degradation and not synthesis.
Mol Cell Biochem 1992 Jan 15
PMID:Intracellular cAMP determines the extent of degradation and not the synthesis of collagen by rat hepatocytes. 131 51


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>