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At least 757 records · Page 42Linked to original sources
PubMed1990

GTPase-inhibiting mutations activate the alpha-chain of Gs in human tumours.

Several trophic hormones transmit their signals by coupling to G-proteins. A subset of human growth hormone (GH)-secreting pituitary tumours, characterized by elevated GH secretion, cyclic AMP levels, and adenylyl cyclase activity, carries mutations in the gene that encodes the alpha-chain of Gs. By substitution of a single amino acid (Arg-201 or Gln-227), these mutations inhibit the intrinsic GTPase activity of alpha s, thus stabilizing the protein in its active conformation. The discovery of mutant alpha s proteins in human tumours suggests that the alpha s gene can be converted into an oncogene, called gsp, in cells that proliferate in response to cyclic AMP. Most likely, oncogenes can result from similar mutations in other G-protein alpha-chain genes.

GTP Phosphohydrolases↗
PubMed1990

[Glyphosate utilization by Pseudomonas spec. GS].

Pseudomonas spec. GS grown in the presence of 14C-labelled glyphosate metabolizes the herbicide to inorganic phosphate as well as in respect of utilization of the C-skeleton. Furthermore it was shown that the primary step of degradation is the breakdown of the C-P-bond, because sarcosine was found as a transient metabolite.

Amino Acids↗
PubMed1985

Use dependent depression of fast sodium current in heart muscle by the new antiarrhythmic substance Bonnecor (AWD 19-166, GS 015).

A new antiarrhythmic compound (Bonnecor AWD 19-166, GS 015) was studied in its effects on the fast sodium channel in two types of cardiac preparations (rabbit ventricular myocardium, frog atrial trabecules). AWD 19-166 depressed the maximum upstroke velocity of the action potentials in ventricular myocardium (50% inhibition at 5 X 10(-6) mol/l) and decreased the fast inward current in frog atrial myocardium. High frequent trains of pacing pulses produced a pacing dependent extra block of both the maximal upstroke velocity and the fast inward current. It is concluded that AWD 19-166 blocks the fast sodium channel in a use dependent manner.

Action Potentials↗
PubMed1989

Cholera toxin induces cAMP-independent degradation of Gs.

Cholera toxin stimulates adenylyl cyclase by catalyzing ADP-ribosylation of the alpha chain (alpha s) of Gs, a guanine nucleotide binding regulatory protein. In a rat pituitary cell line, GH3, the toxin-induced increase in GTP-dependent adenylyl cyclase activity is maximal at 1 h; adenylyl cyclase remains elevated for at least 32 h. Surprisingly, cholera toxin also induces a 74-95% decrease in the amount of immunoreactive alpha s in the same cells, as assessed on immunoblots probed with either of two antisera directed against separate alpha s peptide sequences. The decrease in immunoreactive alpha s, which begins after 1 h of toxin treatment and is complete by 8 h, is accompanied by a comparable decrease in the amount of biochemically active alpha s, as assessed by its ability to complement the biochemical defect of alpha s-deficient S49 cyc- membranes. Cholera toxin induces similar decreases in alpha s in wild type S49 lymphoma cells, in S49 kin- mutants, which lack cAMP-dependent protein kinase, and in S49 H21 a mutants, in which alpha s is unable to assume an active conformation upon binding GTP. The toxin-induced decrease in alpha s is somewhat temperature-dependent, but is not blocked by agents that increase lysosomal pH or by colchicine, which promotes breakdown of microtubules. alpha s in detergent-solubilized GH3 membranes is susceptible to proteolysis by an endogenous protease; this susceptibility is markedly increased in membranes from cells previously exposed to cholera toxin for 1 h. Taken together, these results suggest that cholera toxin-induced covalent modification of alpha s marks the protein for accelerated degradation. In addition, the persistence of elevated GTP-dependent adenylyl cyclase activity despite loss of a substantial fraction of alpha s suggests that the amount of alpha s membranes is greater than the amount necessary for maximal activation of cAMP synthesis by cholera toxin.

Adenylyl Cyclases↗
PubMed1989

Mutations in the GTP-binding site of GS alpha alter stimulation of adenylyl cyclase.

Mutational replacements of specific residues in the GTP-binding pocket of the 21-kDa ras proteins (p21ras) reduce their GTPase activity. To test the possibility that the cognate regions of G protein alpha chains participate in GTP binding and hydrolysis, we compared signaling functions of normal and mutated alpha chains (termed alpha s) of Gs, the stimulatory regulator of adenylyl cyclase. alpha s chains were expressed in an alpha s-deficient S49 mouse lymphoma cell line, cyc-. alpha s in which leucine replaces glutamine 227 (corresponding to glutamine 61 of p21ras) constitutively activates adenylyl cyclase and reduces the kcat for GTP hydrolysis more than 100-fold. There is a smaller reduction in GTPase activity in another mutant in which valine replaces glycine 49 (corresponding to glycine 12 of p21ras). This mutant alpha s is a poor activator of adenylyl cyclase. Moreover, the glycine 49 protein, unlike normal alpha s, is not protected against tryptic cleavage by hydrolysis resistant GTP analogs; this finding suggests impairment of the mutant protein's ability to attain the active (GTP-bound) conformation. We conclude that alpha s residues near glutamine 227 and glycine 49 participate in binding and hydrolysis of GTP, although the GTP binding regions of alpha s and p21ras are not identical.

Adenylyl Cyclases↗
PubMed1988

Reticulocyte lysates synthesize an active alpha subunit of the stimulatory G protein Gs.

We placed the cDNAs encoding one of the short types of alpha s (alpha s-1) with Asp-Ser in positions 70 and 71 and one of the long types of alpha s (alpha s-2) in which Asp-Ser are substituted with a string of 16 amino acids, into the pGEM-3 transcription vector downstream from its T7 RNA polymerase promoter, obtained transcripts and translated the mRNAs using a rabbit reticulocyte lysate system, to determine if the molecules would be synthesized and, if so, whether they would be active as assessed in cyc- reconstitution assays. The translation products obtained from both alpha s RNAs were a mixture of primarily three polypeptides of which one (approximately 40-50% of total) represented the complete translation product and the other two appeared to be due to internal translation starts at Met60, before the splice difference between the RNAs, and the other at the first Met after the splice difference. Lysates incubated with short or long alpha s RNA when added to cyc- membranes reconstituted fluoride and GTP[gamma S]-stimulated activities. Thus, in vitro synthesized alpha s subunits are active in interacting both with guanine nucleotides and the adenylyl cyclase enzyme. On incubation without and with the receptor agonist isoproterenol, using GTP as sole added guanine nucleotide, both types of alpha s subunits reconstituted the isoproterenol-stimulated adenylyl cyclase activity. Thus, the synthetic alpha s also interact with receptors, and by inference with beta-gamma dimers, shown previously to be needed for activation by receptor. Quantitative assays in which the activity of the synthetic alpha s-1 was compared to that of native purified human erythrocyte type-1 Gs, indicated that the two products are equipotent within a 2-fold margin of error. Thus, the lysate made fully active alpha s subunits, and alpha s subunits require no post-translational modifications dependent on microsomal processes. This approach may be useful in studying biological functions of other cloned alpha subunits of G proteins.

Animals↗
PubMed1985

[Antiarrhythmic action of 3-carbethoxyamino-5-dimethylaminoacetyl-iminodibenzyl hydrochloride (Bonnecor, AWD 19-166, GS -15) on experimental arrhythmia induced by aconitin, ouabain, calcium chloride and barium chloride].

In comparison with detajmium, prajmalium, ajmaline, quinidine, lidocaine, disopyramide, propranolol, and Ethmozin the substance GS 015 is tested on the aconitin-induced arrhythmia of the rat, the auricular fibrillation by aconitin of the dog, the ventricular arrhythmia induced by ouabaine of the dog, the arrhythmia caused by calcium chloride of the rat, and the arrhythmia induced by barium chloride of the rabbit. The particular qualities of effect in the special forms of arrhythmia are discussed in connection with the study of the antiarrhythmic action in case of coronary occlusion, with the increase of the electric fibrillatory threshold, and with the electrophysiologic tests.

Aconitine↗
PubMed1985

Electrophysiological effects of a new antiarrhythmic substance Bonnecor (AWD 19-166, GS 015) on mammalian myocardium.

A new antiarrhythmic acting drug (Bonnecor, AWD 19-166, GS 015, 3- Carbethoxyamino-5-dimethyl-amino-acetyl-iminodibenzyl -hydrochloride) was tested electrophysiologically on isolated sinus node preparations and right ventricular papillary muscle of the rabbit. Transmembrane potentials were measured using a standard microelectrode technique. The substance was tested within a concentration range between 0.1 and 10 mg/l (2.5 X 10(-7) to 2.5 X 10(-5) mol/l). AWD 19-166 exerted negative chronotropic effects due to decreasing of the slope of the diastolic depolarization in sinus node cells. In ventricular myocardium AWD 19-166 increased the threshold strength of stimulation, prolonged the duration of action potentials at 90% but shortened it at 25% repolarization. The maximum upstroke velocity was found to be depressed. The maximum overshoot potential was diminished inspite of a drug-induced hyperpolarization of the resting transmembrane potential. Analysis of membrane responsiveness due to premature stimulation showed a concentration-dependent delay in the restitution of the maximum upstroke velocity of the premature action potentials and both a flattening and a shift towards more negative potentials of the potential-responsiveness relationship. AWD 19-166 decreased both the maximum upstroke velocity and the duration of Ca-mediated action potentials in concentrations between 2 and 8 mg/l. It is concluded that AWD 19-166 is able to exert potent antiarrhythmic effects in influencing both the fast and slow channel activity.

Action Potentials↗
PubMed1985

Electrical effects of the new antiarrhythmic compound Bonnecor (AWD 19-166, GS 015) under arrhythmogenic conditions.

Electrical effects of the new antiarrhythmic compound Bonnecor (AWD 19-166, GS 015) were studied under conditions known to increase the intracellular Ca2+ activity (extracellular K+-deprivation, application of glycosides). In K+ free solutions right ventricular papillary muscles showed an extreme acceleration of the initial repolarization indicating an intracellular Ca overload. AWD 19-166 could not prevent the initial shortening of action potentials and seems to be less effective in K+ free than in K+ containing solutions. In partially depolarized (22 mmol/lK+) right ventricular papillary muscles oscillatory afterdepolarizations were not suppressed by AWD 19-166. However, the compound prevented the generation of irregular action potentials triggered by the afterdepolarizations.

Action Potentials↗
PubMed1985

[Pharmacokinetics and metabolism of 3-carbethoxyamino-5-dimethylamino-acetyl-iminodibenzyl hydrochloride (Bonnecor, AWD 19-166, GS 015) in the rat].

Excretion, blood level, distribution, and metabolite samples were studied on the rat after application of GS 015 marked by 14C. The compound is quickly and completely absorbed and metabolized from an aqueous solution. The marked substances form a broad blood level maximum, at the occasion of which a main metabolite distinguishes itself apart from the initial compound at first provable yet. The elimination half-life from the blood is 6 h. An intense influx of radioactive substance into the tissues takes place. The excretion of the marked metabolites occurs mainly renally making appear a second main metabolite. Striking sex differences are partly observed in the parameters tested.

Animals↗
PubMed1985

[Pharmacokinetics and pharmacodynamics of 3-carbethoxyamino-5-dimethylamino-acetyl-iminodibenzyl hydrochloride (Bonnecor, AWD 19-166, GS 015) in the dog].

The antiarrhythmic action of GS 015 was studied in proportion to its plasma concentrations ascertained in parallel, making use of the model of the two-step coronary ligature in the conscious dog. Blood levels of 1.0 microgram/ml (2 mg/kg i.v.) or of 0.8 microgram/ml (5 mg/kg orally) brought about a complete suppression of ectopic arrhythmias. The limit concentration for this effect is about 0.5 microgram/ml.

Animals↗
PubMed1985

[Pharmacologic studies on the effect of 3-carbethoxyamino-5-dimethylamino-acetyl-iminodibenzyl hydrochloride (Bonnecor, AWD 19-166, GS 015) on the vegetative and central nervous system].

The substance GS 015 was tested as to its influence on the vegetative and the central nervous system. The total of side effects is low. In spite of the structural resemblance the aspect of action does not correspond to that of tricyclic psychopharmaceutical agents. Pharmacologically relevant doses bring about: local anaesthesia, musculotropic spasmolysis, inhibition of the serotonin-induced oedema, antiulcerative effect, intensification of the toxicity of amphetamine. The results do not oppose to a clinical use.

Animals↗
PubMed1994

Nerve growth factor rapidly prolongs the action potential of mature sensory ganglion neurons in culture, and this effect requires activation of Gs-coupled excitatory kappa-opioid receptors on these cells.

Application of low concentrations (pM-nM) of NGF to mouse dorsal root ganglion (DRG)-spinal cord explants in long-term organotypic cultures rapidly prolongs the duration of the Ca(2+)-dependent component of the action potential (APD) in a major subset of DRG neurons that were previously shown to have characteristic responsiveness to exogenous opioids. These NGF-elicited excitatory modulating effects are blocked by pretreatment of the DRG neurons with monoclonal antibodies to rodent NGF receptors. NGF-induced APD prolongation is also prevented by the opioid receptor antagonist naloxone and the specific kappa-opioid antagonist nor-binaltorphimine (but not by specific mu- and delta-opioid antagonists). The results suggest that NGF stimulates the release of endogenous opioids (e.g., dynorphin) from DRG neurons and that prolongation of the APD occurs secondarily by activation of excitatory kappa-opioid receptor functions on these same or nearby cells. NGF-induced release of small quantities of opioids by DRG neurons would be expected to prolong the APD in view of the remarkable sensitivity of these neurons to the excitatory effects of extremely low (fM-nM) concentrations of exogenous opioid agonists. NGF-induced APD prolongation is blocked by the same cholera toxin A or B subunit treatments previously shown to block Gs coupling and GM1 ganglioside regulation of excitatory opioid receptors, respectively. These in vitro studies suggest that excitatory opioid receptor-mediated functions may play a role in mediating some types of rapid NGF-induced hyperalgesic and other physiologic effects on the nervous system.

Action Potentials↗
PubMed1993

[Localization of segments in the Gs protein interacting with adenylate cyclase using a Gs/G0-chimera].

cDNAs coding for three types of alpha-subunits of GTP-binding proteins Gs and G0 (a short form of alpha s with Asp-Ser in positions 71 and 72, a long form of alpha s with the insertion of 16 amino acid residues instead of Asp-Ser (71-72)--both from bovine brain, and alpha 0 from bovine cerebellum as well as some chimeric alpha s/alpha 0 genes were cloned into a modified pGEM-2 plasmid vector under the control of the SP6 promoter. All the genes were in vitro transcribed and translated, and some functional properties of the resulting proteins were determined, such as adenylyl cyclase activation, ADP-ribosylation with pertussis toxin, limited nucleotide-dependent trypsin proteolysis. Parts of the alpha s polypeptide chain necessary for the activation of adenylyl cyclase were mapped. The alpha s domain interacting with adenylyl cyclase is formed by the alpha s polypeptide chain fragments 235-294 and 337-356 (numbering as of the alpha s long form).

Adenosine Diphosphate Ribose↗
PubMed1993

Increased palmitoylation of the Gs protein alpha subunit after activation by the beta-adrenergic receptor or cholera toxin.

The alpha subunit of the heterotrimeric Gs protein that couples the beta-adrenergic receptor to adenylyl cyclase undergoes post-translational palmitoylation. We examined the dynamics of this modification of alpha s by metabolic labeling of COS and S49 lymphoma cells under different conditions. The endogenous alpha s proteins were immunoprecipitated with a peptide-specific antibody, separated by SDS-polyacrylamide gel electrophoresis, and analyzed by fluorography and densitometry. A pulse-chase study of COS cells incubated with [3H]palmitate or [35S]methionine showed that for alpha s the palmitate turnover (t1/2 approximately 50 min) was significantly faster than the protein degradation. Treatment of cells with 10 microM isoproterenol, a beta-adrenergic receptor agonist, in the presence of [3H]palmitate led to a rapid 4-10-fold increase in the palmitoylation of alpha s. This increase in palmitoylation was concentration-dependent (EC50 approximately 0.9 microM) and blocked by the antagonist propranolol. The mutant alpha s proteins in the unc and H21a S49 cell lines did not show an increase in [3H]palmitate incorporation with isoproterenol treatment. Cholera toxin treatment of COS cells increased the [3H]palmitate incorporation into the alpha s subunits. These data indicate that palmitoylation of the alpha s subunit is dynamic and regulated by activation of the alpha s subunit.

Animals↗
PubMed1993

Stimulation of specific types of Gs-stimulated adenylyl cyclases by phorbol ester treatment.

Regulation of adenylyl cyclases by protein kinase C was studied. Types 1-6 were transiently expressed in 293 cells. The cells were treated with 4 beta-phorbol 12-myristate 13-acetate (PMA), and adenylyl cyclase activity was measured. PMA treatment of untransfected or vector-transfected cells resulted in a 35% (mean) increase in basal and forskolin-stimulated activity. PMA treatment of type 2 enzyme-transfected cells showed a 3-fold increase in basal activity. This stimulation varied with Mg2+ concentration with up to 4.5-fold increases at low Mg2+. PMA treatment resulted in about a 60% increase in fluoride or GTP gamma S (guanosine 5'-O-(thiotriphosphate))-stimulated activity in type 2 enzyme-transfected cells. Expression of the type 1 and 3 enzymes resulted in pronounced Ca2+/calmodulin stimulation, which was significantly increased upon pretreatment with PMA. No effects of PMA treatment were observable on other activities of the type 1 enzyme, whereas all activities of the type 3 enzymes were stimulated. Expressed types 4, 5, and 6 enzymes showed modest (approximately 30%) or no increases in basal and forskolin-stimulated activities upon PMA treatment. These data indicate that specific types of adenylyl cyclases can be functional targets for protein kinase C. The pronounced effects on the basal activity of the type 2 enzyme suggest this adenylyl cyclase could in the appropriate environment be an intracellular signal generator for external signals not routed through the activation of Gs.

Adenylyl Cyclases↗
PubMed1996

Antibody penetration of tumor GS-7 xenografts in nude mice: a model for mucinous adenocarcinoma of the colon.

A new cell line derived from a human adenocarcinoma of the colon, GS-7, was propagated as a s.c. tumor in nude mice. This tumor histologically is a mucinous adenocarcinoma (also designated mucoid or colloid) with characteristic large mucin pools that are not lined by an epithelial layer but may contain scattered, randomly distributed cancer cells. Ten to 20% of human colorectal adenocarcinomas are of this histological type, but rapidly growing xenografts with this histology have been rarely used experimentally. This tumor, therefore, constitutes a useful model for similar human tumors. The mucin pools contain large amounts of carcinoembryonic antigen and tumor-associated glycoprotein 72, and the cells express epithelial glycoprotein 2 on their surface. The ability of antibodies injected i.v. to penetrate this tumor was investigated, using both biotinylated and radioiodinated antibodies (Abs). The results demonstrate that Abs can effectively penetrate the mucin pools, and that large amounts of Ab can localize there. This tumor type may have advantages as a target for certain forms of experimental immunotherapy.

Adenocarcinoma, Mucinous↗