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PubMed1989

[Effects of pingyangmycin on the growth and differentiation of the transplantable squamous cell carcinoma of forestomach (GS-742) in mice].

The effect of pinyangmycin (PYM) on the growth and differentiation of the transplantable squamous cell carcinoma of forestomach (GS-742) in mice was studied by optical and electron microscope. The results showed that PYM had marked growth inhibitory effect on GS-742. Histopathologically, tumor necrosis, fibrosis and infiltration of lymphocytes were obvious in mice treated with PYM. A decrease in mitosis and increase in keratinization were also conspicuous. Ultrastructurally, microvilli on tumor cells were significantly reduced in number with formation of blebs and ruffles; mitochondria were swollen; endoplasmic reticulum was often decreased and dilated; and desmosome and tonofibrils were increased. The results indicate that PYM exerts its growth-inhibitory effect on tumor cells by cell damage, inhibition of mitosis and promotion of differentiation.

Animals↗

PubMed1989

Deficient erythrocyte membrane Gs alpha activity and resistance to trophic hormones of multiple endocrine organs in two cases of pseudohypoparathyroidism.

Patients with pseudohypoparathyroidism (PHPT) have been shown to have deficient cell membrane stimulatory GTP-binding regulatory protein activity (Gs activity) and other defects, which cause renal resistance to parathyroid hormone (PTH). Those patients also have a higher incidence of deficiency of other endocrine axes. To investigate the mechanisms of this syndrome on 2 patients, we measured erythrocytic ghost Gs activity by ADP-ribosylation method, which showed low and low normal levels for the 2 patients who were from the same family, suggesting type Ia of this syndrome. While basal T3, T4 and TSH were compatible with mild primary hypothyroidism in both patients, a TRH test showed exaggerated TSH responses. The estrogen, progesterone and testosterone levels were low and LH and FSH responses were marginally high, indicating gonadal resistance to gonadotropins. ACTH levels were also elevated and ACTH response to CRH was exaggerated in one patient, suggesting additional primary adrenal insufficiency. This finding indicates that systemic endocrine tests are essential for the management of PHPT patients who may have a deficiency of multiple endocrine axes subclinically.

Adolescent↗

PubMed1989

Agonist-promoted sequestration of the beta 2-adrenergic receptor requires regions involved in functional coupling with Gs.

The molecular basis for the desensitization of beta 2-adrenergic receptors was investigated by oligonucleotide-directed mutagenesis. beta-Adrenergic receptor mutants containing deletions within the sixth hydrophilic domain that failed to couple to Gs and stimulate adenylyl cyclase did not undergo agonist-mediated sequestration. In contrast, all receptor mutants that displayed Gs coupling were sequestered away from the cell surface in response to isoproterenol. Progressive truncation of the C-terminus of the receptor resulted in decreases in the initial rates of receptor sequestration and functional uncoupling, although the final extent of these desensitization processes was not affected by the mutations. These data suggest that structural features of the beta 2-adrenergic receptor that are involved in receptor activation are also essential for mediating the subsequent inactivation caused by the sequestration of the receptor from the cell surface.

Adenylyl Cyclases↗

PubMed1987

Forskolin-resistant Y1 mutants harbor defects associated with the guanyl nucleotide-binding regulatory protein, Gs.

The properties of the adenylate cyclase from forskolin-resistant mutants of Y1 adrenocortical tumor cells was compared with the properties of the enzyme from parental Y1 cells in order to localize the site of mutation. In parental Y1 cells, forskolin stimulated adenylate cyclase activity with kinetics suggestive of an interaction at two sites; in mutant cells, forskolin resistance was characterized by a decrease in enzymatic activity at both sites. Forskolin potentiated the enzyme's responses to NaF and guanyl-5'-yl imidodiphosphate (Gpp(NH)p) in parent and mutant clones, and the mutant enzyme showed the same requirements for Mg2+ and Mn2+ as did the parent enzyme. The adenylate cyclase associated with forskolin-resistant mutants was insensitive to ACTH and was less responsive to Gpp(NH)p than was the parent enzyme. In parental Y1 cells and in the forskolin-resistant mutants, cholera toxin catalyzed the transfer of [32P]ADP-ribose from [32P]NAD+ into three membrane proteins associated with the alpha subunit of Gs; however, the amount of labeled ADP-ribose incorporated into mutant membranes was reduced by as much as 70%. Both parent and mutant membranes were labeled by pertussis toxin to the same extent. The insensitivity of the mutant adenylate cyclase to ACTH and Gpp(NH)p and the selective resistance of the mutant membranes to cholera toxin-catalyzed ADP-ribosylation suggest that a specific defect associated with Gs is involved in the mutation to forskolin resistance in Y1 cells.

Adenosine Diphosphate Ribose↗

PubMed1986

The protein cofactor necessary for ADP-ribosylation of Gs by cholera toxin is itself a GTP binding protein.

A membrane-bound protein cofactor (ARF) is required for the cholera toxin-dependent ADP-ribosylation of the stimulatory regulatory component (Gs) of adenylate cyclase. Improved methods for the purification of ARF from bovine brain are described. ARF has a high-affinity binding site for guanine nucleotides. Binding of GTP or GTP gamma S to ARF is necessary for the activity of the cofactor; GDP X ARF does not support ADP-ribosylation of Gs. Although the protein as purified contains stoichiometric amounts of GDP, GTPase activity of isolated ARF was not detected. Cholera toxin-dependent activation of adenylate cyclase thus requires two guanine nucleotide binding proteins.

Adenosine Diphosphate Ribose↗

PubMed1985

[Antifibrillatory and anti-arrhythmic action of 3-carbethoxyamino-5-dimethylamino-acetyl iminodibenzyl hydrochloride (Bonnecor, AWD 19-166, GS 015) on models of myocardial ischemia].

The intense antifibrillatory and antiarrhythmic actions of GS 015, a derivative of the novel structure series of the 5-(beta-aminoacyl)-3-carbalkoxyamino-iminodibenzyles, are demonstrated on models of myocardial ischaemia: antifibrillatory action on the acute coronary occlusion in the conscious rat (1.0 mg/kg i.v.) and antiarrhythmic action on the two-step coronary ligature in the conscious dog according to Harris (2.0 mg/kg i.v. and 5.0 mg/kg orally). On the strength of the present results it is discussed that GS 015 is first of all suitable for influencing "early arrhythmias" with re-entry mechanism and only in the second line for taking an influence on "late arrhythmias" which arise from the occurrence of ectopic focuses. The substance seems suitable for preventing terminal arrhythmias in patients with ischaemic heart disease as well as for treating arrhythmias in the acute stage of myocardial infarction.

Animals↗

PubMed1985

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

The antiarrhythmic action of GS 015 was studied proportionally to its plasma concentrations ascertained in parallel, using the model of the electrofibrillation of the cat's heart. Blood levels of about 1.3 micrograms/ml after i.v. injection of 2 mg/kg caused a marked short-term increase of the fibrillation threshold which then remained at the increased level for a longer period yet, observing GS 015 concentrations between 0.8 and 0.5 micrograms/ml. Individual differences existed in the height of the blood level as well as in its proportion to the effect.

Animals↗

PubMed1993

A novel expression system for Gs-coupled receptors.

Renal epithelial cells LLC-PK1 secrete high amounts of urokinase-type plasminogen activator (uPA) when stimulated through the cAMP-signaling pathway. Here we show that this characteristic can be used to identify rapidly LLC-PK1-derived cell lines expressing heterologous receptors that couple to Gs-proteins. A genomic clone encoding the mouse beta 2 adrenergic receptor (beta 2AR) was transfected stably into LLC-PK1 cells. Drug-resistant colonies were subsequently stimulated with isoproterenol and screened for the secretion uPA activity. Positive clones displayed dose-dependent uPA activity in response to isoproterenol, specifically bound the beta 2AR-agonist iodocyanopindolol and expressed beta 2AR-mRNA. The functional coupling of beta 2AR expression to the inducible uPA gene establishes that LLC-PK1 cells can be used to assay for beta 2AR (and beta 2AR-agonist) function. Our data suggest that this system can be used for functional expression of other heterologous receptors that couple to Gs-proteins.

Animals↗

PubMed1993

Developmental changes of protein kinase C and Gs alpha in hypertrophic cardiomyopathic hamster hearts.

Protein kinase C (PKC) and GTP-binding proteins (G-proteins) are known to be major determinants in the modulation of cardiac function. In this study, we examined the developmental changes of PKC and the alpha-subunit of the stimulatory guanosine triphosphate binding protein (Gs alpha) in hypertrophic cardiomyopathic Syrian hamster (BIO 14.6) hearts, before the onset of hypertrophy (30 days old) and at the peak of hypertrophy (6 months old) and compared these with age-matched control hamster (BIO RB) hearts. At 30 days, cardiac PKC activity was similar between BIO 14.6 and BIO RB both in the membrane (117.1 +/- 9.9 pmol/min/mg vs 131.2 +/- 12.7 pmol/min/mg in controls, n = 8) and in the cytosolic fractions (213.1 +/- 22.0 pmol/min/mg vs 186.6 +/- 23.9 pmol/min/mg in controls, n = 9). At 6 months, PKC activity was significantly higher in BIO 14.6 than in controls, both in the cardiac membrane (131.9 +/- 7.1 pmol/min/mg vs 40.7 +/- 4.7 pmol/min/mg in controls, n = 8, p < 0.00001) and cytosol (77.9 +/- 2.1 pmol/min/mg vs 54.6 +/- 3.3 pmol/min/mg in controls, n = 6, p < 0.0005). In BIO RB hearts, membrane and cytosolic PKC activities were significantly reduced at 6 months compared with those at 30 days of age (p < 0.001). However, the membrane PKC activity in 6-month-old BIO 14.6 was maintained at the level of the 30-day-old hearts. On the other hand, the relative immunoreactive amounts of Gs alpha were similar between BIO RB and BIO 14.6 hearts at 30 days and at 6 months of age.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Coupling of endothelin B receptors to the calcium pump and phospholipase C via Gs and Gq in rat liver.

We have demonstrated in liver from male rats that both endothelin A (ETA) and ETB receptors coexist in equal proportion and that ETA receptors mediate a calcium-dependent activation of glycogenolysis. We describe here a sex difference in endothelin action in hepatocytes because, in female rats, 80% of the ET receptors are of ETB type and, accordingly, activation of glycogenolysis is an ETB-mediated process (EC50 = 0.03 pM). ET-1 stimulation of glycogenolysis in female rats was consecutive to activation of phosphatidylinositol 4,5-bisphosphate hydrolysis (EC50 = 0.03 pM) and to inhibition of the calcium extrusion pump (IC50 = 0.03 pM) in plasma membranes, with ET-1 approximately sarafotoxin S6C approximately ET-3. Endothelin regulation of each effector was potentiated by GTP gamma S. ET-1 did not stimulate adenylyl cyclase activity. To identify the nature of the guanine nucleotide regulatory proteins (G protein(s)) coupling ETB receptors to each effector, we used antibodies against the COOH terminus of different G protein alpha subunits. Antibodies reactive with Gs alpha (RM) blocked ET-1 inhibition of the calcium pump, while they did not affect ET-1 stimulation of phospholipase C. Antibodies reactive with Gq alpha (QL) dose-dependently antagonized stimulation of phospholipase C by ET-1 and vasopressin, without affecting ET-1 inhibition of the calcium pump. Antibodies reactive with Gi1 alpha/Gi2 alpha (AS) had no effect on either system. We conclude that the calcium signal provoked by endothelins in hepatocyte is not only consecutive to activation of phospholipase C but also to inhibition of the plasma membrane calcium pump, each effector being coupled to ETB receptors by different G proteins, Gq, and Gs.

Amino Acid Sequence↗

PubMed1999

Desensitization of cardiac beta-adrenoceptor signaling with heart failure produced by myocardial infarction in the rat. Evidence for the role of Gi but not Gs or phosphorylating proteins.

This study examined mechanisms of beta-adrenergic (AR) desensitization in a myocardial infarction (MI) model of heart failure in the rat. Inotropic responses to isoproterenol (non-selective beta-AR agonist) and RO 363 (selective beta1-AR agonist), in left atria and left papillary muscle, were reduced by up to 65%, while chronotropic responses in right atria were unaffected. beta1- and beta2-AR density did not change after MI, suggesting that changes in beta-AR responsiveness are due to changes occurring downstream of the receptor. Inotropic and chronotropic responses to forskolin were not altered in right and left atria and left papillary muscle after MI, suggesting changes at the level of the G-proteins. Pertussis toxin treatment of animals restored inotropic responses to isoproterenol in left atria and left papillary muscle to levels seen in the sham group, indicating that inactivation of Gi-proteins improves inotropic function in MI rats, and that beta-ARs couple to Gi in cardiac failure. Expression of G-protein receptor kinase 2 (GRK2), beta-arrestin1 and the regulatory subunits of cAMPdPK (RI alpha and RII alpha), showed no change after MI. However the expression of Gi alpha2 was significantly increased in left ventricle (sham 0.888+/-0.140, MI 1. 759+/-0.352 P=0.026), right ventricle (sham 0.031+/-0.004, MI 0. 037+/-0.002 P=0.006) and atria (sham 0.107+/-0.006, MI 0.138+/-0.006 P=0.004), with no changes observed in the expression of Gs alpha. These results suggest that increases in Gi play an important role in the decreased beta-AR responsiveness in the rat model of MI.

Adrenergic beta-Agonists↗

PubMed2001

Gi- and Gs-coupled receptors up-regulate the cAMP cascade to modulate HCN2, but not HCN1 pacemaker channels.

A hallmark of native pacemaker channels is their regulation by neurotransmitters and hormones acting through the second messenger cAMP. In this study, we investigated the modulation of two cloned pacemaker channels, HCN1 and HCN2, by activation of coexpressed inhibitory G protein (Gi)-coupled (p-opioid) or stimulatory G protein (Gs)-coupled [serotonin 5-HT4(a)] receptors in Xenopus oocytes. Both receptors enhanced HCN2, but not HCN1 currents. Receptor activation increased HCN2 current amplitude, increased the activation rate sixfold and decreased the deactivation rate two-fold. In addition, the fully-activated current for HCN2 increased due to a receptor-induced increase of the maximal conductance. These effects were inhibited by 9-(tetrahydro-2'-furyl)adenine (SQ22536), were independent of protein kinases A and C and could be explained by a cAMP-induced shift of the voltage dependence of activation by 15 mV to more positive potentials. The pathway through which these effects occurred involved Gbetagamma-activation of adenylyl cyclase and, in the case of the p-opioid receptor, required co-expression of Galphas. The effect of the 5-HT4(a)-receptor, in part caused by its constitutive activity, occurred directly through Galphas-activation. This suggests that 5-HT4(a) receptors may contribute to functional heterogeneity of pacemaker currents (Ih) in those neurons in which 5-HT4(a)R and HCN2 coexist.

Adenine↗

PubMed2006

Effects of dopamine receptor type 1 and Gs protein alpha subunit gene polymorphisms on blood pressure at rest and in response to stress.

BACKGROUND: Exaggerated blood pressure (BP) response to behavioral stress has been recognized as an independent predictor of increased BP levels in human subjects. The coupling of dopamine receptor type 1 (DRD1) with stimulatory G-proteins plays an important role in BP regulation. Therefore, we investigated whether the A-48G polymorphism of the DRD1 gene and the T393C polymorphism of the Gs protein alpha subunit (GNAS) gene influenced BP levels at rest and in response to stress. METHODS: A total of 912 white and African American (44.5%) twin subjects (17.4 +/- 3.4 years) were genotyped. Systolic and diastolic BP were measured at rest and during two 10-min stress tasks (a social competence interview and a virtual reality car driving simulation test). Regular association analyses and sibpair transmission disequilibrium tests were performed. The two polymorphisms, A-48G (DRD1) and T393C (GNAS), were genotyped by polymerase chain reaction with restriction digestion enzymes. RESULTS: A dominant BP-lowering effect of the -48G allele was observed on diastolic BP at rest (P = .032) and in response to the car driving simulation test (P = .046). The 393C allele was associated with higher systolic BP levels during the social competence interview (P = .024) and the car driving simulation test (P = .016). There were no statistical significances between the two loci for systolic and diastolic BP at rest and during stress. CONCLUSIONS: Our findings suggest that DRD1 and GNAS loci contribute to BP regulation at rest, and in particular, in response to behavioral stress. The BP measurement during behavioral stress may have added value for gene association studies.

Adolescent↗

PubMed2005

G protein betagamma11 complex translocation is induced by Gi, Gq and Gs coupling receptors and is regulated by the alpha subunit type.

G protein activation by Gi/Go coupling M2 muscarinic receptors, Gq coupling M3 receptors and Gs coupling beta2 adrenergic receptors causes rapid reversible translocation of the G protein gamma11 subunit from the plasma membrane to the Golgi complex. Co-translocation of the beta1 subunit suggests that gamma11 translocates as a betagamma complex. Pertussis toxin ADP ribosylation of the alphai subunit type or substitution of the C terminal domain of alphao with the corresponding region of alphas inhibits gamma11 translocation demonstrating that alpha subunit interaction with a receptor and its activation are requirements for the translocation. The rate of gamma11 translocation is sensitive to the rate of activation of the G protein alpha subunit. alpha subunit types that show high receptor activated rates of guanine nucleotide exchange in vitro support high rates of gamma11 translocation compared to alpha subunit types that have a relatively lower rate of guanine nucleotide exchange. The results suggest that the receptor induced translocation of gamma11 is controlled by the rate of cycling of the G protein through active and inactive forms. They also demonstrate that imaging of gamma11 translocation can be used as a non-invasive tool to measure the relative activities of wild type or mutant receptor and alpha subunit types in a live cell.

Animals↗

PubMed1998

GTP analogue hydrolysis by the Gs protein: implication for the role of catalytic glutamine in the GTPase reaction.

Hydrolysis of GTP, bound to members of the G-protein superfamily, terminates their downstream signaling activity. A conserved glutamine serves a critical role in this pivotal guanosine triphosphatase (GTPase) reaction. However, the role of the catalytic glutamine in GTP hydrolysis is still not well understood. We have employed substrate-assisted catalysis to probe the catalytic mechanism of Gs alpha using GTP analogues. These GTP analogues, each having different functional groups, were designed to support or refute particular putative GTPase mechanisms. We have found that a hydrogen donor group, in close proximity to the gamma-phosphate of GTP, is necessary and sufficient to substitute for the function of the catalytic glutamine in the GTPase reaction.

Adenosine Triphosphate↗

PubMed2002

Contribution of the two Gs-coupled PGE2-receptors EP2-receptor and EP4-receptor to the inhibition by PGE2 of the LPS-induced TNFalpha-formation in Kupffer cells from EP2-or EP4-receptor-deficient mice. Pivotal role for the EP4-receptor in wild type Kupffer cells.

BACKGROUND/AIMS: Prostaglandin E2 (PGE2) is known to inhibit the lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNFalpha) formation in Kupffer cells via an increase in cAMP. Four receptor-subtypes have been cloned for PGE2 so far. Two of them, the EP2-receptor and the EP4-receptor are linked to stimulatory Gs-proteins and could mediate the inhibition by PGE2 of TNFalpha-formation. METHODS: The significance of both receptors for PGE2-dependent inhibition of LPS-induced TNFalpha-formation was studied using Kupffer cells of mice in which either one of the two receptors had been eliminated by homologous recombination. RESULTS: The mRNAs of both receptors were expressed in wild type mouse Kupffer cells. Exogenous PGE2 inhibited TNFalpha-formation in Kupffer cells lacking either EP2-receptor or EP4-receptor to a similar extent as in control cells, however, 10-fold higher PGE2 concentrations were needed for half maximal inhibition in cells lacking the EP4-receptor than in control or EP2-receptor-deficient cells. The response to endogenous PGE2 was blunted in EP4-receptor-deficient mice only and especially after prolonged incubation. CONCLUSIONS: The data indicate, that PGE2 can inhibit TNFalpha-formation via both the EP2- and the EP4-receptor and that, however, the EP4-receptor appears to be physiologically more relevant in Kupffer cells since it conferred a high affinity response to PGE2.

Animals↗

PubMed1997

Beta-adrenoreceptor coupling to GS protein in alcohol dependence, panic disorder, and patients with both conditions.

Ethanol may downregulate G-protein-coupled beta-adrenoreceptors (beta AR). beta AR may also be dysregulated in panic disorder (PD). In clinical samples, many patients have comorbid alcohol dependence (AD) and PD. Therefore, we investigated beta AR coupling in patients with these disorders. We harvested polymorphonuclear leukocytes from 24 healthy volunteers (Vs), and from 22 abstinent AD patients, 7 PD patients, and 9 patients with comorbid AD/PD. beta AR were assayed using saturation and agonist-displacement experiments. Group differences were tested using one-way analysis of variance (ANOVA). All beta AR binding parameters were similar in AD patients and Vs. The ratio of the agonists' dissociation constant from the receptor in the low affinity state (KL) to that in the high affinity state (KH) was significantly higher in PD patients than in AD patients and Vs (930.97 +/- 440.80 vs. 226.2 +/- 94.47 vs. 197.05 +/- 61.03, respectively, p < .01). This finding suggests that beta AR are supercoupled to GS in patients with PD. There was a trend for higher total receptor density (RT) in AD/PD and PD patients (Vs = 39.06 +/- 42.57 vs. AD = 27.93 +/- 23.07 vs. AD/PD = 66.85 +/- 79.02 vs. PD = 68.36 +/- 49.20, p < .08). There were no differences between AD/PD and PD patients, who combined had a significantly higher RT than Vs and AD patients (Vs = 38.95 +/- 8.81 vs. AD = 29.63 +/- 5.07 vs. AD/PD = 67.51 +/- 17.00, fmol/mg protein, p < .04). Finally, AD/PD patients had a significantly higher receptor density in the low-affinity conformational state than Vs and AD patients, but not PD patients (25.96 +/- 11.59 vs. 10.69 +/- 1.53 vs. 7.62 +/- 1.08 vs. 17.07 +/- 5.26 fmol/mg protein, respectively, p < .005). beta AR function in polymorphonuclear leukocytes is normal in abstinent alcoholics. The previously reported abnormal beta AR regulation in alcoholism may be state dependent. The higher RT and KL/KH ratio in AD/PD and PD, but not in AD patients, suggest that increased beta AR function may be important in the pathophysiology of PD.

Adrenergic beta-Agonists↗

PubMed2005

Mutational analysis of PRKAR1A and Gs(alpha) in sporadic adrenocortical tumors.

Little is known about the pathogenesis of adrenocortical tumors. The cAMP-dependent pathway is physiologically activated by ACTH in adrenocortical cells and different components of this cascade may be altered in some functioning adrenocortical tumors. Recently, mutations of the gene encoding the PKA type 1 A regulatory subunit (R1 A), PRKAR1A, associated with loss of heterozygosity (LOH) at PRKAR1A locus, have been demonstrated in primary pigmented nodular adrenocortical disease (PPNAD), either isolated or associated with Carney complex. Moreover, activating mutations of the Gs(alpha) gene (the gsp oncogene) have also been found in a small number of adrenocortical cortisol-secreting adenomas. Aim of this study was to investigate the presence of such genetic alterations on a series of 10 ACTH-independent Cushing syndrome due to non-PPNAD adrenocortical adenomas. The coding sequence of PRKAR1A, evaluated by PCR and direct sequencing analysis, revealed the absence of mutations while heterozygosity for at least 1 polymorphism excluded LOH in most tumors. In one single adenoma gsp mutation was detected. In conclusion, we provide additional evidence that the only mutational changes able to activate the cAMP pathway so far identified, i.e. PRKAR1A mutations and gsp oncogene, are a rare event in adrenocortical tumors.

Adrenal Cortex Neoplasms↗