Gs Protein Function Research Articles

Allergic Airway Inflammation and β-Adrenoceptor Dysfunction

A reduced response to beta-adrenoceptor agonists has been reported in airways of asthmatic subjects. Mechanisms for beta-adrenoceptor dysfunction are (1) inactivation or downregulation of beta-adrenoceptors by specific agonists (homologous desensitization) or by inflammatory mediations (heterologous desensitization), (2) inactivation or downregulation of second messengers of beta-adrenoceptor pathway. Studies from our laboratory have shown that allergen challenge of passively sensitized human bronchi causes a beta-adrenoceptor dysfunction as a result of reduced activity of the receptor-coupled Gs protein. The dysfunction was prevented by leukotriene-receptor antagonists or a cell membrane stabilizer, but not by an antihistimine or indomethacin, suggesting a central role of leukotrienes released from resident inflammatory cells in its genesis. Furthermore, the beta-adrenoceptor response and Gs protein function after allergen exposure were restored by short-term (3 h) incubation with beclomethasone dipropionate, suggesting an effect independent of gene transcription. Restoration of the intracellular beta-adrenoceptor effector pathway may contribute to the efficacy of corticosteroids in the acute treatment of asthma.

Influence of relaxin and insulin on the activity of adenylate cyclase in the myometrium of pregnant women with type 1 diabetes mellitus

The study of the influence of relaxin and insulin on the activity of adenylate clase (AC) in the myometrium of pregnant women showed that human relaxin 2 (10-9-10-8 M) causes a stronger stimulating effect on the enzyme activity than insulin. The activating effect of relaxin and insulin was potentiated by a non-hydrolyzable GTP analogue, guanylylimidodiphosphate (GIDP), in the myometrium of pregnant women, which indicates the participation of the stimulating GTP-binding protein (Gs-protein) in the realization of the effect of peptides. In the myometrium of pregnant women with type 1 diabetes mellitus under the action of relaxin, insulin and isoproterenol, a decrease in the sensitivity of AC activity to these hormones was found in comparison with these indicators in the myometrium of pregnant women. With the combined effect of peptides and GIDP in the myometrium of pregnant women with type 1 diabetes mellitus, a weakening of the potentiation of the GIDP effect of hormones on AC activity was observed, which may indicate a decrease in the coupling between receptors, Gs protein, and AC. The studies carried out allow us to conclude that in pregnant women with type 1 diabetes mellitus, the decrease in AC stimulation under the influence of these hormones is associated with a weakening of the Gs-protein function.

A decrease in the amount and function of the stimulatory GTP-binding protein in the small resistance arteries of spontaneously hypertensive rats.

We investigated the concentration of stimulatory GTP-binding protein (Gs protein) in the peripheral resistance arteries of spontaneously hypertensive rats (SHR), normotensive Wistar-Kyoto rats (WKY), and renovascular hypertensive rats (RHR). Changes in the function of Gs protein in SHR and WKY were also investigated by microcannulation techniques. The localization and abundance of Gs protein were determined immunohistochemically in 4-, 10- and 20-week-old SHR and age-matched WKY (control), as well as in RHR. Sections of the cremaster artery were stained with polyclonal antibodies to Gs protein. The concentration of Gs protein-like immunoreactivity in the cremaster artery was significantly lower in SHR at 4, 10, and 20 weeks of age, relative to that in age-matched WKY. In contrast, no significant differences were detected in the abundance of Gs between RHR and control rats. The dilatory response by isoproterenol in the presence of beta1-adrenoceptor blocker was lower in 4- and 10-week-old SHR than in age-matched WKY. The dilatory response by cholera toxin was also lower in SHR than in WKY for these two age groups. These results indicated that the amount and function of Gs protein in the peripheral resistance vessels in SHR was reduced. Since this change occurred before the onset of hypertension and no changes were seen in the secondary hypertensive rats, this change was not a secondary change due to hypertension. The impaired receptor-Gs protein-mediated signal transduction in the peripheral resistance arteries may be one of the possible mechanisms responsible for the pathogenesis of hypertension in SHR.

Developmental enhancement of secretory response to isoproterenol coupled with increases in β-adrenoceptor density and Gs protein function in rat parotid tissues

The β-adrenergic agonist, isoproterenol (IPR), stimulated more significantly and sensitively amylase secretion from both the tissues of 7- and 56-day-old rats than a cholinergic agonist, carbachol, at the same concentration. The EC 50 value of amylase secretion with IPR decreased significantly during development but that with carbachol did not change. Estimation by measuring bindings of [ 3H]dihydroalprenolol and [ 3H]quinuclidynylbenzylate indicated the marked increases in the numbers of both β-adrenoceptors and muscarinic receptors in the tissues during development. The affinity of β-adrenoceptors for the agonist was also enhanced during development, but that of muscarinic receptors for the agonist was not. These developmental changes in the number and affinity of β-adrenoceptors and muscarinic receptors paralleled those in amylase secretory response of the tissues to their agonists. The response of adenylate cyclase (AC) of the tissues to 1 μM IPR was steadily enhanced after birth. In contrast, the response of AC to 1 μM forskolin was high until 14 days old, but markedly decreased at 28 days old and thereafter maintained this level. The increase in cholera toxin-catalyzed ADP-ribosylation (AR) of stimulatory GTP binding proteins (Gs proteins) in the tissues was apparent at 14 days old, reaching a maximum at 56 days old and thereafter decreasing with age. On the other hand, pertussis toxin-catalyzed AR of inhibitory GTP binding proteins (Gi proteins) did not change after birth. Thus, the ratio of apparent levels of Gs to Gi proteins increased significantly after birth, reaching a maximum at 56 days old, but decreased rapidly till 84 days old and thereafter maintained this level. These changes in the ratio paralleled those in the response of AC to IPR. These results showed that the rapid and marked increases in the number and affinity of β-adrenoceptors and the ratio of apparent levels of Gs to Gi proteins in rat parotid tissues during development had a key role in the enhancement of the secretory response of the tissues to β-agonists.

An anaesthetic dose of ethanol inhibits cyclic AMP formation by altering the function of GS protein.

The effects of ethanol on cerebral gamma-aminobutyric acid (GABA)B receptor and its related signal transduction systems were studied. [3H]GABA binding to cerebral GABAB receptors in crude synaptic membranes from rat brain was unaffected by ethanol at concentrations up to 100 mM, although 100 mM ethanol suppressed not only the basal but also forskolin-stimulated cAMP formation in cerebral cortical membrane vesicles. The suppressive effect of baclofen and the stimulatory effect of forskolin on cAMP formation was also found to be unaltered by ethanol. Pretreatment of the membrane vesicles with islet-activating protein (IAP), which eliminates the function of Gi protein by its ribosylation, had no effect on the suppressive effects of ethanol on basal and forskolin-stimulated cAMP formation. In contrast, pretreatment of membrane vesicles with 'low pH', which is known to suppress the function of Gs protein, eliminated the suppressive effect of ethanol on cAMP formation. These results suggest that an anaesthetic dose of ethanol, which is below that necessary to cause changes in receptor binding, may not affect the functions of GABAB/Gi, G(O) protein/adenylate cyclase system, but suppresses cAMP formation systems which are coupled with Gs protein.

Reduced Gs protein function and Gαs levels in leukocytes of patients with Parkinson's disease

Early events in signal information transduction beyond dopamine, beta-adrenergic, and muscarinic receptors, involving receptor-coupled G-protein function and G alpha subunit immunoreactive levels were measured in mononuclear leukocytes (MNLs) of 12 never-treated patients with Parkinson's disease in comparison with 10 age- and sex-matched healthy control subjects. Both beta-adrenergic and dopamine receptor-coupled Gs protein function as measured by cholera toxin-sensitive, isoproterenol- and dopamine-induced increases in Gpp(NH)p-binding capacity, in MNLs of patients with Parkinson's disease were found to be significantly reduced in comparison with those in the control group. Muscarinic receptor-coupled non-Gs (Gi or G(o)) protein function: pertussis toxin-sensitive, carbamylcholine-induced increase in Gpp(NH)p-binding capacity, was not found to be significantly different between patients with Parkinson's disease and control subjects. G protein alpha subunits were measured through immunoblotting analyses with specific polyclonal antibodies against G alpha s, G alpha i, and G alpha q subunits. MNL levels of the 45-kDa species of G alpha s were found to be significantly reduced in patients with Parkinson's disease in comparison with control subjects. Other non-Gs proteins (Gi, Gq) did not show any significant quantitative differences between patients with Parkinson's disease and control subjects. The reductions in G alpha s levels in MNLs of patients with Parkinson's disease may explain the beta-adrenergic and dopamine receptor-coupled Gs protein hypofunction detected in MNLs of these patients. As previous studies have failed to observe significant changes in receptor levels in MNLs of patients with Parkinson's disease, our findings of reduced dopaminergic and beta-adrenergic receptor-coupled Gs function and of G alpha s immunoreactive levels in MNLs of Parkinson's patients point to alterations distal to these receptors at the level of the signal-transducing Gs protein.

P.2.018 - Reduced Gs protein function and Gαs levels in leukocytes of patients with Parkinson's disease

P.2.018 - Reduced Gs protein function and Gαs levels in leukocytes of patients with Parkinson's disease

Functional and quantitative measures of receptor-coupled G proteins in human mononuclear leukocytes: no change with age.

Aging has been associated with alterations in signal transduction for a number of neurotransmitter receptors in human tissues. Heterotrimeric G proteins play a pivotal role in postreceptor information transduction, by coupling a variety of hormone and neurotransmitter receptors to several intracellular effector functions. Developmental and age-related changes in the abundance of specific G alpha subunits have been shown in the human brain. In the present study, functional and quantitative measures of G proteins were conducted in human mononuclear leukocytes obtained from 19 healthy subjects of increasing age. Gs protein function, assessed through cholera toxin-sensitive beta-adrenergic and dopaminergic agonists induced increases in 3H-Gpp(NH)p binding capacities to membranes of mononuclear leukocytes, and Gi protein function, assessed through pertussis toxin-sensitive muscarinic agonist induced increase in guanine nucleotide binding capacity, were found to be unaltered by increasing age. Immunobloting analyses with specific polyclonal antibodies against G alpha s, G alpha i, and G alpha q subunit proteins in mononuclear leukocyte membranes obtained from the same subjects showed that the quantities of these proteins in mononuclear leukocytes were as well independent of age. Insofar as age-related alterations in cellular information transduction mechanisms in peripheral tissues are important from the etiological, diagnostic, and pharmacological aspects of age-related disorders, it is important to know that both the coupling of receptors to G proteins, the function of these proteins, and their abundance in human peripheral mononuclear leukocytes stays unaltered by the aging process.

Reduced β-adrenergic receptor-coupled Gs protein function and Gsα immunoreactivity in mononuclear leukocytes of patients with depression

beta-Adrenergic receptor-coupled Gs protein function was measured in 26 depressed patients through cholera toxin-sensitive, isoproterenol-induced increases in 3H-Gpp(NH)p binding capacity to mononuclear leukocytes (MNL). Highly significant reductions in receptor-coupled Gs protein function were observed in the depressed patients: 2.0 +/- 1.3% increases in guanine nucleotide-binding capacity, in comparison with the control group values of 28.3 +/- 6.9%. Similar reductions in Gs protein function were detected in both uni- and bipolar depressed patients. A significant negative correlation was found between receptor-coupled Gs protein measures and the severity of depression. Adding semiquantitative measures of MNL Gs alpha through immunoblot analysis by use of polyclonal antibodies against Gs alpha subunit, it was found that Gs alpha relative immunoreactivity was reduced from 100 +/- 2.0% in the control group of subjects to 75.9 +/- 2.3% in the depressed patients. We have previously described hyperfunctional Gs proteins in leukocytes of patients with mania. The present findings of reduced function of Gs in depressed patients suggests receptor-coupled Gs protein activity as a biochemical parameter indicatory of the affective state. Reduced receptor-coupled Gs protein function may reflect reduced levels of the beta-adrenergic receptor previously shown in leukocytes of depressed patients; however, our complementary immunoblot studies suggest a direct, postreceptor, quantitative, and functional reduction in Gs protein in MNL of depressed patients.

The guanine nucleotide-binding regulatory proteins (G proteins) in myocardium with ischemia.

Guanine nucleotide-binding regulatory proteins (G proteins) play a major role in the regulation of a number of physiological processes, such as stimulation or Inhibition of adenylate cyclase activity or gaiting of ionic channels. Myocardial ischemia could induce the changes in receptor-G protein signal transduction system in the heart. Therefore, this article will focus on the role and alterations of G proteins (especially, Gs and Gi) in myocardial ischemia. The Gi protein rapidly loses functional activity during very early myocardial ischemia. In contrast to Gi protein, the function of Gs protein during this phase has not been evaluated. Moreover, the changes in Gs protein after 30 min of ischemia are contradictory. However, the sensitization of the adenylate cyclase activity in the very early phase of acute ischemia is gradually replaced by a decrease in adenylate cyclase activity with prolonged ischemia. The decrease in the function and amount of Gs protein may be one of the factors that induce these changes. The function of Gs protein was also decreased in the canine hearts with ischemia and reperfusion. In contrast to ischemia and reperfusion, there are no significant alterations in G proteins and modulation of adenylate cyclase in the stunned myocardium. It has become increasingly evident that Gi protein may play an important role in the cardioprotective effects of preconditioning. When beta-adrenoreceptor densities are reduced in chronic myocardial ischemia, decreased in the amount and function of Gi protein and increased amount of Gs protein may play the role in preservation of the adenylate cyclase activity. These alterations in G proteins may play the important role in the myocardial function during myocardial ischemia.

Drug-Induced Taste and Smell Disorders

Drugs in every major pharmacological category can impair both taste and smell function and do so more commonly than presently appreciated. Impairment usually affects sensory function at a molecular level, causing 2 major behavioural changes--loss of acuity (i.e. hypogeusia and hyposmia) and/or distortion of function (i.e. dysgeusia and dysosmia). These changes can impair appetite, food intake, cause significant lifestyle changes and may require discontinuation of drug administration. Loss of acuity occurs primarily by drug inactivation of receptor function through inhibition of tastant/odorant receptor: (i) binding; (ii) Gs protein function; (iii) inositol trisphosphate function; (iv) channel (Ca++,Na++) activity; (v) other receptor inhibiting effects; or (vi) some combination of these effects. Distortions occur primarily by a drug inducing abnormal persistence of receptor activity (i.e. normal receptor inactivation does not occur) or through failure to activate: (i) various receptor kinases; (ii) Gi protein function; (iii) cytochrome P450 enzymes; or other effects which usually (iv) turn off receptor function; (v) inactivate tastant/odorant receptor binding; or (vi) some combination of these effects. Termination of drug therapy is commonly associated with termination of taste/smell dysfunction, but occasionally effects persist and require specific therapy to alleviate symptoms. Treatment primarily requires restoration of normal sensory receptor growth, development and/or function. Treatment which restores sensory acuity requires correction of steps initiating receptor and other pathology and includes zinc, theophylline, magnesium and fluoride. Treatment which inhibits sensory distortions requires reactivation of biochemical inhibition at the receptor or inactivation of inappropriate stimulus receptor binding and/or correction of other steps initiating pathology including dopaminergic antagonists, gamma-aminobutyric acid (GABA)-ergic agonists, calcium channel blockers and some orally active local anaesthetic, antiarrhythmic drugs.

Manganous chloride stimulation of adenylate cyclase responsiveness in ocular ciliary process membranes

Manganous chloride was compared with magnesium chloride in supporting maximal stimulations of the adenylate cyclase system in ocular ciliary process membranes by isoproterenol, vasoactive intestinal peptide, sodium fluoride, guanosine 5'-(beta, gamma-imino) triphosphate (GppNHp), or forskolin, and in supporting synergism between isoproterenol and forskolin. The primary effect of Mn2+ (2 mM) was due to an interaction at the catalytic unit. Mn2+ had no significant effect on the function of the GTP-binding stimulatory G-protein (Gs) which couples beta-adrenergic receptors to the catalytic unit of adenylate cyclase. However, Gs-protein function was impaired by Mn2+ relative to Mg2+ when GppNHp was used instead of GTP as the ligand for the Gs-protein. Compared with Mg2+, Mn2+ caused a 4-5.5-fold increase in adenylate cyclase responsiveness to all the activators tested (except GppNHp, where the increase was 2.5-3.5-fold). Thus Mn2+ ions appeared to be intrinsically more effective at divalent cation binding sites on the catalytic unit that control its enzymatic activity. Ciliary process membranes differ from erythrocyte and S49 lymphoma cell membranes where 2 mM Mn2+ strongly inhibits hormone-Gs-protein-mediated stimulations of adenylate cyclase. Divalent cations bound to the catalytic unit also affected the degree of synergism between hormone-activated Gs and forskolin to stimulate adenylate cyclase activity. In the presence of MgCl2 all effective doses of isoproterenol and forskolin in combination showed marked synergism. In contrast, with MnCl2 there was no synergism with high-dose isoproterenol-forskolin combinations, which gave only additive responses.