Inhibitory GTP-binding Protein Research Articles

Functional interaction of H2-receptors and 5HT4-receptors in atrial tissues isolated from double transgenic mice and from human patients

In the past, we generated transgenic mice that overexpress the human histamine 2 (H2)-receptor (H2-TG) or that overexpress the human serotonin 4 (5-HT4)-receptor (5-HT4-TG) in the heart. Here, we crossbred these lines of mice to generate double transgenic mice that overexpress both receptors (DT). This was done to study a conceivable interaction between these receptors in the mouse heart as a model for the human heart. When in left atria, initially, force of contraction was elevated maximally with 1 µM serotonin, and subsequently, histamine was cumulatively applied; a biphasic effect of histamine was noted: the force of contraction initially decreased, maximally at 10 nM histamine, and thereafter, the force of contraction increased again at 1 µM histamine. Notably, functional interaction between 5-HT and histamine was also identified in isolated electrically stimulated trabeculae carneae from human right atrium (obtained during cardiac surgery). These functional and biochemical data together are consistent with a joint overexpression of inotropically active H2-receptors and 5-HT4-receptors in the same mouse heart. We also describe an antagonistic interaction on the force of contraction of both receptors in the mouse atrium (DT) and in the human atrial muscle strips. We speculate that via this interaction, histamine might act as a “brake” on the cardiac actions of 5-HT via inhibitory GTP-binding proteins acting on the activity of adenylyl cyclase.

Mutation analysis of inhibitory guanine nucleotide binding protein alpha (GNAI) loci in young and familial pituitary adenomas.

Pituitary adenomas are neoplasms of the anterior pituitary lobe and account for 15–20% of all intracranial tumors. Although most pituitary tumors are benign they can cause severe symptoms related to tumor size as well as hypopituitarism and/or hypersecretion of one or more pituitary hormones. Most pituitary adenomas are sporadic, but it has been estimated that 5% of patients have a familial background. Germline mutations of the tumor suppressor gene aryl hydrocarbon receptor-interacting protein (AIP) predispose to hereditary pituitary neoplasia. Recently, it has been demonstrated that AIP mutations predispose to pituitary tumorigenesis through defective inhibitory GTP binding protein (Gαi) signaling. This finding prompted us to examine whether germline loss-of-function mutations in inhibitory guanine nucleotide (GTP) binding protein alpha (GNAI) loci are involved in genetic predisposition of pituitary tumors. To our knowledge, this is the first time GNAI genes are sequenced in order to examine the occurrence of inactivating germline mutations. Thus far, only somatic gain-of-function hot-spot mutations have been studied in these loci. Here, we have analyzed the coding regions of GNAI1 , GNAI2, and GNAI3 in a set of young sporadic somatotropinoma patients (n = 32; mean age of diagnosis 32 years) and familial index cases (n = 14), thus in patients with a disease phenotype similar to that observed in AIP mutation carriers. In addition, expression of Gαi proteins was studied in human growth hormone (GH), prolactin (PRL), adrenocorticotropic hormone (ACTH)-secreting and non-functional pituitary tumors. No pathogenic germline mutations affecting the Gαi proteins were detected. The result suggests that loss-of-function mutations of GNAI loci are rare or nonexistent in familial pituitary adenomas.

Abstract 4452: Regulation of histone deacethylase 8 expression by inhibitory GTP binding proteins signaling via JNK pathway in H1299 non-small-cell lung cancer cells

Abstract Histone deacetylases (HDACs) have been reported to regulate the cell growth, proliferation, and apoptosis, and HDAC8 seems to be involved in tumor growth and progression. Inhibitory heterotrimeric GTP-binding proteins (Gαi) are activated by seven transmembrane receptors, and regulate cell proliferation by regulating the activity of adenylate cyclase, phospholipase C beta, and ion channels. This study investigated the effects of Gαi signaling system on the expression of HDAC8 and the underlying mechanism in lung cancer cells. When Gi signaling system was activated by expressing Gαi1Q204L, a constitutively active mutant Gαi1, the expression of HDAC8 protein and mRNA was increased in H1299 non-small-cell lung cancer (NSCLC) cells. Inhibition of transcription by treatment with Actinomycin D did not block the increase in mRNA, and the degradation of HDAC8 mRNA was facilitated in Gαi1Q204L -expressing cells, suggesting Gαi1 signaling system increases HDAC8 expression by stabilizing mRNA. In addition, treatment with adenosine or an adenosine receptor agonist, CCPA (2-chloro-N(6)-cyclopentyladenosine), which activate Gαi-coupled receptors, also increased HDAC8 mRNA and protein levels. The effect of adenosine was blocked by treatment with DPCPX (8-Cyclopentyl-1,3-dipropylxanthine), an adenosine A1 receptor antagonist. JNK phosphorylation was inhibited by treatment with the adenosine and CCPA. Treatment with a JNK inhibitor, SP600125 increased HDAC8 expression. Adenosine inhibited the activation of MKK4 and CDC 42, which act upstream of JNK. Activation of Gαi signaling stimulated proliferation of H1299 cells, and the stimulating effect was abolished by knockdown of Gαi1. Thus, we conclude that Gαi signaling increases HDAC8 expression via JNK pathway, resulting in stimulation of cell proliferation of H1299 lung cancer cells. This study suggests that Gαi signaling may be involved in epigenetic control of gene expression by regulating HDAC8 expression. Citation Format: Hwa-jeong Hahn, Yong-Sung Juhnn. Regulation of histone deacethylase 8 expression by inhibitory GTP binding proteins signaling via JNK pathway in H1299 non-small-cell lung cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4452. doi:10.1158/1538-7445.AM2014-4452

Amrinone, Forerunner of Novel Cardiotonic Agents, Caused Paradigm Shift of Heart Failure Pharmacotherapy

Amrinone has been a great forerunner of noncatechol and nonglycoside novel cardiotonic agents and given an extraordinary strong impact in the development of novel cardiotonic agents. Unexpectedly, amrinone failed to rescue the patients with chronic heart failure, thereby produced a prominent paradigm shift from inotropic to cardioprotective therapy, and contributed to advances in the current pharmacotherapy of chronic heart failure. In this study, Alousi et al showed that amrinone is a potent, long-acting positive inotropic agent when given orally or intravenously to either anesthetized or unanesthetized dogs. Amrinone was orally active with a rapid onset of action and duration of >5 hours and showed a wide separation between the positive inotropic and chronotropic effect. Amrinone increased contractile force and the rate of force development without changes in duration of the contractile cycle or time-to-peak tension, and in action potential or excitability even when it was given in relatively high concentrations. The toxicological studies attested to a low toxicity of amrinone ≤10 mg/kg, with less serious side effects such as lowering in blood pressure and an increase in heart rate compared with cardiac glycosides and catecholamines. The therapeutic index of amrinone was wide in contrast to that of cardiac glycosides that have a therapeutic index of ≈2 to 3, with life-threatening arrhythmias. Of greater interest was that amrinone did not increase cardiac output of the normal (nonfailing), but increased that of the failing heart model. The mechanism of action of amrinone, however, was shown only by exclusion criteria. Amrinone did not seem to act via a catecholamine mechanism because its inotropic effect was not blocked by the β-receptor blocking agent propranolol or by depletion of cardiac norepinephrine with reserpine. It was reported that there were no significant changes in the level of cardiac cAMP or phosphodiesterase (PDE) that could be responsible …

Diastolic dysfunction following HIV infection

The development of myocardial dysfunction in patients following HIV infection continues to be widely reported [1–6]. Compelling evidence of surprisingly frequent diastolic dysfunction followed by systolic dysfunction is observed in both the pediatric and adult populations [1–6]. Basic mechanisms proposed to explain these clinical observations include direct effects of HIV proteins, or indirect effects of cytokines, co-infection, autoimmunity and antiretroviral toxicity [1,7]. The advent of highly active antiretroviral therapies (HAART) has prolonged survival, but has been considered a potential source of cardiovascular complications of HIV infection [1]. Recent clinical studies in countries lacking universal access to HAART have provided some insights into the relative importance of HIV infection as opposed to its pharmacotherapy in HIV cardiomyopathy. These recent studies indicate that HIV cardiomyopathy is more prevalent among patients with HIV alone compared with those patients with HIV who also received HAART [1–6]. Thus, HIV infection appears to contribute more to HIV cardiomyopathy than does its treatment. Human autopsy data identifying gp120 in the hearts of HIV cardiomyopathy patients prior to HAART also support its potential pathogenic role in this condition [8]. The study by Kelly et al.[9] in this issue of AIDS provides an exciting and timely addition to the literature supporting a pathogenic role for HIV proteins in myocardial dysfunction following HIV infection. The authors use the most elegant and definitive hemodynamic and echocardiographic techniques to convincingly demonstrate the presence of diastolic dysfunction in their simian immunodeficiency virus (SIV) model. Their data are quite consistent with diastolic dysfunction in HIV patients that has been reported using echocardiographic methods. The authors found up-regulation of macrophage markers in the myocardium of SIV-infected animals that did not correlate with functional decline. On the contrary, SIV-induced diastolic dysfunction was strongly correlated with the extent of SIV replication in the myocardium. The authors concluded that their data support a pathogenic role for HIV virus or viral proteins in myocardial dysfunction. These clinical reports stimulated efforts to study the direct effects of recombinant HIV proteins in animal models to appreciate their potential pathogenic role in HIV cardiomyopathy [10–15]. The HIV coat protein, gp120, was reported to enhance IL-1beta-induced inducible nitric oxide synthase expression and nitric oxide production in neonatal rat cardiac myocytes in vitro[10]. A direct, negative inotropic effect of HIV gp120 on adult rat ventricular myocytes (ARVMs) was subsequently shown to be mediated through a novel CXCR4-iPLA2-p38 mitogen activated protein (MAP) kinase-troponin I signaling pathway [11–13]. Specifically relevant to Kelly et al. was a report that the injection of HIV gp120 resulted in a selective defect in the diastolic relaxation (lusitropic) response to adrenergic stimulation in vivo[14]. Defective adrenergic signaling is a common characteristic of both ischemic and nonischemic cardiomyopathies [16]. Previously described ‘classical’ mechanisms include decreased beta adrenergic receptor density, decreased adenylyl cyclase/protein kinase A activity, uncoupling the beta adrenergic receptor from stimulatory guanosine-5′-triphosphate (GTP)-binding protein, Gs, in conjunction with an increase in beta adrenergic receptor kinase (BARK) activity, as well as an increase in the content of the inhibitory GTP-binding protein, Gi [16]. Additional molecular insights into adrenergic signaling and diastolic function may provide an alternative mechanism for diastolic dysfunction in HIV [16]. Investigators using elegant experimental designs have provided compelling evidence for a role for p38 MAP kinase and troponin I as molecular components of signaling pathways participating in adrenergic-mediated enhancement of diastolic function. In particular, p38 MAP kinase has been reported to regulate pathways involved in the Ser 23/24 phosphorylation of troponin I [15,16]. Interestingly, a direct correlation was reported between the attenuation of the lusitropic effect of isoproterenol by the p38 MAP kinase inhibitor with blocking the phosphorylation of both p38 MAP kinase and troponin I [14]. These data are most consistent with a selective effect of HIV gp120 on p38 MAP kinase-troponin I signaling that results in attenuated adrenergic enhancement of diastolic relaxation. These clinical and experimental data are consistent with the hypothesis that HIV proteins may directly contribute to myocardial dysfunction. Kelly et al.[9] in this issue of AIDS now provide an excellent animal model with which to further explore potential molecular mechanisms contributing to myocardial dysfunction following HIV infection. Further studies into the signaling pathways stimulated by HIV proteins in the myocardium may provide novel insights and therapeutic targets relevant to HIV, as well, as other human cardiomyopathies. Acknowledgements The work was supported by NIH RO-1 #HL70565 and Department of Veterans Affairs MERIT Award. Conflicts of interest There are no conflicts of interest.

Abstract LB-388: Regulation of histone deacethylase 8 expression by inhibitory GTP binding proteins in H1299 non-small-cell lung cancer cells

Abstract Histone deacetylases (HDACs) have been reported to regulate the cell growth, cell proliferation, cell cycle, and apoptosis, and increased expression of HDAC (classI) was observed in many kinds of cancer. HDAC8, most recently discovered subtype of HDAC classI, seems to be involved in tumor growth and progression because it is highly expressed in several cell lines and human cancer tissues. Inhibitory heterotrimeric GTP-binding proteins (Gi) are activated by seven transmembrane receptors bound with ligands, and regulate cellular metabolism and proliferation by regulating the activity of adenylate cyclase, activate phospholipase C beta, and ion channels. Thus, we made a hypothesis that Gi signaling system may regulate the expression of HDAC8, and this study investigated the effects of Gi signaling system on the expression of HDAC8 and the underlying mechanism in lung cancer cells. When Gi signaling system was activated by expressing Gαi1Q204L, a constitutively active mutant Gαi1, the expression of both HDAC8 protein and mRNA was increased in H1299 non-small-cell lung cancer (NSCLC) cells. However, the HDAC8 promoter activity did not increased significantly, and treatment with Actinomycin D did not block the increase in mRNA. The degradation of HDAC8 mRNA was facilitated in Gαi1Q204L-expressing cells, indicating Gi signaling system increases HDAC8 expression by stabilizing mRNA. Furthermore, treatment with adenosine or CCPA (2-chloro-N(6)cyclopentyladenosine), which bind to adenosine receptors leading to Gαi1 activation, also increased both HDAC8 mRNA and protein levels in H1299 cells. The effect of adenosine was blocked by treatment with DPCPX (8-Cyclopentyl-1,3-dipropylxanthine), an adenosine A1 receptor antagonist. These results indicate that Gi signaling system increases the HDAC8 expression by stabilizing its mRNA in H1299 NSCLC cells Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-388. doi:1538-7445.AM2012-LB-388

Sonic Hedgehog-induced Proliferation Requires Specific Gα Inhibitory Proteins

Proliferation of cerebellar granular neuronal precursors (CGNPs) is mediated by Sonic Hedgehog (Shh), which activates the Patched and Smoothened (Smo) receptor complex. Although its protein sequence suggests that Smo is a G protein coupled receptor (GPCR), the evidence that this receptor utilizes heterotrimeric G proteins as downstream effectors is controversial. In Drosophila, Gα(i) is required for Hedgehog (Hh) activity, but the involvement of heterotrimeric G proteins in vertebrate Shh signaling has not yet been established. Here, we show that Shh-induced proliferation of rat CGNPs is enhanced strongly by the expression of the active forms of Gα(i/o) proteins (Gα(i1), Gα(i2), Gα(i3), and Gα(o)) but not by members of another class (Gα(12)) of heterotrimeric G proteins. Additionally, the mRNAs of these different Gα(i) members display specific expression patterns in the developing cerebellum; only Gα(i2) and Gα(i3) are substantially expressed in the outer external granular layer, where CGNPs proliferate. Consistent with this, Shh-induced proliferation of CGNPs is reduced significantly by knockdowns of Gα(i2) and Gα(i3) but not by silencing of other members of the Gα(i/o) class. Finally, our results demonstrate that Gα(i2) and Gα(i3) locate to the primary cilium when expressed in CGNP cultures. In summary, we conclude that the proliferative effects of Shh on CGNPs are mediated by the combined activity of Gα(i2) and Gα(i3) proteins.

Regulation of meiotic maturation1

Mammalian oocytes are arrested at prophase of the first meiotic division before induction of maturation by the preovulatory LH surge. In vitro, oocyte maturation occurs spontaneously. The first meiotic arrest is characterized by a large nucleus called the germinal vesicle. One important signaling molecule for resumption of meiosis is cyclic AMP (cAMP). High levels of cAMP block spontaneous meiotic resumption. Research investigating the regulation of oocyte cAMP has led to the discovery of new receptors, guanosine 5'-triphosphate-binding (G) proteins, cyclases, and phosphodiesterases. Leydig insulin-like 3, a polypeptide growth factor of the insulin family, is expressed in thecal cells. Leydig insulin-like 3 activates the Leu-rich, repeat-containing, G protein-coupled receptor 8, which is expressed in the oocyte. Coupled to the inhibitory GTP binding protein, this receptor leads to a decrease in cAMP production. Treatment with Leydig insulin-like 3 polypeptide initiates meiotic progression of oocytes in preovulatory follicles, demonstrating the importance of cAMP management for meiotic resumption. Furthermore, microinjection of an antibody against stimulatory G protein (Gs) into mouse oocytes results in meiotic resumption, suggesting that meiotic arrest of the oocyte is dependent on Gs activity. The orphan Gs-linked receptor, GPR3, is expressed in the oocyte. The oocytes of GPR3-null mice resume meiosis when still in their follicles, suggesting that GPR3 is involved in the control of cAMP production and thus meiotic arrest. Cyclic nucleotides are synthesized by cyclases and degraded by phosphodiesterases. Mouse and rat oocytes express isoform 3 of adenylyl cyclase. In the mouse, the null mutation results in approximately 50% of the oocytes resuming meiosis, demonstrating the importance of the synthesis of cAMP in controlling nuclear maturation. The null mutation of the major phosphodiesterase expressed in mouse oocytes results in female sterility due to ovulation of meiotically arrested oocytes that cannot be fertilized. Maintenance of meiotic arrest is explained by constitutive cAMP signaling associated with undetectable cAMP-phosphodiesterase activity. Collectively, these results are beginning to illuminate the key signaling molecules involved in the control of intraoocyte cAMP levels, thus regulating the arrest and resumption of meiosis.

Dopaminergic effects on encoding of a motor memory in chronic stroke

The effects of a single oral dose of levodopa administered in a randomized, double-blind, placebo-controlled cross-over design on formation of a motor memory were studied by a training protocol in patients with chronic stroke. Levodopa enhanced the ability of motor training to encode an elementary motor memory relative to placebo. Up-regulation of dopaminergic function may enhance motor memory formation, crucial for successful rehabilitative treatments in patients with chronic stroke.

Mechanism of galanin-inhibited insulin release

In the insulin-secreting beta cell line Rin m 5F, galanin, a newly discovered ubiquitous neuropeptide, inhibited, by 50%, the stimulation of insulin release induced by gastric inhibitory polypeptide (GIP) or forskolin, i.e. two cAMP-generating effectors. In contrast, it failed to decrease the stimulation of insulin release elicited by either the Ca2+-mobilizing agent, carbamoylcholine, or by dibutyryl-cAMP. Concomitantly, galanin inhibited the GIP-and forskolin-stimulated cAMP production. Furthermore, adenylate cyclase in membranes from Rin m 5F cells was highly sensitive to galanin, which exerted a marked inhibitory effect on the forskolin-stimulated enzyme activity. All these galanin effects were observed at low physiological doses, in the nanomolar range. Overnight treatment of the Rin m 5F cells with pertussis toxin completely abolished the inhibitory effect of galanin on insulin release, cAMP production and adenylate cyclase activity. Moreover, pertussis toxin specifically ADP-ribosylated a 39-kDa protein present in membranes from those cells. Taken together, these data show that the galanin inhibition of insulin release most likely occurs through the inhibition of adenylate cyclase, involving a petussis-toxin-sensitive inhibitory GTP-binding regulatory protein.

Study of the functional organization of a novel adenylate cyclase signaling mechanism of insulin action.

In this study we continued decoding the adenylate cyclase signaling mechanism that underlies the effect of insulin and related peptides. We show for the first time that insulin signal transduction via an adenylate cyclase signaling mechanism, which is attended by adenylate cyclase activation, is blocked in the muscle tissues of the rat and the mollusk Anodonta cygnea in the presence of: 1) pertussis toxin, which impairs the action of the inhibitory GTP-binding protein (Gi); 2) wortmannin, a specific blocker of phosphatidylinositol 3-kinase; and 3) calphostin C, an inhibitor of different isoforms of protein kinase C. The treatment of sarcolemmal membrane fraction with cholera toxin increases basal adenylate cyclase activity and decreases the sensitivity of the enzyme to insulin. We suggest that the stimulating effect of insulin on adenylate cyclase involves the following stages of hormonal signal transduction cascade: receptor tyrosine kinase --> Gi protein (betagamma) --> phosphatidylinositol 3-kinase --> protein kinase C (zeta?) --> Gs protein --> adenylate cyclase --> cAMP.

Chronic opioid treatment of the mouse thymoma cell lines R1.G1 and R1EGO leads to down-regulation of the kappa opioid receptor without desensitization of adenylyl cyclase activity

Kappa opioid agonists alter some immune functions of macrophages, and T- and B-lymphocytes. The mouse thymoma cell lines R1.G1 and R1EGO express only κ-opioid receptors and these κ-opioid receptors are coupled to an inhibitory GTP-binding regulatory protein. Binding of κ-opioid agonists to the opioid receptor leads to the inhibition of adenylyl cyclase activity in these cells. In this study, an acute (15 min) and chronic (24 h) treatment of R1.G1 and R1EGO cell with a potent κ-opioid agonist (−)U50,488 (100 nM) was studied to determine if a κ-opioid agonist altered receptor number and/or desensitization of adenylyl cyclase activity in these two cell lines. Chronic treatment of both R1.G1 and R1EGO cells with (−)U50,488 lead to down-regulation of the κ-opioid receptor, measured as a decrease of approximately 50% in the B max value for the binding of [ 3H]U69,593. The binding affinity ( K d value) was not affected after chronic treatment either in R1.G1 or R1EGO cells. There was no difference in the magnitude of inhibition of adenylyl cyclase activity by (−)U50,488 between the acute (15 min) and chronic (24-h) treatment in both cell lines R1.G1 and R1EGO. This study indicates that chronic opioid treatment of mouse thymoma R1.G1 and R1EGO cell lines leads to down-regulation of the receptor, without desensitization. This phenomenon was observed in R1.1 parent mouse thymoma cell line and recently in CHO cells expressing κ-opioid receptor. This study demonstrates that unlike some neuronal preparations, chronic opioid treatment of the thymoma cell lines resulted in receptor down-regulation without desensitization.

Arg60 Leu Mutation in the First Cytoplasmic Loop of the Platelet Thromboxane A2 Receptor Is Not Essential for Mediating Inhibitory Coupling between the Receptor and Adenylyl Cyclase

Human platelet thromboxane A₂ (TXA₂) receptor (TXR) has been reported to functionally couple to the inhibitory GTP-binding protein for adenylyl cyclase (G_i). However, it still remains unclear which portions of the TXR structure are critical determinants in that coupling. We have previously reported several patients with platelet dysfunction, whose platelets showed impaired coupling between TXR and phospholipase C caused by an Arg⁶⁰ to Leu mutation in the first cytoplasmic loop. To investigate whether this portion is essential for mediating inhibitory coupling between TXR and adenylyl cyclase, we analyzed the inhibition by the TXA₂ analog of the PGE₁ or forskolin-induced platelet cAMP increase in patients’ platelets, and found that the inhibition occurred normally. This suggests that Arg⁶⁰ in the first cytoplasmic loop of the TXR is not involved in TXR-G_i coupling.

Adenylyl Cyclase Regulates Signal Onset via the Inhibitory GTP-binding Protein, Gi

Adenylyl cyclase, the enzyme that converts ATP to cAMP, is regulated by its stimulatory and inhibitory GTP-binding proteins, G(s) and G(i), respectively. Recently, we demonstrated that besides catalyzing the synthesis of cAMP, type V adenylyl cyclase (ACV) can act as a GTPase-activating protein for Galpha(s) and also enhance the ability of activated receptors to stimulate GTP-GDP exchange on heterotrimeric G(s) (Scholich, K., Mullenix, J. B., Wittpoth, C., Poppleton, H. M., Pierre, S. C., Lindorfer, M. A., Garrison, J. C., and Patel, T. B. (1999) Science 283, 1328-1331). This latter action of ACV would facilitate the rapid onset of signaling via G(s). Because the C1 region of ACV interacts with the inhibitory GTP-binding protein Galpha(i), we investigated whether the receptor-mediated activation of heterotrimeric G(i) was also regulated by ACV and its subdomains. Our data show that ACV and its C1 domain increased the ability of a muscarinic receptor mimetic peptide (MIII-4) to enhance activation of heterotrimeric G(i) such that the amount of peptide required to stimulate G(i) in steady-state GTPase activity assays was 3-4 orders of magnitude less than without the C1 domain. Additionally, the MIII-4-mediated binding of guanosine 5'-(gamma-thio)triphosphate (GTPgammaS) to G(i) was also markedly increased in the presence of ACV or its C1 domain. In contrast, the C2 domain of ACV was not able to alter either the GTPase activity or the GTPgammaS binding to G(i) in the presence of MIII-4. Furthermore, in adenylyl cyclase assays employing S49 cyc(-) cell membranes, the C1 (but not the C2) domain of ACV enhanced the ability of peptide MIII-4 as well as endogenous somatostatin receptors to activate endogenous G(i) and to inhibit adenylyl cyclase activity. These data demonstrate that adenylyl cyclase and its C1 domain facilitate receptor-mediated activation of G(i).

Beta-adrenergic receptor signaling: an acute compensatory adjustment-inappropriate for the chronic stress of heart failure? Insights from Gsalpha overexpression and other genetically engineered animal models.

Under normal physiological conditions, the heart must be able to increase its output 5-fold to supply the required blood flow to the coronary circulation and skeletal muscles during severe stress. This is normally met by ≈5-fold increases in myocardial contractility, ≈3-fold increases in heart rate, and additional increases in stroke volume.1 This increased load requires a commensurate increase in myocardial blood flow, because oxygen extraction across the heart is nearly complete, even under normal conditions. Accordingly, the design of the cardiovascular system evolved to conserve myocardial metabolic demand, and consequently coronary blood flow, at rest, but with considerable reserve that can be called on rapidly in times of stress. There is a host of compensatory adjustments, including changes in metabolic substrates and kinetics, as well as oxygen-carrying capacity, that may be recruited in response to stress. However, none is more important than the autonomic nervous system in general, and the sympathetic arm in particular, in terms of providing large, rapid changes in cardiac function. When this compensatory mechanism is unavailable, eg, after treatment with propranolol, the 3-fold increases in heart rate and 5-fold increases in myocardial contractility in response to exercise cannot be achieved.1 In this connection, it is recognized that heart failure is a state characterized by enhanced sympathetic tone, but when the failing myocardium is challenged by β-adrenergic stimulation in vivo or in vitro, the most frequent result is β-adrenergic downregulation or desensitization.2 3 4 5 An impairment of cardiac function leads to autocrine, paracrine, and neurohormonal adjustments, including a strong sympathetic component (Figure 1⇓); under acute conditions, these reflex adjustments are beneficial, as noted above. However, when the sympathetic nervous system is chronically and tonically stimulated, as occurs in the pathogenesis of heart failure, desensitization mechanisms are called into play, such that the …

Inhibition of protein phosphatase 2A (PP2A) mimics suckling-induced sensitization of mammotropes: Involvement of a pertussis toxin (PTX) sensitive G-protein and the adenylate cyclase (AC)

In lactating rats, suckling renders mammotropes more responsive to prolactin (PRL)-releasing stimuli and less responsive to PRL-inhibiting secretagogues. We have previously shown that a decrease in the activity of protein phosphatase 2A (PP2A) may be responsible for the decrease in responsiveness to the inhibitory secretagogue dopamine (DA). In our present experiments, we have studied the involvement of the adenylate cyclase (AC), stimulatory and inhibitory GTP-binding proteins and also the role of PP2A in the sensitization phenomenon. Pituitary cells obtained from mother rats separated from their pups for 4 h prior to dispersion (non-suckled), suckled for 10 or 30 min after the separation period (suckled) and without separation (continual suckling) were incubated in the presence of different doses of forskolin to activate AC and DA. In a further study, pituitary cells of non-suckled rats were pretreated with cholera toxin (CTX) or pertussis toxin (PTX) and tested for the stimulatory action of forskolin or TRH on PRL release. Ocadaic acid (OA) pretreatment has been used to investigate the involvement of PP2A. Hormone secretion was measured by the reverse hemolytic plaque assay (RHPA). Our results have shown that cells from non-suckled rats were unresponsive to forskolin. A 10-min suckling stimulus sensitizes pituitary mammotropes to respond with a PRL release to a dose-dependent activation of AC by forskolin. This sensitization of AC becomes a permanent feature of the cells when suckling continues for an additional 20 min. We have also found that pituitary mammotropes from non-suckled dams respond to forskolin or TRH with PRL release when they were preincubated with either PTX or the PP2A inhibitor OA. It clearly indicates that the non-responsive pituitary can be shifted to the responsive stage by uncoupling of inhibitory G-protein from its receptor as well as by inhibition of PP2A. This latter finding, consonant with our previous results, suggests that suckling may cause selective changes in the function of G i of mammotropes due to a rapid phosphorylation which can remove tonic, GTP-dependent inhibitory function.

A Decrease in the Amount and Function of Inhibitory GTP-Binding Protein in the Resistance Small Artery From Spontaneously Hypertensive Rats

The inhibitory GTP-binding protein (Gi protein) plays an important role in regulation of vascular tone. Many studies have implicated the role of Gi protein in conduit vessels. However, the physiological role of Gi protein in the control of peripheral microvascular tone in hypertension has not been established yet. Therefore, we investigated the concentration of Gi protein in the peripheral resistance arteries and aorta in the spontaneously hypertensive rats (SHR), normotensive Wistar-Kyoto rats (WKY) and renovascular hypertensive rats (RHR), using immunohistochemical methods semiquantitatively. Changes in the function of Gi protein in relation to α2-adrenoceptor were also investigated by microcannulation techniques. We have shown that the amount of α2 subunits of Gi protein in the cremaster small artery was significantly lower in SHR aged 4 weeks and older than in age-matched WKY and that there were no significant differences between RHR and WKY. We also demonstrated that the function of Gi protein in relation to a2-adrenoceptor was already lower in SHR before the onset of hypertension. The quantitative and functional decline in Gi protein in the smooth muscle cells of peripheral small arteries were observed in SHR even before the onset of hypertension, whereas rats with secondary hypertension did not exhibit this finding.

Regulation of the GTP-binding protein-based antilipolytic system of sheep adipocytes by growth hormone.

Chronic exposure of sheep adipose tissue to growth hormone (GH) in vitro decreases the ability of the adenosine analogue, N6-phenylisopropyladenosine (PIA), to inhibit isoprenaline-stimulated lipolysis by a mechanism which is dependent on both gene transcription and protein serine/threonine phosphorylation. The inhibition is not due to a change in ligand binding to the adenosine receptor, the amounts of the three isoforms of the inhibitory GTP-binding protein, Gi, or the maximum (forskolin-stimulated) adenylate cyclase activity. The ability of GH to modulate the PIA-activated adenosine receptor to stimulate dissociation of heterotrimeric Gi was assessed by measurement of pertussis toxin-catalysed ADP-ribosylation of Gi; GH does not appear to alter the interaction between the activated receptor and Gi. The ability of GH to alter the ability of activated Gi to inhibit adenylate cyclase activity was assessed by measuring the ability of a GTP analogue, guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG), to inhibit forskolin-stimulated adenylate cyclase activity; chronic exposure to GH prevented this effect of p[NH]ppG. Thus the attenuation of the inhibition of lipolysis by PIA by chronic exposure of adipocytes to GH appears to be due to an impairment in the interaction between adenylate cyclase and the alpha subunit of one or more isoforms of Gi.

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.

Autoreceptor-induced inhibition of neuropeptide Y release from PC-12 cells is mediated by Y2 receptors.

Pheochromocytoma (PC)-12 cells express Y1, Y2, and Y3 neuropeptide Y (NPY) receptors when differentiated with nerve growth factor (NGF). The present work evaluated NGF-differentiated PC-12 cells as a model system to study modulation of NPY release by NPY autoreceptors. We demonstrated that both K+ and nicotine stimulated concomitant release of NPY and dopamine from differentiated PC-12 cells. We also showed in this study that NPY release from PC-12 cells was attenuated in a concentration-dependent manner by peptide YY (PYY)-(13-36), a selective agonist for the Y2 type of NPY receptors. This result demonstrated that NPY release could be modulated by NPY autoreceptors of the Y2 subtype. The inhibitory action of PYY-(13-36) may be mediated at least in part by inhibition of N-type Ca2+ channels, because PYY-(13-36) could not produce further inhibitory effects in the presence of a maximum effective concentration of omega-conotoxin, an N-type Ca2+-channel blocker. The inhibition by PYY-(13-36) could be blocked by pretreatment of cells with pertussis toxin, suggesting that an inhibitory GTP-binding protein was involved. Furthermore, the function of NPY autoreceptors could be modulated by other receptors such as beta-adrenergic and ATP receptors. The evoked release of NPY was also attenuated by ATP and adenosine, which have been shown to be colocalized and coreleased with NPY from sympathetic nerve terminals. These results suggest that PC-12 cells differentiated with NGF may be an ideal model to study regulatory mechanisms of NPY release and that autoreceptor-mediated regulation of NPY release appears to act through the Y2 subtype of the NPY receptor.