Inhibitory Guanine Nucleotide Research Articles

Activity of cAMP-Dependent Protein Kinase is Reduced in Protein-Energy Malnourished Rats

Glucagon decreases glutathione synthesis in hepatocytes from well-nourished rats. However, in hepatocytes from malnourished rats, glucagon does not inhibit glutathione synthesis, suggesting a desensitization of cAMP-mediated signal transduction. We investigated the mechanism for this desensitization of cAMP-mediated responsiveness in malnourished rats by comparing the signal transduction pathways in rats fed very low protein diets (0.5 g protein/100 g diet) with those of rats fed diets adequate in protein (15 g protein/100 g diet) for 2 wk. Glucagon receptor and forskolin-stimulated cAMP production were greater in hepatocytes from malnourished rats. Stimulation of adenylyl cyclase with forskolin, guanine nucleotides or manganese in hepatic membranes was also enhanced after malnutrition. Moreover, quantity of the stimulatory guanine nucleotide regulatory protein was 70–80% greater in hepatocytes from malnourished rats but the inhibitory guanine nucleotide regulatory protein was not different. These results suggested that desensitization of cAMP-mediated signal transduction after malnutrition occurred at a site distal to cAMP production. Maximal activity of cAMP-dependent protein kinase was 60% lower in liver homogenates from malnourished rats compared with controls. This difference in activity was confined to the cytosolic compartment, with no difference in activity observed in the particulate fraction. Lower activity of cAMP-dependent protein kinase in the cytosol of malnourished rats was associated with a 43% reduction in the quantity of regulatory subunit type I, with no difference in the regulatory subunit type II. These data indicate that desensitization of cAMP signal transduction in rat liver after malnutrition is due to a decrease in the quantity and activity of cAMP-dependent protein kinase.

Very Low Protein Diets Induce a Rapid Decrease in Hepatic cAMP-Dependent Protein Kinase Followed by a Slower Increase in Adenylyl Cyclase Activity in Rats

Recent evidence indicates that cAMP-mediated responses are desensitized in liver during malnutrition. While receptor-stimulated production of cAMP is increased in hepatocytes from rats fed very low protein diets for 14 d, activity of cAMP-dependent protein kinase (PKA) is decreased in liver cytosol. The present study investigated the time course for this desensitization. Weanling rats were fed either a 0.5 (malnourished) or 15% protein (control) diet for 1, 3, 7 or 14 d. Total PKA activity decreased after only 3 d of feeding the low protein diet. This decrease was confined to the cytosolic compartment and was associated with a lower quantity of immunoreactive RI regulatory subunit of PKA, with no difference in the quantity of immunoreactive RII regulatory subunit. In contrast, basal-, MnCl2- and guanine nucleotide regulatory protein-stimulated adenylyl cyclase activities were not greater in liver membranes of malnourished rats than in those of the control rats until the 2nd wk of feeding. Greater activity was paralleled by an increase in the quantity of the stimulatory guanine nucleotide regulatory protein at d 14. The inhibitory guanine nucleotide regulatory protein quantity did not differ between dietary groups. Greater cAMP production was not mediated by changes in PKA phosphorylation of adenylyl cyclase because preincubation of membranes with purified PKA catalytic subunit decreased MnCl2-stimulated cAMP production equally in liver membranes of both control and malnourished rats. Similarly, treatment with alkaline phosphatase decreased adenylyl cyclase activity but did not eliminate the difference in adenylyl cyclase activity between control and malnourished rats. These data demonstrate that loss of PKA activity is an early response to a low protein diet and that, subsequently, a number of molecular adaptations occur which increase cAMP production. These changes may be adaptive responses to malnutrition that maintain essential cAMP-dependent functions.

Knockdown of Inhibitory Guanine Nucleotide Binding Protein Giα-2 by Antisense Oligodeoxynucleotides Attenuates the Development of Hypertension and Tachycardia in Spontaneously Hypertensive Rats.

BackgroundWe previously showed that the levels of both Giα‐2 and Giα‐3 proteins were augmented in spontaneously hypertensive rats (SHRs) before the onset of hypertension. In addition, intraperitoneal injection of pertussis toxin, which inactivates both Giα proteins, prevented the development of hypertension in SHRs. The aim of the present study was to determine the specific contributions of Giα‐2 and Giα‐3 proteins to the development of hypertension.Methods and ResultsAntisense oligodeoxynucleotide of Giα‐2 and Giα‐3 encapsulated in PEG/DOTAP/DOPE cationic liposomes were administrated intravenously into 3‐week‐old prehypertensive SHRs and Wistar Kyoto rats, whereas the control Wistar Kyoto rats and SHRs received PBS, empty liposomes, or sense. The knockdown of Giα‐2 but not Giα‐3 protein attenuated tachycardia and prevented the development of hypertension up to age 6 weeks; thereafter, blood pressure started increasing and reached the same level as that of untreated SHRs at 9 weeks. Furthermore, Giα‐2 and Giα‐3 antisense oligodeoxynucleotide treatments significantly decreased the enhanced levels of Giα‐2 and Giα‐3 proteins, respectively, and enhanced levels of superoxide anion and NADPH oxidase activity in heart, aorta, and kidney and hyperproliferation of vascular smooth muscle cells from SHRs aged 6 weeks. In addition, antisense oligodeoxynucleotide treatment with Giα‐2 but not Giα‐3 restored enhanced inhibition of adenylyl cyclase by oxotremorine to WKY levels.ConclusionsThese results suggested that the enhanced expression of Giα‐2 but not Giα‐3 protein plays an important role in the pathogenesis of hypertension and tachycardia in SHRs.

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.

Is Natriuretic Peptide Receptor C a New Target for Hypertension Therapeutics?

See related article, pp 846–855 Natriuretic peptides (NPs) include atrial NP (ANP), B-type NP, and C-type NP. NPs exert cardiovascular protective effects through interaction with their receptors. There are 2 major classes of NP receptors (NPRs): guanylyl cyclase–coupled receptors A and B (NPR-A and NPR-B) and inhibitory guanine nucleotide regulatory protein (inhibitory G protein)–coupled receptor C (NPR-C; Figure). The cardiovascular protective effects of NPs have been primarily attributed to the elevation of intracellular cGMP via NPR-A and NPR-B.1 NPR-C, initially identified as a clearance receptor,2 has been shown to inhibit adenylyl cyclase via the α subunit of the inhibitory G protein and to stimulate phospholipase C signaling via the βγ subunits of the same inhibitory G protein.3 NPR-C is widely distributed in many tissues and cell types and has higher density than …

The significance of differential expression of genes and proteins in human primary cells caused by microgrooved biomaterial substrata

We demonstrate that etched microgrooves, with truncated V-shape in cross-section and subsequent acid etching, on titanium substrata alter the expression of various genes and proteins in human primary cells. Etched microgrooves with 30 or 60 μm width and 10 μm depth promoted human gingival fibroblast proliferation and significantly enhanced the osteoblast differentiation of human bone marrow-derived mesenchymal stem cells and human periodontal ligament cells by inducing differential expression of various genes involved in cell adhesion, migration, proliferation, mitosis, cytoskeletal reorganization, translation initiation, vesicular trafficking, proton transportation, transforming growth factor-β signaling, mitogen-activated protein kinase signaling, simvastatin’s anabolic effect on bone, inhibitory guanine nucleotide binding protein (G protein)’s action, sumoylation pathway, survival/apoptosis, mitochondrial distribution, type I collagen production, osteoblast differentiation, and bone remodeling that were verified by the differential display PCR and quantitative real-time PCR. The most influential genes on the enhancement of fibroblast proliferation or osteoblast differentiation were determined by multiple regression analysis, and the expression of relevant proteins was confirmed by western blotting and protein quantitation.

Editorial [Hot topic: Crucial Role of Redox Signaling in the Regulation of Heart Health (Guest Editor: Dipak K. Das)

Editorial [Hot topic: Crucial Role of Redox Signaling in the Regulation of Heart Health (Guest Editor: Dipak K. Das)

Role of Natriuretic Peptide Family in Cardiovascular Medicine

The natriuretic peptides (NP) are a group of structurally similar but genetically distinct peptides with many favorable physiological properties that have emerged as important candidates for development of diagnostic tools and therapeutic agents in cardiovascular diseases. The NP family includes atrial natriuretic peptide (ANP, 28AA), urodilatin (INN: Ularitide, 32 AA), B-type natriuretic peptide (BNP, 32AA), C-type natriuretic peptide (CNP, 22AA), and D-type natriuretic peptide (DNP, 38AA). They share common features and exhibit tissue distribution of gene expression as well as functional and pharmacological characteristics. The primary sites of synthesis of the NP are the heart and brain; additional extra cardiac and extra cranial sites include intestine and kidney. Membrane-bound guanyl cyclase-coupled NP receptors (NPR) (A- and B- types) are generally implicated in mediating NP effects via the production of cyclic GMP as the intracellular messenger. NPR-C lacking the guanyl cyclase domain may influence the target cell function through inhibitory guanine nucleotide (Gi) protein, and they likely also act as clearance receptors for circulating peptides. NPs are identified as regulatory diuretic-natriuretic substances responsible for salt and water homeostasis and as hormones lowering blood pressure. This review discusses the essential biochemistry, physiological properties of NP and their manifold functional implications in cardiovascular medicine.

Role of oxidative stress in high glucose-induced decreased expression of Giα proteins and adenylyl cyclase signaling in vascular smooth muscle cells

We have recently shown that aorta from streptozotocin (STZ)-induced diabetic rats and A10 vascular smooth muscle cells (VSMCs) exposed to high glucose exhibited decreased levels of inhibitory guanine nucleotide regulatory protein (Gi)alpha proteins. In the present studies, we investigated the implication of oxidative stress in the hyperglycemia/diabetes-induced decreased expression of the Gialpha protein and adenylyl cyclase signaling in VSMCs by using antioxidants. The levels of Gialpha proteins were significantly decreased in A10 VSMCs exposed to high glucose and in aortic VSMCs from STZ-diabetic rats compared with control cells and were restored to control levels by antioxidants. In addition, (111)Mn-tetralis(benzoic acid porphyrin) and uric acid, scavengers of peroxynitrite, and NG-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase but not catalase, also restored the high glucose-induced decreased expression of Gialpha proteins to the control levels in A10 VSMCs. Furthermore, the enhanced production of superoxide anion (O2-) and increased activity of NADPH oxidase in these cells were also restored to control levels by diphenyleneiodonium, an inhibitor of NADPH oxidase. In addition, the diminished inhibition of adenylyl cyclase activity by inhibitory hormones and forskolin-stimulated adenylyl cyclase activity by low concentrations of GTPgammaS as well as the enhanced stimulation of adenylyl cyclase by stimulatory agonists in hyperglycemic cells were restored to control levels by antioxidant treatments. These results suggest that high glucose-induced decreased levels of Gialpha proteins and associated signaling in A10 VSMCs may be attributed to the enhanced oxidative stress due to augmented levels of peroxynitrite and not to H2O2.

Targeted Inhibition of Cardiomyocyte Gi Signaling Enhances Susceptibility to Apoptotic Cell Death in Response to Ischemic Stress

A salient characteristic of dysfunctional myocardium progressing to heart failure is an upregulation of the adenylyl cyclase inhibitory guanine nucleotide (G) protein alpha subunit, G alpha(i2). It has not been determined conclusively whether increased Gi activity in the heart is beneficial or deleterious in vivo. Gi signaling has been implicated in the mechanism of cardioprotective agents; however, no in vivo evidence exists that any of the G alpha subunits are cardioprotective. We have created a novel molecular tool to specifically address the role of Gi proteins in normal and dysfunctional myocardium. We have developed a class-specific Gi inhibitor peptide, GiCT, composed of the region of G alpha(i2) that interacts specifically with G protein-coupled receptors. GiCT inhibits Gi signals specifically in vitro and in vivo, whereas Gs and Gq signals are not affected. In vivo expression of GiCT in transgenic mice effectively causes a "functional knockout" of cardiac G alpha(i2) signaling. Inducible, cardiac-specific GiCT transgenic mice display a baseline phenotype consistent with nontransgenic mice. However, when subjected to ischemia/reperfusion injury, GiCT transgenic mice demonstrate a significant increase in infarct size compared with nontransgenic mice (from 36.9+/-2.5% to 50.9+/-4.3%). Mechanistically, this post-ischemia/reperfusion phenotype includes increased myocardial apoptosis and resultant decreased contractile performance. Overall, our results demonstrate the in vivo utility of GiCT to dissect specific mechanisms attributed to Gi signaling in stressed myocardium. Our results with GiCT indicate that upregulation of G alpha(i2) is an adaptive protective response after ischemia to shield myocytes from apoptosis.

Abstract 2492: Clenbuterol, Unlike Other B 2 -Adrenoceptor Agonists, Predominantly Activates the G i Protein in Isolated Rat Ventricular Myocytes

Clenbuterol (CLEN), a β 2 -adrenoceptor (AR) agonist, has been employed in combination with other pharmacological agents and LVADs to treat patients with severe heart failure (HF). Chronic administration of CLEN increases expression of Ca regulatory proteins in isolated cardiac myocytes and improves their contractility. However, the acute effects of CLEN on myocyte contractility compared to other β 2 -AR agonists and the signalling pathway of CLEN remain undefined. Here we compared the acute effects of CLEN to two other β 2 -AR agonists, fenoterol (FEN) and salbutamol (SAL). Isolated rat ventricular myocytes were superfused with β 2 AR agonists and sarcomere shortening was measured. CLEN produced a negative inotropism (30 μM: 65.3 ± 9.7% of control, n=9; p<0.05; 100 μM: 37.9 ± 8.0%, n=9; p<0.01) whereas FEN showed a positive inotropic response (30 μM: 184.8 ± 19.1% of control, n=9; p<0.01; 100 μM: 198.1 ± 20.5%, n=9; p<0.01). SAL had no significant effect. Selective β 1 AR blockade with 300 nM CGP 20712A did not affect CLEN’s action on sarcomere shortening but significantly reduced the contractile response to FEN and SAL (p<0.05). Additional blockade of β 2 AR-Gs with 50 nM ICI 118,551 unveiled a negative inotropic response to FEN (100 μM: 61.5 ± 7.3% of control, n=7; p<0.001) and SAL (100 μM: 67.4 ± 3.6% of control, n=7; p<0.01) and did not alter the response to CLEN. Incubation with 2 μg/ml pertussis toxin (PTX) almost abolished the negative inotropic effects of CLEN: 30 μM (PTX: 95.1 ± 4.9%, n=6 vs control: 73.9 ± 5.5%, n=7; p<0.05) and 100 μM (PTX: 96.2 ± 9.6%, n=6 vs control: 21.7 ± 5.5%, n=7; p<0.001), suggesting the involvement of the inhibitory guanine nucleotide binding protein (G i ). In addition, overexpression of G i by adenoviral (AdV) transfection for 48 hrs induced a stronger negative inotropic effect upon application of CLEN compared with control cultured cells (AdV-Gi: clen 30 μM: 36.8 ± 8.3% of control, n=9; p<0.01; 100 μM: 19.7 ± 6.5%, n=9; p<0.01). In summary, CLEN significantly depresses contractility of ventricular myocytes, an effect not seen with FEN or SAL. CLEN predominantly acts through G i and the consequent downstream signaling pathways activation may explain the beneficial effects observed during administration of CLEN in patients treated with LVADs.

Enhanced expression of Giα protein and adenylyl cyclase signaling in aortas from 1 kidney 1 clip hypertensive ratsThis paper is one of a selection of papers published in this Special issue, entitled Second Messengers and Phosphoproteins—12th International Conference.

We have previously shown the augmented levels of Gialpha-2 and Gialpha-3 proteins (isoforms of inhibitory guanine nucleotide regulatory protein (G-protein)), and not of Gsalpha, in the hearts and aortas of spontaneously and experimentally induced hypertensive rats. The increased expression of Gialpha and blood pressure was restored toward WKY levels by captopril treatment, suggesting a role for angiotensin (Ang) II in the enhanced expression of Gialpha protein and blood pressure. This study was undertaken to investigate whether 1 kidney 1 clip (1K-1C) hypertensive rats that exhibit enhanced levels of Ang II also express enhanced levels of Gialpha proteins. Aortas from 1K-1C hypertensive rats were used. The expression of G-proteins was determined at protein levels with immunoblotting techniques, using specific antibodies for different isoforms of G-proteins. The levels of Gialpha-2 and Gialpha-3 proteins were significantly higher in aortas from 1K-1C hypertensive rats than in control rats; Gsalpha levels were unchanged. The inhibitory effect of low concentrations of guanosine 5'-[gamma-thio]triphosphate (GTPgammaS) on forskolin (FSK)-stimulated adenylyl cyclase (AC) activity was significantly enhanced in aortas from 1K-1C hypertensive rats; the inhibitory effect of C-ANP(4-23), which specifically interacts with the atrial natriuretic peptide (ANP)-C receptor, and Ang II on AC was attenuated. GTPgammaS, isoproterenol, glucagon, NaF, and FSK stimulated the AC activity in aortas from control and hypertensive rats to varying degrees; however, the stimulations were significantly lower in hypertensive rats than in control rats. These data suggest that aortas from 1K-1C hypertensive rats exhibit enhanced expression of Gialpha proteins and associated functions.

Oxidative stress contributes to the enhanced expression of Giα proteins and adenylyl cyclase signaling in vascular smooth muscle cells from spontaneously hypertensive rats

We have previously shown an enhanced expression of Gialpha proteins in spontaneously hypertensive rats (SHR) that precedes the development of hypertension. Since oxidative stress has been shown to be increased in SHR, the present studies were undertaken to examine the role of oxidative stress in enhanced expression of Gialpha proteins in SHR. Aortic vascular smooth muscle cells (VSMC) from 12-week-old SHR and Wistar-Kyoto (WKY) rats were used for the present studies. The levels of inhibitory guanine nucleotide regulatory proteins (Gialpha-2 and Gialpha-3) and stimulatory proteins (Gsalpha) were determined by western blotting techniques. Adenylyl cyclase activity was determined by measuring [32P]cAMP formation from [alpha-32P]ATP. VSMC from SHR exhibited enhanced expression of Gialpha-2 and Gialpha-3 proteins as compared with age-matched WKY rats; however, the levels of Gsalpha proteins were not different between the two groups. The levels of superoxide anion (O2-) were also increased in SHR as compared with WKY rats, and losartan, an AT1 receptor antagonist, restored the enhanced levels to control WKY rat levels. Treatment of VSMC with antioxidants such as N-acetyl-L-cysteine (NAC) or diphenyleneiodonium (DPI) for 24 h decreased the enhanced expression of Gialpha-2 and Gialpha-3 proteins in a concentration-dependent manner in VSMC from SHR. In addition, the inhibition of forskolin-stimulated enzyme activity by low concentrations of GTPgammaS (receptor-independent Gi functions) and C-ANP4-23-mediated inhibition of adenylyl cyclase (receptor-dependent Gi functions) that were significantly enhanced in SHR were restored to WKY rat levels by NAC and DPI treatments. Similarly, diminished stimulation of adenylyl cyclase by GTPgammaS, isoproterenol and sodium fluoride in SHR was also restored towards control WKY rat levels by NAC and DPI treatments. Furthermore, PD98059, a selective inhibitor of mitogen-activated protein kinase, was able to restore the enhanced expression of Gialpha proteins in VSMC from SHR towards WKY rat levels. In addition, the enhanced activity of extracellular signal-regulated kinase 1/2 in SHR as compared with WKY rats, as demonstrated by enhanced phosphorylation of extracellular signal-regulated kinase 1/2, was also restored to WKY rat levels by NAC or DPI. These results suggest that enhanced levels of Gialpha proteins and associated functions in SHR may be attributed to the enhanced oxidative stress present in SHR, which exerts its effects through the mitogen-activated protein kinase signaling pathway.

Natriuretic peptide receptor-C signaling and regulation

The natriuretic peptides (NP) are a family of three polypeptide hormones termed atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). ANP regulates a variety of physiological parameters by interacting with its receptors present on the plasma membrane. These are of three subtypes NPR-A, NPR-B, and NPR-C. NPR-A and NPR-B are guanylyl cyclase receptors, whereas NPR-C is non-guanylyl cyclase receptor and is coupled to adenylyl cyclase inhibition or phospholipase C activation through inhibitory guanine nucleotide regulatory protein (Gi). ANP, BNP, CNP, as well as C-ANP 4–23, a ring deleted peptide that specifically interacts with NPR-C receptor inhibit adenylyl cyclase activity through Gi protein. Unlike other G-protein-coupled receptors, NPR-C receptors have a single transmembrane domain and a short cytoplasmic domain of 37 amino acids, which has a structural specificity like those of other single transmembrane domain receptors. A 37 amino acid cytoplasmic peptide is sufficient to inhibit adenylyl cyclase activity with an apparent Ki similar to that of ANP 99–126 or C-ANP 4–23. In addition, C-ANP 4–23 also stimulates phosphatidyl inositol (PI) turnover in vascular smooth muscle cells (VSMC) which is attenuated by dbcAMP and cAMP-stimulatory agonists, suggesting that NPR-C receptor-mediated inhibition of adenylyl cyclase and resultant decreased levels of cAMP may be responsible for NPR-C-mediated stimulation of PI turnover. Furthermore, the activation of NPR-C receptor by C-ANP 4–23 and CNP inhibits the mitogen-activated protein kinase activity stimulated by endothelin-3, platelet-derived growth factor, phorbol-12 myristate 13-acetate, suggesting that NPR-C receptor might also be coupled to other signal transduction system or that there may be an interaction of the NPR-C receptor and some other signaling pathways. In this review article, NPR-C receptor coupling to different signaling pathways and their regulation will be discussed.

Mechanisms of alcohol-associated cancers: introduction and summary of the symposium

Chronic alcohol consumption is associated with an increased risk for cancers of many organs, such as oral cavity, pharynx, larynx, and esophagus; breast; liver; ovary; colon; rectum; stomach; and pancreas. An understanding of the underlying mechanisms by which chronic alcohol consumption promotes carcinogenesis is important for development of appropriate strategies for prevention and treatment of alcohol-associated cancers. The National Institute on Alcohol Abuse and Alcoholism, Office of Dietary Supplements, Office of Rare Diseases, National Cancer Institute, National Institute on Drug Abuse, and National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, sponsored an international symposium on Mechanisms of Alcohol-Associated Cancers in Bethesda, Maryland, USA, October 2004. The following is a summary of the symposium. Chronic ethanol consumption may promote carcinogenesis by (1) production of acetaldehyde, which is a weak mutagen and carcinogen; (2) induction of cytochrome P450 2E1 and associated oxidative stress and conversion of procarcinogens to carcinogens; (3) depletion of S-adenosylmethionine and, consequently, induction of global DNA hypomethylation; (4) induction of increased production of inhibitory guanine nucleotide regulatory proteins and components of extracellular signal-regulated kinase–mitogen-activated protein kinase signaling; (5) accumulation of iron and associated oxidative stress; (6) inactivation of the tumor suppressor gene BRCA1 and increased estrogen responsiveness (primarily in breast); and (7) impairment of retinoic acid metabolism. Nicotine may promote carcinogenesis through activation of extracellular signal-regulated kinase/cyclooxygenase-2/vascular endothelial growth factor signaling pathway.

Beta-adrenoceptor blockers as agonists: coupling of beta2-adrenoceptors to multiple G-proteins in the failing human heart.

Beta blockers have been shown in clinical trials to improve cardiac function and reduce mortality of heart failure patients. However, these agents require careful titration since they can produce an initial decrease in cardiac output. The authors have recently shown that beta blockers, including some used clinically, can directly depress contraction of myocardium from the failing (but not nonfailing) human heart. This occurs on single ventricular myocytes and is therefore completely independent of any inhibition of endogenous catecholamines. The effect appears to be mediated primarily by the beta2-adrenoceptor (AR) and is dependent on the inhibitory guanine nucleotide binding protein, Gi. Using a transgenic mouse model, as well as adenoviral vectors to overexpress Gi or the human beta2AR in adult myocytes of various species, the authors demonstrate that agents that are blockers for bAR/Gs coupling can be agonists at a beta2AR/Gi-coupled form of the receptor. The negative effect of beta blockers could contribute to the initial cardiodepression that is observed when introducing these agents in heart failure patients. However, in the long term, beta2AR/Gi coupling may enhance the ability of beta blockers to protect and improve the function of the failing heart.

Implications of intrathecal pertussis toxin animal model on the cellular mechanisms of neuropathic pain syndrome.

Like opioid tolerance, neuropathic pain syndrome manifested by hyperalgesia and allodynia responds poorly to opioids. Hitherto, its development is still not clear and its treatment and prevention are still disputable. Pertussis toxin (PTX) which ADP-ribosylates the alpha-subunit of inhibitory guanine nucleotide binding regulatory proteins (Gi/Go), is used to induce morphine tolerance through intrathecal (i.t.) injection. It decreases the antinociceptive effect of opioid receptor agonists, and produces a thermal hyperalgesia as well. With treatment of PTX the inhibitory Gi- and Go-proteins signal transduction is inactivated. Inhibition of the inhibitory system would likely lead to a predominance of the excitatory system. Intrathecal PTX administration has also been suggested as a model for study of the central mechanisms of neuropathic pain. In our previous studies, with intrathecal microdialysis and drug delivery techniques, we correlated the biochemical and pharmacological effects on the behavioral expressions of i.t. PTX-treated rats. Intrathecal PTX administration would induce thermal hyperalgesia in rats, with accompaniments of a prolonged increase in the concentrations of excitatory amino acids (EAAs), glutamate and aspartate, and a decrease in the concentration of the inhibitory amino acid (IAA) glycine in the spinal CSF dialysates. The PTX-induced thermal hyperalgesia peaked between day 2 and 4, but no cold allodynia is observed; i.t. administration of N-methyl-D-aspartate (NMDA) receptor antagonist, D-2-amino-5-phosponovaleric acid (D-AP5), glycine and protein kinase C (PKC) inhibitor chelerythrine attenuated the thermal hyperalgesia. The PKC gamma content of both synaptosomal and cytosolic fractions were significantly increased in PTX-treated rats. In contrast, the levels of PKC alpha, beta I, or beta II isozymes in these fractions were unaffected. Infusion of NMDA antagonist D-AP5 prevented both the thermal hyperalgesia and the increase in PKC gamma expression in PTX-treated rats. Similar to our previous report, i.t. PTX reduced morphine's analgesic effect. PKC inhibitor chelerythrine attenuated this reduction of morphine's analgesia, and an inhibition of the morphine-evoked EAAs release was observed in PTX-treated rats as well. Taken together, i.t. PTX-induced neuropathic pain syndrome is accompanied by increasing of EAAs, decreasing of IAA release, and a selective increasing of PKC gamma expression in the spinal cord. Inhibition of PKC not only blocked thermal hyperalgesia, but also reversed the reduction of morphine's analgesic effect in PTX-rats. These results suggest that PTX-induced neuropathic pain syndromes are involved in EAAs, IAAs and PKC alternations.

Angiotensin-II-induced enhanced expression of Gi proteins is attenuated by losartan in A10 vascular smooth muscle cells: role of AT1 receptors.

We have previously shown that treatment of A10 vascular smooth muscle cells (VSMCs) with angiotensin II (Ang II) enhanced the expression of inhibitory guanine nucleotide regulatory proteins (Gi alpha2 and Gi alpha3). In the present studies, we have investigated the role of type 1 angiotensin receptors (AT1) in the Ang-II-induced enhanced expression of Gi alpha proteins and their functions in A10 SMCs. Ang II enhanced the levels of Gi alpha2 and Gi alpha3 proteins and their mRNA, as determined by Western and Northern blot analysis, respectively; losartan treatment attenuated the enhanced expression of Gi alpha2 and Gi alpha3 proteins and their mRNA in a concentration-dependent manner. In addition, the inhibition of adenylyl cyclase induced by Ang II and des(Glu18,Ser19,Glu20,Leu21,Gly22)ANP(4-23)-NH2 (C-ANP(4-23)), which was attenuated by Ang-II treatment, was partially restored by losartan treatment. Similarly, losartan was also able to restore the Ang-II-induced stimulatory responses of isoproterenol and N-ethylcarboxamide adenosine (NECA) on adenylyl cyclase activity. These results suggest a role for AT1 receptors in Ang-II-evoked increases in Gi alpha protein expression and Gs-mediated stimulation in VSMCs.

Beta-Arrestin-mediated PDE4 cAMP phosphodiesterase recruitment regulates beta-adrenoceptor switching from Gs to Gi.

Phosphorylation of the beta(2) adrenoreceptor (beta(2)AR) by cAMP-activated protein kinase A (PKA) switches its predominant coupling from stimulatory guanine nucleotide regulatory protein (G(s)) to inhibitory guanine nucleotide regulatory protein (G(i)). beta-Arrestins recruit the cAMP-degrading PDE4 phosphodiesterases to the beta(2)AR, thus controlling PKA activity at the membrane. Here we investigate a role for PDE4 recruitment in regulating G protein switching by the beta(2)AR. In human embryonic kidney 293 cells overexpressing a recombinant beta(2)AR, stimulation with isoprenaline recruits beta-arrestins 1 and 2 as well as both PDE4D3 and PDE4D5 to the receptor and stimulates receptor phosphorylation by PKA. The PKA phosphorylation status of the beta(2)AR is enhanced markedly when cells are treated with the selective PDE4-inhibitor rolipram or when they are transfected with a catalytically inactive PDE4D mutant (PDE4D5-D556A) that competitively inhibits isoprenaline-stimulated recruitment of native PDE4 to the beta(2)AR. Rolipram and PDE4D5-D556A also enhance beta(2)AR-mediated activation of extracellular signal-regulated kinases ERK12. This is consistent with a switch in coupling of the receptor from G(s) to G(i), because the ERK12 activation is sensitive to both inhibitors of PKA (H89) and G(i) (pertussis toxin). In cardiac myocytes, the beta(2)AR also switches from G(s) to G(i) coupling. Treating primary cardiac myocytes with isoprenaline induces recruitment of PDE4D3 and PDE4D5 to membranes and activates ERK12. Rolipram robustly enhances this activation in a manner sensitive to both pertussis toxin and H89. Adenovirus-mediated expression of PDE4D5-D556A also potentiates ERK12 activation. Thus, receptor-stimulated beta-arrestin-mediated recruitment of PDE4 plays a central role in the regulation of G protein switching by the beta(2)AR in a physiological system, the cardiac myocyte.

Inhibition of alcohol dehydrogenase blocks enhanced Gi-protein expression following ethanol treatment in experimental hepatocellular carcinoma in vitro.

Chronic alcohol abuse is one of the major contributors to the onset and progression of hepatocellular carcinoma (HCC). We have previously identified increased expression and function of inhibitory guanine nucleotide regulatory proteins (Gi-proteins) in primary human and animal models of HCC. Stimulation of Gi-proteins in HCC stimulates cell mitogenesis, an effect not observed in hepatocytes. The aim of this study was to determine the effect of ethanol and ethanol metabolism on Gi-protein expression in an experimental model of HCC. Pharmacological agents that inhibit alcohol metabolism were used in conjunction with ethanol or ethanol metabolites. We were also able to assess the relative contribution of alcohol and acetaldehyde, the major metabolite of alcohol, on Gi-protein expression in HCC and hepatocytes. These studies used the rat hepatic tumorigenic H4IIE cell line in conjunction with isolated rat hepatocytes. Cells were cultured in vitro and exposed to ethanol, ethanol in the presence of an alcohol dehydrogenase (ADH) inhibitor, or acetaldehyde for varying lengths of time. Ethanol metabolism and changes in Gi-protein expression were subsequently determined by assay. Exposure to ethanol alone led to significant dose and time dependent increases in Gialpha1/2 and Gialpha3 protein and mRNA expression in HCC cells. In contrast, ethanol failed to alter Gialpha1/2, and only moderately affected Gialpha3 protein expression in isolated cultured hepatocytes. Pretreatment of HCC cells and hepatocytes with 4-methyl pyrazole (4-MP, 10 microm) significantly inhibited alcohol metabolism. Treatment of HCC cells with 4-MP inhibited changes in Gi-protein expression following exposure to ethanol (25 mm, 24 h). In addition, the increased expression of Gi-proteins observed after exposure to ethanol in HCC were mimicked by direct exposure of HCC cells to acetaldehyde in a dose and time dependent manner. These data suggest that alcohol metabolites, not alcohol, lead to increased Gi-protein expression in HCC in vitro. Ethanol and ethanol metabolites, in contrast, fail to significantly alter Gialpha1/2 protein expression in hepatocytes. These data may have significant implications in HCC progression in vivo.