G-protein Expression Research Articles

Angiotensin II-induced changes in G-protein expression and resistance of renal microvessels in young genetically hypertensive rats.

Altered regulation of cAMP may contribute to enhanced renal reactivity to angiotensin II (Ang II) in spontaneously hypertensive rats (SHR). Such a phenomenon may occur in renal preglomerular arterioles and may involve changes in expression of GTP-binding regulatory proteins. We have examined the effects of Ang II on steady state levels of G(alpha i-1,2), G(alpha i-3), G(alpha s) and G(alpha q) in preglomerular arterioles from young marginally hypertensive SHR and on mean arterial pressure (MAP), renal vascular resistance (RVR) and renal cAMP excretion (UcAMP.V). Young (5-6 week old) SHR and Wistar Kyoto (WKY) rats received Ang II (35 ng/kg/min, s.c.) or vehicle for 7 days via osmotic minipumps. Urine was collected over the last 24 h. On day seven, MAP and renal blood flow were measured in anesthetized rats and RVR was determined. Preglomerular arterioles were isolated by perfusing the kidneys with iron oxide and using a series of mechanical steps coupled with the use of a magnet to retain iron-laden vessels. Membranes were prepared and the expressions of G(alpha i-1,2), G(alpha i-3), G(alpha s) and G(alpha q) were evaluated by Western immunoblotting. Baseline MAP (124 +/- 6 mmHg) was only marginally (p > 0.05) higher in SHR when compared with WKY rats (110 +/- 4 mmHg). RBF (3.04 +/- 0.16 mL/min) was significantly lower and RVR (41.10 +/- 1.37 mmHg.min/mL) was significantly higher in SHR when compared to age-matched WKY rats (4.36 +/- 0.30 mL/min and 25.79 +/- 1.58 mmHg.min/mL, respectively). Ang II significantly increased MAP in SHR (17 mmHg) but not in WKY rats. These increases in MAP were accompanied by significant increases in RVR in SHR (48% over control) but not in WKY rats. Compared to WKY rats, preglomerular arterioles from SHR exhibited significantly higher basal expression of G(alpha i-1,2) (11- fold), G(alpha 1-3) (13-fold) and G(alpha s) (3-fold). Chronic infusion of Ang II, however, downregulated the expression of G(alpha s) (by 53%; p < 0.05), G(alpha i-1,2) (by 72%; p < 0.05) and G(alpha i-3) (by 35%; p > 0.05) in SHR preglomerular arterioles but significantly upregulated the expression of these proteins in WKY by 3-, 8- and 15-fold, respectively. Basal levels of G(alpha q) were not different in preglomerular arterioles from the two strains but were downregulated by Ang II in both WKY (74% of basal) and SHR (52% of control). Baseline UcAMP.V was significantly lower in SHR (31.22 +/- 6.51 nmol/24 h) compared with WKY rats (65.33 +/- 3.60 nmol/24 h). Chronic Ang II infusion significantly increased UcAMP.V in SHR as well as WKY rats. These data clearly demonstrate that expressions of Gi isoforms as well as Gs in renal microvessels are elevated during early stages of hypertension and suggest that the elevated levels of Gi proteins may be directly associated with a blunted adenylyl cyclase-cAMP cascade in the renal microvasculature. Furthermore, Ang II appears to directly downregulate the expression of Gs in young SHR but not in young WKY renal microvessels. Such diversity in its effect on G-protein expression may be important for enhanced renal sensitivity to Ang II in SHR.

7] Reduction of neurotransmitter receptor and G-protein expression in vivo and in vitro by antisense oligodeoxynucleotide treatment

The use of antisense oligodeoxynucleotides (ODNs) to reduce or eliminate the expression of specific proteins has become an increasingly important research tool. Although there is some disagreement about the precise mechanism of its action, the exquisite specificity of the ODN for its target is hardly disputed. The increased popularity and utilization of this approach have also spurred the establishment of many new businesses dedicated to the custom synthesis of antisense ODNs, in any number of shapes, forms, and styles, all at increasingly more affordable rates. This combination of specificity, availability, and affordability has allowed more and more investigators to utilize this molecular approach without the added expense of establishing a costly traditional molecular biology dimension within their laboratories. The use of ODNs has been especially fruitful when applied to neurotransmitter receptors and components of second-messenger systems and has allowed addressing questions about receptor/second-messenger interactions and second-messenger-mediated downstream events that have been difficult to answer. This chapter describes how antisense ODNs may be used in cultured cell lines and in intact animals to reduce the expression of neurotransmitter receptors and glyco (G) proteins.

10] Use of antisense expression plasmids to attenuate G-protein expression in primary neurons

Unraveling signaling pathways in primary neurons has been a central issue for neurobiologists and pharmacologists, but the analysis has been hampered by the paucity of specific molecular tools with which to dissect signal transduction cascades. The advent of gene cloning demonstrated a degree of molecular diversity unknown, and largely unsuspected, in most gene families. This was nowhere more apparent than in G protein–coupled receptor (GPCR)-mediated signal transduction. This signaling cascade starts with the activation of a GPCR and consequent catalytic guanosine diphosphate/guanosine triphosphate (GDP/GTP) exchange on the α subunit of a trimeric G protein, thereby causing dissociation into a GTP-bound α subunit and a βγ dimer, either of which can act on a myriad of channels or enzymes. Currently, genetic diversity accounts for more than 1,000 GPCRs, 17 G α subunits, 5 G β subunits, and 12 G γ subunits, as well as hundreds of ion channel and enzyme effectors. This diversity is further increased by the existence of splice variants. The fact that the recognition of this molecular diversity largely relied on molecular cloning bears testament to the degree of similarity among the gene products, which was not discernible using conventional biochemical or pharmacological agents. This similarity offers a challenge to develop specific molecular tools, because the degree of amino acid identity among cognate members of a gene family can be so great as to preclude the design of agents that will readily distinguish individual relatives.

The role of cAMP-MAPK signalling in the regulation of human hepatocellular carcinoma growth in vitro.

We have previously identified that primary human hepatocellular carcinoma (HCC) is associated with altered guanine nucleotide regulatory protein (G-protein) expression concomitant with decreased adenylyl cyclase (AC) and increased mitogen activated protein kinase (MAPK) activity in vivo. This study aims to address the potential link between Gs protein regulation of AC activity/ cyclic adenosine monophosphate (cAMP) production and the subsequent downstream regulation of MAPK activity and mitogenesis. Pharmacological agents which selectively interact with specific target proteins involved in signal transduction via the Gs-AC-cAMP-MAPK signalling pathway were employed in cultured human HCC cell lines in these studies. These agents allow us to address the role of individual components of these pathways in the regulation of mitogenesis in HCC. These studies utilized three distinct human HCC cell lines (HepG2, Hep3B and SKHep) in the absence and presence of agents that alter AC-cAMP dependent signalling. De novo DNA synthesis was determined as a marker of altered cellular proliferation, and MAPK activity was determined as the ability to catalyse myelin basic protein (MBP) phosphorylation. 8-Bromo-cAMP (8-Br-cAMP; a cell-permeable cAMP analogue) and forskolin (AC activator) dose-dependently decreased thymidine incorporation in all three cell lines. In addition, serum-stimulated [3H] thymidine incorporation was significantly decreased in HepG2, Hep3B and SKHep cell lines following treatment with either 8-Br-cAMP or forskolin. By contrast, MDL12330A (MDL; irreversible AC inhibitor) enhanced thymidine incorporation in all three cell lines. Treatment with either 8-Br-cAMP or forskolin significantly decreased serum-stimulated MAPK activity. These data suggest that cAMP acts as an anti-mitogenic agent in these hepatic tumorigenic cell lines in vitro such that inhibition of AC activity promotes MAPK activity and cellular mitogenesis in HCC.

Volume-overload induced changes in G-protein expression in rat heart

Volume-overload induced changes in G-protein expression in rat heart

Gene Expressions of Opioid Receptors and G-Proteins in Pineal Glands

In our previous studies, the opioid receptors located on pinealocytes have been identified and characterized, and these receptors have been found to play a stimulatory role in melatonin synthesis by activating the rate limiting enzyme, N-acetyltransferase (NAT). In the present study, by using reverse transcriptase polymerase chain reaction (RT-PCR) followed by nested-PCR, segments of delta and mu opioid receptors have been amplified from mRNA of rat pineal gland and cerebral cortex. In addition, segments of delta and mu opioid receptors have also been amplified from mRNA of human pineal gland. Furthermore, Gαi/o- and Gβ-protein-coupled receptor mRNAs have been amplified and identified from rat pineal gland. The regulatory effects of morphine on Gαi/o and Gβ mRNA levels have been semiquantitatively analyzed. Acute morphine administration caused significant increase in Gαi/o, and Gβ, mRNA levels in rat pineal gland, but not in other brain regions. Further studies are needed in order to elaborate the mechanisms of these opioid receptors in regulating G-protein expression in pineal gland.

New insights into the in vivo function of heterotrimeric G-proteins through gene deletion studies.

A huge number of receptors signals through heterotrimeric G-proteins to regulate a wide variety of physiological processes. The ability to modify G-protein expression in vivo provides a powerful new tool to analyze the function of G-protein-mediated signalling pathways. Genetic ablation of G-protein alpha-subunit genes is continuing to give new insights into the physiological roles of heterotrimeric G-proteins. This review highlights recent advances resulting from such genetic approaches to the study of G-protein-mediated signalling.

Perinatal PTX-sensitive G-protein expression and regulation of conductive 22Na + transport in lung apical membrane vesicles

Using apical membrane vesicles (AMV) prepared from mature foetal and early neonatal guinea pig lung we show that pertussis toxin (PTX)-sensitive G-protein regulation of conductive 22Na + uptake undergoes rapid changes following birth. Thus, G-protein activation by intravesicular incorporation of 100 μM GTPγS into vesicles resuspended in NaCl, which in late gestation stimulated uptake, consistently induced inhibition of conductive Na + uptake into AMV prepared from neonatal lung at 4 days of age (N4) (52±9%, n=8, P<0.05). This response was not significantly different in the presence of the relatively impermeant anion isethionate (Ise −) (69±9%, n=7, P<0.05). Changes in the regulation of uptake were already detectable on the day of birth (N0) in AMV resuspended in NaCl, with GTPγS inducing both stimulatory and inhibitory responses. These data indicate that the processes by which 22Na + uptake into AMV is regulated by G-proteins undergoes a change at birth and by 4 days of age, G-protein regulation of uptake occurs predominantly via modulation of co-localised Na + channels. Intravesicular incorporation of GDPβS or pre-treatment with PTX did not significantly alter conductive 22Na + uptake in the presence of NaCl or NaIse suggesting that constitutively active G-proteins are not involved in this process. Pre-treatment of AMV with PTX prevented the inhibition of conductive 22Na + uptake by GTPγS (105±16% n=7) indicating that a PTX-sensitive G-protein mediates the inhibition of channels in neonatal AMV. Western blotting demonstrated enrichment of G iα1, G iα2, G iα3 and G oα in the apical membrane preparations. We also show that there is a significant rise in the levels of G iα3 during the early neonatal period providing a potential candidate for the G-protein mediated changes in regulation of conductive 22Na + uptake in neonatal AMV.

Enhanced Gi-protein-mediated mitogenesis following chronic ethanol exposure in a rat model of experimental hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is associated with increased expression and function of inhibitory guanine nucleotide regulatory proteins (Gi-proteins). This study addresses the effects of chronic ethanol exposure on the expression and function of adenylyl cyclase (AC)-linked G-proteins (Gs and Gi) and growth in experimental HCC. G-protein expression and function was determined by immunoblot in the hepatic tumorigenic H4IIE cell line and isolated cultured hepatocytes in the absence or presence of ethanol (5-100 mmol/L). Chronic exposure (24 hours) to ethanol dose-dependently increased Gialpha1/2 expression in the H4IIE cell line, but not in cultured hepatocytes. Gsalpha-protein expression remained unchanged in both H4IIE cells and cultured hepatocytes following ethanol treatment. In addition, ethanol directly activated a Gi-protein, because pertussis toxin (PTx)-catalyzed, adenosine diphosphate (ADP)-dependent ribosylation of Gialpha substrates decreased following ethanol treatment. The increased functional activity of Gialpha1/2-protein expression was confirmed by demonstrating that ethanol dose-dependently inhibited basal and stimulated AC activity in H4IIE cells, while not significantly altering basal AC activity in isolated cultured hepatocytes. Furthermore, while ethanol had no significant effect on basal mitogenesis in H4IIE cells or hepatocytes, increased mitogenesis caused by direct Gialpha-protein stimulation (mastoparan M7; 10-5,000 nmol/L) was further enhanced in the presence of ethanol, an effect that was completely blocked following Gi-protein inhibition (PTx; 100 ng/mL). In contrast, activation of Gi-proteins using M7 failed to alter cellular mitogenesis in isolated cultured hepatocytes, whether in the absence or presence of ethanol. Finally, analysis of mitogen-activated protein kinase (MAPK) activity demonstrated that chronic ethanol treatment further enhanced Gi-protein-stimulated MAPK activity in hepatic tumorigenic cells. In conclusion, these data demonstrate that ethanol enhances cellular mitogenesis in experimental HCC as a result of, at least in part, a Gi-MAPK-dependent pathway. Furthermore, this effect may be caused by ethanol's direct up-regulation of the expression and activity of Gi-proteins in HCC.

Diverse expression of G-proteins in human sarcoma cell lines with different osteogenic potential: evidence for the involvement of Gi2 in cell proliferation.

Previously, it has been shown that the GTP-binding protein Gi2 is implicated in cellular growth [1,2] and differentiation [2,3]. In the present paper we demonstrate that this is also the case for human sarcoma cells. Six human osteosarcoma and three soft tissue sarcoma clonal cell lines were analyzed for levels of G-protein mRNA and polypeptide expression and effector enzyme (i.e., adenylate cyclase and phospholipase C) activation, which were all compared with individual growth rates. Unexpectedly, it appeared that the various strains exhibited large inter-individual variations in G-protein expression and signaling system activation. However, cell doubling time in the exponential phase of growth was inversely correlated (r = 0.71, P < 0.05) to immunodetected levels of intrinsic Gi2 alpha. Furthermore, cells stably transfected with a retroviral (pZipNeo(SV)X) construct containing the activating or inactivating Gi2 alpha-R179E or Gi2 alpha-G204A point mutations consistently reduced or enhanced individual cell strain doubling time, respectively. It appeared that other parameters investigated, including cellular alkaline phosphatase and monoclonal antibody epitope binding, both being markers of the proliferating osteoblast, did not correlate with cell doubling times.

Altered expression of inhibitory guanine nucleotide regulatory proteins (Gi-proteins) in experimental hepatocellular carcinoma.

Guanine nucleotide regulatory proteins (G-proteins) play an important role in the onset and progression of malignancy. We hypothesized that alterations in inhibitory G-protein (Gi) expression and/or function may contribute to cellular invasion and formation of hepatocellular carcinoma (HCC). H4IIE hepatoma cells were inoculated directly into the liver parenchyma of ACI strain rats, and membranes were prepared from HCC livers and adjacent nonneoplastic livers 12 days following the initial inoculation. Expression of inhibitory Gialpha proteins was determined by Western blot analysis and changes in the functional activity of these proteins confirmed by pertussis toxin catalyzed ADP ribosylation and adenylyl cyclase activity. Inhibitory Gialpha1, Gialpha1/2, and Gialpha3 protein expression was significantly elevated in HCC when compared to adjacent nonneoplastic liver and sham-operated hepatic tissue. Pertussis toxin catalyzed ADP ribosylation of Gialpha substrates was significantly enhanced in HCC concomitant with increased basal and stimulated adenylyl cyclase activity following uncoupling of Gi-proteins with manganese ions. The role of Gi-proteins in cellular proliferation was confirmed using cultured H4IIE cells and normal hepatocytes. In quiescent H4IIE cells, mastoparan (Gialpha activator) increased [3H] thymidine incorporation and cell growth in a dose-dependent manner, whereas both pertussis toxin (a Gi-protein inhibitor) and 8-bromo-cAMP inhibited mitogenesis. In contrast, in isolated cultured hepatocytes, mastoparan inhibited [3H] thymidine incorporation, while pertussis toxin and 8-bromo-cAMP were mitogenic. We conclude that HCC is associated with marked changes in Gialpha-protein expression in vivo and in vitro, direct activation of which leads to increased mitogenesis in H4IIE cells in vitro.

SUBSTANCE P-INDUCED MAST CELL MEDIATOR RELEASE IS ASSOCIATED WITH STEM CELL FACTOR-DEPENDENT UPREGULATION OF Gαi-2 AND Gαi-3 PROTEINS AND DOWNSTREAM SIGNALING THROUGH A PROTEIN KINASE C-DEPENDENT PATHWAY

51 Aims: Substance P (SP)-induced mast cell activation has been implicated in the pathogenesis of gastrointestinal inflammation, but the precise nature of mast cell/SP interactions has not been completely defined. We previously reported that stem cell factor (SCF) conferred reactivity to mast cells stimulated by substance P through a signaling pathway involving G-proteins (Gastroenterology 1996; 110: A911). We therefore assessed whether SCF influenced the expression of G-proteins in mast cells and examined the role of protein kinase C (PKC) in SP-induced mast cell activation. Methods: Bone marrow cultured mast cells (BMCMCs) were maintained in either SCF (50 ng/ml) or 20% Concanavalin A-stimulated spleen conditioned medium (CM) for 4-6 weeks. Western blot analysis of G-protein expression was then performed on BMCMCs in SCF or CM. BMCMCs in SCF or CM were incubated with3 H-5-hydroxytryptamine (5-HT) in the presence or absence of the PKC inhibitors calphostin C (CC; 1 μM) or bisindolylmaleimide I (BIM; 10μM), and then challenged with either SP (10-4 M) or medium alone. Radioactive counts in the supernatants and cell pellets were measured and specific 5-HT release calculated. Results: SP induced significant 5-HT release from BMCMCs in SCF compared with medium alone (12.5 ± 1.5% vs. 1.6 ± 0.2%, respectively, p < 0.05), while no 5-HT release occurred from BMCMCs in CM. Western blot analysis demonstrated a marked increase in the expression of the G-proteins Gαi-2 (by 5 fold) and Gαi-3 (by 20 fold) in BMCMCs in SCF compared with BMCMCs in CM. Furthermore, the PKC inhibitors CC or BIM inhibited SP-induced 5-HT release from BMCMCs in SCF (by 74% and 100%, respectively). Conclusions: We found that mast cells maintained in SCF acquire the ability to release serotonin upon stimulation with SP and express increased levels of Gαi-2 and Gαi-3 proteins. Furthermore, our data demonstrate that SP-induced mast cell pre-formed mediator release involves a protein kinase C-dependent pathway.

The Effect of Prenatal Cocaine Exposure on Myometrial α- andβ-Adrenergic Receptor Kinetics and G-Protein Expression in Guinea Pigs† 1078

Maternal cocaine abuse is associated with increased uterine contractions which may lead to preterm labor and prematurity. One mechanism of cocaine action is through uptake inhibition of norepinephrine (an α- andβ-adrenergic agonist). As adrenergic stimulation plays an important role in uterine contractility, we hypothesized that cocaine exposure during pregnancy alters adrenergic receptor kinetics and its G-protein-mediated transmembrane signal transduction. Therefore, we studied the effect of cocaine exposure on α- and β-adrenergic receptor kinetics and G-protein expression in pregnant guinea pig myometrium. Methods: Pregnant guinea pigs were injected daily with cocaine (5mg/kg SC) or saline (control) throughout the latter half of gestation (term: 65 days). Guinea pigs were killed at 90% gestation, the uteri removed, and membrane protein fraction obtained. Saturation binding assays were performed for β,α1, and α2 adrenergic receptors using the tritiated radioligands DHA, prazosin, and rauwolscine, respectively, to determine receptor concentration, Bmax, in fmol/mg of protein, and affinity, kD, in nmol/mg of protein. Gi α and Gs α subunit expression was determined by immunoblotting techniques. Membrane protein fractions were resolved by SDS-PAGE and probed using a polyclonal antibody against the inhibitory (i) and stimulatory (s) Gα-protein subunits and compared using optical densitometry. Results: Myometrium from cocaine-exposed guinea pigs showed a significant decrease in the concentration of β-adrenergic receptors as compared with the control group (Bmax 78.5±6.97 (cocaine) vs 143±23.1(control), p<0.02) without significant difference in kD (2.58 vs 3.47). There was no significant difference in the concentration or affinity ofα1-adrenergic receptors (Bmax: 98.7(cocaine) vs 91.5(control) and kD: 0.523 (cocaine) vs 2.34 (control) orα2-adrenergic receptors (Bmax: 162 (cocaine) vs 133(control) and kD: 4.06 (cocaine) vs 2.55 (control)). Optical densitometry measurements failed to demonstrate a significant difference in either Giα or Gs α protein subunit expression.Conclusion: This study demonstrates that prenatal cocaine exposure down-regulates myometrial β-adrenergic receptor concentration in pregnant guinea pigs without significant effect on α-adrenergic receptor kinetics or G-protein expression. Decreased β-adrenergic activity may result in unopposed α-adrenergic stimulation and increased uterine contractions. These findings provide a mechanism for the relationship between maternal cocaine abuse and prematurity.

Flow-mediated regulation of G-protein expression in cocultured vascular smooth muscle and endothelial cells.

G-proteins have been implicated in the transduction of a number of flow-induced responses. We determined whether flow can modulate vascular endothelial or smooth muscle cell G-protein signaling. By use of a perfused transcapillary coculture system that permits the chronic exposure of cultured endothelial cells (ECs) and smooth muscle cells (SMCs) to physiological shear stresses, cocultures were exposed to stepwise increases in flow up to (1) 2 mL/min (low flow: 0.5 dyne/cm2), or (2) 44 mL/min (high flow: 15 dyne/cm2) and maintained for 72 hours before SMCs and ECs were harvested separately. Using Western blot analysis, EC Gi alpha3 expression was significantly increased (41+/-2.9%) by high-flow conditions compared with low-flow. The changes in G-protein expression were associated with a significant increase in endothelial nitric oxide synthase (eNOS) activity, elevated prostacyclin levels in the perfusing media, increased pertussis toxin-catalyzed ADP ribosylation of Gi alpha substrates, and enhanced agonist-stimulated GTPase activity in cocultured ECs. In contrast, high flow induced a significant decrease in Gi alpha1-2 expression (57 5%) in SMCs cocultured with ECs, an effect that was endothelium dependent, inhibited by indomethacin, and correlated with a decrease in pertussis toxin-catalyzed ADP ribosylation of Gi alpha substrates, reduced agonist-stimulated GTPase activity, and enhanced basal and G-protein-stimulated adenylyl cyclase activity. These data demonstrate that flow mediates selective changes in EC and SMC G-protein expression concomitant with changes in G-protein functionality and cellular signaling capacity. Moreover, flow-induced changes in SMC G-protein signaling capacity are endothelium dependent and require a cyclooxygenase product. G-protein modulation may thus represent an important mechanism whereby hemodynamic forces regulate vessel wall function.

Alterations in guanine nucleotide regulatory protein expression and activity in human hepatocellular carcinoma

Alterations in the expression and activity of guanine nucleotide regulatory proteins (G proteins) have been linked to the growth of several human tumors. We hypothesized that the expression and activity of G proteins are altered in human hepatocellular carcinoma (HCC). The expression of Gi and Gs proteins was determined in six human tumors and six normal controls (adjacent nonneoplastic liver) by Western blotting using specific antisera raised against the alpha subunit of G proteins Gi1, Gi1-2, Gi3, and Gs. Differences in G-protein expression were quantified by densitometry and expressed as percentage change from normal controls. The expression of Gi alpha1 was significantly increased in 80% of tumors (Gi alpha1, 284% +/- 77%; P < .05 percent of normal tissue), whereas Gi alpha1-2 and Gi alpha3 expression was increased in 67% of tumors (Gi alpha1-2, 218% +/- 21%; Gi alpha3, 154% +/- 6%; P < .05 percent of normal tissue). The functional activity of Gi alpha proteins as determined by pertussis toxin-catalyzed adenosine diphosphate (ADP)-ribosylation was also significantly increased in these tumors. In contrast, Gs alpha-protein expression was significantly reduced in all tumors examined (74% +/- 8% of normal tissue, P < .05). The functional activity of Gs alpha, as determined by adenylyl cyclase (AC) activity, was significantly decreased in tumor as compared to normal liver under both basal and agonist stimulated (guanosine triphosphate gamma S and forskolin) conditions. In summary, these data show for the first time a significant alteration in G-protein expression and functional activity in human HCC tissue. These alterations indicate a down-regulation of the AC-linked enzyme effector system in HCC that may be of critical importance to the formation and progression of human hepatocellular carcinoma.

Alterations in guanine nucleotide regulatory protein expression and activity in human hepatocellular carcinoma

Alterations in the expression and activity of guanine nucleotide regulatory proteins (G proteins) have been linked to the growth of several human tumors. We hypothesized that the expression and activity of G proteins are altered in human hepatocellular carcinoma (HCC). The expression of Gi and Gs proteins was determined in six human tumors and six normal controls (adjacent nonneoplastic liver) by Western blotting using specific antisera raised against the alpha subunit of G proteins Gi1, Gi1-2, Gi3, and Gs. Differences in G-protein expression were quantified by densitometry and expressed as percentage change from normal controls. The expression of Gi α1 was significantly increased in 80% of tumors (Gi α1, 284% +/- 77%; P < .05 percent of normal tissue), whereas Gi α1-2 and Gi α3 expression was increased in 67% of tumors (Gi α1-2, 218% +/- 21%; Gi α3, 154% +/- 6%; P < .05 percent of normal tissue). The functional activity of Giα proteins as determined by pertussis toxin-catalyzed adenosine diphosphate (ADP)-ribosylation was also significantly increased in these tumors. In contrast, Gs α-protein expression was significantly reduced in all tumors examined (74% +/- 8% of normal tissue, P < .05). The functional activity of Gs α, as determined by adenylyl cyclase (AC) activity, was significantly decreased in tumor as compared to normal liver under both basal and agonist stimulated (guanosine triphosphate γ S and forskolin) conditions. In summary, these data show for the first time a significant alteration in G-protein expression and functional activity in human HCC tissue. These alterations indicate a down-regulation of the AC-linked enzyme effector system in HCC that may be of critical importance to the formation and progression of human hepatocellular carcinoma. (Hepatology 1997 Nov;26(5):1189-94)

G-protein-mediated signaling in cholesterol-enriched arterial smooth muscle cells. 1. Reduced membrane-associated G-protein content due to diminished isoprenylation of G-gamma subunits and p21ras.

Mechanisms contributing to altered heterotrimeric G-protein expression and subsequent signaling events during cholesterol accretion have been unexplored. The influence of cholesterol enrichment on G-protein expression was examined in cultured smooth muscle cells that resemble human atherosclerotic cells by exposure to cationized LDL (cLDL). cLDL, which increases cellular free and esterified cholesterol 2-fold and 10-fold, respectively, reduced the cell membrane content of Galphai-1, Galphai-2, Galphai-3, Gq/11, and Galphas. The following evidence supports the premise that the mechanism by which this occurs is due to reduced isoprenylation of the Ggamma-subunit. First, the inhibitory effect of cholesterol enrichment on the membrane content of Galphai subunits was found to be post-transcriptional, since the mRNA steady-state levels of Galphai(1-3) were unchanged following cholesterol enrichment. Second, the membrane expression of alpha and beta subunits was mimicked by cholesterol and 17-ketocholesterol, both of which inhibit HMG-CoA reductase. Third, inhibition of Galphai and Gbeta expression in cholesterol-enriched cells was overcome by mevalonate, the immediate product of HMG-CoA reductase. Fourth, pulse-chase experiments revealed that cholesterol enrichment did not reduce the degradation rate of membrane-associated Galphai subunits. Fifth, cholesterol enrichment also reduced membrane expression of Ggamma-5, Ggamma-7upper; these gamma subunits are responsible for trafficking of the heterotrimeric G-protein complex to the cell membrane as a result of HMG-CoA reductase-dependent post-translational lipid modification (geranylgeranylation) and subsequent membrane association. Cholesterol enrichment did not alter expression of G-gamma-5 mRNA, as assessed by reverse transcriptase polymerase chain reaction, supporting a post-transcriptional defect in Ggamma subunit expression. Fifth, cholesterol enrichment also reduced the membrane content of p21ras (a low molecular weight G-protein requiring farnesylation for membrane targeting) but did not alter the membrane content of the two proteins that do not require isoprenylation for membrane association&sbd;PDGF-receptor or p60-src. Reduced G-protein content in cholesterol-laden cells was reflected by reduced G-protein-mediated signaling events, including ATP-induced GTPase activity, thrombin-induced inhibition of cyclic AMP accumulation, and MAP kinase activity. Collectively, these results demonstrate that cholesterol enrichment reduces G-protein expression and signaling by inhibiting isoprenylation and subsequent membrane targeting. These results provide a molecular basis for altered G-protein-mediated cell signaling processes in cholesterol-enriched cells.

Acanthocheilonema viteae:Stage-Specific Expression of G-Protein α-Subunits

We have previously demonstrated by Western blot analysis that the adult stage of the filarial nematodeAcanthocheilonema viteaeexpresses the α-subunits of heterotrimeric G-proteins corresponding to GSand Gq. We now show, using the same technique, that these two α-subunits are not detectable in the microfilaria stage of the parasite. Conversely, microfilariae contain Go, an α-subunit not expressed by the adult worm. No other G-protein α-subunits were found in microfilariae by Western blotting. However, reverse transcriptase-polymerase chain reaction (RT-PCR) with degenerate G-protein oligonucleotide primers, followed by hybridisation analysis, using oligonucleotides specific for individual G-protein α-subunits, not only confirmed expression of Go, but also detected Gi1and G11α-subunits. G-protein expression in infective larvae was also investigated by RT-PCR analysis: this stage of the organism was found to resemble the adult more than the microfilaria but differed from the adult in that GSwas absent and Gi3was present. The significance of these stage-specific differences in G-protein expression is discussed with respect to their possible role in parasite development and survival.

G-protein expression in melanotropes changes coincident with innervation of the developing rat pituitary intermediate lobe

The two isoforms of the dopamine D2 receptor, the D2short and the D2long differ in a 29 amino acid insert in the third cytoplasmic loop with which G proteins interact. We have previously reported that in rat melanotropes, expression of D2short increases markedly at the end of the first postnatal week which is concurrent with innervation of the intermediate lobe. Using immunohistochemistry, this study examined expression of Gαi1/2, Gαi3, Gαo and Gαs proteins before and after dopaminergic innervation. Gαi3 increased through gestational day 20, and then remained level to postnatal day 6. At this time, coinciding with the induction of D2short expression, Gαi3 immunoreactive intensity increased markedly, possibly indicating co-regulation of these receptors. On postnatal day 6, Gαs immunoreactive intensity increased in some, but not all, melanotropes. The resulting heterogeneity in Gs expression persisted in the adult. Gαi1/2 immunoreactivity did not change and Gαo was detected only subsequent to the event of innervation. Thus, dopamine released from axons and acting through D2 receptor stimulation could increase Gαi3 immunoreactivity and decrease Gαs immunoreactive intensity in some melanotropes.

Nitric oxide synthase inhibition by N(omega)-nitro-L-arginine methyl ester modulates G-protein expression and adenylyl cyclase activity in rat heart.

We have recently shown an enhanced expression of inhibitory guanine nucleotide regulatory proteins Gi alpha-2 and Gi alpha-3 and their respective mRNA in hearts from DOCA-salt hypertensive rats. However, it is not known whether these changes are due to the expressed hypertrophy or hypertension. The present studies were therefore undertaken to investigate this possibility. Hypertension in Sprague-Dawley rats was induced by the oral administration of the arginine analog N(omega)-nitro-L-arginine methyl ester (L-NAME) in their drinking tap water for a period of 4 weeks. The control rats were given plain tap water only. L-NAME-treated rats showed an enhanced blood pressure (190 +/- 9.23 mm Hg; n = 20) compared to control rats (121 +/- 6.3 mm Hg; n = 20). However, heart to body weight ratio was not different in the two groups. Guanosine 5'-o-(3-thiotriphosphate) (GTPgammaS) stimulated adenylyl cyclase activity in heart membranes from both groups, but the extent of stimulation was significantly decreased in L-NAME-treated rats. Similarly, stimulations exerted by isoproterenol, glucagon, NaF, and forskolin on adenylyl cyclase were also diminished in L-NAME-treated rats. On the other hand, the inhibitory effect of low concentrations of GTPgammaS on forskolin-stimulated enzyme activity was significantly enhanced. The extent of oxotremorine-mediated inhibition of adenylyl cyclase was unaltered in both control and L-NAME-induced hypertensive rats. The levels of Gi alpha-2 and Gi alpha-3, but not of stimulatory guanine nucleotide regulatory protein Gs alpha, as determined by immunoblotting, were significantly augmented in L-NAME-treated rats. Northern blot studies revealed a significant increase in Gi alpha-2 and Gi alpha-3 mRNA with no changes in Gs alpha mRNA. These results suggest that the altered expression of Gi alpha proteins and adenylyl cyclase activity in L-NAME-treated rats may be attributed to hypertension and not to hypertrophy.