Novel formula for cell kinetics in xenograft model of hepatocellular carcinoma using histologically calculable parameters

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.     

RGS4 inhibits Gq-mediated activation of mitogen-activated protein kinase and phosphoinositide synthesis

Recombinant regulators of G protein-signaling (RGS) proteins stimulate hydrolysis of GTP by alpha subunits of the Gi family but have not been reported to regulate other G protein alpha subunits. Expression of recombinant RGS proteins in cultured cells inhibits Gi-mediated hormonal signals probably by acting as GTPase-activating proteins for Galphai subunits. To ask whether an RGS protein can also regulate cellular responses mediated by G proteins in the Gq/11 family, we compared activation of mitogen-activated protein kinase (MAPK) by a Gq/11-coupled receptor, the bombesin receptor (BR), and a Gi-coupled receptor, the D2 dopamine receptor, transiently co-expressed with or without recombinant RGS4 in COS-7 cells. Pertussis toxin, which uncouples Gi from receptors, blocked MAPK activation by the D2 dopamine receptor but not by the BR. Co-expression of RGS4, however, inhibited activation of MAPK by both receptors causing a rightward shift of the concentration-effect curve for both receptor agonists. RGS4 also inhibited BR-stimulated synthesis of inositol phosphates by an effector target of Gq/11, phospholipase C. Moreover, RGS4 inhibited inositol phosphate synthesis activated by addition of AlF4- to cells overexpressing recombinant alphaq, probably by binding to alphaq.GDP.AlF4-. These results demonstrate that RGS4 can regulate Gq/11-mediated cellular signals by competing for effector binding as well as by acting as a GTPase-activating protein.     

Gonadotropin and gonadal steroid release in response to a gonadotropin-releasing hormone agonist in Gqalpha and G11alpha knockout mice

In this study, we used mice lacking the G11alpha [G11 knockout (KO)] or Gqalpha gene (Gq KO) to examine LH release in response to a metabolically stable GnRH agonist (Buserelin). Mice homozygous for the absence of G11alpha and Gqalpha appear to breed normally. Treatment of (5 wk old) female KO mice with the GnRH agonist Buserelin (2 microg/100 microl, sc) resulted in a rapid increase of serum LH levels (reaching 328 +/- 58 pg/25 microl for G11 KO; 739 +/- 95 pg/25 microl for Gq KO) at 75 min. Similar treatment of the control strain, 129SvEvTacfBr for G11 KO or the heterozygous mice for Gq KO, resulted in an increase in serum LH levels (428 +/- 57 pg/25 microl for G11 KO; 884 +/- 31 pg/25 microl for Gq KO) at 75 min. Both G11 KO and Gq KO male mice released LH in response to Buserelin (2 microg/100 microl of vehicle; 363 +/- 53 pg/25 microl and 749 +/- 50 pg/25 microl 1 h after treatment, respectively). These values were not significantly different from the control strain. In a long-term experiment, Buserelin was administered every 12 h, and LH release was assayed 1 h later. In female G11 KO mice and control strain, serum LH levels reached approximately 500 pg/25 microl within the first hour, then subsided to a steady level (approximately 100 pg/25 microl) for 109 h. In male G11 KO mice and in control strain, elevated LH release lasted for 13 h; however, LH levels in the G11 KO male mice did not reach control levels for approximately 49 h. In a similar experimental protocol, the Gq KO male mice released less LH (531 +/- 95 pg/25 microl) after 13 h from the start of treatment than the heterozygous male mice (865 +/- 57 pg/25 microl), but the female KO mice released more LH (634 +/- 56 pg/25 microl) after 1 h from the start of treatment than the heterozygous female mice (346 +/- 63 pg/25 microl). However, after the initial LH flare, the LH levels in the heterozygous mice never reached the basal levels achieved by the KO mice. G11 KO mice were less sensitive to low doses (5 ng/per animal) of Buserelin than the respective control mice. Male G11 KO mice produced more testosterone than the control mice after 1 h of stimulation by 2 microg of Buserelin, whereas there was no significant difference in Buserelin stimulated testosterone levels between Gq KO and heterozygous control mice. There was no significant difference in Buserelin stimulated estradiol production in the female Gq KO mice compared with control groups of mice. However, female G11 KO mice produced less estradiol in response to Buserelin (2 microg) compared with control strain. Although there were differences in the dynamics of LH release and steroid production in response to Buserelin treatment compared with control groups of mice, the lack of complete abolition of these processes, such as stimulated LH release, and steroid production, suggests that these G proteins are either not absolutely required or are able to functionally compensate for each other.     

Transformation of Rat-1 fibroblasts with the v-src oncogene increases the tyrosine phosphorylation state and activity of the alpha subunit of Gq/G11

Two major intermediaries in signal transduction pathways are pp60v-sre family tyrosine kinases and heterotrimeric guanine nucleotide-binding proteins. In Rat-1 fibroblasts transformed by the v-src oncogene, endothelin-1 (ET-1)-induced inositol 1,4,5-trisphosphate accumulation is increased 6-fold, without any increases in the numbers of ET-1 receptors or in the response to another agonist, thrombin. This ET-1 hyperresponse can be inhibited by an antibody directed against the carboxyl terminus of the Gq/G11 alpha subunit, suggesting that the Gq/G11 protein couples ET-1 receptors to phospholipase C (PLC). While v-src transformation did not increase the expression of the Gq/G11 alpha subunit, immunoblotting with anti-phosphotyrosine antibodies and phosphoamino acid analysis demonstrated that the Gq/G11 alpha subunit becomes phosphorylated on tyrosine residues in v-src-transformed cells. Moreover, when the Gq/G11 protein was extracted from control and transformed cell lines and reconstituted with exogenous PLC, AIF*4-stimulated Gq/G11 activity was markedly increased in extracts from v-src-transformed cells. Our results demonstrate that the process of v-src transformation can increase the tyrosine phosphorylation state of the Gq/G11 alpha-subunit in intact cells and that the process causes an increase in the Gq/G11 alpha-subunit's ability to stimulate PLC following activation with AIF-4.     

Multidrug resistance gene expression in rodents and rodent hepatocytes treated with mitoxantrone

Overexpression of P-glycoprotein in tumor cells can represent a severe drawback for cancer chemotherapy. P-glycoprotein acts as an efflux transporter for a variety of chemotherapeutic agents. It is encoded by multidrug resistance (mdr) genes of the subfamily 1 in humans (MDR1) and rodents (mdr1a and 1b). Because mdr1 gene expression is inducible in cultured rat hepatocytes and in rat liver with chemical carcinogens such as 2-acetylaminofluorene or aflatoxin B1, which form DNA-binding electrophiles during their metabolism, we investigated whether the DNA-damaging chemotherapeutic drug mitoxantrone may induce multidrug resistance in rodents and in hepatocytes in primary culture. In H4IIE rat hepatoma cells stably transfected with a luciferase construct containing the rat mdr1b promoter, mitoxantrone caused a concentration-dependent increase in promoter activity. Mdr1 gene expression in cultured rat hepatocytes was enhanced at mitoxantrone concentrations greater than or equal to 0.1 microM and in mouse hepatocytes at 5 microM. In hepatocytes from both species, a correlation was found between mdr1 induction and the inhibition of protein synthesis. In vivo, mitoxantrone was a very powerful inducer of mdr1 gene expression in rat liver and small intestine. In rat kidney, induction of mRNA was lower, and a marginal effect was seen in lung. In contrast with rats, no significant induction of mdr1 gene expression was obtained in mouse liver. Probably as a consequence of inhibition of protein synthesis, mitoxantrone did not lead to a pronounced elevation of P-glycoprotein levels in rat liver and kidney.     

Gq/11 and PLC-beta 1 mediate the substance P-induced inhibition of an inward rectifier K+ channel in brain neurons

1. Substance P (SP) induces a slow neuronal excitation in cholinergic neurons from the nucleus basalis by suppressing an inwardly rectifying K+ current (Kir). We have determined which G protein alpha-subunit mediates this SP effect. 2. After intracellularly injecting antibody against each alpha-subunit of G proteins (Gq alpha/11 alpha, G12 alpha, and G13 alpha) with an Eppendorf microinjector, we examined, by using the whole cell patch-clamp and the ON-cell mode of single-channel recording, the effect of SP on Kir in cultured neurons of the nucleus basalis. The effect of SP on Kir was substantially reduced in neurons injected with antibodies to Gq alpha/11 alpha but not with antibodies to G12 alpha or G13 alpha. 3. The effects of antibodies against three isozymes of phospholipase C (PLC-beta 1, PLC-beta 2, and PLC-beta 3) were tested. The SP-induced suppression of Kir was reduced by antibody against PLC-beta 1 but not by antibodies against PLC-beta 2 or PLC-beta 3. 4. We conclude that the SP-induced inhibition of Kir in nucleus basalis neurons is mediated by Gq/11 and PLC-beta 1.     

A novel mitogenic signaling pathway of bradykinin in the human colon carcinoma cell line SW-480 involves sequential activation of a Gq/11 protein, phosphatidylinositol 3-kinase beta, and protein kinase Cepsilon

The signaling routes connecting G protein-coupled receptors to the mitogen-activated protein kinase (MAPK) pathway reveal a high degree of complexity and cell specificity. In the human colon carcinoma cell line SW-480, we detected a mitogenic effect of bradykinin (BK) that is mediated via a pertussis toxin-insensitive G protein of the Gq/11 family and that involves activation of MAPK. Both BK-induced stimulation of DNA synthesis and activation of MAPK in response to BK were abolished by two different inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY 294002, as well as by two different inhibitors of protein kinase C (PKC), bisindolylmaleimide and Ro 31-8220. Stimulation of SW-480 cells by BK led to increased formation of PI3K lipid products (phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3, 4-bisphosphate) and to enhanced translocation of the PKCepsilon isoform from the cytosol to the membrane. Both effects of BK were inhibited by wortmannin, too. Using subtype-specific antibodies, only the PI3K subunits p110beta and p85, but not p110alpha and p110gamma, were detected in SW-480 cells. Finally, p110beta was found to be co-immunoprecipitated with PKCepsilon. Our data suggest that in SW-480 cells, (i) dimeric PI3Kbeta is activated via a Gq/11 protein; (ii) PKCepsilon is a downstream target of PI3Kbeta mediating the mitogenic signal to the MAPK pathway; and (iii) PKCepsilon associates with the p110 subunit of PI3Kbeta. Thus, these results add a novel possibility to the emerging picture of multiple pathways linking G protein-coupled receptors to MAPK.     

Early inhibition of protein phosphatases preferentially blocks phorbol ester-stimulated mitogenic signalling in melanocytes: increase in specific tyrosine phosphoproteins

Inhibition of protein phosphatases has been suggested as an alternative mechanism of tumor promotion (H. Fujiki, Mol. Carcinog. 5:91, 1992). We have now used early melanocyte passages dependent on phorbol esters and serum for growth and later passages with partial phorbol ester independence, to investigate the role of protein phosphatases on melanocyte DNA synthesis. Neither okadaic acid, an inhibitor of ser/thr protein phosphatases, nor vanadate, an inhibitor of tyrosine phosphatases, can stimulate basal or serum-stimulated mitogenesis in contrast to phorbol esters. Moreover, both phosphatase inhibitors are able to suppress serum and phorbol ester-stimulated mitogenesis, if added within 4 hours of growth activation. Inhibition of mitogenesis by either inhibitor correlated with an early increase in a common set of tyrosine phosphoproteins, which included a major 33 Kd species. Our data suggest that protein phosphatase inhibitors are growth suppressors and antagonize phorbol ester effects in cells of melanocytic origin, implying an early requirement for protein phosphatase activity during mitogenic signalling in these cells.     

Differential intracellular signaling of the GalR1 and GalR2 galanin receptor subtypes

The diverse physiological functions exerted by the neuropeptide galanin may be regulated by multiple G protein-coupled receptor subtypes and intracellular signaling pathways. Three galanin receptor subtypes (GalRs) have been recently cloned, but the G protein coupling profiles of these receptors are not completely understood. We have generated GalR1- and GalR2-expressing Chinese hamster ovary (CHO) cell lines and systematically examined the potential for these two receptors to couple to the Gs, Gi, Go, and Gq proteins. Galanin did not stimulate an increase in cAMP levels in GalR1/CHO or GalR2/CHO cells, suggesting an inability of either receptor to couple to Gs. Galanin inhibited forskolin-stimulated cAMP production in GalR1/CHO cells by 70% and in GalR2/CHO cells by 30%, suggesting a strong coupling of GalR1 to Gi and a more modest coupling between GalR2 and Gi. GalR1 and GalR2 both mediated pertussis toxin-sensitive MAPK activity (2-3-fold). The stimulation mediated by GalR1 was inhibited by expression of the C-terminus of beta-adrenergic receptor kinase (beta ARKct), which specifically inhibits G beta gamma signaling, but was not affected by the protein kinase C (PKC) inhibitor, bis[indolylmaleimide], or cellular depletion of PKC. In contrast, GalR2-mediated MAPK activation was not affected by beta ARKct expression but was abolished by inhibition of PKC activity. The data demonstrate that GalR1 is coupled to a Gibetagamma signaling pathway to mediate MAPK activation. In contrast, GalR2 utilizes a distinct signaling pathway to mediate MAPK activation, which is consistent with Go-mediated MAPK activation in CHO cells. Galanin was unable to stimulate inositol phosphate (IP) accumulation in CHO or COS-7 cells expressing GalR1. In contrast, galanin stimulated a 7-fold increase in IP production in CHO or COS-7 cells expressing GalR2. The GalR2-mediated IP production was not affected by pertussis toxin, suggesting a linkage of GalR2 with Gq/G11. Thus, the GalR1 receptor appears to activate only the Gi pathway. By contrast, GalR2 is capable of stimulating signaling which is consistent with activation of Go, Gq/G11, and Gi. The differential signaling profiles and the tissue distribution patterns of GalR1 and GalR2 may underlie the functional spectra of galanin action mediated by these galanin receptors and regulate the diverse physiological functions of galanin.     

G proteins of the Gq family couple the H2 histamine receptor to phospholipase C

In several cell systems histamine has been shown to stimulate both adenylyl cyclase and phospholipase C through activation of a G protein-coupled H2 receptor. To analyze the bifurcating signal emanating from the activated H2 receptor and to identify the G proteins involved, H1 and H2 histamine receptors were functionally expressed in baculovirus-infected insect cells. Histamine challenge lead to concentration-dependent cAMP formation and Ca2+ mobilization in Sf9 cells infected with a virus encoding the H2 receptor, whereas H1 receptor stimulation only resulted in pronounced phospholipase C activation. To analyze the G protein coupling pattern of histamine receptors, activated G proteins were labeled with [alpha-32P]GTP azidoanilide and identified by selective immunoprecipitation. In insect cell membranes expressing H1 histamine receptors, histamine led to incorporation of the label into alpha q-like proteins, whereas activation of the H2 receptor resulted in labeling of alpha q- and alpha s-like G protein alpha-subunits. In COS cells transfected with H2 receptor complementary DNA, histamine caused concentration-dependent accumulation of cAMP and inositol phosphates; the latter effect was insensitive to pertussis toxin treatment. Histamine stimulation led to a pronounced increase in inositol phosphate production when complementary DNAs coding for alpha q, alpha 11, alpha 14, or alpha 15 G protein alpha-subunits were cotransfected. This increase was specific for Gq family members, as overexpression of alpha 12 or alpha s did not enhance histamine-stimulated phospholipase C activation. In membranes of guinea pig heart, addition of [alpha-32P]GTP azidoanilide resulted in labeling of alpha q and alpha 11 via the activated H1 and also via H2 receptors. These data demonstrate that dual signaling of the activated H2 histamine receptor is mediated by coupling of the receptor to Gs and Gq family members.     

cAMP and beta gamma subunits of heterotrimeric G proteins stimulate the mitogen-activated protein kinase pathway in COS-7 cells

Mitogen-activated protein kinases (MAPKs) are activated by a variety of extracellular stimuli, including agonists for G protein-coupled receptors. Using transient transfection of COS-7 cells, we have studied the stimulation of a hemagglutinin-tagged p44mapk (p44HA-mapk) by receptors coupled to Gs, Gq, and Gi. Agonists that act via all three G proteins stimulated p44HA-mapk activity. A constitutively activated alpha s mutant, forskolin, and a cAMP analog also increased p44HA-mapk activity, indicating that cAMP in COS-7 cells, in contrast to other cell types, activates the MAPK pathway. Similarly, a constitutively activated alpha q mutant, overexpression of phospholipase C-beta 2, and a phorbol ester also stimulated p44HA-mapk, suggesting that Gq-coupled receptors stimulate the MAPK pathway by increasing phosphatidylinositol turnover and probably stimulating protein kinase C. In COS-7 cells, in contrast to Rat-1 cells, mutationally activated alpha i did not stimulate the MAPK pathway. G protein beta and gamma subunits, overexpressed together, did activate p44HA-mapk; this finding suggests that in COS-7 cells Gi-coupled receptors may stimulate the MAPK pathway through beta gamma. These unexpected results in COS-7 cells show that G proteins and second messengers regulate the MAPK pathway differently in different cell types.     

Histone modification governs the cell cycle regulation of a replication-independent chromatin assembly pathway in Saccharomyces cerevisiae

We describe a replication-independent, cell cycle-regulated chromatin assembly pathway in budding yeast. The activity of this pathway is low in S phase extracts but is very high in G2, M, and G1 cell extracts, with peak activity in late M/early G1. The cell cycle regulation of this pathway requires a specific pattern of posttranslational modification of histones H3 and/or H4, which is distinct for H3/H4 present in S phase versus M and G1 phase cell extracts. Histone H3/H4 modification is therefore important for the reciprocal control of replication-dependent and -independent chromatin assembly pathways during the cell cycle.     

Cyclic adenosine monophosphate-mediated protection against bile acid-induced apoptosis in cultured rat hepatocytes

Cyclic adenosine monophosphate (cAMP) has been shown to modulate apoptosis. To evaluate the role of cAMP in bile acid-induced hepatocyte apoptosis, we studied the effect of agents that increase cAMP on the induction of apoptosis by glycochenodeoxycholate (GCDC) in cultured rat hepatocytes. GCDC induced apoptosis in 26.5%+/-1.1% of hepatocytes within 2 hours. Twenty-minute pretreatment of hepatocytes with 100 micromol/L 8-(4-chlorothiophenyl) cAMP (CP-cAMP) resulted in a reduction in the amount of apoptosis to 35.2%+/-3.8% of that seen in hepatocytes treated with GCDC alone. Other agents that increase intracellular cAMP, including dibutyryl cAMP (100 micromol/L), glucagon (200 nmol/L), and a combination of forskolin (20 micromol/L) and 3-isobutyl-1-methylxanthine (20 micromol/L), also inhibited GCDC-induced apoptosis to a similar extent. Pretreatment with the protein kinase A (PKA) inhibitor, KT5720, prevented the protective effect of CP-cAMP and inhibited CP-cAMP-induced activation of PKA activity. Inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin (50 nmol/L), or Ly 294002 (20 micromol/L) also prevented the cytoprotective effect of cAMP. PI3K assays confirmed that wortmannin (50 nmol/L) inhibited PI3K activity, while CP-cAMP had no effect on the activity of this lipid kinase. GCDC increased mitogen-activated protein kinase (MAPK) activity, but had no effect on stress-activated protein kinase (SAPK) activity in hepatocytes. cAMP decreased basal and GCDC-induced MAPK activity and increased SAPK activity. The MAPK kinase inhibitor, PD 98059, inhibited both GCDC-mediated MAPK activation and GCDC-induced apoptosis. In conclusion: 1) agents that increase intracellular cAMP protect against hepatocyte apoptosis induced by hydrophobic bile acids; 2) activation of MAPK by GCDC may be involved in bile acid-induced apoptosis; and 3) cAMP-mediated cytoprotection against bile acid-induced apoptosis appears to involve PKA, MAPK, and PI3K.     

Cytoprotective effects of adrenomedullin in glomerular cell injury: central role of cAMP signaling pathway

Activation of cAMP signaling pathway was shown to inhibit some pathobiologic processes in mesangial cells (MC). We investigated whether adrenomedullin (ADM), a potent agonist of adenylate cyclase, is synthesized in MC and whether it can, via cAMP, suppress the generation of reactive oxygen metabolites (ROM) and proliferation of cells in glomeruli. With the use of an immunohistologic technique ADM was detected in mesangial and microvascular areas of rat glomeruli. MC grown in primary culture synthesized ADM, and the synthesis was stimulated by TNF alpha and IL-1 beta but not by PDGF and EGF. ADM inhibited ROM generation in MC dose-dependently and caused in situ activation of protein kinase A (PKA). In macrophages (cell line J774) ROM generation was about four times higher than in MC and was inhibited by ADM in a similar way as in MC. The rate of MC proliferation, measured by [3H]-incorporation, and the activity of mitogen-activated protein kinase (MAPK) stimulated by PDGF and EGF were dose-dependently inhibited by ADM; the maximum inhibition (at 10 nM ADM) was about -80%. Mitogenesis of MC and MAPK activity when stimulated to a similar extent by endothelin (ET-1) was inhibited by ADM to a significantly (P < 0.01) lesser degree (-30%). Further, ADM inhibited PDF-stimulated mitogenesis and activation of MAPK in cultured vascular smooth muscle cells (VSMC). The inhibition of PDGF-activated MAPK by ADM in VSMC was reversed by the protein kinase A (PKA) inhibitor, H89. Taken together, results indicate the adrenomedullin (ADM) generated in mesangial cells (MC) can suppress, via activation of the cAMP-protein kinase A (PKA) signaling pathway, reactive oxygen metabolites (ROM) generation in MC and infiltrating macrophages as well as mitogen-activated protein kinase (MAPK)-mediated mitogenesis in MC and vascular smooth muscle cells (VSMC). We suggest that introglomerular ADM may serve as a cytoprotective autoacoid that suppresses pathobiologic processes evoked by immuno-inflammatory injury of glomeruli.     

RGS4 inhibits G-protein signaling in cardiomyocytes

BACKGROUND: RGS family members are GTPase-activating proteins for heterotrimeric Gq and Gi proteins. RGS genes are expressed in heart tissue and in cultured cardiomyocytes. There is evidence that altered RGS gene expression may contribute to the pathogenesis of cardiac hypertrophy and failure. METHODS AND RESULTS: We investigated the ability of RGS proteins to block G-protein signaling in vivo by using a cultured cardiomyocyte transfection system. Endothelin-1, angiotensin II, and phenylephrine signal through Gq or Gi family members and promote the hypertrophy of cardiomyocytes. We found that phenylephrine-mediated and endothelin-1-mediated induction of the atrial natriuretic factor and myosin light chain-2 genes was inhibited in cells that were transfected with RGS4. Phenylephrine-mediated gene induction was not inhibited in cells that were transfected with N128A-RGS4, a point mutant form that lacks GTPase-activating protein activity. Phenylephrine-mediated myofilament organization and cell growth were also blocked in cells by RGS4. CONCLUSIONS: These results demonstrate that RGS protein can inhibit G-protein-mediated signaling in vivo and suggest that increased expression of RGS protein may be a counterregulatory mechanism to inhibit G protein signaling.     

Evidence that Gq family G proteins do not function in mouse egg activation at fertilization

Embryonic development is initiated after the fertilizing sperm contacts the egg and triggers a process termed "egg activation," resulting in calcium release, cortical granule exocytosis, recruitment of maternal mRNAs, and cell cycle resumption. Heterotrimeric guanine nucleotide-binding proteins (G proteins) may be involved in mouse egg activation since inhibition of G protein beta gamma subunits partially inhibits sperm-induced cell cycle resumption. In addition, specific events of egg activation can be initiated in the absence of sperm by acetylcholine stimulation of mouse eggs overexpressing the human m1 muscarinic receptor, a G protein-coupled receptor. In somatic cell, G proteins in the Gq family couple ligand stimulation of the m1 muscarinic receptor to activation of phospholipase C, resulting in the production of inositol 1,4,5-trisphosphate (IP3) and IP3-mediated release of intracellular calcium. Since IP3-mediated calcium release is involved in egg activation at fertilization, we have examined the role of Gq family G proteins in both sperm-independent (muscarinic receptor-mediated) and sperm-induced egg activation using a function-blocking antibody raised against the common C-terminal region of Gq and G11 proteins. We show that this antibody effectively inhibits Gq family G proteins in mouse eggs by demonstrating that the antibody inhibits egg activation in response to stimulation of the m1 muscarinic receptor. This same antibody, however, does not inhibit sperm-induced egg activation events. These results indicate that although activation of Gq family G proteins can result in egg activation in the mouse, it is unlikely that these proteins are used by the sperm to initiate egg activation at fertilization.