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Differential Expression of Guanine Nucleotide-Binding Proteins Enhances Camp Synthesis in Regenerating Rat Liver

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Differential Expression of Guanine Nucleotide-Binding Proteins Enhances Camp Synthesis in Regenerating Rat Liver Differential expression of guanine nucleotide-binding proteins enhances cAMP synthesis in regenerating rat liver. A M Diehl, … , D Wolfgang, G Wand J Clin Invest. 1992;89(6):1706-1712. https://doi.org/10.1172/JCI115771. Research Article Events leading to cAMP accumulation after partial hepatectomy (PH) and effects of cAMP on hormonal induction of DNA synthesis in hepatocytes were characterized. Hepatic cAMP peaked biphasically post-PH and paralleled changes in adenylyl cyclase activity. Fluctuations in cyclase activity were not explained by variations in glucagon receptor kinetics, but reflected altered G-protein expression. Membrane levels of the stimulatory G-protein, Gs alpha, increased early after PH and were sustained. Levels of the inhibitory G-protein, Gi2 alpha, increased more slowly, peaked later, and quickly fell. Levels of both G-proteins correlated poorly with levels of their mRNAs, suggesting posttranscriptional factors modify their membrane concentrations. When growth factor-induced DNA synthesis was compared in hepatocyte cultures grown with or without agents that increase intracellular cAMP, DNA synthesis was inhibited by sustained high levels of cAMP but was enhanced when high cAMP levels fell. In both regenerating liver and hepatocyte cultures, the expression of a "differentiated" hepatocyte gene, phosphoenolpyruvate carboxykinase, correlated with elevated cAMP levels. These data suggest that the differential expression of G-proteins integrates signals initiated by several growth factors so that the accumulation of cAMP is tightly regulated post-PH. The ensuing variations in cAMP levels modulate both growth and differentiated functions during liver regeneration. Find the latest version: https://jci.me/115771/pdf Differential Expression of Guanine Nucleotide-binding Proteins Enhances cAMP Synthesis in Regenerating Rat Liver Anna Mae Diehl, Shi Qi Yang, David Wolfgang, and Gary Wand Department ofMedicine, The Johns Hopkins University, Baltimore, Maryland 21205 Abstract diated stimulation of liver cell proliferation is scant (6). Indeed, in several nonhepatic cell lines, cAMP clearly inhibits cellular Events leading to cAMP accumulation after partial hepatec- proliferation (7, 8). To clarify the role ofcAMP-dependent pro- tomy (PH) and effects of cAMP on hormonal induction of DNA cesses in compensatory hepatic growth, we sought to character- synthesis in hepatocytes were characterized. Hepatic cAMP ize the cellular events that result in hepatic cAMP accumula- peaked biphasically post-PH and paralleled changes in ade- tion after PH and to determine whether similar levels ofcAMP nylyl cyclase activity. Fluctuations in cyclase activity were not alter hormonal induction of DNA synthesis in normal adult explained by variations in glucagon receptor kinetics, but re- hepatocytes. flected altered G-protein expression. Membrane levels of the stimulatory G-protein, Gs alpha, increased early after PH and Methods were sustained. Levels of the inhibitory G-protein, Gi2 alpha, increased more slowly, peaked later, and quickly fell. Levels of Materials. Unless noted below, all chemicals used in these experiments both G-proteins correlated poorly with levels of their mRNAs, were purchased from Sigma Chemical Co. (St. Louis, MO). Reagents suggesting posttranscriptional factors modify their membrane for determination of hepatic protein content were purchased from Bio- concentrations. When growth factor-induced DNA synthesis Rad Laboratories (Richmond, CA). Guanidine isothiocyanate, sarco- was compared in hepatocyte cultures grown with or without syl, and agarose were obtained from Bethesda Research Laboratories agents that increase intracellular cAMP, DNA synthesis was (Life Technologies, Gaithersburg, MD). Phenol was purchased from Fluka (Ronkonkoma, All inhibited by sustained high levels of cAMP but was enhanced NY). radioactive isotopes were obtained when high cAMP levels fell. In both from Amersham Corp. (Arlington Heights, IL). Nylon membranes for regenerating liver and Northern analysis were from ICN Biomedicals, Inc. (Irvine, CA). hepatocyte cultures, the expression of a "differentiated" hepa- Membranes (Immobolin-P) for Western analysis were purchased from tocyte gene, phosphoenolpyruvate carboxykinase, correlated Millipore Corp. (Bedford, MA). Kodak XAR-5 film was used for all with elevated cAMP levels. These data suggest that the differ- autoradiographs. Tissue culture media (Medium 199) and type I colla- ential expression of G-proteins integrates signals initiated by gen (Vitrogen) were purchased from Gibco Laboratories (Grand Is- several growth factors so that the accumulation of cAMP is land, NY). 60-mm Falcon culture plates came from Becton Dickinson tightly regulated post-PH. The ensuing variations in cAMP Labware (Lincoln Park, NJ). levels modulate both growth and differentiated functions during Primary antisera to specific G-protein subunits were raised in rab- liver regeneration. (J. Clin. Invest. 1992. 89:1706-1712.) Key bits against synthetic peptides corresponding to defined regions of G- protein subunits (generously supplied by Dr. Janet Robishaw, Gei- words: G-proteins * liver - AMP regeneration cyclic singer Clinic, Danville, PA). Antiserum C584, which was raised against a synthetic carboxy-terminal peptide specific for Gs alpha, recognized Introduction the 45- and 52-kD forms ofGs alpha (9). Antiserum A54, which specifi- cally recognizes GI alpha-2, was raised against a synthetic peptide The role of cAMP-dependent signal transduction in regulation unique to the middle of Gi alpha-2 protein and recognized a 40-kD of hepatocellular proliferation is uncertain. Hepatic concentra- protein. B2 antiserum was raised to a specific carboxy-terminal peptide tions of cAMP increase dramatically in the prereplicative phase of the B2 subunit and recognized a 35-kD protein. of liver regeneration after partial hepatectomy (PH)' (1, 2). cDNA probes for the mRNAs of GTP-binding proteins were pro- Furthermore, hepatic sensitivity to beta-adrenergic agents and vided by Dr. Levine (The Johns Hopkins University, School of Medi- glucagon, factors that induce cAMP is cine, Baltimore, MD) (10). The cDNA probe for phosphoenolpyruvate accumulation, height- carboxykinase (PEPCK) was provided by Dr. Axel Kahn ened in such circumstances in the mRNA for (Institut Co- (3). Changes the chin de Genetique Molecularie, INSERM, Paris, France) (1 1). cDNA cAMP-dependent kinase, protein kinase A, also occur during for 18S was provided by Dr. Barbara Sollner-Webb, The Johns Hop- the prereplicative phase of liver regeneration (4, 5). These data kins University (12). suggest that cAMP-dependent processes may modulate hepatic Animals. Adult male Sprague-Dawley rats (weight - 300 g) were regenerative growth. However, direct evidence for cAMP-me- purchased from Charles River Breeding Laboratories, Inc. (Wilming- ton, MA). Partial hepatectomy experiments. 64 rats were housed with a 12-h light/12-h dark cycle and permitted ad lib. access to a standard rat Address reprint requests to A. M. Diehl, M.D., Gastroenterology Divi- chow, pellet-type diet for I wk. At the end of this equilibration period, sion, 912 Ross Research Building, The Johns Hopkins University 12 animals underwent sham laparotomy. During this procedure, the School of Medicine, 720 Rutland Avenue, Baltimore, MD 21205. liver was manipulated, but not resected. animals 9 1991 Sham-operated were Receivedfor publication July and in revisedform 29 January killed at 1, 3, 6, or 24 h and liver was 1992. postoperatively tissue removed and saved for subsequent analysis. 52 animals underwent a 70% PH J. Clin. Invest. © The American Society for Clinical Investigation, Inc. 1. Abbreviations used in this paper: AC, adenylyl cyclase; IBMX, isobu- 0021-9738/92/06/1706/07 $2.00 tylmethylxanthine; LDH, lactate dehydrogenase; PEPCK, phosphoe- Volume 89, June 1992, 1706-1712 nolpyruvate carboxykinase; PH, partial hepatectomy. 1706 A. M. Diehl, S. Q. Yang, D. Wolfgang, and G. Wand with light ether anesthesia in midmorning (8-10 a.m.) (13). The re- had viabilities > 90%. Cells suspended in 3 ml of serum-free media sected tissues were saved at -70'C until analysis. Oral intake was not were plated on collagen-coated plastic dishes at a density of 1.5 X 106 restricted before or after surgery. Perioperative mortality was < 5%. cells/60-mm plate in the presence or absence of growth factors (dexa- Rats were killed at various times (1, 3, 6, 12, 24, 36, 48, and 72 h) after methasone [1 g/ml], glucagon [10-9 M], epidermal growth factor [10 PH. Resected liver tissue was immediately freeze clamped in liquid ng/ml], and insulin [500 U/liter]) (20) and in the presence or absence of nitrogen and then stored at -70'C for up to 4 wk. agents (10MM 8-Br-cAMP or 100MgM isobutylmethylxanthine [IBMX] Adenylyl cyclase (AC) assay and Western analysis. Liver mem- plus 1 MM forskolin) known to induce cAMP accumulation (21). 52 branes were prepared from at least four rats killed at each time point plates/experiment were used to test each condition. Three separate per- after PH. The livers were homogenized in 1 mM Tris-HCl, 1 mM fusion experiments were performed. In each experiment, eight plates EDTA, 10% sucrose (pH 7.4). The homogenates were passed through a were harvested at 24, 42, 48, 66, and 72 h and immediately homoge- 200-Mm nylon mesh and then centrifuged for 5 min at 500 gto remove nized in RNA lysis buffer (18) so that RNA could be isolated for subse- debris and nuclei. Supernatants were centrifuged at 170,000 g for
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