Gut 1993; 34:1601-1606 1601 Down regulation of epidermal growth factor receptors in liver proliferation induced by a mixture of triiodothyronine, amino acids, glucagon, and heparin (TAGH) D A Vesey, A C Selden, H J F Hodgson Abstract results in EGF receptor down regulation; in This study investigated the mechanisms by in vitro hepatocyte cultures TAGH does not which TAGH solution (a mixture of triiodo- induce EGF receptor down regulation, although thyronine, amino acids, glucagon, and it was capable of inducing phosphorylation of heparin) induces DNA synthesis in hepato- tyrosine residues of the EGF receptor in liver cytes in the liver of intact rats, with particular plasma membrane preparations in vitro. reference to events at the epidermal growth factor (EGF) receptor. Both partial hepatec- tomy and infusion of TAGH stimulated DNA Materials and methods synthesis at 24 hours and both procedures resulted in a reduction of EGF receptors ANIMALS assessed in plasma membranes isolated from All experiments were performed on male August rat liver at this time. In cell cultures, while rats (250-300 g) (National Institute of Medical EGF strongly stimulated DNA synthesis and Research, Mill Hill, London, UK), and all started EGF receptor down regulation, TAGH procedures were carried out between 9 00 am had only a minor effect (1.5xbasal) on DNA and noon. synthesis and did not interact with or down Seventy per cent hepatectomy was performed regulate the EGF receptor. Membrane phos- as described by Higgins and Anderson9 under phorylation studies, however, showed that ether anaesthetic. Sham operations consisted of TAGH induced phosphorylation of tyrosine laparotomy and gentle manipulation ofthe liver. residues in the EGF receptor. The in vivo The TAGH solution (9 ml) was made up fresh action ofTAGH seems to entail recruitment of at the time of the experiment and infused by similar changes in the EGF receptor to those the tail vein from 20 ml hyperdermic syringes that occur after partial hepatectomy. mounted in horizontal syringe pumps set to (Gut 1993; 34: 1601-1606) deliver 3 ml/h. The TAGH solution consisted of 100 ,tg of triiodothyronine (200 rd), 1 mg of glucagon (1 ml), 100 units of heparin (100 >d) The factors controlling hepatocyte proliferation and 7 7 ml ofan amino acid mixture (Synthamin are complex and incompletely understood.' 14*). Synthamin 14* is a commercial mixture of Prominent events at the hepatocyte membrane nine essential and six non-essential L-amino after induction of DNA synthesis in rat livers by acids (8 5% wt/vol). The solution was adjusted to partial hepatectomy include down regulation pH 7-2 with 0 01 M sodium hydroxide. and phosphorylation of the epidermal growth factor (EGF) receptor.2 3 These events can also be seen when EGF is used to start DNA synthesis in MATERIALS hepatocytes in vitro."7 Ligand binding to the [6-3H]-Thymidine (specific activity 25-30 EGF receptor therefore seems to be integrally Ci/mmol) and carrier free Na125I were obtained associated with liver cell proliferation. from Amersham International PLC, Amersham, Short et all described the action of TAGH Bucks, UK. William's medium E, fetal solution - a mixture of triiodothyronine, amino bovine serum (FBS), penicillin/streptomycin, acids, glucagon and heparin - as an initiator of fungizone (amphotericin B), gentamycin, and DNA synthesis, hepatocyte mitosis, and also Nunclon microwell plates were obtained from causing an increase in liver size in intact rats. The Gibco, Paisley, Renfrewshire, Scotland. Insulin time course of DNA synthesis after the start of (human monocomponent, actrapid) and Gastroenterology Unit, the infusion was the same as that following 70% glucagon were purchased from Novo, Copen- Department of Medicine, Royal Postgraduate hepatectomy, enhancement of DNA formation hagen, Denmark. Triiodothyronine was from Medical School, London occurring within 15 hours. The mechanism of Sigma. Heparin was from Leo Labs Ltd, D A Vesey action of TAGH has not been elucidated. As the Princes Risborough, Bucks, UK. Collagenase A C Selden constituents of TAGH solution are readily avail- (Clostridium histolyticum) was purchased from H J F Hodgson as this offers a Boehringer Mannheim Ltd, Lewes, East Sussex, Correspondence to: able pharmaceutical preparations, Professor H J F Hodgson, potential treatment for severe liver diseases in UK. The amino acid solution, Synthamin 14, Gastroenterology Unit, Laboratories Department of Medicine, which hepatic regeneration would be beneficial. was purchased from Travenol Ltd, Royal Postgraduate Medical We therefore investigated the mode of action of Compton, Berks, UK. Collaborative Research School, Du Cane Road, TAGH by comparing in vivo and in vitro pheno- mouse EGF (tissue culture grade) was obtained London W12 ONN. Other Accepted for publication mena induced in hepatocytes by EGF and from Universal Biologicals, London, UK. 10 March 1993 TAGH. The findings show that in vivo TAGH reagents and chemicals were from Sigma 1602 Vesey, Selden, Hodgson Chemical Company or BDH Chemicals, both of 60 minutes." Measurements were made in tripli- Poole, Dorset, UK. cate. Binding data were analysed and curves fitted using the GraphPAD Inplot computer program (San Diego, CA, USA). IN VIVO 3H-THYMIDINE INCORPORATION Rats received an intraperitoneal injection of 3H-thymidine (05 ,uCi/g body weight) one hour IN VITRO 3H-THYMIDINE INCORPORATION before death. Twenty four hours after partial DNA synthesis was assessed in cultured hepato- hepatectomy or the start of the TAGH infusion, cytes by measurement of 3H-thymidine incor- rats were anaesthetised and the liver perfused in poration into cell DNA after stimulation with place with ice cold saline to remove excess blood. insulin (10-7 M) and EGF (20 ng/ml) or plating The liver was rapidly removed and placed on ice. medium containing the dilutions of TAGH for A small section of liver was frozen for measure- 24 hours (see Tables)." The amount, in quad- ment of thymidine incorporation. The remain- ruplicate, of 3H-thymidine incorporated was ing tissue was used to prepare liver cell plasma assessed biochemically as described by Selden membranes. 3H-thymidine incorporation into and Hodgson.'6 liver DNA was measured biochemically by a modification of the method of Munro and Fleck,'0 as described in Vesey et al. " EGF RECEPTOR PHOSPHORYLATION STUDIES Phosphorylation studies were carried out essen- tially as Rubin et al.3 The reaction was performed ISOLATION AND CULTURE OF HEPATOCYTES at 0°C in a total volume of 50 [1, containing Adult rat hepatocytes were isolated using a 50 tg of membrane protein, 50 mM Pipes at pH modification of the Berry and Friend pro- 7-0, 30 mM MgCl2, 10 mM 2-mercaptoethanol, cedure,'2 as described in Vesey et al 1992."l Cells and 0-32 mM ethylene glycol BIS-(,3-aminoethyl were resuspended at 3 x i05 or 2 x i05 viable ether) N,N,N',N'-tetraacetic acid. Factors, cells/ml in William's medium E, without EGF (5 Fg/ml in H20), or TAGH solution L-glutamine, supplemented with penicillin (200 (10 >d), or water (10 ,ul) as control were added to IU/ml); streptomycin (200 IU/ml); gentamycin the tubes. After 15 minutes p reincubation at 0°C (80 .tg/ml); fungizone (1 25 .tg/ml); 5% the reaction was started by addition of 15 iCi (vol/vol) heat inactivated fetal bovine serum and (15 [L) of y 32P ATP to a final concentration of dexamethazone (10-8 M) (plating medium) and 2 mM. It was stopped after one minute with 25 seeded on rat tail collagen (type-i) coated ,ul of 9% SDS sample buffer (0-0015% bromo- Nunclon 24-well cluster trays or 96-well micro- phenol blue, 10 ml of2% SDS, 25 mM TRIS pH titre tissue culture plates, at densities of 7 5 x 6-8, and 20% sucrose). Tubes were sealed and 104/cm2 and at 6-25 x 104/cm2 respectively. heated for three minutes at 100°C. Aliquots (40 1tl) were subjected to electrophoresis on 5% SDS PAGE slab gels. PLASMA MEMBRANE PREPARATION Rat liver plasma membranes prepared by the method of Aronson and Touster'3 were PHOSPHOAMINO ACID ANALYSIS resuspended in 5 mM HEPES buffer pH 7-5 at The band corresponding to the EGF receptor approximately 5 mg/ml and stored in liquid was excised and the protein eluted from the gel nitrogen until used. Protein concentrations were strip in 0-05 M ammonium bicarbonate contain- determined by a modification of the method of ing SDS (0-1%) and ,3-mercaptoethanol (5%) at Lowry'4 using BSA as a standard. TABLE I DNA synthesis in the liver ofrats after TAGH infusion IODINATION OF EGF EGF was iodinated by a modification of the 3H-Thymidine incorporation chloramine-T method to give a specific activity Treatment (dpmrx 10-4Ig liver)* No of between 20 to 60 [tCi/tg as previously Saline 7-9(4-8-11-2) 4 Hepatectomy 469 (283-711) 8 described." TAGH 63-4(39-118) 8 *Mean (range). 3H-thymidine incorporation in rat liver 24 hours after infusion of EGF BINDING STUDIES TAGH compared with the response to hepatectomy. Thymidine On hepatocytes - binding studies on whole cells was injected 1 hour before death at 24 hours. Data represent the were carried out in 24 well cluster trays."I For means (range). analysis of receptor numbers and affinity, cells were incubated with increasing amounts of TABLE II EGF bindingcharacteristics ofplasma membranes isolatedfrom whole liver 24 hours after 70% partial 125I-EGF (30-4000 fmoles) for 2 5 hours, and hepatectomy or TAGH infusion non-specific binding determined at each point by inclusion of a 1-2 Rg/ml of unlabelled EGF in High affinity site replicate cultures. This was subtracted from Treatment Bmax KD) total counts bound to obtain specific binding. Sham 1138 (132) 1799 (385) Points represent the means oftriplicate measure- Hepatectomy 627 (278) 1690 (278) ments.