Analysis of the adenylate cyclase signalling system, and alterations induced by culture with insulin, in a novel SV40-DNA-immortalized hepatocyte cell line (P9 cells)

Author:

Livingstone C1,MacDonald C2,Willett B2,Houslay M D1

Affiliation:

1. Molecular Pharmacology Group, Department of Biochemistry, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.,

2. Department of Biological Sciences, University of Paisley, Paisley PAl 2BE, Scotland, U.K.

Abstract

An immortalized cell line, called P9, was derived from hepatocytes by transfection with SV40 DNA. These cells expressed enzyme activities characteristic of hepatocytes, namely glucose-6-phosphatase, glycogen phosphorylase, bilirubin glucuronyltransferase and both glucagon- and prostaglandin E1 (PGE1)-stimulated adenylate cyclase activities, albeit at decreased levels compared with native hepatocytes. Levels of the G-protein subunits alpha-Gi-2, alpha-Gi-3, G beta and the ‘long’ form of alpha-G2 (45 kDa) were approximately 4-fold higher relative to native hepatocytes, whereas those of the ‘short’ form of alpha-G2 (42 kDa) were lower by approximately 40%. Associated with this were marked alterations in the guanine nucleotide regulation of adenylate cyclase. Receptor-mediated stimulation, achieved by either PGE1 or glucagon, was apparent in P9 cells, although the latter was only evident upon amplification with forskolin. Glucagon-stimulated cyclic AMP accumulation in P9 cells did not exhibit desensitization, as in hepatocytes, nor was the phosphorylation of alpha-Gi-2 evident. Culture of P9 cells with insulin led to a dose-dependent decrease (EC50 0.2 +/- 0.1 nM) in the ability of PGE1 to stimulate adenylate cyclase activity, with the maximum effect attained after approximately 6 h. A comparable attenuation of stimulation was seen for glucagon- and guanine-nucleotide-stimulated adenylate cyclase activities. In cells cultured with insulin, lower levels of GTP were required to stimulate adenylate cyclase, ADP-ribosylation of the 45 kDa form of alpha-Gs with cholera toxin was attenuated, and the expression of both alpha Gi-2 and alpha-Gi-3 was increased. It is suggested that the expression of alpha-Gi-2 and alpha-Gi-3 may be directly regulated by the action of insulin in hepatocytes and P9 cells.

Publisher

Portland Press Ltd.

Subject

Cell Biology,Molecular Biology,Biochemistry

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