Abstract

Glucagon binding has, to date, generally been measured using membrane-fragment preparations [3]. Such investigations have been complemented by studies using isolated intact cells [5], and experiments of this kind can be considered as rather more relevant with respect to the in vivo situation. Furthermore, use of the intact cellular system permits the correlation of extracellular hormone binding with resulting intracellular metabolic effects. In order to obtain large quantities of defined populations of isolated, functionally and morphologically intact cells reproducibly from rat liver, we developed a simple separation procedure: a) Parenchymal liver cells of adult (-~ 3 months) male rats were prepared by the perfusion technique with 80 U/ml collagenase I (Sigma) in HEPES buffer [4]. b) Neonatal liver cells were isolated from young ( 1 5 days) rats by digestion with 1.8 U/ml dispase I (Boehringer) or 80 U/ml collagenase I [6]. The suspensions of neonatal or adult rat liver cells were further purified by isopycnic centrifugation through a continuous Percoll gradient [1]. For this purpose, a solution of 40.5% (v/v) Percoll in Eagle's MEM was centrifuged at 15,000 g for 20 rain in a fixed-angle rotor 9 After layering the cell suspensions on top of the resulting gradient, a further centrifugation was carried out for 30 min at 100 g in a swingbucket rotor. Intact parenchymal cells of neonatal and adult rat liver banded at a density (determined refractometrically or by marker beads) of 1.075 1.090 g/ml, whereas non-viable cells banded at a lower density 9 After removal from the gradient, the cells were washed twice with buffer and were then suspended in Eagle's MEM supplemented with 10% calf serum. About 9 0 95 % of the cells proved to be viable as judged by Trypan-blue exclusion. The interaction between glucagon and specific receptors was studied in isolated hepatocytes of neonatal and adult rats using radioactively-labelled glucagon. In order to determine the dissociation constant Ko for the glucagon binding and the 0.030-

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