Isolation of Rat Hepatocytes for Pharmacological Studies on Metabotropic Glutamate Receptor (mGluR) Subtype 5: A Comparison Between Collagenase I Versus Collagenase IV

Abstract

Isolated hepatocytes can be obtained from the liver by collagenase infusion, a procedure that could affect cell isolation as well as the integrity of membrane receptors. In this respect we compared metabotropic glutamate subtype 5 receptor (mGluR5) protein expression and activity in rat hepatocytes isolated by two collagenases, type I and type IV. Hepatocytes were isolated from male Wistar rats (200-250 g) using collagenase I or colla-genase IV and after isolation, viability and morphology of rat hepatocytes were assessed measuring mGluR5 protein expression by Western blot analyses. mGluR5 activation was evaluated by inositol-1-phosphate (IP-1) accumulation after treatment with the mGluR5 orthosteric agonist ACPD or the selective antagonist MPEP. No difference in cellular viability and morphology was observed using collagenase I when compared with collage-nase IV. An increase in mGluR5 protein expression was observed in hepatocytes isolated using collagenase IV with respect to collagenase I. Moreover, hepatocytes treated with ACPD and with MPEP presented higher levels of IP-1 when isolated using collagenase IV compared tocollagenase I. These results indicate that collage-nase IV better preserves the activity of surface proteins such as mGluR5in isolated rat hepatocytes, an in vitro model useful to reduce the use of experimental animals, in line with the 3R ethical framework.