Internalization study using EDTA-prepared hepatocytes for receptor-mediated endocytosis of haemoglobin-haptoglobin...

We have demonstrated the internalization of haemoglobin-haptoglobin (Hb-Hp) complex using rat hepatocytes prepared by EDTA perfusion, followed by Percoll. The isolated hepatocytes exhibited a saturation curve of the binding of fluorescein isothiocyanate-labelled haemoglobin-haptoglobin complex (FITC-Hb-Hp. Furthermore, competition between the binding of FITC-Hb-Hp and unlabelled Hb to the hepatocytes, was observed. The cells exhibited approximately 9 x 10(4) 'high affinity sites' (Kd approximately 1.2 microM) for the Hb-Hp complex. The data in toto suggest the presence of only one type of receptor i.e. the high affinity receptor (in both affinity and number of sites per cell). The results were similar to those obtained from rat hepatocytes prepared by collagenase digestion [1]. In order to verify whether EDTA-prepared hepatocytes could be used for the study of receptor-mediated endocytosis, the internalization of pre-bound Hb-Hp in the isolated hepatocytes was assessed by two methods. First, acid-insensitive FITC-Hb-Hp time-dependently increased following incubation at 37 degrees C. Secondly, Hb-Hp became inaccessible to the exogenous FITC-anti-haemoglobin antibody. These processes were dependent on ATP, but independent of Ca2+ and stimulated by GTP. The results demonstrate that the receptor-mediated endocytosis of Hb-Hp occurred in the EDTA-prepared hepatocytes.