Int. J. Biochem. Vol. 20, No, 12, pp. 1435-1441, !988 0020-71 IX/88 $3.00 + 0.00 Printed in Great Britain. All rights reserved Copyright © 1988 Pergamon Press plc CHARACTERIZATION OF SPECIFIC INSULIN BINDING SITES ON CHROMAFFIN CELLS FROM BOVINE ADRENAL MEDULLA GULDBORG SERCK-HANSSEN l, ODDMUND SOVIK 2 and ROLV T. LIE3 t Department of Physiology, 2Department of Pediatrics and 3Norwegian Medical Birth Registry, University of Bergen, Bergen, Norway [Tel. (05)29 1700] (Received 18 April 1988) Abstract--1. Insulin receptors were investigated in isolated chromaffin cells from bovine adrenal medulla. 2. The cells were incubated with [~25I]insulin in HEPES buffer, pH 7.8 at 15°C for 180 min to obtain steady state binding. Specific binding was linearly related to the number of cells in the range 0.5-10 × 10 6 cells/ml. Insulin and proinsulin caused half maximal displacement of specifically bound tracer in concentrations of 0.18 and 2.46nM, respectively. 3. Computer analysis of the binding data gave a linear Scatchard plot, consistent with a single class of non-interacting receptors with an affinity constant of 5.6 nM - ~, the total number of receptors per cell being 1700. 4. The apparent MW of the insulin binding subunit of the receptor was 135,000, determined by affinity crosslinking and SDS gel electrophoresis under reducing conditions. INTRODUCTION It is now generally accepted that the initial step in insulin action is the binding to specific recognition sites on the cell membrane of the target cell (Czech, 1981). Specific insulin binding sites have been identified in a great variety of tissues not regarded as classical targets of this hormone (Czech et al., 1981). Insulin receptors have also been detected in the central nervous system, with a widespread distribu- tion throughout the brain (Havrankova et al., 1978). The functional role of insulin receptors in the brain is not well understood. Evidence for uptake of plasma insulin by the CNS by transcytosis across the brain endothelial cells has recently been presented by Duffy and Pardridge (1987), while the issue of insulin synthesis within the brain is more controversial (Baskin et al., 1988). Likewise, the functional role of insulin in the peripheral nervous system is largely unknown. The adrenal medulla has been widely applied as a model in studies related to biochemical and physiological functions in the sympathetic nervous system (Smith and Winkler, 1972). Isolated chromaffin cells kept in primary culture maintain many of the properties of chromaftin cells in vivo, such as the ability to syn- thesize and secrete catecholamines (CA) and opioids (Livett et al., 1983; Chang et al., 1982). These cells therefore provided an attractive model system for studying the role of insulin in the function of the autonomic nervous system. Since specific cellular binding is a prerequisite for hormone action, the work was initiated by characterizing insulin binding sites in chromaffin cells in terms of kinetics, specificity and molecular structure. The results obtained demon- strate that the binding of insulin in these cells fulfil the criteria of a true hormone-receptor interaction and thus confirm and extend the work of Bergeron et al. (1980), displaying specific insulin binding in rat adrenal medulla by autoradiography. A preliminary report of some of this work has been presented elsewhere (Serck-Hanssen and S~vik, 1985). MATERIALS AND METHODS Materials Mono [125I-(Tyr Al4)]insulin (spec. act. 218mCi/mg), bovine insulin and porcine insulin and proinsulin were obtained from Novo Research Lab., Denmark, and di- succinimidyl suberate (DSS) from Pierce Chemical Co., Rockford, U.S.A. Bovine serum albumin (BSA), col- lagenase type I and deoxyribonuclease 1 were purchased from Sigma Chemical Co., St Louis, U.S.A., and Percoll from Pharmacia Fine Chemicals, Sweden. Cell preparation Chromaffin cells were isolated from bovine adrenal me- dulla as described by Wilson and Viveros (1981) except that all procedures were carried out at room temperature. The glands obtained while still warm were carried to the labora- .tory at ambient temperature. In each experiment 8 glands were initially perfused retrogradely for 30 rain at 37°C with Ca-free Krebs Henseleit (KH) buffer gassed with 95% 02/5% CO 2 and containing 0.05% collagenase. The medul- lae were carefully removed, minced and further digested 3 times with 0.05% collagenase and 15/~g DNAase/ml in KH under a 95% 02/5% CO 2 atmosphere. The dissociated cells were purified by Percoll gradient centrifugation, washed in KH containing 2.2 mM CaCI2 (KH/Ca) and stored over- night at 4°C in capped bottles in KH/Ca supplemented with 0.5% BSA. On the second day cells were washed twice in KH/Ca, resuspended in insulin binding buffer (see below), filtered through 50 pm nylon mesh and counted in a Burker haemocytometer. Viability was assessed by Trypan blue exclusion (0.2% final conc.) and the percentage of chromaffin cells was estimated by neutral red staining (Role and Perlman, 1980). Preparation of crude rat liver plasma membranes Livers from Wistar female rats (approx. 250g) were homogenized in 1 mM NaHCO3, containing 2 mM phenyl- methylsulfonyl fluoride (PMSF) as described by Hav- rankova et al. (1978), using Ultra Turrax stainless-steel 1435