Melvin Blecher

Hormone receptors 7. Characteristics of insulin receptors in a new line of cloned neonatal rat hepatocytes

1. A new line of cloned, differentiated rat hepatocytes (RL-PR-C) was evaluated for its usefulness as an in vitro system for studying the regulation of the insulin receptor. 2. Insulin rapidly reversibly and specifically bound to RL-PR-C hepatocytes. Binding of tracer 125I-labeled insulin, which was competitively inhibited by native insulin as well as by proinsulin and analogs of insulin and proinsulin in proportion to their biological activity, was not influenced by glucagon, corticotropin, or human growth hormone. Anti-insulin receptor serum from a patient with Acanthosis Nigricans Type B competed with 125I-labeled insulin for binding to cell surface sites. 3. Trypsinization destroyed insulin binding sites, but these were restored by incubation under growth conditions; a 75% restoration of binding sites was achieved by one cell population doubling. 4. RL-PR-C hepatocytes responded to insulin binding by an increase in glycogen synthesis from glucose. The insulin effect was maximal at 85 nM, but was detectable at lower, more physiological, concentrations. 5. Chronic exposure (for at least 3h) of hepatocytes to insulin (10(-10)--(10(-8) M) reduced by up to 60% the number of binding sites for insulin (down-regulation). Down-regulation was prevented by cycloheximide at concentration (10 micron) sufficient to inhibit markedly protein synthesis from tracer isoleucine. Recovery from down-regulation induced by native insulin at 10(-7 M or lower concentrations was complete by 18 h under growth conditions. 6. Although RL-PR-C hepatocytes spontaneously transform after about 90 population doublings, no significant differences between normal and transformed cells were observed in insulin binding characteristics and in interaction of cells with anti-insulin receptor serum. However, transformed cells exhibited a substantially reduced (maximum of 20%) down-regulation response to insulin. 7. RL-PR-C rat hepatocytes appear, for these reasons, to be a useful model system for studying the regulation of the insulin receptor.

Kinetics of activation of ADP-ribosylation and adenylate cyclase by cholera toxin in cloned differentiated hepatocytes

Cholera toxin activates adenylate cyclase in a wide variety of eukaryotic cells. This process can be shown to depend upon exogenous NAD’ in purified rat liver plasma membranes [l], lysed avian erythrocytes [2], mouse neuroblastoma cells [3] and sarcoma 180 membranes [4]. This NAD’ requirement suggested that ADPribosylation might be involved in the activation of adenylate cyclase by cholera toxin, similar to the ADP-ribosylation of elongation factor 2 by diphtheria toxin [.5]. Cholera toxin has very recently been shown to catalyze the transfer of the ADP-ribose moiety of NAD’ to the guanidino moiety of arginine [6], as well as to several membrane components of lysed pigeon erythrocytes, one of which contains the guanyl nucleotide regulatory site of adenylate cyclase [7]. This report examines the kinetics of cholera toxin stimulation of ADP-ribosylation of macromolecules and adenylate cyclase activity in cloned, differentiated, diploid hepatocytes [8] and in a crude membrane preparation derived from these cells, and concludes that there may be a cause-and-effect relationship between both cholera toxin effects.

Isoproterenol-induced ADP-ribosylation of a single plasma membrane protein of cultured differentiated RL-PR-C hepatocytes

Abstract We present here what we believe to be the first report of the stimulation of NAD + -dependent ADP-ribosyltransferase activity by a hormone. Isoproterenol stimulated the ADP-ribosylation of RL-PR-C hepatocyte membranes in a concentration-dependent fashion; the effect was abolished by the β-adrenergic antagonist, propranolol. Although hepatocyte plasma membrane ADP-ribosyltransferase and adenylate cyclase activities differed in their sensitivity to isoproterenol, the kinetics of both effects were quite similar. PAGE separation of membrane proteins after ADP-ribosylation from [2,8- 3 H-adenine]NAD + identified the acceptor for isoproterenol-enhanced ADP-ribosylation as the same 55,000 dalton guanyl nucleotide regulatory protein serving for both endogenous and cholera toxin-stimulated processes in these cells.

Alterations of cAMP metabolism and hormone responsiveness of cloned differentiated rat liver cells (RL-PR-C) upon spontaneous transformation

Abstract In normal Rat Liver Primary Culture (RL-PR-C) liver cells, cAMP was low prior to confluency, then rose continuously as cells became contact inhibited. In contrast, spontaneously transformed RL-PR-C cells did not become contact inhibited, and cAMP decreased steadily with increasing cell density. Normal cells released large amounts of cAMP into the extracellular fluid at all densities, while transformed cells did not do so at any density. Neither exogenous db-cAMP nor phosphodiesterase inhibitors reversed the uncontrolled growth of transformed cells, nor did conditioned media from contact-inhibited normal cells. While both normal and transformed RL-PR-C hepatocytes produced large amounts of cAMP in response to epinephrine and cholera toxin, transformed cells were much more sensitive to these agents; however, only normal cells responded to glucagon. Although the plasma membrane adenylate cyclase of transformed hepatocytes responded better than did that of normal cells to epinephrine, cholera toxin and fluoride, the basal cyclase activity of transformed cells was only about half that of normal cells. The adenylate cyclase of transformed cells did not respond to glucagon, although the number of glucagon receptors of such cells far exceeded that of normal cells. The V max of cyclic nucleotide phosphodiesterase of normal hepatocytes was five times that of transformed cells, although the K m was unchanged. The data indicate that spontaneous transformation of diploid differentiated RL-PR-C hepatocytes leads to cultural hormone receptor and cAMP changes similar to those seen in undifferentiated fibroblasts and other cells transformed by viruses and chemical carcinogens. Although there are significant changes in various parameters of cAMP metabolism upon transformation, decreased cAMP per se does not seem to be responsible for transformation. Furthermore, it is possible that following transformation, these hepatocytes lose some factor necessary for coupling of the glucagon receptor to adenylate cyclase.

Insulin receptors and functions in normal and spontaneously transformed cloned rat hepatocytes

Abstract Spontaneous transformation of late passages of a cloned line of normal, differentiated, diploid rat hepatocytes (RL-PR-C) leads, not only to a loss of contact-inhibition of growth and ultimately to oncogenicity, but also to a variety of characteristics suggestive of a loss of involvement with insulin: lack of a serum requirement for growth; reductions in numbers and affinity of insulin receptors; increased rate of dissociation of insulin; loss of insulin stimulation of protein synthesis; and, a reduction in insulins ability to activate glycogen synthase and to stimulate glycogen synthesis from glucose.

The Endocrine System: Interaction of Prostaglandins with Adenylyl Cyclase Cyclic AMP Systems

Higgins and Braunwald (1972) in a review of the biochemical, physiological, and clinical considerations of the prostaglandins made the observation that “prostaglandins … are ubiquitous in mammalian tissues and have potent physiologic activities, probably through an as yet incompletely characterized interaction with adenylyl cyclase in the endocrine, reproductive, nervous, digestive, hemostatic, respiratory, cardiovascular and renal systems.” It is the intent of this chapter to review those recent advances pertaining to this “incompletely characterized interaction” of prostaglandins and the adenylyl cyclase-cyclic AMP systems in various of the organs comprising the endocrine system. The reader should be forewarned that although progress has been made in anwering some of the questions relating to this interaction, much is still unknown and consequently no satisfactory hypothesis has been formulated to unify all of the divergent findings.

Hormone receptors: VI. on the nature of the binding of glucagon and insulin to human circulating mononuclear leukocytes

Several characteristics of the binding of insulin and glucagon to human circulating mononuclear leukocytes have been studied. Functional analysis (latex bead ingestion) revealed that cell mixtures, as prepared according to Boyum and used generally in studies of insulin resistance in humans, consist of 20-29% phagocytic monocytes, with the remainder being lymphocytes. Partial separation of monocytes from lymphocytes on columns of Sephadex G-10, followed by correlation of insulin binding with cell type, confirms that the monocyte is the binding species. Insulin influenced neither glucose uptake nor the further conversion of glucose to lipids and CO2 by the leukocytes. The transport of alpha-aminoisobutyrate, a nonmetabolizable amino acid, into these cells was also unaffected by insulin. Monocyte/lymphocyte mixtures specifically bound glucagon and prostaglandin E1. At physiological concentrations of these hormones, steady states were reached in 15 min and 45 min, respectively. In contrast to the 8-10-fold increases in cellular cyclic AMP produced by prostaglandins, the effect of glucagon was very small but apparently real. Under appropriate preincubation conditions, sodium azide and iodoacetamide inhibited phagocytosis and insulin binding in parallel. The binding of glucagon was unaffected by these agents. Although both antimycin A and actinomycin D inhibited phagocytosis of the monocytes, only the former inhibited insulin binding; there was only a slight effect on glucagon binding. We would conclude that the binding of insulin to human circulating monocytes, although reflective of insulin resistance in diabetes mellitus and obesity, may not be to traditional receptors. In contrast, the binding of glucagon to lymphocyte/monocyte mixtures may be to function-linked receptors.

Characterization of dopamine binding sites in standard preparations of brain synaptic membranes.

Abstract Brain synaptic membranes, prepared according to DeRobertis and to Whittaker, were compared morphologically and biochemically. Electron microscopy revealed that DeRobertis preparations were heavily contaminated with synaptosomes, mitochondria, storage vesicles, and a variety of extraneous membrane structures; Whittaker preparations appeared to consist of synaptic membranes, with a relatively small number of presynaptic storage vesicles. Substantial dopamine β-hydroxylase, monoamine oxidase, and catecholamine- O -methyl transferase activities were present in DeRobertis preparations; Whittaker membrane preparations contained low-to-undetectable activities of these enzymes. Although dopamine bound to membranes derived from both brain cortex and corpus striatum, only membranes from the latter area contained dopamine-sensitive adenylate cyclase. Binding of dopamine to Whittaker striatal synaptic membrane preparations was relatively rapid, saturable, partially reversible, and did not chemically alter the ligand. A variety of dopamine acceptors appeared to be present in this preparation since the ed 50 (from Klotz plots) of dopamine binding was 25 times that for activation of adenylate cyclase; Scatchard plots revealed both high and low affinity binding sites for dopamine; and, binding and adenylate cyclase activation studies with dopamine, carried out in the presence of fluphenazine, cocaine, pargyline, and reserpine, reveal that even the more-pure Whittaker preparations of striatal synaptic membranes contain at least three major dopamine-binding components: postsynaptic (and perhaps presynaptic as well) membrane receptors linked to adenylate cyclase, a presynaptic synaptosomal reuptake receptor, and storage vesicle sequestration.

Partial purification of a water-soluble liver protein that regulates adenylate cyclase activity (basal, hormone- and cholera-toxin-activated) and cholera-toxin-catalyzed ADP-ribosylation of the membrane G protein

We have found in water-soluble extracts of rat liver (and RL-PR-C cloned rat hepatocytes), prepared in the absence of detergent, a factor that markedly enhances basal, isoproterenol and cholera toxin activation of adenylate cyclase of rigorously washed hepatocyte membranes, in the absence of added GTP. The factor, which has characteristics of a protein with an Mr of approx. 35000, has been fractionated from crude cytosol by gel filtration, and then further purified over 50-fold by sequential ion-exchange chromatography. The site of action of the protein appears to be at the level of the guanine nucleotide regulatory (G) protein of the plasma membrane adenylate cyclase complex, as the factor, cooperatively with GTP, also permitted cholera toxin to ADP-ribosylate (from 32P-labeled NAD) two integral membrane proteins that migrated on SDS-polyacrylamide gel electrophoresis gels with the mobilities (Mr approx. 46 000 and 48 000) generally observed for the guanine nucleotide regulator protein subunits. In this system, isoproterenol did not stimulate ADP-ribosylation, in either the presence or absence of the liver protein factor.

Uncoupling of the Glucagon Receptor-Adenylate Cyclase System by Glucagon in Cloned Differentiated Rat Hepatocytes

The ability of glucagon to induce a state of desensitization to glucagon responsiveness has been examined in a cloned line of normal, differentiated, diploid rat hepatocytes (RL-PR-C). These cells, which respond to glucagon with increased production of cyclic AMP, become refractory to further stimulation of cyclic AMP synthesis following a 4 hour exposure period of the cells to the hormone. Refractoriness to glucagon was demonstrated over a wide range of hormone concentrations (10(-12) to 10(-6) M). In desensitized cells that were subsequently washed free of the hormone, recovery from refractoriness was complete by 20 hours. The mechanism underlying this desensitization does not appear to involve decreased receptor numbers, increased efflux of cyclic AMP from the cells, increased degradation of cyclic AMP by phosphodiesterase, or an alteration in the catalytic unit of the adenylate cellular cyclic AMP responsiveness to glucagon in normal RL-PR-C hepatocytes may involve glucagon a reversible uncoupling of glucagon receptors from adenylate cyclase. In addition, late passage, spontaneously transformed RL-PR-C hepatocytes were found to exist in a state in which glucagon receptors are permanently uncoupled from adenylate cyclase.