Effect of monensin and diabetes on asialoglycoprotein degradation in rat hepatocytes.

We have studied the effects of two modulations--streptozotocin-induced diabetes in vivo, and the presence of the carboxylic proton ionophore monensin in vitro--on the degradation of 3H-asialoorosomucoid ligand in isolated rat hepatocytes. The ligand was internalized by means of a synchronous wave procedure. Diabetes was associated with a marked decrease in the amount of total degraded radioactive ligand compared to that in normal cells (3.6% and 37.3% of internalized ligand respectively, at 60 min), together with increased secretion of degradation products into the incubation medium (87% and 46.3% of the total degraded ligand was secreted by diabetic and normal cells, respectively). Monensin induced similar effects in normal cells, but had no apparent effect in diabetic cells.     

The effect of monensin and chloroquine on the endocytosis and toxicity of chimeric toxins

The toxicity of two conjugates containing ribosome-inactivating proteins (RIPs, i.e. saporin and ricin-A chain x-linked to transferrin) has been measured on a prostatic cancer line (PC3) naturally overexpressing the transferrin receptor, in the presence of monensin and chloroquine. This paper investigates whether the increased toxicity of Tf-RIPs induced by monensin and chloroquine may be due to alterations of the normal endocytotic pathway of the complexes mediated by the transferrin receptor. Monensin, besides inducing alkalinization of normally acid intracellular compartments, causes an accumulation of the receptor-bound Tf-RIP in a perinuclear region contiguous to the cisternae of the trans-Golgi network. Chloroquine, though increasing the intracellular pH, seems not to modify the endocytotic pathway of these chimeric molecules. We believe that the enhanced toxicity of the Tf-RIPs may be related to intracellular alkalinization (i.e. endosomal or lysosomal pH) rather than to the effects on the recycling of transferrin receptor-bound toxins. We conclude that the efficacy of chimeric toxins may be modulated not only by the carrier used for their engineering but also by addition of drugs able to influence the stability and activation of the toxins inside the cell. Received 22 December 1997; received after revision 30 March 1998; accepted 15 May 1998     

Effect of diabetes on the enzymes of the cholinergic system of the rat brain

Summary Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities were determined in several brain regions of normal and streptozotocin-induced diabetic rats. The diabetic rats exhibited significant increase in ChAT activity (p<0.05) in all brain regions studied except for the cortex and the midbrain. Meanwhile, the diabetes condition was associated with significant increase (p<0.05) in AChE activity of the bulbus olfactorius, medulla oblongata and cerebellum. These data suggest that uncontrolled diabetes is associated with significant alterations in the brain cholinergic systems.To whom requests of reprints should be addressed.This work was supported by grants from the National Aeronautics and Space Administration (NSG 2183 and NAG-2-411), a grant from the National Institutes of Health (NIH Grant RR0811) and a grant from the Division of Research Resources, National Institutes of Health (NIH Grant RR03020).     

Effect of spironolactone on p-nitrophenol glucuronidation in isolated rat hepatocytes

The effect of spironolactone (SP) on p-nitrophenol (PNP) glucuronidation was studied in isolated rat hepatocytes with appropriate viability conditions. A significant increase of protein concentration and PNP glucuronidation was found in the hepatocytes from SP-treated rats. Increased enzyme activity apparently was related to the SP dose. The results favor the conclusion that SP may induce PNP glucuronidation in the hepatocyte.     

Effect of nonprotein thiols on protein synthesis in isolated rat hepatocytes

The ability of nonprotein thiols to modulate rates of protein synthesis was investigated in isolated rat hepatocytes. Addition of cysteine stimulates protein labelling by [¹⁴C] Leucine. Glutahione depletion, induced by in vivod administration of L-buthionine sulfoximine and diethylmaleate, did not alter the effect of cysteine, although it decreased the rate of protein synthesis by 32%. The effect of cysteine on protein synthesis does not seem to be related to a perturbatin of the redox state of the NAD⁺/NADH system or to changes in the rate of gluconeogenic pathway. The following observations indicate that cysteine may stimulate protein syntheis by increasing intracellular levels of aspartate: 1. Amino-oxyacetate, an inhibitor of pyridoxyal-dependent enzymes, inhibits protein labelling and decreases aspartate cellular content, whereas most amino acids accumulate or remain unchanged; 2. Cysteine, in the absence or in the presence of amino-ocycetate, stimulates protein labelling and induces aspartate accumulation, although mot amino acids diminish or remain unchanged.     

Effect of lipophilic cations on thiamine transport system in isolated rat hepatocytes

The effect of lipophilic cations such as triphenylmethylphosphonium, tetraphenylphosphonium and tetraphenylarsonium in addition to dibenzyldimethylammonium on thiamine transport in isolated rat hepatocytes was studied. Lipophilic cations at the concentration 10 microM almost completely inhibited thiamine uptake. Kinetic studies showed that these compounds were competitive inhibitors with a very high affinity. These results suggest that lipophilic cations in addition to quaternary ammonium compounds also share a common binding site for thiamine in isolated rat hepatocytes.     

Decreased number of asialoglycoprotein receptors in diabetic BB Wistar rat

Summary The binding of asialoglycoproteins by hepatic binding protein was studied in freshly isolated hepatocytes from genetically diabetic BB Wistar rats. The number of cell surface asialoglycoprotein receptors was dramatically decreased (58,000±38,000 for diabetic rats compared to 267,000±70,000 for normal rats), while the association equilibrium constant was not changed. These results parallel those obtained with streptozotocin-diabetic rats and support the hypothesis that insulin deprivation is responsible for the decrease in the receptor number.     

Effect of glucocorticoids on the appearance of gamma-glutamyl transpeptidase activity in primary cultures of adult rat hepatocytes

Summary Primary cultures of adult rat liver parenchymal cells showed a progressive rise of gamma-glutamyl transpeptidase (GGT) activity (E.C. 2.3.2.2.) after the first 5 days of culture. The presence of dexamethasone and other synthetic glucocorticoids in the culture medium partially prevented this increase.Supported by a research grant (No. 12/180/76) from the Spanish Instituto Nacional de la Salud, Ministerio de Sanidad y Seguridad Social.     

Effect of glucocorticoids on the appearance of gamma-glutamyl transpeptidase activity in primary cultures of adult rat hepatocytes

Primary cultures of adult rat liver parenchymal cells showed a progressive rise of gamma-glutamyl transpeptidase (GGT) activity (E.C. 2.3.2.2) after the first 5 days of culture. The presence of dexamethasone and other synthetic glucocorticoids in the culture medium partially prevented this increase.     

Effect of diabetes on the enzymes of the cholinergic system of the rat brain

Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities were determined in several brain regions of normal and streptozotocin-induced diabetic rats. The diabetic rats exhibited significant increase in ChAT activity (p less than 0.05) in all brain regions studied except for the cortex and the midbrain. Meanwhile, the diabetes condition was associated with significant increase (p less than 0.05) in AChE activity of the bulbus olfactorius, medulla oblongata and cerebellum. These data suggest that uncontrolled diabetes is associated with significant alterations in the brain cholinergic systems.     

Location of lectin receptors on rat hepatocytes by transmission and scanning electron microscopy

Summary Rexeptors for various lectins have been located on isolated hepatocytes by transmission and scanning electron microscopy, using gold markers of variable sizes. Quantitative data indicated that binding of some lectin markers depended upon their sizes.Acknowledgments. The authors thank Miss E. Bujard and Mr. A. Isely for the rat liver perfusion and Mrs M. Weber for the photographic work.     

Ontogeny of the sex steroid and prolactin receptors in the male rat adrenal gland

Summary Cytosolic estrogen and androgen receptors and membrane prolactin-binding sites in the male adrenal glands showed a definite pattern during sexual development. The level of sexual steroid receptors paralleled adrenal growth, whereas prolactin binding reached its maximum value in mature rats.Lüthy, I. A., Predoctoral Fellow from the Consejo Nacional de Investigaciones Cientificas y Técnicas and Calandra, R. S., Research Career Awardee from the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.Acknowledgments. We would like to thank Mrs D. Bas and Mrs D. B. Destéfano for the skillful technical assistance and the secretarial work, respectively. This work was partially supported by the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina (CONICET), and the Comisión Nacional de Energía Atómica.     

Analysis of translation products synthesized in isolated rat hepatocytes treated with diethylnitrosamine

Summary Isolated rat hepatocytes were labeled with³⁵S-methionine in the presence of 25 mM diethylnitrosamine (DENA). The intrinsically labeled proteins were analyzed by one-and two-dimensional gel electrophoresis and the fluorographic patterns were compared with those obtained from untreated hepatocytes. The results of short term experiments (2 h) show that, in the presence of 25 mM DENA, protein synthesis is inhibited by 50%. This reduction encompasses all protein species without selective inhibition of certain proteins.This work was supported by CNR (Project Control of Neoplastic Growth) grant No. 810132696 and partially by AIRC.