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Biomedical subjects

R A Roth

Publications and source records attributed to R A Roth.

At least 289 records · Page 16Linked to original sources
PubMed1985

Expression of a functional human insulin receptor from a cloned cDNA in Chinese hamster ovary cells.

We have placed human insulin receptor cDNA into a vector under the control of the simian virus 40 (SV40) early promoter and tested its function by transient expression in microinjected Xenopus oocytes and by expression in stably transformed CHO cells. The precursor and the alpha and beta subunits of the receptor were detected by immunoprecipitation from extracts of these cells. The human insulin receptor expressed in CHO cells specifically binds 125I-labeled insulin but not insulin-like growth factor I, displays insulin-stimulated autophosphorylation of the beta subunit, and mediates insulin-stimulated 2-deoxyglucose uptake. We conclude that the human insulin receptor is synthesized, processed normally, and functional in this heterologous cell system.

Animals↗
PubMed1985

Increased vascular responsiveness in lungs of rats with pulmonary hypertension induced by monocrotaline pyrrole.

Monocrotaline (MCT) is a natural product that causes pulmonary hypertension in rats after metabolic activation to a pyrrole form (MCTP). To examine the vascular reactivity of the pulmonary bed, blood-perfused isolated lungs from MCTP- or vehicle-treated rats were challenged with angiotensin II (AII) and 5-hydroxytryptamine (5HT), and the resultant increases in perfusion pressure were measured. Fourteen days after a single exposure, the pressor responses to AII (0 25 or 0.50 microgram) and 5 HT (12.5 to 50 microgram) were approximately 3 times as great in isolated lungs of MCTP-treated rats as in those of control rats. When examined 7 days after exposure, the response to 25 micrograms 5HT but not to 0.25 microgram AII was enhanced by MCTP; MCT is known to decrease 5HT uptake by pulmonary capillary endothelial cells. Imipramine, a 5HT uptake inhibitor, did not alter the vascular responses to 25 micrograms 5HT in lungs from either control or MCTP-treated rats. The increased responsiveness of the pulmonary vasculature to AII and 5HT after MCTP exposure could play a role in the development and/or maintenance of pulmonary hypertension in this rat model.

Angiotensin II↗
PubMed1985

Plate binding assay for monoclonal anti-receptor antibodies.

A new procedure has been developed for rapid identification of monoclonal antibodies to the insulin receptor. The technique uses the ability of polyvinyl chloride plates coated with anti-mouse antibodies to specifically adsorb antibodies in hybridoma supernatants. Detection of anti-receptor antibodies is accomplished by allowing the adsorbed anti-receptor antibodies to bind radiolabeled hormone-receptor complexes. The method does not require pure receptor and can be adapted for studies of any receptor or binding protein where a labeled ligand is available. With small amounts of the insulin receptor (20-50 ng), the method could detect antibody concentrations as low as 300 pM. In summary, the assay is sensitive, rapid, and requires small amounts of impure receptor.

Animals↗
PubMed1985

Injury to the isolated, perfused lung by exposure in vitro to monocrotaline pyrrole.

Monocrotaline pyrrole (MCTP) is a reactive metabolite of the pyrrolizidine alkaloid monocrotaline. It causes pulmonary lesions associated with pulmonary hypertension and right ventricular hypertrophy. Conditions of exposure to MCTP that result in early lung injury were examined in isolated rat lungs perfused with buffered medium containing 4% bovine serum albumin. When a high, acutely lethal dose (8.1 mg) of chemically synthesized MCTP was added to the 50-ml perfusion medium reservoir, no effects on the perfused lung occurred. However, when the same quantity of MCTP was injected directly into the pulmonary arterial (PA) cannula, the lungs accumulated considerable fluid within 1 h, and this was accompanied by elevated perfusion pressure and elevated lactate dehydrogenase (LDH) activity in the perfusion medium. A lower dose (1.2 mg) of MCTP that is pulmonary hypertensive in vivo also produced edema and elevated perfusion pressure when injected into the PA cannula. Removal of perfused 5-hydroxytryptamine, a function of pulmonary endothelium, was unaltered compared to vehicle-treated control lungs, as was perfusate LDH activity. These results indicate that injury to isolated lungs occurs soon after direct exposure to MCTP, even at a moderate dose that produces primarily delayed effects in vivo.

Animals↗
PubMed1985

Biochemistry, physiology and drug metabolism--implications regarding the role of the lungs in drug disposition.

Blood flow and its distribution may influence the functioning of drug metabolizing enzymes in vivo, and also determine the degree to which various organs participate in the metabolic clearance of agents from the body. 'Physiological' pharmacokinetic modeling suggests that in some situations the lung, because of its greater blood flow, may have a significant role in metabolic drug clearance in vivo, despite its low content of drug-metabolizing enzymes relative to the liver. For example, rat liver has much greater microsomal benzo[a]pyrene (BP) hydroxylase (AHH) activity than lung, both in control rats and in rats pretreated with the enzyme inducer, 3-methylcholanthrene (3MC). However, studies using AHH enzyme kinetics, as well as studies in isolated perfused organs and in vivo, indicate that the lungs' contribution to total body metabolic clearance of BP is substantial despite the lungs' relatively low AHH activity compared to liver. Studies with 5-hydroxytryptamine similarly indicate that the lung is important in the metabolic disposition of this amine. These results emphasize that the role of an organ in metabolic drug disposition in vivo cannot be predicted directly from enzyme activity in that organ. By accounting for both biochemical and physiological influences, useful predictions regarding drug disposition may be made for normal and diseased individuals.

Amines↗
PubMed1984

Influence of red blood cells, serum albumin, and serum lipoproteins on the clearance of benzo[alpha]pyrene by isolated livers of 3-methylcholanthrene-treated rats.

Red blood cells, serum albumin, and serum lipoproteins transport benzo[alpha]pyrene and other xenobiotic compounds in the circulation. The distribution of benzo[alpha]pyrene and its metabolites among these blood components was examined, and the effect of their presence in the perfusion medium on the ability of isolated livers from 3-methylcholanthrene-pretreated rats to clear circulating benzo[alpha]pyrene was determined. A large fraction (45%) of the benzo[alpha]pyrene in rat blood was associated with the serum lipoproteins. However, only 8% of the benzo[alpha]pyrene metabolites was associated with this component. Forty to forty-five percent of each was associated with red blood cells. Benzo[alpha]pyrene clearance by isolated rat livers was 1.8 +/- 0.2 ml/min when the medium contained only red blood cells and buffer. Addition of serum lipoproteins or serum albumin increased benzo[alpha]pyrene clearance to 5.1 +/- 0.5 or 8.5 +/- 0.9 ml/min respectively. Appearance of benzo[alpha]pyrene metabolites in perfusions medium and bile was similarly altered by the changes in medium composition. These results indicate that the clearance of benzo[alpha]pyrene by rat liver depends on the composition of the medium perfusing the organ and suggest that alterations in blood components in vivo may influence the metabolic disposition of this carcinogen.

Animals↗
PubMed1984

Production and characterization of a monoclonal antibody to rat liver thiol: protein-disulfide oxidoreductase/glutathione-insulin transhydrogenase.

Rat liver thiol:protein-disulfide oxidoreductase/glutathione-insulin transhydrogenase (glutathione:protein disulfide oxidoreductase, EC 1.8.4.2) was purified and found to give two bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis. A monoclonal antibody was produced against this enzyme preparation and found to remove all the insulin degrading activity of purified preparations of the enzyme. This monoclonal antibody was also found to react with the two different forms of the enzyme observed on gel electrophoresis. These results suggest that glutathione-insulin transhydrogenase can exist in more than one state.

Animals↗
PubMed1984

Effects of covalently linked insulin dimers on receptor kinase activity and receptor down regulation.

Certain covalently linked insulin dimers have previously been found to have a greater ability to bind to the insulin receptor than to stimulate lipogenesis in adipocytes. The present report presents data indicating that the same insulin dimers also have a greater ability to bind to the receptor than to stimulate the kinase activity of the insulin receptor. In particular, one such covalently linked insulin dimer had less than 1% the potency of native insulin in stimulating the receptor kinase although it could bind to the solubilized receptor with 30% the potency of native insulin. In contrast, this dimer could down regulate the insulin receptor with approximately 30% the potency of native insulin. These results suggest that stimulation of the receptor kinase may require more than simple occupancy of the receptor binding site whereas down regulation of the receptor may require only the binding of ligand to the receptor.

Dose-Response Relationship, Drug↗
PubMed1984

Morphologic and cytochemical characteristics of amine-containing globule leukocytes in rat tracheal epithelium.

Amine-containing cells in the tracheal epithelium are typically of the small-granule type (diameter approximately 100 nm). However, in the rat, another amine-containing cell type has been identified that possesses the amine-handling features of the APUD-series of cells (amine precursor uptake and decarboxylation) but not the ultrastructural characteristics. It has been postulated that these cells may be related to cutaneous melanocytes. In this study, fluorescent cells were present in the laryngeal and tracheal epithelial lining of adult Sprague-Dawley rats following freeze-drying and exposure to formaldehyde vapor (FIF or formaldehyde-induced fluorescence). Microspectrofluorimetry revealed an emission maximum at 493 nm. The excitation maximum could not be calculated but appeared to be around or below 350 nm (to record spectra below requires the use of quartz optics). Yellow fluorescence also emanated from serotonin-containing mast cells (excitation and emission maxima: 401/515 nm). Tracheal segments processed according to the aqueous formaldehyde ( AFIF ) technique, for the demonstration of 5- hydroxytryptophan (5-HTP) or serotonin (5-HT), failed to identify fluorescent cells in the epithelial lining even though connective-tissue mast cells were evident. Subsequent treatment of AFIF -fixed sections with formaldehyde and HCl vapors ( AFIF -HCl) resulted in the formation of a fluorogenic compound within numerous cells in the tracheal lining (455/537 nm). This spectral shift and increase in intensity of fluorescence following acidification are characteristic for standards and/or cells that contain tryptamine, tryptophan, or peptides with NH2-terminal tryptophan and are markedly different from microspectrofluorimetric data reported for the phenylethylamines or serotonin. It is therefore postulated that these cells contain a closely related beta-(3-indolyl) ethylamine-like compound, serotonin excluded. The morphology of the fluorescent cells was similar when prepared according to the FIF or AFIF -HCl techniques. Conjunctive staining, the examination of a single section first by fluorescence microscopy and subsequently by other histochemical and cytochemical methods, demonstrated that the fluorescent granules were also methylene blue, alcian blue, periodic-acid Schiff, and ferric- fericyanide positive. Subsequent correlative electron microscopic examination of Epon-embedded AFIF -HCl-treated tracheal sections demonstrated that these amine-containing cells were globule leukocytes.

Animals↗
PubMed1984

ATP and other nucleoside triphosphates inhibit the binding of insulin to its receptor.

ATP, in a dose-dependent manner, inhibited the binding of 125I-insulin to its receptor in rat liver and human placental membranes. With rat liver plasma membranes an effect of ATP was detected at concentrations between 1.0 and 2.5 mmol/L, and maximal effects were seen at 10.0 mmol/L where binding was decreased by approximately 40%. The effect of ATP was one half-maximal within 10 minutes and maximal within 60 minutes. Scatchard analysis indicated that ATP was acting primarily to change the binding affinity of the insulin receptor. The effect of ATP was mimicked by CTP, GTP, and UTP, but not by ADP, 5'-AMP, 3'-AMP, 3'5'-cyclic AMP and adenosine. The ATP analog AMP-PNP had a potency approximately 10% that of ATP. The effect of ATP was not significantly influenced by inhibitors of phosphoprotein kinases and phosphoprotein phosphatases. In human placental membranes, ATP had a similar effect in inhibiting 125I-insulin binding to its receptor. Moreover, ATP was active in inhibiting insulin binding to purified human placental insulin receptors at 0.01 mmol/L, a concentration 1/100 of that needed for inhibiting binding to intact membranes. These studies indicate, therefore, that ATP and other nucleoside triphosphates influence the ability of the insulin receptor to bind insulin.

Adenosine Monophosphate↗
PubMed1984

Alteration of monocrotaline pyrrole-induced cardiopulmonary effects in rats by hydrallazine, dexamethasone or sulphinpyrazone.

The effects of intraperitoneal hydrallazine, dexamethasone, or sulphinpyrazone on the toxicity of monocrotaline pyrrole (MCTP) were examined in rats 14 days after injection of MCTP (5 mg kg-1, i.v.). MCTP alone caused increases in lung weight, and of both lactate dehydrogenase activity and protein concentration in bronchopulmonary lavage fluid. Right ventricular hypertrophy also occurred. Hydrallazine (3 mg kg-1, daily), a vasodilator and platelet prostaglandin synthesis inhibitor, reduced the degree of right ventricular hypertrophy and the elevation in the concentration of protein in lavage fluid. Dexamethasone (27 micrograms kg-1, daily), an anti-inflammatory agent and inhibitor of phospholipase, also reduced the right ventricular hypertrophy and the increased protein concentration in lavage fluid caused by MCTP. Sulphinpyrazone (100 mg kg-1, twice daily), an inhibitor of platelet prostaglandin biosynthesis, prevented right ventricular hypertrophy in the MCTP treated rats without affecting any of the indices of lung injury. These results provide further support for the hypothesis that platelets and vasoconstrictor agents play a role in the development of MCTP-induced pulmonary hypertension.

Animals↗
PubMed1984

Effects of thrombocytopenia on monocrotaline pyrrole-induced pulmonary hypertension.

Monocrotaline pyrrole (MCTP) causes lung injury, pulmonary hypertension, and right ventricular hypertrophy in rats. To determine if platelets are involved in the cardiopulmonary effects of MCTP, the response to MCTP was determined in thrombocytopenic rats. Blood platelet count was reduced to 10-20% of normal for 48 h by ip administration of an antirat platelet serum (PAS) prepared in the goat. Rats were treated iv with either MCTP 5 mg/kg or dimethylformamide vehicle and with either PAS or preimmune serum. Fourteen days after MCTP, right ventricular hypertrophy and several indexes of lung injury were measured. MCTP treatment produced right ventricular hypertrophy, increased lactate dehydrogenase activity and protein concentration in bronchopulmonary lavage fluid, increased perfusion pressure in isolated lungs, and decreased pulmonary clearance and metabolism of perfused 5-hydroxytryptamine. Thrombocytopenia did not influence the changes in these indexes of lung injury produced by MCTP in this protocol. When PAS was given 12 h before MCTP, it did not affect right ventricular hypertrophy, but when PAS treatment was begun 3 or 6 days after MCTP, right ventricular hypertrophy was decreased by 19 or 41%, respectively. These results suggest that platelets help to mediate the development of pulmonary hypertension and the hypertrophic response of the right heart following MCTP administration.

Animals↗
PubMed1984

Dual regulation of glycogen metabolism by insulin and insulin-like growth factors in human hepatoma cells (HEP-G2). Analysis with an anti-receptor monoclonal antibody.

Insulin and the insulinlike growth factors (IGF-I and IGF-II) are members of a family of hormones that regulate the metabolism and growth of many tissues. Cultured HEP-G2 cells (a minimal deviation human hepatoma) have insulin receptors and respond to insulin by increasing their glycogen metabolism. In the present study with HEP-G2 cells, we used 125I-labeled insulin, IGF-I, and IGF-II to identify distinct receptors for each hormone by competition-inhibition studies. Unlabeled insulin was able to inhibit 125I-IGF-I binding but not 125I-IGF-II binding. A mouse monoclonal antibody to the human insulin receptor that inhibits insulin binding and blocks insulin action inhibited 75% of 125I-insulin binding, but inhibited neither 125I-IGF-I nor 125I-IGF-II binding. When glycogen metabolism was studied, insulin stimulated [3H]glucose incorporation into glycogen in a biphasic manner; one phase that was 20-30% of the maximal response occurred over 1-100 pM, and the other phase occurred over 100 pM-100 nM. The anti-receptor monoclonal antibody inhibited the first phase of insulin stimulation but not the second. Both IGF-I and IGF-II stimulated [3H]glucose incorporation over the range of 10 pM-10 nM; IGF-I was three to fivefold more potent. The monoclonal antibody, however, was without effect on IGF regulation of glycogen metabolism. Therefore, these studies indicate that insulin as well as the IGFs at physiological concentrations regulate glycogen metabolism in HEP-G2 cells. Moreover, this regulation of glycogen metabolism is mediated by both the insulin receptor and the IGF receptors.

Animals↗
PubMed1984

Degradation of insulin by isolated mouse pancreatic acini. Evidence for cell surface protease activity.

In the present study, we have used isolated mouse pancreatic acini to investigate the relationship between 125I-insulin binding and its degradation in order to probe the nature and cellular localization of the degradative process. In these cells, the proteolysis of 125I-insulin was dependent on time and cell concentration, and was saturated by unlabeled insulin with a Km of 290 nM. Since this value was much higher than the Kd for insulin binding to its receptor (1.1 nM), the data indicated that 125I-insulin degradation by acini occurred primarily via nonreceptor mechanisms. Several lines of evidence suggested that insulin was being degraded by the neutral thiol protease, insulin degrading enzyme (IDE). First, insulin degradation was inhibited by thiolreacting agents such as N-ethylmaleimide and p-chloromercuribenzoate. Second, the Km for degradation in acini was similar to the reported Km for IDE in other tissues. Third, the enzyme activity had a relative mol wt of approximately 130,000 by gel filtration, a value similar to that reported for purified IDE. Fourth, the degrading activity was removed with a specific antibody to IDE. Other lines of evidence suggested that enzymes located on the cell surface played a role in insulin degradation by acini. First, the nonpenetrating sulfhydryl reacting agent 5,5' dithiobis-2-nitrobenzoic acid blocked 125I-insulin degradation. Second, a specific antibody to IDE identified the presence of the enzyme on the cell surface. Third, chloroquine, leupeptin and antipain, agents that inhibit lysosomal function, did not influence 125I-insulin degradation. Fourth, highly purified pancreatic plasma membranes degraded 125I-insulin.

Animals↗