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Biomedical subjects

L C Harrison

Publications and source records attributed to L C Harrison.

At least 217 records · Page 12Linked to original sources
PubMed1984

Absence of covalent binding by insulin to erythrocyte and reticulocyte insulin receptors.

We investigated whether insulin forms covalent bonds with its receptors on erythrocytes and reticulocytes, as it does in adipocytes (1). Of the [125I]-insulin specifically bound at 37 degrees C to human and rat erythrocytes and rat reticulocytes, only 1.5-2.3% was non-dissociable on extensive washing. When ghosts prepared from the washed cells were solubilized in Triton X-100, only 0.6-1.5% of the specifically bound radioactivity appeared in the void volume of a Sephadex G-50 column. Moreover in contrast to adipocytes, this high molecular weight radioactivity was not immunoprecipitable by antibodies to the insulin receptor and was dissociated during chromatography in sodium dodecyl sulphate. Thus we have been unable to demonstrate the formation of covalent bonds between insulin and its receptors on erythrocytes and reticulocytes. This finding is consistent with the hypothesis that covalent binding of insulin is a necessary receptor modification for insulin's metabolic effects.

Animals↗
PubMed1984

Precipitation of the thyrotropin receptor and identification of thyroid autoantigens using Graves' disease immunoglobulins.

The thyrotropin (TSH) receptor is a putative target for autoantibodies in Graves' hyperthyroidism and therefore, should be capable of being identified, isolated, and structurally characterized by immunological means. To this end, four sera from patients with hyperthyroidism, three of which inhibited the binding of 125I-TSH to Triton-solubilized human thyroid membranes, were used to isolate TSH receptors by immunoprecipitation. To account for an effect of TSH binding or receptor occupancy on the ability of Graves' immunoglobulins to precipitate TSH receptors, two approaches were taken: (a) specific 125I-TSH binding activity was measured after solubilized thyroid membranes had been incubated with Graves' sera followed by precipitation with Staphylococcus protein A ("receptor depletion"); (b) TSH binding sites were labeled with 125I-TSH and the complexes were precipitated using Graves' sera and Staphylococcus protein A ("receptor precipitation"). The three sera which inhibited 125I-TSH binding depleted 125I-TSH binding activity between 30-80%. Preformed complexes between Staphylococcus protein A and immunoglobulins in these sera were also able to deplete 125I-TSH binding activity. However, after receptor depletion, the one serum that did not inhibit 125I-TSH binding was associated with a significant increase in 125I-TSH binding. All four sera specifically precipitated 80-100% of receptors identified by prelabeling with 125I-TSH. The dilutions of sera that precipitated 50% of 125I-TSH-receptor complexes ranged from 1:150-1:20. Complexes were partially precipitated by high concentrations of control sera (1:20), but the relative potency of control sera was at least fourfold less than Graves' sera. Immunoprecipitates of 125I-labeled thyroid membranes were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography to reveal Graves'-specific bands of reduced molecular weights of 100-110,000, 80-90,000, and 70-75,000. These bands were similar to those obtained from 125I-labeled thyroid membranes purified by TSH affinity chromatography. Thus, Graves' immunoglobulins: (a) precipitate unoccupied and occupied TSH receptors, (b) in one case, neither inhibit binding nor immunodeplete the unoccupied receptor but immunoprecipitate 125I-TSH-receptor complexes, suggesting that binding of TSH may initiate an interaction between the binding site and a separate immunoreactive molecule, and (c) identify the molecular structure of Graves' autoantigens, putatively, the TSH receptor.

Animals↗
PubMed1984

Exacerbation of bronchial asthma following treatment with amiodarone. Demonstration of an antiadrenergic effect in vitro.

We describe a patient with bronchial asthma whose respiratory symptoms worsened during treatment and rechallenge with amiodarone. Laboratory studies on cultured pulmonary cells demonstrated an anti-beta-adrenergic effect of amiodarone not due to inhibition of beta-adrenergic receptor binding. Amiodarone should be used with caution in patients with obstructive pulmonary disease.

Amiodarone↗
PubMed1984

A postbinding inhibitor of insulin action. Increased concentrations in the plasma of non-insulin-dependent diabetic subjects.

"Postreceptor" insulin resistance in persons with non-insulin-dependent diabetes (NIDDM) could be due to an intrinsic defect in insulin-sensitive pathways or to the action of a circulating inhibitor. Since evidence for the former is lacking, we have addressed the question of a circulating inhibitor by examining the effect of plasma and plasma extracts from NIDDM subjects on the lipogenic response of rat adipocytes to insulin. A majority (77%) of plasma samples (1:20 dilution) from unselected, treated NIDDM subjects (N = 69) inhibited insulin-stimulated conversion of 3-3H-glucose to 3H-lipid in rat adipocytes to a greater extent than did control samples (N = 24). The mean +/- SD inhibition by NIDDM plasma (81 +/- 21%) was significantly greater (P less than 0.01) than by control plasma (50 +/- 14%). Diabetic and, to a lesser degree, control plasma both caused a significant decrease in the maximal response of lipogenesis to insulin. Inhibitory activity was extracted into acid/ethanol, present in the flow of a Sep-pak C18 column, heat-stable (56 degrees C for 30 min [plasma], 80 degrees C for 30 min [acid/ethanol]), resistant to proteases, and dialyzable through 1000-dalton-mol wt exclusion dialysis tubing. The inhibition by NIDDM plasma or partially purified inhibitor could not be explained by the presence of insulin antibodies, insulin receptor antibodies, other inhibitors of insulin binding, or the concentrations of known counterregulatory factors. There was no correlation between inhibitory activity and plasma glucose (r = 0.26), insulin (r = 0.33), C-peptide (r = 0.26), or HbA1c (r = 0.26).(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗
PubMed1983

Disulfide exchange between insulin and its receptor. A possible post-binding step in insulin action.

A fraction of the insulin specifically bound to adipocytes undergoes a disulfide interchange with its receptor (Clark, S., and Harrison, L. C. (1982) J. Biol. Chem. 257, 12239-12244). In order to test the hypothesis that this covalent modification is a relevant step in insulin action, we have examined the relationship between disulfide binding of insulin and several insulin bioeffects, using sulfhydryl group blocking reagents as probes. The half-time of disappearance of disulfide linked insulin-receptor complexes (I-(S-S)-R) was rapid (4.5 min), consistent with their hypothesized role. A cell-impermeable reagent 5,5'-dithiobis(nitrobenzoic acid) (DTNB) had no effect on specific insulin binding but caused a dose-dependent decrease in both I-(S-S)-R and insulin-stimulated glucose transport. DTNB also inhibited the effect of insulin to stimulate glucose oxidation and to inhibit epinephrine-stimulated cyclic AMP production. In cultured IM-9 lymphocytes, insulin-induced down regulation of its receptor was decreased by 75% in the presence of 1 mM DTNB. Receptor antibodies stimulated adipocyte glucose transport maximally but their effect, unlike that of insulin, was not inhibited by DTNB. These findings suggest that receptor sulfhydryl groups are required for insulin action and support the notion that their interchange with insulin is a necessary step in activation of postreceptor pathways.

1-Methyl-3-isobutylxanthine↗
PubMed1983

Calcium-dependence of insulin receptor phosphorylation.

Phosphorylation of the insulin receptor of isolated rat adipocytes in response to insulin has been studied. Immunoprecipitation of adipocyte membrane protein demonstrated increased incorporation of 32P after exposure to insulin for 15 min, but this was dependent on the presence of physiological concentrations of Ca2+ and Mg2+. Autoradiography of solubilized immunoprecipitated membrane protein after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed that most of the 32P incorporation occurred in a band corresponding to Mr 95 000, which has been identified previously as the beta-subunit of the insulin receptor. 32P incorporation was inhibited by 2,4-dinitrophenol and trifluoperazine. It is suggested that insulin-receptor phosphorylation is an energy-requiring process that is Ca2+-dependent and may be modulated by calmodulin. Phosphorylation may proceed independently of glucose transport.

Adipose Tissue↗
PubMed1982

Atopy, autonomic function and beta-adrenergic receptor autoantibodies.

Atopic individuals (with asthma, allergic rhinitis or atopic eczema) have impaired sensitivity to beta-adrenergic agents. After the finding of antibodies to the beta-adrenergic receptor in the serum of a subject with allergic rhinitis, coded sera from atopic and control subjects were assayed for immunoglobulins that inhibited the specific binding of 125I-labelled hydroxybenzylpindolol to beta-receptors in mammalian lung membranes. Antibodies were present in nine of 60 subjects: 3/19 normal control subjects, 1/9 pre-allergic, 4/17 asthma, 0/8 allergic rhinitis, and 1/7 cystic fibrosis patients. Antibodies of the IgG class in these sera were also demonstrated by indirect precipitation of solubilized lung beta-receptors. The autonomic sensitivity of the nine antibody-positive subjects (Ab+) was compared with that of antibody-negative subjects (Ab-). The Ab+ subjects required 15.0 +/- 1.9 ng isoprenaline (isoproterenol) kg-1 min-1 i.v. to increase pulse pressure by at least 22 mmHg (Ab-, 7.7 +/- 0.4; n = 20; P less than 0.001), and 12.4 +/- 1.8 ng isoprenaline kg-1 min-1 i.v. to increase plasma cyclic AMP concentrations by 50% (Ab-, 8.08 +/- 0.62; n = 13; P less than 0.02). Ab+ subjects required 2.06 +/- 0.3% phenylephrine to dilate their pupils (Ab-, 2.55 +/- 0.08; n = 57; P less than 0.05) and 0.61 +/- 0.08% carbachol to constrict their pupils (Ab-, 0.78 +/- 0.03%; n = 57; P less than 0.05). A role for autoantibodies as beta-receptor antagonists was further supported by showing that human lung cells (VA-13 line) cultured in the presence of globulins from Ab+ subjects had a markedly impaired cyclic AMP response to isoprenaline. These results suggest that autoantibodies to beta-receptors play a pathogenetic role in asthma and related disorders. They have important implications for the concept of autoimmunity.

Autoantibodies↗
PubMed1982

Subpopulations of antibodies directed against evolutionarily conserved regions of the insulin molecule in insulin-treated patients.

In the present study, we attempted to define possible subpopulations of antibodies which theoretically could be directed against evolutionarily conserved regions of the insulin molecule in sera from insulin-treated diabetic patients using a variety of labelled and unlabelled insulins which differ widely in structure but are very similar in functional properties. Ten high titre human insulin antisera from patients treated with mixed beef-pork insulin were examined. All sera were found to bind 125I-pork insulin better than labelled chicken insulin which bound better than labelled fish insulin. Detailed studies were conducted with four of the antisera using the pork and fish tracers. With two of the antisera, a subpopulation of antibody could be detected with 125I-fish insulin which had similar affinity for both fish and pork insulin, but reacted much less well with guinea pig insulin and the desoctapeptide derivative of porcine insulin. Based on the known properties of these four insulins, the data provide suggestive evidence consistent with the hypothesis that there are subpopulations of antibodies recognizing regions on the insulin molecule that are well conserved, possibly the region involved in the formation of insulin dimers or receptor binding.

Animals↗
PubMed1982

The insulin receptor on the human lymphocyte: insulin-induced down-regulation of 126,000 and 90,000 glycosylated subunits.

Cultured human lymphoblastoid B lymphocytes were surface-labelled with iodine125 and solubilized in 1% Triton X-100 in the presence of protease inhibitors. After purification of labelled glycoproteins by elution from immobilized wheat germ lectin with 0.3 mol/l N-acetyl-D-glucosamine, insulin receptors were quantitatively immunoprecipitated using IgG receptor auto-antibodies. The overall recovery of labelled glycoprotein was 0.02-0.04%; analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography under reducing conditions revealed two major bands with molecular weights of 126,000 and 90,000, and a minor band of 67,000 daltons. The mobilities of both major receptor subunits were increased after treatment with neuraminidase. When lymphocyte receptor binding was 'down-regulated' before surface labelling, there was a concomitant decrease in the recovery of both the 126,000 and 90,000 subunits. These data indicate that 'down-regulation' of binding probably involves degradation of the receptor molecule.

Autoantibodies↗
PubMed1982

Autoantibodies to the insulin receptor and insulin-resistant diabetes.

Autoantibodies to the insulin receptor are a rare cause of insulin-resistant diabetes, but when they occur they produce a profound clinical syndrome. These antibodies block insulin binding, immunoprecipitate solubilized insulin receptors, and their acute effect is to mimic the biological effects of insulin. However, prolonged exposure of cells to these antibodies produces a state of insulin resistance. Since the antigen to which the antibody is directed is relatively well-characterized, many of the observations in this syndrome can serve as a model for elucidating molecular mechanisms in other diseases with antibodies against membrane components. The autoantibodies to the insulin receptor have also provided valuable probes in the study of insulin receptor structure and insulin action.

Adipose Tissue↗