The effect of nitric oxide on glucose metabolism

Nitric oxide (NO) is an important bioactive signaling molecule that mediates a variety of normal physiological functions, which, if altered, could contribute to the genesis of many pathological conditions, including diabetes. In this study, we examined the possible diabetogenicity of NO by noting differences in the cellular binding of insulin in dogs treated with the NO donor, S-nitrosoglutathione (GSNO) compared to captopril-treated controls. GSNO administration resulted in an abnormality in glucose metabolism which was attributed to decreased binding of insulin to its receptor on the cell membrane of mononuclear leucocytes, 11.60 +/- 0.60% in GSNO-treated dogs compared with 18.10 +/- 1.90% in captopril-treated control (p < 0.05). The decreased insulin binding was attributed to decreased insulin receptor sites per cell, 21.43 +/- 2.51 x 10(4) in GSNO-treated dogs compared with 26.60 +/- 1.57 x 10(4) in captopril-treated controls (p < 0.05). Average affinity analysis of the binding data demonstrated that this decrease in insulin binding was also due to a decrease in average affinity of the receptor on mononuclear leucocytes for insulin. This was evident by a decrease in empty and filled site affinities in GSNO-treated dogs compared with that of captopril-treated dogs (p < 0.05). It appears that GSNO is exerting its effect by decreasing the number of insulin receptor sites and/or decreasing the average receptor affinity. These results provide evidence for a novel role of NO as a modulator of insulin binding and the involvement of NO in the aetiology of diabetes mellitus.     

Increased insulin receptor binding in erythrocytes from growth hormone-deficient children

Erythrocytes from growth hormone-deficient children (GHd-children) (n=10) showed a statistically significant increase in insulin binding at low unlabeled insulin concentrations, together with a threefold decrease in apparent receptor affinity, as compared to control children (C) (n=11). Scatchard analysis of the binding data using the two-site model revealed that both the receptor concentration R₁ [GHd-children 0.10±0.01 ng/ml and C 0.03±0.002 ng/ml] and the dissociation constant K_D1 [GHd-children (0.48±0.05)×10^–9M and C (0.19±0.01)×10^–9M] for high affinitylow capacity sites were significantly increased in erythrocytes from GHd-children, while neither receptor concentrations (R₂) nor the dissociation constant (K_D2) for low affinity-high capacity sites proved to be altered. These events were accompanied by a normal sensitivity to insulin as well as glucose tolerance in the GHd-group. The meaning of the increased insulin binding with normal insulin sensitivity in GH-deficiency is discussed.     

Effect of alterations in membrane lipid unsaturation on the properties of the insulin receptor of Ehrlich ascites cells

We have altered the phospholipid composition of the plasma membranes of Ehrlich ascites cells grown in mice and studied the effects on the properties of the insulin receptor of this cell. The insulin receptor of the Ehrlich cell demonstrated all of the binding characteristics of mammalian insulin receptors: specificity for insulin and insulin analogs, saturability, inverse relationship of steady-state binding levels to temperature, and negative cooperativity. Cellular phospholipids enriched in monounsaturated fatty acyl groups were produced by growth in animals that were maintained on a diet rich in coconut oil; cellular phospholipids enriched in polyunsaturated fatty acyl groups were produced in animals fed sunflower oil. Insulin receptors were present in the normal cells at 180 000 sites/cell but this fell to 125 000 (p <0.001) in cells enriched in monounsaturated fatty acids and rose to 386 000 (p <0.001) in cells enriched in polyunsaturated fatty acids. The normal cells had affinity constants ( and ) of 0.03 and 0.01 nM⁻¹. The cells enriched in monounsaturated fatty acids had an increase in these affinity constants to 0.06 and 0.03 nM⁻¹ whereas values of 0.01 and 0.005 nM⁻¹ were obtained in the cells enriched in polyunsaturated fatty acids (all comparison p <0.001). Thus, increased unsaturation of plasma membrane phospholipids, produced by dietary manipulations, was associated with an increase in insulin receptor number but a decrease in binding affinity. In contrast, increased saturation of the phospholipids of the plasma membrane was associated with a decrease in receptor number and an increase in affinity. The results can be explained by a model in which the insulin receptor is assumed to be multimeric.     

Insulin receptor binding from mid-term and full-term placentas of patients with gestational diabetes mellitus and normal pregnant women

Insulin receptor binding was examined in the microvillous membranes of mid-term (20–22 weeks of gestation, MT) and full-term (FT) placentas from patients with gestational diabetes mellitus (GDM) and in normal pregnant control (N). Mid-term placentas were obtained from patients who have had spontaneous abortion. The maximum per cent specific binding (%SB) in MT placenta for GDM was significantly lower (4.8%) compared with the FT placenta (22%, p<0.001), while in the N group the maximum per cent specific binding for MT placenta was 14.1% compared with 26% for the FT placneta (p<0.001). Binding data from FT placenta of well-controlled GDM patients were similar with the FT placenta from N group (22%SB for GDM VS 26% SB for N). Even as there were similarities in the binding characteristics of FT placentas from both groups the placental membrane protein content in the GDM group was lower by 50% compared with the N control (2.5±0.11 VS 4.8±0.15 mg protein/g placenta respectively, p<0.001) suggesting that in the GDM group achieving a tight glycemic control could improve receptor affinities. Data from the competitive binding assay of GDM patients showed that the insulin necessary to achieve 50% inhibition (ID₅₀) was significantly lower in MT compared with the FT placenta (0.9×10^–9 M VS 3.8×10^–9 M, p<0.001) but in the N placenta there was no alteration in the ID₅₀ of MT and FT placentas (3.1×10^–9 M VS 4×10^–9 M, p<0.01, respectively). The present study demonstrated that in GDM the placental insulin receptor binding was significantly lower in spontaneously aborted placenta compared with placentas collected at full-term. Furthermore, these data suggest that the objective to achieve a tight glycemic control in GDM patients could optimize insulin receptor function similar to that of a normal pregnancy. Thus a full term placenta from GDM patients under a well managed glycemic control throughout the entire duration of pregnancy would result in an optimum insulin receptor function.     

The Hyperglycemic Effect of S-Nitrosoglutathione in the Dog

The present study investigates the pharmacological activity of the nitric oxide (NO) donor S-nitrosoglutathione (GSNO) on the plasma glucose and insulin levels in healthy normoglycemic dogs. The plasma nitrate and nitrite concentrations were measured by a commercial autoanalyzer and taken as the biochemical markers of in vivo nitric oxide formation. Plasma glucose levels were measured by the glucose oxidase method, while the insulin levels were determined by radioimmunoassay. The possible effect of the coadministration of ascorbic acid (vitamin C) and GSNO on plasma glucose levels was also examined. In healthy normoglycemic dogs, administration of 35 and 50 mg/kg of GSNO caused a dose-dependent increase in postprandial plasma glucose levels. The plasma glucose levels were significantly elevated at the 1.5-, 2.0-, and 2.5-h time intervals of the oral glucose tolerance test at both concentrations of GSNO (P < 0.05). These values were significantly higher than those obtained using captopril (control). Furthermore, coadministration of 35 mg/kg of GSNO and 50 mg/kg ascorbic acid enhanced the postprandial hyperglycaemic effect observed for the administration of only 35 mg/kg of GSNO. There was a 35–100% increase in plasma nitrate concentration on administration of both doses of GSNO. Intravenous administration of GSNO (35 mg/kg) and captopril (20 mg/kg) significantly decreased the mean arterial blood pressure and increased the heart rate. The blood pressure-lowering effect of these drugs was more pronounced on systolic than on diastolic blood pressure (P < 0.05). These results suggests that in healthy normoglycaemic dogs: (a) nitric oxide released from GSNO increases postprandial plasma glucose levels and inhibits glucose-stimulated insulin secretion, (b) ascorbic acid enhances the postprandial hyperglycaemic effect of GSNO, probably by increasing the release of NO, and (c) GSNO decreases mean arterial blood pressure and increase heart rate in normotensive dogs.     

Decreased insulin binding and antilipolytic response in adipocytes from patients with Cushing's syndrome

Human adipocytes from patients with chronic endogenous hypercortisolism (Cushing's syndrome) showed a statistically significant decrease in insulin binding at low unlabelled-insulin concentrations but no change in receptor numbers (Cushing's 180,000±48,000 (3) receptors/cell and controls 189,000±30,000 (7)) together with a fourfold decrease in apparent receptor affinity (ED50: Cushing's 2.25×10^–9 M and controls 0.57×10^–9 M) and a decreased sensitivity to the antilipolytic effect of insulin. These events could represent the final situation of a chronic and endogenous regulation by high levels of cortisol of insulin receptors in human adipose tissue.     

Endothelin receptor in microvessels isolated from human meningiomas: Quantification with radioluminography

Summary 1. We characterized specific¹²⁵I-endothelin-1 (¹²⁵I-ET-1) binding sites in microvessels isolated from human meningiomas, using anin vitro quantitative receptor autoradiographic technique coupled to a radioluminographic imaging plate system.2. This newly developed and highly sensitive method revealed high-affinity ET receptors present in pellet sections of the microvessels from all the meningiomas studied, regardless of histological subtypes (dissociation constant, 1.2 ± 0.3 nM; maximum binding capacity, 185 ± 56 fmol/mg; means ± SE for nine tumors).3. In five cases of meningiomas, ET-3 competed for¹²⁵I-ET-1 binding to microvessels from those tumors with a low affinity [50% inhibiting concentration (IC₅₀) of 1.6 ± 0.4 × 10^–6 M], and a selective ET_B receptor agonist, sarafotoxin S6c, up to 10^–6 M, did not displace ET binding from the sections.4. In the sections of microvessels from four other tumors, biphasic competition curves were obtained in the case of incubation in the presence of increasing concentrations of ET-3, with an IC₅₀ of 1.1 ± 0.2 × 10^–9 M for the high-affinity component and 1.6 ± 0.3 × 10^–6 M for the low-affinity component, respectively. In addition, S6c competed for ET binding to those sections (IC₅₀=2.3 ± 0.2 × 10^–10 M) and 10^–6 M S6c displaced 30% of the control, corresponding to the high-affinity component of competition curves obtained in the presence of ET-3.5. Our results suggest that (a) capillaries in human meningiomas express a large number of high-affinity ET_A (non-ET_B) receptors with a small proportion of ET_B receptors, and (b) ET may have a role in neovascularization, tumor blood flow, and/or function of the blood-tumor barrier in meningioma tissues by interacting with specific receptors present on the surface of the endothelium.     

Kainate receptor modification in the fetal guinea pig brain during hypoxia

The present study tests the hypothesis that hypoxia alters the high-affinity kainate receptors in fetal guinea pig brain. Experiments were conducted in normoxic and hypoxic guinea pig fetus at preterm (45 days of gestation) and term (60 days of gestation). Hypoxia in the guinea pig fetus was induced by exposure to maternal hypoxia (FiO₂=7%) for 60 min. Brain tissue hypoxia in the fetus was documented biochemically by decreased levels of ATP and phosphorreatine. [³H]-Kainate binding characteristics (Bmax=number of receptors, Kd=dissociation constant) were used as indices of kainate receptor modification. P₂ membrane fractions were prepared from the cortex of normoxic and hypoxic fetuses and were washed six times prior to performing the binding assays. [³H]kainate binding was performed at 0°C for 30 min in a 500 l medium containing 50 mM Tris-HCl buffer, 0.1 mM EDTA (pH 7.4), 300 g protein and varying concentrations of radiolabelled kainate ranging from 1 to 200 nM. Non-specific binding was determined in the presence of 1.0 mM glutamate. During brain development from 45 to 60 days gestation, Bmax value increased from 330±16 to 417±10 fmoles/mg protein; however, the Kd was unchanged (8.2±0.4 vs 8.8±0.5 nM, respectively). During hypoxia at 60 days, the Kd value significantly increased as compared to normoxic control (15.5±0.7 vs 8.8±0.5 nM, respectively), whereas the Bmax was not affected (435±12 vs 417±10 fmol/mg protein, respectively). At 45 days, hypoxia also increased the Kd (11.9±0.6 vs 8.2±0.4 nM) without affecting the Bmax (290±15 vs 330±16 fmol/mg protein, respectively). The results show that the number of kainate receptors increase during gestation without change in affinity and demonstrate that hypoxia modifies the high-affinity kainate receptor sites at both ages; however the effect is much stronger at 60 days (term). The decreased affinity of the site could decrease the kainate receptor-mediated fast kinetics of desensitization and provide a longer period for increased Na⁺-influx, leading to increased accumulation of intracellular Ca²⁺ by reversal of the Na⁺–Ca²⁺ exchange mechanism. In addition, Kd values for kainate-type glutamate receptor sites are 30–40 fold lower (i.e. higher affinity) than those for NMDA-displaceable glutamate sites. The higher affinity suggests that the activation of the kainate-type glutamate receptor during hypoxia could precede initiation of NMDA receptormediated excitotoxic mechanisms. We propose that hypoxia-induced modification of the high affinity kainate receptor in the fetus is a potential mechanism of neuroexcitotoxicity.     

Receptor of Insulin-Like Growth Factor 1 in Tissues of the Mollusc Anodonta cygnea

To identify insulin-like receptors in the mollusc Anodonta cygnea, specific binding of ¹²⁵I-insulin and ¹²⁵I-IGF-1 by WGA-purified glycoprotein fractions of foot muscles and neural ganglia is studied. The binding sites for IGF-1 are detected for the first time in invertebrates, both in the muscles, and in the neural tissue of the mollusc. The level of ¹²⁵I-IGF-1 binding in the muscle tissue was equal to 2.8 ± 0.1, in the neural tissue, to 4.0 ± 0.2% per 5 µg of protein. The equilibrium dissociation constant (K _d) was equal to 4.8 ± 0.3 and 4.3 ± 0.2 nM, respectively. The relative affinity of the binding sites to insulin did not exceed 1% of their affinity to IGF-1. Binding of ¹²⁵I-insulin in the muscle tissue was not detected; the level of labeled insulin binding in the neural tissue was equal to 0.5% per 5 µg of protein. In the sarcolemmal fraction of the mollusc foot, IGF-1 and, to a lesser degree, insulin at a dose of 100 nM initiated phosphorylation of tyrosine in a protein with mol. mass of 70 kDa. The minor band of the phosphorylation was also detected in the zone of protein of 80 kDa. The conclusion is made about the existence in molluscan tissues of high-conserved receptors-tyrosine kinases identical by functional parameters to the mammalian receptor of IGF-1. From this, it is suggested that the peptides close by structure to vertebrate IGF-1 may be involved in physiological processes in A. cygnea. The problem of the nature of the insulin-binding sites in the molluscan neural tissue is discussed.     

Decreased 5-HT2C receptor binding in the cerebral cortex and brain stem during pancreatic regeneration in rats

The purpose of this study was to investigate the role of central 5-HT_2C receptor binding in rat model of pancreatic regeneration using 60–70% pancreatectomy. The 5-HT and 5-HT_2C receptor kinetics were studied in cerebral cortex and brain stem of sham operated, 72 h pancreatectomised and 7 days pancreatectomised rats. Scatchard analysis with [³H] mesulergine in cerebral cortex showed a significant decrease (p < 0.05) in maximal binding (B_max) without any change in K_d in 72 h pancreatectomised rats compared with sham. The decreased B_max reversed to sham level by 7 days after pancreatectomy. In brain stem, Scatchard analysis showed a significant decrease (p < 0.01) in B_max with a significant increase (p < 0.01) in K_d. Competition analysis in brain stem showed a shift in affinity towards a low affinity. These parameters were reversed to sham level by 7 days after pancreatectomy. Thus the results suggest that 5-HT through the 5-HT_2C receptor in the brain has a functional regulatory role in the pancreatic regeneration.     

High affinity beta-2-adrenergic receptors in mononuclear leucocytes: similar density in young and old normal subjects

In normal subjects beta-adrenergic responsiveness in the cardiovascular system has been shown to be impaired with increasing age. In order to correlate reduced hormonal responsiveness to an age-related defect at the receptor level, high affinity beta-adrenergic receptors in homogenates of human mononuclear leucocytes have been studied with a (?)-³H-dihydroalprenolol (³H-DHA) binding assay. The binding sites have been characterized by rapid kinetics, saturability, structural and sterospecificity. Binding equilibrium was obtained within 16 minutes at 37° and was reversed by 50% within 10.6 minutes. In 22 healthy subjects a binding capacity of 60 ± 8 fmol/mg protein and an equilibrium dissociation constant (K_D) of 0.6 ± 0.05 nM was found. Beta-adrenergic agonists displaced ³H-DHA binding with a potency order of isoproterenol > adrenaline > noradrenaline. The (?) isomers of beta-adrenergic agonists and antagonists were one to two orders of magnitude more potent as inhibitors of ³H-DHA binding than their corresponding (+) isomers. The binding capacity and affinity of the beta-adrenergic receptors did not differ in old, as compared to young normal subjects. Leucocytes from 14 individuals 18–40 years old had an average density of 53 ± 4 fmol/mg protein, while the average density in leucocytes from 8 individuals aged 53–65 years was 67 ± 8 fmol/mg protein. The K_D was 0.6 ± 0.05 nM in both groups. In conclusion, an age-related decrease of beta-adrenergic receptor-mediated cardiovascular functions does not seem to be reflected in the properties of beta-adrenergic receptors of mononuclear leucocytes.     

Effects of S-nitroso-N-acetyl-penicillamine administration on glucose tolerance and plasma levels of insulin and glucagon in the dog.

It has been suggested that nitric oxide (NO, nitrogen monoxide) is a regulator of carbohydrate metabolism in skeletal muscle. The present study was undertaken to investigate the acute effects of the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) on blood glucose levels and on the gluco-regulatory hormones insulin and glucagon in healthy dogs. The acute effects of SNAP on mean arterial pressure and heart rate were also investigated. The drug was administered intravenously and the pre- and postprandial blood glucose, plasma insulin, and glucagon concentrations were determined at half-hour time intervals postadministration after a glucose challenge. The plasma nitrate and nitrite concentrations were measured and taken as the biochemical markers of in vivo NO formation. The oral glucose tolerance test revealed an impaired glucose tolerance in SNAP-treated dogs as reflected by the area under the glucose curve, 1150.50 +/- 63.00 mmol x 150 min and 1355.25 +/- 102.01 mmol/L x 150 min in dogs treated with 10 and 20 mg/kg of SNAP, respectively, compared with 860.25 +/- 60.68 mmol/L x 150 min in captopril-treated controls (P < 0.05). The 2-h blood glucose concentration in dogs treated with 20 mg/kg body wt of SNAP was 9.17 +/- 1.10 mmol/L compared with 5.59 +/- 0.26 mmol/L for captopril-treated controls (P = 0.015). The oral glucose tolerance test also confirmed an impaired insulin secretion in the SNAP-treated dogs. While the plasma insulin concentration increased gradually in the captopril-treated controls to a peak value of 39.50 +/- 2.55 microIU/ml, 1.5 h after a glucose challenge there was a decrease in the plasma insulin concentration in SNAP-treated dogs to a low value of 20.67 +/- 0.88 microIU/ml (P = 0.006). In contrast, there were no significant differences in plasma glucagon concentration in SNAP-treated dogs and captopril-treated dogs at any time points. Using the Griess reaction, we found that there was a 27-95% increase in plasma nitrate/nitrite concentration on administration of SNAP. The sustained hyperglycemic effect observed in SNAP-treated dogs was accompanied by a marginal decrease in the mean arterial blood pressure and a significant increase in heart rate (P < 0.05). We conclude that acute administration of SNAP in the oral glucose tolerance test releases NO that modulates the parameters of carbohydrate metabolism.     

Epigallocatechin gallate protects U937 cells against nitric oxide-induced cell cycle arrest and apoptosis

Ingesting phenolic phytochemicals in many plant products may promote health, but the effects of phenolic phytochemicals at the cellular level have not been fully examined. Thus, it was determined if the tea phenolic phytochemical, epigallocatechin gallate (EGCG), protects U937 human pro-monocytic cells against the nitrogen free radical, nitric oxide (*NO). Cells were incubated for 4-6 h with 500 microM S-nitrosoglutathione (GSNO), which generates *NO, but this did not induce single-strand breaks in DNA. Nevertheless, 82 +/- 4% of GSNO-treated cells, compared to only 39 +/- 1% of untreated cells, were arrested in the G(1)-phase of the cell cycle. However, dosing the GSNO-treated cells with 9, 14, or 18 microg/ml of EGCG resulted in only 74 +/- 8%, 66 +/- 1%, and 43 +/- 3% of the cells, respectively, in the G(1)-phase. Exposing cells to GSNO also resulted in the emergence of a sub-G(1) apoptotic cell population numbering 14 +/- 3%, but only 5 +/- 2%, 5 +/- 1%, and 2 +/- 0% upon dosing of the GSNO-treated cells with 9, 14, and 18 microg/ml of EGCG, respectively. Furthermore, exposing cells to GSNO resulted in greater cell surface binding of annexin V-FITC, but binding was 41-89% lower in GSNO-treated cells dosed with EGCG. Collectively, these data suggest that *NO or downstream products induced cell cycle arrest and apoptosis that was not due to single-strand breaks in DNA, and that EGCG scavenged cytotoxic *NO or downstream products, thus reducing the number of cells in a state of cell cycle arrest or apoptosis.     

Regulation of heart insulin receptor tyrosine kinase activity by magnesium and spermine

Insulin action and aspects of the insulin-signaling pathway have been studied in the heart although the direct regulation of the heart’s insulin receptor has not been explored. This study describes the first purification and characterization of the mammalian (rabbit, rat and bovine) heart insulin receptor. The rabbit heart IR showed maximum insulin binding of 18 μg/mg (~1 mole insulin/mole (α₂β₂) receptor) and a curvilinear Scatchard plot with a high affinity K_D for insulin binding of ~4 nM at optimal pH (7.8) and NaCl concentration (150 mM). The insulin receptor tyrosine kinase activity was stimulated by insulin, Mg²⁺ (half-maximum response at ~5.6–10.6 nM and ~8.5 mM, respectively) and by the physiological polyamines, spermine and spermidine. The stimulation by Mg²⁺ and the polyamines occurred with and without insulin. These characteristics of the heart insulin receptor provide a mechanism for regulating the activity of the receptor’s tyrosine kinase activity by the intracellular free Mg²⁺ concentration and the polyamines in the absence and presence of insulin.     

Nitric Oxide Modulates Muscarinic Acetylcholine Receptor Binding in the Cerebral Cortex of Gerbils

To determine whether nitric oxide (NO) acts as a modulator of muscarinic acetylcholine receptor (mACh-R) function, we performed a radioligand receptor assay using [³H]quinuclidinyl benzylate ([³H]QNB), the NO radical (NO·) donor 3-(2-Hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC7) and a gerbil brain cortical membrane preparation. NOC7 (at 10 M, 100 M or 1 mM concentrations) significantly reduced the [³H]QNB binding K_d values (from 0.196 ± 0.009 nM in the control, to 0.151 ± 0.013, 0.144 ± 0.012 and 0.153 ± 0.007 nM respectively). NOC7 did not alter the displacement curves of atropine or carbachol. Reduction of SH groups with dithiothreitol, in the presence of the NO donor, significantly increased [³H]QNB binding affinity whereas alkylation by N-ethylmaleimide markedly decreased it. The observed enhancing effect on mACh-R binding affinity for [³H]QNB, may reflect conformational changes in the receptors mediated by the NO generated, and these changes might be explained by NO reactions with such groups through conditions supporting redox reactions intrinsic to the NO molecule, similar to those occurring in redox regulatory sites reported for other neurotransmitter pathways in the CNS.     

Effect of Allopurinol on Hypoxia-Induced Modification of the NMDA Receptor in Newborn Piglets

The present study tests the hypothesis that pretreatment with allopurinol, a xanthine oxidase inhibitor, will prevent modification of the NMDA receptor during cerebral hypoxia in newborn piglets. Eighteen newborn piglets were studied. Six normoxic control animals were compared to six untreated hypoxic and six allopurinol (20 mg/kg i.v.) pretreated hypoxic piglets. Cerebral hypoxia was induced by lowering the FiO₂ to 0.05–0.07 for 1 hour and tissue hypoxia was confirmed biochemically by the measurement of ATP and phosphocreatine. Brain cell membrane Na⁺,K⁺-ATPase activity was determined to assess membrane function. Na⁺,K⁺-ATPase activity was decreased from control in both the untreated and treated hypoxic animals (46.0 ± 1.0 vs 37.9 ± 2.5 and 37.3 ± 1.4 mol Pi/mg protein/hr, respectively, p < 0.05). [³H]MK-801 binding was determined as an index of NMDA receptor modification. The receptor density (Bmax) in the untreated hypoxic group was decreased compared to normoxic control (1.09 ± 0.17 vs 0.68 ± 0.22 pmol/mg protein, p < 0.01). The dissociation constant (Kd) was also decreased in the untreated group (10.0 ± 2.0 vs 4.9 ± 1.4 nM, p < 0.01), indicating an increase in receptor affinity. However, in the allopurinol treated hypoxic group, the Bmax (1.27 ± 0.09 pmol/mg protein) was similar to normoxic control and the Kd (8.1 ± 1.2 nM, p < 0.05) was significantly higher than in the untreated hypoxic group. The data show that the administration of allopurinol prior to hypoxia prevents hypoxia-induced modification of the NMDA receptor-ion channel binding characteristics, despite neuronal membrane dysfunction. By preventing NMDA receptor-ion channel modification, allopurinol may produce a neuromodulatory effect during hypoxia and attenuate NMDA receptor mediated excitotoxicity.     

Exogenous nitric oxide inhibits IRS-1 expression in rat hepatocytes and skeletal myocytes

Summary Accumulative evidence has supported the role of nitric oxide (NO) in a variety of normal physiological functions as well as many pathological conditions. In this study, we examined the possible diabetogenicity of NO by measuring the expression of the insulin receptor substrate (IRS)-1 in rat hepatocytes and skeletal myocytes. IRS-1 is important in the insulin-mediated signal transduction pathway in both liver and skeletal muscle. Exogenous NO donated by S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione (GSNO) resulted in significant reduction in levels of IRS-1 in both cells, when compared to the insulin-stimulated control (p<0.001). Reversal to near normal levels was achieved using the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO). SNAP was the more potent drug, and the skeletal myocytes were the more sensitive cells to the inhibitory effects of NO released from the drugs. These results provide further evidence that exogenous NO is a potent modulator of insulin-mediated signal transduction and may play a significant role in the pathogenesis of type 2 diabetes mellitus.     

Detailed analysis of heterogeneity of [3H]naloxone binding sites in rat brain synaptosomes

The experiments reported in this paper address the question of heterogeneity of [³H]naloxone binding sites in rat brainstem synaptosomal preparations at 23°C in the presence of 100 mM sodium chloride. Kinetic analysis in the presence of 0.4, 4 and 10 nM [³H]naloxone gave pseudo-first order association rate of 0.9±0.04, 1.23±0.08 and 1.06±0.08 min^–1, respectively. The dissociation of a 1 nM [³H]naloxone receptor complex was biphasic with dissociation rate constants of 1.8 and 0.4 min^–1. On the other hand, dissociation of 10 nM [³H]naloxone was monophasic with ak _d of 1.1 min^–1. Two subpopulations of binding sites were also observed by steady state binding studies, with Kd values of 0.5 and 3.4 nM and a ratio of high to low affinity sites of 1:9. Heterogeneity of [³H]naloxone binding sites could be seen by displacement studies performed with opioid eptides and alkaloids. We suggest that our data best fits a model with two independent naloxone binding sites wherein either one or both undergo a multi-step interaction with ligand.     

Properties of the insulin receptor of rat pancreatic islet

Rat pancreatic islets have been shown to possess specific binding sites for ¹²⁵I-labeled insulin. Enzymatic and chemical modification of islets are used to reveal important structures and chemical groups for insulin binding. Pretreatment with trypsin, neuraminidase, 1-ethyl-3(3-dimethylamino)carbodiimide (a carboxyl reagent), tetranitromethane (a tyrosyl and thiol reagent), and 1,3-difluoro-4,6-dinitrobenze (modification of protein functional groups) decreased binding of insulin. This was due to the diminuation of the receptor number; in the case of trypsin-pretreatment also the receptor affinity was decreased. Inhibition of insulin binding was in each case associated with a decrease of the inhibitory effect of exogenous insulin on glucose-induced insulin secretion (not measured in the case of difluorodinitrobenzene and tetranitromethane). Phospholipase A₂ (cleavage of phospholipids) did not affect these parameters. 5,5′-dithiobis(2-nitrobenzoic acid) (Ellman's reagent) and possibly p-chloromercuribenzoate (both thiol reagents) increased the number of receptors and decreased receptor affinity, but did not influence the inhibitory effect of insulin on insulin release. It is concluded that protein functional groups, sialic acid, carboxyl and tyrosyl groups, but not phospholipids and probably not sylfhyryl groups are important for the interaction of insulin with insulin receptors of rat pancreatic islets.     

Enhancement of [m-methoxy3H]MDL100907 binding to 5HT2A receptors in cerebral cortex and brain stem of streptozotocin induced diabetic rats

5-Hydroxytryptamine_2A (5-HT_2A) receptor kinetics was studied in cerebral cortex and brain stem of streptozotocin (STZ) induced diabetic rats. Scatchard analysis with [³H] (±) 2,3dimethoxyphenyl-1-[2-(4-piperidine)-methanol] ([³H]MDL100907) in cerebral cortex showed no significant change in maximal binding (B_max) in diabetic rats compared to controls. Dissociation constant (K_d) of diabetic rats showed a significant decrease (p < 0.05) in cerebral cortex, which was reversed to normal by insulin treatment. Competition studies of [³H]MDL100907 binding in cerebral cortex with ketanserin showed the appearance of an additional low affinity site for 5-HT_2A receptors in diabetic state, which was reversed to control pattern by insulin treatment. In brain stem, scatchard analysis showed a significant increase (p < 0.05) in B_max accompanied by a significant increase (p < 0.05) in K_d. Competition analysis in brain stem also showed a shift in affinity towards a low affinity State for 5-HT_2A receptors. All these parameters were reversed to control level by insulin treatment. These results show that in cerebral cortex there is an increase in affinity of 5-HT_2A receptors without any change in its number and in the case of brain stem there is an increase in number of 5HT_2A receptors accompanied by a decrease in its affinity during diabetes. Thus, from the results we suggest that the increase in affinity of 5-HT_2A receptors in cerebral cortex and upregulation of 5-HT_2A receptors in brain stem may lead to altered neuronal function in diabetes.