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D P Udrisar

Publications and source records attributed to D P Udrisar.

11 recordsLinked to original sources
PubMed1996

Propranolol stimulates testicular interstitial fluid formation and testosterone secretion in rats.

We investigated the effect of intratesticularly injected propranolol on testicular interstitial fluid (TIF) formation and on testosterone levels in the TIF of intact adult male Wistar rats (4-9 rats per group). dl-propranolol at doses of 0.6, 1.2, or 6.0 mg/kg was injected into the left (L) testis whereas the right (R) testis (control testis) received vehicle. dl-propranolol (6.0 mg/kg) caused a significant increase in both TIF volume (329%) and TIF levels of testosterone (257%) in the L testis but not in the R (control) testis 3 h post-injection. In rats treated simultaneously with human chorionic gonadotropin (hCG, 5 IU/rat, sc) the same dose or propranolol (6.0 mg/kg) significantly increased the stimulatory effect of hCG on testosterone secretion by 1.8-fold, but hCG did not modify the stimulatory effect of propranolol on TIF volume. These results demonstrate a direct stimulatory effect of propranolol on TIF volume and testosterone secretion, both under basal and hCG-stimulated conditions.

Animals↗
PubMed1994

Inhibitory action of in vitro ethanol and acetaldehyde exposure on LHRH-and phorbol ester-stimulated testosterone secretion by rat testicular interstitial cells.

Ethanol and acetaldehyde have been shown to inhibit testicular steroidogenesis. However the mechanism(s) of signal transduction involved in their action is still unclear. We examined the possible involvement of phospholipid-sensitive, calcium-dependent protein kinase (protein Kinase C, PK-C) in the intracellular mechanism of action of ethanol and acetaldehyde by stimulating testosterone production in rat testicular interstitial cells with LHRH and the phorbol ester PDBu, both of which activate PK-C at receptor (LHRH) and post-receptor (PDBu) sites. Ethanol (2000 mg %) inhibited 10(-7) M LHRH and 200 nM PDBu-stimulated testosterone production by 81 +/- 4.7% and 60 +/- 20.4%, respectively. Acetaldehyde (20 mg %) reduced the amount of testosterone produced by 10(-7) M LHRH and 200 nM PDBu by 59.4 +/- 1.2% and 52.5 +/- 5.4% respectively. Basal testosterone levels were unaffected by ethanol and reduced by acetaldehyde. However, the functional test of cell viability by preincubating cells with these doses of ethanol and acetaldehyde did not decrease their ability to respond appropriately to subsequent stimulation with LHRH, demonstrating that cell viability was unaffected by incubation with these drugs. The data presented here suggest that direct ethanol and acetaldehyde exposure results in a reduced ability of the testicular interstitial cells to respond to stimulation of PK-C pathway.

Acetaldehyde↗
PubMed1992

Fluoride and phosphatidylserine induced inhibition of cytosolic insulin-degrading activity.

Cytosol (C) (100,000 x g/60 min, supernatant) from liver, brain and testis (Wistar male rats) are shown to contain insulin degrading activity (C-IDA). The regulation of C-IDA in these fractions by ligands that activate G protein and PKC were examined C-IDA from liver, brain and testis was inhibited 76%; 64% and 50% by 50 mM F- respectively. Chromatography of C fraction from liver on Sephadex G-50 in presence of 1 M (NH4)2SO4 and 20% (v/v) glycerol (experimental condition to remove guanine nucleotides from G proteins) decreased in about 3-fold aluminum fluoride effect on C-IDA. Mg++ (from 5mM to 10 mM) enhanced fluoride effects by inhibiting fully C-IDA. Phosphatidylserine in presence of ATP completely inhibited C-IDA; this inhibition was 31.3% mediated by a phosphorylation reaction. It is concluded that cytosol from different tissues contain proteins capable to associate ligands as aluminum fluoride and PS to regulate C-IDA. It is proposed a mechanism of protein-protein interaction to modulate C-IDA.

Adenosine Triphosphate↗
PubMed1984

Liver cells binding with high insulin doses at 37 C.

Insulin binding and receptor mediated insulin degradation were studied in isolated rat hepatocytes under physiological conditions (37 C, 100% oxygen, Krebs improved Ringer III with glutamate, pyruvate and fumarate, 150 mg% glucose, 1% bovine albumin). 10(6) rat hepatocytes/tube were incubated with various doses of insulin. Steady state binding with low insulin doses (0.05, 0.5 and 66 ng/tube) was reached in 15 minutes, that state being kept for the rest of the experimental time (75 min). Receptor mediated degradation (Kap) at 15 minutes was 0.0479 min-1, including doses of 5 000 and 50 000 ng/tube. Direct correlation was found between degradation and low doses of insulin, being the slope value equal to Kap. Intracellular accumulation of insulin was found at pharmacological concentrations of insulin (5 000 and 50 000 ng/tube) from the first 15 minutes. That accumulation was dose and time dependent. At 75 minutes, with a 0.2 microM insulin concentration, at least 53% of insulin was estimated as insulin accumulated in the cell, since it was not filtrable with acid medium on Sephadex G 50 superfine. When Triton or dodecyl sulphate were used to solubilize the cells, insulin recovery was complete after binding. Intracellular accumulation, however, was not demonstrated at the first two minutes. Binding studies with 16.67 microM insulin in the presence of degradation inhibitors, such as 2 mM N-ethylmaleimide and 5 mM tetracaine hydrochloride, demonstrated that intracellular accumulation of the hormone occurs when degradation is blocked. On the contrary, after trypsin digestion of receptors, degradation was not observed, while increases in binding were abolished, resembling non-specific binding. Under the experimental conditions reported here, neither intracellular accumulation of insulin nor extracellular release of insulin degradation products can be demonstrated at 2 minutes; insulin accumulation is dose dependent, and it is suggested by the fact that the velocity of insulin internalization exceeds its velocity of degradation.

Animals↗
PubMed1984

Insulin processing. Its correlation with glucose conversion to CO2.

Insulin-receptor binding, insulin degradation and biologic response (14C-glucose conversion into 14CO2) were studied in adipocytes of control (CG), fasted (FG-88 hr) and hyperinsulinic rats (HG-exogenous hyperinsulinism). The number of cells normalized to 3.5 X 10(5) cells/tube in all three groups. Insulin binding and degradation were studied at 5, 15, 30, 60 and 120 minutes of incubation with 3.5 X 10(-11) M, 6.66 X 10(-11) M, 1.0 X 10(-9) M, 6.66 X 10(-9) and 6.66 X 10(-6) M insulin. The net increments of 14CO2 taken into account (delta U-14C-glucose converted into 14CO2) ranged from the basal value to 10(6) microU in each case (30, 60 and 120 minutes). Quantitative analysis of results was performed with the Terris and Steiner degradation equation (formula; see text) (IR). Differences in insulin binding, comparing the three groups, lacked statistical significance, though FG data were systematically plotted above those of CG, occurring the opposite with HG. Degradation studies showed HG to have values statistically higher than the controls, while FG values were lower. HG also showed higher amounts of 14CO2, with basal levels more elevated than CG, while FG showed the inverse behavior. 14CO2 increased in the three groups along the 120-minutes incubation period (30, 60 and 120 minutes). Receptor-mediated degradation at 30 minutes, when binding is in steady state, showed a Kap value very close to that found by linear regression for the 2 and 10 microU doses (Kap min-1 CG: 0.1654, FG: 0.0824, HG: 0.5045; slope values for the 2 and 10 microU doses CG: 0.2181, FG: 0.0824, HG: 0.3718). The degradation velocity, considered as function of IR, was constant in each group at 30, 60 and 120 minutes. Since Kap values in the FG and HG indicate differences in their degradation velocities, this constant can be considered as indicative of the metabolic situations under study. At the same time, the biologic response (14C-glucose conversion into 14CO2) depends as well on the metabolic conditions. Glucose consumption and Kap value were then compared. All the groups showed linear correlation between the binding dependent velocity of degradation (Kap) and the net conversion of U-14C-glucose into 14CO2 at 30, 60 and 120 minutes, with ordinate close to zero (30 min: 0.1539; 60 min: -0.3812; 120 min: 0.1311). The slope increased along the incubation period, indicating that 14CO2 accumulation is time dependent.(ABSTRACT TRUNCATED AT 400 WORDS)

Adipose Tissue↗
PubMed1981

Preparation of biologically active mono-125I-insulin of high specific activity.

Pork insulin was labeled by the chloramine T technique (phosphate buffer 0.25 M; pH 7.5; EDTA 0.001 M; chloramine T: 0.2625 mg/ml; sodium metabisulfite 2.4 mg/ml) in a reaction volume of 50 microliters, adding chloramine T every 30 seconds twice (2.1 micrograms in 1 minute) and halting the reaction with 5 microliters metabisulfite. Three fractions were separated in preparative starch gel: F1 (mono-125I-insulin contaminated with cold insulin), F2 (mono-125I-insulin free from cold insulin), and F3 (di-125I-insulin). Insulins with low and high specific activity (iodine/insulin ratios 0.1/1 and 1/1 respectively) were prepared for study purposes, and quality was assessed by means of dose-response curves with antibodies and with liver cells. Specific activity of F2 as obtained from dose-response curves utilizing Scatchard's plot was 323 and 382 mCi/mg. Specific activity of F1 varied according to the extent of contamination with cold insulin. A reduction in the F2 B/F ratio was observed upon iodination with iodine/insulin ratios of 1/1 or in the neighborhood. The mass and immunoreactivity of F3, as well as its B/F ratios were constant, regardless of specific activity. The behavior with antibodies was ratified upon observations on uptake by liver cells and glucose consumption by isolated fat cells. In conclusion, F2 labeled with 0.1/1 iodine/insulin ratios was separated from cold insulin in preparative starch gel, thus increasing its specific activity (360 mCi/mg approximately) without alteration of its immunologic or biologic properties.

Animals↗
PubMed1981

Increase of ionic strength for charcoal separation of B/F fractions in radioimmunoassays.

The increase in protein adsorption by charcoal as ionic strength increases (salting-out adsorption), was used to separate the bound and free fractions of glucagon, insulin, hGH, hLH and hPRL in the radioimmunoassay. The hormones were labelled with 125I and to express the immunocomplex, gamma-globulin was labelled with 125I. The charcoal used to produce the separation was suspended in magnesium sulfate 3 M (charcoal-SO4Mg). The optimum amount of charcoal and the final concentration of magnesium sulfate determined for each hormone were: glucagon (charcoal 5 mg/tube, 0.125 M); insulin (charcoal 5 mg/tube, 0.131 M); hGH (charcoal 40 mg/tube, 0. 447 M); hLH (charcoal 40 mg/tube, 0.447 M) and hPRL (charcoal 60 mg/tube, 0.321 M). The serum concentration was 1/20 for all hormones, excepting glucagon, where 1/10 was used. The stability of the immunocomplex was studied and it was shown that, under suitable conditions, increased ionic strength does not cause the dissociation of the bound fraction.

Adsorption↗
PubMed1981

Insulin binding in mouse liver cells isolated with chelating agents.

Mouse liver cells were isolated with Ca2+ and K+ chelating agents. Cell concentrations in all experiments ranged from 2.5 X 10(5) to 1.44 X 10(6) cells/tube. The kinetics of insulin-receptor binding was studied at 2 C and 20 C. Binding of 1.67 X 10(-11) M 125I-insulin reached equilibrium at 2 C at 180 min; Ka at 50% binding was 0.736 X 10(7) M-1 sec-1. At 20 C equilibrium occurred at 30 min; Ka at 50% binding was 7.519 X 10(7) M-1 sec-1. Non-specific binding was measured by adding 16.6 microM native insulin. Kinetics studies of association point to a pure bimolecular reaction since the constant remains unaltered at different times. In studies of bound complex dissociation, insulin release from the receptor involves first order kinetics, 50% of the bound insulin becoming released during the experimental period. Dissociation was studied at 20 C only, either by dilution or addition of 16.6 microM native insulin. Both methods yielded the same result, showing the dissociation kinetics to be a first order reaction with a half-life of 101 min and Kd: 2.5 X 10(-4) sec-1. Competitive inhibition of native insulin (1.67 X 10(-10), 3.33 X 10(-10), 1.67 X 10(-9), 3.33 X 10(-9), 1.67 X 10(-8), 3.33 X 10(-8), 1.67 X 10(-7), 3.33 X 10(-7) M) against 1.67 X 10(-11) M 125I-insulin was studied in equilibrium. Heterogeneity among active binding sites was found: one population of high affinity and low capacity (2 C: K = 4.64 X 10(7) L/M, Ro = 213 X 10(-11) M; 20 C: K = 2.90 X 10(8) L/M Ro = 28.5 X 10(-11) M) and one of low affinity and high capacity (2 C: K = 6.81 X 10(7) L/M Ro: 836 X 10(-11) M; 20 C: K = 2.63 X 10(6) L/M, Ro: 1080 X 10(-11) M). The results show the use of chelating agents in the separation of liver cells to be of value in physicochemical studies of insulin-receptor interaction.

Animals↗
PubMed1981

Isolation of liver cells with Ca2+ and K+ chelating agents. Biochemistry and cell morphology.

Cell morphology, glutamic pyruvic (GTP) and glutamic oxalacetic transaminases (GOT) concentrations, and the ability to produce glucose or urea from different substrates (pyruvate, alanine, fructose, lactate and glutamine) were studied in isolated mouse and rat liver cells in the presence of Ca2+ and K+ chelating agents (0.1 M sodium perchlorate and 0.027 M sodium citrate with 1 mg/ml bovine albumin; ionic strength: 0.198, pH: 7.4). The chelating agent is perfused through the portal vein of an in situ liver, at low pressure (8 ml/min) at 20 C for 15 min. Cell dispersion is obtained by cutting liver lobes and "massaging" the tissue with a plastic spatula. Wash and cell concentration may be obtained by sedimentation or centrifugation in Krebs III, glucose 150 mg %, improved with 0.16 M pyruvate, 0.1 M fumarate and 0.16 M glutamate. This procedure furnished 53.06 +/- 3.33 X 10(6) cells, which was highly significant (p less than 0.001) with respect to saline controls: 6.11 +/- 1.91 X 10(6). After staining with Papanicolaou, hematoxylin-eosin, and PAS, the cellular material obtained was classified optically into: normal isolated parenchymal liver cells, hepatocyte clumps, "burst" cells, normal blood or reticuloendothelial cells, cellular debris and non-cellular material. Cell morphology showed that a constant perfusion (8 ml/min) with a minimal mechanical treatment, 82.5% of the liver cells appears normal. Biochemical study showed that transaminases are indeed lost, but this loss is below the amount capable of effecting metabolic blockade (3/4 of transaminases remain in liver cells; GOT in cells: 692 +/- 218; GPT in cells. 264 +/- 94; GOT in supernatant: 152 +/- 29; GPT in supernatant: 79 +/- 12 mUI/10(6) cells, after recovering 60 min at 37 C) (means +/- SEM). Conversion of substrates (sodium pyruvate 10 mM, 20 mM D-L alanine, 10 mM fructose and 20 mM D-L sodium lactate) into glucose was statistically significant with respect to the baseline when the liver cells were isolated and recovered (rat liver cells, basal: 25.37 +/- 3.73; pyruvate: 54.04 +/- 7.98; DL-alanine: 62 +/- 10.07; fructose: 264.67 +/- 20.51; DL-lactate: 78.05 +/- 17.99 mmoles/10(6) cels, means +/- SEM). Urea production from 5 mM DL-glutamine was statistically highly significant to the basal with rat liver cell isolated and recovered (basal: 160.60 +/- 3.76; DL-glutamine: 608.47 +/- 16.15 mmoles/10(6) cells; means +/- SEM). The results obtained suggest that liver cells isolated with Ca2+ and K+ chelating agents used as described above are of value for biochemical studies.

Alanine Transaminase↗
PubMedDate not supplied

The influence of pentoxyfylline [1-(5-oxohexyl-) 3,7-dimethylxanthine] (BL 191) on the insulin secretion induced by glibenclamide and by arginine/glucose in the perfused pancreas.

Pharmacodynamic characteristics of pentoxyfylline (BL 191) related to insulin secretion by the isolated perfused rat pancreas are studied. The results obtained show that: 1) BL 191 (5 mM) is capable of stimulating insulin secretion, even in the presence of another stimulator; 2) BL 191 increases both phases of the secretion produced by constant arginine 20 mM/glucose 5 mM perfusion; 3) BL 191 significantly increases and turns biphasic the monophasic insulin secretion pattern produced by 1 microgram/ml glibenclamide; 4) the effects mentioned in points 2) and 3) are inhibited if the phosphodiesterase activator imidazole (300 mg/100 ml) is present in the perfusion medium; 5) the phosphodiesterase inhibitor theophylline has the same effects as BL 191, except for its inability to stimulate insulin release in the absence of another stimulator; 6) somatostatin (100 ng/ml) significantly inhibits insulin secretion produced by arginine/glucose or glibenclamide, as well as by arginine, glucose plus theophylline or BL 191, and by glibenclamide plus theophylline or BL 191, in both cases the inhibitory effect of somatostatin is reduced by the presence of BL 191 or theophylline.

Animals↗