Computer analysis reveals changes in renal Na+-glucose cotransporter in diabetic rats

1989 ◽  
Vol 257 (2) ◽  
pp. C385-C396 ◽  
Author(s):  
M. E. Blank ◽  
F. Bode ◽  
K. Baumann ◽  
D. F. Diedrich
Keyword(s):  
Rate Constants ◽  
Time Course ◽  
Operation Mode ◽  
High Capacity ◽  
Membrane Vesicles ◽  
Diabetic Rats ◽  
Hill Coefficient ◽  
Computer Assisted ◽  
Major Operation ◽  
Kinetic Plots

A novel, computer-assisted program was developed to analyze the time course of Na+-glucose cotransport by rat renal cortical brush-border membrane vesicles (BBMV). Transporter characteristics can be measured, which routine kinetic analyses fail to distinguish: cotransporter membrane density is derived from the picomoles of D-glucose bound per milligram of protein. Binding is stereospecific, blocked by phlorizin, and supported equally well by Na+ or K+ (but not Cs+). Quasi-first-order influx and efflux rate constants for the composite Na+-driven influx and the (presumed) Na+-independent efflux processes were highly dependent on glucose concentration. Either two Na+-glucose transporters exist in proximal tubules or a single mechanism abruptly changes rate when glucose falls to low levels. The major operation mode is slow, has a high capacity but low affinity, and may have a 2 Na+:2 glucose stoichiometry (Hill coefficient is unity). The minor system is a fast, smaller-capacity, higher-affinity operation with a 2 Na+:1 glucose stoichiometry that was not distinguishable when the same data were analyzed in conventional kinetic plots. Results with streptozocin-induced diabetic rats illustrate the method's utility. Low-glucose-affinity cotransporters were upregulated in hyperglycemic, but not in cachectic, ketoacidotic animals. Rate constants, especially for efflux, were decreased in diabetes.

scholarly journals Glutamine transport by vesicles isolated from tumour-cell mitochondrial inner membrane

1995 ◽  
Vol 308 (2) ◽  
pp. 629-633 ◽  
Author(s):  
M Molina ◽  
J A Segura ◽  
J C Aledo ◽  
M A Medina ◽  
I Núnez de Castro ◽  
...  
Keyword(s):  
Transport System ◽  
Time Course ◽  
Ph Dependence ◽  
High Capacity ◽  
Glutamine Metabolism ◽  
Hill Coefficient ◽  
Ph Optimum ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Glutamine Transport

Mitochondrial-inner-membrane vesicles, isolated from Ehrlich ascites carcinoma cells by titration with detergents, accumulated L-glutamine by a very efficient transport system. The vesicles lack any phosphate-activated glutaminase activity, allowing measurement of transport rates without interference by L-glutamine metabolism. The time course of the transport was linear for the first 60 s, reaching a steady state after 120 min. L-Glutamine transport showed co-operativity, with a Hill coefficient of 2.2; the kinetic parameters S0.5 and Vmax had values of 5 mM and 26 nmol/30 s per mg of protein respectively. The pH-dependence curve showed a bell shape, with a pH optimum about 8.0. The uptake of L-glutamine was not affected by the presence of a 50-fold molar excess of D-glutamine, L-cysteine, L-histidine, L-alanine, L-serine and L-leucine, whereas L-glutamate behaved as a poor inhibitor. The structural analogue L-glutamate gamma-hydroxamate (5mM) inhibited the net uptake by 68%; interestingly, other analogues (6-diazo-5-oxo-L-norleucine, acivicin and L-glutamate gamma-hydrazide) were ineffective. The impermeant thiol reagent p-chloromercuriphenylsulphonic acid (0.5mM) completely abolished the mitochondrial L-glutamine uptake; in contrast, other thiol reagents (mersalyl and N-ethylmaleimide) did not significantly affect the transport. These data confirm the existence of a specific transport system with high capacity for L-glutamine in the mitochondrial inner membrane, a step preceding the highly operative glutaminolysis in tumour cells.

Effect of Calcium ion on the interaction between Thrombin and Heparin. Thermal Denaturation

1977 ◽  
Vol 38 (03) ◽  
pp. 0677-0684 ◽  
Author(s):  
Raymund Machovich ◽  
Péter Arányi
Keyword(s):  
Calcium Ions ◽  
Rate Constants ◽  
Thermal Denaturation ◽  
Time Course ◽  
Complex Analysis ◽  
Heat Inactivation ◽  
Second Phase ◽  
Denaturation Kinetics ◽  
Enzyme Protection ◽  
Enzyme Denaturation

SummaryHeat inactivation of thrombin at 54° C followed first order kinetics with a rate constant of 1.0 min−1 approximately. Addition of heparin resulted in protection against thermal denaturation and, at the same time, rendered denaturation kinetics more complex. Analysis of the biphasic curve of heat inactivation in the presence of heparin revealed that the rate constants of the second phase changed systematically with heparin concentrations. Namely, at 4.5 × 10−6M, 9 × 10−6M, 1.8 × 10−5M and 3.6 × 10−5M heparin concentrations, the rate constants were 0.27 min−1, 0.17 min−1, 0.11 min−1 and 0.06 min−1, respectively.Sulfate as well as phosphate ions displayed also enzyme protection against heat inactivation, however, the same effect was obtained already at a heparin concentration, lower by three orders of magnitude.The kinetics of enzyme denaturation was not affected by calcium ions, whereas in the presence of heparin the inactivation rate of thrombin changed, i. e. calcium ions abolished the biphasic character of time course of thermal denaturation.Thus, the data suggest that calcium ions contribute to the effect of heparin on thrombin.

scholarly journals Regression of gestational diabetes induced cardiomegaly in offspring of diabetic rat

2009 ◽  
Vol 24 (4) ◽  
pp. 251-255 ◽  
Author(s):  
Honório Sampaio Menezes ◽  
Cláudio Galeano Zettler ◽  
Alice Calone ◽  
Jackson Borges Corrêa ◽  
Carla Bartuscheck ◽  
...  
Keyword(s):  
Body Weight ◽  
Wistar Rats ◽  
Weight Ratio ◽  
Diabetic Rats ◽  
Heart Weight ◽  
Diabetic Rat ◽  
Control Group ◽  
Computer Assisted ◽  
Significant Difference ◽  
Levene Test

PURPOSE: To compare body weight and length, heart weight and length, heart-to-body weight ratio, glycemia, and morphometric cellular data of offspring of diabetic rats (ODR) and of normal rats (control). METHODS: Diabetes was induced in 3 pregnant Wistar rats, bearing 30 rats, on the 11th day after conception by intraperitoneal injection of 50 mg/kg of streptozotocin. Six normal pregnant Wistar rats, bearing 50 rats, made up the control group. Morphometric data were obtained using a scale for the weight, length, heart and body measurements. Morphometric cellular data were obtained by a computer assisted method applied to the measurements of myocytes. Statistical analysis utilized Student's t-test, ANOVA and Levene test. RESULTS: Control offspring had greater mean body weight and length than offspring of diabetic rats (p < 0.001). Heart weight and length and heart-to-body ratios of newborn rats differed between groups at birth (p < 0.001), but showed no difference at 21 days. Mean nuclei area and perimetric value of the myocytes decrees throughout the first 21 days of life (p < 0.01) in the diabetic group. CONCLUSIONS: Heart hypertrophy on the offspring of diabetic rats at birth was demonstrated by the significant difference between the groups. After the eleventh day, no difference was found, which confirmed regression of cardiomegaly. The significant difference between the first and the 21th day of life, for nuclei area feature, demonstrate regression of cardiac hypertrophy in the offspring of diabetic rats.

Effect of diabetes and insulin treatment of diabetic rats on total RNA, poly(A)+ RNA, and mRNA in skeletal muscle

1991 ◽  
Vol 260 (3) ◽  
pp. C409-C416 ◽  
Author(s):  
J. D. Kent ◽  
S. R. Kimball ◽  
L. S. Jefferson
Keyword(s):  
Skeletal Muscle ◽  
Ribosomal Rna ◽  
Time Course ◽  
Diabetic Rats ◽  
In Vitro Translation ◽  
Specific Gene ◽  
Insulin Administration ◽  
Tissue Mass ◽  
Total Rna

We have assessed the time course of alterations in several biochemical parameters and expression of specific mRNAs in gastrocnemius muscle following both the induction of diabetes and the administration of insulin to diabetic rats. Muscle mass, total RNA, and total protein were reduced, whereas poly(A)+ RNA relative to total RNA was increased following the induction of diabetes. All the above parameters, with the exception of poly(A)+ RNA, were reciprocally and rapidly altered following administration of insulin to 3-day diabetic animals. These changes suggest that during the induction of diabetes 1) total cellular protein is reduced at a rate that is less than the reduction in gastrocnemius mass, whereas RNA is reduced at a rate 1.5 times the reduction in tissue mass, and 2) poly(A)+ RNA is elevated relative to total RNA. After insulin administration, there appears to be coordinate synthesis of both poly(A)+ RNA and ribosomal RNA, assuming 85% of total RNA is ribosomal. Therefore, we conclude that poly(A)+ RNA is more stable than ribosomal RNA during diabetes, whereas the amounts of poly(A)+ RNA and ribosomal RNA are increased at the same rates following insulin administration to diabetic animals. Analysis of expression of specific gene products over the same time course, as assessed by in vitro translation of total RNA followed by two-dimensional gel analysis, suggests that there are a few mRNAs that are very rapidly altered in response to insulin administration. The mRNAs that are altered demonstrate variable temporal patterns of either repression or full or transient expression. These rapid, but limited, alterations in gene expression may prove important in the development of the defects that occur in skeletal muscle in response to diabetes.

scholarly journals Mechanism of methaemoglobin reduction by human erythrocytes

1980 ◽  
Vol 188 (2) ◽  
pp. 535-540 ◽  
Author(s):  
A Tomoda ◽  
M Ida ◽  
A Tsuji ◽  
Y Yoneyama
Keyword(s):  
Methylene Blue ◽  
Isoelectric Focusing ◽  
Rate Constants ◽  
Time Course ◽  
Reaction Rate ◽  
Polyacrylamide Gel ◽  
Human Erythrocytes ◽  
Redox Potentials ◽  
Reaction Rate Constants ◽  
Beta 2

The time course of methaemoglobin reduction in human erythrocytes treated with nitrite was studied at pH 7.4, 37 degrees C, in the presence or absence of Methylene Blue, and the changes in methaemoglobin, intermediate haemoglobins and oxyhaemoglobin during the reaction were analysed by isoelectric-focusing on Ampholine/polyacrylamide-gel plates. In both cases, with or without the dye, the intermediate haemoglobins were found to be present at (alpha 3+beta 2+)2 and (alpha 2+beta 3+)2 valency hybrids from their characteristic position on electrophoresis, but amounts changed consecutively with time. The amount of (alpha 3+beta 2+)2 was always greater than that of the (alpha 2+beta 3+)2 valency hybrid. This result is explained by the differences in redox potentials between alpha- and beta-chains in methaemoglobin tetramer. It was concluded that methaemoglobin was reduced in human erythrocytes through these two different pats: methaemoglobin leads to k+3 (alpha 2+beta 3+)2 leads to k+3 oxyhaemoglobin. The reaction rate constants k'+1 (= k+1+k+3) and k'+2(=k+2+k+4) were estimated from the changes in each component methaemoglobin, intermediate haemoglobins [(alpha 3+beta 2+)2+(alpha 2+beta 3+)2] and oxyhaemoglobin.

scholarly journals Contribution of the intra- and intermolecular routes in autocatalytic zymogen activation: application to pepsinogen activation.

2006 ◽  
Vol 53 (2) ◽  
pp. 407-420 ◽  
Author(s):  
Ramón Varón ◽  
Matilde E Fuentes ◽  
Manuela García-Moreno ◽  
Francisco Garcìa-Sevilla ◽  
Enrique Arias ◽  
...  
Keyword(s):  
Kinetic Parameters ◽  
Rate Constants ◽  
Time Course ◽  
Initial Conditions ◽  
Reaction Scheme ◽  
Complete Analysis ◽  
Zymogen Activation ◽  
Relative Contribution ◽  
Starting Point ◽  
Definition Of

Taking as the starting point a recently suggested reaction scheme for zymogen activation involving intra- and intermolecular routes and the enzyme-zymogen complex, we carry out a complete analysis of the relative contribution of both routes in the process. This analysis suggests the definition of new dimensionless parameters allowing the elaboration, from the values of the rate constants and initial conditions, of the time course of the contribution of the two routes. The procedure mentioned above related to a concrete reaction scheme is extrapolated to any other model of autocatalytic zymogen activation involving intra- and intermolecular routes. Finally, we discuss the contribution of both of the activating routes in pepsinogen activation into pepsin using the values of the kinetic parameters given in the literature.

Sodium and calcium share the electrogenic 2 Na(+)-1 H+ antiporter in crustacean antennal glands

1990 ◽  
Vol 259 (5) ◽  
pp. F758-F767
Author(s):  
G. A. Ahearn ◽  
P. Franco
Keyword(s):  
Driving Forces ◽  
Electrical Potential ◽  
Membrane Vesicles ◽  
Homarus Americanus ◽  
Competitive Inhibitor ◽  
Hill Coefficient ◽  
Affinity Constant ◽  
Electrical Potential Difference ◽  
Static Head ◽  
Transmembrane Electrical Potential

Na uptake by short-circuited epithelial brush-border membrane vesicles of Atlantic lobster (Homarus americanus) antennal gland labyrinth was Cl independent, amiloride sensitive, and stimulated by a transmembrane H+ gradient [( H]i greater than [H]o; i is internal, o is external). Na influx (2.5-s uptake) was a sigmoidal function of [Na]o (25-400 mM) when pHi = 5.0 and pHo = 8.0 and followed the Hill equation for binding cooperatively [apparent maximal influx (Jmax) = 271 nmol.mg protein-1.s-1, apparent affinity constant for Na (KNa) = 310 mM Na, and Hill coefficient (n) = 2.41]. Amiloride acted as a competitive inhibitor of Na binding to two external sites with markedly dissimilar apparent amiloride affinities (Ki1 = 14 microM; Ki2 = 1,340 mM). Electrogenic Na-H antiport by these vesicles was demonstrated by equilibrium-shift experiments in which an imposed transmembrane electrical potential difference was the only driving force for exchange. A transport stoichiometry of 2 Na to 1 H was demonstrated with the static-head technique in which a balance of driving forces was attained with 10:1 Na gradient and 100:1 H gradient. External Ca, like amiloride, was a strong competitive inhibitor of Na-H exchange, acting at two sites on the outer vesicular face with markedly different apparent divalent cation affinities (Ki1 = 20 microM; Ki2 = 500 microM). Ca-H exchange by electrogenic Na-H antiporter was demonstrated in complete absence of Na by use of an outward H gradient in presence and absence of amiloride. Both external amiloride (Ki1 = 70 microM; Ki2 = 500 microM) and Na (Ki1 = 12 mM; Ki2 = 380 mM) were competitive inhibitors of Ca-H exchange. These results suggest that the electrogenic 2 Na-1 H exchanger characterized for this crustacean epithelium may also have a role in organismic Ca balance.

Time course of changes in albumin synthesis and mRNA in diabetic and insulin-treated diabetic rats

1985 ◽  
Vol 248 (6) ◽  
pp. E656-E663 ◽  
Author(s):  
D. E. Peavy ◽  
J. M. Taylor ◽  
L. S. Jefferson
Keyword(s):  
Total Protein ◽  
Time Course ◽  
Insulin Treatment ◽  
Relative Rate ◽  
Cdna Probe ◽  
Diabetic Rats ◽  
Rate Of Change ◽  
Albumin Synthesis ◽  
Albumin Mrna

Albumin synthesis in rat liver in vivo decreased from 12.7 to 2.2% of total protein synthesis during the first 3 days after the induction of diabetes and then remained relatively constant at this depressed rate for another 3 days. Insulin treatment begun on the 3rd day after the induction of diabetes restored albumin synthesis to control values within 3 days. Hybridization of total polyadenylate-containing RNA with a specific albumin cDNA probe revealed a close correspondence between the relative abundance of albumin mRNA and the relative rate of albumin synthesis after induction of diabetes and in response to insulin treatment. The apparent half-life of albumin mRNA, based on the rate of change of the message from one steady-state level to another, was approximately 22 h in both diabetic and insulin-treated diabetic rats. Diabetes of 3-day duration had no effect on the average sizes of total and albumin-synthesizing polysomes or on the ribosomal half-transit time for total protein and albumin. However, the number of albumin-synthesizing polysomes decreased as a result of diabetes to approximately one-third the number found in control livers. Taken together the results indicate that albumin synthesis was regulated by the availability of albumin mRNA and not by alterations in degradation, sequestration, or translation of message.

Metabolic and hormonal changes following endotoxin administration to diabetic rats

1982 ◽  
Vol 243 (1) ◽  
pp. R77-R81
Author(s):  
D. L. Kelleher ◽  
B. C. Fong ◽  
G. J. Bagby ◽  
J. J. Spitzer
Keyword(s):  
Plasma Glucose ◽  
Time Course ◽  
Glycogen Content ◽  
Metabolic Response ◽  
Insulin Response ◽  
Liver Glycogen ◽  
Diabetic Rats ◽  
Hormonal Changes ◽  
Glycogen Depletion ◽  
Endotoxin Administration

The aim of these investigations was to study the time course and cause of the altered metabolic response of diabetic rats to endotoxin administration. Escherichia coli endotoxin was administered to streptozotocin-diabetic and control normoglycemic rats. At 1, 2, 5, 8, and 24 h following endotoxin, animals were decapitated. Plasma samples were analyzed for glucose, lactate, insulin, glucagon, and corticosteroids. In addition, tissue glycogen content of liver and skeletal muscle was determined. Endotoxin caused an elevation of plasma glucose in both diabetic and normoglycemic rats by 1 h postinjection. The elevation was prolonged in diabetic rats for 8 h but lasted only 2 h in nondiabetic rats. Both endotoxin-treated groups demonstrated hyperlactacidemia following endotoxin. Endotoxin led to liver glycogen depletion in both diabetic and normoglycemic rats, whereas muscle glycogen content was only slightly affected. Plasma glucagon and corticosteroids rose immediately and remained elevated in both endotoxin-treated groups. A significant insulin response to rising plasma glucose was observed in nondiabetic but not in diabetic rats following endotoxin. These results suggest that the exaggerated and prolonged hyperglycemia observed in diabetic endotoxin-treated rats is due to hypersecretion of glucose-mobilizing hormones and elevated gluconeogenesis, unmatched by an adequate secretion of insulin to promote glucose uptake and utilization.

Time course and vectorial nature of albumin metabolism in isolated perfused rabbit PCT

1988 ◽  
Vol 255 (3) ◽  
pp. F520-F528 ◽  
Author(s):  
C. H. Park
Keyword(s):  
Time Course ◽  
Hydrolysis Product ◽  
High Capacity ◽  
Intact Protein ◽  
Renal Metabolism ◽  
First Order ◽  
First Order Kinetics ◽  
Hydrolysis Products ◽  
Renal Tubular ◽  
Hydrolysis Of

The time course and vectorial nature of renal metabolism of albumin (Alb) were studied. The tubular absorption, accumulation, and hydrolysis of Alb and the release of the hydrolysis products were determined in the isolated rabbit proximal convoluted tubule (PCT) perfused with tritiated Alb ([3H3C]Alb) at 36.4 micrograms/ml. The Alb absorption across the apical membrane was constant (99.9 +/- 4.9 x 10(-3) ng.min-1.mm-1). In contrast, the accumulation and hydrolysis of Alb in the cells increased nonlinearly with time. The bulk of the tritium that accumulated in the cells was associated with intact [3H3C]Alb. Only the final hydrolysis products were released from the cells and these first appeared in the peritubular bath 6–7 min after the start of perfusion of the tubule with [3H3C]Alb. The hydrolysis product was not detectable in the tubule lumen. The proteolytic activity correlated linearly with the protein load to the cells, characteristic of first-order kinetics and a high-capacity system. The results suggest that the renal tubular handling of proteins proceeds from the apical to the basolateral aspect of the cell. The transcellular processing of Alb is rapid and can occur in 6–7 min. The accumulation of intact protein in the cell and the first-order kinetics of hydrolysis of the absorbed protein suggest that the rate-limiting step in proximal tubular handling of proteins may include the initial hydrolysis of protein or reside in steps that precede the hydrolysis.

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