Cross-linking of proteins by Maillard processes: characterization and detection of lysine-arginine cross-links derived from glyoxal and methylglyoxal

Alpha-dicarbonyl compounds, such as glyoxal and methylglyoxal, are crucial intermediates in the browning and cross-linking of proteins by reducing sugars in the course of the Maillard reaction. The cross-linking units 2-ammonio-6-([2-[(4-ammonio-5-oxido-5-oxopentyl)amino]-4,5-dihydro - 1H-imidazol-5-ylidene]amino)hexanoate (9) and 2-ammonio-6-([2-[(4-ammonio-5-oxido-5-oxopentyl) amino]-4-methyl-4,5-dihydro-1H-imidazol-5-ylidene]amino)hexanoate (10), designated as GODIC and MODIC, are identified and quantified from glyoxal/methylglyoxal-bovine serum albumin (BSA) incubations. Independent syntheses and unequivocal structural characterization are given for 9 and 10. A protocol was established for their determination by liquid chromatography-mass spectrometry (LC-MS) with electrospray ionization (ESI). BSA and the respective alpha-dicarbonyl compound were incubated at 37 degrees C, pH 7.4 for 1 week, and the time-dependent formation of 9 and 10 was observed. The maximum value obtained from a solution containing 50 g/L BSA and 2 mM glyoxal or methylglyoxal after a 7-day incubation period corresponds to an arginine derivatization quota of 13.0 +/- 0.32 mmol 9/mol Arg or 3.0 +/- 0.12 mmol 10/mol Arg. The cross-links 9 and 10 were also detected in a D-glucose-BSA incubation. From these results, it seems justified to assign an important role to 9 and 10 in the cross-linking of proteins in vivo as well as in foodstuffs. In an additional model study, formation of 9 and 10 was compared to that of the imidazolium cross-links GOLD 3 and MOLD 4.     

In vivo covalent binding of retronecine-labelled [3H]seneciphylline and [3H]senecionine to DNA of rat liver, lung and kidney

Retronecine-labelled [3H]seneciphylline ([3H]SPH) and [3H]senecionine ([3H]SON) of high specific radioactivity (22 and 49 mCi/mmol, respectively) were prepared biosynthetically with seedlings of Senecio vulgaris L. using [2,3-3H]putrescine as precursor. [2,3-3H]Putrescine was synthesized by Gabriel synthesis of 1,4-diamino-2-butene from 1,4-dibromo-2-butene and catalytic hydrogenation of the product with tritium gas. Rats of both sexes were treated with the labelled pyrrolizidine alkaloids (PAs) (75-215 microCi SPH or 40-485 microCi SON/kg body wt.) and killed after 6 h or 4-5 days. SON-treated females excreted 83.4 +/- 0.2% of applied radioactivity in faeces and urine within 4 days whereas equally treated males excreted 90.9 +/- 3.2% in the same time. Excretion of 3H-activity from SPH-treated females was completed within 5 days (104.7 +/- 6.4%). Corresponding with these results, tissue levels were highest in SON-treated females. DNA and proteins were isolated from liver, lungs and kidneys and covalent binding of the alkaloids to DNA was determined. A Covalent Binding Index (CBI, mumol alkaloid bound per mol nucleotides/mmol alkaloid administered per kg body wt.) of 210 +/- 12 was found for the liver from SON-treated females whereas binding to liver DNA of males was lower by a factor of 4. The DNA damage determined six hours after treatment persisted during the following 4 days. Administration of [3H]SPH to female and male rats resulted in a CBI of 69 +/- 7 and 73/92, respectively, for the liver DNA. Furthermore we found binding of both alkaloids to DNA of lungs and kidneys in male and female rats. The in vivo formation of [3H]SON derived DNA adducts could be proved by HPLC analysis of hydrolyzed DNA.     

The contribution of polar group burial to protein stability is strongly context-dependent

We previously suggested that proteins gain more stability from the burial and hydrogen bonding of polar groups than from the burial of nonpolar groups (Pace, C. N. (2001) Biochemistry 40, 310-313). To study this further, we prepared eight Thr-to-Val mutants of RNase Sa, four in which the Thr side chain is hydrogen-bonded and four in which it is not. We measured the stability of these mutants by analyzing their thermal denaturation curves. The four hydrogen-bonded Thr side chains contribute 1.3 +/- 0.9 kcal/mol to the stability; those that are not still contribute 0.4 +/- 0.9 kcal/mol to the stability. For 40 Thr-to-Val mutants of 11 proteins, the average decrease in stability is 1.0 +/- 1.0 kcal/mol when the Thr side chain is hydrogen-bonded and 0.0 +/- 0.5 kcal/mol when it is not. This is clear evidence that hydrogen bonds contribute favorably to protein stability. In addition, we prepared four Val-to-Thr mutants of RNase Sa, measured their stability, and determined their crystal structures. In all cases, the mutants are less stable than the wild-type protein, with the decreases in stability ranging from 0.5 to 4.4 kcal/mol. For 41 Val-to-Thr mutants of 11 proteins, the average decrease in stability is 1.8 +/- 1.3 kcal/mol and is unfavorable for 40 of 41 mutants. This shows that placing an [bond]OH group at a site designed for a [bond]CH3 group is very unfavorable. So, [bond]OH groups can contribute favorably to protein stability, even if they are not hydrogen-bonded, if the site was selected for an [bond]OH group, but they will make an unfavorable contribution to stability, even if they are hydrogen-bonded, when they are placed at a site selected for a [bond]CH3 group. The contribution that polar groups make to protein stability depends strongly on their environment.     

Formation pathways for lysine-arginine cross-links derived from hexoses and pentoses by Maillard processes: unraveling the structure of a pentosidine precursor

Covalently cross-linked proteins are among the major modifications caused by the advanced Maillard reaction. So far, the chemical nature of these aggregates and their formation pathways are largely unknown. Synthesis and unequivocal structural characterization are reported for the lysine-arginine cross-links N(6)-(2-([(4S)-4-ammonio-5-oxido-5-oxopentyl]amino)-5-[(2S,3R)-2,3,4- trihydroxybutyl]-3,5-dihydro-4H-imidazol-4-ylidene)-l-lysinate (DOGDIC 12), N(6)-(2-([(4S)-4-ammonio-5-oxido-5-oxopentyl]amino)-5-[(2S)-2,3-dihydroxypropyl]-3,5-dihydro-4H-imidazol-4-ylidene)-l-lysinate (DOPDIC 13), and 6-((6S)-2-([(4S)-4-ammonio-5-oxido-5-oxopentyl] amino)-6-hydroxy-5,6,7,7a-tetrahydro-4H-imidazo[4,5-b] pyridin-4-yl)-l-norleucinate (pentosinane 10). For these compounds, as well as for glucosepane 9 and pentosidine 11, the formation pathways could be established by starting from native carbohydrates, Amadori products, and 3-deoxyosones, respectively. Pentosinane 10 was unequivocally proven to be an important precursor of pentosidine 11, which is a well established fluorescent indicator for advanced glycation processes in vivo. The Amadori products are shown to be the pivots in the formation of the various cross-links 9-13. The bicyclic structures 9-11 are directly derived from aminoketoses, whereas 12 and 13 stem from reaction with the 3-deoxyosones. All products 9-13 were identified and quantified from incubations of bovine serum albumin with the respective 3-deoxyosone or carbohydrate. From these results it seems fully justified to expect both glucosepane 9 and DOGDIC 12 to constitute important in vivo cross-links.     

Anomeric specificity of D-glucose production by rat hepatocytes

The anomeric specificity of D-glucose metabolism in intact hepatocytes remains a matter of debate. This issue was further investigated in the present study, which is based on the quantification of the alpha- and beta-anomers of the 13C-enriched isotopomers of D-glucose generated by rat liver cells exposed to either D-[1-13C] fructose or D-[2-13C] fructose in the presence of D2O. The D-[1-13C]glucose/D-[6-13C]glucose paired ratios found in the cells exposed to D-[1-13C] fructose and the D-[2-13C]glucose/D-[5-13C]glucose paired ratios found in the cells exposed to D-[2-13C] fructose yielded a paired beta/alpha ratio averaging (mean +/- S.E.M.) 79.3 +/- 6.1%. In the case of the isotopomers of D-glucose formed by gluconeogenesis, the D-[2-13C]glucose/D-[5-13C]glucose and D-[3-13C]glucose/D-[4-13C]glucose paired ratios found in cells exposed to D-[1-13C] fructose, as well as the D-[1-13C]glucose/D-[6-13C]glucose and D-[3-13C]glucose/D-[4-13C]glucose paired ratios found in cells exposed to D-[2-13C]fructose, yielded an alpha/beta paired ratio averaging 75.0 +/- 5.8%. Last, in the cells exposed to D-[2-13C]fructose, the beta/alpha ratio for the C2-deuterated isotopomers of D-[2-13C]glucose represented 78.9 +/- 3.7% of that for the C5-deuterated isotopomers of D-[5-13C]glucose. The three values representative of the anomeric specificity of D-glucose production by liver cells were not significantly different from one another, with an overall mean value of 76.9 +/- 3.6%. These findings unambiguously document that the anomeric specificity of phosphoglucoisomerase is operative in intact hepatocytes, resulting in a preferential output of the alpha-anomer of 13C-enriched D-glucose under the present experimental conditions.     

Interactions of sodium pentobarbital with D-glucose and L-sorbose transport in human red cells.

Pentobarbital acts as a mixed inhibitor of net D-glucose exit, as monitored photometrically from human red cells. At 30 degrees C the Ki of pentobarbital for inhibition of Vmax of zero-trans net glucose exit is 2.16+/-0.14 mM; the affinity of the external site of the transporter for D-glucose is also reduced to 50% of control by 1. 66+/-0.06 mM pentobarbital. Pentobarbital reduces the temperature coefficient of D-glucose binding to the external site. Pentobarbital (4 mM) reduces the enthalpy of D-glucose interaction from 49.3+/-9.6 to 16.24+/-5.50 kJ/mol (P<0.05). Pentobarbital (8 mM) increases the activation energy of glucose exit from control 54.7+/-2.5 kJ/mol to 114+/-13 kJ/mol (P<0.01). Pentobarbital reduces the rate of L-sorbose exit from human red cells, in the temperature range 45 degrees C-30 degrees C (P<0.001). On cooling from 45 degrees C to 30 degrees C, in the presence of pentobarbital (4 mM), the Ki (sorbose, glucose) decreases from 30.6+/-7.8 mM to 14+/-1.9 mM; whereas in control cells, Ki (sorbose, glucose) increases from 6.8+/-1.3 mM at 45 degrees C to 23.4+/-4.5 mM at 30 degrees C (P<0.002). Thus, the glucose inhibition of sorbose exit is changed from an endothermic process (enthalpy change=+60.6+/-14.7 kJ/mol) to an exothermic process (enthalpy change=-43+/-6.2 7 kJ/mol) by pentobarbital (4 mM) (P<0.005). These findings indicate that pentobarbital acts by preventing glucose-induced conformational changes in glucose transporters by binding to 'non-catalytic' sites in the transporter.     

Effect of dihydroxyacetone and pyruvate on plasma glucose concentration and turnover in noninsulin-dependent diabetes mellitus

Consumption of dihydroxyacetone and pyruvate (DHP) increases muscle extraction of glucose in normal men. To test the hypothesis that these three-carbon compounds would improve glycemic control in diabetes, we evaluated the effect of DHP on plasma glucose concentration, turnover, recycling, and tolerance in 7 women with noninsulin-dependent diabetes. The subjects consumed a 1,500-calorie diet (55% carbohydrate, 30% fat, 15% protein), randomly containing 13% of the calories as DHP (1/1) or Polycose (placebo; PL), as a drink three times daily for 7 days. On the 8th day, primed continuous infusions of [6-3H]-glucose and [U-14C]-glucose were begun at 05.00 h, and at 09.00 h a 3-hour glucose tolerance test (75 g glucola) was performed. Two weeks later the subjects repeated the study with the other diet. The fasting plasma glucose level decreased by 14% with DHP (DHP = 8.0 +/- 0.9 mmol/l; PL = 9.3 +/- 1.0 mmol/l, p less than 0.05) which accounted for lower postoral glucose glycemia (DHP = 13.1 +/- 0.8 mmol/l, PL = 14.7 +/- 0.8 mmol/l, p less than 0.05). [6-3H]-glucose turnover (DHP = 1.50 +/- 0.19 mg.kg-1.min-1, PL = 1.77 +/- 0.21 mg.kg-1.min-1, p less than 0.05) and glucose recycling, the difference in [6-3H]-glucose and [U-14C]-glucose turnover rates, decreased with DHP (DHP = 0.25 +/- 0.07 mg.kg-1.min-1, PL = 0.54 +/- 0.10 mg.kg-1.min-1, p less than 0.05). Fasting and postoral glucose, plasma insulin, glucagon, and C peptide levels were unaffected by DHP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Collagen, cross-linking, and advanced glycation end products in aging human skeletal muscle.

We examined intramuscular endomysial collagen, cross-linking, and advanced glycation end products, as well as the general and contractile protein concentration of 20 young (25 +/- 3 yr) and 22 old (78 +/- 6 yr, range: 70-93 yr) sedentary men and women to better understand the underlying basis of changes in skeletal muscle mass and function that occur with aging. The old individuals had an impaired ability (increased time) (P < 0.05) to climb stairs (80%), rise from a chair (56%), and walk (44%), as well as lower (P < 0.05) quadriceps muscle volume (-29%), muscle strength (-35%), muscle power (-48%), and strength (-17%) and power (-33%) normalized to muscle size. Vastus lateralis muscle biopsies revealed that intramuscular endomysial collagen (young: 9.6 +/- 1.1, old: 10.2 +/- 1.2 microg/mg muscle wet wt) and collagen cross-linking (hydroxylysylpyridinoline) (young: 395 +/- 65, old: 351 +/- 45 mmol hydroxylysylpyridinoline/mol collagen) were unchanged (P > 0.05) with aging. The advanced glycation end product, pentosidine, was increased (P < 0.05) by approximately 200% (young: 5.2 +/- 1.3, old: 15.9 +/- 4.5 mmol pentosidine/mol collagen) with aging. While myofibrillar protein concentration was lower (-5%, P < 0.05), the concentration of the main contractile proteins myosin and actin were unchanged (P > 0.05) with aging. These data suggest that the synthesis and degradation of proteins responsible for the generation (myosin and actin) and transfer (collagen and pyridinoline cross-links) of muscle force are tightly regulated in aging muscle. Glycation-related cross-linking of intramuscular connective tissue may contribute to altered muscle force transmission and muscle function with healthy aging.     

Direct regulation of Na(+)-dependent myo-inositol transport by sugars in retinal pigment epithelium: role of phorbol ester and staurosporin

An Na(+)-dependent active process for myo-inositol (MI) uptake, sharing a common carrier system with glucose and sensitive to phlorizin, was previously established in primary cultures of bovine retinal pigment epithelial (RPE) cells (26, 32). The present report further examines the nature of glucose-induced inhibition of MI transport in primary cultures of RPE cells. RPE cells were grown in supplemented Dulbecco's modification of Eagle's medium (DMEM) containing 5 mM D-glucose (basic growth media) or 40 mM D-glucose or its nonmetabolizable analogue, alpha-methyl-D-glucoside (alpha MG); 1-5 mM nonradioactive MI, pyruvate, or lactate; or 0.2-20 microM phorbol 12-myristate 13-acetate (TPA) or straurosporin (modified growth media), for up to 4 weeks. The capacity of RPE cells to accumulate 3H-MI (ratios of intracellular transported radioactive MI, [MI]i, to external free MI concentration, [MI]i/[MI]o) decreased by up to 41% or 34% when cells were grown for 10 days or longer with 40 mM D-glucose or 40 mM alpha MG, respectively, compared to cells grown in basic growth media. The rate of uptake of 3H-MI also was reduced to 63 +/- 15% or 48 +/- 8% of the control values when cells were fed 1 or 5 mM nonradioactive MI, respectively. In addition, cellular capacity to bind to [3H]phlorizin was reduced to 52 +/- 7%, 61 +/- 5%, or 38 +/- 6% of the controls when RPE cells were fed 40 mM D-glucose, 40 mM alpha MG, or 5 mM nonradioactive MI, respectively. Growth media containing either pyruvate or lactate, the glucose metabolites, did not suppress the ability of RPE cells to accumulate MI. An 18 +/- 8% reduction in [3H]thymidine incorporation into DNA occurred when cells were grown in 40 mM glucose for 12-14 days, compared to cells grown with 5 mM glucose. Chronic treatment (12-14 days) of the cells with phorbol ester, an activator of protein kinase C, caused up to twofold increase in MI uptake, [3H]phlorizin binding, cell number, and DNA synthesis. However, when the rates of MI uptake into cells grown in basic growth media or TPA-treated media were normalized to cell number, no significant difference in MI uptake was found between the treated and untreated cells. Addition of staurosporin, a protein kinase C inhibitor, together with TPA, in the growth media reversed the phorbol-induced increase of MI uptake.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phosphorylation of microtubule-associated proteins regulates their interaction with actin filaments

We have determined the absolute phosphate content of microtubule-associated proteins (MAPs) and established that phosphorylation inhibits the actin filament cross-linking activity of MAPs and both of the major MAP components, MAP-2 and tau. Similar results were obtained with actin from rabbit muscle, hog brain, and Acanthamoeba castellanii. We used the endogenous phosphatases and kinases in hog brain microtubule protein to modulate MAP phosphate level before isolating heat-stable MAPs. MAPs isolated directly from twice-cycled microtubule protein contain 7.1 +/- 0.1 (S.E.) mol of phosphate/300,000 g protein. After incubating microtubule protein without ATP, MAPs, had 4.9 +/- 0.6 phosphates. After incubating microtubule protein with 1 mM ATP and 5 microM cAMP in 2 mM EGTA, MAPs had 8.6 +/- 0.5 phosphates but there was also exchange of three more [32P]phosphates from gamma-labeled ATP for preexisting MAP phosphate. Incubation of microtubule protein with ATP and cAMP in 5 mM CaCl2 resulted in exchange but no net addition of phosphate to MAPs. We fractionated the MAP preparations by gel filtration and obtained MAP-2 with 4.3 to 7.5 and tau with 1.5 to 2.2 mol of phosphate/mol of protein depending on how we treated the microtubule protein prior to MAP isolation. The actin filament cross-linking activity of whole MAPs, MAP-2, and tau depended on the MAP-phosphate content. In all cases, phosphorylation of MAPs inhibited actin filament cross-linking activity. The concentration of high phosphate MAPs required to form a high viscosity solution with actin filaments was 2 to 4 times more than that of low phosphate. MAPs. During incubation of microtubule protein with [gamma-32P]ATP, only MAP peptides are labeled. Treatment of these MAPs with either acid or alkaline phosphatase removes phosphate mainly from MAP-2, with an increase in actin filament cross-linking activity. Thus, both MAP phosphorylation and the effect of phosphorylation on actin cross-linking activity of MAPs are reversible.     

Glucose turnover in compensated hepatic cirrhosis

Glucose turnover and recycling from glucose derived 3-carbon intermediates were examined in overnight fasted patients with compensated hepatic cirrhosis and in age- and weight-matched normal control subjects. Fasting blood concentrations of glucose, lactate and glycerol were similar in both groups but blood pyruvate (60 +/- 10 vs. 80 +/- mumol/l, P less than 0.05), blood alanine (0.23 +/- 0.02 vs 0.34 +/- 0.02 mmol/l, P less than 0.01) were decreased and serum insulin increased (19 [13-24]v 7 [4-11] mU/l, P less than 0.01) in cirrhotic subjects. Absolute glucose turnover, assessed by analysis of decay of [3H]-3-glucose specific activity was decreased in cirrhotic patients (8.1 +/- 0.6 v 12.1 +/- 0.7 mol/kg-1 min-1). Glucose "recycling", assessed by the difference between absolute glucose turnover and that given by [14C]-1-glucose data, was normal in cirrhotic patients suggesting that Cori cycle (glucose-lactate-glucose) activity was normal. These data support previous findings of decreased peripheral glucose utilisation and insulin resistance in cirrhotic patients.     

Characterization of Rb uptake into Sf9 cells using cation chromatography: evidence for a K-Cl cotransporter

To assess cation-chloride cotransporter activity in Sf9 cells, cation chromatography was used to measure initial uptake rates of Rb. Rb exchanged with cellular K, with 30% of cellular K replaced after a 40 min exposure to Rb. Rb uptake into Sf9 cells was not inhibited by 50 μmol l(-1) ouabain. Rb uptake was approximately 65% inhibited by 250 μmol l(-1) bumetanide added to the assay solution, and was more than 95% inhibited when cells were pre-incubated for 20 min with bumetanide (100 and 1000 μmol l(-1)). Uptake of Rb and Cl followed simple Michaelis-Menten kinetics, with a K(m) for Rb of 17.1+/-2.2 mmol l(-1) and a K(m) for Cl of 93.7+/-5.6 mmol l(-1). Rb uptake was not dependent upon extracellular Na. Two min exposures to solutions with reduced [Na] or [Cl] produced small but significant changes in cellular Na content. We conclude that the primary Rb uptake pathway in Sf9 cells is a K-Cl cotransporter and that cation chromatography can be used to effectively study kinetic parameters of cotransporter function in tissue culture cells. Characterization of baseline cation-chloride cotransporter activity in Sf9 cells strengthens their utility as a tool for expression and characterization of exogenous proteins.     

Glucose transfer across the intact guinea-pig placenta

Experiments were carried out in anaesthetized pregnant guinea-pigs. Following the maternal injection of a bolus containing 14C-hexose and 3H2O, blood was sampled from the fetal umbilical vein during a single circulatory transit. A placental transfer index was calculated from the ratio of the tracers in the fetal whole blood divided by that in maternal plasma. The transfer index for D-glucose, 0.66 +/- 0.03 (SEM), greatly exceeded that for L-glucose, 0.013 +/- 0.004. Elevation of the maternal plasma D-glucose concentration, with unlabelled D-glucose, resulted in saturation of D-glucose transfer with an apparent Km of 1.2 x 10(-2) mol/l mean maternal plasma D-glucose. Phlorizin at maternal plasma concentrations of approximately 10(-3) mol/l inhibited D-glucose transfer by 40%. Phloretin did not affect D-glucose transfer at levels estimated to be 10(-4) mol/l. Specificity studies with substituted D-glucose analogues showed that alpha-methyl-D-glucoside is not transported by a facilitated pathway; 2-deoxy-D-glucose and 3-O-methyl-D-glucose share the D-glucose carrier and D-galactose has a partial affinity for the D-glucose carrier.     

Clearance, metabolic fate and tissue distribution of an injected bolus of photoaffinity-labeled rat androgen binding protein

An injected bolus of tritiated photolabeled rat androgen binding protein (ABP) is cleared from the circulation in a biphasic manner. The rapid component of the clearance curve indicates a clearance half time (t1/2) of 4.24 +/- 0.20 h and the slow component indicates a t1/2 of 13.27 +/- 2.92 h. After injection, photolabeled ABP is rapidly degraded as determined by electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate. This method dissociates ABP into subunits of approximately 48,000 daltons. One-half hour after injection of photolabeled ABP, 90% of the radioactivity migrates as a 20,000- rather than as a 48,000-dalton moiety; at the 24-h point only 20,000- and 16,000-dalton species were detectable. Chromatography of plasma on Sephadex G-150 indicated that, under nondenaturing conditions, radioactivity was still associated with proteins having a molecular weight similar to the native ABP dimer. These results suggest that the dimer is substrate for the proteolytic processes affecting ABP. Small (congruent to 7000 dalton) fragments of ABP could be detected in urine by G-150 chromatography 2 h after injection. The control protein [3H] bovine serum albumin (BSA) was cleared from the blood with a time course identical to that of photolabeled ABP (t1/2's 4.69 +/- 0.09 h and 13.56 +/- 1.03 h). [3H] BSA was degraded to peptides that were similar in size to those formed from photolabeled ABP. The uptake of photolabeled ABP and [3H] BSA by potential target tissues (testis, epididymis and ventral prostate) was equivalent, suggesting that the uptake of ABP was a nonspecific phenomenon rather than a receptor-mediated event. The liver and kidneys contained more radioactivity that did the other tissues and there was a differential uptake and clearance of photolabeled ABP and [3H] BSA by these organs. Whether this difference is related to specific uptake processes or to differences in the size and/or structure of the proteins or other factors remains to be determined.     

Evidence of multiple operational affinities for D-glucose inside the human erythrocyte membrane.

1. The Michaelis-Menten parameters of labelled D-glucose exit from human erythrocytes at 2 degrees C into external solution containing 50 mM D-galactose were obtained. The Km is 3.4 +/0 0.4 mM, V 17.3 +/- 1.4 MMOL . 1(-1) cell water . min-1 for this infinite-trans exit procedure. 2. The kinetic parameters of equilibrium exchange of D-glucose at 2 degrees C are Km = 25 +/- 3.4 mM, V 30 +/- 4.1 mmol . 1(-1) cell water . min-1. 3. The Km for net exit of D-glucose into solutions containing zero sugar is 15.8 +/- 1.7 mM, V 9.3 +/- 3.3 mmol . 1(-1) cell water . min-1. 4. This experimental evidence corroborates the previous finding of Hankin, B.L., Lieb, W.R. and Stein, W.D. [(1972) Biochim. Biophys. Acta 255, 126--132] that there are sites with both high and low operational affinities for D-glucose at the inner surface of the human erythrocyte membrane. This result is inconsistent with current asymmetric carrier models of sugar transport.     

Dopamine-induced epithelial K(+) and Na(+) movements in the salivary ducts of Periplaneta americana

K(+)- and Na(+)-selective double-barrelled microelectrodes were used for intracellular and luminal measurements in salivary ducts of Periplaneta americana. The salivary ducts were stimulated with dopamine (10(-6) mol l(-1)). Dopamine decreased intracellular [K(+)] from 112+/-17 mmol l(-1) to 40+/-13 mmol l(-1) (n=6) and increased intracellular [Na(+)] from 22+/-19 mmol l(-1) to 92+/-4 mmol l(-1) (n=6). Luminal [K(+)] was 15+/-3 mmol l(-1) in the unstimulated salivary ducts and increased to 26+/-11 mmol l(-1) upon stimulation with dopamine (n=10). Luminal [Na(+)] was insignificantly increased from 105+/-25 mmol l(-1) to 116+/-22 mmol l(-1) (n=12) by stimulation with dopamine. The potential difference across the basolateral membrane (PD(b)) was depolarized from -65+/-6 mV to -31+/-13 mV (n=12) and the transepithelial potential difference (PD(t)) was hyperpolarized from -13+/-6 mV to -22+/-7 mV (n=22, lumen negative) upon stimulation with dopamine. The re-establishment of prestimulus values of intracellular [K(+)] and [Na(+)] and PD(b) was inhibited by basolateral addition of ouabain (10(-4) mol l(-1)). Furosemide (10(-4) mol l(-1)) in the bath inhibited the dopamine-induced increase in intracellular [Na(+)], the decrease in intracellular [K(+)] and the depolarization of PD(b). We propose a model for dopamine-stimulated ion transport in the salivary ducts involving basolateral Na(+)-K(+)-2Cl(-) cotransport and active extrusion of K(+) via the apical membrane.     

Experimental determination of net protein charge, [A]tot, and Ka of nonvolatile buffers in bird plasma.

The quantitative mechanistic acid-base approach to clinical assessment of acid-base status requires species-specific values for [A]tot (the total concentration of nonvolatile buffers in plasma) and Ka (the effective dissociation constant for weak acids in plasma). The aim of this study was to determine [A]tot and Ka values for plasma in domestic pigeons. Plasma from 12 healthy commercial domestic pigeons was tonometered with 20% CO2 at 37 degrees C. Plasma pH, Pco2, and plasma concentrations of strong cations (Na, K, Ca), strong anions (Cl, L-lactate), and nonvolatile buffer ions (total protein, albumin, phosphate) were measured over a pH range of 6.8-7.7. Strong ion difference (SID) (SID5=Na+K+Ca-Cl-lactate) was used to calculate [A]tot and Ka from the measured pH and Pco2 and SID5. Mean (+/-SD) values for bird plasma were as follows: [A]tot=7.76+/-2.15 mmol/l (equivalent to 0.32 mmol/g of total protein, 0.51 mmol/g of albumin, 0.23 mmol/g of total solids); Ka=2.15+/-1.15x10(-7); and pKa=6.67. The net protein charge at normal pH (7.43) was estimated to be 6 meq/l; this value indicates that pigeon plasma has a much lower anion gap value than mammals after adjusting for high mean L-lactate concentrations induced by restraint during blood sampling. This finding indicates that plasma proteins in pigeons have a much lower net anion charge than mammalian plasma protein. An incidental finding was that total protein concentration measured by a multianalyzer system was consistently lower than the value for total solids measured by refractometer.     

Impaired enzyme-to-enzyme channelling between hexokinase isoenzyme(s) and phosphoglucoisomerase in rat pancreatic islets incubated at a low concentration of D-glucose

It was recently proposed that in rat pancreatic islets exposed to 8.3 mM D-glucose, alpha-D-glucose-6-phosphate undergoes enzyme-to-enzyme channelling between hexokinase isoenzyme(s) and phosphoglucoisomerase. To explore the identity of the hexokinase isoenzyme(s) involved in such a tunnelling process, the generation of 3HOH from the alpha- and beta-anomers of either D-[2-3H]glucose or D-[5-3H]glucose was now measured over 60 min incubation at 4 degrees C in pancreatic islets exposed only to 2.8 mM D-glucose, in order to decrease the relative contribution of glucokinase to the phosphorylation of the hexose. Under these experimental conditions, the ratio for 3HOH production from D-[2-3H]glucose/D-[5-3H]glucose at anomeric equilibrium (39.7 +/- 11.6%) and the beta/alpha ratios for the generation of 3HOH from either the D-[2-3H]glucose anomers (70.9 +/- 12.6%) or the D-[5-3H]glucose anomers (59.6 +/- 12.4%) indicated that a much greater fraction of alpha-D-glucose-6-phosphate escapes from the process of enzyme-to-enzyme channelling in the islets exposed to 2.8 mM, rather than 8.3 mM D-glucose. These findings suggest, therefore, that the postulated process of enzyme-to-enzyme channelling involves mainly glucokinase.     

Effect of insulin, prostaglandin E1 and uptake inhibitors on glucose transport in the perfused guinea-pig placenta

The effects of insulin, prostaglandin E1 (PGE1) and uptake inhibitors on unidirectional D-glucose influx at brush border (maternal) and basal (fetal) sides of the guinea-pig syncytotrophoblast were investigated in the intact, perfused guinea-pig placenta by rapid, paired-tracer dilution. Experiments were performed in either an in situ preparation artificially perfused through the umbilical vessels (intact maternal circulation) or in the fully isolated dually-perfused placenta in which both interfaces were studied simultaneously. Kinetic characterization of unidirectional D-glucose influx gave apparent Km values (mean +/- SEM) at maternal and fetal sides of 70 +/- 6 and 87 +/- 16 mM respectively; corresponding Vmax values were 53 +/- 3 and 82 +/- 6 mumol min-1g-1. At the fetal side (singly-perfused placenta) cytochalasin B (50 microM), ethylidene-D-glucose (100 mM) and PGE1 (1 microM) partially inhibited D-glucose uptake whereas cortisol (50 microM) and progesterone (100 microM) had no effect. Abolition of the sodium gradient across the fetal interface did not modulate the kinetics of influx. In the presence of 150 mu units ml-1 insulin (dually-perfused placenta), unidirectional uptake into the trophoblast and transplacental D-[3H]glucose transfer were unaltered. In contrast, prostaglandin E1 (1 microM) markedly reduced the Km and Vmax for D-glucose at both interfaces and the inhibitory effect was reflected in a reduction in specific transplacental D-glucose transfer. Further experiments showed that the isolated placenta releases prostaglandins (PGE; PGF2 alpha) into both circulations. Bilateral insulin perfusion did not affect either lactate release by the placenta or rapid metabolism of D-[14C]glucose to [3H]lactate (usually less than 10% effluent [14C]lactate in 5 min). An asymmetric degradation of exogenous insulin was observed in the dually-perfused placenta: uterine venous samples contained 24 +/- 7 microunits ml-1 immunoreactive insulin when compared to the arterial concentration (151 +/- 3 microU ml-1 perfusate) while no change was measureable in the fetal circulation within the same time period (152 +/- 5 microU ml-1). This asymmetry was confirmed in experiments employing [125I]insulin. These results demonstrate that glucose transport in the intact guinea-pig placenta occurs by a sodium-independent, cytochalasin B-inhibitable system which is insulin-insensitive. Prostaglandin E1 appeared to be a potent transport inhibitor which suggests that prostaglandins may be involved in the 'down' regulation of placental glucose transport in vivo.     

Erythrocyte ion regulation across inactive muscle during leg exercise.

Ion concentration changes in whole blood, plasma, and erythrocytes across inactive muscle were examined in eight healthy males performing four 30-s bouts of maximal isokinetic cycling with 4 min rest between each bout. Blood was sampled from the arm brachial artery and deep antecubital vein during the intermittent exercise period and for 90 min of recovery. Arterial and venous erythrocyte lactate concentration ([Lac-]) increased from 0.3 +/- 0.1 to 12.5 +/- 1.3 (p < 0.01) and 1.1 +/- 0.4 to 8.5 +/- 1.5 mmol/L (p < 0.01), respectively, returning to control values during recovery. Arterial and venous plasma [Lac-] increased from 1.5 +/- 0.2 to 27.7 +/- 1.8 and from 1.3 +/- 0.4 to 25.7 +/- 3.5 mmol/L, respectively, and was greater than erythrocyte [Lac-] throughout exercise and recovery. Arterial and venous [K+] increased in erythrocytes from 119.5 +/- 5.1 to 125.4 +/- 4.6 (p < 0.01) and from 113.6 +/- 1.7 to 120.6 +/- 7.1 mmol/L, respectively, decreasing to control during recovery. In arterial and venous plasma, [K+] increased from 4.3 +/- 0.1 to 6.1 +/- 0.2 (p < 0.01) and from 4.5 +/- 0.2 to 5.3 +/- 0.2 mmol/L (p < 0.01), respectively, decreasing to control during recovery. The efflux of Lac- out of erythrocytes against an electrochemical concentration gradient suggests the presence of an active transport system. Efflux of K+ from erythrocytes as blood passes across inactive muscle affords an important adaptation to the K+ release from muscle activated in heavy exercise.