Contribution of leucine to oxidative metabolism of the rat medullary thick ascending limb

It has recently been reported that branched-chain amino acid aminotransferase (BCAATase) is inhomogeneously distributed in the kidney. BCAATase activity is several-fold higher in the medullary thick ascending limb (MTAL) than in other nephron segments. The present work was designed to determine whether leucine, a branched-chain amino acid (AA), is used as metabolic fuel by this nephron segment. MTAL were isolated from the inner stripe of the outer medulla of adult Sprague Dawley rats by mild enzymatic digestion and appropriate sieving. Leucine aminotransferase activity measured in homogenates of MTAL was 653±52 pmol -ketoglutarate formed/g protein per hour, a value threefold higher than that observed in the renal cortex or muscle in the same rats. Substrate oxidation was assessed by measuring¹⁴CO₂ production from tracer amounts of uniformly labeled¹⁴C-amino acids or glucose in isolated MTAL incubated in modified Earle balanced salt solution. When each substrate was offered at a concentration of 1 mM, leucine oxidation was much higher than that of unbranched AA, but fivefold lower than that of glucose. With 1 mM glucose and 1 mM leucine in the medium, leucine oxidation was close to that of glucose (123±8 versus 177±15 pmol CO₂/g protein per hour), probably because glucose contributed to the formation of -ketoglutarate, a cosubstrate for leucine transamination. Inhibition of salt transport by furosemide (0.1 mM) decreased oxidation of both substrates by 60–70%. Inhibition of salt transport by ouabain (1 mM) decreased glucose oxidation markedly. However, it doubled leucine oxidation when glucose was absent from the medium and decreased leucine oxidation by only 28% when glucose was present. This might be due to an ouabain-dependent alteration in membrane permeability to AA. These findings show that leucine is oxidized in rat MTAL and may contribute to supporting active transport in this segment. This contribution could be important after a protein meal or on high protein diet, situations in which plasma level of branched-chain AA is elevated.     

ATPase activity in macula densa cells of the rabbit kidney

Na-K- and Mg-activated ATPase activities were determined in maculae densae and glomeruli dissected from both superficial and juxtamedullary nephrons of normal rabbits, using an ultramicro method including a cycling reaction. Activities were expressed as P_i generated per macula densa or per glomerulus and normalized for tissue volume. Results indicate that the mean volume of superficial and juxtamedullary macula densa samples was not statistically different, while glomeruli from deep nephrons had sample volumes that were 29% larger than those from superficial nephrons (P<0.001). Correcting for volume both superficial and juxtamedullary macula densa samples had an Na-K-ATPase activity of 0.37±0.21 fmol · h^–1 · (m³)^–1. Mg-ATPase activity in both pools was also similar [0.41±0.07 and 0.52±0.1 fmol · h^–1 · (m³)^–1]. Na-K-ATPase activity in macula densa cells is estimated to be about 1/40th the activity of surrounding cortical thick ascending limb cells. Total glomerular ATPase per unit volume was significantly higher in glomeruli from superficial than from deep nephrons [0.41±0.04 vs. 0.28±0.04 fmol · h^–1 · (m³)^–1 P<0.05]. There was no statistically significant activity of Na-K-ATPase in either superficial or deep glomeruli. These results suggest that in contrast to previous reports, the macula densa contains Na-K-ATPase, but at a low level relative to surrounding tubular cells. Further, in normal rabbits, this activity is invariant in superficial and juxtamedullary samples.     

Quick isolation of rat medullary thick ascending limbs

This paper describes a rapid and simple method for isolation of medullary thick ascending limbs (MTAL) from rat kidney. The technique takes advantage of the fact that MTAL represents a high fraction of the inner stripe (IS) tissue in the outer medulla, and that this nephron segment is more resistant than others to mechanical and enzymatic disruption. Special attention was given in the design of each step of the isolation procedure in order to improve purity and yield of the preparation. Major steps are the following: 1) careful dissection of the IS; 2) cutting IS tissue into small pieces of regular size ( 1 mm³); 3) mild and brief enzymatic hydrolysis in a 65 U/ml collagenase solution; 4) separation of long MTAL segments from other tubule fragments and cells, and washing of the collagenase solution, on a nylon sieve (100 m opening). This technique does not require lengthy centrifugations and provides about 6 mg fresh tisue (=1 mg protein) from two rat kidneys in 2 h. Light microscopy and transmission electron microscopy show a good purity (at least 95%) and good preservation of TAL ultrastructural morphology. Adenylate cyclase responsiveness to arginine-vasopressin (AVP), glucagon (GLU) and salmon calcitonin (SCT) of the MTAL suspension is similar to that reported for single microdissected rat MTAL. Viability of the MTALs was demonstrated by the ability to accumulate cyclic AMP in presence of AVP, GLU, SCT and forskolin. Normal oxygen consumption was 45.1±2.4 (SEM) l·mg protein^–1·h^–1 (n=8). It was not enhanced with addition of succinate, indicating that integrity of cellular membrane is preserved. It was decreased by half in presence of either furosemide or ouabain. This indicates that electrolyte transport is maintained in the MTAL tubule suspension. This technique represents an easy and quick way in obtaining milligrams of medullary thick ascending limb from small laboratory animals, with a high purity and viability, and maintenance of cell polarity.     

Gastric emptying and serum insulin levels after intake of glucose-polymer solutions

Summary To examine the gastric emptying characteristics of four drinks varying in carbohydrate composition and concentration, five men ingested 600 ml of one of the different drinks on four separate occasions. All drinks contained Na⁺ 71 mmol · l^–1, Cl^– 60 mmol · l^–1, Mg⁺² 5 mmol · l^–1 and citrate 7 mmol · l^–1; the carbohydrate component was either 3% glucose, 3% glucose-polymer (GP), 5% GP or 10% GP. With ^99mTc-diethylene-triaminepenta-acetic acid (DTPA) as a marker, a scintillation camera and computer were used to measure the rate of gastric emptying. The half-emptying times (T _1/2) were inversely related to the glucose content of the solutions. The T _1/2 for 3% PG was 22.4±4.4 min (mean±SE) and for 10% GP 50±3.3 min (p< 0.005). There was no significant difference in T_1/2 between the 3% glucose and 3% GP solutions. The increments in blood glucose (highest blood levels from 7.4±0.3 mmol · l^–1 to 8.9±0.8 mmol · l^–1), serum insulin (from 28±6 mU · l^–1 to 77±13 mU · l^–1) and C-peptide (from 3.6±0.4 g · l^–1 to 5.8±0.9 g · l^–1) were related to the amount of carbohydrate ingested. In all cases the serum insulin levels were high enough to inhibit the liberation of free fatty acids from the adipose tissue. It is concluded that the amount of carbohydrate in glucosyl units in the solution is a major determinant of gastric emptying. Furthermore, the inhibitory effect of glucose is not modified by replacing monomer glucose with glucose polymer or adding NaCl (about 70 mmol · l^–1) in the solution.     

Lipoprotein lipase activity in skeletal muscle and adipose tissue of marathon runners after simple and complex carbohydrate-rich diets

Summary A comparison of the influence of simple and complex carbohydrate (CHO) consumption on adipose tissue- and skeletal muscle-lipoprotein lipase activity (AT-LPLA, SM-LPLA) was examined. Twenty male marathon runners were divided into two equal dietary groups: simple-CHO and complex-CHO. Half of the subjects in each group consumed either a low-CHO (15% energy [E] intake), or a mixed diet (50% CHO) for 3 days. Immediately following this dietary period, the subjects consumed a CHO-rich diet (70% E intake) predominant in simple-CHO or in complex-CHO for an additional 3 days. Thereafter, all subjects returned to a normal mixed diet. Skeletal muscle biopsies, adipose tissue aspirations, and venous blood samples were obtained prior to dietary manipulation (PRE), upon completion of the 6 day diet (POST I), and 2 weeks after returning to a normal diet (POST II). The samples were analysed for AT-LPLA, SM-LPLA, serum insulin, and free fatty acids (FFA), and blood glucose, and lactate. SM-LPLA fell 71% from PRE values of 0.39±0.30 mol · g^–1 · h^–1 to POST I values of 0.11 ±0.09 mol · g^–1 · h^–1 (means±SD) (p<0.05), after a complex-CHO diet. However, the simple-CHO diet did not alter SM-LPLA. AT-LPLA similarly decreased (p<0.05) after the complex-CHO diet, and no significant decrease was noted after the simple-CHO diet. Serum FFA decreased significantly (p<0.05) after a simple-CHO diet (0.82±0.13 to 0.65±0.10 mmol l^–1) but were unchanged after a complex-CHO diet. Blood glucose and lactate, and serum insulin were not altered following a CHO-rich diet.     

Characteristics of the transport of oxalate and other ions across rabbit proximal colon

In order to characterize oxalate handling by the P2 segment of the rabbit proximal colon, the fluxes of [¹⁴C]oxalate, ²²Na⁺, and ³⁶Cl^– were measured in vitro using conventional short-circuiting techniques. In standard buffer the proximal colon exhibited net secretion of Na⁺ (–2.31±0.64 equiv cm^–2 h^–1), negligible net Cl^– transport, and net secretion of oxalate (–12.7±1.6 pmol cm^–2 h^–1). Replacement of buffer Na⁺ or Cl^– abolished net oxalate secretion, while HCO ₃ ^– -free media revealed a net absorption of oxalate (19.3±4.2 pmol cm^–2 h^–1) and stimulated NaCl absorption. Mucosal amiloride and dimethylamiloride (1 mM) significantly reduced the unidirectional fluxes of oxalate and enhanced sodium secretion by decreasing J _ms ^Na . The anion exchange inhibitor 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS; 0.1 mM, both sides) reduced the unidirectional fluxes of oxalate and chloride. Serosal epinephrine (50 M) stimulated oxalate absorption (21.3±6.3 pmol cm^–2 h^–1) and sodium absorption (5.71±1.20 equiv cm^–2 h^–1), whereas dibutyryl-cAMP enhanced oxalate secretion (–43.4±6.9 pmol cm^–2 h^–1) and stimulated chloride secretion (–7.27 ±0.64 equiv cm^–2 h^–1). These results indicate that the P2 segment of the proximal colon possesses (a) secretory as well as absorptive capacities, (b) oxalate fluxes that are mediated by pathways involving Na⁺, Cl^–, HCO ₃ ^– transport and (c) a net oxalate flux that is sensitive to absorptive and secretory stimuli.     

Tubular transport processes in proximal tubules of hypothyroid rats. Lack of relationship between thyroidal dependent rise of isotonic fluid reabsorption and Na+−K+-ATPase activity

Hypothyroid rats reconstituted with 10 g/kg b.w. per day of tri-iodothironine (T₃) for 4 days resulted in normal free T₃ and TSH levels. FT₃ levels were: 0.53±0.3 pg/ml in hypothyroid rats; 2.78±1.21 pg/ml in hormone reconstituted rats and 2.90±0.90 pg/ml in euthyroid rats. TSH levels were 3,508±513 g/ml in hypothyroid rats; 1,008±204 g/ml in reconstituted rats and 270±184 ng/ml in euthyroid rats.When hypothyroid rats were reconstituted with 50 g T₃/kg b.w. per day, TSH levels were nearly normal after 4 days (1,157±621 ng/ml). However FT₃ levels after 1–4 days were always higher than in euthyroid rats.Hypothyroid rats show a decrease in isotonic fluid reabsorption (J _v) in the proximal tubule (1.50±0.08 versus 4.96±0.23 10^–2 nl·mm^–1·s^–1 in euthyroid animals). 1 day after T₃ (10 g/kg b.w./day) injectionJ _v was increased significantly to 2.05±0.20 10^–2 nl·mm^–1·s^–1 and continued to increase during 4 days of T₃ reconstitution.When 50 g T₃/kg b.w./day was used,J _v increased to 2.75±0.07 after 1 day and to 3.10±0.42 10^–2 nl·mm^–1·s^–1 after 4 days.J _v was never reaching a value close to that of euthyroid rats because the tubular radius in hypothyroid rats (14.7±1.8 m) is less than that of euthyroid rats (19.2±0.5 m). The radius in hypothyroid rats treated with T₃ was unchanged over a 4 day course with either high or low doses of T₃.Na⁺–K⁺-ATPase activity was found to be 2.91±0.16 M P_i/h×mg protein in homogenates of kidney cortex from hypothyroid rats. Treatment of hypothyroid rats with 10 g or 50 g of T₃ resulted in an initial decrease in ATPase activity, followed by an increase to base level in hypothyroid rats with 10 g and a significantly higher level with 50 g. This decrease in ATPase activity was contrasted to the increase inJ _v.These data indicate that there is a dissociation between the effects of physiological doses of thyroid hormones on proximal tubular reabsorption and the effects of T₃ on Na⁺–K⁺-ATPase activity of kidney cortex. This leads to question the relationship between sodium transport and ATPase activity under physiological doses of thyroid hormones. An early effect of physiological doses of thyroid hormones on brush border Na⁺ permeability is suggested.     

Exercise and the oxidation and storage of glucose,maize-syrup solids and sucrose determined from breath13CO2

In order to determine which of maize syrup solids, glucose and sucrose were more readily oxidised during exercise and least readily oxidised afterwards, the rates of oxidation of three almost identical isoenergetic solutions of carbohydrates (330 ml of 18.5% w/v solutions of glucose, maize syrup solids and sucrose, 989–1050 kJ total energy) naturally enriched with¹³C were examined at rest and during and after 1 h uphill walking at 75% maximum oxygen uptake ( ) in nine subjects [mean (SEM) , 45.4 (0.9) ml·kg^–1-min^–1]. Rates of production of expired¹³CO₂ were used to estimate rates of oxidation of each exogenous substrate. Energy expenditure and the contributions from total carbohydrate and fat oxidation were calculated from whole-body gas exchange. At rest, aize syrup solids were oxidised less than sucrose during the 1st h [glucose 2.7 (0.2) g · h^–1, maize syrup solids 1.9 (0.3) g · h^–1, sucrose 3.7 (0.2) g · h^–1; maize syrup solids vs sucroseP < 0.01], but this difference disappeared after a further 3 h at rest [glucose 8.3 (0.5) g · h^–1, maize syrup solids 7.7 (0.5) g · h^–1, sucrose 8.1 (0.4) g · h-1]. During exercise, all the carbohydrates were oxidised to the same extent [glucose 23.0 (2.8) g · h^–1, maize syrup solids 23.9 (3.4) g · h^–1, sucrose 27.5 (2.6) g · h^–1) but during 4 h of recovery after exercise, maize syrup solids were oxidised least [glucose 4.6 (0.1) g · h^–1, maize syrup solids 3.7 (0.1) g · h^–1, sucrose 6.4 (0.1) g · h^–1;P < 0.05] suggesting that it may be stored to a greater extent. The results suggest that 18.5% glucose, maize syrup solids and sucrose solutions were equally well oxidised during exercise. During recovery from exercise maize syrup solids were oxidised less than glucose, which in turn was oxidised less than sucrose.     

Electrophysiological analysis of sodium-transport in the colon of the frog (Rana esculenta)

Sodium transport and apical bioelectrical membrane properties were investigated in frog colonic epithelium in the absence and presence of the antidiuretic hormone arginine-vasotocin (AVT). Apical Na-permeability and intracellular Na-activity were evaluated by analysis of current-voltage relationships in the serosally K-depolarized tissue. Tissue- and apical membrane capacitance were measured by voltages step analysis. The frog colon was found to be a tight epithelium with a transepithelial resistance of 2.63±0.25 k·F (n=17). 85–90% of short circuit current (11.2±1.1 A·F·l^–1;n=17) was related to electrogenic Na-transport from mucosa to serosa. Graded doses of amiloride (<50 mol·l^–1) induced Michaelis-Menten-type inhibition kinetics. Serosal addition of 10^–6 mol·l^–1 AVT induced a significant increase in sodium current (25%), apical sodium permeability (19%) and tissue capacitance (4.3%) whereas intracellular Na-activity remained unchanged. There was a good correlation between increased Na-current and apical Na-permeability. No correlation was found between Na-current and membrane capacitance. Our results demonstrate that in contrast to other species the amphibian colon shows a natriferic reaction to AVT. We suggest that the regulation of Na-transport in frog colon is similar to that in the toad urinary bladder. It is caused by an activation of preexisting apical Na-channels and not by fusion of subapical cytoplasmic vesicles with the apical membrane.     

Effects of muscarinic blockade on the thermic effect of oral or intravenous carbohydrate

Summary Muscarinic blockade by atropine has been shown to decrease the thermic effect of a mixed meal, but not of intravenous glucose. To further delineate the mechanisms involved in the atropine-induced inhibition of thermogenesis after a meal, plasma substrate and hormone concentrations, energy expenditure (EE) and substrate oxidation rates were measured before and during a continuous glucose infusion (44.4 mol·kg^–1·min^–1) with or without atropine. After 2 h of glucose infusion, a 20-g oral fructose load was administered while the glucose infusion was continued. Plasma insulin concentrations attained a plateau at 596 (SEM 100) pmol·l^–1 after 120 min of glucose infusion and were not affected by muscarinic blockade; plasma glucose concentrations peaked at 13.3 (SEM 0.5) mmol·l^–1 at 90 min and decreased progressively thereafter; no difference was observed with or without atropine. Plasma free fatty acid and glucagon concentrations, with or without atropine, were both decreased to 201 (SEM 18) mol·l^–1 and 74 (SEM 4) ng·l^–1, respectively, after 2 h of glucose infusion, and were not further suppressed after oral fructose. Carbohydrate oxidation rates (CHO_ox) increased to 20.8 (SEM 1.4) mol·kg^–1·min^–1 and lipid oxidation rates (L_ox) decreased to 1.5 (SEM 0.3) mol·kg^–1·min^–1 between 90 and 120 min after the beginning of glucose infusion and were not affected by atropine. Glucose-induced thermogenesis was similar with [6.5% (SEM 1.4%) of basal EE] or without [6.0% (SEM 1.0%), NS) muscarinic blockade during the 30 min preceding fructose ingestion. During the second half-hour after fructose ingestion, atropine infusion inhibited markedly the stimulation of CHO_ox [+2.8 (SEM 1.0) mol·kg^–1·min^–1 vs +6.9 (SEM 1.0) mol·kg^–1·min^–1, saline, P<0.02] and the suppression of L_ox [–0.8 (SEM 0.2) mol·kg^–1·min^–1 vs –1.4 (SEM 0.2) mol·kg^–1·min^–1, saline, P<0.05]. Carbohydrate-induced thermogenesis during the second half-hour after fructose ingestion, increased to 13.0% (SEM 2.0%) without atropine and was suppressed to 7.7% (SEM 1.9%) (P< 0.05, vs saline) with atropine. It was concluded that muscarinic blockade suppressed the increase of thermogenesis observed after oral fructose, but not during intravenous glucose infusion and that this suppression occurred independently of alterations of plasma insulin concentrations.     

Erythrocyte,plasma and urinary magnesium in men before and after a marathon

Summary Erythrocyte, plasma and urinary magnesium (Mg²⁺) concentration was measured in 23 runners before and after a marathon race. Blood samples were drawn from an antecubital vein the morning before the race (baseline), at 3 p.m. (2 h before the start), upon finishing and 12 h later. Compared with the baseline values, the intra-erythrocyte and plasma Mg²⁺ were decreased (p<0.05 or less) immediately after the marathon, from 2.13±0.16 to 2.02±0.18 mmol · l^–1 cells and from 0.88±0.06 to 0.81±0.07 mmol · l^–1 respectively. The Mg²⁺ concentration returned to pre-race values 12 h after completion of the marathon. The urinary Mg²⁺ excretion rate decreased (p<0.001) from 29±13 to 5±3 mol · min^–1 during the marathon and increased (p<0.05) 12 h after the race to 38±18 mol · min^–1. It is concluded that the reduction in plasma Mg²⁺ ion concentration during the marathon cannot be attributed to erythrocyte uptake, urinary excretion or loss in sweat. It is suggested that Mg²⁺ may be released from erythrocytes into the extracellular fluids during sustained exercise and taken up from these fluids by the adipose cells.     

Effects of glucagon on Na+, Cl−, K+, Mg2+ and Ca2+ transports in cortical and medullary thick ascending limbs of mouse kidney

The effects of glucagon on transepithelial Na⁺, Cl^–, K⁺, Ca²⁺ and Mg²⁺ net fluxes were investigated in isolated perfused cortical (cTAL) and medullary (mTAL) thick ascending limbs of Henle's loop of the mouse nephron. Transepithelial ion net fluxes (J _Na ⁺,J _Cl ^–,J _K ⁺,J _Ca ²⁺,J _Mg ²⁺) were determined by electron probe analysis of the collected tubular fluid. Simultaneously the transepithelial voltage (PD_te) and the transepithelial resistance (R _te) were recorded. In cTAL-segments (n=8), glucagon (1.2×10^–8 mol · l^–1) stimulated significantly the reabsorption of Na⁺, Cl^–, Ca²⁺ and Mg²⁺J _Na ⁺ increased from 204±20 to 228±23 pmol · min^–1 · mm^–1,J _Cl ^– from 203±18 to 234±21 pmol · min^–1 · mm^–1,J _Ca ²⁺ from 0.52±0.13 to 1.34±0.30 pmol · min^–1 · mm^–1 andJ _Mg ²⁺ from 0.51±0.08 to 0.84±0.08 pmol · min^–1 · mm^–1.J _K+ remained unchanged: 3.2±1.3 versus 4.0±1.9 pmol · min^–1 · mm^–1. Neither PD_te (16.3±1.5 versus 15.9±1.4 mV) norR _te (22.5±3.0 versus 20.3±2.6 cm²) were changed significantly by glucagon. However, in the post-experimental periods a significant decrease in PD_te and increase inR _te were noted. In mTAL-segments (n=9), Mg²⁺ and Ca²⁺ transports were close to zero and glucagon elicited no significant effect. The reabsorptions of Na⁺ and Cl^–, however, were strongly stimulated:J _Na ⁺ increased from 153±17 to 226±30 pmol · min^–1 · mm^–1 andJ _Cl ^– from 151±23 to 243±30 pmol · min^–1 · mm^–1. The rise in NaCl transport was accompanied by an increase in PD_te from 10.3±1.1 to 12.3±1.2 mV and a decrease inR _te from 19.1±2.7 to 17.8±2.0 cm². No net K⁺ movement was detectable either in the absence or in the presence of glucagon. A micropuncture study carried out in hormone-deprived rats indicated that glucagon stimulates Na⁺, Cl^–, K⁺, Mg²⁺ and Ca²⁺ reabsorptions in the loop of Henle. In conclusion our data demonstrate that glucagon stimulates NaCl reabsorption in the mTAL segment and to a lesser extent in the cTAL segment whereas it stimulates Ca²⁺ and Mg²⁺ reabsorptions only in the cortical part of the thick ascending limb of the mouse nephron. These data are in good agreement with, and extend, those obtained in vivo on the rat with the hormone-deprived model.This study was supported by the Commission des Communautés Européennes, Grant no. ST 23, 00951F (CD) and by Wissenschaftsausschuß der Nato über den DAAD     

Transport ofl-cystine in isolated perfused proximal straight tubules

Unidirectional fluxes ofl-³⁵S-cystine and intracellular³⁵S activity were measured in isolated perfused segments of rabbit proximal straight tubule. The absorptive (lumen-to-both) flux ofl-³⁵S-cysteine showed a tendency toward saturation within the concentration limits imposed by the low solubility of cystine (0.3 mmol·l^–1). In contrast, for the bath-to-lumen fluxes, there was a linear relation between the bathing solution concentration ofl-³⁵S-cystine and the rate of³⁵S appearance in the lumen. Nonlinear fitting of both sets of unidirectional flux data gave a maximal cystine transport rate (J _max) of 1.45±0.27 (SEM) pmol min^–1 mm^–1, a Michaelis constant (K _m) of 0.20±0.07 mmol·l^–1, and an apparent permeability coefficient of 0.27±0.11 pmol min^–1 mm^–1 (mmol·l^–1)^–1 (approximately 0.06 m/s). The³⁵S concentration in the cell exceeded that in the lumen by almost 60-fold during the lumen-to-bath flux, and exceeded the bathing solution concentration by 4.7-fold during the bath-to-lumen flux. Thus cystine was accumulated by the cells across either membrane, but over 77% of the intracellular activity was in the form of cysteine. Although the presence of luminall-lysine or cycloleucine inhibited the absorptive flux of cystine, neither amino acid affected the bath-to-lumen flux.Some of the work described here was presented as an abstract at the 8th International Congress of Nephrology, Athens, Greece, 1981     

Effects of glucose ingestion or glucose infusion on fuel substrate kinetics during prolonged exercise

To determine if bypassing both intestinal absorption and hepatic glucose uptake by intravenous glucose infusion might increase the rate of muscle glucose oxidation above 1 g · min^–1, ten endurance-trained subjects were studied during 125 min of cycling at 70% of peak oxygen uptake (VO_{2 peak}). During exercise the subjects ingested either a 15 g · 100 ml^–1 U-¹⁴C labelled glucose solution or H₂O labelled with a U-¹⁴C glucose tracer for the determination of the rates of plasma glucose oxidation (Rox) and exogenous carbohydrate (CHO) oxidation from plasma¹⁴C glucose and¹⁴CO₂ specific activities, and respiratory gas exchange. Simultaneously, 2-³H glucose was infused at a constant rate to measure glucose turnover, while unlabelled glucose (25% dextrose) was infused into those subjects not ingesting glucose to maintain plasma glucose concentration at 5 mmol · l^–1. Despite similar plasma glucose concentrations [ingestion 5.3 (SEM 0.13) mmol · l^–1; infusion 5.0 (0.09) mmol · l^–1], compared to glucose infusion, CHO ingestion significantly increased plasma insulin concentrations [12.9 (1.0) vs 4.8 (0.5) mU · l^–1;P<0.05], raised total Rox values [9.5 (1.2) vs 6.2 (0.7) mmol · 125 min^–1 kg fat free mass^–1 (FFM);P<0.05] and rates of CHO oxidation [37.2 (2.8)vs 24.1 (3.9) mmol · 125 min^–1 kg FFM^–1;P<0.05]. An increased reliance on CHO metabolism with CHO ingestion was associated with a decrease in fat oxidation. Whereas the contribution from fat oxidation to energy production increased to 51 (10)% with glucose infusion, it only reached 18 (4)% with glucose ingestion (P<0.05). Despite these differences in plasma insulin concentration and rates of fat oxidation, the rates of glucose oxidation by muscle were similar after 125 min of exercise for both trials [ingestion 93 (8); infusion 85 (5) mol · min^–1 kg FFM^–1], suggesting that peak rates of muscle glucose oxidation were primarily dependent on blood glucose concentration which, in turn, regulated the hepatic appearance of ingested CHO.     

Effects of pre-exercise carbohydrate feedings on muscle glycogen use during exercise in well-trained runners

Summary The purpose of this study was to examine the effects of pre-exercise glucose and fructose feedings on muscle glycogen utilization during exercise in six well-trained runners ( =68.2±3.4 ml·kg^–1·min^–1). On three separate occasions, the runners performed a 30 min treadmill run at 70% . Thirty minutes prior to exercise each runner ingested 75 g of glucose (trial G), 75 g of fructose (trial F) or 150 ml of a sweetened placebo (trial C). During exercise, no differences were observed between any of the trials for oxygen uptake, heart rate or perceived exertion. Serum glucose levels were elevated as a result of the glucose feeding (P<0.05) reaching peak levels at 30 min post-feeding (7.90±0.24 mmol·l^–1). With the onset of exercise, glucose levels dropped to a low of 5.89±0.85 mmol·l^–1 at 15 min of exercise in trial G. Serum glucose levels in trials F and C averaged 6.21±0.31 mmol·l^–1 and 5.95±0.23 mmol·l^–1 respectively, and were not significantly different (P<0.05). There were also no differences in serum glucose levels between any of the trials at 15 and 30 min of exercise. Muscle glycogen utilization in the first 15 min of exercise was similar in trial C (18.8±8.3 mmol·kg^–1), trial F (16.3±3.8 mmol·kg^–1) and trial G (17.0±1.8 mmol·kg^–1), and total glycogen use was also similar in trial C (25.6±7.9 mmol·kg^–1), trial F (35.4±5.7 mmol·kg^–1) and trial G (24.6±3.2 mmol·kg^–1). In contrast to previous research, these results suggest that pre-exercise feedings of fructose or glucose do not affect the rate of muscle glycogen utilization during 30 min of treadmill running in trained runners.     

The effects of extracellular potassium,ouabain, and prostaglandins on intracellular potassium activity in sheep cardiac Purkinje fibers

(1) Intracellular K activity (a _K ⁱ ) of sheep heart Purkinje fibers was measured using K-selective microelectrodes (liquid ion exchanger).a _K ⁱ in the resting state with an extracellular K of 4 mmol·l^–1 was 112.9±6.1 mmol·l^–1 (n=47) for a membrane potential (V _M) of –73.3±0.9 mV.V _M deviated from the calculated potassium equilibrium potential (E _K=–93 mV). (2) When extracellular K was decreased to 2 mmol·l^–1 or increased to 6 and 10 mmol·l^–1 E _K changed from –114 to –84 and –73 mV, with little change ina _K ⁱ . (3)a _K ⁱ andV _M significantly decreased after administration of 10^–6 mol·l^–1 ouabain. (4) Prostaglandins (PGI₂ 10–100 g·l^–1 and PGE₂ 0.01–1 g·l^–1) decreaseda _K ⁱ without greatly changingV _M. The differences betweenV _M andE _K became smaller. These effects indicate an increase in K permeability and may explain the antiarrhythmic action of prostaglandins.This study was supported by the Deutsche Forschungsgemeinschaft (grant Wi 328). In preliminary form part of the data has been presented (Pflügers Arch. 384: R 13, 1980, and Proc. XXVIII. Int Congr Physiol Sci, Vol XIV: 279, 1980)     

Electrical properties and electrolyte transport in bovine tracheal epithelium: Effects of ion substitutions,transport inhibitors and histamine

The active transport of Na⁺ and Cl^– across the tracheal epithelium of the cow was investigated in vitro, using the short-circuit technique, by means of ion substitutions, transport inhibitors and by measuring²²Na and³⁶Cl fluxes. Under short-circuit conditions, short-circuit current (i _o) was 168±5A cm^–2 (mean±SEM,n=30), i.e. 6.2±0.2 Eq h^–1cm^–2 and resistance (R) was 248±10cm². Net Na⁺ flux toward the submucosa (J _{Na net} ^L-S ) and net Cl^– flux toward the lumen (J _{Cl net} ^S-L ) were of the same magnitude, i.e. 2.7±0.2 and 2.9±0.2 Eq h^–1 cm^–2, respectively. The permeability coefficients were 3.6·10^–6 forP _Na and 7.8·10^–6 cm s^–1 forP _Cl. Under open-circuit conditions, the transepithelial electrical potential difference () was 43±2 mV (lumen negative,n=20).J _{Na net} ^L-S andJ _{Cl net} ^S-L were close to zero.Bilateral substitution of Cl^– with SO ₄ ^2– or isethionate, or administration of furosemide 5·10^–3M or bumetanide 10^–4M in the submucosal bathing medium produced a 40 to 50% decrease ini ₀; furosemide abolishedJ _{Cl net} ^S-L . Bilateral substitution of Na⁺ with choline or Mg²⁺, or addition of ouabain 10^–4M to the submucosal bath abolishedi ₀;J _{Na net} ^L-S andJ _{Cl net} ^S-L were suppressed by ouabain. Amiloride 10^–4M in the luminal bath reducedi ₀ by 23% and diminishedJ _{Na net} ^L-S by 80%. Histamine 10^–4M, added to the submucosal bathing medium, reducedJ _{Na net} ^L-S and increasedJ _{Cl net} ^S-L , under short-circuit conditions. In open-circuit conditions, histamine had little effect on ion fluxes. This substance had no effect on the electrical properties, as shown previously.These results are consistent with the model proposed by Silva et al. [20] for a Cl^–-secreting, Na⁺-reabsorbing epithelium.Supported by the Swiss National Foundation (SNF), grant no. 3.588-0.79     

Preferential distribution of leukocytes in rat mesentery microvessel networks

Distribution of leukocytes in rat mesenteric microvessel networks was studied using intravital fluorescence video microscopy. A digital image analysis system was used to measure vessel diameters, flow velocities and leukocyte fluxes in 306 capillaries of 8 networks. Capillaries were defined as vessel segments connecting divergent to convergent branch points. Their topological position within the network was quantified by a generation number defined as the number of bifurcations between the capillary and the arteriole feeding the network.Proximal capillaries (generation numbers 4 and 5) were slightly but significantly smaller in diameter (8.9±0.4) m, mean ± SEM) than distal ones (generation numbers 20 and 21, 10.1±0.4 m). Average capillary flow velocity decreased markedly from 2.0±1.0 mm·s^–1 in proximal to 0.41 ±0.06 mm·s^–1 in distal capillaries. Average leukocyte concentration was 3.4±0.5·10⁹ l^–1 and thus significantly below systemic values (6.0·10⁹ l^–1) in proximal capillaries, and above in distal ones (11.7±2.6·10⁹l^–1).The analysis of flow and leukocyte flux partition at 138 bifurcations showed preferential distribution of leukocytes to the daughter capillary with higher flow rate. This suggests a tentative explanation for the observed leukocyte accumulation along the microvascular tree: due to their low fractional flow, proximal capillaries draw relatively leukocytepoor blood from the arteriole feeding the network; this leads to an increased leukocyte concentration in distal capillaries. As a consequence of the concomitant increase of capillary diameter with increasing generation number, leukocytes are preferentially flowing through larger capillaries and are excluded from small ones.     

The role of the cardiac nerves in the regulation of sodium excretion in conscious dogs

Conscious, chronically instrumented dogs, maintained on a high sodium intake, were used to investigate whether surgical cardiac denervation impairs the natriuresis associated with left atrial pressure increase produced in three ways: during an increase in left atrial pressure by means of a reversible mitral stenosis (protocol 1); after an i.v. saline load (1.0 ml 0.9%·saline min^–1·kg^–1 over 60 min) (protocol 2); after an oral saline load (14.5 mmol Na·kg^–1 given with the food as an isotonic solution) (protocol 3).During a reversible mitral stenosis, in intact dogs, urine volume and sodium excretion increased markedly (from 34–145 l·min^–1·kg^–1 and from 3–12 mol·min^–1·kg^–1); mean arterial pressure increased by an average of 2 kPa (15 mm Hg) and heart rate by 55 b/min; plasma renin activity fell from 0.37–0.21 ng Al·ml^–1·h^–1. Cardiac denervation eliminated these effects of left atrial distension except for a small increase in heart rate (12 b/min). This indicates that the natriuresis and diuresis during left atrial distension resulted from stimulation of receptors located in the left atrium.In contrast, during protocol 2 and 3, the same amounts of sodium and water were excreted in the cardiac denervated dogs as compared to the intact dogs. A comparable decrease in plasma renin activity also was observed. — Apparently the presence of the cardiac nerves is not a prerequisite for maintenance of sodium and water homeostasis.     

Transport of putrescine in the isolated rabbit intestine

The transepithelial fluxes of putrescine were studied in sections of the three segments of rabbit intestine mounted in an Ussing chamber. The ileum exhibited the highest mucosal-to-serosal (J _ms) and serosal-to-mucosal (J _sm) unidirectional fluxes of 1 mol/l [³H]putrescine. Putrescine net flux (J _net=J _ms–J _sm) was deduced to be positive through the duodenum (J _net=53.40±14.30 pmol h^–1 cm^–2), not significantly different from zero through the jejunum (J _net=8.90±19.20 pmol h^–1 cm^–2) and negative through the ileum (J _net=–34.30± 13.80 pmol h^–1 cm^–2). Increasing putrescine concentration up to 10 mmol/l led to an increase in J _ms, J _sm and J _net without affecting the transport polarity in the ileum. The tissue retention of putrescine after 100 min was higher by the serosal side than by the mucosal side of the ileum. In parallel experiments, isolated pieces of ileum accumulated putrescine to a five- to sixfold concentration gradient by a ouabain-inhibitable mechanism. In contrast with arginine and in order of decreasing potency, putrescine, cystamine (a transglutaminase inhibitor), spermidine and spermine (1 mmol/l) reduced both unidirectional fluxes of putrescine across the ileum in the Ussing chamber. The latter effectors, except spermine, and N,N-dimethylcasein (1 mg/ml) led to an important, if not complete, suppression of putrescine secretion by the ileum, while the calmodulin antagonist melittin (0.3 g/ml) reversed the polarity of polyamine transport, suggesting the involvement of transglutaminase in putrescine transport. We conclude that the heterogeneous pattern of putrescine transport along the small-intestinal epithelium constitutes an important feature of the regulation of polyamine concentrations in this tissue.