Effect of NaCl on kinetics ofd-glucosamine uptake in yeasts differing in halotolerance

The initial rate ofd-glucosamine uptake by the non-halotolerant yeastSaccharomyces cerevisiae was approximately halved as the apparent half saturation constant (K_m) and the apparent maximum velocity (V_max) changed from 6.6mm to 16.4mm and from 22 μmol · g⁻¹ · min⁻¹ to 16 μmol · g⁻¹ · min⁻¹, respectively, when the salinity in the medium was increased from zerom to 0.68m NaCl. Corresponding changes in a high affinity transport system in the halotolerant yeastDebaryomyces hansenii were from 1.1mm to 4.6mm and from 3.1 μmol · g⁻¹ · min⁻¹ to 4.5 μmol · g⁻¹ · min⁻¹, implying a practically unchanged transport capacity. In 2.7m NaCl, K_m and V_max in this system were 24.5mm and 1.1 μmol · g⁻¹ · min⁻¹, respectively, representing a marked decrease in transport capability. Nevertheless, the degree of affinity in this extreme salinity must still be regarded as noteworthy. In addition to the high affinity transport system inD. hansenii, a low affinity system, presumably without relevance ind-glucosamine transport, was observed.     

Characteristics of dipeptide transport in pig jejunum in vitro

 Characteristics of dipeptide transport in pig jejunum were investigated in vitro by applying the Ussing-chamber technique and mucosal uptake studies. Addition of both glycyl-l-glutamine and glycyl-l-sarcosine (20 mmol · l⁻¹) to the mucosal buffer solution significantly increased the short-circuit current by 2.60 ± 0.15 and 1.57 ± 0.20 μeq · cm⁻² · h⁻¹, respectively. Concentration-dependent changes in short-circuit current followed Michaelis-Menten kinetics with similar affinity constants for both dipeptides. From unidirectional flux rates for radiolabelled glycyl-l-sarcosine, a net flux rate for glycyl-l-sarcosine of 49.8 ± 6.7 nmol · cm⁻² · h⁻¹ was calculated. In mucosal uptake experiments, the apical influx of ¹⁴C-labelled glycyl-l-sarcosine into isolated porcine mucosa was pH dependent and significantly inhibited by glycyl-l-glutamine. Moreover, RT-PCR studies with primers derived from rabbit PepT1 identified two PCR fragments of identical size to rabbit PepT1 from pig intestinal mRNA preparations. In conclusion, our studies revealed key features of mammalian intestinal peptide transporters and give evidence for a PepT1-like transporter in the pig jejunum that could significantly contribute to the overall amino acid absorption from the gut. Accepted: 30 June 1999     

Note on the mathematical theory of oxygen consumption at low oxygen pressures

Summary A mathematical analysis of the diffusion of oxygen into a cell is made, under the assumption, that the true rate of consumption of oxygen by protoplasm is constant and independent on the oxygen pressure. The results are compared with experimental data. For unfertilizedArbacia eggs and for bacteria the experimental curves cannot be represented by the theory. Some additional assumptions, like those proposed byGerard, seem unavoidable. For fertilizedArbacia eggs a good agreement may however be obtained, by taking for the diffusion coefficient of oxygen trough the protoplasm 7×10⁻⁷ cm²·min⁻¹, and for the permeability of the cell surface to oxygen 4.25×10⁻⁴cm·min⁻¹. With 1 Text-figure     

In vitro studies on active calcium absorption from ovine rumen

From various in vivo and in vitro studies it has been shown that the rumen represents a significant site of Ca²⁺ absorption in sheep and goats. It was the aim of the present study to further characterize the underlying mechanisms. Unidirectional flux rates of Ca²⁺ across rumen wall epithelia of sheep were measured in vitro by applying the Ussing-chamber technique in the absence of electrochemical gradients. Under these conditions, significant Ca²⁺ net flux rates (J_net) clearly indicate the presence of active mechanisms for Ca²⁺ transport. Short chain fatty acids (SCFAs) caused highest stimulation of Ca²⁺ J_net (6.3 ± 1.9 nmol · cm⁻² · h⁻¹) when used as a mixture of acetate, proprionate and butyrate in physiological proportions (36, 15, 9 mmol · l⁻¹, respectively). The effect of 30 mmol · l⁻¹ butyrate (3.2 ± 0.6 nmol · cm⁻² · h⁻¹) was higher than respective amounts of propionate and acetate (0.6 ± 0.8 nmol · cm⁻² · h⁻¹ and 0.9 ± 0.8 nmol · cm⁻² · h⁻¹, respectively). Eliminating SCFAs resulted in Ca²⁺ J_net of 0.4 ± 1.1 nmol ^. cm^−2 .h⁻¹. Addition of Ca channel blocker verapamil (mucosal 1 mmol · l⁻¹) had no significant effect on SCFA-stimulated J_net of Ca²⁺, whereas application of Na⁺/H⁺ inhibitor amiloride (mucosal 1 mmol · l⁻¹) further enhanced the Ca²⁺ J_net by >65%. The Ca²⁺-pump inhibitor vanadate had no significant effect on J_net of Ca²⁺. Dietary Ca depletion enhanced calcitriol plasma concentrations but had no effect on active Ca²⁺ absorption across the rumen wall of sheep. In addition, no effect on active Ca²⁺ absorption could be observed during early lactation. In conclusion, there is clear evidence for the rumen as a main site for active Ca²⁺ absorption in sheep. Our results suggest the presence of a Ca²⁺/H⁺ exchange mechanism in the apical membrane of rumen epithelial cells which depends on SCFA absorption and which does not seem to be under the control of calcitriol. Basolateral Ca²⁺ extrusion occurs independently from Ca²⁺-pump activity and may be accomplished via Na⁺/Ca²⁺ exchange. Accepted: 29 June 1999     

Effects of Lens Major Intrinsic Protein on Glycerol Permeability and Metabolism

Lens Major Intrinsic Protein (MIP) is a member of a family of membrane transport proteins including the Aquaporins and bacterial glycerol transporters. When expressed in Xenopus oocytes, MIP increased both glycerol permeability and the activity of glycerol kinase. Glycerol permeability (p _Gly ) was 2.3 ± 0.23 × 10⁻⁶ cm sec⁻¹ with MIP vs. 0.92 ± 0.086 × 10⁻⁶ cm sec⁻¹ in control oocytes. The p _Gly of MIP was independent of concentration from 5 × 10⁻⁵ to 5 × 10⁻² m, had a low temperature dependence, and was inhibited approximately 90%, 80% and 50% by 1.0 mm Hg⁺⁺, 0.2 mm DIDS (diisothiocyanodisulfonic stilbene), and 0.1 mm Cu⁺⁺, respectively. MIP-enhanced glycerol phosphorylation, resulting in increased incorporation of glycerol into lipids. This could arise from an increase in the total activity of glycerol kinase, or from an increase in its affinity for glycerol. Based on methods we present to distinguish these mechanisms, MIP increased the maximum rate of phosphorylation by glycerol kinase (0.12 ± 0.03 vs. 0.06 ± 0.01 pmol min⁻¹ cell⁻¹) without changing the binding of glycerol to the kinase (K _M ∼ 10 μm). Received: 23 May 1997/Revised: 4 August 1997     

Reconstitution of a Regulated Transepithelial Water Pathway in Cells Transfected with AQP2 and an AQP1/AQP2 Hybrid Containing the AQP2-C Terminus

Transepithelial water permeability was measured in LLC-PK1 cells stably transfected with aquaporins (AQPs): AQP1, AQP2, and a chimera of AQP1 and AQP2 containing 41 amino acids of the C-terminus of AQP2. Transepithelial water fluxes (Jw) were not previously reported in cells transfected with aquaporins. Jw were now recorded each minute using a specially developed experimental device. A significant increase in Posm after forskolin (FK) plus vasopressin (VP) was found in AQP2 transfected cells (39.9 ± 8.2 vs. 12.5 ± 3.3 cm · sec⁻¹· 10⁻³), but not in cells transfected with AQP1 (15.3 ± 3.6 vs. 13.4 ± 3.6 cm · sec⁻¹· 10⁻³). In the case of the AQP1/2 cells (chimera) the FK plus VP induced Posm was smaller than in AQP2 cells but significantly higher than in mock cells at rest (18.1 ± 4.8 vs. 6.7 ± 1.0 cm · sec⁻¹· 10⁻³). The increases in Posm values were not paralleled by increases in ¹⁴C-Mannitol permeability. HgCl₂ inhibited the hydrosmotic response to FK plus VP in AQP2 transfected epithelia. Results were comparable to those observed, in parallel experiments, in a native ADH-sensitive water channel containing epithelial barrier (the toad urinary bladder). Electron microscopy showed confluent LLC-PK1 cells with microvilli at the mucosal border. The presence of spherical or elongated intracellular vacuoles was observed in AQP2 transfected cells, specially after FK plus VP stimulus and under an osmotic gradient. These results demonstrate regulated transepithelial water permeability in epithelial cells transfected with AQP2. Received: 24 June 1997/Revised: 16 September 1997     

Temperature dependence of habituation of the initial responses to cold-water immersion

The initial responses to cold-water immersion, evoked by stimulation of peripheral cold receptors, include tachycardia, a reflex inspiratory gasp and uncontrollable hyperventilation. When immersed naked, the maximum responses are initiated in water at 10°C, with smaller responses being observed following immersion in water at 15°C. Habituation of the initial responses can be achieved following repeated immersions, but the specificity of this response with regard to water temperature is not known. Thirteen healthy male volunteers were divided into a control (C) group (n = 5) and a habituation (H) group (n = 8). Each subject undertook two 3-min head-out immersions in water at 10°C wearing swimming trunks. These immersions took place at a corresponding time of day with 4 days separating the two immersions. In the intervening period the C group were not exposed to cold water, while the H group undertook another six, 3-min, head-out immersions in water at 15°C. Respiratory rate (f _R), inspiratory minute volume (V˙ _I) and heart rate (f _H) were measured continuously throughout each immersion. Following repeated immersions in water at 15°C, the f _R, V˙ _I and f _H responses of the H group over the first 30 s of immersion were reduced (P < 0.01) from 33.3 breaths · min⁻¹, 50.5 l · min⁻¹ and 114 beats · min⁻¹ respectively, to 19.8 breaths · min⁻¹, 26.4 l · min⁻¹ and 98 beats · min⁻¹, respectively. In water at 10°C these responses were reduced (P < 0.01) from 47.3 breaths · min⁻¹, 67.6 l · min⁻¹ and 128 beats · min⁻¹ to 24.0 breaths · min⁻¹, 29.5 l · min⁻¹ and 109 beats · min⁻¹, respectively over a corresponding period of immersion. Similar reductions were observed during the last 2.5 min of immersions. The initial responses of the C group were unchanged. It is concluded that habituation of the cold shock response can be achieved by immersion in warmer water than that for which protection is required. This suggests that repeated submaximal stimulation of the cutaneous cold receptors is sufficient to attenuate the responses to more maximal stimulation. Accepted: 6 February 1998     

Culture of human hepatocytes from small surgical liver biopsies. Biochemical characterization and comparison with in vivo

Summary High yields of human hepatocytes (up to 23×10⁶ viable cells/g) were obtained from small surgical liver biopsies (1 to 3 g) by a two-step collagenase microperfusion method. Cell viability was about 95%, attachment efficiency of hepatocytes seeded on fibronectin-coated plates was 80% within 1 h after plating, and cells survived for about 2 wk in serum-free Ham’s F12 containing 0.2% bovine serum albumin, 10⁻⁸ M insulin, and 10⁻⁸ M dexamethasone. To evaluate the metabolism of human hepatocytes in serum-free conditions, we measured their most characteristic biochemical functions and compared them to those reported for human liver. After 24 h in culture, glycogen content was 1250±177 nmol glucose/mg cell protein and remained stable for several days. Gluconeogenesis from lactate in hormone-free media was (3.50±0.17 nmol glucose·mg⁻¹·min⁻¹) similar to that reported for human liver. Insulin at 10⁻⁸ M activated glycolysis (×1.40) and glycogenesis (×1.34), and glucagon at 10⁻⁹ M stimulated gluconeogenesis (×1.35) and glycogenolysis (×2.18). Human hepatocytes synthesized albumin, transferrin, fibrinogen, α₁-antitrypsin, α₁-antichymotrypsin, α₁-acid glycoprotein, haptoglobin, α₂-macroglobulin, and plasma fibronectin and excreted them to the culture medium. Maximum protein synthesis was stimulated by 10⁻⁹ M dexamethasone. Basal urea synthesis oscillated between 2.5 and 3.5 nmol·mg⁻¹ cell protein·min⁻¹, about 5 times the value estimated for human liver. Cytochrome P-450 decreased in culture but it was still 20% of freshly isolated hepatocytes by Day 5 in culture. In addition, ethoxycumarin-O-deethylase and aryl hydrocarbon hydroxylase could be induced in vitro by treatment with methyl cholanthrene. Glutathione levels were similar to those reported for human liver (35 nmol·mg⁻¹). The results of our work show that adult human hepatocytes obtained from small surgical biopsies and cultured in chemically defined conditions express their most important metabolic functions to an extent that is similar to that reported for adult human liver.     

Fish Gill Respiratory Cells in Culture: A New Model for Cl−-secreting Epithelia

Primary cultures of sea bass gill cells grown on permeable membranes form a confluent, polarized, functional tight epithelium as characterized by electron microscopy and electrophysiological and ion transport studies. Cultured with normal fetal bovine serum (FBS) and mounted in an Ussing chamber, the epithelium presents a small short-circuit current (I _sc : 1.4 ± 0.3 μA/cm²), a transepithelial voltage (V _t ) of 12.7 ± 2.7 mV (serosal positive) and a high transepithelial resistance (R _t : 12302 ± 2477 Ω× cm²). A higher degree of differentiation and increased ion transport capacities are observed with cells cultured with sea bass serum: numerous, organized microridges characteristic of respiratory cells are present on the apical cell surface and there are increased I _sc (11.9 ± 2.5 μA/cm²) and V _t (25.9 ± 1.7 mV) and reduced R _t (4271 ± 568 Ω× cm²) as compared with FBS-treated cells. Apical amiloride addition (up to 100 μm) had no effect on I _sc . The I _sc , correlated with an active Cl⁻ secretion measured as the difference between ³⁶Cl⁻ unidirectional fluxes, was partly blocked by serosal ouabain, bumetanide, DIDS or apical DPC or NPPB and stimulated by serosal dB-cAMP. It is concluded that the chloride secretion is mediated by a Na⁺/K⁺/2Cl⁻ cotransport and a Cl⁻/HCO₃ ⁻ exchanger both responsible for Cl⁻ entry through the basolateral membrane and by apical cAMP-sensitive Cl⁻ channels. This study gives evidence of a functional, highly differentiated epithelium in cultures composed of fish gill respiratorylike cells, which could provide a useful preparation for studies on ion transport and their regulation. Furthermore, the chloride secretion through these cultures of respiratorylike cells makes it necessary to reconsider the previously accepted sea water model in which the chloride cells are given the unique role of ion transport through fish gills. Received: 12 July 1996/Revised: 5 November 1996     

Cyclic GMP-phosphodiesterase inhibition does not alter cerebral oxygen consumption

The effect of zaprinast, a cyclic guanosine monophosphate inhibitor, on the level of cyclic GMP and cerebral O₂ consumption was determined. Anesthetized male Long-Evans rats were divided into a control group (n=15) and a zaprinast treated group (n=15). Vehicle was applied topically to the left cortex and 3·10⁻³ M zaprinast was applied to the right cortex. A saline treated control group was also studied. Regional cerebral blood flow was determined by [¹⁴C]-iodoantipyrine and regional O₂ extraction was determined by microspectrophotometry. The level of cyclic GMP was measured by radioimmunoassay. There were no hemodynamic or blood gas differences between groups. The level of cyclic GMP was not significantly different between the right and left cerebral cortex of the control group (17.0±4.3 and 17.7±4.6 pmol/g). In the zaprinast treated group, there was a significant (46%) increase in the level of cyclic GMP in the zaprinast treated cortex (20.5±8.1) in comparison to the vehicle treated cortex (14.0±5.7). Zaprinast did not significantly alter cerebral blood flow. There were no significant differences in regional O₂ extraction. The O₂ consumption of the zaprinast treated cortex (8.0±3.3 ml O₂·min⁻¹·100 g⁻¹) was not different from that of the vehicle treated cortex (7.0±2.9) or those of the control group. Thus, our data indicated that the increased level of cyclic GMP had no significant effect on cerebral oxygen consumption.     

Ca2+ regulation of heart contractility in Octopus

Isometric force development of electrically paced preparations isolated from the systemic heart of Octopus vulgaris were utilized to examine the regulation of contractility by Ca²⁺. Increases in extracellular Ca²⁺, to the physiological level, resulted in enhancement of twitch force. For instance, at 36 beats · min⁻¹ an increase in Ca²⁺ from 3 to 9 mmol · l⁻¹ resulted in a threefold increase in twitch force development. When steady-state contraction at 12 beats · min⁻¹ was followed by a rest period of either 5 or 10 min, the first contraction always exhibited either an increase in twitch force or stayed unchanged such that post-rest twitch force was about 133% of the last value in the steady-state train. Ryanodine (12.5 μmol · l⁻¹), which is considered to be a specific inhibitor of the Ca²⁺ storage and release capabilities of the sarcoplasmic reticulum (SR), was applied to further assess Ca²⁺ handling. Twitch force fell to about 22% of the preteatment level in preparations paced at either 12 or 36 beats · min⁻¹. In all preparations the frequency transition from 12 to 36 beats · min⁻¹ was associated with an increase in resting tension. The␣increase␣was 37 ± 14% prior to ryanodine treatment and was significantly elevated to 127 ± 33% following treatment. When steady-state contraction at 36 beats · min⁻¹ was followed by a rest period of 10 s, the first contraction was not significantly different from the last beat in the train prior to ryanodine; however, with ryanodine treatment, post-rest twitch force development significantly decreased. Twitch force development was regular at pacing rates of up to 300 beats · min⁻¹. Twitch force was maintained up to rates of 84 beats · min⁻¹ but␣decreased thereafter and reached a value of about 10% at 300 beats · min⁻¹. Resting tension increased substantially as frequency was elevated from 12 to 36 beats · min⁻¹ and then gradually increased as frequency was further elevated to 180 beats · min⁻¹. In conclusion, the Octopus ventricle is dependent upon extracellular Ca²⁺ for contraction. A post-rest potentiation of force development, the negative impact of ryanodine, and the ability to respond regularly at high pacing rates imply a strong reliance on the SR in Ca²⁺ cycling based on criteria established for vertebrate hearts. Accepted: 19 January 1997     

Transport of hemicellulose monomers in the xylose-fermenting yeastCandida shehatae

Summary Cells ofCandida shehatae repressed by growth in glucose- or D-xylose-medium produced a facilitated diffusion system that transported glucose (K _s±2 mM,V _max±2.3 mmoles g⁻¹ h⁻¹),d-xylose (K _s±125 mM,V _max±22.5 mmoles g⁻¹ h⁻¹) and D-mannose, but neither D-galactose norl-arabinose. Cells derepressed by starvation formed several sugar-proton symports. One proton symport accumulated 3-0-methylglucose about 400-fold and transported glucose (K _s±0.12 mM,V _max ± 3.2 mmoles g⁻¹ h⁻¹) andd-mannose, a second proton symport transportedd-xylose (K _s± 1.0 mM,V _max 1.4 mmoles g⁻¹ h⁻¹) andd-galactose, whilel-arabinose apparently used a third proton symport. The stoicheiometry was one proton for each molecule of glucose or D-xylose transported. Substrates of one sugar proton symport inhibited non-competitively the transport of substrates of the other symports. Starvation, while inducing the sugar-proton symports, silenced the facilitated diffusion system with respect to glucose transport but not with respect to the transport of D-xylose, facilitated diffusion functioning simultaneously with thed-xylose-proton symport.     

l-Arginine Effects on Na+ Transport in M-1 Mouse Cortical Collecting Duct Cells—A Cationic Amino Acid Absorbing Epithelium

The effect of l-arginine on transepithelial ion transport was examined in cultured M-1 mouse renal cortical collecting duct (CCD) cells using continuous short circuit current (I _SC ) measurements in HCO₃ ⁻/CO₂ buffered solution. Steady state I _SC averaged 73.8 ± 3.2 μA/cm² (n= 126) and was reduced by 94 ± 0.6% (n= 16) by the apical addition of 100 μm amiloride. This confirms that the predominant electrogenic ion transport in M-1 cells is Na⁺ absorption via the epithelial sodium channel (ENaC). Experiments using the cationic amino acid l-lysine (radiolabeled) as a stable arginine analogue show that the combined activity of an apical system y⁺ and a basal amino acid transport system y⁺L are responsible for most cationic amino acid transport across M-1 cells. Together they generate net absorptive cationic amino acid flux. Application of l-arginine (10 mm) either apically or basolaterally induced a transient peak increase in I _SC averaging 36.6 ± 5.4 μA/cm² (n= 19) and 32.0 ± 7.2 μA/cm² (n= 8), respectively. The response was preserved in the absence of bath Cl⁻ (n= 4), but was abolished either in the absence of apical Na⁺ (n= 4) or by apical addition of 100 μm amiloride (n= 6). l-lysine, which cannot serve as a precursor of NO, caused a response similar to that of l-arginine (n= 4); neither L-NMMA (100 μm; n= 3) nor L-NAME (1 mm; n= 4) (both NO-synthase inhibitors) affected the I _SC response to l-arginine. The effects of arginine or lysine were replicated by alkalinization that mimicked the transient alkalinization of the bath solution upon addition of these amino acids. We conclude that in M-1 cells l-arginine stimulates Na⁺ absorption via a pH-dependent, but NO-independent mechanism. The observed net cationic amino acid absorption will counteract passive cationic amino acid leak into the CCD in the presence of electrogenic Na⁺ transport, consistent with reports of stimulated expression of Na⁺ and cationic amino acid transporters by aldosterone. Received: 11 September 2000/Revised: 6 December 2000     

Effect of intense interval workouts on running economy using three recovery durations

The purposes of this study were to determine whether running economy (RE) is adversely affected following intense interval bouts of 10 × 400-m running, and whether there is an interaction effect between RE and recovery duration during the workouts. Twelve highly trained male endurance athletes [maximal oxygen consumption; V˙O_{2 max} =72.5 (4.3) ml·kg⁻¹·min⁻¹; mean (SD)] performed three interval running workouts of 10 × 400 m with a minimum of 4 days between runs. Recovery duration between the repetitions was randomly assigned at 60, 120 or 180 s. The velocity for each 400-m run was determined from a treadmill V˙O_{2 max} test. The average running velocity was 357.9 (9.0) m · min⁻¹. Following the workout, the rating of perceived exertion (RPE) increased significantly (P < 0.01) as recovery duration between the 400-m repetitions decreased (14.4, 16.1, and 17.7 at 180s, 120s, and 60 s recovery, respectively). Prior to and following each workout, RE was measured at speeds of 200 and 268 m · min⁻¹. Changes in RE from pre- to post-workout, as well as heart rate (HR) and respiratory exchange ratio (R) were similar for the three recovery conditions. When averaged across conditions, oxygen consumption (V˙O₂) increased significantly (P < 0.01) from pre- to post-test (from 38.5 to 40.5 ml · kg⁻¹ · min⁻¹ at 200 m · min⁻¹, and from 53.1 to 54.5 ml · kg⁻¹ · min⁻¹ at 268 m · min⁻¹, respectively). HR increased (from 124 to 138, and from 151 to 157 beats · min⁻¹ respectively) and R decreased (from 0.90 to 0.78, and from 0.93 to 0.89, respectively) at 200 and 268 m · min⁻¹, respectively (P < 0.01). This study showed that RE can be perturbed after a high-intensity interval workout and that the changes in V˙O₂, HR and R were independent of the recovery duration between the repetitions. Accepted: 23 June 1997     

Relationship between maximal oxygen uptake on different ergometers, lean arm volume and strength in paraplegic subjects

The present experiment was designed to study the importance of strength and muscle mass as factors limiting maximal oxygen uptake (V˙O_{2 max} ) in wheelchair subjects. Thirteen paraplegic subjects [mean age 29.8 (8.7) years] were studied during continuous incremental exercises until exhaustion on an arm-cranking ergometer (AC), a wheelchair ergometer (WE) and motor-driven treadmill (TM). Lean arm volume (LAV) was estimated using an anthropometric method based upon the measurement of various circumferences of the arm and forearm. Maximal strength (MVF) was measured while pushing on the rim of the wheelchair for three positions of the hand on the rim (−30°, 0° and +30°). The results indicate that paraplegic subjects reached a similar V˙O_{2 max} [1.23 (0.34) l · min⁻¹, 1.25 (0.38) l · min⁻¹, 1.22 (0.18) l · min⁻¹ for AC, TM and WE, respectively] and V˙O_{2 max} /body mass [19.7 (5.2) ml · min⁻¹ · kg⁻¹, 19.5 (6.14) ml · min⁻¹ · kg⁻¹, 19.18 (4.27) ml · min⁻¹ · kg⁻¹ for AC, TM and WE, respectively on the three ergometers. Maximal heart rate f _{c max} during the last minute of AC (173 (17) beats · min⁻¹], TM [168 (14) beats · min⁻¹], and WE [165 (16) beats · min⁻¹], were correlated, but f _{c max} was significantly higher for AC than for TM (P<0.03). There were significant correlations between MVF and LAV (P<0.001) and between the MVF data obtained at different angles of the hand on the rim [311.9 (90.1) N, 313.2 (81.2) N, 257.1 (71) N, at −30°, 0° and +30°, respectively]. There was no correlation between V˙O_{2 max} and LAV or MVF. The relatively low values of f _{c max} suggest that V˙O_{2 max} was, at least in part, limited by local aerobic factors instead of central cardiovascular factors. On the other hand, the lack of a significant correlation between V˙O_{2 max} and MVF or muscle mass was not in favour of muscle strength being the main factor limiting V˙O_{2 max} in our subjects. Accepted: 31 January 1997     

The development of an isokinetic squat device: reliability and relationship to functional performance

The aims of the present study were: (1) to assess aerobic metabolism in paraplegic (P) athletes (spinal lesion level, T4–L3) by means of peak oxygen uptake (V˙O_2peak) and ventilatory threshold (VT), and (2) to determine the nature of exercise limitation in these athletes by means of cardioventilatory responses at peak exercise. Eight P athletes underwent conventional spirographic measurements and then performed an incremental wheelchair exercise on an adapted treadmill. Ventilatory data were collected every minute using an automated metabolic system: ventilation (l · min⁻¹), oxygen uptake (V˙O₂, l · min⁻¹, ml · min⁻¹ · kg⁻¹), carbon dioxide production (V˙CO₂, ml · min⁻¹), respiratory exchange ratio, breathing frequency and tidal volume. Heart rate (HR, beats · min⁻¹) was collected with the aid of a standard electrocardiogram. V˙O_2peak was determined using conventional criteria. VT was determined by the breakpoint in the V˙CO₂−V˙O₂ relationship, and is expressed as the absolute VT (V˙O₂, ml · min⁻¹ · kg⁻¹) and relative VT (percentage of V˙O_2peak). Spirometric values and cardioventilatory responses at rest and at peak exercise allowed the measurement of ventilatory reserve (VR), heart rate reserve (HRr), heart rate response (HRR), and O₂ pulse (O₂ P). Results showed a V˙O_2peak value of 40.6 (2.5) ml · min⁻¹ · kg⁻¹, an absolute VT detected at 23.1 (1.5) ml · min⁻¹ · kg⁻¹ V˙O₂ and a relative VT at 56.4 (2.2)% V˙O_2peak. HRr [15.8 (3.2) beats · min⁻¹], HRR [48.6 (4.3) beat · l⁻¹], and O₂ P [0.23 (0.02) ml · kg⁻¹ · beat⁻¹] were normal, whereas VR at peak exercise [42.7 (2.4)%] was increased. As wheelchair exercise excluded the use of an able-bodied (AB) control group, we compared our V˙O_2peak and VT results with those for other P subjects and AB controls reported in the literature, and we compared our cardioventilatory responses with those for respiratory and cardiac patients. The low V˙O_2peak values obtained compared with subject values obtained during an arm-crank exercise may be due to a reduced active muscle mass. Absolute VT was somewhat comparable to that of AB subjects, mainly due to the similar muscle mass involved in wheelchair and arm-crank exercise by P and AB subjects, respectively. The increased VR, as reported in patients with chronic heart failure, suggested that P athletes exhibited cardiac limitation at peak exercise, and this contributed to the lower V˙O_2peak measured in these subjects. Accepted: 22 April 1997     

The dynamics of local kinetic parameters of glutamate dehydrogenase in rat liver

Kinetic parameters of glutamate dehydrogenase (GDH, EC 1.4.1.2) for glutamate were determined in periportal and pericentral zones of adult male and female rat liver lobules under normal fed conditions and after starvation for 24 h. GDH activity was measured as formazan production over time against a range of glutamate concentrations in serial cryostat sections using image analysis. Captured gray value images were transformed to absorbance images and local initial velocities (V_ini) were calculated. A hyperbolic function was used to describe the relationship between substrate concentration and local V_ini. Under fed conditions, V_max values were similar in male and female rats (8±2 and 16±2 μmol min⁻¹ cm⁻³ liver tissue in periportal and pericentral zones, respectively). Starvation increased V_max, especially in pericentral zones of females (to 27±1 μmol min⁻¹ cm⁻³ liver tissue). Under fed conditions, the affinity of GDH for glutamate was similar in male and female rats (2.5±0.5 mM and 3.5±0.8 mM in periportal and pericentral zones, respectively). Starvation had no effect on K_m values in male rats, but in female rats affinity for glutamate decreased significantly in both zones (K_m values of 4.0±0.1 mM and 8.6±0.8 mM, respectively). These local changes in the kinetic parameters of GDH indicate that conversion of glutamate to α-oxoglutarate cannot be predicted on the basis of GDH concentrations or zero-order activity in the different zones of liver lobules alone.     

Indole-3-Acetic Acid Control on Acidic Oat Cell Wall Peroxidases

Incubation of oat coleoptile segments with 40 μm indoleacetic acid (IAA) induced a decrease of 35–60% in peroxidase activity at the cell wall compartment. Treatment with IAA also produced a similar decrease in the oxidation of NADH and IAA at the cell wall. Isoelectric focusing of ionic, covalent, and intercellular wall peroxidase fractions showed that acidic isoforms (pI 4.0–5.5) were reduced preferentially by IAA treatment. Marked differences were found between acidic and basic wall isoperoxidases in relation to their efficacy in the oxidation of IAA. A peroxidase fraction containing acidic isoforms oxidized IAA with a V _max/s_0.5 value of 2.4 × 10⁻² min⁻¹· g fw⁻¹, 4.0 times higher than that obtained for basic peroxidase isoforms (0.6 × 10⁻² min⁻¹· g fw⁻¹). In contrast, basic isoforms were more efficient than acidic isoperoxidases in the oxidation of coniferyl alcohol or ferulic acid with H₂O₂ (5.6 and 2.1 times, respectively). The levels of diferulate and lignin in the walls of oat coleoptile segments were not altered by treatment with IAA. The decrease in cell wall peroxidase activity by IAA was related more to reduced oxidative degradation of the hormone than to covalent cell wall cross-linking. Received November 1, 1998; accepted December 14, 1998     

Swelling-Activated K+ Efflux and Regulatory Volume Decrease Efficiency in Human Bronchial Epithelial Cells

This study describes the correlation between cell swelling-induced K⁺ efflux and volume regulation efficiency evaluated with agents known to modulate ion channel activity and/or intracellular signaling processes in a human bronchial epithelial cell line, 16HBE14o⁻¹. Cells on permeable filter supports, differentiated into polarized monolayers, were monitored continuously at room temperature for changes in cell height (T_c), as an index of cell volume, whereas ⁸⁶Rb efflux was assessed for K⁺ channel activity. The sudden reduction in osmolality of both the apical and basolateral perfusates (from 290 to 170 mosmol/kg H₂O) evoked a rapid increase in cell volume by 35%. Subsequently, the regulatory volume decrease (RVD) restored cell volume almost completely (to 94% of the isosmotic value). The basolateral ⁸⁶Rb efflux markedly increased during the hyposmotic shock, from 0.50 ± 0.03 min⁻¹ to a peak value of 6.32 ± 0.07 min⁻¹, while apical ⁸⁶Rb efflux was negligible. Channel blockers, such as GdCl₃ (0.5 mM), quinine (0.5 mM) and 5-nitro-2-(3-phenyl-propylamino) benzoic acid (NPPB, 100 μM), abolished the RVD. The protein tyrosine kinase inhibitors tyrphostin 23 (100 μM) and genistein (150 μM) attenuated the RVD. All agents decreased variably the hyposmosis-induced elevation in ⁸⁶Rb efflux, whereas NPPB induced a complete block, suggesting a link between basolateral K⁺ and Cl⁻¹ efflux. Forskolin-mediated activation of adenylyl cyclase stimulated the RVD with a concomitant increase in basolateral ⁸⁶Rb efflux. These data suggest that the basolateral extrusion of K⁺ and Cl⁻¹ from 16HBE14o⁻¹ cells in response to cell swelling determines RVD efficiency.     

Cardiorespiratory responses to underwater treadmill walking in healthy females

This study compared the cardiorespiratory responses of eight healthy women (mean age 30.25 years) to submaximal exercise on land (LTm) and water treadmills (WTm) in chest-deep water (Aquaciser). In addition, the effects of two different water temperatures were examined (28 and 36°C). Each exercise test consisted of three consecutive 5-min bouts at 3.5, 4.5 and 5.5 km · h⁻¹. Oxygen consumption (V˙O₂) and heart rate (HR), measured using open-circuit spirometry and telemetry, respectively, increased linearly with increasing speed both in water and on land. At 3.5 km · h⁻¹ V˙O₂ was similar across procedures [χ = 0.6 (0.05) l · min⁻¹]. At 4.5 and 5.5 km · h⁻¹ V˙O₂ was significantly higher in water than on land, but there was no temperature effect (WTm: 0.9 and 1.4, respectively; LTm: 0.8 and 0.9 l · min⁻¹, respectively). HR was significantly higher in WTm at 36°C compared to WTm at 28°C at all speeds, and compared to LTm at 4.5 and 5.5 km · h⁻¹ (P ≤ 0.003). The HR-V˙O₂ relationship showed that at a V˙O₂ of 0.9 l · min⁻¹, HR was higher in water at 36°C (115 beats · min⁻¹) than either on land (100 beats · min⁻¹) or in water at 28°C (99 beats · min⁻¹). The Borg scale of perceived exertion showed that walking in water at 4.5 and 5.5 km · h⁻¹ was significantly harder than on land (WTm: 11.4 and 14, respectively; LTm: 9.9 and 11, respectively; P ≤ 0.001). These cardiorespiratory changes occurred despite a slower cadence in water (the mean difference at all speeds was 27 steps/min). Thus, walking in chest-deep water yields higher energy costs than walking at similar speeds on land. This data has implications for therapists working in hydrotherapy pools. Accepted: 3 September 1997