Characterization of Na+/H+ Exchanger Isoform (NHE1, NHE2 and NHE3) Expression in Prairie Dog Gallbladder

Gallbladder Na⁺ absorption is linked to gallstone formation in prairie dogs. Na⁺/H⁺ exchange (NHE) is one of the major Na⁺ absorptive pathways in gallbladder. In this study, we measured gallbladder Na⁺/H⁺ exchange and characterized the NHE isoforms expressed in prairie dogs. Na⁺/H⁺ exchange activity was assessed by measuring amiloride-inhibitable transepithelial Na⁺ flux and apical ²²Na⁺ uptake using dimethylamiloride (DMA). HOE-694 was used to determine NHE2 and NHE3 contributions. Basal J ^Na _ms was higher than J ^Na _sm with J ^Na _net absorption. Mucosal DMA inhibited transepithelial Na⁺ flux in a dose-dependent fashion, causing J ^Na _ms equal to J ^Na _sm and blocking J ^Na _net absorption at 100 μm. Basal ²²Na⁺ uptake rate was 10.9 ± 1.0 μmol · cm⁻²· hr⁻¹ which was inhibited by ∼43% by mucosal DMA and ∼30% by mucosal HOE-694 at 100 μm. RT-PCR and Northern blot analysis demonstrated expression of mRNAs encoding NHE1, NHE2 and NHE3 in the gallbladder. Expression of NHE1, NHE2 and NHE3 polypeptides was confirmed using isoform-specific anti-NHE antibodies. These data suggest that Na⁺/H⁺ exchange accounts for a substantial fraction of gallbladder apical Na⁺ entry and most of net Na⁺ absorption in prairie dogs. The NHE2 and NHE3 isoforms, but not NHE1, are involved in gallbladder apical Na⁺ uptake and transepithelial Na⁺ absorption. Received: 9 February 2001/Revised: 11 April 2001     

In vitro studies on calcium absorption from the gastrointestinal tract in␣small ruminants

Unidirectional flux rates of Ca²⁺ across gastrointestinal tissues from sheep and goats were measured in vitro by applying the Ussing-chamber technique. Except for the sheep duodenum, mucosal to serosal Ca²⁺ flux rates (J _ms) exceeded respective flux rates in the opposite direction (J _sm) in both species and in all segments of the intestinal tract. This resulted in net Ca²⁺ flux rates␣(J _net = J _ms − J _sm) ranging between −2 and 9 nmol · cm⁻² · h⁻¹ in sheep and between 10 and 15 nmol cm⁻² · h⁻¹ in goats. In sheep, only J _net in jejunum, and in goats, J _netin duodenum and jejunum were significantly different from zero. Using sheep rumen wall epithelia, significant J _net of Ca²⁺ of around 5 nmol · cm⁻² · h⁻¹ could be detected. Since the experiments were carried out in the absence of an electrochemical gradient, significant net Ca²⁺ absorption clearly indicates the presence of active mechanisms for Ca²⁺ transport. Dietary Ca depletion caused increased calcitriol plasma concentrations and induced significant stimulations of net Ca²⁺ absorption in goat rumen. J _net of Ca²⁺ across goat rumen epithelia was significantly reduced by 1 mmol · l ⁻¹ verapamil in the mucosal buffer solution. In conclusion, there is clear evidence for the rumen as a main site for active Ca²⁺ absorption in small ruminants. Stimulation of active Ca²⁺ absorption by increased plasma calcitriol levels and inhibition by mucosal verapamil suggest mechanistic and regulatory similarities to active Ca²⁺ transport as described for the upper small intestines of monogastric species. Accepted: 31 July 1996     

Protective effects of prostaglandins in the isolated gastric mucosa of the eel, Anguilla anguilla

The protective effect of endogenous prostaglandins on the fish gastric mucosa was evaluated by studying the effect of indomethacin and aspirin, known cyclooxigenase inhibitors, on the mucosal ulceration in the isolated gastric sacs of Anguilla anguilla. Gastric sacs devoid of muscle layers were incubated in the presence of indomethacin (10⁻⁴ mol · l⁻¹) or aspirin (10⁻⁴ mol · l⁻¹) in different experimental conditions. Both the anti-inflammatory drugs produced ulcers, but the effects were more severe in the presence of histamine and in the absence of HCO₃ ⁻ in the incubation bath. The effects of prostaglandin E₂ (PGE₂) on acid secretion rate (J_H) and on alkaline secretion rate (J_OH) were evaluated (with the aid of the pH stat method) in isolated gastric mucosa mounted in Ussing chambers. We found that PGE₂ (10⁻⁸–10⁻⁵ mol · l⁻¹) increased J_H in a dose-dependent manner. In tissues pretreated with luminal omeprazole (10⁻⁴ mol · l⁻¹), PGE₂ stimulated gastric alkaline secretion. It was nullified by serosal removal of HCO₃ ⁻ or Na⁺ and by serosal ouabain (10⁻⁴ mol · l⁻¹). These results suggested that prostaglandins also exert their protective effects in fish gastric mucosa. This protection seems partially due to a stimulation of exogenous HCO₃ ⁻ transport from the serosal to the mucosal side. It is likely that this transport is an active transcellular mechanism coupled to Na⁺ transport. Accepted: 14 April 2000     

pH gradients and a mirco-pore filter at the luminal surface affect fluxes of propionic acid across guinea pig large intestine

A neutral pH microclimate had been shown at the luminal surface of the large intestine. The aim was to estimate to what extent fluxes of propionic acid/propionate are affected by changes of the luminal pH when this microclimate is present, largely reduced or absent. Fluxes of propionic acid/propionate (J ^Pr) across epithelia from the caecum, the proximal and the distal colon of guinea pigs were measured in Ussing chambers with and without a filter at the luminal surface. With bicarbonate and with a neutral or an acid pH of mucosal solutions (pH 7.4 or 6.4), mucosal-to-serosal fluxes (J _ms^Pr) were 1.5 to 1.9-fold higher at the lower pH, in bicarbonate-free solutions and carbonic anhydrase (CA) inhibition 2.1 to 2.6-fold. With a filter at the mucosal surface and with bicarbonate containing solutions, J _ms^Pr was not or only little elevated at the lower pH. Without bicarbonate J _ms^Pr was clearly higher. We conclude that the higher J _ms^Pr after luminal acidification is due to vigorous mixing in Ussing chambers resulting in a markedly reduced unstirred layer. Therefore, an effective pH microclimate at the epithelial surface is missing. J _ms^Pr is not or is little affected by lowering of pH because in the presence of bicarbonate the filter maintains the pH microclimate. However, in bicarbonate-free solutions J _ms^Pr was higher at pH 6.4 because a pH microclimate does not develop. Findings confirm that 30–60% of J _ms^Pr results from non-ionic diffusion.     

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     

Luminal hyperosmolarity decreases Na transport and impairs barrier function of sheep rumen epithelium

The effects of luminal hyperosmolarity on Na and Cl transport were studied in rumen epithelium of sheep. An increase of luminal osmotic pressure with mannitol (350 and 450 mosm/l) caused a significant increase of tissue conductance, G _T, which is linearly correlated with flux rates of ⁵¹Cr-EDTA and indicates an increase of passive permeability. Studies with microelectrodes revealed, that an increase of the osmotic pressure caused a significant increase of the conductance of the shunt pathway from 1.23±0.10 (control) to 1.92±0.14 mS cm⁻² (450 mosm/l) without a change of fractional resistance. Hyperosmolarity significantly increased J _sm and reduced J _net Na. The effect of hyperosmolarity on J _ms Na is explained by two independent and opposed effects: increase of passive permeability and inhibition of the Na⁺/H⁺ exchanger. Hypertonic buffer solution induced a decrease of the intracellular pH (pH_i) of isolated ruminal cells, which is consistent with an inhibition of Na⁺/H⁺ exchange, probably isoform NHE-3, because NHE-3-mRNA was detectable in rumen epithelium. These data are in contrast to previous reports and reveal a disturbed Na transport and an impaired barrier function of the rumen epithelium, which predisposes translocation of rumen endotoxins and penetration of bacteria.     

The absorption of calcium ions from the ovine reticulo-rumen

Net Ca²⁺ and Mg²⁺ absorption rates were measured in vivo from buffer solutions placed in the washed reticulo-rumen, isolated in situ in 30 conscious, trained sheep. An increase in concentration of short chain fatty acids (SCFA) in the buffer, over the range 0–50 mM, was shown to stimulate the net rates of absorption of Ca²⁺ and Mg²⁺ ions from the rumen. Similarly, the results of in vitro experiments, carried out with ovine rumen epithelium mounted in short-circuited Ussing chambers, showed that the absence of SCFA from the chamber fluid resulted in a reduction in J_net Ca²⁺ caused by reduced flux of Ca²⁺ ions in the mucosal to serosal direction (J_ms Ca²⁺). The addition of 1 mM acetazolamide, an inhibitor of carbonic anhydrase, to the ruminal buffer used in the in vivo experiments led to significant reductions in the net absorption rates of Ca²⁺and Mg²⁺ ions in the presence of SCFA (50 mmol l⁻¹) but not in the absence of SCFA. However, in the in vitro experiments, the addition of 60 μM ethoxyzolamide had no significant effect on J_net Ca²⁺. A reduction in pH of the intraruminal buffer in vivo from 6.8 to 5.4 led to significant increases in the net absorption rates of Ca²⁺and Mg²⁺ ions, an effect which was duplicated for Ca²⁺ in preliminary in vitro experiments in which the pH of the mucosal buffer was reduced from 7.4 to 5.4. This stimulatory effect was confined to J_ms Ca²⁺ and J_net Ca²⁺. Ussing chambers were also used to demonstrate that J_net Ca²⁺ was reduced by a high transmural potential difference (PD), caused by voltage clamping, independently of the mucosal K⁺ concentration. Both unidirectional Ca²⁺ fluxes consisted of a PD-dependent and a K⁺-insensitive PD-independent component. The latter may be represented by a Ca²⁺/2H⁺ antiporter. It is postulated that SCFA, and to a lesser extent H₂CO₃, can stimulate J_ms Ca²⁺ by activation of an apical Ca²⁺/2H⁺ antiporter through the provision of protons within the ruminal epithelial cell. A mild reduction in ruminal pH may also lead to a similar stimulation of this putative electroneutral exchange. Accepted: 26 July 2000     

Characterisation of <Emphasis Type="SmallCaps">l</Emphasis>-alanine and glycine absorption across the gut of an ancient vertebrate

This study utilised an in vitro technique to characterise absorption of two amino acids across the intestinal epithelium of Pacific hagfish, Eptatretus stoutii. Uptake of l-alanine and glycine conformed to Michaelis–Menten kinetics. An uptake affinity (K _m; substrate concentration required to attain a 50% uptake saturation) of 7.0 mM and an uptake capacity (J _max) of 83 nmol cm⁻² h⁻¹ were described for l-alanine. The K _m and J _max for glycine were 2.2 mM and 11.9 nmol cm⁻² h⁻¹, respectively. Evidence suggested that the pathways of l-alanine and glycine absorption were shared, and sodium dependent. Further analysis indicated that glycine uptake was independent of luminal pH and proline, but a component of uptake was significantly impaired by 100-fold excesses of threonine or asparagine. The presence of a short-term (24 h) exposure to waterborne glycine, similar in nature to that which may be expected to occur when feeding inside an animal carcass, had no significant impact on gastrointestinal glycine uptake. This may indicate a lack of cross talk between absorptive epithelia. These results are the first published data to describe gastrointestinal uptake of an organic nutrient in the oldest extant vertebrate and may provide potential insight into the evolution of nutrient transport systems.     

Physical characterization of high-affinity gastrointestinal Cu transport in vitro in freshwater rainbow trout Oncorhynchus mykiss

This study investigated the transport of copper (Cu) in the gut of trout. Examination of the spatial distribution of Cu along the digestive tract and a physical characterization of the uptake process was carried out using an in vitro gut sac technique and ⁶⁴Cu as a tracer. Unidirectional Cu uptake was highest in the anterior intestine followed in decreasing order by the posterior intestine, mid intestine and the stomach. Cu uptake was resistant to hypoxia and appeared to be fueled equally well by Cu(II) or Cu (I) at Cu concentrations typically found in the fluid phase of the chyme in vivo in the trout intestine. Transport demonstrated saturation kinetics (e.g. K _m = 31.6 μM, J _max = 17 pmol cm⁻² h⁻¹, in mid intestine) at low Cu levels representative of those measured in the chyme in vivo, with a diffusive component at higher Cu concentrations. Q ₁₀ analysis indicated Cu uptake is via diffusion across the apical membrane and biologically mediated across the basolateral membranes of enterocytes. The presence of l-histidine but not d-histidine stimulated both Cu and Na uptake suggesting a common pathway for the transport of Cu/Na with l-histidine.     

Localization of aquaporin-2, renal morphology and urine composition in the bottlenose dolphin and the Baird’s beaked whale

This study examined the distribution pattern of aquaporin-2 (AQP2), relative medullary thickness (RMT) and urine properties in the bottlenose dolphin Tursiops truncatus and Baird’s beaked whale Berardius bairdii. Immunohistochemical studies revealed that AQP2 was localized in the collecting tubules/ducts of both species’ renicules, as in terrestrial mammals. The collecting ducts with AQP2 were thinner and arranged more densely in the dolphin than in the whale. RMT values in the renicule were moderate in both species, but were significantly higher in the dolphin (6.0 ± 0.9) than the whale (4.9 ± 0.7). Urine of the bottlenose dolphin is comparatively concentrated (osmolality: 1715.7 ± 279.4 mOsm kg⁻¹, Na⁺: 490.1 ± 87.9 mmol l⁻¹, Cl⁻: 402.7 ± 79.6 mmol l⁻¹, K⁺: 80.7 ± 25.8 mmol l⁻¹, urea nitrogen: 703.5 ± 253.9 mmol l⁻¹), while urine of the dead Baird’s beaked whale is less concentrated (osmolality: 837.5 ± 293.8 mOsm kg⁻¹, Na⁺: 192.9 ± 81.5 mmol l⁻¹, Cl⁻: 159.9 ± 71.4 mmol l⁻¹, K⁺: 44.3 ± 29.5 mmol l⁻¹, urea nitrogen: 270.7 ± 120.3 mmol l⁻¹). These data suggest it is possible that the differences in these renal morphological features may be related in some way to the difference in urine composition between the species, although further studies are necessary. M. Suzuki and N. Endo are equal contributors to this study.     

Glycolytic pathway and hydrogen yield studies of the extreme thermophile <Emphasis Type="Italic">Caldicellulosiruptor saccharolyticus</Emphasis>

NMR analysis of ¹³C-labelling patterns showed that the Embden–Meyerhof (EM) pathway is the main route for glycolysis in the extreme thermophile Caldicellulosiruptor saccharolyticus. Glucose fermentation via the EM pathway to acetate results in a theoretical yield of 4 mol of hydrogen and 2 mol of acetate per mole of glucose. Previously, approximately 70% of the theoretical maximum hydrogen yield has been reached in batch fermentations. In this study, hydrogen and acetate yields have been determined at different dilution rates during continuous cultivation. The yields were dependent on the growth rate. The highest hydrogen yields of 82 to 90% of theoretical maximum (3.3 to 3.6 mol H₂ per mol glucose) were obtained at low growth rates when a relatively larger part of the consumed glucose is used for maintenance. The hydrogen productivity showed the opposite effect. Both the specific and the volumetric hydrogen production rates were highest at the higher growth rates, reaching values of respectively 30 mmol g⁻¹ h⁻¹ and 20 mmol l⁻¹ h⁻¹. An industrial process for biohydrogen production will require a bioreactor design, which enables an optimal mix of high productivity and high yield.     

H+ and Cl− secretion in the stomach of the teleost fish, Anguilla anguilla : stimulation by histamine and carbachol

The fundus of an eel stomach was mounted in an Ussing chamber and bathed with control Ringer on the serosal side and with unbuffered solution on the mucosal side. The gastric mucosa exhibited a mucosa negative transepithelial voltage (V _t), a “short circuit” current (I _SC) and a small spontaneous acid secretion rate (J _H). All these parameters were abolished by cimetidine treatment. Bilateral ion substitution experiments in tissues lacking spontaneous acid secretion suggested that a net Cl⁻ transport from serosa to mucosa was responsible for the genesis of the I _SC in the absence of H⁺ secretion. Serosal application of histamine (10⁻⁴ mol · l⁻¹) or carbachol (10⁻⁴ mol · l⁻¹) stimulated both I _SC and J _H. The action of carbachol was independent of histamine. The control as well as the histamine-stimulated I _SC was sensitive to both serosal bumetanide (10⁻⁵ mol · l⁻¹), inhibitor of the Na⁺-K⁺-2Cl⁻ cotransport, and 4,4-diisothiocyano-stilbene-2,2-disulphonic acid (DIDS, 5 · 10⁻⁴ mol · l⁻¹), inhibitor of the Cl⁻-HCO⁻ ₃ exchange, while the I _SC stimulated by carbachol was nullified by serosal DIDS. These data suggested that the non-acidic Cl⁻ uptake across the serosal membrane was linked to the activity of both Na⁺-K⁺-2Cl⁻ cotransport and Cl⁻-HCO⁻ ₃ antiporter; histamine stimulated both transporters while carbachol was limited to the anion exchanger. The finding that the acid secretion was strictly dependent on serosal Cl⁻ and was completely blocked by serosal DIDS suggested that the Cl⁻ accompanying H⁺ secretion entered the cell through the serosal membrane by the Cl⁻-HCO⁻ ₃ exchange. In addition, the acid secretion stimulated by carbachol was also dependent on serosal Na⁺ and sensitive to the application of 5-N-N-dimethyl-amiloride in the serosal bath, suggesting that the increased activity of the Cl⁻-HCO⁻ ₃ during carbachol treatment was linked to the activation of serosal Na⁺-H⁺ exchange. The inhibitory effect of luminal omeprazole (10⁻⁴ mol · l⁻¹) on acid secretion suggested the presence of the H⁺-K⁺ pump on the luminal membrane. Accepted: 18 September 1997     

Purification and properties of the cell-associated beta-xylosidase from Aureobasidium

β-Xylosidase was extracted from Aureobasidium sp. ATCC 20524 and purified to homogeneity. The molecular mass was estimated at 411 kDa. The enzyme contained 15.3% (w/w) carbohydrate. The optimum pH and temperature were pH 3.5 and 80°C, respectively. The enzyme was stable at pH 3.5–9 after 3 h and at 80°C after 15 min. The Michaelis constant (K _m) and maximum velocity (V _max) toward p-nitrophenyl-β-D-xyloside were 2.0 mmol l⁻¹ and 0.94 mmol min⁻¹ mg⁻¹ protein, respectively. The enzyme was inhibited strongly by mercury, lead, and copper ions. Journal of Industrial Microbiology & Biotechnology (2001) 26, 276–279. Received 02 August 2000/ Accepted in revised form 15 December 2000     

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     

Histidine Absorption across Apical Surfaces of Freshwater Rainbow Trout Intestine: Mechanistic Characterization and the Influence of Copper

The essential amino acid histidine performs critical roles in health and disease. These functions are generally attributed to the amino acid itself, but could also be mediated by a positive effect on trace element bioavailability. Mechanistic information regarding the absorption of histidine across the gastrointestinal tract is essential for understanding the interplay between amino acid and mineral nutrients and the implications of these interactions for nutrition and toxicology. Using intestinal brush-border membrane vesicles obtained from freshwater rainbow trout, absorption of histidine over the range 0.78–780 μm was found to be saturable, with a maximal transport rate (J _max) of 9.1 ± 0.8 nmol mg protein⁻¹ min⁻¹ and a K _m (histidine concentration required to reach 50% of this level) of 339 ± 68 μm. Histidine uptake was highly specific as 10-fold elevated levels of a variety of amino acids with putative shared transporters failed to significantly inhibit uptake. Elevated levels of d-histidine, however, impaired uptake of the natural l-isomer. The presence of “luminal” copper (8.3 μm) significantly increased both the J _max and K _m of histidine transport. This suggests that chelated copper–histidine species cross the brush-border epithelium through transport pathways distinct from those used by histidine alone.     

Transport of butyrate across the isolated bovine rumen epithelium — interaction with sodium, chloride and bicarbonate

The Ussing chamber technique was used for studying unidirectional fluxes of ¹⁴C-butyrate across the bovine rumen epithelium in vitro. Significant amounts of butyrate were absorbed across the bovine rumen epithelium in vitro, without any external driving force. The paracellular pathway was quantitatively insignificant. The transcellular pathway was predominately voltage-insensitive. The serosal to mucosal (SM) pathway was regulated by mass action, whereas the mucosal to serosal (MS) pathway further includes a saturable process, which accounted for 30 to 55% of the MS flux. The studied transport process for ¹⁴C-butyrate across the epithelium could include metabolic processes and transport of ¹⁴C-labelled butyrate metabolites. The transport of butyrate interacted with Na⁺, Cl⁻ and HCO₃⁻, and there was a linear relationship between butyrate and sodium net transport. Lowering the sodium concentration from 140 to 10 mmol l⁻¹ decreased the butyrate MS flux significantly. Amiloride (1 mmol l⁻¹) did, however, not reduce the butyrate flux significantly. Chloride concentration in itself did not seem to influence the transport of butyrate, but chloride-free conditions tended to increase the MS and SM flux of butyrate by a DIDS-sensitive pathway. DIDS (bilateral 0.5 mmol l⁻¹) did further decrease the butyrate SM flux significantly at all chloride concentrations. Removing bicarbonate from the experimental solutions decreased the MS and increased the SM flux of butyrate significantly, and abolished net butyrate flux. There were no significant effects of the carbonic anhydrase inhibitor Acetazolamide (bilateral 1.0 mmol l⁻¹). The results can be explained by a model where butyrate and butyrate metabolites are transported both by passive diffusion and by an electroneutral anion-exchange with bicarbonate. The model couples sodium and butyrate via CO₂ from metabolism of butyrate, and intracellular pH.     

Facilitated Transport of Lactate by Rat Jejunal Enterocyte

L-lactate transport mechanism across rat jejunal enterocyte was investigated using isolated membrane vesicles. In basolateral membrane vesicles l-lactate uptake is stimulated by an inwardly directed H⁺ gradient; the effect of the pH difference is drastically reduced by FCCP, pCMBS and phloretin, while furosemide is ineffective. The pH gradient effect is strongly temperature dependent. The initial rate of the proton gradient-induced lactate uptake is saturable with respect to external lactate with a K _m of 39.2 ± 4.8 mm and a J _max of 8.9 ± 0.7 nmoles mg protein⁻¹ sec⁻¹. A very small conductive pathway for l-lactate is present in basolateral membranes. In brush border membrane vesicles both Na⁺ and H⁺ gradients exert a small stimulatory effect on lactate uptake. We conclude that rat jejunal basolateral membrane contains a H⁺-lactate cotransporter, whereas in the apical membrane both H⁺-lactate and Na⁺-lactate cotransporters are present, even if they exhibit a low transport rate. Received: 22 October 1996/Revised: 11 March 1997     

Effect of sodium citrate on performance and metabolism of human skeletal muscle during supramaximal cycling exercise

The aim of this study was to examine whether the alkalosis-induced improvement in supramaximal performance could be explained by a less-altered muscle metabolic status. Eight subjects first performed exhausting exercise at 120% peak oxygen uptake after ingesting either a placebo (PLC) or sodium citrate (CIT) at a dose of 0.5 g · kg⁻¹ body mass to determine exhaustion time (t _exh). They then, performed exercise (Lim-EX) at the same relative intensity lasting PLCt _exh minus 20 s in both treatments. Samples were taken from vastus lateralis muscle at rest (90-min after the ingestion) and at the end of Lim-EX. Arterial blood samples were obtained at rest (immediately prior to and 90 min after ingesting the drug) and during the 20-min post-exercise recovery. The t _exh was significantly increased by CIT [PLC 258 (SD 29) s, CIT 297 (SD 45) s]. The CIT raised the rest [citrate] in blood [PLC 0.11 (SD 0.01) mmol · l⁻¹, CIT 0.34 (SD 0.07) mmol · l⁻¹] and in muscle [PLC 0.78 (SD 0.23) mmol · kg⁻¹ dry mass, CIT 1.00 (SD 0.21) mmol · kg⁻¹ dry mass]. Resting muscle pH and buffering capacity were unchanged by CIT. The same fall in muscle pH was observed during Lim-EX in the two conditions. This was associated with similar variations in both the cardio-respiratory response and muscle energy and metabolism status in spite of a better blood acid-base status after CIT. Thus, CIT would not seem to allow the alkalinization of the muscle cytosolic compartment. Though sodium citrate works in a similar way to NaHCO₃ on plasma alkalinization and exercise performance, the exact nature of the mechanisms involved in the delay of exhaustion could be different and remains to be elucidated. Accepted: 26 November 1996     

Effect of volatile fatty acids on Shigella dysenteriae during anaerobic digestion of human night soil

Thein vitro toxic effect of different volatile fatty acids (VFA) on Shigella dysenteriae was studied in pure culture. Volatile fatty acids viz., acetate, propionate, butyrate, valerate, caproate and heptanoate, exerted pH dependent toxic effect on the pathogen, with minimum inhibitory concentration in the range of 10–3000 mg l⁻¹. The effect of high levels of VFA on S. dysenteriae was studied during anaerobic digestion of human night soil in an experimental digester with VFA level ≅ 9000 mg l⁻¹ and pH ≅ 6.5. Another digester, with VFA level ≅ 700 mg l⁻¹ and pH 7.4, served as the control. In the experimental digester, S. dysenteriae was completely eliminated within 18 days. In the control digester, a four-log reduction in pathogen count was achieved however the pathogen was not completely eliminated. T ₉₀ values for the experimental and control digesters were 2.2 and 3.7 days respectively. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of the oxygen supply on pattern of growth and corrinoid and organic acid production of Propionibacterium shermanii

Propionibacterium shermanii CDB 10014 is able to grow even at high oxygen transfer rates (24.0 mmol O₂ l⁻¹ h⁻¹), in contrast to reports in the specialised literature, where all Propionibacteria are considered oxygen-sensitive microorganisms. Propionic acid is the main product in anaerobiosis. The presence of oxygen in the system leads to an inhibition of propionic acid production while acetic acid formation is enhanced. At high oxygen supply rates no propionic acid is produced and acetic acid is the main product. Lactic acid is also produced in reasonable quantities (2.7 g l⁻¹). The growth rate (μ_max) is higher in anaerobiosis (0.19 h⁻¹) than in aerobiosis (0.12–0.15 h⁻¹). The cell yield is higher in aerobiosis (0.18–0.22 g g⁻¹) than in anaerobiosis (0.14 g g⁻¹) suggesting the oxidative metabolism of glucose by Propionibacterium shermanii CDB 10014. No corrinoid production was detected at oxygen transfer rates of more than 13.6 mmol l⁻¹ h⁻¹. Received: 10 September 1997 / Received revision: 6 January 1998 / Accepted: 9 January 1998