Consequences of renal mass reduction on amino acid and biogenic amine levels in nephrectomized mice

Summary. Amino acid and biogenic amine changes were investigated in nephrectomized mice ten days postsurgery. Uremic mice exhibited changes in amino acid concentrations in plasma, urine and brain. Particularly plasma methionine, citrulline and arginine levels were significantly enhanced in nephrectomized mice compared to controls whereas serine was decreased. Urinary excretion of methionine, citrulline and alanine was higher in nephrectomized mice compared to controls whereas many amino acids were increased in brain of nephrectomized mice. Brain and urinary amino acid changes were more pronounced in the 75% than in the 50% nephrectomized mice. Brain norepinephrine and dopamine and its metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid were significantly increased whereas serotonin was decreased comparing the 75% nephrectomized mice to the sham-operated mice. This study demonstrates that at very early stages of renal insufficiency, specific amino acid and biogenic amine changes occur in plasma, urine and brain. These alterations might depend qualitatively and quantitatively on the degree of functional renal mass reduction. Received April 5, 1999     

High Protein Diet and Huntington's Disease

Huntington’s disease (HD) is a neurodegenerative disorder caused by the huntingtin (HTT) gene with expanded CAG repeats. In addition to the apparent brain abnormalities, impairments also occur in peripheral tissues. We previously reported that mutant Huntingtin (mHTT) exists in the liver and causes urea cycle deficiency. A low protein diet (17%) restores urea cycle activity and ameliorates symptoms in HD model mice. It remains unknown whether the dietary protein content should be monitored closely in HD patients because the normal protein consumption is lower in humans (~15% of total calories) than in mice (~22%). We assessed whether dietary protein content affects the urea cycle in HD patients. Thirty HD patients were hospitalized and received a standard protein diet (13.7% protein) for 5 days, followed by a high protein diet (HPD, 26.3% protein) for another 5 days. Urea cycle deficiency was monitored by the blood levels of citrulline and ammonia. HD progression was determined by the Unified Huntington’s Disease Rating Scale (UHDRS). The HPD increased blood citrulline concentration from 15.19 μmol/l to 16.30 μmol/l (p = 0.0378) in HD patients but did not change blood ammonia concentration. A 2-year pilot study of 14 HD patients found no significant correlation between blood citrulline concentration and HD progression. Our results indicated a short period of the HPD did not markedly compromise urea cycle function. Blood citrulline concentration is not a reliable biomarker of HD progression.     

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     

Low-Carbohydrate,High-Protein Diet Affects Rhythmic Expression of Gluconeogenic Regulatory and Circadian Clock Genes in Mouse Peripheral Tissues

Recent studies have demonstrated that metabolic changes in mammals induce feedback regulation of the circadian clock. The present study evaluates the effects of a low-carbohydrate high-protein diet (HPD) on circadian behavior and peripheral circadian clocks in mice. Circadian rhythms of locomotor activity and core body temperature remained normal in mice fed with the HPD diet (HPD mice), suggesting that it did not affect the central clock in the hypothalamus. Two weeks of HPD feeding induced mild hypoglycemia without affecting body weight, although these mice consumed more calories than mice fed with a normal diet (ND mice). Plasma insulin levels were increased during the inactive phase in HPD mice, but increased twice, beginning and end of the active phase, in ND mice. Expression levels of the key gluconeogenic regulatory genes PEPCK and G6Pase were significantly induced in the liver and kidneys of HPD mice. The HPD appeared to induce peroxisome proliferator-activated receptor α (PPARα) activation, since mRNA expression levels of PPARα and its typical target genes, such as PDK4 and Cyp4A10, were significantly increased in the liver and kidneys. Circadian mRNA expression of clock genes, such as BMAL1, Cry1, NPAS2, and Rev-erbα, but not Per2, was significantly phase-advanced, and mean expression levels of BMAL1 and Cry1 mRNAs were significantly elevated, in the liver and kidneys of HPD mice. These findings suggest that a HPD not only affects glucose homeostasis, but that it also advances the molecular circadian clock in peripheral tissues. (Author correspondence: k-ooishi@aist.go.jp)     

DMPS and N-acetylcysteine induced renal toxicity in mice exposed to mercury

Acute effects of mercuric chloride (HgCl₂) were evaluated on mice. Mice received a single dose of HgCl₂ (4.6 mg/kg, subcutaneously) for three consecutive days. Thirty minutes after the last injection with HgCl₂, mice received one single injection of 2,3-dimercapto-1-propanesulfonic acid (DMPS) or N-acetylcysteine (NAC) or diphenyl diselenide (PhSe)₂. DMPS, NAC and (PhSe)₂ were utilized as therapy against mercury exposure. At 24 h after the last HgCl₂ injection, blood, liver and kidney samples were collected. δ-Aminolevulinate dehydratase (δ-ALA-D) and Na⁺, K_-⁺ ATPase activities, thiobarbituric acid-reactive substances (TBARS), non-protein thiols (NPSH) and ascorbic acid concentrations were evaluated. Plasma aspartate (AST) and alanine (ALT) aminotransferase activities, as well as urea and creatinine levels were determined. The group of mice exposed to Hg + (PhSe)₂ presented 100% of lethality. Exposure with HgCl₂ caused a decrease on the body weight gain and treatments did not modify this parameter. δ-ALA-D, AST and ALT activities, TBARS, ascorbic acid levels and NPSH (hepatic and erythrocytic) levels were not changed after HgCl₂ exposure. HgCl₂ caused an increase in renal NPSH content and therapies did not modify these levels. Mice treated with (PhSe)₂, Hg + NAC and Hg + DMPS presented a reduction in plasma NPSH levels. Creatinine and urea levels were increased in mice exposed to Hg + NAC, while Hg + DMPS group presented an increase only in urea level. Na⁺, K_-⁺ ATPase activity was inhibited in mice exposed to Hg + DMPS and Hg + NAC. In conclusion, therapies with (PhSe)₂, DMPS and NAC following mercury exposure must be better studied because the formation of more toxic complexes with mercury, which can mainly damage renal tissue.     

Betaine activates a hyperpolarizing chloride conductance in squid olfactory receptor neurons

Isolated olfactory receptor neurons from the squid Lolliguncula brevis respond to betaine, a repellent odorant, with hyperpolarizing receptor potentials. Using perforated-patch techniques, we determined that the hyperpolarizing conductance was selective for Cl⁻ and could be reversibly blocked by the Cl⁻ channel blockers 4-acetamido-4′-isothio-cyanatistilbene-2,2′disulfonic acid and niflumic acid. Gramicidin-patch recordings revealed that [Cl⁻]_i in squid olfactory receptor neurons is normally very low compared to vertebrate olfactory receptor neurons, and that activating a Cl⁻ conductance would hyperpolarize the cell in vivo. The lack of dependence on internal or external K⁺ or Na⁺ ruled out the possibility that the Cl⁻ conductance was generated by a cation-dependent cotransporter or pump. Common G-protein-dependent signalling pathways, including phospholipase C, arachidonic acid, and cyclic nucleotides, do not appear to be involved. Ca²⁺ imaging experiments showed that betaine did not affect [Ca²⁺]_i, suggesting that the Cl⁻ current is not Ca²⁺ dependent. Our findings represent the first report of an odorant-activated, hyperpolarizing chloride conductance in olfactory receptor neurons. Accepted: 20 March 1998     

Chloride Conductance and P i Transport are Separate Functions Induced by the Expression of NaPi-1 in Xenopus Oocytes

Expression of the protein NaPi-1 in Xenopus oocytes has previously been shown to induce an outwardly rectifying Cl⁻ conductance (G_Cl), organic anion transport and Na⁺-dependent P _i -uptake. In the present study we investigated the relation between the NaPi-1 induced G_Cl and P _i -induced currents and transport. NaPi-1 expression induced P _i -transport, which was not different at 1–20 ng/oocyte NaPi-1 cRNA injection and was already maximal at 1–2 days after cRNA injection. In contrast, G_Cl was augmented at increased amounts of cRNA injection (1–20 ng/oocyte) and over a five day expression period. Subsequently all experiments were performed on oocytes injected with 20 ng/oocytes cRNA. P _i -induced currents (Ip) could be observed in NaPi-1 expressing oocytes at high concentrations of P _i (≥ 1 mm P _i ). The amplitudes of Ip correlated well with G_Cl. Ip was blocked by the Cl⁻ channel blocker NPPB, partially Na⁺-dependent and completely abolished in Cl⁻ free solution. In contrast, P _i -transport in NaPi-1 expressing oocytes was not NPPB sensitive, stronger depending on extracellular Na⁺ and weakly affected by Cl⁻ substitution. Endogenous P _i -uptake in water-injected oocytes amounted in all experiments to 30–50% of the Na⁺-dependent P _i -transport observed in NaPi-1 expressing oocytes. The properties of the endogenous P _i -uptake system (K _m for P _i > 1 mm; partial Na⁺- and Cl⁻-dependence; lack of NPPB block) were similar to the NaPi-1 induced P _i -uptake, but no Ip could be recorded at P _i -concentrations ≤3 mm. In summary, the present data suggest that Ip does not reflect charge transfer related to P _i -uptake, but a P _i -mediated modulation of G_Cl. Received: 22 October 1997/Revised: 24 March 1998     

Regulatory volume decrease after swelling induced by urea in fibroblasts: prominent role of organic osmolytes

Cell swelling, regulatory volume decrease (RVD), volume-sensitive Cl⁻ (Cl⁻ _swell) current and taurine efflux after exposure to high concentrations of urea were characterized in fibroblasts Swiss 3T3, and results compared to those elicited by hyposmotic (30%) swelling. Urea 70, 100, and 150 mM linearly increased cell volume (8.25%, 10.6%, and 15.7%), by a phloretin-inhibitable process. This was followed by RVD by which cells exposed to 70, 100, or 150 mM urea recovered 27.6%, 38.95, and 74.1% of their original volume, respectively. Hyposmolarity (30%) led to a volume increase of 25.9% and recovered volume in 32.5%. ³H-taurine efflux was increased by urea with a sigmoid pattern, as 9.5%, 18.9%, 71.5%, and 89% of the labeled taurine pool was released by 70, 100, 150, or 200 mM urea, respectively. Only about 11% of taurine was released by 30% hyposmolarity reduction in spite of the high increase in cell volume. Urea-induced taurine efflux was suppressed by NPPB (100 μM) and markedly reduced by the tyrosine kinase-general blocker AG18. The Cl⁻ _swell current was more rapidly activated and higher in amplitude in the hyposmotic than in the isosmotic/urea condition (urea 150 mM), but this was not sufficient to accomplish an efficient RVD. These results showed that at similar volume increase, cells swollen by urea showed higher taurine efflux, lower Cl⁻ _swell current and more efficient RVD, than in those swollen by hyposmolarity. The correlation found between RVD efficiency and taurine efflux suggest a prominent role for organic over ionic osmolytes for RVD evoked by urea in isosmotic conditions.     

Electrophysiological characterization of chloride secretion across the jejunum and colon of pigs as affected by age and weaning

Hypersecretion of chloride can cause diarrhea, a disease frequently occurring in young pigs, particularly around weaning. We investigated the contribution of different channels to intestinal Cl⁻ secretion as influenced by age and weaning. Jejunal and colonic epithelia from 4-month-old pigs and 4-week-old piglets were incubated in Ussing chambers and stimulated by carbachol and forskolin. Changes in short-circuit currents were taken as measure of electrogenic net Cl⁻ secretion. DIDS or NPPB served to inhibit Ca-activated Cl⁻-channels and outwardly rectifying Cl⁻-channels (ORCC) or cystic fibrosis transmembrane regulator (CFTR), respectively. Depolarizing the basolateral membrane allowed to examine the influence of K⁺-channels on Cl⁻ secretion. Forskolin-stimulated Cl⁻ secretion was mediated by CFTR. ORCC were not involved. Carbachol-induced Cl⁻ secretion could be ascribed to an enhanced driving force due to the opening of K⁺-channels, whereas Ca-dependent Cl⁻ channels seemed not to be involved. In jejunum, piglets showed higher Cl⁻ secretion than pigs. Two days after weaning forskolin induced an I _sc overshoot and a faster increase in G _t. In colon, Cl⁻ secretion was neither influenced by age nor by weaning. The data suggest a disposition of porcine jejunum for a higher Cl⁻ secretion in young and freshly weaned piglets, which might be a natural defense mechanism as well as a predisposing factor for diarrhea.     

Tetrachloroethene dechlorination kinetics by Dehalospirillum multivorans immobilized in upflow anaerobic sludge blanket reactors

Tetrachloroethene (C₂Cl₄) dechlorination kinetics in upflow anaerobic sludge blanket (UASB) reactors was determined after introducing de novo activities into the granular sludge. These activities were introduced by immobilizing Dehalospirillum multivorans in a test reactor containing unsterile granular sludge, and in a reference reactor, R1, containing sterile granular sludge. A second reference reactor, R2, contained only unsterile granular sludge and served as a control. The kinetic experiments were performed by pulsing the reactors with C₂Cl₄ in a recirculating batch mode. Formate and acetate were added as electron donor and carbon source. Both reactors inoculated with D. multivorans dechlorinated C₂Cl₄ to an equimolar amount of C₂H₂Cl₂ with only traces of C₂HCl₃ in the effluent. In the control reactor, C₂HCl₃ accumulated before C₂H₂Cl₂ was produced. A computer simulation program (AQUASIM) was used to estimate the kinetic parameters. The half-saturation constants (K _s) for C₂Cl₄ and C₂HCl₃ were almost equal in the reactors containing D.␣multivorans (17 μM and 18 μM for C₂Cl₄; 26 μM and 28 μM for C₂HCl₃), indicating no influence of sludge bacteria on the affinity of D. multivorans for C₂Cl₄ and C₂HCl₃. The maximum dechlorination rates (k _m X _B) were about twice as high in the reactor containing D.␣multivorans immobilized in sterile sludge (11 mmol C₂Cl₄ l sludge⁻¹ day⁻¹ and 27 mmol C₂HCl₃ l sludge⁻¹ day⁻¹) than in the test reactor (4.4 mmol C₂Cl₄ l sludge⁻¹ day⁻¹ and 15 mmol C₂HCl₃ l sludge⁻¹ day⁻¹). Compared to other C₂Cl₄-degrading systems, the dechlorination rates of the inoculated reactors and their affinities for C₂Cl₄ and C₂HCl₃ were high. Therefore, introduction of de novo activity is promising for the use of anaerobic reactors to bioremediate C₂Cl₄-polluted water. Received: 5 November 1998 / Received revision: 25 January 1999 / Accepted: 31 January 1999     

Effects of NEM on Voltage-activated Chloride Conductance in Toad Skin

The regulation of the voltage-activated chloride current conductance (G _Cl ) in toad skin was investigated by the use of the SH reagents N-ethylmaleimide (NEM) and p-chloro-mercuricbenzenesulfonic acid PCMBS. This anion pathway is controlled by a voltage-sensitive gating regulator. Mucosal application of NEM decreased the voltage-activation in a time and concentration dependent manner, half-maximal inhibition being exerted at a concentration of 30 μm within 20 min. At concentrations higher than 100 μm, the voltage-activated G _Cl was near-completely and irreversibly inhibited in less than 10 min. Resting, deactivated conductance was essentially unaffected. NEM had no effect on active sodium transport (measured as I _sc ) under conditions, which fully dissipated the voltage-activated G _Cl . After complete inhibition of the voltage-activated G _Cl with NEM, chloride conductance could still be stimulated by CPT-cAMP as in control tissues. Under these conditions, NEM at concentrations above 1 mm decreased G _Cl reversibly. Mucosal application of PCMBS at 500 μm inhibited the activated conductance by 35%, which was slightly reversible. Inhibition of voltage-activated G _Cl , which was observed after mucosal addition of the membrane-impermeable NEM analogue, eosin-5-maleimide, was completely reversible after washout. This suggests that the binding site for the maleimide is not accessible from the external face of the apical membrane. Brief application of NEM at lower concentrations (1–3 min, ≤100 μm) led to partial inhibition of G _Cl , followed by occasionally complete recovery upon washout of NEM. Recovery of voltage-activated G _Cl was progressively attenuated and eventually disappeared after subsequent brief applications of NEM. This could reflect recruitment of permeation/control sites from a finite pool. The data are discussed in the frame of a working model for the voltage-activated Cl⁻-pathway, that contains two principle components, i.e., an anion-selective permeation path which is controlled by regulatory protein(s). Received: 18 December 1996/Revised: 28 April 1997     

Concurrent and Independent HCO− 3 and Cl− Secretion in a Human Pancreatic Duct Cell Line (CAPAN-1)

The present study investigated both HCO⁻ ₃ and Cl⁻ secretions in a human pancreatic duct cell line, CAPAN-1, using the short-circuit current (I _sc ) technique. In Cl⁻/HCO⁻ ₃-containing solution, secretin (1 μm) or forskolin (10 μm) stimulated a biphasic rise in the I _sc which initially reached a peak level at about 3 min and then decayed to a plateau level after 7 min. Removal of external Cl⁻ abolished the initial transient phase in the forskolin-induced I _sc while the plateau remained. In HCO⁻ ₃/CO₂-free solution, on the contrary, only the initial transient increase in I _sc was prominent. Summation of the current magnitudes observed in Cl⁻-free and HCO⁻ ₃-free solutions over a time course of 10 min gave rise to a curve which was similar, both in magnitude and kinetics, to the current observed in Cl⁻/HCO⁻ ₃-containing solution. Removal of external Na⁺ greatly reduced the initial transient rise in the forskolin-induced I _sc response, and the plateau level observed under this condition was similar to that obtained in Cl⁻-free solution, suggesting that Cl⁻-dependent I _sc was also Na⁺-dependent. Bumetanide (50 μm), an inhibitor of the Na⁺-K⁺-2Cl⁻ cotransporter, and Ba²⁺ (1 mm), a K⁺ channel blocker, could reduce the forskolin-induced I _sc obtained in Cl⁻/HCO⁻ ₃-containing or HCO⁻ ₃-free solution. However, they were found to be ineffective when external Cl⁻ was removed, indicating the involvement of these mechanisms in Cl⁻ secretion. On the contrary, the HCO⁻ ₃-dependent (in the absence of external Cl⁻) forskolin-induced I _sc could be significantly reduced by carbonic anhydrase inhibitor, acetazolamide (45 μm). Basolateral application of amiloride (100 μm) inhibited the I _sc ; however, a specific Na⁺-H⁺ exchanger blocker, 5-N-methyl-N-isobutylamiloride (MIA, 5–10 μm) was found to be ineffective, excluding the involvement of the Na⁺-H⁺ exchanger. However, an inhibitor of H⁺-ATPase, N-ethylmaleimide did suppress the I _sc (IC₅₀= 22 μm). Immunohistochemical studies also confirmed the presence of a vacuolar type of H⁺-ATPase in these cells. H₂DIDS (100 μm), an inhibitor of Na⁺-HCO⁻ ₃ cotransporter, was without effect. Apical addition of Cl⁻ channel blocker, diphenylamine-2,2′-dicarboxylic acid (DPC, 1 mm), but not disulfonic acids, DIDS (100 μm) or SITS (100 μm), exerted an inhibitory effect on both Cl⁻ and HCO⁻ ₃-dependent forskolin-induced I _sc responses. Histochemical studies showed discrete stainings of carbonic anhydrase in the monolayer of CAPAN-1 cells, suggesting that HCO⁻ ₃ secretion may be specialized to a certain population of cells. The present results suggest that both HCO⁻ ₃ and Cl⁻ secretion by the human pancreatic duct cells may occur concurrently and independently. Received: 17 October 1997/Revised: 3 April 1998     

Effects of Hyperthermia on Intracellular Chloride

Hyperthermia induces transient changes in [Na⁺] _i and [K⁺] _i in mammalian cells. Since Cl⁻ flux is coupled with Na⁺ and K⁺ in several processes, including cell volume control, we have measured the effects of heat on [Cl⁻] _i using the chloride indicator, MQAE, with flow cytometry. The mean basal level of [Cl⁻] _i in Chinese hamster ovary cells was 12 mm. Cells heated at 42.0° or 45.0°C for 30 min had about a 2.5-fold increase in [Cl⁻] _i above unheated control values when measured immediately after heating. There was about a 3-fold decrease in [Na⁺] _i under the same conditions, as measured by Sodium Green. The magnitude of the increase in [Cl⁻] _i depended upon time and temperature. The [Cl⁻] _i recovered in a time-dependent fashion to control values by 30 min after heating. When cells were heated at 45.0°C for 30 min in the presence of 1.5 mm furosemide, the heat-induced [Cl⁻] _i increase was completely blocked. Since furosemide inhibits the Na⁺/K⁺/2Cl⁻ cotransporter, Cl⁻ channels, and even Cl⁻HCO₃ exchange, these ion transporters may be involved in the heat-induced increase in [Cl⁻] _i . Received: 15 June 1995/Revised: 9 April 1996     

Co-inoculation of Urea and DAP Tolerant <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> and <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> as Integrated Approach for Growth Enhancement of <Emphasis Type="Italic">Brassica juncea</Emphasis>

Two plant growth promoting rhizobacteria––Sinorhizobium meliloti RMP1 and Pseudomonas aeruginosa GRC₂ were studied for integrated nutrient management to obtain improved yield of Brassica juncea. Low concentrations of urea and diammonium phosphate (DAP) stimulated the growth of both S. meliloti RMP1 and P. aeruginosa GRC₂. 1 M of urea and 0.35 M of DAP was found lethal for RMP1, while 1.3 M and 0.37 M concentrations of urea and DAP proved to be toxic for GRC₂. Lc₅₀ was observed as 0.49 M of urea and 0.15 M of DAP for RMP1, and 0.66 M urea and 0.18 M of DAP for GRC₂. Urea and DAP adaptive variants of RMP1 and GRC₂ was isolated. Adaptive bacterial variants had better growth rates at sub-lethal (Lc₅₀) concentrations of urea and DAP as compared to non-adaptive variants. They also retained plant growth promoting attributes similar to non adaptive variants. GRC₂ and RMP1 did not affect the growth of each other and were chemotactically active for DAP, urea as well as root exudates of B. juncea. Both the isolates colonized well in the rhizosphere of B. juncea, as their populations were recorded ≈5 log₁₀ cfu g⁻¹ after 120 days. Interestingly, the colonization ability was found even better when both strains were co-inoculated, as their population was recorded in the range of ≈6 log₁₀ cfu g⁻¹ after 120 days. In field trials, application of RMP1 and GRC₂ resulted in significant increase in biomass and yield of B. juncea as compared to control. However, yield was better with application of half dose and full dose of recommended fertilizers. Interestingly, the biomass as well as yield improved further when both isolates were applied together along with half dose of recommended fertilizers.     

A Basolateral Chloride Conductance in Rat Lingual Epithelium

We used Ussing chamber measurements and whole-cell recordings to characterize a chloride conductance in rat lingual epithelium. Niflumic acid (NFA) and flufenamic acid (FFA), nonsteroidal anti-inflammatory aromatic compounds known to inhibit Cl⁻ conductances in other tissues, reduced transepithelial short-circuit current (I _sc ) in the intact dorsal anterior rat tongue epithelium when added from the serosal side, and reduced whole-cell currents in rat fungiform taste cells. In both Ussing chamber and patch-clamp experiments, the effect of NFA was mimicked by replacement of bath Cl⁻ with methanesulfonate or gluconate. In low Cl⁻ bath solution, the effect of NFA on whole-cell current was reduced. Replacement of bath Ca²⁺ with Ba²⁺ reduced the whole-cell Cl⁻ current. We conclude that a Ca²⁺-activated Cl⁻ conductance is likely present in the basolateral membrane of the rat lingual epithelium, and is present in the taste receptor cells from fungiform papillae. Further experiments will be required to identify the role of this conductance in taste transduction. Received: 8 September 1997/Revised: 27 March 1998     

Complete degradation of tetrachloroethene in coupled anoxic and oxic chemostats

Anaerobic tetrachloroethene(C₂Cl₄)-dechlorinating bacteria were enriched in slurries from chloroethene-contaminated soil. With methanol as electron donor, C₂Cl₄ and trichloroethene (C₂HCl₃) were reductively dechlorinated to cis-1,2-dichloroethene (cis-C₂H₂Cl₂), whereas, with l-lactate or formate, complete dechlorination of C₂Cl₄ via C₂HCl₃, cis-C₂H₂Cl₂ and chloroethene (C₂H₃Cl) to ethene was obtained. In oxic soil slurries with methane as a substrate, complete co-metabolic degradation of cis-C₂H₂Cl₂ was obtained, whereas C₂HCl₃ was partially degraded. With toluene or phenol both of the above were readily co-metabolized. Complete degradation of C₂Cl₄ was obtained in sequentially coupled anoxic and oxic chemostats, which were inoculated with the slurry enrichments. Apparent steady states were obtained at various dilution rates (0.02–0.4 h⁻¹) and influent C₂Cl₄-concentrations (100–1000 μM). In anoxic chemostats with a mixture␣of␣formate and glucose as the carbon and electron source, C₂Cl₄ was transformed at high rates (above␣140 μmol l⁻¹ h⁻¹, corresponding to 145 nmol Cl⁻ min⁻¹ mg protein⁻¹) into cis-C₂H₂Cl₂ and C₂H₃Cl. Reductive dechlorination was not affected by addition of 5 mM sulphate, but strongly inhibited after addition of 5 mM nitrate. Our results (high specific dechlorination rates and loss of dechlorination capacity in the absence of C₂Cl₄) suggest that C₂Cl₄-dechlorination in the anoxic chemostat was catalysed by specialized dechlorinating bacteria. The partially dechlorinated intermediates, cis-C₂H₂Cl₂ and C₂H₃Cl, were further degraded by aerobic phenol-metabolizing bacteria. The maximum capacity for chloroethene (the sum of tri-, di- and monochloro derivatives removed) degradation in the oxic chemostat was 95 μmol l⁻¹ h⁻¹ (20 nmol min⁻¹ mg protein⁻¹), and that of the combined anoxic → oxic reactor system was 43.4 μmol l⁻¹ h⁻¹. This is significantly higher than reported thus far. Received: 17 April 1997 / Received revision: 6 June 1997 / Accepted: 7 June 1997     

Apical Na+-Cl− Symport in Rabbit Gallbladder Epithelium: A Thiazide-Sensitive Cotransporter (TSC)

Cl⁻ apically enters the epithelium of rabbit gallbladder by a Na⁺-Cl⁻ symport, sensitive to hydrochlorothiazide (HCTZ). Since HCTZ also activates an apical SITS-sensitive Cl⁻ conductance (G _Cl ), the symport inhibition might be merely due to a short circuit of the symport by G _Cl rather than to a direct action of HCTZ on the symporter. To examine whether the symport is directly inhibited by HCTZ and whether the symporter belongs to the family of thiazide-sensitive cotransporters (TSC), radiochemical measurements of the apical Cl⁻ uptake, electrophysiological determinations of intracellular Cl⁻ and Na⁺ activities (a _i,Cl and a _i,Na ) with selective theta microelectrodes and molecular biology methods were used. The ³⁶Cl⁻ uptake proved to be a measurement of the apical unidirectional Cl⁻ influx (J _mc ) and of the symport only (without backflux components), with measuring times of 45 sec under all experiment conditions; its inhibition by HCTZ was unaffected by G _Cl activation or abolition. After HCTZ treatment the decrease in a _i,Cl (measured as the initial rate or in 3 min) was larger than the decrease in a _i,Na . The difference was reduced to one third in a group of epithelia in which the elicited G _Cl was reduced to one third; moreover it was abolished in any case when G _Cl was abolished with 10⁻⁴ m SITS. The SITS-insensitive rate of a _i,Cl decrease was equal to that of the a _i,Na decrease in any case. Thus the a _i,Cl decrease displays a component dependent on G _Cl activation and a second component dependent on symport inhibition. Using the RT-PCR technique a cDNA fragment was obtained that was 99% identical to the corresponding region of the rabbit renal TSC isoform. The results indicate that in rabbit gallbladder epithelium HCTZ displays a dual action, namely G _Cl activation and Na⁺-Cl⁻ symport inhibition. This Na⁺-Cl⁻ symporter is the first TSC found to be functionally expressed in a nonrenal or nonrenal-like epithelium. Received: 29 July 1999/Revised: 23 March 2000     

Single-channel properties of a stretch-sensitive chloride channel in the human mast cell line HMC-1

A stretch-activated (SA) Cl⁻ channel in the plasma membrane of the human mast cell line HMC-1 was identified in outside-out patch-clamp experiments. SA currents, induced by pressure applied to the pipette, exhibited voltage dependence with strong outward rectification (55.1 pS at +100 mV and an about tenfold lower conductance at −100 mV). The probability of the SA channel being open (P _o) also showed steep outward rectification and pressure dependence. The open-time distribution was fitted with three components with time constants of τ_1o = 755.1 ms, τ_2o = 166.4 ms, and τ_3o = 16.5 ms at +60 mV. The closed-time distribution also required three components with time constants of τ_1c = 661.6 ms, τ_2c = 253.2 ms, and τ_3c = 5.6 ms at +60 mV. Lowering extracellular Cl⁻ concentration reduced the conductance, shifted the reversal potential toward chloride reversal potential, and decreased the P _o at positive potentials. The SA Cl⁻ currents were reversibly blocked by the chloride channel blocker 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) but not by (Z)-1-(p-dimethylaminoethoxyphenyl)-1,2-diphenyl-1-butene (tamoxifen). Furthermore, in HMC-1 cells swelling due to osmotic stress, DIDS could inhibit the increase in intracellular [Ca²⁺] and degranulation. We conclude that in the HMC-1 cell line, the SA outward currents are mediated by Cl⁻ influx. The SA Cl⁻ channel might contribute to mast cell degranulation caused by mechanical stimuli or accelerate membrane fusion during the degranulation process.     

Pharmacological Evidence that Calcium is Not Required for P2-Receptor-Stimulated Cl− Secretion in HT29-Cl.16E

Extracellular ATP at micro- to millimolar concentrations activates Cl⁻ conductance and increases cytosolic calcium ([Ca] _i ) in many epithelial cells, including the colonic epithelial cell line HT29-Cl.16E. Therefore, [Ca] _i has been postulated to be the intracellular messenger for Cl⁻ channel activation. HT29-Cl.16E is a highly differentiated cell line that forms confluent monolayers and secretes mucins and Cl⁻. The involvement of [Ca] _i in the purinergically-stimulated Cl⁻ secretion was investigated pharmacologically in this cell line by whole-cell patch-clamp and Ussing chamber techniques, as well as [Ca] _i measurements in fura-2 loaded cells. The calmodulin inhibitors W13 (5 μm) and chlorpromazine (50 μm) abolished increases in ATP-stimulated [Ca] _i -increases by 90% and 80%, respectively. However, these inhibitors had no effect on the ATP-stimulated Cl⁻ conductance measured in either individual cells or confluent monolayers. As controls, the effects of W13 and chlorpromazine on Ca²⁺-ionophore stimulated Cl⁻ conductance was measured. In this case, the two compounds inhibited whole cell Cl⁻ conductance and monolayer Isc by 90% and 100%, respectively. These data demonstrate: (1) The purinergically-stimulated increase in Cl⁻ current does not require an increase in [Ca] _i , suggesting the involvement of either another signaling pathway or direct activation of Cl⁻ channels by purinergic receptors. (2) A calmodulin or a calmodulinlike binding site that is sensitive to W13 and chlorpromazine participates in the regulation of the [Ca] _i increase by purinergic receptors in HT29-Cl.16E. Received: 4 December 1995/Revised: 16 August 1996     

A Novel,Volume-correlated Cl− Conductance in Marginal Cells Dissociated from the Stria Vascularis of Gerbils

Using the whole-cell patch-clamp technique, we examined Cl⁻-selective currents manifested by strial marginal cells isolated from the inner ear of gerbils. A large Cl⁻-selective conductance of ∼18 nS/pF was found from nonswollen cells in isotonic buffer containing 150 mm Cl⁻. Under a quasi-symmetrical Cl⁻ condition, the `instantaneous' current-voltage relation was close to linear, while the current-voltage relation obtained at the end of command pulses of duration 400 msec showed weak outward rectification. The permeability sequence for anionic currents was as SCN⁻ > Br⁻≅ Cl⁻ > F⁻ > NO⁻ ₃≅ I⁻ > gluconate⁻, corresponding to Eisenmann's sequence V. When whole-cell voltage clamped in isotonic bathing solutions, the cells exhibited volume changes that were accounted for by the Cl⁻ currents driven by the imposed electrochemical potential gradients. The volume change was elicited by lowered extracellular Cl⁻ concentration, anion substitution and altered holding potentials. The Cl⁻ conductance varied in parallel with cell volume when challenged by bath anisotonicity. The whole-cell Cl⁻ current was only partially blocked by both 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, 0.5 mm) and diphenylamine-2-carboxylic acid (DPC, 1.0 mm), but 4-acetamido-4′-isothiocyanato-stilbene-2,2′-disulfonic acid (SITS, 0.5 mm) was without effect. The properties of the present whole-cell Cl⁻ current resembled those of the single Cl⁻ channel previously found in the basolateral membrane of the marginal cell (Takeuchi et al., Hearing Res. 83:89–100, 1995), suggesting that the volume-correlated Cl⁻ conductance could be ascribed predominantly to the basolateral membrane. This Cl⁻ conductance may function not only in cell volume regulation but also for the transport of Cl⁻ and the setting of membrane potential in marginal cells under physiological conditions. Received: 15 August 1995/Revised: 3 November 1995