Effects of Microdialysis-Perfusion with Anisoosmotic Media on Extracellular Amino Acids in the Rat Hippocampus and Skeletal Muscle

Changes in the levels of amino acids have been implicated as being important in osmoregulation both within and outside the CNS. The present study addressed the question of whether changes in osmolarity affect the extracellular concentration of amino acids in the rat hippocampus and femoral biceps muscle (FBM). Microdialysis probes were implanted in these tissues and perfused with standard physiological saline. Amino acid concentrations in the dialysate were determined with HPLC separation of o-phthaldialdehyde derivatives and fluorescence detection. The osmolarity of the perfusion buffer was gradually decreased by reduction of the concentration of NaCl from 122 to 61 to 0 mM. In other experiments, the osmolarity was increased by elevation of the NaCl level from 122 to 183 to 244 mM or by addition of mannitol. Glutamate, aspartate, gamma-aminobutyrate, and alanine levels in dialysate from the hippocampus increased when the concentration of NaCl was decreased by 61 mM, and they were further elevated when NaCl was omitted. Taurine and phosphoethanolamine (PEA) levels were maximally elevated at the intermediary decrease of NaCl concentration, and glutamine in particular but also methionine and leucine were suppressed by perfusion with hypoosmolar medium. The amino acid response of the FBM differed substantially from that of the hippocampus. The aspartate content increased slightly, and there was a marginal transient increase in PEA level. Perfusion with media containing high concentrations of NaCl induced diminished dialysate levels of taurine, PEA, and glutamate, whereas levels of other amino acids were either unaffected or increased. Mannitol administration via the perfusion fluid led to reduced levels of taurine, PEA, glutamate, and aspartate. In contrast to the effects of high NaCl levels, hyperosmotic mannitol did not induce increases in level of any of the amino acids detected. The results suggest that taurine and PEA are involved in osmoregulation in the mammalian brain. From a quantitative viewpoint, taurine seems to be most important. Transmitter amino acids may also be involved in the maintenance of the volume of neural cells subjected to severe disturbances in osmotic equilibrium.     

Factors affecting the transport of beta-amino acids in rat renal brush-border membrane vesicles. The role of external chloride

The effect of a variety of ions and other solutes on the accumulation of the beta-amino acid, taurine, was examined in rat renal brush-border membrane vesicles. Initial taurine uptake (15 and 30 s) is sodium-dependent with a typical overshoot. This Na+ effect was confirmed by exchange diffusion and gramicidin inhibition of taurine uptake. External K+ or Li+ do not increase taurine accumulation more than Na+-free mannitol, except that the combination of external K+ and Na+ in the presence of nigericin enhances uptake. Of all anions tested, including more permeant (SCN- and NO3-) or less permeant (SO4(2-)), chloride supported taurine accumulation to a significantly greater degree. Preloading vesicles with choline chloride reduced taurine uptake, suggesting that external Cl- stimulates uptake. Since this choline effect could be related to volume change, due to the slow diffusion of choline into vesicles, brush-border membrane vesicles were pre-incubated with LiCl, LiNO3 and LiSO4. Internal LiCl, regardless of the final Na+ anion mixture, reduced initial rate (15 and 60 s) and peak (360 s) taurine uptake. Internal LiNO3 or LiSO4 with external NaCl resulted in similar or higher values of uptake at 15, 60 and 360 s, indicating a role for external Cl- in taurine uptake in addition to Na+ effect. Although uptake by vesicles is greatest at pH 8.0 and inhibited at acidic pH values (pH less than 7.0), an externally directed H+ gradient does not influence uptake. Similarly, amiloride, an inhibitor of the Na+/H+ antiporter, had no influence on taurine accumulation over a wide variety of concentrations or at low Na+ concentrations. Taurine uptake is blocked only by other beta-amino acids and in a competitive fashion. D-Glucose and p-aminohippurate at high concentrations (greater than 10(-3) M) reduce taurine uptake, possibly by competing for sodium ions, although gramicidin added in the presence of D-glucose inhibits taurine uptake even further. These studies more clearly define the nature of the renal beta-amino acid transport system in brush-border vesicles and indicate a role for external Cl- in this uptake system.     

Effects of raised osmolarity on canine tracheal epithelial ion transport function

Evaporation of water from upper airway surfaces increases surface liquid osmolarity. We studied the effects of raised osmolarity of the solution bathing the luminal surface of excised canine tracheal epithelium. Osmolarity was increased by adding NaCl or mannitol. NaCl addition induced a concentration-dependent fall in short-circuit current and a rise in transepithelial conductance (-33% and +14% per 100 mosM, respectively). Unidirectional isotopic fluxes of 22Na, 36Cl, and [14C]mannitol were measured in short-circuited tissues in the base-line state and after addition of NaCl or mannitol to an isotonic mucosal solution. NaCl addition (75 mM) caused a 50% increase in conductance (G) and a parallel increase in [14C]mannitol permeability (Pmann), indicating an increase in paracellular permeability. Net Cl- secretion was reduced 50%, and net Na+ absorption was unchanged despite an increased chemical gradient for absorption, indicating an inhibition of active ion transport. Mannitol addition (150 mM) abolished net Na+ absorption but did not increase G or Pmann or change net Cl- secretion. These results suggest that responses to increased tracheal surface liquid osmolarity during spontaneous breathing may occur in both the cellular (inhibition of active Na+ and Cl- transport) and paracellular (increased [14C]mannitol permeability) compartments of the mucosa.     

A Possible Interrelationship Between Extracellular Taurine and Phosphoethanolamine in the Hippocampus

The effect of guanidinoethane sulfonic acid (GES), an inhibitor of taurine uptake, was examined with respect to endogenous amino acids in the hippocampus of the freely moving rabbit. GES increased the extracellular levels of both taurine and phosphoethanolamine (PEA), other amino acids being unaffected. However, long-term oral administration of GES selectively reduced endogenous taurine levels. The effect of GES on PEA appeared to be a consequence of the elevated extracellular taurine as exogenously administered taurine per se increased PEA levels in the extracellular space. The findings are discussed in conjunction with the proposed membrane-stabilizing effects of taurine.     

Analysis of hyposmolarity-induced taurine efflux pathways in the bullfrog sympathetic ganglia.

Hyposmolarity-induced taurine release was dependent on the decrease in medium osmolarity (5-50%) in the satellite glial cells of the bullfrog sympathetic ganglia. Release of GABA induced by hyposmolarity was much less than that of taurine. Omission of external Cl- replaced with gluconate totally suppressed taurine release, but only slightly suppressed GABA release. Bumetanide and furosemide, blockers of the Na+/K+/2Cl- cotransport system, inhibited taurine release by about 40%. Removal of external Na+ by replacement with choline, or omission of K+, suppressed taurine release by 40%. Antagonists of the Cl-/HCO3 exchange system, SITS, DIDS and niflumic acid, significantly reduced taurine release. The carbonic anhydrase inhibitor, acetazolamide, reduced the taurine release by 34%. Omission of external HCO3 by replacement with HEPES caused a 40% increase in the hyposmolarity-induced taurine release. Hyposmolarity-induced GABA release was not affected by bumetanide or SITS. Chloride channel blockers, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and N-phenylanthranilic acid (DPC), practically abolished taurine release. Blockers of K+ channels, clofilium and quinidine, had no effect on the taurine release. The hyposmolarity-induced taurine release was considerably enhanced by a simultaneous increase in external K+. GABA was not mediated by the same transport pathway as that of taurine. These results indicate that Cl- channels may be responsible for the hyposmolarity-induced taurine release, and that Na+/K+/2Cl- cotransporter and Cl-/HCO3 exchanger may contribute to maintain the intracellular Cl- levels higher than those predicted for a passive thermodynamic distribution in the hyposmolarity-induced taurine release.     

Cl-/HCO3- exchange at the apical membrane of Necturus gallbladder

The hypothesis of Cl-/HCO3- exchange across the apical membrane of the epithelial cells of Necturus gallbladder was tested by means of measurements of extracellular pH (pHo), intracellular pH (pHi), and Cl- activity (alpha Cli) with ion-sensitive microelectrodes. Luminal pH changes were measured after stopping mucosal superfusion with a solution of low buffering power. Under control conditions, the luminal solution acidifies when superfusion is stopped. Shortly after addition of the Na+/H+ exchange inhibitor amiloride (10(-3) M) to the superfusate, alkalinization was observed. During prolonged (10 min) exposure to amiloride, no significant pHo change occurred. Shortly after amiloride removal, luminal acidification increased, returning to control rates in 10 min. The absence of Na+ in the superfusate (TMA+ substitution) caused changes in the same direction, but they were larger than those observed with amiloride. Removal of Cl- (cyclamate or sulfate substitution) caused a short-lived increase in the rate of luminal acidification, followed by a return to control values (10-30 min). Upon re-exposure to Cl-, there was a transient reduction of luminal acidification. The initial increase in acidification produced by Cl- removal was partially inhibited by SITS (0.5 mM). The pHi increased rapidly and reversibly when the Cl- concentration of the mucosal bathing solution was reduced to nominally 0 mM. The pHi changes were larger in 10 mM HCO3-Ringer's than in 1 mM HEPES-Ringer's, which suggests that HCO3- is transported in exchange for Cl-. In both HEPES- and HCO3-Ringer's, SITS inhibited the pHi changes. Finally, intracellular acidification or alkalinization (partial replacement of NaCl with sodium propionate or ammonium chloride, respectively) caused a reversible decrease or increase of alpha Cli. These results support the hypothesis of apical membrane Cl-/HCO3- exchange, which can be dissociated from Na+/H+ exchange and operates under control conditions. The coexistence at the apical membrane of Na+/H+ and Cl-/HCO3- antiports suggests that NaCl entry can occur through these transporters.     

Na/K/Cl cotransport in cultured human fibroblasts

The transport characteristics and regulation of the Na/K/Cl cotransport system were investigated in cultured human fibroblasts (HSWP cells). The existence of the system was documented by the finding that digitoxin-insensitive K+ influx was dependent upon the presence of both Na+ and Cl- in the extracellular milieu. It was found that only Br- could partially substitute for Cl-, with SCN-, I-, acetate, and gluconate being ineffective. Li+ could partially substitute for Na+; however, choline was without effect. The shape of the titration curves for K+ influx versus extracellular Cl- concentration was dependent upon the substituted anion. Furthermore, the apparent Km for Cl- at saturating [K+]o and [Na+]o, was also dependent upon the substituted anion and ranged from 30 mM (gluconate substitution) to 100 mM (acetate substitution). The titration curves for K+ influx versus extracellular Na+ concentration displayed hyperbolic kinetics and the apparent Km = 15 mM at saturating [K+]o. The curve for K+ influx versus extracellular K+ concentration was a hyperbola and the apparent Km for K+ = 3 mM at saturating [Na+]o. The digitoxin-insensitive K+ flux was found to be sensitive to related 5-sulfamoylbenzoic acid derivatives, commonly known as "loop" diuretics and to be insensitive to both: amiloride (3,5-diamino-N-(aminoiminomethyl)-6-chloropyrazinecarboxamide++ +) and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. The Na/K/Cl cotransport system was not stimulated by serum, but was slightly stimulated by two peptide mitogens. Furthermore, agents which cause an elevation in cellular cyclic AMP levels were found to be potent inhibitors of cotransport.     

Anion modulation of taste responses in sodium-sensitive neurons of the hamster chorda tympani nerve

Beidler's work in the 1950s showed that anions can strongly influence gustatory responses to sodium salts. We have demonstrated "anion inhibition" in the hamster by showing that the chorda tympani nerve responds more strongly to NaCl than to Na acetate over a wide range of concentrations. Iontophoretic presentation of Cl- and acetate to the anterior tongue elicited no response in the chorda tympani, suggesting that these anions are not directly stimulatory. Drugs (0.01, 1.0, and 100 microM anthracene-9-carboxylate, diphenylamine-2-carboxylate, 4- acetamido-4'-isothiocyanatostilbene-2,2'-disulfonate, and furosemide) that interfere with movements of Cl- across epithelial cells were ineffective in altering chorda tympani responses to 0.03 M of either NaCl or Na acetate. Anion inhibition related to movements of anions across epithelial membranes therefore seems unlikely. The chorda tympani contains a population of nerve fibers highly selective for Na+ (N fibers) and another population sensitive to Na+ as well as other salts and acids (H fibers). We found that N fibers respond similarly to NaCl and Na acetate, with spiking activity increasing with increasing stimulus concentration (0.01-1.0 M). H fibers, however, respond more strongly to NaCl than to Na acetate. Furthermore, H fibers increase spiking with increases in NaCl concentration, but generally decrease their responses to increasing concentrations of Na acetate. It appears that anion inhibition applies to taste cells innervated by H fibers but not by N fibers. Taste cells innervated by N fibers use an apical Na+ channel, whereas those innervated by H fibers may use a paracellularly mediated, basolateral site of excitation.     

Taurine and cell volume maintenance in the shark rectal gland: cellular fluxes and kinetics

Tissue slices of shark rectal gland are studied to examine the kinetics of the cellular fluxes of taurine, a major intracellular osmolyte in this organ. Maintenance of high steady-state cell taurine (50 mM) is achieved by a ouabain-sensitive active Na+-dependent uptake process and a relatively slow efflux. Uptake kinetics are described by two saturable taurine transport components (high-affinity, Km 60 microM; and low-affinity, Km 9 mM). [14C]Taurine uptake is enhanced by external Cl-, inhibited by beta-alanine and unaffected by inhibitors of the Na+/K+/2Cl- co-transport system. Two cellular efflux components of taurine are documented. Incubation of slices in p-chloromercuribenzene sulfonate (1 mM) reduces taurine uptake, increases efflux of taurine and induces cell swelling. Studies of efflux in isotonic media with various cation and anion substitutions demonstrate that high-K+ markedly enhances taurine efflux irrespective of cell volume changes (i.e. membrane stretching is not involved). Moreover, iso-osmotic cell swelling induced in media containing propionate is not associated with enhanced efflux of taurine from the cells. It is suggested that external K+ exerts a specific effect on the cytoplasmic membrane to increase its permeability to taurine.     

In vitro studies on the release of cortisol from interrenal tissue in trout (Salmo gairdneri)--II. Action of changes in extracellular electrolytes

Secretion of cortisol by the interrenal tissue of the trout Salmo gairdneri was studied in vitro by a perifusion method in relation to the effects of electrolyte concentrations in the medium. An increase in osmotic pressure (produced by adding mannitol or NaCl) induced an immediate, but brief augmentation in cortisol release. Suppression of Na+ had no effect while its reintroduction in the medium led to stimulation of hormone release. By contrast, a sharp peak was obtained whenever Cl- concentration was dropped (by 50 mM fractions). These opposite effects of Na+ and Cl- when they vary independently of each other is interpreted with regard to osmoregulation and acid-base regulation. Raising K+ even to high levels (up to 20 mM) produced no change. The absence of Ca2+ had no obvious effect while its addition induced an immediate peak of cortisol release. In addition, external Ca2+ proved necessary for the action of ACTH to occur. These results establish that cortisol release in trout may be directly affected by changes in electrolyte concentrations in the extracellular space.     

Regulation of internal solute concentrations of marine Vibrio alginolyticus in response to external NaCl concentration.

Slightly halophilic marine Vibrio alginolyticus grown in the range of NaCl from 0.2 to 1.5 M maintained the total internal solute concentration always higher than the external medium by about 0.25 osM. The concentrations of macromolecules such as DNA, RNA, and protein were little affected by the increase in medium NaCl. The internal K+ concentration was kept to about 400 mM in the range of medium NaCl from 0.4 to 0.8 M; it rose to 510 mM when the bacterium was grown in 1.5 M NaCl, indicating that K+ increased only slightly in response to the large increase in medium NaCl. Thus, in contrast to the case of nonhalophilic and extremely halophilic bacteria, K+ was unlikely to act as a major component to regulate the internal solute concentration of marine V. alginolyticus. The internal Na+ and Cl- concentrations were maintained always lower than those in the growth medium, but they increased in response to the increase in medium NaCl. The concentration of internal Na+ was close to that of K+ at the concentration of medium NaCl that supports the optimal growth of this organism. The total amino acid content of V. alginolyticus increased from 76 to 413 mM by the increase in medium NaCl from 0.2 to 1.5 M. The concentrations of glutamic acid and prolined were 254 and 72 mM, respectively, when grown in 1.5 M NaCl. These results indicated that Na+, Cl- and amino acids, especially glutamic acid and proline, contributed to the regulation of internal solute concentration of V. alginolyticus in response to the increased external NaCl.     

Na+ + Cl- -gradient-driven, high-affinity, uphill transport of taurine in human placental brush-border membrane vesicles

Uptake of taurine in human placental brush-border membrane vesicles was greatly stimulated in the presence of an inwardly-directed Na+ + Cl- -gradient and uphill transport of taurine could be demonstrated under these conditions. Na+ as well as Cl- were obligatory for this uptake and both ion gradients could energize the uphill transport. This Na+ + Cl- -gradient-dependent taurine uptake was stimulated by an inside-negative membrane potential, demonstrating the electrogenicity of the process. The uptake system was highly specific for beta-amino acids and the Km of the system for taurine was 6.5 +/- 0.4 microM.     

The cellular mechanism of active chloride secretion in vertebrate epithelia: studies in intestine and trachea

The cellular mechanism of active chloride secretion, as it is manifested in the intestine and trachea, appears to possess the following elements: (1)NaCl cl-transport across the basolateral membrane; (2) Cl- accumulation in the cell above electrochemical equilibrium due to the Na+ gradient; (3) a basolateral Na+-K+ pump that maintains the Na+ gradient; (4) a hormone-regulated Cl- permeability in the apical membrane; (5) passive Na/ secretion through a paracellular route, driven by the transepithelial potential difference; and (6) an increase in basolateral membrane K+ permeability occurring in conjunction with an increase in Na+-K+ pump rate. Electrophysiological studies in canine trachea support this model. Adrenalin, a potent secretory stimulus in that tissue, increases apical membrane conductance through a selective increase in Cl- permeability. Adrenalin also appears to increase basolateral membrane K+ permeability. Whether or not adrenalin also increases paracellular Na+ permeability is unclear. Some of the testable implications of the above secretion model are discussed.     

Elevation of Taurine in Hippocampal Extracellular Fluid and Cerebrospinal Fluid of Acutely Hypoosmotic Rats: Contribution by Influx from Blood?

Previous work has demonstrated that there is a selective increase in extracellular taurine in the brain during acute water intoxication. One aim of the present study was to investigate whether plasma taurine contributes to this increase. To this end, the concentrations of taurine, other amino acids, and ethanolamine (EA) were measured in plasma and CSF of urethane-anesthetized rats injected with 150 ml/kg body weight of distilled water. Blood pressure, blood gases, and pH, as well as plasma and CSF osmolality, were also measured. The CSF level of albumin was quantitated to study the function of the blood-CSF barrier. In separate experiments, hippocampal microdialysis was performed to determine the effects of acute plasma hypoosmolality on extracellular amino acids. Finally, the effect of water injection on hippocampal specific gravity and tissue amino acids was assessed. Blood gases and pH were essentially unchanged after water administration. Mean arterial blood pressure increased to peak levels approximately 50 mm Hg above control. Plasma osmolality decreased rapidly, whereas the depression of CSF osmolality was slower and less pronounced. The average volume of the hippocampus increased by 8%. Water injection was accompanied by a 25-fold elevation of taurine in plasma, whereas phosphoethanolamine (PEA) and EA increased moderately. A small fraction of the increase in plasma taurine might derive from blood cells because dilution of blood in vitro led to doubled plasma levels of the amino acid. Taurine, PEA, and EA increased consistently in CSF and hippocampal microdialysates. Plasma hypoosmolality transiently opened the blood-CSF barrier is reflected by augmented CSF concentrations of albumin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Renal transport of taurine in luminal membrane vesicles from rabbit proximal tubule

The uptake of taurine by luminal membrane vesicles from pars convoluta and pars recta of rabbit proximal tubule was examined. In pars convoluta, the transport of taurine was characterized by two Na(+)-dependent (Km1 = 0.086 mM, Km2 = 5.41 mM) systems, and one Na(+)-independent (Km = 2.87 mM) system, which in the presence of an inwardly directed H(+)-gradient was able to drive the transport of taurine into these vesicles. By contrast, in luminal membrane vesicles from pars recta, the transport of taurine occurred via a dual transport system (Km1 = 0.012 mM, Km2 = 5.62 mM), which was strictly dependent on Na+. At acidic pH with or without a H(+)-gradient, the Na(+)-dependent flux of taurine was drastically reduced. In both kind of vesicles, competition experiments only showed inhibition of the Na(+)-dependent high-affinity taurine transporter in the presence of beta-alanine, whereas there was no significant inhibition with alpha-amino acids, indicating a beta-amino acid specific transport system. Addition of beta-alanine, L-alanine, L-proline and glycine, but not L-serine reduced the H(+)-dependent uptake of taurine to approx. 50%. Moreover, only the Na(+)-dependent high-affinity transport systems in both segments specifically required Cl-. Investigation of the stoichiometry indicated 1.8 Na+: 1 Cl-: 1 taurine (high affinity), 1 Na+: 1 taurine (low affinity) and 1 H+: 1 taurine in pars convoluta. In pars recta, the data showed 1.8 Na+: 1 Cl-: 1 taurine (high affinity) and 1 Na+: 1 taurine (low affinity).     

N-Methylaspartate-Evoked Liberation of Taurine and Phosphoethanolamine In Vivo: Site of Release

The effect of N-methyl-D,L-aspartic acid (NMA) on extracellular amino acids was studied in the rabbit hippocampus with the brain dialysis technique. Administration of 0.5 or 5 mM NMA caused a concentration-dependent liberation of taurine and phosphoethanolamine (PEA). Taurine increased by 1,200% and PEA by 2,400% during perfusion with 5 mM NMA whereas most other amino acids rose by 20-100%. The effect of NMA appeared to be receptor-mediated, as coperfusion with D-2-amino-5-phosphonovaleric acid curtailed the NMA response by some 90%. The NMA-stimulated release of taurine and PEA was suppressed when Ca2+ was omitted and further inhibited when Co2+ was included in the perfusion medium. The effect of NMA was mimicked by the endogenous NMA agonist quinolinic acid and the partial NMA agonist D,L-cis-2,3-piperidine dicarboxylic acid. Although the NMA-evoked release of taurine and PEA was Ca2+-dependent in vivo, NMA had no effect on Ca2+ accumulation in hippocampal synaptosomes. The previously reported NMA-induced activation of dendritic Ca2+ spikes and the lack of effect on synaptosomal Ca2+ uptake suggest that taurine and PEA are released from sites other than nerve terminals, possibly from dendrosomatic sites. This notion was strengthened by the absence of an effect of NMA on the efflux of radiolabelled taurine from hippocampal synaptosomes. In contrast, high K+ stimulated synaptosomal uptake of Ca2+ and release of taurine.     

Evidence for extensive methylation of ribosomal RNA genes in a rat XC cell line

The effects of extracellular Na+, K+ and Cl- on neurite outgrowth of PC12 pheochromocytoma cells were studied. Nerve growth factor (NGF)-induced neurite formation was inhibited upon substitution of choline chloride for NaCl under normal culture conditions. It was found that neurite formation increased proportionately with the concentration of Na+ in medium up to 150 mM. When PC12 cells were exposed to NGF in suspension culture followed by transfer to new dishes, they showed neurite extention in response to NGF in an RNA- and protein synthesis-independent manner. Under these conditions, neurite outgrowth occurred normally in 60-150 mM Na+, whereas it decreased significantly at lower concentrations of Na+. Na+ dependency was also observed for cyclic AMP-mediated neurite formation of PC12 cells. In contrast neurite outgrowth was independent of K+ in the range 5-106 mM, suggesting that membrane potential did not play a role in this process. No alterations were observed in neurite outgrowth with Cl- replaced by NO3-, SO2-4, or 2-hydroxyethanesulfonate. Thus, extracellular Na+ plays a role in controlling neurite formation of these cells. An attempt was made to relate this effect to a decrease in cytoplasmic Ca2+ concentration monitored by a fluorescent dye sensitive to Ca2+.     

Independence of apical membrane Na+ and Cl- entry in Necturus gallbladder epithelium

Transepithelial fluid transport (Jv) and intracellular Na+ and Cl- activities (aNai, aCli) were measured in isolated Necturus gallbladders to establish the contribution of different proposed apical membrane entry mechanisms to transepithelial salt transport. In 10 mM HCO3- Ringer's, Jv was 13.5 +/- 1.1 microliter X cm-2 X h-1, and was significantly reduced by a low bicarbonate medium and by addition of amiloride (10(-3)M) or SITS (0.5 X 10(-3)M) to the mucosal bathing solution. Bumetanide (10(-5)M) was ineffective. Bilateral Na+ removal abolished Jv. The hypothesis of NaCl cotransport was rejected on the basis of the following results, all obtained during mucosal bathing solution changes: during Na+ removal, aNai fell 4.3 times faster than aCli; during Cl- removal, aCli fell 7.5 times faster than aNai; amiloride (10(-3) M) reduced aNai at a rate of 2.4 +/- 0.3 mM/min, whereas aCli was not changed; bumetanide (10(-5) M) had no significant effects on Jv or aCli. The hypothesis of Na-K-Cl cotransport was rejected for the same reasons; in addition, K+ removal from the mucosal bathing solution (with concomitant Ba2+ addition) did not alter aNai or aCli. The average rate of NaCl entry under normal transporting conditions, estimated from Jv, assuming that the transported fluid is an isosmotic NaCl solution, was 22.5 nmol X cm-2 X min-1. Upon sudden cessation of NaCl entry, assuming no cell volume changes, aNai and aCli should fall at an average rate of 4.8 mM/min. To compare this rate with the rates of Na+ and Cl- entry by ion exchange, the Na+ or Cl- concentration in the mucosal bathing solution was reduced rapidly to levels such that electroneutral cation or anion exchange, respectively, should cease. The rate of Na+ or Cl- entry before this maneuver was estimated from the initial rate of fall of the respective intracellular ionic activity upon the mucosal solution substitution. aNai and aCli decreased at initial rates of 3.7 +/- 0.4 and 5.9 +/- 0.8 mM/min, respectively. The rate of fall of aNai upon reduction of external [Na] was not affected by amiloride (10(-3) M), and the rate of fall of aCli upon reduction of external [Cl] was unchanged by SITS (0.5 X 10(-3) M), which indicates that net cation or anion exchange was, in fact, abolished by the changes in Na+ and Cl- gradients, respectively. I conclude that double exchange (Na+/H+ and Cl-/HCO-3) is the predominant or sole mechanism of apical membrane NaCl entry in this epithelium.     

Effect of chloride on pH microclimate and electrogenic Na+ absorption across the rumen epithelium of goat and sheep

Active Na+ absorption across rumen epithelium comprises Na+/H+ exchange and a nonselective cation conductance (NSCC). Luminal chloride is able to stimulate Na+ absorption, which has been attributed to an interaction between Cl-/HCO3- and Na+/H+ exchangers. However, isolated rumen epithelial cells also express a Cl- conductance. We investigated whether Cl- has an additional effect on electrogenic Na+ absorption via NSCC. NSCC was estimated from short-circuit current (Isc) across epithelia of goat and sheep rumen in Ussing chambers. Epithelial surface pH (pHs) was measured with 5-N-hexadecanoyl-aminofluorescence. Membrane potentials were measured with microelelectrodes. Luminal, but not serosal, Cl- stimulated the Ca2+ and Mg2+ sensitive Isc. This effect was independent of the replacing anion (gluconate or acetate) and of the presence of bicarbonate. The mean pHs of rumen epithelium amounted to 7.47 +/- 0.03 in a low-Cl- solution. It was increased by 0.21 pH units when luminal Cl- was increased from 10 to 68 mM. Increasing mucosal pH from 7.5 to 8.0 also increased the Ca2+ and Mg2+ sensitive Isc and transepithelial conductance and reduced the fractional resistance of the apical membrane. Luminal Cl- depolarized the apical membrane of rumen epithelium. 5-Nitro-2-(3-phenylpropylamino)-benzoate reduced the divalent cation sensitive Isc, but only in low-Cl- solutions. The results show that luminal Cl- can increase the microclimate pH via apical Cl-/HCO3- or Cl-/OH- exchangers. Electrogenic Na+ absorption via NSCC increases with pH, explaining part of the Cl- effects on Na+ absorption. The data further show that the Cl- conductance of rumen epithelium must be located at the basolateral membrane.     

Effect of chloride and glutamate ions on in vitro protein synthesis by the moderate halophile Vibrio costicola.

Vibrio costicola grown in the presence of different NaCl concentrations contains cell-associated Na+ and K+ ions whose sum is equal to or greater than the external Na+ concentration. In the presence of 0.5 M NaCl, virtually no in vitro protein is synthesized in extracts of cells grown in 1.0 M NaCl. However, we report here that active in vitro protein synthesis occurred in 0.6 M or higher concentrations of Na2SO4, sodium formate, sodium acetate, sodium aspartate, or sodium glutamate, whereas 0.6 M NaF, NaCl, or NaBr completely inhibited protein synthesis as measured by polyuridylic acid-directed incorporation of [14C]phenylalanine. Sodium glutamate, sodium aspartate, and betaine (0.3 M) counteracted the inhibitory action of 0.6 M NaCl. The cell-associated Cl- concentration was 0.22 mol/kg in cells grown in 1.0 M NaCl. Of this, the free intracellular Cl- concentration was only 0.02 mol/kg. Cells contained 0.11 mol of glutamate per kg and small concentrations of other amino acids. All of the negative counterions for cell-associated Na+ and K+ have not yet been determined. In vitro protein synthesis by Escherichia coli was inhibited by sodium glutamate. Hybridization experiments with ribosomes and the soluble (S-100) fractions from extracts of E. coli and V. costicola showed that the glutamate-sensitive fraction was found in the soluble, not the ribosomal, part of the system. The phenylalanyl-tRNA synthetase of V. costicola was not inhibited by 0.5 M or higher concentrations of NaCl; it was slightly more sensitive to high concentrations of sodium glutamate. Therefore, this enzyme was not responsible for the salt response of the V. costicola in vitro protein-synthesizing system.