Both seawater acclimation and environmental ammonia exposure lead to increases in mRNA expression and protein abundance of Na⁺:K⁺:2Cl⁻ cotransporter in the gills of the climbing perch, Anabas testudineus

The freshwater climbing perch, Anabas testudineus, is an obligatory air-breathing teleost which can acclimate to seawater, survive long period of emersion, and actively excrete ammonia against high concentrations of environmental ammonia. This study aimed to clone and sequence the Na⁺:K⁺:2Cl⁻ cotransporter (nkcc) from the gills of A. testudineus, and to determine the effects of seawater acclimation or exposure to 100 mmol l⁻¹ NH₄Cl in freshwater on its branchial mRNA expression. The complete coding cDNA sequence of nkcc from the gills of A. testudineus consisted of 3,495 bp, which was translated into a protein with 1,165 amino acid residues and an estimated molecular mass of 127.4 kDa. A phylogenetic analysis revealed that the translated Nkcc of A. testudineus was closer to fish Nkcc1a than to fish Nkcc1b or Nkcc2. After a progressive increase in salinity, there were significant increases in the mRNA expression and protein abundance of nkcc1a in the gills of fish acclimated to seawater as compared with that of the freshwater control. Hence, it can be concluded that similar to marine teleosts, Cl⁻ excretion through basolateral Nkcc1 of mitochondrion-rich cells (MRCs) was essential to seawater acclimation in A. testudineus. Exposure of A. testudineus to 100 mmol l⁻¹ NH₄Cl for 1 or 6 days also resulted in significant increases in the mRNA expression of nkcc1a in the gills, indicating a functional role of Nkcc1a in active ammonia excretion. It is probable that NH₄ ⁺ enter MRCs through basolateral Nkcc1a before being actively transported across the apical membrane. Since the operation of Nkcc1a would lead to an increase in the intracellular Na⁺ concentration, it can be deduced that an upregulation of basolateral Na⁺/K⁺-ATPase (Nka) activity would be necessary to compensate for the increased influx of Na⁺ into MRCs during active NH₄ ⁺ excretion. This would imply that the main function of Nka in active NH₄ ⁺ excretion is to maintain intracellular Na⁺ and K⁺ homeostasis instead of transporting NH₄ ⁺ directly into MRCs as proposed previously. In conclusion, active salt secretion during seawater acclimation and active NH₄ ⁺ excretion during exposure to ammonia in freshwater could involve similar transport mechanisms in the gills of A. testudineus.     

Vasopressin alters the mechanism of apical Cl− entry from Na+:Cl− to Na+:K+:2Cl− cotransport in mouse medullary thick ascending limb

Summary Experiments were performed usingin vitro perfused medullary thick ascending limbs of Henle (MTAL) and in suspensions of MTAL tubules isolated from mouse kidney to evaluate the effects of arginine vasopressin (AVP) on the K⁺ dependence of the apical, furosemide-sensitive Na⁺:Cl^– cotransporter and on transport-related oxygen consumption (QO₂). In isolated perfused MTAL segments, the rate of cell swelling induced by removing K⁺ from, and adding onemm ouabain to, the basolateral solution [ouabain(zero-K⁺)] provided an index to apical cotransporter activity and was used to evaluated the ionic requirements of the apical cotransporter in the presence and absence of AVP. In the absence of AVP cotransporter activity required Na⁺ and Cl^–, but not K⁺, while in the presence of AVP the apical cotransporter required all three ions.⁸⁶Rb⁺ uptake into MTAL tubules in suspension was significant only after exposure of tubules to AVP. Moreover,²²Na⁺ uptake was unaffected by extracellular K⁺ in the absence of AVP while after AVP exposure²²Na⁺ uptake was strictly K⁺-dependent. The AVP-induced coupling of K⁺ to the Na⁺:Cl^– cotransporter resulted in a doubling in the rate of NaCl absorption without a parallel increase in the rate of cellular²²Na⁺ uptake or transport-related oxygen consumption. These results indicate that arginine vasopressin alters the mode of a loop diuretic-sensitive transporter from Na⁺:Cl^– cotransport to Na⁺:K⁺:2Cl^– cotransport in the mouse MTAL with the latter providing a distinct metabolic advantage for sodium transport. A model for AVP action on NaCl absorption by the MTAL is presented and the physiological significance of the coupling of K⁺ to the apical Na⁺:Cl^– cotransporter in the MTAL and of the enhanced metabolic efficiency are discussed.     

The fluted giant clam (Tridacna squamosa) increases nitrate absorption and upregulates the expression of a homolog of SIALIN (H+:2NO3− cotransporter) in the ctenidium during light exposure

Coral Reefs - Giant clams flourish in nutrient-poor waters of tropical Indo-Pacific because they live in symbiosis with extracellular dinoflagellates (zooxanthellae) and receive photosynthates from...     

Na+-coupled Cl− transport in the gastric mucosa of the guinea pig

The Cl^–/HCO ₃ ^– exchange mechanism usually postulated to occur in gastric mucosa cannot account for the Na⁺-dependent electrogenic serosal to mucosal Cl^– transport often observed. It was recently suggested that an additional Cl^– transport mechanism driven by the Na⁺ electrochemical potential gradient may be present on the serosal side of the tissue. To verify this, we have studied Cl^– transport in guinea pig gastric mucosa. Inhibiting the (Na⁺, K⁺) ATPase either by serosal addition of ouabain or by establishing K⁺-free mucosal and serosal conditions abolished net Cl^– transport. Depolarizing the cell membrane potential with triphenylmethylphosphonium (a lipid-soluble cation), and hence reducing both the Na⁺ and Cl^– electrochemical potential gradients, resulted in inhibition of net Cl^– flux. Reduction of short-circuit current on replacing Na⁺ by choline in the serosal bathing solution was shown to be due to inhibition of Cl^– transport. Serosal addition of diisothiocyanodisulfonic acid stilbene (an inhibitor of anion transport systems) abolished net Cl^– flux but not net Na⁺ flux. These results are compatible with the proposed model of a Cl^–/Na⁺ cotransport mechanism governing serosal Cl^– entry into the secreting cells. We suggest that the same mechanism may well facilitate both coupled Cl^–/Na⁺ entry and coupled HCO ₃ ^– /Na⁺ exit on the serosal side of the tissue.     

Appearance of cuboidal cells in relation to salinity in gills of Fundulus heteroclitus, a species exhibiting branchial Na+ but not Cl− uptake in freshwater

Fundulus heteroclitus (killifish) is a model organism for ionoregulatory studies, particularly because of its opercular epithelium, although the gills are the major sites of ion exchange. Whereas Na⁺ and Cl⁻ are excreted through the gills in seawater (SW), the killifish is unusual in taking up only Na⁺ and not Cl⁻ at the gills in freshwater (FW). We describe morphological changes in the branchial epithelium following transfer from an acclimation medium of 10% SW to 100% SW or FW. In 10% SW, mitochondria-rich cells resemble typical seawater chloride cells (SWCCs) with accessory cells. After transfer to 100% SW, no change occurs in pavement cell (PVC) morphology or mitotic rate (measured by bromo-deoxyuridine technique), although the density of SWCC apertures increases several fold because of the uncovering of buried SWCCs by PVCs, in accord with increased rates of Na⁺ and Cl⁻ efflux. After transfer to FW, PVC morphology remains unchanged, but SWCCs and accessory cells are quickly covered by PVCs, with many undergoing apoptosis or necrosis. The mitotic rate doubles by 10–14 h but typical freshwater chloride cells (FWCCs) do not appear. Instead, a wedge-shaped cell type that is moderately rich in apically oriented mitochondria, with a large ovoid nucleus, thin cytoplasmic layer, paucity of vesicular-tubular network, and variably villous surface rapidly (by 3 h) and progressively appears in the filament epithelium, by both uncovering and mitosis. This cell type is similar to that recently identified as the site of Na⁺ uptake in the FW trout gill. We propose the new term “cuboidal cell” for this cell, based on its morphology, to avoid confusion with traditional terminology (of PVC). We hypothesize that the cuboidal cells are the sites of active Na⁺ uptake in FW F. heteroclitus and suggest that the lack of Cl⁻ uptake is attributable to the absence of typical FWCCs previously described in teleosts.This work was supported by NSERC Discovery grants (to C.M.W.) and by an NSERC International Fellowship (to P.L.). C.M.W. is supported by the Canada Research Chair Program.     

Glutamate induces Cl− and K+ currents in the olfactory interneurons of a terrestrial slug

Glutamate-induced responses in the procerebral neurons of the terrestrial slug Limax marginatus were examined using the nystatin-perforated patch recording technique applied in the voltage-clamp mode and local application of drugs. The procerebrum contains two types of interneurons with different spontaneous activities, bursting and nonbursting neurons. In the bursting neurons, a puff of glutamate evoked a rapidly desensitizing current followed by a smaller sustained current. The reversal potential of the early component showed that the current was mediated by Cl⁻ ions, while the late component was presumed to be mediated by K⁺ ions. In the nonbursting neurons, glutamate evoked a sustained current with a strong outward rectification, and the current was mediated by K⁺ ions. Ibotenate selectively evoked the rapidly desensitizing response in the bursting neurons, whereas quisqualate evoked a non-desensitizing K⁺ current both in the bursting and nonbursting neurons. The glutamate-induced K⁺ current had similar characteristics with the spontaneous synaptic activities in the procerebrum neurons, suggesting the possibility that glutamate receptors are involved in the spontaneous oscillatory activity. Accepted: 10 February 1999     

The nature of the neutral Na+−Cl−-coupled entry at the apical membrane of rabbit gallbladder epithelium: II. Na+−Cl− symport is independent of K+

Summary In the epithelium of rabbit gallbladder, in the nominal absence of bicarbonate, intracellular Cl^– activity is about 25mm, about 4 times higher than intracellular Cl^– activity at the electrochemical equilibrium. It is essentially not affected by 10^–4 m acetazolamide and 10^–4 m 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate (SITS) even during prolonged exposures; it falls to the equilibrium value by removal of Na⁺ from the lumen without significant changes of the apical membrane potential difference. Both intracellular Cl^– and Na⁺ activities are decreased by luminal treatment with 25mm SCN^–; the initial rates of change are not significantly different. In addition, the initial rates of change of intracellular Cl^– activity are not significantly different upon Na⁺ or Cl^– entry block by the appropriate reduction of the concentration of either ion in the luminal solution. Luminal K⁺ removal or 10^–5 m bumetanide do not affect intracellular Cl^– and Na⁺ activities or Cl^– influx through the apical membrane. It is concluded that in the absence of bicarbonate NaCl entry is entirely due to a Na⁺–Cl^– symport on a single carrier which, at least under the conditions tested, does not cotransport K⁺.     

Intracellular compartmentation of Na+, K+ and Cl− in the Ehrlich ascites tumor cell: Correlation with the membrane potential

Summary The intracellular distribution of Na⁺, K⁺, Cl^– and water has been studied in the Ehrlich ascites tumor cell. Comparison of the ion and water contents of whole cells with those of cells exposed to La³⁺ and mechanical stress indicated that La³⁺ treatment results in selective damage to the cell membrane and permits evaluation of cytoplasmic and nuclear ion concentrations. The results show that Na⁺ is sequestered within the nucleus, while K⁺ and Cl^– are more highly concentrated in the cell cytoplasm. Reduction of the [Na⁺] of the incubation medium by replacement with K⁺ results in reduced cytoplasmic [Na⁺], increased [Cl^–] and no change in [K⁺]. Nuclear concentrations of these ions are virtually insensitive to the cation composition of the medium. Concomitant measurements of the membrane potential were made. The potential in control cells was –13.7 mV. Reduction of [Na⁺] in the medium caused significant depolarization. The measured potential is describable by the Cl^– equilibrium potential and can be accounted for in terms of cation distributions and permeabilities. The energetic implications of the intracellular compartmentation of ions are discussed.     

Functional role of a putative carbonic anhydrase II-binding domain in the electrogenic Na+-HCO3− cotransporter NBCe1 expressed in Xenopus oocytes

The electrogenic Na⁺-HCO₃^? cotransporter NBCe1 plays essential roles in the regulation of systemic and/or local pH. Homozygous inactivating mutations in NBCe1 cause proximal renal tubular acidosis associated with ocular abnormalities. We recently showed that defective membrane expression of NBCe1, caused by several mutations such as Delta65bp (S982NfsX4), is also associated with familial migraine. The Delta65bp mutant is quite unique in that it lacks a putative carbonic anhydrase (CA) II-binding domain but still shows an apparently normal transport activity in Xenopus oocytes. In this addendum, we show that the co-expression of CAII together with the wild-type NBCe1 or the Delta65bp mutant does not enhance the NBCe1 activities in oocytes. Moreover, a carbonic anhydrase inhibitor acetazolamide fails to inhibit the wild-type or the Delta65bp activities co-expressed with CAII. These results indicate that a bicarbonate transport metabolon proposed for the interaction between CAII and NBCe1 does not work at least in Xenopus oocytes.     

Nature of the neutral Na+-Cl− coupled entry at the apical membrane of rabbit gallbladder epithelium: IV. Na+/H+, Cl−/HCO 3 − double exchange,hydrochlorothiazide-sensitive Na+-Cl− symport and Na+-K+-2Cl− cotransport are all involved

Transepithelial fluid transport was measured gravimetrically in rabbit gallbladder (and net Na+ transport was calculated from it), at 27 degrees C, in HCO(3-)-free bathing media containing 10(-4) M acetazolamide. Whereas luminal 10(-4) M bumetanide or 10(-4) M 4-acetamido-4'-iso-thiocyanostilbene-2,2'-disulfonate (SITS) did not affect fluid absorption, 25 mM SCN- abolished it; hydrochlorothiazide (HCTZ) in the luminal medium reduced fluid absorption from 28.3 +/- 1.6 (n = 21) to 8.6 +/- 1.6 microliters cm-2 hr-1 (n = 10), i.e., to about 30%. This maximum effect was already obtained at 10(-3) M concentration; the apparent IC50 was about 2 x 10(-4) M. The residual fluid absorption, again insensitive to SITS, was completely inhibited by SCN- or bumetanide. Cl- influx at the luminal border of the epithelium, measured under the same conditions and corrected for the extracellular space and paracellular influx, proved insensitive to 10(-4) M bumetanide, but was slowly inhibited by 10(-3) M HCTZ, with maximum inhibition (about 54%) reached after a 10-min treatment; it subsequently rose again, in spite of the presence of HCTZ. However, if the epithelium, treated with HCTZ, was exposed to 10(-4) M bumetanide during the measuring time (45 sec), inhibition was completed and the subsequent rise of Cl- influx eliminated. Intracellular Cl- accumulation with respect to the predicted activity value at equilibrium decreased significantly upon exposure to 10(-3) M HCTZ, reached a minimum within 15-30 min of treatment, then rose again significantly at 60 min. Simultaneous exposure to HCTZ and bumetanide decreased the accumulation to a significantly larger extent as compared to HCTZ alone, already in 15 min, and impeded the subsequent rise. Intracellular K+ activity rose significantly within 30 min treatment with HCTZ; the increase proved bumetanide dependent. The results obtained show that Na(+)-Cl- symport, previously detected under control conditions, is the HCTZ-sensitive type; its inhibition elicits bumetanide-sensitive Na(+)-K(+)-2Cl- cotransport. Thus, the three forms of neutral Na(+)-Cl(-)-coupled transport so far evidenced in epithelia, Na+/H+, Cl-/HCO3- double exchange (in the presence of exogenous bicarbonate), HCTZ-sensitive Na(+)-Cl- symport and bumetanide-sensitive Na(+)-K(+)-2Cl- cotransport, are all present in the apical membrane of rabbit gallbladder.     

Intracellular Na+, K+ and Cl− activities in Ehrlich ascites tumor cells

Ehrlich ascites tumor cell membrane potential (V_m) and intracellular Na⁺, K⁺ and Cl⁻ activities were measured under steady-state conditions in normal saline medium (Na⁺ = 154, K⁺ = 6, Cl⁻ = 150 mequiv./l). Membrane potential was estimated to be −23.3 ± 0.8 mV using glass microelectrodes. Intracellular ion activities were estimated with similar glass electrodes rendered ion-selective by incorporation of ion-specific ionophores. Measurements of V_m and ion-activity differences were made in the same populations of cells. Under these conditions the intracellular Na⁺, K⁺ and Cl⁻ activities are 4.6 ± 0.5; 68.3 ± 8.0; and 43.6 ± 2.1 mequiv./l, respectively. The apparent activity coefficients for Na⁺ and K⁺ are 0.18 ± 0.02 and 0.41 ± 0.05 respectively. These are significantly lower than the activity coefficients expected for the ions in physiological salt solutions (0.71 and 0.73, respectively). The activity coefficient for intracellular Cl⁻ (0.67 ± 0.03), however, is close to that of the medium (0.73), and the transmembrane electrochemical potential difference for Cl⁻ is not different from zero. The results establish that the energy available from the Na⁺ electrochemical gradient is much greater than previously estimated from chemical measurements.     

Fluoxetine Inhibits K+ Transport Pathways (K+ Efflux, Na+-K+-2Cl− Cotransport, and Na+ Pump) Underlying Volume Regulation in Corneal Endothelial Cells

We have studied regulatory volume responses of cultured bovine corneal endothelial cells (CBCEC) using light scattering. We assessed the contributions of fluoxetine (Prozac) and bumetanide-sensitive membrane ion transport pathways to such responses by determining K⁺ efflux and influx. Cells swollen by a 20% hypo-osmotic solution underwent a regulatory volume decrease (RVD) response, which after 6 min restored relative cell volume by 98%. Fluoxetine inhibited RVD recovery; 20 μm by 26%, and 50 μm totally. Fluoxetine had a triphasic effect on K⁺ efflux; from 20 to 100 μm it inhibited efflux 2-fold, whereas at higher concentrations the efflux first increased to 1.5-fold above the control value, and then decreased again. Cells shrunk by a 20% hyperosmotic solution underwent a regulatory volume increase (RVI) which also after 6 min restored the cell volume by 99%. Fluoxetine inhibited RVI; 20 μm by 25%, and 50 μm completely. Bumetanide (1 μm) inhibited RVI by 43%. In a Cl⁻-free medium, fluoxetine (50–500 μm) progressively inhibited bumetanide-insensitive K⁺ influx. The inhibitions of RVI and K⁺ influx induced by fluoxetine 20 to 50 μm were similar to those induced by 1 μm bumetanide and by Cl⁻-free medium. A computer simulation suggests that fluoxetine can interact with the selectivity filter of K⁺ channels. The data suggest that CBCEC can mediate RVD and RVI in part through increases in K⁺ efflux and Na-K-2Cl cotransport (NKCC) activity. Interestingly, the data also suggest that fluoxetine at 20 to 50 μm inhibits NKCC, and at 100–1000 μm inhibits the Na⁺ pump. One possible explanation for these findings is that fluoxetine could interact with K⁺-selective sites in K⁺ channels, the NKC cotransporter and the Na⁺ pump.     

Studies on chloride permeability of the skin ofLeptodactylus ocellatus: II. Na+ and Cl− effect of inward movements of Cl−

Summary At low concentration (1mm) of Cl^– in the outer solution, the influx of chloride through the isolated skin (J ₁₃ ^Cl ) of the South American frogLeptodactylus ocellatus (L.) seems to be carried by two mechanisms: (i) a passive one that exhibits the characteristics of an exchange diffusion process, and (ii) an active penetration. Studies of the influx and efflux of chloride (J ₁₃ ^Cl andJ ₃₁ ^Cl ) indicate, that the presence of a high (107mm) concentration of Cl^– in the outer solution activates the translocation of this ion through the cells. Studies of the unidirectional flux of Cl^– across the outer barrier (J ₁₂ ^Cl ) indicate that Na⁺ out stimulates the penetration of Cl^– at this level. Cl^– out, in turn, stimulates, theJ ₁₂ ^Na , but this effect is only detected at low concentrations of Na⁺ out.     

Na+ and Cl− conductances are controlled by cytosolic Cl− concentration in the intralobular duct cells of mouse mandibular glands

Our previously published whole-cell patch-clamp studies on the cells of the intralobular (granular) ducts of the mandibular glands of male mice revealed the presence of an amiloride-sensitive Na⁺ conductance in the plasma membrane. In this study we demonstrate the presence also of a Cl^– conductance and we show that the sizes of both conductances vary with the Cl^– concentration of the fluid bathing the cytosolic surface of the plasma membrane. As the cytosolic Cl^– concentration rises from 5 to 150 mmol/liter, the size of the inward Na⁺ current declines, the decline being half-maximal when the Cl^– concentration is approximately 50 mmol/liter. In contrast, as cytosolic Cl^– concentration increases, the inward Cl^– current remains at a constant low level until the Cl^– concentration exceeds 80 mmol/liter, when it begins to increase. Studies in which Cl^– in the pipette solution was replaced by other anions indicate that the Na⁺ current is suppressed by intracellular Br^-, Cl^– and NO ₃ ^- but not by intracellular I^-, glutamate or gluconate. Our studies also show that the Cl^– conductance allows passage of Cl^– and Br^- equally well, I-less well, and NO ₃ ^- , glutamate and gluconate poorly, if at all. The findings with NO ₃ ^- are of particular interest because they show that suppression of the Na⁺ current by a high intracellular concentration of a particular anion does not depend on actual passage of that anion through the Cl^– conductance. In mouse granular duct cells there is, thus, a reciprocal regulation of Na⁺ and Cl^– conductances by the cytosolic Cl^– concentration. Since the cytosolic Cl^– concentration is closely correlated with cell volume in many epithelia, this reciprocal regulation of Na⁺ and Cl^– conductances may provide a mechanism by which ductal Na⁺ and Cl transport rates are adjusted so as to maintain a stable cell volume.This project was supported by the National Health and Medical Research Council of Australia. We thank Professor P. Barry (University of New South Wales) for assistance with the junction potential measurements.     

Stoichiometry and ion affinities of the Na−K−Cl cotransport system in the intestine of the winter flounder (Pseudopleuronectes americanus)

Summary Na–K–Cl cotransport stoichiometry and affinities for Na, K and Cl were determined in flounder intestine. Measurement of simultaneous NaCl and RbCl influxes resulted in ratios of 2.2 for Cl/Na and 1.8 for Cl/Rb. The effect of Na and Rb on Rb influx showed first order kinetics withK _1/2 values of 5 and 4.5mm and Hill coefficients of 0.9 and 1.2, respectively. The effect of Cl on rubidium influx showed a sigmoidal relationship withK _1/2 of 20mm and a Hill coefficient of 2.0. The effects of variations in Na and Cl concentration on short-circuit current (I _sc) were also determined. TheK _1/2 for Na was 7mm with a Hill coefficient of 0.9 and theK _1/2 for Cl was 46mm with a Hill coefficient of 1.9. Based on the simultaneous influx measurements, a cotransport stoichiometry of 1Na1K2Cl is concluded. The Hill coefficients for Cl suggest a high degree of cooperativity between Cl binding sites. Measurements of the ratio of net Na and Cl transepithelial fluxes under short-circuit conditions (using a low Na Ringer solution to minimize the passive Na flux) indicate that the Cl/Na flux ratio is approximately 21. Therefore Na recycling from serosa to mucosa does not significantly contribute to theI _sc. Addition of serosal ouabain (100 m) inhibited Rb influx, indicating that Na–K–Cl cotransport is inhibited by ouabain. This finding suggests that a feedback mechanism exists between the Na–K-ATPase on the basolateral membrane and the apical Na–K–2Cl cotransporter.