Localisation autoradiographique des ions Cl− et Na+ dans les cellules à chlorure de la branchie d'anguille (Anguilla anguilla L.) adaptée à l'eau de mer

Résumé Nous avons montré par une technique autoradiographique que les ions Cl^– et Na⁺ sont concentrés dans les cellules à chlorure de la branchie d'anguille adaptée à l'eau de mer. La signification de cette accumulation ionique plus marquée vers le pôle apical de ces cellules a été discutée par rapport à l'excrétion branchiale de ces ions en eau de mer.

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Autoradiographic localization of Cl^– and Na⁺ ions in the chloride cells of sea water adapted eel (Anguilla anguilla L.) gills

Summary With an autoradiographic technique Cl^– and Na⁺ ions have been shown to be localized in the chloride cells of sea water eel gills. The significance of this accumulation, more marked towards the apical pole of these cells, is discussed with regard to branchial excretion of these ions in sea water.

     

Branchial and renal ion fluxes and transepithelial electrical potential differences in rainbow trout,Oncorhynchus mykiss: effects of aluminium at low pH

Synopsis Adult rainbow trout, Oncorhynchus mykiss, were acutely exposed for 4 hours to low pH (4.4) and elevated Al-concentrations (300 µgI^–1) in soft water (Ca²⁺ + Mg²⁺ = 25 µmolI^–1). Comparison of branchial and renal ion fluxes (Na⁺, Cl^–, Mg²⁺, Ca²⁺ and NH₄ ⁺) gave evidence that pH and Al effects were primarily localized at the gill site. The negative whole body ion balance seemed to be caused by stimulatory effects on Na and Cl efflux especially under Al stress and to a lesser extent by inhibition of influx. Measurements of gill potentials indicated positive shifts, which were similar in response to increasing levels of H⁺ ions and Al. It is suggested that Al-induced changes of branchial potentials causes high diffusable loss of ions through interference with membrane-bound Ca²⁺ at the gill site.     

The effects of experimentally altered gill chloride cell surface area on acid-base regulation in rainbow trout during metabolic alkalosis

To evaluate the role of the gill chloride cells in regulating metabolic alkalosis in rainbow trout (Oncorhynchus mykiss), the surface area of branchial chloride cells was altered experimentally using combined cortisol/ovine growth hormone injections. Long-term (10-day) treatment of fish with cortisol/ovine growth hormone caused an increase in the two-dimensional chloride cell fractional surface area when compared to uninjected fish (from 8.4 to 29.7%). This was the combined result of an increase in the size of individual cells (from 34.6 to 59.2 m²) and increased numbers of cells (from 2368 to 5006 cells · mm^-2). Metabolic alkalosis was induced by intra-arterial infusion of 140 mmol · l^-1 NaHCO₃; control fish were infused with 140 mmol · l^-1 NaCl. Blood pH and plasma [HCO₃ ^-] increased in both the untreated and the cortisol/ovine growth hormone-treated fish. However, the increases in pH (from 8.05 to 8.53) and [HCO₃ ^-] (from 5.9 to 22.2 mmol · l^-1) in the untreated fish were significantly greater than in the cortisol/ovine growth hormone-treated fish (pH increased from 7.78 to 8.11; [HCO₃ ^-] increased from 5.5 to 13.9 mmol · l^-1). In all fish, NaHCO₃ infusion elicited an increase in the rate of branchial basic equivalent excretion (acidic equivalent uptake) which, in turn, was caused by decreases and increases in branchial Na⁺ uptake and Cl^- uptake, respectively. In the untreated fish, there was a pronounced increase (75%) in chloride cell surface area during NaHCO₃ infusion. The attenuation of the metabolic alkalosis during HCO₃ ^- infusion in the cortical/ovine growth hormone-treated fish was caused, at least in part, by an enhancement of branchial basic equivalent excretion. In these fish that already displayed a proliferation of chloride cells, there was no further increase in chloride cell surface area. The changes in Na⁺ influx and Cl^- influx were quantitatively similar during NaHCO₃ infusion in both groups. This suggests that the greater rate of base excretion in the cortisol/ovine growth hormone-treated fish was caused by a greater percentage of Cl^- uptake being coupled to HCO₃ ^- excretion and less to Cl^- excretion (Cl^- exchange diffusion).Abbreviations Amm total ammonia - bw body weight - CC chloride cell - CCFA chloride cell fractional area - cort/oGH cortisol/ovine growth hormone - dpm disintegrations per minute - J ^Amm net flux of total ammonia - J _in unidirectional influx - J _inCl^- chloride ion uptake - J _inNa⁺ sodium ion uptake - J _netH⁺ net acidic equivalent flux - J ^TA net flux of titrable alkalinity - MS 222 ethyl-m-aminobenzoate - oGH ovine growth hormone - PVC pavement cell - SEM scanning electron microscope - TA titrable alkalinity     

The Effect of Ration on Acclimation to Environmental Acidity in Rainbow Trout

Freshwater salmonids exposed to low environmental pH typically suffer a net loss of ions, primarily Na⁺ and Cl^–, across the gills, resulting in reduced plasma and tissue ion concentrations. However, in recent experiments in our laboratory, juvenile rainbow trout, Oncorhynchus mykiss, fed a ration of 1% body weight d^–1 or greater showed no ionoregulatory disturbance during chronic, sublethal acidification. This raised the possibility that these fish had acclimated to low pH in that they would be better able to withstand further, more severe acidification than fish that had no prior experience of acid conditions: previous studies had concluded that such acclimation does not occur. This hypothesis was tested by measuring unidirectional ion fluxes during a 24h acute acid challenge (pH 4.2) in juvenile rainbow trout that had previously been exposed to either ambient pH 6.2 (naive fish) or sublethal low pH 5.2 (acid pre-exposed fish) for 90 days, and fed a ration of either 1.0 or 0.25% d^–1 (wet basis). No mortalities were observed during the acute acid challenge in the fish fed the higher ration and no differences between the two groups in the response of Na⁺ fluxes were observed. Sodium influx in both groups was significantly inhibited throughout the challenge and Na⁺ net flux was significantly stimulated over the first 6h. Prior to the acute acid challenge, the fish fed the lower ration that had previously been exposed to pH 5.2 had significantly lower plasma ion concentrations than those fish previously exposed to pH 6.2. Both groups suffered mortalities; those of the naive fish (22% by 24h) being markedly lower than those of the acid pre-exposed fish (68% by 24h). However, there were no significant differences in either Na⁺ or Cl^– fluxes between the two groups of fish during the acid challenge: both showed significant inhibition of ion influxes and significantly greater net ion losses, resulting in reduced plasma ion concentrations. These results indicate that rainbow trout are unable to acclimate to environmental acidification irrespective of the availability of dietary salts.     

Chloride transport activation by plasma osmolarity during rapid adaptation to high salinity of Fundulus heteroclitus

Transition from low salt water to sea water of the euryhaline fish, Fundulus heteroclitus, involves a rapid signal that induces salt secretion by the gill chloride cells. An increase of 65 mOsm in plasma osmolarity was found during the transition. The isolated, chloridecell-rich opercular epithelium of sea-water-adapted Fundulus exposed to 50 mOsm mannitol on the basolateral side showed a 100% increase in chloride secretion, which was inhibited by bumetanide 10^–4 m and 10^–4 m DPC (N-Phenylanthranilic acid). No effect of these drugs was found on apical side exposure. A Na⁺/H⁺ exchanger, demonstrated by NH₄Cl exposure, was inhibited by amiloride and its analogues and stimulated by IBMX, phorbol esters, and epithelial growth factor (EGF). Inhibition of the Na⁺/H⁺ exchanger blocks the chloride secretion increase due to basolateral hypertonicity. A Cl^–/HCO ₃ ^– exchanger was also found in the chloride cells, inhibited by 10^–4 m DIDS but not involved in the hyperosmotic response. Ca²⁺ concentration in the medium was critical for the stimulation of Cl^– secretion to occur. Chloride cell volume shrinks in response to hypertonicity of the basolateral side in sea-water-adapted operculi; no effect was found on the apical side. Freshwater-adapted fish chloride cells show increased water permeability of the apical side. It is concluded that the rapid signal for adaptation to higher salinities is an increased tonicity of the plasma that induces chloride cell shrinkage, increased chloride secretion with activation of the Na⁺K⁺2Cl^– cotransporter, the Na⁺/H⁺ exchanger and opening of Cl^– channels.The work was supported by the National Institutes of Health, Research Grant EYO1340 to J.A.Z. Part of this research was performed while Dr. Zadunaisky was a Scholar In Residence at the Fogarty International Center of The National Institutes of Health in Bethesda, Maryland. Ms. Dawn Roberts was a fellow of the Grass Foundation and Pew Foundation during this work. Grants from the National Science Foundation and the National Institutes of Health to the Mount Desert Island Biological Laboratory also provided assistance for this research.     

Distinct Na+/K+/2Cl- cotransporter localization in kidneys and gills of two euryhaline species, rainbow trout and killifish

The kidney is an organ playing an important role in ion regulation in both freshwater (FW) and seawater (SW) fish. The mechanisms of ion regulation in the fish kidney are less well studied than that of their gills, especially at the level of transporter proteins. We have found striking differences in the pattern of Na⁺/K⁺/2Cl^- cotransporter (NKCC) expression between species. In the killifish kidney, NKCC is apically localized in the distal and collecting tubules and basolaterally localized in the proximal tubules. However, in the SW killifish gill, NKCC is basolaterally co-localized with Na⁺/K⁺-ATPase, whereas in FW, NKCC immunoreactivity is primarily apical, although still colocalized within the same mitochondria-rich cell with basolateral Na⁺/K⁺-ATPase. Rainbow trout kidney has NKCC only in the apical membrane of the distal and collecting tubules in both environments, with no signal being detected in the proximal tubule. On the other hand, in the trout gill, NKCC is found basolaterally in both FW and SW environments. An important observation is that, in the gills of rainbow trout, the trailing edge of the filament possesses mostly Na⁺/K⁺-ATPase-positive but NKCC-negative mitochondria-rich cells, whereas in the region between and at the roots of the gill lamellae, most mitochondria-rich cells exhibit both Na⁺/K⁺-ATPase- and NKCC-positive immunoreactivity. These results suggest that the differential localization of transporters between the two species represents differences in function between these two euryhaline fishes with different life histories and strategies. Funding for this research was provided by NSERC Discovery Grants to G.G.G. and W.S.M., an Alberta Ingenuity Fund PDF, and a fellowship from the NSERC Research Capacity Development Grant to F.K.     

On the interrelations of the Na+-and Cl?-influxes in the pulmonate freshwater snailLymnaea stagnalis

Summary In the freshwater snailLymnaea stagnalis the influxes of Na⁺ and Cl^– were studied at different external concentrations of these ions. The characteristies of the Na⁺- and Cl^–-influxes are similar with respect to saturation kinetics,K _m (0.1 mM) and activation by low-salt adaptation. In short-term experiments the Na⁺- and Cl^–-influxes are independent. Because of the counter-ions (H⁺ and HCO ₃ ^– ) involved, this indicates a potential acid-base regulatory capacity. Low-salt adaptation, due to either Na⁺-or Cl^–-depletion, activates both the Na⁺- and the Cl^–-influx. It is suggested that under both conditions the number of active integumental pumps, involved in Na⁺- as well as in Cl^–-uptake, is increased.     

Na+ uptake through the brush border membranes of intestine from fresh water and sea-water adapted trout (Salmo gairdneri,R.)

Summary A comparative study of the mechanisms of Na⁺ absorption through brush border membranes of enterocytes from freshwater (FW) and seawater (SW) adapted trout were carried out using purified vesicle preparations. In contrast to FW trout, SW trout were found to possess a Na⁺–K⁺–Cl^– cotransport process. This finding is regarded as a major adaptation to SW since this cotransport allows an increase of ions and water absorption. Both FW and SW trout were equipped with a Na⁺–H⁺ exchange. In FW, the intestine of the trout had both a Na⁺–Na⁺ exchange and a Na⁺ conductance which may be responsible for enterocyte Na⁺ uptake along the potential gradient.     

Growth characteristics and ion contents of non-selected and salt-selected callus lines of highbush blueberry (Vacdnium corymbosum) cultivars Blue Crop and Denise Blue

Non-selected and sodium chloride selected callus lines of Vacdnium corymbosum L.cv Blue Crop and cv. Denise Blue were grown on media supplemented with 0–100 mM NaCl. For both cultivars, fresh weight and dry weight yields were greater in selected lines on all levels of NaCl. Selected lines of Blue Crop displayed better growth than selected lines of Denise Blue at most concentrations of NaCl. Internal Na⁺ and Cl^– concentrations in selected and non-selected lines of both cultivars increased as external concentration was raised. However, selected lines of Blue Crop and Denise Blue accumulated more Na⁺ and Cl^– than non-selected lines. Selected lines of both cultivars maintained higher levels of K⁺ than non-selected lines on all external NaCl levels. Selected lines of Blue Crop had higher levels of Na⁺ and Cl^– than that of Denise Blue. The results suggest Na⁺ and Cl^– accumulation could be a mechanism allowing better growth in selected lines at moderate salinity levels (50–75 mM NaCl).     

Passive sodium movements across the opercular epithelium: The paracellular shunt pathway and ionic conductance

Summary The unidirectional Na⁺, Cl^–, and urea fluxes across isolated opercular epithelia from seawater-adaptedFundulus heteroclitus were measured under different experimental conditions. The mean Na⁺, Cl^–, and urea permeabilities were 9.30×10^–6 cm·sec^–1, 1.24×10^–6 cm·sec^–1, and 5.05×10^–7 cm·sec^–1, respectively. The responses of the unidirectional Na⁺ fluxes and the Cl^– influx (mucosa to serosa) to voltage clamping were characteristic of passively moving ions traversing only one rate-limiting barrier. The Na⁺ conductance varied linearly with, and comprised a mean 54% of, the total tissue ionic conductance. The Cl^– influx and the urea fluxes were independent of the tissue conductance. Triaminopyrimidine (TAP) reduced the Na⁺ fluxes and tissue conductance over 70%, while having no effect on the Cl^– influx or urea fluxes. Mucosal Na⁺ substitution reduced the Na⁺ permeability 60% and the tissue conductance 76%, but had no effect on the Cl^– influx or the urea fluxes. Both the Na⁺ and Cl^– influxes were unaffected by respective serosal substitutions, indicating the lack of any Na⁺/Na⁺ and Cl^–/Cl^– exchange diffusion.The results suggest that the unidirectional Na⁺ fluxes are simple passive fluxes proceeding extracellularly (i.e., movement through a cation-selective paracellular shunt). This pathway is dependent on mucosal (external) Na⁺, independent of serosal (internal) Na⁺, and may be distinct from the transepithelial Cl^– and urea pathways.     

Effects of cytochalasin B on water, Na+ and Cl? exchanges in the gill of seawater adapted mulletMugil capito

Summary Electron microscopy study shows that cytochalasin treatment of the mullet damages the microfilaments system in the apex of gill ionocytes: the microfilaments are reduced in number and shortened. Cytochalasin causes a reduction of transgill potential difference and an increase of the Na⁺ and Cl^– blood concentration, of the diffusional water permeability of the gill, of the Na⁺ branchial influx and of Cl^– efflux. The increase of the Na⁺ influx may result in a reduction of the Na⁺ net excretion flux compared to the control. The increased permeability in cytochalasin treated fish facilitates the Cl^– entry probably leading to a reduction of the net Cl^– excretion. The partial inhibition of the K⁺ dependent components of Na⁺ and Cl^– effluxes also contributes to the reduction of Na⁺ and Cl^– excretion. The role of microfilaments in the mechanisms of ionic excretion by the gill is discussed.     

Ouabain-induced cell swelling in rabbit cortical collecting tubule: NaCl transport by principal cells

Summary Ouabain had no effect on the volume of intercalated cells of DOCA-stimulated rabbit cortical collecting tubules, but caused principal cells to swell rapidly at an initial rate of 67% min., Principal cells swelled 133% then activated regulatory volume decrease mechanisms and shrank at an initial rate of –3%/min to a new volume 13% above control. The initial rate of ouabain swelling was completely inhibited by perfusate Na⁺ removal or reduced 95% by luminal addition of 10^–5 m amiloride. Luminal peritubular, or bilateral Cl^– removal each caused cell shrinkages of 10% and reduced the rate of ouabain swelling by 70, 85, and 99%, respectively. The presence of an apical Cl^– transport step in principal cells was confirmed by increasing luminal K⁺ from 5 to 53mm, which caused cell swelling of 22%. This volume increase was completely blocked by luminal Cl^– removal, but was unaffected by peritubular Cl^– substitution. Perfusion of CCT with 0.1mm acetazolomide, 0.1mm DPC or 0.5mm SITS caused principal cell shrinkages of 7–9% and reduced the rate of ouabain swelling by 60, 70, and 40%, respectively. The initial rate of ouabain swelling was inhibited 70% by bilateral CO₂/HCO₃ removal and 50% by whole animal acid loading. Taken together these results demounstrate that ouabain swelling is due to cellular NaCl accumulation and that Na⁺ enters the cell primarily through apical Na⁺ channels. Cellular Cl^– entry occurs at least partially through the apical membrane and may be mediated by a Cl^–/HCO ₃ ^– exchanger. Brief (45–90 sec) exposure of principal cells to ouabain is associated with a rapid inhibition of Na⁺ and/or Cl^– entry steps, whereas long-term (>5min) ouabvain exposure completely blocks one or both of these transport pathways.     

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.     

Stimulation by HCO 3 − of Na+ transport in rabbit gallbladder

Summary Bicarbonate presence in the bathing media doubles Na⁺ and fluid transepithelial transport and in parallel significantly increases Na⁺ and Cl^– intracellular concentrations and contents, decreases K⁺ cell concentration without changing its amount, and causes a large cell swelling. Na⁺ and Cl^– lumen-to-cell influxes are significantly enhanced, Na⁺ more so than Cl^–. The stimulation does not raise any immediate change in luminal membrane potential and cannot be due to a HCO ₃ ^– -ATPase in the brush border. The stimulation goes together with a large increase in a Na⁺-dependent H⁺ secretion into the lumen. All of these data suggests that HCO ₃ ^– both activates Na⁺–Cl^– cotransport and H⁺–Na⁺ countertransport at the luminal barrier.Thiocyanate inhibits Na⁺ and fluid transepithelial transport without affecting H⁺ secretion and HCO ₃ ^– -dependent Na⁺ influx. It reduces Na⁺ and Cl^– concentrations and contents, increases the same parameters for K⁺, causes a cell shrinking, and abolishes the lumen-to-cell Cl^– influx. It enters the cell and is accumulated in the cytoplasm with a process which is Na⁺-dependent and HCO ₃ ^– -activated. Thus, SCN^– is likely to compete for the Cl^– site on the cotransport carrier and to be slowly transferred by the cotransport system itself.     

Growth hormone and cortisol treatment stimulate seawater tolerance in both anadromous and landlocked Arctic charr

In a comparative experiment the effect of cortisol and growth hormone (GH) on the hypo-osmoregulatory ability of a landlocked and an anadromous strain of Arctic charr (Salvelinus alpinus) was investigated. Cortisol and GH were implanted either alone or in combination, and the fish were exposed to a 24 h seawater challenge test (SWT) on days 14 and 28 after implantation. Hypo-osmoregulatory ability, measured as plasma osmolality and chloride concentration after the SWTs, was better in the anadromous than in the landlocked strain, irrespective of treatment. However, cortisol provided a strong stimulation of hypo-osmoregualtory ability in both strains, and this stimulation seemed to be potentiated by GH in an additive manner. Improved hypo-osmoregulatory ability in GH + cortisol treated anadromous Arctic charr was accompanied by increased gill Na⁺, K⁺-ATPase activity and Na⁺–K⁺–2Cl⁻ cotransporter protein abundance, but no changes in gill Na⁺,K⁺-ATPase α1a and α1b mRNA levels. For landlocked charr the improved hypo-osmoregulatory ability in GH +cortisol treated fish was accompanied only with an increase in gill Na⁺–K⁺–2Cl⁻ cotransporter protein abundance. Hormone treatment caused an improvement of hypo-osmoregulatory ability that was of approximately the same magnitude in the landlocked as in the anadromous Arctic charr. This suggests that the lack of spontaneous development of hypo-osmoregulatory ability often seen in landlocked populations of Arctic charr may depend, at least partly, on a lack of the hormonal activation seen in anadromous populations.     

Effects of antidiuretic hormone on cellular conductive pathways in mouse medullary thick ascending limbs of Henle: I. ADH increases transcellular conductance pathways

Summary This paper reports experiments designed to assess the relations between net salt absorption and transcellular routes for ion conductance in single mouse medullary thick ascending limbs of Henle microperfusedin vitro. The experimental data indicate that ADH significantly increased the transepithelial electrical conductance, and that this conductance increase could be rationalized in terms of transcellular conductance changes. A minimal estimate (G _c ^min ) of the transcellular conductance, estimated from Ba⁺⁺ blockade of apical membrane K⁺ channels, indicated thatG _c ^min was approximately 30–40% of the measured transepithelial conductance. In apical membranes, K⁺ was the major conductive species; and ADH increased the magnitude of a Ba⁺⁺-sensitive K⁺ conductance under conditions where net Cl^– absorption was nearly abolished. In basolateral membranes, ADH increased the magnitude of a Cl^– conductance; this ADH-dependent increase in basal Cl^– conductance depended on a simultaneous hormone-dependent increase in the rate of net Cl^– absorption. Cl^– removal from luminal solutions had no detectable effect onG _e , and net Cl^– absorption was reduced at luminal K⁺ concentrations less than 5mm; thus apical Cl^– entry may have been a Na⁺,K⁺,2Cl^– cotransport process having a negligible conductance. The net rate of K⁺ secretion was approximately 10% of the net rate of Cl^– absorption, while the chemical rate of net Cl^– absorption was virtually equal to the equivalent short-circuit current. Thus net Cl^– absorption was rheogenic; and approximately half of net Na⁺ absorption could be rationalized in terms of dissipative flux through the paracellular pathway. These findings, coupled with the observation that K⁺ was the principal conductive species in apical plasma membranes, support the view that the majority of K⁺ efflux from cell to lumen through the Ba⁺⁺-sensitive apical K⁺ conductance pathway was recycled into cells by Na⁺,K⁺,2Cl^– cotransport.     

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.     

Regulation of intracellular chloride activity during perfusion with hypertonic solutions in theNecturus proximal tubule

Summary In a previous study we presented evidence that chloride transport across the basolateral membrane inNecturus proximal tubule cells occurs predominantly via exchange for both Na⁺ and HCO ₃ ^– . In this study the regulation of intracellular chloride was further examined in the doubly-perfused kidney preparation using conventional and chloride-sensitive microelectrodes. Application of hypertonic basolateral solutions containing 80mm raffinose stimulated an efflux of chloride such that chloride activity remained unchanged at control levels. Membrane potential did not change in these experiments. Inhibition of Cl^– exit across the basolateral cell membrane by removal of either HCO ₃ ^– or Na⁺ from the perfusion solution resulted in a significant increase in intracellular chloride activity,a _Cl ⁱ , when basolateral osmolarity was raised. Hypertonic basolateral solutions also produced a significant rise ina _Cl ⁱ in the presence of SITS.This study provides further evidence that chloride is transported across the basolateral cell membrane in exchange for both Na⁺ and HCO ₃ ^– . Since this exchange mechanism is activated in response to hypertonic solutions, these studies suggest a functional role for this exchanger in the regulation ofa _Cl ⁱ in theNecturus proximal tubule cell during volume changes.     

Intracellular and luminal ion concentrations in sea turtle salt glands determined by x-ray microanalysis

Summary The lachrymal salt glands of hatchlings of the green sea turtle (Chelonia mydas) secrete a hyperosmotic (up to 2000 mosmol·kg^–1) NaCl solution. X-ray microanalysis of frozen-hydrated glands showed that during secretion intracellular Na⁺ concentration in the principal cells increased from 13 to 34 mmol·l^–1 of cell water, whilst Cl^– and K⁺ concentrations remained unchanged at 81 mmol·l^–1 and 160–174 mmol·l^–1, respectively. The high Cl^– concentration and the change in Na⁺ concentration are consistent with the prevailing paradigm for secretion by the structurally and functionally similar elasmobranch rectal gland. Concentrations of Na⁺, Cl^– and K⁺ in the lumina of secretory tubules of secreting (Na⁺ 122, Cl^– 167, K⁺ 38 mmol·l^–1) and non-secreting (Na⁺ 114, Cl^–1 174, K⁺ 44 mmol·l^–1) glands were similar and the fluid was calculated to be approximately isosmotic with blood. In the central canals Na⁺ and Cl^– concentrations were similar but K⁺ concentration was lower (11–15 mmol·l^–1). It is concluded that either a high transepithelial NaCl gradient in secretory tubules and central canals is very rapidly dissipated during the short time between gland excision and freezing, or that ductal modification of an initial isosmotic secretion occurs.     

Effects of caecal ligation and saline acclimation on plasma concentration and organ mass in male and female Pekin ducks,Anas platyrhynchos

Summary The intestinal caeca reabsorb urinary sodium chloride (NaCl) and water (Rice and Skadhauge 1982). Free water may be generated if the reabsorbed NaCl is secreted via salt gland secretion (Schmidt-Nielsen et al. 1958). Therefore ceacal ligation should (a) reduce hingut NaCl and water reabsorption, (b) enhance the increase in plasma osmolality during saline acclimation, and (c) affect drakes more than ducks. Twelve Pekin drakes and 13 Pekin ducks, Anas platyrhynchos, were caecally ligated or sham operated before acclimation to 450 mmol · 1 NaCl. Body mass, hematocrit, plasma osmolality, and inonic concentrations of plasma, cloacal fluid, and salt gland secretion were measured after each increase in drinking water salinity. Osmoregulatory organ masses were determined. Caecal ligation did not effect plasma osmolality or ion concentrations of plasma, cloacal fluid, or salt gland secretion, but reduced salt gland size in ducks. Drakes and ducks drinking fresh water had the same hematocrit, plasma osmolality, and plasma concentrations of Na⁺ and Cl^–. In both sexes exposure to 75 mmol · 1^-1 NaCl significantly decreased plasma [Na⁺] and doubled cloacal fluid [Na⁺]. Exposure to 450 mmol · 1^-1 NaCl decreased body mass and increased hematocrit, plasma [Na⁺], [Cl^–], and plasma osmolality (more in drakes than in ducks); cloacal fluid osmolality nearly doubled compared to freshwater-adapted ducks, due mainly to osmolytes other than Na⁺ and Cl^–. The [Cl^–] in salt gland secretion only slightly exceeded drinking water [Cl^–].Abbreviations AVT antiduretic hormone - CF cloacal fluid - ECFV extraoellular fluid volume - FW freshwater acclimated - Hct hematocrit - MDWE mean daily water flux - [Na ⁺]_cf cloacal fluid sodium concentration - [Na ⁺]_pl plasma sodium concentration - Osm _cf cloacal fluid osmolality - Osm _pl plasma osmolality - SGS salt gland secretion - TBW total body water