Requirement of Cl− and Na+ for the ouabain-resistant control of cell volume in slices of rat liver

Summary The ability of liver cells to control their volume in the presence of ouabain has been studied in tissue slices that were recovering at 38°C from a period of swelling at 1°C. Morphological observations were made in conjunction with measurements of the net movements of water and ions. Extrusion of water in the presence of ouabain (2mm) was accompanied by a net loss of Na⁺ and Cl^– and by the formation of characteristic, rounded vesicles in the peri-canalicular regions of the hepatocytes; bile canaliculi were patent. When incubation was carried out in a medium in which either NO ₃ ^– or SO ₄ ^2– replaced Cl^–, ouabain-resistant water extrusion was prevented and the cytoplasmic vesicles normally found with ouabain were almost totally absent. When these slices were subsequently transferred to Cl^– medium with oubain, extrusion of intracellular water was initiated and cytoplasmic vesicles reappeared. Replacement of medium Na⁺ by Li⁺ mimicked the effects of ouabain on water and ion movements and ultrastructure. In addition, the ouabain-resistant extrusion of water and Cl^– was reduced and there was some diminution in the number of vesicles induced by ouabain. Furosemide (2mm) had little effect on water movement or ultrastructure in the absence of ouabain, but it slowed the net water loss and substantially reduced the formation of cytoplasmic vesicles in the presence of ouabain. The results show a close relationship between ouabain-resistant water extrusion and the formation of the cytoplasmic vesicles that are characteristic of treatment with ouabain. They further suggest that a cotransport of Na⁺ and Cl^– forms an important part of the mechanism underlying ouabain-resistant water extrusion and, specifically, that this cotransport may take place across the membranes of the cytoplasmic vesicles.     

Morphological and physiological studies of rat kidney cortex slices undergoing isosmotic swelling and its reversal: A possible mechanism for ouabain-resistant control of cell volume

Summary Slices of rat kidney cortex were induced to swell by preincubation at 1°C in an isotonic Ringer's solution, and their capacity to reverse swelling, by net extrusion of cellular water, was studied during subsequent incubation at 25°C. The recovery from swelling was prevented by the respiratory inhibitor, antimycin A. On the other hand, extrusion of water was little affected by ouabain. The extrusion of water continuing in the presence of ouabain (but not that in its absence) was significantly reduced when furosemide was added or when medium Cl^– was replaced by NO ₃ ^– , or I^–. There was substantial variability in the morphological appearance of cells within the cortical slices. Different segments of the nephron showed different structural changes during swelling and its reversal, the proximal tubules being most markedly affected. Proximal tubular cells of swollen slices showed disorganization of brush borders and expansion of their apical surfaces, and contained vesicles in their apical cytoplasm. Upon recovery at 25°C, the apical portions of these cells showed reversal of the expansion, but some apical vesicles remained. These vesicles were much more numerous after recovery in the presence of ouabain, but they were much reduced in numbers, or totally absent, when recovery took place in the presence of furosemide or absence of Cl^–, with or without ouabain. The vesicles seen in the presence of ouabain alone appeared to fuse with each other and with infoldings of the basolateral plasma membrane. Rather similar results were obtained with distal tubular cells in the slices. We suggest that volume regulation in the proximal and distal tubular cells proceeds by way of two mechanisms. The first consists of extrusion of water coupled to the ouabain-sensitive transport of Na⁺ and K⁺. The other proceeds by way of an ouabain-resistant entry of water into apical cytoplasmic vesicles, following furosemide-sensitive movements of Cl^– and Na⁺; the vesicles then expel their contents by exocytosis at the basolateral cell borders.     

Characterization of cell ion exchange in the sea anemoneCondylactis gigantea

Summary Maintenance of intracellular ion contents and their relations to transmembrane potential were studied in tentacles ofCondylactis gigantea. Tentacles leached at 2°C in 10 mMK⁺ and 2 mM K⁺ artificial seawaters (10K ASW and 2K ASW) with and without 2 MM ouabain, and in 0K ASW, lost cell K⁺ and gained Na⁺. Rewarming to 25°C in 10K ASW resulted in a marked accumulation of K⁺ and extrusion of Na⁺ in tentacles leached in 10K ASW and in 0K ASW. Initial rate of Na⁺ extrusion was twice the initial rate of K⁺ accumulation, suggesting a pump coupling ratio of 2. In tentacles leached and rewarmed in 2K ASW, no net reaccumulation of K⁺ and little net extrusion of Na⁺ was observed; i.e., the pump just kept pace with the leaks. Ouabain inhibited K⁺ reaccumulation and Na⁺ extrusion. This effect was less marked in 10K ASW than in 2K ASW confirming, in anemone tentacles, the well documented ouabain-K⁺ antagonism observed in other systems. In no case did K _i ⁺ /K ₀ ⁺ equal Cl ₀ ^– /Cl _i ^– ; therefore, the distribution of these ions did not fit a Donnan distribution. Transmembrane potential difference was –22±3 mV in 10K ASW at 25°C. It fitted a modified Nernst equation which includes the pump coupling ratio and a Na⁺ to K⁺ permeability ratio of 0.31.A moderately high permeability of the cell membrane to Na⁺, and a ouabain and K⁺ sensitive ion pump, exchanging 2 Na⁺ for 1 K⁺, appear to be responsible for the observed ionic distribution and transmembrane potential in anemone cells.     

Relationship between coupled Na+−K+−Cl− transport and water absorption across the seawater eel intestine

Summary Simultaneous measurements of net ion and water fluxes were made in the stripped intestine of the seawater eel, and the relationship between Na⁺, K⁺, Cl^– and water transport were examined in the presence of mucosal KCl and serosal NaCl Ringer (standard condition). When Cl^– was removed from both sides of the intestine, net K⁺ flux from mucosa to serosa was reduced, accompanied by complete blockage of water absorption. Since it has been shown that net Cl^– and water fluxes depend on K⁺ transport under the standard condition (Ando 1983), the interdependence of K⁺ and Cl^– transport suggests the existence of a coupled KCl transport system, while the parallelism between the net Cl^– and water fluxes suggests that water absorption is linked to the coupled KCl transport. The coupled KCl and water transport were inhibited by treatment with ouabain or with Na⁺-free Ringer solutions, suggesting the existence of a Na⁺-dependent KCl transport system and linkage of water absorption to the coupled Na⁺–K⁺–Cl^– transport. Since ouabain blocked the active Na⁺–K⁺–Cl^– transport almost completely, the permeability coefficients for K⁺ and Na⁺ through the paracellular shunt pathway were estimated as P_K=0.076 and P_Na=0.058 cm/h, and P_Cl was calculated as 0.005 cm/h. Although Na⁺-independent K⁺ and Cl^tt- fluxes were observed again in the present study, these fluxes were not inhibited by CN^–, ouabain or diuretics, and evoked even after blocking the Na⁺–K⁺–Cl^– transport completely with ouabain. These results indicate that the Na⁺-independent K⁺ and Cl^– fluxes are distinct from the active Na⁺–K⁺–Cl^– transport and are not themselves active.     

Ion and water balance in isolated epithelial cells of the abdominal skin of the frogLeptodactylus ocellatus

Summary Isolated epithelial cells were obtained from abdominal skin of the frogLeptodactylus ocellatus by a trypsination-dissection method. As estimated by nigrosin staining, the amount of damaged cells is only 6.6±0.7 per cent. When washed briefly after incubation the ionic concentrations in these cells were (mm): K⁺ 14.20±4.0; Na⁺ 15.8±1.8; and Cl^– 57.2±5.3. If they are not washed, the concentration of K⁺ remains essentially the same (131.2±1.4mm) but the Na⁺ concentration is much higher (38.5±0.9mm). It is shown that a large fraction of Na⁺ is contained in a compartment that is freely connected with the bathing solution. Ouabain (10^–4 m) elicits a marked decrease of K⁺, a slight decrease of Cl^–, and an increase of Na⁺ content. In an equal period, low temperature (3°C) produces a similar effect, although less marked than ouabain.     

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.     

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.     

Effects of ouabain on chloride movements across the seawater eel intestine

Summary Simultaneous measurements of transepithelial potential difference (PD) and net water flux were made in the stripped intestine of seawater eels, and the effects of ouabain on these two parameters were examined in normal Ringer solution or under a chloride concentration gradient. Ouabain reduced the serosa-negative PD and the net water flux in normal Ringer solution with a linear relationship between the PD and the net water flux. Removal of K⁺ from the Ringer solution on both serosal and mucosal sides also reduced the PD and the net water flux to approximately zero. On the other hand, blocking the Na⁺–K⁺ pump by ouabain, K⁺-free or Na⁺-free Ringer solution increased the diffusion potential for Cl^–. Inhibition of Cl^– transport and increment in Cl^– permeability by ouabain occurred almost simultaneously. It is likely, therefore, that Cl^– transport as well as Cl^– permeability is dependent on Na⁺–K⁺ pump activity. A possible mechanism of dependence of Cl^– transport on the Na⁺–K⁺ pump is discussed in relation to the increment in Cl^– permeability.     

Nuclear microanalysis of the monovalent ions distribution in the human amnion

Summary The effect of taurine on the Na⁺, K⁺, Cl^– concentration and distribution in epithelial and compact layers of the human amniotic membrane had been investigated using the Bordeaux nuclear microprobe. Particle induced X-ray emission and Rutherford backscattering spectrometry techniques had been used to provice quantitative measurements. In physiological medium, the monovalent ions concentrations were identical in epithelial and compact layers. The addition of taurine in Hanks' physiological fluid had no effect on Na⁺ concentration, but decreased K⁺ and Cl^– concentration in both layers. The quantitative results were related to electrophysiological observations on the effect of taurine on ionic exchanges through channels and paracellular pathways.     

Current-voltage relationships of a sodium-sensitive potassium channel in the tonoplast ofChara corallina

Summary The membrane of mechanically prepared vesicles ofChara corallina has been investigated by patch-clamp techniques. This membrane consists of tonoplast as demonstrated by the measurement of ATP-driven currents directed into the vesicles as well as by the ATP-dependent accumulation of neutral red. Addition of 1mm ATP to the bath medium induced a membrane current of about 3.2 mA·m^–2 creating a voltage across the tonoplast of about –7 mV (cytoplasmic side negative). On excised tonoplast patches, currents through single K⁺-selective channels have been investigated under various ionic conditions. The open-channel currents saturate at large voltage displacements from the equilibrium voltage for K⁺ with limiting currents of about +15 and –30 pA, respectively, as measured in symmetric 250mm KCl solutions. The channel is virtually impermeable to Na⁺ and Cl^–. However, addition of Na⁺ decreases the K⁺ currents. TheI–V relationships of the open channel as measured at various K⁺ concentrations with or without Na⁺ added are described by a 6-state model, the 12 parameters of which are determined to fit the experimental data.     

Measurement and stoichiometry of bumetanide-sensitive (2Na∶1K∶3Cl) cotransport in ferret red cells

Summary The bumetanide-sensitive uptake of Na⁺, K^–(Rb^–) and Cl^– has been measured at 21°C in ferrent red cells treated with (SITS+DIDS) to minimize anion flux via capnophorin (Band 3). During the time course of the influx experiments tracer uptake was a first-order rate process. At normal levels of external Na⁺ (150mm) the bumetanide-sensitive uptake of K⁺ was dependent on Cl^– and represented almost all of the K⁺ uptake, the residual flux demonstrating linear concentration dependence. The uptake of Na⁺ and Cl^– was only partially inhibited by bumetanide indicating that pathways other than (Na+K+Cl) cotransport participate in these fluxes. The diuretic-sensitive uptake of Na⁺ or Cl^– was, however, abolished by the removal of K⁺ or the complementary ion indicating that bumetanide-sensitive fluxes of Na⁺, K⁺ and Cl^– are closely coupled. At very low levels of [Na] _o (<5mm) K⁺ influx demonstrated complex kinetics, and there was evidence of the unmasking of a bumetanide-sensitive Na⁺-independent K⁺ transport pathway. The stoichiometry of bumetanide-sensitive tracer uptake was 2Na1K3Cl both in cells suspended in a low and a high K⁺-containing medium. The bumetanide-sensitive flux was markedly reduced by ATP depletion. We conclude that a bumetanide-sensitive cotransport of (2Na1K3Cl) occurs as an electroneutral complex across the ferret red cell membrane.     

[14C]Carbon-dioxide fixation by isolated leaf epidermes with stomata closed or open

Following small hypo-osmotic shocks, ion concentrations (Na⁺, K⁺, Cl^-) in Platymonas subcordiformis decreased; this was due mainly to an increase of cell volume. With larger hypo-osmotic stresses, the decrease of ion concentration continued and, additionally, extrusion of mannitol was observed. The ion and mannitol concentrations were not regained after 240 min. In contrast, following hyperosmotic shocks, the ion concentrations increased transitorily during the first 20–40 min. The same was true for K⁺ following small hyperosmotic stresses and for Na⁺ and — partially — Cl^- with larger shocks. Large hyperosmotic stresses caused permanent accumulation of mannitol, which levelled off after 60–80 min. Thus the transient increase of ions bridged the concentration gap until mannitol was accumulated to a high enough concentration to account for the osmotic adaptation of Platymonas, together with a basal level of the ions K⁺, Na⁺, Cl^-.Abbreviations PS photosynthesis - Resp respiration     

Localization of Na+, K+-ATPase to the inside of the basolateral cell membranes of epithelial cells of proximal and distal tubules in rabbit kidney

Summary Cysteine-sensitive alkaline phosphatase and/or ouabain-sensitive Na⁺, K⁺-ATPase were studied by ultrastructure cytochemistry in epithelial cells of proximal and distal kidney tubules. Alkaline phosphatase reactivity was confined to the surface of the microvillous luminal cell membrane of proximal tubule cells, whereas distal tubules and collecting ducts were unreactive. The Na⁺, K⁺-ATPase reactivity was localized evenly along the cytoplasmic side of the basolateral cell membrane of cells of proximal and distal tubules and in collecting ducts. In the proximal tubules, where the activity was strongest, the Na⁺, K⁺-ATPase deposits were also found in the 10–50 nm gap between the cell membrane and the cisternae of tubulo-cisternal endoplasmic reticulum (TER) underlying a major part of the basolateral cell membrane. The restriction of Na⁺, K⁺-ATPase sites, which are involved in extrusion of Na⁺ from the cell, to a narrow cytoplasmic compartment located between the cell membrane and the cisternae of TER, is consistent with a transport role for the TER.     

Plasma membrane potential of Lettré cells does not depend on cation gradients but on pumps

Summary The plasma membrane potential of Lettré cells has been determined with the optical indicator oxonol-V and found to be –57 mV at 37°C (range –20 to –80 mV depending on the physiological condition of the cells). Increasing extracellular K⁺ does not depolarize cells: even in the presence of 155mM K⁺ the potential is –41 mV; membrane potential is also insensitive to the chemical gradient of Na⁺,Mg²⁺, Ca²⁺ or Cl^–. Ouabain depolarizes the cells; H⁺ efflux from cells is stimulated by extracellular Na⁺. We propose that in Lettré cells the plasma membrane potential is generated by electrogenic cation pumps. The balancing fluxes of Na⁺ and K⁺ are mainly through electroneutral cation exchanges (Na⁺/K⁺ and Na⁺/H⁺) and the magnitude of the potential is limited by organic anion leaks. Such a mechanism may operate in other biological membranes also.     

Distribution of Na+, K+ and Cl− between nucleus and cytoplasm inChironomus salivary gland cells

Summary Previous studies of the electrochemical activity coefficients of intracellular Na⁺ and K⁺ have suggested that the free form of these ions may be unevenly distributed within the intracellular fluids. One possible site of such subcellular compartmentalization is the cell nucleus. In order to examine this possibility, the cells ofChironomus salivary glands were studied with conventional liquid ion-exchange microelectrodes sensitive to K⁺ and Cl^–, with a new liquid ion-exchange microelectrode sensitive to Na⁺, and with open-tipped micropipets. Both the electrochemical activities for Na⁺, K⁺ and Cl^–, and the electrical potential were the same on both sides of the nuclear membrane. The possibility was considered that a difference in the junction potentials within the nucleoplasm and cytoplasm might have masked a real difference in electrical potential between these two phases. To study that possibility, changes were induced in the junction potentials by altering the composition of the fluid filling the exploring micropipets. The results have suggested that the magnitudes of the junction potentials are the same on both sides of the nuclear envelope. The simplest interpretation of the data is that the chemical activities of Na⁺, K⁺ and Cl^– are the same within the nucleus and cytoplasm. This suggests that other subcellular structures, possibly the endoplasmic reticulum and mitochondria, are responsible for subcellular compartmentalization.     

Chloride transport and the membrane potential in the marine alga,Halicystis parvula

Summary The relationship between the rate of Cl^– transport and the electrical properties ofHalicystis parvula was investigated. Three metabolic inhibitors-darkness, cyanide (2mm), and low temperature (4°C)-all rapidly and reversibly reduce both the short circuit current (SCC), which is a measure of net Cl^– transport, and the vacuole electrical potential (PD). Plotting thePD vs. SCC for inhibited cells yields a linear regression with ay-intercept of zero. ThePD is also greatly reduced when the [Cl^–] of the external medium is lowered. Raising the external [K⁺] produces an appreciable, but less than Nernstian, depolarization, while increasing the external [H⁺] tenfold has no net effect on thePD. Decreasing the external [Na⁺] by tenfold produces only a slight depolarization. Thus, the outer plasma membrane appears to be moderately selective for K⁺ over Na⁺ or H⁺. The effects of ion substitutions in the vacuolar perfusing solutions on thePD reveal that the vacuolar membrane does not discriminate electrically between Cl^– and the much larger anions, isethionate and benzenesulfonate, or between Na⁺ and K⁺. The data suggest that in internally perfused cells ofH. parvula generation of thePD of –50 to –60 mV by a transport system involving only electroneutral pumps is unlikely and that most of thisPD is generated by an electrogenic Cl^– pump.     

Identification and reconstitution of a Na/K/Cl cotransporter and K channel from luminal membranes of renal red outer medulla

Electrophysiological studies on renal thick ascending limb segments indicate the involvement of a luminal Na⁺/K⁺/Cl⁻ cotransport system and a K⁺ channel in transepithelial salt transport. Sodium reabsorption across this segment is blocked by the diuretics furosemide and bumetanide. The object of our study has been to identify in intact membranes and reconstitute into phospholipid vesicles the Na⁺/K⁺/Cl⁻ cotransporter and K⁺ channel, as an essential first step towards purification of the proteins involved and characterization of their roles in the regulation of transepithelial salt transport. Measurements of ⁸⁶Rb⁺ uptake into membrane vesicles against large opposing KCl gradients greatly magnify the ratio of specific compared to non-specific isotope flux pathways. Using this sensitive procedure, it has proved possible to demonstrate in crude microsomal vesicle preparations from rabbit renal outer medulla two ⁸⁶Rb⁺ fluxes. (A) A furosemide-inhibited ⁸⁶Rb⁺ flux in the absence of Na⁺ (K⁺-K⁺ exchange). This flux is stimulated by an inward Na⁺ gradient (Na⁺/K⁺ cotransport) and is inhibited also by bumetanide. (B) A Ba²⁺-inhibited ⁸⁶Rb⁺ flux, through the K⁺ channel. Luminal membranes containing the Na⁺/K⁺/Cl⁻ cotransporter and K⁺ channels, and basolateral membranes containing the Na⁺/K⁺ pumps were separated from the bulk of contaminant protein by metrizamide density gradient centrifugation. The Na⁺/K⁺/Cl⁻ cotransporter and K⁺ channel were reconstituted in a functional state by solubilizing both luminal membranes and soybean phospholipid with octyl glucoside, and then removing detergent on a Sephadex column.     

Na+, K+, Cl− cotransport and its regulation in Ehrlich ascites Tumor cells. Ca2+/Calmodulin and protein kinase C dependent pathways

Summary Net Cl^– uptake as well as unidirectional³⁶Cl influx during regulatory volume increase (RVI) require external K⁺. Half-maximal rate of bumetanide-sensitive³⁶Cl uptake is attained at about 3.3mm external K⁺. The bumetanide-sensitive K⁺ influx found during RVI is strongly dependent on both Na⁺ and Cl^–. The bumetanide-sensitive unidirectional Na⁺ influx during RVI is dependent on K⁺ as well as on Cl^–. The cotransporter activated during RVI in Ehrlich cells, therefore, seems to transport Na⁺, K⁺ and Cl^–. In the presence of ouabain and Ba⁺ the stoichiometry of the bumetanide-sensitive net fluxes can be measured at 1.0 Na⁺, 0.8 K⁺, 2.0 Cl^– or approximately 1 : Na, 1 : K, 2 : Cl. Under these circumstances the K⁺ and Cl^– flux ratios (influx/efflux) for the bumetanide-sensitive component were estimated at 1.34 ±0.08 and 1.82 ± 0.15 which should be compared to the gradient for the Na⁺, K⁺, 2Cl^– cotransport system at 1.75 ± 0.24.Addition of sucrose to hypertonicity causes the Ehrlich cells to shrink with no signs of RVI, whereas shrinkage with hypertonic standard medium (all extracellular ion concentrations increased) results in a RVI response towards the original cell volume. Under both conditions a bumetanide-sensitive unidirectional K⁺ influx is activated. During hypotonic conditions a small bumetanide-sensitive K⁺ influx is observed, indicating that the cotransport system is already activated.The cotransport is activated 10–15 fold by bradykinin, an agonist which stimulates phospholipase C resulting in release of internal Ca²⁺ and activation of protein kinase C.The anti-calmodulin drug pimozide inhibits most of the bumetanide-sensitive K⁺ influx during RVI. The cotransporter can be activated by the phorbol ester TPA. These results indicate that the stimulation of the Na⁺, K⁺, Cl^– cotransport involves both Ca²⁺/calmodulin and protein kinase C.     

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.     

Control of canine kidney cortex slice volume and ion distribution at hypothermia by impermeable anions

Hypothermia induces swelling of dog kidney cortex slices. Swelling of cells during hypothermia is related to a number of factors including the permeability of Cl⁻. By substituting lactobionate for Cl⁻, while maintaining isoosmotic conditions, swelling is prevented. Lactobionate is an impermeable anion and its presence in the suspending fluid prevents swelling of dog kidney cortex slices in salts of Na⁺, K⁺ or combinations of Na⁺ and K⁺ even in the presence of metabolic inhibitors. By maintaining a ratio of 80 mM lactobionate: 60 mM chloride and an appropriate ratio of Na⁺:K⁺ (80 mM:60 mM), both the total tissue H₂O and ratio of intracellular K⁺/Na⁺ are kept within normal ranges during hypothermic incubation of tissue slices. Kidney cortex slices suspended in this medium at 30 °C respire at a rate 30–40% slower than that of control slices suspended in saline. A similar result is obtained by adding ouabain to slices suspended in saline. This suggests that the Na⁺-pump activity is suppressed under these conditions and results in a reduced energy demand on the cell. These results are discussed in relation to utilizing this type of solution for long-term perfusion preservation of kidneys for transplantation.