Prostaglandin E2-stimulated glandular ion and water secretion in isolated frog skin (Rana esculenta)

Summary Prostaglandins are known to stimulate the active sodium absorption in frog skin. In this paper it is shown that prostaglandin E₂ (PGE₂) stimulates an active secretion of Cl^–, Na⁺, and K⁺ from the skin glands inRana esculenta. The active Cl secretion is enhanced more than the Na and K secretion. Therefore, in skins where the Na absorption is inhibited by amiloride, the addition of PGE₂ results in an increase in the short-circuit current (SCC). The PGE₂-stimulated Cl secretion could be inhibited by the presence of ouabain or furosemide in the basolateral solution or diphenylamine-2-carboxylate in the apical solution. The PGE₂-stimulated Cl secretion was enhanced by the phosphodiesterase inhibitor, theophylline, indicating that the effect of PGE₂ was caused by an increase in the intracellular cAMP level in the gland cells. The calcium ionophore A23187, which increases the PGE₂ synthesis in frog skin, stimulated the glandular Cl secretion. This secretion could be blocked by the prostaglandin synthesis inhibitor indomethacin, indicating that A23187 acts by increasing the prostaglandin synthesis and not by a direct action of Ca²⁺ ionsper se. The net water flow (J _w) and the Cl secretion were measured simultaneously under the conditions outlined above. The stimulation, inhibition, and the time-course of the outward-directedJ _w were similar to the change observed for the Cl secretion. These results show that PGE₂ stimulates a glandular secretion of Cl and water in frog skin, probably by increasing the cAMP level in the gland cells.     

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.     

Effects of anions on cellular volume and transepithelial Na+ transport across toad urinary bladder

Summary The effects of complete substitution of gluconate for mucosal and/or serosal medium Cl^– on transepithelial Na⁺ transport have been studied using toad urinary bladder. With mucosal gluconate, transepithelial potential difference (V _T) decreased rapidly, transepithelial resistance (R _T) increased, and calculated short-circuit current (I _sc) decreased. CalculatedE _Na was unaffected, indicating that the inhibition of Na⁺ transport was a consequence of a decreased apical membrane Na⁺ conductance. This conclusion was supported by the finding that a higher amiloride concentration was required to inhibit the residual transport. With serosal gluconateV _T decreased,R _T increased andI _sc fell to a new steady-state value following an initial and variable transient increase in transport. Epithelial cells were shrunken markedly as judged histologically. CalculatedE _Na fell substantially (from 130 to 68 mV on average). Ba²⁺ (3mm) reduced calculatedE _Na in Cl^– Ringer's but not in gluconate Ringer's. With replacement of serosal Cl^– by acetate, transepithelial transport was stimulated, the decrease in cellular volume was prevented andE _Na did not fall. Replacement of serosal isosmotic Cl^– medium by a hypo-osmotic gluconate medium (one-half normal) also prevented cell shrinkage and did not result in inhibition of Na⁺ transport. Thus the inhibition of Na⁺ transport can be correlated with changes in cell volume rather than with the change in Cl^– per se. Nystatin virtually abolished the resistance of the apical plasma membrane as judged by measurement of tissue capacitance. With K⁺ gluconate mucosa, Na⁺ gluconate serosa, calculated basolateral membrane resistance was much greater, estimated basolateral emf was much lower, and the Na⁺/K⁺ basolateral permeability ratio was much higher than with acetate media. It is concluded the decrease in cellular volume associated with substitution of serosal gluconate for Cl^– results in a loss of highly specific Ba²⁺-sensitive K⁺ conductance channels from the basolateral plasma membrane. It is possible that the number of Na⁺ pump sites in this membrane is also decreased.     

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.     

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.     

Chloride movement across the basolateral membrane of proximal tubule cells

Summary Electrophysiologic and tracer experiments have shown that Cl^– entersNecturus proximal tubule cells from the tubule lumen by a process coupled to the flow of Na⁺, and that Cl^– entry is electrically silent. The mechanism of Cl^– exit from the cell across the basolateral membrane has not been directly studied. To evaluate the importance of the movement of Cl^– ions across the basolateral membrane, the relative conductance of Cl^– to K⁺ was determined by a new method. Single-barrel ion-selective microelectrodes were used to measure intracellular Cl^– and K⁺ as a function of basolateral membrane PD as it varied normally from tubule to tubule. Basolateral membrane Cl^– conductance was about 10% of K⁺ conductance by this method. A second approach was to voltage clamp the basolateral PD to 20 mV above and below the spontaneous PD, while sensing intracellular Cl^– activity with the second barrel of a double-barrel microelectrode. An axial wire electrode in the tubule lumen was used to pass current across the tubular wall and thereby vary the basolateral membrane PD. Cell Cl^– activity was virtually unaffected by the PD changes. We conclude that Cl^– leavesNecturus proximal tubule cells by a neutral mechanism, possibly coupled to the efflux of Na⁺ or K⁺.     

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.     

Identification of a high affinity taurine transporter which is not dependent on chloride

Taurine transport by lactating gerbil mammary tissue has been examined. Taurine uptake is, mediated by a high-affinity system which is specific for -amino acids. The uptake of taurine is Na⁺-dependent but appears not to be obligatorly dependent upon Cl^–. Thus, replacing Na⁺ with choline almost abolished taurine uptake. Substituting Cl^– with NO ₃ ^– had no effect whereas SCN^– induced a small but significant increase in taurine influx. Taurine uptake was Na⁺-dependent under conditions where Cl^– had been replaced with NO ₃ ^– . However, it is apparent that the Na⁺-dependent taurine transport system requires the presence of a permeable anion because replacing Cl^– with gluconate markedly reduced taurine uptake. Cell-swelling, induced by a hyposmotic challenge, increased the efflux of taurine from gerbil mammary tissue via a pathway sensitive to niflumic acid.Abbreviations Tris (Tris(hydroxymethyl)aminomethane - BES (N,N-bis[2-hydroxyethyl]-2-aminoethane sulphonic acid)     

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.     

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.     

Apical Na+ permeability of frog skin during serosal Cl− replacement

Summary Gluconate substitution for serosal Cl^– reduces the transepithelial short-circuit current (I _sc) and depolarizes shortcircuited frog skins. These effects could result either from inhibition of basolateral K⁺ conductance, or from two actions to inhibit both apical Na⁺ permeability (P _Na ^ap ) and basolateral pump activity. We have addressed this question by studying whole-and split-thickness frog skins. Intracellular Na⁺ concentration (C _Na ^c ) andP _Na ^ap have been monitored by measuring the currentvoltage relationship for apical Na⁺ entry. This analysis was conducted by applying trains of voltage pulses, with pulse durations of 16 to 32 msec. Estimates ofP _Na ^ap ) and CNa/c were not detectably dependent on pulse duration over the range 16 to 80 msec. Serosal Cl^– replacement uniformly depolarized short-circuited tissues. The depolarization was associated with inhibition ofI _sc across each split skin, but only occasionally across the whole-thickness preparations. This difference may reflect the better ionic exchange between the bulk medium and the extracellular fluid in contact with the basolateral membranes, following removal of the underlying dermis in the split-skin preparations.P _Na ^ap was either unchanged or increased, and CNa/c either unchanged or reduced after the anionic replacement. These data are incompatible with the concept that serosal Cl^– replacement inhibitsP _Na ^ap and Na, K-pump activity. Gluconate substutition likely reduces cell volume, triggering inhibition of the basolateral K⁺ channels, consistent with the data and conclusions of S.A. Lewis, A.G. Butt, M.J. Bowler, J.P. Leader and A.D.C Macknight (J. Membrane Biol. 83:119–137, 1985) for toad bladder. The resulting depolarization reduces the electrical force favoring apical Na⁺ entry. The volume-conductance coupling serves to conserve volume by reducing K⁺ solute loss. Its molecular basis remains to be identified.     

Basolateral membrane potential of a tight epithelium: Ionic diffusion and electrogenic pumps

Summary The contribution of specific ions to the conductance and potential of the basolateral membrane of the rabbit urinary bladder has been studied with both conventional and ion-specific microelectrode techniques. In addition, the possibility of an electrogenic active transport process located at the basolateral membrane was studied using the polyene antibiotic nystatin. The effect of ion-specific microelectrode impalement damage on intracellular ion activities was examined and a criterion set for acceptance or rejection of intracellular activity measurements. Using this criterion, we found (K⁺)=72mm and (Cl^–)=15.8mm. Cl^– but not K⁺ was in electrochemical equilibrium across the basolateral membrane. The selective permeability of the basolateral membrane was measured using microelectrodes, and the data analyzed using the Goldman, Hodgkin-Katz equation. The sodium to potassium permeability ratio (P _Na/P _K) was 0.044, and the chloride to potassium permeability ratio (P _Cl/P _K) was 1.17. Since K⁺ was not in electrochemical equilibrium, intracellular (K⁺) is maintained by active metabolic processes, and the basolateral membrane potential is a diffusion potential with K⁺ and Cl^– the most permeable ions. After depolarizing the basolateral membrane with high serosal potassium bathing solutions and eliminating the apical membrane as a rate limiting step for ion movement using the polyene antibiotic nystatin, we found that the addition of equal aliquots of NaCl to both solutions caused the basolateral membrane potential to hyperpolarize by up to 20 mV (cell interior negative). This popential was reduced by 80% within 3 min of the addition of ouabain to the serosal solution. This hyperpolarization most probably represents a ouabain sensitive active transport process sensitive to intracellular Na⁺. An equivalent electrical circuit for Na⁺ transport across rabbit urinary bladder is derived, tested, and compared to previous results. This circuit is also used to predict the effects that microelectrode impalement damage will have on individual membrane potentials as well as time-dependent phenomena; e.g., effect of amiloride on apical and basolateral membrane potentials.     

Cl−, Na+, and H+ fluxes during the acidification of rabbit reticulocyte endocytic vesicles

The ionic fluxes associated with the ATP-dependent acidification of endocytic vesicles were studied in a preparation isolated from rabbit reticulocytes enriched for transferrin-transferrin receptor complexes. No vesicle acidification was observed in the absence of intra- and extravesicular ions (sucrose_in/sucrose_out), while maximal acidification was observed with NaCl_in/KCl_out·K _in ⁺ was a poor substitute for Na _in ⁺ , and Cl _out ^– could be replaced by other anions with the following efficacy of acidification: Cl^–>Br^–>I^–>PO ₄ ^3– >gluconate>SO ₄ ^2– . Flux studies using³⁶Cl^– and²²Na⁺ showed that the vesicles had a permeability for Cl^– and Na⁺, and that ATP-dependent H⁺ pumping was accompanied by a net influx of Cl^– and a net efflux of Na⁺ provided that there was a Na⁺ concentration gradient. After 3 mins, the time necessary to maximal acidification, the electrical charge generated by the entrance of H⁺ was countered to about 45% by the Cl^– influx and to about 42% by the Na⁺ efflux. These studies demonstrated that both Cl^– and Na⁺ fluxes are necessary for optimal endocytic vesicle acidification.     

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.     

Renal cortical basolateral Na+/HCO 3 − cotransporter: I. Partial purification and reconstitution

The renal basolateral Na⁺/HCO ₃ ^– cotransporter is the main system responsible for HCO ₃ ^– transport from proximal tubule cells into the blood. The present study was aimed at purifying and functionally reconstituting the Na⁺/HCO ₃ ^– cotransporter protein from rabbit renal cortex. Highly purified rabbit renal cortical basolateral membrane vesicles (hereafter designated as original basolateral membrane), enriched 12-fold in Na-K-ATPase, were solubilized in 2% octylglucoside, and then reconstituted in l--phosphatidylcholine (proteoliposomes). Na⁺/HCO ₃ ^– cotransporter activity was assessed as the difference in ²²Na uptake in the presence of HCO ₃ ^– and gluconate. The activity of the Na⁺/HCO ₃ ^– cotransporter was enhanced 18-fold in the solubilized protein reconstituted into proteoliposomes compared to the original basolateral membranes. The reconstituted solubilized purified protein exhibited kinetic properties similar to the cotransporter from original basolateral membranes. In addition, it was like the original cotransporter, inhibited by disulfonic stilbene SITS, and was eleetrogenic. The catalytic subunit of protein kinase A significantly inhibited Na⁺/HCO ₃ ^– cotransporter activity in proteoliposomes. The octylglucoside-solubilized protein was further purified by hydroxylapatite column chromatography, and this resulted in an additional enhancement of Na⁺/HCO ₃ ^– cotransporter activity of 80-fold over the original basolateral membranes. The fractions containing the highest activity were further processed by glycerol gradient centrifugation, resulting in a 124- to 300-fold increase in Na⁺/HCO ₃ ^– cotransporter activity compared to the original basolateral membranes. SDS-PAGE analysis showed an enhancement of a protein doublet of 56 kD MW in the glycerol gradient fraction. Our results demonstrate that we have partially purified and reconstituted the renal Na⁺/HCO ₃ ^– cotransporter and suggest that the 56 kD doublet protein may represent the Na⁺/HCO ₃ ^– cotransporter.This work was supported by the Merit Review Program from the Veterans Administration Central Office (J.A.L.A.), and the National Kidney Foundation of Illinois (A.A.B.).     

Transepithelial Taurine Transport in Caco-2 Cell Monolayers

Here we characterized transepithelial taurine transport in monolayers of cultured human intestinal Caco-2 cells by analyzing kinetic apical and basolateral uptake and efflux parameters. Basolateral uptake was Na⁺- and Cl⁻- dependent and was inhibited by β-amino acids. Uptake by this membrane showed properties similar to those of the apical TauT system. In both membranes, taurine uptake fitted a model consisting of a non-saturable plus a saturable component, with a higher half-saturation constant and transport capacity at the apical membrane (K_m, 17.1 μmol/L; V_max, 28.4 pmol·cm⁻²·5 min⁻¹) than in the basolateral domain (K_m, 9.46 μmol/L; V_max, 5.59 pmol·cm⁻²·5 min⁻¹). The non-saturable influx component, estimated in the absence of Na⁺ and Cl⁻, showed no significant differences between apical and basolateral membranes (K_D, 89.2 and 114.7 nL·cm⁻² · 5 min⁻¹, respectively). Taurine efflux from the cells is a diffusive process, as shown in experiments using preloaded cells and in trans-stimulation studies (apical K_D,72.7 and basolateral K_D, 50.1 nL·cm⁻²·5 min⁻¹). Basolateral efflux rates were significantly lower than passive influx rates. We conclude that basolateral taurine uptake in Caco-2 cells is mediated by a transport mechanism that shares some properties with the apical system TauT. Moreover, calculation of unidirectional and transepithelial taurine fluxes reveals that apical influx of this amino acid is higher than basolateral efflux rates, thereby enabling epithelial cells to accumulate taurine against a concentration gradient.     

pHi regulation in Ehrlich mouse ascites tumor cells: Role of sodium-dependent and sodium-independent chloride-bicarbonate exchange

pH _i recovery in acid-loaded Ehrlich ascites tumor cells and pH _i maintenance at steady-state were studied using the fluorescent probe BCECF.Both in nominally HCO ₃ ^– -free media and at 25 mm HCO ₃ ^– , the measured pH _i (7.26 and 7.82, respectively) was significantly more alkaline than the pH _i . value calculated assuming the transmembrane HCO ₃ ^– gradient to be equal to the Cl^– gradient. Thus, pH _i in these cells is not determined by the Cl^– gradient and by Cl^–/HCO ₃ ^– exchange.pH _i recovery following acid loading by propionate exposure, NH ₄ ⁺ withdrawal, or CO₂ exposure is mediated by amiloride-sensitive Na⁺/H⁺ exchange in HCO₃ ^– free media, and in the presence of HCO ₃ ^– (25 mm) by DIDS-sensitive, Na⁺-dependent Cl^–/HCO ₃ ^– exchange. A significant residual pH _i recovery in the presence of both amiloride and DIDS suggests an additional role for a primary active H⁺ pump in pH _i regulation. pH _i maintenance at steady-state involves both Na⁺/H⁺ exchange and Na⁺-dependent Cl^–/HCO ₃ ^– exchange.Acute removal of external Cl^– induces a DIDS-sensitive, Na⁺-dependent alkalinization, taken to represent HCO ₃ ^– influx in exchange for cellular Cl^–. Measurements of ³⁶Cl^– efflux into Cl^–-free gluconate media with and without Na⁺ and/or HCO ₃ ^– (10 mm) directly demonstrate a DIDS-sensitive, Na⁺ dependent Cl^–/HCO ₃ ^– exchange operating at slightly acidic pH _i (pH_o 6.8), and a DIDS-sensitive, Na⁺-independent Cl^–/HCO ₃ ^– exchange operating at alkaline pH _i (pH _o 8.2).The excellent technical assistance of Marianne Schiødt and Birgit B. Jørgensen is gratefully acknowledged. The work was supported by the Carlsberg Foundation (B.K.) and by a grant from the Danish Natural Science Foundation (E.K.H. and L.O.S.).     

A Model for Fluid Secretion in <Emphasis Type="Italic">Rhodnius</Emphasis> Upper Malpighian Tubules (UMT)

We have measured fluid secretion rate in Rhodnius prolixus upper Malpighian tubules (UMT) stimulated to secrete with 5-OH-tryptamine. We used double perfusions in order to have access separately to the basolateral and to the apical cell membranes. Thirteen pharmacological agents were applied: ouabain, Bafilomycin A₁, furosemide, bumetanide, DIOA, Probenecid, SITS, acetazolamide, amiloride, DPC, BaCl₂, pCMBS and DTT. These agents are known to block different ion transport functions, namely ATPases, co- and/or counter-transporters and ion and water channels. The basic assumption is that water movement changes reflect changes in ion transport mechanisms, which we localize as follows: (i) At the basolateral cell membrane, fundamental are a Na⁺-K⁺-2Cl^– cotransporter and a Cl^–-HCO₃^– exchanger; of intermediate importance are the Na⁺-K⁺-ATPase, Cl^– channels and Rp-MIP water channels; K⁺ channels play a lesser role: (ii) At the apical cell membrane, most important are a K⁺-Cl^– cotransport that is being located for the first time, a V-H⁺-ATPase; and a Na⁺-H⁺ exchanger; a urate-anion exchanger and K⁺ channels are less important, while Cl^– channels are not important at all. A tentative model for the function of the UMT cell is presented.Symbols and abbreviations:ACTZ, acetazolamide; cAMP, cyclic adenosine-mono-phosphate; DIOA, [(dihydroindenyl)oxy] alkanoic acid; DPC, diphenylamine-2-carboxylate; DTT, dithiothreitol; 5-HT, 5-hydroxy-tryptamine; IR, Insects Ringer; J_v, secretion rate [nl/cm².s]; pCMBS, parachloro-mercuri-benzene-sulphonate; Rp-MIP, Rhodnius prolixus water channels; SITS, 4-acetamido-4-isothiocyanatostilbene -2,2-disulfonic Acid; UMT, upper malpighian tubules.     

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.     

Intestinal HCO 3 − secretion inAmphiuma: Stimulation by mucosal Cl− and serosal Na+

Summary The requirement for Na⁺ and Cl^– in the bathing media to obtain a maximal HCO ₃ ^– secretory flux ( ) across isolated short-circuitedAmphiuma duodenum was investigated using titration techniques and ion substitution. Upon substitution of media Na⁺ with choline, HCO ₃ ^– secretion was markedly reduced. Replacement of media Cl^– produced a smaller reduction of . The presence of Cl^– enhanced HCO ₃ ^– secretion only if Na⁺ was also in the media. Elevation of media Na⁺ or Cl^– in the presence of the other ion produced a saturable increase of . In the presence of Na⁺, Cl^– stimulated when added to the mucosal but not the serosal medium. In the presence of Cl^–, Na⁺ elevated when added to the serosal but not the mucosal medium. The ability of mucosal Cl^– to stimulate was not apparently dependent on mucosal Na⁺. Simultaneous addition of 10mm Cl^– to the Na⁺-free mucosal medium and 10mm Na⁺ to the Cl^–-free serosal medium stimulated above levels produced by serosal Na⁺ alone. In conclusion, intestinal HCO ₃ ^– secretion required mucosal Cl^– and serosal Na⁺ and did not involve mucosal NaCl cotransport. The results are consistent with a mucosal Cl^– absorptive mechanism in series with parallel basolateral Na⁺–H⁺ and Cl^––HCO ₃ ^– exchange mechanisms.