Ion fluxes inAcetabularia: Vesicular shuttle

Summary Ion flux relations in the unicellular marine algaAcetabularia have been investigated by uptake and washout kinetics of radioactive tracers (²²Na⁺,⁴²K⁺,³⁶Cl^– and⁸⁶Rb⁺) in normal cells and in cell segments with altered compartmentation (depleted of vacuole or of cytoplasm). Some flux experiments were supplemented by simultaneous electrophysiological recordings. The main results and conclusions about the steady-state relations are: the plasmalemma is the dominating barrier for translocation of K⁺ with influx and efflux of about 100 nmol·m^–2·sec^–1×K⁺ passes three- to sevenfold more easily than Rb⁺ does. Under normal conditions, Cl^– (the substrate of the electrogenic pump, which dominates the electrical properties of the plasmalemma in the resting state) shows two efflux components of about 17 and 2 mol·m^–2·sec^–1, and a cytoplasmic as well as vacuolar [Cl^–] of about 420mm ([Cl^–] _o =529mm). At 4°C, when the pump is inhibited, both influx and efflux, as well as the cellular [Cl^–], are significantly reduced. Na⁺ ([Na⁺] _i : about 70mm, [Na⁺] _o : 461mm), which is of minor electrophysiological relevance compared to K⁺, exhibits rapid and virtually temperature-insensitive (electroneutral) exchange (two components with about 2 and 0.2 mol·m^–2·sec^–1 for influx and efflux). Some results with Na⁺ and Cl^– are inconsistent with conventional (noncyclic) compartmentation models: (i) equilibration of the vacuole (with the external medium) can be faster than equilibration of the cytoplasm, (ii) absurd concentration values result when calculated by conventional compartmental analysis, and (iii) large amounts of ions can be released from the cell without changes in the electrical potential of the cytoplasm. These observations can be explained by the particular compartmentation of normalAcetabularia cells (as known by electron micrographs) with about 1 part cytoplasm, 5 parts central vacuole, and 5 parts vacuolar vesicles. These vesicles communicate directly with the central vacuole, with the cytoplasm and with the external medium.     

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.     

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.     

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.     

Function and regulation of the avian caecal bulb: influence of dietary NaCl and aldosterone on water and electrolyte fluxes in the hen (Gallus domesticus) perfused in vivo

Summary The function of the caecal bulb, and its adaptation to chronic high- or low-Na⁺ intake, was investigated by in vivo perfusion of anaesthetised birds. Effects of acute aldosterone injection (125 g·kg^–1 body mass) were also measured.Evidence was found for primary active net absorption of Na⁺, inducing parallel Na-linked absorption of water and Cl^– and secretion of K⁺. Around 20–35% of total Cl^– absorption and K⁺ secretion were independent of Na⁺ fluxes, and these components appear to be driven by passive processes with apparent conductances of 6.3×10^–3 (G _Cl) and 1.1×10^–3 (G _K) S·cm^–2.Acetate (40mM) stimulated Na⁺ fluxes (8.5–9.9 Eq·cm^–2·h^–1) and Na-linked water fluxes (27–44 l·cm^–2·h^–1). Increased coupling ratios (2.9–4.6 l·Eq^–1) and other data indicate that these effects may be due to increased osmotic permeabilities of barriers involved in the Na-linked water transfer pathway.Low-Na⁺ maintenance enhanced EPD (49–69 mV, serosa positive) and all net fluxes:J _Na (6.8–11.6);J _K (–3.2––4.3);J _Cl (4.3–5.6 Eq·cm serosal area^–2·h^–1);J _v (28–43 l·cm^–2·h^–1) (mucosal-serosal fluxes positive).Acute aldosterone enhancedJ _Na (10.8–14.0 Eq·cm^–2·h^–1) and EPD (54–66 mV) by 3 h after injection, but had no effect on the Na-linked components ofJ _K orJ _Cl.Abbreviations ECPD, EPD Electrochemical or electrical potential difference - G _Cl ,G _K ionic conductances (Cl^–, K⁺) - J _v ,J _ion net volume or ion flux rate, mucosa-serosa positive;P _d (Cl) diffusive permeability coefficient (of Cl^–) - SEDM standard error of difference between means     

Characterization of esophageal desalination in the seawater eel,Anguilla japonica

To characterize mechanisms of esophageal desalination, osmotic water permeability and ion fluxes were measured in the isolated esophagus of the seawater eel. The osmotic permeability coefficient in the seawater eel esophagus was 2·10^-4 cm·s^-1. This value was much lower than those in tight epithelial, although the eel esophagus is a leaky epithelium with a tissue resistance of 77 ohm·cm^-2. When the esophagus was bathed in normal Ringer solutions on both sides no net ion and water fluxes were observed. However, when mucosal NaCl concentration was increased by a factor of 3, Na⁺ und Cl^- ions were transferred from mucosa to serosa (desalination). If only Na⁺ or Cl^- concentration in the mucosal fluid was increased by a factor of 3, net Na⁺ and Cl^- fluxes were reduced to 30–40%, indicating that 60–70% of the net Na⁺ and Cl^- fluxes are coupled mutually. The coupled NaCl transport seems to be effective in desalting the luminal high NaCl. The remaining 30–40% of the total Na⁺ and Cl^- fluxes seems to be due to a simple diffusion, because these components are independent of each other and follow their electrochemical gradients, and also because these fluxes remain even after treatment with NaCN or ouabain. A half of the coupled NaCl transport could be explained by a Na⁺/H⁺–Cl^-/HCO ₃ ^- double exchanger on the apical membrane of the esophageal epithelium, because mucosal amiloride and 4.4-diisothiocyanatostilbene-2,2-disulphonic acid inhibited the net Na⁺ and Cl^- fluxes by approximately 30%. The other half of the coupled NaCl transport, which follows their electrochemical gradients, still remains to be explained.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulphonic acid - NMDG N-methyl-d-glucosamine - P _Cl Cl^- permeability coefficient - PD transepithelial potential difference - P _Na Na⁺ permeability coefficient - P _osm osinotic permeability coefficient - TALH thick ascending limb of Henle's loop     

Calculations of K+, Na+, and Cl? fluxes across cell membrane with Na+/K+ pump, NKCC, NC cotransport and ionic channels with non-Goldman rectification in K+-channels: Normal and apoptotic cells

The balance of K⁺, Na⁺, and Cl⁻ fluxes across the cell membrane with the Na⁺/K⁺ pump, ion channels, and Na⁺K⁺2Cl⁻ (NKCC) and Na⁺-Cl⁻ (NC) cotransport was calculated to determine the mechanism of cell shrinkage in apoptosis. It is shown that all unidirectional K⁺, Na⁺, and Cl⁻ fluxes; the ion channel permeability; and the membrane potential can be found using the principle of the flux balance if the following experimental data are known: K⁺, Na⁺, and Cl⁻ concentrations in cell water; total Cl⁻ flux; total K⁺ influx; and the ouabain-inhibited pump component of the Rb⁺(K⁺) influx. The change in different ionic pathways during apoptosis was estimated by calculations based on the data reported in the preceded paper (Yurinskaya et al., 2010). It is found that cell shrinkage and the shift in ion balance in U937 cells induced to apoptosis with 1 μM staurosporine occur due to the coupling of reduced pump activity with a decrease in the integral permeability of Na⁺ channels, whereas K⁺ and Cl⁻ channel permeability remains almost unchanged. Calculations show that only a small part of the total fluxes of K⁺, Na⁺, and Cl⁻ account for the fluxes mediated by NKCC and NC cotransporters. Despite the importance of cotransport fluxes for maintaining the nonequilibrium steady-state distribution of Cl⁻, they cannot play a significant role in apoptotic cell shrinkage because of their minority and cannot be revealed by inhibitors.     

Relations between intracellular ion activities and extracellular osmolarity inNecturus gallbladder epithelium

Summary The interactions between ion and water fluxes have an important bearing on osmoregulation and transepithelial water transport in epithelial cells. Some of these interactions were investigated using ion-selective microelectrodes in theNecturus gallbladder. The intracellular activities of K⁺ and Cl^– in epithelial cells change when the epithelium is adapted to transport in solutions of a low osmolarity. In order to achieve new steady states at low osmolarities, cells lost K⁺, Cl^– and some unidentified anions. Surprisingly, the apparent K⁺ concentration remained high: at an external osmolartity of 64 mOsm the intracellular K⁺ concentration averaged 95mm. This imbalance was sensitive to anoxia and ouabain. The effects of abrupt changes in the external osmolarities on the intracellular activities of Na⁺, K⁺ and Cl^– were also investigated. The gradients were effectuated by mannitol. The initial relative rates of change of the intracellular activities of Na⁺ and Cl^– were equal. The data were consistent with Na⁺ and Cl^– ions initially remaining inside the cell and a cell membraneL _p of 10^–3 cm sec^–1 osm^–1, which is close to the values determine by Spring and co-workers (K.R. Spring, A. Hope & B.-E. Persson, 1981.In: Water Transport Across Epithelia. Alfred Benzon Symposium 15. pp. 190–200. Munskgaard, Copenhagen). The initial rate of change of the intracellular activity of K⁺ was only 0.1–0.2 times the change observed in Na⁺ and Cl^– activities, and suggests that K⁺ ions leave the cell during the osmotically induced H₂O efflux and enter with an induced H₂O influx. The coupling is between 98 and 102 mmoles liter^–1. Various explanations for the anomalous behavior of intracellular K⁺ ions are considered. A discussion of the apparent coupling between K⁺ and H₂O, observed in nonsteady states, and its effects on the distribution of K⁺ and H₂O across the cell membrane in the steady states, is presented.     

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 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.     

Urea uptake and translocation in toad urinary bladder: The effect of antidiuretic hormone

Summary The uptake of C¹⁴-urea into everted and noneverted bladder sacs was compared, over short time periods (up to 2 min), with the transepithelial urea fluxes. This method allowed the study of the time course of urea uptake and distribution, while previously this problem was only studied in steady-state conditions. When mucosal uptake was studied no accumulation of C¹⁴-urea inside the tissue was observed, indicating that the mucosal border could be the limiting step. Comparative studies of urea and inulin uptake from the serosal side showed that urea equilibrated with the water epithelial cells in less than 30 sec. This accumulation suggested again that the mucosal border is an effective barrier for urea translocation. The kinetics of the increase in urea permeability induced by antidiuretic hormone was also studied and it was similar (T1/2:4.3 min) to the kinetics of the increase in water permeability induced by the hormone (T1/2:5.6 min). A strong parallelism was also observed between the time course of the increases in water and urea permeabilities induced by medium hypertonicity (T1/2 25 and 26 min, respectively). The values obtained for the permeability coefficientk _trans), either at rest or under ADH were similar to those previously reported employing steady-state techniques (28±8 and 432±25 cm·sec^–1·10^–7, respectively).     

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.     

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.     

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.     

Prostaglandin E2 enhances the sodium conductance of exocrine glands in isolated frog skin (Rana esculenta)

Summary Prostaglandins are known to stimulate the active transepithelial Na⁺ uptake and the active secretion of Cl^– from the glands of isolated frog skin. In the present work the effect of prostaglandin E₂ (PGE₂) on the glandular Na⁺ conductance was examined. In order to avoid interference from the Na⁺ uptake and the glandular Cl^– secretion the experiments were carried out on skins where the Cl^– secretion was inhibited (the skins were bathed in Cl^– Ringer's solution in the presence of furosemide, or in NO ₃ ^– Ringer's solution), and the active Na⁺ uptake was blocked by the addition of amiloride. Transepithelial current, water flow and ion fluxes were measured. A negative current was passed across the skins (the skins were clamped at –100 mV, basolateral solution was taken as reference). When PGE₂, was added to the skins under these experimental conditions, the current became more negative; this was mainly due to an increase in the Na⁺ efflux. Together with the increase in Na⁺ efflux a significant increase of the water secretion was observed. The water secretion was coupled to the efflux of Na⁺, and when one Na⁺ was pulled from the basolateral to the apical solution via this pathway 230 molecules of water follwed. From the data presented it is suggested that this pathway for Na⁺ is confined to the exocrine glands.     

Kinetic studies on the stimulation of Na+−H+ exchange activity in renal brush border membranes isolated from thyroid hormone-treated rats

Summary Na⁺–H⁺ exchange activity in renal brush border membrane vesicles isolated from hyperthyroid rats was increased. When examined as a function of [Na⁺], treatment altered the initial rate of Na⁺ uptake by increasingV _m (hyperthyroid, 18.9±1.1 nmol Na⁺ · mg^–1 · 2 sec^–1; normal, 8.9±0.3 nmol Na⁺ · mg^–1 · 2 sec^–1), and not the apparent affinityK _Na ⁺ (hyperthyroid, 7.3±1.7mm; normal, 6.5±0.9mm). When examined as a function of [H⁺] and at a subsaturating [Na⁺] (1mm), hyperthyroidism resulted in the proportional increase in Na⁺ uptake at every intravesicular pH measured. A positive cooperative effect on Na⁺ uptake was found with increased intravesicular acidity in vesicles from both normal and hyperthyroid rats. When the data were analyzed by the Hill equation, it was found that hyperthyroidism did not change then (hyperthyroid, 1.2±0.06; normal, 1.2±0.07) or the [H⁺]_0.5 (hyperthyroid, 0.39±0.08 m; normal, 0.44±0.07 m) but increased the apparentV _m (hyperthyroid, 1.68±0.14 nmol Na⁺ · mg^–1 · 2 sec^–1; normal 0.96±0.10 nmol Na⁺ · mg^–1 · 2 sec^–1). The uptake of Na⁺ in exchange for H⁺ in membrane vesicles from normal and hyperthyroid animals was not influenced by membrane potential. H⁺ translocation or debinding was rate limiting for Na⁺–H⁺ exchange since Na⁺–Na⁺ exchange activity was greater than Na⁺–H⁺ exchange activity. Hyperthyroidism caused a proportional increase and hypothyroidism caused a proportional decrease in Na⁺–Na⁺ and Na⁺–H⁺ exchange. We conclude that hyperthyroidism leads to either an increase in the number of functional exchangers in the membrane or exactly proportional increases in the rate-limiting steps for Na⁺–Na⁺ and Na⁺–H⁺ exchange activity.     

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.     

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.     

Effect of pH on transepithelial electrical parameters in seawater-adapted eel intestine

The sensitivity to external pH of Cl^- absorption was studied in isolated stripped intestinal mucosa of the eel, Anguilla anguilla, mounted in Ussing chambers. Short-circuit current, transepithelial potential difference and conductance were measured in bathing solutions containing various combinations of HCO₃ ^--concentration (0–25 mmol·l^-1), partial pressure of CO₂ (0–76 mm Hg) and pH (6.9–7.9). A linear relationship was found between pH and short-circuit current in the range of pH studied both in HCO₃ ^-/CO₂ Ringer and in Hepes Ringer. The pH effect was almost completely reversible. It was not affected by the presence of mucosal Ba²⁺ (10^-3 mol·l^-1) but it was inhibited by the presence of luminal (10^-5 mol·l^-1) or serosal (10^-4 mol·l^-1) bumetanide. The results obtained suggest that the Cl^- absorption in the European eel intestine is pH sensitive. The data do not indicate whether the pH affects directly the Na⁺–K⁺–Cl^- cotransport and/or the basolateral Cl^- conductance or other mechanisms indirectly linked to Cl^- absorption.Abbreviations g _t transepithelial conductance - Hepes N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid - I _sc short circuit current - R _t transepithelial resistence - V _t transepithelial potential difference