Na+ channel activity in cultured renal (A6) epithelium: Regulation by solution osmolarity

Summary Solution osmolarity is known to affect Na⁺ transport rates across tight epithelia but this variable has been relatively ignored in studies of cultured renal epithelia. Using electrophysiological methods to study A6 epithelial monolayers, we observed a marked effect of solution tonicity on amiloride-sensitive Na⁺ currents (I _sc).I _sc for tissues bathed in symmetrical hyposmotic (170 mOsm), isosmotic (200 mOsm), and hyperosmotic (230 or 290 mOsm) NaCl Ringer's solutions averaged 25±2, 9±2, 3±0.4, and 0.6±0.5 A/cm², respectively. Similar results were obtained following changes in the serosal tonicity; mucosal changes did not significantly affectI _sc. The changes inI _sc were slow and reached steady-state within 30 min. Current fluctuation analysis measurements indicated that single-channel currents and Na⁺ channel blocker kinetics were similar for isosmotic and hyposmotic conditions. However, the number of conducting Na⁺ channels was approximately threefold higher for tissues bathed in hyposmotic solutions. No channel activity was detected during hyperosmotic conditions. The results suggest that Na⁺ channels in A6 epithelia are highly sensitive to relatively small changes in serosal solution tonicity. Consequently, osmotic effects may partly account for the large variability in Na⁺ transport rates for A6 epithelia reported in the literature.     

Correlation Between Transepithelial Na+ Transport and Transepithelial Water Movement Across Isolated Frog Skin (Rana esculenta)

In the present work the coupling under short-circuited conditions between the net Na⁺-influx across isolated frog skin and the transepithelial transport of water was examined i.e., the short-circuit current (I _sc ) and the transepithelial water movement (TEWM) were measured simultaneously. It has been shown repeatedly that the I _sc across isolated frog skin is equal to the net transepithelial Na⁺ transport. Furthermore the coupling between transepithelial uptake of NaCl under open-circuit conditions and TEWM was also measured. The addition of antidiuretic hormone (AVT) to skins incubated under short-circuited conditions resulted in an increase in the I _sc and TEWM. Under control conditions I _sc was 9.14 ± 2.43 and in the presence of AVT 45.9 ± 7.3 neq cm⁻² min⁻¹ (n= 9) and TEWM changed from 12.45 ± 4.46 to 132.8 ± 15.8 nL cm⁻² min⁻¹. The addition of the Na⁺ channel blocking agent amiloride resulted in a reduction both in I _sc and TEWM, and a linear correlation between I _sc and TEWM was found. The correlation corresponds to that 160 ± 15 (n= 7) molecules of water follow each Na⁺ across the skin. In another series of experiments it was found that there was a linear correlation between I _sc and the increase in apical osmolarity needed to stop the TEWM. The data presented indicate that the observed coupling between the net transepithelial Na⁺ transport and TEWM is caused by local osmosis. Received: 16 October 1996/Revised: 6 March 1997     

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.     

Mechanisms of liquid flux across pulmonary alveolar epithelial cell monolayers

Summary Active transport of sodium by pulmonary alveolar epithelial cells (AEC) is believed to be an important component of edema clearance in the normal and injured lung. Data supporting this premise have come from measurements of sodium movement across AEC monolayers or from perfused lung model systems. However, direct measurement of fluid flux across AEC monolayers has not been reported. In the present work, AEC were studied with an experimental system for the measurement of fluid flux (Jv) across functionally intact cell monolayers. Primary adult rat type II alveolar epithelial cells were cultured on 0.8 μm nuleopore filters previously coated with gelatin and fibronectin. Intact monolayers were verified by high electrical resistance (> 1000 Θ) at 4–5 d of primary culture. At the same time interval, transmission electron microscopy revealed cells with type I cell-like morphology throughout the monolayer. These were characterized by both adherens and tight junctional attachments. Fluid flux across the monolayers was measured volumetrically over a period of 2 h in the presence of HEPES-buffered DMEM containing 3% fatty acid-free bovine serum albumin. Flux (Jv) was inhibited 39% by 1 × 10⁻⁴ M ouabain (P < 0.01) and 27% by 5 × 10⁻⁴ M amiloride (P < 0.05). These data support the concept that AEC Na⁺/K⁺-ATPase and Na⁺ transport systems are important determinants of AEC transepithelial fluid movement in vitro.     

Stimulation of Transepithelial Na<Superscript>+</Superscript> Current by Extracellular Gd<Superscript>3+</Superscript> in <Emphasis Type="Italic">Xenopus laevis</Emphasis> Alveolar Epithelium

In the present study we investigated the effect of extracellular gadolinium on amiloride-sensitive Na⁺ current across Xenopus alveolar epithelium by Ussing chamber experiments and studied its direct effect on epithelial Na⁺ channels with the patch-clamp method. As observed in various epithelia, the short-circuit current (I _sc) and the amiloride-sensitive Na⁺ current (I _ami) across Xenopus alveolar epithelium was downregulated by high apical Na⁺ concentrations. Apical application of gadolinium (Gd³⁺) increased I _sc in a dose-dependent manner (EC ₅₀ = 23.5 µM). The effect of Gd³⁺ was sensitive to amiloride, which indicated the amiloride-sensitive transcellular Na⁺ transport to be upregulated. Benz-imidazolyl-guanidin (BIG) and p-hydroxy-mercuribenzonic-acid (PHMB) probably release apical Na⁺ channels from Na⁺-dependent autoregulating mechanisms. BIG did not stimulate transepithelial Na⁺ currents across Xenopus lung epithelium but, interestingly, it prevented the stimulating effect of Gd³⁺ on transepithelial Na⁺ transport. PHMB increased I _sc and this stimulation was similar to the effect of Gd³⁺. Co-application of PHMB and Gd³⁺ had no additive effects on I _sc. In cell-attached patches on Xenopus oocytes extracellular Gd³⁺ increased the open probability (NP _o) of Xenopus epithelial sodium channels (ENaC) from 0.72 to 1.79 and decreased the single-channel conductance from 5.5 to 4.6 pS. Our data indicate that Xenopus alveolar epithelium exhibits Na⁺-dependent non-hormonal control of transepithelial Na⁺ transport and that the earth metal gadolinium interferes with these mechanisms. The patch-clamp experiments indicate that Gd³⁺ directly modulates the activity of ENaCs.     

Fish Gill Respiratory Cells in Culture: A New Model for Cl−-secreting Epithelia

Primary cultures of sea bass gill cells grown on permeable membranes form a confluent, polarized, functional tight epithelium as characterized by electron microscopy and electrophysiological and ion transport studies. Cultured with normal fetal bovine serum (FBS) and mounted in an Ussing chamber, the epithelium presents a small short-circuit current (I _sc : 1.4 ± 0.3 μA/cm²), a transepithelial voltage (V _t ) of 12.7 ± 2.7 mV (serosal positive) and a high transepithelial resistance (R _t : 12302 ± 2477 Ω× cm²). A higher degree of differentiation and increased ion transport capacities are observed with cells cultured with sea bass serum: numerous, organized microridges characteristic of respiratory cells are present on the apical cell surface and there are increased I _sc (11.9 ± 2.5 μA/cm²) and V _t (25.9 ± 1.7 mV) and reduced R _t (4271 ± 568 Ω× cm²) as compared with FBS-treated cells. Apical amiloride addition (up to 100 μm) had no effect on I _sc . The I _sc , correlated with an active Cl⁻ secretion measured as the difference between ³⁶Cl⁻ unidirectional fluxes, was partly blocked by serosal ouabain, bumetanide, DIDS or apical DPC or NPPB and stimulated by serosal dB-cAMP. It is concluded that the chloride secretion is mediated by a Na⁺/K⁺/2Cl⁻ cotransport and a Cl⁻/HCO₃ ⁻ exchanger both responsible for Cl⁻ entry through the basolateral membrane and by apical cAMP-sensitive Cl⁻ channels. This study gives evidence of a functional, highly differentiated epithelium in cultures composed of fish gill respiratorylike cells, which could provide a useful preparation for studies on ion transport and their regulation. Furthermore, the chloride secretion through these cultures of respiratorylike cells makes it necessary to reconsider the previously accepted sea water model in which the chloride cells are given the unique role of ion transport through fish gills. Received: 12 July 1996/Revised: 5 November 1996     

Effects of aldosterone on Na transport in the toad bladder II. The anaerobic response

The action of aldosterone on active Na⁺ transport was assessed under aerobic and anaerobic conditions in the isolated urinary bladder of the toad, BUfo marinus. Aldesterone augmented the short-circuit current (I_sc) under rigorous anaerobiosis. Four lines of evidence indicate that the increase in anaerobic I_sc does not represent an equivalent increase in active Na⁺ transport: 1. Net Na⁺ transport, determined by isotopic fluxes, was the same in the aldosterone-treated and control quarter-bladders, and significantly greater than the simultaneously measured I_sc. 2. Amiloride, an inhibitor of the apiral entry of Na⁺, did not reduce the steroid-dependent increase in the anaerobic I_sc. 3. Substitution of choline for Na⁺ in the mucosal medium reduced the magnitude of the anaerobic I_sc values did not eliminate the effect of aldosterone. 4. Addition of ouabain, a potent inhibitor of the Na⁺ pump, partially inhibited the effect of aldosterone on the anerobic I_sc but a significant hormonal increment remained. The source of the anaerobic I_sc was not identified; an effort was made, however, to determine the dependence of this current on glycolysis. During anaerobics, aldosterone increased the integral I_sc by 42% but did not alter lactate production. These results suggest that the steroid-dependent increase in the anaerobic I_sc may involve effects on permeability properties of the epithelium rather than on active tranport systems.     

Concurrent and Independent HCO− 3 and Cl− Secretion in a Human Pancreatic Duct Cell Line (CAPAN-1)

The present study investigated both HCO⁻ ₃ and Cl⁻ secretions in a human pancreatic duct cell line, CAPAN-1, using the short-circuit current (I _sc ) technique. In Cl⁻/HCO⁻ ₃-containing solution, secretin (1 μm) or forskolin (10 μm) stimulated a biphasic rise in the I _sc which initially reached a peak level at about 3 min and then decayed to a plateau level after 7 min. Removal of external Cl⁻ abolished the initial transient phase in the forskolin-induced I _sc while the plateau remained. In HCO⁻ ₃/CO₂-free solution, on the contrary, only the initial transient increase in I _sc was prominent. Summation of the current magnitudes observed in Cl⁻-free and HCO⁻ ₃-free solutions over a time course of 10 min gave rise to a curve which was similar, both in magnitude and kinetics, to the current observed in Cl⁻/HCO⁻ ₃-containing solution. Removal of external Na⁺ greatly reduced the initial transient rise in the forskolin-induced I _sc response, and the plateau level observed under this condition was similar to that obtained in Cl⁻-free solution, suggesting that Cl⁻-dependent I _sc was also Na⁺-dependent. Bumetanide (50 μm), an inhibitor of the Na⁺-K⁺-2Cl⁻ cotransporter, and Ba²⁺ (1 mm), a K⁺ channel blocker, could reduce the forskolin-induced I _sc obtained in Cl⁻/HCO⁻ ₃-containing or HCO⁻ ₃-free solution. However, they were found to be ineffective when external Cl⁻ was removed, indicating the involvement of these mechanisms in Cl⁻ secretion. On the contrary, the HCO⁻ ₃-dependent (in the absence of external Cl⁻) forskolin-induced I _sc could be significantly reduced by carbonic anhydrase inhibitor, acetazolamide (45 μm). Basolateral application of amiloride (100 μm) inhibited the I _sc ; however, a specific Na⁺-H⁺ exchanger blocker, 5-N-methyl-N-isobutylamiloride (MIA, 5–10 μm) was found to be ineffective, excluding the involvement of the Na⁺-H⁺ exchanger. However, an inhibitor of H⁺-ATPase, N-ethylmaleimide did suppress the I _sc (IC₅₀= 22 μm). Immunohistochemical studies also confirmed the presence of a vacuolar type of H⁺-ATPase in these cells. H₂DIDS (100 μm), an inhibitor of Na⁺-HCO⁻ ₃ cotransporter, was without effect. Apical addition of Cl⁻ channel blocker, diphenylamine-2,2′-dicarboxylic acid (DPC, 1 mm), but not disulfonic acids, DIDS (100 μm) or SITS (100 μm), exerted an inhibitory effect on both Cl⁻ and HCO⁻ ₃-dependent forskolin-induced I _sc responses. Histochemical studies showed discrete stainings of carbonic anhydrase in the monolayer of CAPAN-1 cells, suggesting that HCO⁻ ₃ secretion may be specialized to a certain population of cells. The present results suggest that both HCO⁻ ₃ and Cl⁻ secretion by the human pancreatic duct cells may occur concurrently and independently. Received: 17 October 1997/Revised: 3 April 1998     

A role for ERK1/2 in EGF- and ATP-dependent regulation of amiloride-sensitive sodium absorption

Receptor-mediated inhibition of amiloride-sensitive sodium absorption was observed in primary and immortalized murine renal collecting duct cell (mCT12) monolayers. The addition of epidermal growth factor (EGF) to the basolateral bathing solution of polarized monolayers reduced amiloride-sensitive short-circuit current (I_sc) by 15–25%, whereas the addition of ATP to the apical bathing solution decreased I_sc by 40–60%. Direct activation of PKC with phorbol 12-myristate 13-acetate (PMA) and mobilization of intracellular calcium with 2,5-di-tert-butyl-hydroquinone (DBHQ) reduced amiloride-sensitive I_sc in mCT12 monolayers by 46 ± 4% (n = 8) and 22 ± 2% (n = 8), respectively. Exposure of mCT12 cells to EGF, ATP, PMA, and DBHQ caused an increase in phosphorylation of p42/p44 (extracellular signal-regulated kinase; ERK1/2). Pretreatment of mCT12 monolayers with an ERK kinase inhibitor (PD-98059; 30 µM) prevented phosphorylation of p42/p44 and significantly reduced EGF, ATP, and PMA-induced inhibition of amiloride-sensitive I_sc. In contrast, pretreatment of monolayers with a PKC inhibitor (bisindolylmaleimide I; GF109203x; 1 µM) almost completely blocked the PMA-induced decrease in I_sc, but did not alter the EGF- or ATP-induced inhibition of I_sc. The DBHQ-mediated decrease in I_sc was due to inhibition of basolateral Na⁺-K⁺-ATPase, but EGF-, ATP-, and PMA-induced inhibition was most likely due to reduced apical sodium entry (epithelial Na⁺ channel activity). The results of these studies demonstrate that acute inhibition of amiloride-sensitive sodium transport by extracelluar ATP and EGF involves ERK1/2 activation and suggests a role for MAP kinase signaling as a negative regulator of electrogenic sodium absorption in epithelia. mitogen-activated protein kinase; epithelial ion transport; epithelial sodium channel     

Na,K-ATPase and the development of Na+ transport in rat distal colon

Summary Na, K-ATPase function was studied in order to evaluate the mechanism of increased colonic Na⁺ transport during early postnatal development. The maximum Na⁺-pumping activity that was represented by the equivalent short-circuit current after addition of nystatin (I _sc ^N ) did not change during postnatal life or after adrenalectomy performed in 16-day-old rats.I _sc ^N was entirely inhibited by ouabain; the inhibitory constant was 0.1mm in 10-day-old (young) and 0.4mm in 90-day-old (adult) rats. The affinity of the Na, K pump for Na⁺ was higher in young (11mm) than in adult animals (19mm). The Na, K-ATPase activity (measured after unmasking of latent activity by treatment with sodium dodecylsulfate) increased during development and was also not influenced by adrenalectomy of 16-day-old rats. The inhibitory constant for ouabain (K _I ) was not changed during development (0.1–0.3mm). Specific [³H]ouabain binding to isolated colonocytes increased during development (19 and 82 pmol/mg protein), the dissociation constant (K _D ) was 8 and 21 m in young and adult rats, respectively. The Na⁺ turnover rate per single Na, K pump, which was calculated fromI _sc ^N and estimated density of binding sites per cm² of tissue was 500 in adult and 6400 Na⁺/min·site in young rats. These data indicate that the very high Na⁺ transport during early postnatal life reflects an elevated turnover rate and increased affinity for Na⁺ of a single isoform of the Na, K pump. The development of Na⁺ extrusion across the basolateral membrane is not directly regulated by corticosteroids.     

Modifications of current properties by expression of a foreign potassium channel gene in Xenopus embryonic cells

The pH-sensitivity of transepithelial K⁺ transport was studied in vitro in isolated vestibular dark cell epithelium from the gerbil ampulla. The cytosolic pH (pH _iwas measured microfluorometrically with the pH-sensitive dye 2,7-bicarboxyethyl-5(6)-carboxyfluorescein (BCECF) and the equivalent short-circuit current (I _sc), which is a measure for transepithelial K⁺ secretion, was calculated from measurements of the transepithelial voltage (V _t)and the transepithelial resistance (R _t) in a micro-Ussing chamber. All experiments were conducted in virtually HCO ₃ ^– -free solutions. Under control conditions, pH _iwas 7.01±0.04 (n=18), V _twas 9.1±0.5 mV, R _t16.7±0.09 cm², and I _sc was 587±30 A/cm² (n=49). Addition of 20 mm propionate^– caused a biphasic effect involving an initial acidification of pH _i, increase in V _tand I _sc and decrease in R _tand a subsequent alkalinization of pH _i, decrease of V _tand increase of R _t. Removal of propionate^– caused a transient effect involving an alkalinization of pH _i, a decrease of V _tand I _sc and an increase in R _t. pH _iin the presence of propionate^– exceeded pH _iunder control conditions. Effects of propionate ^– on V _t, R _tand I _sc were significantly larger when propionate^– was applied to the basolateral side rather than to the apical side of the epithelium. The pH _i-sensitivityof I _sc between pH 6.8 and 7.5 was –1089 A/(cm² · pH-unit) suggesting that K⁺ secretion ceases at about pH _i7.6. Acidification of the extracellular pH (pH _o)caused an increase of V _tand I _sc and a decrease of R _tmost likely due to acidification of pH _i. Effects were significantly larger when the extracellular acidification was applied to the basolateral side rather than to the apical side of the epithelium. The pH _osensitivity of I _sc between pH 7.4 and 6.4 was –155 A/(cm² · pH unit). These results demonstrate that transepithelial K⁺ transport is sensitive to pH _iand pH _oand that vestibular dark cells contain propionate^– uptake mechanism. Further, the data suggest that cytosolic acidification activates and that cytosolic alkalinization inactivates the slowly activating K⁺ channel (I _sK)in the apical membrane. Whether the effect of pH _ion the I _sK channel is a direct or indirect effect remains to be determined.The authors wish to thank Drs. Daniel C. Marcus, Zhjiun Shen and Hiroshi Sunose for helpful discussions. This work was supported by grants NIH-R29-DC01098 and NIH-R01-DC00212.     

K<Superscript>+</Superscript> transport in the caterpillar intestine epithelium: role of osmolytes for the K<Superscript>+</Superscript>-secretory capacity of the tobacco hornworm midgut

The midgut of the tobacco hornworm, Manduca sexta, actively secretes potassium ions. This can be measured as short-circuit current (I_sc) with the midgut mounted in an Ussing chamber and superfused with a high-K⁺ saline containing as its major osmolyte 166 mM sucrose. Iso-osmotic substitution of sucrose by non-metabolisable compounds (mannitol, urea, NaCl and the polyethylene glycols 200, 400 and 600) led to a dramatic, though reversible, drop in the current. Acarbose, a specific inhibitor of invertase (sucrase) in vertebrates and insects, had no detectable influence on I_sc. Unexpectedly, after replacing sucrose iso-osmotically with the saccharides glucose, fructose, trehalose or raffinose, the K⁺ current could no longer be supported. However, all osmolytes smaller than sucrose (except for NaCl), metabolisable or not, initiated an immediate, quite uniform but transient, increase in I_sc by about 20%, before its eventual decline far below the control value. Hypo-osmotic treatment by omission of sucrose also transiently increased the K⁺ current. Small osmolytes substituted for sucrose caused no transient I_sc stimulation when the epithelium had been challenged before with hypo-osmolarity; however, the eventual decline in I_sc could not be prevented. Our data seem inconsistent with a role of sucrose as energiser or simple osmolyte. Rather, we discuss here its possible role as analogous to that of sucrose in lower eukaryotes or plants, as an extra- and/or intracellular compatible osmolyte that stabilises structure and/or function of the proteins implicated in K⁺ transport.Communicated by G. Heldmaier     

Effects of tryptamine on active sodium and chloride transport in the isolated bullfrog cornea

The effects of the serotonin analogue, tryptamine, on the active transepithelial transport of Na⁺ and Cl⁻ in the in vitro bullfrog cornea were studied. Tryptamine, 1 mM, inhibited both the short-circuit current (I_sc) and potential difference (PD) of corneas transporting either Na⁺ alone or both Na⁺ and Cl⁻. The electrical resistance, R, increased in all cases. Both unidirectional Na⁺ and Cl⁻ fluxes were decreased by tryptamine and these changes accounted for the inhibitory effects on the I_sc. The effects of tryptamine were considered along with with those of 2 mM theophylline and 0.1 mM ouabain. Tryptamine inhibited the I_sc and both undirectional Cl⁻ fluxes which were previously stimulated by theophylline. Theophyline addition, after tryptamine preincubation, increased the Cl⁻ undirectional fluxes but did not restore the inhibited I_sc. The inhibitory effects of tryptamine on active Na⁺ and Cl⁻ transport were different from those of ouabain. While both drugs inhibited the forward Na⁺ and Cl⁻ fluxes, their backfluxes decreased with tryptamine and increased with ouabain. The addition to the bathing solution of tryptamine after ouabain preincubation reduced the ouabain-increased backward Cl⁻ flux and further increased the electrical resistance. These results are analyzed in terms of an electrical model from which it appears that tryptamine's mechanism of action was to decrease cellular permeability to the transepithelial movement of Na⁺ and Cl⁻.     

Characterization of K+ Channels in the Basolateral Membrane of Rat Tracheal Epithelia

To study K⁺ channels in the basolateral membrane of chloride-secreting epithelia, rat tracheal epithelial monolayers were cultured on permeable filters and mounted into an Ussing chamber system. The mucosal membrane was permeabilized with nystatin (180 μg/ml) in the symmetrical high K⁺ (145 mm) Ringer solution. During measurement of the macroscopic K⁺ conductance properties of the basolateral membrane under a transepithelial voltage clamp, we detected at least two types of K⁺ currents: one is an inwardly rectifying K⁺ current and the other is a slowly activating outwardly rectifying K⁺ current. The inwardly rectifying K⁺ current is inhibited by Ba²⁺. The slowly activating K⁺ current was potentiated by cAMP and inhibited by clofilium, phorbol 12-myristae 13-acetate (PMA) and lowering temperature. This is consistent with the biophysical characteristics of I _SK channel. RT-PCR analysis revealed the presence of I _SK cDNA in the rat trachea epithelia. Although 0.1 mm Ba²⁺ only had minimal affect on short-circuit current (I _sc) induced by cAMP in intact epithelia, 0.1 mm clofilium strongly inhibited it. These results indicate that I _SK might be important for maintaining cAMP-induced chloride secretion in the rat trachea epithelia. Received: 1 March 1996/Revised: 5 August 1996     

cAMP Increases Apical IsK Channel Current and K+ Secretion in Vestibular Dark Cells

Adenosine 3′,5′-cyclic monophosphate (cAMP) is known to stimulate exogenous I_sK channel current in the Xenopus oocyte expression system. The present study was performed to determine whether elevation of cytosolic cAMP in a native mammalian epithelium known to secrete K⁺ through endogenously expressed I_sK channels would stimulate K⁺ secretion through these channels. The equivalent short circuit current (I _sc ) across vestibular dark cell epithelium in gerbil was measured in a micro-Ussing chamber and the apical membrane current (I _IsK ) and conductance (g _IsK ) of I_sK channels was recorded with both the on-cell macro-patch and nystatin-perforated whole-cell patch-clamp techniques. It has previously been shown that I _sc can be accounted for by transepithelial K⁺ secretion and that the apical I_sK channels constitute a significant pathway for K⁺ secretion. The identification of the voltage-dependent whole-cell currents in vestibular dark cells was strengthened by the finding that a potent blocker of I_sK channels, chromanol 293B, strongly reduced I _IsK from 646 ± 200 to 154 ± 22 pA (71%) and g _IsK from 7.5 ± 2.6 to 2.8 ± 0.4 nS (53%). Cytoplasmic cAMP was elevated by applying dibutyryl cyclic AMP (dbcAMP), or the phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine (IBMX) and Ro-20-1724. dbcAMP (1 mm) increased I _sc and I _IsK from 410 ± 38 to 534 ± 40 μA/cm² and from 4.3 ± 0.8 to 11.4 ± 2.2 pA, respectively. IBMX (1 mm) caused transient increases of I _sc from 415 ± 30 to 469 ± 38 μA/cm² and Ro-20-1724 (0.1 mm) from 565 ± 43 to 773 ± 58 μA/cm². IBMX increased I _IsK from 5.5 ± 1.5 to 16.9 ± 5.8 pA in on-cell experiments and from 191 ± 31 to 426 ± 53 pA in whole-cell experiments. The leak conductance due to all non-I_sK channel sources did not change during dbcAMP and IBMX while 293B in the presence of dbcAMP reduced I _IsK by 84% and g _IsK by 62%, similar to unstimulated conditions. These results demonstrate that the cAMP pathway is constitutively active in vestibular dark cells and that the cAMP pathway stimulates transepithelial K⁺ secretion by increasing I_sK channel current rather than by altering another transport pathway. Received: 9 June 1995/Revised: 17 October 1996     

Na+-independent,electrogenic Cl- uptake across the posterior gills of the Chinese crab (Eriocheir sinensis): Voltage-clamp and microelectrode studies

Summary Single gill lamellae from posterior gills of Chinese crabs (Eriocheir sinensis) were isolated, separated into halves and mounted in a modified Ussing chamber. Area-related short-circuit current (I_sc) and conductance (G_tot) of this preparation were measured. Epithelial cells were impaled with microelectrodes through the basolateral membrane and cellular potentials (V_i under open- and V_sc under short-circuit conditions) as well as the voltage divider ratios (F_i, F_o) were determined.With NaCl salines on both sides an outside positive PD_te (22±2 mV) and an I_sc (-64±13 A·cm^-2) with a polarity corresponding to an uptake of negative charges (inward negative) were obtained. Trough-like potential profiles were recorded across the preparation under open- as well as short-circuit conditions (V_o=-101±5 mV, external bath as reference; V_i=-78±2 mV, internal bath as reference; V_sc=-80±2 mV, extracellular space as reference). The voltage divider ratios of the external (apical membrane plus cuticle) and internal (basolateral membrane) barrier were F_o=0.92±0.01 and F_i=0.08±0.01, respectively. To investigate a Cl^--related contribution to the above parameters, Na⁺-free solutions in the external bath (basolateral NaCl-saline) were used. Inward negative I_sc under these conditions almost completely depended on external Cl^-. Elimination of Cl^- in the external bath reversed I_sc, and G_tot decreased substantially. Concomitantly, V_sc depolarised and F_o increased. Cl^--dependent current and conductance showed saturation kinetics with increasing external [Cl^-]. Addition of 20 mmol·1^-1 thiocyanate to the external bath had similar, although less pronounced, effects as Cl^- substitution. Equally, external SITS (1 mmol·1^-1) inhibited the current and, concomitantly, G_tot decreased substantially. Addition of 1 mmol·1^-1 acetazolamide to, and omission of NaHCO₃ from, the basolateral bath resulted in a decrease of I_sc while G_tot remained unchanged. The Cl^--channel blocker DPC inhibited I_sc almost completely when added to the basolateral saline, whereas G_tot decreased moderately; however, V_sc depolarised without significant change of F_i. Ouabain had no influence on I_sc and G_tot. Increasing the basolateral [K⁺] resulted in a decrease in I_sc, while G_tot was not affected. At the same time V_sc largely depolarised and F_i decreased. Addition of the K⁺-channel blocker Ba⁺⁺ (5 mmol·1^-1) to the basolateral solution resulted in a two-step alteration of the transepithelial (I_sc, Gtot) and cellular (V_sc, F_i) parameters. The results are discussed with regard to (i) the mechanisms responsible for active transbranchial Cl^- uptake, and (ii) the technical improvement of being able to perform transport studies with crab gill preparations in an Ussing chamber.Abbreviations DMSO dimethylsulfoxide - DPC diphenylamine-2-carboxylate - F _{o, i} voltage divider ratio for external (o) and internal (i) barrier, respectively - G _Cl conductance related to the external [Cl^-] - G _tot total tissue conductance - I _Cl short-circuit current related to the external [Cl^-] - I _sc short-circuit current - PD _te transepithelial potential difference - R _ME resistance of the microelectrode - SITS 4-acetamido-4-isothiocyanato-stilbene-2,2-disulfonic acid - V _{o, i} open-circuit voltage across the external (o) and internal (i) barrier, respectively - V _sc intracellular potential under short-circuit conditions     

P2Y(2) receptor-mediated inhibition of amiloride-sensitive short circuit current in M-1 mouse cortical collecting duct cells

Extracellular nucleotides modulate renal ion transport. Our previous results in M-1 cortical collecting duct cells indicate that luminal and basolateral ATP via P2Y₂ receptors stimulate luminal Ca²⁺-activated Cl⁻ channels and inhibit Na⁺ transport. Here we address the mechanism of ATP-mediated inhibition of Na⁺ transport. M-1 cells had a transepithelial voltage (V _te ) of −31.4 ± 1.3 mV and a transepithelial resistance (R _te ) of 1151 ± 28 Ωcm². The amiloride-sensitive short circuit current (I _sc ) was −28.0 ± 1.1 μA/cm². The ATP-mediated activation of Cl⁻ channels was inhibited when cytosolic Ca²⁺ increases were blocked with cyclopiazonic acid (CPA). Without CPA the ATP-induced [Ca²⁺]_i increase was paralleled by a rapid and transient R _te decrease (297 ± 51 Ωcm²). In the presence of CPA, basolateral ATP led to an R _te increase by 144 ± 17 Ωcm² and decreased V _te from −31 ± 2.6 to −26.6 ± 2.5 mV. I _sc dropped from −28.6 ± 2.4 to −21.6 ± 1.9 μA/cm². Similar effects were observed with luminal ATP. In the presence of amiloride, ATP was without effect. This reflects ATP-mediated inhibition of Na⁺ absorption. Lowering [Ca²⁺]_i by removal of extracellular Ca²⁺ did not alter the ATP effect. PKC inhibition or activation were without effect. Na⁺ absorption was activated by pH_i alkalinization and inhibited by pH_i acidification. ATP slightly acidified M-1 cells by 0.05 ± 0.005 pH units, quantitatively not explaining the ATP-induced effect. In summary this indicates that extracellular ATP via luminal and basolateral P2Y₂ receptors inhibits Na⁺ absorption. This effect is not mediated via [Ca²⁺]_i, does not involve PKC and is to a small part mediated via intracellular acidification. Received: 9 February 2001/Revised: 17 May 2001     

Na+ transport by rabbit urinary bladder,a tight epithelium

Summary By in vitro experiments on rabbit bladder, we reassessed the traditional view that mammalian urinary bladder lacks ion transport mechanisms. Since the ratio of actual-to-nominal membrane area in folded epithelia is variable and hard to estimate, we normalized membrane properties to apical membrane capacitance rather than to nominal area (probably 1 F 1 cm² actual area). A new mounting technique that virtually eliminates edge damage yielded resistances up to 78,000 F for rabbit bladder, and resistances for amphibian skin and bladder much higher than those usually reported. This technique made it possible to observe a transport-related conductance pathway, and a close correlation between transepithelial conductance (G) and short-circuit current (I _sc) in these tight epithelia.G andI _sc were increased by mucosal (Na⁺) [I _sc0 when (Na⁺)0], aldosterone, serosal (HCO ₃ ^– ) and high mucosal (H⁺); were decreased by amiloride, mucosal (Ca⁺⁺), ouabain, metabolic inhibitors and serosal (H⁺); and were unaffected by (Cl^–) and little affected by antidiuretic hormone (ADH). Physiological variation in the rabbits' dietary Na⁺ intake caused variations in bladderG andI _sc similar to those caused by the expectedin vivo changes in aldosterone levels. The relation betweenG andI _sc was the same whether defined by diet changes, natural variation among individual rabbits, or most of the above agents. A method was developed for separately resolving conductances of junctions, basolateral cell membrane, and apical cell membrane from thisG–I _sc relation. Net Na⁺ flux equalledI _sc. Net Cl^– flux was zero on short circuit and equalled only 25% of net Na⁺ flux in open circuit. Bladder membrane fragments contained a Na⁺–K⁺-activated, ouabain-inhibited ATPase. The physiological significance of Na⁺ absorption against steep gradients in rabbit bladder may be to maintain kidney-generated ion gradients during bladder storage of urine, especially when the animal is Na⁺-depleted.     

Effects of aldosterone on Na transport in the toad bladder I. Glycolysis and lactate production under aerobic conditions

Previous studies indicated that aldosterone enhances active Na⁺ transport, glycolysis, lactate production and respiration of the toad bladder. Evidence was also presented that the changes in glycolysis and lactate production were secondary to the changes in active Na⁺ transport. Further analysis of the relationships between metabolism and Na⁺ transport was undertaken with the aid of two inhibitors of pyruvate metabolism, oxythiamine and phenylpyruvate. These inhibitors prevented the aldosterone-induced increase in oxidation of [6-¹⁴C]glucose but had little effect on the increase in lactate production. In contrast, the effect on Na⁺ transport (i.e., I_sc) was completely inhibited by oxythiamine plus phenylpyruvate with glucose as substrate. The effect on Na⁺ transport, however, was obtained wth the by-pass substrates, oxaloacetate plus ß-hydroxybutyrate, in the presence of these inhibitors. These results implied that steroidal enhancement of lactate production and Na⁺ transport were independent effects. To evaluate whether an increase in Na⁺ transport, per se would augment lactate production, the responses were evaluated under conditions of an imposed Na⁺ gradient (mucosal Na⁺ = 5 mM; serosal Na⁺ = 110 mM). Addition of NaCl to the mucosal media evoked the same increase in I_sc as the addition of aldosterone; both additions increased I_sc more than two-fold. Aldosterone reduced lactate production under these conditions while the re-addition of NaCl had no effect on lactate formation. These results are consistent with an action of aldosterone on pathways involved in oxidative energy metabolism, and suggest that the activation of glycolysis may be a function of the net balance between energy production and utilization.