Membrane potentials and intracellular Cl− activity of toad skin epithelium in relation to activation and deactivation of the transepithelial Cl− conductance

Summary The potential dependence of unidirectional³⁶Cl fluxes through toad skin revealed activation of a conductive pathway in the physiological region of transepithelial potentials. Activation of the conductance was dependent on the presence of Cl^– or Br^– in the external bathing solution, but was independent of whether the external bath was NaCl-Ringer's, NaCl-Ringer's with amiloride, KCl-Ringer's or choline Cl-Ringer's To partition the routes of the conductive Cl^– ion flow, we measured in the isolated epithelium with double-barrelled microelectrodes apical membrane potentialV _a , and intracellular Cl^– activity,a _Cl ^c , of the principal cells indentified by differential interference contrast microscopy. Under short-circuit conditionsI _sc=27.0±2.0 A/cm², with NaCl-Ringer's bathing both surfaces,V _a was –67.9±3.8mV (mean ±se,n=24, six preparations) anda _Cl ^c was 18.0±0.9mM in skins from animals adapted to distilled water. BothV _a anda _Cl ^a were found to be positively correlated withI _sc (r=0.66 andr=0.70, respectively). In eight epithelia from animals adapted to dry milieu/tap waterV _a anda _Cl ^c were measured with KCl Ringer's on the outside during activation and deactivation of the transepithelial Cl^– conductance (G _Cl) by voltage clamping the transepithelial potential (V) at 40 mV (mucosa positive) and –100 mV. AtV=40 mV; i.e. whenG _Cl was deactivated,V _a was –70.1±5.0 mV (n=15, eight preparations) anda _Cl ^c was 40.0±3.8mm. The fractional apical membrane resistance (fR _a) was 0.69±0.03. Clamping toV=–100 mV led to an instantaneous change ofV _a to 31.3±5.6 mV (cell interior positive with respect to the mucosal bath), whereas neithera _Cl ^c norfR _a changed significantly within a 2 to 5-min period during whichG _Cl increased by 1.19±0.10 mS/cm². WhenV was stepped back to 40 mV,V _a instantaneously shifted to –67.8±3.9 mV whilea _Cl ^c andfR _a remained constant during deactivation ofG _Cl. Similar results were obtained in epithelia impaled from the serosal side. In 12 skins from animals adapted to either tap water or distilled water the density of mitochondria-rich (D _MRC) cells was estimated and correlated with the Cl current (I _Cl though the fully activated (V=–100mV) Cl^– conductance). A highly significant correlation was revealed (r=–0.96) with a slope of –2.6 nA/m.r. (mitochondria-rich cell and an I-axis intercept not significantly different from zero. In summary, the voltage-dependent Cl currents were not reflected infR _a anda _Cl ^a of the principal cells but showed a correlation with the m.r. cell density. We conclude that the pricipal cells do not contribute significantly to the voltage-dependent Cl conductance.     

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     

Electrophysiological studies of forskolin-induced changes in ion transport in the human colon carcinoma cell line HT-29 cl.19A: Lack of evidence for a cAMP-activated basolateral K+ conductance

Summary Forskolin (i.e, cAMP)-modulation of ion transport pathways in filter-grown monolayers of the Cl^–-secreting subclone (19A) of the human colon carcinoma cell line HT29 was studied by combined Ussing chamber and microimpalement experiments.Changes in electrophysiological parameters provoked by serosal addition of 10^–5 m forskolin included: (i) a sustained increase in the transepithelial potential difference (3.9±0.4 mV). (ii) a transient decrease in transepithelial resistance with 26±3 · cm² from a mean value of 138±13 · cm² before forskolin addition, (iii) a depolarization of the cell membrane potential by 24±1 mV from a resting value of –50±1 mV and (iv) a decrease in the fractional resistance of the apical membrane from 0.80±0.02 to 0.22±0.01. Both, the changes in cell potential and the fractional resistance, persisted for at least 10 min and were dependent on the presence of Cl^– in the medium. Subsequent addition of bumetanide (10^–4 m), an inhibitor of Na/K/2Cl cotransport, reduced the transepithelial potential, induced a repolarization of the cell potential and provoked a small increase of the transepithelial resistance and fractional apical resistance. Serosal Ba²⁺ (1mm), a known inhibitor of basolateral K⁺ conductance, strongly reduced the electrical effects of forskolin. No evidence was found for a forskolin (cAMP)-induced modulation of basolateral K⁺ conductance.The results suggest that forskolin-induced Cl^– secretion in the HT-29 cl.19A colonic cell line results mainly from a cAMP-provoked increase in the Cl^– conductance of the apical membrane but does not affect K⁺ or Cl^– conductance pathways at the basolateral pole of the cell. The sustained potential changes indicate that the capacity of the basolateral transport mechanism for Cl^– and the basal Ba²⁺-sensitive K⁺ conductance are sufficiently large to maintain the Cl^– efflux across the apical membrane. Furthermore, evidence is presented for an anomalous inhibitory action of the putative Cl^– channel blockers NPPB and DPC on basolateral conductance rather than apical Cl^– conductance.     

Membrane potential,anion and cation conductances in Ehrlich ascites tumor cell

Summary The fluorescence intensity of the dye 1,1-dipropyloxadicarbocyanine (DiOC₃-(5)) has been measured in suspensions of Ehrlich ascites tumor cells in an attempt to monitor their membrane potential (V _m ) under different ionic conditions, after treatment with cation ionophores and after hypotonic cell swelling. Calibration is performed with gramicidin in Na⁺-free K⁺/choline⁺ media, i.e., standard medium in which NaCl is replaced by KCl and cholineCl and where the sum of potassium and choline is kept constant at 155mm. Calibration by the valinomycin null point procedure described by Lariset al. (Laris, P.C., Pershadsingh, A., Johnstone, R.M., 1976,Biochim. Biophys. Acta 436:475–488) is shown to be valid only in the presence of the Cl^–-channel blocker indacrinone (MK196). Distribution of the lipophilic anion SCN^– as an indirect estimation of the membrane potential is found not to be applicable for the fast changes inV _m reported in this paper. Incubation with DiOC₃-(5) for 5 min is demenstrated to reduce the Cl^– permeability by 26±5% and the NO ₃ ^– permeability by 15±2%, while no significant effect of the probe could be demonstrated on the K⁺ permeability. Values forV _m , corrected for the inhibitory effect of the dye on the anion conductance, are estimated at –61±1 mV in isotonic standard NaCl medium, –78±3 mV in isotonic Na⁺-free choline medium and –46±1 mV in isotonic NaNO₃ medium. The cell membrane is depolarized by addition of the K⁺ channel inhibitor quinine and it is hyperpolarized when the cells are suspended in Na⁺-free choline medium, indicating thatV _m is generated partly by potassium and partly by sodium diffusion. Ehrlich cells have previously been shown to be more permeable to nitrate than to chloride. Substituting NO ₃ ^– for all cellular and extracellular Cl^– leads to a depolarization of the membrane, demonstrating thatV _m is also generated by the anions and that anions are above equilibrium. Taking the previously demonstrated single-file behavior of the K⁺ channels into consideration, the membrane conductances in Ehrlich cells are estimated at 10.4 S/cm² for K⁺, 3.0 S/cm² for Na⁺, 0.6 S/cm² for Cl^– and 8.7 S/cm² for NO ₃ ^– . Addition of the Ca²⁺-ionophore A23187 results in net loss of KCl and a hyperpolarization of the membrane, indicating that the K⁺ permeability exceeds the Cl^– permeability also after the addition of A23187. The K⁺ and Cl^– conductances in A23187-treated Ehrlich cells are estimated at 134 and 30 S/cm², respectively. The membrane potential is depolarized in hypotonically swollen cells, confirming that the increase in the Cl^– permeability following hypotonic exposure exceeds the concommitant increase in the K⁺ permeability. In control experiments where the membrane potentialV _m =E _K =E _Cl =E _Na , it is demonstrated that cell volume changes has no significant effect on the fluorescence signal, apparently because of a large intracellular buffering capacity. The increase in the Cl^– conductances is 68-fold when cells are transferred to a medium with half the osmolarity of the standard medium, as estimated from the net Cl^– efflux and the change inV _m . The concommitant increase in the K⁺ conductance, as estimated from the net K⁺ efflux, is only twofold.     

Effect of Metabolic Inhibitors on Membrane Potential and Ion Conductance of Rat Astrocytes

1. The aim of this study was to elucidate the effect of metabolic inhibition on the membrane potential and ion conductance of rat astrocytes. The metabolic inhibitors investigated were dinitrophenol (DNP), carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP), cyanide, and oligomycin.2. Primary cultures of astroglial cells from newborn rat cerebral cortex were cultivated for 13–20 days on chamber slides. The effect of metabolic inhibitors on the cellular ATP concentration was estimated from the decrease in peak chemiluminescence from the luciferin/luciferase reaction. The membrane potential and ion conductances were measured from whole-cell recordings with the patch-clamp technique.3. After 2.0 min of incubation ATP decreased from the control level to 43%with cyanide (2 mM), 58% with DNP (1 mM), 47% with FCCP (1 M), and 69% with oligomycin (10 M).4. Under normal conditions V was –74.4±1.0 mV. DNP and FCCP both caused a rapid and reversible depolarization equivalent to a shift in the I/V curve of 8.2±1.3 and 19.7±3.8 mV, respectively. DNP decreased the slope conductance (g) by 22.1% but FCCP had no significant effect on g. In contrast, neither oligomycin nor cyanide had any significant effect on the I/V curve.5. Tetraethylammonium (TEA; 10 mM) depolarized the cells by 7.1±2.0 mV but had no significant effect on g. In the presence of TEA, DNP caused a depolarization of 52.8±3.5 mV and increased g by 45.5±9.6%. The action of FCCP was not affected by the presence of TEA.6. Perfusion of the astrocytes with a Cl^– free solution inhibited the action of DNP and FCCP. Thus the depolarization was only 4.2±1.5mV in DNP and 3.7±0.3 mV in FCCP, which were significantly smaller effects than in the presence of a high intracellular [Cl^–].7. Block of tentative K_ATP channels with tolbutamide (1 mM) or Cl^– channels with Zn²⁺ (1 mM) did not inhibit the depolarization caused by DNP or FCCP.8. In conclusion, DNP and FCCP have specific effects on the plasmalemma in rat astrocytes which may be due to opening of Cl^– channels. This effect was not seen with cyanide or oligomycin and should be considered as a possible complication when DNP and FCCP are used for metabolic inhibition.     

Basolateral membrane potential and conductance in frog skin exposed to high serosal potassium

Summary In studies of apical membrane current-voltage relationships, in order to avoid laborious intracellular microelectrode techniques, tight epithelia are commonly exposed to high serosal K concentrations. This approach depends on the assumptions that high serosal K reduces the basolateral membrane resistance and potential to insignificantly low levels, so that transepithelial values can be attributed to the apical membrane. We have here examined the validity of these assumptions in frog skins (Rana pipiens pipiens). The skins were equilibrated in NaCl Ringer's solutions, with transepithelial voltageV _t clamped (except for brief perturbations V _t) at zero. The skins were impaled from the outer surface with 1.5m KCl-filled microelectrodes (R _el>30 M). The transepithelial (short-circuit) currentl _i and conductanceg _t=–I _t/V _t, the outer membrane voltageV _o (apical reference) and voltage-divider ratio (F _o=V _o/V _t), and the microelectrode resistanceR _el were recorded continuously. Intermittent brief apical exposure to 20 m amiloride permitted estimation of cellular (c) and paracellular (p) currents and conductances. The basolateral (inner) membrane conductance was estimated by two independent means: either from values ofg _i andF _o before and after amiloride or as the ratio of changes (–I _c/V _i) induced by amiloride. On serosal substitution of Na by K, within about 10 min,I _c declined andg _t increased markedly, mainly as a consequence of increase ing _p. The basolateral membrane voltage (V _i(=–V _o) was depolarized from 75±4 to 2±1 mV [mean±sem (n=6)], and was partially repolarized following amiloride to 5±2 mV. The basolateral conductance increased in high serosal K, as estimated by both methods. Essentially complete depolarization of the basolateral membrane and increase in its conductance in response to high [K] were obtained also when the main serosal anion was SO₄ or NO₃ instead of Cl. On clampingV _t over the range 0 to +125 mV in K₂SO₄-depolarized skins, the quasi-steady-stateV _o V _t relationship was linear, with a mean slope of 0.88±0.03. The above results demonstrate that, in a variety of conditions, exposure to high serosal K results in essentially complete depolarization of the basolateral membrane and a large increase in its conductance.     

Electrical characteristics of stomatal guard cells: The ionic basis of the membrane potential and the consequence of potassium chlorides leakage from microelectrodes

The membrane electrical characteristics of stomatal guard cells in epidermal strips from Vicia faba L. and Commelina communis L. were explored using conventional electrophysiological methods, but with double-barrelled microelectrodes containing dilute electrolyte solutions. When electrodes were filled with the customary 1–3 M KCl solutions, membrane potentials and resistances were low, typically decaying over 2–5 min to near-30 mV and <0.2 k·cm² in cells bathed in 0.1 mM KCl and 1 mM Ca²⁺, pH 7.4. By contrast, cells impaled with electrodes containing 50 or 200 mM K⁺-acetate gave values of-182±7 mV and 16±2 k·cm² (input resistances 0.8–3.1 G, n=54). Potentials as high as (-) 282 mV (inside negative) were recorded, and impalement were held for up to 2 h without appreciable decline in either membrane parameter. Comparison of results obtained with several electrolytes indicated that Cl^- leakage from the microelectrode was primarily responsible for the decline in potential and resistance recorded with the molar KCl electrolytes. Guard cells loaded with salt from the electrodes also acquired marked potential and conductance responses to external Ca²⁺, which are tentatively ascribed to a K⁺ conductance (channel) at the guard cell plasma membrane.Measurements using dilute K⁺-acetate-filled electrodes revealed, in the guard cells, electrical properties common to plant and fungal cell membranes. The cells showed a high selectivity for K⁺ over Na⁺ (permeability ratio P_Na/P_K=0.006) and a near-Nernstian potential response to external pH over the range 4.5–7.4 (apparent P_H/P_K=500–600). Little response to external Ca²⁺ was observed, and the cells were virtually insensitive to CO₂. These results are discussed in the context of primary, charge-carrying transport at the guard cell plasma membrane, and with reference to possible mechanisms for K⁺ transport during stomatal movements. They discount previous notions of Ca²⁺-and CO₂-mediated transport control. It is argued, also, that passive (diffusional) mechanisms are unlikely to contribute to K⁺ uptake during stomatal opening, despite membrane potentials which, under certain, well-defined conditions, lie negative of the potassium equilibrium potential likely prevailing.Abbreviations and symbols EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - Mes 2-(N-morpholino) propanesulfornic acid - E equilibrium potential - G_m membrane conductance - R_in input resistance - V_m membrane potential     

Active Cl− absorption by the Chinese crab (Eriocheir sinensis) gill epithelium measured by transepithelial potential difference

Summary Isolated posterior gills from Chinese crabs (Eriocheir sinensis) acclimated to tap-water were perfused and bathed with full, physiological saline (containing Na⁺ and Cl^–). Under these conditions they developed an outside positive transepithelial potential difference (PD_te). Substitution of Na⁺ by choline on both sides of the epithelium resulted in a substantial hyperpolarization of the PD_te, while substitution of Cl^– by gluconate reverses PD_te to outside negative values.The magnitudes of the potential differences were strongly related to the adaptation media (artificial seawater or tap-water).The KCN-sensitive, outside positive PD_te was shown to be strongly dependent on Cl^–. Application of thiocyanate and 4-acetamido-4-isothiocyanato-stilbene-2,2 disulfonic acid (SITS) to the bath solution resulted in a reduction of the PD_te, while the Cl^–-channel blocker, diphenylamine-2-carboxylic acid (DPC), showed no effect. The PD_te was markedly reduced by acetazolamide, an inhibitor of carbonic anhydrase (CA), and these results are discussed with reference to the presence of a Cl^–/HCO ₃ ^– -exchanger in the apical membrane.Chloride was shown to pass the basolateral membrane via Cl^–-channels: Diphenylamine-2-carboxylic acid (DPC) reduced the PD_te with an IC₅₀ of 3.7×10^–5 mol/l when added to the perfusion saline. A basolateral K⁺-channel and its linkage to Cl^– uptake could be demonstrated by using the K⁺-channel blocker, Ba²⁺, or increased K⁺ concentrations in the perfusion saline (PD_te decrease). Ouabain did not reduce the PD_te under nominally Na⁺-free conditions, indicating that the Cl^– transport is independent of the Na⁺/K⁺-ATPase. In this paper we shall discuss the possible energy sources and linkages between pH regulation and active Cl^– absorption under these experimental conditions.Abbreviations A9C anthracene-9-carboxylic acid - CA carbonic anhydrase - DMSO dimethylsulfoxide - DPC diphenylamino-2-carboxylic acid - PD _te transepithelial potential difference - SITS 4-acetamido-4-isothiocyanato-stilbene-2,2-disulfonic acid - TEA tetraethyl-ammoniumchloride     

Short-chain fatty acids and CO2 as regulators of Na+ and Cl− absorption in isolated sheep rumen mucosa

Summary Unidirectional ²²Na⁺ and ³⁶Cl^– fluxes were determined in short-circuited, stripped rumen mucosa from sheep by using the Ussing chamber technique. In both CO₂/HCO _– ³ -containing and CO₂/HCO _– ³ -free solutions, replacement of gluconate by short-chain fatty acids (SCFA, 39 mM) significantly enhanced mucosal-toserosal Na⁺ absorption without affecting the Cl^– transport in the same direction. Short-chain fatty acid stimulation of Na⁺ transport was at least partly independent of Cl^– and could almost completely be abolished by 1 mM mucosal amiloride, while stimulation of Na⁺ transport was enhanced by lowering the mucosal pH from 7.3 to 6.5. Similar to the SCFA action, raising the PCO₂ in the mucosal bathing solution led to an increase in the amiloride-sensitive mucosal-to-serosal Na⁺ flux. Along with its effect on sodium transport, raising the PCO₂ also stimulated chloride transport. The results are best explained by a model in which undissociated SCFA and/or CO₂ permeate the cell membrane and produce a raise in intracellular H⁺ concentration. This stimulates an apical Na⁺/H⁺ exchange, leading to increased Na⁺ transport. The stimulatory effect of CO₂ on Cl^– transport is probably mediated by a Cl^–/HCO _– ³ exchange mechanism in the apical membrane. Binding of SCFA anions to that exchange as described for the rat distal colon (Binder and Mehta 1989) probably does not play a major role in the rumen.Abbreviations DIDS 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid - G _t transepithelial conductance (mS·cm^-2) - HSCFA undissociated short-chain fatty acids - J _ms mucosal-to-serosal flux (Eq · cm^-2 · h^-1) - J _net net flux (Eq · cm^-2 · h^-1) - J _sm serosal-to-mucosal flux (Eq · cm^-2 · h^-1) - PD transepithelial potential difference (mV) - SCFA dissociated short-chain fatty acids - SCFA short-chain fatty acids     

Thiocyanate transport across fish intestine (Pleuronectes platessa)

Summary When bathed on both sides with identical chloride-containing salines thein vitro preparation of the plaice intestine maintains a negative (serosa to mucosa) short-circuit current of 107±11 A/cm², a transepithelial potential difference of 5.5±0.6 mV (serosa negative), and a mean mucosal membrane potential of –45.4±0.6 mV. Under these conditions the intracellular chloride activity is 32mm.If chloride in the bathing media is partially, or completely substituted by thiocyanate the measured electrical parameters do not change but transepithelial flux determinations show a reduction in chloride fluxes and the presence of a significant thiocyanate flux. The addition of piretanide (10^–4 m) reduced the short-circuit current and the mucosa-to-serosa fluxes of chloride and thiocyanate; this inhibition is similar to the effect of piretanide on chloride transport in this tissue.The results indicate that thiocyanate is transported in this tissue via the piretanide-sensitive chloride pathway and are compared with the effects of thiocyanate on other tissues reported in the literature.     

Ion transport across the early chick embryo: I. Electrical measurements,ionic fluxes and regional heterogeneity

The chick blastoderm at the stage of late gastrula is a flat disc formed by three cell layers and exhibiting epithelial properties. Blastoderms were cultured in miniature chambers and their electrophysiological characteristics were determined under Ussing conditions.Under open-circuit condition and identical physiological solutions on both sides, spontaneous transblastodermal potential difference (V _oc) of –7.5±3.3 mV (ventral side positive) was measured. Under short-circuit condition (transblastodermal V = 0 mV), the blastoderm generated short-circuit current (I _sc) of 21±8 A/cm², which was entirely dependent on extracellular sodium, sensitive to ouabain applied ventrally and independent of extracellular chloride. The net transblastodermal Na⁺ flux fully accounted for the measured I _sc, both under control conditions and with ouabain. The total transblastodermal resistance (R _tot) was 390±125 cm².Frequently, the V _oc, I _sc and R _tot showed spontaneous oscillations with a period of 4–5 min. Removal of endoderm and mesoderm did not significantly affect the electrical properties, indicating that the electrogenic sodium transport is generated by the ectoderm.The V _oc and I _sc measured in the area pellucida (–1.3±0.8 mV, 9.3±4.4 A/cm²) and extraembryonic area opaca (–7.8±1.1 mV, 31.2±12.7 A/cm²) were significantly different. Such a heterogeneous distribution of electrical properties can explain the presence in the blastoderm of extracellular electrical currents found by using a vibrating probe.This work was supported by the Swiss National Research Foundation (grant. 3.418-0.86 to P.K.) and by Roche Research Foundation (grant. to U.K.). We thank Drs. E. Raddatz and Y. de Ribaupierre for helpful discussions.     

Intracellular potassium activity in mammalian proximal tubule: Effect of perturbations in transepithelial sodium transport

Summary Intracellular potassium activity (a _{K i} ) was measured in control conditions in mid-cortical rabbit proximal convoluted tubule using two methods: (i) by determination of the K⁺ equilibrium potential (E _K) using Ba²⁺-induced variations in the basolateral membrane potential (V _BL) during transepithelial current injections and (ii) with double-barrel K-selective microelectrodes. Using the first method, the meanV _BL was –48.5±3.2 mV (n=16) and the meanE _K was –78.4±4.1 mV corresponding to aa _{K i} of 68.7mm. With K-selective microelectrodes,V _BL was –36.6±1.1 mV (n=19),E _K was –64.0±1.1 mV anda _{K i} averaged 40.6±1.7mm. While these lastE _K andV _BL values are significantly lower than the corresponding values obtained with the first method (P<0.001 andP<0.01, respectively), the electrochemical driving force for K transport across the basolateral membrane ( _K =V _BL –E _K) is not significantly different for both techniques (30.1±3.3 mV for the first technique and 27.6±1.8 mV for ion-selective electrodes). This suggests an adequate functioning of the selective barrel but an underestimation ofV _BL by the reference barrel of the double-barrel microelectrode. Such double-barrel microelectrodes were used to measure temporal changes ina _{K i} and _K in different experimental conditions where Na reabsorption rate (J _Na) was reduced.a _{K i} was shown to increase by 12.2±2.7 (n=5) and 14.1±4.4mm (n=5), respectively, whenJ _Na was reduced by omitting in the luminal perfusate: (i) 5.5mm glucose and 6mm alanine and (ii) glucose, alanine, other Na-cotransported solutes and 110mm Na. In terms of the electrochemical driving force for K exit across the basolateral membrane, _K, a decrease of 5.4±2.0 mV (P<0.05,n=5) was measured when glucose and alanine were omitted in the luminal perfusate while _K remained unchanged whenJ _Na was more severely reduced (mean change =–1.7±2.1 mV, NS,n=5). In the latter case, this means that the electrochemical driving force for K efflux across the basolateral membrane has not changed while both the active influx through the Na–K pump and the passive efflux in steady state are certainly reduced. If the main pathway for K transport is through the basolateral K conductance, this implies that this conductance must have decreased in the same proportion as that of the reduction in the Na–K pump activity.     

Voltage dependence of current through the Na,K-exchange pump ofRana oocytes

Summary We have studied current (I _Str) through the Na, K pump in amphibian oocytes under conditions designed to minimize parallel undesired currents. Specifically,I _Str was measured as the strophanthidin-sensitive current in the presence of Ba^2–, Cd²⁺ and gluconate (in place of external Cl^–). In addition,I _Str was studied only after the difference currents from successive applications and washouts of strophanthidin (Str) were reproducible. The dose-response relationship to Str in four oocytes displayed a meanK _0.5 of 0.4 m, with 2–5 m producing 84–93% pump' block. From baseline data with 12 Na⁺-preloaded oocytes, voltage clamped in the range [–170, +50 mV] with and without 2–5 m Str, the averageI _Str depended directly onV _m up to a plateau at 0 mV with interpolated zero current at –165 mV. In three oocytes, lowering the external [Na⁺] markedly decreased the voltage sensitivity ofI _p , while producing only a small change in the maximal outwardI _Str. In contrast, decreasing the external [K⁺] from 25 to 2.5mm reducedI _Str at 0 mV without substantially affecting its voltage dependence. At K⁺ concentrations of 1mm, both the absolute value ofI _Str at 0 mV and the slope conductance were reduced. In eight oocytes, the activation of the averagedI _Str by [K⁺] _o over the voltage interval [–30, +30 mV] was well fit by the Hill equation, with K=1.7±0.4mm andnH (the minimum number of K⁺ binding sites) =1.7±0.4. The results unequivocally establish that the cardiotonic-sensitive current ofRana oocytes displays only a positive slope conductance for [K⁺] _o >1mm. There is therefore no need to postulate more than one voltage-sensitive step in the cycling of the Na, K pump under physiologic conditions. The effects of varying external Na⁺ and K⁺ are consistent with results obtained in other tissues and may reflect an ion-well effect.     

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     

Cellular mechanism of HCO 3 − and Cl− transport in insect salt gland

Summary Active HCO ₃ ^t- secretion in the anterior rectal salt gland of the mosquito larva,Aedes dorsalis, is mediated by a 11 Cl^–/HCO ₃ ^– exchanger. The cellular mechanisms of HCO ₃ ^– and Cl^– transport are examined using ion- and voltage-sensitive microelectrodes in conjunction with a microperfused preparation which allowed rapid saline changes. Addition of DIDS or acetazolamide to, or removal of CO₂ and HCO ₃ ^– from, the serosal bath caused large (20 to 50 mV) hyperpolarizations of apical membrane potential (V _a) and had little effect on basolateral potential (V _bl). Changes in luminal Cl^– concentration alteredV _a in a repid, linear manner with a slope of 42.2 mV/decaloga _Cl ^l –. Intracellular Cl^– activity was 23.5mm and was approximately 10mm lower than that predicted for a passive distribution across the apical membrane. Changes in serosal Cl^– concentration had no effect onV _bl, indicating an electrically silent basolateral Cl^– exit step. Intracellular pH in anterior rectal cells was 7.67 and the calculated was 14.4mm. These results show that under control conditions HCO₃ enters the anterior rectal cell by an active mechanism against an electrochemical gradient of 77.1 mV and exits the cell at the apical membrane down a favorable electrochemical gradient of 27.6 mV. A tentative cellular model is proposed in which Cl enters the apical membrane of the anterior rectal cells by passive, electrodiffusive movement through a Cl^–-selective channel, and HCO ₃ ^– exits the cell by an active or passive electrogenic transport mechanism. The electrically silent nature of basolateral Cl^– exit and HCO₃ entry, and the effects of serosal addition of the Cl^–/HCO₃ exchange inhibitor, DIDS, on and transepithelial potential (V _ic) suggest strongly that the basolateral membrane is the site of a direct coupling between Cl^– and HCO ₃ ^– movements.     

Separation of sodium and calcium channels in the surface membrane of molluscan nerve cells

By intracellular dialysis of isolated neurons of the mollusksHelix pomatia andLimnaea stagnalis and by a voltage clamp technique the characteristics of transmembrane ionic currents were studied during controlled changes in the ionic composition of the extracellular and intracellular medium. By replacing the intracellular potassium ions by Tris ions, functional blocking of the outward potassium currents was achieved and the inward current distinguished in a pure form. Replacement of Ringer's solution in the extracellular medium with sodium-free or calcium-free solution enabled the inward current to be separated into two additive components, one carried by sodium ions, the other by calcium ions. Sodium and calcium inward currents were found to have different kinetics and different potential-dependence: _mNa=1±0.5 msec, _mCa=3±1 msec, _hNa=8±2 msec, _hCa=115±10 msec (V_m=0), G_Na=0.5 (V_m=–21±2 mV), G_Ca=0.5 (V_m=–8±2 mV). Both currents remained unchanged by tetrodotoxin, but the calcium current was specifically blocked by cadmium ions (2·10^–3 M), verapamil, and D=600, and also by fluorine ions if injected intracellularly. All these results are regarded as evidence that the soma membrane of the neurons tested possesses separate systems of sodium and calcium ion-conducting channels. Quantitative differences are observed in the relative importance of the systems of sodium and calcium channels in different species of mollusks.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 8, No. 2, pp. 183–191, March–April, 1976.     

Outwardly rectifying chloride channels in lymphocytes

Summary Outwardly rectifying Cl^– channels in cultured human Jurkat T-lymphocytes were activated by excising a patch of membrane using the inside-out (i/o) patch-clamp configuration and holding at depolarized voltages for prolonged periods of time (1–6 min at +80 mV, 20°C). The single-channel current at +80 mV was 4.5 ± 0.3 pA and at –80 mV, it was 1.0 ± 0.4 pA. After activation, the probability of being open (P ₀)for the lymphocyte channel was voltage independent. Activation of the Cl^– channel in lymphocytes was temperature dependent. Nineteen percent of i/o recordings from lymphocytes made at 20°C exhibited Cl^– channel activity. In contrast, 49% of recordings made at 30°C showed channel activity. The number of channels in an active patch was not significantly different at the two temperatures. Channel activation in excised, depolarized patches also occurred 20-fold faster at 30°C than at 20°C. There was no marked change in the single-channel conductance at 30°C. Open-channel conductance was blocked by 200 m indanyloxyacetic acid (IAA) or 1 mm SITS when applied to the intracellular side of the patch. The characteristics of this channel are similar to epithelial outwardly rectifying Cl^– channels thought to be involved in fluid secretion     

Sialic acid and the surface charge associated with hyperpolarization-activated,inward rectifying channels

Summary The whole-cell configuration of the patch-clamp technique was used with cultured pacemaker cells from the rabbit sinoatrial node to test the hypothesis that sialic acid residues (NANA) constitute much of the negative surface charge associated with hyperpolarization-activated, inward rectifying channels. Activation-voltage relationships (between –70 and –140 mV) were determined for hyperpolarization-activated (inward rectifying) current (i _f). Addition of 10mm Ca²⁺ shifted the half-activation potential (V _1/2) from –89.5±0.9 mV to –77.9±2.6 mV (P<0.01), confirming the presence of negative fixed charges on the myocytes after 3 to 5 days in culture. Addition of 20mm dimethonium, an organic divalent cation that screens but does not bind to negative surface charge, shiftedV _1/2 from –86.8±1.4 mV to –75.0±1.7 mV (P<0.001) without affecting the amplitude of the current. In contrast, 10mm Ca²⁺ reduced the amplitude ofi _f significantly. Incubation of cells with a highly purified preparation of neuraminidase (0.1–2.0 U/ml, 1 hr, 37°C), an enzyme that selectively removes NANA from glycoproteins and glycolipids, failed to alterV _1/2 or the amplitude ofi _f significantly. Pretreatment of cells with neuraminidase (1.0 U/ml, 1 hr, 37°C) failed to alter the positive shift ofV _1/2 produced by dimethonium. The results suggest that NANA does not constitute the negative surface charge associated with hyperpolarization-activated, inward rectifying channels.     

Characteristics of L-Type Ca2+ Channels in the Membrane of Mesenteric Artery Myocytes from Genetically Hypertensive Rats

Using the standard voltage-clamp technique in the whole-cell mode, we studied the characteristics of barium currents (I _Ba; Ba²⁺ concentration in the external solution was 5 mM) carried through L-type Ca²⁺ channels in the membrane of myocytes of the resistive mesenteric artery from normotensive and genetically hypertensive rats (NR and GHR, respectively). To perforate the membrane, we used amphotericin B. The arbitrary density of I _Ba through the plasma membrane of GHR myocytes significantly exceeded this parameter in the NR group. For both animal groups, activation curves plotted as the dependence of the membrane conductance (G _Ba) on the membrane potential were not significantly different: the membrane potential for half activation (V _0.5) of I _Ba in the NR myocytes was equal to 1.0 ± 0.3 mV with slope factor k = 6.3 ± 0.4 mV, whereas in the GHR myocytes V _0.5 = -1.6 ± 0.2 mV and k = 6.2 ± 0.5 mV. The stationary inactivation curves for I _Ba differed significantly: in the NR myocytes, V _0.5 = -24.2 ± 0.4 mV and k = 8.3 ± 0.2 mV, whereas in the GHR myocytes such parameters were, respectively, -21.4 ± 0.4 and 8.7 ± 0.3 mV. The pattern of intersection of stationary activation and stationary inactivation curves for I _Ba was indicative of the existence of a window current, i.e., the non-inactivating component of I _Ba within the -40 to ±20 mV range; the phenomenon was clearly pronounced in the GHR myocytes. Differences in the arbitrary density of integral I _Ba and window current were observed. These differences can cause an increased tone of the blood vessels in hypertensive animals.     

Cellular and transepithelial responses of goldfish intestinal epithelium to chloride substitutions

Summary In goldfish intestine chloride was substituted by large inorganic anions (gluconate or glucuronate) either mucosally, serosally or bilaterally. Changes in intracellular activities of chloride (a _i Cl^–), sodium (a _i Na⁺) and potassium (a _i K⁺), pH_i, relative volume, membrane and transepithelial potentials, transepithelial resistance and voltage divider ratio were measured. Control values were:a _i Cl^–=35 meq/liter, a _i Na⁺=11 meq/liter and a _i K⁺=95 meq/liter. During bilateral substitution the latter two did not change while a _i Cl^– dropped to virtually zero.Mucosal membrane potentials (_ms) were: control,-53 mV; serosal substitution,-51 mV; bilateral substitution,-66 mV; while during mucosal substitution a transient depolarization occurred and the final steady state _ms was-66 mV.During control and bilateral substitution the transepithelial potentials (_ms) did not differ from zero. During unilateral substitutions _ms was small, in the order of magnitude of the errors in the liquid junction potentials near the measuring salt bridges.During bilateral substitution pH _i increased 0.4 pH units. Cellular volume decreased during mucosal substitution to 88% in 40 min; after serosal substitution it transiently increased, but the new steady-state value was not significantly above its control value.Three minutes after mucosal substitution ana _i Cl^– of approx. 10 meq/liter was measured.Chemical concentrations of Na, K and Cl were determined under control conditions and bilateral substitution. Cl concentrations were also measured as a function of time after unilateral substitutions.The data indicate an electrically silent chloride influx mechanism in the brush border membrane and an electrodiffusional chloride efflux in the basolateral membrane. A substantial bicarbonate permeability is present in the basolateral membrane. The results are in agreement with the observed changes in membrane resistances, volume changes and pH changes.