Effects of acetylcholine, serotonin and noradrenalin on ion transport in the middle and posterior part of Anguilla anguilla intestine

The effects of acetylcholine analogues, serotonin and catecholamines on ion transport were studied in both the middle and the posterior intestine of Anguilla anguilla, mounted in an Ussing chamber, with the aim of understanding whether these regulators affect different mechanisms in the different tracts. In the middle intestine, acetylcholine analogues and serotonin decreased the serosa negative transepithelial potential and short-circuit current without altering the transepithelial resistance; catecholamines reversed the inhibitory effects of both regulators. Similar opposite effects were produced by both the acetylcholine analogues and noradrenalin in the posterior intestine. However, the lowering of the short-circuit current elicited by serotonin was paralleled by the decrease of the transepithelial resistance, whilst noradrenalin had the opposite effects on both parameters. These observations, together with the results of experiments performed by measuring the dilution potential in the control condition and in the presence of either serotonin or serotonin plus noradrenalin, led us to hypothesize that serotonin increases the anion conductance of the paracellular pathway while noradrenalin decreases it. In both the middle and posterior intestine, these regulators probably affect transcellular transport mechanisms by acting on the Na-K-Cl transporter; both acetycholine and serotonin decrease its activity while noradrenalin increases it. Accepted: 22 May 1999     

Ion transport across leech integument

The dorsal skin of the leech Hirudo medicinalis was used for electrophysiological measurements performed in Ussing chambers. The leech skin is a tight epithelium (transepithelial resistance = 10.5±0.5 k· cm^-2) with an initial short-circuit current of 29.0±2.9 A·cm^-2. Removal of Na⁺ from the apical bath medium reduced short-circuit current about 55%. Ouabain (50mol·l^-1) added to the basolateral solution, depressed the short-circuit current completely. The Na⁺ current saturated at a concentration of 90 mmol Na⁺·l^-1 in the apical solution (K _M=11.2±1.8 mmol·l^-1). Amiloride (100 mol·l^-1) on the apical side inhibited ca. 40% of the Na⁺ current and indicated the presence of Na⁺ channels. The dependence of Na⁺ current on the amiloride concentration followed Michaclis-Menten kinetics (K _i=2.9±0.4 mol·l^-1). The amiloride analogue benzamil had a higher affinity to the Na⁺ channel (K _i=0.7±0.2 mol·l^-1). Thus, Na⁺ channels in leech integument are less sensitive to amiloride than channels known from vertebrate epithelia. With 20 mmol Na⁺·l^-1 in the mucosal solution the tissue showed an optimum amiloride-inhibitable current, and the amiloride-sensitive current under this condition was 86.8±2.3% of total short-circuit current. Higher Na⁺ concentrations lead to a decrease in amiloride-blockade short-circuit current. Sitmulation of the tissue with cyclic adenosine monophosphate (100 mol·l^-1) and isobutylmethylxanthine (1 mmol·l^-1) nearly doubled short-circuit current and increased amiloride-sensitive Na⁺ currents by 50%. By current fluctuation analysis we estimated single Na⁺ channel current (2.7±0.9 pA) and Na⁺ channel density (3.6±0.6 channels·m^-2) under control conditions. After cyclic adenosine monophosphate stimulation Na⁺ channel density increased to 5.4±1.1 channels·m^-2, whereas single Na⁺ channel current showed no significant change (1.9±0.2 pA). These data present a detailed investigation of an invertebrate epithelial Na⁺ channel, and show the similarities and differences to vertebrate Na⁺ channels. Whereas the channel properties are different from the classical vertebrate Na⁺ channel, the regulation by cyclic adenosine monophosphate seems similar. Stimulation of Na⁺ uptake by cyclic adenosine monophosphate is mediated by an increasing number of Na⁺ channels.Abbreviations slope of the background noise component - ADH antidiuretic hormone - cAMP cyclic adenosine monophosphate - f frequency - f _c coner frequency of the Lorentzian noise component - Hepes N-hydroxyethylpiperazine-N-ethanesulphonic acid - BMX isobutyl-methylxanthine - i _Na single Na⁺ channel current - I _Na max, maximal inhibitable Na⁺ current - I _SC short circuit current - K _i half maximal blocker concentration - K _M Michaelis constandard error of the mean - S _(f) power density of the Lorentzian noise component - S ₀ plateau value of the Lorentzian noise component - TMA tetramethylammonium - Trizma TRIS-hydroxymethyl-amino-methane - V _max maximal reaction velocity - V _T transepithelial potential - K half maximal blocker concentration     

Further evidence for the regulation of the tight junction ion selectivity by cAMP in goldfish intestinal mucosa

Summary It has been reported that cAMP controls the transepithelial Cl^– conductance in fish intestine (Bakker, R., Groot, J.A., 1984,Am. J. Physiol. 246:G213–G217; Krasny, E.J., Madara, J.L., DiBona, D.L., Frizzell, R.A., 1983,Fed. Proc. 42:1100). In both studies, the cAMP effect was interpreted as an increase in tight junction Cl^– conductance, because cAMP did not change the membrane potential or membrane resistance ratio. However, the activation of a Cl^– conductance in the membranes of a subset of the epithelial cells might be difficult to discern from an increase in tight junction Cl^– conductance. Here we report experiments that were designed to distinguish a tight junction Cl^– conductance from a membrane Cl^– conductance in a subpopulation of the epithelial cells. The effect of hypotonicity on the cAMP-induced increase in transepithelial conductance showed that cAMP-induced conductance is located in series with the lateral intercellular spaces. Transepithelial serosa to mucosa direct current caused an increase in resistance due to so-called transport number effects. Forskolin abolished the transport number effects, indicating that cAMP increases the Cl^– conductance of the tight junctions. Increasing cAMP did not change mannitol fluxes, whereas Cl^– fluxes more than doubled. Changes in dilution potential and transepithelial resistance demonstrated that the cAMP-induced conductance is specific for Cl^– and Br^– as opposed to I^–, NO ₃ ^– , SO ₄ ^2– and gluconate^–. In contranst, cytochalasin D also decreased the transepithelial resistance and dilution potential in Nagluconate Ringer's. This demonstrates that cAMP acts on the tight junctions in a more specific manner than cytochalasin D.     

Aldosterone regulates paracellular pathway resistance in rabbit distal colon

Summary Regulation of the paracellular pathway in rabbit distal colon by the hormone aldosterone was investigated in vitro in Ussing chambers by means of transepithelial and microelectrode techniques. To evaluate the cellular and paracellular resistances an equivalent circuit analysis was used. For the analysis the apical membrane resistance was altered using the antibiotic nystatin. Under control conditions two groups of epithelia were found, each clearly dependent on the light: dark regime. Low-transporting epithelia (LT) were observed in the morning and high-transporting epithelia (HT) in the afternoon. Na⁺ transport was about 3-fold higher in HT than in LT epithelia. Incubating epithelia of both groups with 0.1 mol·1^-1 aldosterone on the serosal side nearly doubled in LT epithelia the short circuit current and transepithelial voltage but the transepithelial resistance was not influenced. Maximal values were reached after 4–5 h of aldosterone treatment. In HT epithelia due to the effect of aldosterone all three transepithelial parameters remained constant over time. Evaluation of the paracellular resistance revealed a significant increase after aldosterone stimulation in both epithelial groups. This increase suggests that tight junctions might have been regulated by aldosterone. The hormonal effect on electrolyte transport was also dependent on the physiological state of the rabbit colon. Since net Na⁺ absorption in distal colon is, in addition to transcellular absorption capacity, also dependent on the permeability of the paracellular pathway, the regulation of tight junctions by aldosterone may be a potent mechanism for improving Na⁺ absorption during hormone-stimulated ion transport.Abbreviations V _t transepithelial potential difference (mV) - R _t transepithelial resistance (·cm²) - G _t transepithelial conductance (mS·cm^-2) - I_sc calculated short circuit current (A·cm^-2) - V _a apical membrane potential difference (mV) - V _bl basolateral membrane potential difference (mV) - voltage divider ratio - R _a apical membrane resistance (·cm²) - R _bl basolateral membrane resistance (·cm²) - R _c cellular resistance ( of apical and basolateral resistance) (·cm²) - R _p resistance of the paracellular pathway (·cm²) - G _a apical membrane conductance (mS·cm^-2) - G _bl basolateral membrane conductance (mS·cm^-2) - G _p paracellular conductance (mS·cm^-2) - G _t transepithelial conductance (mS·cm^-2) - HT _contr high transporting control epithelia - LT _contr low transporting control epithelia - HT _aldo aldosterone incubated high transporting epithelia - LT _aldo aldosterone incubated low transporting epithelia     

Ionic conductance pathways in the mouse medullary thick ascending limb of Henle. The paracellular pathway and electrogenic Cl- absorption

Net Cl- absorption in the mouse medullary thick ascending limb of Henle (mTALH) involves a furosemide-sensitive Na+:K+:2 Cl- apical membrane symport mechanism for salt entry into cells, which occurs in parallel with a Ba++-sensitive apical K+ conductance. The present studies, using the in vitro microperfused mouse mTALH, assessed the concentration dependence of blockade of this apical membrane K+-conductive pathway by Ba++ to provide estimates of the magnitudes of the transcellular (Gc) and paracellular (Gs) electrical conductances (millisiemens per square centimeter). These studies also evaluated the effects of luminal hypertonicity produced by urea on the paracellular electrical conductance, the electrical Na+/Cl- permselectivity ratio, and the morphology of in vitro mTALH segments exposed to peritubular antidiuretic hormone (ADH). Increasing luminal Ba++ concentrations, in the absence of luminal K+, produced a progressive reduction in the transcellular conductance that was maximal at 20 mM Ba++. The Ba++-sensitive transcellular conductance in the presence of ADH was 61.8 +/- 1.7 mS/cm2, or approximately 65% of the total transepithelial conductance. In phenomenological terms, the luminal Ba++-dependent blockade of the transcellular conductance exhibited negative cooperativity. The transepithelial osmotic gradient produced by luminal urea produced blebs on apical surfaces, a striking increase in shunt conductance, and a decrease in the shunt Na+/Cl- permselectivity (PNa/PCl), which approached that of free solution. The transepithelial conductance obtained with luminal 800 mM urea, 20 mM Ba++, and 0 K+ was 950 +/- 150 mS/cm2 and provided an estimate of the maximal diffusion resistance of intercellular spaces, exclusive of junctional complexes. The calculated range for junctional dilution voltages owing to interspace salt accumulation during ADH-dependent net NaCl absorption was 0.7-1.1 mV. Since the Ve accompanying ADH-dependent net NaCl absorption is 10 mV, lumen positive, virtually all of the spontaneous transepithelial voltage in the mouse mTALH is due to transcellular transport processes. Finally, we developed a series of expressions in which the ratio of net Cl- absorption to paracellular Na+ absorption could be expressed in terms of a series of electrical variables. Specifically, an analysis of paired measurement of PNa/PCl and Gs was in agreement with an electroneutral Na+:K+:2 Cl- apical entry step. Thus, for net NaCl absorption, approximately 50% of Na+ was absorbed via a paracellular route.     

Effect of oxytocin on transepithelial transport of water and Na+ in distinct ventral regions of frog skin (Rana catesbeiana)

Thoracic, abdominal, and pelvic fragments of ventral skin of Rana catesbeiana were analysed regarding the effect of oxytocin on: (1) transepithelial water transport; (2) short-circuit current; (3) skin conductance and electrical potential difference; (4) Na⁺ conductance and electrical potential difference; (4) Na⁺ conductance, the electromotive force of Na⁺ transport mechanism, and shunt conductance; (5) short-circuit current responses to fast Na⁺ by K⁺ replacement in the outer compartment, and (6) epithelial microstructure. Unstimulated water and Na⁺ permeabilities were low along the ventral skin. Hydrosmotic and natriferic responses to oxytocin increased from thorax to pelvis. Unstimulated Na⁺ conductance was greater in pelvis than in abdomen, the other electrical parameters being essentially similar in both skin fragments. Contribution of shunt conductance to total skin conductance was higher in abdominal than in pelvic skin. Oxytocin-induced increases of total skin conductance, Na⁺ conductance, and shunt conductance in pelvis were significantly larger than in abdomen. An oscillatory behaviour of the short-circuit current was observed only in oxytocin-treated pelvic skins. Decrease of epithelial thickness and increase of mitochondria-rich cell number were observed from thorax to pelvis. Oxytocin-induced increases of interspaces were more conspicuous in pelvis and abdomen than in thorax.Abbreviations E _Na electromotive force of sodium transport mechansim - G _KCI skin conductance with external KCI Ringer - G _Na sodium conductance (series conductance) - G _shunt shunt pathway conductance - G _total total skin conductance - J _v water flux (in units of volume per area per time) - MRC mitochondria-rich cells - PD potential difference across skin - R _shunt resistance of the shunt pathway - SCC short-circuit current     

Ca2+ transport by opercular epithelium of the fresh water adapted euryhaline teleost,Fundulus heteroclitus

We examined transepithelial transport of Ca²⁺ across the isolated opercular epithelium of the euryhaline killifish adapted to fresh water. The opercular epithelium, mounted in vitro with saline on the serosal side and fresh water (0.1 mmol·l^–1 Ca²⁺) bathing the mucosal side, actively transported Ca²⁺ in the uptake direction; net flux averaged 20–30 nmol·cm^–2·h^–1. The rate of Ca²⁺ uptake varied linearly with the density of mitochondria-rich cells in the preparations. Ca²⁺ uptake was saturable, apparent K _1/2 of 0.348 mmol·l^–1, indicative of a multistep transcellular pathway. Ca²⁺ uptake was inhibited partially by apically added 0.1 mmol·l^–1 La³⁺ and 1.0 mmol·l^–1 Mg²⁺. Addition of dibutyryl-cyclic adenosine monophosphate (0.5 mmol·l^–1)+0.1 mmol·l^–1 3-isobutyl-l-methylxanthine inhibited Ca²⁺ uptake by 54%, but epinephrine, clonidine and isoproterenol were without effect. Agents that increase intracellular Ca²⁺, thapsigargin (1.0 mol·l^–1, serosal side), ionomycin (1.0 mol·l^–1, serosal side) and the calmodulin blocker trifluoperazine (50 mol·l^–1, mucosal side) all partially inhibited Ca²⁺ uptake. In contrast, apically added ionomycin increased mucosal to serosal unidirectional Ca²⁺ flux, indicating Ca²⁺ entry across the apical membrane is rate limiting in the transport. Verapamil (10–100 mol·l^–1, mucosal side), a Ca²⁺ channel blocker, had no effect. Results are consistent with a model of Ca²⁺ uptake by mitochondria rich cells that involves passive Ca²⁺ entry across the apical membrane via verapamil-insensitive Ca²⁺ channels, intracellular complexing of Ca²⁺ by calmodulin and basolateral exit via an active transport process. Increases in intracellular Ca²⁺ invoke a downregulation of transcellular Ca²⁺ transport, implicating Ca²⁺ as a homeostatic mediator of its own transport.Abbreviations DASPEI 2-(4-dimethylaminostyryl)-N-ethylpyridinium iodide - db-cAMP dibutyryl-cyclic adenosine monophosphate - FW fresh water - G _t transepithelial conductance - I _sc short-circuit current - IBMX 3-isobutyl-1-methylxanthine - SW sea water - TFP trifluoperazine - V _t transepithelial potential     

Epithelial Barrier Modulation by a Channel Forming Peptide

NC-1059 is a synthetic channel-forming peptide that provides for ion transport across, and transiently reduces the barrier integrity of, cultured epithelial monolayers derived from canine kidney (MDCK cells). Experiments were conducted to determine whether epithelial cells derived from other sources were similarly affected. Epithelial cells derived from human intestine (T-84), airway (Calu-3), porcine intestine (IPEC-J2) and reproductive duct (PVD9902) were grown on permeable supports. Basal short circuit current (I _sc) was <3 μA cm⁻² for T-84, IPEC-J2 and PVD9902 cell monolayers and <8 μA cm⁻² for Calu-3 cells. Apical NC-1059 exposure caused, in all cell types, an increase in I _sc to >15 μA cm⁻², indicative of net anion secretion or cation absorption, which was followed by an increase in transepithelial conductance (in mS cm⁻²: T-84, 1.6 to 62; PVD9902, 0.2 to 51; IPEC-J2, 0.3 to 26; Calu-3, 2.3 to 13). These results are consistent with the peptide affecting transcellular ion movement, with a likely effect also on the paracellular route. NC-1059 exposure increased dextran permeation when compared to basal permeation, which documents an effect on the paracellular pathway. In order to evaluate membrane ion channels, experiments were conducted to study the dose dependence and stability of the NC-1059-induced membrane conductance in Xenopus laevis oocytes. NC-1059 induced a dose-dependent increase in oocyte membrane conductance that remained stable for greater than 2 h. The results demonstrate that NC-1059 increases transcellular conductance and paracellular permeation in a wide range of epithelia. These effects might be exploited to promote drug delivery across barrier epithelia.     

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.     

Effects of Ag+ on ion transport by the corneal epithelium of the rabbit

Summary Exposure of thein vitro rabbit corneal epithelium to Ag⁺ by the addition of AgNO₃ (10^–7–10^–5)m) to the apical surface or by the use of imperfectly chlorided Ag/AgCl half-cells in Ussing-style membrane chambers, greatly increases short-circuit current and transepithelial potential. The early phase (the first 30 min) of the short-circuit current stimulation by Ag⁺ is linearly dependent on tear-side sodium concentration, is largely a result of a tenfold increase in net Na⁺ uptake and is incompletely inhibited by ouabain, suggesting that Ag⁺ increases cation (primarily Na⁺) conductance of the apical membrane. This mechanism for the Ag⁺ effect is supported by microelectrode experiments, wherein Ag⁺ depolarizes specifically the apical barrier potential and increases apical barrier conductance. A later phase in the effect (0.5–3 hr) is characterized by a gradual increase in³⁶Cl^– and¹⁴C-mannitol unidirectional fluxes, by a decline in epithelial resting potential and short-circuit current, by complete ouabain inhibition and by fit to saturation kinetics with respect to Na⁺ concentration in the bathing media. This pahse of the effect apparently reflects a nonselective opening of the paracellular pathway in the epithelium and is rate-limited by Na⁺ pump activity at the basolateral membrane. Both phases are associated with swelling of the corneal stroma and may be rapidly reversed using thiol agents (reduced glutathione and dithiothreitol). The results suggest that Ag⁺ may be useful in the study of cation transport by epithelia and the work provides basic physiological information that is pertinent to the prophylactic use of AgNO₃ in clinical ophthalmology.     

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.     

Ion transport across the isolated intestinal mucosa of the winter flounder,Pseudopleuronectes americanus: II. Effects of cyclic AMP

Summary Addition of cyclic AMP and theophylline to the intestinal mucosa of the winter flounder,Pseudopleuronectes americanus decreased short-circuit current and net Na and Cl absorption and increased total conductance and the serosa-to-mucosa unidirectional Cl flux (J _sm ^Cl ). The last two changes were independent of the original rate of NaCl absorption and persisted even when net absorption of Na and Cl had been abolished by ouabain. Voltageclamp experiments revealed that the increment inJ _sm ^Emphasis>/Cl produced by cyclic AMP is PD-insensitive and therefore not due to an increase in the Cl conductance of the paracellular shunt. Cyclic AMP appears, therefore, both to inhibit net NaCl absorption and to increase the Cl permeability and total conductance of the intestinal epithelial cells; its failure to stimulate secretion (in contrast to its action on mammalian intestine) may be related to the absence of crypts in flounder intestinal epithelium.     

Voltage dependence of cellular current and conductances in frog skin

Summary Knowledge of the voltage dependencies of apical and basolateral conductances is important in determining the factors that regulate transcellular transport. To gain this knowledge it is necessary to distinguish between cellular and paracellular currents and conductances. This is generally done by sequentially measuring transepithelial current/voltage (I _t /V _t ) and conductance/voltage (g _t /V _t ) relationships before and after the abolition of cellular sodium transport with amiloride. Often, however, there are variable time-dependent and voltage-dependent responses to voltage perturbation both in the absence and presence of amiloride, pointing to effects on the paracellular pathway. We have here investigated these phenomena systematically and found that the difficulties were significantly lessened by the use of an intermittent technique, measuringI _t andg _t before and after brief (<10 sec) exposure to amiloride at each setting ofV _t .I/V relationships were characterized by these means in frog skins (Rana pipiens, Northern variety, andRana temporaria). Cellular current,I _c , decreased with hyperpolarization (larger serosa positive clamps) ofV _t . DerivedI _c /V _t relationships betweenV _t =0 and 175 mV (serosa positive) were slightly concave upwards. Because values of cell conductance,g _c , remained finite, it was possible to demonstrate reversal ofI _c . Values of the reversal potentialV' averaged 156±14 (sd,n=18) mV. Simultaneous microelectrode measurements permitted also the calculation of apical and basolateral conductances,g _a andg _b . The apical conductance decreased monotonically with increasing positivity ofV _t (andV _a ). In contrast, in the range in which the basolateral conductance could be evaluated adequately (V _t <125 mV),g _b increased with more positive values ofV _t (andV _b ). That is, there was an inverse relation betweeng _b and cellular current at the quasi-steady state, 10–30 sec after the transepithelial voltage step.     

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     

Intracellular sodium activity and sodium transport inNecturus gallbladder epithelium

Summary Ion-sensitive glass microelectrodes, conventional microelectrodes and isotope flux measurements were employed inNecturus gallbladder epithelium to study intracellular sodium activity, [Na] _i , electrical parameters of epithelial cells, and properties of active sodium transport. Mean control values were: [Na] _i : 9.2 to 12.1mm; transepithelial potential difference, _ms : –1.5 mV (lumen negative); basolateral cell membrane potential, _es : –62 mV (cell interior negative); sodium conductance of the luminal cell membrane,g _Na: 12 mho cm^–2; active transcellular sodium flux, 88 to 101 pmol cm^–2 sec^–1 (estimated as instantaneous short-circuit current). Replacement of luminal Na by K led to a decrease of the intracellular sodium activity at a rate commensurate to the rate of active sodium extrusion across the basolateral cell membrane. Mucosal application of amphotericin B resulted in an increase of the luminal membrane conductance, a rise of intracellular sodium activity, and an increase of short-circuit current and unidirectional mucosa to serosa sodium flux. Conclusions: (i) sodium transport across the basolateral membrane can proceed against a steeper chemical potential difference at a higher rate than encountered under control conditions; (ii) the luminal Na-conductance is too low to accommodate sodium influx at the rate of active basolateral sodium extrusion, suggesting involvement of an electrically silent luminal transport mechanism; (iii) sodium entry across the luminal membrane is the rate-limiting step of transcellular sodium transport and active sodium extrusion across the basolateral cell membrane is not saturated under control conditions.     

Cell K activity in frog skin in the presence and absence of cell current

Summary Cell K activity,a _k, was measured in the short-circuited frog skin by simultaneous cell punctures from the apical surface with open-tip and K-selective microelectrodes. Strict criteria for acceptance of impalements included constancy of the open-tip microelectrode resistance, agreement within 3% of the fractional apical voltage measured with open-tip and K-selective microelectrodes, and constancy of the differential voltage recorded between the open-tip and the K microelectrodes 30–60 sec after application of amiloride or substitution of apical Na. Skins were bathed on the serosal surface with NaCl Ringer and, to reduce paracellular Cl conductance and effects of amiloride on paracellular conductance, with NaNO₃ Ringer on the apical surface.Under control conditionsa _k ^r was nearly constant among skins (mean±SD=92±8mM, 14 skins) in spite of a wide range of cellular currents (5 to 70 A/cm²). Cell current (and transcellular Na transport) was inhibited by either apical addition of amiloride or substitution of Na by other cations. Although in some experiments the expected small increase ina _k ^r after inhibition of cell current was observed, on the average the change was not significant (98±11mM after amiloride, 101±12mM after Na substitution), even 30 min after the inhibition of cell current. The membrane potential, which in the control state ranged from –42 to –77 mV, hyperpolarized after inhibition of cell current, initially to –109±5mV, then depolarizing to a stable value (–88±5mV) after 15–25 min. At this time K was above equilibrium (E _k=98±2mV), indicating that the active pump mechanism is still operating after inhibition of transcellular Na transport.The measurement ofa _k ^r permitted the calculation of the passive K current and pump current under control conditions. assuming a constant current source with almost all of the basolateral conductance attributable to K. We found a significant correlation between pump current and cell current with a slope of 0.31, indicating that about one-third of the cell current is carried by the pump, i.e., a pump stoichiometry of 3Na/2K.     

Passive and active transport properties of a gill model, the cultured branchial epithelium of the freshwater rainbow trout (Oncorhynchus mykiss)

Branchial epithelia of freshwater rainbow trout were cultured on permeable supports, polyethylene terephthalate membranes ("filter inserts"), starting from dispersed gill epithelial cells in primary culture. Leibowitz L-15 media plus foetal bovine serum and glutamine, with an ionic composition similar to trout extracellular fluid, was used. After 6 days of growth on the filter insert with L-15 present on both apical and basolateral surfaces, the cultured preparations exhibited stable transepithelial resistances (generally 1000-5000 ohms cm2) typical of an electrically tight epithelium. Under these symmetrical conditions, transepithelial potential was zero, and unidirectional fluxes of Na+ and Cl- across the epithelium and permeability to the paracellular marker polyethylene glycol-4000 (PEG) were equal in both directions. Na+ and Cl- fluxes were similar to one another and linearly related to conductance (inversely related to resistance) in a manner indicative of fully conductive passive transport. Upon exposure to apical fresh water, transepithelial resistance increased greatly and a basolateral-negative transepithelial potential developed. At the same time, however, PEG permeability and unidirectional effluxes of Na+ and Cl- increased. Thus, total conductance fell, and ionic fluxes and paracellular permeability per unit conductance all increased greatly, consistent with a scenario whereby transcellular conductance decreases but paracellular permeability increases upon dilution of the apical medium. In apical fresh water, there was a net loss of ions from the basolateral to apical surfaces as effluxes greatly exceeded influxes. However, application of the Ussing flux ratio criterion, in two separate series involving different methods for measuring unidirectional fluxes, revealed active influx of Cl- against the electrochemical gradient but passive movement of Na+. The finding is surprising because the cultured epithelium appears to consist entirely of pavement-type cells.     

Na and Cl transport across the isolated turtle colon: Parallel pathways for transmural ion movement

Summary Transmural fluxes of³H-mannitol and²²Na or³⁶Cl were measured simultaneously in portions of isolated turtle colon stripped of serosal musculature. The relationships between mannitol flux and the flux of Na or Cl are characteristic of simple diffusion and suggest that transmural mannitol flow is largely confined to a paracellular pathway where Na, Cl and mannitol move much as in free solution. The contribution of edge damage to the transmural mannitol flow appears to be minimal. Mucosal hyperosmolarity causes blisters in epithelial tight junctions and increases the diffusional permeability to Na and mannitol, suggesting that the rate-limiting barrier in the shunt path is the tight junction. If the total mucosa to serosa flux of Na is corrected for the portion traversing the shunt pathway it is apparent that changes in the short-circuit current are completely accounted for by the mucosa to serosal movement of Na through a cellular path. In addition, the serosa to mucosa flux of Na appears to be restricted to the shunt. These observations suggest that there is no appreciable backflux of Na through the active, cellular path. In the presence of 10^–4 m amiloride the short-circuit current is markedly reduced and the mucosa to serosa Na flux is restricted to the shunt, so that the net Na flux is abolished. The small amiloride-insensitive short-circuit current is consistent with HCO₃ secretion. Mucosa to serosa and serosa to mucosa fluxes of Cl appear to be largely restricted to the paracellular shunt path and there is no evidence for any net flow of Cl under short-circuit conditions. The total tissue conductance can be described as the sum of three components: a shunt conductance which is linearly related to the transmural mannitol flow, an active conductance which is linearly related to the short-circuit current and a small residual conductance. The shunt conductance is attributable to the diffusive movements of Na and Cl through the paracellular path. Variations in the active Na transport from tissue to tissue are largely attributable to variations in the apparent conductance of the active Na transport path. The driving force for active Na transport can be described as an apparent emf of approximately 130 mV. These results suggest that transmural mannitol flux provides a quantitative estimate of the ion permeability and electrical conductance of a paracellular shunt path across the isolated turtle colon and thereby facilitates the study of the transport characteristics and electrical properties of cellular paths for transepithelial solute movement.     

Chloride conductance and mitochondria-rich cell density in isolated skin of Rana catesbeiana acclimated to various environments

The Cl⁻ conductance in isolated skin of frogs (Rana catesbeiana) acclimated to 30 mM solutions of NaCl, Na₂SO₄, MgCl₂ and distilled water (DW) was studied. Transepithelial potential difference (PD_trans), short-circuit current (I_SC) and total conductance (G_t) were measured under conditions such that there was Cl⁻ flux in the presence and absence of Na⁺ transport. The Cl⁻ content of the mucosal solution was acutely replaced with SO₄²⁻ or gluconate to evaluate the effect of removal of Cl⁻ conductance on electrophysiological parameters. Mitochondria-rich cell density (D_MRC) was also measured. Skins from frogs acclimated to NaCl and Na₂SO₄ showed the lowest and the highest D_MRC, respectively, but no difference could be found between the skins from frogs acclimated to DW and MgCl₂ indicating that D_MRC is not unconditionally dependent on environmental Cl⁻ in this species. Frogs acclimated to NaCl showed marked differences when compared to the other groups: the highest G_t, probably represented by a higher paracellular conductance; the lowest transepithelial electrical potential difference which remained invariant after replacement of mucosal Cl⁻ with SO₄²⁻ or replacement of mucosal Cl⁻ with gluconate and an inwardly oriented positive current in the absence of bilateral Na⁺.     

Alanine-stimulated exocytosis in<Emphasis Type="Italic"> Aplysia</Emphasis> enterocytes: effect of Na<Superscript>+</Superscript> transport and requirement for actin filaments

We used the Aplysia californica intestinal epithelium to investigate the effect of alanine-stimulated Na⁺ absorption on apical membrane exocytosis and whether stimulated exocytosis requires intact actin filaments. The fluid-phase marker fluorescein dextran was used to determine rates of apical membrane exocytosis. L-alanine significantly increased apical exocytosis by ~30% compared to controls, and there is a modest, positive correlation between alanine-stimulated exocytosis and short-circuit current (I _SC). Thus, apical exocytosis is modulated to some extent by the magnitude of Na⁺ and alanine entry across the apical membrane. Apical exocytosis is also responsive to virtually any increase in Na⁺ and alanine entry because increments in alanine-stimulated I _SC as small as 1 A/cm² stimulated exocytosis. We used D-alanine to determine which parameter (sensitivity to transport vs. magnitude of transport) was most important in activation of apical exocytosis. D-alanine-stimulated I _SC was one-sixth that of L-alanine, but stimulated exocytosis was only 29% less than that of L-alanine. Therefore, the apical exocytic system is more responsive to small increases in transport than to the magnitude of transport. Latrunculin A (Lat-A) disrupts the actin cytoskeleton and reduced constitutive apical exocytosis by ~65% and completely abolished alanine-stimulated exocytosis. Hence, constitutive exocytosis and alanine-stimulated exocytosis require actin filaments for recruitment of vesicles to the apical membrane. During nutrient absorption, actin filament-regulated apical exocytosis may represent a negative feedback system that modulates apical membrane tension.Abbreviations alaASW ASW containing alanine - C _m membrane capacitance - ASW artificial seawater - ETOH ethanol - fCa apical membrane fractional capacitance - FD fluorescein dextran - G _K plasma membrane potassium conductance - G _K,a apical membrane potassium conductance - HRP horseradish peroxidase - I _SC short-circuit current - J _Na transcellular net sodium flux - K _D dissociation constant - Lat-A latrunculin A - manASW ASW containing mannitol - PT proximal tubule - RFU relative fluorescence units - V _a apical membrane potential Communicated by L.C.-H. Wang