Paths of ion transport across canine fetal tracheal epithelium

Fluid secretion by the fetal sheep lung is thought to be driven by secretion of Cl- by the pulmonary epithelium. We previously demonstrated Cl- secretion by tracheal epithelium excised from fetal dogs and sheep. In this study we characterized the ion transport pathways across fetal canine tracheal epithelium. The transport of Na+ and Cl- across trachea excised from fetal dogs was evaluated from transepithelial electrical properties and isotope fluxes. Under basal conditions the tissues were characterized by a lumen-negative potential difference (PD) of 11 mV and conductance of 5.2 mS/cm2. The short-circuit current (Isc) was 43 microA/cm2 (1.6 mueq.cm-2.h-1). Basal Na+ flows were symmetrical, but net Na+ absorption (1.1 mueq.cm-2.h-1) could be induced by exposure of the luminal surface to amphotericin B (10(-6) M). Bilateral replacement of Na+ reduced Isc by 85%. Replacement of submucosal Na+ or exposure to submucosal furosemide (10(-4) M) reduced net Cl- secretion by 60-70%. Luminal exposure to indomethacin (10(-6) M) induced a 50% decrease in Isc, whereas isoproterenol (10(-6) M) increased Isc by 120%. The properties of the Cl- secretory pathway across fetal dog trachea are consistent with the model proposed for Cl- secretion across adult dog trachea and other Cl- -secreting tissues (e.g., bullfrog cornea and shark rectal gland). The absence of basal Na+ absorption by fetal dog trachea probably reflects limited apical membrane Na+ permeability.     

Bioelectric properties and ion flow across excised human bronchi

Bioelectric properties and ion transport of excised human segmental/subsegmental bronchi were measured in specimens from 40 patients. Transepithelial electric potential difference (PD), short-circuit current (Isc), and conductance (G), averaged 5.8 mV (lumen negative), 51 microA X cm-2, and 9 mS X cm-2, respectively. Na+ was absorbed from lumen to interstitium under open- and short-circuit conditions. Cl- flows were symmetrical under short-circuit conditions. Isc was abolished by 10(-4) M ouabain. Amiloride inhibited Isc (the concentration necessary to achieve 50% of the maximal effect = 7 X 10(-7) M) and abolished net Na+ transport. PD and Isc were not reduced to zero by amiloride because a net Cl- secretion was induced that reflected a reduction in Cl- flow in the absorptive direction (Jm----sCl-). Acetylcholine (10(-4) M) induced an electrically silent, matched flow of Na+ (1.7 mueq X cm-1 X h-1) and Cl- (1.9 mueq X cm-12 X h-1) toward the lumen. This response was blocked by atropine. Phenylephrine (10(-5) M) did not affect bioelectric properties or unidirectional ion flows, whereas isoproterenol (10(-5) M) induced a small increase in Isc (10%) without changing net ion flows significantly. We conclude that 1) Na+ absorption is the major active ion transport across excised human bronchi, 2) Na+ absorption is both amiloride and ouabain sensitive, 3) Cl- secretion can be induced by inhibition of the entry of luminal Na+ into the epithelia, and 4) cholinergic more than adrenergic agents modulate basal ion flow, probably by affecting gland output.     

Effects of raised osmolarity on canine tracheal epithelial ion transport function

Evaporation of water from upper airway surfaces increases surface liquid osmolarity. We studied the effects of raised osmolarity of the solution bathing the luminal surface of excised canine tracheal epithelium. Osmolarity was increased by adding NaCl or mannitol. NaCl addition induced a concentration-dependent fall in short-circuit current and a rise in transepithelial conductance (-33% and +14% per 100 mosM, respectively). Unidirectional isotopic fluxes of 22Na, 36Cl, and [14C]mannitol were measured in short-circuited tissues in the base-line state and after addition of NaCl or mannitol to an isotonic mucosal solution. NaCl addition (75 mM) caused a 50% increase in conductance (G) and a parallel increase in [14C]mannitol permeability (Pmann), indicating an increase in paracellular permeability. Net Cl- secretion was reduced 50%, and net Na+ absorption was unchanged despite an increased chemical gradient for absorption, indicating an inhibition of active ion transport. Mannitol addition (150 mM) abolished net Na+ absorption but did not increase G or Pmann or change net Cl- secretion. These results suggest that responses to increased tracheal surface liquid osmolarity during spontaneous breathing may occur in both the cellular (inhibition of active Na+ and Cl- transport) and paracellular (increased [14C]mannitol permeability) compartments of the mucosa.     

Bacterial pneumonia stimulates macromolecule secretion and ion and water fluxes in sheep trachea

In vivo instillation of Pasteurella haemolytica (greater than or equal to 10(7) colony-forming units/kg) into a lobar bronchus of sheep produced bacterial pneumonia by 7 days postinoculation. Infection was verified bacteriologically and histologically. Macromolecule secretion and ion and water fluxes were subsequently measured in tracheal tissues in vitro and were compared with values from sham-infected sheep. Macromolecules were radiolabeled with 35SO4 and [3H]threonine, and we measured the secretion of macromolecule-bound radiolabel onto the mucosa. Unidirectional fluxes of Cl-, Na+, and water were measured with radioactive tracers under open-circuit and short-circuit conditions. Lung infection increased basal secretion of bound 35SO4 (by 189%) and bound [3H]-threonine (by 110%). It significantly increased net Na+ absorption under open- and short-circuit conditions and induced open-circuit net absorption of Cl- and water (16 +/- 29 microliters X cm-2 X h-1). These changes were associated with specific recruitment of neutrophils and elevated levels of arachidonate metabolites (thromboxane B2 and leukotriene B4) in the airways. Thus the bacterial pneumonia-induced changes in tracheal mucus secretion may be the result of airway inflammation.     

Serotonin receptors for chloride secretion in hen colon

1. The effect of serotonin on chloride secretion in hen colon was studied under short circuit conditions. 2. Serotonin added to the serosal side induced a short-lived peak increase in Cl(-)-secretion (6.2 +/- 1.0 mumole.cm-2.h-1), in short circuit current (5.4 +/- 0.7 mumole.cm-2.h-1) and in cord conductance (8.1 +/- 0.7 mS.cm-2) with an apparent EC50 around 8 microM, and a more prolonged rise in chloride secretion of around 3.0 mumole.cm-2.h-1. 3. The short circuit current is a reasonable measure of net chloride secretion at the peak. 4. Several specific and non-specific serotonin receptor antagonists were studied for their influence on the serotonin induced peak response in short circuit current and cord conductance. 5. These antagonists covered the whole range of currently defined serotonin receptor types and subtypes: 5-HT1A, 5-HT1B, 5-HT1C, 5-HT1D, 5-HT2, and 5-HT3. 6. Adrenergic, cholinergic and histaminergic receptor antagonists were also tested for an interaction at the serotonin receptor involved in Ca(-)-secretion. 7. None of the antagonists had any influence on the serotonin response in short circuit current or cord conductance.     

Characterization of Na-K-ATPase in dog tracheal epithelium: enzymatic and ion transport measurements

The dog tracheal epithelium actively secretes Cl and absorbs Na. The possible dependency of this electrolyte transport on a Mg-dependent, Na-K-activated adenosine triphosphatase (Na-K-ATPase, EC 3.6.1.3) was examined. The characteristics of this enzyme system were investigated using homogenates of tracheal epithelium. The electrical properties and ion fluxes of this epithelium were determined in tissues mounted in Ussing chambers. Addition of Na and K produced an approximate 50% activation of basal Mg-ATPase activity. The apparent Km values for ATP, Na, K, and Mg were 0.4, 12.7, 1.9, and 1.6 mM, respectively. The total specific ATPase activity was 8.1 +/- 0.4 and that of the Mg-ATPase 4.3 +/- 0.1 mumol Pi. mg protein -1.h-1. Addition of ouabain (1 muM) or omission of K from the submucosal bathing solution reduced potential difference (PD) and short-circuit current (SCC) significantly. Relatively low concentrations (0.1 mM or less) of ethacrynic acid, furosemide, or 2,4-dinitrophenol (2,4-DNP) depressed SCC and PD significantly, i.e., at concentrations that were without effect on the Na-K-ATPase activity. Ethacrynic acid inhibited Cl secretion, whereas 2,4-DNP lowered both Na and Cl transport. These data demonstrate that 1) the tracheal mucosa of dogs contains a Na-K-ATPase at relatively high specific activity, 2) this enzyme is likely contained in the basal aspect of this membrane, 3) it appears to be essential for maintenance of Cl secretion, and 4) Cl secretion can be reduced (by ethacrynic acid, furosemide, and 2,4-DNP) without Na-K-ATPase inhibition.     

Movement of Na+ and Cl- across the isolated fetal rat stomach

Unidirectional and net isotopic fluxes of Na+ and Cl- were determined at steady state across isolated stomach of rat fetuses on days 19 and 21. On day 19, when parietal cells are not yet functional, net absorptions of Na+ and Cl- (respectively: 3.8 +/- 1.1 and 5.2 +/- 1.7 mu eq X h-1 X cm-2) were observed. By contrast, active secretion of Cl- (-2.1 +/- 1.8 mu eq X h-1 X cm-2) associated with decreased absorption of Na+ (45%) was noted on day 21, and both Na+ and Cl- net movements accounted for the short-circuit current, as observed on adult gastric mucosa. These results show that Na+ active absorption precedes Cl- secretion in fetal rat at the time of parietal cells differenciation.     

Chloride transport in the ferret trachea

A net secretion of chloride stimulated by carbamylcholine was observed in whole trachea. Luminal anthracene-9-carboxylic acid inhibited the net secretion of chloride and the transepithelial potential difference across the isolated trachea. Submucosal ouabain inhibited the net secretion of chloride and submucosal ouabain, or submucosal bumetanide inhibited the transepithelial potential difference across the isolated trachea. Short-circuited flat sheets of trachea manifested a net secretion of chloride induced by carbamylcholine. Serosal ouabain inhibited the short-circuit current and net chloride flux across isolated flat sheets of trachea.     

Effects of calcitonin gene-related peptide on airway epithelial functions in dogs

We studied the effects of calcitonin gene-related peptide (CGRP) on ciliary beat frequency (CBF) and electrical properties of canine tracheal epithelium by a photoelectric method and Ussing's short-circuit technique, respectively. CGRP dose dependently increased CBF, an effect that was accompanied by elevation of intracellular cyclic AMP but not affected by blockade of either Ca2+-influx or arachidonic acid metabolism. In contrast, CGRP elicited only a small and transient increase in short-circuit current without significant alterations in transepithelial potential difference or tissue conductance. These results suggest that CGRP may play a role in regulating airway mucociliary transport function.     

Common channels for water and protons at apical and basolateral cell membranes of frog skin and urinary bladder epithelia. Effects of oxytocin, heavy metals, and inhibitors of H(+)-adenosine triphosphatase

We have compared the response of proton and water transport to oxytocin treatment in isolated frog skin and urinary bladder epithelia to provide further insights into the nature of water flow and H+ flux across individual apical and basolateral cell membranes. In isolated spontaneous sodium-transporting frog skin epithelia, lowering the pH of the apical solution from 7.4 to 6.4, 5.5, or 4.5 produced a fall in pHi in principal cells which was completely blocked by amiloride (50 microM), indicating that apical Na+ channels are permeable to protons. When sodium transport was blocked by amiloride, the H+ permeability of the apical membranes of principal cells was negligible but increased dramatically after treatment with antidiuretic hormone (ADH). In the latter condition, lowering the pH of the apical solution caused a voltage-dependent intracellular acidification, accompanied by membrane depolarization, and an increase in membrane conductance and transepithelial current. These effects were inhibited by adding Hg2+ (100 microM) or dicyclohexylcarbodiimide (DCCD, 10(-5) M) to the apical bath. Net titratable H+ flux across frog skin was increased from 30 +/- 8 to 115 +/- 18 neq.h-1.cm-2 (n = 8) after oxytocin treatment (at apical pH 5.5 and serosal pH 7.4) and was completely inhibited by DCCD (10(-5) M). The basolateral membranes of the principal cells in frog skin epithelium were found to be spontaneously permeable to H+ and passive electrogenic H+ transport across this membrane was not affected by oxytocin. Lowering the pH of the basolateral bathing solution (pHb) produced an intracellular acidification and membrane depolarization (and an increase in conductance when the normal dominant K+ conductance of this membrane was abolished by Ba2+ 1 mM). These effects of low pHb were blocked by micromolar concentrations of heavy metals (Zn2+, Ni2+, Co2+, Cd2+, and Hg2+). Lowering pHb in the presence of oxytocin (50 mU/ml) produced a transepithelial current (3 microA.cm-2 at pHb 5.5) which was blocked by 100 microM of Hg2+, Zn2+, or Ni2+ at the basolateral side, and by DCCD (10(-5) M) or Hg2+ (100 microM) from the apical side. The net hydroosmotic water flux (JH2O) induced by oxytocin in frog bladder sacs was blocked by inhibitors of H(+)-adenosine triphosphatase (ATPase). Diethylstilbestrol (DES 10(-5) M), oligomycin (10(-8) M), and DCCD (10(-5) M) prevented JH2O when present in the lumen. These effects cannot be attributed to inhibition of metabolism since cyanide (10(-4) M), or 2-deoxyglucose (10(-3) M) had no effect on JH2O.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hypo-osmolar stimulation of transepithelial Cl- secretion in cultured human T84 intestinal epithelial layers.

Intact epithelial monolayers of T84 human colonic adenocarcinoma cells were exposed from the basolateral surfaces to hypo-osmotic media; in responsive tissues this resulted in a transient stimulation of inward short-circuit current (SCC) to a peak of 12.9 +/- 1.5 (S.E., n = 10) microA/cm2 which declined to prestimulation values of SCC (2.1 microA/cm2) within 5 min. Exposure of T84 cells to hypo-osmotic media results in an increase in cytosolic [Ca2+]i, dependent on extracellular Ca2+ influx. The cell-swelling activated SCC is abolished upon medium Cl- replacement and by 100 microM bumetanide applied to the basal-surfaces, consistent with the inward SCC resulting from transepithelial Cl- secretion. 100 microM DIDS (4,4'-diisothiocyanantostilbene-2,2'-disulphonic acid) also abolished the cell-swelling activated increase in SCC; DIDS is without effect upon the VIP-stimulated SCC, suggesting distinct Cl- channels are involved in the two responses.     

Bioelectric properties and ion transport across excised canine fetal and neonatal airways

Knowledge of liquid secretion by fetal lung stems from studies of sheep. We extended these studies to dogs and examined the persistence of the fetal pattern of airway epithelial permeability and ion transport in the neonatal animal. Plasma and lung liquid from fetal dogs were analyzed for Na+, K+, Cl-, and HCO3-. Only the Cl- concentration of fetal lung liquid (129 meq/l) was significantly different from that of fetal plasma (111 meq/l). Segments of trachea from fetal and neonatal (less than 1, 7-10, and 21-46 days after birth) dogs were excised and mounted in flux chambers. The transepithelial potential difference (PD) of all tissues was oriented lumen negative (9.8-14.8 mV). Under short-circuit conditions, unidirectional Na+ flows were symmetrical. Cl- was secreted, and the secretion was equivalent to short-circuit current (Isc). Cl- secretion persisted under open-circuit conditions. Lobar bronchi from 21- to 46-day neonates absorbed Na+ (1.9 mueq.cm-2.h-1), but unidirectional flows of Cl- were symmetrical. Amiloride (10(-4) M) reduced Isc of neonatal bronchi by 47% but did not affect fetal bronchi. Isoproterenol increased Isc of both fetal (33%) and neonatal (40%) bronchi. These responses suggest that fetal bronchi do not absorb Na+ but can be stimulated to secrete Cl-. We conclude that Cl- secretion by epithelium of large airways may contribute to fetal lung liquid production, but it is unlikely that the tracheal epithelium is involved in fluid absorption at birth. Whereas fetal bronchi appear to secrete Cl-, neonatal bronchi absorb Na+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid transport by the helicoidal colon of the new-born pig.

The proximal colon of the new-born pig maintains a stable short-circuit current which is partly dependent upon the presence of methionine. This interaction betweeen methionine and short-circuit current shows Michaelis-Menten knetics with a Km of 0.24 mM and a V of 27 muA.cm-2. The net flux of methionine to the serosal surface of proximal colons also shows a hyperbolic relation to the external concentration of methionine (Km 0.38 mM; V 10.4 nmol.cm-2. min-1). The proximal colon concentrates methionine within its epithelium giving a mucosal to medium ratio of 11.2 +/- 1.9 (90 min incubation in 1 mM methionine). The ability of the colon to transport methionine across and concentrate methionine within its mucosa is maintained for at least 24 h after birth. Colonic transport of amino acids could be physiologically important in the pig, where the immediate post-natal transfer of immune globulins has been shown to cause a temporary inhibition of normal intestinal function.     

Anthracene-9-carboxylic acid inhibits an apical membrane,chloride conductance in canine tracheal epithelium

Summary Canine tracheal epithelium secretes Cl from the submucosal to the mucosal surface via an electrogenic transport process that appears to apply to a wide variety of secretory epithelia. Cl exit across the apical membrane is thought to be a passive, electrically conductive process. To examine the cellular mechanism of Cl secretion we studied the effect of anthracene-9-carboxylic acid (9-AC), an agent known to inhibit the Cl conductance of muscle membrane. When added to the mucosal solution, 9-AC rapidly and reversibly decreases short-circuit current and transepithelial conductance, reflecting a reduction in electrogenic Cl secretion. The inhibition is concentration-dependent and 9-AC does not appear to compete with Cl for the transport process. The decrease in current and conductance results from a decrease in the net and both unidirectional transepithelial Cl fluxes without substantial alterations of Na fluxes. Furthermore, 9-AC specifically inhibits a Cl conductance: tissues bathed in Cl-free solutions showed no response to 9-AC. Likewise, when the rate of secretion and Cl conductance were minimized with indomethacin, addition of 9-AC did not alter transepithelial conductance. In contrast, neither removal of Na from the media nor blockade of the apical Na conductance with amiloride prevented a 9-AC-induced decrease in transepithelial conductance. We also found that the effect of 9-AC is independent of transepithelial transport: 9-AC decreases transepithelial conductance despite inhibition of Cl secretion with ouabain or furosemide. Intracellular electrophysiologic techniques were used to localize the effect of 9-AC to a reduction of the electrical conductance of the apical cell membrane: 9-AC hyperpolarizes the electrical potential difference across the apical membrane and decreases its relative conductance. 9-AC also prevents the characteristic changes in the cellular electrical potential profile, transepithelial conductance, and the ratio of membrane conductances produced by a reduction in mucosal bathing solution Cl concentration. These results indicate that 9-AC inhibits Cl secretion in tracheal epithelium by blocking an electrically conductive Cl exit step in the apical cell membrane. Thus, they support a cellular model of Cl secretion in which Cl leaves the cell across a Cl permeable apical membrane driven by its electrochemical gradient.     

Interdependence between sodium transport, external chloride, and sodium/calcium exchanger in the isolated skin of the Rana pipiens

The aim of this study was to analyze the relationship of the Na+/Ca2+ exchanger, cytosolic calcium, and chloride to the transepithelial transport of sodium in isolated frog skin. Sodium transport was measured as amiloride-inhibitable short circuit current (SCC). We studied the effect of variations in the concentrations of external chloride and of the manipulation of calcium on sensitive amiloride SCC. Modifications in the movement of Ca2+ were induced by an ionophore, A23187, and a Ca2+ channel blocker, nifedipine. Calcium ionophore A23187 (5 and 20 microM), in a normal Ringer's solution, increased SCC and transepithelial potential difference (PD). In contrast, nifedipine (20 microM) reduced SCC and PD. The role of the Na+/Ca2+ exchanger was studied using dichlorobenzamil (DCB, 50 microM) and quinacrine (1 mM), inhibitors of this exchanger. They selectively increased SCC and PD on the mucosal side of the skin, with no effect on the serosal side. This response occurred only in the presence of extracellular calcium. Replacement of NaCl by sodium methanesulfonate or the addition of furosemide (1 mM) at the serosal compartment, decreased basal SCC and PD and blocked the response to A23187 and the mucosal effect of DCB and quinacrine. These results suggest the presence of an Na+/Ca2+ exchanger located on the mucosal side of the frog skin, which participates in the transepithelial sodium transport. The action of this exchanger may be modulated by external chloride and calcium. J. Exp. Zool. 289:23-32, 2001.     

The action of epinephrine on Madin-Darby canine kidney cells

We have used cultured monolayers of Madin-Darby canine kidney (MDCK) cells, which form epithelial layers of high transepithelial resistance, grown on Millipore filters, for transport studies. In the absence of hormones net ion transport is of small magnitude and is consistent with a net absorptive flow (apical to basal) of Na+. Epinephrine, effective only from the basolateral cell surface, stimulates a net secretion (basal to apical) of Cl-. A substantial portion of net Cl- secretion is inhibited by loop diuretics such as furosemide applied to the basolateral cell aspects. The participation of a diuretic-sensitive cotransport system for Na+, K+, and Cl-, similar to that found in other cells, in transepithelial Cl- flux is postulated. The action of catecholamines on MDCK cell adenylate cyclase and on a Ca2+-activated K+ conductance is described.     

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.     

Uncoupled basal sodium absorption and chloride secretion in prairie dog (Cynomys ludovicianus) gallbladder.

1. Prairie dog gallbladders mounted in a Ussing-type chamber and bathed with symmetrical Ringer's solutions exhibited a transepithelial resistance (Rt) of 51 +/- 5 omega cm2, a lumen negative potential difference (Vms) of 11.5 +/- 0.7 mV and a short-circuit current (Isc) of 6.9 +/- 0.3 microEq/hr/cm2. 2. Radioisotopic ion flux experiments revealed that the basal Isc of 6.9 +/- 0.3 microEq/hr/cm2 was mostly accounted for by net Na+ absorption of 3.2 +/- 0.5 microEq/hr/cm2 and net Cl- secretion of 2.9 +/- 0.3 microEq/hr/cm2. 3. In HCO3- free Ringer's, net Na+ flux was virtually abolished, net Cl- flux decreased by 50% and Isc was reduced by 77%. 4. 10(-3) M mucosal amiloride and DIDS reduced Isc by 28 and 24%, respectively. 5. Mucosal NaCl diffusion potentials indicated that the paracellular pathway was cation selective. 6. Thin section electron micrographs showed a single cell population in this epithelium suggesting that net Na+ absorption and Cl- secretion may emerge from the same cells. 7. We conclude that prairie dog gallbladder epithelium is an electrogenic tissue and, in contrast to gallbladders of most other species, simultaneously but independently absorbs Na+ and secretes Cl-.     

Cl- transport by gastric mucosa: cellular Cl- activity and membrane permeability

The mechanism of Cl- secretion in the isolated, resting (i.e. cimetidine-treated) gastric mucosa of Necturus has been investigated with radioisotopic and electrophysiological techniques. Measurement of transepithelial 36Cl- fluxes (mucosal to serosal (M leads to S), Jms Cl-; S leads to M, Jsm Cl-) during control conditions show that at open circuit, when the transepithelial potential difference psi ms = 20 mV (S ground), Jms Cl- = Jsm Cl-, i.e. Jnet Cl- = 0, but during short-circuit current conditions Jnet Cl- = I sc = 2 mu equiv cm-2 h. Experiments with low [Cl-] solutions indicate that Cl- exchange diffusion does not contribute significantly to either Jms Cl- or Jsm Cl-. Double-barrelled, Cl- -selective microelectrodes showed that in open circuit, the cellular (C) chemical potential for Cl-, psi c Cl- = 31 mV (apparent [Cl-] = 29 mM), the electrical potential across the M membrane, psi m = -34 mV (mucosa ground) while that across the S membrane, psi s = -52 mV (serosa ground). During short-circuit current conditions, psi m = psi s = -49 mV and [Cl-]c = 30 mM. The permeability of the M membrane to Cl- (Pm Cl-) was calculated both from the tracer experiments and the electrode measurements by using the constant-field equation. Short-term (45 s) uptake of 36Cl- at [Cl]m = 96 mM during short circuit conditions gave Pm Cl- = 2.6 x 10(-5) cm s-1. Measurement of [Cl-]c by means of the electrodes when [Cl-]m was changed from 96 to 2 mM or from 2 to 96 mM gave Pm Cl- = 2.9-5.7 x 10(-5) cm s-1. Our results indicate that during open circuit conditions Cl- is accumulated across the S membrane into gastric cells in an energy-requiring step, but since Jnet Cl- = 0, Cl- must leak back into the S solution at a rate equal to the entry rate. When the tissue is short-circuited, Cl- secretion occurs (Jnet Cl- = Isc) owing to the same energy-requiring accumulation of Cl- by the cells and a passive (apparently electrodiffusive) movement across the mucosal membrane.     

Microelectrode studies of amphotericin B on Na+ and K+ conductance in bullfrog cornea

Addition of 10(-5) M amphotericin B to the tear solution of an in vitro preparation of the frog cornea increased the transepithelial conductance, gt, and decreased the apical membrane fractional resistance, f(R0), in the presence or absence of tear Na+ and Cl-. In the presence of tear Na+ and Cl-, amphotericin B increased the short-circuit current, Isc, from 3.9 to 8.8 microA.cm-2 and changed the intracellular potential, V0, from -48.5 to -17.9 mV probably due to a higher increase in the Na+ than in the K+ conductance. In the absence of tear Na+ and Cl-, amphotericin B decreased Isc from 5.5 to about 0 microA.cm-2 due to K+ (and possibly Na+) flux from cell to tear and changed V0 from -35.4 to -63.6 mV due to the increase in conductance of both ions. Increase in the tear K+ from 4 to 79 mM (in exchange for choline), in the presence of amphotericin B and absence of tear Na+ and Cl-, decreased f(R0) from 0.09 to 0.06, increased gt from 0.23 to 0.31 mS, increased Isc from 0.63 to 7.3 microA.cm-2, and changed V0 from -65.5 to -17.3 mV due to the change in EK in the presence of a high conductance in the tear membrane. Similar effects were observed with an increase of tear Na+. Results support the concept that the Na+ conductance opened by amphotericin B in the apical membrane is greater than the K+ conductance. Previously observed transepithelial effects of the ionophore may be explained mostly on the basis of its effect on the apical membrane.