Model of bicarbonate secretion by resting frog stomach fundus mucosa. II. Role of the oxyntopeptic cells

In the present publication we report mainly electrophysiological studies on oxyntopeptic cells of frog gastric mucosa which aim at clarifying a possible involvement of these cells in the process of resting gastric alkali (HCO₃ ^–) secretion, described in the preceding publication. The experiments were performed on intact gastric fundus mucosa of Rana esculenta mounted in Ussing chambers. After removal of the muscle and connective tissue layer oxyntopeptic cells were punctured from the serosal surface with conventional or pH-sensitive microelectrodes to measure, besides transepithelial voltage and resistance, the basolateral cell membrane potential, the voltage divider ratio, and the cell pH in response to secretagogues and/or changes in serosal ion concentration. Carbachol (10^–4 mol/l), which transiently stimulated HCO₃ ^– secretion by 0.22 mol·cm^–2·h^–1, transiently acidified the cells by 0.09±SEM 0.03 pH units (n=6) and transiently induced an apical cell membrane anion conductance. According to the model of gastric HCO₃ ^– secretion presented in the preceding publication, this anion conductance could be involved in gastric HCO₃ ^– secretion, mediating, besides Cl^– efflux, also apical HCO₃ ^– efflux. In addition carbachol stimulated basolateral Na⁺(HCO₃ ^–)_n-cotransport, which according to the results from the preceding publication mediates basolateral HCO₃ ^– uptake for secretion. By contrast, cAMP-mediated secretagogues, such as histamine or others, which stimulate HCl secretion and transiently alkalinize the oxyntopeptic cells, were found to down-regulate the basolateral Na⁺(HCO₃ ^–)_n-cotransporter. The data indicate that the oxyntopeptic cells may play a role in HCO₃ ^– secretion across resting frog fundus mucosa.     

Expression of rat renal Na+HCO3-cotransporter in Xenopus laevis oocytes

The goal of this study was to determine whether Na⁺HCO₃ ^– cotransport from rat renal cortex can be functionally expressed in Xenopus laevis oocytes. Using a two electrode voltage-clamp device, HCO₃ ^–-dependent currents were determined as the difference in current measured in a nominally HCO₃ ^–-free buffer, pH 7.5 and a buffer with 30 mmol/l HCO₃ ^–, pCO₂ 5.33 kPa, pH 7.5. The size of renal cortex mRNA required to express maximum HCO₃ ^–-dependent current was 2–3 kb. The expressed current depended on Na⁺ and was sensitive to 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS) and acetazolamide (each 1 mmol/l), inhibitors known to block the native Na⁺HCO₃ ^– cotransporter in several tissues including rat proximal tubule. Water-injected oocytes did not show any measurable response either to increased HCO₃ ^– nor to Na⁺-free perfusion or to the inhibitors, indicating the absence of an endogenous electrogenic HCO₃ ^– transporter. Our data indicate that the rat renal mRNA of 2–3 kb size contains the message for the Na⁺HCO₃ ^– cotransporter.     

Selective blockage of cell membrane K conductance by an antisecretory agent in guinea-pig gallbladder epithelium

Loperamide inhibits PGE₁-induced electrogenic HCO₃ secretion in guinea-pig gallbladder. Underlying changes in epithelial cell membrane properties were investgated using intracellular microelectrode techniques in vitro. In the absence of PGE₁, mucosal loperamide (10^–4 mol/l) reversibly depolarized both cell membranes by 6 mV. The apparent ratio of membrane resistances (R _a/R _b) remained unchanged and so did voltage responses to luminal Cl removal and Na reduction. The depolarizing response to elevation of luminal K concentration from 5 to 76 mmol/l was decreased from 13 to 8 mV. In the presence of 1 PGE₁, the apical membrane is mainly permeable to Cl and HCO₃. Under these conditions, loperamide reduced membrane potentials by 10 mV,R _a/R _b remaining constant at 0.4. Effects on voltage responses to changes in luminal Na or K concentration were unchanged. Responses to luminal Cl removal (transient depolarization) were greatly enhanced (from 22 to 42 mV) as predictable from the fall in K permeability that hinders Cl efflux from cell into lumen. Less marked but significant effects were obtained with 10^–5 mol/l (mucosal side) and serosal loperamide (10^–4 mol/l). We suggest that loperamide inhibits electrogenic HCO₃ secretion by reducing apical membrane K permeability. The resulting depolarization diminishes the driving force for conductive anion efflux from cell into lumen. This conclusion is supported by the ability of luminal K elevation to mimick loperamide inhibition of the secretory flux of HCO₃ (pH-stat experiments).     

Evidence for a Na+/H+ exchanger at the basolateral membranes of the isolated frog skin epithelium: Effect of amiloride analogues

We have investigated the possible existence of a Na⁺/H⁺ ion exchanger in the frog skin epithelium by using isotopic methods and two amiloride analogues: 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and phenamil. We found phenamil to be a specific blocker of sodium entry to its cellular transport compartment since it inhibited both the transepithelial Na⁺ influxes (J ₁₃) with aK _I of 4·10^–7 mol/l and the Na⁺ pool (control: 77±4 neq·h^–1·cm^–2; phenamil: 21±1 neq·h^–1·cm^–2). On the contrary EIPA (10^–5 mol/l) had no effect onJ ₁₃ nor on the apical Na⁺ conductance. Acidification of the epithelium by passing from a normal Ringer (25 mmol/l HCO ₃ ^– , 5% CO₂, pH 7.34) to a HCO ₃ ^– -free Ringer (5% CO₂, pH 6.20) while blocking the Na⁺ conductance with phenamil, produced a large stimulation of Na⁺ influxes exclusively across the basolateral membranes (J ₃₂), after return to a normal Ringer (J ₃₂=706±76 and 1635±199 neq·h^–1·cm^–2 in control and acid-loaded epithelia respectively). The stimulation ofJ ₃₂ was initiated when the epithelia were acid-loaded with Ringer of pH lower than 6.90 and was blocked by amiloride (K _I=7·10^–6 mol/l) and EIPA (K _I=5·10^–7 mol/l) whereas phenamil had no effect. In na⁺-loaded epithelia (ouabain treated) the Na⁺ efflux across the basolateral membranes was stimulated by an inwardly directed proton gradient and was blocked by EIPA (10^–5 mol/l) or amiloride (10^–4 mol/l), a result suggesting reversibility of the mechanism. We conclude that a Na⁺ permeability mediated by a Na⁺/H⁺ ion exchanger exists in the basolateral membranes, which is stimulated by intracellular acidification and is sensitive to amiloride or EIPA. This exchanger is proposed to be involved in intracellular pH regulation.     

Mechanisms of oxalate absorption and secretion across the rabbit distal colon

To further evaluate the mechanisms of oxalate (Ox^2–) transport in the intestine the following studies were performed using isolated, short-circuited segments of the rabbit distal colon (DC). In control buffer, the DC absorbed Ox^2– (net Ox^2– flux, J _Net ^Ox =5.4±0.7 pmol · cm^–2 · h^–1). Replacement of Na⁺ with N-methyl-d-glucamine (NMDG⁺) abolished Ox^2– absorption by decreasing mucosal to serosal Ox^2– flux (J _ms ^Ox ), without affecting Cl^– transport, while gluconate substitution for Cl^– did not affect J _Net ^Ox or net Na⁺ flux (J _Net ^Na ). Addition of Na⁺ to the serosal side of tissues bathed by NMDG⁺ buffer increased J _ms ^Ox 40% without altering mucosal to serosal Cl^– flux (J _ms ^Cl ). Serosal amiloride or dimethyl amiloride (10^–3 M) abolished J _Net ^Ox by decreasing J _ms ^Ox , it increased serosal to muscosal Cl^– flux (J _sm ^Cl ) and it gradually inhibited short-circuit current (I _sc). Mucosal amiloride (10^–4 M) abolished I _sc but had no effect on Ox^2– or Cl^– fluxes. Serosal 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS, 10^–6 M) reduced J _ms ^Ox by 20% and J _Net ^Ox by 43% without affecting J _ms ^Cl or J _Net ^Cl . Dibutyryl cyclic adenosine monophosphate (dB-cAMP, 5×10^–4 M, both sides) stimulated Ox^2– secretion (J _Net ^Ox = –12.6±3.3 pmol · cm^–2 · h^–1). The dB-cAMP-induced secretion of Ox^2– and Cl^– were fully abolished by serosal furosemide (10^–4 M) and partially inhibited (35%) by 5×10^–4 M mucosal NPPB [5-nitro-2-(3-phenylpropylamino)-benzoic acid], a putative Cl^– channel blocker. It is proposed that: (1) basal absorption of Ox^2–, but not Cl^–, is dependent upon a previously undescribed basolateral Na⁺-H⁺ exchanger that may be coupled to a DIDS-sensitive, basolateral anion exchange system that mediates Ox^2– flux; (2) the DC secretes Ox^2– in response to dB-cAMP by a mechanism that is indistinguishable from the pathway for Cl^– secretion.     

Effect of secretin and inhibitors of HCO3 −/H+ transport on the membrane voltage of rat pancreatic duct cells

The aim of the present study was to study the effect of secretin on the electrophysiological response of pancreatic ducts. Furthermore, we investigated the effects of lipid-soluble buffers and inhibitors of HCO₃ ^–/H⁺ transport. Ducts obtained from fresh rat pancreas were perfused in vitro. Secretin depolarized the basolateral membrane voltage, V _bl, by up to 35 mV (n=37); a halfmaximal response was obtained at 3×10^–11 mol/l. In unstimulated ducts a decrease in the luminal Cl^– concentration (120 to 37 mmol/l) had a marginal effect on V _bl, but after maximal secretin stimulation it evoked a 14±2 mV depolarization (n=6), showing that a luminal Cl^– conductance G _Cl- was activated. The depolarizing effect of secretin on V _bl was often preceded by about a 6 mV hyperpolarization, most likely due to an increase in the basolateral G _K+. Perfusion of ducts with DIDS (4,4 — diisothiocyanatostilbene — 2,2 — disulphonic acid, 0.01 mmol/l) or addition of ethoxzolamide (0.1 mmol/l) to the bath medium diminished the effect of secretin. Acetate or pre-treatment of ducts with NH₄ ⁺/NH₃ (10 mmol/l in the bath) depolarized the resting V _bl of –65±2 mV by 16±4 mV (n=7) and 19±3 mV (n=10), respectively. The fractional resistance of the basolateral membrane (FR _bl) doubled, and the depolarizing responses to changes in bath K⁺ concentrations (5 to 20 mmol/l) decreased from 22±1 to 11±2 mV. The Na⁺/H⁺ antiporter blocker EIPA (5-[N-ethyl-N-isopropyl]-amiloride, 0.1 mmol/l) also depolarized V _bl by 10±1 mV, FR_bl increased and the response to K⁺ concentration changes decreased (n=7). Effects of EIPA and ethoxzolamide on V _bl were greater in ducts deprived of exogenous HCO₃ ^–/CO₂. Taken together, the present study shows that secretin increased the basolateral G _K+ and the luminal G _Cl-. The depolarizing effect of secretin was diminished following inhibition of HCO₃ ^– transport (DIDS), or HCO₃ ^–/H⁺ generation (ethoxzolamide). Manoeuvres that presumably led to lowered intracellular pH (NH₄ ⁺/NH₃ removal, acetate, EIPA) decreased the basolateral G _K+. The present data support our previously published model for pancreatic HCO₃ ^– secretion, and indicate that the basolateral membrane possesses a pH-sensitive G _K+.     

A new class of inhibitors of cAMP-mediated Cl− secretion in rabbit colon,acting by the reduction of cAMP-activated K+ conductance

Previously we have shown that arylamino-benzoates like 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), which are very potent inhibitors of NaCl absorption in the thick ascending limb of the loop of Henle, are only poor inhibitors of the cAMP-mediated secretion of NaCl in rat colon. This has prompted our search for more potent inhibitors of NaCl secretion in the latter system. The chromanole compound 293 B inhibited the equivalent short-circuit current (I _sc) induced by prostaglandin E₂ (n=7), vasoactive intestinal polypeptide (VIP,n=5), adenosine (n=3), cholera toxin (n=4) and cAMP (n=6), but not by ionomycin (n=5) in distal rabbit colon half maximally (IC₅₀) at 2 mol/l from the mucosal and at 0.7 mol/l from the serosal side. The inhibition was reversible and paralleled by a significant increase in transepithelial membrane resistance [e.g. in the VIP series from 116±16 ·cm² to 136±21 ·cm² (n=5)]. A total of 25 derivatives of 293 B were examined and structure activity relations were obtained. It was shown that the racemate 293 B was the most potent compound with-in this group and that its effect was due to the enantiomer 434 B which acted half maximally at 0.25 mol/l. Further studies in isolated in vitro perfused colonic crypts revealed that 10 mol/l 293 B had no effect on the membrane voltage across the basolateral membrane (V _bl) in non-stimulated crypt cells: –69±3 mV versus –67±3 mV (n=10), whilst in the same cells 1 mmol/l Ba²⁺ depolarised (V _bl) significantly. However, 293 B depolarised (V _bl) significantly in the presence of 1 mol/l forskolin: –45±4mV versus –39±5 mV (n=7). Similar results were obtained with 0.1 mmol/l adenosine. 293 B depolarised (V _bl) from –40±5 mV to –30±4 mV (n=19). This was paralleled by an increase in the fractional resistance of the basolateral membrane. VIP had a comparable effect. The hyperpolarisation induced by 0.1 mmol ATP was not influenced by 10 mol/l 293 B: –75±6 mV versus –75±6 mV (n=6). Also 293 B had no effect on basal K⁺ conductance (n=4). Hence, we conclude that 293 B inhibits the K⁺ conductance induced by cAMP. This conductance is apparently relevant for Cl^– secretion and the basal K⁺ conductance is insufficient to support secretion.     

Na+-HCO3 − cotransporter and intracellular pH regulation in chicken enterocytes

The current studies examine the presence of the Na⁺-HCO₃ ^– cotransporter in chicken enterocytes and its role in cytosolic pH (pH_i) regulation. The pH-sensitive dye 2,7-bis(carboxyethyl)-5,6-carboxy-fluorescein (BCECF) was used to monitor pH_i. Under resting conditions, pH_i was 7.25 in solutions buffered with bis(2-hydroxyethyl)-1-piperazine ethanesulphonic acid (HEPES) and 7.17 in those buffered with HCO₃ ^–. Removal of external Na⁺ decreased pH_i and readdition of Na⁺ rapidly increased pH_i towards the control values. These Na⁺-dependent changes were greater in HCO ₃ ^– than in HEPES-buffered solutions. In HCO ₃ ^– - free solutions the Na⁺-dependent changes in pH_i were prevented by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and unaffected by 4,4-diisothiocyanatostilbene disulphonic acid (H₂-DIDS). In the presence of HCO ₃ ^– , the Na⁺-induced changes in pH_i were sensitive to both EIPA and H₂-DIDS. In the presence of EIPA, cells partially recovered from a moderate acid load only when both Na⁺ and HCO ₃ ^– were present. This pH_i recovery, which was EIPA resistant, and dependent on Na⁺ and HCO ₃ ^– , was inhibited by H₂-DIDS and occurred at equal rates in both Cl^–-containing and Cl^–-free solutions. Kinetic analysis of the rate of HCO ₃ ^– - and Na⁺- dependent pH_i recovery from an acid load as a function of the Na⁺ concentration revealed first-order kinetics with a Michaelis constant, K _m, of 11 mmol/l Na⁺. It is concluded that in HCO₃ ^/– buffered solutions both the Na⁺/H⁺ exchanger and the Na⁺-HCO₃ ^– cotransporter participate in setting the resting pH_i in isolated chicken enterocytes and help the recovery from acid loads.     

The Na+-HCO 3 − cotransporter operates with a coupling ratio of 2 HCO 3 − to 1 Na+ in isolated rabbit renal proximal tubule

All the relevant literature reports indicate that net rates of salt and water absorption and cell membrane potentials (V _b) are lower, but intracellular Na⁺ concentration is higher in rabbit renal proximal tubule in vitro than in rat proximal tubule in vivo. Since the different driving forces should influence basolateral Na⁺-HCO₃ ^– cotransport we have studied the operation of the cotransporter in isolated rabbit renal proximal tubule in vitro with special emphasis on the stoichiometry of flux coupling (q). Using conventional and ion-selective intracellular microelectrodes three series of experiments were performed: (a) we determined the V _b response to a 21 reduction of bath HCO₃ ^– or Na⁺ concentration, (b) we determined initial efflux rates of HCO₃ ^– or Na⁺ ions in response to a sudden 101 reduction of bath HCO₃ ^– concentration, and (c) we collapsed the tubules and determined electrochemical driving forces of Na⁺ and HCO₃ ^– across the basolateral cell membrane under conditions approaching zero net flux in the control state in the presence of Ba²⁺- and in Cl^–-free solutions. All measurements concurrently yielded a coupling ratio of approximately two HCO₃ ^– ions to one Na⁺ ion (q=2). This result contrasts with the ratio q=3, which we have previously observed in similar experiments on rat renal proximal tubule in vivo [Yoshitomi et al. (1985) Pflügers Arch 405:360] and which was also observed on rabbit renal basolateral cell membrane vesicles in vitro [Soleimani et al. (1987) J Clin Invest 79:1276]. This indicates that — depending on the functional state of the cell — the Na⁺-HCO₃ ^– cotransporter can operate with variable stoichiometry and suggests that it transports either 1 Na⁺ + 1 HCO₃ ^– + 1 CO₃ ^2– ion [Soleimani and Aronson (1989) J Biol Chem 264:18 302] or 1 Na⁺ + 2 HCO₃ ^– ions.     

Characterization of potassium channels in respiratory cells

In a previous study [26] we described the properties of potassium channels in cultured respiratory cells derived from cystic fibrosis patients (CF) and normal individuals (N). In the present study we examine the regulatory mechanisms of these channels by the patch clamp technique. Since there were no apparent differences in the properties of CF and N K⁺ channels the results were pooled. In the excised inside/out configuration the channel was blocked by different K⁺ channel blockers. Barium (5 · 10^–3 mol/l), retraethylammoniumchloride (5 · 10^–3 mol/l), quinidine 10^–3 mol/l) and lidocaine (5 · 10^–3 mol/l), when added to the cytosolic side, inhibited K⁺ channels reversibly. An increase in the calcium concentration from 10^–7 mol/l to 10^–6 mol/l led to a marked increase in the open channel probability (P _o). Further increases in Ca²⁺ concentration increasedP _o only slightly. No pH effects on the cytosolic side of the channel were observed. The channel open probability was reduced when ATP was present on the cytosolic side at a concentration of 10^–4 mol/l to 10^–3 mol/l. Non hydrolysable adenosine 5-[,-methylene] triphosphate had the same inhibitory effect as ATP. The inhibition by ATP was blunted by the simultaneous addition of 1 mmol/l ADP. The inhibition of K⁺ channels by cytosolic ATP may represent a channel regulatory mechanism in the intact cell. This would allow for coupling between the activity of the (Na⁺+K⁺)-pump and the basolateral K⁺ conductance.     

K+-induced swelling of vestibular dark cells is dependent on Na+ and Cl− and inhibited by piretanide

Epithelial cell height was measured in order to estimate the cell volume of dark cells from the ampullae of the semicircular canal of the gerbil. Under control conditions, addition of 10^–4 mol/l piretanide, 10^–5 mol/l 5-nitro-2(3-phenylpropylamino)-benzoic acid (NPPB), 5 mmol/l barium or 10^–3 mol/l quinidine had no significant effect on cell height. Addition of 10^–4 mol/l NPPB or 10^–3 mol/l ouabain led to a small significant decrease in cell height which was not reversible. Substitution of Na⁺ by N-methyl-d-glucamine or of Cl^– by gluconate led to a significant and reversible reduction in cell height. Isotonic elevation of [K⁺] from 3.6 to 25 mmol/l in a PO₄-buffered, HCO₃-free solution led to an increase in cell height from 5.8±0.1 (SEM) to 8.7±0.2 (n= 62) during the first 40 s. During prolonged exposure to elevated [K⁺] (3–5 min; n=19), some tissue samples underwent a regulatory volume decrease. K⁺-induced swelling was absent in both isotonic Cl^– -free and isotonic Na⁺-free solutions and was inhibited by the loop diuretic piretanide (10^–5 and 10^–4 mol/l) or by the (Na⁺+ K⁺) ATPase inhibitor ouabain (10^–3 mol/l) or by 10^–4mol/l NPPB. After the removal of ouabain or 10^–4 mol/l NPPB, K⁺-induced swelling under control conditions was enhanced and was less reversible as compared to control conditions before the experiment. K⁺-induced swelling was not altered by NPPB (10^–5 mol/l) or barium (5 mmol/l); however, barium slowed shrinking upon return of [K⁺] to control level. In the presence of 10^–3 mol/l quinidine, K⁺-induced swelling was enhanced and not reversible. These data suggest that dark cells from the semicircular canal possess an Na⁺2Cl^–K⁺ cotransporter as a solute uptake mechanism and a solute efflux mechanism which is sensitive to barium and inhibited by quinidine.     

Mechanism of NaCl secretion in the rectal gland of spiny dogfish (Squalus acanthias)

Rectal gland tubules (RGT) of spiny dogfish were dissected and perfused in vitro. Transepithelial PD (PD_te), resistance (R_te), the PD across the basolateral membrane (PD_bl) and intracellular chloride and potassium activities (a _Cl– ^cell ,a _K+ ^cell ) were measured. In a first series, 67 RGT segments were perfused with symmetric shark Ringers solution. The bath perfusate contained in addition db-cAMP 10^–4, forskolin 10^–6, and adenosine 10^–4 mol · l^–1. PD_te was –11±1 (n=67) mV lumen negative, R_te 27±2 (n=47) cm². PD_bl –75±0.4 (n=260) mV.a _K+ ^cell anda _Cl– ^cell were 109±22 (n=4) and 38±4 (n=36) mmol · l^–1 respectively. These data indicate that Cl^– secretion across the RGT must be an uphill transport process, whereas secretion of Na⁺ could be driven by the lumen negative PD_te. Intracellular K⁺ is 14 mV above equilibrium with respect to the basolateral membrane PD and Cl^– is 23 mV above equilibrium across the apical membrane. In series 2, the conductivity properties of the apical and basolateral membrane as well as that of the paracellular pathway were examined in concentration step experiments. Decrease of the basolateral K⁺ concentration led to a rapid hyperpolarization of PD_bt with a mean slope of 19 mV per decade of K⁺ concentration change. Addition of 0.5 mmol · l^–1 Ba²⁺ to the bath solution lead to a marked depolarization and abolished the response to K⁺ concentration steps. In the lumen a Cl^– concentration downward step led to a depolarization of the lumen membrane; resulting in a mean slope of 18 mV per decade of Cl^– concentration change. When dilution potentials were generated across the epithelium, the polarity indicated that the paracellular pathway is cation selective. In series 3 the equivalent short circuit current (I_sc=PD_te/R_te) was determined as a function of symmetrical changes in Na⁺ concentration, with Cl^– held at 276 mmol · l^–1, and as a function of symmetrical changes in Cl^– concentration, with Na⁺ held at 278 mmol · l^–1 I_sc was a saturable function of Na⁺ concentration (Hill coefficient 0.9±0.1,K _1/2 4.4 mmol · l^–1,n=7) and also a saturable function of Cl^– concentration (Hill coefficient 2.0±0.1,K _1/2 75 mmol · l^–1,n=11). These data are compatible with the assumption that the carrier responsible for NaCl uptake has a 1 Na⁺ per 2 Cl^– stoichiometry. In series 4, the effect of a K⁺ concentration downward step on PD_bl anda _Cl– ^cell transients was followed with high time resolution in the presence and absence of basolateral furosemide (5 · 10^–5 to 10^–4 mol · l^–1) in an attempt to examine whether K⁺ reduction on the bath side inhibits Na⁺Cl^– uptake by the carrier system as does e.g. furosemide. The data indicate that removal of K⁺ from the bath side exerts an effect comparable to that of furosemide, i.e. it inhibits the carrier. We conclude that NaCl secretion in the RGT cell comprises at the least the following components: In the basolateral membrane, the (Na⁺+K⁺)-ATPase, probably the Na⁺ 2 Cl^–K⁺ carrier, and a K⁺ conductance. In the apical membrane a Cl^– conductance; and a Na⁺ conductive paracellular pathway.Supported by Deutsche Forschungsgemeinschaft DFG-Gr 480/8-1. Parts of this study have been presented at the 3rd International Symposium on Ion Selective Electrodes, Burg Rabenstein 1983, 16th Annual Meeting American Society of Nephrology, Washington DC 1983, 49th Tagung der Deutschen Physiologischen Gesellschaft, Dortmund 1984. A summary of the present study was published in Bulletin Mount Desert Island Biological Laboratory (Vol. 83)     

Chloride and potassium conductances of cultured human sweat ducts

The purpose of this study was to characterize the ion conductances, in particular those for Cl^– and K⁺, of human sweat duct cells grown in primary culture. Sweat duct cells from healthy individuals were grown to confluence on a dialysis membrane, which was then mounted in a mini-Ussing chamber and transepithelial and intracellular potentials were measured under open-circuit conditions. Under control conditions the epithelia developed mucosa-negative transepithelial potentials, V _te, of about –10mV. The apical membrane potential, V _a, was –25 mV to –30 mV (n=97) in most cells, but several cells had a higher potential of about –55 mV (n=29). Mucosal amiloride (10 mol/l) hyperpolarized V _a from –31±1 mV to a new sustained level of –46±2 mV (n=36). These changes were accompanied by increase in the fractional resistance of the apical membrane, fR _a, and decreases of V _te and the equivalent short-circuit current, I _sc. In amiloride-treated tissues an increase in mucosal K⁺ concentration (5 mmol/l to 25 mmol/l) depolarized V _a by 5±1 mV (n=8), while the same step on the serosal side depolarized V _a by 20±2 mV (n=8). A Cl^– channel blocker 3,5-dichloro-diphenylamine-2-carboxylate DCl-DPC; 10 mol/l) depolarized V _a by 5±1 mV (n=6), an effect that was lost after amiloride application. The blocker had no effect from the serosal side. Reduction of mucosal Cl^– (from 120 to 30 or 10 mmol/l) depolarized V _a by 9–11 mV (n=35), an effect that was often followed by a secondary hyperpolarization of 10–30 mV (n=27). Isoproterenol (5 mol/l) increased the V _a responses to low Cl^– such that the depolarizing response was increased from 10±1 mV to 19±2 mV (n=8); the hyperpolarizing response seemed to be reduced. With changes in Cl^– concentration on the serosal side, V _a remained relatively constant at –25 mV, while V _te decreased from –8 mV to–3 mV; hence, V _bl depolarized by about 5 mV. Taken together, our results show that the human sweat duct epithelium possesses Na⁺, K⁺ and Cl^– conductances on the luminal membrane and Cl^– and K⁺ conductances on the basolateral membrane. The Cl^– conductances on the luminal membrane is sensitive to DCl-DPC, and can be activated by isoproterenol. The small K⁺ conductance on the luminal membrane could account for some K⁺ secretion in sweat glands.     

The chloride/base exchanger in the basolateral cell membrane of rabbit renal proximal tubule S3 segment requires bicarbonate to operate

Isolated microperfused S3 segments of rabbit renal proximal tubule were investigated with pH-sensitive double-barrelled intracellular microelectrodes to determine whether the Cl^–/base exchanger, which we have previously identified in the basolateral cell membrane of this segment requires HCO₃ ^– or can also work in CO₂/HCO₃ ^– free conditions. Cell pH (pH_i) was measured in response to sudden substitution of bath Cl^– by gluconate. In control solutions containing 25 mmol/l HCO₃ pH_i increased initially by 5.0±0.3 × 10^–3 unit/s but after perfusion with CO₂/HCO₃ ^–-free solutions pH_i of the same cells increased only by 1.3±0.2 × 10^–3 unit/s in response to Cl^– substitution. From measurements of the cellular buffering power it was calculated that the control base flux had fallen drastically from 3.7±0.3 to 0.3±0.1 × 10^–12 mols/s·cm tubule length. To test whether the remaining flux might have resulted from metabolic CO₂, oxidative metabolism was poisoned with cyanide (5 mmol/l). This abolished the pH change (pH_i) in CO₂/HCO₃ ^–-free solutions, but did not affect the pH shift in the presence of HCO₃ ^–. The data indicate that basolateral Cl^–/base exchange in S3 segment requires HCO₃ ^– to operate. A model in which HCO₃ ^– absorption proceeds in form of OH^– and CO₂ can be largely excluded.     

Electrophysiological investigation of microdissected gastric glands of bullfrog I. Basolateral membrane properties in the resting state

In the present experiments we have made a new attempt to characterize the ion transport properties of H⁺-secreting cells of the gastric mucosa using electrophysiological techniques. Individual gastric glands of bullfrog fundus mucosa were manually dissected, mounted in holding pipettes and superfused with various test solutions while individual cells were punctured with conventional or H⁺-sensitive double-barrelled microelectrodes. All measurements were performed in the resting state (0.1 mmol/l cimetidine). In HCO₃ ^–-containing control Ringer solution the cell membrane potential (V_b) averaged –45.6±0.9 mV (±SEM, n=54). From the fast initial V _b responses to changing bath K⁺, Na⁺, Cl^– or HCO₃ ^– concentrations we deduced that the basolateral cell membrane contains conductances for K⁺, Na⁺, and Cl^– but not for HCO₃ ^–, and that a Na⁺-HCO₃ ^– cotransporter is not present. The K⁺ conductance was inhibited by Ba²⁺ (3 mmol/l), but the Cl^– conductance was not inhibited by 4,4 diisothiocyanato-stilbene-2,2disulphonic acid (DIDS, 0.3 mmol/l), nor selectively inhibited by 5-nitro-2-(3)-phenylpropyl-aminobenzoate (NPPB, 10 (mol/l). In a great number of cells the V_b response to Cl^– substitution revealed two components: an initial spiking depolarization which reflected conductive Cl^– efflux and a secondary slow hyperpolarization, the origin of which was not immediately evident. Since the latter response could be mimicked by CO₂-free perfusion, strongly depressed by Ba²⁺ and eliminated by DIDS, we conclude that it reflects HCO₃ ^– uptake into the cells via a DIDS sensitive Cl^–/HCO₃ ^– exchanger which alkalinizes the cells and stimulates the basolateral K⁺ conductance. Our results confirm, revise and extend the results of previous, less direct, investigations of gastric cell ion transport.     

Effect of external sodium on intracellular chloride activity in the surface cells of frog gastric mucosa

The intracellular chloride activity and its dependence on ionic substitutions in the bathing media was studied in individual surface cells of resting gastric mucosa using conventional and Cl^– selective microelectrodes. When the tissue was perfused with control NaCl-Ringer the cell membrane p.d.'s, cell-lumen (_cm) and cell-serosa (_cs) were –40.9±0.6 mV and –66.8±0.5 mV (n=175) respectively specitively and the p.d. measured by the Cl^– selective microelectrodes across the serosal membrane ( _cs ^Cl- ) averaged –32.4±0.7 mV (n=138). From these values an intracellular Cl^– activity (a _c ^Cl– ) of 15.3 mmol/l can be estimated. The data indicate that chloride ion is distributed close to equilibrium at the luminal membrane while it is accumulated by an energy requiring step at the serosal membrane. Reduction (2 mmol/l) or absence of chloride from the luminal bath did not result in any detectable change ofa _c ^Cl– ; on the other hand, after removal of Cl^– from the serosal bath the intracellular Cl^– activity fell to 7.1 mmol/l.When the tissue was exposed to serosal Na⁺-free Ringer (Na⁺ replaced by choline or TMA), although thea _c ^Cl– remained unaffected, a marked reduction of the electrochemical gradient for Cl^– at the serosal membrane was observed.These data indicate that: (i) chloride is accumulated in the surface cells against its electrochemical potential difference at the serosal membrane; (ii) the luminal membrane has a negligible conductance to Cl^–, while the serosal membrane represents a conductive pathway to chloride; (iii) the uphill entry of chloride at the serosal membrane seems to be, at least partially, Na⁺-dependent.Parts of this work were presented at the International Symposium on the Theory and Application of Ion-Selective Electrodes in Physiology and Medicine, Erlangen, 1983, and at the SIBS-SIFSINU Congress, Saint Vincent, 1983. The study was supported by C.N.R. (Rome)-Grants CT 81.00173. 04 and CT 82.00147.04     

A novel synergistic stimulation of Na+-transport across frog skin (Xenopus laevis) by external Cd2+- and Ca2+-ions

Isolated skin of the clawed frogXenopus laevis was mounted in an Ussing-chamber. The transcellular sodiumcurrent (I _Na) was identified either as amiloride-blockable (10^–3 mol/l) short-circuit current (I _SC), or by correctingI _SC for the shunt-current obtained with mucosal Tris. A dose of 10 mmol/l Cd²⁺ applied to the mucosal side increased the current by about 70%. The half-maximal effect was reached at a Cd²⁺-concentration of 2,6 mmol/l (in NaCl-Ringer). The quick and fully reversible effect of Cd²⁺ could not be seen when 10^–3 mol/l amiloride was placed in the outer, Na⁺-containing solution, nor when Na⁺ was replaced by Tris. This suggests that Cd²⁺ stimulatesI _Na. Cd²⁺ intefered with the Na⁺-current self-inhibition, and therefore with the saturation ofI _Na by increasing the apparent Michaelis constant (K _Na) of this process. The I _Na recline after stepping up mucosal [Na⁺] was much reduced in presence of Cd²⁺. Ca²⁺-ions on the mucosal side had an identical effect to Cd²⁺, and 10 mmol/l Ca²⁺ increaseI _Na by about 100%. The half-maximal effect was obtained with 4.4 mmol/l Ca²⁺. The mechanism ofI _Na-stimulation by Ca²⁺ did not seem to differ from that of Cd²⁺. Thus, although of low Na⁺-transport capacity,Xenopus skin appears to be as good a model for Na⁺-transporting epithelia asRanidae skin, with the exception of the calcium effect which, so far, has not been reported forRanidae.     

Electrophysiological study of transport systems in isolated perfused pancreatic ducts: properties of the basolateral membrane

In order to study the mechanism of pancreatic HCO ₃ ^– transport, a perfused preparation of isolated intra-and interlobular ducts (i.d. 20–40 m) of rat pancreas was developed. Responses of the epithelium to changes in the bath ionic concentration and to addition of transport inhibitors was monitored by electrophysiological techniques. In this report some properties of the basolateral membrane of pancreatic duct cells are described. The transepithelial potential difference (PD_te) in ducts bathed in HCO ₃ ^– -free and HCO ₃ ^– -containing solution was –0.8 and –2.6 mV, respectively. The equivalent short circuit current (I_sc) under similar conditions was 26 and 50 A·cm^–2. The specific transepithelial resistance (R_te) was 88 cm². In control solutions the PD across the basolateral membrane (PD_bl) was –63±1 mV (n=314). Ouabain (3 mmol/l) depolarized PD_bl by 4.8±1.1 mV (n=6) within less than 10 s. When the bath K⁺ concentration was increased from 5 to 20 mmol/l, PD_bl depolarized by 15.9±0.9 mV (n=50). The same K⁺ concentration step had no effect on PD_bl if the ducts were exposed to Ba²⁺, a K⁺ channel blocker. Application of Ba²⁺ (1 mmol/l) alone depolarized PD_bl by 26.4±1.4 mV (n=19), while another K⁺ channel blocker TEA⁺ (50 mmol/l) depolarized PD_bl only by 7.7±2.0 mV (n=9). Addition of amiloride (1 mmol/l) to the bath caused 3–4 mV depolarization of PD_bl. Furosemide (0.1 mmol/l) and SITS (0.1 mmol/l) had no effect on PD_bl. An increase in the bath HCO ₃ ^– concentration from 0 to 25 mmol/l produced fast and sustained depolarization of PD_bl by 8.5±1.0 mV (n=149). It was investigated whether the effect of HCO ₃ ^– was due to a Na⁺⁺-dependent transport mechanism on the basolateral membrane, where the ion complex transferred into the cell would be positively charged, or whether it was due to decreased K⁺ conductance caused by lowered intracellular pH. Experiments showed that the HCO ₃ ^– effect was present even when the bath Na⁺ concentration was reduced to a nominal value of 0 mmol/l. Similarly, the HCO ₃ ^– effect remained unchanged after Ba²⁺ (5 mmol/l) was added to the bath. The results indicate that on the basolateral membrane of duct cells there is a ouabain sensitive (Na⁺+K⁺)-ATPase, a Ba²⁺ sensitive K⁺ conductance and an amiloride sensitive Na⁺/H⁺ antiport. The HCO ₃ ^– effect on PD_bl is most likely due to rheogenic anion exit across the luminal membrane.     

Effects of glucagon on Na+, Cl−, K+, Mg2+ and Ca2+ transports in cortical and medullary thick ascending limbs of mouse kidney

The effects of glucagon on transepithelial Na⁺, Cl^–, K⁺, Ca²⁺ and Mg²⁺ net fluxes were investigated in isolated perfused cortical (cTAL) and medullary (mTAL) thick ascending limbs of Henle's loop of the mouse nephron. Transepithelial ion net fluxes (J _Na ⁺,J _Cl ^–,J _K ⁺,J _Ca ²⁺,J _Mg ²⁺) were determined by electron probe analysis of the collected tubular fluid. Simultaneously the transepithelial voltage (PD_te) and the transepithelial resistance (R _te) were recorded. In cTAL-segments (n=8), glucagon (1.2×10^–8 mol · l^–1) stimulated significantly the reabsorption of Na⁺, Cl^–, Ca²⁺ and Mg²⁺J _Na ⁺ increased from 204±20 to 228±23 pmol · min^–1 · mm^–1,J _Cl ^– from 203±18 to 234±21 pmol · min^–1 · mm^–1,J _Ca ²⁺ from 0.52±0.13 to 1.34±0.30 pmol · min^–1 · mm^–1 andJ _Mg ²⁺ from 0.51±0.08 to 0.84±0.08 pmol · min^–1 · mm^–1.J _K+ remained unchanged: 3.2±1.3 versus 4.0±1.9 pmol · min^–1 · mm^–1. Neither PD_te (16.3±1.5 versus 15.9±1.4 mV) norR _te (22.5±3.0 versus 20.3±2.6 cm²) were changed significantly by glucagon. However, in the post-experimental periods a significant decrease in PD_te and increase inR _te were noted. In mTAL-segments (n=9), Mg²⁺ and Ca²⁺ transports were close to zero and glucagon elicited no significant effect. The reabsorptions of Na⁺ and Cl^–, however, were strongly stimulated:J _Na ⁺ increased from 153±17 to 226±30 pmol · min^–1 · mm^–1 andJ _Cl ^– from 151±23 to 243±30 pmol · min^–1 · mm^–1. The rise in NaCl transport was accompanied by an increase in PD_te from 10.3±1.1 to 12.3±1.2 mV and a decrease inR _te from 19.1±2.7 to 17.8±2.0 cm². No net K⁺ movement was detectable either in the absence or in the presence of glucagon. A micropuncture study carried out in hormone-deprived rats indicated that glucagon stimulates Na⁺, Cl^–, K⁺, Mg²⁺ and Ca²⁺ reabsorptions in the loop of Henle. In conclusion our data demonstrate that glucagon stimulates NaCl reabsorption in the mTAL segment and to a lesser extent in the cTAL segment whereas it stimulates Ca²⁺ and Mg²⁺ reabsorptions only in the cortical part of the thick ascending limb of the mouse nephron. These data are in good agreement with, and extend, those obtained in vivo on the rat with the hormone-deprived model.This study was supported by the Commission des Communautés Européennes, Grant no. ST 23, 00951F (CD) and by Wissenschaftsausschuß der Nato über den DAAD     

Mechanism of hydrogen ion transport in the diluting segment of frog kidney

Transepithelial H⁺ transport was studied in diluting segments of the isolated-perfused kidney ofrana esculenta. The experiments were performed in controls as well as in K⁺-adapted and Na⁺-adapted animals (exposed to 50 mmol/l KCl or NaCl, resp. for at least 3 days). Conventional and single-barreled, liquid ion-exchanger H⁺-sensitive microelectrodes were applied in the tubule lumen to evaluate transepithelial H⁺ net flux (J _te ^H ) as well as limiting transepithelial electrical and H⁺ electrochemical potential differences (PD _te ,E _te ^H ) and luminal pH at zero net flux conditions. The measurements were made in absence (control) and presence of furosemide (5·10^–5 mol/l) or amiloride (10^–3 mol/l). E _te ^H (lumen positive vs ground) was 19±3 mV in controls, 43±3 mV in K⁺ adapted but about zero in Na⁺ adapted animals. Using the correspondingPD _te -values, steady state luminal pH of 7.63±0.05, 7.13±0.05 and 8.02±0.02 was calculated for the respective groups of animals (peritubular pH 7.80). In parallel, significant secretoryJ _te ^H (from blood to lumen) was found in controls (14±2 pmol·cm^–2·S^–1) which was stimulated by K⁺ adaptation (61±8 pmol·cm^–2·s^–1) but reversed in direction by Na⁺-adaptation (–8±1 pmol·cm^–2·s^–1). Amiloride inhibited secretoryJ _te ^H . Elimination of the lumen positivePD _te by furosemide did not affect significantlyE _te ^H andJ _te ^H in control and K⁺ adapted animals but abolished reabsorptiveJ _te ^H in Na⁺ adapted animals.We conclude that in frog diluting segment H⁺ secretion is an active, amiloride-sensitive, furosemide-insensitive transport process. The data are consistent with luminal Na⁺/H⁺ exchange. The activity of this system depends critically on the metabolic state of the animal.Parts of the data were presented at the 16th Ann. Meeting of the Am. Soc. Nephrol., Washington (1983)This work was supported by österr. Forschungsrat, Proj. No.: 4366 and by Dr. Legerlotz Stiftung