Functional heterogeneity in the hamster medullary thick ascending limb of Henle's loop

Cellular heterogeneity was examined in the hamster medullary thick ascending limb (MAL) perfused in vitro by electrophysiological measurements with an intracellular microelectrode. Random measurements of fractional resistance of basolateral membrane (Rf _B) revealed two cell populations, high basolateral conductance (HBC) cells havingRf _B of 0.05±0.01 (n=24) and low basolateral conductance (LBC) cells havingRf _B of 0.80±0.03 (n=32). Basolateral membrane potentials (V _B) were not different between HBC cells and LBC cells (–72.6±1.2,n=43 vs. –70.0±1.2,n=35). Addition of 2 mmol/l Ba²⁺ to the bath depolarized the basolateral membrane in the HBC cells from –70.4±3.2 to –20.9±5.9 mV (n=8) but not in the LBC cells (from –74.4±1.9 to –72.0±2.1 mV). Increasing K⁺ or decreasing Cl^– in the bathing solution caused marked positive deflection ofV _B in the HBC cells but little or no change inV _B in the LBC cells. Elimination of Cl^– from the lumen or addition of furosemide to the lumen enhanced the potential response of the HBC cells to basolateral application of Ba²⁺. Accordingly, with Ba²⁺ present in the bath, the potential response of the HBC cells to a decrease in bath Cl^– concentration was enhanced. These observations suggest that a K⁺ conductance exists in the basolateral membrane of HBC cells in paralled with a Cl^– conductance. The basolateral cell membrane of LBC cells also contains a Cl^– conductance. In these cells, but not in HBC cells, the potential response to decreasing bath Cl^– concentration increased when bath pH was decreased from 7.4 to 6.0 Apparent K⁺ transference numbers of the luminal membrane were higher in LBC cells (0.74±0.05,n=7) than in HBC cells (0.20±0.02,n=5). From these data, we conclude: (1) there are two distinct cell types in the hamster medullary thick ascending limb; (2) there is a low Cl^– conductance in basolateral membrane of LBC cells which is stimulated by low pH.     

Isolated perfused rabbit colon crypts: stimulation of Cl− secretion by forskolin

The aim of this study was to characterize ion conductances and carrier mechanisms of isolated in vitro perfused rabbit colonic crypts. Crypts were isolated from rabbit colon mucosa and mounted on a pipette system which allowed controlled perfusion of the lumen. In non-stimulated conditions basolateral membrane voltage (V _b1) was –65±1 mV (n=240). Bath Ba²⁺ (1 mmol/ l) and verapamil (0.1 mmol/l) depolarized V _b1 by 21±2 mV (n=7) and 31±1 (n=4), respectively. Lowering of bath Cl^– concentration hyperpolarized V _b1 from –69±3 to –75±3 mV (n=9). Lowering of luminal Cl^– concentration did not change V _b1. Basolateral application of loop diuretics (furosemide, piretanide, bumetanide) had no influence on V _b1 in non-stimulated crypts. Forskolin (10^–6 mol/l) in the bath depolarized V _b1 by 29±2 mV (n=54) and decreased luminal membrane resistance. In one-third of the experiments a spontaneous partial repolarization of V _b1 was seen in the presence of forskolin. During forskolin-induced depolarization basolateral application of loop diuretics hyperpolarized V _b1 significantly and concentration dependently with a potency sequence of bumetanide > piretanide furosemide. Lowering bath Cl^– concentration hyperpolarized V _b1. Lowering of luminal Cl^– concentration from 120 to 32 mmol/l during forskolin-induced depolarization led to a further depolarization of V_b1 by 7±2 mV (n=10). We conclude that V_b1 of rabbit colonic crypt cells is dominated by a K⁺ conductance. Stimulation of the cells by forskolin opens a luminal Cl^– conductance. Basolateral uptake of Cl^– occurs via a basolateral Na⁺ : 2Cl^– : K⁺ cotransport system.     

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.     

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+.     

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.     

Effect of NH4 +/NH3 on cytosolic pH and the K+ channels of freshly isolated cells from the thick ascending limb of Henle's loop

The conductance properties of the luminal membrane of cells from the thick ascending limb of Henle's loop of rat kidney (TAL) are dominated by K⁺. In excised membrane patches the luminal K⁺ channel is regulated by pH changes on the cytosolic side. To examine this pH regulation in intact cells of freshly isolated TAL segments we measured the membrane voltage (V _m) in slow-whole-cell (SWC) recordings and the open probability (P _o) of K⁺ channels in the cell-attached nystatin (CAN) configuration, where channel activity and part of V _m can be recorded. The pipette solution contained K⁺ 125 mmol/l and Cl^– 32 mmol/l. Intracellular pH was determined by 2,7 bis(2-carboxyethyl)-5,(6)-carboxyfluorescein (BCECF) fluorescence. pH changes were induced by the addition of 10 mmol/l NH₄ ⁺/NH₃ to the bath. In the presence of NH₄ ⁺/NH₃ intracellular pH acidified by 0.53±0.11 units (n=7). Inhibition of the Na⁺2Cl^– K⁺ cotransporter by furosemide (0.1 mmol/l) reversed this effect and led to a transient alkalinisation by 0.62±0.14 units (n=7). In SWC experiments V _m of TAL cells was -72±1 mV (n=70). NH₄ ⁺/NH₃ depolarised V _m by 22±2 mV (n=25). In 11 SWC experiments furosemide (0.1 mmol/l) attenuated the depolarising effect of NH₄ ⁺ from 24±3 mV to 7±3 mV. Under control conditions the single-channel conductance of TAL K⁺ channels in CAN experiments was 66±5 pS and the reversal voltage for K⁺ currents was 70±2 mV (n=35). The P _o of K⁺ channels in CAN patches was reduced by NH₄ ⁺/NH₃ from 0.45±0.15 to 0.09±0.07 (n=7). NH₄ ⁺/NH₃ exposure depolarised the zero current voltage of the permeabilised patches by-9.7±3.6 mV (n=5). The results show that TAL K⁺ channels are regulated by cytosolic pH in the intact cell. The cytosolic pH is acidified by NH₄ ⁺/NH₃ exposure at concentrations which are physiologically relevant because Na⁺2Cl^–K⁺(NH₄ ⁺) cotransporter-mediated import of NH₄ ⁺ exceeds the rate of NH₃ diffusion into the TAL. K⁺ channels are inhibited by this acidification and the cells depolarise. In the presence of furosemide TAL cells alkalinise proving that NH₄ ⁺ uptake occurs by the Na⁺2Cl^–K⁺ cotransporter. The findings that, in the presence of NH₄ ⁺/NH₃ and furosemide, V _m is not completely repolarised and that K⁺ channels are not activated suggest that the respective K⁺ channels may in addition to their pH regulation be inhibited directly by NH₄ ⁺/NH₃.     

Membrane potential measurements of transitional cells from the crista ampullaris of the Gerbil

Transitional cells of the crista ampullaris were impaled with microelectrodes in order to record the membrane potential (PD) and to investigate membrane properties. In control solution the PD was –87±1 mV (n=103). This value is not significantly different from –83±2 mV (n=24) measured in Cl^– free solution. [Cl^–] steps from 150 to 15 mmol/l (n=24) depolarized the membrane by about 2 mV, indicating a minor Cl^– conductance. The transference number for K⁺ was 0.75±0.01 (n=79) obtained from the PD responses to K⁺ steps from 3.6 to 25 mmol/l. The cell membrane depolarized and the amplitude of PD responses to [K⁺] steps was reduced by Ba²⁺ (2·10^–6 to 10^–3 mol/l), quinidine (10^–3 mol/l), quinine (10^–3 mol/l), Rb⁺ (20 mmol/l), Cs⁺ (20 mmol/l), NH₄ ⁺ (20 mmol/l) and Tl⁺ (0.5 mmol/l), whereas tetraethylammonium (TEA, 20 mmol/l) had no effect. The dose-response curve for Ba²⁺ in the presence of 3.6 mmol/l K⁺ was shifted to the right by approximately three decades in the presence of 25 mmol/l K⁺ and by a factor of about 4 in the presence of 135 mmol/l gluconate as a substitute for Cl^–. Transitional cells were depolarized by ouabain, suggesting the presence of (Na⁺+K⁺-ATPase.This work was supported by grants from the Deafness Research Foundation to PhW and the National Institute of Health (NS 19490) to DCM     

Crypt base cells show forskolin-induced Cl− secretion but no cation inward conductance

Whole-cell patch-clamp studies in base cells of isolated colonic crypts of rats pretreated with dexamethasone were performed to examine the effects of stimulation by forskolin (10 mol/1). The experiments were designed in order to distinguish between two postulated effector mechanisms: the activation of a non-selective cation channel and the activation of Cl^– channels. As shown in an accompanying report, forskolin depolarizes the membrane voltage (V _m) by some 40–50 mV and enhances the whole-cell membrane conductance (G _m) substantially in these cells. In this report all experiments were performed in the presence of forskolin. A reduction of the bath Na⁺ concentration from 145 to 2 mmol/1 led to a hyperpolarization ofV _m by some 20–30 mV This hyperpolarization occurred very slowly suggesting that the hyperpolarization produced by the low-Na⁺ solution was caused indirectly and not by a change in the equilibrium potential for Na⁺,E _Na ⁺. A complete kinetic analysis of the effect on voltage of bath Na⁺ revealed a saturation-type relation with a high apparent affinity for Na⁺ of around 5–10 mmol/1. A reduction in bath Cl^– concentration from 145 to 32 mmol/1 caused a depolarization ofV _m from –34 ± 3 to –20 ± 4 mV (n = 13) in the presence of a high bath Na⁺ concentration, but had the opposite effect at low (5 mmol/1) Na⁺ concentrations:V _m was hyperpolarized from –46 ± 4 to –62 ± 6 mV (n = 13). If the effect of Na⁺ onV _m was caused by a non-selective cation channel the opposite would have been expected. To test directly whether the Na⁺2Cl^–K⁺ cotransporter was responsible for the effects of changes in bath Na⁺ onV _m, the effects of increasing concentrations of several loop diuretics were examined. Furosemide, piretanide, torasemide and burnetanide (up to 0.1–0.5 mmol/1) all hyperpolarizedV _m, albeit only by less than 10 mV. Another subclass of loop diuretics containing a tetrazolate in position 1 [e.g. azosemide, no. 19A and no. 20A from Schlatter E, Greger R, Weidtke C (1983) Pflüger Arch 396: 210–217] were much more effective. Azosemide hyperpolarizedV _m from –46 ± 3 to –74 ± 2 mV (n = 18) and reducedG _m from 11 ± 1 to 4 ± 1 nS (n = 14). These data indicate that forskolin stimulates Cl^– secretion in these cells by a mechanism fully compatible with the current scheme for exocrine secretion involving the Na⁺2Cl^–K⁺ cotransporter.     

Effect of high NaCl intake on Na+ and K+ transport in the rabbit distal convoluted tubule

Morphological studies have demonstrated that a chronic increase in distal Na⁺ delivery causes hypertrophy of the distal convoluted tubule (DCT). To examine whether high NaCl-intake also causes functional changes in the well defined DCT, we measured transmural voltage (V _T), lumen-to-bath Na⁺ flux (J _Na(LB)), and net K⁺ secretion (J _K(net)) in DCTs obtained from control rabbits and those on high NaCl-intake diets. The lumen negativeV _T was significantly greater in the high NaCl group than in the control group. The net K⁺ secretion (pmol mm^–1 min^–1) was greater in the high NaCl-intake group (54.1±13.0 vs 14.7±5.6). The K⁺ permeabïlities in both luminal and basolateral DCT membranes, as assessed by the K⁺-induced transepithelial voltage deflection inhibitable with Ba²⁺, were increased in the experimental group. The lumen-to-bath²²Na flux (pmol mm^–1 min^–1) was also greater in the experimental group (726±119 vs 396±65). TheV _T component inhibitable with amiloride was also elevated in the high NaCl-intake group. Furthermore, Na⁺–K⁺-ATPase activity of the DCT was higher in the experimental than in the control group. We conclude that high NaCl intake increases both Na⁺ reabsorption and K⁺ secretion by the DCT. This phenomenon is associated with an increased Na⁺–K⁺-ATPase activity along with increased Na⁺ and K⁺ permeabilities of the luminal membrane, and an increase in the K⁺ permeability of the basolateral membrane. Cellular mechanisms underlying these functional changes remain to be established.     

Ba2+ and amiloride uncover or induce a pH-sensitive and a Na+ or non-selective cation conductance in transitional cells of the inner ear

The membrane potential V _m the cytosolic pH (pH_i), the transference numbers (t) for K⁺, Cl^– and Na⁺/ non-selective cation (NSC) and the pH-sensitivity of V _m were investigated in transitional cells from the vestibular labyrinth of the gerbil. V _m, pH_i, , and the pH_i sensitivity of V _m were under control conditions were –92±1 mV (n=89 cells), pH_i 7.13±0.07 (n=11 epithelia), 0.87±0.02 (n=22), 0.02±0.01 (n=19), 0.01±0.01 (n=24) and –5 mV/pH unit (n=13 cells/n=11 epithelia), respectively. In the presence of 100 mol/l Ba²⁺ the corresponding values were: –70±1 mV (n=32), pH_i 7.16±0.08 (n=6), 0.31±0.05 (n=4), 0.06±0.01 (n=6), 0.20±0.03 (n=10) and -16 mV/pH-unit (n=15/n=6). In the presence of 500 mol/l amiloride the corresponding values were: –72±2mV (n=34), pH_i 7.00±0.07 (n=5), 0.50±0.04 (n=6), 0.04±0.01 (n=11), 0.28±0.04 (n=9) and –26 mV/pH-unit (n=20/n=5). In the presence of 20 mmol/l propionate plus amiloride the corresponding values were: –61±2 mV (n=27), pH_i 6.72±0.06 (n=5), 0.30±0.02 (n=6), 0.06±0.01 (n=5) and 0.40±0.02 (n=8), respectively. V _m was depolarized and and pH_i decreased due to (a) addition of 1 mmol/l amiloride in 150 mmol/l Na⁺ by 38±1 mV (n=8), from 0.82±0.02 to 0.17±0.02 (n=8) and by 0.13±0.01 pH unit (n=6), respectively; (b) reduction of [Na⁺] from 150 to 1.5 mmol/l by 3.3±0.5 mV (n=30), from 0.83±0.02 to 0.75±0.04 (n=9) and by 0.33±0.07 pH unit (n=4), respectively and (c) addition of 1 mmol/l amiloride in 1.5 mmol/l Na⁺ by 20±1 mV (n=11) and from 0.83±0.03 to 0.53±0.02 (n=5), respectively. These data suggest that the K⁺ conductance is directly inhibited by amiloride and Ba²⁺ and that Ba²⁺ and amiloride uncover or induce a pH-sensitive and a Na⁺/NSC conductance which may or may not be the same entity.Some of the data have been presented at various meetings and appear in abstract form in [31, 35, 37]     

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.     

Effect of bradykinin and histamine on the membrane voltage,ion conductances and ion channels of human glomerular epithelial cells (hGEC) in culture

The effects of bradykinin (BK) and histamine (Hist) on the membrane voltage (V _m), ion conductances and ion channels of cultured human glomerular epithelial cells (hGEC) were examined with the nystatin patch clamp technique. Cells were studied between passage 3 and 20 in a bath rinsed with Ringer-like solution at 37°C. The mean value of V _m was –41±0.5 mV (n=189). BK (10^–6 mol/l, n=29) and Hist (10^–5 mol/l, n= 55) induced a rapid transient hyperpolarization by 15±1 mV and 18±1 mV, respectively. The hyperpolarization was followed by a long lasting depolarization by 6±1 mV (BK 10^–6 mol/l) and 7±1 mV (Hist 10^–5 mol/l). The ED₅₀ was about 5×10^–8 mol/l for BK and 5×10^–7 mol/l for Hist. In the presence of both agonists, increases of outward and inward currents were observed. A change in the extracellular K⁺ concentration from 3.6 to 30 mmol/l depolarized V _m by 8±1 mV and completely inhibited the hyperpolarizing effect of both agents (n=11). Reduction of extracellular Cl^– concentration from 145 to 30 mmol/l led to a depolarization by 2 ±1 mV (n=25). In 30 mmol/l Cl^– the depolarizations induced by BK (10^–7 mol/l) and Hist (10^–6 mol/l) were augmented to 9±2 mV (n=14) and to 10±2 mV (n=11), respectively. Ba²⁺ (5 mmol/l) depolarized V _m by 19±5 mV (n=6) and completely inhibited the hyperpolarization induced by BK (10^–6 mol/l, n=3) and reduced that of Hist (10^–5 mol/l) markedly (n=3). Preincubation with the K⁺ channel blocker charybdotoxin (1–10 nmol/l) for 3 min had no significant effect on V _m, but reduced markedly the BK(10^–6 mol/l, n=11) and Hist-(10^–5 mol/l, n=6) induced hyperpolarizations. In 10 out of 31 experiments in the cell attached nystatin patch configuration big K⁺ channels with a conductance of 247±17 pS were found. The open probability of these K⁺ channels was increased 3- to 5-fold during the hyperpolarization induced by BK (10^–7 mol/l) or Hist (10^–5 mol/l, both n= 4). In excised inside/out patches this K⁺ channel had a mean conductance of 136±8.5 pS (n=10, clamp voltage 0 mV). The channel was outwardly rectifying and its open probability was increased when Ca²⁺ on the cytosolic side was greater than 0.1 mol/l. The data indicate that BK and Hist activate a and a in hGEC. The hyperpolarization is induced by the activation of a Ca²⁺-dependent maxi K⁺ channel.     

The effect of secretagogues on ion conductances of in vitro perfused,isolated rabbit colonic crypts

Several secretagogues were used in this study, including those which enhance intracellular cyclic adenosine monophosphate (cAMP) production, as well as others which elevate intracellular Ca²⁺ activity and are known to increase Cl^– secretion in the intact colon and in colonic carcinoma cell lines. They were examined with respect to their effects on electrophysiological properties in isolated rabbit distal colonic crypts. Crypts were dissected manually and perfused in vitro. Transepithelial voltage (V _te), transepithelial resistance (R _te), membrane voltage across the basolateral membrane (V _bl), and fractional basolateral membrane resistance (FR _bl), were estimated. Basolateral prostaglandin E₂ (PGE₂, 0.1 mol/l), vasoactive intestinal peptide (VIP, 1 nmol/l) and adenosine (0.1 mmol/l) induced an initial depolarisation and a secondary partial repolarisation of (V _bl). In the case of adenosine, the initial depolarization of (V _bl) was by 31±2 mV (n=47).R _te fell significantly from 16.4±3.6 to 14.2±3.7 ·cm² (n= 6), andFR _blincreased significantly from 0.11±0.02 to 0.51±0.10 (n=6). In the second phase the repolarisation of (V _bl) amounted 11±2 mV (n=47) and a steadystate (V _bl) of –51±2 mV (n=47) was reached.R _te fell further and significantly to a steady-state value of 12.4±3.8 ·cm² (n=6) andFR _bl fell significantly to 0.42±0.13 (n=6). In 30% of the experiments, a transient hyperpolarisation of (V _bl) by 8±2 mV (n=14) was seen during wash out of adenosine. In the presence of adenosine, but not under control conditions, lowering of luminal Cl^– concentration from 120 to 32 mmol/l depolarised (V _bl) significantly by 8±1 mV (n=9). Basolateral ATP and ADP (0.1 mmol/l) led to a short initial depolarisation followed by a sustained and significant hyperpolarisation by 6±2 mV (n=27) and 5±4 mV (n=8), respectively. Carbachol (CCH) hyperpolarised (V _bl) in a concentration-dependent manner. At 100 mol/l (bath) the hyperpolarisation was by 14±2 mV (n=11) andFR _bl fell slightly. Neurotensin (10 nmol/l), isoproterenol (10 mol/l) and uridine 5-triphosphate (UTP, 0.1 mmol/l) had no effect. It is concluded that PGE₂, VIP and adenosine upregulate sequentially a luminal Cl^– conductance and a basolateral K⁺ conductance by increasing intracellular cAMP concentration. Ca²⁺ mobilising hormones such as ATP, ADP, and CCH increase the basolateral K⁺ conductance, while the effect on luminal Cl^– conductance appears to be very limited.     

Macula densa cells sense luminal NaCl concentration via furosemide sensitive Na+2Cl−K+ cotransport

The macula densa cells of the juxtaglomerular apparatus probably serve as the sensor cells for the signal which leads to the appropriate tubuloglomerular feedback response. The present study reports basolateral membrane voltage (PD_bl) measurements in macula densa cells. We isolated and perfused in vitro thick ascending limb segments with the glomerulus, and therefore the macula densa cells, and the early distal tubule still attached. Macula densa cells were impaled with microelectrodes under visual control. PD_bl was recorded in order to examine how these cells sense changes in luminal NaCl concentrations. The addition of furosemide, a specific inhibitor of the Na⁺2Cl^–K⁺ cotransporter in the thick ascending limb, to the lumen of the perfused thick ascending limb hyperpolarized PD_bl from –55±5 mV to –79±4 mV (n=7). Reduction of NaCl in the lumen perfusate from 150 mmol/l to 30 mmol/l also hyperpolarized PD_bl from –48±3 mV to –66±5 mV (n=4). A Cl^– concentration step in the bath from 150 mmol/l to 30 mmol/l resulted in a 24±4 mV (n=4) depolarization of PD_bl. This depolarization of PD_bl was absent when furosemide was present during the Cl^– concentration step. These data suggest that the macula densa cells sense changes in luminal NaCl concentration via coupled uptake of Na⁺ and Cl^–. The transport pathways for NaCl transport in macula densa cells are probably identical to those in the thick ascending limb: the (Na⁺+K⁺)-ATPase in the basolateral membrane drives Na⁺ and Cl^– uptake via the luminal Na⁺2Cl^–K⁺ cotransport, Cl^– leaves the cell via basolateral Cl^– channels and K⁺ recycles across the apical membrane via K⁺ channels. Changes in intracellular Cl^– activity as a result of altered luminal NaCl uptake, and thus voltage changes of the basolateral membrane are probably the first signal in the tubuloglomerular feedback regulation.This study was supported by Deutsche Forschungsgemeinschaft Gr. 480/9     

Properties of single K+ channels in the basolateral membrane of rabbit proximal straight tubules

The basolateral membrane of rabbit straight proximal tubules, which were cannulated and perfused on one side, was investigated with the patch clamp technique. Properties of inward and outward directed single K⁺ channel currents were studied in cell-attached and insideout oriented cell-excised membrane patches. In cell-attached patches with NaCl Ringer solution both in pipette and bath, outward K⁺ currents could be detected after depolarization of the membrane patch by about 20–30 mV. The current-voltage (i/V) relationship could be fitted by the Goldman-Hodgkin-Katz (GHK) current equation, with the assumption that these channels were mainly permeable for K⁺ ions. A permeability coefficientP _K of (0.17±0.04) · 10^–12 cm³/s was obtained, the single channel slope conductance at infinite positive potentialg(V ) was 50±12 pS and the single channel conductance at the membrane resting potentialg(V _bl) was 12±3 pS (n=4). In cell-excised patches, with NaCl in the pipette and KCl in the bath, the data could also be fitted to the GHK equation and yieldedP _K = (0.1 ±0.01) ·10^–12 cm³/s,g(V ) = 40 ± 4 pS andg(V _bl) = 7 ± 1 pS (n=8). In cell-attached patches with KCl in the pipette and NaCl in the bath, inward K⁺ channels occurred at clamp potentials 60 mV, whereas outward K⁺ channel current was detected at more positive voltages. The current-voltage curves showed slight inward rectification. The single channel conductance, obtained from the linear part of the i/V curve by linear regression, was 46±3 pS and the reversal potential was 59±6 mV (n=9). In cell-excised patches with KCl in the pipette and NaCl in the bath, inward directed K⁺ channel currents could again be described by the GHK equation. The single channel parameters were similar to those recorded for outward K⁺ currents (see above). In inside-out oriented cell-excised patches with NaCl in the pipette and KCl in the bath, reducing bath (i.e. cytosolic) Ca²⁺ concentration from 10^–6 mol/l to less than 10^–9 mol/l did not affect the open state probability of single channel currents. These results demonstrate that the observed channels are permeable for K⁺ ions in both directions and that these basolateral K⁺ channels in rabbit proximal straight tubule are not directly dependent on Ca²⁺ ions.     

Electrophysiological study of inner medullary collecting duct of hamsters

The electrophysiological properties of the hamster mid-inner medullary collecting duct (IMCD₂) cells were examined in isolated and perfused preparations by intracellular impalement with conventional 1 mol/l KCl microelectrodes and cable analysis. The transmural voltage (V _T) was not different from 0 mV, while the basolateral transmembrane voltage (V _B) was –81.7±0.91 mV (n=221). The transmural resistance (R _T) was 109 cm², indicating that the IMCD₂ is composed of tight epithelia. The fractional apical membrane resistance (fR _A) was 0.98±0.003 (n=10). Abrupt changes in the luminal concentration of Na⁺, K⁺ or Cl^– did not alter the apical membrane voltage (V _A) or V _T, and neither 2 mmol/l Ba²⁺ nor 10 mol/l amiloride in the lumen affected V _A and V _T. Moreover, pretreatment of hamsters with deoxycorticosterone acetate (5 mg/ kg, s.c.) for 10–14 days caused only a very small change in V _T in the negative direction. Amiloride in the lumen increased R _T and increased the voltage divider ratio very slightly. However, an abrupt increase in K⁺ concentration in the bath from 5 mmol/l to 50 mmol/l or addition of 2 mmol/l Ba²⁺ to the bath depolarized the basolateral membrane by 39 mV and 29 mV, respectively. In the presence of 2 mmol/l Ba²⁺ in the bath, a reduction of HCO₃ ^– concentration from 25 mmol/l to 2.5 mmol/l depolarized V _B by 20.4 mV. No Cl^– conductance was demonstrated in the basolateral membrane. Addition of ouabain to the bath or elimination of K⁺ from the bath caused only very small changes in V _B of the IMCD₂ as compared to the marked responses ovserved in the medullary thick ascending limb of the loop of Henle and in the upper portion of the descending limb of the long-looped nephron. These findings are compatible with the view that either a weak Na⁺-K⁺ pump or an ouabain-resistant pump in combination with high K⁺ conductance in the basolateral membrane mainly accounts for the maintenance of the intracellular concentration of Na⁺ and K⁺. The IMCD₂ may contribute little to the transmural transport of Na⁺ and K⁺. The physiological significance of HCO₃ ^– conductance in the basolateral membrane remains to be established.     

Effects of ouabain and temperature on cell membrane potentials in isolated perfused straight proximal tubules of the mouse kidney

In isolated perfused segments of the mouse proximal tubule, the potential difference across the basolateral cell membrane (PDbl) was determined with conventional microelectrodes. Under control conditions with symmetrical solutions it amounted to –62±1 mV (n=118). The potential difference across the epithelium (PDte) was –1.7±0.1 mV (n=45). Transepithelial resistance amounted to 1.82±0.09 k cm (n=28), corresponding to 11.4±0.6 cm². Increasing bath potassium concentration from 5 to 20 mmol/l depolarized PDbl by +24±1 mV (n=103), and PDte by +1.6±0.1 mV (n=19). Thus, the basolateral cell membrane is preferably conductive to potassium. Rapid cooling of the bath perfusate from 38°C to 10°C led to a transient hyperpolarization of PDbl from –60±1 to –65±1 mV (n=21) within 40 s followed by gradual depolarization by +18±1% (n=14) within 5 min. The transepithelial resistance increased significantly from 1.78±0.11 k cm to 2.20±0.21 k cm (n=15). Rapid rewarming of the bath to 38°C caused a depolarization from –61±2 mV (n=17) to –43±2 mV (n=16) within 15 s followed by a repolarization to –59±2 mV (n=10) within 40 s. Ouabain invariably depolarized PDbl. During both, sustained cooling or application of ouabain, the sensitivity of PDbl to bath potassium concentration decreased in parallel to PDbl pointing to a gradual decrease of potassium conductance. Phlorizin hyperpolarized the cell membrane from –59±2 to –66±1 mV (n=13), virtually abolished the transient hyperpolarization under cooling, and significantly reduced the depolarization after rewarming from +17±2 mV (n=16) to +9±3 mV (n=9).The present data indicate that the contribution of peritubular potassium conductance to the cell membrane conductance decreases following inhibition of basolateral (Na⁺+K⁺)-ATPase. Apparently, cooling from 37° to 10°C does not only reduce (Na^–+K⁺)-ATPase activity but in addition luminal sodium uptake mechanisms such as the sodium glucose cotransporter. As a result, cooling leads to an initial hyperpolarization of the cell followed by depolarization only after some delay.Parts of this study have been presented at the 60th and 61th Meeting of the Deutsche Physiologische Gesellschaft, Dortmund 1984 and Berlin 1985     

Transport properties of the basolateral membrane of the oxyntic cells in frog fundic gastric mucosa

The conductive properties of the basolateral membrane of oxyntic cells (OC) of frog fundic gastric mucosa were investigated by utilizing the microelectrode technique. By examining the response of the basolateral cell membrane potential difference,V _cs, to sudden ion concentration changes in the serosal bath it was concluded that the basolateral membrane of OC has a high Ba²⁺-sensitive K⁺-conductance, and no Cl^–-conductance both in resting (cimetidine) and in stimulated (histamine) state. The response ofV _cs to serosal Cl^–-removal, consisting in a slight hyperpolarization (anomalous Nernst response), could not be explained by possible permeability changes to K⁺ and Na⁺ since the potential response to Cl^– was essentially preserved by blocking K⁺-permeability with Ba²⁺ and replacing all Na⁺ by choline. Conversely, hyperpolarization ofV _cs after Cl^–-free perfusion was abolished by exposure to HCO ₃ ^– -free solution, indicating that HCO ₃ ^– -ions are required at the serosal bath for Cl^– to get his effect. It was investigated wether the effect of Cl^– was due to an electrogenic Na⁺(HCO ₃ ^– ) _n /Cl^– exchange mechanism on the basolateral membrane. Experiments showed that the potential response to HCO ₃ ^– -removal and to Na⁺-removal, consisting in a depolarization ofV _cs, was similar both in presence and in absence of Cl^–. Furosemide (0.5 mmol/l) had no effect on steadyV _cs andV _t. The electrophysiological analysis of the data led to excluding the involvement of Na-Cl, Na-2Cl and NaK-2Cl cotransports, and to including the existence of an electrogenic Na⁺(HCO ₃ ^– ) _n /Cl^– exchange process, while suggests the presence of an electroneutral Cl^–/HCO ₃ ^– exchange mechanism to explain Cl^–-transport across the basolateral membrane of OC.This work was supported by a research grant from Ministero della Pubblica Istruzione, Rome, Italy     

Small and maxi K+ channels in the basolateral membrane of isolated crypts from rat distal colon: single-channel and slow whole-cell recordings

The patch-clamp technique was used to characterize K⁺ channel activity in the basolateral membrane of isolated crypts from rat distal colon. In cell-attached patches with KCl in the pipette, channels with conductances ranging from 6 pS to 80 pS appeared. With NaCl in the pipette and KCl in the bath in excised inside-out membrane patches a small-conductance channel with a mean conductance of 12±6 pS (n=18) was observed. The channel has been identified as K⁺ channel by its selectivity for K⁺ over Na⁺ and by its sensitivity to conventional K⁺ channel blockers, Ba²⁺ and tetraethylammonium (TEA⁺). Changes of cytosolic pH did not attenuate channel activity. Activity of the 12-pS channel was increased by membrane depolarization and elevated cytosolic Ca²⁺ concentration. In addition, a maxi K⁺ channel with a mean conductance of 187±15 pS (n=4) in symmetrical KCl solutions was only occasionally recorded. The maxi K⁺ channel could be blocked by Ba²⁺ (5 mmol/l) on the cytosolic side. Using the slow-whole cell recording technique under control conditions, a cell membrane potential of –70±10mV (n=18) was measured. By application of various K⁺ channel blockers such as glibenclamide, charybdotoxin, apamin, risotilide, Ba²⁺ and TEA⁺ in the bath, only Ba²⁺ and TEA⁺ depolarized the cell membrane. The present data suggest that the small K⁺ channel (12 pS) is involved in the maintenance of the cell membrane resting potential.