cAMP increases the basolateral Cl−-conductance in the isolated perfused medullary thick ascending limb of Henle's loop of the mouse

The effect of cAMP on transepithelial and transmembrane potential differences and resistances was examined in isolated in vitro perfused mouse medullary thick ascending limbs of Henle's loop (mTAL). The effects of furosemide and barium were tested. Stimulation of NaCl transport by ADH 10^–9+dbcAMP 4·10^–4+forskolin 10^–6 mol·l^–1 (paired experiments) resulted in: a) an increase in transepithelial potential difference, referenced to the grounded bath, from +6.7±0.3 mV to +12.0±0.4 mV (n=47); b) a decrease in transepithelial resistance from 25±1 cm² to 20±1 cm² (n=47); c) a depolarization of the basolateral membrane by 12 mV and of the apical membrane by 7 mV (n=36); d) a decrease in the fractional resistance of the basolateral membrane from 0.27±0.005 to 0.15±0.06 (n=12). Furosemide (10^–4 mol·l^–1) abolished the active transepithelial transport potential and hyperpolarized the basolateral membrane potential to values which were similar in both control and cAMP treated mTAL segments. Barium increased the transepithelial resistance and depolarizedPD _bl to similar values in both functional states. An increase in the fractional conductance of the basolateral membrane was also seen, if, prior to the cAMP treatment, the luminal Na⁺2Cl^–K⁺ contransport was inhibited by furosemide. Thus, we propose that stimulation of active NaCl reabsorption in the mTAL segment of the mouse by ADH, mediated via cAMP, increases primarily the basolateral chloride conductance.Supported by Deutsche Forschungsgemeinschaft Gr 480/6-2Parts of this study have been presented at the 59th Meeting of the German Physiological Society in Dortmund 1984 and at the 69th FASEB Meeting in Anaheim 1985     

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.     

Electrical properties of cultured renal tubular cells (OK) grown in confluent monolayers

OK cells grown to confluent monolayers were investigated by microelectrode techniques and microinjection. Cell membrane potential difference (PD_m) in bi-carbonate-free solution is –61.8±0.6 mV (n=208), cell membrane resistance (R_m) amounts to 1.4±0.2k · cm² (n=8). The apparent transference number for potassium (t_K ⁺) is 71±3% (n=28) and can be reduced by 3 mmol/l BaCl₂ to 7.5±4.0%; (n=8). In the presence of extracellular CO₂ and HCO ₃ ^– (pH 7.4) the cells acidify by 0.34±0.05 pH units (n=12). This leads to a depolarization of PD_m by 8.4±1.8 mV (n=8), an increase in R_m by 49±10% (n= 10), and a reduction of K⁺-conductance to 63±5% (n= 13). Intracellular acidification by the NH₄Cl-prepulse technique also inhibits K⁺-conductance and depolarizes the membrane. Recovery from an intracellular acid load is reflected by cell membrane repolarization. This recovery can be inhibited by amiloride (10^–3 mol/l). Na⁺- and Cl^–-conductances could not be detected.The transepithelial resistance (R _te) of OK cell monolayers 1 day after plating is 41±6 ·cm² and decreases with time after plating. Intercellular communication (electrical or dye coupling) was not observed.Conclusions: 1. The membrane potential of OK cells is largely determined by a pH-sensitive, barium-blockable K⁺-conductance. 2. Amiloride-blockable Na⁺/H⁺-exchange is reflected by membrane potential changes via this K⁺-conductance. 3. Monolayers of OK cells are electrically leaky.Parts of this study were presented at the 66th meeting of the Deutsche Physiologische Gesellschaft, Würzburg, September 1988 [Pflügers Arch 412 (Suppl 1):R55].     

Electrophysiology of cell volume regulation in proximal tubules of the mouse kidney

The present study has been designed to test for the influence of cell swelling on the potential difference and conductive properties of the basolateral cell membrane in isolated perfused proximal tubules. During control conditions the potential difference across the basolateral cell membrane (PD_bl) is –65±1 mV (n=74). Decrease of peritubular osmolarity by 80 mosmol/l depolarizes the basolateral cell membrane by +7.8±0.5 mV (n=42). An increase of bath potassium concentration from 5 to 20 mmol/l depolarizes the basolateral cell membrane by +25±1 mV (n=11), an increase of bath bicarbonate concentration from 20 to 60 mmol/l hyperpolarizes the basolateral cell membrane by –3.2±0.5 mV (n=13). A decrease of bath chloride concentration from 79.6 to 27 mmol/l hyperpolarizes the basolateral cell membrane by –1.8±0.7 mV (n=6). During reduced bath osmolarity, the influence of altered bath potassium concentration on PD_bl is decreased ( PD_bl=+16±2 mV,n=11), the influence of altered bicarbonate concentration on PDbl is increased ( PD_bl=–6.0±0.8 mV,n=13), and the influence of altered bath chloride concentration on PD_bl is unaffected ( PD_bl=–1.8±0.6 mV,n=6). Barium depolarizes the basolateral cell membrane to –28±2 mV (n=16). In the presence of 1 mmol/l barium, decrease of peritubular osmolarity by 80 mosmol/l leads to a transient hyperpolarization of the basolateral cell membrane by –5.9±0.5 mV (n=16). This transient hyperpolarization is blunted in the absence of extracellular bicarbonate. In conclusion, cell swelling depolarizes straight proximal tubule cells and increases bicarbonate selectivity of the basolateral cell membrane at the expense of potassium selectivity. The data reflect either incrases of bicarbonate conductance or decrease of potassium conductance during exposure of proximal tubule cells to hypotonic media.Parts of this work were presented at the 18th Congress of the Gesellschaft für Nephrologie, Frankfurt/M. 1986 and at the 8th International Symposium on Biochemical Aspects of Kidney Function, Dubrovnik 1986     

Effect of bicarbonate on potassium conductance of isolated perfused rat pancreatic ducts

The aim of this study was to investigate the role of the K⁺ conductance in unstimulated and stimulated pancreatic ducts and to see how it is affected by provision of exogenous HCO ₃ ^– /CO₂. For this purpose we have applied electrophysiological techniques to perfused pancreatic ducts, which were dissected from rat pancreas. The basolateral membrane potential PD_bl of unstimulated duct cells was between –60mV and –70mV, and the cells had a relatively large K⁺ conductance in the basolateral membrane as demonstrated by (a) 20–22 mV depolarization of PD_bl in response to increase in bath K⁺ concentration from 5 mmol/l to 20mmol/l and (b) the effect of a K⁺ channel blocker, Ba²⁺ (5 mmol/l), which depolarized PD_bl by 30–40mV. These effects on unstimulated ducts were relatively independent of bath HCO ₃ ^– /CO₂. The luminal membrane seemed to have no significant K⁺ conductance. Upon stimulation with secretin or dibutyryl cyclic AMP, PD_bl depolarized to about –35 mV in the presence of HCO ₃ ^– /CO₂. Notably, the K⁺ conductance in the stimulated ducts was now only apparent in the presence of exogenous HCO ₃ ^– /CO₂ in the bath solutions. Upon addition of Ba₂₊, PD_bl depolarized by 13±1 mV (n=7), the fractional resistance of the basolateral membrane, FR_bl increased from 0.66 to 0.78 (n=6), the specific transepithelial resistance, R _te, increased from 52±13 cm² to 59±15 cm² (n=11), and the whole-cell input resistance, R _c, measured with double-barrelled electrodes, increased from 20 M to 26 M (n=3). These results are consistent with Ba²⁺ inhibition of the K⁺ conductance. Following removal of exogenous HCO ₃ ^– /CO₂ in the same ducts, stimulation led to a larger depolarization on PD_bl to about –25 mV, and Ba²⁺ had a smaller effect on PD_bl and no significant effect on the resistances. The individual resistances in the duct epithelium were estimated from equivalent circuit analysis. The luminal membrane resistance, R ₁ decreased from about 2000 cm² to 80 cm² upon stimulation. The basolateral membrane resistance, R _bl, remained at 90–120 cm², and the paracellular shunt resistance, R _s, at 50–80 cm². Ba²⁺ increased R _bl of stimulated ducts to about 200 cm², an effect present only if the ducts were provided with exogenous HCO ₃ ^– /CO₂. Taken together, the present results indicate that the basolateral K⁺ conductance of pancreatic ducts is sensitive to exogenous HCO ₃ ^– /CO₂, i.e. without HCO ₃ ^– /CO₂ the conductance becomes very low although the ducts are undergoing stimulation.A preliminary report of the present study has been presented at the XXXI International Congress of Physiological Sciences, Helsiniki, Finland, July 1989     

Taurocholate depolarizes rat hepatocytes in primary culture by increasing cell membrane Na+ conductance

Rat hepatocytes in primary culture were impaled with conventional microelectrodes. Addition of 5–100 mol/l taurocholate led to a slowly developing depolarization that was maximal at 50 mol/l (10.5±1.5 mV, n=15) and not reversible. The effect was Na⁺ dependent and decreased in cells preincubated with 1 mol/l taurocholate. Increasing external K⁺ tenfold depolarized the cells by 12.3±2.3 mV under control conditions and by 6.3±1.2 mV with 50 mol/l taurocholate present (n=7). Depolarization by 1 mmol/l Ba²⁺ was 7.6±0.8 mV and 6.0±0.7 mV (n=9) before and after addition of taurocholate, respectively. Cable analysis and Na⁺ substitution experiments reveal that this apparent decrease in K⁺ conductance reflects an actual increase in Na⁺ conductance: in the presence of taurocholate, specific cell membrane resistance decreased from 2.8 to 2.3 k · cm² · Na⁺ substitution by 95% depolarized cell membranes by 8.9±2.9 mV (n=9), probably due to indirect effects on K⁺ conductance via changes in cell pH. With taurocholate present, the same manoeuvre changed membrane voltages by –0.8±2.6 mV. When Na⁺ concentration was restored to 100% from solutions containing 5% Na⁺, cells hyperpolarized by 3.5±3.6 mV (n=7) under control conditions and depolarized by 4.4±2.9 mV in the presence of taurocholate, respectively. In Cl^– substitution experiments, there was no evidence for changes in Cl^– conductance by taurocholate. These results show that taurocholate-induced membrane depolarization is due to an increase in Na⁺ conductance probably via uptake of the bile acid.     

Electrophysiological characterization of rabbit distal convoluted tubule cell

The distal convoluted tubule (DCT) from rabbit kidney were perfused in vitro to study the conductive properties of the cell membranes by using electrophysiological methods. When the lumen and the bath were perfused with a biearbonate free solution buffered with HEPES, the transepithelial voltage (V _T) averaged –2.8±0.6 mV (n=20), lumen negative. The basolateral membrane voltage (V _B) averaged –77.8±1.1 mV (n=33) obtained by intracellular impalement of microelectrodes. Cable analysis performed by injecting a current from perfusion pipette revealed that the transepithelial resistance was 21.8±1.7 ·cm² and the fractional resistance of the luminal membrane was 0.78±0.03 (n=8), indicating the existence of ionic conductances in the luminal membrane. Addition of amiloride (10^–5 mol/l) to the luminal perfusate or Na⁺ removal from the lumen abolished the lumen negativeV _T and hyperpolarized the apical membrane. An increase in luminal K⁺ concentration from 5 to 50 mmol/l reduced the apical membrane potential (V _A) by 37.5±2.6 mV (n=7), whereas a reduction of Cl^– in the luminal perfusate did not changeV _A significantly (0.5±0.5 mV,n=4). Addition of Ba²⁺ to the lumen reducedV _A by 42.6±1.0 mV (n=4). When the bathing fluid was perfused with 50 mmol/l K⁺ solution, the basolateral membrane voltage (V _B) fell from –76.8±1.5 to –31.0±1.3 mV (n=18), and addition of Ba²⁺ to the bath reducedV _B by 18.3±4.8 mV (n=7). Although a reduction of Cl^– in the bathing fluid from 143 to 5 mmol/l did not cause any significant fast initial depolarization (1.8±1.7 mV,n=8), a spike like depolarization (14.0±2.5 mV,n=4) was observed, upon Cl^– reduction in the presence of Ba²⁺ in the bath. From these results, we conclude that the apical membrane of DCT has both K⁺ and Na⁺ conductances and the basolateral membrane has a K⁺ conductance and a small Cl^– conductance.     

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.     

Active NaCl transport in the cortical thick ascending limb of Henle's loop of the mouse does not require the presence of bicarbonate

The aim of the present study was to investigate whether bicarbonate buffer (CO₂ + HCO ₃ ^– ) is required to sustain maximal NaCl transport in the cortical thick ascending limb of Henle's loop (cTAL) of the mouse. Transepithelial Na⁺ and Cl^– net fluxes (J _Na, J _Cl, pmol min^–1 mm^–1), measured by electron microprobe analysis, were similar irrespective of the presence or absence of CO₂ + HCO ₃ ^– in luminal and bathing solutions J _NaCl with CO₂ + HCO ₃ ^– =203±25 pmol min^–1 mm^–1; J NaCl without CO₂ + HCO ₃ ^– =213±13 pmol min^–1 mm^–1, n=14). Furthermore the transepithelial potential difference, V _te, the transepithelial resistance, R _te, and the basolateral membrane potential, V _bl, were unaffected by CO₂ + HCO ₃ ^– . In the absence of CO₂ + HCO ₃ ^– , V _te was +17.0±1.7 mV(n=9) (lumen positive), R _te was 28±2 cm² (n=9) and V _bl was –76±4 mV (n=6). In the presence of CO₂ + HCO ₃ ^– , V _te, R _te and V _bl were +15.9±1.5 mV, 29±1 cm² and –73±5 mV, respectively. 4-Acetamido-4-isothiocyanatostilbene-2,2-disulphonic acid (SITS; 0.1 mmol l^–1) and amiloride (1 mmol l^–1) added to the (CO₂ + HCO ₃ ^– )-containing lumen perfusate were without effect on V _te and R _te. Finally, the effect of furosemide (0.1 mmol l^–1) on V _te and V _bl in the presence of CO₂ + HCO ₃ ^– was investigated. Furosemide reversibly decreased V _te from +13.7±1.1 mV to +1.7±0.7 mV (n=6) and hyperpolarized V_bl from –70±1 to –89±3 mV (n=5), suggesting passive distribution of Cl^– across the basolateral membrane. In conclusion, these data suggest that active NaCl transport in the cTAL of the mouse does not require the presence of CO₂ + HCO ₃ ^– .     

Electrophysiological studies in principal cells of rat cortical collecting tubules ADH increases the apical membrane Na+-conductance

The mechanism of ion transport across principal cells of rat cortical collecting tubules (CCT) and its regulation by vasopressin (ADH) has been studied in the isolated perfused tubule. To amplify the response to ADH rats were treated with 5 mg I. M. desoxycorticosterone 4–9 days prior to the experiments. Addition of 2·10^–10 mol·1^–1 ADH increased the transepithelial voltage from –5.1 ±0.7 mV to –16.1±1.4 mV (n=37) and decreased the transepithelial resistance from 51±4 cm² to 39±2 cm² (n=33). Optical and functional differentiation of impalements of principal and intercalated cells was made and only data of principal cells are presented. ADH depolarized the apical membrane from 79±1 mV to 66±2 mV (n=26) and decreased the fractional resistance of the apical membrane from 0.76±0.04 to 0.70±0.04 (n=13). These ADH effects were prevented by 10^–5 or 10^–4 mol·1^–1 luminal amiloride which hyperpolarized the apical membrane when added in the presence or absence of ADH. Apical and basolateral membranes were dominated by large K⁺ conductances and addition of 3 mmol·1^–1 barium to bath or lumen perfusates increased transepithelial resistance almost two-fold, whereas luminal amiloride increased the transepithelial resistance only by 26–35%. Ouabain (0.5 mmol·1^–1, bath) depolarized the basolateral membrane and decreased its K⁺ conductance. These effects were prevented by the simultaneous presence of apical amiloride suggesting that the only route of Na⁺ entry into the principal cells occurred via the amiloride sensitive Na⁺ conductance. We conclude that ADH stimulates Na⁺ reabsorption and K⁺ secretion in the rat CCT primarily by increasing the Na⁺ conductance in the apical cell membrane.Parts of this study have been presented at the 19th ASN meeting in Washington, DC, USA 1986     

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.     

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]     

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.     

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.     

Intracellular potentials of microperfused human sweat duct cells

Intracellular potentials of cells from isolated segments of microperfused human sweat ducts were measured in order to determine the electrical profiles of these cells under resting, transporting, and inhibited conditions. Even though the cells are relatively small (ca. 6–8 m), continuous recordings of intracellular potentials from the same impalement were stable for up to 2 h. In the resting condition in normal Ringer's solution when the lumen of the duct was collapsed and not perfused, the intracellular potential measured across the basal membrane was 34.6±1.5 mV (n=31; mean±SE). In the same bathing medium, when the duct lumen was also perfused with normal Ringer's solution, the basolateral membrane potential (V _b), the apical membrane potential (V _a) and transepithelial potential (V _t) was –33.8±0.47 mV, –23.7±0.48 mV and –9.6±0.9 mV (n=73), respectively. The average input impedence (R _i) of these cells was 19.6±0.4 M (n=36). The frequency distribution ofV _b was unimodal suggesting that only one functional cell type exists in this tissue. Amiloride (0.1 mM) in the lumen hyperpolarized bothV _a andV _b by –40.5±3.6 mV and –33.2±3.7 mV (n=15), respectively, with a slight but significant increase inR _i (15%), while abolishingV _t. Removing luminal Cl^– depolarizedV _a by +37.0±4.2 mV and hyperpolarizedV _b by –19.0±4.2 mV (n=11). Removing Cl^– from the bath hyperpolarizedV _a by –3.3±2.3 mV and depolarizedV _b by +24.3±2.7 mV (n=15). Ouabain caused an initial fast depolarization (+8 mV) followed by a prolonged slow depolarization ofV _b, and an increase inR _i of about 84%. These results not only provide the first electrical profile of the human sweat duct tissue, but they also show that its cell membrane potentials are unusually low. This unusual property of this epithelium appears to be due to the combination of a significant Na⁺ conductance at the apical membrane and a remarkably high tissue Cl^– conductance.     

Effects of diadenosine polyphosphates,ATP and angiotensin II on membrane voltage and membrane conductances of rat mesangial cells

Diadenosine polyphosphates have been shown to influence renal perfusion pressure. As mesangial cells may contribute to these effects we investigated the effects of diadenosine triphosphate (Ap₃A), diadenosine tetraphosphate (Ap₄A), diadenosine pentaphosphate (Ap₅A) and diadenosine hexaphosphate (Ap₆A) on membrane voltage (V _m) and membrane conductance (g _m) in mesangial cells (MC) of normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats in primary and long-term culture. We applied the patch-clamp technique in the fast-whole-cell configuration to measure V _m and g _m. To compare the effects of diadenosine polyphosphates with hitherto known agonists we also tested adenosine 5-triphosphate (ATP) and angiotensin II (Ang II). As there was no significant difference in the V _m values in MC of WKY (–42±1 mV, n=70) and SHR rats (–45±2 mV, n=99) as well as in the agonist-induced changes of V _m, all data were pooled. The V _m of all the cells was –44±1 mV (n=169) and g _m was 15.9±1.8 nS (n=141). Ion-exchange experiments showed the presence of a K⁺ and a non-selective cation conductance in resting MC whereas a Cl^– conductance or a Na⁺selective conductance could not be observed. Ap₃A, Ap₄A, Ap₅A, AP₆A and ATP each at a concentration of 5 mol/l, led to a significant depolarization of V _m by 5±2 mV (n=14), 7±1 mV (n=25), 3±1 mV (n=23), 2±1 mV (n=16), and 14±2 mV (n=23), respectively. For Ap₄A, the most potent diadenosine polyphosphate, we determined the half-maximally effective concentration (EC ₅₀) as 6 mol/l (n=5–25), for ATP as 2 mol/l (n=9–37), and for Ang II as 8 nmol/l (n=6–18). Ap₄A 100 mol/l increased g _m significantly by 55±20% (n=16), 100 mol/l ATP by 135±60% (n=18). The diadenosine polyphosphates examined were able to depolarize V _m (Ang II >ATP> Ap₄A>Ap₃A>Ap₅A>Ap₆A) by activation of a Cl^– conductance and a non-selective cation conductance, as do ATP or Ang II.     

Intracellular potassium activity in epithelial cells of frog fundic gastric mucosa

Microelectrodes were used to measure membrane potential and intracellular potassium activity in surface epithelial cells (SEC) of frog (Rana esculenta) fundic gastric mucosa in vitro. Separate measurements were carried out by applying fine-tipped, single barrelled, KCl filled non-selective electrodes and liquid K⁺-selective electrodes. Membrane potentials with respect to the mucosal and serosal surfaces, measured with non-selective electrodes, were –54.5±1.0 S.E. mV (n=59) and –73.0±1.1 S.E. mV (n=59) respectively. The electrical potential difference referred to the mucosal surface, when measured with K⁺-sensitive electrodes, was +21.2±0.8 S.E. mV (n=35), and intracellular K⁺ activity was 98.5 mmol/l. Assuming that intracellular and extracellular K⁺ activity coefficients are equal (_K=_K), the K⁺ concentration is 135.0 mmol/l. The K⁺ equilibrium potential,E _K, was calculated as –90.0 mV i.e. more negative than both membrane potentials. This result indicates active potassium accumulation in the SEC and provides direct evidence of the presence of an active K⁺ pump in either both or in only one of the cell membranes.     

A new oil-gap method for internal perfusion and voltage clamp of single cardiac cells

(1.) We designed a new technique to achieve fast voltage clamp, combined with internal perfusion. The single guinea-pig cardiac cell, dissociated by collagenase treatment, was stretched across an oil-gap (30–40 m wide) from a pool of Tyrode solution to a pool of internal solution. Part of the cell membrane was disrupted in the internal solution by crushing on the cell, a tapered tip of a glass capillary. Through the open end, the intracellular medium was equilibrated with test solutions and electrical current was injected for the voltage clamp of the membrane in the Tyrode pool. (2.) The capacitive transient on stepping the membrane potential decayed with a time constant of 10–60 s, depending on the capacitive area (20–80 pF). The time course was a single exponential in 46% of the atrial cells and in 66% of the ventricular cells. In these tissues the series resistance, approximated by a ratio of the time constant andC _m, was 686±180 k (n=37) in the ventricular cells or 812±143 k (n=18) in the atrial cells. The stable seal resistance (R _seal) established in the oil-gap was around 33 M in the ventricular cells and 100 M in the atrial cells. (3.) A rapid increase in the inward current followed by a slow decay was observed on repolarization over the range negative to the potassium equilibrium potential. From the inward rectification of both peak and late currents and suppressive effects of Cs⁺ on the current, the current changes were atrributed to activation and inactivation of the inward rectifier K channel. (4.) The Na current was activated by depolarization from a holding potential of –100 mV across a threshold of about –60 mV. In normal external (145 mM Na⁺) and internal (15 mM Na⁺) solutions, peak amplitude was obtained around –25 mV. The maximum chord conductance was 6.2±1.6 mS/F in 15 ventricular cells and 3.0±0.90 mS/F in 9 atrial cells in normal Tyrode solution. The process of inactivation was fitted with a sum of two exponential functions. (5.) The reversal potential of the Na current agreed well with that predicted from the Nernst equation during perfusion of 15 and 100 mM Na⁺ internal solutions in the presence of external 140 mM Na⁺. The shift of the reversal potential was completed within 30 s of switching the internal solution. (6.) This oil-gap voltage clamp technique facilitates control of the composition of both the intra- and extra-cellular media and should prove suitable for use in studies of intracellular mechanisms controlling the membrane current of enzymatically dissociated elongated cells.     

Small-conductance chloride channels induced by cAMP,Ca2+, and hypotonicity in HT29 cells: ion selectivity,additivity and stilbene sensitivity

Previous studies in HT₂₉ cells utilizing the cellattached nystatin (CAN) method [Greger R, Kunzelmann K (1991) Pflügers Arch 419:209–211] have revealed that the Cl^– channels induced by cAMP or by increasing cytosolic Ca²⁺, e.g. by addition of ATP, and by hypotonic cell swelling share in common their conductance, which was so small in our studies [Kunzelmann et al. (1992) Pflügers Arch (in press)] that we could not resolve it at the single-channel level. This prompted the question whether these Cl^– conductances can be distinguished in terms of their ion selectivity and sensitivity towards inhibitors. Whether these pathways are additive or not was also examined. The present study utilized the whole-cell patch-clamp and the CAN methods. A total of 160 patches were studied. In whole-cell patches 8-(4-chlorophenylthio)-cAMP (cAMP, 0.1±1 mmol/l) induced a significant depolarization by 5 mV and a twofold increase in conductance (G) from 6.2±1.5 nS to 11.7±3.2 nS (n=15). Total replacement of Cl^– by Br^– and I^– in cAMP-treated cells hyperpolarized the membrane voltage (V) significantly from –35±2.8 to –39±3.4 and –45± 3.3 mV respectively, but had no detectable effect on G, which was 11.9±3.3 nS in the case of Br^– and 11.8± 3.3 nS in the case of I^–. Hence, the permselectivity of the cAMP pathway was I^–>Br^–>Cl^–, but the conductances for these anions were all indistinguishable. For ATP at 10–100 mol/l the depolarization was least with I^–: from –41±1.1 to –36±2.4mV, intermediate for Br^– to –25±1.6 mV, and largest for Cl^– to –20±1.8 mV (n=18). ATP increased G from 3.4±0.3 nS to 12.9±2.8 nS (Cl^–), to 12.9±2.8 nS (Br^–) and to 12.9±2.7 (I^–) (n=18). These data indicate that the ATP-induced anion channel has a permeability sequence of I^–>Br^–>Cl^–. The conductance for all three anions was identical. Hypotonic cell swelling by 160 mosmol/l induced a depolarization that was smallest for I^–, from –42±4 to –32±2.1 mV, intermediate for Br^–: –29±1.8mV, and similar for Cl^–: –28±2 mV (n=20). G was increased from 2.8±0.8 nS to 15±2.5nS in the case of Cl^–, to 15±2.5 nS for Br^– and to 16±2.6 nS for I^– (n=20). Therefore, all three pathways are indistinguishable with respect to their anion selectivity. All three pathways are insensitive towards low concentrations of 4-nitro-2-(3-phenylpropylamino)benzoate, but are all blocked by 4,4-diisothiocyanatostilbene-2,2-disulphonic acid, with a half-maximal inhibition around 0.6 mmol/l. Finally, the possible additivity was examined in three permutations. ATP (0.1 mmol/l) alone (n=14) had a slightly but not significantly larger effect on conductance than the combination of ATP and cAMP (1 mmol/l, n=14) and the combination of ATP and hypotonicity (193 mosmol/l, n=13). Similarly, the effects of hypotonicity and cAMP (n=11) were not additive. These data indicate that all three pathways share common properties. Hence, it is suggested that all three pathways converge on the same small Cl^– channel.Supported by DFG Gr 480/10 and BMFT 01 GA 8816     

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