The disulfonic stilbene DIDS and the marine poison maitotoxin activate the same two types of endogenous cation conductance in the cell membrane of Xenopus laevis oocytes.

In the present experiments we exposed the intra- or extracellular surface of excised giant membrane patches of Xenopus laevis oocytes bathed in 140 mmol/l Na-aspartate solution to the anion transport inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS, 250 micromol/l). We observed that DIDS activated at least two cation conductances: (1) a non-selective cation (NSC) conductance that was mediated by channels of approximately 27 pS and resembled the stretch-activated cation conductance that has been observed in the oocyte cell membrane previously, and (2) a Na+-selective conductance, the single-channel events of which could not be resolved and which resembled the depolarization-induced Na+ conductance that has also been observed in the oocyte cell membrane previously. Both conductances were blocked by 1 mmol/l amiloride from the intra- and extracellular surfaces but inhibition of the NSC conductance by extracellular amiloride was less pronounced. Both conductances activated only slowly with a delay of 15-60 s after application of DIDS and remained active even after DIDS was washed off. This suggests that DIDS caused the exocytosis of preformed channels and this interpretation was supported by our additional observation that extracellular application of maitotoxin (MTX) mimicked the effects of DIDS. MTX is a marine toxin that has recently been reported to induce exocytosis in Xenopus laevis oocytes. The fact that DIDS and MTX each carry two sulfonyl groups suggests that they act on the same positively charged binding sites of an exocytosis-inducing protein. Our observations demonstrate that using DIDS to inhibit heterologously expressed anion transporters in the cell membrane of Xenopus laevis oocytes may compromise proper determination of the transporter currents. This effect can be prevented if the DIDS-activated endogenous cation conductances are suppressed by application of amiloride to the cytoplasmic surface of the cell membrane.

The renal Na-HCO3-cotransporter expressed in Xenopus laevis oocytes: inhibition by tenidap and benzamil and effect of temperature on transport rate and stoichiometry.

In the present experiments we expressed the rat kidney Na+-HCO3- cotransporter (rkNBC) in Xenopus laevis oocytes to reinvestigate the flux coupling ratio under improved measuring conditions. Essentially the current/voltage (I/V) relationship of isolated inside-out giant membrane patches was measured and the stoichiometric ratio was calculated from the reversal potential (VI=0) of the cotransport current (INBC). INBC was defined as that part of the total current that was suppressed when rkNBC was inhibited. Previously we have used the disulfonic stilbene DIDS to inhibit rkNBC, but we now found that tenidap or benzamil are better suited as inhibitors. Tenidap blocked rkNBC rapidly and reversibly both from the intra- and extracellular surface with half maximal inhibition at 13 micromol/l and it did not cause the same potentially disturbing side effects as DIDS. In addition, we found that the endogenous depolarization-induced Na+ conductance of the oocyte, which may compromise the I/V analysis, can be suppressed by applying 1 mmol/l amiloride to the cytosolic surface of the patch. The new measuring conditions greatly increased the yield of successful experiments. The distribution of 27 measurements of VI=0 obtained at near physiological Na+ and HCO3- concentrations and in absence of Cl-, K+ and cytosolic Ca2+ showed that the calculated stoichiometric ratios closely approached the value of 2 HCO3-:1 Na+ if the expression density of rkNBC was high. This result fully confirms our previous observations. Further experiments showed that the difference between the stoichiometric ratio of 3:1 observed in rat proximal tubule in vivo and the present value is not due to the temperature difference. We conclude that, depending on local modulatory influences, rkNBC can operate with different stoichiometric ratios and the present data and those reported in an accompanying publication [Müller-Berger et al., Pflügers Arch (2001) DOI 10.1007/s004240100592] show that these ratios are integer numbers i.e. either 2:1 or 3:1.

The renal Na-HCO3-cotransporter expressed in Xenopus laevis oocytes: change in stoichiometry in response to elevation of cytosolic Ca2+ concentration.

The Na+-HCO3- cotransporter of rat kidney (rkNBC) was expressed in Xenopus laevis oocytes to test whether cytosolic Ca2+ ([Ca2+]i) affects the cotransport stoichiometry. The current/voltage relationship of giant inside-out membrane patches of rkNBC-expressing oocytes was measured at near-physiological Na+ and HCO3- concentrations and the cotransport current, INBC, was defined as the current inhibited by 0.25 mmol/l tenidap. Essentially, we determined the reversal potential (VI=0) of INBC and the slope conductance (gNBC). The coupling ratio of (HCO3-) to Na+ (q) was calculated from VI=0. As reported in the preceding publication [Ducoudret et al., Pflügers Arch (2001) DOI 10.1007/s004240100594], in Ca2+-free solutions q was 2:1. This did not change when [Ca2+]i was increased to 0.1 micromol/l. At 0.5 micromol/l, however, only a few patches showed q=2:1, while most patches exhibited q=3:1. This indicates that [Ca2+]i affected the transport function of membrane-resident rkNBC molecules, and the bimodal distribution of VI=0 points to an indirect effect possibly mediated by differently expressed Ca2+-dependent protein kinases. The shift in q was associated with the predicted near twofold increase in gNBC and was confirmed by measurements of VI=0 at different Na+ and HCO3- concentrations. Because we previously observed that the cotransport in proximal tubule cells is susceptible to carbonic anhydrase (CA) inhibition, but only if it works at q=3:1, we propose that kNBC has three transport sites: when working at q=2:1 it binds 2 (HCO3-)+1 Na+, and while at q=3:1 it binds 1 CO3(2-)+1 HCO3- +1 Na+. The latter is equivalent to the transfer of 3 (HCO3-) +1 Na+, because in the presence of CA the generation of 1 CO3(2-) on one side of the membrane and its disintegration on the other transiently liberates 1 CO2, which follows by diffusion. This model explains the increase in (HCO3-) transport that is associated with the change in q from 2:1 to 3:1 by a selectivity change of a binding site from HCO3- to CO3(2-). This is more likely than the induction of a new transport pouch for a third (HCO3-) ion, which would require exceedingly large conformational changes of the transport protein.

Incubation in tissue culture media allows isolated rabbit proximal tubules to regain in-vivo-like transport function: response of HCO3-absorption to norepinephrine.

Using a new stop-flow perfusion technique with microspectrofluorometric determination of luminal fluid pH, we have studied which substrates or incubation conditions allow isolated rabbit proximal tubules to attain in-vivo-like rates of HCO3- absorption (J(HCO3)) and maximal responses of J(HCO3) to norepinephrine (NE). Essentially three incubation media were tested: plasma-like HCO(3-)-Ringer solution containing 5 mmol/l D-glucose (G-Ringer sol.), the same solution also containing 10 mmol/l lactate and 5 mmol/l L-alanine, (LAG-Ringer sol.), and two tissue culture media (DMEM and RPMI 1640). Compared to G-Ringer sol., application of LAG-Ringer sol. in the bath and/or lumen, or application of DMEM or RPMI 1640 in the bath either slightly increased or decreased J(HCO3) with borderline significance. However, RPMI 1640 plus 1 mmol/l pyruvate stimulated J(HCO3) by 55%. While NE (10(-5) mol/l), if applied in G-Ringer sol., had no effect, in the presence of LAG-Ringer sol. it increased J(HCO3) by approximately =40%, and in the presence of DMEM or RPMI 1640 it increased J(HCO3) by approximately =100%. This stimulation by NE followed Michaelis-Menten kinetics with an EC50 value of 0.25 micromol/l and was probably mediated by alpha1-adrenergic receptors. Additional cell pH measurements suggest that NE stimulates the basolateral Na+-HCO3- cotransporter which then becomes susceptible to inhibition by cAMP. We conclude that incubation in tissue culture media allows isolated proximal tubules to maintain a better functional state than the commonly used solutions with unphysiologically high substrate concentrations.

Micromolar concentrations of steroids and of aldosterone antagonists inhibit the outwardly rectifying chloride channel with different kinetics.

We used the patch-clamp technique to analyse the open/close kinetics of single, outwardly rectifying, intermediate-conductance (ORIC) Cl- channels from cultured epithelial cells under control conditions and in presence of different inhibitors. As observed previously in excised inside/out patches under control conditions, the switching kinetics were characterized by one open-state time constant (tau0 is approximately 30 ms) and three closed-state time constants (tau(cl)is approximately = to 0.2 ms, tau(c2) is approximately = 2 ms and tau(c3) is approximately = 60 ms). Aldosterone, six further steroids and two aldosterone antagonists inhibited channel open probability (NPo) concentration dependently with the potency at 10 micromol/l increasing in the sequence: hydrocortisone, corticosterone, P-oestradiol, cortisone, aldosterone, testosterone, progesterone, canrenone, spironolactone. Although all substances decreased tau(o), neither the steroids nor the aldosterone antagonists affected tau(cl), tau(c2) or tau(c3) or induced additional transitions with additional time constants. Instead, the steroids increased the prevalence of tau(c2) in the dwell-time histograms and the aldosterone antagonists increased the prevalence of tau(c3), both in a concentration-dependent manner. These observations may be explained by a model in which one open state leads to one of three closed states with rate constants alpha, beta and gamma, and in which beta or gamma increase under the influence of steroids or aldosterone antagonists, respectively. Cytosol, which contains a Cl- channel inhibitor of unknown molecular structure, (Krick et al., Pflügers Arch 418:491, 1991) was also tested, but the results did not conform to the blocker mechanisms described above. This shows that there are even further modes of channel inhibition and argues against the cytosolic Cl- channel inhibitor being a steroid.

Basolateral proteinase-activated receptor (PAR-2) induces chloride secretion in M-1 mouse renal cortical collecting duct cells.

1. Using RT-PCR, Northern blot analysis, and immunocytochemistry, we confirmed renal expression of proteinase-activated receptor (PAR-2) and demonstrated its presence in native renal epithelial and in cultured M-1 mouse cortical collecting duct (CCD) cells. 2. We investigated the effects of a PAR-2 activating peptide (AP), corresponding to the tethered ligand that is exposed upon trypsin cleavage, and of trypsin on M-1 cells using patch-clamp, intracellular calcium (fura-2) and transepithelial short-circuit current (ISC) measurements. 3. In single M-1 cells, addition of AP elicited a concentration-dependent transient increase in the whole-cell conductance. Removal of extracellular Na+ had no effect while removal of Cl- prevented the stimulation of outward currents. The intracellular calcium concentration increased significantly upon application of AP while a Ca2+-free pipette solution completely abolished the electrical response to AP. 4. In confluent monolayers of M-1 cells, apical application of AP had no effect on ISC whereas subsequent basolateral application elicited a transient increase in ISC. This increase was not due to a stimulation of electrogenic Na+ absorption since the response was preserved in the presence of amiloride. 5. The ISC response to AP was reduced in the presence of the Cl- channel blocker diphenylamine-2-carboxylic acid on the apical side and abolished in the absence of extracellular Cl-. 6. Trypsin elicited similar responses to those to AP while application of a peptide (RP) with the reverse amino acid sequence of AP had no effect on whole-cell currents or ISC. 7. In conclusion, our data suggest that AP or trypsin stimulates Cl- secretion by Ca2+-activated Cl- channels in M-1 CCD cells by activating basolateral PAR-2.

Activation of an apical Cl- conductance by extracellular ATP in Necturus gallbladder is mediated by cAMP and not by [Ca2+]i.

Necturus gallbladder epithelium (NGE) expresses a CFTR-like apical Cl- conductance that can be activated by cAMP. Here, we show that extracellular ATP (100 microM), which is known to elevate intracellular Ca2+ and to hyperpolarize cells by stimulating apical and basolateral K+ conductances, also stimulates an apical Cl- conductance (Ga,Cl), however with a much slower time course. The selectivity sequence of Ga,Cl was SCN- > I- > NO3- > Br- > Cl- >> isethionate (ISE-), but SCN- and I- partially blocked it, which is analogous to observations of CFTR Cl- channels. To disclose a possible role for intracellular Ca2+, gallbladders were incubated with the Ca2+ chelator BAPTA/AM or bathed in solutions containing only submicromolar Ca2+ concentrations. BAPTA partially inhibited the Ca(2+)-mediated hyperpolarization, but did not reduce the ATP-dependent activation of Ga,Cl and the latter was also seen in low extracellular Ca2+. On the other hand, the cAMP-antagonist Rp-8-Br-cAMPS strongly inhibited the stimulation of Ga,Cl by ATP (as well as by forskolin), but left the ATP-induced hyperpolarization unchanged. Preincubation with a low concentration of forskolin markedly enhanced the stimulatory effect of ATP, and this effect was not modified by the selective inhibition of protein kinase C. These data suggest the involvement of different signal transduction pathways in the ATP-dependent activation of K+ and Cl- conductances in NGE. The stimulation of the Ga,Cl appears to be mediated by cAMP but not by elevation of intracellular Ca2+.

The activation of an apical Cl- conductance by extracellular ATP is potentiated by genistein in Necturus gallbladder epithelium.

Necturus gallbladder epithelium (NGE) expresses a CFTR-homologous apical Cl- conductance (Ga,Cl) which can be activated either by elevation of intracellular cAMP or by extracellular ATP. Here we show by microelectrode experiments and impedance analysis that genistein (50 microM), which is known to potentiate the stimulation of Ga,Cl in several cell culture models, also potentiates the stimulation of Ga,Cl by low doses of forskolin in NGE. Moreover, we show that genistein also potentiates the stimulation of Ga,Cl by ATP. In addition genistein renders gallbladders that initially do not respond to ATP sensitive to this stimulant, and it delays the conductance inactivation after ATP removal. Under control conditions Ga,Cl inactivates within < 5 min, but in the presence of genistein a significant Ga,Cl persists even after 60 min. These effects of genistein are not related to inhibition of protein tyrosine kinases, since structurally different inhibitors of the tyrphostin family do not mimic the genistein effects. The data support our conclusion that stimulation of Ga,Cl by ATP is mediated by activation of the cAMP pathway and involves a CFTR-homologous protein. They also favour the view that genistein acts via inhibition of protein phosphatases which dephosphorylate CFTR, but cannot exclude the possibility of a direct interaction with CFTR.

Stoichiometry of the rat kidney Na+-HCO3- cotransporter expressed in Xenopus laevis oocytes.

The rat kidney Na+-HCO3- cotransporter (rkNBC) was expressed in Xenopus laevis oocytes and transport via rkNBC was studied with the patch-clamp technique in giant inside/out (i/o) or outside/out (o/o) membrane patches. The current/voltage (I/V) relation(s) of individual patches was(were) determined in solutions containing only Na+ and HCO3- as permeable ions. The current carried by rkNBC (INBC) was identified by its response to changing bath Na+ concentration(s) and quantified as the current blocked by 4, 4'-diisothiocyanatostilbene disulfonate (DIDS). The stoichiometric ratio (q) of HCO3- to Na+ transport was determined from zero-current (reversal) potentials. The results and conclusions are as follows. First, DIDS (250 micromol/l) blocks INBC irreversibly from both the extracellular and the intracellular surface. Second, in the presence of Na+ and HCO3- concentration gradients similar to those which rkNBC usually encounters in tubular cells, q was close to 2. The same value was also observed when the HCO3- concentration was 25 mmol/l throughout, but the Na+ concentration was either high (100 mmol/l) or low (10 mmol/l) on the extracellular or intracellular surface of the patch. These data demonstrate that in the oocyte cell membrane rkNBC works with q=2 as previously observed in a study of isolated microperfused tubules (Seki et al., Pflügers Arch 425:409, 1993), however, they do not exclude the possibility that in a different membrane and cytoplasmic environment rkNBC may operate with a different stoichiometry. Third, in most experiments bath application of up to 2 mmol/l ATP increased the DIDS-inhibitable conductance of i/o patches by up to twofold with a half saturation constant near 0.5 mmol/l. This increase was not associated with a change in q, nor with a shift in the I/V relationship which would suggest induction of active transport (pump current). Since the effect persisted after ATP removal and was not observed with the non-hydrolysable ATP analogue AMP-PNP, it is possible that rkNBC is activated by phosphorylation via protein kinases that might adhere to the cytoplasmic surface of the membrane patch.

A stop-flow microperfusion technique for rapid determination of HCO3- absorption/H+ secretion by isolated renal tubules.

In the present experiments on microdissected tubules of rabbit kidney we present a refined stop-flow method for determining the rate of HCO3- absorption (J(HCO3)) or H+ secretion (JH) that can be applied to isolated microperfused tubules. Using the pH-sensitive indicator dye BCECF (2',7'-bis [2-carboxyethyl]-5[6]-carboxyfluorescein) the luminal perfusate pH is continuously measured with a microspectrofluorometric set-up, and the pH change following a sudden stop of perfusion is analysed. Because the tubules partially collapse after stop-flow the calculation of fluxes requires a correction for volume loss. This is achieved by referring all fluxes to the remaining luminal volume, which can be estimated from the decay of the 440 nm reference fluorescence. During perfusion of the lumen with pure HCO3- Ringer solution, and of the bath with the same solution but containing 5.5 mmol/l D-glucose as metabolic substrate, J(HCO3) averaged 4.4+/-0.2 pmol cm(-1) x s(-1) (n=40) and 13.4+/-0.8 pmol x cm(-1) x s(-1) (n=5) in proximal straight tubules (PST) and in proximal convoluted tubules respectively. These values agree very well with data obtained in other laboratories with the picapnotherm technique. The present method has the advantage of requiring fewer micromanipulations and a shorter measuring time, thus allowing regulatory changes in J(HCO3) to be analysed. Moreover it does not involve measurements of radioactivity, and it also allows J(H) to be measured in HCO3(-) free solutions which in PST averaged 0.9 pmol x cm(-1) x s(-1) (n=8) in the present experiments.

Alkaline secretion by frog gastric glands measured with pH microelectrodes in the gland lumen.

1. In the present work we have measured the pH of the secreted fluid within the gland lumen of isolated but intact gastric mucosa of Rana esculenta. Tissues were mounted in a double chamber allowing continuous perfusion of the mucosal and serosal compartment, and the measurements were made with double-barrelled pH glass microelectrodes inserted into the glands from the serosal surface under microscopic inspection. 2. During inhibition of H+ secretion by cimetidine (100 microM) the luminal gland pH (pHgl) averaged 7.60 +/- 0.05 pH units (mean +/- s.e.m.; n = 35), a value significantly higher than bath solution pH (7.45 +/- 0.02; P < 0.001) and also higher than intracellular pH of oxyntopeptic cells (pHi), which averaged 7.53 +/- 0.06 (n = 18). 3. Stimulation of acid secretion with histamine (500 microM) reversibly decreased pHgl to values which could be as low as 2.5. Together with electrophysiological criteria this response was routinely used to verify the proper location of the microelectrode tip within the gland lumen. 4. Stimulation with carbachol (100 microM) or pentagastrin (50 microM) in the presence of cimetidine rapidly and reversibly increased pHgl by 0.10 +/- 0.01 pH units (n = 24; P < 0.001) and 0.09 +/- 0.02 pH units (n = 6; P < 0.05), respectively. 5. The observation that gastric gland fluid is more alkaline than the bath solutions and that carbachol or pentagastrin further alkalinize it strongly suggests that oxyntopeptic cells participate in gastric alkaline secretion at least under cholinergic stimulation.

Partial recovery of in vivo function by improved incubation conditions of isolated renal proximal tubule. I. Change of amiloride-inhibitable K+ conductance.

Isolated microperfused rabbit renal proximal tubule S2 segments, if incubated in conventional substrate containing HCO3- Ringer solution, exhibit lower cell membrane potentials (Vb) and elevated intracellular Na+ concentrations ([Na]i) compared to rat tubules in vivo. Assuming that these and other differences reflect insufficient metabolic and/or hormonal stimulation of the cells, we have used microelectrode techniques to test whether improving substrate supply and applying norepinephrine (NE, to compensate for the missing nerve supply) reverts Vb and [Na]i to values observed in vivo. Application of D-glucose (5.5 mmol/l) and additional application of pyruvate, lactate, or L-alanine (each 10 mmol/l), or bathing the tubules in Dulbecco's modified Eagle's tissue culture medium (DMEM) significantly increased Vb and, whenever tested, reduced [Na]i as compared to substrate-free or D-glucose-containing control solution and these effects could be prevented - as tested in the case of pyruvate - by inhibition of the Na/K pump with ouabain. However, high concentrations of acetate, beta-hydroxybutyrate, or L-glutamine had no significant effect. The largest effect was obtained with joint application of DMEM and NE (10 micromol/l) which increased Vb from -42.8 +/- 1.3 mV (SEM) to -55.3 +/- 2.5 mV (n = 11). Interestingly we noticed that under the latter conditions the Vb response to bath application of 1 mmol/l amiloride virtually disappeared, i.e. it changed from a depolarization of +14.6 +/- 1.4 mV (in D-glucose Ringer solution) to +0.6 +/- 0.7 mV (in DMEM plus NE) (n = 8), with some tubules showing even a small hyperpolarization. The latter implies partial restoration of the in vivo behaviour, since in experiments on rat proximal tubules in vivo amiloride regularly hyperpolarized the cells (by -3.4 +/- 0.76 mV, n = 5). Obviously under conventional in vitro conditions an amiloride-inhibitable K+ conductance is activated which is inactive in vivo and also inactivates under improved conditions in vitro. In agreement with observations reported in the subsequent publication our results demonstrate that isolated proximal tubules undergo functional alterations which may be largely prevented by improved metabolic and stimulatory incubation conditions.

Partial recovery of in vivo function by improved incubation conditions of isolated renal proximal tubule. II. Change of Na-HCO3 cotransport stoichiometry and of response to acetazolamide.

In the preceding publication we reported that some transport properties of proximal tubules perfused in vitro differ from those of tubules perfused in vivo, and that the in vivo function can be largely recuperated by improved metabolic substrate supply and stimulation with norepinephrine (NE). Since we have previously observed that the basolateral Na-HCO3-cotransporter operates with an overall stoichiometric ratio of q approximately 3 HCO3- :1 Na+ in vivo, but with q approximately 2 HCO3- :1 Na+ in vitro and that it responds differently in both cases to acetazolamide (ACZ), we have now tested whether the cotransporter can regain its in vivo function in vitro if the incubation conditions are improved. Cell membrane potentials (Vb) and cell pH (pHi) were measured with microelectrodes and microfluorimetric techniques on isolated S2 segments of rabbit proximal tubule and the instantaneous Vb response to a 2:1 reduction of bath HCO3- or Na+ concentration was determined. (DeltaVb)HCO3 and (deltaVb)Na averaged 13.1 +/- 0.9 mV (SEM) and 6.9 +/- 0.5 mV in D-glucose-containing control HCO3-Ringer solution and decreased respectively to 10.1 +/- 0.5 mV and 3.8 +/- 0.2 mV (n = 8) after incubation in tissue culture medium and NE (10(-5) mmol/l). These data imply that q increased from 1.9 to 2.7. Concomitantly the tubules became susceptible to ACZ (1 mmol/l), which reduced (deltaVb)HCO3 in control conditions only to 94.6 +/- 1.2% but under improved incubation conditions to 64.5 +/- 2.4%. As verified in voltage divider measurements the latter reduction was not caused by activation of a basolateral K+ conductance. The results indicate that improved incubation conditions can at least partially revert cotransport function towards that of the in vivo state. The effect of ACZ may be explained if in the improved state 1 CO32- + 1 HCO3- + 1 Na+ are cotransported, in which case inhibition of carbonic anhydrase (CA) may cause a CO32-/pH disequilibrium to develop in the basal labyrinth which impedes the cotransport. Under conventional incubation conditions, however, when only 2 HCO3- + 1 Na+ are cotransported no such disequilibrium should develop irrespective of whether CA is active or inhibited.

Is resting state HCO3- secretion in frog gastric fundus mucosa mediated by apical Cl(-)-HCO3- exchange?

1. We have tested the widely accepted hypothesis that resting-state bicarbonate secretion of gastric fundus mucosa is mediated by Cl(-)-HCO3- exchange in the apical membrane of surface epithelial cells (SECs). To this end, SECs of isolated fundus mucosa of Rana esculenta were punctured with double-barrelled microelectrodes to measure intracellular pH (pHi). 2. No significant pHi changes were observed in response to changing luminal HCO3- and/or Cl- concentrations. The change in pHi (delta pHi) in response to luminal chloride substitution averaged 0.00 +/- 0.01 pH units (mean +/- S.E.M.; n = 48), and did not change after blocking putative basolateral acid/base transporters which could have masked the pHi response. 3. On the other hand, pHi responded readily and reversibly to luminal perfusion with either low-pH (pH 2.5) solution (delta pHi = -0.36 +/- 0.05; n = 4; P < 0.01) or CO2-free HCO3- Ringer solution (delta pHi = +0.10 +/- 0.01; n = 29; P < 0.001). These observations demonstrate that the solution change was effective and complete within 1 min and show that the apical membrane of SECs is permeable to CO2. 4. The apical membrane of frog SECs could not be stained with an antibody against the C-terminal end of the mouse Cl(-)-HCO3- exchanger isoform AE2, although this antibody readily stained the basolateral membrane of the oxyntopeptic cells (OCs). 5. In conclusion, the presence of a Cl(-)-HCO3- exchanger in the apical membrane of SECs of frog gastric fundus mucosa in the resting state could not be confirmed, but other models of HCO3- secretion cannot be fully excluded. Observations from electrical measurements, favouring a model of conductive HCO3- secretion, point to the OCs rather than the SECs as a site of origin of HCO3- secretion.

[Chloride conduction of nasal fibroblasts in polyposis patients with cystic fibrosis and in patients without cystic fibrosis. Relevance for the ENT physician]. / Chloridleitfähigkeit nasaler Fibroblasten von Polyposis-Patienten mit zystischer Fibrose und Patienten ohne zystische Fibrose. Relevanz für den HNO-Arzt.

Cystic fibrosis (CF) is a complex systemic disease that has pathological alterations in the upper airways, including the recurrent formation of nasal polyps. Although the fibroblast is the predominant cell type in nasal stroma and nasal polyps, little is known about the electrophysiological properties of nasal fibroblasts. We investigated whether fibroblasts possess a cAMP-regulated chloride conductance which is impaired in patients with CF. Thus far the few studies concerning conductance in fibroblasts have been performed on skin fibroblasts using indirect methods and have yielded conflicting results. Therefore we studied chloride conductance in fused nasal fibroblasts by employing conventional microelectrodes. We have demonstrated that a cAMP-regulated chloride conductance is present in fibroblasts. However, this chloride conductance cannot be activated in fibroblasts from CF-patients. Thus, we present direct evidence that the impairment of the cAMP-regulated chloride conductance in CF is not confined to epithelial cells but also affects the fibroblast. We discuss how this conductance might modulate fibroblast proliferation to produce polyp formation.

Contribution of surface epithelial cells to total conductance of Necturus gastric fundus mucosa.

Microelectrode techniques were used to quantify the contribution of surface epithelial cells (SEC) to transepithelial conductance (gt) of Necturus gastric fundus mucosa. Transepithelial voltage (Vt) and resistance (Rt) as well as the basolateral cell membrane potential (Vb) and voltage divider ratio of SEC were measured. Freshly mounted preparations did not respond to luminal amiloride (10 microM), but within 2-3 h a significant response developed (delta Vt = 3.8 +/- 1.2 mV, delta Rt = 63 +/- 23 omega cm2, and delta Vb = -6.9 +/- 1.3 mV), indicating activation of an apical Na+ conductance in SEC. Using circuit analysis equations, we calculate that SEC contribute 10.4% to gt under control conditions and 13.0% after Na+ conductance activation. Histamine (0.1 mM), which stimulates the oxyntopeptic cells (OC), increased Vt and decreased Rt but did not significantly alter the membrane resistances of SEC. As a result, the contribution of SEC to gt fell to 7.4 or 9.3%, respectively. The data confirm that SEC are poorly permeable and that the major conductance path across gastric mucosa leads through OC in the glands. The reason for the protracted in vitro activation of the apical Na+ conductance in SEC is not known.

On the mechanism of bicarbonate exit from renal proximal tubular cells.

We compare here the results of electrophysiological measurements on proximal tubular cells performed on rat kidney in vivo and on isolated rabbit and rat tubules in vitro. Based on different effects of carbonic anhydrase inhibitors in the in vivo and in vitro preparation, we conclude that NaHCO3 cotransport across the basolateral cell membrane functions as Na(+)-CO3(2-)-HCO3- cotransport in vivo, but as Na(+)-HCO3(-)-HCO3- cotransport in the classical in vitro preparation. The former, but not the latter, transport mode is characterized by generation of local disequilibrium pH/CO3(2-) concentrations that oppose fluxes if membrane-bound carbonic anhydrase is inhibited. In support of this conclusion, we find that overall transport functions with a HCO3- to Na+ stoichiometry of 3:1 in vivo (since each transported CO3(2-) eventually generates 2 HCO3- ions), but 2:1 in vitro. This has been deduced from various measurements, among them super-Nernstian and reverse nernstian, potential responses to changing ion concentrations which are characteristic of obligatorily coupled cation-anion cotransporters, but are not known in classical electrochemistry. The different transport modes in vivo and in vitro suggest that isolated proximal tubules have functional deficits compared to proximal tubules in vivo.

Hypertonicity activates nonselective cation channels in mouse cortical collecting duct cells.

We investigated the effect of cell shrinkage on whole-cell currents of M-1 mouse cortical collecting duct cells. Addition of 100 mM sucrose to an isotonic NaCl bath solution induced cell shrinkage and increased whole-cell currents within 5-10 min by approximately 12-fold. The effect was reversible upon return to isotonic solution and could also be elicited by adding 100 mM urea or 50 mM NaCl. Replacement of bath Na+ by K+, Cs+, Li+, or Rb+ did not significantly affect the stimulated inward current, but replacement by N-methyl-D-glucamine reduced it by 88.1 +/- 1.3% (n = 34); this demonstrates that hypertonicity activates a nonselective alkali cation conductance. The activation was independent of extra- and intracellular Ca2+, but 1 or 10 mM ATP in the pipette suppressed it in a concentration-dependent manner, indicating that intracellular ATP levels may modulate the degree of channel activation. Flufenamic acid (0.1 mM) and gadolinium (0.1 mM) inhibited the stimulated current by 68.7 +/- 5.9% (n = 9) and 32.4 +/- 11.7% (n = 6), respectively, whereas 0.1 mM amiloride had no significant effect. During the early phase of hypertonic stimulation single-channel transitions could be detected in whole-cell current recordings, and a gradual activation of 30 and more individual channels with a single-channel conductance of 26.7 +/- 0.4 pS (n = 29) could be resolved. Thus, we identified the nonselective cation channel underlying the shrinkage-induced whole-cell conductance that may play a role in volume regulation.

A calcium-activated and nucleotide-sensitive nonselective cation channel in M-1 mouse cortical collecting duct cells.

We recently reported that M-1 mouse cortical collecting duct cells show nonselective cation (NSC) channel activity (Proc. Natl. Acad. Sci. USA 89:10262-10266, 1992). In this study, we further characterize the M-1 NSC channel using single-channel current recordings in excised inside-out patches. The M-1 NSC channel does not discriminate between Na+, K+, Rb+, Cs+, and Li+. It has a linear I-V relation with a conductance of 22.7 +/- 0.5 pS (n = 78) at room temperature. The Pcation/P(anion) ratio is about 60 and there is no measurable conductance for NMDG, Ca2+, Ba2+, and Mn2+. Cytoplasmic calcium activates the M-1 NSC channel at a threshold of 10(-6) M and depolarization increases channel activity (NPo). Cytoplasmic application of adenine nucleotides inhibits the M-1 NSC channel. At doses of 10(-4) M and 10(-3) M, ATP reduces NPo by 23% and 69%, respectively. Furthermore, since ADP (10(-3) M) reduces NPo by 93%, the inhibitory effect of adenine nucleotides is not dependent on the presence of a gamma-phosphoryl group and therefore does not involve protein phosphorylation. The channel is not significantly affected by 8-Br-cGMP (10(-4) M) or by cGMP-dependent protein kinase (10(-7) M) in the presence of 8-Br-cGMP (10(-5) M) and ATP (10(-4) M). The NSC channel is not sensitive to amiloride (10(-4) M cytoplasmic and/or extracellular) but flufenamic acid (10(-4) M) produces a voltage-dependent block, reducing NPo by 35% at depolarizing voltages and by 80% at hyperpolarizing voltages. We conclude that the NCS channel of M-1 mouse cortical collecting duct cells belongs to an emerging family of calcium-activated and nucleotide-sensitive nonselective cation channels. It does not contribute to amiloride-sensitive sodium absorption and is unlikely to be a major route for calcium entry. The channel is normally quiescent but may be activated under special physiological conditions, e.g., during volume regulation.