Christoph Korbmacher

ATP stimulates Cl− secretion and reduces amiloride-sensitive Na+ absorption in M-1 mouse cortical collecting duct cells

1 Using equivalent short circuit current (ISC) measurements we examined the effect of extracellular ATP on transepithelial ion transport in M‐1 mouse cortical collecting duct cells. Apical addition of ATP produced a rapid transient peak increase in ISC. This was followed by a fall below basal ISC due to a reduction in the amiloride‐sensitive ISC component. 2 The ATP‐induced ISC increase was preserved in the presence of apical amiloride while it was reduced in the absence of extracellular Cl− and in the presence of the apical Cl− channel blockers diphenylamine‐2‐carboxylic acid (DPC, 1 mM), DIDS (300 μM) and niflumic acid (100 μM). 3 The stimulatory effect of apical ATP on ISC was concentration dependent with an EC50 of about 0.6 μM. Basolateral ATP elicited a similar ISC response. Experiments using the ATP scavenger hexokinase demonstrated that the ATP effects were elicited via separate apical and basolateral receptors. 4 ATP and UTP applied to either the apical or the basolateral bath equi‐potently stimulated ISC while ‘purified’ ADP and UDP had no effect consistent with P2Y2 purinoceptors, the expression of which was confirmed using RT‐PCR. 5 Intracellular calcium concentration ([Ca2+]i) measurements using fura‐2 demonstrated that ATP and UTP elicited a rise in [Ca2+]i with EC50 values of 1.1 and 0.6 μM, respectively. The shape and time course of the calcium response were similar to those of the ISC response. The peak ISC response was preserved in the nominal absence of extracellular calcium but was significantly reduced in cells pre‐incubated with the calcium chelator BAPTA AM. 6 We conclude that in M‐1 cells extracellular ATP reduces amiloride‐sensitive Na+ absorption and stimulates Cl− secretion via calcium‐activated Cl− channels through activation of P2Y2 purinoreceptors located in the apical and basolateral membrane.

Additional Disruption of the ClC-2 Cl- Channel Does Not Exacerbate the Cystic Fibrosis Phenotype of Cystic Fibrosis Transmembrane Conductance Regulator Mouse Models

Cystic fibrosis is a fatal inherited disease that is caused by mutations in the gene encoding a cAMP-activated chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR). It has been suggested that the cystic fibrosis phenotype might be modulated by the presence of other Cl- channels that are coexpressed with CFTR in some epithelial cells. Because the broadly expressed plasma membrane Cl- channel, ClC-2, is present in the tissues whose function is compromised in cystic fibrosis, we generated mice with a disruption of both Cl- channel genes. No morphological changes in their intestine, lung, or pancreas, tissues affected by cystic fibrosis, were observed in these mice. The mortality was not increased over that observed with a complete lack of functional CFTR. Surprisingly, mice expressing mutant CFTR (deletion of phenylalanine 508), survived longer when ClC-2 was disrupted additionally. Currents across colonic epithelia were investigated in Ussing chamber experiments. The disruption of ClC-2, in addition to CFTR, did not decrease Cl- secretion. Colon expressing wild-type CFTR even secreted more Cl- when ClC-2 was disrupted, although CFTR transcript levels were unchanged. It is concluded that ClC-2 is unlikely to be a candidate rescue channel in cystic fibrosis. Our data are consistent with a model in which ClC-2 is located in the basolateral membrane.

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.

Osmotic Shrinkage Activates Nonselective Cation (NSC) Channels in Various Cell Types

Abstract. Osmotic cell shrinkage activates a nonselective cation (NSC) channel in M-1 mouse cortical collecting duct cells (Volk, Frömter & Korbmacher, 1995, Proc. Natl. Acad. Sci. USA92: 8478-8482). To see whether shrinkage-activated NSC channels are an ubiquitous phenomenon, we tested the effect of hypertonic extracellular solution on whole-cell currents of HT29 human colon carcinoma cells, BSC-1 renal epithelial cells, A10 vascular smooth muscle cells, and Neuro-2a neuroblastoma cells. Addition of 100 mm sucrose to an isotonic NaCl bath solution induced cell shrinkage of HT29 cells as evidenced by a decrease in cell diameter from 18 ± 1 μm to 12 ± 1 μm (n= 13). Upon cell shrinkage whole-cell currents of HT29 cells increased within 8 ± 1 min by about 30-fold (n= 13). Cell shrinkage and current activation were reversible upon return to isotonic solution. Replacement of bath Na+ by K+ or Li+ had almost no effect on the stimulated inward current. In contrast, replacement by N-methyl-d-glucamine (NMDG) completely abolished it and shifted the reversal potential from −4.5 ± 0.7 mV to −57 ± 4.1 mV (n= 10). Thus, the stimulated conductance is nonselective for alkali cations but highly selective for cations over anions with a cation-to-anion permeability ratio of about 13. Flufenamic acid (100 μm) inhibited the stimulated current by 84 ± 4.7% (n= 8). During the early phase of hypertonic stimulation single-channel transitions could be detected in whole-cell current recordings, and a gradual activation of 12 and more individual channels with a single-channel conductance of 17.6 ± 0.9 pS (n= 4) could be resolved. In analogous experiments similar shrinkage-activated NSC channels were also observed in BSC-1 renal epithelial cells, A10 vascular smooth muscle cells, and Neuro-2a neuroblastoma cells. These findings indicate that shrinkage-activated NSC channels are an ubiquitous phenomenon and may play a role in volume regulation.

Regulation of cytoplasmic pH of cultured bovine corneal endothelial cells in the absence and presence of bicarbonate

SummaryIntracellular pH (pHi) in confluent monolayers of cultured bovine corneal endothelial cells was determined using the pH-dependent absorbance of intracellularly trapped 5(and 6)carboxy-4′,5′-dimethylfluorescein. Steady-state pH was 7.05±0.1 in the nominal absence of bicarbonate, and 7.15±0.1 in the presence of 28mm HCO3−/5% CO2. Following an acid load imposed by a NH4Cl prepulse, pHi was regulated in the absence of HCO3− by a Na+-dependent process inhibitable to a large extent by 1mm amiloride and 0.1mm dimethylamiloride. In the presence of 28mm HCO3−/5% CO2, this regulation was still dependent on Na+, but the inhibitory potency of amiloride was less. DIDS (1mm) partially inhibited this regulation in the presence, but not in the absence of bicarbonate. With cells pretreated with DIDS, amiloride was as effective in inhibiting recovery from acid load as in the absence of HCO3−. The presence of intracellular Cl− did not appreciably affect this recovery, which was still sensitive to DIDS in the absence of Cl−. Removal of extracellular Na+ led to a fall of pHi, which was greatly attenuated in the absence of HCO3−. This acidification was largely reduced by 1mm DIDS, but not by amiloride. Cl removal led to an intracellular alkalinization in the presence of HCO3−. The presence of a Cl−/HCO3− exchanger was supported by demonstrating DIDS-sensitive36Cl− uptake into confluent cell monolayers. Thus, bovine corneal endothelial cells express three processes involved in intracellular pH regulation: an amiloride-sensitive Na+/H− antiport, a Na−−HCO3− symport and a Cl−/HCO3− exchange, the latter two being DIDS sensitive.

Maitotoxin (MTX) activates a nonselective cation channel in Xenopus laevis oocytes

Abstractu2002Maitotoxin (MTX) may exert its toxic effect by activating ion conductances and has been shown to elicit a fertilization-like response in Xenopus laevis oocytes. In the present study we investigated the electrophysiological response of stage V–VI Xenopus oocytes to MTX using the two-microelectrode voltage-clamp technique. Membrane voltage (Vm) measurements demonstrated that MTX (50 pM to 1 nM) depolarized the oocytes from –49±7 to –14±1 mV. Subsequent replacement of bath Na+ by the impermeant cation NMDG (N-methyl-d-glucamine) shifted Vm from –14±1 to –53±5 mV (n=29). This indicates that MTX activates a cation conductance. Indeed, current measurements at a holding potential of –60 or –100 mV showed that within 10 s of MTX application an inward current component developed which was largely abolished by extracellular Na+ removal. After a 1-min application of 1 nM MTX the NMDG-sensitive current increased more than 100-fold from 0.14±0.03 μA to a peak value of 21±3 μA (n=11). The effect of MTX was concentration dependent with an EC50 of about 250 pM but only slowly reversible. Ion substitution experiments indicated that the stimulated conductance was nonselective for monovalent cations with a slight preference for NH4+ (2.1) > K+ (1.5) > Na+ (1.0) > Li+ (0.7). Regarding divalent cations, a complex biphasic response to extracellular Na+ replacement by Ca2+ was observed, which suggests that the stimulated channels may have a small Ca2+ permeability but that exposure to high extracellular Ca2+ enhances recovery from MTX stimulation. No significant conductance for Mn2+ was observed. Application of 1 mM benzamil, 1 mM amiloride, or 100 μM 1-(β-[3-(4-Methoxyphenyl)-propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SK&F 96365) reduced the MTX-stimulated inward current by 81%, 62%, or 65%, respectively. Gd3+ had an inhibitory effect of 29% and 38% at concentrations of 10 μM or 100 μM, respectively. Flufenamic acid, niflumic acid, (RS)-(3,4-dihydro-6,7-dimethoxyisoquinoline-1-γ1)-2-phenyl-N,N-di-[2-(2,3,4-trimethoxyphenyl)-ethyl]-acetamide (LOE908), and 3′,5′-dichlorodiphenylamine-2-carboxylic acid (DCDPC), known blockers of other nonselective cation channels, had no significant effect. We conclude that MTX activates a nonselective cation conductance in Xenopus oocytes. The underlying channels may be involved in changes in Vm that occur during the early stages of fertilization.

Aldosterone responsiveness of the epithelial sodium channel (ENaC) in colon is increased in a mouse model for Liddle's syndrome

Liddles syndrome is an autosomal dominant form of human hypertension, caused by gain‐of‐function mutations of the epithelial sodium channel (ENaC) which is expressed in aldosterone target tissues including the distal colon. We used a mouse model for Liddles syndrome to investigate ENaC‐mediated Na+ transport in late distal colon by measuring the amiloride‐sensitive transepithelial short circuit current (ΔISC‐Ami) ex vivo. In Liddle mice maintained on a standard salt diet, ΔISC‐Ami was only slightly increased but plasma aldosterone (PAldo) was severely suppressed. Liddle mice responded to a low or a high salt diet by increasing or decreasing, respectively, their PAldo and ΔISC‐Ami. However, less aldosterone was required in Liddle animals to achieve similar or even higher Na+ transport rates than wild‐type animals. Indeed, the ability of aldosterone to stimulate ΔISC‐Ami was about threefold higher in Liddle animals than in the wild‐type controls. Application of aldosterone to colon tissue in vitro confirmed that ENaC stimulation by aldosterone was not only preserved but enhanced in Liddle mice. Aldosterone‐induced transcriptional up‐regulation of the channels β‐ and γ‐subunit (βENaC and γENaC) and of the serum‐ and glucocorticoid‐inducible kinase 1 (SGK1) was similar in colon tissue from Liddle and wild‐type animals, while aldosterone had no transcriptional effect on the α‐subunit (αENaC). Moreover, Na+ feedback regulation was largely preserved in colon tissue of Liddle animals. In conclusion, we have demonstrated that in the colon of Liddle mice, ENaC‐mediated Na+ transport is enhanced with an increased responsiveness to aldosterone. This may be pathophysiologically relevant in patients with Liddles syndrome, in particular on a high salt diet, when suppression of PAldo is likely to be insufficient to reduce Na+ absorption to an appropriate level.

K+-conductance and electrogenic Na+/K+ transport of cultured bovine pigmented ciliary epithelium

SummaryUsing intracellular microelectrode technique, we investigated the changes in membrane voltage (V) of cultured bovine pigmented ciliary epithelial cells induced by different extracellular solutions. (1)V in 213 cells under steady-state conditions averaged −46.1±0.6 mV (sem). (2) Increasing extracellular K+ concentration ([K+]o) depolarizedV. Addition of Ba2+ could diminish this response. (3) Depolarization on doubling [K+]o was increased at higher [K+]o (or low voltage). (4) Removing extracellular Ca2+ decreasedV and reduced theV amplitude on increasing [K+]o. (5)V was pH sensitive. Extra-and intracellular acidification depolarizedV; alkalinization induced a hyperpolarization.V responses to high [K+]o were reduced at acidic extracellular pH. (6) Removing Ko+ depolarized, Ko+ readdition after K+ depletion transiently hyperpolarizedV. These responses were insensitive to Ba2+ but were abolished in the presence of ouabain or in Na+-free medium. (7) Na+ readdition after Na+ depletion transiently hyperpolarizedV. This reaction was markedly reduced in the presence of ouabain or in K+-free solution but unchanged by Ba2+. It is concluded that in cultured bovine pigmented ciliary epithelial cells K+ conductance depends on Ca2+, pH and [K+]o (or voltage). An electrogenic Na+/K+-transport is present, which is stimulated during recovery from K+ or Na+ depletion. This transport is inhibited by ouabain and in K+-or Na+-free medium.

Evidence for Na/H exchange and Cl/HCO3 exchange in A10 vascular smooth muscle cells

In the present study we used the pH sensitive absorbance of 5(and6)-carboxy-4′,5′-dimethylfluorescein to investigate intracellular pH (pHi) regulation in A10 vascular smooth muscle cells: (1) The steady state pHi in A10 cells averaged 7.01±0.1 (mean±SEM,n=26) at an extracellular pH of 7.4 (28 mM HCO3/5% CO2). (2) Removal of extracellular sodium led to an intracellular acidification of 0.36±0.07 pH-units (mean±SEM,n=8). (3) pHi-Recovery after an acute intracellular acid load (by means of NH4Cl-prepulse) was reversibly blocked by 1 mM amiloride and was dependent on the presence of sodium. The velocity of pHi recovery increased with increasing sodium concentrations with an apparentKm for external sodium of about 30 mM and aVmax of about 0.35 pH units/min. These findings are compatible with a Na/H exchanger being responsible for pHi recovery after an acid load. (4) Removal of extracellular chioride induced an intracellular alkalinization of 0.23±0.03 pH-units (mean±SEM,n=10). The alkalinization was dependent on the presence of extracellular bicarbonate (5) Removal of chloride during pHi recovery from an alkaline load (imposed by acetate prepulse) stopped and reversed pHi backregulation. Chloride removal had no effect in the absence of bicarbonate or in the presence of 10−4 M DIDS, suggesting that the effects were mediated by a Cl/HCO3 exchanger. In conclusion we have demonstrated evidence for a Na/H exchanger and a Cl/HCO3 exchanger in A10 vascular smooth muscle cells.

Sulfonylurea receptors inhibit the epithelial sodium channel (ENaC) by reducing surface expression.

Abstract. In the kidney the epithelial Na+ channel (ENaC) is co-expressed with the sulfonylurea receptor (SUR), an ABC protein that shares a high degree of homology with the cystic fibrosis transmembrane conductance regulator (CFTR) and reportedly modifies ENaC in various preparations. To investigate a possible regulatory relationship between SUR and ENaC, we performed co-expression studies on Xenopuslaevis oocytes, which were assayed for amiloride-sensitive currents (ΔIami). Moreover, a chemiluminescence assay was used to investigate the surface expression of extracellular hemagglutinin-tagged SUR1 (SUR1-HA) or HA-tagged ENaC (ENaC-HA). In oocytes co-injected with SUR1/ENaC (or SUR2B/ENaC) ΔIami was reduced by ≅53% (or ≅45%) compared to ΔIami measured in matched control oocytes injected with ENaC alone. The inhibitory effect of SUR on ΔIami was preserved in oocytes expressing ENaC with C-terminally truncated subunits. Co-expression of SURs did not confer sensitivity of ΔIami to diazoxide, pinacidil, tolbutamide, or glibenclamide. ENaC does not facilitate the surface expression of SUR1-HA, which is known to be retained in the endoplasmatic reticulum (ER) by an ER-retention/retrieval signal. SUR1-HAAAA, a mutant that lacks this signal, still inhibits ENaC currents. Chemiluminescence was reduced by ≅49% in oocytes co-expressing ENaC-HA/SUR1 compared to that in control oocytes expressing ENaC-HA alone. We conclude that SUR does not interact with ENaC at the level of the plasma membrane but that it inhibits ΔIami by reducing surface expression of the channel.