Cl− channels in smooth muscle cells

In smooth muscle cells (SMCs), the intracellular chloride ion (Cl^?) concentration is high due to accumulation by Cl^?/HCO₃ ^? exchange and Na⁺–K⁺–Cl^? cotransportation. The equilibrium potential for Cl^? (E _Cl) is more positive than physiological membrane potentials (E _m), with Cl^? efflux inducing membrane depolarization. Early studies used electrophysiology and nonspecific antagonists to study the physiological relevance of Cl^? channels in SMCs. More recent reports have incorporated molecular biological approaches to identify and determine the functional significance of several different Cl^? channels. Both “classic” and cGMP-dependent calcium (Ca²⁺)-activated (Cl_Ca) channels and volume-sensitive Cl^? channels are present, with TMEM16A/ANO1, bestrophins, and ClC-3, respectively, proposed as molecular candidates for these channels. The cystic fibrosis transmembrane conductance regulator (CFTR) has also been described in SMCs. This review will focus on discussing recent progress made in identifying each of these Cl^? channels in SMCs, their physiological functions, and contribution to diseases that modify contraction, apoptosis, and cell proliferation.     

Role of the cytoskeleton in the regulation of Cl− channels in human embryonic skeletal muscle cells

The effects of volume change and cytoskeleton manipulation on the Cl^– channels in human embryonic skeletal muscle cells were studied. Trypsination, used for production of myoballs, changes the channel properties only a little. When the external osmolarity was reduced from 300 to 270 mosmol/l, the specific Cl^– conductance,g _Cl, (at &-80 mV) of myoballs increased from 5.1±1.9 to 30.4±12.2 S/cm² (SD;n=6) within 15 min. Concomitantly, the kinetics of Cl^– currents, elicited by clamping the membrane potential from a negative to positive values, changed from activation and subsequent slow inactivation to instantaneous activation with fast inactivation. G protein activation, protein kinase action or [Ca²⁺]_i elevation seemed not to be involved in these effects. Similar changes were produced in the absence of a transmembrane osmotic gradient by 500 nM intracellular cytochalasin D (g _Cl=34.3 ±10.3 S/cm²;n=6) or 12.5 M colchicine (g _Cl= 15.4±1.4 S/cm²;n=5). When the external osmolarity was increased to 418 mosmol/l, 1 M cytochalasin D did not affectg _Cl. In four of six cell-attached patches the open probability of the intermediate Cl^– channel was increased after reduction of the bath osmolarity. In inside-out patches, the drugs increased the open probability of the channels. It is concluded that the Cl^– channels are under control of the cytoskeleton.     

Gi2 proteins couple somatostatin receptors to low-conductance K+ channels in rat pancreatic α-cells

Somatostatin hyperpolarized rat pancreatic alpha-cells and inhibited spontaneous electrical activity by activating a low-conductance K+ channel (0.9 pS with physiological ionic gradients). This channel was insensitive to tolbutamide (a blocker of ATP-sensitive K+ channels) and apamin (an inhibitor of small-conductance Ca(2+)-activated K+ channels). Channel activation was prevented by pre-treating the cells with pertussis toxin, indicating the involvement of G-proteins. A direct interaction between an inhibitory G-protein and the somatostatin-activated K+ channel is suggested by the finding that intracellular application of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma-S) and the G beta gamma subunit of G-proteins resulted in a transient stimulation of the current. Activation of the K+ current by somatostatin was inhibited by intracellular dialysis with specific antibodies to Gi1/2 and was not seen in cells treated with antisense oligonucleotides against G-proteins of the subtype Gi2. We conclude that somatostatin suppresses alpha-cell electrical activity by a Gi2-protein-dependent mechanism, which culminates in the activation of a sulphonylurea- and apamin-insensitive low-conductance K+ channel.     

Effects of hydroxyl radicals on outwardly rectifying chloride channels in a cultured human bronchial cell line (16HBE14o–)

Respiratory pathologies can result from the exposure of airway epithelial cells to oxidative stress. We studied the effects of the hydroxyl radical *OH, for which there is no natural intra- or extracellular scavenger, on an outwardly rectifying chloride channel (ORCC). In the human bronchial cell line 16HBE14o-, the cytoplasmic side of ORCC in inside-out excised membrane patches was exposed to *OH created by simultaneously superfusing Fe2+ and H2O2 in front of the patch-pipette. ORCC was activated by depolarizing voltage steps. Its open probability (Po) increased with bath [Ca2+] above 1 microM. Upon brief exposure to *OH, ORCC first closed and then alternated between periods of closure and normal activity. The duration of closure increased with the duration of *OH exposure but voltage steps could reopen the channel. After 10 min exposure to *OH, however, the channel closed irreversibly, regardless of the number of subsequent voltage steps or the duration of washing. Low [Ca2+] in the bath accelerated the irreversible closure of the channel in the presence of *OH. Intracellular application of *OH progressively inhibited ORCC activity by inducing long closure periods that increased with time. This might have important pathophysiological implications in the process of inflammation.     

Voltage-dependent inactivation of volume-regulated Cl− current in human T84 colonic and B-cell myeloma cell lines

The voltage-dependent inactivation of a volume-regulated Cl^– current (VRChlC) in an epithelial (T84), and a B cell myeloma (RPMI-8226) cell lines were compared. Both cell types exhibited similar time and voltage-dependent current inactivation when recorded using standard whole cell patch technique, with a NaCl external solution that was 90 mOsm hypotonic to the Cs-Glutamate internal solution. The time course of voltage-dependent current inactivation was fit by the sum of a slow, dominant voltage-dependent and a faster voltage-independent components. Recovery from inactivation was approximated by a single exponential process and was dependent on the recovery voltage. The major difference was that the voltage-dependent current inactivation in myeloma cells was shifted to a 30mV more depolarized value than that in T84 cells although the effective charge movement of inactivation was similar. These results indicate that the macroscopic volume-regulated Cl^– current in these two cell lines is functionally similar.     

cAMP-dependent activation of small-conductance Cl− channels in HT29 colon carcinoma cells

The present study was performed to examine the conductance properties in the colon carcinoma cell line HT₂₉ and the activation of Cl^– channels by cAMP. A modified cell-attached nystatin patch-clamp technique was used, allowing for the simultaneous recording of the cell membrane potential (PD) and the conductance properties of the cell-attached membrane. In resting cells, PD was –56±0.4 mV (n=294). Changing the respective ion concentrations in the bath indicate that these cells possess a dominating K⁺ conductance and a smaller Cl^– conductance. A significant non-selective cation conductance, which could not be inhibited by amiloride, was only observed in cells examined early after plating. The K⁺ conductance was reversibly inhibited by 1–5 mmol/l Ba²⁺. Stimulation of the cells by the secretagogues isoproterenol and vasointestinal polypeptide (VIP) depolarized PD and induced a Cl^– conductance. Similar results were obtained with compounds increasing cytosolic cAMP: forskolin, 3-isobutyl-1-methylxanthine, cholera toxin and 8-bromoadenosine cyclic 3,5-monophosphate (8-Br-cAMP). VIP (1 nmol/l, n=10) and isoproterenol (1 umol/l, n=12) depolarized the cells dose-dependently and reversibly by 12±2 mV and 13±2 mV. The maximal depolarization was reached after some 20 s. The depolarization was due to increases in the fractional Cl^– conductance. Simultaneously the conductance of the cellattached membrane increased from 155±31 pS to 253±40 pS (VIP, n=4) and from 170±43 pS to 268±56 pS (isoproterenol, n=11), reflecting the gating of Cl^– channels in the cell-attached membrane. 5-Nitro-2-(3-phenylpropylamino)-benzoate (1 mol/l) was without significant effects in resting and in forskolin-stimulated HT₂₉ cells. The agonist-induced conductance increase of the cell-attached nystatin patches was not paralleled by the appearance of detectable single-channel events in these membranes. These data suggest activation of small, non-resolvable Cl^– channels by cAMP.Supported by DFG Gr 480/10 and BMFT 01 GA 88/6     

Regulatory volume decrease in a renal distal tubular cell line (A6) I. Role of K+ and Cl−

Changes in volume of A6 epithelial cells were monitored by recording cell thickness (T _c). The response ofT _c to a reduction of the basolateral osmolality from 260 to 140 mosmol/kg was recorded while transepithelial Na⁺ transport was inhibited by 20 M amiloride. With Cl^–-containing bathing media, this osmotic challenge elicited a rapid rise inT _c followed by a regulatory volume decrease (RVD). Substitution of SO₄ ^2– or gluconate for Cl^– markedly reduced the RVD, whereas cells completely maintained their ability to regulate their volume after replacing Cl^– by NO₃ ^–. A conductive pathway for Cl^– excretion is suggested, which is insensitive to NPPB [5-nitro-2-(3-phenylpropylamino)benzoic acid], an inhibitor of some types of Cl^– channels. Ba²⁺ (5 or 20 mM) reduced the RVD. A more pronounced inhibition of the RVD was obtained with 500 M quinine, a potent blocker of volume-activated K⁺ channels. K⁺-induced depolarization of the basolateral membranes of tissues incubated with SO₄ ^2–-containing solutions completely abolished the RVD. Noise analysis in the presence of Ba²⁺ showed the activation of an apical K⁺ conductive pathway. These results demonstrate that cell volume regulation is controlled by processes involving Cl^– and K⁺ excretion through conductive pathways.     

Small-conductance Cl− channels in HT29 cells: activation by Ca2+, hypotonic cell swelling and 8-Br-cGMP

The present study demonstrates the activation of Cl^– channels in HT₂₉ cells by agonist (ATP, neurotensin, carbachol) increasing cytosolic Ca²⁺, by hypotonic cell swelling and by cGMP. Cell-attached nystatin patch-clamp (CAN) as well as slow and fast wholecell recordings were used. The cell membrane potential was depolarized in a dose-dependent manner with halfmaximal effects at 0.4 umol/l for ATP, 60 pmol/l for neurotensin and 0.8 mol/l for carbachol. The depolarization, which was caused by Cl^– conductances increases, occurred within 1 s and was accompanied by a simultaneous and reversible increase of the input conductance of the cell-attached membrane from 295±32 pS to 1180±271 pS (ATP; 10 mol/l, n=21) and 192±37 pS to 443±128 pS (neurotensin; 1 nmol/l, n=8). The effects of the agonists could be mimicked by ionomycin (0.2 umol/l), suggesting that an increase in intracellular Ca²⁺ was responsible for the activation of Cl^– channels. The depolarization was followed by a secondary hyperpolarization. Hypotonic cell swelling also depolarized the cells and induced an increase in the membrane conductance. With 120 mmol/l NaCl the depolarization was 10±0.8 mV and the cell-attached conductance increased from 228±29 pS to 410±65 (n=26) pS. NaCl at 90 mmol/l and 72.5 mmol/l had even stronger effects. Comparable conductance increases were also obtained when the different agonists or hypotonic cell swelling were examined in whole cell experiments.5-Nitro-2-(3-phenylpropylamino)-benzoate (1 mol/l) did not prevent the effects of Ca²⁺-increasing hormones and of hypotonic solutions. An increase in Cl^– conductance was also induced by 8-Br-cGMP (1 mmol/l) but not by heat-stable Escherichia coli toxin. In contrast to their conductance-increasing effects in CAN patches, the different agonists and cell swelling did not activate resolvable single channels in these cell-attached membranes. This indicates that the Cl^– channels involved have a single-channel conductance too small ( 4 pS, 150 Hz) to be resolved by our techniques.     

The outwardly rectifying Cl− channel is not involved in cAMP-mediated Cl− secretion in HT-29 cells: evidence for a very-low-conductance Cl− channel

The patch-clamp technique and transepithelial current measurements in conjunction with analysis of transepithelial current noise were employed in order to clarify the role of the outwardly rectifying, depolarization-induced Cl^– channel (ORDIC) during cAMP-mediated Cl^– secretion in HT-29/B6 cells. Confluent monolayers growing on permeable supports were used in order to ensure the apical location of measured Cl^– channels. The ORDIC needed to be activated by excision and/or depolarization, and was found in both cAMP-stimulated and non-stimulated cells. Both 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and 4,4-dinitro-2,2-stilbenedisulphonate (DNDS) induced fast flickery-type blocks of the ORDIC at low, micromolar blocker concentrations and were used as a probe for ORDIC. However, these substances were ineffective in blocking transepithelial forskolin-induced Cl^– secretion of monolayers in Ussing chambers. No inhibitory effect at all was detected for DNDS up to 1 mmol/l. NPPB blocked the ORDIC at low concentrations (IC₅₀=0.5±0.3 mol/l) by reducing its open probability, but NPPB did not block forskolin-induced Cl^– secretion unless high concentrations were used (IC₅₀=240±10 mol/l). In order to exclude effects of NPPB other than on the apical Cl^– channel, trans-epithelial measurements were performed in basolaterally amphotericin-permeabilized, forskolin-stimulated preparations, and a serosal-to-mucosal Cl^– gradient was applied as a driving force. Under these conditions, NPPB's inhibitory effects were also very small. Noise analysis of this gradient-driven Cl^– current showed a very-low-frequency Lorentzian noise component (f _c=1.4±0.2 Hz), which was not compatible with Lorentzians predicted from single-channel gating of ORDIC. As revealed from fura-2 fluorescence measurements, forskolin-stimulated Cl^– secretion occurred in the absence of changes in intracellular Ca²⁺. Thus, we conclude that there is an apical Cl^– channel in HT-29/B6 that is activated through the cAMP-mediated pathway and is insensitive to NPPB and DNDS, and the kinetics of which are incompatible with ORDIC kinetics. Therefore, despite its prevalence in isolated patches and even in cell-attached recordings, the ORDIC appears not to be involved in cAMP-mediated Cl^– secretion by HT-29/B6 cells. From noise analysis, a very-small-conductance (probably below 1 pS), slow-gating Cl^– channel was calculated as the conductive site in the apical membrane during forskolin stimulation.     

Influence of extracellur Ca2+ on endogenous Cl− channels in Xenopus oocytes

Removal of Ca²⁺ from the external bath solution evoked marked depolarization and large currents (up to several microamperes) in voltage-clamped defolliculated oocytes of Xenopus laevis. The resulting current was not carried by a cation influx but was due to a huge Cl^– efflux, which could be strongly inhibited by the Cl^– channel blockers flufenamic acid and niflumic acid. Removal of Mg²⁺ or Ba²⁺ from the solutions had the same effects as removing Ca²⁺. The reversal potential of –12 mV also indicated that Cl^– channels were responsible for the large currents. Patch-clamp studies revealed a single-channel slope conductance of 90 pS. During oocyte maturation these channels remained active. The half-maximal Ca²⁺ concentration of about 20 M showed that quite low doses of extracellular Ca²⁺ profoundly influence the electrical properties of the oocyte membrane.     

Characterization of the Cl− conductance in the granular duct cells of mouse mandibular glands

We have previously shown that mouse mandibular granular ducts contain a hyperpolarization-activated Cl^– conductance. We now show that the instantaneous current/voltage (I/V) relation of this Cl^– conductance is inwardly rectifying with a slope conductance of 15.4±1.8 nS (n=4) at negative potentials and of 6.7±0.9 nS (n=4) at positive potentials. Thus, the inward rectification seen in the steady-state I/V relation is due, not only to voltage activation of the Cl^– conductance, but also to the intrinsic conductance properties of the channel. We show further that the ductal Cl^– conductance is not activated by including ATP (10 mmol/l) in the pipette solution. Finally, we show that the conductance is not blocked by the addition of any of the following compounds to the extracellular solution: anthracene-9-carboxylate (A9C, 1 mmol/l), diphenylamine-2-carboxylate (DPC, 1 mmol/l), 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB, 100 mol/l), 4,4-diisothiocyanato-stilbene-2,2-disulphonate (DIDS, 100 mol/l), indanyloxyacetic acid (IAA-94, 100 mol/l), verapamil (100 mol/l), glibenclamide (100 mol/l) and Ba²⁺ (5 mmol/l). The properties of the ductal Cl^– conductance most nearly resemble those of the ClC-2 channel. Both channel types have instantaneous I/V relations that are slightly inwardly rectifying, are activated by hyperpolarization with a time-course in the order of hundreds of milliseconds, have a selectivity sequence of Br^–>Cl^–>I^–, and are insensitive to DIDS. The only identified difference between the two is that the ClC-2 channel is 50% blocked both by DPC and A9C (1 mmol/l), whereas the ductal Cl^– conductance is insensitive to these compounds.     

Activation of basolateral Cl− channels in the rat colonic epithelium during regulatory volume decrease

Exposure to a hypotonic medium caused an increase in the diameter of isolated crypts from the rat colon. The increase in cell volume was only transient and lasted about 7 min. Despite of the continuous presence of the hypotonic medium, cell volume decreased again. This regulatory volume decrease (RVD) was inhibited by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), a Cl^– channel blocker, and by Ba²⁺, a K⁺ channel blocker. Cell-attached patch-clamp recordings revealed that the RVD was associated with the activation of previously silent basolateral channels. These channels were identified after excision of the patch as Cl^– channels (28 pS) and as K⁺ channels (45–60 pS). The RVD was dependent on the presence of external Ca²⁺. The phospholipase A₂ inhibitor, quinacrine, and the lipoxygenase blocker, nordihydroguaiaretic acid, inhibited RVD, while indomethacin had no effect. In Ussing chamber experiments an exposure to hypotonic media caused an initial, transient increase in tissue conductance (G _t), followed by a prolonged decrease in short-circuit current (I _sc) and the potential difference (V). The height of the electrical response was dependent on the decrease in the osmolarity in a range from 20 mosmol l^–1 to 90 mosmol l^–1. The increase in G _t was blocked by NPPB and Ba²⁺, whereas the decrease in I _sc or V was inhibited by NPPB but enhanced by Ba²⁺. This suggests that in the later phase the osmotically induced Cl^– conductance exceeds the K⁺ conductance leading to an electrogenic response, while the initial response of the RVD is an opening of Cl^– and K⁺ channels in a ratio of about 11. With respect to the inhibitory efficacy of nordihydroguaiaretic acid and the inefficacy of indomethacin, leukotrienes seem to be involved in the mediation of this response.     

Characterization of two distinct Cl− conductances in fused human respiratory epithelial cells

The present microelectrode experiments on fused respiratory epithelial cells of cystic fibrosis (CF) origin and non-CF origin aim at characterizing the molecular basis of the Cl^– conductances regulated by cyclic adenosine monophosphate (cAMP) or respectively Ca²⁺, as described in the preceding publication. Cell membrane potential (V _m) and resistance (R _m) were recorded as well as their response to substitution of 90% of bath Cl^– by isethionate (V _m,ISE), by I^– (V _m,I), or by other halide anions. Fused CF cells had significantly (P<0.05) higher control V _m values (P–18.0 ±9.4 mV, ±SD, n=68) than fused non-CF cells (–12.5±6.6 mV, n=69) and responded to the Ca²⁺ ionophore A23187 with an increase in the V _m response to Cl^– substitution, but did not respond to forskolin. This indicates that CF cells express only the Ca²⁺-stimulated Cl^– conductance. Injection of the antibody M3A7 against a fusion protein containing amino acids 1195 to 1480 of the CF gene product into young, forskolin-stimulated or old non-CF cells decreased V _m,ISE and V _m,I within 15 min to values observed in CF cells. This indicates inhibition of the cAMP-stimulated Cl^– conductance and supports the molecular identity of this conductance with the CF gene product. However, the slow onset of inhibition does not allow secondary effects to be excluded and a slight fall in R _m remains unexplained. Stimulation of the Ca²⁺-regulated Cl^– conductance was not impaired. Injection of M3A7 into CF cells or of a control antibody in non-CF cells had no effect. In the search for the single-channel equivalent of the Ca²⁺-stimulated Cl^– conductance we injected a concentrated placental cytosol fraction containing a cytosolic inhibitor of the outwardly rectifying intermediate conductance (ORIC) Cl^– channel into fused non-CF cells stimulated either with A23187 or forskolin. However, no effect was observed. This speaks against a role of the ORIC Cl^– channel in the Ca²⁺-activated Cl^– conductance, although it cannot definitely be excluded.     

Patch-clamp studies of K+ and Cl− channel currents in canine pancreatic acinar cells

K⁺ and Cl^– channel currents in the plasma membrane of isolated canine pancreatic acinar cells were studied by patch-clamp single-channel and whole-cell current recording techniques. In excised inside-out patches, we found a Ca⁺-activated (control range 0.01–0.4 M) and voltage-activated K²⁺-selective channel with a unit conductance of approximately 40 pS in symmetrical K⁺-rich solutions. In intact cells, addition of acetylcholine (1 M) or bombesin tetradecapeptide (0.1 nM) to the bath evoked an increase in frequency of K⁺ channel opening. In whole-cell recordings on cells dialyzed with K⁺-rich and Ca²⁺-free solution containing 0.5 mM EGTA, the resting potential was about –40mV. Depolarizing voltage pulses activated outward K⁺ currents, which were blocked by 10 mM tetraethylammonium, whereas hyperpolarizing pulses evoked smaller inward currents. Acetylcholine or bombesin activated the K⁺ current and enhanced the inward current, which was reduced by a low Cl^– (10 mM) intracellular solution at –90 mV holding potential. These results suggest that both Ca²⁺- and voltage-activated K⁺ channels and Ca²⁺-activated Cl^– channels exist in the plasma membrane of canine pancreatic acinar cells.     

Blockers of volume-activated Cl− currents inhibit endothelial cell proliferation

Volume-activated Cl^– currents (I_Cl,vol) and cell growth have been measured in cultured endothelial cells from bovine pulmonary artery (CPAE) in the absence and presence of compounds which block these currents. The anti-oestrogen drug tamoxifen, which efficiently arrests the growth of breast cancer cells (l), inhibits both I_Cl,vol and cell proliferation with IC₅₀ of 3.8 and 4.8 mol/l respectively. NPPB and quinine, which also block I_Cl,vol, inhibit the growth of CPAE cells as well. Current and cell growth were closely correlated under all these conditions. We conclude that I_Cl,vol might be involved in the control of endothelial cell growth and thus might be important for the modulation of vascularisation and vascular remodelling.     

Effects of interferon-γ on cell differentiation and cytokine production of a human monoblast cell line,U937

The U937 cell, a human monoblast cell line, has been used as a model to study the function of human monocytes. We investigated the effects of interferon- (IFN-) on Superoxide anion (O ₂ ^– ) production, cell surface antigens, and cytokine production of U937 cells. IFN- treatment enhanced O ₂ ^– production of fMLP or PMA-stimulated U937 cells. IFN- increased the ratio of CD23 and CD 11b positive cells. The fluorescence intensity of CD14 and CD25 was enhanced by IFN- treatment. U937 cells produced IL-1, IL-1, IL-6, and TNF- by lipopolysaccharide (LPS) stimulation. IFN- treatment enhanced TNF- production, but decreased IL-6 production. These results suggest that IFN- differentiates U937 cells to monocytelike cells and it regulates the production systems of IL-6 and TNF- separately in U937 cells.