Effects of NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid) on chloride transport in intestinal tissues and the T84 cell line.

NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid) has been reported to block Cl- channels in isolated rabbit nephrons with high potency (IC50 = 80 nM). The effects of this compound on Cl(-)-mediated transport processes in intestinal tissues have been studied using agonist-stimulated short-circuit current (T84) in Ussing chamber experiments and 36Cl- fluxes in monolayers of a colonic cell line (T84). NPPB inhibited PGE1-stimulated Isc in rabbit distal colon and ileum at concentrations in the range 20 to 100 microM. However, NPPB at the same concentrations also inhibited glucose-stimulated Isc in rabbit ileum, suggesting that its effects were not restricted to those on Cl- transport. Consistent with this, exposure of rabbit distal colon to 100 microM NPPB was found to reduce endogenous ATP levels by 69%, implying that, at these concentrations, NPPB could impair active transport processes by an effect on cellular energy metabolism. Clear evidence for a direct effect of NPPB on epithelial chloride channels was found in studies on Cl- fluxes in T84 cell monolayers. NPPB inhibited VIP-stimulated Cl- uptake into T84 cells with an IC50 of 414 microM. NPPB (1 mM) also inhibited Cl- efflux from pre-loaded cells confirming its effect as a weak Cl- channel blocker in this system.     

Volume-activated chloride currents in interstitial cells of Cajal

Interstitial cells of Cajal (ICC) undergo marked morphological changes on contraction of the musculature, making it essential to understand properties of mechanosensitive ion channels. The whole cell patch-clamp technique was used to identify and to characterize volume-activated Cl- currents in ICC cultured through the explant technique. Hypotonic solutions (approximately 210 mosM) activated an outwardly rectifying current, which reversed near the equilibrium potential for Cl-. Time-dependent inactivation occurred only at pulse potentials of +80 mV, with a time constant of 478 +/- 182 ms. The degree of outward rectification was calculated using a rectification index, the ratio between the slope conductances of +65 and -55 mV, which was 13.9 +/- 1.5 at 76 mM initial extracellular Cl- concentration. The sequence of relative anion permeability of the outwardly rectifying Cl- channel was I- > Cl- > aspartate-. The chloride channel blockers, DIDS and 5-nitro-2-(3-phenlypropl-amino)benzoic acid, caused a voltage-dependent block of the outwardly rectifying Cl- current, inhibition occurring primarily at depolarized potentials. On exposure to hypotonic solution, the slope conductance significantly increased at the resting membrane potential (-70 mV) from 1.2 +/- 0.2 to 2.0 +/- 0.4 nS and at the slow-wave plateau potential (-35 mV) from 2.1 +/- 0.3 to 5.0 +/- 1.0 nS. The current was constitutively active in ICC and contributed to the resting membrane potential and excitability at the slow-wave plateau. In conclusion, swelling or volume change will depolarize ICC through activation of outwardly rectifying chloride channels, thereby increasing cell excitability.     

Inhibition of prostaglandin E2 synthesis by a blocker of epithelial chloride channels

Arginine-vasopressin (AVP) elicits a variety of responses in cultured rat mesangial cells, among them stimulation of prostaglandin biosynthesis and activation of Cl- channels. AVP produced an 11-fold increase over basal levels in prostaglandin E2 release from cultured mesangial cells. This response was completely inhibited by 25 microM indomethacin and 82 +/- 5% inhibited by 25 microM 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) which is a potent blocker of epithelial Cl- channels. The IC50 for NPPB inhibition of prostaglandin E2 release was 8 microM. Indomethacin and NPPB at 25 microM also inhibited AVP-stimulated cellular accumulation of prostaglandin E2 by 98% and 79 +/- 7% respectively. The inhibitory effect of NPPB was not due to interference with the cellular response to AVP since at 50 microM it did not block AVP-stimulated release of arachidonate metabolites from cells metabolically labeled with [3H]-arachidonic acid. It is suggested that NPPB inhibition of prostaglandin E2 synthesis is at the cyclooxygenase level on the basis of its structural similarity to the fenamic acid type of cyclooxygenase inhibitors.     

Basolateral Cl- Transport Is Stimulated by Terbutaline in Adult Rat Alveolar Epithelial Cells

Stimulation of adult rat alveolar epithelial cells with terbutaline was previously shown to activate Cl- channels in the apical membrane. In this study, we show that terbutaline stimulates net transepithelial (apical-to-basolateral) Cl- absorption from 0.19 +/- 0.13 to 1.43 +/- 0.31 mmol x cm-2 x hr-1. Terbutaline also increases net Cl- efflux across the basolateral membrane under conditions where an outward [K+] gradient exists and the membrane voltage is clamped at zero mV. When the [K+] gradient is eliminated, the effect of terbutaline on net Cl- efflux is inhibited to the extent that no significant Cl- efflux can be detected across the basolateral membrane. RT-PCR experiments detected mRNA for three KCl cotransport isoforms (KCC1, KCC3 and KCC4) in monolayer cultures of alveolar epithelial cells. Western blot analysis using antibodies to the four cloned isoforms of KCl cotransporters revealed the presence of KCC1 and KCC4 isoforms in monolayer cultures of these cells. These results provide evidence suggesting a role for KCl cotransport in terbutaline-stimulated transepithelial Cl- absorption.     

Prostaglandin E1 activates a chloride current in Jurkat T lymphocytes via cAMP-dependent protein kinase.

Patch-clamp studies have identified a cAMP-dependent Cl- conductance in lymphocytes that is defectively regulated in cystic fibrosis. In this study we used 125I efflux and whole-cell patch-clamp studies to investigate whether prostaglandin E1 (PGE1), an agonist that generates intracellular cAMP in Jurkat T lymphocytes, activates a Cl- conductance. Stimulation of T cells by externally applied PGE1 stimulated 125I efflux and activated a slowly developing membrane current. When external and internal Cl- were about equal, the current reversed at about zero mV, but when external Cl- was lowered from 157 to 7 mM the reversal potential shifted 75 mV in the positive direction, demonstrating that the current carrier was Cl-. In addition, the current was blocked by 10 microM 5-nitro-2(3-phenylpropylamino) benzoic acid (NPPB), a potent Cl- channel blocker. A membrane-permeable cAMP analog mimicked the effect of PGE1, whereas intracellular application of a cAMP antagonist Rp-cAMP blocked the effect of PGE1. Addition of purified catalytic subunit of cAMP-dependent protein kinase (PKA) plus ATP to the recording pipette also activated a similar current, whereas internally applied Walsh inhibitor, the synthetic peptide inhibitor of PKA, blocked the PGE1 effect. These results suggest that PGE1, acting through PKA, activates a Cl- current in Jurkat T cells.     

ATP激活鼻咽癌细胞氯电流并减小细胞容积

采用全细胞膜片钳技术和细胞容积测量技术,在低分化鼻咽癌细胞株CNE-2Z上观察ATP 诱导的Cl- 电流的特性及其对细胞容积的影响。细胞外微摩尔水平的ATP 以剂量依赖性的方式激活一个具有弱外向整流特性,没有时间依赖性失活的电流,此电流的反转电位 [(-0.05 ± 0.03) mV]接近Cl- 的平衡电位(-0.9 mV)。用葡萄糖酸置换细胞外液Cl- 后, ATP 激活的电流明显减小并且反转电位发生改变。氯通道抑制剂NPPB (200 μmol/L)可以抑制这一电流 [(81.03 ± 9.3)%] 。此电流亦可被嘌呤受体(P2Y) 拮抗剂反应蓝 2 抑制 [(67.39 ± 5.06)%]。50 μmol/L 的 ATP 使在等渗状态下的细胞容积缩小, 替代和耗竭细胞外、内的Cl- 后, ATP 的这一作用消失。这些结果提示细胞外微摩尔水平的 ATP 可通过兴奋 P2Y 受体激活氯通道而产生与细胞容积调节相关的Cl- 电流。     

Single-cell image analysis to assess ABC-transporter-mediated efflux in highly purified hematopoietic progenitors

BACKGROUND: Normal and malignant hematopoietic stem cells are characterized by their capacity to actively extrude fluorescent dyes. The contribution of different ATP-binding cassette (ABC) transporters to this phenomenon is largely unknown due to the small stem cell numbers limiting the use of standard methods to assess functional efflux. METHODS: We used epifluorescence microscopy (EFM) in combination with single-cell image analysis to study ABC-transporter-mediated efflux in highly purified, viable, CD34+CD38- cells sorted on an adhesive biolayer. P-glycoprotein and multidrug-resistant protein (MRP)-mediated efflux were quantitated using fluorescent substrates (rhodamine-123 and calcein acetoxymethyl ester [calcein-AM]) and specific inhibitors (verapamil and probenecid, respectively). RESULTS: The feasibility, sensitivity, and reproducibility of rhodamine-123 efflux quantitation using single-cell EFM was shown in cell lines and compared with standard flow cytometric assessment. P-glycoprotein-mediated transport was higher in CD34+CD38- cells than in more differentiated progenitors (mean efflux index = 2.24 +/- 0.35 and 1.14 +/- 0.11, respectively; P = 0.01). P-glycoprotein-mediated transport was the main determinant of the rhodamine "dull" phenotype of these cells. In addition, significant MRP-mediated efflux was demonstrated in CD34+CD38- and CD38+ cells (mean efflux index = 1.42 +/- 0.19 and 1.28 +/- 0.18, respectively). CONCLUSION: The described method is a valuable tool for assessing ABC-transporter-mediated efflux in highly purified single cells. Both P-glycoprotein and MRP-mediated efflux are present in human CD34+CD38- hematopoietic stem cells.     

Immunologically activated chloride channels involved in degranulation of rat mucosal mast cells.

Crosslinking of type I Fc epsilon receptors (Fc epsilon RI) on the surface of basophils or mast cells initiates a cascade of processes leading to the secretion of inflammatory mediators. We report here a correlation between mediator secretion and the activation of Cl- channels in rat mucosal-type mast cells (line RBL-2H3). Stimulation of RBL cells by either IgE and antigen or by a monoclonal antibody specific for the Fc epsilon RI, resulted in the activation of Cl- ion channels as detected by the patch-clamp technique. Channel activation occurred slowly, within minutes after stimulation. The channel has a slope conductance of 32 pS at potentials between 0 and -100 mV, and an increasing open-state probability with increasing depolarization. Activation of apparently the same Cl- channels could be mimicked without stimulation by isolating inside-out membrane patches in tyrode solution. Parallel inhibition of both Cl- channel activity and mediator secretion, as monitored by serotonin release, was observed by two compounds, the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and the anti-allergic drug cromolyn. NPPB inhibited both the antigen-induced Cl- current and the serotonin release, where half-maximal inhibition occurred at similar doses, at 52 microM and 77 microM, respectively. The drug cromolyn, recently found to inhibit immunologically induced mediator secretion from RBL cells upon intracellular application, also blocks Cl- channels (IC50 = 15 microM) when applied to the cytoplasmic side of an inside-out membrane patch. The observed Cl- channel activation upon immunological stimulation and the parallel inhibition of channel current and of serotonin release suggests a functional role for this Cl- channel in mediator secretion from the mast cells studied.     

Pharmacological and biophysical properties of swelling-activated chloride channel in mouse cardiac myocytes

The present study was designed to observe the properties of swelling-activated chloride channel (ICl.swell) in mouse cardiac myocytes using patch clamp techniques. In whole-cell recordings, hypotonic solution activated a chloride current that exhibited outward rectification, weak voltage-dependent inactivation, and anion selectivity with permeability sequence of I- > Br- > Cl-. The current was sensitive to Cl- channel blockers tamoxifen, NPPB and DIDS. In single-channel recordings, cell swelling activated a single channel current which showed outward rectification with open probability of 0.76 +/- 0.08 and conductance of 38.1 +/- 2.5 pS at +100 mV under [Cl-] symmetrical condition. I-V relation revealed the reversal potential as expected for a Cl(-)-selective channel. These results suggested that in mouse cardiac myocytes, swelling-activated, outward rectifying chloride channel with a single channel conductance of 38.1 +/- 2.5 pS (at +100 mV under [Cl-] symmetrical condition) underlies the volume regulatory Cl- channel.     

Characterization of the taurine transport pathway in A6 kidney cells

We investigated the role of taurine in cell homeostasis and characterized the taurine transport pathway in cultured kidney cells (A6). The taurine concentration in A6 cells varies with the osmolarity of the culture medium, suggesting that taurine participates in cell osmolarity. Under isosmotic conditions, 14C-taurine efflux through the apical membranes (aJtaur) was 6-7 times lower than that through the basolateral membranes (bJtaur). Under hyposmotic conditions, aJtaur remained almost unchanged. On the contrary, bJtaur increased 8 times in comparison with isosmotic conditions. In hyposmotic conditions, bJtaur was inhibited by 500 microM DIDS, 50 microM NPPB, 10 microM of the two oxonol derivatives DISBAC(2)3 and WW-791, and 100 microM ketoconazole. Conversely, 100 microM 1,9-dideoxyforskolin, 10 microM tamoxifen, 100 microM niflumic acid and 50 microM verapamil had no inhibitory effects. Cell volume regulation upon hyposmotic stress was also found to be inhibited by DISBAC(2)3 (K0.5 of 5+/-1 microM) and by ketoconazole. Nystatin was used to permeabilize the apical membranes with the aim to further characterize bJtaur. 14C-taurine transepithelial fluxes in nystatin-treated cells were found to be linear over taurine concentrations ranging from 3.5 microM to 35 mM. Clamping the transepithelial voltage at positive values (serosal side) slightly stimulated the 14C-taurine transport. Similar time courses of 14C-taurine, 36Cl and 86Rb transepithelial fluxes were found under osmotic stimulation followed by DIDS inhibition in nystatin-treated cells. In whole cell patch-clamp experiments, DISBAC(2)3 application resulted in a strong and reversible decrease of the global Cl- current which was stimulated by hyposmotic stress. Our study indicates that taurine participates in the control of A6 cell osmolarity and that the transporting taurine pathway (efflux) is on the basolateral membranes. In addition to usual chloride channel blockers, oxonol was found to be a potent blocker of the taurine transport and of the swelling-activated chloride current. Using a pharmacological approach, we could not distinguish between a common or different pathway for Cl- and taurine.     

Assessment of swelling-activated Cl- channels using the halide-sensitive fluorescent indicator 6-methoxy-N-(3-sulfopropyl)quinolinium.

This study describes a quantitative analysis of the enhancement in anion permeability through swelling-activated Cl- channels, using the halide-sensitive fluorescent dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). Cultured bovine corneal endothelial monolayers perfused with NO3- Ringer's were exposed to I- pulses under isosmotic and, subsequently, hyposmotic conditions. Changes in SPQ fluorescence due to I- influx were significantly faster under hyposmotic than under isosmotic conditions. Plasma membrane potential (Em) was -58 and -32 mV under isosmotic and hyposmotic conditions, respectively. An expression for the ratio of I- permeability under hyposmotic condition to that under isosmotic condition (termed enhancement ratio or ER) was derived by combining the Stern-Volmer equation (for modeling SPQ fluorescence quenching by I-) and the Goldman flux equation (for modeling the electrodiffusive unidirectional I- influx). The fluorescence values and slopes at the inflection points of the SPQ fluorescence profile during I- influx, together with Em under isosmotic and hyposmotic conditions, were used to calculate ER. Based on this approach, endothelial cells were shown to express swelling-activated Cl- channels with ER = 4.9 when the hyposmotic shock was 110 +/- 10 mosM. These results illustrate the application of the SPQ-based method for quantitative characterization of swelling-activated Cl- channels in monolayers.     

Swelling-activated K+ efflux and regulatory volume decrease efficiency in human bronchial epithelial cells

This study describes the correlation between cell swelling-induced K+ efflux and volume regulation efficiency evaluated with agents known to modulate ion channel activity and/or intracellular signaling processes in a human bronchial epithelial cell line, 16HBE14o(-1). Cells on permeable filter supports, differentiated into polarized monolayers, were monitored continuously at room temperature for changes in cell height (T(c)), as an index of cell volume, whereas (86)Rb efflux was assessed for K+ channel activity. The sudden reduction in osmolality of both the apical and basolateral perfusates (from 290 to 170 mosmol/kg H(2)O) evoked a rapid increase in cell volume by 35%. Subsequently, the regulatory volume decrease (RVD) restored cell volume almost completely (to 94% of the isosmotic value). The basolateral (86)Rb efflux markedly increased during the hyposmotic shock, from 0.50 +/- 0.03 min(-1) to a peak value of 6.32 +/- 0.07 min(-1), while apical (86)Rb efflux was negligible. Channel blockers, such as GdCl(3) (0.5 mM), quinine (0.5 mM) and 5-nitro-2-(3-phenyl-propylamino) benzoic acid (NPPB, 100 microM), abolished the RVD. The protein tyrosine kinase inhibitors tyrphostin 23 (100 microM) and genistein (150 microM) attenuated the RVD. All agents decreased variably the hyposmosis-induced elevation in (86)Rb efflux, whereas NPPB induced a complete block, suggesting a link between basolateral K(+) and Cl(-1) efflux. Forskolin-mediated activation of adenylyl cyclase stimulated the RVD with a concomitant increase in basolateral (86)Rb efflux. These data suggest that the basolateral extrusion of K+ and Cl(-1) from 16HBE14o(-1) cells in response to cell swelling determines RVD efficiency.     

Role of Ca2+-activated Cl- channels in the mechanism of apoptosis induced by cyclosporin A in a human hepatoma cell line

The mechanism of apoptosis induced by cyclosporin A (CsA) in a human hepatoma cell line was investigated. CsA induced apoptosis in a dose- and time-dependent manner in HepG2 human hepatoma cells. CsA induced Cl- efflux, which was significantly blocked by niflumic acid (NA), a specific inhibitor, and flufenamic acid (FA), 5-nitro-2-(3-phenyl-propylamino)-benzoate (NPPB), and 4,4'-diisothiocyanoto-stibene-2,2'-disulfonic acid (DIDS), non-specific inhibitors of Ca2+-activated Cl- channels (CaCCs), not by calyculin A, an inhibitor of K+,Cl- -cotransport. In addition, CsA did not alter intracellular K+ concentration. Moreover, CsA increased intracellular Ca2+ concentration, and treatment with BAPTA/AM, an intracellular Ca2+ chelator, significantly inhibited the CsA-induced Cl- efflux, indicating that CsA induced Cl- efflux through the activation of CaCCs. Treatment with these CaCC inhibitors (NA, FA, NPPB, and DIDS) markedly prevented the CsA-induced apoptosis. Taken together, these results suggest that CaCCs may mediate apoptosis induced by CsA in HepG2 cells. Furthermore, these results provide a new insight into the novel function of CaCCs in the regulation of cancer cell apoptosis associated with perturbation of intracellular Ca2+ signal.     

Whole-cell chloride conductances in cultured brushed human nasal epithelial cells.

Human airway epithelial cells were obtained by nasal brushing, thus avoiding the use of proteolytic enzymes for cell isolation. Whole-cell Cl- conductances were studied in these cells by means of the patch-clamp technique. During whole-cell recordings, cell swelling activated a Cl- conductance that was blocked by indanyloxyacetic acid (48 +/- 10% inhibition at 50 microM). The swelling-induced current outwardly rectified and showed inactivation at depolarizing voltages (> or = +60 mV) and activation at hyperpolarizing voltages (< or = -30 mV). The voltage sensitivity of current activation was approximately twice that of inactivation. Another Cl- current with different kinetics was observed when nonswollen airway cells were stimulated with ionomycin (2 microM) in the presence of 1 mM Ca2+. The Ca(2+)-induced current exhibited activation during depolarizing voltage steps (> or = +40 mV) and inactivation during hyperpolarizing voltage steps (< or = -40 mV). In contrast to the swelling-induced current, the activation of Ca(2+)-induced current was less sensitive to voltage compared with its inactivation. Tail current analysis suggested that Cl- channels having a linear current-voltage relation mediate the response to Ca2+. This study indicates that brushed human nasal epithelial cells possess Cl- conductances that are regulated by cell swelling and Ca2+ and that they represent a useful in vitro model for studying ion transport in epithelia.     

Effects of N-ethylmaleimide on ouabain-insensitive cation fluxes in human red cell ghosts

In red cells of several species, the sulfhydryl reagent N-ethylmaleimide activates a Cl- -dependent, ouabain-resistant K+ transport pathway. Here we report our attempts to demonstrate ouabain-resistant Cl- -dependent K+ fluxes stimulated by N-ethylmaleimide in resealed human red cell ghosts using Rb+ as a K+ analogue. In contrast to intact cells, the rate constants of the base level Rb+ efflux in ghosts were similar in NaNO3 and NaCl (okRb = 0.535 +/- 0.079 h-1 and 0.534 +/- 0.085 h-1, respectively), while 1 mM N-ethylmaleimide stimulated Rb+ efflux strongly in NaNO3 (okRb = 14.26 +/- 1.32 h-1) and moderately in NaCl (okRb = 2.73 +/- 0.54 h-1). This effect was dependent on the presence of internal ATP. Stimulation of Rb+ efflux was observed in the presence of greater than or equal to 0.2 mM N-ethylmaleimide and increased at pH values approaching 8.0, consistent with titration of SH groups. N-Ethylmaleimide-stimulated Rb+ efflux was approx. 50% inhibited by 100 microM quinine sulfate whereas 1 microM bumetanide had no effect. In NaCl the N-ethylmaleimide-stimulated efflux saturated with initial internal ghost Rb+ concentration, but rates increased linearly in NaNO3. Replacement of external Na+ with glucamine or choline decreased the N-ethylmaleimide-stimulated Rb+ efflux, suggesting a role for external Na+. N-Ethylmaleimide-stimulated Rb+ efflux was greater in buffers with lipophilic anions such as SCN- or NO3- than in solutions with Cl- or acetate. However, the cation selectivity of the pathway studied was low, as Li+ efflux was also stimulated by N-ethylmaleimide. We conclude that the effect of N-ethylmaleimide on ouabain-resistant cation effluxes of human red cell ghosts is very different from the selective action of N-ethylmaleimide on Rb+ influxes in intact red cells.     

A kinetic study of Rhodamine123 pumping by P-glycoprotein

The MDR1 P-glycoprotein (P-gp) actively extrudes a wide variety of structurally diverse cytotoxic compounds out of the cell, is widely expressed in the epithelial cells of kidney, liver and intestine, and in the endothelial cells of brain and placenta, and plays an important role in drug resistance. We measured the accumulation of Rhodamine 123 (Rho123), a substrate of P-gp, into a drug sensitive and a drug resistant strain of the human leukemia cell line K562, as function of Rho123 concentration. With the aid of a mathematical transformation, we used the accumulation of Rho123 into the sensitive cells as a surrogate measure for the internal concentration of the probe in the resistant cells, and were thus able to measure the kinetic parameters of drug efflux pumping by P-gp. Drug pumping was half-saturated at an external Rho123 concentration of 7.2E-06+/-1.1E-06 M, and displayed a co-operative behaviour with a Hill number of 1.94+/-0.32. Verapamil could be shown to inhibit Rho123 efflux uncompetitively.     

Elimination of clonogenic tumor cells from bone marrow using methylprednisolone (MP) and etoposide VP16: an in vitro pharmacologic study

The ability to eliminate malignant cells from bone marrow (BM) while retaining sufficient numbers of normal progenitors to ensure engraftment, may well establish the future of autologous BM transplantation (ABMT) for hematologic malignancies. In this study, we describe the effects of methylprednisolone (MP) and etoposide (VP16) alone or in combination on 5 tumor cell lines (HL-60, a promyelocytic cell line; Molt-4, a T cell leukemia; Daudi, a Burkitt's lymphoma and R10/8226 and R40/8226, doxorubicin-resistant myeloma cell lines). The tumor cell kill efficiency of the drugs was assayed using the limiting dilution assay. We determined the toxic effect on progenitor cells by assaying granulocyte-macrophage colony-forming units (CFU). With a combination of MP at 10(-3) M and VP16 at 75 microM, we observed the following log reduction in tumor cell clones: HL-60, 4.695 +/- 0.001; Molt-4, 3.626 +/- 0.036; Daudi, 5.633 +/- 0.001; R10/8226, 3.052 +/- 0.544; R40/8226, 3.126 +/- 0.080. CFU recovery was 24% +/- 5%. Mixing tumor cell lines with a 20-fold excess of normal irradiated BM cells did not eliminate the inhibitory effect of the drug combination. We propose that MP and VP16 used in concert produce effective purging of malignant hematopoietic cells from BM while sparing normal progenitors needed for engraftment.     

Cl- channel blockers inhibit transition of quiescent (G0) fibroblasts into the cell cycle

Modulation of ion permeability during the cell cycle is one of the key events in cell cycle progression. We have compared the effects of K+ and Cl- channel blockers on the cell cycle in synchronous and asynchronous NIH3T3 cells. The Cl- channel blocker 5-N-2-(3-phenylpropylamino) benzoic acid (NPPB; 0.2 mM) inhibited entry into S phase in synchronous cells but not in asynchronous cells, while the K+ channel blocker 4-aminopyridine (4-AP) showed similar inhibitory effects in both conditions. In NIH3T3 cells synchronized by serum deprivation/replenishment, G0-to-G1 transition occurred within 8 h after serum addition, and the G1/S checkpoint at 10-14 h. NPPB applied only at 0-8 or 8-14 h after serum addition inhibited entry into S phase. Cl- permeability measured as 125I efflux increased at 4 and 10 h after serum addition. Ki-67-negative cells, which represent quiescent G0 phase cells, progressively decreased in number until 8 h after serum addition. The Cl- channel blockers (NPPB and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid [DIDS]) but not the K+ channel blocker (4-AP) significantly decreased the rate of reduction in number of Ki-67-negative cells. These data indicate that an increase in Cl- permeability plays an important role in reentry of quiescent cells into the proliferating phase, in addition to the known effects on passage through the G1/S checkpoint.     

Characterisation of the effects of anthranilic and (indanyloxy) acetic acid derivatives on chloride transport in membrane vesicles.

The effects of the Cl- channel blockers, NPPB, IAA94/95 and a number of related compounds on 36Cl- transport in membrane vesicles from bovine kidney cortex and rabbit ileum mucosa brush borders have been studied. These vesicles have been previously shown to be enriched in Cl- channel and Cl-/anion cotransport activity, respectively. Chloride transport was assayed in both types of vesicles by measuring the uptake of 36Cl- in response to an outwardly-directed Cl- concentration gradient. In kidney microsomes, a large proportion of the observed 36Cl- uptake was mediated by an electrogenic uniport and could be substantially reduced by clamping the membrane potential at zero mV using K+ and valinomycin. Chloride uptake was inhibited by both NPPB and IAA94/95 with apparent IC50 values of around 10 microM under optimal conditions (i.e., 4 min uptake at 4 degrees C). Under other conditions (e.g., 10 min uptake at 25 degrees C), where uptake had reached a steady-state level, much higher concentrations of inhibitor were required to cause inhibition. Therefore, previous differences in the reported potency of these compounds may, in part, have been due to the conditions under which Cl- uptake was measured. In addition, both NPPB and, to a lesser extent, IAA94/95 were found to have other effects on the vesicles, in that, when added at a concentration of 100 microM, they induced a leakage of pre-accumulated 36Cl-. This was probably caused by either dissipation of membrane potential or damage to the vesicle membranes. The sulphonic acid derivatives of NPPB and IAA94/95 (NPPB-S and ISA94/95, respectively) blocked 36Cl- uptake with around the same potency as NPPB and IAA94/95, but did not cause any non-specific Cl- leakage, when added at concentrations up to 100 microM. Inhibition of 36Cl- uptake by all four compounds was almost completely reversible. However, when vesicles were incubated with the inhibitors in the presence of an outward Cl- concentration gradient, or if vesicles were freeze/thawed in the presence of the compounds, inhibition could be only partially reversed. In rabbit brush border membrane vesicles, 36Cl- uptake was not reduced when the vesicles were voltage clamped using valinomycin and K+, and was therefore probably mediated by Cl-/Cl- exchange. However, despite the lack of effect of valinomycin, 36Cl- uptake was inhibited by both NPPB (approx. 80% inhibition at 100 microM) and, to a lesser extent, by IAA94/95 (approx. 30% inhibition at 100 microM).(ABSTRACT TRUNCATED AT 400 WORDS)     

Roles of volume-activated Cl- currents and regulatory volume decrease in the cell cycle and proliferation in nasopharyngeal carcinoma cells

OBJECTIVES: Previously it has been shown, that the volume-activated plasma membrane chloride channel is associated with regulatory volume decrease (RVD) of cells and may play an important role in control of cell proliferation. We have demonstrated that both expression of the channel and RVD capacity are actively regulated in the cell cycle. In this study, we aimed to further study the role of the volume-activated chloride current and RVD in cell cycle progression and overall in cell proliferation. MATERIALS AND METHODS: Whole-cell currents, RVD, cell cycle distribution, cell proliferation and cell viability were measured or detected with the patch-clamp technique, the cell image analysis technique, flow cytometry, the MTT assay and the trypan blue assay respectively, in nasopharyngeal carcinoma cells (CNE-2Z cells). RESULTS: The Cl- channel blockers, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and tamoxifen, inhibit the volume-activated chloride current, RVD and proliferation of CNE-2Z cells in a dose-dependent manner. Analysis of relationships between the current, RVD and cell proliferation showed that both the current and RVD were positively correlated with cell proliferation. NPPB (100 microM) and tamoxifen (20 microM) did not significantly induce cell death, but inhibited cell proliferation, implying that the blockers may inhibit cell proliferation by affecting cell cycle progression. This was verified by the observation that tamoxifen (20 microM) and NPPB (100 microM) inhibited cell cycle progress and arrested cells at the G0/G1 phase boundary. CONCLUSIONS: Activity of the volume-activated chloride channel is one of the important factors that regulate the passage of cells through the G1 restriction point and that the Cl- current associated with RVD plays an important role in cell proliferation.