Volume-sensitive Cl Research Articles

Endothelin signalling regulates volume-sensitive Cl− current via NADPH oxidase and mitochondrial reactive oxygen species

We assessed regulation of volume-sensitive Cl(-) current (I(Cl,swell)) by endothelin-1 (ET-1) and characterized the signalling pathway responsible for its activation in rabbit atrial and ventricular myocytes. ET-1 elicited I(Cl,swell) under isosmotic conditions. Outwardly rectified Cl(-) current was blocked by the I(Cl,swell)-selective inhibitor DCPIB or osmotic shrinkage and involved ET(A) but not ET(B) receptors. ET-1-induced current was abolished by inhibiting epidermal growth factor receptor (EGFR) kinase or phosphoinositide-3-kinase (PI-3K), indicating that these kinases were downstream. Regarding upstream events, activation of I(Cl,swell) by osmotic swelling or angiotensin II (AngII) was suppressed by ET(A) blockade, whereas AngII AT(1) receptor blockade failed to alter ET-1-induced current. Reactive oxygen species (ROS) produced by NADPH oxidase (NOX) stimulate I(Cl,swell). As expected, blockade of NOX suppressed ET-1-induced I(Cl,swell), but blockade of mitochondrial ROS production with rotenone also suppressed I(Cl,swell). I(Cl,swell) was activated by augmenting complex III ROS production with antimycin A or diazoxide; in this case, I(Cl,swell) was insensitive to NOX inhibitors, indicating that mitochondria were downstream from NOX. ROS generation in HL-1 cardiomyocytes measured by flow cytometry confirmed the electrophysiological findings. ET-1-induced ROS production was inhibited by blocking either NOX or mitochondrial complex I, whereas complex III-induced ROS production was insensitive to NOX blockade. ET-1-ET(A) signalling activated I(Cl,swell) via EGFR kinase, PI-3K, and NOX ROS production, which triggered mitochondrial ROS production. ET(A) receptors were downstream effectors when I(Cl,swell) was elicited by osmotic swelling or AngII. These data suggest that ET-1-induced ROS-dependent I(Cl,swell) is likely to participate in multiple physiological and pathophysiological processes.

Regulation of Volume-Sensitive Chloride Current in Cardiac HL-1 Myocytes

HL-1 cells derived from mouse atrial myocytes retain many features of differentiated adult cardiomyocytes, continuously divide, and are emerging as a useful experimental tool. Features of several HL-1 cation channels have been described, but the characteristics of its Cl− channels are unknown. We studied regulation of volume-sensitive Cl− current, ICl,swell, under conditions that isolate anion currents. Modest osmotic swelling (0.85T; T, times-isosmotic) elicited robust outwardly-rectifying Cl− currents in virtually every HL-1 cell (typically, 15 - 30 pA/pF at +60 mV; ECl = −40 mV). As expected for ICl,swell, Cl− current in 0.85T was fully inhibited by DCPIB (10 μM) and was outwardly rectifying in both physiological and symmetrical Cl− gradients. Regulation of HL-1 ICl,swell matched that in enzymatically dissociated adult cardiomyocytes. In 0.85T, HL-1 ICl,swell was fully blocked by both the NADPH oxidase inhibitor gp91ds-tat (500 nM) and the mitochondrial ETC inhibitor rotenone (10 μM), and in isosmotic bath solution (1T), DCPIB fully suppressed H2O2-induced (100 μM) ICl,swell. Furthermore, as in adult cardiomyocytes, endothelin-1 (ET-1; 10 nM) activated a DCPIB-sensitive current in 1T that was outwardly-rectifying in HL-1 cells with physiological and symmetrical Cl− gradients. ET-1-induced HL-1 ICl,swell was suppressed by the ETA receptor blocker BQ123 (1 μM) and by blocking ROS production with gp91ds-tat. HL-1 ICl,swell also was activated by bacterial sphingomyelinase (0.03 U/mL) that produces ceramide. These findings in HL-1 cells recapitulated the biophysical and pharmacological features of ICl,swell and its regulation by ROS, endothelin, and ceramides in adult myocytes. Our data indicate that HL-1 cells are a useful tool for dissecting the regulation and role of ICl,swell in cardiac myocytes.

Involvement of volume‐sensitive Cl− channels in the proliferation of human subcutaneous pre‐adipocytes

1. Obesity is a significant challenge in terms of public health and preventive medicine. Inhibition of pre-adipocyte proliferation is believed to be important in the proposed anti-obesity mechanism. The aim of the present study was to examine the interplay between Cl(-) channels and their possible involvement in the proliferation of undifferentiated human pre-adipocytes. 2. Pre-adipocytes were isolated from human abdominal subcutaneous adipose tissue. Membrane ion currents were recorded using the whole-cell patch-clamp technique. Expression of the Cl(-) channel ClC-3 gene and protein was determined by reverse transcription-polymerase chain reaction (RT-PCR) and western blot, respectively. Cell proliferation was evaluated using the [(3)H]-thymidine incorporation assay. 3. Electrophysiological recordings revealed a volume-sensitive Cl(-) current (I(Cl.vol)) expressed in pre-adipocytes that was activated under hyposmotic conditions (external osmolarity decreased by 80%) and inhibited by the Cl(-) channel blocker tamoxifen. 4. Expression of the ClC-3 channel gene and protein was confirmed by RT-PCR and western blot analysis. Blocking I(Cl.vol) with tamoxifen supressed the proliferation of pre-adipocytes in a concentration-dependent manner. 5. Collectively, the results of the present study indicate that the volume-sensitive Cl(-) channel participates in regulation of the proliferation of human subcutaneous pre-adipocytes.

Exogenous and endogenous ceramides elicit volume-sensitive chloride current in ventricular myocytes

Because ceramide accumulates in several forms of cardiovascular disease and ceramide-induced apoptosis may involve the volume-sensitive Cl(-) current, I(Cl,swell), we assessed whether ceramide activates I(Cl,swell). I(Cl,swell) was measured in rabbit ventricular myocytes by whole-cell patch clamp after isolating anion currents. Exogenous C(2)-ceramide (C(2)-Cer), a membrane-permeant short-chain ceramide, elicited an outwardly rectifying Cl(-) current in both physiological and symmetrical Cl(-) solutions that was fully inhibited by DCPIB, a specific I(Cl,swell) blocker. In contrast, the metabolically inactive C(2)-Cer analogue C(2)-dihydroceramide (C(2)-H(2)Cer) failed to activate Cl(-) current. Bacterial sphingomyelinase (SMase), which generates endogenous long-chain ceramides as was confirmed by tandem mass spectrometry, also elicited an outwardly rectifying Cl(-) current that was inhibited by DCPIB and tamoxifen, another I(Cl,swell) blocker. Bacterial SMase-induced current was partially reversed by osmotic shrinkage and fully suppressed by ebselen, a scavenger of reactive oxygen species. Outward rectification with physiological and symmetrical Cl(-) gradients, block by DCPIB and tamoxifen, and volume sensitivity are characteristics that identify I(Cl,swell). Insensitivity to C(2)-H(2)Cer and block by ebselen suggest involvement of ceramide signalling rather than direct lipid-channel interaction. Exogenous and endogenous ceramide elicited I(Cl,swell) in ventricular myocytes. This may contribute to persistent activation of I(Cl,swell) and aspects of altered myocyte function in cardiovascular diseases associated with by ceramide accumulation.

Investigation of ion channel mechanism for the regulatory volume decrease in lung adenocarcinoma cells

This study was designed to observe the regulatory volume decrease (RVD) process in human lung adenocarcinoma cells (A549) and to investigate its ion channel mechanism. Electric measurement system of cell volume was used to detect the cell volume changes following exposure to hypotonic solution. Whole-cell patch clamp recordings were applied to investigate the characteristics of the volume-sensitive Cl(-) channel in A549 cells. Extracellular hypotonicity induced cell swelling followed by a typical RVD process, which can be inhibited by Cl(-) channel blocker (NPPB 100 micromol/L) and K(+) channel blocker (CsCl 5 mmol/L). Meanwhile, a outward-rectifying chloride currents which was sensitive to NPPB and DIDs was recorded in A549 using the whole cell patch clamp. The human lung adenocarcinoma cells has RVD process which is dependent on the parallel activation of Cl(-) channel and K(+) channel. The volume-sensitive Cl(-) channel is involved in volume regulation of lung adenocarcinoma cells.

Roles of Volume-sensitive Cl- Channel on Apoptosis and Drug Resistance in Cancer Cells.

Roles of Volume-sensitive Cl- Channel on Apoptosis and Drug Resistance in Cancer Cells.

Microglia processes block the spread of damage in the brain and require functional chloride channels

Microglia cells exhibit two forms of motility, constant movement of filopodia probing surrounding brain tissue, and outgrowth of larger processes in response to nearby damage. The mechanisms and functions of filopodia sensing and process outgrowth are not well characterized but are likely critical for normal immune function in the brain. Using two photon laser scanning microscopy we investigated microglia process outgrowth in response to damage, and explored the relationship between process outgrowth and filopodia movement. Further, we examined the roles of Cl(-) or K(+) channel activation, as well as actin polymerization in these two distinct processes, because mechanistic understanding could provide a strategy to modulate microglia function. We found that volume sensitive Cl(-) channel blockers (NPPB, tamoxifen, DIDS) prevented the rapid process outgrowth of microglia observed in response to damage. In contrast, filopodia extension during sensing was resistant to Cl(-) channel inhibitors, indicating that these motile processes have different cellular mechanisms. However, both filopodia sensing and rapid process outgrowth were blocked by inhibition of actin polymerization. Following lesion formation under control conditions, rapidly outgrowing processes contacted the damaged area and this was associated with a 37% decrease in lesion volume. Inhibition of process outgrowth by Cl(-) channel block, prevention of actin polymerization, or by selectively ablating microglia all allowed lesion volume to increase and spread into the surrounding tissue. Therefore, process outgrowth in response to focal brain damage is beneficial by preventing lesion expansion and suggests microglia represent a front line defence against damage in the brain.

Endothelin-1 Regulates Volume-Sensitive Chloride Current in Rabbit Atrial Myocytes via Reactive Oxygen Species from Mitochondria and NADPH Oxidase

Angiotensin II (AngII) signaling and reactive oxygen species (ROS) produced by NADPH oxidase (NOX) are implicated in the activation of volume-sensitive Cl current (ICl,swell) by both beta1-integrin stretch and osmotic swelling. Because endothelin-1 (ET-1) is a potential downstream mediator of AngII and ET-1 blockade abrogates AngII-induced ROS generation, we studied how ET-1 signaling regulates ICl,swell. Under isosmotic conditions, ET-1 (10 nM) elicited an outwardly rectifying Cl current that was fully blocked by the highly selective ICl,swell inhibitor DCPIB (10 μM) and by osmotic shrinkage. Selective ETA (BQ-123, 1 μM) but not ETB blockade (BQ-788, 100 nM) fully suppressed ET-1-induced current. ET-1-induced ICl,swell also was abolished by inhibitors of EGFR kinase (AG1478, 10 μM) and PI-3K (LY294002, 20 μM; wortmannin, 500 nM), which also suppress stretch- and swelling-induced ICl,swell. ERK inhibitors (PD 98059, 10 μM; U0216, 1 μM) partially and fully blocked ET-1- and EGF- induced currents, respectively, but did not effect ICl,swell elicited by H2O2. ET-1 acts downstream from AngII. ETA blockade (BQ-123) abolished ICl,swell elicited by both AngII and osmotic swelling, whereas AT1 blockade (losartan, 5 μM) did not effect ET-1-induced ICl,swell. Both NOX and mitochondria are important sources of ROS in cardiomyocytes. Blocking NOX with apocynin (500 μM) or mitochondrial complex I with rotenone (10 μM) both completely suppressed ET-1-induced ICl,swell. In contrast, ICl,swell elicited by antimycin A (10 μM), which stimulates superoxide production by mitochondrial complex III, was insensitive to apocynin and the NOX fusion peptide inhibitor gp91ds-tat (500 nM). These data suggest that ET-1 and ETA receptors are required intermediates in AngII-, swelling-, and stretch-induced activation of ICl,swell. Moreover, enhancement of mitochondrial ROS production by ROS from NOX is likely to contribute to activation of ICl,swell by ET-1.

HIV Protease Inhibitors Activate Volume-Sensitive Chloride Current in Ventricular Myocytes by Generating Mitochondrial Reactive Oxygen Species

HIV protease inhibitors (HIV PI) have been successfully used to reduce morbidity and mortality of HIV infection. However, their long-term use causes significant side effects including cardiac arrhythmias. Previously we showed that outwardly-rectifying, volume-sensitive Cl current (ICl,swell) is regulated by signaling pathways that elicit production of reactive oxygen species (ROS). Because certain HIV PI recently were reported to augment ROS production, we tested whether HIV PI stimulate ICl,swell in rabbit ventricular myocytes. Under isosmotic conditions, ritonavir (15 μM, 20 min) and lopinavir (15 μM, 25 min) induced outwardly-rectifying Cl currents (1.5 ± 0.3 pA/pF and 1.9 ± 0.3 pA/pF at +60 mV, respectively) that were fully inhibited by the highly selective ICl,swell-blocker DCPIB (10 μM). In contrast, amprenavir (15 μM, 30 min) and nelfinavir (15 μM, 30 min) did not modulate ICl,swell, and raltegravir (MK-0518, 15 μM, 30 min), an HIV integrase inhibitor, also was ineffective. Two major sources of ROS in cardiomyocytes are sarcolemmal NADPH oxidase and mitochondria. The specific NADPH oxidase inhibitor apocynin (500 μM) failed to inhibit the ritonavir- or lopinavir-induced currents, although we previously found apocynin blocks ICl,swell activation upon osmotic swelling and stretch. In contrast, rotenone (10 μM, 30 min), a mitochondrial complex I inhibitor that limits electron flux to and ROS production by complex III, blocked 102 ± 4% of ritonavir- and 82 ± 12% of lopinavir-induced ICl,swell. Furthermore, the membrane-permeant, glutathione peroxidase mimetic ebselen (15 μM, 15 min) suppressed ICl,swell elicited by ritonavir (102 ± 3%) and lopinavir (93 ± 6%). These results suggest that ritonavir and lopinavir activate ICl,swell via mitochondrial ROS production by complex III. Activation of ICl,swell by certain HIV PI may contribute to their untoward effects in heart and potentially other tissues.

P2Y1 receptor‐mediated glutamate release from cultured dorsal spinal cord astrocytes

P2 receptors have been implicated in the release of neurotransmitter and proinflammatory cytokines by the response to neuroexcitatory substances in astrocytes. In the present study, we examined the mechanisms of ADP and adenosine 5'-O-2-thiodiphosphate (ADPbetaS, ADP analogue) on glutamate release from cultured dorsal spinal cord astrocytes by using confocal laser scanning microscopy and HPLC. Immunofluorescence activity showed that P2Y(1) receptor protein is expressed in cultured astrocytes. ADP and ADPbetaS-induced [Ca(2+)](i) increase and glutamate release are mediated by P2Y(1) receptor. Ca(2+) release from IP(3)-sensitive calcium stores and protein kinase C (PKC) activation is important for glutamate release from astrocytes. Furthermore, P2Y(1) receptor-evoked glutamate release is regulated by volume-sensitive Cl(-) channels and anion co-transporter, which open up the possibility that P2Y(1) receptor activation causes the increase of cell volume. Release of glutamate by ADPbetaS was abolished by 5-nitro-2 (3-phenyl propy lamino)-benzoate plus furosemide but was unaffected by botulinum toxin A. These observations indicate that P2Y(1) receptor-evoked glutamate may be mediated via volume-sensitive Cl(-) channel but not via exocytosis of glutamate containing vesicles. We speculate that P2Y(1) receptors-evoked glutamate efflux, occurring under pathological condition, may modulate the activity of synapses in spinal cord.

Roles of volume-sensitive Cl− channels in cell volume regulation and AVP secretion in vasopressin neurons

Roles of volume-sensitive Cl− channels in cell volume regulation and AVP secretion in vasopressin neurons

Thrombin increases hyposmotic taurine efflux and accelerates % MathType!Translator!2!1!AMS LaTeX.tdl!TeX -- AMS-LaTeX! % MathType!MTEF!2!1!+- % feaaeaart1ev0aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbbjxAHX % garmWu51MyVXgatuuDJXwAK1uy0HwmaeHbfv3ySLgzG0uy0Hgip5wz % aebbnrfifHhDYfgasaacH8qrps0lbbf9q8WrFfeuY-Hhbbf9v8qqaq % Fr0xc9pk0xbba9q8WqFfea0-yr0RYxir-Jbba9q8aq0-yq-He9q8qq % Q8frFve9Fve9Ff0dmeaabaqaciGacaGaaeqabaWaaeWaeaaakeaaca % qGjbGaae4qaiaabMeadaqhaaWcbaGaae4CaiaabEhacaqGLbGaaeiB % aiaabYgaaeaacqGHsislaaaaaa!3FBE! $$ {\text{ICI}}^{ - }_{{{\text{swell}}}} $$ and RVD in 3T3 fibroblasts by a src-dependent EGFR transactivation

The present study in Swiss3T3 fibroblasts examines the effect of thrombin on hyposmolarity-induced osmolyte fluxes and RVD, and the contribution of the src/EGFR pathway. Thrombin (5 U/ml) added to a 30% hyposmotic medium markedly increased hyposmotic 3H-taurine efflux (285%), accelerated the volume-sensitive Cl- current (ICI-swell) and increased RVD rate. These effects were reduced (50-65%) by preventing the thrombin-induced intracellular Ca2+ [Ca2+]i rise with EGTA-AM, or with the phospholipase C (PLC) blocker U73122. Ca2+calmodulin (CaM) and calmodulin kinase II (CaMKII) also participate in this Ca2+-dependent pathway. Thrombin plus hyposmolarity increased src and EGFR phosphorylation, whose blockade by PP2 and AG1478, decreased by 30-50%, respectively, the thrombin effects on hyposmotic taurine efflux, ICI-swell and RVD. Ca2+- and src/EGFR-mediated pathways operate independently as shown by (1) the persistence of src and EGFR activation when [Ca2+]i rise is prevented and (2) the additive effect on taurine efflux, ICI-swell or RVD by simultaneous inhibition of the two pathways, which essentially suppressed these events. PLC-Ca2+- and src/EGFR-signaling pathways operate in the hyposmotic condition and because thrombin per se failed to increase taurine efflux and ICI-swell under isosmotic condition it seems that it is merely amplifying these previously activated mechanisms. The study shows that thrombin potentiates hyposmolarity-induced osmolyte fluxes and RVD by increasing src/EGFR-dependent signaling, in addition to the Ca2+-dependent pathway.

Regulation of swelling-activated Cl- current by angiotensin II signalling and NADPH oxidase in rabbit ventricle

We assessed whether hypoosmotic swelling of cardiac myocytes activates volume-sensitive Cl(-) current (I Cl,swell) via the angiotensin II (AngII)-reactive oxygen species (ROS) signalling cascade. The AngII-ROS pathway previously was shown to elicit I(Cl,swell) upon mechanical stretch of beta(1D) integrin. Integrin stretch and osmotic swelling are, however, distinct stimuli. For example, blocking Src kinases stimulates swelling-induced but inhibits stretch-induced I Cl,swell. I Cl,swell was measured in rabbit ventricular myocytes by whole-cell voltage clamp. Swelling-induced I Cl,swell was completely blocked by losartan and eprosartan, AngII type I receptor (AT1) antagonists. AT1 stimulation transactivates epidermal growth factor receptor (EGFR) kinase. Blockade of EGFR kinase with AG1478 abolished both I Cl,swell and AngII-induced Cl(-) current, whereas exogenous EGF evoked a Cl(-) current that was suppressed by osmotic shrinkage. Phosphatidylinositol 3-kinase (PI-3K) is downstream of EGFR kinase, and PI-3K inhibitors LY294002 and wortmannin blocked I Cl,swell. Ultimately, AngII signals via NADPH oxidase (NOX) and superoxide anion, O2*. NOX inhibitors, diphenyleneiodonium, apocynin and gp91ds-tat, eliminated I Cl,swell, whereas scramb-tat, an inactive gp91ds-tat analogue, was ineffective. O2* rapidly dismutates to H2O2. Consistent with H2O2 being a downstream effector, catalase inhibited I Cl,swell, and exogenous H2O2 overcame suppression of I Cl,swell by AT1 receptor, EGFR kinase, and PI-3K blockers. H2O2-induced current was not blocked by osmotic shrinkage, however. Activation of I Cl,swell by osmotic swelling is controlled by the AngII-ROS cascade, the same pathway previously implicated in I Cl,swell activation by integrin stretch. This in part explains why I Cl,swell is persistently activated in several models of cardiac disease.

Regulatory volume decrease after swelling induced by urea in fibroblasts: prominent role of organic osmolytes

Cell swelling, regulatory volume decrease (RVD), volume-sensitive Cl(-) (Cl(-) (swell)) current and taurine efflux after exposure to high concentrations of urea were characterized in fibroblasts Swiss 3T3, and results compared to those elicited by hyposmotic (30%) swelling. Urea 70, 100, and 150 mM linearly increased cell volume (8.25%, 10.6%, and 15.7%), by a phloretin-inhibitable process. This was followed by RVD by which cells exposed to 70, 100, or 150 mM urea recovered 27.6%, 38.95, and 74.1% of their original volume, respectively. Hyposmolarity (30%) led to a volume increase of 25.9% and recovered volume in 32.5%. (3)H-taurine efflux was increased by urea with a sigmoid pattern, as 9.5%, 18.9%, 71.5%, and 89% of the labeled taurine pool was released by 70, 100, 150, or 200 mM urea, respectively. Only about 11% of taurine was released by 30% hyposmolarity reduction in spite of the high increase in cell volume. Urea-induced taurine efflux was suppressed by NPPB (100 microM) and markedly reduced by the tyrosine kinase-general blocker AG18. The Cl(-) (swell) current was more rapidly activated and higher in amplitude in the hyposmotic than in the isosmotic/urea condition (urea 150 mM), but this was not sufficient to accomplish an efficient RVD. These results showed that at similar volume increase, cells swollen by urea showed higher taurine efflux, lower Cl(-) (swell) current and more efficient RVD, than in those swollen by hyposmolarity. The correlation found between RVD efficiency and taurine efflux suggest a prominent role for organic over ionic osmolytes for RVD evoked by urea in isosmotic conditions.

Anion channel blockers attenuate delayed neuronal cell death induced by transient forebrain ischemia

Chloride efflux is known to be involved in the progression of apoptosis in various cell types. We have recently shown that the volume-sensitive outwardly rectifying (VSOR) anion channel serves as the pathway for apoptotic chloride efflux in some cells. In the present study, we tested the neuroprotective effects of drugs that can block the VSOR anion channel, on delayed neuronal death (DND) induced by transient forebrain ischemia. The functional expression of the VSOR anion channel was first examined in hippocampal neurons in both primary culture and hippocampal slice preparations, by the whole-cell patch-clamp technique. We then tested the channel's sensitivity to an anion channel blocker, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and a tyrosine kinase blocker, genistein. By histological examinations and cytochrome c release assessments, the protective effects of these drugs on the DND of hippocampal CA1 neurons in mice subjected to transient ischemia were examined. Drugs were administered via the jugular vein prior to ischemic treatment and into the peritoneal cavity after reperfusion. Hippocampal neurons were found to express the volume-sensitive Cl(-) channel, which exhibits outward rectification and is sensitive to DIDS and genistein. Administration of DIDS or genistein reduced cytochrome c release and the number of damaged neurons in the CA1 region after transient forebrain ischemia. This fact suggests that the DND induction mechanism involves the activity of the VSOR anion channel and that this channel may provide a therapeutic target for the treatment of stroke.

Differential dependence of regulatory volume decrease behavior in rabbit corneal epithelial cells on MAPK superfamily activation

We characterized the dependence of hypotonicity-induced regulatory volume decrease (RVD) responses on mitogen-activated protein kinase (MAPK) pathway signaling in SV40-immortalized rabbit corneal epithelial cells (RCEC). Following calcein-AM loading, RVD was monitored using a microplate fluorescence reader. Western blot analysis determined MAPK activation. After 30 min, the RVD response restored the relative cell volume to nearly isotonic values, whereas it was inhibited when cells were bathed either in a Cl −-free solution or with the Cl −-channel inhibitors: 5-nitro-2-(3-phenylpropylamino)benzoic acid or niflumic acid. Similar declines occurred with either a high-K + (20 mM) supplemented solution or the K + channel inhibitor 4-aminopyridine. Activation of extracellular signal-regulated kinase (ERK), p38, and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) was time and tonicity-dependent. Stimulation of ERK and SAPK/JNK was maximized earlier than that of p38. Activation of ERK and SAPK/JNK was insensitive to Cl − and K + channel inhibitors, whereas inhibition with either PD98059 or SP600125, respectively, blocked RVD. However, inhibition of p38 with SB203580 had no effect on RVD. Suppression of RVD instead blocked p38 activation. Differences in the dependence of RVD activation on Erk1/2 and p38 signaling were validated in dominant negative (d/n)-Erk1 and d/n-p38 cells. Volume-sensitive Cl − and K + channel activation contributes, in concert, to RVD in RCEC. Therefore, swelling-induced ERK and SAPK/JNK stimulation precedes Cl − and K + channel activation, whereas p38 activation occurs as a consequence of RVD.

EGFR Kinase Regulates Volume-sensitive Chloride Current Elicited by Integrin Stretch via PI-3K and NADPH Oxidase in Ventricular Myocytes

Stretch of β1 integrins activates an outwardly rectifying, tamoxifen-sensitive Cl− current (Cl− SAC) via AT1 receptors, NADPH oxidase, and reactive oxygen species, and Cl− SAC resembles the volume-sensitive Cl− current (ICl,swell). Epidermal growth factor receptor (EGFR) kinase undergoes transactivation upon stretch, integrin engagement, and AT1 receptor activation and, in turn, stimulates NADPH oxidase. Therefore, we tested whether Cl− SAC is regulated by EGFR kinase signaling and is volume sensitive. Paramagnetic beads coated with mAb for β1 integrin were attached to myocytes and pulled with an electromagnet. Stretch activated a Cl− SAC that was 1.13 ± 0.10 pA/pF at +40 mV. AG1478 (10 μM), an EGFR kinase blocker, inhibited 93 ± 13% of Cl− SAC, and intracellular pretreatment with 1 μM AG1478 markedly suppressed Cl− SAC activation. EGF (3.3 nM) directly activated an outwardly rectifying Cl− current (0.81 ± 0.05 pA/pF at +40 mV) that was fully blocked by 10 μM tamoxifen, an ICl,swell blocker. Phosphatidylinositol 3-kinase (PI-3K) is downstream of EGFR kinase. Wortmannin (500 nM) and LY294002 (100 μM), blockers of PI-3K, inhibited Cl− SAC by 67 ± 6% and 91 ± 25% respectively, and the EGF-induced Cl− current also was fully blocked by LY294002. Furthermore, gp91ds-tat (500 nM), a cell-permeable, chimeric peptide that specifically blocks NADPH oxidase assembly, profoundly inhibited the EGF-induced Cl− current. Inactive permeant and active impermeant control peptides had no effect. Myocyte shrinkage with hyperosmotic bathing media inhibited the Cl− SAC and EGF-induced Cl− current by 88 ± 9% and 127 ± 11%, respectively. These results suggest that β1 integrin stretch activates Cl− SAC via EGFR, PI-3K, and NADPH oxidase, and that both the Cl− SAC and the EGF-induced Cl− currents are likely to be the volume-sensitive Cl− current, ICl,swell.

Regulation of volume-sensitive Cl − channels in multi-drug resistant MCF7 cells

The P-glycoprotein (P-gp) is thought to be involved in the regulation of volume-sensitive chloride channels. In this study, the possible coupling between P-gp and swelling-activated chloride channels has been examined in MCF7 cells with sensitive (MDR −), resistant (MDR +), and reversed resistant (MDR REV) phenotypes. Western blot analysis showed that incubation of cells with doxorubicin induced P-gp expression in a reversible manner. Exposure of MDR + cells to hypotonicity resulted in an inhibition of P-gp activity while hypotonic challenges induced swelling-activated chloride currents ( I Cl-swell) in MDR −, MDR +, and MDR REV MCF7 cells. While verapamil inhibited I Cl-swell in all cell types, doxorubicin and vincristine rapidly and reversibly inhibited I Cl-swell uniquely in MDR +. Intracellular dialysis of MDR + cells with C219 anti-P-gp antibody abolished the sensitivity of I Cl-swell to doxorubicin and led to a response pattern very close to that of MDR − cells. Taken together, these results strongly suggest that the P-glycoprotein regulates I Cl-swell in resistant MCF7.

Alterations in the regulatory volume decrease (RVD) and swelling-activated Cl-current associated with neuroendocrine differentiation of prostate cancer epithelial cells

Neuroendocrine (NE) differentiation of prostate epithelial/basal cells is a hallmark of advanced, androgen-independent prostate cancer, for which there is no successful therapy. Here we report for the first time on alterations in regulatory volume decrease (RVD) and its key determinant, swelling-activated Cl- current (I(Cl,swell)), associated with NE differentiation of androgen-dependent LNCaP prostate cancer epithelial cells. NE-differentiating regimens, namely, chronic cAMP elevation or androgen deprivation, resulted in generally augmented I(Cl,swell) and enhanced RVD. This occurred as a result of both the increased endogenous expression of ClC-3, which is a volume-sensitive Cl- channel involved, as we show, in I(Cl,swell) in LNCaP (lymph-node carcinoma of the prostate) cells and the weaker negative I(Cl,swell) control from Ca2+ entering via store-dependent pathways. The changes in the RVD of NE-differentiated cells generally mimicked those reported for Bcl-2-conferred apoptotic resistance. Our results suggest that strengthening the mechanism that helps to maintain volume constancy may contribute to better survival rates of apoptosis-resistant NE cells.

Roles of Volume-sensitive Cl− Channel in Cisplatin-induced Apoptosis in Human Epidermoid Cancer Cells

The anti-cancer drug cisplatin induces apoptosis by damaging DNA. Since a stilbene-derivative blocker of Cl-/HCO3- exchangers and Cl- channels, SITS, is known to induce cisplatin resistance in a manner independent of intracellular pH and extracellular HCO3-, we investigated the relation between cisplatin-induced apoptosis and Cl- channel activity in human adenocarcinoma KB cells. A stilbene derivative, DIDS, reduced cisplatin-induced caspase-3 activation and cell death, which were detected over 18 h after treatment with cisplatin. DIDS was also found to reduce sensitivity of KB cells to 5-day exposure to cisplatin. Whole-cell patch-clamp recordings showed that KB cells functionally express volume-sensitive outwardly rectifying (VSOR) Cl- channels which are activated by osmotic cell swelling and sensitive to DIDS. Pretreatment of the cells with cisplatin for 12 h augmented the magnitude of VSOR Cl- current. Thus, it is concluded that cisplatin-induced cytotoxicity in KB cells is associated with augmented activity of a DIDS-sensitive VSOR Cl- channel and that blockade of this channel is, at least in part, responsible for cisplatin resistance induced by a stilbene derivative.