Role of Calcium in Volume-Activated Chloride Currents in a Mouse Cholangiocyte Cell Line

Volume-activated Cl(-) channels (VACCs) play vital roles in many cells including cholangiocytes. Previously, we characterized the VACCs in mouse cholangiocytes. Since calcium plays an important role in VACC regulation in many cells, we have studied the effect of calcium modulation on the regulatory volume decrease (RVD) and VACC currents in mouse bile duct cells (MBDCs). Cell volume measurements were assessed by a Coulter counter with cell sizer, and conventional whole-cell patch-clamp techniques were used to study the role of calcium on RVD and VACC currents. Cell volume study indicated that MBDCs exhibited RVD, which was inhibited by 5-nitro-2'-(3-phenylpropylamino)-benzoate (NPPB), 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra-acetoxymethyl ester (BAPTA-AM) but not by removal of extracellular calcium. During hypotonic challenge, MBDCs exhibited an outwardly rectified current, which was significantly inhibited by administration of classical chloride channel inhibitors such as NPPB and tamoxifen. Chelation of the intracellular calcium with BAPTA-AM or removal of extracellular calcium and calcium channel blocker had no significant effect on VACC currents during hypotonic challenge. In addition to VACC, MBDC had a calcium-activated chloride channel, which was inhibited by NPPB. The present study is the first to systemically study the role of calcium on the VACC and RVD in mouse cholangiocytes and demonstrates that a certain level of intracellular calcium is necessary for RVD but the activation of VACC during RVD does not require calcium. These findings suggest that calcium does not have a direct regulatory role on VACC but has a permissive role on RVD in cholangiocytes.     

Electrophysiological characterization of volume-activated chloride currents in mouse cholangiocyte cell line

Recent electrophysiological and radioisotope efflux studies have demonstrated various Cl(-) channels in cholangiocytes including volume-activated Cl(-) channels (VACC). Because VACCs play prominent roles in many vital cellular functions and physiology in cholangiocytes, we have examined their electrophysiological characteristics in mouse cholangiocytes to provide an important framework for studying in the future. The present study is to characterize VACCs expressed in the mouse bile duct cell (MBDC) line, conditionally immortalized by SV40 virus. Conventional whole cell patch-clamp techniques were used to study the electrophysiological characteristics of VACC in MBDC. When the MBDCs were exposed to hypotonic solution, they exhibited an outwardly rectified current, which was significantly inhibited by replacing chloride in the bath solution with gluconate or glutamate and by administration of classic chloride channel inhibitors 5-nitro-2-(3-phenylpropylamino)-benzoate, glybenclamide, DIDS, and tamoxifen. These inhibitory effects were reversible with washing them out from the bath solution. Moreover, the ion selectivity of the volume-activated channel to different anions indicates that it is more permeable to SCN(-) > I(-) >/= Cl(-) > F(-) >/= acetate >/= glutamate >/= gluconate. These electrophysiological characteristics demonstrate that the volume-activated current observed is a VACC. In addition, the VACC in MBDC has electrophysiological characteristics similar to those of the VACC in human cholangiocarcinoma cell line. The present study is the first to characterize the VACC in mouse cholangiocyte and will provide an important framework for further studies to examine and understand the role of the VACC in biliary secretion and ion-transport physiology.     

Volume-Sensitive Chloride Channels are Involved in Maintenance of Basal Cell Volume in Human Acute Lymphoblastic Leukemia Cells

Chloride channels are expressed ubiquitously in different cells. However, the activation and roles of volume-activated chloride channels under normal isotonic conditions are not clarified, especially in lymphatic cells. In this study, the activation of basal and volume-activated chloride currents and their roles in maintenance of basal cell volume under isotonic conditions were investigated in human acute lymphoblastic leukemia Molt4 cells. The patch-clamp technique and time-lapse image analysis were employed to record whole-cell currents and cell volume changes. Under isotonic conditions, a basal chloride current was recorded. The current was weakly outward-rectified and volume-sensitive and was not inactivated obviously in the observation period. A 47% hypertonic bath solution and the chloride channel blockers NPPB and tamoxifen suppressed the current. Exposure of cells to 47% hypotonic bath solution activated further the basal current. The hypotonicity-activated current possessed properties similar to those of the basal current and was inhibited by NPPB, tamoxifen, ATP and hypertonic bath solution. Furthermore, extracellular hypotonic challenges swelled the cells and induced a regulatory volume decrease (RVD). Extracellular applications of NPPB, tamoxifen and ATP swelled the cells under isotonic conditions and inhibited the RVD induced by hypotonic cell swelling. The results suggest that some volume-activated chloride channels are activated under isotonic conditions, resulting in the appearance of the basal chloride current, which plays an important role in the maintenance of basal cell volume in lymphoblastic leukemia cells. Chloride channels can be activated further to induce a regulatory volume recovery when cells are swollen.     

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.     

Volume-activated taurine permeability in cells of the human erythroleukemic cell line K562

The effects of hypotonic shock on cell volume, taurine influx and efflux were examined in the human erythroleukemic cell line K562. Cells exposed to hypotonic solutions exhibited a regulatory volume decrease (RVD) following rapid increases in cell volume. Cell swelling was associated with a increased taurine influx and efflux. The volume-activated taurine pathway was Na⁺-independent, and increased in parallel with increasing cell volume. The chloride channel blocker, 2,5-dichlorodiphenylamine-2-carboxylic acid (DCDPC), completely blocked the volume-activated taurine influx and efflux, while [dihydroin-denyl]oxy]alkanoic acids (DIOA) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), an anion exchanger and anion channel blocker, respectively, also inhibited significantly. These results suggest that taurine transport is increased in response to hypotonic stress, which may be mediated via a volume-activated, DCDPC-sensitive anion channel. © 1996 Wiley-Liss, Inc.     

Involvement of regulatory volume decrease in the migration of nasopharyngeal carcinoma cells

The transwell chamber migration assay and CCD digital camera imaging techniques were used to investigate the relationship between regulatory volume decrease (RVD) and cell migration in nasopharyngeal carcinoma cells (CNE-2Z cells). Both migrated and non-migrated CNE-2Z cells, when swollen by 47% hypotonic solution, exhibited RVD which was inhibited by extracellular application of chloride channel blockers adenosine 5‘-triphosphate (ATP), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and tamoxifen. However, RVD rate in migrated CNE-2Z cells was bigger than that of non-migrated cells and the sensitivity of migrated cells to NPPB and tamoxifen was higher than that of nonmigrated cells. ATP, NPPB and tamoxifen also inhibited migration of CNE-2Z cells. The inhibition of migration was positively correlated to the blockage of RVD, with a correlation coefficient (r) = 0.99, suggesting a functional relationship between RVD and cell migration. We conclude that RVD is involved in cell migration and RVD may play an important role in migratory process in CNE-2Z cells.     

Hypotonically activated chloride current in HSG cells

Hypotonically induced changes in whole-cell currents and in cell volume were studied in the HSG cloned cell line using the whole-cell, patch clamp and Coulter counter techniques, respectively. Exposures to 10 to 50% hypotonic solutions induced dose-dependent increases in whole-cell conductances when measured using K⁺ and Cl^– containing solutions. An outward current detected at 0 mV, corresponded to a K⁺ current which was transiently activated, (usually preceding activation of an inward current and had several characteristics in common with a Ca²⁺-activated K⁺ current we previously described in these cells. The hypotonically induced inward current had characteristics of a Cl^– current. This current was inhibited by NPPB (5-nitro-2-(3-phenyl-propylamino)-benzoate) and SITS (4-acetamido-4-isothiocyanostilbene), and its reversal potentials corresponded to the Cl^– equilibrium potentials at high and low external Cl^– concentrations. The induced current inactivated at voltages greater than +80 mV, and the I-V curve was outwardly rectifying. The current was unaffected by addition of BAPTA or removal of GTP from the patch pipette, but was inhibited by removal of ATP or by the presence of extracellular arachidonic acid, quinacrine, nordihydroguairetic acid, and cytochalasin D. Moreover, exposure of HSG cells to hypotonic media caused them to swell and then to undergo a regulatory volume decrease (RVD) response. Neither NPPB, SITS or quinine acting alone could inhibit RVD, but NPPB and quinine together totally inhibited RVD. These properties, plus the magnitudes of the induced currents, indicate that the hypotonically induced K⁺ and Cl^– currents may underlie the RVD response. Cytochalasin D also blocked the RVD response, indicating that intact cytoskeletal F-actin may be required for activation of the present currents. Hence, our results indicate that hypotonic stress activates K⁺ and Cl^– conductances in these cells, and that the activation pathway for the K⁺ conductance apparently involves [Ca²⁺], while the activation pathway for the Cl^– conductance does not involve [Ca²⁺] nor lipoxygenase metabolism, but does require intact cytoskeletal F-actin.We thank Mr. Louis Stamps for excellent technical support. Thanks also to Dr. Mitsunobu Sato from the Second Department of Oral and Maxillofacial Surgery, Tokushima University, Japan for sending us the HSG-PA cells, and to Dr. Englert from Hoechst company for providing us with NPPB. This work was supported by National Institute of Dental Research grants R01 DE09812 and R03 DE10535.     

Impaired regulatory volume decrease in freshly isolated cholangiocytes from cystic fibrosis mice: implications for cystic fibrosis transmembrane conductance regulator effect on potassium conductance

Various K(+) and Cl(-) channels are important in cell volume regulation and biliary secretion, but the specific role of cystic fibrosis transmembrane conductance regulator in cholangiocyte cell volume regulation is not known. The goal of this research was to study regulatory volume decrease (RVD) in bile duct cell clusters (BDCCs) from normal and cystic fibrosis (CF) mouse livers. Mouse BDCCs without an enclosed lumen were prepared as described (Cho, W. K. (2002) Am. J. Physiol. 283, G1320-G1327). The isotonic solution consisted of HEPES buffer with 40% of the NaCl replaced with isomolar amounts of sucrose, whereas hypotonic solution was the same as isotonic solution without sucrose. The cell volume changes were indirectly assessed by measuring cross-sectional area (CSA) changes of the BDCCs using quantitative videomicroscopy. Exposure to hypotonic solutions increased relative CSAs of normal BDCCs to 1.20 +/- 0.01 (mean +/- S.E., n = 50) in 10 min, followed by RVD to 1.07 +/- 0.01 by 40 min. Hypotonic challenge in CF mouse BDCCs also increased relative CSA to 1.20 +/- 0.01 (n = 53) in 10 min but without significant recovery. Coadministration of the K(+)-selective ionophore valinomycin restored RVD in CF mouse BDCCs, suggesting that the impaired RVD was likely from a defect in K(+) conductance. Moreover, this valinomycin-induced RVD in CF mice was inhibited by 5-nitro-2'-(3-phenylpropylamino)-benzoate, indicating that it is not from nonspecific effects. Neither cAMP nor calcium agonists could reverse the impaired RVD seen in CF cholangiocytes. Our conclusion is that CF mouse cholangiocytes have defective RVD from an impaired K(+) efflux pathway, which could not be reversed by cAMP nor calcium agonists.     

迁移的鼻咽癌细胞容积激活性氯电流

用膜片钳技术研究了Transwell小室趋化迁移后的鼻咽癌CNE-2Z细胞容积激活性CT电流。47％低渗刺激迁移后的CNE-2Z细胞诱发容积激活性氯电流,与未迁移细胞相比,其特性以及其对氯通道阻断剂的敏感性发生明显的变化,此电流的密度明显高于未迁移细胞,而且该电流几乎完全被氯通道阻断剂adenosine-5'-triphosphate(ATP,10 mmol/L)、5-nitro-2-3-phenylpropylamino benzoic acid(NPPB,100μmol/L)和他莫昔芬(30μmol/L)抑制,其中NPPB和他莫昔芬对迁移细胞的抑制作用明显强于未迁移细胞。迁移后的CNE-2Z细胞容积激活性氯通道对阴离子的通透性为:Br>Cl>I>葡萄糖酸,与未迁移细胞(I>Br>Cl>葡萄糖酸)不同。结果提示,容积激活性氯通道可能参与CNE-2Z细胞的迁移过程。     

Characterization of regulatory volume decrease in freshly isolated mouse cholangiocytes

Cell volume regulation plays a vital role in many cell functions. Recent study indicates that both K(+) and Cl(-) channels are important for the regulatory volume decrease (RVD) of cholangiocarcinoma cells, but its physiological significance is unclear due to the tumorous nature of the cells used. This present study reports the RVD of normal mouse cholangiocytes by using freshly isolated bile duct cell clusters (BDCC). A relatively simple and practical method of measuring the cross-sectional area of BDCCs by quantitative videomicroscopy was used to indirectly measure their volumes. Mouse cholangiocytes exhibited RVD, which was inhibited by 5-nitro-2'-(3-phenylpropylamino)-benzoate, DIDS, and glibenclamide, suggesting its dependence on certain chloride channels, such as volume-activated chloride channels. It is also inhibited by barium chloride but not by tetraethylammonium chloride, indicating its dependence on certain potassium channels. However, cAMP agonists had no significant effect on the RVD of BDCCs. This indirect method described can be used to study the RVD of cholangiocytes from normal as well as genetically altered mouse livers.     

Inhibitory effects of epoxyeicosatrienoic acids on volume-activated chloride channels in rat mesenteric arterial smooth muscle

Epoxyeicosatrienoic acids (EETs) are synthesized from arachidonic acid by cytochrome P450 epoxygenases in endothelial cells. It has previously been shown that EETs activate K(+) channels, which are important for the hyperpolarization and dilation of blood vessels. However, the effects of EETs on other ion channels have been less well studied. We investigated the effects of EETs on volume-activated Cl(-) channels (VACCs) in rat mesenteric arterial smooth muscle cells. Whole-cell patch clamp recording demonstrated that hypotonic solution and guanosine 5'-[gamma-thio]triphosphate (GTPgammaS) induced a 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB)- and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS)-sensitive VACC current in the primary cultured rat mesenteric arterial smooth muscle cells. The VACC current was inhibited by EETs and the order of potency was 8,9-EET>5,6-EET>11,12-EET>14,15-EET. The inhibitory effects of EETs could be reversed by 14,15 epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, an EET analog), Rp-cGMP and KT-5823 (protein kinase G inhibitors). Interestingly, the inhibitory effects of EETs on VACCs were not influenced by Rp-cAMP (a protein kinase A antagonist) but it could be abolished by NF-449 (a Gs protein inhibitor), indicating the involvement of cAMP but not protein kinase A. In conclusion, our results demonstrate that EETs inhibit VACCs in rat mesenteric arterial smooth muscle cells through a cGMP-dependent pathway, which is probably due to the cross-activation by cAMP. This mechanism may be involved in the regulation of cell volume and membrane potential.     

Volume-Activated Chloride Currents in Fetal Human Nasopharyngeal Epithelial Cells

Volume-activated chloride channels have been studied by us extensively in human nasopharyngeal carcinoma cells. However, the chloride channels in the counterpart of the carcinoma cells have not been investigated. In this study, volume-activated chloride currents (I_cl,vol) were characterized in normal fetal human nasopharyngeal epithelial cells using the whole-cell patch-clamp technique. Under isotonic conditions, nasopharyngeal epithelial cells displayed only a weak background current. Exposure to 47% hypotonic solution activated a volume-sensitive current. The reversal potential of the current was close to the calculated equilibrium potential for Cl⁻. The peak values of the hypotonicity-activated current at +80 mV ranged from 0.82 to 2.71 nA in 23 cells. Further analysis indicated that the density of the hypotonicity-activated current in most cells (18/23) was smaller than 60 pA/pF. Only five cells presented a current larger than 60 pA/pF. The hypotonicity-activated current was independent of the exogenous ATP. Chloride channel inhibitors ATP, tamoxifen and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), inhibited the current dramatically. The anion permeability of the hypotonicity-activated chloride channels was I⁻ > Br⁻ > Cl⁻ > gluconate. Unexpectedly, in isotonic conditions, ATP (10 mM) activated an inward-rectified current, which had not been observed in the nasopharyngeal carcinoma cells. These results suggest that, under hypotonic challenges, fetal human nasopharyngeal epithelial cells can produce I_cl,vol, which might be involved in cell volume regulation.     

Extracellular ATP activates a P2 receptor in necturus erythrocytes during hypotonic swelling

We recently reported that ATP is released from Necturus erythrocytes via a conductive pathway during hypotonic swelling and that extracellular ATP potentiates regulatory volume decrease (RVD). This study was designed to determine whether extracellular ATP exerts its effect via a purinoceptor. This was accomplished using three different experimental approaches: 1) hemolysis studies to examine osmotic fragility, 2) a Coulter counter to assess RVD, and 3) the whole-cell patch-clamp technique to measure membrane currents. We found extracellular ATP and ATPγS, two P2 agonists, decreased osmotic fragility, enhanced cell volume recovery in response to hypotonic shock, and increased whole-cell currents. In addition, 2-methylthio-ATP potentiated RVD. In contrast, UTP, α,β-methylene-ATP, and 2′-& 3′-O-(4-benzoyl-benzoyl) adenosine 5′-triphosphate and the P1 agonist adenosine had no effect regardless of experimental approach. Furthermore, the P2 antagonist suramin increased osmotic fragility, inhibited RVD, and reduced whole-cell conductance in swollen cells. Consistent with a previous study that indicated cell swelling activates a K⁺ conductance, suramin had no effect in the presence of gramicidin (a cationophore used to maintain a high K⁺ permeability). We also found the P2 antagonist pyridoxal-5-phosphate-6-azophenyl-2′4-disulfonic acid (PPADS) increased osmotic fragility; however, reactive blue 2 and the P1 antagonists caffeine and theophylline had no effect. Our results show that extracellular ATP activated a P2 receptor in Necturus erythrocytes during hypotonic swelling, which in turn potentiated RVD by stimulating K⁺ efflux. Pharmacological evidence suggested the presence of a P2X receptor subtype. Received: 6 January 2001/Revised: 17 April 2001     

Regulatory volume decrease is actively modulated during the cell cycle

Nasopharyngeal carcinoma cells, CNE-2Z, when swollen by 47% hypotonic solution, exhibited a regulatory volume decrease (RVD). The RVD was inhibited by extracellular applications of the chloride channel blockers tamoxifen (30 microM; 61% inhibition), 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 100 microM; 60% inhibition), and ATP (10 mM; 91% inhibition). The level and time constant of RVD varied greatly between cells. Most cells conducted an incomplete RVD, but a few had the ability to recover their volume completely. There was no obvious correlation between cell volume and RVD capacity. Flow cytometric analysis showed that highly synchronous cells were obtained by the mitotic shake-off technique and that the cells progressed through the cell cycle synchronously when incubated in culture medium. Combined application of DNA synthesis inhibitors, thymidine and hydroxyurea arrested cells at the G1/S boundary and 87% of the cells reached S phase 4 h after being released. RVD capacity changed significantly during the cell cycle progression in cells synchronized by shake-off technique. RVD capacity being at its highest in G1 phase and lowest in S phase. The RVD capacity in G1 (shake-off cells sampled after 4 h of incubation), S (obtained by chemical arrest), and M cells (selected under microscope) was 73, 33, and 58%, respectively, and the time constants were 435, 769, and 2,000 sec, respectively. We conclude that RVD capacity is actively modulated in the cell cycle and RVD may play an important role in cell cycle progress.     

Inhibitory effects of epoxyeicosatrienoic acids on volume-activated chloride channels in rat mesenteric arterial smooth muscle

Epoxyeicosatrienoic acids (EETs) are synthesized from arachidonic acid by cytochrome P450 epoxygenases in endothelial cells. It has previously been shown that EETs activate K⁺ channels, which are important for the hyperpolarization and dilation of blood vessels. However, the effects of EETs on other ion channels have been less well studied. We investigated the effects of EETs on volume-activated Cl⁻ channels (VACCs) in rat mesenteric arterial smooth muscle cells. Whole-cell patch clamp recording demonstrated that hypotonic solution and guanosine 5′-[γ-thio]triphosphate (GTPγS) induced a 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB)- and 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS)-sensitive VACC current in the primary cultured rat mesenteric arterial smooth muscle cells. The VACC current was inhibited by EETs and the order of potency was 8,9-EET > 5,6-EET > 11,12-EET > 14,15-EET. The inhibitory effects of EETs could be reversed by 14,15 epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, an EET analog), Rp-cGMP and KT-5823 (protein kinase G inhibitors). Interestingly, the inhibitory effects of EETs on VACCs were not influenced by Rp-cAMP (a protein kinase A antagonist) but it could be abolished by NF-449 (a Gs protein inhibitor), indicating the involvement of cAMP but not protein kinase A. In conclusion, our results demonstrate that EETs inhibit VACCs in rat mesenteric arterial smooth muscle cells through a cGMP-dependent pathway, which is probably due to the cross-activation by cAMP. This mechanism may be involved in the regulation of cell volume and membrane potential.     

Fundamental role of ClC-3 in volume-sensitive Cl- channel function and cell volume regulation in AGS cells

Volume regulation is essential for cell function, but it is unknown which channels are involved in a regulatory volume decrease (RVD) in human gastric epithelial cells. Exposure to a hypotonic solution caused the increase in AGS cell volume, followed by the activation of a current. The reversal potential of the swelling-induced current suggested that Cl- was the primary charge carrier. The selectivity sequence for different anions was I- > Br- > Cl- > F- > gluconate. This current was inhibited by flufenamate, DIDS, tamoxifen, and 5-nitro-2-(3-phenylpropylamino)benzoate. Intracellular dialysis of three different anti-ClC-3 antibodies abolished or attenuated the Cl- current and disrupted RVD, whereas the current and RVD was unaltered by anti-ClC-2 antibody. Immunoblot studies demonstrated the presence of ClC-3 protein in Hela and AGS cells. RT-PCR analysis detected expression of ClC-3, MDR-1, and pICln mRNA in AGS cells. These results suggest a fundamental role of endogenous ClC-3 in the swelling-activated Cl- channels function and cell volume regulation in human gastric epithelial cells.     

Swelling-Induced Ca<Superscript>2+</Superscript> Influx and K<Superscript>+</Superscript> Efflux in American Alligator Erythrocytes

The American alligator can hibernate during winter, which may lead to osmotic imbalance because of reduced kidney function and lack of food consumption during this period. Accordingly, we hypothesized that their red blood cells would have a well-developed regulatory volume decrease (RVD) to cope with the homeostatic challenges associated with torpor. Osmotic fragility was determined optically, mean cell volume was measured by electronic sizing, and changes in intracellular Ca²⁺ concentration were visualized using fluorescence microscopy and fluo-4-AM. Osmotic fragility increased and the ability to regulate volume was inhibited when extracellular Na⁺ was replaced with K⁺, or when cells were exposed to the K⁺ channel inhibitor quinine, indicating a requirement of K⁺ efflux for RVD. Addition of the ionophore gramicidin to the extracellular medium decreased osmotic fragility and also potentiated volume recovery, even in the presence of quinine. In addition, hypotonic shock (0.5× Ringer) caused an increase in cytosolic Ca²⁺, which resulted from Ca²⁺ influx because it was not observed when extracellular Ca²⁺ was chelated with EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid). Furthermore, cells loaded with BAPTA-AM (1,2-bis(2-aminophenoxymethyl)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl) ester) or exposed to a low Ca²⁺-EGTA hypotonic Ringer had a greater osmotic fragility and also failed to recover from cell swelling, indicating that extracellular Ca²⁺ was needed for RVD. Gramicidin reversed the inhibitory effect of low extracellular Ca²⁺. Finally, and surprisingly, the Ca²⁺ ionophore A23187 increased osmotic fragility and inhibited volume recovery. Taken together, our results show that cell swelling activated a K⁺ permeable pathway via a Ca²⁺-dependent mechanism, and this process mediated K⁺ loss during RVD.     

睫状体色素上皮细胞容积激活性氯电流

为研究睫状体色素上皮 (pigmentedciliaryepithelial,PCE)细胞容积激活性Cl-电流的特性 ,用膜片箝全细胞记录技术记录了猪的低渗液诱发的容积激活性Cl-电流。此电流外向占优势 ,几乎没有时间依赖性失活 ,电流 电压曲线显示此电流反转电位 (- 6 3± 0 5mV)很接近氯离子平衡电位的计算值 (ECl=0mV)。电流的激活依赖于细胞内ATP ,细胞外ATP抑制外向电流和内向电流 ,但外向电流抑制率大于内向电流抑制率 (92 %比 74% ,P <0 0 1)。氯离子通道阻断剂tamoxifen抑制外向电流和内向电流 ,两个抑制率几乎相等 (85 %比 87% ,P >0 0 5 )。此电流特性与其他类型细胞的P糖蛋白相关电流很相似。结果提示PCE细胞容积激活性Cl-电流的形成可能与P糖蛋白有关     

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%. ³H-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 μM) 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.