P-glycoprotein and cell volume-activated chloride channels

The multidrug resistance P-glycoprotein (P-gp) is an active drug transporter which can expel hydrophobic compounds from cells. Expression of P-gp has many effects on cells and tissues and the physiological function, or functions, of P-gp are still unclear. Recently, expression of P-gp has been associated with altered activity of chloride channels which play a role in regulating cell volume of response to osmotic shock or nutrient uptake. The nature and physiological role of this association has been a subject of some debate. In this article, mechanisms by which P-gp might influence cell volume-activated chloride currents is discussed, and the potential physiological role of this regulation considered.     

Cell cycle-dependent expression of volume-activated chloride currents in nasopharyngeal carcinoma cells

Patch-clamping and cell imageanalysis techniques were used to study the expression of thevolume-activated Cl current,I_Cl(vol), and regulatory volume decrease (RVD)capacity in the cell cycle in nasopharyngeal carcinoma cells (CNE-2Z). Hypotonic challenge caused CNE-2Z cells to swell and activated aCl current with a linear conductance, negligibletime-dependent inactivation, and a reversal potential close to theCl equilibrium potential. The sequence of anionpermeability was I > Br > Cl > gluconate. The Cl channelblockers tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB),and ATP inhibited I_Cl(vol). Synchronous cultures of cells were obtained by the mitotic shake-off technique and by adouble chemical-block (thymidine and hydroxyurea) technique. Theexpression of I_Cl(vol) was cell cycle dependent,being high in G₁ phase, downregulated in S phase, butincreasing again in M phase. Hypotonic solution activated RVD, whichwas cell cycle dependent and inhibited by the Cl channelblockers NPPB, tamoxifen, and ATP. The expression of I_Cl(vol) was closely correlated with the RVDcapacity in the cell cycle, suggesting a functional relationship.Inhibition of I_Cl(vol) by NPPB (100 µM)arrested cells in G₀/G₁. The data also suggest that expression of I_Cl(vol) and RVD capacity areactively modulated during the cell cycle. The volume-activatedCl current associated with RVD may therefore play animportant role during the cell cycle progress.

     

MDR1基因在睫状体色素上皮细胞容积激活性氯电流中的作用

用膜片钳,反义寡核苷酸,免疫荧光及激光共聚焦显微镜等技术,研究MDR1基因在牛睫状体色素上皮（pigmented ciliary epithelial,PCE)细胞容积激活性氯电流中的作用,PCE细胞表达MDR1基因产物-P糖蛋白（P-gp),反义MDR1寡核苷酸抑制MDR1基因的表达（P-gp免疫荧光减少93%）,延缓容积激活性氯电流的出现（潜伏期延长109%）,并导致激活率降低62%及电流峰值减小56%,而核酸转染剂阳离子脂质体和非配对性的寡核苷酸对电流没有显著性影响,上述观察结果表明,睫状体色素上皮细胞容积激活性氯电流与内源性MDR1表达有关。     

Electrophysiological and molecular identification of hepatocellular volume-activated K channels

Although K⁺ channels are essential for hepatocellular function, it is not known which channels are involved in the regulatory volume decrease (RVD) in these cells. We have used a combination of electrophysiological and molecular approaches to describe the potential candidates for these channels. The dialysis of short-term cultured rat hepatocytes with a hypotonic solution containing high K⁺ and low Cl⁻ concentration caused the slow activation of an outward, time-independent current under whole-cell configuration of the patch electrode voltage clamp. The reversal potential of this current suggested that K⁺ was the primary charge carrier. The swelling-induced K⁺ current (I_Kvol) occurred in the absence of Ca²⁺ and was inhibited with 1 μM Ca²⁺ in the pipette solution. The activation of I_Kvol required both Mg²⁺ and ATP and an increasing concentration of Mg-ATP from 0.25 through 0.5 to 0.9 mM activated I_Kvol increasingly faster and to a larger extent. The KCNQ1 inhibitor chromanol 293B reversibly depressed I_Kvol with an IC₅₀ of 26 μM. RT-PCR detected the expression of members of the KCNQ family from KCNQ1 to KCNQ5 and of the accessory proteins KCNE1 to KCNE3 in the rat hepatocytes, but not KCNQ2 and KCNE2 in human liver. Western blotting showed KCNE3 expression in a plasma membrane-enriched fraction from rat hepatocytes. The results suggest that KCNQ1, probably with KCNE2 or KCNE3 as its accessory unit, provides a significant fraction of I_Kvol in rat hepatocytes.     

Electrophysiological and molecular identification of hepatocellular volume-activated K+ channels

Although K+ channels are essential for hepatocellular function, it is not known which channels are involved in the regulatory volume decrease (RVD) in these cells. We have used a combination of electrophysiological and molecular approaches to describe the potential candidates for these channels. The dialysis of short-term cultured rat hepatocytes with a hypotonic solution containing high K+ and low Cl- concentration caused the slow activation of an outward, time-independent current under whole-cell configuration of the patch electrode voltage clamp. The reversal potential of this current suggested that K+ was the primary charge carrier. The swelling-induced K+ current (IKvol) occurred in the absence of Ca2+ and was inhibited with 1 microM Ca2+ in the pipette solution. The activation of IKvol required both Mg2+ and ATP and an increasing concentration of Mg-ATP from 0.25 through 0.5 to 0.9 mM activated IKvol increasingly faster and to a larger extent. The KCNQ1 inhibitor chromanol 293B reversibly depressed IKvol with an IC50 of 26 microM. RT-PCR detected the expression of members of the KCNQ family from KCNQ1 to KCNQ5 and of the accessory proteins KCNE1 to KCNE3 in the rat hepatocytes, but not KCNQ2 and KCNE2 in human liver. Western blotting showed KCNE3 expression in a plasma membrane-enriched fraction from rat hepatocytes. The results suggest that KCNQ1, probably with KCNE2 or KCNE3 as its accessory unit, provides a significant fraction of IKvol in rat hepatocytes.     

Ultrastructural and biochemical characterization of a cloned mouse keratinocyte cell line

A cloned population of mouse C3H/He keratinocytes was obtained from the 14th passage of an epidermal cell line. A two-step cloning procedure using Petriperm dishes was performed. The cloned population, grown at 34 °C, was subcultured more than 30 times over a one year period. By day 14, three cell layers were formed; the ultrastructural morphology and immunofluorescence characterization of these layers showed numerous tonofilament bundles and well organized desmosome tonofilament structures. They thereby resemble the proliferative compartment of the epidermis. High resolution acrylamide gel electrophoresis of the keratins extracted from the cloned cells showed the presence of many keratin subunits. The tonofilaments extracted from the cell layers, as well as from the supernatant cells, contained a small quantity of high MW keratins (rel. MW 63 000; apparent isoelectric point 5.5–6.2). These results indicate that the cloned keratinocyte cell line had retained a certain maturation capacity in culture.     

Establishment and characterization of a spontaneously immortalized mouse ameloblast-lineage cell line

Tooth development was cooperatively regulated by the epithelial ameloblasts and mesenchymal odontoblasts. Ameloblasts secrete enamel matrix, critical for enamel formation. While there are several reports about establishment of immortalized ameloblast-like cells by introducing viral oncogene, we tried to establish a spontaneously immortalized ameloblast-lineage cell line, maintaining the cell type specific character, including the ability to induce in vitro bio-mineralization. The established cell line (ameloblast-lineage cell; ALC) maintained the expression of several ameloblast specific genes (Amelogenin, Tuftelin, and Enamelin) in long-term culture. They formed calcified nodules after the induction by medium switching from SMEM to DMEM, having high-level alkaline-phosphatase activity. The size and number of calcified nodule formation were enhanced by TGF-beta treatment. Six weeks after sub-cutaneous implantation of ALC to athymic nude mice, we ectopically observed enamel epithelium like structure formation, chondrogenesis, and calcification. These data indicate that ALC is a useful experimental tool to analyze ameloblast character.     

Isolation and characterization of cholangiocyte primary cilia

Primary cilia are distinct organelles expressed by many vertebrate cells, including cholangiocytes; however, their functions remain obscure. To begin to explore the physiological role of these organelles in the liver, we described the morphology and structure of cholangiocyte cilia and developed new approaches for their isolation. Primary cilia were present only in bile ducts and were not observed in hepatocytes or in hepatic arterial or portal venous endothelial cells. Each cholangiocyte possesses a single cilium that extends from the apical membrane into the bile duct lumen. In addition, the length of the cilia was proportional to the bile duct diameter. We reproducibly isolated enriched fractions of cilia from normal rat and mouse cholangiocytes by two different approaches as assessed by scanning electron, transmission electron, and confocal microscopy. The purity of isolated ciliary fractions was further analyzed by Western blot analysis using acetylated tubulin as a ciliary marker and P2Y(2) as a nonciliary cell membrane marker. These novel techniques produced enriched ciliary fractions of sufficient purity and quantity for light and electron microscopy and for biochemical analyses. They will permit further assessment of the role of primary cilia in normal and pathological conditions.     

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.     

Protein kinase C-mediated phosphorylation of the human multidrug resistance P-glycoprotein regulates cell volume-activated chloride channels.

The multidrug resistance P-glycoprotein (P-gp), which transports hydrophobic drugs out of cells, is also associated with volume-activated chloride currents. It is not yet clear whether P-gp is a channel itself, or whether it is a channel regulator. Activation of chloride currents by hypotonicity in cells expressing P-gp was shown to be regulated by protein kinase C (PKC). HeLa cells exhibited volume-activated chloride currents indistinguishable from those obtained in P-gp-expressing cells except that they were insensitive to PKC. HeLa cells did not express detectable P-gp but, following transient transfection with cDNA encoding P-gp, the volume-activated channels acquired PKC regulation. PKC regulation was abolished when serine/threonine residues in the consensus phosphorylation sites of the linker region of P-gp were replaced with alanine. Replacement of these residues with glutamate, in order to mimic the charge of the phosphorylated protein, also mimicked the effects of PKC on channel activation. These data demonstrate that PKC-mediated phosphorylation of P-gp regulates the activity of an endogenous chloride channel and thus indicate that P-gp is a channel regulator.     

Isolation and molecular characterization of a mouse renal microvascular endothelial cell line

Murine endothelial cells (ECs) have proven difficult to obtain and maintain in culture. Long-term maintenance of normal ECs remains a difficult task. In this article we report the establishment of the first cellular line of renal microvascular endothelium obtained from normal tissue. Cells were isolated, cloned, and maintained by serial passages for longer than 24 mo, using endothelial cell growth supplement (ECGS) and gelatin-coated plates. Their morphology and ultrastructure, expression of von Willebrand factor, presence of smooth muscle alpha-actin, vimentin, cytokeratin filaments, capillary structures formed on Matrigel, and some typical ECs surface molecules were the criteria used to characterize cultured ECs. When examined for responsiveness to Shiga toxin-1, 13-20% of cytotoxicity was observed when coincubated with lipopolysaccharides. This cytotoxicity was not observed for normal lung ECs (1G11). Consequently, REC-A4 line retains characteristics of resting microvascular ECs and represents a useful in vitro model to study biological and physiopathological properties of renal endothelium.     

Isolation and genetic characterization of dibucaine-resistant variants of a mouse lymphocytic cell line

A series of dibucaine-resistant (Dib^R) variants of the mouse lymphoid cell line L5178Y have been induced by mutagen (EMS) and isolated by selection for resistance to a short (48 h), high concentration (0.045 mM) drug pulse. Dib^R isolates grow exponentially in the presence of 0.025-– 0.030 mM dibucaine, drug concentrations that are toxic to the parent cell line. Like L5178Y, these variants are pseudodiploid. The dibucaine-resistant phenotype has remained stable in four independently derived populations, subcultured for 7 or 11 months in growth medium without drug. Also, the frequency of Dib^R variants increases as the concentration of inducing mutagen is increased. These latter two findings suggest, but do not prove, that the dibucaine-resistant phenotype occurs because of gene mutation. All Dib^R isolates were found to be cross-resistant to the growth-inhibiting effects of tetracaine, but sensitive to procaine and benzocaine. Chromosome number or cell size is an important consideration in evaluating the cytotoxicity of dibucaine because normal pseudotetraploid cells are more tolerant to the toxic effects of this drug than are wild-type pseudodiploid cell populations. Hybridization studies indicate that the dibucaine-resistant phenotype of one variant may be dominant, and that of another recessive. Dib^R variants will be important for future studies of the mechanism of local anesthetic action.