Cyto B dependent and ouabain insensitive Regulatory Volume Decrease in bicellular mouse embryo

Mouse single-cell embryos exhibit robust Regulatory Volume Decrease (RVD). In what manner the very early mammalian embryo following zygote stage is appreciably altered by the anisotonic extracellular solution is, as yet, totally unclear. Little attention was paid to this direction since there was no way to determine the blastomere volume. This work has served to quantitatively investigate the osmotic response of bicellular mouse embryos employing Laser Scanning Microtomography (LSM) followed with three-dimensional reconstruction (3 DR). We have shown that bicellular mouse embryos in hypotonic Dulbecco’s experience RVD. Embryonic cells subjected to hyposmolar exhibit rapid osmotic swelling followed by gradual shrinking back toward their original volume. The van’ t Hoff law defines swelling phase with the effective hydraulic conductivity of 0.3 micron · min⁻¹ · atm⁻¹. Water release during RVD in bicellular mouse embryos is abolished by Cytochalasin B (Cyto B) and the volume recovery is insensitive to ouabain treatment.     

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.     

Different physiological mechanisms control isovolumetric regulation and regulatory volume decrease in chick embryo cardiomyocytes

Cultured chick embryo cardiac myocytes submitted to a 180 mOsm/kg hyposmotic solution swell present a regulatory volume decrease (RVD). This RVD is mediated by a Ca(2+)influx followed by a 40% loss of total taurine content accompanied by the loss of lesser amounts of other osmolytes. Kidney cells respond to a gradual change in osmolality by maintaining their volume at the initial level. This is termed isovolumetric regulation (IVR), which may activate regulatory processes other than those observed with sudden changes in osmolality. When cardiac myocytes were exposed to a gradual change in osmolality, they show a partial IVR which is not dependent upon extracellular Ca(2+). Potassium channel blockers, quinidine and Ba(2+), and the chloride channel blocker, diphenylamine-2-carboxylate (DPC), compromise IVR in our model. Tritiated taurine loss and total intracellular K(+)contents were analyzed in cultured cardiomyocytes submitted to a gradual change in osmolality. The cultured cells lost approximately 10% of their taurine and 35% of their total K(+). These findings suggest that different compensatory mechanisms are activated when cells are exposed to stepwise and gradual changes in osmolality. Inorganic osmolytes (through conductive pathways) are preferentially mobilized during the physiological and/or patho-physiological IVR situation, perhaps reflecting energetic conservation in response to a less traumatic event for the cardiac myocytes.     

Substrate stiffness-dependent regulatory volume decrease and calcium signaling in chondrocytes

The pericellular matrix stiffness is strongly associated with its biochemical and structural changes during the aging and osteoarthritis progress of articular c...     

Osmotic water permeability and regulatory volume decrease of rat thymocytes

Rat thymocytes displayed robust regulatory volume decrease (RVD) when suspended in NaCl-based hypotonic Ringer solutions. The RVD of thymocytes was completely abolished upon replacement of external Na+ ions with K+, indicating a role of coupled efflux of K+ and Cl- ions as a driving force of regulatory volume decrease. Osmotic water permeability (Pf) measured in KCl-based hypotonic solutions was (1.3 +/- 1.0 x 10(-4) cm/s at 25 degrees C and was temperature-dependent with low activation energy (Ea = 4.65 +/- 0.77 kcal/mol) characteristic to water transport through pores. HgCl2 and a sulfhydryl-blocking reagent, methyl methanethiosulphonate (MMTS), modulated the water permeability of thymocytes in a biphasic manner: inhibited at low dose (0.1-1 micromol/l) and restored or even enhanced at higher (10-100 micromol/l) concentrations. RVD paralleled the Pf: it was greatly suppressed at low dose of MMTS (sufficient to attenuate the water transport), but recovered at higher dose, when the water movement was restored. Therefore we suggest that thymocytes require the effective water transport for functional regulatory volume decrease.     

低渗条件下小肠上皮细胞调节性细胞容积减小的离子通道机制

目的:观察人小肠上皮细胞调节性细胞容积减小(RVD)的过程,探讨参与RVD过程的离子通道机制.方法:将培养的人小肠上皮细胞暴露于低渗溶液, 利用电子细胞体积测量系统测定细胞平均容积变化过程和离子通道的参与过程;采用RT-PCR方法检测人小肠上皮细胞上离子通道的表达.结果:人小肠上皮细胞具有良好的RVD功能; 其RVD过程可被氯通道阻断剂NPPB 和钾通道阻断剂四乙铵所阻断; 进一步的研究发现, 中等电导钙激活性钾通道(IK)的特异性阻断剂Clotrimazole (CLT) (1μmol/L)可以明显抑制细胞的RVD过程,而大电导钙激活性钾通道(BK)和小电导钙激活性钾通道(SK)的特异阻断剂iberiotoxin (100 nmol/L)和apamin (100 nmol/L)对RVD过程无任何抑制作用.RT-PCR的结果也显示, 人小肠上皮细胞只有IK表达, 而无SK和BK的表达.结论:人小肠上皮细胞具有RVD功能,RVD过程的完成有赖于氯通道和钾通道的平行激活, 而其中参与容积调节的钾通道是中等电导钙激活型钾通道IK.     

Separate taurine and chloride efflux pathways activated during regulatory volume decrease

Organic osmolyte and halide permeability pathways activated inepithelial HeLa cells by cell swelling were studied by radiotracer efflux techniques and single-cell volume measurements. The replacement of extracellular Cl byanions that are more permeant through the volume-activated Cl channel, as indicated byelectrophysiological measurements, significantly decreasedtaurine efflux. In the presence of less-permeant anions, an increase intaurine efflux was observed. Simultaneous measurement of the¹²⁵I, used as a tracer forCl, and[³H]taurine effluxshowed that the time courses for the two effluxes differed. InCl-rich medium the increasein I efflux was transient,whereas that for taurine was sustained. OsmosensitiveCl conductance, assessed bymeasuring changes in cell volume, increased rapidly after hypotonicshock. The influx of taurine was able to counteractCl conductance-dependentcell shrinkage but only ~4 min after triggering cell swelling. Thistaurine-induced effect was blocked by DIDS. Differences in anionsensitivity, the time course of activation, and sensitivity to DIDSsuggest that the main cell swelling-activated permeability pathways fortaurine and Cl are separate.

     

The multidrug resistance P-glycoprotein modulates cell regulatory volume decrease.

Cell volume is frequently down-regulated by the activation of anion channels. The role of cell swelling-activated chloride channels in cell volume regulation has been studied using the patch-clamp technique and a non-invasive microspectrofluorimetric assay for changes in cell volume. The rate of activation of these chloride channels was shown to limit the rate of regulatory volume decrease (RVD) in response to hyposmotic solutions. Expression of the human MDR1 or mouse mdr1a genes, but not the mouse mdr1b gene, encoding the multidrug resistance P-glycoprotein (P-gp), increased the rate of channel activation and the rate of RVD. In addition, P-gp decreased the magnitude of hyposmotic shock required to activate the channels and to elicit RVD. Tamoxifen selectively inhibited both chloride channel activity and RVD. No effect on potassium channel activity was elicited by expression of P-gp. The data show that, in these cell types, swelling-activated chloride channels have a central role in RVD. Moreover, they clarify the role of P-gp in channel activation and provide direct evidence that P-gp, through its effect on chloride channel activation, enhances the ability of cells to down-regulate their volume.     

Activation of paternally derived regulatory mechanism in early mouse embryo

A stage-specific occurrence of retrovirus-like elements (intracisternal particles) is regularly observed within the endoplasmic reticulum of early mouse embryos. A quantitative electron microscopic study was carried out on hybrid embryos issued from the matings between high- and low-producer mouse strains of intracisternal particles. The low-producer character behaved as dominant in all these crosses, and this enabled us to demonstrate the activation of paternally introduced regulatory mechanism in two-cell embryos and morulae. There was no sign of maternal effect in the results of reciprocal crosses.     

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.     

Inactivation of regulatory volume decrease in human peripheral blood lymphocytes by N-ethylmaleimide

The sulfhydryl group reagent N-ethylmaleimide was found to inhibit in a dose dependent manner regulatory volume decrease of human peripheral lymphocytes swollen in buffered hyposmotic NaCl media. In hyposmotic KCl media NEM treated lymphocytes prevented an additional secondary swelling seen in control lymphocytes. The data suggest that N-ethylmaleimide acts on ion transport mechanisms involved in volume regulatory changes. This effect contrasts with the stimulation by N-ethylmaleimide of apparently volume sensitive K/Cl fluxes in certain mammalian red cells.     

Leishmania donovani amastigotes mobilize organic and inorganic osmolytes during regulatory volume decrease

The protozoan parasite Leishmania donovani encounters large fluctuations in osmolality as it cycles between its insect vector and human host. The flagellated promastigote exhibits regulatory volume responses involving organic and inorganic osmolytes, but little is known about volume regulation in the clinically relevant amastigote that multiplies within the parasitophorous vacuoles of mammalian host cells. Using a combination of morphological, X-ray microanalytical, and biochemical approaches we determined that non-motile amastigotes respond to hypotonic stress with (1) an amino acid and l-alanine-mediated regulatory volume decrease, and (2) a parallel release of Na+, K+, P (presumably as negatively charged phosphates), and subsequently Cl- from cytoplasm and the cell as a whole. In addition P, Zn2+, and subsequently Ca2+ increase in acidocalcisomes as Cl- content declines in this compartment. This evidence is the first to document subcellular translocation of, and thus a potential role for, zinc in volume regulatory responses. These coordinated changes in organic and inorganic osmolytes demonstrate that amastigote subcellular compartments, particularly acidocalcisomes, function in maintaining ionic homeostasis in the response of Leishmania amastigotes to hypo-osmotic stress.     

IK channels are involved in the regulatory volume decrease in human epithelial cells

Parallel activation ofCa²⁺-dependent K⁺ channels and volume-sensitiveCl channels is known to be responsible for KCl effluxduring regulatory volume decrease (RVD) in human epithelial Intestine407 cells. The present study was performed to identify theK⁺ channel type. RT-PCR demonstrated mRNA expression ofCa²⁺-activated, intermediate conductance K⁺(IK), but not small conductance K⁺ (SK1) or largeconductance K⁺ (BK) channels in this cell line. Whole cellrecordings showed that ionomycin or hypotonic stress activated inwardlyrectifying K⁺ currents that were reversibly blocked by IKchannel blockers [clotrimazole (CLT) and charybdotoxin] but not by SKand BK channel blockers (apamin and iberiotoxin). Inside-out recordingsrevealed the existence of CLT-sensitive single K⁺-channelactivity, which exhibited an intermediate unitary conductance (30 pS at100 mV). The channel was activated by cytosolic Ca²⁺ ininside-out patches and by a hypotonic challenge in cell-attached patches. The RVD was suppressed by CLT, but not by apamin oriberiotoxin. Thus we conclude that the IK channel is involved in theRVD process in these human epithelial cells.