Inhibition Of Regulatory Volume Decrease Research Articles

Novel quartz crystal microbalance cytosensor for real-time monitoring of polystyrene nanospheres effect on cell apoptosis

• A convenient quartz crystal microbalance (QCM) cytosensor was first developed for dynamical evaluating of polystyrene (PS) nanospheres effect on cell apoptosis. • The proposed QCM cytosensor was capable of monitoring cell volume regulatory of MDA-MB-231 cell for the evaluation of the cytotoxicity of PS nanospheres. • The study established a convenient model to evaluate the cytotoxicity of different-sized PS nanospheres. A convenient quartz crystal microbalance (QCM) cytosensor was fabricated to monitor cell volume regulatory of MDA-MB-231 cells in real time for evaluating cytotoxicity of polystyrene (PS) nanospheres of different sizes. Herein poly-l-lysine and mercaptosuccinic acid were modified onto gold electrode to capture MDA-MB-231 cells. Cell volume and cell mass are tightly linked and thus the processes of regulatory volume decrease (RVD) could be measured by monitoring the changes of cell mass dynamically. Preliminary result showed that RVD of MDA-MB-231 cells was inhibited by PS nanospheres of different sizes. And the inhibition of PS nanospheres on RVD in MDA-MB-231 cells was size-dependent. In addition, flow cytometry assay showed that apoptosis of MDA-MB-231 cells was induced by PS nanospheres in a size-dependent manner. Analysis result showed that inhibition of RVD was proportionate to apoptosis rate, correlation coefficient R = 0.9622, and it kept the good linear relationship in a large range. Therefore, the fabricated QCM cytosensor was able to monitor RVD for evaluation of the cytotoxicity of PS nanospheres of different sizes. The study established a convenient QCM cytosensor to evaluate the cytotoxicity of different-sized PS nanospheres, which was expected to provide a tool for evaluation of the cytotoxicity mechanism of PS nanospheres.

Evidence that reduction in volume protects in situ articular chondrocytes from mechanical impact

ABSTRACTChondrocytes, the resident cells in articular cartilage, carry the burden of producing and maintaining the extracellular matrix (ECM). However, as these cells have a low proliferative capacity and are not readily replaced, chondrocyte death due to extreme forces may contribute to the pathogenesis of osteoarthritis (OA) after injury or may inhibit healing after osteochondral transplantation, a restorative procedure for damaged cartilage that requires a series of mechanical impacts to insert the graft. Consequently, there is a need to understand what factors influence the vulnerability of in situ chondrocytes to mechanical trauma. To this end, the objective of this study was to investigate how altering cell volume by different means (hydrostatic pressure, uniaxial load, and osmotic challenge with and without inhibition of regulatory volume decrease) affects the vulnerability of in situ chondrocytes to extreme mechanical forces. Using a custom experimental platform enabling testing of viable and intact murine cartilage-on-bone explants, we established a strong correlation between chondrocyte volume and vulnerability to impact injury wherein reduced volume was protective. Moreover, we found that the volume-perturbing interventions did not affect cartilage ECM mechanical properties, suggesting that their effects on chondrocyte vulnerability occurred at the cellular level. The findings of this study offer new avenues for novel strategies aimed at preventing chondrocyte loss during osteochondral grafting or to halting the progression of cell death after a joint destabilizing injury.

Inhibition of Regulatory Volume Decrease Enhances the Cytocidal Effect of Hypotonic Shock in Hepatocellular Carcinoma.

Background: Hypotonic shock induces cytocidal effects through cell rupture, and cancer therapy based on this mechanism has been clinically administered to hepatocellular carcinoma patients. We herein investigated the effectiveness of hypotonic shock combined with the inhibition of regulatory volume decrease as cancer therapy for hepatocellular carcinoma.Methods: Morphological changes in human hepatocellular carcinoma cell lines were observed under a differential interference contrast microscope connected to a high-speed digital video camera. Cell volume changes under hypotonic shock with or without chloride, potassium, or water channel blockers were observed using a high-resolution flow cytometer. In order to investigate cytocidal effects, the number of surviving cells was compared after exposure to hypotonic solution with and without each channel blocker (re-incubation experiment).Results: Video recordings showed that cells exposed to distilled water rapidly swelled and then ruptured. Cell volume measurements revealed regulatory volume decrease under mild hypotonic shock, whereas severe hypotonic shock increased the number of broken fragments as a result of cell rupture. Moreover, regulatory volume decrease was inhibited in cells treated with each channel blocker. Re-incubation experiments showed the cytocidal effects of hypotonic shock in cells exposed to hypotonic solution, and additional treatments with each channel blocker enhanced these effects.Conclusion: The inhibition of regulatory volume decrease with chloride, potassium, or water channel blockers may enhance the cytocidal effects of hypotonic shock in hepatocellular carcinoma. Hypotonic shock combined with the inhibition of regulatory volume decrease was a more effective therapy than hypotonic shock alone.

Pleural lavage with distilled water during surgery for esophageal squamous cell carcinoma

This study aimed to investigate cytocidal effects of hypotonic shock on esophageal squamous cell carcinoma (ESCC) cell lines, and to apply pleural lavage with distilled water to surgery for ESCC. Three human ESCC cell lines, TE5, TE9 and KYSE170 were exposed to distilled water, and morphological changes in ESCC cells were closely observed under a differential interference contrast microscope connected to a high-speed digital video camera. Further, serial cell volume changes after hypotonic shock were measured using a high-resolution flow cytometer. To investigate the cytocidal effects of hypotonic shock on ESCC cells, re-incubation of ESCC cells was performed after hypotonic shock. Additionally, the effects of 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), a Cl- channel blocker, during hypotonic shock were analyzed. Video recordings by high-speed digital camera demonstrated that hypotonic shock with distilled water induced cell swelling followed by cell rupture. Measurements of cell volume changes using a high-resolution flow cytometer indicated that severe hypotonicity with distilled water increased broken fragments of ESCC cells within 5 min. Re-incubation experiments demonstrated cytocidal effects of hypotonic shock on ESCC cells. Treatment of cells with NPPB increased cell volumes by the inhibition of regulatory volume decrease, which is observed during hypotonic shock, and enhanced cytocidal effects. These findings demonstrated the cytocidal effects of hypotonic shock on ESCC cells, and clearly support the efficacy of pleural lavage with distilled water during surgery for ESCC.

Copper inhibits P2Y 2-dependent Ca 2+ signaling through the effects on thapsigargin-sensitive Ca 2+ stores in HTC hepatoma cells

Purinergic P2Y 2 G-protein coupled receptors play a key role in the regulation of hepatic Ca 2+ signaling by extracellular ATP. The concentration of copper in serum is about 20 μM. Since copper accumulates in the liver in certain disease states, the purpose of these studies was to assess the effects of copper on P2Y 2 receptors in a model liver cell line. Exposure to a P2Y 2 agonist UTP increased [Ca 2+] i by stimulating Ca 2+ release from thapsigargin-sensitive Ca 2+ stores. Pretreatment of HTC cells for several minutes with copper did not affect cell viability, but potently inhibited increases in [Ca 2+] i evoked by UTP and thapsigargin. During this pretreatment, copper was not transported into the cytosol, and inhibited P2Y 2 receptors in a concentration-dependent manner with the IC 50 of about 15 μM. These results suggest that copper inhibits P2Y 2 receptors through the effects on thapsigargin-sensitive Ca 2+ stores by acting from an extracellular side. Further experiments indicated that these effect of copper may lead to inhibition of regulatory volume decrease (RVD) evoked by hypotonic solution. Thus, copper may contribute to defective regulation of purinergic signaling and liver cell volume in diseases associated with the increased serum copper concentration.

Expression and functional role of potassium-chloride cotransporters (KCC) in astrocytes and C6 glioma cells

Brain edema formation following brain injury is a serious but still poorly treatable medical condition. The understanding of volume regulation in astrocytes, the main cells involved in the formation of cytotoxic brain edema, is key for the development of novel treatment strategies. This study investigates the role of potassium-chloride cotransporters (KCC) for cell volume regulation in glial cells. PCR revealed the expression of KCC isoforms in a glial cell line (C6) and primary cultured astrocytes. Specific inhibition of KCCs caused glial cell swelling and resulted in a complete inhibition of regulatory volume decrease upon hypotonic medium-induced cell swelling. Therefore, our results show that KCCs play an important role in the maintenance and regulation of cell volume in astrocytes.

TRPM7 is a stretch- and swelling-activated cation channel involved in volume regulation in human epithelial cells

Stretch- and swelling-activated cation (SSAC) channels play essential roles not only in sensing and transducing external mechanical stresses but also in regulating cell volume in living cells. However, the molecular nature of the SSAC channel has not been clarified. In human epithelial HeLa cells, single-channel recordings in cell-attached and inside-out patches revealed expression of a Mg(2+)- and Gd(3+)-sensitive nonselective cation channel that is exquisitely sensitive to membrane stretch. Whole cell recordings revealed that the macroscopic cationic currents exhibit transient receptor potential (TRP) melastatin (TRPM)7-like properties such as outward rectification and sensitivity to Mg(2+) and Gd(3+). The whole cell cation current was augmented by osmotic cell swelling. RT-PCR and Western blotting demonstrated molecular expression of TRPM7 in HeLa cells. Treatment with small interfering RNA (siRNA) targeted against TRPM7 led to abolition of single stretch-activated cation channel currents and of swelling-activated, whole cell cation currents in HeLa cells. The silencing of TRPM7 by siRNA reduced the rate of cell volume recovery after osmotic swelling. A similar inhibition of regulatory volume decrease was also observed when extracellular Ca(2+) was removed or Gd(3+) was applied. It is thus concluded that TRPM7 represents the SSAC channel endogenously expressed in HeLa cells and that, by serving as a swelling-induced Ca(2+) influx pathway, it plays an important role in cell volume regulation.

Loss of cell volume regulation during metabolic inhibition in renal epithelial cells (A6): role of intracellular pH.

In renal ischemia, tubular obstruction induced by swelling of epithelial cells might be an important mechanism for reduction of the glomerular filtration rate. We investigated ischemic cell swelling by examining volume regulation of A6 cells during metabolic inhibition (MI) induced by cyanide and 2-deoxyglucose. Changes in cell volume were monitored by recording cell thickness (T(c)). Intracellular pH (pH(c)) measurements were performed with the pH-sensitive probe 5-chloromethyl-fluoresceine diacetate. T(c) measurements showed that MI increases cell volume. Cell swelling during MI is proportional to the rate of Na(+) transport and is not followed by a volume regulatory response. Furthermore, MI prevents the regulatory volume decrease (RVD) elicited by a hyposmotic shock. MI induces a pronounced intracellular acidification that is conserved during a subsequent hypotonic shock. A transient acidification induced by a NH(4)Cl prepulse causes a marked delay of the RVD in response to a hypotonic shock. On the other hand, acute lowering of external pH to 5, simultaneously with the hypotonic shock, allowed the onset of RVD. However, this RVD was completely arrested approximately 10 min after the initiation of the hyposmotic challenge. The inhibition of RVD appears to be related to the pronounced acidification that occurred within this time period. In contrast, when external pH was lowered 20 min before the hyposmotic shock, RVD was absent. These data suggest that internal acidification inhibits cellular volume regulation in A6 cells. Therefore, the intracellular acidification associated with MI might at least partly account for the failure of volume regulation in swollen epithelial cells.

The role of bicarbonate in regulatory volume decrease (RVD) in the epithelial-derived human breast cancer cell line ZR-75-1.

This study investigates the mechanisms involved in the regulatory volume decrease (RVD) in ZR-75-1 epithelial-derived human breast cancer cells. Cell volume changes were measured during osmotic shock using video imaging. In HEPES-buffered hypotonic solutions no RVD was observed; however, RVD was observed in HCO(3)(-)-buffered hypotonic solutions. Inhibition of RVD by 10 microM tamoxifen and 100 microM DIDS (inhibitors of volume-regulated anion channels; VRAC) and 2 mM TEA(+) (inhibitor of K(+) channels) indicates a role for these channels. In HCO(3)(-)-buffered Cl(-)-free solutions RVD was partially abolished indicating that HCO(3)(-) efflux can support RVD but also may have another role. Further experiments investigated whether HCO(3)(-) assists in the accumulation of Cl(-) via Cl(-)-HCO(3)(-) exchange. Regulatory volume increase (RVI) was also HCO(3)(-)-dependent and was inhibited by 500 microM DIDS and 10 microM 5-( N, N-dimethyl)-amiloride (DMA) indicating a role for coupled Cl(-)-HCO(3)(-) and Na(+)-H(+) exchange. Finally, in the presence of 10 microM DMA, RVD was partially inhibited providing further evidence for a role of Cl(-)-HCO(3)(-) exchange. Thus RVD in ZR-75-1 cells involves the activation of VRAC and K(+) channels. RVD is HCO(3)(-)-dependent and HCO(3)(-) efflux through VRAC appears to contribute directly to RVD. HCO(3)(-), however, also has another role in facilitating Cl(-) accumulation via Cl(-)-HCO(3)(-) exchange.

Volume regulation following hypotonic shock in isolated crypts of mouse distal colon.

1. A video-imaging technique of morphometry was used to measure the diameter as an index of cell volume in intact mouse distal colon crypts submitted to hypotonic shock. 2. Transition from isotonic (310 mosmol l-1) to hypotonic (240 mosmol l-1) saline caused a pronounced increase in crypt diameter immediately followed by regulatory volume decrease (RVD). 3. Exposure of crypts to Cl--free hyposmotic medium increased the rapidity of both cell swelling and RVD. Exposure of crypts to Na+-free hyposmotic medium reduced the total duration of swelling. Return to initial diameter was followed by further shrinkage of the crypt cells. 4. The chloride channel inhibitor NPPB (50 microM) delayed the swelling phase and prevented the subsequent normal decrease in diameter. 5. The K+ channel blockers barium (10 mM), charybdotoxin (10 nM) and TEA (5 mM) inhibited RVD by 51, 44 and 32 %, respectively. 6. Intracellular [Ca2+] rose from a baseline of 174 +/- 17 nM (n = 8) to 448 +/- 45 nM (n = 8) during the initial swelling phase 7. The Ca2+ channel blockers verapamil (50 microM) and nifedipine (10 microM), the chelator of intracellular Ca2+ BAPTA AM (30 microM), or the inhibitor of Ca2+ release TMB-8 (10 microM), dramatically reduced volume recovery, leading to 51 % (n = 9), 25 % (n = 7), 37 % (n = 6), 32 % (n = 8) inhibition of RVD, respectively. TFP (50 microM), an antagonist of the Ca2+-calmodulin complex, significantly slowed RVD. The Ca2+ ionophore A23187 (2 microM) provoked a dramatic reduction of the duration and amplitude of cell swelling followed by extensive shrinkage. The release of Ca2+ from intracellular stores using bradykinin (1 microM) or blockade of reabsorption with thapsigargin (1 microM) decreased the duration of RVD. 8. Prostaglandin E2 (PGE2, 5 microM) slightly delayed RVD, whereas leukotriene D4 (LTD4, 100 nM) and arachidonic acid (10 microM) reduced the duration of RVD. Blockade of phospholipase A2 by quinacrine (10 microM) inhibited RVD by 53 %. Common inhibition of PGE2 and LTD4 synthesis by ETYA (50 microM) or separate blockade of PGE2 synthesis by 1 microM indomethacin reduced the duration of RVD. Blockade of LTD4 synthesis by nordihydroguaiaretic acid (NDGA) did not produce any significant effect on cell swelling or subsequent RVD. 9. Staurosporine (1 microM), an inhibitor of protein kinases, inhibited RVD by 58 %. Taken together the experiments demonstrate that the RVD process is under the control of conductive pathways, extra- and intracellular Ca2+ ions, protein kinases, prostaglandins and leukotrienes.

Protein kinase C and regulatory volume decrease in mudpuppy red blood cells.

This study examined whether protein kinase C (PKC) stimulates K+ efflux during regulatory volume decrease (RVD) in Necturus maculosus (mudpuppy) red blood cells (RBCs). The limit of osmotic fragility increased with the general protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7, 10 micrometer), but not with the cyclic nucleotide-dependent kinase antagonists N-(2'-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004, 10 micrometer) and N-2-(methylamino)ethyl-5-isoquinoline-sulfonamide (H-8, 5 micrometer). Consistent with these results, osmotic fragility also increased with the PKC antagonists bisindolylmaleimide I (GF-109203X or bis I, 100 nm), bisindolylmaleimide II (bis II, 100 nm), and chelerythrine (10 micrometer). The effect of these three antagonists and H-7 was reversed with gramicidin (5 micrometer in a choline Ringer), indicating PKC was linked to K+ efflux (gramicidin is a cationophore that was used to ensure a high K+ permeability). We also measured cell volume recovery from hypotonic shock (0.5x Ringer) with a Coulter counter and estimated cell volume from the hematocrit. The percent RVD compared to control decreased with H-7 (10 micrometer), sphingosine (100 nm), chelerythrine (10 micrometer), bis I (100 nm), and bis II (100 nm), but not with HA-1004 (10 micrometer) nor H-8 (5 micrometer). Inhibition of RVD by H-7, chelerythrine, bis I, and bis II was reversed with gramicidin (5 micrometer). Furthermore, using the patch clamp technique, we found H-7 (10 micrometer) reduced a whole cell conductance that was activated during cell swelling. In addition, a conductance responsible for K+ efflux during cell swelling was inhibited by bis I (100 nm) and bis II (100 nm). These results indicate that a conductive pathway mediating K+ loss during RVD is regulated, at least in part, by protein kinase C.

Methylmercury-induced inhibition of regulatory volume decrease in astrocytes: characterization of osmoregulator efflux and its reversal by amiloride

Swelling of neonatal rat primary astrocyte cultures by hypotonic media leads to regulatory volume decrease (RVD) and the resumption of resting cell volume. RVD is associated with activation of conductive K + and Cl − channels, allowing for the escape of KCl, as well as the release of osmoregulators, such as taurine and myoinositol. As we have previously shown [D. Vitarella, H.K. Kimelberg, M. Aschner, Inhibition of RVD in swollen rat primary astrocyte cultures by methylmercury (MeHg) is due to increase amiloride-sensitive Na + uptake, Brain Res. 732 (1996) 169–178.], MeHg, when added to hypotonic buffer inhibits RVD, primarily due to increased cellular permeability to Na + via the Na +/H + antiporter. The present study was, therefore, undertaken to assess the ability of cation–anion cotransport blockers to reverse the inhibitory effect of MeHg on RVD in swollen astrocytes, and to further characterize MeHg-induced changes in astrocytic osmoregulatory release processes. The studies demonstrate the following: (1) MeHg-induced inhibition of RVD is partially inhibited by the Na +/H + antiporter blocker, amiloride, but not SITS (4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid), DIDS (4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid), furosemide or bumetanide; (2) exposure of swollen astrocytes to MeHg is associated with specific effects on osmoregulatory release, leading to significant inhibition of taurine release and a significant increase in potassium and myoinositol release compared with release in hypotonic conditions.

The role of calcium in the volume regulation of rat lacrimal acinar cells.

Earlier studies have suggested a role for Ca2+ in regulatory volume decrease (RVD) in response to hypotonic stress through the activation of Ca2+-dependent ion channels (Kotera & Brown, 1993; Park et al., 1994). The involvement of Ca2+ in regulating cell volume in rat lacrimal acinar cells was therefore examined using a video-imaging technique to measure cell volume. The trivalent cation Gd3+ inhibited RVD, suggesting that Ca2+ entry is important and may be via stretch-activated cation channels. However, Fura-2 loaded cells did not show an increase in [Ca2+]i during exposure to hypotonic solutions. The absence of any changes in [Ca2+]i resulted from the buffering of cytosolic Ca2+ by Fura-2 during hypotonic shock and therefore inhibition of RVD. The intracellular Ca2+ chelator, BAPTA, also inhibited the RVD response to hypotonic shock. An increase in [Ca2+]i induced by either acetylcholine or ionomycin, was found to decrease cell volume under isotonic conditions in lacrimal acinar cells. Cell shrinkage was inhibited by tetraethylammonium ion, an inhibitor of Ca2+-activated K+ channels. On the basis of the presented data, we suggest an involvement of intracellular Ca2+ in controlling cell volume in lacrimal acinar cells.

Cell volume regulation in proximal renal tubules from trout (Salmo trutta).

Regulatory volume decrease (RVD) following hypo-osmotic stimulation was studied using videometric methods in isolated proximal renal tubules from trout (Salmo trutta). The relative tubule diameter increased by 132.0+/-4.8 % (maximum swelling within 1 min at 15 and 25 degrees C and within 4 min at 10 degrees C) following a change from iso-osmotic (290 mosmol kg-1) to hypo-osmotic (160 mosmol kg-1) Ringer's solution. The tubule diameter subsequently decreased to approximately one-quarter of the maximal value. Ouabain (1 mmol l-1) reduced cell swelling and inhibited the RVD response by 28.0+/-10.5 %. Furthermore, increasing the bath K+ concentration by 30 mmol l-1 inhibited RVD by 76.5+/-3.6 %. The K+ channel blocker quinine, but not Ba2+ (1 and 2 mmol l-1), significantly decreased the RVD response (by 25.0+/-5.4 and 72.3+/-5.1 % at 0.1 and 0.5 mmol l-1, respectively). Similarly, increasing the Cl- concentration in the bath from 47 to 102 mmol l-1 induced a significant reduction (45. 2+/-7.9 %) in RVD. The RVD response was also markedly reduced (by 54. 7+/-5.3 %) by the Cl- channel blocker indacrinone (MK-196; 0.5 mmol l-1), but only marginally by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 1, 5, 8 and 10 micromol l-1). Addition of the K+/Cl- symport inhibitor furosemide (0.1 mmol l-1) resulted in a 39.8+/-3.9 % inhibition of RVD. This inhibition could be completely overcome by simultaneous administration of 1 micromol l-1 tributyltin (anion exchanger) and furosemide. Chelation of either extracellular (1 mmol l-1 EGTA) or both extra- and intracellular Ca2+ (1 mmol l-1 EGTA, 10 micromol l-1 A23187) had no effect on this RVD process. Furthermore, as measured using the fluorescent dye Fura-2/AM, there was no increase in the intracellular free Ca2+ concentration upon hypo-osmotic stimulation. Administration of the 5-lipoxygenase antagonist ETH 615-139 (20 micromol l-1), however, induced a 60 % inhibition of RVD. Simultaneous addition of ETH-615 and either the K+ ionophore gramicidin (0.5 mmol l-1) or the anion exchanger tributyltin (1 micromol l-1) could not reverse the ETH 615-139 inhibition. Finally, administration of the cycloxygenase inhibitor indomethacin had only a small, but significant, effect on RVD. We conclude that RVD following hypo-osmotic swelling is in these cells a temperature- and ouabain-sensitive process that appears to be the result of K+ efflux through quinine-sensitive, Ba2+-insensitive K+ channels and Cl- efflux through an MK-196- and furosemide-sensitive Cl- conductance that is relatively unaffected by NPPB. This KCl efflux seems to be regulated by eicosanoids produced by the 5-lipoxygenase. Arachidonic acid metabolites from the cycloxygenase pathway are not involved in this process. Similarly, neither extra- nor intracellular Ca2+ appears to be important for the signalling of RVD.

Metallothionein induction protects swollen rat primary astrocyte cultures from methylmercury-induced inhibition of regulatory volume decrease

Metallothionein (MT) proteins have been postulated to play a role in the detoxification of heavy metals. Since methylmercury (MeHg) preferentially accumulates in astrocytes, and MT-1 and MT-2 are astrocyte-specific MT isoforms, we investigated the ability of MTs to attenuate MeHg-induced cytotoxicity. The toxic effects of MeHg on astrocytes were investigated in a model of regulatory volume decrease (RVD) in which the cells are swollen by exposure to a hypotonic buffer. Preexposure to CdCl 2 (1 μM) for 72, 96 or 120 h, prior to acute exposure to hypotonic buffer and McHg (10 μM) led to a time-dependent increase in the intracellular levels of astrocyte MT proteins. The acute MeHg-induced inhibition of RVD was significantly, and almost fully reversed by preexposure to CdCl 2. This reversal was time-dependent, 120-h preexposure to CdCl 2 producing the greatest reversibility. Furthermore, the ability of astrocytes to efficiently volume regulate in the presence of MeHg-containing hypotonic buffer was highly correlated ( r = 0.99) with the intracellular levels of MT proteins. The release of [ 3H]taurine, an osmolyte involved in the RVD process was also measured. The inhibitory effect of MeHg on [ 3H]taurine in swollen cells was significantly, and fully reversed by CdCl 2 preexposure. The study suggests that astrocytes induced to express high levels of MT proteins are resistant to the acute inhibitory effect of McHg on RVD.

Inhibition of regulatory volume decrease in swollen rat primary astrocyte cultures by methylmercury is due to increased amiloride-sensitive Na + uptake

Primary astrocyte cultures from neonatal rats were swollen by exposure to hypotonic buffer with and without 10 μM methylmercury (MeHg). We investigated the effects of MeHg on K + (using 86Rb), taurine, d-aspartate (a non metabolizable analogue of glutamate) and Na + fluxes during regulatory volume decrease (RVD), with an electrical impedance method for determination of cell volume, coupled with on-line measurements of efflux of radioactive ions and amino acids. Addition of 10 μM MeHg completely inhibited RVD in swollen astrocytes, increased the uptake of 22Na +, increased 86Rb release, and decreased 3H-taurine release. There was no effect on the rate of release of 3H- d-aspartate from swollen astrocytes. 0.5 mM amiloride completely inhibited MeHg-induced increased Na + influx during RVD, while 1 mM furosemide had no effect. When Na + in the hypotonic buffer was replaced with N-methyl- d-glucamine (NMDG), RVD in the presence of MeHg was indistinguishable from controls. These results indicate that MeHg increases cellular permeability to ions such as Na + and K +, and that an increase in Na + permeability via Na +/H + exchange, offsetting K + loss, is the primary mechanism in its inhibition of RVD in swollen astrocytes.

Cl channel blockers inhibit the volume-activated efflux of Cl and taurine in cultured neurons.

The effects of the Cl channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), 1,9-dideoxyforskolin (DDF), dipyridamole, and niflumic acid and of the polyunsaturated fatty acids arachidonic, linolenic, and linoleic acids on regulatory volume decrease (RVD) and associated 125I and [3H]taurine fluxes in cultured rat cerebellar granule neurons were examined. Dose-response curves of NPPB, DDF, and dipyridamole showed 20-100% inhibition of RVD and osmolyte fluxes. Niflumic acid was less potent, requiring 150-600 microM to show effects of this magnitude. The polyunsaturated fatty acids (5-20 microM) inhibited 80-90% RVD and osmolyte fluxes, with arachidonic acid exhibiting the most potent effect. The volume-associated taurine efflux was somewhat higher in younger neurons, but the pharmacological sensitivity was essentially the same in immature and mature cells. The effects of all tested drugs on 125I and [3H]taurine fluxes were remarkably similar, indicating a close pharmacological sensitivity of the transport mechanism for the two osmolytes. This is in line with the suggestion of a common pathway for the volume-associated release of Cl and amino acids functioning as osmolytes.

Regulation of cell volume and intracellular pH in hyposmotically swollen rat osteosarcoma cells.

The maintenance of cell volume involves transduction of a volume-sensing signal into effectors of volume-regulatory transporters. After exposure to anisotonic conditions, cells undergo compensatory volume changes that are mediated by active transport and passive movement of ions and solutes. Intracellular pH (pHi) homeostasis may be compromised during these processes. We have studied pHi and some of the signal transduction mechanisms involved in the regulatory volume decrease (RVD) that occurs after exposure to hypoosmolar conditions in rat osteosarcoma cells, ROS 17/2.8. Cells were loaded with BCECF; pHi and cell volume were estimated by dual excitation ratio fluorimetry. Swelling of cells in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered hypotonic medium induced a rapid cell swelling followed by an incomplete RVD of approximately 30% in suspended (i.e., round) cells and approximately 60% in attached (i.e., spread) cells that was independent of subpassage number. RVD was inhibited by ouabain, valinomycin, and high external [K+], all of which should reduce the cell membrane electrochemical gradient for K+. Inhibition of RVD was induced also by decreasing intracellular [Ca2+] with BAPTA-AM and by depletion of Cl-, indicating the role of calcium-regulated K+ and Cl- efflux during RVD. Depolymerization of actin filaments by cytochalasin D prolonged the RVD three-fold and nonspecific activation of GTP-binding proteins up-regulated RVD. In attached cells the hypoosmolar-induced swelling caused a large reduction in pHi (approximately 0.7 units), which was sustained as long as cells were in hypoosmotic medium. The reduction of pHi induced by cell swelling was inhibited by Na(+)-free extracellular medium, ouabain, the tyrosine kinase inhibitor genistein, and to a lesser extent by Cl(-)-free medium. However, amiloride failed to inhibit the hypoosmolar-induced reduction of pHi. Collectively these data indicate that RVD of ROS 17/2.8 cells in HEPES-buffered medium is dependent on conductive efflux of K+ and Cl- that is regulated by cell shape, actin, and GTP-binding proteins. The sustained inhibition of pHi homeostasis induced by cell swelling may reflect the existence of cell volume sensing mechanisms that operate through tyrosine kinases to regulate pHi.

Mechanisms of regulatory volume decrease in nonpigmented human ciliary epithelial cells

To study the net solute and water efflux pathways of the ciliary epithelium we employed a cultured human NPE cell line. Because of the possible relationship between transepithelial ion and water flux and cell volume regulation, the ion efflux pathways mediating regulatory volume decrease (RVD) were investigated. Osmotic swelling of NPE cells was followed by a volume recovery. Volume recovery was K+ dependent and inhibited by K+ channel blockers such as quinine (1 mM). After osmotic swelling, a Cl(-)-dependent membrane depolarization occurred that was inhibited by Cl- channel blockers such as 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 microM) or Ca2+ chelators such as ethylene glycolbis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA, 2.0 mM). Cell swelling was also accompanied by an increase in intracellular Ca2+ concentration ([Ca2+]i) of approximately 200 nM. The swelling-induced rise in [Ca2+]i and RVD were diminished in the presence of 10 microM La3+, 50 nM 12-O-tetradecanoylphorbol 13-acetate, and nominally Ca(2+)-free medium. Near total blockage of RVD occurred after pretreatment of NPE cells with Ca(2+)-free EGTA-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) acetoxymethyl ester-containing solutions. The inhibition of RVD by EGTA-BAPTA treatment was overcome by increasing K+ conductance with gramicidin. The above findings indicate that RVD in NPE cells is mediated by separate K+ and Cl- conductances (channels). These data also show that swelling-induced increases in [Ca2+]i help modulate net ion efflux during regulation.

Volume-activated K+ and Cl- pathways of dissociated eccrine clear cells.

In isolated rhesus eccrine clear cells, regulatory volume decrease (RVD) occurs after osmotic swelling. RVD was completely inhibited by 1 mM quinidine, 200 nM charybdotoxin, 1 mM diphenylamine-2-carboxylic acid (DPC), or 0.1 mM 4-nitro-2(3-phenylpropyl-amino)benzoate. RVD was also inhibited in Ca(2+)-free medium by vinblastine (antimicrotubular agent), N-(6-aminohexyl)-5-chloro-1- naphthalenesulfonamide (W-7), or 0.1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Valinomycin reversed quinidine- and DIDS-induced inhibition of RVD but not the inhibition caused by Ca(2+)-free medium, DPC, vinblastine, or W-7. The cytosolic free Ca2+ concentration, as determined by the fura 2 method, increased from 220 nM in the control to 435 nM during RVD. Activation of both K+ and Cl-currents was also directly demonstrated with the whole cell current-voltage clamp method. DIDS inhibited swelling-induced K+, but not Cl-, currents and depolarized the membrane potential during RVD, further supporting the notion that DIDS inhibited swelling-activated K+, but not Cl-, pathways. We conclude that the observed RVD is mediated by the activation of conductive Ca(2+)-dependent K+ and Cl- pathways.