In Vitro Characterization of a Threonine-Ligated Molybdenyl-Sulfide Cluster as a Putative Cyanide Poisoning Antidote; Intracellular Distribution, Effects on Organic Osmolyte Homeostasis, and Induction of Cell Death.

Binuclear molybdenum sulfur complexes are effective for the catalytic conversion of cyanide into thiocyanate. The complexes themselves exhibit low toxicity and high aqueous solubility, which render them suitable as antidotes for cyanide poisoning. The binuclear molybdenum sulfur complex [(thr)Mo2O2(µ-S)2(S2)]- (thr - threonine) was subjected to biological studies to evaluate its cellular accumulation and mechanism of action. The cellular uptake and intracellular distribution in human alveolar (A549) cells, quantified by inductively coupled plasma mass spectrometry (ICP-MS) and cell fractionation methods, revealed the presence of the compound in cytosol, nucleus, and mitochondria. The complex exhibited limited binding to DNA, and using the expression of specific protein markers for cell fate indicated no effect on the expression of stress-sensitive channel components involved in cell volume regulation, weak inhibition of cell proliferation, no increase in apoptosis, and even a reduction in autophagy. The complex is anionic, and the sodium complex had higher solubility compared to the potassium. As the molybdenum complex possibly enters the mitochondria, it is considered as a promising remedy to limit mitochondrial cyanide poisoning following, e.g., smoke inhalation injuries.

Stress-induced modulation of volume-regulated anions channels in human alveolar carcinoma cells.

Shift in the cellular homeostasis of the organic osmolyte taurine has been associated with dysregulation of the volume-regulated anion channel (VRAC) complex, which comprises leucine-rich repeat-containing family 8 members (LRRC8A-E). Using SDS-PAGE, western blotting, qRT-PCR, and tracer technique ([3 H]taurine) we demonstrate that reactive oxygen species (ROS) and the cell growth-associated kinases Akt/mTOR, play a role in the regulation of VRAC in human alveolar cancer (A549) cells. LRRC8A is indispensable for VRAC activity and long-term exposure to hypoosmotic challenges and/or ROS impairs VRAC activity, not through reduction in total LRRC8A expression or LRRC8A availability in the plasma membrane, but through oxidation/inactivation of kinases/phosphatases that control VRAC activity once it has been instigated. Pursuing Akt signaling via the serine/threonine kinase mTOR, using mTORC1 inhibition (rapamycin) and mTORC2 obstruction (Rictor knockdown), we demonstrate that interference with the PI3K-mTORC2-Akt signaling-axes obstructs stress-induced taurine release. Furthermore, we show that an increased LRRC8A expression, following exposure to cisplatin, ROS, phosphatase/lipoxygenase inhibitors, and antagonist of CysLT1-receptors, correlates an increased activation of the proapoptotic transcription factor p53. It is suggested that an increase in LRRC8A protein expression could be taken as an indicator for cell stress and limitation in VRAC activity.

Downregulation of Leucine-Rich Repeat-Containing 8A Limits Proliferation and Increases Sensitivity of Glioblastoma to Temozolomide and Carmustine.

BACKGROUND: Glioblastoma (GBM) is the most common primary malignant brain tumor in adults. Ubiquitously expressed volume-regulated anion channels (VRAC) are thought to play a role in cell proliferation, migration, and apoptosis. VRAC are heteromeric channel complexes assembled from proteins belonging to the leucine-rich repeat-containing 8A (LRRC8A through E), among which LRRC8A plays an indispensable role. In the present work, we used an RNAi approach to test potential significance of VRAC and LRRC8A in GBM survival and sensitivity to chemotherapeutic agents. METHODS: Primary GBM cells were derived from a human surgical tissue sample. LRRC8A expression was determined with quantitative RT-PCR and downregulated using siRNA. The effects of LRRC8A knockdown on GBM cell viability, proliferation, and sensitivity to chemotherapeutic agents were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and Coulter counter assays. Cell cycle progression was further explored using fluorescence-activated cell sorting analysis of propidium iodide-stained cells. RESULTS: Temozolomide (TMZ), carmustine, and cisplatin reduced GBM cell survival with the IC50 values of ~1,250, 320, and 30 µM, respectively. Two of three tested gene-specific siRNA constructs, siLRRC8A_3 and siLRRC8A_6, downregulated LRRC8A expression by >80% and significantly reduced GBM cell numbers. The most potent siLRRC8A_3 itself reduced viable cell numbers by ≥50%, and significantly increased toxicity of the sub-IC50 concentrations of TMZ (570 µM) and carmustine (167 µM). In contrast, the effects of siLRRC8A_3 and cisplatin (32 µM) were not additive, most likely because cisplatin uptake is VRAC-dependent. The results obtained in primary GBM cells were qualitatively recapitulated in U251 human GBM cell line. CONCLUSION: Downregulation of LRRC8A expression reduces GBM cell proliferation and increases sensitivity to the clinically used TMZ and carmustine. These findings indicate that VRAC represents a potential target for the treatment of GBM, alone or in combination with the current standard-of-care.

Development and validation of an ICP-MS method for quantification of total carbon and platinum in cell samples and comparison of open-vessel and microwave-assisted acid digestion methods.

Cisplatin is a widely used chemotherapeutic drug. Due to severe side effects and intrinsic or acquired resistance, there is a great interest in developing new platinum-based anticancer agents and a need for robust and validated analytical methods for determination of platinum accumulation in biological samples. A validated ICP-MS method for quantification of total carbon and platinum in cell samples is presented, applicable for cellular drug accumulation studies of platinum-based drugs, enabling estimation of drug accumulation while simultaneously determining carbon to monitor the sample digestion efficiency. Adequate precision (RSD <6%), accuracy and sensitivity were achieved for carbon and platinum determinations. Limits of detection were 0.9-3.0 mg/L for carbon and 0.11-0.50 µg/L for platinum. Determination of platinum by ICP-MS in cell samples digested applying either open-vessel or microwave-assisted acid digestion produced similar concentrations, although the residual carbon content in the sample solutions were significantly higher following open-vessel acid digestion compared to microwave-assisted acid digestion. Experiments showed that the residual carbon content after acid digestion did not have an influence on determination of total platinum by ICP-MS.

In vitro evaluation of the enantiomeric R- and S-1,1'-binaphthyl-2,2'-diaminodichlorido-Pt(ii) complexes in human Burkitt lymphoma cells: emphasis on cellular accumulation, cytotoxicity, DNA binding, and ability to induce apoptosis.

The aim of this project is to gain insights into the uptake and cellular actions of the enantiomeric R- and S-1,1'-binaphthyl-2,2'-diaminodichlorido-Pt(ii) complexes (R- and S-[Pt(DABN)Cl2]) in the cisplatin-sensitive human Burkitt lymphoma cell line (Gumbus, IC50: 1.3 ± 0.2 µM) and its cisplatin-resistant sub-line (CDDPrGB, IC50: 6.6 ± 1.2 µM). The cellular uptakes of R- and S-[Pt(DABN)Cl2] are ca. 4-fold higher than cisplatin, and involve a transport mechanism independent of the volume-sensitive, organic anion-channel complex, which facilitates cisplatin accumulation. The cisplatin-resistant CDDPrGB cells are not cross-resistant to either S- or R-[Pt(DABN)Cl2]. We also find that even though R-[Pt(DABN)Cl2] has a higher maximal cellular uptake and binds at higher levels to calf-thymus DNA than S-[Pt(DABN)Cl2], it appears that S-[Pt(DABN)Cl2] is more cytotoxic for Gumbus (IC50: 0.4 ± 0.1 µM) compared to R-[Pt(DABN)Cl2] (IC50: 0.7 ± 0.3 µM). The cellular action of R- and S-[Pt(DABN)Cl2] involves G0/G1 cell cycle arrest and cell death involving the extrinsic and intrinsic apoptotic pathways.

MCT1 and MCT4 Expression and Lactate Flux Activity Increase During White and Brown Adipogenesis and Impact Adipocyte Metabolism.

Adipose tissue takes up glucose and releases lactate, thereby contributing significantly to systemic glucose and lactate homeostasis. This implies the necessity of upregulation of net acid and lactate flux capacity during adipocyte differentiation and function. However, the regulation of lactate- and acid/base transporters in adipocytes is poorly understood. Here, we tested the hypothesis that adipocyte thermogenesis, browning and differentiation are associated with an upregulation of plasma membrane lactate and acid/base transport capacity that in turn is important for adipocyte metabolism. The mRNA and protein levels of the lactate-H+ transporter MCT1 and the Na+,HCO3- cotransporter NBCe1 were upregulated in mouse interscapular brown and inguinal white adipose tissue upon cold induction of thermogenesis and browning. MCT1, MCT4, and NBCe1 were furthermore strongly upregulated at the mRNA and protein level upon differentiation of cultured pre-adipocytes. Adipocyte differentiation was accompanied by increased plasma membrane lactate flux capacity, which was reduced by MCT inhibition and by MCT1 knockdown. Finally, in differentiated brown adipocytes, glycolysis (assessed as ECAR), and after noradrenergic stimulation also oxidative metabolism (OCR), was decreased by MCT inhibition. We suggest that upregulation of MCT1- and MCT4-mediated lactate flux capacity and NBCe1-mediated HCO3-/pH homeostasis are important for the physiological function of mature adipocytes.

Role of volume-regulated and calcium-activated anion channels in cell volume homeostasis, cancer and drug resistance.

Volume-regulated channels for anions (VRAC) / organic osmolytes (VSOAC) play essential roles in cell volume regulation and other cellular functions, e.g. proliferation, cell migration and apoptosis. LRRC8A, which belongs to the leucine rich-repeat containing protein family, was recently shown to be an essential component of both VRAC and VSOAC. Reduced VRAC and VSOAC activities are seen in drug resistant cancer cells. ANO1 is a calcium-activated chloride channel expressed on the plasma membrane of e.g., secretory epithelia. ANO1 is amplified and highly expressed in a large number of carcinomas. The gene, encoding for ANO1, maps to a region on chromosome 11 (11q13) that is frequently amplified in cancer cells. Knockdown of ANO1 impairs cell proliferation and cell migration in several cancer cells. Below we summarize the basic biophysical properties of VRAC, VSOAC and ANO1 and their most important cellular functions as well as their role in cancer and drug resistance.

Ion channels and transporters in the development of drug resistance in cancer cells.

Multi-drug resistance (MDR) to chemotherapy is the major challenge in the treatment of cancer. MDR can develop by numerous mechanisms including decreased drug uptake, increased drug efflux and the failure to undergo drug-induced apoptosis. Evasion of drug-induced apoptosis through modulation of ion transporters is the main focus of this paper and we demonstrate how pro-apoptotic ion channels are downregulated, while anti-apoptotic ion transporters are upregulated in MDR. We also discuss whether upregulation of ion transport proteins that are important for proliferation contribute to MDR. Finally, we discuss the possibility that the development of MDR involves sequential and localized upregulation of ion channels involved in proliferation and migration and a concomitant global and persistent downregulation of ion channels involved in apoptosis.

Double mutation at the putative protein kinase C phosphorylation sites Thr151 plus Thr323 in the mouse leukotrieneD4 receptor eliminates homologous desensitization.

BACKGROUND/AIMS: Signalling via CysLT1 is involved in activation of volume sensitive K(+) channels and homologous desensitization of the LTD4 receptor impairs regulatory volume decrease (RVD). The aim is to illustrate the effect of mutation of putative PKC consensus phosphorylation sites in the CysLT1R on desensitization and RVD. METHODS: mCysLT1 contains 4 putative PKC consensus phosphorylation sites, and four mutants were created: Thr151Gly, Thr323Gly, Thr151Gly plus Thr323Gly, and Thr236Gly plus Ser243Gly. Functional mCysLT1 receptor activity after injection of in vitro transcribed cRNA into Xenopus laevis oocytes was visualized as a LTD4-evoked, Ca(2+)-activated Cl(-) currents recorded by two-electrode voltage clamp. RESULTS: Repetitive LTD4 administration (100 nM) desensitized the LTD4-evoked currents in oocytes expressing wild type CysLT1. Single mutations as well as the double mutation Thr236Gly plus Ser243Gly had no or a slight effect on the LTD4 induced desensitization. However, double mutation Thr323Gly plus Thr151Gly prevented the desensitization. As a functional consequence we find that inhibition of PKC accelerates RVD and prevents the inhibitory effect of LTD4-pretreatment on RVD in Ehrlich ascites tumour cells. CONCLUSION: These data indicate that simultaneous PKC-mediated phosphorylation at the 2(nd) inner loop (Thr(151)) and at the C-terminal domain (Thr(323)) leads to mCysLT1 receptor desensitization and abrogates the RVD response following osmotic cell swelling.

Cell volume regulation and signaling in 3T3-L1 pre-adipocytes and adipocytes: on the possible roles of caveolae, insulin receptors, FAK and ERK1/2.

Caveolae have been implicated in sensing of cell volume perturbations, yet evidence is still limited and findings contradictory. Here, we investigated the possible role of caveolae in cell volume regulation and volume sensitive signaling in an adipocyte system with high (3T3-L1 adipocytes); intermediate (3T3-L1 pre-adipocytes); and low (cholesterol-depleted 3T3-L1 pre-adipocytes) caveolae levels. Using large-angle light scattering, we show that compared to pre-adipocytes, differentiated adipocytes exhibit several-fold increased rates of volume restoration following osmotic cell swelling (RVD) and osmotic cell shrinkage (RVI), accompanied by increased swelling-activated taurine efflux. However, caveolin-1 distribution was not detectably altered after osmotic swelling or shrinkage, and caveolae integrity, as studied by cholesterol depletion or expression of dominant negative Cav-1, was not required for either RVD or RVI in pre-adipocytes. The insulin receptor (InsR) localizes to caveolae and its expression dramatically increases upon adipocyte differentiation. In pre-adipocytes, InsR and its effectors focal adhesion kinase (FAK) and extracellular signal regulated kinase (ERK1/2) localized to focal adhesions and were activated by a 5 min exposure to insulin (100 nM). Osmotic shrinkage transiently inhibited InsR Y(146)-phosphorylation, followed by an increase at t=15 min; a similar pattern was seen for ERK1/2 and FAK, in a manner unaffected by cholesterol depletion. In contrast, cell swelling had no detectable effect on InsR, yet increased ERK1/2 phosphorylation. In conclusion, differentiated 3T3-L1 adipocytes exhibit greatly accelerated RVD and RVI responses and increased swelling-activated taurine efflux compared to pre-adipocytes. Furthermore, in pre-adipocytes, Cav-1/caveolae integrity is not required for volume regulation. Given the relationship between hyperosmotic stress and insulin signaling, the finding that cell volume regulation is dramatically altered upon adipocyte differentiation may be relevant for the understanding of insulin resistance and metabolic syndrome.

HL-1 mouse cardiomyocyte injury and death after simulated ischemia and reperfusion: roles of pH, Ca2+-independent phospholipase A2, and Na+/H+ exchange.

The Ca(2+)-independent phospholipase A(2) VI (iPLA(2)-VI) and the Na(+)/H(+) exchanger isoform 1 (NHE1) are highly pH-sensitive proteins that exert both protective and detrimental effects in cardiac ischemia-reperfusion. Here, we investigated the role of extracellular pH (pH(o)) in ischemia-reperfusion injury and death and in regulation and function of iPLA(2)-VI and NHE1 under these conditions. HL-1 cardiomyocytes were exposed to simulated ischemia (SI; 0.5% O(2), 8 mM K(+), and 20 mM lactate) at pH(o) 6.0 and 7.4, with or without 4 or 8 h of reperfusion (SI/R). Cytochrome c release and caspase-3 activation were reduced after acidic compared with neutral SI, whereas necrotic death, estimated as glucose-6-phosphate dehydrogenase release, was similar in the two conditions. Inhibition of iPLA(2)-VI activity by bromoenol lactone (BEL) elicited cardiomyocyte necrosis during normoxia and after acidic, yet not after neutral, SI. The isoform-selective enantiomers R- and S-BEL both mimicked the effect of racemic BEL after acidic SI. In contrast, inhibition of NHE activity by EIPA had no significant effect on necrosis after SI. Both neutral and acidic SI were associated with a reversible loss of F-actin and cortactin integrity. Inhibition of iPLA(2)-VI disrupted F-actin, cortactin, and mitochondrial integrity, whereas inhibition of NHE slightly reduced stress fiber content. iPLA(2)-VIA and NHE1 mRNA levels were reduced during SI and upregulated in a pH(o)-dependent manner during SI/R. This also affected the subcellular localization of iPLA(2)-VIA. Thus, the mode of cell death and the roles and regulation of iPLA(2)-VI and NHE1 are at least in part determined by the pH(o) during SI. In addition to having clinically relevant implications, these findings can in part explain the contradictory results obtained from previous studies of iPLA(2)-VIA and NHE1 during cardiac I/R.

Physiology of cell volume regulation in vertebrates.

The ability to control cell volume is pivotal for cell function. Cell volume perturbation elicits a wide array of signaling events, leading to protective (e.g., cytoskeletal rearrangement) and adaptive (e.g., altered expression of osmolyte transporters and heat shock proteins) measures and, in most cases, activation of volume regulatory osmolyte transport. After acute swelling, cell volume is regulated by the process of regulatory volume decrease (RVD), which involves the activation of KCl cotransport and of channels mediating K(+), Cl(-), and taurine efflux. Conversely, after acute shrinkage, cell volume is regulated by the process of regulatory volume increase (RVI), which is mediated primarily by Na(+)/H(+) exchange, Na(+)-K(+)-2Cl(-) cotransport, and Na(+) channels. Here, we review in detail the current knowledge regarding the molecular identity of these transport pathways and their regulation by, e.g., membrane deformation, ionic strength, Ca(2+), protein kinases and phosphatases, cytoskeletal elements, GTP binding proteins, lipid mediators, and reactive oxygen species, upon changes in cell volume. We also discuss the nature of the upstream elements in volume sensing in vertebrate organisms. Importantly, cell volume impacts on a wide array of physiological processes, including transepithelial transport; cell migration, proliferation, and death; and changes in cell volume function as specific signals regulating these processes. A discussion of this issue concludes the review.

Casein kinase 2 regulates the active uptake of the organic osmolyte taurine in NIH3T3 mouse fibroblasts.

Inhibition of the constitutively active casein kinase 2 (CK2) with 2-dimethyl-amino-4,5,6,7-tetrabromo-1H-benzimidasole stimulates the Na(+)-dependent taurine influx via the taurine transporter TauT in NIH3T3 cells. CK2 inhibition reduces the TauT mRNA level and increases the localization of TauT to ER but has no detectable effect on TauT protein expression. On the other hand, CK2 inhibition increases the affinity of TauT towards Na(+ )and reduces the Na(+)/taurine stoichiometry for active taurine uptake. It is suggested that CK2 controls the cellular taurine uptake in unperturbated NIH3T3 cells, i.e., inhibition of CK2 increases the affinity of TauT towards Na(+) and hence Na(+)-dependent taurine uptake.

Homologous desensitisation of the mouse leukotriene B4 receptor involves protein kinase C-mediated phosphorylation of serine 127.

Murine leukotriene B(4) (LTB(4)) receptor (mBLT1) cDNA was identified by searching the EST database using human LTB(4) receptor as the query sequence. Expression of functional mBLT1 after injection of in vitro transcribed cRNA into Xenopus laevis oocytes was demonstrated as LTB(4)-evoked, Ca(2+)-activated Cl(-) currents recorded by two-electrode voltage clamp. From mBLT1-expressing oocytes, a dose-dependent relationship between the Ca(2+)-activated Cl(-) current and LTB(4) concentration was demonstrated with an apparent EC(50) of 6.7 nM. Following LTB(4) stimulation of mBLT1, we observed two transient, spatially distinct Ca(2+)-activated, inwardly directed Cl(-) currents in the oocytes: a fast peak current requiring relatively high LTB(4) concentrations, and a slowly progressing Cl(-) current. Nucleotides, PGE(2), 12R-hydroxy-5, 8, 14-cis-10-trans-eicosatetraenoic acid, and LTD(4) did not activate mBLT1. U75302, specifically targeting BLT1, significantly reduced LTB(4)-evoked Cl(-) currents. Repetitive LTB(4) administration desensitized the LTB(4)-evoked currents. Activation of protein kinase C (PKC) by PMA addition completely eliminated the LTB(4)-evoked currents, whereas down-regulation of PKC by prolonged PMA exposure (20 h) impaired mBLT1 desensitisation. In addition, Ser-127-Ala substitution of the PKC consensus phosphorylation site on the second intracellular loop prevented the mBLT1 desensitisation. These data indicate that PKC-mediated phosphorylation at Ser-127 leads to mBLT1 desensitisation.

Role of phospholipase A2 in the induction of drip loss in porcine muscle.

The role of phospholipase A2 in the induction of drip loss from pig muscle has been investigated. In samples from porcine M. longissimus dorsi, total PLA2 activity as well as mRNA and protein levels of the group VIA iPLA2 (iPLA2-VIA) increased during the initial 4 h post-mortem period. Morphological studies of porcine muscle showed that at 4 h post-mortem, gaps had formed between muscle fibers and that the sarcolemma membrane borders appeared blurred. At the same time iPLA2-VIA protein levels were increased inside muscle fibers and at the sarcolemma. iPLA2-VIA mRNA abundance in samples from different breeds of pigs with variations in drip loss revealed no clear correlation between drip loss level and iPLA2-VIA expression. Together, these data indicate that during the post-mortem period, iPLA2-VIA expression and activity is increased at the muscle fiber membranes. PLA2 activity may affect membrane permeability and consequently the progression of drip formation in porcine muscle.

Roles of phospholipase A2 isoforms in swelling- and melittin-induced arachidonic acid release and taurine efflux in NIH3T3 fibroblasts.

Osmotic swelling of NIH3T3 mouse fibroblasts activates a bromoenol lactone (BEL)-sensitive taurine efflux, pointing to the involvement of a Ca(2+)-independent phospholipase A(2) (iPLA(2)) (Lambert IH. J Membr Biol 192: 19-32, 2003). We report that taurine efflux from NIH3T3 cells was not only increased by cell swelling but also decreased by cell shrinkage. Arachidonic acid release to the cell exterior was similarly decreased by shrinkage yet not detectably increased by swelling. NIH3T3 cells were found to express cytosolic calcium-dependent cPLA(2)-IVA, cPLA(2)-IVB, cPLA(2)-IVC, iPLA(2)-VIA, iPLA(2)-VIB, and secretory sPLA(2)-V. Arachidonic acid release from swollen cells was partially inhibited by BEL and by the sPLA(2)-inhibitor manoalide. Cell swelling elicited BEL-sensitive arachidonic acid release from the nucleus, to which iPLA(2)-VIA localized. Exposure to the bee venom peptide melittin, to increase PLA(2) substrate availability, potentiated arachidonic acid release and osmolyte efflux in a volume-sensitive, 5-lipoxygenase-dependent, cyclooxygenase-independent manner. Melittin-induced arachidonic acid release was inhibited by manoalide and slightly but significantly by BEL. A BEL-sensitive, melittin-induced PLA(2) activity was also detected in lysates devoid of sPLA(2), indicating that both sPLA(2) and iPLA(2) contribute to arachidonic acid release in vivo. Swelling-induced taurine efflux was inhibited potently by BEL and partially by manoalide, whereas the reverse was true for melittin-induced taurine efflux. It is suggested that in NIH3T3 cells, swelling-induced taurine efflux is dependent at least in part on arachidonic acid release by iPLA(2) and possibly also by sPLA(2), whereas melittin-induced taurine efflux is dependent on arachidonic acid release by sPLA(2) and, to a lesser extent, iPLA(2).

Cell shrinkage as a signal to apoptosis in NIH 3T3 fibroblasts.

Cell shrinkage is a hallmark of the apoptotic mode of programmed cell death, but it is as yet unclear whether a reduction in cell volume is a primary activation signal of apoptosis. Here we studied the effect of an acute elevation of osmolarity (NaCl or sucrose additions, final osmolarity 687 mosmol l(-1)) on NIH 3T3 fibroblasts to identify components involved in the signal transduction from shrinkage to apoptosis. After 1.5 h the activity of caspase-3 started to increase followed after 3 h by the appearance of many apoptotic-like bodies. The caspase-3 activity increase was greatly enhanced in cells expressing a constitutively active G protein, Rac (RacV12A3 cell), indicating that Rac acts upstream to caspase-3 activation. The stress-activated protein kinase, p38, was significantly activated by phosphorylation within 30 min after induction of osmotic shrinkage, the phosphorylation being accelerated in fibroblasts overexpressing Rac. Conversely, the activation of the extracellular signal-regulated kinase (Erk1/2) was initially significantly decreased. Subsequent to activation of p38, p53 was activated through serine-15 phosphorylation, and active p53 was translocated from the cytosol to the nucleus. Inhibition of p38 in Rac cells reduced the activation of both p53 and caspase-3. After 60 min in hypertonic medium the rate constants for K+ and taurine efflux were increased, particular in Rac cells. We suggest the following sequence of events in the cell shrinkage-induced apoptotic response: cellular shrinkage activates Rac, with activation of p38, followed by phosphorylation and nuclear translocation of p53, resulting in permeability increases and caspase-3 activation.

High expression of the taurine transporter TauT in primary cilia of NIH3T3 fibroblasts.

Taurine, present in high concentrations in various mammalian cells, is essential for regulation of cell volume, cellular oxidative status as well as the cellular Ca2+ homeostasis. Cellular taurine content is a balance between active uptake through the saturable, Na(+)-dependent taurine transporter TauT, and passive release via a volume-sensitive leak pathway. Here we demonstrate that: (i) TauT localizes to the primary cilium of growth-arrested NIH3T3 fibroblasts, (ii) long-term exposure to TNF(alpha) or hypertonic sucrose medium, i.e., growth medium supplemented with 100 mM sucrose, increases ciliary TauT expression and (iii) long-term exposure to hypertonic taurine medium, i.e., growth medium supplemented with 100 mM taurine, reduces ciliary TauT expression. These results point to an important role of taurine in the regulation of physiological processes located to the primary cilium.

Regulation of the expression and subcellular localization of the taurine transporter TauT in mouse NIH3T3 fibroblasts.

The cellular level of the organic osmolyte taurine is a balance between active uptake and passive leak via a volume sensitive pathway. Here, we demonstrate that NIH3T3 mouse fibroblasts express a saturable, high affinity taurine transporter (TauT, Km = 18 microm), and that taurine uptake via TauT is a Na+- and Cl(-)-dependent process with an apparent 2.5 : 1 : 1 Na+/Cl-/taurine stoichiometry. Transport activity is reduced following acute administration of H2O2 or activators of protein kinases A or C. TauT transport activity, expression and nuclear localization are significantly increased upon serum starvation (24 h), exposure to tumour necrosis factor alpha (TNFalpha; 16 h), or hyperosmotic medium (24 h); conditions that are also associated with increased localization of TauT to the cytosolic network of microtubules. Conversely, transport activity, expression and nuclear localization of TauT are reduced in a reversible manner following long-term exposure (24 h) to high extracellular taurine concentration. In contrast to active taurine uptake, swelling-induced taurine release is significantly reduced following preincubation with TNFalpha (16 h) but unaffected by high extracellular taurine concentration (24 h). Thus, in NIH3T3 cells, (a) active taurine uptake reflects TauT expression; (b) TauT activity is modulated by multiple stimuli, both acutely, and at the level of TauT expression; (c) the subcellular localization of TauT is regulated; and (d) volume-sensitive taurine release is not mediated by TauT.