Differences in Action of PACAP-27 and PACAP-38 on Guinea Pig Gallbladder Smooth Muscle Using Synthetic C-terminally Modified PACAP Peptides

Pituitary adenylate cyclase activating polypeptide (PACAP) occurs in two bioactive forms, PACAP-38 and PACAP-27 that have identical N-terminal sequences but differ by the presence of a C-terminal 11 residue elongation in the former. Although VIP and PACAP have several similar biological actions due to their amino acid sequence similarity, we have found that they evoke opposite responses in the guinea pig gallbladder smooth muscle, where PACAP induces contraction while VIP causes relaxation. In addition the response to PACAP-38 is four times lower than that of PACAP-27. In a previous study we have reported the role of the N-terminal α-helical regions of PACAP-27 which play a key role in gallbladder contraction. In the present study the biological action on the guinea pig gallbladder was investigated using a synthetic mini-library of C-terminally deleted peptides related to PACAP-38. The effects caused by residues within the C-terminus are not a result of a response via the M-receptor or Na⁺ channel, but most likely arise from a delicate balance between the differential effects of PACAP-38 on specific PAC1 and VPACs receptors.     

C-terminal amidation of PACAP-38 and PACAP-27 is dispensable for biological activity at the PAC1 receptor

PACAP-27 and PACAP-38 are the exclusive physiological ligands for the mammalian PAC1 receptor. The role of C-terminal amidation of these ligands at that receptor was examined in neuroendocrine cells expressing the PAC1 receptor endogenously and in non-neuroendocrine cells in which the human and rat PAC1 receptors were expressed from stable single-copy genes driven by the CMV promoter, providing stoichiometrically appropriate levels of this Gs-coupled GPCR in order to examine the potency and intrinsic activity of PACAP ligands and their des-amidated congeners. We found that replacement of the C-terminal glycine residues of PACAP-27 and -38 with a free acid; or extension of either peptide with the two to three amino acids normally found at these positions in PACAP processing intermediates in vivo following endoproteolytic cleavage and after exoproteolytic trimming and glycine-directed amidated, were equivalent in potency to the fully processed peptides in a variety of cell-based assays. These included real-time monitoring of cyclic AMP generation in both NS-1 neuroendocrine cells and non-neuroendocrine HEK293 cells; PKA-dependent gene activation in HEK293 cells; and neuritogenesis and cell growth arrest in NS-1 cells. The specific implications for the role of amidation in arming of secretin-related neuropeptides for biological function, and the general implications for neuropeptide-based delivery in the context of gene therapy, are discussed.     

Involvement of ERK MAP kinase in endoplasmic reticulum stress in SH-SY5Y human neuroblastoma cells

Endoplasmic reticulum (ER) stress has increasingly come into focus as a factor contributing to neuronal injury. Although caspase-dependent mechanisms have been implicated in ER stress, the signaling pathways involved remain unclear. In this study, we examined the role of the extracellular signal-regulated kinase (ERK), a mitogen-activated protein (MAP) kinase pathway that is highly conserved in many systems for balancing cell survival and death. Prolonged treatment of the human neuroblastoma cell line SH-SY5Y with thapsigargin, an inducer of ER stress, increased cell death over 24-48 h, as measured by LDH release. Caspases were involved; increased levels of active caspase-3 and cleaved caspase substrate PARP were detected, and treatment with Z-VAD-FMK reduced thapsigargin-induced cytotoxicity. In contrast, inhibition of calpain was not protective, although calpain was activated following thapsigargin treatment. An early and transient phosphorylation of ERK1/2 occurred after thapsigargin-induced ER stress, and targeting this pathway with the MEK inhibitors U0126 or PD98059 significantly reduced cell death. Similar cytoprotection was obtained against brefeldin A, another ER stress agent. However, protection against ER stress via ERK inhibition was not accompanied by amelioration of caspase-3 activation, PARP cleavage, or DNA laddering. These data indicate that ERK may contribute to non-caspase-dependent pathways of injury after ER stress.     

Docosahexaenoic acid induces ERK1/2 activation and neuritogenesis via intracellular reactive oxygen species production in human neuroblastoma SH-SY5Y cells

Docosahexaenoic acid (22: 6n-3; DHA) is a long chain polyunsaturated fatty acid that exists highly enriched in fish oil, and it is one of the low molecular weight food chemicals which can pass a blood brain barrier. A preliminary survey of several fatty acids for expression of growth-associated protein-43 (GAP-43), a marker of axonal growth, identified DHA as one of the most potent inducers. The human neuroblastoma SH-SY5Y cells exposed to DHA showed significant and dose-dependent increases in the percentage of cells with longer neurites. To elucidate signaling mechanisms involved in DHA-enhanced basal neuritogenesis, we examined the role of extracellular signal-regulated kinase (ERK)1/2 and intracellular reactive oxygen species (ROS) production using SH-SY5Y cells. From immunoblotting experiments, we observed that DHA induced the ROS production, protein tyrosine phosphatase inhibition, mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) phosphorylation, and sequentially ERK1/2 phosphorylation, the last of which was significantly reduced by MEK inhibitor U0126. Both antioxidants and MEK inhibitor affected DHA-induced GAP-43 expression, whereas the specific PI3K inhibitor LY294002 did not. We found that total protein tyrosine phosphatase activity was also downregulated by DHA treatment, which was counteracted by antioxidant pretreatment. These results suggest that the ROS-dependent ERK pathway, rather than PI3K, plays an important role during DHA-enhanced neurite outgrowth.     

Peroxynitrite induces GADD34, 45, and 153 VIA p38 MAPK in human neuroblastoma SH-SY5Y cells

Peroxynitrite, one of the most reactive radicals, is produced from superoxide anion and nitric oxide. A peroxynitrite generator, 3-morpholinosydonimine (SIN-1), was found to induce the expression of three different growth arrest and DNA damage-inducible (GADD) mRNA, GADD34, GADD45, and GADD153, at the early phase during cell death in human neuroblastoma SH-SY5Y cells. In addition, peroxynitrite activated p38 MAPK just before induction of three GADD mRNA. A specific inhibitor of p38 MAPK, SB202190, markedly suppressed peroxynitrite-induced expression of three GADD mRNA in SH-SY5Y cells. The expression of three GADD genes and also p38 MAPK phosphorylation were suppressed by treatment with radical scavengers, superoxide dismutase plus catalase and glutathione. Glutathione depletion by L-buthionine-S, R-sulfoximine (BSO), increased the vulnerability of the cells to peroxynitrite. These findings indicate that peroxynitrite-mediated oxidative stress activated p38 MAPK to induce three GADD genes.     

Eclalbasaponin I causes mitophagy to repress oxidative stress-induced apoptosis via activation of p38 and ERK in SH-SY5Y cells

Oxidative stress accompanying excessive accumulation of reactive oxygen species (ROS) and mitochondrial dysfunction leads to the occurrence of neurodegenerative diseases. Our previous study showed that Eclalbasaponin I (EcI), a triterpene saponin isolated from Aralia elata (Miq.) Seem. (A. elata), repressed oxidative stress in human neuroblastoma SH-SY5Y cells. However, the detailed mechanism remains unclear. In this study, pretreatment with EcI in SH-SY5Y cells significantly activated the p38-mitogenactivated protein kinase (p38), the extracellular regulated protein kinase (ERK), whereas it did not affect the c-jun NH2 terminal kinases (JNK). In accordance with the initial findings, EcI-induced neuroprotective effect was attenuated by SB203580 (SB, a p38 inhibitor) or FR180204 (FR, an ERK inhibitor), being further confirmed by specific small interfering RNA (siRNA). Inhibition of either p38 or ERK up-regulated the apoptosis induction in EcI- and H₂O₂-cotreated cells. Furthermore, p38 or ERK suppression enhanced intracellular and mitochondrial ROS generation, decreased the activities of endogenous antioxidant defences as well as the mitochondrial membrane potential (MMP), resulting in dysfunction of mitochondria. In addition, EcI-induced autophagy and mitophagy were obviously down-regulated when p38 or ERK activation was blocked. Cumulatively, these findings supported that EcI-caused mitophagy contributed to the neuroprotective effect through p38 or ERK activation. Mitophagy induction might be an effective therapeutic intervention in neurodegenerative diseases.     

IGF2 derived from SH-SY5Y neuroblastoma cells induces the osteoclastogenesis of human monocytic precursors

The insulin-like growth factors 2 (IGF2) is a peptide hormone that binds to the insulin-like growth factor 1 receptor (IGF1R) and is abundantly stored in bone. IGF1R is deeply involved in the pathogenesis of many cancers that growth within bone and is also involved in osteoclast biology. Among different cell lines representative of osteolytic tumors, we found a very high expression of IGF2 in SH-SY5Y cells derived from neuroblastoma (NB). We previously showed that NB cells induce an osteolytic process through the Osteoprotegerin/RANKL/RANK and the canonical Wnt pathway system. Here, we hypothesized that NB promotes osteoclastogenesis also via IGF2. First, we demonstrated the presence of IGF1R on the osteoclast basolateral membrane, and we observed a cyclic IGF1R activation along with the differentiation process, also when induced by SH-SY5Y. Moreover, we found that IGF2 mRNA expression in SH-SY5Y cells was further increased when co-cultured with mesenchymal stromal cells, suggesting that IGF2 is important for NB interaction with the bone microenvironment. Finally, the treatment of SH-SY5Y cells with an anti-IGF2 siRNA or the addition of anti-IGF1R molecules impaired NB-induced osteoclastogenesis, even though the chemoattraction of monocytes by NB cells was unaffected. Our findings suggest that in IGF2-producing osteolytic tumors IGF1R is a good candidate for targeted therapies in combination with conventional drugs.     

p38 MAPK and ERK1/2 pathways are involved in the pro-apoptotic effect of notoginsenoside Ft1 on human neuroblastoma SH-SY5Y cells

Aims

This study aims to investigate the effect and the mechanisms of notoginsenoside Ft1, a natural compound exclusively found in P. notoginseng, on the proliferation and apoptosis of human neuroblastoma SH-SY5Y cells.

Main methods

CCK-8 assay was used to assess the cell proliferation. Flow cytometry was performed to measure the cell cycle distribution and cell apoptosis. Hoechst 33258 staining was conducted to confirm the morphological changes of apoptotic cells. Protein expression was detected by western blot analysis and caspase 3 activity was measured by colorimetric assay kit.

Key findings

Among the saponins examined, Ft1 showed the best inhibitory effect on cell proliferation of SH-SY5Y cells with IC₅₀ of 45 μM. Ft1 not only arrested the cell cycle at S, G₂/M stages, but also promoted cell apoptosis, which was confirmed by Hoechst 33258 staining. Further studies demonstrated that Ft1 up-regulated the protein expressions of cleaved caspase 3, phospho-p53, p21, and cyclin B1, but down-regulated that of Bcl-2. Moreover, Ft1 enhanced the phosphorylation of ERK1/2, JNK and p38 MAPK. However, the phosphorylation of Jak2 and p85 PI3K was reduced by Ft1. Inhibitors of p38 MAPK and ERK1/2 but not JNK abrogated the up-regulated protein expressions of cleaved caspase 3, p21 and down-regulated protein expression of Bcl-2 as well as elevated caspase 3 activity induced by Ft1.

Significance

Ft1 arrested the proliferation and elicited the apoptosis of SH-SY5Y cells possibly via p38 MAPK and ERK1/2 pathways, which indicates the potential therapeutic effect of it on human neuroblastoma.     

Tissue transglutaminase mediates activation of RhoA and MAP kinase pathways during retinoic acid-induced neuronal differentiation of SH-SY5Y cells

All-trans-retinoic acid (RA) plays a crucial role in survival and differentiation of neurons. For elucidating signaling mechanisms involved in RA-induced neuronal differentiation, we have selected SH-SY5Y cells, which are an established in vitro cell model for studying RA signaling. Here we report that RA-induced neuronal differentiation of SH-SY5Y cells is coupled with increased expression/activation of TGase and in vivo transamidation and activation of RhoA. In addition, RA promotes formation of stress fibers and focal adhesion complexes, and activation of ERK1/2, JNK1, and p38alpha/beta/gamma MAP kinases. Using C-3 exoenzyme (RhoA inhibitor) or monodansylcadaverine (TGase inhibitor), we show that transamidated RhoA regulates cytoskeletal rearrangement and activation of ERK1/2 and p38gamma MAP kinases. Further, by using stable SH-SY5Y cell lines (overexpressing wild-type, C277S mutant, and antisense TGase), we demonstrate that transglutaminase activity is required for activation of RhoA, ERK1/2, JNK1, and p38gamma MAP kinases. Activated MAP kinases differentially regulate RA-induced neurite outgrowth and neuronal marker expression. The results of our studies suggest a novel mechanism of RA signaling, which involves activation of TGase and transamidation of RhoA. RA-induced activation of TGase is proposed to induce multiple signaling pathways that regulate neuronal differentiation.     

Cry1Ac toxin induces macrophage activation via ERK1/2, JNK and p38 mitogen-activated protein kinases

The Cry1Ac toxin from Bacillus thuringiensis is used commercially as a bio-insecticide and is expressed in transgenic plants that are used for human and animal consumption. Although it was originally considered innocuous for mammals, the Cry1Ac toxin is not inert and has the ability to induce mucosal and systemic immunogenicity. Herein, we examined whether the Cry1Ac toxin promotes macrophage activation and explored the signalling pathways that may mediate this effect. Treatment of primary and RAW264.7 macrophages with the Cry1Ac toxin resulted in upregulation of the costimulatory molecules CD80, CD86 and ICOS-L and enhanced production of nitric oxide, the chemokine MCP-1 and the proinflammatory cytokines TNF-α and IL-6. Remarkably, the Cry1Ac toxin induced phosphorylation of the mitogen-activated protein kinases (MAPKs) ERK1/2, JNK and p38 and promoted nuclear translocation of nuclear factor-kappa B (NF-κB) p50 and p65. p38 and ERK1/2 MAPKs were involved in this effect, as indicated by the Cry1Ac-induced upregulation of CD80 and IL-6 and TNF-α abrogation by the p38 MAPK inhibitor SB203580. Furthermore, treatment the MEK1/2 kinase inhibitor PD98059 blocked increases in MCP-1 secretion and augmented Cry1Ac-induced ICOS-L upregulation. These data demonstrate the capacity of the Cry1Ac toxin to induce macrophage activation via the MAPK and NF-κB pathways.     

Protein kinase C remains functionally active during TPA induced neuronal differentiation of SH-SY5Y human neuroblastoma cells.

SH-SY5Y human neuroblastoma cells can be induced to differentiate into a neuronal phenotype by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). In other cell systems, TPA treatment frequently leads to down-regulation of protein kinase C (PKC). However, we now report that TPA-treated and non-treated SH-SY5Y cells express PKC-alpha, but not PKC-beta and PKC-gamma, mRNA. Furthermore, only a slight down-regulation of the PKC-alpha protein could be seen during prolonged treatment with 16 nM TPA, the concentration giving optimal differentiation. In contrast, a higher concentration of TPA (1.6 microM) results in a poor neuronal differentiation and a complete down-regulation of PKC-alpha. PKC-alpha was rapidly translocated to the particulate fraction and remained membrane bound for at least 4 days during treatment with 16 nM TPA. In such cells a sustained increased level of the phosphorylated form of a 80,000 Dalton PKC-substrate was found. In addition to this sustained augmented phosphorylation, administration of fresh TPA at day 4 caused a small but reproducible further increased level of phosphorylated substrate. When the PKC activity was measured by the histone phosphorylation assay a substantial fraction of the initial enzyme activity could still be detected after 4 days of TPA treatment. Taken together, the data demonstrate that PKC remains functionally active during TPA induced differentiation of SH-SY5Y cells, which may suggest a continuous role for the enzyme during the differentiation process.     

Protein kinase C activation and down-regulation in relation to phorbol ester-induced differentiation of SH-SY5Y human neuroblastoma cells

The role of protein kinase C activation in changes in muscarinic receptor functions and in the appearance of biochemical properties characteristic of neuronal cells was studied in SH-SY5Y human neuroblastoma cells induced to differentiate with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). A decrease in muscarinic receptor sensitivity with respect to agonist induced Ca2+ mobilization and receptor number parallelled the increase in membrane-associated protein kinase C (PK-C) activity. These changes occurred during the first 6 h of culture, and they were associated with rounding-up of cells. A subsequent decrease in particulate PK-C activity was followed by an increase in noradrenaline content, the appearance of an electrically excitable membrane, and an increase in the level of neuron-specific enolase. These changes were accompanied by a pronounced neurite outgrowth. 1-(5-Isoquinolinesulphonyl)-2-methylpiperazine (H-7), an inhibitor of PK-C and cyclic nucleotide-dependent protein kinases, enhanced the morphological differentiation induced by TPA, whereas N-(2-guanidinoethyl)-5-isoquinolinesulphonamide (HA-1004), which primarily inhibits cyclic nucleotide-dependent protein kinases, had no effect on the TPA-induced phenotypic differentiation. H-7 inhibited the decrease in muscarinic receptor sensitivity and receptor number, but had no effect on the appearance of the electrically excitable membrane or on the increase in the neuron-specific enolase level. Both H-7 and HA-1004 inhibited the TPA-induced increase in noradrenaline content.     

Differential expression and signaling of the human histamine H3 receptor isoforms of 445 and 365 amino acids expressed in human neuroblastoma SH-SY5Y cells

In stably-transfected human neuroblastoma SH-SY5Y cells, we have compared the effect of activating two isoforms of 445 and 365 amino acids of the human histamine H₃ receptor (hH₃R₄₄₅ and hH₃R₃₆₅) on [³⁵S]-GTPγS binding, forskolin-induced cAMP formation, depolarization-induced increase in the intracellular concentration of Ca²⁺ ions ([Ca²⁺]i) and depolarization-evoked [^3?H]-dopamine release. Maximal specific binding (B_max) of [^3?H]-N-methyl-histamine to cell membranes was 953?±?204 and 555?±?140?fmol/mg protein for SH-SY5Y-hH₃R₄₄₅ and SH-SY5Y-hH₃R₃₆₅ cells, respectively, with similar dissociation constants (K_d, 0.86?nM and 0.81?nM). The mRNA of the hH₃R₃₆₅ isoform was 40.9?±?7.9% of the hH₃R₄₄₅ isoform. No differences in receptor affinity were found for the H₃R ligands histamine, immepip, (R)(-)-α-methylhistamine (RAMH), A-331440, clobenpropit and ciproxifan. Both the stimulation of [³⁵S]-GTPγS binding and the inhibition of forskolin-stimulated cAMP accumulation by the agonist RAMH were significantly larger in SH-SY5Y-hH₃R₄₄₅ cells ([³⁵S]-GTPγS binding, 158.1?±?7.5% versus 136.5?±?3.6% for SH-SY5Y-hH₃R₃₆₅ cells; cAMP accumulation, ?74.0?±?4.9% versus ?43.5?±?5.3%), with no significant effect on agonist potency. In contrast, there were no differences in the efficacy and potency of RAMH to inhibit [^3?H]-dopamine release evoked by 100?mM K⁺ (?18.9?±?3.0% and ?20.5?±?3.3%, for SH-SY5Y-hH₃R₄₄₅ and SH-SY5Y-hH₃R₃₆₅ cells), or the inhibition of depolarization-induced increase in [Ca²⁺]i (S2/S1 ratios: parental cells 0.967?±?0.069, SH-SY5Y-hH₃R₄₄₅ cells 0.639?±?0.049, SH-SY5Y-hH₃R₃₆₅ cells 0.737?±?0.045). These results indicate that in SH-SY5Y cells, hH₃R₄₄₅ and hH₃R₃₆₅ isoforms regulate in a differential manner the signaling pathways triggered by receptor activation.     

Translocation and phosphorylation of calcyclin binding protein during retinoic acid-induced neuronal differentiation of neuroblastoma SH-SY5Y cells

For better understanding of functions of the Calcyclin Binding Protein (CacyBP) and exploring its possible roles in neuronal differentiation, the subcellular localization of human CacyBP was examined in retinoic acid(RA)-induced and uninduced neuroblastoma SH-SY5Y cells. Immunostaining indicated that CacyBP was present in the cytoplasm of uninduced SH-SY5Y cells, in which the resting Ca(2+) concentration was relatively lower than that of RA-induced cells. After the RA induction, immunostaining was seen in both the nucleus and cytoplasm. In the RA-induced differentiated SH-SY5Y cells, CacyBP was phosphorylated on serine residue(s), while it existed in a dephosphorylated form in normal (uninduced) cells. Thus, the phosphorylation of CacyBP occurs when it is translocated to the nuclear region. The translocation of CacyBP during the RA-induced differentiation of SH-SY5Y cells suggested that this protein might play a role in neuronal differentiation.     

Critical role of ASK1 in the 6-hydroxydopamine-induced apoptosis in human neuroblastoma SH-SY5Y cells

6-hydroxydopamine (6-OHDA)-induced apoptosis in dopaminergic neuronal cells is a common cell model of Parkinson's disease (PD). The role of apoptosis signal-regulating kinase 1 (ASK1) in this model has not been well studied. We observed significant activation of ASK1, p38 and JNK, as well as apoptosis in human dopaminergic neuroblastoma SH-SY5Y cells exposed to 6-OHDA. Over-expressing kinase-dead mutant ASK1(K709M) or knock-down of endogenous ASK1 by its small interfering RNA (siRNA) greatly suppressed activation of these kinases and apoptosis in the cells. It was found that the activation of p38 and JNK was suppressed to almost the same extent as that of ASK1 in the ASK1-knock-down cells, suggesting that activated ASK1 is almost totally responsible for activation of p38/JNK. It was also observed that the 6-OHDA-induced cell apoptosis could be effectively prevented by over-expressing the dominant-negative mutant of p38 or p38 inhibitor SB203580, demonstrating that activation of p38/JNK signalling is required for initiating the programmed cell death. Furthermore, suppression of the 6-OHDA-generated reactive oxygen species (ROS) by pre-incubation of cells with N-acetyl-L-cysteine effectively inhibited the 6-OHDA-induced activation of ASK1, p38 and JNK, and protected the cells from apoptosis. This study clearly shows the route from ROS generation by 6-OHDA to initiation of p38/JNK signalling via activation of ASK1 in the studied PD model.     

Carbachol enhances forskolin-stimulated cyclic AMP accumulation via activation of calmodulin system in human neuroblastoma SH-SY5Y cells.

We have investigated the modulatory action of carbachol on intracellular cAMP levels in human neuroblastoma SH-SY5Y cells. Carbachol enhanced forskolin-stimulated cAMP levels in a dose-dependent manner (EC50 = 3 microM). The enhancing effect of carbachol was completely inhibited by pirenzepine and atropine. Pertussis toxin treatment of the cells partially affected the ability of carbachol. Furthermore, carbachol also enhanced the effect of vasoactive intestinal peptide (EC50 = 3 microM)-, adenosine- and prostaglandin E1-stimulated cAMP levels. The enhancing response of carbachol was sensitive to trifluoperazine but insensitive to calphostin C. These results suggest that the mechanism for carbachol-induced cAMP levels may act, at least in part, through the activation of calmodulin system in SH-SY5Y cells. Hence we describe for the first time a synergistic interaction between calmodulin- and cAMP-dependent signal transduction pathway mediated by carbachol in neuron-derived cell line.     

Muscarinic receptor regulation of osmosensitive taurine transport in human SH-SY5Y neuroblastoma cells

The ability of G protein‐coupled receptors to regulate osmosensitive uptake of the organic osmolyte, taurine, into human SH‐SY5Y neuroblastoma cells has been examined. When monitored under isotonic conditions and in the presence of physiologically relevant taurine concentrations (1–100 μM), taurine influx was mediated exclusively by a Na⁺‐dependent, high‐affinity (K_m = 2.5 μM) saturable transport mechanism (V_max = 0.087 nmol/mg protein/min). Reductions in osmolarity of > 20% (attained under conditions of a constant NaCl concentration) resulted in an inhibition of taurine influx (> 30%) that could be attributed to a reduction in V_max, whereas the K_m for uptake remained unchanged. Inclusion of the muscarinic cholinergic agonist, oxotremorine‐M (Oxo‐M), also resulted in an attenuation of taurine influx (EC₅₀～0.7 μM). Although Oxo‐M‐mediated inhibition of taurine uptake could be observed under isotonic conditions (～25–30%), the magnitude of inhibition was significantly enhanced by hypotonicity (～55–60%), a result that also reflected a reduction in the V_max, but not the K_m, for taurine transport. Oxo‐M‐mediated inhibition of taurine uptake was dependent upon the availability of extracellular Ca²⁺ but was independent of protein kinase C activity. In addition to Oxo‐M, inclusion of either thrombin or sphingosine 1‐phosphate also attenuated volume‐dependent taurine uptake. The ability of Oxo‐M to inhibit the influx of taurine was attenuated by 4‐[(2‐butyl‐6,7‐dichloro‐2‐cyclopentyl‐2,3‐dihydro‐1‐oxo‐1H‐inden‐5‐yl)oxy]butanoic acid, an inhibitor of the volume‐sensitive organic osmolyte and anion channel. 4‐[(2‐Butyl‐6,7‐dichloro‐2‐cyclopentyl‐2,3‐dihydro‐1‐oxo‐1H‐inden‐5‐yl)oxy]butanoic acid also prevented receptor‐mediated changes in the efflux and influx of K⁺ under hypoosmotic conditions. The results suggest that muscarinic receptor activation can regulate both the volume‐dependent efflux and uptake of taurine and that these events may be functionally coupled.     

Gliotoxin induces caspase-dependent neurite degeneration and calpain-mediated general cytotoxicity in differentiated human neuroblastoma SH-SY5Y cells

In this study, a significant increase by 50% in intracellular free calcium concentration ([Ca(2+)](i)) was observed in differentiated human neuroblastoma (SH-SY5Y) cells after exposure to 0.25microM of the fungal metabolite gliotoxin for 72h. Further, the involvement of caspases and calpains was demonstrated to underlie the gliotoxin-induced cytotoxic and neurite degenerative effects. The caspase inhibitor Z-VAD-fmk almost completely reduced the neurite degeneration from 40% degeneration of neurites to 5% as compared to control. Inhibition of calpains with calpeptin significantly attenuated gliotoxin-induced cytotoxicity, determined as reduction in total cellular protein content, from 43% to 14% as compared to control cells. Western blot analyses of alphaII-spectrin breakdown fragments confirmed activity of the proteases, and that alphaII-spectrin was cleaved by caspases in gliotoxin-exposed cells. These results show that calpains and caspases have a role in the toxicity of gliotoxin in differentiated SH-SY5Y cells and that the process may be Ca(2+)-mediated.