间尼索地平对5-羟色胺诱导大鼠肺动脉平滑肌细胞增殖和迁移的影响及其机制

目的探讨间尼索地平(m-Nis)对5-羟色胺(5-HT)诱导的大鼠肺动脉平滑肌细胞(PASMCs)增殖和迁移的影响,并深入研究其作用机制。方法采用植块法培养大鼠PASMCs。实验分为6组:空白对照组、5-HT(1μmol·L-1)组,m-Nis(10-5、10-6、10-7、10-8mol.L-1)组。噻唑蓝(MTT)比色法、Transwell迁移小室法分别测定PASMCs的增殖和迁移,Western blot法检测大鼠PASMCs中增殖细胞核抗原(PCNA)的蛋白表达及细胞外信号调节激酶1和2(ERK1/2)的磷酸化水平,研究m-Nis对ERK1/2/MAPK信号通路的影响。结果不同浓度m-Nis明显抑制了5-HT诱导的大鼠PASMCs增殖(P<0.05或P<0.01)和迁移(P<0.01),并呈现一定的浓度依赖性;另外,Western blot结果显示m-Nis对5-HT诱导的大鼠PASMCs中PCNA的蛋白表达有不同程度的抑制作用(P<0.05或P<0.01),10-5,10-6,10-7mol.L-1m-Nis还明显抑制了5-HT诱导的大鼠PASMCs中ERK1/2磷酸化水平的升高,p-ERK1/2/ERK1/2比值均有不同程度地降低(P<0.05或P<0.01)。结论m-Nis对5-HT诱导的大鼠PASMCs增殖和迁移有明显的抑制作用,可能与其抑制PCNA蛋白表达及ERK1/2/MAPK信号通路有关。     

Sildenafil inhibits human pulmonary artery smooth muscle cell proliferation by decreasing capacitative Ca2+ entry

Ca(2+) is a pivotal signal in human pulmonary artery smooth muscle cells (PASMCs) proliferation. Capacitative Ca(2+) entry (CCE) via the store-operated channel (SOC), which encoded by the transient receptor potential (TRP) gene, is an important mechanism for regulating intracellular Ca(2+) concentration ([Ca(2+)](i)) in PASMCs. Sildenafil, a potent type 5 nucleotide-dependent phosphodiesterase (PDE) inhibitor, has been proposed as a therapeutic tool to treat or prevent pulmonary arterial hypertension (PAH); however, the mechanism of its antiproliferative effect on PASMCs remains unclear. This study was designed to investigate the possible antiproliferative mechanism of sildenafil on human PASMCs, namely, its effect on the Ca(2+)-signal pathway. Cultured normal PASMCs were treated with endothelin-1 (ET-1) or ET-1 plus sildenafil separately. Cell number and viability were determined with a hemocytometer or MTT assay. [Ca(2+)](i) was measured by loading PASMCs with fura 2-AM. Expression of the TRPC1 gene and protein was detected by RT-PCR and Western blot, respectively. The results show that sildenafil dose-dependently inhibited the proliferation of PASMCs, the enhancement of basal [Ca(2+)](i) level, increase of CCE, and upregulation of TRPC expression induced by ET-1. These results suggest that sildenafil potently inhibits ET-1-induced PASMCs proliferation and downregulation of CCE and TRPC expression may be responsible for its antiproliferative effect.     

间尼索地平对5-羟色胺诱导的大鼠肺动脉平滑肌细胞增殖中Ca2+/CaM/CaN通路的影响

探讨Ca²⁺/CaM/CaN信号通路在5-羟色胺 (5-HT) 诱导的大鼠肺动脉平滑肌细胞 (PASMCs) 增殖中的作用以及间尼索地平 (m-Nis) 对此的影响。采用细胞培养、噻唑蓝 (MTT) 比色检测、激光共聚焦显微镜及反转录聚合酶链反应 (RT-PCR) 等方法, 研究5-HT (1 μmol·L⁻¹) 对大鼠PASMCs细胞增殖的影响以及m-Nis对此的抑制作用, 并通过检测m-Nis对5-HT诱导的大鼠PASMCs增殖中Ca²⁺/CaM/CaN通路的影响深入探讨其作用机制。结果显示, 5-HT (1 μmol·L⁻¹) 可明显诱导大鼠PASMCs增殖 (P < 0.01), m-Nis对此有明显的抑制作用, 并呈现一定的浓度依赖性 (P < 0.05或P < 0.01); 另外, m-Nis还明显抑制了5-HT引起的[Ca²⁺]_i的升高 (P < 0.01)、CaM和CaN mRNA的表达以及CaN活性的升高 (P < 0.05或P < 0.01)。结果表明, Ca²⁺/CaM/CaN信号通路在5-HT诱导的大鼠PASMCs增殖中起重要作用, m-Nis抗5-HT诱导的增殖作用可能与抑制[Ca²⁺]_i增高从而阻断了Ca²⁺/CaM/CaN信号通路有关。     

5-Hydroxytryptamine induces cyclooxygenase-2 in rat vascular smooth muscle cells: Mechanisms involving Src, PKC and MAPK activation [corrected

Considering the importance of 5-hydroxytryptamine (5-HT) and cyclooxygenase (COX) products in vascular pathology, we investigated the effects of 5-HT on COX expression in rat vascular smooth muscle cells (VSMCs), and to provide mechanistic insights into these effects. VSMCs were enzymatically isolated from aortic media of Wistar rats. Incubation of VSMCs with 5-HT for 24h stimulated prostaglandin I(2) production, but this stimulation was completely suppressed by NS-398, a selective COX-2 inhibitor. 5-HT induced transient COX-2, but not COX-1, protein and mRNA expression in concentration- and time-dependent manners. This effect of 5-HT was completely inhibited by sarpogrelate, a 5-HT(2A) receptor antagonist. 5-HT-induced COX-2 expression was markedly blunted by Ca(2+) depletion; GF 109203X, a protein kinase C (PKC) inhibitor; PP2, an inhibitor of Src-family tyrosine kinase (Src); PD 98059, an inhibitor of extracellular signal-regulated kinase (ERK) activation; SB 203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK); and SP 600125, an inhibitor of c-Jun N-terminal kinase (JNK). 5-HT activated ERK and p38 MAPK, followed by JNK activation. PP2 inhibited these activations, while GF 109203X inhibited only JNK activation. Furthermore, PD 98059 inhibited JNK activation. These results suggest that 5-HT induces COX-2 expression in rat VSMCs, and that PKC, Src, and MAPK activation are each essential for the full expression of COX-2 pathways.     

Serotonin induces pulmonary artery smooth muscle cell migration

The chronic phase of pulmonary arterial hypertension (PAH) is associated with vascular remodeling, especially thickening of the smooth muscle layer of large pulmonary arteries and muscularization of small pulmonary vessels, which normally have no associated smooth muscle. Serotonin (5-hydroxytryptamine, 5-HT) has been shown to induce proliferation and hypertrophy of pulmonary artery smooth muscle cells (PASMC), and may be important for in vivo pulmonary vascular remodeling. Here, we show that 5-HT stimulates migration of pulmonary artery PASMC. Treatment with 5-HT for 16h increased migration of PASMC up to four-fold as monitored in a modified Boyden chamber assay. Increased migratory responses were associated with cellular morphological changes and reorganization of the actin cytoskeleton. 5-HT-induced alterations in morphology were previously shown in our laboratory to require cAMP [Lee SL, Fanburg BL. Serotonin produces a configurational change of cultured smooth muscle cells that is associated with elevation of intracellular cAMP. J Cell Phys 1992;150(2):396-405], and the 5-HT4 receptor was pharmacologically determined to be the primary activator of cAMP in bovine PASMC [Becker BN, Gettys TW, Middleton JP, Olsen CL, Albers FJ, Lee SL, et al. 8-Hydroxy-2-(di-n-propylamino)tetralin-responsive 5-hydroxytryptamine4-like receptor expressed in bovine pulmonary artery smooth muscle cells. Mol Pharmacol 1992;42(5):817-25]. We examined the role of the 5-HT4 receptor and cAMP in 5-HT-induced bovine PASMC migration. PASMC express 5-HT4 receptor mRNA, and a 5-HT4 receptor antagonist and a cAMP antagonist completely blocked 5-HT-induced cellular migration. Consistent with our previous report that a cAMP-dependent Cl(-) channel is required for 5-HT-induced morphological changes in PASMC, phenylanthranilic acid, a Cl(-) channel blocker, inhibited actin cytoskeletal reorganization and migration produced by 5-HT. We conclude that 5-HT stimulates PASMC migration and associated cytoskeletal reorganization through the 5-HT4 receptor and cAMP activation of a chloride channel.     

Effect of xanthine oxidase-catalyzed reactive oxygen species generation on secretagogue-evoked calcium mobilization in mouse pancreatic acinar cells.

In the present study we have employed fura-2 loaded isolated mouse pancreatic acinar cells to monitor the effect that xanthine oxidase (XOD)-catalyzed reactive oxygen species generation presents on Ca(2+) mobilization by the secretagogue cholecystokinin octapeptide (CCK-8). Our results show that perfusion of pancreatic acinar cells with CCK-8 at a physiological concentration (20 pM) induced low frequency oscillations in intracellular free calcium concentration ([Ca(2+)](i)) at a rate of 1 per minute; this oscillatory pattern was completely inhibited by the introduction in the perifusion medium of 20 mU/mL XOD to generate reactive oxygen species. In addition, perfusion of pancreatic acinar cells with 20 mU/mL XOD in the absence of extracellular calcium led to a transient increase in [Ca(2+)](i,) that blocked the initiation of the Ca(2+) signals in response to 20 pM CCK-8. Similarly, XOD was also able to block acetylcholine evoked Ca(2+) spikes. However, reactive oxygen species had no effect either on Ca(2+) extrusion or on re-uptake into intracellular stores, but CCK-8-evoked Ca(2+) entry was reduced by XOD. In conclusion, our results show that XOD-evoked reactive oxygen species generation leads to a reduction either of Ca(2+) mobilization, following stimulation of pancreatic acinar cells with the Ca(2+)-mobilizing agonists CCK-8 and acetylcholine, and Ca(2+) influx evoked by CCK-8 depletion of intracellular stores. The possible XOD inhibitory mechanism on Ca(2+) mobilization by agonists is discussed.     

The calcium-sensing receptor mediates hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells through MEK1/ERK1,2 and PI3K pathways

Activation of the calcium-sensing receptor (CaSR) leads to an increase of intracellular calcium concentration and alteration of cellular activities. High level of intracellular calcium is involved in hypoxia-induced proliferation of pulmonary arterial smooth muscle cells (PASMCs). However, whether the CaSR is expressed in PAMSCs and is related to the hypoxia-induced proliferation of PASMCs is unclear. In this study, the expression and distribution of CaSRs were detected by RT-PCR, western blotting and immunofluorescence; the intracellular concentration of free calcium ([Ca(2+) ](i) ) was determined by confocal laser scanning microscopy; cell proliferation was tested using an MTT and BrdU incorporation assay; cell cycle analysis was carried out using a flow cytometric assay; and the expression of proliferating cell nuclear antigen (PCNA), extracellular signal-regulated protein kinase 1,2 (ERK1,2) and AKT were analysed by western blotting. We observed that both CaSR mRNA and protein were expressed in rat PASMCs. Lowering of oxygen from 21% to 2.5% led to increased [Ca(2+) ](i) and CaSR expression. This condition of hypoxia also stimulated PASMCs proliferation accompanying with increased phosphorylation of ERK1,2 and AKT. GdCl(3) (an agonist of CaSR) or NPS2390 (an antagonist of CaSR) amplified or weakened the effect of hypoxia, respectively. PD98059 (a MEK1 inhibitor) or LY294002 (a PI3K inhibitors) decreased the up-regulation of PCNA expression and the increase of the cell proliferation index induced by hypoxia and GdCl(3) in PASMCs. Our results suggest that CaSR is expressed in rat PASMCs, and that CaSR activation through MEK1/ERK1,2 and PI3 kinase pathways is involved in hypoxia-induced proliferation of PASMCs.     

Calcium-mediated activation of c-Jun NH2-terminal kinase (JNK) and apoptosis in response to cadmium in murine macrophages.

Cadmium is a well-known carcinogenic and immunotoxic metal commonly found in cigarette smoke and industrial effluent. An altered intracellular calcium ([Ca(2+)](i)) level has been implicated in the pathophysiology of immune dysfunction. The present study was designed to determine the possible involvement of calcium (Ca(2+)) and mitogen-activated protein kinases (MAPKs) signaling pathways on cadmium-induced cell death in J774A.1 murine macrophage cells. Cadmium caused a low-amplitude [Ca(2+)](i) elevation at 20 microM and rapid and high-amplitude [Ca(2+)](i) elevation at 500 microM. Exposure to cadmium dose-dependently induced phosphorylation of c-Jun NH(2)-terminal kinase (JNK) and deactivated p38 MAPK. Use of the selective JNK inhibitor SP600125 suggested that activation of JNK is pro-apoptotic and pro-necrotic. Buffering of the calcium response with 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxy-methyl) ester (BAPTA-AM) and ethylene glycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) completely blocked cadmium-induced apoptotic response. The pretreatment of cells with BAPTA-AM and EGTA suppressed the cadmium-induced cell injury, including growth arrest, mitochondrial activity impairment, and necrosis, and it also recovered the cadmium-altered JNK and p38 MAPK activity. Chelating [Ca(2+)](i) also reversed cadmium-induced hydrogen peroxide generation, suggesting that production of reactive oxygen species (ROS) is related to [Ca(2+)](i). The present study showed that cadmium induces a [Ca(2+)](i)-ROS-JNK-caspase-3 signaling pathway leading to apoptosis. Furthermore, cadmium-induced [Ca(2+)](i) regulates phosphorylation/dephosphorylation of JNK and p38, and it modulates signal transduction pathways to proliferation, mitochondrial activity, and necrosis.     

Copper-induced stimulation of extracellular signal-regulated kinase in trout hepatocytes: the role of reactive oxygen species, Ca2+, and cell energetics and the impact of extracellular signal-regulated kinase signaling on apoptosis and necrosis.

The present study investigated if copper (Cu) exposure of trout hepatocytes, which stimulates formation of reactive oxygen species (ROS) and increases intracellular free Ca(2+) (Ca(2+)i), leads to an activation of extracellular signal-regulated kinase (ERK), the mechanisms underlying this activation, and the role of ERK signaling in cell death. Cu stimulated a time- and dose-dependent increase of phosphorylated extracellular signal-regulated kinase (pERK), and preventing the associated Ca(2+) influx or radical formation diminished or inhibited ERK activation, respectively. Furthermore, Cu enhanced caspase 3/7 activity and necrosis, and both effects were inhibited by treatments diminishing radical production and by chelating extracellular Ca(2+). In addition, ERK activity, and to a lesser extent caspase activity, was reduced by inhibiting mitochondrial ATP production, suggesting ATP dependence of the process. Inhibition of the ERK activator MEK, as well as of p38, significantly reduced caspase activation and necrosis, whereas c-Jun N-terminal kinase (JNK) inhibition diminished only caspase activity. Likewise, inhibition of MEK and p38, but not of JNK, prevented Cu-induced ROS production. In summary, we found that stimulation of ERK by Cu exposure of trout hepatocytes is dependent on radical formation and ATP, whereas Ca(2+) only modulates ERK activity. At the same time, activated ERK, as well as p38, contributes to enhanced ROS formation, whereas JNK did not. All three mitogen-activated protein kinases appear to promote apoptotic cell death upon Cu exposure, and ERK and p38 also stimulate necrosis.     

Inhibitory effect of calcitonin gene-related peptide on hypoxia-induced rat pulmonary artery smooth muscle cells proliferation: role of ERK1/2 and p27

Calcitonin gene-related peptide (CGRP) inhibits angiotensin II-induced proliferation of aortic smooth muscle cells via inactivation of extracellular signal-regulated protein kinase 1/2 (ERK1/2). ERK1/2 is necessary for the degradation or down-regulation of the cell cycle inhibitor p27, and is also crucial in mediating proliferation of pulmonary artery smooth muscle cells (PASMCs). Whether ERK1/2/p27 signal pathway is involved in CGRP-mediated pathogenesis of pulmonary hypertension and vascular remodeling remains unknown. Pulmonary hypertension was induced by hypoxia in rats, and capsaicin (50 mg/kg, s.c.) was used to deplete endogenous CGRP. Proliferation of cultured PASMCs was determined by BrdU incorporation method and flow cytometry. The expression/level of CGRP, p27, ERK1/2, c-fos and c-myc was analyzed by radioimmunoassay, immunohistochemistry, real-time PCR or Western blot. Sensory CGRP depletion by capsaicin exacerbated hypoxia-induced pulmonary hypertension in rats, as shown by an increase in right ventricle systolic pressure, mean pulmonary artery pressure and vascular hypertrophy, accompanied with decreased p27 expression and increased expression of phosphorylated ERK1/2, c-fos and c-myc. Exogenous application of CGRP significantly inhibited hypoxia-induced proliferation of PASMCs concomitantly with increased p27 expression and decreased expression of phosphorylated ERK1/2, c-fos and c-myc. These effects of CGRP were abolished in the presence of CGRP(8-37). Knockdown of p27 also reversed the inhibitory effect of CGRP on proliferation of PASMCs and expression of c-fos and c-myc, but not on ERK1/2 phosphorylation. These results suggest that CGRP inhibits hypoxia-induced proliferation of PASMCs via ERK1/2/p27/c-fos/c-myc pathway. Down-regulation of CGRP may contribute to remodeling of pulmonary arteries in hypoxia-induced pulmonary hypertension.     

5-Hydroxytryptamine-induced proliferation of pulmonary artery smooth muscle cells are extracellular signal-regulated kinase pathway dependent

AIM: To investigate the effect of 5-hydroxytryptamine transporter (5-HTT) inhibitor fluoxetine and antisense oligodeoxynucleotide (ODN) to extracelluar signal-regulated kinases (ERKs) on pulmonary arterial smooth muscle cells (PASMCs) proliferation induced by 5-HT. METHODS: Liposomal transfection was used to introduce ODNs to ERK1/2 into cultured rat PASMCs and the transfection efficiency was measured by observing the uptake of the fluorecein isothiocynate (FITC)-labeled antisense ODN in PASMCs. The effects of 5-HTT selective inhibitor fluoxetine and ODNs on the proliferation of PASMCs were evaluated by cell number counting and cell cycle analysis, and measured by microculture tetrazolium (MTT) assay and flow cytometry (FCM), respectively. RESULTS: Liposomes mediated the transfection of ODNs into PASMCs with high efficiency. MTT assay showed fluoxetine (10 micromol/L, 1 micromol/L, and 100 nmol/L) concentration dependently inhibited the proliferation of PASMCs induced by 5-HT (1 micromol/L) in vitro. The proliferation rate of PASMCs by 5-HT was significantly inhibited by pretreatment with ERK1/2 antisense ODN (0.2 micromol/L) from 251%+/-18% to 86%+/-5% (P<0.01). Flow cytometric analysis of cell cycle distribution showed that the increase of 5-HT induced S phase fraction (SPF) and proliferation index (PI) were significantly inhibited by fluoxetine (1 micromol/L) or antisense ODN with SPF from 36%+/-4% to 26%+/-3% and 24%+/-4%, and PI from 34%+/-2% to 29%+/-2% and 24%+/-2%, respectively. CONCLUSION: 5-HTT mediates the mitogenic effect of 5-HT on PASMCs and the proliferation of PASMCs induced by 5-HT is dependent on ERKs signal pathway.     

KMUP-1 inhibits pulmonary artery proliferation by targeting serotonin receptors/transporter and NO synthase,inactivating RhoA and suppressing AKT/ERK phosphorylation

KMUP-1 inhibits monocrotaline (MCT)-induced pulmonary artery (PA) proliferation by targeting serotonin (5-HT) receptors, inactivating RhoA and reducing phosphorylation of AKT/ERK. In MCT-treated rats, KMUP-1 f (5 mg/kg p.o.; 1 mg/kg i.p. × 21 days) decreased proliferation (PCNA-positive) cells and 5-HTT-expression in lung and 5-HT levels in plasma. In isolated PA, KMUP-1 and simvastatin (0.1–100 μM) inhibited 5-HT (10 μM)-induced PA constriction. l-NAME-pretreatment reduced KMUP-1-induced relaxation. In pulmonary arterial smooth muscle cells (PASMCs), KMUP-1 (1–100 μM) and simvastatin (10 μM) inhibited 5-HT-induced cell migration and proliferation and KMUP-1 (1–100 μM) inhibited 5-HT-induced Ca²⁺ influx. Similar to Y27632, KMUP-1 (1–100 μM) inhibited 5-HT-induced RhoA/ROCK expression, while KMUP-1, Y27632 and simvastatin at 10 μM inhibited 5-HT-induced 5-HTT expression and KMUP-1 inhibited 5-HT-induced phosphorylation of AKT and ERK1/2 in PASMCs. In human pulmonary arterial endothelial cell (HPAEC), KMUP-1 (1–100 μM) increased the expression of eNOS and 5-HT_2B and also at 10 μM augmented eNOS expression and production of nitric oxide (NO) in 5-HT-treated HPAEC. In radioligand binding, the IC₅₀/K_i values of KMUP-1 for 5-HT_2A, 5-HT_2B and 5-HT_2C receptors were 0.34/0.0971, 0.04/0.0254, and 0.408/0.214 μM respectively. In conclusion, KMUP-1 inhibits MCT-induced PA proliferation by binding to 5-HT_2A, 5-HT_2B and 5-HT_2C receptors, increasing endothelial eNOS/5-HT_2B receptor expression and NO release and inhibiting 5-HTT/RhoA/ROCK expression and AKT/ERK phosphorylation. KMUP-1 is suggested to be useful in the treatment of 5-HT-induced pulmonary artery proliferation.     

Introduction of the 5-HT3B subunit alters the functional properties of 5-HT3 receptors native to neuroblastoma cells

The identification of a second 5-HT(3) (5-HT(3B)) subunit provides an explanation for 5-HT(3) receptor heterogeneity. We investigated whether introduction of recombinant 5-HT(3B) subunits would alter the functional properties of mouse neuroblastoma 5-HT(3) receptors. RT-PCR analysis revealed that NB41A3 cells contain mRNAs encoding 5-HT(3A) and 5-HT(3B) subunits. 5-HT increased intracellular Ca(2+) concentration ([Ca(2+)](i)) and caused the concentration-dependent activation of inward currents recorded at -60 mV. Both actions of 5-HT were antagonized by ondansetron. The 5-HT concentration-response relationship of NB41A3 cells was indistinguishable from that of the related NG108-15 cell line. The selective 5-HT(3)-receptor agonist mCPBG also elevated [Ca(2+)](i) and activated inward currents. 2-M-5HT was less efficacious than 5-HT as an activator of 5-HT(3) receptors in NB41A3 cells and did not significantly increase [Ca(2+)](i). The 5-HT induced increase in [Ca(2+)](i) did not involve caffeine- or thapsigargin-sensitive intracellular Ca(2+) stores. The introduction of the 5-HT(3B) subunit by transient transfection of NB41A3 cells caused 5-HT to become less potent as an activator of 5-HT(3) receptors and altered the kinetics of 5-HT activated currents so that they resembled currents mediated by 5-HT(3AB) receptors. The 5-HT(3B) subunit also abolished the 5-HT induced [Ca(2+)](i) increase seen in untransfected NB41A3 cells. These data are consistent with the hypothesis that NB41A3 cells predominantly express homomeric 5-HT(3A) receptors that become heteromeric 5-HT(3AB) receptors upon introduction of the recombinant 5-HT(3B) subunit.     

Reciprocal effects of glucose on the process of cell death induced by calcium ionophore or H2O2 in rat lymphocytes

We have examined the effects of glucose at high concentrations on the process of cell death induced by excessive increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) or oxidative stress in rat lymphocytes. The cell death elicited by the excessive increase in [Ca(2+)](i) seemed to be induced by an activation of Ca(2+)-dependent K(+) channels because the inhibitors for Ca(2+)-dependent K(+) channels attenuated the decrease in cell viability. Glucose at 30-50mM augmented the decrease in cell viability by the excessive increase in [Ca(2+)](i). It was not specific for glucose because it was the case for sucrose or NaCl, suggesting an involvement of increased osmolarity in adverse action of glucose. On the contrary, glucose protected the cells suffering from oxidative stress induced by H(2)O(2), one of reactive oxygen species. It was also the case for fructose or sucrose, but not for NaCl. The process of cell death induced by H(2)O(2) started, being independent from the presence of glucose. Glucose delayed the process of cell death induced by H(2)O(2). Sucrose and fructose also protected the cells against oxidative stress. The reactivity of sucrose to reactive oxygen species is lower than those of glucose and fructose. The order in the reactivity cannot explain the protective action of glucose. Glucose at high concentrations exerts reciprocal actions on the process of cell death induced by the oxidative stress and excessive increase in [Ca(2+)](i).     

Insulin induces internalization of the 5-HT2A receptor expressed in HEK293 cells

To visualize the 5-hydroxytryptamine2A (5-HT2A) receptor, we developed a 5-HT2A receptor fused with yellow fluorescent protein (5-HT2A-YFP) and expressed this receptor in HEK293 cells. In 5-HT2A-YFP-expressing cells, but not in YFP-expressing or non-expressing cells, 5-HT induced a transient increase in the intracellular Ca(2+) concentration (Ca(2+) transient) in the Fluo 3 assay, suggesting that 5-HT2A-YFP possesses a function similar to the wild-type 5-HT2A receptor. Interestingly, not only 5-HT but also insulin induced the internalization of 5-HT2A-YFP. Insulin also inhibited the 5-HT-induced Ca(2+) transient. Genistein, an inhibitor of tyrosine kinase, blocked these insulin effects. Our results provide the first evidence that insulin receptor signaling via tyrosine kinase activation induces internalization of the plasma membrane 5-HT2A receptor, and demonstrate crosstalk between the 5-HT2A receptor and the insulin receptor.     

Novel effect of Y-24180, a presumed specific platelet-activating factor receptor antagonist, on Ca2+ levels and growth of human osteosarcoma cells.

In human osteosarcoma MG63 cells, the effect of Y-24180, a presumed specific platelet-activating factor (PAF) receptor antagonist, on intracellular Ca(2+) concentration ([Ca(2+)](i)) and proliferation was measured by using fura-2 and tetrazolium as fluorescent dyes, respectively. Y-24180 (1-5 microM) caused a rapid and sustained [Ca(2+)](i) rise in a concentration-dependent manner. The [Ca(2+)](i) rise was inhibited by 35% by dihydropyridines or removal of extracellular Ca(2+), but was not altered by verapamil and diltiazem. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca(2+)](i) rise, after which 5 microM Y-24180 failed to increase [Ca(2+)](i); conversely, depletion of Ca(2+) stores with 5 microM Y-24180 abolished thapsigargin-induced [Ca(2+)](i) rise. U73122, an inhibitor of phoispholipase C, inhibited histamine-induced, but not 5 microM Y-24180-induced [Ca(2+)](i) rise. Overnight treatment with 0.1-5 microM Y-24180 inhibited cell proliferation in a concentration-dependent manner. Together, these findings suggest that Y-24180 acts as a potent and cytotoxic Ca(2+) mobilizer in human osteosarcoma cells, by inducing both extracellular Ca(2+) influx and intracellular Ca(2+) release. Alterations in cytosolic Ca(2+) regulation may lead to interferences of various cellular functions; thus, attention should be exercised in using Y-24180 as a selective PAF receptor antagonist.     

Stimulation of 5-HT1A receptor with 8-OH-DPAT inhibits hydrogen peroxide-induced neurotoxicity in cultured rat cortical cells.

We investigated the effect of 8-hydroxy-2-(N,N-dipropylamino)tetralin (8-OH-DPAT), a specific 5-HT(1A) receptor agonist, on H(2)O(2)-induced neuronal cell death in cultured rat cortical cells. H(2)O(2) produced a concentration-dependent reduction of cell viability, which was significantly reduced by (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine (MK-801), an N-methyl-d-aspartate (NMDA) receptor antagonist. Pretreatment of 8-OH-DPAT over the concentration range of 1-100 microM significantly inhibited the H(2)O(2) (100 microM)-induced neuronal cell death as assessed by a MTT assay and the number of apoptotic nuclei, evidenced by Hoechst 33342 staining. The protective effect of 8-OH-DPAT (100 microM) was completely blocked by the simultaneous treatment of 1-(2-methoxyphenyl)-4-[4-(2-phthalimideo)butyl]piperazine (NAN-190, 10muM), a selective 5-HT(1A) receptor antagonist, but not in the presence of the dopamine receptor blocker spiperone (10 microM), indicating that the protective effect of 8-OH-DPAT was mediated via 5-HT(1A) receptors. In addition, 8-OH-DPAT inhibited the H(2)O(2)-induced elevation of glutamate release into the medium and cytosolic Ca(2+) concentration ([Ca(2+)](c)), generation of reactive oxygen species (ROS), and caspase-3 activity. These results suggest that the activation of 5-HT(1A) receptor with 8-OH-DPAT may ameliorate an oxydative stress-induced apoptosis of neuronal cell by interfering with the increase of [Ca(2+)](c), and then by inhibiting glutamate release, generation of ROS and caspase activity.     

Serotonin-induced proliferation of pulmonary arterysmooth muscle cells is serotonin transporter and ERK pathway dependent

AIM: To investigate the effect of serotonin transporter (5-HTT)inhibitor fluoxetine and antisense oligodeoxynucleotide (ODN)to extracelluar signal-regulated kinases (ERKs) on pulmonary arterial smooth muscle cells (PASMCs) proliferation induced by 5-HT. METHODS: Liposomal transfection was used to introduce ODNs to ERK1/2 into cultured rat PASMCs and the transfection efficiency was measured by observing the uptake of the