Chi-Tso Chiu

P2Y2 receptor‐mediated proliferation of C6 glioma cells via activation of Ras/Raf/MEK/MAPK pathway

Extracellular purine and pyrimidine nucleotides have been implicated in the regulation of several cellular functions including mitogenesis. In this study, experiments were conducted to characterize the P2Y receptor on C6 glioma cells responsible for stimulating cell proliferation associated with mitogen‐activated protein kinase (MAPK) activation. UTP and ATP produced a similar effect on [3H]‐thymidine incorporation in a time‐ and concentration‐dependent manner, suggesting the involvement of P2Y2 receptor in mediating proliferation of C6 glioma cells. In response to UTP, both p42 and p44 MAPK were activated in a time‐ and concentration‐dependent manner using Western blot analysis with an anti‐phospho‐p42/p44 MAPK antibody. The phosphorylation reached maximal levels after 5 min and declining by 30 min. Pretreatment with pertussis toxin (PTX) did not change these responses to UTP. Both DNA synthesis and phosphorylation of MAPK in response to UTP were attenuated by tyrosine kinase inhibitors, genistein and herbimycin A, protein kinase C (PKC) inhibitors, staurosporine and GF109203X, and removal of Ca2+ by addition of BAPTA/AM plus EGTA. UTP‐induced [3H]‐thymidine incorporation and p42/p44 MAPK phosphorylation was completely inhibited by PD98059 (an inhibitor of MEK1/2). Furthermore, we showed that overexpression of dominant negative mutants of Ras (RasN17) and Raf (Raf‐301) completely suppressed MEK1/2 and p42/p44 MAPK activation induced by ATP and UTP. These results conclude that the mitogenic effect of UTP is mediated through a P2Y2 receptor that involves the activation of Ras/Raf/MEK/MAPK pathway. UTP‐mediated MAPK activation was modulated by Ca2+, PKC, and tyrosine kinase associated with cell proliferation in cultured C6 glioma cells.

Mitogenic effect of oxidized low-density lipoprotein on vascular smooth muscle cells mediated by activation of Ras/Raf/MEK/MAPK pathway

It has been demonstrated that oxidized low‐density lipoprotein (OX‐LDL) is a risk factor in atherosclerosis by stimulating vascular smooth muscle cell (VSMC) proliferation. However, the mechanisms of OX‐LDL‐induced cell proliferation are not completely understood. Therefore, we investigated the effect of OX‐LDL on cell proliferation associated with mitogen‐activated protein kinase (MAPK) activation in rat cultured VSMCs. Both native‐LDL (N‐LDL) and OX‐LDL induced a time‐ and concentration‐dependent incorporation of [3H]‐thymidine in VSMCs. OX‐LDL induced time‐ and concentration‐dependent phosphorylation of p42/p44 MAPK. Pretreatment of these cells with pertussis toxin or U73122 attenuated the OX‐LDL‐induced responses. Pretreatment with PMA for 24 h, preincubation with a PKC inhibitor staurosporine or the tyrosine kinase inhibitors, genistein and herbimycin A for 1 h, substantially reduced [3H]‐thymidine incorporation and p42/p44 MAPK phosphorylation induced by OX‐LDL. Removal of Ca2+ by BAPTA/AM or depletion of the internal Ca2+ pool by thapsigargin significantly inhibited OX‐LDL‐induced [3H]‐thymidine incorporation and p42/p44 MAPK phosphorylation. OX‐LDL‐induced [3H]‐thymidine incorporation and p42/p44 MAPK phosphorylation was inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 MAPK) in a concentration‐dependent manner. Overexpression of dominant negative mutants of Ras (H‐Ras‐15A) and Raf (Raf‐N4) significantly suppressed MEK1/2 and p42/p44 MAPK activation induced by OX‐LDL and PDGF‐BB, indicating that Ras and Raf may be required for activation of these kinases. These results suggest that the mitogenic effect of OX‐LDL is mediated through a PTX‐sensitive G protein‐coupled receptor that involves the activation of the Ras/Raf/MEK/MAPK pathway similar to that of PDGF‐BB in rat cultured VSMCs.

Tumour necrosis factor‐α‐ and interleukin‐1β‐stimulated cell proliferation through activation of mitogen‐activated protein kinase in canine tracheal smooth muscle cells

The elevated levels of inflammatory cytokines such as tumour necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) have been found in the fluid of airways in symptomatic asthmatics. These cytokines have been considered as mitogens to stimulate cell proliferation in tracheal smooth muscle cells (TSMCs). We therefore investigated the effects of TNF‐α and IL‐1β on cell proliferation and activation of p42/p44 mitogen‐activated protein kinase (MAPK) in these cells. TNF‐α and IL‐1β induced [3H]‐thymidine incorporation in a time‐ and concentration‐dependent manner. The maximal stimulation of [3H]‐thymidine incorporation induced by TNF‐α and IL‐1β was seen 12 h after incubation with these cytokines. In response to TNF‐α and IL‐1β, p42/p44 MAPK was activated with a concentration‐dependent manner in TSMCs. Pretreatment of TSMCs with pertussis toxin did not change DNA synthesis and phosphorylation of MAPK induced by TNF‐α and IL‐1β. These responses were attenuated by a tyrosine kinase inhibitor herbimycin, a phosphatidyl choline (PC)‐phospholipase C (PLC) inhibitor D609, a phosphatidyl inositide (PI)‐PLC inhibitor U73122, a protein kinase C inhibitor staurosporine, and removal of Ca2+ by addition of BAPTA/AM plus EGTA. TNF‐α‐ and IL‐1β‐induced [3H]‐thymidine incorporation and phosphorylation of p42/p44 MAPK was completely inhibited by PD98059 (an inhibitor of MEK1/2), indicating that activation of MEK1/2 was required for these responses. These results suggest that the mitogenic effects of TNF‐α and IL‐1β were mediated through the activation of MEK1/2 and p42/p44 MAPK pathway. TNF‐α‐ and IL‐1β‐mediated responses were modulated by PLC, Ca2+, PKC, and tyrosine kinase associated with cell proliferation in TSMCs.

Interleukin-1beta enhances bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilization in canine tracheal smooth-muscle cells: involvement of the Ras/Raf/mitogen-activated protein kinase (MAPK) kinase (MEK)/MAPK pathway.

Elevated levels of several cytokines including interleukin-1beta (IL-1beta) have been detected in airway fluid of asthmatic patients. Inhalation of IL-1beta induced a bronchial hyper-reactivity to contractile agonists. However, the implication of IL-1beta in the pathogenesis of bronchial hyper-reactivity is not completely understood. Therefore, we investigated the effect of IL-1beta on bradykinin (BK)-induced inositol phosphate [Ins(X)P] accumulation and Ca2+ mobilization, and up-regulation of BK receptor density in canine cultured tracheal smooth-muscle cells (TSMCs). Treatment of TSMCs with IL-1beta potentiated BK-induced Ins(X)P accumulation and Ca2+ mobilization. However, there was no effect on the Ins(X)P response induced by endothelin-1, 5-hydroxytryptamine or carbachol. Treatment with platelet-derived growth factor B-chain homodimer (PDGF-BB) also enhanced the BK-induced Ins(X)P response. These enhancements by IL-1beta and PDGF-BB might be due to an up-regulation of BK B(2) receptor density (B(max)), since [(3)H]BK binding to TSMCs was inhibited by the B(2)-selective agonist and antagonist, BK and Hoe 140, but not by B(1)-selective reagents. The enhancing effects of IL-1beta and PDGF-BB on Ins(X)P accumulation, Ca2+ mobilization and B(max) were attenuated by PD98059 [an inhibitor of activation of mitogen-activated protein kinase (MAPK) kinase, MEK] and cycloheximide (an inhibitor of protein synthesis), suggesting that IL-1beta may share a common signalling pathway with PDGF-BB via protein synthesis. Furthermore, overexpression of dominant negative mutants, H-Ras-15A and Raf-N4, significantly suppressed the up-regulation of BK receptors induced by IL-1beta, indicating that Ras and Raf may be required for activation of these kinases. These results suggest that the augmentation of BK-induced responses produced by IL-1beta might be, at least in part, mediated through activation of the Ras/Raf/MEK/MAPK pathway in TSMCs.

Mechanisms of thrombin-induced MAPK activation associated with cell proliferation in human cultured tracheal smooth muscle cells

The elevated level of thrombin has been detected in the airway fluids of asthmatic patients. However, the implication of thrombin in the pathogenesis of bronchial hyperreactivity was not completely understood. Therefore, in this study we investigated the effect of thrombin on cell proliferation and p42/p44 mitogen-activated protein kinase (MAPK) activation in human tracheal smooth muscle cells (TSMCs). Thrombin stimulated [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in a time- and concentration-dependent manner in TSMCs. Pretreatment of TSMCs with pertussis toxin (PTX) significantly inhibited [3H]thymidine incorporation and phosphorylation of MAPK induced by thrombin. These responses were attenuated by tyrosine kinase inhibitors genistein and herbimycin A, phosphatidyl inositide (PI)-phospholipase C (PLC) inhibitor U73122, protein kinase C (PKC) inhibitor GF109203X, removal of Ca(2+) by addition of BAPTA/AM plus EGTA, and PI 3-kinase inhibitors wortmannin and LY294002. In addition, thrombin-induced [3H]-thymidine incorporation and p42/p44 MAPK phosphorylation was completely inhibited by PD98059 (an inhibitor of MEK1/2), indicating that activation of MEK1/2 was required for these responses. Furthermore, overexpression of dominant negative mutants, RasN17 and Raf-301, significantly suppressed p42/p44 MAPK activation induced by thrombin and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. These results conclude that the mitogenic effect of thrombin was mediated through the activation of Ras/Raf/MEK/MAPK pathway. Thrombin-mediated MAPK activation was modulated by PI-PLC, Ca(2+), PKC, tyrosine kinase, and PI 3-kinase associated with cell proliferation in cultured human TSMCs.

Mechanisms underlying the inhibitory effect of propofol on the contraction of canine airway smooth muscle.

BACKGROUND Propofol has been shown to produce relaxation of preconstricted airway smooth muscle. Although the inhibition of calcium mobilization is supposed to be the major mechanism of action, the whole picture of the mechanisms is not completely clear. METHODS Contractile response was performed using canine tracheal rings. The effects of propofol on carbachol-induced mobilization of intracellular Ca2+ and phosphoinositide hydrolysis were measured using cultured canine tracheal smooth muscle cells by monitoring fura-2 signal and assessing the accumulation of [3H]-inositol phosphates. To detect the effect of propofol on muscarinic receptor density and affinity, [3H]N-methyl-scopolamine was used as a radioligand for receptor binding assay. RESULTS Pretreatment with propofol shifts the concentration-response curves of carbachol-induced smooth muscle contraction to the right in a concentration-dependent manner without changing the maximal response. Propofol not only decreased the release of Ca2+ from internal stores but also inhibited the calcium influx induced by carbachol. In addition, carbachol-induced inositol phosphate accumulation was attenuated by propofol; the inhibitory pattern was similar to the contractile response. Moreover, propofol did not alter the density of muscarinic receptors. The dissociation constant value was not altered by pretreatment with 100 microM propofol but was significantly increased by 300 microM (propofol, 952+/-229 pM; control, 588+/-98 pM; P<0.05). CONCLUSIONS Propofol attenuates the muscarinic receptor-mediated airway muscle contraction. The mechanism underlying these effects was attenuation of inositol phosphate generation and inhibition of Ca2+ mobilization through the inhibition of the receptor-coupled signal-transduction pathway.

Thrombin-stimulated cell proliferation mediated through activation of Ras/Raf/MEK/MAPK pathway in canine cultured tracheal smooth muscle cells.

The elevated level of thrombin has been detected in the airway fluids of asthmatic patients and shown to stimulate cell proliferation in tracheal smooth muscle cells (TSMCs). However, the implication of thrombin in the cell proliferation was not completely understood. In this study, thrombin stimulated [3H]thymidine incorporation and p42/p44 mitogen-activated protein kinase (MAPK) phosphorylation in a time- and concentration-dependent manner in TSMCs. Pretreatment of TSMCs with pertussis toxin (PTX) significantly inhibited [3H]thymidine incorporation and phosphorylation of MAPK induced by thrombin. These responses were attenuated by tyrosine kinase inhibitors genistein and herbimycin A, phosphatidyl inositide (PI)-phospholipase C (PLC) inhibitor U73122, protein kinase C inhibitor GF109203X, removal of Ca2+ by addition of BAPTA/AM plus EGTA, PI 3-kinase inhibitors wortmannin and LY294002, and inhibitor of MEK1/2 PD98059. Furthermore, overexpression of dominant negative mutants, H-Ras-15A and Raf-N4, significantly suppressed p42/p44 MAPK activation induced by thrombin and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. These results conclude that the mitogenic effect of thrombin was mediated through the activation of Ras/Raf/MEK/MAPK pathway. Thrombin-mediated MAPK activation was modulated by PI-PLC, Ca2+, PKC, tyrosine kinase, and PI 3-kinase associated with cell proliferation in canine cultured TSMCs.

Tumour necrosis factor-α enhances bradykinin-induced signal transduction via activation of Ras/Raf/MEK/MAPK in canine tracheal smooth muscle cells

Abstract Inhalation of tumour necrosis factor-α (TNF-α) induced a bronchial hyperreactivity to contractile agonists. However, the mechanisms of TNF-α involved in the pathogenesis of bronchial hyperreactivity were not completely understood. Therefore, we investigated the effect of TNF-α on bradykinin (BK)-induced inositol phosphate (IP) accumulation and Ca2+ mobilization, and up-regulation of BK receptor density in canine cultured tracheal smooth muscle cells (TSMCs). Pretreatment of TSMCs with TNF-α potentiated BK-induced IP accumulation and Ca2+ mobilization. However, there was no effect on the IP response induced by endothelin-1 (ET-1), 5-hydroxytryptamine (5-HT), and carbachol. Pretreatment with PDGF B-chain homodimer (PDGF-BB) also enhanced BK-induced IP response. These enhancements induced by TNF-α and PDGF-BB might be due to an increase in BK B2 receptor density (Bmax), since [3H]BK binding to TSMCs was inhibited by the B2 selective agonist and antagonist, BK and Hoe 140, but not by the B1 selective reagents. The enhancing effects of TNF-α and PDGF-BB were attenuated by PD98059 (an inhibitor of activation of MAPK kinase, MEK) and cycloheximide (an inhibitor of protein synthesis), suggesting that TNF-α may share a common signalling pathway with PDGF-BB via protein(s) synthesis in TSMCs. Furthermore, overexpression of dominant negative mutants, H-Ras-15A and Raf-N4, significantly suppressed p42/p44 mitogen-activated protein kinase (MAPK) activation induced by TNF-α and PDGF-BB and attenuated the effect of TNF-α on BK-induced IP response, indicating that Ras and Raf may be required for activation of these kinases. These results suggest that the augmentation of BK-induced responses produced by TNF-α might be, at least in part, mediated through activation of Ras/Raf/MEK/MAPK pathway in TSMCs.

Effects of CAMP elevating agents on carbachol-induced phosphoinositide hydrolysis and calcium mobilization in cultured canine tracheal smooth muscle cells

Abstract The effects of increases in intracellular adenosine 3′,5′-cyclic monophosphate (CAMP) on carbachol-induced generation of inositol phosphates (IPs) and increases in intracellular Ca 2+ ([Ca 2+ ] i ) were investigated in canine cultured tracheal smooth muscle cells (TSMCs). The CAMP elevating agents, cholera toxin (CTX) and forskolin, induced concentration- and time-dependent CAMP formation with half-maximal effects (-IogEC 50 ) at concentrations of 7.6 ± 1.3 g/ml and 4.8 ± 0.9 M, respectively. Forskolin caused a concentration-dependent inhibition of carbachol-induced increase in [Ca 2+ ] i with half-maximal inhibition (-IogEC 50 at 5.2 ± 0.7 M. Pretreatment of TSMCs with either CTX (10 μ/ml, 4 h), forskolin (10–100 μM, 30 min), or dibutyryl CAMP (1 mM, 30 min) inhibited carbachol-stimulated Ca 2+ mobilization and IPs accumulation. The inhibitory effects of these agents produced both depression of the maximal response and a shift to the right of the concentration-response curve of carbachol without changing the EC 50 values. After treatment with forskolin for 24 h, carbachol-induced IPs accumulation and Ca 2+ mobilization were close to those of control group. SQ-22536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine, 10 μM], an inhibitor of adenylate cyclase, and HA-1004 [N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride, 50 μM], an inhibitor of CAMP-dependent protein kinase (PKA), attenuated the ability of forskolin to inhibit carbachol-induced IPs accumulation. Moreover, the inactive analogue of forskolin, 1,9-dideoxy forskolin, did not inhibit these responses evoked by carbachol, suggesting that activation of CAMP/PKA was involved in these inhibitory effects of forskolin. The K D and B max values of the muscarinic receptor (mAChR) for [ 3 H]-N-methyl scopolamine binding were not significantly changed by forskolin treatment for 30 min and 24 h, suggesting that the inhibitory effect of forskolin is distal to the mAChR. The locus of this inhibition was further investigated by examining the effect of forskolin treatment on AIF 4 -stimulated IPs accumulation in canine TSMCs. The AIF 4 -induced response was inhibited by forskolin, supporting the notion that G protein(s) are directly activated by AIF4 and uncoupled to phospholipase C by forskolin treatment. We conclude that CAMP elevating agents inhibit carbachol-stimulated generation of IPs and Ca 2+ mobilization in canine cultured TSMCs. Since generation of IPs and increases in [Ca 2+ ]; are very early events in the activation of mAChRs, attenuation of these events by cAMP elevating agents might well contribute to the inhibitory effect of CAMP on tracheal smooth muscle function.

Inhibition of 5‐hydroxytryptamine‐induced phosphoinositide hydrolysis and Ca2+ mobilization in canine cultured tracheal smooth muscle cells by phorbol ester

Regulation of the increase in inositol‐1,4,5‐trisphosphate (IP3) production and intracellular Ca2+ concentration ([Ca2+]i by protein kinase C (PKC) was investigated in canine cultured tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by 5‐hydroxytryptamine (5‐HT) caused an initial transient [Ca2+]i peak followed by a sustained elevation of [Ca2+]i in a concentration‐dependent manner. Pretreatment of TSMCs with phorbol 12‐myristate 13‐acetate (PMA, 1 μM) for 30 min blocked the 5‐HT‐induced IP3 formation and Ca2+ mobilization. This inhibition was reduced after the cells had been incubated with PMA for 8 h, and within 48 h the 5‐HT‐induced Ca2+ mobilization reached the same extent as control cells. The concentration of PMA that gave half‐maximal inhibition of 5‐HT‐induced increase in [Ca2+]i was 4 nM. Pretreatment of TSMCs with staurosporine (1 μM) of GF109203X (0.1 μM), PKC inhibitors, inhibited the ability of PMA to attenuate 5‐HT‐induced responses, suggesting that the inhibitory effect of PMA was mediated through the activation of PKC. In parallel with the effect of PMA on 5‐HT‐induced IP3 formation and Ca2+ mobilization, the translocation and down‐regulation of PKC isozymes were determined by Western blot analysis in TSMCs. Analysis of cell extracts by Western blotting with antibodies against different PKC isozymes revealed that TSMCs expressed PKC‐α, βI, βII, δ, ε, θ and ζ. With PMA treatment of the cells for various times, translocation of PKC‐α, βI, βII, δ, ε and θ from the cytosol to the membrane was seen after 5 min, 30 min, 2 h, and 4 h treatment. However, 24 h treatment caused a partial down‐regulation of these PKC isozymes PKC‐ζ was not significantly translocated and down‐regulated at any of the times tested. In conclusion, these results suggest that activation of PKC may inhibit the receptor‐mediated phosphoinositide hydrolysis and consequently attenuate the [Ca2+]i increase or inhibit both responses independently. The translocation of PKC‐α, βI, βII, δ, ε, and θ induced by PMA caused an attenuation of 5‐HT‐stimulated IP3 accumulation and Ca2+ mobilization in TSMCs.