Chuen Mao Yang

Lipopolysaccharide enhances bradykinin‐induced signal transduction via activation of Ras/Raf/MEK/MAPK in canine tracheal smooth muscle cells

Bacterial lipopolysaccharide (LPS) was found to induce inflammatory responses and to enhance bronchial hyperreactivity to several contractile agonists. However, the implication of LPS in the pathogenesis of bronchial hyperreactivity was not completely understood. Therefore, in this study, we investigated the effect of LPS on mitogen‐activated protein kinase (MAPK) activation associated with potentiation of bradykinin (BK)‐induced inositol phosphates (IPs) accumulation and Ca2+ mobilization in canine cultured tracheal smooth muscle cells (TSMCs). LPS stimulated phosphorylation of p42/p44 MAPK in a time‐ and concentration‐dependent manner using a Western blot analysis against a specific phosphorylated form of MAPK antibody. Maximal stimulation of the p42 and p44 MAPK isoforms occurred after 7 min‐incubation and the maximal effect was achieved with 100 μg ml−1 LPS. Pretreatment of TSMCs with LPS potentiated BK‐induced IPs accumulation and Ca2+ mobilization. However, there was no effect on the IPs response induced by endothelin‐1, 5‐hydroxytryptamine, and carbachol. In addition, pretreatment with PDGF‐BB enhanced BK‐induced IPs response. These enhancements by LPS and PDGF‐BB might be due to an increase in BK B2 receptor density (Bmax) in TSMCs, characterized by competitive inhibition of [3H]‐BK binding using B1 and B2 receptor‐selective reagents. The enhancing effects of LPS and PDGF‐BB were attenuated by PD98059, an inhibitor of MAPK kinase (MEK), suggesting that the effect of LPS may share a common signalling pathway with PDGF‐BB in TSMCs. Furthermore, overexpression of dominant negative mutants, H‐Ras‐15A and Raf‐N4, significantly suppressed p42/p44 MAPK activation induced by LPS and PDGF‐BB, 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 LPS might be, at least in part, mediated through activation of Ras/Raf/MEK/MAPK pathway in TSMCs.

Activation of mitogen-activated protein kinase by oxidized low-density lipoprotein in canine cultured vascular smooth muscle cells

Oxidized low-density lipoprotein (OX-LDL) contributes significantly to the development of atherosclerosis. However, the mechanisms of OX-LDL-induced vascular smooth muscle cell (VSMC) proliferation are not completely understood. Therefore, we investigated the effect of OX-LDL on cell proliferation associated with a specific pattern of mitogen-activated protein kinase (MAPK) by [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in canine cultured VSMCs. OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in a time- and concentration-dependent manner in VSMCs. Pretreatment of these cells with pertussis toxin (PTX) for 24 hours attenuated the OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation, indicating that these responses were mediated through a receptor coupled to a PTX-sensitive G protein. In cells pretreated with PMA for 24 h and with either the PKC inhibitor staurosporine or the tyrosine kinase inhibitor genistein for 1h, substantially reduced the [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in response to OX-LDL. Removal of Ca(2+) by addition of BAPTA/AM plus EGTA significantly inhibited OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation, indicating the requirement of Ca(2+) for these responses. OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation was completely inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 MAPK). Furthermore, we also 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 OX-LDL and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. Taken together, these results suggest that the mitogenic effect of OX-LDL is mediated through a PTX-sensitive G-protein-coupled receptor that involves the activation o Ras/Raf/MEK/MAPK pathway similar to those of PDGF-BB in canine cultured VSMCs.

Pharmacological and functional characterization of bradykinin receptors in rat cultured vascular smooth muscle cells

The pharmacological properties of bradykinin receptors were characterized in rat cultured vascular smooth muscle cells (VSMCs) using [3H]-bradykinin as a ligand. Analysis of binding isotherms gave an apparent equilibrium dissociation constant (K(D)) of 1.2 +/- 0.2 nM and a maximum receptor density (Bmax) of 47.3 +/- 4.4 fmol/mg protein. The specific binding of [3H]-bradykinin to VSMCs was inhibited by the B2 receptor-selective agonists (bradykinin and kallidin) and antagonists ([D-Arg0, Hyp3, Thi5, D-Tic7, Oic8]-bradykinin (Hoe 140) and [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin) with an order of potency as kallidin = bradykinin = Hoe 140 > [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin, but not by a B1 receptor-selective agonist (des-Arg9-bradykinin) and antagonist ([Leu8, des-Arg9]-bradykinin). Stimulation of VSMCs by bradykinin produced a concentration-dependent inositol phosphate (IP) accumulation, and initial transient peak of [Ca2+]i with half-maximal responses (pEC50) were 7.53 and 7.69, respectively. B2 receptor-selective antagonists (Hoe 140 and [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin) significantly antagonized the bradykinin-induced responses with pK(B) values of 8.3-8.7 and 7.2-7.9, respectively. Pretreatment of VSMCs with pertussis toxin (100 ng/ml, 24 h) did not alter the bradykinin-induced inositol phosphate accumulation and [Ca2+]i changes in VSMCs. Removal of external Ca2+ led to a significant attenuation of responses induced by bradykinin. Influx of external Ca2+ was required for the bradykinin-induced responses, since Ca2+-channel blockers, nifedipine, verapamil, and Ni2+, partially inhibited the bradykinin-induced IP accumulation and Ca2+ mobilization. These results demonstrate that bradykinin stimulates phosphoinositide hydrolysis and Ca2+ mobilization via a pertussis toxin-insensitive G-protein in rat VSMCs. Bradykinin B2 receptors may be predominantly mediating IP accumulation and subsequently induction of Ca2+ mobilization may function as the transducing mechanism for bradykinin-stimulated contraction of vascular smooth muscle.

Bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilization in canine cultured tracheal epithelial cells

Experiments were designed to differentiate the mechanisms and subtype of kinin receptors mediating the changes in intracellular Ca2+ concentration ([Ca2+]i) induced by bradykinin (BK) in canine cultured tracheal epithelial cells (TECs). BK and Lys‐BK caused an initial transient peak of [Ca2+]i in a concentration‐dependent manner, with half‐maximal stimulation (pEC50) obtained at 7.70 and 7.23, respectively. Kinin B2 antagonists Hoe 140 (10 nM) and [D‐Arg0, Hyp3, Thi5,8, D‐Phe7]‐BK (1 μM) had high affinity in antagonizing BK‐induced Ca2+ response with pKB values of 8.90 and 6.99, respectively. Pretreatment of TECs with pertussis toxin (100 ng ml−1) or cholera toxin (10 μg ml−1) for 24 h did not affect the BK‐induced IP accumulation and [Ca2+]i changes in TECs. Removal of Ca2+ by the addition of EGTA or application of Ca2+‐channel blockers, verapamil, diltiazem, and Ni2+, inhibited the BK‐induced IP accumulation and Ca2+ mobilization, indicating that Ca2+ influx was required for the BK‐induced responses. Addition of thapsigargin (TG), which is known to deplete intracellular Ca2+ stores, transiently increased [Ca2+]i in Ca2+‐free buffer and subsequently induced Ca2+ influx when Ca2+ was re‐added to this buffer. Pretreatment of TECs with TG completely abolished BK‐induced initial transient [Ca2+]i, but had slight effect on BK‐induced Ca2+ influx. Pretreatment of TECs with SKF96365 and U73122 inhibited the BK‐induced Ca2+ influx and Ca2+ release, consistent with the inhibition of receptor‐gated Ca2+ ‐channels and phospholipase C in TECs, respectively. These results demonstrate that BK directly stimulates kinin B2 receptors and subsequently phospholipase C‐mediated IP accumulation and Ca2+ mobilization via a pertussis toxin‐insensitive G protein in canine TECs. These results also suggest that BK‐induced Ca2+ influx into the cells is not due to depletion of these Ca2+ stores, as prior depletion of these pools by TG has no effect on the BK‐induced Ca2+ influx that is dependent on extracellular Ca2+ in TECs.

Purinoceptor-stimulated phosphoinositide hydrolysis in Madin-Darby canine kidney (MDCK) cells

Extracellular nucleotides, acting through P2-purinoceptors, have been implicated in the regulation of ion transport in epithelia, including Madin-Darby canine kidney (MDCK) cells. In this study, experiments were conducted to characterize the P2-purinoceptor subtype on MDCK cells responsible for stimulating inositol phosphate (IP) accumulation using a range of nucleotide analogues. In Ca2+- and Mg2+-free Krebs-Henseleit solution (KHS), ATP, UTP, and ATPγS caused an increase in IP accumulation as a function of concentration with comparable kinetics. The order of potency for the nucleotide analogues was UTP = ATPγS > ATP = 2-chloro ATP (Cl-ATP) >> α,β-methylene ATP (α,β-MeATP) = 2-methylthio ATP (2MeSATP). Selective agonists for P1-, P2X- and P2Y-purinoceptors, such as N6-cyclopentyl adenosine, AMP, α,β-MeATP, and 2MeSATP, had little effect. Stimulation of MDCK cells with maximally effective concentrations of ATP and UTP showed no additive effect and furthermore, ATP, UTP, and ATPγS induced cross-desensitization of the IP response, suggesting that ATP and UTP act upon a common nucleotide receptor, i.e. a P2U-purinoceptor. In Ca2+- and Mg2+-containing KHS, the concentration-response curves of ATP, UTP, and ATPγS were shifted to the right of those obtained in Ca2+- and Mg2+-free buffer, and asymptotic maxima were not reached, indicating that ATP4- and not MgATP2- or CaATP2- was the active agonist. Pretreatment of MDCK cells with pertussis toxin (PTX) inhibited ATP- and UTP-induced IP accumulation in a concentration-dependent fashion but did not completely abolish the IP accumulation, indicating that a PTX-sensitive G protein was partially involved in the IP response. In conclusion, ATP- and UTP-stimulated IP accumulation in MDCK cells appears to be mediated through the activation of P2U-purinoceptors coupled to a G protein that is partially sensitive to PTX. A form of nucleotide uncomplexed with divalent ions such as ATP4- seems to be the preferential agonist form for the purinoceptors on MDCK cells.

Pharmacological and functional characterization of bradykinin receptors in canine cultured tracheal epithelial cells

1 A direct [3H]‐bradykinin ([3H]‐BK) binding assay has been used to characterize the BK receptors in canine cultured tracheal epithelial cells (TECs). Based on receptor binding assay, TECs have specific, saturable, high‐affinity binding sites for [3H]‐BK. 2 The specific [3H]‐BK binding was time‐ and temperature‐dependent. Equilibrium of association of [3H]‐BK with the BK receptors was attained within 30 min at room temperature and 1 h at 4°C, respectively. 3 Analysis of binding isotherms yielded an apparent equilibrium dissociation constant (KD) of 1.5 ± 0.2 nM and a maximum receptor density (Bmax) of 53.2 ± 5.2 fmol mg−1 protein. The Hill coefficient for [3H]‐BK binding was 1.00 ± 0.02. The association (K1 and dissociation (K‐1) rate constants were (7.6 ± 1.1) × 106 M−1 min−1 and (9.2 ± 1.5) × 10 M−3 min−1, respectively. KD, calculated from the ratio of K‐1 and K1 was 1.2 ± 0.3 nM, a value close to that calculated from Scatchard plots of binding isotherms. 4 Neither a B1 receptor selective agonist (des‐Arg9‐BK, 0.1 nM‐10 μm) nor antagonist ([Leu8, des‐Arg9]‐BK, 0.1 nM‐10 μm) significantly inhibited [3H]‐BK binding to TECs, which excludes the presence of B1 receptors in canine TECs. 5 The specific binding of [3H]‐BK to canine TECs was inhibited by the B2 receptor selective antagonists ([D‐Arg0, Hyp3, Thi5, D‐Tic7, Oic8]‐BK (Hoe 140, 0.1 nM‐10 μm) and [D‐Arg0, Hyp3, Thi5,8, D‐Phe7]‐BK, 0.1 nM‐10 μm) and agonists (BK and kallidin, 0.1 nM‐10 μm) with a best fit by a one‐binding site model. The order of potency for the inhibition of [3H]‐BK binding was kallidin = BK = Hoe 140 > [D‐Arg0, Hyp3, Thi5,8, D‐Phe7]‐BK. 6 BK and kallidin significantly induced concentration‐dependent accumulation of IPs with a half‐maximal response (EC50) at 17.6 ± 3.5 and 26.6 ± 5.3 nM, respectively, while the B1‐selective agonist, des‐Arg9‐BK did not stimulate IPs accumulation and the B1‐selective antagonist [Leu8, des‐Arg9‐BK did not inhibit BK‐induced IPs accumulation. Two B2‐selective antagonists, Hoe 140 and [D‐Arg0, Hyp3, Thi5,8, D‐Phe7]‐BK, inhibited BK‐stimulated IPs accumulation with apparent pKB values of 8.8 ± 0.3 and 7.0 ± 0.3, respectively. 7 It is concluded that the pharmacological characteristics of the BK receptors in canine cultured TECs are primarily of the B2 receptor subtype which might regulate the function of tracheal epithelium through the activation of this receptor subtype coupling to PI hydrolysis.

Inhibition of Bradykinin-Induced Phosphoinositide Hydrolysis and Ca2+ Mobilisation by Phorbol Ester in Rat Cultured Vascular Smooth Muscle Cells

Regulation of the increase in inositol phosphate (IP) production and intracellular Ca2+ concentration ([Ca2+]i by protein kinase C (PKC) was investigated in cultured rat vascular smooth muscle cells (VSMCs). Pretreatment of VSMCs with phorbol 12-myristate 14-acetate (PMA, 1 microM) for 30 min almost abolished the BK-induced IP formation and Ca2+ mobilisation. This inhibition was reduced after incubating the cells with PMA for 4 h, and within 24 h the BK-induced responses were greater than those of control cells. The concentrations of PMA giving a half-maximal (pEC50) and maximal inhibition of BK induced an increase in [Ca2+]i, were 7.8 +/- 0.3 M and 1 microM, n = 8, respectively. Prior treatment of VSMCs with staurosporine (1 microM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Paralleling the effect of PMA on the BK-induced IP formation and Ca2+ mobilisation, the translocation and downregulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of the cells with PMA for various times, translocation of PKC-alpha, betaI, betaII, delta, epsilon, and zeta isozymes from the cytosol to the membrane were seen after 5 min, 30 min, 2 h, and 4 h of treatment. However, 24-h treatment caused a partial downregulation of these PKC isozymes in both fractions. Treatment of VSMCs with 1 microM PMA for either 1 or 24 h did not significantly change the K(D) and Bmax of the BK receptor for binding (control: K(D) = 1.7 +/- 0.2 nM; Bmax = 47.3 +/- 4.4 fmol/mg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. In conclusion, these results demonstrate that translocation of PKC-alpha, betaI, betaII, delta, epsilon, and zeta induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca2+ mobilisation in VSMCs.

Uncoupling of ATP-induced inositol phosphate formation and Ca2+ mobilization by phorbol ester in canine cultured tracheal epithelial cells

The regulation of the increase in inositol phosphates (IPs) production and intracellular Ca(2+) concentration ([Ca(2+)](i)) by protein kinase C (PKC) was investigated in canine cultured tracheal epithelial cells (TECs). Pretreatment of TECs with phorbol 12-myristate 13-acetate (PMA, 1 microM) for 30 min attenuated the ATP- and UTP-induced IPs formation and Ca(2+) mobilization. The concentrations of PMA that gave half-maximal (EC(50)) inhibition of ATP- and UTP-induced IPs accumulation and an increase in [Ca(2+)](i) were 5-10 and 4-12 nM, respectively. Prior treatment of TECs with staurosporine (1 microM), a PKC inhibitor, partially inhibited the ability of PMA to attenuate ATP- and UTP-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Furthermore, analysis of cell extracts by Western blotting with antibodies against different PKC isozymes revealed that TECs expressed PKC-alpha, -betaI, -betaII, -gamma, -delta, -epsilon, -theta, and -zeta. With PMA treatment of the cells for various times, translocation of PKC-alpha, -betaI, -betaII, -gamma, -delta, -epsilon, and -theta from the cytosol to the membrane was seen after 5- and 30-min and 2- and 4-h treatment. However, 6-h treatment caused a partial down-regulation of these PKC isozymes. PKC-zeta 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 phosphoinositide (PI) hydrolysis and consequently attenuate the [Ca(2+)](i) increase or inhibit independently both responses to ATP and UTP. The translocation of PKC-alpha, -betaI, -betaII, -delta, -epsilon, -gamma, and -theta induced by PMA caused an attenuation of ATP- and UTP-induced IPs accumulation and Ca(2+) mobilization in TECs.

Uncoupling of bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilization by phorbol ester in canine cultured tracheal epithelial cells.

1 Regulation of the increase in inositol phosphates (IPs) production and intracellular Ca2+ concentration ([Ca2+]i by protein kinase C (PKC) was investigated in canine cultured tracheal epithelial cells (TECs). Stimulation of TECs by bradykinin (BK) led to IPs formation and caused an initial transient [Ca2+]i peak in a concentration‐dependent manner. 2 Pretreatment of TECs with phorbol 12‐myristate 13‐acetate (PMA, 1 μM) for 30 min attenuated the BK‐induced IPs formation and Ca2+ mobilization. The maximal inhibition occurred after incubating the cells with PMA for 2 h. 3 The concentrations of PMA that gave half‐maximal (pEC50) inhibition of BK‐induced IPs accumulation and an increase in [Ca2+]i were 7.07 M and 7.11 M, respectively. Inactive phorbol ester, 4α‐phorbol 12,13‐didecanoate at 1 μM, did not inhibit these responses. Prior treatment of TECs with staurosporine (1 μM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK‐induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. 4 In parallel with the effect of PMA on the BK‐induced IPs formation and Ca2+ mobilization, the translocation and down‐regulation of PKC isozymes were determined. Analysis of cell extracts by Western blotting with antibodies against different PKC isozymes revealed that TECs expressed PKC‐α, βI, βII, γ, δ, ε, Θ and ζ. With PMA treatment of the cells for various times, translocation of PKC‐α, βI, βII, γ, δ, ε and Θ from cytosol to the membrane was seen after 5 min, 30 min, 2 h, and 4 h treatment. However, 6 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. 5 Treatment of TECs with 1 μM PMA for either 30 min or 6 h did not significantly change the KD and Bmax receptor for BK binding (control: KD=1.7±0.3 nM; Bmax=50.5±4.9 fmol/mg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK‐induced responses. 6 In conclusion, these results suggest that activation of PKC may inhibit the phosphoinositide hydrolysis and consequently attenuate the [Ca2+]i increase or inhibit independently both responses to BK. The translocation of pKC‐α, βI, βII, δ, ε, γ, and Θ induced by PMA caused an attenuation of BK‐induced IPs accumulation and Ca2+ mobilization in TECs.