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ABSTRACT: Inhalation of tumour necrosis factor-alpha (TNF-alpha) induced a bronchial hyperreactivity to contractile agonists. However, the mechanisms of TNF-alpha involved in the pathogenesis of bronchial hyperreactivity were not completely understood. Therefore, we investigated the effect of TNF-alpha on bradykinin (BK)-induced inositol phosphate (IP) accumulation and Ca(2+) mobilization, and up-regulation of BK receptor density in canine cultured tracheal smooth muscle cells (TSMCs). Pretreatment of TSMCs with TNF-alpha potentiated BK-induced IP accumulation and Ca(2+) 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-alpha and PDGF-BB might be due to an increase in BK B(2) receptor density (B(max)), since [3H]BK binding to TSMCs was inhibited by the B(2) selective agonist and antagonist, BK and Hoe 140, but not by the B(1) selective reagents. The enhancing effects of TNF-alpha 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-alpha 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-alpha and PDGF-BB and attenuated the effect of TNF-alpha 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-alpha might be, at least in part, mediated through activation of Ras/Raf/MEK/MAPK pathway in TSMCs.
Cellular Signalling 10/2001; 13(9):633-43. · 4.06 Impact Factor
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ABSTRACT: 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.
Biochemical Journal 04/2001; 354(Pt 2):439-46. · 4.90 Impact Factor
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ABSTRACT: 1. The effects of increase in intracellular adenosine 3':5'-cyclic monophosphate (cAMP) on endothelin-1 (ET-1)-induced generation of inositol phosphates (IPs) and increase in intracellular Ca2+ ([Ca2+]i) were investigated in canine cultured tracheal smooth muscle cells (TSMCs). 2. Pretreatment of TSMCs with either cholera toxin (CTX; 10 microg ml(-1), 4 h), forskolin (10 microM, 30 min), or dibutyryl cAMP (1 mM, 30 min) inhibited ET-1-stimulated Ca2+ mobilization (by 23 +/- 5%, n = 8) and IPs accumulation (by 32 +/- 6%, n = 4). While after treatment with forskolin for 24 h, the cells retained the ability to respond to ET-1-induced Ca2+ mobilization to the same extent as the control group. 3. Forskolin (1-100 microM) inhibited the ET-1-induced increase in [Ca2+]i, but the lower concentrations had little effect on this response. 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 ET-1 without changing the -logEC50 values. 4. The water-soluble forskolin analogue L-858051, 7-deacetyl-7beta-(gamma-N-methylpiperazino)-butyryl forskolin, significantly inhibited ET-1-stimulated IPs accumulation. In contrast, the addition of 1,9-dideoxy forskolin, an inactive analogue of forskolin, had little effect on stimulated responses. Moreover, SQ-22536, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine, an inhibitor of adenylate cyclase, and both H-89, N-(2-aminoethyl)-5-isoquinolinesulfonamide, and HA-1004, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide, inhibitors of cAMP-dependent protein kinase (PKA), attenuated the ability of forskolin to inhibit ET-1-induced IPs accumulation. These results suggest that activation of cAMP/PKA was involved in these inhibitory effects of forskolin. 5. The locus of this inhibition of forskolin treatment on AlF4(-)-stimulated IPs accumulation was investigated in canine TSMCs. The AlF4(-)-induced IPs accumulation was inhibited by forskolin, supporting that G protein(s) are directly activated by AlF4- and uncoupled to phospholipase C by forskolin treatment. 6. We conclude that cAMP elevating agents inhibit ET-1-stimulated generation of IPs and Ca2+ mobilization in canine cultured TSMCs. Since generation of IPs and increases in [Ca2+]i are very early events in the activation of ET-1 receptors, attenuation of these events by cAMP elevating agents might well contribute to the inhibitory effect of cAMP on tracheal smooth muscle function.
Journal of Autonomic Pharmacology 09/1998; 18(4):213-21.
