Histamine Antagonizes Serotonin and Growth Factor-induced Mitogen-activated Protein Kinase Activation in Bovine Tracheal Smooth Muscle Cells

Abstract

We examined the effects of the bronchoconstrictor agonists serotonin (5-hydroxytryptamine; 5-HT) and histamine on mitogen-activated protein (MAP) kinase activation in cultured bovine tracheal myocytes. Kinase renaturation assays demonstrated activation of the 42- and 44-kDa MAP kinases within 2 min of 5-HT exposure. MAP kinase activation was mimicked by alpha-methyl-5-HT and reduced by pretreatment with either phorbol 12,13-dibutyrate or forskolin, suggesting activation of the 5-HT2 receptor, protein kinase C, and Raf-1, respectively. Raf-1 activation was confirmed by measurement of Raf-1 activity, and the requirement of Raf-1 for 5-HT-induced MAP kinase activation was demonstrated by transient transfection of cells with a dominant-negative allele of Raf-1. Histamine pretreatment significantly inhibited 5-HT and insulin-derived growth factor-1-induced MAP kinase activation. Attenuation of MAP kinase activation was reversed by cimetidine, mimicked by forskolin, and accompanied by cAMP accumulation and inhibition of Raf-1, suggesting activation of the H2 receptor and cAMP-dependent protein kinase A. However, histamine treatment inhibited Raf-1 but not MAP kinase activation following treatment with either platelet-derived growth factor or epidermal growth factor, implying a Raf-1-independent MAP kinase activation pathway. In summary, our data suggest a model whereby 5-HT activates MAP kinase via a protein kinase C/Raf-1 pathway, and histamine attenuates MAP kinase activation by serotonin via activation of cAMP-dependent protein kinase A and inhibition of Raf-1.

Phenotypic diversity and molecular mechanisms of airway smooth muscle proliferation in asthma

“…Various diverse stimuli have now been demonstrated to increase the proliferation of airway smooth muscle cells in cell culture, and have been reviewed in detail elsewhere [13,47,48]. Broadly, the substances now implicated in mitogenesis of airway smooth muscle, in addition to the action of serum itself (discussed in [49]), include pro-inflammatory mediators (histamine [46], 5-hydroxytryptamine (5-HT) [50], phenylephrine [51], tachykinins [52], endothelin (ET)-1 [53±56] and leukotriene D 4 [57]), several inflammatory cell-and plasma-derived enzymes (a-thrombin [58], tryptase [59], b-hexosaminidase and b-glucuronidase [60]), polypeptide growth factors (platelet-derived growth factor (PDGF) [61], epidermal growth factor (EGF) [62], fibroblast growth factor-2 (FGF-2) [63] and insulin-like growth factors (IGFs) [64]) and thromboxanes [65]. Pro-inflammatory cytokines (interleukin-1b [66], interleukin-6 [67], and tumour necrosis factor-a [68]) also induce a proliferative response in airway smooth muscle, which is revealed only under conditions of cyclo-oxygenase inhibition [69], in which the production and action of inhibitory prostanoids such as prostaglandin E 2 is limited.…”

“…MEK-1 activates both p44 ERK1 and p42 ERK2 by direct phosphorylation of tyrosine and threonine residues. In bovine airway smooth muscle, ERK activation is induced by a wide variety of stimuli, including PDGF, EGF, IGF-1, 5-HT [50,74], bradykinin [75], a-thrombin [75,76], ET-1 [56,76] and hydrogen peroxide [77]. Several independent groups have reported that the duration of ERK activation is critical for transduction of the mitogenic stimulus.…”

“…Activated MEK-1 directly phosphorylates (on tyrosine and threonine residues) and activates both of the ERK isoforms p44 ERK1 and p42 ERK2 which translocate to the cell nucleus where they can phosphorylate and activate various transcription factors such as the constituent regulatory elements of activation protein 1 (AP-1), retinoblastoma protein (pRb) and cyclin D 1 . A Raf-1-independent pathway to ERK activation that is mediated by an unknown MEK-1 activator, proposed by HERSHENSON and coworkers [50,73], is shown in grey Pharmacological inhibitors are shown in italics. PDGF: platelet-derived growth factor; FGF-2: fibroblast growth factor-2; EGF: epidermal growth factor.…”

“…However, HERSHENSON et al [50] have provided evidence in bovine airway smooth muscle that ERK activation by polypeptide growth factors may occur independently of the Raf-1 intermediary. In support of this, activation of adenylyl cyclase activity by pretreatment with forskolin significantly reduced both EGF-and PDGF-stimulated activation of Raf-1, but had no effect on ERK activity [50,76].…”

“…However, HERSHENSON et al [50] have provided evidence in bovine airway smooth muscle that ERK activation by polypeptide growth factors may occur independently of the Raf-1 intermediary. In support of this, activation of adenylyl cyclase activity by pretreatment with forskolin significantly reduced both EGF-and PDGF-stimulated activation of Raf-1, but had no effect on ERK activity [50,76]. Since these [50,76] and other groups [80] have demonstrated that MEK-1 is necessary and sufficient for ERK activity, other pathways that are independent of Raf-1 must exist, representing alternative routes to MEK-1 activation.…”