Up-Regulation of Airway Smooth Muscle Histamine H1 Receptor mRNA, Protein, and Function by b2-Adrenoceptor Activation

Histamine, released from activated mast cells, causes bronchoconstriction mediated by H1 receptors, whereas b2-agonists are widely used for the relief of bronchoconstriction. In this study, we examined the effects of the b2-adrenoceptor agonist, fenoterol, on the expression of H1 receptors at the mRNA and protein levels, and functional responses. Incubation of bovine tracheal smooth muscle with fenoterol (10 M) for 2 h increased H1 receptor mRNA (maximum ;190%). The number of H1 receptors was increased after 12 and 18 h without any change in binding affinity. In the contraction experiments, the concentration-response curves for histamine-induced contraction were shifted significantly to the left after 18-h exposure to fenoterol, consistent with the increase in receptor number. The fenoterol-induced increase in H1 receptor mRNA was concentration-dependent and was abolished by propranolol and ICI 118551, but not by CGP 20712A, indicating that fenoterol acts via b2-adrenoceptors. These effects were mimicked by other cAMP-elevating agents forskolin and prostaglandin E2, and by the stable cAMP analog 8-bromo-cAMP. Cycloheximide alone produced superinduction of H1 receptor mRNA and augmented the fenoterol-induced increase in H1 receptor mRNA. Fenoterol increased both the stability and the transcription rate of H1 receptor mRNA. Pretreatment with dexamethasone did not prevent fenoterol-induced up-regulation of H1 receptor mRNA. Thus, fenoterol increases the expression of airway smooth muscle H1 receptors via activation of the cAMP system through increased gene transcription and mRNA stability. This mechanism may be involved in the adverse responses encountered with the clinical use of short-acting b2-agonists. Histamine is an important mediator of airway smooth muscle contraction, which is mediated via H1 receptors (Chand and Sofia, 1995). H1 receptors have now been cloned from several species (Yamashita et al., 1991; Fujimoto et al., 1993; Horio et al., 1993; Fukui et al., 1994; Inoue et al., 1996). Activation of histamine H1 receptors leads to the formation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and diacylglycerol from phosphatidylinositol 4,5-bisphosphate hydrolysis (Leurs et al., 1995). Ins(1,4,5)P3 binds to a specific receptor on endoplasmic/sarcoplasmic reticulum, which leads to the release of Ca from intracellular stores, resulting in an initial transient contractile response, whereas diacylglycerol activates protein kinase C (PKC), which is believed to be responsible for the sustained phase of the smooth muscle contraction (Rasmussen et al., 1987). H1 receptors are expressed in airway smooth muscle and may be regulated at the transcriptional level. PKC phosphorylation sites have recently been identified in the third intracellular loop of the histamine H1 receptors. We have shown that phorbol esters, which activate PKC, result in a marked down-regulation of H1 receptors due to uncoupling of the receptor and a reduction in transcription (Pype et al., 1998). b2-Adrenoceptor agonists have been widely used as bronchodilators for the relief of the symptoms of asthma, because they act as functional antagonists that counteract multiple bronchoconstrictors. b2-Adrenoceptors are localized to airway smooth muscle of all airways from trachea to terminal bronchioles (Barnes, 1995). However, the use of high doses of b2-agonists has been linked to the development of bronchial hyper-responsiveness (Taylor et al., 1993; Wahedna et al., 1993) and an increase in asthma exacerbations and mortality (Suissa et al., 1994; Barrett and Strom, 1995). Sustained use of b2-adrenoceptor agonists is associated with loss of protection to bronchoconstrictors, such as adenosine, allergen, This work was funded by a GlaxoWellcome research grant, an Imperial College Initiative Award, UK, and the Netherlands Asthma Foundation. 1 Present address: Department of Allergy, National Children’s Research Center 3-35-31, Taishido, Setagaya-ku, Tokyo, 154-8509, Japan. ABBREVIATIONS: Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; PKA, protein kinase A; PKC, protein kinase C; PGE2, prostaglandin E2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DMEM, Dulbecco’s modified Eagle’s medium; SSC, standard sodium citrate. 0026-895X/00/050857-08$3.00/0 MOLECULAR PHARMACOLOGY Copyright © 2000 The American Society for Pharmacology and Experimental Therapeutics MOL 57:857–864, 2000 /13116/821224 857 at A PE T Jornals on O cber 8, 2017 m oharm .aspeurnals.org D ow nladed from methacholine, and histamine (O’Connor et al., 1992; Cockcroft and Swystun, 1996). b2-Adrenoceptors are coupled via Gs protein to the membrane-bound enzyme adenylyl cyclase to increase production of cAMP. The formation of cAMP leads to the characteristic cellular response via the activation of a specific protein kinase, protein kinase A (PKA), by dissociating a regulatory (inhibitory) subunit. PKA then phosphorylates serine and threonine residues on specific proteins, such as regulatory proteins within the cell, which results in relaxation of airway smooth muscle. Evidence has emerged to support the concept of regulatory “cross talk” between the b2-adrenoceptor/cAMP signaling pathway and the phospholipase C-coupled/Ins(1,4,5)P3 transmembrane signaling mechanism (Madison and Brown, 1988; Schramm et al. 1995; Zaagsma et al., 1997). This study was thus undertaken to investigate the effects of the b2-adrenoceptor agonist fenoterol on the expression of the histamine H1 receptor gene and functional receptors in bovine tracheal smooth muscle. We have previously shown that this tissue expresses H1 receptors with no detection of H2 receptors (Pype et al., 1998). Because glucocorticoids counteract the effect of b2-adrenoceptor agonists, the effects of steroid in the presence of fenoterol on the expression of the histamine H1 receptor gene were also investigated. Experimental Procedures Materials. [a-P]dCTP (3000 Ci/mmol) was purchased from Amersham International (Amersham, UK). [P]UTP (800 Ci/mmol) and [pyridimyl-5-H]mepyramine (pyrilamine; 20 Ci/mmol) were from DuPont/NEN (Boston, MA). Fenoterol hydrobromide, (2)-isoproterenol hydrochloride, histamine dihydrochloride, and triprolidine were from Sigma. Methacholine chloride and HEPES-buffered Dulbecco’s modified Eagle’s medium (DMEM) were either from Sigma or ICN Biomedicals (Costa Mesa, CA). Scintillant Filtron-X was from National Diagnostics (Hull, UK). Tissue culture supplies were from Life Technologies (Paisley, UK). All other chemicals were of reagent grade and from standard sources. Tissue Preparation and Incubation. Fresh bovine tracheae were obtained from the abattoir and placed in oxygenated KrebsHenseleit solution [(in millimolar): NaCl 118, KCl 5.9, MgSO4 1.2, CaCl2 2.5, NaH2PO4 1.2, NaHCO3 25.5, and glucose 5.6] at room temperature. The tracheal smooth muscle layer was dissected after stripping off epithelium; mucosa and connective tissue and smooth muscle pieces were prepared in Krebs-Henseleit solution at room temperature. After washing, incubation was performed in HEPESbuffered DMEM supplemented with 10% fetal calf serum, 2 mM L-glutamate, 100 I.U./ml penicillin, 100 mg/ml streptomycin, and 0.25 mg/ml amphotericin B at 37°C in a shaker incubator. For molecular and binding studies, pieces of bovine tracheal smooth muscle were placed in T-75 flasks and incubated in the absence or presence of 10 to 10 M fenoterol for the indicated periods of time. The tissues were then frozen and kept at 270°C for RNA extraction and membrane preparation. To examine whether the effects of fenoterol were mediated via specific receptor subtypes, propranolol (nonselective b-adrenoceptor antagonist; 10 M), ICI 118551 (selective b2-adrenoceptor antagonist; 10 M), or CGP 20712A (selective b1-adrenoceptor antagonist; 10 M) were added for 1 h before 10 M fenoterol for a further 2 h. Preincubation of bovine tracheal smooth muscle with the protein synthesis inhibitor cycloheximide (10 mg/ml) for 1 h before addition of 10 M fenoterol for a further 2 h was carried out to assess whether new synthesis of protein(s) was required. Incubation of bovine tracheal smooth muscle with a direct adenylyl cyclase activator, forskolin (10 M), a nonhydrolyzable analog of cAMP, 8-bromo-cAMP (10 M), or other cAMP-elevating agents such as prostaglandin E2 (PGE2; 10 26 M) for 2 h was carried out to see whether the effect of fenoterol is mediated by the elevation of cAMP. The half-life of the H1 receptor mRNA in control and fenoterol-treated tissues was measured by incubation in the absence and presence of 10 M fenoterol for 2 h before the addition of 5 mg/ml actinomycin D for various times (Rodgers et al., 1985). In some experiments, incubation of bovine tracheal smooth muscle with 10 M fenoterol for 2 h was carried out in the presence of various concentrations of dexamethasone (10 to 10 M). For contraction experiments, smooth muscle strips (;5 3 2 3 1 mm) were washed in sterile DMEM and then exposed for 18 h to 10 M or 10 M fenoterol, to 10 M dexamethasone alone or in combination with 10 M fenoterol, or to vehicle (three to four smooth muscle strips in 10 ml of the same medium used for the incubation of muscle pieces in a 25-cm cell culture flask) at 37°C in a shaking