Hypotonic stimulation induces airway constriction in normal and asthmatic airways. However, the osmolarity sensor in the airway has not been characterized. TRPV4 (also known as VR-OAC, VRL-2, TRP12, OTRPC4), an osmotic-sensitive cation channel in the transient receptor potential (TRP) channel family, was recently cloned. In the present study, we show that TRPV4 mRNA was expressed in cultured human airway smooth muscle cells as analyzed by RT-PCR. Hypotonic stimulation induced Ca2+influx in human airway smooth muscle cells in an osmolarity-dependent manner, consistent with the reported biological activity of TRPV4 in transfected cells. In cultured muscle cells, 4α-phorbol 12,13-didecanoate (4-αPDD), a TRPV4 ligand, increased intracellular Ca2+level only when Ca2+was present in the extracellular solution. The 4-αPDD-induced Ca2+response was inhibited by ruthenium red (1 μM), a known TRPV4 inhibitor, but not by capsazepine (1 μM), a TRPV1 antagonist, indicating that 4-αPDD-induced Ca2+response is mediated by TRPV4. Verapamil (10 μM), an L-type voltage-gated Ca2+channel inhibitor, had no effect on the 4-αPDD-induced Ca2+response, excluding the involvement of L-type Ca2+channels. Furthermore, hypotonic stimulation elicited smooth muscle contraction through a mechanism dependent on membrane Ca2+channels in both isolated human and guinea pig airways. Hypotonicity-induced airway contraction was not inhibited by the L-type Ca2+channel inhibitor nifedipine (1 μM) or by the TRPV1 inhibitor capsazepine (1 μM). We conclude that functional TRPV4 is expressed in human airway smooth muscle cells and may act as an osmolarity sensor in the airway.

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Optical difference spectroscopy was used to identify and quantify human adrenal microsomal and mitochondrial cytochrome P450 enzyme interactions with the histamine H₃ receptor antagonists thioperamide, clobenpropit and ciproxifan. Addition of these structurally diverse imidazole H₃ receptor antagonists to cytochrome-P450-containing human adrenal microsomal and mitochondrial preparations resulted in concentration-dependent type II optical difference spectra. Respective spectral dissociation constants (K_S) for the drug interactions with human adrenal microsomal and mitochondrial cytochrome P450 were 1.5 and 1.6 µmol/l for thioperamide, 3.1 and 0.28 µmol/l for clobenpropit and 0.10 and 0.11 µmol/l for ciproxifan. The three compounds demonstrated a similar activity profile in cytochrome-P450-containing bovine adrenal microsomal and mitochondrial preparations. Findings indicate direct coordination of these imidazole-containing H₃ receptor antagonists with the heme moiety of human adrenal cytochrome P450 isozymes.

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Asthma is characterized by acute episodes of nonspecific airway hyperreactivity and chronic pulmonary inflammation exacerbated by stimuli including allergen exposure. In order to reproduce the physiologic and immunologic responses that occur in asthmatic patients, we have characterized a model of antigen-induced inflammation in which allergic mice (B6D2F1) that had been challenged once with aerosolized ovalbumin and had developed a pulmonary cellular infiltrate were rechallenged 1 wk later. Pulmonary inflammation in rechallenged mice was substantially greater than that in single-challenged mice. Eosinophils and activated-memory T cells (CD44+, CD45RBlo) in bronchoalveolar lavage (BAL) fluid accumulated to higher levels and with faster kinetics in response to the second challenge than in response to the first challenge. Eosinophils in lung tissue also accumulated to higher levels but with similar kinetics in response to the second challenge than in response to the first challenge. Similarly, interleukin (IL)-4 and IL-5 steady-state mRNA levels in lung tissue increased after the second challenge and were higher than those measured after a single challenge. Furthermore, treatment of mice with an anti-IL-5 monoclonal antibody 2 h prior to rechallenge inhibited antigen induced eosinophil accumulation in the lungs. In mice challenged twice, peak in vivo bronchoconstrictor responsiveness to acetylcholine was increased following the second challenge compared with that observed following the initial challenge. In contrast, ex vivo tracheal smooth muscle contractile responsiveness to acetylcholine was not altered. Although mucus accumulation and epithelial damage in pulmonary tissue were evident in mice challenged twice, these parameters were slightly reduced compared with those seen at similar times in mice challenged once. Therefore, although these mice exhibit only slight bronchial epithelial damage, the presence of significant inflammation and airway hyperreactivity to acetylcholine as well as slightly increased baseline reactivity demonstrate important similarities with the pathophysiology of asthma.