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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1. Pig nasal mucosal strips were incubated with alpha-adrenoceptor antagonists followed by alpha2-adrenoceptor agonist concentration-response curves. 2. Contractions elicited by the alpha2-adrenoceptor agonists BHT-920 (pD2 = 6.16 +/- 0.07), UK 14,304 (pD2 = 6.89 +/- 0.13) and PGE-6201204 (pD2 = 7.12 +/- 0.21) were blocked by the alpha2-adrenoceptor antagonist yohimbine (0.1 microm). In contrast, the alpha1-adrenoceptor antagonist prazosin (0.03 microm) had no effect on the BHT-920-, UK 14,304- and PGE-6201204-induced contractions, but blocked the contractile response to the alpha(1)-adrenoceptor agonist phenylephrine (pD2 = 5.38 +/- 0.04) and the mixed alpha1- and alpha2-adrenoceptor agonist oxymetazoline (pD(2) = 6.30 +/- 0.22). 3. The alpha2-adrenoceptor antagonist yohimbine (0.01-0.1 microm, pA2 = 8.04), alpha2B/C-adrenoceptor antagonist ARC 239 (10 microm, pK(b) = 6.33 +/- 0.21), alpha2A/C-adrenoceptor antagonist WB 4101 (0.3 microm, pK(b) = 8.01 +/- 0.24), alpha2A-adrenoceptor antagonists BRL44408 (0.1 microm, pK(b) = 6.82 +/- 0.34) and RX 821002 (0.1 microm, pKb = 8.31 +/- 0.35), alpha2C-adrenoceptor antagonists spiroxatrine (1 microm, pKb = 7.32 +/- 0.32), rauwolscine (0.1 microm, pKb = 8.16 +/- 0.14) and HV 723 (0.3 microm, pKb = 7.68 +/- 0.14) inhibited BHT-920-induced contractions in pig nasal mucosa. 4. The present antagonist potencies showed correlations with binding affinity estimates (pKi) obtained for these antagonists at the human recombinant alpha2A- and alpha2C-adrenoceptors (r = 0.78 and 0.83, respectively) and with binding affinity estimates (pKd) obtained in pig native alpha2A- and alpha2C-monoreceptor assays (r = 0.85 and 0.78, respectively). No correlation was observed for the alpha2B-subtype. 5. In conclusion, contractile responses to phenylephrine, BHT-920, UK 14,304, PGE-6201204 and oxymetazoline indicate that alpha1- and alpha2-adrenoceptors are present and mediate vasoconstriction in pig nasal mucosa. Furthermore, correlation analysis comparing antagonist potency in pig nasal mucosa with affinities for human recombinant alpha2-adrenoceptors and native pig alpha2-adrenoceptors suggest that alpha2A- and alpha2C-adrenoceptor subtypes constrict pig nasal mucosa vasculature.

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We present the pharmacological and pharmacokinetic profiles of a novel histamine H 3 receptor antagonist, N- (3,phenyl]-methyl]-urea (SCH 79687). The H 3 -receptor binding K i values for SCH 79687 were 1.9 and 13 nM in the rat and guinea pig (GP), respectively. The K i values for SCH 79687 at histamine H 1 and H 2 receptors were greater than 1 M. SCH 79687 showed a 41-and 82-fold binding selectivity for the H 3 receptor over ␣ 2A -adrenoceptors and imidazoline I 2 , and Ͼ500-fold H 3 selectivity compared with over 60 additional receptors. The pA 2 value for SCH 79687 in the GP ileum electrical field-stimulated (EFS) contraction was 9.6 Ϯ 0.3. Similar H 3 antagonist activity was observed in the EFS cryopreserved and fresh tissue isolated human saphenous vein (HSV) assays (pK b ϭ 9.4 Ϯ 0.3 and 10.1 Ϯ 0.4). SCH 79687 (30 nM) did not block clonidine-induced inhibition of EFSinduced contractions in HSV. SCH 79687 (ED 50 ϭ 0.3 mg/kg i.v.) attenuated (R)-␣-methylhistamine inhibition of sympathetic hypertensive responses in the GP. At the time of activity evaluation, the GP plasma SCH 79687 concentration was 25 ng/ml at the dose of 0.3 mg/kg i.v. In feline nasal studies, combined administration of SCH 79687 (3 mg/kg i.v.) and the H 1 -antagonist loratadine (3 mg/kg i.v.), at individual doses that do not produce decongestion, inhibited the compound 48/80-induced congestion by 47%. The ␣-adrenergic agonist phenylpropanolamine (PPA; 1 mg/kg i.v.) also attenuated compound 48/80 nasal responses by 42%. Unlike the H 3 /H 1 combination that did not affect blood pressure (BP), PPA (1 mg/kg i.v.) significantly increased BP compared with control animals by a maximum of 31 mm Hg. Orally, SCH 79687 (10 mg/kg) plus loratadine (10 mg/kg) also produced decongestion without effects on BP. In pharmacokinetic studies, oral dosing with SCH 79687 in the rat (10 mg/kg) and monkey (3 mg/kg) achieved plasma C max and area under the curve values greater than 1.5 and 12.1 g ⅐ h/ml, respectively. SCH 79687 is an orally active H 3 antagonist with a good pharmacokinetic profile that, in combination with an H 1 antagonist, demonstrates decongestant efficacy comparable with oral sympathomimetic decongestants but without hypertensive liabilities.

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