Abstractacid -dihydroxyeicosatrienoic acid * hydroxyeicosatetraenoic acid Cytochrome P450 metabolizes arachidonic acid to several unique and biologically active compounds in rabbit liver and kidney. Microsomal fractions prepared from rabbit lung homogenates metabolized arachidonic acid through cytochrome P450 pathways, yielding cis-epoxyeicosatrienoic acids (EETs) and their hydration products, vic-dihydroxyeicosatrienoic acids, mid-chain cis-trans conjugated dienols, and 19-and 20-hydroxyeicosatetraenoic acids. Inhibition studies using polyclonal antibodies prepared against purified CYP2B4 demonstrated 100% inhibition of arachidonic acid epoxide formation. Purified CYP2B4, reconstituted in the presence of NADPH-cytochrome P450 reductase and cytochrome b5, metabolized arachidonic acid, producing primarily EETs. EETs were detected in lung homogenate using gas chromatography/mass spectroscopy, providing evidence for the in vivo pulmonary cytochrome P450 epoxidation of arachidonic acid. Chiral analysis of these lung EETs demonstrated a preference for the 14(R),15(S)-, 11(S),12(R)-, and 8(S),9(R)-EET enantiomers. Both EETs and vic-dihydroxyeicosatrienoic acids were detected in bronchoalveolar lavage fluid. At micromolar concentrations, methylated 5,6-EET and 8,9-EET significantly relaxed histamine-contracted guinea pig hilar bronchi in vitro. In contrast, 20-hydroxyeicosatetraenoic acid caused contraction to near maximal tension. We conclude that CYP2B4, an abundant rabbit lung cytochrome P450 enzyme, is the primary constitutive pulmonary arachidonic acid epoxygenase and that these locally produced, biologically active eicosanoids may be involved in maintaining homeostasis within the lung. (J. Clin. Invest. 1995. 95:2150-2160

We examined the role of sensory nerves in mediating nonadrenergic inhibitory responses in airway segments isolated from male Sprague-Dawley rats. In the presence of adrenergic blockade, capsaicin (Cap; 1 microM) elicited marked relaxation responses in isolated bronchi precontracted with bethanechol (Beth). Cap-induced inhibitory responses were unaffected by tetrodotoxin (TTX), were attenuated by incubation of the airway with indomethacin (Indo), phosphoramidon, or RP-67580, but were abolished by previous exposure of the airway to Cap and by denuding the epithelium. Substance P (SP; 1 microM), neurokinins A and B (1 microM), and calcitonin gene-related peptide (0.1 microM) relaxed Beth-contracted airway segments to a similar extent. The SP-induced responses were unaffected by adrenergic blockade or by pretreatment with either TTX, phosphoramidon, or Cap, but were attenuated by RP-67580 and abolished by Indo and by denuding the epithelium. In anesthetized mechanically ventilated rats, Cap (50 and 100 micrograms/kg i.v.) elicited a dose-dependent reversal of the increase in lung resistance induced by an infusion of Beth. The Cap-induced bronchodilation was unaffected by pretreatment with propranolol alone or in combination with hexamethonium. SP (44 nmol/kg iv) also evoked bronchodilatory responses in intact animals, which were unaffected by propranolol and hexamethonium but were abolished by treatment of the animals with Indo. Electrical-field stimulation (EFS) evoked nonadrenergic noncholinergic relaxation responses in contracted airway segments. These EFS-induced inhibitory responses were markedly attenuated by treatment of the airway segment with TTX, Cap, or RP-67580. We conclude that neuropeptides released from Cap-sensitive sensory nerves have potent inhibitory effects in rat airways that are mediated, in part, by activation of neurokonin NK1 receptors on epithelium and subsequent release of an inhibitory prostaglandin(s).

We hypothesized that substance P and capsaicin would cause the release of prostaglandin E2(PGE2) from intrapulmonary bronchi isolated from Sprague-Dawley rats. Substance P (1 μM) caused the release of PGE2, measured with enzyme immunoassay, from the isolated airway segments; PGE2 release was inhibited by the neurokinin (NK)1-receptor antagonist, RP-67580, by inhibition of cyclooxygenase with meclofenamate, and by removal of the epithelium. The release of PGE2 caused by capsaicin (1 μM) was similar in magnitude to that caused by substance P. The capsaicin-induced release of PGE2was inhibited by desensitization of sensory nerves with capsaicin and by RP-67580, meclofenamate, and epithelial denudation. We conclude that activation of NK1 receptors on epithelium causes release of PGE2, which most likely represents the ultimate mediator of airway smooth muscle relaxation, produced by exogenous neuropeptides and by activation of the sensory nerve inhibitory system. Epithelial damage, such as that seen in asthmatic airways, would disrupt this protective system in the lungs, which could contribute to the development of airway disease.

A set of novel tachykinin-like peptides has been isolated from bullfrog brain and gut. These compounds, ranatachykinin A (RTKA), ranatachykinin B (RTKB), and ranatachykinin C (RTKC), were named for their source, Rana catesbeiana, and their homology to the tachykinin peptide family. We present the first report of the micelle-bound structures and pharmacological actions of the RTKs. Generation of three-dimensional structures of the RTKs in a membrane-model environment using (1)H NMR chemical shift assignments, two-dimensional NMR techniques, and molecular dynamics and simulated annealing procedures allowed for the determination of possible prebinding ligand conformations. RTKA, RTKB, and RTKC were determined to be helical from the midregion to the C-terminus (residues 4-10), with a large degree of flexibility in the N-terminus and minor dynamic fraying at the end of the C-terminus. The pharmacological effects of the RTKs were studied by measuring the elevation of intracellular Ca(2+) in Chinese hamster ovarian cells stably transfected with the bullfrog substance P receptor (bfSPR). All of the RTKs tested elicited Ca(2+) elevations with a rank order of maximal effect of RTKA >/= SP > RTKC >/= RTKB. A high concentration (1 microM) of the neuropeptides produced varying degrees of desensitization to a subsequent challenge with the same or different peptide, while a low concentration (1 pM) produced sensitization at the bfSPR. Our data suggest differences in amino acid side chains and their charged states at the C-terminal sequence or differences in secondary structure at the N-terminus, which do not overlap according to the findings in this paper, may explain the differing degree and type of receptor activation seen at the bfSPR.