Identification of a novel 7-cis-11-trans-lipoxin A4 generated by human neutrophils: total synthesis, spasmogenic activities and comparison with other geometric isomers of lipoxins A4 and B4.

Addition of (15S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid (15-HETE) and the ionophore A23187 (2.5 microM) to human neutrophils led to the formation of both lipoxin A4 and lipoxin B4 as well as a novel 5,6,15-trihydroxyeicosatetraenoic acid. The new compound was identified using an improved isolation and detection system and its basic structure was determined by physical methods. On the basis of biosynthetic considerations, geometric isomers of lipoxin A4 and lipoxin B4 were prepared by total synthesis. Comparison of these synthetic materials with the neutrophil-derived product showed that the new compound is (5S,6R,15S)-trihydroxy-9,11,13-trans-7-cis-eicosatetraenoic acid or the 7-cis-11-trans-isomer of LXA4 (7-cis-11-trans-LXA4). LXA4, 11-trans-LXA4, 7-cis-LXA4 and 7-cis-11-trans-LXA4 all evoked dose-dependent (0.1-10 microM) contractions of the guinea pig lung strip, whereas 6-cis-LXB4 and 6-cis-8-trans-LXB4 relaxed this preparation. LXA4 and 7-cis-LXA4 were approx. 10-times more potent than the compounds with 11-trans geometry. However, all four double-bond isomers of LXA4 caused contractions which, based upon pharmacological evidence, appeared to involve specific activation of the same site as cysteinyl-containing leukotrienes. In conclusion, 7-cis-11-trans-LXA4 was isolated and identified as a novel biologically active eicosanoid formed by human neutrophils.

Evidence for a 5(6)-epoxytetraene intermediate in the biosynthesis of lipoxins in human leukocytes. Conversion into lipoxin A by cytosolic epoxide hydrolase.

The existence of a 15(S)-hydroxy-5,6-oxido-7,9,13-trans-11-cis-eicosatetraenoic acid intermediate in the biosynthesis of lipoxins A and B has recently been proposed. In the present study, human leukocytes were exposed to 15-HETE and the divalent cation ionophore A23187 and alcohol trapping studies were performed. The products containing alkyltetraenes were isolated and characterized. HPLC analysis, UV spectroscopy and GC/MS of the products showed that 5,15-dihydroxy-14-O-alkyleicosatetraenoic acids were formed, indicating that 5(6)-epoxytetraenes (precursor of the trapping product) were formed in human leukocytes. To gain further evidence for the role of 5(6)-epoxytetraene intermediate in the biosynthesis of lipoxins, (15)-hydroxy-5,6-oxido-7,9,13-trans-11-cis-eicosatetraenoic acid was prepared by total chemical synthesis. When added to purified human liver cytosolic epoxide hydrolase, the epoxide was rapidly and quantitatively converted into LXA. The results provide further evidence for the role of a 5(6)epoxytetraene intermediate in the biosynthesis of lipoxins.

Design of thymidylate synthase inhibitors using protein crystal structures: the synthesis and biological evaluation of a novel class of 5-substituted quinazolinones.

The design, synthesis, and biological evaluation of a new class of inhibitors of thymidylate synthase (TS) is described. The molecular design was carried out by a repetitive crystallographic analysis of protein-ligand structures. At the onset of this project, we focused on the folate cofactor binding site of a high-resolution ternary crystal complex of Escherichia coli TS, 5'-fluorodeoxyuridylate (5-FdUMP) and a classical glutamate-containing folic acid analog. A preliminary ternary crystal structure of a novel compound was successfully solved. Upon analysis of this initial complex, further structural elaborations were made, and a series of active 5-(arylthio)quinazolinones was developed. The synthetic strategy was based on the displacement of a halogen at the 5-position of a quinazolinone by various aryl thioanions. The compounds were tested for inhibition of purified E. coli and/or human TS, and were assayed for cytotoxicity against three tumor cell lines in vitro. Significant thymidine protection effects were observed with several of the inhibitors, indicating that TS was the intracellular locus of activity.

Design, synthesis, and evaluation of nonpeptidic inhibitors of human rhinovirus 3C protease.

The design, synthesis, and biological evaluation of reversible, nonpeptidic inhibitors of human rhinovirus (HRV) 3C protease (3CP) are reported. A novel series of 2,3-dioxindoles (isatins) were designed that utilized a combination of protein structure-based drug design, molecular modeling, and structure-activity relationship (SAR). The C-2 carbonyl of isatin was envisioned to react in the active site of HRV 3CP with the cysteine responsible for catalytic proteolysis, thus forming a stabilized transition state mimic. Molecular-modeling experiments using the apo crystal structure of human rhinovirus-serotype 14 (HRV-14) 3CP and a peptide substrate model allowed us to design recognition features into the P1 and P2 subsites, respectively, from the 5- and 1-positions of isatin. Attempts to optimize recognition properties in the P1 subsite using SAR at the 5-position were performed. In addition, a series of ab initio calculations were carried out on several 5-substituted isatins to investigate the stability of sulfide adducts at C-3. The inhibitors were prepared by general synthetic methods, starting with commercially available 5-substituted isatins in nearly every case. All compounds were tested for inhibition of purified HRV-14 3CP. Compounds 8, 14, and 19 were found to have excellent selectivity for HRV-14 3CP compared to other proteolytic enzymes, including chymotrypsin and cathepsin B. Selected compounds were assayed for antiviral activity against HRV-14-infected HI-HeLa cells. A 2.8 A cocrystal structure of derivative 19 covalently bound to human rhinovirus-serotype 2 (HRV-2) 3CP was solved and revealed that the isatin was situated in essentially the same conformation as modeled.

Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 4. Incorporation of P1 lactam moieties as L-glutamine replacements.

The structure-based design, chemical synthesis, and biological evaluation of various human rhinovirus (HRV) 3C protease (3CP) inhibitors which incorporate P1 lactam moieties in lieu of an L-glutamine residue are described. These compounds are comprised of a tripeptidyl or peptidomimetic binding determinant and an ethyl propenoate Michael acceptor moiety which forms an irreversible covalent adduct with the active site cysteine residue of the 3C enzyme. The P1-lactam-containing inhibitors display significantly increased 3CP inhibition activity along with improved antirhinoviral properties relative to corresponding L-glutamine-derived molecules. In addition, several lactam-containing compounds exhibit excellent selectivity for HRV 3CP over several other serine and cysteine proteases and are not appreciably degraded by a variety of biological agents. One of the most potent inhibitors (AG7088, mean antirhinoviral EC90 approximately 0.10 microM, n = 46 serotypes) is shown to warrant additional preclinical development to explore its potential for use as an antirhinoviral agent.

Structure-based design of lipophilic quinazoline inhibitors of thymidylate synthase.

To develop novel lipophilic thymidylate synthase (TS) inhibitors, the X-ray structure of Escherichia coli TS in ternary complex with FdUMP and the inhibitor 10-propargyl-5,8-dideazafolic acid (CB3717) was used as a basis for structure-based design. A total of 31 novel lipophilic TS inhibitors, lacking a glutamate residue, were synthesized; 26 of them had in common a N-((3,4-dihydro-2-methyl-6-quinazolinyl)methyl)-N-prop-2-ynylaniline+ ++ structure in which the aniline was appropriately substituted with simple lipophilic substituents either in position 3 or 4, or in both. Compounds were tested for their inhibition of E. coli TS and human TS and also for their inhibition of the growth in tissue culture of a murine leukemia, a human leukemia, and a thymidine kinase-deficient human adenocarcinoma. The crystal structures of five inhibitors complexed with E. coli TS were determined. Five main conclusions are drawn from this study. (i) A 3-substituent such as CF(3), iodo, or ethynyl enhances binding by up to 1 order of magnitude and in the case of CF(3) was proven to fill a nearby pocket in the enzyme. (ii) A simple strongly electron-withdrawing substituent such as NO(2) or CF(3)SO(2) in the 4-position enhances binding by 2 orders of magnitude; it is hypothesized that the transannular dipole so induced interacts favorably with the protein. (iii) Attempts to combine the enhancements of i and ii in the same molecule were generally unsuccessful (iv) A 4-C(6)H(5)SO(2) substituent provided both electron withdrawal and a van der Waal's interaction of the phenyl group with a hydrophobic surface at the mouth of the active site. The inhibition (K(is) = 12 nM) of human TS by this compound, 7n, showed that C(6)H(5)SO(2) provided virtually as much binding affinity as the CO-glutamate which it had replaced. (v) The series of compounds were poorly water soluble, and also the potent TS inhibition shown by several of them did not translate into good cytotoxicity. Compounds with large cyclic groups linked to position 4 by an SO or SO(2) group did, however, have IC(50)'s in the range 1-5 microM. Of these, 4-(N-((3,4-dihydro-2-methyl-6-quinazolinyl)methyl)-N-prop-2-ynylamino )phenyl phenyl sulfone, 7n, had IC(50)'s of about 1 microM and was chosen for further elaboration.

Tripeptide aldehyde inhibitors of human rhinovirus 3C protease: design, synthesis, biological evaluation, and cocrystal structure solution of P1 glutamine isosteric replacements.

The investigation of tripeptide aldehydes as reversible covalent inhibitors of human rhinovirus (HRV) 3C protease (3CP) is reported. Molecular models based on the apo crystal structure of HRV-14 3CP and other trypsin-like serine proteases were constructed to approximate the binding of peptide substrates, generate transition state models of P1-P1' amide cleavage, and propose novel tripeptide aldehydes. Glutaminal derivatives have limitations since they exist predominantly in the cyclic hemiaminal form. Therefore, several isosteric replacements for the P1 carboxamide side chain were designed and incorporated into the tripeptide aldehydes. These compounds were found to be potent inhibitors of purified HRV-14 3CP with Kis ranging from 0.005 to 0.64 microM. Several have low micromolar antiviral activity when tested against HRV-14-infected H1-HeLa cells. The N-acetyl derivative 3 was also shown to be active against HRV serotypes 2, 16, and 89. High-resolution cocrystal structures of HRV-2 3CP, covalently bound to compounds 3, 15, and 16, were solved. These cocrystal structures were analyzed and compared with our original HRV-14 3CP-substrate and inhibitor models.

Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 1. Michael acceptor structure-activity studies.

The structure-based design, chemical synthesis, and biological evaluation of peptide-derived human rhinovirus (HRV) 3C protease (3CP) inhibitors are described. These compounds incorporate various Michael acceptor moieties and are shown to irreversibly bind to HRV serotype 14 3CP with inhibition activities (kobs/[I]) ranging from 100 to 600 000 M-1 s-1. These inhibitors are also shown to exhibit antiviral activity when tested against HRV-14-infected H1-HeLa cells with EC50's approaching 0.50 microM. Extensive structure-activity relationships developed by Michael acceptor alteration are reported along with the evaluation of several compounds against HRV serotypes other than 14. A 2.0 A crystal structure of a peptide-derived inhibitor complexed with HRV-2 3CP is also detailed.

Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 2. Peptide structure-activity studies.

The structure-based design, chemical synthesis, and biological evaluation of various peptide-derived human rhinovirus (HRV) 3C protease (3CP) inhibitors are described. These compounds are comprised of an ethyl propenoate Michael acceptor moiety and a tripeptidyl binding determinant. The systematic modification of each amino acid residue present in the binding determinant as well as the N-terminal functionality is described. Such modifications are shown to provide irreversible HRV-14 3CP inhibitors with anti-3CP activities (kobs/[I]) ranging from 60 to 280 000 M-1 s-1 and antiviral EC50's which approach 0.15 microM. An optimized inhibitor which incorporates several improvements identified by the structure-activity studies is also described. This molecule displays very rapid irreversible inhibition of HRV-14 3CP (kobs/[I] = 800 000 M-1 s-1) and potent antiviral activity against HRV-14 in cell culture (EC50 = 0.056 microM). A 1.9 A crystal structure of an S-alkylthiocarbamate-containing inhibitor complexed with HRV-2 3CP is also detailed.

Structure-based design of substituted diphenyl sulfones and sulfoxides as lipophilic inhibitors of thymidylate synthase.

Six new diphenyl sulfoxide and five new diphenyl sulfones were designed, synthesized, and tested for their inhibition of human and Escherichia coli thymidylate synthase (TS) and of the growth of cells in tissue culture. The best sulfoxide inhibitor of human TS was 3-chloro-N-((3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl)-4- (phenylsulfinyl)-N-(prop-2-ynyl)-aniline (7c) that had a Ki of 27 nM. No sulfone improved on TS inhibition by the previously reported 4-(N-((3,4-dihydro-2-methyl-6-quinazolinyl)methyl)-N-prop-2- ynylamino)phenyl phenyl sulfone (Ki = 12 nM). Nevertheless, one sulfone, 4-((2-chlorophenyl)sulfonyl)-N-((3,4-dihydro-2-methyl-4-oxo-6- quinazolinyl)methyl)-N-(prop-2-ynyl)aniline, was selected, on the basis of its inhibition of both TS and cell growth, for antitumor testing; it gave a 61% increase in life span to mice bearing the thymidino kinase-deficient L5178Y (TK-) lymphoma. A crystal structure of N-((3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl)-4-((2- methylphenyl)sulfinyl)-N-(prop-2-ynyl)aniline complexed with E. coli TS was solved and revealed selective binding of one sulfoxide enantiomer. AMBER calculations showed that the enantioselection was due to asymmetric electrostatic effects at the mouth of the active site. In contrast, a similar crystal structure of the sulfoxide 7c, along with AMBER calculations, indicated that both enantiomers bound, but with different affinities. The side chain of Phe176 shifted in order to structurally accommodate the chlorine of the more weakly bound enantiomer.

The effects of peptide histidine isoleucine and neuropeptide Y on mucus volume output from the ferret trachea.

1. The effects of peptide histidine isoleucine (PHI) and neuropeptide Y (NPY) were examined on the mucus volume output produced by methacholine and phenylephrine in the ferret whole trachea in vitro. 2. Sustained application of methacholine (5 microM) or phenylephrine (20 microM) produced a maintained volume output of mucus from the trachea. Both these agonists also increased the output of lysozyme (a marker for serous cell secretion). 3. PHI inhibited the maintained mucus volume output produced by methacholine but had no effect on that due to phenylephrine. The output of lysozyme produced by methacholine or phenylephrine was not significantly changed by PHI. 4. NPY enhanced the volume output of mucus produced by methacholine or phenylephrine; however, the rate of output of lysozyme in mucus produced by both agonists was reduced by NPY. 5. We suggest that PHI has no effect on serous cell secretion but inhibits secretion from another source, possibly mucous cells. NPY inhibits serous cell secretion but has a stronger stimulant action on secretion from another source, again possibly mucous cells. 6. PHI and NPY may be important physiological modulators of mucus volume output in the ferret trachea.

Receptors mediating the effects of substance P and neurokinin A on mucus secretion and smooth muscle tone of the ferret trachea: potentiation by an enkephalinase inhibitor.

1. The effects of substance P (SP) and neurokinin A (NKA) were examined on tracheal smooth muscle tone, mucus volume output, lysozyme output and albumin transport across the ferret in vitro whole trachea in the presence and absence of the enkephalinase inhibitor, thiorphan. 2. SP (0.001-3 microM) and NKA (0.01-10 microM) contracted the tracheal smooth muscle and increased mucus volume, lysozyme and albumin outputs into the tracheal lumen. The EC50 values for SP and NKA for all of the variables measured were significantly reduced, and all of the maximum responses were significantly enhanced by thiorphan (10 microM). 3. In the presence of thiorphan, SP (1 microM) and NKA (10 microM) produced albumin concentrations in the secreted mucus (8.9 and 7.2 micrograms microliters-1) which were greater than those in the submucosal buffer (4.2 micrograms microliters-1). 4. In the presence of thiorphan, NKA was approximately 5 times more potent than SP at contracting the tracheal smooth muscle. Conversely SP was 23, 15 and 22 times more potent than NKA at stimulating mucus volume, lysozyme and albumin outputs respectively. 5. Thus, there is neutral endopeptidase in the ferret trachea in vitro which cleaves exogenously applied SP and NKA, thereby reducing the magnitude and potency of their actions. SP and NKA contract the ferret tracheal muscle probably by an action at NK2 (or NK3)-receptors but stimulate mucus volume output, lysozyme output and albumin transport across the tracheal wall probably by an action on NK1 receptors.

Endothelin-1 inhibits pre-stimulated tracheal submucosal gland secretion and epithelial albumin transport.

1. Endothelin-1 potently contracts smooth muscle, including that in the airways. However, its effect on airway mucosal function has not so far been studied. 2. We have used the ferret whole trachea in vitro to examine the effect of endothelin-1 on tracheal smooth muscle tone, transepithelial potential difference (p.d.), submucosal gland secretion (including lysozyme secretion from serous cells) and active epithelial albumin transport. In addition we have examined the effects of endothelin on submucosal gland secretion and albumin transport pre-stimulated with the muscarinic agonist methacholine and the alpha-adrenoceptor agonist phenylephrine. The effects of the Ca2+ channel blocker nifedipine on the responses to endothelin have also been assessed. 3. Endothelin (0.1-100 nM) produced concentration-dependent increases in intraluminal tracheal pressure indicating smooth muscle contraction, and in the negativity of the transepithelial p.d. These effects were partially inhibited by nifedipine (10 microM). 4. Endothelin (0.01-100 nM) had no significant effect on baseline rates of mucus, lysozyme or albumin outputs, but produced concentration-dependent reductions in maintained methacholine- and phenylephrine-induced mucus, lysozyme and albumin outputs. In general endothelin was more potent against methacholine-induced effects. All of the concentration-response curves for endothelin were shallow and some appeared to be biphasic, suggesting the possibility of more than one mechanism of action of endothelin. 5. The effects of endothelin (at concentrations greater than 1 nM) on phenylephrine-induced mucus volume, lysozyme and albumin outputs were significantly inhibited by nifedipine. Similarly the effect of endothelin (greater than 1 nM) on methacholine-induced mucus volume and albumin outputs (but not lysozyme output) was attenuated by nifedipine. Similarly the effect of endothelin (>1 nM) on methacholine-induced mucus volume and albumin outputs (but not lysozyme output) was attenuated by nifedipine. The effects of endothelin (at concentrations <1 nM) on methacholine and phenylephrine-induced responses were generally not affected by nifedipine.6. Thus, endothelin contracts ferret tracheal smooth muscle and increases transepithelial p.d. at least in part by opening dihydropyridine-sensitive Ca2+ channels. Endothelin does not directly stimulate submucosal gland secretion or epithelial albumin transport, but inhibits methacholine- and phenylephrineinduced secretion and transport. The inhibitory effects produced by higher concentrations of endothelin may be mediated partially by activation of dihydropyridine-sensitive Ca2+ channels, although the explanation for this is not clear. The mechanism of action of endothelin in attenuating stimulated secretion and epithelial transport at lower concentrations is unknown.

The effect of vasoactive intestinal peptide on smooth muscle tone and mucus secretion from the ferret trachea.

The effect of vasoactive intestinal peptide (VIP) was examined on the smooth muscle contraction and mucus secretion produced by methacholine and phenylephrine in the ferret whole trachea in vitro. VIP (0.5 to 800 nM) produced a concentration-dependent relaxation of the ferret trachea contracted by methacholine (1 microM) and phenylephrine (10 microM). The concentration-response curves for methacholine- and phenylephrine-induced contractions were both shifted to the right by VIP (0.1 microM). Methacholine-induced secretion was inhibited in a concentration-dependent manner by VIP, whereas that due to phenylephrine was enhanced. The concentration-response curve for methacholine-induced secretion was shifted to the right by VIP, whereas the curve for phenylephrine was shifted to the left. Methacholine produced a concentration-dependent increase in the rate of output of lysozyme from the ferret trachea with no corresponding increase in the concentration of lysozyme in the mucus. Phenylephrine produced a concentration-dependent increase in the rate of output and in the concentration of lysozyme. VIP (0.1 microM) significantly increased the concentration of lysozyme in the mucus produced by methacholine with no increase in the rate of lysozyme output. However, the rate of lysozyme output due to phenylephrine was significantly increased by VIP (0.1 microM) with no increase in concentration. We suggest that VIP inhibits secretion from mucous cells stimulated by methacholine, and enhances the secretion produced by phenylephrine from serous cells.

Evidence for an atypical, or beta 3-adrenoceptor in ferret tracheal epithelium.

1. A preparation of the ferret trachea in vitro was used to examine the effects of three selective beta-adrenoceptor agonists on lysozyme secretion from submucosal gland serous cells and epithelial albumin transport into tracheal mucus following sustained, submaximal stimulation of mucus production with methacholine (20 microM). 2. Prenalterol, salbutamol and BRL 37344 all enhanced methacholine-induced albumin output. BRL 37344 was 10,000 times more potent than salbutamol, and salbutamol was slightly more potent than prenalterol. The concentrations required to increase albumin output by 100% (EC100%) were 1.4 nM, 0.7 mM and approximately 1.0 mM for BRL 37344, salbutamol and prenalterol, respectively. All three agonists inhibited methacholine-induced lysozyme output, with salbutamol being 60 times more potent than BRL 37344, and BRL 37344 being approximately 100 times more potent than prenalterol. 3. The selective beta 2-adrenoceptor antagonist, ICI 118551, inhibited the increase in albumin output produced by BRL 37344, but much more potent at inhibiting the response to salbutamol; the pA2 for ICI 118551 was 5.55 and 7.18 (P less than 0.001) when the agonist was BRL 37344 and salbutamol, respectively. ICI 118551 also attenuated the inhibition of lysozyme output produced by the two agonists, but was 10-30 times more potent at inhibiting this response than the albumin response to BRL 37344 and salbutamol. 4. The greater potency (4-5 orders of magnitude) of BRL 37344, compared to the beta 1- (prenalterol) and beta 2- (salbutamol) adrenoceptor selective agonists, in stimulating methacholine-induced albumin transport suggests that tracheal epithelium possess an atypical, or beta 3-adrenoceptor similar to that previously reported for adipocytes and gastrointestinal smooth muscle. The weak antagonism of the response to BRL 37344 by ICI 118551 would also be consistent with an atypical adrenoceptor mediating the albumin transport response. Inhibition of methacholine-induced serous cell lysozyme output would appear to be mediated predominantly by beta2-adrenoceptors.5. In view of the possible beneficial protective effects of albumin in airway surface liquid, selective beta3-agonists like BRL 37344 might have potential value in the prevention and/or treatment of inflammatory airway disease.

Platelet-activating factor relaxes ferret tracheal smooth muscle and reduces transepithelial potential difference in vitro.

1. The effects of platelet activating factor (PAF) were examined on the smooth muscle tone, mucus volume, lysozyme and albumin outputs and potential difference (PD) across the ferret tracheal wall. 2. PAF (0.1-10 microM) had no direct effect on mucus volume, lysozyme or albumin output from the ferret trachea. PAF produced concentration-dependent relaxations of the tracheal smooth muscle and reductions in PD across the tracheal wall. There was no change in the histological appearance of the trachea after exposure to PAF. 3. The PAF-induced smooth muscle relaxation was not affected by FPL55712, a combination of mepyramine and cimetidine, or by a combination of the oxygen free-radical scavengers catalase and superoxide dismutase (SOD); but was abolished by indomethacin or the PAF-receptor antagonist WEB2086. 4. The PAF-induced reduction in PD was not affected by indomethacin, FPL55712 or mepyramine and cimetidine, but was prevented by catalase and SOD, and by WEB2086. 5. We conclude that PAF relaxes ferret tracheal smooth muscle in vitro by receptor-mediated release of a bronchodilator prostaglandin, possibly PGE2. PAF also reduces PD across the trachea suggesting changes in epithelial function; however, there is no histological epithelial damage after PAF. The reduction in PD with PAF is probably produced by receptor-mediated release of oxygen free-radicals. The cellular source of these free-radicals and of the dilator prostaglandin is unclear.

PAF-induced muscarinic cholinoceptor hyperresponsiveness of ferret tracheal smooth muscle and gland secretion in vitro.

1. The effects of exposure of the ferret trachea in vitro to platelet activating factor (PAF) were examined on methacholine-induced smooth muscle contraction, mucus volume and lysozyme outputs, and albumin transport across the tracheal epithelium. 2. Methacholine (0.1-30 microM) produced concentration-dependent increases in tracheal smooth muscle tone and mucus volume, lysozyme and albumin outputs from the trachea. 3. The concentration-response curves for methacholine-induced smooth muscle contraction, mucus volume and lysozyme outputs were all shifted upwards after exposure of the trachea to PAF (1 microM) with a significant increase in maximum response for each variable. The EC50 values for methacholine-induced smooth muscle contraction and mucus volume output were significantly reduced after PAF exposure suggesting an increase in the potency of methacholine. The concentration-response curve for methacholine-induced albumin output was shifted downwards after PAF exposure with a greatly reduced maximum but no change in the EC50 for methacholine. 4. PAF-induced hyperresponsiveness of methacholine-induced smooth muscle contraction, mucus volume and lysozyme outputs was not affected by indomethacin, FPL55712, or mepyramine and cimetidine, but was prevented by catalase and superoxide dismutase (SOD), and by WEB2086. Similarly, PAF-induced inhibition of methacholine-stimulated albumin output was prevented by catalase and SOD, and by WEB2086. 5. We conclude that PAF induces hyperresponsiveness of ferret tracheal smooth muscle and submucosal gland secretion (including lysozyme secretion from serous cells) to methacholine. This hyperresponsiveness is probably produced by receptor-mediated release of oxygen free-radicals. The inhibition of methacholine-induced albumin flux suggests a loss of epithelial function which is also probably mediated by release of free-radicals. The mechanism by which the free-radicals produce the changes in responsiveness to methacholine, and the cellular source of the free-radicals, remain to be established.

Effects of neuropeptides and capsaicin on the canine tracheal vasculature in vivo.

1. The nonadrenergic, noncholinergic nervous system may control the airway vasculature via various neuropeptides. We have perfused the cranial tracheal arteries of the anaesthetized dog and investigated the effects of neuropeptides and capsaicin (which is supposed to release neuropeptides from sensory nerve endings) on the tracheal vasculature by injecting them locally into the perfusion system. 2. Neurokinin A (NKA, 0.02-20 pmol), calcitonin gene-related peptide (CGRP, 2-200 pmol) and peptide histidine isoleucine (PHI, 0.02-2 nmol) dose-dependently decreased tracheal vascular resistance (Rtv). NKA was 10 and 100 times more potent than CGRP and PHI, respectively. The duration of the response to CGRP was greatly prolonged with larger doses. Galanin (0.2-2 nmol) had no appreciable effect on Rtv. 3. Neuropeptide Y (NPY 0.02-2 nmol) and bombesin (0.02-10 nmol) dose-dependently increased Rtv. However, the dose-response curve for bombesin was bell-shaped suggesting the development of tachyphylaxis with larger doses. In smaller doses, bombesin was twice as potent as NPY. The duration of the response to NPY was prolonged with larger doses. 4. With the exception of PHI no neuropeptide altered tracheal smooth muscle tone; PHI (1 and 2 nmol) caused small dilatations of the trachea. 5. The effects of capsaicin (2-100 nmol) were complex. Usually, the vascular response had two dose-dependent phases: a rapid vasoconstriction followed by a small, longer-lasting vasodilatation. The tracheal smooth muscle response was usually biphasic, a contraction followed by a relaxation. 6. According to previous and present data, the order of potency of the neuropeptides on the canine tracheal vasculature is for the vasodilators : NKA > vasoactive intestinal peptide (VIP) > CGRP > substance P > PHI, and for the vasoconstrictors: bombesin > NPY. The longer-acting neuropeptides (VIP, CGRP and NPY) may be more important than the shorter-acting neuropeptides (substance P, NKA, PHI and bombesin) as regulators of the airway wall blood flow.

H1- and H2-receptor characterization in the tracheal circulation of sheep.

1. The effects of histamine, the specific H1-agonist SKF 71481-A2 and the H2-agonist dimaprit were examined on tracheal vascular resistance in sheep anaesthetized with pentobarbitone. Tracheal vascular resistance was determined by perfusing the cranial tracheal arteries at constant flows and measuring inflow pressures. Changes in tracheal smooth muscle tone were also measured. 2. Histamine and SKF 71481-A2 contracted the tracheal smooth muscle and this effect was blocked by the H1-antagonist mepyramine. Stimulation of H2-receptors with dimaprit had no effect on tracheal smooth muscle tone. 3. Histamine had a complex action on the tracheal vasculature producing either a triphasic change (early dilatation then constriction followed by late dilatation) or just a constriction. SKF 71481-A2 always produced a biphasic change in vascular resistance (dilatation followed by constriction). Dimaprit dilated the tracheal vasculature. 4. The late dilatation produced by histamine in some sheep was blocked by bilateral cervical vagotomy but the mechanism for this effect is not known. No other responses to histamine, SKF 71481-A2 or dimaprit were affected by vagotomy. 5. The vasoconstriction produced by histamine and SKF 71481-A2 was antagonized by mepyramine indicating a H1-receptor-mediated effect. Cimetidine had no effect on the vasoconstriction to histamine suggesting a lack of involvement of H2-receptors. 6. The vasodilatation produced by histamine and SKF 71481-A2 was also antagonized by mepyramine, again suggesting a H1-receptor-mediated action. Cimetidine had no effect on the vasodilator response to histamine indicating no involvement of H2-receptors in this response. 7. The dilator effect of dimaprit was antagonized by cimetidine suggesting this effect was mediated by H2-receptors. 8. We conclude that H1-receptors in the various parts of the sheep tracheal vasculature can cause increases and decreases in total tracheal vascular resistance; that H2-receptors decrease resistance; and that the tracheal smooth muscle contracts on activation of H1-receptors but has no response to H2-agonists.

A study of the histamine H2-receptor mediating relaxation of the parenchymal lung strip preparation of the guinea-pig.

The relaxation produced by several H2-receptor agonists and forskolin was investigated on strips of guinea-pig lung parenchyma. Dimparit, 1 microM to 10 mM, 4-methyl histamine, 0.5 microM to 100 microM and impromidine, 10 nM to 1 microM, had no effect on the tone of the unstimulated strips of lung parenchyma but caused a dose-dependent relaxation of strips that were contracted by 2-pyridylethylamine (2-PEA), 15 microM. Forskolin, 10 nM to 4 microM, produced a dose-dependent relaxation of both the stimulated and unstimulated lung strips. The muscarinic antagonist atropine, 1 microM, and the beta 2-adrenoceptor antagonist propranolol, 10 microM, had no effect on the dose-response curve for dimaprit-induced relaxation of the lung strip. The dose-response curve for dimaprit was shifted to the right in a dose-dependent manner by increasing concentrations of a variety of H2-antagonists. Schild plots produced a straight line for all the H2-antagonists with slopes not significantly different from unity. The equilibrium dissociation constants for the H2-antagonists on the lung strip preparation were similar to those previously reported for inhibition of the chronotropic activity of histamine on guinea-pig right atria and inhibition of [3H]-tiotidine binding to homogenates of guinea-pig lung parenchyma.