European Journal of Pharmacology, 212 (lY92) 187-194 8 1992 Elsevier Science Publishers B.V. All rights reserved

187 0014-2999/92/$05.00

EJP 52304

Epithelial

modulation of the relaxant activity of atriopeptides and guinea-pig tracheal smooth muscle Lynette

B. Fernandcs,

Department Received

in rat

Janet M.H. Preuss and Roy G. Goldie

ofPharmacolo&~, Unkersiiy of Western Australia, Nedlands 6009, Australia

1 July 1991, revised MS received

22 October

1991, accepted

3 December

1991

Three pcptide components of atrial natriuretic factor (ANF) caused relaxation of carbachol-contracted guinea-pig isolated tracheal smooth muscle. These were the I-28, 5-28 and 5-27 pcptide sequences (ANF(l-28), ANF-(5-28) and ANF-(5-27)). The peptides were lo-30 times more potent in epithclium-denuded than in cpithelium-intact preparations. In the absence of airway epithelium, ANF-(l-28) was the most potent relaxant (mean pD, = 7.40 k 0.08), with ANF-(S-27) and ANF-(5-28) 2-3 fold less potent. The neutral endopeptidase inhibitor phosphoramidon (1 PM) increased the potency of ANF-(5-27) in both c?ithelium-intact and cpithelium-denuded guinea-pig tracheal rings. In contrast, removal of the epithelium from rat trachea, or pretreatment with phosphoramidon (1 PM) decreased relaxant rcsponsivencss to ANF-(5-27). Thus, in rat trzchca, epithclial cndopeptidase may convert ANF-(5-27) to a more active relaxant pcptide. Human bronchial preparations with or without epithclium, obtained from non-diseased lung samples and from a single sample of asthmatic lung, were virtually unresponsive to ANF-(5-27). Consistent with the spasmolytic effects of ANF in guinea-pig trachea, autoradiographic analysis revealed the presence of a sparse population of specific binding sites for [ “‘I]ANF-(I -28) over both tracheal smooth muscle and epithclium. The present study shows that the relaxant effects of atriopcptins in rat and guinea-pig airway smooth muscle were modulated by the epithclium and the activity of neutral endopcptidasc. However, marked species differences in airway smooth muscle responsiveness to ANF and in the modulatory role of the airway cpithelium were evident. ANF (atrial natriurctic

factor);

Relaxation; Smooth

Trachea (guinea-pig); Trachea (rat); Bronchus muscle (airway); Autoradiography

1. Introduction The

peptide

substances

collectively

known

as atria1

(De Bold, 1982, Trippodo ct al., 1982) cause endothclium-independent relaxation of vascular smooth muscle (Scivoletto and Carvalho, 1984). The 1-28 amino acid sequence (ANF-(l-28), atriopeptin I), as well as ANF-(5-27) (atriopeptin II) and ANF-(5-28) (atriopeptin III), all caused relaxation of isolated intestinal smooth muscle preparations (Thibault et al., 1984) and relaxed bovine trachea (Ishii and Murad, 1989) and guinea-pig trachea (O’Donnell et al., 1985; Watanabe et al., 1988). Synthetic ANF (ANF-(3-28)) also caused concentration-dependent relaxation of guinea-pig tracheal preparations (Hamcl and Ford-Hutchinson, 1986). However, marked species differences have been demonstrated with respect to the relaxant effects of natriurctic

factor

(ANF)

-Correspondence to: R.G. Goldie, Department of Pharmacology, University of Western Australia, Perth, Nedlands 6009, Western Australia. Tel. 61.9.389 2812, fax 61.9.389 3469.

(human);

Epithelium;

ANF in airway smooth muscle, with only weak responses seen in rat trachea or rat or guinea-pig lung parenchyma (Hamel and Ford-Hutchinson, 1986). Furthcrmore, human isolated bronchial preparations were insensitive to ANF (Labat et al., 1988). Several factors may influence the responsiveness of airway smooth muscle preparations to ANF. These include the activity of dcgradative enzymes such as neutral cndopeptidase which is known to inactivate these peptides (Olins et al., 1987; Bertrand and Doblc, 1988) and to exist in the airway epithelium and submucosa (Sckizawa et al., 1987; Borson et al., 1986). It might be expected that removal of the airway cpithelium would increase tracheal responsiveness to ANF, as is the case for other pcptides which are metabolised by neutral endopeptidase, including the relaxant vasoactive intestinal peptide (VIP) (Farmer and Togo, 1990) and for the spasmogcnic tachykinins substance P, neurokinin A and neurokinin B (Fine et al., 1989; Frossard et al., 1989). However, O’Donnell et al. (1985) observed no significant effect of epithclium removal on the potency of ANF-(5-27) in guinea-pig tracheal preparations with spontaneous airway smooth muscle tone.

188 We were interested to determine whether airway epithelial neutral cndopcptidasc might have a modulatory influence on relaxant responsiveness to atriopeptins in carbachol-contracted airway preparations from rat, guinea-pig and man. In addition, the distribution of specific binding sites for ANF in guinea-pig trachea was examined.

2. Materials and methods

2.1. Tissue preparation for organ bath studies Male Wistar rats (220-250 g) were killed by stunning and exsanguination and the trachea removed. Male guinea-pigs ( S R / C Tricolour) weighing 450-500 g were killed by cervical dislocation and the trachea removed. Macroscopically normal specimens of human bronchi (2-3 mm i.d.) were obtained from lung tissue taken at thoracotomy from lung cancer patients. Bronchi were also obtained 12 h post-mortem from a single 37 year old asthmatic subject who died before the arrival of medical assistance following an asthma attack. Bronchial tube segments and guinea-pig and rat trachea were dissected free of surrounding tissue and cut into rings (approximately 2 mm in width). Alternate rings served as control (epithelium-intact) or test (epithelium-denuded) preparations. The epithelium was removed from test preparations using a cx)tton woolcoated probe. Histological examination of haematoxylin and cosin stained, paraffin-embedded 6 tzm transverse sections of these tracheal rings aftcr organ bath experimentation, confirmed the removal of virtually all epithelial cells down to the basement membrane, as previously demonstrated for both human bronchial rings (Fernandes et al., 1990) and for guineapig tracheal ring preparations (Goldie et al., 1986). All preparations were suspended under 0.5 g tension and equilibrated for 1 h in Krebs solution maintained at 37°C and aerated with 5% CO 2 in oxygen. Relaxant responses to cumulative concentrations of ANF-(5-27) were assessed in carbachol-contracted rat isolated trachea (EC25), guinea-pig isolated trachea (EC.~0) and in human non-diseased and asthmatic bronchial preparations (ECs0). ECzs or ECs~ concentrations of carbachol were determined from cumulative concentration-effect curves. In addition, the effect of epithelium removal on responses to ANF-(1-28) and ANF-(5-28) was determined in guinea-pig isolated trachea. In airway preparations in which ANF peptides reversed carbachol-induced tone by > 50%, relaxant responses were measured as percent maximal relaxation (Emax). Potency was then determined by interpolation as the pcptidc concentration causing 50% Em~~ i.e. ECs0. The relaxant potencies of ANF-(1-28), ANF-(5-28) and ANF-(5-27) in thesc preparations

were also expressed as pD 2 values, where pD 2 = -logmECs0. Relaxant responses were also measured as percent carbachol-induced tone and presented graphically as concentration-effect curves. In some expcriments, the effects of indomethacin (5 >M) or phosphoramidon (1 or 10/zM) on responses to ANF-(5-27) in epithelium-intact or epithelium-denuded guinea-pig and rat isolated trachea were also determined. Airway preparations were pretreatcd with these agents for 45 rain prior to and during exposure to carbachol and the construction of relaxant concentration-effect curves. ( - ) - I s o p r e n a l i n e (5 /xM) a n d / o r theophylline (1 raM) were uscd to induce rclaxation in some airway preparations precontracted with carbachol.

2.2. Radioligand binding and autoradiography Slide-mounted sections of guinea-pig trachea were incubated with [12~I]ANF-(I-28) (0.3-5 nM) in TrisHCI buffer (170 mM, pH 7.6, 22°C) containing bovine serum albumin (0.25%) and the protease inhibitor phenylmethylsulfonylfluoride (10 /xM). Unless otherwise stated, each slide contained four tracheal sections from each of three animals. Non-specific binding was determined in the presence of non-radiolabelled ANF(1-28) (1 /zM). After incubation periods of 90 min, which were found to be optimal for this tissue, unbound radioligand was removed by 2 consecutive 5 min washes in buffer at 22°C. Tissue sections werc then wiped from slides onto G F / A glass-fibre filter paper (Whatman) and counted in a Packard gamma counter. Autoradiograms were produced using slide-mounted sections of guinea-pig trachea incubated as for binding studies with [125I]ANF-(1-28) (1 riM). Following incubation and washing, tracheal sections were dried rapidly under a stream of cold, dry. air. Emulsion-coated coverslips (Kodak NTB-2) were attached with cyanoacrylate adhesive to one end of the slides and thc preparations exposed for 10 days at 4°C. The emulsion was developed in Dektol (Kodak) diluted 1:1 for 3 rain, rinsed for 15 s in 1% acetic acid containing 2.5% Hypam hardener (llford), fixed for 2:5 min with Hypam rapidfix (Ilford) diluted 1:4 with water containing 2.5% Hypam hardener and then washed in water for a minimum of 3(} rain to remove photographic fixatives. Tissue sections were stained with haematoxylin, dehydrated with ethanol, cleared in xylcne and mounted in DePeX (BDH) for light microscopy.

2.3. Drugs Unless otherwise stated, rat atriopeptide fractions were used. Drugs used were: bovine serum albumin, carbachol chloride, (-)-isoprenaline hydrochloride, theophylline, indomethacin, ANF-(5-27), phospho-

189

r.amidon (Sigma); ANF-(1-28), [~25I]ANF-(1-28), ANF-(5-28) (Auspep) and phenylmethylsulfonylfluor:de (Calbiochem). All drugs tested in organ bath studies were prepared daily in 0.9% w / v NaC1 solution (saline). ( - ) - l s o p r e n a l i n e was prepared daily in saline containing 20 ~ g / m l ascorbic acid. Stock solutions of all atriopeptides were prepared in distilled water. Solutions of phenylmcthylsulfonylfluoride were prepared in absolute ethanol and indomethacin was dissolved in Na~_CO 3 (10 m g / m l ) and diluted [br use in Krebs solution. The composition of Krcbs solution was (mM): NaCI 117.6, KCI 5.4, NaHCO,~ 25, K H z P O 4 1.03, MgSO 4 0.57, D-glucose 11.1 and CaCI 2 2.5.

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Numerical data are presented as means + S.E.M. "lhc probability of differences between mean results was determined using either Student's two-tailed t-test (non-paired) or two-way analysis of variance (Microcomputer Applied Statistics System; MASS7) as appropriate and was considered significant if P < 0.05.

140 I

10

3, Results

3.1. Organ bath studies 3.1.1. Guinea-pig trachea At EC~0, carbachol caused mean (_+S.E.M.) increases in resting tracheal tension of 2.62_+_ 0.25 g (n = 14) and 2.59 _+ 0.22 g (n = 14) in epithelium-intact and epithelium-denuded preparations, respectively. These values arc not significantly different ( P > 0.2). All three rat atriopcptide fractions caused concentration-dependent relaxation of carbacho[-contracted guinea-pig tracheal ring preparations. However, significantly greater relaxation responses were obtained in epithelium-denuded than in epithelium-intact guineapig tracheal preparations at all concentrations tested (fig. 1) as assessed by analysis of variance (P < 0.05). lr_domethacin (5 p~M) was without apparcnt effect in these preparations. Em~× values obtained to each of the atriopcptides tested were significantly lower in epithelium-intact than in epithelium-denuded tissue (table 1, P 0.2). ANF-(5-27) produced only weak relaxation responses in these preparations. Furthermorc, the weak responses obtained to this peptidc were apparently independent of the prcsencc of an intact epithelium (P > 0.05, analysis of variance). ( - ) - l s o p r e n a l i n e (5 p~M) causcd complete reversal of carbachol-induccd b:ronchial tone. Carbachol also caused concentration-dependent contraction in bronchial tissue obtained post-mortem from a single sample of asthmatic lung (pD2 = 6.13 + 0.17, n = 3). As with bronchial rings from non-asthmatic lung, ANF-(5-27) failed to relax these carbachol (ECs0) contracted preparations. I [owever, complete relaxation was obtained in response to theophylline (1

respectivcly. Specific binding of this radiolabcl accounted for only 33% of total binding at 1 nM. Data describing the concentration dependence of total and non-specific binding were subjccted to an analysis of variance, incorporating Fisher's least significant difference test. This indicated that these two curves were significantly differcnt (P < 0.05).

3.3. Autoradiography Autoradiograms were produced following incubation of slide-mounted sections of guinea-pig trachea with 1 nM [~2Sl]ANF-(1-28)in the abscnce (total bind-

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3.2. Radioligand b#zding studies

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Low levels of concentration-dependent specific binding of [~251]ANF-(I-28) werc detected in slidemounted sections of guinea-pig trachea. This specific [ ~25I]ANF-(1-28) binding analyzcd by non-lincar, least squares regression involved a saturable population of high affinity sites. The maximum number of specific binding sites (B ..... ) and the dissociation constant (K,~) fcr this ligand werc 1.78 fmol/slidc and 1.30 nM respectively. Scatchard analysis yielded similar values t'~r B,,~,~x and K,~, namely 2.60 fmol/slide and 0.97 nM

Fig. 4 (a) Darkficld photomicrograph of a 10 /xm thaw-rnot, nted frozen section of guinea-pig trachea showing the distribution of autoradiographic grains representing total ['25I]ANF-(1-28) (1 nM) binding. (b) Brightficld photomicrograph of the above section. Ep = cpithelium, SM = smooth muscle, C = cartilage. (c) Darkficld photomicrograph showing the distribution of autoradiographic grains in a section incubated with [ ~ I ] A N F - ( I - 2 8 ) (1 nM) in the presence of A N F - ( I - 2 8 ) (1 p.M), represcnting non-spccific binding. Bar = 100 ,tt nq.

192 ing) and presence (non-specific binding) of 1 /zM nonradiolabelled ANF-(1-28) (fig. 4). Relatively high levels of non-specific binding were revealed over tissue structures, with very low densities of specific autoradiographic grains detected over tracheal smooth muscle and epithelium.

4. Discussion

In the present study, all three atriopeptins tested caused relaxation of guinea-pig and rat tracheal preparations. Relaxant responsiveness to ANF was significantly modulated by epithelium removal in both species. However, whereas responsiveness of guinea-pig isolated trachea was markedly increased by removal of the epithelium, relaxation was significantly attenuated in epithelium-denuded rat tracheal preparations. In epithelium-intact guinea-pig trachea, the peptides were approximately equipotent and produced similar, low Em, ~ values. They were also approximately equipotent when tested in epithelium-denuded guineapig trachea. However, comparison of their effects in preparations with and without epithelium shows that removal of this tissue produced marked increases in their airway smooth muscle maximal relaxant effects and potencies. Thus, the epithelium may act as a site of metabolism/clearance of lhese peptides in guinea-pig. It has been demonstrated that ANF is degraded by an enzyme similar to neutral endopeptidasc (Bertrand and Doblc, 1988; Olins et al., 1987). The neutral endopeptidase inhibitor phosphoramidon was shown to inhibit the hydrolysis of this peptide (Stephenson and Kenny, 1987). In the present study, the influence of phosphoramidon was assessed with respect to guineapig and rat tracheal responsiveness to only ANF-(5-27). Our results show that phosphoramidon produced changes in responsiveness of epithelium-intact trachea to this peptide in each species that were similar to those caused by epithelium removal. Thus, relaxant responsiveness to ANF-(5-27) was markedly increased in guinea-pig trachea and significantly reduced in rat trachea in the presence of phosphoramidon. These data are consistent with previous reports of the presence of neutral endopeptidase in the epithelium of trachea from the guinea-pig (Djokic et al., 1989) and rat (Umeno ct al., 1989). However, phosphoramidon failed to exactly mimic the effects of epithelium removal in epithelium-intact guinea:pig trachea, as it also increased the potency of this peptidc in epithelium-denuded preparations, suggesting that this enzyme was present in the epithelium and elsewhere in the tracheal wall. Similarly, phosphoramidon, increased the potency of substance P in both epithelium intact and denuded guinea-pig trachea (Fine ct aI., 1989). These findings are in line with reports of

the presence of neutral endopeptidasc in non-epithelial structures including nerves, glands and airway smooth muscle (Borson et al., 1986; Sekizawa ct al., 1987). In contrast Devillier et al. (1988) did not observe any additional increase in smooth muscle sensitivity to neuropeptides in epithelium-denuded guinea-pig trachea in the presence of the neutral endopeptidasc inhibitor thiorphan. Similarly, no increase in VIP potency was seen in the presence of phosphoramidon in epithelium-denuded trachea (Farmer and Togo, 1990), suggesting that the cndopeptidase metabolizing this peptide was largely located in the epithelium. It might be expected that 10/zM phosphoramidon would inhibit neutral endopeptidase throughout the tracheal wall, resulting in maximal leftward shifts and overlapping concentration-effect curves in preparations with and without epithelium. We have not fully evaluated the reason for the additional increase in ANF potency caused by phosphoramidon in epithelium-denuded preparations. In epithelium-denuded guinea-pig trachea in which airway smooth muscle tone was first raised with carbachol (present study), the potency of ANF-(5-27) was 2-3 fold lower than that observed by O'Donncll et aI. (1985) in similar preparations with natural tone. This difference was to be expected, given the inverse relationship between the level of airway tone and the potency of relaxant agonists including /3-adrenoceptor agonists (Van den Brink, 1973) and ANF (Watanabe et al., t988). However, ..we did not observe the very high sensitivity of epithelium-intact trachea to ANF-(5-27) reported by O'Donnell et al. (1985). Furthermore, the failure of epithelium removal, with consequent rem~>val of epithelial cndopeptidasc, to increase the relaxant potency of ANF in guinea-pig tracheal preparations with only natural tone, is apparently at odds with data from the present study in carbachol-contracted trachea. The reasons for this remain unclear. We and others have previously reported that removaI of the airway epithelivm had no significant influence on the potency or maximal contractile effect of carbachol in rat trachea (Frossard and Muller, 1986), guinea-pig trachea (Goldie ct al., 1986; Hisayama et al., 1988) or human bronchus (Fernandes et al., 1990). Thus, epithelium-dependent changes in relaxant potencies of the peptidcs were not the result of altered carbachol-induced airway smooth muscle tone. All three atriopeptins caused incomplete reversal of carbachoI-induced tone in rat trachea, even though these preparations were only precontracted with an EC2.s concentration of this spasmogen. In contrast with guinea-pig trachea, greater E , ~ values were obtained in epithelium-intact than in epithelium-denuded rat tracheal preparations. Furthermore, ANF-induced relaxation responses in epithelium-in~.act rat trachea were virtually abolished in the presence of phosphoramidon,

193

s~s.ggesting that the airway epithelium in this species may act as a site of endopeptidasc-mediated conversion of ANF-(5-27) to a more active airway smooth muscle relaxant pcptide. Possibilities include peptides resulting from clcavage at Cys-7-Phc-8, Arg-14-11e-15, Gly-16-Aia-17 and Scr-25-Phc-26 (Olins et al., 1987; Stephcnson and Kenny, 1987). The greater relaxant activity of ANF-(5-27) in epithelium-intact rat trachea was not due to the release of cyclooxygcnasc products, as indomethacin had no marked effect on responsivencss to this peptide in these preparations. lnfused ANF has been shown to cause bronchodilatation in asthmatic subjects (Hulks et al., 1989). In the present study however, bronchial preparations obtained post-mortem from a single asthmatic subject failed to respond to ANF-(5-27). In accord with a previous study (Labat ct al., 1988), bronchial tissue from non-diseased samples of human lung were also unresponsive to ANF-(5-27) and this was independent of the presence of epithelium. The influence of phosphoramidon on responsiveness of human bronchus to this pcptide was not tested in the present study due to the paucity of tissue. The failure of ANF-(5-27) to induce marked relaxation of human bronchial preparations was not duc to any significant damage to airway smooth muscle, since isoprenaline and thcophylline caused complete relaxation of this tissue. The present d~.ta suggest that bronchodilatation in man following intravenous administration of ANF was not due to a direct relaxant effect on bronchial smooth muscle. Other explanations include activation of inhibitory neuronal pathways, the sccondary release of spasmolytic mediators such as prostacyclin within the airway wall and a centrally mediated reflex bronchodilatation. At present, these remain untested speculations. A single class of specific binding sites for ANF has been described by radioligand binding techniques in a variety of tissues including membrane preparations from rat lung (lwasa et al., 1988) and type 11 alveolar el~ithelial cells (Ishii et al., 1989). While previous autoradiographic studies have demonstrated specific binding of [~SI]ANF-(5-28) associated with pulmonary vascular smooth muscle and endothe[ium, specific autocadiographic grains were not detected over either airway smooth musclc or epithelium in rat lung (Bianchi et al., 1985). However, in the present study, consistent with the spasmolytic activity of ANF in guinea-pig trachea, specific binding sites for this peprice were detected over both smooth muscle and epitl~.elium in this tissue. The present study has demonstrated clear species differences with respect to the spasmolytic effect of ANF and to the influence of thc epithelium on airway responsiveness to these pcptides.

Acknowledgements This research was supported by the National [lealth and Medical Research Council of Australia aud the TVW Telethon Foundation of Western Australia. The authors also wish to thauk the Office of the Coroner for the City of Perth and thc Forensic Pathologists of the State Health Department, for making available posl-mortem specimens of human lung tissue.

References Bertrand, P. and A. Doblc, 1988, Degradation of atrial natriuretic peptides by an enzyme in rat kidney resembling neutral endopeptidase, Biochem. Pharmacol. 37, 3817. Bianchi, C., J. Gutkowska, G. Thibault, R. Garcia, J. Genest and M. Cantin, 1985, Radioautographic localization of ~251-atrial natriuretic factor (ANF) in rat tissues, Histochemistry 82, 441. Borson, D.B., B. Malfroy. M. Gold, J. Ramachandran and J.A. Nadel, 1986, Tachykinins inhibit enkephalinase activity from tracheas and lungs of ferrets, Physiologist 29, 174. De Bold, A.J., 1982, Atrial natriuretic factor of the rat heart. Studies on isolation and properties, Proc. Soc. Exp. Biol. Mcd. 170, 133. Dcvillier, P., C. Advenier, G. Drapcau, J. Marsac and D. Rcgoli, 1988, Comparison of the effects of epithelium removal and of an enkephalinase inhibitor on the ncurokinin-induced contractions of guinea-pig isolated trachea, Br. J. Pharmacol. 94, 675. Djokic, T.D., J.A. Nadcl, I').J. l)usser, K. Sekizawa, P.D. Graf and D.B. Borson, 1989, lnhibitors of neutral endopcptidase potentiate electrically and capsaicin-induccd non-cholinergic contraction in guinea-pig bronchi, J. Pharmacol. Exp. Thor. 248, 7. Farmer, S.G. and J. Togo, 1990, Effects of epithelium removal on relaxation of airway smooth muscle induced by vasoactive intestinal pcptide and electrical field stimulation, Br. J. Pharmacol. 100, 73. Fernandes, L.B., J.M.It. l'reuss, J.W. Paterson and R.G. Goldie, 1990, Epithelium-derived inhibitory factor in human bronchus, Eur. J. Pharmacol. 187, 331. Fine, J.M., T. Gordon and D. Shcppard, 1989, Epithelium removal alters responsiveness of guinea-pig trachea to substance P, J. Appl. Physiol. 66, 232. Frossard, N. and F. Muller, 1986, Epithelial modulation of tracheal smooth muscle responses to antigenic stimulation, J. Appl. Physiol. 61, 1449. Frossard, N., K.J. Rhoden and P.J. Barnes, 1989, Influence of epithelium on guinea-pig airway responses to tachykinins: Role of endopeptidasc and c3,'cloox3.genase,J. Pharmacol. Exp. Ther. 248, 292. Goldie, R.G., J.M. Papadimitriou, J.W. Paterson, P..I. Rigby, G.J. Self, and D. Spina, 1986, Influence of the epithelium on responsivcuess of guinea-pig isolated trachea to contractile and relaxant agonists, Br. J. Pharmacol. 87, 5. Hamcl, R. and A.W. Ford-Hutchinson, 1986, Relaxant profile of synthetic atrial natriurctic factor on guinea-pig pulmonary, tissues, Eur. J. Pharmacol. 121, 151. Hisayama, T., I. Takayanagi, F. Nakazato aud K. Ilirano, 1988, Epithelium selectively controls hypersensitivity of the response of smooth muscle to leukotriene D 4 by endogenous prostanoid(s) in guinea-pig trachea, Naunyn-Schmiedeb. Arch. Pharmacol. 337, 296. llulks, G., A. Jardine, J.M.C. Connell and N.C. Thomson, 1989, Bronchodilator effccts of atrial natriuretic peptidc in asthma, Br. Med. J. 299, 1081.

194

lshii, K. and F. Murad, 1989, ANP relaxes bovine tracheal smooth muscle and increases cGMP, Am. J. Physiol. 256, 495. lshii, Y., T. Watanabc, M. Watanabc, S. Hasegawa and Y. Uchiyama, 1989, Effiects of atrial natriurctic peptide on Type II alveolar epithelial cells of the rat lung. Autoradiographic and morphomctric studies, J. Anat. 166. 85. lwasa, F., M. Furuya, Y. ltayashi and N. Ohuuma, 1988, Stimt, lation of guauylatc cyclase activiLv by irreversible binding of atrial natriurclic peptidc to its receptor, Biochcm. Pharmacol. 37, 2757. Labat, C., X. Norel, J. Benvcnistc and C. Brink, 1988, Vasorclaxant effects of atrial peptidc II on isolated human pulmona~s muscle, preparations, Eur. J. Pbarmacol. 150, 397. O'Donnell, M., R. Garippa and A.F. Wclton, 1985, Relaxant activity of atriopeptins in isolated guinea-pig airway and vascular smooth muscle, Peptidcs 6. 597. Olins, G.M., K.L. Spear, N.R. Siegel and H.A. Zurchcr-Nccly, 1987, Inactivation of atrial natriurctic factor by the renal brush border, Biochim. Biophys. Acta 901, 97. Scivolctto, R. and M.H.C. Carvalho, 1984, Cardionatrin causes vasodilation in vitro which is not dependent on the presence of endothclial cells, Eur. J. Pharmacol. 101, 143. Sckizawa, K.J., J. Tamaoki. P.D. Graf, C.B. Basbaum, D.B. Borson and J.A. Nadel, 1987, Enkephalinase inhibitor potentiates mammalian tachykinin-induccd contraction in ferret trachea. J. Pha,-macol. Exp. Ther. 243, 1211.

Stcphenson, S.L. and A.J. Keuny, 1987, The hydn~lysis of c~-human atrial natriurctic pcptide by pig kidney microvillar membranes is initiated by endopcptidasc-24.11, Biochcm. J. 243, 183. Thibault, G., R. Garcia, F. Carrier, N.G. Scidah, N.G., C. Lazt, re, M. Chretien, M. Cantin and J. (icncst, 1984, Structure-activity relationships of atrial natriuretic factor (ANF). I. Natriurctic activity and relaxatkm of intestinal smooth muscle. Biochem. Biophys. Res. Commun. 125, 938. Trippodo, N.C., A.A. Macphcc, F.E. Cole and H.L. Blakeslcy, 1982~ Partial characterization of a natriuretic substance in rat atrial heart tisst, e, Proc. Soc. Exp. Biol. Mcd. 170, 502. Umeno, E., J.A. Nadel, H.T. lluang and D.M. McDonald, 19~0, Inhibition of neutral endopeptidase potentiates net, rogenic inflammation in rat trachea, J. Appl. Physiol. 66, 26A-7. Van Dcu Brink, F.G., 1973, The model of functional interaction II. Experimental verification of a new model: the antagonism of /3-adrcnoceptor stimulants and other agonists, Eur. J. Pharmacol. 22, 279. Watanabe, H., H. Furui, K. Yamaki, R. Suzuki, -l'. Takagi and T. Sataki. 1988, Atrial natriuretic polypeptidc causes dose-dependent relaxant effect ou guinea pig tracheal smooth muscle, Am. Rev. Rcspir. Dis. 137, 1112.

Epithelial modulation of the relaxant activity of atriopeptides in rat and guinea-pig tracheal smooth muscle.

Three peptide components of atrial natriuretic factor (ANF) caused relaxation of carbachol-contracted guinea-pig isolated tracheal smooth muscle. Thes...
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