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Pharmacological evaluation of the lung strip of neonatal swine
Gel,. Pharmac.. Vol. 10. pp. 381 to 384 © Pergamon Press Ltd 1979. Printed in Great Britain
0306-3623/79/0901-0381502.00/0
PHARMACOLOGICAL EVALUATION OF THE LUNG STRIP O F N E O N A T A L SWINE N. CHAND, L. DEROTH and L. P. PHANEUF
D6partement d'Anatomie et Physiologic animales, Facult6 de Medecine v6terinaire, Universit6 de Montr6al, Saint-Hyacinthe, Quebec J2S 7C6 (Received 9 January 1979)
Abstract--1. Isolated lung parenchymal strips (LPS) of neonatal swine contract to PGF:,, histamine, carbachol, norepinephrine (NE) and phenylephrine (PE). 2. Relaxations to sympathomimetic agents (isoproterenol and epinephrine), bradykinin, 5-HT and dimaprit (H2-agonist) were respectively blocked by propranolol, indomethacin, methysergide and metiamide. 3. Contractions to histamine were blocked by mepyramine, but were unaffected by metiamide. 4. Contractions to NE and PE, enhanced by propranolol, were inhibited by phentolamine and dibenzyline. 5. LPS of neonatal piglets possess :t and fl adrenoceptor mediating contraction and relaxation respectively. INTRODUCTION The knowledge of the actions of the autonomic and autacoid agents on the airway smooth muscles of man and animals constitutes the essential requirement for the better understanding of the pathophysioiogical mechanisms in the development of respiratory diseases (Widdicombe & Sterling, 1970). Neonatal piglets (pigs in general) have been extensively used as an experimental animal model for studying the pathophysiologicai processes in human neonates (Book & Bustad, 1974; Cooper, 1975; DeRoth & Downie, 1978; DeRoth & Chand, 1979). The airway pharmacology in man has been carried out on tissues obtained at necropsy or during lung surgery (Mathe et al., 1971; Dunlop & Smith, 1977; Kneussl & Richardson, 1978; Thulesius et al., 1978). As far as could be ascertained no information is available on the actions of autonomic and autacoid drugs on airway smooth muscles of human neonates, obviously due to the nonavailabi!ity of such tissues. Lulich et al. (1976) have introduced lung parenchymal strips for the pharmacological evaluation of the peripheral airways (bronchiolar-alveolar ducts and alveoli). The present investigation des.tribes the actions of some selected autonomic and autacoid agents on the lung strips of neonatal piglets.
Recordall Series 5000 pen recorder via a Narco Physiograph. It was observed that both strips of each pair produced approximately equal responses to the agonists. After establishing dose-response-curves to agonists, a predetermined concentration of an antagonist was added to one of the tissues. Responses to agonists were re-estabfished 60 min later. The second strip of each pair served as control to monitor any time related changes in the sensitivity of the tissues to agonists during the course of each experiment. The effectiveness and specificity of the antagonist was determined by measuring the "dose-ratio": the ratio of equieffective doses of agonist in the presence and absence of antagonist (Gaddum et al., 1955). Drugs used in this study were: histamine diphosphate, bradykinin triacetate, isoproterenol, epinephrine (E), norepinephrine (NE) bitartrate, phenylephrine HCI (PE), 5-hydroxytryptamine (serotonin: 5-HT) creatinine sulphate, carbamyl choline chloride (carbachol), atropine sulphate (Sigma Chemical Co., St. Louis, MO); PGE1, E2, F2, (The Upjohn Co., Kalamazoo, MI); terbutaline sulphate (Astra Pharmaceutical Products, Inc., MA); dibenzyline, dimaprit, metiamide and cimetidine (S.K.F., Weiwyn Garden City, England); salbutamol sulphate (Allen & Hanburys Research Ltd, U.K.); mepyramine maleate (Poulenc Ltd, Montreal, Quebec); propranolol (Ayerst Laboratories, NY); methysergide hydrogen maleinate (Sandoz Canada Ltd, Montreal); indomethacin (Merck Frost Laboratories) and phentolamine mesylate (Ciba). Drug concentrations are expressed as final molar (M) bath concentration.
MATERIALS AND METHODS
RESULTS
Eighteen piglets of either sex, weighing 700-1400 g were lightly anaesthetized, 6-24 hr after birth, by intraperitoneal injection of pentobarbitone sodium (30 mg/kg), followed by thoracotomy and exsanguination. Lungs were immediately removed in ice-cold oxygenated Krebs' solution. Minimum of 12 lung strips (of about 25 x 3 × 2 ram) were dissected out following the method of Lulich et al. (1976). Three pairs of lung strips were simultaneously mounted in different isolated tissue baths containing Krebs-Henseleit solution, bubbled with 5% CO2 in 02, at 37°C. Tissues were allowed to equilibrate for at least 1 hr under a resting tension of 1.5 g. Single or cumulative dose-response curves to each agonist were recorded on lung strip from 7 to II piglets with an E & M Isotonic Myograph Transducer (Narco Instrument, Houston, TX) connected to a Fisher 381
Neonatal piglet lung strips responded to PGF2, > histamine > carbachol > norepinephrine (NE) > phenylephrine (PE) with contractions i n concentration related fashion (Fig. 1). 5-HT and bradykinin (10 -6 to 10 -4) were either inactive or produced weak relaxations (n = 7). Lung strips under resting tension relaxed to isoproterenol (10-7-10 -4) and to low concentrations of epinephrine (E: 10- 6 10- s). Further increase in the concentrations of isoproterenol ( > l0 -a) and E ( > 10-'*) produced contractions of varying magnitudes (Fig. la). Indomethacin (10-6-10 -s) did not relax lung strips.
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Fig. la. Dose-response curves of PGF2~ (A), histamine (e), carbachol (®), epinephrine (I), norepinephrine (D) and phenylephrine (~) on lung strips from 7 to 9 neonatal piglets. Each point represents the mean _+ SEM. b. Dose-response curves of histamine (e), epinephrine (1), norepinephrine (O), and phenylephrine (I)) on neonatal piglet lung strips in the presence of propranolol (1 x 10-5). Each point represents the mean + SEM of strips from 6 to 9 piglets.
I0 -2
Lung strip pharmacology The lung strips which were contracted to PGF2~, histamine or carbachol responded to isoproterenol (10-7-10-5), PGEI, PGE2 (10-6-5 x 10-s), terbutaline (10-5-10-4), salbutamoi (10-6-10 -'~) and dimaprit (I0- 5-10- 3) with relaxations. The strips maximally contracted to carbachol and histamine responded to PE and NE with superimposed contractions and to epinephrine (10-6-10 -4 ) with relaxations. Mepyramine (10-7) selectively antagonized contractions to histamine (dose-ratio = 25 + 5, mean + SEM, n = 7) without altering responses to carbachol. Metiamide and cimetidine (10-6-10 -5) did not modify histamine-induced contractions. At higher concentrations (5 × 10 -5) metiamide and cimetidine equally antagonized contractions to histamine and carbachol (dose-ratios = 3 + 2, n = 5). Atropine (10 -7 ) blocked carbachol responses (doseratio = 100 + 30, n = 5), but at higher concentrations (10-6-10 -5, n = 6) it also completely antagonized histamine responses. Propranolol (1 x 10 -5) markedly enhanced contractions to epinephrine > NE > PE (Fig. lb) and, blocked or reversed relaxations to isoproterenol. After fl-blockade, the order of the efficacy of these drugs was as follows: N E > E > P G F 2 ~ > P E > h i s t a mine > carbachol > isoproterenol. Phentolamine and dibenzyline (10-s-5 x 10 -4) reversed or inhibited contractions to E, NE and PE and also antagonized histamine and carbachol responses at higher concentrations. The relaxations to 5-HT (10-5-10-4), bradykinin (10-6-10 -4) and dimaprit (10-5-5 x 10 -4) were antagonized by methysergide (10-6-10-s), indomethacin (10-6-10 -5) and metiamide (10-s-5 x 10-s), respectively.
383
Although metiamide and cimetidine did not potentiate histamine-induced contractions, however relaxations to dimaprit (a selective H2-antagonist) (Parsons et al., 1977) on the lung strips of the piglets were antagonized by metiamide. These observations seem to suggest the presence of some dimaprit sensitive H2-receptors in the peripheral airways of neonatal piglets (this study) and guinea-pigs (Chand & DeRoth, 1978b). The blockade of the contractile responses to histamine by mepyramine showed the occurrence of H:histamine receptors in peripheral airways of neonatal piglets (this study), cat (Lulich et al., 1976), rabbit, horse and guinea-pig (Chand & DeRoth, 1978a,b; 1979a,c) and tracheobronchial smooth muscles of several species (Persson & Ekman, 1976; Dunlop & Smith, 1977; Chand & Eyre, 1977a, 1978; Chand & DeRoth, 1978a,b; 1979a,b). Indomethacin (a potent PG-synthetase inhibitor) (Vane,. 1971) was found to inhibit relaxations to bradykinin on the lung strip of neonatal piglets (this study) and tracheobronchial smooth muscles of swine (Chand & DeRoth, 1978a,b; DeRoth & Chand, 1979) and of several other animal species (Chand & Eyre, 1977b). These investigations support the contention that bradykinin relaxes constricted airway smooth muscles by releasing inhibitory prostaglandins (Chand & Eyre, 1977b). Prostaglandins of E and F series are well known to have strong and opposite actions on the airway smooth muscles of several species (Kadowitz et al., 1975; Chand & Eyre, 1977a, 1978). PGEI and E2 relaxed piglet lung strips contracted to PGF2,, histamine or carbachol. As opposed to the lung strips, the tracheobronchial smooth muscles of adult and neonatal swine are unresponsive to PGF2, (Chand & DeRoth, 1978a,c: DeRoth & Chand, 1979). Among all the autonomic (carbachoi and NE, E, pE) and autacoid (histamine, 5=HT, bradykinin) DISCUSSION agents, PGF2, was found to produce maximal conThe lung parenchyma chiefly possesses bronchio- striction on the lung strip in the neonatal piglets. iar-alveolar ducts and alveoli (Lulich et al., 1976). The Therefore it may not be unreasonable to postulate lung strips of the piglets (this study), horse and rabbit that this vasoactive lipid (prostaglandins, and likeact(Chand & DeRoth, 1978a; 1979a,b) contract to carba- ing substances, endoperoxides) may be playing an imchol, histamine, PGF2,, E, NE and PE. However, pig- portant role in the development of the clinical symplet trachea partially contracted to carbachol invari- toms and in the pathogenesis of the respiratory maniably relax to E, NE and PE (DeRoth & Chand, 1979). festations in maladaptation syndrome and otlrer airPropranolol enhanced contractions to E, NE and PE way diseases in the neonates. and reversed isoproterenol- and E-induced relaxSerotonin (5-HT) is known to contract airway ations in the neonatal piglet strip (this study) and NE- smooth muscles of several mammalian species (Brockinduced relaxations in cat and guinea-pig lung strip lehurst, 1958; Chand & Eyre, 1977a, 1978). However, (Lulich et al., 1976; Chand & DeRoth, 197.8b). These 5-HT is either inactive or relaxes the entire airways observations coupled with the antagonism of contrac- of adult and neonatal swine (this study; Chand & tions to sympathomimetic agents by dibenzyline and DeRoth, 1978a,b; DeRoth & Chand, 1979). Simiphentolamine appear to suggest the presence of both lar to its action on the neonatal piglet lung strip, :t- and fl-adrenoceptors respectively mediating con- 5-HT has been described to relax the bronchioles in striction and relaxation in the peripheral airways of man and monkey (Brocklehurst, 1958) and bronchus neonatal piglets (this study), horse, rabbit, guinea-pig of man (Mathe et al., 1971). Methysergide, a 'D' mus(Chand & DeRoth, 1978a,b; 1979a,b) and cat (Lulich cular tryptamine receptor antagonist (Gaddum & et al., 1976). Picarelli, 1957) was found to antagonize relaxations Metiamide and cimetidine (H2-antagonists) (Black to 5-HT on the peripheral airways of neonatal swine et aL, 1973; Brimblecombe et aL, 1975) have earlier (this study) and human bronchus (Mathe et al., 1971). been described to potentiate contractile responses to Thus these observations seem to suggest the presence histamine or to block histamine-induced relaxations of"D"-muscular serotonin receptors in the peripheral in the airways of several species including man (Dun- airways of the piglets. lop & Smith, 1977; Chand & Eyre, 1977a, 1978; It may be concluded from this investigation that Okpako et al., 1978; Chand & DeRoth, 1978b,c). neonatal piglet lung possesses a- and fl-adrenoceptors
N. CHAND, L. DEROTH and I. P. PHANFUF
384
mediating constriction and relaxation respectively; a small population of mepyramine sensitive H~-histamine receptors and dimaprit-sensitive H2-receptors as well as some " D " tryptamine receptors. SUMMARY The lung p a r e n c h y m a l strips of the neonatal piglets responded to PGF2~ > histamine > carbachol > norepinephrine (NE) > phenylephrine (PE) with contractions. 5-HT, bradykinin (BK), 4-methylhistamine a n d dimaprit were either inactive or produced weak relaxations. At low concentrations isoproterenol and epinephrine (E) relaxed lung strips, but further increase in their concentrations produced contractions of varying magnitudes. Strips contracted to PGF2~, histamine or carbachol responded to P G E 1 , P G E 2 , isoproterenol, terbutaline a n d salbutamoi (fl-agonists) with relaxations. Relaxations to BK, 5-HT and dimaprit were inhibited by indomethacin, methysergide a n d metiamide, respectively. Metiamide a n d cimetidine (selective Hz-antagonists) did n o t alter responses to histamine a n d carbachol. Mepyramine, atropine a n d p h e n t o l a m i n e or dibenzyline antagonized contractions to histamine, carbachol a n d sympathomimetic agents, respectively. P r o p r a n o l o l (a fl-adrenoceptor antagonist) blocked or reversed relaxations to E a n d isoproterenol, and markedly enhanced contractions to E > N E > PE. Thus, it may be concluded that there are ct- a n d fl-adrenergic and H i - h i s t a m i n e receptors in the neonatal piglet lung strip. Acknowledgements--The authors thank the following companies for the generous gifts of drugs: Poulenc Ltd, Montreal, Canada for mepyramine maleate; Smith, Kline and French Canada Ltd, Montreal, Quebec for dibenzyline, dimaprit, cimetidine and metiamide; the Upjohn Co., Kalamazoo, MI, U.S.A. for prostaglandins; Ayerst Laboratories, New York for propranolol; Astra Pharmaceutical Products Inc., MA for terbutaline; Allen & Hanburys Research Ltd, U.K. for salbutamol sulphate; Merck Frost Laboratories, Quebec for indomethacin; Sandoz (Canada) Ltd, Quebec, for methysergide. Supported by grants from Agriculture Canada and the Conseil des Recherches et Services Agricoles du Qu6bec. REFERENCES
ANTHRACITE R. F., VACHON L. & KNOPP P. H. (1971) Alpha-adrenergic receptors in the human lung. Psychosore. Med. 33, 481-488. BLACK J. S., DUNCAN W. A. M., EMMETr J. C., GANELLIN C. R., HESSELBO T., PARSONS M. E. & WYLLIE J. H. (1973) Metiamide, an orally active histamine H2-receptor antagonist. Agents & Actions 3, 133-137. BOOK S. A. & BUSTAD L. K. (1974) The fetal and neonatal pig in biomedical research. J. Anita. Sci. 38, 997-1002. BRIMRLECOMBE R. W., DUNCAN W. A., DURANT G. J., EMMETT J. C., GANELLIN C. R. & PARSONS M. E. (1975) Cimetidine--A nonthiourea H2-receptor antagonist. J. Int. Med. Res. 3, 86-92. BROCKLEHURST W. (1958) The action of 5-hydroxytryptamine on smooth muscle. In: 5-Hydroxytryptamine (Edited by LEWIS J. P.), pp. 172-176. Pergamon Press, Oxford. CHAND N. & DEROTH L. (1978a) In vitro comparisons of drug responses on swine and rabbit airways. The Physiologist 21, 19. CHAND N. & DEROTH L. (1978b) Occurrence of H2-inhibitory receptors and or-excitatory adrenoceptors in guineapig lung. XI Annual Conf. of Indian Pharmacol. Society -p. 78.
CHAND N. & DEROTH L. (1978c) Actions of histamine and other substances on the airway smooth muscle of swine (in vitro). Vet. Sci. Commun. 2, 151 155. CHAND N. ~,~ DEROTH L. (1979a) Pharmacological characterization of the rabbit lung strip. Res. Commun. chem. Path. Pharmac. 23, 223 232. CHAND N..~ DEROTH L. (1979b) Responses to autonomic and autacoid agent on horse lung strip. J. Vet. Pharmac. Ther. In press. CHAND N. t~¢ LYRE P. (1977a) Spasmolytic actions of histamine in airway smooth muscle: an atypical H-receptor. Fedn. Proc. 36, 1022. CHAND N. & EYRE P. (1977b) Bradykinin relaxes contracted airways through prostaglandin production. J. Pharm. Pharmac. 27, 387-388. CHAND N. & EYRE P. (1978) Spasmolytic action of histamine in airway smooth muscle of horse. Agents & Actions g, 191-198. COLLIER H. O., HOLGATE J. A., SCHACHTER M. & SHORLEY P. G. (1960) The bronchoconstrictor action of bradykinin in the guinea-pig. Br. J. Pharmac. Chemother. tS, 290-297. COOPER J. E. (1975) The use of the pig as an animal model to study problems associated with low birthweight. Lab. " Anita. 9, 329 336. DEROTH L. & CHAND N. (1979) Pharmacological study of tracheal smooth muscle in neonatal swine. Zentbl. Vet Med. A. 26, 393-401. DEROTH L. & DOWNIE H. G. (1978) Basic cardiovascular parameters in the 'underweight neonatal swine. Biol. Neonate. 34, 155-160. DUNLOP L. S. & SMITH A. P. (1977) The effect of histamine antagonists on antigen-induced contractions of sensitized human bronchus in vitro. Br. J. Pharmac. 59, 475P. GADDUM J. n. • PICARELLI Z. P. (1957) Two kinds of tryptamine receptors. Br. J. Pharmac. 12, 323-328. GADDUM J. H., HAMEED K. A., HATHWAY D. E. & STEPHENS F. F. (1955) Quantitative studies of antagonists for 5-hydroxytryptamine. Q.J. exp. Physiol. 40, 49-74. KADOWITZ P. J., JOINER P. D. & HYMAN A. L. (1975) Physiological and pharmacological roles of prostaglandins. An. Rev. Pharmac. IS, 285-306. KNEUSSEL M. P. & RICHARDSONJ. B. (1978) Alpha-adrenergic receptors in human and canine tracheal and bronchial smooth muscle. J. appl. Physiol. 45, 307-311. LULICH K. M., MITCHELL H. W. & SPARROW M. P. (1976) The cat lung strip as an in vitro preparation of peripheral airways : a comparison of ~-adrenoceptor agonists, autacoids and anaphylactic challenge on the lung strip and trachea. Br. J. Pharmac. 58, 71-79. MATHE A. A., ASTR/3~ A. & PERSSON N. A. (1971) Some bronchoconstricting and bronchodilating responses of human isolated bronchi. Evidence for the existence of ~-adrenoceptors. J. Pharm. Pharmac. 23, 905-910. OKPAKO D. T., CHAND N. & LYRE P. (1978) The presence of inhibitory histamine H2-receptors in guinea-pig tracheobronchial muscle. J. Pharm. Pharmac. 30, 280-282. PARSONS M. E., OWEN D. A. A., GANELLIN C. R. & DURANT G. J. (1977) Dimaprit-S-[3(N,N-dimethylamino)propyl]isothiourea]--A highly specific histamine H2-receptot agonist. Part I. Pharmacology. Agents & Actions 7, 31-37. PERSSON C. G. A. & EKMAN M. (1976) Contractile effects of histamine in large and small respiratory airways. Agents & Actions 6, 389-393. VANE J. R. (1971) Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature N e w Biol. 231, 323-335. THULESIUS O., BOE J. & BERLIN E. (1978) Physiological and pharmacological studies on isolated human bronchial preparations. Scand. J. Resp. Dis. 59, 66-74. WIDDICOMBE J. G. t~ STERLING G. (1970) The autonomic nervous system and breathing. Archs intern. Med. 126, 31 !-329.
0306-3623/79/0901-0381502.00/0
PHARMACOLOGICAL EVALUATION OF THE LUNG STRIP O F N E O N A T A L SWINE N. CHAND, L. DEROTH and L. P. PHANEUF
D6partement d'Anatomie et Physiologic animales, Facult6 de Medecine v6terinaire, Universit6 de Montr6al, Saint-Hyacinthe, Quebec J2S 7C6 (Received 9 January 1979)
Abstract--1. Isolated lung parenchymal strips (LPS) of neonatal swine contract to PGF:,, histamine, carbachol, norepinephrine (NE) and phenylephrine (PE). 2. Relaxations to sympathomimetic agents (isoproterenol and epinephrine), bradykinin, 5-HT and dimaprit (H2-agonist) were respectively blocked by propranolol, indomethacin, methysergide and metiamide. 3. Contractions to histamine were blocked by mepyramine, but were unaffected by metiamide. 4. Contractions to NE and PE, enhanced by propranolol, were inhibited by phentolamine and dibenzyline. 5. LPS of neonatal piglets possess :t and fl adrenoceptor mediating contraction and relaxation respectively. INTRODUCTION The knowledge of the actions of the autonomic and autacoid agents on the airway smooth muscles of man and animals constitutes the essential requirement for the better understanding of the pathophysioiogical mechanisms in the development of respiratory diseases (Widdicombe & Sterling, 1970). Neonatal piglets (pigs in general) have been extensively used as an experimental animal model for studying the pathophysiologicai processes in human neonates (Book & Bustad, 1974; Cooper, 1975; DeRoth & Downie, 1978; DeRoth & Chand, 1979). The airway pharmacology in man has been carried out on tissues obtained at necropsy or during lung surgery (Mathe et al., 1971; Dunlop & Smith, 1977; Kneussl & Richardson, 1978; Thulesius et al., 1978). As far as could be ascertained no information is available on the actions of autonomic and autacoid drugs on airway smooth muscles of human neonates, obviously due to the nonavailabi!ity of such tissues. Lulich et al. (1976) have introduced lung parenchymal strips for the pharmacological evaluation of the peripheral airways (bronchiolar-alveolar ducts and alveoli). The present investigation des.tribes the actions of some selected autonomic and autacoid agents on the lung strips of neonatal piglets.
Recordall Series 5000 pen recorder via a Narco Physiograph. It was observed that both strips of each pair produced approximately equal responses to the agonists. After establishing dose-response-curves to agonists, a predetermined concentration of an antagonist was added to one of the tissues. Responses to agonists were re-estabfished 60 min later. The second strip of each pair served as control to monitor any time related changes in the sensitivity of the tissues to agonists during the course of each experiment. The effectiveness and specificity of the antagonist was determined by measuring the "dose-ratio": the ratio of equieffective doses of agonist in the presence and absence of antagonist (Gaddum et al., 1955). Drugs used in this study were: histamine diphosphate, bradykinin triacetate, isoproterenol, epinephrine (E), norepinephrine (NE) bitartrate, phenylephrine HCI (PE), 5-hydroxytryptamine (serotonin: 5-HT) creatinine sulphate, carbamyl choline chloride (carbachol), atropine sulphate (Sigma Chemical Co., St. Louis, MO); PGE1, E2, F2, (The Upjohn Co., Kalamazoo, MI); terbutaline sulphate (Astra Pharmaceutical Products, Inc., MA); dibenzyline, dimaprit, metiamide and cimetidine (S.K.F., Weiwyn Garden City, England); salbutamol sulphate (Allen & Hanburys Research Ltd, U.K.); mepyramine maleate (Poulenc Ltd, Montreal, Quebec); propranolol (Ayerst Laboratories, NY); methysergide hydrogen maleinate (Sandoz Canada Ltd, Montreal); indomethacin (Merck Frost Laboratories) and phentolamine mesylate (Ciba). Drug concentrations are expressed as final molar (M) bath concentration.
MATERIALS AND METHODS
RESULTS
Eighteen piglets of either sex, weighing 700-1400 g were lightly anaesthetized, 6-24 hr after birth, by intraperitoneal injection of pentobarbitone sodium (30 mg/kg), followed by thoracotomy and exsanguination. Lungs were immediately removed in ice-cold oxygenated Krebs' solution. Minimum of 12 lung strips (of about 25 x 3 × 2 ram) were dissected out following the method of Lulich et al. (1976). Three pairs of lung strips were simultaneously mounted in different isolated tissue baths containing Krebs-Henseleit solution, bubbled with 5% CO2 in 02, at 37°C. Tissues were allowed to equilibrate for at least 1 hr under a resting tension of 1.5 g. Single or cumulative dose-response curves to each agonist were recorded on lung strip from 7 to II piglets with an E & M Isotonic Myograph Transducer (Narco Instrument, Houston, TX) connected to a Fisher 381
Neonatal piglet lung strips responded to PGF2, > histamine > carbachol > norepinephrine (NE) > phenylephrine (PE) with contractions i n concentration related fashion (Fig. 1). 5-HT and bradykinin (10 -6 to 10 -4) were either inactive or produced weak relaxations (n = 7). Lung strips under resting tension relaxed to isoproterenol (10-7-10 -4) and to low concentrations of epinephrine (E: 10- 6 10- s). Further increase in the concentrations of isoproterenol ( > l0 -a) and E ( > 10-'*) produced contractions of varying magnitudes (Fig. la). Indomethacin (10-6-10 -s) did not relax lung strips.
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CONCENTRATION ( M )
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Fig. la. Dose-response curves of PGF2~ (A), histamine (e), carbachol (®), epinephrine (I), norepinephrine (D) and phenylephrine (~) on lung strips from 7 to 9 neonatal piglets. Each point represents the mean _+ SEM. b. Dose-response curves of histamine (e), epinephrine (1), norepinephrine (O), and phenylephrine (I)) on neonatal piglet lung strips in the presence of propranolol (1 x 10-5). Each point represents the mean + SEM of strips from 6 to 9 piglets.
I0 -2
Lung strip pharmacology The lung strips which were contracted to PGF2~, histamine or carbachol responded to isoproterenol (10-7-10-5), PGEI, PGE2 (10-6-5 x 10-s), terbutaline (10-5-10-4), salbutamoi (10-6-10 -'~) and dimaprit (I0- 5-10- 3) with relaxations. The strips maximally contracted to carbachol and histamine responded to PE and NE with superimposed contractions and to epinephrine (10-6-10 -4 ) with relaxations. Mepyramine (10-7) selectively antagonized contractions to histamine (dose-ratio = 25 + 5, mean + SEM, n = 7) without altering responses to carbachol. Metiamide and cimetidine (10-6-10 -5) did not modify histamine-induced contractions. At higher concentrations (5 × 10 -5) metiamide and cimetidine equally antagonized contractions to histamine and carbachol (dose-ratios = 3 + 2, n = 5). Atropine (10 -7 ) blocked carbachol responses (doseratio = 100 + 30, n = 5), but at higher concentrations (10-6-10 -5, n = 6) it also completely antagonized histamine responses. Propranolol (1 x 10 -5) markedly enhanced contractions to epinephrine > NE > PE (Fig. lb) and, blocked or reversed relaxations to isoproterenol. After fl-blockade, the order of the efficacy of these drugs was as follows: N E > E > P G F 2 ~ > P E > h i s t a mine > carbachol > isoproterenol. Phentolamine and dibenzyline (10-s-5 x 10 -4) reversed or inhibited contractions to E, NE and PE and also antagonized histamine and carbachol responses at higher concentrations. The relaxations to 5-HT (10-5-10-4), bradykinin (10-6-10 -4) and dimaprit (10-5-5 x 10 -4) were antagonized by methysergide (10-6-10-s), indomethacin (10-6-10 -5) and metiamide (10-s-5 x 10-s), respectively.
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Although metiamide and cimetidine did not potentiate histamine-induced contractions, however relaxations to dimaprit (a selective H2-antagonist) (Parsons et al., 1977) on the lung strips of the piglets were antagonized by metiamide. These observations seem to suggest the presence of some dimaprit sensitive H2-receptors in the peripheral airways of neonatal piglets (this study) and guinea-pigs (Chand & DeRoth, 1978b). The blockade of the contractile responses to histamine by mepyramine showed the occurrence of H:histamine receptors in peripheral airways of neonatal piglets (this study), cat (Lulich et al., 1976), rabbit, horse and guinea-pig (Chand & DeRoth, 1978a,b; 1979a,c) and tracheobronchial smooth muscles of several species (Persson & Ekman, 1976; Dunlop & Smith, 1977; Chand & Eyre, 1977a, 1978; Chand & DeRoth, 1978a,b; 1979a,b). Indomethacin (a potent PG-synthetase inhibitor) (Vane,. 1971) was found to inhibit relaxations to bradykinin on the lung strip of neonatal piglets (this study) and tracheobronchial smooth muscles of swine (Chand & DeRoth, 1978a,b; DeRoth & Chand, 1979) and of several other animal species (Chand & Eyre, 1977b). These investigations support the contention that bradykinin relaxes constricted airway smooth muscles by releasing inhibitory prostaglandins (Chand & Eyre, 1977b). Prostaglandins of E and F series are well known to have strong and opposite actions on the airway smooth muscles of several species (Kadowitz et al., 1975; Chand & Eyre, 1977a, 1978). PGEI and E2 relaxed piglet lung strips contracted to PGF2,, histamine or carbachol. As opposed to the lung strips, the tracheobronchial smooth muscles of adult and neonatal swine are unresponsive to PGF2, (Chand & DeRoth, 1978a,c: DeRoth & Chand, 1979). Among all the autonomic (carbachoi and NE, E, pE) and autacoid (histamine, 5=HT, bradykinin) DISCUSSION agents, PGF2, was found to produce maximal conThe lung parenchyma chiefly possesses bronchio- striction on the lung strip in the neonatal piglets. iar-alveolar ducts and alveoli (Lulich et al., 1976). The Therefore it may not be unreasonable to postulate lung strips of the piglets (this study), horse and rabbit that this vasoactive lipid (prostaglandins, and likeact(Chand & DeRoth, 1978a; 1979a,b) contract to carba- ing substances, endoperoxides) may be playing an imchol, histamine, PGF2,, E, NE and PE. However, pig- portant role in the development of the clinical symplet trachea partially contracted to carbachol invari- toms and in the pathogenesis of the respiratory maniably relax to E, NE and PE (DeRoth & Chand, 1979). festations in maladaptation syndrome and otlrer airPropranolol enhanced contractions to E, NE and PE way diseases in the neonates. and reversed isoproterenol- and E-induced relaxSerotonin (5-HT) is known to contract airway ations in the neonatal piglet strip (this study) and NE- smooth muscles of several mammalian species (Brockinduced relaxations in cat and guinea-pig lung strip lehurst, 1958; Chand & Eyre, 1977a, 1978). However, (Lulich et al., 1976; Chand & DeRoth, 197.8b). These 5-HT is either inactive or relaxes the entire airways observations coupled with the antagonism of contrac- of adult and neonatal swine (this study; Chand & tions to sympathomimetic agents by dibenzyline and DeRoth, 1978a,b; DeRoth & Chand, 1979). Simiphentolamine appear to suggest the presence of both lar to its action on the neonatal piglet lung strip, :t- and fl-adrenoceptors respectively mediating con- 5-HT has been described to relax the bronchioles in striction and relaxation in the peripheral airways of man and monkey (Brocklehurst, 1958) and bronchus neonatal piglets (this study), horse, rabbit, guinea-pig of man (Mathe et al., 1971). Methysergide, a 'D' mus(Chand & DeRoth, 1978a,b; 1979a,b) and cat (Lulich cular tryptamine receptor antagonist (Gaddum & et al., 1976). Picarelli, 1957) was found to antagonize relaxations Metiamide and cimetidine (H2-antagonists) (Black to 5-HT on the peripheral airways of neonatal swine et aL, 1973; Brimblecombe et aL, 1975) have earlier (this study) and human bronchus (Mathe et al., 1971). been described to potentiate contractile responses to Thus these observations seem to suggest the presence histamine or to block histamine-induced relaxations of"D"-muscular serotonin receptors in the peripheral in the airways of several species including man (Dun- airways of the piglets. lop & Smith, 1977; Chand & Eyre, 1977a, 1978; It may be concluded from this investigation that Okpako et al., 1978; Chand & DeRoth, 1978b,c). neonatal piglet lung possesses a- and fl-adrenoceptors
N. CHAND, L. DEROTH and I. P. PHANFUF
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mediating constriction and relaxation respectively; a small population of mepyramine sensitive H~-histamine receptors and dimaprit-sensitive H2-receptors as well as some " D " tryptamine receptors. SUMMARY The lung p a r e n c h y m a l strips of the neonatal piglets responded to PGF2~ > histamine > carbachol > norepinephrine (NE) > phenylephrine (PE) with contractions. 5-HT, bradykinin (BK), 4-methylhistamine a n d dimaprit were either inactive or produced weak relaxations. At low concentrations isoproterenol and epinephrine (E) relaxed lung strips, but further increase in their concentrations produced contractions of varying magnitudes. Strips contracted to PGF2~, histamine or carbachol responded to P G E 1 , P G E 2 , isoproterenol, terbutaline a n d salbutamoi (fl-agonists) with relaxations. Relaxations to BK, 5-HT and dimaprit were inhibited by indomethacin, methysergide a n d metiamide, respectively. Metiamide a n d cimetidine (selective Hz-antagonists) did n o t alter responses to histamine a n d carbachol. Mepyramine, atropine a n d p h e n t o l a m i n e or dibenzyline antagonized contractions to histamine, carbachol a n d sympathomimetic agents, respectively. P r o p r a n o l o l (a fl-adrenoceptor antagonist) blocked or reversed relaxations to E a n d isoproterenol, and markedly enhanced contractions to E > N E > PE. Thus, it may be concluded that there are ct- a n d fl-adrenergic and H i - h i s t a m i n e receptors in the neonatal piglet lung strip. Acknowledgements--The authors thank the following companies for the generous gifts of drugs: Poulenc Ltd, Montreal, Canada for mepyramine maleate; Smith, Kline and French Canada Ltd, Montreal, Quebec for dibenzyline, dimaprit, cimetidine and metiamide; the Upjohn Co., Kalamazoo, MI, U.S.A. for prostaglandins; Ayerst Laboratories, New York for propranolol; Astra Pharmaceutical Products Inc., MA for terbutaline; Allen & Hanburys Research Ltd, U.K. for salbutamol sulphate; Merck Frost Laboratories, Quebec for indomethacin; Sandoz (Canada) Ltd, Quebec, for methysergide. Supported by grants from Agriculture Canada and the Conseil des Recherches et Services Agricoles du Qu6bec. REFERENCES
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