TiPS- February 1990 [Vol. 11] evaluating the reliability of affinity estimates obtained by pharmacological analysis.
References 1 Colquhoun, D. (1987) in Perspectives on Receptor Classification (Black, J. W., Jenkinson, D. H. and Gerskowitch, V. P., eds), pp. 103-114, Alan R. Liss 2 Mackay, D. (1988) Trends Pharmacol. Sci.
67 9, 156-157 3 Kenakin, T. P. (1989) Trends Pharmacol. Sci. 10, 18-22 4 Left, P. and Harper, D. H. (1989) J. Theor. Biol. 140, 381-397 5 Furchgott, R. F. (1966) Adv. Drug Res. 3, 21-55 6 Barlow, R. B., Scott, N. C. and Stephenson, R. P. (1967) Br. J. Pharmacol. 31, 188-196 7 Stephenson, R. P. (1956) Br. J. Pharmacol. 11, 379-393
8 del Castillo, J. and Katz, B. (1957) Proc. R. Soc. London Ser. B 146, 369-381 9 de Lean, A., Stadel, J. M. and Lefkowitz, R. J. (1978) J. Biol. Chem. 255, 7108-7117 10 Mackay, D. (1990) Trends Pharmacol. Sci. 11, 17-22 11 Waud, D. R. (1969) J. Pharmacol. Exp. Ther. 170, 117-122 12 Besse, J. C. and Furchgott, R. F. (1976) J. Pharmacol. Exp. Ther. 197, 66-78 13 Kenakin, T. P. and Beek, D. (1980) J. Pharmacol. Exp. Ther. 213, 406-413
Airway epithelium and smooth muscle responsiveness
Airway epithelium-derived inhibitory factor Roy G. Goldie, Lynette B. Fernandes, Stephen G. Farmer and Douglas W. P. Hay Various bronchoactive agents can induce the release from the airway epithelium of an inhibitory substance that is able to relax certain tissues including rat aorta and possibly also airway smooth muscle. This substance, whose existence has recently been confirmed using a new bioassay system, is distinct from nitric oxide (EDRF) and is also known to be non-prostanoid in nature. Roy Goldie and colleagues describe the properties of this factor, and its potential clinical significance. One of the cardinal diagnostic symptoms of bronchial asthma is the exquisite 'twitchiness' of the airways to pharmacological, environmental and allergic stimuli 1. The mechanisms underlying this nonspecific airway hyperresponsiveness, although the subject of a great deal of scientific and clinical investigation, are not yet established. Although its precise role in the pathogenesis of asthma has not been elucidated, it has long been known that the airway epithelium in asthmatic lungs is abnormal 2. Indeed, epithelial damage associated with airway inflammation is a characteristic feature in both mild and severe asthma and may be causally re-
R. G. Goldie is a Senior Research Fellow at the Department of Pharmacology, University of Western Australia, Perth, Nedlands, W A 6009, Australia, and L. B. Fernandes has recently completed PhD studies in this Department. S. G. Farmer is a Staff Scientist at Nova Pharmaceutical Corporation, 6200 Freeport Centre, Baltimore, MD 21224-2788, USA. D. W. P. Hay is a Senior Investigator at the Department of Pharmacology, SmithKline Beecham Laboratories, King of Prussia, PA 19406-0939, USA.
lated to the accompanying airway hyperresponsiveness 3'4. Until recently, the epithelium was regarded generally as having a largely passive role in airways, providing a barrier to the penetration of stimulatory solutes, including allergens, to the underlying smooth muscle, submucosal mast cells, irritant receptors and C-fiber afferent nerve endings involved in axon reflexes. However, epithelial secretions and the activity of cilia are important to the effective clearance of irritant particles from the bronchi 5. Inflammatory events, possibly involving the cytotoxic effects of eosinophil-derived major basic protein, disrupt epithelial cell tight junctions, thereby compromising the protective function of this tissue s and perhaps contributing to the airway hyperresponsiveness. For some years now, it has been known that at least 12 different epithelial cell t y p e s 7 h a v e profound effects on abnormal and normal lung function. This review focuses on recent evidence that the epithelium can modulate airway smooth muscle responsiveness.
Mechanical removal of the airway epithelium increases responsiveness of airway smooth muscle to a variety of spasmogens. This phenomenon was first demonstrated in canine bronchus 8 and was confirmed in airways from cattle 9, guinea-pigs w-12, rabbits ~3 and rats 14. Deliberate epithelial removal from human bronchial preparations causes increases in spasmogen potency similar to those obtained in these animal models 15. Clearly, such models, which involve complete removal of the epithelium, can only approximate to the sometimes patchy epithelial damage seen in human asthma. It is not established whether shedding of the epithelium in vivo in humans results in enhanced airway contractility to spasmogens in vitro. The mechanisms underlying the phenomenon remain the subject of controversy. For some agonists the epithelium may act as a site of loss (i.e. site of metabolism) from the biophase, limiting the access of the agent to the smooth muscle, and thereby reducing its effect as an agonist. This has been reported for isoprenaline is, adenosine 17, substance P and related tachykinins 18'19, vasoactive intestinal peptide 2°, and arachidonic acid 21-23. It is also possible that removal of the epithelium facilitates access of bronchoconstrictor substances to submucosal structures including the smooth muscle, resulting in increased responsiveness to spasmogens 24. However, this may not explain the selective enhancement of sensitivity to only some airway spasmogens 14'2s, or reduced responsiveness to some relaxant agonists such as isoprenaline, or the effects of epithelium removal on neurogenic contractions 8. Conversely, there is evidence that
1990,ElsevierSciencePublishersLtd. (UK) 0165- 6147/90/$02.00
68
TiPS - February 1990 [Vol. 11]
i #i
rat ao~a
m MI-
guinea-pig tracheal tube
Fig. 1. A co-axial assembly for the bioassay of EpDIF released from donor airway tissue, as first described by lid. Ilhan and L Sahin (1986) Eur. J. Pharmacol. 131, 293-296.
the effect of epithelium removal on responsiveness of perfused trachea from sensitized guineapigs to antigen challenge in vitro is due, at least partly, to loss of a diffusion barrier 26. Another explanation is that the airway epithelium may release one or more inhibitory factors, either basally or in response to some agonists, which are capable of attenuating airway responsiveness 26-28. Such a system may function in a manner analogous to that described for the influence of an endothelium-derived relaxing factor (EDRF) on vascular smooth muscle 29. Bronchoconstriction to a particular spasmogen in nonasthmatics is limited, reaching a plateau level at high doses, whereas asthmatics continue to bronchoconstrict in a dosedependent manner. This suggests that there is a lack of a 'braking' mechanism which would otherwise prevent an excessive response 3°. It is possible then that, in asthma, epithelial damage or dysfunction causes a reduction in the influence of an epithelium-derived inhibitory factor(s) (EpDIF) and thus contributes to the characteristic airway hyperresponsiveness associated with this chronic disease.
EpDIF: fact or fiction? Lundblad and Persson 31 concluded that the presence or absence of epithelium was of little
consequence to the pharmacology of the guinea-pig tracheal open ring preparation in vitro. It is important to note, however, that this study examined responsiveness to carbachol, the potency of which is known to be unaltered by epithelium stripping 1°'14, and is in contrast to the effect of epithelium removal on responsiveness to other cholinomimetics such as methacholine 11'13"1s'32 as well as to acetylcholine itselfs'12. Furthermore, responsiveness to histamine, which has been confirmed in many studies to be enhanced by epithelium stripping 8'13'24'26'32, was not tested 31. In addition to the notable species differences that have been reported, appreciable differences in the magnitude of the influence of the epithelium are evident, and various methodological differences have perhaps contributed to apparently conflicting results within some species. A large body of independent research has, however, confirmed the phenomenon of epithelium-dependent modulation of mammalian airway smooth muscle responsiveness to several agonists in vitro, although the majority of these data, while consistent with the existence of EpDIF, provide only indirect support for its existence. Descriptions of the transfer of this putative inhibitory autacoid from epithelium and its subsequent bio-
assay would provide convincing direct evidence in support of its existence, in addition to contributing to its eventual identification. The first attempt to detect EpDIF using a superfusion cascade bioassay technique failed to produce any evidence for the release of such a factor from guineapig tracheal epithelium 24. The possibility of preferential abluminal secretion of EpDIF, or of dilution and rapid degradation of putative inhibitory substances(s) in such transfer systems, has encouraged other workers to use 'sandwich' preparations (as described by Furchgott and Zawads k i 29 in studies of EDRF), or variants of them in attempts to detect EpDIF.
Bioassay of EpDIF A co-axial sandwich assembly, in which the recipient or assay preparation is mounted within the lumen of a donor airway tube segment was first described by Ilhan and Sahin (Fig. 1). This study provided the first direct evidence for the release and transfer of an epithelium-derived inhibitory substance capable of causing relaxation of endothelium-denuded rabbit aorta: acetylcholine produced relaxation of aortic tissue when it was positioned within a guinea-pig tracheal tube segment with an intact epithelium. No relaxation
a
b
c
l
/" r
histamine 100 W¢
50 mg [
\
10 rain
phenylephrine0.05 ~
•
•
•
Fig. 2. Responses induced by histamine (100 [JM; •) in phenylephrine-precontracted ( • ) rat aorta strips, a: with an intact endothelium; b: with the endothelium removed; ¢: with the endothelium removed and the strip mounted co-axially within the lumen of an epithelium-intact guinea-pig tracheal tube segment.
69
TiPS - February 1990 [Vol. 11]
was seen in the absence of epithelium. Using co-axial preparations, the ability of EpDIF released from guinea-pig trachea to relax precontracted rat anococcygeus muscle 33 and rat aorta 34 (Fig. 2) in the presence of indometacin or propranolol has recently been demonstrated. The contractile potency of ovalbumin in tracheal preparations from sensitized guinea-pigs had been shown to be greater in epithelium-denuded than in epithelium-intact tissue 32. This effect was attributed to the loss of EpDIF and resultant enhancement of the potency of histamine and leukotrienes released in response to ovalbumin, although increased release of mediators from mast cells might also be involved 26. Indeed, when similar epitheliumdenuded preparations were mounted co-axially within an epithelium-intact guinea-pig tracheal tube, sensitivity to ovalbumin was reduced to levels observed in intact preparations 3s. These data suggest that a smooth muscle inhibitory factor (EpDIF) is released, either passively or in response to spasmogens, and transferred from donor airway epithelium; this results in attenuation of the contractile effect of the spasmogens (histamine and leukotrienes) released by ovalbumin.
Nature and mechanism of action of EpDIF Smooth muscle spasmogens including histamine stimulate the production and release of relaxant prostaglandins such as PGE2 from airway preparations in vitro 36. The epithelium is a source of various prostanoids including PGE2. The possibility that EpDIF is a relaxant prostaglandin is suggested by studies showing that inhibition of epithelial cyclooxygenase to some extent mimics the effect of epithelium stripping on contractions of guinea-pig trachea induced by leukotriene C4 (Ref. 25) or methacholine 11, and on bethanecholinduced contraction in rabbit b r o n c h u s 22.
However, in many studies increased responsiveness of airway preparations to some spasmogens caused by epithelium stripping remained unaltered in the presence of inhibitors of cyclooxygenase and/or lipoxygenases,9,11,24.
Furthermore, the production of guinea-pig tracheal and h u m a n bronchial EpDIF and its direct coaxial bioassay on rat anococcygeus or aorta have been demonstrated in the presence of inhibitors of these enzymes 33"34. Moreover, PGE2 causes contraction of rat aorta 37. Thus, while the epithelium is capable of releasing relaxant as well as constrictor prostaglandins, it is clear that a non-prostanoid inhibitory factor is also released from mammalian airway epithelium. The identity of EpDIF awaits clarification. The evidence indicates that EpDIF is not the same as EDRF. Epithelium-dependent relaxation of vascular34 or gastrointestinal33 smooth muscle was not affected by methylene blue, which inhibits the effects of EDRF on soluble guanylyl cyclase. EpDIF must therefore be an agent that produces endothelium-independent relaxation in these systems. The inflammatory mediator platelet activating factor (PAF) was inactive in rat aorta 34, showing that it is not EpDIF. Preliminary evidence suggests that EpDIF does not act via an alteration in extracellular Ca 2+ translocation 3s. []
[]
[]
There is a growing body of evidence in support of the idea that several bronchoactive agents can induce the release of a nonprostanoid inhibitory substance from the airway epithelium. It has now been shown that EpDIF, an epithelium-derived factor from guinea-pig trachea, relaxes vascular 34 and gastrointestinal smooth muscle 33 and may also relax airway smooth muscle 39. It remains to be determined whether relaxation of airway and non-airway smooth muscle is produced by the same epithelium-derived factor: is there more than one EpDIF? It is also possible that this autacoid can reduce spasmogen potency in the airway by means additional to functional antagonism at airway smooth muscle. Vasodilation of the bronchial circulation in vivo, following release of EpDIF, may enhance the clearance of inhaled chemical stimulants diffusing through the mucosa towards airway smooth muscle, mast cells
and nerve fibers. The marked epithelium-dependent relaxation of vascular tissue may indicate that the effects of EpDIF are more profound and physiologically relevant in the vascular beds of the airway wall than in the airway smooth muscle per se. EpDIF might exert inhibitory effects on some or all of these submucosal tissues, as well as on inflammatory cells. Challenges to be addressed include the elucidation of the nature and mechanisms of action of EpDIF. This will require the development of a reliable, sensitive system for the demonstration of the release of measurable quantities of EpDIF. Furthermore, and although a difficult task, it will be important to assess the pathophysiological relevance of EpDIF in lung function in vivo, both in animals and in humans. Identification of EpDIF may lead to development of novel agents (e.g. EpDIF analogues, stimulants of its release, or inhibitors of its degradation) in the therapy of airway disease.
References 1 Boushey, H. A., Holtzman, M. J., Sheller, J. R. and Nadel, J. A. (1980) Am. Rev. Respir. Dis. 121, 389-413 2 Cutz, E., Levison, H. and Cooper, D. M. (1978) Histopathology 2, 407-421 3 Boushey, H. A. and Holtzman, M. J. (1985) Am. Rev. Respir. Dis. 131, 312-313 4 Laitinen, L. A., Heino, M., Laitinen, A. and Haahtela, T. (1985) Am. Rev. Respir. Dis. 131, 599-606 5 Sleigh, M. A., Blake, J. R. and Liron, N. (1988) Am. Rev. Respir. Dis. 137, 726-731 6 Frigas, E. and Gleich, G. J. (1986) J. Allergy Clin. Immunol. 77, 527-537 7 Breeze, R. G. and Wheeldon, E. B. (1977) Am. Rev. Respir. Dis. 116, 705--777 8 Flavahan, N. A., Aarhus, L. L., Rimele, T. J. and Vanhoutte, P. M. (1985) J. Appl. Physiol. 58, 834-838 9 Barnes, P. J., Cuss, F. M. and Palmer, I. B. (1985) Br. J. Pharmacol. 86, 685-691 10 Goldie, R. G. et al. (1986) Br. J. Pharmacol. 87, 5-14 11 Hay, D. W. P. et al. (1986) Eur. J. Pharmacol. 129, 11-18 12 Murlas, C. (1986) Clin. Sci. 70, 571-575 13 Raeburn, D. et al. (1986) Life Sci. 38, 809-816 14 Frossard, N. and Muller, F. (1986) ]. Appl. Physiol. 61. 1449-1456 15 Raeburn, D., Hay, D. W. P., Farmer, S. G. and Fedan, J. S. (1986) Eur. J. Pharmacol. 123, 451-453 16 Farmer, S. G., Fedan, J. S., Hay, D. W. P. and Raeburn, D. (1986) Br. J. Pharmacol. 89, 407-414 17 Advenier, C., Devillier, P., Matran, R. and Naline, E. (1988) Br. J. Pharmacol. 93, 295-302 18 Fine, I. M., Gordon, T. and Sheppard, D. (1989) J. Appl. Physiol. 66, 232-237
TiPS
70 19 Frossard, N., Rhoden, K. J. and Barnes, P. J. (1989) J. Pharmacol. Exp. Ther. 248, 292-298 20 Farmer, S. G. and Togo, J. (1989) Br. ]. Pharmacol. 98, 784P 21 Nijkamp, F. P. and Folkerts, G. (1986) Eur. J. Pharmacol. 131, 315-316 22 Butler, G. B., Adler, K. B., Evans, J. N., Morgan, D. W. and Szarek, J. L. (1987) Am. Rev. Respir. Dis. 135, 1099-1104 23 Farmer, S. G., Hay, D. W. P., Raeburn, D. and Fedan, J. S. (1987) Br. J. Pharmacol. 92, 231-236 24 Holroyde, M. C. (1986) Br. J. Pharmacol. 87, 501-507 25 Hay, D. W. P. et al. (1987) Br. J. Pharmacol. 92, 381-388 26 Undem, B. J., Raible, D. G., Adkinson,
27 28 29 30 31 32 33
N. F. and Adams, G. K. (1988)J. Pharmacol. Exp. Ther. 244, 659-665 Farmer, S. G. (1987) Trends Pharmacol. Sci. 8, 8-10 Vanhoutte, P. M. (1988) Am. Rev. Respir. Dis. 138, $24-$30 Furchgott, R. F. and Zawadzki, J. V. (1980) Nature 288, 373-376 Woolcock, A. J., Salome, C. M. and Yan, K. (1984) Am. Rev. Respir. Dis. 130, 71-75 Lundblad, K. A. L. and Persson, C. G. A. (1988) Br. J. Pharmacol. 93, 909-917 Hay, D. W. P., Raeburn, D., Farmer, S. G., Fleming, W. W. and Fedan, J. S. (1986) Life Sci. 38, 2461-2468 Guc, M. O., Ilhan, M. and Kayaalp, S. O. (1988) Eur. J. Pharmacol. 148, 405--409
K-Opioid receptors and analgesia Mark J. Millan Over the past decade, opioids have attracted great attention. One important reason for this is the need for novel, strong analgesics free of the abuse potential and side-effects of narcotics such as morphine. Because morphine acts at ~t-opioid receptors, efforts have been made to characterize analgesia mediated by non-~t sites, in particular K-opioid receptors. There is now good evidence that K-receptors do indeed mediate analgesia. H o w e v e r , K-agonists display properties that could curtail their therapeutic exploitation. Since the first selective K-agonists are now entering clinical trials, this is an opportune moment for M a r k Millan to review the pharmacology of drugs of this type in the control of nociception and their therapeutic potential as analgesics. T h e n a r c o t i c o p i o i d analgesics, of w h i c h t h e p r o t o t y p e is m o r p h i n e , are of e n o r m o u s t h e r a p e u t i c i m p o r t a n c e in the c o n t r o l of m o d e r ate to severe p a i n ~. U n f o r t u n a t e l y , s i g n i f i c a n t p r o b l e m s remain1'2: c e r t a i n t y p e s of p a i n , for e x a m p l e deafferentation pain, respond p o o r l y to their a d m i n i s t r a t i o n ; they possess a high abuse potential a n d d e p e n d e n c e liability, w h i l s t r e p e a t e d a d m i n i s t r a t i o n is a s s o c i a t e d w i t h tolerance; a n d t h e y i n d u c e several side-effects s u c h as r e s p i r a t o r y d e p r e s s i o n a n d n a u s e a (see Table I). N a r c o t i c analgesics are the exo g e n o u s c o u n t e r p a r t s of the end o g e n o u s o p i o i d p e p t i d e s . Like these, t h e y exert their a c t i o n s via o p i o i d receptors, of w h i c h at least t h r e e m a j o r t y p e s exist: ~, 6 a n d i; (see Refs 2 a n d 3). This m u l t i plicity offers the p o t e n t i a l for the d i s c o v e r y of n o v e l analgesics. M o r p h i n e is a relatively selective l i g a n d of ~ - o p i o i d r e c e p t o r s (see
M. J. Millan is Team Leader at Fondax, Neurobiology Division, Groupe de Recherches Servier, 7 rue Ampere, 92800 Puteaux, Paris, France.
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February 1990 [Vol. 11]
34 Fernandes, L. B., Paterson, J. W. and Goldie, R. G. (1989) Br. J. Pharmacol. 96, 117-124 35 Hay, D. W. P., Muccitelli, R. M., Horstemeyer, D. L, Wilson, K. M. and Raeburn, D. (1987) Eur. ]. Pharmacol. 136, 247-250 36 Orehek, J., Douglas, J. S. and Bouhuys, A. (1975) J. Pharmacol. Exp. Ther. 194, 554-564 37 Karanian, J. W., Moran, F. M., Ramey, E. R. and Ramwell, P. W. (1981) Br. J. Pharmacol. 72, 10-12 38 Raeburn, D., Hay, D. W. P. and Fedan, J. S. (1987) Cell Calcium 8, 429-436 39 Munakata, M., Mitzner, W. and Menkes, H. (1988) J. Appl. Physiol. 64, 466--471
Table I). C e r t a i n a u t h o r s have a r g u e d for the existence of ~treceptor s u b t y p e s 2, b u t most interest has c e n t r e d o n the h o p e t h a t non-~t t y p e s of o p i o i d receptor m i g h t m e d i a t e analgesia in the a b s e n c e of o t h e r u n d e s i r a b l e actions. T h e r e is n o w substantial e v i d e n c e that 6 - r e c e p t o r s can m e d i a t e a n t i n o c i c e p t i o n (see Box 1) at spinal a n d s u p r a s p i n a l sites (see Ref. 3). H o w e v e r , in this respect, it is K-receptors that have c o m m a n d e d b y far the greatest a t t e n t i o n : their i n v o l v e m e n t in the c o n t r o l of n o c i c e p t i o n a n d the p o t e n t i a l of K-agonists as novel
TABLE I. Comparative pharmacology of ~- and K-opioid receptors
Receptor type Selective agonists Selective antagonists Noxious stimulus quality modulated
Site of action
p,
K
DAMGO; morphiceptin; morphine; fentanyl
U50488H; U69593; PD117302; ICI907607
CTAP; 13-funaltrexamine
nor-binaltorphimine
chemical pressure heat electrical
+ + + +
+ + + (ID)
brain
+ +
+ +
+ (INF)
+ (INF)
spinal cord periphery
activation of
inhibition of
K + channels
Ca+channels
Tolerance
+
+
Dependence/withdrawal
+
+ (mild)
euphorigenic
dysphoric/psychotomimetic
high
low
sedation; respiratory; depression; constipation; nausea/vomiting; pruritus
sedation; diuresis
Neuronal mechanism
Subjective effects Abuse potential Side-effects
Order of stimulus qualities may correspond to their relative (increasing) intensities. ID, action is intensity dependent; INF, action is expressed under conditions of inflammation. Pre-clinical data indicate that like p,-opioid receptor agonists, K-agonists induce tolerance; however, the withdrawal syndrome for K-agonists differs from (and is milder than) that for i~-agonists [Cowan, A. et al. (1988) J. Pharmaco/. Exp. Ther. 246, 950-955; Gmerek, D. E. et a/. (1987) J. Pharmaco/. Exp. Ther. 242, 428-436; Vonvoigtlander, P. F. et a/. (1983) J. Pharmaco/. Exp. Ther. 224, 7-12].
1990, Elsevier Science Publishers Ltd. (UK) 0165- 6147/90/$02.00
References 1 Colquhoun, D. (1987) in Perspectives on Receptor Classification (Black, J. W., Jenkinson, D. H. and Gerskowitch, V. P., eds), pp. 103-114, Alan R. Liss 2 Mackay, D. (1988) Trends Pharmacol. Sci.
67 9, 156-157 3 Kenakin, T. P. (1989) Trends Pharmacol. Sci. 10, 18-22 4 Left, P. and Harper, D. H. (1989) J. Theor. Biol. 140, 381-397 5 Furchgott, R. F. (1966) Adv. Drug Res. 3, 21-55 6 Barlow, R. B., Scott, N. C. and Stephenson, R. P. (1967) Br. J. Pharmacol. 31, 188-196 7 Stephenson, R. P. (1956) Br. J. Pharmacol. 11, 379-393
8 del Castillo, J. and Katz, B. (1957) Proc. R. Soc. London Ser. B 146, 369-381 9 de Lean, A., Stadel, J. M. and Lefkowitz, R. J. (1978) J. Biol. Chem. 255, 7108-7117 10 Mackay, D. (1990) Trends Pharmacol. Sci. 11, 17-22 11 Waud, D. R. (1969) J. Pharmacol. Exp. Ther. 170, 117-122 12 Besse, J. C. and Furchgott, R. F. (1976) J. Pharmacol. Exp. Ther. 197, 66-78 13 Kenakin, T. P. and Beek, D. (1980) J. Pharmacol. Exp. Ther. 213, 406-413
Airway epithelium and smooth muscle responsiveness
Airway epithelium-derived inhibitory factor Roy G. Goldie, Lynette B. Fernandes, Stephen G. Farmer and Douglas W. P. Hay Various bronchoactive agents can induce the release from the airway epithelium of an inhibitory substance that is able to relax certain tissues including rat aorta and possibly also airway smooth muscle. This substance, whose existence has recently been confirmed using a new bioassay system, is distinct from nitric oxide (EDRF) and is also known to be non-prostanoid in nature. Roy Goldie and colleagues describe the properties of this factor, and its potential clinical significance. One of the cardinal diagnostic symptoms of bronchial asthma is the exquisite 'twitchiness' of the airways to pharmacological, environmental and allergic stimuli 1. The mechanisms underlying this nonspecific airway hyperresponsiveness, although the subject of a great deal of scientific and clinical investigation, are not yet established. Although its precise role in the pathogenesis of asthma has not been elucidated, it has long been known that the airway epithelium in asthmatic lungs is abnormal 2. Indeed, epithelial damage associated with airway inflammation is a characteristic feature in both mild and severe asthma and may be causally re-
R. G. Goldie is a Senior Research Fellow at the Department of Pharmacology, University of Western Australia, Perth, Nedlands, W A 6009, Australia, and L. B. Fernandes has recently completed PhD studies in this Department. S. G. Farmer is a Staff Scientist at Nova Pharmaceutical Corporation, 6200 Freeport Centre, Baltimore, MD 21224-2788, USA. D. W. P. Hay is a Senior Investigator at the Department of Pharmacology, SmithKline Beecham Laboratories, King of Prussia, PA 19406-0939, USA.
lated to the accompanying airway hyperresponsiveness 3'4. Until recently, the epithelium was regarded generally as having a largely passive role in airways, providing a barrier to the penetration of stimulatory solutes, including allergens, to the underlying smooth muscle, submucosal mast cells, irritant receptors and C-fiber afferent nerve endings involved in axon reflexes. However, epithelial secretions and the activity of cilia are important to the effective clearance of irritant particles from the bronchi 5. Inflammatory events, possibly involving the cytotoxic effects of eosinophil-derived major basic protein, disrupt epithelial cell tight junctions, thereby compromising the protective function of this tissue s and perhaps contributing to the airway hyperresponsiveness. For some years now, it has been known that at least 12 different epithelial cell t y p e s 7 h a v e profound effects on abnormal and normal lung function. This review focuses on recent evidence that the epithelium can modulate airway smooth muscle responsiveness.
Mechanical removal of the airway epithelium increases responsiveness of airway smooth muscle to a variety of spasmogens. This phenomenon was first demonstrated in canine bronchus 8 and was confirmed in airways from cattle 9, guinea-pigs w-12, rabbits ~3 and rats 14. Deliberate epithelial removal from human bronchial preparations causes increases in spasmogen potency similar to those obtained in these animal models 15. Clearly, such models, which involve complete removal of the epithelium, can only approximate to the sometimes patchy epithelial damage seen in human asthma. It is not established whether shedding of the epithelium in vivo in humans results in enhanced airway contractility to spasmogens in vitro. The mechanisms underlying the phenomenon remain the subject of controversy. For some agonists the epithelium may act as a site of loss (i.e. site of metabolism) from the biophase, limiting the access of the agent to the smooth muscle, and thereby reducing its effect as an agonist. This has been reported for isoprenaline is, adenosine 17, substance P and related tachykinins 18'19, vasoactive intestinal peptide 2°, and arachidonic acid 21-23. It is also possible that removal of the epithelium facilitates access of bronchoconstrictor substances to submucosal structures including the smooth muscle, resulting in increased responsiveness to spasmogens 24. However, this may not explain the selective enhancement of sensitivity to only some airway spasmogens 14'2s, or reduced responsiveness to some relaxant agonists such as isoprenaline, or the effects of epithelium removal on neurogenic contractions 8. Conversely, there is evidence that
1990,ElsevierSciencePublishersLtd. (UK) 0165- 6147/90/$02.00
68
TiPS - February 1990 [Vol. 11]
i #i
rat ao~a
m MI-
guinea-pig tracheal tube
Fig. 1. A co-axial assembly for the bioassay of EpDIF released from donor airway tissue, as first described by lid. Ilhan and L Sahin (1986) Eur. J. Pharmacol. 131, 293-296.
the effect of epithelium removal on responsiveness of perfused trachea from sensitized guineapigs to antigen challenge in vitro is due, at least partly, to loss of a diffusion barrier 26. Another explanation is that the airway epithelium may release one or more inhibitory factors, either basally or in response to some agonists, which are capable of attenuating airway responsiveness 26-28. Such a system may function in a manner analogous to that described for the influence of an endothelium-derived relaxing factor (EDRF) on vascular smooth muscle 29. Bronchoconstriction to a particular spasmogen in nonasthmatics is limited, reaching a plateau level at high doses, whereas asthmatics continue to bronchoconstrict in a dosedependent manner. This suggests that there is a lack of a 'braking' mechanism which would otherwise prevent an excessive response 3°. It is possible then that, in asthma, epithelial damage or dysfunction causes a reduction in the influence of an epithelium-derived inhibitory factor(s) (EpDIF) and thus contributes to the characteristic airway hyperresponsiveness associated with this chronic disease.
EpDIF: fact or fiction? Lundblad and Persson 31 concluded that the presence or absence of epithelium was of little
consequence to the pharmacology of the guinea-pig tracheal open ring preparation in vitro. It is important to note, however, that this study examined responsiveness to carbachol, the potency of which is known to be unaltered by epithelium stripping 1°'14, and is in contrast to the effect of epithelium removal on responsiveness to other cholinomimetics such as methacholine 11'13"1s'32 as well as to acetylcholine itselfs'12. Furthermore, responsiveness to histamine, which has been confirmed in many studies to be enhanced by epithelium stripping 8'13'24'26'32, was not tested 31. In addition to the notable species differences that have been reported, appreciable differences in the magnitude of the influence of the epithelium are evident, and various methodological differences have perhaps contributed to apparently conflicting results within some species. A large body of independent research has, however, confirmed the phenomenon of epithelium-dependent modulation of mammalian airway smooth muscle responsiveness to several agonists in vitro, although the majority of these data, while consistent with the existence of EpDIF, provide only indirect support for its existence. Descriptions of the transfer of this putative inhibitory autacoid from epithelium and its subsequent bio-
assay would provide convincing direct evidence in support of its existence, in addition to contributing to its eventual identification. The first attempt to detect EpDIF using a superfusion cascade bioassay technique failed to produce any evidence for the release of such a factor from guineapig tracheal epithelium 24. The possibility of preferential abluminal secretion of EpDIF, or of dilution and rapid degradation of putative inhibitory substances(s) in such transfer systems, has encouraged other workers to use 'sandwich' preparations (as described by Furchgott and Zawads k i 29 in studies of EDRF), or variants of them in attempts to detect EpDIF.
Bioassay of EpDIF A co-axial sandwich assembly, in which the recipient or assay preparation is mounted within the lumen of a donor airway tube segment was first described by Ilhan and Sahin (Fig. 1). This study provided the first direct evidence for the release and transfer of an epithelium-derived inhibitory substance capable of causing relaxation of endothelium-denuded rabbit aorta: acetylcholine produced relaxation of aortic tissue when it was positioned within a guinea-pig tracheal tube segment with an intact epithelium. No relaxation
a
b
c
l
/" r
histamine 100 W¢
50 mg [
\
10 rain
phenylephrine0.05 ~
•
•
•
Fig. 2. Responses induced by histamine (100 [JM; •) in phenylephrine-precontracted ( • ) rat aorta strips, a: with an intact endothelium; b: with the endothelium removed; ¢: with the endothelium removed and the strip mounted co-axially within the lumen of an epithelium-intact guinea-pig tracheal tube segment.
69
TiPS - February 1990 [Vol. 11]
was seen in the absence of epithelium. Using co-axial preparations, the ability of EpDIF released from guinea-pig trachea to relax precontracted rat anococcygeus muscle 33 and rat aorta 34 (Fig. 2) in the presence of indometacin or propranolol has recently been demonstrated. The contractile potency of ovalbumin in tracheal preparations from sensitized guinea-pigs had been shown to be greater in epithelium-denuded than in epithelium-intact tissue 32. This effect was attributed to the loss of EpDIF and resultant enhancement of the potency of histamine and leukotrienes released in response to ovalbumin, although increased release of mediators from mast cells might also be involved 26. Indeed, when similar epitheliumdenuded preparations were mounted co-axially within an epithelium-intact guinea-pig tracheal tube, sensitivity to ovalbumin was reduced to levels observed in intact preparations 3s. These data suggest that a smooth muscle inhibitory factor (EpDIF) is released, either passively or in response to spasmogens, and transferred from donor airway epithelium; this results in attenuation of the contractile effect of the spasmogens (histamine and leukotrienes) released by ovalbumin.
Nature and mechanism of action of EpDIF Smooth muscle spasmogens including histamine stimulate the production and release of relaxant prostaglandins such as PGE2 from airway preparations in vitro 36. The epithelium is a source of various prostanoids including PGE2. The possibility that EpDIF is a relaxant prostaglandin is suggested by studies showing that inhibition of epithelial cyclooxygenase to some extent mimics the effect of epithelium stripping on contractions of guinea-pig trachea induced by leukotriene C4 (Ref. 25) or methacholine 11, and on bethanecholinduced contraction in rabbit b r o n c h u s 22.
However, in many studies increased responsiveness of airway preparations to some spasmogens caused by epithelium stripping remained unaltered in the presence of inhibitors of cyclooxygenase and/or lipoxygenases,9,11,24.
Furthermore, the production of guinea-pig tracheal and h u m a n bronchial EpDIF and its direct coaxial bioassay on rat anococcygeus or aorta have been demonstrated in the presence of inhibitors of these enzymes 33"34. Moreover, PGE2 causes contraction of rat aorta 37. Thus, while the epithelium is capable of releasing relaxant as well as constrictor prostaglandins, it is clear that a non-prostanoid inhibitory factor is also released from mammalian airway epithelium. The identity of EpDIF awaits clarification. The evidence indicates that EpDIF is not the same as EDRF. Epithelium-dependent relaxation of vascular34 or gastrointestinal33 smooth muscle was not affected by methylene blue, which inhibits the effects of EDRF on soluble guanylyl cyclase. EpDIF must therefore be an agent that produces endothelium-independent relaxation in these systems. The inflammatory mediator platelet activating factor (PAF) was inactive in rat aorta 34, showing that it is not EpDIF. Preliminary evidence suggests that EpDIF does not act via an alteration in extracellular Ca 2+ translocation 3s. []
[]
[]
There is a growing body of evidence in support of the idea that several bronchoactive agents can induce the release of a nonprostanoid inhibitory substance from the airway epithelium. It has now been shown that EpDIF, an epithelium-derived factor from guinea-pig trachea, relaxes vascular 34 and gastrointestinal smooth muscle 33 and may also relax airway smooth muscle 39. It remains to be determined whether relaxation of airway and non-airway smooth muscle is produced by the same epithelium-derived factor: is there more than one EpDIF? It is also possible that this autacoid can reduce spasmogen potency in the airway by means additional to functional antagonism at airway smooth muscle. Vasodilation of the bronchial circulation in vivo, following release of EpDIF, may enhance the clearance of inhaled chemical stimulants diffusing through the mucosa towards airway smooth muscle, mast cells
and nerve fibers. The marked epithelium-dependent relaxation of vascular tissue may indicate that the effects of EpDIF are more profound and physiologically relevant in the vascular beds of the airway wall than in the airway smooth muscle per se. EpDIF might exert inhibitory effects on some or all of these submucosal tissues, as well as on inflammatory cells. Challenges to be addressed include the elucidation of the nature and mechanisms of action of EpDIF. This will require the development of a reliable, sensitive system for the demonstration of the release of measurable quantities of EpDIF. Furthermore, and although a difficult task, it will be important to assess the pathophysiological relevance of EpDIF in lung function in vivo, both in animals and in humans. Identification of EpDIF may lead to development of novel agents (e.g. EpDIF analogues, stimulants of its release, or inhibitors of its degradation) in the therapy of airway disease.
References 1 Boushey, H. A., Holtzman, M. J., Sheller, J. R. and Nadel, J. A. (1980) Am. Rev. Respir. Dis. 121, 389-413 2 Cutz, E., Levison, H. and Cooper, D. M. (1978) Histopathology 2, 407-421 3 Boushey, H. A. and Holtzman, M. J. (1985) Am. Rev. Respir. Dis. 131, 312-313 4 Laitinen, L. A., Heino, M., Laitinen, A. and Haahtela, T. (1985) Am. Rev. Respir. Dis. 131, 599-606 5 Sleigh, M. A., Blake, J. R. and Liron, N. (1988) Am. Rev. Respir. Dis. 137, 726-731 6 Frigas, E. and Gleich, G. J. (1986) J. Allergy Clin. Immunol. 77, 527-537 7 Breeze, R. G. and Wheeldon, E. B. (1977) Am. Rev. Respir. Dis. 116, 705--777 8 Flavahan, N. A., Aarhus, L. L., Rimele, T. J. and Vanhoutte, P. M. (1985) J. Appl. Physiol. 58, 834-838 9 Barnes, P. J., Cuss, F. M. and Palmer, I. B. (1985) Br. J. Pharmacol. 86, 685-691 10 Goldie, R. G. et al. (1986) Br. J. Pharmacol. 87, 5-14 11 Hay, D. W. P. et al. (1986) Eur. J. Pharmacol. 129, 11-18 12 Murlas, C. (1986) Clin. Sci. 70, 571-575 13 Raeburn, D. et al. (1986) Life Sci. 38, 809-816 14 Frossard, N. and Muller, F. (1986) ]. Appl. Physiol. 61. 1449-1456 15 Raeburn, D., Hay, D. W. P., Farmer, S. G. and Fedan, J. S. (1986) Eur. J. Pharmacol. 123, 451-453 16 Farmer, S. G., Fedan, J. S., Hay, D. W. P. and Raeburn, D. (1986) Br. J. Pharmacol. 89, 407-414 17 Advenier, C., Devillier, P., Matran, R. and Naline, E. (1988) Br. J. Pharmacol. 93, 295-302 18 Fine, I. M., Gordon, T. and Sheppard, D. (1989) J. Appl. Physiol. 66, 232-237
TiPS
70 19 Frossard, N., Rhoden, K. J. and Barnes, P. J. (1989) J. Pharmacol. Exp. Ther. 248, 292-298 20 Farmer, S. G. and Togo, J. (1989) Br. ]. Pharmacol. 98, 784P 21 Nijkamp, F. P. and Folkerts, G. (1986) Eur. J. Pharmacol. 131, 315-316 22 Butler, G. B., Adler, K. B., Evans, J. N., Morgan, D. W. and Szarek, J. L. (1987) Am. Rev. Respir. Dis. 135, 1099-1104 23 Farmer, S. G., Hay, D. W. P., Raeburn, D. and Fedan, J. S. (1987) Br. J. Pharmacol. 92, 231-236 24 Holroyde, M. C. (1986) Br. J. Pharmacol. 87, 501-507 25 Hay, D. W. P. et al. (1987) Br. J. Pharmacol. 92, 381-388 26 Undem, B. J., Raible, D. G., Adkinson,
27 28 29 30 31 32 33
N. F. and Adams, G. K. (1988)J. Pharmacol. Exp. Ther. 244, 659-665 Farmer, S. G. (1987) Trends Pharmacol. Sci. 8, 8-10 Vanhoutte, P. M. (1988) Am. Rev. Respir. Dis. 138, $24-$30 Furchgott, R. F. and Zawadzki, J. V. (1980) Nature 288, 373-376 Woolcock, A. J., Salome, C. M. and Yan, K. (1984) Am. Rev. Respir. Dis. 130, 71-75 Lundblad, K. A. L. and Persson, C. G. A. (1988) Br. J. Pharmacol. 93, 909-917 Hay, D. W. P., Raeburn, D., Farmer, S. G., Fleming, W. W. and Fedan, J. S. (1986) Life Sci. 38, 2461-2468 Guc, M. O., Ilhan, M. and Kayaalp, S. O. (1988) Eur. J. Pharmacol. 148, 405--409
K-Opioid receptors and analgesia Mark J. Millan Over the past decade, opioids have attracted great attention. One important reason for this is the need for novel, strong analgesics free of the abuse potential and side-effects of narcotics such as morphine. Because morphine acts at ~t-opioid receptors, efforts have been made to characterize analgesia mediated by non-~t sites, in particular K-opioid receptors. There is now good evidence that K-receptors do indeed mediate analgesia. H o w e v e r , K-agonists display properties that could curtail their therapeutic exploitation. Since the first selective K-agonists are now entering clinical trials, this is an opportune moment for M a r k Millan to review the pharmacology of drugs of this type in the control of nociception and their therapeutic potential as analgesics. T h e n a r c o t i c o p i o i d analgesics, of w h i c h t h e p r o t o t y p e is m o r p h i n e , are of e n o r m o u s t h e r a p e u t i c i m p o r t a n c e in the c o n t r o l of m o d e r ate to severe p a i n ~. U n f o r t u n a t e l y , s i g n i f i c a n t p r o b l e m s remain1'2: c e r t a i n t y p e s of p a i n , for e x a m p l e deafferentation pain, respond p o o r l y to their a d m i n i s t r a t i o n ; they possess a high abuse potential a n d d e p e n d e n c e liability, w h i l s t r e p e a t e d a d m i n i s t r a t i o n is a s s o c i a t e d w i t h tolerance; a n d t h e y i n d u c e several side-effects s u c h as r e s p i r a t o r y d e p r e s s i o n a n d n a u s e a (see Table I). N a r c o t i c analgesics are the exo g e n o u s c o u n t e r p a r t s of the end o g e n o u s o p i o i d p e p t i d e s . Like these, t h e y exert their a c t i o n s via o p i o i d receptors, of w h i c h at least t h r e e m a j o r t y p e s exist: ~, 6 a n d i; (see Refs 2 a n d 3). This m u l t i plicity offers the p o t e n t i a l for the d i s c o v e r y of n o v e l analgesics. M o r p h i n e is a relatively selective l i g a n d of ~ - o p i o i d r e c e p t o r s (see
M. J. Millan is Team Leader at Fondax, Neurobiology Division, Groupe de Recherches Servier, 7 rue Ampere, 92800 Puteaux, Paris, France.
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February 1990 [Vol. 11]
34 Fernandes, L. B., Paterson, J. W. and Goldie, R. G. (1989) Br. J. Pharmacol. 96, 117-124 35 Hay, D. W. P., Muccitelli, R. M., Horstemeyer, D. L, Wilson, K. M. and Raeburn, D. (1987) Eur. ]. Pharmacol. 136, 247-250 36 Orehek, J., Douglas, J. S. and Bouhuys, A. (1975) J. Pharmacol. Exp. Ther. 194, 554-564 37 Karanian, J. W., Moran, F. M., Ramey, E. R. and Ramwell, P. W. (1981) Br. J. Pharmacol. 72, 10-12 38 Raeburn, D., Hay, D. W. P. and Fedan, J. S. (1987) Cell Calcium 8, 429-436 39 Munakata, M., Mitzner, W. and Menkes, H. (1988) J. Appl. Physiol. 64, 466--471
Table I). C e r t a i n a u t h o r s have a r g u e d for the existence of ~treceptor s u b t y p e s 2, b u t most interest has c e n t r e d o n the h o p e t h a t non-~t t y p e s of o p i o i d receptor m i g h t m e d i a t e analgesia in the a b s e n c e of o t h e r u n d e s i r a b l e actions. T h e r e is n o w substantial e v i d e n c e that 6 - r e c e p t o r s can m e d i a t e a n t i n o c i c e p t i o n (see Box 1) at spinal a n d s u p r a s p i n a l sites (see Ref. 3). H o w e v e r , in this respect, it is K-receptors that have c o m m a n d e d b y far the greatest a t t e n t i o n : their i n v o l v e m e n t in the c o n t r o l of n o c i c e p t i o n a n d the p o t e n t i a l of K-agonists as novel
TABLE I. Comparative pharmacology of ~- and K-opioid receptors
Receptor type Selective agonists Selective antagonists Noxious stimulus quality modulated
Site of action
p,
K
DAMGO; morphiceptin; morphine; fentanyl
U50488H; U69593; PD117302; ICI907607
CTAP; 13-funaltrexamine
nor-binaltorphimine
chemical pressure heat electrical
+ + + +
+ + + (ID)
brain
+ +
+ +
+ (INF)
+ (INF)
spinal cord periphery
activation of
inhibition of
K + channels
Ca+channels
Tolerance
+
+
Dependence/withdrawal
+
+ (mild)
euphorigenic
dysphoric/psychotomimetic
high
low
sedation; respiratory; depression; constipation; nausea/vomiting; pruritus
sedation; diuresis
Neuronal mechanism
Subjective effects Abuse potential Side-effects
Order of stimulus qualities may correspond to their relative (increasing) intensities. ID, action is intensity dependent; INF, action is expressed under conditions of inflammation. Pre-clinical data indicate that like p,-opioid receptor agonists, K-agonists induce tolerance; however, the withdrawal syndrome for K-agonists differs from (and is milder than) that for i~-agonists [Cowan, A. et al. (1988) J. Pharmaco/. Exp. Ther. 246, 950-955; Gmerek, D. E. et a/. (1987) J. Pharmaco/. Exp. Ther. 242, 428-436; Vonvoigtlander, P. F. et a/. (1983) J. Pharmaco/. Exp. Ther. 224, 7-12].
1990, Elsevier Science Publishers Ltd. (UK) 0165- 6147/90/$02.00
