Br. J. Pharmacol. (1992), 106, 978-985
'."
Macmillan Press Ltd, 1992
Adenosine-induced bronchoconstriction of isolated lung and trachea from sensitized guinea-pigs Julian R. Thorne & 'Kenneth J. Broadley Department of Pharmacology, Welsh School of Pharmacy, University of Wales College of Cardiff, P.O. Box 13, Cardiff CF1
3XF
1 The bronchoconstriction of airway-perfused lungs and contraction of superfused tracheal spirals from guinea-pigs in response to adenosine were examined. 2 In lungs from untreated animals, adenosine had little effect unless the perfusion pressure was raised with carbachol (1.1 tlM), when it caused a fall in perfusion pressure. However, if removed from guinea-pigs sensitized with ovalbumen (5 mg and 10 mg i.p. 14 and 12 days before use), adenosine was bronchoconstrictor, exerting bronchodilator effects only at high (1 mg) doses. The constrictor response to adenosine (300 lig) was significantly greater than that in lungs from untreated or sham-injected animals. 3 In superfused trachea from untreated animals, adenosine exerted only relaxant responses. In tissues from ovalbumen-sensitized guinea-pigs adenosine produced contracile responses, with relaxation appearing only at high (1 mg) doses. 4 Thus sensitization by antigen challenge revealed a bronchoconstrictor response of isolated airway preparations to adenosine. This is related to the clinical situation where only asthmatic subjects respond to adenosine by bronchoconstriction and suggests that the sensitization may destabilize inflammatory cells for mediator release by adenosine. 5 The response to a second exposure to adenosine was consistently reduced (lungs) or converted to a relaxation (trachea) indicating tachyphylaxis and consistent with a mediator release mechanism. 6 The P,-purinoceptor antagonist, 8-phenyltheophylline (3.9 jiM), antagonized the relaxant responses to higher doses of adenosine. However, it did not affect the contractile responses to lower doses of adenosine. Whether this is due to P,-purinoceptors not being involved in the contractile response, or whether preferential blockade of the relaxant response leaves the contraction unopposed and apparently unblocked, remains to be established. Keywords: Adenosine; airway perfused lungs of guinea-pig; superfused trachea; bronchoconstriction; ovalbumen sensitization
Introduction The predominant response of airway smooth muscle to adenosine is relaxation (Farmer & Farrar, 1976; Karlsson et al., 1982; Darmani & Broadley, 1986) which is mediated via P,-purinoceptors of the A2-subtype (Brown & Collis, 1982). In human non-asthmatic subjects, however, adenosine has no effect upon pulmonary function (Cushley et al., 1983) while in asthmatics it produces a bronchoconstriction (Cushley et al., 1983; 1984; Cushley & Holgate, 1985; Mann et al., 1985; Crimi et al., 1988; 1989). To evaluate the relevance of this bronchoconstrictor response to adenosine in asthma and in its treatment with the Pi-receptor antagonist theophylline, it was considered desirable to demonstrate a bronchoconstriction by adenosine in animal tissues. A bronchoconstrictor response to adenosine or its analogues has been demonstrated in vivo in anaesthetized rats (Pauwels & Van Der Straeten, 1987) and guinea-pigs (Manzini & Ballati, 1990), the latter being attributed to vagally-mediated bronchospasm. We have also demonstrated bronchoconstriction to inhaled adenosine in conscious guinea-pigs sensitized to ovalbumen, but not in unsensitized animals (Thorne & Broadley, 1990a). Small contractile responses of isolated tracheal preparations have also been demonstrated which have been attributed to the presence of longitudinal bands of smooth muscle and favoured by spiral cutting of the tissue (Satchell & Smith, 1984). The small contractile component precedes the predominant relaxation (Coleman, 1976; Karlsson et al., 1982) and appears to depend upon the degree of resting tone (Fredholm et al., 1979; Advenier et al., 1982). There is some
Author for correspondence.
disagreement regarding whether the contractile response to the adenosine analogue L-N6-phenylisopropyladenosine (LP1A) is antagonized by PI-receptor antagonists (Caparrotta et al., 1984; Ghai et al., 1987; Farmer et al., 1988). However, the possibility exists for it being mediated via the release of arachidonic acid derivatives (Advenier et al., 1982; Caparrotta et al., 1984). Because the contractile response to adenosine in these studies was dominated by the relaxation response, the present study was undertaken to establish measurable and reproducible bronchoconstrictor responses to adenosine in guinea-pig isolated lung tissue. Preliminary accounts of some of these results have been given to the British Pharmacological Society (Thorne & Broadley, 1988a,b; 1990b).
Methods Male Dunkin-Harley guinea-pigs (Halls, Stafford, UK) weighing 300-400g at purchase, 400-500g at termination, were used throughout. Groups of 6 animals were housed in grid-bottom cages and kept at an ambient temperature of 22 ± 1VC under 12 h normal phase light-dark cycle. The guinea-pigs were fed on Special Rabbit Pellet (plain) 680 supplied by Grain Harvesters, Canterbury, Kent or Diet TR2 with vitamin C, supplied by Pilsbury's Ltd., Birmingham. Drinking water was supplemented with ascorbic acid and allowed ad libitum.
Sensitization procedures Guinea-pigs were actively sensitized with ovalbumen administered by intraperitoneal (i.p.) injections (5 mg) on day 0 and
ADENOSINE-INDUCED BRONCHOCONSTRICTION
10 mg on day 2. They were used 14-15 days after the start of immunization. Sham pretreatment was performed, in which the injections were made identically but with vehicle containing no ovalbumen.
Airway-perfused lung preparation Guinea-pigs were killed by cervical dislocation and trachea and lungs excised. The trachea was removed 5-10 mm above the bifurcation. The entire lung was perfused via the airways by cannulating the trachea. Half-lung preparations were perfused via the bronchi. Perfusion was with warmed (370C) and gassed (5% CO2 in oxygen) Krebs-bicarbonate solution of composition in twice-distilled water (mM): NaCl 118, NaHCO3 24.9, KCI 4.6, CaCI2 2.5, MgSO4 1.15, KH2PO4 1.15 and glucose 5.5. A constant flow rate of 5mlmin-' was maintained by a Watson-Marlow peristaltic pump (tube internal diameter 0.5 mm) and changes in back-pressure were measured with a Bell and Howell physiological pressure transducer (type 4-327-L221) located at the side-arm on the perfusion cannula. A Condon mercury manometer was included in the system in series with the pressure transducer to accommodate some degree of volume change during drug responses. Perfusion pressure (resting level approximately 15 mmHg) was recorded on a Devices M19 polygraph (Ormed, Welwyn Garden, UK). The surface of the lungs was not scarified to aid outflow of perfusate.
Superfused tracheal spirals After removal of the trachea from the lungs, it was cut spirally (Constantine, 1965). Lengths of 3-4cm were suspended in a heated jacket (37TC) and superfused with prewarmed and gassed (5% CO2 in oxygen) Krebs-bicarbonate solution at a constant flow rate of 5 ml min-'. Changes in isometric tension were measured by attaching the upper end of the spiral to a Devices UFl transducer (2 oz sensitivity range) and recorded on a Devices M19 polygraph. Intrinsic tone was induced by allowing the spirals to equilibrate under an applied tension of 1 g for 45-60 min.
unpaired t test, a significant difference being assumed at the 5% probability level.
Drugs The following drugs were used: adenosine, carbachol (carbamylcholine chloride) and 8-phenyltheophylline were obtained from Sigma UK, and ovalbumen and aluminium hydroxide from BDH, Poole, Dorset, UK. All stock solutions were prepared in 0.9% saline. 8-Phenyltheophylline was initially dissolved in 1 ml 1 M NaOH.
Results
Effect of adenosine in perfused lungs from untreated and ovalbumen-sensitized guinea-pigs In airway-perfused lungs from untreated guinea-pigs constricted by a constant infusion of carbachol (1.1 jM), bolus doses of adenosine (300 jig and 1 mg) caused dose-related bronchodilatation (Figure 1). However, in the absence of background constriction, adenosine (300 pg) had no effect or produced a small constriction. In lungs from guinea-pigs previously sensitized to ovalbumen, adenosine (300 fig) produced a more marked constriction (Figure 1). Dose-response curves obtained in carbachol-constricted lungs showed a more marked constrictor-effect of adenosine in sensitized tissues at doses up to 300 jig but thereafter a bronchodilator action equivalent to that of untreated tissues (Figure 2). Single doses of carbachol (10 pg), adenosine (300 ,ug), adenosine (300 ptg), ovalbumen (200 jLg) and ovalbumen (200 gLg) were next administered, in that order, to lungs from untreated and ovalbumen-sensitized guinea-pigs (Figure 3). The carbachol responses did not differ but there was a significantly greater constriction to adenosine after ovalbumen sensitization (Figure 4). The response to adenosine after ovalbumen sensitization was also significantly greater
Experimental protocol After a 45-60 min equilibration period, agonists were added to lung or tracheal preparations as 0.1 ml bolus injections made into the connecting rubber tubing immediately prior to the warming coil. Antagonists were examined usually in paired tissues from the same animal, one half serving as the control the other being exposed to antagonist from 30 min before and thereafter throughout agonist exposure. For lung halves, left and right lungs were alternated for antagonist exposure. When resting perfusion pressure of the lung was raised, carbachol (1.1-LM) was perfused throughout. Dose-response curves were constructed by half-logarithmic increments in dose, each dose being added when the perfusion pressure had returned to the resting level following changes from the previous dose. For evaluating different agonists in the same preparation, single bolus doses were usually given in the order: carbachol (10 jig lung, 30 ng trachea), adenosine (300 ltg lung, 100 jig trachea), adenosine repeated, ovalbumen (200 gg lung, 500 ng trachea), ovalbumen repeated. The estimated concentration of adenosine after a dose of 300 fig to the lung is 2-3 mM.
a
Carbachol-constricted untreated 90-
I
El El 50- * 300 g
1mg
Responses were measured as the peak change in perfusion pressure (lungs) or isometric tension (trachea) induced by each bolus dose of agonist. Mean responses ± s.e.mean were calculated. Statistical comparisons were made between responses for each dose of agonist by Student's paired or
mi
b 60-
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0-
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300 jig
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Analysis of results
979
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Figure 1 Typical responses to adenosine (0) of isolated airwayperfused lungs from guinea-pigs. Animals were either untreated (a and b) or sensitized with ovalbumen at 14 (5 mg i.p.) and 12 days (10 mg i.p.) before use. In (a) the resting perfusion pressure was raised by perfusion with carbachol (l.1IjM) throughout.
980
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Dose of adenosine (pug) Figure 2 Mean ( ± s.e.mean, vertical bars) dose-response curves for the effects of bolus doses of adenosine on the perfusion pressure (mmHg) of airway-perfused lungs from untreated (0, n = 8) or ovalbumen-sensitized guinea-pigs (U, n = 6, 5 mg i.p., 14 days and 10 mg i.p., 12 days before use). Perfusion pressure was raised by perfusion with carbachol (1.1 pM) throughout. A significant difference, by Student's unpaired t test, between responses in untreated and sensitized tissues is indicated by *.
50-
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Ovalbumen-sensitized lung
OA
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Figure 4 Mean (± s.e.mean, vertical bars) increases in perfusion pressure (mmHg) of isolated airway-perfused lungs in response to bolus doses of carbachol (CCh, 10 pg), adenosine (ADO, 300 pg), repeated adenosine, ovalbumen (OA, 200 jig) and repeated ovalbumen. Doses were added in the above order, as illustrated in Figure 3, to lungs from untreated (open columns, n = 8) and ovalbumensensitized guinea-pigs (solid columns, n = 11) (5 mg i.p., 14 days and 10 mg i.p., 12 days before use). A significant difference, by Student's unpaired t test, between untreated and sensitized tissues is indicated by * and between the first and second doses of adenosine or ovalbumen by **.
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Figure 3 Typical responses of isolated airway-perfused lungs from ovalbumen-sensitized guinea-pigs (5 mg i.p., 14 days and 10 mg i.p., 12 days before use). Bolus doses of carbachol (CCh, 10jig), adenosine (ADO, 300 fig) and ovalbumen (OA, 200 jig) were added at (0). The lower trace is a continuation from above, but with a change in scale for the vertical axis.
than after sham injection. Ovalbumen induced constriction only after sensitization. The constrictor responses of sensitized lungs to the second administrations of adenosine and ovalbumen were significantly less than to the first exposure (Figures 3 and 4).
Effects of adenosine in superfused tracheae from untreated and ovalbumen-sensitized guinea-pigs Dose-response curves for bolus injections of adenosine to superfused tracheae revealed only relaxant responses of preparations from untreated animals whereas contractile responses were evident at doses up to 100 fig in preparations from ovalbumen-sensitized guinea-pigs. These were followed by a small relaxation at 1 mg (Figure 5). A single dose of adenosine (100 jig) was also relaxant in untreated tissues but after ovalbumen sensitization, it was contractile in common with ovalbumen (Figure 6).
Effect of 8-phenyltheophylline
on
constrictor responses to
adenosine Single doses of carbachol, adenosine, adenosine, ovalbumen and ovalbumen, in that order, were examined in paired lung-
200
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Figure 5 Mean ( ± s.e.mean, vertical bars) dose-response curves for the effects of bolus doses of adenosine on the tension (mg) of superfused tracheal spirals from untreated (0, n = 4) or ovalbumensensitized guinea-pigs (U, n = 4, 5 mg i.p., 14 days and 10 mg i.p., 12 days before use). A significant difference, by Student's unpaired t test, between responses in untreated and sensitized tissues is indicated by *.
halves from sensitized guinea-pigs. One half acted as the control and the other was perfused throughout with 8phenyltheophylline (8-PT, 3.9 jM). 8-PT had no effect upon any of the responses (Figure 7). The responses to the second exposures to adenosine (in the presence of 8-PT only) and to ovalbumen were significantly less than the first exposures. In superfused tracheae from sensitized guinea-pigs, 8-PT (3.9 gM) was also without effect upon the contractions induced by the first exposures to adenosine and ovalbumen (Figure 8). The second exposures to adenosine and ovalbumen in the absence of 8-PT produced relaxation response of the trachea, but in the present of 8-PT, these relaxations were restored to contractile responses which, in the case of adenosine, were no different from those obtained on the first exposure. Dose-response curves for adenosine were obtained in superfused tracheae from ovalbumen-sensitized guinea-pigs.
ADENOSINE-INDUCED BRONCHOCONSTRICTION
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CCh ADO OA Figure 6 Mean ( ± s.e.mean, vertical bars) changes in tension (mg) of superfused tracheal spirals in response to bolus doses of carbachol (CCh, 30 ng), adenosine (ADO, 100 pg), and ovalbumen (OA, 500 ng) added in that order. Tissues were removed from untreated (open columns, n = 7) and ovalbumen-sensitized guinea-pigs (solid columns, n = 6) (5 mg i.p., 14 days and 10mg i.p., 12 days before use). A significant difference, by Student's unpaired t test, between untreated and sensitized tissues is indicated by * .
ADO
ADO
OA
OA
Figure 8 Effects of 8-phenyltheophylline (8-PT) on the mean ( ± s.e.mean, vertical bars) changes in tension (mg) of superfused tracheal spirals from ovalbumen-sensitized guinea-pigs (5 mg i.p., 14 days and 10 mg i.p. 12 days before use). Responses to bolus doses of adenosine (ADO, 100 ptg), repeated adenosine, ovalbumin (OA, 500 ng) and repeated ovalbumen, added in that order, were obtained in the absence (open columns, n = 5) and present of 8-PT (solid columns, n = 8, 3.9 jiM). A significant difference between first and second exposures to ADO and OA is indicated by * and between values in the absence and presence of 8-PT by **, as determined by Student's paired and unpaired t tests respectively.
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OA ADO ADO OA Figure 7 Effects of 8-phenyltheophylline (8-PT) on the mean (± s.e.mean, vertical bars, n = 7) increases in perfusion pressure (mmHg) of isolated airway-perfused lung halves from ovalbumensensitized guinea-pigs (5 mg i.p., 14 days and 10 mg i.p. 12 days before use). Responses to bolus doses of carbachol (CCh, 10 gLg), adenosine (ADO, 300 pg), repeated adenosine, ovalbumen (OA, 200 fig) and repeated ovalbumen, added in that order, were obtained in the absence (open columns) and, in the contralateral lung half, presence of 8-PT (solid columns, 3.9 PM). A significant difference, by Student's paired t test, between first and second exposures to ADO and OA is indicated by *. Values in the absence and presence of 8-PT were not significantly different. CCh
In the absence of 8-PT a biphasic curve was obtained with prominent contractile responses followed, at the higher dose used (1 mg), by a relaxation. In the presence of 8-PT
(3.9 pM), the contractions were not blocked but no relaxation occurred at the highest dose used (Figure 9a). A similar result was obtained in lung halves from ovalbumen-sensitized guinea-pigs that were perfused throughout with carbachol (1.1 jaM). The constrictions induced by lower doses of adenosine were unaffected by 8-PT (3.9 laM) whereas the dilator responses to higher doses were significantly antagonized (Figure 9b).
Discussion Adenosine produced predominantly bronchodilator responses in airway-perfused lungs preconstricted with carbachol and in
lo-,
105 Dlo, 1o 104 lo2 Dose of adenosine (>9g) Figure 9 Effects of 8-phenyltheophylline (8-PT) on the mean ( ± s.e.mean, vertical bars) dose-response curves for (a) changes in tension (mg) of superfused tracheal spirals and (b) changes in perfusion pressure (mmHg) of airway-perfused lung halves, in response to adenosine. Both tissues were obtained from ovalbumen-sensitized guinea-pigs (5 mg i.p., 14 days and 10 mg i.p., 12 days before use). Perfusion pressure of lungs was raised by perfusing with carbachol (1.1 LM) throughout. Increasing bolus doses of adenosine were added to tracheal spirals either in the absence (0, n = 4) or presence (U, n = 11) of 8-PT (3.9 IM) and to lung halves from the same animals (n = 9), one half in the absence (0) and the other in the presence of 8-PT (-). A significant difference, by Student's t test (trachea unpaired; lung paired) between values in the absence and presence of 8-PT is indicated by *. 10
982
J.R. THORNE & K.J. BROADLEY
superfused trachea from untreated guinea-pigs. This confirms numerous reports in the literature of relaxant responses of guinea-pig immersed tracheal preparations (Farmer & Farrar, 1976; Jones et al., 1980; Brown & Collis, 1982; Karlsson et al., 1982; Darmani & Broadley, 1986; Advenier et al., 1988). A small constriction by lower doses of adenosine could be distinguished in the lung but not the trachea. Contractile responses of the immersed trachea have been observed previously to precede the dominant relaxation (Coleman, 1976; Fredholm et al., 1979; Advenier et al., 1982; Karlsson et al., 1982; Farmer et al., 1988), and have been attributed to arachidonic acid breakdown products (Advenier et al., 1982; Caparrotta et al., 1984). When tissues were removed from ovalbumen-sensitized guinea-pigs, a bronchoconstrictor response to adenosine was revealed in both tissues. In the lungs, the response to a single bolus dose was significantly greater than that of lungs from untreated animals, while the contraction of the trachea compared with relaxation of trachea from untreated animals. Similarly, dose-response curves revealed contractile responses at low doses followed by bronchorelaxation at higher doses. Thus we had achieved our objective of producing reproducible bronchoconstrictor responses of isolated respiratory tissues to adenosine. Why should sensitization to an antigen such as ovalbumen promote a bronchconstrictor response to adenosine? Sensitized guinea-pigs have long been used as an animal model of asthma, in which aerosol challenge with antigen provokes airway inflammation and bronchoconstriction similar to that of the human early asthmatic response (Kallos & Kallos, 1984) but whether sensitization per se induces another feature of asthma, namely airway hyperreactivity, is doubtful. There are reports that tissues removed from sensitized guinea-pigs display raised sensitivity to spasmogens (see Cortijo et al., 1989). However, in the present study there was no apparent increase in the response size to carbachol in tissues from sensitized animals. This agrees with most studies which fail to demonstrate a non-specific increase in sensitivity to histamine (Brink et al., 1981; Saad & Burka, 1983; Hay et al., 1986; Mansour & Daniel, 1987). To observe non-specific hyperreactivity to the bronchoconstrictor effect of spasmogens in vivo or in vitro, it is necessary to expose the ovalbumen-sensitized animal to antigen several hours before provocation by the
bronchoconstrictor. Increased sensitivity to 5-hydroxytryptamine (5-HT), histamine or prostaglandin F2
'."
Macmillan Press Ltd, 1992
Adenosine-induced bronchoconstriction of isolated lung and trachea from sensitized guinea-pigs Julian R. Thorne & 'Kenneth J. Broadley Department of Pharmacology, Welsh School of Pharmacy, University of Wales College of Cardiff, P.O. Box 13, Cardiff CF1
3XF
1 The bronchoconstriction of airway-perfused lungs and contraction of superfused tracheal spirals from guinea-pigs in response to adenosine were examined. 2 In lungs from untreated animals, adenosine had little effect unless the perfusion pressure was raised with carbachol (1.1 tlM), when it caused a fall in perfusion pressure. However, if removed from guinea-pigs sensitized with ovalbumen (5 mg and 10 mg i.p. 14 and 12 days before use), adenosine was bronchoconstrictor, exerting bronchodilator effects only at high (1 mg) doses. The constrictor response to adenosine (300 lig) was significantly greater than that in lungs from untreated or sham-injected animals. 3 In superfused trachea from untreated animals, adenosine exerted only relaxant responses. In tissues from ovalbumen-sensitized guinea-pigs adenosine produced contracile responses, with relaxation appearing only at high (1 mg) doses. 4 Thus sensitization by antigen challenge revealed a bronchoconstrictor response of isolated airway preparations to adenosine. This is related to the clinical situation where only asthmatic subjects respond to adenosine by bronchoconstriction and suggests that the sensitization may destabilize inflammatory cells for mediator release by adenosine. 5 The response to a second exposure to adenosine was consistently reduced (lungs) or converted to a relaxation (trachea) indicating tachyphylaxis and consistent with a mediator release mechanism. 6 The P,-purinoceptor antagonist, 8-phenyltheophylline (3.9 jiM), antagonized the relaxant responses to higher doses of adenosine. However, it did not affect the contractile responses to lower doses of adenosine. Whether this is due to P,-purinoceptors not being involved in the contractile response, or whether preferential blockade of the relaxant response leaves the contraction unopposed and apparently unblocked, remains to be established. Keywords: Adenosine; airway perfused lungs of guinea-pig; superfused trachea; bronchoconstriction; ovalbumen sensitization
Introduction The predominant response of airway smooth muscle to adenosine is relaxation (Farmer & Farrar, 1976; Karlsson et al., 1982; Darmani & Broadley, 1986) which is mediated via P,-purinoceptors of the A2-subtype (Brown & Collis, 1982). In human non-asthmatic subjects, however, adenosine has no effect upon pulmonary function (Cushley et al., 1983) while in asthmatics it produces a bronchoconstriction (Cushley et al., 1983; 1984; Cushley & Holgate, 1985; Mann et al., 1985; Crimi et al., 1988; 1989). To evaluate the relevance of this bronchoconstrictor response to adenosine in asthma and in its treatment with the Pi-receptor antagonist theophylline, it was considered desirable to demonstrate a bronchoconstriction by adenosine in animal tissues. A bronchoconstrictor response to adenosine or its analogues has been demonstrated in vivo in anaesthetized rats (Pauwels & Van Der Straeten, 1987) and guinea-pigs (Manzini & Ballati, 1990), the latter being attributed to vagally-mediated bronchospasm. We have also demonstrated bronchoconstriction to inhaled adenosine in conscious guinea-pigs sensitized to ovalbumen, but not in unsensitized animals (Thorne & Broadley, 1990a). Small contractile responses of isolated tracheal preparations have also been demonstrated which have been attributed to the presence of longitudinal bands of smooth muscle and favoured by spiral cutting of the tissue (Satchell & Smith, 1984). The small contractile component precedes the predominant relaxation (Coleman, 1976; Karlsson et al., 1982) and appears to depend upon the degree of resting tone (Fredholm et al., 1979; Advenier et al., 1982). There is some
Author for correspondence.
disagreement regarding whether the contractile response to the adenosine analogue L-N6-phenylisopropyladenosine (LP1A) is antagonized by PI-receptor antagonists (Caparrotta et al., 1984; Ghai et al., 1987; Farmer et al., 1988). However, the possibility exists for it being mediated via the release of arachidonic acid derivatives (Advenier et al., 1982; Caparrotta et al., 1984). Because the contractile response to adenosine in these studies was dominated by the relaxation response, the present study was undertaken to establish measurable and reproducible bronchoconstrictor responses to adenosine in guinea-pig isolated lung tissue. Preliminary accounts of some of these results have been given to the British Pharmacological Society (Thorne & Broadley, 1988a,b; 1990b).
Methods Male Dunkin-Harley guinea-pigs (Halls, Stafford, UK) weighing 300-400g at purchase, 400-500g at termination, were used throughout. Groups of 6 animals were housed in grid-bottom cages and kept at an ambient temperature of 22 ± 1VC under 12 h normal phase light-dark cycle. The guinea-pigs were fed on Special Rabbit Pellet (plain) 680 supplied by Grain Harvesters, Canterbury, Kent or Diet TR2 with vitamin C, supplied by Pilsbury's Ltd., Birmingham. Drinking water was supplemented with ascorbic acid and allowed ad libitum.
Sensitization procedures Guinea-pigs were actively sensitized with ovalbumen administered by intraperitoneal (i.p.) injections (5 mg) on day 0 and
ADENOSINE-INDUCED BRONCHOCONSTRICTION
10 mg on day 2. They were used 14-15 days after the start of immunization. Sham pretreatment was performed, in which the injections were made identically but with vehicle containing no ovalbumen.
Airway-perfused lung preparation Guinea-pigs were killed by cervical dislocation and trachea and lungs excised. The trachea was removed 5-10 mm above the bifurcation. The entire lung was perfused via the airways by cannulating the trachea. Half-lung preparations were perfused via the bronchi. Perfusion was with warmed (370C) and gassed (5% CO2 in oxygen) Krebs-bicarbonate solution of composition in twice-distilled water (mM): NaCl 118, NaHCO3 24.9, KCI 4.6, CaCI2 2.5, MgSO4 1.15, KH2PO4 1.15 and glucose 5.5. A constant flow rate of 5mlmin-' was maintained by a Watson-Marlow peristaltic pump (tube internal diameter 0.5 mm) and changes in back-pressure were measured with a Bell and Howell physiological pressure transducer (type 4-327-L221) located at the side-arm on the perfusion cannula. A Condon mercury manometer was included in the system in series with the pressure transducer to accommodate some degree of volume change during drug responses. Perfusion pressure (resting level approximately 15 mmHg) was recorded on a Devices M19 polygraph (Ormed, Welwyn Garden, UK). The surface of the lungs was not scarified to aid outflow of perfusate.
Superfused tracheal spirals After removal of the trachea from the lungs, it was cut spirally (Constantine, 1965). Lengths of 3-4cm were suspended in a heated jacket (37TC) and superfused with prewarmed and gassed (5% CO2 in oxygen) Krebs-bicarbonate solution at a constant flow rate of 5 ml min-'. Changes in isometric tension were measured by attaching the upper end of the spiral to a Devices UFl transducer (2 oz sensitivity range) and recorded on a Devices M19 polygraph. Intrinsic tone was induced by allowing the spirals to equilibrate under an applied tension of 1 g for 45-60 min.
unpaired t test, a significant difference being assumed at the 5% probability level.
Drugs The following drugs were used: adenosine, carbachol (carbamylcholine chloride) and 8-phenyltheophylline were obtained from Sigma UK, and ovalbumen and aluminium hydroxide from BDH, Poole, Dorset, UK. All stock solutions were prepared in 0.9% saline. 8-Phenyltheophylline was initially dissolved in 1 ml 1 M NaOH.
Results
Effect of adenosine in perfused lungs from untreated and ovalbumen-sensitized guinea-pigs In airway-perfused lungs from untreated guinea-pigs constricted by a constant infusion of carbachol (1.1 jM), bolus doses of adenosine (300 jig and 1 mg) caused dose-related bronchodilatation (Figure 1). However, in the absence of background constriction, adenosine (300 pg) had no effect or produced a small constriction. In lungs from guinea-pigs previously sensitized to ovalbumen, adenosine (300 fig) produced a more marked constriction (Figure 1). Dose-response curves obtained in carbachol-constricted lungs showed a more marked constrictor-effect of adenosine in sensitized tissues at doses up to 300 jig but thereafter a bronchodilator action equivalent to that of untreated tissues (Figure 2). Single doses of carbachol (10 pg), adenosine (300 ,ug), adenosine (300 ptg), ovalbumen (200 jLg) and ovalbumen (200 gLg) were next administered, in that order, to lungs from untreated and ovalbumen-sensitized guinea-pigs (Figure 3). The carbachol responses did not differ but there was a significantly greater constriction to adenosine after ovalbumen sensitization (Figure 4). The response to adenosine after ovalbumen sensitization was also significantly greater
Experimental protocol After a 45-60 min equilibration period, agonists were added to lung or tracheal preparations as 0.1 ml bolus injections made into the connecting rubber tubing immediately prior to the warming coil. Antagonists were examined usually in paired tissues from the same animal, one half serving as the control the other being exposed to antagonist from 30 min before and thereafter throughout agonist exposure. For lung halves, left and right lungs were alternated for antagonist exposure. When resting perfusion pressure of the lung was raised, carbachol (1.1-LM) was perfused throughout. Dose-response curves were constructed by half-logarithmic increments in dose, each dose being added when the perfusion pressure had returned to the resting level following changes from the previous dose. For evaluating different agonists in the same preparation, single bolus doses were usually given in the order: carbachol (10 jig lung, 30 ng trachea), adenosine (300 ltg lung, 100 jig trachea), adenosine repeated, ovalbumen (200 gg lung, 500 ng trachea), ovalbumen repeated. The estimated concentration of adenosine after a dose of 300 fig to the lung is 2-3 mM.
a
Carbachol-constricted untreated 90-
I
El El 50- * 300 g
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Figure 1 Typical responses to adenosine (0) of isolated airwayperfused lungs from guinea-pigs. Animals were either untreated (a and b) or sensitized with ovalbumen at 14 (5 mg i.p.) and 12 days (10 mg i.p.) before use. In (a) the resting perfusion pressure was raised by perfusion with carbachol (l.1IjM) throughout.
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Dose of adenosine (pug) Figure 2 Mean ( ± s.e.mean, vertical bars) dose-response curves for the effects of bolus doses of adenosine on the perfusion pressure (mmHg) of airway-perfused lungs from untreated (0, n = 8) or ovalbumen-sensitized guinea-pigs (U, n = 6, 5 mg i.p., 14 days and 10 mg i.p., 12 days before use). Perfusion pressure was raised by perfusion with carbachol (1.1 pM) throughout. A significant difference, by Student's unpaired t test, between responses in untreated and sensitized tissues is indicated by *.
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Figure 4 Mean (± s.e.mean, vertical bars) increases in perfusion pressure (mmHg) of isolated airway-perfused lungs in response to bolus doses of carbachol (CCh, 10 pg), adenosine (ADO, 300 pg), repeated adenosine, ovalbumen (OA, 200 jig) and repeated ovalbumen. Doses were added in the above order, as illustrated in Figure 3, to lungs from untreated (open columns, n = 8) and ovalbumensensitized guinea-pigs (solid columns, n = 11) (5 mg i.p., 14 days and 10 mg i.p., 12 days before use). A significant difference, by Student's unpaired t test, between untreated and sensitized tissues is indicated by * and between the first and second doses of adenosine or ovalbumen by **.
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Figure 3 Typical responses of isolated airway-perfused lungs from ovalbumen-sensitized guinea-pigs (5 mg i.p., 14 days and 10 mg i.p., 12 days before use). Bolus doses of carbachol (CCh, 10jig), adenosine (ADO, 300 fig) and ovalbumen (OA, 200 jig) were added at (0). The lower trace is a continuation from above, but with a change in scale for the vertical axis.
than after sham injection. Ovalbumen induced constriction only after sensitization. The constrictor responses of sensitized lungs to the second administrations of adenosine and ovalbumen were significantly less than to the first exposure (Figures 3 and 4).
Effects of adenosine in superfused tracheae from untreated and ovalbumen-sensitized guinea-pigs Dose-response curves for bolus injections of adenosine to superfused tracheae revealed only relaxant responses of preparations from untreated animals whereas contractile responses were evident at doses up to 100 fig in preparations from ovalbumen-sensitized guinea-pigs. These were followed by a small relaxation at 1 mg (Figure 5). A single dose of adenosine (100 jig) was also relaxant in untreated tissues but after ovalbumen sensitization, it was contractile in common with ovalbumen (Figure 6).
Effect of 8-phenyltheophylline
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adenosine Single doses of carbachol, adenosine, adenosine, ovalbumen and ovalbumen, in that order, were examined in paired lung-
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Figure 5 Mean ( ± s.e.mean, vertical bars) dose-response curves for the effects of bolus doses of adenosine on the tension (mg) of superfused tracheal spirals from untreated (0, n = 4) or ovalbumensensitized guinea-pigs (U, n = 4, 5 mg i.p., 14 days and 10 mg i.p., 12 days before use). A significant difference, by Student's unpaired t test, between responses in untreated and sensitized tissues is indicated by *.
halves from sensitized guinea-pigs. One half acted as the control and the other was perfused throughout with 8phenyltheophylline (8-PT, 3.9 jM). 8-PT had no effect upon any of the responses (Figure 7). The responses to the second exposures to adenosine (in the presence of 8-PT only) and to ovalbumen were significantly less than the first exposures. In superfused tracheae from sensitized guinea-pigs, 8-PT (3.9 gM) was also without effect upon the contractions induced by the first exposures to adenosine and ovalbumen (Figure 8). The second exposures to adenosine and ovalbumen in the absence of 8-PT produced relaxation response of the trachea, but in the present of 8-PT, these relaxations were restored to contractile responses which, in the case of adenosine, were no different from those obtained on the first exposure. Dose-response curves for adenosine were obtained in superfused tracheae from ovalbumen-sensitized guinea-pigs.
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CCh ADO OA Figure 6 Mean ( ± s.e.mean, vertical bars) changes in tension (mg) of superfused tracheal spirals in response to bolus doses of carbachol (CCh, 30 ng), adenosine (ADO, 100 pg), and ovalbumen (OA, 500 ng) added in that order. Tissues were removed from untreated (open columns, n = 7) and ovalbumen-sensitized guinea-pigs (solid columns, n = 6) (5 mg i.p., 14 days and 10mg i.p., 12 days before use). A significant difference, by Student's unpaired t test, between untreated and sensitized tissues is indicated by * .
ADO
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OA
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Figure 8 Effects of 8-phenyltheophylline (8-PT) on the mean ( ± s.e.mean, vertical bars) changes in tension (mg) of superfused tracheal spirals from ovalbumen-sensitized guinea-pigs (5 mg i.p., 14 days and 10 mg i.p. 12 days before use). Responses to bolus doses of adenosine (ADO, 100 ptg), repeated adenosine, ovalbumin (OA, 500 ng) and repeated ovalbumen, added in that order, were obtained in the absence (open columns, n = 5) and present of 8-PT (solid columns, n = 8, 3.9 jiM). A significant difference between first and second exposures to ADO and OA is indicated by * and between values in the absence and presence of 8-PT by **, as determined by Student's paired and unpaired t tests respectively.
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OA ADO ADO OA Figure 7 Effects of 8-phenyltheophylline (8-PT) on the mean (± s.e.mean, vertical bars, n = 7) increases in perfusion pressure (mmHg) of isolated airway-perfused lung halves from ovalbumensensitized guinea-pigs (5 mg i.p., 14 days and 10 mg i.p. 12 days before use). Responses to bolus doses of carbachol (CCh, 10 gLg), adenosine (ADO, 300 pg), repeated adenosine, ovalbumen (OA, 200 fig) and repeated ovalbumen, added in that order, were obtained in the absence (open columns) and, in the contralateral lung half, presence of 8-PT (solid columns, 3.9 PM). A significant difference, by Student's paired t test, between first and second exposures to ADO and OA is indicated by *. Values in the absence and presence of 8-PT were not significantly different. CCh
In the absence of 8-PT a biphasic curve was obtained with prominent contractile responses followed, at the higher dose used (1 mg), by a relaxation. In the presence of 8-PT
(3.9 pM), the contractions were not blocked but no relaxation occurred at the highest dose used (Figure 9a). A similar result was obtained in lung halves from ovalbumen-sensitized guinea-pigs that were perfused throughout with carbachol (1.1 jaM). The constrictions induced by lower doses of adenosine were unaffected by 8-PT (3.9 laM) whereas the dilator responses to higher doses were significantly antagonized (Figure 9b).
Discussion Adenosine produced predominantly bronchodilator responses in airway-perfused lungs preconstricted with carbachol and in
lo-,
105 Dlo, 1o 104 lo2 Dose of adenosine (>9g) Figure 9 Effects of 8-phenyltheophylline (8-PT) on the mean ( ± s.e.mean, vertical bars) dose-response curves for (a) changes in tension (mg) of superfused tracheal spirals and (b) changes in perfusion pressure (mmHg) of airway-perfused lung halves, in response to adenosine. Both tissues were obtained from ovalbumen-sensitized guinea-pigs (5 mg i.p., 14 days and 10 mg i.p., 12 days before use). Perfusion pressure of lungs was raised by perfusing with carbachol (1.1 LM) throughout. Increasing bolus doses of adenosine were added to tracheal spirals either in the absence (0, n = 4) or presence (U, n = 11) of 8-PT (3.9 IM) and to lung halves from the same animals (n = 9), one half in the absence (0) and the other in the presence of 8-PT (-). A significant difference, by Student's t test (trachea unpaired; lung paired) between values in the absence and presence of 8-PT is indicated by *. 10
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superfused trachea from untreated guinea-pigs. This confirms numerous reports in the literature of relaxant responses of guinea-pig immersed tracheal preparations (Farmer & Farrar, 1976; Jones et al., 1980; Brown & Collis, 1982; Karlsson et al., 1982; Darmani & Broadley, 1986; Advenier et al., 1988). A small constriction by lower doses of adenosine could be distinguished in the lung but not the trachea. Contractile responses of the immersed trachea have been observed previously to precede the dominant relaxation (Coleman, 1976; Fredholm et al., 1979; Advenier et al., 1982; Karlsson et al., 1982; Farmer et al., 1988), and have been attributed to arachidonic acid breakdown products (Advenier et al., 1982; Caparrotta et al., 1984). When tissues were removed from ovalbumen-sensitized guinea-pigs, a bronchoconstrictor response to adenosine was revealed in both tissues. In the lungs, the response to a single bolus dose was significantly greater than that of lungs from untreated animals, while the contraction of the trachea compared with relaxation of trachea from untreated animals. Similarly, dose-response curves revealed contractile responses at low doses followed by bronchorelaxation at higher doses. Thus we had achieved our objective of producing reproducible bronchoconstrictor responses of isolated respiratory tissues to adenosine. Why should sensitization to an antigen such as ovalbumen promote a bronchconstrictor response to adenosine? Sensitized guinea-pigs have long been used as an animal model of asthma, in which aerosol challenge with antigen provokes airway inflammation and bronchoconstriction similar to that of the human early asthmatic response (Kallos & Kallos, 1984) but whether sensitization per se induces another feature of asthma, namely airway hyperreactivity, is doubtful. There are reports that tissues removed from sensitized guinea-pigs display raised sensitivity to spasmogens (see Cortijo et al., 1989). However, in the present study there was no apparent increase in the response size to carbachol in tissues from sensitized animals. This agrees with most studies which fail to demonstrate a non-specific increase in sensitivity to histamine (Brink et al., 1981; Saad & Burka, 1983; Hay et al., 1986; Mansour & Daniel, 1987). To observe non-specific hyperreactivity to the bronchoconstrictor effect of spasmogens in vivo or in vitro, it is necessary to expose the ovalbumen-sensitized animal to antigen several hours before provocation by the
bronchoconstrictor. Increased sensitivity to 5-hydroxytryptamine (5-HT), histamine or prostaglandin F2
