Airway Hyperresponsiveness in Anaesthetised Guinea-Pigs 18–24 Hours after Antigen Inhalation Does Not Occur with All Intravenously Administered Spasmogens

0 Pharmacology & Toxicology 1999, 84, 281-287. Printed in Denmark . All rights reserved

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Airway Hyperresponsiveness in Anaesthetised Guinea-pigs 1%24 Hours after Antigen Inhalation Does Not Occur with

All Intravenously Administered Spasmogens Alexia Johnson and Kenneth J. Broadley

Division of Pharmacology, Welsh School of Pharmacy, Cardiff University, Cardiff CFI 3XF, U.K.

(Received November 10, 1998; Accepted December 22, 1998)

Abstract: Actively sensitised guinea-pigs were exposed to inhalation challenges with ovalbumin aerosol (macro- and microshock) and airway responsiveness to six intravenously administered spasmogens was evaluated 18 to 24 hr later in the anaesthetised animal. An increase in airway sensitivity was defined as a significant leftward shift of the dose-response curve when compared with saline-challenged control sensitized animals. After ovalbumin-macroshock ( I % ovalbumin for 2 min. with mepyramine cover against fatal anaphylaxis), airway hyperresponsiveness was seen to 5-HT, the thromboxane A2-mimetic, U-46619, and bradykinin but not to methacholine, histamine or substance I? A similar pattern was seen after ovalbumin-microshock (0.01% ovalbumin for 60 min.). with induction of airway hyperreactivity to 5-HT and U-46619 but not methacholine or histamine. When the U-46619 dose-response curve was constructed following treatment of the animals with atropine (1 mgikg. intravenously), airway hyperresponsiveness was no longer significant. As an index of airway inflammation, lung weights were significantly heavier in ovalbumin-challenged animals, than in saline-challenged controls. The results of this study with intravenously administered spasmogens does not support claims that ovalbumin- induced airway hyperreactivity in the guinea-pig is a ‘non-specific’ phenomenon.

Asthma is characterised by a so-called non-specific airway hyperreactivity or hyperresponsiveness (Boushey et al. 1980). Inhaled histamine and cholinergic agonists are the most commonly used test spasmogens for the demonstration of ‘non-specific’ airway hyperresponsiveness following antigen challenge of asthmatic subjects (Cockcroft et al. 1977; Cartier et af. 1982; Lai et al. 1989).

Guinea-pig models have been developed to investigate the mechanisms involved in the pathogenesis of allergic asthma and such models have shown most of the major character- istics of the disease. These include antigen-induced early and late asthmatic responses, airway inflammation and airway hyperresponsiveness. Antigen-induced airway inflammation is characterised by the influx of inflammatory cells into the airways and detailed evaluations of this have been carried out in guinea-pig models of allergic asthma (San- jar & Morley 1990; Tarayre et al. 1990; Banner & Page 1995; Lewis & Broadley 1995; Lewis et al. 1996).

Daffonchio et al. (1 989) showed that airway hyperresponsiveness to intravenous methacholine and 5-hydroxytryptamine (5-HT), and also inhaled 5-HT, occurred 1 hr after sensitised anaesthetised guinea-pigs were exposed to an ovalbumin-microshock inhalation (0.03‘1/0). No change in reactivity was seen at 30 min. when the initial anaphylactic bronchoconstriction had subsided. The airway hyperre-

Author for correspondence: K. J. Broadley, Division of Pharma- cology, Welsh School of Pharmacy, Cardiff University, Cathays Park, Cardiff CFI 3XE U.K. (fax 01222 874149, e-mail Broad- ley KJ@Cardiff .ac.uk).

sponsiveness could not therefore be attributed to any re- sidual reduction in airway calibre.

Despite numerous studies using anaesthetised allergic guinea-pig models to investigate airway hyperresponsiveness following the early asthmatic response (Anderson 1982; Daffonchio et ul. 1988 & 1989; Nieri et al. 1992; Under- wood et al. 1992) or that which may follow the late asthmatic response (Coyle et al. 1988; Havill et al. 1990; Sanjar et a1. 1990; Noonan et al. 1991; Farmer et al. 1992; Handley et ul. 1992; Underwood et ul. 1992; Howell et al. 1992 & 1993; Arimura et al. 1994), none appears to have presented data using more than 2 different agonists. The majority of studies have opted for agonists which act predominantly through a direct action on bronchial smooth muscle recep- tors e.g. acetylcholine, methacholine or histamine.

The concept of non-specific airway hyperresponsiveness in guinea-pigs has been questioned by a lack of airway hyperresponsiveness to inhaled acetylcholine (Heuer et al. 1994) or histamine (Banner et al. 1996) at 24 hr after an antigen challenge. With no more than two spasmogens under study, however, it is difficult to assess to what extent the airway hyperresponsiveness is non-specific. Similarly, the hyperresponsiveness in anaesthetised guinea-pigs measured immediately after an infusion of the antigen has been described as heterogeneous since there was a variable degree of increased reactivity to a wider range of intravenously administered spasmogens (Crowther et al. 1997). The aim of the present study was therefore to investigate whether an aerosolised antigen-challenge leads to airway hyperresponsiveness (measured 18 to 24 hr after challenge) to a wide

282 ALEXIA JOHNSON AND KENNETH J. BROADLEY

variety of intravenously administered spasmogens in sensitised, anaesthetised guinea-pigs.

Materials and Methods

Sensitisation and challenge with ovalbumin. Male Dunkin-Hartley guinea-pigs (Halls, Staffordshire, UK) weighing 350 to 400 g at pur- chase were sensitised to ovalbumin with a single intraperitoneal injection of ovalbumin (10 pg) and Al(OH), (100 mg) in Iml normal saline. This sensitisation protocol has been shown by passive cu- taneous anaphylaxis to generate both IgE and IgG, antibodies (An- dersson 1980).

At 14 to 21 days after sensitisation, the animals were challenged by exposure to a nebulised solution of ovalbumin in a sealed per- spex box (35OX20OX150 mm). The aerosol was generated by a Wright nebuliser (Wright 1958) using medical air at 10 or 20 psi. The mean droplet diameter was less than 2 pm. The aerosol was passed into the box for 2 min. to reach an initial steady-state before introducing the animal.

Two types of ovalbumin-challenge were used which utilised different doses of antigen. A high dose challenge (1% w/v in normal saline for 2 min. delivered at 20 psi) referred to as a macroshock, in which mepyramine (10 mgikg intraperitoneally) was given 30 min. beforehand to protect against fatal anaphylaxis. In spite of the mepyramine cover, animals still experienced a severe anaphylactic reaction manifest as exaggerated inhalations and/or cough usually with some degree of cyanosis and, rarely, convulsions. Those animals not responding to antigen-challenge with apparent bronchoconstriction (less than 5%) were excluded from the study.

A milder challenge (ovalbumin 0.01% w/v in normal saline for 60 min. delivered at 10 psi) referred to as a microshock, abolished the need for protection with mepyramine. These animals also experienced an anaphylactic reaction which manifested as exaggerated inhalations and rarely cyanosis but no convulsive diaphragmatic breathing was seen. As the degree of anaphylaxis for this group was typically less than that seen after an ovalbumin-macroshock, verification of sensitisation was obtained by giving an intravenous bolus of ovalbumin (40 pg) after the determination of airway reactivity under general anaesthesia. Less than 5%) of animals failed to respond. This method of verification was also used after an ovalbumin-macroshock.

Control animals were sensitised by intraperitoneal injections of ovalbumin as described above but were subsequently given nebulised normal saline instead of ovalbumin for either 2 or 60 min. Comparison of the ovalbumin challenged sensitised animals with unsensitised animals that had received either nebulised saline or ovalbumin was not considered to be appropriate. Such animals would not serve as controls for the effects of the ovalbumin challenge alone since sensitisation itself may induce some degree of reactivity change as reported in vitro (Cortijo el al. 1989).

Determination of airway reactivity. The method developed by Di- xon & Brodie (1903) was used for the assessment of airway calibre in the anaesthetised guinea-pig. The lungs were ventilated artificially with an open side arm just above the tracheal cannula which allowed escape of excess air. The pulmonary inflation pressure, which is known to increase approximately in proportion to the degree of bronchoconstriction (at constant volume), was measured. Baseline pulmonary inflation pressure typically ranged from 10-13 cm H20, measured by means of a water manometer, which was 20 to 30% of maximum (-50 cm H20) and did not significantly differ between treatment groups.

Animals were taken 18 to 24 hr after ovalbumin-challenge and anaesthetised with sodium pentobarbitone (60 mg/kg) by intraperitoneal injection and placed in the dorsal recumbent position on a heated small animal operating table. A cannula was inserted into the trachea (Portex size 8; O.D. 2.76 mm) and the lungs mechanic- ally ventilated by a Harvard constant volume respiration pump (58

strokeshin. of 1 ml laboratory air per 100 g body weight). Pulmon- ary inflation pressure was measured from a lateral port in the affer- ent limb of the ventilator circuit, with a pressure transducer (Pioden Controls Ltd.). Systemic arterial blood pressure measurements were made via a polythene catheter (Portex; O.D. 0.96 mm) placed in a carotid artery (usually the right) and connected to a Bell and How- ell physiological pressure transducer. A cannula (Portex; O.D. 0.96 mm) was also placed in the right jugular vein for intravenous drug administration.

A period of equilibration, typically 10 min., was allowed after surgical intervention. During this time the lungs were hyperinflated with 3 tidal volumes of room air, (by occluding outflow) and allowed to reach a stable resting pulmonary inflation pressure value.

Drug administration. Dose-response curves to intravenously administered methacholine, 5-HT, U-46619, histamine, substance P and bradykinin were obtained between 18 and 24 hr after either ovalbumin-macroshock or ovalbumin-microshock (with the excep- tion of bradykinin and substance P). Increasing doses of spasmogen were given at -3 min. intervals, providing pulmonary inflation pressure had returned to approximate baseline values. In some animals, atropine was given intravenously ( I mgikg) -15 min. before constructing dose-response curves to histamine or U-46619. This dose of atropine was sufficient to block the effect of an intravenous dose (6 mg/kg) of methacholine that was supramaximally effective in causing bronchoconstriction. Atropine failed to significantly af- fect the baseline pulmonary inflation pressure.

The dose-response curves to 5-HT were obtained after the com- pletion of a dose-response curve to methacholine. A minimum period of 15 min. was allowed between dose-response curves and the second dose-response curve was not started until pulmonary inflation pressure had returned to resting baseline level and blood pressure was stable. Although it is possible that the prior exposure to methacholine may have affected 5-HT responses, this was not considered to be a limitation of the study since both controls and ovalbumin-exposed animals were treated identically. Also, this approach was selected so that a direct comparison of the degree of any airway hyperresponsiveness to two spasmogens could be made in the same animal. Dose-response curves to histamine, U-46619, bradykinin and substance P were obtained in sep- arate experiments.

All drug solutions were in normal saline. The volume of each dose was between 0.1 and 0.5 ml and was washed in by -0.2 ml normal saline (a volume which exceeded that of the cannula).

Lung wet-dry weight measurements. Following determination of airway reactivity, the lungs were removed from anaesthetised guinea- pigs (after cervical dislocation) blotted dry and weighed before pla- cing in an oven with silica gel at 80". They remained in the oven until constant weight was reached -24 hr later. The weight difference before and after drying was calculated. This value was then standardised according to the weight of the guinea-pig. So for a 650 g animal with a 5 g wet lung weight which was reduced to 0.7 g through drying, the standard difference ratio would be 6.62 g/kg body weight. Thus, the larger the standard difference ratio the greater the weight loss and hence the greater the water content at the time of measurement.

Data analysis. The peak developed pulmonary inflation pressure above resting level after administration of each dose of spasmogen was measured. This was expressed as a percentage of the maximum pulmonary inflation pressure which was determined at the start of each experiment by manual occlusion of the tracheal cannula before connection to the animal. During this occlusion, the excess air was diverted through the open side arm. EDSo values (i.e. the dose required to cause an increase to 50% maximal pulmonary inflation pressure) were determined from individual dose-response curves by linear interpolation between points on either side. For bradykinin, the ED2, values (i.e. the dose required to cause a doubling of base-

AIRWAY HYPERREACTIVITY TO INTRAVENOUS SPASMOGENS

E >loo- 2 80- E a c 60- 0 + 2 40- -

283

40 - U-

F 2d I I , 201 2 011 1 10 100 160 1000

I

E r U - 4661 9 HISTAMINE 2100, 1001 P

20' I I I 1 20J 10 100 1000 10000 10 MO 1000

BRABYKININ SUBSTANCE P Fig. 1. Dose-response curves for the peak pulmonary inflation pressure induced by intravenous doses (ng/100 g) of (A) 5-HT, (B) U-46619, (C) bradykinin, (D) methacholine. (E) histamine and (F) substance P in sensitised, anaesthetised guinea-pigs 18 to 24 hr after inhalation challenge with saline (0) or an ovalbumin-macroshock (I%, 2 min., W). Points show the mean pulmonary inflation pressure expressed as a percentage of the maximum possible pulmonary inflation pressure, +S.E.M., n = 4 7 animals for each curve.

line pulmonary inflation pressure) were used since the maximal bronchoconstriction achieved was not 10O1%. Geometric mean EDTo or ED2, values were calculated together with their 95% confidence limits. To determine airway sensitivity differences between ovalbumin-challenged animals and saline-challenged controls, logEDSO or EDzx values were compared by an unpaired Student's t-test. Airway hyperresponsiveness is defined as a significant leftward shift of the dose-response curve after ovalbumin-challenge and hence a lower EDSo value when compared with controls. For multiple compari- sons between spasmogens under the same experimental conditions e.g. potency data, Duncan's multiple range test was used. A prob- ability level of P<0.05 was considered statistically significant in all experiments.

Drugs used. Atropine sulphate, bradykinin, heparin sodium (por- cine). histamine diphosphate, 5-hydroxytryptamine (creatinine sulphate complex), methacholine bromide (acetyl-P-methylcholine bromide), ovalbumin, substance P and U-46619 (9,l I-dideoxy-1 la,9a- epoxymethano-prostaglandin F2() were all purchased from Sigma. Poole, Dorset, UK. Sagatal (pentobarbitone sodium B.P. 60 mg/ml) was purchased from RhBne MBrieux, U.K. Aluminium hydroxide and mepyramine maleate were gifts from RhBne Poulenc-Rorer, Dagenham, Essex. UK.

Results

Efects of spasmogens. Intravenous injections of SHT, U-46619, bradykinin, methacholine, histamine and substance P caused dose-related in- creases in pulmonary inflation pressure (fig. 1). U-46619 was the most potent agonist, causing bronchoconstriction at significantly lower doses than any other spasmogen both in control and ovalbumin-challenged guinea-pigs (table 1).

Effect of ovalbumin-macroshock on airway responsiveness. Exposure of actively sensitised guinea-pigs to an aerosol of ovalbumin (10 mg/ml, 2 min.) induced an increase in airway responsiveness to the bronchoconstrictor responses to 5- HT, U-46619 and bradykinin (fig. lA, B and C, respectively), the EDs0 and ED2, values being significantly reduced compared with saline-challenged controls (table 1 ) .

Tuble I Sensitivity to various intravenous spasmogens in anaesthetised guinea-pigs after saline (control) or ovalbumin challenge. EDs0 values are the geometric mean doses for a 50% of maximum pulmonary inflation pressure except for bradykinin where the ED2, value (dose needed to double baseline pulmonary inflation pressure) is given. Values in parenthesis are 95% confidence limits. A significant leftwards shift (hyperresponsiveness)* or rightwards shift (hyporesponsiveness)" of dose-response curves as measured from ED5,, values and determined by Student's unpaired t-test. NS not significantly difference from the ED," value in the absence of atropine.

Spasmogen Control n Challenged n

Ovalbumin-macroshock methacholine 3 64 6 193 6

(40-5 52) (77484)

( I 18-31 6) (43- 143) 5-HT 193 6 78* 6

U-46619 5.6 5 2.4* 7

Histamine 258 5 226 7

Substance P 177 4 258 4

(3.7-8.6) (1.9-3.0)

(174382) (167-305)

(25-1225) (1 32-504)

Brad ykinin 5323 4 522* 5 (2897-9780) (1 4 1- 19 33)

Ovalbumin-microshock Methacholine 384 4

5-HT 171 4

(3 16-346)

( 152-193)

(4.0-7.0) U-46619 5.3 4

(with atropine) 9.2NS 4 (4.3- 19.9)

Histamine 299 4 (170-526)

(with atropine) 21gNS 4 ( 140-340)

318 7

48* 7

2.6* 4

4ANS 4

315 4

386#NS 4

(224453)

( 19- 123)

(1.64.3)

(3.1-7.4)

(213-465)

(299497)


100-

80-

60 - - X

40-

A 100 1

C

R

I I 1 10 100

20'1 0 1

U-46619 HISTAMINE

Fig. 2. Dose-response curves for the peak pulmonary inflation pressure induced by intravenous doses (ng/100 g) of (A) 5-HT, (B) U-46619, ( C ) methacholine and (D) histamine in sensitised, anaesthetised guinea-pigs 18 to 24 hr after inhalation challenge with saline (0) or an ovalbumin-microshock (0.01%, 60 min., m). Points show the mean pulmonary inflation pressure expressed as a percentage of the maximum possible pulmonary inflation pressure, tS.E.M., n = 4 7 animals for each curve.

In contrast, there was no significant increase in airway responsiveness to methacholine, histamine or substance P (fig. lD, E and F, respectively) with respect to saline-challenged controls.

Eflect of ovalbumin-microshock on airway responsiveness. Exposure of sensitised guinea-pigs to a n aerosol of ovalbumin (100 pg/ml, 60 min.) also induced an increase in airway responsiveness to 5-HT and U-46619 (fig. 2A and B, respectively), with significant reduction of the EDso values (table 1). Again there was no hyperresponsiveness to methacholine or histamine (fig. 2C and D, respectively) with respect to saline-challenged controls.

Responses to intravenous U-466 19 and histamine were further examined in the presence of atropine (1 mg/kg). In both the control animals and those subjected to ovalbumin microshock, atropine produced no significant change in the EDso values for histamine or U-46619 (table 1 ) . However, the dose-response curve for histamine in the atropine- treated animals subjected to ovalbumin microshock was dis- placed to the right of that observed in the atropine-treated control animals (fig. 3B) and the EDso value was significantly greater (table 1). In the case of U-46619, the EDs0 values in ovalbumin-challenged and control atropine- treated animals were no longer significantly different (fig. 3A, table I ) .

Lung wet-dry weight measurements. The lungs were removed after the determination of airway reactivity from three of the groups of guinea-pigs receiving ovalbumin-microshock exposures (methacholine/S-HT and

U-466 19 and histamine both after atropine). A significant increase in lung weight was seen in the ovalbumin-challenged animals (6.13(5.6&6.60) g/kg body weight, n = 15) compared with the saline-challenged controls (5.49(5.26- 5.71)g/kg body weight, n = 12).

The effect of intravenous spasmogens on blood pressure. The spasmogens caused changes in blood pressure, with dose-related falls in diastolic pressure occurring with methacholine, histamine, bradykinin and substance P. and an increase with U-46619. Changes in blood pressure with 5-HT were inconsistent and could not be quantified. The changes in diastolic blood pressure were similar in control and ovalbumin-challenged guinea-pigs. For example, the falls in diastolic blood pressure in saline and ovalbumin- macroshock challenged guinea-pigs after substance P, gave ED2" values (i.e. the dose required for a drop in diastolic blood pressure of 20 mmHg) of 6055232 and 6882242 ng/lOO g, respectively which were not significantly different (P= 0.8 1 ) .

20-1 r I I i 0.1 1 10 100

U-46619

2

e, n

?I

20J 1 I I 1 10 100 1000 10000

HISTAMINE Fig. 3. Dose-response curves for the peak pulmonary inflation pressure induced by intravenous doses (ngi100 g) of (A) U-46619 and (B) histamine 15 min. after atropine treatment (1 mg/kg intravenously) in sensitised, anaesthetised guinea-pigs 18 to 24 hr after inhalation challenge with saline (0) or an ovalbumin-microshock (O.Ol'Yu, 60 min., m). Points show the mean pulmonary inflation pressure expressed as a percentage of the maximum possible pulmonary inflation pressure, ZS.E.M., n=4 animals for each curve.

AIRWAY HYPERREACTIVITY TO INTRAVENOUS SPASMOGENS 285

Discussion

Airway responsiveness was assessed in anaesthetised, ovalbumin-sensitised guinea-pigs between 18 and 24 hr after the ovalbumin challenge, a time that we have shown to coincide with the late phase response in the conscious guinea-pig (Lewis & Broadley 1995; Lewis et d. 1996). The present study has demonstrated that when a wide range of spasmogens was used to evaluate airway responsiveness, it was not always possible to show airway hyperresponsiveness. Ovalbumin-induced airway hyperresponsiveness was evident with bradykinin, U-46619 and 5-HT, but not with methacholine, histamine or substance €? These results show that the airway hyperresponsiveness is not a non-specific phenomenon in guinea-pigs.

The ability of ovalbumin challenge to induce airway hyperresponsiveness to a particular spasmogen did not ap- pear to depend on the bronchoconstrictor potency (on a weight for weight basis) of that spasmogen. U-46619 was a significantly more potent bronchoconstrictor agent than bradykinin and yet both agents revealed a similar level of ovalbumin-induced airway hyperresponsiveness. Interest- ingly, the nature of bradykinin-induced bronchoconstriction appeared different to that of the other spasmogens. Bradykinin-induced bronchoconstriction may be mediated indirectly through the release of cyclooxygenase products (Rossoni et al. 1980; Zhang et al. 1991), or the stimulation of sensory C-fibres to cause the antidromic release of neuropeptides e.g. neurokinin A and substance P (Barnes 1987). The slopes of the dose-response curves to bradykinin after both saline and ovalbumin challenge were much shallower than for the other spasmogens. Airway hyperresponsiveness was therefore measured as a vertical rather than a horizon- tal shift of the dose-response curve, which was the preferred method of measuring changes in sensitivity.

Two types of ovalbumin-challenge were used, one which caused severe anaphylactic bronchoconstriction (ovalbumin-macroshock) and one which did not (ovalbumin- microshock). Both types of challenge have been shown to produce early and late phase bronchoconstriction in conscious guinea-pigs (Lewis et al. 1996). The degree of airway hyperresponsiveness was not directly related to the magnitude of the antigen-induced early phase bronchoconstriction; ovalbumin-micro and macroshocks typically produced similar effects on reactivity yet the degree of early phase bronchoconstriction by the ovalbumin-macroshock was substantially greater. The possibility exists that mepyramine treatment before ovalbumin-macroshock could interfere with the development of the late asthmatic response (Sant- ing c’t al. 1994), although we have shown a similar degree of late phase response in guinea-pigs after ovalbumin-macro and microshock (Lewis et al. 1996). Neither challenge produced airway hyperresponsiveness to histamine or methacholine. Both types of challenge induced airway hyperresponsiveness to 5-HT and U-46619. 5-HT was examined after obtaining a dose-response curve to methacholine in the same guinea-pig. The animals could therefore be con-

sidered to be hyperresponsive but such hyperresponsiveness was not demonstrable with methacholine. The degree of airway hyperresponsiveness was similar for micro- and macroshock challenges and suggests that a mild challenge is as effective in causing airway hyperresponsiveness as a severe one and thus supports the widely held view that bronchoconstriction per se is not directly linked to airway hyperresponsiveness.

In some of the microshock experiments, atropine was used to determine whether vagal pathways were involved in the bronchoconstriction induced by U-466 19 and histamine. In the presence of atropine, the leftward shift of the dose-response curve to U-46619 due to ovalbumin challenge was no longer significant. In the case of histamine, ovalbumin challenge appeared to cause a significant degree of hyporesponsiveness in the presence of atropine. The loss of significant airway hyperresponsiveness to U-466 19 and the revelation of hyporesponsiveness to histamine after atropine administration would suggest that cholinergic pathways may have a role in the appearance of airway hyperresponsiveness in this model. One mechanism for a cholinergic involvement may be the activation of sensory pathways by the antigen challenge, which may facilitate cholinergic transmission. The increase in lung eosinophils that we have shown following both the macro- and microshock antigen challenges in this model (Lewis et al. 1996) may be respon- sible for the activation of sensory nerve endings through release of cytotoxic compound such as major basic protein, eosinophil cationic protein and reactive oxygen species and epithelial shedding (Dahl et al. 1992). Depletion of neuropeptides from sensory nerves by capsaicin has been shown to inhibit airway hyperresponsiveness after repeated antigen exposure (Matsuse et al. 1991).

The lack of airway hyperresponsiveness to certain spasmogens supports previous studies which have examined responsiveness at -24 hr after antigen challenge in anaesthetised guinea-pigs. Sanjar et al. (1990) were unsuccessful in demonstrating ovalbumin-induced airway hyperresponsiveness after 24 hours when using the spasmogen prostaglandin Fz, given as an intravenous bolus. The same study, however, showed airway hyperresponsiveness when intravenous histamine was used. Similarly, single spasmogen studies have failed to reveal airway hyperresponsiveness to iv histamine at any time up to 72 hr after ovalbumin challenge (Banner et al. 1996) or to acetylcholine (Heuer et al. 1994). We have found only one other study which examined a wide range of spasmogens (Crowther et al. 1997). In common with our results, these authors also claimed that antigen induced a hyperresponsiveness that was said to be heterogeneous. In their studies, however, responsiveness was measured immediately after an ovalbumin infusion.

Drugs given parenterally may be inactivated to varying degrees during their passage through the lungs (Ferreira & Vane 1967) and thus explain differences in degree of airway hyperresponsiveness. The possibility of rapid metabolism of acetylcholine did not prevent the demonstration of airway hyperresponsiveness to this spasmogen in both IgG- and


IgE-based models (Coyle et al. 1988; Havill et al. 1990; Noonan et al. 1991; Arimura et al. 1994). Methacholine was used as the muscarinic agonist, in the present study. This non-selective muscarinic agonist is not susceptible to degra- dation by blood-borne butyrylcholinesterases (Broadley 1996). Ovalbumin-macroshock and ovalbumin-microshock challenges both failed to provoke airway hyperresponsiveness to methacholine. Two studies (Howell et al. 1992 & 1993) report airway hyperresponsiveness to methacholine in guinea-pigs 18 to 24 hr after an ovalbumin challenge in an IgG-based model. There is no published data demonstrating ovalbumin-induced airway hyperresponsiveness to intravenous methacholine -24 hr after ovalbumin challenge in the guinea-pig model used here, which is IgE-based (And- e rson 1980). Likewise, we have found no airway hyperresponsiveness to inhaled methacholine in anaesthetised guinea-pigs (Johnson & Broadley 1997). This contrasts with studies in asthma patients where airway hyperresponsiveness to inhaled methacholine is readily demonstrated.

There are no studies using the intravenous route for spasmogen delivery in humans, but an increasing number of studies using the inhaled route have found evidence in support of a more selective type of hyperresponsiveness. Djukanovic (1993) stated that close correlations between responses to histamine and methacholine alone do not consti- tute ‘non-specific’ airway responsiveness. Allergen challenge has been shown to cause a greater and more prolonged increase in responsiveness to bradykinin than to methacholine (Berman et al. 1995), which supports the idea that hyperresponsiveness is a selective pathophysiological abnormality (Djukanovic 1993). Aspirin-sensitive asthma patients display a selective hyperresponsiveness to leukotriene E4 but not leukotriene C4 or histamine (Christie et al. 1993), whilst Adelroth et al. (1986) found that asthma patients who were most responsive to methacholine were least responsive to leukotrienes C4 and D4.

The wet-dry weights of the lungs were used as an index of airways inflammation and/or mucus production. They were significantly greater in ovalbumin-challenged animals than the saline controls. Increase in wet weight could be a conse- quence of tissue oedema due to plasma extravasation aris- ing from airways inflammation (Evans et al. 1988). Likely mediators of this response are the leukotrienes (Woodward et al. 1983) and bradykinin (Rogers et ul. 1990). Plugging of the airways with increased mucus secretion may also ac- count for an increase in lung weight. Airway wall thickening by oedema and obstruction of the airway lumen with mucus may contribute to the airway hyperresponsiveness (James et al. 1989; Wiggs et al. 1992), since increased wall thickness reduces the amount of smooth muscle shortening required to occlude the airway lumen. However, since not all spasmogens display airway hyperresponsiveness, this cannot be the sole mechanism.

In conclusion, these results suggest that exposure of ovalbumin-sensitized guinea-pigs to inhaled ovalbumin induces airway hyperresponsiveness to 5-HT, U-46619 and bradykinin but not to methacholine, histamine or substance I?

The airway hyperresponsiveness induced by sensitization and subsequent challenge with ovalbumin in this species cannot therefore be described as non-specific.

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Airway Hyperresponsiveness in Anaesthetised Guinea-Pigs 18–24 Hours after Antigen Inhalation Does Not Occur with All Intravenously Administered Spasmogens