Differential effects of nitric oxide synthase inhibitors in an in vivo allergic rat model.

The in vivo role of nitric oxide in inflammatory cell migration, vascular permeability and the development of hyperresponsiveness to methacholine (MCh) was studied in rats 24 h following ovalbumin (OVA) challenge. The NO synthase (NOS) inhibitors N(G)-mono-methyl-L-arginine (L-NMMA; nonselective), aminoguanidine (two-fold inducible NOS-selective), N(omega)-nitro-L-arginine methyl ester (L-NAME; 2000-fold endothelial cell NOS-selective) or S-methyl-L-thiocitrulline (100-fold neuronal NOS-selective) were administered (100 mg x kg(-1) s.c.) to OVA-sensitized Piebald-Virol-Glaxo rats on 3 consecutive days during which they were challenged with allergen (1% OVA). Responses to inhaled MCh were measured in anaesthetized animals 24 h after OVA challenge. Cellular inflammation and vascular permeability were assessed using bronchoalveolar lavage (BAL) fluid collected 30 min after administration of Evans blue (50 mg x kg(-1) i.v.). OVA challenge in sensitized animals induced hyperresponsiveness to MCh, inflammatory cell influx and increased leakage of Evans blue into the BAL fluid (n=9, p<0.001). Aminoguanidine was effective in inhibiting the allergen-induced cellular influx and microvascular leakage (n=9, p<0.001) without altering responses to MCh. This effect was reserved by L-arginine. L-NAME (n=5, p<0.01) and S-methyl-L-thiocitrulline (n=6, p<0.001) further potentiated the allergen-induced hyperresponsiveness without altering cellular inflammation. L-NMMA attenuated both the OVA-induced cellular influx and Evans blue leakage (n=8, p<0.001) as well as further potentiating the hyperresponsiveness to MCh (p<0.05). From these studies, it is suggested that, in allergic Piebald-Virol-Glaxo rats, nitric oxide production by inducible nitric oxide synthase plays a role in the migration of inflammatory cells and increase in vascular permeability following allergen challenge, whereas nitric oxide produced by the constitutively expressed neuronal nitric oxide synthase limits hyperresponsiveness to methacholine.

Modification of the inflammatory response to allergen challenge after exposure to bacterial lipopolysaccharide.

The potential role of respiratory infections in altering the development of atopy and asthma is complex. Infections have been suggested to be effective in preventing the induction of T-helper 2-polarized allergen-specific immunity in early life, but also to exacerbate asthma in older, sensitized individuals. The mechanism(s) underlying these effects are poorly defined. The aim of this work was to determine the influence of lipopolysaccharide (LPS) exposure on the development of sensitization to allergen and the response to allergen challenge in vivo. Piebald-Virol-Glaxo rats were exposed to a single aerosol of LPS 1 d before or 1, 2, 4, 6, 8, or 10 d after sensitization with ovalbumin (OVA). On Day 11 animals were exposed to 1% OVA and responses to allergen were measured 24 h later, monitoring inflammatory cell influx and microvascular leakage into bronchoalveolar lavage (BAL) fluid as well as pulmonary responses to methacholine using the forced oscillation technique. Histologic analysis was included to complement the BAL results. Single aerosol exposure to LPS 1 d before and up to 4 d after intraperitoneal injection of OVA protected against the development of OVA-specific immunoglobulin (Ig) E. LPS exposure 6, 8, or 10 d after sensitization further exacerbated the OVA-induced cellular influx, resulting in neutrophilia and increased Evans Blue dye leakage with no effect on serum IgE levels. In addition, LPS abolished the OVA-induced hyperresponsiveness in sensitized animals when given 18 h after OVA challenge. This study demonstrates that exposure to LPS can modify the development of allergic inflammation in vivo by two independent mechanisms. Exposure early in the sensitization process, up to Day 6 after exposure to allergen, prevented allergen sensitization. Exposure to LPS after allergen challenge in sensitized animals abolished the hyperresponsiveness and modified the inflammatory cell influx characteristic of late-phase response to allergen.

Effects of salbutamol and Ro-20-1724 on airway and parenchymal mechanics in rats.

We investigated the effects of a selective beta(2)-agonist, salbutamol, and of phosphodiesterase type 4 inhibition with 4-(3-butoxy-4-methoxy benzyl)-2-imidazolidinone (Ro-20-1724) on the airway and parenchymal mechanics during steady-state constriction induced by MCh administered as an aerosol or intravenously (iv). The wave-tube technique was used to measure the lung input impedance (ZL) between 0.5 and 20 Hz in 31 anesthetized, paralyzed, open-chest adult Brown Norway rats. To separate the airway and parenchymal responses, a model containing an airway resistance (Raw) and inertance (Iaw), and a parenchymal damping (G) and elastance (H), was fitted to ZL spectra under control conditions, during steady-state constriction, and after either salbutamol or Ro-20-1724 delivery. In the Brown Norway rat, the response to iv MCh infusion was seen in Raw and G, whereas continuous aerosolized MCh challenge produced increases in G and H only. Both salbutamol, administered either as an aerosol or iv, and Ro-20-1724 significantly reversed the increases in Raw and G when MCh was administered iv. During the MCh aerosol challenge, Ro-20-1724 significantly reversed the increases in G and H, whereas salbutamol had no effect. These results suggest that, after MCh-induced changes in lung function, salbutamol increases the airway caliber. Ro-20-1724 is effective in reversing the airway narrowings, and it may also decrease the parenchymal constriction.

Muscarinic blockade of methacholine induced airway and parenchymal lung responses in anaesthetised rats.

BACKGROUND: It has previously been shown that M1 cholinergic receptors are involved in the parenchymal response to inhaled methacholine in puppies using the M1 selective antagonist pirenzepine. Although M3 receptors are responsible for acetylcholine induced bronchoconstriction in isolated rat lung, the role of M1 receptors has not been determined in the rat in vivo. METHODS: Anaesthetised, paralysed, open chested Brown Norway rats were mechanically ventilated and the femoral vein cannulated for intravenous injection of drugs. Low frequency forced oscillations were applied to measure lung input impedance (ZL) and computerised modelling enabled separation of ZL into airway and parenchymal components. Atropine (500 microg/kg iv) and pirenzepine (50, 100 or 200 microg/kg iv) were administered during steady state constriction generated by continuous inhalation (1 mg/ml) or intravenous (10 or 15 microg/kg/min) administration of methacholine. RESULTS: Continuous inhalation of methacholine produced a 185% increase in frequency dependent tissue resistance (G) which was effectively inhibited by atropine 500 microg/kg iv (p<0.01, n = 6). Pirenzepine (50, 100 or 200 microg/kg) had a minimal effect on the parenchymal response to inhaled methacholine. A 258% increase in airway resistance (Raw) was induced by continuous intravenous infusion of methacholine and this response was effectively abolished by pirenzepine (p<0.001, n = 5). Cutting the vagi in the cervical region did not alter baseline airway mechanics. Vagotomy did not affect lung responses to intravenous methacholine nor the ability of pirenzepine to reduce these responses. CONCLUSIONS: In the rat, M1-subtype receptors are functional in airways but not in the tissue.

The pharmacology of epanolol (ICI 141292)--a new beta 1-selective adrenoceptor partial agonist.

The clinical benefit of beta-adrenoceptor partial agonists is still debated. To clarify the situation, epanolol, ICI 141,292 [N-[-2-(3-o-cyanophenoxy-2-hydroxypropylamino)ethyl]-4- hydroxyphenylactamide], has been developed to assess the role of modest beta-adrenoceptor partial agonist activity in humans. Animal studies have shown that epanolol is a potent beta-adrenoceptor partial agonist with a greater affinity for beta 1- than beta 2-adrenoceptors. In vitro, the PA2 values obtained for espanolol at atrial and tracheal beta-adrenoceptors were 8.42 and 6.33, respectively (isoproterenol as agonist), giving a selectivity ratio of 123. The potency was studied in vivo in the dog, where it was also shown that as an antagonist at the cardiac beta 1-adrenoceptor, it was 18 and 40 times more potent than atenolol and practolol, respectively. Espanolol has less partial agonist activity in the rat than pindolol, but more than practolol. In this species, it is also a classical partial agonist, exhibiting agonist activity at all beta-adrenoceptor blocking doses. This is in contrast to pindolol, which caused predominantly beta-adrenoceptor blockade at low doses and partial agonist activity at higher doses. These differences were confirmed in haemodynamic studies in the dog. In contrast to many other partial agonists, the partition coefficient, log P, of epanolol in octanol and water is low (0.92).

Classification of beta-adrenoceptors in human isolated bronchus.

(+/-)-Isoprenaline (Iso), (-)-adrenaline (Ad), (-)-noradrenaline (NA), (+/-)-phenylephrine (Phe) and the beta 2-selective adrenoceptor agonist (+/-)-fenoterol (Fen) caused a concentration-dependent relaxation of human isolated bronchial preparations. Iso, Ad and NA caused complete relaxation of both spontaneous and carbachol-induced bronchial tone. Fen, which was only tested in preparations where tone was induced with carbachol, also caused complete relaxation. However, Phe was a partial agonist in all preparations tested. When relaxation responses to these amines were calculated as a % of their maximal effects, comparison of EC50 values showed that the order of potency was Iso greater than Ad = Fen greater than NA greater than Phe (92:27:25:1:0.2) in preparations with carbachol-induced tone and Iso greater than Ad greater than NA greater than Phe (112:38:1:0.3) in preparations with spontaneous tone. pA2 values determined for the beta-adrenoceptor antagonists propranolol (non-selective), atenolol (beta-selective) and ICI-118, 551 (beta 2-selective), using Iso as an agonist were, 9.3, 5.3 and 9.1 respectively. These results indicate that beta 2-adrenoceptors mediate relaxation of human isolated bronchus to sympathomimetic amines in preparations obtained 4-14 h post-mortem from non-diseased lung. alpha-Adrenoceptors were apparently sparse or absent in this tissue.

The pharmacology of a beta 2-selective adrenoceptor antagonist (ICI 118,551).

While specific antagonists of the beta 1-adrenoceptor, such as atenolol and betaxolol, are widely available, a potent specific antagonist selective for the beta 2-adrenoceptor has yet to be described. Previously described beta 2-selective antagonists such as butoxamine, H 35/25, and IPS 339 are lacking in potency, specificity, or appropriate beta 2-selectivity. ICI 118,551 [erythro-dl-1-(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol] possesses a high degree of selectivity and specificity for the beta 2-adrenoceptor. The affinity of propranolol and ICI 118,551 for beta-adrenoceptors has been determined by comparing their antagonist potencies, expressed as pA2 values, against the actions of isoproterenol on the guinea pig atrium and uterus. ICI 118,551 had a higher affinity for the uterine beta 2-receptor than did propranolol (pA2 9.26 and 8.64, respectively) but a lower affinity for the atrial beta 1-receptor (pA2 7.17 and 8.30, respectively). Thus, the beta 2/ beta 1-selectivity ratios, in vitro, were 123 for ICI 118,551 and 2.2 for propranolol. The potency and selectivity of ICI 118,551 and atenolol on the chronotropic and vasodilator actions of isoproterenol were compared in anaesthetised dogs. The apparent K' B values at the vascular beta-adrenoceptor were 2.1 micrograms/kg for ICI 118,551 and 253 micrograms/kg for atenolol, and the potency ratio for antagonism of vascular versus atrial actions of isoproterenol was greater than 250:1. In regard to ancillary pharmacological properties, ICI 118,551 has no partial agonist activity but has a membrane-stabilising action similar to that of propranolol.

Classification of beta-adrenoceptors in isolated bronchus of the pig.

1 (+/-)-Isoprenaline (Iso), (-)-adrenaline (Ad), (-)-noradrenaline (NA), the beta 2-selective adrenoceptor agonist (+/-)-fenoterol (Fen) and the beta 1-selective adrenoceptor agonist (+/-)-RO363 caused concentration-dependent relaxation of preparations of pig bronchus pre-contracted with carbachol 40-ng/ml (0.22 microM). Iso, Ad, NA and Fen caused complete relaxation of carbachol-induced tone, but RO363 caused relaxation equivalent to only 59% of the maximal response to Iso. 2 When relaxation responses to these amines were plotted as a % of their maximal effects, comparison of EC50 values showed that the order of potency was RO363 greater than Iso greater than NA greater than Fen greater than Ad (14.4:4.6:1:0.4:0.3). 3 pA2 values determined for the beta-adrenoceptor antagonists propranolol (non-selective) and atenolol (beta 1-selective), or the partial agonist salbutamol (beta 2-selective) using Iso as agonist were 8.3, 7.3 and 4.4 respectively. The pA2 value for atenolol using RO363 as the agonist was 7.6. 4 These results indicate that porcine bronchus contains a homogeneous population of beta 1-adrenoceptors.

Pharmacological responses of human and porcine lung parenchyma, bronchus and pulmonary artery.

1 Responses of preparations of human and porcine isolated bronchus and pulmonary artery to carbachol (CCh), methacholine, histamine, 5-hydroxytryptamine (5-HT), (-)-noradrenaline (NA), (-)adrenaline (Adr) and (+/-)-isoprenaline (Iso) were compared with responses to the same agonists in isolated lung parenchyma strips. 2 All preparations from both human and porcine lung contracted in response to histamine and all, except preparations of porcine pulmonary artery, contracted in response to CCh. Human and porcine pulmonary artery and parenchyma strip contracted in response to NA while bronchial preparations invariably relaxed. Iso caused relaxation of human and porcine bronchus and parenchyma strip. Although 5-HT was completely inactive in tissues isolated from pig lung, this amine was a powerful spasmogen in human pulmonary artery, relaxed human bronchus and caused variable responses in human parenchyma. 3 Results indicate that the pharmacological characteristics of human and porcine parenchyma strips may be explained in terms of responses of vascular or airways smooth muscle.

A comparative study of beta-adrenoceptors in human and porcine lung parenchyma strip.

1. Responses to (+/-)-isoprenaline (Iso), (-)-adrenaline (Adr) and (-)-noradrenaline (NA) were compared in isolated preparations of human and porcine lung parenchyma strip. 2. The order of relaxant potencies of these catecholamines in both human and porcine lung parenchyma was Iso greater than Adr greater than NA (1:0.24:0.01, human; 1:0.21:0.01.pig). These results suggest that beta 2-adrenoceptors predominate in both types of lung parenchyma strip. 3. pA2 values for the beta-adrenoceptor antagonist, propranolol (non-selective), with Iso as the agonist, in human and porcine lung strips were 7.84 and 7.83 respectively and for atenolol were 6.50 and 5.35 respectively. Taken as a whole results indicate the existence of an apparently homogeneous population of beta 2-adrenoceptors in porcine parenchyma strip, while both beta 1 and beta 2-adrenoceptors were revealed in human lung parenchyma.

A study of the relationship between cardiac beta-adrenoceptor blockade and intrinsic sympathomimetic activity in rats depleted of catecholamines.

1. The intrinsic sympathomimetic activity of a range of beta-adrenoceptor antagonists and its relationship to beta-adrenoceptor blockade was studied in pentobarbitone-anaesthetized, vagotomized rats which had been depleted of catecholamines by pretreatment with syrosingopine. Dichlorisoprenaline, practolol, oxprenolol, pindolol and acebutolol, produced dose-dependent positive chronotropic responses in this preparation. 2. The relationship between the dose requirements for this intrinsic sympathomimetic activity and beta-adrenoceptor-blocking activity was not the same for all drugs: (i) dichlorisoprenaline and practolol had intrinsic activity at all beta-adrenoceptor-blocking doses; and (ii) oxprenolol, pindolol and acebutolol had predominantly beta-adrenoceptor blockade at the lower dose levels and agonist activity only became significant at high doses relative to those producing beta-adrenoceptor blockade. 3. The positive chronotropic response to both practolol and pindolol was observed in rats which had been pithed and was antagonized by propranolol (0.1-3.0 mg/kg, i.v.), indicating that beta-adrenoceptors were involved. 4. It was concluded that the intrinsic sympathomimetic activity of beta-adrenoceptor antagonists was not a simple property as it was described by the relationship between the dose requirements for intrinsic sympathomimetic activity and for beta-adrenoceptor blockade as well as the degree of partial agonist activity.

The influence of the intrinsic sympathomimetic activity of beta-adrenoceptor antagonists on haemodynamic effects in anaesthetized dogs.

1. The effects of propranolol, atenolol (ICI 66,082), practolol and pindolol on heart rate and maximal left ventricular dp/dt, atrioventricular conduction time, mean aortic flow and diastolic blood pressure during cardiac pacing were investigated over a wide dose range (0.025-4.0 mg/kg, i.v.) in dogs anaesthetized with pentobarbitone.2. Propranolol and atenolol produced similar reductions in haemodynamic parameters. Propranolol had no further effect in dogs pretreated with atenolol. 3. Practolol tended to cause smaller reductions in the haemodynamic parameters than either propranolol or atenolol. Subsequent administration of propranolol still had some depressant activity. 4. Pindolol produced a biphasic response, with depression of cardiac function at the low doses (0.025 and 0.1 mg/kg), but a reversal of effect as the dose was increased. 5. It is therefore concluded that, in anaesthetized dogs, the intrinsic activity of practolol and pindolol limits the fall in heart rate, cardiac conduction, aortic flow and maximal dp/dt observed with beta-adrenoceptor blockade. With pindolol, however, the influence of intrinsic activity is observed only in high doses related to beta-adrenoceptor blockade.

An application of receptor models to the analysis of data on beta-adrenoceptor blockade.

1. The classical single receptor competitive occupancy model accurately describes the joint action of an agonist (isoprenaline) and a beta-adrenoceptor antagonist (propranolol) or some partial agonists (dichlorisoprenaline, practolol) on the positive chronotropic response in rats which have been depleted of catacholamines. 2. The mathematical form of the model suggests that the dissociation constants of classical competitive partial agonists may be assessed using dose ratios by exactly the same method as that currently used for agonist-antagonist interactions, provided that the log dose-response curves are first suitably normalized. 3. Close agreement between the theoretical mathematical models and the experimental data can be demonstrated by statistical fitting for certain beta-adrenoceptor antagonists (propranolol, dichlorisoprenaline, practolol). 4. The model fails to describe the behaviour of other beta-adrenoceptor antagonists (oxprenolol, pindolol). A possible extension of the model to include these drugs is proposed.