Pär Ewing
email: par.ewing@astrazeneca.com

Åke Ryrfeldt

Carl-Olof Sjöberg

Paul Andersson

Vasoconstriction after inhalation of budesonide: a study in the isolated and perfused rat lung

Keywords: ; corticosteroids, budesonide, nongenomic, pulmonary circulation, vasoconstriction

Clinical studies have shown that inhaled corticosteroids can induce rapid vasoconstriction in the airways, leading to decreased mucosal blood flow. The aim of this study was to investigate whether vasoconstriction of the pulmonary circulation after short inhalation of a corticosteroid can be detected in the isolated and perfused rat lung (IPL) -a model which could serve as a substitute or a complement to clinical models. Methods: IPLs were briefly exposed to dry powder aerosol of budesonide. The pulmonary perfusate flow rate was assessed during 100 minutes post exposure. A reduction in perfusion flow rate was interpreted as vasoconstriction. Main Results: Vasoconstriction was more pronounced after brief inhalation of 10 and 50 μg budesonide than 2 μg. The onset of vasoconstriction became statistically significant within 10-40 minutes after inhalation. Co-administration of a selective α 1 -adrenoceptor antagonist (prazosin 50 nM added to the perfusate) reduced vasoconstriction by approximately 50% during 100 minutes of perfusion (p=0.003).

Conclusions: Inhaled budesonide rapidly induces pulmonary vasoconstriction suggesting a nongenomic mechanism probably related to disposition of noradrenaline at the neuro-muscular junction. This ex vivo model could serve as a substitute or a complement to clinical models for investigating rapid effects of glucocorticoid receptor agonists on the pulmonary/bronchial circulation.

Introduction

Corticosteroids applied topically on the skin give rise to a blanched area due to vasoconstriction. Skin blanching tests have therefore frequently been used for screening of potency of corticosteroids in drug development [START_REF] Kumar | Transient effect of inhaled fluticasone on airway mucosal blood flow in subjects with and without asthma[END_REF][START_REF] Barnes | Relative safety and efficacy of inhaled corticosteroids[END_REF]. Inhaled corticosteroids (ICS) have prominent effects on inflammatory changes of the tracheobronchial airway vasculature in patients with bronchial asthma. ICS are known to exert their effect on airway vasculature through a delayed genomic [START_REF] Barnes | Molecular mechanisms of cortcosteroids in allergic diseases[END_REF] and a non-genomic phenomena, in common with topical steroids, thereby causing significant vasoconstriction in the larger airways and modifying, at least in part, some of the pathophysiological components of airway narrowing in bronchial asthma [START_REF] Mendes | Comparative bronchial vasoconstrictive efficacy of inhaled glucocorticosteroids[END_REF]. These clinically observed effects include suppression of proinflammatory responses such as hyperperfusion, microvasculature hyperpermeability, oedema formation, inflammatory cell recruitment and the formation of new blood vessels [START_REF] Charan | Bronchial vascular congestion and angiogenesis[END_REF][START_REF] Rodrigo | Rapid effects of inhaled corticosteroids in acute asthma: an evidence-based evaluation[END_REF]. Inhaled fluticasone propionate (FP) has been shown to reduce blood flow in airway mucosa of asthmatics and healthy volunteers [START_REF] Kumar | Transient effect of inhaled fluticasone on airway mucosal blood flow in subjects with and without asthma[END_REF]. Vasoconstrictive effect of ICS has also been demonstrated by inhalation of beclomethasone dipropionate and budesonide [START_REF] Mendes | Comparative bronchial vasoconstrictive efficacy of inhaled glucocorticosteroids[END_REF]. High doses of ICS have shown to significantly improve lung function in patients with asthma within 1-2 h of treatment. This rapid improvement leading to an improved discharge rate of asthma patients from the emergency rooms suggests that a rapid nongenomic mechanism of ICS is involved [START_REF] Rodrigo | Rapid effects of inhaled corticosteroids in acute asthma: an evidence-based evaluation[END_REF]. The commonly accepted genomic mode-of-action for corticosteroids involves the formation of a cytosolic corticosteroid-receptor complex that is subsequently translocated to the nucleus for its mode of action [START_REF] Barnes | Molecular mechanisms of cortcosteroids in allergic diseases[END_REF]. However, few studies have brought mechanistic insight to the rapid non-genomic action of corticosteroids. Iversen was first to demonstrate a role in vasomotor tone for corticosteroid-sensitive extraneuronal uptake of noradrenaline, mediated through saturable membrane transporters [START_REF] Iversen | The uptake of adrenaline by the rat isolated heart[END_REF][START_REF] Iversen | Inhibition of catecholamine Uptake-2 by steroids in the isolated rat heart[END_REF]. Recently, pretreatment

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5 with the selective α 1 -adrenoceptor antagonist terazosin was suggested to abolish the effect of fluticasone propionate on mucosal blood flow. This would indicate that noradrenergic signalling was involved in the corticosteroid sensitive regulation of smooth muscle tone in airway vasculature [START_REF] Brieva | Adrenergic airway vascular smooth muscle responsiveness in healthy and asthmatic subjects[END_REF]10].

The isolated and perfused rat lung (IPL) has been used in our laboratory as a model for studying effects of various pharmacological interventions on lung mechanics and vasoconstriction [START_REF] Selg | Antagonism of thromboxane receptors by diclofenac and lumiracoxib[END_REF][START_REF] Svens | A study of mechanisms underlying amitriptyline-induced acute lung function impairment[END_REF]. Recently, the DustGun aerosol technology has been used to expose the IPL, via dry powder inhalation, to aerosols of drugs and toxicants [START_REF] Gerde | A novel method to aerosolize powder for short inhalation exposures at high concentrations: isolated rat lungs exposed to respirable diesel soot[END_REF][START_REF] Ewing | Increasing exposure levels cause an abrupt change in the absorption and metabolism of acutely inhaled benzo(a)pyrene in the isolated, ventilated, and perfused lung of the rat[END_REF][START_REF] Ewing | Short inhalation exposures of the isolated and perfused rat lung to respirable dry particle aerosols; the detailed pharmacokinetics of budesonide, formoterol and terbutaline[END_REF].

In the present study we combined these two techniques to achieve three specific aims; i) to establish a method for accurately measuring the perfusate flow rate of the rat IPL perfused in the single-pass mode at a constant hydrostatic pressure, ii) to measure the perfusion flow rate for up to 100 minutes after short inhalation exposures of the IPL to air control, lactose and budesonide, iii) to study the influence of noradrenergic selective antagonist on budesonideexposed lungs.

Material and methods

Isolated perfused lungs

The experiments were approved by the local animal ethical committee. Thirty female Sprague-Dawley rats weighing 285-310 g were used in this study. The surgical procedure and lung mechanical measurements were performed as described in detail elsewhere [START_REF] Gerde | A novel method to aerosolize powder for short inhalation exposures at high concentrations: isolated rat lungs exposed to respirable diesel soot[END_REF]. The lungs were perfused with a 2% bovine serum albumin Krebs-Ringer physiological buffer. Briefly, each animal was anesthetised by injecting pentabarbital intraperitoneally and the chest was opened. A perfusate inlet catheter was attached to the pulmonary artery and perfusate was exiting the lung

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through a left heart catheter. A tracheoctomy was perfomed and lungs and heart were dissected en bloc and placed in an artificial thoracic chamber. A negative pressure of 0.5 kPa was applied in the chamber and lungs were ventilated at 75 breaths/min, and thus, an alternating negative pressure was established inside the chamber. Lung mechanical data was collected throughout the experiment using an EMKA system for monitoring the IPL (EMKA technologies, Paris, France).

The following lung mechanics were measured at a baseline: conductance (Gaw) = 66 ± 21 mL/s/kPa, dynamic compliance (Cdyn) = 2.7 ± 0.4 mL/kPa, and tidal volume (TV) = 1.2 ± 0.2 mL; (SD, n = 30).

Measurement of perfusion flow rate

Criteria for constant hydrostatic pressure perfusion; i) the vertical distance between the free liquid table in the perfusate reservoir and the apex of the heart of the IPL inside the thoracic chamber was set to approximately 100 mm H 2 O, equal to a hydrostatic pressure of 1.0 kPa, ii) the peristaltic pump speed was adjusted so that the reservoir was continually overflowed with perfusate, in order to keep the free liquid table at a constant level. Single-pass perfusion of the IPL was used. The perfusate flow rate was repeatedly measured five times per minute for up to 120 minutes, providing 600 discrete data points per exposure. The exposure cycle of the DustGun aerosol device was synchronized to allow for a pre-exposure flow measurement period at baseline [START_REF] Ewing | Short inhalation exposures of the isolated and perfused rat lung to respirable dry particle aerosols; the detailed pharmacokinetics of budesonide, formoterol and terbutaline[END_REF]. The exposures were made during the 95

Short inhalation exposures of the IPL to dry powder aerosols

The characterization of aerosols generated by the DustGun apparatus and the short inhalation exposures of the IPL to respirable aerosols in high concentrations have been previously

described [START_REF] Gerde | A novel method to aerosolize powder for short inhalation exposures at high concentrations: isolated rat lungs exposed to respirable diesel soot[END_REF][START_REF] Ewing | Increasing exposure levels cause an abrupt change in the absorption and metabolism of acutely inhaled benzo(a)pyrene in the isolated, ventilated, and perfused lung of the rat[END_REF][START_REF] Ewing | Short inhalation exposures of the isolated and perfused rat lung to respirable dry particle aerosols; the detailed pharmacokinetics of budesonide, formoterol and terbutaline[END_REF]. In the present study the IPL was exposed to budesonide in a series of doses delivering respectively, 2, 10 or 50 microgram budesonide (BUD-2, BUD-10 and BUD-50, respectively) in different groups of lungs (Table I). Lactose is commonly used as an excipient in inhalation drugs and was used a particle aerosol control (Table I). Budesonide and lactose were provided by AstraZeneca R&D, Lund. Measurements of the perfusion flow rate in air exposed IPL preparations were used as a negative control. In each experiment an air control period was

M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS 8
used during which the perfusion flow rate was recorded for 20 minutes prior to any exposure. The lungs were exposed to air by the DustGun apparatus and the perfusion flow rate was sampled 100 minutes after the air control period.

Co-exposure of α

-adrenoceptor antagonist and BUD-10

Prazosin, a selective α 1 -adrenoceptor antagonist, was used to test the effect of the antagonist on pulmonary vascular tone in IPLs co-exposed to budesonide. Prazosin hydrochloride (Sigma-Aldrich Sweden AB, Stockholm, Sweden) at 50 nM was administrated to the perfusate throughout the total time of perfusion in combination with short inhalation exposures to BUD-10.

Data and Statistical evaluation

To remove noise from the flow rate measurements, the raw data was low-pass filtered by calculating the running median of a window of ten consecutive measurements (Q n ) [START_REF] Bangham | Data-sieving hydrophobicity plots[END_REF]. The flow rate ( lung q ˆ) was calculated for the three subdivided periods of perfusion and for the whole period of perfusion post exposure. In figure 2, for the purpose of clarifiaction, lung q ˆwas expressed as a reduction in % of the average pulmonary perfusion flow rate (%qlung), (Eqn. 3).

A two-way analysis of variance (ANOVA) was applied for comparisons between treatments (using Sigmastat ver 3.0). Pair-wise multiple comparisons were performed with a Bonferroni correction for treatment, time and the effect parameter lung q ˆ. The differences were regarded statistically significant when the p-value was less than 0.05. Values are presented as mean±SD.

Eqn. The perfusion of the IPL, with constant hydrostatic pressure, provided a stable baseline in all experiments. The measured perfusate flow rate during the 20 minute long baseline interval was 20.0 ±1.8 mL/min (mean±SD, n=30). During ventilation of the air control lungs for 120 minutes, we observed a linear deterioration in the lung mechanics of 5-10 % for Cdyn and of 2-4 % for Gaw. This level of deterioration in lung mechanics in the IPL was also seen in lungs treated with lactose and budesonide and has also been reported in the literature [START_REF] Uhlig | Methods in Pulmonary Research[END_REF].

2 baseline n nlung Q Q q = for Q n 1…500 q nlung = relative
The perfusate flow rate of air control lungs decreased slightly over time. At 100 minutes post exposure, the perfusate flow rate (q nlung ) was 94.5 ±3.7 % (mean±SD, n=5) of the baseline value, and the average perfusate flow rate ( lung q ˆ) for the entire perfusion period was 97.9±3.3 % (mean±SD, n=5) of the baseline value (figure 1).

The mass median aerodynamic diameter (MMAD) of budesonide and lactose aerosols was 2.3 μm and 5.5 μm, respectively [START_REF] Ewing | Short inhalation exposures of the isolated and perfused rat lung to respirable dry particle aerosols; the detailed pharmacokinetics of budesonide, formoterol and terbutaline[END_REF]. The predicted lung deposition in μg and the average initial concentration of BUD in the lungs immediately after exposure were estimated for each exposure group (Table I) [START_REF] Ewing | Short inhalation exposures of the isolated and perfused rat lung to respirable dry particle aerosols; the detailed pharmacokinetics of budesonide, formoterol and terbutaline[END_REF].

Lactose exposures were chosen as a control for lungs exposed to a particle aerosol without apparent pharmacology, to compare with budesonide exposed lungs. The estimated deposited lung dose of lactose was 40±11 μg (mean±SD, n=5). The average flow rate of lungs exposed to lactose ( lung q ˆ) was compared with air control lungs. None of the pair-wise comparisons of air control lungs and lactose differed significantly (table II and figure 2). At 100 minutes post exposure, the perfusate flow rate (q nlung ) of lactose exposed lungs was 86.6±3. ) for the entire perfusion period was 95.0 ±1.7 % (mean±SD, n=5) of the baseline value (figure 1 and2).

After exposures of the IPL to budesonide, the average perfusate flow rate ( 100 0 ˆlung q )

was measured for 100 minutes post exposure (figure 1a,b). In comparison to lactose-exposed lungs, there was a gradual reduction in lung q ˆfor BUD-10 and BUD-50 (figure 1a). The onset of reduction of perfusion flow rate became significant for BUD-10 and BUD-50 in comparison to lactose during the 10 to 40 minute period ( 40 10 ˆlung q

), (p= 0.012 and 0.049, respectively). The perfusion flow rate for the BUD-2 exposures did not differ significantly from that of the lactose exposures. Hence, these comparisons demonstrate a dose-dependent onset of the perfusate flow rate reduction (figure 2). Further, while the flow rate of the BUD-2 exposures showed no effect on overall qlung ( 100 0

ˆlung q

), the BUD-10 exposures substantially reduced the perfusion flow rate of the lungs. Then at the BUD-50 exposures there was a smaller reduction in 100 0 ˆlung q , causing an unexpected bell-shaped dose-response for the budesonide sensitive pulmonary flow rate reduction. This smaller reduction for BUD-50 was as significant as noted for BUD-10 during the last 60 minutes of perfusion (p<0.001), but for the total period the difference in perfusion flow rate was non-significant (p= 0.185) (figure 2). The co-administration of BUD-10 with 50 nM prazosin given via the perfusate resulted in a decreased reduction of the perfusate flow rates ( lung q ˆ) and delayed the onset of reduction in comparison to BUD-10 (figure 1b). Within the 40 to 100 minute period, as well as for the whole time period, co-exposure to prazosin decreased significantly the flow reduction induced by the BUD-10 exposure (p<0.001 and =0.003, respectively). The flow reduction induced by the co-exposure of BUD-10 with prazosin 50 nM was roughly half that of the BUD-10 exposure alone (figure 2). The administration of 50 nM

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12 prazosin hydrochloride by itself had no affect of the pulmonary perfusion flow rate. For all comparisons of BUD with the positive control (lactose), a significant reduction of perfusate flow rate compared to the positive control was interpreted as vasoconstriction of pulmonary vessels.

M A N U S C R I P T A C C E P T E D

Discussion

This study, performed in the isolated and perfused rat lung, demonstrates a significant reduction in the pulmonary perfusion flow rate within 40 minutes after inhalation of sufficiently high doses of budesonide. It has been known that after administration of corticosteroids, a minimum of one to two hours are required for induction of their genomic effects, i.e. appearance of de novo synthesised proteins. The rapid reduction of perfusion flow rate observed in this study may be a result of a non-transcriptional and corticosteroid-sensitive disposition of noradrenaline (NA) causing vasoconstriction of pulmonary vessels [START_REF] Horvath | Norepinephrine transport by the extraneuronal monoamine transporter in human bronchial arterial smooth muscle cells[END_REF].

Several studies in humans have addressed the effects of ICS on blood flow in the bronchial mucosa [START_REF] Kumar | Transient effect of inhaled fluticasone on airway mucosal blood flow in subjects with and without asthma[END_REF][START_REF] Wanner | A simplified noninvasive method to measure airway blood flow in humans[END_REF], where data on perfusion of the bronchial tree have been derived from the measured disappearance of an inhaled inert gas from the airways (exhaled air) into the tracheo-bronchial circulation [START_REF] Kumar | Transient effect of inhaled fluticasone on airway mucosal blood flow in subjects with and without asthma[END_REF]. However, fewer clinical studies have addressed a possible effect of ICS on the pulmonary circulation. In asthmatics, inhalation of platelet aggregation factor (PAF) has been used for acute induction of a perfusion-ventilation mismatch in asthmatics [19]. Within 45 minutes after inhalation of fluticasone propionate, the corticosteroid ameliorated the PAF induced gas exchange mismatch, measured as improved systemic arterial oxygen tension. The authors proposed that this increase in systemic oxygen tension most likely reflected a corticosteroid-dependent effect on pulmonary circulation, leading to a better alveolar ventilation-blood perfusion balance. To our knowledge, this is the only study to show an effect of ICS on the pulmonary circulation in a clinical model.

M A N U S C R I P T A C C E P T E D

Yet, an important question is whether the presently described results derived from measurements on the pulmonary circulation in rats have any bearings on clinical findings in the bronchial circulation as discussed above. Thus, in order to bring together our findings of rapid corticosteroid-sensitive reduction of perfusion flow rates in the IPL with corticosteroid-sensitive vasoconstriction of bronchial vessels of the human lung, the discussion will be narrowed down to the following topics: 1. mechanism for rapid vascular effects of ICS in humans; 2. viability, disposition and uptake of NA in the IPL; 3. sympathetic innervation of the pulmonary and bronchial circulation and; 4. the applied corticosteroid dose to target tissue e.g. ex vivo doses compared to clinical doses.

1. Horvath and co-workers have, in addition to clinical studies, brought deep mechanistic insight into the topic of vascular effects of ICS [START_REF] Mendes | Comparative bronchial vasoconstrictive efficacy of inhaled glucocorticosteroids[END_REF][START_REF] Brieva | Adrenergic airway vascular smooth muscle responsiveness in healthy and asthmatic subjects[END_REF][10] . They have demonstrated, in isolated human bronchial arterial smooth muscle cells [START_REF] Horvath | Norepinephrine transport by the extraneuronal monoamine transporter in human bronchial arterial smooth muscle cells[END_REF], that corticosterone, budesonide and methylprednisolone dose-dependently inhibit -by an unknown but rapid mechanism -the extraneuronal uptake of NA. By doing so, the corticosteroids increase the concentration of NA at the neuro-muscular junction that may result in increased α 1 -adrenoceptor occupancy, noradrenergic signalling and vasomotor tone. This mechanism can certainly explain the acute bronchial vasoconstrictive action of inhaled ICS in humans [START_REF] Horvath | Norepinephrine transport by the extraneuronal monoamine transporter in human bronchial arterial smooth muscle cells[END_REF].

2. Few investigators have previously studied the mechanisms of disposition of NA in the isolated and perfused rat lung. In a tracer study by Metting et al [START_REF] Metting | Compartmental analysis of the efflux of l-[3H]norepinephrine from isolated perfused rat lungs[END_REF], where NA was administered as a bolus dose into the pulmonary artery, the authors concluded that NA is mainly retained in the lung tissues in a slow intra-cellular compartment that is sensitive to inhibition of both neuronal and extraneuronal reuptake of NA. This study not only illustrates the utility of a simple adrenoceptor within the lung vessels, and thus, renders the pressor effect induced by budesonide less pronounced. Thus, in the IPL, a corticosteroid-sensitive redisposition of NA in lung tissuesthrough an unknown mechanism -may facilitate an increase in α 1 -adrenoceptor occupancy, noradrenergic signalling and vasomotor tone [START_REF] Horvath | Norepinephrine transport by the extraneuronal monoamine transporter in human bronchial arterial smooth muscle cells[END_REF]. In future studies, we will continue to address the role of a corticosteroid-sensitive disposition of NA, in the light of both neuronal and extraneuronal uptake.

3. We surveyed the literature for a cross-species comparison of the anatomy of sympathetic nervous system, and more specifically, the distribution of nor-adrenergic fibers in bronchial and pulmonary vasculature. The bronchial vessels in rats, monkeys and humans are sustained by a network noradrenergic fibers [START_REF] El-Bermani | Pulmonary noradrenergic innervation of rat and monkey: a comparative study[END_REF][START_REF] Sheppard | Neurone specific enolase and S-100: new markers for delineating the innervation of the repsiratory tract in man and other mammals[END_REF]. Pulmonary vessels of the rat have been demonstrated by some workers to have a low frequency of noradrenergic innervation. El-Bermani [START_REF] Sheppard | Neurone specific enolase and S-100: new markers for delineating the innervation of the repsiratory tract in man and other mammals[END_REF] has reported that the branched pulmonary arteries and the small pulmonary veins with surrounding smooth muscles in Wistar rats were frequently innervated with nor-adrenergic fibers.

The main flow resistance of the pulmonary circulation is considered to stem from these richly innervated plexuses of pulmonary veins. Thus, there seems to be some similarities between both the frequency and distribution of the nor-adrenergic innervation in the pulmonary veins and in the

M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS 16
bronchial arteries. This insight suggests that, after stimulation of noradrenergic nerve-endings, similar physiological responses such as vasoconstriction are to be expected in both types of neuro-muscular plexuses, i.e. pulmonary bronchial arteries and pulmonary veins.

4. An important aspect of comparing vasoconstriction in clinical and preclinical models is whether the applied exposures in preclinical models are relevant for the clinical situation. In humans, after inhalation of a nominal dose of 800 μg of budesonide -a dose that significantly reduced mucosal blood flow in healthy subjects as well as in asthmatics [START_REF] Mendes | Comparative bronchial vasoconstrictive efficacy of inhaled glucocorticosteroids[END_REF] -typically 20% of the nominal dose is expected to deposit in the lung [START_REF] Derom | Lung deposition and efficacy of inhaled formoterol in patients with moderate to severe COPD[END_REF]. If this mass is dissolved and distributed in 500 g human lung tissue, this would correspond to an average initial tissue concentration in the lung of approximately 1 μM directly after inhalation (it should be kept in mind, however, that budesonide is not likely to be homogenously distributed, and that concentrations locally in parts of the mucosa can be much higher). For comparison, we have calculated that in the rat IPL, exposures to 10 μg budesonide (that caused a rapid reduction of flow rate) results in an initial average tissue concentration of 15 μM, or about one order of magnitude higher than observed in the clinic. This difference in initial target tissue concentration is not unreasonable considering: i) there is at least a five-fold difference in airway air/blood barrier thickness between the two species (human >rat) [START_REF] Ewing | Increasing exposure levels cause an abrupt change in the absorption and metabolism of acutely inhaled benzo(a)pyrene in the isolated, ventilated, and perfused lung of the rat[END_REF], and, ii) there is most likely a more peripheral deposition of particles in the IPL setup in comparison to administration of budesonide to central airways in humans, which is likely to give a much more rapid extraction of the drug to the circulation from the rat lung than from the human lung. It is reasonable to assume that soon after inhalation, a faster extraction of higher tissue concentrations from the rat lung leads to local lung tissue concentrations similar to those of corresponding human lung exposures. Some support for this argument comes from the measured time to reach the maximum concentration (tmax) of budesonide in the blood in the two cases. For budesonide inhaled by healthy male subjects, the tmax in the systemic circulation range from 10-24 min [START_REF] Thorsson | Pharmacokinetics and systemic activity of fluticasone via Diskus and pMDI, and of budesonide via Turbuhaler[END_REF], whereas we have measured the tmax in the single-pass perfusate of the rat IPL to be 0.7-1.1 min [START_REF] Ewing | Short inhalation exposures of the isolated and perfused rat lung to respirable dry particle aerosols; the detailed pharmacokinetics of budesonide, formoterol and terbutaline[END_REF]. Even if the clinical tmax is somewhat delayed because of being sampled on the venous side of the circulation, whereas the arterial side is sampled in case of the IPL, it is reasonable to assume that the thicker air/blood barriers of human lung would retain the budesonide at least one order of magnitude longer than the thinner barriers of the rat IPL. A significant reduction of the perfusion flow rate in our IPL model should therefore require a higher initial concentration than in case of clinical exposures.

Hence, studies from our laboratory and others have shown that the rat IPL can serve as model to study the disposition of noradrenaline and the corticosteroid-sensitive vasoconstriction of the lung. Therefore, it is reasonable to suggest that the effect of corticosteroid exposures on perfusion flow rates as observed in our IPL model after administration of sufficiently high doses of inhaled budesonide, is most likely controlled by the retention of noradrenaline at the neuromuscular junction, similar to the vascular effects of ICS observed in humans and attributed to a nongenomic corticosteroid effect [START_REF] Kumar | Transient effect of inhaled fluticasone on airway mucosal blood flow in subjects with and without asthma[END_REF][START_REF] Iversen | Inhibition of catecholamine Uptake-2 by steroids in the isolated rat heart[END_REF][START_REF] Horvath | Norepinephrine transport by the extraneuronal monoamine transporter in human bronchial arterial smooth muscle cells[END_REF]. Moreover, similar noradrenergic innervations of pulmonary veins and bronchial arteries in rats and primates allow us to suggest that the rat IPL can serve as model to study corticosteroid sensitive vasoconstriction of the bronchial circulation in humans. The model can, thus, serve as a valuable substitute or complement to more laborious clinical models for investigating rapid, corticosteroid-dependent effects on the pulmonary/bronchial circulation, which may have important implications for the disposition and efficacy of these drugs in the lung, alone or in combination with other inhaled drugs [START_REF] Horvath | The effect of corticosteroids on the disposal of long-acting beta(2)-agonists by airway smooth muscle cells[END_REF].

  was terminated. The flow rate was measured by collecting the perfusate in a container placed directly on an electronic scale (Sartorius ED3202S-CW, Sartorius AG, Germany). By directing the flow through a 3-way valve for 4s to the sample container and for 8 s to drain, discrete weight gain intervals could be recorded by the scale. A data acquisition protocol M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS 7 written in Labview (National Instruments AB, Solna, Sweden) was used to record the step-wise increase in cumulative weight of the sampled perfusate. An algorithm was designed to identify the time point and weight at the beginning and end of each weight gain interval. The pulmonary perfusion flow rate (mL/min) of each sample interval was calculated by dividing the weight gain of each sampling interval with its corresponding time duration (eqn 1at the beginning of the n th interval (g) m n+1 = recorded mass at the end of the n th interval (g) ρ = density of perfusate (g/mL) t sample =sample interval (min)

  window was advanced with one sample for each calculation of Q n from the first to the 600 th sample. The first 100 samples, corresponding to the air control period, were used to assure that a steady baseline was maintained throughout the first 20 minutes of lung perfusion. For treatments and controls, each perfusion flow rate measurement was normalized to its 20 minute low-pass filtered reading by assigning it there the value 100% (Q baseline ). The relative perfusion flow rate (q nlung ) from time 0-100 minutes post exposure of each sample n was calculated by dividing each Q n of that series with the corresponding Q baseline (Eqn 2). The reduction of perfusate flow rate following treatment was analysed by dividing the period of perfusion post-exposure into three different time periods; from time zero to 10 minutes, from 10 minutes to 40 minutes, and from 40 minutes to 100 minutes. For each control and treatment, the average relative pulmonary perfusion M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS 9

  perfusion flow rate of the n th sample Q baseline = baseline perfusion flow rate at 20 minutes (mL/min) average relative perfusion flow rate (averaged over any given interval (0-10, 10-40, 40-100 or 0-100 minutes) %qlung = percentage reduction of average relative perfusion flow rate (averaged over any given interval (0-10, 10-40, 40-100 or 0-100 minutes) M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS 10 3 Results

11 (

 11 mean±SD, n=5) of the baseline value and the average perfusate flow rate (

15 ex 1 - 1 - 1 -

 15111 vivo model compared to a much more complex in vivo situation, but certainly provides evidence that uptake mechanisms of NA are functional in the IPL. Furthermore, in the present study we have demonstrated that the perfusion flow rate in the IPL is sensitive to high concentrations of ICS and that the selective α adrenoceptor antagonist prazosin reduced the budesonide-induced vasoconstriction by about 50%, which suggest that the effect of budesonide is mediated through the α adrenoceptor[START_REF] Brieva | Adrenergic airway vascular smooth muscle responsiveness in healthy and asthmatic subjects[END_REF]10]. Hence, in our model we hypothesise that the selective α adrenoceptor antagonist prazosin competes with NA at the binding sites of the α 1 -

  The authors thank Anna-Miller Larsson for advice and valuable comments. This study was funded by AstraZeneca R&D as a part of a studentship to Pär Ewing.

25 7

 25 Figure 1a and b) Relative perfusate flow rate (qlung) in the lungs plotted versus time.

Figure 2 )

 2 Figure 2) Reduction of relative perfusate flow rate %qlung (Eqn 3) in the lungs,

  FIGURE 2

Table I )

 I The deposited mass (μg) after inhalation of budesonide and lactose in the IPL and the calculated initial average lung concentration in μM of budesonide in 1.5 g lung tissue.

		ARTICLE IN PRESS
	Table II	0-10 minutes	10-40 minutes	40-100 minutes	0-100 minutes
	Table I Treatment	n Mass deposited (μg) Lung average conc. (μM) 10 0 ˆ-lung q 40 10 ˆ-lung q 100 40 ˆ-lung q 100 0 ˆ-lung q
	Drug BUD-2 BUD-10 BUD-50 BUD-10 +Prazosin 5 5 5 5 Lactose 5 Air Control 1.0±0.02 Lactose 1.0±0.01 BUD-2 0.99±0.01 BUD-10 0.98±0.03 BUD-10 praz 0.99±0.01 BUD-50 0.97±0.02	2.5±0.3 11±0.8 48±9.3 12±0.2 40±11 M 0.99±0.03 0.98±0.01 0.98±0.01 0.93±0.03 * 0.97±0.02 A N U S C R I P T 4 15 75 15 80 0.97±0.04 0.98±0.03 0.92±0.03 0.95±0.02 0.91±0.03 0.94±0.02 0.79±0.06 ** 0.85±0.04 ** 0.87±0.02*, § § 0.91±0.02 § § 0.93±0.02 * 0.85±0.03 ** 0.89±0.02**
	A C C E P T E D
					24

Table II )

 II . The mean relative pulmonary perfusate flow rate ( lung q ˆ) for the different


Maria Gabriella Matera

Eugenio Martuscelli

Mario Cazzola
email: mario.cazzola@uniroma2.it

Pharmacological modulation of β-adrenoceptor function in patients with coexisting chronic obstructive pulmonary disease and chronic heart failure

published or not. The documents may come

Pharmacological modulation of β-adrenoceptor function

in patients with coexisting chronic obstructive pulmonary disease and chronic heart failure

Introduction

The prevalence of chronic heart failure (CHF) in patients with chronic obstructive pulmonary disease (COPD) is known to range from 20% to 32% [START_REF] Rutten | Unrecognized heart failure in elderly patients with stable chronic obstructive pulmonary disease[END_REF][START_REF] Havranek | Spectrum of heart failure in older patients: results from the National Heart Failure Project[END_REF][START_REF] O'connor | Demographics, clinical characteristics, and outcomes of patients hospitalized for decompensated heart failure: observations from the IMPACT-HF registry[END_REF][START_REF] Braunstein | Noncardiac comorbidity increases preventable hospitalizations and mortality among Medicare beneficiaries with chronic heart failure[END_REF][START_REF] Gustafsson | Female sex is associated with a better long-term survival in patients hospitalized with congestive heart failure[END_REF][START_REF] Dahlstrom | Frequent noncardiac comorbidities in patients with chronic heart failure[END_REF][START_REF] Render | Left ventricular dysfunction in deteriorating patients with chronic obstructive pulmonary disease[END_REF]. This high prevalence of CHF in patients with COPD is not surprising, because COPD and CHF share smoking as a common risk factor, and, moreover, it is likely that low-grade systemic inflammation accelerates progression of coronary atherosclerosis, which ultimately results in ischemic cardiomyopathy [START_REF] Fabbri | Complex chronic comorbidities of COPD[END_REF].

Although the coexistence of COPD and CHF is common, it is often unrecognized because dyspnea and functional intolerance are common presenting symptoms and this makes the coexistence of both conditions difficult to diagnose regardless of patients age [START_REF] Padeletti | Coexistent chronic obstructive pulmonary disease and heart failure in the elderly[END_REF]. Moreover, patients with coexisting COPD and CHF may have therapeutic constraints that have not been comprehensively investigated. Pharmacological modulation of β-adrenoceptor (β-AR) function is one of the critical issues in the treatment of these patients [START_REF] Cazzola | Inhaled β 2 -adrenoceptor agonists: cardiovascular safety in patients with obstructive lung disease[END_REF].

Interfering with β-AR function is central in the treatment of both COPD and CHF. In fact, inhaled β 2 -agonists are one of the mainstays of COPD therapy [START_REF] Rabe | Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: GOLD executive summary[END_REF], whereas β-antagonists, also called β-blockers, reduce mortality and morbidity in patients with CHF [START_REF] Dickstein | ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure 2008: the Task Force for the Diagnosis and Treatment of Acute and Chronic Heart Failure 2008 of the European Society of Cardiology. Developed in collaboration with the Heart Failure Association of the ESC (HFA) and endorsed by the European Society of Intensive Care Medicine (ESICM)[END_REF]. Consequently, pharmacological modulation of β-AR function is recommended by current COPD [START_REF] Rabe | Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: GOLD executive summary[END_REF] and CHF guidelines [START_REF] Dickstein | ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure 2008: the Task Force for the Diagnosis and Treatment of Acute and Chronic Heart Failure 2008 of the European Society of Cardiology. Developed in collaboration with the Heart Failure Association of the ESC (HFA) and endorsed by the European Society of Intensive Care Medicine (ESICM)[END_REF], respectively.

Traditionally, the treatment of COPD is based on the use of long-acting bronchodilators, mainly long-acting β 2 -agonists (LABAs) and/or long-acting antimuscarinic agents, as well as the use of short-acting β-agonists (SABAs) as rescue medication, but it has been suggested that the use of β 2 -agonists might lead to an increased risk for adverse events in patients suffering from COPD [START_REF] Salpeter | Cardiovascular effects of βagonists in patients with asthma and COPD: a meta-analysis[END_REF] as well as in those suffering from CHF [START_REF] Cazzola | Inhaled β 2 -adrenoceptor agonists: cardiovascular safety in patients with obstructive lung disease[END_REF][START_REF] Au | Risk of mortality and heart failure exacerbations associated with inhaled βadrenoceptor agonists among patients with known left ventricular systolic dysfunction[END_REF]. On the other hand, despite clear evidence of the effectiveness of β-blockers in the management of patients with CHF, the use of these agents has traditionally been contraindicated in COPD, mainly because of anecdotal evidence and case reports citing acute bronchospasm [START_REF] Cazzola | The pharmacologic treatment of uncomplicated arterial hypertension in patients with airway dysfunction[END_REF] and increased airway hyperresponsiveness (AHR) [START_REF] Van Der Woude | Detrimental effects of β-blockers in COPD: a concern for nonselective β-blockers[END_REF] after their administration.

The objective of this review is to verify whether the evidence supports or refutes the pharmacological modulation of β-AR function in patients with coexisting COPD and CHF.

β-ARs in human airways and heart

β-ARs are subdivided into three types; β 1 , β 2 and β 3 . They are members of the seven-transmembrane spanning family of G-protein-coupled receptors related to bacteriorhodopsin. There is a 65% to 70% homology between β 1 -β 3 and β 2 -ARs.

β-ARs and human airways

β 2 -ARs are widely distributed in human airway smooth muscle [START_REF] Ruffin | Assessment of β-adrenoceptor antagonists in asthmatic patients[END_REF].

Radioligand binding studies on lobectomy specimens have shown β 2 -AR density to increase with increasing airway generation, with high levels in the alveolar region [START_REF] Spina | Autoradiographic localization of β-adrenoceptors in asthmatic human lung[END_REF]. β 2 -ARs are also expressed on many pro-inflammatory and immune cells, including mast cells, macrophages, neutrophils, lymphocytes, eosinophils, epithelial and endothelial cells, and both type I and type II alveolar cells. β 2 -AR stimulation produces airway relaxation, but prolonged β 2 -AR activation leads to a decrease in receptor responsiveness (i.e.

desensitization) that differs depending on the cell type [START_REF] Johnson | The β-adrenoceptor[END_REF]. The rate at which a β-agonist drives receptor desensitisation parallels its intrinsic efficacy [START_REF] Cazzola | Inhaled β 2 -adrenoceptor agonists: cardiovascular safety in patients with obstructive lung disease[END_REF].

Intrinsic efficacy refers to the ability of a drug, independent of tissue conditions, to interact with a receptor to activate its downstream signal transduction pathway. The intrinsic efficacies of commonly used β-agonists are listed in table 1.

Also β 1 -ARs are present in the lungs . They account for 10% and 30% of the ARs on submucosal glands and alveolar walls, respectively [START_REF] Ruffin | Assessment of β-adrenoceptor antagonists in asthmatic patients[END_REF]. Reasons for modulating β-AR function in patients with COPD and chronic heart failure

β-ARs and the heart

M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS 6
The presence of β-ARs in human airways and heart explains why the modulation of β-AR function is important in influencing pulmonary and cardiac functions. In COPD patients, β 2 -agonists interact with the β 2 -ARs and relax airway smooth muscle. On the other side, overwhelming evidence supports a major role for the β-AR system in heart failure. While this system is ideally suited for short-term increases in cardiac performance, its long-term activation is apparently detrimental. These damaging effects appear to be mainly due to stimulation of the β 1 -AR subtype, but the responsible signalling pathways need to be identified.

In the failing heart, the respective proportions of A meta-analysis of 9 randomized clinical trials that lasted at least one year, with > 3,500 patients, found that when salmeterol was used instead of placebo or usual therapy, patients were less likely to withdraw early and suffer an exacerbation, and had a better health status and a greater increase in FEV 1 [27]. There was no tachyphylaxis or death reported although some papers fatal "cardiac failure" in the bambuterol group during the first month, and a lower but increased incidence during the second to sixth months, whereas there was no excess in the salmeterol group. This finding raised the question why bambuterol and not salmeterol caused CHF. It is likely that this was linked to the route of administration (oral instead that inhaled) and the different dose, significantly higher with bambuterol that induced a high systemic bioavailability and, consequently, higher risk of side effects.

M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS

β-AR blocking drugs

Chronic heart failure

When β-blocking drugs are used for coronary artery disease or hypertension, they acutely produce a decrease in contractility in patients with heart failure.

Also, patients with recently decompensated heart failure are very susceptible to worsening even with low doses of this class of drugs. The initiation of βblockade in these patients might be expected to cause worsening heart failure by interfering with the positive inotropic and lusitropic actions of endogenous catecholamines [START_REF] Haber | Why do patients with congestive heart failure tolerate the initiation of β-blocker therapy?[END_REF]. Consequently, the use of β-blocker therapy was once thought to be contraindicated in patients with heart failure with systolic dysfunction.

Paradoxically, the chronic use of certain β-blockers has been the treatment that has had the most success for decreasing mortality in CHF. Since the pioneering work in the 1970s [START_REF] Swedberg | Prolongation of survival in congestive cardiomyopathy by β-receptor blockade[END_REF], it took two decades until β-blockers turned from contraindication to standard treatment in heart failure [START_REF] Bristow | β-adrenergic receptor blockade in chronic heart failure[END_REF] So far there is not a conclusive explanation that is able to simultaneously support a short-term detrimental effect with a chronic beneficial effect.

However, the fact that the beneficial effect of the antagonist is not observed for weeks or even months after equilibrium of

β

Discussion

Regarding the utilization of β-agonists, there is no solid proof at present time that supports or denies their use in the treatment of CHF, although some evidence indicates that therapy with inhaled β-agonists is associated with an increased risk for CHF decompensation and all-cause mortality in patients with CHF [38,39]. In effect, CHF has a tremendously high mortality and morbidity.

Therefore, it is not surprising that any adverse effect of a drug on survival would be much easier to observe and document in this disease [START_REF] Bond | Getting to the heart of asthma: can "β blockers" be useful to treat asthma?[END_REF]. It must be mentioned that inhaled β 2 -agonists induce adverse cardiac effects in COPD patients with pre-existing cardiovascular disease [START_REF] Cazzola | Cardiac effects of formoterol and salmeterol in patients suffering from COPD with preexisting cardiac arrhythmias and hypoxemia[END_REF]. In particular, the adverse effects of β 2 -agonists are likely to be exacerbated in COPD patients with coexistent CHF [START_REF] Jemtel | Diagnostic and therapeutic challenges in patients with coexistent chronic obstructive pulmonary disease and chronic heart failure[END_REF].

Regarding the utilization of β-blockers, the information from trials and metaanalyses of patients with COPD exposed to continued treatment with those βblockers that are more selective for the β 1 -AR is reassuring, but there are two

M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS 14 fundamental concerns.
First of all, the long-term safety of β 1 -blockers in airways disease has not been proven. In general, the studies were limited to few weeks, excluded patients who had recent exacerbations and were deemed to be at higher-risk, included younger patients, and may have been subject to publication bias [START_REF] Ashrafian | Beta-blocker therapy of cardiovascular diseases in patients with bronchial asthma or COPD: the pro viewpoint[END_REF]. The second concern is linked to the fact that recent analyses that examined the affinity of a wide range of so called β 1 -blockers to a homogenous population of human β-AR subtypes expressed in cell lines, showed a more limited preference for β 1 -AR than previously thought, suggesting that many β 1 -blockers block β 2 -ARs in bronchi in addition to blocking β 1 -ARs in the heart [START_REF] Jemtel | Diagnostic and therapeutic challenges in patients with coexistent chronic obstructive pulmonary disease and chronic heart failure[END_REF]. This finding could explain why the effects of the β 1 -blocker metoprolol on AHR are the same as those of the nonselective βblocker propranolol in patients with COPD [START_REF] Van Der Woude | Detrimental effects of β-blockers in COPD: a concern for nonselective β-blockers[END_REF].

Therefore, the advice is that prescribing β-blockers for COPD patients should be restricted to specialists who are able to monitor patients closely. In reality, many general practitioners (GPs) may feel reticent about providing repeat prescriptions to COPD patients started on β-blockers by cardiology specialists.

A commonsense approach would be only to prescribe these drugs when the GP knows the patient is stabilised on them, when they are shown to be beneficial to the patient, and when the consultant and GP agree on required monitoring and follow-up, and the responsibility for it [START_REF] Panting | Asthma and beta-blocker prescription: a medico-legal view[END_REF].

This issue needs careful consideration. Traditionally, the treatment of COPD is based on the use of long-acting bronchodilators although the potential risk for adverse cardiovascular events in patients with COPD that is present even when inhaled anticholinergic are used [START_REF] Cazzola | Inhaled β 2 -adrenoceptor agonists: cardiovascular safety in patients with obstructive lung disease[END_REF][START_REF] Singh | Inhaled anticholinergics and risk of major adverse cardiovascular events in patients with chronic obstructive pulmonary disease: a systematic review and meta-analysis[END_REF]. β-Blockers might neutralize the actions of β-agonists, so that the risk of adverse cardiovascular events associated with these agents might be different in subjects already using βblockers than among nonusers of β-blockers. However, there is evidence that This is to ensure that no patient is denied the benefits of β-blockers. Therefore, although β-blockers can be introduced in any medical setting for treating patients with concurrent COPD and CHF, we believe that it still seems appropriate to only use those β-blockers that are more selective for the β 1 -AR but without ISA, at the lowest dose and to titrate them slowly (table 5) with attention to lung function and symptoms, adding an inhaled antimuscarinic agent when bronchodilation is needed [START_REF] Cazzola | The pharmacologic treatment of uncomplicated arterial hypertension in patients with airway dysfunction[END_REF]99].

β-Blockers with β 2 -agonist activity (celiprolol) might preferable to conventional β 1 -blockers, but there is some risk that bronchospasm may occur in certain individuals and the bronchodilator response to inhaled β 2 -agonists might be impaired [START_REF] Cazzola | The pharmacologic treatment of uncomplicated arterial hypertension in patients with airway dysfunction[END_REF]. Finally, it must be mentioned that β1and β2-AR responses to drug treatment exhibit marked interindividual variability [112][113][114]. At present, it seems to be clear that β-AR polymorphisms do not play a role as disease-causing genes, although they might be risk factors, might modify disease, and/or might influence progression of disease. In addition, β-AR polymorphisms might influence drug responses (Table 6).

The β 2 -AR variations Gly 16 Arg and Gln 27 Glu and, more recently, haplotypic variations, have been the focus of numerous pharmacogenetic studies looking at responses to SABAs and LABAs in subjects with chronic airway obstruction • Chronically reduce mortality by 30 -65%

• Chronically reduce hospitalizations, MI and sudden cardiac death.

• Start with low dose and increase dose very slowly (months) to tolerable and effective dose. Some patients get worse before they start to improve.

• Not recommended in patients in Class I or IV.

5 Although β 1 - 2 -

 512 ARs are classically described as the key participants in triggering and regulating cardiac contractility, stimulation of both β 1 -ARs and β 2 -ARs leads to positive inotropy via coupling to adenylyl cyclase and generation of cAMP[20].Bristow et al. [20] demonstrated that the proportions of β 1 -AR and β 2 -AR in normal hearts are 77 and 23%, respectively. Functional β 1 -ARs and β ARs coexist on atria and ventricles with β 1 /β 2 -AR ratio of about 60 to 70%/40 to 30% in the atria and about 70 to 80%/30 to 20% in the ventricles[21]. Interestingly, Rodefeld et al.[22] showed that in human sinoatrial nodes, total β-AR density is about 3-fold higher than that in the adjacent atrial myocardium; although the β 1 -AR subtype predominates in both regions, the β 2 -AR density is about 2.5-fold higher in the sinoatrial node than in the right atrial myocardium and this is consistent with physiologic studies that implicate this receptor in regulating cardiac chronotropism. β 2 -ARs are also present on adrenergic nerve terminals in the heart, where they facilitate noradrenaline release [23]. The functional responses mediated by β 1 -ARs and β 2 -ARs are not necessarily different, likely because human cardiac β 2 -ARs are more effectively coupled to adenylyl cyclase than are β 1 -ARs [24]. There is solid documentation of directly β-AR-mediated positive inotropic and chronotropic effects in humans in vivo using β 2 -agonists such as terbutaline or salbutamol. These effects are slightly antagonized by β-blockers that are more selective for the β 1 -AR such as bisoprolol or atenolol [24]. The activation of prejunctional β 2 -ARs might contribute to the cardiac effects of β-agonists because it leads to enhanced noradrenaline release, at least at high doses [24]. β-AR stimulation provides the most powerful mechanism to augment cardiac contractility in response to a 'fight-or-flight' situation, but prolonged exposure of β-ARs to an agonist causes desensitization [25].

β 1 -

 1 ARs and β 2 -ARs are 60 and 38% [21]. This decrease in the proportion of β 1 -ARs and the relative increase in the proportion of β 2 -ARs have been postulated to be a result of selective down-regulation of the β 1 -ARs, with little or no change in β 2 -ARs [21]. It is a protective mechanism that counteracts deleterious effects induced by chronic sympathetic activation [21]. Consequently, the failing heart becomes more dependent on the β 2 -ARs for inotropic support. However, the activation of these receptors may also alter the cardiac electrical stability, increasing the propensity for the formation of malignant arrhythmias in the diseased heart.β 2 -agonists COPD A volume of published evidence supports the role of LABAs in the treatment of stable COPD. They induce prolonged bronchodilation and translate this action into other health-outcome measures related to quality of life, such as the severity of dyspnoea, exercise capacity, and, mainly, number and severity of exacerbations [26].

  important finding because many patients with underlying cardiovascular diseases such as CHF have concomitant obstructive airway disease[40].Whatever the case may be, the observation that β 2 -agonists may exacerbate heart failure is supported by physiologic observations. β 1 -ARs are downregulated and desensitized among patients with LV systolic dysfunction and β 2 -ARs, although desensitized, are found in normal numbers and represent a higher proportion of total β-ARs [41, 42]. Among patients with heart failure, β 2 -agonists augment cardiac function but, with regular exposure to β 2agonists, myocardial β 2 -ARs become desensitized and down-regulated. [43, 44]. Moreover, chronic β 2 -ARs stimulation elevates G i expression [45]. The coupling of β 2 -ARs to G i proteins negatively regulates the G s -mediated contractile response in the heart of many mammalian species [45]. Intriguingly, the Ile 164 polymorphism of the β 2 -AR, which leads to a dysfunctional receptor, has been demonstrated to be associated with decreased exercise tolerance and a 5-fold increase risk of death among patients with CHF [46]. The possibility that chronic β-AR stimulation induces myocardial, but not systemic, elaboration of TNF-α, IL-1β, and IL-6 [47] is another important finding. In fact, evidence suggests that proinflammatory cytokines are capable of modulating cardiovascular function by a variety of mechanisms, including promotion of LV remodelling [48], induction of contractile dysfunction [49], and uncoupling of myocardial β 2 -ARs [50]. Although the possibility that β 2 -agonists might exacerbate heart failure, Ng et al. [51] explored if chronic inhaled salmeterol therapy, 100 µg bid, improved pulmonary function without augmentation of neurohormonal systems or ventricular ectopy in symptomatic heart failure subjects with LV ejection fraction <40%. Salmeterol significantly increased mean rate-pressure product by 5%, and FEV 1 without producing measurable effects on neuroactivation or ventricular ectopy. The safe profile of salmeterol was also confirmed by the Drug and Safety Research Unit and the School of Medicine at Southampton (UK) [52]. Using the prescription-event monitoring technique, researchers from this Unit re-examined the safety issue for salmeterol, and bambuterol, a M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS 10 long-acting oral agent and prodrug of terbutaline. There was an excess of non-

β

  -blockers lead to an increase in the density of β-ARs[START_REF] Callaerts-Vegh | Effects of acute and chronic administration of beta-adrenoceptor ligands on airway function in a murine model of asthma[END_REF]. This effect might suggest an bigger risk associated with the concomitant use of β-agonists and β-blockers in COPD. Apparently, β 1 -blockers are associated with an augmented bronchodilator response to β-agonists[START_REF] Ashrafian | Beta-blocker therapy of cardiovascular diseases in patients with bronchial asthma or COPD: the pro viewpoint[END_REF]. This is a clear indication of facilitated β-AR stimulation, but facilitated β-AR stimulation would also M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS 15 increase heart rate and myocardial oxygen demand [10]. Case-control studies demonstrated an association between β 2 -agonist use and an increased risk for myocardial infarction, CHF, cardiac arrest, and acute cardiac death. Intriguingly, the absence of a concurrent possible protective effect of βblockers has been suggested in this situation [95] but, unfortunately, the efficacy of concomitant β-blockers to offset the adverse cardiovascular effects of β 2 -agonists in COPD patients with coexistent CHF has not yet been assessed in clinical trials. If the above considerations lead to fear of the unconditional use of β-blockers in COPD, one should also consider a pharmacologic phenomenon that is opposite of what we have just described. In effect, exposure to β-agonists may alter β-ARs such that the affinity for ligands is reduced 10-fold [96]. Consequently, prior exposure to β-agonists may reduce binding of antagonists to β 2 -ARs. This relationship may explain the suggested high tolerance for βblockers in patients with COPD, who routinely inhale β 2 -agonists [83-86]. The lack of certainties about how to treat a patient with concurrent COPD and CHF leaves us still doubt about what to do, even for the obvious ethical, but also legal (malpractice), implications of our choices. Unfortunately, there are currently no prospective long-term studies on the safety of β-blockers in COPD and, moreover, the beneficial effect of β-AR blockade in patients with coexistent CHF and COPD is difficult to ascertain because such patients have been excluded from all large efficacy trials to date. Nonetheless, the literature is reassuring, although a retrospective analysis assessed the use of β-blockers in CHF patients with COPD showed that the long-term use of β-blockers did not increase the risk of respiratory complications and there was no difference in outcomes with the use of so called 'cardioselective' or nonselective β-blockers and the conclusion was that 'cardioselective' β-blockers without ISA are preferred until future studies resolve this issue [97]. In the recent publication of the Heart Failure Society of America on heart failure practice guidelines [98], β-blocker therapy is recommended in the great majority of patients with LV systolic dysfunction, even in the presence of concomitant COPD. They recommend that β-blockers are initiated at a low dose and uptitrated gradually M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS 16in two weeks intervals. The guidelines also recommend that patients with difficulties in initiating, uptitrating, or maintaining β-blocker therapy to be referred to a physician with expertise in the management of these patients.

β 1 -

 1 blockers that modulate the endogenous production of nitric oxide (nebivolol) are another interesting option. Nebivolol seems to be more useful than other β-blockers in African American patients and those suspected of having decreased nitric oxide bioavailability as an underlying pathophysiology of disease [100], and, moreover, in seniors with heart failure [101]. Nonetheless, more research is needed to better assess the impact of these drugs on patients with COPD [102]. Elderly is a particular issue in clinical practice since the prevalence of both CHF and COPD increases with age [103, 104]. Human aging is associated with an increase in the activity of the sympathetic nervous system [105, 106] and a diminished β-AR response to catecholamine [107, 108]. As a consequence, an alteration of bronchodilator response to β-agonists in elderly people has been suggested [109], and also treatment with β-blockers can be more challenging due to desensitization of β-ARs and variable pharmacokinetic responses that occur with age (potentially decreased absorption, metabolism and excretion) [110]. It is clear that it is imperative to individualize therapy for each patient [111] but the implementation of a safe and effective treatment may be more difficult in elderly. Senescence, concurrent diseases, malnutrition, polipharmacy may influence presentation and clinical course of CHF and COPD M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS 17 and interfere with their management. For this reason, a careful monitoring of treatment is recommended when dealing with this population.

[

  115]. Thus far, a consensus on the effects of β 2 -AR genetic variations has not been reached, although there does appear to be a reproducible adverse effect in subjects homozygous for Arg16 that are regularly treated with SABAs [115]. The complexity of the genotype by response effects observed makes clinical application of β 2 -AR genetic variations limited, and may require the use of detailed haplotypic variation to fully understand the role that β 2 -AR plays in regulating β-agonist response [115]. Studies have examined whether the Ser 49 Gly or Arg 389 Gly β 1 -AR polymorphisms influence responses to β-blockade during CHF treatment. Apparently, a better response to these drugs is observed in individuals with the Gly 49 variant [116]. On the other hand, in vitro and ex vivo results from the heart that showed that the Arg 16 Gly and Gln 27 Glu β 2 -AR polymorphisms exert little influence on the functional activity of β 2 -AR. When investigating the association between the treatment efficacy of β-blockers in CHF patients and β 2 -AR polymorphisms, most studies failed to detect a positive association [117]. Further studies must confirm or deny that β-AR polymorphisms might affect drug responsiveness because this might lead to a more individualized drug therapy. MR, Ginsburg R, Umans V, Fowler M, Minobe W, Rasmussen R, et al. β 1 -and β 2 -adrenergic-receptor subpopulations in nonfailing and failing human ventricular myocardium: coupling of both receptor subtypes to muscle contraction and selective β 1 -receptor down-regulation in heart failure. Circ Res 1986;59:297-309 21. Brodde OE. β 1 -and β 2 -adrenoceptors in the human heart: properties, function and alterations in chronic heart failure. Pharmacol Rev 1991;43:203-42 22. Rodefeld MD, Beau SL, Schuessler RB, Boineau JP, Saffitz JE. β-adrenergic and muscarinic cholinergic receptor densities in the human sinoatrial node: identification of a high β 2 -adrenergic receptor density. J Cardiovasc Electrophysiol 1996;7:1039-49 23. Newton GE, Parker JD. Acute effects of β 1 -selective and nonselective βadrenergic receptor blockade on cardiac sympathetic activity in congestive heart failure. Circulation 1996;94:353-58 24. Brodde OE, Michel MC. Adrenergic and muscarinic receptors in the human heart. Pharmacol Rev 1999;51:651-90 25. Xiao RP, Zhu W, Zheng M, Chakir K, Bond R, Lakatta EG et al. Subtypespecific β-adrenoceptor signaling pathways in the heart and their potential clinical implications. Trends Pharmacol Sci 2004;25:358-65 26. Appleton S, Poole P, Smith B, Veale A, Lasserson TJ, Chan MM. Longacting β 2 -agonists for poorly reversible chronic obstructive pulmonary disease. Cochrane Database Syst Rev 2006;3:CD001104 27. Stockley RA, Whitehead PJ, Williams MK. Improved outcomes in patients with chronic obstructive pulmonary disease treated with salmeterol compared with placebo/usual therapy: results of a meta-analysis. Respir Res 2006;7:147-56 28. Donohue JF, Menjoge S, Kesten S. Tolerance to bronchodilating effects of salmeterol in COPD. Respir Med 2003;97:1014-20 29. Tsagaraki V, Amfilochiou A, Markantonis SL. Evidence of tachyphylaxis associated with salmeterol treatment of chronic obstructive pulmonary disease patients. Int J Clin Pract 2006;60:415-21 M A N U S C R I P T A C C E P T E D

•

  Acutely depress myocardial function (pharmacological)• Chronically improve myocardial function (biological)

o

  Significant heart block without pacemaker o Severe bronchospastic pulmonary disease • Not contraindications:

  β2-AR No reported interactions with β-blockers Gln 27 Glu (Gln to Glu switch at codon 27) Glu 27 β2-AR is resistant to receptor downregulation Thr 164 Ile (Thr to Ile switch at codon 164) Ile 164 β2-AR presents loss of function, decreased agonist binding, decreased cAMP Arg = arginine, Gln = glutamine; Glu = glutamic acid; Gly = glicine, Ile = isoleucine, Ser = serine, Thr = threonine

  (table2). Metoprolol and bisoprolol are 75-fold and 120-fold more selective for β 1 -ARs compared with β 2 -ARs in humans, respectively[START_REF] Bristow | Changes in myocardial and vascular receptors in heart failure[END_REF], and have modest inverse activity (i.e., they decrease the spontaneous activity of the receptor in the absence of agonist)[START_REF] Engelhardt | Constitutive activity of the human β 1 -adrenergic receptor in β 1 -receptor transgenic mice[END_REF], whereas carvedilol is nonselective, shows no

				ARTICLE IN PRESS
				11
	M failure. inverse activity [60], dissociates slowly from the receptor [61], and is a radical scavenger and α 1 -antagonist. All three compounds led to similar clinical results, but carvedilol was superior to metoprolol in the COMET trial [62]. Methodological criticism aside, this trial does not answer the questions of possibly protective β 2 -ARs and whether the additional properties of carvedilol are important. A role for intrinsic A N U S C R I P T sympathomimetic activity (ISA), an ancillary pharmacologic property of some
	Blocking β-ARs when cardiac function depends on sympathoadrenergic drive long appeared counterintuitive. Today, studies with bisoprolol, metoprolol, and carvedilol (Cardiac Insufficiency Bisoprolol Study (CIBIS II) [56], Metoprolol A C C E P T E D
	CR/XL Randomised Interventional Trial in Congestive Heart Failure (MERIT-HF)
	[57]	and	Carvedilol	Prospective	Randomised	Cumulative	Survival

(COPERNICUS)

[START_REF] Krum | Effects of initiating carvedilol in patients with severe chronic heart failure: results from the COPERNICUS Study[END_REF]

, respectively,) show a similar reduction in the risk of death by a third or more in patients with CHF who were already receiving ACE inhibitors and diuretics, a benefit greater than of any other drug used in heart β-blockers, is supported by the observations that the strong partial agonist xamoterol was detrimental in heart failure

[63]

, and the weak partial agonist bucindolol

[64]

was ineffective in the BEST trial

[65]

. The beneficial β-blockers are neutral (carvedilol) or inverse agonistic (bisoprolol, metoprolol)

[START_REF] Engelhardt | Constitutive activity of the human β 1 -adrenergic receptor in β 1 -receptor transgenic mice[END_REF]

.

However, because of the low constitutive activity of the β 1 -AR, it is uncertain how important inverse agonism is. Taken together, the question is still open which pharmacological properties of β-blockers make them effective in CHF (table

3

).

  in the impact on airway function, depending on whether a β-blocker is β 1 -AR selective or β 1 -AR nonselective[67][68][69][70][71][72]. β 1blockade does not attenuate β 2 -agonist induced bronchodilation. However, also β 1 -blockers can adversely affect airways. In fact, all β-blockers that apparently are more selective for the β 1 -AR block β 2 -ARs when their concentrations are high enough[73]. Moreover, the so-called β 1 -blockers are not completely β 1selective, which could mean that they might have some affinity for β 2 -ARs on airway smooth muscle[74]. This mean that with large doses of all β-blockers β 1 -AR selectivity is lost and all β-ARs are at risk for being blocked. Therefore, the classification into 'cardioselective' β 1 -blockers and nonselective β-blockers is important, but oversimplified.These observations indicate that pindolol is potentially dangerous at least in asthmatic patients. There is also evidence that treatment with β-blockers that have ISA is associated with down-regulation of β 2 -AR [78]. This finding is consistent with data showing that β-antagonists with ISA did not produce the increase in β 2 -agonist response that was seen with β-antagonists without ISA In any case, the cumulative evidence from trials and meta-analyses indicates that 'cardioselective' β 1 -blockers should not be routinely withheld from patients with COPD because the benefits of β 1 -blockers in patients with COPD who also have cardiac disease far outweigh the risks [82]. Systematic Cochrane Database Reviews and further meta-analyses by Salpeter et al. of RCTs of patients with reversible airway disease and COPD exposed to single dose or continued treatment with what they called 'cardioselective' β-antagonists have been published [83-86]. Although they found that acute (single dose) βblockade resulted in a small reduction in FEV 1 , this was mitigated by a modest increase in FEV 1 with β 2 -agonists. Moreover, chronic treatment (3-28 days) produced no change in FEV 1 , symptoms, or inhaler use, whilst inducing a modest response to β 2 -agonists. In addition, no consistent exacerbation of airways disease was recorded in the 80 trials identified. Recently, van Gestel et

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	M A N U S C R I P T identified relevant differences M A N U S C R I P T
	A C C E P T E D A C C E P T E D

-blockade is achieved suggests this is a compensatory mechanism, indirectly caused by the β-AR blockade and not due to the β-blockade per se

[66]

.

COPD

β-blockers are known to worsen FEV 1 and AHR in asthma, and therefore reluctance exists to prescribe these agents to COPD patients as well, despite the anticipated beneficial cardiovascular effects (table

4

). Most of the adverse pulmonary effects exerted by β-blockers in COPD patients are related to interference with β 2 -AR-mediated bronchodilation. Various studies have

The therapeutic importance of ISA in COPD patients is questionable.

Apparently, ISA seems to be at least as important as β 1 -selectivity in reducing the increase in airway resistance that results from β-blockade both at rest and during exertion

[75]

. Moreover, it reduces the bronchoconstrictor response to inhaled histamine during β-blockade in asthma patients

[76]

. Decalmer et al.

[77] examined the effects of approximately equipotent single oral doses of β 1antagonists (atenolol, 100 mg; metoprolol, 100 and 300 mg) and four nonselective β-(β 1 + β 2 ) blockers (propranolol, 100 mg; oxprenolol, 100 mg; timolol, 10 mg; and pindolol, 5 mg) on FEV 1 in 10 asthmatic patients. Pindolol is the only agent with ISA. All drugs caused a fall in FEV 1 , but only atenolol did not differ significantly from placebo in this respect. Moreover, the four nonselective β-blockers inhibited the bronchodilator response to inhaled isoprenaline, whereas the selective β 1 -blockers allowed some bronchodilation.

[79-81]. Since agents with ISA offer less cardioprotection than do βantagonists without this ancillary property, the use of these drugs should be severely restricted.

al.

[START_REF] Van Gestel | Impact of cardioselective β-blockers on mortality in patients with chronic obstructive pulmonary disease and atherosclerosis[END_REF]

reinforced this view by providing solid proof that, in carefully selected patients with COPD undergoing vascular surgery, β 1 -blockers are safe and beneficial in prolonging survival.

Table 1 -Summary of some pharmacological properties of selected β 2 - agonists

 1

	β2-agonist	Affinity for	Efficacy	Potency	Selectivity	Intrinsic	Approximate	Approximate
		β2-AR (Ki,	at β2-AR	at β2-	ratio (β1-	efficacy	onset of	duration of
		nmol/L)	(a)	AR (b)	AR/β2-AR)	(%)	action (min)	action
	Isoprenaline	200	(100)	(1)	1/1	100	2-5	<20 min
	Salbutamol	2500	88	1/120	1/1375	4.9	2-3	4-6 h
	Fenoterol	ND	100	ND	1:120	42	2-4	4-6 h
	Terbutaline	ND	65-85	ND	ND	ND	2-4	4-6 h
	Salmeterol	53	63	8.5	1/85000	<2	30	>12 h
	Formoterol	76	100	20	1/120	20	2-3	>12 h
	a Relative to isoprenaline as 100%.
	b Relative to isoprenaline.
	ND = no data available.

Table 5 -β-blockers for CHF

 5

	Drug	β-AR selectivity	Starting Dose	Target Dosage
	Bisoprolol	It is 120-fold more	2.5 mg/day	10mg/day
		selective for the β 1 -AR
		compared with β 2 -AR
		in humans
	Carvedilol	In addition to blocking	6.25 mg/day	50 mg/day (100
		β 1 -and β 2 -ARs, it also		mg/day if >85 kg)
		blocks α 1 -ARs and has
		antioxidant and
		antiendothelin
		properties
	Metoprolol	It is 75-fold more	12.5-25mg/day	200mg/day
		selective for the β 1 -AR
		compared with β 2 -AR
		in humans

Table 6 -Effect of gene polymorphisms on the pharmacological treatment of COPD and CHF β1-AR Polymorphism Arg

 6 389 Gly (Arg to Gly switch at codon 389) Arg 389 β1-AR is three to four times more responsive to agonist-evoked stimulation than Gly 389 β1-AR Responses to β1-AR blockers are larger in subjects homozygous for Arg 389 Ser 49 Gly (Ser to Gly switch at codon 49) Ser 49 β1-AR has decreased agonistpromoted downregulation and decreased adrenergic coupling Gly 49 β1-AR variant is linked to greater benefit from β1-AR blockade β2-AR Polymorphism Arg 16 Gly (Arg to Gly switch at codon 16) Gly 16 β2-AR has greater agonist-promoted downregulation Larger vasodilation in subjects homozygous for the Arg 16