1 Departament de Farmacologia, Facultat de Medicina i Odontologia, Universitat de València, 46010 València, Spain; 2 Faculté de Médecine Paris-Ouest, Unité de Formation et de Recherche Biomédicale des Saint Pères, and Centre Hospitalier de Versailles, 78157 Le Chesnay, France; and 3 Shanghai Second Medical University, 200025 Shanghai, China
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ABSTRACT |
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The
muscarinic functional antagonism of isoproterenol relaxation and the
contribution of muscarinic M2 receptors were examined in
human isolated bronchus. In intact tissues, acetylcholine (ACh) precontraction decreased isoproterenol potency and maximal relaxation (log EC50 shift =
1.49 ± 0.16 and
Emax inhibition for 100 µM ACh = 30%) more than the
same levels of histamine contraction. The M2
receptor-selective antagonist methoctramine (1 µM) reduced this
antagonism in ACh- but not histamine-contracted tissues. Similar
results were obtained for forskolin-induced relaxation. After selective
inactivation of M3 receptors with
4-diphenylacetoxy-N-(2-chloroethyl)piperadine hydrochloric
acid (30 nM), demonstrated by abolition of contractile and inositol
phosphate responses to ACh, muscarinic recontractile responses were
obtained in U-46619-precontracted tissues fully relaxed with
isoproterenol. Methoctramine antagonized recontraction, with
pKB (6.9) higher than in intact tissues (5.4), suggesting participation of M2 receptors. In
M3-inactivated tissues, methoctramine augmented the
isoproterenol relaxant potency in U-46619-contracted bronchus and
reversed the ACh-induced inhibition of isoproterenol cAMP accumulation.
These results indicate that M2 receptors cause indirect
contraction of human bronchus by reversing sympathetically mediated
relaxation and contribute to cholinergic functional antagonism.
airway smooth muscle; muscarinic receptors; methoctramine; 4-diphenylacetoxy-N-(2-chloroethyl)piperadine hydrochloric acid
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INTRODUCTION |
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HUMAN AIRWAYS ARE
DENSELY innervated with parasympathetic nerves, which provide the
dominant neural constrictor pathway. Previous studies using in situ
hybridization and Northern blot analysis have demonstrated the presence
of muscarinic M2 and M3 receptors in human
airway smooth muscle cells but no detectable mRNA for M1,
M4, or M5 receptors (10). The
postjunctional muscarinic receptor subtype mediating contraction of
human airways is the M3 receptor (16, 25).
Functional studies have provided evidence of the existence of
prejunctional M2 receptors in human airways that modulate
acetylcholine (ACh) release from postganglionic prejunctional
cholinergic nerve endings (12, 20). By contrast, the
functional role of postjunctional M2 receptors localized to the human airway smooth muscle is unclear. Functional experiments do
not support their direct contribution to the contractile response elicited by muscarinic stimulation in human isolated airways
(25). However, a number of studies have implicated these
M2 receptors in the functional antagonism between airway
smooth muscle contraction by muscarinic agonists and the relaxation
elicited by -adrenoceptor agonists in canine (7, 13)
and guinea pig (24, 30) trachea, but not in bovine trachea
(17).
The involvement of M2 receptors in the impairment of the
isoproterenol-mediated relaxation of cholinergic tone may be explained by the finding that the activation of these receptors inhibits -agonist- or forskolin-stimulated cAMP production in airway smooth muscle of different animal species (18, 28). These
observations are consistent with findings in other tissues. Thus,
although no direct contractile response to M2 receptor
activation can be demonstrated in rat intestinal muscle, guinea pig
ileum, rat esophageal muscularis mucosae, or pig urinary bladder, an
indirect influence on contraction via inhibition of cAMP-mediated
smooth muscle relaxation by
-adrenoceptor agonists, forskolin,
5-hydroxytryptamine, or vasoactive intestinal peptide has been reported
(5, 8, 15, 29).
The functional antagonism by muscarinic agonists of the
-adrenoceptor-mediated relaxation has been scarcely studied in human isolated bronchus (23, 26). Therefore, the aim of the
present study was to examine further the functional antagonism between the relaxation produced by the cAMP pathway and the contraction produced by ACh and histamine in human isolated bronchus and the contribution of M2 receptors to this functional antagonism.
Relaxation was obtained with isoproterenol, a
-adrenoceptor agonist,
and with a direct activator of adenylyl cyclase, forskolin. ACh and histamine were chosen as spasmogens, because both are relevant mediators in allergic asthma. Methoctramine was chosen as a relatively selective antagonist of M2 receptors (11). To
explore better the contribution of M2 receptors,
experiments were also performed on tissues after selective inactivation
of muscarinic M3 receptors with the alkylating agent
4-diphenylacetoxy-N-(2-chloroethyl)piperadine hydrochloric
acid (4-DAMP mustard) (22).
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METHODS |
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Preparation of human tissues for pharmacomechanical experiments. Lung tissue was obtained from patients who were undergoing surgery for lung carcinoma. None of the patients had a history of asthma. The experiments were approved by the local ethics committee. A piece of macroscopically normal tissue at a distance from the malignancy was obtained, and rings (3-4 mm long, 2-4 mm ID) were prepared as previously described (19). Preparations were stored in physiological salt solution (PSS) composed of (mM) 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.1 KH2PO4, 25.0 NaHCO3, and 11.7 glucose and equilibrated with 5% CO2 in O2 at 4°C. Experiments were completed within 24 h of initiation of storage (19).
Bronchial rings were suspended for isometric recording of tension changes under an initial load of 2.0 g and equilibrated for 60-90 min with changes in bath PSS every 15-20 min before any pharmacological intervention; at the end of the equilibration period, the resting load was stable at 1-2 g. Under these conditions, responses were optimal and reproducible according to data from this and other laboratories (19, 27).Experimental protocol in intact tissues. Human bronchi were first contracted with 1 mM ACh and then relaxed with 3 mM theophylline to obtain reference values for maximal contraction and relaxation of the preparation (19). After the samples were washed and re-equilibrated, relaxation-response curves were obtained at basal tone or at three levels of plateau contraction, which corresponded to ~20, 50, and 70% of the maximal contraction to ACh. Preliminary experiments showed that the concentrations required to obtain such plateau levels of contractions were 1, 10, and 100 µM for ACh and 0.3, 1, and 10 µM for histamine, respectively.
Cumulative concentration-response curves to isoproterenol (1 nM to 1-3 µM) or forskolin (0.01-1 µM) were obtained in the absence or presence of methoctramine (0.3 or 1 µM) added 30 min before tone was built up with ACh or histamine. This range of methoctramine concentrations was chosen on the basis of to previous data (26, 28). Because interindividual variation in drug sensitivity was large in human isolated bronchus (4), paired bronchial rings obtained from the same patients were used for experiments comparing shifts ofExperimental protocol in M3 receptor-inactivated tissues. Functional and biochemical experiments were first performed to determine whether 4-DAMP mustard induces a maximal inhibition of the responses to ACh. For functional studies, bronchi were contracted after equilibration with 1 mM ACh to obtain reference values for maximal contraction. After the samples were washed and re-equilibrated, 0.1-30 nM 4-DAMP mustard or its vehicle was added to the bath for 60 min. The drug was then removed by washing with PSS at 10-min intervals over 30 min, and a cumulative concentration-response curve to ACh was constructed. Only one concentration-response curve to ACh was obtained in each ring. For biochemical studies, the inhibition by 4-DAMP mustard of inositol phosphate formation produced by ACh was determined (see below).
Once the concentration of 4-DAMP mustard that virtually abolished the ACh-induced contraction and inositol phosphate production in human isolated bronchus was established, the protocol to achieve selective M3 receptor inactivation was essentially as previously outlined (9, 29). After 1 mM ACh was added to obtain a reference value for maximal contraction and the tissues were washed, the tissues were incubated with the alkylating agent 4-DAMP mustard (30 nM) for 60 min in the presence of 1 µM methoctramine to protect M2 receptors, with methoctramine added 30 min before the 4-DAMP mustard. The drugs were then removed from the tissues by washing for 60 min with PSS. Then two different protocols were carried out: one to assess the presence of functional antagonism in M3 receptor-inactivated tissues and its reversal by methoctramine and another to detect the presence of recontraction (i.e., reversal of relaxation) and its antagonism by methoctramine under conditions of stimulated adenylyl cyclase activity (i.e., isoproterenol-induced relaxation of precontracted tissue) (9). In the first protocol, paired tissues were incubated with or without 1 µM methoctramine for 30 min, 100 µM ACh was added, and preparations were contracted with 0.1 µM U-46619 to a plateau level of ~70%; then a concentration-response curve to isoproterenol was constructed. In the second protocol, paired tissues were incubated with or without 1 µM methoctramine and contracted 30 min later with 0.1 µM U-46619; then the stable contractile tone was relaxed to baseline with 10 µM isoproterenol, and 10 min later a concentration-response curve to ACh was constructed.Inositol phosphate measurement. Total inositol phosphate accumulation was determined as previously reported (2). Briefly, human bronchi were incubated in PSS containing 50 µCi of myo-[3H]inositol for 4 h at 37°C in 95% O2-5% CO2. 4-DAMP mustard (1, 10, or 30 nM) or its vehicle (control tissues) was added for 60 min, and the preparation was stimulated for 15 min at 37°C with PSS (control) or 1 mM ACh. Inositol phosphates were separated by an HPLC ion-exchange system, and radioactivity was measured in a Flow-One on-line radioactivity detector (Packard, Meriden, MA). Inositol phosphate production was expressed as a percentage of the production induced by 1 mM ACh in paired control tissues.
Measurement of cAMP. The content of cAMP in human isolated bronchus was measured as previously outlined (3). Briefly, bronchial rings were subjected to the M3 receptor inactivation process by incubation with 30 nM 4-DAMP as indicated above; then the preparation was exposed for 30 min to 100 µM ACh or its vehicle, and 10 µM isoproterenol or its vehicle was added for 10 min. At the end of experiment, tissues were homogenized and centrifuged, and the amount of cAMP in the soluble fraction was estimated (RPN 225 enzyme immunoassay kit, Amersham Life Sciences) following the instructions of the manufacturer without acetylation.
Drugs and statistical analysis of results. Drugs were obtained from Sigma-Aldrich (St. Louis, MO), except 4-DAMP mustard and methoctramine tetrahydrochloride, which were purchased from Research Biochemical (Natick, MA). Solutions of 4-DAMP mustard were freshly prepared in dimethyl sulfoxide and subsequently diluted in distilled water. Stock solutions of U-46619 were prepared in dimethyl sulfoxide and those of forskolin in ethanol, and these solutions were subsequently diluted in PSS. Isoproterenol solutions contained 22 µM ascorbic acid as an antioxidant. Vehicle controls (drug solvent only) were run in parallel, and no significant vehicle effects were observed. Drug concentrations are expressed as final bath concentrations of the active species.
Contractile and relaxant responses are expressed as a percentage of the reference contractile (1 mM ACh) and relaxant (3 mM theophylline) responses. The EC50 of spasmogens and relaxants, i.e., that producing half-maximal response (Emax), was calculated by nonlinear regression analysis of log concentration-effect curves (GraphPad, San Diego, CA) and expressed as pD2 (i.e., ![]() |
RESULTS |
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Methoctramine did not influence the level of basal or induced tone in intact tissues. There was no significant effect of 0.3 or 1 µM methoctramine on the basal tone of the preparations (not shown). The level of plateau contraction produced by ACh before the concentration-relaxation curves were performed for isoproterenol or forskolin did not differ between control and methoctramine-treated tissues (not shown). The contractile responses to 0.3, 1, and 10 µM histamine were not significantly different from the corresponding values observed after 1, 10, and 100 µM ACh, and the contraction in response to 10 µM histamine was not reduced by 1 µM methoctramine (not shown). In the experiments where 0.1 µM U-46619 was used to raise the tissue tone, contractions were not affected by pretreatment with 1 µM methoctramine (not shown).
Incubation with 0.3 and 1 µM methoctramine did not significantly displace the concentration-response curve to ACh (not shown), but 10 µM methoctramine produced a small parallel rightward shift from control curves, with a pKB of 5.4 ± 0.3 (n = 4), which is consistent with values reported in this preparation with carbachol as agonist (25).Methoctramine reduced the functional antagonism of the relaxation
to isoproterenol and forskolin produced by ACh in intact tissues.
When the level of contraction was increased with ACh, a rightward and
downward shift of the concentration-response curves to isoproterenol
was observed. This indicates a functional antagonism between the
isoproterenol relaxation and the ACh contraction. The displacement of
the concentration-response curves of isoproterenol produced by ACh was
partially reversed in the presence of methoctramine (Fig.
1).
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Hexamethonium and tetrodotoxin did not influence the
ACh-isoproterenol functional antagonism or the reduction of this
antagonism by methoctramine in intact tissues.
Pretreatment with 10 µM hexamethonium or 1 µM tetrodotoxin, in the
absence or presence of 1 µM methoctramine, did not depress the
contraction in response to 10 µM ACh (not shown). Hexamethonium and
tetrodotoxin did not modify the functional antagonism of the isoproterenol relaxation exerted by ACh: shifts of isoproterenol pD2 values of 0.61 ± 0.09 and
0.46 ± 0.03 were obtained in the presence of hexamethonium and tetrodotoxin,
respectively, which did not differ from the corresponding control
values of
0.58 ± 0.07 and
0.48 ± 0.03 (n = 5 per group). The facilitatory effect of 1 µM
methoctramine on the isoproterenol relaxation of ACh-contracted tissues
was not altered by hexamethonium and tetrodotoxin (not shown).
Methoctramine failed to increase the reduced relaxation to
isoproterenol and forskolin in histamine-contracted intact tissues.
When the level of tone was increased with histamine, the
concentration-response curves to isoproterenol were shifted rightward, but the maximal relaxation to isoproterenol and its potency were scarcely affected compared with similar levels of contraction elicited
by ACh (cf. Figs. 1 and 3). Similar
results were obtained with forskolin (cf. Figs. 2 and 3). Methoctramine
(1 µM) did not increase the relaxations elicited by isoproterenol and
forskolin in preparations contracted with 10 µM histamine (Fig. 3).
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Contraction and inositol phosphate production in response to ACh
were virtually abolished in M3 receptor-inactivated
tissues.
4-DAMP mustard produced a concentration-dependent inhibition of the
contractions evoked by ACh, with a progressive reduction of maximal
responses without significant changes in potency (Fig. 4). 4-DAMP mustard (30 nM) produced a
virtually complete inhibition of the ACh contraction without affecting
the contraction elicited by U-46619: Emax and
pD2 values were 89 ± 6% and 7.40 ± 0.06, respectively, in the absence and 83 ± 8% and 7.37 ± 0.07, respectively, in the presence of 30 nM 4-DAMP mustard
(n = 3 for each group).
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Methoctramine reduced the antagonism produced by ACh in the
relaxation and cAMP elevation elicited by isoproterenol in
M3 receptor-inactivated tissues.
Because 100 µM ACh produced no contraction in M3
receptor-inactivated tissues (see above), tone was increased with 0.1 µM U-46619 in the presence of 100 µM ACh to a level of contraction (71 ± 6% of 1 mM ACh, n = 5) similar to that
produced by 100 µM ACh in intact tissues. In this situation, a
rightward displacement of the concentration-response curves to
isoproterenol was observed (Fig. 5).
These results indicate the persistence of a functional antagonism
produced by ACh against the relaxant effects of isoproterenol in
M3 receptor-inactivated tissues. This antagonism was
partially reversed by methoctramine (Fig. 5).
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ACh-induced recontraction in M3 receptor-inactivated tissues is antagonized by methoctramine. In tissues subjected to selective M3 receptor alkylation, precontracted with 0.1 µM U-46619 (71 ± 5%), and fully relaxed with 10 µM isoproterenol, ACh produced a concentration-dependent recontraction with Emax of 55 ± 3% and pD2 of 4.23 ± 0.23 (n = 4). These values were significantly lower than those obtained in paired intact tissues (Emax = 98 ± 2% and pD2 = 5.05 ± 0.08). The maximal recontraction was close to the level of contraction produced by U-46619, thus indicating near-complete reversal of relaxations to isoproterenol. The curves for recontraction were antagonized in a surmountable fashion by 1 µM methoctramine, with a pKB of 6.92 ± 0.41 (n = 4). This value is significantly higher than that found in intact tissues (see above), and it would be consistent with activation of M2 receptors.
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DISCUSSION |
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Increased functional antagonism of cholinergic tone against
isoproterenol and forskolin relaxation of human isolated bronchus.
This study was carried out in human isolated bronchus to assess the
functional antagonism between spasmogens, such as ACh and histamine,
and relaxants, such as isoproterenol (a -adrenoceptor agonist) and
forskolin (an adenylyl cyclase activator), and the role that
M2 receptors may play in this antagonism. We found that a
gradual increase of tone promoted by ACh from low to half- or near-maximal levels resulted in a pronounced, ACh
concentration-dependent decrease in the ability of isoproterenol and
forskolin to relax the airway smooth muscle. However, when tone was
increased with histamine to levels similar to those obtained with ACh,
the ability of isoproterenol or forskolin to relax the airway smooth
muscle was decreased to a lesser extent.
Methoctramine enhanced relaxation by isoproterenol and forskolin in ACh- but not histamine-contracted human bronchus. Whether M2 receptors contribute to the functional antagonism between ACh- and isoproterenol- and forskolin-induced relaxation of human isolated bronchus was addressed in the present study by using methoctramine, a relatively selective antagonist of M2 receptors (11) that is well characterized in human bronchus (25, 26). Because 0.3-1 µM methoctramine did not depress contraction to spasmogens, any effect of this antagonist cannot be attributed to differences in the contraction level before the relaxation curves were obtained (17).
Isoproterenol and forskolin relaxations of ACh-contracted human bronchus were facilitated in the presence of methoctramine, both in terms of pD2 values and maximal effects. This finding suggests that M2 receptors play a role in the above-mentioned functional antagonism that is in keeping with results obtained in canine, rabbit, and guinea pig trachea with the use of methoctramine and other selective M2 receptor antagonists (7, 13, 24, 30). Consistent with these results is our finding that the relaxation by isoproterenol or forskolin of histamine-induced tone, which does not involve M2 receptors, was not facilitated in the presence of methoctramine.Hexamethonium and tetrodotoxin did not influence the effect of methoctramine on functional antagonism between ACh and isoproterenol. Human bronchus contains intramural parasympathetic ganglia, and exogenously administered ACh could activate nicotinic receptors located on ganglionic neurons. On the other hand, micromolar concentrations of methoctramine show a significant antagonist activity at nicotinic receptors (11). We found that 10 µM hexamethonium did not depress the contraction produced by 10 µM ACh in human isolated bronchus, nor did it influence the facilitation produced by 1 µM methoctramine of the isoproterenol relaxation in preparations contracted with ACh. Similar results were obtained by inhibiting nerve conduction with the sodium channel blocker tetrodotoxin (1 µM). These results indicate that, consistent with other studies (1), neural mechanisms do not contribute to the contraction to exogenously added ACh and that interaction with nicotinic receptors is not likely involved in the action of methoctramine on the functional antagonism between ACh and isoproterenol in human isolated bronchus.
Methoctramine augmented isoproterenol-induced relaxation and antagonized cholinergic recontraction in M3 receptor-inactivated tissues. Because the augmentation by an M2 receptor antagonist of the relaxant potencies of isoproterenol and forskolin in ACh-contracted tissues provides somewhat ambiguous evidence for an involvement of the M2 receptor in this functional antagonism (5), we performed additional studies in M3 receptor-inactivated tissues. Selective alkylation of M3 receptors after incubation of tissues with 4-DAMP mustard, in the presence of methoctramine to protect M2 receptors, has successfully unmasked a functional role for M2 receptors in a number of previous studies (5, 6, 9, 14, 15, 21, 29).
Incubation of human isolated bronchus with the alkylating agent 4-DAMP mustard inhibited the ACh-induced contraction and inositol phosphate production in a concentration-related manner, with responses virtually abolished for 30 nM 4-DAMP. Therefore, we demonstrate that alkylation conditions can be achieved in human bronchus that selectively and extensively deplete the muscarinic M3 receptor population, thus facilitating exploration of a functional role for M2 receptors. After M3 receptor inactivation, the relaxant potency of isoproterenol in U-46619-contracted human bronchus was reduced in the presence of 100 µM ACh, indicating the persistence of a cholinergic functional antagonism in these experimental conditions. ACh was also able to reduce the increase of cAMP elicited by isoproterenol in M3 receptor-inactivated tissues. The isoproterenol relaxation and the cAMP accumulation were augmented in the presence of methoctramine, suggesting the participation of M2 receptors in this functional antagonism. These results confirm and extend previous findings in cultured human airway smooth muscle cells where carbachol was shown to inhibit the isoproterenol-induced augmentation of cAMP, and methoctramine reversed this effect by blockade of M2 receptors (28). Furthermore, in U-46619-contracted tissues that were exposed to isoproterenol to provide full relaxation via elevation of cAMP, ACh induced concentration-dependent recontractions (i.e., indirect contraction or reversal of relaxation). The maximal recontraction to ACh, while reversing the contracture to U-46619, remained less than that mediated by direct M3 receptor activation. However, the magnitude of the response was markedly larger than that in the alkylated tissues. This provides some evidence that M2, rather than M3, receptors mediate the response. Further evidence to implicate M2 receptors was obtained from studies with methoctramine, because this drug antagonized the recontractions, with pKB values (6.9) that are higher than those obtained in intact tissues (5.4) and more consistent with activation of muscarinic M2 receptors than with activation of M3 receptors. These findings would be consistent with results obtained in guinea pig ileum (15, 21) and in rat (9) and pig (29) urinary bladder, in which an M2 receptor-mediated contraction to muscarinic stimulation has been demonstrated after M3 receptor inactivation and elevation of cAMP levels. In conclusion, these results indicate that, in addition to mechanisms derived from the activation of M3 receptors, M2 receptors appear to be involved in the functional antagonism of relaxant responses to isoproterenol produced by cholinergic stimulation. This effect may be related to the inhibition of adenylyl cyclase, because methoctramine was able also to reduce the functional antagonism elicited by ACh against forskolin-induced relaxation and reversed the muscarinic inhibition of isoproterenol cAMP accumulation in M3 receptor-inactivated tissues. These findings may have relevance in the pathophysiology and pharmacology of the human airway smooth muscle. Thus activation of postjunctional M2 receptors may gain influence under conditions of high sympathoinhibitory tone acting in concert with M3 receptors to reinforce bronchoconstriction. The possibility that M2 receptor activation inhibits cAMP accumulation promoted by some inhibitory neurotransmitters widens the potential modulation of this receptor over bronchial tone. Also, nonselective muscarinic antagonists, by blocking the postjunctional M2 receptor-mediated inhibition of relaxation, might be more efficient for treating bronchospasm than M3 receptor-selective antagonists, yet the relative clinical importance of this mechanism over the blockade of autoinhibitory M2 receptors remains to be determined. ![]() |
ACKNOWLEDGEMENTS |
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The authors are indebted to the teams of the Services of Thoracic Surgery and Pathology of the Hospital La Fe and Hospital Clínico of the University of Valencia for making the human lung tissue available to us. The authors also thank P. Santamaría and S. Martí for expert technical assistance.
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FOOTNOTES |
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The present work was supported in part by Comisíon de Investigacíon Cientifíca y Técnica Grants SAF1999-0111 and SAF2000-0144 (Spain) and funds from Generalitat Valenciana.
Address for reprint requests and other correspondence: E. J. Morcillo, Dept. de Farmacología, Facultad de Medicina y Odontología, Av Blasco Ibáñez 15, 46010 Valencia, Spain (E-mail: Esteban.Morcillo{at}uv.es).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
June 21, 2002;10.1152/ajplung.00084.2002
Received 22 March 2002; accepted in final form 12 June 2002.
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