Facilitatory beta 2-adrenoceptors on cholinergic and adrenergic nerve endings of the guinea pig trachea

Jan Roelof A. de Haas, J. Saskia Terpstra, Monica van der Zwaag, Pieter G. E. Kockelbergh, Ad F. Roffel, and Johan Zaagsma

Department of Molecular Pharmacology, University of Groningen, 9713 AV Groningen, The Netherlands


    ABSTRACT
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Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

Using electrical field stimulation of epithelium-denuded intact guinea pig tracheal tube preparations, we studied the presence and role of prejunctional beta 2-adrenoceptors by measuring evoked endogenous acetylcholine (ACh) and norepinephrine (NE) release directly. Analysis of ACh and NE was through two HPLC systems with electrochemical detection. Electrical field stimulation (150 mA, 0.8 ms, 16 Hz, 5 min, biphasic pulses) released 29.1 ± 2.5 pmol ACh/g tissue and 70.2 ± 6.2 pmol NE/g tissue. Preincubation for 15 min with the selective beta 2-adrenoceptor agonist fenoterol (1 µM) increased both ACh and NE overflow to 178 ± 28 (P < 0.01) and 165 ± 12% (P < 0.01), respectively, of control values, increases that were abolished completely by the selective beta 2-adrenoceptor antagonist ICI-118551 (1 µM). Further experiments with increasing fenoterol concentrations (0.1-100 µM) and different preincubation periods (1, 5, and 15 min) showed a strong and concentration-dependent facilitation of NE release, with maximum response levels decreasing (from nearly 5-fold to only 2.5-fold of control value) with increasing agonist contact time. In contrast, sensitivity of facilitatory beta 2-adrenoceptors on cholinergic nerves to fenoterol gradually increased when the incubation period was prolonged; in addition, a bell-shaped concentration-response relationship was found at 15 min of preincubation. Fenoterol concentration-response relationships (15-min agonist preincubation) in the presence of atropine and yohimbine (1 µM each) were similar in the case of NE release, but in the case of ACh release, the bell shape was lost. The results indicate a differential capacity and response time profile of facilitatory prejunctional beta 2-adrenoceptors on adrenergic and cholinergic nerve terminals in the guinea pig trachea and suggest that the receptors on adrenergic nerves are more susceptible to desensitization.

facilitatory prejunctional beta 2-adrenoceptors; acetylcholine release; norepinephrine release


    INTRODUCTION
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Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

THE AIRWAYS OF MANY SPECIES are innervated by both parasympathetic and sympathetic neural pathways. The physiological effect of the parasympathetic nerves, which use acetylcholine (ACh) as a neurotransmitter, is excitatory, causing airway constriction. On the other hand, the sympathetic innervation for which norepinephrine (NE) is the neurotransmitter is inhibitory, causing airway dilatation (21). As in other tissues, these airway neural systems may be subject to prejunctional control of neurotransmitter release.

Prejunctional beta -adrenoceptors were described for the first time by Adler-Graschinsky and Langer (1) and by Stjärne (17), who found that isoproterenol caused an increase of tritiated NE overflow evoked by nerve stimulation of postganglionic adrenergic nerves in guinea pig isolated atria and vas deferens, respectively. Although facilitatory prejunctional beta -adrenoceptors have been described in many central and peripheral tissues since, both in vitro and in vivo (6, 13, 14), conclusive information about such receptors in airways has been somewhat controversial.

Originally, it was concluded that prejunctional beta 2-adrenoceptors exerted an inhibitory influence on cholinergic neurotransmission in human airways. This viewpoint emerged from indirect studies (2, 16) in which the effects of beta -adrenoceptor agonists on the contractile responses of bronchial smooth muscle to electrical field stimulation (EFS) and exogenous ACh were compared to discriminate between pre- and postjunctional adrenoceptor effects. This technique, although useful, is essentially indirect because it does not measure the actual release of the neurotransmitter. With the use of the same technique, it has been recently demonstrated in our laboratory that, at least in guinea pig main bronchi, inhibitory beta 2- or beta 3-adrenoceptors are not functional on cholinergic nerve endings (18).

A more direct approach to investigate the existence and function of prejunctional receptors is to measure the actual overflow of neurotransmitters. With such a technique, inhibition of [3H]ACh release mediated by beta -adrenoceptors has been reported for isolated rat and guinea pig tracheae, but this response required an intact epithelium (20). On the other hand, direct, i.e., epithelium-independent, beta 2-adrenoceptor-mediated facilitation of endogenous ACh release was observed in the equine trachea (22, 23) and, more recently, in the guinea pig trachea (4). beta -Adrenoceptor-mediated facilitation of NE release in the airways has hitherto only been described in one report; i.e., in rat isolated tracheae, an epithelium-dependent facilitation was observed (5).

In the present study, for the first time, the existence and function of prejunctional beta 2-adrenoceptors on both parasympathetic and sympathetic nerve endings in epithelium-denuded guinea pig tracheae have been explored simultaneously by measuring the EFS-evoked release of endogenous (i.e., not prelabeled) ACh and NE with two sensitive HPLC systems equipped with electrochemical detection (ECD) that have been developed over the past years (6, 14, 19). In these experiments, the nature and magnitude of the response to a single concentration of the beta 2-adrenoceptor agonist fenoterol were first established; subsequently, the concentration-response relationships for fenoterol were characterized in the absence and presence of auto- and heteroreceptor antagonists (i.e., atropine and yohimbine) and at various agonist contact times.


    METHODS
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Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

Tissue preparation. Adult guinea pigs of either sex (Charles River, Kisslegg, Germany) weighing 600-800 g were killed by a sharp blow on the head, after which the tracheae were rapidly removed. The tracheae were placed in Krebs-Henseleit (KH) buffer solution (37°C) of the following composition (in mM): 117.50 NaCl, 5.60 KCl, 1.18 MgSO4, 2.52 CaCl2, 1.28 NaH2PO4, 25.00 NaHCO3, and 5.55 D-glucose, pH 7.4, aerated with 5% CO2-95% O2. The tracheae were prepared free of connective tissue, and the airway epithelium was carefully removed by moving a 15-cm woolen thread up and down the trachea twice. Subsequently, the tracheae were cut into four segments. To ensure comparable levels of neurotransmitter release, the proximal and distal parts provided one set of preparations and the two middle parts provided one set. The two tracheal segments were positioned over two platinum rod electrodes placed 0.7 cm apart in a water-jacketed tissue bath (37°C) in such a way that the smooth muscle layers were located between the electrodes as described by Ten Berge et al. (19).

Experimental protocol. All release experiments were performed in the presence of 10 µM L-tyrosine. No reuptake inhibitors, acetylcholinesterase inhibitors, or auto- and heteroreceptor antagonists were used unless stated otherwise. The tracheal preparations were allowed to equilibrate for four 15-min periods in 10.0 ml of fresh KH buffer, after which the tissues were placed in 4.0 ml of fresh KH buffer. After 15 min, three 500-µl samples were taken to determine the spontaneous release of ACh and NE. In the remaining 2.5 ml of KH buffer, EFS was applied (16 Hz, 150 mA, 0.8 ms, biphasic square-wave pulses) for 5 min [first episode of stimulation (S1)]. After EFS, three 500-µl samples were taken. Subsequently, the preparations were allowed to recover for 30 min in fresh KH buffer. All samples were stored immediately at -20°C until analysis.

The first set of experiments started with three control episodes of stimulation (S1-S3). After the 30-min recovery from the previous stimulation episode, the selective beta 2-adrenoceptor agonist fenoterol (1 µM) was added and incubated for 15 min before the fourth episode of stimulation (S4). The fifth episode of stimulation (S5) was a control stimulation. Before the sixth episode of stimulation (S6), the selective beta 2-adrenoceptor antagonist ICI-118551 (1 µM) was added and incubated for 30 min, and fenoterol (1 µM) was administered 15 min later. The seventh episode of stimulation (S7) was a control stimulation. In each experiment, control stimulations were run in parallel in tracheal preparations that did not receive any beta 2-adrenoceptor agent.

In the second set of experiments, the concentration and incubation time dependence of the prejunctional effects of fenoterol were explored. As before, S1-S3 were performed in the absence of drugs. S4-S7 were performed in the presence of increasing concentrations of fenoterol (0.1-100 µM), with incubation times being 1, 5, or 15 min. Parallel control experiments in the absence of fenoterol were performed regularly.

The effects of 0.1-100 µM fenoterol (15-min agonist preincubation) were also studied under conditions where auto- and heteroregulation of neurotransmitter release, through prejunctional muscarinic and alpha 2-adrenoceptors, was eliminated by coincubation with atropine and yohimbine (both at 1 µM). The antagonists were present from 30 min before S3 onward.

At the end of the experiments, the preparations were blotted and weighed. The amount of neurotransmitter released during stimulation is expressed in picomoles per gram of tissue per 5 min.

Measurement of released ACh. KH samples (300 µl) were injected into an HPLC-ECD system equipped with a reverse phase-based anion-exchange analytic column. The mobile phase contained 100 mM potassium phosphate buffer (pH 8.0) in ultrapure water filtered through a 0.2-µm membrane filter (Schleicher & Schuell, Dassel, Germany), and the flow rate was 0.35 ml/min. After separation, ACh was hydrolyzed in an immobilized enzyme reactor by acetylcholinesterase to choline, and the choline was subsequently oxidized by choline oxidase to betaine and hydrogen peroxide. Hydrogen peroxide was then detected with an electrochemical detector (Antec Leyden, Leiden, The Netherlands) set to an oxidation potential of +500 mV. The analytic column and immobilized enzyme reactor were prepared according to Damsma and Westerink (7) and Ten Berge et al. (19), with slight modification. Briefly, a guard and an analytic column (both 75 × 2.1 mm; Chrompack, Middelburg, The Netherlands) were prepared from Chromspher 5 C18 (Chrompack). The analytic column was then loaded with 0.5% sodium dodecyl sulfate. After being washed with water, the column was equilibrated overnight with the mobile phase. To prepare the enzyme reactor, an enzyme solution containing 80 U of acetylcholinesterase and 40 U of choline oxidase dissolved in 1 ml of the mobile phase was passed through a column containing Lichrosorb-NH2 (Merck, Darmstadt, Germany) activated with glutaraldehyde. Before use, the reactor was washed for 30 min with the mobile phase. The detection limit of the HPLC-ECD system was ~20 fmol ACh/injection.

Measurement of released NE. After extraction of 450-µl KH samples according to the method previously described (15), catecholamines were determined by HPLC-ECD. The column was an Alltech Adsorbosphere Catecholamine C18 3-µm cartridge (100 × 4.6 mm), and the mobile phase (pH 3.8) was composed of ultrapure water-methanol (97.0:3.0 vol/vol), 0.78% citric acid, 0.68% NaH2PO4, 0.02% octane sulfonic acid, and 0.01% EDTA. This solution was filtered through a 0.2-µm membrane filter (Schleicher & Schuell). The flow rate was set at 1.00 ml/min. An Environmental Sciences Associates Coulochem 5100 A electrochemical detector with a 5011 high-sensitivity analytic cell set to an oxidation potential of +350 mV was used for the detection of catecholamines. Correction for incomplete recovery was applied with 3,4-dihydroxybenzylamine as an internal standard. The detection limit of the HPLC-ECD system was ~4 fmol NE/injection.

Drugs. The following substances were used: ICI-118551 hydrochloride (Zeneca Pharmaceuticals, Macclesfield, UK), 3,4-dihydroxybenzylamine hydrobromide, ACh chloride, acetylcholinesterase (EC 3.1.1.7), choline oxidase (EC 1.1.3.17), fenoterol hydrobromide, and NE hydrochloride (Sigma, St. Louis, MO). All other chemicals were of reagent grade and obtained from standard sources.

Statistical analysis. ACh and NE release are expressed as a percentage of the release after S3, and means ± SE were calculated for all data. Single-dose fenoterol effects were evaluated by one-way analysis of variance with a post hoc Student-Newman-Keuls test. When below the 0.05 level of probability, the actual level of significance was calculated by Student's t-test. Cumulative effects of fenoterol were evaluated by repeated-measures analysis of variance with a post hoc Student-Newman-Keuls test. When below the 0.05 level of probability, the actual level of significance was calculated by Student's paired t-test.


    RESULTS
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Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

Basal levels of EFS-induced ACh and NE release. EFS (150 mA, 16 Hz, and 0.8 ms for 5 min) evoked the release of ACh and NE from autonomic nerve endings in guinea pig tracheal smooth muscle. Control ACh release, i.e., during S3, averaged 29.1 ± 2.5 pmol · g tissue-1 · 5 min-1 (n = 46 experiments), and control NE release averaged 70.2 ± 6.2 pmol · g tissue-1 · 5 min-1 (n = 48 experiments; see Fig. 1 for chromatograms). Spontaneous release of either neurotransmitter was not observed.


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Fig. 1.   HPLC-electrochemical detection (ECD) chromatograms showing measurement of 0.42 pmol ACh (A) and 0.59 pmol norepinephrine (NE; B) per injection as released during the 3rd episode of electrical field stimulation (EFS; S3).

Effects of fenoterol and ICI-118551 on EFS-induced ACh and NE release. The release of both ACh and NE remained fairly constant throughout the seven stimulation episodes in control preparations, the ACh and NE release at S7 being 93 ± 6 (n = 6 experiments) and 93 ± 14% (n = 6 experiments), respectively, of the release at S3, indicating negligible exhaustion of neurotransmitter pools. Pretreatment of guinea pig tracheal segments with 1 µM fenoterol for 15 min before S4 resulted in an enhanced overflow of both ACh and NE (Fig. 2). The EFS-evoked overflow of ACh was augmented by 78.1 ± 27.5% (P < 0.01; n = 6 experiments), whereas the overflow of NE was augmented by 65.3 ± 11.8% (P < 0.01; n = 6 experiments) compared with S3. To confirm that these facilitations were induced by activation of beta 2-adrenoceptors, the effect of 1 µM ICI-118551 was studied. It was found that ICI-118551 completely abolished the facilitatory effect of fenoterol alone and that the remaining overflow of both ACh and NE was not significantly different from that in control tracheal preparations (Fig. 2). ICI-118551 by itself did not affect neurotransmitter overflow (data not shown).


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Fig. 2.   Effects of fenoterol (1 µM) in absence and presence of ICI-118551 (1 µM) on evoked overflow of ACh (A) and NE (B) from epithelium-denuded guinea pig tracheae. EFS (16 Hz, 150 mA, 0.8 ms) was applied during 5-min stimulation periods. Values are means ± SE of 6 experiments, each performed in duplicate. ** Significantly different compared with parallel control stimlations, P < 0.01.

Effects of agonist exposure time and concentration on EFS-induced ACh and NE release. The relative sensitivity of the facilitatory prejunctional beta 2-adrenoceptors was investigated by studying the concentration dependency to fenoterol (0.1-100 µM) at different preexposure times (1, 5, and 15 min; Figs. 3 and 4). With a 1-min preincubation, fenoterol from 1 to 100 µM concentration dependently enhanced evoked ACh overflow, whereas 0.1 µM fenoterol was still without effect; however, with 10 and 100 µM fenoterol, facilitation reached significance. With the 5-min preincubation period, the facilitatory responsiveness of the prejunctional beta 2-adrenoceptors had increased; the maximum effect was already reached at 1 and 10 µM fenoterol, whereas with 100 µM fenoterol, facilitation tended to diminish. With a 15-min exposure time, sensitivity was further increased; significant facilitation of a magnitude similar to the maximum effect reached with the 5-min incubation of 1 µM of the agonist was already seen with 0.1 µM fenoterol. Facilitation after 15 min increased further with 1 µM fenoterol, whereas at higher fenoterol concentrations, particularly 100 µM, the facilitatory effect diminished, resulting in a clear bell-shaped concentration-response relationship (Fig. 3).


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Fig. 3.   Effects of increasing concentrations of fenoterol at different preincubation times on EFS-evoked ACh overflow from epithelium-denuded guinea pig tracheal preparations. C, control. Values are means ± SE from 6-10 experiments. Significantly different compared with control values preceding agonist administration: * P < 0.05; *** P < 0.005.


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Fig. 4.   Effects of increasing concentrations of fenoterol at different preincubation times on EFS-evoked NE overflow from epithelium-denuded guinea pig tracheal preparations. Values are means ± SE from 6-10 experiments. Significantly different compared with control values preceding agonist administration: * P < 0.05; *** P < 0.005.

NE overflow showed a concentration-dependent facilitation after pretreatment with fenoterol at all agonist exposure periods. In contrast to ACh overflow, highest facilitation was observed with the shorter preincubation times; moreover, the capacity of the prejunctional beta 2-adrenoceptors to facilitate NE overflow was much stronger, reaching levels of ~500% of the control level at the highest fenoterol concentration (100 µM, 1 min; Fig. 4).

Effect of a 15-min agonist exposure on EFS-induced ACh and NE release in the presence of auto- and heteroreceptor inhibitors. In the presence of atropine and yohimbine (both 1 µM), fenoterol facilitated the evoked ACh release to a similar extent as in the absence of these receptor antagonists (Fig. 5). The decreased facilitation with higher agonist concentrations was no longer observed, however. NE release again showed a concentration-dependent facilitation in response to fenoterol, the effect being slightly greater in the presence of atropine and yohimbine (Fig. 5).


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Fig. 5.   Effects of a 15-min preincubation with increasing concentrations of fenoterol on EFS-evoked ACh (A) and NE (B) overflow from epithelium-denuded guinea pig tracheal preparations in absence (open bars) and presence (solid bars) of auto- and heteroreceptor inhibition through atropine and yohimbine (1 µM each). Values are means ± SE from 6-11 experiments. Significantly different compared with control values preceding agonist administration: * P < 0.05; *** P < 0.005.


    DISCUSSION
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Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

The present study was designed to establish whether and to what extent endogenous ACh and NE release from parasympathetic and sympathetic nerve endings, respectively, are under prejunctional control of beta 2-adrenoceptors in epithelium-denuded guinea pig tracheae. With the use of the beta 2-adrenoceptor agonist fenoterol and the beta 2-adrenoceptor antagonist ICI-118551, we demonstrated that both parasympathetic and sympathetic nerve endings in the guinea pig trachea are equipped with facilitatory beta 2-adrenoceptors (Fig. 2). Among these, especially those on the adrenergic nerve endings were able to induce a large facilitation of neurotransmitter (NE) release (Fig. 4). Concentration-response and agonist contact time relationships were markedly different between ACh and NE release (Figs. 3-5).

In these experiments, we measured the simultaneous release of endogenous ACh and NE in the absence of acetylcholinesterase inhibitors and inhibitors of neuronal or extraneuronal norepinephrine uptake. Considering the relatively long duration, the experiments were performed in the presence of tyrosine, however (compare with Ref. 5). Choline preincubation was not applied because in a recent study, Ten Berge et al. (19) found this precursor to have no influence at all on the enhancement of endogenous ACh release induced by atropine even after repeated administration.

The present findings of facilitatory beta 2-adrenoceptors on cholinergic nerve endings differ from earlier reports (2, 16, 20) where inhibitory beta 2-adrenoceptor-mediated control over ACh release was described. Such inhibitory adrenoceptors were demonstrated in guinea pig and rat tracheae and appeared to be located in the airway mucosa because the isoproterenol-induced inhibition of stimulated [3H]ACh release was no longer present after removal of this mucosa (20). In the present experiments, such indirect effects can be excluded because the tracheal preparations were carefully denuded of epithelium, which was checked histologically on several occasions. Our finding of prejunctional facilitatory beta 2-adrenoceptors on cholinergic nerve endings in the guinea pig trachea is in line, however, with observations in the equine trachea, where isoproterenol concentration dependently increased EFS-evoked endogenous ACh release (22, 23). Recently, beta 2-adrenoceptor-mediated facilitation of [3H]ACh release from the guinea pig trachea has also been described (4).

A direct facilitatory effect of beta -adrenoceptor agonist on EFS-evoked NE overflow from sympathetic nerves in the guinea pig trachea has not been previously reported. An indirect effect was observed in the isolated rat trachea, however, where isoproterenol augmented the EFS-induced NE release in the presence but not in the absence of the mucosa in an indomethacin-sensitive fashion (5). As mentioned above, our experimental setup ruled out the possibility of such indirect effects.

Interesting differences in the facilitatory responses to fenoterol were observed between cholinergic and adrenergic nerves. When the agonist preincubation time was prolonged from 1 to 15 min, the response of the prejunctional beta 2-adrenoceptors to facilitate ACh overflow increased; at the same time, the concentration-response relationship gradually attained a bell-shaped nature. To investigate whether counterregulation by prejunctional M2 muscarinic and alpha 2-adrenergic auto- and heteroreceptors was responsible for or contributed to the diminished facilitation seen with a 15-min preincubation with 10 and 100 µM compared with 1 µM fenoterol, concentration-response relationships were also established in the presence of atropine and yohimbine. As shown in Fig. 5, the facilitation obtained with the two higher fenoterol concentrations remained very similar to that of 1 µM fenoterol under these conditions, indicating that auto- and/or heteroreceptor-mediated counterregulation is indeed involved.

The facilitation of NE overflow by increasing concentrations of fenoterol was most prominent at the shortest preincubation time; particularly with a 15-min preincubation with 10 and 100 µM fenoterol, facilitation was diminished compared with that at 1 and 5 min. In the presence of yohimbine and atropine, similar facilitatory effects of the agonist were observed, although at the highest fenoterol concentration, some enhancement of the facilitation was observed. However, the extent of facilitation with 10 and 100 µM fenoterol (15-min incubation) under auto- and heteroreceptor blockade was clearly less than that with 1- and 5-min preincubations. These data would suggest that the beta 2-adrenoceptors on adrenergic nerve terminals in the guinea pig trachea are readily susceptible to desensitization.

It should be mentioned that exhaustion of endogenous neurotransmitter pools is an unlikely explanation for the time-related effects observed with fenoterol because 1) facilitation of NE release increased with all fenoterol concentrations, being most pronounced at S7 after a 1-min fenoterol preincubation, and 2) repeated atropine incubations result in equal facilitations of ACh release (19; unpublished results).

The observation that prejunctional beta -adrenoceptors on adrenergic nerve endings are sensitive to desensitization is not unexpected, although it has not been analyzed as such in the guinea pig trachea. Rapid desensitization of these receptors has been previously demonstrated in isolated rat kidney preparations, with isoproterenol producing a bell-shaped concentration-response curve with 20- and 5-min but not with 2-min exposure times (10). Our results do not show a bell-shaped curve of NE overflow when tracheae are exposed to increasing fenoterol concentrations. This difference in response may be due to a different receptor density or coupling efficiency on sympathetic nerve endings in the renal artery compared with that in the trachea or simply to differences in experimental setup and design.

The observations by Belvisi et al. (4) and Zhang et al. (24) that both isoproterenol and forskolin facilitate ACh release indicate that the prejunctional beta 2-adrenoceptor signaling, at least in cholinergic nerve endings, is through adenylyl cyclase and cAMP. Two different protein kinases, beta -adrenergic-receptor kinase (beta -ARK) and cAMP-dependent protein kinase A (PKA), are known to be involved in short-term beta -adrenoceptor desensitization. beta -ARK has been shown to phosphorylate the agonist-activated beta -adrenoceptor rapidly; as a consequence, another cytosolic protein, beta -arrestin, binds to the phosphorylated receptor leading to uncoupling from the Gs protein (see Ref. 3 for a review). In contrast, PKA-mediated phosphorylation of the beta -adrenoceptor proceeds at a slower rate (9, 11). Recently, McGraw and Liggett (12) have shown that the desensitization susceptibility of beta -adrenoceptors in different pulmonary cell types is clearly related to cellular levels of beta -ARK rather than of PKA. It is tempting to speculate that the differential desensitization susceptibilities of the two prejunctional beta 2-adrenoceptor populations are related to differences in the involvement of beta -ARK, this kinase having a more prominent role in adrenergic nerve terminals. However, the differential time frame in which the facilitatory responses develop and the differential susceptibility to desensitization of the facilitatory beta 2-adrenoceptors on cholinergic and adrenergic nerves may also suggest the involvement of distinct intracellular signaling pathways.

In conclusion, the results presented in this paper are the first demonstration that EFS-evoked overflow of both endogenous ACh and NE from guinea pig trachealis autonomic nerves are augmented by prejunctional beta 2-adrenoceptor activation. The physiological and therapeutic significance of these facilitatory beta 2-adrenoceptors is not completely clear. ACh release appears under dual (epithelium and nerve) and opposite (inhibition and facilitation) control of beta -adrenergic receptors, at least in guinea pig airways (20; present study). Adrenergic innervation in the human lung is generally believed to project to the vasculature and glands rather than to airway smooth muscle. Enhanced NE release from vascular nerves might cause vasoconstriction, thereby reducing vasocongestion and decreasing the tendency for edema formation; alternatively, or in addition, NE might overflow to relax airway smooth muscle, and there may be direct (although sparse) adrenergic innervation of airway smooth muscle in the human lung as well (8). Bearing this in mind, it should be noted that, although the overflow of both the bronchoconstricting neurotransmitter ACh and the bronchodilating neurotransmitter NE is augmented by fenoterol, the capacity of prejunctional beta 2-adrenoceptors on adrenergic nerves is markedly higher compared with that on cholinergic nerves after short but also after long incubation periods and both with and without functional auto- and heteroreceptor counterregulation. As a result, beta 2-adrenoceptor agonists might have indirect bronchodilatory effects, through the enhancement of NE overflow, in addition to the direct relaxant action on bronchial smooth muscle that occurs through postjunctional beta 2-adrenoceptors.


    ACKNOWLEDGEMENTS

We thank Frans Brouwer for experimental support. Zeneca Pharmaceuticals (Macclesfield, UK) is acknowledged for the donation of ICI-118551.


    FOOTNOTES

This work was supported by Netherlands Asthma Foundation Grant 93.70.

Address for reprint requests: J. R. A. de Haas, Department of Molecular Pharmacology, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.

Received 27 May 1997; accepted in final form 30 November 1998.


    REFERENCES
Top
Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

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Am J Physiol Lung Cell Mol Physiol 276(3):L420-L425
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