Role of cAMP and neuronal K+ channels on alpha 2-AR-induced inhibition of ACh release in equine trachea

Xiang-Yang Zhang, Feng-Xia Zhu, and N. Edward Robinson

Departments of Large Animal Clinical Sciences and Physiology, Michigan State University, East Lansing, Michigan 48824-1314

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

To investigate the effects of changes in intracellular cAMP on alpha 2-adrenoceptor (AR)-induced inhibition of airway acetylcholine (ACh) release, we examined the effects of the alpha 2-AR agonist clonidine on electrical field stimulation-evoked ACh release from equine tracheal parasympathetic nerves before and after treatment with 8-bromo-cAMP or forskolin. We also tested whether charybdotoxin (ChTX)- or iberiotoxin (IBTX)-sensitive Ca2+-activated K+ channels mediate alpha 2-AR-induced inhibition by examining the effect of clonidine in the absence and presence of ChTX or IBTX on ACh release. The amount of released ACh was measured by HPLC coupled with electrochemical detection. Clonidine (10-7 to 10-5 M) dose dependently inhibited ACh release before and after treatment with 8-bromo-cAMP (10-3 M) or forskolin (3 × 10-5 M). ChTX and IBTX, both at the concentration of 5 × 10-7 M, significantly increased ACh release; however, they did not alter the magnitude of clonidine-induced inhibition. These results indicated that in equine tracheal parasympathetic nerves, alpha 2-AR-induced inhibition of ACh release is via an intracellular cAMP-independent pathway. Activation of both ChTX- and IBTX-sensitive Ca2+-activated K+ channels inhibits the electrical field stimulation-evoked ACh release, but these channels are not involved in the alpha 2-AR-induced inhibition of ACh release.

adenosine 3',5'-cyclic monophosphate; potassium ion; alpha 2-adrenoceptor; acetylcholine; clonidine; charybdotoxin; iberiotoxin; 8-bromoadenosine 3',5'-cyclic monophosphate; forskolin; cholinergic neurotransmission

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

ACETYLCHOLINE (ACh) release from airway parasympathetic nerves is modulated prejunctionally by autoreceptors and a variety of heteroreceptors. By direct measurement of ACh release, our laboratory and other investigators have confirmed that muscarinic autoreceptors, the M2 subtype in most species, provide negative feedback that normally inhibits ACh release (6, 18) and that inhibitory alpha 2-adrenoceptors (ARs) (3, 22, 24) inhibit ACh release more potently in the trachea than in the smaller bronchi (22). Zhang and colleagues (23, 24, 26) have also discovered an excitatory beta 2-AR, activation of which increases ACh release.

In comparison with information available from other parts of the nervous system, the intracellular transduction pathways coupling these prejunctional receptors to ACh release are largely unexplored in the airway. Baker et al. (2) have shown that ACh release is dependent on N-type, omega -conotoxin-sensitive Ca2+ channels. We (25) have recently demonstrated that increasing the intracellular concentration of cAMP by means of the adenylyl cyclase (AC) activator forskolin, the lipid-soluble cAMP analog 8-bromo-cAMP, or the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) facilitates ACh release. Furthermore, by examining the additive effects of 8-bromo-cAMP or forskolin with the beta -AR agonist isoproterenol, we (25) confirmed that beta 2-AR-induced augmentation of ACh release is via a cAMP-dependent pathway.

In many tissues, alpha 2-receptors are coupled to AC via an inhibitory G protein so that their activation can decrease intracellular levels of cAMP (7, 9, 16). It is therefore possible that alpha 2-receptors inhibit ACh release via a decrease in cAMP. However, recent studies (11, 15) demonstrated that activation of alpha 2-receptors inhibits norepinephrine (NE) release from the sympathetic nervous system via a cAMP-independent mechanism. For example, activation of the alpha 2-AR inhibits NE release from cultured rat ganglion cells even when intracellular levels of cAMP are fixed by use of analogs of cAMP (15). In addition, it has been clearly demonstrated by patch-clamping techniques that the inhibition of the N-type Ca2+ channels and of NE release that follow activation of the alpha 2-AR in sympathetic neurons is independent of a soluble, diffusible intracellular messenger such as cAMP (11). Therefore, one of the purposes of our present study was to investigate the role of intracellular cAMP on alpha 2-AR-induced inhibition of ACh release from airway parasympathetic nerves. We wanted to determine whether the alpha 2-AR-mediated inhibition is via intracellular cAMP-dependent pathways by examining the effect of the alpha 2-AR agonist clonidine on ACh release before and after the intracellular concentration of cAMP was fixed by 8-bromo-cAMP (10-3 M) or increased by forskolin (3 × 10-5 M). If inhibition of ACh release by alpha 2-AR agonists is primarily via cAMP-independent pathways, clonidine should inhibit ACh release before and after the intracellular concentration of cAMP is elevated. If a significant component of alpha 2-AR-mediated inhibition is via cAMP-dependent pathways, inhibition should be significantly reduced by forskolin or abolished after the intracellular concentration of cAMP is clamped by 8-bromo-cAMP.

Measurement of smooth muscle tension in guinea pig airway has suggested that blockade of Ca2+-activated K+ (KCa) channels with charybdotoxin (ChTX) prevents prejunctional inhibition of cholinergic neurotransmission by the alpha 2-AR agonist clonidine (13). However, by direct measurement of ACh release, Baker et al. (3) demonstrated that alpha 2-AR-induced inhibition of ACh release from guinea pig airway parasympathetic nerves is not mediated via iberiotoxin (IBTX)-sensitive K+ channels. One possible explanation for the above conflicting results may be the different pharmacological characteristics of ChTX and IBTX. Therefore, in the second protocol, we investigated whether the alpha 2-AR-mediated inhibition is via KCa channels by examining the effect of clonidine before and after the blockade of KCa channels with ChTX or IBTX. If the alpha 2-AR-mediated inhibition is via these K+ channels, ChTX and/or IBTX should attenuate the inhibition.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Tissue. Tissue was collected from 15 horses (aged 7.9 ± 1.5 yr, body wt 415.1 ± 15.5 kg) for this study, which was approved by the All-University Committee on Animal Use and Care of Michigan State University (East Lansing). Other investigators also used tissues from the same animals for a variety of studies. The horses had no clinical signs of respiratory disease for several weeks before they were killed by injection of an overdose of pentobarbital sodium through the jugular vein. Postmortem examination revealed that the lungs and airways were normal in gross appearance. A segment of trachea between the 6th and 30th cartilaginous rings above the carina was quickly collected, immersed in Krebs-Henseleit (KH) solution (composition in mM: 118.4 NaCl, 25.0 NaHCO3, 11.7 dextrose, 4.7 KCl, 2.6 CaCl2 · 2H2O, 1.19 MgSO4 · 7H2O, and 1.16 KH2PO4), and gassed with 95% O2-5% CO2 during the whole experiment. The trachea was opened longitudinally by dissection of the cartilages in its anterior aspect and was pegged flat on a paraffin block submerged in KH solution. Tracheal smooth muscle strips with epithelium intact were cut with a template along the fiber direction before being suspended in tissue baths. The temperature within the baths was maintained at 37°C, and the KH solution was changed every 15 min. Square-wave electrical impulses were produced by a stimulator (S88, Grass Instruments, Quincy, MA) and passed through a stimulus power booster (Stimu-Splitter II, Med-Lab Instruments, Loveland, CO) to electrodes in the tissue baths.

Measurement of electrical field stimulation-induced ACh release. Four tissue strips (each measuring 2 × 15 mm) were cut and tied together at both of their ends with 3-0 surgical silk thread. Each tracheal-strip bundle (wet weight 198.0 ± 3.0 mg; n = 156 strips) was suspended in a 2-ml tissue bath with a pair of parallel platinum wire electrodes built against the wall of the bath in the vertical direction (Radnoti Glass Technology, Monrovia, CA). One end of the tissue strip was secured to the bottom of the bath with a glass tissue holder; the other end was attached to an 8-g weight via a surgical thread that passed over a steel bar above the tissue bath (17). After an ~120-min equilibration period, the tissues were incubated for 60 min with the cholinesterase inhibitor neostigmine (10-6 M), the sympathetic nerve blocker guanethidine (10-5 M), and the muscarinic-autoreceptor antagonist atropine (10-7 M). These agents were present during the remainder of the experiment. Endogenous prostanoids and nitric oxide from an inhibitory nonadrenergic noncholinergic system do not affect electrical field stimulation (EFS)-induced ACh release from our tissue preparations (23, 26).

The ACh concentration in the tissue bath liquid was measured by HPLC coupled with electrochemical detection. The mobile phase contained 100 mM Na2HPO4 (pH 8.0), and the flow rate was 0.35 ml/min. The samples were filtered through 0.2-µm nylon membrane filters (Acrodiscs 13, Gelman Sciences, Ann Arbor, MI) and injected into the HPLC column at a volume of 25 µl/injection. An external ACh standard (2.5 pmol in 25 µl) was injected every six samples, and the concentration of ACh in the samples was calculated based on the bracketed calibration (for details of this technique, see Refs. 17, 24).

Study design. EFS (0.5 Hz, 0.5 ms, 20 V) was applied to all the tissues for four 15-min periods, with a 30-min resting interval between consecutive stimuli. During the first EFS, we determined the baseline release of ACh. Then the tested drugs were added to the baths 10-30 min (see protocols 1 and 2 for details) before subsequent EFS. To eliminate any ACh that may have been released during the incubation period (17), the tissue baths were drained and refilled with fresh KH solution containing the tested drugs immediately before the EFS was begun. The tissue bath solution was collected on the completion of each EFS for the measurement of ACh. The tissues were rinsed four times with the KH solution immediately after collection of the samples. At the end of the experiment, the tissues were blotted dry and weighed.

Protocol 1: effect of alpha 2-AR-agonist clonidine on EFS-induced ACh release before and after increasing the intracellular concentration of cAMP with 8-bromo-cAMP or forskolin. Data analysis in this study involved calculation and comparison of the percent inhibition of ACh release by clonidine in the absence and presence of 8-bromo-cAMP or forskolin. Both EFS frequency and baseline ACh release may affect the magnitude of alpha 2-AR-agonist-induced inhibition of ACh release (11, 22). In our preliminary experiments, we demonstrated that clonidine-induced inhibition of ACh release did not significantly differ at 0.5- and 1-Hz EFS frequencies. Both 8-bromo-cAMP (10-3 M) and forskolin (5 × 10-5 M) alone doubled the EFS-evoked ACh release (25). To keep the baseline ACh release the same before the alpha 2-AR-agonist was added, we stimulated the control tissues (in the absence of cAMP analogs) at 1 Hz and the 8-bromo-cAMP- and forskolin-treated tissues at 0.5 Hz.

Three sets of tissue-strip bundles were used in this protocol. Each set consisted of two tissue-strip bundles; one tissue bundle did not receive clonidine and served as a time control. The remaining one received clonidine (10-7 to 10-5 M) 10 min before subsequent EFS after the first (baseline) stimulation (20 V, 0.5 ms). The first set of two tissue-strip bundles (control) received neither 8-bromo-cAMP nor forskolin and was stimulated at 1 Hz. The second and third sets of tissue-strip bundles were incubated with 8-bromo-cAMP (10-3 M) or forskolin (5 × 10-5 M), respectively, 20 min before the application of clonidine or its vehicle and were stimulated at 0.5 Hz. The percent inhibition of ACh release by clonidine was calculated and compared in the absence and presence of 8-bromo-cAMP or forskolin.

Protocol 2: effect of the alpha 2-AR-agonist clonidine on EFS-induced ACh release in the absence and presence of the KCa-channel blockade with ChTX or IBTX. Three tissue-strip bundles were used; one did not receive any drug treatment and served as a time control. After the first stimulation (20 V, 0.5 ms, 0.5 Hz), the second tissue-strip bundle received ChTX (5 × 10-7 M) 30 min before the second EFS, and ChTX remained in the tissue bath during the subsequent EFS. Clonidine (10-6 to 10-5 M) was then applied 10 min before the third and fourth EFS. A comparison of ACh release rate before (first EFS) and after (second EFS) ChTX was made to evaluate the effect of blockade of KCa channels on basal EFS-evoked ACh release. The ACh release rate during the second EFS was then used as a baseline to calculate the clonidine-induced percent inhibition of ACh release in the presence of ChTX. A similar protocol was performed in the third set of tissue-strip bundles except in the absence of ChTX. The percent inhibition of ACh release by clonidine was compared in the absence and presence of ChTX. This protocol was repeated with IBTX (5 × 10-7 M) instead of ChTX.

Drugs. ACh chloride, atropine sulfate, 8-bromo-cAMP, ChTX, clonidine hydrochloride, omega -conotoxin GVIA, guanethidine monosulfate, IBTX, and neostigmine methyl sulfate were dissolved in deionized water. Forskolin was dissolved in DMSO. The appropriate working solutions of all the drugs were made with KH buffer on the day of the experiment. The drug solution was pipetted into the tissue bath at 1% of the bath volume. The final concentration of the drugs is expressed as their bath molar concentration. All drugs were from Sigma (St. Louis, MO).

Statistical analysis. The ACh release rate is expressed as both picomoles per gram per minute and a percentage of baseline (first EFS without drug treatment). Means ± SE for all parameters were calculated. The ACh release rates before and after ChTX or IBTX treatment were compared by paired t-test. The other data were evaluated by repeated-measures ANOVA and ANOVA with contrasts by Statview II (Abacus Concepts, Calabasas, CA) for the Macintosh computer. Means were compared by Fisher's (protected) least significant difference test. Except where otherwise specified, n is the number of animals. P < 0.05 was considered statistically significant.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Protocol 1: effect of alpha 2-AR-agonist clonidine on EFS-induced ACh release before and after increasing the intracellular concentration of cAMP with 8-bromo-cAMP or forskolin. Baseline ACh release, i.e., release during first EFS period, averaged 6.53 ± 1.24 and 11.46 ± 1.76 pmol · g-1 · min-1 at 0.5 and 1 Hz, respectively. In 8-bromo-cAMP (10-3 M)- and forskolin (5 × 10-5 M)-incubated tissues, EFS (0.5 Hz)-evoked ACh release was 11.59 ± 2.17 and 12.92 ± 2.277 pmol · g-1 · min-1, which is in the similar release level as that evoked by 1-Hz EFS. In all time-control tissues, ACh release remained constant throughout the four stimulations. The alpha 2-AR-agonist clonidine (10-7 to 10-5 M) inhibited ACh release in a concentration-dependent manner in both the absence (control; Fig. 1A) and presence of 8-bromo-cAMP (Fig. 1B) or forskolin (Fig. 1C). At 10-5 M, clonidine caused 50.1 ± 8.0, 41.2 ± 6.6, and 50.0 ± 7.4% inhibition of ACh release in control, 8-bromo-cAMP-pretreated, and forskolin-pretreated tissues, respectively (Fig. 2). When the percent inhibition was compared at the concentrations of clonidine of 10-7, 10-6, and 10-5 M, there were no significant differences between the control and 8-bromo-cAMP- or forskolin-pretreated groups (n = 6 animals; Fig. 2).


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Fig. 1.   Effect of alpha 2-adrenoceptor agonist clonidine (10-7 to 10-5 M) on electrical field stimulation-evoked ACh release from control (A) and 10-3 M 8-bromo-cAMP (B)- and 5 × 10-5 M forskolin (C)-preincubated equine tracheal-strip bundles (n = 6). * Significantly different from time control.


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Fig. 2.   Comparison of clonidine-induced inhibition of ACh release from control tracheal-strip bundles with that measured in presence of 10-3 M 8-bromo-cAMP or 5 × 10-5 M forskolin. n = 6 animals.

Protocol 2: effect of the alpha 2-AR-agonist clonidine on EFS-induced ACh release in the absence and presence of a KCa-channel blockade with ChTX or IBTX. Application of ChTX (5 × 10-7 M) increased EFS-induced ACh release from 6.40 ± 0.51 to 14.73 ± 2.42 pmol · g-1 · min-1 (n = 6 animals; Fig. 3A). Iberiotoxin (5 × 10-7 M) also significantly increased EFS-induced ACh release from 5.81 ± 0.78 to 9.35 ± 0.98 pmol · g-1 · min-1 (n = 4 animals; Fig. 4A). Even though both ChTX and IBTX significantly facilitated ACh release, they did not alter the magnitude of clonidine-induced inhibition. The percent inhibition of ACh release by clonidine was identical in the absence and presence of ChTX (n = 6 animals; Fig. 3B) or IBTX (n = 4 animals; Fig. 4B), indicating clonidine-induced inhibition of ACh release is independent of both ChTX- and IBTX-sensitive KCa channels.


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Fig. 3.   Effect of alpha 2-adrenoceptor agonist clonidine (Clo) on electrical field stimulation (EFS)-evoked ACh release in absence and presence of charybdotoxin (ChTX). Even though 5 × 10-7 M ChTX doubled EFS-evoked ACh release rate (A), percent inhibition of ACh release (B) after clonidine was identical in absence and presence of ChTX. n = 6 animals. * Significantly different from tissues without ChTX.


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Fig. 4.   Effect of alpha 2-adrenoceptor agonist Clo on EFS-evoked ACh release in absence and presence of iberiotoxin (IBTX). Even though 5 × 10-7 M IBTX significantly facilitated EFS-evoked ACh release rate (A), percent inhibition of ACh release (B) after Clo was identical in absence and presence of IBTX. n = 4 animals. * Significantly different from tissues without IBTX.

    DISCUSSION
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Abstract
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Materials & Methods
Results
Discussion
References

When intracellular cAMP levels are elevated by activation of stimulatory G proteins with cholera toxin, activation of AC by forskolin, administration of membrane-permeable cAMP analogs, or inhibition of phosphodiesterase, there is an increased release of ACh from equine (25) and guinea pig (4) airway parasympathetic and phrenic nerves (21) as well as from cerebral cholinergic neurons (10), rat superior cervical ganglion (5), and guinea pig ileum myenteric plexus (1, 20). It is therefore well established that increasing intracellular cAMP increases ACh release. In many tissues, alpha 2-ARs are coupled to AC via an inhibitory G protein so that their activation can decrease intracellular levels of cAMP (7, 9, 16). It is therefore possible that the inhibition of ACh release from airway parasympathetic nerves by activation of alpha 2-ARs is mediated via a decrease in cAMP. However, the results of our first experiments do not support this hypothesis. 8-Bromo-cAMP (10-3 M) had no significant effect on clonidine-induced inhibition of ACh release, and the magnitude of inhibition was similar in control tissues and in the presence of forskolin (Fig. 2). These results indicate that the majority of alpha 2-AR-induced inhibition of ACh release from airway parasympathetic nerves is via mechanisms that are independent of intracellular cAMP. This is similar to the situation in the sympathetic nervous system, in which alpha 2-AR-induced inhibition of NE release is not affected by cAMP analogs and therefore is independent of the intracellular cAMP pathway (11, 15). We cannot totally exclude a minor role for cAMP-dependent pathways, however, because there was a tendency for 8-bromo-cAMP to attenuate the effect of clonidine at 10-6 M.

Activation of prejunctional K+ channels leads to hyperpolarization of the plasma membrane, reduces Ca2+ influx through voltage-activated channels, and decreases neurotransmitter release (12, 14). In guinea pig airway, Miura et al. (13) demonstrated that ChTX (10-8 M), a blocker of KCa and voltage-dependent K+ channels (8), potentiates EFS-induced smooth muscle contraction without affecting the response to exogenously applied ACh. These observations were interpreted to suggest that activation of ChTX-sensitive K+ channels inhibits cholinergic neurotransmitter release. Our direct measurement of ACh release confirms the above conclusion. When K+ channels were blocked with ChTX, EFS-induced ACh release was potentiated. Thus in airway parasympathetic nerves as in other parts of the nervous system, neuronal ChTX-sensitive KCa channels are inhibitory to ACh release. This finding is also in agreement with a recent demonstration that NS- 1619, a selective large-conductance KCa-channel opener, inhibits EFS-evoked [3H]ACh release from guinea pig trachea (14). In contradiction to this conclusion is the observation by Baker et al. (3) that a more selective blocker of fast-conductance KCa [KCa(f)] channel (8), IBTX, at a concentration (10-7 M) that increased tracheal tension, failed to alter EFS-evoked ACh release from guinea pig trachea. The possible explanations for these conflicting observations have been carefully discussed by Baker et al. (3). In addition to the methodological difference, ChTX is pharmacologically less selective than IBTX for KCa(f) channels; it also inhibits several other classes of KCa channels (8). It is possible that although IBTX-sensitive KCa(f) channels have been excluded, other classes of ChTX-sensitive KCa channels may be involved in the control of ACh release from guinea pig airway parasympathetic nerves. However, our results demonstrated that IBTX significantly increased ACh release, indicating that activation of prejunctional IBTX-sensitive KCa channels inhibits ACh release from equine airway parasympathetic nerves. Both Baker et al. (3) and we used HPLC to directly measure the ACh release. Therefore, one possible explanation for the differences between our results and those of Baker et al. (3) might be the species difference. The average increases in ACh released from our tissue preparations by ChTX and IBTX at the concentration of 5 × 10-7 M were 8.33 and 3.54 pmol · g-1 · min-1, respectively. In agreement with the literature (8), these results suggest that ChTX may inhibit several other classes of KCa channels in addition to those that are IBTX sensitive.

If activation of ChTX- or IBTX-sensitive KCa channels inhibits ACh release, it is possible that alpha 2-ARs may exert their effect on ACh release via activation of such channels. Previous investigations have reached conflicting conclusions on this point. Miura et al. (13) demonstrated reversal of clonidine-induced inhibition of EFS-induced smooth muscle contraction by ChTX and concluded that ChTX-sensitive K+ channels are involved in the alpha 2-AR-induced inhibition of ACh release from guinea pig airway parasympathetic nerves. However, in direct measurements of neurotransmitter release, IBTX (10-7 M) failed to alter clonidine-induced inhibition of ACh release from guinea pig trachea (3). Again, the different results may be due to the different pharmacological characteristics of ChTX and IBTX. Therefore, in the present study, we tested the effects of ChTX and IBTX on clonidine-induced inhibition of EFS-evoked ACh release. Our results clearly demonstrated that even though both ChTX and IBTX potentiate EFS-induced ACh release, they do not alter the alpha 2-AR-induced inhibition of ACh release. The reasons for these conflicting conclusions obtained by direct measurement of ACh release and from functional evaluations (13) are currently unclear. Nevertheless, as our laboratory (17, 22-24, 26) and other investigators (3) have previously mentioned, functional studies that compare the effect of an agent on EFS- and ACh-induced contractions rely on several assumptions, and a differential effect on the two contractions can be explained by several possibilities. For example, a greater number of muscarinic receptors on the airway smooth muscle cells may be activated by exogenous ACh than by EFS, which, in turn, could decrease the response to inhibitory agonists. Furthermore, EFS activates several types of airway nerves and releases several neurotransmitters in addition to ACh that can affect the response of the muscle. Finally, many agents, such as K+-channel blockers, act both prejunctionally to modulate ACh release and postjunctionally to alter the response of smooth muscle to the released ACh. For these reasons, it is essential to directly measure ACh release to accurately investigate prejunctional modulation of cholinergic neurotransmitter release.

On the basis of our above observations, we conclude that in equine tracheal parasympathetic nerves, alpha 2-AR-induced inhibition of ACh release is via an intracellular cAMP-independent pathway. Both ChTX- and IBTX-sensitive KCa channels inhibit the EFS-evoked ACh release but are not involved in the alpha 2-AR-induced inhibition. Therefore, the intracellular mechanisms of alpha 2-AR-induced inhibition of ACh release are still unclear. In many neurons, the inhibitory regulation of neurotransmitter release is mediated via direct coupling of prejunctional receptors to Ca2+ channels (12). The alpha 2-AR-induced inhibition of NE release from sympathetic neurons is associated with a reduction in the activity of N-type Ca2+ channels (11). N-type, omega -conotoxin-sensitive Ca2+ channels regulate ACh release in the rat myenteric plexus (19) and guinea pig (2) and horse (unpublished data) airway parasympathetic nerves. Therefore, it is likely that N-type, omega -conotoxin-sensitive Ca2+-channels may mediate the alpha 2-AR-induced inhibition of ACh release from airway parasympathetic nerves. Further investigations with an electrophysiological approach are needed to directly measure Ca2+ influx in airway parasympathetic ganglion cells.

    ACKNOWLEDGEMENTS

We thank Cathy Berney for technical assistance and Victoria Hoelzer-Maddox and MaryEllen Shea for manuscript preparation.

    FOOTNOTES

This work was supported in part by an endowment from the Matilda R. Wilson Fund.

X.-Y. Zhang is a fellow of the American Lung Association of Michigan.

Address for reprint requests: X.-Y. Zhang, Dept. of Large Animal Clinical Sciences, Michigan State Univ., East Lansing, MI 48824-1314.

Received 11 August 1997; accepted in final form 11 February 1998.

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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Lung Cell Mol Physiol 274(5):L827-L832
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