Pulmonary Pharmacology (1992) 5,25 l-255
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Atypical Presynaptic Alpha-adrenoceptor Modulation of Neurally-mediated Cholinergic Responsesin Guinea-pig Tracheal Smooth Muscle D. C. Thompson*, L. Diamond, R, J. Altiere University of Colorado School of Pharmacy, Boulder, Colorado 80309-0297, USA
SUMMARY: Cholinergic excitatory nerves in guinea-pig trachea are subject to inhibitory control by presynaptic q-adrenoceptors. Recently, the nature of these receptors has come into question insofar as the presynaptic inhibitory effects of the oc,-adrenoceptor agonist, clonidine, in the guinea-pig trachea have been shown to be antagonized by the a,-adrenoceptor antagonist, yohimbine, as well as the a,-adrenoceptor antagonist, prazosin. This inhibitory action of prazosin had not been described previously in the airways and may relate to the use of norepinephrine rather than clonidine as the a-adrenoceptor agonist in earlier studies. The present study evaluates the susceptibility of norepinephrine-induced inhibition of neurally-mediated cholinergic excitatory responses to antagonism by prazosin and yohimbine under conditions identical to those which showed clonidine to be sensitive to these antagonists. In tissues pretreated with guanethidine, propranolol and indomethacin, norepinephrine (1 PM) induced a 37-fold rightward shift of the frequency-response curve for neurally-mediated cholinergic contractions which was reversed partially by pretreatment of tissues with yohimbine. Norepinephrine also caused a concentrationdependent inhibition of choline@ ‘twitch’ responses induced by intermittent (1 Hz) nerve stimulation. This action of norepinephrine was antagonized in a concentrationdependent manner by yohimbine but was unaffected by prazosin. These results indicate that in guinea-pig trachea the presynaptic inhibitory actions of norepinephrine on cholinergic nerves are mediated via classical q-adrenoceptors, i.e. receptors that can be blocked by yohimbine but not by prazosin. This distinguishes the action of norepinephrine from that shown previously for clonidine and provides support for the contention that these agonists do not act on the same population of u,-adrenoceptors.
Recent experiments in our laboratory have revealed that presynaptic a,-adrenoceptors in guinea-pig trachea exhibit a unique characteristic, i.e. they are sensitive to antagonism by the a,-adrenoceptor antagonist, prazosin, when clonidine, the a,-adrenoceptor agonist, is used for their activation.4 In general, the actions of a,-adrenoceptor agonists such as clonidine are blocked by a,-adrenoceptor antagonists but not by a,-adrenoceptor antagonists. However, in pithed rats in which heart rate has been elevated by stimulation of the nervi accelerantes, clonidine-induced bradycardia is inhibited by prazosin,5 suggesting that prazosin may act as an antagonist of presynaptic a*adrenoceptors on sympathetic nerves in this tissue. Vizi and colleagues6 also demonstrated an antagonistic effect of prazosin on the presynaptic inhibitory effect of the a,-adrenoceptor agonist, xylazine, on sympathetic nerves innervating the isolated rat vas deferens. No such antagonistic effect of prazosin was exerted against the non-specific a-adrenoceptor agonist, norepinephrine.6 Based upon these observations
INTRODUCTION Cholinergic nerves innervating airway smooth muscle have been shown to be inhibited presynaptically by aadrenoceptor agonists. I9293The presynaptic receptors mediating these inhibitory responses have been characterized through the use of subtype-specific antagonists (e.g. prazosin, the a,-adrenoceptor subtype-specific antagonist and yohimbine, the a,-adrenoceptorspecific antagonist). The response most typically studied is norepinephrine-induced inhibition of neurally-mediated choline& contraction.‘gZ Additional studies have examined the relative inhibitory potencies of various a-adrenoceptor subtype-specific agonists.3T4 Results of such studies have shown the presynaptic a-adrenoceptor on cholinergic nerves innervating guinea-pig tracheal smooth muscle to be of the a,-adrenoceptor subtype.
* For correspondence. 09524600/92/040251+
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and results obtained using the combination of prazosin and yohimbine, Vizi and colleagues6 proposed the existence of two different presynaptic a?-adrenoceptors. Previous studies investigating presynaptic a-adrenoceptors on cholinergic nerves in guinea-pig airways have used norepinephrine as the agonist and have found prazosin to possess little antagonistic activity.’ In light of the results of Vizi et al, it is conceivable that the disparate antagonistic actions of prazosin against clonidine4 and norepinephrine’ in the guinea-pig trachea are related to the agonist itself. The present study was undertaken to re-examine the inhibitory effects of norepinephrine on neurally-mediated cholinergic nerve responses in the guinea-pig trachea and to assess the susceptibility of these effects to antagonism by prazosin and yohimbine.
MATERIALS
AND METHODS
Male guinea-pigs, weighing between 350 and 500 g, were anaesthetized with pentobarbital sodium (60 mg/ kg, i.p.). The trachea was removed and placed in Krebs-Henseleit solution (KHS) of the following composition (mM): NaCl, 118.2; KCl, 4.74; CaCl,, 2.54; KH,PO,, 1.19; NaHCO,, 26.2; MgSO,, 1.19; D( + )-glucose, 11.1; sodium ascorbate, 1.O; CaNa,EDTA, 0.03. Stainless steel tissue hooks were placed through the lumen of ring segments (46 mm in length) and, upon mounting in the organ bath, care was taken to ensure that the tissue hooks were not resting upon the smooth muscle layer. All preparations were mounted between platinum plate electrodes7 in 10 ml glass jacketed organ baths containing KHS maintained at 37°C and gassed with 5% CO, in 0,. An initial load of 3 g was placed on each segment. Tissues were allowed to equilibrate for a period of 60min, during which time the bathing solution was exchanged at 10 min intervals. Acetylcholine (1 mM) was then applied to all tissues. Upon attainment of the peak response, a 60-min wash-out period ensued after which the experimental procedures were initiated. Each trachea was disected to yield four ring segments such that one segment acted as a control (no treatment) and the other three were subjected to treatment. Responses were measured isometrically using forcedisplacement transducers (Grass FT.03c) and displayed on a polygraph (Grass model 79D). All preparations were continually exposed to propranolol (1 C(M) and guanethidine (10 PM) to block adrenergically-mediated inhibitory responses and indomethacin (3 PM) to prevent the development of spontaneous resting tone. Tissues were subjected to continuous intermittent electrical field stimulation (1 Hz, 0.3 ms pulse duration, 10 V pulse amplitude, 5 s stimulation period at 60 s intervals) following wash-
out of the acetylcholine response. A period of 30 min was allowed for the ‘twitch’ responses to become reproducible after which tissues were incubated with vehicle (10 pl distilled water) or an a-adrenoceptor antagonist (prazosin, a,-adrenoceptor antagonist; a,-adrenoceptor antagonist). After yohimbine, 15 min, norepinephrine or norepinephrine vehicle (equivalent volume of distilled water) was administered to tissues in a cumulative manner. In addition. frequency-response curves for cholinergic nerve stimulation were induced in resting tissues by electrical field stimulation (0.1-80 Hz, 0.3 ms pulse duration, 10 V pulse amplitude, 5 s stimulation period at 60 s intervals). Electrical field stimulation was applied to preparations using square wave pulses generated by a Grass S-88 stimulator in series with a signal conditioner (Stimu-Splitter, Med Lab Instruments, Ft. Collins, CO. USA). The following pharmacological agents were used in the present study: indomethacin, norepinephrine bitartrate, d,l-propranolol hydrochloride, yohimbine hydrochloride (Sigma Chemical Company, St. Louis, MO, USA), guanethidine monosulfate (Ciba Geigy, Summit, NJ, USA) and prazosin (Pfizer, Groton, CT, USA). All drugs were dissolved in distilled water and diluted subsequently in KHS. Indomethacin was initially dissolved in 200 mM Na,CO, and subsequently diluted in KHS such that the final bath concentration of Na,CO, was 20 FM. Data are presented as the means f SEM. The negative logarithm of the concentration of norepinephrine which induced 50% inhibition of the cholinergic ‘twitch’ response was designated as the pDL. The logarithm of the frequency of electrical field stimulation which elicited a 25% maximal acetylcholine response was designated as the log EC,,. This value was determined by regression analysis of the linear portion of the log frequency-response curve. Statistical comparisons were performed using unpaired Student’s ttests, with differences of P-C 0.05 being considered significant.
RESULTS Frequency-dependent contractile responses were elicited by electrical field stimulation with each individual response returning to resting baseline tone prior to induction of a subsequent response. In tissues treated with 10 PM norepinephrine, the frequencyresponse relationship for cholinergic nerve stimulation was significantly shifted 37-fold to the right and the maximal contractions were reduced significantly (Fig. 1). This inhibitory action of norepinephrine was reversed to a small but significant extent by yohimbine (Fig. 1) and was unaffected by prazosin (data not shown).
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Fig. 1 Modulation of neurally-mediated responses by norepinephrine in the guinea-pig trachea. Neurally-mediated cholinergic excitatory responses were induced by electrical field stimulation (0. I-80 Hz, 0.3 ms, 10 V for 5 s) in guinea-pig tracheal preparations treated with guanethidine (10 PM), propranolol (1 PM) and indomethacin (3 PM). Responses expressed as a percentage of the response to 1 mM acetylcholine (ACh) were obtained in the absence (0), presence (e) of norepinephrine (10 PM) or the combined presence of yohimbine (0.1 PM) and norepinephrine (10 pM) (W) or yohimbine (1 PM) and norepinephrine (10 PM) (A). Data represent the mean f SEM from four experiments.
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Fig. 2 Concentration-dependent inhibition by norepinephrine of neurally-mediated chohnergic responses in guinea-pig trachea: sensitivity to inhibition by yohimbine. Guinea-pig tracheal tissues treated with guanethidine (10 PM), propranolol (1 HIM)and indomethacin (3 pM) were subjected to continuous trains of intermittent electrical field stimulation (1 Hz, 0.3 ms pulse duration, 10 V amplitude, 5 s period at 60 s intervals) and ‘twitch’ responses were obtained. Control (time-control) twitch responses were treated with norepinephrine vehicle (distilled water; Cl) over the duration of the experiment. Other tissues received cumulative additions of norepinephrine. Preparations treated with norepinephrine were subjected to pretreatment with yohimbine vehicle (norepinephrine control) (10 pl distilled water; 0), 0.1 pM yohimbine (W) or 1 PM yohimbine (0). Twitch responses obtained following addition of norepinephrine or norepinephrine vehicle are expressed as a percentage of the magnitude of the twitch responses prior to norepinephrine administration. The pD, values for norepinephrine in the absence of yohimbine or presence of 0.1 PM and 1 PM yohimbine were 6.67 f 0.14, 5.98 f 0.12 and 5.54 f 0.22, respectively. Data represent the mean with 1 SEM from four experiments. * P < 0.05, unpaired Student’s r-test, compared to time-control response (0). + Pi 0.05, unpaired Student’s t-test, compared to norepinephrine control response (0).
The ability of norepinephrine to attenuate contractile responses induced by intermittent electrical field stimulation (1 Hz, 5 s) (‘twitch’ responses) was used to estimate the potency of norepinephrine in repressing neurally-mediated cholinergic responses. Norepinephrine induced a concentration-dependent inhibition of cholinergic ‘twitch’ responses with a pD, of
6.67 f 0.14 (Fig. 2). This action of norepinephrine was antagonized in a concentration-dependent manner by yohimbine (Fig. 2) to the extent that yohimbine shifted the norepinephrine concentration-response curve to the right in a parallel manner but did not affect the maximal inhibitory action of norepinephrine. Prazosin, on the other hand, failed to influence
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3 Concentration-dependent inhibition by norepinephrine of neurally-mediated cholinergic responses in guinea-pig trachea: sensitivity to inhibition by prazosin. Guinea-pig tracheal tissues treated with guanethidine (10 pi), propranolol (1 PM) and indomethacin (3 FM) were subjected to continuous trains of intermittent electrical field stimulation (1 Hz, 0.3 ms pulse duration, 10 V amplitude, 5 s period at 60 s intervals) and ‘twitch’ responses were obtained. Control (time-control) twitch responses were treated with norepinephrine vehicle (distilled water; Cl) over the duration of the experiment. Other tissues received cumulative additions of norepinephrine. Preparations treated with norepinephrine were subjected to pretreatment with prazosin vehicle (norepinephrine control) (10 ul distilled water; 0), 0.1 PM prazosin (m) or 1 PM prazosin (0). Twitch responses obtained following addition of norepinephrine or norepinephrine vehicle are expressed as a percentage of the magnitude of the twitch responses prior to norepinephrine administration. The pD, values for norepinephrine in the absence of prazosin or presence of 0.1 PM and I KM prazosin were 6.71 f 0.13, 6.65 f 0.24 and 6.44 f 0.08, respectively. Data represent the mean with 1 SEM from four experiments. * P< 0.05, unpaired Student’s t-test, compared to time-control response (0). Fig.
the concentration-response relationship for norepinephrine (Fig. 3). Neither yohimbine nor prazosin influenced the magnitude of the cholinergic ‘twitch’ responses. Further, norepinephrine per se had no effect on the resting tone of tracheal preparations.
DISCUSSION
In the present study, the inhibitory action of norepinephrine on electrical field stimulation-induced (i.e. neurally-mediated) cholinergic contractile responses was examined. Norepinephrine elicited concentrationdependent inhibition of ‘twitch’ responses induced by 1 Hz intermittent field stimulation. The pD, for this effect was 6.67 kO.14. This value is greater than the 5.89 f 0.12 reported by Kamikawa and Shimo3 for norepinephrine-induced inhibition of neurallymediated cholinergic responses. This difference most likely relates to the frequency-dependence of a-adrenoceptor-mediated presynaptic inhibition.4 Kamikawa and Shimo3 used a higher frequency of stimulation (8 Hz) than that employed in the present study (1 Hz). It also is worth noting that the potency of norepinephrine might have been underestimated in the present experiments since sites of loss, such as neuronal and extraneuronal uptake, were not blocked. Norepinephrine (10 l.~) caused a 37-fold rightward shift of the frequency-response curve for cholinergic
nerve-mediated contractile responses and also depressed the maximal contractile response induced by cholinergic nerve stimulation. These results differ from those obtained previously4 with an equimolar concentration of clonidine in which the agonist induced a la-fold rightward shift of the frequencyresponse curve but did not depress the maximal response to cholinergic nerve stimulation. The decreased efficacy of clonidine relative to norepinephrine may relate to the fact that the former agent acts as a partial agonist at a,-adrenoceptors whereas the latter acts as a full agonist at these same receptors. All experiments were conducted in tissues pretreated with the @-adrenoceptor antagonist, propranolol. Hence, the possibility can be excluded that the inhibitory effects of norepinephrine were related to an action on post-synaptic /Ladrenoceptors. That norepinephrine was acting via a,-adrenoceptors to inhibit cholinergic responses was indicated by the ability of the a,-adrenoceptor antagonist, yohimbine, to antagonize its inhibitory effects on cholinergic ‘twitch’ responses. In these studies, the nature of the antagonism appeared to be competitive, i.e. parallel rightward shift with no depression of the norepinephrine maximum. Such results are consistent with those described previously by Grundstrom et al.’ The a,-adrenoceptor antagonist, prazosin, had no effect on the modulation of neurally-mediated cholinergic responses by norepinephrine. Grundstriim and colleagues’ showed that the presynaptic inhibitory
Atypical Presynaptic a-Adrenoceptor
actions of norepinephrine in the guinea-pig trachea were only weakly antagonized by prazosin. It is conceivable that higher concentrations of prazosin than those used in the present study (i.e. greater than 1 PM) may have antagonized the actions of norepinephrine given that the pA, for prazosin as estimated by Grundstrlim et al was 5.89.’ Results of the present investigation using norepinephrine can be compared directly with those obtained previously using clonidine4 since identical experimental procedures were followed in both studies. The comparison reveals the following information. First, the presynaptic inhibitory actions of norepinephrine and clonidine are antagonized in a competitive manner by yohimbine, indicating that both exert their effects through activation of aZadrenoceptors. Second, the presynaptic inhibitory actions of clonidine but not norepinephrine are antagonized by prazosin. This observation, coupled with the documented lack of effect of the a,-adrenoceptor agonist, phenylephrine, on neurally-mediated cholinergic responses4 suggests that clonidine acts on an qadrenoceptor population distinct from that acted upon by norepinephrine. Alternatively, both clonidine and norepinephrine may act on the same population of cqadrenoceptors but prazosin may bind to an accessory site which hinders the attachment of clonidine but not norepinephrine. Whatever the case might be, it is apparent that the results of the present study, taken together with the results of previous studies, support the proposal of Vizi and colleagues6 for the existence
of different
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qadrenoceptors.
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Acknowledgements This work was supported by a grant from the National Heart Lung and Blood Institute (HL27025). Ralph J. Altiere is the recipient of a Research Career Development Award from the National Heart Lung and Blood Institute (HL 02356). References 1. Grundstrom N, Andersson R G G, Wikberg J E S. Prejunctional alpha 2 adrenoceptors inhibit contraction of tracheal smooth muscle by inhibiting cholinergic neurotransmission. Life Sci 1981; 28: 2981-2986. 2. Grundstrom N, Andersson R G G. Inhibition of the cholinergic neurotransmission in human airways via prejunctional alpha-2-adrenoceptors. Acta Physiol Stand 1985; 125: 513-517. 3. Kamikawa Y, Shimo Y. Inhibitory effects of sympathomimetic drugs on cholinergically mediated contractions of guinea-pig isolated tracheal muscle. J Pharm Pharmacol 1986; 38: 742-747. 4. Thompson D C, Diamond L, Altiere R J. Presynaptic alpha adrenoceptor modulation of neurally mediated cholinergic and nonadrenergic noncholinergic inhibitory responses in guinea pig trachea. J Pharmacol Exp Ther 1990, 254: 3063 11. 5. Timmermans P B M W M, Lam E, Van Zwieten P. The interaction between prazosin and clonidine at a-adrenoceptors in rats and cats. Eur.J Pharmacol 1979; 55: 57-66. 6. Vizi E S. Ludvie N. Rbnai A Z. Follv G. Dissociation of presynaptic cqadrenoceptors followmg prazosin administration: presynaptic effect of prazosin. Eur J Pharmacol 1983; 95: 287-290. 7. Altiere R J, Szarek J L, Diamond L. Neural control of relaxation in cat airways smooth muscle. J Appl Physiol: Respir Eviron Exercise Physiol 1984; 57: 15361544.
Date received: 8 July 1991 Date accepted: 12 November 1991
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Atypical Presynaptic Alpha-adrenoceptor Modulation of Neurally-mediated Cholinergic Responsesin Guinea-pig Tracheal Smooth Muscle D. C. Thompson*, L. Diamond, R, J. Altiere University of Colorado School of Pharmacy, Boulder, Colorado 80309-0297, USA
SUMMARY: Cholinergic excitatory nerves in guinea-pig trachea are subject to inhibitory control by presynaptic q-adrenoceptors. Recently, the nature of these receptors has come into question insofar as the presynaptic inhibitory effects of the oc,-adrenoceptor agonist, clonidine, in the guinea-pig trachea have been shown to be antagonized by the a,-adrenoceptor antagonist, yohimbine, as well as the a,-adrenoceptor antagonist, prazosin. This inhibitory action of prazosin had not been described previously in the airways and may relate to the use of norepinephrine rather than clonidine as the a-adrenoceptor agonist in earlier studies. The present study evaluates the susceptibility of norepinephrine-induced inhibition of neurally-mediated cholinergic excitatory responses to antagonism by prazosin and yohimbine under conditions identical to those which showed clonidine to be sensitive to these antagonists. In tissues pretreated with guanethidine, propranolol and indomethacin, norepinephrine (1 PM) induced a 37-fold rightward shift of the frequency-response curve for neurally-mediated cholinergic contractions which was reversed partially by pretreatment of tissues with yohimbine. Norepinephrine also caused a concentrationdependent inhibition of choline@ ‘twitch’ responses induced by intermittent (1 Hz) nerve stimulation. This action of norepinephrine was antagonized in a concentrationdependent manner by yohimbine but was unaffected by prazosin. These results indicate that in guinea-pig trachea the presynaptic inhibitory actions of norepinephrine on cholinergic nerves are mediated via classical q-adrenoceptors, i.e. receptors that can be blocked by yohimbine but not by prazosin. This distinguishes the action of norepinephrine from that shown previously for clonidine and provides support for the contention that these agonists do not act on the same population of u,-adrenoceptors.
Recent experiments in our laboratory have revealed that presynaptic a,-adrenoceptors in guinea-pig trachea exhibit a unique characteristic, i.e. they are sensitive to antagonism by the a,-adrenoceptor antagonist, prazosin, when clonidine, the a,-adrenoceptor agonist, is used for their activation.4 In general, the actions of a,-adrenoceptor agonists such as clonidine are blocked by a,-adrenoceptor antagonists but not by a,-adrenoceptor antagonists. However, in pithed rats in which heart rate has been elevated by stimulation of the nervi accelerantes, clonidine-induced bradycardia is inhibited by prazosin,5 suggesting that prazosin may act as an antagonist of presynaptic a*adrenoceptors on sympathetic nerves in this tissue. Vizi and colleagues6 also demonstrated an antagonistic effect of prazosin on the presynaptic inhibitory effect of the a,-adrenoceptor agonist, xylazine, on sympathetic nerves innervating the isolated rat vas deferens. No such antagonistic effect of prazosin was exerted against the non-specific a-adrenoceptor agonist, norepinephrine.6 Based upon these observations
INTRODUCTION Cholinergic nerves innervating airway smooth muscle have been shown to be inhibited presynaptically by aadrenoceptor agonists. I9293The presynaptic receptors mediating these inhibitory responses have been characterized through the use of subtype-specific antagonists (e.g. prazosin, the a,-adrenoceptor subtype-specific antagonist and yohimbine, the a,-adrenoceptorspecific antagonist). The response most typically studied is norepinephrine-induced inhibition of neurally-mediated choline& contraction.‘gZ Additional studies have examined the relative inhibitory potencies of various a-adrenoceptor subtype-specific agonists.3T4 Results of such studies have shown the presynaptic a-adrenoceptor on cholinergic nerves innervating guinea-pig tracheal smooth muscle to be of the a,-adrenoceptor subtype.
* For correspondence. 09524600/92/040251+
05 $08.00/O
251
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252
D. C. Thompson et al
and results obtained using the combination of prazosin and yohimbine, Vizi and colleagues6 proposed the existence of two different presynaptic a?-adrenoceptors. Previous studies investigating presynaptic a-adrenoceptors on cholinergic nerves in guinea-pig airways have used norepinephrine as the agonist and have found prazosin to possess little antagonistic activity.’ In light of the results of Vizi et al, it is conceivable that the disparate antagonistic actions of prazosin against clonidine4 and norepinephrine’ in the guinea-pig trachea are related to the agonist itself. The present study was undertaken to re-examine the inhibitory effects of norepinephrine on neurally-mediated cholinergic nerve responses in the guinea-pig trachea and to assess the susceptibility of these effects to antagonism by prazosin and yohimbine.
MATERIALS
AND METHODS
Male guinea-pigs, weighing between 350 and 500 g, were anaesthetized with pentobarbital sodium (60 mg/ kg, i.p.). The trachea was removed and placed in Krebs-Henseleit solution (KHS) of the following composition (mM): NaCl, 118.2; KCl, 4.74; CaCl,, 2.54; KH,PO,, 1.19; NaHCO,, 26.2; MgSO,, 1.19; D( + )-glucose, 11.1; sodium ascorbate, 1.O; CaNa,EDTA, 0.03. Stainless steel tissue hooks were placed through the lumen of ring segments (46 mm in length) and, upon mounting in the organ bath, care was taken to ensure that the tissue hooks were not resting upon the smooth muscle layer. All preparations were mounted between platinum plate electrodes7 in 10 ml glass jacketed organ baths containing KHS maintained at 37°C and gassed with 5% CO, in 0,. An initial load of 3 g was placed on each segment. Tissues were allowed to equilibrate for a period of 60min, during which time the bathing solution was exchanged at 10 min intervals. Acetylcholine (1 mM) was then applied to all tissues. Upon attainment of the peak response, a 60-min wash-out period ensued after which the experimental procedures were initiated. Each trachea was disected to yield four ring segments such that one segment acted as a control (no treatment) and the other three were subjected to treatment. Responses were measured isometrically using forcedisplacement transducers (Grass FT.03c) and displayed on a polygraph (Grass model 79D). All preparations were continually exposed to propranolol (1 C(M) and guanethidine (10 PM) to block adrenergically-mediated inhibitory responses and indomethacin (3 PM) to prevent the development of spontaneous resting tone. Tissues were subjected to continuous intermittent electrical field stimulation (1 Hz, 0.3 ms pulse duration, 10 V pulse amplitude, 5 s stimulation period at 60 s intervals) following wash-
out of the acetylcholine response. A period of 30 min was allowed for the ‘twitch’ responses to become reproducible after which tissues were incubated with vehicle (10 pl distilled water) or an a-adrenoceptor antagonist (prazosin, a,-adrenoceptor antagonist; a,-adrenoceptor antagonist). After yohimbine, 15 min, norepinephrine or norepinephrine vehicle (equivalent volume of distilled water) was administered to tissues in a cumulative manner. In addition. frequency-response curves for cholinergic nerve stimulation were induced in resting tissues by electrical field stimulation (0.1-80 Hz, 0.3 ms pulse duration, 10 V pulse amplitude, 5 s stimulation period at 60 s intervals). Electrical field stimulation was applied to preparations using square wave pulses generated by a Grass S-88 stimulator in series with a signal conditioner (Stimu-Splitter, Med Lab Instruments, Ft. Collins, CO. USA). The following pharmacological agents were used in the present study: indomethacin, norepinephrine bitartrate, d,l-propranolol hydrochloride, yohimbine hydrochloride (Sigma Chemical Company, St. Louis, MO, USA), guanethidine monosulfate (Ciba Geigy, Summit, NJ, USA) and prazosin (Pfizer, Groton, CT, USA). All drugs were dissolved in distilled water and diluted subsequently in KHS. Indomethacin was initially dissolved in 200 mM Na,CO, and subsequently diluted in KHS such that the final bath concentration of Na,CO, was 20 FM. Data are presented as the means f SEM. The negative logarithm of the concentration of norepinephrine which induced 50% inhibition of the cholinergic ‘twitch’ response was designated as the pDL. The logarithm of the frequency of electrical field stimulation which elicited a 25% maximal acetylcholine response was designated as the log EC,,. This value was determined by regression analysis of the linear portion of the log frequency-response curve. Statistical comparisons were performed using unpaired Student’s ttests, with differences of P-C 0.05 being considered significant.
RESULTS Frequency-dependent contractile responses were elicited by electrical field stimulation with each individual response returning to resting baseline tone prior to induction of a subsequent response. In tissues treated with 10 PM norepinephrine, the frequencyresponse relationship for cholinergic nerve stimulation was significantly shifted 37-fold to the right and the maximal contractions were reduced significantly (Fig. 1). This inhibitory action of norepinephrine was reversed to a small but significant extent by yohimbine (Fig. 1) and was unaffected by prazosin (data not shown).
Atypical Presynaptic a-Adrenoceptor
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Fig. 1 Modulation of neurally-mediated responses by norepinephrine in the guinea-pig trachea. Neurally-mediated cholinergic excitatory responses were induced by electrical field stimulation (0. I-80 Hz, 0.3 ms, 10 V for 5 s) in guinea-pig tracheal preparations treated with guanethidine (10 PM), propranolol (1 PM) and indomethacin (3 PM). Responses expressed as a percentage of the response to 1 mM acetylcholine (ACh) were obtained in the absence (0), presence (e) of norepinephrine (10 PM) or the combined presence of yohimbine (0.1 PM) and norepinephrine (10 pM) (W) or yohimbine (1 PM) and norepinephrine (10 PM) (A). Data represent the mean f SEM from four experiments.
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Fig. 2 Concentration-dependent inhibition by norepinephrine of neurally-mediated chohnergic responses in guinea-pig trachea: sensitivity to inhibition by yohimbine. Guinea-pig tracheal tissues treated with guanethidine (10 PM), propranolol (1 HIM)and indomethacin (3 pM) were subjected to continuous trains of intermittent electrical field stimulation (1 Hz, 0.3 ms pulse duration, 10 V amplitude, 5 s period at 60 s intervals) and ‘twitch’ responses were obtained. Control (time-control) twitch responses were treated with norepinephrine vehicle (distilled water; Cl) over the duration of the experiment. Other tissues received cumulative additions of norepinephrine. Preparations treated with norepinephrine were subjected to pretreatment with yohimbine vehicle (norepinephrine control) (10 pl distilled water; 0), 0.1 pM yohimbine (W) or 1 PM yohimbine (0). Twitch responses obtained following addition of norepinephrine or norepinephrine vehicle are expressed as a percentage of the magnitude of the twitch responses prior to norepinephrine administration. The pD, values for norepinephrine in the absence of yohimbine or presence of 0.1 PM and 1 PM yohimbine were 6.67 f 0.14, 5.98 f 0.12 and 5.54 f 0.22, respectively. Data represent the mean with 1 SEM from four experiments. * P < 0.05, unpaired Student’s r-test, compared to time-control response (0). + Pi 0.05, unpaired Student’s t-test, compared to norepinephrine control response (0).
The ability of norepinephrine to attenuate contractile responses induced by intermittent electrical field stimulation (1 Hz, 5 s) (‘twitch’ responses) was used to estimate the potency of norepinephrine in repressing neurally-mediated cholinergic responses. Norepinephrine induced a concentration-dependent inhibition of cholinergic ‘twitch’ responses with a pD, of
6.67 f 0.14 (Fig. 2). This action of norepinephrine was antagonized in a concentration-dependent manner by yohimbine (Fig. 2) to the extent that yohimbine shifted the norepinephrine concentration-response curve to the right in a parallel manner but did not affect the maximal inhibitory action of norepinephrine. Prazosin, on the other hand, failed to influence
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CM)]
3 Concentration-dependent inhibition by norepinephrine of neurally-mediated cholinergic responses in guinea-pig trachea: sensitivity to inhibition by prazosin. Guinea-pig tracheal tissues treated with guanethidine (10 pi), propranolol (1 PM) and indomethacin (3 FM) were subjected to continuous trains of intermittent electrical field stimulation (1 Hz, 0.3 ms pulse duration, 10 V amplitude, 5 s period at 60 s intervals) and ‘twitch’ responses were obtained. Control (time-control) twitch responses were treated with norepinephrine vehicle (distilled water; Cl) over the duration of the experiment. Other tissues received cumulative additions of norepinephrine. Preparations treated with norepinephrine were subjected to pretreatment with prazosin vehicle (norepinephrine control) (10 ul distilled water; 0), 0.1 PM prazosin (m) or 1 PM prazosin (0). Twitch responses obtained following addition of norepinephrine or norepinephrine vehicle are expressed as a percentage of the magnitude of the twitch responses prior to norepinephrine administration. The pD, values for norepinephrine in the absence of prazosin or presence of 0.1 PM and I KM prazosin were 6.71 f 0.13, 6.65 f 0.24 and 6.44 f 0.08, respectively. Data represent the mean with 1 SEM from four experiments. * P< 0.05, unpaired Student’s t-test, compared to time-control response (0). Fig.
the concentration-response relationship for norepinephrine (Fig. 3). Neither yohimbine nor prazosin influenced the magnitude of the cholinergic ‘twitch’ responses. Further, norepinephrine per se had no effect on the resting tone of tracheal preparations.
DISCUSSION
In the present study, the inhibitory action of norepinephrine on electrical field stimulation-induced (i.e. neurally-mediated) cholinergic contractile responses was examined. Norepinephrine elicited concentrationdependent inhibition of ‘twitch’ responses induced by 1 Hz intermittent field stimulation. The pD, for this effect was 6.67 kO.14. This value is greater than the 5.89 f 0.12 reported by Kamikawa and Shimo3 for norepinephrine-induced inhibition of neurallymediated cholinergic responses. This difference most likely relates to the frequency-dependence of a-adrenoceptor-mediated presynaptic inhibition.4 Kamikawa and Shimo3 used a higher frequency of stimulation (8 Hz) than that employed in the present study (1 Hz). It also is worth noting that the potency of norepinephrine might have been underestimated in the present experiments since sites of loss, such as neuronal and extraneuronal uptake, were not blocked. Norepinephrine (10 l.~) caused a 37-fold rightward shift of the frequency-response curve for cholinergic
nerve-mediated contractile responses and also depressed the maximal contractile response induced by cholinergic nerve stimulation. These results differ from those obtained previously4 with an equimolar concentration of clonidine in which the agonist induced a la-fold rightward shift of the frequencyresponse curve but did not depress the maximal response to cholinergic nerve stimulation. The decreased efficacy of clonidine relative to norepinephrine may relate to the fact that the former agent acts as a partial agonist at a,-adrenoceptors whereas the latter acts as a full agonist at these same receptors. All experiments were conducted in tissues pretreated with the @-adrenoceptor antagonist, propranolol. Hence, the possibility can be excluded that the inhibitory effects of norepinephrine were related to an action on post-synaptic /Ladrenoceptors. That norepinephrine was acting via a,-adrenoceptors to inhibit cholinergic responses was indicated by the ability of the a,-adrenoceptor antagonist, yohimbine, to antagonize its inhibitory effects on cholinergic ‘twitch’ responses. In these studies, the nature of the antagonism appeared to be competitive, i.e. parallel rightward shift with no depression of the norepinephrine maximum. Such results are consistent with those described previously by Grundstrom et al.’ The a,-adrenoceptor antagonist, prazosin, had no effect on the modulation of neurally-mediated cholinergic responses by norepinephrine. Grundstriim and colleagues’ showed that the presynaptic inhibitory
Atypical Presynaptic a-Adrenoceptor
actions of norepinephrine in the guinea-pig trachea were only weakly antagonized by prazosin. It is conceivable that higher concentrations of prazosin than those used in the present study (i.e. greater than 1 PM) may have antagonized the actions of norepinephrine given that the pA, for prazosin as estimated by Grundstrlim et al was 5.89.’ Results of the present investigation using norepinephrine can be compared directly with those obtained previously using clonidine4 since identical experimental procedures were followed in both studies. The comparison reveals the following information. First, the presynaptic inhibitory actions of norepinephrine and clonidine are antagonized in a competitive manner by yohimbine, indicating that both exert their effects through activation of aZadrenoceptors. Second, the presynaptic inhibitory actions of clonidine but not norepinephrine are antagonized by prazosin. This observation, coupled with the documented lack of effect of the a,-adrenoceptor agonist, phenylephrine, on neurally-mediated cholinergic responses4 suggests that clonidine acts on an qadrenoceptor population distinct from that acted upon by norepinephrine. Alternatively, both clonidine and norepinephrine may act on the same population of cqadrenoceptors but prazosin may bind to an accessory site which hinders the attachment of clonidine but not norepinephrine. Whatever the case might be, it is apparent that the results of the present study, taken together with the results of previous studies, support the proposal of Vizi and colleagues6 for the existence
of different
presynaptic
qadrenoceptors.
Modulation
of Cholinergic Responses
255
Acknowledgements This work was supported by a grant from the National Heart Lung and Blood Institute (HL27025). Ralph J. Altiere is the recipient of a Research Career Development Award from the National Heart Lung and Blood Institute (HL 02356). References 1. Grundstrom N, Andersson R G G, Wikberg J E S. Prejunctional alpha 2 adrenoceptors inhibit contraction of tracheal smooth muscle by inhibiting cholinergic neurotransmission. Life Sci 1981; 28: 2981-2986. 2. Grundstrom N, Andersson R G G. Inhibition of the cholinergic neurotransmission in human airways via prejunctional alpha-2-adrenoceptors. Acta Physiol Stand 1985; 125: 513-517. 3. Kamikawa Y, Shimo Y. Inhibitory effects of sympathomimetic drugs on cholinergically mediated contractions of guinea-pig isolated tracheal muscle. J Pharm Pharmacol 1986; 38: 742-747. 4. Thompson D C, Diamond L, Altiere R J. Presynaptic alpha adrenoceptor modulation of neurally mediated cholinergic and nonadrenergic noncholinergic inhibitory responses in guinea pig trachea. J Pharmacol Exp Ther 1990, 254: 3063 11. 5. Timmermans P B M W M, Lam E, Van Zwieten P. The interaction between prazosin and clonidine at a-adrenoceptors in rats and cats. Eur.J Pharmacol 1979; 55: 57-66. 6. Vizi E S. Ludvie N. Rbnai A Z. Follv G. Dissociation of presynaptic cqadrenoceptors followmg prazosin administration: presynaptic effect of prazosin. Eur J Pharmacol 1983; 95: 287-290. 7. Altiere R J, Szarek J L, Diamond L. Neural control of relaxation in cat airways smooth muscle. J Appl Physiol: Respir Eviron Exercise Physiol 1984; 57: 15361544.
Date received: 8 July 1991 Date accepted: 12 November 1991
