Inhibitory effects of catecholamines on cholinergically and non-cholinergically mediated contractions of guinea-pig isolated bronchial muscle

The actions of catecholamines on the responses evoked by electrical field stimulation or by acetylcholine and substance P in guinea-pig bronchial strip chain have been examined. Electrical field stimulation evoked a biphasic contraction, consisting of a cholinergically-mediated fast contraction followed by a non-cholinergically-mediated slow contraction. All catecholamines tested caused a concentration-dependent reduction in the height of the biphasic contraction, where non-cholinergic contractions were more potently inhibited. The inhibitory effect of isoprenaline was largely prevented by propranolol (2 microM) alone, whereas those of noradrenaline and adrenaline were prevented by treatment with both propranolol (2 microM) and yohimbine (2 microM). The inhibitory effect of dopamine was unaffected either by propranolol (2 microM), yohimbine (2 microM) or haloperidol (10 microM). Submaximal contractions of bronchial muscle evoked by exogenous acetylcholine (2 microM) or substance P (0.2 microM) were also inhibited by catecholamines, except dopamine, but the effects were antagonized by propranolol (2 microM) alone. The results suggest that in guinea-pig isolated bronchial muscle, catecholamines can inhibit both cholinergic and non-cholinergic excitatory neurotransmissions not only by postjunctional beta-adrenoceptors but also by prejunctional alpha 2-adrenoceptors.     

Stimulation of alpha 1-adrenoceptors increases electrically evoked [3H]acetylcholine release from the rat phrenic nerve

The modulation by sympathomimetic amines of the electrically evoked [3H]acetylcholine release from the motor nerve was investigated. Phenylephrine (10 mumol/l), alpha-methylnoradrenaline (10 mumol/l) and adrenaline (1 mumol/l) enhanced the electrically evoked [3H]acetylcholine release from the rat phrenic nerve. The enhancing effect of phenylephrine was completely prevented by low concentrations of prazosin (0.1 or 0.01 mumol/l) whereas a high concentration of yohimbine (1 mumol/l) was necessary to abolish the effect of phenylephrine. The simultaneous blockade of alpha- and beta-adrenoceptors, i.e. propranolol (0.1 mumol/l) together with prazosin (0.01 mumol/l) or yohimbine (1 mumol/l), was required to abolish the enhancing effect of alpha-methylnoradrenaline. Likewise, the enhancing effect of adrenaline could be abolished only by a combination of two antagonists (propranolol together with yohimbine or prazosin). Neither clonidine nor oxymetazoline (1 or 10 mumol/l) modulated the evoked [3H]acetylcholine release. The experiments showed the existence of alpha-adrenoceptors which are present on the motor nerve and whose stimulation mediates an increase in evoked transmitter release. The different potencies found for prazosin and yohimbine indicate that an alpha 1-subtype of the receptors was involved. Increased sympathetic activity may facilitate neuromuscular transmission by stimulation of presynaptic alpha- and beta-adrenoceptors.     

Alpha 2-adrenoceptor mechanisms in guinea-pig trachea

In the presence of propranolol tracheal strips from guinea-pigs were markedly contracted by norepinephrine and clonidine but only slightly by phenylephrine. The contractile responses to clonidine and norepinephrine were inhibited by yohimbine but not by prazosin. The specific binding of [3H]p-aminoclonidine to the microsomal fractions from guinea-pig tracheal muscle was much greater than that of [125I]BE2254. These results indicate that alpha 2-adrenoceptors are the predominant subtype of alpha-adrenoceptor in the guinea-pig trachea. The response to norepinephrine was abolished by cyclooxygenase inhibitors, suggesting the release of excitatory prostaglandins by norepinephrine. These results raise the possibility that norepinephrine interacts with alpha 2-adrenoceptors and releases prostaglandins to contract tracheal muscle.     

Interaction between clonidine and histamine on the guinea-pig isolated trachea

In experiments on guinea-pig isolated tracheal spirals, clonidine, in concentrations of 10^?6 to 3 times 10^?4 M. had a contracting effect which was strongly inhibited by prazosin but not significantly modified by yohimbine. Moreover, clonidine (3 times 10^?5 to 3 times 10^?4 M) potentiated histamine-induced contractions; this latter effect was inhibited specifically by α₁-adrenoceptor antagonists (e.g. prazosin, AR-C 239) but unmodified by yohimbine, nicardipine or agents acting on the arachidonic acid cascade. It would therefore appear that clonidine in high concentrations contracts the guinea-pig trachea by stimulating α₁-adrenoceptors and that, contrary to what has been reported with other animal species, notably the dog, the guinea-pig trachea is devoid of α₂-adrenoceptors that mediate contractions.     

Inhibitory effects of imidazolines on histamine liberation from human leukocytes and on tracheal smooth muscle tone.

Antigen-induced IgE-mediated release of histamine from human leukocytes, an in vitro model of allergic reactions, was blocked by imidazole and imidazole-compounds such as oxymetazoline and clonidine. The H-2-antihistamines antagonized this effect of imidazolines. Alpha- and H-1-receptor blocking agents did not antagonize the effect. The contractile effects of the imidazolines were tested on tracheal preparations from the cow and guinea-pig. Imidazole was found to be a rather potent contracting agent, while oxymetazoline only caused weak contractions. Clonidine relaxed the tracheal muscles, when used in the concentration range which were inhibitory in the leukocyte experiments. The contractions caused by imidazolines were non-competitively inhibited by clemastine, while the relaxing effects were blocked by a combination of propranolol, phentolamine and cimethidine. The results suggest that imidazolines which inhibit histamine release and relax bronchial smooth muscles may be of therapeutic importance in the treatment of human allergic disorders.     

Subtype classification of the presynaptic alpha-adrenoceptors which regulate [3H]-noradrenaline secretion in guinea-pig isolated urethra.

1. The following experiments were carried out to investigate the presence and type of functional presynaptic receptors in adrenergic nerves of the guinea-pig urethra. 2. The urethra from male guinea-pigs was incubated with [3H]-noradrenaline and superfused with Tyrode solution in vitro. The fractional secretion of [3H]-noradrenaline evoked by 300 electrical pulses was measured. 3. The [3H]-noradrenaline secretion was positively frequency-dependent, yielding a half-maximal secretion at 8 +/- 5 Hz. Stimulation was usually applied at 5 Hz. 4. The [3H]-noradrenaline secretion was not altered by noradrenaline (1 or 100 microM), norephedrine (1 microM), isoprenaline (0.1 microM), 5-hydroxytryptamine (10 microM), oxotremorine (10 microM), adenosine (0.2 mM), propranolol (1 microM), atropine (1 microM) or 8-phenyltheophylline (10 microM). 5. The [3H]-noradrenaline secretion was enhanced by clonidine (3 microM), chlorpromazine (10 microM), metitepine (1 microM), 4-aminopyridine (0.5 mM), tetraethylammonium (2 mM), 3-isobutyl-1-methylxanthine (4 mM), 8-bromo cyclic AMP (1 mM) and forskolin (25 microM). 6. The alpha-adrenoceptor antagonists rauwolscine, yohimbine, phentolamine, prazosin and AR-C 239 maximally enhanced the [3H]-noradrenaline secretion to about 300% of control. The partial alpha-adrenoceptor agonist oxymetazoline maximally enhanced the secretion to about 200% of control. The order of apparent EC50 values was rauwolscine less than yohimbine less than phentolamine less than oxymetazoline less than prazosin less than AR-C 239.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of the beta-receptor mediated effect on slow-contracting skeletal muscle in vitro.

Subtetanic contractions of the guinea-pig isolated soleus, a slow-contracting skeletal muscle, were evoked by transmural field-stimulation. Isoprenaline caused a dose-dependent depression of the contractions. This effect was inhibited by propranolol and H 35/25 (1-(p-tolyl-2-isopropylamino-1-propanol) but not by practolol. Similar results were obtained for terbutaline. Tazolol and H 80/62 (1-isopropylamino-3-(p-hydroxyphenoxy)-2-propanol (HCl), selective beta1-agonists, had no effect per se but inhibited the effect of terbutaline. Adrenaline, noradrenaline, and dopamine all caused a dose-dependent decrease in the force of the soleus contractions, their potencies being in that order. Tyramine did not appreciably affect the contractions nor did it inhibit the effect of terbutaline. Pretreatment with reserpine, if anything, increased the response to terbutaline. It is concluded, in conformity with previous in vivo studies, that the adrenergic receptor mediating the effect on the soleus muscle contractions is of the beta2-type. Indirect sympathomimetic effects do not contribute to the responses observed on the isolated soleus muscle.     

Inhibitory Effects of Imidazolines on Histamine Liberation from Human Leukocytes and on Tracheal Smooth Muscle Tone

Abstract Antigen-induced IgE-mediated release of histamine from human leukocytes, an in vitro model of allergic reactions, was blocked by imidazole and imidazole-compounds such as oxymetazoline and clonidine. The H-2-antihistamines antagonized this effect of imidazolines. Alpha- and H-1-receptor blocking agents did not antagonize the effect. The contractile effects of the imidazolines were tested on tracheal preparations from the cow and guinea-pig. Imidazole was found to be a rather potent contracting agent, while oxymetazoline only caused weak contractions. Clonidine relaxed the tracheal muscles, when used in the concentration range which were inhibitory in the leukocyte experiments. The contractions caused by imidazolines were non-competitively inhibited by clemastine, while the relaxing effects were blocked by a combination of propranolol, phentolamine and cimethidine. The results suggest that imidazolines which inhibit histamine release and relax bronchial smooth muscles may be of therapeutic importance in the treatment of human allergic disorders.     

Analysis of the β-receptor mediated effect on slow-contracting skeletal muscle in vitro

Subtetanic contractions of the guinea-pig isolated soleus, a slow-contracting skeletal muscle, were evoked by transmural field-stimulation. Isoprenaline caused a dose-dependent depression of the contractions. This effect was inhibited by propranolol and H 35/25 (1-(p-tolyl-2-isopropylamino-1-propanol) but not by practolol. Similar results were obtained for terbutaline. Tazolol and H 80/62 (1-isopropylamino-3-(p-hydroxyphenoxy)-2-propanol HCl), selective β₁-agonists, had no effect per se but inhibited the effect of terbutaline. Adrenaline, noradrenaline, and dopamine all caused a dose-dependent decrease in the force of the soleus contractions, their potencies being in that order. Tyramine did not appreciably affect the contractions nor did it inhibit the effect of terbutaline. Pretreatment with reserpine, if anything, increased the response to terbutaline. It is concluded, in conformity with previous in vivo studies, that the adrenergic receptor mediating the effect on the soleus muscle contractions is of the β₂-type. Indirect sympathomimetic effects do not contribute to the responses observed on the isolated soleus muscle.     

Pharmacological characterization of the adrenoceptors in the muscularis mucosae of the guinea-pig esophagus

Pharmacological characteristics of the post-junctional adrenoceptors in the muscularis mucosae of the guinea-pig esophagus were examined in vitro. In the presence of propranolol (3 microM), all catecholamines, phenylephrine and methoxamine produced a weak contraction of the isolated muscularis mucosae. The order of potency of the contractile response was adrenaline greater than noradrenaline greater than methoxamine greater than phenylephrine and much weaker isoproterenol, dopamine and clonidine. The contractile responses to noradrenaline (10 microM) and adrenaline (10 microM) were markedly inhibited by phentolamine (3 microM), prazosin (3 microM), indomethacin (1 microM), aspirin (100 microM) and polyphloretin (30 microM), and slightly by yohimbine (3 microM). On the other hand, all catecholamines and terbutaline produced relaxation of the isolated muscularis mucosae which was maximally contracted with carbachol (3 microM) in the presence of phentolamine (10 microM), in a concentration-dependent manner. The order of potency of the inhibitory response was isoproterenol greater than noradrenaline greater than adrenaline and much weaker terbutaline and dopamine. The inhibitory responses to catecholamines were competitively antagonized by three beta blockers, being in the following order of potency as estimated by their pA2 values: propranolol greater than atenolol much greater than butoxamine. The pretreatment of the tissue with indomethacin (1 microM) did not modify the inhibitory responses to catecholamines. These results suggest that the guinea-pig esophageal muscularis mucosae has excitatory alpha 1 adrenoceptors and inhibitory beta 1 adrenoceptors, and the alpha 1, but not beta 1, adrenoceptors may link with the endogenous arachidonic acid cascade.     

Inhibition of adrenomedullary catecholamine release by propranolol isomers and clonidine involving mechanisms unrelated to adrenoceptors.

1 Transmural electrical stimulation (10 Hz, 40 V, 1 ms for 60s) increased total catecholamine secretion from perfused cat adrenal glands; this response was enhanced by neostigmine and inhibited by mecamylamine, suggesting that release of acetylcholine from splanchnic nerve terminals was stimulating nicotinic receptors and enhancing catecholamine secretion. 2 Isoprenaline, (-)-propranolol and (+)-propranolol (10(-7)-10(-5)M) inhibited the electrically-evoked secretory response by 40-70%; similar reductions were obtained with clonidine and yohimbine. Neither, (+)-propranolol nor (-)-propranolol inhibited K-evoked secretion from cat adrenals; in contrast, nimodipine potently inhibited it (IC50 = 24 nM). 3 Either, racemic propranolol or the (+)- or (-)-isomers (1-10 microM) equally inhibited [3H]-noradrenaline release evoked by nicotine or acetylcholine from cultured bovine adrenal chromaffin cells; clonidine (10 microM) inhibited secretion by 50% and yohimbine or isoprenaline did not affect it. 4 The results indicate that adrenomedullary catecholamine release evoked by splanchnic nerve stimulation is not modulated by alpha- or beta-adrenoceptors and suggest that propranolol may inhibit secretion by blocking ion fluxes through the acetylcholine receptor ionophore. Clonidine may inhibit secretion by this same mechanism, and/or by interfering with some intracellular event in the secretory mechanism.     

Atypical presynaptic alpha-adrenoceptor modulation of neurally-mediated cholinergic responses in guinea-pig tracheal smooth muscle

Cholinergic excitatory nerves in guinea-pig trachea are subject to inhibitory control by presynaptic α₂-adrenoceptors. Recently, the nature of these receptors has come into question insofar as the presynaptic inhibitory effects of the α₂-adrenoceptor agonist, clonidine, in the guinea-pig trachea have been shown to be antagonized by the α₂-adrenoceptor antagonist, yohimbine, as well as the α₁-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 α-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 μm) 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 concentration-dependent inhibition of cholinergic ‘twitch’ responses induced by intermittent (1 Hz) nerve stimulation. This action of norepinephrine was antagonized in a concentration-dependent 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 α₂-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 α₂-adrenoceptors.     

Effects of alpha-adrenoceptor antagonists on clonidine-induced inhibition of insulin secretion by isolated pancreatic islets.

The effects of clonidine, yohimbine, corynanthine and prazosin on glucose-induced insulin secretion by incubated or perifused mouse pancreatic islets were investigated. Clonidine (0.1 microM) inhibited glucose-induced insulin secretion alone and in the presence of yohimbine (0.1 microM), corynanthine (10 microM) or prazosin (1 microM). In higher concentrations, yohimbine (1-10 microM) antagonized the inhibitory effect of clonidine (0.1 microM) upon glucose-induced insulin secretion by incubated islets and by perifused islets. The results support the view that adrenergic inhibition of insulin secretion is mediated by alpha 2-adrenoceptors on pancreatic beta-cells.     

Presynaptic muscarinic and alpha-adrenoceptor-mediated regulation of GABA release from myenteric neurones of the guinea-pig small intestine.

The effects of cholinomimetic and sympathomimetic drugs on the release of [3H]-gamma-aminobutyric acid ([3H]-GABA) evoked by high K+ from the isolated small intestine of the guinea-pig were investigated, in the presence of tetrodotoxin. Acetylcholine and oxotremorine, at concentrations ranging from 10(-9) to 10(-6) M inhibited the evoked release of [3H]-GABA in a concentration-dependent manner, while nicotine was without effect. Scopolamine and pirenzepine inhibited the effect of oxotremorine, while hexamethonium had no effect. The IC50 values for scopolamine and pirenzepine of the oxotremorine (3 X 10(-8) M)-induced inhibition were 1.02 X 10(-9) M and 9.78 X 10(-10) M, respectively. Noradrenaline, but not isoprenaline inhibited the evoked release of [3H]-GABA. Clonidine (10(-10)-10(-6) M) reduced the evoked release of [3H]-GABA in a concentration-dependent manner, but phenylephrine had no effect. The inhibitory effect of clonidine was antagonized by yohimbine but not by prazosin. These findings provide evidence for the localization of M1-muscarinic and alpha 2-adrenoceptors on GABAergic nerve terminals and their involvement in the presynaptic control of the release of GABA from the guinea-pig small intestine.     

Dual action of clonidine on ethanol-induced gastric lesions: is the imidazoline-preferring receptor involved?

The imidazole alpha 2-adrenoceptor agonist, clonidine, exerts a dual action on ethanol-induced gastric lesions. At lower doses, it has a gastroprotective effect which is also seen with two other alpha 2-adrenoceptor agonists - the catecholamine, alpha-methyldopa and the guanidine, guanabenz. The gastroprotective action of the three drugs is prevented by the selective alpha 2-adrenoceptor antagonist, yohimbine, suggesting that the action is mediated by alpha 2-adrenoceptors. However, at higher doses clonidine aggravates ethanol-induced gastric lesions, an effect also seen with another imidazole, oxymetazoline. The aggravating action is not prevented by yohimbine and is not seen with alpha-methyldopa and guanabenz. This suggests that it involves a receptor/mechanism other than alpha 2- possibly an imidazoline-preferring receptor but further work, including radioligand binding studies, is needed to confirm this.     

Interactions between the effects of endothelin-1, clonidine and yohimbine on electrically-induced contractions in rat vas deferens.

1. The relationship between endothelin-1(ET-1)-induced effects on the contractile responses of epididymal portion of rat vas deferens elicited by field electrical stimulation (FES: 80 V, 1 msec, 0.1 Hz) and the effects of the alpha 2-adrenoceptor agonist clonidine and the alpha 2-adrenoceptor antagonist yohimbine were studied. 2. ET-1 (0.01 nM-0.1 microM) concentration-dependently increased the FES-induced contractions. 3. ET-1 (0.1 nM-0.1 microM) reversed the inhibitory effect of clonidine on the FES-evoked contractions whereas ET-1 applied before clonidine exerted a dual effect on the clonidine-induced inhibition of the FES-evoked contractions. 4. The ET-1-induced enhancement of FES-induced contractions was potentiated in the presence of 1 microM yohimbine and was not observed at all in the presence of 10 microM yohimbine. Yohimbine, applied at concentrations of 1 and 10 microM exerted similar blocking effects on the alpha 1-adrenoceptor agonistic effects of phenylephrine. However, yohimbine at a concentration of 10 microM markedly potentiated the contractile effect of exogenous adenosine 5'-triphosphate (ATP), 30 microM. Tetrodotoxin abolished this effect of yohimbine. 5. The results presented here suggest the existence of modulating interactions between the ET-1-evoked increase of FES-induced contractions of rat vas deferens and the alpha 2-adrenoceptor drugs clonidine and yohimbine.     

REVERSAL OF ADRENALINE-INDUCED CONTRACTION IN THE RABBIT PULMONARY ARTERY BY CLONIDINE

Characterization of clonidine which reversed the adrenaline-induced contractions was assessed using the rabbit pulmonary arterial segment. Clonidine in high concentrations (10(-5)-10(-4) mol/l) contracted this vascular tissue. Nevertheless, an adrenaline (3 X 10(-6)mol/l)-induced contraction, which can be markedly depressed by phentolamine, was reversed to relaxation by pre-addition of 3 X 10(-5)mol/l clonidine. This reversal was abolished by 10(-5) mol/l propranolol, a beta-adrenoceptor antagonist. The cumulative concentration-contraction curve for adrenaline was shifted to the right by 10(-5) mol/l clonidine without further alteration at 10(-4) mol/l, suggesting a partial agonistic feature of clonidine for alpha-adrenoceptors. Contractions evoked by 5 X 10(-5) mol/l clonidine alone were effectively reduced by prazosin (IC50 = 1.7 X 10(-7) mol/l) or yohimbine, (IC50 = 3.3 X 10(-5) mol/l) and those by phenylephrine (10(-5) mol/l) were suppressed as well (prazosin, IC50 = 3.4 X 10(-8) mol/l; yohimbine, IC50 = 5.0 X 10(-6) mol/l). However, the depolarization-induced contractions with 30 mmol/l KCl were virtually unaltered by both antagonists. These results suggest that the reversal action of clonidine on the adrenaline-induced contraction is primarily mediated by a partial agonistic property for postsynaptic alpha 1-adrenoceptors.     

The contractions induced in rat and guinea-pig aortic strips by the alpha 2-adrenoceptor selective agonists B-HT 920 and UK 14,304 are mediated by alpha 1-adrenoceptors

The alpha-adrenergic action of the selective alpha 2-adrenoceptor agonists B-HT 920 and UK 14,304 was investigated on helically cut preparations of rat and guinea-pig isolated aorta. The alpha 1-adrenoceptor selective stimulant (-)-phenylephrine was included for comparison. All agonists induced concentration-dependent contractions in both preparations. Calcium entry blockade by D 600 almost abolished the contractions evoked by B-HT 920 and UK 14,304 in rat aorta while those evoked in guinea-pig aorta were less strongly affected. Contractions elicited by (-)-phenylephrine were moderately impaired by D 600 in rat aorta whereas there was only a limited effect in guinea-pig aorta. Analysis of the prazosin and yohimbine antagonism of B-HT 920- and UK 14,304-evoked contractions showed the involvement of alpha 1-like adrenoceptors in rat and guinea-pig aorta, prazosin being approximately 1000 times more potent that yohimbine. The results show that B-HT 920 and UK 14,304 contract rat and guinea-pig aorta via alpha 1-like adrenoceptors which are not identical. It is submitted that rat and guinea-pig alpha 1-adrenoceptors activate different contractile processes.     

Effects of imidazoline derivatives on cholinergic motility in guinea-pig ileum: involvement of presynaptic α2-adrenoceptors or imidazoline receptors?

The present study investigates the possibility that imidazoline receptors mediate modulation of cholinergic motor functions of the guinea-pig ileum. For this purpose, the effects of a series of compounds with known affinity for α₂-adrenoceptors and/or imidazoline recognition sites were examined on the cholinergic twitch contractions evoked by electrical field stimulation (0.1 Hz) of longitudinal muscle-myenteric plexus preparations. Additional experiments were carried out on ileal strips preincubated with [³H]choline, superfused with physiological salt solution containing hemicholinium-3, and subjected to electrical field stimulation (1 Hz). The stimulation-induced outflow of radioactivity was taken as an index of endogenous acetylcholine release. α-Methyl-noradrenaline, noradrenaline, clonidine, medetomidine, oxymetazoline and xylazine caused a concentration-dependent inhibition of twitch responses (IC₅₀ from 0.13 to 1.05 μM; E_max from 85.9 to 92.5%). Rilmenidine and agmatine were less potent in reducing the twitch activity, and the latter compound acted also with low intrinsic activity (IC₅₀=44.9 μM; E_max=35.5%). In interaction experiments, the inhibitory action of clonidine on twitch responses was competitively antagonized by RX 821002 (2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline), idazoxan, rauwolscine, yohimbine and BRL 44408 (2-[2H-(1-methyl-1,3-dihydroisoindole)-methyl]-4,5-dihydroimidazoline), whereas prazosin (10 μM), ARC 239 (2-(2,4-(O-methoxy-phenyl)-piperazin-1-yl)-ethyl-4,4-dimethyl-1,3-(2H,4H)-isoquinolindione; 10 μM) and BRL 41992 (1,2-dimethyl-2,3,9,13b-tetrahydro-1H-dibenzo[c,f]imidazol[1,5-a]azepi-ne; 10 μM) were without effect. Rauwolscine antagonized the inhibitory effects of various agonists on ileal twitch activity in a competitive manner and with similar potency. Agmatine and idazoxan did not significantly modify the twitch contractions when tested in the presence of α₂-adrenoceptor blockade by rauwolscine (3 μM) or RX 821002 (1 μM). Linear regression analysis showed that the affinity values of antagonists correlated with their affinity at the α_2A and α_2D binding sites as well as at previously classified α_2A/D adrenoceptor subtypes, whereas no significant correlation was obtained when comparing the potency estimates of agonists and antagonists with the affinity at I₁ or I₂ binding sites. When tested on the electrically induced outflow of tritium, α-methyl-noradrenaline, noradrenaline, clonidine, medetomidine, oxymetazoline, xylazine and rilmenidine yielded inhibitory concentration-response curves which were shifted rightward to a similar extent in the presence of rauwolscine (3 μM). In the absence of further drugs, agmatine significantly reduced the evoked tritium outflow at the highest concentrations tested (10 and 100 μM), whereas idazoxan (up to 100 μM) was without effect. When RX 821002 (1 μM) was added to the superfusion medium, neither agmatine nor idazoxan modified the evoked outflow of radioactivity. The results argue against modulation by imidazoline receptors of acetylcholine release from myenteric plexus nerve terminals. They provide evidence that compounds endowed with imidazoline-like structures affect the cholinergic motor activity of the guinea-pig ileum by interacting with presynaptic α₂-adrenoceptors belonging to the α_2D subtype. Received: 10 October 1997 / Accepted: 14 March 1998     

Clonidine-induced nitric oxide-dependent vasorelaxation mediated by endothelial alpha(2)-adrenoceptor activation

1. To assess the involvement of endothelial alpha(2)-adrenoceptors in the clonidine-induced vasodilatation, the mesenteric artery of Sprague Dawley rats was cannulated and perfused with Tyrode solution (2 ml min(-1)). We measured perfusion pressure, nitric oxide (NO) in the perfusate using chemiluminescence, and tissue cyclic GMP by RIA. 2. In phenylephrine-precontracted mesenteries, clonidine elicited concentration-dependent vasodilatations associated to a rise in luminal NO. One hundred nM rauwolscine or 100 microM L(omega)-nitro-L-arginine antagonized the clonidine-induced vasodilatation. Guanabenz, guanfacine, and oxymetazoline mimicked the clonidine-induced vasorelaxation. 3. In non-contracted mesenteries, 100 nM clonidine elicited a maximal rise of NO (123+/-13 pmol); associated to a peak in tissue cyclic GMP. Endothelium removal, L(omega)-nitro-L-arginine, or rauwolscine ablated the rise in NO. One hundred nM aminoclonidine, guanfacine, guanabenz, UK14,304 and oxymetazoline mimicked the clonidine-induced surge of NO. Ten microM ODQ obliterated the clonidine-induced vasorelaxation and the associated tissue cyclic GMP accumulation; 10 - 100 nM sildenafil increased tissue cyclic GMP accumulation without altering the clonidine-induced NO release. 4. alpha(2)-Adrenergic blockers antagonized the clonidine-induced rise in NO. Consistent with a preferential alpha(2D)-adrenoceptor activation, the K(B)s for yohimbine, rauwolscine, phentolamine, WB-4101, and prazosin were: 6.8, 24, 19, 165, and 1489 nM, respectively. 5. Rat pretreatment with 100 mg kg(-1) 6-hydroxydopamine reduced 95% tissue noradrenaline and 60% neuropeptide Y. In these preparations, 100 nM clonidine elicited a rise of 91.9+/-15.5 pmol NO. Perfusion with 1 microM guanethidine or 1 microM guanethidine plus 1 microM atropine did not modify the NO surge evoked by 100 nM clonidine. 6. Clonidine and congeners activate endothelial alpha(2D)-adrenoceptors coupled to the L-arginine pathway, suggesting that the antihypertensive action of clonidine involves an endothelial vasorelaxation mediated by NO release, in addition to presynaptic mechanisms.