Prejunctional Alpha 2-adrenoceptors Research Articles

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.

Inhibitory effects of sympathomimetic drugs on cholinergically mediated contractions of guinea-pig isolated tracheal muscle.

The experiments examine the actions of sympathomimetic drugs on the responses evoked by electrical field stimulation or by acetylcholine in guinea-pig tracheal strip chains. Electrical field stimulation evoked contractions which were cholinergically mediated, in the presence of guanethidine (10 microM) and indomethacin (2 microM). All the sympathomimetic drugs tested caused a concentration-dependent reduction in the height of these contractions. Inhibitory effects of isoprenaline and terbutaline were largely prevented by propranolol (2 microM) alone, whereas those of clonidine, oxymetazoline, lidamidine and WHR1370 were prevented by yohimbine alone (2 microM). Treatments with both propranolol and yohimbine were required to prevent the inhibitory effects of noradrenaline, adrenaline and dopamine. Contractions evoked by exogenous acetylcholine (0.1-3 microM) were also inhibited by all catecholamines and terbutaline, but not by clonidine, oxymetazoline, lidamidine and WHR1370. The inhibitory effects were antagonized by propranolol (2 microM) alone. The results suggest that in guinea-pig isolated tracheal muscle, sympathomimetic drugs can inhibit cholinergic neurotransmission not only by postjunctional beta 2-adrenoceptors but also by prejunctional alpha 2-adrenoceptors.

Pre-junctional alpha 2-adrenoceptor activity of B-HT920.

An in-vitro study has been carried out on the pre-junctional alpha 2-adrenoceptor activity of the thiazoloazepine derivative B-HT 920 (6-allyl-2-amino-5,6,7,8-tetrahydro-4H-thiazolo[4,5-d]-azepine) using field-stimulated rat vas deferens and guinea-pig ileum. The alpha 2-selective agonists clonidine (an imidazoline derivative) and alpha-methyl noradrenaline (a beta-phenethylamine derivative) were compared. Results show that B-HT 920 is a potent agonist on pre-junctional alpha 2-adrenoceptors and is competitively antagonized by the selective alpha 2-adrenoceptor antagonist yohimbine. The characteristics of the pharmacological responses obtained with B-HT920 indicate that it interacts with the receptor in an imidazoline-like, rather than a beta-phenethylamine-like, manner.

The prejunctional inhibitory effect of alpha-methylnoradrenaline in the rat vas deferens is not mediated via alpha 2-adrenoceptors.

In field-stimulated rat vas deferens, clonidine (10(-9)-10(-7) M) and alpha-methylnoradrenaline (10(-7)-3 x 10(-5) M) concentration-dependently inhibited twitch response to electrical field stimulation. The inhibitory effects of clonidine and alpha-methylnoradrenaline were similarly reduced by phenoxybenzamine (10(-6) M). However, idazoxan (3 x 10(-7) M) antagonized clonidine but not alpha-methylnoradrenaline-induced responses. The inhibitory effect of alpha-methylnoradrenaline was also not antagonized by phentolamine (3 x 10(-7) M), SK&F 104856 (2-vinyl-7-chloro-3,4,5,6-tetrahydro-4-methylthienol[4,3,2 ef][3]benzazepine) 3 x 10(-6) M) or yohimbine 3 x 10(-7) M). These antagonists however, attenuated the twitch-inhibiting effect of clonidine. The -logKB values were 8.02 +/- 0.05, 6.91 +/- 0.10, and 7.58 +/- 0.07 for phentolamine, SK&F 104856 and yohimbine respectively. In-vivo treatment of rats with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (6 mg kg-1), to inactivate prejunctional alpha 2-adrenoceptors, attenuated clonidine, but not alpha-methylnoradrenaline-induced responses. It was concluded that adrenoceptors are not involved in the prejunctional inhibitory effects of alpha-methylnoradrenaline in the rat vas deferens.

Alpha-adrenoceptor subtypes and Ca2+ mobilization in the rabbit ear artery.

The mobilization of cellular and extracellular Ca2+ pools by selective alpha 1-adrenoceptor (phenylephrine) and alpha 2-adrenoceptor (xylazine) agonists as well as noradrenaline was evaluated in rabbit ear artery. Noradrenaline and phenylephrine possess full intrinsic activity for both types of Ca2+ mobilization whereas xylazine up to 1 mM had only a limited contractile effect, being more effective in inducing extracellular Ca2+-dependent response. However, extracellular Ca2+ was mobilized by xylazine in a concentration 20 times higher than that required to stimulate pre-junctional alpha 2-adrenoceptors. Noradrenaline (5 microM) and xylazine (1 mM) induced cellular and extracellular Ca2+-dependent contractions which were prazosin-sensitive and yohimbine-resistant. Xylazine-induced contractile activity, particularly that dependent upon the extracellular Ca2+ pool, was markedly reduced by selective adrenergic denervation with 6-hydroxydopamine, but the actions of noradrenaline were unaffected. These results suggest that: (1) rabbit ear artery contain post-junctional alpha 1-adrenoceptor but not alpha 2-adrenoceptors; (2) stimulation of these alpha 1-adrenoceptors can account for the overall contractile activity of exogenously added noradrenaline and (3) stimulation of alpha 1-adrenoceptors results in mobilization of cellular as well as extracellular Ca2+ pools.

Different sites of action for alpha 2-adrenoceptor antagonists in the modulation of noradrenaline release and contraction response in the vas deferens of the rat.

Rat vas deferens was prepared, loaded with [3H]noradrenaline, and superfused to measure the release of tritium in resting conditions and in response to electrical field stimulation. The alpha 2-adrenoceptor antagonists yohimbine, CH-38083 (7,8-(methylenedioxi)-14 alpha-hydroxyalloberbane HCl), and idazoxan increased the electrically induced release of tritium in a concentration-dependent manner, whereas noradrenaline and the alpha 2-adrenoceptor agonist xylazine exerted opposite effects. The inhibitory effect of noradrenaline on electrically induced tritium release was antagonized by yohimbine, CH-38083, and idazoxan. Of the alpha 2-adrenoceptor antagonists tested, yohimbine and CH-38083 reversed the xylazine-induced inhibition of tritium release, and idazoxan was found to be completely ineffective against xylazine. Idazoxan, yohimbine and CH-38083 antagonized the inhibitory effect of xylazine on electrical stimulation-induced contractions of the vas deferens, as was evidenced by the apparent pA2 values. We conclude from the present experiments that noradrenaline and xylazine inhibit noradrenaline release by acting on distinct prejunctional alpha 2-adrenoceptors and that the receptor subtype that responds to xylazine is insensitive to idazoxan. In addition, inhibition by xylazine of contractility but not of noradrenaline release was antagonized by idazoxan, suggesting that besides noradrenergic neurotransmission, other motor transmitter systems (purinergic) may also be involved in the inhibition by alpha 2-adrenoceptor antagonists of mechanical responses in the rat vas deferens.

Noradrenergic vasoconstriction of pig prostatic small arteries

The current study investigated the distribution of adrenergic nerves and the action induced by noradrenaline (NA) in pig prostatic small arteries. Noradrenergic innervation was visualized using an antibody against dopamine-beta-hydroxylase (DBH), and the NA effect was studied in small arterial rings mounted in microvascular myographs for isometric force recordings. DBH-immunoreactive nerve fibers were located at the adventitia and the adventitia-media border of the vascular wall. Electrical field stimulation (EFS, 1-32 Hz) evoked frequency-dependent contractions that were reduced by guanethidine and prazosin (adrenergic neurotransmission and alpha1-adrenoceptors blockers, respectively) and by the alpha2-adrenoceptor agonist UK 14,304. The alpha2-adrenoceptor antagonist rauwolscine reversed the UK 14,304-produced inhibition. NA produced endothelium-independent contractions that were antagonized with low estimated affinities and Schild slopes different from unity by prazosin and the alpha1A-adrenoceptor antagonist N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-alpha-alpha-dimethyl-1H-indole-3-ethanamine (RS 17053). The alpha1A-adrenoceptor antagonist 5-methyl-3-[3-[4-[2-(2,2,2,-trifluoroethoxy) phenyl]-1-piperazinyl]propyl]-2,4-(1H)-pyrimidinedione (RS 100329), which also displays high affinity for alpha1L-adrenoceptors, and the alpha1L-adrenoceptor antagonist tamsulosin, which also has high affinity for alpha1A- and alpha1D-adrenoceptors, induced rightward shifts with high affinity of the contraction-response curve to NA. The alpha1D-adrenoceptor antagonist 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]-ethyl]8-azaspiro[4,5]decane-7,9-dione dihydrochloride (BMY 7378) failed to modify the NA contractions that were inhibited by extracellular Ca2+ removal and by voltage-activated (L-type) Ca2+ channel blockade. These data suggest that pig prostatic resistance arteries have a rich noradrenergic innervation; and NA, whose release is modulated by prejunctional alpha2-adrenoceptors, evokes contraction mainly through activation of muscle alpha1L-adrenoceptors coupled to extracellular Ca2+ entry via voltage (L-type)- and non-voltage-activated Ca2+ channels.

α1A‐ and α1D‐adrenoceptors are the major functional subtypes of renal α1‐adrenoceptors in streptozotocin‐induced diabetic and normal Sprague–Dawley rats

1 The present study investigated the effect of streptozotocin-induced diabetes on alpha(1)-adrenoceptor subtypes in rat renal resistance vessels. 2 Studies on renal haemodynamics were carried out 7 days after the last streptozotocin. Changes in renal blood flow were recorded in response to electrical stimulation of the renal nerve (RNS) and a range of adrenergic agonists; noradrenaline (NA), phenylephrine (PE) and methoxamine (MTX), either in the absence or the presence of nitrendipine (Nit), 5-methylurapidil (MEU), chlorethylclonidine (CEC) or BMY 7378. 3 In non-diabetic animals, Nit, MEU and BMY 7378 significantly attenuated renal vasoconstriction induced by adrenergic agonists, while CEC showed a significant accentuation in RNS-induced responses without having a significant effect on responses to adrenergic agonists. In diabetic rats, renal vasoconstriction was also significantly reduced in Nit-, MEU- and BMY 7378-treated groups and CEC potentiated RNS-induced contractions caused a change similar to that observed in non-diabetic rats. BMY 7378 significantly (P < 0.05) attenuated the PE- and MTX-induced vasoconstrictions but did not cause any significant (P > 0.05) alteration in the RNS- and NA-induced responses. 4 The results showed functional co-existence of alpha(1A)- and alpha(1D)-adrenoceptors in the renal vasculature of SD rats irrespective of the presence of diabetes. A possible minor contribution of prejunctional alpha-adrenoceptor subtype has also been suggested in either experimental group, particularly possible functional involvement of alpha(1B)-adrenoceptor subtypes in non-diabetic SD rats.

ATP release and its prejunctional modulation.

We studied some properties of the release of noradrenaline and ATP in isolated sympathetically innervated tissues. Release was elicited by electric stimulation and assessed as overflow of tritiated compounds (after labelling with [3H]noradrenaline) and enzymically measured ATP, respectively. Evans blue, which inhibits ectonucleotidases, greatly increased the evoked overflow of ATP, indicating that a major part of the ATP was metabolized after release. Much of the ATP was postjunctional in origin. The neural fraction was isolated when postjunctional release was suppressed by prazosin (alpha 1-adrenoceptor antagonist) and suramin (P2 purinoceptor antagonist). Comparison of neural ATP and [3H]-noradrenaline release showed that prostaglandin E2 reduced the release of both co-transmitters to a similar extent. Activation of prejunctional alpha 2-adrenoceptors, however, preferentially reduced the release of [3H]noradrenaline, and activation of prejunctional A1 purinoceptors reduced preferentially the release of ATP. Nucleotides such as ATP depressed the release of [3H]noradrenaline through two receptors: the well-known prejunctional A1 receptors and a separate group of prejunctional P2 purinoceptors. P2 antagonists increased the release of [3H]-noradrenaline. Overall, the results indicate differential storage, release and modulation of release of the two sympathetic co-transmitters. They also indicate that postganglionic sympathetic axons possess receptors for both co-transmitters: alpha 2 and P2 autoreceptors.

Pre‐junctional α2‐adrenoceptors modulation of the nitrergic transmission in the pig urinary bladder neck

To investigate the nitric oxide (NO)-mediated nerve relaxation and its possible modulation by pre-junctional alpha2-adrenoceptors in the pig urinary bladder neck. Urothelium-denuded bladder neck strips were dissected, and mounted in isolated organ baths containing a physiological saline solution (PSS) at 37 degrees C and continuously gassed with 5% CO2 and 95% O2, for isometric force recording. The relaxations to transmural nerve stimulation (electrical field stimulation [EFS]) or exogenously applied NO were carried out on strips pre-contracted with 1 microM phenylephrine (PhE) and treated with guanethidine (10 microM) and atropine (0.1 microM), to block noradrenergic neurotransmission and muscarinic receptors, respectively. EFS (0.2-1 Hz, 1 msec duration, 20 sec trains, current output adjusted to 75 mA) evoked frequency-dependent relaxations which were abolished by the neuronal voltage-activated Na+ channel blocker tetrodotoxin (TTX, 1 microM). These responses were potently reduced by the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine (L-NOARG, 30 microM) and further reversed by the NO synthesis substrate L-arginine (L-ARG, 3 mM). The alpha2-adrenoceptor agonist BHT-920 (2 microM) reduced the electrically evoked relaxations, its effectiveness being higher on the responses induced by low frequency stimulation. BHT-920-elicited reductions were fully reversed by the alpha2-adrenoceptor antagonist rauwolscine (RAW, 1 microM). Exogenous NO (1 microM-1 mM) induced concentration-dependent relaxations which were not modified by BHT-920, thus eliminating a possible post-junctional modulation. These results indicate that NO is involved in the non-adrenergic non-cholinergic (NANC) inhibitory neurotransmission in the pig urinary bladder neck, the release of NO from intramural nerves being modulated by pre-junctional alpha2-adrenoceptor stimulation.

Purinergic and adrenergic cotransmission in canine isolated and perfused gastroepiploic arteries.

1. The vasoconstrictor responses of canine gastroepiploic artery to periarterial electrical nerve stimulation (PNS; 30 s trains of pulses at a frequency of 2, 4 or 8 Hz) were observed in a frequency dependent manner. The PNS-induced vasoconstrictions were abolished by tetrodotoxin (1 micromol/L) and mostly depressed but not completely by guanethidine (10 micromol/L). 2. Vasoconstrictor responses to administered noradrenaline were antagonized significantly by prazosin (0.1 micromol/L), an alpha1-adrenoceptor antagonist, but were not significantly affected by suramin (100 micromol/L), a P2 purinoceptor antagonist, or alpha,beta-methylene ATP (1 micromol/L), a P2X receptor desensitizing agent. Exogenous ATP-induced responses were clearly depressed by suramin or alpha,beta-methylene ATP, but were not significantly affected by prazosin. 3. The vasoconstrictor responses to PNS at a low frequency (2 and 4 Hz) of stimulation were markedly inhibited by suramin (100 micromol/L) and by alpha,beta-methylene ATP (1 micromol/L). The remaining responses after suramin or alpha,beta-methylene ATP were abolished by subsequent application of prazosin (0.1 micromol/L). At a high frequency (8 Hz) of stimulation, the vascular response was not significantly inhibited by suramin or alpha,beta-methylene ATP, but it was abolished by prazosin. 4. Injection of xylazine (0.3-30 nmol/L), an alpha2-adrenoceptor agonist, did not induce any clear vasoconstriction. The exposure of tissues to rauwolscine (0.1-0.3 micromol/L), an alpha2-adrenoceptor antagonist, dose-dependently increased PNS-induced vasoconstrictions at all frequencies tested. 5. The present results indicate that ATP acts as a cotransmitter with noradrenaline and is responsible for post-junctional vasoconstrictor responses at low frequencies of sitmulation, whereas the effect of noradrenaline is dominant at high-frequency stimulation in canine gastroepiploic artery. Prejunctional alpha2-adrenoceptor autoinhibition may modulate the release of either noradrenaline or ATP from sympathetic nerve terminals.

Presence, distribution and physiological function of adrenergic and muscarinic receptor subtypes in the human heart.

The sympathetic and parasympathetic nervous system play a powerful role in controlling cardiac function by activating adrenergic and muscarinic receptors. In the human heart there exist alpha1-, beta1- and beta2-adrenoceptors and M2-muscarinic receptors and possibly also (prejunctional) alpha2-adrenoceptors. Beta1- and beta2-adrenoceptors are quite evenly distributed in the human heart while M2-receptors are heterogeneously distributed (more receptors in atria than in ventricles). Stimulation of beta1- and beta2-adrenoceptors causes increases in heart rate and force of contraction while stimulation of M2-receptors decreases heart rate (directly in atria) and force of contraction (indirectly in ventricles). Pathological situations (such as heart failure) or pharmacological interventions (for example, beta-blocker treatment) can alter the distribution of beta1- and beta2-adrenoceptors in the human heart, while M2-receptors are only marginally affected. On the other hand, relatively little is known on distribution and functional role of alpha1- and alpha2-adrenoceptor subtypes in the human heart.

Presynaptic alpha2-adrenoceptor-mediated modulation of adenosine 5' triphosphate and noradrenaline corelease: differences in canine mesenteric artery and vein.

1. The modulatory effects of agonists and antagonists of prejunctional alpha2-adrenoceptors on the electrical field stimulation (EFS, 0.3 ms, 12 V)-induced release of endogenous noradrenaline (NA) and the cotransmitter adenosine 5' triphosphate (ATP) were measured in endothelium-denuded segments of canine inferior mesenteric artery and compared with effects in mesenteric vein. The overflow of NA and ATP was evoked by long-duration (2 min) EFS at low frequency (4 Hz) and high frequency (16 Hz) of stimulation and was analysed using HPLC techniques with electrochemical detection and fluorescence detection, respectively. 2. The EFS-evoked overflow of both NA and ATP was significantly reduced by tetrodotoxin (1 microM) and guanethidine (10 microM) in the artery and vein. Desipramine (10 microM), a blocker of neuronal uptake of NA, increased the EFS (4 and 16 Hz)-evoked overflow of NA in both artery and vein. EFS-evoked overflow of NA in vein exceeded the NA overflow in artery at both 4 and 16 Hz in control preparations as well as in the presence of desipramine. However, the EFS-evoked overflow of ATP was equal in the artery and vein. 3. Stimulation of alpha2-adrenoceptors with clonidine (0.1 microM) and oxymethazoline (0.3 microM) reduced the EFS evoked overflow of NA in both artery and vein at 4 Hz, whereas the NA overflow at 16 Hz remained unchanged in both blood vessels. The overflow of ATP as well as of ADP (and hence ATP:ADP ratio) was unaffected by the alpha2-adrenoceptor agonists in the artery and vein. 4. In artery, blockade of alpha2-adrenoceptors with yohimbine at a concentration of 0.1 microM caused no effect on the NA overflow neither at 4 Hz nor at 16 Hz of EFS. Yohimbine at a concentration of 1 microM increased the overflow of NA at 4 Hz but not 16 Hz of EFS. In vein, however, yohimbine (0.1 and 1 microM) increased NA overflow at both 4 and 16 Hz of stimulation. Idazoxan (1 microM) increased the NA overflow in artery only at 4 Hz, whereas in vein idazoxan increased the NA overflow at both 4 and 16 Hz. No changes of EFS-evoked ATP overflow were observed in the presence of 0.1 microM yohimbine in both artery and vein. Greater concentration of yohimbine (i.e. 1 microM) increased the overflow of ATP in both the artery and vein only at 4 Hz EFS. Idazoxan (1 microM) enhanced the ATP overflow only at 16 Hz in vein. The overflow of ADP was affected by both yohimbine and idazoxan in a similar manner to the ATP overflow so that the ATP:ADP ratios were not changed. 5. In conclusion, sympathetic nerves in both mesenteric arteries and veins appear to release ATP along with NA. Release of NA in veins exceeds release of NA in arteries, whereas both the canine artery and vein release equal amount of ATP. At long-duration nerve stimulation (as might occur during stress) the alpha2-adrenoceptors appear to rather modulate release of NA than release of the cotransmitter ATP. The prejunctional autoinhibition of NA release is more effective at lower frequencies of nerve stimulation. The alpha2-adrenoceptor-mediated neuromodulation plays a greater role in veins than arteries. Quantitative differences in alpha2-adrenoceptor-mediated neuromodulation in the arteries and veins may participate to differing contributions of mesenteric blood vessels to the control of blood flow and volume distribution in splanchnic circulation.

Prejunctional muscarinic receptor modulation of noradrenaline release from sympathetic neurones in rabbit aorta.

The prejunctional muscarinic modulation of stimulation-evoked release of 3H-noradrenaline from sympathetic neurones in rabbit aorta was examined. The role of transmitter uptake, alpha-adrenoceptor blockade, stimulation frequency and endothelium on the modulation was investigated. Rings of aorta were incubated with (-)-3H-noradrenaline and subsequently subjected to electrical-field stimulation. Fractional 3H-overflow was determined by liquid scintillation counting. Acetylcholine (10(-8)-3 x 10(-6) M) added cumulatively, reduced the stimulation-evoked 3H-overflow up to 80%. The effect of acetylcholine was the same in intact and endothelium-free aorta. The inhibitory effect of acetylcholine was inversely related to the frequency of stimulation (1-10 Hz). The maximal inhibition (%) was 80 (1 Hz), 53 (3 Hz) and 14 (10 Hz). The inhibitory effect of acetylcholine (10(-6) M) and carbachol (10(-5) M) reached a maximum 15 min. after addition and then remained almost constant. Cocaine (3 x 10(-5) M) did not alter the effect of acetylcholine. Desipramine (10(-6) M) and corticosterone (4 x 10(-5) M) attenuated the inhibition seen with low concentrations (10(-8)-10(-7) M) of acetylcholine. The acetylcholine-induced inhibition was antagonized by desipramine. Cocaine plus corticosterone attenuated the inhibition seen with high concentrations (10(-6)-3 x 10(-6) M) of acetylcholine. Rauwolscine (10(-6) M) enhanced the maximal inhibitory effect of acetylcholine. We conclude that the inhibitory effect of acetylcholine on 3H-overflow from rabbit aorta preloaded with 3H-noradrenaline is (1) inversely related to stimulation frequency; (2) independent of endothelium; (3) unaffected by neuronal and extraneuronal transmitter uptake; (4) that cocaine is not a prejunctional muscarinic antagonist; (5) that cocaine, but not desipramine, is suited as a neuronal uptake inhibitor in studies of prejunctional muscarinic receptor subtypes; and (6) and that there is an inverse interaction between prejunctional alpha2-adrenoceptors and muscarinic receptors.

Regulation of pre-synaptic alpha adrenergic activity in the corpus cavernosum.

The neurotransmitters and vasoactive substances regulating tone in the smooth muscle of the penile arteries/arterioles and the trabeculae of the corpora cavernosa are critical mediators of the state of penile erection. Contemporary research reveals a coordinated, intricate interplay between the pathways of vasorelaxation and vasoconstriction representing a most efficient physiological mechanism to initiate and maintain penile erection. This paper will focus on the role of the adrenergic constrictor pathways in penile erection and, more specifically, on the pre-junctional adrenergic mechanisms that regulate smooth muscle constriction. All neurogenic constrictor responses are related to the release of norepinephrine from adrenergic nerves that act on post-junctional alpha-1 and pre-junctional and post-junctional alpha-2 receptor subtypes. Based on the current state of knowledge, there are at least three pre-junctional mechanisms regulating penile smooth muscle tone. First, norepinephrine release from the adrenergic nerves binds to the pre-junctional alpha-2 adrenoceptor on the adrenergic nerves and negatively regulates norepinephrine release. Blockade of this reaction by selective alpha-2 receptor antagonists (e.g. yohimbine or delequamine) will enhance norepinephrine release. Second, norepinephrine release from the adrenergic nerves binds to the pre-junctional alpha-2 adrenoceptor on the non-adrenergic, non-cholinergic (NANC) nerves and inhibits nitric oxide synthesis and release. Blockade of this reaction by selective alpha-2 receptor antagonists (e.g. yohimbine or delequamine) will enhance nitric oxide release, facilitating erection. Finally, cholinergic nerves pre-junctionally inhibit norepinephrine release from the adrenergic nerve and stimulate the NANC nerve to increase nitric oxide synthesis and release. These observations indicate that different neurotransmitters regulate the adrenergic neurotransmission pathway. Based on the above concepts for pre-junctional and post-junctional regulation of smooth muscle tone, the most efficacious strategy to reduce adrenergic activity and facilitate penile erection is to combine alpha-1 and alpha-2 adrenergic receptor antagonists. In this case, any enhancement of norepinephrine release is of little importance because the alpha-1 receptor antagonist will impede this vasoconstrictor response. This will also enhance the release of nitric oxide, which increases smooth muscle relaxation and decreases contraction resulting in penile erection.

Investigation of the prejunctional alpha2-adrenoceptor mediated actions of MDMA in rat atrium and vas deferens.

1. We have investigated the effects of methylenedioxymethamphetamine (MDMA, 'ecstasy') on peripheral noradrenergic neurotransmission in the rat. 2. In rat atrial slices pre-incubated with [3H]-noradrenaline and in the presence of desipramine (1 micronM) to prevent effects of MDMA on basal outflow of tritium, MDMA (10 micronM) significantly inhibited the release of tritium evoked by short trains of six pulses at 100 Hz every 10 s for 3 min. This effect did not occur in the presence of the alpha2-adrenoceptor antagonist yohimbine (1 micronM). 3. In epididymal portions of rat vas deferens in the presence of nifedipine (10 micronM), MDMA produced a concentration-dependent inhibition of single pulse nerve stimulation-evoked contractions with a pD2 of 5.88+/-0.16 (n=4). Inhibitory effects of MDMA were antagonized by the alpha2-adrenoceptor antagonist yohimbine (0.3 micronM), but not by the 5-hydroxytryptamine receptor antagonist cyanopindolol in a concentration (1 micronM) which markedly antagonized the inhibitory actions of the 5-HT-1 receptor agonist 5-carboxamidotryptamine. 4. In prostatic portions of rat vas deferens in the presence of cocaine (3 micronM), MDMA produced a concentration-dependent inhibition of single pulse nerve stimulation-evoked contractions with a pD2 of 5. 12+/-0.21 (n=4). In the absence of cocaine, only the highest concentration of MDMA (30 micronM) produced an inhibition, but the alpha2-adrenoceptor antagonist yohimbine (0.3 micronM) converted the response to MDMA from inhibition to potentiation of the stimulation-evoked contraction. 5. In radioligand binding studies, MDMA showed similar affinities for alpha2B, alpha2C and alpha2D-adrenoceptor sites, with pKi values of 5.14+/-0.16, 5.11+/-0. 05 and 5.31+/-0.14, respectively. 6 It is concluded that MDMA has significant alpha2-adrenoceptor agonist actions.

Effects of some imidazolidine alpha2-adrenoceptor agonists in rat isolated atria.

1. The prejunctional alpha2-adrenoceptor agonist activity of four imidazolidines (UK14819, UK14304, UK15121 and UK11957) were compared to that of clonidine in rat isolated atrial preparations. 2. The preparations consisted of spontaneously beating left and right atrial pairs that were incubated with [3H]-noradrenaline. The efflux of radioactivity induced by electrical field stimulation of intramural sympathetic nerves was used as an index of neurotransmitter release and inotropic and chronotropic responses were recorded. 3. Two quinoxalinyl imidazolidines (UK14819 and UK14304 which have chloride and bromide substitution, respectively, in the phenyl moiety) caused decreases in the efflux of radioactivity with stimulation at 2 Hz and 10 Hz but not at 20 Hz. These effects were antagonised by the alpha2-adrenoceptor antagonist idazoxan (0.3 microM) but they were not affected by the alpha1-adrenoceptor antagonist prazosin (0.1 microM). 4. The third halogenated quinoxalinyl imidazolidine analogue (UK15121, which has an iodide substitution in the phenyl ring) and a quinolinyl imidazolidine (UK11957) have actions similar to clonidine. They decreased the efflux of radioactivity with stimulation at 1 Hz (for UK11957) or 2 Hz (for UK15121) and enhanced it at higher frequencies of stimulation. Both the inhibitory and enhancing effects were antagonised by idazoxan but they were not affected by prazosin. 5. Unlike the other three imidazolidines in the present study, the quinolinyl imidazolidine (UK11957), caused a decrease in resting release of radioactivity and this effect was prevented by the monoamine oxidase inhibitor pargyline (30 microM). 6. These findings suggest that the 5-chloro-or 5-bromo substituted quinoxalinyl imidazolidines (UK14819 and UK14304) are full agonists at prejunctional alpha2-adrenoceptors, but the 5-iodo-substituted quinoxalinyl imidazolidine (UK15121) and the quinolinyl analogue of UK14304 (UK11957), like clonidine, appear to be partial agonists at these receptors.

ORL1 receptor-mediated inhibition by nociceptin of noradrenaline release from perivascular sympathetic nerve endings of the rat tail artery.

In the present study the effect of the opioid heptadecapeptide nociceptin, also termed orphanin FQ, an endogenous ligand for the orphan receptor named ORL1 (opioid receptor-like 1) receptor, was investigated on [3H]noradrenaline release induced by electrical field stimulation (24 pulses at 0.4 Hz, 200 mA, 0.3 ms duration) in the rat tail artery in the absence and presence of an alpha2-adrenoceptor antagonist, rauwolscine 3 microM. Nociceptin inhibited the electrically-evoked tritiated noradrenaline release in a concentration-dependent manner from rat tail arteries. This inhibitory effect of nociceptin was enhanced in the presence of the alpha2-adrenoceptor antagonist rauwolscine (maximum inhibition by 25% and 50% in the absence and presence of rauwolscine, respectively). At a supramaximal concentration (10 microM), the inhibitory action of DAGO, a selective micro-opioid receptor agonist, was less pronounced than that of nociceptin. The inhibitory effect of nociceptin was counteracted by naloxone benzoylhydrazone (3 microM) which by itself did not change the stimulation-evoked noradrenaline overflow. Naloxone (10 microM), a non-selective opioid receptor antagonist, did not affect the inhibitory effect of nociceptin whereas it abolished that of DAGO. In conclusion, these results suggest that nociceptin modulates noradrenergic neurotransmission by acting on prejunctional ORL1 receptors located on nerve terminals innervating the rat tail artery. They also demonstrate that prejunctional ORL1 receptors interact with prejunctional alpha2-adrenoceptors. The physiological significance of this phenomenon remains to be determined.

Investigation of the subtypes of alpha2-adrenoceptor mediating prejunctional inhibition in rat atrium and cerebral cortex.

We have investigated the subtype of alpha2-adrenoceptor mediating prejunctional inhibition of neurotransmission in rat atrium in comparison with the alpha2-adrenoceptor mediating prejunctional inhibition in rat cerebral cortex. In rat atrium and cerebral cortex, prejunctional alpha2-adrenoceptors were investigated in terms of the ability of alpha2-adrenoceptor antagonists to increase the stimulation-evoked overflow of tritium in tissues pre-incubated with [3H]-noradrenaline. The relatively non-selective alpha2-adrenoceptor antagonist yohimbine and the alpha2D-adrenoceptor selective antagonist BRL 44408 had potencies in rat atrium which were similar to their potencies in rat cerebral cortex. The antagonists ARC 239, HV 723, WB 4101, prazosin, chlorpromazine and abanoquil, which have low affinity for alpha2D-adrenoceptors, significantly increased stimulation-evoked overflow at lower concentrations in rat atrium than rat cerebral cortex. Antagonist potency at prejunctional alpha2-adrenoceptors was correlated with antagonist affinity at alpha2-adrenoceptor ligand binding sites in membranes of rat kidney (alpha2B) and submandibular gland (alpha2D), and human recombinant alpha2C-adrenoceptors labelled with [3H]yohimbine. The correlation between ligand binding sites and the functional receptor in the rat cerebral cortex was significant only for the alpha2D-adrenoceptor ligand binding site (r=0.87, n=8, P<0.01) as compared to the alpha2B-adrenoceptor (r=0.32, n.s.) or alpha2C-adrenoceptor (r=0.12, n.s.) ligand binding sites. The correlation between ligand binding sites and the functional receptor in the rat atrium was not significant for any ligand binding site, with r=0.64, 0.68 and 0.67 for the alpha2D-, the alpha2B- and the alpha2C-adrenoceptor ligand binding sites, respectively. It is concluded that the functional prejunctional alpha2-adrenoceptor of rat cerebral cortex closely resembles the alpha2D-adrenoceptor ligand binding site of rat submandibular gland, but the rat atrium may contain two subypes of prejunctional alpha2-adrenoceptor, alpha2D and another subtype, possibly alpha2B or alpha2C.

Prejunctional alpha2-adrenoceptors and peroxide-induced potentiation of norepinephrine release from the bovine iris.

Peroxides can enhance field-stimulated [3H]norepinephrine ([3H]NE) release in isolated irides from several mammalian species. In the present study, we investigated the role of prejunctional alpha2-adrenoceptors in peroxide-induced potentiation of sympathetic neurotransmission in bovine isolated irides. Isolated hemi-irides were incubated in a Krebs buffered-solution containing [3H]NE and prepared for studies of neurotransmitter release using the superfusion method. Alpha2-adrenoceptor agonists, oxymetazoline, UK-14304 and clonidine inhibited field-stimulated [3H]NE overflow without affecting basal tritium efflux. Pretreatment of tissues with H2O2 (300 microM) had no effect on inhibition of evoked [3H]NE release caused by the alpha2-adrenergic agonists. However, H2O2 (300 microM) caused significant (P < 0.01) leftward shifts of excitatory concentration-response curves to yohimbine (10 nM-1 microM). In contrast, yohimbine (1 microM) did not prevent the enhancement of evoked [3H]NE overflow induced by H2O2 (300 microM). In conclusion, excitatory effects of peroxides on sympathetic neurotransmission in bovine irides are not mediated by prejunctional alpha2-adrenoceptors.