Prejunctional Alpha 2-adrenoceptors Research Articles

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.

Interaction of the renin-angiotensin system, bradykinin and sympathetic nerves with cholinergic transmission in the rat isolated trachea.

1. The present study was undertaken to investigate the interaction of the renin-angiotensin system (RAS), bradykinin and the sympathetic nervous system with cholinergic transmission in the rat airways. Experiments were performed on epithelium-intact and epithelium-denuded preparations of rat isolated trachea which had been incubated with [3H]-choline to incorporate [3H]-acetylcholine into the cholinergic transmitter stores. Tracheal preparations were subjected to electrical field stimulation (trains of 1 ms pulses, 5 Hz, 15 V) and the stimulation-induced (S-I) efflux taken as an index of transmitter acetylcholine release. 2. In both epithelium-intact and epithelium-denuded tracheal preparations, the alpha 2-adrenoceptor agonist UK14304 (0.1 and 1 microM) inhibited the S-I efflux, in a concentration-dependent manner. The inhibition of S-I efflux produced by UK14304 (1 microM) was antagonized by the selective alpha 2-adrenoceptor antagonist idazoxan (0.3 microM). Idazoxan (0.3 microM) alone had no effect on the S-I efflux. 3. Angiotensin II (0.1 and 1 microM) was without effect on the S-I efflux in either epithelium-intact or epithelium-denuded tracheal preparations. When angiotensin-converting enzyme was inhibited by perindoprilat (10 microM), angiotensin II (1 microM) was also without effect on the S-I efflux. Similarly, in the presence of idazoxan (0.3 microM), to block prejunctional alpha 2-adrenoceptors, angiotensin II (0.1 and 1 microM) did not alter the S-I efflux. When added alone, perindoprilat (10 microM) did not alter the S-I efflux. 4. In epithelium-denuded preparations, bradykinin (0.01-1 microM) inhibited the S-I efflux. In epithelium-intact preparations, there was also a tendency for bradykinin (0.1 and 1 microM) to inhibit the S-I efflux but this was not statistically significant. However, when angiotensin-converting enzyme and neutral endopeptidase were inhibited by perindoprilat (10 microM) and phosphoramidon (1 microM), respectively, bradykinin (1 microM) significantly inhibited the S-I efflux in epithelium-intact preparations as well as in epithelium-denuded preparations. The inhibition of the S-I efflux produced by bradykinin, in the combined presence of perindoprilat (10 microM) and phosphoramidon (1 microM), was unaffected by the additional presence of the cyclo-oxygenase inhibitor indomethacin (10 microM) and/or the nitric oxide synthase inhibitor NG-nitro-L-arginine (100 microM), in either epithelium-intact or epithelium-denuded preparations. 5. In conclusion, the findings of the present study suggest that airway parasympathetic nerves are endowed with alpha 2-adrenoceptors which subserve inhibition of transmitter acetylcholine release. Under the present conditions, however, transmitter acetylcholine release is not subject to transneuronal modulation by noradrenaline released from adjacent sympathetic nerves in the airways. Moreover, angiotensin II and perindoprilat do not appear to modulate acetylcholine release from parasympathetic nerves of the airways. In contrast, bradykinin inhibits acetylcholine release from airway parasympathetic nerves but this action of bradykinin is limited by the activity of epithelial angiotensin-converting enzyme and/or neutral endopeptidase. The inhibitory action of bradykinin on cholinergic transmission in the airways does not appear to involve the liberation of prostaglandins or nitric oxide.

Prejunctional alpha sub 2-Adrenoceptors Inhibit Nitrergic Neurotransmission in Horse Penile Resistance Arteries

Purpose To study the influence of alpha-adrenergic stimuli on non-adrenergic non-cholinergic (NANC) neurogenic relaxation in isolated horse penile resistance arteries. Materials and Methods Deep intracavernous penile arteries with an internal lumen diameter of 200-500 micro m., isolated from the corpus cavernosum of young horses, were mounted in microvascular myographs for isometric tension recording and electrical field stimulation (EFS) of autonomic nerve terminals. Results In the presence of guanethidine (10 sup -5 M) and atropine (10 sup -7 M) tone of the arteries was raised by the thromboxane analogue, U46619. EFS (1, 4 and 32 Hz) induced frequency-dependent relaxations, which were abolished in the presence of tetrodotoxin, while N G-nitro-L-arginine (L-NOARG, 10 sup -4 M) abolished the relaxations to EFS at 1 Hz, and significantly reduced the relaxations at 4 Hz and 32 Hz by 82.5 +/- 10.2% and 52.9 +/- 4.7%, respectively (n = 6). EFS induced relaxations of a similar magnitude in penile arteries contracted with U46619 or the alpha 1-adrenoceptor agonist, phenylephrine, while the alpha 2-adrenoceptor agonist, BHT920 (10 sup -6 M), produced an inhibitory effect on the EFS-evoked relaxations which was inversely related to the stimulus frequency (1, 4 and 32 Hz). BHT920 had no effect on the relaxations induced by exogenous nitric oxide (NO), added as acidified sodium nitrite (10 sup -6-10 sup -3 M). The inhibitory effect of BHT920 on NANC relaxations was reversed by 10 sup -7 M rauwolscine. Conclusion These results suggest that the release of a NANC neurotransmitter primarily thought to be NO is inhibited by stimulation of prejunctional alpha 2-adrenoceptors in horse penile resistance arteries.

Intracellular Calcium Buffering Declines in Aging Adrenergic Nerves

Stimulation-evoked norepinephrine release from rat tail artery adrenergic nerves increased with advancing age in the Fischer-344 rat when function of norepinephrine uptake mechanisms and prejunctional alpha-2 adrenoceptors were blocked. When calcium channels were bypassed with the ionophore, ionomycin (4 μM), norepinephrine release from aged nerves (20 months) was still elevated as compared to 6-month-old nerves. Norepinephrine release stimulated by high K + was also higher in 20-month nerves. The intracellular calcium chelator, 1,2 bis(2-aminophenoxy)ethane- N,N,N′, N′-tetraacetomethylester (BAPTA/AM), was used to determine whether age-related increases in norepinephrine release could be reversed with the addition of an artificial intracellular calcium buffer. Exposure to BAPTA/AM decreased stimulation-evoked norepinephrine release in both old and young tail arteries; however, the effect was significantly greater in older arteries. When mitochondrial calcium uptake was compromised using the uncoupler of mitochondrial oxidative phosphorylation, dinitrophenol, BAPTA caused a further decrease in stimulation-evoked norepinephrine release in 20-month tail arteries with much less effect in 6-month-old nerves. These results suggest that intracellular calcium buffering is less efficient in older nerves.

Hypertension due to chronic blockade of P1-purinoceptors.

1. Long-term treatment of rats with 1,3-dipropyl-8-sulphophenylxanthine (DPSPX), a non-selective antagonist of adenosine receptors, causes a hypertensive state. 2. In DPSPX-hypertensive rats, prejunctional alpha 2-adrenoceptors become supersensitive to the selective alpha 2-adrenoceptor agonist UK-14,304, while postjunctional adrenoceptor-mediated responses are not changed; furthermore, prejunctional beta-adrenoceptor-mediated facilitation of noradrenaline release is also enhanced. 3. In DPSPX-hypertensive rats, there are important morphological alterations of the small arteries, their lumina appearing strongly reduced and occasionally occluded by proliferation of the intimal cells. 4. In DPSPX-hypertensive rats, there is an increase in plasma renin, and captopril prevents not only the development of the hypertension but also the morphological changes in the arteries. 5. Other important changes occur in DPSPX-hypertensive rats: an alteration of the adrenergic regulation of the cardiac functions and an enhancement of perivascular neurotransmission. 6. These results suggest that adenosine may play an important role in the development of some kinds of human hypertension.

Peripheral alpha 2-adrenoceptor-mediated sympathoinhibitory effects of mivazerol.

Mivazerol is a new compound that could potentially reduce perioperative cardiovascular morbidity and mortality in patients with or at risk of coronary disease and submitted to surgery. This action of mivazerol depends on a well documented centrally mediated reduction in sympathetic nerve activity, but a direct peripheral decrease in sympathetic neurotransmitter release induced by activation of prejunctional alpha 2-adrenoceptors located on sympathetic nerve endings could also contribute. To investigate this issue, the effects of mivazerol on the pressor, systemic and regional hemodynamic (pulsed Doppler technique) as well as on the cardiac responses to electrical stimulation of the spinal cord (SCS) were measured in pithed rats in the absence and in the presence of mivazerol. Mivazerol exerted strong sympathoinhibitory effects: SCS-induced increases in blood pressure, total peripheral resistance and heart rate were dose-dependently reduced by mivazerol, but among the regional vascular beds investigated, only the hindlimb vasoconstrictor responses were significantly drug-affected. All these sympathoinhibitory effects of mivazerol were abolished by prior yohimbine administration. Simultaneously, mivazerol did not induce any postjunctional adrenoceptor blockade as it did not affect noradrenaline cardiac and hemodynamic effects. On the contrary, through postjunctional alpha 2-adrenoceptor stimulation, mivazerol, in this pithed preparation, dose-dependently increased blood pressure, total peripheral and hindlimb vascular resistances, but heart rate was not affected. We conclude that, in the pithed rat, mivazerol exerts strong peripheral sympathoinhibitory effects. The mechanism involved is prejunctional alpha 2-adrenoceptor activation as i) mivazerol does not display any postsynaptic alpha-adrenoceptor blocking effect--it even behaves as as postsynaptic alpha 2-adrenoceptor agonist--and ii) yohimbine abolishes mivazerol's sympathoinhibitory effects. Thus, direct peripheral together with central mechanisms contribute to mivazerol's sympathoinhibitory effects and ultimately to its cardioprotective action.

Agmatine, an endogenous modulator of noradrenergic neurotransmission in the rat tail artery.

1. We investigated the vascular effects of agmatine (decarboxylated arginine), an endogenous ligand for alpha 2-adrenoceptors and non-adrenoceptor imidazoline (I-) receptors, present in endothelium, smooth muscle and plasma, using the rat tail artery as a model. 2. While by itself agmatine (10 nM-1 mM) was without effect on isolated arterial rings, at the highest concentration used (1 mM) it slightly increased EC50 values for contractions elicited respectively by the alpha 1- and alpha 2- adrenoceptor agonists methoxamine and clonidine. 3. Agmatine (0.03-1 mM) produced a concentration-dependent transient inhibition of the contractions induced by transmural nerve stimulation (TNS; 200 mA, 0.2 ms, 1 Hz, 10 s). This effect was abolished by the alpha 2-adrenoceptor antagonists, rawolscine and idazoxan. 4. In the presence of rawolscine or idazoxan, agmatine produced a concentration-dependent delayed facilitation of TNS-induced contractions, which was prevented by cocaine. 5. Neither inhibitory nor potentiating actions were produced by agmatine on contractions induced by noradrenaline (NA) administration. 6. Agmatine did not directly affect [3H]-NA uptake in bovine cultured chromaffin cells. 7. Agmatine can regulate vascular function by two opposing actions at sympathetic nerve terminals, with different latencies: a transient inhibition of NA release mediated by prejunctional alpha 2-adrenoceptors and a cocaine-sensitive delayed facilitation the mechanism of which is undetermined at present. 8. The results reveal the existence of a novel endogenous amine modulating NA release in the perivascular sympathetic terminals.