Activation Of Prejunctional Alpha 2-adrenoceptors Research Articles

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.

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.

Prejunctional interaction of α2‐adrenoceptors and D2 dopamine receptors on perivascular sympathetic nerves

1. Potential interaction between release-inhibiting prejunctional alpha 2-adrenoceptors and D2 dopamine receptors was investigated by measuring D2 receptor-mediated inhibition of stimulation-evoked tritium overflow from perfused rat tail arteries incubated with [3H]-noradrenaline. 2. In the presence of cocaine (10(-5)M), which enhanced stimulation-evoked tritium overflow, the D2 dopamine agonist N-0923 [(S)-(-) 2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin] (10(-8)M) was less effective at inhibiting stimulation-evoked tritium overflow compared to inhibition in the absence of cocaine. 3. In the presence of cocaine, yohimbine (10(-6)M) enhanced stimulation-evoked [3H]-noradrenaline release. Under these conditions, inhibition produced by N-0923 was enhanced compared to tissues without yohimbine. 4. In the presence of cocaine, the alpha 2-adrenoceptor agonist UK-14,304 (10(-7)M) reduced stimulation-evoked tritium overflow. However, in this case, N-0923-mediated inhibition was not significantly altered. 5. In most circumstances increasing or reducing activation of prejunctional alpha 2-adrenoceptors resulted in attenuation or enhancement, respectively, of D2 receptor activation. However, in the case of UK-14,304 this relationship did not hold. Thus, most but not all the evidence supports an interaction between prejunctional alpha 2-adrenoceptors and D2 dopamine receptors.

Chloroethylclonidine: an irreversible agonist at prejunctional alpha 2-adrenoceptors in rat vas deferens.

1. The possibility that chloroethylclonidine (CEC) activates prejunctional alpha 2-adrenoceptors was studied in the isolated vas deferens of the rat. Tissues were stimulated electrically and both the stimulation-evoked overflow of tritium (after preincubation with [3H]-noradrenaline) and the purinergic contraction component (isolated by prazosin 0.3 microM) were measured. 2. CEC (0.1-3 microM) concentration-dependently reduced the overflow of tritium evoked by trains of 6 pulses/100 Hz. The inhibition by CEC was not altered by prazosin (0.3 microM) but was prevented by pre-exposure to rauwolscine (0.3 microM). The inhibition, once established, did not fade upon washout of CEC, even when the washout fluid contained rauwolscine (0.3 microM). 3. CEC (0.1-3 microM) concentration-dependently reduced the purinergic component of contractions elicited by single pulses. The inhibition, again, was prevented by pre-exposure to rauwolscine (0.3 microM) and once established, did not fade upon washout of CEC, even when the washout fluid contained rauwolscine (0.3 microM). 4. CEC (3 microM) reduced the overflow of tritium evoked by 20 pulses/10 Hz, did not alter the overflow evoked by 100 pulses/10 Hz and increased the overflow evoked by 500 pulses/10 Hz. 5. CEC (3 microM) reduced the early peak, but increased the late plateau phase, of purinergic contractions elicited by 100 pulses/10 Hz. 6. It is concluded that CEC reduces the release of noradrenaline and a purinergic co-transmitter by irreversible activation of prejunctional alpha 2-adrenoceptors. CEC seems to be a partial alpha 2-agonist with an efficacy lower than that of noradrenaline. The prejunctional inhibitory effect limits the suitability of CEC for the characterization of postjunctional alpha 1-adrenoceptors mediating responses to sympathetic nerve stimulation.

Role of cyclic AMP in the release of noradrenaline from isolated rat atria. Effect of pretreatment with clenbuterol.

The possible role of cyclic AMP (cAMP) on tritium overflow evoked by stimulation of the cardio-accelerant nerves was studied in rat atria preincubated with [3H]-noradrenaline. Addition of the activator of adenylate cyclase forskolin (1 mumol/l), or of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 100 mumol/l), did not affect both basal and evoked overflow. However, in the presence of the alpha 2-adrenoceptor antagonist yohimbine (0.03 mumol/l) both forskolin and IBMX increased the stimulation-induced transmitter overflow by 49% and 141%, respectively (compared to yohimbine 0.03 mumol/l). Thus, in rat atria the cAMP-dependent facilitation of noradrenaline release is only present when the autoinhibition exerted by activation of prejunctional alpha 2-adrenoceptors is blocked. Propranolol (0.1 mumol/l) that did not produce any effect on noradrenaline release markedly reduced the facilitatory response induced by forskolin in the presence of yohimbine. When rats were pretreated with the beta 2-adrenoceptor agonist clenbuterol (0.3 mg.kg-1, s.c., twice daily, 14 days), a treatment which desensitizes beta-adrenoceptor-mediated facilitation of noradrenaline release (Kazanietz and Enero 1989), the facilitatory effect of forskolin and IBMX in the presence of yohimbine was abolished. The results indicate that in rat atria the effect of forskolin and IBMX on noradrenaline release are only to be observed after blockade of presynaptic alpha 2-adrenoceptor autoinhibition. beta-adrenoceptor blockade or clenbuterol pretreatment decreases the facilitatory response to forskolin and hence prejunctional beta-adrenoceptor-mediated enhancement of noradrenaline release is linked to the stimulation of adenylate cyclase.

Omega conotoxin and prejunctional modulation of the biphasic response of the rat isolated urinary bladder to single pulse electrical field stimulation

1. Single pulse electrical field stimulation (EFS) produces a biphasic response of muscle strips of the rat isolated urinary bladder consisting of an early and a late contraction which were atropine-resistant and atropine-sensitive, respectively. Repeated application of desensitizing doses of the P2 purinoceptor agonist, alpha, beta-methylene ATP (mATP) inhibited the early response while leaving unaffected the late component. 2. Omega conotoxin (CTX, 0.1 microM) inhibited both the early and the late response either in control conditions or after enhancement by physostigmine (0.1 microM). The effect of CTX was, in both cases, more pronounced on the late than the early response to EFS. CTX (0.1 microM) failed to affect contraction produced by ATP or acetylcholine at concentrations (0.3 mM and 0.5 microM) which produced a response similar to that to EFS. 3. The effect of physostigmine was more intense for the late than the early response and was abolished by atropine. In the presence of CTX, physostigmine enhanced both the early and the late components of the mechanical response to EFS. 4. Nifedipine (0.1-1 microM) reduced to a similar extent both the early and late responses. Bay K 8644 (1 microM) produced a marked enhancement of the response to EFS, which, however, did not have a distinct late peak. In the presence of Bay K 8644, either atropine (3 microM) or tetrodotoxin (1 microM) had minor inhibitory effects indicating the myogenic origin of the response. 5. Neurokinin A (0.1-1 nM) enhanced both the early and late responses to EFS without affecting the contraction produced by exogenous acetylcholine or ATP. A consistent potentiation was evident also in the presence of CTX and for the early response, in the presence of atropine. Clonidine (3 microM) inhibited the response to EFS either in the absence or the presence of physostigmine. The inhibitory effect of clonidine, shown previously to depend upon activation of prejunctional alpha 2-adrenoceptors, was still observed in presence of CTX or atropine. 6. It is concluded that CTX-sensitive voltage dependent calcium channels play a more important role in determining the cholinergic rather than the non-cholinergic, putatively purinergic, component of the biphasic response of the rat bladder to single pulse EFS. The action of CTX is likely to be exerted on N-type rather than L-type (dihydropyridine-sensitive) calcium channels. Prejunctional modulation (enhancement by neurokinin A, inhibition by clonidine) occurs even in the presence of CTX-sensitive channels blockade.

No influence of prejunctional α2‐adrenoceptors on the effects of nicotine and tyramine in guinea‐pig atria

1. Guinea-pig left atria were stimulated at a rate of 0.5 Hz and preloaded with 10 microCi 7-[3H]noradrenaline. In the presence of cocaine, 3 x 10(-6) mol l-1, and atropine, 10(-7) mol l-1, noradrenaline release was stimulated twice in each atrium, either by electrical field stimulation (one pulse, 0.2 ms 30 V, during each refractory period for 5 min) or by addition of either nicotine, 3 x 10(-5) mol l-1, or tyramine 10(-5) mol l-1. 2. The release of radioactivity caused by field stimulation and the associated positive inotropic effect were almost abolished by 3 x 10(-8) mol l-1 tetrodotoxin. Pretreatment with 3 x 10(-7) mol l-1 idazoxan increased the effects of field stimulation, while they were attenuated in the presence of clonidine, 3 x 10(-7) mol l-1. 3. The inotropic effect and the release of radioactivity caused by nicotine were greatly attenuated by tetrodotoxin, but were not significantly influenced by idazoxan or clonidine. 4. The release of radioactivity caused by tyramine and the inotropic effect of this drug were resistant to tetrodotoxin pretreatment and were not modified by idazoxan or clonidine. 5. It is concluded that nicotine releases noradrenaline by initiating a propagated action potential. But activation of prejunctional alpha 2-adrenoceptors does not seem to attenuate the transmitter release by limiting the spread of the action potential in the prejunctional sympathetic neuronal network.

Effect of /-ephedrine on cholinergic neurotransmission in the isolated guinea pig ileum.

In the isolated guinea pig ileum, the effects of I-ephedrine on the twitch response to field stimulation were investigated in the presence of propranolol. Ephedrine and clonidine inhibited the twitch response but not the contraction to exogenous acetylcholine. The inhibitory effect of clonidine was significantly diminished by yohimbine pretreatment. However, the inhibitory effect of ephedrine was not influenced by yohimbine, sulpiride or 6-hydroxydopamine (6-OHDA) pretreatment. Most of this action of ephedrine appeared to be cholinergic prejunctional in nature, but unrelated to activation of prejunctional alpha 2-adrenoceptors and dopamine sensitive receptors on the cholinergic nerves in this preparation.

Alpha-2 adrenergic modulation of norepinephrine secretion in the perfused rabbit iris-ciliary body.

Clonidine and other selective alpha-2 adrenergic agonists have been found to lower intraocular pressure in the eyes of rabbits and primates, including humans. It has been suggested that the ocular hypotensive response to alpha-2 agonists may be mediated, in part, by prejunctional inhibition of norepinephrine secretion at intraocular synapses. In this study, we have investigated the effects of adrenergic agonists and antagonists on field-stimulated, Ca++-dependent release of 3H-norepinephrine (3H-NE) from isolated, perfused rabbit iris-ciliary bodies and have utilized radioligand binding methods to identify prejunctional adrenoceptors in this tissue. Clonidine (10(-9)-10(-5) M) produced a dosage-dependent inhibition of stimulation-evoked 3H-NE secretion (EC50 approximately equal to 3 X 10(-8) M), but did not alter basal secretion. Other adrenergic agonists capable of activating alpha-2 adrenoceptors (e.g., epinephrine, norepinephrine and xylazine) also significantly depressed 3H-NE secretion, whereas selective alpha-1 adrenergic or beta adrenergic agonists were without effect. Clonidine-mediated inhibition of 3H-NE release was reversed by the selective alpha-2 antagonist yohimbine (10(-7) M), but was unaffected by prazosin or timolol. Yohimbine alone markedly enhanced 3H-NE secretion, indicating tonic activation of prejunctional alpha-2 adrenoceptors by endogenous released norepinephrine. Forskolin or 8-bromo-cAMP, which alone enhanced norepinephrine secretion, failed to attenuate the inhibitory responses to alpha-2 agonists. 3H-rauwolscine binding measurements showed a small decrease in alpha-2 receptor sites in iris-ciliary body membranes following surgical sympathetic denervation. It is concluded that the rabbit iris-ciliary body contains functional, prejunctional alpha-2 adrenoceptors which may play an autoregulatory role in vivo and contribute to the ocular effects of adrenergic drugs.

Adrenergic transmission in the dog mesenteric vein and its modulation by alpha-adrenoceptor antagonists.

Adrenergic transmission was investigated in the dog mesenteric vein by recording electrical responses of single smooth muscle cells to perivascular nerve stimulation. Perivascular nerve stimulation generated an excitatory junction potential (e.j.p.) and a slow depolarization of the membrane. The amplitude of the e.j.p. was increased by increasing the stimulus intensity, and at high intensity, a spike potential was generated. Repetitive stimulation of the nerves showed facilitation of e.j.ps and enhanced the amplitude of slow depolarization. A linear relationship was observed between the amplitude of the e.j.p. and of slow depolarization. The slow depolarization was inhibited by application of yohimbine or phentolamine, but not by prazosin. The amplitude of e.j.p. was increased by prazosin and was decreased by yohimbine. Both e.j.p. and slow depolarization were inhibited by guanethidine or tetrodotoxin. Exogenously applied noradrenaline depolarized the muscle membrane and, in high concentrations (greater than 10(-7)M), generated slow waves. These effects of noradrenaline were blocked by yohimbine. High concentrations of prazosin (greater than 10(-6)M) showed weak inhibitory effects on the noradrenaline actions. The amplitude of e.j.p. was decreased by exogenously applied noradrenaline in a dose-dependent manner. The inhibitory effect of noradrenaline on the e.j.p. was suppressed by yohimbine, but not by prazosin or phentolamine. Phentolamine, but not prazosin, enhanced the facilitation process of e.j.ps. This effect was not suppressed by exogenously applied noradrenaline. Application of neostigmine but not of atropine, reduced the e.j.p. amplitude without affecting the slow depolarization. It was concluded that, in the dog mesenteric vein, perivascular nerve stimulation produced three types of electrical responses of the smooth muscle membrane, i.e., e.j.p., slow depolarization and spike potential. The slow depolarization was generated by activation of alpha 2-adrenoceptors. Exogenously applied noradrenaline reduced the e.j.p. amplitude through activation of prejunctional alpha 2-adrenoceptors, but the reduction may not involve alpha-autoinhibitory mechanisms.

A study of stimulation-evoked activation of alpha 2-adrenoceptors in the rat isolated vas deferens.

Experiments were performed with prostatic and epididymal segments of rat vas deferens to determine whether alpha 2-adrenoceptors in this organ were activated during field stimulation of sympathetic terminals with short trains of pulses applied at low frequencies. In prostatic segments the magnitude of twitches evoked by trains of ten field pulses at 1 or 2 Hz declined after 2-3 s of stimulation. In contrast, facilitation of twitches and fusion of contractions occurred when similar stimulation was applied to epididymal segments. In prostatic segments from rats treated with reserpine (2.5 mg/kg i.p.) 24 h previously, there was no decline in the magnitude of twitches produced by successive impulses in a train of stimulating pulses. In epididymal segments from reserpine-treated rats facilitation of twitches in response to successive impulses in each train still occurred. In prostatic segments cocaine (5 and 10 mumol/l) enhanced twitch fade with stimulation at 1 and 2 Hz without altering the time for onset of this effect. In epididymal segments cocaine led to enhancement and prolongation of contractile responses. In prostatic segments yohimbine (0.01-0.06 mumol/l) reduced or reversed the effect of cocaine in enhancing twitch fade. In preparations where the reversal of the effect of cocaine by yohimbine was incomplete, subsequent addition of phentolamine (1 mumol/l) produced complete reversal. In epididymal segments yohimbine (0.01 mumol/l) produced a further enhancement in the twitch responses to stimulation at 1 and 2 Hz. Subsequent addition of phentolamine (1 mumol/l) reversed the facilitatory effects of cocaine and yohimbine. Propranolol (10 mumol/l) was without effect upon responses to stimulation in either segment of rat vasa deferentia. These experiments indicate that noradrenaline, released by short trains of impulses applied at low frequency to hypogastric nerve terminals activates prejunctional alpha 2-adrenoceptors in the prostatic segment of the vas deferens. In the epididymal portion the effects arising from activation of prejunctional alpha 2-adrenoceptors are outweighed by the consequences of activation of extrajunctional alpha 1-adrenoceptors located on longitudinally arranged muscle.