Alpha 2-autoreceptors Research Articles

Modulation of neurotransmitter release via histamine H3 heteroreceptors.

Presynaptic H3 receptors occur on histaminergic neurones of the CNS (autoreceptors) and on non-histaminergic neurones of the central and autonomic nervous system (heteroreceptors). H3 heteroreceptors, most probably located on the postganglionic sympathetic nerve fibres innervating the resistance vessels and the heart, have been identified in the model of the pithed rat. Furthermore, we could show in superfusion experiments that H3 heteroreceptors also occur on the sympathetic neurones supplying the human saphenous vein and the vasculature of the pig retina and on the serotoninergic, dopaminergic and noradrenergic neurones in the brain of various mammalian species, including man. The effects of three recently described H3 receptor ligands were studied in superfused mouse brain cortex slices. The potency of the novel H3 receptor agonist imetit exceeded that of R-(-)-alpha-methylhistamine (the reference H3 receptor agonist) by one log unit and that of histamine by almost two log units. Clobenpropit was shown to be a competitive H3 receptor antagonist, exhibiting a pA2 as high as 9.6 (exceeding the pA2 of the reference H3 receptor antagonist thioperamide by one log unit). The irreversible antagonism of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was also studied. Interactions of the H3 heteroreceptor with the dopamine autoreceptor in mouse striatal slices and the alpha 2-autoreceptor in mouse brain cortex slices could be demonstrated. Activation of alpha 2-autoreceptors decreases the H3 receptor-mediated effect. Blockade of alpha 2-autoreceptors increases the H3 receptor-mediated effect only if the alpha 2-autoreceptors are simultaneously activated by endogenous noradrenaline. The H3 receptor-mediated inhibition of noradrenaline release in mouse brain cortex slices was attenuated by the K+ channel blocker tetraethylammonium but this attenuation was abolished by reduction of the Ca2+ concentration in the medium (to compensate for the facilitatory effect of tetraethylammonium on noradrenaline release). Accordingly, we assume that the H3 receptors are not coupled to voltage-sensitive K+ channels. Pertussis toxin and N-ethylmaleimide attenuated the H3 receptor-mediated effect in the mouse brain cortex, suggesting that the H3 receptors are coupled to a G protein (eg Gi or Go). However, negative coupling to an adenylate cyclase does not appear to exist since an H3 receptor-mediated inhibition of cAMP accumulation was not obtained in mouse brain cortex membranes. H3 receptor ligands are currently undergoing clinical testing and might become new remedies for the treatment of disease of the gastrointestinal and bronchial system and the CNS.

Modulation of noradrenaline release from the sympathetic nerves of the human saphenous vein and pulmonary artery by presynaptic EP3- and DP-receptors.

1. Spirally cut strips of the human saphenous vein and pulmonary artery were used to determine the pharmacological properties of the presynaptic prostanoid receptors involved in the modulation of sympathetic [3H]-noradrenaline release. Strips preincubated with [3H]-noradenaline were superfused with physiological salt solution containing inhibitors of uptake1 and uptake2 and rauwolscine to eliminate involvement of presynaptic alpha 2-adrenoceptors. Tritium overflow was evoked by transmural electrical stimulation (standard frequency: 2 Hz). 2. In the saphenous vein, prostaglandin E2 (PGE2) inhibited the electrically-evoked tritium overflow; at the highest concentration investigated, tritium overflow was inhibited by more than 75% and the pEC50 value was 7.00. These effects were mimicked by prostaglandin E1, the EP1/EP3 receptor agonist, sulprostone and the EP2/EP3 receptor agonist, misoprostol with the rank order (pEC50): sulprostone (8.60) > PGE1 (7.25) > misoprostol (6.96). This rank order of potency suggests that the inhibitory effect of the drugs is mediated by presynaptic EP3-receptors. In contrast, PGF2 alpha did not inhibit evoked tritium overflow; the IP/EP1 receptor agonist iloprost and the stable thromboxane A2 analogue U 46619 (9, 11-dideoxy-11 alpha,9 alpha-epoxy-methanoprostaglandin F2 alpha) produced inhibition only at concentrations above 1 microM. 3. The EP1-receptor antagonist, AH 6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid) had no effect on the evoked tritium overflow nor did it modify the inhibitory effect of PGE2, further excluding involvement of inhibitory presynaptic EP1-receptors. 4. PGD2 caused a facilitation of evoked tritium overflow in the saphenous vein; this facilitation is probably mediated by presynaptic DP-receptors, since it was abolished by the selective DP-receptor antagonist, BW A868C (3-benzyl-5-(6-carboxyhexyl)-1-(2-cyclohexyl-2-hydroxyethylamino)hydantoin).5. In the pulmonary artery, sulprostone (pECm value 8.35), misoprostol (7.70) and PGE2 (6.80)inhibited electrically-evoked tritium overflow. This rank order of potency is consistent with the involvement of inhibitory presynaptic EP3-receptors.6. These results suggest that the sympathetic nerve fibres of both human saphenous vein and pulmonary artery are endowed with presynaptic inhibitory EP3 receptors. The EP3-receptors do not interact with the alpha 2-autoreceptors. In addition, the human saphenous vein seems to be endowed with presynaptic facilitatory DP-receptors.

Release of acetylcholine and noradrenaline from the cholinergic and adrenergic afferents in rat hippocampal CA1, CA3 and dentate gyrus regions.

An attempt was made to study the release of acetylcholine (ACh) and noradrenaline and their presynaptic modulation in isolated slice preparations dissected from different subfields of the hippocampus: CA1, CA3 and the dentate gyrus. The slices were perfused and loaded with [3H]choline or with [3H]noradrenaline. The release in response to field stimulation was determined radiochemically and the content of transmitters was assayed by a chemiluminescent method or by HPLC combined with electrochemical detection. After 30 min of loading with [3H]choline there were marked subregional differences in the specific activity of [3H]ACh content. The highest concentration was measured in the dentate gyrus and the lowest in CA3. Evidence was obtained that in all three subfields the cholinergic axon terminals are equipped with inhibitory muscarinic autoreceptors and the noradrenergic terminals with alpha 2-autoreceptors, as indicated by an increase in transmitter release when the tissue was exposed to selective muscarinic or alpha 2-adrenoceptor antagonists. In contrast, the cholinergic boutons are not equipped with alpha 2-adrenoceptors, and noradrenergic terminals do not possess inhibitory muscarinic receptors. It is therefore concluded that while the release of both ACh and noradrenaline is controlled by negative feedback modulation, there is no possibility of establishing a presynaptic inhibitory interaction between the two.

The effect of two different types of stress on locus coeruleus alpha-2 receptor binding

The central noradrenergic neurons of the locus coeruleus (LC) are important in regulating an animal's response to a Stressor. Changes in alpha-2 autoreceptors on LC neurons during stress could alter the functional state of the LC. We investigated the changes in binding of LC alpha-2 receptors immediately following l h and 4 h of two Stressors: novelty + cold and prone restraint. We measured the specific binding of the alpha-2 receptor antagonist [ 3H]idazoxan in the LC of rats using in vitro autoradiography. Specific binding was increased after l h of novelty + cold exposure but decreased after 4 h, and after both 1 and 4 h of prone restraint. The increase after l h of stress may be due to the ability of LC neurons to regulate their own activity because this would produce a dampening of the high LC activity produced by the Stressor. However, extended time and/or intensity of a Stressor may overwhelm this initial response and produce an agonist-induced decrease in receptor binding.

Modulation of N-methyl-D-aspartate (NMDA)-stimulated noradrenaline release in rat brain cortex by presynaptic alpha 2-adrenoceptors.

Rat brain cortex slices and synaptosomes preincubated with [3H]noradrenaline were used to investigate whether the NMDA-evoked noradrenaline release is modulated by agonists or antagonists at presynaptic alpha 2-adrenoceptors. In experiments on slices, noradrenaline and the preferential alpha-adrenoceptor agonists talipexole (former B-HT 920) and clonidine inhibited the NMDA-evoked tritium overflow whereas the selective alpha 1-adrenoceptor agonists cirazoline and methoxamine were ineffective. The alpha 2-adrenoceptor antagonists rauwolscine and idazoxan facilitated the NMDA-evoked tritium overflow whereas the preferential alpha 1-adrenoceptor antagonist prazosin was ineffective. The concentration-response curve of talipexole for its inhibitory effect on NMDA-evoked overflow was shifted to the right by idazoxan (apparent pA2 = 7.5). The EC50 of NMDA (97 mumol/l) for its stimulating effect on tritium overflow was not substantially changed by blockade of alpha 2-autoreceptors with 1 mumol/l rauwolscine (EC50 of NMDA in the presence of the alpha 2-adrenoceptor antagonist, 155 mumol/l), but the maximal overflow of tritium was increased 2.5 fold by this rauwolscine concentration. In experiments on synaptosomes, talipexole and noradrenaline inhibited the NMDA-evoked tritium overflow. The inhibitory effect of talipexole was abolished by idazoxan which, given alone, was ineffective, as was prazosin. Talipexole did also not produce an inhibition when tritium overflow was evoked by NMDA in the presence of omega-conotoxin GVIA 0.1 mumol/l; the latter, by itself, decreased the response to NMDA by about 55%.(ABSTRACT TRUNCATED AT 250 WORDS)

Desensitization of inhibitory prejunctional ?2-adrenoceptors and putative imidazoline receptors on rabbit heart sympathetic nerves

To find out whether sympathetic nerves of the rabbit heart possess pharmacologically relevant prejunctional imidazoline receptors different from alpha-autoreceptors, the inhibition by oxymetazoline, aganodine and BDF 6143 (4-chloro-2-[2-imidazoline-2-ylamino]-isoindoline hydrochloride) of endogenous noradrenaline overflow evoked by stimulation of extrinsic postganglionic sympathetic nerves (0.66 Hz, 80 pulses) was investigated. In addition we wanted to find out whether either type of these prejunctional receptors undergoes desensitization upon pre-exposure to respective agonists. The alpha 2-adrenoceptor agonist oxymetazoline inhibited the evoked noradrenaline overflow (2.9 nmol/l, IC50; about 90%, maximum inhibition). The inhibition was antagonized by rauwolscine (-log KB 8.20). This confirms the presence of alpha 2-autoreceptors. Endogenous noradrenaline activated autoinhibition to a small extent as indicated by a rauwolscine-induced increase in evoked overflow by less than 2-fold. The alpha 2- and imidazoline receptor agonist aganodine inhibited the evoked noradrenaline overflow (2.4 nmol/l, IC50; about 80%, maximum inhibition). The inhibition was antagonized by rauwolscine with a potency (-log KB 6.75), about 1/30 of that found at the alpha 2-autoreceptor. Neither an alpha 2-selective low concentration of rauwolscine nor the alpha 1-adrenoceptor antagonist prazosin, nor SKF 104078, a mixed alpha 1/2-antagonist, reduced the aganodine effect. The alpha 2-adrenoceptor antagonist and imidazoline receptor agonist BDF 6143 inhibited the evoked noradrenaline overflow (18 nmol/l, IC50; about 70% maximum inhibition). The inhibition was insensitive to a low rauwolscine concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Central α2‐autoreceptors: agonist dissociation constants and recovery after irreversible inactivation

1. Rats received an intraperitoneal injection of 1.6 mg kg-1 N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) to achieve an irreversible inactivation of presynaptic release-modulating alpha 2-autoreceptors. Cerebral cortex slices were prepared at different times after the injection (24, 48, 96, 192, 336, 774 h), incubated with [3H]-noradrenaline ([3H]-NA), superfused and stimulated electrically with 4 pulses at 100 Hz (= autoinhibition-free condition). Overflow of radioactivity was used to measure release. Furchgott analysis was used to estimate agonist dissociation constants (KA) and pool size of resynthesized receptors (q). 2. The KA values of the three alpha 2-autoreceptor agonists, bromoxidine (UK-14304), clonidine and noradrenaline (NA) were 187 nM, 72 nM, and 1202 nM, respectively. 3. The release-inhibiting effects of the agonists returned considerably faster than the receptor pool. The calculated half-lives for the recovery of the maximal release-inhibiting effects of bromoxidine, clonidine and NA were 30.7, 63.6 and 20.8 h, respectively, whereas the half-life for the recovery of the receptor pool was 445 h. 4. The data indicate a large receptor reserve at presynaptic alpha 2-autoreceptors for the agonists used and validate the use of EEDQ as a tool for the determination of agonist dissociation constants.

Subclassification of release-regulating alpha 2-autoreceptors in human brain cortex.

1. Release-regulating alpha 2-autoreceptors in human brain were characterized pharmacologically in cortical slices from patients undergoing neurosurgery to remove subcortical tumours; the slices were prelabelled with [3H]-noradrenaline ([3H]-NA) and stimulated electrically (3 Hz, 2 ms, 24 mA) under superfusion conditions. 2. The stimulus-evoked tritium overflow was almost totally Ca(2+)-dependent and tetrodotoxin-sensitive. 3. Clonidine and oxymetazoline 0.01 to 1 microM inhibited in a concentration-dependent manner the evoked overflow of tritium. The two drugs were equipotent (EC50 = 0.03 microM) and their maximal effect was approx. 45%. Phenylephrine and methoxamine, up to 1 microM, did not affect tritium overflow. 4. Yohimbine (0.01-0.1 microM) shifted the concentration-response curve of clonidine to the right. The calculated pA2 value was 8.29. 5. Prazosin and 2-[2-[4-(o-methoxyphenyl)piperazine-1-yl]ethyl]-4,4- dimethyl-1,3(2H,4H)-isoquinolinedione (AR-C 239), tested at 0.3 microM, did not modify the concentration-response curve of clonidine. 6. The effect of clonidine was antagonized by (+)-mianserin (pA2 = 7.74), but not by up to 0.3 microM of the (-)-enantiomer. The concentration-response curve of clonidine was shifted to the right by the novel alpha 2-adrenoceptor antagonist, 5-chloro-4-(1-butyl-1,2,5,6-tetrahydropyridin-3-yl)-thiazole-2-ami ne (Z)-2-butenedioate (1:1) salt (ORG 20350) (pA2 = 7.55). 7. Yohimbine, (+)-mianserin and ORG 20350, but not prazosin and (-)-mianserin, increased the electrically-evoked tritium overflow, suggesting that autoreceptors may be tonically activated by endogenous NA. 8. Desipramine (1 microM) increased evoked tritium overflow from human cortex slices. The effect of clonidine (0.01- 1 g1M) on the evoked overflow of tritium was reduced in presence of 1 muM desipramine.9. It is proposed that autoregulation of NA release can occur in human cerebral cortex. The process involves activation of alpha 2-adrenoceptors which may be either the alpha2X or the alpha2D subtype.

Alpha 2-autoreceptor-mediated modulation of tyrosine hydroxylase activity in noradrenergic regions of the rat brain in vivo.

The physiological importance of brain alpha 2-adrenoceptors in controlling the activity of tyrosine hydroxylase in noradrenergic regions was investigated using the accumulation of 3,4-dihydroxyphenylalanine (DOPA) after decarboxylase inhibition as a measure of the rate of tyrosine hydroxylation (and synthesis of noradrenaline) in vivo. In the hypothalamus and cerebral cortex, clonidine (0.025-1 mg/kg, i.p.) decreased (18%-43%) and idazoxan (0.1-80 mg/kg, i.p.) increased (20%-73%) the synthesis of DOPA in a dose-dependent manner. Moreover, pretreatment with idazoxan (0.1 mg/kg) antagonized the effect of clonidine (0.1 mg/kg) in the hypothalamus. After treatment with reserpine (5 mg/kg, s.c., 18 h before decapitation) and depletion of noradrenaline, clonidine (0.5 mg/kg) continued to decrease (50%-55%) but idazoxan (20 mg/kg) failed to increase the synthesis of DOPA, which suggested the involvement of an alpha-auto-receptor mechanism. Acute treatments of rats (not exposed to reserpine) with a wide variety of adrenoceptor agonists such as guanfacine 6, B-HT920, xylazine, bromoxidine (1 mg/kg) and antagonists such as yohimbine, phentolamine, prazosin (10 or 20 mg/kg) resulted in significant decreases (15%-55%) or increases (21%-99%) in the synthesis of DOPA in both brain regions. However, other agonists (oxymetazoline, azepexole, tramazoline, methoxamine) and antagonists (tolazoline, dihydroergotamine, phenoxybenzamine, propranolol) did not modify the synthesis of DOPA. In the hypothalamus and cerebral cortex the effects of the drugs were consistent with the selectivity of alpha-adrenoceptor agonists and antagonists (except prazosin) for an alpha 2-adrenoceptor. The results also suggest that the alpha 2-autoreceptor that modulates the synthesis of noradrenaline in the rat brain appears to belong to the prazosin-sensitive alpha 2B-subtype.

Presynaptic alpha 2-adrenoceptors mediating inhibition of noradrenaline and 5-hydroxytryptamine release in rat cerebral cortex: further characterization as different alpha 2-adrenoceptor subtypes.

In a previous investigation it was suggested that the alpha 2-adrenoceptors regulating 3H-noradrenaline (3H-NA) release and the alpha 2-heteroreceptors regulating the release of 3H-5-hydroxytryptamine (3H-5-HT) from rat cerebrocortex synaptosomes represent different subtypes of the alpha 2-adrenoceptor in that (-)-mianserin potently blocked the receptors sited on 5-HT terminals but was ineffective at the autoreceptors (Raiteri et al. 1983). In this work a number of alpha 2-adrenoceptor antagonists were tested against NA as an inhibitor of the K+ (15 mmol/l)-evoked release of 3H-NA or 3H-5-HT (in presence of 1 mumol/l desipramine or citalopram, respectively) from superfused rat neocortex synaptosomes. The order of apparent affinity of the antagonists was: idazoxan greater than or equal to ORG 20769 (2-amino-4-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)-thiazole-5-ca rbonitrile (Z)-2-butenedioate (1:1) salt) greater than ORG 20350 (5-chloro-4-(1-butyl-1,2,5,6-tetrahydropyridin-3-yl)-thiazole-2- amine (Z)-2-butenedioate (1:1) salt) greater than or equal to ORG 20091 (5-chloro-4-(1-methyl-1,2,5,6-tetrahydropyridin-3-yl)-thiazole-2- amine (Z)-2-butenedioate (2:1) salt) at the alpha 2-autoreceptor and idazoxan greater than or equal to ORG 20769 greater than ORG 20091 much greater than ORG 20350 at the alpha 2-heteroreceptor. Prazosin (1 mumol/l) or AR-C 239 (1 mumol/l) (2-[2-[4-(o-methoxyphenyl)piperazine-1-yl]ethyl]-4,4-dimethyl- 1,3(2H,4H)-isoquinolinedione) were ineffective in both systems.(ABSTRACT TRUNCATED AT 250 WORDS)

Subclassification of the presynaptic α2‐autoreceptors in rabbit brain cortex

1. alpha 2-Adrenoceptor binding sites have been subclassified into alpha 2A sites of which a main characteristic is very low affinity for prazosin, and alpha 2B sites with relatively high affinity for prazosin. The presynaptic alpha 2-autoreceptors in rabbit brain cortex were studied in order to classify them in terms of alpha 2A and alpha 2B. Release of [3H]-noradrenaline in cortical slices was elicited by trains of 4 pulses delivered at 100 Hz. 2. Clonidine caused concentration-dependent inhibition of the stimulation-evoked overflow of tritium, with an EC50 of 7.5 nM and a maximal inhibition by 96%. 3. The following alpha-adrenoceptor antagonists shifted the concentration-response curve of clonidine to the right (antagonist-receptor dissociation constants KD in brackets): yohimbine (14 nM), 2-[2H-(1-methyl-1,3-dihydroisoindole)methyl]-4,5-dihydroimidazo le (BRL 44408; 15 nM) and 1,2-dimethyl-2,3,9,13betetrahydro-1H-dibenzo[c,f]imidazo[1,5-a]aze pine (BRL 41992; 630 nM). Prazosin 1 microM and 2-[2-[4-(o-methoxyphenyl)piperazine-1-yl]-ethyl]-4,4-dimethyl-1,3 (2H,4H)-isoquinolinedione (AR-C 239) 1 microM failed to antagonize the effect of clonidine. Higher concentrations of prazosin and AR-C 239 greatly accelerated the basal efflux of tritium. 4. The method used permits the functional determination of antagonist affinities undistorted by endogenous alpha 2-autoinhibition. A comparison with affinities derived from radioligand binding experiments indicates that the presynaptic alpha 2-autoreceptors in rabbit brain cortex are markedly different from the alpha 2B-subtype and probably belong to the prazosin-insensitive alpha 2A-subtype.

Involvement of presynaptic imidazoline receptors in the ?2-adrenoceptor-independent inhibition of noradrenaline release by imidazoline derivatives

An involvement of imidazoline recognition sites in the modulation of transmitter release was investigated in the rabbit pulmonary artery and aorta preincubated with [3H]noradrenaline and superfused with physiological salt solution containing cocaine, corticosterone and propranolol. Electrical impulses were applied transmurally at 0.66 or 2 Hz. In the absence of further drugs, rauwolscine as well as the imidazoline derivatives BDF 6143 [4-chloro-2-(2-imidazoline-2-ylamino)-isoindoline], idazoxan and phentolamine increased the 3H overflow from the pulmonary artery, evoked by electrical stimulation at 2 Hz; the effect was due to the alpha 2-autoreceptor blocking property of these drugs. The maximum increase in overflow obtainable with the imidazolines was considerably lower than with rauwolscine. The concentration-response curves of the imidazolines were bell-shaped. At 0.66 Hz, BDF 6143 did not facilitate, but concentration-dependently inhibited, whereas idazoxan failed to change the evoked 3H overflow. When, at the stimulation frequency of 2 Hz, presynaptic alpha 2-adrenoceptors were blocked by rauwolscine and/or pre-exposure to phenoxybenzamine, the electrically evoked 3H overflow from the pulmonary artery and/or aorta was inhibited by the following imidazoline derivatives: the alpha 2-adrenoceptor antagonists BDF 6143, idazoxan and phentolamine, the alpha 1-adrenoceptor agonist with alpha 2-blocking property cirazoline as well as the alpha 2-adrenoceptor agonists clonidine and moxonidine. The maximum inhibition caused by BDF 6143 was greater than that due to clonidine and moxonidine; the latter two, hence, behaved as partial agonists. At the stimulation frequency of 0.66 Hz, the imidazolines exhibited a higher potency than, but a similar intrinsic activity to that at 2 Hz. Noradrenaline did not affect the evoked 3H overflow. The BDF 6143-induced inhibition of evoked 3H overflow was not modified by metitepine, atropine, theophylline, dipyridamole and indometacin, but was counteracted by the partial agonists clonidine and moxonidine. The results exclude the possibility that alpha 1- and alpha 2-adrenoceptors, 5-HT1 receptors, muscarine receptors, P1 purinoceptors and prostaglandin receptors are involved in the imidazoline-induced inhibition of noradrenaline release. They provide evidence indicating that the inhibitory effect is mediated by imidazoline receptors on the postganglionic sympathetic nerve terminals of the rabbit pulmonary artery and aorta.

Heterogeneity of Presynaptic Serotonin Receptors on Sympathetic Neurones in Blood Vessels

Presynaptic serotonin (5-HT) receptors on the postganglionic sympathetic nerves, which mediate inhibition of noradrenaline release in blood vessels of various species and which interact with the presynaptic alpha 2-autoreceptors, are heterogeneous. In the rat vena cava, they are of the 5-HT1B subtype, in the pig coronary artery they belong to a novel, so far unknown class of 5-HT receptors, and in the human saphenous vein they could be classified as 5-HT1D. These results point to marked species differences and the need to carry out experiments in human vascular preparations. Presynaptic 5-HT receptors may be involved in the mechanism of action of the new antimigraine drug sumatriptan.

Regulatory Proteins in Presynaptic Function

Activation of alpha 2-adrenoceptors, opioid, A1-adenosine, and PGE receptors inhibited the stimulation-induced [3H]noradrenaline release in brain tissue in a concentration-dependent manner. Under experimental conditions (360 pulses/3 Hz) where the released noradrenaline activated the presynaptic alpha 2-autoreceptors, the effects of the heteroreceptor (k-opioid, A1-adenosine, PGE) agonists were decreased. By avoiding autoinhibition by either blockade of the alpha 2-autoreceptors with yohimbine or stimulating the tissue with four pulses/100 Hz, the heteroreceptor-mediated inhibition of [3H]noradrenaline release was markedly increased. The dependence of the heteroreceptor-mediated inhibition of evoked noradrenaline release on the extent of alpha 2-autoreceptor activation suggests a common postreceptor signal transduction pathway. PTX-catalyzed [32P]ADP ribosylation of synaptosomal membrane proteins revealed three bands of polypeptides with molecular weights corresponding to the alpha subunits of Go (39,000) and the Gi proteins (40,000, 41,000). Pretreatment with NEM reduced the PTX-induced 32P labeling by alkylating the alpha subunits at or near the site that is ADP ribosylated by PTX in a concentration-dependent manner. K(+)-evoked release of [3H]noradrenaline from synaptosomes indicated the presynaptic localization of the PTX-sensitive G proteins coupled to alpha 2-, k-, and A1-receptors of noradrenergic nerve terminals. Electrically evoked [3H]noradrenaline release was only increased by PTX or NEM in a time- and concentration-dependent manner when autoinhibition was present. The alpha 2-, opioid, and A1-adenosine receptor-mediated inhibition of [3H]noradrenaline release was impaired similarly by PTX or NEM treatment. In contrast, the inhibitory effect of PGE2 remained unaffected. These results indicate that presynaptic alpha 2-, opioid, and A1-receptors but not PGE receptors of noradrenergic nerve terminals are linked to PTX-sensitive G proteins. The interaction between the alpha 2-autoreceptors and the PGE receptors therefore does not occur at the level of a common pool of G proteins but at some subsequent step of the signal transduction mechanism.

Peripheral presynaptic and central effects of clonidine, yohimbine and rauwolscine on the sympathetic nervous system in rabbits.

The function of presynaptic alpha 2-autoreceptors at postganglionic sympathetic neurones under conditions of normal, ongoing sympathetic impulse traffic was studied in anaesthetized rabbits (alfadolone + alfaxalone). Clonidine was used as an alpha 2-adrenoceptor agonist, and yohimbine and rauwolscine were used as antagonists. Mean arterial pressure, postganglionic renal sympathetic firing rate, arterial plasma noradrenaline concentration and heart rate were measured before (basal values) and at the end of 3-min infusions of sodium nitroprusside and phenylephrine, which were given to modulate efferent activity in the sympathetic nervous system through the baroreflex. The nitroprusside- and phenylephrine-induced changes of mean arterial pressure produced the expected changes in sympathetic nerve activity, plasma noradrenaline and heart rate. Clonidine (5 micrograms kg-1 + 0.5 micrograms kg-1 min-1) reduced the basal mean arterial pressure, sympathetic nerve activity and heart rate. It also reduced the nitroprusside-induced increase in the plasma noradrenaline level without changing the nitroprusside-induced increase in sympathetic firing. These results, as well as the mean arterial pressure-sympathetic nerve activity and the sympathetic nerve activity-plasma noradrenaline function curves indicate that clonidine inhibited both sympathetic tone centrally and the average release of noradrenaline per action potential peripherally. Yohimbine (1 mg kg-1 + 0.1 mg kg-1 h-1) and rauwolscine (0.5 mg kg-1 + 0.1 mg kg-1 h-1) increased the basal plasma noradrenaline level without any increase of renal sympathetic nerve activity. They also enhanced the nitroprusside-induced increase in plasma noradrenaline without any enhancement of the nitroprusside-induced increase in sympathetic firing. The hypotensive response to nitroprusside was attenuated, whereas the heart rate response was augmented. These results, as well as the mean arterial pressure-sympathetic nerve activity and the sympathetic nerve activity-plasma noradrenaline function curves indicate that the main effect of yohimbine and rauwolscine was to increase the average release of noradrenaline per action potential. The simultaneous measurement of postganglionic sympathetic nerve activity and the arterial plasma noradrenaline concentration proved suitable to differentiate central (or ganglionic; this distinction was not possible) effects of alpha 2-adrenoceptor ligands from peripheral presynaptic effects. The results show that endogenous presynaptic, alpha 2-adrenergic autoinhibition of noradrenaline release from postganglionic sympathetic neurones operates physiologically in anaesthetized rabbits with ongoing, uninterrupted sympathetic nerve activity. The results also indicate that blockade of alpha 2-autoreceptors enhances the sympathetic reflex compensatory response to a hypotensive stimulus.

Estimation ofpA2 values at presynaptic ?2-autoreceptors in rabbit and rat brain cortex in the absence of autoinhibition

An attempt was made to determine pA2 values of antagonists at the presynaptic, release-inhibiting alpha 2-autoreceptors of rabbit and rat brain cortex under conditions when there was very little released noradrenaline in the autoreceptor biophase and, hence, pA2 values were not distorted by endogenous autoinhibition. Cortex slices were preincubated with 3H-noradrenaline and then superfused and stimulated by trains of 4 pulses delivered at 100 Hz or, in a few cases, by trains of 36 pulses at 3 Hz. The alpha-adrenoceptor agonists clonidine, noradrenaline, and alpha-methylnoradrenaline concentration-dependently decreased the stimulation-evoked overflow of tritium. The alpha-adrenoceptor antagonists yohimbine, rauwolscine and idazoxan did not increase the overflow of tritium elicited by 4 pulses/100 Hz in rabbit brain slices and increased it only slightly in rat brain slices. In contrast, the antagonists increased markedly the overflow at 36 pulses/3 Hz. All antagonists caused parallel shifts to the right of the concentration-response curves of clonidine, noradrenaline, and alpha-methylnoradrenaline. pA2 values were calculated either from linear regression of log [agonist concentration ratio - 1] on log [antagonist concentration] or from sigmoid curve fitting. The slopes of the linear regression lines were close to unity, and the pA2 values calculated by the two methods agreed well. There was no consistent preferential antagonism of any antagonist to any agonist. pA2 values determined with stimulation by 4 pulses/100 Hz were by 0.53-0.80 log units higher than those determined with stimulation by 36 pulses/3 Hz.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuropeptide Y increases the inhibitory effects of clonidine on potassium evoked 3H-noradrenaline but not 3H-5-hydroxytryptamine release from synaptosomes of the hypothalamus and the frontoparietal cortex of the male Sprague-Dawley rat.

The release of 3H-noradrenaline (3H-NA) and of 3H-5-hydroxytryptamine (3H-5-HT) evoked by high-K+ (15 mM) was studied in synaptosomes isolated from the hypothalamus and the frontoparietal cortex of the male Sprague-Dawley rat using a superfusion apparatus. Based on concentration-response curves obtained by analyzing the full-time course of the inhibitory effects of clonidine on 3H-NA and on 3H-5-HT release neuropeptide Y (NPY) (1 nM) was shown to significantly increase the ability of clonidine to inhibit 3H-NA release in synaptosomes isolated from the hypothalamus and from the frontoparietal cortex. NPY (1 nM) alone had no effect on K+-evoked 3H-NA release from these regions. In contrast, NPY (1 nM) did not modulate the inhibitory effects of clonidine on 3H-5-HT release in the above mentioned regions. These results indicate that NPY can increase the sensitivity of the alpha 2-autoreceptors belonging to hypothalamic NA and/or to adrenaline nerve terminals and to cortical NA nerve terminals, while the alpha 2-heteroreceptors inhibiting 3H-HT release in the same brain regions appear not to be regulated by high affinity NPY receptors. Thus, alpha 2-autoreceptors and alpha 2-heteroreceptors appear to be differentially controlled by high affinity NPY receptors at least with regard to regulation of 3H-NA and 3H-5-HT release, respectively.

Effects of chlorpromazine in the nucleus reticularis lateralis on the cat cerebellar potentials.

In the experiments reported here, we investigated chlorpromazine (CPZ)-induced (CPZ)-induced enhancement of the cat cerebellar potentials evoked by peripheral nerve stimulation, with regard to the subtypes of adrenoceptor in the nucleus reticularis lateralis (LRN). Electrical stimulus of the locus coeruleus (LC) at high frequencies enhanced cerebellar potentials evoked by peripheral nerve stimulation. Although similar stimulus increased them after pretreatment with an alpha 2-antagonist, yohimbine, these enhancements were not recognized by pretreatment with an alpha 1-antagonist, prazosin. Microinjection of norepinephrine (NE: 10 micrograms) into LRN decreased cerebellar potentials, and conversely, 30 micrograms of NE significantly increased them. Although microinjection of an alpha 2-adrenoceptor agonist, clonidine, into the LRN depressed cerebellar potentials, clonidine-induced decrease was obviously antagonized by pretreatment with CPZ. Furthermore, an alpha 1-adrenoceptor agonist, phenylephrine, into the LRN increased cerebellar potentials. Pretreatment with CPZ hardly changed phenylephrine-induced enhancement. We though that CPZ blocked alpha 2-autoreceptors in adrenergic terminals from the LC rather than alpha 1-adrenoceptors within the LRN. As a result, NE released from the LC terminals may act on alpha 1-adrenoceptors in the LRN and may attenuate activities of the LRN. Therefore, it was clear that previous CPZ-induced enhancement may be due to depression of the descending inhibitory adrenergic system via CPZ-induced blockade of alpha 2-autoreceptors.

Only activated but not non-activated presynaptic alpha 2-autoreceptors interfere with neighbouring presynaptic receptor mechanisms.

Experiments were carried out in rabbit cerebrocortical slices in order to find out whether the attenuation by presynaptic alpha 2-autoreceptors of effects mediated by presynaptic opioid kappa- and adenosine A1-receptors requires activation of the alpha 2-receptors. The slices were preincubated with 3H-noradrenaline and then superfused with medium containing desipramine 1 mumol/l. They were stimulated electrically either with single pulses or with trains of 32 pulses at 1 Hz. The overflow of tritium elicited by a single pulse amounted to 0.21% of the tritium content of the tissue. It was Ca2+-dependent and tetrodotoxin-sensitive and not changed by rauwolscine 1 mumol/l or yohimbine 0.3 mumol/l. Ethylketocyclazocine (EK; 0.1-10 nmol/l) and R-(-)-N6-phenylisopropyladenosine (PIA; 1-1,000 nmol/l) potently inhibited the overflow evoked by a single pulse, and their effects were not changed by yohimbine. - The overflow of tritium elicited by trains of 32 pulses at 1 Hz amounted to 0.92% of the tritium content of the tissue and was increased approximately fourfold by yohimbine 0.3 mumol/l. EK and PIA were less potent inhibitors than in the one pulse experiments. Yohimbine greatly enhanced the effects of EK and PIA. The enhancement was even more pronounced when the Ca2+ concentration in the medium was reduced in order to obtain a control tritium overflow similar to that evoked by 32 pulses in the absence of yohimbine. The results demonstrate that there is no alpha 2-adrenergic autoinhibition when noradrenaline release is elicited by a single pulse. Under these conditions, the non-activated presynaptic alpha 2-adrenoceptor does not interfere with presynaptic opioid kappa- and adenosine A1-receptor mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Alpha 2-adrenoceptor blocking properties of idazoxan stereoisomers: stereoselectivity for presynaptic alpha 2-adrenoceptors.

The alpha 2-adrenoceptor blocking properties of idazoxan enantiomers were evaluated at pre- and postsynaptic level. The antagonism of the two idazoxan stereoisomers was assessed, at presynaptic level, by their ability to antagonize clonidine at the alpha 2-adrenoceptors regulating noradrenaline release. The antagonist (+)-idazoxan showed an affinity towards the alpha 2-autoreceptors 40 times higher than that showed by (-)-idazoxan. Binding studies revealed (+)-idazoxan to be 7-8 times more potent than (-)-idazoxan in inhibiting the p-[3H]aminoclonidine binding. These results indicate a different affinity of alpha 2-adrenoceptors for the two idazoxan stereoisomers, thus suggesting that the alpha 2-adrenoceptors located pre- and postsynaptically may be of two stereochemically different subtypes.