Alpha 2-autoreceptors Research Articles

Agomelatine-induced akathisia in a 38-year-old woman with depression.

To the Editor: Akathisia is linked to dopamine deficiency most often associated with antipsychotic medication. Newer antipsychotic medication is thought to reduce akathisia due to its serotonin (5-HT) 2(2A/2C) receptor blockade activity.1 Akathisia may also be related to increased adrenergic neurotransmission.2 Mirtazapine in low doses (15 mg) is a 5-HT 2A/2C antagonist and is antiakathisic, while in higher doses (> 30 mg/d) it may induce akathisia due to stimulation of adrenergic neurotransmission via alpha2 autoreceptor blockade.2 Agomelatine is a novel antidepressant that is both a melatonergic agonist and a 5-HT 2C antagonist.3 Agomelatine also enhances dopaminergic and adrenergic input to the frontal cortex. There is 1 previous report of agomelatine-induced akathisia with concomitant duloxetine treatment.4 That report suggested a pharmacodynamic drug-drug interaction causing noradrenergic overstimulation as an explanatory model.4 We present a novel case of likely agomelatine-induced akathisia. Case report. Ms A is a 38-year-old woman who presented with a several-month history of depression without anxiety or suicidal ideation. Many years previously, she had been prescribed sertraline without experiencing akathisia. There was no significant past medical history. Agomelatine was started at the first visit at a dose of 25 mg. Immediately after starting agomelatine, Ms A noticed an inability to sit still, restless movement of the legs, and a constant urge to move. Over the ensuing weeks, she became more significantly distressed and experienced insomnia, loss of appetite and weight, and suicidal ideation. After 5 weeks of taking agomelatine, a psychiatric consultation was sought and a provisional diagnosis of agomelatine-induced akathisia was made on the basis of a global Barnes Akathisia Scale5 score of 4 (marked akathisia). Within 3 days of stopping agomelatine, Ms A noticed significant improvement; in 5 days, all symptoms of akathisia had ceased with no ongoing anxiety, insomnia, or suicidal ideation. In this case, agomelatine was likely causing akathisia on the basis of a Naranjo Adverse Drug Reaction Probability Scale6 score of 7 (probable adverse drug reaction). This case is the first report without a concomitant drug-drug interaction. The induction of akathisia by agomelatine in this patient was somewhat unexpected, given that agomelatine is a 5-HT 2C antagonist and increases dopamine transmission in the frontal cortex. However, there is debate as to whether 5-HT 2A antagonism is more related to antiakathisic effect as opposed to 5-HT 2C antagonism. It may be that an increase in adrenergic stimulation induced by agomelatine may cause akathisia. However, akathisia is a complex phenomenon, and a clear explanatory model for what we observed is unknown. Clinicians should therefore be alert to the possibility of akathisia in patients prescribed agomelatine.

Noradrenergic modulation of vicarious trial-and-error behavior during a spatial decision-making task in rats

Deliberation between possible options before making a decision is crucial to responding with an optimal choice. However, the neural mechanisms regulating this deliberative decision-making process are still unclear. Recent studies have proposed that the locus coeruleus-noradrenaline (LC-NA) system plays a role in attention, behavioral flexibility, and exploration, which contribute to the search for an optimal choice under uncertain situations. In the present study, we examined whether the LC-NA system relates to the deliberative process in a T-maze spatial decision-making task in rats. To quantify deliberation in rats, we recorded vicarious trial-and-error behavior (VTE), which is considered to reflect a deliberative process exploring optimal choices. In experiment 1, we manipulated the difficulty of choice by varying the amount of reward pellets between the two maze arms (0 vs. 4, 1 vs. 3, 2 vs. 2). A difficulty-dependent increase in VTE was accompanied by a reduction of choice bias toward the high reward arm and an increase in time required to select one of the two arms in the more difficult manipulation. In addition, the increase of c-Fos-positive NA neurons in the LC depended on the task difficulty and the amount of c-Fos expression in LC-NA neurons positively correlated with the occurrence of VTE. In experiment 2, we inhibited LC-NA activity by injection of clonidine, an agonist of the alpha2 autoreceptor, during a decision-making task (1 vs. 3). The clonidine injection suppressed occurrence of VTE in the early phase of the task and subsequently impaired a valuable choice later in the task. These results suggest that the LC-NA system regulates the deliberative process during decision-making.

GABA induces norepinephrine exocytosis from hippocampal noradrenergic axon terminals by a dual mechanism involving different voltage-sensitive calcium channels

GABA can evoke norepinephrine (NE) release by activating GABAA receptors or GABA transporters on noradrenergic terminals. The heterocarrier-induced release occurs by conventional exocytosis. We here characterized the mechanism of the GABAA receptor-induced release and investigated what type(s) of voltage-sensitive Ca2+ channels (VSCCs) are involved in the GABA heterocarrier and GABA(A) receptor-evoked release. The effect of GABA in superfused rat hippocampal synaptosomes prelabeled with [(3)H]-NE was partially prevented by bicuculline or the GABA uptake inhibitor SKF 89976A and abolished by blocking both GABAA receptors and GABA transporters. The release elicited through GABAA receptors was Ca2+-dependent, prevented by Cd2+ or by botulinum toxin C, and modulated through alpha2 autoreceptors. The GABAA receptor-evoked release was insensitive to nifedipine and to omega-conotoxin MVIIC, but was inhibited ( approximately 50%) by omega-conotoxin GVIA. The heterocarrier-evoked release, nifedipine-insensitive, was inhibited approximately 30% either by omega-conotoxin GVIA or by omega-conotoxin MVIIC; the combined toxins produced approximately 60% inhibition. To conclude: a) the releases of NE evoked by activation of GABA(A) receptors and GABA heterocarriers are additive, although they both occur by conventional exocytosis; b) the heterocarrier-induced release requires activation of N and P/Q type channels, whereas the GABAA receptor-induced release only involves channels of the N type.

Presynaptic imidazoline receptors. New developments in characterization and classification.

Presynaptic imidazoline receptors (IRs) which inhibit norepinephrine (NE) release from sympathetic nerve endings have been identified in cardiovascular tissue of man, rabbit, rat, and guinea pig. They do not belong to one of the classical presynaptic inhibitory receptor classes such as alpha 2-adrenoceptors or H3 histamine receptors, and there is also no relation to I1- and I2-imidazoline binding sites. Segments of human right atrial appendages preincubated with [3H]NE were used to determine unknown pharmacologic properties of the presynaptic IRs. In the presence of 1 microM rauwolscine, S23230, the (-)-enantiomer of the racemic oxazoline derivative S22687 (5-(2(methyl-phenoxy-methyl)-1,3-oxazoline-2-yl)amine) exhibited low potency in inhibiting the electrically evoked [3H]NE release (pIC30% = 4.96), whereas the (+)-enantiomer S23229 and the racemate S22687 were ineffective. The IR-mediated inhibitory effect of the imidazoline BDF 6143 (4-chloro-2-(2-imidazolin-2-ylamino)-isoindoline) and the guanidine aganodine on evoked [3H]NE release from sympathetic nerves in human atrial appendages was counteracted by rauwolscine (with very low potency) and by the cannabinoid CB1-receptor antagonist SR141715A (N-[piperdin-1-yl]-5-[4-chlorophenyl]-2,4-dichlorophenyl]-4- methyl-1H-pyrazole-3-carboxamide). The inhibitory effect of moxonidine on evoked [3H]NE release (which is exclusively mediated via activation of alpha 2-autoreceptors) was antagonized with high potency by rauwolscine, but not by SR141716A. The cannabinoid CB1 receptor agonists CP55,940([(-)-Cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl] -trans-4- (3-hydroxy-propyl)cyclohexane]) and anandamide inhibited evoked [3H]NE release. Inhibition by CP55,940 and anandamide was abolished by 1 microM SR141716A as well as by 30 microM rauwolscine. In radioligand binding experiments on membranes from human atrial appendages (alpha 2- and sigma-binding sites were masked), cannabinoid receptor ligands and IR agonists displaced the radiolabeled guanidine derivative [3H]DTG (1,3-di-o-tolyguanidine, an agonist at presynaptic IRs) from its binding sites. Comparison of the potencies of these drugs determined in the competition experiments with [3H]DTG with those in inhibiting NE release via activation of the presynaptic IRs in the same tissue revealed a correlation. The present results suggest, e.g., that the presynaptic IRs may have certain binding domains in common with presynaptic cannabinoid receptors or that both receptors are different proteins which interact with each other in an unknown manner.

Presynaptic α2A/D-autoreceptors in the brain cortex of Cercopithecus aethiops

The existence of presynaptic alpha 2-autoreceptors, and the subtype to which the receptors belong, were studied in the brain cortex from two African green monkeys (Cercopithecus aethiops). Slices of the brain cortex were preincubated with 3H-noradrenaline. The slices were then superfused with medium containing desipramine (1 microM) and stimulated electrically with trains of 72 pulses. 3 Hz. Concentration-response curves of nine alpha-adrenoceptor antagonists were determined. All antagonists enhanced the stimulation-evoked overflow of tritium, i.e. the release of noradrenaline, indicating the existence of presynaptic alpha 2-autoreceptors. The EC30%, values of the antagonists (concentrations that increased the evoked overflow of tritium by 30%) were taken as a measure of affinity for the auto-receptors. The subclassification was based on a comparison of these affinity estimates with affinities for alpha 2A, alpha 2B, alpha 2C and alpha 2D radioligand binding sites and for previously classified alpha 2A- and alpha 2D-autoreceptors. The comparison indicates that the alpha 2-autoreceptors in the brain cortex of Cercopithecus aethiops belong to the genetic alpha 2A/D and not the alpha 2B or alpha 2C subtype. In their pharmacological properties, the autoreceptors are closest to the pharmacological alpha 2D subtype. The results support the notion that alpha 2-autoreceptors belong at least predominantly to the alpha 2A/D subtype of alpha 2-adrenoceptors.

Presynaptic imidazoline receptors and alpha 2-adrenoceptors in the human heart: discrimination by clonidine and moxonidine.

The involvement of presynaptic alpha 2-autoreceptors and imidazoline receptors in the modulation of noradrenaline release was investigated in strips from human atrial appendages preincubated with [3H]noradrenaline and superfused with medium containing desipramine and corticosterone. Electrical impulses were applied transmurally at 2 Hz. The imidazoline derivatives moxonidine and clonidine reduced to evoked tritium overflow in a concentration-dependent manner. Moxonidine was 18-fold more potent than clonidine. The concentration-response curve for moxonidine, but not for clonidine was shifted to the right by the alpha 2-adrenoceptor antagonist rauwolscine. The apparent pA2 value of rauwolscine against moxonidine was 8.41. An inhibitory effect was also observed for the imidazoline derivative BDF 6143 (4-chloro-2-(2-imidazolin-2-ylamino)-isoindoline), a mixed alpha 2-adrenoceptor antagonist/imidazoline receptor agonist; BDF 6143 was about 2-fold more potent than clonidine. Rauwolscine (1 microM) did not substantially shift the concentration-response curve of BDF 6143. It is concluded that noradrenaline release in the human atrium is inhibited not only via presynaptic alpha 2-autoreceptors but also via presynaptic non-I1, non-I2 imidazoline receptors. The failure of rauwolscine to antagonize the inhibitory effect of clonidine suggests that clonidine preferentially stimulates the presynaptic imidazoline receptors; the alpha 2-adrenoceptor component of its action is probably suppressed by an inhibitory interaction between the two receptors. In contrast, moxonidine acts via presynaptic alpha 2-autoreceptors, leaving the presynaptic imidazoline receptor unaffected.

Presynaptic alpha 2-autoreceptors in mouse heart atria: evidence for the alpha 2D subtype.

Presynaptic alpha 2-autoreceptors in mouse atria were characterized in terms of the alpha 2A, alpha 2B, alpha 2C and alpha 2D subtypes. Segments of the atria were preincubated with 3H-noradrenaline and then superfused and stimulated electrically. The affinity of up to 16 antagonists for the autoreceptors was assessed as (1) pEC30% values. i.e. concentrations that increased previously autoinhibited release of 3H-noradrenaline (120 pulses, 3 Hz) by 30%, and (2) pKd values against the release-inhibiting effect of 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) under conditions of no or little autoinhibition (2 trains of 20 pulses, 50 Hz, train interval 120 s). The pKd values correlated well with the pEC30% values (r = 0.98; P < 0.001; slope of regression line 0.93), indicating that UK 14,304 and released noradrenaline modulated the release of noradrenaline through pharmacologically identical receptors. Comparison with antagonist affinities for (1) prototypic native alpha 2 radioligand binding sites, (2) radioligand binding sites in COS cells transfected with alpha 2 subtype genes, and (3) previously classified presynaptic alpha 2-autoreceptors-all taken from the literature-indicated that the mouse atrial autoreceptors corresponded to the alpha 2D subtype. For example, the pKd values at mouse atrial auto-receptors correlated closely with pKd values at native alpha 2D binding sites in the bovine pineal gland (r = 0.96; P < 0.001); with pKd values at alpha 2D binding sites in COS cells transfected with the rat alpha 2D gene (r = 0.85; P < 0.01); and with pKd values at guinea-pig cerebral and atrial and mouse cerebral alpha 2D-autoreceptors (r = 0.96-0.98; P < 0.001). The antagonist pKd values at mouse atrial autoreceptors correlated less with pKd values at alpha 2A, alpha 2B and alpha 2C sites. It is concluded that the presynaptic alpha 2-autoreceptors in mouse atria are alpha 2D. This identification supports the hypothesis that at least the majority of alpha 2-autoreceptors belong to the alpha 2A/D pair of orthologous alpha 2-adrenoceptors.

Cholera toxin induces cyclic AMP-independent down-regulation of Gs alpha and sensitization of alpha 2-autoreceptors in chick sympathetic neurons.

The role of the stimulatory GTP-binding protein (Gs) in the alpha 2-autoinhibitory modulation of noradrenaline release was investigated in cultured chick sympathetic neurons. The alpha 2-adrenoceptor agonist UK 14,304 caused a concentration-dependent reduction of electrically evoked [3H] noradrenaline release with half-maximal effects at 14.0 +/- 5.5 nM. In neurons treated with 100 ng/ml cholera toxin for 24 h, the half-maximal concentration was lowered to 3.2 +/- 1.4 nM without changes in the maximal effect of UK 14,304. The pretreatment with cholera toxin also increased the inhibitory action of 10 nM UK 14,304 when compared with the inhibition of noradrenaline release in untreated cultures derived from the same cell population. In cultures treated with either 10 microM forskolin or 100 microM 8-bromo-cyclic AMP, neither the half-maximal concentration nor the maximal effect of UK 14,304 was altered. Cholera toxin, forskolin, and 8-bromo-cyclic AMP all induced an increase in spontaneous outflow and a reduction in electrically evoked overflow, effects not observed after a pretreatment with dideoxyforskolin. Exposure of neurons to cholera toxin, but not to forskolin or 8-bromo-cyclic AMP, induced a translocation of alpha-subunits of Gs (Gs alpha) from particulate to soluble fractions and led ultimately to a complete loss of Gs alpha from the neurons. In contrast, no effect was seen on the distribution of either alpha-subunits of Gi- or Ga-type G proteins or of beta-subunits. These results indicate that cholera toxin causes a selective, cyclic AMP-independent down-regulation of Gs alpha. This down-regulation of Gs alpha is associated with the sensitization of alpha 2-autoreceptors.

α2c‐Adrenoceptor‐modulated release of noradrenaline in human right atrium

1. The aim of the present study was to characterize the presynaptic alpha 2-autoreceptors in human right atrium in terms of the alpha 2A-D system. Segments of atrial appendages were preincubated with [3H]-noradrenaline and then superfused in the presence of cocaine and stimulated electrically. pEC30% values of eight alpha-adrenoceptor antagonists with discriminatory power were determined. pEC30% is the negative logarithm of the antagonist concentration that increased the stimulation-induced overflow of tritium by 30%. For four antagonists, the dissociation constant KD was determined, in addition to pEC30%, against the overflow-inhibiting effect of 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) under autoinhibition-free conditions. 2. pEC30% and KD values yielded identical rank orders of antagonist affinity (rauwolscine > WB 4101 > phentolamine > prazosin) suggesting that both released noradrenaline and the exogenous agonist UK 14,304 activated the same receptor to inhibit release. 3. The eight antagonist pEC30% values obtained in right atrium correlated significantly with their pEC30% values, reported in the literature, at the presynaptic alpha 2C-autoreceptors in human kidney (r = 0.817; slope of the regression line 1.03). No significant correlation was obtained between pEC30% values at atrial autoreceptors and pKD values at previously characterized alpha 2A-autoreceptors in rabbit and alpha 2D-autoreceptors in rat, mouse and guinea-pig tissues. 4. Comparison of antagonist pEC30% values with their pKD values at native alpha 2 binding sites in cells or tissues that express a single subtype only, and with pKD values at alpha 2 binding sites in membranes of COS cells transfected with human alpha 2 subtype genes confirms the alpha 2C character of the atrial autoreceptors: significant correlations were obtained exclusively with the alpha 2C binding sites. 5. Ratios of KD values were computed for alpha 2-autoreceptors in human right atrium and for binding sites in COS cells transfected with human alpha 2 subtype genes. The autoreceptor ratios corresponded well with the respective ratios for the alpha 2C binding sites (maximal three fold deviation) but were, in part, markedly different from ratios calculated for alpha 2A and alpha 2B binding sites (up to 166 fold deviation). This outcome supports the alpha 2C designation of the autoreceptors. 6. In conclusion, the presynaptic alpha 2-autoreceptors in human right atrium are alpha 2C. In this they agree with the previously characterized alpha 2-autoreceptors in human kidney. The alpha 2C classification possibly separates, in general, human alpha 2-autoreceptors from those in lagomorph (rabbit) and rodent (rat, mouse, guinea pig) species that have been proposed to be predominantly alpha 2A or alpha 2D.

Effects of 5-HT receptor agonists on depolarization-induced [3H]-noradrenaline release in rabbit hippocampus and human neocortex.

1. The present study attempted to determine whether noradrenaline (NA) release in rabbit hippocampus and human neocortex is modulated by presynaptic 5-hydroxytryptamine (5-HT) receptors. 2. Slices of rabbit hippocampus and human neocortex, loaded with [3H]-noradrenaline ([3H]-NA) were superfused and the effects of 5-hydroxytryptamine (5-HT) receptor ligands on electrically evoked [3H]-NA release were investigated. 3. In rabbit hippocampus, 5-HT, 5-carboxamidotryptamine (5-CT; 32 microM) and 2-CH3-5-HT (32 microM) increased [3H]-NA release elicited with 360 pulses/3 Hz. Facilitation of transmitter release was not influenced by the 5-HT3 receptor antagonist, tropisetron but was prevented by the alpha 2-adrenoceptor antagonist, rauwolscine. When autoinhibition was avoided by stimulating the tissue with 4 pulses/100 Hz (pseudo-one pulse-(POP) stimulation), 2-CH3-5-HT decreased evoked transmitter release, whereas 5-HT and 5-CT had no effect. Inhibition caused by 2-CH3-5-HT was not affected by tropisetron but counteracted by the alpha 2-adrenoceptor ligands, clonidine and rauwolscine. Inhibition caused by clonidine was diminished in the presence of 5-CT or 2-CH3-5-HT. 4. In human neocortex, [3H]-NA release elicited with 360 pulses/3 Hz was increased by 10 microM 5-HT and 32 microM 5-CT, whereas 2-CH3-5-HT was ineffective. [3H]-NA release evoked with a modified POP stimulation (2 bursts of 4 pulses/100 Hz, 3.5 min apart) was not affected by 2-CH3-5-HT or 5-CT. 5. The present results indicate that 5-HT, 2-CH3-5-HT and 5-CT can act on presynaptic alpha 2-autoreceptors as partial agonists (2-CH3-5-HT; in rabbit hippocampal tissue) or antagonists (5-HT and 5-CT; in tissue of rabbit hippocampus and human neocortex). Furthermore the existence of autoinhibition dictates whether these drugs cause facilitation of release, inhibition or have no effect.

Subclassification of presynaptic alpha 2-adrenoceptors: alpha 2A-autoreceptors in rabbit atria and kidney.

The study was devised to classify, by means of antagonist affinities, the presynaptic alpha 2-autoreceptors of rabbit atria and kidney in terms of alpha 2A, alpha 2B, alpha 2C and alpha 2D. A set of antagonists was chosen that was able to discriminate between the four subtypes. Small pieces of the left atrium and slices of the kidney cortex were preincubated with 3H-noradrenaline and then superfused and stimulated electrically. In one series of experiments, tissue pieces were stimulated by relatively long pulse trains (1 or 2 min) leading to alpha 2-autoinhibition. All 11 (atria) or 10 (kidney) antagonists increased the evoked overflow of tritium. pEC30% values (concentrations causing 30% increase) were interpolated from concentration-response curves. In a second series of experiments, tissue pieces were stimulated by brief pulse trains (0.4 s) that did not lead to alpha 2-autoinhibition, and concentration-inhibition curves of the alpha 2-selective agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) were determined. Most of the 11 (atria) or 8 (kidney) antagonists shifted the concentration-inhibition curve of UK 14,304 to the right. pKd values of the antagonists were calculated from the shifts. pEC30% values correlated with pKd values, both in atria (r = 0.728) and in the kidney (r = 0.930). pEC30% values in atria correlated with pEC30% values in the kidney (r = 0.988) and pKd values in atria correlated with pKd values in kidney (r = 0.923). It is concluded that the alpha 2-autoreceptors in atria and the kidney are the same. Comparison with antagonist affinities for prototypic native alpha 2 binding sites, alpha 2 binding sites in cells transfected with alpha 2 subtype genes, and previously classified presynaptic alpha 2-adrenoceptors--all taken from the literature--indicates that both autoreceptors are alpha 2A. This conclusion is reached with either of the two independent estimates of autoreceptor affinity, pEC30% and pKd. The results are compatible with the hypothesis that at least the majority of alpha 2-autoreceptors belong to the alpha 2A/D branch of the alpha 2-adrenoceptor tree, across mammalian or at least rodent and lagomorph species.

Subclassification of presynaptic ?2-adrenoceptors: ?2D-autoreceptors and ?2D-adrenoceptors modulating release of acetylcholine in guinea-pig ileum

The study was designed to classify in terms of alpha 2A, alpha 2B, alpha 2C and alpha 2D the presynaptic alpha 2-autoreceptors, as well as the alpha 2-receptors modulating the release of acetylcholine, in the myenteric plexus-longitudinal muscle (MPLM) preparation of the guinea-pig ileum. A set of antagonists was chosen that was able to discriminate between the four subtypes. Small pieces of the MPLM preparation were preincubated with 3H-noradrenaline or 3H-choline and then superfused and stimulated electrically. The stimulation periods used (3H-noradrenaline: 3 trains of 20 pulses, 50 Hz, train interval 60 s; 3H-choline: single trains of 30 pulses, 0.2 Hz) did not lead to alpha 2-autoinhibition or inhibition of 3H-acetylcholine release by endogenous noradrenaline. The alpha 2-selective agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) reduced the evoked overflow of tritium in both 3H-noradrenaline and 3H-choline experiments. Most (3H-noradrenaline) or all (3H-choline) of the 10 antagonists shifted the concentration-inhibition curves of UK 14,304 to the right. pKd values of the antagonists were calculated from the shifts. pKd values from 3H-noradrenaline experiments correlated with pKd values from 3H-choline experiments (r = 0.981). It is concluded that alpha 2-autoreceptors and alpha 2-heteroreceptors modulating the release of acetylcholine in the MPLM preparation are of the same subtype. Comparison with antagonist affinities for prototypic native alpha 2 binding sites, binding sites in cells transfected with alpha 2 subtype genes, and previously classified presynaptic alpha 2-adrenoceptors--all taken from the literature--indicates that both are alpha 2D.(ABSTRACT TRUNCATED AT 250 WORDS)

Subclassification of presynaptic alpha 2-adrenoceptors: alpha 2D-autoreceptors in guinea-pig atria and brain.

The study was devised to classify, by means of antagonist and agonist affinities, the presynaptic alpha 2-autoreceptors in guinea-pig heart atria and brain cortex in terms of alpha 2A, alpha 2B, alpha 2C and alpha 2D. A set of antagonists and agonists was chosen that was able to discriminate between the four subtypes. Small pieces of the atria and slices of the brain cortex were preincubated with 3H-noradrenaline and then superfused and stimulated electrically. In one series of experiments (atria only), tissue pieces were stimulated by relatively long pulse trains (1 min) leading to marked alpha 2-autoinhibition. All 10 antagonists increased the evoked overflow of tritium. pEC30% values (concentrations causing 30% increase) were interpolated from concentration-response curves. In a second series of experiments (atria and brain slices), tissue pieces were stimulated by brief pulse trains (0.4 s or 40 ms) that led to little (atria) or no (brain slices) alpha 2-autoinhibition, and antagonist effects against the alpha 2-selective agonist 5-bromo-6-(2-imidazolin-2- ylamino)-quinoxaline (UK 14,304) were examined. All 10 (atria) or 8 (brain) antagonists shifted the concentration-inhibition curve of UK 14,304 to the right. pKd values of the antagonists were calculated from the shifts. In a third series of experiments (brain slices only), also with brief pulse trains (40 ms), pKa values (negative logarithms of dissociation constants of agonist-alpha 2-adrenoceptor complexes) were determined by comparison of concentration-inhibition curves of UK 14,304, guanoxabenz and oxymetazoline in normal tissue and in tissue in which a fraction of the receptors had been blocked by phenoxybenzamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory presynaptic imidazoline receptors on sympathetic nerves in the rabbit aorta differ from I1- and I2-imidazoline binding sites

The involvement of imidazoline receptors in modulation of noradrenaline release was investigated in the rabbit aorta preincubated with [3H]noradrenaline and superfused with physiological salt solution containing cocaine, corticosterone and propranolol. After blockade of alpha 2-autoreceptors by rauwolscine, the electrically evoked tritium overflow was inhibited by various imidazolines and guanidines. The rank order of potency was BDF 7579 (4-chloro-2-isoindolinyl) guanidine) > or = BDF 6143 (4-chloro-2-(2-imidazolin-2-ylamino)-isoindoline) > BDF 6100 [2-(2-imidazolin-2-ylamino)-isoindoline] > clonidine > ST587 (2-(2-chloro-5-trifluoromethylphenylimino) imidazolidine nitrate) > or = cirazoline > tolazoline > idazoxan > phentolamine. Comparison of the potencies of these drugs with those previously found for the presynaptic imidazoline receptors in the rabbit pulmonary artery revealed a very good correlation. In contrast, no positive correlation was found with their affinities for the I1- and I2-imidazoline binding sites in bovine adrenal medullary membranes and with their lipophilicity (log P values). The electrically evoked tritium overflow was also inhibited by the recently identified endogenous imidazoline receptor ligand agmatine, but was not affected by amiloride. In further series of experiments, the ability of putative antagonist at presynaptic imidazoline receptors to counteract the inhibitory effect of imidazoline derivatives was determined. Amiloride, imidazole-4-acetic acid and 1-benzylimidazole did not attenuate the inhibitory effect of BDF 6143 on the electrically evoked tritium overflow. In contrast, rauwolscine antagonized the inhibitory effect of various imidazolines; rauwolscine was clearly less potent in antagonizing the effect of clonidine, BDF 6143 and cirazoline (apparent pA2 6.48-7.32) than in antagonizing that of oxymetazoline and moxonidine (apparent pA2 8.33 and 8.12, respectively). In a final series of experiments, BDF 6143 (under the conditions applied a selective agonist at presynaptic imidazoline receptors) proved to be considerably less potent in inhibiting tritium overflow evoked by high K+ than by electrical stimulation, whereas moxonidine (in rabbit aorta a selective agonist at presynaptic alpha 2-adrenoceptors) exhibited similar potency in inhibiting the overflow evoked by both methods of stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)

Cardiovascular effects of agmatine, a "clonidine-displacing substance", in conscious rabbits.

Agmatine has been identified as a "clonidine-displacing substance" in extracts from bovine brain. We studied its effect on cardiovascular regulation and the role played in this effect by alpha 2-adrenoceptors. In conscious rabbits, agmatine 10 micrograms kg-1 injected intracisternally (i.c.) caused no change, whereas agmatine 30, 100 and 300 micrograms kg-1 i.c. increased renal sympathetic nerve firing, the plasma concentration of noradrenaline and adrenaline and arterial blood pressure. Heart rate tended to be decreased. Yohimbine 1.5 micrograms kg-1 i.c. caused no change, whereas yohimbine 5, 15 and 50 micrograms kg-1 increased renal sympathetic nerve activity, the plasma concentration of noradrenaline and adrenaline, blood pressure and heart rate. In rabbit brain cortex slices preincubated with [3H]-noradrenaline, agmatine 1 to 100 microM did not modify the electrically evoked overflow of tritium (either 4 pulses at 100 Hz or 36 pulses at 3 Hz). The evoked overflow was reduced by 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14304) 0.03 to 30 nM (4 pulses at 100 Hz), and this inhibition was not affected by agmatine 10 and 100 microM. Agmatine did not change the basal efflux of tritium. The results show that agmatine, like yohimbine, causes central sympathoexcitation when given i.c., but agmatine differs from yohimbine in that it does not increase heart rate. Agmatine acts neither as an agonist nor as an antagonist at the alpha 2-autoreceptors in rabbit brain cortex. alpha 2-Adrenoceptors, therefore, are probably not involved in its cardiovascular effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Glial ?1-receptors probably inhibit the high-affinity uptake of noradrenaline into astrocytes in the rat brain in vivo

The effect of alpha 2-receptor blockage on the extraneuronal turnover of noradrenaline (NA) has been studied in the intact rat brain. Tropolone and yohimbine, along with reserpine or desmethylimipramine, were given 30 min after intracerebroventricular injection of [7-3H]NA, i.e. after the tracer had been stored or inactivated. Tropolone given alone did not change the fractions of 3H-activity recovered as [3H]NA from hypothalamus, septum, striatum and pons-medulla, but in the presence of yohimbine improved the [3H]NA recovery in all areas except pons-medulla. The maximum effect was seen in the hypothalamus of reserpine-treated rats. Since the alpha 2-autoreceptors were blocked, the increased [3H]NA recovery does not reflect a down-regulated neuronal NA turnover. Instead it seems to show that a fraction greater than normal of neuronally released NA had been taken up into astrocytes and remained unmetabolized if catechol-O-methyltransferase was inactive. It is assumed that yohimbine enabled the protective tropolone effect by blocking astrocytic alpha 2-receptors that otherwise, either by itself or by antagonizing beta-receptor-induced hyperpolarization or cAMP formation, had impaired parameters that stimulate the high-affinity NA Uptake 1 of astrocytes (e.g. membrane potential, Na+,K(+)-ATPase) or control the gap junction permeability in the glial syncytium.

Rapid, agonist-induced desensitization of alpha 2-autoreceptors modulating transmitter release.

1. The release of previously incorporated [3H]-noradrenaline was investigated in cultures of dissociated chick or rat sympathetic neurones and in cerebrocortical slices from neonatal or adult rats. Noradrenaline, in the presence of 10 mumol l-1 of the uptake inhibitor, cocaine, or the selective alpha 2-adrenoceptor agonist, 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK 4,304), was applied for different periods of time in order to detect a possible time-dependence of the alpha 2-adrenoceptor-mediated inhibition of electrically evoked tritium outflow. 2. In chick sympathetic neurones, stimulation-evoked overflow was reduced to 30%, 42%, or 56% of control when noradrenaline (1 mumol l-1) was present for 2, 8, or 16 min, respectively. Likewise, UK 14,304 (1 mumol l-1) present for these periods of time reduced 3H overflow to 35%, 51%, and 53% of control, respectively. Addition of 1 nmol l-1 to 10 mumol l-1 UK 14,304 for either 2 or 16 min did not produce significantly different IC50 values, but the inhibitory effects were smaller with 16 min as compared to 2 min exposure at concentrations > or = 10 nmol l-1. 3. In rat sympathetic neurones, noradrenaline (100 nmol l-1) reduced stimulation-evoked overflow to 33%, 56%, or 57% of control, when present for 2, 8, or 16 min, respectively. Addition of UK 14,304 (1 mumol l-1) for these periods of time caused inhibition to 11%, 41%, and 46% of control. Applying UK14,304 for either 2 or 16 min did not result in significantly different IC5o values, but the inhibition induced by 16 min as compared to 2 min exposure was smaller at concentrations > 10 nmol 1-1.4. In cerebrocortical slices from either neonatal or adult rats, exposure to 0.1 to 1.0 micromol 1-1 UK14,304 for 16 min never caused a smaller inhibition than a corresponding 3 min exposure, although various experimental conditions were investigated.5 The results demonstrate that alpha 2-adrenoceptors which regulate noradrenaline release from sympathetic neurones undergo agonist-induced desensitization within minutes. Such rapid desensitization of alpha 2-autoreceptors was not detected in brain slice preparations.

New insights into receptor theory, as provided by an artificial partial agonist made-to-measure

In the present study a mixture of a full agonist (noradrenaline) and a full antagonist (yohimbine) was used to mimic the effects of a partial agonist (clonidine) on alpha 2-autoreceptor-mediated regulation of noradrenaline release in order to learn more about the shape of concentration-response curves in the absence and presence of spare receptors. The sigmoidal shape of the cloud of single experimental data points may be reflected by different curve fits based on either descriptive or mechanistic mathematical models. Only mechanistic models allow the interpretation of the relationship between occupancy of receptors and induced response. The experiments were performed in rat neocortex and in rabbit hippocampus tissue where electrical field stimulation with 4 pulses/100 Hz of slices prelabelled with [3H]noradrenaline elicited the release of noradrenaline. A receptor reserve was found in the rabbit hippocampus and quantified from the concentration-response curve of noradrenaline in this tissue using a mechanistic general response function, developed to reflect the condition of spare receptors. The mixture of noradrenaline and yohimbine, NA-Yoh, (three parts to one part), corrected by the different affinities to the alpha 2-autoreceptors, was designed to mirror the quantified proportion of 75% non-spare and 25% spare alpha 2-autoreceptors. In the spare receptor-free rat cortex NA-Yoh acted like a typical partial agonist, as clonidine, with nearly the same EC50 (= Kd in this case) as the full agonist noradrenaline, but with a maximum effect significantly lower than that of noradrenaline. In the rabbit hippocampus, however, the same maximum effect was obtained with NA-Yoh, noradrenaline and clonidine.(ABSTRACT TRUNCATED AT 250 WORDS)

P1-purinoceptor-mediated modulation of neural noradrenaline and ATP release in guinea-pig vas deferens.

The effect of P1-purinoceptor activation on contractions, release of noradrenaline and release of ATP elicited by electrical field stimulation (210 pulses, 7 Hz) was studied in the superfused vas deferens of the guinea pig. Release of noradrenaline was assessed as overflow of total tritium after preincubation with [3H]-noradrenaline. ATP was measured by means of the luciferin-luciferase technique. Electrical stimulation elicited reproducible contraction, tritium overflow and ATP overflow. In the absence of other drugs, adenosine (10-100 microM) did not change evoked contractions but reduced the evoked overflow of tritium and ATP. In subsequent experiments alpha 1-adrenoceptors were blocked by prazosin, P2-purinoceptors by suramin and alpha 2-adrenoceptors by rauwolscine. No or almost no contraction remained under these conditions. The evoked overflow of tritium was 505% and the evoked overflow of ATP 34% of that observed in the absence of prazosin, suramin and rauwolscine. Adenosine (1-100 microM) again reduced the evoked overflow of tritium and ATP, and so did the A1-selective agonist 2-chloro-N6-cyclopentyladenosine (CCPA; 0.032-0.32 microM). Adenosine and CCPA decreased the evoked overflow of ATP to a greater extent than the evoked overflow of tritium. It is concluded that neural release of both postganglionic sympathetic cotransmitters, noradrenaline and ATP, is decreased upon activation of prejunctional P1- (A1-) purinoceptors in guinea-pig vas deferens. The A1-receptor-mediated inhibition of the release of ATP is more marked than the inhibition of the release of noradrenaline, a pattern opposite to the inhibition produced by activation of prejunctional alpha 2-autoreceptors.

Direct observation of the effect of autoreceptors on stimulated release of catecholamines from adrenal cells

The direct effect of alpha 2-autoreceptors was studied by measuring the effects of piperoxan, an alpha 2-autoreceptor antagonist, and clonidine, an agonist on catecholamine exocytosis, from single bovine chromaffin cells in culture. Catecholamine release was elicited by stimulation with 100 microM nicotine and was monitored electrochemically with a carbon-fiber microelectrode placed adjacent to the cell. These electrodes allowed the number of exocytotic release events to be monitored and reported as total charge for release following a specific stimulus. Repeated stimulation with 100 microM nicotine showed that total release caused by the second exposure to nicotine was 32% of the first, and release caused by the third exposure to nicotine was 80% of the second. Total release of catecholamine increased significantly after application of 20 microM piperoxan relative to a control application of balanced salt solution. Application of 20 microM piperoxan alone did not cause release. After the cells were incubated in culture medium containing 20 microM clonidine, a significant decrease in nicotine-stimulated catecholamine release was observed. These results confirm that there are autoreceptors on chromaffin cells and, when relatively high levels of catecholamine are released, the catecholamine stimulates the alpha 2-autoreceptors, which inhibits subsequent release through a negative feedback mechanism. In addition to piperoxan, the sympathomimetic drug amphetamine also increases quantal release after application of nicotine. Amphetamine increases the extracellular concentration of catecholamine, and these data appear to indicate that at least part of the pharmacology of amphetamine might involve blocking catecholamine autoreceptors.