Antagonists that differentiate between α2A- and α2D-adrenoceptors

Four antagonists were examined for their ability to differentiate _2A from the orthologous _2Dadrenoceptors. The antagonists were (2S,12bS) 1, 3-di-methylspiro(1, 3, 4, 5, 6, 6, 7,12b-octahydro-2H-benzo[b]furo[2,3-a]quinolizine)-2,4-pyrimidin-2-one (MK 912), 2-[2-(methoxy-1, 4-benzodioxanyl)imidazoline (RX 821002), efaroxan and benoxathian. The ₂-autoreceptors in rabbit brain cortex were chosen as _2A- and the a₂-autoreceptors in guinea-pig brain cortex as _2D-adrenoceptors. Slices of the brain cortex were preincubated with ³H-noradrenaline and then superfused and stimulated electrically by brief pulse trains (4 pulses, 100 Hz) that led to little, if any, ₂-autoinhibition. 5-Bromo-6-(2-imidazolin-2-ylamino)quinoxaline (UK 14,304) was used as an ₂-adrenoceptor agonist.UK 14, 304 decreased the stimulation-evoked overflow of tritium. The antagonists shifted the concentration-inhibition curve of UK 14, 304 to the right in an apparently competitive manner. Dissociation constants of the antagonists were calculated from the shifts. MK 912, RX 821002 and efaroxan had markedly higher affinity for (guinea-pig) _2D-adrenoceptors (pK _d values 10.0, 9.7 and 9.1, respectively) than for (rabbit) _2A-adrenoceptors (pK _d 8.9, 8.2 and 7.6, respectively). Benoxathian had higher affinity for _2A- (pK _d 7.4) than for _2D-adrenoceptors (pK _d 6.9). Ratios calculated from the K _d values of the four compounds differentiated between _2A and _2D up to 100 fold. It is concluded that MK 912, RX 821002, efaroxan and benoxathian are antagonists with high power to differentiate _2A- from _2D-adrenoceptors.     

Binding sites of α1- and α2-adrenoreceptors

Conclusions The models constructed for the binding sites of rat brain ₁-AR and ₂-AR satisfy all the steric requirements and energy characteristics of interaction with known ligands, cited in [5–7, 14]. The differences detected in the arrangement and orientation of the functional groups of the binding sites permit an explanation of a whole series of typical differences in the interaction of adrenoactive substances with both subtypes of-AR.Our analysis showed that the greatest contribution to the interaction with the receptor is made by ionic, donor-acceptor, and hydrophobic bonds. The role of van der Waals forces in the interactions examined is evidently extremely negligible. The most effective and specific preparations prove to be compounds that not only form the maximum number of donor-acceptor bonds with the receptor but also orient their own hydrophobic fragments in such a way that the ionic and donor-acceptor bonds formed between the molecule and the receptor are shielded from contact with the aqueous phase. The energy effects of hydrophobic interactions of this type may be rather substantial (3.9–3.12).The production of new synthetic preparations, for which the conditions of complementarity to the-AR will be most fully satisfied, can be carried out taking the requirements of structural correspondence of the topography of the binding sites into account.Translated from Khimiko-farmatsevticheskii Zhurnal, Vol. 18, No. 8, pp. 904–912, August, 1984.     

Stimulation frequency-noradrenaline release relationships examined in α2A-, α2B- and α2C-adrenoceptor-deficient mice

The stimulation frequency-noradrenaline release relationship was studied in the vas deferens and the cerebral cortex of NMRI mice, mice in which the alpha2A-, the alpha2B-, the alpha2C- or both the alphaCA- and the alpha2C-adrenoceptor gene had been disrupted (alpha2AKO, alpha2BKO, alpha2CKO and alpha2ACKO), and the wildtype mice from which the knockout animals had been generated. Tissue pieces were preincubated with 3H-noradrenaline and then superfused and stimulated electrically with a constant number of pulses (30 in vas deferens and 50 in brain cortex) at frequencies between 0.03 and 100 Hz. The frequency-evoked tritium overflow curves ascended monophasically in the vas deferens of wildtype and NMRI mice. Disruption of the alpha2B-adrenoceptor gene caused no change. In the vas deferens of alpha2CKO mice, the overflow evoked by low frequencies (0.3 and 1 Hz) was slightly increased. In the vas deferens of alpha2AKO and alpha2ACKO mice, the evoked overflow was increased to a greater extent. Rauwolscine (1 microM) caused a marked increase of the evoked overflow of tritium from the vas deferens of NMRI, wildtype, alpha2BKO and alpha2CKO mice. Rauwolscine also increased the evoked overflow of tritium from the vas deferens of alpha2AKO and alphaC2ACKO mice, but to a smaller extent. The gene disruptions and rauwolscine slightly steepened the slope of the vas deferens frequency-overflow curve. In the brain cortex of wildtype and NMRI mice, the frequency-evoked tritium overflow curves were U-shaped. In the brain cortex of alpha2BKO and alpha2CKO mice, the evoked overflow was slightly reduced. In the brain cortex of alpha2AKO and alpha2AcKO mice, in contrast, the evoked overflow was increased. Rauwolscine (1 microM) caused a marked increase of the evoked overflow of tritium from the brain cortex of NMRI, wildtype, Q2BKO and alpha2CKO mice. Rauwolscine also increased the evoked overflow of tritium from the brain cortex of alpha2AKO and alpha2ACKO mice, but to a smaller extent. The gene disruptions and rauwolscine flattened the U shape of the brain cortex frequency-overflow curve. It is concluded that alpha2-autoinhibition is one factor that shapes the frequency-noradrenaline release relationships in the mouse vas deferens and cerebral cortex. The autoreceptors are mainly alpha2A and to a minor extent, and well detectable in the vas deferens only, alpha2C. When both the alpha2A- and the alpha2C-adrenoceptor have been deleted, alpha2B-adrenoceptors may be expressed as autoreceptors in noradrenergic neurons. It seems possible that alpha2C-autoreceptors depress mainly release at low (around 1 Hz) whereas alpha2A-autoreceptors depress mainly release at high (around 10 Hz) frequencies.     

Subclassification of presynaptic α2-adrenoceptors: α2D-autoreceptors and α2D-adrenoceptors modulating release of acetylcholine in guinea-pig ileum

The study was designed to classify in terms of _2A, _2B, _2C and _2D the presynaptic ₂-autoreceptors, as well as the 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 ₂-autoinhibition or inhibition of ³H-acetylcholine release by endogenous noradrenaline. The ₂-selective agonist 5-bromo6-(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. pK_d values of the antagonists were calculated from the shifts. pK_d values from ³H-noradrenaline experiments correlated with pK_d values from ³H-choline experiments (r = 0.981).It is concluded that ₂-autoreceptors and ₂-heteroreceptors modulating the release of acetylcholine in the MPLM preparation are of the same subtype. Comparison with antagonist affinities for prototypic native ₂ binding sites, binding sites in cells transfected with ₂ subtype genes, and previously classified presynaptic ₂-adrenoceptors — all taken from the literature — indicates that both are _2D. The results are consonant with the hypothesis that at least the majority of ₂-autoreceptors belong to the _2A/D branch of the ₂-adrenoceptor tree, across mammalian or at least across rodent and lagomorph species. The same may hold true for ₂-adrenoceptors on non-noradrenergic neurones.     

Presynaptic α2-autoreceptors in brain cortex: α2D in the rat and α2A in the rabbit

Summary Presynaptic ₂-autoreceptors in rat and rabbit brain cortex were compared by means of antagonists and agonists. Brain cortex slices were preincubated with [³H]-noradrenaline and then superfused and stimulated by 3 (rat) or 4 (rabbit) pulses at a frequency of 100 Hz.The ₂-adrenoceptor agonist bromoxidine (UK 14 304) reduced the electrically evoked overflow of tritium with EC₅₀ values of 4.5 nmol/l in the rat and 0.7 nmol/l in the rabbit. The antagonists phentolamine, 2-[2H-(1-methyl-1,3-dihydroisoindole)methyl]-4,5-dihydroimidazole (BRL 44408), rauwolscine, 1,2-dimethyl-2,3,9,13b-tetrahydro-1H-dibenzo(c,f)imidazo(1,5-a)azepine (BRL 41992), 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101), 6-chloro-9-[(3-methyl-2-butenyl)oxy]-3-methyl-1H-2,3,4, 5-tetrahydro-3-benzazepine (SKF 104078), imiloxan, prazosin and corynanthine did not per se increase the evoked overflow of tritium but shifted the concentration-inhibition curve of bromoxidine to the right in a manner compatible with competitive antagonism. Up to 4 concentrations of each antagonist were used to determine its dissociation constant K_D. The K_D values correlated only weakly between the rat and the rabbit. Dissociation constants K_A of bromoxidine were calculated from equieffective concentrations in unpretreated brain slices and slices in which part of the ₂-adrenoceptors had been irreversibly blocked by phenoxybenzamine. The K_A value was 123 nmol/l in the rat and 7.2 nmol/l in the rabbit.The results confirm the species difference between rat and rabbit brain presynaptic ₂-autoreceptors. Comparison with data from the literature indicates that the rat brain autoreceptors can be equated with the _2D subtype as defined by radioligand binding, whereas the rabbit brain autoreceptors conform to the _2A subtype. For example, the antagonist affinities for the rat autoreceptors correlate with their binding affinities for the gene product of ₂-RG20, the putative rat _2D-adrenoceptor gene (r = 0.97; P<0.01), but not with their binding affinities for the gene product of ₂-C10, the putative human _2A-adrenoceptor gene. Conversely, the rabbit autoreceptors correlate with the ₂-C10 (r = 0.98; P<0.001) but not with the ₂-RG20 gene product. Since presynaptic ₂-autoreceptors are also _2D in rat submaxillary gland and perhaps vas deferens and _2A in rabbit pulmonary artery, the possibility arises that the majority of ₂-autoreceptors generally are _2D in the rat and _2A in the rabbit. Moreover, receptors of the _2A/D group generally may be the main mammalian ₂-autoreceptors.Correspondence to: N. Limberger at the above address     

Subclassification of presynaptic α2-adrenoceptors: α2D-autoreceptors in guinea-pig atria and brain

The study was devised to classify, by means of antagonist and agonist affinities, the presynaptic ₂-autoreceptors in guinea-pig heart atria and brain cortex in terms of _2A, _2B, _2C and _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 ₂-autoinhibition. All 10 antagonists increased the evoked overflow of tritium. pEC_30% 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) ₂-autoinhibition, and antagonist effects against the 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. pK_d 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), pK_a values (negative logarithms of dissociation constants of agonist-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. pEC_30% values in atria correlated with pK_d values (r = 0.942). pK_d values in atria correlated with pK_d values in the brain cortex (r = 0.970).It is concluded that the ₂-autoreceptors in atria and the brain cortex are the same. Comparison with antagonist affinities for prototypic native ₂ binding sites, binding sites in cells transfected with ₂ subtype genes, and previously classified presynaptic ₂-adrenoceptors — all taken from the literature - indicates that both autoreceptors are _2D. In atria, this identification is reached with either of the two independent estimates of autoreceptor affinity, pEC_30% and pK_d, and in the brain cortex it is supported by the agonist pK_a values. The results are compatible with the hypothesis that at least the majority of ₂-autoreceptors belong to the _2AD branch of the ₂-adrenoceptor tree.     

Subclassification of presynaptic α2-adrenoceptors: α2A-autoreceptors in rabbit atria and kidney

The study was devised to classify, by means of antagonist affinities, the presynaptic ₂-autoreceptors of rabbit atria and kidney in terms of _2A, _2B, _2C and _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-Noadrenaline 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 ₂-autoinhibition. All 11 (atria) or 10 (kidney) antagonists increased the evoked overflow of tritium. pEC_30% 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 ₂-autoinhibition, and concentration-inhibition curves of the 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. pK_d values of the antagonists were calculated from the shifts. pEC_30% values correlated with pK_d values, both in atria (r = 0.728) and in the kidney (r = 0.930). pEC_30% values in atria correlated with pEC_30% values in the kidney (r = 0.988) and pK_d values in atria correlated with pK_d values in kidney (r = 0.923).It is concluded that the ₂-autoreceptors in atria and the kidney are the same. Comparison with antagonist affinities for prototypic native ₂ binding sites, ₂ binding sites in cells transfected with ₂ subtype genes, and previously classified presynaptic ₂-adrenoceptors — all taken from the literature — indicates that both autoreceptors are _2A. This conclusion is reached with either of the two independent estimates of autoreceptor affinity, pEC_30% and pK_d. The results are compatible with the hypothesis that at least the majority of ₂-autoreceptors belong to the _2A/D branch of the ₂-adrenoceptor tree, across mammalian or at least rodent and lagomorph species.     

Analgesic synergy between opioid and α2-adrenoceptors

Opioid and α₂-adrenoceptor agonists are potent analgesic drugs and their analgesic effects can synergize when co-administered. These supra-additive interactions are potentially beneficial clinically; by increasing efficacy and/or reducing the total drug required to produce sufficient pain relief, undesired side effects can be minimized. However, combination therapies of opioids and α₂-adrenoceptor agonists remain underutilized clinically, in spite of a large body of preclinical evidence describing their synergistic interaction. One possible obstacle to the translation of preclinical findings to clinical applications is a lack of understanding of the mechanisms underlying the synergistic interactions between these two drug classes. In this review, we provide a detailed overview of the interactions between different opioid and α₂-adrenoceptor agonist combinations in preclinical studies. These studies have identified the spinal cord as an important site of action of synergistic interactions, provided insights into which receptors mediate these interactions and explored downstream signalling events enabling synergy. It is now well documented that the activation of both μ and δ opioid receptors can produce synergy with α₂-adrenoceptor agonists and that α₂-adrenoceptor agonists can mediate synergy through either the α_2A or the α_2C adrenoceptor subtypes. Current hypotheses surrounding the cellular mechanisms mediating opioid–adrenoceptor synergy, including PKC signalling and receptor oligomerization, and the evidence supporting them are presented. Finally, the implications of these findings for clinical applications and drug discovery are discussed.

LINKED ARTICLES

This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2     

Modulation of motor activity by α1 and α2 stimulation in mice

Summary The influence of two -adrenoceptor agonists, clonidine and B-HT 920, on motor activity was tested in mice. Both, clonidine and B-HT 920 (2-amino-6-allyl-5,6,7,8-tetrahydro-4H-thiazolo-[4,5-d]-azepine) in the dose range 30–300 g/kg s.c. equieffectively inhibited exploratory activity. On the other hand only clonidine, which stimulates ₂- and ₂-adrenoceptors increased locomotor activity in mice treated with reserpine (5 mg/kg) and apomorphine (3 mg/kg) in the doses of 0.3 and 1 mg/kg i.p. The highly selective ₂-agonist B-HT 920 was ineffective under these conditions up to 30 mg/kg i.p. It is concluded, that in mice sedative -adrenoceptors are of the ₂- and excitatory of the ₁-type.     

Occurrence, pharmacology and function of presynaptic α2-autoreceptors in α2 A/D-adrenoceptor-deficient mice

The occurrence, pharmacological properties and function of alpha2-autoreceptors were studied in hippocampal slices, occipito-parietal cortex slices, segments of heart atria and segments of the vas deferens of wildtype (WT) mice and mice in which the alpha2 A/D-adrenoceptor gene had been disrupted (alpha2 A/D(KO)). Tissues were preincubated with [3H]-noradrenaline and then superfused and stimulated electrically. Stimulation periods for brain slices consisted either of 1 pulse or of 2-64 pulses delivered at 1-s intervals; stimulation periods for peripheral tissues consisted either of 1 POP (pseudo-one-pulse; brief burst of 20 pulses/50 Hz) or of 2-4 POPs delivered at 1-s intervals. Single pulses or POPs were used to study the effect of medetomidine and its interaction with antagonists. One or more pulses or POPs per stimulation period were used to study alpha2-autoinhibition. Medetomidine decreased the evoked overflow of tritium in WT tissues. In alpha2 A/D(KO) tissues, the inhibition was slightly (peripheral tissues) or greatly (brain slices) attenuated but not abolished. Phentolamine, rauwolscine, spiroxatrine, 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101), tolazoline and prazosin antagonized the effect of medetomidine in all tissues. Their pKd values against medetomidine were compared with pKd values at prototypical alpha2 binding sites by means of a correlation analysis. For WT brain and atrial autoreceptors, the correlations indicated an alpha2D pharmacology, whereas for WT vas deferens autoreceptors they favoured an alpha2B pharmacology. In the KO tissues, any correlation with alpha2D was lost, and the non-alpha2 A/D-autoreceptors displayed alpha2B or alpha2C pharmacology. When 2 or more pulses or POPs were applied to WT tissues per stimulation period, the pulse number-overflow curve (POP number-overflow curve) was flat, indicating that overflow elicited by p pulses (POPs) was much smaller than p times the overflow elicited by a single pulse (POP); moreover, rauwolscine caused a pulse (POP) number-dependent and, at high pulse (POP) numbers, large increase in evoked tritium overflow. In alpha2 A/D(KO) tissues, the pulse (POP) number-overflow curve was much steeper, indicating that overflow elicited by p pulses (POPs) was closer to p times the overflow elicited by a single pulse (POP); moreover, rauwolscine caused no (atria) or only a small increase in overflow, and did so in brain slices only at high pulse numbers (16 and 64). In conclusion, the predominant alpha2D pharmacology of the autoreceptors in WT tissues supports the idea that the main mammalian presynaptic alpha2-autoreceptors belong to the alpha2 A/D subtype. However, alpha2 A/D-deficient animals also possess autoreceptors. As expected, these non-alpha2 A/D-autoreceptors display alpha2B or alpha2C pharmacology. In WT animals, alpha2B- or alpha2C-autoreceptors or both may coexist with alpha2 A/D-autoreceptors, at least in peripheral tissues. Little autoinhibition by released noradrenaline in trains of pulses remains when the alpha2 A/D-adrenoceptor is lacking, again in accord with a predominance of alpha2 A/D-autoreceptors.     

Minimal α1- and α2-adrenoceptor-mediated coronary vasoconstriction in the anaesthetized swine

Summary -Adrenoceptor-mediated coronary vasoconstriction contributes to the initiation and aggravation of experimental and clinical myocardial ischaemia. However, the extent of ₁- and ₂-adrenoceptor-mediated constriction has not been characterized in the porcine coronary circulation despite the frequent use of this experimental model.Fifteen swine were anaesthetized with either -chloralose, enflurane or isoflurane to determine the amount of -adrenoceptor-mediated coronary constriction elicited by either the selective ₁-adrenoceptor agonist methoxamine or the selective ₂-adrenoceptor agonist azepexole. The left anterior descending coronary artery was cannulated and perfused by an external pump delivering constant blood flow from the carotid artery. Following bilateral cervical vagotomy and ß-adrenoceptor blockade with propranolol (2 mg kg^–1), graded dosages of either one of the -adrenoceptor agonists (9–45 g kg^–1 min^–1) were infused into the coronary perfusion line while coronary arterial pressure (CAP) was measured through a distal side arm of the cannula to detect changes in coronary vascular resistance. Infusion of the -adrenoceptor agonists was terminated when systemic arterial pressure increased. Sonomicrometers were used to measure anterior left ventricular wall thickening for the assessment of regional contractile function. During methoxamine infusion, no increase in vascular resistance was observed during -chloralose, enflurane or isoflurane anaesthesia, whereas the infusion of azepexole increased CAP from 103 ± 31 mmHg to 120 ± 35 mmHg (-chloralose), from 101 ± 16 mmHg to 122 ± 11 mmHg (enflurane) and from 84 ± 20 mmHg to 94 ± 19 mmHg (isoflurane), respectively. In four additional swine anaesthetized with enflurane, the intracoronary infusion of the full catecholamine agonist noradrenaline in the presence of propranolol (6 mg kg^–1) increased CAP from 98 ± 10 to 105 ± 10 mmHg prior to an increase in regional left ventricular function or systemic arterial pressure.These results indicate that there are no ₁- and relatively little ₂-adrenoceptor-mediated coronary constrictive effects in swine. Furthermore, neither -adrenoceptor agonist produced any detectable change in regional myocardial contractile function, regardless of the anaesthesia used.Supported by the German Research Foundation (He 1320/3-2). Dr. Guth is the recipient of a scholarship from the Alexander von Humboldt-Foundation. Send offprint requests to G. Heusch at the above address     

Comparison between thermoregulatory effects mediated by α1- and α2-adrenoceptors in normothermic and febrile rabbits

• 1.1. In order to better delineate the profile of thermoregulatory action of α₁-adrenoceptor antagonists; corynanthine (CRN) and BMY 20064 (BMY), and α₂-adrenoceptor agonist; medetomidine (MDT) and B-HT 920 (BHT), the effect of intravenous administration of two doses of these drugs on rectal (T_re) and ear skin (T_e) temperatures, metabolic rate (M), respiratory evaporative heat loss (E_res) and respiratory rate (R_r) were examined in febrile and non-febrile rabbits.
• 2.2. Results indicate that α₁-adrenoceptor antagonists as well as α₂-adrenoceptor agonists markedly lowered body temperature exhibiting antipyretic and hypothermic actions. The hypothermic and antipyretic effect after the CRN or BMY, and BHT or MDT, treatment was associated with inhibition of metabolic rate and/or with body heat redistributed to peripheral tissues and an increase of the potential for heat loss to the environment.
• 3.3. BMY also abolished the thermogenic response to cold. However, BMY did not affect metabolic heat production on exposure to a cold ambient temperature. This unexpected phenomenon is difficult to explain at the present moment. Possible mechanisms responsible for the thermoregulatory activity of BMY are discussed.
• 4.4. These results indicate that α₁- and α₂-adrenoceptor drugs' thermoregulatory actions are similar in event and suggest that both subtypes of α-adrenoceptor might be implicated functionally in a variety of thermoregulatory processes.

     

Acute dilation to α2-adrenoceptor antagonists uncovers dual constriction and dilation mediated by arterial α2-adrenoceptors

Background and purpose:

In mouse tail arteries, selective α₂-adrenoceptor antagonism with rauwolscine caused powerful dilation during constriction to the α₁-adrenoceptor agonist phenylephrine. This study therefore assessed phenylephrine''s selectivity at vascular α-adrenoceptors and the mechanism(s) underlying dilation to rauwolscine.

Experimental approach:

Mouse isolated tail arteries were assessed using a pressure myograph.

Key results:

The α₂-adrenoceptor agonist UK14,304 caused low-maximum constriction that was inhibited by rauwolscine (3 × 10⁻⁸ M) but not by the selective α₁-adrenoceptor antagonist prazosin (10⁻⁷ M). Concentration–effect curves to phenylephrine, cirazoline or noradrenaline were unaffected by rauwolscine but were inhibited by prazosin, which was more effective at high compared with low levels of constriction. In the presence of prazosin, rauwolscine inhibited the curves and was more effective at low compared with high levels of constriction. Although rauwolscine alone did not affect concentration–effect curves to phenylephrine, noradrenaline or cirazoline, it caused marked transient dilation when administered during constriction to these agonists. Dilation was mimicked by another α₂-adrenoceptor antagonist (RX821002, 3 × 10⁻⁸ M), was dependent on agonist selectivity, and did not occur during adrenoceptor-independent constriction (U46619). During constriction to UK14,304 plus U46619, rauwolscine or rapid removal of UK14,304 caused transient dilation that virtually abolished the combined constriction. Endothelial denudation reduced these dilator responses.

Conclusions and implications:

Inhibition of α₂-adrenoceptors caused transient dilation that was substantially greater than the contribution of α₂-adrenoceptors to the constriction. This reflects a slowly reversing α₂-adrenoceptor-mediated endothelium-dependent dilation and provides a rapid, sensitive test of α₂-adrenoceptor activity. This approach also clearly emphasizes the poor selectivity of phenylephrine at vascular α-adrenoceptors.     

Pregabalin is a potent and selective ligand for α(2)δ-1 and α(2)δ-2 calcium channel subunits

Pregabalin, a synthetic branched chain γ-amino acid with anticonvulsant, anxiolytic, and analgesic activities, has been shown to bind with high affinity to the voltage-gated calcium channel α(2)δ subunit. Given the broad therapeutic utility of pregabalin, a series of experiments was undertaken to determine the potency, selectivity, and specificity of pregabalin's receptor-binding profile at α(2)δ-1 and α(2)δ-2 subunits of voltage-gated calcium channels along with 38 widely studied receptors and channels. Receptor autoradiography was used to assess regional-binding density of pregabalin throughout the rat spinal cord and brain. In addition, a series of studies using in vivo electrophysiological recordings of γ-aminobutyric acid (GABA)(A)- and GABA(B)-evoked currents was undertaken to determine the interaction of pregabalin with GABAergic receptor subtypes. Together, the results of these studies demonstrate potent and selective binding of pregabalin to α(2)δ-1 and α(2)δ-2 subunits in native and recombinant human and porcine systems. Pregabalin did not interact with any of the 38 receptors and ion channels evaluated, and a variety of central nervous system (CNS)-targeted therapeutic drugs did not show activity at the α(2)δ subunits of voltage-gated calcium channels. Receptor autoradiography demonstrated extensive [(3)H]-pregabalin binding throughout the CNS, with high-level binding in the cortex, hippocampus, cerebellum, dorsal horn of the spinal cord, and amygdala. Finally, receptor-binding and electrophysiological techniques failed to show evidence of an interaction between pregabalin and GABA(A) or GABA(B) receptors. These studies suggest that the clinical effects of pregabalin are likely due to direct and selective interactions with α(2)δ-1 and α(2)δ-2 subunits of voltage-gated calcium channels.     

Differential Roles of α6β2* and α4β2* Neuronal Nicotinic Receptors in Nicotine- and Cocaine-Conditioned Reward in Mice

Mesolimbic α6* nicotinic acetylcholine receptors (nAChRs) are thought to have an important role in nicotine behavioral effects. However, little is known about the role of the various α6*-nAChRs subtypes in the rewarding effects of nicotine. In this report, we investigated and compared the role of α6*-nAChRs subtypes and their neuro-anatomical locus in nicotine and cocaine reward-like effects in the conditioned place preference (CPP) paradigm, using pharmacological antagonism of α6β2* nAChRs and genetic deletion of the α6 or α4 subunits in mice. We found that α6 KO mice exhibited a rightward shift in the nicotine dose–response curve compared with WT littermates but that α4 KO failed to show nicotine preference, suggesting that α6α4β2*-nAChRs are involved. Furthermore, α6β2* nAChRs in nucleus accumbens were found to have an important role in nicotine-conditioned reward as the intra-accumbal injection of the selective α6β2* α-conotoxin MII [H9A; L15A], blocked nicotine CPP. In contrast to nicotine, α6 KO failed to condition to cocaine, but cocaine CPP in the α4 KO was preserved. Intriguingly, α-conotoxin MII [H9A; L15A], blocked cocaine conditioning in α4 KO mice, implicating α6β2* nAChRs in cocaine reward. Importantly, these effects did not generalize as α6 KO showed both a conditioned place aversion to lithium chloride as well as CPP to palatable food. Finally, dopamine uptake was not different between the α6 KO or WT mice. These data illustrate that the subjective rewarding effects of both nicotine and cocaine may be mediated by mesolimbic α6β2* nAChRs and that antagonists of these receptor subtypes may exhibit therapeutic potential.     

Ventral hippocampal α7 and α4β2 nicotinic receptor blockade and clozapine effects on memory in female rats

Rationale Nicotinic systems in the hippocampus play important roles in memory function. Decreased hippocampal nicotinic receptor concentration is associated with cognitive impairment in schizophrenia and Alzheimer’s disease.Methods We modeled in rats the cognitive effects of chronic decrease in hippocampal α7 or α4β2 receptors with 4-week continuous bilateral local infusions of the α7 nicotinic antagonist methyllycaconitine (MLA) or the α4β2 antagonist dihydro-β-erythroidine (DHβE). The working memory effects of these infusions were assessed by performance on the radial-arm maze. To test the effect of antipsychotic medication, we gave acute injections of clozapine and to determine the impact of nicotine, which is widely used by people with schizophrenia approximately half of the rats received chronic systemic infusions of nicotine.Results Chronic ventral hippocampal DHβE infusion caused a significant (p<0.001) working memory impairment. Acute systemic clozapine (2.5 mg/kg) caused a significant (p<0.005) working memory impairment in rats given control aCSF hippocampal infusions. Clozapine significantly (p<0.025) attenuated the memory deficit caused by chronic hippocampal DHβE infusions. Chronic ventral hippocampal infusions with MLA did not significantly affect the working memory performance in the radial-arm maze, but it did significantly (p<0.05) potentiate the memory impairment caused by 1.25 mg/kg of clozapine. Chronic systemic nicotine did not significantly interact with these effects.Conclusions The state of nicotinic receptor activation in the ventral hippocampus significantly affected the impact of clozapine on working memory with blockade of α7 nicotinic receptors potentiating clozapine-induced memory impairment and blockade of α4β2 receptors reversing the clozapine effect from impairing to improving memory.     

Atypical antipsychotics as noncompetitive inhibitors of α4β2 and α7 neuronal nicotinic receptors

It has been suggested that the interaction of antipsychotic medications with neuronal nicotinic receptors may increase the cognitive dysfunction associated with schizophrenia and may explain why current therapies only partially address this core feature of the illness. In the present studies we compared the effects of the atypical antipsychotics quetiapine, clozapine and N-desmethylclozapine to those of the typical antipsychotics haloperidol and chlorpromazine on the α4β2 and α7 nicotinic receptor subtypes. The binding of [³H]-nicotine to rat cortical α4β2 receptors and [³H]-methyllycaconitine to rat hippocampal α7 receptors was not affected by any of the compounds tested. However, Rb⁺ efflux evoked either by nicotine or the selective α4β2 agonist TC-1827 from α4β2 receptors expressed in SH-EP1 cells and nicotine-evoked [³H]-dopamine release from rat striatal synaptosomes were non-competitively inhibited by all of the antipsychotics. Similarly, α-bungarotoxin-sensitive epibatidine-evoked [³H]-norepinephrine release from rat hippocampal slices and acetylcholine-activated currents of α7 nicotinic receptors expressed in oocytes were inhibited by haloperidol, chlorpromazine, clozapine and N-desmethylclozapine. The inhibitory effects on nicotinic receptor function produced by the antipsychotics tested occurred at concentrations similar to plasma levels achieved in schizophrenia patients, suggesting that they may lead to clinically relevant effects on cognition.     

α2-autoreceptors and α2-heteroreceptors modulating tyrosine and tryptophan hydroxylase activity in the rat brain in vivo: an investigation into the α2-adrenoceptor subtypes

The subtype determination of auto- and hetero-₂-adrenoceptors modulating the synthesis of noradrenaline (NA) and serotonin (5-HT), respectively, was assessed using the accumulation of 3,4-dihydroxyphenylalanine (dopa) and 5-hydroxytryptophan (5-HTP) after decarboxylase inhibition as a measure of the rate of tyrosine and tryptophan hydroxylation in the rat brain in vivo.In the cerebral cortex and hippocampus, Org 3770 (non-selective a2-adrenoceptor antagonist, 0.5–10 mg/kg, i.p.) increased (43%–58%) and clonidine (non-selective ₂-adrenoceptor agonist, 1 mg/kg) decreased (37%–49%) the synthesis of dopa. Also the antagonist ARC 239 (_2B/C selective, 5–40 mg/kg) increased the synthesis of dopa in cortex (39%–46%) and hippocampus (17%–85%). In contrast, the antagonist BRL 44408 (_2D selective, 1–10 mg/kg) did not increase the synthesis of dopa in cortex, and increased it modestly in hippocampus only. The agonist guanoxabenz (_2B/C selective, 0.03–3 mg/kg) decreased the synthesis of dopa in both brain regions (20%–65%), whereas the agonist oxymetazoline (_2D selective, 0.1–3 mg/kg) failed to do so. These results indicated that the ₂-autoreceptors that modulate the synthesis of dopa/NA are probably associated with the _2B/C-subtypes.In cortex and hippocampus, clonidine decreased (35%–53%) the synthesis of 5-HTP but Org 3770 failed to induce the opposite effect (except the 2 mg/kg dose in cortex). BRL 44408 markedly increased the synthesis of 5-HTP in cortex (113%–148%) but not in hippocampus. Similarly, also ARC 239 increased the formation of 5-HTP in cortex (36%–48%) but not in hippocampus, where it was decreased (30%–55%). Oxymetazoline decreased the synthesis of 5-HTP in hippocampus (28%–30%) but failed to do so in cortex. Guanoxabenz in the low dose range (0.03–0.3 mg/kg) did not decrease the synthesis of 5-HTP in any brain region. These results indicated that the ₂-heteroreceptors that modulate the synthesis of 5-HTP/5-HT may well be different from the proposed _2B/C-autoreceptors modulating the synthesis of dopa/NA. These ₂-heteroreceptors appear to be associated with the _2D-subtype.     

Both α2B- and α2C-adrenoceptor subtypes are involved in the mediation of centrally induced gastroprotection in mice

α(2)-adrenoceptors are known to mediate gastroprotective effect in both acid-dependent and acid-independent ulcer models. The aim of the present study was to determine, which of the three α(2)-adrenoceptor subtypes (α(2A), α(2B) or α(2C)) is responsible for this protection. Various α(2)-adrenoceptor agonists and antagonists were administered intracerebroventricularly (i.c.v.) to C57BL/6 mice with deletion of genes encoding the different subtypes. The gastric mucosal damage was induced by orally injected acidified ethanol. Both the non-selective α(2)-adrenoceptor agonist clonidine (0.3-2.8 nmol) and the α(2B/C)-adrenoceptor subtype preferring agonist ST-91 (0.5-11.5 nmol) induced dose-dependent gastroprotective effect in wild type, α(2A)-, α(2B)- and α(2C)-KO mice. In contrast, the α(2A)-adrenoceptor subtype agonist oxymetazoline (0.07-84 nmol i.c.v.) reduced only slightly the development of ethanol-induced ulcers. The effect of clonidine was antagonized by the non-selective antagonist yohimbine (25 nmol) and the α(2B/C)-adrenoceptor antagonist ARC 239 (10.4 nmol), but not by the α(2A)-adrenoceptor antagonist BRL 44408 (7.5 nmol). ARC 239 also reversed the effect of clonidine in α(2A)-, α(2B)- and α(2C)-KO mice, while the selective α(2C)-adrenoceptor antagonist JP 1302 (52 nmol) antagonized that only in α(2B)-KO, but not in α(2A)- and α(2C)-KO mice. These results suggest that α(2B)- and α(2C)-adrenoceptor subtypes can equally contribute to the mediation of gastroprotective effect induced by α(2)-adrenoceptor agonists in mice.