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Unlabelled: 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 α2A or the α2C 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., (© 2014 The British Pharmacological Society.)

Opposite age-dependent changes in alpha2-adrenoceptor and imidazoline I2 receptor (I2-IRs) density have been related to brain gliosis development with aging. To check this hypothesis we applied in rats a model of reactive gliosis induced by heat. The specific binding of [3H]idazoxan (0.5-20 nM) in the presence of (-)adrenaline (5 x 10(-6) M) to membranes from rat brain cortex showed that the density of I(2)-IRs was significantly higher in membranes of injured cortex (Bmax=60+/-6 fmol/mg protein; n=9) than in control (Bmax=38+/-3 fmol/mg protein; n=9; p=0.0053). Conversely, the density of alpha2-adrenoceptors, measured by [3H]clonidine (0.25-16 nM), in the injured cortex (Bmax=75+/-4 fmol/mg protein; n=9) was significantly lower than in sham membranes (Bmax=103+/-7 fmol/mg protein; n=9; p=0.0035). No significant differences in receptor's affinity were observed between both groups. These results support the hypothesis that gliosis induces opposite changes in alpha2-adrenoceptor and I2-IR density.

Atipamezole is an alpha2-adrenoceptor antagonist with an imidazole structure. Receptor binding studies indicate that its affinity for alpha2-adrenoceptors and its alpha2/alpha1 selectivity ratio are considerably higher than those of yohimbine, the prototype alpha2-adrenoceptor antagonist. Atipamezole is not selective for subtypes of alpha2-adrenoceptors. Unlike many other alpha2-adrenoceptor antagonists, it has negligible affinity for 5-HT1A and I2 binding sites. Atipamezole is rapidly absorbed and distributed from the periphery to the central nervous system. In humans, atipamezole at doses up to 30 mg/subject produced no cardiovascular or subjective side effects, while at a high dose (100 mg/subject) it produced subjective symptoms, such as motor restlessness, and an increase in blood pressure. Atipamezole rapidly reverses sedation/anesthesia induced by alpha2-adrenoceptor agonists. Due to this property, atipamezole is commonly used by veterinarians to awaken animals from sedation/anesthesia induced by alpha2-adrenoceptor agonists alone or in combination with various anesthetics. Atipamezole increased sexual activity in rats and monkeys. In animals with sustained nociception, atipamezole increased pain-related responses by blocking the noradrenergic feedback inhibition of pain. In tests assessing cognitive functions, atipamezole at low doses has beneficial effects on alertness, selective attention, planning, learning, and recall in experimental animals, but not necessarily on short-term working memory. At higher doses atipamezole impaired performance in tests of cognitive functions, probably due to noradrenergic overactivity. Recent experimental animal studies suggest that atipamezole might have beneficial effects in the recovery from brain damage and might potentiate the anti-Parkinsonian effects of dopaminergic drugs. In phase I studies atipamezole has been well tolerated by human subjects.

Oxytocin induces long-term changes in, for example, blood pressure, spontaneous motor activity and corticosterone levels in rats. Previous studies in male rats have suggested a role for alpha(2)-adrenoceptors within the central nervous system in these effects. The aim of the present study was to investigate if oxytocin treatment in female rats would influence alpha(2)-adrenoceptors within the hypothalamus, the amygdala and the nucleus of the solitary tract (NTS). For this purpose, female ovariectomized (OVX) rats were treated with oxytocin (1 mg/kg s.c.) or saline once a day for 10 days. Rats were decapitated 5 days after the last injection, and brains and plasma were collected. Quantitative receptor autoradiography for characterization of high affinity alpha(2)-adrenoceptor agonist binding and radioimmunoassay for corticosterone were performed. Oxytocin increased the B(max) values of the alpha(2)-adrenoceptor agonist [3H]UK14.304 binding sites significantly in all the analyzed areas (P<0.05). K(d) values were unchanged. Plasma levels of corticosterone were significantly decreased in the oxytocin-treated rats (P<0.05). These findings are in further support of an interaction between oxytocin receptors and alpha(2)-adrenoceptors and show that oxytocin treatment may increase alpha(2)-adrenoceptor recognition probably leading to an increase in alpha(2)-adrenoceptor signaling in several parts of the brain.

There has been much evidence showing that the central sympathetic nervous system may be involved in the control of blood pressure. In the present study, we investigated the role of the presynaptic alpha2-adrenergic receptors and the cyclic adenosine monophosphate-dependent protein kinase (protein kinase A) in the regulation of norepinephrine release in the central nervous system in hypertension. The alpha2-adrenergic receptor agonists UK 14, 304 and clonidine inhibited the stimulation-evoked [3H]norepinephrine release in a dose-dependent manner in the medulla oblongata of Sprague-Dawley rats. Pretreatment of pertussis toxin (a potent inhibitor of the Gi-protein) attenuated the suppression of NE release by UK 14, 304. The protein kinase A inhibitor H-8 also reduced the stimulation-evoked [3H]norepinephrine release in rat medulla oblongata. In spontaneously hypertensive rats, the inhibitory effect of UK 14, 304 on the stimulation-evoked norepinephrine release was significantly less than in age-matched normotensive Wistar-Kyoto rats. By contrast, the protein kinase A inhibitor H-8 reduced the stimulation-evoked norepinephrine release to a greater extent in hypertension than in normotensive controls. The results of the present study showed that the alteration in the presynaptic alpha2-receptor-protein kinase A system might actively participate in the regulation of norepinephrine release in the central nervous system in hypertension.

The aim of this study was to investigate imidazoline(2) (I(2)) binding site- and alpha(2)-adrenoceptor-mediated control of central noradrenergic and HPA axis activity in control rats and chronically stressed rats with adjuvant-induced arthritis (AA). Basal levels of extracellular nonadrenaline (NA) in the region of the hypothalamic paraventricular nucleus (PVN) of AA rats were significantly greater than controls. Both the I(2) binding site selective ligand BU224 (10 mg kg(-1) i.p.) and the alpha(2)-adrenoceptor antagonist RX821002 (2.5 mg kg(-1) i.p.) significantly elevated extracellular levels of NA in the PVN region and plasma corticosterone (CORT) in a rapid and transient manner in both control and AA rats. The noradrenergic response of AA rats to BU224 was significantly enhanced compared with drug treated controls. There was a significant correlation between extracellular NA in the PVN region and plasma CORT following BU224 and RX821002. In conclusion, central noradrenergic and HPA axis activity in control and chronically stressed AA rats appear to be under the control of both I(2) binding sites and alpha(2)-adrenoceptors. Increased basal levels of extracellular NA in the PVN region of AA rats suggests increased noradrenergic activity in these animals which is modulated to a greater extent by I(2) binding sites than by alpha(2)-adrenoceptors.

Interactions between brain alpha2- and beta-adrenoceptors are of interest in physiological (aging) and pathological (major depression) processes involving both receptors. In this study, total beta-adrenoceptors and beta1/2-subtypes were quantitated in postmortem human brains to investigate their relationships with alpha2A-adrenoceptors and specific G proteins during the process of aging and in brains of suicide victims. Analysis of [3H]CGP12177 binding, in the presence of CGP20712A (beta1-antagonist), indicated that the predominant beta-adrenoceptor in the frontal cortex is the beta1-subtype (65-75%). The density of total beta- (r=-0.60, n=44) or beta1-adrenoceptors (r=-0.78, n=22), but not the beta2-subtype, declined with aging (3-80 years). The density of total beta- or beta1-adrenoceptors, but not the beta2-subtype, correlated with the number of alpha2-adrenoceptors quantitated in the same brains with the agonist [3H]UK14304 (r=0.71-0.81) or the antagonist [3H]RX821002 (r=0.61-0.66). Interestingly, the ratios alpha2/beta- or alpha2/beta1-adrenoceptors did not correlate with the age of the subject at death, indicating that the proportion of alpha2/beta-adrenoceptors in brain remains rather constant during the process of aging. The density of beta-adrenoceptors correlated with the immunodensity of G(alpha)s (r=0.55) and Gbeta (r=0.61) proteins, and that of alpha2-adrenoceptors with those of G(alpha)i1/2 (r=0.88) and Gbeta (r=0.65). In brains of suicides, compared to controls, the ratio between alpha2- and beta- or beta1-adrenoceptors (alpha2-full agonist sites/beta-sites) was greater (1.3- to 2.0-fold; P<0.05). The results demonstrate a close interdependence between brain alpha2- and beta-adrenoceptors during aging, and in brains of suicides. The quantitation of the alpha2A/beta-adrenoceptor ratio could represent a relevant neurochemical index in the study of brain pathologies in which both receptors are involved.

Systemic subchronic oxytocin treatment significantly and substantially increased the B(max) values of the alpha 2 agonist [(3)H]UK14.304 binding sites in the hypothalamus, the amygdala and the paraventricular thalamic nucleus of the rat as shown by quantitative receptor autoradiography. These results suggest that long-term modulation of autonomic and neuroendocrine functions and emotional behaviours elicited by brain oxytocin may involve enhancement of central alpha 2-adrenoceptor function.

The metabolism of NS-49 ((R)-(-)-3'-(2-amino-1-hydroxyethyl)-4'-fluoro-methanesulfonanilide++ + hydrochloride, CAS 137431-04-0), a phenethylamine class alpha 1A-adrenoceptor agonist, was investigated in rats, rabbits, dogs, and monkeys after single intravenous or oral administration of 14C-NS-49 or unlabeled NS-49. The N-acetylated form, the only metabolite was identified in the urine of all the species tested by thin layer chromatography and mass spectrometry. The unchanged drug and N-acetylated form accounted for almost all the radioactivity in the urine. The order of the percentage of the N-acetylated form in the urinary radioactivity after oral administration was monkeys (66%) > rabbits (22%) > rats (14%) > dogs (1.3%). There were no marked differences between the intravenous and oral routes in the percentages of NS-49 and its N-acetylated form for the rats, rabbits, and dogs, indicative of negligible first-pass metabolism. In the monkeys, however, the percentage of NS-49 in the urinary radioactivity after oral administration was about half that after intravenous injection due to first-pass metabolism, and the N-acetylated form showed an inverse relation. The N-acetylated form also was the only metabolite in the plasma and tissues (liver, kidney, heart, and lung) 1 and 4 h after the oral administration of 14C-NS-49 to rats. NS-49 and its N-acetylated form accounted for more than 80% of the radioactivity in the plasma and the four tissues. The percentages of NS-49 and its N-acetylated form in the total radioactivity did not differ markedly among the plasma and tissues. Repeated oral administrations of 1, 10, and 100 mg/kg of NS-49 to male rats once a day for 7 days had no effects on their hepatic drug-metabolizing enzyme activities.

Spirals of endothelially denuded equine saphenous vein were used to study the pre- and post-junctional effects of medetomidine in vitro. The pD2 values were calculated for noradrenaline (6.7 +/- 0.1), phenylephrine (5.6 +/- 0.1), BHT 920 (6.2 +/- 0.2) and UK 14304 (5.7 +/- 0.2). Medetomidine produced a biphasic response, with a pD(2)1 of 8.2 +/- 0.1 and a pD(2)2 of 5.7 +/- 0.1 in the equine saphenous vein (n = 6). Prazosin (10(-7) M) significantly shifted the second phase of the medetomidine concentration-response curve to the right (pD(2)1 was 8.1 +/- 0.2 and pD(2)2 was 5.0 +/- 0.2, P < 0.05). Rings of equine saphenous vein were electrically stimulated to investigate the pre-junctional effects of medetomidine. Increasing concentrations of the alpha 2-adrenoceptor agonist BHT 920 reduced the response to electrical stimulation in a concentration dependent manner to a maximum of 40 +/- 5%, whereas medetomidine (0.1-100 nM) caused a concentration dependent enhancement to a maximum of 490 +/- 150%. These results suggest alpha 1- and alpha 2-adrenoceptors are functional in the equine saphenous vein, but that medetomidine is not acting exclusively as an alpha 2-adrenoceptor agonist.

An association between dysphoric premenstrual syndromes (PMS) and a lifetime history of major depressive disorders has previously been documented. Other studies have demonstrated an increase in the binding of radiolabeled imidazoline compounds to platelets of depressed patients. Clonidine and related imidazoline compounds interact with alpha 2 adrenoceptors to inhibit neuronal noradrenergic activity and in higher concentrations, they stimulate noradrenergic activity through their interaction with imidazoline receptors. Here we report increased 3H para-aminoclonidine binding to high affinity alpha 2 adrenoceptor sites as well as to nonadrenergic imidazoline binding sites in platelets of women with dysphoric PMS. This higher binding was most pronounced during the late-luteal-symptomatic phase of the menstrual cycle and, to a lesser degree, during the non-symptomatic mid-follicular phase. Binding to the imidazoline site distinguished women with dysphoric PMS from women with no such symptoms, was highly positively correlated with the severity of symptoms, and was negatively correlated with plasma levels of progesterone. These findings suggest that platelet imidazoline binding sites might be a biological marker for dysphoric states in PMS or for the vulnerability to develop them. These findings also point to a possible biological link between dysphoric PMS and major depressive disorders.