Jesús A. García-Sevilla

α2-Adrenoceptors in the brain of suicide victims: increased receptor density associated with major depression

To examine directly in the brain the status of the alpha 2-adrenoceptor in major depression, the specific binding of the agonists [3H]clonidine and [3H]UK 14304 was quantitated in various brain regions of suicide victims with a retrospective diagnosis of depression or other psychiatric disorders. In depressed suicides, the binding capacity of [3H]clonidine was found to be increased in the hypothalamus (Bmax 35%-55% greater), and to a lesser extent in the frontal cortex, as compared with that in matched controls, schizophrenic suicides, or suicides with various diagnosis. The binding capacity of [3H]UK 14304 also was found increased in the frontal cortex (Bmax 30% greater), and to a lesser extent in the hypothalamus, of depressed suicides. In other brain regions such as the amygdala, hippocampus, and cerebellum there also was a tendency for an increased receptor density associated with suicide. Moreover, in the frontal cortex of suicides, the potency of norepinephrine in displacing the binding of the antagonist [3H]idazoxan also was found increased (Ki decreased eight-fold). The results indicate that the density and affinity of alpha 2A-adrenoceptors in the high-affinity state are increased in the brain of depressed suicides.

Protection by imidazol(ine) drugs and agmatine of glutamate-induced neurotoxicity in cultured cerebellar granule cells through blockade of NMDA receptor.

This study was designed to assess the potential neuroprotective effect of several imidazol(ine) drugs and agmatine on glutamate‐induced necrosis and on apoptosis induced by low extracellular K+ in cultured cerebellar granule cells. Exposure (30 min) of energy deprived cells to L‐glutamate (1–100 μM) caused a concentration‐dependent neurotoxicity, as determined 24 h later by a decrease in the ability of the cells to metabolize 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazoliumbromide (MTT) into a reduced formazan product. L‐glutamate‐induced neurotoxicity (EC50=5 μM) was blocked by the specific NMDA receptor antagonist MK‐801 (dizocilpine). Imidazol(ine) drugs and agmatine fully prevented neurotoxicity induced by 20 μM (EC100) L‐glutamate with the rank order (EC50 in μM): antazoline (13)>cirazoline (44)>LSL 61122 [2‐styryl‐2‐imidazoline] (54)>LSL 60101 [2‐(2‐benzofuranyl) imidazole] (75)>idazoxan (90)>LSL 60129 [2‐(1,4‐benzodioxan‐6‐yl)‐4,5‐dihydroimidazole] (101)>RX821002 (2‐methoxy idazoxan) (106)>agmatine (196). No neuroprotective effect of these drugs was observed in a model of apoptotic neuronal cell death (reduction of extracellular K+) which does not involve stimulation of NMDA receptors. Imidazol(ine) drugs and agmatine fully inhibited [3H]‐(+)‐MK‐801 binding to the phencyclidine site of NMDA receptors in rat brain. The profile of drug potency protecting against L‐glutamate neurotoxicity correlated well (r=0.90) with the potency of the same compounds competing against [3H]‐(+)‐MK‐801 binding. In HEK‐293 cells transfected to express the NR1‐1a and NR2C subunits of the NMDA receptor, antazoline and agmatine produced a voltage‐ and concentration‐dependent block of glutamate‐induced currents. Analysis of the voltage dependence of the block was consistent with the presence of a binding site for antazoline located within the NMDA channel pore with an IC50 of 10–12 μM at 0 mV. It is concluded that imidazol(ine) drugs and agmatine are neuroprotective against glutamate‐induced necrotic neuronal cell death in vitro and that this effect is mediated through NMDA receptor blockade by interacting with a site located within the NMDA channel pore.

Selective Increase of α2A‐Adrenoceptor Agonist Binding Sites in Brains of Depressed Suicide Victims

Abstract: The α2A‐ and α2C‐adrenoceptor subtypes were evaluated in postmortem brains from suicides with depression (n = 22), suicides with other diagnoses (n = 12), and controls (n = 26). Membrane assays with the antagonist [3H]RX821002 (2‐[3H]methoxyidazoxan) suggested the presence of α2A‐adrenoceptors in the frontal cortex and both α2C‐adrenoceptors and α2A‐adrenoceptors in the caudate. The proportions in caudate were similar in controls (α2A, 86%; α2C, 14%), depressed suicides (α2A, 91%; α2C, 9%), and suicides with other diagnoses (α2A, 88%; α2C, 12%). Autoradiography of [3H]RX821002 binding under α2B/C‐adrenoceptor‐masking conditions confirmed the similar densities of α2A‐adrenoceptors in the cortex, hippocampus, and striatum from controls and suicides. In the frontal cortex of depressed suicides, competition of [3H]RX821002 binding by (−)‐adrenaline revealed a greater proportion (61 ± 9%) of α2A‐adrenoceptors in the high‐affinity conformation for agonists than in controls (39 ± 5%). Simultaneous analysis with the agonists [3H]clonidine and [3H]UK14304 and the antagonist [3H]RX821002 in the same depressed suicides confirmed the enhanced α2A‐adrenoceptor density when evaluated by agonist, but not by antagonist, radioligands. The results indicate that depression is associated with a selective increase in the high‐affinity conformation of the brain α2A‐adrenoceptors.

Increased mRNA expression of α2A-adrenoceptors, serotonin receptors and μ-opioid receptors in the brains of suicide victims

The development of new therapies for the treatment of psychiatric disorders requires an in-depth knowledge of the molecular bases underlying these pathologies, which remain largely unknown. Alterations in adrenoceptors, serotonin receptors, and other G protein-coupled receptors (GPCRs) have been associated with suicide and depression. However, to date, there is little information about mRNA expression of the GPCRs in the frontal cortex of suicide victims. Our goal was to study the expression in the brain of these receptors. For this purpose, we measured mRNA levels by RT-PCR. We found that the expressions of α2A-adrenoceptors, 5-HT1A, 5-HT2A serotonin receptors, and μ-opioid receptors were elevated in the post-mortem brains of these suicide victims with respect to matched controls. Moreover, in the case of α2A-adrenoceptors (the only for which these data were available), a significant correlation was observed between the level of mRNA and protein quantified in the brain of the same subjects, indicating that protein synthesis of this receptor was not influenced by post-translational regulatory mechanisms. In addition, the degree of adrenoceptor and 5-HT receptor expressions appeared to be correlated in the brains of suicide victims and control subjects. Alterations in the expression of adrenoceptors, serotonin, and opioid receptors indicate that these signaling proteins might be related to the etiopathology of suicidal and depressive behaviors. Alternatively, such changes may represent adaptive mechanisms to compensate for other as yet unknown alterations. The results also suggest that these receptors could share common regulatory mechanisms.

Opposite Age-Dependent Changes of α2A-Adrenoceptors and Nonadrenoceptor [3H]Idazoxan Binding Sites (I2-Imidazoline Sites) in the Human Brain: Strong Correlation of I2 with Monoamine Oxidase-B Sites

Abstract: In the postmortem human brain (27 specimens of frontal cortex, Brodmann area 9), the specific binding of the antagonists [3H]RX 821002 (2‐methoxyidazoxan) to α2A‐adrenoceptors and that of [3H]idazoxan to l2‐imidazo‐line sites (a nonadrenoceptor mitochondrial site) were determined in parallel to study the effect of aging (range, 4–89 years) on both brain proteins. The density of α2A‐adrenoceptors and age were negatively correlated (r=‐0.71; p < 0.001). In contrast, the density of l2‐imidazo‐line sites was positively correlated with aging (r= 0.59; p < 0.005). The ratio of receptor densities (α2A/l2) also showed a marked negative correlation with age (r=‐0.76; p < 0.001). In an age‐selected group (range, 10–89 years), the density of monoamine oxidase (MAO)‐B sites labeled by [3H]Ro 19–6327 (lazabemide) also showed a positive correlation with age (r= 0.80; p < 0.005). In these subjects, the density of l2‐imidazoline sites correlated well with the density of MAO‐B sites (r= 0.70; p < 0.005). The ratio of the density of these sites (MAO‐B/l2) did not correlate with the age of the subject at death (r=‐0.15). In the human frontal cortex, idazoxan displayed very low affinity (Ki= 89 μM) against the binding of [3H]Ro 19–6327 to MAO‐B, which discounted a direct interaction of [3H]idazoxan with the active center of the enzyme and indicated that the l2‐imidazoline site cannot be identified with MAO‐B. However, l2‐imidazoline sites and MAO‐B show a clear coexpression not only in the human frontal cortex during the process of aging, but also in various brain regions of the human and rat brains. It is suggested that the l2‐imidazoline site has a specific location on glial (astrocyte) cells.

Activation of I2‐imidazoline receptors enhances supraspinal morphine analgesia in mice: a model to detect agonist and antagonist activities at these receptors

This work investigates the receptor acted upon by imidazoline compounds in the modulation of morphine analgesia. The effects of highly selective imidazoline ligands on the supraspinal antinociception induced by morphine in mice were determined. Intracerebroventricular (i.c.v.) or subcutaneous (s.c.) administration of ligands selective for the I2‐imidazoline receptor, 2‐BFI, LSL 60101, LSL 61122 and aganodine, and the non selective ligand agmatine, increased morphine antinociception in a dose‐dependent manner. Neither moxonidine, a mixed I1‐imidazoline and α2‐adrenoceptor agonist, RX821002, a potent α2‐adrenoceptor antagonist that displays low affinity at I2‐imidazoline receptors, nor the selective non‐imidazoline α2‐adrenoceptor antagonist RS‐15385‐197, modified the analgesic responses to morphine. Administration of pertussis toxin (0.25 μg per mouse, i.c.v.) 6 days before the analgesic test blocked the ability of the I2‐imidazoline ligands to potentiate morphine antinociception. The increased effect of morphine induced by I2‐imidazoline ligands (agonists) was completely reversed by idazoxan and BU 224. Identical results were obtained with IBI, which alkylates I2‐imidazoline binding sites. Thus, both agonist and antagonist properties of imidazoline ligands at the I2‐imidazoline receptors were observed. Pre‐treatment (30 min) with deprenyl, an irreversible inhibitor of monoamine oxidase B (IMAO‐B), produced an increase of morphine antinociception. Clorgyline, an irreversible IMAO‐A, given 30 min before morphine did not alter the effect of the opioid. At longer intervals (24 h) a single dose of either clorgyline or deprenyl reduced the density of I2‐imidazoline receptors and prevented the I2‐mediated potentiation of morphine analgesia. These results demonstrate functional interaction between I2‐imidazoline and opioid receptors. The involvement of Gi‐Go transducer proteins in this modulatory effect is also suggested.

Chronic treatment with the monoamine oxidase inhibitors clorgyline and pargyline down‐regulates non‐adrenoceptor [3H]‐idazoxan binding sites in the rat brain

1 The binding of [3H]‐idazoxan in the presence of 10−6m (−)‐adrenaline was used to quantitate non‐adrenoceptor idazoxan binding sites (NAIBS) in the rat brain after treatment with various psychotropic drugs. 2 Chronic treatment (14 days) with the monoamine oxidase (MAO) inhibitors clorgyline (0.3–10 mg kg−1, i.p.) and pargyline (10 mg kg−1, i.p.), but not with Ro 41–1049 (1 mg kg−1, i.p.), markedly decreased (30–50%) the density of NAIBS in the cerebral cortex without any apparent change in the affinity of the radioligand. 3 Acute (1 day) and/or chronic treatments (14 days) with other psychotropic drugs such as desipramine (3 mg kg−1, i.p.), cocaine (10 mg kg−1, i.p.), reserpine (0.12 mg kg−1, s.c.), haloperidol (1 mg kg−1, i.p.) and diazepam (10 mg kg−1, i.p.) did not alter the density of NAIBS in the cerebral cortex. 4 In vitro, the propargylamines clorgyline, pargyline and deprenyl displaced the binding of [3H]‐idazoxan to NAIBS from two distinct sites, but only clorgyline displayed an apparent very high affinity for a relevant population of NAIBS (KiH = 40 pm; KiL = 10.6 μm). The structurally diverse MAO inhibitors Ro 16–6491 (selective for MAO‐B) and Ro 41–1049 (selective for MAO‐A), as well as the other psychotropic drugs (desipramine, cocaine, reserpine and haloperidol) displaced the binding of [3H]‐idazoxan to NAIBS monophasically and with very low potencies. As expected, the MAO inhibitors clorgyline and Ro 41–1049 displaced the binding of [3H]‐Ro 41–1049 to MAO‐A monophasically and with high potencies (Ki values: 0.18 nm and 22 nm, respectively). In contrast, idazoxan displayed very low affinity (Ki = 40 μm) against the binding of [3H]‐Ro 41–1049 to MAO‐A. These results disprove a direct interaction between [3H]‐idazoxan and the enzyme MAO. 5 Preincubation of cortical membranes with clorgyline (10−9 m or 10−6 m for 30 min) or pargyline (10−6 m or 10−5 m for 30 min), reduced by 30–50% and by 17–30%, respectively, the total density of NAIBS without any apparent change in the affinity of the radioligand. Preincubation with 10−6 m clorgyline did not alter the affinity of cirazoline for the two populations of NAIBS, but reduced by 60% the binding of [3H]‐idazoxan to the high affinity site without affecting the binding of the radioligand to the low affinity site. These results indicate that the two MAO inhibitors irreversibly block the binding of [3H]‐idazoxan to NAIBS. 6 In vivo, however, various acute treatments with clorgyline (1–20 mg kg−1, i.p.) for different time intervals (6–48 h) did not alter the density of NAIBS. In vivo, only very high doses of clorgyline (40 and 80 mg kg−1, i.p.) induced modest decreases (21–28%) in the density of NAIBS in the cerebral cortex. 7 Together the results indicate that the irreversible binding of clorgyline and pargyline to NAIBS found in vitro does not fully explain the marked decreases in the density of NAIBS found in vivo after the chronic treatments. It is suggested that the down‐regulation of NAIBS induced in vivo by clorgyline and pargyline, through a direct or indirect mechanism, may have functional implications.

Attenuation of tolerance to opioid-induced antinociception and protection against morphine-induced decrease of neurofilament proteins by idazoxan and other I2-imidazoline ligands

1 Agmatine, the proposed endogenous ligand for imidazoline receptors, has been shown to attenuate tolerance to morphine‐induced antinociception ( Kolesnikov et al., 1996 ). The main aim of this study was to assess if idazoxan, an α2‐adrenoceptor antagonist that also interacts with imidazoline receptors, could also modulate opioid tolerance in rats and to establish which type of imidazoline receptors (or other receptors) are involved. 2 Antinociceptive responses to opioid drugs were determined by the tail‐flick test. The acute administration of morphine (10 mg kg−1, i.p., 30 min) or pentazocine (10 mg kg−1, i.p., 30 min) resulted in marked increases in tail‐flick latencies (TFLs). As expected, the initial antinociceptive response to the opiates was lost after chronic (13 days) treatment (tolerance). When idazoxan (10 mg kg−1, i.p.) was given chronically 30 min before the opiates it completely prevented morphine tolerance and markedly attenuated tolerance to pentazocine (TFLs increased by 71–143% at day 13). Idazoxan alone did not modify TFLs. 3 The concurrent chronic administration (10 mg kg−1, i.p., 13 days) of 2‐BFI, LSL 60101, and LSL 61122 (valldemossine), selective and potent I2‐imidazoline receptor ligands, and morphine (10 mg kg−1, i.p.), also prevented or attenuated morphine tolerance (TFLs increased by 64–172% at day 13). This attenuation of morphine tolerance was still apparent six days after discontinuation of the chronic treatment with LSL 60101‐morphine. The acute treatment with these drugs did not potentiate morphine‐induced antinociception. These drugs alone did not modify TFLs. Together, these results indicated the specific involvement of I2‐imidazoline receptors in the modulation of opioid tolerance. 4 The concurrent chronic (13 days) administration of RX821002 (10 mg kg−1, i.p.) and RS‐15385‐197 (1 mg kg−1, i.p.), selective α2‐adrenoceptor antagonists, and morphine (10 mg kg−1, i.p.), did not attenuate morphine tolerance. Similarly, the concurrent chronic treatment of moxonidine (1 mg kg−1, i.p.), a mixed I1‐imidazoline receptor and α2‐adrenoceptor agonist, and morphine (10 mg kg−1, i.p.), did not alter the development of tolerance to the opiate. These results discounted the involvement of α2‐adrenoceptors and I1‐imidazoline receptors in the modulatory effect of idazoxan on opioid tolerance. 5 Idazoxan and other imidazol(ine) drugs fully inhibited [3H]‐(+)‐MK‐801 binding to N‐methyl‐D‐aspartate (NMDA) receptors in the rat cerebral cortex with low potencies (Ki: 37–190 μM). The potencies of the imidazolines idazoxan, RX821002 and moxonidine were similar, indicating a lack of relationship between potency on NMDA receptors and ability to attenuate opioid tolerance. These results suggested that modulation of opioid tolerance by idazoxan is not related to NMDA receptors blockade. 6 Chronic treatment (13 days) with morphine (10 mg kg−1, i.p.) was associated with a marked decrease (49%) in immunolabelled neurofilament proteins (NF‐L) in the frontal cortex of morphine‐tolerant rats, suggesting the induction of neuronal damage. Chronic treatment (13 days) with idazoxan (10 mg kg−1) and LSL 60101 (10 mg kg−1) did not modify the levels of NF‐L proteins in brain. Interestingly, the concurrent chronic treatment (13 days) of idazoxan or LSL 60101 and morphine, completely reversed the morphine‐induced decrease in NF‐L immunoreactivity, suggesting a neuroprotective role for these drugs. 7 Together, the results indicate that chronic treatment with I2‐imidazoline ligands attenuates the development of tolerance to opiate drugs and may induce neuroprotective effects on chronic opiate treatment. Moreover, these findings offer the I2‐imidazoline ligands as promising therapeutic co‐adjuvants in the management of chronic pain with opiate drugs.

Inhibition of monoamine oxidase A and B activities by imidazol(ine)/guanidine drugs, nature of the interaction and distinction from I2-imidazoline receptors in rat liver

I2‐Imidazoline sites ([3H]‐idazoxan binding) have been identified on monoamine oxidase (MAO) and proposed to modulate the activity of the enzyme through an allosteric inhibitory mechanism ( Tesson et al., 1995 ). The main aim of this study was to assess the inhibitory effects and nature of the inhibition of imidazol(ine)/guanidine drugs on rat liver MAO‐A and MAO‐B isoforms and to compare their inhibitory potencies with their affinities for the sites labelled by [3H]‐clonidine in the same tissue. Competition for [3H]‐clonidine binding in rat liver mitochondrial fractions by imidazol(ine)/guanidine compounds revealed that the pharmacological profile of the interaction (2 ‐ styryl ‐ 2 ‐ imidazoline, LSL 61112>idazoxan>2 ‐ benzofuranyl ‐ 2 ‐ imidazoline, 2‐BFI=cirazoline>guanabenz>oxymetazoline>>clonidine) was typical of that for I2‐sites. Clonidine inhibited rat liver MAO‐A and MAO‐B activities with very low potency (IC50s: 700 μM and 6 mM, respectively) and displayed the typical pattern of competitive enzyme inhibition (Lineweaver‐Burk plots: increased Km and unchanged Vmax values). Other imidazol(ine)/guanidine drugs also were weak MAO inhibitors with the exception of guanabenz, 2‐BFI and cirazoline on MAO‐A (IC50s: 4–11 μM) and 2‐benzofuranyl‐2‐imidazol (LSL 60101) on MAO‐B (IC50: 16 μM). Idazoxan was a full inhibitor, although with rather low potency, on both MAO‐A and MAO‐B isoenzymes (IC50s: 280 μM and 624 μM, respectively). Kinetic analyses of MAO‐A inhibition by these drugs revealed that the interactions were competitive. For the same drugs acting on MAO‐B the interactions were of the mixed type inhibition (increased Km and decreased Vmax values), although the greater inhibitory effects on the apparent value of Vmax/Km than on the Vmax value indicated that the competitive element of the MAO‐B inhibition predominated. Competition for [3H]‐Ro 41‐1049 binding to MAO‐A or [3H]‐Ro 19‐6327 binding to MAO‐B in rat liver mitochondrial fractions by imidazol(ine)/guanidine compounds revealed that the drug inhibition constants (Ki values) were similar to the IC50 values displayed for the inhibition of MAO‐A or MAO‐B activities. In fact, very good correlations were obtained when the affinities of drugs at MAO‐A or MAO‐B catalytic sites were correlated with their potencies in inhibiting MAO‐A (r=0.92) or MAO‐B (r=0.99) activity. This further suggested a direct drug interaction with the catalytic sites of MAO‐A and MAO‐B isoforms. No significant correlations were found when the potencies of imidazol(ine)/guanidine drugs at the high affinity site (pKiH, nanomolar range) or the low‐affinity site (pKiL, micromolar range) of I2‐imidazoline receptors labelled with [3H]‐clonidine were correlated with the pIC50 values of the same drugs for inhibition of MAO‐A or MAO‐B activity. These discrepancies indicated that I2‐imidazoline receptors are not directly related to the site of action of these drugs on MAO activity in rat liver mitochondrial fractions. Although these studies cannot exclude the presence of additional binding sites on MAO that do not affect the activity of the enzyme, they would suggest that I2‐imidazoline receptors represent molecular species that are distinct from MAO.

Angiotensin II facilitates the potassium-evoked release of 3H-noradrenaline from the rabbit hypothalamus.

Antiotensin II facilitated in a concentration-dependent manner the potassium-evoked 3H-noradrenaline over-flow from the rabbit hypothalamus. This effect which is probably mediated through presynaptic angiotensin facilitatory receptors on noradrenergic nerve terminals was blocked by the specific angiotensin receptor antagonist, saralasin. These results demonstrate that angiotensin II also facilitates the stimulation-evoked release of noradrenaline in the central nervous system.