Suicide and depression are associated with an increased density of ␣ 2 -adrenoceptors (radioligand receptor binding) in specific regions of the human brain. The function of these inhibitory receptors involves various regulatory proteins (G i coupling proteins and G proteincoupled receptor kinases, GRKs), which work in concert with the receptors. In this study we quantitated in parallel the levels of immunolabeled ␣ 2A -adrenoceptors and associated regulatory proteins in brains of suicide and depressed suicide victims. Specimens of the prefrontal cortex (Brodmann area 9) were collected from 51 suicide victims and 31 control subjects. Levels of ␣ 2A -adrenoceptors, G␣ i1/2 proteins, and GRK 2/3 were assessed by immunoblotting techniques by using specific polyclonal antisera and the immunoreactive proteins were quantitated by densitometry. Increased levels of ␣ 2A -adrenoceptors (31-40%), G␣ i1/2 proteins (42-63%), and membrane-associated GRK 2/3 (24 -32%) were found in the prefrontal cortex of suicide victims and antidepressantfree depressed suicide victims. There were significant correlations between the levels of GRK 2/3 (dependent variable) and those of ␣ 2A -adrenoceptors and G␣ i1/2 proteins (independent variables) in the same brain samples of suicide victims (r ϭ 0.56, p ϭ 0.008) and depressed suicide victims (r ϭ 0.54, p ϭ 0.041). Antemortem antidepressant treatment was associated with a significant reduction in the levels of G␣ i1/2 proteins (32%), but with modest decreases in the levels of ␣ 2A -adrenoceptors (6%) and GRK 2/3 (18%) in brains of depressed suicide victims. The increased levels in concert of ␣ 2A -adrenoceptors, G␣ i1/2 proteins, and GRK 2/3 in brains of depressed suicide victims support the existence of supersensitive ␣ 2A -adrenoceptors in subjects with major depression. Key Words: ␣ 2A -Adrenoceptors-G proteins-G protein-coupled receptor kinase-Human brain-Suicide-Depression.

Albeit anthracyclines are widely used in the treatment of solid tumors and leukemias, their mechanism of action has not been elucidated. The present study gives relevant information about the role of nonlamellar membrane structures in signaling pathways, which could explain how anthracyclines can exert their cytocidal action without entering the cell [Tritton, T. R. & Yee, G. (1982) Science 217,[248][249][250]. The anthracycline daunomycin reduced the formation of the nonlamellar hexagonal (Hn) phase (i.e., the hexagonal phase propensity), stabilizing the bilayer structure of the plasma membrane by a direct interaction with membrane phospholipids. As a consequence, various cellular events involved in signal transduction, such as membrane fusion and membrane association of peripheral proteins [e.g., guanine nucleotide-binding regulatory proteins (G proteins and protein kinase C-a*3)], where nonlamellar structures (negative intrinsic monolayer curvature strain) are required, were altered by the presence of daunomycin. Functionally, daunomycin also impaired the expression of the high-affinity state of a G protein-coupled receptor (ternary complex for the a2-adrenergic receptor) due to G-protein dissociation from the plasma membrane. In vivo, daunomycin also decreased the levels of membrane-associated G proteins and protein kinase C-a.3 in the heart. The occurrence of such nonlamellar structures favors the association of these peripheral proteins with the plasma membrane and prevents daunomycin-induced dissociation. These results reveal an important role of the lipid component of the cell membrane in signal transduction and its alteration by anthracyclines.Anthracyclines are potent antitumor drugs. They bind with high affinity to DNA (1), causing a stabilization of the DNA double helix (2). Although a mechanism of anthracycline cytotoxicity based on their interaction with DNA has been proposed, strong evidence has accumulated suggesting the involvement of the plasma membrane in the cytotoxic action of these antitumor drugs. The plasma membrane is the first cellular target found by anthracyclines, and it is able to bind a large number of drug molecules (3, 4), so that a primary action on the physicochemical properties of the cell membrane could trigger a series of events responsible for their cytotoxic effect (5). Anthracyclines with little or no ability to bind to DNA, such as N-trifluoroacetyladriamycin 14-valerate (AD 32), exhibit a good antitumor activity, which argues against DNAmediated effects (6). A number of physicochemical properties of the plasma membrane are affected by anthracyclines (3, 5, 7), and agarose-linked doxorubicin (former generic name, adriamycin) is able to kill cells, without penetrating them, solely by its interaction with the plasma membrane (8). Despite all these indications that the antitumor activity of anthracyclines originates from their interaction with cellular membranes, the mechanism of cytotoxicity has not been elucidated. Here we present evidence that nonlamellar membra...

Heterotrimeric G proteins (peripheral proteins) conduct signals from membrane receptors (integral proteins) to regulatory proteins localized to various cellular compartments. They are in excess over any G protein-coupled receptor type on the cell membrane, which is necessary for signal amplification. These facts account for the large number of G protein molecules bound to membrane lipids. Thus, the protein-lipid interactions are crucial for their cellular localization, and consequently for signal transduction. In this work, the binding of G protein subunits to model membranes (liposomes), formed with defined membrane lipids, has been studied. It is shown that although G protein ␣-subunits were able to bind to lipid bilayers, the presence of nonlamellarprone phospholipids (phosphatidylethanolamines) enhanced their binding to model membranes. This mechanism also appears to be used by other (structurally and functionally unrelated) peripheral proteins, such as protein kinase C and the insect protein apolipophorin III, indicating that it could constitute a general mode of protein-lipid interactions, relevant in the activity and translocation of some peripheral (amphitropic) proteins from soluble to particulate compartments. Other factors, such as the presence of cholesterol or the vesicle surface charge, also modulated the binding of the G protein subunits to lipid bilayers. Conversely, the binding of G protein-coupled receptor kinase 2 and the G protein ␤-subunit to liposomes was not increased by hexagonally prone lipids. Their distinct interactions with membrane lipids may, in part, explain the different cellular localizations of all of these proteins during the signaling process.

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1 This study was designed to assess the in¯uence of activation and blockade of the endogenous opioid system in the brain on two key proteins involved in the regulation of programmed cell death: the pro-apoptotic Fas receptor and the anti-apoptotic Bcl-2 oncoprotein. 2 The acute treatment of rats with the m-opioid receptor agonist morphine (3 ± 30 mg kg 71 , i.p., 2 h) did not modify the immunodensity of Fas or Bcl-2 proteins in the cerebral cortex. Similarly, the acute treatment with low and high doses of the antagonist naloxone (1 and 100 mg kg 71 , i.p., 2 h) did not alter Fas or Bcl-2 protein expression in brain cortex. These results discounted a tonic regulation through opioid receptors on Fas and Bcl-2 proteins in rat brain.

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 a 2 -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-¯ick test. The acute administration of morphine (10 mg kg 71 , i.p., 30 min) or pentazocine (10 mg kg 71 , i.p., 30 min) resulted in marked increases in tail-¯ick latencies (TFLs). As expected, the initial antinociceptive response to the opiates was lost after chronic (13 days) treatment (tolerance). When idazoxan (10 mg kg 71 , 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 71 , i.p., 13 days) of 2-BFI, LSL 60101, and LSL 61122 (valldemossine), selective and potent I 2 -imidazoline receptor ligands, and morphine (10 mg kg 71 , 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 morphineinduced antinociception. These drugs alone did not modify TFLs. Together, these results indicated the speci®c involvement of I 2 -imidazoline receptors in the modulation of opioid tolerance. 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 71 , i.p.) was associated with a marked decrease (49%) in immunolabelled neuro®lament 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 71 ) and LSL 60101 (10 mg kg 71 ) 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 I 2 -imidazoline ligands attenuates the development of tolerance to opiate drugs and may induce neuroprotective eects on chronic opiate treatment. Moreover, these ®ndings oer the I 2 -imidazoline ligands as promising therapeutic coadjuvants in the management of chronic pain with opiate drugs.

The results support a role for the newly discovered imidazoline receptors (mainly the 45-kd receptor expressed in the brain and platelets) in the pathogenesis of depression.

1 This work investigates the receptor acted upon by imidazoline compounds in the modulation of morphine analgesia. The eects of highly selective imidazoline ligands on the supraspinal antinociception induced by morphine in mice were determined. 2 Intracerebroventricular (i.c.v.) or subcutaneous (s.c.) administration of ligands selective for the I 2 -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 I 1 -imidazoline and a 2 -adrenoceptor agonist, RX821002, a potent a 2 -adrenoceptor antagonist that displays low anity at I 2 -imidazoline receptors, nor the selective non-imidazoline a 2 -adrenoceptor antagonist RS-15385-197, modi®ed the analgesic responses to morphine. 3 Administration of pertussis toxin (0.25 mg per mouse, i.c.v.) 6 days before the analgesic test blocked the ability of the I 2 -imidazoline ligands to potentiate morphine antinociception. 4 The increased eect of morphine induced by I 2 -imidazoline ligands (agonists) was completely reversed by idazoxan and BU 224. Identical results were obtained with IBI, which alkylates I 2 -imidazoline binding sites. Thus, both agonist and antagonist properties of imidazoline ligands at the I 2 -imidazoline receptors were observed. 5 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 eect of the opioid. At longer intervals (24 h) a single dose of either clorgyline or deprenyl reduced the density of I 2 -imidazoline receptors and prevented the I 2 -mediated potentiation of morphine analgesia. 6 These results demonstrate functional interaction between I 2 -imidazoline and opioid receptors. The involvement of G i -G o transducer proteins in this modulatory eect is also suggested.