LECTURES 5-HT

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5-HT

1A

RECEPTORS. THEIR ROLE IN ANXIETY AND DEPRESSION.

MECHANISM OF ACTIVATION

Zdzis³aw Chilmonczyk

Drug Institute, Che³mska 30/34, PL 00-725 Warszawa, Poland; Institute of Chemistry, University of Bia³ystok, Pi³sudskiego 11/4, PL 15-443 Bia³ystok, Poland

The role of serotonin (5-HT) in anxiety and depression has been recognized long time ago [3, 11, 33]. Although it may be anticipated that the inhibition of 5-HT release can produce anxiolytic-like behavioral effects [33], and antidepressive effects can be achieved by the improving serotonergic neurotransmission, the mechanisms by which those disorders are mediated through serotonergic system still remain unclear [for a review see 8, 25, 30]. The main goal of the present paper is to discuss some aspects of the role of 5-HT_1Areceptors in anxiety and depression in connection with some attempts to better understand ligand binding and activation.

The 5-HT_1Areceptor is a member of the 5-HT₁ receptor family, having a high affinity for 5-HT, and coupled through G proteins to potassium chan- nels or negatively coupled to adenylate cyclase [for a review see 10]. In man, rat, and guinea pig, 5-HT_1Areceptor was found at high concentration in the raphe nuclei, hippocampal pyramidal cell layer, lateral septum, frontal cortex, and dorsal horn of the spinal cord [13, 34]. In the limbic system (e.g.

hippocampus, septum, entorhinal cortex, and amyg- dala) the receptor subtype is thought to play a role in emotional processes. 5-HT_1A receptors exist as two functionally distinct populations: somatoden- dritic autoreceptors located presynaptically on the cell bodies and dendrites of the raphe 5-HT-containing neurons [35], and postsynaptic receptors,

which are located on non-5-HT-containing neurons in various projection sites [for a review see 10].

The discharge activity of central serotonergic neurons and 5-HT release are regulated, in part, by a local negative feedback mechanism [1, 2, 32].

5-HT released within the raphe region from dendrites and possibly axon terminals acts on somato- dendritic 5-HT_1Aautoreceptors to inhibit neuronal activity [7]. The 5-HT_1A receptor agonists were shown to decrease the 5-HT turnover in the rat hippocampus slices [37], cortex, hypothalamus, and striatum [21].

It has been traditional view that the enhanced serotonergic transmission increases anxiety-related behavior and mice with enhanced 5-HT transmission exhibited increased anxiety [19]. It has also been found that rats undergoing elevated maze test (an anxiety paradigm) had increased 5-HT levels in the ventral hippocampus [36]. On the other hand, the lack of anxiolytic (or anxiogenic) effect of depletion of central 5-HT using tryptophan-free amino acid preparation in panic disorder patients [21]

contradicts the view that anxiety is consistently due to an increase in the 5-HT neurotransmission. It was hypothesized that anxiolytic effects of partial 5-HT_1Aagonists were attributed predominantly to the interaction with presynaptic 5-HT_1A receptors, resulting in a decrease in hyperactive serotonergic neurotransmission [31]. The hypothesis was in

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agreement with the observation that the 5-HT_1A knockout mice showed increased anxiety level in plus maze and in open field test [19, 38]. It has also been observed that p-chlorophenylalanine (PCPA)- -induced depletion of neurotoxic destruction of 5-HT neurons, and direct raphe injection of 5-HT_1A receptor antagonists reversed the 5-HT_1Aagonists- induced enhanced punished respondings in behavioral anxiety models [4]. The involvement of only presynaptic receptors remains, however, controversial [14]. For instance, it was reported that ipsapirone, indorenate and buspirone mediated their anxiolytic effects through the stimulation of postsynaptic receptors in rats [17]. Przegaliñski et al.

found that anticonflict effect of ipsapirone in rats was also mediated by postsynaptic 5-HT_1Arecep- tors [29]. It was additionally observed that pre- or postsynaptic activity of some 5-HT_1Aagonists may depend on the anxiety paradigm [26].

According to the 5-HT hypothesis of major depression, a deficit in serotonergic activity is a proximate cause of depression or is important as vulnerability factor [27, 28]. 5-HT_1A receptors have been postulated to be involved in antidepressant drug action [18] and Blier et al. have suggested that partial 5-HT_1A agonists mediate their antidepressant effects through a net increase in serotonergic neurotransmission following adaptive receptor changes [5]. Similar conclusions were drawn by Schreiber and DeVry who postulated that the antidepressant effects of partial 5-HT_1A agonists re- sulted from an enhancement of serotonergic neurotransmission through their interaction with postsynaptic 5-HT_1Asites [15, 31]. Some doubt about the model of the association of low 5-HT level with depression comes from the finding that rapid 5-HT depletion using an oral tryptophan (a 5-HT precursor)-free amino acid preparation caused relapse in 73% of the patients treated with selective 5-HT re- uptake inhibitor (SSRI), but only in 15% desi- pramine-treated patients, and did not acutely worsen depression in untreated subjects [16]. More recent

“monoamine depletion” studies show, however, that depressed patients who responded to the treat- ment with SSRIs experienced depression relapse after a functional depletion of 5-HT [22].

In spite of those encouraging preclinical and clinical observations, out of the 5-HT_1A receptor agonists only buspirone and tandospirone were so far registered for the use in anxiety and depression.

It was thus concluded that this pharmacological

class failed to live up to its preclinical promises of safer, non-sedating and more effective anxiolytics and antidepressants [25].

Anyhow, a lot of effort has been devoted in or- der to investigate 5-HT_1Aligands as potential anxiolytic and antidepressant drugs. We synthesized several receptor ligands, examined their affinity and intrinsic activity in behavioral and biochemical studies [12, 24]. We identified several good ligands of the receptor exhibiting interesting intrinsic activity in behavioral and biochemical models [9]. In or- der to determine crucial structural features respon- sible for the receptor recognition and activation, molecular dynamics (MD) simulations for the ligand-receptor complexes were carried out. During MD simulation, ligand-induced changes in the receptor 3-D structure were observed. The rigid body movements of transmembrane domains 2, 3, 6, and 7, associated with the rearrangement of the receptor interhelical hydrogen bonding network were observed. It should be noted that functional 5-HT_1A receptor agonist 1 induced such conformational changes in 2nd and 3rd intracellular loops (the approaching of the loops) that might be important for a G-protein binding [6].

Taken together, the above MD simulation data suggest that the compound interacted with 5-HT_1A receptor in a way characteristic of the receptor activation (in agreement with the functional activity data) [9]. The open question is to what extent the model may be generalized while some inconsis- tency in the functional activity data of compound 1 has been observed [23].

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SEROTONIN AND PARKINSON’S DISEASE

Richard M. Kostrzewa

, Ryszard Brus

Department of Pharmacology, Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, USA;

Department of Pharmacology, Medical University of Silesia, PL 41-808 Zabrze, Poland

In 1817 James Parkinson wrote his treatise Pa- ralysis agitans, describing cardinal symtomatology of a neuromuscular disorder, now known as Parkin- son’s disease (PD). In 1919 Trétiakoff discovered that the absence of substantia nigra was another characteristic feature of PD. Only 40 years ago, Hornykiewicz determined that PD is largely a dopamine (DA) deficiency disorder, appropriately treated by replacing the missing DA with exogenous high-dose levodopa (L-DOPA) which is metabolized to DA in the brain. With this in mind, it might seem that the title of this article, with a focus on serotonin (5-HT), is a misnomer. However, from experimental findings of the past 10 years, it seems to us that even if DA is and should remain the primary monoamine focus, there is much to be said for taking the 5-HT system into account in approaching PD therapy.

5-HT fiber outgrowth is suppressed by DA fibers. From experiments in animals, it is known that DA has a suppressive effect on outgrowth of 5-HT terminals. When DA neurons are destroyed

in early postnatal ontogeny, 5-HT fibers proliferate and hyperinnervate a DA-denervated brain region.

Both DA and 5-HT receptors are supersensi- tized in DA-denervated brain. In rodents in which nigrostriatal DA axons are largely destroyed, DA D₁ and D₂ receptors become supersensitized, but for some behavioral paradigms a series of priming doses of a DA agonist (or L-DOPA) is needed for expression of the DA receptor supersensitivity.

Significantly, 5-HT receptor supersensitivity also accompanies DA neural destruction, and its expression does not require priming with either DA agonist or 5-HT agonist.

DA receptor supersensitivity is reliant of 5-HT fiber innervation. DA receptor supersensi- tivity appears to be dependent on intact 5-HT fibers, because DA receptor supersensitivity is eliminated by 5,7-dihydroxytryptamine-induced destruction of 5-HT fibers. This effect is observed whether 5-HT fibers are destroyed in early postnatal ontogeny or in adulthood.

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5-HT fibers mediate DA receptor supersensi- tivity. When an antagonist of 5-HT_2C receptors is administered to rats with supersensitive DA receptors, there is immediate blockade of enhanced responses to DA agonists.

Both DA and 5-HT mediate amphetamine ef- fects. Enhanced behavioral effects of amphetamine in DA-lesioned rats are now known to be associated with enhanced release of both DA and 5-HT.

Several presumed DA effects are now recognized as being attributable to 5-HT, not DA. This in- cludes motor behaviors.

Enhancement of amphetamine-induced DA release after a 5-HT lesion. If lesions are made to both DA and 5-HT fiber innervation of the striatum, amphetamine (or its analogs) produces a dis- proportionately increased release of DA.

L-DOPA ® DA conversion in 5-HT fibers.

When L-DOPA is administered to rats with DA- -denervated neostriatum, it appears that much of

L-DOPA is converted to DA in 5-HT fibers, not in DA fibers per se.

Summary. Although PD is largely a DA-defi- ciency disorder, 5-HT neurons appear to 1) mediate conversion of L-DOPA to DA, at least in part, and 2) influence the amount of DA able to be released from DA nerves (for example as in the above case with amphetamine). At the postsynaptic site, 5-HT fibers and receptors have a major influence on the expression of DA receptor supersensitivity, and 5-HT receptor blockers can attenuate enhanced effects of agonists at DA receptors. It is possible that the efficacy of L-DOPA therapy can be increased by blocking one subtype of 5-HT receptor, while dyskinesias accompanying long-term L-DOPA therapy might be eliminated by blocking another subtype of 5-HT receptor. 5-HT antagonists are be- lieved to represent a potential for prolonging the duration of the dyskinesia-free phase of L-DOPA therapy of PD.

REACTIVE OXYGEN SPECIES (ROS) AND REACTIVE NITROGEN SPECIES (RNS) AS ENDOGENOUS TOXICANTS

OF CNS: SOME ASPECTS OF DEFENSE

Diana Metodiewa

, Agata Kochman

Institute of Applied Radiation Chemistry, Technical University of £ód, Wróblewskiego 15, PL 93-590 £ód, Poland;

Department of Pathological Anatomy, Medical University of Wroc³aw, Marcinkowskiego 1, PL 50-368 Wroc³aw, Poland

Reactive oxygen species (ROS), reactive nitrogen species (RNS), and oxidative and nitrosative injury may be involved in a diversity of pathological phenomena including the neurodegenerative diseases. Since 1968, after the crucial discovery of an enzyme, superoxide dismutase (SOD) by McCord and Fridovich, till now, the scientists have been at- tracted by the toxic role of ROS and their irreversible damage to neural cells [11]. However, bearing in mind that there are expanding toxicological roles assigned to ROS and RNS, it is important to note here that CNS is especially vulnerable to the dama- ges caused by these species, because of its abun-

dant lipid content, relative paucity of antioxidant defense and high rate of O₂consumption [5].

Mitochondria are major sources of ROS (Com- plex III and I, glycerol-1-phosphate dehydroge- nase) [10], and ROS appear to be released both in the matrix and in the intermembrane space. Cellu- lar perturbations associated with neural degenera- tion may be the consequence of disrupted mitochondrial function as well as excessive production of ROS [2]. The consequences of mitochondrial dysfunction are: uncoupling of the respiratory chain, hypergeneration of ROS (superoxide), out- flow of matrix calcium and GSH, change in the mi-

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tochondrial transmembrane potential, release of intermembrane proteins, and they can result in necrosis and/or activation of caspases, with secondary endonuclease activation and oligonucleosomal DNA fragmentation (apoptosis) [17].

RNS (reactive nitrogen species; nitric oxide, NO, or its derivatives) have three types of actions on mitochondria: (i) reversible inhibition of mitochondrial respiration by NO or irreversible inhibition by RNS; (ii) stimulation of ROS or peroxynitrite (ONOO) generation by NO; (iii) induction of mitochondrial permeability transition (MPT) and induction of apoptosis or necrosis [1].

ONOOmay induce mitochondrial dysfunction and cell death of neurons, through nitration of mitochondrial complex I subunits [16]. Moreover, superoxide produced during mitochondrial respiration can react with NO inside mitochondria to yield ONOO[16], and 3-NT (3-nitrotyrosine) is the specific marker of protein nitration by produced in this fast, diffusion-limited reaction of superoxide and NO[11].

Oxidative stress (OS), ROS and RNS have been implicated in the mechanism of brain dysfunction [4, 5, 11] involved in Parkinson’s and Alzheimer’s diseases ALS schizophrenia and depression. Both RNS and ROS have very short half-lives, thereby making very difficult to quantify their fluxes in- sight neural cells and to identify them as a specific cause of neurodegeneration [2, 10. 11]. OS, which is generated as a by-product of normal and aberrant brain metabolic processes that use O₂, may initiate various signalling cascades, leading to apoptotic cell death. Apoptosis in the CNS, in contrast to necrosis, is an endogenous neural cell suicide mechanism triggered in response to biological factors of- ten resulting from OS, via a series of responses that include GSH depletion, neurotrophin signaling and DNA repair mechanisms [10, 11]. Because ROS may play a central role in neural apoptotic pathways, it is worth to emphasize here, that the rate of oxidative DNA damage is directly related to the metabolic rate and inversely related to the life span of the organism [7].

There has been an increasing concern in recent years related to the extensive evidence for simulta- neous action of OS and ET (electron transfer reactions/processes) and the new ET-OS theory [9].

On the basis of the neurotoxin(s) action, implicat- ing ET-functionalities (quinones/methides, ArNO₂, metal complexes, peroxides, radiation) or their me-

tabolites giving rise to ROS by redox cycling, it was proposed that ET-OS biomechanisms can be involved as well in the neurodegenerative disorders (ND). Notably, dopamine, while an essential neuro- transmitter, is also a known neurotoxin that poten- tially plays an etiologic role in ND [14] and its metabolism (oxidation) readily produces ROS and quinone via spontaneous, enzyme-catalyzed or me- tal-enhanced reactions [11, 12, 14].

The redox state of the neural cell is a result of the balance between the level of ROS and thiol

“buffers” such as GSH, which protect cells from the OS. The elevation of ROS exceeding compen- satory changes in the level of thiol “buffers”, may result in the activation of signaling pathways and expression of genes that induce apoptosis in the af- fected neural cells [3].

Glutathione tripeptide (GSH) and glutathione- -dependent enzymes represent the major mechanism and multielemental detoxification system of endogenous antioxidants. GSH biosynthesis, glutathione peroxidases, glutathione S-transferases and glutathione S-conjugate efflux pumps act in integrated manner affording the neural cells protection from OS consequences [3, 6, 11, 12, 15]. The antioxidant responsive element (ARE) was found recently in the gene(s) promotors inducible by OS [6]. Re- cently, it has been concluded that Bcl-2, an anti- apoptotic protein located in the outer mitochondrial membrane, affects the cellular level of ROS, by the modulation of either their overproduction or an endogenous antioxidant pathways [8], but its mode of action is still uncertain.

It is important to emphasize that the antioxidant defense mechanisms of the brain include removal of superoxide, scavenging of ROS and RNS, inhibition of ROS formation, binding of metal ions catalyzing of ROS generation and up-regulation of all the defense systems [3, 4, 6, 8, 11, 15].

Now it is clear that any considerations about the unavoidable generations and actions of the toxic ROS and RNS in vivo should take into account their contribution and interconversions/interactions under conditions of OS, in which many physiologi- cal processes may become more or less efficient.

However, the RNS/ROS ratio may be of impor- tance [5, 11], and the state of the complex endogenous antioxidants defense system of CNS should represent another important line of future research and therapeutic strategies into the mechanism un- derlying brain disorders.

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Acknowledgment. This study was supported by a grant No. 6PO4A 086 19 form the State Committee for Scientific Research, Warszawa, Poland.

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DT-DIAPHORASE: A NEUROPROTECTIVE ENZYME OF DOPAMINERGIC SYSTEMS

Paris I., Martinez P., Arriagada C., Dagnino-Subiabre A., J. Montiel, Caviedes, Caviedes, P., Mora S., Diaz-Veliz G., Cassel, B., C. Olea-Azar

F. Aboitiz Segura-Aguilar J.

Programme of Molecular and Clinical Pharmacology, ICBM, Faculty of Medicine, University of Chile, Independencia 1027, Casilla 70000, Santiago 7, Chile

Although it is generally accepted that free radicals are involved in the neurodegenerative process occurring in the nigro-striatal system in Parkin- son’s disease, the exact mechanism of neurodegen-

eration in vivo is still unknown. One possible mechanism for formation of reactive species in the dopaminergic system is dopamine oxidation. The catechol structure of dopamine is able to be oxi-

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dized to aminochrome, precursor of neuromelanin, in the presence of oxidizing agents such as dioxy- gen, superoxide, hydrogen peroxide, transitions metals, peroxynitrite radical, or enzymes such as prostaglandin H synthetase, cytochrome P450 1A2 and 2E, xanthine oxidase and dopamine b-mono- oxygenase. Recently, it has been reported that neuromelanin biosynthesis is driven by excess of cytosolic dopamine, since adenoviral-mediated over- expression of the synaptic vesicle catecholamine transporter VMAT2 inhibited neuromelanin formation [13]. Dopamine oxidation seems to be very strictly regulated by (i) dopamine uptake into vesicles with low pH which prevents autooxidation and later used for neurotransmission and (ii) MAO metabolism. However, under certain conditions, such as the existence of reduced number of vesicles, low levels of MAO or high levels of dopamine synthe- sis, free dopamine is oxidized to aminochrome which has been proposed as an endogenous neurotoxin [1, 4, 9, 10].

The polymerization of aminochrome and formation of neuromelanin depends on three reactions that prevent toxic effects of aminochrome: (i) two- -electron reduction of aminochrome catalyzed by DT-diaphorase to leukoaminochrome [1, 10], which can be conjugated by sulfotransferase; (ii) conjuga- tion of aminochrome with GSH catalyzed by glutathione transferase (GST) M2-2/M1-1 yielding 4-S- -glutathionyl 5,6-dihydroxyindoline, which is resi- stant to biological oxidizing agents [7]; (iii) con- jugation of dopamine o-quinone (precursor of ami- nochrome) with GSH catalyzed by GST M2-2 to form 5-glutathionyl dopamine [3], which is the precursor of 5-cysteinyldopamine [12], found in human cerebrospinal fluid and in all dopaminergic regions [2, 5].

DT-diaphorase [E.C. 1.6.99.2, NAD(P)H: quinone (menadione) oxidoreductase] is an unique enzyme among flavoenzymes, which is mainly local- ized in the cytosol (95%) but around 5% it is also found associated to the mitochondria and endoplas- mic reticulum. DT-diaphorase reduces quinones to hydroquinones by transfering two-electrons, preventing the formation of semiquinone radical, and it is specifically inhibited by dicoumarol. DT-diaphorase catalyzes two-electron reduction of aminochrome to its catechol (leukoaminochrome) which also autooxidizes in the presence of oxygen, form- ing superoxide radicals and hydrogen peroxide [1, 9]. However, the autooxidation rate of leukoamino-

chrome is very low compared to that of leukoami- nochrome o-semiquinone, and it is mainly (96%) dependent on the presence of superoxide radicals and hydrogen peroxide. Therefore, superoxide dismutase and catalase/GSH peroxidase play an antioxidant role during aminochrome reduction by DT-diaphorase, since they remove superoxide and hydrogen peroxide [1, 9]. DT-diaphorase is present in dopaminergic neurons, constituting 98% of the total quinone reductase activity [6]. Recently, we have reported that DT-diaphorase plays a neuroprotective role against aminochrome toxicity by preventing formation of leukoaminochrome o-semi- quinone radical, i.e. (i) CuSO₄ toxicity increased dramatically when DT-diaphorase was inhibited with dicoumarol in RCSN-3 cells [4]; and (ii) con- tralateral rotation behavior similar to that produced by 6-OH-DA was observed only when DT-diaphorase was inhibited with dicoumarol in rats intra- cerebrally injected with manganese³⁺[8]. The pos- sibility that an inactive DT-diaphorase exists in the patients with Parkinson’s disease has been supported by a recent report showing the expression of an inactive form of DT-diaphorase, due to polymorphism with a point mutation (C ® T) in the cDNA 609 [11]. This polymorphism 609 increases the risk for Parkinson’s disease 3.8-fold and the coexistence of the DT-diaphorase polymorphism C® T 609 cDNA and MAO-B 13 intron polymorphism increases the risk for Parkinson’s disease 5.7 fold [11], supporting the proposed role of DT- diaphorase as neuroprotective enzyme.

Acknowledgment. This work was supported by FON- DECYT (1990622, 1980906 & 1020672).

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