Non-Conventional Effects of Nicotinic Agonists and Monoamine Uptake Blockers in the Central Nervous System

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NON-CONVENTIONAL EFFECTS OF NICOTINIC

AGONISTS AND MONOAMINE UPTAKE BLOCKERS IN

THE CENTRAL NERVOUS SYSTEM

Esteban Hennings, M.D.

Thesis Consultant: Dr. E. Sylvester Vizi

Institute of Experimental Medicine

Budapest-New York, 2002

Semmelweis University,

Neurosciences School of PhD Studies

Opponents: Dr. Klára Gyires, Dr. Lóránd Barthó

Summary

Nicotinic acetylcholine receptors have been linked to several important functions of the central nervous system including memory. Their pharmacological manipulation therefore provides future methods of treatment. In our previous experiments we have shown that dimethylphenylpyperazinium (DMPP) has dual effect not only acting directly on the nicotinic receptor but also on the uptake transporter. Therefore the first part of this thesis investigated if other nicotinic agonists have some effect on the uptake transporter by using [3H]noradrenaline [3H]NA release from rat hippocampal slices. Our data indicate that the majority of nicotinic agonists (nicotine, epibatidnine, anatoxin A, cytisine) increase the release of [3H]NAexclusively via stimulation of nicotinic acetylcholine receptors (nAChRs). DMPP, in addition to the stimulation of nAChRs, also evokes a carrier-mediated release. Lobeline has no stimulatory effect on nAChRs, induces a carrier- mediated release and has a further action of unidentified mechanism. Our results suggest that special caution is required for the interpretation of data, when DMPP or lobeline are used as nicotinic agonists. During these studies we observed that monoamine uptake inhibitors are able to block not only the uptake but also the nAChRs. The second part of the thesis focused on this phenomenon. We found that monoamine uptake blockers behave like non-competitive ion channel blocker-type antagonists and inhibit the nAChR-mediated response with the same efficacy than the most effective nAChR anatagonist mecamylamine. This effect is independent of their chemical structure and substrate selectivity and seems to be a general feature of monoamine uptake transporters.Because these compounds are widely used in therapy (and abused in the case of cocaine), our finding may have great importance in the evaluation of their clinical effects and toxicology.

Candidate’s publications related to the thesis

Full papers

E.C.P. Hennings, J.P. Kiss and E. S. Vizi (1997) Nicotinic acetylcholine receptor antagonist effect of fluoxetine in rat hippocampal slices. Brain Research 759 292-294.

E.C.P. Hennings, J.P. Kiss, K. de Oliveira, P. Toth and E. S. Vizi (1999) Nicotinic acetylcholine receptor antagonist effect of monoamine uptake blockers in rat hippocampal slices. Journal of Neurochemistry 73 1043-1050.

J.P. Kiss, K. Windisch, K. de Oliveira, E.C.P. Hennings, A. Mike, B.K. Szasz (2001) Differential effect of nicotinic agonist on the [3H]norepinephrine release from rat hippocampal slices. Neurochemical Research 26 943-950

Abstracts

E.C.P. Hennings, J.P. Kiss and E.S. Vizi (1997) Nicotinic acetylcholine receptor antagonist effect of fluoxetine in rat hippocampal slices Naunyn Schmiedebergs Arch. Pharmacol. 356(S1):R48

E.C.P. Hennings, J.P. Kiss, K. De Oliveira, P.T. Tóth and E.S. Vizi (1999) Nicotinic acetylcholine receptor antagonistic activity of monoamine uptake blockers in rat hippocampal slices Fundam. Clin. Pharmacol. 13(S1):128S

J.P. Kiss, E.C.P. Hennings, K. De oliveira, P.T. Tóth and E.S. Vizi (2000) Nicotinic acetylcholine receptor antagonistic activity of the selective dopmaine uptake blocker GBR-12909 in rat hippocampal slices J. Physiol. (London) 526:69P

Publications not related to the thesis

Full Papers

J. P. Kiss, E.C.P. Hennings, G. Zsilla and E.S. Vizi (1999) A possible role of nitric oxide in the regulation of dopamine transporter function in the striatum. Neurochemistry International 34345-350.

H. Kalasz, T. Bartok, R. Komoroczy, E Szoko, D. Haberle, J.P. Kiss, E.C.P. Hennings, K. Magyar and S. Furst (1999) Analysis of deprenyl metabolites in the rat brain using HPLC-ES-MS. Current Medicinal Chemistry 6 271-278.

Abstracts G. Zsilla, J.P. Kiss, E.C.P. Hennings, B. Szasz and E.S. Vizi (2001) The possible involvement of nitric oxide in the regulation of dopamine transporter function in rat striatum. Fundam. Clin. Pharmacol. 15(S1):78

Table of Contents

1. - Introduction ...... 7

2. –Background ...... 8

2.1. Monoamines uptake transporters...... 8 2.2. Pharmacology of the monoamine uptake blockers...... 10 2.3. Structure of the nicotinic acetylcholine receptor (nAChR) in the central nervous system...... 12 2.3.1. Molecular biology of the nicotinic receptor ...... 12 2.4. Neuronal acetylcholine receptor agonists and antagonists...... 14 2.4.1 Agonists ...... 14 2.4.1.1. Epibatidine ...... 14 2.4.1.2. Anatoxin-A...... 14 2.4.1.3. Dimethylphenylpiperazinium (DMPP) ...... 15 2.4.2. Antagonists...... 15 2.4.2.1. Mecamylamine ...... 15 2.4.2.2. Methyllycaconitine ...... 16 2.5. Nicotine-evoked release of NA from the hippocampus...... 16 2.6. DMPP-evoked release of NA from hippocampus ...... 16 3. – Aims...... 18

3.1. Comparison of the effect of nicotinic agonists on the hippocampal NA release...... 18 3.2. Investigation of the antinicotinic effect of monoamine uptake transporters in the CNS...... 18 3.2.1. Question of selectivity...... 18 3.2.2 Quantitative analysis ...... 20 3.2.3 Search for the possible mechanism of action...... 20 4. – Methods...... 21

Nicotinic agonists-evoked [3H]noradrenaline release from rat hippocampal slices...... 21 4.2. - Electrical stimulation-evoked [3H]NA release from rat hippocampal slices ...... 22 4.3. –Electrophysiological recording of Na+-currents...... 22 4.4. Statistical Analysis ...... 23 4.4.1 Effect of nicotinic agonists on the noradrenaline release from rat hippocampal slices ...... 23 4.4.2. Effect of nicotinic agonists on the resting release of [3H]noradrenaline from rat hippocampal slices...... 24 5. - Results ...... 27

5.1.Differential effect of nicotinic agonists on the [3H]noradrenaline release from rat hippocampal slices ...... 27 5.1.1 Effect of nicotinic agonists on the electrical stimulation-evoked release of [3H]noradrenaline from rat hippocampal slices...... 27 5.1.2 Effect of the nicotinic antagonist mecamylamine on the action of nicotinic agonists...... 27 5.1.3 Effect of the noradrenaline uptake inhibitor desipramine on the action of nicotinic agonists...... 30 5.2. Effect of nicotine on the release of [3H]NA from rat hippocampal slices ...... 32 5.3. Effect of monoamine uptake blockers on the nicotine-evoked release of [3H]NA from rat hippocampal slices ...... 33

5.4. Correlation between the inhibition of nicotine-evoked [3H]NA release and the inhibition of NA uptake...... 34 5.5. Effect of monoamine uptake blockers on Na+-currents of sympathetic neurons from rat superior cervical ganglia...... 35 5.6 Effect of nAChR-agonists on resting and electrical stimulation-evoked release of [3H]NA ...... 36 5.7 Comparison of the inhibitory effect of TTX and DMI on the nicotine- and electrical stimulation -evoked release of [3H]NA from rat hippocampal slices...... 37 6. – Discussion ...... 38

6.1. Differential effect of nicotinic agonists on the [3H]noradrenaline release from rat hippocampal slices ...... 38 6.1.1. Effect of conventional nicotinic agonists ...... 38 6.1.2. Effect of DMPP...... 39 6.1.3. Effect of lobeline...... 39 6.2. Effect of monoamine uptake blockers in nicotine induced noradrenaline release... 41 7. - Conclusion...... 48

7.1. Differential effect of nicotinic agonists on the [3H]noradrenaline release from rat hippocampal slices ...... 48 7.2. Nicotinic antagonist effect of monoamine uptake blockers...... 48 8. – Acknowledgments...... 49

Bibliography ...... 50

1. - Introduction

Depression is one of the most common psychiatric disorders. At any given moment, about 5-6% of the population is depressed, determined by point prevalence. An estimated of 30 % may become depressed during their lifetime. Depression is by no doubt one of the main causes of suicide or attempted suicide. Several drugs have been developed successfully for the treatment of depression including tricyclic antidepressants (like imipramine and DMI) and selective serotonin reuptake inhibitors (SSRIs as fluoxetine and citalopram) being now the drugs most widely used for treatment of depression (1). The main target of these medications is the blockage of monoamine uptake transporter (the molecule that decreases the concentration of monoamines in the extracellular fluid and the synapse). We will refer to these medications as monoamine uptake blockers. It has been shown that the monoamine uptake blockers are able to block different classes of receptorsIn this thesis we provided evidence that there is an interesting connection between nAChRs and monoamine transporters. We found that certain nicotinic agonists influence the function of monoamine transporters and monoamine uptake blockers have a pronunced effect on nAChRs. The thesis will focus on the similarities between the nAChRs and monoamine transporters and the functional pharamacological consequence of this similarity.

2. –Background

2.1. Monoamines uptake transporters The termination of neurochemical transmission in neurons requires the removal of neurotransmitter from the synapse, the persistent presence of neurotransmitters causes a persistent stimulations of the receptors and potentially dangerous for the integrity of the cell. In the nervous system this is primarily carried out by two processes, the first is the enzymatic degradation of the neurotransmitter converting it to an inactive compound, and the second is transporting the neurotransmitter inside the cell by a process called uptake. This is accomplished by a molecule called the uptake transporter. Studies on neurotransmitter uptake indicated the existence of multiple uptake systems, each relatively selective for a specific neurotransmitter (92). Neurotransmitters are transported across membranes by at least four distinct families of transporters: (a) vesicular transporters that function in the uptake of neurotransmitters into synaptic vesicles (93) (b) Na+ and Cl--dependent transporters that operate on the plasma membrane of neuronal and glial cells (94,95) (c) Na+/K+ -dependent transporters that function on the plasma membranes, specially in glutamate transport (96) (d) general amino acid transport systems that participate in controlling the availability of neurotransmitters outside the cell (97). This thesis will deal exclusively with the family of Na+/Cl- transporters. The Na+/Cl- transporters in mammals can be grouped into four subfamilies: GABA transporters, monoamine transporters, amino acid transporters and “orphans” [typified by neurotransmitter transporter 4 (98)]. The transporters of the first three subfamilies show common structures of 12 transmembrane helices with a single large loop in the external face of membrane with potential for glycosylation (see below). The structure of the orphan subfamily transporter deviates from the other by having two glycosylation loops outside the membrane (70). The Na+/Cl- monoaminergic transporter (comprises the transporters of noradrenaline, serotonin and dopamine) is composed of 12 transmembrane

domains with the N-terminal and C-terminal both inside the cell, also presents 6 extracellular loops and 5 intracellular loops (40,41,42), this structure was elucidated using electron microscopy and polyclonal antibodies directed against specific domains of the molecule (43,44). Recent studies using Zn2+ binding showed that the binding of the neurotransmitter would be in an extracellular structure formed by the second extracellular loop and the transmembrane segments 7 and 8 (45). Also studies using mutation and chimeras showed a role of the transmembrane domains 4, 5 and 3 in the efficacy of the dopamine transport (46,47,48). The region between transmembrane domain six to eight seems to be related to cocaine and tricyclic antidepressants binding, also a tyrosine in the transmembrane domain 11 was found to be important in the binding of NA uptake transporter blockers (47,49) (Figure 1).

Figure 1. Schematic structure of monoamine uptake transporter. (A) region between TM domain 6-8 related to uptake blockers binding and (B) neurotransmitter binding region

The neuron membrane potential is primarily maintained by differences in concentration of Na+ and K+, being the extracellular concentration of sodium much greater than its intracellular concentration. The monoamine transporters use this difference in concentration to transport the neurotransmitter inside the

cell. The driving force would be the gradient of Na+ across the membrane coupling the gradient to the transport of neurotransmitter. This was firmly demonstrated in synaptosomes (50,51,52). For that transport to be done the Na+ and Cl- bind first to the transporter and the conformational change induced by this binding enables the substrate binding. Following the transport step, the Na+ ions are released first, and then the substrate and Cl- are also released. The transporter is rendered competent for the next cycle by returning to the original conformation spontaneously or through facilitation by K+ or H+ gradients or by a membrane potential (99). Each transporter has a variation on the above sequence. The serotonin transporter represents one of the extreme deviations, because it has an electroneutral transport that is dependent on an outward K+ gradient (100). There are significant differences in the stoichiometry of ion transport in the different monoaminergic transporters. Dopamine may be cotransported with 2Na+ and 1CL-, noradrenaline is cotransported with 1Na+ and 1 Cl- and serotonin is also cotransported with 1Na+ and 1Cl- but the transport is also dependent on the presence of K+ in the cytoplasmatic side of the transporter (101,102). It is widely known that the psychiatric diseases are related to changes in change in the neurochemistry of the brain. By using drugs that change the concentration of neurotransmitters in the brain those alterations can be equilibrated. It has been shown that in major depression and suicide there is a consistent reduction in serotonin transporter sites especially in the prefrontal cortical area, probably reflecting a reduction in serotoninergic function in the brain (103). This has been corroborated in vivo using positron emission tomography (104). The pharmacological manipulation of these processes will be discussed in the next section.

2.2. Pharmacology of the monoamine uptake blockers. The primary function of the monoamine uptake blockers is to block the inward transport of the neurotransmitter therefore increasing the extracellular concentration of the neurotransmitter. These drugs mainly are classified

pharmacologically in 2 groups, the tricyclic antidepressants, like desipramine, amytriptiline, imipramine, and the selective serotonin reuptake inhibitors, like fluoxetine, citalopram and sertraline. The specificity of the different drugs for each of the monoamine uptake transporters widely vary. DMI has 50 times more potency for blocking the NA uptake transporter than blocking the serotoninergic and more than one hundred times than the dopaminergic uptake transporter. Fluoxetine and citalopram have at least 50 times more specificity for the serotoninergic transporter than for any other monoaminergic transporters, cocaine is not so specific for the serotoninergic as is for the cathecolaminergic transporters (for a review see 53). Even though this is the primary function it has been shown that they can interact and bind different kind of receptors. Both amitriptyline and its main metabolite, nortriptyline, have an appreciable affinity for 5-HT2 receptors and are nonselective with respect to 5-

HT2A and 5-HT2C subtypes (53). Among the selective 5-HT uptake inhibitors fluoxetine shows some affinity for 5-HT2C receptors, whereas its affinity for 5-HT2A receptors is considerably weaker (54). A study on the effect of fluoxetine on phosphoinositide hydrolysis measured in rat choroids plexus suggested that fluoxetine is a 5-HT2C antagonist; the same study also demonstrated weak 5-HT2C receptor antagonist activity of norfluoxetine and citalopram (55). In neuroblastoma cells, cocaine and fluoxetine inhibited the 5-HT3 receptors (56). Uptake blockers have showed a variable affinity for the cathecolaminergic receptors. DMI showed a very high affinity for α1 receptors but than 2 orders of magnitude lesser than the affinity that showed to α2, β, D1 and D2 (53). The affinity of citalopram for cathecolaminergic receptors was extremely low being as high greater 100 000 nM for the adrenergic β receptor (53). Previously we found that nomifensine is able to block the DMPP-evoked noradrenaline release from rat hippocampal slices in a manner similar to the nicotinic antagonists mecamylamine and pancuronium suggesting a nicotinic antagonist effect of monoamine uptake blockers (26, see below).

2.3. Structure of the nicotinic acetylcholine receptor (nAChR) in the central nervous system.

2.3.1. Molecular biology of the nicotinic receptor The molecular biology of the nAChR is complex. Application of recombinant DNA techniques to the nAChR revealed close homologies between nAChR of electrical organ of Torpedo, human skeletal muscle nicotinic receptor and neuronal receptor. To date the sequences of 12 neuronal nAChRs subunits have been established (2,3,4). Each subunit contains N-terminal and C-terminal hydrophilic segments. The N-terminal subunit is located intracellularly and has phosphorylation sites and the C-terminal subunit that is located extracellularly that contains the acetylcholine binding site (5). Also, each subunit having four hydrophobic putative transmembrane domains designated M1-M4. An M2 domain line the walls of the ion channel properly and has indications that is formed by straight transmembrane Q- helixes with no kinks in the residues exposed to the lumen of the channel (6). The cryoelectron microscopy studies indicate that the other putative transmembrane domains (M1, M3 and M4) are relatively featureless, with a large portion of the polypeptide chain in an extended or unresolved - sheet configuration arranged in the form of a large -barrel outside a central rim the central rim of M2 channel-forming rods(7,8). In contrast studies done with photoactivable hydrophobic probes (9), deuterineum-exchange FTIR studies (10), CD and FTIR spectroscopy of isolated and lipid-reconstituted transmembrane peptides (11) indicate Q-helical structure for M1, M3 and M4 segments. The nAChR receptor is a pentamer of 5 subunits. Nine nAChR 8 Q- subunits have been cloned (Q2-Q10), and 3 types of subunits (2-4) each of them sharing the structure described above (2,3,4,12,13,14,15,16,17). Injection of RNA encoding 2 or 4 into Xenopus oocytes in pair wise combination with 2, 3, 4 RNA can produce at least eight different subtypes of nAChRs. Many different nAChR subunits can be expressed in the same cell (12).

The acetylcholine binding sites are located in the extracellular part of the subunits in a pocket about 30-35 A above the surface membrane, these pockets are located in the loop C of N-terminal hydrophilic segment of Q- subunits (18). The relative affinity for the agonist is modified by the neighbor subunit in the neuromuscular nicotinic receptor (19) and for the subunit in the neuronal acetylcholine receptor (20), it is necessary two molecules of the neurotransmitter to activate the receptor each one bound to each one the subunits (Figure 2). The nAChR belongs to the family of ligand gated-ion channel receptors as the 5-HT3, NMDA and GABA-A. The ligand gated channels is characterized by neurotransmitter induced opening of ionic channels allowing the free flux of ions down gradient, in the case of nAChRs, cations. In the central nervous system the selectivity for the cations is determined by the type of α subunit in the receptor. Receptors containing α7 subunit showed a greater permeability for Ca2+ than for Na+ while in the others the permeability is greater for Na+ (106). After ligand binding the opening duration of the channel is around 1 ms. The duration of the channel opening is dependent on the particular agonist, whereas the conductance of the open channel state is agonist-independent. The nAChR show the classic tristate agonist activation with a resting, an activated and an inactivated state (105).

Figure 2. Three dimensional structure of the neuronal nicotinic acetylcholine receptor

2.4. Neuronal acetylcholine receptor agonists and antagonists The sensitivities of the different nAChRs to the different agonists and antagonists are complex and depend on the subunits the receptors are built up. Here we will discuss the pharmacology of selected nicotinic agents.

2.4.1 Agonists

2.4.1.1. Epibatidine It was first isolated from the skin of the frog Epipedobates tricolor. It behaves as a potent agonist of the chick α3β2, α3β4, α4β2, α4β4, α7 and α8 expressed in oocytes. In binding experiments, both of the Epibatidine isomers showed high affinity for all the tested nAChRs, with a Ki of 10-20 pM to the α4β2 subtype and a Ki of 0.23 pM for the high affinity site of the chick α8 subtype. The affinity of the expressed subtypes was approximately 1000 times greater than their affinity for nicotine or acetylcholine (21, 22). Even though epibatidine showed higher affinity for the α4β2 receptor it is only a partial agonist being the responses obtained of smaller amplitude and slower onset and offset than those obtained with acetylcholine (23).

2.4.1.2. Anatoxin-A This is a derivative from the fresh water cyanobacterium Anabaena flos aqua. It has been shown to be an agonist of α4β2, α4β3 and α7. Nicotine currents in hippocampal neurons were activated by Anatoxin-A with an EC50 of 3.9 mM, while α7 oligomers reconstituted in Xenopus oocytes yielded an EC50 value of 0.58 mM showing that Anatoxin-A was between three and 50 times more potent than nicotine(24). Also the EC50 for stimulating the 86 Rb+ in M10 cell was 48 nM which shows the high potency at stimulating the α4β2 receptor (24).

2.4.1.3. Dimethylphenylpiperazinium (DMPP) DMPP is a powerful semi synthetic drug. In recombinant human nAChRs expressed in Xenopus oocytes (α2β2, α3β2, α3β4, α4β4 and α3β2 and α7)

DMPP showed to be 1.2-12 times as potent as the natural agonist, acetylcholine (25). DMPP is not only a pure nicotinic agonist. On resting release of NA from rat hippocampal slices has been shown to have an effect on carrier-mediated neurotransmitter release as well (26).

2.4.2. Antagonists

2.4.2.1. Mecamylamine Mecamylamine is a non competitive nicotinic antagonist. It has been shown in human nAChRs expressed in Xenopus oocytes that has a more prolonged inhibition on the neuronal type as compared with the muscular-type (27). In patch clamp studies the antagonist effect was only present when the receptor was previously stimulated with nicotine and showed dependence on the transmembrane 2 domain of the β subunit for its effect suggesting that the binding site of the mecamylamine is in the ionic channel (28,29).

2.4.2.2. Methyllycaconitine This is a typical competitive antagonist, when applied to hippocampal neurons, it is capable of specifically and reversibly decreasing the peak amplitude of the whole cell current elicited by nicotinic agonists. The Methyllycaconitine is a selective antagonist producing a potent and reversible blockade of the oocyte-expressed α7 but not the α3β2 or α4β2 subtypes (30).

2.5. Nicotine-evoked release of NA from the hippocampus Multiple nicotine receptors have been found in the hippocampus. In situ hybridization studies showed a strong β2 hybridization signal in the granule layer of the dentate gyrus and in the CA2/CA3 (31). Multiple studies clearly suggest that α7-nAChRs are functionally expressed in most hippocampal

interneurons (32, 33).Immunolabelling done in CA1 in the area of the stratum radiatum found that the localization is both presynaptic and postsynaptic (34). Characteristically low density of α3 subunits was found in hippocampus (35). Also significant expression of almost all the nAChR subunits was found CA1 hippocampal neurons (36). Experiments using hippocampal synaptosomes showed that nicotinic agonist nicotine, DMPP, cytisine and Anatoxin-A increased the noradrenalin release, these effect was mecamylamine sensitive showing that the release was mediated by the nicotinic receptor (37). Further experiments in superfused rat hippocampal slices showed that this release was probably mediated by α3β2 subtype of nAChRs. Other study suggests that α6β2 and β3-containing receptors could also play a role (38). Since the noradrenergic hippocampal innervation originates exclusively from the locus coeruleus and the majority of the varicosities do not make synaptic contact therefore this release must be non-synaptic (39).

2.6. DMPP-evoked release of NA from hippocampus In our previous experiments we found that the DMPP increase the release of noradrenaile in a two-phase release of NA from rat hippocampal slices, The first phase was a steep increase followed by a sudden decline to a lower level that was constant in time. Further analysis revealed that the release of noradrenaline in response to DMPP consists of two components. While the nicotinic receptor antagonists mecamylamine, pancuronium, pipericuronium, the nonselective antagonist Cd2+ and tetrodotoxin completely abolished the peak response (phase I), They had no effect on the tail response (phase II). Whereas the noradrenaline uptake blocker desipramine (DMI 1-10 µM), nisoxetine (1-10 µM), and nomifensine (10 µM) inhibited both phases, nomifensine at a concentration of 1 µM selectively blocked only phase II. Our results indicate that DMPP has a dual effect on the hippocampal noradrenaline release: phase I is a transient, nicotinic receptor-mediated exocytotic release , and phase II is a maintained , transporter- mediated process(26).

3. – Aims

3.1. Comparison of the effect of nicotinic agonists on the hippocampal NA release.

The results with DMPP proved that this nicotinic agonist has a dual action (on the nAChR and the transporter). The question was whether other nicotinic agonists share the same properties. The aim was to compare the effect of a number of nicotinic agonists on the hippocampal NA release and also analyze the mechanisms of action of these compounds (nicotine, anatoxin-A, epibatidine, cytisine, DMPP, lobeline).

3.2. Investigation of the antinicotinic effect of monoamine uptake transporters in the CNS

3.2.1. Question of selectivity In our previous work (26) we found that DMI, nisoxetine and nomifensine inhibit the nicotinic agonist-evoked release of NA from rat hippocampal slicesin the low micromolar range. DMI and nisoxetine inhibit primarily the NA uptake while nomifensine blocks the NA and dopamine (DA) transporter. Since the amino acid identity of the NA and DA transporters is 82 % (107) but the serotonin (5- HT) transporter belongs to the same protein family and its homology with the NA transporter is still very high (about 60 %) (95). It seemed reasonable to investigate whether the selective serotonin reuptake inhibitors (SSRIs) share the property of NA and/or DA uptake blockers to inhibit the nAChR-mediated response. However, the 5-HT/NA selectivity ratio of fluoxetine is only about 30 (89), therefore in the present work we decided also to test another SSRI, citalopram with a much higher selectivity (5-HT/NA = 2900) for the 5-HT uptake (89). We also investigated the effect of cocaine, since it has a relative selectivity toward the DA transporter (107) and the drug, due to its addictive potential has a special clinical importance.

3.2.2 Quantitative analysis In our previous work (26) we have observed the inhibitory action of monoamine uptake blockers but the effect was not characterized quantitatively therefore an additional aim of the present study was to obtain the IC50 values of DMI, nisoxetine and nomifensine. And also we quantified of the new drugs tested: fluoxetine and cocaine.

3.2.3 Search for the possible mechanism of action Finally, we tried to find some clues to understand the nature of the effect of monoamine uptake blockers on the function of nAChRs.

4. – Methods

4.1. – Nicotinic agonists-evoked [3H]noradrenaline release from rat hippocampal slices Male Wistar rats (weighing 150-200 g) were killed by decapitation, and the brain was rapidly removed and immediately placed into ice-cold Krebs solution (composed of 113 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 2.5mM CaCl2, 25 mM NaHCO3, 1.2 mM KH2PO4, 115mM glucose, 0.3 mM Na2EDTA and 0.3 mM ascorbic acid) continuously gassed with a mixture of 95% O2 and 5% CO2. Then the hippocampi were prepared and sliced into 0.4-mm sections by a McIlwain chopper. Slices were dissected by shaking and washed with 5 ml Krebs buffer and loaded for 45 minutes [3H]NA (L-7,8-[3H]NA, 37 MBq, 30-50 Ci/mmol) at a concentration of 10 µCi in 1 ml of Krebs solution. After the incubation slices were washed three times with 10 ml of ice-cold Krebs solution and transferred into a four-channel micro volume perfusion system with an internal volume of 100 µl (57). Four slices were put into each chamber. The temperature inside the chambers was kept at 37 °C. The preparation was superfused at a rate of 0.5 ml/min for 60 min (preperfusion period) and the effluent was discarded. Subsequently 12 3-min fractions were collected. Nicotine, at a concentration of 100 µM, was added beginning from the fourth sample; uptake blockers (DMI, nisoxetine, nomifensine, citalopram, fluoxetine and cocaine) were present in the medium from the beginning of the preperfusion period. At the end of the experiments slices were removed from the chamber and mechanically homogenized in 5 ml of 10% trichloroacetic acid. A 0.5 ml aliquot of the supernatant was added to 2 ml of scintillation cocktail (Ultima Gold; Packard). Tritium was measured with a Packard 1900 TR liquid scintillation counter using an internal standard. The radioactivity released from the tissue has been shown to be [3H]NA using high performance liquid chromatography with radiochemical detection (57). Radioactivity was expressed in terms of disintegration per second per gram of tissue (Bq/g). The fractional release was expressed in terms of the percentage of tritium present in the tissue at the beginning of a sample collection period.

4.2. - Electrical stimulation-evoked [3H]NA release from rat hippocampal slices Slices were prepared as described above, after preperfusion period nineteen 3- min samples were collected. Electrical stimulation (20 V, 2 Hz, 1 ms, 360 impulses) was applied during the third (S1) and the thirteenth sample (S2). Nicotinic agonists, Anatoxin-A, DMPP, cytisine and lobeline were also tested and were administered also from tenth sample. The tritium content of the samples was determined as described above. For the demonstration of no significant Na+ channel blocking effect DMI was administered from the tenth sample, TTX was also administered from tenth sample as comparison.

4.3. –Electrophysiological recording of Na+-currents The method used for Na+-current recording was similar to that previously described by Carrier and Ikeda (58). Recording of Na+-currents was made using the whole cell variant of patch clamp technique (118). Na+-currents were evoked every 20 sec by a 20 msec voltage step from –80 mV to –20 mV. Data were acquired using an Axopatch 1D amplifier (Axon Instrument, Foster City, CA), filtered at 5 kHz and digitized at 20 kHz. The leak current was subtracted by using the standard P4 protocol. Patch pipettes (M87, Sutter Instruments, Novato) had resistance of 1-8-3.5 M. Series resistance compensation was routinely applied by about 70-80%. Patch electrodes were filled with Na+-NMG based internal solution (NaCl; 30 mM, N-methyl-D-glucamine; 140 mM, CaCl2; 1 mM, MgCl2; 2 mM, HEPES; 10 mM, EGTA; 11 mM). Neurons were perfused with Na+-TEA+ based solution

(NaCl; 50 mM, TEACl; 100 mM, MgCl2; 10 mM, HEPES; 10 mM). Drugs were dissolved in the extracellular solution and were applied by switching from the control solution to the drug containing solution. The recording chamber (volume: 200 l) was continuously perfused at a flow rate of 2 ml min-1. Drugs were applied at least for 2 min. Current amplitudes were measured at the peak.

4.4. Statistical Analysis

4.4.1 Effect of nicotinic agonists on the electrical stimulation-evoked release of [3H]noradrenaline from rat hippocampal slices According to the conventional method used in electrical stimulation-evoked release experiments, the response (area under curve) to stimulation (S1 [control] in the absence and S2 [treatment] in the presence of a compound) is calculated using the FR data (Figure 1, upper panel) and the effect of drugs is characterized by the ratio of the two areas (FRS2/FRS1). In this work we used a novel data processing technique, which provided a more detailed analysis of experimental results. The new calculation is based on the notion that in electrical stimulation-evoked release experiments the same sequence is repeated twice. First, two resting fractions are collected then the tissue is stimulated electrically (two fractions), finally the sequence is ended with five post-stimulation fractions. Therefore fractions 1 and 11, 2 and 12, etc. can be matched (Figure 3). Because nineteen samples are collected during the experiment, nine corresponding fraction-pairs can be constructed. Fraction 10 remains unpaired, however this is rather an advantage, because the compounds reach the tissue during this sample, that is, this is a transient fractions from the point of view of drug effect. In the new evaluation technique the ratio of corresponding fractions is depicted in percentage (100*11/1, 100*12/2,..., 100*19/9) (Figure 3, lower panel). The first two points represents the resting release before the electrical stimulation (pre-stimulation resting), the second two points correspond to the electrical stimulation, whereas the last five points characterize the release after the electrical stimulation (post-stimulation resting). In a control experiment, when no drug is applied between the two stimulations, the points lay on a straight line, which is parallel with the x-axis (Figure 3, lower panel). This is very favorable because any drug action appears as a deviation from this horizontal line. The new evaluation method has two major advantages over the traditional technique. First, the dynamics of drug effect is clearly visible. When the area under curve calculation is used, we loose very important information, the time course of drug action, while the new

method preserves this parameter. Second, the traditional method carries a subjective element because the area under curve calculation depends on the person who determines the fraction interval i.e. the beginning and the end of response. This vagueness is completely excluded in the new evaluation. The data represent the mean ± S.E.M. of 4-6 independent experiments. The statistical analysis was performed using ANOVA followed by Dunnett's test; the level of significance was set at p<0.05.

4.4.2. Effect of nicotinic agonists on the resting release of [3H]noradrenaline from rat hippocampal slices Fractional release (FR) data were normalized to decrease variance between subjects. The average of three fractions before treatment was taken as 100% and all fractions were expressed relative to this value as normalized fractional release (nFR) data. The nFR data were analyzed using an area under curve (AUC) method. The average basal FR data of different treatment groups were not significantly different. The response to nicotine was calculated as the surplus release over the basal efflux. Inhibition values were determined in a counterbalanced manner. In every experiment we used three different concentrations of an uptake blocker and a control (nicotine alone) that gave the maximal effect (100%). The inhibitory effect of any drug was expressed relative to this control. Values are mean M SEM of 4-6 independent experiments. Dose- response curves were constructed separately for each drug. Comparison of areas was performed by one-way ANOVA followed by Dunnett’s test or by two- tailed Welch’s t test, where appropriate, p<0.05 was considered significant. IC50 values were calculated by a nonlinear regression (GraphPad Prism program).

In experiments using electrical stimulation the area under curve for S1 and S2 were calculated from fractional release data and inhibition values were determined from the ratio of S2/S1 values (S2/S1 in the presence of drug / S2/S1 in the absence of drug; counterbalanced design, every drug effect was compared to the corresponding control value). Statistical analysis of inhibition values was performed one-way ANOVA followed by Dunnett’s t test as mentioned above. When the correlation was investigated between the inhibition

of nicotine-evoked NA release (IC50) and inhibition of NA uptake (Ki), a linear regression analysis was used.

control phase (1-9) drug effect (11-19) 8 7 6 FRS1 FRS2 5 4 3 2 Fractional release (%) release Fractional 1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 (11/1)*100 ..... (12/2)*100

(13/3)*100 (19/9)*100

200 .....

100 % of corresponding fraction 123456789

resting before electrical resting after stimulation stimulation stimulation

Figure 3. Method of corresponding fractions

5. - Results

5.1.Differential effect of nicotinic agonists on the [3H]noradrenaline release from rat hippocampal slices

5.1.1. Effect of nicotinic agonists on the electrical stimulation-evoked release of [3H]noradrenaline from rat hippocampal slices The resting and electrical stimulation-evoked release of [3H]NA was affected by all nicotinic agonists, however their action showed differences. Majority of the nicotinic agonists (nicotine, 100 µM; cytisine, 30 µM; epibatidine, 1 µM; anatoxin-A, 2 µM) increased only the resting release of [3H]NA before electrical stimulation. This action was transient and it declined rapidly. Neither the electrical stimulation, nor the post-stimulation resting release was affected by these drugs (Figure 4A, see next page). In contrast, DMPP (20 µM) increased the release of [3H]NA in all phases, that is, it stimulated both the pre- and post-stimulation resting release and also increased the response to electrical stimulation (Figure 4B, see next page). The effect of lobeline (30 µM) was very similar to that of DMPP, except for the time-course of action on the resting release before stimulation (fraction pairs 1 and 2). While the effect of DMPP on the pre-stimulation release showed decreasing tendency, the response to lobeline increased with time (Figure 4C, see next page). Selection of drug concentrations was based on dose-response curves obtained previously (Sershen et al, 1997).

5.1.2 Effect of the nicotinic antagonist mecamylamine on the action of nicotinic agonists The enhancing effect of nicotine (100 µM) on the release of [3H]NA was completely blocked by the non-competitive nicotinic antagonists mecamylamine at a concentration of 10 µM (Figure 5A). The effect of antagonists

300 control

250 * nicotine 100 µM 200 * cytisine 30 µM 150 *

100 epibatidine 1 µM 50 anatoxin 2 µM % of corresponding fractions 0 12 345678 9 Fraction pairs B

300

250 * 200 * * * control * * * * 150 * DMPP 20 µM 100

% of corresponding fractions 0 12 345678 9 Fraction pairs

300 * 250 * * * * * 200 * control * * 150 lobeline 30 µM 100

% of corresponding fractions 0 12 345 6789 Fraction pairs

Figure 4. Effect of different nicotinic agonists on the electrical stimulation–evoked release of [3H]NA from rat hippocampal slices. A: nicotine-like agonists (nicotine, cytisine, epibatidine, anatoxin-A). B: dimethylphenypiperzinium (DMPP). C: lobeline. First two fractions: pre-stimulation resting release, second two fractions: electrical stimulation, last five fractions: post-stimulation resting release (for further explanation see Figure 3). * p < 0.05

0(cytisine, epibatidine, anatoxin-A), which similarly to nicotine, increased only the pre-stimulation resting release of [3H]NA ('nicotine-like' antagonists), was also completely inhibited by mecamylamine (not shown). The effect of DMPP

A 300 250 control 200 nicotine 100 150 µM 100 * * nicotine 100 µM + 50 mecamylamine 10 µM 0

% of corresponding fractions 123 456789 Fraction pairs B

300 250 control 200 DMPP 20 150 fractions µM 100

% of corresponding * DMPP 20 µM + 50 mecamylamine 10 0 µM 1 234 56789 Fraction pairs C

300

250 control 200 150 lobeline 30 µM fractions 100 lobeline 30 µM +

% of corresponding mecamylamine 10 50 µM 0 123 45678 9 Fraction pairs

Figure 5. Effect of the nicotinic antagonist mecamylamine on the nicotinic agonist-evoked 3 release of [ H]NA from rat hippocampal slices. A: nicotine. B: dimethylphenypiperzinium (DMPP). C: lobeline. First two fractions = pre-stimulation resting release, second two fractions = electrical stimulation, last five fractions = post-stimulation resting release (for further explanation see Figure 3). * p < 0.05

(20 µM) was only partially inhibited by mecamylamine, because a significant reduction of release could be observed only in the pre-stimulation resting phase, but the response to DMPP during the electrical stimulation and the post- stimulation resting phase was not influenced by mecamylamine (Figure 5B). Finally, the effect of lobeline on the release of [3H]NA was not affected at all by mecamylamine (Figure 5C).

5.1.3 Effect of the noradrenaline uptake inhibitor desipramine on the action of nicotinic agonists To investigate the possible role of NA transporters in the action of nicotinic agonists we repeated the experiments in the presence of the NA uptake inhibitor DMI (10 µM). This drug was present in the Krebs solution from the beginning of preperfusion, which caused a slight and non-significant upward shift in the control curve. The control ratios moved from about 90 % to about 100 % (see DMI control, Figure 6). The stimulating effect of nicotine was completely blocked by DMI (Figure 6A). Similarly, the response to DMPP was inhibited by the NA uptake inhibitor in all phases (pre-stimulation resting, electrical stimulation, post-stimulation resting) of the experiment. In contrast, the effect of lobeline was only partially inhibited by DMI. It was completely blocked during the electrical stimulation and in the first post-stimulation sample (Figure 6C) but the action was only partially reduced in other fractions. This residual response was not influenced significantly by pargyline (10 µM), when the MAO- B inhibitor was also present in the Krebs solution from the preperfusion (not shown).

300

250 control

200 nicotine 100 µM 150 ** control (DMI) 100 nicotine 100 µM + 50 DMI 10 µM % of corresponding fractions 0 123456789 Fraction pairs B 300

250 control

200 DMPP 20 µM 150 control (DMI) * * *** * * 100 DMPP 20 µM + 50 DMI 10 µM % of corresponding fractions 0 123456789 Fraction pairs

C 300

250 control

200 * * lobeline 30 µM * * 150 * * * control (DMI) 100 * * lobeline 30 µM + 50 DMI 10 µM

% of corresponding fractions 0 123456789 Fraction pairs

Figure 6. Effect of the NA uptake inhibitor desipramine (DMI) on the nicotinic agonist-evoked release of [3H]NA from rat hippocampal slices. A: nicotine. B: dimethylphenypiperazinium (DMPP). C: lobeline. First two fractions = pre-stimulation resting release, second two fractions = electrical stimulation, last five fractions = post-stimulation resting release (for further explanation see Figure 3). * p < 0.05

5.2. Effect of nicotine on the release of [3H]NA from rat hippocampal slices After a 45-min loading with [3H]NA, followed by a 60 min preperfusion period, the slices contained 4928562 M 347939.3 Bq/g (n=36) radioactivity. Perfusion of nicotine at a concentration of 100 µM produced an excess release 3 over the basal efflux of [ H]NA (AUC4-10 = 323.18 ± 44.24 %, n=28). The response was transient and the release returned to the baseline within four fractions in spite of the presence of nicotine in the solution (Figure 9). Although the time course of the response was not affected, the excess release in response to nicotine (100 µM) was significantly lower (AUC4-10 = 87.19 ± 6.02 %, n=8, p < 0.01 in two-tailed Welch’s t test) when the Krebs solution contained ethanol (3 mg/L) in the control experiments for the citalopram-group. The inhibitory effect of ethanol was not further investigated since the difference did not disturb the evaluation of the effect of monoamine uptake blockers.

Normal Krebs Normal Krebs + ethanol 250

200

150

nFR (%) nFR 100

50 Nicotine (100 µM)

0 12345 7 8 9 101112 Fractions

Figure 7. effect of nicotine on basal release of NA from rat hippocampal slices in normal and ethanol containing solution

5.3. Effect of monoamine uptake blockers on the nicotine-evoked release of [3H]NA from rat hippocampal slices The nicotine-evoked release of [3H]NA was dose-dependently inhibited by all of the tested drugs (DMI, nisoxetine, cocaine, citalopram, nomifensine) in the concentration range of 0.03-30 µM (Figure 8) The IC50 values of the monoamine uptake blockers ranged between 0.36 µM (DMI) and 1.84 µM

(nomifensine) (Table 1). For comparison, Table 1 also contains the IC50 values of the nicotinic antagonist mecamylamine.

100

75 Desipramine

Nisoxetine

50 Cocaine

Inhibition (% ) Citalopram

25 Nomifensine

0 -8 -7 -6 -5 -4 Log concentration

Figure 8. Dose-response curve of the inhibitory effect of monoamine uptake blockers on the nicotine-evoked [3H]NA release from rat hippocampal slices, 50 % inhibition is represented by the dotted line. IC50 values were determined by nonlinear regression

IC50 (µM) Concentration range (µM) Threshold concentration* (µM)

Mecamylamine** 0.19 0.1-10 0.3

Fluoxetine** 0.57 0.1-10 1

DMI 0.36 0.03-10 0.3

Nisoxetine 0.59 0.1-10 0.3

Cocaine 0.81 0.1-10 1

Citalopram 0.93 0.1-30 1

Nomifensine 1.84 0.1-10 3

Table 1. Pharmacological quantification of inhibition of nicotine-evoked NA release by monoamine uptake blockers and mecamylamine. * The lowest concentration which produced significant inhibition.

5.4. Correlation between the inhibition of nicotine-evoked [3H]NA release and the inhibition of NA uptake To explore a possible relationship between the observed nAChR antagonist effect and the ability to block NA uptake, the logarithm of the calculated IC50 values were plotted against the logarithm of the Ki values for the NA uptake transporter and the coefficient of correlation (r) was determined

(Figure 9). Ki values were taken from the literature (88,89,90). The analysis showed no correlation between the two variables (r=0.17, slope 0.02, not significantly different from zero) excluding the possibility of a connection between the uptake transporter blocker effect and nAChR-antagonist effect of these medications.

3.5

3.4

3.3 Nom

3.2

3.1 (nM)

50 3.0 Cit Coc 2.9 Log IC 2.8 Nis Flu

2.7

2.6 DMI

2.5 0.0 1.0 2.0 3.0 4.0 5.0

Log K i (nM)

Figure 9. Correlation between IC50 for NA uptake inhibition of monoamine uptake blockers and their respective EC50 for nAChR inhibition

5.5. Effect of monoamine uptake blockers on Na+-currents of sympathetic neurons from rat superior cervical ganglia In patch clamp experiments the uptake blockers (DMI, nisoxetine, nomifensine, citalopram and cocaine) were used at the concentration range (1 and 10 µM) where the inhibition of nicotine-evoked release was already about 90 % (Figure 10). The amplitudes of control current preceding the drug application and the amplitude of the current by the end of drug application were compared. Steady state maximal inhibition usually developed before the end of the 2 min perfusion period. The inhibitory effects of the applied drugs were reversible. A representative recording of the control current and the inhibition in the presence of DMI (10 µM) is shown on Figure 10 inset. Neither drug inhibited Na+-currents at 1 µM and only DMI displayed a pronounced inhibition (52 %) at

10 µM (Figure 4), therefore the possible involvement of Na+-channel blockade in the effect of monoamine uptake blockers was further studied only in the case of DMI.

nomifensine 60 DMI (10 µM) control 50 nisoxetine

40 -channels (%)

+ cocaine 1 ms 30 citalopram 20

DMI

Inhibition of Na 10

0 110 Concentration (µM)

Figure 10 Inhibitory effect of uptake transporters on sodium channel obtained using patch clamp method on rat sympathetic ganglion neurons

5.6 Effect of nAChR-agonists on resting and electrical stimulation-evoked release of [3H]NA As with nicotine anatoxin-A (2 µM) and cytisine (30 µM) showed increase in the resting release of [3H]NA but not in the electrical evoked release (data not shown). As with nicotine this action was transient and decline rapidly. This effect was also characteristically blocked by mecamylamine. 5.7 Comparison of the inhibitory effect of TTX and DMI on the nicotine- and electrical stimulation -evoked release of [3H]NA from rat hippocampal slices The effect of DMI and the Na+-channel blocker TTX on the nicotine- and electrical stimulation-evoked release of NA were compared to test the possible

involvement of Na+ channels in the action of DMI. TTX blocked both the nicotine- and the electrical stimulation-evoked release with the same efficacy

(IC50 was 0.033 and 0.039 µM, respectively) (Figure 11). In contrast, DMI blocked only the nicotine-evoked release (IC50 = 0.36 µM), whereas the electrical stimulation-evoked release was not inhibited at all (zero inhibition), moreover it was potentiated as expected from its uptake blocking effect (not shown on the figure 11)

100

D M I (nic.) 75

D M I (el. st.) 50 TTX (nic.) Inhibition (%)

25 TTX (el. st.)

0 -8 -7 -6 -5 -4 Log concentration (M)

Figure 11. Dose-response curves of the inhibitory effect of tetrodotoxin (inverted closed triangle ) and desipramine (closed circle) on the nicotine-evoked [3H]NA release and the inhibitory effect of tetrodotoxin (closed square ) and desipramine (closed triangle) on electrical stimulation-evoked release from rat hippocampal slices, 50 % inhibition is represented by the dotted line. IC50 values were determined by nonlinear regression

6. – Discussion

6.1. Differential effect of nicotinic agonists on the [3H]noradrenaline release from rat hippocampal slices In previous works (108, 72) we studied the effect of nicotinic agonists on the release of NA from rat hippocampal slices. We found that the major effect of these compounds is the stimulation of nAChRs located on noradrenergic varicosities (37), which induces a Ca2+-dependent vesicular exocytosis. During these studies, however, we have observed that some of the nicotinic agonists (DMPP and lobeline) are able to stimulate the release of NA also in Ca2+-free medium, indicating that the action of these drugs is more complex than that of the conventional nicotinic agonists. The aim of the present study was to investigate the mechanisms involved in the action of different nicotinic agonists on the [3H]NA release from rat hippocampal slices. We used a newly developed calculation method for the evaluation of experimental data, which proved to be very effective tool for the detection of fine differences between the actions of drugs.

6.1.1. Effect of conventional nicotinic agonists Our data, in line with previous results, demonstrated that nicotine, cytisine, epibatidine and anatoxin–A behaved like conventional nicotinic agonists, that is, they increased the release of [3H]NA from rat hippocampal slices exclusively through stimulation of nAChRs, because their action was completely inhibited by the nicotinic antagonist mecamylamine (Figure 3A). These drugs had only a transient and rapidly declining effect on the resting release before the electrical stimulation but had no effect on the electrical stimulation-evoked or on the post-stimulation resting release (Figure 5A), which was the consequence of the rapid desensitization nAChRs. It may seem to be more surprising that the action of nicotine was also completely blocked by the NA uptake inhibitor DMI (Figure 4A). The nicotinic antagonistic property of DMI, however, has already been described earlier

(64,65,109). Results described for this thesis confirmed the findings and quantified the effect.

6.1.2. Effect of DMPP Our results show that DMPP, in contrast to other nicotinic agonists, increases the hippocampal [3H]NA release in all phases (pre-stimulation resting, electrical stimulation, post-stimulation resting) of the experiment (Figure 4B). The observation that mecamylamine inhibits this action only partially in the pre- stimulation resting phase (Figure 5B) suggests that the effect of DMPP has more than one component. It is obvious that DMPP, similarly to other nicotinic agonists, is able to stimulate nAChRs, which results in vesicular exocytosis of NA. What can be the mechanism of the mecamylamine-insensitive effect? It has been observed that substrates of the NA uptake system are able to induce a reversed transport of NA (110). Accumulating data indicate that DMPP can be such a substrate. It has been shown that DMPP is able to release NA from rat vas deferens through a carrier-mediated mechanism but only if the vesicular uptake and monoaminoxidase (MAO) are inhibited and Ca2+ is omitted from the perfusion medium (111). Under normal conditions this effect is very weak in the periphery. Our data show that in the CNS, however, these manipulations are not necessary for a carrier-mediated efflux of NA. The mecamylamine- insensitive part of the DMPP-response completely disappeared in the presence of DMI (Figure 4B), which provided convincing evidence for the involvement of uptake carriers. In addition, DMI blocked also the mecamylamine-sensitive response because of the nAChR antagonistic property of the uptake blocker (see above).

6.1.3. Effect of lobeline Lobeline, similarly to DMPP, increased the resting release of [3H]NA before and after stimulation and potentiated the response to electrical stimulation as well (Figure 5C). The new evaluation method, however, shed light on a very important difference. While the effect of DMPP on pre-stimulation resting release showed a decreasing tendency (fraction pairs 1-2, Figure 4B),

the response to lobeline gradually increased in this phase of the experiment. Further analysis revealed that the effect of lobeline is qualitatively different from that of DMPP because the response to lobeline was not affected at all by mecamylamine (Figure 5C). This is surprising because lobeline has been widely used as a nicotinic agonist (112). Indeed, it has been reported that lobeline competes for the nicotine binding site with high affinity, the Ki ranges between 4-30 nM (113,114,115). Functional-level analogy and evidence for high affinity binding to nAChRs, however does not prove an agonist effect on nAChRs. Accumulating data indicate that the nature of lobeline is ambiguous, and is dependent on the type of nAChR studied: it is either a partial agonist, typically the weakest of the agonists studied (116,117), or an antagonist (115,118). Our data, in line with these findings, suggest that under our experimental conditions the nAChRs involved in the release of [3H]NA are not stimulated by lobeline. The mecamylamine-insensitive effect of lobeline has already been observed previously. Lobeline increased the release of DA from rat (37) and mouse (119) striatal synaptosomes and slices (120) and the release of NA from rat vas deferens (121) in a mecamylamine-insensitive and Ca2+-independent manner. All of these data indicate that the action of lobeline is independent of nAChRs, and the lobeline-induced release is not a vesicular exocyotis. What kind of mechanism can be involved then? It has been shown that lobeline blocks the vesicular monoamine transporter (VMAT2) with high affinity (120,121). In addition, lobeline displaces the monoamines from synaptic vesicles, which leads to the increase of cytoplasmic transmitter concentration (122). This change may promote the reversal of membrane transporters and the concomitant carrier-mediated release of transmitters (123). Our data obtained with DMI (Figure 4C) indicate that a substantial part of the effect of lobeline is results indeed from the reversal of uptake, however, further factors are also involved. It has been observed that, due to the increased cytoplasmic concentration of transmitters (see above) the efflux of metabolites is also increased (122). We tested, therefore, the hypothesis that the residual tritium efflux could be metabolic. It was not reduced, however, significantly in the presence of the MAO-B inhibitor pargyline, suggesting that the DMI-insensitive

release cannot be explained with the increased metabolite outflow, i.e. it is [3H]NA. It is possible that lobeline increases the efflux of [3H]NA through the cell membrane with a similar mechanism as it displaces transmitters from the vesicles into the cytoplasm. This idea is supported by the fact the lobeline- induced [3H]NA release is completely blocked at 12 °C (121). At this temperature the rigidity of biological membranes is increased, which could prevent the ’leakage’ of transmitters through the membrane. Additional effects of lobeline (e.g. blockade of Ca2+-channels (124)) may also contribute to its action on NA release (figure 12).

R Ch U + nA

Lobeline Nicotine NA Epibatidine NA Anatoxin-A Cytisine DMPP

Figure 12. Differential effect of nicotinic agonists on the hippocampal NA release.

6.2. Effect of monoamine uptake blockers in nicotine induced noradrenaline release In our results we have found that monoamine uptake blockers with different chemical structure and selectivity for NA, DA or 5-HT transporters (59,60,61) are able to inhibit the nicotine-evoked increase of hippocampal NA release in a dose-dependent manner. The calculated IC50 values of DMI, nisoxetine,

nomifensine, citalopram, fluoxetine and cocaine ranged between 0.36 and 1.84 µM. It has already been demonstrated that some of the monoamine uptake blockers (DMI, nisoxetine and cocaine) may influence the function of nAChRs. Almost 30 years ago, Su and Bevan (62) reported that DMI and cocaine inhibited the nicotine-evoked release of [3H]NA from spiral strips of the rabbit pulmonary artery. The nicotine-induced exocytotic release of NA from isolated guinea-pig heart was blocked by DMI and nisoxetine (63). In addition, DMI inhibited the DMPP-evoked depolarization and NA release in SH-SY5Y neuroblastoma cells (64). In the same cell line whole cell patch clamp recordings proved that DMI and imipramine inhibited the nAChR-mediated currents in a non-competitive manner (65). The behavioral effects of nicotine (seizures, tremors, fasciculations, etc.) were antagonized by cocaine and cocaine analogs in mice (66). The nicotine-induced inward current (both peak current amplitude and total charge influx) was inhibited by DMI and imipramine in chromaffin cells (67). Our results confirm these findings and extend the range of monoamine uptake blockers possessing nAChR antagonistic activity with nomifensine and the SSRI citalopram. But what is the mechanism of this antagonism? Some authors suggested that the action of nicotine may require an intact NA uptake mechanism (62), that is, the antinicotinic action of uptake blockers would be mediated through the transporter. Comparing the chemical structures and the pharmacological properties of these drugs, their only common feature is that all of them are able to block certain monoamine uptake transporters. Therefore it is justified to assume that these compounds interact with the NA uptake system and this interaction initiates some intra- and/or extracellular events which, in fact, may lead to a functional blockade of nAChRs. To investigate this hypothesis we compared the IC50 values obtained in our experiments with the inhibitory effect of these compounds on NA uptake (Ki values were taken from literature) (54, 59,68), to investigate a possible connection between these properties. The statistical analysis showed no correlation between Ki and IC50 values (Figure 3) suggesting that the NA uptake system is not involved in the inhibitory effect of

monoamine uptake blockers on the nicotine-evoked NA release. This conclusion is supported by our previous observation that nomifensine at a concentration of 1 µM effectively blocked the carrier-mediated component of DMPP-evoked NA release but did not affect the nAChR-mediated component (26) indicating that the actions on the transporter and on the nAChR are separated. It has been shown that antidepressant drugs like DMI and imipramine are able to suppress fast inward sodium (Na+) current in a variety of neuronal preparations (69,70,71). The TTX-dependency of the nicotine-evoked release of [3H]NA (72,73) indicates that the response to nicotine requires the activation of Na+-channels therefore our next assumption was that the monoamine uptake blockers inhibited the nicotine-evoked release via inhibition of Na+ channels. Thus, in the next series of experiments we tested the inhibitory effect of the uptake blockers on the fast TTX-sensitive inward Na+ current in rat sympathetic neurons from the superior cervical ganglia. The effects of uptake blockers were studied in the concentration range (1-10 µM) which proved to be effective in the hippocampal preparation. Our data showed that only DMI had pronounced inhibitory effect on the Na+ current in the rat sympathetic neuron preparation while the rest of the monoamine uptake blockers were ineffective. Since DMI at a concentration of 10 µM inhibited about 50 % of the Na+-currents, our patch clamp data could not rule out the possibility that DMI blocks the nicotine-evoked NA release via inhibition of Na+-channels. However, if this is the case, DMI should have inhibited also the electrical stimulation-evoked release of NA, since this process is Na+-channel dependent. In contrast, our data showed that DMI blocked only the nicotine-evoked NA release but had no inhibitory effect on the electrical stimulation-evoked release. A possible explanation would be for this discrepancy that the two stimulation protocols activate the Na+-channels with different efficacy. Nevertheless, our results obtained with TTX (Figure 5) indicated that the TTX-sensitivity of the nicotine- and electrical stimulation- evoked release was identical (the IC50 was 0.033 and 0.036 µM, respectively) suggesting that Na+-channels are equally affected in both cases. The differential effect of DMI on the two types of NA release excludes that the nAChR

antagonist effect of DMI would be mediated via inhibition of Na+-channels. Taken together, the patch clamp data and TTX-experiments suggest that Na+- channels are not involved in the inhibitory action of monoamine uptake blockers on the nicotine-evoked NA release. In our experiments we investigated the possible mechanism by which monoamine uptake blockers can inhibit the nicotine-evoked NA release from rat hippocampal slices. Although we excluded some reasonable assumptions (involvement of NA uptake and Na+-channels), our results did not give a final and definite answer. Based on the literature, however, we can propose an explanation. It has been shown that tricyclic antidepressants including DMI bind with high affinity to the ion channel of nAChRs prepared from the electric organ of Torpedo ocellata (74). The Kd value of DMI in the presence of ACh was 0.2

µM, which is very close to the IC50 value (0.36 µM) obtained in our experiments. This binding site within the ion channel of nAChRs is the target of the non- competitive nicotinic antagonist mecamylamine (75). The interaction of monoamine uptake blockers with the mecamylamine binding site was also supported by Lerner-Marmarosh et al. (66) who reported that behavioral effects of nicotine were antagonized by cocaine analogs in mice and demonstrated in receptor binding studies that cocaine analogs compete for the mecamylamine binding site with high affinity. These receptor binding studies suggest that DMI and cocaine may behave like channel blocker-type nAChR antagonists. Another group published similar results to us showing that fluoxetine rapidly reduced the amplitude of membrane currents elicited by stimulation of neuronal (2ß4, 3ß4) and muscular (1ß1) nAChRs expressed in Xenopus oocytes (76). In this patch clamp study single channel recordings showed that fluoxetine directly blocks the receptor channel. Recently it has been published that fluoxetine also blocks the neuronal (7) nAChRs by a similar mechanism (77). These results indicate that monoamine uptake blockers with different chemical structure and selectivity interact directly with the ion channel of nAChRs. It is then conceivable that other monoamine uptake blockers may share the same action mechanism, that is, they bind into the ion channel of nAChRs and act as non- competitive channel-blocker type nicotinic antagonists (figure 13). A recent

study suggests that the inhibitory effect of cocaine on nAChRs is dependent on the subunit composition of the receptor (78).

Nicotine mecamylamine ACh

Figure 13. Binding sites of the nicotinic acetylcholine receptor. Our data and the literature indicate that monoamine uptake blockers bind to the mecamylamine binding site and behave like channel blocker-type anatgonists.

A very interesting consequence of our findings is that it may reveal some structural similarity between nicotinic receptors and monoamine uptake carriers. Accumulating data indicate that the membrane transporter proteins contain functional channels (for a review see 79). If we assume that i.) these channels play a crucial role in the transport of molecules through the membrane, ii.) the general mechanism of uptake blockers is the blockade of these channels, and iii.) there is some structural similarity between nAChR ion channels and transporter channels, then the nAChR antagonist property of monoamine uptake inhibitors would be easily understandable. The model we propose predicts that the channel blocker nicotinic antagonists should block the monoamine uptake. Recent data that the NMDA antagonist MK-801 blocks the monoamine transporters expressed in HEK cells (80) seem to support our

hypothesis since the PCP-like NMDA antagonists have been shown to bind also to the ion channels of nAChRs (74). It is a justified question why the nAChR antagonist property of different monoamine uptake blockers remained hidden in spite of the meticulous pharmaceutical research and development. Previously different uptake blockers did not show high affinity for the nAChR, apparently ruling out that these compounds could act on the receptor (54). However, it must be noted that in the binding studies investigating nAChRs almost exclusively ligands of the nicotine binding site (e.g. [3H]nicotine) were used. The uptake blockers have low affinity toward this binding site. For example, cocaine was without effect on the 3 [ H]nicotine binding (Ki > 1mM) while competed for the mecamylamine site with a Ki of 1 µM (66). Since currently good nicotinic channel radioligands are not available, the affinity of monoamine uptake blockers to the nAChR channel was recognized only occasionally (66,74). Our data suggest that the nAChR antagonism is not a sporadic phenomenon among monoamine uptake blockers but a general characteristic of these compounds. Is there a significance of our finding? It has been shown that long term potentiation (for a review see 81), a mechanism implied in the genesis of memories, has some relationship the nAChR (82,83,84,85,86), the monoamine uptake blockers could then by their antinicotinic effect blockage of the receptor interfere in the mechanism of the long term potentiation and therefore in the generation of new memories. Human studies on depressed patients indicate that the plasma concentration of different monoamine uptake blockers is about 1-2 µM (87,88,89,90). Taking into account that the CSF/plasma ratio for DMI is about 10 % (87) and the concentration of DMI in the brain is much higher than in the plasma (88) these compounds (or some of them) may reach a concentration in the brain which can alter the function of nAChRs. Since these receptors play a crucial role in cognitive functions (91), the effect of monoamine blockers on nAChRs may contribute to the development of their therapeutic and/or side effects.

7. - Conclusion 7.1. Differential effect of nicotinic agonists on the [3H]noradrenaline release from rat hippocampal slices Our data indicate that different mechanisms are involved in the effect of nicotinic agonists on the hippocampal NA release from rat brain slices (Table 1): S The majority of nicotinic agonists (nicotine, cytisine, epibatidine, anatoxin-A) increases the vesicular release of NA through stimulation of nAChRs located on noradrenergic varicosities. S DMPP has a dual action because, in addition to the nAChR-mediated mechanism, it is able to induce also a carrier-mediated release through the reversal of NA transporter. S Lobeline has no effect on nAChRs, i.e. it is not a nicotinic agonist under our experimental conditions, but similarly to DMPP, it is able to induce a carrier- mediated release. In addition lobeline has a further stimulatory effect on the hippocampal NA release, the mechanism of which is presently unknown. S Our results show that DMPP and lobeline, independently of nAChRs, are able to substantially increase the release of monoamines, therefore special caution is required for the interpretation of data, when these compounds are used as nicotinic agonists

7.2. Nicotinic antagonist effect of monoamine uptake blockers S We provided evidence that monoamine uptake blockers with different chemical structure and selectivity are able to inhibit the function of nAChRs in the central nervous system. S Based on our data and literature we propose that the mecahanism of action, similarly to that of mecamylamine, is a channel blocker-type antagonism. S This finding may reveal some structural similarity between the ion channels of transporters and nAChRs. These results may help to understand the functional properties of monoamine transporters and have important clinical implications. The monoamine uptake blockers are widely used in the therapy of depressed patients, therefore our findings may have great importance in the understanding of therapeutic and adverse effects of these drugs.

8. – Acknowledgments I would like to thank so many people for the successful completion of this scientific work. They were always friendly and helpful with. Thanks to the Professor Vizi for his always useful and intelligent insights about my work and for his immense patience with my human defects. An intelligent mind taught so many things about science. Thanks to my immediate boss (and friend) Dr. Janos Kiss for his guidance and teaching. We had interminable and fruitful discussions about science and the route of our research. He taught me that the most important thing in science is thinking. Thanks to all my colleagues in the Institute for being always helpful and for their invaluable and multiple suggestions for my work. And finally thanks to my father and mother for helping me grow up and mature.

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