P.1.g.082 Involvement of melatonin in the serotonin synthesis mediated by CYP2D in the brain Anna Haduch, Ewa Bromek, Jacek Wójcikowski, Krystyna Gołembiowska, Władysława Anna Daniel Polish Academy of Sciences, Institute of Pharmacology, Kraków, Poland BACKGROUND Melatonin ip. Apart from the classic pathway of serotonin formation from L-5-hydroxytryptophan, serotonin may also be Melatonin deacetylation 5-methoxytryptamine synthesized via the cytochrome P450 2D (CYP2D)-catalyzed O-demethylation of 5-methoxytryptamine (5-MT). (5-MT) The reaction of serotonin regeneration from 5-methoxytryptamine closes the cycle of melatonin-serotonin biochemical transformation. The reaction has been demonstrated in vitro for cDNA-expressed CYP2D isoforms 5-MT and brain microsomes derived from different rat brain structures [2, 3] and in vivo in the brain using a microdialysis model [1]. 5-methoxytryptamine, direct substrate for CYP2D to form serotonin is synthesized via CYP2D deacetylation from melatonin , which can be formed endogenously and administered as a drug. 5-MT Serotonin The aim of the present study was to demonstrate that melatonin supported (via deacetylation to 5-MT) CYP2D- O-demethylation mediated synthesis of serotonin from 5-MT. METHODS 1. The effect of intraperitoneal administration of melatonin on tissue serotonin concentration in rats. To diminish the formation of serotonin from tryptophan (the classic pathway), rats were lesioned with 5,7-dihydroxytryptamine (5,7-DHT), administered to the main raphe nuclei projecting to the front of the brain. Ten days after the lesion, melatonin (100 mg/kg ip) was administered to sham-operated and lesioned rats, pretreated or not pretreated with the MAO inhibitor pargyline (75 mg/kg ip) 30 min prior to MEL. 2. The prevention by quinine of the melatonine-produced elevation of tissue serotonin concentration. Rats received the CYP2D inhibitor quinine (150 µg/5µl icv. bilaterally) 30 min prior to MEL. 1 hour after administration of melatonin rats were decapitated and brain structures were isolated. 3. Microdialysis: mesurement of extracellular serotonin concentration. The microdialysis probes were implanted into the striatum. Melatonin (100 mg/kg ip) and/or propafenone – CYP2D inhibitor - (50 µM, given using microdialysis probes), were administered to naïve or pargyline-pretreated rats (pargyline 75 mg/kg i.p., 60 min before melatonin). Dialysate fractions were collected for 2 hours [3]. RESULTS Fig. 1. The effect of intraperitoneal melatonin on tissue serotonin concentration in brain structures. Frontal cortex Rest of cortex Hippocampus Nucleus accumbens Striatum 1500 800 2000 1500 sham lesioned sham lesioned ! sham lesioned sham lesioned sham lesioned ! ! 600 ] ] ] ] ] ! ! ! + + + ^ ! 600 ! 1500 + + + + + + tissue tissue tissue tissue tissue ^ + + + ! + + + 1000 + + + 1000 ! + + + + + 400 / mg / mg / mg / mg / / mg mg / ! $ + 400 1000 pg pg pg pg pg ! $ $ ! $ $ $ $ 500 + 500 $ $ + + + 200 200 $ 500 + + + $ Serotonin [ Serotonin [ Serotonin [ Serotonin [ Serotonin Serotonin [ Serotonin $ $ + + + 0 0 0 0 0 MEL PARG MEL+ MEL PARG MEL+ MEL PARG MEL+ MEL PARG MEL+ MEL PARG MEL+ MEL PARG MEL+ MEL PARG MEL+ MEL PARG MEL+ MEL PARG MEL+ MEL PARG MEL+ PARG PARG PARG PARG PARG PARG PARG PARG PARG PARG Thalamus Hypothalamus Brain stem Medulla oblongata Cerebellum 1500 1500 3000 sham 1000 120 sham lesioned sham lesioned lesioned sham lesioned sham lesioned ] ] ] ] ^ ] 800 ! ^ + + + + + + tissue tissue tissue tissue ! tissue 1000 + + + 2000 80 1000 $ ! $ $ $ + + + + + + 600 $ / mg / mg / mg / mg / / mg / + + + $ $ ! + $ $ ! $ pg pg pg pg pg $ + + + $ + + + 400 + + + $ $ 500 500 1000 $ 40 $ $ + + + + $ $ Serotonin [ Serotonin [ Serotonin [ Serotonin [ Serotonin Serotonin [ Serotonin 200 + + + $ $ + + + $ 0 0 0 0 0 MEL PARG MEL+ MEL PARG MEL+ MEL PARG MEL+ MEL PARG MEL+ MEL PARG MEL+ MEL PARG MEL+ MEL PARG MEL+ MEL PARG MEL+ MEL PARG MEL+ MEL PARG MEL+ PARG PARG PARG PARG PARG PARG PARG PARG PARG PARG +p < 0.05; ++p < 0.001; +++p < 0.001 (versus sham-operated rats) ; ^p < 0.05 (versus sham + PARG) ; $p < 0.05; $$p < 0.001; $$$p < 0.001 (versus lesioned animals); !p < 0.05; !!p < 0.001; !!!p < 0.001 (versus lesion + PARG group) Fig. 2. The effect of intracerebral quinine on the melatonine-induced increase in serotonin tissue content in Fig. 3. The effect of melatonin administration on brain structures. extracellular serotonin level in the striatum. 4000 Microdialysis ) control Hippocampus Striatum Thalamus Hypothalamus Brain stem Cerebellum 3500 PARG 75 mg/kg i.p. 600 500 1500 1000 1200 120 level MEL 100 mg/kg i.p. # # # # # # 3000 # # # ** ** PARG 75 mg/kg i.p. + PFN 50 uM ** * * * ] ] ] ] ] 400 * 1200 * * * ] 750 basal 2500 PARG 75 mg/kg i.p. + MEL 100 mg/kg i.p. 400 800 of tissue tissue tissue 80 tissue tissue tissue PARG 75 mg/kg i.p. + PFN 50 uM + MEL 100 mg/kg i.p. 300 900 2000 / mg mg / mg / mg / / mg / mg / mg / 500 pg pg pg pg pg pg 1500 200 600 PFN and/or MEL 200 400 40 1000 PARG 250 100 300 Serotonin (% Serotonin Serotonin [ Serotonin [ Serotonin [ Serotonin Serotonin [ Serotonin [ Serotonin Serotonin [ Serotonin 500 0 0 0 0 0 0 0 Control QUIN MEL QUIN+MEL Control QUIN MEL QUIN+MEL Control QUIN MEL QUIN+MEL Control QUIN MEL QUIN+MEL Control QUIN MEL QUIN+MEL Control QUIN MEL QUIN + MEL -60 -40 -20 0 20 40 60 80 100 120 140 160 180 200 220 240 min SUMMARY CONCLUSIONS 1. Melatonin increased tissue serotonin content in the brain structures of sham- •The presented results indicate that melatonin deacetylation operated or 5,7-DHT-lesioned rats. Pargyline alone potently increased to 5-methoxytryptamine supports the CYP2D-catalyzed serotonin concentration in the brain. The effect of melatonin given jointly with serotonin synthesis from 5-methoxytryptamine in the brain the MAO inhibitor on serotonin content was observed mainly in the brains of lesioned rats. in vivo. 2. The specific CYP2D inhibitor quinine, prevented the melatonin-evoked elevation in tissue concentration of serotonin in the most of the brain •The metabolism of exogenous melatonin to the structures studied. monoaminergic neurotransmitter serotonin may be 3. Melatonin administered intraperitoneally to animals pretreated with pargyline, regarded as a newly recognized additional component of its markedly increased extracellular concentration of serotonin in the striatum. pharmacological action. The CYP2D inhibitor propafenone given intrastructurally, prevented the melatonin-induced increase in serotonin concentration. REFERENCES [1] Haduch, A., Bromek, E., Kot, M., Kamińska, K., Gołembiowska, K., Daniel, W.A., 2015. The cytochrome P450 2D- mediated formation of serotonin from 5-methoxytryptamine in the brain in vivo: a microdialysis study. J Neurochem 133, 83- ACKNOWLEDGEMENTS 92. Financial support comes from Grant DeMeTer no. POIG010102-12-004/09 and statutory funds from the [2] Haduch, A. Bromek, E., Sadakierska-Chudy, A., Wójcikowski, J., Daniel, W.A., 2013. The catalytic competence of Institute of Pharmacology, PAS. cytochrome P450 in the synthesis of serotonin from 5-methoxytryptamine in the brain: an in vitro study. Pharmacol Res 67, 53-59. [3] Yu, A.M., Idle, J.R., Byrd, L.G., Krausz, K.W., Küpfer, A., Gonzalez, F.J., 2003. Regeneration of serotonin from 5- The authors declare that they are not engaged in any conflict of interest. methoxytryptamine by polymorphic human CYP2D6. Pharmacogenetics 13, 173-181. .