Characterisation of serotonin transport mechanisms in rainbow trout peripheral blood lymphocytes: role in PHA-induced lymphoproliferation

\ PERGAMON

Developmental and Comparative Immunology 12 "0888# 26Ð49

Characterisation of serotonin transport mechanisms in rainbow trout peripheral blood lymphocytes] role in PHA!induced lymphoproliferation Francžois Ferrierea\\ Na(m A[ Khanb\ Jean!Philippe Meynielc\ Pierre Deschauxc a

b

Universite de Rennes!0\ UPRES!A 5915 ! Equipe E[M[R[\ Campus de Beaulieu ! Ba¼t] 02\ 24931 Rennes\ cedex\ France Universite de Bourgogne\ Unite de Nutrition Cellulaire et Me tabolique\ BP 399 ! 8 Av[ Savary\ 10900 Dijon\ cedex\ France c Laboratoire de Physiologie\ Unite d|Immuno!Physiologie Ge ne rale et Compare e\ Universite de Limoges\ Faculte des Sciences\ 012 Av[ Albert Thomas\ Limoges\ 76959\ France Received 0 April 0887^ accepted 0 August 0887

Abstract In this study\ we investigated the serotonin transport mechanisms in rainbow trout "Oncorhynchus mykiss# peripheral blood lymphocytes[ We have observed that the transport of serotonin is a membrane transport process that have the properties of a secondary active transport system[ The binding isotherm of ð2HŁ!paroxetine\ a serotonin transport blocker\ demonstrated a high!a.nity binding site with a positive type of cooperativity\ Hill coe.cient being higher than unity[ Known speci_c inhibitors of the mammalian serotonin transporter signi_cantly inhibited the uptake process in _sh lymphocytes[ In order to demonstrate the physiological relevance of the serotonin transporter in T!cell activation\ we conducted experiments on lymphocytes activated or not by phytohemagglutinin "PHA#\ a T!cell mitogen[ We have observed that addition of PHA for 13 hrs\ increased the Vmax but not the Km of this transporter[ Serotonin uptake inhibitors diminished the PHA!activated proliferation of _sh lymphocytes[ The intracellular concentrations of cAMP were found to regulate the serotonin uptake and the PHA!stimulated proliferation as the agents known to augment cAMP stimulated serotonin uptake\ and inhibited the lymphoproliferation[ Inhibitory e}ects of increased cAMP on the proliferation were reversed by the addition of the nanomolar concentrations of 7!OH!DPAT\ a 4!HT0A receptor agonist which is known to diminish the intracellular cAMP concentrations\ suggesting that serotonin also regulates PHA! induced proliferation via 4!HT0A membrane receptors in an autocrine manner[ These results all together demonstrate that _sh lymphocytes possess an active serotonin transporter that is implicated in the proliferation of these immuno! competent cells[ Þ 0888 Elsevier Science Ltd[ All rights reserved[ Keywords] Serotonin^ Transporter^ Rainbow trout^ Cell proliferation^ 4!HT0A

0[ Introduction Serotonin "4!hydroxytryptamine# has been found to in~uence several functions of the immune system

 Corresponding author[ Tel[] "22#!91!88!17!50!23^ fax] "22#! 91!88!17!56!83^ e!mail] francois[ferriereÝuniv!rennes0[fr[

ð0Ł\ notably activation of NK!cells\ macrophages\ T!cells and pre!B lymphocytes in mammals ð1Ð7Ł and activation of T!cells in _sh species ð8Ł[ Modu! lation of the immune system by serotonin occurs via membrane receptors\ i[e[ 4!HT0A and 4!HT2\ present on lymphocytes ð8Ð01Ł[ Moreover\ the mammalian lymphoid organs such as spleen and thymus are innervated with serotonergic and

9034!294X:88:, ! see front matter Þ 0888 Elsevier Science Ltd[ All rights reserved[ PII] S 9 0 3 4 ! 2 9 4 X " 8 7 # 9 9 9 3 0 ! X

27

F[ Ferriere et al[ : Developmental and Comparative Immunolo`y 12 "0888# 26Ð49

catecholaminergic neurons\ suggesting that leuko! cytes are directly exposed to 4!HT ~ow\ even at early stage of di}erentiation ð02Ł[ Another way to in~uence the functions of immune cells will be the uptake of this biogenic amine from the extracellular environment[ A high a.nity sero! tonin transporter\ sensitive to inhibition by a selec! tive group of antidepressants\ has been reported in di}erent cell types such as platelets ð03Ł\ endothelial cells ð04Ł\ intestinal brush!border vesicles ð05Ł and human placental choriocarcinoma cells ð06Ł[ In platelets\ the serotonin transporter participates in the active uptake of serotonin for intracellular storage[ In immune cells\ the serotonin transporter is expressed in murine splenocytes and peritoneal macrophages ð07Ł[ Recently\ Faraj et al[ ð08Ł have shown that human lymphocytes possess a high a.nity serotonin transporter[ However\ no study is available on the characterisation of serotonin transport mechanisms in _sh lymphocytes[ There! fore\ the present research was undertaken to ascer! tain the characteristics of serotonin transport mechanisms in rainbow trout peripheral blood lym! phocytes[ The possible similarities between activity and regulation of 4!HT in both the central nervous and the immune systems will certainly shed light on the possible role of serotonin in the course of activation:proliferation of the immune cells[

1[ Materials and methods

ð2HŁ!thymidine "19 Ci:mmol# were procured from Amersham Radiochemicals\ France[ ð2HŁ!paroxetine "06[0 Ci:mmol# was purchase from NEN!Dupont\ France[ Ficoll!Paque was obtained from Pharmacia Chemicals\ France[ 1[1[ Experimental animals Rainbow trout "Oncorhynchus mykiss# ranging in weight from 049 to 149 g and length from 07 to 11 cm were obtained from a local hatchery "M:S SKIBA\ Haute!Vienne\ France# and maintained in 0999 l indoor circular glass _ber tanks with a con! stant renewal of water during 3 weeks before experi! mentation[ They were fed 4 times weekly ad libitum[ 1[2[ Isolation of peripheral blood lymphocytes To avoid stress\ _sh were anesthesized by placing into water tank containing 1!phenoxyethanol "9[2 ml:l# and bled from the caudal vein using a heparin! ized syringe[ Blood was then quickly diluted with 4 volumes of culture medium "RPMI 0539 Dutch modi_ed containing 09999 mg:ml streptomycin and 09999 UI:ml penicillin# and layered onto an equal volume of Ficoll!Paque[ After 39 min of centri! fugation at 0999 g\ at 3>C\ the interface containing peripheral blood lymphocytes "PBL# was collected\ washed twice "09 min at 799 g# and resuspended in culture medium[ The cell viability of isolated lymphocytes was assessed by trypan blue exclusion test[

1[0[ Chemicals 1[3[ Serotonin uptake measurement Culture medium Leibovitz "L04#\ RPMI 0539 "Dutch modi_ed#\ foetal bovine serum "FBS#\ streptomycin\ penicillin\ 1!mercaptoethanol were obtained from Gibco\ France[ 4!HT in the form of hydrochloride\ R!"¦#!7!hydroxy!1!"di!n!propyl! amino#!tetralin "7!OH!DPAT#\ alaproclate\ zimeli! dine\ 7!Bromo!cAMP\ parachlorophenylalanine "pCPA#\ and Pindobind!4!HT0A were purchased from Research Biochemicals\ Inc[\ USA[ PHA\ Hepes\ pargyline\ forskolin\ cholera toxin were obtained from Sigma\ USA[ Fluoxetine\ paroxetine and sertraline were procured from Merck\ USA[ 2! isobutyl!0!methylxanthine "IBMX# was purchased from Calbiochem[ ð2HŁ!4!HT "10 Ci:mmol# and

The PBL cell density was adjusted to 0×095 cells: ml in the uptake bu}er containing the following] NaCl\ 039[2 mM^ KCl\ 4 mM^ CaCl1\ 0 mM^ MgCl1\ 0 mM^ Hepes\ 19 mM^ Glucose\ 09 mM and Na1HPO3\ 0 mM\ pH 6[3[ All the assays were per! formed in this bu}er[ To probe the Na¦ dependency of serotonin uptake\ Na¦ was iso!osmotically replaced by choline chloride from the uptake bu}er[ In order to prevent serotonin metabolism by monoamine!oxydase\ pargyline "9[0 mg:ml# was added to the bu}er[ The transport parameters were studied by incubating the cells during 04 min at 19>C in the loading bu}er in the presence of increas!

F[ Ferriere et al[ : Developmental and Comparative Immunolo`y 12 "0888# 26Ð49

ing concentrations of ð2HŁ!4!HT "9 to 53 mM#[ Sero! tonin transport kinetics "Km and Vmax# were obtained by a non!graphical method of Wilkinson "Enzpack Software\ Biosoft# which also provided the standard error:deviation of kinetic constants[ For competition experiments\ the cells were incu! bated during 04 min at 19>C in the uptake bu}er containing ð2HŁ!4!HT "014 nM#\ in the presence or absence of drugs at the concentrations indicated in the legends[ For studies on the implication of cAMP in the uptake process\ the cells were preincubated for overnight in culture medium[ The cell viability was then assessed by trypan blue exclusion test[ Cells were washed twice "799 g\ 09 min# and resus! pended in the uptake bu}er containing ð2HŁ!4!HT "014 nM#[ All the experiments were performed for 04 min in order to shed light on the initial kinetics of the transporter[ These studies were conducted at 19>C as it the optimal temperature to perform experiments on rainbow trout lymphocytes ð19Ł[ The speci_c uptake of ð2HŁ!serotonin was de_ned as the di}erence between the total uptake and non speci_c accumulation of 2H!4!HT during 04 min in the presence of 19 mM of sertraline\ a 4!HT uptake inhibitor[ After incubation\ the cells were quickly washed "799 g×09 min# twice with ice cold bu}er containing alaproclate "19 mM#\ a 4!HT uptake inhibitor[ The _nal pellets were dissolved in 0 M NaOH[ 2 ml of liquid scintillation "Pico Flour 39\ Packard# were added and the radioactivity was rec! orded in a scintillation counter "Packard#[

1[4[ Radioligand binding assays In order to demonstrate the presence of a sero! tonin transporter in the plasma membrane of rain! bow trout PBL\ ð2HŁ!Paroxetine binding was assessed[ ð2HŁ!Paroxetine binding assays were per! formed in ~at bottom 85!well tissue culture plates[ Cells were incubated at 0×095 cells:assay in a total volume of 199 ml of phosphate bu}er saline "PBS Dubelcco|s#\ pH 6[3\ in the presence of increasing concentrations of ð2HŁ!Paroxetine "9 to 09 nM# with "non!speci_c binding# or without "total binding# the speci_c uptake inhibitor\ sertraline "099 mM#[ Incubations were performed at 19>C for 39 min as the equilibrium reached during this period[ Cells

28

were then collected onto _berglass _lters "00624 _lters\ Skatron\ U[S[A[# using a cell harvester "Skatron# with four washes of cold PBS\ pH 6[3[ Washing of each _lter took less than 19 s[ Dried _lters were placed in plastic minivials and the radio! activity was counted "after adding 2 ml of Opti! ~uor!O# in a scintillation counter[ Speci_c binding was measured by subtracting the non!speci_c bind! ing from the total binding[ The dissociation con! stant "Kd#\ the transporter density "Bmax# and the Hill coe.cient "nH# were calculated according to Scatchard calculations using a computer assisted IBM!RADLIG software "Biosoft\ Cambridge\ U[K[#[

1[5[ Cell culture and lymphoproliferation assay The lymphocytes were cultured in the L04 medium\ supplemented with 4) heat!inactivated FBS and 1!mercapto!ethanol "4×09−4 M#[ The PBL cell density was adjusted to 1[4×095 cells:ml[ Lymphocytes were cultured in 85!well tissue culture plates "Nunc# in the presence of PHA at 9[4 mg:ml as we know that this concentration of mitogen is optimal for rainbow trout lymphocytes prolifera! tion "data not shown#[ Cells were distributed in triplicate at 4×094 cells:well[ The total volume per well was 199 ml[ Plates were incubated at 1921>C "89) humidity#[ After 85 hrs\ 19 ml of ð2HŁ!thy! midine "19 Ci:mmol\ 9[7 mCi:well# was added and\ 01 hrs later\ the cells were harvested using an auto! matic cell harvester "Basic 85 Harvester Skatron#\ trapping their DNA onto glass _ltermats[ Dried _lter circles were placed in plastic minivials "Packard#\ 2[4 ml Opti!~uor O "Packard# was added and the radioactivity was recorded in a scintillation counter "Packard#[ The results were expressed as Stimu! lation Index\ calculated for each animal and for each triplicate as follows] S[I[ mean cpm stimu! lated culture:mean cpm unstimulated culture[ For indication\ cpm for PHA!stimulated cultures and unstimulated cultures as mean 2 SD are 2775[422 201[86 and 401[622087[2 respectively[ When cultures where made in L04 medium non complemented with FBS\ cpm for PHA!stimulated cultures and unstimulated cultures as mean 2 SD are 0091[932 026[8 and 024[11207[2 respectively[

39

F[ Ferriere et al[ : Developmental and Comparative Immunolo`y 12 "0888# 26Ð49

2[ Results 2[0[ Serotonin transport is a Na¦!dependent process Figure 0 demonstrates the time!dependent accu! mulation of serotonin in rainbow trout peripheral blood lymphocytes[ When NaCl was present in the uptake bu}er\ the transport of 4!HT at the con! centration of 014 nM was very rapid and increased exponentially[ However\ when NaCl in the bu}er was replaced with choline chloride\ uptake of sero! tonin was reduced markedly at all time points and was linear over 29 min of incubation[ The initial uptake rates measured with a 09 minutes incubation were only 39) in the presence of choline chloride as compared to the rates in the presence of NaCl[ These data demonstrate clearly the dependency of serotonin uptake on extracellular Na¦[ 2[1[ Serotonin transport is adaptively regulated Parachlorophenylalanine "pCPA# is an inhibitor of the tryptophan!hydroxylase and it is used in vivo or in vitro to deplete the intracellular pool of sero! tonin[ The lymphocytes were incubated for 13 hrs in the presence of pCPA "4 mM#[ We observed that the speci_c transport of serotonin was higher in the pCPA treated cells than that in untreated controls\ suggesting that the uptake is adaptively regulated "Figure 1#[

Fig[ 1[ E}ect of preincubation with pCPA on serotonin uptake in rainbow trout PBL[ Rainbow trout PBL were preincubated overnight in culture medium in the presence of pCPA "4 mM#[ The cell viability was then assessed by trypan blue exclusion test[ Cells were washed twice and the uptake of serotonin was measured as described in Materials and Methods[ The con! centration of ð2HŁ!serotonin in the uptake bu}er was 014 nM[ Values are mean 2SD of three separate experiments with 3 _shes per experiment[ Data were analysed by employing Student|s t! test of signi_cance[ Values di}er signi_cantly as compared to the control\ p³9[9994 "#[

2[2[ Serotonin transporter is a membrane\ but not a vesicular\ localised system Two types of serotonin transport mechanisms have been described so far] one is localised in the plasma membrane and the other is present in the cytoplasmic storage granules[ These two mech!

Fig[ 0[ Time course of serotonin uptake in rainbow trout PBL[ Uptake of serotonin was measured in the medium containing either NaCl or choline chloride as described in Materials and Methods[ The concentration of ð2HŁ!serotonin in the uptake bu}er was 014 nM[ Values are mean 2SD of three separate experiments with 3 _shes per experiment[ Data were analysed by employing Student|s t!test of signi_cance[ Values di}er signi_cantly\ p³9[94 "#[

F[ Ferriere et al[ : Developmental and Comparative Immunolo`y 12 "0888# 26Ð49

anisms are distinct and di}er in many aspects[ The plasma membrane serotonin transporter is several fold more sensitive than the storage granule trans! porter to inhibition by antidepressants[ On the con! trary\ reserpine is a speci_c inhibitor of the storage granule serotonin transporter with no or minimal e}ect on the plasma membrane transporter[ In fact\ reserpine is used as a speci_c high a.nity ligand for the transporter in the storage granules ð10Ł[ In order to demonstrate that the uptake of serotonin by _sh lymphocytes is not mediated via a vesicle trans! porter\ we elucidated the e}ect of reserpine on sero! tonin uptake in these cells[ Figure 2 shows that reserpine failed to inhibit signi_cantly the serotonin transport in _sh lymphocytes[ In order to demonstrate the presence of serotonin transporter in the plasma membrane of rainbow trout PBL\ ð2HŁ!paroxetine binding was assessed[ The association equilibrium of ð2HŁ!paroxetine binding to the serotonin transporter was reached after 24 min of incubation at 19>C "data not shown#[ The speci_c binding of ð2HŁ!paroxetine to the serotonin transporter was saturable[ However\ the binding isotherms followed in a sigmoid manner "Figure 3#\ and the Scatchard plot provided a con! cave curve "Figure 3\ Inset#[ The transporter density "Bmax# and the dissociation constant "Kd# were\ respectively\ 0[63×09−0929[85×09−09 mol:0×095 cells and 6[3520[32 nM[ The nH value was higher than the unit "nH1[96529[22#[

30

Fig[ 3[ ð2HŁ!paroxetine binding to _sh PBL[ Fish PBL were isolated and incubated "0×095 cells:assay# with increasing con! centrations of ð2HŁ!paroxetine "9 to 09 nM# with "non speci_c binding# or without "total binding# sertraline "099 mM# for 39 min as described in Materials and Methods[ Cells were then collected onto glass!_bre _lters by using a cell harvester and the radioactivity was counted in a scintillation counter[ The speci_c binding was measured by subtracting the non!speci_c binding from the total binding as described in Materials and Methods[ Values are mean 2SD of three separate experiments with 3 _shes per experiment[ Inset] The Scatchard plot of paroxetine bound "B# vs bound:free "B:F# obtained from a computer assisted IBM!RADLIG software "Biosoft\ Cambridge\ U[K[#[

2[3[ Serotonin uptake inhibitors inhibit the uptake process in _sh lymphocytes

Fig[ 2[ E}ect of reserpine on serotonin transport in rainbow trout PBL[ Serotonin uptake was measured in the presence of di}erent concentrations of reserpine "9\ 4 or 09 mM# as described in Materials and Methods[ The concentration of ð2HŁ!serotonin in the uptake bu}er was 014 nM[ Values are mean 2SD of three separate experiments with 3 _shes per experiment[

Several antidepressant agents such as alaproclate\ zimelidine\ ~uoxetine\ paroxetine and sertraline act as potent inhibitors of serotonin uptake in di}erent cell systems ð03Ð05Ł[ We\ therefore\ assessed the e}ects of these inhibitors on serotonin uptake in rainbow trout PBL[ All of these agents signi_cantly inhibited serotonin uptake in a concentration depen! dent manner "Figure 4#[ Fluoxetine\ paroxetine and sertraline are the most e.cient inhibitors of 4!HT uptake in our cells[ They signi_cantly inhibited the uptake process after the _rst concentration we tested "4 mM#[ Neither alaproclate\ nor zimelidine

31

F[ Ferriere et al[ : Developmental and Comparative Immunolo`y 12 "0888# 26Ð49

Fig[ 4[ Inhibition of serotonin transport by uptake inhibitors in rainbow trout PBL[ The serotonin uptake was measured as described in Materials and Methods in rainbow trout PBL in the presence or absence of various serotonin uptake inhibitors at di}erent concentrations "9^ 4^ 09 or 19 mM#[ The concentration of ð2HŁ!serotonin in the uptake bu}er was 014 nM[ Values are mean 2SD of three separate experiments with 3 _shes per experiment[ Values di}er signi_cantly as compared to the respective control\ p³9[4 "#\ p³9[94 "# and p³9[994 "#[

are able to inhibit the uptake when used at 4 mM and inhibition measured at 09 and 19 mM were lower than the inhibition obtained with ~uoxetine\ paroxetine and sertraline[ The serotonin trans! porter in _sh lymphocytes thus seems to be similar to the transporter described in the plasma mem! brane of platelet\ brain and brush!border mem! brane of the placenta in term of sensibility to the inhibitors ð03Ð05Ł[ 2[4[ Serotonin transport pathway is implicated in PHA!induced lymphoproliferation In _sh lymphocytes\ the serotonin transport pro! cess followed the hyperbolic Michaelis!Menten kin! etics "Figure 5#[ In unstimulated lymphocytes\ the Km and Vmax values\ calculated by the non graphic method of Wilkinson\ are 5[3529[405 mM and 15[129[526 pmol:04 min:095 cells\ respectively[ In lymphocytes stimulated by PHA for 13 hours\ the Vmax but not the Km of serotonin transport is increased "Table 0#[ These results show that sero! tonin transport pathway is upregulated during T! cell activation[ The next set of experiments was performed to determine whether serotonin transport may regu! late rainbow trout T!cell proliferation[ All the sero! tonin uptake inhibitors were found to inhibit PHA! stimulated lymphoproliferation "Figure 6#[ Signi_! cant inhibition was observed with ~uoxetine\ par! oxetine and sertraline at the concentration of

Fig[ 5[ Concentration dependence of serotonin transport in rain! bow trout PBL[ The serotonin uptake was measured on resting PBL in the presence of increasing concentration of ð2HŁ!4!HT "9Ð53 mM# for 04 min as described in Materials and Methods[ Kinetics parameters "Km and Vmax# were obtained by the non! linear graphical method of Wilkinson using Enzpack Software "Biosoft#[ Values are mean 2SD of three separate experiments with 3 _shes per experiment[

9[0 mM whereas the other uptake inhibitors required much higher concentrations to produce signi_cant inhibition[ 2[5[ Inhibition of serotonin synthesis inhibits PHA! induced lymphoproliferation Rainbow trout PBL were cultured in the presence of PHA and pCPA with or without 4!HT for 4 days "Figure 7#[ pCPA was added to the cells after 13 hrs

F[ Ferriere et al[ : Developmental and Comparative Immunolo`y 12 "0888# 26Ð49

32

Table 0 E}ect of PHA stimulation on serotonin transport parameters Uptake parameters Treatment of cells

Km mM

Vmax pmol:04 min:095 cells

Resting cells PHA

5[3529[9405 7[4520[01

15[129[526 30[720[68 "#

The PBL were incubated 13 hours in the presence of PHA "9[4 mg:ml# in culture medium[ The cell viability was then assessed by trypan blue exclusion test[ Cells were washed twice and serotonin uptake was measured in the presence of increasing concentrations of ð2HŁ!4!HT "9Ð53 mM# for 04 min as described in Materials and Methods[ Kinetics parameters "Km and Vmax# were obtained by the non!linear graphical method of Wilkinson using Enzpack Software "Biosoft#[ Values are mean 2SD of three separate experiments with 3 _shes per experiment[ Value di}er signi_cantly as compared to control\ p³9[994 "#[

of culture of the 4 days culture period[ Under these conditions\ pCPA inhibited PHA!dependent lym! phocyte proliferation\ and this inhibition was reversed by the addition of exogenous 4!HT in the culture medium[ 2[6[ Serotonin transport and PHA!induced lympho! proliferation are regulated by intracellular cAMP concentrations ] role of 4!HT0A membrane receptors To elucidate the regulation of serotonin transport\ we investigated the in~uence of agents that are known to elevate intracellular cAMP levels[ We employed the following agents^ cholera toxin\ responsible for the ADP!ribosylation of the a subunit of the stimu! latory G protein "Gs# which results in the stimulation of adenylate!cyclase to generate cAMP^ forskolin\ an activator of adenylate!cyclase^ IBMX\ an inhibitor of cAMP degradation by inhibiting cyclic nucleotide phosphodiesterases and 7!Bromo!cAMP\ a mem! brane!permeable cAMP derivative[ The cells were incubated for 13 hrs in the presence of these agents at the concentrations indicated in the legends and the initial serotonin transport rate was determined in these cells "Figure 8#[ The initial rate of serotonin uptake was found to be stimulated markedly by cholera toxin in a concentration dependent manner "Figure 8A#[ For! skolin also stimulated serotonin transport to the

same extent as cholera toxin when used at 19 ng:ml[ IBMX and 7!Bromo!cAMP also stimulated the serotonin transport "Figure 8B# but the stimulation was lesser as compared to the e}ects of cholera toxin and forskolin[ In mammals\ elevation of intracellular cAMP is known to partially inhibit T!cell proliferation in response to a variety of stimuli[ Figure 09 shows that\ in rainbow trout\ T!cell proliferation "stimu! lated by PHA# was signi_cantly curtailed by addition of forskolin "09 mM#[ This inhibition was reversed by addition of either 4!HT or 7!OH! DPAT[ At 0 nM\ the proliferation was completely restored by 7!OH!DPAT[ 4!HT or 7!OH!DPAT alone did not in~uence the PHA!stimulated T!cell proliferation "Fig[ 09#[ In the absence of the FBS in the culture medium\ 4!HT and 7!OH!DPAT signi_cantly stimulated PHA!induced proliferation when used between 9 to 299 nM "Figure 00#[ Pindobind!4!HT0A is thought to be a selective antagonist of 4!HT0A receptor sub! type ð11Ł[ Figure 00 shows that stimulatory e}ects of 7!OH!DPAT were abolished by pindobind!4! HT0A "09 mM#\ suggesting the implication of the 4! HT0A receptor subtype in this activation of pro! liferation by serotonin[ The PHA!induced stimulation indices of _sh T! lymphocytes were less pronounced in cells cultured in the absence of foetal bovine serum[

33

F[ Ferriere et al[ : Developmental and Comparative Immunolo`y 12 "0888# 26Ð49

Fig[ 6[ E}ects of serotonin uptake inhibitors on PHA!stimulated lymphoproliferation[ Rainbow trout PBL were cultured in the presence of PHA "9[4 mg:ml# for 4 days as described in Materials and Methods with the indicated concentrations of 4!HT uptake inhibitors alaproclate\ zimelidine\ ~uoxetine\ paroxetine and sertraline "9 to 09 mM#[ Cultures were incubated with ð2HŁ!thymidine "9[7 mCi:well# for 01 hrs on day 3 before cells were harvested\ trapping their DNA onto glass _bre _lter mats and the radioactivity was measured in a scintillation counter[ Results are expressed as Stimulation Index "mean 2SD# of three separate experiments with 3 _shes per experiment[ Values di}er signi_cantly as compared to the respective control\ p³9[4 "#\ p³9[94 "# and p³9[994 "#[

Fig[ 7[ E}ects of pCPA on PHA!stimulated lymphoproliferation[ Rainbow trout PBL were cultured in the presence of PHA "9[4 mg:ml# and pCPA "4 mM# for 4 days with or without 29 mM 4!HT[ Cultures were incubated with ð2HŁ!thymidine "9[7 mCi:well# for 01 hrs on day 3 before cells were harvested\ trapping their DNA onto glass _bre _lter mats and the radioactivity was measured in a scintillation counter[ Results are expressed as Stimulation Index "mean 2SD# of three separate experiments with 3 _shes per experiment[ Values di}er signi_cantly as compared to the respective control\ p³9[4 "#\ p³9[94"#[

3[ Discussion The present study shows that rainbow trout "Oncorhynchus mykiss# peripheral blood lympho! cytes possess a serotonin transport system[ 4!HT uptake was found to be Na¦!dependent[ In fact\ the presence of Na¦ in the form of electrochemical

gradient contributes to a possible source of energy required for the transport process as it has been considered as a rule in a number of cells ð00\ 06\ 08\ 12Ł[ The depletion of intracellular pool of 4!HT by an inhibitor of serotonin biosynthesis\ para!chloro! phenylalanine "pCPA#\ increased the uptake of serotonin in these cells\ demonstrating that the

F[ Ferriere et al[ : Developmental and Comparative Immunolo`y 12 "0888# 26Ð49

34

Fig[ 8[ E}ects of cholera toxin\ forskolin\ IBMX and 7!bromo!cAMP on serotonin uptake[ Rainbow trout PBL were preincubated overnight in culture medium in the presence of "A# cholera toxin "9^ 4^ 09 or 19 ng:ml#\ "B# forskolin "Fk# "09^ 099 mM#\ IBMX "099 mM# or 7!bromo!cAMP "099 mM#[ The cell viability was then assessed by tryphan blue exclusion test[ Cells were washed twice and serotonin uptake was measured as described in Materials and Methods[ The concentration of ð2HŁ!4!HT in the uptake bu}er was 014 nM[ Mean values 2SD of three separate experiments with 3 _shes per experiment[ Values di}er signi_cantly as compared to the respective control\ p³9[94 "# and p³9[994 "#[

transport process is adaptively regulated[ It is not the _rst report of an adaptively regulated transport of a biogenic amine] the transport of polyamines has been shown to be enhanced by deprivation of cells of their intracellular polyamine contents by incubating in the presence of polyamine biosyn! thesis inhibitors ð13Ł[ All of these properties\ satu! rable uptake\ sodium dependency and sensitiveness to metabolic inhibition are the characteristics of a secondary active transport system of serotonin in _sh lymphocytes ð14Ł[ Reserpine is used as a speci_c high a.nity ligand for the transporter in the storage granules[ In our

cells\ serotonin uptake was found to be insensitive to reserpine\ indicating that serotonin uptake in _sh lymphocytes was a reserpine!insensitive plasma membrane transporter[ The speci_c binding of ð2HŁ! paroxetine to _sh lymphocytes was saturable\ and this agent labels a high a.nity binding site[ The speci_c binding of ð2HŁ!paroxetine followed in a sigmoid manner "Fig[ 3# as the _xation of a molecule facilitated the _xation of the others[ Fixation was then saturable after 7 nM of ð2HŁ! paroxetine[ The {{bell|| shape Scatchard plot dem! onstrated the density "Bmax# and the dissociation constant "Kd# of the transporter to be

35

F[ Ferriere et al[ : Developmental and Comparative Immunolo`y 12 "0888# 26Ð49

Fig[ 09[ E}ects of forskolin on PHA!stimulated lymphoproliferation[ Rainbow trout PBL were cultured in the presence of PHA "9[4 mg:ml# for 4 days as described in Materials and Methods[ After 13 hrs\ cultures received forskolin "Fk# "09 mM# with or without the indicated concentrations of 4!HT or 7!OH!DPAT[ Cultures were incubated with ð2HŁ!thymidine "9[7 m Ci:well# for 01 hrs on day 3 before cells were harvested\ trapping their DNA onto glass _bre _lter mats and the radioactivity was measured in a scintillation counter[ Results are expressed as Stimulation Index "mean 2SD# of three separate experiments with 3 _shes per experiment[ Values di}er signi_cantly compared with their respective control\ p³9[4 "# and p³9[94 "#[

Fig[ 00[ E}ect of serotonin on PHA!stimulated lymphoproliferation[ Rainbow trout PBL were cultured in the presence of PHA "9[4 mg:ml# for 4 days with or without the indicated concentrations of 4!HT or 7!OH!DPAT or 7!OH!DPAT¦pindobind!4!HT0A "09 mM#[ Cultures were made in L04 medium non complemented with FBS[ Cultures were incubated with ð2HŁ!thymidine "9[7 mCi:well# for 0 hr on day 3 before cells were harvested\ trapping their DNA on glass _bre _lter mats and the radioactivity was measured in a scintillation counter[ Results are expressed as Stimulation Index "mean 2SD# of three separate experiments with 3 _shes per experiment[

F[ Ferriere et al[ : Developmental and Comparative Immunolo`y 12 "0888# 26Ð49

0[63×09−0929[85×09−09 mol:0×095 cells and 6[3520[32 nM\ respectively[ The nH value was higher then unit "nH1[96529[22#\ indicating positive type of cooperativity[ These observations are in accordance with the recent _ndings of Lima and Schmeer ð15Ł who have described the identical pro_le of ð2HŁ!paroxetine binding to serotonin transporter in gold_sh "Carassius auratus# retina[ Similar to mammalian cells ð06Ð16Ł\ known sero! tonin uptake inhibitors which we have tested inhibited signi_cantly the serotonin uptake in rain! bow trout lymphocytes[ As in human lymphocytes\ ~uoxetine and paroxetine are most potent than zimelidine and alaproclate to inhibit ð2HŁ!4!HT uptake ð16Ł[ In fact\ most of the attention on the serotonin transporter has so far been focused on the role of the transporter in serotonergic neural transmission[ Recent studies have indicated that the serotonin transporters of the neuronal and non neuronal tissues may be di}erent[ There are indica! tions that the nonneuronal and the neuronal sero! tonin transporters may be under separate genetic control ð17Ł^ these are di}erent proteins which may be regulated di}erently[ Di}erent intracellular second messengers\ acti! vated during lymphocyte activation\ can modify the 4!HT uptake process[ There is evidence in di}erent cell types for the regulation of serotonin transporter by cellular signalling pathways such as Ca1¦ ð14\ 18Ł\ protein kinase C ð18Ð21Ł and cAMP ð05\ 06\ 22Ł[ In human placental choriocarcinoma cells\ the serotonin transporter is upregulated by cAMP and the regulation involves transcriptional activation as treatment of these cells with cholera toxin has stimulatory e}ects on the serotonin transporter activity\ on the transporter density and on the steady state levels of the transporter speci_c mRNAs ð05\ 06\ 22Ł[ The stimulatory e}ects of cholera toxin and forskolin on serotonin transport activity are e}ectively reduced when cellular transcriptional and translational processes are blocked with actino! mycin D and cycloheximide ð22Ł[ Thus\ the mech! anism of the cAMP!dependent regulation of the serotonin transporter is clearly distinct from that of the PKC!dependent regulation of the trans! porter[ The PKC!regulation of the transporter is faster and implicates phosphorylation:dephosphory! lation of the transporter[ The cAMP!regulation is

36

much slower and involves a transcriptional process[ Two hours exposure of _sh lymphocytes to PHA exerted no in~uence on uptake parameters "data not shown#\ rather stimulation by PHA for 13 hours induced a signi_cant increase in the initial transport rate with an insigni_cant decrease in the a.nity[ These observations suggest that the underlying mechanism is not a phosphorylation of the trans! porter\ rather a stimulation of de novo synthesis of the transporter[ Furthermore\ serotonin uptake activity seems to be under the regulation of cAMP in our cells as cholera toxin\ known to elevate the levels of intracellular cAMP\ stimulated the sero! tonin transport in a dose!dependent manner[ At 19 ng:ml\ cholera toxin increased the uptake by approximately 1[9429[14 fold as compared to con! trol[ The e}ect of cholera toxin on serotonin trans! port can be mimicked by forskolin\ IBMX and 7! Bromo!cAMP[ These results are in agreement with other investigators who have reported that IBMX\ dibutyryl!cAMP and forskolin mimic the action of cholera toxin\ eliciting a 0[5Ð1[4 fold stimulation of the serotonin transport activity in human placental choriocarcinoma cells ð06Ł[ The exact mechanism responsible for the stimulation of serotonin trans! port by cAMP has not been investigated in our study^ however\ since these agents elevate the intra! cellular levels of cAMP\ the protein kinase!A is most likely involved in this regulatory process ð06Ł[ Beside the intracellular synthesis\ another source of serotonin will be the uptake of this biogenic amine from the extracellular environment[ Inhibi! tion of T!cell proliferation by pCPA and its reversal by exogenous 4!HT suggest that 4!HT is implicated in T!cell proliferation stimulated by PHA[ In this study\ _sh T!lymphocyte proliferation was found to be sensitive to each of the serotonin uptake inhibitors[ Signi_cant inhibition of the T!cell pro! liferation were observed with ~uoxetine\ paroxetine and sertraline in nanomolar concentrations\ whereas the other uptake inhibitors required much higher concentrations to produce signi_cant inhibition[ These inhibitions are not due to the toxicity of the drugs "data not shown#[ These results suggest the implication of 4!HT transport in rainbow trout T! cell proliferation[ Fluoxetine and other speci_c serotonin uptake inhibitors have become the drugs of choice for treating a}ective disorders and

37

F[ Ferriere et al[ : Developmental and Comparative Immunolo`y 12 "0888# 26Ð49

depression or its treatment with antidepressant agents may have an impact on the normal function of the immune system[ However\ only a limited number of studies have addressed the e}ects of these inhibitors on immune cell functions[ It has been shown that acute ~uoxetine administration "09 mg:kg# in rats resulted in a dose! and time! dependant decrease in mitogen!induced lympho! cyte proliferation and natural killer cell cytolytic activity in vitro ð23Ł[ These e}ects were no longer observed following chronic ~uoxetine administra! tion suggesting that an apparent tolerence of cell! mediated immunity had been developed ð23Ł[ It has also been demonstrated that tricyclic antidepres! sants inhibit IL!5\ IL!0b and TNFa release in human blood monocytes and IL!1 and INFg in T! cells in vitro ð24Ł[ On human natural killer cell! mediated cytolysis\ antidepressants did not decrease e}ector!target cell conjugaison formation\ nor did they induce target cell resistance to NK lysis but the drugs might interfere with the killing mechanism of the e}ector cells ð25Ł[ More recently\ it has been reported that tricyclic antidepressants induce apoptosis in human peripheral lymphocytes ð26Ł[ So\ this transport system is a potential target for abused drugs and antidepressants[ But\ on the other hand\ antidepressants seem to reverse the stress!induced immunomodulation] Mice exposed to chronic auditory stressor and treated with ~uoxetine showed a reduction in stress!induced suppression of thymus and spleen cellularity and peripheral T lymphocyte population ð27Ł[ It has also been reported that forced swim test exposure in the rat activates the HPA!axis and produces alterations in both cellular and non cellular immunity in rats[ The stress!induced behavioral changes were normalized by tricyclic antidepressants treatment and\ further! more\ there is evidence to suggest that pretreatment with antidepressants has protective e}ect against stress!induced immune changes ð28Ł[ However\ the question to be addressed is that by which mechanism the uptake inhibitors inhibit the T!cell proliferation[ Hence\ it is possible that under normal culture conditions\ the foetal bovine serum may be the source of exogenous serotonin\ and deprivation of the exogenous serotonin by these agents will exert the antiproliferative e}ects[ Gilles Fillion at the Pasteur Institute "Paris# has detected

serotonin in considerable quantities in the foetal bovine serum "unpublished results\ personnel com! munication#[ It is also possible that stimulation of _sh lymphocytes with PHA may stimulate the endogenous serotonin synthesis and its release in the extracellular environment[ Hence\ addition of antidepressants in the medium inhibits the reuptake of serotonin and the accumulation of 4!HT in the environment of the cells will become prejudicial to lymphocyte proliferation[ There are several reports which show that mammalian T!cells\ after stimu! lation by PHA or interferon!gamma\ de novo syn! thesise and secrete serotonin in the extracellular medium ð39Ł[ Whether _sh lymphocytes de novo synthesise and secrete serotonin remains to be eluci! dated in future[ However\ in our study we have observed that addition of exogenous serotonin stimulates the PHA!induced T!cell proliferation in absence of foetal bovine serum[ Under physio! logical conditions\ there are many sources which contribute to augment circulating serotonin] baso! phils and mast cells secrete this biogenic amine in the extracellular medium ð7Ł and stressful con! ditions induce hyperactivity of serotonin metabolism in brain and blood ð30Ł[ We have observed that elevation of intracellular cAMP concentrations was found to reduce rainbow trout T!cell proliferation "see above#[ Forskolin inhibited T!cell proliferation stimulated by PHA by approximately 49)[ Addition of exogenous 4!HT or 7!OH!DPAT\ a 4!HT0A receptors agonist\ at nanomolar concentrations reversed forskolin! mediated inhibition of T!cell proliferation[ It is also noteworthy that these two serotonergic agonists alone at these low concentrations exerted no e}ects on _sh T!cell proliferation[ We have pharmaco! logically demonstrated the presence of 4!HT0A receptors in rainbow trout PBL after mitogenic stimulation ð8Ł[ In the central nervous system as well as in periphery\ 4!HT0A receptors regulate adenylate cyclase activity\ and 4!HT0A agonists like 7!OH! DPAT\ inhibit activation of adenylate cyclase induced by agents such as forskolin ð31Ł[ In our study\ the reversal of the forskolin!induced inhibi! tion of T!cell proliferation by 7!OH!DPAT is pos! sibly mediated via diminution of the adenylate cyclase activity through 4!HT0A high a.nity bind! ing site expressed in PHA!stimulated rainbow trout

F[ Ferriere et al[ : Developmental and Comparative Immunolo`y 12 "0888# 26Ð49

lymphocytes ð8Ł[ The stimulatory action of 7!OH! DPAT is abolished by an antagonist of this recep! tor\ pindobind!4!HT0A\ demonstrating that 4!HT0A receptors are functionally implicated in _sh T!cell proliferation[ The high stimulatory e}ects of 7!OH! DPAT in comparison with 4!HT may be due to the fact that high a.nity agonists such as 7!OH!DPAT stimulate signal transduction at concentrations that only label the high a.nity site[ However\ 4!HT is recognised by a variety of 4!HT receptors and is taken up through speci_c uptake mechanisms[ We can also infer the _ndings of several investigators ð32\ 33Ł who have demonstrated that in pancreatic aciner cells and _broblasts\ serotonin is de novo synthesised and it regulates the growth of these cells via 4!HT0B membrane serotonergic receptors through an autocrine loop[ The present study clearly demonstrates that\ dur! ing T!cell proliferation in rainbow trout lympho! cytes\ there is a close relationship among serotonin transporter activity\ adenylate cyclase and expres! sion of 4!HT0A receptors] serotonin transport mech! anism\ adaptively regulated\ is coupled with in! creases in cAMP concentrations which are decreased by serotonin via 4!HT0A receptors present on _sh lymphocytes[

Acknowledgement Authors are thankful to the French Ministry of Defence for the sanction of a contingent grant[

References ð0Ł Plaut M[ Lymphocyte hormone receptors[ Ann[ Rev[ Immunol[ 0876^4]510Ð518[ ð1Ł Hellstrand K\ Hermodsson S[ Role of serotonin in the regulation of human natural killer cell cytotoxicity[ J[ Immunol[ 0876^028]758Ð764[ ð2Ł Hellstrand K\ Hermodsson S[ Monocyte!mediated sup! pression of IL!1!induced NK!cell activation[ Regulation by 4!HT0A!type serotonin receptors[ Scand[ J[ Immunol[ 0889^21]072Ð081[ ð3Ł Sternberg EM\ Trial J\ Parker CW[ E}ect of serotonin on murine macrophages] suppression of Ia expression by serotonin and its reversal by 4!HT1 serotonergic receptor antagonists[ J[ Immunol[ 0875^026]165Ð171[ ð4Ł Sternberg EM\ Wedner HJ\ Leung MK\ Parker CW[ E}ect

ð5Ł

ð6Ł

ð7Ł

ð8Ł

ð09Ł

ð00Ł

ð01Ł

ð02Ł

ð03Ł

ð04Ł

ð05Ł

ð06Ł

ð07Ł

ð08Ł

ð19Ł

ð10Ł

38

of serotonin "4!HT# and other monoamines on murine macrophages] modulation of interferon!gamma induced phagocytosis[ J[ Immunol[ 0876^027]3259Ð3257[ Choquet D\ Korn H[ Dual e}ects of serotonin on a voltage! gated conductance in lymphocytes[ Proc[ Natl[ Acad[ Sci[ USA 0877^74]3446Ð3452[ Bonnet M\ Lespinats G\ Burtin C[ Histamine and serotonin suppression of lymphocyte response to phytohemagglutinin and allogenic cells[ Cell[ Immunol[ 0873^72]179Ð180[ Aune TM\ Kelley KA\ Ranges GE\ Bombara MP[ Sero! tonin!activated signal transduction via serotonin receptors on Jurkat cells[ J[ Immunol[ 0889^034]0715Ð0720[ Ferriere F\ Khan NA\ Troutaud D\ Deschaux P[ Serotonin modulation of lymphocyte proliferation via 4!HT0A recep! tors in rainbow trout "Oncorhynchus mykiss#[ Dev[ Comp[ Immunol[ 0885^19]162Ð172[ Jackson JC\ Cross RJ\ Walker RF\ Markesbery WR\ Brooks WJ\ Roszman TL[ In~uence of serotonin on the immune response[ Immunology 0874^43]494Ð401[ Khan NA\ Ferriere F\ Deschaux P[ Serotonin!induced cal! cium signalling via 4!HT0A receptors in human leukemia "K 451# cells[ Cell Immunol[ 0885^054]037Ð041[ Meyniel JP\ Khan NA\ Ferriere F\ Deschaux P[ Identi! _cation of lymphocyte 4!HT2 receptor subtype and its implications in _sh T!cell proliferation[ Immunol[ Lett[ 0886^44]040Ð059[ Bellinger DL\ Felten SY\ Felten DL[ Maintenance of non! adrenergic sympathetic innervation in the involuted thymus of the aged Fisher 233 rat[ Brain Behav[ Immunol[ 0877^1]022Ð049[ Olson PS\ Ljungvist U\ Bergentz SE[ Analysis of platelet\ red cell and _brin content in experimental arterial and venous thrombi[ Thromb[ Res[ 0863^4]0Ð3[ Myers CL\ Lazo JS\ Pitt BR[ Translocation of protein kinase C is associated with inhibition oh 4!HT uptake by cultured endothelial cells[ Am[ J[ Physiol[ 0878^146]L142Ð L147[ Ramamoorthy S\ Cool DR\ Mahesh VB\ Leibach FH\ Gan! apathy V[ Regulation of the human serotonin transporter[ J[ Biol[ Chem[ 0882^157]10515Ð10520[ Cool DR\ Leibach FH\ Bhalla VB\ Mahesh VB\ Ganapathy V[ Expression and cyclic AMP!dependant regulation of a high a.nity serotonin transporter in the human placental choriocarcinoma cell line "JAR#[ J[ Biol[ Chem[ 0880^155] 04649Ð04646[ Jackson JC\ Walker RF\ Brooks WH\ Roszman TL[ Spec! i_c uptake of serotonine by murine macrophages[ Life Sci[ 0877^31]0530Ð0542[ Faraj BA\ Olkowski ZL\ Jackson RT[ A high a.nity sero! tonin transporter in human lymphocytes[ FASEB J[ 0882^6]A153ÐA161[ Ferriere F\ Khan NA\ Meyniel JP\ Deschaux P[ Serotonin induced calcium!channel gating in rainbow trout "Oncorhynchus mykiss# peripheral blood lymphocytes[ Biochem[ J[ 0886^212]140Ð147[ Stern!Bach Y\ Greenberg!Ofrath N\ Flechner I\ Schuldiner S[ Identi_cation and puri_cation of a functional amine

49

ð11Ł

ð12Ł

ð13Ł

ð14Ł

ð15Ł

ð16Ł

ð17Ł

ð18Ł

ð29Ł

ð20Ł

ð21Ł

ð22Ł

F[ Ferriere et al[ : Developmental and Comparative Immunolo`y 12 "0888# 26Ð49 transporter from bovine chroma.n granules[ J[ Biol[ Chem[ 0889^154]2850Ð2855[ Liau LM\ Sleight AJ\ Pitha J\ Peroutka SJ[ Charac! terisation of a novel and potent 4!hydroxytryptamine 0A receptor antagonist[ Pharmacol[ Biochem[ Behav[ 0880^27] 444Ð448[ Declan WA\ Orchard I[ The uptake and release of serotonin and dopamine associated with locust "Locusta migratoria# salivary glands[ J[ Exp[ Biol[ 0885^088]588Ð698[ Khan NA\ Quenemer V\ Moulinoux JP[ Characterization of polyamine transport pathways[ In] Carter C\ editor[ Neuropharmacology of Polyamines[ Academic Press\ 0883\ pp[ 26Ð42[ Takayanagi S\ Hanai H\ Kameko E[ Ca1¦:calmodulin and protein kinase C!dependent signal transduction a}ect sero! tonin uptake in rat jejunal enterocytes[ Biomed[ Res[ 0882^03]092Ð094[ Lima L\ Schmeer C[ Characterization of serotonin trans! porter in gold_sh retina by the binding of ð2HŁ!paroxetine and the uptake of ð2HŁ!serotonin] modulation by light[ J[ Neurochem[ 0882^51]417Ð424[ Faraj AB\ Olkowski ZL\ Jackson RT[ Active 2H!dopamine uptake by human lymphocytes] correlates with serotonin transporter activity[ Pharmacology 0883^37]219Ð216[ Ieni JR\ Tobach E\ Zukin SR\ Barr GA\ Van Pragg HM[ Multiple 2H!Imipramine binding sites in brains of male and femal Fawn!Hooded and Long!Evans rats[ Eur[ J[ Pharmacol[ 0874^001]150Ð153[ Khan NA\ Meyniel JP\ Deschaux P[ Ca1¦:calmodulin and protein kinase C regulation of serotonin transport in human K451 lymphocytes[ Cell[ Immunol[ 0885^061]158Ð163[ O|Brien TG\ Saladik D[ Regulation of hexose transport in BALB:C 2T2 preadipose cells] e}ects of glucose con! centration and 01!O!tetradecanoylphorbol!02!acetate[ J[ Cell[ Physiol[ 0871^001]265Ð273[ O|Brien TG\ George K\ Prettyman R[ Protein!kinase C and membrane transport] divergent responses of Na¦:K ¦:Cl− cotransport and sugar transport to exogenous diacyl! glycerols[ Biochem[ Biophys[ Acta 0877^834]30Ð49[ Nishizuka Y[ The molecular heterogeneity of protein kinase C and its implications for cellular regulation[ Nature 0877^223]550Ð554[ Ramamoorthy JD\ Ramamoorthy S\ Papapetropoulos A\

ð23Ł

ð24Ł

ð25Ł

ð26Ł

ð27Ł

ð28Ł

ð39Ł

ð30Ł

ð31Ł

ð32Ł

ð33Ł

Catravas JD\ Leibach FH\ Ganapathy V[ Cyclic AMP! independent up!regulation of the human serotonin trans! porter by staurosporine in choriocarcinoma cells[ J[ Biol[ Chem[ 0884^169]06078Ð06084[ Pellegrino TC\ Bayer BM[ Modulation of immune cell func! tion following ~uoxetine administration in rats[ Pharmacol[ Biochem[ Behav[ 0887^48]040Ð046[ Xia Z\ DePierre JW\ Nassberger L[ Tricyclic anti! depressants inhibit IL!5\ IL!0 beta and TNF!alpha release in human blood monocytes and IL!1 and interferon!gamma in T cells[ Immunopharmacol[ 0885^23]16Ð26[ Xiao L\ Eneroth P[ Tricyclic antidepressants inhibit human natural killer cells[ Toxicol[ Appl[ Pharmacol[ 0885^026] 046Ð051[ Xia Z\ DePierre JW\ Nassberger L[ Modulation of apop! tosis induced by tricyclic antidepressants in human peri! pheral lymphocytes[ J[ Biochem[ Mol[ Toxicol[ 0887^01] 004Ð012[ Freire!Garabal M\ Nunez MJ\ Losada C\ Pereiro D\ Riv! eiro MP\ Gonzalez!Patino E\ Mayan JM\ Rey!Mendez M[ E}ects of ~uoxetine on the immunosuppressive response to stress in mice[ Life Sci[ 0886^59]PL392ÐPL302[ Connor TJ\ Kelly JP\ Leonard BE[ Forced swim test! induced endocrine and immune changes in the rat] e}ect of subacute desipramine treatment[ Pharmacol[ Biochem[ Behav[ 0887^48]060Ð066[ Finocchiaro LM\ Arzt ES\ Fernandes!Castelo S\ Criscuolo M\ Finkielman S\ Nahmod VE[ Serotonin and melatonin synthesis in peripheral blood mononuclear cells] stimu! lation by interferon!g as part of an immunomodulatory pathway[ J[ Interferon Res[ 0877^7]694Ð605[ Fritsche R\ Reid SG\ Thomas S\ Perry SF[ Serotonin! mediated release of catecholamines in the rainbow trout Oncorhynchus mykiss[ J[ Exp[ Biol[ 0882^067]080Ð193[ Aune TM\ McGrath KM\ Sarr T\ Bombara MP\ Kelley KA[ Expression of 4!HT0A receptors on activated human T!cells[ J[ Immunol[ 0882^040]0064Ð0072[ Ishizuka L\ Beauchamp RD\ Towensen JC\ Greley GH\ Thompson JC[ Receptor!mediated autocrine growth stimu! latory e}ects of 4!hydroxytryptamine on cultured human pancreatic carcinoid cells[ J[ Cell[ Physiol[ 0881^049]0Ð4[ Seuwen K\ Magnaldo J\ Pouyssegur J[ Serotonin stimulates DNA synthesis in _broblasts acting through 4!HT0B recep! tors coupled to a Gi protein[ Nature 0877^224]143Ð145[