FK506 and rapamycin: novel pharmacological probes of the immune response

TiPS - \une 1991 /Vol. 221 whereas FKS06 (and cyclosporin

FK506 and rapamycin:novel pharmacological probesof the immuneresponse Joseph Y. Chang, Suren N. Sehgal and Catherine C. Bansbach T/wirtwrrtr~c systmisrrnintricate arrderstood.

Novel

network of circuitry which is incompletely

tools are needed to unravel the

releuance of even the

smallest componerrb of this network. While the clinical potential ofFK.506 and rapamyci,l as selective inrnarnoslrppressanb is the major reuson for their rltrrent imporfance, preclinical studies described here by Joseph Chang and colleagues have already suggested several provocative ideas which may revise Our biochemical concepts of T-cell activation. With the combination o/ nrolccular and cellular studies, the insightsaained with FKSO6/rapamycin may lead to a better understanding oj the biochemical circuits that are inaolord in the imnnrne response. Ultimately, studies may lead to the identification of un cndogenrxts bamlrnostrppressiue ligand that mimics FK506 or rapamycin. Within the past two years, two ma&ides with potent immunosuppressive activity, FK% and

rapamycin, have been shown to be extremely effective in prolonging the survival of organ allografts and preventing the onset of autoimmune disease in animals (see Box). Both drugs inhibit T-cell proliferation but appear to exert their actions at different steps of T-cell activation. FK506,like cyclosporin A, counteracts mitogenic or antigenic stimulation at an early stage, preventing transcription of IL-2 and other ear!y response genes, whereas rapamycin intervenes in events more closely related to DNA synthesis. The cyclosporin A binding protein, cyclophilin, has recently been shown to possess cis-trans peptidy1 prolyl isomerase activity’,’ and a similar, but not identical, isomaase that binds both FK5@’ and rapamycin has subsequently been described5. On the basis of these data, it has been proposed that a class of binding proteins with isomerase activity (immunophilins) may account for the

A) czn ccmpMely inhibit T-cc!; activation following a mitogenic stimulus only when added during the early stages of activation (O-3 hours), rapamycin is effective even when added 6 hours after stimulation”. This suggests that cyclosporIn A and IX!%6 specifically inhibit celIs from proceeding fmm resting (&) into G1 of the cell cycle while rapamycln inhibits cell division later in G,, prior to S phase12. Immunosupptession at the Go phase by cyclospori~ A can be correlated with inhibition of a T-cell cytoplasmic activation signal. It has been proposed that this cell activator of DNA replication (ADR) in T cells serves as an intracellular

mitogenic

Si&pP?

However, FICSU and rapw\ycin’s minimally affect ADR, suggesting that these macroIldes are most likely n&mining ceII cycIe division by a mechanism(s) independent of ADR. Regardless of the mechanism, it would appear that after commitment to activation, T cells become relatively insensitive to cycIosporin A and FK%l6whereas rapamycln is still able to exert an immunosuppressive activity of cyclosporin A, FK!506 and rapaeffect. mycin. This review attempts to Apart from acting at different stages of cell division, the imsynthesize and integrate recent molecular and cellular data into a munosuppressive effect of cydosporin A, FK506 and rapamyc!In mechanistic framework that may guide future studies of this invaries considerably, depending on the manner by which T and B triguing class of agent. Where cells are activated. Rapamycin relevant, reference to cyclosporin suppresses a wider spectrum of TA is made to provide an appropriate perspective for the pharmaand B-cell activation pathways cology of the macrolides, FK506 than FK!506 or cydosporln A. and rapamycin. While Cd+-dependent T-cell proliferation (e.g. ionom ‘n plus the T-cell proliferation Induced by phorbol ester, PMA)X? and B-cell agonists proliferation (antl-IgM)*6am sensiFKSO6and rapamycin potently tive to cycIosporln A, FK506 and inhibit the proliferation of animal rapamycin, inhibition of both and human T lymphocytes and TCa2+-dependent and Caz+-indecell clonesin responseto a variety pendent (e.g. PMA alone, antiof stimuli. For example, concanCD28 or lipoPoIysac&rlde, LPS) avalin A-induced clonal expanpathways could be achieved only sion is markedly suppressed at with rapamycin 0. E. Kay, pers. subnanomolar concentrations that commun.). On the other hand, are several orders of magnitude T-cell proliferation induced by a lower than that of cyclosporin Abe’. high supraoptimal concanavalin A T-cell responses to specific anticoncentration (5 pg ml-‘) has gens, allogeneic lymphocytes and been shown to be incompletely other Wins an also reduced by inhibited by rapamycin, whereas these macroKdes7-‘0. In addition FK!% and cydosporin A -will to having a direct inhibitory completely abrogate the T-cell action on responding cytotoxic response’0. This apparent difT-lymphocyte precursors, FK506 ference in efficacy between rapaalso impairs the abflity of alIomycin and FK506 appears to antigen presentir cells to activate partially depend on the agonist these precursors *% . Interestingly, concentrations used to activate T

TiPS - lune 7991[Vol. 121

219

In vho phtmmcokqy

ofFK506and rapamycin

+amydn sod FKu)6 we pmduced by Streptomyces hygrmwpicussnd Sfmptomycestsukubtunsis, respectively (Fig.).lnbrestin mpamycinand FK!U ta the dir& result of the remarkabledfecta these ager.ta dirpkyinmodctsofoqankan+antationandaw i--&b.. lul--rl -ccI--. & _--I uLuuuBU.y. YI”Vx.zn, Vr “1wCMlD*UIU.uru, U3--_ LIczq .“E paaweastran@antationhaabeendemonstratedin aarIycIinka!t&Is. At l’ittsbwgh’, FK!%b was sllccesb)fullyusedinl4livergraftrecspia&iwherenoneof thesepatientssI&redreJsction.AdditioMlJy,thesame studyshowedthatnoreJectionwasobserWdinpstients whohadreceivedakidneyorpanaeasgnift.Alarger study of 36 renal transplant mcipientsr continned the potencyofPKslb;&sesaslowss0.l5mgLg-’were effective.Arecentnumograph&cussesthe&nical a@encewithPK5Mmoreexttnsi~.Todate,no dir&al data are available with rcqxunycir~ Since nprmrclnhWlIOtkmd~hthedini~thiS raaQn~inrtcrdOncDmpathnnimrlphumrCdogybehvenmo6andRpwlrlntoplwideabrief

PemPaWe0ftheimmunaauPPresdwactivityofthe hvo macroltdss under similsr in ufvoconditions.

by rapamycin or lX.506 at doses that are, again, markedly lower than those ofcydosPorin A. However, the dog is ~ecuWy sensitive to FK% and npamyrin in terms of side effects;gastrointestinal ubxmtions and vasclditishavebeenseeninthiasPeciesathighdoses. I ___. LL 1._ _Il__i -- .--- . ..- - h .---WI. ~pUt-.I8 BlecsonoqJan~,rlwuosnd rsPamydnsmak3oe&ctiveinsnimalmod&3of autobnmune dkieawsgu. The mauoitdes inhibit Prokinurir, Prevent pqwion of nvphroprtfiy and bnprovelikspaninrmutinernodeidqmtaniclupua eIytbenlatoaua(MiuJipralouse). In addition,rapmnytin, but not FKSW,reduceathe devatad auwnt%ody titra in theaa animals~‘. Spontan~ devdqing dtabetaain tha non-obaae diabetic mouaa baa been BothmacrolideapmducadwelatedinhiMtibnd expe&untaUy induced arthritis in rata (adjuvant arthritis)*Id mice (type n c&gan-induced a&&s). Hist&$wevi~frvmowatudiesaa~that =stiVta wt.*.? aa judgd by the Inwamcnt m the lolnt patho&y of rapamyciwtreakdantmala.Notably,rapamycinis not ananM&mmaWyagentsincent~paw edemaLminimdyafkctedbyRpunycin.

Refemlcet3 1 Star&T. E.cf a/.(19@)Lanntii,MOD-1001 2 stvd T. E.d d. (1390)I. Am.Mrd.&WC. 244,63-67 3 StamJ,T. I, Todo, S., Fun&J. J. and Croth,C. (1991) _ .__ Tma+ani.kc. a 1-w 4 ~R.&,HqnEC.,Murphp,M.P.adShorthourc,R (19s9)Tranqkl;.Pnw.21,1042-1044 5 Mtd&~a~i&l. aadSho&mw. R.(1990)Trannspf~nt. bwtakgrafbhssbcanach&vadat+kmicand&al &ass less thrn slngkg” for both drugs”‘. when ~d7tinua&fa14(*YI~sn~PUmPI suwivalofratheartgraftsatdoses u&llo.o2mgq “(Rct.7). Theanti-rejWansctivityofPK5O5andraPamycin canabobeachkvadinlarger~.survwof =ganwf@~dog(kid=y),pig~~d~)~d Prima&s (kidney and haart) is significantly incrassed

cells. For example, rapamycin com..l,r,l.. pwsry

:rl.:h~be .lllll”LID

a.“A_r.m.-...1:r u15 ~“II~(I~IP”I.111

A response in mouse splenocytes if a s&optimal concentration is used to induce T-cell proliferation. Similariy, mouse thymocyte proliferation can be reduced totally by rapamycin when phytohemagglutinin plus IL-1 are added at suboptimal concentrations. In any event, despite the structural similarities of FKSO6and rapamycin, of FKso6 paralthe pharmxol lels that of cyY osporin A rather than that of rapamycin. The data

6 C&k, R Y. et at.(1X89) LaM ii, 227 7 Stqkow&, M, Ck, H., Dabze,P. and R&WI.6. D. (WI) rm#QM&a 51.22-26 8 StJobn.CotUar, 0. d al.(Isee)Traa&rl.Pwc.20,226-228 9 Taio#9. d d. (l!Hs)sqry 104,-49 10 T~l, R. tf al. (1989)Clin.Innmcttol. IntmBaoprtkd. Sl,ll&ll7 I1 wwaar,L.M..cummolu,T. A.,Chang,J.Y.,Set+, S.N. andAdaow,L.M.(WIlJFASEB j. 4, A358

hrrther predict that rapamyciu a-., (IIIR. .LcaJ LSL~I,, .-r-.&.&rCa.CI,FY c,aba@%I--r_rdl .UI~ V. I-\=.. dysfunction that are resistant to cyclosporin A and FK506. T-cell activation and second messengers One of the fundamental mechanisms of T-cell activation involves the generation of inositol hiphosphate (IPd and Ca” mobilization”, and the possibility that the immunosuppressive effects of cyclosporin A, FK!% and rapamycin are mediated through a

;ItreC engw:

actio:. 011tiu~e XC9AiC! km

heen

::WfR-

i!wcQpt?d,

However, the data to date s%qgest that &is is unlikely. I;\ Jurkat cells’B and murine splenocytes”, inhibition of IL-2 synthesis by FK506 (and cydosporin A) is not accompanied by a reduction in intmceflular Ca*+ and 14. Even when It-2 production is completely inhibited, studies have shown that Caz+ fluxar 1P3levels remain unchanged, indicating that the site of action is subsequent to the generation of these

TiPS - \une 1991 IVol. 121

220

second messengers. While comparable data have not been generated for rapamycin, there is circumstantial evidence to suggest a similar lack of effect on early second messengers. For example, the fact that rapamycin can inhibit lymphocyte Ca2+-independent proliferation would argue against a site of action focused on IPa generation or Ca2+ flux. Moreover, since IF’s and intracellular Ca’+ must be elevated for at least 2-4 hours in T cells for optimal signal transduction2”*“, the ability of rapamycin to inhibit lymphocyte proliferation as late as 6 hours after stimulation again suggests that rapamycin must interfere at a site downstream from the generation of early second messengers in signal transduction. Although T-cell receptor occupancy and the accompanying biochemical events are necessary for T-cell proliferation, they are not sufficient. Additional co-stimulatory signals are necessary to drive the T cell to full activation”. Indeed, there are a large number of studies demonstrating that mitogens, anti-T-cell receptor and anti-CD3 do not induce T-cell proliferation in the absence of accessory signals provided by antigen presenting cells or protein kinase C activators such as PMA which may substitute for lymphokines secreted by these cells. More recently, the activation of the cell surface molecule, CD28, has also been proposed to be an additional co-stimula!ory signa123~24. Signals fmm these accessory pathways are transmitted via a biochemical pathway that does not involve IPs and Ca2+ mobilization. Since rapamycin, but not FR506 or cyclosporin A, is particularly effective agains; T-cell pmliferation induced by these signals, it is highly suggestive that one of the major targets for rapamyrin action may be the co-stimu%ry signal(s) required for fu!l acrivation. Role of cytokines The synthesis of cytokines, especially IL-2, is a prerequisite for perpetuating the complex network of events that lead to T-cell proliferation and activation. Therefore, the ability of FR506and cyclosporin A to suppress the synthesis of IL-2, IL-2 receptor, IL-3, IL-4, y-interferon, TNF-u and

GM-CSF as measured either b& bioassay9 or mRNA analysis suggests a basis for their immunosuppressive effects. The mechanism by which cyclosporin A26 and FR50@ block transcription of the IL-2 gene has been elucidated. IL-2 gene expression is dependent on the combinatorial effects of at least five proteins that bind to regulatory sequences upstream of the promoter site. FK596 and cyclosporin A have the greatest inhibitory effect on transcription driven by the interaction of the binding nuclear regulatory proteins NF-AT, NF-IL-2A and NF-IL-2B with their respective DNA binding sites. Gene activation induced by the NF-KB protein is less sensitive to these drugs, and they have no effect on genetic response to the PMAresponsive nuclear factor AP-ln. These effects are not due to direct interference with DNA binding, as addition of cyclosporin A to nuclear extracts of untreated lyrnphocytes does not interfere with the usual association of transcription factor with target DNA. However, when NF-AT from cyclosporin A-treated cells is used in this assay, no binding is observed%. This indicates that cyclosporin A affects cellular processes involved in the generation of nuclear factors that activate the IL-2 gene following T-cell stimulation. Deletion analyses have also demonstrated that the inhibitory action of cyclosporin A (and by analogy FR506) is likely to affect more than one of these transcription factors, as no single mutation within the IL-2 enhancer was able , ~n~~~~tZ3Z$X~o~~f E nucleotides from the NF-AT binding site, however, most strongly affected transcriptional sensitivity to cyclosporin A, and a deletion within the promoter-distal Ott-1 binding site decreased response to cyclosporin A as well. Unlike FR506 and cyclosporin A, rapamycin does not consistently inhibit the synthesis of IL-2 and other cytokines25, indicating that enhancer function at the level of the IL-2 gene is not likely to be affected by rapamycin. Instead, rapamycin appears to act more like a functional (but not a receptor) antagonist of cytokine action, For example, rapamycin markedly

inhibits proliferation of T cells, and T-cell clones such as CTLL-2 cells and DiO.G4 cells, in response to exogenously added IL-2 or IL-41°. In addition, y-interfemninduced LydE antigen expression in YAC ctllszB and anti-IgM- and LPSdrivtn &cell response (1. E. Ray, pens. commun.) are inhibited by rapamycin. On a cautionary note, rapamycin may inRuence in Pi00 cytokine levels through indirect secondary mechanisms even though it appears unable to directly inhibit cytokint synthesis in vitro. These pharmacological data clearly show that cyclosporin A and FKSO6, but not rapamycin, inhibit IL-2 synthesis. This observation is particularly revealing since it buttresses the apnmt that FK!506and cydosporin A are unlikely to exert an immunosuppressive effect once T cells are fully committed to activation. By contrast, the commitment step in T-cell activation is predic&d to have no impact on the ability of raparnycin to reduce T-cell pmliferation (Fig. 1). DrtlR interacR’oas While FU% and mpamycin individually pnniuce a consistent, albeit different, inhibitoxy tfftct on T-cell functional nsponsce, the combined etTects of FK!R?6 and rapamycin are more complex. For example, rapamydn anta@zes FR506-induced inhibition of T-cell prolifemtion, IL-2 synthesis and IL-2 receptor expmaion in T cells stimulated with ionomycin and PhiA=. Indeed, the aqonism by rapamycin is a&&able even when the agmt is added as late as 6 hours after the addition of FR506. It has abo been determined that a lOO-fold excess of mpamydn counteracts the Mibitory effects of FR506 on IL-2 syn thesis and DNA fmgment&ion in an anti-CD3 stimulated T-cell hybridoma, and a 3f&fold excess of rapamycin prevented FRSO6 from affecting NF-AT-dependent transcription . Reciprocally, lX506 antagonizes the ability of mpamycin to inhibit those activation pathways sensitive to mpamycin, namely the pt0BferatiW m%ponsc! by spltnic T cells to c-2 plus PMA, y-in&&on-medtated induction of Ly-6E expression in YAC and IL-2 dependent proliferation of

TiPS - lune 1991 [Vol. 121

221

antigens Ca*+ ionophores \

mitogens / PMA

t

t

0

0

activafed rcen

ectmed TceU

.... ..F-b h+ ,.*.. . . .-• *. ??? ? ? ? ?? ?

activated ? T&i

+-

.pJ.@.@ T-CELLPROLFERATtON

CTLL-2 T cell#. Competition between FK!W and rapamycin can only occur if FK506 and rapamycin share a common receptor, and there is evtdence from binding studies in YAC cells that FK506 and mpamycin do indeed compete for a common binding site28. However, the combination of equimotar concentrations of the two agents can result in considerably greater inhibition of the T-cell response than either drug alone, white antagomsm could be achieved when either macrolide is present in marked molar excess over the othw. Indeed, in stidies

and rapamycin (J. E. Kay, pets. commun.). Interestingly, cyclosporin Amediated inhibition of T-cell responses is consistently enhanced by FKEG6or rapamycin, regardless of concentrationrs. Combinations of cyclosportn A and FKSOEor rapamycin invariably result in a greater inhibition of mitogen and altoantigen-induced T-cell responses. Again this is functional evidence to suggest that the cellular element that binds cyclosporin A is distinct from the rapamycin/FKSO6 binding site.

where the eff&ct~ of FK506 and rapamycin were evahrated against the CoIIQnavalin Ares se, both potentiation and inhi r ition have been obaerv&, dependin on the relative concentrations oPI FK506

Inmmnasuppression

and

‘~first demonstrated that cyctosportn A binds to a cytoptasmic protein (subsequently named cyclophilin)29, the func-

tional significance of this binding was not known. Now cyclophilin has been shown to possess peptidy1 pr~lyl ci&rans isomerase (or rotamase) activity’,2 that catalyses the slow cis-trms isomer& ation of proline peptide bonds and accelerates slow rate limiting steps in the folding of protinecontaining proteins. Cydosporin A inhibits this rotamase activity, and further speculation that rotamase activity has physiological relevance has been fueled by the observation that FKSO6also binds to an intracellular rotamase, tentatively named FK506 binding protein (FKBPP”. Again, rotamase activity in this protein is markedly reduced in the presence of FKXW or rapamycins. Cyclophilin and FKBP do not share amino acid sequence homology and neither cyclosporin A nor FK506 binds to its non-respective protein1*2*3’. Both proteins are quite abundant (close to 0.4% of total cellular protein in Jurkat cells) and are highly conserved among species. There is evidence to indicate that cyclophilin is present in Neurospora crassa and Saccharomyces cerevisiap, and a periplasmic homolog of cyclophilin exists in Escherichia col?‘. Additionally, smaller molecular weight binding proteins for cyclosporin A and FKSO6 have been reported’-. Taken together, these data suggest the possibility that there may be a superfamily of rotamases (immunophilins) which mediates the of proline-containing folding proteins involved in cell growth. The mechanism by which rotamases catalyse the interconversion of cis and km proline dmide rotamers may involve the formation of either an enzyme nucleophile-substrate tetrahedral adduct or an enzyme-substrate twisted peptide-prolyl amide bond. Conceivably, FKBOE-,rapamycin- and cycloeporin A-induced inhibition of rotamase activity could involve the formation of a stable complex with the enzyme by either of these two mechanisms. For example, the C-8 or c-9 of rapamycin and FK!X%could form a long-lived tetrahedra! adduct in the FKBP active site. Recent NMR specWacopv studies of the FKSWFKBP comple~5, however, suggest that the a&to carbony of FK!5B6at gnnrnd state possesses the optimal chemical

TiPS - ]une 1991 !Vof. 121

-

/ h I

inactive complex X

tran5dlJctii

protein X

conformation to occupy the site of the twisted carbonyl cf ,a bound peptide substrate. Therefore, it has been hypothesized that the uketo amide of FK506 and rapamycin serves as a surrogate of the twisted amide bond of a bound proline-containing peptide. While mtamase activity of cyclophilin is inhibited by cyclosporin A, and that of FKBP by FK506 and rapamycin, it is becoming clear that the inhibition of rotamase activity does not mediate immunosuppression. The synthetic molecule, 5MBD, which contains the putative FKBP-binding domain of FK506 and rapamy&, was found to bind to FKBP and inhibit its rotamase activity without any inhibitory effect on T-cell activation%. This synthetic immunophilin @and, however, inhibited the actions of both FK506 and rapamycin at concentrations that would be predicted by their relative affinities for FKgP. These data indicate that the binding of FK506 and rapamycin to immunophilins, resulting in :onformatfonal change andlor the abtlity to acquire critical intracellular targets, is necessary for their immunosuppressive

action,

Despite the ill-defined role of rotamases in T-cell activation, the ability of FK506 and rapamycin to bind to an immunophilin while exerting different effects on functional T-cell responses is intriguing. Clearly, as described in previous sections, the effects of FK506 on functional responses resemble those of cyclosporin A rather than those of rapamycin. In an attempt to reconcile both molecular and cellular data, the following model has been proposed which argues that the immunophilin-dnrg complex and not the reduction in rotamase activity per se is critical for immunosuppression5,a7 (Fig. 2). While the result of FK506 binding is an interference in antigen receptor signals that regulate IL-2 synthesis, rapamycin binding affects signals that are triggered by IL-2 and other cytokines. This hy pothesis woultl further imply that the complex is an intermediary step in the expression of drug activity and the unique functional properties of each macrolide must be dependent on separate target proteins that are affected by FK506-immunophilin and rapamycin-immunophilin complexes.

The elucidation of these secondary proteins will comprise an interesting area for future research. Acknow~mb The autbom thank Dr B. Weichman for critical reading of the manuscript. We are also grateful to our academic coUaM&rs, especially Drs J. Kay, P, M&la, S. Metcalfe, for sharing their thoughts and data More publication. Finally, we thank WyethAyerst chemists and biologists who have played an integral part in the development of rapamycin. Refereaees

1 Takahashi, N., Hayano, T. and Suzuki, M. (19B9)Naturr 337.4n-475 2 Fischer, C., Wittmann-tiebdd, B., Lang K., Kiefhatw, 7. and Schmid, F. X. (1989)N~trwt337,476-478 3 Siekierka,J.J.,Hunll, S. H. Y.. Poe,M., Lin,C.S. and Sigal,N. H. (19es)Nature 341,~737 4 Harding. M. W., Galat, A., Uehling, D.lE.a&d_S$iber, S. L. (198R)N&we 5 B&r, B.k et al. (1990)Pmt. Nu!l AcQ~. Sri. USA 07.923l-!G35 ~.~~ 6 Kino, T. et 01. (1967) J. Antibiot.40, 124%125.5 7 Kino, T. et al. f19B7)J. Antlbiot.40, 1256-1263 8 Kay,J. E.. Ben&, C. R., Goodier,M. R., Wick, C. J. and Doe, S. A. E (19B9)

TiPS - june 1991 /Vol. 121 fmmunofogy67 473-477 9 Sawada. S., Suzuki, G., Kawase. Y..and Takaku, F. (1987) I. Inmumol. 139. 1797-1883 10 Dumont, F. J.. Staruch, M. I., Koprak. S. L., Melino, M. R. and Sigal, N. H. (1998) 1. fmertmo/.144,251-258 11 Thomas, J., Matthews, C., Carroll. R.. Loreth, R. and Thomas, F. (1990) TmnspfQntatfon49.390-396 12 MetcaRe, S. M. and Richards, F. M. (1990) Transpfantatfon49,789-802 13 Kimball, P. M., Kman, R. H. and Kahan, B. D. (1990) Tnmsplantation49, 186-191 14 Gitowski, j. K. and Cohen, S. (1983) Cell. hamsno/. 75,3fk3311 15 Kimbafl, P. M., Kerman, R. H. and K&an, 8. D. Traanspfuntation (in press) 16 Waffiser, P., Benrie, C. R. and Kay, J. E. (1989) lmmur&gy 68,434-435 17 Nisbet-Brown, E., Chcung, R. K. and Grinstein, S. (1985) Nature 316.545-5147

223 18 Bierer, B., Schreiber, S. L. and Burakoff, S. I. (1990) Transpfnrrtatiorr 49,1168-1202 19 Fujii, Y., Fujii, S. and Kaneko. f. (1990) rransphtatio~1 47, 1081-1082 20 Goldsmith, M. A. and Weiss, A. (1988) Science248,1029-1031 21 Kimball, P. M., Gamar, N. and Sell, S. (1988) Cell. fa~n~anof.113, 107-116 22 Jenkins, M. K. and Mueller, D. L. (1989) in Aduancesin Regufstionof Cell Growtb (Vol. 1) (Mond, J. J., Cambier, J. C. and Weiss, A., eds), pp. 141-172, Raven Press 23 June, C. H., Ledbetter, J. A., Gillespie, M. M., Lindstein, T. and Thompson, C. B. (1987) Mol. Cell. Biol. 7,447241 24 Ledbetter, J. A. et al. (19%) 1. famunol. 137,3299-3X5 25 Tocci, M. J. ct al. (1989) 1. Immsr~ol.143, 718-726 26 Emmel. E. A. et al. (1989) Science246, 1617-1620 27 Mattila, P. S. et nl. (1990) EMBO /. 9.

5-Hydroxytryptamine: a chameleonin the heart Pramod R. Saxena and Carlos M. Villal6n 5.HT has profound effects on cardiac rate and force in a variety of animal species, including humans. The main initial response to 5-HT is a shortlastin bradycardia, mediated via a Bezold-Jarisch-like reflex, and initiated by stimuf&ion of 5-HTz receptors present on cardiac vagal afferents. Once this bradycardia is suppressed, 5-HT induces cardiac stimulation which, true to its chat&conic nature described here by Pramod Saxena and Carlos VillaI% is mediated by different mechanisms and receptors in different vecies. in several species, including humans, coronary vasodilatation is mediated by 5-HTr-like nccptors, while both 5-HTI-like and 5-HTz receptors mediate vasoconstriction. This knowledge may lead to a better assessment of the possible role of S-HT in cardiovascular pathologies and to the development of selective 5-HT receptor ugonisfs and antagonists for therapottic usefulness in heart failure, coronary vasospasm and to avoid potential cardicc side-effects. Although an appreciable amount (0.4 pg g- *) of 5-hydroxytryptamine (5-HT) is present in the mammalian heart, the amine does not seem to play any significant physiological role’. However, in a variety of animals, including humans, 5-HT can elicit either 5vadycardia or tachycardia, and either coronary vasodilatation or vasoconstriction, depending on factors such as the presence or absence of intact autonomic nervous system or endothelium. It is perhaps not generally recognized’ that these effects are mediated in different species by different P. R. Saxena is Pro/cs.wrand Chaimmn and C. M. Villa&t is d Senior rcsrarch fellow itt Ihe Dfpwtmnit of Phvmarol~y, Frrrrlty of Medicfnc,Eraanus Unitkvsfty.PostBox 1738, 3008 DR Rottrrdrmr,Tke Netbcrtnrrds.

mechanisms, employing receptor types (Fig. 1).

several

!MTreceptors There has been dramatic progress in the classification of 5-HT receptors in the recent past. Until recently, three main types of 5-HT receptors (IHTr-like, 5-HTz and 5-HTz) had been describeds5, but now there is also evidence for the putative 5-HTI another P : &HTt-like receptors :Ef%e agonist: S-carboxamidotryptamine; antagonists: mcthiothepin and methysergide, which also block 5-fiTz receptors), !%iTz receptors (agonists: cu-methyl5-HT and DOl; antagonist: ketanserin), 5-HTJ receptors (selective agonist: 2-methyl+HT; antagonists: MDL72222, ICS205930). and ~-HT., receptors (agonists: 5-meth-

44tE5533 28 Dumont, F. 1. rt nl. (1996) 1. /rrrrrrrrrrol. 144.1418-1424 29 Handschumacher. R. E.. Hardine. M. W., Rice. J., Drugge. R. 1. a2 Speicher. D. W. (1984) Sorsrr 226, 544446 30 Harrison, R. K. and Stein, R. L. (1999) 8ioshemislrv29._~.~ 38134816 __~_ 31 Maki, N. it nf. (1990) Pmr. Nat/ Ad. Sri. USA 87,54408443 32 Tropschug, M., Barthelmess, I. B. and Neupert. W. (1989) Nrttare 342,%3-955 33 Liu, J. and Walsh, C. T. (1990) Proc.Not/ Acad. Sd. USA 87,4028-4032 34 Donnelly, j. G., Paiaszynski, E. W. and Soldin, 5.1. (19%) Clin. Clrcn. 36, 1034 35 Rosen?M. K., Stanbdaerf, R. F, Calat, A., Nakatsuka, M. and Schreiber, 5. L. (1990) Science248,863866 36 Bierer. B. E., Somers, P. K.. Wandless, T. I.. Burakoff, S. 1. and Schreiber, S. L. (1990) Srience250, 556-559

oxytryptamine, or-methyld-HT and some benzamide derivatives, including renzapride; antagonist: high concentrations of 1CS2O5930). The 5-HTr-like receptors are heterogeneous in nature as several S-HTr binding-site subtyp& have ‘been recognized (S-HTIa, 5.HT,s, 5-HTrc, S-HTm and 5-HTtz), but these may not correspond with some 5-HT,-like receptor-mediated functional response~~.~. 5-HT-induced bradycardia The initial response to 5-HT in most species with intact autonomic nervous system is an intense, but transient, bradycardia. Intravenous, intracoronary or local epicardial administration c.f 5-HT. I-phenylbiguanide and 2-methyi5-f-D elicit a short-lasting bradycardia that is effectively antagonized by destruction of the CNS, vagotomy, ganglion blockers, muscarine antagonists as well as MDL72222, ICS205930 and a variety of other 5-HTa receptor antagonists%‘“. Therefore, the 5-HTinduced bradycardia is mainly due to a Bezold-Jarisch-like reflex originating from the depolarization of afferent cardiac neurons by an effect on cardiac sensorv receptors belonging to the 5-l-iT3-type.’ Bradvcardia can also result from ather mechanisms. For example, the 5-HTIA receptor agonists N,N,dipropyl-5&OH-D?AT, carboxamidotryptamine and flesinoxan increase vagal and decrease sympathetic tone to elicit bradycardia and hypotension; such CNS effects can be inhibited by drugs that act as antagonists at