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Chapter 61: Neurohypophyseal hormone receptors: relation to behavior
A. Ermisch. R. Landgraf and H.-J. Ruhle (Eds.) Progress in Brain Research, Vol. 91
459
0 1992 Elsevier Science Publishers B.V. All rights reserved.
CHAPTER 61
Neurohypophyseal hormone receptors: relation to behavior J . Elands, E.R. de Kloet' and D. de Wied2 Max PIanck Institute for Psychiatry, 8000 Munich 40, Germany; I Department of Medical Pharmacology, Center f o r BioPharmaceutical Sciences, Leiden, The Netherlands; and Department of Pharmacology, Rudolf Magnus Institute, Utrecht, The Netherlands
Introduction Arginine vasopressin (AVP) and oxytocin (OT) exert a multitude of effects in the CNS. It is believed that these peptides are distributed via a divergent neuronal network, consisting of AVP and OT containing fibers and terminals and are mediated by specific receptors for AVP and OT. The involvement of AVP and OT in centrally mediated effects has been investigated by applying the peptides AVP, OT or their structural analogs either intracerebroventricularly (icv.) or directly into certain brain areas, or by observing regional biochemical changes following systemic, or icv. treatment. Well known examples are the antipyretic effects of AVP in the septa1 area (Kovacs and De Wied, 1983; Ruwe et al., 1985; Naylor et al., 1986), the AVP-induced cardiovascular effects mediated via the nucleus tractus solitarii (NTS) (Vallejo et al., 1984), sexual behavior induced by OT in the nucleus ventromedialis of the hypothalamus (NVM) (Arletti and Bertolini, 1985; Caldwell et al., 1986; Elands et al., 1991) and the modulation of avoidance behavior by both AVP and OT (Kovacs et al., 1979; De Wied et al., 1987, 1991). To get insight in the mechanism underlying these processes we have studied the AVP
and OT receptors in the CNS, in order to correlate the pharmacology of these receptors with their physiological or behavioral effects. The importance of this approach may be illustrated by the apparent lack of AVP and OT receptors in the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei (Elands et al., 1988d; Tribollet et al., 1988), regions where a positive feedback action of OT on its own release has been reported (Freund-Mercier and Richard, 1984). The first observation towards the characterization of AVP and OT receptors in the CNS, made by Barberis and colleagues (Audigier and Barberis, 1985), revealed the presence of two classes of binding sites on hippocampal membranes. One class was designated as AVP binding sites since it had binding characteristics comparable to the classical AVP V,, receptors in the liver and blood vessels. The other class, having a more complex binding profile due to an interaction with both AVP and OT, constituted the OT binding sites. This class appeared to have an equally high affinity for AVP as for OT. These observations prompted us to develop ligands that were more selective (Elands et al., 1988a,b,c) in order to characterize these receptors and then, by having the appropriate ligands, to examine the ap-
460
and mammary gland. Using OT, AVP and a series of structural analogs we were able to confirm that CNS OT receptors recognize AVP (Ki = 1.6 nM) and AVT (Ki = 1.9 nM) with similar affinity as OT (Ki = 1.9 nM) (Elands et al., 1988a); this property Development of selective ligands confers to these OT receptors an apparent nonselectivity in binding of the naturally occurring Essentially two lines have been followed: tritium neurohypophyseal hormones AVP and OT. In spite labeling of analogs that have a high selectivity and of this non-selective nature this receptor site is termradioiodination of analogs that have been modified ed an OT receptor. The peripheral type O T receptors in order to incorporate an iodine molecule without in the uterus and the mammary gland also had an loss of affinity. Using the first approach [Thr4equally high affinity for AVP and OT (Elands et al., Gly7]0T (Elands et al., 1988a) was tritiated. In the 1988a). OT receptors in the rat brain are generally rat this OT receptor agonist expresses a lo4 times comparable to the classical uterine type, although lower affinity for V, and V, receptors and is thus OT receptors in the NVM were found to be slightly extremely selective for OT receptors. It appeared to different (Elands et al., 1991); a somewhat lower aflabel OT receptors with high affinity (Kd = 1-2 finity for OT (Ki = 6 nM vs. 1.0 nM for uterine nM). For the other approach antagonists of the receptors), [1251]-OTA(Ki = 0.15 nM vs. 0.05 nM) AVP V, and OT receptors have been used that and [Thr4Gly7]0T (Ki = 20 nM vs. 1 nM), may could incorporate an iodine molecule without possibly indicate an OT receptor heterogeneity. dramatic changes in affinity. Thus potent anFinally, AVP receptors in the CNS seem to be extagonists (Elands et al., 1988b,c) were substituted at clusively of the V,, type; they bind AVP with a high the ninth position with a tyrosylamide residue. This affinity and OT with a 10 - 30-fold lower affinity. resulted (i) in a highly radioactive, selective OT Moreover, these AVP receptors recognize the receptor antag~nist(['~~I]-d(CH,)~[Tyr(Me)~Thr~classical pressor antagonist d(cH,)&T~r(Me)~]OrnsTyr-NH~]VT,[1251]-OTA) that did bind with AVP (which has an enhanced pressor versus anan extremely high affinity (Kd = 0.05 nM) to OT tidiuretic potency) with a high affinity (Ki = 0.5 receptors and displayed a very low non-specific nM) and the antidiuretic agonist dDAVP with a low binding and (ii) in an equally radioactive V,, anaffinity tagonist [1251]-d(CH2)5[Tyr(Me)2Tyr-NH2]AVP, (Ki > 500 nM) (Voorhuis and Elands, unpublished observations). [1251]-AVA),that did bind with a high affinity (Kd = 0.3 nM) to V,, receptors and also had a moderate Models for AVP and OT action in the brain non-specific binding. The benefit of these analogs has led to a worldA VP and temperature regulation wide use of the radioiodinated O T antagonist and to AVP induces an antipyretic response in febrile the rapid development of other radioiodinated rats (Kovacs and De Wied, 1983; Ruwe et al., 1985; analogs. Caution is, however, needed since these Naylor et al., 1986), and this effect is mediated by compounds do not necessarily bind to receptor subAVP receptors in the brain. The pharmacology of types for the endogenous hormones that have not the hypothermic action correlates well with the been demonstrated yet. binding specificity of V, receptors. OT is ineffective in doses where AVP potently affects temperature Receptor pharmacology (Kovacs and De Wied, 1983). Moreover, dDAVP is not effective (Naylor et al., 1987), while d(CH,),Using the newly developed analogs we set out to ex[Tyr(Me),]AVP potentiates the febrile response amine and compare the pharmacology of OT bind(Naylor et al., 1986) or blocks the antipyretic effect ing sites in (i) the hippocampus and (ii) the uterus
parently complex pharmacology of AVP and OT receptors in relation to their putative function (De Wied et al., 1991; Elands et al., 1991).
46 1
of AVP (Naylor et al., 1987).In this model AVP acts as an agonist . OT and dDAVP are considerably less active, while the V, antagonist d(CH2)5[Tyr(Me)2]AVP potently antagonizes the AVP induced effect (Fig. 1). These observations strongly suggest the implication of a V,, receptor in AVP mediated antipyresis. Moreover, an excellent correlation between the pharmacology of receptor binding and regulation of body temperature is evident.
OT
AVP
+
+
V, ant.
?
V,
ant.
OT ant. NVM
l
"OT" receptor
OT, A VP and sexual behavior ILordosis behavior As has already been discussed OT receptors Fig. 2. Model of the effects of OT, AVP and three antagonists recognize AVP and AVT with similar affinity as on lordosis behavior in the female rat. Arrows represent activity OT. OT receptors, both in the NVM and the hipon the OT receptor in the NVM. The blocking effect of the OT antagonist has been assessed on both OT' and AVP stimulated pocampus, are selectively labeled by the selective sexual behavior, the effects of the AVP antagonists have only OT antagonist de~Gly(NH~)~d(CH~)&Tyr(Me)~been demonstrated for the OT-induced lordosis behavior. V, Thr40rn8]VT, which has a 500-fold lower affinity ant., d(CH2)5[Tyr(Me)2]AVP;V, ant., d(CH,),[D-Phe211e4Alagfor hippocampal V, receptors (Voorhuis and ElNH2]AVP; OT ant., desGly (NH,)9d(CH,),[Tyr(Me)2Thr4] ands, unpublished observations). OVT. In the NVM AVP and OT have been demonstrated to increase excitability of neurons (Kow and 1988d; Johnson et al., 1989).A component of sexual behavior (lordosis) of female rats primed with proPfaff, 1986), however, only OT receptors could be demonstrated in this nucleus (Elands et al., 1988d; gesterone and estrogen is also affected by OT inTribollet et al., 1988). Estrogen treatment induces jected icv. (Arletti and Bertolini, 1985; Caldwell et the expression of OT receptors in this brain region al., 1986; Elands et al., 1991), or directly in the many fold (De Kloet et al., 1986; Elands et al., NVM (Schumacher et al., 1990). Conflicting reports appeared about a stimulatory or inhibitory action of AVP 1 OTI dDAVP AVP (Sodersten et al., 1983; Caldwell et al., 1986), which may well be a time-dependent phenomenon (Caldwell et al., 1986).After icv. injection we found a strong stimulation by OT of the lordosis response V, ant. that could be reduced below control levels by the OT ant. selective OT receptor antagonist de~Gly(NH3~d(CH2)5[Tyr(Me)2Thr4]0VT; AVP stimulated lordosis behavior with a comparable potency as OT (Elands et al., 1991). Although the direction of the AVP effect may not be unequivocallydetermined by the different investigators, the pharmacology of the Hypotherrnia NVM OT receptors corresponds nicely to the Fig. 1. Model for the interaction of AVP, OT and structural analogs with V, receptors in the septa1 area in the regulation of behavioral potency of the structural analogs (Fig. body temperature. + + represents a high and + /O a low activity 2). OT, AVP and de~Gly(NH~)~d(CH~)~[Tyr(Me)~on the V, receptor-mediated temperature regulation. The Thr4]0VT have a relatively high affinity for the horizontal bars represent the antagonistic action of AVPIOT anNVM receptor, while the V2 antagonist d(CH2)&~tagonists. V, ant., d(CH2)5[Tyr(Me)2AVP;V, ant., d(CH,),[DPhe211e4Alag-NH2]AVP (Caldwell et al., 1990) and Ile211e4]AVP; OT ant., desGly (NH2)9d(CH2)5[Tyr(Me)2Thr4] OVT. the V, antagonist d(CH2)5[Tyr(Me)2]AVPhave a
462
receptor. In this case it can be predicted that the effects on passive avoidance behavior are blocked by a selective OT receptor antagonist, regardless whether they are evoked by OT, AVP or AVT. InV , l V , ant. terestingly, the binding specificity of the nonselective OT receptor corresponds well with the efOT ant. fects of these peptides on conditioned avoidance behavior. We have demonstrated that the effects of “OT“ receptors AVP, OT and AVT on passive avoidance behavior are blocked by the selective OT antagonist desGly J . 4 (NH2)9d(CH2)5[Tyr(Me)2Thr40rn8]VT (De Wied Avoidance Behavior et al., 1991). Moreover, using three different antagFig. 3. InteractionofOT, AVPand twoantagonists with thenononists, selective for OT receptors (de~Gly(NH,)~selective OT receptors in the hippocampal area and the possible d(CH2)5[Tyr(Me)2Thr40rn8]VT), AVP V,, vs. V, implication for the peptide effects on retention of conditioned (d(CH,)5[Tyr(Me)2]AVP) or V, vs. V, receptors avoidance behavior. The arrows represent the activity on the non-selective OT receptor. + , Agonist; - , “inverse” agonist. (d(CH2)5[~-Ile211e4]AVP)we investigated the inThe horizontal bars indicate antagonist activity. All three anvolvement of AVP and OT receptors in passive tagonists are able to attenuatethe AVP and OT effects. Only the avoidance behavior. The V,, and V, antagonists OT antagonist fully antagonizes the AVP,,-,, effects. V,/V, that we used are not selective for AVP vs. OT recepant., d(CH2)5[Tyr(Me)2]AVP; V,/V, ant., d(CH2),[D-lle211e4] AVP; OT ant., desGly(NHz)gd(CH2),[Tyr(Me)2Thr4]0VT.tors, giving them a relatively high affinity for OT V,/V, (or V,/V,) means an enhanced selectivity for V, vs. V, (or receptors. All three antagonists used appeared to v, vs. V]). block the AVP and OT effects on passive avoidance behavior (Fig. 3). The OT antagonist, selective for low affinity for NVM OT receptor sites (Elands et OT receptors, is not very likely to interfere in low al., 1991) and have no effect on lordosis behavior. doses with V,, receptors; however, the relative high The high affinity for AVP of the NVM OT recepaffinity of the AVP antagonists may explain the tors may be reflected in a high potency of AVP in the ability to act on OT receptors. We concluded that modulation of sexual behavior. the OT receptor is very likely involved in the mediation of neurohypophyseal peptide effects on passive A VP, OT and conditioned avoidance behavior avoidance behavior. Behavioral observations show that the hippocamOpposite behavioral effects have been reported pus is strongly involved in the mediation of condifor AVP and OT. AVP induces a delay and OT a tioned (passive) avoidance behavior affected by facilitation of the extinction of avoidance behavior neurohypophyseal peptides (Kovacs et al., 1986). In (De Wied et al., 1989). In passive avoidance tests these peptides facilitate, respectively attenuate the the hippocampus both OT receptors and AVP V, receptors are present (Audigier and Barberis, 1985; retention of this behavior. This could imply that, usElands et al., 1988a; Tribollet et al., 1988). Passive ing behavioral performance as end point, AVP and avoidance behavior is stimulated by AVP and atOT may be considered as agonists and “inverse” tenuated by OT (Fig. 3). It seems reasonable to exagonists, respectively, acting on the non-selective pect that in the hippocampus responses to these horOT receptors. The opposite modes of action may mones are mediated by hippocampal V, receptors, take place via a heterogeneous class of OT receptors respectively by the non-selective hippocampal OT or via a complex underlying physiological mechreceptors. There is, however, also the possibility anism. that AVP (and AVT) also interact with the OT Fragments of AVP and OT, that may be metareceptor due to the non-selective nature of this bolically derived, exert a very powerful action on
+I
I-
463
conditioned avoidance behavior (De Wied et al., De Kloet, E.R., Voorhuis, Th.A.M., Burbach, J.P.H. and De Wied, D. (1985) Autoradiographic localization of binding 1989). The AVP fragments are virtually devoid of sites for the arginine vasopressin (VP) metabolite VP,- in rat antidiuretic or pressor activity (De Wied et al., brain. Neurosci. Lett., 56: I- 12. 1987), a finding that corresponds with the lack of afDe Kloet, E.R., Voorhuis, Th.A.M., Boschma, Y.and Elands, finity of these fragments for VP V,, receptors. J. (1986) Estradiol modulates density of putative “oxytocin Body temperature is not affected by such fragments receptors” in discrete rat brain regions. Neuroendocrinology, 44: 415-421. (desGlyNH2 AVP) (Kovacs and De Wied, 1983). De Wied, D., Gaffori, O., Burbach, P., Kovacs, G.L. and Van The AVP and OT receptors in the brain, however, Ree, J.M. (1987) Structure activity relationship studies with C display no affinity for fragments like [pGlu4Cyt6]terminal fragments of vasopressin and oxytocin on avoidance AVP(,-,) and [ ~ G ~ u ~ C ~ ~ ~8)] (De A VKloet P ( ~ etbehaviors of rats. J. Pharmacol. Exp. Ther., 241: 268- 274. De Wied, D., Joels, M., Burbach, J.P.H., De Jong, W., De al., 1985; Barberis, personal communication, Kloet, E.R., Gaffori, 0.W. J., Urban, I. J.A., Van Ree, J.M., Voorhuis and Elands, unpublished observations), Van Wimersma Greidanus, Tj.B., Veldhuis, H.D., Versteeg, suggesting the presence of separate interaction sites D.H.G. and Wiegant, V.M. (1989) Vasopressin effects on the for AVP and OT and their fragments. However, the central nervous system. In: Peptide Hormones: Effects and interference of the AVP fragment [pGlu4Cyt6]Mechanisms of Action, I , 3: 97 - 140. AVP(, - 8) with passive avoidance behavior is blockDe Wied, D., Elands, J. and Kovacs, G . (1991)Interactiveeffects of neurohypophyseal neuropeptides with receptor antagonists ed by all three antagonists used, although the OT anon passive avoidance behavior: mediation by a cerebral tagonist desGly (NH2)9d(CH2)5[Tyr(Me)2Thr4]neurohypophyseal hormone receptor? Proc. Natl. Acad. Sci. OVT, that binds selectively to OT receptors, was the U.S.A., 88: 1494- 1498. most potent (De Wied et al., 1991), somehow sugElands, J., Barberis, C. and Jard, S. (1988a) [3H][Thr4,Gly7]0T: a highly selective ligand for central and gesting that the fragment exerts its effects via a peripheral OT receptors. Am. J. Physiol., 254: E31 -E38. signal transduction pathway triggered by OT recepElands, J . , Barberis, C., Jard, S., Lammek, B., Manning, M., tor stimulation. These observations again suggest Sawyer, W.H. and De Kloet, E.R. (1988b) [12’1]that the non-selective nature of the O T receptors is d(CH2),[Tyr(Me)2,Tyr(NH2)9]AVP: iodination and binding reflected in the potency with which analogs interfere characteristics of a vasopressin receptor ligand. FEES Lett., via this receptor. 229: 251 -255. Elands, J., Barberis, C., Jard, S., Tribollet, E., Dreifuss, J.-J., In conclusion, the non-selective and putatively Bankowski, K., Manning, M. and Sawyer, W.H. (1988~)12’1heterogeneous (Elands et al., 1991) OT receptor labelled d(CH2)S[Tyr(Me)Z,Thr4,Tyr-NH;]OVT: a selective class with its high affinity for OT as well as AVP oxytocin receptor ligand. Eur. J. Pharmacol., 147: 197 - 207. may be a key site in the brain involved in modulation Elands, J., Beetsma, A., Barberis, C . and DeKloet, E.R. (3988d) of circuits underlying adaptive behavior, Topography of the oxytocin receptor system in rat brain: an
References Arletti, R. and Bertolini, A. (1985) Oxytocin stimulates lordosis behavior in female rats. Neuropeptides, 6: 247 -253. Audigier, S . and Barberis, C. (1985) Pharmacological characterization of two specific binding sites for neurohypophyseal hormones in hippocampal synaptic plasma membranes of the rat. EMBO J., 4: 1407- 1412. Caldwell, J.D., Prange, A.J., Jr. and Pedersen, C.A. (1986) Oxytocin facilitates the sexual receptivity of estrogen treated female rats. Neuropeptides, 7: 175 - 189. Caldwell, J.D., Barakat, AS., Smith, D.D., Hruby, V.J. and Pedersen, C.A. (1990) A uterotonic antagonist blocks the oxytocin-induced facilitation of female sexual receptivity. Brain Reas.,512: 291 -296.
autoradiographical study with a selective radioiodinated oxytocin antagonist. J . Chem. Neuroanat., I : 293 -302. Elands, J., Van Doremalen, E., Spruyt, B. and De Kloet, E.R. (1991) Oxytocin receptors in the rat hypothalamic ventromedial nucleus: a study of possible mediators of female sexual behavior. In: S . Jard and R. Jamison (Eds.), Proceedings of the Third Vasopressin Conference, Montpellier (France), John Libbey, London, pp. 31 1 - 319. Freund-Mercier, M.J. and Richard, P. (1984) Electrophysiological evidence for facilitatory control of oxytocin neurones by oxytocin during suckling in the rat. J. Physiol. (Lond.), 352: 447 - 466. Johnson, A.E., Coirini, H., Bal1,G.F. and McEwen, B.S. (1989) Anatomical localization of the effects of 17-P-estradiol on oxytocin receptor binding in the ventromedial hypothalamic nucleus. Endocrinology, 124: 207 - 21 1. Kovacs, G.L. and De Wied, D. (1983) Hormonally active
464 arginine-vasopressin suppresses endotoxin induced fever in rats: lack of effect of oxytocin and a behaviorally active vasopressin fragment. Neuroendocrinology, 37: 258 - 261, Kovacs, G.L., Bohus, B., Versteeg, D.H.G., De Kloet, E.R. and De Wied, D. (1979) Effect of oxytocin and vasopressin on memory consolidation: sites of action and catecholaminergic correlates after local microinjection into limbic-midbrain structures. Brain Res., 175: 303 - 314. Kovacs, G.L., Veldhuis, H.D.,Versteef, D.H.G. and De Wied, D. (1986) Facilitation of avoidance behavior by vasopressin fragments microinjected into limbic midbrain structures. Brain Res., 371: 17-24. Kow, L.M. and Pfaff, D.W. (1986) Vasopressin excites ventromedial hypothalamic glucose responsive neurons in vitro. Physiol. Behav., 37: 153 - 158. Naylor, A.M., Cooper, K.E. and Veale, W.L. (1986) Vasopressin and fever: evidence supporting the existence of an endogenous antipyretic system in the brain. Can. J. Physiol. Pharmacol., 65: 1333- 1338. Naylor, A.M., Gubitz, G.J., Dinarello, C.A. and Veale, W.L. (1987) Central effects of vasopressin and l-desamino-8-~arginine vasopressin (dDAVP) on interleukin fever in the rat. Brain Rex, 401: 173- 177.
Ruwe, W.D., Naylor, A.M. andVeale, W.L. (1985)Perfusionof vasopressin within the rat brain suppresses prostaglandin E hyperthermia. Brain Res., 338: 219 - 224. Schumacher, M., Coirini, H., Pfaff, D.W. and McEwen, B.S. (1990) Behavioral effects of progesterone associated with rapid modulation of oxytocin receptors. Science, 250: 691 - 694. Sodersten, P., Henning, M., Melin, P. and Ludin, S. (1983) Vasopressin alters female sexual behaviour by acting on the brain independently of alterations in blood pressure. Nature, 301: 608 - 610. Tribollet, E., Barberis, C., Jard, S., Dubois-Dauphin, M. and Dreifuss, J.J. (1988) Localization and pharmacological characterization of high affinity binding sites for vasopressin and oxytocin in the rat brain by light microscopic autoradiography. Brain Res., 442: 105 - 118. Vallejo, M., Carter, D.A. and Lightman, S.L. (1984) Haemodynamic effects of arginine vasopressin microinjections into the nucleus tractus solitarius: a comparative study of vasopressin, a selective vasopressin receptor agonist and antagonist, and oxytocin. Neurosci. Lett., 52: 247 - 252.
459
0 1992 Elsevier Science Publishers B.V. All rights reserved.
CHAPTER 61
Neurohypophyseal hormone receptors: relation to behavior J . Elands, E.R. de Kloet' and D. de Wied2 Max PIanck Institute for Psychiatry, 8000 Munich 40, Germany; I Department of Medical Pharmacology, Center f o r BioPharmaceutical Sciences, Leiden, The Netherlands; and Department of Pharmacology, Rudolf Magnus Institute, Utrecht, The Netherlands
Introduction Arginine vasopressin (AVP) and oxytocin (OT) exert a multitude of effects in the CNS. It is believed that these peptides are distributed via a divergent neuronal network, consisting of AVP and OT containing fibers and terminals and are mediated by specific receptors for AVP and OT. The involvement of AVP and OT in centrally mediated effects has been investigated by applying the peptides AVP, OT or their structural analogs either intracerebroventricularly (icv.) or directly into certain brain areas, or by observing regional biochemical changes following systemic, or icv. treatment. Well known examples are the antipyretic effects of AVP in the septa1 area (Kovacs and De Wied, 1983; Ruwe et al., 1985; Naylor et al., 1986), the AVP-induced cardiovascular effects mediated via the nucleus tractus solitarii (NTS) (Vallejo et al., 1984), sexual behavior induced by OT in the nucleus ventromedialis of the hypothalamus (NVM) (Arletti and Bertolini, 1985; Caldwell et al., 1986; Elands et al., 1991) and the modulation of avoidance behavior by both AVP and OT (Kovacs et al., 1979; De Wied et al., 1987, 1991). To get insight in the mechanism underlying these processes we have studied the AVP
and OT receptors in the CNS, in order to correlate the pharmacology of these receptors with their physiological or behavioral effects. The importance of this approach may be illustrated by the apparent lack of AVP and OT receptors in the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei (Elands et al., 1988d; Tribollet et al., 1988), regions where a positive feedback action of OT on its own release has been reported (Freund-Mercier and Richard, 1984). The first observation towards the characterization of AVP and OT receptors in the CNS, made by Barberis and colleagues (Audigier and Barberis, 1985), revealed the presence of two classes of binding sites on hippocampal membranes. One class was designated as AVP binding sites since it had binding characteristics comparable to the classical AVP V,, receptors in the liver and blood vessels. The other class, having a more complex binding profile due to an interaction with both AVP and OT, constituted the OT binding sites. This class appeared to have an equally high affinity for AVP as for OT. These observations prompted us to develop ligands that were more selective (Elands et al., 1988a,b,c) in order to characterize these receptors and then, by having the appropriate ligands, to examine the ap-
460
and mammary gland. Using OT, AVP and a series of structural analogs we were able to confirm that CNS OT receptors recognize AVP (Ki = 1.6 nM) and AVT (Ki = 1.9 nM) with similar affinity as OT (Ki = 1.9 nM) (Elands et al., 1988a); this property Development of selective ligands confers to these OT receptors an apparent nonselectivity in binding of the naturally occurring Essentially two lines have been followed: tritium neurohypophyseal hormones AVP and OT. In spite labeling of analogs that have a high selectivity and of this non-selective nature this receptor site is termradioiodination of analogs that have been modified ed an OT receptor. The peripheral type O T receptors in order to incorporate an iodine molecule without in the uterus and the mammary gland also had an loss of affinity. Using the first approach [Thr4equally high affinity for AVP and OT (Elands et al., Gly7]0T (Elands et al., 1988a) was tritiated. In the 1988a). OT receptors in the rat brain are generally rat this OT receptor agonist expresses a lo4 times comparable to the classical uterine type, although lower affinity for V, and V, receptors and is thus OT receptors in the NVM were found to be slightly extremely selective for OT receptors. It appeared to different (Elands et al., 1991); a somewhat lower aflabel OT receptors with high affinity (Kd = 1-2 finity for OT (Ki = 6 nM vs. 1.0 nM for uterine nM). For the other approach antagonists of the receptors), [1251]-OTA(Ki = 0.15 nM vs. 0.05 nM) AVP V, and OT receptors have been used that and [Thr4Gly7]0T (Ki = 20 nM vs. 1 nM), may could incorporate an iodine molecule without possibly indicate an OT receptor heterogeneity. dramatic changes in affinity. Thus potent anFinally, AVP receptors in the CNS seem to be extagonists (Elands et al., 1988b,c) were substituted at clusively of the V,, type; they bind AVP with a high the ninth position with a tyrosylamide residue. This affinity and OT with a 10 - 30-fold lower affinity. resulted (i) in a highly radioactive, selective OT Moreover, these AVP receptors recognize the receptor antag~nist(['~~I]-d(CH,)~[Tyr(Me)~Thr~classical pressor antagonist d(cH,)&T~r(Me)~]OrnsTyr-NH~]VT,[1251]-OTA) that did bind with AVP (which has an enhanced pressor versus anan extremely high affinity (Kd = 0.05 nM) to OT tidiuretic potency) with a high affinity (Ki = 0.5 receptors and displayed a very low non-specific nM) and the antidiuretic agonist dDAVP with a low binding and (ii) in an equally radioactive V,, anaffinity tagonist [1251]-d(CH2)5[Tyr(Me)2Tyr-NH2]AVP, (Ki > 500 nM) (Voorhuis and Elands, unpublished observations). [1251]-AVA),that did bind with a high affinity (Kd = 0.3 nM) to V,, receptors and also had a moderate Models for AVP and OT action in the brain non-specific binding. The benefit of these analogs has led to a worldA VP and temperature regulation wide use of the radioiodinated O T antagonist and to AVP induces an antipyretic response in febrile the rapid development of other radioiodinated rats (Kovacs and De Wied, 1983; Ruwe et al., 1985; analogs. Caution is, however, needed since these Naylor et al., 1986), and this effect is mediated by compounds do not necessarily bind to receptor subAVP receptors in the brain. The pharmacology of types for the endogenous hormones that have not the hypothermic action correlates well with the been demonstrated yet. binding specificity of V, receptors. OT is ineffective in doses where AVP potently affects temperature Receptor pharmacology (Kovacs and De Wied, 1983). Moreover, dDAVP is not effective (Naylor et al., 1987), while d(CH,),Using the newly developed analogs we set out to ex[Tyr(Me),]AVP potentiates the febrile response amine and compare the pharmacology of OT bind(Naylor et al., 1986) or blocks the antipyretic effect ing sites in (i) the hippocampus and (ii) the uterus
parently complex pharmacology of AVP and OT receptors in relation to their putative function (De Wied et al., 1991; Elands et al., 1991).
46 1
of AVP (Naylor et al., 1987).In this model AVP acts as an agonist . OT and dDAVP are considerably less active, while the V, antagonist d(CH2)5[Tyr(Me)2]AVP potently antagonizes the AVP induced effect (Fig. 1). These observations strongly suggest the implication of a V,, receptor in AVP mediated antipyresis. Moreover, an excellent correlation between the pharmacology of receptor binding and regulation of body temperature is evident.
OT
AVP
+
+
V, ant.
?
V,
ant.
OT ant. NVM
l
"OT" receptor
OT, A VP and sexual behavior ILordosis behavior As has already been discussed OT receptors Fig. 2. Model of the effects of OT, AVP and three antagonists recognize AVP and AVT with similar affinity as on lordosis behavior in the female rat. Arrows represent activity OT. OT receptors, both in the NVM and the hipon the OT receptor in the NVM. The blocking effect of the OT antagonist has been assessed on both OT' and AVP stimulated pocampus, are selectively labeled by the selective sexual behavior, the effects of the AVP antagonists have only OT antagonist de~Gly(NH~)~d(CH~)&Tyr(Me)~been demonstrated for the OT-induced lordosis behavior. V, Thr40rn8]VT, which has a 500-fold lower affinity ant., d(CH2)5[Tyr(Me)2]AVP;V, ant., d(CH,),[D-Phe211e4Alagfor hippocampal V, receptors (Voorhuis and ElNH2]AVP; OT ant., desGly (NH,)9d(CH,),[Tyr(Me)2Thr4] ands, unpublished observations). OVT. In the NVM AVP and OT have been demonstrated to increase excitability of neurons (Kow and 1988d; Johnson et al., 1989).A component of sexual behavior (lordosis) of female rats primed with proPfaff, 1986), however, only OT receptors could be demonstrated in this nucleus (Elands et al., 1988d; gesterone and estrogen is also affected by OT inTribollet et al., 1988). Estrogen treatment induces jected icv. (Arletti and Bertolini, 1985; Caldwell et the expression of OT receptors in this brain region al., 1986; Elands et al., 1991), or directly in the many fold (De Kloet et al., 1986; Elands et al., NVM (Schumacher et al., 1990). Conflicting reports appeared about a stimulatory or inhibitory action of AVP 1 OTI dDAVP AVP (Sodersten et al., 1983; Caldwell et al., 1986), which may well be a time-dependent phenomenon (Caldwell et al., 1986).After icv. injection we found a strong stimulation by OT of the lordosis response V, ant. that could be reduced below control levels by the OT ant. selective OT receptor antagonist de~Gly(NH3~d(CH2)5[Tyr(Me)2Thr4]0VT; AVP stimulated lordosis behavior with a comparable potency as OT (Elands et al., 1991). Although the direction of the AVP effect may not be unequivocallydetermined by the different investigators, the pharmacology of the Hypotherrnia NVM OT receptors corresponds nicely to the Fig. 1. Model for the interaction of AVP, OT and structural analogs with V, receptors in the septa1 area in the regulation of behavioral potency of the structural analogs (Fig. body temperature. + + represents a high and + /O a low activity 2). OT, AVP and de~Gly(NH~)~d(CH~)~[Tyr(Me)~on the V, receptor-mediated temperature regulation. The Thr4]0VT have a relatively high affinity for the horizontal bars represent the antagonistic action of AVPIOT anNVM receptor, while the V2 antagonist d(CH2)&~tagonists. V, ant., d(CH2)5[Tyr(Me)2AVP;V, ant., d(CH,),[DPhe211e4Alag-NH2]AVP (Caldwell et al., 1990) and Ile211e4]AVP; OT ant., desGly (NH2)9d(CH2)5[Tyr(Me)2Thr4] OVT. the V, antagonist d(CH2)5[Tyr(Me)2]AVPhave a
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receptor. In this case it can be predicted that the effects on passive avoidance behavior are blocked by a selective OT receptor antagonist, regardless whether they are evoked by OT, AVP or AVT. InV , l V , ant. terestingly, the binding specificity of the nonselective OT receptor corresponds well with the efOT ant. fects of these peptides on conditioned avoidance behavior. We have demonstrated that the effects of “OT“ receptors AVP, OT and AVT on passive avoidance behavior are blocked by the selective OT antagonist desGly J . 4 (NH2)9d(CH2)5[Tyr(Me)2Thr40rn8]VT (De Wied Avoidance Behavior et al., 1991). Moreover, using three different antagFig. 3. InteractionofOT, AVPand twoantagonists with thenononists, selective for OT receptors (de~Gly(NH,)~selective OT receptors in the hippocampal area and the possible d(CH2)5[Tyr(Me)2Thr40rn8]VT), AVP V,, vs. V, implication for the peptide effects on retention of conditioned (d(CH,)5[Tyr(Me)2]AVP) or V, vs. V, receptors avoidance behavior. The arrows represent the activity on the non-selective OT receptor. + , Agonist; - , “inverse” agonist. (d(CH2)5[~-Ile211e4]AVP)we investigated the inThe horizontal bars indicate antagonist activity. All three anvolvement of AVP and OT receptors in passive tagonists are able to attenuatethe AVP and OT effects. Only the avoidance behavior. The V,, and V, antagonists OT antagonist fully antagonizes the AVP,,-,, effects. V,/V, that we used are not selective for AVP vs. OT recepant., d(CH2)5[Tyr(Me)2]AVP; V,/V, ant., d(CH2),[D-lle211e4] AVP; OT ant., desGly(NHz)gd(CH2),[Tyr(Me)2Thr4]0VT.tors, giving them a relatively high affinity for OT V,/V, (or V,/V,) means an enhanced selectivity for V, vs. V, (or receptors. All three antagonists used appeared to v, vs. V]). block the AVP and OT effects on passive avoidance behavior (Fig. 3). The OT antagonist, selective for low affinity for NVM OT receptor sites (Elands et OT receptors, is not very likely to interfere in low al., 1991) and have no effect on lordosis behavior. doses with V,, receptors; however, the relative high The high affinity for AVP of the NVM OT recepaffinity of the AVP antagonists may explain the tors may be reflected in a high potency of AVP in the ability to act on OT receptors. We concluded that modulation of sexual behavior. the OT receptor is very likely involved in the mediation of neurohypophyseal peptide effects on passive A VP, OT and conditioned avoidance behavior avoidance behavior. Behavioral observations show that the hippocamOpposite behavioral effects have been reported pus is strongly involved in the mediation of condifor AVP and OT. AVP induces a delay and OT a tioned (passive) avoidance behavior affected by facilitation of the extinction of avoidance behavior neurohypophyseal peptides (Kovacs et al., 1986). In (De Wied et al., 1989). In passive avoidance tests these peptides facilitate, respectively attenuate the the hippocampus both OT receptors and AVP V, receptors are present (Audigier and Barberis, 1985; retention of this behavior. This could imply that, usElands et al., 1988a; Tribollet et al., 1988). Passive ing behavioral performance as end point, AVP and avoidance behavior is stimulated by AVP and atOT may be considered as agonists and “inverse” tenuated by OT (Fig. 3). It seems reasonable to exagonists, respectively, acting on the non-selective pect that in the hippocampus responses to these horOT receptors. The opposite modes of action may mones are mediated by hippocampal V, receptors, take place via a heterogeneous class of OT receptors respectively by the non-selective hippocampal OT or via a complex underlying physiological mechreceptors. There is, however, also the possibility anism. that AVP (and AVT) also interact with the OT Fragments of AVP and OT, that may be metareceptor due to the non-selective nature of this bolically derived, exert a very powerful action on
+I
I-
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conditioned avoidance behavior (De Wied et al., De Kloet, E.R., Voorhuis, Th.A.M., Burbach, J.P.H. and De Wied, D. (1985) Autoradiographic localization of binding 1989). The AVP fragments are virtually devoid of sites for the arginine vasopressin (VP) metabolite VP,- in rat antidiuretic or pressor activity (De Wied et al., brain. Neurosci. Lett., 56: I- 12. 1987), a finding that corresponds with the lack of afDe Kloet, E.R., Voorhuis, Th.A.M., Boschma, Y.and Elands, finity of these fragments for VP V,, receptors. J. (1986) Estradiol modulates density of putative “oxytocin Body temperature is not affected by such fragments receptors” in discrete rat brain regions. Neuroendocrinology, 44: 415-421. (desGlyNH2 AVP) (Kovacs and De Wied, 1983). De Wied, D., Gaffori, O., Burbach, P., Kovacs, G.L. and Van The AVP and OT receptors in the brain, however, Ree, J.M. (1987) Structure activity relationship studies with C display no affinity for fragments like [pGlu4Cyt6]terminal fragments of vasopressin and oxytocin on avoidance AVP(,-,) and [ ~ G ~ u ~ C ~ ~ ~8)] (De A VKloet P ( ~ etbehaviors of rats. J. Pharmacol. Exp. Ther., 241: 268- 274. De Wied, D., Joels, M., Burbach, J.P.H., De Jong, W., De al., 1985; Barberis, personal communication, Kloet, E.R., Gaffori, 0.W. J., Urban, I. J.A., Van Ree, J.M., Voorhuis and Elands, unpublished observations), Van Wimersma Greidanus, Tj.B., Veldhuis, H.D., Versteeg, suggesting the presence of separate interaction sites D.H.G. and Wiegant, V.M. (1989) Vasopressin effects on the for AVP and OT and their fragments. However, the central nervous system. In: Peptide Hormones: Effects and interference of the AVP fragment [pGlu4Cyt6]Mechanisms of Action, I , 3: 97 - 140. AVP(, - 8) with passive avoidance behavior is blockDe Wied, D., Elands, J. and Kovacs, G . (1991)Interactiveeffects of neurohypophyseal neuropeptides with receptor antagonists ed by all three antagonists used, although the OT anon passive avoidance behavior: mediation by a cerebral tagonist desGly (NH2)9d(CH2)5[Tyr(Me)2Thr4]neurohypophyseal hormone receptor? Proc. Natl. Acad. Sci. OVT, that binds selectively to OT receptors, was the U.S.A., 88: 1494- 1498. most potent (De Wied et al., 1991), somehow sugElands, J., Barberis, C. and Jard, S. (1988a) [3H][Thr4,Gly7]0T: a highly selective ligand for central and gesting that the fragment exerts its effects via a peripheral OT receptors. Am. J. Physiol., 254: E31 -E38. signal transduction pathway triggered by OT recepElands, J . , Barberis, C., Jard, S., Lammek, B., Manning, M., tor stimulation. These observations again suggest Sawyer, W.H. and De Kloet, E.R. (1988b) [12’1]that the non-selective nature of the O T receptors is d(CH2),[Tyr(Me)2,Tyr(NH2)9]AVP: iodination and binding reflected in the potency with which analogs interfere characteristics of a vasopressin receptor ligand. FEES Lett., via this receptor. 229: 251 -255. Elands, J., Barberis, C., Jard, S., Tribollet, E., Dreifuss, J.-J., In conclusion, the non-selective and putatively Bankowski, K., Manning, M. and Sawyer, W.H. (1988~)12’1heterogeneous (Elands et al., 1991) OT receptor labelled d(CH2)S[Tyr(Me)Z,Thr4,Tyr-NH;]OVT: a selective class with its high affinity for OT as well as AVP oxytocin receptor ligand. Eur. J. Pharmacol., 147: 197 - 207. may be a key site in the brain involved in modulation Elands, J., Beetsma, A., Barberis, C . and DeKloet, E.R. (3988d) of circuits underlying adaptive behavior, Topography of the oxytocin receptor system in rat brain: an
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autoradiographical study with a selective radioiodinated oxytocin antagonist. J . Chem. Neuroanat., I : 293 -302. Elands, J., Van Doremalen, E., Spruyt, B. and De Kloet, E.R. (1991) Oxytocin receptors in the rat hypothalamic ventromedial nucleus: a study of possible mediators of female sexual behavior. In: S . Jard and R. Jamison (Eds.), Proceedings of the Third Vasopressin Conference, Montpellier (France), John Libbey, London, pp. 31 1 - 319. Freund-Mercier, M.J. and Richard, P. (1984) Electrophysiological evidence for facilitatory control of oxytocin neurones by oxytocin during suckling in the rat. J. Physiol. (Lond.), 352: 447 - 466. Johnson, A.E., Coirini, H., Bal1,G.F. and McEwen, B.S. (1989) Anatomical localization of the effects of 17-P-estradiol on oxytocin receptor binding in the ventromedial hypothalamic nucleus. Endocrinology, 124: 207 - 21 1. Kovacs, G.L. and De Wied, D. (1983) Hormonally active
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Ruwe, W.D., Naylor, A.M. andVeale, W.L. (1985)Perfusionof vasopressin within the rat brain suppresses prostaglandin E hyperthermia. Brain Res., 338: 219 - 224. Schumacher, M., Coirini, H., Pfaff, D.W. and McEwen, B.S. (1990) Behavioral effects of progesterone associated with rapid modulation of oxytocin receptors. Science, 250: 691 - 694. Sodersten, P., Henning, M., Melin, P. and Ludin, S. (1983) Vasopressin alters female sexual behaviour by acting on the brain independently of alterations in blood pressure. Nature, 301: 608 - 610. Tribollet, E., Barberis, C., Jard, S., Dubois-Dauphin, M. and Dreifuss, J.J. (1988) Localization and pharmacological characterization of high affinity binding sites for vasopressin and oxytocin in the rat brain by light microscopic autoradiography. Brain Res., 442: 105 - 118. Vallejo, M., Carter, D.A. and Lightman, S.L. (1984) Haemodynamic effects of arginine vasopressin microinjections into the nucleus tractus solitarius: a comparative study of vasopressin, a selective vasopressin receptor agonist and antagonist, and oxytocin. Neurosci. Lett., 52: 247 - 252.