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S.16.04 Histamine H3-receptor ligands: Effects of inverse agonists and protean ligands
S.16.
The orexin-aminergic
mechanism to fiue tuuiug the activation for the adequate behavioural response.
of discrete
connection braiu
in the control
regions
References [l] [2] [3] [4] [5]
Pannentier, R. et al. J Neumsci 22, 7695-7711 (2002). Passani, M.B. & Blandina, P. The neuronal histaminergic cognition. Cur Med Chem, in press (2003). Passani, M. B. et al. Eur J Neurosci 14, 1522-1532 (2001). Cangioli, I. et al. Eur J Neurosci 16, 521-528 (2002). Bacciottini, L. et al. Eur J Neurosci 15, 1669*1680 (2002).
IS 16 04
Histamine inverse
Hs-receptor agonists and
ligands: protean
Effects ligands
system
in
of
J.M. Arrau ’ S. Morisset’, A. Rouleau’, F. Gbahou’, X. Ligueau !P, .I. Tardivel-Lacombe’, H. Stark3, W. Schuuack3, C.R. Gauelliu4, J.-C. Schwartz’. ‘Centre Paul Broca, Unit& de Neurobiologie et Pharmacologic, Paris, France; 2LaDoratoire Bioprojet, Paris, France; 3Freie UniversitZit Berlin, Institut fiir Pharmazie, Berlin, Germany; 4 University College London, Department of Chemistry, London, United Kingdom The histamine H3 receptor was detected as au autoreceptor coutrolling histamine release in braiu [ 11. The use of Hj-receptor autagouists in vivo showed au activation of histamine ueurous [2] that promotes arousal aud attention This activation has beeu proposed as a therapeutic approach in humau atteutioual aud ageiug disorders. Data accumulated over the last years suggested that G-proteiucoupled receptors (GPCRs) could be active eveu in the absence of au agonist. This coustitutive (or spoutaueous) activity was mainly evidenced for recombiuaut receptors overexpressed a&or mutated. Cousisteut with the physiological relevance of the process, we recently showed that coustitutive activity of H3 autoreceptors regulates histamine ueurous in vivo. Most autagouists are in fact acting as iuverse agonists at H3 receptors aud euhauce histamine ueurou activity by abrogatiug the brake triggered by coustitutive activity of autoreceptors [3]. Followiug clouiug of the H3 receptor, evidence was obtained for the existence in the braiu of fuuctioual isoforms geuerated by pseudo-iutrou reteutioilideletiou in the third intracellular loop [341. Wheu expressed at physiological densities in CHO cells, these isoforms display coustitutive activity aud standard H3 -receptor autagouists, such as thioperamide aud ciproxifau, act as poteut iuverse agonists 011 [35S]GTPy[S] binding, [3H]arachidouic acid release aud iuhibitiou of CAMP accumulation In addition, 011 these three fuuctioual assays, the level of coustitutive activity is depeudeut 011 the isoform. It is higher for the louger isoform H3(455) thau with the shorter isofonn H3(4t3) of the rat receptor. Curreut models for GPCR activation assume that coustitutive activity results from au equilibrium betweeu iuactive aud active coufonnatious of the receptor. However, for a giveu GPCR, the efficacies of active coufonnatious are expected to be depeudeut 011 the fuuctioual test system. In agreement, coustitutive activity of the rat H3 receptor expressed in the same cell is strongly depeudeut 011 the signaling pathway. The differeut efficacies of active coufonnatious in equilibrium also lead to the existence of “protean agonists, a class of drugs displayiug a spectrum of activity from agouism to iuverse agouism at the same GPCR. We could observe proteau agouism with proxyfau which behaves as a neutral autagouist in vitro aud in vivo [3]. In addition to neutral autagouism, we found that proxyfau cau induce either agouism or iuverse agouism 011 various responses coupled to the recombiuaut
of vigilance
and food
intake
s141
or native H3 receptor. For example, wheu the rat H3(413) isofonn is expressed at moderate densities in CHO cells, proxyfau behaves as a neutral autagouist 011 [3H]arachidouic acid release [3], but wheu the same isofonn is expressed at a high density in the same cells, proxyfau displays partial agouism 011 MAP kiuase activity, [35S]GTPy[S] biudiug aud iuhibitiou of CAMP fonnatiou, aud partial iuverse agouism 011 [3H]arachidouic acid release. [35S]GTPy[S] biudiug demonstrates the coustitutive activity of native H3 receptors present in mouse or rat braiu [3,5]. This coustitutive activity of the native H3R is observed in all regions of the rat brain Moreover, it appears to be oue of the highest among GPCRs. Although au appareut lack of iuverse agouism does uot furuish defiuitive evidence for the absence of coustitutive activity, it is worth uotiug that several compounds classified as iuverse agonists at overexpressed or mutated GPCRs, fail to decrease [35S]GTPy[S] biudiug to membranes of rat cerebral cortex or striatum. However, we observed that besides H3 receptors, native At adeuosiue receptors display high coustitutive activity in rat braiu [5]. The humau H3 receptor expressed at moderate densities also displays high coustitutive activity [5]. We found that coustitutive activity is of similar amplitude in humau aud rat wheu arachidouic acid release is monitored. However, 011 [35S]GTPy[S] binding, the human receptor displays lower coustitutive activity. Whereas the agonist imetit similarly iucreases [35S]GTPy[S] biudiug to membraues of CHO cells expressing the rat aud humau receptor, the effect of ciproxifau is lower for the human H3 receptor. As showu for the rat receptor [3], coustitutive activity of the recombiuaut human receptor is correlated to receptor density. However, the maximal iucrease in [35S]GTPy[S] biudiug observed at the highest densities remaius substantially lower thau the maximal response produced by au agonist, suggesting that the amount of receptor may be limiting for coustitutive activity of the humau H3 receptor. Coustitutive activity of the humau receptor is also evidenced using iuhibitiou of [35S]GTPy[S] biudiug by uulabelled GTPyS. The expression of the receptor in CHO cells generates a high affinity biudiug for GTPyS which is uot observed in wild type cells. The capacity of this biudiug site indicates that -25% of humau H3 receptors exist in a precoupled aud coustitutively active state. Ciproxifau attenuates but does uot abolish totally this high affinity binding, iudicatiug that it acts as a partial iuverse agonist at the human H3 receptor. Our fiudiugs obtained with proxyfau in the rat show that the therapeutic iuterest of proteau ligauds is hard to predict. The observatiou that coustitutive activity of the human H3 receptor is easily detected at moderate densities suggests that it occurs in human brain This observatiou may have importaut implications in tenns of drug development. The role of coustitutive activity of H3 autoreceptors in the regulation of histamine ueurous in vivo suggests that iuverse agonists should find therapeutic applications as iiootropic ageiits. References [l] [2] [3] [4] [5]
Arrang JM et al. Nature 1983; 302: 832-7. Arrang JM et al. Nature 1987; 327: 117-23. Morisset S et al. Nature 2000; 408: 86G4. Morisset S et al. B&hem Biophys Res Comnun 2001; Rouleau A et al. Brit J Pharmacol 2002; 135: 383-92.
280: 75-80.
The orexin-aminergic
mechanism to fiue tuuiug the activation for the adequate behavioural response.
of discrete
connection braiu
in the control
regions
References [l] [2] [3] [4] [5]
Pannentier, R. et al. J Neumsci 22, 7695-7711 (2002). Passani, M.B. & Blandina, P. The neuronal histaminergic cognition. Cur Med Chem, in press (2003). Passani, M. B. et al. Eur J Neurosci 14, 1522-1532 (2001). Cangioli, I. et al. Eur J Neurosci 16, 521-528 (2002). Bacciottini, L. et al. Eur J Neurosci 15, 1669*1680 (2002).
IS 16 04
Histamine inverse
Hs-receptor agonists and
ligands: protean
Effects ligands
system
in
of
J.M. Arrau ’ S. Morisset’, A. Rouleau’, F. Gbahou’, X. Ligueau !P, .I. Tardivel-Lacombe’, H. Stark3, W. Schuuack3, C.R. Gauelliu4, J.-C. Schwartz’. ‘Centre Paul Broca, Unit& de Neurobiologie et Pharmacologic, Paris, France; 2LaDoratoire Bioprojet, Paris, France; 3Freie UniversitZit Berlin, Institut fiir Pharmazie, Berlin, Germany; 4 University College London, Department of Chemistry, London, United Kingdom The histamine H3 receptor was detected as au autoreceptor coutrolling histamine release in braiu [ 11. The use of Hj-receptor autagouists in vivo showed au activation of histamine ueurous [2] that promotes arousal aud attention This activation has beeu proposed as a therapeutic approach in humau atteutioual aud ageiug disorders. Data accumulated over the last years suggested that G-proteiucoupled receptors (GPCRs) could be active eveu in the absence of au agonist. This coustitutive (or spoutaueous) activity was mainly evidenced for recombiuaut receptors overexpressed a&or mutated. Cousisteut with the physiological relevance of the process, we recently showed that coustitutive activity of H3 autoreceptors regulates histamine ueurous in vivo. Most autagouists are in fact acting as iuverse agonists at H3 receptors aud euhauce histamine ueurou activity by abrogatiug the brake triggered by coustitutive activity of autoreceptors [3]. Followiug clouiug of the H3 receptor, evidence was obtained for the existence in the braiu of fuuctioual isoforms geuerated by pseudo-iutrou reteutioilideletiou in the third intracellular loop [341. Wheu expressed at physiological densities in CHO cells, these isoforms display coustitutive activity aud standard H3 -receptor autagouists, such as thioperamide aud ciproxifau, act as poteut iuverse agonists 011 [35S]GTPy[S] binding, [3H]arachidouic acid release aud iuhibitiou of CAMP accumulation In addition, 011 these three fuuctioual assays, the level of coustitutive activity is depeudeut 011 the isoform. It is higher for the louger isoform H3(455) thau with the shorter isofonn H3(4t3) of the rat receptor. Curreut models for GPCR activation assume that coustitutive activity results from au equilibrium betweeu iuactive aud active coufonnatious of the receptor. However, for a giveu GPCR, the efficacies of active coufonnatious are expected to be depeudeut 011 the fuuctioual test system. In agreement, coustitutive activity of the rat H3 receptor expressed in the same cell is strongly depeudeut 011 the signaling pathway. The differeut efficacies of active coufonnatious in equilibrium also lead to the existence of “protean agonists, a class of drugs displayiug a spectrum of activity from agouism to iuverse agouism at the same GPCR. We could observe proteau agouism with proxyfau which behaves as a neutral autagouist in vitro aud in vivo [3]. In addition to neutral autagouism, we found that proxyfau cau induce either agouism or iuverse agouism 011 various responses coupled to the recombiuaut
of vigilance
and food
intake
s141
or native H3 receptor. For example, wheu the rat H3(413) isofonn is expressed at moderate densities in CHO cells, proxyfau behaves as a neutral autagouist 011 [3H]arachidouic acid release [3], but wheu the same isofonn is expressed at a high density in the same cells, proxyfau displays partial agouism 011 MAP kiuase activity, [35S]GTPy[S] biudiug aud iuhibitiou of CAMP fonnatiou, aud partial iuverse agouism 011 [3H]arachidouic acid release. [35S]GTPy[S] biudiug demonstrates the coustitutive activity of native H3 receptors present in mouse or rat braiu [3,5]. This coustitutive activity of the native H3R is observed in all regions of the rat brain Moreover, it appears to be oue of the highest among GPCRs. Although au appareut lack of iuverse agouism does uot furuish defiuitive evidence for the absence of coustitutive activity, it is worth uotiug that several compounds classified as iuverse agonists at overexpressed or mutated GPCRs, fail to decrease [35S]GTPy[S] biudiug to membranes of rat cerebral cortex or striatum. However, we observed that besides H3 receptors, native At adeuosiue receptors display high coustitutive activity in rat braiu [5]. The humau H3 receptor expressed at moderate densities also displays high coustitutive activity [5]. We found that coustitutive activity is of similar amplitude in humau aud rat wheu arachidouic acid release is monitored. However, 011 [35S]GTPy[S] binding, the human receptor displays lower coustitutive activity. Whereas the agonist imetit similarly iucreases [35S]GTPy[S] biudiug to membraues of CHO cells expressing the rat aud humau receptor, the effect of ciproxifau is lower for the human H3 receptor. As showu for the rat receptor [3], coustitutive activity of the recombiuaut human receptor is correlated to receptor density. However, the maximal iucrease in [35S]GTPy[S] biudiug observed at the highest densities remaius substantially lower thau the maximal response produced by au agonist, suggesting that the amount of receptor may be limiting for coustitutive activity of the humau H3 receptor. Coustitutive activity of the humau receptor is also evidenced using iuhibitiou of [35S]GTPy[S] biudiug by uulabelled GTPyS. The expression of the receptor in CHO cells generates a high affinity biudiug for GTPyS which is uot observed in wild type cells. The capacity of this biudiug site indicates that -25% of humau H3 receptors exist in a precoupled aud coustitutively active state. Ciproxifau attenuates but does uot abolish totally this high affinity binding, iudicatiug that it acts as a partial iuverse agonist at the human H3 receptor. Our fiudiugs obtained with proxyfau in the rat show that the therapeutic iuterest of proteau ligauds is hard to predict. The observatiou that coustitutive activity of the human H3 receptor is easily detected at moderate densities suggests that it occurs in human brain This observatiou may have importaut implications in tenns of drug development. The role of coustitutive activity of H3 autoreceptors in the regulation of histamine ueurous in vivo suggests that iuverse agonists should find therapeutic applications as iiootropic ageiits. References [l] [2] [3] [4] [5]
Arrang JM et al. Nature 1983; 302: 832-7. Arrang JM et al. Nature 1987; 327: 117-23. Morisset S et al. Nature 2000; 408: 86G4. Morisset S et al. B&hem Biophys Res Comnun 2001; Rouleau A et al. Brit J Pharmacol 2002; 135: 383-92.
280: 75-80.