Neuropeptides (1992) 22,99- IO1 0 Longman Group UK Ltd 1992
Interaction of Amyloid Tachykinin Receptors
p Protein (25-35) with
P. ROVERO, R. PATACCHINI, A. R. RENZETTI*, M. BROWN*, J. MIZRAHI”, C. A. MAGGI and A. GIACHEl-TI Pharmacology and Chemistry Department, A. Menarini Pharmaceuticals, Via Sette Santi 3, 50131, Florence, Italy and *Pharmacology Department, Guidotti Pharmaceuticals, Pisa, Italy
Abstract - Amyloid 5 protein (25-35) failed to significantly interact with tachykinin NK, (rat forebrain, guinea-pig ileum) NK, (rabbit pulmonary artery, hamster trachea) or NK-3 (guinea-pig cortex) receptors, as determined by radioligand binding and functional assays. A weak interaction (Ki 14.8 PM) was detected with NK, receptors in rat small intestine. It appears unlikely that direct interaction with tachykinin receptors may account for the reported ability of amyloid 5 protein (25-35) to affect neuronal survival.
Ala-Ile-Ile-Gly-Leu-Met, bears a striking homology with peptides of the tachykinin (TK) family, particularly with the common C-terminal region of TKs which is critical for interaction with NK,, NK, and NK, TK receptors (see ref. 3 for review). Yankner et al. (2) reported that addition of TKs reverse the early neutrotrophic or late neurotoxic actions of amyloid p protein, while addition of TK receptor antagonists (e.g. spantide) mimics the dual action of the protein on neuronal survival. From these results, the involvement of the TK system in the neuronal actions of the amyloid p protein has been suggested but its direct interaction with the TK receptor has never been proven. In this study we have addressed such a question by investigating the ability of the undecapeptide ‘active sequence’ (25-35) of amyloid /3 protein to interact with different TK receptors by radioligand binding and functional studies.
Introduction Deposition of amyloid p protein is thought to be the central event leading to tangle formation and neuronal death in Alzheimer disease (see ref. 1 for review). Recent reports have suggested that the protein exerts a dual action on the survival of hippocampal neurons in culture: neurotrophic for differentiating neurones at low concentrations, neurotoxic for mature neurons at high concentrations (2). These effects are mediated by a discrete partial sequence of the amyloid p protein, encompassing aminoacids from 25 to 35. The amyloid p protein (25-35) undecapeptide or Gly-Ser-Asn-Lys-GlyDate received 2 December 199 1 Date accepted 30 January 1992 Address correspondence to: Dr C. A. Maggi, Pharmacology Department, A. Menarini Pharmaceuticals, Via Sette Santi 3, 5013 1, Florence, Italy
99
100 Methods Radioligand binding Interaction with TK receptors was investigated in membranes from rat forebrain using [3H]substance P, 0.8-l nM at 20°C for 20 min as ligand for NKI receptors, as described by Iwamoto et al. (4), in membranes from rat small intestine using [12sI]neurokinin A, 50-70 pM at 24°C for 30 min as ligand for NK2 receptors as described by Bergstrom et al. (5) and membranes from guinea-pig brain cortex using [3H]senktide 1.8-2 nM at 2O’C for 60 min as ligand for NK3 receptors, as described by Renzetti et al. (6). Bioassay Interaction of amyloid p protein (25-35) with NK, receptors was assessed in the guinea-pig isolated ileum in the presence of atropine (1 pM) using substance P methylester as an agonist and, for NK, receptors, in the endothelium-deprived rabbit pulmonary artery and hamster isolated trachea using neurokinin A as an agonist, as described in Maggi et al. (7). Statistical analysis All data in the text are mean f s.e. of the mean. Ki values were calculated using the LIGAND program (8). Drugs The C-terminal amidated form of amyloid p protein (25-35) or Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-GlyLeu-Met-NH, was synthesized by conventional solid phase methods. Substance P, substance P methylester and neurokinin A were from Peninsula. Atropine HCI was from Serva. Results Amyloid 8 protein (25-35) (10 PM) failed to displace radiolabeled substance P or senktide from NK, (rat forebrain) or NK> receptors (guinea-pig cortex), respectively. In the same experimental conditions Ki of substance P was 0.53 f 0.18 and 523 & 100 nM for NK, and NK, receptors, respectively. The fragment of amyloid p protein displaced [lzsI] neurokinin A from NK, receptors in rat small intestine with a Ki of 14.8 f 2 PM. In the same assay conditions, the Ki for substance P was 0.98 f 0.37 PM.
NEUROPEPTIDES
Amyloid p protein (25-35) had no agonist activity in the isolated guinea-pig ileum, rabbit pulmonary artery or hamster trachea, nor did antagonize the contractile response to substance P methylester (guinea-pig ileum, NK, receptor-mediated) or neurokinin A rabbit pulmonary artery and hamster trachea, NK, receptor-mediated).
Discussion Interaction of the active sequence of the amyloid p protein with NK, or NK, receptors could not be demonstrated in the present experiments. This fragment of amyloid p protein is responsible for the neuronotrophic and neurotoxic activity of amyloid p protein and shares at least 56% homology with various peptides of the TK family, including the C-terminal end, Gly-Leu-MetNH, which is crucial for interaction with NK,, NK, or M(, receptors (3). According to the data of Yanlmer et al. (2), the hypothesis may be advanced that amyloid p protein acts as a TK antagonist: in fact its neuronotrophic and neurotoxic actions are mimicked by TK antagonists and reversed by TK agonists, such as substance P. In particular, the observation that substance P and physalaemin but not neurokinin A or eledoisin were able to revert the action of amyloid j3protein on neuronal survival, led to the suggestion that substance P-preferring (NK,) receptors may be involved. Such an hypothesis is not supported by the present findings. The only significant action of amyloid p protein (25-35) unraveled by the present experiments is a low affinity (pM concentrations) interaction with NK, receptors in membranes of the rat small intestine. As the effect of amyloid p protein on neuronal survival are not reverted by neurokinin A, eledoisin or kassinin (2), which are powerful ligands for NK, receptors (3), it is unlikely that the weak interaction observed here may be involved in the ability of amyloid p protein to affect neuronal survival. In conclusion, the present findings do not support the idea that direct interaction with TKreceptors may be held responsible for the ability of amyloid p protein (2535) to affect neuronal survival. Other mechanisms should be sought to account for its actions on neuronal survival in the context of the pathogenesis of Alzheimer disease.
INTERACTION
OF AMYLOID
p PROTEIN
(Z-35)
WITH TACHYKININ
References 1. Hardy, J. and Allsop, D. (1991). Amyloid deposition as the
2.
3.
4.
5.
central event in the aetiology of Alzheimer’s disease, Trends in Pharmacol. Sci. 12: 383-388. Yankner, B. A., DufQ, L. K. and Kirschner, D. A. (1990). Neurotrophic and neurotoxic effects of amyloid 8 protein: reversal by tachykinin neuropeptides, Science 250: 279-282. Guard. S. and Watson. S. P. (1991). Tachvkininrecentortwes: classification and membrane signallingmechanisms, N&othem. bit. 18: 149-165. Iwamoto, I., Ueki, I and Nadel, J. A. (1988). Effect ofneutral endopeptidase inhibitors on [3H]substance P binding in rat ileum, Neuropeptides 11: 185- 193. Bergstrom, L., Beaujouan, J. J., Torrens, Y., Saffroy, M.,
RECEPTORS
101
Glowinski, J., Lavielle, S., Chassaing, G., Marquet, A., D’Grleans-Juste, P., Dion, S. and Regoli, D. (1988). [3H]neurokinin A labels a specific tachyki& binding site in the rat duodenal smooth muscle, Mol. Pharmacol. 32: 764-771. 6. Renzetti, A. R., Bsrsacchi, P., Criscuoli, M. and Lucacchini. A. (1991). Characterization of NI& binding sites in rat and guinea-pig cortical membranes by the selective ligand [3H]Senktide, Neuropeptides 18: 107-l 14. 7. Maggi, C. A., Patacchini, R., Feng, D. M. and Folkers, K. (1991). Activity of Spantide I and II at various tachykinin receptors and NK* tachykinin receptor subtypes, European Journal of Pharmacology 199: 127-129. 8. Munson, P. J. and Rodbard, D. (1980). LIGAND: a versatile computerized approach for characterization of ligand-binding systems, Anal. Biochem. 107: 220-239.
Interaction of Amyloid Tachykinin Receptors
p Protein (25-35) with
P. ROVERO, R. PATACCHINI, A. R. RENZETTI*, M. BROWN*, J. MIZRAHI”, C. A. MAGGI and A. GIACHEl-TI Pharmacology and Chemistry Department, A. Menarini Pharmaceuticals, Via Sette Santi 3, 50131, Florence, Italy and *Pharmacology Department, Guidotti Pharmaceuticals, Pisa, Italy
Abstract - Amyloid 5 protein (25-35) failed to significantly interact with tachykinin NK, (rat forebrain, guinea-pig ileum) NK, (rabbit pulmonary artery, hamster trachea) or NK-3 (guinea-pig cortex) receptors, as determined by radioligand binding and functional assays. A weak interaction (Ki 14.8 PM) was detected with NK, receptors in rat small intestine. It appears unlikely that direct interaction with tachykinin receptors may account for the reported ability of amyloid 5 protein (25-35) to affect neuronal survival.
Ala-Ile-Ile-Gly-Leu-Met, bears a striking homology with peptides of the tachykinin (TK) family, particularly with the common C-terminal region of TKs which is critical for interaction with NK,, NK, and NK, TK receptors (see ref. 3 for review). Yankner et al. (2) reported that addition of TKs reverse the early neutrotrophic or late neurotoxic actions of amyloid p protein, while addition of TK receptor antagonists (e.g. spantide) mimics the dual action of the protein on neuronal survival. From these results, the involvement of the TK system in the neuronal actions of the amyloid p protein has been suggested but its direct interaction with the TK receptor has never been proven. In this study we have addressed such a question by investigating the ability of the undecapeptide ‘active sequence’ (25-35) of amyloid /3 protein to interact with different TK receptors by radioligand binding and functional studies.
Introduction Deposition of amyloid p protein is thought to be the central event leading to tangle formation and neuronal death in Alzheimer disease (see ref. 1 for review). Recent reports have suggested that the protein exerts a dual action on the survival of hippocampal neurons in culture: neurotrophic for differentiating neurones at low concentrations, neurotoxic for mature neurons at high concentrations (2). These effects are mediated by a discrete partial sequence of the amyloid p protein, encompassing aminoacids from 25 to 35. The amyloid p protein (25-35) undecapeptide or Gly-Ser-Asn-Lys-GlyDate received 2 December 199 1 Date accepted 30 January 1992 Address correspondence to: Dr C. A. Maggi, Pharmacology Department, A. Menarini Pharmaceuticals, Via Sette Santi 3, 5013 1, Florence, Italy
99
100 Methods Radioligand binding Interaction with TK receptors was investigated in membranes from rat forebrain using [3H]substance P, 0.8-l nM at 20°C for 20 min as ligand for NKI receptors, as described by Iwamoto et al. (4), in membranes from rat small intestine using [12sI]neurokinin A, 50-70 pM at 24°C for 30 min as ligand for NK2 receptors as described by Bergstrom et al. (5) and membranes from guinea-pig brain cortex using [3H]senktide 1.8-2 nM at 2O’C for 60 min as ligand for NK3 receptors, as described by Renzetti et al. (6). Bioassay Interaction of amyloid p protein (25-35) with NK, receptors was assessed in the guinea-pig isolated ileum in the presence of atropine (1 pM) using substance P methylester as an agonist and, for NK, receptors, in the endothelium-deprived rabbit pulmonary artery and hamster isolated trachea using neurokinin A as an agonist, as described in Maggi et al. (7). Statistical analysis All data in the text are mean f s.e. of the mean. Ki values were calculated using the LIGAND program (8). Drugs The C-terminal amidated form of amyloid p protein (25-35) or Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-GlyLeu-Met-NH, was synthesized by conventional solid phase methods. Substance P, substance P methylester and neurokinin A were from Peninsula. Atropine HCI was from Serva. Results Amyloid 8 protein (25-35) (10 PM) failed to displace radiolabeled substance P or senktide from NK, (rat forebrain) or NK> receptors (guinea-pig cortex), respectively. In the same experimental conditions Ki of substance P was 0.53 f 0.18 and 523 & 100 nM for NK, and NK, receptors, respectively. The fragment of amyloid p protein displaced [lzsI] neurokinin A from NK, receptors in rat small intestine with a Ki of 14.8 f 2 PM. In the same assay conditions, the Ki for substance P was 0.98 f 0.37 PM.
NEUROPEPTIDES
Amyloid p protein (25-35) had no agonist activity in the isolated guinea-pig ileum, rabbit pulmonary artery or hamster trachea, nor did antagonize the contractile response to substance P methylester (guinea-pig ileum, NK, receptor-mediated) or neurokinin A rabbit pulmonary artery and hamster trachea, NK, receptor-mediated).
Discussion Interaction of the active sequence of the amyloid p protein with NK, or NK, receptors could not be demonstrated in the present experiments. This fragment of amyloid p protein is responsible for the neuronotrophic and neurotoxic activity of amyloid p protein and shares at least 56% homology with various peptides of the TK family, including the C-terminal end, Gly-Leu-MetNH, which is crucial for interaction with NK,, NK, or M(, receptors (3). According to the data of Yanlmer et al. (2), the hypothesis may be advanced that amyloid p protein acts as a TK antagonist: in fact its neuronotrophic and neurotoxic actions are mimicked by TK antagonists and reversed by TK agonists, such as substance P. In particular, the observation that substance P and physalaemin but not neurokinin A or eledoisin were able to revert the action of amyloid j3protein on neuronal survival, led to the suggestion that substance P-preferring (NK,) receptors may be involved. Such an hypothesis is not supported by the present findings. The only significant action of amyloid p protein (25-35) unraveled by the present experiments is a low affinity (pM concentrations) interaction with NK, receptors in membranes of the rat small intestine. As the effect of amyloid p protein on neuronal survival are not reverted by neurokinin A, eledoisin or kassinin (2), which are powerful ligands for NK, receptors (3), it is unlikely that the weak interaction observed here may be involved in the ability of amyloid p protein to affect neuronal survival. In conclusion, the present findings do not support the idea that direct interaction with TKreceptors may be held responsible for the ability of amyloid p protein (2535) to affect neuronal survival. Other mechanisms should be sought to account for its actions on neuronal survival in the context of the pathogenesis of Alzheimer disease.
INTERACTION
OF AMYLOID
p PROTEIN
(Z-35)
WITH TACHYKININ
References 1. Hardy, J. and Allsop, D. (1991). Amyloid deposition as the
2.
3.
4.
5.
central event in the aetiology of Alzheimer’s disease, Trends in Pharmacol. Sci. 12: 383-388. Yankner, B. A., DufQ, L. K. and Kirschner, D. A. (1990). Neurotrophic and neurotoxic effects of amyloid 8 protein: reversal by tachykinin neuropeptides, Science 250: 279-282. Guard. S. and Watson. S. P. (1991). Tachvkininrecentortwes: classification and membrane signallingmechanisms, N&othem. bit. 18: 149-165. Iwamoto, I., Ueki, I and Nadel, J. A. (1988). Effect ofneutral endopeptidase inhibitors on [3H]substance P binding in rat ileum, Neuropeptides 11: 185- 193. Bergstrom, L., Beaujouan, J. J., Torrens, Y., Saffroy, M.,
RECEPTORS
101
Glowinski, J., Lavielle, S., Chassaing, G., Marquet, A., D’Grleans-Juste, P., Dion, S. and Regoli, D. (1988). [3H]neurokinin A labels a specific tachyki& binding site in the rat duodenal smooth muscle, Mol. Pharmacol. 32: 764-771. 6. Renzetti, A. R., Bsrsacchi, P., Criscuoli, M. and Lucacchini. A. (1991). Characterization of NI& binding sites in rat and guinea-pig cortical membranes by the selective ligand [3H]Senktide, Neuropeptides 18: 107-l 14. 7. Maggi, C. A., Patacchini, R., Feng, D. M. and Folkers, K. (1991). Activity of Spantide I and II at various tachykinin receptors and NK* tachykinin receptor subtypes, European Journal of Pharmacology 199: 127-129. 8. Munson, P. J. and Rodbard, D. (1980). LIGAND: a versatile computerized approach for characterization of ligand-binding systems, Anal. Biochem. 107: 220-239.
