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Amyloid β protein does not interact with tachykinin receptors coupled to inositol phospholipid hydrolysis in human astrocytoma cells
166
Brain Research, 600 (1993) 166-168 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 25498
Amyloid/3 protein does not interact with tachykinin receptors coupled to inositol phospholipid hydrolysis in human astrocytoma cells M. Di Stefano, G. Aleppo, G. Casabona, A.A. Genazzani, U. Scapagnini and F. Nicoletti Institute of Pharmacology, Universityof Catania, School of Medicine, Catania (Italy) (Accepted 6 October 1992) Key words: Amyloid/3 protein; Substance P; Phosphoinositide hydrolysis;UC-11MG astrocytoma cell We have tested the interaction between amyloid /3 protein (A/3P) and tachykinin receptors in cultured UC-11MG astrocytoma cells, which express high affinity substance P receptors and respond to substance P with an unusually large stimulation of polyphosphoinositide hydrolysis. Both the full-length A/3P (A/3Pl_40) and the fragment 25-35 (A/3P25_35) did not affect the stimulation of [3H]inositolmonophosphate (InsP) formation by substance P. A/3P25_35 was also inactive when applied to the cultures 18 or 72 h prior to the assay. In addition, A/3P25_35 did not displace specificallybound [3H]SarMet substance P from its recognition sites in intact UC-11MG cells. These results suggest that, at least in this specific cell type, amyloid peptides do not interact with substance P receptors. Amyloid /3 protein (A/3P), a 39-42 amino acid polypeptide, is the major constituent of amyloid plaques and cerebrovascular deposits characteristic of Alzheimer's disease and Down's syndrome 2,3. H o w A/3P contributes to the pathophysiology of neuronal degeneration in Alzheimer's disease is unclear at present. A/3P added to differentiated hippocampal neurons causes dendritic and axonal retraction followed by neuronal death t4. These effects are mediated by a portion of A/3P (amino acids 25-35), which is structurally homologous to neuropeptides of the tachykinin family, including substance p14. Hence, it has been suggested that tachykinin receptors are involved in the toxic effects of A/3P in the CNS. Accordingly, substance P and physalaemin prevent, whereas tachykinin receptor antagonists mimic, the toxic action of A/3P in cultured neurons ~4. However, based on the evidence that A/3P does not affect substance P-induced Ca2 + mobilization, Mitsuhashi et al. 8 argued that, at least in virustransformed rat kidney cells, substance P receptors do not recognize A/3P as a ligand. We have addressed this problem in U C - 1 1 M G human astrocytoma cells, which express a high density of substance P receptors and respond to substance P with an unusually large stimulation of polyphosphoinositide (PPI) hydrolysis 4. Cell culture The U C - 1 1 M G human astrocytoma cells were kindly provided by Dr. Eric Gruenstein (University of Cincin-
nati). Ceils were suspended in R P M I 1640 (Gibco, Grand Island, NY) (supplemented with 4 m M glutamine and 0.1 m g / m l gentamycin sulfate) containing 10% fetal calf serum, and plated on Nunc plastic Petri dishes at an initial density of 8,000 and 20,000 c e l l s / m l in 35-ram and 24-well dishes, respectively. Ceils became confluent within 4 - 6 days of plating.
Measurement of PPI hydrolysis Stimulation of PPI hydrolysis was estimated by measuring the formation of [3H]inositolmonophosphate (InsP) in the presence of Li +. Cultures grown in 35-mm dishes were preincubated for 16-18 hours with 2 /zCi of myo-[3H]inositol for the labeling of m e m b r a n e inositol phospholipids. Then, cultures were rinsed with K r e b s - H e n s e l e i t buffer (equilibrated with 95% 0 2 / 5 % CO 2 to p H 7.4) containing 10 m M LiC1 with or without 0.1% BSA, and preincubated at 37°C for 15 min. After the addition of substance P or other receptor agonists, the incubation was continued for 60 min. Amyloid peptides, when present, were added at different times prior to the addition of substance P. The reaction (release of [3H]inositolphosphates from m e m b r a n e phospholipids) was terminated by addition of m e t h a n o l / w a t e r (1:1). Ceils were harvested and the suspension was centrifuged at low speed after further addition of c h l o r o f o r m / m e t h a n o l / w a t e r (final proportion 1 : 1 : 1 ) . The [3H]InsP extracted in the
Correspondence: F. Nicoletti, Institute of Pharmacology, University of Catania, Viale A. Doria 6, 95125, Catania, Italy. Fax: (39) (95) 333 219.
167 800
TABLE I
• control v + AOP(25-85)
Stimulation of PPI hydrolysis by substance P in cultures treated with amyloid peptides
r, o •.,~ -
600 e
/
E ~~ L O
""
~
400
Values are means_+S.E.M. of 3-6 individual determinations, n.d. = not done. Amyloid peptides or DMSO have been added 10 rain (in the incubation buffer) or 18 or 72 h (in the culture medium) prior to the addition of substance P.
•/ /
[3H]InsP formation (cpm / dish)
200
0
Control
,
10
g
8
7
- l o g [ S u b s t a n c e P], M Fig. 1. Concentration-dependent stimulation of [3H]InsP formation by substance P in UC-11MG astrocytoma cells incubated in the absence or presence of A/3P25_35 (10 txM). Values are expressed as % over basal, and calculated from the means of 4 individual determinations. For each point, S.D. was less than 10% of the mean value. Basal [3H]InsP formation=2,800_+48 cpm/dish. AflP25_35 was added l0 rain prior to substance P. aqueous phase was separated by a n i o n exchange chromatography, as described previously tl. M e a s u r e m e n t o f [ 3 H ] S a r M e t - s u b s t a n c e P binding
[3H]SarMet-substance P binding was m e a s u r e d in intact cells plated onto 24-well culture dishes at 4°C, to minimize any effect of ligand degradation, ligand internalization, or receptor desensitization 4. Cultures were rinsed with K r e b s - H e n s e l c i t buffer ( p H 7.4) with or without 0.1% BSA, and preincubated for 35 min at 4°C. Then, [3H]SarMet-substance P (2 nM) was a d d e d in the presence of buffer (total binding), 500 nM substance P (non-specific binding) or A/3P25_35. After 50 min of incubation at 4°C (for the kinetics of [3H]SarMet-substance P binding in intact cells, see ref. 4), cultures were extensively washed with ice-cold K r e b s - H e n s e l e i t buffer and cells were solubilized with 0.5 N N a O H . Materials
myo-[3H]Inositol (spec. act. 16.5 C i / m m o l ) and [3H]SarMet-substance P (spec. act. 32.5 C i / m m o l ) were from N E N - D u P o n t (Florence, Italy), substance P, [DPro 2, D-Trp7'9)-substance P and carbamylcholine were from Sigma (St Louis, MO), and quisqualate from Tocris N e u r a m i n (Essex, UK). AflP25_35 and A/3Pl_40 were purchased from B a c h e m (CA) and dissolved in D M S O at an initial concentration of 2 mM. Amyloid peptides maintained their solubility w h e n diluted into K r e b s - H e n s e l e i t buffer or in the culture medium. Peptides in D M S O were diluted at least 200-fold so that the final concentration of D M S O in the incubation buffer or in the culture m e d i u m was never greater than 0.5%. W h e n cultures were incubated in buffer containing 0.1% BSA, addition of substance P led to a concentra-
0.5% DMSO 10 min 2,500 -+250 18 h 2,200_+310 72 h n.d. A/3P25_3s (10 txM) 10 min 2,600 -+320 ]8 h n.d. 72 h 2,000 -+190 A/3PI-40 (10/xM) 10 rain 2,700 -+280
Substance P (1 nM)
5,800 _+110 5,500_+ 98 5,700 -+210 6,600 _+410 6,200_+360 5,500 -+330 5,900 _+960
t i o n - d e p e n d e n t increase in [3H]InsP formation, with an a p p a r e n t ECs0 value of 1.5 n M and a maximal stimulation ranging from 7 - 1 2 fold at 10 n M (Fig. 1). Carbamylcholine (a muscarinic receptor agonist) and quisqualate (a metabotropic glutamate r e c e p t o r agonist) were inactive (data not shown). T h e putative substance P antagonist, [D-Pro z, D-TrpV'9]-substance P, hardly inhibited substance P-stimulated [3H]InsP formation ( c p m / m l - basal 3,000 _+ 350; substance P (2 nM) 20,000 + 500; [D-Pro 2, D-Trpy'9]-substance P (10 IzM) 2,800 _+ 150; substance P + [D-Pro 2, D-TrpV'9]-sub stance P 17,000 _+ 430). AflP25 35 and A/3P1_40 (0.1-10 /zM) did not increase the basal formation of [3H]InsP and did not affect the stimulation of PPI hydrolysis by substance P (Table I and Fig. 1). A/3P25_35 was inactive when applied to the cultures 10 min, 18 or 72 h prior to the addition of substance P (Table I). To exclude the possibility that AC3P could complex with BSA, experiments were also p e r f o r m e d in BSA-free buffer. U n d e r these conditions, substance P was less potent in stimulating PPI hydrolysis, and AflP25_35 (10 /zM) was still TABLE II [ 3H]SarMet-substance P binding to intact UC-11MG cells incubated in the absence or presence of 0.1% BSA and various concentrations of substance P or AflP25_35
Values are means +_S.E.M. of 4 determinations. [3H]SarMet-substance P binding (fmol / mg prot.) + BSA
Total Substance P 10 nM 500 nM A/3P 100 nM 500 nM 10/~ M
- BSA
980 +_36
790 + 40
130+ 13 110 _+13
190_+23 140_+30
980 _+49 880_+58 970 + 70
1,000 _+77 1,100_+27 1,000 _+39
168 inactive in antagonizing the stimulation of [3H]InsP formation induced by 2 or 10 nM substance P (data not shown). As expected (see ref. 4) [3H]SarMet-substance P bound with high affinity to specific recognition sites present on intact UC-11MG astrocytoma cells. Addition of 10 nM substance P displaced the total amount of specifically bound [3H]SarMet-substance P (see Table II), which is consistent with the reported value of about 450 pM for the K D of [3H]SarMet-substance P in intact astrocytoma cells incubated at 4°C4. A/3P25 .~5 failed to displace specifically bound [3H]SarMet-substance P from its recognition sites both in the absence and presence of BSA (Table II). Deposition of A/3P within brain parenchyma and cerebral vessels is considered as an initial event in the pathological cascade leading to tangle formation and neuronal death in Alzheimer's disease. Little is known about the mechanism by which diffuse amyloid plaques evolve into typical 'senile plaques', characterized by dystrophic neurites surrounding the amyloid core (for a review, see ref. 3). One possibility is that A/3P enables the potential toxicity of excitatory amino acids 6 and this increases the vulnerability of neurons that are weakened by an energy deficit, as may occur in Alzheimer's disease brain ~. If the toxic effect of A/3P is receptor mediated, the characterization of A/~P receptor will be crucial for the design of drugs that block the pathological sequelae in Alzheimer's disease. The discovery that substance P and physalaemin antagonize the toxic response of differentiated hippocampal neurons to A/~P 14 suggests that A/3P may interact with tachykinin receptors. We did not confirm this interaction in UC-11MG astrocytoma cells, which express a high density of substance P receptors coupled to PPI hydrolysis 4. Both A/~Pj 41) and A/3P25_35 (the fragment which shows structural analogy with tachykinin neuropeptides) had no intrinsic activity and did not antagonize substance P-stimulated PPI hydrolysis even when applied at concentrations 10,000-fold greater than those of substance P. A/3P25_35 was also inactive when applied 72 h prior to the assay, a time that, in other cell models, is sufficient for peptide precipitation and formation of aggregates in culture 12. The presence of BSA (which facilitated the response to substance P) did not mask a potential effect of A/3P25_35, which was still inactiv e in BSA-free buffer. These results, combined with the lack of displacement of specifically bound [3H]SarMet-substance P by A/3P, suggest that amyloid peptides do not interact with substance P receptors in this specific cell line, unless the affinity of the receptor for A/3P is so low that it requires a ligand concentration greater than 10 IxM. Knowing that there are at least three distinct
tachykinin receptors 7'm'~3 coupled to multiple signal transduction pathways '), we cannot exclude the possibility that A/3P interacts with a specific subtype that is not expressed by UC-11MG astrocytoma cells. It is consistent with this hypothesis that [D-Pro 2, D-TrpT'9) substance P did not prevent substance P-stimulated [3H]InsP formation in UC-11MG astrocytoma cells, whereas it mimics the toxic action of A/3P in mature hippocampal neurons ~4. As an alternative explanation, A/3P and substance P may be recognized by targets other than classical tachykinin receptors, such as the serpin-enzyme complex receptor 5. The role of the serpin-enzyme complex receptor in the neurotoxic activity of A/~P remains to be established. We are grateful to Dr. Eric Gruenstein and Michael J. Lucherini (University of Cincinnati, Medical Center) for kindly providing the UC-11MG astrocytoma cell line. 1 Copani, A., Koh, J.Y. and Cotman, C.W., /3-Amyloid increases neuronal susceptibility to injury by glucose deprivation, NeuroReport, 2 (1991) 763-765. 2 Glenner, G.G. and Wong, C.W., Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein, Biochem. Biophys. Res. Commun., 120 (1984) 885-890. 3 Hardy, J. and Allsop, D., Amyloid deposition as the central event in the aetiology of Alzheimer's disease, Trends Pharmacol. Sci., 12 (1991) 383-388. 4 Johnson, C.L. and Johnson C.G., Characterization of receptors for substance P in human astrocytoma cells: radioligand binding and inositol phosphate formation, J. Neurochem., 58 (1992) 471477. 5 Joslin, G., Krause, J.E., Hershey, A.D., Adams, S.P., Fallon, RJ. and Perlmutter, D.H., Amyloid-/3 peptide, substance P and bombesin bind to the serpin-enzyme complex receptor, J. Biol. Chem., 266 (1991) 21897-21902. 6 Koh, J.-y., Yang, L.L. and Cotman, C.W., /3-Amyloid protein increases the vulnerability of cultured cortical neurons to excitotoxic damage, Brain Res., 533 (1990) 315-320. 7 Liang, T. and Cascieri, M.A., Substance P receptor on parotid cell membranes, J. Neurosci., 1 (1981) 1133-1141. 8 Mitsubashi, M., Akitays, T., Turk, C.W. and Payan, D.G., Amyloid /3 protein substituent peptides do not interact with the substance P receptor expressed in cultured cells, Mot. Brain Res., 11 (1991) 177-180. 9 Nakajima, Y., Tsuchida, K., Negishi, M., Ito, S. and Nakanishi, S., Direct linkage of three tachykinin receptors to stimulation of both phosphatidylinositol hydrolysis and cyclic AMP cascades in transfected chinese hamster ovary cells, J. Biol. Chem., 267 (1992) 2437-2442. 10 Nakanishi, S., Mammalian tachykinin receptors, Annu. Rec. Neurosci., 14 (1991) 123-136. 11 Nicoletti, F., Wroblewski, J.T., Novelii, A., Alho, H., Guidotti, A. and Costa, E., The activation of inositol phosphotipid metabolism as a signal-transducing system for excitatory amino acids in primary cultures of cerebellar granule cells, J. Neurosci., 6 (1986) 1905-1911. 12 Pike, C., Walencewicz, A.J., Glabe, C.G. and Cotman, C.W., In vitro aging of/3-amyloid protein causes peptide aggregation and neurotoxicity, Brain Res., 563 (1991) 311-314. 13 Torrens, Y., Beaujouan, J.C., Saffroy, M., Daguet de Montety, M.C., Bergstrom, L. and Glowinsky, J., Substance P receptors in primary cultures of cortical astrocytes from the mouse, Proc. Natl. Acad. Sci. USA, 83 (1986) 9216-9220. 14 Yankner, B.A., Duffy, L.K. and Kirschner, D.A., Neurotrophic and neurotoxic effects of amyloid/3 protein: reversal by tachykinin neuropeptides, Science, 250 (1990) 279-282.
Brain Research, 600 (1993) 166-168 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 25498
Amyloid/3 protein does not interact with tachykinin receptors coupled to inositol phospholipid hydrolysis in human astrocytoma cells M. Di Stefano, G. Aleppo, G. Casabona, A.A. Genazzani, U. Scapagnini and F. Nicoletti Institute of Pharmacology, Universityof Catania, School of Medicine, Catania (Italy) (Accepted 6 October 1992) Key words: Amyloid/3 protein; Substance P; Phosphoinositide hydrolysis;UC-11MG astrocytoma cell We have tested the interaction between amyloid /3 protein (A/3P) and tachykinin receptors in cultured UC-11MG astrocytoma cells, which express high affinity substance P receptors and respond to substance P with an unusually large stimulation of polyphosphoinositide hydrolysis. Both the full-length A/3P (A/3Pl_40) and the fragment 25-35 (A/3P25_35) did not affect the stimulation of [3H]inositolmonophosphate (InsP) formation by substance P. A/3P25_35 was also inactive when applied to the cultures 18 or 72 h prior to the assay. In addition, A/3P25_35 did not displace specificallybound [3H]SarMet substance P from its recognition sites in intact UC-11MG cells. These results suggest that, at least in this specific cell type, amyloid peptides do not interact with substance P receptors. Amyloid /3 protein (A/3P), a 39-42 amino acid polypeptide, is the major constituent of amyloid plaques and cerebrovascular deposits characteristic of Alzheimer's disease and Down's syndrome 2,3. H o w A/3P contributes to the pathophysiology of neuronal degeneration in Alzheimer's disease is unclear at present. A/3P added to differentiated hippocampal neurons causes dendritic and axonal retraction followed by neuronal death t4. These effects are mediated by a portion of A/3P (amino acids 25-35), which is structurally homologous to neuropeptides of the tachykinin family, including substance p14. Hence, it has been suggested that tachykinin receptors are involved in the toxic effects of A/3P in the CNS. Accordingly, substance P and physalaemin prevent, whereas tachykinin receptor antagonists mimic, the toxic action of A/3P in cultured neurons ~4. However, based on the evidence that A/3P does not affect substance P-induced Ca2 + mobilization, Mitsuhashi et al. 8 argued that, at least in virustransformed rat kidney cells, substance P receptors do not recognize A/3P as a ligand. We have addressed this problem in U C - 1 1 M G human astrocytoma cells, which express a high density of substance P receptors and respond to substance P with an unusually large stimulation of polyphosphoinositide (PPI) hydrolysis 4. Cell culture The U C - 1 1 M G human astrocytoma cells were kindly provided by Dr. Eric Gruenstein (University of Cincin-
nati). Ceils were suspended in R P M I 1640 (Gibco, Grand Island, NY) (supplemented with 4 m M glutamine and 0.1 m g / m l gentamycin sulfate) containing 10% fetal calf serum, and plated on Nunc plastic Petri dishes at an initial density of 8,000 and 20,000 c e l l s / m l in 35-ram and 24-well dishes, respectively. Ceils became confluent within 4 - 6 days of plating.
Measurement of PPI hydrolysis Stimulation of PPI hydrolysis was estimated by measuring the formation of [3H]inositolmonophosphate (InsP) in the presence of Li +. Cultures grown in 35-mm dishes were preincubated for 16-18 hours with 2 /zCi of myo-[3H]inositol for the labeling of m e m b r a n e inositol phospholipids. Then, cultures were rinsed with K r e b s - H e n s e l e i t buffer (equilibrated with 95% 0 2 / 5 % CO 2 to p H 7.4) containing 10 m M LiC1 with or without 0.1% BSA, and preincubated at 37°C for 15 min. After the addition of substance P or other receptor agonists, the incubation was continued for 60 min. Amyloid peptides, when present, were added at different times prior to the addition of substance P. The reaction (release of [3H]inositolphosphates from m e m b r a n e phospholipids) was terminated by addition of m e t h a n o l / w a t e r (1:1). Ceils were harvested and the suspension was centrifuged at low speed after further addition of c h l o r o f o r m / m e t h a n o l / w a t e r (final proportion 1 : 1 : 1 ) . The [3H]InsP extracted in the
Correspondence: F. Nicoletti, Institute of Pharmacology, University of Catania, Viale A. Doria 6, 95125, Catania, Italy. Fax: (39) (95) 333 219.
167 800
TABLE I
• control v + AOP(25-85)
Stimulation of PPI hydrolysis by substance P in cultures treated with amyloid peptides
r, o •.,~ -
600 e
/
E ~~ L O
""
~
400
Values are means_+S.E.M. of 3-6 individual determinations, n.d. = not done. Amyloid peptides or DMSO have been added 10 rain (in the incubation buffer) or 18 or 72 h (in the culture medium) prior to the addition of substance P.
•/ /
[3H]InsP formation (cpm / dish)
200
0
Control
,
10
g
8
7
- l o g [ S u b s t a n c e P], M Fig. 1. Concentration-dependent stimulation of [3H]InsP formation by substance P in UC-11MG astrocytoma cells incubated in the absence or presence of A/3P25_35 (10 txM). Values are expressed as % over basal, and calculated from the means of 4 individual determinations. For each point, S.D. was less than 10% of the mean value. Basal [3H]InsP formation=2,800_+48 cpm/dish. AflP25_35 was added l0 rain prior to substance P. aqueous phase was separated by a n i o n exchange chromatography, as described previously tl. M e a s u r e m e n t o f [ 3 H ] S a r M e t - s u b s t a n c e P binding
[3H]SarMet-substance P binding was m e a s u r e d in intact cells plated onto 24-well culture dishes at 4°C, to minimize any effect of ligand degradation, ligand internalization, or receptor desensitization 4. Cultures were rinsed with K r e b s - H e n s e l c i t buffer ( p H 7.4) with or without 0.1% BSA, and preincubated for 35 min at 4°C. Then, [3H]SarMet-substance P (2 nM) was a d d e d in the presence of buffer (total binding), 500 nM substance P (non-specific binding) or A/3P25_35. After 50 min of incubation at 4°C (for the kinetics of [3H]SarMet-substance P binding in intact cells, see ref. 4), cultures were extensively washed with ice-cold K r e b s - H e n s e l e i t buffer and cells were solubilized with 0.5 N N a O H . Materials
myo-[3H]Inositol (spec. act. 16.5 C i / m m o l ) and [3H]SarMet-substance P (spec. act. 32.5 C i / m m o l ) were from N E N - D u P o n t (Florence, Italy), substance P, [DPro 2, D-Trp7'9)-substance P and carbamylcholine were from Sigma (St Louis, MO), and quisqualate from Tocris N e u r a m i n (Essex, UK). AflP25_35 and A/3Pl_40 were purchased from B a c h e m (CA) and dissolved in D M S O at an initial concentration of 2 mM. Amyloid peptides maintained their solubility w h e n diluted into K r e b s - H e n s e l e i t buffer or in the culture medium. Peptides in D M S O were diluted at least 200-fold so that the final concentration of D M S O in the incubation buffer or in the culture m e d i u m was never greater than 0.5%. W h e n cultures were incubated in buffer containing 0.1% BSA, addition of substance P led to a concentra-
0.5% DMSO 10 min 2,500 -+250 18 h 2,200_+310 72 h n.d. A/3P25_3s (10 txM) 10 min 2,600 -+320 ]8 h n.d. 72 h 2,000 -+190 A/3PI-40 (10/xM) 10 rain 2,700 -+280
Substance P (1 nM)
5,800 _+110 5,500_+ 98 5,700 -+210 6,600 _+410 6,200_+360 5,500 -+330 5,900 _+960
t i o n - d e p e n d e n t increase in [3H]InsP formation, with an a p p a r e n t ECs0 value of 1.5 n M and a maximal stimulation ranging from 7 - 1 2 fold at 10 n M (Fig. 1). Carbamylcholine (a muscarinic receptor agonist) and quisqualate (a metabotropic glutamate r e c e p t o r agonist) were inactive (data not shown). T h e putative substance P antagonist, [D-Pro z, D-TrpV'9]-substance P, hardly inhibited substance P-stimulated [3H]InsP formation ( c p m / m l - basal 3,000 _+ 350; substance P (2 nM) 20,000 + 500; [D-Pro 2, D-Trpy'9]-substance P (10 IzM) 2,800 _+ 150; substance P + [D-Pro 2, D-TrpV'9]-sub stance P 17,000 _+ 430). AflP25 35 and A/3P1_40 (0.1-10 /zM) did not increase the basal formation of [3H]InsP and did not affect the stimulation of PPI hydrolysis by substance P (Table I and Fig. 1). A/3P25_35 was inactive when applied to the cultures 10 min, 18 or 72 h prior to the addition of substance P (Table I). To exclude the possibility that AC3P could complex with BSA, experiments were also p e r f o r m e d in BSA-free buffer. U n d e r these conditions, substance P was less potent in stimulating PPI hydrolysis, and AflP25_35 (10 /zM) was still TABLE II [ 3H]SarMet-substance P binding to intact UC-11MG cells incubated in the absence or presence of 0.1% BSA and various concentrations of substance P or AflP25_35
Values are means +_S.E.M. of 4 determinations. [3H]SarMet-substance P binding (fmol / mg prot.) + BSA
Total Substance P 10 nM 500 nM A/3P 100 nM 500 nM 10/~ M
- BSA
980 +_36
790 + 40
130+ 13 110 _+13
190_+23 140_+30
980 _+49 880_+58 970 + 70
1,000 _+77 1,100_+27 1,000 _+39
168 inactive in antagonizing the stimulation of [3H]InsP formation induced by 2 or 10 nM substance P (data not shown). As expected (see ref. 4) [3H]SarMet-substance P bound with high affinity to specific recognition sites present on intact UC-11MG astrocytoma cells. Addition of 10 nM substance P displaced the total amount of specifically bound [3H]SarMet-substance P (see Table II), which is consistent with the reported value of about 450 pM for the K D of [3H]SarMet-substance P in intact astrocytoma cells incubated at 4°C4. A/3P25 .~5 failed to displace specifically bound [3H]SarMet-substance P from its recognition sites both in the absence and presence of BSA (Table II). Deposition of A/3P within brain parenchyma and cerebral vessels is considered as an initial event in the pathological cascade leading to tangle formation and neuronal death in Alzheimer's disease. Little is known about the mechanism by which diffuse amyloid plaques evolve into typical 'senile plaques', characterized by dystrophic neurites surrounding the amyloid core (for a review, see ref. 3). One possibility is that A/3P enables the potential toxicity of excitatory amino acids 6 and this increases the vulnerability of neurons that are weakened by an energy deficit, as may occur in Alzheimer's disease brain ~. If the toxic effect of A/3P is receptor mediated, the characterization of A/~P receptor will be crucial for the design of drugs that block the pathological sequelae in Alzheimer's disease. The discovery that substance P and physalaemin antagonize the toxic response of differentiated hippocampal neurons to A/~P 14 suggests that A/3P may interact with tachykinin receptors. We did not confirm this interaction in UC-11MG astrocytoma cells, which express a high density of substance P receptors coupled to PPI hydrolysis 4. Both A/~Pj 41) and A/3P25_35 (the fragment which shows structural analogy with tachykinin neuropeptides) had no intrinsic activity and did not antagonize substance P-stimulated PPI hydrolysis even when applied at concentrations 10,000-fold greater than those of substance P. A/3P25_35 was also inactive when applied 72 h prior to the assay, a time that, in other cell models, is sufficient for peptide precipitation and formation of aggregates in culture 12. The presence of BSA (which facilitated the response to substance P) did not mask a potential effect of A/3P25_35, which was still inactiv e in BSA-free buffer. These results, combined with the lack of displacement of specifically bound [3H]SarMet-substance P by A/3P, suggest that amyloid peptides do not interact with substance P receptors in this specific cell line, unless the affinity of the receptor for A/3P is so low that it requires a ligand concentration greater than 10 IxM. Knowing that there are at least three distinct
tachykinin receptors 7'm'~3 coupled to multiple signal transduction pathways '), we cannot exclude the possibility that A/3P interacts with a specific subtype that is not expressed by UC-11MG astrocytoma cells. It is consistent with this hypothesis that [D-Pro 2, D-TrpT'9) substance P did not prevent substance P-stimulated [3H]InsP formation in UC-11MG astrocytoma cells, whereas it mimics the toxic action of A/3P in mature hippocampal neurons ~4. As an alternative explanation, A/3P and substance P may be recognized by targets other than classical tachykinin receptors, such as the serpin-enzyme complex receptor 5. The role of the serpin-enzyme complex receptor in the neurotoxic activity of A/~P remains to be established. We are grateful to Dr. Eric Gruenstein and Michael J. Lucherini (University of Cincinnati, Medical Center) for kindly providing the UC-11MG astrocytoma cell line. 1 Copani, A., Koh, J.Y. and Cotman, C.W., /3-Amyloid increases neuronal susceptibility to injury by glucose deprivation, NeuroReport, 2 (1991) 763-765. 2 Glenner, G.G. and Wong, C.W., Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein, Biochem. Biophys. Res. Commun., 120 (1984) 885-890. 3 Hardy, J. and Allsop, D., Amyloid deposition as the central event in the aetiology of Alzheimer's disease, Trends Pharmacol. Sci., 12 (1991) 383-388. 4 Johnson, C.L. and Johnson C.G., Characterization of receptors for substance P in human astrocytoma cells: radioligand binding and inositol phosphate formation, J. Neurochem., 58 (1992) 471477. 5 Joslin, G., Krause, J.E., Hershey, A.D., Adams, S.P., Fallon, RJ. and Perlmutter, D.H., Amyloid-/3 peptide, substance P and bombesin bind to the serpin-enzyme complex receptor, J. Biol. Chem., 266 (1991) 21897-21902. 6 Koh, J.-y., Yang, L.L. and Cotman, C.W., /3-Amyloid protein increases the vulnerability of cultured cortical neurons to excitotoxic damage, Brain Res., 533 (1990) 315-320. 7 Liang, T. and Cascieri, M.A., Substance P receptor on parotid cell membranes, J. Neurosci., 1 (1981) 1133-1141. 8 Mitsubashi, M., Akitays, T., Turk, C.W. and Payan, D.G., Amyloid /3 protein substituent peptides do not interact with the substance P receptor expressed in cultured cells, Mot. Brain Res., 11 (1991) 177-180. 9 Nakajima, Y., Tsuchida, K., Negishi, M., Ito, S. and Nakanishi, S., Direct linkage of three tachykinin receptors to stimulation of both phosphatidylinositol hydrolysis and cyclic AMP cascades in transfected chinese hamster ovary cells, J. Biol. Chem., 267 (1992) 2437-2442. 10 Nakanishi, S., Mammalian tachykinin receptors, Annu. Rec. Neurosci., 14 (1991) 123-136. 11 Nicoletti, F., Wroblewski, J.T., Novelii, A., Alho, H., Guidotti, A. and Costa, E., The activation of inositol phosphotipid metabolism as a signal-transducing system for excitatory amino acids in primary cultures of cerebellar granule cells, J. Neurosci., 6 (1986) 1905-1911. 12 Pike, C., Walencewicz, A.J., Glabe, C.G. and Cotman, C.W., In vitro aging of/3-amyloid protein causes peptide aggregation and neurotoxicity, Brain Res., 563 (1991) 311-314. 13 Torrens, Y., Beaujouan, J.C., Saffroy, M., Daguet de Montety, M.C., Bergstrom, L. and Glowinsky, J., Substance P receptors in primary cultures of cortical astrocytes from the mouse, Proc. Natl. Acad. Sci. USA, 83 (1986) 9216-9220. 14 Yankner, B.A., Duffy, L.K. and Kirschner, D.A., Neurotrophic and neurotoxic effects of amyloid/3 protein: reversal by tachykinin neuropeptides, Science, 250 (1990) 279-282.