Inhibitory effects of β-amyloid peptides on nicotine-induced Ca2+ influx in PC12h cells in culture

ELSEVIER

Neuroscience Letters 173 (1994) 147 150

Inhibitory effects of fl-amyloid peptides on nicotine-induced PC12h cells in culture

,

C a 2+

influx in

Takato Takenouchi and Eisuke Munekata* Institute of Applied Biochemistry, University of Tsukuba, Tsukuba 305, Japan Received 26 January 1994; Revised version received 11 April 1994; Accepted 11 April 1994

Abstract

Syntheticfl-amyloidpeptides and the neuropeptide substance P (SP) were examined for their ability to modulate nicotinic response in PCI2h cells, a subclone of PC12 cells. SP, flA1--40 and its peptide fragment flA25-35-NH2 significantlyinhibited an increase in cytoplasmic calcium concentrations ([Ca2+]0 induced by nicotine in a dose-dependent manner. Furthermore, flA1-40 was found to inhibit the [Ca2+]iincrease induced by depolarization with a high concentration of potassium. These findings show that bothflA 1 and flA25-35-NH2 may mimic the function of SP on inhibition of nicotinic response through different mechanisms. Key words."fl-Amyloid peptide; Substance P; PC12h cell; Nicotinic response

fl-Amyloid protein (fl-AP) containing 39-43 amino acid residues is one of the major constituent of senile plaques in Alzheimer disease (AD). fl-AP has been known to show a neurotrophic or neurotoxic effect on cultured neuronal cells. Yankner et al. reported that the undecapeptide fragment offl-AP (amino acid residues 25 to 35, flA25"35) had activities similar to the full-length fl-AP in undifferentiated and differentiated neurons [22]. Since flA25-35 shares the C-terminal structure, Gly-LeuMet, with the tachykinin family peptides, tachykinins were tested for their effects on trophic and toxic actions offl-AE In particular, substance P (SP) which is a representative tachykinin peptide could reverse the effect of fl-AP on hippocampal neurons in culture [22] and prevent the neuronal death caused by fl, AP in adult rat brain [12]. From these findings, the relationship between SP and fl-AP on neuronal cells has attracted much interest. Initially, fl-AP was assumed to exhibit these activities through the tachykinin receptors on the surface of the neurons. However, fl-AP and minimal active sequence flA25-35 were not seen to significantly interact with the

* Corresponding author. 0304-3940/94/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3940(94)00309-X

NK-1 receptor known as the SP receptor and other tachykinin receptors from some studies using tissue contraction and radiofigand binding assays [16], or cultured cell lines responding to tachykinin [15, 19]. Hence, there should be unknown pathways to account for these flAP effects as distinguished from conventional tachykinin receptor mediation. Although SP is known to have several pharmacological properties, such as contracting activity on various smooth muscles, hypotensive effects and sialogogic effects, most of these activities could not be explained by the mediation of the NK- 1 receptor. One of the functions of SP that is thought not to be mediated by the NK-1 receptor is the modulation of the nicotinic response on adrenal chromaffin cells [4] or PC12 cell lines [17], because antagonists of SP on the NK-1 receptor did not display antagonistic activities against this action [1,10]. Also, Geraghty et al. suggested the existence of other SP binding site as distinguished from the NK-1 receptor in the bovine adrenal medulla [5]. SP has been suggested to enhance and stabilize a desensitized state of the nicotinic acetylcholine receptor at lower concentrations of nicotine. In the present study, we examined whether a peptide corresponding to the first 40 amino acids of/~AP (flA1 40), flA25-35 peptides. C-terminal carboxyl-free form

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T Takenouchi, E. Munekata/Neuroscience

Letters 173 (1994) 147 150

(A) [Ca2-]i

nM

117

T

*

nicotine

SP nicotine

T

T

13A25-35-NH= nicotine

T

I"

T

Imln

~A1-40 nicotine 13A25-35-OH nlcollne

(B) [Ca2*]i

02

nM

_2

T nicotine

T NKA

T nicotine

-J

'~T- T NKB nicotine

=1rain i

Fig. i. A: effects of SR flA25 35-NH2, f l A l 4 0 and flA25-35-OH on the 32 y M nicotine-induced [Ca>]i increase in PCI2h cells at a peptide concentration of 10/~M. B: NKA and NKB did not significantly affect the [Ca2+]~ increase induced by 32 ruM nicotine at 10 yM.

flA25-35-OH (NH2-GSNKGAIIGLM-COOH) and amidated derivative flA25-35-NH2 modulated the nicotinic response as well as did SP on the PC12h cells, a subclone of PC I2 cells [7], as a model system for studying SP function. To clearly investigate these actions, we examined the inhibitory activity of these peptides against the intracellular increase in free Ca > concentrations ([Ca2+]0 induced by nicotine using a calcium sensitive indicator dye, fura-2 [6]. PC12h cells were a gift from Dr. H. Hatanaka (Osaka University, Japan) and routinely grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with F-12 Nutrient Mixture (Ham) containing 5% horse serum and 5% precolostrum newborn calf serum, and incubated in a humidified 90% air/10% CO2 incubator at 37°C. The peptides used in this study were manually synthesized with a glass vessel by the Fmoc solid phase method. Coupling reactions in chain elongations were carried out by the BOP-HOBt method [8]. All couplings were checked by Kaiser's ninhydrin test [9]. Peptides were cleaved by trifluoroacetic acid and purified by reverse phase H P L C on a Nucleosil 300 7C18. The purity of the peptides was confirmed by analytical HPLC and amino acid analysis. The molecular weight of f l A l ~ 0 was also checked by fast atom bombardment mass spectrometry (FAB-MS). [Ca>]~ measurement was carried out according to the method ofTakuwa et al. [20]. The PC12h cell suspension was centrifuged, and the cells were resuspended, and washed in HEPES-NaOH buffer (140 mM NaC1, 4 mM KCI, 1 mM MgC12, 1.25 mM CaC12, 1 mM NaH2PO4, 11 mM glucose, 0.2% bovine serum albumin, pH 7.4). Fura 2-AM was added to the cell suspension (5/IM final concentration) and loaded for 1 h at 20°C. After two washings in the buffer, approximately 10 6 cells were used

in each set of experiments, monitoring with a CAF-100 spectrofluorometer (Japan Spectroscopy Inc., Tokyo, Japan) at 30°C. The free Ca 2+ concentration was calculated from the measurements of the ratio of fluorescence intensities with an excitation wavelength of 340 nm and 380 nm, emission wavelength of 500 nm as described by Grynkiewicz et al. [6]. These peptides were dissolved and immediately used for the experiments after lyophilization. f l A l ~ 0 peptide was dissolved in dimethylsulfoxide (DMSO) solution since this peptide had poor solubility in aqueous solution. To examine the precise effect of the peptides on [Ca2+]i increase stimulated by nicotine in PC12h cells, 32 p M of nicotine was used because the nicotinic acetylcholine receptor was rapidly desensitized by nicotine itself and resulted in a decrease of [CaZ+]~ at concentrations higher than 32 ¢tM. Fig. 1A illustrates the inhibitory effects of SP and fl-amyloid peptides on PCI2h cells. Addition of 32 p M nicotine evoked a rapid [Ca>]~ increase due to extracellular Ca >, followed by a gradual decrease of Ca 2+ concentration and came to a sustained phase that was a higher Ca 2+ concentration than the basal level, When the cells were pretreated with 10/~M SP, this nicotinic response pattern was changed accompanying the desensitization. The inhibition pattern offlA25-35-NH 2 on the nicotinic response was very similar to that of SE This suggests that flA25-35-NH2 acts via the same mechanism as SP and does not involve competition with nicotine for the receptor recognition site on the PC12h cells [18]. Although flAl~40 significantly inhibited the nicotinic response, the inhibition pattern was different from that of SP in rapid desensitization and the sustained phase, f l A 1 4 0 seems to directly block the nicotine-induced extracellular Ca > flux into cells rather than enhance the desensitization of the nicotinic acetylcholine

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T Takenouchi, E. Munekata/Neuroscience Letters 173 (1994) 147-150

o

80

O O O

6O

o~

40' 2O

~

10"10

,6A1-40

10"9

10"8

~ ,

10"7

10"6

10"5

10"4

PEPTIDE C O N C E N T R A T I O N (M)

Fig. 2. Concentration dependence of the effects of SP, flA25-35-NH 2 and flA1M0 on the [Ca2+]i increase induced by nicotine. The maximal [Ca2÷]~ induced by 32 ,uM nicotine at one minute after each peptide injection was indicated as a percentage of the positive control value, [Ca2+]i induced by 32/IM nicotine alone. Values are means + S.E.M. (n = 3-5). *P < 0.001, **P < 0.01 (vs. pretreatment with each peptides at 1 nM).

receptor, flA25-35-OH did not cause a measurable inhibitory response in [Ca2+]i increase stimulated by 32/~M nicotine at a peptide concentration of 10/IM. Therefore, the structure of the C-terminal is important to conserve the inhibitory potency offlA25-35 peptide to say nothing of SP [2]. Other mammalian tachykinins, neurokinin A (NKA) and neurokinin B (NKB), could not significantly inhibit the [Ca2+]i increase induced by 32 FtM nicotine at 10 tiM (Fig. 1B). NKB was dissolved in DMSO. Fig. 2 shows the dose-dependent inhibitory actions of SP and fl-amyloid peptides on the nicotinic response. SP inhibited the nicotine-induced [Ca2+]i increase from approximately 1/IM, and showed nearly 70% inhibition at 10/IM. flA25-35-NH2 caused a dose-dependent inhibition to the nicotine-induced [Ca2+]i increase, flA 1-40 also had the same dose-dependent inhibitory activity. The dose-dependent inhibition curves of flA25-35-NH 2 and flA1-40 were shifted slightly to the right compared with SE The difference in the inhibition patterns between flAl40 and flA25-35-NH 2 prompted us to investigate the effect offlAl-40 on the [Ca2+]i increase induced by depolarization with a high concentration of potassium. The rapid [Ca2÷]i increase stimulated by 60 mM K ÷ was due to extracellular Ca 2+ influx through the voltage-dependent calcium channels on the surface of PC 12h cells, since this [Ca2+]i increase disappeared by the addition of 4 mM EGTA and was blocked by 100nM nifedipine, which is an L-type voltage-dependent calcium channel blocker (Fig. 3A). SP and flA25-35-NH2 could not significantly inhibit the [Ca2+]i increase induced by 60 mM K + at 10 HM (Fig. 3A). flAI-40 inhibited the 60 mM K + induced [Ca2+]i increase in a dose-dependent manner (Fig. 3B), suggesting that it blocks the Ca 2+ influx from extracellu-

lar in PC12h cells, probably caused by direct interaction with the cell membranes because of its hydrophobicity. Cheung et al. reported that ]3A25-35-NH2 inhibited the 10/IM nicotine-induced catecholamine secretion in a manner analogous to SP in cultured bovine adrenal chromaffin cells [3]. Concerning these inhibitory actions offlA25-35-NH2 on the nicotinic response, our findings were in agreement with their findings because the catecholamine release was necessary to increase the [Ca2+]i in these cells [21]. Although they could not test the effect of/~A1-40 for its low solubility in aqueous buffers, we observed the inhibitory effect offlA 1-40 on the nicotinic response using the addition of 1.0% DMSO in buffer (A) [Ca2*], nM

t high-K +

? nlfedipine

135

~ high-K+

T I~,A1-40

l high-K +

i~ SP

T high.K*

-- -

1"

T

high-K + [~A25-35-NH2

~ high-K*

(B)

120 100

B0 O fJ

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20

0

1

10

32

~A~-40 (pM) Fig. 3. A: effects of 100 nM nifedipine and 10/IMflAI-40 on the [Ca2+]i increase induced by 60 m M K + in PC12h cells, flA25-35-NH2 and SP could not inhibit the [Ca2+]~ increase induced by 60 m M K ÷ at 10/~M. B: concentration dependence of the effect of flA1-40 on the [CaZ+], increase induced by 60 m M K +. The [Ca2+]~ increase induced by 60 m M K ÷ at one minute after addition offlAI-40 was indicated as a percentage of the positive control value, [Ca2+]~ induced by 60 m M K* alone. Values are means + S.E.M. (n = 3). *P < 0.001 (vs. pretreatment with f l A l ~ 0 at 1/2M).

150

77 fakenouchi. E, Munekata / N2,uroscience Letters 173 f1994) 147 150

because the low concentration of DMSO had no influence on the [Ca>]i induced by nicotine. One common feature of some identified neurotoxins is the unusual activation of [Ca=+]i [11,14]. fl-Amyloid peptides also has been reported to elevate rest levels of calcium and enhance calcium responses to excitatory amino acids and calcium ionophore in human cortical neurons [13]. Though it is difficult to consider that this inhibitory effects offl-amyloid peptides are directly connected with its neurotoxicity, our findings indicate that flA25 35-NH= may interact with unknown SP binding site on PCl2h cells as distinguished from the NK-1 receptor and provide a way to characterize the inhibitory activity of SP. In conclusion, we have shown that both flA1-40 and flA25 35-NH2 could inhibit the [Ca>]~ increase caused by nicotine on PC I2h cells as well as SR but the actions of both peptides might be mediated by different mechanisms. This also raises the possibility that high concentrations of/~AP prevent normal neuronal transmissions. [1] Boksa, P. and Livett, B.G., The substance P receptor subtype modulating catecholamine release from adrenal chromaffin cells, Brain Res., 332 (1985) 29-38. [2] Boyd, N.I). and Leeman, S.E,, Multiple actions of substance P that regulate the functional properties of acetylcholine receptors of clonal rat PC12 cells, J. Physiol., 389 (1987) 69-97. [3] Cheung, N.S., Small, D.H. and Livett, B.G., An amyloid peptide, flA4-25 35, mimics the function of substance P on modulation of nicotine-evoked secretion and desensitization in cultured bovine adrenal chromaffin cells, J. Neurochem., 60 (1993) 1163 1166. [4] Clapham: D.E. and Neher, E., Substance P reduces acetylcholineinduced currents in isolated bovine chromaffin ceils, J. Physioh, 347 (19841 255 277. [5] Geraghty, D.P., Livett, B.G., Rogerson, E.M. and Burcher, E., A novel substance P binding site in bovine adrenal medulla, Neurosci. Lett., 112 (1990) 276 28 I. [6] Grynkiewicz, G., Poenie, M. and Tsien, R. Y., A new generation of Ca > indicators with greatly improved fluorescence properties, J. Biol. Chem., 260 (1985) 3440 3450. [7] Hatanaka. H.. Nerve growth factor-mediated stimulation of tyrosine hydroxylase activity in a clonal rat pheochromocytoma cell line, Brain Res., 222 (1981) 225 233. [8] Hudson, D., Methodological imprecations of simultaneous solidphase peptide synthesis. 1. comparison of different coupling procedures, J. Org. Chem.. 53 (1988) 617-624.

[9] Kaiser, E., Colescott, R.L., Bossinger, C.D. and Cook, El., Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides, Anal. Biochem., 34 (1970) 595 598. [10] Khalil, Z., Marley, P.D. and Liven, B.G., Effect of substanceP on nicotine-induced desensitization of cultured bovine adrenal chromarlin cells: possible receptor subtypes, Brain Res., 459 (1988) 282-288. [11] Kornecki, E. and Ehrlich, Y,H., Neuroregulatory and neuropathorogical actions of the ether-phospholipid platelet-activating factor, Science, 240 (1988) 1792 1794. [12] Kowall, N.W., Beal, M.F., Busciglio, J.. Dully, L.K. and Yankner, B.A., An in vivo model for the neurodegenerative effects of fl-amyloid and protection by substance P, Proc. Natl. Acad. Sci. USA, 88 (199l) 7247 7251. [13] Mattsom M.E, Cheng, B., Davis, D., Bryant, K., Lieberburg, 1. and Rydel, R.E., fl-Amyloid peptides destabilize calcium homeostasis and render human cortical neurons vulnerable to excitotoxicity, J. Neurosci., 12 (1992) 376 389. [14] Mattson, M.P., Rychlik, B., You, J,S. and Sisken, J.E., Sensitivity of cultured human embryonic cerebral cortical neurons to excitatory amino acid-induced calcium influx and neurotoxicity, Brain Res., 542 (1991) 97 106. [15] Mitsuhashi, M., Akitaya, T., Turk, C.W. and Payan, D.G., Amyloid fl protein substituent peptides do not interact with the substance P receptor expressed in cultured cells, Mol. Brain Res., I I (1991) 177 180. [16] Rovero, P., Patacchini, R., Renzetti, A.R., Brown, M., Mizrahi, J., Maggi, C.A. and Giacheni, A., Interaction ofamyloidfl protein (25 35) with tachykinin receptors, Neuropeptides, 22 (1992) 99 101. [17] Sirnasko, S.M., Durkin, J.A. and Weiland, G.A., Effects of substance P on nicotinic acetyleholine receptor function in PC12 cells, J. Neurochem.. 49 (1987) 253 260. [18] Stallcup, W.B. and Patrick, J., Substance P enhances cholinergic receptor desensitization in a clonal nerve cell line, Proc. Natl. Acad. Sci. USA, 77 (1980) 634 638 [19] Stefano, M.D., Aleppo, G., Casabona. G., Genazzani, A.A., Scapagnini, U. and Nicoletti, F., Amyloid fl protein does not interact with tachykinin receptors coupled to inositol phospholipid hydrolysis in human astrocytoma cells, Brain Res., 600 (1993) 166 168. [20] Takuwa, N., Takuwa, Y,, Yanagisawa, M., Yamashita, K. and Masaki, T,, A novel vasoactive peptide endothelin stimulates mitogenesis through inositol lipid turnover in Swiss 3T3 fibroblasts, J. Biol. Chem., 264 (1989) 7856 7861. [21] Wilson, S.E and Kirschner, N., Calcium-evoked secretion from digitonin-permeabilized adrenal medullary chromaffin cells, J. Biol. Chem., 258 (1983) 4994 5000. [22] Yankner, B.A., Duffy, L.K. and Kirschner, D.A., Neurotrophic and neurotoxic effects ofamyloid fl protein: reve.'sal by tachykinin neuropeptides, Science, 250 (1990) 279 282.