Peptides. Vol. 16. No. 2. pp. 21 I-214, 1995 Copyright 0 1995 ElsevierScience Ltd Printedin the USA. All rights reserved
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Pharmacological Evidence for Neurokinin Receptors in Murine Neuroblastoma C 1300 Cells SHIGETOMO
Institute of Applied Biochemistry. University of Tsukuba, Tsukuba, Ibaraki 305, Japan Received
23 May 1994
FUKUHARA, S., H. MUKAI AND E. MUNEKATA. Pharmacological evidence for neurokinin receptors in murine neuroblastoma C1.300 cells. PEPTIDES 16(2) 21 I-214, 199.5.-We found that neurokinin A (NKA) and neurokinin B (NKB) induce an increase in the concentration of intracellular free Ca*+ ([Ca”],) in murine neuroblastoma C 1300 cells (E&,: NKA 87 2 13 nM. NKB 97 ? 15 nM). Substance P (SP) also caused a transient Ca ‘+ increase, although the potency of SP was much less than that of NKA and NKB. The increase in [Ca*‘], induced by NKA and NKB was inhibited by SR 48,968, a selective antagonist for NKI, and [gAla’]NKA(4-IO), a selective agonist for NK2. did not stimulate the increase in [Ca’+],. NKA- and NKB-induced Ca2+ mobilization was not inhibited by CP-96,345 and [Trp’$Ala”]NKA(4- IO), selective antagonists for NK, and NK,, respectively. These results suggested that C 1300 cells express endogenous NK2 neurokinin receptors that have different features from known NK? receptors. Tachykinin
Intracellular free Ca’+
C 1300 cells
are highly metastatic (2), suggesting that Cl300 cells have neurokinin receptors.
NEUROKININ peptides consisting of substance P (SP) (25), neurokinin A (NKA) (9), neurokinin B (NKB) (9). neuropeptide K (NPK) (24), and neuropeptide y (NPy) (8) have a common Phe-X-Gly-Leu-Met-NH2 structure at the C-terminus. These peptides have a wide variety of pharmacological activities such as the contraction of smooth muscle, hypotension, a sialogogic effect, and the depolarization of primary sensory neurons (12). These functions are thought to be mediated by three pharmacologically distinct cell-surface receptors classified as NK,, NK2, and NKS that display limited selectivity toward SP, NKA, and NKB, respectively (12). Recently, neurokinin-related signaling has been investigated using several cell lines transfected with the cDNA of each neurokinin receptor (3,5,13). These studies indicate that the activation of neurokinin receptors induces the stimulation of phospholipase C to cause phosphatidylinositol hydrolysis and Ca*’ mobilization. However, various cells have different signaling modes, except for that of phosphatidylinositol hydrolysis. For example, each rat neurokinin receptor stably expressed in Chinese hamster ovary cells can directly couple to both phospholipase C and adenylate cyclase (13). In C6-2B rat glioma cells transfected with bovine NK2 receptor cDNA, NKA negatively modulates agonist-stimulated adenylate cyclase activity due to the elevation of the intracellular Ca2’ level by activation of the receptor (3). To study the intracellular signal transduction that induces the functions of neurokinins under physiological conditions, we searched for cell lines expressing endogenous neurokinin receptors. We found that neurokinins induce an increase in the concentration of intracellular free Ca2+ ([Ca*+], ) in neuroblastoma Cl300 cells derived from the A/J mouse, which
METHOD Materials Materials were obtained as follows: RPM1 1640 medium and trypsin, from GIBCO (Grand Island, NY); murine neuroblastoma Cl300 cells, from RIKEN Cell Bank (Tsukuba, Japan); fetal calf serum (FCS), from Boehringer Mannheim (Tokyo, Japan); fura2-AM, from DOJIN (Kumamoto, Japan). CP-96,345 (21) and SR 48,968 (1,6) were provided by Dr. A. Nagahisa and Dr. X. Emonds-Ah, respectively. All other chemicals were of reagent grade. Synthetic Peptides
All peptides were synthesized by means of solid-phase chemistry and purified by reverse-phase high performance liquid chromatography (RP-HPLC). The purity of synthetic peptides was confirmed by analytical RP-HPLC. The composition of the peptides was determined by amino acid analysis. The molecular weight of peptides was assessed by fast atom bombardment mass spectrometry. Cell Culture Murine neuroblastoma Cl300 cells were cultured in a humidified atmosphere of 5% CG2/95% air at 37°C in RPM1 1640 me-
’ Requests for reprints should be addressed to Professor Eisuke Munekata. 211
dium supplemented with 10% (v/v) FCS, 100 pg/ml streptomycin, and 100 U/ml penicillin.
The Measurement of [Ca”],
[Ca’ ‘I i (nM)
[Ca2’] i (nM)
Cl 300 cells cultured in 75cm* culture flasks were detached by a brief exposure to 0.02% ethylenediaminetetraacetic acid/ 0.25% trypsin and immediately neutralized with 5 ml of RPM1 1640 medium containing 10% (v/v) FCS. The cells were harvested and washed twice with HEPES-NaOH buffer (140 m&I NaCl, 4 mM KC], 1 m&I MgC12, 1.25 truI4 CaCl,, 1 mM NaH2P04, 11 mM glucose, 0.2% bovine serum albumin, pH 7.4). The [Ca”], was measured using fura-2, a sensitive luminescent calcium chelator, according to the method of Takuwa et al. (22,23). To incorporate fura- into cells, fura-2-AM was added to 4 ml of cell suspension (4 /.LMfinal concentration), followed by an incubation for 1 h at 20°C. After two washes, the cells were placed in buffer at 20°C until use. About 1.0 x 10” cells (1 ml) were transferred to a quartz cuvette and were stirred continuously. The cells were stimulated by the peptides at 30°C instead of 37”C, because intracellular fura- mostly leaked and the exact calculation of the [Ca”], level was difficult at 37°C (22). The responses at 30°C were a little lower than those at 37”C, but the concentration dependence of the peptides at 30°C was same as that at 37°C. Fluorescence was recorded in a CAF-100 spectrofluorometer (Japan Spectroscopy Inc., Tokyo, Japan) with excitation wavelengths of 340 and 380 nm, and an emission wavelength of 500 nm. The [Ca”], was calculated as described by Grynkiewicz et al. (7). We confirmed that the peptides in the cell suspension were not degraded by the residual trypsin after detaching the cells during the stimulation. All peptides were dissolved in dimethyl sulfoxide (DMSO) because of the poor solubility of several of them in distilled water. The final concentration of DMSO was 1% after the addition of peptides, which had no effect upon the induced increase in [Ca*‘],. To examine the effect of antagonists for neurokinin receptors on the increase in [Ca”], induced by NKA and NKB, the cells were stimulated with the peptides after an incubation with antagonists for 1 min.
[Ca2’ ‘I i (nM) 88 58
NKB lO%l lmin
.c 6 T
[Peptide] (M) FIG. 1.Effects of neurokinins on [Ca*‘], in Cl 300 cells. (A) Time course of the neurokinin-induced [Ca”], increase in fura-2-loaded Cl 300 cells. Arrowheads indicate the addition of peptides. Fluorimetric recordings of representative experiments are shown. (B) Concentration-response curves of the [Ca”], increase in Cl300 cells induced by neurokinin and related peptides. Cells were stimulated by the indicated concentrations of SP (0). NKA (N), NKB (0), [gAla’]NKA(4-10) (Cl), and senktide (A). The increase in [Ca”], at each concentration is plotted as a percentage relative to the maximal response induced by NKA at 1.O X lo-’ M. The values are the means + SD of three separate experiments.
Figure l(A) shows typical traces representing the effects of SP, NKA, and NKB on [Ca*‘], in Cl300 cells. Both NKA and NKB at 1.0 X 10m5 h4 induced an immediate but transient increase in [Ca*+],, followed by a sustained increase. SP at 1.0 X 1Om5M also induced a transient increase in Ca*‘, although it was smaller than that induced by NKA and NKB. A sustained Ca*’ increase by SP was not evident from Fig. l(A). The [Ca”], reached a maximal concentration within 20 s after the addition of NKA and NKB. The effects of NKA and NKB on [Ca*‘], were concentration-dependent (E&: NKA 87 ? 13 nM, NKB 97 ? 15 nM) [Fig. l(B)]. The maximal increase in [Ca*+], induced by NKA and NKB was about 200 nM. Figure l(B) shows the concentration-dependent increase in [Ca”], induced by neurokinins and related peptides. Senktide (27) and [pAla*]NKA(410) (18), which are selective agonists for NK2 and NK3, respectively, did not stimulate an increase in [Ca*+], at a concentration of 1.0 X 10-5 M. To characterize the subtype of neurokinin receptor expressed in Cl 300 cells, the inhibitory effects of individual selective antagonists for NK,, NK2, and NK3 on the increase in [Ca*+]i induced by NKA and NKB were examined. NKA- and NKB-stimulated Ca*+ mobilization was inhibited by SR 48,968, a selective antagonist for NK2, in a concentration-dependent manner (It&: about 40 and 50 nM when stimulated by 1.O X lo-’ M NKA and
NKB, respectively) (Fig. 2). However, CP-96,345, a selective antagonist for NK,, affected neither the NKA- nor the NKBinduced Ca*’ mobilization at a concentration of 1.0 x 10m5 M (data not shown). In addition, the increase in [Ca*‘], caused by NKA or NKB was not affected by [Trp7,/3Alaa]NKA(4- 10) (4), a selective antagonist for NK3, at 1.O X 10e6 M (data not shown). DISCUSSION
We demonstrated that NKA and NKB increased the [Ca”], in Cl300 cells. SP also induced Ca*+ mobilization, although to a much lesser extent than NKA and NKB. These results suggest that Cl300 cells endogenously express neurokinin receptors. We examined the effects of selective antagonists for NK,, NK2, and NK3 on the increase in [Ca”], induced by NKA and NKB to characterize the subtype(s) of neurokinin receptors expressed in C 1300 cells. The increase in [Ca*‘], induced by NKA and NKB was inhibited by SR 48,968 (Fig. 2). However, CP96,345 and [Trp7,pAla8]NKA(410) did not affect the increase in [Ca*+], by NKA and NKB. We also examined the effects of selective agonists for NK2 and NKJ on the [C$‘]i, and it was shown that [PAla’]NKA(4-10) and senktide did not stimulate the increase in [Ca”]i [Fig. l(B)]. The subtype(s) of neurokinin
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