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Journal of Oral Science, Vol. 45, No. 3, 139-144, 2003 Original
Cyclopiazonic acid discharges intracellular Ca2+ stores and stimulates Ca2+ influx in cultured human gingival fibroblasts Hiroko Matsumoto§, Reiri Takeuchi§, Takafumi Arai§, Naoto Sato§, Yoshiaki Akimoto† and Akira Fujii§ §Department
of Pharmacology and †Second Department of Oral Surgery, Nihon University School of Dentistry at Matsudo, Chiba 271-8587 (Received 12 June and accepted 7 August 2003)
Abstract: The effect of cyclopiazonic acid (CPA) on changes in the intracellular free Ca2+ concentration ([Ca2+]i) evoked by bradykinin (BK), histamine (HIST) and thapsigargin (TG) was investigated in human gingival fibroblasts. CPA itself dose-dependently stimulated [Ca2+]i responses in both the absence and presence of extracellular Ca2+. Pretreatment with CPA (< 5 µM) enhanced the [Ca2+]i responses evoked by 5 nM BK and 1 mM HIST. However, CPA-pretreatment depressed the [Ca2+]i response evoked by 1 µM TG in a dose-dependent manner. Moreover, CPA accelerated the Ca2+ influx caused by 5 nM BK and 1mM HIST, but did not alter that caused by 1 µM TG. These results indicate that CPA discharges intracellular Ca2+ stores, resulting in their depletion, and enhances Ca2+ influx across the plasma membrane. (J. Oral Sci. 45, 139-144, 2003) Key words: cyclopiazonic acid; bradykinin; histamine; thapsigargin; intracellular free Ca 2+ concentration ([Ca2+]i); human gingival fibroblast.
Introduction Cyclopiazonic acid (CPA), a mycotoxin derived from the Aspergillus and Penicillium genera, has been reported to inhibit the Ca2+-ATPase of the sarcoplasmic reticulum Correspondence to Dr. Akira Fujii, Department of Pharmacology, Nihon University School of Dentistry at Matsudo, 2-870-1 Sakaecho-Nishi, Matsudo, Chiba 271-8587, Japan Tel: +81-47-360-9344 Fax: +81-47-360-9348 E-mail:
[email protected]
(SR) of striated muscle (1-3), smooth muscle (4-8) and cardiac muscle (9), the endoplasmic reticulum of lymphocytes (10), and aortic endothelial cells (11). CPA has also been reported to stimulate Ca2+ influx through nonspecific cation channels in endothelial cells (12,13), in an inositol 1,4,5-trisphosphate (IP3) -independent manner in cultured bovine pulmonary arterial endothelial cells (14) and in urinary bladder smooth muscle (15). However, the effect of CPA on the intracellular free Ca2+ concentration ([Ca2+]i) in gingival fibroblasts has not been elucidated. In the course of a study to clarify the effects of calcium channel blockers such as nifedipine on gingival overgrowth, we demonstrated that fibroblasts originating from nifedipine responders (nifedipine responder, NIFr) tended to exhibit a higher cell proliferation rate and increased synthesis of DNA and collagen than those from nifedipine nonresponders (nifedipine non-responder, NIFn) in the presence of 1 µM nifedipine (16). Furthermore, gingival fibroblasts originating from NIFrs showed a greater cytosolic calcium response to bradykinin (BK), thrombin, prostaglandins E2 and F2α and platelet-derived growth factor, but not to histamine (HIST) and bombecin, than ones originating from NIFns (17). We also demonstrated that tenidap caused discharge of intracellular Ca2+ stores and inhibited Ca2+ influx in cultured human gingival fibroblasts (18). Tenidap also inhibited DNA and collagen synthesis, depressed cell proliferation and lowered the intracellular pH in these cells (19). From these recent data, we concluded that tenidap might be useful in preventing gingival overgrowth caused by calcium channel blockers, especially nifedipine. Since CPA has quite similar properties to tenidap, it was thought worthwhile to investigate its effects on human gingival fibroblasts. The objectives of the present study were therefore to
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determine how CPA affects the [Ca2+]i responses to BK, HIST and thapsigargin (TG) in cultured human gingival fibroblasts.
Materials and Methods Chemicals and Reagents CPA, BK, ionomycin, ethylene glycol-bis (-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) and TG were purchased from Sigma Chemical Co., MO, USA. Fura-2/acetoxymethyl ester (Fura-2/AM) and N,N,N’,N’tetrakis-(2-pyridilmethyl)-ethylenediamine (TPEN) were purchased from Dojin Laboratories, Japan. All the chemicals and reagents used in tissue culturing were purchased from Invitrogen, Carlsbad, CA, USA.
Cells The cell culture was prepared according to a previously reported method (16,20). Briefly, cultures of fibroblast-like cells were established from a gingival specimen obtained from a NIFr patient (a 47 year-old woman who had been treated with nifedipine 40 mg/day for 2 years 8 months) during the clearance of her remaining teeth. The use of the gingival samples was approved by the Committee on Studies Involving Human Beings of Nihon University School of Dentistry at Matsudo (No. EC99-001). Fibroblasts were obtained by trypsinization of the primary outgrowth of cells using a previously described procedure (16,18,21). After establishment, the cells were maintained in DMEM-10 medium (Dulbecco’s modified Eagle’s medium [DMEM] supplemented with 10% fetal calf serum [FCS], streptomycin 100 µg/ml, penicillin G 200 U/ml and amphotericin B 0.2 µg/ml), and routinely passaged using 0.25% trypsin and 0.02% ethylenediamine-N, N, N’, N’tetraacetic acid (EDTA) in Dulbecco’s phosphate-buffered saline (DPBS). The homogeneity of the fibroblasts was determined by flow cytometry (FACS Vantage, Nippon Becton Dickinson, Japan). The fibroblasts used in the experiments exhibited logarithmic-phase proliferation between the 5th and the 8th passage.
Determination of [Ca2+]i The determination of [Ca2+]i was performed in essentially the same manner as reported previously (17,18,20). The fibroblasts were harvested by treatment with 0.25% trypsin and 0.02% EDTA in DPBS, and washed three times with physiological salt solution (PSS) supplemented with 10% FCS. The PSS contained (in mM) NaCl, 136.9; KCl, 15.4; CaCl 2 , 1.5; MgCl 2 , 1.0; 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES), 20; EDTA, 0.01; TPEN, 0.01 and glucose, 5.5 (22). Fura-2/AM (50 µg) was dissolved in 25 µl of dimethylsulfoxide and 187 µl of FCS
was mixed in. PSS supplemented with 10% FCS (6 ml) was added and the resulting mixture was sonicated for 20 s using an Ultrasonic Processor (VP-5, Taitech, Japan). Cell suspension (4 ml in PSS supplemented with 10% FCS) was added to the sonicated solution to give a final Fura-2/AM concentration of 5 µM and a cellular concentration of approximately 106 cells/ml. The cells were loaded with Fura-2/AM at 37°C for 30 min, then washed three times with PSS. The Fura-2-loaded cell suspension (5 × 105 cells/ml in PSS) was kept on ice until use. For the experiments, a portion (2 ml) of the cell suspension was placed in a cuvette and preincubated for 3 min at 37°C. Its fluorescence emission (at 500 nm) was then measured at two different excitation wave lengths, 340 and 380 nm, using a CAF-110 Intracellular Ion Analyzer (JASCO, Japan) at 37°C (18). The [Ca2+]i was estimated from the formula described by Grynkiewicz et al. (23).
Results Homogeneity of the fibroblasts The cell strain used in the present experiment was subjected to flow cytometry to ensure homogeneity in appearance, size (scatter forward, SCF), surface characteristics (FCS) and cytoplasmic features (scatter side, SSC).
Increase in the intracellular Ca2+ response to BK, HIST and TG after CPA-pretreatment The dose-response curve for CPA is shown in Figure 1. In both the presence and absence of 1.5 mM extracellular Ca2+, CPA evoked a [Ca2+]i response in a dose-dependent manner. The [Ca2+]i response was greater in the presence of extracellular Ca2+ than in its absence, indicating that CPA causes Ca2+ influx through the plasma membrane. The effects of pretreatment with CPA for 2 min on the [Ca2+]i responses evoked by BK (5 nM), HIST (1 mM) and TG (1 µM) are shown in Figures 2a, 2b and 2c, respectively. In the presence of 1.5 mM of extracellular Ca2+, the [Ca2+]i response evoked by 5 nM BK was increased by pretreatment with CPA (< 5 µM) in a dosedependent manner (Fig. 2a). In the absence of extracellular Ca2+, CPA pretreatment did not alter the BK-induced [Ca2+]i response. The same trend was seen with the [Ca2+]i response evoked by HIST (1 mM) (Fig. 2b). However, CPApretreatment dose-dependently depressed the [Ca2+]i response evoked by TG (1 µM) (Fig. 2c).
Effect of CPA on Ca2+ influx after BK-, HISTor TG-pretreatment The effect of CPA on Ca2+ influx across the plasma membrane after depletion of intracellular Ca2+ stores by
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5 nM BK and 1 mM HIST in the absence of extracellular Ca2+ is summarized in Figures 3a and 3b, respectively. Ca2+ influx at 0 and 5 min after the addition of CPA was increased in line with the CPA dose by both BK (Fig. 3a) and HIST (Fig. 3b) pretreatment. However, Ca2+ influx was CPA dose-dependently depressed by TG pretreatment (Fig. 3c). The peak [Ca2+]i response is summarized in Table 1.
Discussion As previously reported by others (1-7,10), CPA depleted cytoplasmic Ca2+ stores in gingival fibroblasts. Thus, the [Ca2+]i was increased CPA dose-dependently in the absence of extracellular Ca2+ (Fig. 1). The difference in [Ca2+]i values between experiments carried out in the presence and absence of extracellular Ca2+ indicates that CPA induced the influx of Ca2+ through the plasma membrane. In the presence of 5 nM BK or 1 mM HIST plus extracellular Ca2+, the highest [Ca2+]i response and Ca2+ influx was observed at 5 µM CPA (Figs. 2a and 2b). However, in the presence of 1 µM TG plus extracellular Ca2+, the [Ca2+]i
Fig. 1 Dose-response curve for the effect of CPA on the [Ca2+]i response. CPA depleted intracellular Ca2+ stores dose-dependently (over the range 0 to 50 µM) in both the absence (substitution of 0.5 mM EGTA for CaCl2 in the PSS) and presence (1.5 mM of Ca2+ added to the PSS) of extracellular Ca2+. CPA also increased Ca2+ influx through the plasma membrane. CPA treatment (20 µl; final concentration 0, 1, 5 or 50 µM) was performed at 37°C. : [Ca2+]i response evoked by CPA in the presence of 1.5 mM of extracellular Ca2+. : [Ca2+]i response evoked by CPA in the absence of extracellular Ca2+.
Fig. 2 Effects of CPA pretreatment on [Ca2+]i responses evoked by various stimulants (BK, HIST or TG). The [Ca2+]i responses of gingival fibroblasts evoked by BK (5 nM), HIST (1 mM) or TG (1 µM) after a 2-min pretreatment with CPA (0, 1, 5 or 50 µM) in the absence or presence of 1.5 mM of extracellular Ca2+ were examined at 37°C. (a) [Ca2+]i response evoked by BK (5 nM). (b) [Ca2+]i response evoked by HIST (1 mM). (c) [Ca2+]i response evoked by TG (1 µM). : [Ca2+]i responses evoked by the stimulants after pretreatment with CPA in the presence of 1.5 mM of extracellular Ca2+. : [Ca2+]i responses evoked by the stimulants after pretreatment with CPA in the absence of extracellular Ca2+.
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Fig. 3 Effects of CPA on Ca2+ influx after pretreatment with various stimulants (BK, HIST or TG) for 2 min. Pretreatment with either 5 nM BK, 1 mM HIST or 1 µM TG was carried out for 2 min in the absence of extracellular Ca2+ in order to deplete intracellular stores. This was followed by the addition of Ca2+ (final concentration of 1.5 mM) to the extracellular solution at 0 and 5 min after the addition of CPA (1, 5 or 50 µM). (a) [Ca2+]i response evoked by BK (5 nM). (b) [Ca2+]i response evoked by HIST (1 mM). (c) [Ca2+]i response evoked by TG (1 µM).
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Table 1 Effects of CPA on Ca2+ influx: Cells were subjected to various pretreatments before the addition of 1.5 mM Ca2+ to the extracellular solution. All values represent the [Ca2+]i (nM) after the addition of Ca2+ Pretreatment
0 min
5 min
-
49.0
35.8
1 µM CPA
72.5
57.5
5 µM CPA
131.5
87.8
50 µM CPA
216.0
212.8
5 nM BK + 1 µM CPA
133.0
99.0
+ 5 µM CPA
163.0
154.0
+ 50 µM CPA
358.0
231.0
1 mM HIST + 1 µM CPA
129.0
108.0
+ 5 µM CPA
144.0
141.0
+ 50 µM CPA
340.0
231.0
246.5
349.5
1 µM TG + 1 µM CPA + 5 µM CPA
258.0
356.0
+ 50 µM CPA
193.5
203.5
response and Ca2+ influx decreased in line with the CPA dose (Fig. 2c). This indicates that the effect of CPA is the same as that of TG (i.e., inhibition of Ca2+-ATPase), but not completely the same as those of BK and HIST (which produce IP3, thereby resulting in the depletion of Ca2+ stores). The Ca2+ influx at 0 and 5 min after the addition of CPA was dose-dependently accelerated by CPA when 5 nM BK or 1 mM HIST was used for pretreatment (Figs. 3a and 3b). However, pretreatment with 1 µM TG did not produce a CPA dose-dependent alteration in Ca2+ influx (Fig. 3c). This also indicates that CPA possess the same function as TG. In our previous study, we demonstrated that tenidap depleted intracellular Ca2+ stores and decreased Ca2+ influx in gingival fibroblasts (18,19). Since CPA has the same effect as tenidap in depleting intracellular Ca2+ stores, we were interested to clarify whether it shared the same ability to inhibit Ca2+ influx through the plasma membrane. We found that CPA depletes intracellular Ca2+ stores by inhibiting Ca2+-ATPase, and that it enhances Ca2+ influx; these properties are exactly the same as those of TG. However, CPA did not inhibit Ca2+ influx through the plasma membrane. Therefore, unlike tenidap, CPA might not be suitable for depressing gingival overgrowth (19).
Acknowledgments This work was supported in part by a Suzuki Grant for Joint Research 1997 (A.F.) from Nihon University School
of Dentistry at Matsudo, and by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (09671908 [A.F.], 1997-1998; 10557173 [A.F.], 1998-1999) and from the Japanese Society for the Promotion of Science (11671885 [H.M.], 1999-2000).
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