Pharmacology & Pharmacy, 2012, 3, 427-432
http://dx.doi.org/10.4236/pp.2012.34057 Published Online October 2012 (http://www.SciRP.org/journal/pp)
1
Pharmacological Evidence for the Involvement of Calcium
Entry through TRPV1 Channels in Nifedipine-Induced
[Ca2+]i Elevation in Gingival Fibroblasts*
Toshimi Hattori#, Toshiaki Ara, Yoshiaki Fujinami
Department of Dental Pharmacology, Matsumoto Dental University, Shiojiri, Japan.
Received July 4th, 2012; revised August 20th, 2012; accepted September 14th, 2012
ABSTRACT
Background: Among anti-hypertension drugs, calcium (Ca2+) antagonists cause gingival overgrowth as a side effect.
We previously discovered that this side effect was due to elevation of the calcium concentration in the cytosol ([Ca2+]i).
Ca2+ entry through non-selective cation channels (NSCCs) and Ca2+ release from intracellular Ca2+ stores are involved
in this [Ca2+]i elevation. Furthermore, we discovered that calcium-sensing receptors (CaSRs) participate in nifedipine-
induced [Ca2+]i elevation. Transient receptor potential (TRP) channels have been identified as NSCCs. In the present
study, we undertook experiments to determine if TRPV1 channels are present in gingival fibroblasts and to ascertain if
nifedipine-activated NSCCs are TRPV1 channels. Methods: Normal human gingival fibroblast Gin-1 cells were used.
The [Ca2+]i was measured using a video-imaging analysis system with the Ca2+-sensitive fluorescent dye fura-2/AM.
Results: The NSCC inhibitor SKF96365 significantly inhibited nifedipine-induced [Ca2+]i elevation. TRPV1 channel
agonists such as capsaicin, olvanil and resiniferatoxin concentration-dependently elevated the [Ca2+]i. The TRPV1
channel activator anandamide concentration-dependently increased the [Ca2+]i. The TRPV1 channel antagonists cap-
sazepine, AMG9810, iodoresiniferatoxin, ruthenium red, and SB366791 significantly inhibited nifedipine-induced
[Ca2+]i elevation. Conclusion: These results suggest that Ca2+ entry through TRPV1 channels is involved in the nifedi-
pine-induced [Ca2+]i elevation seen in gingival fibroblasts. We describe here a modified version of our “calcium trigger
theory”.
Keywords: Nifedipine; Gingival Overgrowth; Gingival Fibroblast; TRPV1 Channel
1. Introduction
Among calcium (Ca2+) antagonists, anti-hypertension drugs
cause gingival overgrowth as a side effect [1]. We pre-
viously discovered that this side effect is due to elevation
of the calcium concentration in the cytosol ([Ca2+]i) and
advocated the “calcium trigger theory” as the developing
mechanism [2]. This theory was based on results show-
ing that Ca2+ antagonists enhance Ca2+ entry through non-
selective cation channels (NSCCs) [3] and Ca2+ release
from the intracellular Ca2+ stores (endoplasmic reticula)
[4].
Furthermore, we discovered that calcium-sensing re-
ceptors (CaSRs) participate in nifedipine (typical Ca2+
antagonist)-induced [Ca2+]i elevation. This hypothesis
was based upon results demonstrating that CaSR agonists
(gentamicin, neomycin, spermine, and LaCl3) elevated
the [Ca2+]i and that the CaSR antagonist NPS2390 inhib-
ited nifedipine-induced [Ca2+]i elevation [5]. It has been
reported that signals from CaSR stimulation activate
NSCCs [6] and Ca2+ release from Ca2+ stores [7].
Transient receptor potential (TRP) channels have been
identified as NSCCs based upon their function [8-10]. In
particular, there are several reports describing transient
receptor potential vanilloid type-I (TRPV1) protein chan-
nels or capsaicin receptors as NSCCs [11-15] and that
TRP channels are expressed in most tissues [16]. These
include smooth muscles in the portal vein [11], oocytes
[12], peripheral sensory neurons [13,14], and central
neurons [17]. Inoue et al. [11] found that the NSCC in-
hibitor SKF96365 blocked the permeability of divalent
cations, and stated that TRP channels could be Ca2+-
permeable, non-selective cation channels. Furthermore,
Stelt and Marzo [17] claimed that the transient receptor
potential vanilloid type I (TRPVI) protein is a non-se-
lective cation channel that belongs to a large family of
TRP ion channels. In the present study, we undertook
*This work was supported by Japan Society for the Promotion of Sci-
ence [Grant-in-Aid for Scientific Research (C) 22592321].
#Corresponding author.

Pharmacological Evidence for the Involvement of Calcium Entry through TRPV1 Channels
in Nifedipine-Induced [Ca2+]i Elevation in Gingival Fibroblasts
428
experiments to determine if TRPV1 channels are present
in gingival fibroblasts and whether nifedipine-activated
NSCCs are TRPV1 channels.
2. Materials and Methods
2.1. Cell Culture
Normal human gingival fibroblast Gin-1 cells were ob-
tained from Dainippon Pharmaceutical Co. Ltd. (Osaka,
Japan). Cells were cultured for 3 - 6 days in Dulbecco’s
modified Eagle medium (Medium 41; Dainippon Phar-
maceutical Co. Ltd.). Cells (5 × 103 per cm2) were plated
on fibronectin-coated glass cover slips adhered to a flexi-
perm disc (Greiner Bio-One GmbH, Göttingen, Germany).
The medium was supplemented with 10% fetal bovine
serum in a humidified atmosphere of 95% air and 5%
CO2 at 37˚C. The medium also contained antibiotics (50
U/ml penicillin and 50 μg/mL streptomycin; Sigma-Al-
drich, St. Louis, MO, USA) and was changed every 2 - 3
days.
2.2. Measurement of the [Ca2+]i
The [Ca2+]i was measured with the Ca2+-sensitive fluores-
cent dye fura-2/AM (Dojindo Laboratories, Kumamoto,
Japan). Cells were kept in a buffer comprising 135 mM
NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM
glucose, and 20 mM HEPES-NaOH (pH 7.4). They were
loaded with the dye by incubation in 5 μM fura-2/AM for
45 min at 37˚C. Cells were then washed to remove ex-
cess fura-2/AM and then incubated in fresh buffer
(without fura-2/AM) for 15 min after incubation to allow
intracellular cleavage of the acetoxymethyl ester conju-
gate (and thus activation) of fura-2. Excitation light from
a xenon lamp was passed through a filter (340 nm or 360
nm). The emission wavelength for analyses was 500 nm.
Changes in the fluorescence intensity of fura-2 in cells
were recorded with a video-imaging analysis system
(FC-400, Furusawa Laboratory Appliance, Kawagoe,
Japan). The [Ca2+]i was determined as the ratio of the
fluorescence stimulated by excitation at 340 nm or 360
nm compared with a standard calibration curve obtained
using a Calcium Calibration Buffer Kit I (Molecular
Probes, Eugene, OR, USA).
To minimise leakage of fura-2, cells were kept at 32˚C
during fluorescence measurements using a bath tempera-
ture controller (DTC-100A; DIA Medical Systems, Ku-
nitachi, Japan). Cells were soaked in a flexiperm cham-
ber containing 0.5 ml of saline and perfused at 8.0 ml/min
with a tubing pump system (Master flex 7524-10; Cole-
Parmer Instrument Company, Barrington, IL, USA). Drugs
at appropriate concentrations were added to the perfusate.
The time of treatment with CaSR agonists was 30 s. To
ensure that fura-2 fluorescence was maintained within
the linear range (i.e., did not become saturated), we se-
lected for analyses cells with a basal [Ca2+]i in the range
50 - 200 nM.
2.3. Chemicals
Tissue culture reagents were purchased from Gibco BRL
(Rockville, MD, USA). Nifedipine, capsaicin, olvanil,
resiniferatoxin, anandamide, capsazepine, AMG9810,
ruthenium red, iodoresiniferatoxin, and SB366791 were
purchased from Sigma-Aldrich. SKF96365 was obtained
from Calbiochem (San Diego, CA, USA). All other
chemicals were supplied by Nacalai Tesque (Kyoto, Ja-
pan). These chemicals were dissolved in dimethyl sul-
foxide (Sigma-Aldrich) as stock solutions, and thereafter
added to the perfusate.
2.4. Statistical Analyses
Data are the mean ± standard error of the mean (SEM)
and the number of observations (N). Statistical analyses
of the data were undertaken by the Student’s two-sided
paired t-test. Differences between mean values were con-
sidered significant if the probability of error (p) was less
than 0.05.
3. Results
The anti-hypertension drug isradipine is a dihydropyri-
dine derivative, just like nifedipine. Isradipine has been
shown to be antagonised by NSCC inhibitors such as
SKF96365, GdCl3, HgCl2, and flufenamic acid [3]. To
confirm that NSCCs are involved in nifedipine-induced
[Ca2+]i elevation, the interaction of SKF96365 with nife-
dipine was examined. SKF96365 significantly inhibited
the effect of nifedipine (nifedipine alone, 60.24 ± 5.38
nM; SKF96365+nifedipine: 32.36 ± 5.59 nM (N = 32, p <
0.001)).
The effects of agonists of TRPV1 channels (capsaicin,
olvanil, and resiniferatoxin) on the [Ca2+]i were exam-
ined to confirm the existence of TRPV1 channels in gin-
gival fibroblasts. Capsaicin (1 - 10 μM, Figures 1(a) and
(b)), olvanil (1 - 20 μM, Figure 1(c)) and resiniferatoxin
(0.5 - 5.0 μM, Figure 1(d)) concentration-dependently
elevated the [Ca2+]i.
We investigated the effect of an activator of TRPV1
channels, anandamide, on the [Ca2+]i. Anandamide (1 -
50 μM) concentration-dependently raised the [Ca2+]i (Fig-
ure 2).
The effects of antagonists of TRPV1 channels (cap-
sazepine, AMG9810, iodoresiniferatoxin, ruthenium red,
and SB366791) were tested. Capsazepine (10 μM),
AMG9810 (10 μM), iodoresiniferetoxin (5 μM), ruthenium
red (50 μM), and SB366791 (10 μM) significantly inhibited
nifedipine-induced elevation of the [Ca2+]i (Figure 3).

Pharmacological Evidence for the Involvement of Calcium Entry through TRPV1 Channels
in Nifedipine-Induced [Ca2+]i Elevation in Gingival Fibroblasts
429
(a)
(b)
(c)
(d)
Figure 1. Elevation of the [Ca2+]i by agonists of the TRPV1
channel. (a) The trace shows a representative time-course of
the [Ca2+]i in the case of capsaicin application; (b) [Ca2+]i
measurements were made in the presence (hatched or black
bars) or absence (clear bars) of capsaicin (1, 2, 5, and 10
μM); (c) In the case of resiniferatoxin application (0.5, 1, 2,
and 5 μM). (d) In the case of olvanil application (1, 5, 10,
and 20 μM). N = 30 (capsaicin), 16 (resiniferatoxin), or 28
(olvanil). Data are mean ± SEM. *p < 0.05, ***p < 0.005, and
****p < 0.001 compared with corresponding pretreatment
values.
Figure 2. Elevation of the [Ca2+]i by an activator of the
TRPV1 channel, anandamide. The upper trace shows a re-
presentative time-course of the [Ca2+]i in the case of anan-
damide application. [Ca2+]i measurements were made in the
presence (hatched and black bars) or absence (clear bars) of
anandamide (1, 5, 10, and 50 μM). Data are mean ± SEM. N =
35. ****p < 0.001 compared with corresponding pretreat-
ment values.
4. Discussion
We previously observed the involvement of CaSRs in
nifedipine-induced elevation of the [Ca2+]i [5]. There are
reports that CaSR stimulation induces NSCC activation
[18,19] and that NSCCs are TRPV1 channels [11-14,
17,20,21]. With respect to TRPV1 channels in non-ex-
citable cells, Myrdal and Steyger [11] reported that there
are more than 20 members of a newly described group of
membrane proteins that act as receptors and ion channels:
the TRP family. They are non-selective, calcium-per-
meant cation channels. Most of them are non-voltage-
gated and are involved in calcium homeostasis (espe-
cially in non-electrically active cells). Thus, the present
study was conducted to determine if TRPV1 channels are
involved in nifedipine-induced [Ca2+]i elevation in gin-
gival fibroblasts.
The effect of the NSCC inhibitor SKF96365 on nife-
dipine-induced [Ca2+]i elevation was investigated to con-
firm a relationship between the actions of Ca2+ antago-
nists and NSCCs in gingival fibroblasts. Nifedipine-in-
duced [Ca2+]i elevation was inhibited by SKF96365,
confirming that Ca2+ entry though NSCCs is involved in
nifedipine-induced elevation of the [Ca2+]i.
To ensure the expression of TRPV1 channels in gin-
gival fibroblasts, effects of agonists (capsaicin, olvanil,
and resiniferatoxin) and an activator of TRPV1 channels

Pharmacological Evidence for the Involvement of Calcium Entry through TRPV1 Channels
in Nifedipine-Induced [Ca2+]i Elevation in Gingival Fibroblasts
430
Figure 3. Inhibition of nifedipine-induced [Ca2+]i elevation by antagonists of the TRPV1 channel. The upper trace shows a
representative time-course of the [Ca2+]i in the case of capsazepine application. [Ca2+]i measurements were made in the pres-
ence (hatched or black bars) or absence (clear bars) of capsazepine (10 μM), AMG9810 (10 μM), iodoresiniferatoxin (5 μM),
ruthenium red (50 μM), and SB366791 (10 μM). Data are mean ± SEM. N = 23 (capsazepine), 35 (AMG9810), 18 (iodores-
iniferatoxin), 24 (ruthenium red), or 29 (SB366791). ****p < 0.001 compared with corresponding pretreatment values.
(anandamide) were tested. All elevated the [Ca2+]i con-
firming, on a functional level, that TRPV1 channels were
present in gingival fibroblasts.
The effects of antagonists of TRPV1 channels (cap-
sazepine, AMG9810, iodo-resiniferatoxin, ruthenium red,
and SB366791) on nifedipine-induced elevation of the
[Ca2+]i were examined to ascertain if nifedipine enhances
Ca2+ entry through TRPV1 channels. All of the antago-
nists of TRPV1 channels significantly inhibited nifedip-
ine-induced [Ca2+]i elevation. These results suggested
that Ca2+ entry through TRPV1 channels was involved in
nifedipine-induced [Ca2+]i elevation in gingival fibro-
blasts.
From the results obtained from our previous studies
[4,5], a “modified calcium trigger theory” regarding the
mechanism of Ca2+ antagonist-induced gingival over-
growth could be postulated (Figure 4). The mechanism
of [Ca2+]i elevation involved three main steps. First, Ca2+
antagonists directly act on a CaSR-linked Gq protein,
which activates phospholipase Cβ to produce inositol
1,4,5-triphosphate (IP3) and diacylglycerol (DAG). Sec-
ond, IP3 and DAG induce the activation of IP3 receptors
in Ca2+ stores and activation of protein kinase C, respec-
tively. Third, the [Ca2+]i is elevated by Ca2+ release from
Ca2+ stores and by Ca2+ entry through TRPV1 channels.
In addition, sustained elevation of [Ca2+]i, which is nec-
Figure 4. Mechanism of Ca2+ antagonist-induced gingival
overgrowth: the “modified calcium trigger theory”. At first,
Ca2+ antagonists elevate the [Ca2+]i by directly stimulating
CaSRs, which activate TRPV1 channels and enhance Ca2+
release from endoplasmic reticula. These actions finally
become an indispensable “trigger” of gingival overgrowth.
CaSR: calcium-sensing receptor; TRPV1: transient recep-
tor potential V1 channel; bFGF: basic fibroblast growth
factor; FGFR: basic fibroblast growth factor receptor; TK:
tyrosine kinase; PLCγ: phospholipase Cγ: IP3: inositol
1,4,5-triphosphate; ER: endoplasmic reticulum; and MAPK:
mitogen-activated protein kinase.

Pharmacological Evidence for the Involvement of Calcium Entry through TRPV1 Channels
in Nifedipine-Induced [Ca2+]i Elevation in Gingival Fibroblasts
431
essary in cell proliferation [22], is caused by positive
feedback related to the secretion of basic fibroblast growth
factor and activation of tyrosine kinase.
5. Conflicts of Interests
The authors declare that they have no conflict of interests
to disclose.
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