Full text of DOI:10.1038/sj.bjp.0704286

British Journal of Pharmacology (2001) 134, 233 ± 236
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SPECIAL REPORT
Selective inhibition of thapsigargin-induced contraction and
capacitative calcium entry in mouse anococcygeus by
tri¯uoromethylphenylimidazole (TRIM)
1
1
1
*,1Alan Gibson, Filomena Fernandes, Pat Wallace & Ian McFadzean
1Centre for Cardiovascular Biology & Medicine, School of Biomedical Sciences, King's College London, Hodgkin Building,
Guys Campus, London SE1 9RT
This study examined the e€ects of tri¯uoromethylphenylimidazole (TRIM) on tone, and calcium
entry, in mouse anococcygeus stimulated by either thapsigargin (Tg; 100 nM) which activates
capacitative calcium entry (CCE), or high K (60 mM) which activates voltage-operated calcium
channels. TRIM (1 ± 333 mM) produced concentration-related relaxation of Tg-induced tone (EC50,
42 mM) but was much less e€ective against high K. In single smooth muscle cells loaded with
FURA-2, TRIM reduced the increase in ¯uorescence ratio produced by Tg but had no e€ect on that
produced by high K. The relaxations of Tg-induced tone, and reduction in ¯uorescence ratio, were
obtained in the presence of L-NG-nitroarginine and were thus independent of nitric oxide synthase
inhibition; further, TRIM had no discernible e€ect on nitrergic responses. TRIM provides a novel
drug for the selective inhibition of CCE and a template for the development of more potent
inhibitors.
British Journal of Pharmacology (2001) 134, 233 ± 236
Keywords: Anococcygeus (mouse); capacitative calcium entry; potassium; smooth muscle; thapsigargin; TRIM
Abbreviations: CCE, capacitative calcium entry; ER/SR, endoplasmic/sarcoplasmic reticulum; L-NOARG, L-NG-nitroarginine;
SOC, store-operated channel; Tg, thapsigargin; TRIM, tri¯uoromethylphenylimidazole; TRP, transient receptor
potential
Introduction In many cell types, including smooth muscle
(Gibson et al., 1998), depletion of the calcium stores within
the endoplasmic/sarcoplasmic reticulum (ER/SR) activates
calcium-permeable, store-operated channels (SOCs) in the
plasma membrane, initiating so-called capacitative calcium
entry (CCE; Putney, 1990; Berridge, 1995; Parekh & Penner,
1997; Putney & McKay, 1999). There is currently intense
interest in CCE directed mainly towards identifying the signal
between the ER/SR and the plasma membrane, the molecular
identity of the SOC proteins, and the cellular functions
dependent on calcium entry via this route (Putney & McKay,
1999; Harteneck et al., 2000). However, although drugs are
available for the selective activation of CCE, including
blockers of the ER/SR Ca-ATPase such as thapsigargin
(Tg), research is hindered by a paucity of selective inhibitors
of CCE. The most commonly used drugs, including
SKF96365 (Merritt et al., 1990), inhibit calcium entry
through both voltage-dependent and voltage-independent
channels at similar concentrations (Clementi & Meldolesi,
1996). There is therefore a pressing need for the identi®cation
of more selective inhibitors of CCE. Here, we report that
tri¯uoromethylphenylimidazole (TRIM) produces selective
inhibition of Tg-induced CCE in the mouse anococcygeus
muscle and that this action of TRIM is independent of its
previously reported ability to inhibit nitric oxide synthase
(Handy et al., 1995; Moore & Handy, 1997).
Methods Tension studies Male mice (LACA; 25 ± 35 g)
were killed by stunning and exsanguination. Anococcygeus
muscles were dissected and set up for the recording of
isometric tension responses to drugs and nitrergic ®eld
stimulation (10 Hz; 10 s trains every 100 s) as described
previously (Gibson et al., 1994). The composition of the
Krebs solution was (mM): NaCl 118.1, KCl 4.7, MgSO4 1.0,
KH2PO4 1.0, glucose 11.1, NaHCO3 25.0, CaCl2 2.5; the
solution was maintained at 378C and gassed continuously
with 95%O2:5%CO2. High K (60 mM) Krebs solution was
prepared by increasing the amount of KCl and reducing
NaCl appropriately to maintain isotonicity. In all
experiments,
the
Krebs
solution
contained
1 mM
phentolamine and 50 mM L-NG-nitroarginine (L-NOARG),
with the exception that the latter was omitted in experiments
involving nitrergic stimulation. When Tg was used to raise
tone the Krebs solution also contained 10 mM verapamil to
block Ltype voltage-operated calcium channels; at this
concentration, verapamil completely prevented contraction
to 60 mM K solution (not shown). To measure the e€ects of
relaxant drugs (TRIM, papaverine, SKF96365, miconazole),
muscle tone was raised with either 100 nM Tg or 60 mM K;
once a stable increase in tension had been established, the
relaxants were applied and the per cent relaxation of induced
tone calculated after 4 min.
Fura-2 fluorescence Single smooth muscle cells were isolated
from the mouse anococcygeus by enzymic digestion, loaded
with FURA-2, and ¯uorescence recorded by a method fully
described elsewhere (Wallace et al., 1999). In all experiments,
*Author for correspondence; E-mail: alan.gibson@kcl.ac.uk

234
A. Gibson et al
Special Report
the perfusion ¯uid contained 50 mM L-NOARG, and 10 mM
verapamil was present in experiments involving Tg. To
determine the e€ect of TRIM, the cells were initially
perfused with calcium-free medium prior to the addition of
either 100 nM Tg or 60 mM K. After 10 min in the presence
of these agents, 2.5 mM calcium was then applied; once a
stable increase in ¯uorescence ratio had been established,
TRIM was added and the per cent change in ¯uorescence
ratio calculated after 4 min. Because of the recognized
uncertainties inherent in the measurement of absolute
intracellular calcium concentrations, the results throughout
this study are expressed as the ratio of ¯uorescence at 340
and 380 nm (R340/380). However, in some cells, the absolute
values of cytosolic calcium concentration were obtained using
the equation of Grynkiewicz et al. (1985); cells were
permeabilized with ionomycin (50 mM) and exposed to
bathing medium containing 10 mM calcium followed by
medium containing zero added calcium plus 1,2-bis(2-
aminophenoxy)ethane-N,N,N',N'-tetraaecetic acid (BAPTA;
1 mM). Using this procedure, the resting calcium
concentration was estimated as 54+11 nM (n=6).
Statistics Results are expressed as mean+s.e.mean and
statistical analysis was by Student's t-test with a P value of
0.05 or less taken as signi®cant.
Drugs used All drugs were obtained from Sigma, except
TRIM (Lancaster Synthesis); they were dissolved in distilled
water, except Tg (1 mM stock solution in dimethylsulphoxide)
and miconazole (10 mM stock solution in dimethylsulphoxide).
Figure 1 (a) Trace showing the e€ect of TRIM on tone and
nitrergic relaxations (10 Hz; 10 s trains every 100 s) of the mouse
anococcygeus following contraction by 100 nM thapsigargin (in the
presence of 10 mM verapamil). TRIM was added at the times
indicated by the arrows. Transient decreases in tone are in response
to activation of nitrergic nerves by electrical ®eld stimulation. (b)
Control experiment for the above, showing that Tg-induced tone and
nitrergic relaxations remained relatively constant in the absence of
TRIM.
Results The initial aim of this study was to investigate the
e€ects of TRIM, an inhibitor of neuronal nitric oxide
synthase (Handy et al., 1995; Moore & Handy, 1997), on
nitrergic relaxations of the mouse anococcygeus; tone was
raised with 100 nM Tg, which (in the presence of 10 mM
verapamil) contracts the mouse anococcygeus by activating
CCE (Wallace et al., 1999). Under these conditions,
relaxation to ®eld stimulation is completely blocked by the
general nitric oxide synthase inhibitor L-NOARG (Ayman et
al., 2001). As shown in Figure 1, TRIM had no detectable
e€ect on nitrergic relaxations; rather, it caused clear,
concentration-related relaxation of Tg-induced tone. To
determine the selectivity of this relaxation, we compared the
e€ects of TRIM against tone induced by either 100 nM Tg
(via CCE) or high K (60 mM; via voltage-operated calcium
channels). In these experiments, ®eld stimulation was not
applied, and 50 mM L-NOARG was included in the bathing
medium to inhibit nitric oxide synthase. Sustained contrac-
(Gibson et al., 1994; Wayman et al., 1996; Wallace et al.,
1999), relaxed Tg-induced tone by 88+4% (n=7) but also
relaxed high K-induced tone to the same extent (79+4%;
n=7; P40.05). Similarly, 100 mM miconazole was equi-
e€ective against Tg (52+7% relaxation; n=6) and high K
(46+6% relaxation; n=6; P40.05).
In single smooth muscle cells, loaded with FURA-2 and
bathed in calcium-free medium, Tg (in the presence of 10 mM
verapamil) caused an initial transient increase in R340/380
re¯ecting depletion of calcium from the SR; subsequent
addition of 2.5 mM calcium resulted in a sustained increase in
¯uorescence ratio as a result of CCE (Figure 3; Wallace et al.,
1999). As expected, high K (in the absence of verapamil) did
not produce an initial transient increase in ¯uorescence ratio,
but the subsequent addition of 2.5 mM calcium resulted in a
sustained increase similar to that observed with Tg (Figure 3;
increase in R340/380 of 0.27+0.02 with high K, n=7, and
0.23+0.02 with Tg, n=6). Whilst it is perhaps surprising that
the increase in ¯uorescence ratio was the same when calcium
in¯ux was activated by either CCE (Tg) or voltage-operated
tions to Tg and high
K were similar in magnitude
(550+50 mg and 440+40 mg tension respectively; n=8 in
both cases) and were equally sensitive to relaxation by the
general smooth muscle relaxant papaverine (pEC50 M,
5.55+0.07 and 5.75+0.09 respectively; n=6 in both cases).
As observed in the initial experiments with ®eld stimulation,
TRIM relaxed Tg-induced tone (pEC50 M, 4.38+0.07; n=6;
Figure 2) but it was much less e€ective against high K,
producing only a small, but signi®cant, relaxation at the
highest concentration used.
calcium channels (high K), TRIM produced
a clear
di€erentiation between the two pathways. Thus, application
of 200 mM TRIM during the sustained phase caused a
signi®cant reduction of ¯uorescence ratio in the case of Tg,
but had no e€ect with high K (Figure 3).
For comparison, the e€ects of SKF96365 and miconazole
were examined. 20 mM SKF96365, which we have used in
previous studies to inhibit CCE in the mouse anococcygeus
British Journal of Pharmacology vol 134 (2)

A. Gibson et al
Special Report
235
Figure 2 Concentration-response curves for relaxations of the
mouse anococcygeus muscle in response to TRIM when tone is
raised by either 100 nM thapsigargin (Tg; in the presence of 10 mM
verapamil) or high K (60 mM). Measurements were taken 4 min after
addition of TRIM to the bath. Each point is the mean+s.e.mean
from a minimum of ®ve individual muscle preparations. *P50.05,
signi®cant relaxation.
Discussion The important ®nding of the present study is
that TRIM produced marked inhibition of contractions and
calcium entry induced by Tg, which are mediated via CCE in
the mouse anococcygeus (Wallace et al., 1999), in concentra-
tions which had no e€ect on responses to high K, which are
mediated via voltage-operated calcium channels (Gibson et
al., 1994). Thus, TRIM represents a novel agent for the
selective inhibition of store-operated, as opposed to voltage-
operated, calcium entry. TRIM is an N1-substituted
imidazole and several of the agents that have been employed
to date as inhibitors of CCE also fall into this class, including
SKF96365 and miconazole (Clementi & Meldolesi, 1996).
However, these latter drugs also inhibit voltage-operated
calcium entry in similar concentrations and are therefore
non-selective. Although TRIM was less potent than
SKF96365 against Tg-induced tone, its degree of selectivity
represents an important advance. As noted previously with
SKF96365 (Wallace et al., 1999), the inhibitory e€ect of
TRIM on Tg-induced calcium in¯ux was relatively small
compared with its e€ect on Tg-induced tone; two possible
explanations for this are, (i) there is a threshold concentration
above which cytoplasmic calcium must rise in order to
initiate contraction and, (ii) a fraction of the increase in
¯uorescence ratio observed on calcium re-admission is due to
the small non-CCE `leak' observed in control cells not
exposed to Tg (Wallace et al., 1999).
Figure 3 (a and b) Traces showing the ¯uorescence ratio from
single smooth muscle cells freshly dispersed from the mouse
anococcygeus and loaded with FURA-2. The cells were bathed
initially in calcium-free medium and exposed to either 100 nM
thapsigargin (Tg; a) or 60 mM K (b) followed by 2.5 mM calcium
and then 200 mM TRIM. Verapamil (10 mM) was present throughout
in the experiments with Tg, but omitted in those with high K. The
combined results from at least ®ve such experiments are contained in
the histogram (c) which shows the mean+s.e.mean reduction in
¯uorescence ratio obtained in each case 4 min after addition of
TRIM. *P50.05, signi®cant reduction.
TRIM was initially shown to be an inhibitor of neuronal
nitric oxide synthase (Handy et al., 1995; Moore & Handy,
1997). However, this action cannot explain the e€ects
observed here. First, TRIM did not produce a discernible
inhibition of nitrergic relaxations; whilst interpretation of
these results was complicated by the marked loss of tone in
the presence of TRIM, they agree with a similar lack of e€ect
on nitrergic relaxations reported in rabbit corpus cavernosum
(Teixeira et al., 1998). Secondly, relaxations of Tg-induced
tone were obtained in the presence of the nitric oxide
synthase inhibitor L-NOARG which completely blocks
British Journal of Pharmacology vol 134 (2)

236
A. Gibson et al
Special Report
nitrergic relaxations of mouse anococcygeus contracted by Tg
(Ayman et al., 2001). Thirdly, nitric oxide itself has been
found to inhibit, not activate, CCE in smooth muscle (Cohen
et al., 1999; Ayman et al., 2001). Thus, inhibition of CCE
represents an additional action of TRIM, independent of its
ability to inhibit nitric oxide synthase.
It is not yet possible to determine whether TRIM inhibits
CCE by a direct e€ect on the SOC or by interference with the
signal generated by depletion of the SR. There is mounting
evidence that SOCs may be comprised of protein subunits
similar to the transient receptor potential (TRP) proteins ®rst
detected in Drosophila. Several mammalian TRP homologues
have been identi®ed to date (Harteneck et al., 2000) raising
the possibility that the subunit composition and functional
characteristics of SOCs may vary widely among tissues. In
preliminary studies, using the polymerase chain reaction in
whole mouse anococcygeus, we have detected expression of
TRPs 1, 2, 3 and 6 (communicated to the Joint British/
Canadian Pharmacological Societies Meeting, Vancouver,
2001); more de®nitive studies using single smooth muscle
cells, freshly dispersed from the tissue, are now in progress.
Given this diversity of TRP proteins, it will be important to
determine whether the inhibitory action of TRIM reported
here is a general e€ect or whether it is tissue speci®c; if the
latter, then TRIM may prove an important tool for the
characterization of CCE in di€erent cell types.
In conclusion, TRIM provides a novel, selective inhibitor
of CCE and should be useful in improving our understanding
of this ubiquitous calcium entry pathway. In addition, it may
act as a template for the development of analogues which are
both more potent and free from the complication of nitric
oxide synthase inhibition.
The authors thank the Wellcome Trust for support.
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(Received May 14, 2001
Revised June 29, 2001
Accepted July 10, 2001)
British Journal of Pharmacology vol 134 (2)