Pflgers Arch Eur J Physiol (1998) 437:158160
S H O R T C O M M U N I C AT I O N
A. Shmigol D.A. Eisner Susan Wray
Carboxyeosin decreases the rate of decay of the [Ca2+]i transient
in uterine smooth muscle cells isolated from pregnant rats
Received: 17 July 1998 / Received after revision: 23 September 1998 / Accepted: 25 September 1998
Abstract In myometrial smooth muscle cells the rate of
decline of intracellular calcium ([Ca2+]i) is determined
by Ca2+ extrusion from the cell and uptake into intra-
cellular stores. The relative quantitative contribution of
these processes however, has not been established. We
therefore examined the effect of the sarcolemmal Ca2+
pump inhibitor, carboxyeosin, on the rate of the [Ca2+]i
transient decline in myocytes isolated from pregnant rat
uterus. Indo-1 was used in conjunction with the whole-
cell patch-clamp technique to measure [Ca2+]i simulta-
neously with transmembrane calcium current (ICa).
[Ca2+]i transients were elicited by repetitive membrane
depolarization to simulate the natural pattern of uterine
electrical activity. The rate of [Ca2+]i removal was calcu-
lated from the falling phase of the [Ca2+]i transient. Pre-
treatment of the cells with 2 M carboxyeosin led to a
marked decrease in the rate of [Ca2+]i transient decay,
suggesting that the sarcolemmal Ca2+ pump is involved
in the calcium extrusion process. Removal of the extra-
cellular Na also decreased the rate of [Ca2+]i decay, indi-
cating an important role for the Na+/Ca2+ exchange. When
both the sarcolemmal Ca2+ pump and Na+/Ca2+ exchange
were inhibited the cell failed to restore [Ca2+]i after the
stimulation. Comparison of the rate constants of [Ca2+]i
decay in control conditions and after carboxyeosin treat-
ment shows that approximately 30% of [Ca2+]i decay is
due to the sarcolemmal calcium pump activity. The re-
maining 70% can be attributed to the activity of Na+/Ca2+
exchanger and the intracellular calcium stores.
Key words Ca-pump Ca-ATPase Indo-1
[Ca2+]i decline
A. Shmigol S. Wray ()
Department of Physiology, University of Liverpool,
Crown Street, Liverpool, L69 3BX, UK
(e-mail: s.wray@liverpool.ac.uk,
Tel.: +44 151 794 5306, Fax: +44 151 794 5321)
D.A. Eisner
Department of Veterinary Preclinical Sciences,
University of Liverpool, Liverpool, L69 3BX, UK
Springer-Verlag 1998
Introduction
Contraction of smooth muscle requires an elevation of
intracellular [Ca2+] ([Ca2+]i). The main source of Ca2+
for activation of uterine smooth muscle cells is the extra-
cellular fluid, from which Ca2+ enters the cytosol
through L-type calcium channels. Ca2+-induced Ca2+ re-
lease from sarcoplasmic reticulum (SR) may also con-
tribute to the [Ca2+]i transient [9, 10]. Several Ca2+-trans-
porting systems are responsible for the subsequent low-
ering of [Ca2+]i during relaxation of smooth muscle cells.
Although intracellular Ca2+ stores can contribute to the
rate of [Ca2+]i decay, long-term regulation of [Ca2+]i re-
quires Ca2+ extrusion by sarcolemmal mechanisms, since
the intracellular Ca2+ stores have a finite capacity. Ca2+
extrusion is therefore crucial for ensuring adequate re-
laxation. In uterine and other smooth muscle cells, Ca2+
extrusion is due to the activity of two major systems an
adenosine triphosphate (ATP)-driven Ca2+ pump and a
Na+/Ca2+ exchanger. Both of these have been studied us-
ing subcellular preparations (mainly plasmalemmal vesi-
cles, see [7] for review) but their roles under physiologi-
cal conditions are not well characterised; e.g. their rela-
tive contribution to overall extrusion of calcium is not
known. This is, in part, due to the lack of specific inhibi-
tors of the Ca2+ pump or Na+/Ca2+ exchanger. In red
blood cells the fluorescein derivative carboxyeosin has
been shown to inhibit the plasma membrane Ca2+ pump
directly [5], and to decrease the rate of [Ca2+]i decay in
cardiac cells [2]. There are no data available on the effect
of carboxyeosin in smooth muscle cells. We have there-
fore investigated the effect of carboxyeosin on the rate of
[Ca2+]i decay and used this substance to assess the contri-
bution of the plasmalemmal Ca2+ pump to Ca2+ extrusion
in smooth muscle cells isolated from pregnant rat uterus.
Materials and methods
Experiments were performed on acutely isolated uterine smooth
muscle cells. Female Wistar rats at the end of gestation (day
1921) were killed by cervical dislocation under CO2 anaesthesia.
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The method of cell isolation was essentially the same as previous-
ly described [9] except that collagenase type IA was used and the
incubation time with enzyme was increased to 45 min. After en-
zyme treatment the cells were washed in physiological saline and
then Kraftbrhe (KB) solution (see below).
The methods used for [Ca2+]i measurement and voltage clamp
were the same as used previously [9]. Indo-1 (Molecular Probes,
Eugene, Ore., USA) was used as a Ca2+-sensitive indicator. The
indicator was loaded into the cell via a patch electrode. The
[Ca2+]i was calculated from the ratio of the fluorescence signal in-
tensities at 400 and 500 nm (F400:F500) using a standard procedure
[6, 9]. To measure the rate of Ca2+ removal from the cytosol we
fitted the [Ca2+]i transient decay to a single exponential.

Solutions

Cells were superfused continuously with pre-warmed extracellular

solution containing (in mM): NaCl, 140; KCl, 5.4; CaCl2, 2; MgCl2,
1.2; glucose, 10; 4-(2-hydroxyethyl)-1-piperazineethanesulphonic
acid (HEPES), 10; adjusted to pH 7.4 with NaOH. The nominally
Ca-free solution was prepared by omitting Ca2+ from the above
solution and increasing the [Mg2+] to 5 mM. The Na-free solution
was prepared by equimolar substitution of Na+ by tris(hydroxy-
methyl)aminomethane (TRIS+) in normal extracellular saline. The
Hanks solution used for cell isolation was composed as follows (in
mM): NaCl, 137; KCl, 5.1; KH2PO4, 0.44; Na2HPO4, 0.26; glucose,
5.5; HEPES, 10; adjusted to pH 7.2 with NaOH. The KB medium
contained (in mM): KCl, 40; K2HPO4, 10; KOH, 105; taurine, 10;
glucose, 11; ethyleneglycolbis(-aminoethylether)-N,N,N,N-tetra-
acetic acid (EGTA), 0.1; HEPES, 5. Methanesulphonic acid was
used to adjust the pH of this solution to 7.2. The pipette solution
contained CsCl, 130; MgATP 2; K5Indo-1 0.1; HEPES 10 adjusted
to pH 7.2 with CsOH. Carboxyeosin (Calbiochem, San Diego, Calif.,
USA) was dissolved in methanol to yield a 20-mM stock solution.
Carboxyeosin-containing solution was prepared by adding 10 l
stock solution to 100 ml nominally Ca-free saline. To inhibit the
plasmalemmal Ca2+ pump, the cells were incubated in a carboxy-
eosin-containing solution for 5 min at room temperature and washed
by bath perfusion with normal extracellular solution for 510 min.
All experiments were performed at 35 C. All chemicals were ob-
tained from Sigma, unless stated otherwise.
Where appropriate, the results are presented as meansSEM.
Otherwise, the traces shown represent typical results from at least
five similar experiments on cells, obtained from at least two dif-
ferent rats. Statistical differences were tested using ANOVA.
Results
In the present study, we used repetitive membrane depo-
larization to elevate [Ca2+]i. Figure 1A shows an exam-
ple of [Ca2+]i transients (top traces) elicited by a train of
ten voltage pulses from 80 mV to 0 mV (the accompany-
ing records of membrane current are shown as spikes on
the bottom trace). The left-hand trace in Fig. 1A was re-
corded from a control cell. Upon cessation of stimulation
[Ca2+]i declined toward the resting level of 10925 nM.
The rate constant of [Ca2+]i decay in control conditions
was 0.6330.024 s1 (n=8). Pre-treatment of the cells
with carboxyeosin caused significant elevation in resting
[Ca2+]i to 20611 nM (n=5, P<0.01). The right-hand
panel in Fig. 1A shows the [Ca2+]i transient and current
recorded from a carboxyeosin-treated cell. There was no
detectable change in the current after carboxyeosin treat-
ment. From Fig. 1A, it is clear that the cell was capable of
restoring [Ca2+]i after the stimulation, despite the inhibi-
tion of the plasmalemmal calcium pump. However, the
Fig. 1 A Intracellular [Ca2+] ([Ca2+]i) transient (upper traces) and
membrane current (Im, lower traces) elicited by trains of voltage-
clamp pulses (seen as spikes in the lower traces). [Ca2+]i transients
were recorded from a control cell (left) and a cell pre-treated with
carboxyeosin (right). B Normalized [Ca2+]i transient decay ob-
tained from the cells shown in A. The traces were normalized to
the peak amplitude of corresponding [Ca2+]i transients after sub-
traction of the steady-state [Ca2+]i level. Solid lines were obtained
by fitting a single exponential equation to the data
rate of this decay was slowed. This can be seen more
clearly in Fig. 1B, where the normalized-to-peak ampli-
tude records of [Ca2+]i transient decay are superimposed.
After inhibition of the Ca2+ pump with carboxyeosin, the
mean value of the rate constant of decay of the [Ca2+]i
transient was 0.4350.01 s1 (n=5), which is about 70%
of control. This difference was statistically significant
(P<0.05) indicating a 30% contribution from sarcolemmal
calcium pump to the extrusion of Ca2+ from the cytosol.
To estimate the contribution from Na+/Ca2+ exchange
to the [Ca2+]i removal we compared the rate of [Ca2+]i
decay in control with that in Na+-free extracellular solu-
tion. Figure 2A shows the effect of Na+ removal on the
basal level of [Ca2+]i and the rate of [Ca2+]i transient de-
cay. Bath application of Na+-free solution elevated basal
[Ca2+]i from 10925 to 17819 nM (n=8, P<0.05). This
elevation was abolished when Ca2+ was omitted from the
Na+-free solution (not illustrated). The train of ten volt-
age pulses from 80 to 0 mV elicited a transient increase
of [Ca2+]i which decayed to a steady-state level after the
end of stimulation. [Ca2+]i decreased further when the
normal [Na+] in the bath was restored. Abolition of
Na+/Ca2+ exchange had a greater effect on the rate of
[Ca2+]i transient decay than did inhibition of the Ca2+
pump. On average, the rate constant of calcium decay
was decreased from 0.6330.024 to 0.2580.02 s1 upon
160
Fig. 2A, B The effect of sodium removal (solid bars) on [Ca2+]i
transients during repetitive membrane depolarization (spikes on
the Im records, lower traces). [Ca2+]i transient from a control cell
(A) and a carboxyeosin-treated cell (B)
the abolition of the Na+/Ca2+ exchange (P<0.05, n=7) in-
dicating that up to 60% of [Ca2+]i decay is due to the
Na+/Ca2+ exchange. When the Ca2+ pump was inhibited
with carboxyeosin, the increase of [Ca2+]i in response to
Na+-free solution was substantially augmented (Fig. 2B).
Upon Na+ removal from the bath solution, the basal
[Ca2+]i level was increased to 50948 nM (n=5). It can
be seen from Fig. 2B that the carboxyeosin treated cell
was unable to restore [Ca2+]i after the train of depolariz-
ing pulses as long as Na+ was absent from the extracellu-
lar fluid. Upon return of Na+, however, there was a resto-
ration of [Ca2+]i to the initial level.
Discussion
In the present study we have investigated the inhibitory
action of carboxyeosin on the rate of [Ca2+]i transient de-
cay in smooth muscle cells isolated from myometrium of
pregnant rats. We found that carboxyeosin treatment
causes an increase in basal [Ca2+]i and a significant
slowing of the decay of the [Ca2+]i transient, which can
be attributed to the inhibition of the sarcolemmal Ca2+
pump [2, 5]. Comparing the rate of [Ca2+]i decay in con-
trol and after carboxyeosin treatment one can calculate
the relative contribution of the sarcolemmal Ca2+ pump
to overall [Ca2+]i decay. We have estimated that the Ca2+
pump contributes 30% to [Ca2+]i decay in uterine smooth
muscle cells.
The rate of [Ca2+]i decay was also substantially de-
creased upon abolition of the Na+/Ca2+ exchange indicat-
ing its important role in [Ca2+]i removal from the cytosol
at physiological [Ca2+]. It should be pointed out howev-
er, that cell dialysis with a [Na+-free pipette solution, as
used in this study, can lead to overestimation of the
Na+/Ca2+ exchanger contribution to overall Ca2+ extru-
sion since a low [Na+]i would facilitate Ca2+ extrusion
via the Na+/Ca2+ exchanger (compare [8]). We found that
the rate of [Ca2+]i decay was decreased by 60% when
Na+/Ca2+ exchange was abolished. Our data are in agree-
ment with those obtained on toad gastric smooth muscle
[8], but in contrast to results in guinea-pig ureter [1] where
authors observed little effect of Na+ removal on the decay
of [Ca2+]i. When interpreting data on relative contribution
of different mechanisms to [Ca2+]i extrusion it should be
noted that abolition of one mechanism may lead to the
compensatory increase in another [3].
Our results show that the cells were capable of remov-
ing the Ca2+ from the cytosol, although at a slower rate
provided that at least one of the two sarcolemmal Ca2+
extrusion mechanisms was still functional (compare right-
hand panel in Figs. 1A and 2A). However, when both the
Ca2+ pump and Na+/Ca2+ exchange were inhibited there
was little if any [Ca2+]i decay (see Fig. 2B) These data
suggest that the sarcolemmal mechanisms of Ca2+ extru-
sion play a crucial role in the regulation of [Ca2+]i, and
that the intracellular stores or organelles do not make a
noticeable contribution, unless they are working in series
with sarcolemmal mechanisms [4].
Acknowledgements This work was supported by the Medical Re-
search Council.
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