Na+/Ca2+ exchange and its role in
intracellular Ca2+ regulation in guinea pig detrusor
smooth muscle
C.
Wu1 and
C. H.
Fry2
1 Department of Medicine and 2 Institute of Urology
and Nephrology, University College London, London W1P 7PN, United
Kingdom
 |
ABSTRACT |
The role of Na+/Ca2+ exchange in
regulating intracellular Ca2+ concentration
([Ca2+]i) in isolated smooth muscle cells
from the guinea pig urinary bladder was investigated. Incremental
reduction of extracellular Na+ concentration resulted in a
graded rise of [Ca2+]i; 50-100 µM
strophanthidin also increased [Ca2+]i. A
small outward current accompanied the rise of
[Ca2+]i in low-Na+ solutions
(17.1 ± 1.8 pA in 29.4 mM Na+). The quantity of
Ca2+ influx through the exchanger was estimated from the
charge carried by the outward current and was ~30 times that which is
necessary to account for the rise of [Ca2+]i,
after correction was made for intracellular Ca2+ buffering.
Ca2+ influx through the exchanger was able to load
intracellular Ca2+ stores. It is concluded that the level
of resting [Ca2+]i is not determined by the
exchanger, and under resting conditions (membrane potential
50 to
60 mV), there is little net flux through the exchanger. However, a
small rise of intracellular Na+ concentration would be
sufficient to generate significant net Ca2+ influx.
urinary bladder; intracellular calcium; sodium/calcium exchange
 |
INTRODUCTION |
THE SODIUM/CALCIUM
EXCHANGE protein in the plasma membrane is a bidirectional,
electrogenic ion antiporter that couples translocation of three
Na+ ions against one Ca2+ ion (25,
31). This facilitated transport is driven by
transmembrane electrochemical gradients, particularly the
Na+ gradient (4). It plays an important role
in regulating the intracellular Ca2+ concentration
([Ca2+]i) by extruding Ca2+ from
the cell in the forward mode and by mediating Ca2+ entry in
its reverse mode. Na+/Ca2+ exchange proteins
have various isoforms and are expressed in myocardium and other cells
(30); the gene for coding these proteins has been
identified and the primary structure elucidated (19, 29).
The physiological and pathological roles of
Na+/Ca2+ exchange have been intensively studied
in several cell types, particularly cardiac muscle (19, 28,
32), but their importance in smooth muscle is less clear. The
exchanger plays a key role in regulating [Ca2+]i and contractile function in vascular
smooth muscle (1, 19, 33), in particular when the
intracellular Na+ concentration
([Na+]i) is altered (3). A role
has also been proposed in visceral smooth muscles such as stomach,
uterus, and ureter (20, 22, 33). However, in airway smooth
muscle, despite expression of the exchanger proteins (26),
the functional contribution of Na+/Ca2+
exchange to Ca2+ homeostasis is insignificant
(14).
In detrusor smooth muscle, the mechanisms by which the cell membrane
regulates transmembrane Ca2+ movement are unclear, and the
presence of a functional Na+/Ca2+ exchange is
controversial. The bell-shaped voltage dependence of
depolarization-induced [Ca2+]i transients
(8, 37) suggests that Ca2+ influx occurs
mainly via L-type Ca2+ channels, and entry via reverse mode
of Na+/Ca2+ exchange is limited. In addition,
the recovery rate of Ca2+ transients is independent of
membrane potential (8), also suggesting that
Ca2+ extrusion via Na+/Ca2+
exchange during this phase is limited. However, reduction of extracellular Na+ causes an increase of resting
[Ca2+]i in detrusor muscle (27),
which could be explained by the existence of
Na+/Ca2+ exchange. This study was, therefore,
undertaken to clarify the role for Na+/Ca2+
exchange in intracellular Ca2+ regulation in detrusor muscle.
 |
METHODS |
Preparation of single cells.
Single detrusor smooth muscle cells were isolated from the urinary
bladder of adult guinea pigs of either sex (400-900 g). Animals
were killed by cervical dislocation, in accordance with procedures
approved by the United Kingdom Animals (Scientific Procedures) Act of
1986. The bladder was rapidly removed and placed in
Ca2+-free solution containing (in mM) 105.4 NaCl, 20.0 NaHCO3, 3.6 KCl, 0.9 MgCl2, 0.4 NaH2PO4, 5.5 glucose, 4.5 sodium pyruvate, and
4.9 HEPES, pH 7.1. Small pieces of detrusor muscle were cut from the
dome of the bladder and digested in a collagenase-based enzyme mixture
dissolved in the same Ca2+-free solution. Cells were
dissociated as described previously (21).
Solutions.
Detrusor myocytes were continuously superfused with an extracellular
solution (Tyrode) containing (in mM) 118 NaCl, 24.0 NaHCO3, 4.0 KCl, 1.0 MgCl2, 0.4 NaH2PO4,
1.8 CaCl2, 6.1 glucose, and 5.0 sodium pyruvate (pH 7.4) at
37°C, gassed with 95% O2-5% CO2.
Low-Na+ solutions were made by substituting
Tris · HCl for NaCl, while NaHCO3 and sodium
pyruvate were not altered to maintain a normal cellular pH regulation
and metabolism. An intracellular filling solution for patch pipettes
contained (in mM) 20 KCl, 100 aspartic acid, 5.45 MgCl2,
5.0 Na2ATP, 0.2 Na3GTP, 0.05 EGTA, and 5.0 HEPES, pH 7.1, adjusted with 1 M KOH. For measurement of the
Ca2+ current and determination of sarcoplasmic
Ca2+ buffer capacity, KCl was replaced by CsCl and pH was
adjusted with 1 M CsOH.
Measurement of
[Ca2+]i.
[Ca2+]i was measured by epifluorescence
microscopy with the fluorescent indicator fura 2. Cells were loaded by
incubation with 5 µM fura 2 acetoxymethyl ester in
Ca2+-free HEPES-buffered solution at 25°C for 30-60 min
and then kept at 4°C for use later the same day. An aliquot of cell
suspension was placed in a perfusion chamber mounted on the stage of an
inverted microscope. Cells were allowed to settle and adhere to the
glass coverslip base of the chamber before they were superfused with Tyrode solution at 37°C at a rate of ~2 ml/min. Cells were
illuminated alternately at 340 and 380 nm; the emitted light was split
by a dichroic mirror centered at 410 nm and collected by a
photomultiplier between 410 and 510 nm.
The fura 2 ratio signal was converted to
[Ca2+]i values using an in vitro calibration
method. The relationship between [Ca2+]i and
the ratio (fluorescence ratio at 340/380 excitation) is given
(11)
|
(1)
|
where Rmin and Rmax refer to ratio (R)
values at 0 [Ca2+]i and saturating
[Ca2+]i, respectively;
is the ratio at 0 and saturating [Ca2+]i excited at 380 nm
alone; and Kd (224 nM) is the dissociation constant of fura 2 for Ca2+.
Voltage-clamp recordings.
Ionic currents were recorded with patch-type electrodes in a whole cell
configuration. An Axopatch-1D system (Axon Instruments) was used to
perform voltage clamp with an IBM-compatible computer to generate
voltage-clamp protocols and record membrane currents through an
analog-to-digital converter (Digidata 1200; Axon Instruments) with a
sampling frequency of 4 kHz and a cut-off frequency of 2 kHz.
Voltage-clamp protocols were supported by pCLAMP software (Axon
Instruments). Patch pipettes were made from borosilicate glass and had
a resistance of 3-5 M
when filled with intracellular solutions
(above). For simultaneous recording of
[Ca2+]i and membrane, current cells were
dialyzed with fura 2 via patch pipettes filled with the intracellular
filling solution plus 100 µM K5-fura 2 (Calbiochem); this
gave sufficient signal-to-noise ratio without significant effect on the
Ca2+ transient (37).
Statistics.
Numerical data are presented as means ± SE since experimental
observations were generally repeated several times on the same cell.
Student's two-tailed paired t-tests were used to examine the significance of difference between two data sets; the null hypothesis was rejected when P < 0.05.
 |
RESULTS |
The dependence of
[Ca2+]i on low
extracellular Na+ concentration and
strophanthidin.
Figure 1A shows an
experimental recording of the [Ca2+]i in
an undialyzed cell when extracellular Na+
concentration ([Na]o) was lowered from control (147.4 mM)
to 90, 60, or 29.4 mM. A small reversible rise of
[Ca2+]i occurred in each case, with the
largest changes in the solutions of lowest Na+
concentration: reduction from 147.4 to 29.4 mM increased
[Ca2+]i from 124 ± 14 to 190 ± 16 nM (n = 14, P < 0.05). Figure
1B plots the change of [Ca2+]i
(
[Ca2+]i) as a function of
[Na+]o.

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Fig. 1.
A: effect of reduced extracellular
Na+ concentration ([Na+]o) on
intracellular Ca2+ concentration
([Ca2+]i) in an isolated guinea pig detrusor
cell. The [Na+]o, reduced from a control
value of 147.4 mM to 90, 60, and 29.4 mM, was shown. B: mean
data showing the effect of low-Na+ solutions on the change
of [Ca2+]i
( [Ca2+]i) from that in control solution.
Mean data ± SD. C: the action of 50 µM
strophanthidin on [Ca2+]i. All experiments
were in 1.8 mM extracellular Ca2+.
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An increase of the [Na+]i should also raise
[Ca2+]i via Na+/Ca2+
exchange. Thus 50-100 µM strophanthidin was added to the
superfusate, which would raise [Na+]i by
blockade of the sarcolemmal Na+-K+-ATPase
(18). Figure 1C shows that a small, maintained
rise of [Ca2+]i was generated by 50 µM
strophanthidin, which slowly reversed when the inhibitor was removed.
In 12 cells, 100 µM strophanthidin increased
[Ca2+]i from 122 ± 11 to 139 ± 11 mM (P < 0.01).
Outward current in low-Na+ solutions.
Transmembrane movement of ions via Na+/Ca2+
exchange is electrogenic and is thereby able to generate membrane
current. Influx of one Ca2+ ion as three
Na+ ions leave the cell should be accompanied by a net
outward current. Figure
2A shows that when
[Na+]o was lowered to 29.4 mM around a cell
voltage clamped at
60 mV, a rise of [Ca2+]i
was accompanied by an outward current. In 28 cells, reducing [Na+]o to 29.4 mM generated a current of
17.1 ± 1.8 pA. The generation of outward current preceded the
rise of [Ca2+]i in all cells and is seen in
Fig. 2 as the interval between the two vertical dotted lines. It is
hypothesized that the initial Ca2+ influx through the
exchanger is absorbed by intracellular buffers so that the bulk
sarcoplasm [Ca2+] will not alter initially, but
that membrane current will alter immediately, reflecting transmembrane
ion flux.

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Fig. 2.
The effect of reduced [Na+]o
from 147.2 to 29.4 mM on [Ca2+]i and membrane
current. A: the vertical dotted lines (left) show
the onset of outward current generation; the horizontal dotted lines
(right) show the onset of a rise of
[Ca2+]i. B: 2 exposures to a
low-Na+ solution; 10 µM nifedipine was added before the
second intervention. Cells were voltage clamped at 60 mV.
I, current; Vm, membrane
potential.
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|
The rise of [Ca2+]i in the
low-Na+ solutions was measured when the membrane potential
was clamped to
60 mV. This renders unlikely the involvement of L-type
Ca2+ channels in this rise of
[Ca2+]i. However, this was confirmed by
repeating the above experiments in the presence of 10 µM nifedipine
(Fig. 2B). The L-type Ca2+ channel antagonist
had no effect on the magnitude of changes to
[Ca2+]i and membrane current induced by the
low-Na+ solutions.
A more quantitative approach was undertaken to examine whether the
magnitude of the Ca2+ influx carried by the
Na+/Ca2+ exchanger could account for the
increase of [Ca2+]i associated with the
outward current. This required estimation of the sarcoplasmic
Ca2+ buffering power, as shown below.
Determination of sarcoplasmic Ca2+ buffering.
Most Ca2+ entering the cell is bound by sarcoplasmic
binding sites, and only a small fraction remains as free ions. The
sarcoplasmic Ca2+ buffer power (BCa) can be
defined as a dimensionless quotient, 
Cac/
[Ca2+]i, where

Cac is an increment of total sarcoplasmic
Ca2+ concentration (i.e., the sum of free and bound
Ca2+) and
[Ca2+]i is the
change of ionized [Ca2+]i.

Cac was calculated in a voltage-clamp experiment by
integrating a Ca2+ current (ICa)
generated by membrane depolarization and
[Ca2+]i by the associated rise of
[Ca2+]i. It was important that concomitant
Ca2+ release from intracellular stores did not supplement
the rise of [Ca2+]i. This was achieved by
adding ryanodine to either the patch pipette solution (20 µM) or to
the superfusate (50 µM); results were quantitatively similar wherever
ryanodine was initially included. Cs+ also replaced
K+ in the pipette solution to block outward current, which
was necessary for accurate quantification of
ICa.
Figure 3 illustrates an experiment in
which a cell was depolarized from a holding potential of
60 to +10
mV; this triggered an inward current followed by an increase of
[Ca2+]i. The inward current and the
Ca2+ rise were due to activation of L-type Ca2+
channels, because both could be blocked completely by 5-10 µM nifedipine or verapamil. Two superimposed pairs of records are obtained
from one cell; one in the absence (dotted lines) and, subsequently, one
in the presence (solid lines) of 50 µM ryanodine. Although the
magnitude of ICa was similar in both sets, the
rise of [Ca2+]i was smaller in the presence
of ryanodine, implying a small component of Ca2+-induced
Ca2+ release (CICR).

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Fig. 3.
The time course of membrane Ca2+ current
(ICa), charge movement as the integral of
ICa ( ICa), and the
rise of [Ca2+]i during a step depolarization
from 60 to +10 mV. The dotted lines are traces obtained in the
absence of ryanodine, solid lines in the presence of 50 µM ryanodine
in the superfusate. The shaded box indicates the time interval over
which ICa was integrated.
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The value of BCa was determined over the first 300 ms of
depolarization, since this has been shown in cells of similar size to
be long enough to allow spatial homogeneity of
[Ca2+]i, while short enough to minimize the
influence of other Ca2+ removal systems (35),
and as evidenced by the steady level of
[Ca2+]i in Fig. 3. Furthermore, the measured
[Ca2+]i has been shown to be approximately
proportional to 
Cac during this period
(15). Total Ca2+ influx was calculated from
|
(2)
|
where 
ICadt is the
total charge entry, zCa is the valency of the
Ca2+ ion, and F is the Faraday's constant.
ICa was integrated by measuring the area under
the current curve with reference to the current level at the holding
potential. The integrals are also plotted in Fig. 3, superimposed on
the [Ca2+]i traces. It is evident that the
rise of [Ca2+]i is seen only after a delay,
when a significant Ca2+ influx has occurred.
Average cell volume was estimated to be ~2.2 pl, assuming that the
detrusor cell is modeled as two cones joined at their bases, each 7.5 µm in diameter and 75 µm long. In 12 cells in the presence of
ryanodine, 
ICadt was calculated
to be 72.9 ± 9.8 µmol/l cell volume (ignoring the volume
contribution from intracellular organelles) and
[Ca2+]i was 143 ± 27 nM (i.e., an
increase from 153 ± 30 to 296 ± 50 nM). This yields an
average BCa of 510.
This calculated value of BCa is an overestimate, since a
contribution to the total Ca2+ buffering comes from
intracellular fura 2 (100 µmol/l) and EGTA (50 µmol/l) in the patch
pipette filling solution, if it is assumed they are fully equilibrated
with the sarcoplasm. Their respective Kd has
been measured as 283 and 363 nM from in vitro calibrations (7,
37). From the law of mass action, the amount of Ca2+
binding to the two buffers when the [Ca2+]i
increased from 153 to 296 nM is 16.2 and 7.6 µmol/l, respectively. This leaves a total of 49.1 µmol/l from cellular buffers and a cellular BCa value of 343.
Charge influx in low
[Na+]o solutions is
sufficient to account for the rise of
[Ca2+]i.
The integral of the outward current (in coulombs) generated on
reduction of [Na+]o from 147.4 to 29.4 mM was
used as a measure of Ca2+ influx. This assumes that the
current represents turnover of a 3:1 Na+/Ca2+
exchange, so that one mole of charge (coulombs/F) is equivalent to an
influx of one mole of Ca2+. The integral was measured over
a 30-s interval from the onset of outward current to when the rise of
[Ca2+]i was near maximal. In 12 cells, this
represented an influx of 789 ± 173 µmol/l cell volume. The
increment in sarcoplasmic Ca2+ content would be 1.55 µmol/l cell volume over this interval using the above value for
BCa. The increment of Ca2+ in the sarcoplasm
can be likewise estimated from the concomitant rise of
[Ca2+]i, and in the same cells this was
51 ± 8.0 nmol/l cell volume over the same interval. The
difference between the estimated and measured increments of
sarcoplasmic Ca2+ will be due to removal of
Ca2+ from the sarcoplasm by organelles and other surface
membrane processes. Thus Ca2+ influx through
Na+/Ca2+ exchange is sufficient to account for
the rise of [Ca2+]i. The implications of
these calculations for Ca2+ regulation by the detrusor cell
are considered in DISCUSSION.
Reduction of [Na+]o
enhances the refilling of functional intracellular
Ca2+ stores.
The possible role of Na+/Ca2+ exchange in
regulating intracellular Ca2+ was investigated. The reverse
mode of the exchange, driven by low [Na+]o,
increases the resting level of [Ca2+]i. One
consequence is that accumulation of the Ca2+ by
intracellular stores may be increased. To test this, the effect of a
low [Na+]o solution on the refilling of
intracellular stores was investigated; the maximum caffeine-induced
[Ca2+]i transient was used as an index of the
amount of releasable Ca2+ in the store. In these refilling
experiments, cells were voltage clamped at
60 mV, and, therefore, any
contributions from possible secondary changes to L-type
Ca2+ channel activity were minimized. Figure
4A shows that a reduction of
[Na+]o to 29.4 mM during a refilling interval
of 5 min increased the subsequent caffeine-induced
[Ca2+]i transient, which indicates that
refilling of the Ca2+ store was facilitated. Preexposure to
the low [Na+]o solution increased the
caffeine-induced Ca2+ transient to 118 ± 6% of
control (n = 12, P < 0.05). Control experiments showed that an interval of 5 min between successive caffeine exposures gave reproducible [Ca2+]i
transients, indicating that refilling was complete.

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Fig. 4.
The influence of low-Na+ solution
(A) and 50 µM strophanthidin (B) on the
caffeine (caff)-induced intracellular Ca2+ transient. The
cell was exposed to 20 mM caffeine where indicated by horizontal bars
in the records. Between the first and second caffeine exposures, the
cell was exposed to the low-Na+ solution or strophanthidin.
The Vm was voltage clamped at 60 mV throughout
the recordings.
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Strophanthidin also facilitates refilling of functional
intracellular Ca2+ stores.
An increase of the [Na+]i should also enhance
filling of intracellular stores via the reverse mode of
Na+/Ca2+ exchange. Refilling experiments were,
therefore, repeated in the presence of strophanthidin as an
intervention to raise [Na+]i. Figure
4B shows that application of strophanthidin for 5 min between successive applications of caffeine enhanced the second Ca2+ transient. In 11 cells, 50 µM strophanthidin
increased the caffeine-induced Ca2+ transient to 120 ± 7% of control values (P < 0.05).
Lowering [Na+]o or
raising [Na+]i can cause
spontaneous Ca2+ oscillations.
An elevated sarcoplasmic Ca2+ concentration and the
increased accumulation of stored Ca2+ could result in
oscillatory changes of [Ca2+]i via CICR
(32). Singular or multiple spontaneous Ca2+
oscillations could be observed by both lowering
[Na+]o and applying strophanthidin and were
more frequent after longer exposure of these interventions. Figure
5 illustrates an example during exposure
to a low-Na+ solution (29.4 mM) in which the resting
[Ca2+]i was increased with superimposed
Ca2+ oscillations. These observations suggest that
stimulation of the exchanger by either a decrease of
[Na+]o or by an increase of
[Na+]i can lead to oscillatory
Ca2+ waves.
 |
DISCUSSION |
Evidence for
Na+/Ca2+
exchange in detrusor muscle.
This study has demonstrated the functional existence of
Na+/Ca2+ exchange in detrusor smooth muscle
cells. Lowering [Na+]o caused an increase of
[Ca2+]i, which was graded as the
[Na+]o was successively reduced. In
addition, strophanthidin, which should raise
[Na+]i by inhibition of the sodium pump, also
increased resting [Ca2+]i. Both of these
interventions demonstrate a linkage between the transmembrane
Na+ and Ca2+ gradients. That such an increase
of [Ca2+]i occurs via
Na+/Ca2+ exchange was reinforced by the
measurement of an outward current during the rise of
[Ca2+]i in low-Na+ solutions: an
outward current would be expected from an exchanger with a 3:1
stoichiometry during Ca2+ influx. The rise of
[Ca2+]i and the membrane current were
independent of the L-type Ca2+ current, a major
Ca2+ entry pathway in this tissue (8), as
these phenomena were observed when the membrane potential was clamped
at
60 mV and when channel antagonists were added. Further
quantitative analysis, allowing for sarcoplasmic Ca2+
buffering, showed that the charge carried by the outward current was
sufficient to account for the increase of
[Ca2+]i.
The functional existence of Na+/Ca2+ exchange
has also been reported in vascular (1, 24, 39) and some
visceral (20, 22, 33, 34) smooth muscles. In the majority
of these studies, evidence for the exchanger is based mainly on the
effect of altered [Na+]o or
[Na+]i on tissue contractility or
intracellular [Ca2+]i, and relatively few
observations have been made on the exchange current. One study
(20), however, observed a small Na+-dependent
inward tail current on repolarization from +10 to
110 mV in stomach
smooth muscle cells of Bufo marinus, providing first evidence of the current associated with the forward-mode operation of
the exchanger in smooth muscle cells. This study has observed such a
current in reverse-mode operation.
The magnitude of the Na+/Ca2+ exchange current
in smooth muscle is relatively small compared with that in cardiac
muscle (32). In Bufo stomach smooth muscle
cells, the mean inward current was only 2.4 pA
([Ca2+]i, 400 nM;
[Na+]i nominally zero,
[Na+]o = 94 mM;
[Ca2+]o = 20 mM). In this study, only 17 pA of outward current was recorded with a four- to fivefold enhanced
Na+ gradient. These relatively small currents will,
therefore, obscure any role played by the exchanger in regulating the
decline of [Ca2+]i following depolarization,
as well as determining the steady-state [Ca2+]i (20).
Possible role for
Na+/Ca2+
exchange in intracellular Ca2+
regulation.
Compared with the magnitude of other Ca2+ entry mechanisms,
such as the L-type Ca2+ current, it is unlikely that the
magnitude of Ca2+ entry through
Na+/Ca2+ exchange during the brief
depolarization associated with, for example, the action potential is
significant. However, given the relatively noninactivating
characteristic of the current (see Fig. 2), the exchanger may provide
constant Ca2+ fluxes over a longer period of time. The
ability of the exchanger to provide sufficient Ca2+ entry
is genuine, as evidenced by the rise of
[Ca2+]i during superfusion with low
Na+- or strophanthidin-containing solutions.
To provide a more quantitative measure of Ca2+ influx via
the exchanger required an estimate of the buffering capacity of the cell. By integrating the exchange current and using a 3:1 ratio for
Na+:Ca2+ exchange, a buffer ratio of 1:343 was
calculated (i.e., for 343 Ca2+ ions entering the cell, one
remains ionized in the sarcoplasm) for
[Ca2+]i in the range of 100-1,440 nM.
The exact value quoted here should be qualified by the caveats that:
the volume in which Ca2+ is distributed is an overestimate,
since no account was taken for that occupied by intracellular
organelles; some Ca2+ and fura 2 may be accumulated by
organelles where the equilibrium reaction between the reactants may be
different; and no account is taken for Ca2+ efflux through
other mechanisms over the time course of integration. However, the
value is similar to those (between 114 and 250) obtained for vascular
(9, 15) and visceral (16) smooth muscle as well as cardiac muscle (13, 36).
Ca2+ influx through the exchanger was sufficient to
replenish intracellular Ca2+ stores. Enhancement of
caffeine-induced Ca2+ release was possible by stimulating
exchanger influx during the interval between successive exposures to
caffeine, produced by either lowering [Na+]o
or adding strophanthidin. Some other studies have proposed that
Ca2+ influx via Na+/Ca2+ exchange
plays a role in replenishing functional intracellular Ca2+
stores in vascular smooth muscle cells and astrocytes (5, 6,
10) by either directly measuring release of stored
Ca2+ by agonists or indirectly by monitoring the activity
of spontaneous transient outward currents that are sensitive to
subsarcolemmal Ca2+. By measuring the releasable
Ca2+ by caffeine, this study provides the first direct
evidence for a role of the exchange in refilling the functional
intracellular Ca2+ stores in smooth muscle during
successive stimuli.
Oscillatory Ca2+ waves are associated with a raised resting
[Ca2+]i and enhanced store refilling. These
spontaneous Ca2+ spikes could result from enhanced
exchanger activity as they were particularly generated in low
[Na+]o solutions or in the presence of
strophanthidin. This finding may have pathophysiological implications
since the spontaneous Ca2+ waves may underlie uncontrolled
contractions in tissue such as detrusor muscle.
Significance during bladder pathology.
An important conclusion that may be drawn from the calculation of net
Ca2+ through the exchanger, from the integral of the
outward current, is that Na+/Ca2+ exchange
exists in a steady state with other cellular processes, so that net
flux of Ca2+ via the exchanger will be determined by the
prevailing levels of [Na+]o,
[Ca2+]o, [Na+]i,
and the membrane potential. One condition of particular significance, when net Ca2+ entry through the exchanger may be
significant, is during cellular hypoxia. The wall of the bladder can
undergo profound and prolonged periods of hypoxia during filling with
urine, as the wall is stretched and blood flow falls (2).
This may be exacerbated when the bladder wall hypertrophies as a
consequence of outflow tract obstruction. Acute cellular hypoxia
depresses the ability of intracellular stores to accumulate
Ca2+, increases the resting sarcoplasmic
[Ca2+]i (12), and may result
from a depression of ATPase activity associated with store
reaccumulation of Ca2+ by a reduction of cellular ATP
levels. However, it is unknown if the ATPase associated with sodium
pump function is also depressed. If so, this would raise
[Na+]i and hence lead to significant
Ca2+ influx by Na+/Ca2+ exchange,
leading to Ca2+ overload and cellular deterioration. The
significance of the role of [Na+]i in
determining whether the exchanger acts to cause Ca2+ influx
or efflux is illustrated. For a 3:1 exchanger, the relationship between
the transmembrane Na+ and Ca2+ gradients is
|
(3)
|
where Vm is the membrane potential and
R and T have their usual meanings. For different
[Na+]i values, it is possible to calculate
the [Ca2+]i at which the exchanger will exist
in a steady state if the other variables are known. Figure
6 plots these
[Ca2+]i values as a function of
[Na+]i for three different values of
Vm between
60 and
40 mV. The horizontal line
shows the mean value of [Ca2+]i (153 nM)
measured in these experiments. At
60 mV, the resting potential
recorded in these cells (37), an
[Na+]i of 13.7 mM, would ensure that no net
exchange of Ca2+ would occur over the membrane; at
50 and
40 mV, which are achieved during spontaneous oscillations of
Vm in these cells, the
[Na+]i would be smaller, 12.1 and 10.7 mM,
respectively. If the [Na+]i was increased
above these steady-state values, net Ca2+ influx would
follow. Various estimations of [Na+]i have
been made in smooth muscle ranging from 10 to 13 mM (17, 23,
38), which suggests that Na+/Ca2+
exchange may be near a steady state in these tissues. The precise value
of [Na+]i has not been measured in detrusor
smooth muscle, but this model suggests that a small rise would be
sufficient to precipitate net Ca2+ influx, which could lead
to Ca2+ oscillations of the type reported here, and
Ca2+ overload of the tissue, if the influx was prolonged.
The control of [Na+]i and the role of sodium
pump inhibition during hypoxia become, therefore, crucial aspects to
investigate in the pathogenesis of abnormal detrusor function.

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Fig. 6.
The relationship between intracellular Na+
concentration ([Na+]i) and steady-state
[Ca2+]i for a 3:1
Na+:Ca2+ exchange model (Eq. 3). The
lines denote calculations for membrane potentials of 40, 50, and
60 mV. The horizontal line is the measured
[Ca2+]i in these experiments; arrows denote
the [Na+]i at steady state with the measured
[Ca2+]i at the 3 values of membrane
potential. A higher [Na+]i would result in
net Ca2+ influx; a lower [Na+]i
would result in net efflux.
|
|
 |
FOOTNOTES |
Address for reprint requests and other correspondence: C. H. Fry, Institute of Urology and Nephrology, Univ. College London, 48 Riding House St., London W1P 7PN, United Kingdom (E-mail:
c.fry{at}ucl.ac.uk).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 9 June 2000; accepted in final form 4 December 2000.
 |
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