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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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)
[Ca<SUP>2+</SUP>]<SUB>i</SUB><IT>=K</IT><SUB>d</SUB><IT>&bgr; </IT><FR><NU>(R<IT>−</IT>R<SUB>min</SUB>)</NU><DE>(R<SUB>max</SUB><IT>−</IT>R)</DE></FR> (1)
where Rmin and Rmax refer to ratio (R) values at 0 [Ca2+]i and saturating [Ca2+]i, respectively; beta  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 MOmega 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 (Delta [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 (Delta [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+.

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.

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, Delta Sigma Cac/Delta [Ca2+]i, where Delta Sigma Cac is an increment of total sarcoplasmic Ca2+ concentration (i.e., the sum of free and bound Ca2+) and Delta [Ca2+]i is the change of ionized [Ca2+]i. Delta Sigma Cac was calculated in a voltage-clamp experiment by integrating a Ca2+ current (ICa) generated by membrane depolarization and Delta [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 (int 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.

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 Delta [Ca2+]i has been shown to be approximately proportional to Delta Sigma Cac during this period (15). Total Ca2+ influx was calculated from
−<FR><NU>1</NU><DE>z<SUB>Ca</SUB><IT>F</IT></DE></FR> <LIM><OP>∫</OP></LIM><IT> I</IT><SUB>Ca</SUB> d<IT>t</IT> (2)
where -int 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, -int ICadt was calculated to be 72.9 ± 9.8 µmol/l cell volume (ignoring the volume contribution from intracellular organelles) and Delta [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.

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.


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Fig. 5.   Spontaneous [Ca2+]i oscillations induced by low-Na+ solution (29.4 mM [Na+]o).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
(<FR><NU>[Na<SUP>+</SUP>]<SUB>o</SUB></NU><DE>[Na<SUP>+</SUP>]<SUB>i</SUB></DE></FR>)<SUP><IT>3</IT></SUP><IT>=</IT><FR><NU>[Ca<SUP>2+</SUP>]<SUB>o</SUB></NU><DE>[Ca<SUP>2+</SUP>]<SUB>i</SUB></DE></FR><IT>·</IT>exp<FENCE><FR><NU><IT>V</IT><SUB>m</SUB><IT>F</IT></NU><DE><IT>RT</IT></DE></FR></FENCE> (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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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