INTRODUCTION

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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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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.

(1)

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

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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.

View larger version (15K): [in this window] [in a new window]   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+.

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.

View larger version (27K): [in this window] [in a new window]   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.

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 (B_Ca) can be defined as a dimensionless quotient, Ca_c/[Ca²⁺]_i, where Ca_c 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. Ca_c was calculated in a voltage-clamp experiment by integrating a Ca²⁺ current (I_Ca) 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 I_Ca.

View larger version (18K): [in this window] [in a new window]   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.

(2)

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 B_Ca. 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.

View larger version (21K): [in this window] [in a new window]   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.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Spontaneous [Ca2+]i oscillations induced by low-Na+ solution (29.4 mM [Na+]o).

	DISCUSSION

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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.

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.

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 V_m 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 V_m 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 V_m 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.

View larger version (15K): [in this window] [in a new window]   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.