INTRODUCTION

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THE EXTRUSION OF Ca²⁺ maintains Ca²⁺ homeostasis in vena cava smooth muscle cells. Removal of Ca²⁺ from the cytosol is an active process that depends primarily on the plasma membrane Ca²⁺-adenosinetriphosphatase (ATPase; PMCA) and the Na⁺-Ca²⁺ exchanger, which directly extrude intracellular Ca²⁺, and the sarcoendoplasmic reticulum Ca²⁺-ATPase (SERCA). PMCA is an ATP-dependent pump activated by Ca²⁺/calmodulin and represents one of the two Ca²⁺-exporting systems in the plasma membrane (PM). It is referred to as a high-affinity, low-capacity pump. This 130-kDa phosphoprotein consisting of PMCA 1b and 1a isoforms in vascular smooth muscle (VSM) of the rabbit (18) mediates an active transport of Ca²⁺ at a Ca²⁺-to-ATP ratio of one. Hardin et al. (15) found that ATP produced by membrane-associated glycolysis may be the primary fuel source for the PMCA. Na⁺-Ca²⁺ exchange is the second Ca²⁺ translocator in the PM, but, unlike the PMCA, it is known as a high-velocity, low-affinity antiporter. It electrogenically exchanges three Na⁺ for one Ca²⁺, thus it responds to Na⁺ and Ca²⁺ gradients as well as to the transmembrane electrical potential (6). One possible reason why the Na⁺-Ca²⁺ exchanger can be effective in spite of its low affinity is because it is exposed to a localized high intracellular Ca²⁺ concentration ([Ca²⁺]_i; see Ref. 6) that is due to the unloading of Ca²⁺ from the sarcoplasmic reticulum (SR) into the restricted space between the SR and PM.

By convention, the PMCA and Na⁺-Ca²⁺ exchange are thought to be entirely responsible for Ca²⁺ extrusion; however, an additional more complex pathway for the removal of [Ca²⁺]_i in which the SR plays a key role has been proposed. It has already been established that the SR is a transient source and sink for Ca²⁺ during fluctuations in contractile tension (30) and may act as a "buffer barrier" (32) by taking up a fraction of the Ca²⁺ that enters the cells through the plasmalemma before it reaches the myofilaments. It is postulated that, in order for the SR to buffer Ca²⁺ on a steady-state basis, it is required that Ca²⁺ be released from the SR lumen and extruded into the extracellular space (ECS; see Ref. 31). This extrusion process is thought to be mediated by the transport of Ca²⁺ from the SR where ryanodine and inositol 1,4,5-trisphosphate (IP₃) receptor-operated Ca²⁺ channels release Ca²⁺ into the subsarcolemmal space between the SR and the plasmalemma (this process is referred to as "vectorial release") from where Na⁺-Ca²⁺ exchangers complete the extrusion process (22). Thus it is implied that SERCA functions in concert with Na⁺-Ca²⁺ exchangers closely apposed to the peripheral SR, thereby contributing significantly to Ca²⁺ extrusion. The SERCA is an ATP-dependent and azide-insensitive (i.e., nonmitochondrial) protein (105 kDa), like the PMCA, which expresses the SERCA 2a and 2b (and possibly SERCA 3) isoforms in rabbit VSM (25). Endogenous phospholamban, in its dephosphorylated state, inhibits SERCA, but, when phosphorylated by various protein kinases, its activity is blocked, resulting in the activation of SERCA. According to Eggermont et al. (11), stimulated Ca²⁺-ATPase activity in the SR and PM is approximately the same in bovine pulmonary VSM; however, it may differ widely between different smooth muscle types and depends on several factors, including regulation by protein kinases, proteins, lipids, and cytosolic pH.

In VSM, mitochondrial Ca²⁺ uptake plays a minor role in Ca²⁺ homeostasis under physiological conditions (41), but, when [Ca²⁺]_i rises to pathological levels, the mitochondria begin to sequester significant amounts of Ca²⁺ (21). The apparent Michaelis constant for Ca²⁺ uptake in VSM mitochondria, corresponding to a value of 17 µM, is ~200-fold higher than the resting [Ca²⁺]_i in these cells. Thus it is improbable that the mitochondria act as a Ca²⁺ sink even when [Ca²⁺]_i is elevated during stimulation.

In the present study, our objective was to determine the mechanism of Ca²⁺ extrusion using vena cava smooth muscle cells from the rabbit as a model. With the help of various pharmacological tools, we were able to block SERCA, the Na⁺-Ca²⁺ exchanger, and the PMCA. For inactivating the PMCA, it was necessary to prevent ATP from being generated through a membrane-associated glycolytic pathway that preferentially fuels the Ca²⁺ pump (15). Iodoacetate (IAA), a metabolic inhibitor that inhibits ATP synthesis, was used to target the glycolytic machinery associated with the PMCA by way of arresting the glycolytic enzyme glyceraldehyde phosphate dehydrogenase (5). IAA also affects other enzymes due to its capability to alkylate sulfhydryl groups; however, this reaction likely occurs after 1-2 h of incubation with IAA (5). Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) was used in combination with IAA as a mitochondrial poison or protonophoric uncoupler of energy-dependent respiration to further prevent ATP synthesis in the mitochondria and to also inhibit mitochondrial Ca²⁺ sequestration (31) via the dissipation of the mitochondrial proton gradient and membrane potential (2). We employed cyclopiazonic acid (CPA) to inhibit SERCA and prevent refilling of the SR (4). CPA is readily reversible, and its effects are relatively rapid. The Na⁺-Ca²⁺ exchanger was blocked by removing Na⁺ from the perfusate. We then determined the relative contributions of the PMCA, the SERCA, and the Na⁺-Ca²⁺ exchanger to Ca²⁺ extrusion and measured SR refilling after systematically blocking the SERCA and the Na⁺-Ca²⁺ exchanger.

	MATERIALS AND METHODS

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Tissue preparation. New Zealand White rabbits (1.5-2.5 kg) were killed by CO₂ asphyxiation then exsanguinated. The inferior vena cava was promptly excised, and connective tissue and adventitia were removed in a petri dish containing normal physiological saline solution (PSS), pH 7.4, at room temperature (22-25°C). The tissue was opened longitudinally, cut into a 25 by 7-mm rectangular sheet, and the luminal endothelial layer teased off by a piece of wet filter paper. A rectangular plastic frame with a channel of silicon elastomer near each edge was used to pin the smooth muscle strip into place.

[Ca²⁺]_i measurement. Background tissue autofluorescence was measured before loading the tissue with the fluorescent Ca²⁺ indicator fura 2. In a spectrofluorimeter (Spex Fluorolog; Spex Industries), light from a xenon lamp was beamed through a sample chamber where it entered a solution-filled acrylic cuvette (4 ml capacity) containing the smooth muscle tissue on its support frame. The luminal side of the tissue was illuminated with alternating wavelengths of 340 and 380 nm coinciding with the peak of fluorescence of Ca²⁺-bound and -unbound fura 2. Light emitted from the tissue (505 nm) was collected in the front face mode via a photomultiplier. The vena cava was then incubated for 1.5 h in the dark with 5 µM fura 2-acetoxymethyl ester and 5 µM pluronic F-127 in oxygenated, room temperature normal PSS. After loading, the tissue was washed in normal PSS then transferred to a cuvette and placed in the sample chamber of the spectrofluorimeter. The bathing solutions were changed by a peristalstic pump, perfusing the tissue at a rate of 0.37 ml/s, and by vacuuming off the overflow. All experiments were carried out at room temperature.

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View this table: [in this window] [in a new window]   Table 1.   Rate of [Ca2+]i decline fitted to the equation, f = aebx + cedx (see MATERIALS AND METHODS) with blockade of the Na+-Ca2+ exchanger and SERCA

	RESULTS

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Total inhibition of Na⁺-Ca²⁺ exchanger and Ca²⁺ pump activity abolishes Ca²⁺ extrusion. To determine whether Ca²⁺ extrusion could be blocked, the major contributing mechanisms, which include the Na⁺-Ca²⁺ exchanger and the PMCA, as well as the mechanism thought to be partly involved in SR-mediated extrusion, the SERCA, were inhibited (Fig. 1). A control response was obtained by recording changes in [Ca²⁺]_i while perfusing with 80 mM K⁺ PSS and PE (20 µM; loading stimulus) followed by the addition of zero Ca²⁺ PSS and phentolamine (10 µM), a competitive antagonist of PE, to abolish activation and Ca²⁺ entry. The initial rise in [Ca²⁺]_i was due to the Ca²⁺-loading effect of K⁺ and PE, whereas the subsequent fall in [Ca²⁺]_i was assumed to be due to active extrusion in the absence of Ca²⁺ influx. The experimental curve in Fig. 1 was obtained by following the same protocol as in the control, except for the addition of CPA (20 µM), a SERCA blocker, IAA (1 mM), an inhibitor of membrane-associated glycolysis, and FCCP (3 µM), an uncoupler of oxidative metabolism in the mitochondria, at the time of activation of Ca²⁺ influx to allow for preincubation and, in addition, removal of external Na⁺ to block the Na⁺-Ca²⁺ exchanger upon inactivation and Ca²⁺ removal. Within 120 s, the height of the activation curve had reached its peak of 352.6 ± 49.4 nM, n = 6 (compared with the peak control value of 201.8 ± 32.2 nM, n = 6), and then, by blocking Ca²⁺ influx (as in the control), it could be determined whether all of the Ca²⁺-extruding components would be inhibited by the combination of CPA, IAA, FCCP, and zero Na⁺. The resulting plateau, lasting until the end of the experiment, made it clear that the combination of blockers used to target SERCA, the PMCA, and the Na⁺-Ca²⁺ exchange were sufficient to completely block Ca²⁺ efflux. Without an apparent decay in [Ca²⁺]_i, it is obvious that the passive leak of Ca²⁺ out of the cells is negligible. Controls for the effects of FCCP and IAA in a normal PSS and zero Ca²⁺ PSS medium are shown in Fig. 1, inset. There was no change in [Ca²⁺]_i with IAA alone. However, in the FCCP control, a delayed upward deflection in [Ca²⁺]_i led to a sustainable plateau phase that indicated inhibition of active Ca²⁺ removal from the cytoplasm. The FCCP-induced rise in [Ca²⁺]_i was responsible for the higher experimental peak when compared with the control.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Metabolic and transport blockade prevents Ca2+ extrusion. Intracellular Ca2+ concentration ([Ca2+]i) is recorded during stimulation of Ca2+ influx with 80 mM K+ physiological saline solution (PSS) and 20 µM phenylephrine (PE) followed by 0 Ca2+ PSS containing 10 µM phentolamine (left). This experiment is repeated in the presence of ATP inhibitors 1 mM iodoacetate (IAA), 3 µM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor cyclopiazonic acid (CPA; 20 µM) and with Na+ removed (right). In this case, [Ca2+]i remains elevated. Traces are typical of 7 experiments. Inset: controls for the effect of IAA (solid line), and FCCP (broken line). Normal PSS is the initial bathing solution for these controls (solid bar), which was later replaced by 0 Ca2+ PSS (broken bar). Data in inset are typical of 3 experiments.

SERCA and Na⁺-Ca²⁺ exchanger blockade slows the rate of [Ca²⁺]_i decline. Typical responses to inhibitors of SERCA and the Na⁺-Ca²⁺ exchanger are shown in Fig. 2, A-C. After establishment of basal [Ca²⁺]_i (54.1 ± 9.8 nM, n = 6), each strip of smooth muscle was loaded with Ca²⁺ by the addition of 80 mM K⁺ PSS and 20 µM PE (as before) and then washed with zero Ca²⁺ PSS and 10 µM phentolamine to abolish the influx of Ca²⁺. These manipulations caused a sustained increase in [Ca²⁺]_i followed by a decline upon removal of external Ca²⁺, revealing the process of Ca²⁺ extrusion. The data collected from these experiments are summarized in Fig. 3.

View larger version (14K): [in this window] [in a new window]   View larger version (15K): [in this window] [in a new window]   View larger version (14K): [in this window] [in a new window]   Fig. 2.   Blockade of Na+-Ca2+ exchange and SERCA attenuates the rate of decline in [Ca2+]i. Time course (seconds) changes in [Ca2+]i (nM) were monitored during stimulation with 80 mM K+ PSS and PE (20 µM) and then by washing out with 0 Ca2+ PSS and phentolamine (10 µM) as shown on left. On the right, this experiment is repeated (for comparison, the control decay is superimposed as shown by the broken line) with external Na+ removed (A), which reveals Na+-independent Ca2+ extrusion, CPA (20 µM) preincubation to selectively block SERCA (B), and a combination of CPA and 0 Na+ (C). The effect of Na+ removal and CPA on the tissue preparation appears in the insets of A and B, respectively. Exposure to normal PSS (solid bar) induces a rise in [Ca2+]i followed by a fall upon removal of external Ca2+ (broken bar). These results are representative of 6 or more experiments.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Comparison of the effects of CPA, 0 external Na+, and their combination on the rate of [Ca2+]i decline. With blockade of SERCA, the rate of [Ca2+]i decline was 77.72 ± 5.08% of control, n = 7. With blockade of Na+-Ca2+ exchange, the rate of decline in [Ca2+]i was further reduced to 53.35 ± 4.56% of control, n = 8, similar to a value of 48.49 ± 3.52%, n = 6, which was obtained by blocking both of these Ca2+ extrusion mechanisms. § P < 0.0009 vs. control and * P<0.0007 vs. CPA-, 0 Na+-, and 0 Na+ + CPA-treated tissue by analysis of variance (ANOVA) and Newman-Keuls test.

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Summarizing the effects of CPA, zero Na⁺, and CPA plus zero Na⁺ on their ability to block Ca²⁺ extrusion. Figure 3 illustrates the extent to which the SERCA, Na⁺-Ca²⁺ exchange, and the PMCA each contributed to the [Ca²⁺]_i decline. SERCA-mediated Ca²⁺ extrusion accounted for ~23%, whereas the remaining 77% (n = 7) was due to extrusion by the PMCA and the Na⁺-Ca²⁺ exchanger. The Na⁺-Ca²⁺ exchanger, with or without SERCA activity present, contributed to approximately one-half (47%) of the extruded Ca²⁺ as revealed by blockade of the Na⁺-Ca²⁺ exchanger with zero Na⁺. The other one-half, 53% (n = 8), was extruded by the PMCA. This was confirmed by inactivating SERCA and the Na⁺-Ca²⁺ exchanger simultaneously (no significant differences between 53 and 49%, n = 6), which revealed that about one-half of the Na⁺-Ca²⁺ exchanger-mediated Ca²⁺ extrusion involved Ca²⁺ uptake by SERCA (23/47 = 50%). An alternative method for determining the rate of Ca²⁺ extrusion was to calculate the slope of the [Ca²⁺]_i decline curve at a specific elevated [Ca²⁺]_i (e.g., 200 or 250 nM) using statistical tests to compare the slopes of the control decays with those of the experimental decays. With respect to statistically significant differences, the outcomes, regardless of using rate constants or slopes, were the same (data not shown).

SERCA blockade empties the Ca²⁺ store within the SR, an effect that is opposite to that seen with Na⁺-Ca²⁺ exchanger blockade. To investigate the possibility that SR Ca²⁺ accumulation, without Ca²⁺ extrusion, was responsible for a significant decline in [Ca²⁺]_i, we measured caffeine-induced [Ca²⁺]_i increases as an indicator of the SR Ca²⁺ content (Fig. 4). A bolus of caffeine (25 mM) was administered in Ca²⁺-free PSS as a control (peak₁). After the first exposure to caffeine, Ca²⁺ was replenished in the absence of caffeine to readjust the baseline. The tissue was then exposed to 80 mM K⁺ PSS and PE (20 µM) to reproduce the conditions of the previous experiments. After perfusion with zero Ca²⁺ PSS and phentolamine (10 µM; and CPA and/or zero Na⁺ if blockade is required), a second caffeine-induced Ca²⁺ release was elicited (peak₂).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Sarcoplasmic reticulum (SR) refills only during blockade of Na+-Ca2+ exchange (0 Ca2+ PSS or CPA treatment prevents refilling). The first [Ca2+]i transient was induced by caffeine (25 mM) in 0 Ca2+ PSS (downward arrows). Undershoot was readjusted by adding Ca2+ back to the bathing solution (upward arrows), and then stimulation with 80 mM K+ PSS and PE (20 µM) followed by washout with 0 Ca2+ PSS and phentolamine (10 µM) was performed as in previous experiments with a subsequent addition of caffeine to determine the depletory effect on the SR. A control trace (A) was obtained, and then the experiment was repeated with 0 Na+ PSS (B), CPA (C), or CPA + 0 Na+ PSS (D). In the inset, a control for multiple exposures to caffeine was obtained. Each trace is representative of 4 experiments.

Comparing the refilling capacity of the SR in the presence and absence of SERCA and Na⁺-Ca²⁺ exchanger inhibitors. The amplitude of the first and second caffeine-induced Ca²⁺ responses were ratioed (peak₂/peak₁) and are compared in Fig. 5. With the activity of all the extrusion pathways intact (control), peak₂/peak₁ of 13.2 ± 6.7%, n = 4, was very low. SERCA blockade appeared to further reduce SR Ca²⁺ by preventing reuptake (peak₂/peak₁ = 1.7 ± 1.7%, n = 4, though not significantly different from the control, P < 0.3). When SERCA and the Na⁺-Ca²⁺ exchanger were blocked simultaneously, the Ca²⁺ replenishing effect of Na⁺-Ca²⁺ exchanger blockade was nullified by the depletory effect of CPA (peak₂/peak₁ = 5.1 ± 2.5%, n = 4), indicating that SERCA is an upstream effector of SR-mediated Ca²⁺ extrusion. In contrast to the responses seen in the control and those with CPA, the removal of external Na⁺ by itself markedly enhanced Ca²⁺ reuptake into the SR as seen by the large peak₂-to-peak₁ ratio of 85.5 ± 13.5%, n = 4. 

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Comparison of the refilling of SR in 0 Ca2+ solution under conditions of SERCA and Na+-Ca2+ exchange blockade. Amplitude (basal to peak levels) of the second caffeine-induced Ca2+ transient (peak2) was compared with the internal control (peak1). Pooled data are expressed as percent ratio of the amplitude of caffeine-induced Ca2+ release (peak2/peak1). In the absence of external Ca2+ (control: 13.19 ± 6.75%, n = 4), Na+-Ca2+ exchanger blockade induces refilling of the SR, as shown by the caffeine-induced Ca2+ release ratio of 85.53 ± 13.46%, n = 4, whereas, with the addition of CPA, the SR Ca2+ store is completely depleted, corresponding to a value of 5.08 ± 2.51%, n = 4. With Na+-Ca2+ exchange uninhibited, CPA still has the same effect (1.70 ± 1.70%, n = 4). § P < 0.002 vs. 0 Na+-treated tissue and * P < 0.3 vs. 0 Ca2+ PSS- vs. CPA- and 0 Na+ + CPA-treated tissue by ANOVA.

	DISCUSSION

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Ca²⁺ extrusion is a physiological process that is essential for keeping the resting [Ca²⁺]_i low in response to the Ca²⁺ entry that is due to the activity of a variety of Ca²⁺ channels in the presence of a steep electrochemical gradient across the PM. Our aim in designing this study was to better understand the mechanisms involved in Ca²⁺ extrusion. Total impairment of Ca²⁺ pumping activity (i.e., PMCA, SERCA, and mitochondrial) and Na⁺-Ca²⁺ exchange completely abolished Ca²⁺ extrusion as seen by a plateau in the elevated steady-state [Ca²⁺]_i after high K⁺ stimulation and the subsequent perfusion with a Ca²⁺-free bathing solution. PMCA blockade was achieved through the reduction of ATP synthesis (using FCCP and IAA; see Ref. 25), which also inhibits SERCA (29) and is reported to markedly reduce the bidirectional operation of the Na⁺-Ca²⁺ exchanger in the squid axon (9). In determining the relative contribution of each of the distinct Ca²⁺ translocators, we found that, of the total Ca²⁺ extrusion that could be blocked by complete metabolic inhibition, 47% was extruded by the Na⁺-Ca²⁺ exchangers, and 53% was extruded via the PMCA. Similar values were obtained by Furukawa and colleagues (12) and McCarron and co-workers (20), using the stomach smooth muscle of Bufo marinus, observed a Na⁺-dependent [Ca²⁺]_i decline accounting for 38-52% of the total Ca²⁺ removal between a [Ca²⁺]_i of 300 and 500 nM (20). Although there is excellent agreement between the results quoted above, it warrants considering the experimental conditions before extrapolation to physiological conditions in vivo. In our study, temperature was lowered from 37°C to room temperature, which may have had differential effects on the Ca²⁺-ATPases and the Na⁺-Ca²⁺ exchanger (10). Another factor is the removal of external Na⁺, which, when used to block Na⁺-Ca²⁺ exchange, can alter pH_i, leading to a change in the affinity of SERCA, PMCA, and the Na⁺-Ca²⁺ exchanger for Ca²⁺. However, in a study by Motley et al. (23), switching to a Na⁺-free solution was associated with negligible changes in pH_i. An additonal potential problem is whether the Na⁺ substitutes, Li⁺ or N-methyl-D-glucamine, have different effects on the inferior vena cava, although, according to one study, no differences were observed (13). Further complexity is introduced by the use of multicellular preparations, which, unlike single cells, exhibit an averaged response but which, on the other hand, do not require isolation with digesting enzymes that may alter cell physiology. The multicellular preparation may have contributed to the requirement for two exponential components in fitting the rates of [Ca²⁺]_i decline. However, the fact that several mechanisms contribute to Ca²⁺ extrusion and that cellular Ca²⁺ redistribution may take place would also tend to complicate the kinetics. For this reason, we adopted a second method by measuring the instantaneous rate of [Ca²⁺]_i decline at a fixed elevated [Ca²⁺]_i value, which led to conclusions identical to those derived from measuring changes in the fast rate constant.

Aside from the PMCA and the Na⁺-Ca²⁺ exchanger, no other extrusion pathway has been clearly established. Nonetheless, previous work from this laboratory has eluded to a complementary extrusion mechanism that involves the SR and structurally linked Na⁺-Ca²⁺ exchangers (30). It was postulated that the superficial SR acts as a buffer barrier to Ca²⁺ entry and that, to maintain this function, the SR must release Ca²⁺ into the subplasmalemmal space from where it is subsequently extruded (31). Chen and van Breemen (7) also suggested the presence of an extrusion pathway between the SR and the PM when they found that inhibition of Ca²⁺ accumulation by the SR increases [Ca²⁺]_i, which was not accompanied by an increase in Ca²⁺ influx. Suzuki et al. (28) have observed reduced Ca²⁺-activated K⁺ channel activity (an indicator for localized Ca²⁺ increases derived from the SR) in smooth muscle treated with CPA, suggesting that Ca²⁺ uptake is immediately followed by extrusion of Ca²⁺ toward the ECS. Several other studies (24, 33) lead to the conclusion that this unique extrusion pathway does indeed exist. One study supports the idea that Ca²⁺ taken up into the SR is later extruded through an interaction between the SR and the PM and that, when SERCA is blocked, cytosolic Ca²⁺ is removed by the PM extrusion mechanisms not involved in SR-mediated Ca²⁺ extrusion (19).

The proposed participation of the SR in Ca²⁺ extrusion from the cells is consistent with the observations made in this study in which SERCA blockade reduced the rate of [Ca²⁺]_i decline by 23%. An alternative interpretation of this result, which does not involve a multistep Ca²⁺ transport between SERCA and Na⁺-Ca²⁺ exchange, would be that the SR is accumulating and retaining cytoplasmic Ca²⁺ during exposure to zero external Ca²⁺. In this case, SERCA would function additively to Na⁺-Ca²⁺ exchange and the PMCA. However, our finding that the effects of CPA and zero external Na⁺ were not additive is not consistent with this interpretation. Other findings reported in RESULTS also diminish the likelihood of simple SR accumulation and retention as a possible explanation. We found that there was no significant difference in caffeine-sensitive SR Ca²⁺ content when comparing the [Ca²⁺]_i decline under control and CPA conditions (Fig. 4, A and C). Removal of external Na⁺ greatly enhanced SR Ca²⁺ retention, but its abolition by CPA did not significantly slow the rate of [Ca²⁺]_i decline. Results first obtained by Aaronson and van Breemen (1) then later confirmed by Blaustein and colleagues (3) established that, in VSM, the Na⁺-Ca²⁺ exchanger plays a key role in controlling the amount of Ca²⁺ stored in the SR. One reason for the high SR Ca²⁺ content in zero Na⁺ media may be that Ca²⁺ released from the SR into the junctional space is unable to leave the cell because of the blocked Na⁺-Ca²⁺ exchanger and is quickly pumped back into the SR without being detected by cytoplasmic fura 2, as Wolska and Lewartowski (34) demonstrated. Such an Na⁺-sensitive pool has also been described in guinea pig taeni coli (1). In parallel experiments by Chen and van Breemen (8; using thapsigargin as a SERCA inhibitor in place of CPA), similar data were obtained. The results presented here are best explained by the model presented in Fig. 6. This model assumes that a fraction (50%) of the Na⁺-Ca²⁺ exchangers is positioned close to the peripheral SR and functions mainly to remove Ca²⁺ from the junctional space between PM and SR membranes (denoted [Ca²⁺]_j). [Ca²⁺]_j is thought to be elevated above the bulk myoplasmic Ca²⁺ concentration ([Ca²⁺]_m; see Ref. 27) because of asymmetric release or impaired diffusion away from the release sites. The low-affinity, high-velocity Na⁺-Ca²⁺ exchanger transports Ca²⁺ from the junctional space to the ECS. This assumption is supported by our observation that inhibition of this process allows reuptake of Ca²⁺ into the SR via SERCA. Additional evidence in support of SR-mediated Ca²⁺ extrusion is presented by Stehno-Bittel and Sturek (27), who also demonstrated the presence of peripheral Ca²⁺ gradients by simultaneous measurement of outward currents mediated by Ca²⁺-sensitive K⁺ channels and global [Ca²⁺]_i using fura 2. A functional relationship between the peripheral SR and the Na⁺-Ca²⁺ exchanger has also been proposed on the basis of ultrastructure. Moore et al. (22), using digital imaging fluorescence microscropy and a constrained deconvolution algorithm for processing the data, showed colocalization and suggested functional coupling of the Na⁺-Ca²⁺ exchanger and the SR in smooth muscle. This was recently confirmed by Blaustein and co-workers (16, 17). The pathway for SR unloading relies on an asymmetrical arrangement of SERCA and SR Ca²⁺ release sites such that the Ca²⁺ taken up from the myoplasm is preferentially released by Ca²⁺ channels facing the junctional space rather than by those facing the myoplasm. Additional evidence suggests that the Na⁺-Ca²⁺ exchanger may have selective access to SR Ca²⁺ (17). It has also been suggested that local IP₃ gradients may promote the asymmetrical Ca²⁺ release by the peripheral SR. Although experimental data support the interaction between the Na⁺-Ca²⁺ exchanger and SR Ca²⁺ transport, the detailed mechanism of this process is, at present, unclear.

View larger version (37K): [in this window] [in a new window]   Fig. 6.   Schematic diagram illustrating the Ca2+ extrusion process as mediated by the Na+-Ca2+ exchanger (filled ovals), plasma membrane Ca2+-ATPase (PMCA; filled circle), and the SERCA (shaded circles). PMCA extrudes 53% of the Ca2+ in the myoplasm, whereas the remaining Ca2+ is extruded by the Na+-Ca2+ exchangers into the extracellular space (ECS), some of which is activated by SR-released Ca2+. About one-half of the Ca2+ extruded by the Na+-Ca2+ exchange system is in series with Ca2+ uptake and release by the SR, forming a Ca2+ gradient in the junctional space that permits Ca2+j (Ca2+j within the junctional space) to exceed Ca2+m (myoplasmic Ca2+m). The increase in [Ca2+]i activates Na+-Ca2+ exchange; however, if the latter is blocked, Ca2+ is pumped back into the SR.

In conclusion, our findings support a role for the VSM SR in Ca²⁺ extrusion by a sequential coupling of SERCA, Ca²⁺ release channels, and the Na⁺-Ca²⁺ exchanger.