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Hypoxic pulmonary vasoconstriction in intact rat intrapulmonary arteries is not initiated by inhibition of Na⁺-Ca²⁺ exchange

Division of Asthma, Allergy, and Lung Biology, King's College London, London, United Kingdom

Submitted 14 September 2006 ; accepted in final form 3 July 2007

	ABSTRACT

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It has been proposed that a hypoxia-induced inhibition of the Na⁺-Ca²⁺ exchanger (NCX) contributes to hypoxic pulmonary vasoconstriction (HPV). By recording isometric tension development in rat intrapulmonary arteries (IPA), we examined the effect on HPV of maneuvers that reduce the ability of NCX to regulate intracellular Ca²⁺ concentration ([Ca²⁺]_i). In some experiments, fura pentakis(acetoxymethyl) ester-3 (fura PE-3) was also used to monitor [Ca²⁺]_i. HPV was elicited in IPA that were pretreated with 10 µM diltiazem and slightly preconstricted with PGF₂, which enhances the hypoxic response. Substitution of Na⁺ with Li⁺ increased HPV and the associated rise in [Ca²⁺]_i. Pretreatment with ouabain (100 µM) to diminish the Na⁺ gradient or with the reverse-mode NCX inhibitor KB-R7943 (3 or 10 µM) had no significant effect on HPV. Combined treatment with ouabain and low-[Na⁺] (24 mM) solution enhanced HPV strongly. The role of NCX in Ca²⁺ extrusion was examined by assessing the decrease in [Ca²⁺]_i in Ca²⁺-free physiological saline solution either containing or lacking Na⁺ following a high K⁺-induced loading of cellular [Ca²⁺]. Although the large initial rapid fall in [Ca²⁺] was Na⁺ independent, final recovery of [Ca²⁺] to its basal level was delayed in the absence of Na⁺. Therefore, HPV persisted or was increased under conditions in which forward-mode NCX was already attenuated or prevented, demonstrating that inhibition of NCX by hypoxia is unlikely to initiate HPV. Instead, NCX appears to act to inhibit HPV as would be expected if it is functioning to extrude Ca²⁺.

hypoxia; pulmonary artery

HPV in isolated pulmonary arteries (PA) typically consists of a biphasic contractile response, with a rapid transient vasoconstriction (phase 1) that is followed by a slowly developing sustained contraction (phase 2) (4, 9). Although there is general agreement that HPV is associated with a rise in intracellular Ca²⁺ concentration ([Ca²⁺]_i), the underlying mechanisms are still in dispute. Although there is extensive evidence that acute hypoxia inhibits K⁺ channels, leading to membrane depolarization and Ca²⁺ influx via voltage-gated Ca²⁺ channels, it has also been reported that Ca²⁺ release and Ca²⁺ influx through store-operated channels (SOC) play major roles in the development of HPV (1, 4, 7, 13, 17, 19). Furthermore, previous reports suggest that phase 2 is dependent on the presence of an intact endothelium and the release of unknown endothelial mediator(s), which causes Ca²⁺ sensitization of the contractile apparatus (1, 16, 18).

Inhibition of Ca²⁺ extrusion, particularly via the Na⁺-Ca²⁺ exchanger (NCX), has also been proposed to increase [Ca²⁺]_i during hypoxia (20, 25). NCX is a low-affinity, high-capacity Ca²⁺ extrusion mechanism that uses the energy inherent in the inward electrochemical Na⁺ gradient to pump Ca²⁺ out of many types of cells, exchanging 3 Na⁺ for 1 Ca²⁺ during this process (3). NCX has been hypothesized to play a crucial role in the extrusion of intracellular Ca²⁺, especially during smooth muscle stimulation (3). Wang et al. (25) showed that one subtype of the NCX (NCX1) is expressed in rat pulmonary arteries and also presented evidence that inhibition of this process may be crucial for development of HPV (25). They observed that inhibition of NCX by Na⁺ removal mimicked the effect of low PO₂ on [Ca²⁺]_i, that hypoxia inhibited NCX in smooth muscle cells isolated from rat PA, and that the effects of hypoxia and Na⁺ removal were not additive. They therefore proposed that inhibition of NCX by hypoxia leads to a decrease in cytosolic Ca²⁺ extrusion, resulting in the accumulation of intracellular Ca²⁺ and contraction.

However, it is also conceivable NCX could play a more active role in Ca²⁺ entry during HPV. In a variety of cell types, including smooth muscle, NCX1 has been reported to be enriched in areas of the sarcolemma close to the endoplasmic reticulum (ER) (3, 6). Since the opening of SOC leads to Na⁺ as well as Ca²⁺ entry, and since the direction in which NCX moves Ca²⁺ is a function of the electrochemical Na⁺ gradient, it has been suggested that the elevation of [Na⁺]_i in the subsarcolemmal space that occurs upon activation of SOC can inhibit or even reverse Na⁺-Ca²⁺ exchange, thus promoting Ca²⁺ influx, refilling of the ER, and as a consequence, contraction (2, 10, 29).

Given the lack of information regarding whether NCX is involved in HPV in intact pulmonary arteries as opposed to isolated cells, we examined the extent to which manipulating the activity of NCX using Na⁺ substitution, ouabain and KB-R7943, affected HPV in isolated intrapulmonary arteries (IPA) of the rat. Our results suggest that NCX does not contribute importantly to the effects of hypoxia on contraction in this preparation.

	METHODS

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Tension measurements. Adult male Wistar rats (250–275 g) were killed by cervical dislocation in accordance with Schedule 1 as prescribed and approved by the UK Home Office and the Guide for the Care and Use Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996). The heart and lungs were rapidly removed and placed into cold Krebs physiological saline solution (PSS; in mM: 118 NaCl, 24 NaHCO₃, 1 MgSO₄, 0.44 NaH₂PO₄, 4 KCl, 5.5 glucose, and 1.8 CaCl₂). Small IPA (200–600 µm in diameter) were dissected free and cleaned of connective and adipose tissue. They were then mounted on a Mulvany-Halpern myograph, bathed in Krebs solution gassed with 5% CO₂ and balance air, and warmed to 37°C. Vessels were stretched to a tension equivalent to 30 mmHg, which is close to the physiological pulmonary artery blood pressure, and they were stimulated by repeated 2-min exposures to 80 mM K⁺ containing PSS (in mM: 38 NaCl, 24 NaHCO₃, 1 MgSO₄, 0.44 NaH₂PO₄, 80 KCl, 5.5 glucose, and 1.8 CaCl₂) until the resulting contraction reached a stable level.

Experimental protocols. Before eliciting HPV, arteries were preconstricted with prostaglandin F₂ (PGF₂) to a level that was 10–20% of the maximal contraction to 80 mM K⁺. This "pretone" has previously been described to greatly enhance the contractile response of small PA to hypoxia (9). Arteries were then exposed to a hypoxic gas mixture (5% CO₂, balance nitrogen or 1% O₂, 5% CO₂, balance nitrogen) for 40 min. Vessels were allowed to recover in PSS under normoxic conditions for at least 1 h between successive exposures to hypoxia. All experiments were conducted in the presence of diltiazem (10 µM) to eliminate effects caused by changes in membrane potential. Drugs were added to the solution at least 15 min before the imposition of hypoxia.

Tension development during hypoxia was expressed as a percentage of the maximal contraction, measured as the increase in tension over baseline recorded at the beginning of experiments during a 2-min exposure of the artery to 80 mM K⁺. This is referred to as % 80K in the text. In most experiments, the artery was first exposed to hypoxia under control conditions (i.e., with pretone, in normal [Na⁺] PSS) and then an hour later exposed to hypoxia after application of a drug (ouabain, KB-R7943) or a solution change (Na⁺ substitution). Time control experiments, consisting of two exposures to hypoxia under control conditions, were also run to assess time-dependent changes in HPV (n = 11). In all experiments, the concentration of PGF₂ was adjusted if necessary so that the pretone level was the same for both exposures to hypoxia.

Ca²⁺ and pH measurements. For measurement of [Ca²⁺]_i, arteries mounted on a confocal wire myograph (Danish Myo Technology, Aarhus) were loaded with the Ca²⁺-sensitive fluorescent dye fura PE-3/AM (4 µM) for 1 h at 37°C. The dye was flushed from the solution, and the myograph was then transferred onto the stage of an inverted microscope (Nikon Diaphot TMD 200). Vessels were illuminated alternately at 340 and 380 nm using Optoscan monochromator (Cairn Research), and fluorescence at >510 nm was recorded parallel with tension using Acquisition Engine software (Cairn Research). Data were collected and stored on a PC for offline analysis. Changes in [Ca²⁺]_i during HPV are shown as the effect of hypoxia on the F₃₄₀/F₃₈₀ ratio (F₃₄₀/F₃₈₀) normalized to the effect of 80 mM K⁺ on this ratio. Time control experiments showed that F₃₄₀/F₃₈₀ was not significantly different when two 40-min exposures to hypoxia, separated by at least an hour, were carried out (n = 4; data not shown).

For measurement of pH_i, arteries were loaded with 10 µM BCECF/AM for 1 h at 37°C. After removal of the dye, arteries were illuminated alternatively at 490 nm and 440 nm, and the ratio of emission intensities (R_490/440) was recorded parallel with tension. At the end of the experiment, arteries were incubated in a series of solutions containing 140 mM KCl, 10 mM HEPES, 1 mM EGTA, and 2 µg/ml nigericin, in which the pH had been adjusted with 0.5 M KOH to values of between 6 and 8. Steady-state values of R_490/440 were plotted against pH, and the resulting mean data were fitted with a Hill curve that was used to convert R_490/440 values recorded during the experiment to pH_i.

Statistical analysis. Tension and F₃₄₀/F₃₈₀ were measured just before hypoxia was imposed, at the peak of the phase 1 HPV contraction, and thereafter at 5-min intervals throughout HPV and reoxygenation. Results in Figs. 3–7 are presented as means ± SE of these measurements. Student's t-test for paired or unpaired data was performed, as appropriate, to evaluate statistical significance. Differences were assumed to reach statistical significance at P < 0.05 and are indicated by asterisks.

View larger version (15K): [in this window] [in a new window]   Fig. 1. Effects of Li+-PSS and NMDG-PSS in PGF2-preconstricted pulmonary arteries. Intrapulmonary arteries (IPA) were first exposed to 80 mM K+, and endothelial function was assessed by relaxation of PGF2 contractions (10 µM) to acetylcholine (10 µM). Following a period of recovery, IPA were preconstricted to 10–15% 80K using PGF2, and then physiological saline solution (PSS) was replaced by Li+-PSS in the continuing presence of the same concentration of PGF2 in endothelium-intact IPA (Aa, solid circles in B; n = 5), in endothelium-denuded vessels (Ab, open circles in B, n = 7), or in endothelium-intact IPA in the presence of the 300 µM L-NAME (Ac, solid inverted triangles in B, n = 5). In other experiments, PSS was replaced by NMDG-PSS in the presence of PGF2 in endothelium-intact IPA in the presence of 300 µM L-NAME (Ad, open triangles in B, dashed line, n = 8).

View larger version (9K): [in this window] [in a new window]   Fig. 2. Na+-free solution ([Na+]e = 0.44 mM) does not prevent hypoxic pulmonary vasoconstriction (HPV) in isolated rat small pulmonary arteries. Representative trace shows force development to hypoxia in a PGF2-preconstricted IPA in the presence of extracellular Na+ (left) and after substitution of extracellular Na+ (Li+-PSS; [Na+]e = 0.44 mM; right).

View larger version (10K): [in this window] [in a new window]   Fig. 3. Effect of substituting Na+ by Li+ ([Na+]e = 0.44 mM) on tension (A) and [Ca2+]i (B) during HPV. A: IPA were preconstricted and exposed either twice to hypoxia under control conditions or first to hypoxia under control conditions, and then to hypoxia after Na+ substitution. In both cases, the second response is shown, with the closed circles representing the time control and the open circles showing the HPV following Na+ substitution. Contraction was normalized to the maximal contraction (to 80 mM K+) in the same vessel. Values are means ± SE in 11 tissues for both cases. In this and subsequent figures, asterisks indicate time points during hypoxia and reoxygenation (generally every 5 min) at which HPV was significantly (P < 0.05) increased by Na+ substitution, whereas indicates that the PGF2-induced contraction was increased compared with control. B: changes in F340/F380 during hypoxia under control conditions and after Na+ substitution plotted as a percentage of the peak F340/380 during 2-min exposure to 80 mM K+. The increase in F340/F380 caused by PGF2 has been subtracted, with the value of this parameter during HPV estimated using linear interpolation between the values of F340/F380 measured just before hypoxia and just after reoxygenation-induced recovery was complete. Values are means ± SE in 7 tissues. *P < 0.05.

View larger version (8K): [in this window] [in a new window]   Fig. 7. Combined treatment with partial Na+ substitution ([Na+]e = 24 mM) and ouabain (100 µM) significantly increases the sustained phase of HPV and underlying pretone but has no effect on phase 1. Values are means ± SE in 9 tissues (open circles) and are plotted against time controls (n = 11; closed circles). *P < 0.05 vs. time control during HPV, P < 0.05 vs. time control after reoxygenation.

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	RESULTS

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If the inhibition of NCX by hypoxia demonstrated previously (25) in isolated pulmonary artery myocytes is responsible for HPV in intact arteries, it would be predicted that the effect of inhibition of NCX on contraction in IPA should mimic that of hypoxia. We therefore examined the effect of substituting Na⁺ with either Li⁺ or NMDG (Li⁺-PSS or NMDG-PSS, respectively; [Na⁺]_e = 0.44 mM in both cases), which would be expected to rapidly deplete intracellular Na⁺ and block net fluxes of Ca²⁺ via the NCX. Substitution of Li⁺- or NMDG-PSS for Krebs solution under basal conditions generally had no effect on tension (n = 31 and 19, respectively), although in a minority of arteries, a transient contraction occurred (n = 4 and 8, respectively). However, since HPV in these arteries is greatly enhanced in the presence of pretone, we then determined the effect of Na⁺ substitution in IPA preconstricted to 10–15% 80K using PGF₂. Under these conditions, Li⁺-PSS evoked an initial small vasodilatation (6.4 ± 1.5% 80K), which was followed by a larger, slowly developing contraction (26.9 ± 4.0% 80K after 40 min) (Fig. 1Aa, solid circles in Fig. 1B; n = 5). In endothelium-denuded vessels (Fig. 1Ab, open circles in Fig. 1B, n = 7) or in the presence of 300 µM L-NAME (Fig. 1Ac, solid inverted triangles in Fig. 1B, n = 5), the initial vasodilatation was replaced by a transient contraction (29.0 ± 3.6% and 39.3 ± 2.9% 80K, respectively). This was followed by a sustained contraction (37.3 ± 4.2% and 36.6 ± 4.9% of contraction to 80 mM K⁺, respectively), thus resembling HPV in shape and time course (see e.g., Fig. 2, left).

Application of NMDG-PSS in the presence of L-NAME caused a transient contraction in PGF₂-preconstricted IPA (n = 8) that was similar in magnitude to that caused by Li⁺-PSS. However, this was not followed by the progressive development of a slow contraction, so that within 15 min, tension had returned to the level observed in normal PSS (Fig. 1Ad, open triangles in Fig. 1B). Thus, in this case, abrogation of NCX function did not produce a contraction that mimicked HPV.

A second prediction of the hypothesis that HPV can be largely explained by a hypoxia-induced suppression of NCX activity (25) is that hypoxia should have little or no effect on contraction if NCX is already inhibited. We next examined whether this was the case. When arteries were preconstricted with PGF₂ and then exposed to Li⁺-PSS in the continued presence of agonist, subsequent hypoxic challenge consistently evoked HPV (n = 4; Fig. 2). Under these conditions, contractions in response to hypoxia were similar in size or larger than in normal [Na⁺] PSS, and underlying constriction was greatly enhanced. However, because application of Li⁺-PSS tended to raise the pretone level, which could have influenced the response to hypoxia (14), in subsequent experiments arteries were preincubated for at least 15 min with Li⁺-PSS (or under other conditions designed to inhibit NCX) before preconstriction with PGF₂, the concentration of which was then adjusted so that the pretone level closely matched that imposed during the control response.

Using this approach, pretreatment with Li⁺-PSS did not significantly affect the contractile response during phase 1 of HPV. However, tension after phase 1, which normally fell sharply within 5 min, remained markedly elevated. Contraction after 40 min of hypoxia (phase 2) was also substantially increased. However, since the underlying pretone contraction was also enhanced, the fall in tension upon reoxygenation was not significantly different than in normal [Na⁺] PSS (Figs. 3A and 8). Similar results were obtained in two experiments in which diltiazem was not included in the solution (not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 8. Summary of effects of altering Na+-Ca2+ exchanger (NCX) function on phases 1 and 2 of HPV. Bars represent the mean effects of various treatments on phase 1 (white bars, tension measured at the peak of phase 1) and phase 2 (gray bars, measured as the decrease in tension upon reoxygenation) HPV. In each case, an initial HPV was recorded under control conditions, and then a second HPV was recorded 1 h later under control conditions (TC) or after treatment as follows: Li+, Li-PSS; KB-R 3, KB-R7943 (3 µM); KB-R 10, KB-R7943 (10 µM); OUA, ouabain (100 µM); 24 Na+ + OUA, [Na+] = 24 mM and ouabain (100 µM). Phase 1 and phase 2 contractions during the second HPV are shown as a percentage of the initial control HPV. *Significant (P < 0.05) difference from the time control.

When Na⁺ was substituted with NMDG using the same protocol, phase 1 HPV was significantly attenuated, whereas phase 2 was increased to an extent similar to that seen in Li⁺-PSS (Fig. 4). In most experiments, arteries failed to relax following reoxygenation, and in some cases, tension even continued to increase, an effect that was not apparent when Li⁺-PSS was used.

View larger version (9K): [in this window] [in a new window]   Fig. 4. Effect of substituting Na+ by NMDG ([Na+]e = 0.44 mM) on tension during HPV. Experiments were carried out using the same protocol as for Fig. 3. Values are means ± SE, n = 11 for control (open circles) and 9 for NMDG (closed circles). Asterisks represent time points at which HPV was significantly different than control, whereas indicates that that the PGF2-induced contraction was increased compared with control.

View larger version (9K): [in this window] [in a new window]   Fig. 5. KB-R7943 had no significant effect on HPV. Values are means ± SE for time controls (closed circles, n = 11), and arteries are pretreated with 3 µM (open circles, n = 12) or 10 µM (inverted closed triangles, n = 4) KB-R7943.

View larger version (9K): [in this window] [in a new window]   Fig. 6. Ouabain (100 µM; open circles) pretreatment had no significant effect on HPV. Values are means ± SE in 16 tissues and are plotted against time controls (n = 11; closed circles).

To assess the extent to which NCX contributes to Ca²⁺ extrusion in IPA, the recovery of [Ca²⁺]_i following the loading of Ca²⁺ into cells was assessed in fura PE-3-loaded arteries. IPA were incubated in nominally Ca²⁺-free PSS and treated with either thapsigargin (Thg; 1 µM, n = 4) or cyclopiazonic acid (CPA, 30 µM, n = 3) to prevent Ca²⁺ uptake into the sarcoplasmic reticulum. IPA were then exposed to PSS containing 0.44 mM Na⁺, 1.8 mM Ca²⁺, and 30 mM K⁺ to depolarize the smooth muscle cells and load them with Ca²⁺ via L-type Ca²⁺ channels. After 10 min, IPA were then placed into Ca²⁺-free PSS containing either normal or 0.44 mM [Na⁺]. Representative traces of the resulting changes in [Ca²⁺]_i are shown for IPA treated with CPA (Fig. 9A) and Thg (Fig. 9B). Since Thg and CPA work similarly, and since the results obtained with these two drugs were indistinguishable, the data obtained using both drugs were combined and are shown in Fig. 9C. Figure 9 illustrates that although the initial rapid fall in [Ca²⁺]_i that occurred in Ca²⁺-free solution when the depolarizing stimulus was terminated was little affected by the absence of a Na⁺ gradient, the final recovery of [Ca²⁺]_i to a basal level was significantly delayed in the low-[Na⁺] PSS. As shown in Fig. 9B, if normal [Na⁺] was restored during the delayed recovery phase in the continuing absence of Ca²⁺, [Ca²⁺]_i then fell rapidly. These data are consistent with the idea that NCX makes a small but significant contribution to the extrusion of Ca²⁺ from IPA smooth muscle cells.

View larger version (12K): [in this window] [in a new window]   Fig. 9. Ca2+ extrusion in normal and low-[Na+] solutions. A: fura PE-3-loaded IPA was incubated in Ca2+-free PSS and exposed to 30 µM cyclopiazonic acid (CPA) to inhibit SERCA. The artery was then exposed to PSS containing 0.44 mM Na+, 1.8 mM Ca2+, and 30 mM K+ for 5 min to increase [Ca2+]i and subsequently returned to Ca2+-free PSS to observe Ca2+ extrusion. Following a second Ca2+ loading, the artery was placed in Ca2+-free PSS containing 0.44 mM Na+ (with Li+ as the Na+ substitute). B: an experiment similar to that shown in A, except that 1 µM thapsigargin (Thg) was used to inhibit SERCA, and recovery from Ca2+ loading was examined first in 0.44 mM Na+ and then in the normal Na+ concentration. C: results from 4 similar experiments using Thg and 3 experiments using CPA were combined by normalizing the data such that the baseline R340/380 was set to 0% and the R340/380 at the end of the Ca2+ loading period was set to 100%. Means ± SE values for data recorded at 1-min intervals are shown. *The two sets of points indicated by the bracket were significantly different (P < 0.01) as assessed by repeated measures ANOVA.

View larger version (7K): [in this window] [in a new window]   Fig. 10. Representative trace illustrating the effect of Na+ substitution on intracellular pH, which was measured using BCECF as described in METHODS.

	DISCUSSION

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However, it is alternatively possible that the activation rather than inhibition of NCX could contribute to HPV. The opening of nonselective cation channels, either those associated with store emptying or directly activated by agonists acting on receptors, is known to cause Na⁺ influx (2). NCX has been shown to be localized to the areas of the plasmalemma that are closely apposed to peripheral elements of the endoplasmic reticulum (12). It has therefore been proposed that rises in [Na⁺] in the narrow junctional space between the endoplasmic reticulum, whether caused by agonists (10) or by inhibition of the ₂-isoform-based type of the Na⁺-K⁺-ATPase, which is also localized to these regions of the plasmalemma (6), act to reverse NCX, thus causing Ca²⁺ influx and loading the ER with Ca²⁺. Since evidence has been presented that Ca²⁺ release and SOC are important in raising [Ca²⁺]_i during HPV (24), and that these channels are mainly permeable to Na⁺ in pulmonary artery smooth muscle cells (21), it is conceivable that NCX could serve as an important pathway for Ca²⁺ influx during HPV.

In this study, we sought to determine whether NCX might play either of these roles in a well-characterized model of HPV in isolated IPA (16, 17). If inhibition of NCX by hypoxia made an important contribution to the development of HPV, procedures designed to affect NCX would be expected to alter HPV along predictable lines. In particular, inhibition of the ability of NCX to mediate Ca²⁺ extrusion should both mimic HPV and prevent or attenuate subsequent effects of hypoxia on both [Ca²⁺]_i and contraction.

We tested this prediction first by examining the effect of substituting Na⁺ on tension development in IPA that had been slightly preconstricted with PGF₂, under which condition hypoxia causes a biphasic contraction (17). Substitution of Na⁺ by Li⁺ caused a transient relaxation followed by a slow contraction. Because there is evidence that hypoxia inhibits nitric oxide release (1), we repeated these experiments in endothelium-denuded arteries and in the presence of L-NAME to inhibit nitric oxide production and found that in both cases, Na⁺ substitution then caused a biphasic contraction similar to HPV. On the other hand, substitution of Na⁺ by NMDG had no significant effect on sustained contraction in PGF₂-preconstricted IPA, implying that it might have been the elevation in [Li⁺] that was causing this contraction rather than Na⁺ substitution per se.

Na⁺ substitution with Li⁺ had no significant effect on phase 1 HPV. The effect of Li-PSS on phase 2 was more difficult to define precisely, because although the rise in tension after 40 min of hypoxia was significantly enhanced in Li-PSS, Fig. 1 indicates that the underlying contraction to PGF₂ rose by about the same amount, providing an explanation of why the fall in tension upon reoxygenation remained unchanged. However, it is notable that this increase in pretone (Fig. 1) was too slow to explain the observation that tension immediately after phase 1 was greatly increased by Na⁺ substitution. Similarly, measurements of the hypoxia-induced rise in [Ca²⁺]_i also indicated that although substitution of Na⁺ with Li⁺ did not significantly enhance the effect of prolonged hypoxia, it did appear to increase [Ca²⁺]_i soon after phase 1. These results are consistent with the concept that NCX is contributing to the removal of Ca²⁺ from the cytoplasm during HPV, especially after it reaches its highest level during phase 1, but that other Ca²⁺-removing mechanisms are also involved and are able eventually to compensate for its absence.

On the other hand, substituting Na⁺ with NMDG caused a marked attenuation of phase 1 HPV, although significantly increasing tension development during phase 2. Following a transient enhancement, NMDG did not affect the contraction caused by PGF₂ (Fig. 1), implying that its effect on the sustained response to hypoxia reflected a genuine enhancement of phase 2 HPV.

The results observed in experiments utilizing both Na⁺ substitutes demonstrate that phase 2 HPV was not inhibited in the absence of a Na⁺ gradient, under which condition NCX is unable to mediate either the net entry or extrusion of Ca²⁺. It therefore seems that inhibition of NCX does not play an important role in sustained HPV in isolated IPA.

The results of these experiments are less easily interpreted with regard to the role of NCX in phase 1, since Na⁺ substitution with NMDG inhibited phase 1, whereas its replacement by Li⁺ had no effect. Although it is possible that the inhibition of phase 1 seen in NMGD is the "true" effect on this phase of inhibiting NCX, and that the lack of effect on phase 1 in Li⁺-PSS is due to an enhancing effect of Li⁺ on contraction (as, for example, seen in Fig. 1) balancing out an attenuation caused by inhibition of NCX, it is also the case that neither ouabain nor KB-R7943 affected phase 1, implying strongly that it neither requires reverse-mode NCX nor is limited by forward-mode NCX.

The concept that HPV is not initiated by a hypoxia-induced inhibition of NCX was also borne out by experiments utilizing ouabain and the combination of ouabain and partial Na⁺ substitution with Li⁺. Pretreatment with ouabain caused no significant effect on either HPV or the pretone contraction. On the other hand, combined pretreatment with ouabain and low (24 mM) Na⁺ PSS, which in addition to inhibiting NCX-mediated Ca²⁺ extrusion would be expected to promote net Ca²⁺ influx via NCX more strongly than would ouabain alone (because extracellular Na⁺ competes with Ca²⁺ for entry via the exchanger; Ref. 3), increased both phases of HPV, particularly phase 2, thus suggesting that NCX is still active during HPV. Since HPV was either not inhibited or was increased as a result of these maneuvers, all of which would be expected to inhibit NCX-mediated Ca²⁺ extrusion, these results again do not support the concept that HPV is due primarily to inhibition of NCX-mediated Ca²⁺ extrusion.

As described above, recent reports have established that SOC-mediated Ca²⁺ entry is activated by hypoxia and have indicated that this pathway may be largely responsible for causing HPV (9, 24, 27). Our results do not, however, support the suggestion (26) that Na⁺ entry via SOC additionally promotes HPV by enhancing reverse-mode NCX. Substitution of extracellular Na⁺ by Li⁺ (which even if it entered the cell through cation channels could not be used by reverse-mode NCX to enhance Ca²⁺ entry), ouabain (by increasing resting [Na⁺]_i), and KB-R7943 (by inhibiting reverse-mode NCX) would all be expected to interfere with this process, yet none of these maneuvers inhibited HPV.

Substitution of Na⁺ by either Li⁺ or NMDG caused a transient alkalinization followed by a small decrease in the steady state pH_i. The transient initial alkalinization caused by Na⁺ removal was irrelevant with regard to HPV in these experiments because Na⁺ was always substituted at least 25 min before hypoxia was imposed. It also seems unlikely that the slight subsequent acidification could have been contributing to the generally facilitory effect of these solutions on HPV. For example, NMDG-PSS did not have any sustained effect on tension in PGF₂-preconstricted arteries, implying that the acidification per se does not increase tension even in preconstricted arteries. Moreover, Raffestin and McMurtry (15) showed that intracellular acidification caused a decrease in HPV in perfused rat lung, implying that if anything, the fall in pH_i caused by Na⁺ removal might have attenuated the enhancement of HPV we recorded.

Together, these results indicate that NCX does not make an obvious contribution to the regulation of Ca²⁺ homeostasis in these cells, except perhaps when the Na⁺ gradient is removed or reversed and [Ca²⁺]_i is sufficiently elevated. Thus, responses to hypoxia were enhanced in low-Na⁺ PSS, especially if ouabain was also present, but the smaller decrease in the Na⁺ gradient due to ouabain alone, or inhibition of NCX with KB-R7943, had no obvious effect on HPV. Moreover, complete substitution of Na⁺ with Li⁺ or NMDG did not affect basal tone, and even in the presence of PGF₂ replacement of Na⁺ with NMDG did not further increase tension. In accordance with this concept, the absence of a Na⁺ gradient had only a minor effect on the recovery of [Ca²⁺]_i following Ca²⁺ loading of the cells with a depolarizing solution. The rapid initial fall in [Ca²⁺]_i during the recovery from Ca²⁺ loading was most likely due to the plasmalemmal Ca²⁺ ATPase, since IPA in these experiments were pretreated with Thg or CPA to inhibit SERCA. Presumably the plasmalemmal Ca²⁺ ATPase works in parallel with NCX to extrude Ca²⁺, as has been shown in other types of smooth muscle, but is predominant in this respect. The relative importance of these two mechanisms seems to vary widely between different types of smooth muscle (8) and has not previously been examined in intact IPA. It is also of interest that Liu et al. (11) found using a similar protocol that bladder smooth muscle from Pmca1^+/– Pmca^–/– mice in which PMCA protein levels were halved were still able to completely extrude a Ca²⁺ load after pharmacological inhibition of both SERCA and NCX, suggesting that additional mechanisms for Ca²⁺ removal from the cytoplasm, e.g., the mitochondria, may also exist and be more important then has heretofore been appreciated (22).

The results of Fig. 9 differ from those of Wang et al. (25), who showed that Na⁺ removal dramatically slowed the rate at which [Ca²⁺]_i fell in isolated IPA myocytes following a Ca²⁺ load initiated by the application of caffeine and high-K⁺ solution. One possible explanation for these results is that we removed Ca²⁺ from the bath solution to measure Ca²⁺ extrusion, whereas they did not. Therefore, any increase in Ca²⁺ influx during the period when Ca²⁺ was falling (e.g., an enhancement of caffeine-stimulated SOC due to Na⁺ removal) could have been misinterpreted as an inhibition of extrusion. This might also have applied to their finding that hypoxia inhibited Ca²⁺ extrusion, since there is evidence that hypoxia not only releases Ca²⁺ to activate SOC but potentiates SOC when this has previously been activated by Ca²⁺ store depletion (24).

In summary, we have used Na⁺ substitution, Na⁺ pump inhibition, and the pharmacological NCX inhibitor KB-R7943 to assess the possible contribution of inhibition or reversal of NCX to HPV. Our results show that these procedures uniformly failed to inhibit HPV. Thus it seems unlikely that inhibition of NCX by hypoxia, or activation of its reverse mode due to hypoxia-induced Na⁺ entry, contributes significantly to HPV, at least in this model. Instead, NCX is probably functioning as one of several Ca²⁺-removing pathways acting to limit hypoxia-induced increases in [Ca²⁺]_i, a role consistent with the enhancement of the PGF₂-induced contraction in IPA and with previous observations that it functions to promote Ca²⁺ extrusion in vascular smooth muscle under conditions in which the Na⁺ gradient is intact.

	GRANTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
We are grateful to the British Heart Foundation (FS/03/022/15153) and the Wellcome Trust (PG 059564) for support of this study.

	FOOTNOTES

 

Address for reprint requests and other correspondence: P. I. Aaronson, Rm. 3.10, Franklin Wilkins Bldg., Waterloo Campus, King's College London, London SE1 9NH (e-mail: philip.aaronson{at}kcl.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.

	REFERENCES

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