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J Physiol (2003), 549.2, pp. 553-562
© Copyright 2003 The Physiological Society
DOI: 10.1113/jphysiol.2002.037135

An important role for the Na⁺-Ca²⁺ exchanger in the decrease in cytosolic Ca²⁺ concentration induced by isoprenaline in the porcine coronary artery

Jun Yamanaka, Junji Nishimura, Katsuya Hirano and Hideo Kanaide

	ABSTRACT

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The role of the Na⁺-Ca²⁺ exchanger (NCX) in the mechanism of the isoprenaline (Iso)-induced vasorelaxation was investigated by simultaneously monitoring the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and tension of fura-2-loaded medial strips of porcine coronary arteries. Normal physiological salt solution (PSS) contained 137.3 mM Na⁺ and 5.9 mM K⁺. During the sustained phase of contraction, Iso induced only a transient decrease in [Ca²⁺]_i when contraction was induced by depolarization with 118 mM K⁺ solution containing 25.2 mM Na⁺. When contraction was induced with 30 mM K⁺ in PSS containing 113.2 mM Na⁺, Iso induced a sustained decrease in [Ca²⁺]_i, whereas in contractions induced by 30 mM K⁺ in a low Na⁺ (25.2 mM Na⁺) PSS, Iso transiently decreased [Ca²⁺]_i. Replacement of Ca²⁺ with Ba²⁺ (which cannot be extruded by the Ca²⁺ pumps but can be extruded through the NCX) resulted in decreased [Ba²⁺]_i induced by Iso in normal but not in low Na⁺ PSS. On the other hand, Iso induced a sustained decrease in [Ca²⁺]_i when strips were pre-contracted by U46619, a thromboxane A₂ analogue, in PSS. Various types of K⁺ channel blockers (iberiotoxin, 4-aminopyridine, apamin or glibenclamide) or combinations of these blockers failed to completely inhibit the Iso-induced decreases in [Ca²⁺]_i and tension. However, Iso-induced sustained decreases in [Ca²⁺]_i during the contraction induced by U46619 were greatly inhibited in a low Na⁺ PSS. The Iso-induced decrease in tension during contraction by U46619 was greatly inhibited by 2',4'-dichlorobenzamil, a forward- and reverse-mode NCX inhibitor, but not by ouabain, a selective inhibitor of Na⁺,K⁺-ATPase. These results indicate that the NCX is involved in the Iso-induced reduction of [Ca²⁺]_i and tension of the porcine coronary arterial smooth muscle.

(Resubmitted 9 January 2003; accepted after revision 27 February 2003; first published online 9 May 2003)
Corresponding author H. Kanaide: Division of Molecular Cardiology, Research Institute of Angiocardiology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Email: kanaide{at}molcar.med.kyushu-u.ac.jp

	INTRODUCTION

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It is well known that isoprenaline (Iso) induces relaxation of the vascular smooth muscle through the production of adenosine 3',5'-cyclic monophosphate (cAMP). The relaxant effects mediated by cAMP are thought to be due mainly to the decrease in intracellular Ca²⁺ concentration ([Ca²⁺]_i), as well as a decrease in Ca²⁺ sensitivity of the contractile apparatus in smooth muscle cells (Nishimura & van Breemen, 1989; Ushio-Fukai et al. 1993). To explain the cAMP-induced decrease in [Ca²⁺]_i, several mechanisms have been proposed, including an inhibition of Ca²⁺ influx due to a hyperpolarization via stimulation of Ca²⁺-activated K⁺ channels (Sadoshima et al. 1988), a stimulation of Ca²⁺ uptake into the intracellular stores (Mueller & van Breemen, 1979) and an increase in Ca²⁺ extrusion from cells through the sarcolemmal Ca²⁺ pump (Bulbring & Tomita, 1987). Vascular smooth muscles utilize two major systems for Ca²⁺ extrusion, namely, through the sarcolemmal Ca²⁺ pump and the Na⁺-Ca²⁺ exchanger (NCX) (Barnes & Liu, 1995). The involvement of the NCX in the Iso-induced relaxation of the coronary arterial smooth muscle, however, has not been reported.

The NCX plays an important role in maintaining calcium homeostasis in many mammalian tissues (Schulze et al. 1993; Lee et al. 2001). This exchanger catalyses the electrogenic exchange of one Ca²⁺ for three Na⁺ in each reaction cycle. The NCX can transport Ca²⁺ either into or out of the cells, depending on the electrochemical driving force on the exchanger. Previous studies have identified three isoforms of the NCX (NCX1, NCX2 and NCX3) that are encoded by distinct genes in mammalian cells (Nicoll et al. 1990, 1996; Li et al. 1994). NCX1-specific transcripts are most abundant in the heart, although they are found in many other tissues (Komuro et al. 1992; Nicoll et al. 1996). In contrast, expression of NCX2 and NCX3 genes is restricted to the brain and skeletal muscles, respectively (Li et al. 1994; Nicoll et al. 1996). The factors regulating NCX1 activity have been investigated in mammalian cells. NCX1 is activated by [Ca²⁺]_i (Hilgemann, 1990) and external monovalent cations (Gadsby et al. 1991) and is inhibited by high cytoplasmic Na⁺ concentrations (Hilgemann, 1990; Gadsby et al. 1991), low cytoplasmic pH (Doering & Lederer, 1994), and adenosine triphosphate (ATP) depletion (Condrescu et al. 1995; Iwamoto et al. 1996). In addition, the consensus phosphorylation sites have been identified which suggest that the NCX may be a target for cAMP-dependent protein kinase (PKA) and/or protein kinase C (PKC) (Blaustein & Lederer, 1999). However, a variety of conflicting physiological results have been obtained following PKA or PKC activation.

In the present study, we have determined whether or not the NCX is involved in the mechanism for the Iso-induced relaxation of the coronary arterial smooth muscle, by monitoring Iso-induced decreases in [Ca²⁺]_i and tension simultaneously. The results obtained indicated that the increase in NCX activity plays an important role in the decrease in [Ca²⁺]_i during Iso-induced vasorelaxation.

	METHODS

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Tissue preparation

The study protocol was approved by the Animal Care and the Ethical Committee of the Research Institute of Angiocardiology, Faculty of Medicine, Kyushu University. Right coronary arteries (2-3 cm from the origin) were isolated from fresh porcine hearts at a local slaughterhouse immediately after the animals had been killed. All tissue specimens were placed in ice-cold normal physiological salt solution (PSS) and brought to the laboratory. After the segments were cut open longitudinally, the adventitia was trimmed away. Cotton swabs were used to rub off the inner surface of the arteries in order to remove the endothelium. The media was cut into equal-size strips (1 5 0.1 mm). The complete removal of the endothelium was confirmed by the lack of any relaxing response of the strips to 1 µM bradykinin.

Fura-2 loading

Coronary arterial media strips were loaded with the [Ca²⁺]_i indicator dye fura-2 by incubating in oxygenated (a mixture of 95 % O₂ and 5 % CO₂) Dulbecco's modified Eagle's medium containing 25 µM fura-2/AM (an acetoxymethyl ester form of fura-2) and 5 % fetal bovine serum for 4 h at 37 °C (Kanaide, 1999).

Measurement of tension

After being loaded with fura-2, the coronary arterial media strips were mounted vertically in a quartz organ bath and then the isometric tension was measured, as described previously (Ushio-Fukai et al. 1993). In brief, during the fura-2 equilibration period (1 h), the strips were stimulated with 118 mM K⁺ PSS repeatedly and the resting tension was increased stepwise to obtain the maximal force. The developed force was expressed as a percentage, by assigning the values at rest in normal PSS (5.9 mM K⁺) to be 0 % and those at a steady state of contraction in 118 mM K⁺ PSS to be 100 %.

Front-surface fluorometry

Changes in the fluorescence intensity of the fura-2-Ca²⁺ complex were monitored simultaneously with force development, by use of a front-surface fluorometer specifically designed for fura-2 fluorometry (CAM-OF3, Japan Spectroscopic Co., Tokyo, Japan), as previously described (Kanaide, 1999). In brief, the ratio of fluorescence (500 nm) intensities at alternating (400 Hz) 340 nm and 380 nm excitation light was monitored and their ratio (F₃₄₀/F₃₈₀) was recorded as an indicator of [Ca²⁺]_i. The fluorescence ratio was expressed as a percentage, by assigning the values at rest in normal (5.9 mM K⁺) and 118 mM K⁺ PSS to be 0 % and 100 %, respectively.

Replacement of Ca²⁺ with Ba²⁺

In the Ba²⁺ experiments, strips were incubated first in 2 mM EGTA-Ca²⁺-free PSS for 10 min, then in Ca²⁺-free PSS for 5 min, and finally they were incubated in 1.25 mM Ba²⁺ PSS. After this treatment, the strips were stimulated with 118 mM K⁺/Ba²⁺ PSS several times in order to completely replace Ca²⁺ with Ba²⁺. After these procedures, the specific protocol shown in Fig. 5 was carried out.

Drugs and solutions

The composition of normal PSS for fura-2 studies was as follows (mM): NaCl 123, KCl 4.7, NaHCO₃ 15.5, KH₂PO₄ 1.2, MgCl₂ 1.2, CaCl₂ 1.25 and D-glucose 11.5. PSS containing 30 mM K⁺ and 118 mM K⁺ was prepared by replacing NaCl with equimolar KCl, and thus containing 113.2 mM and 25.2 mM Na⁺, respectively. The 5.9 mM K⁺/Ba²⁺ PSS and the 118 mM K⁺/Ba²⁺ PSS were made by an equimolar substitution of BaCl₂ for CaCl₂. Solutions of 5.9 mM K⁺/low Na⁺ (25.2 mM) PSS and 30 mM K⁺/low Na⁺ (25.2 mM) PSS were made by an equimolar substitution of LiCl for NaCl (Table 1). PSS was bubbled with a mixture of 95 % O₂ and 5 % CO₂ and the resulting pH was 7.4. Fura-2/AM was purchased from Dojindo (Kumamoto, Japan). Fetal bovine serum, Iso, iberiotoxin, 4-aminopyridine, apamin, glibenclamide and ouabain were purchased from Sigma (St Louis, MO, USA). U46619 (9,11-dideoxy-9,11-methanoepoxy prostagrandin F₂) and 2',4'-dichlorobenzamil were purchased from Funakoshi (Osaka, Japan) and Molecular Probes (Eugene, OR, USA), respectively. All other chemicals were from Wako (Osaka, Japan).

Data analysis

All data from the simultaneous measurements of [Ca²⁺]_i and tension were collected by a computerized data acquisition system (MacLab, ADInstruments, Castle Hill, Australia, running on an Apple Macintosh computer). The data for the representative traces shown in the figures were printed directly from the computer using a laser printer (Cannon, Japan). The data are expressed as the mean ± S.E.M. of the indicated number of experiments. One strip obtained from one animal was used for each experiment, and therefore the number of experiments (n value) indicates the number of animals. Statistical analysis was performed using Student's unpaired t test and P values of less than 0.05 were considered to be significant.

	RESULTS

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Effects of Iso on the increases in [Ca²⁺]_i and tension induced by 118 mM K⁺ and U46619 in PSS

Figures 1A and B shows representative time courses for the effect of 10 µM Iso on [Ca²⁺]_i and tension induced by 118 mM K⁺ solution and 100 nM U46619, a thromboxane A₂ analogue. Depolarization with 118 mM K⁺ solution induced a rapid increase in [Ca²⁺]_i. After reaching its peak, [Ca²⁺]_i declined to reach a plateau within 10 min. The tension also rapidly elevated to reach a plateau level. The levels of [Ca²⁺]_i and tension at 10 min after stimulation with 118 mM K⁺ solution were designated to be 100 % (Fig. 1A). An application of 100 nM U46619 induced rapid increases in both [Ca²⁺]_i and tension, which reached the steady state within 20 min (Fig. 1B). The level of tension (109.77 ± 3.94 %, n = 30) at 20 min after stimulation by 100 nM U46619 was similar to that obtained with 118 mM K⁺ depolarization (100 % as assigned), while the level of [Ca²⁺]_i (63.63 ± 1.52 %, n = 30) was significantly lower than that obtained with 118 mM K⁺ depolarization (100 %). As shown in Fig. 1A, the application of 10 µM Iso during the steady state contraction induced by 118 mM K⁺ solution caused a small, though significant, transient reduction of [Ca²⁺]_i levels (81.14 ± 1.64 %, n = 12), while the tension decreased to reach a new steady state level (54.06 ± 2.79 %, n = 12). However, when arterial strips were pre-contracted with 100 nM U46619 (Fig. 1B), 10 µM Iso induced much greater decreases in [Ca²⁺]_i and tension ([Ca²⁺]_i, 21.97 ± 2.03 %, n = 16; tension, 5.05 ± 1.08 %, n = 16), compared with the effects of Iso on the 118 mM K⁺-induced increases in [Ca²⁺]_i and tension.

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Figure 1. Effects of Iso on [Ca2+]i and tension when added at the sustained phase of contractions induced by 118 mM K+ solution or 100 nM U46619 A, application of 10 µM isoprenaline (Iso) induced a transient decrease in [Ca2+]i and a sustained relaxation, when the strip was pre-contracted by 118 mM K+ solution. B, application of 10 µM Iso induced sustained decreases in [Ca2+]i and tension, when the strip was pre-contracted by 100 nM U46619. The levels of [Ca2+]i and tension at 10 min after stimulation with 118 mM K+ solution were designated to be 100 %. The traces shown are representative of 12 similar independent experiments.

Effects of K⁺ channel blockers on the Iso-induced decreases in [Ca²⁺]_i and tension during U46619-induced contraction

The finding that the Iso induced a decrease in [Ca²⁺]_i in U46619-induced contraction in PSS (5.9 mM K⁺) (Fig. 1B) which was much greater than in 118 mM K⁺ depolarization (Fig. 1A) indicates that there may be a possible role for K⁺ channels in Iso-induced relaxation. To investigate the relative contribution of K⁺ channels, we examined the effects of 100 nM iberiotoxin (IBTX), an inhibitor of large conductance Ca²⁺-activated K⁺ (BK) channels, 30 µM 4-aminopyridine (4-AP), an inhibitor of voltage-dependent K⁺ (K_V) channels, 3 µM apamin, an inhibitor of small conductance Ca²⁺-activated K⁺ (SK) channels and 3 µM glibenclamide, an inhibitor of ATP-activated K⁺ (K_ATP) channels, on the Iso-induced decreases in [Ca²⁺]_i and tension during U46619-induced contraction. The concentrations of the K⁺ channel blockers were used at the maximum levels that did not affect the increases in [Ca²⁺]_i and tension induced by 100 nM U46619 (Kawasaki et al. 1997). As shown in Fig. 2A and B, the application of 100 nM IBTX or 30 µM 4-AP 20 min after the addition of 10 µM Iso did not affect the tension levels, while [Ca²⁺]_i was slightly reversed by IBTX (25.0 ± 2.27 %, n = 6) and 30 µM 4-AP (23.18 ± 1.84 %, n = 6). Even when 100 nM IBTX, 30 µM 4-AP, 3 µM apamin and 3 µM glibenclamide were used in combination, tension was not affected, although [Ca²⁺]_i was slightly reversed by 33.5 ± 4.53 % (n = 6; Fig. 2C).

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Figure 2. Effects of various K+ channel blockers on Iso-induced relaxation A, iberiotoxin (IBTX, 100 nM), an inhibitor of large conductance Ca2+-activated K+ channels, B, 4-aminopyridine (4-AP, 30 µM), an inhibitor of voltage-dependent K+ channels and C, IBTX (100 nM) + 4-AP (30 µM) + apamin (an inhibitor of small conductance Ca2+-activated K+ channels, 3 µM) + glibenclamide (an inhibitor of ATP-activated K+ channels, 3 µM) were applied during the Iso-induced relaxation of the contraction induced by 100 nM U46619. The traces shown are representative of 6 similar independent experiments.

Effects of low extracellular Na⁺ concentration ([Na⁺]_o) on the Iso-induced decreases in [Ca²⁺]_i and tension during U46619-induced contraction

The experiments shown in Fig. 2 indicated that the observation that Iso was more effective in normal PSS than in 118 mM K⁺ solution (Fig. 1) could not be fully explained by Iso-induced K⁺ channel opening. It was considered, therefore, that the NCX could play an important role in the decreases in [Ca²⁺]_i and tension induced by Iso, because the Na⁺ concentration in 118 mM K⁺ solution was made very low (25.2 mM; Table 1) to maintain the osmolarity of the solution. Figure 3A shows the representative time course for the effect of Iso on [Ca²⁺]_i and tension induced by 100 nM U46619 in low Na⁺ (25.2 mM) PSS, in which most of the Na⁺ was replaced with 121.8 mM Li⁺ (Table 1). The application of low Na⁺ PSS induced a slight sustained increase in [Ca²⁺]_i (9.83 ± 1.19 %; n = 12) without any significant change in tension (0.27 ± 0.16 %; n = 12) (Fig. 3A). The extent of the increases in [Ca²⁺]_i and tension induced by 100 nM U46619 in low Na⁺ PSS was not significantly different from that in normal PSS ([Ca²⁺]_i, 64.89 ± 1.76 %, n = 15; tension, 107.67 ± 2.14 %, n = 15). Under these conditions, the decreases in [Ca²⁺]_i and tension induced by 10 µM Iso during activation by 100 nM U46619 in low Na⁺ PSS were greatly inhibited ([Ca²⁺]_I, 59.95 ± 2.28 %, n = 9 (P < 0.05); tension, 60.36 ± 4.01 %, n = 9 (P < 0.05)). The [Ca²⁺]_i levels 15 min after the application of Iso (59.95 ± 2.28 %, n = 9) were significantly lower than those before the application of Iso (65.32 ± 2.08 %, n = 9). It should be noted that the patterns of [Ca²⁺]_i and tension in Fig. 3A became similar to those in Fig. 1A.

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Figure 3. Effects of 'low [Na+]' on the Iso-induced relaxation A, Iso (10 µM) was applied during U46619-induced contraction in low Na+ solution ([Na+]: 25.2 mM, [K+]: 5.9 mM, [Li+]: 121.8 mM). Iso-induced decreases in [Ca2+]i and tension were greatly inhibited by 'low Na+'. The traces shown are representative of 9 similar independent experiments. B, Iso (10 µM) was applied during the contraction induced by 30 mM K+ in normal Na+ (113.2 mM) solution (the second contraction) and in low Na+ (25.2 mM) solution (the fourth contraction). Iso induced a sustained decrease in [Ca2+]i during the contraction induced by 30 mM K+ solution (the second contraction), while Iso induced a transient decrease in [Ca2+]i during the contraction induced by 30 mM K+/low [Na+] solution (the fourth contraction). The traces shown are representative of 8 similar independent experiments.

Effects of Iso on the increases in [Ca²⁺]_i and tension induced by the 30 mM K⁺ solution and the 30 mM K⁺/low Na⁺ solution

Figure 3B shows the representative time courses of the effect of 10 µM Iso on [Ca²⁺]_i and tension induced by the 30 mM K⁺ solution containing 113.2 mM Na⁺ and the 30 mM K⁺/low Na⁺ (25.2 mM) solution. The application of 10 µM Iso during the steady state contraction induced by 30 mM K⁺ solution (65.08 ± 3.29 %, n = 8) caused a small but sustained reduction of [Ca²⁺]_i levels (55.76 ± 4.76 %, n = 8, P < 0.05), while the tension decreased markedly and rapidly to reach a steady state level (4.56 ± 0.81 %, n = 8). In contrast, when 10 µM Iso was applied to tissues placed in 30 mM K⁺/low Na⁺ solution, there appeared only a small and transient decrease in [Ca²⁺]_i and a sustained reduction of [Ca²⁺]_i was not observed. In addition, the increases in [Ca²⁺]_i and tension induced by 30 mM K⁺ solution ([Ca²⁺]_i, 65.08 ± 3.29 %, n = 8; tension, 20.22 ± 3.50 %, n = 8) were significantly lower than those induced by 30 mM K⁺/low Na⁺ PSS ([Ca²⁺]_i, 78.185 ± 4.29 %, n = 8, P < 0.05); tension, 37.07 ± 4.56 %, n = 8, P < 0.05)).

Effects of low extracellular Na⁺ concentration ([Na⁺]_o) on the forskolin-induced decreases in [Ca²⁺]_i and tension during U46619-induced contraction

To confirm the participation of the cAMP/PKA system in the extracellular Na⁺-dependent decrease in [Ca²⁺]_i, we next used forskolin, an activator of adenylate cyclase. Figure 4A shows the representative time course for the effects of 3 µM forskolin on [Ca²⁺]_i and tension induced by 100 nM U46619. The application of 3 µM forskolin during the steady state contraction induced by 100 nM U46619 induced decreases in [Ca²⁺]_i and tension ([Ca²⁺]_i, 14.43 ± 1.50 %, n = 5; tension, 14.40 ± 5.93 %, n = 5). The patterns of the decreases in [Ca²⁺]_i and tension induced by forskolin were similar to those produced by Iso (Fig. 1B). Figure 4B shows a representative time course for the effect of forskolin on [Ca²⁺]_i and tension induced by 100 nM U46619 in low Na⁺ (25.2 mM) PSS, in which most of the Na⁺ was substituted by 121.8 mM Li⁺ (Table 1). Under these conditions, the decrease in [Ca²⁺]_i and tension induced by 3 µM forskolin during activation by 100 nM U46619 in low Na⁺ PSS was greatly inhibited ([Ca²⁺]_i, 40.31 ± 8.98 %, n = 5, P < 0.05; tension, 50.80 ± 2.39 %, n = 5, P < 0.05).

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Figure 4. Effects of 'low [Na+]' on the forskolin-induced relaxation A, the application of 3 µM forskolin induced sustained decreases in [Ca2+]i and tension when the strip was pre-contracted by 100 nM U46619. The levels of [Ca2+]i and tension 10 min after stimulation with 118 mM K+ solution were designated as 100 %. B, forskolin (3 µM) was applied during the U46619-induced contraction in low Na+ solution ([Na+]: 25.2 mM, [K+]: 5.9 mM, [Li+]: 121.8 mM). Forskolin-induced relaxation was greatly inhibited. The traces shown are representative of 5 similar independent experiments.

Effects of Iso on [Ba²⁺] _i

Figure 5A and B shows the effect of Iso on [Ba²⁺]_i. Ba²⁺ can enter the cells through Ca²⁺ channels, but cannot be extruded by the Ca²⁺ pumps of the sarcolemma and the sarcoplasmic reticulum (Yamaguchi et al. 1989; Schilling et al. 1989; Seguchi et al. 1996; Ushio-Fukai et al. 2000). The NCX is the only pathway for Ba²⁺ to be extruded (Wagner-Mann et al. 1992). Depolarization with 118 mM K⁺ solution containing 25.2 mM Na⁺ and 1.25 mM Ba²⁺ (Ba²⁺ solution) induced a rapid increase in the fluorescence ratio, indicating a rapid elevation of [Ba²⁺]_i. Arterial tension also rapidly increased to reach a plateau level. When the solution was replaced with 5.9 mM K⁺/Ba²⁺ solution containing 137.3 mM Na⁺, the tension rapidly declined to reach a plateau level, while the ratio slowly and gradually declined to reach a plateau level (Fig. 5A). The subsequent application of 10 µM Iso to the 5.9 mM K⁺/Ba²⁺ solution decreased [Ba²⁺]_i to -17.80 ± 1.43 % (n = 6), assuming the levels of [Ba²⁺]_i at 10 min after stimulation with 118 mM K⁺ solution to be 100 %. As shown in Fig. 5B, when the solution was replaced with 5.9 mM K⁺/low Na⁺/Ba²⁺ solution in order to inhibit the NCX, the tension and the ratio slowly and gradually elevated to reach a plateau level ([Ba²⁺]_i, 89.61 ± 9.78 %, n = 6; tension, 67.70 ± 8.21 %, n = 6). However, the subsequent application of 10 µM Iso to tissues exposed to the 5.9 mM K⁺/low Na⁺/Ba²⁺ did not decrease [Ba²⁺]_i although tension was decreased to the resting level.

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Figure 5. Effects of Iso on [Ba2+] i and tension A, Iso (10 µM) was applied in the presence of normal extracellular [Na+] (137.3 mM) after the replacement of Ca2+ with Ba2+. Depolarization with 118 mM K+ solution induced changes in [Ba2+]i and tension which were similar to those in [Ca2+]i and tension before replacement of Ca2+ with Ba2+. Iso induced a decrease in [Ba2+]i with no change in tension when it was applied at rest. B, Iso (10 µM) was applied in the presence of low extracellular Na+ concentration after the replacement of Ca2+ with Ba2+. The application of low Na+ solution induced increases in [Ba2+]i and tension. The subsequent application of Iso induced a relaxation with no decrease in [Ba2+]i. The traces shown are representative of 6 similar independent experiments.

Effects of a NCX blocker on the Iso-induced relaxation U46619-induced contraction

We next used 2',4'-dichlorobenzamil (2,4-DCB), a forward- and reverse-mode inhibitor of the NCX (Lee et al. 2001), to confirm the contribution of the NCX to Iso-induced relaxation. As shown in Fig. 6A, 1 mM 2,4-DCB applied 10 min before and during the application of 100 nM U46619 greatly inhibited the Iso-induced decrease in tension (39.99 ± 5.51 %, n = 6, P < 0.05). We could not examine the change in [Ca²⁺]_i because 2,4-DCB at this concentration interfered with fura-2 fluorometry.

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Figure 6. Effects of 2,4-DCB or ouabain on the Iso-induced relaxation A, Iso (10 µM) was applied during the 100 nM U46619-induced contraction in the presence of 1 mM 2',4'-dichlorobenzamil (2,4-DCB), a forward- and reverse-mode inhibitor of NCX. In comparison with the traces in Fig. 1B, 2,4-DCB apparently inhibited the Iso-induced relaxation. The level of [Ca2+]i could not be measured because 2,4-DCB (1 mM) interfered with the fluorescence signals of fura-2. B, Iso was applied during U46619-induced contraction in the presence of 1 µM ouabain, a selective inhibitor of Na+,K+-ATPase. The change in [Ca2+]i was partly reversed, while tension was not affected by ouabain. The traces shown are representative of 6 similar independent experiments.

Effects of ouabain on the Iso-induced decreases in [Ca²⁺]_i and tension

We next used ouabain, a selective inhibitor of the sodium-potassium adenosine triphosphatase (Na⁺,K⁺-ATPase), to determine the relative contribution of Na⁺,K⁺-ATPase to Iso-induced reduction of [Ca²⁺]_i and tension. As shown in Fig. 6B, 1 µM ouabain did not affect the Iso-induced decrease in tension, while it slightly reversed [Ca²⁺]_i (31.25 ± 1.83 %, n = 6).

	DISCUSSION

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Based on the following observations we have concluded that the NCX plays an important role in the Iso-induced arterial relaxation and decreased [Ca²⁺]_i. (1) Various types of K⁺ channel blockers, even if used in combination, could not completely reverse the decreases in [Ca²⁺]_i and tension induced by Iso after U46619 in normal PSS (137.3 mM Na⁺) (Fig. 2). (2) The sustained decrease in [Ca²⁺]_i due to Iso was inhibited when U46619 was applied in the low Na⁺ (25.2 mM) PSS (Fig. 3A). (3) Although Iso induced a sustained decrease in [Ca²⁺]_i when the concentration of K⁺ was reduced to 30 mM, Iso induced only a transient decrease in [Ca²⁺]_i when the Na⁺ concentration was reduced to 25.2 mM (Fig. 3B). (4) Iso decreased [Ba²⁺]_i in the presence of high [Na⁺] (137.3 mM), while Iso did not decrease [Ba²⁺]_i in the presence of low [Na⁺] (25.2 mM) (Fig. 5). (5) An NCX inhibitor, 2,4-DCB inhibited the Iso-induced relaxation (Fig. 6A). (6) Ouabain had only a partial effect on the Iso-induced decrease in [Ca²⁺]_i (Fig. 6B). (7) These effects were thought to be mediated by the cAMP/PKA system, because forskolin had a similar effect to Iso (Fig. 4).

As we have reported previously (Ushio-Fukai et al. 1993), Iso-induced decreases in [Ca²⁺]_i and tension were much greater in the strips pre-contracted by agonist in normal PSS, compared with those in the strips pre-contracted by 118 mM K⁺ solution. This observation was confirmed and is clearly shown in Fig. 1, in which Iso induced only a transient decrease in [Ca²⁺]_i in 118 mM K⁺ solution, while it induced a sustained decrease in [Ca²⁺]_i of the U46619-induced contraction in normal PSS. In addition, the mechanism for the Iso-induced relaxation that occurs without a sustained decrease in [Ca²⁺]_i as seen in Fig. 1A would be explained by the cAMP-induced decrease in Ca²⁺ sensitivity as we have reported previously (Nishimura & van Breemen, 1989; Ushio-Fukai et al. 1993). Because cAMP has been shown to inhibit Ca²⁺ influx due to a hyperpolarization caused by the stimulation of Ca²⁺-activated K⁺ channels (Sadoshima et al. 1988), we reasoned that the inability of Iso to induce a sustained decrease in [Ca²⁺]_i could be due to the inactivation of this mechanism in a 118 mM K⁺ solution. We thus added various K⁺ channel blockers during the Iso-induced relaxation (Fig. 2). In agreement with this postulated mechanism, changes in [Ca²⁺]_i could be reversed by K⁺ channel blockers. However, the level of [Ca²⁺]_i, recorded even in the presence of various K⁺ channel blockers used in combination, was still much lower than that before the application of Iso, indicating that the above-mentioned observation could not be fully explained by this mechanism alone.

Depolarization with high external [K⁺] solution has been frequently used to activate smooth muscle preparations, where it causes a rapid rise and sustained type of contraction in many smooth muscle types. Substituting KCl for NaCl has traditionally been used to prepare high external [K⁺] solutions. It thus follows that a high external [K⁺] solution has a low Na⁺ concentration. We examined whether a low [Na⁺] solution may negate the ability of Iso to induce a sustained decrease in [Ca²⁺]_i during the contraction induced by a high external [K⁺] solution. In support of this notion is the observation that Iso could induce a sustained decrease in [Ca²⁺]_i when the K⁺ concentration was reduced (and the Na⁺ concentration was simultaneously raised) to near 30 mM (Ushio-Fukai et al. 1993, Fig. 3B). In order to test this hypothesis, we used a protocol similar to that in Fig. 1B and used the low Na⁺ PSS by substituting LiCl for NaCl (Fig. 3A). Under these conditions, Iso-induced sustained decreases in [Ca²⁺]_i and tension were greatly inhibited. In other words, the patterns of [Ca²⁺]_i and tension in Fig. 3A became more similar to those in Fig. 1A. In addition, the Iso-induced sustained decrease in [Ca²⁺]_i observed during activation by 30 mM K⁺ solution could also be inhibited by lowering the Na⁺ concentration (Fig. 3B). These results indicated that an Iso-induced sustained decrease in [Ca²⁺]_i requires the presence of physiological or higher concentrations of extracellular Na⁺.

The potency of Iso-induced decrease in [Ca²⁺]_i was reduced further in 30 mM K⁺ than in U46619. This could be due to the difference in the concentration of [Na⁺]_o, namely 113.2 mM in 30 mM K⁺ PSS vs.137.3 mM in normal PSS. Another reason could be because Iso-induced hyperpolarizing effects do not readily operate in a 30 mM K⁺ solution. Furthermore, forskolin-induced decreases in [Ca²⁺]_i and tension during activation by U46619 in low Na⁺ PSS were also greatly inhibited (Fig. 4). This experiment supports the role of the cAMP/PKA system in the regulation of extracellular Na⁺-dependent decreases in [Ca²⁺]_i and tension.

The Iso-induced decrease in [Ca²⁺]_i could be explained by the following mechanisms: (1) the inhibition of Ca²⁺ influx due to a hyperpolarization (discussed above), (2) the stimulation of Ca²⁺ uptake into the intracellular stores, (3) an increase in Ca²⁺ extrusion from cells through the sarcolemmal Ca²⁺ pump and (4) an increase in Ca²⁺ extrusion from cells through the NCX. Because the Iso-induced sustained decrease in [Ca²⁺]_i was dependent on a physiological or higher concentration of extracellular Na⁺ as mentioned above, Ca²⁺ extrusion through the NCX would be a reasonable possibility. However, since this mechanism has not been described before, we examined the role of the NCX in the Iso-induced decrease in [Ca²⁺]_i. For this purpose, we used Ba²⁺, which can enter the cells through Ca²⁺ channels and can be extruded by the NCX (Wagner-Mann et al. 1992), but not by the Ca²⁺ pumps of the sarcolemma and the sarcoplasmic reticulum (Yamaguchi et al. 1989; Schilling et al. 1989; Seguchi et al. 1996; Ushio-Fukai et al. 2000). Furthermore, as Ba²⁺ binds fura-2 with a resulting increase in emitted fluorescence similar to that for Ca²⁺, this enabled us to monitor [Ba²⁺]_i. Figure 5 clearly shows that Iso can decrease [Ba²⁺]_i in the presence of higher extracellular [Na⁺], but cannot do so when a low extracellular Na⁺ PSS solution is used. These results strongly support the idea that Iso activates NCX to extrude Ca²⁺. The inhibitory effect of 2,4-DCB, an NCX inhibitor, on the Iso-induced relaxation further lends support to this proposal.

NCX activity and gene expression has been reported in blood vessels (Nakasaki et al. 1993; Juhaszova et al. 1996; Quednau et al. 1997; Wakimoto et al. 2000) and functional studies indicate that this exchanger plays an important role in the regulation of cytosolic Ca²⁺ in vascular myocytes (Zhu et al. 1994; Slodzinski & Blaustein, 1998; Nazer & van Breemen, 1998; Blaustein & Lederer, 1999; Wang et al. 2000). Slaughter et al. (1989) reported that the NCX has a 3-6-fold greater transporting capacity than that of the sarcolemmal Ca²⁺ pump. It is thus not surprising that the activity of the exchanger is regulated in several ways and to different extents. Concerning the regulation of the NCX by PKA, the consensus phosphorylation sites have been identified and suggest that the NCX may be a target for PKA (Blaustein & Lederer, 1999). However, a variety of conflicting physiological results have been obtained following PKA activation. Mene et al. (1993) reported that both basal and vasoconstrictor-stimulated NCX activity in human mesangial cells were acutely inhibited by receptor-mediated and by forskolin-induced activation of adenylate cyclase activity and by dibutyryl cAMP. However, it is also reported that the the activity of the neural isoform of the NCX is preferentially increased by PKA activation (He et al. 1998). Our present results strongly indicate that Iso also increases the activity of the NCX.

Although the data obtained in the present study indicate that Iso induces Ca²⁺ extrusion through the NCX, it is still not clear where the site of action is for the Iso-mediated signal transduction molecule, presumably PKA. It is possible that Iso activates Na⁺/K⁺ ATPase to induce activation of NCX by increasing the electrochemical driving force. The sarcolemmal Na⁺-K⁺ ATPase has been implicated in the mechanism of -adrenoceptor agonist-induced relaxation of airway and vascular smooth muscle (Webb & Bohr, 1981; Gunst & Stropp, 1988). Stimulation of enzymatic activity of Na⁺-K⁺ ATPase by cAMP may lead to generation of the Na⁺ gradient necessary to exclude Ca²⁺ via the NCX or hyperpolarization of the membrane, which in turn reduces Ca²⁺ influx through membrane potential-dependent Ca²⁺ channels (Fleming, 1980). However, the present results could not be explained by this mechanism alone, because ouabain, a specific inhibitor of Na⁺-K⁺ ATPase, did not completely inhibit the Iso-induced decrease in [Ca²⁺]_i. In summary, the present results indicate that Iso decreases [Ca²⁺]_i and tension via activation of the NCX and that the NCX is functionally expressed in porcine coronary artery smooth muscle.

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Acknowledgements

We thank Dr I. Laher (UBC, Vancouver) for a critical reading of our manuscript. This study was supported in part by Grants-in-Aid for Scientific Research (No. 13557067, 13470149, 13832006, 13670723, 13671591, 14657174, 14570675) and for Scientific Research on Priority Area (No. 14026038) from the Ministry of Education, Culture, Sports, Science and Technology, Japan, by the Research Grant for Cardiovascular Diseases (12C-2, 13C-4) from the Ministry of Health, Labour and Welfare, Japan, and by grants from the Japan Space Forum, Kanehara Ichiro Memorial Foundation and Mochida Memorial Foundation for Medical and Pharmaceutical Research.

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