airway smooth muscle (ASM) contraction induced by agonist stimulation results from two phenomena: elevation in cytosolic Ca²⁺ concentration and sensitization of the contractile machinery to Ca²⁺ (7). Histamine interacts with the H₁ receptor and activates G_q proteins leading to inositol 1,4,5-trisphosphate (IP₃) generation (21). This, in turn, leads to Ca²⁺ release from the sarcoplasmic reticulum (SR) through the IP₃-sensitive channel receptor (37) and the ryanodine channel receptor (23) followed by extracellular Ca²⁺ influx. The pathways involved in the latter have not been clearly identified (8) and show a complex interaction with the first stages of contraction (19).

Earlier studies have supported a role for extracellular Na⁺ in regulating contraction of guinea pig tracheal muscle (13). Such dependency might reflect involvement of the Na⁺/Ca²⁺ exchanger (NCX), which is an integral membrane protein that transports three Na⁺ in exchange for one Ca²⁺, with transport direction dependent on electrochemical gradient of both ions (12). In its forward mode of operation, NCX mediates Ca²⁺ efflux and Na⁺ influx. However, due to certain events such as membrane depolarization and/or intracellular Na⁺ accumulation, NCX is able to mediate Ca²⁺ influx and Na⁺ efflux (reverse mode of operation; Ref. 4).

We (27) have previously reported that NCX is expressed and could play an important role in Ca²⁺ homeostasis in guinea pig ASM based on several lines of evidence. First, we have detected mRNA of NCX1 in ASM. Second, Na⁺ substitution by N-methyl-d-glucamine (NMDG⁺) or inhibition of Na⁺/K⁺ pump with ouabain produces an increase in intracellular Ca²⁺ in cultured ASM cells (14). Third, inhibition of Na⁺/K⁺ pump with ouabain increases muscle tension (15). Finally, histamine-precontracted guinea pig tracheal rings show a decreased relaxation rate when washed in a Na⁺-free solution (13).

On the other hand, it has been reported that agonists like histamine and carbachol provoke a small and sustained inward cationic current through nonselective cationic channels (NSCC) of unknown molecular identity (16, 37). Functionally, these channels have been referred to as store-operated Ca²⁺ channels (1), and it has been speculated that they could correspond to canonical transient receptor potential channels (TRPC) (2), which have been previously described in ASM (30). The many possible combinations in which these proteins can form tetrameric channels and the lack of specific inhibitors has complicated their precise study (29).

Recently, Rosker et al. (32) reported that in HEK-293 cells overexpressing TRPC3 channels, stimulation with carbachol was associated with an increase of intracellular Ca²⁺ concentration, which depended on extracellular Na⁺ since its substitution or the NCX inhibition with KB-R7943 reduced such effect. In the same cell line, they also showed by coimmunoprecipitation experiments that NCX and TRPC3 are physically associated.

In the present work, we propose that the NCX, working in its reverse mode of operation, mediates part of the Ca²⁺ influx secondary to the NSCC-dependent intracellular Na⁺ increase. By measuring isometric force on guinea pig tracheal rings, we observed that sustained histamine contractions depend on external Na⁺ and inferred that this is due to activation of NSCC and NCX. Also, using fluorescence microscopy on freshly dissociated tracheal cells, we observed that extracellular Na⁺ did influence the rise of Ca²⁺ elicited by histamine. Our results support the view that the NCX-mediated Ca²⁺ entry promoted by histamine stimulation is critical in developing and maintaining the sustained contraction and that possibly the NCX is activated due to depolarization and intracellular Na⁺ increase provoked by NSCC channels.

METHODS

Tissue Preparation and Organ Bath Experiments

Our protocols were approved by the Animal Care Committee of the Universidad Autónoma de San Luis Potosí. Whole tracheas were obtained from male guinea pigs (500–800 g) that had been euthanized with Na⁺ pentobarbital (130 mg/kg ip). Tracheal rings measuring 2 mm wide were cut and dissected free of adipose and connective tissue, and the epithelium was removed by gently rubbing the lumen with a cotton swab. In some force experiments, rings were cut at the opposite side of the muscle so that strips were obtained. Each tracheal strip or ring was mounted into an organ bath either by tying both ends with silk thread or using stainless steel hooks inserted into the lumen. One end was anchored to a support plastic rod, and the other one was suspended from a Grass force transducer (FT.03). Tissues were bathed in normal physiological solution (PS) containing, in mM, 135 NaCl, 4.7 KCl, 1.17 MgSO₄, 1.15 KH₂PO₄, 1.1 CaCl₂, 20 HEPES, and 10 dextrose as well as 3 μM indomethacin (to inhibit formation of prostaglandins) and maintained at 37°C (pH 7.4). Tissues were passively stretched to impose a preload tension of 1 g (to reach maximal responses) and allowed to equilibrate for 1 h before experimental manipulation. Isometric changes in tension were amplified and plotted using a Grass amplifier and chart recorder, respectively, and later digitized with Polyview software (Astro-Med, West Warwick, RI). At the beginning of every experiment, rings were challenged with a maximally effective concentration of histamine (10 μM) to assess their functional state. Rings belonging to the same animal were used, and each one received a different treatment. Thus n represents the number of animals used for the same protocol. Test substances were added directly to the organ bath.

Tracheal rings contain autonomic nervous terminals, so to exclude any effect caused by endogenous neurotransmitters, force experiments were performed in the presence of 1 μM atropine (to inhibit muscarinic receptors), 10 μM prazosin (to inhibit α-adrenergic receptors), 10 μM propranolol (to inhibit β-adrenergic receptors), and 1 mM l-NAME (to inhibit nitric oxide synthase).

Cell Dissociation

Whole tracheas were obtained as described above and dissected free of adipose and connective tissues. A muscle strip was then dissected and placed in a dissociation buffer containing, in mM, 130 NaCl, 5 KCl, 1 MgCl₂, 1 CaCl₂, 10 HEPES, 10 dextrose, 10 taurine, and 0.25 EDTA as well as 0.5 mg/ml F-type collagenase and 1.5 mg/ml papain. Digestion was obtained by overnight incubation at 4°C, followed by 1 h at room temperature and 50 min at 31°C. The muscle strip was then placed in a solution containing, in mM, 135 KCl, 1 CaCl_2, 1 MgCl₂, 10 HEPES, and 10 dextrose. With the help of fire-polished Pasteur pipettes, cells were released from the tissue and studied as described below.

Ca²⁺ Measurements

Cells were loaded by incubation with fura-2 AM (4 μM) at room temperature for 50 min. Cells were allowed to settle onto a glass coverslip that comprised the bottom of a perfusion chamber (∼0.75-ml volume). The chamber was mounted on a Nikon inverted microscope, and physiological bathing solution was superfused (1–3 ml/min at room temperature) during the experiment. The ratio of fluorescence emission at 510 nm with alternate excitation wavelengths of 345 and 380 nm was measured using a DeltaRAM system as previously described (25). Images were collected every 6 s and analyzed using ImageMaster software (Photon Technology International; London, ON, Canada). Fluorescence ratio measurements were quantified at two time-points during stimulation. The peak ratio was measured as the difference between highest ratio observed after stimulation and the basal ratio value. The sustained phase of fluorescence was measured as the difference between the ratio observed before agonist washing and the basal level of fluorescence to which the cell returned at the conclusion of stimulation.

Drugs

KB-R7943 was purchased from Tocris (Ellisville, MO). All other chemicals and salts were purchased from Sigma-Aldrich (St. Louis, MO). KB-R7943, nifedipine, and SKF-96365 were dissolved in DMSO at a final concentration of less than 0.1%.

Statistical Analyses

Residuals of the treatments were tested for homogeneity of variances using Brown-Forsythe test (35) as well as for normal distribution using Shapiro-Wilk test (9). n refers to the number of animals, and P < 0.05 was considered statistically significant.

Data from tension experiments were considered as independent groups since measurements were made on different tracheal rings from the same animal. Accordingly, ANOVA was used to compare several mean values, and these results are presented as means ± SD. Multiple comparisons were tested using Tukey-Kramer honestly significant difference test (5).

Data from fluorescence experiments were considered dependent data since measurements were made within the same cell. Accordingly, data were analyzed using Student's paired t-test for single pairwise comparisons. Such results are described as the mean of differences ± SD of differences.

RESULTS

Isometric Force Experiments

Effect of external Na⁺ substitution on contraction induced by histamine.

To assess the role of external Na⁺ and NCX on histamine-induced contractions of tracheal rings, Na⁺ was replaced by either NMDG⁺ or Li⁺ (Fig. 1). Experiments in which extracellular Na⁺ was replaced by NMDG⁺ showed a small and transitory contraction in 60% of the preparations. After histamine stimulation, we observed three contraction profiles: 1) small and rapidly declining, representing 80% of preparations (Fig. 1A, middle trace); 2) small and slowly declining (data not shown); or 3) small and steady (data not shown). Considering all profiles, the maximum force attained by histamine stimulation was significantly lower than the control as expressed in %force from previous histamine contraction (99 ± 1% for PS vs. 53 ± 1% for NMDG⁺; P < 0.0001; n = 6). In contrast, when Na⁺ was substituted for LiCl, a small reproducible increase in force was produced (Fig. 1A, bottom trace). Once histamine was added, a further sustained contraction was observed, and the peak tension, measured from the previous basal level, showed no significant decrease (91 ± 1%; P = 0.062; n = 6) compared with control.

Effect of NCX Inhibition by KB-R7943

By measuring isometric force generation on histamine-contracted tracheal rings, we built a dose-response curve for the nonspecific NCX inhibitor KB-R7943 and found an IC₅₀ of 55 ± 4 μM and a maximal relaxation using 100 μM (data not shown). Then we used this last concentration of KB-R7943 on tracheal rings to assess the role of the reverse mode of the NCX during histamine contraction. It has been previously reported that KB-R7943 inhibits various channels and transporters: Ca_V1.2 channels (3), NMDA receptor channels (36), nicotinic receptors (31), and the norepinephrine transporter (26). Since our preparation includes neurotransmitter inhibitors and because of the nature of the tissue, we considered that the only relevant interaction of KB-R7943 apart from that with NCX would be with the Ca_V1.2 channels. Therefore, we used 70 nM nifedipine (which we had previously shown to block the contraction induced by 80 mM KCl, data not shown) to exclude their contribution. Preincubation with KB-R7849 led to a significant decrease in peak force during histamine stimulation (92.3 ± 2.5% remaining force for vehicle vs. 52.3 ± 5.6% force for KB-R7943; P < 0.001; n = 4). On the other hand, preincubation with nifedipine led to no significant change in this peak magnitude, discarding nonspecific effects of KB-R7849 on Ca_V1.2 channels at the beginning of contraction (89.7 ± 1.8% remaining force; P > 0.05 compared with vehicle; n = 4; Fig. 2).

We also explored the effect of NCX inhibition on sustaining the contraction by histamine under a different protocol, in which the drugs were applied at the peak of force development. After maximal relaxation with nifedipine, addition of KB-R7943 significantly relaxed the muscle further (49 ± 6% remaining force for nifedipine vs. 1 ± 3% force for nifedipine + KB-R7943; P < 0.0001; n = 6; Fig. 3).

Effect of NSCC Inhibition

To examine the role of NSCC channels during force development induced by histamine, we used two nonspecific NSCC blockers, SKF-96365 and 2-aminoethyldiphenyl borate (2-APB). The protocol consisted of eliciting maximal contraction by 10 μM histamine whereupon addition of 50 μM SKF-96365, the remaining tonic force was significantly reduced as well as after 10 μM 2-APB addition (91 ± 8% force for vehicle, 1 ± 1% force for SKF-96365, and 19 ± 7% force for 2-APB; P < 0.001; n = 6; Fig. 4).

Cell Imaging Experiments

To further understand the mechanisms underlying force development, we focused on studying Ca²⁺ changes in single smooth muscle cells. We then established a cell isolation procedure, which resulted in elongated cells able to contract and show an increase in fura-2 fluorescence ratio after histamine stimulation. We observed that histamine stimulation resulted in a fast increase in fluorescence ratio reaching a peak value circa 5 s, which rapidly decreased (circa 40 s) to a small level over the resting signal and persisted until the agonist was washed out. Then we tested if this response pattern could be repeated on the same cell by stimulating it again after a 20-min resting period so as to establish a suitable model for drug application. Indeed, changes in fluorescence ratio measured at the peak as well as during the sustained phase (just before agonist wash-out) showed no significant difference (mean of differences = 0.07 ± 0.061, P = 0.1078, n = 6; and mean of differences = 0.013 ± 0.0019, P = 0.09, n = 6, respectively; Figs. 5 and 6). Since the second stimulation was statistically comparable to the first one, the protocols described below correspond to drug addition simultaneous with histamine during the second stimulation of the same cell.

Effect of NCX Inhibition by KB-R7943

Addition of 100 μM KB-R7943 significantly decreased the change in peak fluorescence ratio in a second stimulation with histamine (mean of differences = 0.20 ± 0.10; P = 0.0128; n = 6) as well as during the sustained phase (mean of differences = 0.19 ± 0.09; P = 0.0124; n = 6; Figs. 5 and 6).

Effect of Na⁺ Substitution by NMDG⁺

By substituting external Na⁺, we observed no significant difference in peak fluorescence ratio in a second stimulation with histamine (mean of differences = 0.017 ± 0.08; P = 0.057; n = 4). Nevertheless, we found a significant decrease in the ratio during the sustained Ca²⁺ rise period (mean of differences = 0.54 ± 0.25; P = 0.048; n = 4; Figs. 5 and 6).

Effect of NSCC Inhibition by SKF-96365

NSCC inhibitor SKF-96365 (50 μM) decreased the change in peak fluorescence ratio in a second stimulation with histamine (mean of differences = 0.43 ± 0.16; P = 0.0005; n = 7), as well as during the sustained phase (mean of differences = 0.47 ± 0.18; P = 0.020; n = 7; Figs. 5 and 6).

DISCUSSION

The principal aim of this work was to improve our knowledge regarding the mechanisms involved in force generation during histamine stimulation of ASM. Our most important finding was that histamine contraction in guinea pig ASM depends on extracellular Na⁺ and was sensitive to the nonspecific NCX blocker KB-R7943 as well as to NSCC nonspecific blockers SKF-96365 and 2-APB. These findings have led us to propose that histamine causes Ca²⁺ entry mediated by the NCX operating in its reverse mode secondary to a cationic influx (primarily Na⁺) through NSCC. It has been reported previously that during histamine stimulation, an inward current composed mainly of Na⁺ is observed in isolated equine tracheal cells (37). In that respect, we have demonstrated here that Na⁺ can be substituted for Li⁺ generating equivalent contractions induced by histamine. We propose that histamine stimulates Na⁺ influx through NSCC, cell depolarization, and the increase in subplasmalemmal Na⁺ concentration. These conditions might favor first the reverse mode of the NCX and later the activation of Ca_V1.2 channels, which have been characterized in this tissue previously (38). On the other hand, as Na⁺ is replaced by NMDG⁺, neither depolarization nor Na⁺ influx through NSCC could be possible. Thus Ca²⁺ release from SR, Ca²⁺ entry through NSCC, and Ca²⁺ entry through reverse-mode NCX would provide for the small histamine-induced contraction observed.

Preincubation with KB-R7943 allowed us to explore the role of NCX during the beginning of histamine contraction as well as throughout tonic force development. In the presence of KB-R7943, a significant diminishment in maximal force developed was observed, whereas preincubation with 70 nM nifedipine had no effect (Fig. 2). We thus suggest that NCX is active in its reverse mode at an early stage of contraction after Ca²⁺ release from the SR, whereas Ca_V1.2 channels participate somewhat later during stimulation. This contrasts with results from Satake et al. (34) where incubation with 1 μM nifedipine leads to a significant decrease in force generation. We have no explanation for this difference, which could probably be due to a different dose and incubation period.

The transient histamine contraction observed after preincubation with KB-R7943 could be the result of Ca²⁺ release from SR. This is supported by results where histamine stimulation in Ca²⁺-free-EGTA solution show a transient contraction of similar magnitude to the one observed in presence of KB-R7943 (92.3 ± 3% force for PS vs. 47.2 ± 4% force for Ca²⁺-free; P = 0.0025; n = 5; data not shown). In addition, this contraction is also similar to the one observed when Na⁺ is substituted for NMDG. Our interpretation for this is that when NCX has been inhibited, not only is Ca²⁺ entry blocked, but also membrane depolarization does not reach the threshold for Ca_V1.2 channel activation.

When Na⁺ is substituted by NMDG⁺, we would not expect Na⁺/K⁺ pump inhibition since it has been documented that in the presence of saturating concentration of extracellular K⁺, extracellular Na⁺ inhibits pump rate (24). A possible side effect of Na⁺ substitution by NMDG is intracellular acidification due to inactivation of the Na⁺/H⁺ exchanger (33). We addressed this situation by performing experiments in extracellular pH = 6.8 to lower intracellular pH to at least 6.7 and found no significant difference in force development by histamine stimulation (2.2 ± 0.2 g for control vs. 2.1 ± 0.2 g for external pH 6.7; P = 0.2363; n = 4; data not shown).

Regarding Na⁺ substitution by LiCl, we propose that histamine stimulation produces Li⁺ influx through NSCC, membrane depolarization, and activation of Ca_V1.2 channels. It is worth noting that Li⁺ is not transported through the NCX in either direction (4), and therefore we expect that the NCX will not operate under this condition.

Experiments performed in our lab confirmed the presence of the mRNA of NCX 1 (27) in guinea pig tracheal smooth muscle. Also, we had provided functional evidence supporting the expression of NCX in ASM derived from studies where the Na⁺/K⁺ pump was inhibited with ouabain (14, 15). With this evidence in hand, we proceeded to study the role of NCX during tonic contraction under the Ca_V1.2 channel inhibition. We show compelling data supporting that contraction and Ca²⁺ influx rely on both Ca_V1.2 channels and the NCX, since the inhibition of both completely abolished force development. At the time when we were writing this manuscript, Hirota et al. (17) proposed the functional importance of the NCX in ASM during contraction induced by histamine, acetylcholine, or serotonin. Their results support our findings that agonist stimulation produces the conditions for NCX reversal and Ca²⁺ entry. Also, Zhang et al. (39) reported that either inhibition of NCX by KB-R9743 or external Na⁺ substitution by NMDG⁺ attenuated the store depletion-mediated Ca²⁺ entry in pulmonary artery smooth muscle. Both results are in agreement with our present findings. A very interesting approach for future work would be downregulation of the NCX using small interfering RNA in smooth muscle cells, which would highlight medium- and long-term effects of this mechanism of Ca²⁺ handling.

The high dose of KB-R9743 needed to inhibit force development by histamine in our experiment suggests that the NCX isoform in this tissue might be less sensitive to the drug (11). Interestingly, it has been reported that mutation at residue Gly833 renders NCX 1 almost insensitive to inhibition by KB-R7943 (20). This residue is located within the conserved α-2 repeat structure, which could then be a target for analysis of variation between splicing isoforms of NCX.

Using isolated smooth muscle cells, we then tested the effects of NCX and NSCC inhibitors on intracellular Ca²⁺ changes produced by agonist. Histamine stimulation resulted in a rapid and transitory increase in fluorescence ratio followed by a sustained phase just above basal levels (15% of the peak in Ca²⁺ rise), which lasted until histamine was washed out. A similar profile has been previously described on isolated smooth muscle cells (6, 22, 28), although studies performed in whole smooth muscle tissues show a different behavior with a sustained elevation in fluorescence matching force development (6, 19, 34). The reasons that underlie this difference between muscle strips and isolated cells are not clear at the moment, making any comparison between results very difficult. A protocol of two consecutive challenges of histamine allowed us to test the effect of different blockers. Thus the second application of histamine together with the NCX inhibitor KB-R7943 resulted in a significantly lower peak value as well as a lower level of fluorescence ratio in the sustained phase. We suggest that the NCX is operating in the Ca²⁺ influx mode and that the KB-R7943-insensitive component is due to Ca²⁺ release from the stores and perhaps Ca²⁺ entry through NSCC.

We also tested substitution of external Na⁺ by NMDG⁺ in single isolated cells. We observed that the sustained phase of Ca²⁺ entry in single cells also depends on the presence of external Na⁺. Regarding the peak in fluorescence obtained after histamine stimulation in NMDG⁺, we did not detect a significant decrease compared with PS. In single cell experiments, histamine was added simultaneously with Na⁺-free solution. Therefore, the conditions for NCX reversal might be present for Ca²⁺ entry during the peak (4). Regarding the sustained phase of fluorescence elevation, one explanation would be that, assuming that intracellular Na⁺ has reached a very low level, NCX can no longer operate in its reverse mode.

Still, we propose that the underlying event that triggers activation of NCX is a depolarizing inward Na⁺ current entering through NSCC as was previously reported by Hirota et al. (18) for carbachol stimulation. By using nonspecific NSCC inhibitors SKF-96365 and 2-APB, we observed that all the histamine-evoked contraction is abolished and thus depends completely on their activation. On the other hand, our results using the aforementioned experimental approach for isolated cells clearly show that application of SKF-93635 and thus inhibition of NSCC significantly lowers the peak fluorescence ratio and completely abolishes fluorescence in the sustained phase of the curve. This is in agreement with results reported by Dai et al. (10) where SKF-96365 inhibits contraction and Ca²⁺ waves in porcine tracheal smooth muscle cells.

Altogether, our results lead us to propose the following model (Fig. 7). Activation of H₁ receptors by histamine triggers a signaling cascade leading to formation of IP₃ and diacylglycerol. IP₃ produces Ca²⁺ release from SR, and this, in turn, causes NSCC opening. Na⁺ influx through these channels causes some membrane depolarization as well as a local increase in [Na⁺]_i in the vicinity of NCX. These conditions then promote NCX reverse mode of operation but are not sufficient to activate Ca_V1.2 channels. Ca²⁺ entry mediated by the NCX may add to Ca²⁺ released from the SR and activate Ca²⁺-activated Cl⁻ channels (18, 21). This, in turn, should cause enough depolarization to activate the Ca_V1.2 channels and, together with sensitization events, give rise to a characteristic histamine contraction. This model, then, suggests activation of NCX in its reverse mode as a critical pathway for Ca²⁺ influx. We speculate that NCX is a possible target for drug design in the treatment of pathologies such as asthma (4).

GRANTS

This work was supported by CONACyT-Scholarship 65845 to P. Algara-Suárez, Universidad Autónoma de San Luis Potosí Grants C05-FAI-10-3.24 to S. Sanchez-Armass, C03-FRC-06-7.8 and C06-FAI-03-4.7 to U. Meza, and C06-FAI-11-12.49 to R. Espinosa-Tanguma, and Canadian Institutes of Health Research Grant MOP-10019 to S. M. Sims.

FOOTNOTES

• 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.