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Am J Physiol Lung Cell Mol Physiol 293: L191-L198, 2007. First published April 20, 2007; doi:10.1152/ajplung.00485.2006
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Functional coupling between the Na+/Ca2+ exchanger and nonselective cation channels during histamine stimulation in guinea pig tracheal smooth muscle

Paola Algara-Suárez,1 Catalina Romero-Méndez,1 Tom Chrones,2 Sergio Sánchez-Armass,1 Ulises Meza,1 Stephen M. Sims,2 and Ricardo Espinosa-Tanguma1

1Departmento de Fisiología, Facultad de Medicina, Universidad Autónoma de San Luis Potosí, San Luis Potosí, México, and 2Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada

Submitted 20 December 2006 ; accepted in final form 16 April 2007


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Airway smooth muscle (ASM) contracts partly due to an increase in cytosolic Ca2+. In this work, we found that the contraction caused by histamine depends on external Na+, possibly involving nonselective cationic channels (NSCC) and the Na+/Ca2+ exchanger (NCX). We performed various protocols using isometric force measurement of guinea pig tracheal rings stimulated by histamine. We observed that force reached 53 ± 1% of control during external Na+ substitution by N-methyl-D-glucamine+, whereas substitution by Li+ led to no significant change (91 ± 1%). Preincubation with KB-R7943 decreased the maximal force developed (52.3 ± 5.6%), whereas preincubation with nifedipine did not (89.7 ± 1.8%). Also, application of the nonspecific NCX blocker KB-R7943 and nifedipine on histamine-precontracted tracheal rings reduced force to 1 ± 3%, significantly different from nifedipine alone (49 ± 6%). Moreover, nonspecific NSCC inhibitors SKF-96365 and 2-aminoethyldiphenyl borate reduced force to 1 ± 1% and 19 ± 7%, respectively. Intracellular Ca2+ measurements in isolated ASM cells showed that KB-R7943 and SKF-96365 reduced the peak and sustained response to histamine (0.20 ± 0.1 and 0.19 ± 0.09 for KB-R, 0.43 ± 0.16 and 0.47 ± 0.18 for SKF, expressed as mean of differences). Moreover, Na+-free solution only inhibited the sustained response (0.54 ± 0.25). These data support an important role for NSCC and NCX during histamine stimulation. We speculate that histamine induces Na+ influx through NSCC that promotes the Ca2+ entry mode of NCX and CaV1.2 channel activation, thereby causing contraction.

airway smooth muscle; KB-R7943; SKF-96365

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 Gq 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
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RESULTS
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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 {alpha}-adrenergic receptors), 10 µM propranolol (to inhibit beta-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 CaCl2, 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
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 METHODS
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DISCUSSION
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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.


Figure 1
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  		Fig. 1. A: representative traces of isometric force measurements of 10 µM histamine-stimulated guinea pig tracheal rings in PS (physiological solution; top trace), Na+-free solution with N-methyl-D-glucamine (NMDG+; middle trace), and Na+-free solution with LiCl (bottom trace). Three different rings from the same animal were randomly assigned to an external solution containing either NaCl, NMDG+, or LiCl in an isosmolar concentration. Arrow shows basal level considered for tension measurements. Force measurements were considered 5 min after histamine stimulation for comparison between contractions. B: summary results. Data are shown as % of force (means ± SD, n = 6). *P < 0.05 compared with vehicle (ANOVA and Tukey-Kramer HSD).

 
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: CaV1.2 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 CaV1.2 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 CaV1.2 channels at the beginning of contraction (89.7 ± 1.8% remaining force; P > 0.05 compared with vehicle; n = 4; Fig. 2).


Figure 2
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  		Fig. 2. A: representative traces of isometric force measurements of 10 µM histamine-stimulated guinea pig tracheal rings after preincubation with 100 µM KB-R7943 (top trace) or 70 nM nifedipine (bottom trace). Three different rings from the same animal were randomly assigned to incubation with either KB-R7943, nifedipine, or vehicle. Arrows show corresponding points considered for tension measurements. B: summary results. Data are shown as % of force (means ± SD, n = 4). *P < 0.05 compared with vehicle (ANOVA and Tukey-Kramer HSD). CTRL, control.

 
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).


Figure 3
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  		Fig. 3. A: representative traces of isometric force measurements of guinea pig tracheal rings stimulated by 10 µM histamine. Two different rings from the same animal were randomly assigned to either addition of 100 µM KB-R7943 or vehicle after full effect of 70 nM nifedipine was attained. Force measurements were considered just before nifedipine addition and before histamine wash-out for comparison. B: summary results. Data are shown as % of force (means ± SD, n = 6). *P < 0.05 (unpaired t-test).

 
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).


Figure 4
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  		Fig. 4. A: representative traces of isometric force measurements of 10 µM histamine-stimulated guinea pig tracheal rings. Three different rings from the same animal were randomly assigned to either addition of 50 µM SKF-96365 (SKF), 10 µM 2-aminoethyldiphenyl borate (2-APB), or vehicle (data not shown). Force measurements were considered just drug addition and before histamine wash-out for contraction comparison. B: summary results. Data are shown as % of remaining force (means ± SD, n = 6). *P < 0.05 compared with vehicle (ANOVA and Tukey-Kramer HSD).

 
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.


Figure 5
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  		Fig. 5. Representative traces from fluorescence ratio changes observed during stimulation of isolated smooth muscle cells with 10 µM histamine. Cells were stimulated twice with histamine and given a 20-min recovery time between stimulations, and fluorescence ratio was measured as indicated by arrows. During the second stimulation, histamine was added together with PS (A), Na+-free solution (substituting Na+ with NMDG; B), 100 µM KB-R7943 in PS (C), or 50 µM SKF-96365 in PS (D). PSS, saline solution.

 

Figure 6
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  		Fig. 6. Summary of results measured at peak fluorescence (A) and sustained phase of fluorescence (B). Data are shown as difference in fluorescence ratio (mean of differences ± SD of differences for control and KB-R7943, n = 6, for NMDG, n = 4, and for SKF-96365, n = 7). *P < 0.05 compared with a value of 0, where 0 means no difference between treatments (paired t-test).

 
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
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 ABSTRACT
 METHODS
 RESULTS
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GRANTS
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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 CaV1.2 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 CaV1.2 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 CaV1.2 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 CaV1.2 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 CaV1.2 channel inhibition. We show compelling data supporting that contraction and Ca2+ influx rely on both CaV1.2 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 {alpha}-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 CaV1.2 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 CaV1.2 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).


Figure 7
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  		Fig. 7. Model of functional interaction between the Na+/Ca2+ exchanger (NCX) and nonselective cationic channels (NSCC) during histamine (His) stimulation. A: histamine acts on its specific H1 receptor and initiates a signaling cascade leading to formation of inositol 1,4,5-trisphosphate (IP3). IP3 produces Ca2+ release from sarcoplasmic reticulum (S.R.), and this, in turn, causes NSCC opening. Next, a Na+ current entering through these channels partially depolarizes the membrane and locally increases [Na+]i in the vicinity of NCX. B: these conditions would then promote NCX operation in reverse mode. Ca2+ entry through NCX would, in turn, contribute to contraction and activate Ca2+-dependent Cl– channels (ClCa). C: this last event would cause even greater depolarization and activation of CaV1.2 channels, which also contribute importantly to Ca2+ influx that sustains contraction.

 

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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
 

Address for reprint requests and other correspondence: R. Espinosa-Tanguma, Facultad de Medicina, Universidad Autónoma de San Luis Potosí, Av. Venustiano Carranza 2405, Col. Los Filtros, San Luis Potosi, CP 78210, Mexico (e-mail: espinosr{at}uaslp.mx)

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.


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