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Intracellular Cl^– fluxes play a novel role in Ca²⁺ handling in airway smooth muscle

Asthma Research Group, Firestone Institute for Respiratory Health, St. Joseph's Healthcare; and Department of Medicine, McMaster University, Hamilton, Ontario, Canada

Submitted 13 September 2005 ; accepted in final form 12 January 2006

	ABSTRACT

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Intracellular Ca²⁺ is actively sequestered into the sarcoplasmic reticulum (SR), whereas the release of Ca²⁺ from the SR can be triggered by activation of the inositol 1,4,5-trisphosphate and ryanodine receptors. Uptake and release of Ca²⁺ across the SR membrane are electrogenic processes; accumulation of positive or negative charge across the SR membrane could electrostatically hinder the movement of Ca²⁺ into or out of the SR, respectively. We hypothesized that the movement of intracellular Cl^– (Cl) across the SR membrane neutralizes the accumulation of charge that accompanies uptake and release of Ca²⁺. Thus inhibition of SR Cl^– fluxes will reduce Ca²⁺ sequestration and agonist-induced release. The Cl^– channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 10^–4 M), previously shown to inhibit SR Cl^– channels, significantly reduced the magnitude of successive acetylcholine-induced contractions of airway smooth muscle (ASM), suggesting a "run down" of sequestered Ca²⁺ within the SR. Niflumic acid (10^–4 M), a structurally different Cl^– channel blocker, had no such effect. Furthermore, NPPB significantly reduced caffeine-induced contraction and increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i). Depletion of Cl, accomplished by bathing ASM strips in Cl^–-free buffer, significantly reduced the magnitude of successive acetylcholine-induced contractions. In addition, Cl^– depletion significantly reduced caffeine-induced increases in [Ca²⁺]_i. Together these data suggest a novel role for Cl fluxes in Ca²⁺ handling in smooth muscle. Because the release of sequestered Ca²⁺ is the predominate source of Ca²⁺ for contraction of ASM, targeting Cl fluxes may prove useful in the control of ASM hyperresponsiveness associated with asthma.

chloride; 5-nitro-2-(3-phenylpropylamino)benzoic acid; niflumic acid; calcium handling; excitation-contraction coupling

Although the role of intracellular Ca²⁺ in signaling and smooth muscle contraction has been well studied, little is known about the functional or regulatory properties of intracellular Cl^– (Cl). Alterations in Cl, brought about by reducing extracellular Cl^– (Cl) concentrations in experimental situations, have been shown to alter G protein-coupled receptor signaling in non-smooth muscle cells (5): the replacement of Cl with a multitude of large impermeant anions reduced the phasic (early) portion of agonist-induced contraction of ileal longitudinal smooth muscle. The reduction in contractile responses correlated well with a reduction in cellular ⁴⁵Ca²⁺ uptake. Together these data suggested the existence of a Ca²⁺ pool that was sensitive to alterations in Cl(21, 22). More recently, Cl has been implicated in Ca²⁺ handling and EC coupling within gastrointestinal and vascular smooth muscle, respectively. Inhibition of SR Cl^– channels reduced the Ca²⁺ sequestration in saponin-permeabilized gastrointestinal smooth muscle cells (19). Depletion of Cl reduced angiotensin II- and norepinephrine-induced contractions of vascular smooth muscle (12). Furthermore, it has been suggested that Cl^– is the primary ionic species that contributes to charge neutralization during Ca²⁺ uptake in skeletal muscle and isolated smooth muscle SR vesicles (3, 13, 19).

The objective of our study was to determine what role, if any, Cl plays in EC coupling and Ca²⁺ handling in ASM. Upon active pumping of Ca²⁺ into the SR, a positive charge will accumulate that will eventually impede further uptake. We first hypothesized, therefore, that Cl^– flux across the SR membrane during Ca²⁺ reuptake neutralizes the accumulation of positive charge, allowing for maximal sequestration of Ca²⁺. Second, we hypothesized that the release of Ca²⁺ from the SR must also be coupled to a Cl^– efflux pathway that acts to neutralize the negative charge accumulation. To this end, inhibition of SR Cl^– channels would reduce the release of Ca²⁺ upon agonist stimulation. We therefore investigated the effects of Cl^– channel blockers and Cl^– substitution on mechanical and Ca²⁺ release responses in ASM.

	MATERIALS AND METHODS

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Tissue preparation. All experimental procedures were approved by the McMaster University Animal Care Committee and conform to the guidelines set out by the Canadian Council on Animal Care. Tracheae were obtained from cows (136–454 kg) or pigs (20–90 kg) killed at the local abattoir and transported to the lab in ice-cold Krebs buffer (see Solutions and chemicals). When tracheae were received in the lab, the epithelium was removed, and tracheal ASM strips (2–3 mm wide, 10 mm long) were excised and used immediately or stored at 4°C for use up to 48 h.

Cell isolation. Tracheal ASM strips were digested in modified Hanks' balanced salt solution (with NaHCO₃, without CaCl₂ and MgSO₄) containing collagenase (Sigma blend type F, 2 mg/ml) and elastase (type IV, 250 µg/ml). After a 30-min incubation period at 37°C, papain (30 µg/ml) and (–)-1,4-dithio-L-threitol (750 µg/ml) were added, and the tissues were incubated for an additional 20–30 min. Cells were gently triturated with the use of a wide-bore pipette and then centrifuged to form a loose pellet. Supernatant was removed, and cells were resuspended in standard Ringer solution (see Solutions and chemicals).

Organ bath studies. Intact segments of tracheal ASM were mounted in 4-ml organ baths with the use of silk thread (Ethicon 4-0) such that one end of the tissue was anchored and the other fastened to a Grass FT.03 force transducer, and preload tension of 1.0–1.5 g was applied. Isometric tension was digitized at 2 Hz and recorded online using the DigiMed System Integrator program (MicroMed, Louisville, KY). Tissues were bathed in modified Krebs buffer bubbled with 95% O₂-5% CO₂ and heated to 37°C. During a 1-h equilibration, tissues were repeatedly washed with modified Krebs buffer. To test for tissue responsiveness and viability, ASM strips were challenged with 60 mM KCl. The KCl was then washed out, and tissues were allowed to recover before experiments were conducted.

Microelectrode studies. Intact segments of tracheal ASM (5 mm in width) were superfused at a rate of 3 ml/min with the modified Krebs buffer, bubbled with 95% O₂-5% CO₂, and heated to 37°C. Channel blockers and agonists were added directly to the modified Krebs buffer and introduced to the bath via superfusion. Microelectrodes were pulled from borosilicate glass using a P-87 Flaming/Brown micropipette puller (Sutter Instrument, Novato, CA). The tip resistance of the microelectrodes used was within 30–100 M when filled with 3 M KCl. Membrane potential changes were measured at 37°C and amplified on a Duo 773 electrometer (World Precision Instruments, Sarasota, FL), digitally sampled at 5 Hz, and analyzed using WinDaq 700 series data-acquisition software (Dataq Instruments, Akron, OH).

Intracellular Ca²⁺ fluorimetry. Isolated tracheal ASM cells (see Cell isolation) were incubated with fluo-4 AM (2 µM, containing 0.1% Pluronic F-127) for 30 min at 37°C. Cells were then placed in a Plexiglas recording chamber and superfused with Ringer solution for a period of 30 min before experimentation to allow for complete dye hydrolysis. 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and zero-Cl^– Ringer were delivered via the bathing solution, whereas caffeine was delivered via a micropipette (Picospritzer II; General Valve, Fairfield, NJ). Confocal microscopy was performed at room temperature (21–23°C) with the use of a custom-built apparatus (25) based on an inverted Nikon Eclipse TE2000-4 microscope with a x40 S Fluor oil objective. Briefly, 488-nm illumination from a photodiode laser was scanned across an isolated cell in X- and Y-planes by using two mirrors oscillating at 8 kHz and 30 Hz, respectively. The emitted fluorescence (>500 nm) was detected using a photomultiplier. The signal was then digitized, and images were generated (1 frame/s, 480 x 400 pixels); these were stored in TIF stacks of several hundred frames on a local hard drive using image-acquisition software (Video Savant 4.0; IO Industries, London, ON, Canada). Image files were then imported into Scion Image (Scion; free download: http://www.scioncorp.com) for subsequent analysis, using a custom-written macro designed to determine average fluorescence intensity over a defined nonnuclear region of interest.

Solutions and chemicals. Modified Krebs buffer used in organ bath and microelectrode studies consisted of (in mM) 116 NaCl, 4.6 KCl, 1.2 MgSO₄, 2.5 CaCl₂, 1.3 NaH₂PO₄, 23 NaHCO₃, 11 D-glucose, 0.01 indomethacin, 0.0001 propranolol, and 0.1 N-nitro-L-arginine(L-NNA) bubbled with 95% O₂-5% CO₂ to maintain pH 7.4. Zero-Cl^– Krebs buffer consisted of (in mM) 116 sodium isethionate, 4.6 potassium acetate, 1.2 magnesium acetate, 2.5 calcium acetate, 1.3 NaH₂PO₄, 23.0 NaHCO₃, 11 D-glucose, 0.01 indomethacin, 0.0001 propranolol, and 0.1 L-NNA bubbled with 95% O₂-5% CO₂ to maintain pH 7.4.

Dissociation buffer consisted of Ca²⁺-free Hanks' balanced salt solution (Sigma, St. Louis, MO) to which appropriate enzymes, dissolved in distilled water, were added. Ringer buffer, utilized for Ca²⁺ fluorimetry experiments, consisted of (in mM) 130 NaCl, 5 KCl, 1 CaCl₂, 1 MgCl₂, 20 HEPES, and 10 D-glucose; pH 7.4 with NaOH (300 mosM). Zero-Cl^– Ringer consisted of (in mM) 130 sodium isethionate, 5 potassium acetate, 1 calcium acetate, 1 magnesium acetate, 20 HEPES, and 10 D-glucose; pH 7.4 with NaOH (300 mosM).

Acetylcholine (ACh; 10^–1 M in distilled-deionized water), niflumic acid (10^–1 M in DMSO), NPPB (10^–1 M in DMSO), and nifedipine (10^–1 M in ethanol) stock solutions were diluted in Krebs buffer as appropriate. Caffeine was dissolved directly in Krebs buffer to attain a final concentration of 10 mM.

Data analysis. Contractile responses to caffeine and ACh were normalized to a KCl (60 mM) response in each tissue. The effect of Cl^– depletion or ion channel blockers on agonist-induced increases in [Ca²⁺]_i and force generation is expressed as a percent change from within tissue control responses. Agonist-induced changes in [Ca²⁺]_i were derived from averaging fluorescence intensities from regions of interest (30 x 30 pixels) defined in central nonnuclear regions of single tracheal ASM cells.

All responses are reported as means ± SE; n refers to the number of animals. Statistical comparisons were made using Student's t-test (for single pairwise comparisons) or one-way ANOVA (for multiple comparisons of mean values) followed by the appropriate post hoc test. P < 0.05 was considered statistically significant.

	RESULTS

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Agonists that cause contraction of ASM generally increase the open probability of Ca²⁺-dependent Cl^– channels (CDCC) through the release of Ca²⁺ from the SR, causing membrane depolarization. In bovine tracheal ASM, cholinergic contractions induced by increasing concentrations of carbachol (10^–9 to 10^–5 M) were insensitive to pretreatment with niflumic acid (10^–4 M) and NPPB (10^–4 M), two structurally distinct inhibitors of plasmalemmal Cl^– channels (Fig. 1). Furthermore, inhibition of L-type Ca²⁺ channels had little effect on carbachol-induced contractions of bovine tracheal ASM (nifedipine, 10^–6 M) (Fig. 1).

View larger version (20K): [in this window] [in a new window]   Fig. 1. Cholinergic contraction of bovine airway smooth muscle (ASM) triggered by increasing concentrations of the cholinergic agonist carbachol (CCh; 10–9 M to 10–5 M) in the presence of blockers of L-type Ca2+ channels (nifedipine, 10–6 M) and Cl– channels [niflumic acid, 10–4 M; 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), 10–4 M]. Values are means ± SE; n = 5–7.

View larger version (29K): [in this window] [in a new window]   Fig. 2. A–D: representative tracings of repetitive contractions of bovine ASM triggered by the cholinergic agonist ACh (3 x 10–7 M; filled boxes) under control conditions (A) or after treatment with niflumic acid (10–4 M; B), nifedipine (10–6 M; C), or NPPB (10–4 M; D). E: mean changes in magnitude of ACh contractions (bottom). Values are means ± SE; n = 6–7. *P < 0.05 compared with control. #P < 0.05 compared with all groups. Inset (top) illustrates the reproducibility of the responses in the absence of any blockers. R1, response after 1st challenge; Rfinal, response after final challenge.

View larger version (13K): [in this window] [in a new window]   Fig. 3. CCh (left) and caffeine (Caff; right) both depolarized the membrane of porcine ASM under control conditions. Pretreatment with the Cl– channel blocker niflumic acid (10–4 M) had no effect on resting membrane potential but abolished the depolarizing response to CCh and caffeine. Values are means ± SE; n = 4–11. *P < 0.05 compared with control.

Cholinergic stimulation of ASM triggers a number of pathways that may contribute to contraction. To resolve the role of Cl^– channels in Ca²⁺ handling, we used caffeine to trigger Ca²⁺ release independent of muscarinic receptor activation. Reproducible caffeine-induced contractions could be triggered at 15-min intervals (Fig. 4A). Nifedipine and niflumic acid added 10 min before challenges with caffeine significantly reduced caffeine-induced contractions (–25.2 ± 13.2%, n = 4, and –35.5 ± 15.0%, n = 6, respectively), indicating a role for voltage-dependent Ca²⁺ influx in these responses. However, the Cl^– channel blocker NPPB significantly reduced caffeine-induced contractions to a greater extent than either nifedipine or niflumic acid (–68.6 ± 11.1%, n = 5, P < 0.05) (Fig. 4D; summarized in Fig. 4E).

View larger version (22K): [in this window] [in a new window]   Fig. 4. A–D: representative tracings of contractions of bovine ASM triggered by the Ca2+-releasing agonist caffeine (10 mM; filled boxes) in the absence (control; A) or presence of niflumic acid (10–4 M; B), nifedipine (10–6 M; C), or NPPB (10–4 M; D). E: mean changes in caffeine-induced contractions. Values are means ± SE; n = 5–6. *P < 0.05 compared with control. #P < 0.05 compared with all groups.

View larger version (19K): [in this window] [in a new window]   Fig. 5. A: representative tracing of Ca2+ transients in isolated single bovine ASM cells triggered by the Ca2+-releasing agent caffeine (10 mM; filled boxes) in the presence of NPPB (10–4 M), measured using Ca2+ fluorimetry and confocal microscopy. F510, average fluorescence intensity at 510 nm; AU, arbitrary units. B: mean changes in caffeine-evoked Ca2+ transients upon introduction of NPPB or vehicle (control) compared with magnitude of Ca2+ transient evoked immediately before that introduction. Values are means ± SE; n = 4. *P < 0.05 compared with control.

View larger version (21K): [in this window] [in a new window]   Fig. 6. A–C: representative tracings of repetitive contractions of bovine ASM triggered by the cholinergic agonist ACh (3 x 10–7 M; filled boxes) with corresponding velocity traces for control (A) or during continuous (B) or intermittent removal of external Cl– (C). D: mean changes in magnitudes of ACh-induced contractions. E and F: mean changes in peak rate of ACh-induced contraction during continuous (E) and intermittent depletion of external Cl– (F). G and H: mean changes in peak rate of relaxation following ACh-induced contraction during continuous (G) and intermittent depletion of external Cl– (H). W, response was obtained with Cl–-containing buffer. All mean (±SE) values are expressed as percent changes from first ACh-evoked response; n = 4–6. *P < 0.05 compared with control. #P < 0.05 compared with intermittent zero Cl–.

To determine whether long-term Cl^– depletion was in fact altering Ca²⁺ handling within tracheal ASM, we compared caffeine-induced Ca²⁺ transients in isolated ASM cells exposed to normal or zero-Cl^– Ringer buffer. These responses were significantly smaller in peak magnitude in the absence of Cl^– (44.9 ± 7.9% reduction, P < 0.05, n = 4) (Fig. 7A; summarized in Fig. 7B).

View larger version (14K): [in this window] [in a new window]   Fig. 7. A: representative tracing of Ca2+ transients in isolated single bovine ASM cells triggered by the Ca2+-releasing agent caffeine (10 mM; filled boxes) in the presence and absence of external Cl– measured using Ca2+ fluorimetry and confocal microscopy. B: mean changes in caffeine-evoked Ca2+ transients expressed as percent change in magnitude of Ca2+ transient in the presence of Cl–-free Ringer compared with Ca2+ transient evoked just before removal of extracellular Cl–. Values are means ± SE; n = 4. *P < 0.05 compared with control.

	DISCUSSION

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The objectives of our study were to determine what role Cl and membrane Cl^– fluxes have on EC coupling and Ca²⁺ handling in ASM. Many lines of evidence suggest that EC coupling in ASM is unique among smooth muscle types in that it is less reliant on voltage-dependent events. Indeed, our data indicate that inhibition of plasmalemmal Cl^– channels with niflumic acid and NPPB has little effect on cholinergic contraction of tracheal SM (Fig. 1). Furthermore, direct inhibition of L-type Ca²⁺ channels with nifedipine had little effect on cholinergic contractions, suggesting that contraction of ASM might be driven by release of Ca²⁺ from the SR or by other receptor-activated signaling pathways, rather than voltage-dependent Ca²⁺ influx.

Rather than contributing directly to EC coupling in ASM through membrane depolarization, Cl^– channels may instead play a role in Ca²⁺ handling in ASM. After its release from the SR, Ca²⁺ is actively resequestered into the SR (20, 27) to maintain an effective releasable store upon subsequent stimulation with a contractile agonist (26). Ca²⁺ release and uptake are both electrogenic processes, resulting in accumulation of charge on the SR membrane that ultimately hinders further Ca²⁺ flux. As such, Cl^– flux into the SR may be coupled to Ca²⁺ reuptake, and/or Cl^– efflux to Ca²⁺ release, to neutralize that charge accumulation and thereby facilitate Ca²⁺ mobilization. Such compensatory ion fluxes have been described in skeletal muscle (3, 13, 14) and vascular smooth muscle (19) but have not yet been explored in ASM. We therefore performed successive ACh challenges on intact ASM strips in the presence and absence of external Cl^– and/or blockers of Cl^– or Ca²⁺ channels with the intent of probing their effects on Ca²⁺ handling.

In our experiments, NPPB reduced mechanical responses and fluorimetric Ca²⁺ transients to a significantly greater extent than niflumic acid or nifedipine, suggesting that its effect is not due solely to a reduction in voltage-dependent Ca²⁺ influx. Pollock et al. (19) also reported differing efficacies of various Cl^– channel blockers on Ca²⁺ handling: NPPB and IAA-94 inhibited sequestration of Ca²⁺ in saponin-permeabilized gastrointestinal smooth muscle cells but not Ca²⁺ uptake into cardiac muscle SR vesicles, whereas neither niflumic acid nor 4,4'-dinitrostilbene-2,2'-disulfonic acid affected Ca²⁺ sequestration in those gastrointestinal smooth muscles. Different efficacies between Cl^– channel blockers may reflect different selectivities of the Cl^– channels on the SR compared with those on the plasmalemma or may be secondary to varying abilities of the blockers to cross the plasmalemma and interact with the channels on the SR.

Although NPPB significantly reduced both caffeine-induced contractions and increases in [Ca²⁺]_i, it did not completely abolish caffeine-induced contraction or Ca²⁺ transients, suggesting that additional ion fluxes may exist to neutralize charge accumulation during Ca²⁺ movement into and out of the SR. K⁺ and H⁺ channels also have been described in the SR of skeletal muscle (3, 16, 30) and postulated to contribute to neutralization of charges during Ca²⁺ handling.

Because it appears that the SR Cl^– fluxes may be playing a role in the uptake and release of Ca²⁺, we wanted to examine what effect depletion of Cl might have on cholinergic contractions in ASM. We hypothesized that depletion of Cl would reduce SR Ca²⁺ content by hindering uptake and would further reduce agonist-induced Ca²⁺ release by reducing SR Cl^– levels.

We created chloride-free buffers by substituting sodium chloride with sodium isethionate and all other chloride salts with acetates. Isethionate is a large ion that is highly impermeant to membrane anion channels yet elicits biological effects similarly to gluconate when used to substitute for chloride (12, 21, 22).

As per our hypothesis, prolonged removal of external Cl^– reduced the peak magnitudes as well as the velocities of cholinergic contractions in a rapidly reversible fashion (i.e., immediately upon reintroduction of Cl^– at the end of the experiment) and also slowed the rate of relaxation upon washout of ACh, suggesting that depletion of Cl was altering both the rates and magnitudes of Ca²⁺ flux across the SR membrane. Interestingly, when Cl^– was reintroduced during the recovery period (when the SR is presumably refilling) and then removed immediately before addition of ACh, the peak magnitudes and velocities of contraction were normal (i.e., not different from control), but the contractions then decayed to the levels seen when Cl^– had been absent throughout the repeated cholinergic stimulations (cf. Fig. 6, B and C) and the rates of relaxation were significantly slowed (Fig. 6, G and H).

These observations are entirely consistent with our hypothesis that Cl is required for efficient sequestration of Ca²⁺, as described previously in guinea pig ileal smooth muscle (21, 22). Although an alternative explanation involves a Cl^– dependence of G protein-coupled receptor signaling (5), we found that caffeine-evoked Ca²⁺ transients also were significantly decreased in Cl^–-free Ringer buffer.

In conclusion, our data suggest that airway smooth muscle SR membrane expresses an NPPB-sensitive Cl^– channel that functions to neutralize charge accumulation resulting from uptake and release of Ca²⁺. Furthermore, Cl is necessary for refilling of the SR, thus providing a novel role for Cl^– in Ca²⁺ handling and EC coupling in ASM.

	GRANTS

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These studies were supported by Career Awards from the Canadian Institutes of Health Research (to L. J. Janssen), Altana Pharmaceuticals (to S. Hirota), and the Natural Sciences and Engineering Research Council of Canada (to S. Hirota), as well as operating grants from the Canadian Institutes of Health Research and the Ontario Thoracic Society.

	FOOTNOTES

 

Address for reprint requests and other correspondence: L. J. Janssen, L-314, St. Joseph's Healthcare, 50 Charlton Ave. East, Hamilton, Ontario, Canada L8N 4A6 (e-mail: janssenl{at}mcmaster.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	REFERENCES

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1. Ann Twiss M, Harman E, Chresrown S, and Hendeles L. Efficacy of calcium channel blockers as maintenance therapy for asthma. Br J Clin Pharmacol 53: 243–249, 2002.[CrossRef][Web of Science][Medline]
2. Coburn RF, Thornton D, and Arts R. Effect of trachealis muscle contraction on tracheal resistance to airflow. J Appl Physiol 32: 397–403, 1972.[Free Full Text]
3. Fink RHA and Stephenson DG. Ca²⁺-movement in muscle modulate by the state of K⁺-channels in the sarcoplasmic reticulum membranes. Pflügers Arch 409: 374–380, 1987.[CrossRef][Web of Science][Medline]
4. Garty M, Cohen E, Mazar A, Ilfeld DN, Spitzer S, and Rosefeld JB. Effect of nifedipine and theophylline in asthma. Clin Pharmacol Ther 40: 195–198, 1986.[Web of Science][Medline]
5. Higashijima T, Ferguson KM, and Sternweis PC. Regulation of hormone-sensitive GTP-dependent regulatory proteins by chloride. J Biol Chem 262: 3597–3602, 1987.[Abstract/Free Full Text]
6. Janssen LJ and Sims SM. Spontaneous transient inward currents and rhythmicity in canine and guinea-pig tracheal smooth muscle cells. Pflügers Arch 427: 473–480, 1994.[CrossRef][Web of Science][Medline]
7. Janssen LJ and Sims SM. Ca²⁺-dependent Cl^– current in canine tracheal smooth muscle cells. Am J Physiol Cell Physiol 269: C163–C169, 1995.[Abstract/Free Full Text]
8. Janssen LJ. Acetylcholine and caffeine activate Cl^– and suppress K⁺ conductances in human bronchial smooth muscle. Am J Physiol Lung Cell Mol Physiol 270: L772–L781, 1996.[Abstract/Free Full Text]
9. Janssen LJ and Sims SM. Acetylcholine activates non-selective cation and chloride conductances in canine and guinea-pig tracheal myocytes. J Physiol 453: 197–218, 1992.[Abstract/Free Full Text]
10. Janssen LJ and Sims SM. Histamine activates Cl^– and K⁺ currents in guinea-pig tracheal myocytes: convergence with muscarinic signalling pathways. J Physiol 465: 661–677, 1993.[Abstract/Free Full Text]
11. Janssen LJ and Sims SM. Substance P activates Cl^– and K⁺ conductances in guinea-pig tracheal smooth muscle cells. Can J Physiol Pharmacol 72: 705–710, 1994.[Web of Science][Medline]
12. Jensen BL, Ellekvist P, and Skott O. Chloride is essential for contraction of afferent arterioles after agonists and potassium. Am J Physiol Renal Physiol 272: F389–F396, 1997.[Abstract/Free Full Text]
13. Kasai M and Kometani T. Ionic permeability of sarcoplasmic reticulum membrane. In: Cation Flux Across Biomembranes. New York: Academic, 1979, p. 167–177.
14. Kourie J, Laver D, Ahern G, and Dulhunty A. A calcium-activated chloride channel in sarcoplasmic reticulum vesicles from rabbit skeletal muscle. Am J Physiol Cell Physiol 270: C1675–C1686, 1996.[Abstract/Free Full Text]
15. Lidji M, Molho M, Chagnac A, Katz I, and Benzaray S. Therapy with nifedipine in asthma: a randomized double-blind crossover trial. Isr J Med Sci 24: 1–4, 1988.[Web of Science][Medline]
16. Meissner R and McKinley D. Permeability of canine cardiac sarcoplasmic reticulum vesicles to K⁺, Na⁺, H⁺ and Cl^–. J Biol Chem 257: 7704–7711, 1982.[Abstract/Free Full Text]
17. Ono K, Nakao M, and Iijima T. Chloride-sensitive nature of the histamine-induced Ca²⁺ entry in cultured human aortic endothelial cells. J Physiol 511: 837–849, 1998.[Abstract/Free Full Text]
18. Patakas D, Maniki E, Tsara V, and Dascalopoulou E. Nifedipine treatment of patients with bronchial asthma. J Allergy Clin Immunol 79: 959–963, 1987.[CrossRef][Web of Science][Medline]
19. Pollock N, Kargacin M, and Kargacin G. Chloride channel blockers inhibit Ca²⁺ uptake by the smooth muscle sarcoplasmic reticulum. Biophys J 75: 1759–1766, 1998.[Web of Science][Medline]
20. Poulsen JC, Caspersen C, Mathiasen D, East JM, Tunwell RE, Lai FA, Maeda N, Mikoshiba K, and Treiman M. Thapsigargin-sensitive Ca²⁺-ATPases account for Ca²⁺ uptake to inositol 1,4,5-trisphosphate-sensitive and caffeine-sensitive Ca²⁺ stores in adrenal chromaffin cells. Biochem J 307: 749–758, 1995.[Medline]
21. Rangachari PK and Triggle CR. Chloride removal and excitation-contraction coupling in guinea pig ileal smooth muscle. Can J Physiol Pharmacol 64: 1521–1527, 1986.[Web of Science][Medline]
22. Rangachari PK, Triggle CR, and Daniel EE. Chloride removal selectively inhibits phasic contractions of guinea pig ileal longitudinal smooth muscle. Can J Physiol Pharmacol 60: 1741–1744, 1982.[Web of Science][Medline]
23. Roffel AF, Meurs H, Elzinga CR, and Zaagsma J. Characterization of the muscarinic receptor subtype involved in phosphoinositide metabolism in bovine tracheal smooth muscle. Br J Pharmacol 99: 293–296, 1990.[Web of Science][Medline]
24. Saleh SN and Greenwood IA. Activation of chloride currents in murine portal vein smooth muscle cells by membrane depolarization involves intracellular calcium release. Am J Physiol Cell Physiol 288: C122–C131, 2005.[Abstract/Free Full Text]
25. Sanderson MJ and Parker I. Video-rate confocal microscopy. Methods Enzymol 360: 447–481, 2003.[Web of Science][Medline]
26. Sims SM, Jiao Y, and Zheng ZG. Intracellular calcium stores in isolated tracheal smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 271: L300–L309, 1996.[Abstract/Free Full Text]
27. White C and McGeown JG. Regulation of basal intracellular calcium concentration by the sarcoplasmic reticulum in myocytes from the rat gastric antrum. J Physiol 529: 395–404, 2000.[Abstract/Free Full Text]
28. Yang CM, Chou SP, Wang YY, Hsieh JT, and Ong R. Muscarinic regulation of cytosolic free calcium in canine tracheal smooth muscle cells: Ca²⁺ requirement for phospholipase C activation. Br J Pharmacol 110: 1239–1247, 1993.[Web of Science][Medline]
29. Yang CM, Yo YL, and Wang YY. Intracellular calcium in canine cultured tracheal smooth muscle cells in regulated by M₃ muscarinic receptors. Br J Pharmacol 110: 983–938, 1993.[Web of Science]
30. Yu X, Carroll S, Rigaud JL, and Inesi G. H⁺ countertransport and electrogenicity of the sarcoplasmic reticulum Ca²⁺ pump in reconstituted proteoliposomes. Biophys J 64: 1232–1242, 1993.[Medline]

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