HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 288/1/L84    most recent 00446.2003v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (10) Citing Articles via Google Scholar Google Scholar Articles by Carbajal, V. Articles by Montaño, L. M. Search for Related Content PubMed PubMed Citation Articles by Carbajal, V. Articles by Montaño, L. M.

LTD₄ induces hyperresponsiveness to histamine in bovine airway smooth muscle: role of SR-ATPase Ca²⁺ pump and tyrosine kinase

³Departamento de Farmacología, Facultad de Medicina, Universidad Nacional Autónoma de México, Ciudad Universitaria and ²Laboratorio de Investigación en Autoinmunidad and ¹Departamento de Hiperreactividad Bronquial, Instituto Nacional de Enfermedades Respiratorias, México DF, México

Submitted 17 December 2003 ; accepted in final form 13 September 2004

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Airway hyperresponsiveness is a key feature of asthma, but its mechanisms remain poorly understood. Leukotriene D₄ (LTD₄) is one of the few molecules capable of producing airway hyperresponsiveness. In this study, LTD₄, but not leukotriene C₄ (LTC₄), produced a leftward displacement of the concentration-response curve to histamine in bovine airway smooth muscle strips. Neither LTC₄ nor LTD₄ modified the concentration-response curve to carbachol. In simultaneous measurements of intracellular Ca²⁺ ([Ca²⁺]_i) and contraction, histamine or carbachol produced a transient Ca²⁺ peak followed by a plateau, along with a contraction. LTD₄ increased the histamine-induced transient Ca²⁺ peak and contraction but did not modify responses to carbachol. Enhanced responses to histamine induced by LTD₄ were not modified by staurosporine or chelerythrine but were abolished by genistein. Western blot showed that carbachol, but not histamine, caused intense phosphorylation of extracellular signal-regulated kinase 1/2 and that LTD₄ significantly enhanced the phosphorylation induced by histamine, but not by carbachol. L-type Ca²⁺ channel participation in the hyperresponsiveness to histamine was discarded because LTD₄ did not modify the [Ca²⁺]_i changes induced by KCl. In tracheal myocytes, LTD₄ enhanced the transient Ca²⁺ peak induced by histamine (but not by carbachol) and the sarcoplasmic reticulum (SR) Ca²⁺ refilling. Genistein abolished this last LTD₄ effect. Partial blockade of the SR-ATPase Ca²⁺ pump with cyclopiazonic acid reduced the Ca²⁺ transient peak induced by histamine but not by carbachol. These results suggested that LTD₄ induces hyperresponsiveness to histamine through activation of the tyrosine kinase pathway and an increasing SR-ATPase Ca²⁺ pump activity. L-type Ca²⁺ channels seemed not to be involved in this phenomenon.

asthma; intracellular calcium ion; leukotriene D₄; tyrosine kinase; extracellular signal-regulated kinase 1/2; sarcoplasmic reticulum

LTD₄ is a potent in vivo and in vitro contracting agent for human airway smooth muscle (12) and has been implicated in the pathogenesis of asthma (4). The mechanism by which LTD₄ contracts the smooth muscle seems to be a complex phenomenon mediated by several pathways. In this context, it has been proposed that the effect of LTD₄ is linked to cytosolic Ca²⁺ elevation through phosphoinositol hydrolysis (2, 11, 32), the activation of L-type Ca²⁺ channels and nonselective cation channels (11, 32), and a rise in the capacitative Ca²⁺ entry (32). Moreover, Ca²⁺-independent mechanisms such as activation of protein kinase C (PKC)- and protein tyrosine kinase pathways have also been suggested to be involved in the LTD₄-induced smooth muscle contraction (2, 32, 38). Whether these mechanisms involved in the smooth muscle contraction could be also implicated in the development of airway hyperresponsiveness is unclear. Thus the aim of the present study was to evaluate some of these mechanisms, namely the role of L-type Ca²⁺ channels, PKC and tyrosine kinase pathways, in the LTD₄-induced hyperresponsiveness in bovine tracheal smooth muscle. In addition, because sarcoplasmic reticulum (SR)-ATPase Ca²⁺ pump is a major mechanism involved in the regulation of intracellular Ca²⁺ concentration ([Ca²⁺]_i; see Ref. 20), we were also interested in investigating the possible role of the SR-ATPase Ca²⁺ pump in the LTD₄-induced airway smooth muscle hyperresponsiveness.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Bovine tracheal tissue was obtained from a local slaughterhouse and immersed in Krebs solution for its transportation to the laboratory. This bovine tissue was submitted to the following experimental protocols.

Organ bath experiments. Bovine tracheas were dissected free of cartilage, epithelium, and connective tissue, and smooth muscle strips (2–2.5 mm wide, 5 mm long) were suspended in 5-ml organ baths containing Krebs solution (in mM): 118 NaCl, 25 NaHCO₃, 4.6 KCl, 1.2 KH₂PO₄, 1.2 MgSO₄, 11 glucose, and 2 CaCl₂. Preparations were maintained at 37°C and bubbled with 5% CO₂ in oxygen at pH 7.4. Tissues were attached to an isometric force transducer (model FT03; Grass Instruments, West Warwick, RI) connected to a system of signal conditioner (CyberAmp 380, Axon Instruments, Foster City, CA) plus an analog-digital interface (Digidata 1200A; Axon). Recordings were stored in a microcomputer and analyzed using data acquisition and analysis software (AxoScope version 7.0; Axon). Preparations were equilibrated for 30 min under a resting tension of 1–1.5 g before testing. Tissues were then stimulated three times with 60 mM KCl (lasting 20 min each) until maximum stable responses were obtained.

Concentration-response curves to carbachol (3.2 x 10^–9 to 1 x 10^–3 M) and histamine (3.2 x 10^–8 to 1 x 10^–3 M) were constructed with or without LTC₄ (10 nM) or LTD₄ (32 nM) preincubation (35 min). These LTC₄ and LTD₄ concentrations were selected from pilot experiments demonstrating that they caused no contractile response or a small contraction (10% of the response to 60 mM KCl) that returned to baseline level after 30 min. Because it was important to avoid biotransformation of exogenous CysLTs, in those experiments using LTC₄, tissues were also preincubated with serine borate (15 mM), a well-known -glutamyl transpeptidase inhibitor (29, 35), to prevent its biotransformation to LTD₄. Likewise, in all experiments involving LTD₄, we used L-cysteine (6 mM) as inhibitor of the aminopeptidase involved in its biotransformation to LTE₄ (33). Contractile responses to carbachol or histamine were expressed as a percentage of the maximum contraction induced by KCl stimulation.

Simultaneous measurement of [Ca²⁺]_i and contraction. Bovine airway smooth muscle strips (1–1.5 mm wide, 5 mm long) were loaded with fura 2-AM by incubating them under protection from light during 3.5 h at 37°C in a shaking bath at 54 strokes/min with 2 ml aerated (5% CO₂ in O₂) Krebs containing 20 µM fura 2-AM, 1 mM probenecid, and 0.01% pluronic F-127. Afterward, strips were washed during 10–15 min with Krebs solution with 10 µM indomethacin to remove the dye from the extracellular space. Each preparation was mounted vertically in the bottom of a 4-ml fluorimeter cuvette of polymethacrylate using a special adaptor from Photon Technology International (PTI, Princenton, NJ). This cuvette was filled with aerated Krebs (10 µM indomethacin) and put inside of a PTI fluorometer. The lower end of the strip was fixed to the adaptor while the upper end was attached to an isometric force displacement transducer (FSG-01; Experimetria, Budapest, Hungary) connected to an analog-digital interface (PTI) via an EasyGraf recorder (model TA240; Gould Electronics, Cleveland, OH).

Fura 2 loaded in the smooth muscle strips was excited by alternating pulses of 340 and 380 nm light, and emission was collected at 510 nm using a PTI fluorometer. The fluorescence acquisition rate was 0.8/s. [Ca²⁺]_i was calculated according to the formula of Grynkiewicz et al. (17). The K_d of fura 2 was assumed to be 386 nM (22). The mean 340- to 380-nm fluorescence ratios for R_max and R_min were obtained by exposing the tissue to 5 mM Ca²⁺ in the presence of 50 µM ionomycin and in Ca²⁺-free medium with 10 mM EGTA, respectively. R_max was 2.49 and R_min 1.19. The fluorescence ratio at 380-nm light excitation, in Ca²⁺-free medium and Ca²⁺-saturated tissues (), was 1.36. Recordings were stored in a microcomputer and analyzed using data acquisition and analysis software (Felix version 1.21; PTI).

Preparations were equilibrated for 15 min under a resting tension of 1–1.5 g before testing. Tissues were then stimulated two times with 60 mM KCl during 10 min each to corroborate their viability. Afterward, a first stimulation (S₁) with 1.3 µM histamine (approximately the EC₅₀) was done. A second stimulation (S₂) with histamine was performed 45 min later. In some experiments, tissues were incubated with 1, 10, or 32 nM LTD₄ during 30 min before S₂. We corroborated that 32 nM LTD₄ concentration did not modify the resting smooth muscle tension, in spite of causing a small [Ca²⁺]_i increment, which returned to basal levels before starting experiments. In separate experiments, smooth muscle strips submitted to LTD₄ preincubation were also incubated with staurosporine (an unspecific PKC inhibitor, 10 nM), chelerythrine (a PKC inhibitor, 1 µM), genistein (a tyrosine kinase inhibitor, 10 µM), or daidzein (an inactive analog of genistein that was used as negative control for this drug, 10 µM). In other experiments, we used 100 nM carbachol or 30 mM KCl (equimolar) to produce S₁ and S₂ responses with or without 32 nM LTD₄ preincubation, and with or without genistein (10 µM, KCl experiments only). In all experiments in which LTD₄ was added, tissues were also preincubated with L-cysteine (3 mM). To evaluate the role of L-type Ca²⁺ channels in the responses to carbachol (1 µM) or histamine (10 µM), some tissues were incubated during 15 min with 30 µM methoxyverapamil hydrochloride (D-600). Drugs were administered using a special adaptor from PTI. This adaptor had two metallic tubes. The first one was used to add solutions (with or without drugs) to tracheal strips through syringes mounted in a serial system of three-way stopcocks. Under these conditions, drugs added to the tissues always had a uniform concentration. The second tube was used to remove the solution.

Measurement of [Ca²⁺]_i in tracheal myocytes. Airway smooth muscle cells were obtained from bovine tracheas, as described elsewhere (26). Smooth muscle (200 mg) was minced, placed in 5 ml Hanks' solution (GIBCO-BRL, Rockville, MD) containing 2 mg cysteine and 0.05 U/ml papaine, and incubated for 10 min at 37°C. Tissue was washed with Leibovitz's solution (GIBCO) to remove the enzyme excess and then was put in a Krebs solution containing 0.144 mg/ml of a highly purified collagenases and neutral protease mixture (Liberase Blendzyme 2; Roche, Indianapolis, IN) during 12 min until dispersed cells were observed. This procedure allowed us to obtain cells with consistent levels of resting [Ca²⁺]_i. Next, cells were loaded with 0.5 µM fura 2-AM in low Ca²⁺ (0.1 mM) at room temperature (22–25°C). After 1 h, cells were allowed to settle down in a 1.5-ml heated perfusion chamber with a glass cover in the bottom. This chamber was mounted on an inverted microscope (Diaphot 200; Nikon, Tokyo, Japan), and cells that adhered to the glass were continuously perfused at a rate of 2–2.5 ml/min with Krebs solution (37°C, equilibrated with 5% CO₂ in O₂, pH 7.4) containing 1.5 mM Ca²⁺.

Cells loaded with fura 2 were exposed to alternating pulses of 340- and 380-nm excitation light, and emission light was collected at 510 nm using a microphotometer (PTI). Background fluorescence was automatically subtracted and determined by removing the cell from the field before starting the experiments. The fluorescence acquisition rate was 0.5/s. [Ca²⁺]_i was calculated according to the formula of Grynkiewicz et al. (17). The K_d of fura 2 was assumed to be 386 nM (22). The mean 340- to 380-nm fluorescence ratios for R_max and R_min were obtained by exposing the cells to 10 mM Ca²⁺ in the presence of 10 µM ionomycin and in Ca²⁺-free Krebs with 10 mM EGTA, respectively. R_max was 6.06 and R_min 0.39. The fluorescence ratio at 380-nm light excitation in Ca²⁺-free medium and Ca²⁺-saturated cells () was 4.23. Recordings were stored in a microcomputer and analyzed using data acquisition and analysis software (Felix version 1.21; PTI).

The effect of LTD₄ on [Ca²⁺]_i changes induced by histamine (100 µM) or carbachol (10 µM) was evaluated by preincubating the isolated myocytes with or without 32 nM LTD₄ during 20 min.

To indirectly evaluate the activity of the SR-ATPase Ca²⁺ pump, we measured the ability of myocytes to refill their SR Ca²⁺ stores. Before experiments, viability of the single cells was assessed through stimulation with caffeine in Krebs solution. Next, myocytes were perfused with Ca²⁺-free solution, and 1 min later caffeine (S₁) was added during 10 min. The caffeine stimulation in a Ca²⁺-free medium completely depletes the SR Ca²⁺ store (7). Afterward, cells were washed with Ca²⁺-free medium to remove caffeine and perfused with Krebs (2.5 mM Ca²⁺) during 10 min to allow SR Ca²⁺ refilling. The stimulation with caffeine was repeated once again (S₂) under the same Ca²⁺-free conditions. In these experiments, the S₂-to-S₁ ratio corresponded to the degree of SR Ca²⁺ refilling. In some experiments, cells were incubated with 32 nM LTD₄ or 10 µM genistein plus LTD₄ before S₂.

Finally, the role of the SR-ATPase Ca²⁺ pump in the normal responses of myocytes to carbachol (3.1 µM) or histamine (3.1 µM) was evaluated through the partial inhibition of this pump with cyclopiazonic acid (CPA, 1 µM, 20-min preincubation). Because we sought to cause a partial impairment (but not the abolishment) of the SR-ATPase Ca²⁺ pump activity, we chose this CPA concentration, inasmuch as it reduced responses to caffeine in 69%.

Tyrosine kinase pathway activation. Activation of the tyrosine kinase pathway by different drugs was explored by measuring the phosphorylation of its downstream target P44/42 [extracellular signal-regulated kinase (ERK) 1/2] MAP kinase through Western blot analysis. Bovine smooth muscle strips were incubated with 100 nM carbachol or 1.3 µM histamine during 5 min, with or without preincubation with 32 nM LTD₄ during 30 min in the presence of 3 mM L-cysteine (added 15 min before LTD₄). Separate strips were incubated with LTD₄ during 5 min, with or without L-cysteine. Final stimulation was stopped by immersing the tissues in liquid nitrogen, where they remained stored until analysis. After being defrosted on ice, each tissue was homogenized (Polytron PT3100; Kinematica, Luzern, Switzerland) using lysis buffer (1% Triton X-100, 50 mM Tris, pH 7.4, 150 mM NaCl, 0.1 mM EDTA and EGTA, 1.0 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin and leupeptin, 1.0 mM Na₃VO₄, and 50 mM NaF) according to previous reports (8). Tissue protein (40 µg) from each sample was loaded in different lanes of a 12% SDS-polyacrylamide gel. In an additional lane, ERK2 control protein (phosphorylated or total) was also added. After electrophoretic separation under reducing conditions, proteins were transferred to a nitrocellulose membrane and quenched with Tris-buffered saline (TBS) containing 5% nonfat milk and 0.1% Tween 20. Membrane was submitted to overnight incubation (12 h, 4°C) with rabbit polyclonal antibodies raised against phosphorylated or total ERK1 and ERK2 proteins (Cell Signaling, Beverly, MA) and then washed three times with TBS-Tween 20 (0.1%). ERK1/2 were detected by adding horseradish peroxidase-labeled anti-rabbit antibodies. Immunoblots were developed using an enhanced chemiluminescent reactant (LumiGLO; Cell Signaling) and an optimal exposition of the nitrocellulose sheets to X-ray films (Biomax ML Film; Kodak, Rochester, NY). ERK1/2 immunoblots were analyzed by densitometry using Kodak digital science ID software version 2.03 (Eastman Kodak, New Haven, CT).

Drugs. Fura 2-AM, probenecid, pluronic F-127, ionomycin, histamine dihydrochloride, carbamylcholine chloride (carbachol), LTC₄, LTD₄, L-cysteine, L-serine, boric acid, staurosporine, chelerythrine chloride, methoxyverapamil hydrochloride (D-600), indomethacin, and CPA were obtained from Sigma (St. Louis, MO). Fura 2-AM, ionomycin, and CPA were dissolved in DMSO (final concentration in the experiments was 0.025%, a concentration devoid of effects on the contractile and [Ca²⁺]_i responses). Genistein was purchased from GIBCO-BRL. Daidzein was obtained from Biomol (Plymouth Meeting, PA).

Data analysis. Every concentration-response curve to carbachol or histamine in smooth muscle strips was analyzed through the maximum contraction and the –log effective concentration at 50% of the response (–log EC₅₀), calculated by interpolation in a straight line regression of the log concentration vs. the probit-transformed response.

In simultaneous measurements, the evaluation of [Ca²⁺]_i was done by measuring the peak response and the averaged plateau (measured from the visually identified beginning of plateau until the end of the 10-min record), whereas the contractile response was evaluated by the maximum contraction. To minimize the biological variability, responses to histamine, carbachol, KCl, and caffeine were evaluated in S₂ compared with their respective basal responses (S₁). Thus an S₂/S₁ ratio was used in all evaluations. Differences in S₂/S₁ ratios were compared among groups.

Most data were evaluated through one-way ANOVA followed by Dunnett's test or Bonferroni correction. For assessment of ERK1/2 phosphorylation, data from each treatment group were expressed as a percentage of control tissue phosphorylation. Tissues stimulated with LTD₄, carbachol, or histamine were evaluated through one-sample t-test, whereas remaining pairwise comparisons were evaluated through nonpaired Student's t-test. Adjustment of data according to ERK1/2 total protein load was considered not necessary, since these last values did not change with the different treatments. In other experiments, we used paired and nonpaired Student's t-tests. Statistical significance was considered with a P value <0.05 bimarginally. Data in the text and Figs. 1–11 are means ± SE.

View larger version (34K): [in this window] [in a new window]   Fig. 1. Effect of leukotriene C4 (LTC4, 10 nM) and leukotriene D4 (LTD4, 32 nM) on the bovine airway smooth muscle responsiveness to carbachol. Symbols represent means ± SE of n = 8 experiments in each group. CTL, control group.

View larger version (32K): [in this window] [in a new window]   Fig. 11. Densitometry data from Western blot analysis of extracellular signal-regulated kinase (ERK) 1/2 in bovine airway smooth muscle submitted to different treatments. Open bars, tissues stimulated with drugs indicated at bottom; filled bars, L-cysteine (L-Cys) + LTD4; hatched and cross-hatched bars, tissues that were preincubated with L-Cys + LTD4 before administration of carbachol or histamine, respectively. Inset shows an example of the Western blot with the following treatments: lane 1, control phosphorylated ERK2 protein; lane 2, control tissue without any treatment; lane 3, LTD4; lane 4, L-Cys + LTD4; lane 5, CCh; lane 6, L-Cys + LTD4 + CCh; lane 7, His; lane 8, L-Cys + LTD4 + His. All experiments correspond to n = 5. P < 0.05 with respect to the control group without any treatment. *P < 0.05 and **P < 0.02 with respect to tissues stimulated with the agonist alone.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

Organ bath experiments. Carbachol and histamine produced a concentration-dependent contraction of the bovine airway smooth muscle strips. The concentration-response curve to carbachol was not modified by preincubating the tissues for 35 min with LTC₄ or LTD₄. Thus the –log EC₅₀ (M) for carbachol in the control group (7.1494 ± 0.1192) was not statistically different from the –log EC₅₀ in the LTC₄ (7.2118 ± 0.1088) or LTD₄ (7.2101 ± 0.1326) groups, and the same occurred for the maximum contraction (Fig. 1). By contrast, LTD₄ but not LTC₄ caused a leftward displacement of the concentration-response curve to histamine (5.8638 ± 0.0691, P < 0.01 and 5.4511 ± 0.1658, respectively) compared with controls (5.4246 ± 0.0740, Fig. 2). Neither LTD₄ nor LTC₄ changed the maximum contraction to histamine.

View larger version (31K): [in this window] [in a new window]   Fig. 2. Effect of LTC4 (10 nM) and LTD4 (32 nM) on the bovine airway smooth muscle responsiveness to histamine. Symbols represent means ± SE of n = 8–11 experiments in each group.

View larger version (23K): [in this window] [in a new window]   Fig. 3. Typical recordings of simultaneous measurements of intracellular Ca2+ concentration ([Ca2+]; oscillatory recordings) and contraction (smooth lines) in bovine airway smooth muscle strips. Two successive stimulations (S1 and S2) with carbachol (100 nM), histamine (1.3 µM), or KCl (30 mM) were accomplished without (A, C, and E) or with (B, D, and F) LTD4 preincubation (32 nM).

View larger version (24K): [in this window] [in a new window]   Fig. 4. Effect of LTD4 preincubation on histamine (1.3 µM)-, carbachol (100 nM, n = 7)-, or KCl (30 mM, n = 5)-induced changes of intracellular Ca2+ concentration ([Ca2+]i) and contraction in bovine airway smooth muscle strips. Open bars indicate responses to the different agents used, in the absence of LTD4. Hatched, cross-hatched, and filled bars correspond to 1, 10, and 32 nM LTD4 incubation, respectively (n = 4, 5, and 7, respectively, only for the histamine group). **P < 0.01 and *P < 0.05.

View larger version (38K): [in this window] [in a new window]   Fig. 5. Effects of kinase inhibitors on the LTD4 (32 nM, n = 7)-induced increment of intracellular Ca2+ and enhancement in tension development by histamine (His, 1.3 µM) in bovine airway smooth muscle strips. Staurosporine (10 nM, n = 4) and chelerythrine (1 µM, n = 8), two PKC inhibitors, did not modify the effect of LTD4 on intracellular Ca2+ peak and maximum contraction induced by histamine. Genistein (10 µM, n = 5) completely abolished the enhancing effect of LTD4 on the intracellular Ca2+ changes and tension development induced by histamine. **P < 0.01 and *P < 0.05.

To evaluate if extracellular Ca²⁺ plays a role in the development of LTD₄-induced hyperresponsiveness, the effect of LTD₄ incubation on the KCl responses was also evaluated. We found that LTD₄ (n = 5) promoted an enhancement of the S₂/S₁ ratio of KCl-induced contraction (1.66 ± 0.20 vs. control 1.31 ± 0.12, n = 5, P < 0.05) in spite of the lack of effect on the KCl-induced [Ca²⁺]_i change (Fig. 3, E and F, and Fig. 4). Although preincubation with genistein (n = 8) avoided the LTD₄-induced enhancement of the smooth muscle contraction to KCl (1.20 ± 0.11, P < 0.05), this drug also caused a mild but statistically significant diminution (P < 0.05) of the contractile response to KCl alone (1.05 ± 0.05, n = 8, P < 0.05, Fig. 6). Daidzein (n = 6), the negative control for genistein, did not modify the response to KCl alone (S₂/S₁ contraction, 1.36 ± 0.21).

View larger version (31K): [in this window] [in a new window]   Fig. 6. Effect of genistein (GEN, n = 8) on the LTD4-induced hyperresponsiveness to KCl (30 mM, n = 5) in bovine airway smooth muscle strips. *P < 0.05 vs. its respective group without genistein.

View larger version (16K): [in this window] [in a new window]   Fig. 7. Effect of 30 µM methoxyverapamil hydrochloride (open bars) on the responses to carbachol (1 µM, n = 5) and histamine (10 µM, n = 6). **P < 0.01.

View larger version (19K): [in this window] [in a new window]   Fig. 8. Effect of LTD4 (32 nM) on the responses to carbachol (CCh, 10 µM, n = 7) or histamine (100 µM, n = 4) in single bovine airway smooth muscle cells. *P < 0.05 compared with His group.

View larger version (33K): [in this window] [in a new window]   Fig. 9. Effect of LTD4 (32 nM, n = 9) on the sarcoplasmic reticulum (SR) Ca2+ refilling in single bovine airway smooth muscle cells. A: experimental protocol to evaluate SR Ca2+ refilling using caffeine (10 mM). For details see Measurement of [Ca2+]i in tracheal myocytes. B: typical recordings of LTD4 effect on the SR-Ca2+ refilling. C: LTD4 induced an increment in the SR-Ca2+ refilling, expressed as the S2-to-S1 ratio, compared with control (CTL, n = 9) group. Genistein (10 µM, n = 9), completely abolished this LTD4 effect. S1, first caffeine stimulation; S2, second caffeine stimulation. *P < 0.01.

View larger version (29K): [in this window] [in a new window]   Fig. 10. Effect of cyclopiazonic acid (CPA, 1 µM) on the responses to carbachol (CCh, 3.1 µM) or histamine (3.1 µM) in single bovine airway smooth muscle cells (n = 4).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
In the present study, we found that LTD₄ was able to induce hyperresponsiveness to histamine, but not to carbachol, in bovine airway smooth muscle and that activation of tyrosine kinase and an increased SR-ATPase Ca²⁺ pump activity are involved in this hyperresponsiveness. Although other mechanisms could be participating, our results also suggested that PKC activation and L-type Ca²⁺ channels did not participate in this LTD₄-induced effect.

Changes induced by LTD₄ in strips and in myocytes were abolished by genistein (Figs. 5, 6, and 9). Although the latter drug is considered a tyrosine kinase pathway inhibitor, it also possesses other nonspecific effects (13). Thus, to discard such nonspecific actions, we used daidzein as a negative control, a substance having all the effects of genistein, except the capability to inhibit tyrosine kinase. The fact that genistein, but not daidzein, attenuated the LTD₄-induced hyperresponsiveness to histamine suggested the participation of the tyrosine kinase pathway in this phenomenon. This possibility was corroborated by the analysis of phosphorylation of ERK1/2 MAP kinase, a well-known downstream target of tyrosine kinase. In such analysis, we found that preincubation with LTD₄ notably enhanced the amount of phosphorylated ERK1/2 proteins (Fig. 11). Involvement of tyrosine kinase in the activity of LTD₄ has already been postulated for explaining its contractile effect on the smooth muscle (32, 38). Additionally, LTD₄ acts through G protein-coupled receptors (19), and cross talk between the G protein-coupled receptor signaling and the tyrosine kinase pathway has been documented (21, 37). A number of studies in smooth muscles have demonstrated that one consequence of ERK1/2 activation is the phosphorylation of caldesmon (14, 18), a protein that in its nonphosphorylated state inhibits the actomyosin ATPase and reduces smooth muscle force production (24). Phosphorylation of caldesmon abolishes its inhibitory action, thus allowing the smooth muscle contraction. Therefore, if LTD₄-induced ERK1/2 activation can produce caldesmon phosphorylation, the ensuing facilitation of contraction might constitute one mechanism involved in the LTD₄-induced hyperresponsiveness to histamine.

A potential mechanism explaining the linkage between the tyrosine kinase pathway and SR-ATPase Ca²⁺ pump activity has not been described so far. It is known that the SR-ATPase Ca²⁺ pump could be inhibited by phospholamban, but such inhibitory effect ends when this protein is phosphorylated by cAMP-dependent PKA, cGMP-dependent protein kinase, PKC, or calmodulin-dependent kinase (9, 27, 15). On the other hand, it has been postulated that calmodulin-dependent kinase directly phosphorylates the SR-ATPase Ca²⁺ pump and activates it (16). Thus it would be possible that the tyrosine kinase pathway, playing a similar role as the previously mentioned protein kinases, might phosphorylate phospholamban (or by a direct phosphorylation of the pump), thus increasing the SR-ATPase Ca²⁺ pump activity. Nevertheless, this possibility remains to be demonstrated.

An interesting finding of our study was the lack of an LTD₄ effect on the responses to carbachol (Figs. 1, 3, 4, and 8). Airways from asthmatic subjects are extremely sensitive to many contractile agonists, including cholinergic agents (4). In the clinical setting, the degree of airway hyperresponsiveness is commonly measured through a bronchial challenge with increasing concentrations of inhaled methacholine. Thus the inability of LTD₄ to induce hyperresponsiveness to carbachol suggests that, in asthmatic patients, molecules other than LTD₄ must also be involved in the generation of airway hyperresponsiveness, probably through different mechanisms. For example, Deshpande et al. (10) found that tumor necrosis factor--induced hyperresponsiveness to ACh in human airway smooth muscle cells seems to be mediated through an increased production of CD38/cADP-ribose, a pathway that elicits SR-Ca²⁺ release through activation of ryanodine receptor channels.

Possible explanations for the differential effect of LTD₄ on the responses to histamine but not to carbachol are difficult to outline. At least two mechanisms can be postulated.

First, it is well known that muscarinic responses strongly depend on inositol trisphosphate (IP₃) production, with the subsequent Ca²⁺ release from internal SR-Ca²⁺ stores (5, 6). As has been observed by others (23, 34), we found that L-type Ca²⁺ channel participation in contractile responses to muscarinic agonists is minimal, whereas contraction induced by histamine mainly depends on extracellular Ca²⁺ sources (Fig. 7). Although histamine can induce the production of IP₃, the amount of this second messenger is much lower than the quantity produced by muscarinic agents (6, 30). In addition, it has been recently described that SR-ATPase Ca²⁺ pump activity is required for an appropriate histamine response, but not for muscarinic stimulation (3, 30). This requirement is probably explained by a reduced SR-Ca²⁺ content diminishing the IP₃ receptor sensitivity (36), and thus the activity of SR-ATPase Ca²⁺ pump is necessary to maintain high Ca²⁺ fluxes through the IP₃ receptors (25). Moreover, we were able to corroborate in tracheal myocytes the hypothesis that the SR-ATPase Ca²⁺ pump plays an essential role in the response to histamine, inasmuch as partial deterioration of this pump diminished such response without affecting the response to carbachol (Fig. 10). Therefore, in our study, the LTD₄-induced increase in the Ca²⁺ transient peak during the histamine stimulation might be explained by the enhanced activity of SR-ATPase Ca²⁺ pump, promoting a higher sensitivity of IP₃ receptors and a greater SR-Ca²⁺ release. The lack of effect of LTD₄ on the carbachol responses would be explained because muscarinic agonists cause such a high production of IP₃ that it is not necessary for the increased sensitivity of the IP₃ receptors to produce an appropriate response. In addition, we discarded that increased responses to histamine induced by LTD₄ were because of L-type Ca²⁺ channel involvement, since LTD₄ incubation did not modify the Ca²⁺ changes induced by KCl (Figs. 3F and 4).

Furthermore, Western blot analysis demonstrated that carbachol alone was able to induce an intense phosphorylation of ERK1/2, and the presence of LTD₄ did not enhance this action, which is in agreement with the lack of LTD₄ effect on the carbachol-induced contraction. As commented above, histamine, by contrast, did not modify basal levels of ERK1/2 phosphorylation, but a notable enhancement of this effect could be observed when tissues were incubated with LTD₄.

Although LTD₄ did not modify the changes in [Ca²⁺]_i caused by KCl, this leukotriene increased the contractile response to KCl, i.e., provoked hyperresponsiveness to KCl (Figs. 3F and 4). These findings suggest that the augmented contraction of the smooth muscle was not because of an increment of extracellular Ca²⁺ influx and that other mechanisms, probably through phosphorylation processes, might be involved. This enhanced reactivity was abolished by genistein, suggesting the participation of the tyrosine kinase pathway. However, genistein also provoked a significant reduction of the KCl response, and thus it is not clear to what extent the diminution of the LTD₄-induced hyperresponsiveness caused by genistein was the result of a direct effect of this drug on KCl responses. On the other hand, in a recent study, Setoguchi et al. (31) explored for the first time possible mechanisms explaining the hyperresponsiveness induced by LTC₄ in porcine tracheal smooth muscle strips. They found that LTC₄ preincubation almost duplicated the contractile response to 40 mM KCl without affecting the Ca²⁺ fluorescence ratio and that a rho-rho kinase pathway was responsible of this phenomenon. They also found that genistein did not modify the LTC₄-induced hyperresponsiveness to KCl. Although these results differed from ours, differences in both studies concerning animal species and type of leukotriene render them hardly comparable, and further studies are necessary.

In conclusion, our results suggested that LTD₄-induced hyperresponsiveness to histamine in bovine airway smooth muscle is mediated by activation of the tyrosine kinase pathway, probably by activation of the downstream target ERK1/2 proteins, and an increasing SR-ATPase Ca²⁺ pump activity. L-type Ca²⁺ channels seemed not to be involved. The participation of other mechanisms known to be induced by LTD₄ remains to be explored.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was partially supported by a grant from Merck Sharp & Dohme and Dirección General de Asuntos del Personal Académico Grant IN210005.

	ACKNOWLEDGMENTS

 
We thank Diana E. Aguilar Leon from the Departamento de Patología Experimental del Instituto Nacional de Ciencias Médicas y de la Nutrición Salvador Zubirán for technical assistance during Western blot execution.

	FOOTNOTES

 

Address for reprint requests and other correspondence: L. M. Montaño, Instituto Nacional de Enfermedades Respiratorias, Tlalpan 4502, CP 14080, México DF, México (E-mail: lmmr{at}servidor.unam.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.

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 

1. Abela A and Daniel EE. Neural and myogenic effects of leukotrienes C₄, D₄, and E₄ on canine bronchial smooth muscle. Am J Physiol Lung Cell Mol Physiol 266: L414–L425, 1994.[Abstract/Free Full Text]
2. Accomazzo MR, Rovati GE, Vigano T, Hernandez A, Bonazzi A, Bolla M, Fumagalli F, Viappiani S, Galbiati E, Ravasi S, Albertoni C, Di Luca M, Caputi A, Zannini P, Chiesa G, Villa AM, Doglia SM, Folco G, and Nicosia S. Leukotriene D₄-induced activation of smooth-muscle cells from human bronchi is partly Ca²⁺-independent. Am J Respir Crit Care Med 163: 266–272, 2001.[Abstract/Free Full Text]
3. Aguilar-Maldonado B, Gomez-Viquez L, Garcia L, Del Angel RM, Arias-Montano JA, and Guerrero-Hernandez A. Histamine potentiates IP₃-mediated Ca²⁺ release via thapsigargin-sensitive Ca²⁺ pumps. Cell Signal 15: 689–697, 2003.[CrossRef][Web of Science][Medline]
4. Barnes PJ. Inflammatory mediator receptors and asthma. Am Rev Respir Dis 135: S26–S31, 1987.[Web of Science][Medline]
5. Baron CB, Cunningham M, Strauss JF 3rd, and Coburn RF. Pharmacomechanical coupling in smooth muscle may involve phosphatidylinositol metabolism. Proc Natl Acad Sci USA 81: 6899–6903, 1984.[Abstract/Free Full Text]
6. Bazán-Perkins B, Carbajal V, Sommer B, Macías-Silva M, González-Martínez M, Valenzuela F, Daniel EE, and Montaño LM. Involvement of different Ca²⁺ pools during the canine bronchial sustained contraction in Ca²⁺-free medium. Lack of effect of PKC inhibition. Naunyn Schmiedebergs Arch Pharmacol 358: 567–573, 1998.[CrossRef][Web of Science][Medline]
7. Bazán-Perkins B, Flores-Soto E, Barajas-López C, and Montaño LM. Role of sarcoplasmic reticulum Ca²⁺ content in Ca²⁺ entry of bovine airway smooth muscle cells. Naunyn Schmiedebergs Arch Pharmacol 368: 277–283, 2003.[CrossRef][Web of Science][Medline]
8. Breslin JW, Pappas PJ, Cerveira JJ, Hobson RW II, and Durán WN. VEGF increases endothelial permeability by separate signaling pathways involving ERK1/2 and nitric oxide. Am J Physiol Heart Circ Physiol 284: H92–H100, 2003.[Abstract/Free Full Text]
9. Cornwell TL, Pryzwansky KB, Wyatt TA, and Lincoln TM. Regulation of sarcoplasmic reticulum protein phosphorylation by localized cyclic GMP-dependent protein kinase in vascular smooth muscle cells. Mol Pharmacol 40: 923–931, 1991.[Abstract]
10. Deshpande DA, Walseth TF, Panettieri RA, and Kannan MS. CD38/cyclic ADP-ribose-mediated Ca²⁺ signaling contributes to airway smooth muscle hyperresponsiveness. FASEB J 17: 452–454, 2003.[Abstract/Free Full Text]
11. Dumitriu D, Prie S, Bernier SG, Guillemette G, and Sirois P. Mechanism of action of leukotriene D₄ on guinea pig tracheal smooth muscle cells: roles of Ca⁺⁺ influx and intracellular Ca⁺⁺ release. J Pharmacol Exp Ther 280: 1357–1365, 1997.[Abstract/Free Full Text]
12. Ellis JL and Undem BJ. Role of cysteinyl-leukotrienes and histamine in mediating intrinsic tone in isolated human bronchi. Am J Respir Crit Care Med 149: 118–122, 1994.[Abstract]
13. Geissler JF, Traxler P, Regenass U, Murray BJ, Roesel JL, Meyer T, McGlynn E, Storni A, and Lydon NB. Thiazolidine-diones. Biochemical and biological activity of a novel class of tyrosine protein kinase inhibitors. J Biol Chem 265: 22255–22261, 1990.[Abstract/Free Full Text]
14. Gerthoffer WT, Yamboliev IA, Pohl J, Haynes R, Dang S, and McHugh J. Activation of MAP kinases in airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 272: L244–L252, 1997.[Abstract/Free Full Text]
15. Grover AK and Khan I. Calcium pump isoforms: diversity, selectivity and plasticity. Cell Calcium 13: 9–17, 1992.[CrossRef][Web of Science][Medline]
16. Grover AK, Xu A, Samson SE, and Narayanan N. Sarcoplasmic reticulum Ca²⁺ pump in pig coronary artery smooth muscle is regulated by a novel pathway. Am J Physiol Cell Physiol 271: C181–C187, 1996.[Abstract/Free Full Text]
17. Grynkiewicz G, Poenie M, and Tsien RY. A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440–3450, 1985.[Abstract/Free Full Text]
18. Hedges JC, Oxhorn BC, Carty M, Adam LP, Yamboliev IA, and Gerthoffer WT. Phosphorylation of caldesmon by ERK MAP kinases in smooth muscle. Am J Physiol Cell Physiol 278: C718–C726, 2000.[Abstract/Free Full Text]
19. Howard S, Chan-Yeung M, Martin L, Phaneuf S, and Salari H. Polyphosphoinositide hydrolysis and protein kinase C activation in guinea pig tracheal smooth muscle cells in culture by leukotriene D₄ involve a pertussis toxin sensitive G-protein. Eur J Pharmacol 227: 123–129, 1992.[CrossRef][Web of Science][Medline]
20. Janssen LJ, Walters DK, and Wattie J. Regulation of [Ca²⁺]_i in canine airway smooth muscle by Ca²⁺-ATPase and Na⁺/Ca²⁺ exchange mechanisms. Am J Physiol Lung Cell Mol Physiol 273: L322–L330, 1997.[Abstract/Free Full Text]
21. Jin N, Siddiqui RA, English D, and Rhoades RA. Communication between tyrosine kinase pathway and myosin light chain kinase pathway in smooth muscle. Am J Physiol Heart Circ Physiol 271: H1348–H1355, 1996.[Abstract/Free Full Text]
22. Kajita J and Yamaguchi H. Calcium mobilization by muscarinic cholinergic stimulation in bovine single airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 264: L496–L503, 1993.[Abstract/Free Full Text]
23. Kannan MS, Davis C, Ladenius AR, and Kannan L. Agonist interactions at the calcium pools in skinned and unskinned canine tracheal smooth muscle. Can J Physiol Pharmacol 65: 1780–1787, 1987.[Web of Science][Medline]
24. Katsuyama H, Wang CL, and Morgan KG. Regulation of vascular smooth muscle tone by caldesmon. J Biol Chem 267: 14555–14558, 1992.[Abstract/Free Full Text]
25. Lee MG, Xu X, Zeng W, Diaz J, Kuo TH, Wuytack F, Racymaekers L, and Muallem S. Polarized expression of Ca²⁺ pumps in pancreatic and salivary gland cells. Role in initiation and propagation of [Ca²⁺]i waves. J Biol Chem 272: 15771–15776, 1997.[Abstract/Free Full Text]
26. Montaño LM, Carbajal V, Arreola JL, Barajas-López C, Flores-Soto E, and Vargas MH. Acetylcholine and tachykinins involvement in the caffeine-induced biphasic change in intracellular Ca²⁺ in bovine airway smooth muscle. Br J Pharmacol 139: 1203–1211, 2003.[CrossRef][Web of Science]
27. Nobe K, Sutliff RL, Kranias EG, and Paul RJ. Phospholamban regulation of bladder contractility: evidence from gene-altered mouse models. J Physiol 535: 867–878, 2001.[Abstract/Free Full Text]
28. O'Hickey SP, Hawksworth RJ, Fong CY, Arm JP, Spur BW, and Lee TH. Leukotrienes C₄, D₄, and E₄ enhance histamine responsiveness in asthmatic airways. Am Rev Respir Dis 144: 1053–1057, 1991.[Web of Science][Medline]
29. Orning L and Hammarstrom S. Inhibition of leukotriene C and leukotriene D biosynthesis. J Biol Chem 255: 8023–8026, 1980.[Abstract/Free Full Text]
30. Rueda A, Garcia L, and Guerrero-Hernandez A. Luminal Ca²⁺ and the activity of sarcoplasmic reticulum Ca²⁺ pumps modulate histamine-induced all-or-none Ca²⁺ release in smooth muscle cells. Cell Signal 14: 517–527, 2002.[CrossRef][Web of Science][Medline]
31. Setoguchi H, Nishimura J, Hirano K, Takahashi S, and Kanaide H. Leukotriene C₄ enhances the contraction of porcine tracheal smooth muscle through the activation of Y-27632, a rho kinase inhibitor, sensitive pathway. Br J Pharmacol 132: 111–118, 2001.[CrossRef][Web of Science][Medline]
32. Snetkov VA, Hapgood KJ, Mcvicker CG, Lee TH, and Ward JP. Mechanisms of leukotriene D₄-induced constriction in human small bronchioles. Br J Pharmacol 133: 243–252, 2001.[CrossRef][Web of Science][Medline]
33. Snyder DW, Aharony D, Dobson P, Tsai B, and Krell RD. Pharmacological and biochemical evidence for the metabolism of peptide leukotrienes by guinea pig airway smooth muscle in vitro. J Pharmacol Exp Ther 231: 224–229, 1984.[Abstract/Free Full Text]
34. Tao L, Huang Y, and Bourreau JP. Control of the mode of excitation-contraction coupling by Ca²⁺ stores in bovine trachealis muscle. Am J Physiol Lung Cell Mol Physiol 279: L722–L732, 2000.[Abstract/Free Full Text]
35. Tate SS and Meister A. Serine-borate complex as a transition-state inhibitor of -glutamyl transpeptidase. Proc Natl Acad Sci USA 75: 4806–4809, 1978.[Abstract/Free Full Text]
36. Thrower EC, Mobasheri H, Dargan S, Marius P, Lea EJ, and Dawson AP. Interaction of luminal calcium and cytosolic ATP in the control of type 1 inositol (1,4,5)-trisphosphate receptor channels. J Biol Chem 275: 36049–36055, 2000.[Abstract/Free Full Text]
37. Tolloczko B, Tao FC, Zacour ME, and Martin JG. Tyrosine kinase-dependent calcium signaling in airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 278: L1138–L1145, 2000.[Abstract/Free Full Text]
38. Wong WS, Koh DS, Koh AH, Ting WL, and Wong PT. Effect of tyrosine kinase inhibitors on antigen challenge of guinea pig lung in vitro. J Pharmacol Exp Ther 283: 131–137, 1997.[Abstract/Free Full Text]

This article has been cited by other articles:

				P. Algara-Suarez, C. Romero-Mendez, T. Chrones, S. Sanchez-Armass, U. Meza, S. M. Sims, and R. Espinosa-Tanguma Functional coupling between the Na+/Ca2+ exchanger and nonselective cation channels during histamine stimulation in guinea pig tracheal smooth muscle Am J Physiol Lung Cell Mol Physiol, July 1, 2007; 293(1): L191 - L198. [Abstract] [Full Text] [PDF]

				J. M. Dai, K.-H. Kuo, J. M. Leo, P. D. Pare, C. van Breemen, and C.-H. Lee Acetylcholine-Induced Asynchronous Calcium Waves in Intact Human Bronchial Muscle Bundle Am. J. Respir. Cell Mol. Biol., May 1, 2007; 36(5): 600 - 608. [Abstract] [Full Text] [PDF]

				J. Chavez, P. Segura, M. H. Vargas, J. L. Arreola, E. Flores-Soto, and L. M. Montano Paradoxical effect of salbutamol in a model of acute organophosphates intoxication in guinea pigs: role of substance P release Am J Physiol Lung Cell Mol Physiol, April 1, 2007; 292(4): L915 - L923. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 288/1/L84    most recent 00446.2003v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (10) Citing Articles via Google Scholar Google Scholar Articles by Carbajal, V. Articles by Montaño, L. M. Search for Related Content PubMed PubMed Citation Articles by Carbajal, V. Articles by Montaño, L. M.