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The potential interaction of MARCKS-related peptide and diltiazem on acrolin-induced airway mucus hypersecretion in rats
International Immunopharmacology 17 (2013) 625–632
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The potential interaction of MARCKS-related peptide and diltiazem on acrolin-induced airway mucus hypersecretion in rats☆ Peng Chen a,b,1,2, Zhiping Deng b,1,2, Tao Wang b, Lei Chen b, Jiqiong Li b, Yulin Feng b, Shangfu Zhang c, Yunyie Nin b, Daishun Liu b, Yajuan Chen b, Xuemei Ou b, Fuqiang Wen b,⁎ a b c
Department of Respiratory Medicine, The Third People's Hospital of Chengdu, Chengdu, Sichuan 610031, China Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China, Department of Respiratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China Department of Pathology, West China china Hospital, Sichuan University, Chengdu, Sichuan 610041, China
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Article history: Received 15 April 2013 Received in revised form 24 July 2013 Accepted 6 August 2013 Available online 3 September 2013 Keywords: Mucus hypersecretion MARCKS Diltiazem Airway
a b s t r a c t Airway mucus hypersecretion is recognized as a pathophysiological feature of airway inflammation. Ca2+ entry and myristoylated alanine-rich C kinase substrate translocation are considered as important factors in such process. To investigate the potential interaction of myristoylated alanine-rich C kinase substrate (MARCKS)-related peptide and diltiazem on acrolein-induced airway mucus hypersecretion in rats, rat model of airway mucus hypersecretion was established by inhalation of acrolein on 12 consecutive days. MARCKS-related peptide, diltiazem, saline or the combination (MARCKS-related peptide + diltiazem) was intratracheally administered respectively. The rats were received pilocarpine to stimulate mucus release before sacrifices. The expression of Mucin5ac in bronchoalveolar lavage fluid (BALF) was measured by ELISA. Intracellular Muc5ac level was detected by immunohistochemical staining and western-blot. Muc5ac mRNA in lung was analyzed by RT-PCR. Results: Instillation of MARCKS-related peptide attenuated the release of Muc5ac in BALF induced by acrolein(p b 0.05). Diltiazem alone had no effect on mucus hypersecretion induced by acrolein. However, the release of Muc5ac in BALF was further reduced when challenged with simultaneous instillation with MARCKS-related peptide and diltiazem, compared with MARCKS-related peptide alone (p b 0.05). The intracellular level of Muc5ac in lung was increased when treated with MARCKS-related peptide alone or MARCKS-related peptide plus diltiazem (p b 0.05). Nevertheless, diltiazem alone did not take effect as above. Conclusions: In the model of airway mucus hypersecretion induced by acrolein, MARCKS-related peptide attenuated mucus secretion and the inhibitory effect was enhanced by diltiazem, which may be due to a further diminution of the intracellular free calcium concentration and retention of mucin within epithelial goblet cells. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Airway mucus hypersecretion is now recognized as a key pathophysiological feature of inflammatory diseases of the airways [1,2], including asthma, cystic fibrosis and chronic obstructive pulmonary disease (COPD). Especially, COPD patients (N2/y) with chronic bronchial mucus hypersecretion are at greater risk of exacerbations [3]. In
☆ Disclosure of Conflicts of Interest: The authors certify that all our affiliations with or financial involvement in, within the past 5 years and foreseeable future, any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript have been completely disclosed. ⁎ Corresponding author. Tel.: +86 28 8542 2380; fax: +86 28 8558 2944. E-mail addresses: [email protected] (P. Chen), [email protected] (Z. Deng), [email protected] (T. Wang), [email protected] (L. Chen), [email protected] (J. Li), [email protected] (Y. Feng), [email protected] (S. Zhang), [email protected] (Y. Nin), [email protected] (D. Liu), [email protected] (Y. Chen), [email protected] (X. Ou), [email protected] (F. Wen). 1 Lead author. 2 Peng Chen and Zhiping Deng contributed equally to this work. 1567-5769/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.intimp.2013.08.001
AECOPD, previous histopathological studies have suggested that plenty of mucus released into airway acutely, which resulted in obstruction, bacteria colonization, destruction of airway walls and disordered gas exchange [4,5] and ulteriorly contributed to an accelerated rate of decline in pulmonary function and a critical risk factor for mortality [6]. The major components of mucus are mucin glycoproteins, which are constituted by Muc5ac primarily. Currently, the underlying mechanisms involved in hypersecretion remain obscure and conventional therapeutic strategies have limited efficacy in modulating airway mucus secretion. Myristoylated alanine-rich C kinase substrate (MARCKS) protein has been proved to play a key role in regulating mucin secretion [2], which serves as the point of convergence for coordinating the actions of PKC and PKG to control mucin granule release. MARCKS interacts with actin and myosin in the cytoplasm and thus tethers the granule to such cellular contractile apparatus, which mediates subsequent granule movement and exocytosis. MARCKS-related peptide, namely myristoylated N-terminal sequence (MANS), refers to the 24-amino acid fragment of N-terminal
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domain of MARCKS. MANS may competitively inhibit the attachment of MARCKS to membranes of intracellular mucin granules [7]. Upon secretagogue stimulation, MANS peptide inhibits mucin secretion in a concentration-dependent manner in polarized airway epithelial cells [8] and in vivo (mice). Nowadays, MANS has been shown to improve airway obstruction in a mouse model of asthma through the act of inhibiting methacholine (MCh)-induced mucin secretion [9]. However, it is still unknown whether MANS can attenuate mucus secretion in a rat model of mucus hypersecretion induced by acrolein. Acrolein, a low-molecular-weight aldehyde found in photochemical smog and tobacco smoke, can induce mucus hypersecretion, inflammation and airway obstruction [10,11]. Acrolein induced mucus hypersecretion is accompanied by mucous cell differentiation, which may contribute to the pathogenesis of obstructive airway diseases including COPD via inducing the over-production of Muc5ac. Extracellular Ca2+ influx and subsequent elevation of intracellular free calcium ([Ca2+]i)concentration are closely involved in mucin secretion [12,13]. The influx of extracellular Ca2+ rather than the absolute level of [Ca2+]i regulate the rapid onset and extent of exocytotic responses to HNE in airway gland cells [14,15]. Diltiazem, which can inhibit Ca2+ influx by blocking L-type calcium channel, has been verified to inhibit mucus secretion in cultured rabbit gastric mucosal cells [16]. However, whether diltiazem can inhibit airway mucus secretion, or there is any synergistic effects of MANS and diltiazem in such a rat model remains unknown. The present study is to investigate the effects of myristoylated alanine rich C kinase substrate (MARCKS)-related peptide and diltiazem on acrolein-induced airway mucus hypersecretion in rats.
Sprague–Dawley rats (200–250 g) (National Rodent Laboratory Animal Resources, Shanghai Branch, Shanghai, China) at the age of 4 weeks were used for experiments. Rats were randomly divided into ten experimental groups (n = 10 for each group): (a) control group (rats treated with saline); (b) Acrolein group (rats receiving inhalation of acrolein fog alone) (Suzhou Pharmaceutical Co, Suzhou, China); (c) Acrolein + Pilocarpine group (rats receiving inhalation of acrolein fog and intraperitoneal injection (i.p.) with pilocarpine) (Huada Pharmaceutical Co, Ltd. Chengdu, China); (d) Acrolein + Pilocarpine + RNS group (rats receiving acrolein fog and pilocarpine together with RNS (missense control RNS peptide, myristic acid– GTAPAAEGAGAEVKRASAEAKQAF synthesized by Peptide Biotechnology Co. Ltd, Shanghai, China); (e–g) Acrolein + Pilocarpine + MANS group(rats receiving acrolein fog and pilocarpine together with 1, 5 or 10 mg/kg of MANS) (identical to the first 24 amino acids of the N terminus of MARCKS, myristic acid–GAQFSKTAAKGEAAAERPGEAAVA synthesized by Peptide Biotechnology Co. Ltd, Shanghai, China); (h) Acrolein + Pilocarpine + MANS + Diltiazem groups (rats receiving acrolein fog, pilocarpine and MANS (10 mg/kg) together with 0.2 mg/kg of diltiazem) (Tanabe Seiyaku Co., Ltd, Tianjin, China.); (i) Acrolein + Pilocarpine + Diltiazem group (rats receiving acrolein fog and pilocarpine together with 0.2 mg/kg of diltiazem). Rat model of airway mucus hypersecretion was established by inhalation of 3.0 ppm acrolein fog 6 h a day for 12 days [17]. On the 13th day, 0.1 ml saline containing MANS, RNS, diltiazem or the combination was intratracheally administered respectively, which was followed by intraperitoneal injection with pilocarpine (150 mg/Kg) to stimulate mucus release 30 min before sacrifice.
2. Materials and methods
2.2. Bronchoalveolar lavage
2.1. Animals and treatment
Bronchoalveolar lavage was performed through a tracheal cannula with PBS (pH 7.4) on day 13 after pilocarpine injection as described previously [18]. This procedure was repeated two times. Cold sterile PBS (3 ml) was used to inflate the lung, and the lavage fluid was recovered
The Animal Care and Use Committee of West China Hospital of Sichuan University approved this animal study. SPF grade male
Fig. 1. Muc5ac in BALF. The release of Muc5ac in response to pilocarpine and acrolein was attenuated by pretreatment with MANS peptide in a concentration-dependent manner. Compared with MANS group, Muc5ac level was further attenuated in MANS + Diltiazem group, whereas RNS peptide or diltiazem did not affect mucin secretion. *, P b 0.05 compared with pilocarpine stimulation group. $, P b 0.05 compared with MANS group (10 mg/kg).
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with 80% of the original volume. The lavage fluid was centrifuged at 400 × g for 15 min at 4 °C. And the cell-free supernatant was kept at −70 °C until the assay. 2.3. Tissue preparation and airway histopathology After death, the chests were opened and the lungs were excised completely, which were carefully washed off the BALF and fixed by 4% paraformaldehyde subsequently. After being embedded in paraffin, tissue sections were stained with alcian blue-periodic-Schiff (AB-PAS) and hematoxylin. The area of positive staining was determined by an image analyzer.
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Burlingame, CA, USA) which was based on the biotin-avidin system and in accordance with the manufacturer's protocol. The reaction was visualized with DAB kit (Vector Laboratories Ltd, Burlingame, CA, USA). The images were analyzed with Image-Pro plus 4.5 Software (Media Cybernetics Co, USA).
2.5. Biochemical assays Total protein in BALF was evaluated using Bradford method. For mucin and Muc5ac detection, enzyme-linked lectin assay was performed as described previously [9,18].
2.4. Immunohistochemistry for Muc5ac
2.6. Reverse transcription-polymerase chain reaction
Immunohistochemistry for Muc5ac was performed in paraffinembedded tissue sections with the avidin-biotin-peroxidase technique. Antigenic site retrieval was accomplished by microwaving each slide for 15 min in 0.01 M citric acid buffer (pH 6.0). Sections were subsequently incubated with polyclonal anti-Muc5ac (1:100) (Santa Cruz Biotechnology, Inc, USA) antibodies overnight at 4 °C. For control group, sections were incubated with PBS. Antibody binding was detected with the SP kit (Vector Laboratories Ltd,
Total RNA was extracted from lung homogenates with Trizol (Invitrogen Co, USA) and reversely transcribed, and then the complementary DNA was amplified by polymerase chain reaction with the PCR kit (Takara Bio Inc, Dalian, China). Primers: forward, 5′ CAG CCTATGTGAAAGATGCC 3′ reverse 5′ GTAGAGGGAAGTGGAGTTATTGC 3′ for Muc5ac; forward 5′ TGTCACCAACTGGGACGATA 3′ reverse 5′ AGGTCTTTACGGATGTCAACG 3′ for ß-actin. The products amplified were separated by agarose gel electrophoresis and visualized by Bio-
Fig. 2. Level of Muc5ac mRNA in rat lung. The level of Muc5ac mRNA was significantly increased in rats stimulated with acrolein fog, which was not changed by the treatment with RNS, MANS or diltiazem. A: Representative result of RT-PCR for Muc5ac mRNA; B: OD ratio of Muc5ac mRNA. †, P b 0.05 compared with control.
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Rad universalhood system. Relative quantity of Muc5ac mRNA was obtained by a comparative method using ß-actin as an internal control.
2.7. Western blot analysis Tissue homogenate containing 50 mg total protein was suspended in SDS sample buffer and boiled for 5 min. Proteins were separated by SDSPAGE electrophoresis in 6% acrylamide-bisacrylamide (60:1) for 5 h [19]. The proteins were then transferred electrophoretically onto PVDF membranes which were blocked with 5% BSA for 1 h and subsequently incubated overnight with anti-rat Muc5ac (1:100 dilution) (Santa Cruz Biotechnology, Inc, USA) antibody at 4 °C. β-actin served as the constitutive control to confirm equal amount of protein loading. After being washed with TBST, the membranes were incubated with the corresponding horseradish peroxidase-linked anti-mouse antibody (Pierce Biotechnology, Inc, Rockford, USA) diluted 1:20000 in TBST for 1 h at room temperature. After being further washed with TBST, immunoreactive bands were visualized by enhanced chemiluminescence (ECL), and quantified by densitometry with the Bio-Rad Universal Hood (BioRad, Hercules, CA) and Quantity One software (Bio-Rad). All results were normalized to β-actin level in each lane. All experiments were performed two times.
2.8. Statistical analysis All data were expressed as mean ± SD. Statistical analysis was performed with SPSS12.0 software. The differences between each variable were analyzed by One-Way ANOVA. P values of b0.05 were considered significant. 3. Results 3.1. MANS and diltiazem synergistically attenuated the level of Muc5ac in BALF To determine whether MANS or diltiazem attenuated the release of Muc5ac in BALF induced by acrolein and pilocarpine, we measured the level of Muc5ac in BALF by ELISA. After inhalation of 3.0 ppm acrolein fog, the level of Muc5ac in BALF was increased correspondingly. Moreover, treated with acrolein and pilocarpine, the level of Muc5ac in BALF was augmented significantly. In the MANS (5 or 10 mg/kg) or MANS (10 mg/kg) plus diltiazem group, the increased level of Muc5ac was attenuated. Interestingly, co-treatment with MANS (10 mg/kg) and diltiazem appeared to show more obvious effect on decreasing mucin release compared with MANS (10 mg/kg) alone. Treatment with RNS or diltiazem had no effect on levels of Muc5ac in BALF (Fig. 1). These
Fig. 3. Expression of Muc5ac protein in rat lung detected by Western blot. Western blot analysis illuminated that the incremental expression of Muc5ac protein was detected in acrolein fog-stimulated rat lungs, which was remarkably attenuated by i.p pilocarpine. In the MANS or MANS plus diltiazem group, the intracellular protein level was reverted (*p b 0.05). RNS or diltiazem alone had no effect on expression of Muc5ac protein. A: Representative result of Muc5ac protein detected by Western blot; B: OD ratio of Muc5ac protein. †, P b 0.05 compared with control. *, P b 0.05 compared with acrolein fog and pilocarpine stimulation group. $, P b 0.05 compared with MANS group (10 mg/kg).
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Fig. 4. Immunohistochemistry for Muc5ac. A: Representative IHC-stained tissue sections from each group. (a) Control (b) Intratracheal injection of saline. Goblet cells filled with Muc5ac granules populated in bronchial epithelium. (c) Intratracheal injection of saline, followed by i.p. pilocarpine. Goblet cells released Muc5ac and epithelium-staining was reverted to Muc5acnegative mostly. (d) Intratracheal injection of RNS peptide, followed by i.p. pilocarpine. Epithelium-staining was similar to that in c. RNS peptide did not thwart pilocarpine-induced release of Muc5ac positive granules. (e–h) Intratracheal injection of MANS peptide at a dose of 1 mg (e), 5 mg (f), 10 mg (g) respectively or MANS (10 mg) with diltiazem (h), followed by i.p. pilocarpine. Muc5ac was repleted in goblet cells and seemed to be retained in these cells when administered with MANS or administered with MANS and diltiazem simultaneously. (i) Intratracheal injection of diltiazem, followed by i.p. pilocarpine. The interruptive effect of diltiazem against pilocarpine was feeble. Scale bars, 100 μm. Original magnification: ×400. B: OD of Muc5ac staining. †, P b 0.05 compared with control. *, P b 0.05 compared with acrolein fog and pilocarpine stimulation group. $, P b 0.05 compared with MANS group (10 mg/kg).
results suggest that MANS and diltiazem synergistically attenuate the level of Muc5ac in BALF.
3.2. MANS and diltiazem attenuated the level of Muc5ac by preventing Muc5ac from releasing into the airway To ensure whether MANS and diltiazem attenuated the level of Muc5ac in BALF due to their effect on decreasing Muc5ac production or suppressing release of Muc5ac, we analyzed Muc5ac mRNA stability by RT-PCR and measured the main intracellular expression of Muc5ac protein by Western blot. RT-PCR analysis indicated that the level of Muc5ac mRNA significantly increased in rats treated with acrolein. The increased level of Muc5ac mRNA was not affected by the administration of MANS or diltiazem (Fig. 2), which suggested that the effect on arresting the Muc5ac production was not the role that MANS or diltiazem played at least in the initial 30 min (from agent administration to sacrifice). Additionally, Western blot analysis illuminated that the incremental expression of Muc5ac protein was detected in acrolein fog-stimulated rat lungs, which was obviously attenuated by the i.p. pilocarpine. However, after MANS or MANS plus diltiazem was
administered, the intracellular protein level was reverted. The addition of RNS or diltiazem did not affect the expression of Muc5ac protein (Fig. 3). Acrolein significantly augmented Muc5ac production while pilocarpine promoted the release of Muc5ac brimmed in goblet cells into lumens. We conclude that MANS and diltiazem can synergistically attenuate the release of Muc5ac induced by pilocarpine.
3.3. MANS and diltiazem cooperatively retained Muc5ac within epithelial goblet cells Muc5ac is the predominant gel-forming mucin. Immunohistochemistry for Muc5ac was performed to evaluate effects of MANS and diltiazem on retaining Muc5ac in goblet cells. Inhalation of acrolein fog resulted in remarkable positive-staining for Muc5ac in bronchial epithelial cells compared with control group. After administration with pilocarpine, the goblet cells released mucin granules. As a result, the epithelium-staining was most Muc5ac-negative. Muc5ac was repleted in goblet cells and seemed to be prisoned in such cells after administered with MANS. In addition, MANS plus diltiazem group drew our
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Fig. 5. Histopathological examination of lung tissue section stained with AB-PAS. A: Representative lung tissue sections stained with AB-PAS from rats treated with acrolein fog. (a) Control (b) Intratracheal injection of saline. Goblet cells brimmed with PAS-positive granules. (c) Intratracheal injection of saline, followed by i.p. pilocarpine. Mucin granules were set free. (d) Intratracheal injection of RNS peptide, followed by i.p. pilocarpine. Epithelium-staining was semblable to that in c. (e–h) Intratracheal injection of MANS peptide at a dose of 1 mg (e), 5 mg (f), 10 mg (g) respectively or MANS (10 mg) with diltiazem (h), followed by i.p. pilocarpine. Mucin was retained in goblet cells after MANS treatment, and was further blocked after challenged by MANS and diltiazem synchronously. (i) Intratracheal injection of diltiazem, followed by i.p. pilocarpine. The variation was not prominent enough to be cared. Scale bars, 200 μm. Original magnification: ×200 × 600. B: Relative percentage of positive ABPAS-stained area. †, P b 0.05 compared with control. *, P b 0.05 compared with acrolein fog and pilocarpine stimulation group. $, P b 0.05 compared with MANS group (10 mg/kg).
more attention for its more remarkable effect. Muc5ac level was not affected by the treatment with RNS or diltiazem alone (Fig. 4).
3.4. MANS and diltiazem blocked the release of mucous granules in coordination Though Muc5ac is regarded as the principal gel-forming mucin, to assess the secretion of the entire gel-forming mucin in rat's airway, AB/PAS staining of goblet cell for mucous granules was performed additionally. In control group AB-PAS positive cells in airway epithelia were hardly detected, which were highly visualized in acrolein group (Fig. 5A). As goblet cells released mucin granules following treatment with pilocarpine, epithelium became major PAS-negative. Pilocarpine induced release of PAS positive granules was meaningfully restrained in rats treated with MANS. Furthermore, the influence was extended in rats administered with MANS and diltiazem synchronously. No meaningful variation was admitted in rats treated with RNS or diltiazem (Fig. 5). These results implied that MANS and diltiazem suppressed the release of entire mucous granules including Muc5ac in coordination.
4. Discussion In the present study, intratracheal instillation of MARCKS-related peptide lessened the release of Muc5ac in rats stimulated with acrolein and pilocarpine. Diltiazem alone had no effect on mucin release as above. However, the release of Muc5ac in BALF was thwarted to a greater extent via simultaneous instillation of MARCKS-related peptide and diltiazem, compared with MARCKS-related peptide alone. Moreover, the intracellular protein level of Muc5ac in lung visually increased when treated with MARCKS-related peptide or MARCKS-related peptide together with diltiazem though MUC5AC staining was not affected by diltiazem alone. To clarify, in the section of rats' lung tissues preparation, although we had carefully washed such samples off the BALF and fixed them by 4% paraformaldehyde subsequently in order to get rid of the confusion of Muc5ac released in the rat's airway/BALF as far as possible, we still can not guarantee there was no Mac5ac in airway adhered to the lung tissue sample at all during our western test. However, each group challenged with western blot stood at the same baseline, and as the final figure showed, there were obvious differences between each group. At
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least, such values represented the major change of intracellular level of Muc5ac produced by goblet cells. To make it clear, we also measured the Mac5ac level in BALF meanwhile. As the level of Mac5ac mainly confined in the goblet cell was decreasing, the level of Mac5ac released to the airway/BALF was increasing, vice versa, which coincided with our hypothesis. For the last two years, human neutrophil elastase, methacholine aerosol or LPS has been widely used to establish mucus hypersecretion and airway inflammation models. All of these previous studies gradually reveals that myristoylated alanine-rich C-kinase substrate (MARCKS) -related protein, as peptides analogous to the amino (N)-terminus of MARCKS, namely myristoylated N-terminal sequence (MANS), plays an important role in regulating the adhesion, migration, degranulation of inflammatory cell, which eventually leads to the decrease of mucus hypersecretion by airway epithelium and improvement of airway obstruction [1,9]. Part of its further mechanisms is to competitively inhibit the attachment of MARCKS to membranes of intracellular mucin granules [7]. Upon secretagogue stimulation, MANS peptide inhibits mucin secretion in a concentration-dependent manner in polarized airway epithelial cells [8] and in vivo (mice) [9]. Recently, some studies also indicate that there may be some interaction between MARCKS, calpain and HSP70. The inhibition of airway hypersecretion even improves the pulmonary function in response to cumulative aerosol stimulation with serotonin in vivo [9]. However, it is still unknown whether MANS can attenuate mucus secretion in a rat model of mucus hypersecretion induced by acrolein. What's more, now that blocking the Ca2+ influx may inhibit the secretion to some extent, will it helpful to assist the MAN's work? In our present study, we first discovered that MANS also attenuated mucus hypersecretion induced by acrolein inhalation and some interaction between MANS and diltiazem may be involved with such process. The effect of MARCKS-related peptide on mucin secretion and the basic biochemical study about Ca2+ influx have been demonstrated in previous researches especially in acute secretion disorder models involved in asthma and AECOPD [7]. Here we try to discuss the potential mechanisms and the interaction between MANS and diltiazem. The system may be operated by decreasing cytosolic Ca2+ concentration and inhibiting protein kinase C (PKC) activation [20]. MANS possibly down-regulate [Ca2+]i via at least two mechanisms. On one hand, MANS was found to bind calmodulin, which competed with MARCKS [21,22]. On the other hand, MANS appeared to reverse the translocation of MARCKS from plasma membrane to cytosol by competitively occupying the granule membrane binding sites. As a result, MARCKS was confined within the periphery of epicyte and interrupted PIP2 from being hydrolyzed by PLC, which correspondingly disturbed the production of IP3 [23–25]. Coinstantaneous instillation of MANS and diltiazem seemed to inhibit local Ca2+ release from internal stores by decreasing IP3 level [26,27], and meanwhile block extracellular Ca2+ entry via blocking L-type calcium channel. To sum up, Ca2+ entry is a prerequisite for many kinds of airway secretion [28,29]. Inhibit the Ca2+ entry, inhibit the secretion. In this study diltiazem itself had no effect on mucin release. Diltiazem, which can block L-type calcium channel, has been verified to affect mucus secretion by checking extracellular Ca2+ entry [16]. However, Ca2+ influx not only depended on L-type calcium channels, but also voltage-sensitive Ca2+ channels or other potential Ca2+ entry gates, and the subsequent intracellular Ca2+ mobilization as well led to a potentiation of mucus secretion [30]. Maybe that was why cytosolic Ca2+ concentration was not affected by blocking L-type calcium channels singly and led us to find a synergistic reagent to help diltiazem with a further decline of intracellular Ca2+ concentration. As expected, MANS seems to be the one. However, we need further experiments to identify the accurate level of Ca2+ influx inhibited by MARCKS plus diltiazem. In summary, the results demonstrated that in rat model of airway mucus hypersecretion induced by acrolein inhalation, MARCKS-
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related peptide attenuated mucus secretion and the inhibitory effect was enhanced by diltiazem. These findings may lay the foundation for the development of a new therapeutic approach against the acute mucus-secreting disorder involved in asthma and AECOPD.
Acknowledgment This study was supported by grant 30971327 from the National Natural Science Foundation of China.
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
The potential interaction of MARCKS-related peptide and diltiazem on acrolin-induced airway mucus hypersecretion in rats☆ Peng Chen a,b,1,2, Zhiping Deng b,1,2, Tao Wang b, Lei Chen b, Jiqiong Li b, Yulin Feng b, Shangfu Zhang c, Yunyie Nin b, Daishun Liu b, Yajuan Chen b, Xuemei Ou b, Fuqiang Wen b,⁎ a b c
Department of Respiratory Medicine, The Third People's Hospital of Chengdu, Chengdu, Sichuan 610031, China Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China, Department of Respiratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China Department of Pathology, West China china Hospital, Sichuan University, Chengdu, Sichuan 610041, China
a r t i c l e
i n f o
Article history: Received 15 April 2013 Received in revised form 24 July 2013 Accepted 6 August 2013 Available online 3 September 2013 Keywords: Mucus hypersecretion MARCKS Diltiazem Airway
a b s t r a c t Airway mucus hypersecretion is recognized as a pathophysiological feature of airway inflammation. Ca2+ entry and myristoylated alanine-rich C kinase substrate translocation are considered as important factors in such process. To investigate the potential interaction of myristoylated alanine-rich C kinase substrate (MARCKS)-related peptide and diltiazem on acrolein-induced airway mucus hypersecretion in rats, rat model of airway mucus hypersecretion was established by inhalation of acrolein on 12 consecutive days. MARCKS-related peptide, diltiazem, saline or the combination (MARCKS-related peptide + diltiazem) was intratracheally administered respectively. The rats were received pilocarpine to stimulate mucus release before sacrifices. The expression of Mucin5ac in bronchoalveolar lavage fluid (BALF) was measured by ELISA. Intracellular Muc5ac level was detected by immunohistochemical staining and western-blot. Muc5ac mRNA in lung was analyzed by RT-PCR. Results: Instillation of MARCKS-related peptide attenuated the release of Muc5ac in BALF induced by acrolein(p b 0.05). Diltiazem alone had no effect on mucus hypersecretion induced by acrolein. However, the release of Muc5ac in BALF was further reduced when challenged with simultaneous instillation with MARCKS-related peptide and diltiazem, compared with MARCKS-related peptide alone (p b 0.05). The intracellular level of Muc5ac in lung was increased when treated with MARCKS-related peptide alone or MARCKS-related peptide plus diltiazem (p b 0.05). Nevertheless, diltiazem alone did not take effect as above. Conclusions: In the model of airway mucus hypersecretion induced by acrolein, MARCKS-related peptide attenuated mucus secretion and the inhibitory effect was enhanced by diltiazem, which may be due to a further diminution of the intracellular free calcium concentration and retention of mucin within epithelial goblet cells. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Airway mucus hypersecretion is now recognized as a key pathophysiological feature of inflammatory diseases of the airways [1,2], including asthma, cystic fibrosis and chronic obstructive pulmonary disease (COPD). Especially, COPD patients (N2/y) with chronic bronchial mucus hypersecretion are at greater risk of exacerbations [3]. In
☆ Disclosure of Conflicts of Interest: The authors certify that all our affiliations with or financial involvement in, within the past 5 years and foreseeable future, any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript have been completely disclosed. ⁎ Corresponding author. Tel.: +86 28 8542 2380; fax: +86 28 8558 2944. E-mail addresses: [email protected] (P. Chen), [email protected] (Z. Deng), [email protected] (T. Wang), [email protected] (L. Chen), [email protected] (J. Li), [email protected] (Y. Feng), [email protected] (S. Zhang), [email protected] (Y. Nin), [email protected] (D. Liu), [email protected] (Y. Chen), [email protected] (X. Ou), [email protected] (F. Wen). 1 Lead author. 2 Peng Chen and Zhiping Deng contributed equally to this work. 1567-5769/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.intimp.2013.08.001
AECOPD, previous histopathological studies have suggested that plenty of mucus released into airway acutely, which resulted in obstruction, bacteria colonization, destruction of airway walls and disordered gas exchange [4,5] and ulteriorly contributed to an accelerated rate of decline in pulmonary function and a critical risk factor for mortality [6]. The major components of mucus are mucin glycoproteins, which are constituted by Muc5ac primarily. Currently, the underlying mechanisms involved in hypersecretion remain obscure and conventional therapeutic strategies have limited efficacy in modulating airway mucus secretion. Myristoylated alanine-rich C kinase substrate (MARCKS) protein has been proved to play a key role in regulating mucin secretion [2], which serves as the point of convergence for coordinating the actions of PKC and PKG to control mucin granule release. MARCKS interacts with actin and myosin in the cytoplasm and thus tethers the granule to such cellular contractile apparatus, which mediates subsequent granule movement and exocytosis. MARCKS-related peptide, namely myristoylated N-terminal sequence (MANS), refers to the 24-amino acid fragment of N-terminal
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domain of MARCKS. MANS may competitively inhibit the attachment of MARCKS to membranes of intracellular mucin granules [7]. Upon secretagogue stimulation, MANS peptide inhibits mucin secretion in a concentration-dependent manner in polarized airway epithelial cells [8] and in vivo (mice). Nowadays, MANS has been shown to improve airway obstruction in a mouse model of asthma through the act of inhibiting methacholine (MCh)-induced mucin secretion [9]. However, it is still unknown whether MANS can attenuate mucus secretion in a rat model of mucus hypersecretion induced by acrolein. Acrolein, a low-molecular-weight aldehyde found in photochemical smog and tobacco smoke, can induce mucus hypersecretion, inflammation and airway obstruction [10,11]. Acrolein induced mucus hypersecretion is accompanied by mucous cell differentiation, which may contribute to the pathogenesis of obstructive airway diseases including COPD via inducing the over-production of Muc5ac. Extracellular Ca2+ influx and subsequent elevation of intracellular free calcium ([Ca2+]i)concentration are closely involved in mucin secretion [12,13]. The influx of extracellular Ca2+ rather than the absolute level of [Ca2+]i regulate the rapid onset and extent of exocytotic responses to HNE in airway gland cells [14,15]. Diltiazem, which can inhibit Ca2+ influx by blocking L-type calcium channel, has been verified to inhibit mucus secretion in cultured rabbit gastric mucosal cells [16]. However, whether diltiazem can inhibit airway mucus secretion, or there is any synergistic effects of MANS and diltiazem in such a rat model remains unknown. The present study is to investigate the effects of myristoylated alanine rich C kinase substrate (MARCKS)-related peptide and diltiazem on acrolein-induced airway mucus hypersecretion in rats.
Sprague–Dawley rats (200–250 g) (National Rodent Laboratory Animal Resources, Shanghai Branch, Shanghai, China) at the age of 4 weeks were used for experiments. Rats were randomly divided into ten experimental groups (n = 10 for each group): (a) control group (rats treated with saline); (b) Acrolein group (rats receiving inhalation of acrolein fog alone) (Suzhou Pharmaceutical Co, Suzhou, China); (c) Acrolein + Pilocarpine group (rats receiving inhalation of acrolein fog and intraperitoneal injection (i.p.) with pilocarpine) (Huada Pharmaceutical Co, Ltd. Chengdu, China); (d) Acrolein + Pilocarpine + RNS group (rats receiving acrolein fog and pilocarpine together with RNS (missense control RNS peptide, myristic acid– GTAPAAEGAGAEVKRASAEAKQAF synthesized by Peptide Biotechnology Co. Ltd, Shanghai, China); (e–g) Acrolein + Pilocarpine + MANS group(rats receiving acrolein fog and pilocarpine together with 1, 5 or 10 mg/kg of MANS) (identical to the first 24 amino acids of the N terminus of MARCKS, myristic acid–GAQFSKTAAKGEAAAERPGEAAVA synthesized by Peptide Biotechnology Co. Ltd, Shanghai, China); (h) Acrolein + Pilocarpine + MANS + Diltiazem groups (rats receiving acrolein fog, pilocarpine and MANS (10 mg/kg) together with 0.2 mg/kg of diltiazem) (Tanabe Seiyaku Co., Ltd, Tianjin, China.); (i) Acrolein + Pilocarpine + Diltiazem group (rats receiving acrolein fog and pilocarpine together with 0.2 mg/kg of diltiazem). Rat model of airway mucus hypersecretion was established by inhalation of 3.0 ppm acrolein fog 6 h a day for 12 days [17]. On the 13th day, 0.1 ml saline containing MANS, RNS, diltiazem or the combination was intratracheally administered respectively, which was followed by intraperitoneal injection with pilocarpine (150 mg/Kg) to stimulate mucus release 30 min before sacrifice.
2. Materials and methods
2.2. Bronchoalveolar lavage
2.1. Animals and treatment
Bronchoalveolar lavage was performed through a tracheal cannula with PBS (pH 7.4) on day 13 after pilocarpine injection as described previously [18]. This procedure was repeated two times. Cold sterile PBS (3 ml) was used to inflate the lung, and the lavage fluid was recovered
The Animal Care and Use Committee of West China Hospital of Sichuan University approved this animal study. SPF grade male
Fig. 1. Muc5ac in BALF. The release of Muc5ac in response to pilocarpine and acrolein was attenuated by pretreatment with MANS peptide in a concentration-dependent manner. Compared with MANS group, Muc5ac level was further attenuated in MANS + Diltiazem group, whereas RNS peptide or diltiazem did not affect mucin secretion. *, P b 0.05 compared with pilocarpine stimulation group. $, P b 0.05 compared with MANS group (10 mg/kg).
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with 80% of the original volume. The lavage fluid was centrifuged at 400 × g for 15 min at 4 °C. And the cell-free supernatant was kept at −70 °C until the assay. 2.3. Tissue preparation and airway histopathology After death, the chests were opened and the lungs were excised completely, which were carefully washed off the BALF and fixed by 4% paraformaldehyde subsequently. After being embedded in paraffin, tissue sections were stained with alcian blue-periodic-Schiff (AB-PAS) and hematoxylin. The area of positive staining was determined by an image analyzer.
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Burlingame, CA, USA) which was based on the biotin-avidin system and in accordance with the manufacturer's protocol. The reaction was visualized with DAB kit (Vector Laboratories Ltd, Burlingame, CA, USA). The images were analyzed with Image-Pro plus 4.5 Software (Media Cybernetics Co, USA).
2.5. Biochemical assays Total protein in BALF was evaluated using Bradford method. For mucin and Muc5ac detection, enzyme-linked lectin assay was performed as described previously [9,18].
2.4. Immunohistochemistry for Muc5ac
2.6. Reverse transcription-polymerase chain reaction
Immunohistochemistry for Muc5ac was performed in paraffinembedded tissue sections with the avidin-biotin-peroxidase technique. Antigenic site retrieval was accomplished by microwaving each slide for 15 min in 0.01 M citric acid buffer (pH 6.0). Sections were subsequently incubated with polyclonal anti-Muc5ac (1:100) (Santa Cruz Biotechnology, Inc, USA) antibodies overnight at 4 °C. For control group, sections were incubated with PBS. Antibody binding was detected with the SP kit (Vector Laboratories Ltd,
Total RNA was extracted from lung homogenates with Trizol (Invitrogen Co, USA) and reversely transcribed, and then the complementary DNA was amplified by polymerase chain reaction with the PCR kit (Takara Bio Inc, Dalian, China). Primers: forward, 5′ CAG CCTATGTGAAAGATGCC 3′ reverse 5′ GTAGAGGGAAGTGGAGTTATTGC 3′ for Muc5ac; forward 5′ TGTCACCAACTGGGACGATA 3′ reverse 5′ AGGTCTTTACGGATGTCAACG 3′ for ß-actin. The products amplified were separated by agarose gel electrophoresis and visualized by Bio-
Fig. 2. Level of Muc5ac mRNA in rat lung. The level of Muc5ac mRNA was significantly increased in rats stimulated with acrolein fog, which was not changed by the treatment with RNS, MANS or diltiazem. A: Representative result of RT-PCR for Muc5ac mRNA; B: OD ratio of Muc5ac mRNA. †, P b 0.05 compared with control.
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Rad universalhood system. Relative quantity of Muc5ac mRNA was obtained by a comparative method using ß-actin as an internal control.
2.7. Western blot analysis Tissue homogenate containing 50 mg total protein was suspended in SDS sample buffer and boiled for 5 min. Proteins were separated by SDSPAGE electrophoresis in 6% acrylamide-bisacrylamide (60:1) for 5 h [19]. The proteins were then transferred electrophoretically onto PVDF membranes which were blocked with 5% BSA for 1 h and subsequently incubated overnight with anti-rat Muc5ac (1:100 dilution) (Santa Cruz Biotechnology, Inc, USA) antibody at 4 °C. β-actin served as the constitutive control to confirm equal amount of protein loading. After being washed with TBST, the membranes were incubated with the corresponding horseradish peroxidase-linked anti-mouse antibody (Pierce Biotechnology, Inc, Rockford, USA) diluted 1:20000 in TBST for 1 h at room temperature. After being further washed with TBST, immunoreactive bands were visualized by enhanced chemiluminescence (ECL), and quantified by densitometry with the Bio-Rad Universal Hood (BioRad, Hercules, CA) and Quantity One software (Bio-Rad). All results were normalized to β-actin level in each lane. All experiments were performed two times.
2.8. Statistical analysis All data were expressed as mean ± SD. Statistical analysis was performed with SPSS12.0 software. The differences between each variable were analyzed by One-Way ANOVA. P values of b0.05 were considered significant. 3. Results 3.1. MANS and diltiazem synergistically attenuated the level of Muc5ac in BALF To determine whether MANS or diltiazem attenuated the release of Muc5ac in BALF induced by acrolein and pilocarpine, we measured the level of Muc5ac in BALF by ELISA. After inhalation of 3.0 ppm acrolein fog, the level of Muc5ac in BALF was increased correspondingly. Moreover, treated with acrolein and pilocarpine, the level of Muc5ac in BALF was augmented significantly. In the MANS (5 or 10 mg/kg) or MANS (10 mg/kg) plus diltiazem group, the increased level of Muc5ac was attenuated. Interestingly, co-treatment with MANS (10 mg/kg) and diltiazem appeared to show more obvious effect on decreasing mucin release compared with MANS (10 mg/kg) alone. Treatment with RNS or diltiazem had no effect on levels of Muc5ac in BALF (Fig. 1). These
Fig. 3. Expression of Muc5ac protein in rat lung detected by Western blot. Western blot analysis illuminated that the incremental expression of Muc5ac protein was detected in acrolein fog-stimulated rat lungs, which was remarkably attenuated by i.p pilocarpine. In the MANS or MANS plus diltiazem group, the intracellular protein level was reverted (*p b 0.05). RNS or diltiazem alone had no effect on expression of Muc5ac protein. A: Representative result of Muc5ac protein detected by Western blot; B: OD ratio of Muc5ac protein. †, P b 0.05 compared with control. *, P b 0.05 compared with acrolein fog and pilocarpine stimulation group. $, P b 0.05 compared with MANS group (10 mg/kg).
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Fig. 4. Immunohistochemistry for Muc5ac. A: Representative IHC-stained tissue sections from each group. (a) Control (b) Intratracheal injection of saline. Goblet cells filled with Muc5ac granules populated in bronchial epithelium. (c) Intratracheal injection of saline, followed by i.p. pilocarpine. Goblet cells released Muc5ac and epithelium-staining was reverted to Muc5acnegative mostly. (d) Intratracheal injection of RNS peptide, followed by i.p. pilocarpine. Epithelium-staining was similar to that in c. RNS peptide did not thwart pilocarpine-induced release of Muc5ac positive granules. (e–h) Intratracheal injection of MANS peptide at a dose of 1 mg (e), 5 mg (f), 10 mg (g) respectively or MANS (10 mg) with diltiazem (h), followed by i.p. pilocarpine. Muc5ac was repleted in goblet cells and seemed to be retained in these cells when administered with MANS or administered with MANS and diltiazem simultaneously. (i) Intratracheal injection of diltiazem, followed by i.p. pilocarpine. The interruptive effect of diltiazem against pilocarpine was feeble. Scale bars, 100 μm. Original magnification: ×400. B: OD of Muc5ac staining. †, P b 0.05 compared with control. *, P b 0.05 compared with acrolein fog and pilocarpine stimulation group. $, P b 0.05 compared with MANS group (10 mg/kg).
results suggest that MANS and diltiazem synergistically attenuate the level of Muc5ac in BALF.
3.2. MANS and diltiazem attenuated the level of Muc5ac by preventing Muc5ac from releasing into the airway To ensure whether MANS and diltiazem attenuated the level of Muc5ac in BALF due to their effect on decreasing Muc5ac production or suppressing release of Muc5ac, we analyzed Muc5ac mRNA stability by RT-PCR and measured the main intracellular expression of Muc5ac protein by Western blot. RT-PCR analysis indicated that the level of Muc5ac mRNA significantly increased in rats treated with acrolein. The increased level of Muc5ac mRNA was not affected by the administration of MANS or diltiazem (Fig. 2), which suggested that the effect on arresting the Muc5ac production was not the role that MANS or diltiazem played at least in the initial 30 min (from agent administration to sacrifice). Additionally, Western blot analysis illuminated that the incremental expression of Muc5ac protein was detected in acrolein fog-stimulated rat lungs, which was obviously attenuated by the i.p. pilocarpine. However, after MANS or MANS plus diltiazem was
administered, the intracellular protein level was reverted. The addition of RNS or diltiazem did not affect the expression of Muc5ac protein (Fig. 3). Acrolein significantly augmented Muc5ac production while pilocarpine promoted the release of Muc5ac brimmed in goblet cells into lumens. We conclude that MANS and diltiazem can synergistically attenuate the release of Muc5ac induced by pilocarpine.
3.3. MANS and diltiazem cooperatively retained Muc5ac within epithelial goblet cells Muc5ac is the predominant gel-forming mucin. Immunohistochemistry for Muc5ac was performed to evaluate effects of MANS and diltiazem on retaining Muc5ac in goblet cells. Inhalation of acrolein fog resulted in remarkable positive-staining for Muc5ac in bronchial epithelial cells compared with control group. After administration with pilocarpine, the goblet cells released mucin granules. As a result, the epithelium-staining was most Muc5ac-negative. Muc5ac was repleted in goblet cells and seemed to be prisoned in such cells after administered with MANS. In addition, MANS plus diltiazem group drew our
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Fig. 5. Histopathological examination of lung tissue section stained with AB-PAS. A: Representative lung tissue sections stained with AB-PAS from rats treated with acrolein fog. (a) Control (b) Intratracheal injection of saline. Goblet cells brimmed with PAS-positive granules. (c) Intratracheal injection of saline, followed by i.p. pilocarpine. Mucin granules were set free. (d) Intratracheal injection of RNS peptide, followed by i.p. pilocarpine. Epithelium-staining was semblable to that in c. (e–h) Intratracheal injection of MANS peptide at a dose of 1 mg (e), 5 mg (f), 10 mg (g) respectively or MANS (10 mg) with diltiazem (h), followed by i.p. pilocarpine. Mucin was retained in goblet cells after MANS treatment, and was further blocked after challenged by MANS and diltiazem synchronously. (i) Intratracheal injection of diltiazem, followed by i.p. pilocarpine. The variation was not prominent enough to be cared. Scale bars, 200 μm. Original magnification: ×200 × 600. B: Relative percentage of positive ABPAS-stained area. †, P b 0.05 compared with control. *, P b 0.05 compared with acrolein fog and pilocarpine stimulation group. $, P b 0.05 compared with MANS group (10 mg/kg).
more attention for its more remarkable effect. Muc5ac level was not affected by the treatment with RNS or diltiazem alone (Fig. 4).
3.4. MANS and diltiazem blocked the release of mucous granules in coordination Though Muc5ac is regarded as the principal gel-forming mucin, to assess the secretion of the entire gel-forming mucin in rat's airway, AB/PAS staining of goblet cell for mucous granules was performed additionally. In control group AB-PAS positive cells in airway epithelia were hardly detected, which were highly visualized in acrolein group (Fig. 5A). As goblet cells released mucin granules following treatment with pilocarpine, epithelium became major PAS-negative. Pilocarpine induced release of PAS positive granules was meaningfully restrained in rats treated with MANS. Furthermore, the influence was extended in rats administered with MANS and diltiazem synchronously. No meaningful variation was admitted in rats treated with RNS or diltiazem (Fig. 5). These results implied that MANS and diltiazem suppressed the release of entire mucous granules including Muc5ac in coordination.
4. Discussion In the present study, intratracheal instillation of MARCKS-related peptide lessened the release of Muc5ac in rats stimulated with acrolein and pilocarpine. Diltiazem alone had no effect on mucin release as above. However, the release of Muc5ac in BALF was thwarted to a greater extent via simultaneous instillation of MARCKS-related peptide and diltiazem, compared with MARCKS-related peptide alone. Moreover, the intracellular protein level of Muc5ac in lung visually increased when treated with MARCKS-related peptide or MARCKS-related peptide together with diltiazem though MUC5AC staining was not affected by diltiazem alone. To clarify, in the section of rats' lung tissues preparation, although we had carefully washed such samples off the BALF and fixed them by 4% paraformaldehyde subsequently in order to get rid of the confusion of Muc5ac released in the rat's airway/BALF as far as possible, we still can not guarantee there was no Mac5ac in airway adhered to the lung tissue sample at all during our western test. However, each group challenged with western blot stood at the same baseline, and as the final figure showed, there were obvious differences between each group. At
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least, such values represented the major change of intracellular level of Muc5ac produced by goblet cells. To make it clear, we also measured the Mac5ac level in BALF meanwhile. As the level of Mac5ac mainly confined in the goblet cell was decreasing, the level of Mac5ac released to the airway/BALF was increasing, vice versa, which coincided with our hypothesis. For the last two years, human neutrophil elastase, methacholine aerosol or LPS has been widely used to establish mucus hypersecretion and airway inflammation models. All of these previous studies gradually reveals that myristoylated alanine-rich C-kinase substrate (MARCKS) -related protein, as peptides analogous to the amino (N)-terminus of MARCKS, namely myristoylated N-terminal sequence (MANS), plays an important role in regulating the adhesion, migration, degranulation of inflammatory cell, which eventually leads to the decrease of mucus hypersecretion by airway epithelium and improvement of airway obstruction [1,9]. Part of its further mechanisms is to competitively inhibit the attachment of MARCKS to membranes of intracellular mucin granules [7]. Upon secretagogue stimulation, MANS peptide inhibits mucin secretion in a concentration-dependent manner in polarized airway epithelial cells [8] and in vivo (mice) [9]. Recently, some studies also indicate that there may be some interaction between MARCKS, calpain and HSP70. The inhibition of airway hypersecretion even improves the pulmonary function in response to cumulative aerosol stimulation with serotonin in vivo [9]. However, it is still unknown whether MANS can attenuate mucus secretion in a rat model of mucus hypersecretion induced by acrolein. What's more, now that blocking the Ca2+ influx may inhibit the secretion to some extent, will it helpful to assist the MAN's work? In our present study, we first discovered that MANS also attenuated mucus hypersecretion induced by acrolein inhalation and some interaction between MANS and diltiazem may be involved with such process. The effect of MARCKS-related peptide on mucin secretion and the basic biochemical study about Ca2+ influx have been demonstrated in previous researches especially in acute secretion disorder models involved in asthma and AECOPD [7]. Here we try to discuss the potential mechanisms and the interaction between MANS and diltiazem. The system may be operated by decreasing cytosolic Ca2+ concentration and inhibiting protein kinase C (PKC) activation [20]. MANS possibly down-regulate [Ca2+]i via at least two mechanisms. On one hand, MANS was found to bind calmodulin, which competed with MARCKS [21,22]. On the other hand, MANS appeared to reverse the translocation of MARCKS from plasma membrane to cytosol by competitively occupying the granule membrane binding sites. As a result, MARCKS was confined within the periphery of epicyte and interrupted PIP2 from being hydrolyzed by PLC, which correspondingly disturbed the production of IP3 [23–25]. Coinstantaneous instillation of MANS and diltiazem seemed to inhibit local Ca2+ release from internal stores by decreasing IP3 level [26,27], and meanwhile block extracellular Ca2+ entry via blocking L-type calcium channel. To sum up, Ca2+ entry is a prerequisite for many kinds of airway secretion [28,29]. Inhibit the Ca2+ entry, inhibit the secretion. In this study diltiazem itself had no effect on mucin release. Diltiazem, which can block L-type calcium channel, has been verified to affect mucus secretion by checking extracellular Ca2+ entry [16]. However, Ca2+ influx not only depended on L-type calcium channels, but also voltage-sensitive Ca2+ channels or other potential Ca2+ entry gates, and the subsequent intracellular Ca2+ mobilization as well led to a potentiation of mucus secretion [30]. Maybe that was why cytosolic Ca2+ concentration was not affected by blocking L-type calcium channels singly and led us to find a synergistic reagent to help diltiazem with a further decline of intracellular Ca2+ concentration. As expected, MANS seems to be the one. However, we need further experiments to identify the accurate level of Ca2+ influx inhibited by MARCKS plus diltiazem. In summary, the results demonstrated that in rat model of airway mucus hypersecretion induced by acrolein inhalation, MARCKS-
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related peptide attenuated mucus secretion and the inhibitory effect was enhanced by diltiazem. These findings may lay the foundation for the development of a new therapeutic approach against the acute mucus-secreting disorder involved in asthma and AECOPD.
Acknowledgment This study was supported by grant 30971327 from the National Natural Science Foundation of China.
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