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Differential inhibitory effects of CysLT(1) receptor antagonists on P2Y(6) receptor-mediated signaling and ion transport in human bronchial epithelia.

Lau WK, Chow AW, Au SC, Ko WH - PLoS ONE (2011)

Bottom Line: CysLTs exert their biological effects via specific G-protein-coupled receptors.Pranlukast inhibited the UDP-evoked I(SC) potentiated by an Epac activator, 8-(4-Chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-CPT-2'-O-Me-cAMP), while montelukast and zafirlukast had no such effect.In summary, our data strongly suggest for the first time that in human airway epithelia, the three specific CysLT(1) receptor antagonists exert differential inhibitory effects on P2Y(6) receptor-coupled Ca(2+) signaling pathways and the potentiating effect on I(SC) mediated by cAMP and Epac, leading to the modulation of ion transport activities across the epithelia.

View Article: PubMed Central - PubMed

Affiliation: School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China.

ABSTRACT

Background: Cysteinyl leukotriene (CysLT) is one of the proinflammatory mediators released by the bronchi during inflammation. CysLTs exert their biological effects via specific G-protein-coupled receptors. CysLT(1) receptor antagonists are available for clinical use for the treatment of asthma. Recently, crosstalk between CysLT(1) and P2Y(6) receptors has been delineated. P2Y receptors are expressed in apical and/or basolateral membranes of virtually all polarized epithelia to control the transport of fluid and electrolytes. Previous research suggests that CysLT(1) receptor antagonists inhibit the effects of nucleotides acting at P2Y receptors. However, the detailed molecular mechanism underlying the inhibition remains unresolved.

Methodology/principal findings: In this study, western blot analysis confirmed that both CysLT(1) and P2Y(6) receptors were expressed in the human bronchial epithelial cell line 16HBE14o-. All three CysLT(1) antagonists inhibited the uridine diphosphate (UDP)-evoked I(SC), but only montelukast inhibited the UDP-evoked [Ca(2+)](i) increase. In the presence of forskolin or 8-bromoadenosine 3'5' cyclic monophosphate (8-Br-cAMP), the UDP-induced I(SC) was potentiated but was reduced by pranlukast and zafirlukast but not montelukast. Pranlukast inhibited the UDP-evoked I(SC) potentiated by an Epac activator, 8-(4-Chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-CPT-2'-O-Me-cAMP), while montelukast and zafirlukast had no such effect. Pranlukast inhibited the real-time increase in cAMP changes activated by 8-CPT-2'-O-Me-cAMP as monitored by fluorescence resonance energy transfer imaging. Zafirlukast inhibited the UDP-induced I(SC) potentiated by N(6)-Phenyladenosine-3',5'-cyclic monophosphorothioate, Sp-isomer (Sp-6-Phe-cAMP; a PKA activator) and UDP-activated PKA activity.

Conclusions/significance: In summary, our data strongly suggest for the first time that in human airway epithelia, the three specific CysLT(1) receptor antagonists exert differential inhibitory effects on P2Y(6) receptor-coupled Ca(2+) signaling pathways and the potentiating effect on I(SC) mediated by cAMP and Epac, leading to the modulation of ion transport activities across the epithelia.

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Western blotting analysis showing the protein expression of CysLT1 and P2Y6 receptors in 16HBE14o- cells.The expression of CysLT1 (44 kDa) and P2Y6 (41 kDa) receptors was demonstrated (lane 1), and their positions on the blot closely matched their calculated molecular masses of 39 kDa and 36 kDa, respectively. Detection of these protein bands appeared specific, as they were mostly blocked by prior reabsorption of the antibodies with their respective control antigen for 2 h at 4°C (lane 2).
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pone-0022363-g001: Western blotting analysis showing the protein expression of CysLT1 and P2Y6 receptors in 16HBE14o- cells.The expression of CysLT1 (44 kDa) and P2Y6 (41 kDa) receptors was demonstrated (lane 1), and their positions on the blot closely matched their calculated molecular masses of 39 kDa and 36 kDa, respectively. Detection of these protein bands appeared specific, as they were mostly blocked by prior reabsorption of the antibodies with their respective control antigen for 2 h at 4°C (lane 2).

Mentions: To examine the presence of CysLT1 and P2Y6 receptors in 16HBE14o- cells, western blot analysis was conducted. The protein expression of CysLT1 and P2Y6 receptors in 16HBE14o- cells was detected as shown in Fig. 1. The CysLT1 receptor polyclonal antibody identified an intense 44-kDa band in whole cell lysates of 16HBE14o- cell monolayers (Fig. 1A, left lane). The specificity of the band was confirmed by the complete abolishment of the immunoreactive signal in 16HBE4o- cells by the CysLT1 receptor polyclonal antibody that had been preadsorbed with specific blocking peptides. The blocking peptides correspond to amino acid residues 318–337 of the human CysLT1 receptor (Fig. 1A, right lane). On the other hand, the P2Y6 receptor was identified as an intense 41-kDa band (Fig. 1B, left lane). The specificity of the band was confirmed by the complete abolishment of the immunoreactive signal by the P2Y6 receptor antibody preadsorbed with specific blocking peptides. The blocking peptides correspond to amino acid residues 322–343 of the human P2Y6 receptor (Fig. 1B, right lane). Western blot analysis demonstrated that 16HBE14o- cells expressed CysLT1 receptors and confirmed our previous finding on P2Y6 receptor expression in this cell line [11].


Differential inhibitory effects of CysLT(1) receptor antagonists on P2Y(6) receptor-mediated signaling and ion transport in human bronchial epithelia.

Lau WK, Chow AW, Au SC, Ko WH - PLoS ONE (2011)

Western blotting analysis showing the protein expression of CysLT1 and P2Y6 receptors in 16HBE14o- cells.The expression of CysLT1 (44 kDa) and P2Y6 (41 kDa) receptors was demonstrated (lane 1), and their positions on the blot closely matched their calculated molecular masses of 39 kDa and 36 kDa, respectively. Detection of these protein bands appeared specific, as they were mostly blocked by prior reabsorption of the antibodies with their respective control antigen for 2 h at 4°C (lane 2).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3142161&req=5

pone-0022363-g001: Western blotting analysis showing the protein expression of CysLT1 and P2Y6 receptors in 16HBE14o- cells.The expression of CysLT1 (44 kDa) and P2Y6 (41 kDa) receptors was demonstrated (lane 1), and their positions on the blot closely matched their calculated molecular masses of 39 kDa and 36 kDa, respectively. Detection of these protein bands appeared specific, as they were mostly blocked by prior reabsorption of the antibodies with their respective control antigen for 2 h at 4°C (lane 2).
Mentions: To examine the presence of CysLT1 and P2Y6 receptors in 16HBE14o- cells, western blot analysis was conducted. The protein expression of CysLT1 and P2Y6 receptors in 16HBE14o- cells was detected as shown in Fig. 1. The CysLT1 receptor polyclonal antibody identified an intense 44-kDa band in whole cell lysates of 16HBE14o- cell monolayers (Fig. 1A, left lane). The specificity of the band was confirmed by the complete abolishment of the immunoreactive signal in 16HBE4o- cells by the CysLT1 receptor polyclonal antibody that had been preadsorbed with specific blocking peptides. The blocking peptides correspond to amino acid residues 318–337 of the human CysLT1 receptor (Fig. 1A, right lane). On the other hand, the P2Y6 receptor was identified as an intense 41-kDa band (Fig. 1B, left lane). The specificity of the band was confirmed by the complete abolishment of the immunoreactive signal by the P2Y6 receptor antibody preadsorbed with specific blocking peptides. The blocking peptides correspond to amino acid residues 322–343 of the human P2Y6 receptor (Fig. 1B, right lane). Western blot analysis demonstrated that 16HBE14o- cells expressed CysLT1 receptors and confirmed our previous finding on P2Y6 receptor expression in this cell line [11].

Bottom Line: CysLTs exert their biological effects via specific G-protein-coupled receptors.Pranlukast inhibited the UDP-evoked I(SC) potentiated by an Epac activator, 8-(4-Chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-CPT-2'-O-Me-cAMP), while montelukast and zafirlukast had no such effect.In summary, our data strongly suggest for the first time that in human airway epithelia, the three specific CysLT(1) receptor antagonists exert differential inhibitory effects on P2Y(6) receptor-coupled Ca(2+) signaling pathways and the potentiating effect on I(SC) mediated by cAMP and Epac, leading to the modulation of ion transport activities across the epithelia.

View Article: PubMed Central - PubMed

Affiliation: School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China.

ABSTRACT

Background: Cysteinyl leukotriene (CysLT) is one of the proinflammatory mediators released by the bronchi during inflammation. CysLTs exert their biological effects via specific G-protein-coupled receptors. CysLT(1) receptor antagonists are available for clinical use for the treatment of asthma. Recently, crosstalk between CysLT(1) and P2Y(6) receptors has been delineated. P2Y receptors are expressed in apical and/or basolateral membranes of virtually all polarized epithelia to control the transport of fluid and electrolytes. Previous research suggests that CysLT(1) receptor antagonists inhibit the effects of nucleotides acting at P2Y receptors. However, the detailed molecular mechanism underlying the inhibition remains unresolved.

Methodology/principal findings: In this study, western blot analysis confirmed that both CysLT(1) and P2Y(6) receptors were expressed in the human bronchial epithelial cell line 16HBE14o-. All three CysLT(1) antagonists inhibited the uridine diphosphate (UDP)-evoked I(SC), but only montelukast inhibited the UDP-evoked [Ca(2+)](i) increase. In the presence of forskolin or 8-bromoadenosine 3'5' cyclic monophosphate (8-Br-cAMP), the UDP-induced I(SC) was potentiated but was reduced by pranlukast and zafirlukast but not montelukast. Pranlukast inhibited the UDP-evoked I(SC) potentiated by an Epac activator, 8-(4-Chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-CPT-2'-O-Me-cAMP), while montelukast and zafirlukast had no such effect. Pranlukast inhibited the real-time increase in cAMP changes activated by 8-CPT-2'-O-Me-cAMP as monitored by fluorescence resonance energy transfer imaging. Zafirlukast inhibited the UDP-induced I(SC) potentiated by N(6)-Phenyladenosine-3',5'-cyclic monophosphorothioate, Sp-isomer (Sp-6-Phe-cAMP; a PKA activator) and UDP-activated PKA activity.

Conclusions/significance: In summary, our data strongly suggest for the first time that in human airway epithelia, the three specific CysLT(1) receptor antagonists exert differential inhibitory effects on P2Y(6) receptor-coupled Ca(2+) signaling pathways and the potentiating effect on I(SC) mediated by cAMP and Epac, leading to the modulation of ion transport activities across the epithelia.

Show MeSH
Related in: MedlinePlus