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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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Effects of CysLT1 receptor antagonists on Epac activation and PKA activity.(A) The monochrome CFP and FRET images showing the cytosolic distribution of the fluorescent Epac probe in 16HBE14o- cells transfected with CFP-Epac-YFP. (B) Representative pseudocolor images of CFP/FRET emission ratios before and after the addition of 8-CPT-2′-O-Me-cAMP. (D) Real-time cAMP changes (normalized CFP/FRET emission ratio) recorded in cells stimulated with 50 µM 8-CPT-2′-O-Me-cAMP with or without 1 µM pranlukast shown in (B). The agents were added at time zero. (C) Summarized data showing the effect of CysLT1 receptor antagonists on the CFP/FRET emission ratio. Each column represents the mean ± S.E. (n = 8–10). (*, p<0.05, Student's t-test compared with control). (E) Confluent 16HBE14o- cells were treated with either vehicle alone (control), 100 µM UDP, or UDP with different CysLT1 receptor antagonists (1 µM) for 5 min. PKA activity was measured as a function of fluorescence intensity. (F) Summarized data showing the relative fluorescence level as compared with the control level. Each column represents the mean ± S.E. (*, p<0.05, n = 4, one-way ANOVA with Bonferroni post-hoc test).
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pone-0022363-g006: Effects of CysLT1 receptor antagonists on Epac activation and PKA activity.(A) The monochrome CFP and FRET images showing the cytosolic distribution of the fluorescent Epac probe in 16HBE14o- cells transfected with CFP-Epac-YFP. (B) Representative pseudocolor images of CFP/FRET emission ratios before and after the addition of 8-CPT-2′-O-Me-cAMP. (D) Real-time cAMP changes (normalized CFP/FRET emission ratio) recorded in cells stimulated with 50 µM 8-CPT-2′-O-Me-cAMP with or without 1 µM pranlukast shown in (B). The agents were added at time zero. (C) Summarized data showing the effect of CysLT1 receptor antagonists on the CFP/FRET emission ratio. Each column represents the mean ± S.E. (n = 8–10). (*, p<0.05, Student's t-test compared with control). (E) Confluent 16HBE14o- cells were treated with either vehicle alone (control), 100 µM UDP, or UDP with different CysLT1 receptor antagonists (1 µM) for 5 min. PKA activity was measured as a function of fluorescence intensity. (F) Summarized data showing the relative fluorescence level as compared with the control level. Each column represents the mean ± S.E. (*, p<0.05, n = 4, one-way ANOVA with Bonferroni post-hoc test).

Mentions: To examine whether the inhibitory action of pranlukast occurs through Epac, we monitored Epac1 activation by using a cyan fluorescent protein (CFP)-Epac-yellow fluorescent protein (YFP) fusion construct, which has been used by others to detect real-time changes in [cAMP] via a fluorescence resonance energy transfer (FRET)-based approach [25]–[27]. Fig. 6A shows that 16HBE14o- cells were successfully transfected with the fusion construct of CYP-Epac-YFP, which is sensitive to the dynamic changes of [cAMP]. As expected, the Epac activator 8-CPT-2′-O-cAMP (50 µM) evoked about a 20% increase in the CFP/FRET emission ratio, which indicates a global increase in cAMP (Fig. 6B and C). Pranlukast could inhibit the activation of Epac mediated by 8-CPT-2′-O-cAMP. Fig. 6D summarizes the inhibitory effects of montelukast, pranlukast, and zafirlukast. In the presence of pranlukast (1 µM), the increase in the CFP/FRET emission ratio induced by 8-CPT-2′-O-cAMP was reduced to 45.5% of the control (p<0.05, n = 8). Montelukast (1 µM) and zafirlukast (1 µM) exhibited no inhibitory effects (p>0.05, n = 8–10). In the control experiments, addition of montelukast, pranlukast or zafirlukast (1 µM) did not induce any discernible increase in FRET ratio (n = 3).


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)

Effects of CysLT1 receptor antagonists on Epac activation and PKA activity.(A) The monochrome CFP and FRET images showing the cytosolic distribution of the fluorescent Epac probe in 16HBE14o- cells transfected with CFP-Epac-YFP. (B) Representative pseudocolor images of CFP/FRET emission ratios before and after the addition of 8-CPT-2′-O-Me-cAMP. (D) Real-time cAMP changes (normalized CFP/FRET emission ratio) recorded in cells stimulated with 50 µM 8-CPT-2′-O-Me-cAMP with or without 1 µM pranlukast shown in (B). The agents were added at time zero. (C) Summarized data showing the effect of CysLT1 receptor antagonists on the CFP/FRET emission ratio. Each column represents the mean ± S.E. (n = 8–10). (*, p<0.05, Student's t-test compared with control). (E) Confluent 16HBE14o- cells were treated with either vehicle alone (control), 100 µM UDP, or UDP with different CysLT1 receptor antagonists (1 µM) for 5 min. PKA activity was measured as a function of fluorescence intensity. (F) Summarized data showing the relative fluorescence level as compared with the control level. Each column represents the mean ± S.E. (*, p<0.05, n = 4, one-way ANOVA with Bonferroni post-hoc test).
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Related In: Results  -  Collection

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

pone-0022363-g006: Effects of CysLT1 receptor antagonists on Epac activation and PKA activity.(A) The monochrome CFP and FRET images showing the cytosolic distribution of the fluorescent Epac probe in 16HBE14o- cells transfected with CFP-Epac-YFP. (B) Representative pseudocolor images of CFP/FRET emission ratios before and after the addition of 8-CPT-2′-O-Me-cAMP. (D) Real-time cAMP changes (normalized CFP/FRET emission ratio) recorded in cells stimulated with 50 µM 8-CPT-2′-O-Me-cAMP with or without 1 µM pranlukast shown in (B). The agents were added at time zero. (C) Summarized data showing the effect of CysLT1 receptor antagonists on the CFP/FRET emission ratio. Each column represents the mean ± S.E. (n = 8–10). (*, p<0.05, Student's t-test compared with control). (E) Confluent 16HBE14o- cells were treated with either vehicle alone (control), 100 µM UDP, or UDP with different CysLT1 receptor antagonists (1 µM) for 5 min. PKA activity was measured as a function of fluorescence intensity. (F) Summarized data showing the relative fluorescence level as compared with the control level. Each column represents the mean ± S.E. (*, p<0.05, n = 4, one-way ANOVA with Bonferroni post-hoc test).
Mentions: To examine whether the inhibitory action of pranlukast occurs through Epac, we monitored Epac1 activation by using a cyan fluorescent protein (CFP)-Epac-yellow fluorescent protein (YFP) fusion construct, which has been used by others to detect real-time changes in [cAMP] via a fluorescence resonance energy transfer (FRET)-based approach [25]–[27]. Fig. 6A shows that 16HBE14o- cells were successfully transfected with the fusion construct of CYP-Epac-YFP, which is sensitive to the dynamic changes of [cAMP]. As expected, the Epac activator 8-CPT-2′-O-cAMP (50 µM) evoked about a 20% increase in the CFP/FRET emission ratio, which indicates a global increase in cAMP (Fig. 6B and C). Pranlukast could inhibit the activation of Epac mediated by 8-CPT-2′-O-cAMP. Fig. 6D summarizes the inhibitory effects of montelukast, pranlukast, and zafirlukast. In the presence of pranlukast (1 µM), the increase in the CFP/FRET emission ratio induced by 8-CPT-2′-O-cAMP was reduced to 45.5% of the control (p<0.05, n = 8). Montelukast (1 µM) and zafirlukast (1 µM) exhibited no inhibitory effects (p>0.05, n = 8–10). In the control experiments, addition of montelukast, pranlukast or zafirlukast (1 µM) did not induce any discernible increase in FRET ratio (n = 3).

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