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A1 adenosine receptor-stimulated exocytosis in bladder umbrella cells requires phosphorylation of ADAM17 Ser-811 and EGF receptor transactivation.

Prakasam HS, Gallo LI, Li H, Ruiz WG, Hallows KR, Apodaca G - Mol. Biol. Cell (2014)

Bottom Line: Despite the importance of ADAM17-dependent cleavage in normal biology and disease, the physiological cues that trigger its activity, the effector pathways that promote its function, and the mechanisms that control its activity, particularly the role of phosphorylation, remain unresolved.Preventing this phosphorylation event by expression of a nonphosphorylatable ADAM17(S811A) mutant or expression of a tail-minus construct inhibits A1AR-stimulated, ADAM17-dependent HB-EGF cleavage.Furthermore, expression of ADAM17(S811A) in bladder tissues impairs A1AR-induced apical exocytosis.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine and Cell Biology, University of Pittsburgh, Pittsburgh, PA 15261.

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A1AR-stimulated exocytosis is dependent on protein synthesis and secretion. (A) Cryosection of rat bladder epithelium labeled with an A1AR-specific antibody (green), rhodamine–phalloidin to label the actin cytoskeleton (red), and TOPRO-3 (blue) to label the nucleus. Right, merge. An umbrella cell is marked with an asterisk and the white arrows mark the apicolateral borders of the cell. Scale bar, 12 μm. (B) Rabbit uroepithelium was mounted in Ussing chambers and after equilibration left untreated (no drug), or 1 μM adenosine or 500 nM CCPA was added to the mucosal hemichamber. Percentage changes in CT were monitored. (C) Rabbit tissue was left untreated, pretreated with 5 μg/ml brefeldin A (BFA) for 30 min, or pretreated with 100 ng/ml cyclohexamide for 60 min. CCPA (500 nM) was then added to the mucosal hemichamber and CT recorded. Data for CCPA alone are reproduced from B. (B, C) Mean changes in CT ± SEM (n ≥ 4). Significant differences (p < 0.05), relative to no drug in A or CCPA in B, are indicated with an asterisk.
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Figure 1: A1AR-stimulated exocytosis is dependent on protein synthesis and secretion. (A) Cryosection of rat bladder epithelium labeled with an A1AR-specific antibody (green), rhodamine–phalloidin to label the actin cytoskeleton (red), and TOPRO-3 (blue) to label the nucleus. Right, merge. An umbrella cell is marked with an asterisk and the white arrows mark the apicolateral borders of the cell. Scale bar, 12 μm. (B) Rabbit uroepithelium was mounted in Ussing chambers and after equilibration left untreated (no drug), or 1 μM adenosine or 500 nM CCPA was added to the mucosal hemichamber. Percentage changes in CT were monitored. (C) Rabbit tissue was left untreated, pretreated with 5 μg/ml brefeldin A (BFA) for 30 min, or pretreated with 100 ng/ml cyclohexamide for 60 min. CCPA (500 nM) was then added to the mucosal hemichamber and CT recorded. Data for CCPA alone are reproduced from B. (B, C) Mean changes in CT ± SEM (n ≥ 4). Significant differences (p < 0.05), relative to no drug in A or CCPA in B, are indicated with an asterisk.

Mentions: Late-phase exocytosis is triggered when uroepithelial tissue is maximally stretched (Balestreire and Apodaca, 2007; Yu et al., 2009), but it is unknown whether other stimuli promote a similar response. Because adenosine release is significantly increased as the bladder reaches its capacity (Prakasam et al., 2012), and because extracellular adenosine also triggers exocytosis (Yu et al., 2006), we tested the hypothesis that adenosine stimulates exocytosis by way of EGFR transactivation. Adenosine can stimulate exocytosis when added to either the serosal or mucosal surfaces of isolated uroepithelium; however, in this study, we focused our attention on the mucosal events, as these are primarily mediated by the A1AR, and EGFR transactivation occurs at this surface (Yu et al., 2006; Balestreire and Apodaca, 2007). Consistent with our previous reports (Yu et al., 2011; Prakasam et al., 2012), the A1AR was localized to the apical pole of rat umbrella cells (as well as in the underlying lamina propria; Figure 1A). When adenosine was added to the mucosal surface of isolated rabbit tissue, it stimulated increased tissue capacitance (CT; 1 μF ≈ 1 cm2; Figure 1B), which in this tissue correlates well with other measures of apical exocytosis (Truschel et al., 2002; Wang et al., 2003a). Similar results were obtained when the highly selective and high-affinity A1AR agonist 2-chloro-N6-cyclopentyladenosine (CCPA) was used. Unlike adenosine, CCPA is not rapidly converted to inosine or to AMP (Manjunath and Sakhare, 2009; Prakasam et al., 2012) and was thus used in our subsequent studies. We next determined whether A1AR-stimulated exocytosis, like that observed in response to excess stretch, is dependent on protein synthesis or secretion. Indeed, treatment with cycloheximide or brefeldin A significantly blocked CCPA-mediated exocytosis (Figure 1C). The effect of the latter was particularly pronounced.


A1 adenosine receptor-stimulated exocytosis in bladder umbrella cells requires phosphorylation of ADAM17 Ser-811 and EGF receptor transactivation.

Prakasam HS, Gallo LI, Li H, Ruiz WG, Hallows KR, Apodaca G - Mol. Biol. Cell (2014)

A1AR-stimulated exocytosis is dependent on protein synthesis and secretion. (A) Cryosection of rat bladder epithelium labeled with an A1AR-specific antibody (green), rhodamine–phalloidin to label the actin cytoskeleton (red), and TOPRO-3 (blue) to label the nucleus. Right, merge. An umbrella cell is marked with an asterisk and the white arrows mark the apicolateral borders of the cell. Scale bar, 12 μm. (B) Rabbit uroepithelium was mounted in Ussing chambers and after equilibration left untreated (no drug), or 1 μM adenosine or 500 nM CCPA was added to the mucosal hemichamber. Percentage changes in CT were monitored. (C) Rabbit tissue was left untreated, pretreated with 5 μg/ml brefeldin A (BFA) for 30 min, or pretreated with 100 ng/ml cyclohexamide for 60 min. CCPA (500 nM) was then added to the mucosal hemichamber and CT recorded. Data for CCPA alone are reproduced from B. (B, C) Mean changes in CT ± SEM (n ≥ 4). Significant differences (p < 0.05), relative to no drug in A or CCPA in B, are indicated with an asterisk.
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Related In: Results  -  Collection

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Figure 1: A1AR-stimulated exocytosis is dependent on protein synthesis and secretion. (A) Cryosection of rat bladder epithelium labeled with an A1AR-specific antibody (green), rhodamine–phalloidin to label the actin cytoskeleton (red), and TOPRO-3 (blue) to label the nucleus. Right, merge. An umbrella cell is marked with an asterisk and the white arrows mark the apicolateral borders of the cell. Scale bar, 12 μm. (B) Rabbit uroepithelium was mounted in Ussing chambers and after equilibration left untreated (no drug), or 1 μM adenosine or 500 nM CCPA was added to the mucosal hemichamber. Percentage changes in CT were monitored. (C) Rabbit tissue was left untreated, pretreated with 5 μg/ml brefeldin A (BFA) for 30 min, or pretreated with 100 ng/ml cyclohexamide for 60 min. CCPA (500 nM) was then added to the mucosal hemichamber and CT recorded. Data for CCPA alone are reproduced from B. (B, C) Mean changes in CT ± SEM (n ≥ 4). Significant differences (p < 0.05), relative to no drug in A or CCPA in B, are indicated with an asterisk.
Mentions: Late-phase exocytosis is triggered when uroepithelial tissue is maximally stretched (Balestreire and Apodaca, 2007; Yu et al., 2009), but it is unknown whether other stimuli promote a similar response. Because adenosine release is significantly increased as the bladder reaches its capacity (Prakasam et al., 2012), and because extracellular adenosine also triggers exocytosis (Yu et al., 2006), we tested the hypothesis that adenosine stimulates exocytosis by way of EGFR transactivation. Adenosine can stimulate exocytosis when added to either the serosal or mucosal surfaces of isolated uroepithelium; however, in this study, we focused our attention on the mucosal events, as these are primarily mediated by the A1AR, and EGFR transactivation occurs at this surface (Yu et al., 2006; Balestreire and Apodaca, 2007). Consistent with our previous reports (Yu et al., 2011; Prakasam et al., 2012), the A1AR was localized to the apical pole of rat umbrella cells (as well as in the underlying lamina propria; Figure 1A). When adenosine was added to the mucosal surface of isolated rabbit tissue, it stimulated increased tissue capacitance (CT; 1 μF ≈ 1 cm2; Figure 1B), which in this tissue correlates well with other measures of apical exocytosis (Truschel et al., 2002; Wang et al., 2003a). Similar results were obtained when the highly selective and high-affinity A1AR agonist 2-chloro-N6-cyclopentyladenosine (CCPA) was used. Unlike adenosine, CCPA is not rapidly converted to inosine or to AMP (Manjunath and Sakhare, 2009; Prakasam et al., 2012) and was thus used in our subsequent studies. We next determined whether A1AR-stimulated exocytosis, like that observed in response to excess stretch, is dependent on protein synthesis or secretion. Indeed, treatment with cycloheximide or brefeldin A significantly blocked CCPA-mediated exocytosis (Figure 1C). The effect of the latter was particularly pronounced.

Bottom Line: Despite the importance of ADAM17-dependent cleavage in normal biology and disease, the physiological cues that trigger its activity, the effector pathways that promote its function, and the mechanisms that control its activity, particularly the role of phosphorylation, remain unresolved.Preventing this phosphorylation event by expression of a nonphosphorylatable ADAM17(S811A) mutant or expression of a tail-minus construct inhibits A1AR-stimulated, ADAM17-dependent HB-EGF cleavage.Furthermore, expression of ADAM17(S811A) in bladder tissues impairs A1AR-induced apical exocytosis.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine and Cell Biology, University of Pittsburgh, Pittsburgh, PA 15261.

Show MeSH
Related in: MedlinePlus