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Results: 9

1.
FIGURE 2.

FIGURE 2. From: Rap1 Activation Plays a Regulatory Role in Pancreatic Amylase Secretion.

Rap1 is activated by Ca2+, DAG, and cAMP. Isolated pancreatic acini were stimulated by Ca2+ ionophore A23187, phorbol ester PMA, forskolin (FSK), and 8-Br-cAMP and then assayed for activation of Rap1. The upper panel shows a representative immunoblot for both GTP-Rap1 and total Rap1. The lower panel shows quantitative analysis for Rap1 activation. Data shown are means ± S.E. (four experiments) for activation of Rap1 expressed as a percentage of basal. *, p < 0.05; **, p < 0.01 versus basal.

Maria E. Sabbatini, et al. J Biol Chem. 2008 August 29;283(35):23884-23894.
2.
FIGURE 5.

FIGURE 5. From: Rap1 Activation Plays a Regulatory Role in Pancreatic Amylase Secretion.

CalDAG-GEF III is expressed in pancreatic acini. The expression of CalDAG-GEFs was assessed in pancreas as well as pancreatic acini by RT-PCR. The brain and kidney were used as positive control. CalDAG-GEF I (A) and CalDAG-GEF III (B) RT-PCR products yielded bands of the expected size (257 and 414 bp, respectively). Only CalDAG-GEF III was expressed not only in whole pancreas but also in pancreatic acini. Results shown are representative of multiple experiments.

Maria E. Sabbatini, et al. J Biol Chem. 2008 August 29;283(35):23884-23894.
3.
FIGURE 9.

FIGURE 9. From: Rap1 Activation Plays a Regulatory Role in Pancreatic Amylase Secretion.

Proposed model for Rap1-mediated response on stimulated amylase release. Activation of Rap1 occurs after the stimulation by secretagogues CCK, carbachol, and VIP in pancreatic acini. Our findings show that different second messengers, including DAG, Ca2+, and cAMP generated from activation of different receptors, CCK and muscarinic as well as VPAC receptors, are able to mediate the activation of Rap1 via either CalDAG-GEFIII or Epac1 and thereby regulate amylase secretion in pancreatic acinar cells. Note that the broken line indicates a less well defined pathway.

Maria E. Sabbatini, et al. J Biol Chem. 2008 August 29;283(35):23884-23894.
4.
FIGURE 3.

FIGURE 3. From: Rap1 Activation Plays a Regulatory Role in Pancreatic Amylase Secretion.

Epac, but not PKA, is involved in Rap1 activation. Either 8-Br-cAMP or the Epac-selective cAMP analog 8-pCPT-2′-O-Me-cAMP (CPT-Me-cAMP) was added to isolated pancreatic acini, and then both Rap1 activation (A) and CREB phosphorylation (B) were determined. 8-Br-cAMP stimulated both Rap1 activation and CREB phosphorylation, whereas CPT-Me-cAMP only stimulated Rap1 activation. PKA inhibition abolished the stimulated effect of forskolin on CREB phosphorylation (D) but did not modify forskolin (FSK)-stimulated Rap1 activation (C). The upper panels show representative immunoblots for GTP-Rap1, total Rap1, phospho-Ser133-CREB, and CREB-2. The lower panels show quantitative analysis of either Rap1 activation or CREB phosphorylation. Data shown are means ± S.E. (four experiments) for either Rap1 activation or CREB phosphorylation expressed as a percentage of basal. *, p < 0.05; **, p < 0.01 versus basal; ††, p < 0.01 versus H-89; ‡‡, p < 0.01 versus FSK.

Maria E. Sabbatini, et al. J Biol Chem. 2008 August 29;283(35):23884-23894.
5.
FIGURE 1.

FIGURE 1. From: Rap1 Activation Plays a Regulatory Role in Pancreatic Amylase Secretion.

Multiple secretagogues activate Rap1. A, isolated mouse pancreatic acini were treated with CCK for the specified time, after which a pull-down assay was used to detect the active form of Rap1. Activation of Rap1 was detected after 2 min, remained elevated at 5 and 10 min, and declined at 30 min. B–D, acini were stimulated with specific concentrations of CCK (B), carbachol (C), and VIP (D) for 10 min and then assayed for activation of Rap1. The upper panels show representative immunoblots for both GTP-Rap1 and total Rap1. The lower panels show quantitative analysis of Rap1 activation. Data shown are means ± S.E. (4–5 experiments) for activation of Rap1 expressed as a percentage of basal. *, p < 0.05; **, p < 0.01 versus basal.

Maria E. Sabbatini, et al. J Biol Chem. 2008 August 29;283(35):23884-23894.
6.
FIGURE 6.

FIGURE 6. From: Rap1 Activation Plays a Regulatory Role in Pancreatic Amylase Secretion.

The Epac1 pathway and the carbachol-induced pathway act independently to activate Rap1. Acini were pretreated with the PLC inhibitor U-73122 and then stimulated with either forskolin (A) or carbachol (B) for 10 min, and Rap1 activation was analyzed. The presence of the PLC inhibitor decreased carbachol-evoked GTP-Rap1 levels without affecting the response of forskolin. C, in other experiments, pancreatic acini were treated with either carbachol or 8-pCPT-2′-O-Me-cAMP (CPT-Me-cAMP) alone or with a combination of both for 10 min. The results showed that the co-addition of both stimulators induces an additive effect on Rap1 activation. The upper panels show representative immunoblots for GTP-Rap1 and total-Rap1. The lower panels show a quantitative analysis of Rap1 activation. Data shown are means ± S.E. (3–5 experiments) for Rap1 activation expressed as a percentage of basal. FSK, forskolin; CCh, carbachol. *, p < 0.05; **, p < 0.01 versus basal; †, p < 0.01 versus U-73122; ‡‡, p < 0.01 versus carbachol.

Maria E. Sabbatini, et al. J Biol Chem. 2008 August 29;283(35):23884-23894.
7.
FIGURE 8.

FIGURE 8. From: Rap1 Activation Plays a Regulatory Role in Pancreatic Amylase Secretion.

GTP-Rap1 is involved in evoked pancreatic amylase release. β-Galactosidase (β-Gal; vector control) and Rap1GAP-overexpressing acini were stimulated with different concentrations of either CCK (A), carbachol (B), and cAMP-evoked secretagogues (C) for 30 min, and amylase release was measured. An inhibition of amylase secretion (40%) was observed when the Rap1GAP-overexpressing acini were stimulated with high concentrations of either CCK or carbachol. In addition, Rap1GAP overexpression decreased 8-Br-cAMP-, CPT-Me-cAMP-, and VIP-stimulated amylase secretion by 60%. Data shown are means ± S.E. (4–6 experiments) of amylase release expressed as a percentage of total. □, β-galactosidase-expressing cells; ▪, Rap1GAP-overexpressing cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control; †, p < 0.05; ††, p < 0.01 versus CCK, carbachol, 8-Br-cAMP, CPT-Me-cAMP, or VIP response in β-galactosidase-expressing cells. D, pancreatic acini were preincubated for 10 min with the PKA inhibitor H-89 and then stimulated with 8-Br-cAMP, CPT-Me-cAMP, or VIP. Secretagogue-stimulated amylase release was not modified in the presence of the inhibitor. Data shown are means ± S.E. (four experiments) of amylase release expressed as a percentage of the total. *, p < 0.05 versus control; †, p < 0.05 versus 8-Br-cAMP or VIP.

Maria E. Sabbatini, et al. J Biol Chem. 2008 August 29;283(35):23884-23894.
8.
FIGURE 7.

FIGURE 7. From: Rap1 Activation Plays a Regulatory Role in Pancreatic Amylase Secretion.

Rap1GAP overexpression blocks the activation of Rap1 by CCK, carbachol, and VIP. Acini were infected overnight with adenovirus expressing either β-galactosidase (vector control) or Rap1GAP and then stimulated with 300 pm CCK (A), 10 μm carbachol (CCh), and 10 nm VIP (B) for 10 min. An inhibition of Rap1 activation was observed when the Rap1GAP-overexpressing acini were stimulated with CCK, carbachol, and VIP. The upper panel shows a representative immunoblot for GTP-Rap1, total Rap1, or Rap1GAP. The lower panel shows the quantitative analysis of Rap1 activation. Data shown are means ± S.E. (four experiments) for activation of Rap1 expressed as a percentage of basal. **, p < 0.01; ***, p < 0.001 versus basal; ††, p < 0.01; †††, p < 0.001 versus CCK, carbachol, or VIP. C, to study the specificity of Rap1GAP, another set of acini were stimulated with 1 nm CCK, and then a RhoA pull-down assay was carried out; Rap1GAP overexpression did not affect CCK-induced RhoA activation. The upper panel shows a representative immunoblot for GTP-RhoA, total RhoA, or Rap1GAP. The lower panel shows the quantitative analysis of RhoA activation. Data shown are means ± S.E. (three experiments) for activation of RhoA expressed as a percentage of basal. *, p < 0.05 versus basal.

Maria E. Sabbatini, et al. J Biol Chem. 2008 August 29;283(35):23884-23894.
9.
FIGURE 4.

FIGURE 4. From: Rap1 Activation Plays a Regulatory Role in Pancreatic Amylase Secretion.

Epac1 is present in pancreatic acini. Both Epac1 and Epac2 expression were assessed in pancreatic acini by RT-PCR (A) and Western blotting (B) as detailed under “Experimental Procedures.” Brain and kidney were used as positive control for Epac1, whereas only brain was used for Epac2. Epac1 mRNA expression was determined in pancreatic acini and yielded a product of the expected size (267 bp) (A), and Epac1 protein was present in mouse pancreatic acini, as indicated by Western blotting analysis (B). Epac2 protein was not present in acini; a second distinct antibody gave similar results. C, in zymogen granule (ZG) fractionation, Rap1 was only present in ZG membrane fraction, whereas Epac1 was present not only in the ZG supernatant but also in ZG membrane fractions. Note that 15 μg of protein from each fraction was applied to the gel, but most (>90%) of the ZG protein was present in the supernatant following ZG lysis. D, immunohistochemistry was used to localize Epac1 or Rap1 (red) and compare it to amylase (green); nuclei were stained with 4,6-diamino-2-phenylindole (blue). Epac1 as well as Rap1 were localized on ZG in close proximity to amylase.

Maria E. Sabbatini, et al. J Biol Chem. 2008 August 29;283(35):23884-23894.

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