1. Volume 132, Issue 4, Pages 1388-1400 (April 2007)
A Novel Role of VIP in Colonic Motility Function: Induction of Excitation–Transcription Coupling in Smooth Muscle Cells 
Xuan–Zheng Shi, Barun K. Choudhury, Pankaj J. Pasricha, Sushil K. Sarna 
Gastroenterology 
Volume 132, Issue 4, Pages (April 2007)
DOI: /j.gastro
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2. Figure 1
(A) Incubation of HCCSMCs with VIP concentration-dependently enhanced the expression of α1C mRNA determined by qPCR (n = 4). (B) The increase of cytosolic calcium (340/380 ratio) in response to 60 mmol/L KCl was greater in cells preincubated with 100 nmol/L VIP for 24 hours and then loaded with Fura-2 AM than in those incubated with vehicle control only (n = 3). The prior inhibition of Cav1.2 channels with methoxyverapamil completely blocked the increase of Ca2+ influx in response to KCl in control as well as in VIP-treated cells. (C) Incubation of the cells with 100 nmol/L VIP concentration-dependently enhanced the expression of α1C protein determined by Western blotting. The incubation of the cells with VIP had no effect on the expression of BKca channels. β-Actin was used as internal control (n = 4). (D) Time course of increase in α1C protein on incubation of HCCSMCs with 100 nmol/L VIP (n = 4). A significant increase was observed at about 6 hours.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

3. Figure 2
The incubation of HCCSMCs with ACh (A), substance P (B), s-nitrosoglutathione (C), and ATP (D) had little effect on the expression of α1C mRNA determined by qPCR. On the other hand, their incubation with PACAP (E) or 8-bromo cAMP (F) concentration-dependently enhanced the expression of α1C (n = 3).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

4. Figure 3
Incubation of HCCSMCs with 100 nmol/L VIP induced sustained generation of cAMP for 24 hours (A). However, PKA was activated only from about 15 minutes to 3 hours (B). In contrast, peak of CREB phosphorylation occurred at about 15 minutes and it lasted for less than 1 hour (C) (n = 3 or 4).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

5. Figure 4
Pretreatment with PKA inhibitor H-89 suppressed the phosphorylation of CREB in response to incubation of the HCCSMCs with VIP. However, their pretreatment with protein kinase C inhibitor calphostin C had little effect (n = 4).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

6. Figure 5
Immunohistochemical staining of pCREB indicated transient phosphorylation of CREB on the nucleus on incubation of HCCSMCs with 100 nmol/L VIP. The images on the left side show the nuclei.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

7. Figure 6
(A) Luciferase reporter assay showed concentration-dependent increase of α1C promoter activity in cells incubated with VIP for 24 hours (n = 5). (B) The Western blot at the top shows that silencing of the CREB gene with targeted RNAi suppressed the expression of CREB in HCCSMCs (2 control samples and 2 samples transfected with CREB RNAi). The left bar plot shows that incubation of normal control HCCSMCs with VIP increased the luciferase activity; this increase of luciferase activity in response to the same treatment of the cells with VIP was suppressed in cells transfected with RNAi of the CREB gene. The control cells were transfected with scrambled RNAi (n = 3). (C) The inhibition of PKA with H-89 blocked the increase of α1C mRNA in response to 24-hour incubation of the cells with VIP. The addition of H-89 alone had little effect (n = 3). VIP was used at a concentration of 10−7 mol/L in all experiments, unless noted otherwise.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

8. Figure 7
(A) Progressive 5′ deletions were used to determine the role of CRE1 and CRE2 in α1C promoter activity. The basic vector pGL2 had little activity. The constitutive activity of the wild-type promoter was taken as 100%. The first deletion that did not involve any of the binding motifs for CREB had little effect on the increase in promoter activity in response to 24-hour incubation of the cells with VIP. The deletion of CRE1 almost completely abolished the increase of promoter activity in response to VIP. Further deletions had little additional effect. (B) The 24-hour incubation of the cells with VIP significantly enhanced activity of the wild-type promoter. The mutation of CRE1 at 2 indicated sites (M1) abolished this response. The mutation of CRE2 at 2 indicated sites (M2) had less effect on promoter activity than the mutation of similar sites in CRE1. The concurrent mutations of CRE1 and CRE2 (M1 + M2) totally blocked the response to VIP (n = 3 or 4).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

9. Figure 8
Incubation of stretched human (A) and rat (B) colonic circular muscle strips with 100 nmol/L VIP for 24 hours enhanced their contractile response to ACh (n = 3 each). VIP treatment also enhanced the expression of α1C in the rat colonic muscle strips (C).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

10. Figure 9
Incubation of stretched rat colonic circular muscle strips with 100 nmol/L VIP antagonist (p-Chloro-D-Phe6, Leu17)-VIP for 24 hours suppressed their contractile response to ACh and KCl (n = 3) (A and B) and decreased the α1C protein level in muscle strips (C).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

11. Figure 10
Increasing concentrations of VIP were added to muscle bath 2 minutes prior to the addition of 1 mmol/L ACh. The response to ACh alone was taken as 100%. The concentrations at which VIP inhibited ACh induced contractions were 1 to 2 log units greater than those that induced gene expression (n = 4).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions