Submitted on June 22, 2006
Accepted on August 2, 2006
Identification of key residues that cause differential gallbladder response to PACAP and VIP in the guinea pig
Muxin Wei1, Kotoyo Fujiki2, Eiji Ando3, Sumin Zhang2, Tsuyoshi Ozaki4, Hiroshi Ishiguro5, Takaharu Kondo5, Kiyoshi Nokihara3, Victor Wray6, and Satoru Naruse7*
1 Medicine, The First Affiliated Hospital with Nanjing Medical University, Nanjing, Jiangsu, China; Medicine, Nagoya University Graduate School of Medicine, Nagoya, Japan
2 Human Nutrition, Nagoya University Graduate School of Medicine, Nagoya, Japan; National Institute of Physiological Sciences, Okazaki, Japan
3 Biotechnology Instruments, Shimadzu Corporation, Kyoto, Japan
4 National Institute of Physiological Sciences, Okazaki, Japan
5 Human Nutrition, Nagoya University Graduate School of Medicine, Nagoya, Japan
6 Structural Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany
7 Medicine, Nagoya University Graduate School of Medicine, Nagoya, Japan
* To whom correspondence should be addressed. E-mail: snaruse{at}med.nagoya-u.ac.jp.
Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) have opposite actions on the gallbladder; PACAP induces contraction while VIP relaxation. Here we have attempted to identify key residues responsible for their interactions with PACAP (PAC1) and VIP (VPAC) receptors in the guinea pig gallbladder. We synthesized PACAP-27/VIP hybrid peptides and compared their actions on isolated guinea pig gallbladder smooth muscle strips using isotonic transducers. [Ala4]- and [Val5]-PACAP-27 were more potent than PACAP-27 in stimulating the gallbladder. In contrast, [Ala4, Val5]- and [Ala4, Val5, Asn9]-PACAP-27 induced relaxation similarly to VIP. [Asn9]-, [Thr11]-, or [Leu13]-PACAP-27 had 20-70% contractile activity of PACAP27, while [Asn24, Ser25, Ile26]-PACAP-27 showed no change in the activity. All VIP analogues, including [Gly4, Ile5, Ser9]-VIP, induced relaxation. In the presence of a PAC1 receptor antagonist, PACAP(6-38), the contractile response to PACAP-27 was inhibited and relaxation became evident. Reverse transcription-polymerase chain reactions (RT-PCR) analysis revealed abundant expressions of PAC1, 'hop' splice variant, VPAC1 and VPAC2 receptor mRNAs in the guinea pig gallbladder. In conclusion, PACAP-27 induces contraction of the gallbladder via PAC1/hop receptors. Gly4 and Ile5 are the key N-terminal residues of PACAP-27 that distinguish PAC1/hop receptors from VPAC1/VPAC2 receptors. However, both the N-terminal and 
-helical regions of PACAP-27 are required for initiating gallbladder contraction.
