AJP - GI Ad Instruments
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Gastrointest Liver Physiol 292: G76-G83, 2007. First published August 10, 2006; doi:10.1152/ajpgi.00279.2006
0193-1857/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/1/G76    most recent
00279.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wei, M.
Right arrow Articles by Naruse, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wei, M.
Right arrow Articles by Naruse, S.

NEUROREGULATION AND MOTILITY

Identification of key residues that cause differential gallbladder response to PACAP and VIP in the guinea pig

Muxin Wei,1,2 Kotoyo Fujiki,3,4 Eiji Ando,5 Sumin Zhang,3,4 Tsuyoshi Ozaki,4 Hiroshi Ishiguro,3 Takaharu Kondo,3 Kiyoshi Nokihara,5 Victor Wray,6 and Satoru Naruse2

1The First Affiliated Hospital of Nanjing Medical University, Nanjing, China; 2Internal Medicine and 3Human Nutrition, Nagoya University Graduate School of Medicine, Nagoya, Japan; 4National Institute of Physiological Sciences, Okazaki, Japan; 5Biotechnology Instruments Department, Shimadzu Corporation, Kyoto, Japan; and 6Department of Structural Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany

Submitted 22 June 2006 ; accepted in final form 2 August 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) have opposite actions on the gallbladder; PACAP induces contraction, whereas VIP induces 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 PACAP-27, whereas [Asn24,Ser25,Ile26]PACAP-27 showed no change in the activity. All VIP analogs, 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. RT-PCR analysis revealed abundant expressions of PAC1 receptor, "hop" splice variant, and 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 NH2-terminal residues of PACAP-27 that distinguish PAC1/hop receptors from VPAC1/VPAC2 receptors. However, both the NH2-terminal and {alpha}-helical regions of PACAP-27 are required for initiating gallbladder contraction.

PAC1 receptor; VPAC1 receptor; VPAC2 receptor; splice variant

PITUITARY ADENYLATE cyclase-activating polypeptide (PACAP) is a member of the secretin/glucagon/vasoactive intestinal polypeptide (VIP) family of peptides (2, 8). It occurs in two bioactive molecules (PACAP-38 and PACAP-27) with identical NH2-terminal sequences. PACAP-27 has a 68% sequence homology to VIP, and all are expressed in the central as well as peripheral and enteric nervous systems (2, 22). They are released from nerve terminals as neurotransmitters or neuromodulators and regulate the function of the brain and peripheral organs. PACAP exhibits protean biological effects on the gastrointestinal tract, including motility, secretion, and blood flow (6). PACAP and VIP are coexpressed in nerve fibers and neurons in the ganglia of the guinea pig gallbladder (7). Although both peptides act primarily as inhibitory transmitters on most gastrointestinal and vascular smooth muscle cells (5, 6, 12), their actions on the gallbladder are opposite; VIP relaxes the gallbladder, whereas PACAP induces the contraction both in vivo (9) and in vitro (15, 23, 24).

Three receptor subtypes that recognize PACAP and VIP have been identified (4, 22), and all belong to the group of seven transmembrane G protein-coupled receptors. The PACAP-specific (PAC1) receptor has a much higher affinity for PACAP than VIP, whereas the classical VIP (VPAC1) receptor and VPAC2 receptor exhibit similar affinities for PACAP and VIP. VPAC1 and VPAC2 receptors lead to activation of the adenylate cyclase/cAMP pathway in which elevation of intracellular cAMP, together with nitric oxide, mediates relaxation of intestinal and vascular smooth muscle cells (6, 22). PAC1 receptors, on the other hand, can activate the dual-signal transduction pathways involving adenylate cyclase and phospholipase C. The activation of the latter probably leads to inositol trisphosphate (IP3)-mediated Ca2+ mobilization and protein kinase C mediated-gallbladder contraction (14), although until now it was not known which receptor subtype is expressed in the gallbladder.

PACAP and related peptides, except helodermin, show no stable structures in aqueous solution (3, 25). However, in more hydrophobic environments, i.e., in 30–50% trifluoroethanol, PACAP-38 has a stable structure consisting of three well-defined domains: an initial disordered NH2-terminus of eight residues, a central {alpha}-helical region from Ser9 to Val26 with a break between Lys20 and Lys21, and a COOH-terminal region with a short {alpha}-helix between Gly28 and Arg34 (25). The structures of PACAP-27 and VIP resemble closely that of PACAP-38 except for the COOH-terminal region. The two helical structures of VIP involve residues Thr7-Lys15 and Val19-Leu27, and a flexible region exists between Glu16 and Ala18 (20). These structural features define specific spatial arrangements of charged residues when they interact with their receptors. Because PACAP-27 has a 68% sequence homology to VIP, the difference in their interaction with PAC1 receptors must reside in the nine amino acid residues that differentiate the two peptides (Table 1). In this study, using guinea pig gallbladder smooth muscle strips, we have attempted to identify key residues for interaction of PACAP with PAC1 receptors by exchanging amino acid residues of PACAP-27 with those of VIP and vice versa. We have also determined the PACAP and VIP receptor subtypes expressed in the guinea pig gallbladder.


View this table:
[in this window]
[in a new window]

  		Table 1. Amino acid sequence of PACAP-27, VIP, and their analogs

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Peptide synthesis. From a consideration of the sequence homology of PACAP and VIP and the results based on the studies by NMR (25), the positions 4, 5, 6, 8, 9, 13, 21, 24, 25, and 26 of PACAP-27 and the positions 4, 5, 9, 11, and 13 of VIP were selected as substitution points for the present study (Table 1). Peptides were synthesized by a simultaneous multiple solid-phase peptide synthesizer (PSSM-8; Shimadzu, Kyoto, Japan), using the 9-fluorenylmethoxycarbonyl strategy as described previously (1). After simultaneous cleavage with a trifluoroacetic acid cocktail, quantitative amounts of the desired crude peptides were obtained, which were purified by a single step of reverse phase-HPLC and characterized by mass spectrometry, sequence analysis, and amino acid analysis.

Biological actions. This study was approved by the Ethical Committees of Nagoya University and National Institute for Physiological Sciences on Animal Use for Experiment. Male guinea pigs of Hartley strain (450–580 g) were killed by stunning and exsanguination. The gallbladder was excised, and four smooth muscle strips (1.2 x 10 mm) were prepared under a binocular microscope. They were then suspended in a horizontal organ bath containing 4 ml of Krebs-Ringer solution (pH 7.4), maintained at 37 ± 0.5°C, and aerated with 95% O2-5% CO2. One end of each muscle strip was fixed to a Silastic rubber bed. The other end was connected to an isotonic displacement transducer (model 45347; NEC-Sanei, Tokyo, Japan) via an L-arm, to which a piezo electric element (100 Hz with movement of 50 µm) was applied to minimize the friction (10). The length of each strip was recorded on a pen recorder (NEC-Sanei) via a computer-controlled data-acquisition system (MacLab, Analog Digital Instruments, Castle Hill, Australia). After we set the initial tension at 500 mg, we allowed muscle strips to equilibrate for 60 min. ACh (Sigma; 10–9 to 10–4 M) was added to the bathing solution to observe the maximal contraction of each muscle strip. After strips were washed, PACAP-27, PACAP-38, VIP (10–10 to 3 x 10–7 M; Peptide Institute, Osaka, Japan), or their hybrid peptides (10–9 to 3 x 10–7 M) were added to the bath. To understand a possible site of action of PACAP-27, atropine (muscarinic receptor antagonist), tetrodotoxin (Na+ channel blocker), loxiglumide (CCK-A receptor antagonist), or PACAP(6–38) (PAC1 receptor antagonist) was administered before PACAP-27.

RT-PCR and sequence analysis. Total RNA was extracted by the RNeasy-Protect mini kit (Qiagen). We performed reverse transcription using an oligo(dT) primer and TaqMan reverse transcription reagents (Roch, Branchburg, NJ). PCR amplification was performed with the following primers with high sequence homology among human, mouse, and rat receptors: 1) PAC1 receptor (GenBank accession no. NM/20%001118) sense, 5'- GAATGACAGCACAGCTCTGTG-3'; antisense, 5'- AGTAACGGTTCACCTTCCAGC-3' (374 bp); 2) VPAC1 receptor (GenBank accession no. NM/20%004624) sense, 5'- GGAAGTACTTCTGGGGGTACA-3'; antisense, 5'- CCATTGAGGAAGCAGTAGAGG-3' (423 bp); and 3) VPAC2 receptor (GenBank accession no. NM/20%003382) sense, 5'- CAGGAAACATAAGCAAAAACTG-3'; antisense, 5'- GTGCAGCTTCCTGAAGAG-3' (209 bp).

PCR was carried out in a 50-µl reaction mixture containing 50 ng cDNA, 5 µl of 10x ExTaq buffer, 4 µl of 2.5 mM dNTP mix, 50 pmol of each primer, and 1.5 U of ExTaq DNA polymerase (TaKaRa, Otsu, Japan). The conditions were as follows: denaturation for 3 min at 94°C, 30 cycles of denaturation for 30 s at 94°C, annealing for 1 min at 60°C, and extension for 30 s at 72°C; we then followed this by 7 min at 72°C using GeneAmp PCR system 9700 (Applied Biosystems, Foster, CA). PCR products were subjected to electrophoresis on 2% agarose gel. PCR products were purified with the High Pure PCR product purification kit (Roche Diagnostics, Mannheim, Germany) and sequenced directly with an automated sequencer (Beckman Coulter, Fullerton, CA) after terminator reaction using a DNA sequencing kit (CEQ DTCS-Quick Start kit; Beckman Coulter).

Data and statistical analyses. Each muscle strip response to the peptides was standardized as a percentage of the contraction induced by the control: ACh (10–4 M) for PACAP and VIP, PACAP-27 (10–7 M) for PACAP analogs, and VIP (10–7 M) for VIP analogs. All data are presented as means ± SE. The mean of four smooth muscle strips of each animal was used for analysis with n = the number of guinea pigs. The one-way factorial ANOVA, multiple comparison tests (Newman-Keuls), and paired t-test were used to compare the peak responses after peptides were added. P < 0.05 was chosen as the level of significance.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of PACAP-27, PACAP-38, and VIP. PACAP-27 induced a biphasic response, a large contractile response followed by a very weak relaxation, in guinea pig gallbladder smooth muscle strips (Fig. 1). The contractile response was dependent on the concentration of PACAP-27. The peak contraction (1.13 ± 0.16 mm) induced by PACAP-27 (10–7 M) was ~20% of the maximal contraction (5.94 ± 0.80 mm) induced by ACh (10–4 M). PACAP-38 induced a contractile response similar to PACAP-27, but it was much less potent than PACAP-27. VIP, on the other hand, induced concentration-related relaxation of the muscle strips. The contractile responses to PACAP-27 were not affected by atropine (10–7 M), tetrodotoxin (10–7 M), or loxiglumide (10–5 M) (data not shown).


Figure 1
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 1. Effects of pituitary adenylate cyclase-activating polypeptide (PACAP)-38, PACAP-27, and vasoactive intestinal polypeptide (VIP) on isolated guinea pig gallbladder smooth muscle strips. Means ± SE (n = 5 guinea pigs), expressed as the percentage of the maximal response to ACh (10–4 M), are shown. Inset: representative response to PACAP-27 (10–7 M). Arrowhead indicates the time point of administration.

 
Effects of NH2 terminus (1–8)-substituted PACAP-27 analogs. The effects of the NH2 terminus-substituted peptides on the gallbladder smooth muscle strips are shown in Fig. 2. [Ala4]PACAP-27 and [Val5]PACAP-27 were significantly (P < 0.01) more potent (~150% at 10–7M) than PACAP-27 in stimulating the gallbladder. In contrast, [Ala4,Val5]PACAP-27 completely lost the stimulatory effect on the muscle strips and, similarly to VIP, exhibited a dose-related relaxation. [Ala4,Val5,Asn9]PACAP-27 also exhibited only relaxation. [des-His1]PACAP-27 and [Ala6]PACAP-27 had no significant activity. [Gly8]PACAP-27 and [D-Asp8]PACAP-27 were significantly (P < 0.05) less potent (~25%) than PACAP-27.


Figure 2
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 2. Effects of NH2 terminus-substituted PACAP-27 analogs on isolated guinea pig gallbladder (GB) smooth muscle strips. Means ± SE (n = 5), expressed as the percentage of the response to PACAP-27 (10–7 M), are shown. A positive response indicates contraction, and a negative one is relaxation. Right: representative responses to some of the analogs. Arrowheads indicate the time points of peptide administration (10–7 M).

 
Effects of COOH terminus (9–26)-substituted PACAP-27 analogs. The contractile response to [Asn9]PACAP-27 at 10–7 M was similar to that to PACAP-27 (~80%), but no further increase in activity was observed at 3 x 10–7 M (Fig. 3). [Thr11]PACAP-27 caused a relaxation (–14%) at 10–8 M, no response at 3 x 10–8 M, but a contraction at 10–7 M (~30% response to PACAP-27 at 10–7 M) and above. The responses to [Leu13]PACAP-27 were ~70% of those to PACAP-27. Substitutions of Lys21 caused a significant loss of activity; the contractile responses to [Ala21]PACAP-27, [Phe21]PACAP-27, and [Pro21]PACAP-27 were ~75%, ~30%, and ~13% of the response to PACAP-27 at 10–7 M, respectively. In addition, the contraction period was shortened and was associated with longer relaxation. The replacement of the COOH terminus of PACAP-27 with that of VIP, [Asn24, Ser25, Ile26]PACAP-27, showed no change in the activity.


Figure 3
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 3. Effects of COOH terminus-substituted PACAP-27 analogs on isolated guinea pig gallbladder smooth muscle strips. Means ± SE (n = 5), expressed as the percentage of the response to PACAP-27 (10–7 M), are shown. A positive response indicates contraction, and a negative one is relaxation. Right: representative response to some of the analogs. Arrowheads indicate the time points of peptide administration.

 
Effects of VIP analogs. All of the NH2 terminus-substituted VIP analogs induced a concentration-related relaxation of gallbladder smooth muscle strips. Their responses at 10–7 M showed no significant difference from those at 10–7 M VIP (Fig. 4). A complete replacement of the NH2 terminus of VIP with that of PACAP-27, i.e., [Gly4, Ile5, Ser9]VIP or PACAP(1–9)-VIP(10–28), also failed to exhibit a PACAP-27-like contraction. However, at a higher concentration (3 x 10–7 M), the relaxation response was less. Substitution in the central domain, [Ser11]VIP, induced a smaller response at 3 x 10–7 M.


Figure 4
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 4. Effects of VIP analogs substituted with amino acid residues of PACAP-27 on isolated guinea pig gallbladder smooth muscle strips. Means ± SE (n = 5), expressed as the percentage of the response to VIP (10–7 M), are shown. Right: representative response to some of the analogs. Arrowheads indicate the time points of peptide administration (10–7 M).

 
Effects of PACAP(6–38). PACAP(6–38), a partial PAC1 receptor antagonist, caused significant (P < 0.01) concentration-related contraction (0% at 10–8 M, 5.6 ± 1.2% at 10–7 M, and 22.3 ± 1.1% at 10–6 M) of gallbladder smooth muscle strips (Fig. 5). A prior administration of PACAP(6–38) induced a concentration-related reduction of the contractile response to PACAP-27 (10–7 M). Relaxation after the contractile response to PACAP-27 was significant at 10–7 M and 10–6 M PACAP(6–38). Significant relaxant effects of PACAP-27 (10–8 to 3 x 10–7 M) were evident in the presence of PACAP(6–38) (10–6 M) (Fig. 6).


Figure 5
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 5. Effects of a prior administration of PACAP(6–38) (10–8 to 10–6 M) on the response to PACAP-27 (10–7 M). Means ± SE (n = 5), expressed as the percentage of the maximal response to ACh (10–4 M), are shown. Right: representative responses. Arrowheads indicate the time points of administration.

 

Figure 6
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 6. Effects of a prior administration of PACAP(6–38) (10–6 M) on the response to PACAP-27 (10–8 to 3 x 10–7 M). Means ± SE (n = 5), expressed as the percentage of the maximal response to ACh (10–4 M), are shown. Inset: representative responses. Arrowheads indicate the time points of administration. C, contraction; R, relaxation.

 
PAC1, VPAC1, and VPAC2 receptor expression. Figure 7 shows RT-PCR analysis of PAC1, VPAC1, and VPAC2 receptor mRNAs in the guinea pig gallbladder. Compared with the pancreas and pancreatic duct, all three receptor transcripts were abundantly expressed in the gallbladder. Sequence analysis of a region of guinea pig PAC1 receptor encoding from the fifth transmembrane domain to the NH2-terminal intracellular domain revealed a high homology (91% in nucleotide sequence and 99% in amino acid) to human PAC1 receptor (Table 2). In addition, there was an isoform of guinea pig PAC1 receptor that contained an additional 84 nucleotides encoding 28 amino acids at the COOH-terminal end of the third intracellular loop. The nucleotide and amino acid sequences of the regions of guinea pig VPAC1 and VPAC2 receptors also had high homologies to the respective human (90% and 95%), rat (91% and 93%), and mouse (93% and 91%) sequences.


Figure 7
View larger version (29K):
[in this window]
[in a new window]

  		Fig. 7. Top: RT-PCR analysis of PAC1, VPAC1, and VPAC2 receptor mRNAs in the guinea pig gallbladder, pancreas, and pancreatic duct. Bottom: deduced amino acid sequences of PAC1 receptor (a) and its isoform (b). G, gallbladder; P, pancreas; D, pancreatic duct; TM, predicted transmembrane domain; GAPDH, glyceraldehyde 3-phosphate dehydrogenase (positive control using GAPDH-specific primers, 452 bp).

 

View this table:
[in this window]
[in a new window]

  		Table 2. Amino acid sequences of guinea pig, human, and rat PAC1, VPAC1, and VPAC2 receptors

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we used isotonic transducers, which, by choosing an appropriate weight load, allowed us simultaneous measurements of both contractile and relaxant activities of the gallbladder smooth muscle strips. In agreement with previous studies (15, 23, 24), PACAP induced contraction of the muscle strips that was followed by relaxation, whereas VIP consistently induced relaxation (Figs. 1 and 2). The stimulatory effect of PACAP-27 was independent of cholinergic nerves (15) and CCK (this study), the two major regulatory mechanisms of the gallbladder motility, but was significantly inhibited by PACAP(6–38), a PACAP-receptor antagonist (Figs. 5 and 6). It has been shown that the relaxant effect of a high concentration of PACAP-27 (10–6 M) can be partially blocked by a PACAP-receptor antagonist as well as by a VIP-receptor antagonist (15). Taken together, it appears that PACAP-27 induces the gallbladder contraction directly via PACAP receptors and the relaxation via both PACAP receptors and PACAP/VIP common receptors. Abundant expressions of PAC1, VPAC1, and VPAC2 receptor mRNAs in the guinea pig gallbladder (Fig. 7) support this interpretation.

Physiological functions of PAC1, VPAC1, and VPAC2 receptors are not yet fully understood. In the present study, the gallbladder contraction was initiated by a 10-fold higher concentration of PACAP-27 than that of VIP (10–9 M), which causes relaxation (Fig. 1). At lower concentrations (10–9 to 10–8 M), the stimulatory and inhibitory effects of PACAP-27 appear to be balanced, resulting in no gallbladder response. Thus, under physiological conditions, when cholinergic and CCK mechanisms are activated, the inhibitory effects of PACAP and VIP via VPAC1 and VPAC2 receptors may be counterbalanced by the stimulatory effect via PAC1 receptors. At higher concentrations (>3 x 10–8 M), the activation of the stimulatory pathway via PAC1 receptors overcomes the inhibitory one via VPAC1 and VPAC2 receptors, resulting in a biphasic response. Under a sustained contraction, circulation of the gallbladder may be maintained by vasodilator actions of PACAP and VIP via both PAC1 and VPAC1 and VPAC2 receptors on blood vessels.

A substitution of a single amino acid residue of the NH2 terminus of PACAP-27, especially [Ala4]PACAP-27, augmented the contractile action of PACAP-27 on the gallbladder (Fig. 2). Similarly, [Ala4]PACAP-27 induced a greater gastric blood flow response than PACAP-27 (11). A substitution of Gly4 with Ala4 may increase the efficacy of PACAP-27 to interact with PAC1 receptors. However, compared with PACAP-27, [Ala4]PACAP-27 and [Val5]PACAP-27 exhibit much lower binding affinity to rat brain membranes with concomitant reductions in stimulating cAMP productions (1). A similar loss of binding affinity and cAMP or IP3 productions of these peptides has been observed in a rat tumor cell line, AR4–2J cells (18). The binding of PACAP-27 to PAC1 receptors and cAMP production occur in the range of 10–9 to 10–7 M (1, 16, 18). The gallbladder stimulatory effect of PACAP-27 was observed in a higher concentration range (10–8 to 10–6 M) in this study and in earlier studies (15, 23). At these concentrations, intracellular signal transduction pathways were probably fully activated and the reduction of the binding affinity might not have influenced the biological response.

Unexpectedly, when Gly4-Ile5 was replaced with Ala4-Val5, the contractile activity of PACAP-27 was completely lost and only the relaxation was induced (Fig. 2). In gastric blood vessels, where PACAP-27 induces a sixfold larger vasodilatation than VIP, [Ala4,Val5]PACAP-27 behaves like VIP (11). The loss of binding affinity of [Ala4,Val5]PACAP-27 is less than that of [Ala4]PACAP-27, but the cAMP synthesis is decreased to the levels by VIP (1). Thus positions 4 and 5 are the key NH2-terminal residues of PACAP-27 that distinguish interactions with PAC1 receptors from those with VPAC1 and VPAC2 receptors in the gallbladder.

Conversely, when the NH2-terminal residues of VIP were replaced with those of PACAP-27 (Gly4, Ile5, or Gly4-Ile5), their relaxation activities were very similar to those of VIP, indicating that the {alpha}-helical region of PACAP-27 is also important for gallbladder contraction. [Gly4,Ile5,Ser9]VIP showed similar concentration-dependent relaxation to [Gly4,Ile5]VIP and [Ala4,Val5]PACAP-27. However, at a high concentration, the relaxation induced by [Gly4,Ile5,Ser9]VIP was only 46% of [Gly4,Ile5]VIP. [Gly4,Ile5,Ser9]VIP has a higher binding affinity to PAC1 receptors than [Gly4,Ile5]VIP, but its affinity is similar to that of [Ala4,Val5,Asn9]PACAP-27 (1). This probably explains the reduced relaxation observed at a high concentration of [Gly4,Ile5,Ser9]VIP.

A substitution of the {alpha}-helical region of PACAP-27 with the corresponding residue of VIP reduced the contractile action of PACAP-27 by 20–70% (Fig. 3). It is interesting to note that a substitution of Ser11 of PACAP-27 with Thr11 induced a complex response of the gallbladder: relaxation at a low concentration (VIP-like), no response at 3 x 10–8 M, but contraction at a high concentration (PACAP-like). Conversely, a substitution of Thr11 of VIP with Ser11 decreased the relaxation of the gallbladder (Fig. 4). The reduction was evident at higher concentrations (3 x 10–7 M) of [Ser11]VIP. Thus Ser11 of PACAP-27 is important for the contractile activity of this peptide. In addition, Thr11 of VIP has been shown to be important for the binding of VIP to VPAC2 but not VPAC1 receptors (13). Thus, this substitution may have increased the binding affinity of PACAP-27 to VPAC2 receptors and thereby induced an intermediate response to PACAP and VIP in the gallbladder. This possibility requires further analyses of binding affinities of these analogs to VPAC1 and VPAC2 receptors.

In agreement with binding studies (1), where [Asn24, Ser25,Ile26]PACAP-27 exhibits the same binding affinity to PAC1 receptors and cAMP productions as PACAP-27, the replacement of the COOH terminus of PACAP-27 with that of VIP had no effect on the contractile activity on the gallbladder. These observations indicate that the COOH-terminal three residues are not critical for the difference in their actions. However, the COOH-terminal {alpha}-helical region (Gly28-Arg34) may be important for the action of PACAP-38 on the gallbladder. Unexpectedly, the most potent competitive PAC1 receptor antagonist, PACAP(6–38) devoid of the NH2 terminus important for the action of PACAP-27 (17), did exhibit concentration-related contraction (Fig. 5). In AR4–2J cells, substitutions of the NH2-terminal residues of PACAP-38 with Ala4, Val5, or Ala4-Val5, unlike PACAP-27, little affected their binding to PAC1 receptors as well as cAMP and IP3 formation (18). Furthermore, it has been shown previously that shorter fragments, especially PACAP(14–27) and PACAP(14–38), were able to stimulate adenylate cyclase (21). These observations suggest that binding of PACAP(6–38) to PAC1 receptors may activate signal pathways leading to the gallbladder contraction. The presence of the COOH-terminal {alpha}-helical region is probably related to a much weaker effect of PACAP-38 than PACAP-27 (Fig. 1).

As with most of the secretin/glucagon/VIP family of peptides, the NH2-terminal His1 was important for the action of PACAP-27 on the gallbladder. Phe6 and Asp8 were also important because substitutions with Ala caused significant loss of binding affinity (1) and gallbladder contraction. A substitution of Lys21 of PACAP-27 with Ala21 reduced the contractile activity by only 23%, suggesting that this basic residue is of minor importance for its interaction with PAC1 receptors. Although further reductions of contractile activities were observed by a substitution with Phe21 or Pro21, the relaxations were unaffected (Fig. 3). This is in contrast to the interaction of VIP with VPAC1 and VPAC2 receptors, in which a substitution of VIP with Ala21 decreases both the binding affinity and adenylate cyclase activation to only 2–5% of the native peptide (13). This may be related to the difference in the structures of the two peptides; VIP maintains a continuous {alpha}-helix in this region (20), whereas PACAP-27 exhibits a break of {alpha}-helical structure at this point (25).

The present study demonstrates the expression of an isoform of the PAC1 receptor in the guinea pig gallbladder. The nucleotide sequence analysis revealed that the isoform was a splice variant that contained an additional 84 nucleotides encoding 28 amino acids in the third intracellular loop (Fig. 7), the key domain for coupling to phospholipase C via a specific G protein. The deduced amino acid sequence was identical to that of a "hop" variant reported in rats (19) and humans (16). Alternative splicing of two exons of rat PAC1-receptor gene generates four major splice variants, named hip, hop1, hop2, and hip-hop2. Each splice variant can be differentially coupled to two intracellular signal transduction pathways and thus results in variable elevations of cAMP and IP3 in a tissue-specific manner (19). Among the four splice variants of human PAC1 receptors, the hop variant had a fivefold greater efficacy in IP3 production than the authentic PAC1 receptor (16). Because PACAP induces relaxation of smooth muscles in most of the tissues that express PAC1 receptors (6), the expression of the hop variant of PAC1 receptor might be related to the contractile response observed in the gallbladder. Further studies are necessary to identify the cellular localizations of PAC1 receptors, the hop variant, and VPAC1 and VPAC2 receptors in the gallbladder.

In conclusion, our study has demonstrated abundant expressions of PAC1 receptor, its hop variant, and VPAC1 and VPAC2 receptor mRNAs in the guinea pig gallbladder. PACAP-27 induces contraction of the gallbladder smooth muscles via PAC1 and/or its hop variant receptors. The positions 4 and 5 are the key NH2-terminal residues of PACAP-27 that distinguish PAC1/hop receptors from VPAC1/VPAC2 receptors in the gallbladder. However, both the NH2-terminal disordered region and {alpha}-helical region of PACAP-27 are required for initiating gallbladder contraction. Tissue-specific expressions of PACAP and VIP and their receptors determine the net functions of PACAP and VIP.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants from the Ministry of Education, Culture, Science, and Technology, Japan Society for the Promotion of Science, and Japan China Medical Association to S. Naruse. M. Wei and S. Zhang were visiting scientists supported by the government of Jiangsu Province, China.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Naruse, Internal Medicine, Nagoya Univ. Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan (e-mail: snaruse{at}med.nagoya-u.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Ando E, Nokihara K, Naruse S, Wray V. Recognition of pituitary adenylate cyclase-activating polypeptide/vasoactive intestinal polypeptide (PACAP/VIP) hybrids and related peptides by rat brain membranes. Biomed Pept Proteins Nucleic Acids 2: 41–46, 1992.
  2. Arimura A. Pituitary adenylate cyclase activating polypeptide (PACAP): discovery and current status of research. Regul Pept 37: 287–303, 1992.[Web of Science][Medline]
  3. Blankenfeldt W, Nokihara K, Naruse S, Lessel U, Schomburg D, Wray V. NMR spectroscopic evidence that helodermin, unlike other members of the secretin/VIP family of peptides, is substantially structured in water. Biochemistry 35: 5955–5962, 1996.[CrossRef][Medline]
  4. Harmar AJ, Arimura A, Gozes I, Journot L, Laburthe M, Pisegna JR, Rawlings SR, Robberecht P, Said SI, Sreedharan SP, Wank SA, Waschek JA. International Union of Pharmacology. XVIII. Nomenclature of receptors for vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide. Pharmacol Rev 50: 265–270, 1998.[Abstract/Free Full Text]
  5. Ito O, Naruse S, Kitagawa M, Ishiguro H, Ko S, Nakajima M, Hayakawa T. The effect of VIP/PACAP family of peptides on pancreatic blood flow and secretion in conscious dogs. Regul Pept 78: 105–112, 1998.[CrossRef][Web of Science][Medline]
  6. Lauff JM, Modlin IM, Tang LH. Biological relevance of pituitary adenylate cyclase-activating polypeptide (PACAP) in the gastrointestinal tract. Regul Pept 84: 1–12, 1999.[CrossRef][Web of Science][Medline]
  7. Mawe GM, Ellis LM. Chemical coding of intrinsic and extrinsic nerves in the guinea pig gallbladder: distributions of PACAP and orphanin FQ. Anat Rec 262: 101–109, 2001.[CrossRef][Medline]
  8. Miyata A, Arimura A, Dahl RR, Minamino N, Uehara A, Jiang L, Culler MD, Coy DH. Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells. Biochem Biophys Res Commun 164: 567–74, 1989.[CrossRef][Web of Science][Medline]
  9. Mizumoto A, Fujimura M, Ohtawa M, Ueki S, Hayashi N, Itoh Z, Fujino M, Arimura A. Pituitary adenylate cyclase activating polypeptide stimulates gallbladder motility in conscious dogs. Regul Pept 42: 39–50, 1992.[CrossRef][Web of Science][Medline]
  10. Naruse S, Sazi T, Ozaki T, Nokihara K, Wray V. The effect of PACAP and VIP on guinea pig pulmonary artery. Biomed Res 15, Suppl 2: 217–219, 1994.
  11. Naruse S, Nakamura T, Wei M, Ando E, Nokihara K, Wray V, Ozaki T, Kitagawa M, Hayakawa T. Effects of PACAP-VIP hybrid peptides on gastric blood flow in conscious dogs. Ann NY Acad Sci 805: 511–515, 1996.[Web of Science][Medline]
  12. Naruse S, Ito O, Kitagawa M, Ishiguro H, Nakajima M, Hayakawa T. Effects of PACAP/VIP/secretin on pancreatic and gastrointestinal blood flow in conscious dogs. Ann NY Acad Sci 865: 463–465, 1998.[CrossRef][Web of Science][Medline]
  13. Nicole P, Lins L, Rouyer-Fessard C, Drouot C, Fulcrand P, Thomas A, Couvineau A, Martinez J, Brasseur R, Laburthe M. Identification of key residues for interaction of vasoactive intestinal peptide with human VPAC1 and VPAC2 receptors and development of a highly selective VPAC1 receptor agonist. Alanine scanning and molecular modeling of the peptide. J Biol Chem 275: 24003–24012, 2000.[Abstract/Free Full Text]
  14. Pang PK, Kline LW. Protein kinase C mediates the contractile actions of pituitary adenylate cyclase activating polypeptide in guinea pig gallbladder strips. Regul Pept 77:63–67, 1998.[CrossRef][Web of Science][Medline]
  15. Parkman HP, Pagano AP, Ryan JP. Dual effects of PACAP on guinea pig gallbladder muscle via PACAP-preferring and VIP/PACAP-preferring receptors. Am J Physiol Gastrointest Liver Physiol 272: G1433–G1438, 1997.[Abstract/Free Full Text]
  16. Pisegna JR, Wank SA. Cloning and characterization of the signal transduction of four splice variants of the human pituitary adenylate cyclase activating polypeptide receptor. Evidence for dual coupling to adenylate cyclase and phospholipase C. J Biol Chem 271: 17267–17274, 1996.[Abstract/Free Full Text]
  17. Robberecht P, Gourlet P, De Neef P, Woussen-Colle MC, Vandermeers-Piret MC, Vandermeers A, Christophe J. Structural requirements for the occupancy of pituitary adenylate-cyclase-activating-peptide (PACAP) receptors and adenylate cyclase activation in human neuroblastoma NB-OK-1 cell membranes. Discovery of PACAP(6–38) as a potent antagonist. Eur J Biochem 207: 239–246, 1992.[Web of Science][Medline]
  18. Schafer H, Zheng J, Morys-Wortmann C, Folsch UR, Schmidt WE. Structural motifs of pituitary adenylate cyclase-activating polypeptide (PACAP) defining PAC1-receptor selectivity. Regul Pept 79: 83–92, 1999.[CrossRef][Web of Science][Medline]
  19. Spengler D, Waeber C, Pantaloni C, Holsboer F, Bockaert J, Seeburg PH, Journot L. Differential signal transduction by five splice variants of the PACAP receptor. Nature 365: 170–175, 1993.[CrossRef][Medline]
  20. Theriault Y, Boulanger Y, St-Pierre S. Structural determination of the vasoactive intestinal peptide by two-dimensional H-NMR spectroscopy. Biopolymers 31: 459–464, 1991.[CrossRef][Web of Science][Medline]
  21. Vandermeers A, Vandenborre S, Hou X, de Neef P, Robberecht P, Vandermeers-Piret MC, Christophe J. Antagonistic properties are shifted back to agonistic properties by further N-terminal shortening of pituitary adenylate-cyclase-activating peptides in human neuroblastoma NB-OK-1 cell membranes. Eur J Biochem 208: 815–819, 1992.[Web of Science][Medline]
  22. Vaudry D, Gonzalez BJ, Basille M, Yon L, Fournier A, Vaudry H. Pituitary adenylate cyclase-activating polypeptide and its receptors: from structure to functions. Pharmacol Rev 52: 269–324, 2000.[Abstract/Free Full Text]
  23. Wei M, Naruse S, Nakamura T, Nokihara K, Ozaki T. The effect of pituitary adenylate cyclase activating polypeptide (PACAP) 38 on gallbladder smooth muscle in vitro. Biomed Res (Tokyo) 15: 221–223, 1994.
  24. Wei M, Naruse S, Nokihara K, Ozaki T, Ando E, Wray V. A study of structure-activity relationship of PACAP. China Natl J New Gastroenterol 3: 131–133, 1996.
  25. Wray V, Kakoschke C, Nokihara K, Naruse S. Solution structure of pituitary adenylate cyclase activating polypeptide by nuclear magnetic resonance spectroscopy. Biochemistry 32: 5832–5841, 1993.[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/1/G76    most recent
00279.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wei, M.
Right arrow Articles by Naruse, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wei, M.
Right arrow Articles by Naruse, S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.