INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

THREE RECEPTOR TYPES for pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal polypeptide (VIP) have been identified that differ in their relative affinities for the two peptides and in their signaling pathways. The PAC₁ receptor (PAC₁R) exhibits high affinity for PACAP (6, 9, 22) and maxadilan (12), an unrelated peptide present in sand flies, and low affinity for VIP (500- to 1,000-fold less). Several splice variants of the PAC₁ receptor have been identified that differ in their relative ability to activate adenylyl cyclase and phospholipase C- (18, 19). VPAC₁ and VPAC₂ receptors, previously known as VIP₁/PACAP₂ and VIP₂/PACAP₃ receptors, respectively, exhibit equally high affinity for PACAP and VIP (6, 22). Our recent studies (13, 15) identified a heterologous, structurally unrelated receptor, the natriuretic peptide clearance receptor (NPR-C), expressed in gastrointestinal smooth muscle of mammalian species, which exhibits high affinity for VIP and PACAP, as well as for its natural ligand, atrial natriuretic peptide. Smooth muscle of the gut in various species expresses mainly VPAC₂ receptors and NPR-C (14, 15, 20). VIP and PACAP bind with high affinity to gastric muscle cells of rabbit (13) and guinea pig (GP) (1). The binding reflects interaction of VIP and PACAP with VPAC₂ receptors coupled via G_s to adenylyl cyclase and with NPR-C coupled via G_i1 and G_i2 to endothelial nitric oxide synthase (eNOS) (14); both signaling pathways participate in mediating gastric smooth muscle relaxation.

However, in GP tenia coli, which does not express eNOS (21), VIP and PACAP appear to interact with distinct VIP and PACAP receptors and to induce relaxation by distinct mechanisms (8). Thus VIP but not PACAP stimulates adenylyl cyclase activity; consequently, relaxation induced by VIP is inhibited by H-89, a selective inhibitor of cAMP-dependent protein kinase, whereas relaxation induced by PACAP is inhibited by the small-conductance K⁺ channel blocker apamin (IC₅₀ ~2 nM). In GP gastric smooth muscle, the antagonists VIP-(10-28) and PACAP-(6-38) inhibit relaxation induced by either VIP or PACAP; in GP tenia coli, however, VIP-(10-28) inhibits relaxation induced by VIP only, whereas PACAP-(6-38) inhibits relaxation induced by PACAP only. Selective inactivation of PACAP receptors in GP tenia coli preserves the relaxant response to VIP, whereas selective inactivation of VIP receptors preserves the response to PACAP.

On the basis of these functional results, we concluded that GP (or rabbit) gastric muscle expressed VPAC₂ receptors, whereas GP tenia coli expressed a VIP-specific receptor that was distinct from VPAC₁ and VPAC₂ receptors but that like them mediated relaxation by activating adenylyl cyclase. GP tenia coli also expressed a PACAP-specific receptor that did not recognize VIP or activate adenylyl cyclase and was thus different from splice variants of the PAC₁ receptor that activate adenylyl cyclase. Using RT-PCR, Northern blot, and sequence analysis, we have characterized the NH₂-terminal extracellular domain of the VIP receptor in rabbit and GP gastric and tenia coli smooth muscle (20) and have shown that amino acid residues crucial for VIP binding, including aspartate, the tryptophan and glycine residues (corresponding to D68, W73, and G109 of the human VPAC₁ receptor) (2, 5), and all six cysteine residues were conserved in rabbit and guinea pig. The sequence in GP tenia coli differed from that in GP gastric muscle by two adjacent residues, L⁴⁰/L⁴¹ in GP gastric muscle versus F⁴⁰/F⁴¹ in GP tenia coli (see Fig. 1). We postulated that these two residues could account for the ability of the receptors in GP tenia coli to bind VIP but not PACAP.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Extracellular domains of rat VPAC2 receptor and guinea pig (GP) vasoactive intestinal polypeptide (VIP) receptors (VIP-R). Chimeric constructs were derived by replacement of S18-C108 in rat VPAC2 by the corresponding sequence in guinea pig receptors (numbering of residues in accordance with position in rat VPAC2 receptors). Residues L40/L41 (F40/F41 in tenia coli) are a characteristic feature of VPAC2 receptors (compare with sequences in Fig. 7).

In this study, two chimeric receptors were constructed in which the NH₂-terminal extracellular domain of the rat wild-type VPAC₂ receptor was replaced by the NH₂-terminal extracellular domain of either GP gastric or GP tenia coli VIP receptor. In addition, a mutant rat VPAC₂ receptor was constructed in which residues L⁴⁰/L⁴¹ in the extracellular domain were replaced with F⁴⁰/F⁴¹ by site-directed mutagenesis. Wild-type, chimeric, and mutant receptors were transiently expressed in COS-1 cells, and the receptors were characterized by VIP and PACAP binding and cAMP formation. The rat-GP tenia coli chimeric receptor was shown to bind VIP selectively and to respond to VIP but not to PACAP with an increase in cAMP.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Construction of chimeric receptors. Chimeric receptors were constructed in which the NH₂-terminal domain of wild-type rat VPAC₂ receptor was replaced with the corresponding domain (S¹⁸-C¹⁰⁸) of GP gastric or tenia coli smooth muscle receptor (see Fig. 1). The technique involved a two-step overlap PCR approach using the ExSite PCR-based site-directed mutagenesis kit. The coding region of cDNA corresponding to rat wild-type VPAC₂ receptor was subcloned in pGEM-4Z vector at XbaI and EcoRI sites, and nucleotide sequence 120-371 was deleted with primers: 5'-TCAGAGACATTCCCGGATTTCATAGATGCGTGTGG-3' (sense) and 5'-CACCCGCACC AGCAACCAGCA-3' (antisense). PCR was performed at 94°C for 4 min, 50°C for 2 min, 72°C for 2 min for 1 cycle, 94°C for 1 min, 56°C for 2 min, 72°C for 1 min for 15 cycles, followed by 1 cycle at 72°C for 5 min. The cDNAs (275 bp) encoding most of the NH₂-terminal extracellular domain (S¹⁸-C¹⁰⁸) of GP gastric or tenia coli VIP receptor cloned in pCR II vector were amplified with primers: 5'-AGCAGCCATCCACCCAGAATG-3' (sense) and 5'-CCACACGCATCTATGAAATCCG-3' (antisense) at 94°C for 1 min for 1 cycle, followed by 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s for each of 15 cycles, and extended at 72°C for 5 min. The underlined primers consisted of overlap sequences of rat and GP gastric or tenia coli VIP receptor specifying the junction site. The PCR products from the first step were purified by agarose gel and used for overlap extension in the second step, which was performed at 94°C for 4 min, 50°C for 2 min, 72°C for 2 min for 1 cycle; 94°C for 1 min, 56°C for 2 min, and 72°C for 1 min for 12 cycles, followed by 1 cycle at 72°C for 5 min.

Site-directed mutagenesis. The coding region of the cDNA corresponding to rat wild-type VPAC₂ receptor was subcloned into pGEM-4Z vector at the XbaI and EcoRI sites. Site-directed mutant (L40F and L41F) of wild-type rat VPAC₂ was generated using the primers 5'-TGCAGAGTTCTTCAGCAGCCAAATGGAGAAT-3' (sense), where the underlined sequence stands for the phenylalanine code, and 5'-CATTTTGTCTCCTCTTCCTGTATTTCC-3' (antisense) and the ExSite PCR-based site-directed mutagenesis kit. PCR was performed at 94°C for 4 min, 50°C for 2 min, 72°C for 1 min for 1 cycle, 94°C for 1 min, 56°C for 2 min, 72°C for 1 min for 12 cycles, and 72°C for 5 min for 1 cycle. The mutation was confirmed by DNA sequencing.

Expression of wild-type, chimeric, and mutant VPAC₂ receptors in COS-1 cells. The cDNAs for 1) wild-type rat VPAC₂, 2) chimeric rat-GP gastric muscle, 3) chimeric rat-GP tenia coli VPAC₂ receptors, and 4) a mutant rat VPAC₂ receptor (L40F and L41F) were subcloned into the expression vector pCDL/SR at XbaI and EcoRI sites and transfected into COS-1 cells by DEAE-dextran pretreatment using the ProFection mammalian transfection system. Briefly, COS-1 cells (American Type Culture Collection) were plated at a density of 2.5 × 10⁶ and transfected with 10 µg of each construct. Control cells were transfected with pCDL/SR vector under the same conditions. The transfected cells were maintained at 37°C in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 200 µg of penicillin-streptomycin in 10% CO₂ for 48 h.

Expression of chimeric and mutant receptors in COS-1 cells. Receptor expression was identified by RT-PCR, Southern blot, and Northern blot. Total RNA was prepared from transfected COS-1 cells and was treated with RNase-free DNase I (1 U/µg RNA) in 10 mM Tris · HCl (pH 7.4) and 5 mM MgCl₂ reaction mixture at 37°C for 30 min. DNase I was inactivated by heating at 70°C for 10 min.

Ligand binding assay. The binding assay was performed as described previously (13). COS-1 cells transfected with different constructs of receptors were detached from culture dishes by incubation with 0.53 mM EDTA in phosphate-buffered saline at 37°C for 30 min. Cells were collected by centrifugation at 400 g and resuspended in Dulbecco's modified Eagle's medium containing BSA (0.1%), amastatin (10 µM), phosphoramidon (1 µM), and bacitracin (0.7 mM) at 10⁶ cells/ml. Suspended cells (500 µl) were incubated for 5 min at room temperature with 50 pM ¹²⁵I-labeled VIP alone or in the presence of various concentrations of nonlabeled ligand. Bound and free radioligands were separated by rapid filtration under reduced pressure through 5-µm polycarbonate Nucleopore filters followed by repeated washing (3 times) with 5 ml of ice-cold phosphate-buffered saline containing 0.1% BSA. Nonspecific binding was measured as the amount of radioactivity associated with the cells in the presence of 10 µM nonlabeled ligand (30 ± 2% of total binding). Specific binding was calculated as the difference between total and nonspecific binding. IC₅₀ was calculated from competition curves using the P.fit program (Biosoft, Elsevier Publishing, Cambridge, UK).

cAMP assay. cAMP was measured by radioimmunoassay as described previously (13, 14). Transfected COS-1 cells were detached and incubated (10⁶ cells/0.5 ml) in the presence of 200 µM 3-isobutyl-1-methylxanthine with or without the indicated concentration of VIP or PACAP-27 for 60 s. cAMP was measured in duplicate by radioimmunoassay using 100-µl aliquots of reconstituted samples, and the results were expressed in picomoles per milligram of protein.

Materials. pGEM-4Z vector, and ProFection Mammalian Transfection System, and DEAE-Dextran were obtained from Promega (Madison, WI); ExSite PCR-based site-directed mutagenesis kit was from Stratagene (La Jolla, CA); VIP and PACAP-27 were from Bachem (Torrance, CA); and ¹²⁵I-VIP, ¹²⁵I-cAMP, [-³²P]ATP, and [-³²]dCTP were from New England Nuclear (Boston, MA). Primers and oligonucleotide probes were synthesized by Integrated DNA Technologies (Coralville, IA). Complementary DNA for rat VPAC₂ receptor was a gift from Dr. J. Pisegna, University of California, Los Angeles; cDNA for human GAPDH was obtained from Clontech (Palo Alto, CA).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Expression of wild-type, chimeric, and mutant VIP receptors in COS-1 cells. Two chimeric receptors were constructed using a two-step overlap PCR approach in which the NH₂-terminal extracellular domain (S¹⁸-C¹⁰⁸) of rat wild-type VPAC₂ receptor was replaced by the corresponding domain of GP gastric or tenia coli receptor (Fig. 1). These receptors were denoted chimeric-G and chimeric-T, respectively. In addition, a mutant rat VPAC₂ receptor (L40F/L41F) was constructed in which the L⁴⁰ and L⁴¹ residues were replaced by the corresponding residues (F⁴⁰, F⁴¹) found exclusively in the NH₂-terminal domain of the tenia coli receptor (Fig. 1). This mutant rat receptor was denoted VPAC₂-mutant. The constructs for those receptors were transiently transfected into COS-1 cells, and receptor expression was determined by Southern and Northern blot (Fig. 2).

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Expression of wild-type, chimeric, and mutant receptors in COS-1 cells. A: Southern blot analysis. RT-PCR products obtained from COS-1 cells transfected with different constructs were hybridized with 32P-labeled nested oligonucleotide probe derived from conserved region of cDNA encoding rat VPAC2 and guinea pig VIP receptors (top). The hybridization band (275 bp) was detected for wild-type rat VPAC2 receptor (lane 2), chimeric rat-guinea pig gastric receptor (chimeric-G, lane 4), chimeric rat-guinea pig tenia coli receptor (chimeric-T, lane 6), and rat mutant VPAC2 receptor (L40F and L41F, lane 8). No band was obtained in COS-1 cells transfected with vector alone (lane 1) or in the absence of reverse transcriptase (RT) (lanes 3, 5, 7, and 9). With a nested probe derived from cDNA specific for guinea pig VIP receptors, the hybridization bands could be detected only for chimeric-G and chimeric-T (lanes 4 and 6, bottom). B: Northern blot analysis. The specific transcripts (~1.4 kb) for VPAC2 receptor were detected in COS-1 cells transfected with constructs for wild-type rat VPAC2 receptor (lane 2), chimeric-G (lane 3), chimeric-T (lane 4), and rat VPAC2 mutant receptor (lane 5); lane 1 is sham vector. Bottom: transcripts for glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Ligand binding. In COS-1 cells transiently transfected with wild-type rat VPAC₂ receptors or with mutant rat VPAC₂ receptors, specific ¹²⁵I-VIP binding was inhibited by VIP and PACAP-27 in a concentration-dependent fashion (Figs. 3A and 4A). The IC₅₀ values for VIP and PACAP-27 were closely similar (wild-type receptor: IC₅₀ VIP 0.7 ± 0.1 nM, PACAP-27 0.6 ± 0.2 nM; mutant VPAC₂ receptor: IC₅₀ VIP 1.5 ± 0.4 nM, PACAP-27 2.5 ± 0.1 nM). In cells transfected with chimeric-G receptors also, VIP and PACAP-27 inhibited ¹²⁵I-VIP with identical IC₅₀ values (0.3 ± 0.1 and 0.4 ± 0.1 nM, respectively) (Fig. 5A). In cells transfected with chimeric-T receptors, VIP inhibited ¹²⁵I-VIP binding with an IC₅₀ of 0.5 ± 0.1 nM that was similar to that found in cells transfected with rat wild-type and mutant VPAC₂ receptors or chimeric-G receptors (Fig. 6A). In contrast, the IC₅₀ for PACAP-27 in cells transfected with chimeric-T receptors was >1,000-fold higher than for VIP (Fig. 6A).

View larger version (10K): [in this window] [in a new window]   Fig. 3.   125I-VIP binding and stimulation of cAMP in COS-1 cells transfected with wild-type rat VPAC2 receptors. A: VIP and pituitary adenylate cyclase-activating peptide (PACAP)-27 inhibited 125I-VIP binding with equal affinity. Results are expressed as percentage of control specific binding. Values are means ± SE of 5 experiments. B: VIP and PACAP-27 stimulated cAMP formation with equal potency. Results are expressed as pmol/mg protein. Values are means ± SE of 3-5 experiments.

View larger version (11K): [in this window] [in a new window]   Fig. 4.   125I-VIP binding and stimulation of cAMP in COS-1 cells transfected with mutant rat VPAC2 receptors. A: VIP and PACAP-27 inhibited 125I-VIP binding with equal affinity. Results are expressed as percentage of control specific binding. Values are means ± SE of 5 experiments. B: VIP and PACAP-27 stimulated cAMP formation with equal potency. Results are expressed as pmol/mg protein. Values are means ± SE of 3-5 experiments.

View larger version (10K): [in this window] [in a new window]   Fig. 5.   125I-VIP binding and stimulation of cAMP in COS-1 cells transfected with chimeric rat-guinea pig gastric VIP receptors. A: VIP and PACAP-27 inhibited 125I-VIP binding with equal affinity. Results are expressed as percentage of control specific binding. Values are means ± SE of 5 experiments. B: VIP and PACAP-27 stimulated cAMP formation with equal potency. Results are expressed as pmol/mg protein. Values are means ± SE of 3-5 experiments.

View larger version (11K): [in this window] [in a new window]   Fig. 6.   125I-VIP binding and stimulation of cAMP in COS-1 cells transfected with chimeric rat-guinea pig tenia coli VIP receptors. A: only VIP inhibited 125I-VIP binding with high affinity. Affinity for PACAP-27 was >1,000-fold lower. Results are expressed as percentage of control specific binding. Values are means ± SE of 5 experiments. B: VIP stimulated cAMP formation with high potency; PACAP-27 had a minimal effect at high concentrations. Results are expressed as pmol/mg protein. Values are means ± SE of 3-5 experiments.

Activation of adenylyl cyclase. The potency with which VIP and PACAP stimulated cAMP formation paralleled the affinity of the peptides for the transfected receptors. In COS-1 cells transfected with wild-type and mutant VPAC₂ receptors, both VIP and PACAP-27 stimulated cAMP formation in a concentration-dependent fashion (Figs. 3B and 4B). The EC₅₀ values for VIP and PACAP-27 were 48 ± 8 and 31 ± 14 nM, respectively, for VPAC₂ receptors and 14 ± 6 nM and 8 ± 3 nM, respectively, for VPAC₂-mutant receptors. In cells transfected with chimeric-G receptors also, VIP and PACAP-27 stimulated cAMP formation with identical EC₅₀ values of 13 ± 5 nM and 12 ± 6 nM, respectively (Fig. 5B). In cells transfected with chimeric-T receptors, VIP stimulated cAMP formation with an EC₅₀ of 3 ±1 nM that was similar to that found for wild-type and mutant rat VPAC₂ receptors or chimeric-G receptors (Fig. 6B). In contrast, the EC₅₀ for PACAP-27 in cells transfected with chimeric-T receptors was >1,000-fold higher than for VIP (Fig. 6B).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

A series of studies by Laburthe and coworkers (2-5, 16, 17) on the determinants of VIP binding to the human VPAC₁ receptor has culminated in the identification of the three-dimensional structure of the NH₂-terminal domain of the receptor (10). Residues E³⁶, W⁶⁷, D⁶⁸, W⁷³, and G¹⁰⁹ (Fig. 7) known to be crucial for VIP (and PACAP) binding were shown to be gathered around a negatively charged groove. Site-directed mutagenesis showed that other residues (P⁷⁴, P⁸⁷, F⁹⁰, and W¹¹⁰) suggested by the model were also crucial to VIP binding (Ref. 10; Fig. 7). Receptor alignment disclosed that all these residues are also conserved in the rat and pig VPAC₁ receptor, as well as in the human, rat, mouse, rabbit, and GP gastric VPAC₂ receptor and the GP tenia coli VIP-specific receptor (Fig. 7). Furthermore, these residues are also conserved in the human, rat, and bovine PAC₁ receptor (18, 19), except for a glutamate residue corresponding to E36 in the human VPAC₁ receptor. Accordingly, none of these residues could account for selective binding of PACAP to PAC₁ receptors or the selective binding of VIP to the tenia coli VIP receptor.

View larger version (44K): [in this window] [in a new window]   Fig. 7.   Alignment of receptors relative to human VPAC1 receptors. All amino acid residues in the extracellular domain shown to be crucial to binding of VIP (and PACAP) in human VPAC1 receptors are conserved in VPAC1 and VPAC2 receptors of other species and in the VIP-specific receptor of tenia coli. Other residues crucial to VIP binding in human VPAC1 receptors (D196 in the 1st extracellular loop and T343 in the 6th transmembrane segment) are also conserved in rat and human VPAC2 receptors. A pair of adjacent LL or FF residues is present only in VPAC2 receptors (corresponding to residues 53/54 in human VPAC1 receptors).

Inspection of the extracellular domain shows that a pair of adjacent leucine residues are a characteristic feature of VPAC₂ receptors (L⁴⁰/L⁴¹ in rat, mouse, rabbit, and GP gastric muscle and L⁴¹/L⁴² in human) and are absent from VPAC₁ and PAC₁ receptors (compare alignment in Figs. 1 and 7). The extracellular domain of the receptor in tenia coli was identical to that in gastric muscle except for the presence of a pair of adjacent phenylalanine residues (F⁴⁰/F⁴¹). The results of the present study indicate that this unique feature accounts for the ability of the receptor in tenia coli to recognize VIP exclusively. A chimeric receptor in which the extracellular domain of the rat VPAC₂ receptor was replaced by the corresponding domain of the GP gastric VPAC₂ receptor bound VIP and PACAP with equally high affinity and stimulated cAMP formation with equal potency. Binding affinity and functional potency were closely similar to those of the wild-type rat VPAC₂ receptor. In contrast, a chimeric receptor in which the extracellular domain of the rat VPAC₂ receptor was replaced by the corresponding domain of the GP tenia coli VIP receptor bound only VIP with high affinity and stimulated cAMP formation with high potency. Affinity for PACAP was >1,000-fold less, and stimulation of cAMP formation was minimal at high concentrations. Thus the VIP receptor expressed in GP gastric muscle is a characteristic VPAC₂ receptor, whereas the VIP receptor expressed in tenia coli is a distinct receptor that possesses many of the structural features of a VPAC₂ receptor but is otherwise distinct and, therefore, is better labeled as a VIP-specific receptor.

The results obtained in the rat VPAC₂-mutant receptor suggest, however, that expression of a pair of adjacent phenylalanine residues in position 40/41 is not sufficient to alter the characteristics of a rat VPAC₂ receptor and to endow it with specificity for VIP. Although the sequences of the extracellular domains of the receptor in GP gastric muscle and tenia coli are identical except for the pair of leucine/phenylalanine residues in positions 40 and 41, the amino acid sequence in the rat VPAC₂ receptor differs from them in 9 of 23 residues between the second and fourth cysteine (C³⁷-C⁶⁰) (Fig. 1). By themselves, these residues cannot account for VIP specificity, because the rat VPAC₂ receptor and the GP gastric receptor exhibit closely similar high affinities for VIP and PACAP. We postulate that both the presence of F⁴⁰/F⁴¹ and the distinct sequences in this region of the extracellular domain determine the specificity of the receptor in GP tenia coli for VIP.

In previous studies (14, 15), we showed that ¹²⁵I-VIP binding to tenia coli muscle membranes was completely inhibited by VIP and partly inhibited by cANP(4-23), a selective ligand for NPR-C. ¹²⁵I-VIP binding reflected the ability of VIP to interact with a cognate VIP receptor and with the heterologous natriuretic peptide receptor, NPR-C. This dual binding of VIP precluded the use of tenia coli to determine the specificity of VIP binding relative to PACAP, because PACAP also can bind to NPR-C and thus compete with VIP for binding to NPR-C. The binding of VIP to distinct receptors in tenia coli leads to concurrent activation of two signaling pathways. Activation of the VIP receptor induces G_s-dependent activation of adenylyl cyclase and stimulation of cAMP formation, whereas activation of NPR-C induces G_i1/G_i2-dependent activation of phospholipase C-3 and stimulation of phosphoinositide hydrolysis. The net response is relaxation mediated by predominant activation of cAMP-dependent protein kinase. A contractile response that reflects inositol trisphosphate-dependent Ca²⁺ release can be unmasked after blockade of cAMP-dependent protein kinase activity.

It should be emphasized, as noted above, that the PACAP receptor in tenia coli, which exhibits no affinity for VIP and does not activate adenylyl cyclase, is distinct not only from the VIP-specific receptor but also from VPAC₁, VPAC₂, and PAC₁ receptors. It is possible that it represents an unrecognized splice variant of PAC₁ that does not activate adenylyl cyclase.

Tenia coli of the GP has featured prominently in the development of concepts on inhibitory neurotransmission and inhibitory (hyperpolarizing) junction potentials in visceral smooth muscle. Our recent studies (8) and those of others (11) raised doubts about the suitability of this muscle tissue. In smooth muscle from other regions, inhibitory junction potentials and relaxation are mediated by nitric oxide (NO) released from nerve terminals and from smooth muscle by VIP/PACAP-dependent activation of eNOS. In tenia coli, which are devoid of eNOS, nerve-stimulated relaxation is not dependent on release of NO from nerve terminals or on generation of NO from muscle cells by the action of VIP of PACAP. VIP induces relaxation by stimulating cAMP production, whereas PACAP induces relaxation and hyperpolarization by activating apamin-sensitive K⁺ channels.