Division of Gastroenterology and Hepatology, Department of Medicine, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania 19107
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ABSTRACT |
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Despite its widespread distribution and
significance in the gut, the role of pituitary adenylate
cyclase-activating peptide (PACAP) in internal anal sphincter (IAS)
relaxation has not been examined. This study examined the role of PACAP
in nonadrenergic noncholinergic (NANC) nerve-mediated relaxation of IAS
smooth muscle. Circular smooth muscle strips from the opossum IAS were prepared for measurement of isometric tension. The influence of PACAP
and vasoactive intestinal peptide (VIP) antagonists and tachyphylaxis
on the neurally mediated IAS relaxation was examined either separately
or in combination. The release of these neuropeptides in response to
NANC nerve stimulation before and after the nitric oxide (NO) synthase
inhibitor
N-nitro-L-arginine
and NO was also investigated. Both PACAP and VIP antagonists caused
significant attenuation of IAS relaxation by NANC nerve stimulation.
The combination of the antagonists, however, did not have an additive
effect on IAS relaxation. VIP tachyphylaxis caused significant
suppression of IAS relaxation by NANC nerve stimulation. PACAP and VIP
were found to be released by NANC nerve stimulation and exogenous NO.
The data suggest the involvement of PACAP in IAS relaxation primarily
by the activation of PACAP1/VIP
receptor and lack of its independent role in the relaxation.
Furthermore, NO may regulate the presynaptic release of PACAP and VIP.
vasoactive intestinal polypeptide; nonadrenergic noncholinergic; nitric oxide synthase; inhibitory neurotransmitter; smooth muscle
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INTRODUCTION |
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STUDIES from different laboratories have shown that vasoactive intestinal peptide (VIP) plays a significant role in the nonadrenergic noncholinergic (NANC) nerve-mediated relaxation of the internal anal sphincter (IAS) (1, 17). On the other hand, part of the NANC nerve-mediated IAS relaxation is mediated via the nitric oxide synthase (NOS) pathway in different species including humans (3, 6, 13, 18, 21, 24, 27).
Recent studies have shown the presence of another neuropeptide,
pituitary adenylate cyclase-activating peptide (PACAP) in different
regions of the gastrointestinal tract (20, 25, 26, 28). Two forms of
PACAP, PACAP-(138) and PACAP-(1
27), both amidated at
the COOH terminus, have been isolated. The PACAP molecules show a
significant homology with VIP and are considered to be the members of
VIP-glucagon-secretin family of peptides (14). It has been suggested
that PACAP exerts its actions via the activation of at least three
types of receptors that belong to a subfamily of the
seven-transmembrane-spanning G protein-coupled receptors. PACAP1 receptor is selective for
PACAP and has 1,000 times more affinity for PACAP than VIP.
PACAP2/VIP1
and
PACAP2/VIP2
receptors have equal affinity for PACAP and VIP (8). It has also been shown that PACAP plays a significant role in the gastrointestinal smooth muscle relaxation in response to NANC nerve stimulation (7, 9,
11). Furthermore, in humans, PACAP has been associated with the normal
functioning of the gut, since a reduction in the PACAP immunoreactive
neurons and levels have been shown in the gastrointestinal motility
disorders, such as Hirschsprung's disease (25). However, the
role of PACAP in IAS relaxation has not been examined.
The purpose of the present investigation was to determine the role of PACAP in IAS relaxation by NANC nerve stimulation. This was carried out by investigating the influence of PACAP and VIP antagonists and tachyphylaxis in response to these neuropeptides on neurally mediated IAS relaxation either separately or in combination. To determine the regulation of PACAP and VIP release, we examined their release in response to NANC nerve stimulation before and after the NOS inhibitor.
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MATERIALS AND METHODS |
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Preparation of smooth muscle strips. Studies were performed on circular smooth muscle strips obtained from adult opossums (Didelphis virginiana) of either sex after pentobarbital sodium anesthesia (40 mg/kg ip) and subsequent exsanguination. The entire anal canal was removed and transferred to a dissecting tray containing oxygenated (95% O2-5% CO2) Krebs solution. The composition of the Krebs solution was as follows (mM): 118.07 NaCl, 4.69 KCl, 2.52 CaCl2, 1.16 MgSO4, 1.01 NaH2PO4, 25 NaHCO3, and 11.10 glucose. The anal canal was cleaned of extraneous connective tissue and blood vessels and opened flat by an incision along the longitudinal axis. The tissue was then pinned flat, and the mucosa along with the submucosa was removed by sharp dissection. Circular smooth muscle strips were obtained from the whole circumference of the anal canal and divided into two equal strips (~2 × 8 mm). The muscle strips were tied at both ends with silk sutures for the measurement of isometric tension.
Measurement of isometric tension. IAS smooth muscle strips prepared as described above were transferred to thermostatically controlled 2-ml muscle baths (37°C) containing Krebs solution bubbled with a 95% O2-5% CO2 mixture. One end of the muscle strip was fixed to the bottom of the muscle bath with a tissue holder, and the other end was attached to an isometric force transducer (model FT03, Grass Instruments, Quincy, MA) for measurement of isometric tension. The tension of the smooth muscle strips was recorded on a Dynograph recorder (model R411, Beckman Instruments, Schiller Park, IL). After an equilibration period of 1 h, with intermittent washings, the optimal length (Lo) and baseline of each smooth muscle strip were determined as previously described (15). Only those smooth muscle strips that developed spontaneous and steady tension and relaxed in response to electrical field stimulation (EFS) were used in the study. To eliminate binding of the peptides to the glass surface, the muscle baths were pretreated with 2.5% BSA, and the pipette tips were siliconized.
NANC nerve stimulation with EFS.
EFS was delivered from a Grass stimulator (model S88) connected in
series to a Med-Lab Stimu-Splitter II (Loveland, CO). The Stimu-Splitter was used to amplify and measure the stimulus intensity using the optimal stimulus parameters for the neural stimulation (12 V,
0.5-ms pulse duration, 200-400 mA, 4-s train) at varying frequencies of 0.5-20 Hz. The electrodes used for the EFS
consisted of a pair of platinum wires fixed at both sides of the smooth muscle strip. Neurally mediated relaxation of IAS smooth muscle strips
was quantified in response to different frequencies of EFS. The
above-mentioned parameters of EFS are known to cause relaxation of IAS
smooth muscle via the selective activation of NANC myenteric neurons.
All the experiments were done in the presence of atropine
(106 M) and guanethidine (3 × 10
6 M).
Drug responses. To determine the influence of PACAP or VIP antagonist or another neurohumoral blocking agent, the control concentration response to a given peptide or an agonist was tested first, followed by its repetition in the presence of the antagonists in the muscle bath. All the antagonists were allowed to maintain contact with the tissues for 10 min before testing of the effects of the agonists.
Tachyphylaxis with PACAP or VIP was achieved by repeated administration of single doses of PACAP or VIP (10Measurement of PACAP and VIP release during EFS.
For the measurement of the release of VIP and PACAP from the smooth
muscle strips during the EFS, the muscle strips were incubated in 2-ml
muscle bath filled with oxygenated Krebs solution containing 0.1% BSA,
20 mM bacitracin, and 1,000 U/ml aprotonin. After a 1-h equilibration
period at Lo, IAS
smooth muscle strips were washed for another 30 min for stabilization
of the basal tension. After the final wash, the muscle strips were
incubated for 10 min in the Krebs solution, and the perfusate was
collected for measurement of basal release of the peptides. To examine
the release of the peptides in response to EFS, the muscle bath
perfusates were collected immediately after the termination of EFS
(1-min train at 1 and 10 Hz). The smooth muscle strips were allowed to stand for 45 min with intermittent washings before another 10-min sample was collected for the measurement of basal peptide release, which was followed by a stimulation period of 1 min. All the samples were collected in siliconized tubes and immediately stored at 80°C until used for the assay of the peptides.
Binding of VIP to IAS smooth muscle membranes.
Binding of VIP to IAS smooth muscle membranes was carried out according
to the previously described method from our laboratory (4). Briefly,
after its isolation, IAS smooth muscle was cleaned of all adherent
tissues and small blood vessels. The tissue was cut into small pieces
and homogenized in an ice bath in Tris buffer (25 mM, pH 7.4)
containing 0.32 M sucrose by using an Ultraturrax tissue homogenizer
(Teckmar, Cincinnati, OH). The supernatant was separated and
centrifuged at 50,000 g for 30 min at
4°C. The pellet was resuspended in Tris buffer (25 mM, pH 7.4)
containing 2 mM EDTA and centrifuged at 50,000 g for 30 min. The pellet was washed
twice with the same buffer by centrifugation in the same way. The final
pellet was suspended in Tris buffer, and aliquots were stored at
80°C until used for VIP binding experiments. The protein
contents of the membranes were determined by the method of Lowry et al.
(12) using BSA as the standard.
Drugs and chemicals.
VIP (porcine), VIP-(1028) (porcine), PACAP-(1
38) amide (PACAP-38 or
PACAP), PACAP-27 amide, and PACAP-(6
38) amide (human, ovine, and rat)
were from Bachem (Torrance, CA).
L-NNA,
N
-nitro-D-arginine
methyl ester, L-arginine
hydrochloride, D-arginine, TTX,
forskolin, and atropine methyl bromide were from Sigma Chemical (St.
Louis, MO). Guanethidine monosulfate was from Ciba Pharmaceuticals (Summit, NJ). EDTA tetrasodium salt was from Fisher Scientific (Fair
Lawn, NJ). 3-[125I]iodotyrosyl10-labeled
VIP (2,000 Ci/mmol) was obtained from Amersham (Arlington Heights, IL).
All other chemicals were of the highest purity available. Solutions of
all the chemicals except forskolin were prepared in Krebs solution
fresh on the day of the experiment. Solutions of NO were prepared as
described previously (21). Forskolin was dissolved in ethanol and
diluted in Krebs solution.
Data analysis. The responses to EFS and other relaxants were expressed as the percentage of maximal relaxation caused by 5 mM EDTA. The results are expressed as means ± SE. Statistical significance between different groups was determined using the t-test, and P < 0.05 was considered significant.
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RESULTS |
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Effects of PACAP antagonist PACAP-(638) on neurally mediated
relaxation of IAS smooth muscle strips caused by EFS.
EFS caused a frequency-dependent fall in the basal tension of IAS
smooth muscle that was significantly attenuated by PACAP-(6
38). In
the control experiments, 1, 2, and 5 Hz of EFS caused 48.8 ± 4.6, 59.2 ± 3.1, and 67.8 ± 3.1% falls in the basal tension of the
smooth muscle strips. After pretreatment with 3 × 10
5 M PACAP-(6
38), these
relaxation responses were significantly reduced to 19.5 ± 1.0, 36.2 ± 2.9 and 43.7 ± 6.9%, respectively (n = 9;
P < 0.05; Fig.
1A).
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Influence of VIP antagonist VIP-(1028) on EFS-induced fall in
basal tension of IAS smooth muscle strips by EFS.
Similar to the PACAP antagonist, the VIP antagonist VIP-(10
28) also
caused a significant inhibition of IAS relaxation caused by different
frequencies of EFS. In the controls, 0.5, 1, 2, and 5 Hz of EFS caused
34.8 ± 3.3, 48.8 ± 4.6, 59.2 ± 3.1, and 67.8 ± 3.1%
falls in the basal tension of IAS. After pretreatment of the smooth
muscle strips with 3 × 10
5 M VIP-(10
28), the
fall in IAS tension with the same frequencies of EFS was significantly
attenuated to 5.4 ± 1.5, 21.6 ± 7.5, 43.4 ± 6.0, and 54.5 ± 6.0%, respectively (n = 9; P < 0.05; Fig. 1B).
Effect of VIP-(1028) plus PACAP-(6
38) on EFS-induced fall in
basal tension of IAS smooth muscle.
To determine the independent role of VIP and PACAP in IAS relaxation
and the nature of VIP and PACAP receptors in IAS, we examined the
effects of VIP-(10
28) and PACAP-(6
38) individually and in
combination on IAS relaxation by different frequencies of EFS. In the
control experiments, 0.5, 1, 2, and 5 Hz of EFS caused 40.4 ± 2.8, 57.2 ± 4.0, 65.7 ± 3.9, and 71.5 ± 3.6% falls in the basal
IAS tension. After VIP-(10
28) (3 × 10
5 M) pretreatment, the
fall in the basal IAS tension with the same frequencies of EFS was
significantly reduced to 5.4 ± 1.5, 21.6 ± 7.5, 43.9 ± 6.0, and 54.5 ± 6.0%, respectively (P < 0.05; Fig. 1C). These values
after VIP-(10
28) plus PACAP-(6
38) (3 × 10
5 M) were 8.2 ± 0.9, 20.3 ± 3.6, 33.3 ± 6.8, and 42.7 ± 8.4%,
respectively. These relaxations were not significantly different from
those observed in the presence of VIP-(10
28) or PACAP-(6
38) alone (Fig. 1C). The results suggest that
VIP and PACAP released endogenously in response to EFS share a common
receptor in causing IAS smooth muscle relaxation.
Effect of PACAP-(627) on EFS-induced IAS relaxation.
In a separate series of experiments, we examined the effects of
PACAP-(6
27), another PACAP antagonist, on IAS relaxation caused by
different frequencies of EFS. Like PACAP-(6
38), PACAP-(6
27) also
caused a rightward shift of the control EFS frequency-response curve.
In control experiments, 1, 2, and 5 Hz of EFS caused 52.0 ± 7.4, 61.9 ± 3.8, and 69.2 ± 1.4% falls in IAS tension. After pretreatment with 3 × 10
5 M PACAP-(6
27), IAS
smooth muscle relaxation was significantly attenuated to 19.8 ± 6.1, 40.8 ± 8.6, and 57.4 ± 6.1%, respectively (n = 4;
P < 0.05; Fig.
2).
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Effect of VIP and VIP plus PACAP tachyphylaxis on IAS relaxation by
EFS.
To further determine the role of PACAP and VIP on neurally mediated IAS
relaxation, we examined the influence of their respective tachyphylaxis
on IAS relaxation by different frequencies of EFS. First of all, to
determine the specificity of their actions, we investigated the effects
of PACAP and VIP before and after the tachyphylaxis. The fall in IAS
tension by PACAP was nearly abolished by PACAP tachyphylaxis
selectively, since VIP tachyphylaxis had no significant effect on PACAP
responses (Fig.
3A). In
control experiments, the fall in IAS tension with 3 × 107 and
10
6 M PACAP was 49.0 ± 6.5 and 58.7 ± 6.4%, respectively. In the presence of PACAP
tachyphylaxis, the fall in IAS tension with same concentrations of
PACAP was 5.8 ± 1.9 and 7.5 ± 2.2%, respectively. On the other
hand, these responses in the presence of VIP tachyphylaxis were 33.6 ± 6.5 and 51.6 ± 5.9%, respectively (Fig.
3A).
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Effects of antagonists and tachyphylaxis of PACAP and VIP on fall in basal IAS tension by NO and forskolin. To examine the specificity of the actions of PACAP and VIP antagonists and their respective tachyphylaxes, the effects of NO and forskolin on the basal IAS tension were investigated before and after these treatments. Both NO (Fig. 5) and forskolin (Fig. 6) caused concentration-dependent relaxation of IAS smooth muscle that was not modified by either the presence of PACAP and VIP antagonists or tachyphylaxis (Figs. 5 and 6; P > 0.05; n = 6).
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Release of PACAP and VIP by EFS and NO. EFS (1 and 10 Hz) caused release of both VIP and PACAP from IAS smooth muscle strips. PACAP released under the present experimental conditions was primarily of PACAP-38 form, and only a small amount of PACAP-27 was released. Interestingly, the levels of PACAP-38 released in response to 1 and 10 Hz of EFS were significantly higher than those of VIP (Fig. 7).
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Receptor binding studies.
The data showing the displacement of radiolabeled VIP by unlabeled VIP
and PACAP are given in Fig. 10. The data
show displacement of radiolabeled VIP by unlabeled VIP, PACAP-38, and
PACAP-27 in a concentration-dependent manner. VIP and PACAP-38 were
found to be nearly equipotent in causing the displacement of
125I-VIP. PACAP-27 on the other
hand was found to be approximately one-tenth as potent in causing the
displacement of 125I-VIP compared
with VIP and PACAP-38. The IC50
values for VIP and PACAP-38 were (1.3 ± 0.2) × 108 vs. (6.5 ± 1.8) × 10
9 M, respectively
(P > 0.05), and for PACAP-27 the
calculated IC50 was (2.3 ± 0.6) × 10
7 M.
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DISCUSSION |
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This study suggests that PACAP may be involved in the NANC nerve-mediated relaxation of IAS and that endogenously released PACAP activates either a common PACAP/VIP receptor or has a considerable overlap for the actions of both the peptides. This is supported by the findings that binding of radiolabeled VIP to IAS smooth muscle membranes was equally displaced by VIP, PACAP-38, and PACAP-27.
PACAP is partly responsible for IAS relaxation in response to NANC
nerve stimulation. We have previously shown that PACAP causes
relaxation of IAS by its action directly at the smooth muscle cells
(23). The present studies show that selective antagonists of PACAP,
PACAP-(638), and PACAP-(6
27) cause significant attenuation of IAS
relaxation caused by NANC nerve stimulation.
The studies further demonstrate the release of PACAP in response to NANC nerve stimulation. Interestingly, in IAS, PACAP was released primarily in the form of PACAP-38. Although PACAP-immunoreactive neurons in IAS were not examined, a number of studies have shown the presence of these neurons in other regions of the gut (20, 25, 26, 28).
In IAS, the receptors responsible for the PACAP-induced relaxation of IAS appear to share common properties with those of VIP. This is supported by the observations that both VIP and PACAP displaced radiolabeled VIP from IAS smooth muscle membranes receptors with the similar potency. Previous studies from our laboratory (22) have shown that PACAP- or VIP-induced relaxation of IAS was blocked by their respective antagonists. Interestingly, the combination of both antagonists failed to cause an attenuation of the EFS-induced IAS relaxation more than that achieved in the presence of either of the antagonists. The data suggest that, in contrast to the independent role of VIP, the role of PACAP in the NANC nerve stimulation-induced IAS relaxation is limited to the involvement of a common VIP/PACAP receptor.
The data suggest an involvement of VIP/PACAP receptor in the neurally mediated relaxation of IAS. There was a significant suppression of the neurally mediated IAS relaxation by VIP tachyphylaxis. The influence of PACAP tachyphylaxis on the neurally mediated IAS relaxation was far more complex. In the lower frequencies of EFS, PACAP tachyphylaxis was found to cause an augmentation of IAS relaxation. Furthermore, PACAP tachyphylaxis caused the restoration of IAS relaxation suppressed by VIP tachyphylaxis. Although, the exact significance of these findings is not known, the actions may be explained on the basis of the complex actions of PACAP (10, 19, 23). In IAS, PACAP causes not only relaxation but also a contraction (23). IAS smooth muscle contraction in response to PACAP was found to be mediated via substance P. In other gastrointestinal smooth muscles, PACAP has recently been shown to produce contraction either via substance P and acetylcholine release (10) or by its action directly at the smooth muscle (19). Both substance P and muscarinic stimulation are well known to produce gastrointestinal smooth muscle contraction via the activation of protein kinase C (PKC) (2).
The activation of PKC is known to interfere with the NOS pathway (16), which plays a major role in the NANC nerve-mediated IAS relaxation and a part of the VIP-induced relaxation of IAS (5, 21, 24). It would be interesting to examine whether PACAP tachyphylaxis, by inhibiting PKC, unmasks the NOS system leading to the augmentation of IAS relaxation. There is also a possibility of upregulation of VIP and NOS inhibitory neurotransmission during PACAP tachyphylaxis, but it is not known why the actions of PACAP antagonists were different from those of PACAP tachyphylaxis. The experiments with specific PACAP and VIP antibodies may help further in the evaluation of the independent and relative role of PACAP and VIP in IAS relaxation.
PACAP tachyphylaxis caused significant suppression of IAS relaxation in
response not only to PACAP but also to VIP. Conversely, however, VIP
tachyphylaxis blocked the effect of VIP only. Although the exact reason
for these observations and the mechanism of their action are not quite
clear, it is possible that PACAP has multiple sites of actions and that
one of the sites is by the activation of a receptor that is shared by
VIP. At such receptors, PACAP may possess higher affinity compared with
VIP. This is corroborated by our ongoing studies showing that PACAP
antagonists were in fact more potent in displacing membrane bound
125I-VIP than the VIP antagonist
VIP-(1028).
The data also suggest that NO upregulates the release of both PACAP and VIP, since exogenous administration of NO caused an increase in the release of both these peptides. Furthermore, similar regulation was found in response to the endogenous release of NO as the inhibition of NO biosynthesis by the NOS inhibitor caused an opposite effect, i.e., decrease in the release of VIP that was increased by the stimulation of NANC nerves. We conclude that the role of PACAP in the NANC nerve-mediated relaxation of IAS is limited to the activation of a common VIP/PACAP receptor.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-35385 and an institutional grant from Thomas Jefferson University.
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FOOTNOTES |
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Address for reprint requests: S. Rattan, 901 College, Thomas Jefferson University, 1025 Walnut St., Philadelphia, PA 19107.
Received 1 December 1997; accepted in final form 1 June 1998.
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