Department of Medicine, Division of Gastroenterology and Hepatology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The putative heme oxygenase inhibitor zinc protoporphyrin IX (ZnPP IX) is known to exert diverse actions, including inhibitory action on smooth muscle relaxation by vasoactive intestinal polypeptide (VIP). The studies were performed in the opossum lower esophageal sphincter (LES) smooth muscle to determine the site of the inhibitory action of ZnPP IX in the smooth muscle relaxation by VIP. We also examined the effect of a direct Gs protein activator, cholera toxin (CTX), known to stimulate adenylate cyclase (AC). CTX caused relaxation of the LES smooth muscle by its action directly at the smooth muscle cells. The convergence of the common mechanisms of actions of VIP and CTX on AC was determined by the suppression of their effects by the AC inhibitor and CTX desensitization. ZnPP IX caused attenuation of the LES smooth muscle relaxation by VIP but not by CTX. ZnPP IX but not zinc deuteroporphyrin IX caused significant inhibition of VIP binding to the membrane receptor. We conclude that ZnPP IX attenuates VIP-induced LES smooth muscle relaxation by inhibition of VIP binding to G protein-coupled receptors linked to AC at a point proximal to G protein activation.
vasoactive intestinal polypeptide; lower esophageal sphincter; inhibitory neurotransmission; nitric oxide synthase; G protein coupled
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
MOST OF THE NEUROHUMORAL receptor interactions leading to a physiological action occur in the following steps. The binding of an agonist to the specific receptor causes change in the receptor conformation, followed by activation of the specific G protein, interaction with the specific enzyme associated with second messenger, interaction with ion channels or other target protein, and finally the specific action. The specific action in the case of the gastrointestinal smooth muscle may either be a relaxation or contraction. The neurohumoral substance examined in the present study is vasoactive intestinal polypeptide (VIP), which causes relaxation of the lower esophageal sphincter (LES) by the activation of receptors located on the smooth muscle cell membranes.
Carbon monoxide (CO) may be produced endogenously from heme by its interaction with heme oxygenase (HO) (26). Two types of HO have been recognized, HO-2 primarily in neural tissues and HO-1 in nonneural tissues. Zinc protoporphyrin IX (ZnPP IX) has been suggested to be a selective inhibitor of HO in a number of systems (26). Although the exact role of CO in the gastrointestinal smooth muscle is not known, it has been shown to cause a direct relaxation in a number of smooth muscle preparations (16, 31, 43), including the LES (28) and the internal anal sphincter (IAS) (31). Progress on the role of the HO pathway in inhibitory neurotransmission has been limited by the lack of selective HO inhibitors.
The putative HO inhibitor ZnPP IX has been recognized to have multiple actions especially in different smooth muscles, in addition to HO inhibition (14, 21). Because of this, caution should be exercised while using this agent primarily to determine the role of the HO pathway. Among other actions, ZnPP IX has been suggested to cause the blockade of VIP-induced relaxation of the IAS (31) as well as LES (28) and other smooth muscles. The exact site of action of ZnPP IX in blocking VIP response has not been investigated. To analyze this issue, specific tools to block or activate different steps along VIP receptor interaction, direct activator of Gs protein (cholera toxin or CTX), inhibitors of adenylate cyclase (AC), and CTX desensitization were employed. There is substantial evidence to suggest that VIP-induced smooth muscle relaxation is primarily mediated by the G protein-coupled receptor stimulation of AC (4, 35).
The main purpose of the present investigation therefore was to determine the mechanism of the inhibitory action of ZnPP on the LES smooth muscle relaxation caused by VIP. In the process, direct receptor binding studies with VIP before and after ZnPP IX were also performed.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparation of smooth muscle strips. The LES smooth muscle strips from opossums (Didelphis virginiana) of either sex were prepared for the recording of isometric tension as described previously (32). Briefly, after receiving anesthesia with pentobarbital (40-50 mg/kg ip) the animals were killed by exsanguination, and the LES along with a section of the esophagus and stomach was isolated and transferred to oxygenated (95% oxygen plus 5% carbon dioxide) Krebs physiological solution of the following composition (in mM): 118.07 NaCl, 4.69 KCl, 2.52 CaCl2, 1.16 MgSO4, 1.01 NaH2PO4, 25 NaHCO3, and 11.10 glucose. The LES was carefully freed of all extraneous tissues, including the large blood vessels, opened, and pinned flat with the mucosal side up on a dissecting tray containing oxygenated Krebs solution. The mucosal and submucosal layers were removed by sharp dissection, and LES circular smooth muscle strips (1 × 10 mm) were prepared as described previously (32).
Measurement of isometric tension. The smooth muscle strips were tied at both ends with silk sutures (6-0; Ethicon, Sommerville, NJ) and transferred to 2-ml muscle baths containing oxygenated Krebs solution (37°C). One end of the muscle strip was anchored at the bottom of the muscle bath and the other end was attached to a force transducer (model FTO3; Grass Instruments, Quincy, MA) for the measurement of isometric tension on a Dynograph recorder (model R411; Beckman Instruments, Schiller Park, IL). The muscle strips were stretched initially at 1 g of tension and then allowed to equilibrate for at least 1 h with regular washings at 20-min intervals. Only the strips that developed spontaneous steady tension and relaxed in response to electrical field stimulation (EFS) were used. The optimal length and the baseline of the smooth muscle strips were determined as described previously (27).
NANC nerve stimulation with EFS. EFS was delivered from a Grass stimulator (model S88; Quincy, MA) connected in series to a Stimu-Splitter II (Med-Lab Instruments, Loveland, CO). The Stimu-Splitter served an important purpose 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 to 20 Hz. The electrodes used for the EFS consisted of a pair of platinum wires fixed at both sides of the smooth muscle strip. The parameters of EFS stated above are known to selectively cause relaxation of the LES smooth muscle via the activation of nonadrenergic noncholinergic (NANC) myenteric neurons.
Drugs and chemicals. The following chemicals were used in the study: ZnPP IX, isoproterenol hydrochloride, and N-ethylmaleimide (NEM) (Aldrich Chemical, Milwaukee, WI); zinc deuteroporphyrin IX 2,4 bis-ethylene glycol (ZnDP IX) (Porphyrin Products, Logan, UT); NG-nitro-L-arginine (L-NNA) and sodium nitroprusside (SNP) (Sigma Chemical, St. Louis, MO), and VIP (Bachem Bioscience, Torrance, CA); and CTX and 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ-22536) (Research Biochemicals International, Natick, MA) and EDTA tetrasodium (Fisher Scientific, Pittsburgh, PA). 125I-VIP (2,000 Ci/mmol) was obtained from Amersham (Arlington Heights, IL).
VIP and isoproterenol are known to activate AC via Gs protein coupling (21, 33). CTX is well known to cause the smooth muscle relaxation by its direct and selective activation of Gs protein and AC (22-24, 41). To determine smooth muscle relaxation via the AC pathway, AC inhibitors SQ-22536 (18) and NEM (11, 27, 32, 38) were used. All chemicals except ZnPP IX and ZnDP IX were dissolved and diluted in Krebs solution and prepared fresh on the day of the experiment. Stock solutions of ZnPP IX and ZnDP IX were prepared by dissolving in 0.2 N sodium hydroxide and diluting with Krebs solution and kept in the dark. The pH of ZnPP IX and ZnDP IX solutions was adjusted to 7.4 using 0.2 N HCl. The final dilution of sodium hydroxide used as solvent for the porphyrins produced no significant effect on the basal LES tone. The vials and pipette tips were siliconized while the muscle baths were treated with 2.5% BSA to prevent the binding of VIP to the surface.Receptor binding studies.
To determine the influence of ZnPP IX on specific binding of VIP to the
LES smooth muscle receptor, we examined the effects of ZnPP IX on VIP
binding and compared them with those of ZnDP IX. VIP binding
experiments on LES smooth muscle membranes were carried out as
described previously (8). Briefly, after isolation of the LES from the
animals, the smooth muscle was cleaned off the mucosa and other
adhering tissues including the serosa and the small blood vessels. The
tissue was cut into small pieces and homogenized on an ice bath in Tris
buffer (25 mM, pH 7.4) containing 0.32 M sucrose using an Ultraturrax
tissue homogenizer (Tekmar, Cincinnati, OH). The homogenate was
centrifuged at 1,000 g for 20 min
(4°C). The supernatant was transferred to a separate tube and
centrifuged at 50,000 g for 30 min
(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 after resuspending and 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 using the
method of Lowry et al. (25) with BSA as the standard.
Drug responses.
Pretreatment with ZnPP IX and ZnDP IX (1 × 104 M) for 10 min was used
to examine their effects on the basal LES tone and changes in response
to different agonists. The dose was chosen based on previous data that
showed that ZnPP IX in this dose was maximally effective in blocking HO
activity in the gastrointestinal smooth muscle preparations (28). To
examine the influence of AC inhibitors, the smooth muscle strips were
pretreated with SQ-22536 (1 × 10
5 to 3 × 10
4 M) or NEM (1 × 10
4 M) for 10 min before
testing the effects of EFS, VIP, and CTX. The concentrations of
different antagonists were selected in view of the earlier literature
and our own experiments. The doses examined were relatively selective
against the intended effects.
Data analysis. The results are expressed as means ± SE of different experiments. The fall of the resting LES tension is expressed as the percentage of maximal potential response (100%) to a supramaximal concentration (5 mM) of EDTA. Statistical significance between different groups was determined by using paired or unpaired t-test where applicable, and P < 0.05 was considered statistically significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Influence of HO inhibitors ZnPP IX and ZnDP IX on LES smooth muscle
relaxation by VIP.
The data in Fig. 1 show that ZnPP IX caused
a marked and significant (P < 0.05, n = 5) shift in the dose-response
curve of VIP in causing the LES relaxation. ZnDP IX on the other hand
had a relatively limited effect. The values of the fall in the basal tension of LES with 3 × 107 M VIP before and after
ZnPP IX (1 × 10
4 M)
were 75.5 ± 7.8 and 25.1 ± 3.9%, respectively
(P < 0.05, n = 5), and the corresponding values
before and after ZnDP IX (1 × 10
4 M) were 72.2 ± 5.5 and 70.0 ± 3.8%, respectively (P > 0.05, n = 5). However, ZnDP IX did
cause a significant suppression of the LES smooth muscle relaxation in
response to lower concentrations of VIP (1 × 10
8 to 1 × 10
7 M). This suppression by
ZnDP IX was significantly less compared with that by ZnPP IX. ZnPP IX
as well as ZnDP IX had no significant effect on the basal tone of the
LES. The basal LES tone before and after ZnPP IX was 1.8 ± 0.1 and
1.8 ± 0.1 g, and before and after ZnDP IX it was 2.2 ± 0.1 and
2.2 ± 0.1 g, respectively (P > 0.05, n = 5).
|
Influence of ZnPP IX and ZnDP IX on LES smooth muscle relaxation by
NANC nerve stimulation with EFS.
Neither ZnPP IX nor ZnDP IX had any significant effect on the LES
smooth muscle relaxation by the NANC nerve stimulation with EFS (Fig.
2; P > 0.05, n = 9). The fall in
the basal LES tension with 5 and 10 Hz of EFS in these experiments was
71.5 ± 2.7 and 76.5 ± 2.7%, and in the presence of
ZnPP IX and ZnDP IX these values were 68.8 ± 4.6, 75.7 ± 3.3, 70.7 ± 3.9, and 74.3 ± 3.3%, respectively.
|
Effects and site of action of CTX on basal LES tone: influences of
L-NNA, TTX, and
-conotoxin.
The role of Gs protein in the
VIP-induced relaxation of the LES smooth muscle and the influence of
ZnPP IX were examined by the use of CTX, which causes direct activation
of Gs protein. CTX caused a
concentration-dependent fall in the basal tension of the LES (Fig.
3). A maximal fall in the basal LES tension
was observed with 2 µg/ml CTX in the muscle bath. The percent fall in
the basal tension of the LES with 2 µg/ml of CTX was 79.6 ± 2.0. The responses of CTX on the LES were not modified by the NO synthase
(NOS) inhibitor L-NNA, the
neurotoxin TTX (1 × 10
6 M), and the combination
of TTX and the neuronal calcium channel blocker
-conotoxin GVIA (1 × 10
6 M)
(P > 0.05, n = 4; Fig.
4).
|
|
Influence of ZnPP IX on LES smooth muscle relaxation by CTX. The dose-response curves for CTX showing the percent fall in the basal tension of the LES, obtained before and after ZnPP IX were found not to be significantly different (P > 0.05, n = 5). The percent fall in the basal LES tension with 0.5, 1.0, and 2.0 µg/ml in control experiments was 65.2 ± 3.7, 72.9 ± 3.4, and 79.6 ± 2.0, respectively. These values in the presence of ZnPP IX were 67.5 ± 2.5, 75.4 ± 3.0, and 81.7 ± 3.0%, respectively.
Influence of AC inhibitors on LES smooth muscle relaxation by VIP
and CTX.
The commonly used AC inhibitor SQ-22536 was found to be relatively
nonselective and ineffective in blocking AC in the LES smooth muscle.
In a different range of concentrations (1 × 105 to 3 × 10
4 M), previously reported
to be specific for the purpose, SQ-22536 caused a significant fall in
the basal tone of the LES. Additionally, SQ-22536 had no significant
effect on the LES relaxation caused by CTX, known to activate AC.
SQ-22536 (1 × 10
4 M
and 3 × 10
4 M) caused
a fall in the basal tension of the LES from 2.2 ± 0.3 to 1.5 ± 0.2, and 1.1 ± 0.3 g, respectively
(P < 0.05, n = 5).
|
|
|
Influence of CTX desensitization on VIP-induced relaxation of LES.
The frequent administration of CTX caused a significant reduction in
its responses. Furthermore, CTX desensitization also caused a
significant reduction in VIP responses in the LES
(P < 0.05, n = 4; Fig.
8), suggesting that the LES relaxation by CTX and VIP follows the same biochemical pathway.
|
Influence of ZnPP IX on binding of VIP to its receptors in LES.
We compared the effects of different concentrations of unlabeled VIP on
125I-VIP binding to the LES smooth
muscle membranes before and after ZnPP IX and ZnDP IX (1 × 104 M). The data given in
Fig. 9 show that in control experiments, VIP caused a significant and concentration-dependent displacement of
the bound 125I-VIP. Although ZnDP
IX had no significant effect on this control VIP receptor binding
curve, ZnPP IX caused a significant inhibition of the displacement
curve. These data suggest that ZnPP IX interferes with the binding of
VIP to the receptor. Furthermore, the data correspond to the functional
data in Fig. 1 that show the suppression of the VIP-induced fall in the
basal LES tension in the presence of ZnPP IX.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Multiple actions of ZnPP IX are well recognized. In the LES the protoporphyrin caused blockade of the smooth muscle AC and guanylate cyclase (GC) stimulation by VIP and atrial natriuretic factor or peptide, respectively (14, 29). Although the nonspecificity of actions of ZnPP IX may be widespread, in the present study we focused primarily on the mechanism of its inhibitory action on VIP.
The studies show that ZnPP IX caused significant attenuation of the G protein-coupled receptor activation by VIP. It is well known that the major part of the relaxant action of VIP in the LES is mediated by direct action at the VIP receptor on the smooth muscle cells via the activation of AC (1, 9, 32, 36). To determine the site of action of ZnPP IX in blocking VIP-induced relaxation of the LES smooth muscle, we systematically investigated the influence of ZnPP IX on agents that work along different steps to produce VIP receptor-mediated smooth muscle relaxation. This included the examination of the actions of VIP on the basal LES tone, VIP receptor binding, and comparison of the actions of VIP vs. CTX before and after ZnPP IX.
CTX is known to cause direct activation of Gs protein associated with AC (5, 41). The present studies in the LES showed for the first time that CTX causes a concentration-dependent fall in the basal tension by its action directly at the smooth muscle cells since it was not modified by neuronal blockade. The convergence of VIP and CTX on AC activation to produce LES relaxation was verified by their similar attenuation by the AC inhibitor NEM. The convergence of the actions of VIP and CTX on a similar intracellular pathway was further confirmed by the blockade of the inhibitory action of VIP on the LES smooth muscle by CTX desensitization.
The data suggest that the fall in the basal tone in the LES induced by VIP and CTX follows the same final biochemical pathway, i.e., the stimulation of AC. Along this final pathway, however, the original sites of action of VIP and CTX are different in causing the LES smooth muscle relaxation. The action of VIP is mediated via activation of G protein-coupled receptors and that of CTX is downstream, bypassing the receptor interaction, and being exerted directly at the level of G protein activation. This difference in the locus of action of these agents in the present study played an important role in the determination of the site of action of ZnPP IX in blocking the action of VIP in the LES.
The inhibitory action of isoproterenol in the LES especially in the
lower concentrations was also attenuated by ZnPP IX (13). The
suppressant effect of ZnPP IX on isoproterenol-induced smooth muscle
relaxation may involve its interaction with the -adrenoceptors. The
data show that ZnPP IX blocks the action of VIP but has no significant
effect on the fall in the LES tension caused by CTX. This suggests that
the site of action of ZnPP IX in inhibiting the action of VIP lies at a
point between the receptor interaction and G protein activation. The
VIP binding experiments further confirmed that the major mechanism of
action of ZnPP IX in inhibiting VIP action is due to inhibition of the
coupling of VIP with the receptor. In separate studies (14) we have
shown that ZnPP IX causes no significant modification of the LES
relaxation by forskolin, a direct stimulator of AC that bypasses G
protein activation.
As stated above, ZnPP IX exerts multiple actions in the smooth muscle. However, the inhibitory action of ZnPP IX on the G protein-coupled receptor activation leading to LES smooth muscle relaxation cannot be explained simply on the basis of complete nonselectivity for the following reasons. The actions of the muscarinic agonist bethanechol that stimulate a specific G protein-coupled receptor (10, 12, 19), causing an increase in the basal tone of the LES (17), and the fall in the LES tone induced by CTX (a polypeptide), SNP, and forskolin were not modified. Furthermore, in the LES, unlike the IAS, the NANC nerve stimulation-induced relaxation of the sphincteric smooth muscle was also not modified by the HO inhibitor. We suggest that in the LES the predominant pathway for the NANC nerve-induced relaxation is NOS. The predominance of the NOS pathway in the LES relaxation is evident from the previously published data that show the NOS inhibitor nearly abolishes the relaxation by NANC nerve stimulation (30, 40).
In light of the strong evidence in favor of the VIP as an inhibitory neurotransmitter (3, 20), it is rather surprising that ZnPP IX caused near obliteration of VIP response but had no effect on the NANC relaxation. One of the plausible explanations may be NOS upregulation. A leftward shift in the EFS-frequency response curve in the feline LES in the presence of ZnPP IX plus L-NNA compared with L-NNA alone (28) and release of NO by ZnPP IX in the rabbit IAS (7) may lend support to this speculation. This may have important pathophysiological implications in the counterregulation between HO and NOS pathways and in protecting the tissues against the deleterious effects of overproduction of NO. The colocalization of HO and NOS, as recently demonstrated in brain neurons (39) and in the feline LES (28), further suggests this possibility.
Despite multiple and nonselective actions of ZnPP IX in different smooth muscles, in the IAS the interaction between VIP and HO was found to be relatively defined since the actions of a closely related peptide, PHI, were not modified by the HO inhibitor (31). The concentrations of ZnPP IX used in the present studies were similar to those found to be effective in inhibiting HO activity in the LES (28). Furthermore, ZnDP IX, which is known to block HO activity with a greater potency than ZnPP IX, had a limited effect on the VIP-induced relaxation of the LES as well as on VIP receptor binding. The data suggest a lack of correlation between the inhibition of HO activity by the porphyrins and their ability to block the responses to VIP.
Because of the nonselectivity of action of ZnPP IX, the exact role of the HO pathway in the LES relaxation by NANC nerve stimulation cannot be determined at the present time. The direct action of CO on the smooth muscle via direct activation of GC, the presence of basal HO activity, its increase by NANC nerve stimulation, and inhibition by ZnPP IX in certain gastrointestinal tissues (28, 31), the presence of HO-2 immunoreactivity in the myenteric plexuses (6, 6, 28, 34); the electrophysiological correlation between CO and NANC relaxation (15); the colocalization of HO with NOS and VIP immunoreactivities (2, 28, 34); and the inhibition of NANC nerve-mediated relaxation by the selective knock-out of the HO-2 gene in certain gastrointestinal tissues (31, 37, 42) suggest participation of the HO pathway in some way in the gastrointestinal motility.
In conclusion, ZnPP IX causes blockade of the action of VIP at the LES smooth muscle membrane receptor that is G protein coupled to AC. The site of action of ZnPP IX is above the level of activation of G protein since the effects of direct G protein activation by CTX were not modified by ZnPP IX. The failure of ZnPP IX to modify NANC nerve stimulation-induced relaxation of the LES may be explained by the possibility that the smooth muscle relaxation by the NO released on NANC nerve-mediated stimulation uses a unique receptor activation that bypasses the G protein coupling to GC. The data further suggest that ZnPP IX, especially at high concentrations, may not be a specific HO inhibitor and that there is a need for a more selective HO inhibitor. The discovery of such an agent may facilitate investigations of the role of the HO pathway in gastrointestinal motility.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-35385 and an institutional grant from Thomas Jefferson University.
![]() |
FOOTNOTES |
---|
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. §1734 solely to indicate this fact.
Address for reprint requests: S. Rattan, 901 College, Dept. of Medicine, Div. of Gastroenterology and Hepatology, 1025 Walnut St., Philadelphia, PA 19107.
Received 16 July 1998; accepted in final form 7 October 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Barnette, M. S.,
F. C. Barone,
P. J. Fowler,
M. Grous,
W. J. Price,
and
H. S. Ormsbee.
Human lower oesophageal sphincter relaxation is associated with raised cyclic nucleotide content.
Gut
32:
4-9,
1991[Abstract].
2.
Battish, R.,
G. Y. Cao,
R. B. Lynn,
S. Chakder,
and
S. Rattan.
Heme-oxygenase 2 (HO-2) distribution in anorectum of opossum: colocalization with neuronal nitric oxide synthase (nNOS) and vasoactive intestinal polypeptide (VIP) (Abstract).
Gastroenterology
114:
A719,
1998.
3.
Biancani, P.,
J. H. Walsh,
and
J. Behar.
Vasoactive intestinal polypeptide: a neurotransmitter for lower esophageal sphincter relaxation.
J. Clin. Invest.
73:
963-967,
1984[Medline].
4.
Burks, T. F.
Fundamentals of gastrointestinal pharmacology.
In: Gastrointestinal Pharmacology and Therapeutics, edited by G. Friedman,
E. D. Jacobson,
and R. W. McCallum. Philadelphia, PA: Lippincott-Raven, 1997, p. 1-20.
5.
Burleigh, D. E.,
and
R. A. Borman.
Evidence for a nonneural electrogenic effect of cholera toxin on human isolated ileal mucosa.
Dig. Dis. Sci.
42:
1964-1968,
1997[Medline].
6.
Chakder, S.,
G. Y. Cao,
R. B. Lynn,
and
S. Rattan.
Distribution of heme oxygenase-2 (HO-2) in the internal anal sphincter (IAS) of opossum (Abstract).
Gastroenterology
112:
A1138,
1997.
7.
Chakder, S.,
S. Rathi,
X. Ma,
and
S. Rattan.
Heme oxygenase inhibitor zinc protoporphyrin IX causes an activation of nitric oxide synthase in the rabbit internal anal sphincter.
J. Pharmacol. Exp. Ther.
277:
1376-1382,
1996[Abstract].
8.
Chakder, S.,
and
S. Rattan.
The entire vasoactive intestinal polypeptide molecule is required for the activation of the vasoactive intestinal polypeptide receptor: functional and binding studies on opossum internal anal sphincter smooth muscle.
J. Pharmacol. Exp. Ther.
266:
392-399,
1993[Abstract].
9.
Chakder, S.,
and
S. Rattan.
Involvement of cAMP and cGMP in relaxation of internal anal sphincter by neural stimulation, VIP, and NO.
Am. J. Physiol.
264 (Gastrointest. Liver Physiol. 27):
G702-G707,
1993
10.
Chen, Q.,
P. Yu,
G. De Petris,
P. Biancani,
and
J. Behar.
Distinct muscarinic receptors and signal transduction pathways in gallbladder muscle.
J. Pharmacol. Exp. Ther.
273:
650-655,
1995[Abstract].
11.
Duhe, R. J.,
M. D. Nielsen,
A. H. Dittman,
E. C. Villacres,
E. J. Choi,
and
D. R. Storm.
Oxidation of critical cystein residues of type I adenylyl cyclase by o-iodosobenzoate or nitric oxide reversibly inhibits stimulation by calcium and calmodulin.
J. Biol. Chem.
269:
7290-7296,
1994
12.
Eglen, R. M.,
S. S. Hedge,
and
N. Watson.
Muscarinic receptor subtypes and smooth muscle function.
Pharmacol. Rev.
48:
531-565,
1996[Medline].
13.
Fan, Y. P.,
S. Chakder,
and
S. Rattan.
Inhibitory effect of zinc protoporphyrin IX on lower esophageal sphincter smooth muscle relaxation by vasoactive intestinal polypeptide and other receptor agonists.
J. Pharmacol. Exp. Ther.
285:
468-474,
1998
14.
Fan, Y. P.,
S. Chakder,
and
S. Rattan.
Heme oxygenase (HO) inhibitor ZN protoporphyrin IX (ZnPP IX) blocks the actions of vasoactive intestinal polypeptide (VIP) by inhibiting G-protein-coupled receptor binding (Abstract).
Gastroenterology
114:
A750,
1998.
15.
Farrugia, G.,
W. A. Irons,
J. L. Rae,
M. G. Sarr,
and
J. H. Szurszewski.
Activation of whole cell currents in isolated human jejunal circular smooth muscle cells by carbon monoxide.
Am. J. Physiol.
264 (Gastrointest. Liver Physiol. 27):
G1184-G1189,
1993
16.
Furchgott, R. F.,
and
D. Jothianandan.
Endothelium-dependent and -independent vasodilation involving cyclic GMP: relaxation induced by nitric oxide, carbon monoxide and light.
Blood Vessels
28:
52-61,
1991[Medline].
17.
Gilbert, R. J.,
S. Rattan,
and
R. K. Goyal.
Pharmacologic identification, activation, and antagonism of two muscarine receptor subtypes in the lower esophageal sphincter.
J. Pharmacol. Exp. Ther.
230:
284-291,
1984[Abstract].
18.
Gonzalez, C.,
C. Barroso,
C. Martin,
S. Gulbenkian,
and
C. Estrada.
Neuronal nitric oxide synthase activation by vasoactive intestinal peptide in bovine cerebral arteries.
J. Cereb. Blood Flow Metab.
17:
977-984,
1997[Medline].
19.
Goyal, R. K.
Muscarinic receptor subtypes: physiology and clinical implications.
N. Engl. J. Med.
321:
1022-1029,
1989[Medline].
20.
Goyal, R. K.,
S. Rattan,
and
S. I. Said.
VIP as a possible neurotransmitter of non-cholinergic non-adrenergic inhibitory neurones.
Nature
288:
378-380,
1980[Medline].
21.
Grundemar, L.,
and
L. Ny.
Pitfalls using metalloporphyrins in carbon monoxide research.
Trends Pharmacol. Sci.
18:
193-195,
1997[Medline].
22.
Houslay, M. D.,
and
G. Milligan.
Tailoring cAMP-signalling responses through isoform multiplicity.
Trends Biochem. Sci.
22:
217-224,
1997[Medline].
23.
Kahl, R. A.,
and
A. G. Gilman.
Purification of a protein cofactor required for ADP-ribosylation of the stimulatory regulation component of adenylate cyclase by cholera toxin.
J. Biol. Chem.
259:
6228-6234,
1997
24.
Kume, H.,
K. Mikawa,
K. Takagi,
and
M. I. Kotlikoff.
Role of G proteins and KCa channels in the muscarinic and -adrenergic regulation of airway smooth muscle.
Am. J. Physiol.
268 (Lung Cell. Mol. Physiol. 12):
L221-L229,
1995
25.
Lowry, O. H.,
N. J. Rosebrough,
A. L. Farr,
and
R. J. Randall.
Protein measurements with the Folin phenol reagent.
J. Biol. Chem.
193:
265-275,
1951
26.
Maines, M. D.
The heme oxygenase system: a regulator of second messenger gases.
Annu. Rev. Pharmacol. Toxicol.
37:
517-554,
1997[Medline].
27.
Moummi, C.,
and
S. Rattan.
Effect of methylene blue and N-ethylmaleimide on internal anal sphincter relaxation.
Am. J. Physiol.
255 (Gastrointest. Liver Physiol. 18):
G571-G578,
1988
28.
Ny, L.,
P. Alm,
P. Ekstrom,
B. Larsson,
L. Grundemar,
and
K.-E. Andersson.
Localization and activity of haem oxygenase and functional effects of carbon monoxide in the feline lower oesophageal sphincter.
Br. J. Pharmacol.
118:
392-399,
1996[Abstract].
29.
Ny, L.,
K.-E. Andersson,
and
L. Grundemar.
Inhibition by zinc protoporphyrin-IX of receptor-mediated relaxation of the rat aorta in a manner distinct from inhibition of haem oxygenase.
Br. J. Pharmacol.
115:
186-190,
1995[Abstract].
30.
Paterson, W. G.,
and
B. Indrakrishnan.
Descending peristaltic reflex in the opossum esophagus.
Am. J. Physiol.
269 (Gastrointest. Liver Physiol. 32):
G219-G224,
1995
31.
Rattan, S.,
and
S. Chakder.
Inhibitory effect of CO on internal anal sphincter: heme oxygenase inhibitor inhibits NANC relaxation.
Am. J. Physiol.
265 (Gastrointest. Liver Physiol. 28):
G799-G804,
1993
32.
Rattan, S.,
and
C. Moummi.
Influence of stimulators and inhibitors of cyclic nucleotides on lower esophageal sphincter.
J. Pharmacol. Exp. Ther.
248:
703-709,
1989[Abstract].
33.
Rattan, S.,
C. Moummi,
and
S. Chakder.
CGRP and ANF cause relaxation of opossum internal anal sphincter via different mechanisms.
Am. J. Physiol.
260 (Gastrointest. Liver Physiol. 23):
G764-G769,
1991
34.
Reed, D.,
S. M. Miller,
G. Farrugia,
M. G. Sarr,
and
J. H. Szurszewski.
Immunocytochemical localization of heme oxygenase-2 and co-localization with nitric oxide synthase in myenteric neurons in human stomach and jejunum (Abstract).
Gastroenterology
110:
A7664,
1996.
35.
Ross, E. M.
Pharmacodynamics: mechanisms of drug action and the relationship between drug concentration and effect.
In: Goodman & Gilman's The Pharmacological Basis of Therapeutics, edited by J. G. Hardman,
L. E. Limbird,
P. B. Molinoff,
R. W. Ruddon,
and A. G. Gilman. New York: McGraw Hill, 1996, p. 29-41.
36.
Szewczak, S. M.,
J. Behar,
G. Billett,
C. Hillemeier,
B. Y. Rhim,
and
P. Biancani.
VIP-induced alterations in cAMP and inositol phosphates in the lower esophageal sphincter.
Am. J. Physiol.
259 (Gastrointest. Liver Physiol. 22):
G239-G244,
1990
37.
Tottrup, A.,
M. A. Knudsen,
F. H. Sorensen,
and
E. B. Glavind.
Pharmacological identification of different inhibitory mediators involved in the innervation of the internal anal sphincter.
Br. J. Pharmacol.
115:
158-162,
1995[Abstract].
38.
Trigo-Rocha, F.,
G. L. Hsu,
C. F. Donatucci,
and
T. F. Lue.
The role of cyclic adenosine monophosphate, cyclic guanosine monophosphate, endothelium and nonadrenergic, noncholinergic neurotransmission in canine penile erection.
J. Urol.
149:
872-877,
1993[Medline].
39.
Vincent, S. R.,
S. Das,
and
M. D. Maines.
Brain heme oxygenase isoenzymes and nitric oxide synthase are co-localized in select neurons.
Neuroscience
63:
223-231,
1994[Medline].
40.
Yamato, S.,
J. K. Saha,
and
R. K. Goyal.
Role of nitric oxide in lower esophageal sphincter relaxation to swallowing.
Life Sci.
50:
1263-1272,
1992[Medline].
41.
Yang, C. M.,
M. C. Hsu,
H. L. Tsao,
C. T. Chiu,
R. Ong,
J.-T. Hsieh,
and
L. W. Fan.
Effects of cAMP elevating agents on carbachol-induced phosphoinositide hydrolysis and calcium mobilization in cultured canine tracheal smooth muscle cells.
Cell Calcium
19:
243-254,
1996[Medline].
42.
Zakhary, R.,
K. D. Poss,
S. R. Jaffrey,
C. D. Ferris,
S. Tonegawa,
and
S. H. Snyder.
Targeted gene deletion of heme oxygenase 2 reveals neural role for carbon monoxide.
Proc. Natl. Acad. Sci. USA
94:
14848-14853,
1997
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |