Mechanism of internal anal sphincter relaxation by CORM-1, authentic CO, and NANC nerve stimulation

Satish Rattan, Rany Al Haj, and Márcio A. F. De Godoy

Division of Gastroenterology and Hepatology, Department of Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

Submitted 11 February 2004 ; accepted in final form 30 March 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The present studies compared the effects of CO-releasing molecule (CORM-1), authentic CO, and nonadrenergic noncholinergic (NANC) nerve stimulation in the internal anal sphincter (IAS). Functional in vitro experiments and Western blot studies were conducted in rat IAS smooth muscle. We examined the effects of CORM-1 (50–600 µM) and authentic CO (5–100 µM) and NANC nerve stimulation by electrical field stimulation (EFS; 0.5–20 Hz, 0.5-ms pulse, 12 V, 4-s train). The experiments were repeated after preincubation of the tissues with the neurotoxin TTX, the guanylate cyclase inhibitor 1H-(1,2,4)oxadiazolo-(4,3-a)quinoxalin-1-one (ODQ), the selective heme oxygenase (HO) inhibitor tin protoporphyrin IX (SnPP-IX), the nitric oxide synthase inhibitor N{omega}-nitro-L-arginine (L-NNA), and SnPP-IX + L-NNA. We also investigated the effects of the HO substrate hematin (100 µM). CORM-1, as well as CO, produced concentration-dependent IAS relaxation, whereas hematin had no effect. TTX abolished and L-NNA significantly blocked IAS relaxation by EFS without any effect on CORM-1 and CO. ODQ blocked IAS relaxation by CORM-1, authentic CO, and EFS. SnPP-IX had no significant effect on IAS relaxation by CORM-1, CO, or EFS. The presence of neuronal nitric oxide synthase, HO-1, and HO-2 in IAS smooth muscle was confirmed by Western blot studies. CORM-1 and CO, as well as NANC nerve stimulation, produced IAS relaxation via guanylate cyclase/cGMP-dependent protein kinase activation. The advent of CORM-1 with potent effects in the IAS has significant implications in anorectal motility disorders with regard to pathophysiology and therapeutic potentials.

heme oxygenase; inhibitory neurotransmitter; nitric oxide synthase; guanylate cyclase; smooth muscle


CARBON MONOXIDE, similar to nitric oxide (NO), is acknowledged as a gaseous neurotransmitter in certain systems (3, 21). In the gastrointestinal (GI) tract, including the internal anal sphincter (IAS), it has been suggested that CO plays a role in inhibitory neurotransmission (11, 15, 23, 31, 33, 43). Similar to NO, CO is believed to act via activation of soluble guanylate cyclase (GC) (3, 21). Endogenous production of CO is one of the results of heme catabolism by heme oxygenase (HO) (3, 20, 21). In contrast to CO, the role of NO synthase (NOS) and NO in nonadrenergic noncholinergic (NANC) nerve stimulation in the GI tract has been well established (18, 32, 34, 37).

Other than suggestions from different laboratories (8, 31, 33, 43), the role of the HO pathway in NANC relaxation of GI smooth muscle has not been established (6). One of the hurdles has been the availability of a CO donor, such as NO. Administration of CO has a number of pitfalls, such as variability in the preparation and rough estimates of CO concentration in the solution. Recently, the availability of CO-releasing molecules (CORMs), such as the tricarbonyl dichlororuthenium (II) dimer [Ru(CO)3Cl2]2 (CORM-1), has made it possible to precisely examine the effects of CO in the tissues (24). The authors have shown reproducible relaxation of rat aortic smooth muscle. The effects and the mechanism of action of these interesting molecules in the GI smooth muscle are not known.

We used a three-pronged approach to examine the effects of CO in IAS smooth muscle: application of CORM-1, authentic CO, and hematin. Hematin, an HO substrate, is known to produce CO, which is responsible for certain physiological actions (17). The purpose of the present investigation was to examine and compare the effects and mechanism of action of these agents in the IAS. In addition, we compared the effects of these substances with the effects of NANC nerve stimulation while determining the role of CO and the HO pathway in NANC relaxation of the rat IAS. The studies were carried out in the rat IAS, because this animal has been recently considered to be a good model for humans (13, 40).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tissue Preparation

Male Sprague-Dawley rats (300–350 g) were killed by decapitation, and the entire anal canal was quickly removed and transferred to oxygenated (95% O2-5% CO2) Krebs physiological solution (in mM: 118.07 NaCl, 4.69 KCl, 2.52 CaCl2, 1.16 MgSO4, 1.01 NaH2PO4, 25 NaHCO3, and 11.10 glucose) at 37°C. Extraneous adventitia, blood vessels, and skeletal muscle tissues connected to the IAS were removed using sharp dissection. The anal canal was then opened and pinned flat with the mucosal side up on a dissecting tray containing oxygenated Krebs physiological solution. The mucosa was removed using sharp dissection. Circular smooth muscle strips (~0.5 x 7 mm) of the IAS (identified as a thickened circular smooth muscle situated at the lowermost part of the alimentary tract) were prepared. The experimental protocol of the study was approved by the Institutional Animal Care and Use Committee of Thomas Jefferson University and was in accordance with the recommendations of the American Association for the Accreditation of Laboratory Animal Care.

Measurement of Isometric Tension

The smooth muscle strips were transferred to 2-ml muscle baths containing oxygenated Krebs solution at 37°C. One end of the muscle strip was anchored at the bottom of the tissue bath, and the other was connected to a force transducer (model FT03, Grass Instruments, Quincy, MA). Isometric tension was measured by the PowerLab/8SP data acquisition system (ADInstruments) and recorded using Chart 4.1.2 (ADInstruments). Each smooth muscle strip was initially stretched to a tension of 0.7 g and then allowed to equilibrate for 90 min. During this period, the smooth muscle bath was replenished with fresh Krebs solution every 20 min. Only the smooth muscle strips that developed spontaneous tone and relaxed in response to electrical field stimulation (EFS, 0.5–20 Hz, 0.5-ms pulse, 12 V, 4-s train) were used. In the presence of atropine (1 x 10–6 M) and guanethidine (1 x 10–3 M), EFS causes stimulation of NANC nerves in the IAS. The changes in basal IAS tone after different agents were expressed as percent maximal relaxation by EDTA (50 mM) at the end of each experiment (1, 5).

Preparation of CORM-1, CO, and Other Agents

CORM-1 was freshly prepared before each experiment. A stock solution of 10–1 M was obtained by dissolving CORM-1 in DMSO following previously published instructions (24). Aliquots of this solution were then delivered to the tissues as described earlier (24) to obtain the final desired concentrations in the muscle bath.

CO was prepared following the method described by Schröder et al. (35). Briefly, 20 ml of Krebs solution were deoxygenated for 1 h with helium gas in a sealed glass vial. The solution was then bubbled with 99.8% CO for 15 min until a saturated (10–3 M) solution was obtained. Tin protoporphyrin IX (SnPP-IX) was dissolved in 0.2 N NaOH and hematin in 0.1 N NaOH. N{omega}-nitro-L-arginine (L-NNA) was dissolved in distilled water, and 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) was dissolved in DMSO. The final concentration of DMSO in the organ bath did not exceed 0.1%.

Drug Responses

Concentration-response curves with 50–600 µM CORM-1 and 5–100 µM CO were obtained in a cumulative fashion as described elsewhere (25). The O2 supply to the muscle bath was temporarily turned off briefly during such studies. The temporary cessation of oxygenation had no significant effect on basal IAS tone. To investigate the mechanism of action of these molecules, these experiments were repeated 20 min after incubation with different inhibitors: the neurotoxin TTX (1 x 10–6 M), the selective HO inhibitor SnPP-IX (1 x 10–4 M), the NOS inhibitor L-NNA (3 x 10–4 M), and the GC inhibitor ODQ (1 x 10–6 M). NANC nerve stimulation experiments by EFS were done in the presence of guanethidine (1 x 10–3 M) and atropine (1 x 10–6 M).

Western Blot Analysis

The presence of HO-1 and HO-2 and neuronal NOS (nNOS) was determined by Western blot studies as described elsewhere (7, 8, 12). Iso-{beta}-actin expression was used as standard for calculations. Briefly, the smooth muscle tissues were cut in small pieces (~1-mm cubes), rapidly homogenized in five volumes of boiling lysis buffer (1% SDS, 1.0 mM sodium orthovanadate, and 10 mM Tris, pH 7.4), and then microwaved for 10 s. The homogenates were centrifuged (16,000 g at 4°C) for 15 min, and protein contents in the resultant supernatant were determined by the method of Lowry et al. (19) with BSA as the standard. The samples were then mixed with 2x sample buffer (125 mM Tris, pH 6.8, 4% SDS, 10% glycerol, 0.006% bromphenol blue, and 2% {beta}-mercaptoethanol) and placed in a boiling water bath for 3 min. The proteins in an aliquot (20 µl containing 40 µg of protein extract) of each sample were separated by 7.5% SDS-polyacrylamide gel. The proteins thus separated were transferred to a nitrocellulose membrane (NCM) by electrophoresis at 4°C. To block nonspecific antibody binding of the antibodies, the NCMs were soaked overnight at 4°C in Tris-buffered saline-Tween 20 (TBST: 20 mM Tris, pH 7.6, 137 mM NaCl, and 0.1% Tween 20) containing 1% BSA. NCMs were then incubated with the specific primary antibodies [goat polyclonal IgG (1:2,000) for HO-1 and HO-2, rabbit polyclonal IgG (1:2,000) for nNOS, and {beta}-actin] for 1 h at room temperature. After they were washed with TBST, the membranes were incubated with horseradish peroxidase-conjugated donkey anti-goat IgG secondary antibodies (1:25,000) for detection of HO-1 and HO-2 and peroxidase-conjugated secondary anti-rabbit IgG secondary antibodies (1:25,000) for detection of nNOS and {beta}-actin for 1 h at room temperature. The corresponding bands were visualized with enhanced chemiluminescence substrate using SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL) and Hyperfilm MP (Amersham Life Science).

NCMs were then stripped of primary and secondary antibodies by incubation with Restore Western blot stripping buffer (Pierce) for 15 min at room temperature. NCMs were soaked overnight at 4°C in TBST. Immunoblots for {beta}-actin were obtained using specific primary and secondary antibodies as described above. Bands corresponding to different proteins on X-ray films were scanned (SnapScn.310, Agfa, Ridgefield Park, NJ), and the respective areas and optical densities were determined by using Image-Pro Plus 4.0 software (Media Cybernetics, Silver Spring, MD).

Drugs and Chemicals

TTX, L-NNA, CORM-1, and hematin were purchased from Sigma-Aldrich (St. Louis, MO). SnPP-IX was obtained from Frontier Scientific (Logan, UT). All antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). ODQ was purchased from Tocris (Ellisville, MO). All experiments involving SnPP-IX and hematin were performed in the dark. DMSO or NaOH used as solvents for some agents in the final concentrations in the tissue bath did not produce a significant effect on basal IAS tone or its relaxation in response to any of the stimuli.

Data Analysis

Values are means ± SE of different observations. Agonist concentration-response curves were fitted using a nonlinear interactive fitting program (Prism 3; Graph Pad Software). Agonist potencies and maximum response are expressed as the negative logarithm of the molar concentration of agonist producing 50% of the maximum response and the maximum effect elicited by the agonist, respectively, calculated from the concentration-response curves. Statistical significance was determined by one-way ANOVA or Student's t-test where suitable. In all cases, P < 0.05 was used to determine statistical significance.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Effect of CORM-1 vs. CO on Basal Tone of IAS

Influence of different neurohumoral antagonists and the neurotoxin TTX. CORM-1, as well as CO, produced concentration-dependent relaxation in the rat IAS. None of the neurohumoral antagonists (hexamethonium, propranolol, guanethidine, atropine, and indomethacin) had a significant effect on the relaxant actions of CORM-1 or CO (data not shown). The neurotoxin TTX (1 x 10–6 M) also had no significant effect on the IAS relaxation caused by these agents (P > 0.05, n = 5–10 animals; Fig. 1, A and B). TTX, on the other hand, nearly abolished the IAS relaxation with EFS (Fig. 1C). {omega}-Conotoxin (1 x 10–6 M), which causes significant attenuation of EFS-induced IAS relaxation, also had no significant effect on CORM-1- and CO-induced IAS relaxation (not shown).



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 1. Effect of carbon monoxide (CO)-releasing molecule (CORM)-1 (A), authentic CO (B), and electrical field stimulation (EFS; C) on basal tone of internal anal sphincter (IAS) smooth muscle before and after TTX (1 x 10–6 M). TTX causes near obliteration of the fall in IAS tone by EFS (*P < 0.05), whereas it has no significant effect on IAS relaxation caused by CORM-1 or CO (P > 0.05, n = 5–10).

 
Influence of SnPP-IX on IAS smooth muscle relaxation by CORM-1, CO, and EFS. The HO inhibitor SnPP-IX (1 x 10–4 M), previously shown to cause significant suppression of NANC relaxation and HO activity in different systems (2, 8, 31, 33, 43), caused no attenuation of IAS relaxation by the maximal effective concentrations of CORM-1 and CO (P > 0.05, n = 9–10; Fig. 2, A and B).



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2. Effect of the heme oxygenase (HO) inhibitor tin protoporphyrin IX (SnPP-IX, 1–3 x 10–4 M) on the fall in basal tone of IAS caused by CORM-1 (A), CO (B), and EFS (C). SnPP-IX does not modify the fall in IAS tone by any of these maneuvers (P > 0.05, n = 9–10).

 
Unexpectedly, however, 1 x 10–4 or 3 x 10–4 M SnPP-IX had no significant effect on EFS-induced relaxation in rat IAS smooth muscle (P > 0.05, n = 4–6; Fig. 2C). In the presence of 1 x 10–4 SnPP-IX, EFS at 5 and 10 Hz caused a decline of 66.4 ± 4.5 and 66.0 ± 6.3% in basal IAS tone. In control experiments, the decline in basal IAS tone was 63.6 ± 6.1 and 69.5 ± 5.0%, respectively.

Influence of L-NNA on IAS relaxation by CORM-1, CO, and EFS. The NOS inhibitor L-NNA (3 x 10–4 M) failed to modify the relaxant effects of CORM-1 or CO (P > 0.05, n = 6–7; Fig. 3, A and B).



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 3. Effect of the nitric oxide synthase (NOS) inhibitor N{omega}-nitro-L-arginine (L-NNA, 3 x 10–4 M) on IAS relaxation caused by CORM-1 (A), CO (B), and EFS (C). L-NNA causes significant attenuation of IAS relaxation caused by EFS (*P < 0.05, n = 7) but has no significant effect on CORM-1 or CO (P > 0.05, n = 6–7).

 
On the contrary, L-NNA caused a significant attenuation of the IAS relaxation caused by NANC nerve stimulation elicited by EFS (P < 0.05, n = 6–7; Fig. 3C). In the presence of L-NNA, 10 Hz of EFS-induced IAS relaxation was significantly attenuated to 25.4 ± 6.5%.

Effect of L-NNA + SnPP-IX on IAS Relaxation by CORM-1, CO, and EFS

Similar to their effect when used individually, the NOS and HO inhibitors in combination also produced no significant effect on the decline of basal IAS tone caused by CORM-1 or CO (P > 0.05, n = 6–8; Fig. 4, A and B).



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 4. Effect of SnPP-IX (1 x 10–4 M) + L-NNA (3 x 10–4 M) on IAS relaxation caused by CORM-1 (A), CO (B), and EFS (C). SnPP-IX + L-NNA causes significant attenuation of IAS relaxation caused by EFS (*P < 0.05, n = 7); however, it has no significant effect on CORM-1 or CO (P > 0.05, n = 6–8).

 
In contrast, L-NNA + SnPP-IX caused a significant attenuation of the IAS relaxation caused by EFS (P < 0.05, n = 10; Fig. 4C). In the presence of L-NNA + SnPP-IX, however, the EFS-induced relaxations were not significantly different from relaxation induced by L-NNA alone (P > 0.05; Figs. 2C and 3C).

Effect of ODQ on IAS Smooth Muscle Relaxation by CORM-1, CO, and EFS

The GC inhibitor ODQ (1 x 10–6 M) significantly attenuated the IAS relaxation caused by CORM-1, CO, and EFS (P < 0.05, n = 3–4). There were, however, interesting differences in terms of the degree of this attenuation by ODQ. The effects of CO were nearly abolished, whereas the GC inhibitor caused quantitative antagonism of CORM-1 and a rightward shift in the EFS-induced relaxation of IAS smooth muscle. The trends in IAS relaxation with these stimuli with a higher concentration of ODQ (1 x 10–5 M) were similar. The maximal effective concentration of CORM-1, CO, and 10 Hz of EFS in the presence of ODQ resulted in IAS relaxation of 24.7 ± 6.9, 4.40 ± 10.26, and 27.22 ± 6.82%, respectively (P < 0.05, n = 3–4; Fig. 5).



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 5. Effect of the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) on IAS relaxation by CORM-1 (A), CO (B), and EFS (C). ODQ causes significant antagonism of the fall in IAS tone by CORM-1 and EFS (*P < 0.05, n = 6–9 for EFS and n = 9–11 for CORM-1). ODQ abolishes the fall in IAS tone by CO (*P < 0.05, n = 9–10). In some experiments, CO in the presence of ODQ causes a small degree of contraction.

 
Effect of Hematin on Basal IAS Tone and IAS Relaxation With EFS

To further investigate the role of the HO pathway in the rat IAS, we examined the effect of the HO substrate hematin. Unexpectedly, incubation of the tissues with 1 x 10–4 and 1 x 10–3 M hematin for up to 30 min caused no significant change in basal IAS tone or IAS relaxation with EFS (P > 0.05, n = 4; Fig. 6).



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 6. Effect of hematin on basal IAS tone (A) and relaxation by EFS (B). Hematin at 1 x 10–4 and 1 x 10–3 M has no significant effect on basal tone or EFS-induced IAS relaxation (P > 0.05, n = 4).

 
Demonstration of HO-1, HO-2, and nNOS in the IAS

HO-1, HO-2, and nNOS were demonstrated in IAS smooth muscle tissue by the Western blot technique (Fig. 7).



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 7. Immunoblots of HO-1, HO-2, and neuronal NOS (nNOS) in IAS smooth muscle. Data show specific presence of the respective enzyme proteins in IAS smooth muscle. Levels of HO-2 were lower than HO-1 levels.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
These studies, for the first time, report the relaxant effect of CORM-1 in the GI smooth muscle. This facilitates basic research on the role of CO and the HO pathway in the GI tract. The main findings of the study are as follows: 1) CORM-1, similar to CO, causes IAS smooth muscle relaxation by its action directly at the smooth muscle cell, and 2) IAS relaxation by CORM-1, CO, and EFS converges on the same intracellular mechanism, i.e., activation of GC. NANC relaxation in the rat IAS involves NOS, and the role of HO remains unclear.

Direct effects of CORM-1 and CO are evident from their independence from any neurohumoral interaction, because it is not modified by the neurohumoral antagonists, including HO and the NOS inhibitors SnPP-IX and L-NNA, respectively, and the neurotoxins TTX and {omega}-conotoxin. In addition, CORM-1 and CO cause concentration-dependent relaxation of smooth muscle cells isolated from the IAS. The direct effect of CO is in agreement with earlier data from our laboratory in the opossum IAS (33) and data reported by others (14, 26, 27, 42). This fulfills an important criterion for the candidate inhibitory neurotransmitter (16, 30). A direct relaxant effect of CORM-1 has been shown in vascular smooth muscle (24) but not in GI smooth muscle.

Although authentic CO should be an ideal agent for investigation of the HO pathway in NANC relaxation, its preparation is cumbersome, and calculations of CO concentrations may not be precise, because they are based on certain assumptions. In addition, the effects of CO may not exactly match those of endogenously released CO during NANC nerve stimulation. In the present studies, this issue became apparent during examination of the influence of the GC inhibitor. ODQ causes near obliteration of IAS relaxation by all concentrations of CO. The effects of CORM-1 and EFS, on the other hand, were quantitative, and their antagonism by ODQ was concentration and frequency dependent, respectively. The data suggest controlled release and delivery of CO by CORM-1 to the target site. These observations are similar to those obtained by Motterlini et al. (24). These investigators, working on the aortic smooth muscle, also reported that ODQ causes competitive antagonism of vasodilatation by CORM-1.

Our studies provide further data in support of a common mode of IAS relaxation by NANC stimulation and by CORM-1 and CO. The IAS relaxation caused by all these maneuvers converges on activation of GC. The GC inhibitor ODQ significantly attenuates the IAS relaxation caused by these stimuli. This notion is supported by a number of studies that show selective activation of GC responsible for the smooth muscle relaxation after application of CO and NANC nerve stimulation (21, 33, 43). In addition, we found the definitive presence of HO-2 in IAS tissues as shown by Western blot studies. These data are similar to previous results in the opossum and human IAS, where not only by the presence of HO-2 protein, but also by immunocytochemistry and laser capture microdissection-RT-PCR, has HO-2 been shown specifically in the myenteric plexus (8, 10). The exact role of HO-1 in the present and previous studies in the IAS is not clear.

In a number of GI preparations, including opossum and murine IAS and feline, porcine, and canine lower esophageal sphincter (2, 8, 22, 27, 42), the role of the HO pathway in NANC relaxation has been speculated. These speculations are based primarily on the effect of HO inhibitors on NANC nerve stimulation, immunocytochemical localizations of HO-2, and functional data from HO-2–/– mice. There is no such information for the rat IAS. In the present studies, we used the selective HO inhibitor SnPP-IX in the concentrations known to cause inhibition of HO-2 in different systems (2, 31). SnPP-IX causes no significant attenuation of NANC relaxation in the rat IAS. To rule out the issue of difference in affinity for HO in the rat IAS, we determined that even the higher concentration of SnPP-IX did not attenuate NANC relaxation.

One of the speculations for the lack of inhibition of NANC relaxation in the rat IAS by SnPP-IX is that HO inhibition may lead to overexpression of NOS activity, which compensates for the inhibition and masks the effect of SnPP-IX. To overcome this, we examined the effect of SnPP-IX in the presence of the NOS inhibitor on NANC relaxation. Even with this experimental protocol, we found no further attenuation of IAS relaxation.

Failure of hematin to exert any significant effect on IAS tone or NANC relaxation is also unforeseen. Hematin was expected to inhibit basal IAS tone and augment HO-related NANC relaxation via CO production, as shown in certain systems (17). The main explanations for the lack of effect of HO inhibitor and precursor in the rat IAS are as follows. First, the HO pathway may not play a significant role in IAS relaxation. Second, HO inhibition by SnPP-IX upregulates constitutive NOS activity, which in turn compensates for the HO inhibition, with the net results being no effect on NANC relaxation. This concept is supported by studies that show a decrease in NOS activity after CO application (38) and by NOS upregulation after HO inhibitor (9, 36). Third, it is possible that in the rat IAS, under the experimental conditions, because of transport problems, the binding sites of HO are not accessible to the substrate and the HO inhibitor. The same situation has been shown to occur in the porcine gastric fundus (11), in which HO-2 is present in significant amounts in the myenteric plexus and CO meets a number of criteria for the inhibitory neurotransmitter (including smooth muscle relaxation by CO). Surprisingly, in those studies, as in our study, the HO inhibitor SnPP-IX had no significant effect on NANC relaxation and HO activity in the smooth muscle strips. The authors did, in fact, demonstrate that SnPP-IX inhibits HO-2 isolated directly from these tissues.

The present data in the rat IAS are in agreement with findings of previous studies in other species that NO plays a major role in NANC relaxation in the IAS (4, 28, 29, 32, 34, 39). The NOS inhibitor L-NNA in the appropriate concentrations causes significant attenuation of NANC relaxation in the IAS. However, in the presence of L-NNA, we still observed ~25% intact IAS relaxation, which was not further affected by an increase in the concentration of the NOS inhibitor. The role of other candidate inhibitory neurotransmitters such as VIP, pituitary adenylate cyclase-activating peptide, and ATP in residual IAS relaxation remains to be determined.

In summary, the studies identify an important inhibitory effect of CORM-1 in the IAS. The studies further show that, in the rat IAS, as in the human, opossum, feline, porcine, and rabbit IAS, a majority of the NANC relaxation is NO mediated. This is in contrast to the murine IAS, where NANC relaxation has been shown to be mediated primarily via CO (41). In light of the direct effect of CORM-1 and CO in causing relaxation of the smooth muscle via GC, and the presence of significant levels of HO-2 in the IAS, a partial role of CO in the NANC relaxation, however, is difficult to rule out. In the rat IAS, HO may have a neuromodulatory role in NOS inhibitory transmission. Regardless of the role of CO in NANC relaxation, alternative approaches, such as CORM treatment, toward achieving IAS smooth muscle relaxation are important in terms of therapeutic potential in spastic anorectal motility disorders. In this regard, the refined molecules that accurately and safely deliver CO to the target site (in this case, the IAS smooth muscle cell) will be certainly preferable, because the targeted delivery of CO as gas may be neither feasible nor practical.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-35385 and by an institutional grant from Thomas Jefferson University.


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. Rattan, Div. of Gastroenterology and Hepatology, Dept. of Medicine, Thomas Jefferson Univ., 1025 Walnut St., Rm. 901 College, Philadelphia, PA 19107 (E-mail: Satish.Rattan{at}Jefferson.edu)

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. Biancani P, Walsh JH, and Behar J. Vasoactive intestinal polypeptide: a neurotransmitter for relaxation of the rabbit internal anal sphincter. Gastroenterology 89: 867–874, 1985.[ISI][Medline]
  2. Boehning D, Moon C, Sharma S, Hurt KJ, Hester LD, Ronnett GV, Shugar D, and Snyder SH. Carbon monoxide neurotransmission activated by CK2 phosphorylation of heme oxygenase-2. Neuron 40: 129–137, 2003.[ISI][Medline]
  3. Boehning D and Snyder SH. Novel neural modulators. Annu Rev Neurosci 26: 105–131, 2003.[CrossRef][ISI][Medline]
  4. Burleigh DE. Ng-nitro-L-arginine reduces nonadrenergic, noncholinergic relaxations of human gut. Gastroenterology 102: 679–683, 1992.[ISI][Medline]
  5. Cao W, Harnett KM, Behar J, and Biancani P. PGF2{alpha}-induced contraction of cat esophageal and lower esophageal sphincter circular smooth muscle. Am J Physiol Gastrointest Liver Physiol 283: G282–G291, 2002.[Abstract/Free Full Text]
  6. Cary SPL and Marletta MA. The case of CO signaling: why the jury is still out? J Clin Invest 107: 1071–1073, 2001.[Free Full Text]
  7. Chakder S, Bandyopadhyay A, and Rattan S. Neuronal NOS gene expression in gastrointestinal myenteric neurons and smooth muscle cells. Am J Physiol Cell Physiol 273: C1868–C1875, 1997.[Abstract/Free Full Text]
  8. Chakder S, Cao GY, Lynn RB, and Rattan S. Heme oxygenase activity in the internal anal sphincter: effects of nonadrenergic, noncholinergic nerve stimulation. Gastroenterology 118: 477–486, 2000.[ISI][Medline]
  9. Chakder S, Rathi S, Ma X, and Rattan S. 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]
  10. Chen Y, Lui VCH, Sham MH, and Tam PKH. Distribution of carbon monoxide-producing neurons in human colon and in Hirschsprung's disease patients. Hum Pathol 33: 1030–1036, 2002.[CrossRef][ISI][Medline]
  11. Colpaert EE, Timmermans JP, and Lefebvre RA. Investigation of the potential modulatory effect of biliverdin, carbon monoxide and bilirubin on nitrergic neurotransmission in the pig gastric fundus. Eur J Pharmacol 457: 177–186, 2002.[CrossRef][ISI][Medline]
  12. Fan YP, Chakder S, Gao F, and Rattan S. Inducible and neuronal nitric oxide synthase involvement in lipopolysaccharide-induced sphincteric dysfunction. Am J Physiol Gastrointest Liver Physiol 280: G32–G42, 2001.[Abstract/Free Full Text]
  13. Fan YP, Puri RN, and Rattan S. Animal model for angiotensin II effects in the internal anal sphincter smooth muscle: mechanism of action. Am J Physiol Gastrointest Liver Physiol 282: G461–G469, 2002.[Abstract/Free Full Text]
  14. Farrugia G, Irons WA, Rae JL, Sarr MG, and Szurszewski JH. Activation of whole cell currents in isolated human jejunal circular smooth muscle cells by carbon monoxide. Am J Physiol Gastrointest Liver Physiol 264: G1184–G1189, 1993.[Abstract/Free Full Text]
  15. Farrugia G, Lei S, Lin X, Miller SM, Nath KA, Ferris CD, Levitt M, and Szurszewski JH. A major role for carbon monoxide as an endogenous hyperpolarizing factor in the gastrointestinal tract. Proc Natl Acad Sci USA 100: 8567–8570, 2003.[Abstract/Free Full Text]
  16. Goyal RK, Rattan S, and Said SI. VIP as a possible neurotransmitter of non-cholinergic non-adrenergic inhibitory neurones. Nature 288: 378–380, 1980.[ISI][Medline]
  17. Lamar CA, Mahesh VB, and Brann DW. Regulation of gonadotropin-releasing hormone (GnRH) secretion by heme molecules: a regulatory role for carbon monoxide? Endocrinology 137: 790–793, 1996.[Abstract]
  18. Lefebvre RA. Nitric oxide in the peripheral nervous system. Ann Med 27: 379–388, 1995.[ISI][Medline]
  19. Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ. Protein measurements with the Folin phenol reagent. J Biol Chem 193: 265–275, 1951.[Free Full Text]
  20. Maines MD. Heme oxygenase: function, multiplicity, regulatory mechanisms, and clinical applications. FASEB J 2: 2557–2568, 1988.[Abstract/Free Full Text]
  21. Maines MD. The heme oxygenase system: a regulator of second messenger gases. Annu Rev Pharmacol Toxicol 37: 517–554, 1997.[CrossRef][ISI][Medline]
  22. Miller SM, Farrugia G, Schmalz PF, Ermilov LG, Maines MD, and Szurszewski JH. Heme oxygenase 2 is present in interstitial cell networks of the mouse small intestine. Gastroenterology 114: 239–244, 1998.[ISI][Medline]
  23. Miller SM, Reed D, Sarr MG, Farrugia G, and Szurszewski JH. Haem oxygenase in enteric nervous system of human stomach and jejunum and co-localization with nitric oxide synthase. Neurogastroenterol Motil 13: 121–131, 2001.[CrossRef][ISI][Medline]
  24. Motterlini R, Clark JE, Foresti R, Sarathchandra P, Mann BE, and Green CJ. Carbon monoxide-releasing molecules: characterization of biochemical and vascular activities. Circ Res 90: E17–E24, 2002.[ISI][Medline]
  25. Moummi C and Rattan S. Effect of methylene blue and N-ethylmaleimide on internal anal sphincter relaxation. Am J Physiol Gastrointest Liver Physiol 255: G571–G578, 1988.[Abstract/Free Full Text]
  26. Ny L, Alm P, Ekstrom P, Larsson B, Grundemar L, and Andersson KE. 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]
  27. Ny L, Alm P, Larsson B, and Andersson KE. Morphological relations between haem oxygenases, NO-synthase and VIP in the canine and feline gastrointestinal tracts. J Auton Nerv Syst 65: 49–56, 1997.[CrossRef][ISI][Medline]
  28. O'Kelly T, Brading A, and Mortensen N. Nerve-mediated relaxation of the human internal anal sphincter: The role of nitric oxide. Gut 34: 689–693, 1993.[Abstract]
  29. O'Kelly TJ, Davies JR, Brading AF, and Mortensen NJMC. Distribution of nitric oxide synthase containing neurons in the rectal myenteric plexus and anal canal. Dis Colon Rectum 352: 350–357, 1994.
  30. Rattan S. The non-adrenergic non-cholinergic innervation of the esophagus and the lower esophageal sphincter. Arch Int Pharmacodyn 280: 62–83, 1986.[ISI][Medline]
  31. Rattan S and Chakder S. Influence of heme oxygenase inhibitors on the basal tissue enzymatic activity and smooth muscle relaxation of internal anal sphincter. J Pharmacol Exp Ther 294: 1009–1016, 2000.[Abstract/Free Full Text]
  32. Rattan S and Chakder S. Role of nitric oxide as a mediator of internal anal sphincter relaxation. Am J Physiol Gastrointest Liver Physiol 262: G107–G112, 1992.[Abstract/Free Full Text]
  33. Rattan S and Chakder S. Inhibitory effect of CO on internal anal sphincter: heme oxygenase inhibitor inhibits NANC relaxation. Am J Physiol Gastrointest Liver Physiol 265: G799–G804, 1993.[Abstract/Free Full Text]
  34. Rattan S, Sarkar A, and Chakder S. Nitric oxide pathway in rectoanal inhibitory reflex of opossum internal anal sphincter. Gastroenterology 103: 43–50, 1992.[ISI][Medline]
  35. Schröder A, Hedlund P, and Andersson KE. Carbon monoxide relaxes the female pig urethra as effectively as nitric oxide in the presence of YC-1. J Urol 167: 1892–1896, 2002.[CrossRef][ISI][Medline]
  36. Seki T, Naruse M, Naruse K, Yoshimoto T, Tanabe A, Imaki T, Hagiwara H, Hirose S, and Demura H. Interrelation between nitric oxide synthase and heme oxygenase in rat endothelial cells. Eur J Pharmacol 331: 87–91, 1997.[CrossRef][ISI][Medline]
  37. Stark ME and Szurszewski JH. Role of nitric oxide in gastrointestinal and hepatic function and disease. Gastroenterology 103: 1928–1949, 1992.[ISI][Medline]
  38. Thorup C, Jones CL, Gross SS, Moore LC, and Goligorsky MS. Carbon monoxide induces vasodilation and nitric oxide release but suppresses endothelial NOS. Am J Physiol Renal Physiol 277: F882–F889, 1999.[Abstract/Free Full Text]
  39. Vanderwinden JM, De Laet MH, Schiffmann SN, Mailleux P, Lowenstein CJ, Snyder SH, and Vanderhaeghen JJ. Nitric oxide synthase distribution in the enteric nervous system of Hirschsprung's disease. Gastroenterology 105: 969–973, 1993.[ISI][Medline]
  40. Vinograd I, Hanani M, Hadary A, Merguerian P, and Nissan S. Animal model for the study of internal anal sphincter activity. Eur Surg Res 17: 259–263, 1985.[ISI][Medline]
  41. Watkins CC, Boehning D, Kaplin AI, Rao M, Ferris CD, and Snyder SH. Carbon monoxide mediates vasoactive intestinal polypeptide-associated nonadrenergic/noncholinergic neurotransmission. Proc Natl Acad Sci USA 101: 2631–2635, 2004.[Abstract/Free Full Text]
  42. Werkström V, Ny L, Persson K, and Andersson KE. Carbon monoxide-induced relaxation and distribution of haem oxygenase isoenzymes in the pig urethra and lower oesophagogastric junction. Br J Pharmacol 120: 312–318, 1997.[Abstract]
  43. Zakhary R, Poss KD, Jaffrey SR, Ferris CD, Tonegawa S, and Snyder SH. Targeted gene deletion of heme oxygenase 2 reveals neural role for carbon monoxide. Proc Natl Acad Sci USA 94: 14848–14853, 1997.[Abstract/Free Full Text]