Mechanism of internal anal sphincter smooth muscle relaxation by phorbol 12,13-dibutyrate.

We investigated the mechanism of the inhibitory action of phorbol 12,13-dibutyrate (PDBu), one of the typical protein kinase C (PKC) activators, in in vitro smooth muscle strips and in isolated smooth muscle cells of the opossum internal anal sphincter (IAS). The inhibitory action of PDBu on IAS smooth muscle (observed in the presence of guanethidine + atropine) was partly attenuated by tetrodotoxin, suggesting that a part of the inhibitory action of PDBu is via the nonadrenergic, noncholinergic neurons. A major part of the action of PDBu in IAS smooth muscle was, however, via its direct action at the smooth muscle cells, accompanied by a decrease in free intracellular Ca(2+) concentration ([Ca(2+)](i)) and inhibition of PKC translocation. PDBu-induced IAS smooth muscle relaxation was unaffected by agents that block Ca(2+) mobilization and Na+-K+-ATPase. The PDBu-induced fall in basal IAS smooth muscle tone and [Ca(2+)](i) resembled that induced by the Ca(2+) channel blocker nifedipine and were reversed specifically by the Ca(2+) channel activator BAY K 8644. We speculate that a major component of the relaxant action of PDBu in IAS smooth muscle is caused by the inhibition of Ca(2+) influx and of PKC translocation to the membrane. The specific role of PKC downregulation and other factors in the phorbol ester-mediated fall in basal IAS smooth muscle tone remain to be determined.

Inducible and neuronal nitric oxide synthase involvement in lipopolysaccharide-induced sphincteric dysfunction.

We examined the effect of endotoxin lipopolysaccharide (LPS) on the basal tone and on the effects of different stimuli and agonists and transcriptional and translational expression of nitric oxide (NO) synthase (NOS) isozymes in the lower esophageal sphincter (LES), pyloric sphincter (PS), and internal anal sphincter (IAS). NO release was also examined before and after LPS. LPS caused a dose-dependent fall in the basal tone and augmentation of the relaxation caused by nonadrenergic, noncholinergic (NANC) nerve stimulation in the LES and IAS. In the PS, LPS had no significant effect on the basal tone and caused an attenuation of the NANC relaxation and an augmentation of the contractile response of muscarinic agonist. Interestingly, the smooth muscle relaxation by atrial natriuretic factor was suppressed in the LES and IAS but not in the PS. These changes in the sphincteric function following LPS may be associated with increase in the inducible NOS (iNOS) expression since they were blocked by iNOS inhibitor L-canavanine. Augmentation of NANC relaxation in the LES and IAS smooth muscle by LPS may be due to the increased activity of neuronal NOS and NO production.

Influence of heme oxygenase inhibitors on the basal tissue enzymatic activity and smooth muscle relaxation of internal anal sphincter.

We examined the actions of different heme oxygenase (HO) inhibitors on the basal HO activity in the opossum internal anal sphincter (IAS), rectum, and liver tissues and the IAS smooth muscle relaxation in response to nonadrenergic noncholinergic (NANC) nerve stimulation and different agonists. All the tissues examined were found to have significant levels of basal HO activity. Among different HO inhibitors, tin protoporphyrin IX (SnPP IX) was found to be most selective in inhibiting the HO activity in the IAS smooth muscle. Conversely, in the liver, all the HO inhibitors except SnPP IX caused significant inhibition of HO activity. Consistent with HO activity inhibition, the IAS smooth muscle relaxations caused by NANC nerve stimulation and vasoactive intestinal polypeptide also were inhibited by zinc protoporphyrin IX and SnPP IX. Zinc protoporphyrin IX also caused a significant attenuation of the IAS smooth muscle relaxation caused by isoproterenol. The IAS smooth muscle relaxation caused by nitric oxide was not significantly modified by any of the HO inhibitors. The data show the presence of HO activity in the IAS and other gastrointestinal tissues. The differential attenuation of HO activity by different HO inhibitors in the IAS smooth muscle and liver confirms the presence of different isozymes of HO in different tissues. Suppression of basal HO activity and the IAS smooth muscle relaxation induced by NANC nerve stimulation or VIP but not NO suggest that some of the stimuli that cause IAS smooth muscle relaxation may involve HO activity.

Heme oxygenase activity in the internal anal sphincter: effects of nonadrenergic, noncholinergic nerve stimulation.

BACKGROUND & AIMS: To date, the exact role of carbon monoxide (CO) in the nonadrenergic, noncholinergic (NANC) relaxation is not known. This is partly related to the lack of an appropriate method to measure heme oxygenase (HO) activity in the gastrointestinal tissues. METHODS: HO activity of the opossum internal anal sphincter (IAS) smooth muscle was determined using a newly developed assay system that used radiolabeled hemin as a substrate. Enzyme activity of the IAS tissues was measured in the basal state, after electric field stimulation (EFS), ganglionic stimulant dimethyl diphenyl piperazinium iodide (DMPP), and neuropeptide vasoactive intestinal polypeptide (VIP). The presence and localization of HO was examined by Western blot analysis and immunocytochemistry. RESULTS: NANC nerve stimulation of the IAS smooth muscle by EFS (0.25-5 Hz), DMPP, and VIP caused a significant increase in the HO activity of the IAS. The increase in HO activity by EFS was inhibited by the HO inhibitor Tin protoporphyrin (1 x 10(-4) mol/L). Both HO-1 and HO-2 were present in the IAS tissue extracts, and both enzymes were localized in the neurons of the myenteric plexus. The method for HO activity determination used in the present study was found to be reliable and reproducible. CONCLUSIONS: The data suggest that the HO pathway may have a role in neurally mediated relaxation of the IAS. The exact site of involvement and the source of HO activity, however, remains to be determined.

Heme oxygenase-2 distribution in anorectum: colocalization with neuronal nitric oxide synthase.

Recent investigations have suggested carbon monoxide (CO) as a putative messenger molecule. Although several studies have implicated the heme oxygenase (HO) pathway, responsible for the endogenous production of CO, in the neuromodulatory control of the internal anal sphincter (IAS), its exact role is not known. Nitric oxide, produced by neuronal nitric oxide synthase (nNOS) of myenteric neurons, is an important inhibitory neural messenger molecule mediating nonadrenergic noncholinergic (NANC) relaxation of the IAS. The present studies were undertaken to investigate in detail the presence and coexistence of heme oxygenase-2 (HO-2) with nNOS in the opossum anorectum. In perfusion-fixed, frozen-sectioned tissue, HO-2 immunoreactive (IR) and nNOS IR nerves were identified using immunocytochemistry. Ganglia containing HO-2 IR neuronal cell bodies were present in the myenteric and submucosal plexuses throughout the entire anorectum. Colocalization of HO-2 IR and nNOS IR was nearly 100% in the IAS and decreased proximally from the anal verge. In the rectum, colocalization of HO-2 IR and nNOS IR was approximately 70%. Additional confocal microscopy studies using c-Kit staining demonstrated the localization of HO-2 IR and nNOS IR in interstitial cells of Cajal (ICC) of the anorectum. From the high rate of colocalization of HO-2 IR and nNOS IR in the IAS as well as the localization of HO-2 IR and nNOS IR in ICC in conjunction with earlier studies of the HO pathway, we speculate an interaction between HO and NOS pathways in the NANC inhibitory neurotransmission of the IAS and rectum.

Biochemical and functional profile of a newly developed potent and isozyme-selective arginase inhibitor.

An increase in arginase activity has been associated with the pathophysiology of a number of conditions, including an impairment in nonadrenergic and noncholinergic (NANC) nerve-mediated relaxation of the gastrointestinal smooth muscle. An arginase inhibitor may rectify this condition. We compared the effects of a newly designed arginase inhibitor, 2(S)-amino-6-boronohexanoic acid (ABH), with the currently available N(omega)-hydroxy-L-arginine (L-HO-Arg), on the NANC nerve-mediated internal anal sphincter (IAS) smooth-muscle relaxation and the arginase activity in the IAS and other tissues. Arginase caused an attenuation of the IAS smooth-muscle relaxations by NANC nerve stimulation that was restored by the arginase inhibitors. L-HO-Arg but not ABH caused dose-dependent and complete reversal of N(omega)-nitro-L-arginine-suppressed IAS relaxation that was similar to that seen with L-arginine. Both ABH and L-HO-Arg caused an augmentation of NANC nerve-mediated relaxation of the IAS. In the IAS, ABH was found to be approximately 250 times more potent than L-HO-Arg in inhibiting the arginase activity. L-HO-Arg was found to be 10 to 18 times more potent in inhibiting the arginase activity in the liver than in nonhepatic tissues. We conclude that arginase plays a significant role in the regulation of nitric oxide synthase-mediated NANC relaxation in the IAS. The advent of new and selective arginase inhibitors may play a significant role in the discrimination of arginase isozymes and have important pathophysiological and therapeutic implications in gastrointestinal motility disorders.

Mechanism of action of cholera toxin on the opossum internal anal sphincter smooth muscle.

Cholera toxin (CTX), an activator of G(s) protein, is an important pharmacological tool in G protein research. The effect and the mechanism of action of CTX in the gastrointestinal smooth muscle, including the internal anal sphincter (IAS), are not known. The present investigation was carried out to examine the effects of CTX on the signal transduction associated with the adenylate cyclase (AC) pathway on the basal tone of the IAS smooth muscle. CTX caused a prompt and dose-dependent fall in the basal tone of the IAS that was not affected by the neurotoxins TTX and omega-conotoxin or the nitric oxide synthase inhibitor N(G)-nitro-L-arginine. The cyclooxygenase inhibitor indomethacin, cAMP-dependent protein kinase inhibitor Rp-8-bromoadenosine 3',5' cyclic monophosphorothioate inhibited CTX-induced IAS smooth muscle relaxation. Furthermore, CTX caused a concentration-dependent relaxation of the isolated smooth muscle cells (SMC) of the IAS, which was blocked by G(s)alpha antibody (G(s)alpha-Ab). The IAS smooth muscle relaxation was accompanied with an increase in the GTPase activity that was also specifically blocked by G(s)alpha-Ab. We conclude that a major part of the inhibitory action of CTX in the IAS is via the direct response of the SMC that is linked with G(s) protein to the AC pathway. A part of the inhibitory action of CTX on the smooth muscle occurs via the activation of cyclooxygenase pathway. The relative contribution of such actions of CTX in the smooth muscle in the gastrointestinal motility disturbances following cholera infection remains to be determined.

99mTc-labeled vasoactive intestinal peptide receptor agonist: functional studies.

UNLABELLED: Vasoactive intestinal peptide (VIP) is a naturally occurring 28-amino acid peptide with a wide range of biological activities. Recent reports suggest that VIP receptors are expressed on a variety of malignant tumor cells and that the receptor density is higher than for somatostatin. Our aims were to label VIP with 99mTc--a generator-produced, inexpensive radionuclide that possesses ideal characteristics for scintigraphic imaging--and to evaluate 99mTc-VIP for bioactivity and its ability to detect experimental tumors. METHODS: VIP28 was modified at the carboxy terminus by the addition of four amino acids that provided an N4 configuration for a strong chelation of 99mTc. To eliminate steric hindrance, 4-aminobutyric acid (Aba) was used as a spacer. VIP28 was labeled with 1251, which served as a control. Biological activity of the modified VIP28 agonist (TP3654) was examined in vitro using a cell-binding assay and an opossum internal anal sphincter (IAS) smooth muscle relaxivity assay. Tissue distribution studies were performed at 4 and 24 h after injection, and receptor-blocking assays were also performed in nude mice bearing human colorectal cancer LS174T. Blood clearance was examined in normal Sprague-Dawley rats. RESULTS: The yield of 99mTc-TP3654 was quantitative, and the yields of 125I-VIP and 1251-TP3654 were >90%. All in vitro data strongly suggested that the biological activity of 99mTc-TP3654 agonist was equivalent to that of VIP28. As the time after injection increased, radioactivity in all tissues decreased, except in the receptor-enriched tumor (P = 0.84) and in the lungs (P = 0.78). The tumor uptake (0.23 percentage injected dose per gram of tissue [%ID/g]) was several-fold higher than 125I-VIP (0.06 %ID/g) at 24 h after injection in the similar system. In mice treated with unlabeled VIP or TP3654, the uptake of 99mTc-TP3654 decreased in all VIP receptor-rich tissues except the kidneys. The blood clearance was biphasic; the alpha half-time was 5 min and the beta half-time was approximately 120 min. CONCLUSION: VIP28 was modified and successfully labeled with 99mTc. The results of all in vitro examinations indicated that the biological activity of TP3654 was equivalent to that of native VIP28 and tumor binding was receptor specific.

Mechanism of inhibition of VIP-induced LES relaxation by heme oxygenase inhibitor zinc protoporphyrin IX.

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.

Mechanisms and sites of action of endothelins 1 and 2 on the opossum internal anal sphincter smooth muscle tone in vitro.

Endothelins, localized in the enteric nervous system, may play important roles in the morphogenesis of the gastrointestinal (GI) tract and in the regulation of GI motility. However, the role of endothelins in the GI sphincters, including the internal anal sphincter (IAS) have not been examined. We examined the actions of endothelins on the basal tone of the opossum IAS circular smooth muscle strips before and after different neurohumoral antagonists or inhibitors. Endothelins 1 and 2 produced a concentration-dependent biphasic effect on the basal tone of the IAS, an initial brief fall followed by a sustained rise. The fall in the IAS smooth muscle tone was not modified by atropine, guanethidine, or tetrodotoxin but was significantly attenuated by the nitric oxide synthase inhibitor L-NNA, the specific neuronal nitric oxide synthase inhibitor, 1-(2-trifluoromethylphenyl)imidazole, the N-type neuronal Ca++-channel blocker omega-conotoxin GVIA, and by the calmodulin antagonist W-13. Endothelin-induced contraction of the IAS, on the other hand, was not affected by any of the neurohumoral antagonists but was significantly inhibited by the selective protein kinase C inhibitor H-7 or the calmodulin inhibitor W-13. The combination of H-7 and W-13 had no additive effect in attenuating the contractile action of endothelin 1. There was clear evidence of a cross-tachyphylaxis to the actions of endothelin 1 and endothelin 2. We conclude that the endothelins exert important neuromodulatory effects on the basal tone of the IAS. The contractile action occurs directly at the smooth muscle and the relaxant action by the activation of neuronal nitric oxide synthase at the nerve terminals. The contraction and relaxation of the smooth muscle caused by endothelins 1 and 2 may involve distinct receptors that are similar for both endothelins. The excitatory actions of endothelin 1 involve both the protein kinase C and the Ca++-calmodulin pathways that may lie in series.

Involvement of pituitary adenylate cyclase-activating peptide in opossum internal anal sphincter relaxation.

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 Nomega-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.

Inhibitory effect of zinc protoporphyrin IX on lower esophageal sphincter smooth muscle relaxation by vasoactive intestinal polypeptide and other receptor agonists.

This study was performed in the opossum lower esophageal sphincter (LES) smooth muscle strips to determine the action of the heme oxygenase inhibitor zinc protoporphyrin IX (ZnPP IX) on the relaxant effect of vasoactive intestinal polypeptide and isoproterenol, which are known to stimulate adenylate cyclase (AC) via G protein coupling, and of the direct activator of AC catalytic subunit forskolin. To investigate the cGMP pathway, we examined the effect of atrial natriuretic factor known to activate the receptor linked to the particulate guanylate cyclase via G protein coupling and that of sodium nitroprusside [nitric oxide (NO) donor], authentic NO and carbon monoxide, which stimulate the intracellular soluble fraction of GC. The smooth muscle relaxation caused by nonadrenergic noncholinergic (NANC) nerve stimulation also was investigated. ZnPP IX caused concentration-dependent attenuation of the relaxant effect of vasoactive intestinal polypeptide, isoproterenol and atrial natriuretic factor without any effect on that of forskolin, sodium nitroprusside, NO and CO. Interestingly, ZnPP IX had no significant effect on the LES relaxation caused by NANC nerve stimulation and the smooth muscle contraction by bethanechol. From these results, we conclude that ZnPP IX attenuates the LES smooth muscle relaxation caused by the stimulation of G protein-coupled receptors to particulate AC and guanylate cyclase. The lack of effect of ZnPP IX on the NANC nerve-mediated LES relaxation suggests either lack of a role of heme oxygenase pathway in the response or an upregulation of NOS leading to normal LES relaxation.

Sites of actions of contractile and relaxant effects of pituitary adenylate cyclase activating peptide (PACAP) in the internal anal sphincter smooth muscle.

In summary, PACAP exerts a biphasic effect (an initial contraction followed by a relaxation) in the IAS. The initial contractile effect with higher concentrations of PACAP was found to be mediated by the activation of PACAP receptor at the substance P-containing nerve terminals. The PACAP receptor(s) responsible for the inhibitory action of the neuropeptide is(are) hypothesized to be present in the IAS smooth muscle cells and on the myenteric nerve terminals. The exact nature and the role of PACAP and the PACAP receptors in the inhibitory neurotransmission, the relationship of PACAP receptors with substance P-containing neurons and IAS smooth muscle cells, and interactions with the NOS pathway and VIP remain to be determined.

Excitatory and inhibitory actions of pituitary adenylate cyclase-activating peptide (PACAP) in the internal anal sphincter smooth muscle: sites of actions.

Unlike its effects on the rest of the GI tract, the effects of pituitary adenylate cyclase-activating peptide (PACAP) on the internal anal sphincter (IAS) are not known. We examined the actions of PACAP-38 (here PACAP) and PACAP-27 on the basal IAS tone of circular smooth muscle strips before and after the administration of different neurohumoral antagonists. PACAP caused a concentration-dependent fall in the basal tone of the IAS. Interestingly, however, at higher concentrations, PACAP caused a biphasic response: an initial contraction followed by a relaxation. Both the contractile and the relaxant responses were insensitive to atropine, guanethidine, apamin or tetrodotoxin. Both the contractile and the relaxant effects were inhibited by PACAP 6-38 (a selective antagonist of PACAP), vasoactive intestinal polypeptide 10-28 (a vasoactive intestinal polypeptide antagonist) and PACAP tachyphylaxis. The nitric oxide synthase inhibitor Nomega-nitro-L-arginine attenuated the inhibitory but not the excitatory effect of PACAP. Conversely, the contractile but not the relaxant effect of PACAP on the IAS was nearly obliterated by the substance P antagonist spantide. The N-type Ca++-channel blocker omega-conotoxin caused significant suppression of both the contractile and the inhibitory actions of PACAP. We conclude that in the IAS, PACAP has a dual effect: a contraction followed by a relaxation. The contraction of IAS by PACAP is speculated to occur via the activation of PACAP receptor at the substance P-containing nerve terminals. PACAP-induced IAS relaxation, on the other hand, appears to be mediated in large part by its direct action at the smooth muscle cells and in part by its action at the nerve terminals of the myenteric inhibitory neurons.

L-arginine deficiency causes suppression of nonadrenergic noncholinergic nerve-mediated smooth muscle relaxation: role of L-citrulline recycling.

Studies were performed on the internal anal sphincter (IAS) smooth muscle strips obtained from opossums (Didelphis virginiana). Isometric tension and L-arginine levels of the tissues were measured under basal conditions, in the presence of electrical field stimulation (EFS) and after treatment with different concentrations of arginase. For the nonadrenergic noncholinergic nerve stimulation, short trains (4 sec) as well as continuous EFS were used. During continuous EFS, after the initial IAS relaxation, the response began to fade within several min to approximately 80% recovery of the basal tone. We also examined the influence of L-arginine and L-citrulline on these responses. For some studies, the tissues were pretreated with L-glutamine (an inhibitor of L-citrulline uptake), L-glutamate or N(G)-hydroxy-L-arginine (an inhibitor of arginase). Interestingly, the basal levels of L-arginine were found to be significantly higher in the IAS (tonic smooth muscle) than in the rectal (phasic smooth muscle) smooth muscle. Arginase caused a concentration-dependent attenuation of the IAS relaxation caused by EFS. L-Citrulline and L-arginine were equipotent in reversing the attenuation. Both arginase (60 min pretreatment) and continuous EFS (tissues collected at the time of maximal recovery of the basal IAS tone after the initial relaxation) caused significant decreases in L-arginine levels. The decreases in the levels of L-arginine were restored by the exogenous administration of either L-arginine or L-citrulline. The restoration of L-arginine levels by L-citrulline but not by L-arginine was selectively blocked by L-glutamine. Furthermore, the IAS relaxation, attenuated by arginase was unaffected by L-glutamine but was restored by N-hydroxy-L-arginine pretreatment. The studies suggest that L-citrulline-L-arginine recycling may play a significant role in the maintenance of IAS relaxation in response to nonadrenergic noncholinergic nerve stimulation.

Regulation of inducible and neuronal nitric oxide synthase gene expression by interferon-gamma and VIP.

The studies examined the regulation of inducible and neuronal nitric oxide synthases (iNOS and nNOS, respectively) in the rat brain, stomach, rectum, and spleen. Relative expression of iNOS and nNOS mRNAs was quantified by the sensitive method of polymerase amplification reactions. The NOS proteins were determined by Western blot, using specific antibodies. Highest levels of nNOS and iNOS mRNAs were expressed in the rat brain and spleen, respectively. Furthermore, both nNOS and iNOS were expressed in the stomach and rectum. Interestingly, treatment of tissues with lipopolysaccharides or cytokine interferon-gamma (IFN-gamma) induced the expression of iNOS and decreased that of nNOS, representing a shift from one isoform to the other. When the tissues were treated with IFN-gamma followed by vasoactive intestinal polypeptide (VIP), the induction of iNOS was reduced by VIP. The changes in iNOS and nNOS expression at the transcriptional level corresponded to those at the translational level. The data suggest a regulatory role of IFN-gamma and VIP in the expression iNOS and nNOS and a counterregulation of iNOS and nNOS in rat tissues.

Inhibitory neurotransmission in lethal spotted mutant mice: a model for Hirschsprung's disease.

BACKGROUND & AIMS: The pathogenesis of Hirschsprung's disease is not well understood. The suitability of the animal model for the unknown pathogenesis of inhibitory neurotransmission in Hirschsprung's disease was investigated. METHODS: Circular smooth muscle strips from the internal anal sphincter (IAS) and distal colon (2, 6, 8, 16, and 24 mm from the anal verge) from normal and Ls/Ls mice (mice homozygous for the lethal spotting mutation that develop fetal megacolon after aganglionosis of the terminal colon) were prepared to record changes in isometric tensions in response to different agents and nonadrenergic, noncholinergic nerve stimulation by electrical field stimulation. RESULTS: Bethanechol was used to produce contraction of the smooth muscle strips of distal colon to record a decrease in the tension. Conversely, the IAS smooth muscle strips developed spontaneous tone. In the normal homozygous mice, electrical field stimulation caused a biphasic response, an initial decrease followed by an after-contraction, whereas in Ls/Ls mice, the predominant response was contraction. All smooth muscle strips from normal and Ls/Ls mice produced relaxation in response to sodium nitroprusside and vasoactive intestinal polypeptide. CONCLUSIONS: Ls/Ls mice may serve as an appropriate animal model to investigate the pathogenesis of the inhibitory neurotransmission in Hirschsprung's disease in the distal colon and IAS.

L-citrulline recycling in opossum internal anal sphincter relaxation by nonadrenergic, noncholinergic nerve stimulation.

BACKGROUND & AIMS: L-citrulline formed stoichiometrically along with nitric oxide (1:1) from L-arginine may be enzymatically converted to L-arginine. The possibility of L-citrulline recycling in the maintenance of nitrergic neurotransmission in the opossum internal anal sphincter (IAS) smooth muscle strips was investigated. METHODS: Responses to nonadrenergic, noncholinergic (NANC) nerve stimulation by electrical field stimulation (EFS) (either short-train or continuous stimulation) on the basal IAS tension were recorded before and after the NO synthase inhibitor N(omega)-nitro-L-arginine (L-NNA), L-NNA plus L-citrulline, or L-arginine. During continuous EFS, when the basal IAS tone after the initial relaxation had recovered to almost pre-EFS levels, the effects of L-citrulline or L-arginine were examined before and after L-glutamine, which is a putative blocker of L-citrulline uptake. RESULTS: Inhibition of NANC nerve-mediated IAS relaxation by L-NNA was reversed by L-citrulline as well as L-arginine. L-Citrulline and L-arginine caused concentration-dependent relaxation of the IAS tone recovered during the prolonged EFS. L-Glutamine blocked the responses of L-citrulline but not of L-arginine. Furthermore, L-glutamine increased the speed of recovery of IAS tone during continuous EFS. CONCLUSIONS: L-citrulline recycling may be responsible for the maintenance of IAS relaxation during frequent short-train and prolonged NANC nerve stimulation.

Neuronal NOS gene expression in gastrointestinal myenteric neurons and smooth muscle cells.

Nitric oxide synthase (NOS) has been characterized in different tissues, and its localization has been suggested in different neuronal tissues, including the myenteric neurons and other nonneuronal cells. The present study examined the distribution of the neuronal NOS (nNOS) mRNA in different tissues of the opossum gastrointestinal tract, internal anal sphincter (IAS) smooth muscle cells, and myenteric neurons using slot-blot and Northern blot hybridization techniques with a specific rat brain nNOS cDNA probe. Significant levels of nNOS gene expression were found in both smooth muscle cells and myenteric neurons of the opossum IAS. This finding was confirmed by reverse transcriptase-polymerase chain reaction analysis of the RNA obtained from cultured opossum IAS smooth muscle cells and myenteric neurons and also from human intestinal smooth muscle and neuroblastoma cell lines. Pyloric sphincter had the highest level of nNOS gene expression compared with other gastrointestinal tissues. There was no significant difference in the nNOS gene expression between other sphincteric and nonsphincteric tissues examined. The present study shows the presence of nNOS gene expression in both neurons and smooth muscle cells. The higher levels of nNOS gene expression in the pyloric sphincter compared with other tissues may have pathophysiological significance in some disease conditions.

Heme oxygenase inhibitor zinc protoporphyrin IX causes an activation of nitric oxide synthase in the rabbit internal anal sphincter.

The studies were performed in the rabbit internal anal sphincter (IAS) smooth muscle strips to examine the influence of the heme oxygenase inhibitor, [zinc protporphyrin (ZnPP IX)], on basal tone. ZnPP IX produced a concentration-dependent fall in the basal tone and was the focus of our investigation. To examine the mechanism of the fall in IAS tone by ZnPP IX, the effect of different concentrations of ZnPP IX on basal IAS tone and the release of nitric oxide were examined before and after the neurotoxin tetrodotoxin and various nitric oxide synthase (NOS) inhibitors. The inhibitory effect of ZnPP IX was blocked by the NOS inhibitors L-NG-nitroarginine, NG-monomethyl-L- arginine and L-N5-(1-iminoethyl)ornithine and the neurotoxin TTX. The fall in IAS tension by ZnPP IX was accompanied by a release of NO. ZnPP IX(1 x 10(-3)M) caused a fall in IAS tension of 43.7% which was reduced to 16.5% in the presence of L-NG-nitroarginine (1 x 10(-4)M). Furthermore, the fall in IAS tone in the presence of ZnPP IX was restored both by the NOS inhibitors and tetrodotoxin. The basal release of nitric oxide in these experiments was 0.50 +/- 0.07 nmol and in the presence of ZnPP IX (1 x 10(-3)M), it increased to 1.72 +/- 0.28 nmol (more than a 3-fold increase). Thus the fall in the basal IAS tone by ZnPP IX was associated with a release of NO from the myenteric neurons as these effects were significantly blocked by the NOS inhibitors and tetrodotoxin. We conclude that in the rabbit IAS, ZnPP IX causes a fall in the basal IAS tension by the activation of NOS in myenteric neurons. We speculate that in the resting state, the heme oxygenase pathway exerts important counter-regulatory effects on the NOS pathway and when blocked (e.g., by ZnPP IX), the underlying NOS pathway is unmasked leading to a massive and prolonged release of NO. The exact significance of this mechanism remains to be determined.