Sushanta Chakder

Nitric oxide pathway in rectoanal inhibitory reflex of opossum internal anal sphincter

The role of nitric oxide in relaxation of the internal anal sphincter (IAS) in response to the rectoanal reflex was studied in the opossum. Resting pressures in the IAS (IASP) were monitored using low-compliance continuously perfused catheters. The NO-synthase inhibitor L-NG-nitro-arginine (L-NNA) caused significant and dose-dependent suppression of the decrease in IASP in response to the reflex mimicked by the rectal balloon distention. NO-synthase inhibitor blocked IAS relaxation in response not only to rectoanal reflex but also to other neural stimuli such as sacral nerve stimulation, local intramural stimulation, and the nicotinic ganglionic stimulant 1,1-dimethyl-4-phenylpiperazinium. Suppression of the neurally mediated IAS relaxation by L-NNA was stereoselective; D-NNA had no effect on the relaxation. The suppression of the rectoanal reflex-induced IAS relaxation by L-NNA was completely reversed by NO precursor L-arginine stereoselectively as D-arginine failed to reverse the suppressed IAS relaxation. Sodium nitroprusside caused a decrease in IASP that was modified neither by the neurotoxin tetrodotoxin nor by L-NNA. Furthermore, the decrease in IASP by the direct-acting beta-adrenoceptor agonist isoproterenol was also not modified by the inhibitor of NO synthase. It is concluded that NO or an NO-like substance is an important mediator of IAS relaxation in response to noradrenergic, noncholinergic 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.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.

Colocalization of NADPH-diaphorase staining and VIP immunoreactivity in neurons in opossum internal anal sphincter.

Nitric oxide and vasoactive intestinal polypeptide (VIP) are important inhibitory neurotransmitters mediating relaxation of the internal anal sphincter. The location and coexistence of these two neurotransmitters in the internal anal sphincter has not been examined. We performed a double-labeling study to examine the coexistence of nitric oxide synthase and VIP in the oppossum internal anal sphincter using the NADPH-diaphorase technique which is a histochemical stain for nitric oxide synthase. In perfusion-fixed, frozen-sectioned tissue, VIP-immunoreactive neurons were labeled using immunofluorescence histochemistry. After photographing the VIP-immunoreactive neurons, nitric oxide synthase was labeled using the NADPH-diaphorase technique. Ganglia containing neuronal cell bodies were present in the myenteric plexus for the entire extent of the internal anal sphincter. VIP-immunoreactive and NADPH-diaphorase-positive neurons were present in ganglia in the myenteric as well as the submucosal plexuses. Most of the VIP-immunoreactive neurons were also NADPH-diaphorase positive. VIP and nitric oxide synthase are present and frequently coexist in neurons in the internal anal sphincter of the opossum. These neurons may be an important source of inhibitory innervation mediating the rectoanal reflex-induced relaxation of the sphincter. The demonstration of the coexistence of these two neurotransmitters will be of fundamental importance in unraveling their relationship and interaction in the internal anal sphincter as well as other systems.

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.

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

BACKGROUND & AIMS The pathogenesis of Hirschsprungs disease is not well understood. The suitability of the animal model for the unknown pathogenesis of inhibitory neurotransmission in Hirschsprungs 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 Hirschsprungs disease in the distal colon and IAS.

Effects of galanin on the opossum internal anal sphincter: Structure-activity relationship

The present study was carried out to investigate the effects of porcine galanin-(1-29), N-terminal fragment galanin-(1-10), C-terminal fragment galanin-(15-29), and the middle fragment galanin-(7-16) on the spontaneous tension of the opossum internal anal sphincter and on the decrease in the resting internal anal sphincter tension in response to neural stimulation by electrical field stimulation. Galanin and galanin-(1-10) caused a concentration-dependent decrease in the resting tension of internal anal sphincter and an augmentation of the percent decrease in the resting tension with electrical field stimulation. Galanin-(15-29), on the other hand, produced an increase in the resting tension of the internal anal sphincter and had no effect on the electrical field stimulation-induced decrease in the resting tension. Galanin-(7-16) produced no significant effect on the internal anal sphincter. The decrease in the internal anal sphincter tension by galanin and galanin-(1-10) was partially antagonized by tetrodotoxin, whereas the increase in the internal anal sphincter tension caused by galanin-(15-29) was not modified by tetrodotoxin. In contrast to its effect in the internal anal sphincter, galanin caused an increase in the resting tension and suppressed a decrease in the lower esophageal sphincter tension in response to electrical field stimulation. From these findings we conclude that (a) galanin exerts an inhibitory effect on the internal anal sphincter by activating galanin receptors both at the intramural inhibitory neurons and at the internal anal sphincter smooth muscle and that the N-terminal portion of galanin may be responsible for these actions; (b) the contractile action of galanin is produced by its action on the smooth muscle; and (c) the actions of galanin on the gastrointestinal tract are tissue specific.

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.Despite its widespread distribution and significance in the gut, the role of pituitary adenylate cyclase-activating peptide (PACAP) in internal anal sphincter (IAS) relaxation has not been examined. This study examined the role of PACAP in nonadrenergic noncholinergic (NANC) nerve-mediated relaxation of IAS smooth muscle. Circular smooth muscle strips from the opossum IAS were prepared for measurement of isometric tension. The influence of PACAP and vasoactive intestinal peptide (VIP) antagonists and tachyphylaxis on the neurally mediated IAS relaxation was examined either separately or in combination. The release of these neuropeptides in response to NANC nerve stimulation before and after the nitric oxide (NO) synthase inhibitor N ω-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.

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.

Sites of Actions of Contractile and Relaxant Effects of Pituitary Adenylate Cyclase Activating Peptide (PACAP) in the Internal Anal Sphincter Smooth Musclea

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.

Vasoactve intestinal polypeptide gene expression is characteristically higher in opossum gastrointestinal sphincters

BACKGROUND/AIMS Vasoactive intestinal polypeptide (VIP) has been suggested to be an inhibitory neurotransmitter in the sphincteric and nonsphincteric smooth muscles of the gut. However, the relative gene expression of VIP in these functionally diverse regions is not known. METHODS The gastrointestinal smooth muscle sphincters of opossums were excised from the adjoining nonsphincteric smooth muscles. RNAs were isolated and subjected to blot hybridizations with VIP complementary DNA probe. Relative expression of VIP gene was quantitated using the densitometric scanning of the VIP messenger RNA (mRNA) transcripts. The cellular specificity of VIP gene expression was investigated in cultures of neuroblastoma cells and myenteric plexuses and compared with those of the smooth muscle cells. RESULTS The data showed higher levels of VIP mRNA in the sphincteric than the adjoining nonsphincteric tissues. VIP mRNA were found in significantly higher amounts in the myenteric neurons and neuroblastoma cells than in the smooth muscle cells. CONCLUSIONS VIP gene expression was significantly higher in the sphincteric smooth muscle regions than in the nonsphincteric regions of the gut. The studies provide further evidence for the role of VIP in neurotransmission of the gut.