Role of Nitric Oxide in Anorectal Function of Normal and Neuronal Nitric Oxide Synthase Knockout Mice

PURPOSE: In vitro data suggest that nitric oxide is an important inhibitory neurotransmitter in the internal anal sphincter, and morphologic evidence implies that it mediates the rectoanal inhibitory reflex. This study examined the anatomy, physiology, and pharmacology of the internal sphincter in control and neuronal nitric oxide synthase knockout mice. METHODS: Neuronal nitric oxide synthase, nicotinamide adenosine triphosphate dinucleotide phosphate diaphorase histochemistry, and PGP 9.5 immunohistochemistry were compared between knockout and sibling control mice. Anorectal manometry was performed with a balloon-tipped water-perfused catheter. In vitro studies were performed on both whole internal anal sphincter rings and strips. RESULTS: Staining of the myenteric plexus and nerves traversing the internal anal sphincter in sibling control mice demonstrated the presence of neuronal nitric oxide synthase and nicotinamide adenine dinucleotide phosphate diaphorase at these sites. These markers were absent in knockout mice. Maximum anal resting pressure was similar in control and knockout mice (15.6 +/- 2.6 cm H(2)O (n = 4) vs. 14.0 +/- 2.3 cm H(2)O (n = 7)). The rectoanal inhibitory reflex was present in all control mice (n = 4) but in only four of seven knockout mice. Field stimulation with parameters designed to activate inhibitory nerves produced relaxation of internal sphincter tissue from both control and knockout mice, which was partially attenuated in control mice only by pretreatment with the nitric oxide synthase inhibitor N omega-nitro-L-arginine. Further inhibition of nerve-induced relaxation in control mice was achieved with antagonists of vasoactive intestinal peptide, adenosine triphosphate, and heme oxygenase. CONCLUSIONS: Although in the normal mouse, nitric oxide is an inhibitory neurotransmitter in the internal sphincter, other transmitters may play a role in the rectoanal inhibitory reflex. These other inhibitory neurotransmitters can apparently compensate for the absence of nitric oxide synthase in knockout mice to maintain approximately normal function.     

Distribution of nitric oxide synthase containing neurons in the rectal myenteric plexus and anal canal

PURPOSE: Following the demonstration that a novel neurotransmitter, nitric oxide (NO), is released during neurogenic relaxation of the internal anal sphincter in vitro,it has been suggested that NO could mediate the rectoanal inhibitory reflex in vivo.The aim of this study was to establish whether the distribution of NO-producing nerves in the anorectum is consistent with this proposed role. METHODS: NO is synthesized in neurons which contain the enzyme nitric oxide synthase and their presence in the anorectum was determined in tissue obtained from nine abdominoperineal and three anterior resection specimens in patients undergoing surgery for rectal carcinoma. Cryostat sections were stained for nitric oxide synthase immunoreactivity, pan-neuronal/axonal immunoreactivity, and NADPH diaphorase activity. RESULTS: Nitric oxide synthase immunoreactivity is present in a subpopulation of neurons in rectal myenteric ganglia which also contain NADPH diaphorase activity. Use of the latter histochemical technique enabled the structure and distribution of nitric oxide synthase containing neurons to be determined in whole-mount preparations. Individual neurons have Dogiel type 1 morphology and are present throughout the rectal myenteric plexus. In the distal rectum, positively stained axons enter shunt fascicles which descend into the anal canal, where they ramify into and throughout the internal anal sphincter. Within the sphincter, positively stained nerves lie in close proximity to smooth muscle cells. CONCLUSION: These results are consistent with the hypothesis that NO is the neurotransmitter that mediates the rectoanal inhibitory reflex.     

The Role of Nitric Oxide in the Acetylcholine-Induced Relaxation of the Feline Internal Anal Sphincter,in Vitro

Background: The relaxatory effect of acetylcholine was investigated on the feline internal anal sphincter (IAS), in vitro. Results: Acetylcholine (10, 30, 100, and 1000 μM) caused a concentration-dependent relaxation of the same magnitude in strips from the proximal and distal IAS. The antagonist of nitric oxide synthase, N^$oM-nitro-L-arginine (L-NNA; 1, 10, and 100 μM), in a concentration-dependent and stereospecific manner, blocked the acetylcholine-induced relaxation, leaving a residual response of 10–30%. The blocking effect of L-NNA (100 μM) could not be shown in tissues that had been incubated with the substrate for nitric oxide synthase, L-arginine (1 mM). Conclusions: The present results suggest that the acetylcholine-induced relaxation of the IAS to a major extent is due to an activation of nitrergic, inhibitory motor neurons to the IAS.     

Effect of nitroblue tetrazolium on NO synthase and motor function of opossum esophagus

Nitric oxide mediates neuromuscular events in the opossum esophagus. The NADPH diaphorase stain is used to localize nitric oxide synthase-containing enteric neurons. Cells stain by the NADPH diaphorase technique because they reduce nitroblue tetrazolium to the visible formazan. The effects of nitroblue tetrazolium on neuromuscular function and nitric oxide synthase of esophageal muscle were studied. The NADPH diaphorase stain was performed. Nitroblue tetrazolium inhibited lower esophageal sphincter relaxation, abolished the latency gradient of the off response, and inhibited nitric oxide synthase. The NADPH diaphorase technique stained myenteric plexus nerve cell bodies and nerve processes. Nitroblue tetrazolium is not a nonspecific muscle or nerve toxin, as nerve-mediated cholinergic responses, responses to exogenous nitric oxide, and responses to myogenic stimulation were maintained after nitroblue tetrazolium abolished the off response and lower esophageal sphincter relaxation. Nitroblue tetrazolium inhibits nitric oxide-mediated events and nitric oxide synthase. It stains neurons in the esophageal myenteric plexus.This work was supported by a Merit Grant and a Research Career Development Award from the Department of Veterans Affairs, and NIH grant DK 11242.     

Lipopolysaccharide Induces Cell Death in Cultured Porcine Myenteric Neurons

Enteric bacteria execute, via lipopolysaccharide (LPS), a pathogenic role in intestinal inflammation. The effects of LPS on survival and neurotransmitter expression in cultured porcine myenteric neurons were investigated. Myenteric neurons were isolated and cultured for 6 days in medium, in LPS (100 ng/ml) with or without α-ketoglutarate or the nitric oxide synthase (NOS) inhibitor L-NAME, in α-ketoglutarate or in the NO donor SNAP. Neuronal survival and expression of vasoactive intestinal peptide (VIP) and NOS were evaluated by immunocytochemistry. Addition of LPS significantly decreased neuronal survival; only 40% survived, compared to controls run in parallel. The LPS-induced neurotoxic effect was not counteracted by the simultaneous presence of α-ketoglutarate or L-NAME. Either SNAP or α-ketoglutarate influenced neuronal survival. Culturing, particularly in the presence of LPS, markedly increased the proportion of VIP-immunoreactive neurons; NOS-immunoreactive neurons were unchanged. The reported LPS-induced neurotoxicity indicates loss of enteric neurons as a consequence of intestinal inflammation.     

Nitric oxide not carbon monoxide mediates nonadrenergic noncholinergic relaxation in the murine internal anal sphincter

BACKGROUND & AIMS: Inhibitory reflexes in the internal anal sphincter (IAS) are controlled by inhibitory nonadrenergic, noncholinergic innervation (i-NANC). We investigated the roles of 3 different neurohumoral agonists as possible i-NANC neurotransmitters: carbon monoxide (CO), nitric oxide (NO), and vasoactive intestinal peptide (VIP). METHODS: IAS smooth muscle strips were isolated from wild-type (WT), heme oxygenase (HO)-2 knockout (HO-2-/-) and neuronal NO synthase (nNOS) knockout (nNOS-/-) mice. Relaxation of IAS was induced by CO, NO, VIP, and electrical field stimulation (EFS) in the presence and absence of neurohumoral inhibitors (tin protoporphyrin IX [SnPP IX] for CO synthesis, N(omega)-nitro-L-arginine [L-NNA] for NO synthesis, and VIP(10-28) for VIP receptor). Western blot and immunohistochemistry were used to test the presence and localization of HO (for CO synthesis) types 1 (HO-1) and 2 (HO-2), neuronal NO synthase (nNOS, for NO synthesis), and VIP. RESULTS: All 3 neurohumoral agonists produced relaxation (with no difference between WT and HO-2-/- IAS), but CO was over 100 times less potent than NO and VIP. EFS produced relaxation in WT and HO-2-/- IAS with the same intensity. L-NNA and nNOS deletion (approximately 80%) and VIP(10-28) (approximately 15%) significantly inhibited the relaxations, whereas SnPP IX had no effect. Positive immunoreactivities for HO-2, nNOS, and VIP were found in the myenteric plexus of WT IAS. HO-2-/- IAS did not express immunoreactivity for HO-2. CONCLUSIONS: i-NANC relaxations of mouse IAS are primarily mediated via NO (by nNOS activity) and partly via VIP. CO directly relaxes the mouse IAS but does not play any significant role in the i-NANC relaxation.     

Nitric oxide deficiency in the internal anal sphincter of patients with chronic anal fissure

Anal fissure is a common condition affecting young to middle-aged adults. It causes severe pain on defecation and rectal bleeding. The aetiology remains uncertain. Spasm of the internal anal sphincter is a constant feature. Nitric oxide (NO) is the major inhibitory neurotransmitter of the internal anal sphincter (IAS). In other spasmodic conditions of the GI tract a lack of normal nitric oxide synthase (NOS) activity has been reported. The aim of this preliminary study was to compare the presence of NOS in the internal sphincters of patients with and without chronic anal fissure. Internal anal sphincter biopsies were taken under general anaesthesia from patients having lateral internal sphincterotomy for chronic anal fissure and from sphincter of patients having abdominoperineal resections as controls. Sections of IAS were stained to show the presence of NADPH diaphorase (and hence presence of NOS). Internal anal sphincter was taken from 6 patients with chronic anal fissure and 6 controls. IAS taken from patients with chronic anal fissure showed little NOS presence compared with controls. It may be that there is an abnormal failure of relaxation of internal sphincter in those patients who develop chronic anal fissure caused by an intrinsic lack of neural NOS in the internal anal sphincter.     

Oddi括约肌功能障碍患者乳头局部组织中血管活性肠肽和一氧化氮的改变及其意义

目的　探讨血管活性肠肽 (VIP)和一氧化氮 (NOS)在Oddi括约肌功能障碍发病机制中的作用。方法　选择 68例因不明原因的上腹痛、黄疸而进行逆行胰胆管造影及乳头括约肌切开术的患者 ,采用内镜下活检 ,钳取乳头黏膜、Oddi括约肌及胆管内壁组织各 1块。标本常规石蜡包埋 ,连续切片 ,免疫组化链酶亲和素 过氧化物酶法 (SABC法 )观察乳头组织内VIP及NOS的变化。将 68例患者分为Oddi括约肌功能障碍组 (SOD组 )与非Oddi括约肌功能障碍组 (非SOD组 )。结果　SOD患者乳头黏膜及Oddi括约肌内VIP和NOS含量明显减少 (P <0 .0 5 ) ,VIP和NOS含量呈正相关关系 (r =0 .87,P <0 .0 1)。结论　乳头局部组织中VIP和NOS的表达明显降低。VIP和NOS的减少可能在SOD的发生机制中具有一定的作用     

Nerve mediated relaxation of the human internal anal sphincter: the role of nitric oxide.

The aim of this study was to determine if nitric oxide (NO) is the non-adrenergic, non-cholinergic neurotransmitter, released by enteric inhibitory nerves, which mediates relaxation of the human internal anal sphincter. Isolated muscle strips were mounted for isometric tension recording in superfusion organ baths. Sodium nitroprusside, an exogenous donor of NO, relaxed the strips in a concentration dependent manner. In the presence of atropine and guanethidine, transmural field stimulation produced tetrodotoxin sensitive relaxations, which were inhibited in a dose dependent and enantiomer specific manner by antagonists of NO synthase; completely by L-nitroarginine and partially by L-N-monomethyl arginine. The effect of these antagonists was reversed by L-arginine but not D-arginine. Oxyhaemoglobin, a scavenger of nitric oxide, also abolished the relaxations but methaemoglobin had no such effect. These results strongly suggest that NO is, or is very closely associated with, the non-adrenergic, non-cholinergic neurotransmitter mediating neurogenic relaxation of the human internal anal sphincter.     

Nitrinergic and peptidergic innervation of the human oesophagus.

The distribution, colocalisation, and interconnections of nitrinergic and peptidergic neurons and nerves in the human oesophagus were examined. Cryosections of surgically resected tissues from eight subjects were studied with indirect immunofluorescence for the presence of 11 neuropeptides and neuron specific enolase. After immunohistochemistry, nitric oxide synthase was shown on the same sections with the beta nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemical reaction. The histochemical findings were verified immunohistochemically on other sections with an antiserum against nitric oxide synthase. Most myenteric neurons (55%) were nitrinergic. Most (96%) received terminations positive for vasoactive intestinal polypeptide (VIP), calcitonin gene related peptide (CGRP) (80%), and galanin (59%). The neuronal somata of 14% also contained VIP, while 10% had galanin. Of the NADPH-diaphorase containing fibers seen in the muscle layers, many had closely associated VIP and galanin, but only rarely CGRP and substance P. Thus, despite abundant representation of both peptidergic and nitrinergic systems in oesophageal smooth muscle, only VIP and galanin colocalised to any significant extent with the nitrinergic elements. These findings provide morphological support for the role of nitric oxide as the non-adrenergic non-cholinergic inhibitory mediator in the human oesophagus and for its possible interactive role with the peptidergic system.     

Vasoactive intestinal peptide: a neurotransmitter for relaxation of the rabbit internal anal sphincter

The circular smooth muscle of the rabbit internal anal sphincter, tested in vitro, exhibited spontaneous resting tonus and relaxed with neural stimuli of 0.05-0.5-ms duration and 1-10-Hz frequency. The relaxation was abolished by tetrodotoxin (10(-6) M) but not by atropine or propranolol at the same molar concentration, suggesting that the inhibitory neural fibers mediating relaxation are nonadrenergic and noncholinergic. One-hour incubation in physiologic solution containing 8% of rabbit vasoactive intestinal peptide (VIP) antiserum significantly reduced the relaxation induced by electrical stimulation. The reduction was greater for the shorter duration stimuli, ranging from 80% to 50% for 0.05-ms stimuli to 35%-11% for 0.5-ms stimuli. Control strips were relaxed in a dose-dependent fashion by 10(-9)-10(-6) M VIP; the antiserum at the concentration used completely blocked the relaxation produced by 10(-7) M VIP and reduced the relaxation produced by 10(-6) M VIP. Adenosine triphosphate also relaxed the internal anal sphincter in a dose-dependent manner. Prolonged inhibitory nerve stimulation (0.5 ms, 10 Hz, 30-min train) caused significant reduction in the relaxation induced by exogenous VIP, whereas the relaxation induced by adenosine triphosphate was unaffected. These data are consistent with VIP being an inhibitory neurotransmitter responsible for relaxation of the internal anal sphincter.     

Effect of chagas' disease on nitric oxide-containing neurons in severely affected and unaffected intestine

PURPOSE: The pathophysiology of Chagas' disease is incompletely understood. Neuronal nitric oxide has been cited as a candidate neurotransmitter responsible for relaxation of the internal anal sphincter. Neuronal nicotinamide adenine dinucleotide phosphate diaphorase can be used as a marker for neuronal nitric oxide synthase. This study was designed to examine the alterations of the nitric oxidecontaining neurons in the enteric nervous system of the colon of patients who underwent resections for advanced megacolon and to compare these specimens with small-bowel specimens from the same patients and with specimens from control subjects. METHODS: Specimens from resected rectum and extramucosal small-bowel biopsy specimens from 11 patients with Chagas megacolon but no apparent small-bowel clinical involvement were compared with the uninvolved colon and jejunum of 10 control patients with colon cancer. Tissues were fixed in Zamboni solution and evaluated by histochemistry for nicotinamide adenine dinucleotide phosphate diaphorase-containing neurons. Reactivity was evaluated on a 0 to 4 scale in the longitudinal muscle, myenteric plexus, circular muscle, submucosal plexus, and mucosa. RESULTS: Specimens from control patients showed well-stained myenteric and submucosal neurons and an abundant network of terminal nerve fibers in the muscle layers. Chagasic specimens had decreased staining in all layers of the gut. Overall there was a statistically significant decrease in nicotinamide adenine dinucleotide phosphate diaphorase-containing neurons. Biopsy specimens from clinically uninvolved small bowel of patients with Chagas' disease also showed decreased reactivity, but to a lesser degree. CONCLUSIONS: Nicotinamide adenine dinucleotide phosphate diaphorase activity is decreased in patients with advanced megacolon. The alterations are more relevant in the myenteric plexus and the circular muscle. Reactivity is also diminished in the clinically uninvolved small bowel, but to a lesser extent.     

Carbon monoxide mediates vasoactive intestinal polypeptide-associated nonadrenergic/noncholinergic neurotransmission

Carbon monoxide (CO) synthesized by heme oxygenase 2 (HO2) and nitric oxide (NO) produced by neuronal NO synthase (nNOS) mediate nonadrenergic/noncholinergic (NANC) intestinal relaxation. In many areas of the gastrointestinal tract, NO and CO function as coneurotransmitters. In the internal anal sphincter (IAS), NANC relaxation is mediated primarily by CO. Vasoactive intestinal polypeptide (VIP) has also been shown to participate in NANC relaxation throughout the intestine, including the IAS. By using a combination of pharmacology and genetic knockout of the biosynthetic enzymes for CO and NO, we show that the physiologic effects of exogenous and endogenous VIP in the IAS are mediated by HO2-synthesized CO.     

Distribution of nitric oxide synthase in stomach myenteric plexus of rats

AIM:To study the distribution of nitdc oxide synthese(NOS)in rat stomach myenteric plexus.METHODS:The distribution of NOS in gastric wall wasstudied in quantity and location by the NADPH-diaphorase(NDP)histochemical staining method and whole mountpreperation technique.RESULTS:NOS was distributed in whole stomach wall,mostof them were located In myenteric plexus,and distributed insubmucosal plexus.The shape of NOS positive neuronswas baslcally similar,most of them being round and oval inshape.But their density,size and staining intensity variedgreatly in the different parts of stomach.The density was 62±38 cells/mm~2(antrum),43±32 cells/mm~2(body),and 32±28 cells/mm~2(fundus),respectively.The size andstaining intensity of NOS positive neurons in the funduswere basically the same,the neurons being large and darkstained,while they were obviously different in antrum.Inthe body of the stomach,the NOS positive neurons were inan Intermediate state from fundus to antrum.There weresome beedlike structures which were strung together byNOS positiva varicosities in nerve fibers,some were closelyadherent to the outer walls of blood vessels.CONCLUSION:Nitric oxide might he involved in themodulation of motility,secretion and blood circulation ofthe stomach,and the significant difference of NOS positiveneurons in different parts of stomach myenteric plexus maybe related to the physiologic function of stomach.     

Nitric oxide synthase and VIP distribution in enteric nervous system in idiopathic chronic constipation

Idiopathic chronic constipation has been correlated to neural abnormalities that consist of a reduced number of myenteric plexus neurons and a decreased concentration of VIP-positive nerve fibers within the circular muscle. Recent studies hypothesized the involvement of nitric oxide in motility disorders of the human gut. To date, no information is available on nitric oxide involvement in idiopathic chronic constipation. The density of VIP- and nitric oxide-producing neurons was evaluated by immunocytochemistry using anti-VIP and anti-nitric oxide synthase antibodies in five patients with idiopathic chronic constipation. A low total neuron density was found at the myenteric plexus. The density of VIP-positive neurons was low while that of nitric oxide synthase-positive neurons was high at both plexuses. Our data confirm that idiopathic slow-transit chronic constipation is due to abnormal neurogenic factors. The presence of numerous nitric oxide synthase-positive neurons, all along the colon and at both plexuses, supports the hypothesis that an excessive production of nitric oxide may cause the persistent inhibition of contractions.Supported by MURST University Funds.     

Ng-nitro-L-arginine reduces nonadrenergic, noncholinergic relaxations of human gut.

The aim of this study was to determine whether nonadrenergic, noncholinergic inhibitory neurotransmission in human circular sigmoid colonic and internal anal sphincter muscle involves release of a nitric oxide-like substance. Colonic and sphincter muscle respond to electrical field stimulation by giving nonadrenergic, noncholinergic relaxations. After-contractions always occur in colonic muscle but only sometimes in sphincter muscle. Ng-Nitro-L-arginine abolished relaxations of sphincter muscle and partially reduced those of colonic muscle. After-contractions were undiminished as were relaxations of sphincter muscle to sodium nitroprusside. The effects of Ng-nitro-L-arginine were reversed by L-arginine. The results suggest that nitric oxide is possibly the neurotransmitter mediating nonadrenergic, noncholinergic relaxations of the human internal anal sphincter muscle.     

Involvement of the L-arginine-nitric oxide pathway in internal anal sphincter relaxation.

The purpose of this study was to examine the role of the L-arginine-nitric oxide pathway in neurogenic relaxation of the internal anal sphincter. Muscle strips representing the internal anal sphincter were prepared from 17 adult opossums. The preparations were mounted in organ baths for recording of isometric tension. N omega-nitro-L-arginine, an agent known to inhibit the L-arginine-nitric oxide pathway, concentration-dependently reduced relaxations induced by transmural field stimulation. At the highest concentration of N omega-nitro-L-arginine (10(-4) mol/L), no relaxation was evoked at any frequency tested (0.5-40 Hz). The inhibitory response to exogenous vasoactive intestinal polypeptide was unaffected by N omega-nitro-L-arginine pretreatment, indicating that vasoactive intestinal polypeptide relaxation does not use the L-arginine-nitric oxide pathway. In addition, responses to forskolin and sodium nitroprusside were not influenced by N omega-nitro-L-arginine preincubation, suggesting that the effect observed was not caused by a direct influence on the adenylate or the guanylate cyclases. It is concluded that the nonadrenergic, noncholinergic innervation of the internal anal sphincter involves an inhibitory substance generated from the L-arginine-nitric oxide pathway. Whether this substance is nitric oxide or a related nitroso compound remains to be settled.     

Lack of nitrate tolerance in isosorbid dinitrate- and sodium nitroprusside-induced relaxation of rabbit internal anal sphincter

AIM： To investigate the tolerance development against the relaxant effect of nitric oxide donating drug isosorbide dinitrate （ISDN） and sodium nitropruside （SNP） in internal anal sphincter （IAS） smooth muscle. METHODS： Relaxation responses of ISDN, and electrical fi eld stimulation （EFS） were obtained before and after tolerance induction by ISDN incubation. RESULTS： ISDN （10-7-10-4 mol/L） and SNP （10-8-10-4 mol/L） caused a concentration-dependent relaxation on the basal tonus of the isolated rabbit IAS strips. After a period of 2 h incubation of the 6 x 10-4 mol/L ISDN the relaxation effects of ISDN and SNP did not change compared to control strips. EFS evoked frequency-dependent relaxation in internal anal sphincter smooth muscle and Emax obtained from control strips were not changed in ISDN tolerance-inducing condition. In this study nitrate tolerance was not observed in rabbit IAS smooth muscle. CONCLUSION： This result shows that nitric oxide donating drugs relaxes the internal anal sphincter of the rabbits without the development of tolerance.     

Local somatothermal stimulation inhibits motility of the internal anal sphincter through nitrergic neural release of nitric oxide

PURPOSE: A somatoanal reflex had been demonstrated in our previous work. Because nitric oxide plays an important role in mediating relaxation of the internal anal sphincter, our purpose was to examine whether and how local somatothermal stimulation inhibits the function of the internal anal sphincter by stimulating nitric oxide releasevia nitrergic neurons and to elucidate the possible mechanism. METHODS: The activity of the internal anal sphincter in anesthetized rabbits was measured by use of continuously perfused, open-tip manometric methods. Local somatothermal stimulation was achieved by applying an electroheating rod 1 cm away from the skin area at the right popliteal region. The responses were further manipulated by pretreating the rabbits with agonists or antagonists linked to nitric oxide synthesis. RESULTS: The motility of the internal anal sphincter before and during local somatothermal stimulation was significantly different (tonic pressure (mean ± standard error of the mean), 5.4 ± 0.3vs. 4.9 ± 0.3 mmHg,P=0.0195; phasic pressure, 3.9 ± 0.6vs. 2.9 ± 0.4 mmHg,P=0.0002; frequency distribution of the phasic contractions (peak-to-peak interval), 28.9 ± 3.7vs. 65.3 ± 10.4 seconds,P=0.0001). The response began at approximately one minute after local somatothermal stimulation when the skin temperature was 41 ± 0.3°C. No anal response was observed when local somatothermal stimulation was applied at the control area. The local somatothermal stimulation-induced internal anal sphincter relaxation was not inhibited by pretreatment with atropine, propranolol, or phentolamine (tonic pressure, 5.8 ± 1vs. 5.2 ± 0.8 mmHg,P=0.038; phasic pressure, 4.2 ± 0.9vs. 3.1 ± 0.6 mmHg,P=0.020; peak-to-peak interval, 27.2 ± 4.3vs. 52.9 ± 14.5 seconds,P=0.043) but was completely blocked by pretreatment with a nitric oxide synthesis inhibitor. The effect of the nitric oxide synthesis inhibitor could be reversed by pretreatment with L-arginine (tonic pressure, 6 ± 0.7vs. 5.6 ± 0.7 mmHg,P=0.047; phasic pressure, 4.7 ± 0.7vs. 3.9 ± 0.5 mmHg,P=0.048; peak-to-peak interval, 23.8 ± 3vs. 33 ± 3.7 seconds,P=0.048), but not by D-arginine. CONCUSION: Local somatothermal stimulation inhibits internal anal sphincter motility through the activation of nonadrenergic noncholinergic neural release of nitric oxide. This procedure may represent a simplified approach for the treatment of anorectal diseases with hypofunction of the L-arginine/nitric oxide pathway.Presented at the XVIIth Biennial Congress of the International Society of University Colon and Rectal Surgeons, Malmö, Sweden, June 7 to 11, 1998.     

Localization of NADPH-diaphorase in the rat pineal gland: An experimental enzyme histochemical study

Abstract: We have used the NADPH-diaphorase enzyme histochemical technique to localize the enzyme nitric oxide synthase in the rat pineal gland. Some scattered NADPH-diaphorase positive pineal cells were present, mostly in the rostral part of the gland close to the pineal stalk. In addition, NADPH-diaphorase positive nerve fibers were located in the pineal capsule, in the connective tissue septae of the gland, and also intraparenchymally between the pinealocytes. Most nerve fibers were endowed with boutons en passage. These nerve fibers remained in the gland after bilateral removal of the superior cervical ganglia verifying a non-sympathetic nature of the NADPH-diaphorase positive nerve fibers. Pineal blood vessels also exhibited NADPH-diaphorase activity. The number and distribution of NADPH-diaphorase containing cells and nerve fibers were not affected by bilateral superior cervical ganglionectomy. Furthermore, animals sacrificed during day or night exhibited the same NADPH-diaphorase pattern. The present investigation provides the first morphological evidence for the presence of NADPH-diaphorase activity in rat pineal cells, suggesting an influence of nitric oxide on pineal metabolism. Furthermore, the presence of NADPH-diaphorase activity in the pineal blood vessels as well as in the perivascular nerve fibers suggests an influence of nitric oxide on the blood flow to the gland and/or the metabolism of the pineal cells adjacent to the blood vessels.