Investigation of the mechanism of nicotine-induced relaxation in guinea pig gallbladder

BACKGROUND: Muscular contraction of the gallbladder is the primary determinant of bile delivery into duedonum. Gallbladder filling and emptying are influenced by both inhibitory and excitatory stimuli, and NO plays a key role in normal relaxation. In this study, to determine whether nicotine acts on the gallbladder muscle, the mechanism of its effect on strips of guinea pig gallbladder was studied in vitro. MATERIALS AND METHODS: Guinea pig gallbladder muscle strips were mounted in organ bath with modified Krebs-Henseleit solution and aerated with Carbogen. Tension was measured with isometric force transducers, and muscle relaxation was expressed as percent decrease of precontraction induced by carbachol. RESULTS: Nicotine produced concentration dependent relaxation when preparations were precontracted by carbachol (10(-6) M). Nicotine-induced relaxation was 51.6 +/- 3.2% of phenylephrine contraction and was not affected by guanethidine (10(-5) M), propranolol (10(-6) M), hexamethonium (10(-4) M), indomethacin (10(-5) M), N(w)-nitro L-arginine methyl ester (L-NAME) (3 x 10(-5) M), methylene blue (10(-5) M), glibenclamide (10(-5) M), clotrimazole (10(-6) M), tetraethylammonium (3 x 10(-4) M), or 4-aminopyridine (10(-3) M). Nicotine did not exhibit a calcium antagonizing effect. CONCLUSIONS: From these results, we concluded that nicotine-induced relaxation of the guinea pig gallbladder is not mediated by the release of noradrenaline, nitric oxide (NO), prostaglandins, or a related substance, or by the activation of potassium channels, or by the stimulation of nicotinic cholinoceptors. Further work is needed to determine the cellular mechanism(s) of action by which nicotine acts on gallbladder smooth muscle.     

Effects of bacterial endotoxin on the contraction and relaxation responses of the rabbit cavernous smooth muscles

PURPOSE: We evaluated effects of bacterial endotoxin during septicemia on contraction and relaxation responses of cavernous smooth muscles in rabbits. MATERIALS AND METHODS: We performed isometric tension studies with norepinephrine (NE), endothelium-dependent and endothelium-independent vasodilators, and nonadrenergic noncholinergic (NANC)-selective electrical field stimulation on the muscle strips of control and endotoxin (lipopolysaccharide; LPS)-treated rabbits. To determine reversibility of the LPS effects on the cavernous smooth muscle, the contraction and relaxation studies were repeated after resting the strips for 1 day at 4C. We also investigated the effect of the nonspecific nitric oxide synthase (NOS) inhibitor (NW-nitro-L-arginine methyl ester; L-NAME) and the selective immunologic NOS inhibitor (aminoguanidine) on reactivity of the strips to NE and acetylcholine. RESULTS: Contractile response to NE was significantly (p <0.01) reduced in the cavernous smooth muscles from the systemically and locally LPS-treated rabbits, compared with control group. Both aminoguanidine and L-NAME markedly improved the diminished contraction of the strips. Relaxation response to endothelium-dependent agonists (acetylcholine and bradykinin) was significantly (p <0.05) decreased in the LPS-treated groups, compared with the control group but not to endothelium-independent vasodilators (papaverine and verapamil) and NANC-selective electrical field stimulation. L-NAME completely inhibited the relaxation response to acetylcholine in the control and LPS-treated groups but aminoguanidine did not. The impaired contraction and relaxation of the strips was completely restored after resting for 1 day. CONCLUSIONS: Bacterial endotoxin may cause non-endothelial overproduction of NO and inhibition of endothelium-derived NO production, which may contribute to impairment of contraction and relaxation of rabbit cavernous smooth muscles.     

The effect of superoxide dismutase on nitric oxide-mediated and electrical field-stimulated diabetic rabbit cavernosal smooth muscle relaxation

OBJECTIVE: To investigate the effect of superoxide dismutase (SOD, the enzyme that accelerates the breakdown of the superoxide anion, O2- to H2O) on nitric oxide (NO)-mediated and electrical field stimulated (EFS) relaxation in diabetic rabbit cavernosal smooth muscle. Materials and methods Diabetes was induced with alloxan (65 mg/kg) in six adult New Zealand White rabbits. After 6 months, cavernosal smooth muscle strips from age-matched controls and diabetic animals were mounted in organ baths. After precontraction with phenylephrine (10 micromol/L) in the presence of atropine (1 micromol/L), guanethidine (5 micromol/L) and indomethacin (10 micromol/L), relaxation responses to EFS (1-20 Hz), carbachol (10(-8)-10(-4) mol/L) and sodium nitroprusside (SNP, 10(-9)-10(-4) mol/L) were assessed in the presence and absence of SOD (100 IU/mL). RESULTS: SNP- and carbachol-mediated (endothelium-independent and -dependent, respectively) relaxations were impaired in the diabetic cavernosal smooth muscle strips compared with controls (concentration required for 50% inhibition, 1.4 micromol/L for diabetic and 0.75 micromol/L for control with SNP, and 44 micromol/L for diabetic and 0.4 micromol/L for control with carbachol). SOD significantly enhanced both SNP- and carbachol-mediated diabetic cavernosal smooth muscle relaxations (both P < 0.05). EFS-mediated relaxations were also significantly (P < 0.05) impaired in the diabetic cavernosal smooth muscle strips; these relaxations were also significantly (P < 0.05) enhanced by SOD. CONCLUSION: NO- and EFS-mediated cavernosal smooth muscle relaxation is impaired in a rabbit model of diabetes but SOD significantly reversed the impaired relaxation. Therefore, in diabetes, the generation of reactive oxygen species may play an important role in the development of erectile dysfunction.     

Functional innervation of the isolated bowel segment

PURPOSE: The aim of this study is to investigate whether there might be an eventual change in the enteric nerve responses to electrical field stimulation (EFS) of the isolated bowel segment (IBS) created by omentoenteropexy. METHODS: In the experimental group, an IBS was created in 10 rats using the omentum as the host organ by dividing of its mesentery 4 weeks later. In the control group, a jejunal loop of identical site and length to the IBS was studied in another 10 rats as normal jejunal segment. Longitudinal muscle strips were prepared from the IBSs (n 20) and the normal jejunal segments (n = 20). The effects of atropine, tetrodotoxin, L-arginine, and L-nitroarginine methyl ester (L-NAME) on the responses to transmural EFS were examined in both the IBS and normal jejunal segment using the isometric tension recording technique. RESULTS: Transmural EFS of the IBS strips produced contractile responses. Atropine and tetrodotoxin abolished the EFS-induced contractions of the IBS. Moreover, EFS-induced contractions were increased by the application of L-NAME, and were decreased by the application of L-arginine in the IBS. Mean percent change of IBS's contraction was not found statistically different from mean percent change of normal jejunal segment's contraction on the effects of L-NAME and L-arginine. CONCLUSION: These results suggest that the IBS, created by omentoenteropexy, produced enteric nerve responses to EFS as seen as in normal jejunal segment.     

Functional evaluation of the grafted wall with porcine-derived small intestinal submucosa (SIS) to a stomach defect in rats

BACKGROUND: Small intestinal submucosa (SIS) represents a novel bio-scaffolding material that may be used to repair hollow-organ defects. However, it is unclear whether neurophysiologic responses return to SIS-grafted areas in the gut. We evaluated the functional recovery of a stomach defect grafted with the porcine-derived SIS. METHODS: Twelve rats had a full-thickness defect created in the stomach. SIS was secured to the gastric wall. After 6 months, muscle strips were harvested from within the grafted area to perform both a histologic and a functional study. Additional full-thickness muscle strips were harvested from the posterior in the same stomach as controls. A dose response curve was obtained with carbachol (CCH) or sodium nitroprusside (SNP). Activation of intrinsic nerves was achieved by electrical field stimulation (EFS). RESULTS: The response to CCH and amplitude in EFS showed tonic contraction in both controls and SIS strips in a concentration-dependent and frequency-dependent manner. The magnitude after each stimulation was significantly lower in SIS strips compared with controls (P < .01). However, the contraction ratio of EFS to ED(50) of CCH was not significantly different between the groups. Additionally, SNP produced relaxation in both strips in a concentration-dependent manner. Histologic findings revealed that an insufficient amount of smooth-muscle cells existed in the muscularis propria, whereas compensated growth was observed in the submucosa with nerve regeneration. CONCLUSIONS: This study demonstrates that SIS provides a template for nerve migration to the graft in the rodent stomach. Innervations showed a similar distribution to that observed in the controls. The clinical implications of such findings warrant additional investigation.     

Advanced glycation end-products are responsible for the impairment of corpus cavernosal smooth muscle relaxation seen in diabetes

OBJECTIVE: To determine if advanced glycation end-products (AGEs) are responsible for the lower neuronal and endothelial-derived nitric oxide (NO)-mediated relaxation of corpus cavernosum in tissue in diabetic rats than in control rats. MATERIALS AND METHODS: Diabetes was induced in male Wistar rats by an intraperitoneal injection with streptozotocin (60 mg/kg). One group of diabetic rats was given free access to water and standard diet. A second group was given standard diet and aminoguanidine with their water (50 mg/100 mL) from the initiation of diabetes. Two groups of rats that were not diabetic acted as age-matched controls. After 8 weeks animals were killed by cervical dislocation, corpus cavernosal tissue strips harvested and mounted in an organ bath to measure isometric tension. After 90 min of equilibration at optimal resting tension and contraction with 1 micromol/L noradrenaline, the response to either acetylcholine or electrical field stimulation (EFS) after adding guanethidine (5 micromol/L) and atropine (1 micromol/L) was determined for each group. RESULTS: There was no difference between the baseline characteristics of all the experimental groups. After 8 weeks the mean body mass and glycosylated haemoglobin (HbA1c) were significantly greater in the diabetic than in control animals. Aminoguanidine had no effect on the recorded body mass or HbA1c. The in vitro relaxation response to the application of acetylcholine or EFS of tissue strips from age-matched control animals fed a standard diet and those supplemented with aminoguanidine were the same. The administration of aminoguanidine to diabetic animals for 8 weeks reversed the expected impaired relaxation response to acetylcholine; the response to EFS was similar. CONCLUSION: AGEs are more prevalent in erectile tissue from diabetic than in control animals. Aminoguanidine reversed the impairment in neuronal and endothelial NO-mediated penile smooth muscle relaxation seen in diabetes. As aminoguanidine prevents AGE formation, erectile dysfunction in diabetes is probably caused partly by the generation of AGEs.     

Endothelial and neuronal-derived nitric oxide mediated relaxation of corpus cavernosal smooth muscle in a rat, in vitro, model of erectile function

We set out to establish a simple, reproducible, rat in vitro model of erectile function and to use this to demonstrate the functional importance of both neuronal- and endothelial-derived nitric oxide within this animal. Two corpora cavernosal smooth muscle strips were harvested from sexually mature male Wistar rats and mounted in an organ bath for measurement of isometric tension. Following contraction with noradrenaline the strips were relaxed by the addition of either acetylcholine or sodium nitroprusside. Electrical field stimulation was performed in the presence of atropine and guanethidine. Relaxation responses were repeated in the presence of methylene blue, L-arginine, L-NNA and haemoglobin +/- L-arginine. Methylene blue abolishes the relaxation to acetylcholine and EFS; L-NNA and haemoglobin cause a significant impairment in the relaxation response. L-arginine reverses the effect of haemoglobin. In conclusion, the inhibitory, relaxant stimulus of rat corpora cavernosa is due to both neuronal nitric oxide and endothelial-derived nitric oxide released in response to cholinergic stimulation.     

Effect of vasoactive intestinal peptide on the motility of guinea-pig colon in vitro

The effects of vasoactive intestinal peptide (VIP) on longitudinal and circular muscle strips of guinea-pig proximal and distal colons, and on propulsive activity of guinea-pig distal colon were investigated in vitro. VIP (10(-9)-10(-6) M) produced relaxations of longitudinal and circular muscle strips in proximal colon and of circular muscle strip in distal colon, but produced a contraction of longitudinal muscle strip in distal colon. VIP-induced responses of the muscle strips were not influenced by indomethacin (10(-6) M). Tetrodotoxin (10(-6) M) and atropine (10(-6) M) converted VIP-induced contraction into relaxation in longitudinal muscle strip of distal colon, although these nerve blockers did not influence VIP-induced relaxations of longitudinal and circular muscle strips in proximal colon and of circular muscle strip in distal colon. VIP (10(-6) M) inhibited spontaneous and carbachol (10(-8) M)-stimulated propulsive activities of the isolated segment in distal colon. These results suggest that VIP may directly relax colonic smooth muscle cells and may indirectly contract longitudinal muscle strip of distal colon, mainly via stimulation of cholinergic neurones in the myenteric plexus of the muscle strip. It is also suggested that VIP-induced watery diarrhea in WDHA syndrome may not due to a direct stimulation of colonic motility.     

Inhibition by nitric oxide and nonadrenergic, noncholinergic (NANC) nerves is preserved in a canine model of extrinsic denervation: implications for small bowel transplantation

BACKGROUND: Small bowel transplantation (SBT) is complicated by changes in graft motility, especially in the early postoperative period. This dysmotility may be related in part to the extrinsic denervation necessitated by the procedure, but specific neurotransmitter response to SBT is incompletely understood. The aim of this study was to evaluate the role of nitric oxide and nonadrenergic, noncholinergic (NANC) enteric neural input in the nonimmunologic etiology of the dysmotility seen after SBT. METHODS: A technique of jejunoileal extrinsic denervation (without disruption of mesenteric vascular supply) was used as a model of canine jejunoileal autotransplantation to avoid potential confounding factors such as ischemia-reperfusion and postallotransplant immunologic effects. Longitudinal smooth muscle strips from ileum and jejunum were studied with in vitro tissue chamber methodology at 0, 2, and 8 weeks after this experimental model to explore early and late effects of denervation. Effects of exogenous nitric oxide (NO) and electric field stimulation (EFS), which releases native, endogenous enteric neurotransmitters) were evaluated in neurally intact control dogs and those undergoing extrinsic denervation. RESULTS: Exogenous NO caused a dose-dependent inhibition of spontaneous contractile activity and in some muscle strips a decrease in basal tone in both groups of dogs. These effects were unchanged by neural blockade with tetrodotoxin and preserved after extrinsic denervation. EFS produced inhibition of spontaneous contractile activity in ileum and a complex, inconsistent response in jejunum. The response to EFS in both ileum and jejunum was unchanged after extrinsic denervation. CONCLUSIONS: Nitric oxide inhibits contractile activity in canine longitudinal muscle of small bowel. Motility changes seen after this large animal model of extrinsic denervation are not caused by changes in NO or NANC neural function. The variability observed between different segments of intestine is important to consider in the context of SBT.     

Effects of castration on nitric oxide-mediated relaxations in male rat corpus cavernosum smooth muscle

BACKGROUND: The effects of castration on nitric oxide- mediated relaxations and nitric oxide synthase activity in male rat corpus cavernosum smooth muscles. METHODS: Eight-week-old male rats were assigned to two groups: control (sham operated) and castrated animals. After 8 weeks, corpus cavernosum smooth muscle strips were mounted in an organ bath for isometric tension recordings. Electrical field stimulation (EFS) was applied to the strips precontracted with 30 microM phenylephrine. The microdialysis probe was inserted into the strip, and Krebs-Henseleit solution was perfused into the probe. The dialysate during EFS and cholinergic stimulation was collected, and the amount of NO(-)(2)/NO(-)(3) (NOx) released in the dialysate was measured by the Greiss method. Sodium nitroprusside and carbachol were cumulatively added to the strips precontracted with 30 microM phenylephrine. RESULTS: EFS caused frequency-dependent relaxations and NOx releases in the strips. Pretreatment with N(omega)-nitro-L-arginine (100 microM) and tetrodotoxin (1 microM) completely inhibited relaxations and NOx releases. The maximum relaxation in the castration group was significantly greater than that in the control group. The release of NOx was significantly greater in the castration group than in the control group. Sodium nitroprusside relaxed the tissues in both groups similarly. Carbachol failed either to relax the tissue or to increase the amount of NOx production in the tissue. CONCLUSION: The present data suggest that castration enhances nitric oxide synthase activity and nitric oxide-mediated relaxations in the male rat corpus cavernosum.     

Effect of acalculous cholecystitis on gallbladder neuromuscular transmission and contractility

BACKGROUND: Impaired smooth muscle contractility is important in the pathophysiology of acalculous cholecystitis. Common bile duct ligation (CBDL) is a model of acalculous cholecystitis, producing acute inflammatory changes and decrease in gallbladder smooth muscle contractility. The aim of this study was to determine whether there is coexistent dysfunction of neural efferent motor pathways of the gallbladder after CBDL. MATERIALS AND METHODS: Gallbladder muscle contractility was studied in vitro in normal, CBDL, and sham-operated guinea pigs. Electric field stimulation (EFS; 2-16 Hz) was used to activate intrinsic nerves and exogenous acetylcholine (ACh) was used to directly stimulate the muscle. H&E-stained slides of muscle strips were scored for inflammatory changes. RESULTS: After CBDL, there was a progressive increase in the inflammation score and decrease in gallbladder muscle contractility to ACh. There was also a progressive decline in EFS-induced contractility when expressed as absolute force or normalized to the maximal muscle contractile response to ACh. The nitric oxide synthase inhibitor l-NNA (10 microM) increased EFS-induced contractions by 50 +/- 25% (P = 0.05) in CBDL animals but had no effect in sham surgical controls. CONCLUSIONS: CBDL with its acute gallbladder inflammation affects gallbladder contractility by two mechanisms: (1) decreased smooth muscle contractility, and (2) decreased neurally mediated contractions. The neurally mediated alterations result from dysfunction of cholinergic excitatory nerves and upregulation of nitric-oxide-mediated inhibition of smooth muscle contractility.     

Role of ATP and Related Purine Compounds on Urethral Relaxation in Male Rabbits

Background Non-adrenergic, non-cholinergic (NANC) relaxation evoked by electrical field stimulation (EFS) has been observed in the urethra, with nitric oxide (NO) considered the agent most probably mediating this effect. However, Burnstock's purinergic hypothesis suggests that ATP and related purine compounds are neurotransmitters in NANC relaxation, although the physiological and pharmacological effects of ATP and related purine compounds in the urethra have been little studied. Methods The effects of ATP and related purine compounds, N_G-nitro-L -arginine (NOARG; an inhibitor of nitric oxide synthesis from l -arginine), calcitonin gene-related peptide (CGRP), substance P and vasoactive intestinal polypeptide (VIP) on relaxation and smooth muscle tension induced by electrical field stimulation (EFS) were studied in isolated male rabbit circular urethral smooth muscle (functional study). In addition, the outflow of ATP elicited by EFS was measured using the luciferase technique (superfusion study). All experiments were performed in the presence of guanethidine (3 times 10_-3 mol/L) and atropine (10_-6 mol/L). Results In preparations contracted with U46619, a prostaglandin peroxidase inhibitor, ATP had almost no effect on EFS-induced relaxation; however, suramin, a non-selective P₂₎-purinoceptor antagonist, and NOARG each markedly attenuated this relaxation in a concentration-dependent manner. ATP and related purine compounds (adenosine, AMP and ADP) each reduced U46619-induced tonic contraction in a concentration-dependent manner. The potencies of the relaxant effects of ATP and these purine compounds were almost the same. In preparations contracted with U46619, CGRP and substance P had no effect on tonic contraction, but VIP reduced tonic contraction in a concentration-dependent manner. In the superfusion study, the outflow of ATP into the superfusate was markedly increased by EFS. When NOARG or prazosin was added to the superfusate, the increase in outflow of ATP was unchanged, but when suramin was added to the superfusate, no increase in outflow of ATP was observed. Conclusions These findings suggest that P₂₎-purinoceptors exist in the male rabbit urethra, and that ATP and related purine compounds may play a role in non-adrenergic, non-cholinergic neurotransmission. Consequently, the pathways mediating urethral relaxation by ATP, NO and VIP may be different.     

Effects of diabetes on neurotransmission in rat vaginal smooth muscle.

The aim of this work was to characterize the effect of experimental diabetes on neurotransmission in rat vagina. Female Sprague-Dawley rats were divided into two groups: non-diabetic controls (NDM, n=38) and diabetics (DM, n=38). DM was produced by intraperitoneal injection of streptozotocin. Eight weeks later the animals were killed, the distal part of the vagina was removed, and smooth muscle strips were prepared for functional organ bath experiments and for measurement of nitric oxide synthase (NOS) activity. In DM preparations, the EC(50) value for noradrenaline (NA) was significantly increased (P<0.05) and the maximal contractile response decreased (P=0.001). In preparations precontracted with NA, the NO donor SNAP and calcitonin gene-related peptide (CGRP) caused concentration-dependent relaxations, which were significantly decreased (P<0.001) in the DM group. Electrical stimulation of nerves (EFS) caused frequency-dependent contractions, which were significantly lower in DM than in NDM strips (P<0.001). SNAP and CGRP concentration-dependently inhibited EFS evoked contractions in both NDM and DM preparations. The inhibition was significantly lower (P<0.05) in the DM group. In NDM preparations precontracted with NA, EFS evoked frequency-dependent relaxations; such relaxations were inhibited or reduced in DM. Treatment with the NOS inhibitor, L-NOARG 0.1 mM, abolished relaxations in all preparations or produced contraction in DM preparations. Calcium-dependent NOS activity was not significantly different in the DM and NDM groups. However, the DM animals showed a small but significant increase in calcium-independent NOS-activity (P<0.05). Diabetes interferes with adrenergic-, cholinergic- and NANC-neurotransmitter mechanisms in the smooth muscle of the rat vagina. The changes in the nitrergic neurotransmission are not due to reduction in NOS-activity, but seem to be due to interference with later steps in the L-arginine/NO/guanylate cyclase/cGMP system.     

Mechanical properties and innervation of the smooth muscle layers of the urethra of greyhounds

OBJECTIVE: To investigate the properties of the smooth muscle layers in the urethral wall of male and female greyhounds, and to consider their roles in continence and micturition. MATERIALS AND METHODS: The distribution and innervation of the smooth muscle layers of the prostate capsule and membranous urethra of male greyhounds were assessed. Strips of smooth muscle from these regions were used to determine the neuropharmacological properties by assessing the excitatory and inhibitory responses to nerve stimulation, and the effects of blocking agents. These were compared with strips from the proximal urethra and from the female urethra. RESULTS: The smooth muscle of the membranous urethra comprised 9% of the wall and received its innervation exclusively in branches from the pelvic plexus. The cholinergic innervation in the male produced 80% of the total contractile response in the longitudinal membranous urethra, 50% in the prostate capsule and 13% in the circular muscle of the proximal urethra. In the female all areas had poor contractile responses. Inhibitory fibres produced relaxation in all parts of male and female urethrae with the major effect caused by nitric oxide. Adrenergic nerves contributed to both residual excitation (alpha receptors) and inhibition (beta receptors). CONCLUSIONS: The longitudinal smooth muscle of the male membranous urethra probably shortens the urethra during micturition, through the activity of cholinergic nerves, whereas the circular smooth muscle of the proximal urethra, under adrenergic control, may be contracted during continence and ejaculation. In the female, the smooth muscle plays a minor role.     

Nicotine inhibits large conductance Ca(2+)-activated K(+) channels and the NO/-cGMP signaling pathway in cultured human endothelial cells

OBJECTIVE: The effects of nicotine on endothelium-dependent vasorelaxation mediated by nitric oxide (NO) are controversial. Since endothelial NO synthesis has been shown to depend on the activity of large conductance Ca(2 + )-activated K(+) channels (BK(Ca)), the present study investigated whether nicotine alters BK(Ca) single channel activity induced by the K(+) channel opener NS1619, and to examine a possible interaction with the endothelial NO generation. DESIGN: The patch-clamp technique was used to examine the BK(Ca) activity. NO production was measured indirectly using a [(3)H]-cGMP-radioimmunoassay. All experiments were performed using cultured endothelial cells derived from human umbilical cord veins. RESULTS: The BK(Ca) opener NS1619 (10 micromol/l) significantly increased the BK(Ca) open-state probability (NPo) from 0.011+/-0.007 (control) to 0.052+/-0.019. Co-perfusion with nicotine (1 micromol/l) significantly decreased NS1619 induced NPo (n = 14, p < 0.05). Intracellular cGMP levels were significantly increased, if cells were stimulated with NS1619 (+ 225%; n = 10, p < 0.05), which was blocked by Nicotine (1 micromol/l). CONCLUSIONS: The results of the present study demonstrate that BK(Ca) activation by NS1619 plays an important role in the regulation of the NO-/cGMP-signaling-pathway. Endothelial dysfunction caused by nicotine may be connected with a decrease in BK(Ca)-activity.     

Longitudinal muscle shows abnormal relaxation responses to nitric oxide and contains altered levels of NOS1 and elastin in uncomplicated diverticular disease

OBJECTIVE: Recent evidence challenges the 'low-fibre/high-colonic intraluminal pressure' hypothesis of diverticular disease (DD) and raises the possibility that other mechanisms are involved. Although bowel wall smooth muscle is known to be hypercontractile in DD, the nature of its relaxation is unknown. The present study investigated colonic smooth muscle responses to nitric oxide, as well as the smooth muscle contents of neural nitric oxide and elastin associated with the disease. METHOD: Immunohistochemical/image analysis of antibodies to nitric oxide synthase (NOS1), co-localized with protein gene product (PGP) and to elastin, was performed on three histological sections of sigmoid colons from 20 patients (10 DD, 10 controls) following resections for rectal tumours. Organ bath experiments examined smooth muscle responsiveness to nitroprusside, a nitric oxide donor. RESULTS: Uncomplicated diverticular longitudinal muscle showed lower nitric oxide immunoreactivity compared with controls: median percentage surface area of NOS1 over PGP was 26.0% (range 0.5-58.3), controls 45.0% (35.0-70.1; P = 0.018). Median percentage surface area of elastin was elevated, 21.3% (10.6-45.6), controls 8.2% (1.7-13.5; P = 0.0002), together with a low sensitivity to nitroprusside [mean - log EC(50) 5.3 (SD 0.5), controls 6.6 (SD 0.5), difference 1.3, 95% CI 0.8-1.7; P < 0.0001] and there were lower maximum relaxation responses to nitroprusside compared with controls: median percentage (relaxation induced by nitroprussside/contraction induced by bethanecol) was 52.0%, range (20.0-92.0), controls 100.0% (71.0-125.0), P < 0.0001. No statistically significant differences were found in circular muscle, at the sample size studied. CONCLUSION: This study established, for the first time, specific abnormalities in longitudinal muscle relaxation and contents of neural nitric oxide and elastin in uncomplicated DD. These findings may have important implications for both colon structure and function in the disease.     

Nitrergic mechanisms mediating inhibitory control of longitudinal smooth muscle contraction in mouse small intestine

Studies using genetic manipulation to investigate mechanisms of control of physiologic function often necessitate mouse models. However, baseline functional analysis of murine small intestinal motility has not been well defined. Our aim was to define nitrergic mechanisms regulating mouse small intestinal longitudinal muscle. Endogenous nitric oxide (NO) is an important neuroregulatory substance mediating inhibition of contractile activity in murine small bowel. Full-thickness muscle strips of jejunum and ileum from C57BL/6 mice (n ≥6 mice) cut in the direction of longitudinal muscle were studied. Numerous conditions of electrical field stimulation (EFS) and effects of exogenous NO and NO donors were studied in the absence or presence of inhibitors of nitric oxide synthase (NOS) and 1H-[1,2,4]-oxadiazaolo-[4,3-a]-quinoxalin-1-one (ODQ), a downstream inhibitor of guanylyl cyclase. EFS induced a frequencydependent inhibition of contractile activity in both jejunum and ileum (P < 0.05). As the voltage of EFS was increased, inhibition turned to excitation in the jejunum; in contrast, the ileum demonstrated a voltage-dependent increasing inhibition (P < 0.05 each). EFS-induced inhibition was blocked by NOS inhibitors and ODQ. NO donors inhibited spontaneous contractile activity abolished by ODQ. NO appears to be an endogenous inhibitory neurotransmitter in murine longitudinal small bowel muscle. Nitrergic mechanisms mediate inhibitory control of murine longitudinal small intestinal muscle. Differences exist in neuroregulatory control between jejunum and ileum that may be related to their known difference in motor patterns. Presented at the Forty-Fifth Annual Meeting of The Society for Surgery of the Alimentary Tract, New Orleans, Louisiana, May 15–19, 2004 (poster presentation). Parts of this work has been published in abstract form (Gastroenterology 2004; 126(Suppl 2): A784). This work was supported by National Institutes of Health grant R01-DK-39337 (M.G.S.) and by the Mayo Foundation.     

Role of nitric oxide in the internal anal sphincter of Hirschsprung's disease

It is not clear what contribution the internal anal sphincter (IAS) makes to the impaired motility observed in patients with Hirschsprung's disease (HD). Nitric oxide (NO) has recently been shown to be a neurotransmitter in the nonadrenergic noncholinergic (NANC) inhibitory nerves in the human gut. To clarify the physiologic significance of NO in the IAS of HD (aganglionosis), we investigated the enteric nerve responses on lesional (aganglionic) and normal IAS muscle strips above the dentate line. Lesional and normal IAS muscle strips above the dentate line were derived from patients with HD (10 cases) and patients who underwent rectal amputation for low rectal cancer (12 cases). A mechanographic technique was used to evaluate in vitro muscle responses to electrical field stimulation (EFS) before and after treatment with various autonomic nerve blockers, N(G)-L-nitroarginine, and L-arginine. The following results were obtained: (1) Cholinergic nerves are mainly involved in the regulation of enteric nerve responses to EFS in the normal IAS. (2) The aganglionic IAS of patients with HD was more strongly innervated by cholinergic nerves than the normal IAS (p < 0.05). (3) NANC inhibitory nerves were found to act on the normal IAS but had no effect on the enteric nerves in patients with aganglionosis. (4) NO was found to act on normal IAS, but no effect was observed in the aganglionic IAS. These findings suggest that innervation of the cholinergic nerves and a loss of NO mediation of NANC inhibitory nerves play an important role in the impaired motility observed in the IAS with HD.