Satish Rattan

GENESIS OF BASAL SPHINCTER PRESSURE: EFFECT OF TETRODOTOXIN ON LOWER ESOPHAGEAL SPHINCTER PRESSURE IN OPOSSUM IN VIVO

Currently it is believed that lower esophageal sphincter closure is maintained by tonic neurohormonal activity. Studies were performed to evaluate the role of neural and myogenic activity in the genesis of resting sphincter pressure in the opossum. The neural activity in the sphincter was blocked by intravenous administration of the puffer fish poison, tetrodotoxin, in intact animals. The respiration in these animals was assisted with a respirator, their blood pressure was supported by intravenous administration of Ringers solution, and their body temperature was maintained by heating lamp. The lower esophageal sphincter pressures were measured with water-filled and continuously perfused catheters which were anchored to the lower esophageal sphincter using a new technique. Tetrodotoxin blocked the sphincter response to neural stimulation with vagal stimulation, esophageal distention, and local electrical stimulation of the sphincter. The mean resting sphincter pressure before tetrodotoxin was 59.0 +/- 2.6 mm Hg, and it was 64.4 +/- 6.9 mm Hg after tetrodotoxin (P greater than 0.05). During neural block with tetrodotoxin, the sphincter response to administration of isoproterenol or bethanechol was similar to that during the control period (P greater than 0.05). These studies show that: (1) Tetrodotoxin can be administered in intact animals to achieve complete block of neural activity in the lower esophageal sphincter; (2) sphincter muscle is not adversely affected by tetrodotoxin; and (3) block of neural activity does not alter the resting lower esophageal sphincter pressure.

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.

Role of galanin in the gut

The neuropeptide galanin is present in both the central and the peripheral nervous systems (l-10) (Table 1). In the central nervous system galanin is widely distributed throughout the spinal cord and the brain. In the spinal cord it is present at all levels and has been detected in large amounts in dorsal horn cells, nerve cell bodies intrinsic to the spinal cord, including neurons of lamina II, intermediate dorsal laminae, neurons around lamina X, and in subpopulations of lower motor neurons. Galanin-containing fibers are most prominent in the lumbosacral regions of the spinal cord (11). In the brain, galanin is widely distributed in the medulla (caudal trigeminal nucleus, nucleus tractus solitarius, and ventral surface), locus caeruleus, and midbrain. In the forebrain, galanin is present in thalamus, hypothalamus, and magnocellular neurosecretory neurons of supraoptic and paraventricular nuclei. The median eminence and hypothalamus have the highest concentrations of galanin. In the peripheral nervous system galanin is richly present in the terminal innervation of the gastrointestinal, respiratory, and urogenital tracts, the pancreas, and the adrenal medulla (6). Galanin is present in the neurons of the myenteric, submucous, and mucous plexuses of the gut. Galaninimmunoreactive nerves have been shown to be present in all layers of the gut wall and at all levels of the gastrointestinal tract (7,12) in the different species examined. Most of the galanin fibers are present in muscle layers; only a few occur in the mucosa. There are a few species differences regarding the distribution of galanin fibers. Galanin-immunoreactive cell bodies are present only in the submucous plexus of human and porcine gut. In the rat they are present in both the myenteric and submucous plexuses, but they are more numerous in the latter. Surgical and chemical denervation experiments suggest that galanin fibers are primarily intrinsic to the enteric nervous system (9,13). Galanin-containing axons are among the longest, projecting distances of 1.5-2 cm in the anal direction (13). Galanin may coexist with vasoactive intestinal polypeptide (VIP) in ganglion cells of the submucous plexuses of different species examined (7). In some cases, e.g., in porcine duodenum, all VIP-immunoreactive neuronal cell bodies of the submucous plexus may contain galanin. In other regions, two thirds of the VIP-immunoreactive cells may show colocalization with galanin, and only a few cells show galanin immunoreactivity alone. In the myenteric plexus of antrum and in the submucous plexus of the canine small intestine (14), it was reported that galaninimmunoreactive fibers always contained VIP immunoreactivity. In the pig, galanin-containing cell bodies may also contain substance P and calcitonin generelated peptide (15). Galanin has also been reported to coexist with acetylcholine (16), catecholamines, 5-hydroxytryptamine, y-aminobutyric acid and vasopressin, oxytocin, corticotropin-releasing factor, neurotensin, and VIP (17-22). Galanin immunoreactivity has also been shown to be present in the primary afferent fibers (3,21). Studies by Ch’ng et al. (11) showing the accumulation of galanin in the sacral regions of the spinal cord following thoracic transection suggest that galanin is present in the ascending pathway, originating from the sacral segments. These observations have been further confirmed by capsaicin pretreatment (23) and surgical deafferentation. Such findings suggest the

Effect of Dopamine on the Esophageal Smooth Muscle in Vivo

Intravenous administration of dopamine caused a dose-dependent reduction in the lower esophageal sphincter pressure and contractions in the lowermost part of the body of the esophagus in the opossum. A dose of 5 mug per kg produced maximal response. The onset of contractions in the body occurred around 20 sec after the onset of the sphincter response. The effect of dopamine was different from isoproterenol, which caused no contractions in the body and caused reduction in sphincter pressure, and from phenylephrine, which also caused no contractions in the body but caused contraction of the sphincter. Moreover, the effect of 5 mug per kg of dopamine was blocked by haloperidol but not by phentolamine, propranolol, bilateral cervical vagotomy, or tetrodotoxin. These studies suggested the presence of selective dopamine receptors which mediated contraction of the esophageal body and relaxation of the sphincter muscle. Since vagal stimulation also causes contraction in the body and relaxation of the sphincter, it was possible that vagal effect on the esophagus was mediated by dopamine receptors. However, haloperidol did not block the effect of electrical stimulation of vagus nerve on body of the esophagus or the lower esophageal sphincter.

Esophageal muscle physiology and morphogenesis require assembly of a collagen XIX–rich basement membrane zone

Collagen XIX is an extremely rare extracellular matrix component that localizes to basement membrane zones and is transiently expressed by differentiating muscle cells. Characterization of mice harboring null and structural mutations of the collagen XIX (Col19a1) gene has revealed the critical contribution of this matrix protein to muscle physiology and differentiation. The phenotype includes smooth muscle motor dysfunction and hypertensive sphincter resulting from impaired swallowing-induced, nitric oxide–dependent relaxation of the sphincteric muscle. Muscle dysfunction was correlated with a disorganized matrix and a normal complement of enteric neurons and interstitial cells of Cajal. Mice without collagen XIX exhibit an additional defect, namely impaired smooth-to-skeletal muscle cell conversion in the abdominal segment of the esophagus. This developmental abnormality was accounted for by failed activation of myogenic regulatory factors that normally drive esophageal muscle transdifferentiation. Therefore, these findings identify collagen XIX as the first structural determinant of sphincteric muscle function, and as the first extrinsic factor of skeletal myogenesis in the murine esophagus.

Impaired rectoanal inhibitory response in scleroderma (systemic sclerosis): an association with fecal incontinence.

Gastrointestinal abnormalities in systemic sclerosis (SSc) involve both myogenic and neural mechanisms. The aims of this study were to evaluate the rectoanal inhibitory response (RAIR) in SSc patients and to correlate RAIR with duration and subtype of disease, antibody status, and lower gastrointestinal symptoms. Thirty-five patients with SSc completed a questionnaire and underwent anorectal manometry (ARM). Forty-five patients without SSc served as controls. In the 35 SSc patients, 62.3% reported diarrhea, 57.1% reported constipation, and 37.1% reported fecal incontinence. Twenty-five of the 35 scleroderma patients (71.4%) demonstrated an impaired or absent RAIR, compared with none of the 45 controls (P < 0:001). Eleven of 13 incontinent SSc patients (84%) had an impaired RAIR. No correlation was found between RAIR and duration or subtype of SSc, antibody status, or presence of diarrhea or constipation. Impaired RAIR was closely correlated with fecal incontinence, suggesting a possible neural mechanism for maintenance of continence.

Involvement of rho and rho-associated kinase in sphincteric smooth muscle contraction by angiotensin II.

The tonic smooth muscles of lower esophageal sphincter (LES) and internal anal sphincter (IAS) are subject to modulation by the neurohumoral agents. We report that angiotensin (Ang) II-induced contraction of rat IAS and LES smooth muscle cells (SMC) was Inhibited by Clostridium botulinum C3 exozyme, HA 1077 and Y 27632, suggesting a role for Rho kinase and a Rhoassociated kinase (ROK). Ang II-induced contraction of the SMC was also attenuated by genistein, antibodies to the pp60c-arc, p190 RhoGTPase-activating protein (p190 RhoGAP), carboxyl terminus of Gα13, carboxyl terminus peptide, and ADP ribosyiation factor (ARF) antibody. Ang II-induced increase in p190 RhoGAP tyrosine phosphorylation was attenuated by genistein. Furthermore, Ang II-induced increase in smooth muscle tone and phosphorylation of myosin light chain (MLC; 20 kDa; MLC20-P) were attenuated by Y 27632 and genlstein. The results suggest an important role for Gα13 and pp60c-arc in the Intracellular events responsible for the activation of RhoA/ROK in Ang II-induced contraction of LES and IAS SMC.

Immunoglobulins from scleroderma patients inhibit the muscarinic receptor activation in internal anal sphincter smooth muscle cells

Systemic sclerosis (SSc) IgGs affecting the M(3)-muscarinic receptor (M(3)-R) have been proposed to be responsible for the gastrointestinal (GI) dysmotility in this disease. However, the effect of SSc IgGs on smooth muscle cell (SMC) function has not been studied. We determined the effect of SSc IgGs on the muscarinic receptor activation by bethanechol (BeCh; methyl derivate of carbachol) in SMC and smooth muscle strips from rat internal anal sphincter. IgGs were purified from GI-symptomatic SSc patients and normal volunteers, with protein G-Sepharose columns. SMC lengths were determined via computerized digital micrometry. The presence of M(3)-R and IgG-M(3)-R complex was determined by Western blot. IgGs from SSc patients but not from normal volunteers caused significant and concentration-dependent inhibition of BeCh response (P < 0.05). The maximal shortening of 22.2 +/- 1.2% caused by 10(-4) M BeCh was significantly attenuated to 8.3 +/- 1.2% by 1 mg/ml of SSc IgGs (P < 0.05). Experiments performed in smooth muscle strips revealed a similar effect of SSc IgG that was fully reversible. In contrast to the effect on BeCh, the SSc IgGs caused no significant effect (P > 0.05) on K(+) depolarization and alpha(1)-adrenoceptor activation by phenylephrine. Western blot studies revealed the specific presence of SSc IgG-M(3)-R complex. SSc IgGs attenuated M(3)-R activation, which was reversible with antibody removal. These data suggest that SSc GI dysmotility may be caused by autoantibodies that inhibit the muscarinic neurotransmission. Future treatment of SSc patients may be directed at the removal or neutralization of these antibodies.

PET Imaging of VPAC1 Expression in Experimental and Spontaneous Prostate Cancer

Among U.S. men, prostate cancer (PC) accounts for 29% of all newly diagnosed cancers. A reliable scintigraphic agent to image PC and its metastatic or recurrent lesions and to determine the effectiveness of its treatment will contribute to the management of this disease. All PC overexpresses VPAC1 receptors. This investigation evaluated a probe specific for a 64Cu-labeled receptor for PET imaging of experimental human PC in athymic nude mice and spontaneously grown PC in transgenic mice. Methods: The probe, TP3939, was synthesized, purified, and labeled with 64Cu and 99mTc. Using a muscle relaxivity assay, biologic activity was assessed and inhibitory concentrations of 50% calculated. Receptor affinity (Kd) for human PC3 cells was determined using 99mTc-TP3939 and 64CuCl2. Blood clearance and in vivo stability were studied. After intravenous administration of either 64Cu-TP3939 or 64CuCl2 in PC3 xenografts and in transgenic mice, PET/CT images were acquired. Prostate histology served as the gold standard. Organ distribution studies (percentage injected dose per gram [%ID/g]) in normal prostate were performed. The ratios of tumor to muscle, tumor to blood, normal prostate to muscle, and tumor to normal prostate were determined. Results: Chemical and radiochemical purities of TP3939 were 96.8% and 98% ± 2%, respectively. Inhibitory concentrations of 50% and affinity constants were 4.4 × 10−8 M and 0.77 × 10−9 M, respectively, for TP3939 and 9.1 × 10−8 M and 15 × 10−9 M, respectively, for vasoactive intestinal peptide 28. Binding of 64CuCl2 to PC3 was nonspecific. Blood clearance was rapid. In vivo transchelation of 64Cu-TP3939 to plasma proteins was less than 15%. 64Cu-TP3939 uptake in PC was 7.48 ± 3.63 %ID/g at 4 h and 5.78 ± 0.66 %ID/g at 24 h after injection and was significantly (P < 0.05) greater than with 64CuCl2 (4.79 ± 0.34 %ID/g and 4.03 ± 0.83 %ID/g at 4 and 24 h, respectively). The ratios of PC to normal prostate at 4 and 24 h were 4 and 2.7, respectively. 64Cu-TP3939 distinctly imaged histologic grade IV prostate intraepithelial neoplasia in transgenic mice, but 18F-FDG and CT did not. Conclusion: Data indicate that TP3939, with its uncompromised biologic activity, delineated xenografts and cases of occult PC that were not detectable with 18F-FDG. 64Cu-TP3939 is a promising probe for PET imaging of PC. It may also be useful for localizing recurrent lesions and for determining the effectiveness of its treatment.

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.