Colonic Muscularis Mucosae Research Articles

Pediatric Gastrointestinal Histopathology in Patients With Tetratricopeptide Repeat Domain 7A (TTC7A) Germline Mutations: A Rare Condition Leading to Multiple Intestinal Atresias, Severe Combined Immunodeficiency, and Congenital Enteropathy.

Mutations in the tetratricopeptide repeat domain 7A (TTC7A) gene are a rare cause of congenital enteropathy that can result in significant morbidity. TTC7A deficiency leads to disruption of the intestinal epithelium. The histopathology of this condition has been partly described in case reports and clinical studies. This manuscript describes an in-depth investigation of the pediatric gastrointestinal pathology of the largest histologically examined cohort with confirmed TTC7A mutations reported to date and, for the first time, compared the findings to age-matched and sex-matched control patients with intestinal atresia not thought to be associated with TTC7A mutations. Hematoxylin and eosin-stained slides of endoscopically obtained mucosal biopsies and surgical resection specimens from 7 patients with known TTC7A mutations were examined retrospectively. The microscopic findings were found to be on a spectrum from atresia-predominant to those with predominantly epithelial abnormalities. Several unique histopathologic characteristics were observed when compared with controls. These included neutrophilic colitis and prominent lamina propria eosinophilia throughout the gastrointestinal tract. Striking architectural abnormalities of the epithelium were observed in 4 of the 7 patients. The 5 patients with intestinal atresia demonstrated hypertrophy and disorganization of the colonic muscularis mucosae accompanied by bland spindle cell nodules within the intestinal wall. The components of the latter were further elucidated using immunohistochemistry, and we subsequently hypothesize that they represent obliterated mucosa with remnants of the muscularis mucosae. Finally, atrophic gastritis was noted in 4 patients. In conclusion, the unique histopathologic characteristics of TTC7A mutation-associated enteropathy described herein more fully describe this novel disease entity in infants who present with congenital enteropathy or enterocolitis.

Adrenergic responses of rat colonic muscularis mucosae

We have investigated adrenoceptor-mediated responses of muscularis mucosae from the proximal, mid and distal regions of the rat colon. Noradrenaline-induced relaxations of the muscularis mucosae in each region were unaffected by atenolol, butoxamine or propranolol, but they were attenuated by the selective beta3-adrenoceptor antagonist cyanopindolol. The beta3-adrenoceptor agonist CL216343 elicited concentration-dependent relaxation of the muscularis mucosae in all regions of the colon. Isoprenaline, a non-selective beta-adrenoceptor agonist, evoked concentration-dependent relaxations of the muscularis mucosae in all regions, but only in the proximal colon were these significantly larger than the maximum noradrenaline-induced relaxation. The alpha1-adrenoceptor agonist methoxamine caused large contractions of the proximal colonic muscularis mucosae. When proximal tissue was pretreated with phentolamine, an alpha1-adrenoceptor antagonist, maximal noradrenaline- and isoprenaline-induced relaxations did not differ significantly. Although the mid colonic muscularis mucosae was also found to possess excitatory alpha1-adrenoceptors, these were associated with small contractions and did not modify the muscle's inhibitory responses to noradrenaline. Distal colonic muscularis mucosae lacked excitatory adrenoceptors and only responded to noradrenaline with beta3-adrenoceptor-mediated relaxations. No evidence was obtained for functional alpha2-adrenoceptors on the muscularis mucosae in any region of the rat colon. These data demonstrated that noradrenaline-induced relaxation of the rat colonic muscularis mucosae was mediated via beta3-adrenoceptors throughout, but in the proximal region this was modified by concurrent excitatory alpha1-adrenoceptor activation. Based upon these observations it appeared unlikely that noradrenaline-induced relaxation of rat colonic muscularis mucosae would be functionally linked to the secretory responses of the corresponding mucosa during periods of increased sympathetic activity.

Prevalence and Outcome of Allergic Colitis in Healthy Infants With Rectal Bleeding: A Prospective Cohort Study

Purpose of the Study. To determine the prevalence of allergic colitis (AC) in a cohort of healthy infants with rectal bleeding. A secondary purpose was to determine if bleeding would resolve in untreated infants with rectal bleeding without biopsy-proven AC. Study Population. There were 22 infants ≤6 months of age with rectal bleeding recruited from the referral area of Cincinnati Children’s Hospital Medical Center (Cincinnati, OH). All subjects had a negative history of bleeding disorders, negative stool cultures, positive hemoccult, and a negative history and physical examination for signs of infection, Hirschsprung disease, and inflammatory bowel disease. Methods. AC was defined histologically as colonic mucosa with ≥6 eosinophils per high-power field and/or eosinophils in the colonic crypts or muscularis mucosae. Formula or maternal diet was changed only for infants with histologic findings of AC. Formula-fed infants were switched to an extensively hydrolyzed formula and were rebiopsied at 3 weeks. If the biopsy was normal, they were continued on the formula and managed clinically. Those with continued histologic evidence of colitis were changed to an amino acid–based formula and rebiopsied at 6 weeks. Breastfed infants continued breastfeeding while mothers followed a milk-protein–free diet. Those with resolution of bleeding and normal biopsies at 3 weeks continued with breastfeeding and a restricted maternal diet. Those with persistent histologic evidence of colitis were rebiopsied at 6 weeks with no further dietary change. Those with persistent bleeding were changed to hydrolysate and rebiopsied at 6 weeks. Those with persistent bleeding and histologic evidence of AC at 6 weeks were changed to an amino acid–based formula. Results. Of 22 subjects, 14 (63.6%) had histologic evidence of AC. Five had normal biopsies and 3 had nonspecific colitis. Seven of the 14 with AC were formula fed. Six of the 7 had resolution of bleeding, on average, in 1.8 weeks (range: 1–5 weeks). One of the 7 was changed to an amino acid formula at 3 weeks and had resolution of bleeding at 5 weeks. The remainder of the 14 were breastfed. Six were followed to completion of the study. One had a delayed diagnoses because of development of worsening rectal bleeding and an abnormal biopsy at week 3 despite a normal biopsy at the onset of the study. The infant failed to improve with hydrolyzed formula but had resolution of bleeding by week 8 after initiation of an amino acid formula. Of the remaining 5, 2 had normal histology at week 3 with maternal elimination of cow’s milk. Two had improvement by week 3, and 1 had no change. The average time for resolution in the breastfed group was 5.6 weeks (range: 2–8 weeks). For the 5 infants without histologic evidence of colitis, the average time for resolution of bleeding was 3.25 weeks. In those with nonspecific colitis, 2 had resolution by week 6, and the third was ultimately diagnosed with inflammatory bowel disease. Conclusions. A significant proportion of infants with rectal bleeding may not have AC and may undergo unnecessary, expensive formula or maternal diet changes that may discourage breastfeeding. Reviewer Comments. This small study provides important insights about the prevalence and natural course of proctocolitis. A much larger prospective placebo-controlled study that compares treatment versus no treatment would be very helpful.

Regional Variations in Neurokinin Receptor Subtype Contributions to Muscularis Mucosae and Epithelial Function in Rat Colon

It is known that the muscularis mucosae and mucosa are not pharmacologically homogeneous throughout the rat colon. The aim of this study was to simultaneously characterize all three neurokinin (NK) receptors in the muscularis mucosae and mucosa along the length of the rat colon. Strips of proximal, mid, and distal colonic muscularis mucosae were prepared for isometric recording or sheets of muscle-free mucosa were mounted in Ussing chambers for measurement of short-circuit current. In both muscularis mucosae and mucosa the greatest responses to substance P were found in the proximal region. Use of selective agonists revealed the presence of all three NK receptors in both structures, however, selective antagonism suggests that only NK2 receptors in the muscularis mucosae and NK1 receptors in the mucosa are physiologically relevant. In conclusion, substance P-induced responses in the rat colon are region-specific and not mediated by a single NK receptor subtype common to both structures.

Prevalence and Outcome of Allergic Colitis in Healthy Infants with Rectal Bleeding: A Prospective Cohort Study

Allergic colitis is often diagnosed clinically in healthy infants with rectal bleeding and often treated with costly hypoallergenic formula. The true prevalence of allergic colitis is unknown. We tested the hypothesis that allergic colitis is overdiagnosed in healthy infants with rectal bleeding. The authors also determined whether rectal bleeding in infants without allergic colitis would resolve without diet change. For the purposes of this study, allergic colitis was defined histologically as colonic mucosa with >or= 6 eosinophils per high power field and/or eosinophils in colonic crypts or muscularis mucosae. We surveyed all 56 Ohio NASPGHAN members to determine standard practice regarding the evaluation of rectal bleeding in infants. In addition, infants <or= 6 months old with rectal bleeding were recruited from the referral area of Cincinnati Children's Hospital Medical Center. All infants underwent flexible sigmoidoscopy with biopsies at 5, 10 and 15 cm. Formula or maternal diet was changed only for infants with histologic findings of allergic colitis as defined. Study subjects were followed for 9 weeks. In the survey of NASPGHAN members, 84% indicated they would empirically change the diet of an infant with rectal bleeding to treat presumed allergic colitis. In our study population, however, only 14 of 22 (64%; 95% confidence interval, 41-83) infants with rectal bleeding had allergic colitis. Five (23%) had normal biopsies and three (14%) had nonspecific colitis. Rectal bleeding in all infants with normal biopsies or nonspecific colitis resolved without diet change except for 1 infant subsequently diagnosed with infantile inflammatory bowel disease. A significant proportion of infants with rectal bleeding may not have allergic colitis and may undergo unnecessary, expensive formula or maternal diet changes that may discourage breast-feeding.

Mesenchymal Tumors of Muscularis Mucosae of Colon and Rectum are Benign Leiomyomas that Should be Separated from Gastrointestinal Stromal Tumors—A Clinicopathologic and Immunohistochemical Study of Eighty-Eight Cases

Most mesenchymal tumors of the gastrointestinal tract are currently classified as specific gastrointestinal stromal tumors. However, true leiomyomas are more common in the esophagus, and they have been occasionally noted in the colon and rectum, but the small number of reported cases does not allow for clinicopathologic profiling. This study was undertaken to characterize 88 tumors of the muscularis mucosae of the colon and rectum. Seventy tumors were obtained form the files of AFIP and 18 cases from the Department of Pathology of the Haartman Institute of the University of Helsinki. The lesions, except one, were removed by snare polypectomy as incidental lesions at cancer or polyp surveillance; one small tumor was an incidental finding in the rectal resection specimen. The tumors had a significant male predominance in both institutions (overall 2.4:1). They occurred in age range of 38 to 85 years (median 62 years). The lesions were typically small (range 1 to 22 mm, median 4 mm) and located predominantly in the rectum and sigmoid (72%). All tumors were composed of well-differentiated, eosinophilic smooth muscle cells that were seen immediately beneath the mucosa obliterating the muscularis mucosae layer and merging with it. Two tumors had significant atypia (“symplastic leiomyoma”); mitotic activity was seen in one of these tumors, but not in others. The lesional cells were uniformly positive for smooth muscle actin and desmin and negative for CD34, CD117 and S100-protein, based on immunohistochemical studies on 20 to 24 cases with each marker. No gastrointestinal stromal tumors were identified among the tumors of muscularis mucosae, and no CD117-positive cells, except mast cells, were seen in the muscularis mucosae layer. None of the patients had morbidity related to the tumor. Based on follow-up data on 29 patients, leiomyomas of muscularis mucosae are benign. They should be separated from gastrointestinal stromal tumors that have a clinicopathologic spectrum including frequent disease-related mortality. Snare polypectomy is an adequate treatment, but ensuring the complete removal and follow-up are necessary precautions for tumors with any atypia or mitotic activity.

Tachykinin-induced responses of rat colonic muscularis mucosae are governed by regional variations in neurokinin receptor subtypes

Tachykinin-induced responses of rat colonic muscularis mucosae are governed by regional variations in neurokinin receptor subtypes

Effects of allopurinol, erythro-9-(2-hydroxy-3-nonyl)adenine and S-(4-nitrobenzyl)-6-thioinosine on the degradation of adenosine 5'-triphosphate in the rat colon muscularis mucosae.

The effects on ATP breakdown of some modulators of adenosine transport or metabolism were studied in the rat colon muscularis mucosae, a tissue which contracts to ATP and is thought to contain P2Y1 receptors. The compounds tested were the xanthine oxidase inhibitor allopurinol, the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and the adenosine uptake blocker S-(4-nitrobenzyl)-6-thioinosine (NBTI). The degradation of adenosine 5'-triphosphate (ATP) (100 microM) and the appearance of metabolites was followed by high pressure liquid chromatography during incubation of isolated tissue preparations alone or in the presence of the drugs, following preincubation with the drugs for 1 h. In the absence of drugs ATP was rapidly degraded by the rat colon muscularis mucosae with a half-life of 6.1 +/- 0.7 min, the major breakdown product being inosine rather than adenosine. Allopurinol (1 microM) and NBTI (10 microM) had no effect on the rate of breakdown of ATP or on the pattern of metabolites produced. EHNA (1 or 10 microM) also had no effect on the half-life of ATP, but in the presence of EHNA (1 microM) the rate of production of inosine was significantly reduced and some adenosine was detected, while in the presence of 10 microm EHNA the production of inosine was abolished and adenosine became the final breakdown product. These results indicate that allopurinol (1 microM) and NBTI (10 microM) have no detectable effect on extracellular purine metabolism in this tissue, and that the build-up of adenosine produced by treatment with EHNA does not have a feedback effect on ATP breakdown.

Ontogeny of adenosine receptors in the longitudinal muscle and muscularis mucosae of the rat distal colon.

The development of adenosine A1 and A2B receptors on the longitudinal muscle and muscularis mucosae of the neonatal rat distal colon has been investigated using homogenate binding, quantitative autoradiography and functional studies. In homogenate binding studies 1,3-[3H]-dipropyl-8-cyclopentylxanthine ([3H]DPCPX) bound with high affinity to A1 receptors in the muscularis mucosae and intact colon from rats aged 10, 15, 20, 25 and 30 days. The affinity of [3H]DPCPX was similar to that in the adult at all ages, but the density of binding sites was higher in the neonatal tissues. Quantitative autoradiography showed a higher density of [3H]DPCPX binding sites in the longitudinal muscle than in the muscularis mucosae at all ages studied (day 10 to adult), and this binding was concentration-dependently displaced by N6-cyclopentyladenosine (CPA). In functional studies the longitudinal muscle relaxed in response to 5'-N-ethylcarboxamidoadenosine (NECA) and CPA at all ages studied (15-30 days), NECA being more potent than CPA. The potency of NECA remained constant and it was antagonised by 1 microM DPCPX at all ages with pA2-values consistent with activation of A2 receptors. The inactivity of 2-[p-(carboxyethyl)-phenylethylamino]-5'-N-ethylcarboxamidoadenosi ne (CGS 21680) indicated that the A2 receptors were of the A2B subtype. The muscularis mucosae contracted in response to CPA at all ages studied (day 15 to adult) and the antagonism by DPCPX (10 nM) were consistent with activation of A1 receptors. In conclusion, binding, autoradiographic and functional studies identified A1 receptors on the rat colon muscularis mucosae at all ages studied. However, while binding and autoradiographic localisation showed the presence of A1 receptors in the longitudinal muscle at all ages studied, functional data only revealed the presence of A2B receptors.

Effects of adenosine 5'-triphosphate, uridine 5'-triphosphate, adenosine 5'-tetraphosphate and diadenosine polyphosphates in guinea-pig taenia caeci and rat colon muscularis mucosae.

The functional effects of adenosine 5'-triphosphate (ATP), uridine 5'-triphosphate (UTP), adenosine 5'-tetraphosphate (AP4) and the diadenosine polyphosphates P1,P3-diadenosine triphosphate (Ap3A), P1,P4-diadenosine tetraphosphate (Ap4A) and P1,P5-diadenosine pentaphosphate (Ap5A) were studied in two isolated smooth muscle preparations thought to contain P2Y (P2Y1) receptors, the guinea-pig taenia caeci (which relaxes to ATP) and the rat colon muscularis mucosae (which contracts to ATP). In addition, the breakdown of these compounds by the rat colon muscularis mucosae was investigated by high pressure liquid chromatography. In the guinea-pig taenia caeci all the purine nucleotides caused relaxation with a potency order of Ap3A=Ap4A> ATP>AP4=Ap5A, and these relaxations were antagonised by suramin with apparent pA2 values in the region of 5, consistent with activation of a P2Y1 receptor. In the rat colon muscularis mucosae the nucleotides caused contraction with a potency order of Ap3A = Ap4A>ATP=AP4 =Ap5A >UTP. However, while suramin (100 microM) inhibited responses to ATP and UTP at all concentrations of agonist, it only inhibited contractions induced by the higher concentrations of AP4, Ap3A and Ap4A and had little effect on contractions induced by Ap5A. A higher concentration of suramin (1 mM) enhanced contractions induced by ATP but greatly inhibited those induced by UTP and had no effect on responses to the other agonists. The A1 adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 10 nM) had no effect on responses to ATP or UTP but inhibited responses to Ap3A, Ap4A, Ap5A and AP4. A combination of suramin (1 mM) and DPCPX (10 nM) almost abolished responses to all the agonists. ATP and UTP were rapidly degraded by the rat colon muscularis mucosae while AP4, Ap3A, Ap4A and Ap5A were degraded more slowly, and the major product detected after breakdown of the purine nucleotides was inosine rather than adenosine. The breakdown of all the nucleotides was inhibited by suramin (1 mM), although this inhibition did not achieve statistical significance in the case of ATP. These results show that while the diadenosine polyphosphates appear to act as P2 agonists in the taenia caeci, in the rat colon muscularis mucosae their major action is via adenosine A1 receptors rather than via P2 receptors. In addition, although they are more stable than ATP or UTP, their action in this tissue is clearly affected by their degradation which complicates the effects of suramin.

Distribution of glial cell line-derived neurotrophic factor mRNA in human colon suggests roles for muscularis mucosae in innervation

Background/Purpose: Glial cell line-derived neurotrophic factor (GDNF) is a ligand for the receptor complex of GDNF family receptor alphas (GFRαs) and Ret receptor tyrosine kinase, the product of a known Hirschsrung's disease gene. The aim of this study was to analyze the mRNA distribution of these genes in the developing human intestine to understand their roles in enteric innervation. Methods: Cryosections of fetal and newborn stomach, ileum, and colon were hybridized in situ with S 35-labeled cRNA probes to GDNF, Ret, GFRα-1 or GFRα-2. GDNF mRNA levels in fetal ileum and colon were compared by reverse transcription-polymerase chain reaction (PCR). Results: GDNF mRNA expression was abundant in the muscularis mucosae of both fetal and newborn colon but was found neither in the neural plexuses nor in other regions of the intestine. Accordingly, by reverse transcription-PCR, GDNF mRNA level was many times higher in colon than ileum. Ret, GFRα-1 and GFRα-2 mRNA were expressed in the ganglionic cells of both myenteric and submucosal plexuses throughout the intestine. Conclusions: The highly restricted distribution of GNDF mRNA suggests an important role for muscularis mucosae in the development of human enteric nervous system. Ret, GFRα-1, and GFRα-2 most likely act as GDNF receptors in colon but may have alternative ligands in other enteric segments.

In vitro evidence that rabbit distal colonic muscularis mucosae has aClostridium difficiletoxin A receptor

In the rabbit ileum Clostridium difficile toxin A causes inflammation and mucosal damage via a specific glycoprotein receptor that contains alpha-D-galactose. In rabbit colon toxin A also causes inflammation, and this is associated with increased myoelectric activity and eicosanoid production. The present in vitro study was undertaken to determine if a toxin A receptor on one or more layers of colonic smooth muscle could mediate the motor effects of this agent. Toxin A (20-100 microg/ml) was without effect on longitudinal and circular muscle but had two different effects on the muscularis mucosae. Initial exposure to the toxin caused increased numbers of spontaneous contractions and a small, atropine-, tetrodotoxin-, and indomethacin-resistant increase in resting tone. More importantly, however, 30-min exposure to toxin A resulted in attenuated muscularis mucosae responses to acetylcholine and K+. Both the small excitatory and the larger inhibitory effects of toxin A were abolished by pretreatment with the lectin BS-1, which binds to toxin A receptors, but not by the nonreceptor-binding lectin DBA. These data strongly suggest that toxin A causes significant motor effects on the distal colonic muscularis mucosae via a receptor-mediated mechanism. These mechanical data were supported by the presence of histologically demonstrable toxin A and BS-1 binding sites on the muscularis mucosae but not on either the longitudinal or circular muscle layers, both of which were unresponsive to the toxin. By depressing muscularis mucosae function and, ultimately, mucosal movement as a result of toxin A production, C. difficile may promote its own proliferation, thus further contributing to the development of antibiotic-associated colitis.

Platelet-derived growth factor (PDGF) induces tyrosine phosphorylation of L-type Ca2+ channels via pp60c-src in rabbit colonic muscularis mucosae (CMM)

Platelet-derived growth factor (PDGF) induces tyrosine phosphorylation of L-type Ca2+ channels via pp60c-src in rabbit colonic muscularis mucosae (CMM)

Types of adrenoreceptors mediating relaxation of rat colonic muscularis mucosae In vitro

Types of adrenoreceptors mediating relaxation of rat colonic muscularis mucosae In vitro

Distribution of several gut neuropeptides and their effects on motor activity in muscularis mucosae of guinea-pig proximal colon

The distribution of peptide-containing nerve fibers and the effect of their neuropeptides on motor activity were studied in the muscularis mucosae of the guinea-pig proximal colon. In the immunohistochemical study, it was shown that tachykinin (TK)-containing nerve fibers densely innervated the muscularis mucosae. In the superfusion study, three kinds of TKs, i.e., neurokinin A (NK-A), neurokinin B (NK-B) or substance P (SP), enhanced the spontaneous activity on the strips of muscularis mucosae with a tetrodotoxin (TTX)-insensitive manner. Their potency was in the rank order of NK-A>NK-B>SP. This suggests that the muscle has a predominant NK2 receptor. Calcitonin gene-related peptide (CGRP)-immunoreactive fibers were commonly observed in the muscle. CGRP induced a potent inhibition on spontaneous activity and a concentration-dependent inhibition on the NK-A-elicited excitation in the presence of TTX, indicating its direct effect on the receptor in the muscle. On the other hand, gastrin releasing peptide (GRP), galanin, neuropeptide Y or somatostatin were more or less immunopositive in nerve fibers, but they had no effect on the motility of the muscle except that GRP sometimes showed a faint increase in spontaneous activity. Neither methionine-enkephalin nor gastrin-17/cholecystokinin was immunoreactive and had any effect on the muscle. These neuropeptides other than TKs and CGRP do not seem to be neuromediators of motor activity of muscularis mucosae. The results suggest the possibility that TK-, especially NK-A- and CGRP-containing neurons, participate in the regulation of motor activity of the muscularis mucosae in the guinea-pig proximal colon.

Tyrosine kinase-dependent modulation of calcium entry in rabbit colonic muscularis mucosae.

We studied the role of tyrosine kinase in the regulation of Ca2+ entry in single smooth muscle cells of the rabbit colonic muscularis mucosae using the whole cell patch-clamp technique. Step depolarization to +10 mV from a holding potential of -60 mV produced inward currents that were abolished by 1 microM nifedipine, consistent with the activation of L-type Ca2+ channels. The tyrosine kinase inhibitors, genistein and tyrphostin B42, dose dependently inhibited these Ca2+ currents. The inactive analogue of tyrphostins, tyrphostin A1, did not affect the currents at concentrations of up to 100 microM. Conversely, the tyrosine phosphatase inhibitor, orthovanadate, enhanced peak Ca2+ currents by 30%. Spontaneous transient outward currents (STOCs) (50-600 pA) were elicited with high K+ in the pipette and at 0-mV holding potential. STOCs were activated due to release of Ca2+ from intracellular stores, required the presence of extracellular Ca2+ concentration, and were insensitive to nifedipine. Genistein abolished STOCs; however, in its presence, outward currents activated by caffeine or carbachol were not affected. The refilling of the Ca2+ stores was studied by first depleting intracellular Ca2+ with carbachol in Ca(2+)-free media followed by reperfusing with a Ca(2+)-containing solution for 3-5 min. Under these conditions, a second application of carbachol evoked an outward current due to Ca2+ release. However, this effect was abolished when the refilling of the stores was carried out in the presence of genistein. Carbachol-evoked currents were not attenuated when the refilling was examined in the presence of orthovanadate. Epidermal growth factor (200 ng/ml) enhanced Ca2+ currents by 60% and markedly increased STOCs by over 200%. Western blot analysis, using an anti-phosphotyrosine antibody, showed a tyrosine phosphorylated protein of 60 kDa in control conditions. This was markedly increased after treatment with epidermal growth factor and carbachol. These results suggest that 1) tyrosine kinase modulates the entry of Ca2+ through L-type channels and through nifedipine-resistant pathways involved in refilling of intracellular stores and 2) stimulation of the kinase by agonists enhances Ca2+ entry in the smooth muscle cells of the rabbit colonic muscularis mucosae.

Characteristics of rat colonic muscularis mucosae responses to inflammatory mediators in vitro: [formula omitted] & LM Anderson, Department of Physiology & Pharmacology, School of Medicine, University of South Dakota, Vermillion, SD 57069

Characteristics of rat colonic muscularis mucosae responses to inflammatory mediators in vitro: [formula omitted] & LM Anderson, Department of Physiology & Pharmacology, School of Medicine, University of South Dakota, Vermillion, SD 57069

Further investigations into adenosine A1 receptor‐mediated contraction in rat colonic muscularis mucosae and its augmentation by certain alkylxanthine antagonists

1. The alkylxanthine antagonists, 8-phenyltheophylline (8-PT), 8-p-sulphophenyltheophylline (8-SPT) and 1,3,7-trimethylxanthine (caffeine) produced rightward displacements of contractile concentration-effect curves to 5'-N-ethylcarboxamidoadenosine (NECA) in rat isolated colonic muscularis mucosae (RCMM) with concentration-ratios consistent with adenosine receptor blockade. The non-xanthine antagonist, 9 fluro-2-(2-furyl)-5,6-dihydro [1,2,4] triazo to [1,5-c]-quinazin-imine (CGS15943A) also antagonized contractions to NECA with an affinity (pKB8.1-8.5) consistent with adenosine A1 receptor blockade. 2. In addition to producing rightward shifts of the concentration-response curves, the maximum contractions to 5'-N-ethylcarboxamidoadenosine (NECA) were also markedly increased in the presence of 8-PT (by 83 +/- 16% at 1 microM), 8-SPT (by 37 +/- 7% at 10 microM) and caffeine (by 45 +/- 5% at 100 microM) but were unaffected by CGS15943A (at 0.01 and 0.03 microM). 3. As with NECA, the maximum contractions to the adenosine A1 receptor agonists R-phenylisopropyladenosine (R-PIA) and N-[(1S, trans)-2-hydroxyclopentyl] adenosine (GR79236) were both antagonized and augmented by 8-PT. In addition, the contractions to NECA in the presence of 8-PT (1 microM) were inhibited by nanomolar concentrations of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). 4. The non-selective phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (1 microM) produced a marked increase in the NECA maximum without producing a rightward shift in the NECA curve, whereas a higher concentration (10 microM) virtually abolished responses. The PDE type III inhibitor,milrinone (1 microM), the type IV inhibitor, rolipram (10 microM), and the type V PDE inhibitor, zaprinast(3 microM), were all without effect on NECA responses in RCMM.5. Partial inhibitions of contractions to NECA were produced by indomethacin (at 3 or 10 micro M) or piroxicam (at 3 microM). Responses to GR79236 were also partially inhibited by indomethacin. In the presence of indomethacin, 8-PT was still able to enhance markedly the maximum contractions obtained to NECA in RCMM.6. The present study has shown that certain alkylxanthine antagonists (but not the non-xanthineCGS15943A) produced a marked augmentation of adenosine Al receptor-mediated contractions inRCMM. The mechanism of this augmentation is, as yet, not known but is unlikely to result from inhibition of PDE. This study has also shown that adenosine Al receptor-induced contractions inRCMM are mediated, in part, via products of the cyclo-oxygenase pathway.

The binding of 1,3-[3H]-dipropyl-8-cyclopentylxanthine to adenosine A1 receptors in rat smooth muscle preparations.

1. The binding of 1,3-[3H]-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX), an antagonist radioligand selective for adenosine A1 receptors, was studied in rat duodenum, colon muscularis mucosae and longitudinal muscle, urinary bladder and vasa deferentia. 2. [3H]-DPCPX bound with high affinity to a single site in all membrane preparations studied with the exception of the rat urinary bladder in which no specific binding was detected. The affinity (Kd) of the binding site for [3H]-DPCPX was similar in all membrane preparations, the colon longitudinal muscle (1.18 +/- 0.47 nM), colon muscularis mucosae (0.84 +/- 0.15 nM), duodenum (1.59 +/- 0.18 nM) and vasa deferentia (0.93 +/- 0.17 nM). The density of [3H]-DPCPX binding sites was similar in the duodenum (38.8 +/- 4 fmol mg-1 protein), muscularis mucosae (43 +/- 3.5 fmol mg-1 protein) and vasa deferentia (43.3 +/- 12.2 fmol mg-1 protein), but in the longitudinal muscle 6-7 fold more binding sites (295 +/- 70 fmol mg-1 protein) were identified. 3. Inhibition studies using DPCPX (0.1-100 nM), N6-cyclopentyladenosine (CPA) (0.1-100 nM), 5'-N-ethylcarboxamidoadenosine (NECA) (2 nM-10 microM) and (R)-N6-phenylisopropyladenosine (R-PIA) (1 nM-1 microM) to displace the binding of [3H]-DPCPX at a concentration around the Kd value (1 nM), demonstrated an order of potency of displacement in all tissues of DPCPX > or = CPA > R-PIA > NECA. This potency order is characteristic of an A1 receptor, indicating that [3H]-DPCPX binds to adenosine A1 receptors in the rat duodenum, colon and vasa deferentia. Two site analysis revealed that the agonists bind to both a high and low affinity state of the receptor.4. The existence of Al binding sites in the rat vasa deferentia, colon muscularis mucosae and duodenum, and their absence in the urinary bladder, is consistent with previous functional studies.However, in contrast to the findings of the [3H]-DPCPX binding assay, no functional response mediated by adenosine Al receptors could be detected by measuring contractile or relaxant responses to CPA in the colon longitudinal muscle. The functional significance of the binding sites in this tissue has therefore yet to be determined.

Comparison of NK‐2 selective peptide and non‐peptide antagonists in rat distal colon muscularis mucosae

Comparison of NK‐2 selective peptide and non‐peptide antagonists in rat distal colon muscularis mucosae