Circular Smooth Muscle Layer Research Articles

5-HT(2B) receptors play a key role in mediating the excitatory effects of 5-HT in human colon in vitro.

1. 5-Hydroxytryptamine (5-HT) is known to produce a number of different effects in the gastrointestinal tract of various species, and has been proposed to play a key role in a number of intestinal disorders in man, including irritable bowel syndrome (IBS), although the receptors involved have yet to be established. The aim of the present study was to investigate the distribution and function of 5-HT(2B) receptors in human colon, and to establish their possible role in the aetiology of IBS. 2. The distribution of 5-HT(2B) receptor mRNA and protein were investigated by quantitative RT - PCR, Western analysis and immunocytochemistry. High levels of both mRNA and protein for 5-HT(2B) receptors were found throughout the human gastrointestinal tract, and in particular in colon, where 5-HT(2B) receptors were found predominantly in the longitudinal and circular smooth muscle layers within the muscularis externa, and in the myenteric nerve plexus lying between these two layers. 3. Electrical field stimulation of longitudinal muscle preparations of human colon mounted in organ baths resulted in neuronally-mediated contractile responses, that were significantly potentiated by application of 5-HT (up to 10(-7) M), with a pEC(50) of 8.2 +/- 0.1 (n=49 donors). The response to 5-HT was inhibited by a number of selective 5-HT(2B) receptor antagonists. 4. This study has shown for the first time that, in contrast to animal studies, the excitatory effects of 5-HT in human colon are mediated by 5-HT(2B) receptors. It is proposed that these receptors contribute to the putative 5-HT-induced colonic smooth muscle hypersensitivity associated with IBS.

Use of an extracorporeal circuit to evaluate effects of intraluminal distention and decompression on the equine jejunum

To use an extracorporeal circuit to evaluate effects of intraluminal distention on the jejunum of healthy horses. 2 jejunal segments from each of 5 horses. Jejunal segments were harvested and maintained in an extracorporeal circuit. One segment was subjected to distention (intraluminal pressure, 25 cm H2O) followed by decompression, and 1 segment was maintained without distention. The influence of distention-decompression on vascular resistance was calculated. Mucosal permeability was evaluated by measuring the clearance of albumin from blood to lumen. After distention and decompression, tissue specimens were collected for histomorphologic evaluation. In addition, the contractile response of the circular smooth muscle layer was determined following incubation with 3 prokinetic agents. Intestinal vascular resistance increased during intraluminal distention and returned to baseline values after decompression. Albumin clearance rate increased after distention, compared with baseline and control values. Histologic examination of the distended segments revealed grade-1 and -2 lesions of the mucosal villus. Edema and hemorrhage were evident in the submucosa and muscular layers. Mesothelial cell loss, edema, and hemorrhage were also evident in the serosa. Mucosal surface area and villus tip height decreased and submucosal volume increased in the distended tissue. Compared with responses in control specimens, distention decreased the contractile response induced by cisapride, erythromycin, and metoclopramide. Intraluminal distention of the jejunum followed by decompression increased mucosal permeability and injury and decreased responses to prokinetic agents. Horses with intraluminal intestinal distention may have a decreased response to prokinetic agents.

Protein expression of phospholipase C in pregnant and nonpregnant rat uterine tissue

Objective: The phosphatidylinositol signaling pathway appears to play a significant role in the intracellular events leading to agonist-stimulated phasic myometrial contractions. The studies described in this report were performed to characterize phospholipase C isoform expression at the protein level and to confirm histologic localization of these proteins within the myometrial smooth muscle layers of the uterus. Methods: For these studies, uterine tissue was obtained from timed- pregnant and spontaneously cycling adult female Sprague-Dawley rats. After isolation of myometrial cell membranes and cytosolic proteins, Western blots were performed by using phospholipase C isoform-specific antibodies. Tissue cross-sections of near-term pregnant rat uterus were used with the phospholipase C isoform-specific antibodies for immunohistochemical studies. Results: The Western blot studies confirmed expression of the phospholipase C-β3, -γ1, -γ2, and -δ1 proteins in both the membrane and cytosolic fractions of rat myometrium; in contrast, only trace amounts of the phospholipase C-β1 protein was observed in this tissue. The immunohistochemical studies demonstrated localization of the phospholipase C-β3, -γ1, -γ2, -δ1 and to a lesser degree phospholipase C-β1 isoforms within the longitudinal and circular smooth muscle layers of the near-term pregnant rat uterus. Conclusion: These studies have confirmed the simultaneous expression of several phospholipase C proteins within the smooth muscle cells of the pregnant and nonpregnant rat uterus, thereby providing support for the possible redundant role of these signal transduction enzymes during the generation of cytosolic calcium oscillations and phasic myometrial contractions. (Am J Obstet Gynecol 2001;185:1191-7.)

Function and localization of oxytocin receptors in the reproductive tissue of rams

Oxytocin is present in the male reproductive tract and has been shown to increase contractility in the epididymis and to modulate steroidogenesis. This study investigated the effects of oxytocin in the testis in vivo, and the presence and cellular localization of oxytocin receptors in the reproductive tract of rams. During the breeding season, mature rams underwent efferent duct ligation before injection of either oxytocin (20 microg) or oxytocin plus an oxytocin antagonist (20 microg) into the testicular artery; the contralateral testicular artery received saline. Injection of oxytocin caused a significant increase (P < 0.05) in the concentration of spermatozoa collected from the rete testis. This effect was not observed after treatment with the oxytocin antagonist plus oxytocin. Western blot analysis performed using a specific oxytocin receptor antibody (020) identified a single immunoreactive band of 66 kDa in testicular and epididymal tissue. This band was present in uterine tissue but not in liver or muscle. Immunocytochemistry identified oxytocin receptors on Leydig and Sertoli cells of the testis, on epithelial cells throughout the epididymis, on peritubular smooth muscle cells in the cauda epididymidis, and on the epithelial cells and circular smooth muscle layer of the ductus deferens. These findings indicate that oxytocin can modulate sperm transport in the ram testis. A role for oxytocin in promoting sperm transit is supported by the localization of oxytocin receptors in the cauda epididymis and ductus deferens, and the presence of receptors on Leydig, Sertoli and epididymal epithelial cells provides further evidence that oxytocin may be involved in the local regulation of steroidogenesis.

Papel da óxido nítrico sintase na etiopatogenia da estenose hipertrófica do piloro na infância

OBJECTIVE: To experimentally reproduce, in rats, the findings corresponding to the histopathology of infantile hypertrophic pyloric stenosis (IHSP), using nitric oxide synthase (NOS) inhibitor (L-NAME). METHODS: L-NAME was administered to pregnant rats (L-NAME group), from the 14th gestational day on in order to reproduce the model of NOS inhibition in the production of IHSP. This group was then compared to control animals. After birth, all the animals in the L-NAME group were maintained under NOS inhibition until the 42nd day of life, when they were sacrificed. The control animals, which did not receive any kind of drug, were also sacrificed on the 42nd day of life. The animals and their internal organs were analyzed and weighed. The pyloric region was technically prepared and observed through light microscopy. RESULTS: The L-NAME group presented lower body and intestinal weight and higher gastric weight than the control group. Light microscopy revealed hypertrophy of the circular smooth muscle layer of the pyloric muscle in L-NAME animals. Conclusions: This work reproduced an experimental model of an IHSP study, confirming the effect of NOS blockade on the pyloric musculature. CONCLUSIONS: this work reproduced an experimental model of an IHSP study, confirming the effect of NOS blockade on the pyloricmusculature.

Role of nitric oxide synthase in the etiopathogenesis of hypertrophic pyloric stenosis in infants

OBJECTIVE: To experimentally reproduce, in rats, the findings corresponding to the histopathology of infantile hypertrophic pyloric stenosis (IHPS), using nitric oxide synthase (NOS) inhibitor (L-NAME). METHODS: L-NAME was administered to pregnant rats (L-NAME group), from the 14th gestational day on in order to reproduce the model of NOS inhibition in the production of IHPS. This group was then compared to control animals. After birth, all the animals in the L-NAME group were maintained under NOS inhibition until the 42nd day of life, when they were sacrificed. The control animals, which did not receive any kind of drug, were also sacrificed on the 42nd day of life. The animals and their internal organs were analyzed and weighed. The pyloric region was technically prepared and observed through light microscopy. RESULTS: The L-NAME group presented lower body and intestinal weight and higher gastric weight than the control group. Light microscopy revealed hypertrophy of the circular smooth muscle layer of the pyloric muscle in L-NAME animals. CONCLUSIONS: This work reproduced an experimental model of an IHPS study, confirming the effect of NOS blockade on the pyloric musculature.

A gap in the murine enteric nervous plexus: A new insight into the neuronal network supplying the colonic circular muscle layer

BACKGROUND. Development of modern gene-targeting technology has made mice an attraCtive species for neurnscience research. The aim was to investigate the functional neuronal input to the colonic circular smooth muscle layer. METHODS. Intracellular recordings of smooth muscle cells (SMCs) of the murine large bowel (caecum, proximal and distal colon) were performed with standard microelectrode technique. Whole-mounts and the complete bowel 3D anatomy of the enteric nervous system (ENS) were stained using NADPH-diaphorase and methylene-blue vital staining. RESULTS.Electrical field stimulation (EFS) of murine large bowel elicited TlX-sensitive inhibitory junction potentials (IJPs) in SMCs of the circular muscle layer which showed characteristic regional differences. In the caecum, EFS evoked monophasic UPs resistant to NG-nitro-L-arginine (L-NNA, IO0/~M). In the proximal and distal colon, UPs consisted of a L-NNA-insensitive fast component followed by a slow component completely blocked by L-NNA (IO0/LM). The slow component was maximal in the oral proximal colon and decreased aborally (16_+2.2 mV, 13.1 _+2.1 mV and 3.8 _+1.5 mV for oral and aboml proximal colon, distal colon, respeCtively, n = 6, p distal colon>caecum. CONCLUSION.The NO-dependent electrical input to the circular muscle layer and the density of nitrergic neurons were maximal in proximal colon and decreased in the aborat direction. The few NADPH-diaphorase-positive neurons in the caecum did not result in NO-depaodent electrical responses in the circular muscle layer. The area devoid of ganglia in the proximal colon did not reveal impaired functional neuronal supply. This region offers the opportunity to investigate the nerve fibers independent of nerve cell bodies.

Perivenous support reduces early changes in human vein grafts: Studies in whole blood perfused human vein segments

Background: Patency of vein grafts in coronary artery bypass grafting procedures is generally less favorable than those of selected arterial grafts. However, vein grafts still are needed in cardiac operations. It would be desirable to find measures to improve the patency of vein grafts next to antithrombotic regimens. Animal studies demonstrated that arterial pressure induces overdistention of the thin-walled vein grafts and that prevention of this overdistention with extravascular support ameliorates the arterialization process with, subsequently, more favorable patency. To evaluate whether perivenous stenting of the rather muscular human vein grafts is also beneficial, we designed an in vitro model to study the early effects of perivenous support in human vein grafts. Methods: Seven paired segments of human vein graft obtained during coronary artery bypass grafting procedures were placed in a perfusion circuit and perfused simultaneously with autologous whole blood, with a pressure of 60 mm Hg (nonpulsatile flow). After 30 minutes of perfusion, one segment, and after 60 minutes of perfusion, the remaining segment were taken for histologic and immunohistochemical examination. In the next experiments 7 segments of human vein graft were placed in the circuit and supported with a polytetrafluoroethylene graft to prevent overdistention with 7 unstented segments as controls. Results: In unsupported vein grafts perfused with autologous blood under a pressure of 60 mm Hg, a complete de-endothelialization was shown after 1 hour of perfusion. In the study vein grafts, with a perivenous polytetrafluoroethylene graft preventing overdistention (n = 7), the endothelium remained intact. Electron microscopic investigation of the media showed severe damage in the circular smooth muscle layer in the unstented group, whereas in the stented group almost no injury was found. Conclusion: In our in vitro closed-loop model, reproducible vessel wall changes were observed in all human vein graft specimens studied. The beneficial effect of perivenous support could also be established for the human greater saphenous vein, providing a basis for clinical application. (J Thorac Cardiovasc Surg 2001;121:290-7)

A Light Microscope Study of the Distribution of Muscle in the Frog Esophagus and Stomach.

The present study reports light microscopical observations of the distribution of muscle in the esophagus and stomach of both the bull frog (Rana catesbeiana) and the African clawed frog (Xenopus laevis). The external muscle coat of the upper half of the esophagus in both species had several collagen coated bundles of striated muscle fibres around the circumference. These striated muscle bundles ran longitudinally from the pharynx to around the vicinity of the center of the esophagus. Beneath these striated muscle bundles was an inner circular layer of smooth muscle. In both species, the inner circular layer of smooth muscle was particularly thick in the region close to the pharynx. In the bull frog, the lower half of the esophagus lacked striated muscle. However, the circular smooth muscle layer, extending from the upper half of the esophagus, was also observed throughout the lower half of the esophagus. An outer longitudinal layer of smooth muscle developed towards the terminal portion of the esophagus such that in this region, both outer longitudinal and inner circular layers of smooth muscle were observed. Similarly in the African clawed frog, the inner circular layer of smooth muscle was continuous along the full length of the esophagus. Again, no striated muscle bundles were observed in the lower half of the esophagus. However, the outer longitudinal layer of smooth muscle was seen to develop in the middle region of the esophagus. Its muscle layer extended to the terminal portion of the esophagus. Thus, both outer longitudinal and inner circular layers of smooth muscle were observed throughout the lower half of the esophagus. In both frogs, the thickness of the outer longitudinal and inner circular layers of smooth muscle changed before and after the esophago-gastric junction. In both frogs, no muscularis mucosa was observed in the esophageal wall. However, in the lower half of the esophagus of the African clawed frog, small bundles of smooth muscle were observed here and there in the submucosa. A fully developed muscularis mucosa with both outer longitudinal and inner circular layers was observed in the upper stomach of both frogs.

Transmission of Rectal Electric Waves: Is it through Circular or Longitudinal Smooth Muscle Layers or Both?

The rectum possesses electric activity in the form of pacesetter (PPs) and action potentials (APs). In recent studies we suggested that the waves are not initiated by the extrarectal autonomic innervation but might be triggered by a ‘rectosigmoid pacemaker’ and are transmitted in the rectal wall through the rectal musculature and not the enteric nerve plexus. To investigate whether the rectal waves are transmitted through the circular or longitudinal muscle layer, the rectum of 18 mongrel dogs was exposed under anesthesia through an abdominal incision. Three electrodes were applied to the rectal wall (longitudinal muscle layer) and another 3 electrodes to the circular muscle; the latter was exposed by splitting apart the fibers of the longitudinal muscle. Rectal electric activity and pressure were recorded from the 6 electrodes before and after performing individual myotomy of the rectal longitudinal (9 dogs), circular (9 dogs), and then the whole muscle layers (18 dogs). The myotomy was performed proximal to and between the electrodes. Pacesetter (PPs) and action potentials (APs) were recorded from the 3 electrodes on the longitudinal muscle but no waves were registered from those on the circular muscle. After longitudinal muscle myotomy was performed between electrodes 1 and 2, PPs and APs were recorded from electrode 1 but not 2 and 3 and when performed proximally to electrode 1, no waves were registered. The rectal pressure increased concomitantly with occurrence of APs. Circular muscle myotomy effected no change in the rectal electric activity recorded from the 3 electrodes applied to the longitudinal muscle. In total muscle myotomy, the electric waves were recorded from the electrodes proximal but not distal to the myotomy. We propose that the motile activity of the rectal longitudinal muscle is initiated by the electric activity which appears to be triggered by the rectosigmoid pacemaker, while that of the circular muscle fibers is believed to be initiated by the stretch reflex induced by rectal distension. This concept is evidenced not only by the current findings but also by the histologic structure of the rectal musculature being of the unitary type of smooth muscles.

Properties of gastric smooth muscles obtained from mice which lack inositol trisphosphate receptor.

Membrane potential recordings, made from the circular smooth muscle layer of the gastric antrum taken from mutant mice which lacked the inositol trisphosphate (InsP3) type 1 receptor, were compared with those obtained from the stomach of control (wild-type) mice. Immunostaining of gastric muscles indicated that the distribution and form of c-kit positive cells were similar in wild-type and mutant mice. Smooth muscles from wild-type mice generated slow waves that in turn initiated spike potentials, while those from mutant mice were either quiescent or generated irregular bursts of spike potentials. In the presence of nifedipine, slow waves with reduced amplitude were generated in wild-type mice, while all electrical activity was abolished in mutant mice. Acetylcholine depolarized and sodium nitroprusside hyperpolarized the membrane in muscles from both types of mice, being more effective in wild-type mice. Noradrenaline produced similar hyperpolarizations in both types of mice. Transmural nerve stimulation evoked inhibitory junction potentials (IJPs) in both wild-type and mutant mice. In wild-type mice, the IJPs were reduced in amplitude by nitroarginine and converted to a cholinergic excitatory junction potential (EJP) by apamin. In mutant mice, the IJPs were unaffected by nitroarginine or atropine but were abolished by apamin. It is concluded that in antral smooth muscle, the expression of InsP3 type 1 receptors may be causally related to the generation of slow waves but not to the generation of action potentials. A lack of InsP3 receptors attenuates cholinergic excitatory and nitrergic inhibitory responses but does not alter the response to noradrenaline.

Selective changes in the G-protein subtypes expression in the antral circular smooth muscle of the diabetic rat

Gastroparesis is a common complication in diabetes. Previous studies indicate a change in signal transduction pathway of antral smooth muscle response to excitatory neurotransmitters such as acetylcholine or neurokinins. Changes in the expression of G proteins in different organs of diabetic animals have been described but there is no data on the stomach in diabetes. Aim: To determine the expression of the G-protein subtypes in the antral circular smooth muscle from STZ-diabetic rats over time. Methods: The circular smooth muscle layer was separated from the antrum of 3 rats at each time point at 2, 4 and 8 month of STZ-induced diabetes (and from 3 littermate controls at each time point). Tissue was homogenized, ultracentrifuged at 40,000 rpm and solubilized to prepare membrane fractions. Western blots of 100 /-Lg samples of purified tissue were probed using specific antibodies to G alpha protein subunits: Gai2 (41.7 kD), Gas (48.5 kD) and Gao (41.2 kD) proteins. Tests were done in duplicate or triplicate. Results: There was a decrease in the expression of Gao in diabetic antral circular smooth muscles compared to the control, at 2, 4 and 8 months: 69.3±0.5% of control (p<O.01), 85.1± 16% (NS) and 73.9±3.4% (p<O.01), respectively. The expression of Gai2 was significantly decreased at 8 month (61.9±8%, p<0.05). There was no difference in the expression of the Gas at either point. Conclusions: There are selective changes in G protein subtype expression in diabetic antral circular smooth muscle over time. These changes may contribute to the altered contractile smooth muscle response which may in turn contribute to diabetic gastroparesis.

Decreased interstitial cell of Cajal volume in patients with slow-transit constipation

Background & Aims: The cause of slow-transit constipation is incompletely understood. Recent observations suggest a central role for interstitial cells of Cajal in the control of intestinal motility. The aim of this study was to determine the volume of interstitial cells of Cajal in the normal sigmoid colon and in the sigmoid colon from patients with slow transit constipation. Methods: Sigmoid colonic samples were stained with antibodies to protein gene product 9.5, c-Kit, and α–smooth muscle actin. Three-dimensional reconstruction of regions of interest was performed using consecutive images collected on a laser scanning confocal microscope and ANALYZE software. Results: Volume of interstitial cells of Cajal was significantly decreased in all layers of sigmoid colonic specimens from patients with slow-transit constipation compared with normal controls. Neuronal structures within the colonic circular smooth muscle layer were also decreased. Conclusions: A decrease in the volume of interstitial cells of Cajal may play an important role in the pathophysiology of slow-transit constipation. GASTROENTEROLGY 2000;118:14-21

Development of interstitial cells of Cajal and pacemaking in mice lacking enteric nerves

Background & Aims: Development of interstitial cells of Cajal (ICC) requires signaling via Kit receptors. Kit is activated by stem cell factor (SCF), but the source of SCF in the bowel wall is unclear and controversy exists about whether enteric neurons express the SCF required for ICC development. Methods: Glial cell line–derived neurotrophic factor (GDNF) knockout mice, which lack enteric neurons throughout most of the gut, were used to determine whether neurons are necessary for ICC development. ICC distributions were determined with Kit immunofluorescence, and function of ICC was determined by intracellular electrical recording. Results: ICC were normally distributed throughout the gastrointestinal tracts of GDNF−/− mice. Intracellular recordings from aganglionic gastrointestinal muscles showed normal slow wave activity at birth in the stomach and small intestine. Slow waves developed normally in aganglionic segments of small bowel placed into organ culture at birth. Quantitative polymerase chain reaction showed similar expression of SCF in the muscles of animals with and without enteric neurons. Expression of SCF was demonstrated in isolated intestinal smooth muscle cells. Conclusions: These data suggest that enteric neurons are not required for the development of functional ICC. The circular smooth muscle layer, which develops before ICC, may be the source of SCF required for ICC development. GASTROENTEROLOGY 1999;117:584-594

Localization of bradykinin B2 receptors in the endometrium and myometrium of rat uterus and the effects of estrogen and progesterone.

In the uterus, bradykinin is a potent inducer of smooth muscle contraction, which is mediated by the bradykinin B2 receptor subtype. However, little is known about the distribution or regulation of this receptor in this tissue. The aim of this study was to localize the B2 receptor in the uterus and determine whether the levels of this receptor were altered during the estrous cycle and modulated by estrogen and/or progesterone in ovariectomized rats. At diestrus, uterine B2 receptors were localized to both the circular and longitudinal smooth muscle layers of the myometrium, the endometrial stroma, the glandular epithelium, and the layer subjacent to the luminal epithelium. B2 receptor levels in both myometrium and endometrium were lowest during early proestrus, when estrogen levels are low, whereas myometrial B2 receptor protein and messenger RNA levels were highest during late proestrous, when estrogen levels peak. Similar findings were observed for the estrogen-supplemented group after ovariectomy, with progesterone appearing to inhibit the estrogen-induced rise in bradykinin B2 receptor density in estrogen/progesterone-treated animals. Using in vitro receptor autoradiography employing the specific B2 receptor antagonist analog, HPP-HOE140, immunostaining with specific antipeptide antibodies generated against the B2 receptor, and in situ hybridization using a specific bradykinin B2 receptor riboprobe, our findings show a discrete distribution of the bradykinin B2 receptor throughout the different layers of the uterus and suggest that bradykinin B2 receptor levels in the rat uterus are regulated by estrogen, and possibly progesterone, in both myometrium and endometrium.

Developmental Expression and Cellular Origin of the Laminin α2, α4, and α5 Chains in the Intestine

Laminins are extracellular matrix glycoproteins that are involved in various cellular functions, including adhesion, proliferation, and differentiation. In this study, we examine the expression patterns and the cellular origins of the laminin α2, α4, and α5 chains in the developing mouse intestine and inin vitromouse/chick or chick/mouse interspecies hybrid intestines.In situhybridization and Northern blot analysis revealed that mRNA levels for all three laminin α chains are highest in the fetal intestine undergoing intense morphogenetic movements. Laminin α4 mRNA and polypeptide are associated with mesenchyme-derived cell populations such as endothelium and smooth muscle. In contrast, laminin α2 and α5 chains participate in the structural organization of the subepithelial basement membrane and, in the mature intestine, show a complementary pattern of expression. All three laminin α chains occur in the smooth muscle basement membrane, with a differential expression of laminin α5 chain in the circular and longitudinal smooth muscle layers. The cellular origin of laminin α2 and α5 chains found in the subepithelial cell basement membrane was studied by immunocytochemical analysis of mouse/chick or chick/mouse interspecies hybrid intestines at various stages of development using mouse-specific antibodies. Laminin α2 was found to be deposited into the basement membrane exclusively by mesenchymal cells, while the laminin α5 chain was deposited by both epithelial and mesenchymal cells in an apparently developmentally regulated pattern. We conclude that (1) multiple laminin α chains are expressed in the intestine, implying specific roles for individual laminin isoforms during intestinal development, and (2) reciprocal epithelial/mesenchymal interactions are essential for the formation of a structured subepithelial basement membrane.

Regenerative potentials evoked in circular smooth muscle of the antral region of guinea-pig stomach.

1. Slow waves recorded from the circular smooth muscle layer of guinea-pig antrum consisted of two components, an initial component and a secondary regenerative component. Whereas both components persisted in the presence of nifedipine, the secondary component was abolished by a low concentration of caffeine. 2. Short segments of single bundles of circular muscle were isolated and impaled with two microelectrodes. Depolarizing currents initiated regenerative responses which resembled those initiated during normal slow waves. These responses had partial refractory periods of 20-30 s and were initiated about 1 s after the onset of membrane depolarization. 3. The regenerative responses persisted in the presence of either nifedipine or cobalt ions but were abolished by caffeine, BAPTA or cyclopiazonic acid. 4. The observations suggest that depolarizing membrane potential changes trigger the release of Ca2+ from intracellular stores and this causes a depolarization by activating sets of unidentified ion channels in the membranes of smooth muscle cells of the circular layer of guinea-pig antrum.

Histological studies of the dorsal nasal, angularis oculi, and facial veins of sheep (Ovis aries).

Selective brain cooling (SBC) requires vasoactivity in the superficial veins of the face of the animal. This vasoactivity is possible because of an adequate amount of smooth muscle in the tunica media of each of these superficial vessels, enabling it to act as a "muscle sphincter". In this study, the angularis oculi, dorsal nasal, distal, and proximal parts of the facial veins in sheep were examined histologically to describe an anatomical basis for SBC. Measurements of the tunica media thickness, the lumen diameter, and the ratio of these measurements showed that the relative tunica media thicknesses in the angularis oculi vein and the dorsal nasal vein are statistically smaller (P < 0.001) than in the distal or the proximal parts of the facial vein. In the angularis oculi, dorsal nasal, and distal part of the facial vein, the tunicae mediae were composed of five to seven circularly arranged smooth muscle layers, suggesting their ability to vasoconstrict. The proximal part of the facial vein possesses both circularly and longitudinally arranged smooth muscle layers. The circular smooth muscle layers suggest a vasoconstrictory function, whereas the longitudinal smooth muscle layers suggest a vasoconstrictory function in this part of the facial vein. Both the dorsal nasal and the proximal part of the facial vein, but not the angularis oculi or the distal part of the facial vein, possess endothelial valves near their confluences with other veins. It was concluded from this study that the angularis oculi and the distal part of the facial vein vasoconstrict, whereas the proximal part of the facial vein vasodilates, enabling the necessary changes in blood flow in SBC.

Spatial pattern of smooth muscle differentiation is specified by the epithelium in the stomach of mouse embryo.

Smooth muscle induction in the stomach of mouse embryo was analyzed experimentally by tissue recombination and organ culture technique. When observed immunohistochemically with anti-alpha-smooth muscle actin antibody, smooth muscle myoblasts first differentiated and formed the circular muscle layer at 13 days of gestation. The longitudinal muscle layer appeared at 15 days in the forestomach and at neonatal period in the glandular stomach. The lamina muscularis mucosae was observed by the neonatal period. When the undifferentiated 11-day stomach was cultivated in vitro for 3 days, an alpha-smooth muscle actin-positive cell layer was formed and the explant exhibited rhythmical contraction. The mesenchyme alone remained as an undifferentiated cell mass. When the mesenchyme was recombined with the epithelium, a distinct smooth muscle layer was formed. These demonstrated that the alpha-smooth muscle actin expression was induced by the epithelium. Next, we examined by heterologous recombination the roles of the epithelium in formation of the organ-specific spatial pattern of smooth muscle differentiation. The stomach mesenchyme recombined with the tracheal epithelium exhibited a partially tracheal type of alpha-smooth muscle actin expression. The uterine epithelium induced only a small number of smooth muscle myoblasts and the spatial organization was disordered. Finally, we assayed if the mode of smooth muscle induction by the stomach epithelium changes during development. Heterochronic recombination proved that the 14-day forestomach epithelium induced a muscularis mucosae-like smooth muscle layer that was contiguous to the epithelium, whereas the 11-day stomach and the 13-day forestomach epithelium induced a circular muscle-like smooth muscle layer that was distant from the epithelium. As a whole, our data suggest that the spatial pattern of smooth muscle differentiation is regulated by organ-specific and stage-specific influences from the epithelium.

Increased insulin-like growth factor-I mRNA expression in pyloric muscle in infantile hypertrophic pyloric stenosis.

The etiology of infantile hypertrophic pyloric stenosis (IHPS) is unknown. Insulin-like growth factor-I (IGF-I) is a polypeptide hormone that elicits various biological activities (cellular growth, replication, and differentiation) by binding to its receptors. IGF-I has been suggested to play an important role in both gastrointestinal (GI) maturation and smooth-muscle-cell (SMC) hypertrophy. Full-thickness muscle biopsy specimens were obtained from 8 IHPS patients (age range 14-64 days, mean 28.1 days) at pyloromyotomy and from 8 age-matched controls (15-60 days, mean 33.8 days) without GI disease at autopsy. In-situ hybridization was performed using an IGF-I-specific and digoxigenin (DIG)-labeled oligonucleotide probe and visualized by nitroblue tetrazolium staining. In normal controls, IGF-I mRNA expression was absent or weak in both circular and longitudinal smooth-muscle layers of pyloric muscle. In contrast, the pyloric muscle in IHPS patients demonstrated strong IGF-I mRNA expression in the circular smooth-muscle layer and moderate expression in the longitudinal smooth-muscle layer. The increase in IGF-I mRNA in pyloric muscle in IHPS suggests that SMCs are actively synthesizing IGF-I, contributing to the development of pyloric muscle hypertrophy.