Messenger RNA Levels and Binding Sites of Muscarinic Acetylcholine Receptors in Gastrointestinal Muscle Layers from Healthy Dairy Cows

Acetylcholine interacts with muscarinic receptors (M) to mediate gastrointestinal (GI) smooth muscle contractions. We have compared mRNA levels and binding sites of M₁to M₅ in muscle tissues from fundus abomasi, pylorus, ileum, cecum, proximal loop of the ascending colon (PLAC), and external loop of the spiral colon (ELSC) of healthy dairy cows. The mRNA levels were measured by quantitative RT-PCR. The inhibition of [³H]-QNB (1-quinuclidinyl-[phenyl-4-³H]-benzilate) binding by M antagonists [atropine (M_{1 ? 5}), pirenzepine (M₁), methoctramine (M₂), 4-DAMP (M₃), and tropicamide (M₄)] was used to identify receptors at the functional level. Maximal binding (B_max) was determined through saturation binding with atropine as a competitor. The mRNA levels of M₁, M₂, M₃, and M₅ represented 0.2, 48, 50, and 1.8%, respectively, of the total M population, whereas mRNA of M₄ was undetectable. The mRNA levels of M₂ and of M₃ in the ileum were lower (P < 0.05) than in other GI locations, which were similar among each other. Atropine, pirenzepine, methoctramine, and 4-DAMP inhibited [³H]-QNB binding according to an either low- or high-affinity receptor pattern, whereas tropicamide had no effect on [³H]-QNB binding. The [³H]-QNB binding was dose-dependent and saturable. B_max in fundus, pylorus, and PLAC was lower (P < 0.05) than in the ELSC, and in the pylorus lower (P < 0.05) than in the ileum. B_max and mRNA levels were negatively correlated (r = -0.3; P < 0.05). In conclusion, densities of M are different among GI locations, suggesting variable importance of M for digestive functions along the GI tract.     

雌激素和孕激素对不同发情方式小鼠子宫内膜中孕激素受体分布的影响

目的探讨雌（Estrogen,E2）、孕激素（Progesterone,P4）对同期发情与自然发情小鼠子宫内膜中孕激素受体（Progesterone receptor,PR）分布的影响。方法45只同日龄雌鼠,根据处理方式的不同随机分为5组：自然发情组（对照组）、同期发情组、卵巢摘除组、P4处理组和E2处理组,5组小鼠在见栓后第4、6、8天分别取样后,采用免疫组织化学法观察小鼠子宫内膜中PR的分布变化情况。结果免疫组织化学染色结果显示,5个处理组小鼠子宫内膜的三种细胞中都有PR存在;同期发情组小鼠子宫内膜中三种类型细胞PR的表达与自然发情组差异有显著性（P〈0.05）;P4处理组小鼠子宫内膜中三种类型细胞PR的表达在见栓第4、6天显著低于卵巢摘除组（P〈0.05）;E2处理组小鼠子宫内膜腺上皮和间质中PR在第4、6、8天时都显著高于卵巢摘除组（P〈0.05）,而在腔上皮中则显著低于卵巢摘除组（P〈0.05）。结论同期发情处理与自然发情小鼠的子宫内膜上PR的分布,都受E2和P4的特异诱导而变化。     

Further observations on upper urinary tract smooth muscle

Summary Light and electron microscopic techniques have been employed to study the arrangement and distribution of two types of muscle in the upper urinary tract of the rat. An outer layer of cells has been identified in the wall of the renal calix and pelvis. These cells are separated by connective tissue but possess numerous processes which make close contacts with adjacent cells. A layer of similar cells has not been observed in the wall of the upper ureter. The inner layer of muscle in the calix and pelvis is composed of larger cells similar to and apparently continuous with ureteric muscle. These cells are closely related to one another without intervening connective tissue and possess numerous bundles of myofilaments which extend along the length of the cell. The two types of muscle are closely related and, in the junctional region, cells of the outer layer are arranged along the length and make close contacts with one or more of the inner smooth muscle cells. A quantitative estimation has been made of nerve bundles associated with smooth muscle forming the outer layer of the calix and pelvis and with the muscle of the ureter. The results have shown a five fold increase in nerves associated with the caliceal muscle when compared with the ureter. The results are discussed in relation to the concept of a ureteric pacemaker.The authors wish to thank Professor G. A. G. Mitchell for his useful advice and encouragement.     

Action of FMRFamide-like Peptides on Porcine Gastrointestinal Motility In Vitro

Decker, B., B. Vadokas, U. Kutschenreuter, K. Golenhofen, K. Voigt, G. P. Mcgregor and K. Mandrek. Action of FMRFamide-like peptides on porcine gastrointestinal motility in vitro. Peptides 18(10) 1531–1537, 1997.—Mechanical activity was recorded in circular and longitudinal smooth muscle preparations isolated from extensive regions of the porcine gastrointestinal tract in response to the FMRFamide-like neuropeptides F8Famide and A18Famide. In all preparations, the peptides were about equipotent in producing phasic contractions or enhancing spontaneous activity. The most prominent responses were observed in jejunal longitudinal strips which were on the average 91% (±4% SEM, n = 15; 10⁻⁶ M) of the histamine (10⁻⁵ M) responses. The peptide-induced phasic activity was completely abolished by nifedipine but was unaffected by tetrodotoxin, atropine, phentolamine, yohimbine, phenoxybenzamine, propranolol, methysergide, cimetidine, indomethacin, levallorphane or naloxone. Both peptides enhanced acetylcholine-induced contractions. However, bovine ileum and guinea-pig taenia coli was not affected by these peptides. The results indicate that F8F- and A18F-amide contract porcine gastrointestinal smooth muscle by acting directly via non-opioid receptors on L-type calcium channels. In addition an increase of the sensitivity to cholinergic stimulation occurs.     

Development of Novel Biodegradable Polymeric Nanoparticles-in-Microsphere Formulation for Local Plasmid DNA Delivery in the Gastrointestinal Tract

There is a critical need for development of novel delivery systems to facilitate the translation of nucleic acid-based macromolecules into clinically-viable therapies. The aim of this investigation was to develop and evaluate a novel nanoparticles-in-microsphere oral system (NiMOS) for gene delivery and transfection in specific regions of the gastrointestinal (GI) tract. Plasmid DNA, encoding for the enhanced green fluorescent protein (EGFP-N1), was encapsulated in type B gelatin nanoparticles. NiMOS were prepared by further protecting the DNA-loaded nanoparticles in a poly(epsilon-caprolactone) (PCL) matrix to form microspheres of less than 5.0 μm in diameter. In order to evaluate the biodistribution following oral administration, radiolabeled (¹¹¹In-labeled) gelatin nanoparticles and NiMOS were administered orally to fasted Balb/C mice. The results of biodistribution studies showed that, while gelatin nanoparticles traversed through the GI tract fairly quickly with more than 54% of the administered dose per gram localizing in the large intestine at the end of 2 h, NiMOS resided in the stomach and small intestine for relatively longer duration. Following oral administration of EGFP-N1 plasmid DNA at 100 μg dose in the control and test formulations, the quantitative and qualitative results presented in this study provide the necessary evidence for transfection potential of NiMOS upon oral administration. After 5 days post-administration, transgene expression in the small and large intestine of mice was observed. Based on these results, NiMOS show significant potential as novel gene delivery vehicle for therapeutic and vaccination purposes.     

Viscoelastic properties of isolated rat colon smooth muscle cells

The measurement of the biomechanical properties of gastrointestinal smooth muscle cells is important for the basic understanding of digestive function and the interaction of muscle cells with the matrix. Externally applied forces will deform the cells depending upon their mechanical properties. Hence, the evoked response mediated through stretch-sensitive ion-channels in the smooth muscle cell membrane will depend upon membrane properties and the magnitude of the external force. The aim of this study was to test the hypothesis that gastrointestinal smooth muscle cells behave in a viscoelastic manner. Smooth muscle cells were dissociated from the muscle layers of the descending colon. The viscoelastic properties of the isolated cells were characterized by measuring the mechanical deflection response of the cell membrane to a negative pressure of 1cm H(2)O applied across the cell through a micropipette and fitting the response to a theoretical viscoelastic solid model. The viscoelastic mechanical constants of the isolated cells (N=9) were found to be as follows: k(1)=19.99+/-2.86 Pa, k(2)=7.19+/-1.21 Pa, mu=25.36+/-6.14 Pas and tau=4.84+/-0.95 s. This study represents, to the best of our knowledge, the first quantitative mechanical properties of isolated living smooth muscle cells from the gastrointestinal tract. The mechanical properties determined in this study will be of use in future analytical and numerical smooth muscle cell models to better predict the mechanism between the magnitude of mechanical stimuli, mechanosensitivity and the evoked afferent responses.     

Gastrointestinal stress as innate defence against microbial attack

The human gastrointestinal (GI) tract has been bestowed with the most difficult task of protecting the underlying biological compartments from the resident commensal flora and the potential pathogens in transit through the GI tract. It has a unique environment in which several defence tactics are at play while maintaining homeostasis and health. The GI tract shows myriad number of environmental extremes, which includes pH variations, anaerobic conditions, nutrient limitations, elevated osmolarity etc., which puts a check to colonization and growth of nonfriendly microbial strains. The GI tract acts as a highly selective barrier/platform for ingested food and is the primary playground for balance between the resident and uninvited organisms. This review focuses on antimicrobial defense mechanisms of different sections of human GI tract. In addition, the protective mechanisms used by microbes to combat the human GI defence systems are also discussed. The ability to survive this innate defence mechanism determines the capability of probiotic or pathogen strains to confer health benefits or induce clinical events respectively.     

Gastrointestinal zygomycosis: a report of three cases

Three cases of gastrointestinal zygomycosis, probably caused by Basidiobolus ranarum, are described. The diagnosis was based on morphology of the fungal elements in infected tissues and histopathologic findings. All the three patients responded favorably to management strategy that included surgical resection of the infected portion of the bowel and institution of specific antifungal therapy.     

Expression pattern of the homeotic gene Bapx1 during early chick gastrointestinal tract development

Regulation of the Bone Morphogenetic Protein (BMP) signaling pathway is essential for the normal development of vertebrate gastrointestinal (GI) tract, but also for the differentiation of the digestive mesenchymal layer into smooth muscles and submucosal layer. Different studies demonstrated that Bapx1 (for bagpipe homeobox homolog 1) negatively regulates the BMP pathway, but its precise expression pattern during the development and the differentiation of the GI tract mesenchyme actually remains to be examined. Here, we present the spatio-temporal expression profile of Bapx1 in the chick GI tract. We show that Bapx1 is first expressed in the undifferentiated mesenchyme of the gizzard and the colon. After the differentiation of the digestive mesenchyme, we found Bapx1 strongly expressed in the gizzard smooth muscle and in the submucosa layer of the colon. This expression pattern provides new insights into the roles of Bapx1 during the regionalization of the GI tract and the differentiation of the digestive mesenchyme of the colon and the stomach.     

Expression pattern of the homeotic gene Bapx1 during early chick gastrointestinal tract development

Regulation of the Bone Morphogenetic Protein (BMP) signaling pathway is essential for the normal development of vertebrate gastrointestinal (GI) tract, but also for the differentiation of the digestive mesenchymal layer into smooth muscles and submucosal layer. Different studies demonstrated that Bapx1 (for bagpipe homeobox homolog 1) negatively regulates the BMP pathway, but its precise expression pattern during the development and the differentiation of the GI tract mesenchyme actually remains to be examined. Here, we present the spatio-temporal expression profile of Bapx1 in the chick GI tract. We show that Bapx1 is first expressed in the undifferentiated mesenchyme of the gizzard and the colon. After the differentiation of the digestive mesenchyme, we found Bapx1 strongly expressed in the gizzard smooth muscle and in the submucosa layer of the colon. This expression pattern provides new insights into the roles of Bapx1 during the regionalization of the GI tract and the differentiation of the digestive mesenchyme of the colon and the stomach.     

长爪沙鼠血清雌性激素的比较研究

目的测定长爪沙鼠、NIH小鼠和SD大鼠的血清雌二醇(E2)和孕酮(P)水平,为研究长爪沙鼠的生殖和胚胎工程提供基础资料。方法用放射免疫分析法测定上述动物生产前后血清雌二醇(E2)和孕酮(P)水平,进行统计学处理和分析。结果不同阶段雌性沙鼠E2值差异无显著性(P>0.05),P值差异有显著性(P<0.01);E2值比较,处女期沙鼠与同期NIH和SD差异有显著性(P<0.01),而在经产期的动物间水平接近(P>0.05);P值比较,处女期沙鼠与NIH接近,与SD差异有显著性(P<0.01);而经产期沙鼠在三种动物中是最高的(P<0.01)。结论长爪沙鼠血清E2、P的含量具种属特异性,并随动物的生理发育时期而变化。     

Alterations of Gastrointestinal Motility in Obesity

All nutrients are absorbed in the gastrointestinal (GI) system, and GI motility plays a very critical role in the consumption of foods, digestion, and absorption of nutrients. Various segments of the GI tract (esophagus, stomach, and intestines) coordinate in a complex yet precise way to control the process of food consumption, digestion, and absorption of nutrients. GI motility not only regulates the rates at which nutrients are processed and absorbed in the gut but also participates in the control of appetite and satiety. Altered GI motility has been associated with various disease conditions (gastroparesis, etc.) and has been frequently observed in obese patients. The significance of these GI motility alterations in obesity is not fully understood, but they have been considered as potential contributing factors in the development and maintenance of obesity and changed eating behavior. Therapies aimed at regulating GI motility are being actively explored and applied clinically for the management of obese patients. To better understand the pathophysiology of obesity, we systematically reviewed GI motility changes observed in obese conditions. The relationship and pathological significance of these findings, as well as the potential therapies by modification of GI motility, are also discussed.     

恒河猴血清孕酮含量测定方法

目的建立恒河猴血清中孕酮含量测定方法。方法本文采用放射免疫测定技术。结果孕酮的回收率为94％,批内CV为5．1％～8．3％,批间CV为4．5％～7．7％,灵敏度为5～10Pg。说明该方法具有较高的灵敏度、特异性、准确性。分别测定了幼年组、成年组和老年组的雌性恒河猴的血清中的孕酮含量分别为：（0．20±0．04）ng／mL、（6．26±0．17）ng,mL和（0．35±0．06）ng/mL;成年雌性恒河猴月经周期孕酮的变化范围为：滤泡期为（1．10±0．12）ng／mL,排卵期（2．36±0．18）ng/mL,黄体期（6．17±0．15）ng/mL,妊娠期随着妊娠月份的增加,孕酮浓度也增加,最高可达50ng／mL。结论经实验验证,该方法灵敏、可靠、适用,可作为恒河猴血清中孕酮含量测定的一种方法。     

碱性调宁蛋白的研究进展

碱性调宁蛋白是一个首先从鸡砂囊和牛主动脉中分离出的相对分子质量为34×103的碱性蛋白。它在平滑肌中特异表达,结合钙调蛋白,肌动蛋白,肌球蛋白,抑制肌球蛋白的ATP酶活性,参与平滑肌收缩、细胞信号转导、维持细胞骨架、抑制细胞增生等。     

Progesterone and progestins: effects on brain, allopregnanolone and beta-endorphin

The increased use of hormonal therapies over the last years has led to improve the knowledge of pharmacological, biochemical and metabolic properties of several progestins and their effects in target tissues, such as the central nervous system. Progesterone and synthetic progestational agents are able to modulate the synthesis and release of several neurotransmitters and neuropeptides in response to specific physiological and pathological stimuli. While these actions may relay on differential activation of progesterone receptor or recruitment of intracellular pathways, some of the differences found between synthetic progestins may depend on the specific conversion to neuroactive steroids, such as the 3-, 5- reduced metabolite, allopregnanolone. This is a potent endogenous steroid that rapidly affects the excitability of neurons and glia cells through direct modulation of the GABA-A receptors activity exerting hypnotic/sedative, anxiolytic, anaesthetic and anticonvulsive properties. Estrogens increase the CNS and serum levels of allopregnanolone and the addition of certain but not all synthetic progestins determines a further increase in allopregnanolone levels, suggesting that the metabolism into this reduced product is related to the chemical structure of progestin molecule used. In addition, depending on specific progestin molecule used, different interaction are found with the estradiol-induced beta-endorphin synthesis and release, showing that diverse progestins have specific and divergent actions on the opiatergic system. These results highlight the concept that natural and synthetic progesterone receptor agonists may systematically induce different biological actions in CNS. This may have far-reaching implications for the clinical effects and related indications of each compound.     

Role of vascular smooth muscle cell in the inflammation of atherosclerosis

Atherosclerosis is a pathologic process occurring within the artery, in which many cell types, including T cell, macrophages, endothelial cells, and smooth muscle cells, interact, and cause chronic inflammation, in response to various inner- or outer-cellular stimuli. Atherosclerosis is characterized by a complex interaction of inflammation, lipid deposition, vascular smooth muscle cell proliferation, endothelial dysfunction, and extracellular matrix remodeling, which will result in the formation of an intimal plaque. Although the regulation and function of vascular smooth muscle cells are important in the progression of atherosclerosis, the roles of smooth muscle cells in regulating vascular inflammation are rarely focused upon, compared to those of endothelial cells or inflammatory cells. Therefore, in this review, we will discuss here how smooth muscle cells contribute or regulate the inflammatory reaction in the progression of atherosclerosis, especially in the context of the activation of various membrane receptors, and how they may regulate vascular inflammation. [BMB Reports 2014; 47(1): 1-7]     

Progesterone enhances in vitro development of bovine embryos

Increased pregnancy rates in cattle given progesterone (P₄) prior to 5 d after breeding have recently been reported. The objective was to determine if this increase in pregnancy rate could be attributed to a direct positive effect of P₄ on the developing embryo. In Experiment 1, 280 bovine oocytes were inseminated in vitro and at Day 3 (insemination = Day 0), good quality 8 cell embryos (n = 206) were randomly allocated to be cultured in either CR1aa+serum with 0 or ∼15 ng/mL (n = 102 and n = 104, respectively). In Experiment 2, 881 bovine oocytes were used; on Day 3, good quality 8 cell embryos (n = 511) were randomly allocated to either the control (CR1aa+FCS, n = 168), vehicle (CR1aa + FCS + ethanol, n = 170), or P₄ treatment (CR1aa + FCS + ∼15 ng/mL P₄ in ethanol, n = 173). On Day 7, in both experiments, there were increased numbers of blastocysts developing in the P₄ group (Experiment 1, 59% and Experiment 2, 71%) compared to the vehicle (Experiment 2, 53%) or control (40 and 62% in Experiments 1 and 2, respectively). The addition of P₄ (8%) stimulated the rate of embryo development (early blastocysts or more advanced stages on Day 6) compared to vehicle (3%) and control (0%) and the P₄ group had more hatched or hatching blastocysts (33%) on Day 9 compared to the control or vehicle group (21 or 22%). Additionally, the P₄ group had greater embryo diameter and significantly more Grade 1 blastocysts on Day 7. In conclusion, P₄ had a direct, positive effect on developing bovine embryos cultured in vitro.     

纤维蛋白（原）在动脉粥样硬化发病中的作用及机制

在高胆固醇饮食诱发的家兔动脉粥样硬化模型上,发现ＡＳ早期即出现高纤维蛋白原血症,血浆Ｆｇ和胆固醇水平呈正相关。组织免疫化学检查发现,Ｆｇ密布于血管内皮下和平滑肌细胞间质。     

Novel model for “calcium paradox” in sympathetic transmission of smooth muscles: Role of cyclic AMP pathway

It is well established that reduction of Ca²⁺ influx through L-type voltage-dependent Ca²⁺ channel (L-type VDCC), or increase of cytosolic cAMP concentration ([cAMP]c), inhibit contractile activity of smooth muscles in response to transmitters released from sympathetic nerves. Surprisingly, in this work we observed that simultaneous administration of L-type VDCC blocker (verapamil) and [cAMP]c enhancers (rolipram, IBMX and forskolin) potentiated purinergic contractions evoked by electrical field stimulation of rat vas deferens, instead of inhibiting them. These results, including its role in sympathetic transmission, can be considered as a “calcium paradox”. On the other hand, this potentiation was prevented by reduction of [cAMP]c by inhibition of adenylyl cyclase (SQ 22536) or depletion of Ca²⁺ storage of sarco-endoplasmic reticulum by blockade of Ca²⁺ reuptake (thapsigargin). In addition, cytosolic Ca²⁺ concentration ([Ca²⁺]_c) evaluated by fluorescence microscopy in rat adrenal medullary slices was significantly reduced by verapamil or rolipram. In contrast, simultaneous incubation of adrenal slices with these compounds significantly increased [Ca²⁺]_c. This effect was prevented by thapsigargin. Thus, a reduction of [Ca²⁺]_c due to blockade of Ca²⁺ influx through L-type VDCC could stimulate adenylyl cyclase activity increasing [cAMP]c thereby stimulating Ca²⁺ release from endoplasmic reticulum, resulting in augmented transmitter release in sympathetic nerves and contraction.