Localization and characterization of prostaglandin E1 receptors in rat small intestine

While prostaglandins of the E series are known to affect several small intestinal functions, their cellular mechanisms are poorly understood. The purposes of our study were to determine whether receptors for PGE are present in rat small intestine and to locate and characterize the receptor binding in the subcellular fractions. Small intestinal binding of prostaglandin E1 was significantly higher than that of prostaglandin E2. Highest receptor binding for prostaglandin E1 was found in the plasma membrane fraction of isolated small intestinal enterocytes. Curvilinearity of prostaglandin E1 binding in plasma membranes upon Scatchard analysis indicated two receptor binding sites in rat small intestine. Competitive binding studies demonstrated that receptor binding was highest for prostaglandins of the E series. These studies are the first to demonstrate specific prostaglandin E1 receptors in different subcellular fractions of rat small intestine. We suggest that receptor binding of prostaglandin E may be an important initial step in the mechanism of prostaglandin-E-induced responses in the small intestine.     

Expression and motor effects of secretin in small and large intestine of the rat

Immunocytochemistry and in situ hybridization revealed abundant secretin expressing cells on duodenal villi with a gradual decrease throughout the small intestines of the rat. They were absent in pancreas, stomach and colon. Secretin caused relaxation of rat intestinal longitudinal muscle in vitro. Studies on colon revealed that the secretin-evoked response was unaffected by apamin, tetrodotoxin, L-NAME, VIP or PACAP pretreatment; secretin itself caused desensitization. Addition of VIP or PACAP when the secretin-evoked relaxation was maximal evoked a further relaxation suggesting the presence of distinct receptors. Secretin causes relaxation via activation of secretin receptors located on the smooth muscle and not via any of the related VIP/PACAP receptors.     

Autoradiographic distribution of vasoactive intestinal polypeptide receptors in rabbit and rat small intestine

Vasoactive intestinal peptide (VIP) is found in the enteric nervous system of all layers of the small intestine. In the gastrointestinal tract, VIP receptors coupled to adenylate cyclase are present on epithelial, smooth muscle and possibly mononuclear cells. This study analyzes the distribution of VIP binding using in vitro autoradiographic techniques. VIP binding was present in high density in the mucosal layer of rabbit duodenum, jejunum and ileum. Low VIP binding was noted over the smooth muscle layers or the lymphoid follicles. Similar results were obtained in rat small intestine. The density of VIP binding was greatest in duodenal mucosa but was present in lower density in jejunal and ileal mucosa. Again, low VIP binding was noted in the smooth muscle layers or lymphoid follicles. Thus, autoradiographic maps of small intestine indicate that VIP receptors are found primarily in the small intestinal mucosa.     

Distribution of immunoglobulin G receptors in the small intestine of the young rat

Conjugates of horseradish peroxidase (HRP) and immunoglobulin G (IgG) were used to map the distribution of cell surface receptors that can bind IgG at 0 degrees C within the small intestine of 10-12-d-old rats. Luminal receptors are present only within the duodenum and proximal jejunum. In these locations, receptors are limited to absorptive cells that line the upper portion of individual villi. Near villus tips, receptors are relatively evenly distributed over the entire luminal plasmalemma. In the midregion of villi, receptors are unevenly distributed over the luminal surface. Receptors (a) specifically bind rat and rabbit IgG, (b) recognize the Fc portion of the immunoglobulins, and (c) bind at pH 6.0 but not pH 7.4. To determine whether IgG receptors are confined to the luminal portion of the plasmalemma, intact epithelial cells were isolated from the proximal intestine of 10-12-d-old rats and incubated with HRP conjugates at 0 degree C. The specific binding of rat IgG-HRP to cells at pH 6.0 indicates that IgG receptors, which are functionally similar to those found on the luminal surface, are also present over the entire abluminal surface of absorptive cells. These results are consistent with the transport of IgG to the abluminal plasma membrane in the form of IgG-receptor complexes on the surface of vesicles. Exposure of these complexes to the serosal plasma, which is presumably at pH 7.4, would cause release of IgG from the receptors. To assess possible inward movement of vesicles from the abluminal surface after discharge of IgG, intravenously injected HRP was used as a space-filling tracer in the serosal plasma. HRP could be visualized within the coated and tubular vesicles responsible for transport of IgG in the opposite direction. These vesicles may, therefore, provide a pathway whereby receptors shuttle between the luminal and abluminal surfaces of cells.     

Slow-waves in rat small intestine

Rat small intestine generates rhythmic slow-wave activity. The slow-waves are not eliminated by ouabain application or incubation in potassium free solution. Exposure to low sodium or calcium free solution decreases slow-wave activity. Incubation in sodium and calcium free solution eliminates activity. It is concluded that rat small intestinal slow-waves may not result from the same mechanism as in the cat.     

Motilin receptors in rabbit stomach and small intestine

Motilin receptors in rabbit antral and duodenal smooth muscle tissue were characterized by direct binding technique using 125I-labeled porcine motilin as a tracer ligand. Binding at 30 degrees C was maximal at 90 min, was saturable and partially reversible. Displacement studies with natural porcine motilin, synthetic leucine-motilin or norleucine-motilin indicated a dissociation constant (Kd) of 1.1 +/- 0.3 nM and a maximal binding capacity (Bmax) of 42 +/- 10 fmol/mg protein. Binding was unaffected by glucagon, pancreatic polypeptide and somatostatin, but substance P interfered via an unknown mechanism. By density gradient centrifugation motilin receptors were shown to be present in plasma membranes. Binding could only be demonstrated in preparations from antrum and upper duodenum. These observations provide evidence for a localized target region for motilin in the gastrointestinal tract, and for a direct interaction of motilin with gastrointestinal smooth muscle tissue.     

Expression of galectin 4 in the rat small intestine during postnatal development

We determined the expression of an endogenous lectin, galectin 4, in the rat small intestine during postnatal development. The mRNA levels of galectin 4 did not change significantly between birth and adulthood. In contrast, the protein was present at higher levels after than before weaning, and the potential ligands for galectin 4 were more highly represented in the enterocyte microvilli of weaned than of suckling rats. These results suggest possible differences either in galectin 4 mRNA stability, in its translation regulation or in the protein stability.     

Expression of arginase II and related enzymes in the rat small intestine and kidney

Arginase, which catalyzes the conversion of arginine to urea and ornithine, and consists of a liver-type (arginase I) and a non-hepatic type (arginase II). Arginine is also used for the synthesis of nitric oxide and creatine phosphate, while ornithine is used for the synthesis of polyamines and proline, and thus collagen. Arginase II mRNA and protein are abundant in the intestine (most abundant in the jejunum and less abundant in the ileum, duodenum, and colon) and kidney of the rat. In the kidney, the levels of arginase II mRNA do not change appreciably from 0 to 8 weeks of age. In contrast, arginase II mRNA and protein in the small intestine are not detectable at birth, appear at 3 weeks of age, the weaning period, and their levels increase up to 8 weeks. On the other hand, mRNAs for ornithine aminotransferase (OAT), ornithine decarboxylase, and ornithine carbamoyltransferase (OCT) are present at birth and their levels do not change much during development. Arginase II is elevated in response to a combination of bacterial lipopolysaccharide, dibutyryl cAMP, and dexamethasone in the kidney, but is not affected by these treatments in the small intestine. Immunohistochemical analysis of arginase II, OAT, and OCT in the jejunum revealed their co-localization in absorptive epithelial cells. These results show that the arginase II gene is regulated differentially in the small intestine and kidney, and suggest different roles of the enzyme in these two tissues. The co-localization of arginase II and the three ornithine-utilizing enzymes in the small intestine suggests that the enzyme is involved in the synthesis of proline, polyamines, and/or citrulline in this tissue.     

Stimulation of neurons in rat ARC inhibits gastric acid secretion via hypothalamic CRF1/2- and NPY-Y1 receptors

Neuropeptide Y (NPY) neuronal projections from the arcuate nucleus (ARC) have been proposed to target corticotropin-releasing factor (CRF)-positive neurons in the paraventricular nucleus (PVN) as part of the ARC-PVN axis. The existence of a positive feedback loop involving CRF receptors in the PVN has been suggested. Exogenous NPY and CRF in the PVN have been shown to inhibit gastric acid secretion. Recently, we have demonstrated that activation of ARC neurons inhibits gastric acid secretion via vagal pathways. To what extent NPY- and CRF-mediated mechanisms in the PVN contribute to the CNS modulation of gastric acid secretion is still an open question. In the present study, we performed consecutive bilateral microinjections of antagonists to NPY receptor subtypes Y1 and Y2 and to CRF1/2 receptors in the PVN and of the excitatory amino acid kainate in the ARC to assess the role of NPY- and CRF-mediated mechanisms in the kainate-induced effects on gastric acid secretion. Gastric acid secretion was measured at the basal condition and during pentagastrin (16 microg/kg body wt) stimulation. Microinjection of vehicle in the PVN and kainate in the ARC decreased gastric acid secretion. Microinjection of the specific NPY-Y1 receptor antagonist BIBP-3226 (200 pmol) and the nonspecific CRF1/2 antagonist astressin (30 pmol) in the PVN abolished the inhibitory effect of neuronal activation in the ARC by kainate on gastric acid secretion. The CRF antagonist astressin was more effective. Pretreatment with the NPY-Y2 receptor antagonist BIIE-0246 (120 pmol) in the PVN had no significant effect. Our results indicate that activation of neurons in the ARC inhibits gastric acid secretion via CRF1/2 and NPY-Y1 receptor-mediated pathways in the PVN.     

代谢型谷氨酸受体在突触可塑性中的作用研究进展

突触可塑性是近 30年来神经科学领域的研究热点之一 ,它主要包括长时程增强 (long termpotentiation ,LTP)和长时程抑制 (long termdepression ,LTD)。以往的研究已经证实 ,离子型谷氨酸受体 (iGluRs)中的NMDA受体和AMPA受体 ,在LTP和LTD的诱导和维持中通过阳离子内流 ,引起细胞内的级联反应而起作用。新近的研究发现 ,代谢型谷氨酸受体 (mGluRs)与G蛋白偶联 ,通过细胞内的多种信使系统介导慢突触传递。本文主要就mGluRs在不同脑区LTP和LTD中的作用进行综述     

Adenosine A1 receptor agonist treatment up-regulates rat brain metabotropic glutamate receptors

Chronic R-N(6)-phenylisopropiladenosine (R-PIA) subcutaneous injection for 6 days significantly increased total glutamate receptor number (180% of control) in rat brain synaptic plasma membranes (SPM), without affecting receptor affinity. A higher increase in metabotropic glutamate (mGlu) receptor number (258% of control) was also detected, indicating that mGlu is the main type of glutamate receptor affected by this treatment. On the other hand, the observed increase in basal and calcium- and Gpp(NH)p-stimulated phospholipase C (PLC) activity after treatment was associated with a significant increase in PLC beta(1) isoform, detected in SPM by immunoblotting assays. Moreover, an increase in PLC activity stimulation with trans-ACPD, in the absence and in the presence of Gpp(NH)p, was detected after R-PIA treatment. These results show that mGlu receptors and its effector system, PLC activity, are up-regulated by chronic exposure to an adenosine A(1) receptor agonist and suggest the existence of a cross-talk mechanism between both signal transduction pathways in rat brain.     

PGE(2) receptors and their intracellular mechanisms in rabbit small intestine

The effects of PGE(2) on longitudinal smooth muscle, the intracellular mechanisms involved, and the localization of EP receptors were investigated in rabbit small intestine. PGE(2) evoked contractions in small intestine that were reduced by tetrodotoxin and hexamethonium. 17-Phenyl trinor PGE(2), sulprostone, misoprostol and 16,16-dimethyl PGE(2) evoked contractions. Butaprost did not modify spontaneous motility. AH 6809 reduced PGE(2) and 17-phenyl trinor PGE(2)-induced contractions. Verapamil, Ca(2+) free medium, staurosporine, forskolin, theophylline, and rolipram diminished, while IP-20 and H-89 increased PGE(2)-induced contractions. Western blot analysis showed protein bands of 41kDa for EP(1), 71kDa for EP(2) and 62kDa for EP(3) receptors. EP(1), EP(2) and EP(3) receptors were detected in neurons of the myenteric and submucosal ganglia, but only EP(3) receptors were found in smooth muscle layers. This study did not detect EP(4) receptor. PGE(2)-induced contractions would be mediated through EP(1) and EP(3) receptors, and voltage-dependent Ca(2+) channels, protein kinase C, and cAMP would be implicated in these responses.     

Expression and localization of the multidrug resistance-associated protein 3 in rat small and large intestine

Multidrug resistance-associated protein 3 (MRP3; symbol ABCC3), has been shown to mediate ATP-dependent transport of organic anions including 17beta-glucuronosyl estradiol, glucuronosyl bilirubin, monovalent, and sulfated bile salts. MRP3 mRNA expression was reported in rat intestine suggesting a role of MRP3 in the intestinal transport. We examined the expression and localization of MRP3 in rat small and large intestine by RT-PCR, immunofluorescence, and immunoblot analysis. MRP3 was identified in all intestinal segments by RT-PCR. MRP3 expression was low in duodenum and jejunum but markedly increased in ileum and colon. With the use of a rat MRP3 specific antibody, MRP3 was localized to the basolateral domains of enterocytes. Immunofluorescence analysis and immunoblot analysis confirmed a strong expression of rat MRP3 in ileum and colon. In contrast, MRP2 was predominantly expressed in the proximal segments of rat small intestine. Our findings demonstrate a high expression of rat MRP3 in ileum and colon and provide evidence for an involvement of MRP3 in the ATP-dependent transport of organic anions, including bile salts from the enterocyte into blood.     

PAR-2 agonists induce contraction of murine small intestine through neurokinin receptors

Protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor and is expressed throughout the gut. It is well known that PAR-2 participates in the regulation of gastrointestinal motility; however, the results are inconsistent. The present study investigated the effect and mechanism of PAR-2 activation on murine small intestinal smooth muscle function in vitro. Both trypsin and PAR-2-activating peptide SLIGRL induced a small relaxation followed by a concentration-dependent contraction. The sensitivity to trypsin was greater than that to SLIGRL (EC50 = 0.03 vs. 40 microM), but maximal responses were similar (12.3 +/- 1.6 vs. 13.7 +/- 1.3 N/cm2). Trypsin-evoked contraction (1 microM) exhibited a rapid desensitization, whereas the desensitization of response to SLIGRL was less even at high concentration (50 microM). Atropine had no effect on PAR-2 agonist-induced contractions. In contrast, TTX and capsaicin significantly attenuated those contractions, implicating a neurogenic mechanism that may involve capsaicin-sensitive sensory nerves. Furthermore, contractions induced by trypsin and SLIGRL were reduced by neurokinin receptor NK1 antagonist SR-140333 or NK2 antagonist SR-48968 alone or were further reduced by combined application of SR-140333 and SR-48968, indicating the involvement of neurokinin receptors. In addition, desensitizing neurokinin receptors with substance P and/or neurokinin A decreased the PAR-2 agonist-evoked contraction. We concluded that PAR-2 agonists induced a contraction of murine intestinal smooth muscle that was mediated by nerves. The excitatory effect is also dependent on sensory neural pathways and requires both NK1 and NK2 receptors.     

AMP deaminases of rat small intestine

Phosphocellulose column chromatography revealed the existence of two forms of AMP deaminase both in whole tissue and in the intestinal epithelium. AMP deaminase I, which eluted from the column as a first activity peak, exhibited hyperbolic, nonregulatory kinetics. The substrate half-saturation constants were determined to be 0.3 and 0.7 mM at pH 6.5 and 7.2, respectively, and did not change in the presence of ATP, GTP and Pi. AMP deaminase II, which eluted from the column as a second activity peak, was strongly activated by ATP and inhibited by GTP and Pi. The S0.5 constants were 3.5 and 7.1 at pH 6.5 and 7.2, respectively. At pH 7.2 ATP (1 mM) S0.5 decreased to 2.5 mM and caused the sigmoidicity to shift to hyperbolic. The ATP half-activation constant was increased 9-fold in the presence of GTP and was not affected by Pi. Mg2+ significantly altered the effects exerted by nucleotides. The S0.5 value was lowered 10-fold in the presence of MgATP and 5-fold in the presence of MgATP, MgGTP and Pi. When MgATP was present, AMP deaminase II from rat small intestine was less susceptible to inhibition by GTP and Pi. A comparison of the kinetic properties of the enzyme, in particular the greater than 100% increase in Vmax observed in the presence of MgCl2 at low (1 mM) substrate concentration, indicates that MgATP is the true physiological activator. GuoPP[NH]P at low concentrations, in contrast to GTP, did not affect the enzyme and even activated it at concentrations above 0.2 mM. We postulate that AMP deaminase II may have a function similar to that of the rat liver enzyme. The significance of the existence of an additional, non-regulatory form of AMP deaminase in rat small intestine is discussed.