Vagus nerve modulates secretin binding sites in the rat forestomach.
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Secretin is well known for its inhibitory action on gastric motility. It has been reported that secretin in a physiological dose inhibits gastric motility through mediation by the vagal afferent pathway. Secretin also elicited relaxation of carbachol-stimulated rat forestomach muscle strips by binding to its receptors, suggesting a direct action on this peripheral tissue. We hypothesized that vagal input may affect the action of secretin by modulating the level of secretin receptor in the forestomach. Several treatments, including vagal ligation, vagotomy, perivagal application of capsaicin or colchicine, intravenous infusion of tetrodotoxin, and intraperitoneal injection of atropine, were performed to investigate their effects on secretin receptor binding to forestomach membranes. Specific binding of 125I-labeled secretin to forestomach membranes was significantly decreased (45%) by vagal ligation, vagotomy (50%), or perivagal colchicine treatment (40%). On the contrary, specific binding of 125I-secretin was not affected by perivagal capsaicin treatment, intravenous infusion of tetrodotoxin, or intraperitoneal injection of atropine. By Scatchard analysis of the binding data, the capacity of the high-affinity binding sites in forestomach membranes was found to decrease significantly after vagal ligation compared with membranes from the sham-operated group. However, the affinity at the high-affinity binding sites, the binding parameters of the low-affinity binding sites, and binding specificity were not changed. Vagal ligation but not perivagal capsaicin treatment reduced the inhibitory effect of secretin on bethanechol-stimulated contraction of isolated forestomach muscle strips, causing a right shift in the dose-response curve. These results suggest that vagal input through axonal transport plays a significant role on secretin action by modulating the capacity of secretin binding sites (but not affinity or specificity), at least in rat forestomach. (+info)
FMRFamide-activated Ca2+ channels in Lymnaea heart cells are modulated by "SEEPLY," a neuropeptide encoded on the same gene.
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The cell-attached, patch-clamp technique was used to investigate the modulatory role of the neuropeptide SEQPDVDDYLRDVVLQSEEPLY ("SEEPLY") on FMRFamide-activated Ca2+ channels in isolated Lymnaea heart ventricular cells. Both SEEPLY and FMRFamide are encoded on the same neuropeptide gene and are coexpressed in a pair of excitatory motor neurons that innervate the heart. FMRFamide applied alone was capable of significantly increasing the P(open) time of a Ca2+ channel in isolated heart muscle cells. However, SEEPLY applied alone did not significantly alter the basal level of Ca2+ channel activity in the same cells. Repeated applications of FMRFamide (15 s every min) resulted in a progressive reduction in the number of Ca2+ channel openings and the overall P(open) time of the channel. The fifth successive 15-s application of FMRFamide failed to cause the Ca2+ channels to open in the majority of cells tested. When FMRFamide and SEEPLY were repeatedly applied together (2-min applications every 4 min) the FMRFamide-activated Ca2+ channels continued to respond after the fifth application of the two peptides. Indeed channel activity was seen to continue after repeated 2-min applications of FMRFamide and SEEPLY for as long as the patch lasted (+info)
Selective inhibition of transient K+ current by La3+ in crab peptide-secretory neurons.
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Although divalent cations and lanthides are well-known inhibitors of voltage-dependent Ca2+ currents (ICa), their ability to selectively inhibit a voltage-gated K+ current is less widely documented. We report that La3+ inhibits the transient K+ current (IA) of crab (Cardisoma carnifex) neurosecretory cells at ED50 approximately 5 microM, similar to that blocking ICa, without effecting the delayed rectifier K+ current (IK). Neurons were dissociated from the major crustacean neuroendocrine system, the X-organ-sinus gland, plated in defined medium, and recorded by whole cell patch clamp after 1-2 days in culture. The bath saline included 0.5 microM TTX and 0.5 mM CdCl2 to eliminate inward currents. Responses to depolarizing steps from a holding potential of -40 mV represented primarily IK. They were unchanged by La3+ up to 500 microM. Currents from -80 mV in the presence of 20 mM TEA were shown to represent primarily IA. La3+ (with TEA) reduced IA and maximum conductance (GA) by approximately 10% for 1 microM and another 10% each in 10 and 100 microM La3+. Normalized GA-V curves were well fit with a single Boltzmann function, with V1/2 +4 mV and slope 15 mV in control; V1/2 was successively approximately 15 mV depolarized and slope increased approximately 2 mV for each of these La3+ concentrations. Cd2+ (1 mM), Zn2+ (200 microM), and Pb2+ (100 microM) or removal of saline Mg2+ (26 mM) had little or no effect on IA. Steady-state inactivation showed similar right shifts (from V1/2 -39 mV) and slope increases (from 2.5 mV) in 10 and 100 microM La3+. Time to peak IA was slowed in 10 and 100 microM La3+, whereas curves of normalized time constants of initial decay from peak IA versus Vc were right-shifted successively approximately 15 mV for the three La3+ concentrations. The observations were fitted by a Woodhull-type model postulating a La3+-selective site that lies 0.26-0.34 of the distance across the membrane electric field, and both block of K+ movement and interaction with voltage-gating mechanisms; block can be relieved by depolarization and/or outward current. The observation of selective inhibition of IA by micromolar La3+ raises concerns about its use in studies of ICa to evaluate contamination by outward current. (+info)
Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide prevent inducible nitric oxide synthase transcription in macrophages by inhibiting NF-kappa B and IFN regulatory factor 1 activation.
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High-output nitric oxide (NO) production from activated macrophages, resulting from the induction of inducible NO synthase (iNOS) expression, represents a major mechanism for macrophage cytotoxicity against pathogens. However, despite its beneficial role in host defense, sustained high-output NO production was also implicated in a variety of acute inflammatory diseases and autoimmune diseases. Therefore, the down-regulation of iNOS expression during an inflammatory process plays a significant physiological role. This study examines the role of two immunomodulatory neuropeptides, the vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP), on NO production by LPS-, IFN-gamma-, and LPS/IFN-gamma-stimulated peritoneal macrophages and the Raw 264.7 cell line. Both VIP and PACAP inhibit NO production in a dose- and time-dependent manner by reducing iNOS expression at protein and mRNA level. VPAC1, the type 1 VIP receptor, which is constitutively expressed in macrophages, and to a lesser degree VPAC2, the type 2 VIP receptor, which is induced upon macrophage activation, mediate the effect of VIP/PACAP. VIP/PACAP inhibit iNOS expression and activity both in vivo and in vitro. Two transduction pathways appear to be involved, a cAMP-dependent pathway that preferentially inhibits IFN regulatory factor-1 transactivation and a cAMP-independent pathway that blocks NF-kappa B binding to the iNOS promoter. The down-regulation of iNOS expression, together with previously reported inhibitory effects on the production of the proinflammatory cytokines IL-6, TNF-alpha, and IL-12, and the stimulation of the anti-inflammatory IL-10, define VIP and PACAP as "macrophage deactivating factors" with significant physiological relevance. (+info)
Trefoil peptide TFF2 (spasmolytic polypeptide) potently accelerates healing and reduces inflammation in a rat model of colitis.
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BACKGROUND: The trefoil peptides are major secretory products of mucus cells of the gastrointestinal tract and show increased expression after inflammatory or ulcerative damage. Recombinant human TFF2 (spasmolytic polypeptide) has been shown to be cytoprotective, and enhances repair in models of gastric injury. AIMS: To test the healing effects of recombinant human (h)TFF2 in a rat model of chronic colitis. METHODS: Colitis was induced by intracolonic administration of dinitrobenzene sulphonic acid in ethanol. Mucosal repair was quantified macroscopically, microscopically by image analysis of tissue histology, and by measuring myeloperoxidase activity. RESULTS: Initial validation studies showed that maximal injury and inflammation occurred at the end of the first week after colitis induction (active phase), and that spontaneous healing was complete by eight weeks. Once daily intrarectal application of hTFF2 (2.5 mg/kg; approximately 0.5 mg/rat) for five days after maximal damage had been sustained, reduced both microscopic and macroscopic injury by 80% and inflammatory index by 50% compared with vehicle controls. In addition, endogenous concentrations of rat TFF2 and TFF3 (intestinal trefoil factor) were increased in the active phase of colitis and were reduced to basal levels by hTFF2 treatment. CONCLUSIONS: This study has shown that hTFF2 enhances the rate of colonic epithelial repair, and reduces local inflammation in a rat model of colitis, and suggests that luminal application of trefoil peptides may have therapeutic potential in the treatment of inflammatory bowel disease. (+info)
A PDZ-containing scaffold related to the dystrophin complex at the basolateral membrane of epithelial cells.
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Membrane scaffolding complexes are key features of many cell types, serving as specialized links between the extracellular matrix and the actin cytoskeleton. An important scaffold in skeletal muscle is the dystrophin-associated protein complex. One of the proteins bound directly to dystrophin is syntrophin, a modular protein comprised entirely of interaction motifs, including PDZ (protein domain named for PSD-95, discs large, ZO-1) and pleckstrin homology (PH) domains. In skeletal muscle, the syntrophin PDZ domain recruits sodium channels and signaling molecules, such as neuronal nitric oxide synthase, to the dystrophin complex. In epithelia, we identified a variation of the dystrophin complex, in which syntrophin, and the dystrophin homologues, utrophin and dystrobrevin, are restricted to the basolateral membrane. We used exogenously expressed green fluorescent protein (GFP)-tagged fusion proteins to determine which domains of syntrophin are responsible for its polarized localization. GFP-tagged full-length syntrophin targeted to the basolateral membrane, but individual domains remained in the cytoplasm. In contrast, the second PH domain tandemly linked to a highly conserved, COOH-terminal region was sufficient for basolateral membrane targeting and association with utrophin. The results suggest an interaction between syntrophin and utrophin that leaves the PDZ domain of syntrophin available to recruit additional proteins to the epithelial basolateral membrane. The assembly of multiprotein signaling complexes at sites of membrane specialization may be a widespread function of dystrophin-related protein complexes. (+info)
A functional analysis of inscuteable and its roles during Drosophila asymmetric cell divisions.
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Cellular diversity in the Drosophila central nervous system is generated through a series of asymmetric cell divisions in which one progenitor produces two daughter cells with distinct fates. Asymmetric basal cortical localisation and segregation of the determinant Prospero during neuroblast cell divisions play a crucial role in effecting distinct cell fates for the progeny sibling neuroblast and ganglion mother cell. Similarly asymmetric localisation and segregation of the determinant Numb during ganglion mother cell divisions ensure that the progeny sibling neurons attain distinct fates. The most upstream component identified so far which acts to organise both neuroblast and ganglion mother cell asymmetric divisions is encoded by inscuteable. The Inscuteable protein is itself asymmetrically localised to the apical cell cortex and is required both for the basal localisation of the cell fate determinants during mitosis and for the orientation of the mitotic spindle along the apical/basal axis. Here we define the functional domains of Inscuteable. We show that aa252-578 appear sufficient to effect all aspects of its function, however, the precise requirements for its various functions differ. The region, aa288-497, is necessary and sufficient for apical cortical localisation and for mitotic spindle (re)orientation along the apical/basal axis. A larger region aa288-540 is necessary and sufficient for asymmetric Numb localisation and segregation; however, correct localisation of Miranda and Prospero requires additional sequences from aa540-578. The requirement for the resolution of distinct sibling neuronal fates appears to coincide with the region necessary and sufficient for Numb localisation (aa288-540). Our data suggest that apical localisation of the Inscuteable protein is a necessary prerequisite for all other aspects of its function. Finally, we show that although inscuteable RNA is normally apically localised, RNA localisation is not required for protein localisation or any aspects of inscuteable function. (+info)
Murine Gcm1 gene is expressed in a subset of placental trophoblast cells.
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The gcm gene of Drosophila melanogaster encodes a transcription factor that is an important component in cell fate specification within the nervous system. In the absence of a functional gcm gene, progenitor cells differentiate into neurons, whereas when the gene is ectopically expressed the cells produce excess glial cells at the expense of neuronal differentiation. Recent searches of databases have uncovered high sequence similarity between the Drosophila gem gene and an anonymous human placental cDNA clone (Altschuller et al., 1996; this communication). Here we report the molecular organization of the murine Gcm1, its spatio-temporal pattern of expression in developing placenta, and its map position at E1-E3 on murine chromosome 9. The murine gene is composed of at least 6 exons. The promoter region contains an "initiation sequence" and is GC rich, characteristics of the promoters of several transcription factors. The mRNA has a modest 5'UTR (ca. 200 bases) but an extensive 3' UTR (ca. 2 kb). Northern blot and mRNA in situ hybridization studies showed that Gcm1 expression was readily detectable only in the placenta. It began at embryonic day 7.5 within trophoblast cells of the chorion and continued to about embryonic day 17.5 within a subset of labyrinthine trophoblast cells. Comparison with other transcription factors revealed that Gcm1 expression defines a unique subset of trophoblast cells. (+info)