ABSTRACT
Elevation in the intracellular Ca(2+) concentration stimulates glucagon secretion from pancreatic α-cells. The Transient Receptor Potential Melastatin 4 channel (TRPM4) is critical for Ca(2+) signaling. However, its role in glucagon secreting α-cells has not been investigated. We identified TRPM4 gene expression and protein in the αTC1-6 cell line using RT-PCR and immunocytochemistry. Furthermore, we performed a detailed biophysical characterization of the channel using the patch-clamp technique to confirm that currents typical for TRPM4 were present in αTC1-6 cells. To investigate TRPM4 function, we generated a stable knockdown clone using shRNA and a lentiviral vector. Inhibition of TRPM4 significantly reduced the responses to different agonists during Ca(2+) imaging analysis with Fura-2AM. The reduction in the magnitude of Ca(2+) signals resulted in decreased glucagon secretion. These results suggested that depolarization by TRPM4 may play an important role in controlling glucagon secretion from α-cells and perhaps glucose homeostasis.
Subject(s)
Arginine Vasopressin/pharmacology , Calcium Signaling , Glucagon-Secreting Cells/metabolism , Glucagon/metabolism , TRPM Cation Channels/metabolism , Animals , Calcium/pharmacology , Cell Line , Cricetinae , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Meglumine/pharmacology , Membrane Potentials/drug effects , Mice , Patch-Clamp Techniques , RNA Interference , Rats , TRPM Cation Channels/agonists , TRPM Cation Channels/antagonists & inhibitors , TRPM Cation Channels/geneticsABSTRACT
Escherichia coli RDEC-1 (serotype O15:H-) is a rabbit enteropathogen in which in vivo enteroadherence is both site specific and age related. To determine whether these differences could be related to mucins, we evaluated inhibition of binding of AF/R1 piliated RDEC-1 by mucins isolated from various segments of intestine of rabbits at different ages. Mucin was purified from intestinal crude mucus by cesium chloride serial ultracentrifugation. RDEC-1 was grown to promote the expression of hydrophobic mannose-resistant AF/R1 pili. Quantitation of in vitro bacterial binding was determined using a crystal violet colorimetric assay. In postweanling rabbits, inhibition of RDEC-1 binding by purified mucin derived from ileal segments (45.1 +/- 2.6%, mean +/- SEM) and proximal colonic segments (46.0 +/- 5.5%) was less than purified mucins derived from jejunal segments (70.0 +/- 2.0%) and distal colonic segments (71.0 +/- 3.7%, p < 0.05) of the intestinal tract. In all age groups, mucins derived from jejunal segments inhibited RDEC-1 binding to a greater level than mucins derived from ileal segments. In addition, inhibition of binding by mucin derived from proximal small intestine of postweanling rabbits (70.0 +/- 2.0%) was greater than that of weanling rabbits (55.2 +/- 3.5%, p < 0.05) with suckling rabbit inhibition (62.1 +/- 3.5%) between these two levels. We conclude that mucin inhibition of RDEC-1 adhesion is both age and region related and therefore may contribute to both age-related and site localization of bacterial infections of the intestinal tract.
Subject(s)
Bacterial Adhesion/drug effects , Escherichia coli/pathogenicity , Mucins/pharmacology , Age Factors , Animals , Animals, Suckling , Escherichia coli/isolation & purification , Escherichia coli/physiology , Escherichia coli Infections/etiology , Escherichia coli Infections/prevention & control , In Vitro Techniques , Intestinal Diseases/etiology , Intestinal Diseases/prevention & control , Intestines/chemistry , Intestines/microbiology , Male , Mucins/chemistry , Mucins/isolation & purification , Organ Specificity , RabbitsABSTRACT
Anionic polyacrylic acid polymers, such as polycarbophil, have a number of properties that would make them suitable carriers for sustained antibiotic release formulations in the intestinal tract. However, little is known with regards to possible microbial adhesion to polycarbophil. The aim of this study was to evaluate for such an interaction using the rabbit enteric pathogen Escherichia coli RDEC-1 (serotype O15:H-). RDEC-1 mediates attaching and effacing binding to intestinal epithelium in a manner morphologically identical to that observed in both human enteropathogenic E. coli and enterohemorrhagic E. coli infections. RDEC-1 bacteria were grown to promote the expression of the mannose-resistant AF/R1 adhesion pili. A nonpiliated mutant, strain M34, was used as a negative control. Using radioactive labeling of bacteria, we quantitated adhesion of piliated RDEC-1 in the presence of polycarbophil using an in vitro adhesion assay system. Binding of piliated RDEC-1 in the adhesion assay was greater than for nonpiliated M34 for all concentrations of bacteria greater than 10(9) (P < .05). Polycarbophil did not cause concentration-dependent inhibition of piliated RDEC-1 binding (P > .05). We conclude polycarbophil does not interfere with the AF/R1 adhesin ligand of RDEC-1. Use of this polymer as a mucoadhesive drug delivery vehicle for nonabsorbable antibiotics in the treatment of gastrointestinal infections would not be expected to interfere with the protective effects of intestinal mucins.
Subject(s)
Acrylic Resins/pharmacology , Antidiarrheals/pharmacology , Bacterial Adhesion/drug effects , Digestive System/microbiology , Animals , Disease Models, Animal , Humans , RabbitsABSTRACT
A modified nomenclature for designating the auxiliary electrodes of the 10% system is described.
