Expression of IRBIT Along the Rat Gastrointestinal Tract

IRBIT (inositol-1,4,5-trisphosphate (IP3) receptors binding protein released with IP3) regulates fluid transport in the pancreatic duct and other epithelial cells. Most of the research done on IRBIT focuses on what transporters and channels IRBIT regulates. Much less is known about what regulates IRBIT and its distribution along the gastrointestinal tract. In the present study, we aim to determine the expression of IRBIT in the gastrointestinal (GI) tract and accessory glands such as the liver and pancreas. IRBIT has two isoforms- a short and a long. To date, antibodies are unable to differentiate between the two, so a different approach is required. PCR (polymerase chain reaction) with specific primers for each isoform will be used. We have harvested stomach, duodenum, jejunum, ileum, proximal and distal colon from male rats. Using a phosphate buffered saline solution containing zero calcium, we have selectively isolated the epithelial cells from each of the aforementioned segments of the GI tract. Using western blot, we found that IRBIT is expressed in duodenum, jejunum, ileum, proximal and distal colon. To test the expression of the short and long IRBIT, using a similar technique we have isolated epithelial cells from stomach, duodenum, jejunum, ileum, proximal and distal colon. Using Trizol-chloroform we have selectively isolated the total mRNA from each tissue. In future experiments, we will make a cDNA library from each tissue to perform PCR and determine the expression of short and long IRBIT

Segovia republicana: Año I Número 123 - 25 Septiembre 1931

Gut clearance (i.e., fluid secretion) is one of the first lines of defense of the intestine against toxins and opportunistic bacteria. In the colon, fluid secretion is coupled to chloride secretion. Previous work has demonstrated that the basolateral Na-K-2Cl cotransporter 1 (NKCC1) represents a site for regulating fluid secretion independently of the apical chloride channels such as the cystic fibrosis transmembrane regulator. In addition, the lab has shown that protein kinase C (PKC) activation causes internalization of NKCC1, which in turn blocks chloride secretion. To date, the post-translational signal responsible for NKCC1 internalization during PKC activation remains unknown. Similarly, the fate of NKCC1 in the endocytic pathway has not been elucidated. In the present study, we investigated the role of ubiquitin as post-translational signal responsible for NKCC1 internalization. Experiments were performed on the human colonic T84 epithelial. Cells were incubated with or without 100 nM phorbol 12-myristate 13-acetate (PMA), a PKC activator, or in presence of 100 µM carbachol, a M3 muscarinic receptor agonist, for 15 or 30 min. NKCC1 ubiquitination was tested by western blot after immunoprecipitating NKCC1. Our preliminary results show that PMA and carbachol induced an increase of NKCC1 ubiquitination compare to control. In addition, blocking the lysosome with 5 µM NH4Cl did not prevent NKCC1 degradation during PKC activation by PMA. Our results suggest that PKC induces NKCC1 internalization through a ubiquitin-dependent pathway and may target NKCC1 for degradation in a lysosomal independent-manner

Determining the Fate of NKCC1 in the Endocytic Pathway and its Role in Host Defense

Fluid secretion represents an important defense mechanism of the gut. Driven by transepithelial chloride section, it flushes pathogens from the intestines. The basolateral Na-K-2Cl cotransporter 1 (NKCC1) is the main mechanism for loading the cell with chloride. Activation of the protein kinase C (PKC) causes internalization of NKCC1 and consequently, a decrease in fluid secretion. However, it is unknown whether the internalization pathway is one of degradation or recycling. In the colon, opportunistic pathogens, such as the fungus Candida albicans (C. albicans) invade the body by shifting its morphology from yeast to filamentous. We hypothesize that when pathogenic, C.albicans decreases fluid secretion by causing NKCC1 internalization. Our research seeks to (1) determine the fate of NKCC1 in the internalization pathway; and (2) explore how C. albicans, when made pathogenic (i.e., filamentous), interferes with the intestine’s secretory defenses. Previously, the lab has attempted to map NKCC1 in the endocytic pathway using microscopy and endosomal markers. The results suggested that NKCC1 may recycle to the plasma membrane. Since then, we have adopted a biochemical approach using western blotting with T84 and MDCK cells. Our experiments blocked possible degradation pathways (lysosome and proteasome), and the amount of NKCC1 remaining after PKC activation was determined using quantitative immunoblotting. Our preliminary results indicate that blocking the lysosomal and proteasomal degradation pathways do not prevent NKCC1 degradation during PKC activation. We have also determined conditions which cause filamentous growth in C.albicans, and will continue searching for others external cues that induce this shift

Determining the Fate of NKCC1 in the Endocytic Pathway and its Role in Host Defense

Fluid secretion represents an important defense mechanism of the gut. Driven by transepithelial chloride section, it flushes pathogens from the intestines. The basolateral Na-K-2Cl cotransporter 1 (NKCC1) is the main mechanism for loading the cell with chloride. Activation of the protein kinase C (PKC) causes internalization of NKCC1 and consequently, a decrease in fluid secretion. However, it is unknown whether the internalization pathway is one of degradation or recycling. In the colon, opportunistic pathogens, such as the fungus Candida albicans (C. albicans) invade the body by shifting its morphology from yeast to filamentous. We hypothesize that when pathogenic, C.albicans decreases fluid secretion by causing NKCC1 internalization. Our research seeks to (1) determine the fate of NKCC1 in the internalization pathway; and (2) explore how C. albicans, when made pathogenic (i.e., filamentous), interferes with the intestine’s secretory defenses. Previously, the lab has attempted to map NKCC1 in the endocytic pathway using microscopy and endosomal markers. The results suggested that NKCC1 may recycle to the plasma membrane. Since then, we have adopted a biochemical approach using western blotting with T84 and MDCK cells. Our experiments blocked possible degradation pathways (lysosome and proteasome), and the amount of NKCC1 remaining after PKC activation was determined using quantitative immunoblotting. Our preliminary results indicate that blocking the lysosomal and proteasomal degradation pathways do not prevent NKCC1 degradation during PKC activation. We have also determined conditions which cause filamentous growth in C.albicans, and will continue searching for others external cues that induce this shift

Mapping NKCC1 in the Endocytic Pathway During PKC Activation in Mardin Darby Canine Kidney Cells

Gut clearance represents an important defense mechanism of the gut by flushing out luminal bacteria and toxins. Transepithelial chloride fluid secretion is what drives gut clearance. The basolateral Na-K-2Cl cotransporter 1 (NKCC1) is the main mechanism for loading cells with chloride for its secretion by apical chloride channels (e.g., cystic fibrosis transmembrane regulator). We have previously shown that protein kinase C (PKC) activation causes internalization of NKCC1, thus blunting chloride secretion. However, the fate of NKCC1 remains unknown. To determine if NKCC1 is recycled or degraded, we used Mardin Darby Canine Kidney (MDCK) cells that stably expresses eGFP-NKCC1 to map the endocytic pathway. For immunocytochemistry, MDCK cells were cultured on coverslips in a six-well plate until confluence. Cells were exposed to either phorbol 12-myristate 13-acetate (PMA), an activator of PKC, or DMSO (vehicle). Cells were fixed with 1% paraformaldehyde, incubated with specific primary antibody against endosomal markers, and mounted for immunofluorescence. Images were acquired with an Olympus compound microscope equipped for fluorescence and processed using ImageJ. In these experiments, we did not find colocalization of NKCC1 with Rab5, a marker of the early endosome. We found occasional colocalization of NKCC1 and Rab11, a marker of vesicles recycling to the plasma membrane. Finally, we did not find colocalization of NKCC1 with either LAMP1 or P20S markers of the lysosome and the proteasome. Our results suggest that some NKCC1, after internalization recycles to the membrane in MDCK. Further investigation will be needed to determine the fate on NKCC1 in the endocytic pathway

Mapping NKCC1 in the Endocytic Pathway During PKC Activation in Mardin Darby Canine Kidney Cells

Gut clearance represents an important defense mechanism of the gut by flushing out luminal bacteria and toxins. Transepithelial chloride fluid secretion is what drives gut clearance. The basolateral Na-K-2Cl cotransporter 1 (NKCC1) is the main mechanism for loading cells with chloride for its secretion by apical chloride channels (e.g., cystic fibrosis transmembrane regulator). We have previously shown that protein kinase C (PKC) activation causes internalization of NKCC1, thus blunting chloride secretion. However, the fate of NKCC1 remains unknown. To determine if NKCC1 is recycled or degraded, we used Mardin Darby Canine Kidney (MDCK) cells that stably expresses eGFP-NKCC1 to map the endocytic pathway. For immunocytochemistry, MDCK cells were cultured on coverslips in a six-well plate until confluence. Cells were exposed to either phorbol 12-myristate 13-acetate (PMA), an activator of PKC, or DMSO (vehicle). Cells were fixed with 1% paraformaldehyde, incubated with specific primary antibody against endosomal markers, and mounted for immunofluorescence. Images were acquired with an Olympus compound microscope equipped for fluorescence and processed using ImageJ. In these experiments, we did not find colocalization of NKCC1 with Rab5, a marker of the early endosome. We found occasional colocalization of NKCC1 and Rab11, a marker of vesicles recycling to the plasma membrane. Finally, we did not find colocalization of NKCC1 with either LAMP1 or P20S markers of the lysosome and the proteasome. Our results suggest that some NKCC1, after internalization recycles to the membrane in MDCK. Further investigation will be needed to determine the fate on NKCC1 in the endocytic pathway