Publicación: ESCHERICHIA COLI HEAT-STABLE ENTEROTOXIN MEDIATES NA+/H+ EXCHANGER 4 INHIBITION INVOLVING CAMP IN T-84 HUMAN INTESTINAL EPITHELIAL CELLS
Fecha
2015
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PLoS One
Resumen
THE ENTEROTOXIGENIC ESCHERICHIA COLI STRAINS LEAD TO DIARRHOEA IN HUMANS DUE TO HEAT-LABILE AND HEAT-STABLE (STA) ENTEROTOXINS. STA INCREASES CL-RELEASE IN INTESTINAL CELLS, INCLUDING THE HUMAN COLONIC CARCINOMA T84 CELL LINE, INVOLVING INCREASED CGMP AND MEMBRANE ALKALIZATION DUE TO REDUCED NA+/H+ EXCHANGERS (NHES) ACTIVITY. SINCE NHES MODULATE INTRACELLULAR PH (PHI), AND NHE1, NHE2, AND NHE4 ARE EXPRESSED IN T84 CELLS, WE CHARACTERIZED THE STA ROLE AS MODULATOR OF THESE EXCHANGERS. PHI WAS ASSAYED BY THE NH4CL PULSE TECHNIQUE AND MEASURED BY FLUORESCENCE MICROSCOPY IN BCECF-PRELOADED CELLS. PHI RECOVERY RATE (DPHI/DT) WAS DETERMINED IN THE ABSENCE OR PRESENCE OF 0.25 ?MOL/L STA (30 MINUTES), 25 ?MOL/L HOE-694 (CONCENTRATION INHIBITING NHE1 AND NHE2), 500 ?MOL/L SODIUM NITROPRUSSIDE (SNP, SPONTANEOUS NITRIC OXIDE DONOR), 100 ?MOL/L DIBUTYRYL CYCLIC GMP (DB-CGMP), 100 NMOL/L H89 (PROTEIN KINASE A INHIBITOR), OR 10 ?MOL/L FORSKOLIN (ADENYLYL CYCLASE ACTIVATOR). CGMP AND CAMP WERE MEASURED IN CELL EXTRACTS BY RADIOIMMUNOASSAY, AND BUFFERING CAPACITY (ßI) AND H+ EFFLUX (JH+) WAS DETERMINED. NHE4 PROTEIN ABUNDANCE WAS DETERMINED BY WESTERN BLOTTING. STA AND HOE-694 CAUSED COMPARABLE REDUCTION IN DPHI/DT AND JH+ (~63%), WITHOUT ALTERING BASAL PHI (RANGE 7.144-7.172). STA DID NOT ALTER ßI VALUE IN A RANGE OF 1.6 PHI UNITS. THE DPHI/DT AND JH+ WAS ALMOST ABOLISHED (~94% INHIBITION) BY STA + HOE-694. STA EFFECT WAS UNALTERED BY DB-CGMP OR SNP. HOWEVER, STA AND FORSKOLIN INCREASED CAMP LEVEL. STA-DECREASED DPHI/DT AND JH+ WAS MIMICKED BY FORSKOLIN, AND STA + HOE-694 EFFECT WAS ABOLISHED BY H89. THUS, INCUBATION OF T84 CELLS WITH STA RESULTS IN REDUCED NHE4 ACTIVITY LEADING TO A LOWER CAPACITY OF PHI RECOVERY REQUIRING CAMP, BUT NOT CGMP. STA EFFECT RESULTS IN A CAUSAL PHENOMENON (STA/INCREASED CAMP/INCREASED PKA ACTIVITY/REDUCED NHE4 ACTIVITY) ENDING WITH INTRACELLULAR ACIDIFICATION THAT COULD HAVE CONSEQUENCES IN THE GASTROINTESTINAL CELLS FUNCTION PROMOTING HUMAN DIARRHOEA
