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083 CITROBACTER RODENTIUM INFECTION CAUSES A DECREASE IN CYCLIC-AMP MEDIATED ION TRANSPORT IN MOUSE INTESTINAL EPITHELIAL CELLS CL Alexander, WK MacNaughton Citrobacter rodentium infection of mice, which mimics the lesions of enteropathogenic and enterohaemorrhagic Escherichia coli infections in humans, was previously shown to decrease ion secretion in response to the Ca++-dependent secretagogue carbachol (CCh). In order to determine the cellular mechanism underlying these observations, we used an in vitro model of the mouse intestinal cell line, CMT-93. CMT-93 monolayers were cultured on semi-permeable membrane supports and infected with log phase C. rodentium grown in LB broth at a multiplicity of infection of 100 for 2 to 8 hr. Membrane monolayers were mounted in Ussing chambers to measure the change in ion secretion by short circuit current (deltaIsc) and area under the curve (AUC), representative of the maximum rate and the total ion secretion respectively. Both CCh and the cAMP-dependent secretagogue, forskolin (FSK) caused a concentration-dependent increase in deltaIsc and AUC when added to the basolateral side of uninfected CMT-93 monolayers. The response to CCh was transient, whereas that to FSK was prolonged. CCh (20 µM) and FSK (10 µM) gave maximal responses, both of which were abolished when chloride-free buffer was used in the chambers. In separate experiments, monolayers grown on culture dishes were similarly infected with C. rodentium for 2 to 8 hr with subsequent RT-PCR performed to assess expression of the cAMP-dependent chloride channel, cystic fibrosis transmembrane conductance regulator (CFTR). The CCh response was not altered by infection with C. rodentium. However, the response to FSK was significantly (p<0.05) decreased at 6 and 8 hr of infection in terms of both deltaIsc (control, 4.5±0.3 µA/cm2; 6 hr, 3.1±0.2 µA/cm2; 8 hr, 1.9±0.1 µA/cm2) and AUC (control, 3.4±0.3 mC/cm2; 6 hr, 1.5±0.1 mC/cm2; 8 hr, 0.8±0.1 mC/cm2). Incubation of CMT-93 cells for 24 hr with LB broth alone (vehicle control) did not alter secretagogue responses. RT-PCR showed no difference in cellular CFTR mRNA levels after 8 hr of infection suggesting that the C. rodentium-induced change in the response to FSK was not due to changes in CFTR gene expression.
Mucosal Inflammation Research Group, University of Calgary, Calgary, Alberta
In conclusion, C. rodentium infection decreases cAMP-dependent, but not Ca++-dependent, ion secretion in a mouse cell line, without decreasing CFTR gene expression.