Figure 1.
Cell model summarizing the hypothetical inhibitory effect of AMPK activation on CTX induced chloride secretion.
CTX binds to the surface ganglioside GM1. Following internalization, the A subunit of the toxin stimulates adenylate cyclase, leading to increased intracellular levels of cAMP and CFTR opening. Secretion of chloride increases luminal osmolarity, resulting in water secretion. Activation of AMPK by AICAR or metformin has an inhibitory effect on CFTR and may therefore abrogate the CTX induced hypersecretion of chloride.
Figure 2.
Intracellular chloride measurements in isolated rat colonic crypts.
A. Sample tracing of an intracellular chloride measurement using real-time fluorescence microscopy. Switching to a chloride-free superfusate causes chloride efflux and an increase in intracellular fluorescence (quenching dye). The kinetics of the change in the fluorescence signal (ΔAFU/sec) correspond to the number of open chloride efflux pathways. The insert shows an image of an isolated colonic crypt loaded with the chloride indicator dye MQAE. B. Bar graphs summarizing chloride efflux under the individual experimental conditions. Activation of AMPK decreases FSK stimulated chloride efflux from isolated rat colonic crypts. C. CFTR is the main chloride efflux pathway affected by AMPK. D Activation of AMPK decreases CTX stimulated chloride.
Figure 3.
WB analysis of AMPK phosphorylation in BBE monolayers.
Phosphorylation of AMPK at Thr172 is a prerequisite for AMPK activation. AICAR treatment resulted in increased phosphorylation of AMPK in polarized BBE monolayers. CTX treatment did not alter the levels of phosphorylated AMPK.
Figure 4.
Detection of CFTR by WB analysis of rat jejunum mucosal lysates.
Equivalent protein (20 µg) from each condition was analyzed by SDS PAGE. A. CTX does not increase CFTR levels in rat jejunum. B. AICAR treatment does not significantly decrease the total amount of CFTR protein in CTX stimulated jejunum.
Figure 5.
Ussing chamber measurements in mouse colonic mucosal sections.
A. Sample tracing demonstrating blunting of FSK stimulated SCC following metformin exposure. SCC is sensitive to bumetanide, indicating that the majority of the current is attributable to chloride secretion B. Bar graphs summarizing changes in SCC following FSK and AMPK activator treatment. C. Sample tracing demonstrating reduction of CTX stimulated SCC after AICAR treatment. D. Bar graphs summarizing changes in SCC following CTX and AICAR treatment.
Figure 6.
Ussing chamber measurements in human colonic mucosal sections.
A. Sample tracing demonstrating blunting of FSK stimulated SCC following AICAR exposure. B. Bar graphs summarizing changes in SCC following FSK and AMPK activator treatment. C. Sample tracing demonstrating reduction of CTX stimulated SCC after AICAR treatment. D. Bar graphs summarizing changes in SCC following CTX and AMPK activator treatment.
Figure 7.
Fluid accumulation in isolated jejunal loops.
Left: Note the increased loop swelling of CTX treated loops (left) vs. AICAR+CTX treated loops (right). Right: Quantification of intestinal fluid accumulation.