Characterization of intestinal brush border guanylate cyclase activation by Escherichia coli heat-stable enterotoxin.
Intestinal brush border guanylate cyclase was previously reported to be activated by the Escherichia coli enterotoxin (STa). This system was reexamined in order to develop a hypothesis for the mechanism of activation. The extent of activation was previously underestimated, since by using sodium azide to inhibit competing reactions and ethylene glycol bis(beta-aminoethyl ether) N,N-tetraacetic acid to chelate Ca2+, which is inhibitory, maximal activations of 30- to 50-fold were obtained. Ca2+ inhibition was only partially relieved by the calmodulin inhibitor calmidazolium. Inhibitors of the O2-dependent activation of soluble guanylate cyclase had no effect on STa activation; hence, it was concluded that STa activation did not involve arachidonate release and oxidation. STa was able to further increase activity already elevated by the nonionic detergent Lubrol PX. The membrane-active agent filipin, which was previously reported to inhibit both basal and agonist-stimulated adenylate cyclase, did not inhibit STa activation of guanylate cyclase. Digitonin, another cholesterol binder, inhibited STa activation at low concentrations, which disappeared at higher concentrations. Both of these agents stimulated basal activity. Dimethyl sulfoxide produced a concentration-dependent inhibition of STa activation, while increasing basal activity 7-fold. Ethanol inhibited both basal and STa-stimulated activity, with the former being more affected. Benzyl alcohol, like ethanol, a "fluidizer" of cell membranes, also inhibited both basal and activated enzymes. We concluded that STa directly activates this guanylate cyclase and, because of the differential effects of inhibitors on basal and STa-stimulated activity, propose a receptor-mediated mechanism.
ElDeib
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Parker
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Veum
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Zinn
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White
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D000818:Animals; D001427:Bacterial Toxins / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology; D006152:Cyclic GMP / Q000494:pharmacology; D004072:Digitonin / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004768:Enterotoxins / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D029968:Escherichia coli Proteins; D005372:Filipin / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D006162:Guanylate Cyclase / Q000378:metabolism*; D066298:In Vitro Techniques; D007413:Intestinal Mucosa / Q000201:enzymology*; D008297:Male; D008871:Microvilli / Q000201:enzymology; D013552:Swine
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; C034151:heat stable toxin (E coli); D006160:Guanosine Triphosphate; D005372:Filipin; D006162:Guanylate Cyclase; D006152:Cyclic GMP; D004072:Digitonin; D002118:Calcium; D004121:Dimethyl Sulfoxide