Guanosine 3',5'-cyclic monophosphate-dependent protein kinase II mediates heat-stable enterotoxin-provoked chloride secretion in rat intestine.
OBJECTIVE
Escherichia coli heat-stable enterotoxins (STa) provoke electrogenic Cl- secretion in the intestine through a guanosine 3',5'-cyclic monophosphate (cGMP)-dependent signal transduction pathway. The cGMP receptor involved in the activation of the Cl- channel is not known with certainty but may comprise either adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (cAK) or cGMP-dependent protein kinase (cGK) type II. The aim of this study was to discriminate between these possibilities using specific kinase inhibitors.


METHODS
Intestinal electrogenic Cl- secretion was determined by measuring short-circuit current (Isc) in a Ussing chamber.


RESULTS
The general protein kinase inhibitors staurosporine and H-8 inhibited rat cGK II activity in vitro with 50% inhibitory concentration values of 4 nmol/L and 3 mumol/L, respectively, which are lower than those reported for cAK. Both staurosporine and H-8, when added to rat proximal colon at concentrations that did not affect the Isc response to 8-bromo-cAMPS, inhibited the STa- and 8-bromo-cGMP-provoked Isc response for more than 80%. Furthermore, the relative specific cGK inhibitor Rp isomer of 8-(chlorophenylthio)-cGMP, but not the cAK inhibitor RP isomer of (Rp) 8-bromo-cAMPS, inhibited the Isc response to submaximal levels of STa in rat proximal colon.


CONCLUSIONS
These data provide further evidence for an important role of cGK II in STa-mediated Cl- secretion in native rat intestinal epithelium.
Vaandrager
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AB
Department of Biochemistry, Cardiovascular Research Institute COEUR, Medical Faculty, Erasmus University, Rotterdam, The Netherlands.
Bot
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AG
De Jonge
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HR
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D002712:Chlorides / Q000378:metabolism* / Q000502:physiology; D003106:Colon / Q000187:drug effects / Q000201:enzymology; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D004355:Drug Stability; D004553:Electric Conductivity; D004768:Enterotoxins / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli; D006358:Hot Temperature; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000187:drug effects*; D007546:Isoquinolines / Q000494:pharmacology; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D019311:Staurosporine / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology
D002712:Chlorides; D004768:Enterotoxins; D004791:Enzyme Inhibitors; D007546:Isoquinolines; D013873:Thionucleotides; D015124:8-Bromo Cyclic Adenosine Monophosphate; C016276:8-bromocyclic GMP; C055656:8-((4-chlorophenyl)thio)cyclic-3',5'-GMP; C053177:N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP; D019311:Staurosporine