INTESTINE INFLAMMATION

Nitric oxide inhibitable isoforms of adenylate cyclase mediate epithelial secretory dysfunction following exposure to ionising radiation

S L Freeman, W K MacNaughton

Mucosal Inflammation Research Group and Department of Physiology and Biophysics, University of Calgary, Calgary, Canada

Correspondence to:
Correspondence to:
Dr W MacNaughton
Department of Physiology and Biophysics, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1; wmacnaug{at}ucalgary.ca

Background: Hyporesponsiveness of the intestinal epithelium to secretagogues occurs in different models of intestinal injury, including radiation enteropathy, and in human disease. While this impairment of barrier function has been linked to increased inducible nitric oxide synthase (iNOS) activity, the cellular target of NO in this phenomenon is not known, although recent studies suggest that some isoforms of adenylate cyclase are inhibited by NO.

Aims: To determine adenylate cyclase isoform distribution in colonic epithelial cells and, in particular, the physiological significance of NO inhibitable adenylate cyclase isoforms 5 and 6 in radiation induced epithelial secretory dysfunction.

Methods: Reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry, and immunohistochemistry were used to examine adenylate cyclase expression. The responsiveness of mouse colon to secretagogues 72 hours post-15 Gy gamma radiation or following in vitro exposure to NO donors was measured in Ussing chambers. Also, cAMP, cGMP, and ATP levels were measured.

Results: RT-PCR, immunocytochemistry, and immunohistochemistry showed that adenylate cyclase 5 was expressed in mouse colon, and isoforms 5 and 6 were expressed in human biopsies and intestinal epithelium. Pharmacological studies showed that these isoforms are functionally important in chloride secretion. NO mediated hyporesponsiveness to secretagogues is primarily a result of decreased adenylate cyclase activity, and not G_i activation or decreased cellular ATP levels.

Conclusions: NO inhibitable isoforms of adenylate cyclase are expressed in mouse and human secretory colonic epithelia, and appear to be the target of radiation induced NO to reduce the responsiveness to cAMP dependent secretagogues.

Keywords: ionising radiation; nitric oxide synthase; nitric oxide; chloride secretion

Abbreviations: Isc, short circuit current; AC, adenylate cyclase; SNP, sodium nitroprusside; PAPA NONOate, 1-propyl-1'-(aminopropyl)-2-hydroxy-2-nitrohydraxine; NO, nitric oxide; iNOS, inducible nitric oxide synthase; RT-PCR, reverse transcription-polymerase chain reaction; LPS, lipopolysaccharide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; TBS, Tris buffered saline; PBS, phosphate buffered saline; 1400w, N-(3-(aminomethyl(benzyl)acetamide; L-NIL, L-N⁶-(1-iminoethyl)lysine; PKA, protein kinase A; PTX, pertussis toxin

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