Rectal epithelial expression of protein kinase A phosphorylation of cystic fibrosis transmembrane conductance regulator.

BACKGROUND/AIMS: Human rectal epithelium in cystic fibrosis (CF) shows impaired ion transport in response to theophylline or bethanechol, although it possesses regulatory subunits of adenosine cyclic 3',5'-monophosphate (cAMP)-dependent protein kinase (protein kinase A). Protein kinase A-specific phosphorylation of CF transmembrane conductance regulator (CFTR) in rectal tissues of control and CF volunteers was examined in this study.

METHODS

CFTR was evaluated using a polyclonal antiserum (pre-NBF) raised against a peptide corresponding to residues 415-427 of CFTR. Microsomal membranes from normal and CF rectal mucosa and from T-84 cells were incubated with [gamma 32P]-adenosine triphosphate +/- protein kinase A and subjected to immunoblotting with pre-NBF and autoradiography.

RESULTS

Pre-NBF recognized a single band of 180 kilodaltons. Protein kinase A altered phosphorylation of this 180-kilodalton band 1.4-, 2.2- and 0.9-fold in T-84, normal, and CF rectal membranes, respectively. Catalytic activities of protein kinase A, Ca2+ calmodulin protein kinase, or protein kinase C in control and CF tissues were similar.

CONCLUSIONS

cAMP and Ca(2+)-signaling pathways are normal up to the kinases in CF rectal mucosa. Our results suggest differences in CFTR phosphorylation in normal and CF rectal mucosal membranes.