ANTISENSE OLIGONUCLEOTIDE TO PRC-EPSILON ALTERS CAMP-DEPENDENT STIMULATION OF CFTR IN CALU-3 CELLS

Protein kinase C (PKC) regulates cystic fibrosis transmembrane conduct ance regulator (CFTR) channel activity but the PKC signaling mechanism is not yet known. The goal of these studies was to identify PKC isoty pe(s) required for control of CFTR function. CFTR activity was measure d as Cl-36 efflux in a Chinese hamster ovary cell line stably expressi ng wild-type CFTR (CHO-wtCFTR) and in a Calu-3 cell line. Chelerythrin e, a PKC inhibitor, delayed increased CFTR activity induced with phorb ol 12-myristate 13-acetate or with the cAMP-generating agents (-)-epin ephrine or forskolin plus 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate. Immunoblot analysis of Calu-3 cells revealed that PKC- alpha, -beta(II), -delta, -epsilon, and -zeta were expressed in conflu ent cell cultures. Pretreatment of cell monolayers with Lipofectin plu s antisense oligonucleotide to PKC-E for 48 h prevented stimulation of CFTR with (-)epinephrine, reduced PKC-epsilon activity in unstimulate d cells by 52.1%, and decreased PKC-epsilon mass by 76.1% but did not affect hormone-activated protein kinase A activity. Sense oligonucleot ide to PKC-epsilon and antisense oligonucleotide to PKC-delta and -zet a did not alter (-)-epinephrine-stimulated CFTR activity. These result s demonstrate the selective regulation of CFTR function by constitutiv ely active PKC-epsilon.