ANTISENSE OLIGODEOXYNUCLEOTIDE TO PKC-DELTA BLOCKS ALPHA(1)-ADRENERGIC ACTIVATION OF NA-K-2CL COTRANSPORT

A role for protein kinase C (PKC)-delta and -zeta: isotypes in alpha(1 )-adrenergic regulation of human tracheal epithelial Na-K-2Cl cotransp ort was studied with the use of isotype-specific PKC inhibitors and an tisense oligodeoxynucleotides to PKC-delta or -zeta mRNA. Rottlerin, a PKC-delta inhibitor, blocked 72% of basolateral-to-apical, bumetanide -sensitive Cl-36 flux in nystatin-permeabilized cell monolayers stimul ated with methoxamine, an alpha(1)-adrenergic agonist, with a 50% inhi bitory concentration of 2.3 mu M. Methoxamine increased PKC activity i n cytosol and a particulate fraction; the response was insensitive to PKC-alpha and -beta(II) isotype-specific inhibitors, but was blocked b y general PKC inhibitors and rottlerin. Rottlerin also inhibited metho xamine-induced PKC activity in immune complexes of PKC-delta, but not PKC-zeta. At the subcellular level, methoxamine selectively elevated c ytosolic PKC-delta activity and particulate PKC-zeta activity. Pretrea tment of cell monolayers with antisense oligodeoxynucleotide to PKC-de lta for 48 h reduced the amount of whole cell and cytosolic PKC-delta, diminished whole cell and cytosolic PKC-delta activity, and blocked m ethoxamine-stimulated Na-K-2Cl cotransport. Sense oligodeoxynucleotide to PKC-delta and antisense oligodeoxynucleotide to PKC-zeta did not a lter methoxamine-induced cotransport activity. These results demonstra te the selective activation of Na-K-2Cl cotransport by cytosolic PKC-d elta.