Am J Physiol Cell Physiol AJP: Regulatory, Integrative and Comparative Physiology
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Am J Physiol Cell Physiol 273: C1632-C1640, 1997;
0363-6143/97 $5.00
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Vol. 273, Issue 5, C1632-C1640, November 1997

Antisense oligodeoxynucleotide to PKC-delta blocks alpha 1-adrenergic activation of Na-K-2Cl cotransport

Carole M. Liedtke and Thomas Cole

The Cystic Fibrosis Center and Departments of Pediatrics and of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106

A role for protein kinase C (PKC)-delta and -zeta isotypes in alpha 1-adrenergic regulation of human tracheal epithelial Na-K-2Cl cotransport was studied with the use of isotype-specific PKC inhibitors and antisense oligodeoxynucleotides to PKC-delta or -zeta mRNA. Rottlerin, a PKC-delta inhibitor, blocked 72% of basolateral-to-apical, bumetanide-sensitive 36Cl flux in nystatin-permeabilized cell monolayers stimulated with methoxamine, an alpha 1-adrenergic agonist, with a 50% inhibitory concentration of 2.3 µM. Methoxamine increased PKC activity in cytosol and a particulate fraction; the response was insensitive to PKC-alpha and -beta II isotype-specific inhibitors, but was blocked by general PKC inhibitors and rottlerin. Rottlerin also inhibited methoxamine-induced PKC activity in immune complexes of PKC-delta , but not PKC-zeta . At the subcellular level, methoxamine selectively elevated cytosolic PKC-delta activity and particulate PKC-zeta activity. Pretreatment of cell monolayers with antisense oligodeoxynucleotide to PKC-delta for 48 h reduced the amount of whole cell and cytosolic PKC-delta , diminished whole cell and cytosolic PKC-delta activity, and blocked methoxamine-stimulated Na-K-2Cl cotransport. Sense oligodeoxynucleotide to PKC-delta and antisense oligodeoxynucleotide to PKC-zeta did not alter methoxamine-induced cotransport activity. These results demonstrate the selective activation of Na-K-2Cl cotransport by cytosolic PKC-delta .

bumetanide; immunoprecipitation; tracheal epithelial cells; subcellular fractionation; nystatin permeabilization; transepithelial chloride flux


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