Anti sense DNA down-regulates proteins kinase C-epsilon and enhances vasopressin-stimulated Na+ absorption in rabbit cortical collecting duct.
Hormonal activation of protein kinase C (PKC) is a major signaling mechanism regulating salt and water transport in the distal nephron. We used antisense DNA to down-regulate a PKC isoform in the rabbit cortical collecting duct (CCD) and examined its role in mediating arginine vasopressin's (AVP) effect on salt transport in the CCD. Immunoblots demonstrate that PKC-epsilon (diacylglycerol sensitive) and PKC-zeta (diacylglycerol insensitive) are the major PKC isoforms in both freshly isolated and primary cultures of rabbit CCDs. Rabbit CCDs grown on semi-permeable supports, displayed a positive baseline short circuit current (Isc), which was abolished by amiloride, demonstrating active Na+ absorption. Both AVP and 8-chloro-phenylthio-cAMP (8CPTcAMP) transiently increased Isc, however, within 40 min Isc fell below baseline. Down-regulation of PKC-epsilon, as confirmed by immunoblot, was achieved either by treatment with a PKC-epsilon-specific antisense oligonucleotide or 48 h of 1 microM PMA. In PKC-epsilon down-regulated cells, 8CPTcAMP produced a sustained, rather than transient, increase in Isc. We suggest cAMP stimulates Na+ transport, but secondary activation of PKC-epsilon results in the sustained inhibition of Na+ transport seen in response to vasopressin in the CCD.
DeCoy
D L
DL
Department of Medicine, Vanderbilt University, Nashville, Tennessee 37232, USA.
Snapper
J R
JR
Breyer
M D
MD
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000494:pharmacology; D016373:DNA, Antisense / Q000737:chemistry; D004553:Electric Conductivity; D005260:Female; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007685:Kidney Tubules, Collecting / Q000378:metabolism*; D008969:Molecular Sequence Data; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D051744:Protein Kinase C-epsilon; D011817:Rabbits; D012964:Sodium / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014667:Vasopressins / Q000494:pharmacology*
D016373:DNA, Antisense; D007527:Isoenzymes; D014667:Vasopressins; D012964:Sodium; D000242:Cyclic AMP; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon; D013755:Tetradecanoylphorbol Acetate