Expression of mRNA for δ, η and θ PKC isoforms in the rabbit cortical collecting duct (CCD)

Academic Article

Abstract

  • We previously demonstrated the presence of the PKC α, ε and ζ isoforms in the rabbit CCD and at least two other isoforms which appeared to be η and θ based both on the restriction pattern of RT-PCR products and immunoblotting. The putative θ isoform protein is present at high levels in freshly immunodissected rabbit CCD, but barely detectable after culture of these cells on permeable supports in vitro. Conversely, the η isoform is more heavily expressed in the cultured than in fresh cells (Am J Physiol 270:F766, 1996). Because growth in culture is associated with a change in the phenotype of Na+ transport regulation, we wished to confirm the identities of the isoforms by sequencing the RT-PCR products. The PCR products formed with a degenerate sense (cysteine-rich region) and antisense (ATP-binding region) primers were cloned into the plasmid vector pCR 2.1 using TA cloning (Invitrogen) and were analyzed by automated DNA sequencing. The 433 bp product from fresh CCD was found to be 83% and 86% identical at the nucleotide (N) and amino acid (A) levels to the mouse PKC-θ, whereas the 332 bp product of cultured CCD showed 84% (N) and 94% (A) sequence identities to the rat PKC-η. An unexpected 359 bp product from fresh CCD displayed 82% (N) and 81% (A) sequence identities to the rat PKC-δ. We conclude that the rabbit CCD expresses the δ, η, and θ PKC isoforms (not previously sequenced in the rabbit), and that their expression levels change in culture.
  • Published In

  • The FASEB Journal  Journal
  • Author List

  • Wilborn TW; Schafer JA
  • Volume

  • 11
  • Issue

  • 3