PMA- and ANG II-induced PKC regulation of the renal Na+-HCO3- cotransporter (hkNBCe1).
The renal electrogenic Na(+)-HCO3- cotransporter (hkNBCe1) plays a major role in the bicarbonate reabsorption by the kidney. We examined how PMA- and ANG II-activated PKCs regulate hkNBCe1 expressed with or without the ANG II receptors AT(1B) in Xenopus laevis oocytes. We found that 10 nM PMA halved the hkNBCe1 current detected in voltage-clamped oocytes. A PKC-specific inhibitor GF-109203X, and a specific inhibitor of Ca-dependent conventional PKCalphabetagamma, GO-6976, significantly reduced PMA inhibition. PMA did not alter surface expression of the cotransporters, but it significantly increased hkNBCe1-PKCalphabetagamma membrane association. We found that at 10(-6) M, ANG II halved the hkNBCe1 current detected in oocytes coexpressing cotransporters with AT(1B). A PKC-specific inhibitor GF-109203X, and a PKCepsilon translocation inhibitor epsilonV1-2 peptide as well as BAPTA-AM (but not GO-6976), significantly reduced ANG II inhibition. At 10(-6) M, ANG II significantly decreased surface expression of the cotransporters and increased hkNBCe1-PKCepsilon membrane association. Additionally, we found that at 10(-11) and 10(-10) M ANG II stimulated hkNBCe1 current. This effect was blocked by BAPTA-AM and partially reduced by GF-109203X. We also found that ANG II increased intracellular Ca2+ in fluo 4-loaded oocytes. Our results suggest that 1) PMA inhibition of hkNBCe1 is mediated by Ca-dependent PKCalphabetagamma and 10 nM PMA does not induce downregulation of cotransporter surface expression. 2) ANG II (10(-6) M) inhibition of hkNBCe1 is mediated by both Ca-independent PKCepsilon and downregulation of cotransporter surface expression, possibly triggered by intracellular Ca2+ mobilization. 3) Similar to proximal tubule, acute ANG II has a biphasic effect on hkNBCe1 coexpressed with AT(1B) in X. laevis oocytes.
Perry
Clint
C
Department of Physiology and Biophysics, University of Colorado Health Sciences Center, Mail Stop 8307, P.O. Box 6511, Aurora, CO 80045, USA.
Blaine
Judith
J
Le
Hong
H
Grichtchenko
Irina I
II
D000804:Angiotensin II / Q000494:pharmacology*; D047228:Angiotensin II Type 1 Receptor Blockers / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism / Q000502:physiology; D005260:Female; D006801:Humans; D008954:Models, Biological; D009865:Oocytes / Q000378:metabolism; D011493:Protein Kinase C / Q000494:pharmacology*; D044140:Receptor, Angiotensin, Type 1 / Q000378:metabolism; D027982:Sodium-Bicarbonate Symporters / Q000235:genetics / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014162:Transfection; D014982:Xenopus laevis / Q000235:genetics
D047228:Angiotensin II Type 1 Receptor Blockers; D044140:Receptor, Angiotensin, Type 1; C498898:SLC4A4 protein, human; D027982:Sodium-Bicarbonate Symporters; D000804:Angiotensin II; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium