PMA- and ANG II-induced PKC regulation of the renal Na⁺-HCO₃⁻cotransporter (hkNBCe1)

Abstract

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.

Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) stimulates the electrogenic Na/HCO 3 cotransporter NBCe1-A expressed in Xenopus oocytes

“…Investigators have reported that phosphorylation by activated PKA (3,4) or an increase in cytosolic Ca 2ϩ (5) can change the Na:HCO 3 Ϫ stoichiometry of the transporter. In addition, both cAMP (3) and ATP (6) appear to stimulate an NBC-mediated current, whereas PKC isoforms inhibit transporter activity (7).…”

Electrogenic NBCe1 (SLC4A4), but not electroneutral NBCn1 (SLC4A7), cotransporter undergoes cholinergic-stimulated endocytosis in salivary ParC5 cells

“…We (43,44) reported earlier that signaling via the angiotensin receptor (AT 1 R) stimulates endocytosis and inhibits functional activity of NBCe1 in Xenopus oocytes. Here, we expanded our early observations to investigate regulation of the surface expression of NBCe1 and NBCn1 in untreated and cholinergically stimulated parotid acinar cells.…”

“…Female Xenopus laevis frogs (NASCO) were anesthetized with 1.5 mg/ml tricaine. The ovarian lobes were surgically removed, dissected, and treated with 2 mg/ml collagenase type IA, and oocytes were incubated as described previously (43,44). The cDNAs encoding human NBCe1 and M3R receptors were each subcloned into the pGH19 expression vector.…”

“…Oocytes were injected with 50 nl of 0.5 ng/nl NBCe1 mRNA; 25 nl of 1 ng/nl NBCe1 mRNA plus 25 nl of 1 ng/nl M3R mRNA; or 50 nl of dH2O. Three days later, oocytes were subjected to electrophysiological experiments as described before (43,44). Briefly, oocytes were voltage-clamped at room temperature using a twoelectrode oocyte clamp (Warner Instruments, New Haven, CT) and microelectrodes made by pulling borosilicate glass capillary tubing (Warner Instruments) on a microelectrode puller.…”

“…The currents were filtered at 20 Hz (4-pole Bessel filter) and digitized. An oocyte was placed in a chamber with a 4 ml/min constant superfusion using bath solutions described previously (43,44). Bath solutions were delivered with syringe pumps (Harvard Apparatus, South Natick, MA), and solutions were switched with pneumatically operated valves (Clippard Instrument Laboratory, Cincinnati, OH).…”