AMP-activated protein kinase inhibits alkaline pH- and PKA-induced apical vacuolar H+-ATPase accumulation in epididymal clear cells

Abstract

Acidic luminal pH and low [HCO(3)(-)] maintain sperm quiescent during maturation in the epididymis. The vacuolar H(+)-ATPase (V-ATPase) in clear cells is a major contributor to epididymal luminal acidification. We have shown previously that protein kinase A (PKA), acting downstream of soluble adenylyl cyclase stimulation by alkaline luminal pH or HCO(3)(-), induces V-ATPase apical membrane accumulation in clear cells. Here we examined whether the metabolic sensor AMP-activated protein kinase (AMPK) regulates this PKA-induced V-ATPase apical membrane accumulation. Immunofluorescence labeling of rat and non-human primate epididymides revealed specific AMPK expression in epithelial cells. Immunofluorescence labeling of rat epididymis showed that perfusion in vivo with the AMPK activators 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) or A-769662 induced a redistribution of the V-ATPase into subapical vesicles, even in the presence of a luminal alkaline (pH 7.8) buffer compared with that of controls perfused without drug. Moreover, preperfusion with AICAR blocked the PKA-mediated V-ATPase translocation to clear cell apical membranes induced by N(6)-monobutyryl-cAMP (6-MB-cAMP). Purified PKA and AMPK both phosphorylated V-ATPase A subunit in vitro. In HEK-293 cells [(32)P]orthophosphate in vivo labeling of the A subunit increased following PKA stimulation and decreased following RNA interference-mediated knockdown of AMPK. Finally, the extent of PKA-dependent in vivo phosphorylation of the A subunit increased with AMPK knockdown. In summary, our findings suggest that AMPK inhibits PKA-mediated V-ATPase apical accumulation in epididymal clear cells, that both kinases directly phosphorylate the V-ATPase A subunit in vitro and in vivo, and that AMPK inhibits PKA-dependent phosphorylation of this subunit. V-ATPase activity may be coupled to the sensing of acid-base status via PKA and to metabolic status via AMPK.

Fig. 1.

AMP-activated protein kinase (AMPK) is expressed in epididymal and vas deferens epithelia in rat and non-human primate by Western blot. A: Western blots of epididymal cauda homogenates from the rhesus monkey (Macaca mulatta) and rat revealed the expression of AMPK-α in these tissues. B: specificity of this antibody labeling in these tissues was demonstrated by the lack of signal when the primary antibody was preincubated with the immunizing peptide. Western blot of recombinant AMPK-α using the same antibody revealed one band at ∼63 kDa, and this band was completely competed off by preincubation of the antibody with the peptide (not shown).

Fig. 2.

AMPK is expressed in epididymal and vas deferens epithelia in rat and non-human primate (Macaca mulatta) by immunofluorescence labeling. A and B: confocal images of immunofluorescence staining image of rat epididymal epithelium in the caput (A) and cauda (B) using an anti-AMPK-α antibody demonstrated diffuse cytoplasmic staining in epididymal epithelial cells, with a higher intensity of staining in clear cells expressing the V-ATPase (E subunit, red). Scale bar = 125 μm. C: higher magnification confocal image of immunofluorescecne staining of rat cauda epididymal epithelium using the anti-AMPK-α antibody. Scale bar = 5 μm. D: preincubation of the anti-AMPK-α antibody with its immunizing peptide substantially reduced the staining in adult rat cauda epididymis, demonstrating the specificity of this antibody for immunofluorescence labeling. E: confocal immunofluorescence labeling of epididymal cauda from adult Macaca mulatta using the same anti-AMPK-α antibody demonstrated diffuse cytoplasmic staining in epithelial cells (green). The epithelial clear cells were labeled with an antibody against the vacuolar ATPase (V-ATPase) E subunit (red). Scale bar = 10 μM.

Fig. 3.

AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) inhibits the pH-mediated V-ATPase accumulation at the apical membrane of clear cells. A and B: confocal images of double immunofluorescence labeling for V-ATPase distribution (green) and for the endocytic marker horseradish peroxidase (HRP, red) in clear cells perfused for 60 min with phosphate-buffered saline (PBS) pH 7.8 (A) or PBS pH 7.8 + AICAR (2 mM) (B). Arrows demarcate the base of the microvilli in clear cells. Treatment of cells with the AMPK activator AICAR prevented the elongation of apical microvilli observed upon exposure to pH 7.8 and prevented accumulation of the V-ATPase at the apical pole. C and D: level of V-ATPase accumulation in microvilli in the cells from A and B was quantified by measuring the area occupied by V-ATPase-labeled microvilli (enclosed in the white line), normalized for the width of the cells at the apical pole (blue line) for each cell. E: quantification of the surface occupied by V-ATPase-labeled microvilli of clear cells normalized by the apical width of each cell under the conditions shown in A and B. Data shown are means ± SE from three tissues and at least 30 cells per condition (*P < 0.05). Scale bar = 5 μm.

Fig. 4.

The AMPK activator A-769662 inhibits the pH-mediated V-ATPase accumulation at the apical membrane of clear cells. A and B: confocal images of double immunofluroscence labeling of the V-ATPase distribution (green) and the endocytic marker HRP (red) in clear cells perfused for 60 min with PBS pH 7.8 (A) or PBS pH 7.8 containing AMPK activator Abbott compound A-769662 (200 μM) (B). Arrows demarcate the base of the microvilli in clear cells. Treatment of cells A-769662 prevented the elongation of apical microvilli observed upon exposure to pH 7.8 and prevented accumulation of the V-ATPase at the apical pole. C: quantification of the surface occupied by V-ATPase-labeled microvilli of clear cells normalized by the apical width of each cell under the conditions shown in A and B. Data shown are means ± SE from three tissues and at least 30 cells per condition (*P < 0.05). Scale bar = 5 μm.

Fig. 5.

AMPK activator AICAR inhibits the PKA-mediated V-ATPase accumulation at the apical membrane of clear cells. A, B, and C: confocal images of the distribution of V-ATPase (green) and of the endocytic marker HRP (red) by immunofluorescence labeling in clear cells perfused with PBS pH 6.5 for 75 min (A); PBS pH 6.5 for 45 min, followed by PBS pH 6.5 containing PKA activator N⁶-monobutyryl-cAMP (6-MB-cAMP, 100 μM) for an additional 30 min (B); or PBS (pH 6.5) containing AICAR (2 mM) for 45 min, followed by PBS (pH 6.5) plus AICAR plus 6-MB-cAMP for an additional 30 min (C). Arrows demarcate the bases of the microvilli. Treatment of cells with AMPK activator AICAR prevented the elongation of apical microvilli observed upon exposure to a specific PKA activator 6-MB-cAMP and prevented accumulation of the V-ATPase at the apical pole. D: quantification of V-ATPase accumulation in apical microvilli for at least three independent perfusions and at least 30 cells per condition. Data shown are the means ± SE (*P < 0.05, relative to PBS, pH 6.5; #P < 0.05, relative to PBS, pH 6.5; **P < 0.05, relative to + 6-MB-cAMP). Scale bar = 7.5 μm.

Fig. 6.

PKA and AMPK phosphorylate the V-ATPase A subunit in vitro. Recombinant FLAG-tagged A subunit was expressed in HEK-293 cells, immunoprecipitated, and incubated with [γ-³²P]ATP in the presence or absence of PKA catalytic subunit or in the presence or absence of AMPK holoenzyme. A phosphoscreen image (top) and immunoblot (bottom) of the same membrane are shown (representative of three experiments). In the top, the V-ATPase A subunit becomes phosphorylated in the presence of PKA and also in the presence of AMPK (first and third lanes). Both the PKA catalytic subunit and the AMPK-α subunit become autophosphorylated, as indicated. The immunoblot (bottom) reveals similar loading of V-ATPase A subunit in all lanes.

Fig. 7.

Tetracycline-inducible knockdown of AMPK-α1 in HEK-293 cells. Immunoblots for AMPK-α1 (top band) and β-actin (bottom band) of lysates derived from HEK-293 cells that were stably transfected with either a control short hairpin RNA (shRNA) that does not silence any known mammalian gene (CON) or an shRNA designed to specifically knock down AMPK-α (KD). Upon exposure to doxycycline for 3 days a ∼50% decrease in AMPK-α1 protein expression normalized to β-actin was observed.

Fig. 8.

PKA- and AMPK-dependent in vivo phosphorylation of the V-ATPase A subunit in HEK-293 cells. FLAG-tagged A subunit was transfected into HEK-293 cells expressing an irrelevant mammalian shRNA (Fig. 7, “CON” cells) or in AMPK KD cells expressing an shRNA for AMPK-α1. Cells were then incubated with [³²P]orthophosphate for 2 h in the presence of a PKA activator (6-MB-cAMP, last 20 min of labeling period) or in the presence of PKA inhibitor myristoylated protein kinase inhibitor (mPKI) (for entire labeling period), followed by lysis of the cells, immunoprecipitation using an anti-FLAG antibody, SDS-PAGE, and immunoblotting using an anti-FLAG antibody. A: typical phospho-screen image (top) revealing the signal of phosphorylated A subunit under the indicated conditions in CON or AMPK KD cells. The Western blot (bottom) confirms similar protein expression and loading of the gel for the different conditions. B: quantification of V-ATPase A subunit phosphorylation signal normalized for protein expression in vivo (*P = 1.6 × 10⁻⁵; **P = 1.3 × 10⁻⁴; #P = 3.7 × 10⁻⁴; ##P = 3.2 × 10⁻⁶; §P = 4.9 × 10⁻⁶, and §§P = 0.016) indicates that the differences between the indicated conditions were statistically significant by analysis of variance; (n = 4 experiments). C: relative PKA-dependent A subunit phosphorylation in CON and AMPK KD cells as calculated by the difference in phosphorylation signal between 6-MB-cAMP- and mPKI-treated cells over the total phosphorylation signal with 6-MB-cAMP treatment (comparing lanes 2 and 3 for CON and lanes 5 and 6 for AMPK KD). *P < 0.05, unpaired t-test.