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1.
Fig. 2

Fig. 2. sAC localization in the kidney. From: Bicarbonate-regulated Adenylyl Cyclase (sAC) Is a Sensor That Regulates pH-dependent V-ATPase Recycling.

Immunolocalization of sAC in kidney cortex (A), inner stripe of the outer medulla (B) and inner medulla (C). Cortical (cTAL) and medullary (mTAL) thick ascending limb of Henle, and distal tubules (DT) show significant expression of sAC. Collecting ducts (CD), which were identified by positive double labeling using V-ATPase antibodies (not shown), also show significant sAC immunolabeling. G identifies glomeruli, and PT identifies proximal tubules. Bars, 25 μm.

Nuria Pastor-Soler, et al. J Biol Chem. 2003 December 5;278(49):49523-49529.
2.
Fig. 6

Fig. 6. Direct inhibition of purified rsACt by 2-hydroxyestradiol. From: Bicarbonate-regulated Adenylyl Cyclase (sAC) Is a Sensor That Regulates pH-dependent V-ATPase Recycling.

NaHCO3-stimulated sAC activity was assayed in the presence of the indicated amounts of estradiol (closed squares), or 2-hydroxyestradiol (2-OH estradiol; open squares). Values represent averages of at least three independent determinations with standard deviations indicated. Inset, sAC activity was assayed as a function of varying ATP-Mg2+ in the presence of 40 mM NaHCO3 and excess (15 mM) MgCl2 in the absence of inhibitor, or in the presence of 3, 6, or 12 μM 2-hydoxyestradiol. Values shown in inset represent single determinations.

Nuria Pastor-Soler, et al. J Biol Chem. 2003 December 5;278(49):49523-49529.
3.
Fig. 3

Fig. 3. V-ATPase recycling at physiological luminal pH. From: Bicarbonate-regulated Adenylyl Cyclase (sAC) Is a Sensor That Regulates pH-dependent V-ATPase Recycling.

A and B, cauda epididymis tubule perfused with PBS containing HRP at the physiological luminal pH of 6.8. Anti-HRP staining revealed high endocytic activity of clear cells (A, arrows) compared with adjacent principal cells. Clear cells were identified by their positive immunofluorescence staining for V-ATPase (B, arrows). The box indicates the apical region of clear cells, which is examined in more detail in panel C. C, Electron microscopy double immunogold labeling for HRP and V-ATPase showed that a sub-population of HRP-containing endosomes located in the sub-apical region (15 nm gold, arrowheads) also contain V-ATPase (8 nm gold, arrows). Bars, 50 μ m (A and B), 120 nm (C).

Nuria Pastor-Soler, et al. J Biol Chem. 2003 December 5;278(49):49523-49529.
4.
Fig. 1

Fig. 1. sAC detection and localization in the epididymis. From: Bicarbonate-regulated Adenylyl Cyclase (sAC) Is a Sensor That Regulates pH-dependent V-ATPase Recycling.

A, RT-PCR detection of mRNA transcripts specific for sAC and markers specific for clear cells in epididymal epithelial cells isolated by laser capture microdissection. B1, B1 subunit of the V-ATPase; E, E subunit of the V-ATPase; CAII, carbonic anhydrase II; bp, 20-bp DNA ladder. B, detection of sAC protein by Western blot in total homogenates from rat cauda epididymidis using a monoclonal anti-sAC antibody. A strong band at around 48 kDa was detected indicating the presence of the active form of sAC. C, double immunofluorescence staining showing V-ATPase (green) and sAC (red) in rat cauda epididymidis. A strong sAC staining is observed in clear cells, identified by their positive staining for V-ATPase. sAC immunoreactivity is also observed in sperm, consistent with its previously described localization. Sub-epithelial muscle cells are also stained for sAC. Bar, 15 μm.

Nuria Pastor-Soler, et al. J Biol Chem. 2003 December 5;278(49):49523-49529.
5.
Fig. 5

Fig. 5. Role of cAMP, intracellular HCO3− and sAC in V-ATPase recycling. From: Bicarbonate-regulated Adenylyl Cyclase (sAC) Is a Sensor That Regulates pH-dependent V-ATPase Recycling.

Confocal images showing the apical regions of clear cells exposed to different luminal conditions and double-stained for V-ATPase (green) and HRP (red). A, PBS, pH 6.5, containing cpt-cAMP. V-ATPase is mainly located in apical microvilli and no co-localization with HRP is observed, despite the acidic luminal pH. B, PBS pH 7.8 containing acetazolamide. V-ATPase staining appears in the sub-apical and cytoplasmic regions of the cell. Very few V-ATPase-positive apical microvilli are visualized, despite the alkaline luminal pH of 7.8. C, perfusion with PBS, pH 7.8, containing acetazolamide followed by cpt-cAMP. V-ATPase-positive microvilli are clearly present, indicating that cAMP induced the apical translocation of V-ATPase. D, perfusion with PBS, pH 7.8, containing the sAC inhibitor, 2-hydroxyestra-diol. V-ATPase is distributed between apical microvilli and sub-apical vesicles. A large amount of V-ATPase co-localizes with HRP-containing endosomes (yellow), indicating that inhibition of sAC prevents the usual alkaline pH-induced translocation of V-ATPase into apical microvilli. E, perfusion with PBS, pH 7.8, containing 2-hydroxyestradiol followed by cpt-cAMP (in the presence of 2-hydroxyestradiol). V-ATPase is present in apical microvilli and minimal co-localization with HRP is observed, indicating that cpt-cAMP induced a translocation of V-ATPase into apical microvilli, despite sAC inhibition. F, perfusion with PBS, pH 7.8, containing estradiol, which does not inhibit sAC. Predominant apical microvilli localization of V-ATPase is seen indicating that estradiol failed to inhibit the apical translocation of V-ATPase caused by alkaline pH. The arrows point to the frontier between the apical microvilli membrane domain and the subapical region of the cells. Bar, 5 μm.

Nuria Pastor-Soler, et al. J Biol Chem. 2003 December 5;278(49):49523-49529.
6.
Fig. 4

Fig. 4. Effect of luminal pH on V-ATPase recycling. From: Bicarbonate-regulated Adenylyl Cyclase (sAC) Is a Sensor That Regulates pH-dependent V-ATPase Recycling.

A, A′, and A″, confocal images showing the apical region of three clear cells from different epididymis perfused with PBS at pH 6.5 and double-labeled for V-ATPase (green) and HRP (red). V-ATPase is distributed between the apical membrane and intracellular sub-apical vesicles. The yellow staining indicates partial co-localization of HRP-containing endosomes with V-ATPase-containing vesicles. B, B′, and B″, three confocal microscope examples of the apical region of clear cells from different epididymis perfused with PBS at pH 7.8. V-ATPase is mainly located in apical microvilli (green) and not in HRP-containing endosomes (red), which are present in the sub-apical region. The absence of yellow staining indicates no co-localization of V-ATPase and HRP. The arrows indicate the frontier (often visible as a dark line) between apical microvilli and the sub-apical region. C and D, immunogold electron microscope images showing V-ATPase staining in clear cells at pH 6.5 (C) and pH 7.8 (D). At luminal pH 6.5, V-ATPase is present largely in intracellular vesicles, and few gold particles are seen in apical microvilli. At luminal pH 7.8, V-ATPase is located mainly in well developed apical microvilli and fewer particles are seen inside the cell. E, histogram showing the mean effect of luminal pH on the number of V-ATPase-associated gold particles per unit length of apical membrane. At least 10 clear cells were analyzed for each perfusion condition. Data are expressed as means ± S.E. (*, p < 0.005). Bars, 5 μm (A and B), 720 nm (C and D).

Nuria Pastor-Soler, et al. J Biol Chem. 2003 December 5;278(49):49523-49529.
7.
Fig. 7

Fig. 7. Effect of luminal bicarbonate concentration on sAC-mediated V-ATPase recycling. From: Bicarbonate-regulated Adenylyl Cyclase (sAC) Is a Sensor That Regulates pH-dependent V-ATPase Recycling.

Confocal images showing the apical region of clear cells from cauda epididymidis perfused with either PBS, pH 7.1 (A), or modified Hanks buffer, pH 7.1, containing 12 mM bicarbonate (5% CO2) in the absence (B) or presence of 2-hydroxyestradiol (C). Double staining for V-ATPase (green) and HRP (red) was performed. A, V-ATPase is distributed between apical microvilli and sub-apical vesicles. The yellow staining indicates partial co-localization of HRP-containing endosomes with V-ATPase-containing vesicles. B, in the presence of bicarbonate, microvilli are more developed, and the V-ATPase is mainly located in apical microvilli. C, 2-hydroxyestradiol induced a significant reduction of the size of microvilli, and V-ATPase is distributed between apical microvilli and sub-apical vesicles. Thus, inhibition of sAC prevents the bicarbonate-induced apical V-ATPase translocation. D, E, and F, the level of V-ATPase in microvilli was quantified by measuring the area occupied by V-ATPase-labeled microvilli (enclosed by the white line), normalized for the width of the cells at the apical pole (blue line) for each cell. G, histogram showing the mean values obtained from at least 10 cells from three to four different epididymis (n = 30 –50 cells per condition). The area occupied by V-ATPase-positive microvilli is significantly higher in the presence of bicarbonate (Bic) than in the absence of bicarbonate at the same pH (PBS). In the presence of the sAC inhibitor 2-hydroxyestradiol (Bic + 2HE), the microvilli area is significantly lower compared with the bicarbonate control (Bic). This result demonstrates that 2-hydroxyestradiol prevented the bicarbonate-mediated V-ATPase translocation into the apical microvilli. Results are expressed as mean ± S.E. *, p < 0.001 versus PBS or Bic + 2HE. Bar, 5 μm.

Nuria Pastor-Soler, et al. J Biol Chem. 2003 December 5;278(49):49523-49529.

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