JBC Ideal method for primary cell transfection

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M309543200 on September 25, 2003

J. Biol. Chem., Vol. 278, Issue 49, 49523-49529, December 5, 2003
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/49/49523    most recent
M309543200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Pastor-Soler, N.
Right arrow Articles by Breton, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pastor-Soler, N.
Right arrow Articles by Breton, S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Bicarbonate-regulated Adenylyl Cyclase (sAC) Is a Sensor That Regulates pH-dependent V-ATPase Recycling*

Nuria Pastor-Soler{ddagger}, Valérie Beaulieu{ddagger}, Tatiana N. Litvin§, Nicolas Da Silva{ddagger}, Yanqiu Chen§, Dennis Brown{ddagger}, Jochen Buck§, Lonny R. Levin§, and Sylvie Breton{ddagger}||

From the {ddagger}Program in Membrane Biology and Renal Unit, Massachusetts General Hospital, Charlestown, Massachusetts 02129, the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215, and the §Department of Pharmacology, Weill Medical College of Cornell University, New York, New York 10021

Received for publication, August 27, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Modulation of environmental pH is critical for the function of many biological systems. However, the molecular identity of the pH sensor and its interaction with downstream effector proteins remain poorly understood. Using the male reproductive tract as a model system in which luminal acidification is critical for sperm maturation and storage, we now report a novel pathway for pH regulation linking the bicarbonate activated soluble adenylyl cyclase (sAC) to the vacuolar H+ATPase (V-ATPase). Clear cells of the epididymis and vas deferens contain abundant V-ATPase in their apical pole and are responsible for acidifying the lumen. Proton secretion is regulated via active recycling of V-ATPase. Here we demonstrate that this recycling is regulated by luminal pH and bicarbonate. sAC is highly expressed in clear cells, and apical membrane accumulation of V-ATPase is triggered by a sAC-dependent rise in cAMP in response to alkaline luminal pH. As sAC is expressed in other acid/base transporting epithelia, including kidney and choroid plexus, this cAMP-dependent signal transduction pathway may be a widespread mechanism that allows cells to sense and modulate extracellular pH.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We recently identified bicarbonate-activated soluble adenylyl cyclase (sAC)1 as a chemosensor mediating bicarbonate-dependent elevation of cAMP (1), defining a potential transduction pathway for cells to sense variations in bicarbonate, as well as the closely related parameters, pCO2 and pH (1–3). sAC is distinct from transmembrane adenylyl cyclases. It is insensitive to regulation by forskolin or heterotrimeric G proteins (2) but is directly activated by bicarbonate ions. It does not have predicted transmembrane domains and is present in both soluble and particulate fractions of cellular extracts (4–6). Mammalian sAC is similar to bicarbonate-regulated adenylyl cyclases present in cyanobacteria (1, 2), suggesting there may be a unifying mechanism for the bicarbonate regulation of cAMP signaling in many biological systems.

sAC is highly expressed in spermatozoa (7) where it is proposed to mediate the bicarbonate-dependent cAMP elevation that precedes capacitation, hyperactivated motility, and acrosome reaction needed for fertilization (1). While spermatozoa mature and are stored along the epididymal lumen, they are kept in a quiescent state by an acidic pH of 6.5–6.8 and a low bicarbonate concentration of 2–7 mM (8). We have previously shown (9, 10) that a sub-population of epithelial cells, the so-called clear cells, are important players in the acidification capacity of the epididymis. Clear cells express high levels of the V-ATPase in their apical pole, and are responsible for the bulk of proton secretion in the vas deferens. Proton secretion by clear cells occurs in a chloride-independent but bicarbonate-dependent manner (11). Similarly to kidney intercalated cells, epididymal clear cells regulate their rate of proton secretion via V-ATPase recycling between intracellular vesicles and the apical plasma membrane (12). In these cells, as well as proton-secreting cells in the turtle bladder, an increase in V-ATPase surface expression and in apical surface area (including microvilli) closely correlates with an increase in proton secretion (13–15). Proton-secreting epithelial cells actively regulate their rate of proton secretion in response to variations in the pH of their immediate environment (15). However, the molecular entities underlying this response still remain unknown. In the present study, we tested whether bicarbonate-regulated sAC might play a role in the dynamic V-ATPase recycling that occurs in these cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Laser Capture Microdissection and RT-PCR—Epithelial cells from rat cauda epididymidis were harvested by laser capture microdissection, and mRNA was extracted and amplified in vitro following a T7-based amplification procedure, as we recently described (16). For RTPCR, oligonucleotide primer pairs were designed to amplify a short sequence in the 3' end of the cDNA. Primers were synthesized by Sigma-Genosys (The Woodlands, TX) and are listed in Table I. The identity of PCR products was confirmed by direct sequencing (MGH, Molecular Biology DNA Sequencing Core Facility).


View this table:
[in this window]
[in a new window]
  		TABLE I
Sequence of the primers used for PCR PPB1, B1 subunit of the V-ATPase; PPE, E subunit of the V-ATPase; CAII, carbonic anhydrase II.

 
Western Blotting—Adult rats were anesthetized and perfused through the left ventricle with PBS (10 mM phosphate buffer containing 0.9% NaCl), pH 7.4, containing protease inhibitors (Complete, Roche). The epididymis was removed and the cauda region was dissected and homogenized. Electrophoresis and immunoblotting were performed as described previously (16), using a monoclonal antibody (R21) raised against the catalytic regions (C1 and C2) of sAC (6). Protein was added at 20 µg/lane, and the antibody was used at a concentration of 1:250.

Tissue Fixation and Immunofluorescence—Adult male Sprague-Dawley rats were anesthetized with sodium pentobarbital (65 mg/kg body weight, intraperitoneally) and perfused via the left ventricle with PBS (pH 7.4) followed by a fixative containing 4% paraformaldehyde, 10 mM sodium periodate, 70 mM lysine, and 5% sucrose (PLP) as described previously (11, 12). Kidney and male reproductive tract organs were harvested and further fixed by immersion in the same fixative overnight. Immunofluorescence was performed on 4 µm cryostat sections. An affinity-purified chicken polyclonal antibody against the E subunit of the V-ATPase was used. This antibody has been characterized previously (17, 18). To localize sAC, two antibodies were used. An affinity-purified polyclonal rabbit antiserum described previously (1) and monoclonal antibody R21 (6), both against the catalytic regions (C1 and C2) of sAC.

In Vivo Perfusion of the Distal Cauda Epididymidis—Adult male Sprague-Dawley rats were anesthetized with sodium pentobarbital as described above. The vas deferens was cannulated through the lumen with a micro cannula (0.4 mm OD, 0.2 mm ID; Kent Scientific Coorporation, Torrington, CT) connected to a 10-ml syringe. A small incision was made in the distal cauda epididymal region to allow the perfusate to exit the tubule. Perfusion was performed retrogradely at a rate of 45 µl/min using a syringe pump (Model 341B, Fisher). The lumen was initially washed free of sperm with PBS (10 mM sodium phosphate, 2 mM potassium phosphate, 137 mM NaCl, 2.7 mM KCl) adjusted to different pH values (6.5, 6.8, 7.1, or 7.8), as indicated under "Results." Horseradish peroxidase (HRP, Sigma) was added to the perfusate at a concentration of 5 mg/ml to detect endocytosis in the absence or presence of inhibitors and/or cpt-cAMP as indicated under "Results." At the end of the experimental period, the luminal solution was changed for HRP-free ice-cold PBS for 3 min (in the continued presence of agonists or inhibitors, if applicable) to wash the lumen free of HRP. The vas deferens and cauda epididymidis were harvested and fixed by immersion in a solution containing 4% paraformaldehyde, 10 mM sodium periodate, 75 mM lysine, and 5% sucrose (PLP) for 5 h at room temperature, or overnight at 4 °C (11, 12). Tissues were then washed in PBS, pH 7.4, and stored in PBS containing 0.02% sodium azide. At least three vas deferens and cauda epididymidis were perfused for each condition described. A minimum of 10 cells per tissue were examined by confocal microscopy for a total of at least 30 cells per condition.

Immunogold Electron Microscopy—Small pieces of PLP-fixed epididymis were post-fixed by immersion in phosphate buffer containing 4% paraformaldehyde and 0.05% glutaraldehyde for 4 h at room temperature. For V-ATPase immunogold staining, pieces of tissues were embedded at low temperature with HM20 resin as described previously (10). Double labeling for V-ATPase and HRP was performed on LR White Resin embedded sections.

Quantification of Gold Particle Labeling—The relative amount of V-ATPase present in the apical microvilli of clear cells was determined from the number of V-ATPase-associated gold particles per unit length of apical membrane. Gold particles present in the apical membrane including microvilli were counted for each cell, and the number was divided by the total length of apical membrane and microvilli for that cell. Using a Wacom graphics tablet and NIH Image software, at least ten cells were analyzed in each group. Data are presented as mean ± S.E.

Cyclase Assay—Cyclase assays were performed in 100 µl reaction volume with 100 ng of His6-tagged rat sACt purified over a nickelnitrilotriacetic acid affinity column as described (1) in the presence of 50 mM Tris-HCl buffer, pH 7.5, 20 mM creatinephosphate, 100 units/ml creatine phosphokinase, 1 mM cAMP, 2.5 mM ATP, 5 mM MgCl2, and 50 mM NaHCO3 for 30 min at 30 °C. Estrogen compounds were purchased from Steraloids, Inc. (Newport, RI).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
sAC Expression and Localization in the Epididymis and Kidney—Epididymal epithelial cells were harvested by laser capture microdissection, and mRNA was amplified in vitro. RT-PCR detected sAC mRNA, and the presence of other clear cell markers confirmed the validity of this preparation (Fig. 1A). Western blot analysis using a specific monoclonal antibody, R21, against the N-terminal portion of sAC (6) detected a major band at around 48 kDa, the predicted molecular mass of the sACt splice variant (2, 19), in the cauda epididymidis (Fig. 1B). sAC was localized by immunofluorescence using two different anti-sAC antibodies. Monoclonal antibody R21 gave a strong staining in clear cells, which were identified by their positive staining for the V-ATPase (Fig. 1C). No specific immunoreactivity was detected in adjacent principal cells, but surrounding muscle tissue and, as previously described (1, 7), spermatozoa were positive. The same pattern of staining was obtained using an affinity-purified anti-sAC rabbit polyclonal antiserum, but no staining was detected using control pre-immune serum (data not shown). sAC is also expressed in the kidney, where it is located in epithelial cells of distal tubules (Fig. 2A), thick ascending limb of Henle (Fig. 2, A and B) and collecting ducts (Fig. 2, B and C), consistent with previous results showing bicarbonate-stimulated adenylyl cyclase activity in rat kidney (20). Proton secretion is a common function of epididymal clear cells and of renal distal tubules, thick ascending limb of Henle, and collecting duct intercalated cells. Thus, sAC is highly expressed in specialized proton-secreting cells of epididymal and renal epithelia, where it might play a role in regulating acid/base transport in these tissues. To test for a potential role of sAC in modulating proton secretion, we used the epididymis as a model system.



View larger version (55K):
[in this window]
[in a new window]
  		FIG. 1.
sAC detection and localization in the epididymis. 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.

 



View larger version (86K):
[in this window]
[in a new window]
  		FIG. 2.
sAC localization in the kidney. 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.

 
V-ATPase Recycling Is Modulated by Luminal pH—Cauda epididymidis was perfused in vivo through the lumen with HRP, a marker of endocytosis, in PBS adjusted to different pH values. Double immunofluorescence labeling for HRP and VATPase was performed on PLP-fixed cryostat sections. At the physiological luminal pH of 6.8, clear cells, identified by their positive immunoreactivity for V-ATPase, show a high endocytic activity compared with adjacent principal cells (Fig. 3, A and B, arrows). Electron microscopy double immunogold labeling for HRP and V-ATPase showed that a sub-population of HRP-containing endosomes clearly contain V-ATPase, indicating that the proton pump is actively recycled at physiological luminal pH (Fig. 3C). The effect of luminal pH variations on V-ATPase recycling was examined using confocal microscopy. At the acidic pH of 6.5, V-ATPase is distributed between the apical microvilli and intracellular sub-apical vesicles (Fig. 4, A, A', and A"). Double labeling for HRP indicated that the VATPase-containing vesicles partially co-localize with HRP-containing endosomes (yellow staining), indicating that a significant amount of V-ATPase is located in the endocytic compartment. In contrast, at the alkaline luminal pH of 7.8, V-ATPase is mainly located in apical microvilli (Fig. 4, B, B', and B", green) and not in sub-apical HRP-containing endosomes (red staining). Almost no co-localization of V-ATPase and HRP is observed, indicating a very low rate of V-ATPase internalization. In addition, clear cells exposed to luminal pH 7.8 exhibit more developed V-ATPase-positive microvilli compared with cells exposed to pH 6.5. The lack of co-localization of V-ATPase with HRP-labeled endosomes and the increased length of V-ATPase positive microvilli indicate that translocation of V-ATPase from sub-apical vesicles to the apical membrane occurs at alkaline luminal pH. These results were confirmed by immunogold electron microscopy showing intracellular vesicle localization of V-ATPase at luminal pH 6.5 (Fig. 4C), and its predominant localization in extensive apical microvilli at pH 7.8 (Fig. 4D). Quantification of the number of V-ATPase-associated gold particles showed a significant increase in the apical membrane density of V-ATPase molecules at alkaline pH compared with acidic pH (Fig. 4E; p < 0.05). Thus, apical V-ATPase amplification occurs via both microvilli extension and an increase in V-ATPase density in the membrane. These results show that clear cells respond to variations in luminal pH by inducing a rapid (within 15 min), pH-dependent shuttling of V-ATPase between the intracellular HRP-positive endocytic compartment and apical microvilli. This alkaline-induced apical membrane V-ATPase accumulation is a potential mechanism to restore luminal pH to its physiological acidic value.



View larger version (52K):
[in this window]
[in a new window]
  		FIG. 3.
V-ATPase recycling at physiological luminal pH. 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 µm (A and B), 120 nm (C).

 



View larger version (110K):
[in this window]
[in a new window]
  		FIG. 4.
Effect of luminal pH on VATPase 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 VATPase 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).

 
cAMP Induces the Apical Translocation of V-ATPase—Previous studies have shown that cAMP stimulates proton secretion in a variety of acidifying epithelial cells (21–23) and induces the exocytic insertion of many actively recycling membrane proteins, including AQP2, CFTR, and GLUT4 (24–26). To determine whether the pH-related translocation of V-ATPase was modulated by cAMP, cauda epididymidis were perfused at luminal pH of 6.5 with PBS containing the cAMP permeant analogue, 8-(4-chorophenylthio)-cAMP, (cpt-cAMP, 1 mM; Sigma) for 15 min. Under these conditions, V-ATPase was present in the apical microvilli and no co-localization of VATPase and HRP was observed, indicating that cAMP induced a translocation of V-ATPase from intracellular vesicles to the apical membrane, even at acidic luminal pH (Fig. 5A).



View larger version (85K):
[in this window]
[in a new window]
  		FIG. 5.
Role of cAMP, intracellular HCO3- and sAC in 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-hydroxyestradiol. 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.

 
Intracellular Bicarbonate Is Essential for the Alkaline pH-induced V-ATPase Translocation—Because sAC is directly activated by bicarbonate ions, we examined the potential contribution of intracellular HCO3- in the pH- and cAMP-dependent V-ATPase recycling. CO2/HCO3- is the main buffer in biological systems, and the equilibrium between HCO3- and H+ with CO2 and H2O is catalyzed by carbonic anhydrases. Clear cells express very high levels of the cytosolic carbonic anhydrase type II (11, 27), and we have shown that the carbonic anhydrase inhibitor, acetazolamide, markedly decreases the rate of proton secretion in isolated vas deferens (11). We, therefore, hypothesized that intracellular bicarbonate production via carbonic anhydrase type II in response to an alkaline luminal pH (and subsequent increase in intracellular pH) could activate sAC. Elevation of cAMP in response to sAC stimulation would then induce an accumulation of V-ATPase in the apical membrane. Cauda epididymidis were perfused in vivo with PBS at pH 7.8, in the presence of acetazolamide (100 µM; Sigma) for 15 min, to inhibit intracellular bicarbonate production via carbonic anhydrase type II. Under these conditions, clear cells showed an almost complete absence of apical microvilli, and V-ATPase was present in the cytoplasmic and sub-apical regions (Fig. 5B). It, therefore, appears that bicarbonate production via carbonic anhydrase is an essential step in the translocation of the V-ATPase from intracellular vesicles into the apical membrane at alkaline luminal pH. Addition of cpt-cAMP (1 mM) to the luminal perfusate for 10 min at the end of the acetazolamide treatment, but in the continued presence of acetazolamide, induced a reappearance of V-ATPase in apical microvilli (Fig. 5C). Because almost all V-ATPase molecules had been internalized at the end of the acetazolamide treatment (Fig. 5B), the apical V-ATPase localization observed upon subsequent addition of cAMP clearly demonstrates that cAMP induced exocytosis of the pump.

sAC Is Involved in the pH-regulated V-ATPase Recycling—To determine whether sAC was involved in this intracellular bicarbonate- and cAMP-dependent response, we examined the effect of luminal alkaline pH on V-ATPase distribution after inhibition of sAC. Catechol derivatives of estrogen, but not the parent estrogen compounds, were thought to inhibit soluble adenylyl cyclase activity from testis (28), and we confirmed these observations using recombinant purified rat sAC protein (Fig. 6). Rat sAC activity was inhibited by 1,3,5(10)-estratrien-2,3,17{beta}-diol (2-hydroxyestradiol; 2-OH estradiol) with an IC50 of 10 µM, and inhibition was non-competitive with substrate ATP. The parent compound 1,3,5(10)-estratrien-3,17{beta}-diol (estradiol) was essentially inert up to 100 µM. Cauda epididymidis were perfused at pH 7.8 in the presence of the sAC inhibitor, 2-hydroxyestradiol (20 µM) for a period of 20 min. Under these conditions, V-ATPase was distributed between apical microvilli and sub-apical vesicles (Fig. 5D) in a manner similar to that observed at pH 6.5 (compare with Fig. 4A). A significant amount of V-ATPase co-localized with HRP-containing endosomes (Fig. 5D, yellow), indicating that inhibition of sAC prevents the usual alkaline pH-induced response (compare with Fig. 4B). Addition of cpt-cAMP for 10 min at the end of the 2-hydroxyestradiol period and in the continued presence of 2-hydroxyestradiol induced a redistribution of V-ATPase into apical microvilli (Fig. 5E), demonstrating that clear cells had retained their ability to respond to cAMP despite the presence of catechol estrogens. Negative controls were conducted by using estradiol (20 µM), which does not inhibit sAC. Under these conditions, clear cells underwent the same V-ATPase translocation to the apical membrane (Fig. 5F) as that observed normally at pH 7.8 or in the presence of cpt-cAMP. In addition, no effect of estradiol was observed on V-ATPase localization at luminal pH 6.5 (data not shown), demonstrating that estradiol is not an activator of V-ATPase exocytosis. These results demonstrate that blockage of V-ATPase apical translocation by catechol estrogens was specifically attributed to inhibition of sAC.



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 6.
Direct inhibition of purified rsACt by 2-hydroxyestradiol. 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 ATPMg2+ 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.

 
Luminal Bicarbonate Induces the Apical Translocation of V-ATPase—Finally, we examined whether clear cells had the ability to respond to variations in luminal bicarbonate in addition to variations in pH. The effect of an increase in luminal bicarbonate at constant pH on V-ATPase localization and recycling was examined. Cauda epididymidis were perfused with either PBS adjusted to pH 7.1 or a solution containing 12 mM bicarbonate and equilibrated with 5% CO2 (pH 7.1). Clear cells exposed to luminal bicarbonate exhibited more developed VATPase-positive apical microvilli, indicating higher apical membrane translocation of the pump, compared with clear cells exposed to the same pH in absence of bicarbonate (compare Fig. 7, A and B). The sAC inhibitor 2-hydroxyestradiol prevented the bicarbonate-induced V-ATPase apical translocation (Fig. 7C). Quantification of the area occupied by the V-ATPase-labeled microvilli, normalized for the width of the cells at the apical membrane border, indicated a significant increase in apical microvilli extension in the presence of bicarbonate (Fig. 7, E and G) compared with the absence of bicarbonate (Fig. 7, D and G), and confirmed that sAC inhibition prevented the bicarbonate-mediated V-ATPase microvilli translocation (Fig. 7, F and G).



View larger version (41K):
[in this window]
[in a new window]
  		FIG. 7.
Effect of luminal bicarbonate concentration on sAC-mediated 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.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The present data show that the distribution of the V-ATPase in specialized proton-secreting cells of the epididymis, the so-called clear cells, is closely related to luminal pH. At alkaline luminal pH, the V-ATPase is mainly located in well developed apical microvilli, and at acidic pH, it is actively recycling between sub-apical endosomes and the apical membrane. We showed that intracellular production of bicarbonate is essential for the alkaline pH-induced response and that cAMP induces an apical translocation of V-ATPase identical to that induced by alkaline pH. Catechol estrogens, which are inhibitors of the newly identified soluble adenylyl cyclase, sAC, inhibit the alkaline-dependent V-ATPase redistribution, identifying sAC as the sensor responsible for the pH-dependent V-ATPase recycling. We propose that alkalinization of luminal pH, followed by an increase in intracellular pH in clear cells, leads to an elevation of intracellular bicarbonate concentration. Bicarbonate elevation activates sAC and triggers cAMP production, which in turn leads to the accumulation of V-ATPase in apical microvilli. The exact mechanism(s) responsible for the cAMP-induced apical translocation of V-ATPase remain unknown, and further studies will be required to identify the downstream target proteins involved in this process. Clear cells have also the ability to respond to an increase in luminal bicarbonate concentration at constant pH, presumably due to entry of bicarbonate across the apical membrane, and subsequent elevation in intracellular bicarbonate concentration followed by sAC activation. A potential candidate for the apical entry of bicarbonate might be NBC3, which is expressed in the apical membrane of clear cells (29).

Previous studies have demonstrated that primary cultures of epididymal principal cells respond to a variety of agonists by secreting bicarbonate into the lumen (30–32). It was then proposed that bicarbonate secretion occurs during sexual arousal to "prime" spermatozoa before ejaculation (32). Although this finding was interesting, early micropuncture measurements had demonstrated that the luminal fluid of the epididymis is acidic and contains a low bicarbonate concentration (8, 33), indicating the presence of acidification mechanisms in this tissue. We have shown that clear cells, which express high levels of the V-ATPase in their apical pole, are key players in luminal acidification (9, 10) and that proton secretion by these cells is abolished upon inhibition of intracellular bicarbonate production by acetazolamide (11). Thus, the marked intracellular redistribution of V-ATPase observed in the present study in the presence of acetazolamide is in agreement with our previous results showing inhibition of proton secretion under these conditions. We propose that clear cells respond to a rise in luminal bicarbonate concentration following agonist-induced principal cell bicarbonate secretion, by increasing their rate of proton secretion. This would re-establish the pH of the lumen to its resting acidic value. The enrichment of sAC in clear cells compared with principal cells provides an exquisite mechanism for such a concerted interaction between principal and clear cells.

The present study also shows high expression of sAC in kidney thick ascending limbs, distal tubules, and collecting ducts. All epithelial cells from the thick ascending limbs and distal tubules, and intercalated cells from the collecting duct also express the V-ATPase (34). The presence of sAC in these segments, as well as the previously reported sAC expression in other acid/base transporting systems including the choroid plexus (1), indicate that the sAC-dependent cAMP signaling pathway may represent a general mechanism allowing these cells to regulate their rate of proton secretion.

This study is the first demonstration of the role of sAC in mediating V-ATPase-recycling in a native epithelium and provides the missing link between extracellular pH (or bicarbonate) signaling and intracellular response. Because sAC is expressed in many biological systems involved in acid/base transport, we propose that this bicarbonate-dependent cAMP pathway may represent a more universal mechanism allowing cells to sense and modulate the pH of their environment. The molecular entities underlying the response of cells to variations in the pH of their immediate environment still remain unknown. Our study unravels key components of a potentially widespread signaling pathway that could be applicable to many biological systems.


   FOOTNOTES
 
* This study was supported by National Institutes of Health Grants HD40793 (to S. B.), DK38452 (to D. B. and S. B.), DK42956 (to D. B.), HD38722 (to L. R. L.), and GM62328 and HD42060 (to J. B.) and by NIH NRSA HD08684 (to N. P.-S.). The Microscopy Core Facility of the MGH Program in Membrane Biology is additionally supported by a Center for the Study of Inflammatory Bowel Disease (CSIBD) Center Grant DK43351 and Boston Area Diabetes and Endocrinology Research Center (BADERC) Award DK57521. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: Massachusetts General Hospital East, Renal Unit, 149 13th St., 149-8000, Charlestown, MA 02129. Tel.: 617-726-5785; Fax: 617-726-5669; E-mail: sbreton{at}receptor.mgh.harvard.edu.

1 The abbreviations used are: sAC, bicarbonate-activated soluble adenylyl cyclase; RT, reverse transcriptase; PBS, phosphate-buffered saline; HRP, horseradish peroxidase. Back


   ACKNOWLEDGMENTS
 
We thank the excellent technical assistance of Mary McKee.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Chen, Y., Cann, M. J., Litvin, T. N., Iourgenko, V., Sinclair, M. L., Levin, L. R., and Buck, J. (2000) Science 289, 625-628[Abstract/Free Full Text]
  2. Buck, J., Sinclair, M. L., Schapal, L., Cann, M. J., and Levin, L. R. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 79-84[Abstract/Free Full Text]
  3. Buck, J., Sinclair, M. L., and Levin, L. R. (2002) Methods Enzymol. 345, 95-105[CrossRef][Medline] [Order article via Infotrieve]
  4. Wuttke, M. S., Buck, J., and Levin, L. R. (2001) JOP 2, 154-158[Medline] [Order article via Infotrieve]
  5. Zippin, J. H., Levin, L. R., and Buck, J. (2001) Trends Endocrinol. Metab. 12, 366-370[CrossRef][Medline] [Order article via Infotrieve]
  6. Zippin, J. H., Chen, Y., Nahirney, P., Kamenetsky, M., Wuttke, M. S., Fischman, D. A., Levin, L. R., and Buck, J. (2003) FASEB J. 17, 82-84[Abstract/Free Full Text]
  7. Sinclair, M. L., Wang, X. Y., Mattia, M., Conti, M., Buck, J., Wolgemuth, D. J., and Levin, L. R. (2000) Mol. Reprod. Dev. 56, 6-11[CrossRef][Medline] [Order article via Infotrieve]
  8. Levine, N., and Marsh, D. J. (1971) J. Physiol. (Lond) 213, 557-570[Abstract/Free Full Text]
  9. Breton, S., Smith, P. J., Lui, B., and Brown, D. (1996) Nat. Med. 2, 470-472[CrossRef][Medline] [Order article via Infotrieve]
  10. Brown, D., Lui, B., Gluck, S., and Sabolic, I. (1992) Am. J. Physiol. Cell Physiol. 263, C913-916[Abstract/Free Full Text]
  11. Breton, S., Hammar, K., Smith, P. J. S., and Brown, D. (1998) Am. J. Physiol. Cell Physiol. 275, C1134-1142[Abstract/Free Full Text]
  12. Breton, S., Nsumu, N. N., Galli, T., Sabolic, I., Smith, P. J., and Brown, D. (2000) Am. J. Physiol. Renal Physiol. 278, F717-F725[Abstract/Free Full Text]
  13. Madsen, K. M., Verlander, J. W., Kim, J., and Tisher, C. C. (1991) Kidney Int. 33, (suppl.) S57-S63
  14. Steinmetz, P. R. (1986) Am. J. Physiol. Renal Physiol. 251, F173-187[Abstract/Free Full Text]
  15. Brown, D., and Breton, S. (1996) J. Exp. Biol. 199, 2345-2358[Abstract]
  16. Isnard-Bagnis, C., Da Silva, N., Beaulieu, V., Yu, A. S., Brown, D., and Breton, S. (2003) Am. J. Physiol. Cell Physiol. 284, C220-C232[Abstract/Free Full Text]
  17. Breton, S., Wiederhold, T., Marshansky, V., Nsumu, N. N., Ramesh, V., and Brown, D. (2000) J. Biol. Chem. 275, 18219-18224[Abstract/Free Full Text]
  18. Fisher, J. S., Pastor-Soler, N., Sharpe, R. M., and Breton, S. (2002) Biol. Reprod. 67, 1106-1114[Abstract/Free Full Text]
  19. Jaiswal, B. S., and Conti, M. (2001) J. Biol. Chem. 276, 31698-31708[Abstract/Free Full Text]
  20. Mittag, T. W., Guo, W. B., and Kobayashi, K. (1993) Am. J. Physiol. 264, F1060-1064[Medline] [Order article via Infotrieve]
  21. Hudson, R. L. (1993) Am. J. Physiol. 264, R946-951[Medline] [Order article via Infotrieve]
  22. Gekle, M., Wunsch, S., Oberleithner, H., and Silbernagl, S. (1994) Pflugers Arch. 428, 157-162[CrossRef][Medline] [Order article via Infotrieve]
  23. Hays, S., Kokko, J. P., and Jacobson, H. R. (1986) J. Clin. Invest. 78, 1279-1286[Medline] [Order article via Infotrieve]
  24. Kelada, A. S., Macaulay, S. L., and Proietto, J. (1992) J. Biol. Chem. 267, 7021-7025[Abstract/Free Full Text]
  25. Kleizen, B., Braakman, I., and de Jonge, H. R. (2000) Eur. J. Cell Biol. 79, 544-556[CrossRef][Medline] [Order article via Infotrieve]
  26. Brown, D. (2003) Am. J. Physiol. Renal Physiol. 284, F893-F901[Abstract/Free Full Text]
  27. Breton, S., Tyszkowski, R., Sabolic, I., and Brown, D. (1999) Histochem. Cell Biol. 111, 97-105[CrossRef][Medline] [Order article via Infotrieve]
  28. Braun, O. (1990) Proc. Soc. Exp. Biol. Med. 194, 56-63
  29. Pushkin, A., Clark, I., Kwon, T. H., Nielsen, S., and Kurtz, I. (2000) J. Androl. 21, 708-720[Abstract]
  30. Sedlacek, R. L., Carlin, R. W., Singh, A. K., and Schultz, B. D. (2001) Am. J. Physiol. Renal Physiol. 281, F557-F570[Abstract/Free Full Text]
  31. Wong, P. Y. (1988) Am. J. Physiol. 254, F121-F133
  32. Carlin, R. W., Lee, J. H., Marcus, D. C., and Schultz, B. D. (2003) Biol. Reprod. 68, 1027-1034[Abstract/Free Full Text]
  33. Levine, N., and Kelly, H. (1978) J. Reprod. Fertil. 52, 333-335[Abstract]
  34. Brown, D., Hirsch, S., and Gluck, S. (1988) J. Clin. Invest. 82, 2114-2126[Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
P. Mardones, J. F. Medina, and R. P. J. O. Elferink
Activation of Cyclic AMP Signaling in Ae2-deficient Mouse Fibroblasts
J. Biol. Chem., May 2, 2008; 283(18): 12146 - 12153.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
J. Liu, W. Lu, D. Reigada, J. Nguyen, A. M. Laties, and C. H. Mitchell
Restoration of Lysosomal pH in RPE Cells from Cultured Human and ABCA4-/- Mice: Pharmacologic Approaches and Functional Recovery
Invest. Ophthalmol. Vis. Sci., February 1, 2008; 49(2): 772 - 780.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
N. M. Pastor-Soler, K. R. Hallows, C. Smolak, F. Gong, D. Brown, and S. Breton
Alkaline pH- and cAMP-induced V-ATPase membrane accumulation is mediated by protein kinase A in epididymal clear cells
Am J Physiol Cell Physiol, February 1, 2008; 294(2): C488 - C494.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
J. Rein, M. Voss, W. Blenau, B. Walz, and O. Baumann
Hormone-induced assembly and activation of V-ATPase in blowfly salivary glands is mediated by protein kinase A
Am J Physiol Cell Physiol, January 1, 2008; 294(1): C56 - C65.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
T. G. Paunescu, N. Da Silva, L. M. Russo, M. McKee, H. A. J. Lu, S. Breton, and D. Brown
Association of soluble adenylyl cyclase with the V-ATPase in renal epithelial cells
Am J Physiol Renal Physiol, January 1, 2008; 294(1): F130 - F138.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
T. G. Paunescu, L. M. Russo, N. Da Silva, J. Kovacikova, N. Mohebbi, A. N. Van Hoek, M. McKee, C. A. Wagner, S. Breton, and D. Brown
Compensatory membrane expression of the V-ATPase B2 subunit isoform in renal medullary intercalated cells of B1-deficient mice
Am J Physiol Renal Physiol, December 1, 2007; 293(6): F1915 - F1926.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Voss, O. Vitavska, B. Walz, H. Wieczorek, and O. Baumann
Stimulus-induced Phosphorylation of Vacuolar H+-ATPase by Protein Kinase A
J. Biol. Chem., November 16, 2007; 282(46): 33735 - 33742.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
N. Da Silva, W. W. C. Shum, J. El-Annan, T. G. Paunescu, M. McKee, P. J. S. Smith, D. Brown, and S. Breton
Relocalization of the V-ATPase B2 subunit to the apical membrane of epididymal clear cells of mice deficient in the B1 subunit
Am J Physiol Cell Physiol, July 1, 2007; 293(1): C199 - C210.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
M. Tresguerres, S. K. Parks, C. M. Wood, and G. G. Goss
V-H+-ATPase translocation during blood alkalosis in dogfish gills: interaction with carbonic anhydrase and involvement in the postfeeding alkaline tide
Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2007; 292(5): R2012 - R2019.
[Abstract] [Full Text] [PDF]

Home page
 J. Exp. Biol.Home page
C. Hille and B. Walz
A vacuolar-type H+-ATPase and a Na+/H+ exchanger contribute to intracellular pH regulation in cockroach salivary ducts
J. Exp. Biol., April 15, 2007; 210(8): 1463 - 1471.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
S. Breton and D. Brown
New insights into the regulation of V-ATPase-dependent proton secretion
Am J Physiol Renal Physiol, January 1, 2007; 292(1): F1 - F10.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
J. Codina, J. Liu, A. J. Bleyer, R. B. Penn, and T. D. DuBose Jr.
Phosphorylation of S955 at the Protein Kinase A Consensus Promotes Maturation of the {alpha} Subunit of the Colonic H+,K+-ATPase
J. Am. Soc. Nephrol., July 1, 2006; 17(7): 1833 - 1840.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. M. Stessin, J. H. Zippin, M. Kamenetsky, K. C. Hess, J. Buck, and L. R. Levin
Soluble Adenylyl Cyclase Mediates Nerve Growth Factor-induced Activation of Rap1
J. Biol. Chem., June 23, 2006; 281(25): 17253 - 17258.
[Abstract] [Full Text] [PDF]

Home page
 J. Exp. Biol.Home page
N. Satake, R. M. A. Elliott, P. F. Watson, and W. V. Holt
Sperm selection and competition in pigs may be mediated by the differential motility activation and suppression of sperm subpopulations within the oviduct
J. Exp. Biol., April 15, 2006; 209(8): 1560 - 1572.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
M. D. Phillips and G. H. Thomas
Brush border spectrin is required for early endosome recycling in Drosophila
J. Cell Sci., April 1, 2006; 119(7): 1361 - 1370.
[Abstract] [Full Text] [PDF]