Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Bicarbonate-regulated adenylyl cyclase (sAC) is a sensor that regulates pH-dependent V-ATPase recycling. 1451 17

The role of the actin cytoskeleton in regulating membrane protein trafficking is complex and depends on the cell type and protein being examined. Using the epididymis as a model system in which luminal acidification is crucial for sperm maturation and storage, we now report that modulation of the actin cytoskeleton by the calcium-activated actin-capping and -severing protein gelsolin plays a key role in regulating vacuolar H(+)-ATPase (V-ATPase) recycling. Epididymal clear cells contain abundant V-ATPase in their apical pole, and an increase in their cell-surface V-ATPase expression correlates with an increase in luminal proton secretion. We have shown that apical membrane accumulation of V-ATPase is triggered by an elevation in cAMP following activation of bicarbonate-regulated soluble adenylyl cyclase in response to alkaline luminal pH (Pastor-Soler, N., Beaulieu, V., Litvin, T. N., Da Silva, N., Chen, Y., Brown, D., Buck, J., Levin, L. R., and Breton, S. (2003) J. Biol. Chem. 278, 49523-49529). Here, we show that clear cells express high levels of gelsolin, indicating a potential role in the functional activity of these cells. When jasplakinolide was used to overcome the severing action of gelsolin by polymerizing actin, complete inhibition of the alkaline pH- and cAMP-induced apical membrane accumulation of V-ATPase was observed. Conversely, when gelsolin-mediated actin filament elongation was inhibited using a 10-residue peptide (PBP10) derived from the phosphatidylinositol 4,5-bisphosphate-binding region (phosphoinositide-binding domain 2) of gelsolin, significant V-ATPase apical membrane mobilization was induced, even at acidic luminal pH. In contrast, the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) and the phospholipase C inhibitor U-73122 inhibited the alkaline pH-induced V-ATPase apical accumulation. Thus, maintenance of the actin cytoskeleton in a depolymerized state by gelsolin facilitates calcium-dependent apical accumulation of V-ATPase in response to luminal pH alkalinization. Gelsolin is present in other cell types that express the V-ATPase in their plasma membrane and recycling vesicles, including kidney intercalated cells and osteoclasts. Therefore, modulation of the actin cortex by this severing and capping protein may represent a common mechanism by which these cells regulate their rate of proton secretion.
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PMID:Modulation of the actin cytoskeleton via gelsolin regulates vacuolar H+-ATPase recycling. 1559 Oct 47

This review explores the origins, diversity and functions of guanylyl cyclases in cellular organisms. In eukaryotes both cGMP and cAMP are produced by the conserved class III cyclase domains, while prokaryotes use five more unrelated catalysts for cyclic nucleotide synthesis. The class III domain is found embedded in proteins with a large variety of membrane topologies and other functional domains, but the vertebrate guanylyl cyclases take only two forms, the receptor guanylyl cyclases with single transmembrane domain and the soluble enzymes with heme binding domain. The invertebrates additionally show a soluble guanylyl cyclase that cannot bind heme, while the more basal metazoans may lack the heme binding enzymes altogether. Fungi, the closest relatives of the metazoans, completely lack guanylyl cylases, but they appear again in the Dictyostelids, the next relative in line. Remarkably, the two Dictyostelid guanylyl cyclases have little in common with the vertebrate enzymes. There is a soluble guanylyl cyclase, which shows greatest sequence and structural similarity to the vertebrate soluble adenylyl cyclase, and a membrane-bound form with the same configuration as the dodecahelical adenylyl cyclases of vertebrates. There is a difference, the pseudosymmetric C1 and C2 catalytic domains have swapped position in the Dictyostelium enzyme. Unlike the vertebrate guanylyl cyclases, the Dictyostelium enzymes are activated by heterotrimeric G-proteins. Swapped C1 and C2 domains are also found in the structurally similar guanylyl cyclases of ciliates and apicomplexans, but these enzymes additionally harbour an amino-terminal ATPase module with ten transmembrane domains. G-protein regulation could not be demonstrated for these enzymes. Higher plants lack class III cyclase domains, but an unexplored wealth of guanylyl cyclases is present in the green alga Chlamydomonas. Progenitors of all structural variants of the eukaryote guanylyl cyclases are found among the prokaryote adenylyl cyclases. This and the close similarity of many guanylyl cyclases to adenylyl cyclases suggests a paraphyletic origin for the eukaryote enzymes with multiple events of conversion of substrate specificity.
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PMID:Guanylyl cyclases across the tree of life. 1576 39

Activation of soluble adenylyl cyclase (sAC) by bicarbonate causes local cAMP generation, indicating that sAC might act as a pH and/or bicarbonate sensor in kidney cells involved in acid-base homeostasis. Therefore, we examined the expression of sAC in renal acid-base transporting intercalated cells (IC) and compared its distribution to that of the vacuolar proton pumping ATPase (V-ATPase) under different conditions. In all IC, sAC and V-ATPase showed considerable overlap under basal conditions, but sAC staining was also found in other cellular locations in the absence of V-ATPase. In type A-IC, both sAC and V-ATPase were apically and subapically located, whereas in type B-IC, significant basolateral colocalization of sAC and the V-ATPase was seen. When apical membrane insertion of the V-ATPase was stimulated by treatment of rats with acetazolamide, sAC was also concentrated in the apical membrane of A-IC. In mice that lack a functional B1 subunit of the V-ATPase, sAC was colocalized apically in A-IC along with V-ATPase containing the alternative B2 subunit isoform. The close association between these two enzymes was confirmed by coimmunoprecipitation of sAC from kidney homogenates using anti-V-ATPase antibodies. Our data show that sAC and the V-ATPase colocalize in IC, that they are concentrated in the IC plasma membrane under conditions that "activate" these proton secretory cells, and that they are both present in an immunoprecipitated complex. This suggests that these enzymes have a close association and could be part of a protein complex that is involved in regulating renal distal proton secretion.
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PMID:Association of soluble adenylyl cyclase with the V-ATPase in renal epithelial cells. 1795 50

In the epididymis, low luminal bicarbonate and acidic pH maintain sperm quiescent during maturation and storage. The vacuolar H(+)-ATPase (V-ATPase) in epididymal clear cells plays a major role in luminal acidification. We have shown previously that cAMP, luminal alkaline pH, and activation of the bicarbonate-regulated soluble adenylyl cyclase (sAC) induce V-ATPase apical accumulation in these cells, thereby stimulating proton secretion into the epididymal lumen. Here we examined whether protein kinase A (PKA) is involved in this response. Confocal immunofluorescence labeling on rat epididymis perfused in vivo showed that at luminal acidic pH (6.5), V-ATPase was distributed between short apical microvilli and subapical endosomes. The specific PKA activator N(6)-monobutyryl-3'-5'-cyclic monophosphate (6-MB-cAMP, 1 mM) induced elongation of apical microvilli and accumulation of V-ATPase in these structures. The PKA inhibitor myristoylated-PKI (mPKI, 10 microM) inhibited the apical accumulation of V-ATPase induced by 6-MB-cAMP. Perfusion at pH 6.5 with 8-(4-chlorophenylthio)-2-O-methyl-cAMP (8CPT-2-O-Me-cAMP; 10 microM), an activator of the exchange protein activated by cAMP (Epac), did not induce V-ATPase apical accumulation. When applied at a higher concentration (100 microM), 8CPT-2-O-Me-cAMP induced V-ATPase apical accumulation, but this effect was completely inhibited by mPKI, suggesting crossover effects on the PKA pathway with this compound at high concentrations. Importantly, the physiologically relevant alkaline pH-induced apical V-ATPase accumulation was completely inhibited by pretreatment with mPKI. We conclude that direct stimulation of PKA activity by cAMP is necessary and sufficient for the alkaline pH-induced accumulation of V-ATPase in clear cell apical microvilli.
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PMID:Alkaline pH- and cAMP-induced V-ATPase membrane accumulation is mediated by protein kinase A in epididymal clear cells. 1816 Apr 85

Alkalosis impairs the natriuretic response to diuretics, but the underlying mechanisms are unclear. The soluble adenylyl cyclase (sAC) is a chemosensor that mediates bicarbonate-dependent elevation of cAMP in intracellular microdomains. We hypothesized that sAC may be an important regulator of Na(+) transport in the kidney. Confocal images of rat kidney revealed specific immunolocalization of sAC in collecting duct cells, and immunoblots confirmed sAC expression in mouse cortical collecting duct (mpkCCD(c14)) cells. These cells exhibit aldosterone-stimulated transepithelial Na(+) currents that depend on both the apical epithelial Na(+) channel (ENaC) and basolateral Na(+),K(+)-ATPase. RNA interference-mediated 60-70% knockdown of sAC expression comparably inhibited basal transepithelial short circuit currents (I(sc)) in mpkCCD(c14) cells. Moreover, the sAC inhibitors KH7 and 2-hydroxyestradiol reduced I(sc) in these cells by 50-60% within 30 min. 8-Bromoadenosine-3',5'-cyclic-monophosphate substantially rescued the KH7 inhibition of transepithelial Na(+) current. Aldosterone doubled ENaC-dependent I(sc) over 4 h, an effect that was abolished in the presence of KH7. The sAC contribution to I(sc) was unaffected with apical membrane nystatin-mediated permeabilization, whereas the sAC-dependent Na(+) current was fully inhibited by basolateral ouabain treatment, suggesting that the Na(+),K(+)-ATPase, rather than ENaC, is the relevant transporter target of sAC. Indeed, neither overexpression of sAC nor treatment with KH7 modulated ENaC currents in Xenopus oocytes. ATPase and biotinylation assays in mpkCCD(c14) cells demonstrated that sAC inhibition decreases catalytic activity rather than surface expression of the Na(+),K(+)-ATPase. In summary, these results suggest that sAC regulates both basal and agonist-stimulated Na(+) reabsorption in the kidney collecting duct, acting to enhance Na(+),K(+)-ATPase activity.
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PMID:Regulation of epithelial Na+ transport by soluble adenylyl cyclase in kidney collecting duct cells. 1912 49

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.
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PMID:AMP-activated protein kinase inhibits alkaline pH- and PKA-induced apical vacuolar H+-ATPase accumulation in epididymal clear cells. 1921 18

The proton-pumping V-ATPase is a complex, multi-subunit enzyme that is highly expressed in the plasma membranes of some epithelial cells in the kidney, including collecting duct intercalated cells. It is also located on the limiting membranes of intracellular organelles in the degradative and secretory pathways of all cells. Different isoforms of some V-ATPase subunits are involved in the targeting of the proton pump to its various intracellular locations, where it functions in transporting protons out of the cell across the plasma membrane or acidifying intracellular compartments. The former process plays a critical role in proton secretion by the kidney and regulates systemic acid-base status whereas the latter process is central to intracellular vesicle trafficking, membrane recycling and the degradative pathway in cells. We will focus our discussion on two cell types in the kidney: (1) intercalated cells, in which proton secretion is controlled by shuttling V-ATPase complexes back and forth between the plasma membrane and highly-specialized intracellular vesicles, and (2) proximal tubule cells, in which the endocytotic pathway that retrieves proteins from the glomerular ultrafiltrate requires V-ATPase-dependent acidification of post-endocytotic vesicles. The regulation of both of these activities depends upon the ability of cells to monitor the pH and/or bicarbonate content of their extracellular environment and intracellular compartments. Recent information about these pH-sensing mechanisms, which include the role of the V-ATPase itself as a pH sensor and the soluble adenylyl cyclase as a bicarbonate sensor, will be addressed in this review.
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PMID:Regulation of the V-ATPase in kidney epithelial cells: dual role in acid-base homeostasis and vesicle trafficking. 1944 85

Kidney proton-secreting A-intercalated cells (A-IC) respond to systemic acidosis by accumulating the vacuolar ATPase (V-ATPase) in their apical membrane and by increasing the length and number of apical microvilli. We show here that the cell-permeant cAMP analog CPT-cAMP, infused in vivo, results in an almost twofold increase in apical V-ATPase accumulation in AE1-positive A-IC within 15 min and that these cells develop an extensive array of apical microvilli compared with controls. In contrast, no significant change in V-ATPase distribution could be detected by immunocytochemistry in B-intercalated cells at the acute time point examined. To show a direct effect of cAMP on A-IC, we prepared cell suspensions from the medulla of transgenic mice expressing EGFP in IC (driven by the B1-subunit promoter of the V-ATPase) and exposed them to cAMP analogs in vitro. Three-dimensional reconstructions of confocal images revealed that cAMP induced a time-dependent growth of apical microvilli, starting within minutes after addition. This effect was blocked by the PKA inhibitor myristoylated PKI. These morphological changes were paralleled by increased cAMP-mediated proton extrusion (pHi recovery) by A-IC in outer medullary collecting ducts measured using the ratiometric probe BCECF. These results, and our prior data showing that the bicarbonate-stimulated soluble adenylyl cyclase (sAC) is highly expressed in kidney intercalated cells, support the idea that cAMP generated either by sAC, or by activation of other signaling pathways, is part of the signal transduction mechanism involved in acid-base sensing and V-ATPase membrane trafficking in kidney intercalated cells.
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PMID:cAMP stimulates apical V-ATPase accumulation, microvillar elongation, and proton extrusion in kidney collecting duct A-intercalated cells. 2005 93

The vacuolar H(+)-ATPase (V-ATPase) in type A kidney intercalated cells is a major contributor to acid excretion in the collecting duct. The mechanisms of V-ATPase-trafficking regulation in kidney intercalated cells have not been well-characterized. In developmentally related epididymal clear cells, we showed previously that PKA, acting downstream of soluble adenylyl cyclase (sAC), induces V-ATPase apical membrane accumulation. These PKA-mediated effects were inhibited by activators of the metabolic sensor AMP-activated kinase (AMPK) in clear cells. Here, we examined the regulation of V-ATPase subcellular localization in intercalated cells by PKA and AMPK in rat kidney tissue slices ex vivo. Immunofluorescence labeling of kidney slices revealed that the PKA activator N(6)-monobutyryl cAMP (6-MB-cAMP) induced V-ATPase apical membrane accumulation in collecting duct intercalated cells, whereas the V-ATPase had a more cytosolic distribution when incubated in Ringer buffer alone for 30 min. V-ATPase accumulated at the apical membrane in intercalated cells in kidney slices incubated in Ringer buffer for 75 min, an effect that was prevented by treatment with PKA inhibitor (mPKI). The V-ATPase distribution was cytosolic in intercalated cells treated with the carbonic anhydrase inhibitor acetazolamide or the sAC inhibitor KH7, effects that were overridden by 6-MB-cAMP. Preincubation of kidney slices with an AMPK activator blocked V-ATPase apical membrane accumulation induced by 6-MB-cAMP, suggesting that AMPK antagonizes cAMP/PKA effects on V-ATPase distribution. Taken together, our results suggest that in intercalated cells V-ATPase subcellular localization and therefore its activity may be coupled to acid-base status via PKA, and metabolic status via AMPK.
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PMID:Vacuolar H+-ATPase apical accumulation in kidney intercalated cells is regulated by PKA and AMP-activated protein kinase. 2014 66


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