V-ATPase Accumulation Research Articles

The fungus Acremonium alternatum enhances salt stress tolerance by regulating host redox homeostasis and phytohormone signaling.

While endophytic fungi offer promising avenues for bolstering plant resilience against abiotic stressors, the molecular mechanisms behind this biofortification remain largely unknown. This study employed a multifaceted approach, combining plant physiology, proteomic, metabolomic, and targeted hormonal analyses to illuminate the early response of Brassica napus to Acremonium alternatum during the nascent stages of their interaction. Notably, under optimal growth conditions, the initial reaction to fungus was relatively subtle, with no visible alterations in plant phenotype and only minor impacts on the proteome and metabolome. Interestingly, the identified proteins associated with the Acremonium response included TUDOR 1, Annexin D4, and a plastidic K+ efflux antiporter, hinting at potential processes that could counter abiotic stressors, particularly salt stress. Subsequent experiments validated this hypothesis, showcasing significantly enhanced growth in Acremonium-inoculated plants under salt stress. Molecular analyses revealed a profound impact on the plant's proteome, with over 50% of salt stress response proteins remaining unaffected in inoculated plants. Acremonium modulated ribosomal proteins, increased abundance of photosynthetic proteins, enhanced ROS metabolism, accumulation of V-ATPase, altered abundances of various metabolic enzymes, and possibly promoted abscisic acid signaling. Subsequent analyses validated the accumulation of this hormone and its enhanced signaling. Collectively, these findings indicate that Acremonium promotes salt tolerance by orchestrating abscisic acid signaling, priming the plant's antioxidant system, as evidenced by the accumulation of ROS-scavenging metabolites and alterations in ROS metabolism, leading to lowered ROS levels and enhanced photosynthesis. Additionally, it modulates ion sequestration through V-ATPase accumulation, potentially contributing to the observed decrease in chloride content.

Identification of differentially expressed Gpr116 (Adgrf5) transcript variants in mouse kidney

Adhesion G protein-coupled receptors (aGPCRs) are important and understudied modulators of physiological processes. Unlike class A GPCRs that are activated by soluble ligands, aGPCRs are self-activated by a cryptic tethered agonist after holoreceptor dissociation. We recently demonstrated that kidney-specific ADGRF5/Gpr116 knockout causes luminal membrane accumulation of V-ATPase in acid-secreting A-type intercalated cells (AICs) in the collecting ducts and a significant reduction in urine pH (Zaidman et al. 2020). Previous work suggests that aGPCRs, and Gpr116 in particular, undergo significant tissue-specific mRNA processing that results in holoreceptors with unique and variable N-terminal structures (Knierim et al. 2019). These aGPCR splice variants possess singular structure-function relationships that are integral to endogenous self-activation. Therefore, identifying tissue-specific transcript variants is critical to understanding the physiological roles of aGPCRs. Besides robust expression in AICs, Gpr116 is also transcribed in endothelial cells (ECs) in the kidney. We hypothesized that unique Gpr116 transcript variants are expressed in renal AICs and ECs. To test this hypothesis, we enzymatically digested kidneys from Gpr116flag V1B1eGFP mice and enriched for ICs (CD31−, GFP+) and ECs (CD31+, GFP−) by FACS. Gpr116 was not detected in CD31− GFP− sorted cells. IC enrichment was confirmed by qPCR by detection of Atp6v0d2, c-kit (CD117), Slc4a1, and Slc4a9 transcripts. EC enrichment was confirmed by detection of Kdr and Nrp1 transcripts. Next, we assayed cDNA from these cell populations for exon variants by PCR, and subsequent nanopore sequencing. We detected and aligned three exons that undergo differential expression in the kidney: exons 2, 12, and 22. Both IC and EC populations were positive for all three differentially expressed exons, but also expressed Gpr116 transcripts without the variable regions, suggesting multiple Gpr116 isoforms in each cell type. However, EC markers were detected in GFP+ ICs, demonstrating some EC contamination in the sorted ICs. To test the tissue specificity of these differentially expressed exons, we assayed 7 other tissues from Gpr116flag V1B1eGFP mice. We detected exon 2 and 12 in lung, heart and fat; we did not detect it in pancreas, spleen, thymus or liver. We detected exon 22 in lung and fat; we did not detect it in pancreas, heart, spleen, thymus or liver. We detected Gpr116flag in the lung, heart, spleen, fat, thymus, kidney and liver, but not pancreas. In summary, we have identified three variable exons within the open reading frame of Gpr116. Future studies will characterize the roles of differentially expressed exons on the endogenous activation of Gpr116 and determine if transcript variants are cell-type specific. This study is supported by the National Institute of Diabetes and Digestive and Kidney Diseases (4R00DK127215). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

DDRGK1, a crucial player of ufmylation system, is indispensable for autophagic degradation by regulating lysosomal function

DDRGK domain-containing protein 1 (DDRGK1) is an important component of the newly discovered ufmylation system and its absence has been reported to induce extensive endoplasmic reticulum (ER) stress. Recently, emerging evidence indicates that the ufmylation system is correlated with autophagy, although the exact mechanism remains largely unknown. To explore the regulation mechanism of DDRGK1 on autophagy, in this study, we established an immortalized mouse embryonic fibroblast (MEF) cell lines harvested from the DDRGK1F/F:ROSA26-CreERT2 mice, in which DDRGK1 depletion can be induced by 4-hydroxytamoxifen (4-OHT) treatment. Here, we show that DDRGK1 deficiency in MEFs has a dual effect on autophagy, which leads to a significant accumulation of autophagosomes. On one hand, it promotes autophagy induction by impairing mTOR signaling; on the other hand, it blocks autophagy degradation by inhibiting autophagosome–lysosome fusion. This dual effect of DDRGK1 depletion on autophagy ultimately aggravates apoptosis in MEFs. Further studies reveal that DDRGK1 loss is correlated with suppressed lysosomal function, including impaired Cathepsin D (CTSD) expression, aberrant lysosomal pH, and v-ATPase accumulation, which might be a potential trigger for impairment in autophagy process. Hence, this study confirms a crucial role of DDRGK1 as an autophagy regulator by controlling lysosomal function. It may provide a theoretical basis for the treatment strategies of various physiological diseases caused by DDRGK1 deficiency.

The Involvement of the Chemokine RANTES in Regulating Luminal Acidification in Rat Epididymis.

BackgroundA complex interplay between different cell types in the epithelium leads to activation of the luminal acidifying capacity of the epididymis, a process that is crucial for sperm maturation and storage. Basal cells sense the luminal angiotensin II (ANG II) and stimulate proton secretion in clear cells through nitric oxide (NO). Our previous study has shown the chemokine regulated upon activation normal T-cell expressed and secreted (RANTES) was expressed in the F4/80 positive macrophages of human epididymis. The objective of this study was to explore the involvement of RANTES in regulating the luminal acidification in the rat epididymis.MethodsThe role of RANTES was investigated by in vivo perfusion with recombinant RANTES, Met-RANTES, and PBS of different pH values. Furthermore, rats vasectomy was performed to alter the epididymal luminal pH. RIA was used to measure the tissue homogenate ANG II concentration. Real time-PCR and western blot were employed to examine the expression levels of AGTR2, RANTES, CCR1, CCR5, and iNOS in epididymis.ResultsRANTES was restricted to the basal macrophages of epididymal ducts and co-localized with its receptors CCR1 and CCR5. Both V-ATPase and iNOS were up-regulated in the cauda epididymis after perfused with recombinant RANTES, while the antagonist Met-RANTES perfusion led to a complete abrogation of the increased expression of V-ATPase in the apical membrane of clear cells and iNOS in macrophages. Upon alkaline perfusion, RANTES expression was significantly increased and the apical accumulation of V-ATPase in the clear cells was induced in the cauda epididymis. The luminal pH in the cauda epididymis increased after vasectomy. The concentration of the ANG II and the expression levels of AGTR2, RANTES, CCR1, CCR5, and iNOS dropped in the cauda epididymis following vasectomy.ConclusionUpon the activation of basal cells, RANTES might induce the NO release from macrophages by interacting with its receptors, which increases proton secretion by adjacent clear cells. Thus, RANTES is possible to participate in the crosstalk among basal cells, macrophages and clear cells for the fine control of an optimum acidic luminal environment that is critical for male fertility.

Plasma membrane V-ATPase controls oncogenic RAS-induced macropinocytosis.

Oncogenic activation of Ras is associated with the acquisition of a unique set of metabolic dependencies that contribute to tumor cell fitness. Mutant Ras cells are endowed with the capability to internalize and degrade extracellular protein via a fluid–phase uptake mechanism termed macropinocytosis1. There is a growing appreciation for the role of this Ras-dependent process in the generation of free amino acids that can be used to support tumor cell growth under nutrient limiting conditions2. However, little is known about the molecular steps that mediate the induction of macropinocytosis by oncogenic Ras. Here we identify vacuolar ATPase (v-ATPase) as an essential regulator of Ras-induced macropinocytosis. Oncogenic Ras promotes the translocation of v-ATPase from intracellular membranes to the plasma membrane (PM) via a pathway that requires protein kinase A (PKA) activation by a bicarbonate-dependent soluble adenylate cyclase (sAC). PM accumulation of v-ATPase is necessary for the cholesterol-dependent association of Rac1 with the PM, a prerequisite for the stimulation of membrane ruffling and macropinocytosis. These observations identify a link between v-ATPase trafficking and nutrient supply by macropinocytosis that could be exploited to curtail the metabolic adaptation capacity of mutant Ras tumor cells.

Hv1 proton channels differentially regulate the pH of neutrophil and macrophage phagosomes by sustaining the production of phagosomal ROS that inhibit the delivery of vacuolar ATPases

Production of ROS and maintenance of an appropriate pH within the lumen of neutrophil and macrophage phagosomes are important for an effective immune response. Hv1 proton channels sustain ROS production at the plasma membrane, but their role in phagosomes is not known. Here, we tested whether Hv1 channels regulate the pHp and sustain phagosomal ROS production in neutrophils and macrophages. The presence of Hv1 channels on phagosomes of human neutrophils and mouse macrophages was confirmed by Western blot and immunostaining. Phagosomal ROS production, measured with OxyBurst-coupled targets, was reduced in neutrophils and macrophages isolated from Hv1-deficient mice. Ratiometric imaging of FITC-coupled targets showed that phagosomes acidified more slowly in Hv1-deficient macrophages and transiently alkalinized when the V-ATPase was inhibited. In WT neutrophils, 97% of phagosomes remained neutral 30 min after particle ingestion, whereas 37% of Hv1-deficient phagosomes were alkaline (pH>8.3) and 14% acidic (pH<6.3). The subpopulation of acidic phagosomes was eliminated by V-ATPase inhibition, whereas NOX inhibition caused a rapid acidification, independently of Hv1 expression. Finally, V-ATPase accumulation on phagosomes was inversely correlated to intraphagosomal ROS production in neutrophils. These data indicate that Hvcn1 ablation deregulates neutrophil pHp, leading to alkalinization in phagosomes with residual ROS production or to the early accumulation of V-ATPase on phagosomes that fail to mount an oxidative response. Hv1 channels therefore differentially regulate the pHp in neutrophils and macrophages, sustaining rapid acidification in macrophage phagosomes and maintaining a neutral pH in neutrophil phagosomes.

Involvement of CFTR in ATP and HCO3‐ secretion in epididymal principal cells

Luminal ATP and HCO3− induce apical membrane accumulation of V-ATPase in epididymal clear cells. We hypothesized that CFTR might participate in the regulation of luminal acidification by clear cells, via crosstalk with principal cells through ATP and HCO3− secretion. Using the epididymal principal cell lines, DC2 and PC1, and a luciferase assay, we showed that forskolin (Fsk) and adrenaline increased ATP release from DC2 but not PC1 cells. The CFTR inhibitor, CFTRinh172, reduced basal ATP release in both cell lines and blocked the Fsk-induced ATP-release in DC2 cells. The rate of intracellular pH recovery (dpHi/dt) after alkalinization caused by removal of CO2 and HCO3− was measured with BCECF as an assessment of HCO3− secretion. Fsk significantly increased dpHi/dt in DC2 but not PC1 cells. CFTRinh172 abolished Fsk-induced HCO3− secretion in DC2 cells. These results suggest that CFTR mediates basal levels of ATP and HCO3− secretion in both cell lines, and Fsk-activation in DC2 cells. Data are compatible with the hypothesis that ATP and HCO3−, secreted by principal cells, increase V-ATPase-dependent proton secretion by clear cells, allowing restoration of luminal pH to its resting acidic value. This study provides new insights into the pathogenesis of cystic fibrosis-related male infertility.

Establishment of Cell-Cell Cross Talk in the Epididymis: Control of Luminal Acidification

Male infertility is often caused by sperm that have low motility and interact poorly with the oocyte. Spermatozoa acquire these crucial functions in the epididymis. A low luminal bicarbonate (HCO(3)(-)) concentration and low pH keep sperm quiescent during their maturation and storage in this organ. This review describes how epididymal epithelial cells work in a concerted manner, together with spermatozoa, to establish and maintain this acidic luminal environment. Clear cells express the proton-pumping ATPase (V-ATPase) in their apical membrane and actively secrete protons. HCO(3)(-) induces V-ATPase accumulation in apical microvilli in clear cells via HCO(3)(-)-sensitive adenylyl cyclase-dependent cAMP production. HCO(3)(-) is secreted from principal cells following basolateral stimulation, to transiently "prime" spermatozoa before ejaculation. Luminal ATP and adenosine also induce V-ATPase apical accumulation in clear cells via activation of P2 and P1 receptors, respectively. ATP is released into the lumen from sperm and principal cells and is then metabolized into adenosine by local nucleotidases. In addition, the V-ATPase is regulated by luminal angiotensin II via activation of basal cells, which can extend narrow body projections that cross the tight junction barrier. Basal cells then secrete nitric oxide, which diffuses out to stimulate proton secretion in clear cells via activation of the cGMP pathway. Thus, an elaborate communication network is present between principal cells and clear cells, and between basal cells and clear cells, to control luminal acidification. Monitoring and decoding these "intercellular conversations" will help define pathophysiologic mechanisms underlying male infertility.

Regulation of V-ATPase recycling via a RhoA- and ROCKII-dependent pathway in epididymal clear cells

Luminal acidification in the epididymis is critical for sperm maturation and storage. Clear cells express the vacuolar H(+)-ATPase (V-ATPase) in their apical membrane and are major contributors to proton secretion. We showed that this process is regulated via recycling of V-ATPase-containing vesicles. We now report that RhoA and its effector ROCKII are enriched in rat epididymal clear cells. In addition, cortical F-actin was detected beneath the apical membrane and along the lateral membrane of "resting" clear cells using a pan-actin antibody or phalloidin-TRITC. In vivo luminal perfusion of the cauda epididymal tubule with the ROCK inhibitors Y27632 (10-30 μM) and HA1077 (30 μM) or with the cell-permeable Rho inhibitor Clostridium botulinum C3 transferase (3.75 μg/ml) induced the apical membrane accumulation of V-ATPase and extension of V-ATPase-labeled microvilli in clear cells. However, these newly formed microvilli were devoid of ROCKII. In addition, Y27632 (30 μM) or HA1077 (30 μM) decreased the ratio of F-actin to G-actin detected by Western blot analysis in epididymal epithelial cells, and Y27632 also decreased the ratio of F-actin to G-actin in clear cells isolated by fluorescence activated cell sorting from B1-enhanced green fluorescence protein (EGFP) transgenic mice. These results provide evidence that depolymerization of the cortical actin cytoskeleton via inhibition of RhoA or its effector ROCKII favors the recruitment of V-ATPase from the cytosolic compartment into the apical membrane in clear cells. In addition, our data suggest that the RhoA-ROCKII pathway is not locally involved in the elongation of apical microvilli. We propose that inhibition of RhoA-ROCKII might be part of the intracellular signaling cascade that is triggered upon agonist-induced apical membrane V-ATPase accumulation.

Role of AMPK and PKA in the trafficking of V‐ATPase in kidney intercalated cells

Vacuolar ATPases (V‐ATPase) are expressed at the plasma membrane of type A intercalated cells (ICs) that acidify the lumen of kidney collecting duct. V‐ATPase function is important for acid excretion and defective V‐ATPase in ICs causes metabolic acidosis. V‐ATPase regulation by direct phosphorylation in mammalian epithelial cells is not well characterized. We have shown that in epididymal clear cells V‐ATPase accumulation at the apical membrane is regulated by cAMP/PKA and AMP‐activated kinase (AMPK). We hypothesized that V‐ATPase subcellular localization in ICs depends on phosphorylation of V1 sector subunits and that V‐ATPase regulation is coupled to acid‐base status via PKA and to metabolic status via AMPK. Apical V‐ATPase accumulation in ICs requires cAMP/PKA, as evidenced immunofluorescence labeling of the V‐ATPase in kidney slices treated with various agonists and antagonists. In addition, AMPK activators prevented cAMP/PKA‐mediated V‐ATPase apical accumulation in ICs. Through mass spectrometry, mutagenesis and expression in cultured cells, we have identified several candidate PKA and AMPK phosphorylation sites in V1 sector subunits that may have functional effects on V‐ATPase localization. We propose that subcellular localization and activity of the V‐ATPase in ICs depends on the direct phosphorylation of V1 sector subunits by PKA and AMPK.

V‐ATPase membrane accumulation and activity are stimulated by cAMP in A‐type intercalated cells

Recycling of the vacuolar proton (H+) pumping ATPase (V‐ATPase) between intracellular vesicles and the plasma membrane in H+‐secreting cells is central to acid‐base regulation by the kidney. Collecting duct (CD) A‐type intercalated cells (A‐IC) respond to systemic acidosis by accumulating the V‐ATPase in their apical membrane, which correlates with higher rates of H+ secretion and increased microvillar extension. We show that CPT‐cAMP infused in vivo results in an almost two‐fold increase in apical V‐ATPase accumulation in A‐IC within 15 min, and that these cells develop an extensive array of apical microvilli. The cAMP effect on A‐IC is direct, as shown in vitro in cells isolated from the medulla of transgenic mice expressing EGFP in IC (driven by the V‐ATPase B1‐subunit promoter). 3D‐reconstructions of confocal images showed that cAMP induced a dramatic, time dependent growth of microvilli in isolated IC. These morphological changes were paralleled by increased cAMP‐mediated H+ extrusion rates by A‐IC in isolated outer medullary CD measured using the ratiometric probe BCECF. These results support the idea that cAMP, generated by the soluble adenylyl cyclase (sAC ‐ which is enriched in IC) or other effectors, is part of the signal transduction mechanism for acid‐base sensing and V‐ATPase trafficking in IC. Funding source: NIH DK‐73266, DK‐42956, DK‐38452 and HD‐40793.

Role of purinergic signaling pathways in V-ATPase recruitment to apical membrane of acidifying epididymal clear cells.

Extracellular purinergic agonists regulate a broad range of physiological functions via P1 and P2 receptors. Using the epididymis as a model system in which luminal acidification is essential for sperm maturation and storage, we show here that extracellular ATP and its hydrolysis product adenosine trigger the apical accumulation of vacuolar H(+)-ATPase (V-ATPase) in acidifying clear cells. We demonstrate that the epididymis can hydrolyze luminal ATP into other purinergic agonists such as ADP via the activity of nucleotidases located in the epididymal fluid and in the apical membrane of epithelial cells. Alkaline phosphatase activity and abundant ecto-5'-nucleotidase protein were detected in the apical pole of principal cells. In addition, we show that nine nucleotidase genes (Nt5e, Alpl, Alpp, Enpp1, 2, and 3, and Entpd 2, 4, and 5), seven ATP P2 receptor genes (P2X1, P2X2, P2X3, P2X4, P2X6, P2Y2, P2Y5), and three adenosine P1 receptor genes (A1, A2B, and A3) are expressed in epithelial cells isolated by laser cut microdissection (LCM). The calcium chelator BAPTA-AM abolished the apical V-ATPase accumulation induced by ATP, supporting the contribution of P2X or P2Y in this response. The PKA inhibitor myristoylated protein kinase inhibitor (mPKI) inhibited adenosine-dependent V-ATPase apical accumulation, indicating the participation of the P1 A2B receptor. Altogether, these results suggest that the activation of P1 and P2 purinergic receptors by ATP and adenosine might play a significant role in luminal acidification in the epididymis, a process that is crucial for the establishment of male fertility.

CAMP stimulates apical V-ATPase accumulation, microvillar elongation, and proton extrusion in kidney collecting duct A-intercalated cells

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.

Purinergic receptors in mouse and rat epididymis : Role of luminal ATP and adenosine in V‐ATPase activation

In the epididymis, V‐ATPase is expressed in clear cells and acidifies the lumen where sperm mature and are stored. We showed that luminal alkaline pH triggers the apical membrane accumulation of V‐ATPase and that this process is calcium‐dependent. However, the link between alkaline pH and intracellular calcium is not completely elucidated. Some purinergic receptors are sensitive to pH, and we examined their expression by RT‐PCR in rat epithelial cells isolated by laser capture microdissection. mRNA transcripts for P1 adenosine receptors (subtypes A1, A2B and A3) and P2 ATP receptors (subtypes P2X1, P2X2, P2X4, P2X11, P2Y1 and P2Y2) were detected. Ecto‐ATPase and alkaline phosphatase were also observed, indicating potential degradation of ATP into adenosine in the epididymal lumen. In vivo perfusion of rat and mouse epididymis showed that both ATP (600µM) and adenosine (300µM) induced apical membrane V‐ATPase recruitment even in the absence of an alkaline pH stimulus. BAPTA‐AM abolished the ATP‐mediated effect, indicating the participation of calcium in this response. These data show that ATP and adenosine are sufficient to cause the accumulation of V‐ATPase in the apical membrane of clear cells. The paracrine activation of purinergic receptors might, therefore, play a significant role in the regulation of proton secretion in the male reproductive tract. This work is supported by NIH grant HD40793.

Alkaline pH- and cAMP-induced V-ATPase membrane accumulation is mediated by protein kinase A in epididymal clear cells

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.

Protein Kinase A (PKA) Regulates Vacuolar H + ‐ATPase (V‐ATPase) Recycling in Epididymal Clear Cells

In the epididymis, low luminal bicarbonate and acidic pH maintain sperm quiescent during maturation and storage. The V-ATPase, located in clear cells, plays a major role in luminal acidification. We have shown that cAMP and the bicarbonate-regulated soluble adenylyl cyclase induces V-ATPase apical accumulation in these cells. We examined whether or not PKA is involved in this response. Confocal immunofluorescence labeling on epididymis perfused in vivo showed that at luminal acidic pH (6.5), V-ATPase was distributed between short apical microvilli and sub-apical endosomes. The specific PKA activator, N6-monobutyryl-cAMP, induced elongation of apical microvilli and accumulation of V-ATPase in these structures. The PKA inhibitor H89 (10 μM) partially inhibited the apical accumulation of V-ATPase induced by the cAMP permeant analogue, 8-cpt-cAMP. Finally, perfusion at pH 6.5 with 8-pCPT-2′-O-methyl-cAMP, an activator of the exchange factors directly activated by cAMP (Epac), can partially mimic the cAMP-induced V-ATPase apical accumulation. These results indicate that direct stimulation of PKA activity by cAMP leads to the accumulation of V-ATPase in apical microvilli, although direct activation of Epac by cAMP may also participate in V-ATPase recycling in epididymal clear cells.

Modulation of the Actin Cytoskeleton via Gelsolin Regulates Vacuolar H+-ATPase Recycling

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.

Delivering an Acid Response

Pastor-Soler et al. used rat epididymus as a model system in which to investigate the role of soluble adenylyl cyclase (sAC), a form of adenylyl cyclase that is activated by bicarbonate, in the ability of epithelial cells to sense and respond to changes in pH. Clear cells, a subpopulation of epididymal epithelial cells, secrete protons into the epididymal lumen to maintain an acid pH. The rate of proton secretion depends on recycling of vacuolar H + ATPase (V-ATPase) between intracellular vesicles and the apical plasma membrane; however, the mechanisms governing this response remain unclear. Pastor-Soler et al. used reverse transcription polymerase chain reaction on rat epididymal epithelial cells harvested by laser capture microdissection combined with Western and immunofluorescence analysis of rat epididymus to confirm the presence of sAC in clear cells. The authors used confocal microscopy in combination with immunofluorescence and electron microscopy in combination with immunogold to monitor V-ATPase localization. In epididymus perfused with acidic saline, V-ATPase recycled between endosomes and the apical membrane, whereas alkaline pH promoted accumulation of V-ATPase in apical microvilli as did both a permeant analog of adenosine 3′,5′-monophosphate (cAMP) and bicarbonate. Pharmacologic inhibition of carbonic anhydrase indicated that bicarbonate production was essential for the response to alkaline pH but not to cAMP. Inhibition of sAC confirmed that this enzyme mediated the response to pH and to bicarbonate. This research establishes sAC as a critical mediator of a cellular response to changes in ambient pH, which may represent a widespread signaling pathway. N. Pastor-Soler, V. Beaulieu, T. N. Litvin, N. Da Silva, Y. Chen, D. Brown, J. Buck, L. R. Levin, S. Breton, Bicarbonate-regulated adenylyl cyclase (sAC) is a sensor that regulates pH-dependent V-ATPase recycling. J. Biol. Chem. 278 , 49523-49529 (2003). [Abstract] [Full Text]

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

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.