Biomedical subjects

John P Geibel

Publications and source records attributed to John P Geibel.

At least 19 recordsLinked to original sources

PubMed2006

DeltaF508 mutation results in impaired gastric acid secretion.

The cystic fibrosis transmembrane conductance regulator (CFTR) is recognized as a multifunctional protein that is involved in Cl(-) secretion, as well as acting as a regulatory protein. In order for acid secretion to take place a complex interaction of transport proteins and channels must occur at the apical pole of the parietal cell. Included in this process is at least one K(+) and Cl(-) channel, allowing for both recycling of K(+) for the H,K-ATPase, and Cl(-) secretion, necessary for the generation of concentrated HCl in the gastric gland lumen. We have previously shown that an ATP-sensitive potassium channel (K(ATP)) is expressed in parietal cells. In the present study we measured secretagogue-induced acid secretion from wild-type and DeltaF508-deficient mice in isolated gastric glands and whole stomach preparations. Secretagogue-induced acid secretion in wild-type mouse gastric glands could be significantly reduced with either glibenclamide or the specific inhibitor CFTR-inh172. In DeltaF508-deficient mice, however, histamine-induced acid secretion was significantly less than in wild-type mice. Furthermore, immunofluorescent localization of sulfonylurea 1 and 2 failed to show expression of a sulfonylurea receptor in the parietal cell, thus further implicating CFTR as the ATP-binding cassette transporter associated with the K(ATP) channels. These results demonstrate a regulatory role for the CFTR protein in normal gastric acid secretion.

ATP-Binding Cassette Transporters↗

PubMed2006

Hypertonic saline attenuates colonic tumor cell metastatic potential by activating transmembrane sodium conductance.

Hypertonic saline (HTS) suppresses tumor cell-endothelial interactions by reducing integrin expression. This translates into reduced adhesion, migration and metastatic potential. This study determined the relative contributions of hyperosmolarity and sodium-specific hypertonicity on the inhibitory effects of HTS, the intracellular pH and sodium responses to HTS and the role of cytoskeletal remodeling in these changes. Human colonic tumor cells (LS174T) were exposed to lipopolysaccharide under isotonic, hypertonic, sodium-free (N-methyl-D-glucamine), hyperosmolar (mannitol or urea), disrupted cytoskeletal (10 microg/ml cytochalasin D) conditions or in the presence of 5-(N-ethyl-N-isopropyl)amiloride (EIPA). Beta(1) integrin expression was measured flow-cytometrically. Intracellular sodium and pH were measured with confocal laser microscopic imaging. Statistical analysis was performed with analysis of variance, and P < 0.05 was considered significant. Data are represented as mean +/- SEM. Hypertonic exposure attenuated integrin expression (62.03 +/- 4.7% of control, P < 0.04). No discernible effect was observed with sodium-free or hyperosmolar solutions. HTS evoked a cellular alkalinization (by a mean 0.2 pH units) and an increase in cytosolic sodium concentration (by a mean 12.4 mM, P < 0.001) via upregulation of sodium-hydrogen exchange. Disassembly of actin microfilaments by cytochalasin D and antiporter inhibition with EIPA abrogated the effect of hypertonicity on integrin expression and intracellular sodium and pH (P < 0.05). HTS downregulates adhesion molecule expression via a hypertonic, sodium-specific, cytoskeletally mediated mechanism that involves activation of sodium-hydrogen exchange with associated changes in intracellular pH and sodium concentrations.

Cell Line, Tumor↗

PubMed2006

The KCNE2 potassium channel ancillary subunit is essential for gastric acid secretion.

Genes in the KCNE family encode single transmembrane domain ancillary subunits that co-assemble with voltage-gated potassium (Kv) channel alpha subunits to alter their function. KCNE2 (also known as MiRP1) is expressed in the heart, is associated with human cardiac arrhythmia, and modulates cardiac Kv alpha subunits hERG and KCNQ1 in vitro. KCNE2 and KCNQ1 are also expressed in parietal cells, leading to speculation they form a native channel complex there. Here, we disrupted the murine kcne2 gene and found that kcne2 (-/-) mice have a severe gastric phenotype with profoundly reduced parietal cell proton secretion, abnormal parietal cell morphology, achlorhydria, hypergastrinemia, and striking gastric glandular hyperplasia arising from an increase in the number of non-acid secretory cells. KCNQ1 exhibited abnormal distribution in gastric glands from kcne2 (-/-) mice, with increased expression in non-acid secretory cells. Parietal cells from kcne2 (+/-) mice exhibited normal architecture but reduced proton secretion, and kcne2 (+/-) mice were hypochlorhydric, indicating a gene-dose effect and a primary defect in gastric acid secretion. These data demonstrate that KCNE2 is essential for gastric acid secretion, the first genetic evidence that a member of the KCNE gene family is required for normal gastrointestinal function.

Achlorhydria↗

PubMed2005

An amino acid transporter involved in gastric acid secretion.

Gastric acid secretion is regulated by a variety of stimuli, in particular histamine and acetyl choline. In addition, dietary factors such as the acute intake of a protein-rich diet and the subsequent increase in serum amino acids can stimulate gastric acid secretion only through partially characterized pathways. Recently, we described in mouse stomach parietal cells the expression of the system L heteromeric amino acid transporter comprised of the LAT2-4F2hc dimer. Here we address the potential role of the system L amino acid transporter in gastric acid secretion by parietal cells in freshly isolated rat gastric glands. RT-PCR, western blotting and immunohistochemistry confirmed the expression of 4F2-LAT2 amino acid transporters in rat parietal cells. In addition, mRNA was detected for the B(0)AT1, ASCT2, and ATB(0+) amino acid transporters. Intracellular pH measurements in parietal cells showed histamine-induced and omeprazole-sensitive H+-extrusion which was enhanced by about 50% in the presence of glutamine or cysteine (1 mM), two substrates of system L amino acid transporters. BCH, a non-metabolizable substrate and a competitive inhibitor of system L amino acid transport, abolished the stimulation of acid secretion by glutamine or cysteine suggesting that this stimulation required the uptake of amino acids by system L. In the absence of histamine glutamine also stimulated H+-extrusion, whereas glutamate did not. Also, phenylalanine was effective in stimulating H+/K+-ATPase activity. Glutamine did not increase intracellular Ca2+ levels indicating that it did not act via the recently described amino acid modulated Ca2+-sensing receptor. These data suggest a novel role for heterodimeric amino acid transporters and may elucidate a pathway by which protein-rich diets stimulate gastric acid secretion.

Action Potentials↗

PubMed2005

The B1-subunit of the H(+) ATPase is required for maximal urinary acidification.

The multisubunit vacuolar-type H(+)ATPases mediate acidification of various intracellular organelles and in some tissues mediate H(+) secretion across the plasma membrane. Mutations in the B1-subunit of the apical H(+)ATPase that secretes protons in the distal nephron cause distal renal tubular acidosis in humans, a condition characterized by metabolic acidosis with an inappropriately alkaline urine. To examine the detailed cellular and organismal physiology resulting from this mutation, we have generated mice deficient in the B1-subunit (Atp6v1b1(-/-) mice). Urine pH is more alkaline and metabolic acidosis is more severe in Atp6v1b1(-/-) mice after oral acid challenge, demonstrating a failure of normal urinary acidification. In Atp6v1b1(-/-) mice, the normal urinary acidification induced by a lumen-negative potential in response to furosemide infusion is abolished. After an acute intracellular acidification, Na(+)-independent pH recovery rates of individual Atp6v1b1(-/-) intercalated cells of the cortical collecting duct are markedly reduced and show no further decrease after treatment with the selective H(+)ATPase inhibitor concanamycin. Apical expression of the alternative B-subunit isoform, B2, is increased in Atp6v1b1(-/-) medulla and colocalizes with the H(+)ATPase E-subunit; however, the greater severity of metabolic acidosis in Atp6v1b1(-/-) mice after oral acid challenge indicates that the B2-subunit cannot fully functionally compensate for the loss of B1. Our results indicate that the B1 isoform is the major B-subunit isoform that incorporates into functional, plasma membrane H(+)ATPases in intercalated cells of the cortical collecting duct and is required for maximal urinary acidification.

Acidosis, Renal Tubular↗

PubMed2005

The calcium-sensing receptor acts as a modulator of gastric acid secretion in freshly isolated human gastric glands.

Gastric acid secretion is activated by two distinct pathways: a neuronal pathway via the vagus nerve and release of acetylcholine and an endocrine pathway involving gastrin and histamine. Recently, we demonstrated that activation of H(+)-K(+)-ATPase activity in parietal cells in freshly isolated rat gastric glands is modulated by the calcium-sensing receptor (CaSR). Here, we investigated if the CaSR is functionally expressed in freshly isolated gastric glands from human patients undergoing surgery and if the CaSR is influencing histamine-induced activation of H(+)-K(+)-ATPase activity. In tissue samples obtained from patients, immunohistochemistry demonstrated the expression in parietal cells of both subunits of gastric H(+)-K(+)-ATPase and the CaSR. Functional experiments using the pH-sensitive dye 2',7'-bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein and measurement of intracellular pH changes allowed us to estimate the activity of H(+)-K(+)-ATPase in single freshly isolated human gastric glands. Under control conditions, H(+)-K(+)-ATPase activity was stimulated by histamine (100 microM) and inhibited by omeprazole (100 microM). Reduction of the extracellular divalent cation concentration (0 Mg(2+), 100 microM Ca(2+)) inactivated the CaSR and reduced histamine-induced activation of H(+)-K(+)-ATPase activity. In contrast, activation of the CaSR with the trivalent cation Gd(3+) caused activation of omeprazole-sensitive H(+)-K(+)-ATPase activity even in the absence of histamine and under conditions of low extracellular divalent cations. This stimulation was not due to release of histamine from neighbouring enterochromaffin-like cells as the stimulation persisted in the presence of the H(2) receptor antagonist cimetidine (100 microM). Furthermore, intracellular calcium measurements with fura-2 and fluo-4 showed that activation of the CaSR by Gd(3+) led to a sustained increase in intracellular Ca(2+) even under conditions of low extracellular divalent cations. These experiments demonstrate the presence of a functional CaSR in the human stomach and show that this receptor may modulate the activity of acid-secreting H(+)-K(+)-ATPase in parietal cells. Furthermore, our results show the viability of freshly isolated human gastric glands and may allow the use of this preparation for experiments investigating the physiological regulation and properties of human gastric glands in vitro.

Adult↗

PubMed2005

L-type amino acids stimulate gastric acid secretion by activation of the calcium-sensing receptor in parietal cells.

Parietal cells are the primary acid secretory cells of the stomach. We have previously shown that activation of the calcium-sensing receptor (CaSR) by divalent (Ca(2+)) or trivalent (Gd(3+)) ions stimulates acid production in the absence of secretagogues by increasing H(+),K(+)-ATPase activity. When overexpressed in HEK-293 cells, the CaSR can be allosterically activated by L-amino acids in the presence of physiological concentrations of extracellular Ca(2+) (Ca(o)(2+); 1.5-2.5 mM). To determine whether the endogenously expressed parietal cell CaSR is allosterically activated by L-amino acids, we examined the effect of the amino acids L-phenylalanine (L-Phe), L-tryptophan, and L-leucine on acid secretion. In ex vivo whole stomach preparations, exposure to L-Phe resulted in gastric luminal pH significantly lower than controls. Studies using D-Phe (inactive isomer) failed to elicit a response on gastric pH. H(+)-K(+)-ATPase activity was monitored by measuring the intracellular pH (pH(i)) of individual parietal cells in isolated rat gastric glands and calculating the rate of H(+) extrusion. We demonstrated that increasing Ca(o)(2+) in the absence of secretagogues caused a dose-dependent increase in H(+) extrusion. These effects were amplified by the addition of amino acids at various Ca(o)(2+) concentrations. Blocking the histamine-2 receptor with cimetidine or inhibiting system L-amino acid transport with 2-amino-2-norbornane-carboxylic acid did not affect the rate of H(+) extrusion in the presence of L-Phe. These data support the conclusion that amino acids, in conjunction with a physiological Ca(o)(2+) concentration, can induce acid secretion independent of hormonal stimulation via allosteric activation of the stomach CaSR.

Amino Acids↗

PubMed2005

Fluorescent imaging of Cl- in Amphiuma red blood cells: how the nuclear exclusion of Cl- affects the plasma membrane potential.

In this work, we test the idea that most, if not all, cellular Cl- of Amphiuma red blood cells is contained in the cytoplasm. If true, this could resolve the difference between the measured plasma membrane potential (Em) and that expected from the Donnan equilibrium distribution of Cl-. We studied the changes in the fluorescence intensity of the Cl- -sensitive dye, MQAE, entrapped in red cells that occurred when intracellular Cl- was exchanged with NO3-. We could thus monitor the distribution of Cl- between the nuclear and cytoplasmic compartments. We found that essentially all of the cell's Cl- resides in the cytoplasm. Knowing the volume of the cell occupied by the nucleus, we could accordingly correct the measured values of cell Cl-. This resulted in establishing a concordance between the measured values of Em and those calculated from the corrected values of the Cl- ratio, thus explaining the discrepancy. The exclusion of Cl- from the nucleus may result from its unusually high content of "excess" DNA that imposes an imbalance of net negative charge.

Animals↗

PubMed2005

Bicarbonate secretion: a neglected aspect of colonic ion transport.

Understanding of the mechanism of colonic electrolyte transport has markedly increased over the past three decades. This article provides a brief summary of the critical features of Na, Cl, and K transport in the large intestine and how these processes may be altered in diarrhea. Less understood is the mechanism of colonic HCO3 secretion. Recent progress in the regulation of HCO3 secretion in the distal colon is summarized with emphasis on the interrelationship between Cl-dependent, short-chain fatty acid (SCFA)-dependent, and cAMP-induced HCO3 secretion. cAMP down-regulates Cl-dependent HCO3 secretion, while SCFA stimulates HCO3 secretion but also inhibits both Cl-dependent and cAMP-induced HCO3 secretion. As SCFAs are the primary anions in stool, it is likely that SCFA-dependent HCO3 secretion is the primary mechanism of HCO3 secretion in the mammalian colon. Future studies will undoubtedly provide increased understanding of the mechanism of HCO3 secretion in health and disease.

Animals↗

PubMed2005

Secretion and absorption by colonic crypts.

The intestines play an important role in the absorption and secretion of nutrients. The colon is the final area for recapturing electrolytes and water prior to excretion, and in order to maintain this electrolyte homeostasis, a complex interaction between secretory and absorptive processes is necessary. Until recently it was thought that secretion and absorption were two distinct processes associated with either crypts or surface cells, respectively. Recently it was demonstrated that both the surface and crypt cells can perform secretory and absorptive functions and that, in fact, these functions can be going on simultaneously. This issue is important in the complexities associated with secretory diarrhea and also in attempting to develop treatment strategies for intestinal disorders. Here, we update the model of colonic secretion and absorption, discuss new issues of transporter activation, and identify some important new receptor pathways that are important modulators of the secretory and absorptive functions of the colon.

Animals↗

PubMed2005

Insulin receptor (IR) and glucose transporter 2 (GLUT2) proteins form a complex on the rat hepatocyte membrane.

The hepatic glucose transporter, GLUT2, facilitates bidirectional glucose transport across the hepatocyte plasma membrane under insulin regulation. We studied the interactions of IR and GLUT2 proteins to determine whether they are physically coupled in a receptor-transporter complex. By comparing endosome and plasma membrane IR and GLUT2 ratios before and after feeding, it was determined that IR and GLUT2 are internalized in a fixed ratio. When solubilized hepatocytes were immunoprecipitated with antibodies against either IR or GLUT2, both proteins co-precipitated. The association of IR and GLUT2 was further assessed by confocal microscopy. Sections of fed liver were incubated with fluorescein-tagged anti-GLUT2 or Texas Red-tagged anti-IR. Colocalization was observed both at the plasma membrane and in the cytosol. Fluorescence-resonance energy transfer studies further confirmed this association. We conclude that IR and GLUT2 form a receptor-transporter complex in hepatocytes, which forms a mechanism of insulin-mediated hepatic glucose regulation.

Animals↗

PubMed2004

Nongenomic stimulation of vacuolar H+-ATPases in intercalated renal tubule cells by aldosterone.

Renal collecting ducts play a critical role in acid-base homeostasis by establishing steep transepithelial pH gradients necessary for the almost complete reabsorption of bicarbonate and the effective secretion of ammonium into the urine. The mechanisms of urine acidification in collecting ducts involve active, electrogenic hydrogen (H+) secretion and, less importantly, potassium (K+)-H+ exchange. Deranged renal acidification and the inability to lower urine pH are hallmarks of distal tubular acidosis and often result from inborn errors of metabolism involving vacuolar H+-ATPase subunits in the collecting ducts. Three factors regulate H+-ATPase activity in intercalated cells of collecting ducts: the acid-base status, angiotensin II, and aldosterone. Most effects of aldosterone involve activation of the mineralocorticoid receptor and genomic changes in transcription and protein synthesis. Here we demonstrate a nongenomic pathway of vacuolar H+-ATPase activation in intercalated cells of isolated mouse outer medullary collecting ducts (OMCD). In vitro exposure of isolated outer medullary collecting ducts to aldosterone (10 nM) for times as short as 15 min increases vacuolar H+-ATPase activity approximately 2- to 3-fold. Neither inhibition of mineralocorticoid receptors nor of transcription and protein synthesis prevented aldosterone-induced stimulation of H+-ATPase. Incubation with colchicine, however, abolished the stimulatory effect of aldosterone, suggesting a role of the microtubular network for H+-ATPase stimulation. Immunohistochemistry in kidneys from aldosterone-injected mice showed increased apical H+-ATPase staining in OMCD-intercalated cells. The stimulatory effect of aldosterone was associated with a transient rise in intracellular Ca2+ and required intact PKC. Thus, rapid nongenomic modulation of vacuolar H+-ATPase activity in OMCD-intercalated cells by aldosterone may play an additional role in hormonal control of systemic acid-base homeostasis.

Aldosterone↗

PubMed2004

Extracellular polyamines regulate fluid secretion in rat colonic crypts via the extracellular calcium-sensing receptor.

BACKGROUND AND AIMS: Polyamines are essential for the normal postnatal development, maintenance, and function of gastrointestinal epithelia. The extracellular Ca(2+) (Ca(2+)(o)/nutrient)-sensing receptor is expressed on both luminal and basolateral membranes of colonocytes, and, in other cell systems, this receptor has been shown to respond to polyamines. Thus, the Ca(2+)-sensing receptor could provide a mechanism for modulation of colonocyte function by dietary and systemic extracellular polyamines. In the present study, we investigated the interaction of polyamines, particularly spermine, and extracellular Ca(2+) on second messenger generation by, and on function of, rat distal colonic crypts. METHODS: Calcium-sensing receptor activation was assessed in colonic epithelial cells and intact crypts freshly isolated from distal colon by monitoring intracellular IP(3) and Ca(2+) accumulation using radioimmunoassay and Fluo-3 fluorometry, respectively. Interactions of extracellular Ca(2+) and spermine on regulation of both basal and forskolin-stimulated fluid transport were measured in crypts microperfused in vitro. RESULTS: Polyamine (spermine > spermidine > putrescine)-mediated enhancement of intracellular D-myo-inositol 1,4,5-trisphosphate (IP(3)) and Ca(2+) accumulation required extracellular Ca(2+), and the EC(50) for extracellular Ca(2+)-mediated activation of the calcium-sensing receptor was reduced by polyamines. Extracellular spermine modulated both basal and forskolin-stimulated fluid secretion in perfused colonic crypts, and the EC(50) for spermine-induced reduction in forskolin-stimulated fluid secretion was inversely dependent on extracellular Ca(2+) (Ca(2+)(o)). CONCLUSIONS: The interactions of extracellular Ca(2+) and polyamines on second messenger accumulation and fluid secretion support a role for the luminal and basolateral calcium-sensing receptors in mediating some of the effects of polyamines on distal colonic epithelial cells.

Animals↗

PubMed2004

Renal vacuolar H+-ATPase.

Vacuolar H(+)-ATPases are ubiquitous multisubunit complexes mediating the ATP-dependent transport of protons. In addition to their role in acidifying the lumen of various intracellular organelles, vacuolar H(+)-ATPases fulfill special tasks in the kidney. Vacuolar H(+)-ATPases are expressed in the plasma membrane in the kidney almost along the entire length of the nephron with apical and/or basolateral localization patterns. In the proximal tubule, a high number of vacuolar H(+)-ATPases are also found in endosomes, which are acidified by the pump. In addition, vacuolar H(+)-ATPases contribute to proximal tubular bicarbonate reabsorption. The importance in final urinary acidification along the collecting system is highlighted by monogenic defects in two subunits (ATP6V0A4, ATP6V1B1) of the vacuolar H(+)-ATPase in patients with distal renal tubular acidosis. The activity of vacuolar H(+)-ATPases is tightly regulated by a variety of factors such as the acid-base or electrolyte status. This regulation is at least in part mediated by various hormones and protein-protein interactions between regulatory proteins and multiple subunits of the pump.

Animals↗

PubMed2003

Molecular cloning and characterization of Atp6v1b1, the murine vacuolar H+ -ATPase B1-subunit.

The multisubunit vacuolar-type proton-translocating ATPases (H(+)-ATPases) mediate the acidification of various intracellular organelles. In a subset of tissues, they also mediate H(+) secretion at the plasma membrane. Two isoforms of the H(+)-ATPase B-subunit exist in humans; we have shown that mutations in ATP6V1B1, encoding the B1-isoform, cause the clinical condition distal renal tubular acidosis. Here we report the cloning and characterization of murine Atp6v1b1, which encodes a 513-amino acid (aa) protein with 93% identity to human ATP6V1B1. Genomic organization is conserved between the murine and human H(+)-ATPase B1-subunits, and Atp6v1b1 maps to a region of mouse chromosome 6 syntenic to human 2p13, the location of ATP6V1B1. Northern blotting detects a 2.2-kb Atp6v1b1 transcript in the kidney and testis, but not other major organs. In mouse kidney, the B1-subunit localizes to intercalated cells of the cortical and medullary collecting duct. B1 protein levels were not increased in either mouse renal cortex or medulla after either 2 or 7 days of oral acid loading. These results demonstrate that Atp6v1b1 encodes the murine ortholog of human ATP6V1B1 and provides a tool for future development of animal models based on manipulation of the Atp6v1b1 genomic locus.

5' Flanking Region↗

PubMed2003

Demonstration of a functional apical sodium hydrogen exchanger in isolated rat gastric glands.

Previous studies have shown that gastric glands express at least sodium-hydrogen exchanger (NHE) isoforms 1-4. Our aim was to study NHE-3 localization in rat parietal cells and to investigate the functional activity of an apical membrane NHE-3 isoform in parietal cells of rats. Western blot analysis and immunohistochemistry showed expression of NHE-3 in rat stomach colocalizing the protein in parietal cells together with the beta-subunit of the H(+)-K(+)-ATPase. Functional studies in luminally perfused gastric glands demonstrated the presence of an apical NHE isoform sensitive to low concentrations of 5-ethylisopropyl amiloride (EIPA). Intracellular pH measurements in parietal cells conducted in omeprazole-pretreated superfused gastric glands showed an Na+-dependent proton extrusion pathway that was inhibited both by low concentrations of EIPA and by the NHE-3 specific inhibitor S3226. This pathway for proton extrusion had a higher activity in resting glands and was inhibited on stimulation of histamine-induced H(+)-K(+)-ATPase proton extrusion. We conclude that the NHE-3 isoform located on the apical membrane of parietal cells offers an additional pathway for proton secretion under resting conditions. Furthermore, the gastric NHE-3 appears to work under resting conditions and inactivates during periods of H(+)-K(+)-ATPase activity.

Amiloride↗

PubMed2003

A rapid enzymatic method for the isolation of defined kidney tubule fragments from mouse.

The increasing number of available genetically manipulated mice makes it necessary to develop tools and techniques for examining the phenotypes of these animals. We have developed a straightforward and rapid method for the isolation of large quantities of single tubule fragments from the mouse kidney. Immunohistochemistry, electron microscopy, and fluorescence microscopy were used to evaluate the viability, functional characteristics, and morphology of proximal tubules (PT), and collecting ducts from cortex (CCD) and inner stripe of the outer medulla (ISOMCD). Tubules were isolated using a modified collagenase digestion technique, and selected under light microscopy for experimentation. Electron microscopy and trypan blue exclusion showed that a large portion of unselected proximal tubules were damaged by the digestion procedure. The selected tubules, however, all excluded trypan blue, indicating that the plasma membrane had remained intact. Immunocytochemistry on isolated CCD showed normal distribution of H(+)-ATPase, pendrin, and anion exchanger-1 (AE-1) staining. The pH-sensitive dye 2',7'-bis(2-carboxylethyl)-5(6)-carboxyfluorescein (BCECF) was used to measure Na(+)-dependent and -independent intracellular pH (pH(i)) recovery rates in PT, and in single intercalated cells of CCD and ISOMCD fragments. Na(+)-dependent pH(i)-recovery was 0.144+/-0.008 (PT), 0.182+/-0.013 (CCD), and 0.112+/-0.010 pH units/min. (ISOMCD). Na(+)-independent pH(i) recovery was found in all three segments (PT: 0.021+/-0.002, CCD: 0.037+/-0.002, ISOMCD: 0.033+/-0.002 pH units/min) and was sensitive to concanamycin. In summary, we have developed a new technique for rapid and straightforward preparation of large quantities of defined tubule fragments from mouse kidney. Using this technique, the first measurements of plasma membrane vacuolar H(+)-ATPase activities in mouse PT and collecting duct were made. This technique will facilitate further characterization of kidney function in normal and genetically manipulated animals.

Animals↗

PubMed2003

Localization and regulation of the ATP6V0A4 (a4) vacuolar H+-ATPase subunit defective in an inherited form of distal renal tubular acidosis.

Vacuolar-type H(+)-ATPases (V-H(+)-ATPases) are the major H(+)-secreting protein in the distal portion of the nephron and are involved in net H(+) secretion (bicarbonate generation) or H(+) reabsorption (net bicarbonate secretion). In addition, V-H(+)-ATPases are involved in HCO(3)(-) reabsorption in the proximal tubule and distal tubule. V-H(+)-ATPases consist of at least 13 subunits, the functions of which have not all been elucidated. Mutations in the accessory ATP6V0A4 (a4 isoform) subunit have recently been shown to cause an inherited form of distal renal tubular acidosis in humans. Here, the localization of this subunit in human and mouse kidney was studied and the regulation of expression and localization of this subunit in mouse kidney in response to acid-base and electrolyte intake was investigated. Reverse transcription-PCR on dissected mouse nephron segments amplified a4-specific transcripts in proximal tubule, loop of Henle, distal convoluted tubule, and cortical and medullary collecting duct. a4 protein was localized by immunohistochemistry to the apical compartment of the proximal tubule (S1/S2 segment), the loop of Henle, the intercalated cells of the distal convoluted tubule, the connecting segment, and all intercalated cells of the entire collecting duct in human and mouse kidney. All types of intercalated cells expressed a4. NH(4)Cl or NaHCO(3) loading for 24 h, 48 h, or 7 d as well as K(+) depletion for 7 and 14 d had no influence on a4 protein expression levels in either cortex or medulla as determined by Western blotting. Immunohistochemistry, however, demonstrated a subcellular redistribution of a4 in response to the different stimuli. NH(4)Cl and K(+) depletion led to a pronounced apical staining in the connecting segment, cortical collecting duct, and outer medullary collecting duct, whereas NaHCO(3) loading caused a stronger bipolar staining in the cortical collecting duct. Taken together, these results demonstrate a4 expression in the proximal tubule, loop of Henle, distal tubule, and collecting duct and suggest that under conditions in which increased V-H(+)-ATPase activity is required, a4 is regulated by trafficking but not protein expression. This may allow for the rapid adaptation of V-H(+)-ATPase activity to altered acid-base intake to achieve systemic pH homeostasis. The significance of a4 expression in the proximal tubule in the context of distal renal tubular acidosis will require further clarification.

Acid-Base Equilibrium↗