NH4Cl Prepulse Research Articles

Real-time influence of intracellular acidification and Na+ /H+ exchanger inhibition on in-cell pyruvate metabolism in the perfused mouse heart: A 31 P-NMR and hyperpolarized 13 C-NMR study.

Disruption of acid-base balance is linked to various diseases and conditions. In the heart, intracellular acidification is associated with heart failure, maladaptive cardiac hypertrophy, and myocardial ischemia. Previously, we have reported that the ratio of the in-cell lactate dehydrogenase (LDH) to pyruvate dehydrogenase (PDH) activities is correlated with cardiac pH. To further characterize the basis for this correlation, these in-cell activities were investigated under induced intracellular acidification without and with Na+ /H+ exchanger (NHE1) inhibition by zoniporide. Male mouse hearts (n= 30) were isolated and perfused retrogradely. Intracellular acidification was performed in two ways: (1) with the NH4 Cl prepulse methodology; and (2) by combining the NH4 Cl prepulse with zoniporide. 31 P NMR spectroscopy was used to determine the intracellular cardiac pH and to quantify the adenosine triphosphate and phosphocreatine content. Hyperpolarized [1-13 C]pyruvate was obtained using dissolution dynamic nuclear polarization. 13 C NMR spectroscopy was used to monitor hyperpolarized [1-13 C]pyruvate metabolism and determine enzyme activities in real time at a temporal resolution of a few seconds using the product-selective saturating excitation approach. The intracellular acidification induced by the NH4 Cl prepulse led to reduced LDH and PDH activities (-16% and -39%, respectively). This finding is in line with previous evidence of reduced myocardial contraction and therefore reduced metabolic activity upon intracellular acidification. Concomitantly, the LDH/PDH activity ratio increased with the reduction in pH, as previously reported. Combining the NH4 Cl prepulse with zoniporide led to a greater reduction in LDH activity (-29%) and to increased PDH activity (+40%). These changes resulted in a surprising decrease in the LDH/PDH ratio, as opposed to previous predictions. Zoniporide alone (without intracellular acidification) did not change these enzyme activities. A possible explanation for the enzymatic changes observed during the combination of the NH4 Cl prepulse and NHE1 inhibition may be related to mitochondrial NHE1 inhibition, which likely negates the mitochondrial matrix acidification. This effect, combined with the increased acidity in the cytosol, would result in an enhanced H+ gradient across the mitochondrial membrane and a temporarily higher pyruvate transport into the mitochondria, thereby increasing the PDH activity at the expense of the cytosolic LDH activity. These findings demonstrate the complexity of in-cell cardiac metabolism and its dependence on intracellular acidification. This study demonstrates the capabilities and limitations of hyperpolarized [1-13 C]pyruvate in the characterization of intracellular acidification as regards cardiac pathologies.

Renal NHE1 is required for regulation of intracellular pH in principal cells and type B intercalated cells

The sodium-hydrogen exchanger isoform 1 (NHE1) is a plasma membrane transporter with roles in transepithelial Na+/H+ transport, intracellular pH (pHi) and cell volume regulation. Within the kidney, NHE1 is previously documented to be expressed in all nephron segments. However, recent transcriptomic and proteomic analyses, and our staining results in mice, consistently find no expression of NHE1 in the proximal tubule or thick ascending limb, but in the distal nephron, including distal convoluted tubule, connecting tubule, and collecting duct (principal and intercalated cells, PC and IC, respectively). We hypothesized that basolateral NHE1 in IC (co-labelling with H+-ATPase B1 subunit, labeling all 3 types of IC: type A, B, and non-A-non-B) plays an important role in acid-base homeostasis. Due to lack of a suitable animal model, we generated kidney-specific NHE1 knockout (NHE1KS-KO) mice. NHE1KS-KO mice completely lack renal NHE1 protein expression compared to their control littermates (n=6 male mice/genotype). No differences were observed in body weight, food and water intake (determined in their home cages) or concentrations of blood Na+, K+, Ca2+, pH or HCO3-. Urine analysis showed no significant differences in electrolytes, minerals of pH. To mechanistically determine the role of NHE1 for pHi regulation in IC and PC we used confocal microscopy of acutely-split open cortical collecting ducts. This method allows for the analysis of a vast number of PC/IC simultaneously. To study the role of NHE1 we employed a NH4Cl pre-pulse (40 mM) and measured pHi recovery after switching back to a physiological solution. Intracellular pH recovery in PC (n=171) was significantly slower (~50%) in NHE1KS-KO mice compared to controls (n=85). Similar to PC, pHi recovery in type B IC of NHE1KS-KO mice (n=38) was significantly slower (~35%) compared to controls (n=40). No differences in type A IC were observed between NHE1KS-KO mice (n=35) and controls (n=29). Inhibition of ClC-K2 via NPPB (Cl-channel blocker) was used to confirm cell types: PC did not respond with a change in pHi but NPPB increased pHi in type A and decreased pHi in type B ICs. In summary, our results identify for the first time an important role of NHE1 for pHi regulation particularly in PCs and type B ICs. Funding to Dr. Rieg was provided by the NIDDK grant 1R01DK110621, VA Merit Review Award IBX004968A, AHA Transformational Research Award 19TPA34850116, and NIDDK Diabetic Complications Consortium grants DK076169 and DK115255. DK095029 and DK117865 (both to O. Pochynyuk). Dr. Thomas was supported by an AHA postdoctoral grant (828731), V. Tomilin by an AHA Career Development Award 19CDA34660148. This is the full abstract presented at the American Physiology Summit 2023 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.

Effects of Andrographolide on Intracellular pH Regulation, Cellular Migration, and Apoptosis in Human Cervical Cancer Cells †.

Cancer cells have been characterized with alkaline intracellular pH (pHi) values (≥7.2) to enable cancer proliferation, migration, and progression. The aim of the present study was to explore the concentration-dependent effects of Andrographolide, an active diterpenoid compound of herb Andrographis paniculata, on Na+/H+ exchanger isoform 1 (NHE1), cellular migration and apoptosis in human cervical cancer cells (HeLa). The pHi was detected by microspectrofluorometry method, and intracellular acidification was induced by NH4Cl prepulse technique. Viability and protein expression were determined by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and Western blot, respectively. Human normal endocervical cells (End1), ectocervical cells (Ect1), and HeLa were bought commercially. The resting pHi value of HeLa (≈7.47) was significantly higher than that of End1 and Ect1 (≈7.30), and shifted from alkaline to acidic following acid/base impacts. In HEPES (4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid | N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) -buffered superfusate, NHE1 and V-ATPase co-existed functionally for acid extrusion in HeLa, while only NHE1 existed functionally in End/Ect1. Andrographolide (3–1000 μM) concentration-dependently inhibited NHE1 activity. Cell-migration and expressions of NHE1, V-ATPase, PARP (poly-ADP-ribose-polymerase), pro-Caspase-3, and Bcl-2 were significantly reduced by pretreating with Andrographolide (≥100 μM) for 24–48 h in HeLa. Andrographolide inhibited cell viability of End1-cells/Ect1 and HeLa (≥100 and ≥30 μM, respectively). The present findings implicate the promising clinical applications of Andrographolide on cervical cancer treatment.

Functional effects of urotensin-II on intracellular pH regulators in human radial artery smooth muscle cells

The regulation of intracellular pH (pHi) plays a vital role in various cellular functions. We previously demonstrated that three different acid extruders, the Na+-H+ exchanger (NHE), Na+−HCO3− co-transporter (NBC) and H+-linked monocarboxylate transporter (MCT), functioned together in cultured human radial artery smooth muscle cells (HRASMCs). However, the functions of acid-loading transporters in HRASMCs remain poorly understood. Urotensin II (U-II), one of the most potent vasoconstrictors, is highly expressed in many cardiovascular diseases. The aim of this present study was to determine the concentration effect of U-II (3 pM∼100 nM) on the functional activity of pHi regulators in HRASMCs. Cultured HRASMCs were derived from segments of human radial arteries obtained from patients undergoing bypass grafting. Changes in pHi recovery due to intracellular acidification and alkalization induced by NH4Cl prepulse and Na-acetate prepulse, respectively, were detected by microspectrofluorimetry with the pH-sensitive fluorescent dye BCECF. Our present study showed that (a) U-II increased the activity of NHE in a concentration-dependent manner but did not change that of NBC or MCT or resting pHi, (b) the Cl−−OH− exchanger (CHE) facilitated base extrusion, and (c) U-II induced a concentration-dependent increase in the activity of CHE. In conclusion, for the first time, our results highlight a concentration-dependent increase in the activity of NHE and CHE, but not NBC and MCT, induced by U-II in HRASMCs.

Functional and molecular mechanism of intracellular pH regulation in human inducible pluripotent stem cells.

AIMTo establish a functional and molecular model of the intracellular pH (pHi) regulatory mechanism in human induced pluripotent stem cells (hiPSCs).METHODShiPSCs (HPS0077) were kindly provided by Dr. Dai from the Tri-Service General Hospital (IRB No. B-106-09). Changes in the pHi were detected either by microspectrofluorimetry or by a multimode reader with a pH-sensitive fluorescent probe, BCECF, and the fluorescent ratio was calibrated by the high K+/nigericin method. NH4Cl and Na-acetate prepulse techniques were used to induce rapid intracellular acidosis and alkalization, respectively. The buffering power (β) was calculated from the ΔpHi induced by perfusing different concentrations of (NH4)2SO4. Western blot techniques and immunocytochemistry staining were used to detect the protein expression of pHi regulators and pluripotency markers.RESULTSIn this study, our results indicated that (1) the steady-state pHi value was found to be 7.5 ± 0.01 (n = 20) and 7.68 ± 0.01 (n =20) in HEPES and 5% CO2/HCO3--buffered systems, respectively, which were much greater than that in normal adult cells (7.2); (2) in a CO2/HCO3--buffered system, the values of total intracellular buffering power (β) can be described by the following equation: βtot = 107.79 (pHi)2 - 1522.2 (pHi) + 5396.9 (correlation coefficient R2 = 0.85), in the estimated pHi range of 7.1-8.0; (3) the Na+/H+ exchanger (NHE) and the Na+/HCO3- cotransporter (NBC) were found to be functionally activated for acid extrusion for pHi values less than 7.5 and 7.68, respectively; (4) V-ATPase and some other unknown Na+-independent acid extruder(s) could only be functionally detected for pHi values less than 7.1; (5) the Cl-/ OH- exchanger (CHE) and the Cl-/HCO3- anion exchanger (AE) were found to be responsible for the weakening of intracellular proton loading; (6) besides the CHE and the AE, a Cl--independent acid loading mechanism was functionally identified; and (7) in hiPSCs, a strong positive correlation was observed between the loss of pluripotency and the weakening of the intracellular acid extrusion mechanism, which included a decrease in the steady-state pHi value and diminished the functional activity and protein expression of the NHE and the NBC.CONCLUSIONFor the first time, we established a functional and molecular model of a pHi regulatory mechanism and demonstrated its strong positive correlation with hiPSC pluripotency.

PH recovery from a proton load in rat cardiomyocytes: effects of chronic exercise.

The ability of cardiomyocytes to recover from a proton load was examined in the hearts of exercise-trained and sedentary control rats in CO2/[Formula: see text]-free media. Acidosis was created by the NH4Cl prepulse technique, and intracellular pH (pHi) was determined using fluorescence microscopy on carboxy-SNARF-1 AM-loaded isolated cardiomyocytes. CO2-independent pHi buffering capacity (βi) was measured by incrementally reducing the extracellular NH4Cl concentration in steps of 50% from 20 to 1.25 mM. βi increased as pHi decreased in both exercise-trained and sedentary control groups. However, the magnitude of increase in βi as a function of pHi was found to be significantly ( P < 0.001) greater in the exercise-trained group compared with the sedentary control group. The rate of pHi recovery from an imposed proton load was found to not be different between the exercise-trained and control groups. The Na+/H+ exchanger-dependent H+ extrusion rate during the recovery from an imposed proton load, however, was found to be significantly greater in the exercise-trained group compared with the control group. By increasing βi and subsequently the Na+/H+ exchanger-dependent H+ extrusion rate, exercise training may provide cardiomyocytes with the ability to better handle an intracellular excess of H+ generated during hypoxia/ischemic insults and may serve in a cardioprotective role. These data may be predictive of two positive outcomes: 1) increased exercise tolerance by the heart and 2) a protective mechanism that limits the degree of myocardial acidosis and subsequent damage that accompanies ischemia-reperfusion stress. NEW & NOTEWORTHY The enhanced ability to deal with acidosis conferred by exercise training is likely to improve exercise tolerance and outcomes in response to myocardial ischemia-reperfusion injury.

Effects of Indomethacin on Intracellular pH and Na⁺/H⁺ Exchanger in the Human Monocytes.

The ability to maintain optimal intracellular pH (pH(i)) is an essential requirement for all cells. Na⁺-H⁺ exchanger (NHE), a ubiquitously expressed transmembrane protein, has been found widely as a major acid extruder in many different cell types, including human monocytes. We therefore investigated the mechanism of the active pH(i) recovery from intracellular acidosis (induced by NH₄Cl prepulse) using intracellular 2',7'-bis (2-carboxethyl)-5(6)-carboxyl-fluorescein (BCECF) fluorescence in cultured human monocytes. Indomethacin is a potent, nonselective inhibitor of cyclooxygenases. Due to its toxicity, the clinical use of indomethacin as an analgesic-antipyretic agent is limited. However, it has recently been found that indomethacin can effectively treat many inflammatory/immune disorders. In this study, we further investigated the effect of indomethacin on the pHi and explored the underlying mechanism. In HEPES (nominally HCO₃⁻-free) Tyrode solution, a pH(i) recovery from induced intracellular acidosis could be blocked completely by 30 μM HOE 694, a specific NHE1 inhibitor, or by removing [Na⁺]₀. Therefore, in the present study, we provided functional evidence, physiologically and pharmacologically, that the HCO₃⁻-independent acid extruder was mostly likely the NHE1 which was involved in acid extrusion in the human monocytes. Moreover, indomethacin (1 μM-1 mM) decreased pH(i) levels in a concentration-dependent manner and significantly suppressed the activity of the NHE1, suggesting that indomethacin-induced intracellular acidosis is caused both by the inhibition of NHE1 activity and the non-specified NHE1-independent acidifying mechanism. In conclusion, our present study demonstrates that NHE1 exists functionally in human monocytes, and the indomethacin-induced pHi decreasing is summation effects on NHE1-dependent and -independent mechanism.

Functional interdependence of NHE1 and merlin in human melanoma cells.

Upregulation of the Na(+)/H(+) exchanger isoform 1 (NHE1) has been correlated with tumor malignancy. In contrast, moesin-radixin-ezrin-like protein (merlin) is a tumor suppressor that protects from cancerogenesis. Merlin is highly related to the members of the ezrin, radixin, and moesin (ERM) protein family that are directly attached to and functionally linked with NHE1. In addition, merlin inhibits the MAPK cascade and the Rho-GTPases known to activate NHE1 activity. The present study investigates whether NHE1 expression and activity affect merlin or, conversely, whether merlin has an impact on NHE1 in human melanoma (MV3) cells. Indeed, features of merlin-deficient MV3 cells point to a functional link: merlin-deficient cells showed a decreased NHE1 expression and, paradoxically, an increase in NHE1 activity as measured upon cytosolic acidification (NH4Cl prepulse method). Loss of merlin also led to an elevated cell motility that could be further increased by NHE1 overexpression, whereas NHE1 overexpression alone had no effect on migration. In contrast, neither NHE1 expression nor its activity had an impact on merlin expression. These results suggest a novel tumor suppressor function of merlin in melanoma cells: the inhibition of the proto-oncogenic NHE1 activity, possibly including its downstream signaling pathways.

HV1 Acts as a Sodium Sensor and Promotes Superoxide Production in Medullary Thick Ascending Limb of Dahl Salt-Sensitive Rats

We previously characterized a H(+) transport pathway in medullary thick ascending limb nephron segments that when activated stimulated the production of superoxide by nicotinamide adenine dinucleotide phosphate oxidase. Importantly, the activity of this pathway was greater in Dahl salt-sensitive rats than salt-resistant (SS.13(BN)) rats, and superoxide production was enhanced in low Na(+) media. The goal of this study was to determine the molecular identity of this pathway and its relationship to Na(+). We hypothesized that the voltage-gated proton channel, HV1, was the source of superoxide-stimulating H(+) currents. To test this hypothesis, we developed HV1(-/-) null mutant rats on the Dahl salt-sensitive rat genetic background using zinc-finger nuclease gene targeting. HV1 could be detected in medullary thick limb from wild-type rats. Intracellular acidification using an NH4Cl prepulse in 0 sodium/BaCl2 containing media resulted in superoxide production in thick limb from wild-type but not HV1(-/-) rats (P<0.05) and more rapid recovery of intracellular pH in wild-type rats (ΔpHI 0.005 versus 0.002 U/s, P=0.046, respectively). Superoxide production was enhanced by low intracellular sodium (<10 mmol/L) in both thick limb and peritoneal macrophages only when HV1 was present. When fed a high-salt diet, blood pressure, outer medullary renal injury (tubular casts), and oxidative stress (4-hydroxynonenal staining) were significantly reduced in HV1(-/-) rats compared with wild-type Dahl salt-sensitive rats. We conclude that HV1 is expressed in medullary thick ascending limb and promotes superoxide production in this segment when intracellular Na(+) is low. HV1 contributes to the development of hypertension and renal disease in Dahl salt-sensitive rats.

The Cardioprotective Effects of Novel Na+/H+ Exchanger Inhibitor TY-51924 on Ischemia/Reperfusion Injury

The inhibitory effects of sodium 3-guanidinocarbonyl-2-methyl-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridine-9-ylmethyl sulfate monoethanolate (TY-51924) are selective for Na(+)/H(+) exchanger (NHE)-1 in PS120 cells expressing human NHE isoforms assayed by NH(4)Cl prepulse technique. The median inhibitory concentrations (micromolar) of TY-51924 were 0.095 ± 0.008 (NHE-1), 0.621 ± 0.093 (NHE-2), and >100 (NHE-3). In anesthetized dogs subjected to 90 minutes ischemia/300 minutes reperfusion, intravenous bolus TY-51924 at 5 and 10 mg/kg administered 5 minutes before reperfusion reduced infarct size. The infarct size reduction ratios of TY-51924 at 5 and 10 mg/kg versus vehicle were 32.8% and 52.4%, respectively. But TY-51924 at 10 mg/kg administered 10 minutes after reperfusion did not reduce infarct size. In 2-step intravenous infusion initiated 15 minutes before reperfusion, TY-51924 at low dose (3.8 mg/kg per 5 minutes, then 6.2 mg/kg per 20 minutes) and at high dose (7.6 mg/kg per 5 minutes, then 12.4 mg/kg per 20 minutes) reduced infarct size. The infarct size reduction ratios of TY-51924 at 10 and 20 mg/kg versus vehicle were 39.2% and 51.7%, respectively; plasma drug concentrations at reperfusion were 16.8 and 38.8 μg/mL, respectively. This indicates that maintaining a plasma drug concentration of >20 μg/mL at reperfusion enables TY-51924 to reduce infarct size by inhibiting the NHE, which is activated during the early period of reperfusion.

Elevated carbon dioxide upregulates NBCn1 Na+/HCO3−cotransporter in human embryonic kidney cells

The NBCn1 Na(+)/HCO3(-) cotransporter catalyzes the electroneutral movement of 1 Na(+):1 HCO3(-) into kidney cells. We characterized the intracellular pH (pHi) regulation in human embryonic kidney cells (HEK) subjected to NH4Cl prepulse acid loading, and we examined the NBCn1 expression and function in HEK cells subjected to 24-h elevated Pco2 (10-15%). After acid loading, in the presence of HCO3(-), ∼50% of the pHi recovery phase was blocked by the Na(+)/H(+) exchanger inhibitors EIPA (10-50 μM) and amiloride (1 mM) and was fully cancelled by 30 μM EIPA under nominally HCO3(-)-free conditions. In addition, in the presence of HCO3(-), pHi recovery after acid loading was completely blocked when Na(+) was omitted in the buffer. pHi recovery after acidification in HEK cells was repeated in the presence of the NBC inhibitor S0859, and the pHi recovery was inhibited by S0859 in a dose-dependent manner (Ki = 30 μM, full inhibition at 60 μM), which confirmed NBC Na(+)/HCO3(-) cotransporter activation. NBCn1 expression increased threefold after 24-h exposure of cultured HEK cells to 10% CO2 and sevenfold after exposure to 15% CO2, examined by immunoblots. Finally, exposure of HEK cells to high CO2 significantly increased the HCO3(-)-dependent recovery of pHi after acid loading. We conclude that HEK cells expressed the NBCn1 Na(+)/HCO3(-) cotransporter as the only HCO3(-)-dependent mechanism responsible for cellular alkaline loading. NBCn1, which expresses in different kidney cell types, was upregulated by 24-h high-Pco2 exposure of HEK cells, and this upregulation was accompanied by increased NBCn1-mediated HCO3(-) transport.

The BK channel in the renal adaptation to chronic metabolic acidosis (CMA) in cortical collecting duct (CCD)

The density of BK channels in CCD intercalated cells (ICs) exceeds that in principal cells. We hypothesized that this channel is upregulated in acid secreting α‐ICs during CMA to recycle K for a luminal H/K‐ATPase. H pump activity was measured in α‐ICs in BCECF‐loaded microperfused CCDs from CMA rabbits (75 mM NH4Cl in drinking water × 5–7 d) as the rate (pH U/min) of Na‐independent pHi recovery from an acute NH4Cl prepulse in the absence (H‐ATPase) and then presence (H/K‐ATPase) of luminal (L) K, in the absence (control) or presence of iberiotoxin (IBX), a BK channel inhibitor. The effect of IBX on pHi was also studied in the presence of bath EIPA and L bafilomycin (Baf) to inhibit Na/H exchange and the H‐ATPase, respectively. Nadir pHi H‐ATPase activity pHi H/K‐ATPase activity Control (n=7) 6.64 ± 0.05 0.057 ± 0.030 6.89 ± 0.08 0.009 ± 0.011 L IBX (n=4) 6.68 ± 0.05 0.043 ± 0.040 6.87 ± 0.10 0.000 ± 0.008 In 5 CCDs, pHi fell from 7.36 ± 0.10 to 7.16 ± 0.06 with EIPA (p&lt;0.05) but was further unchanged by L Baf or IBX. The findings that IBX did not significantly alter (i) H‐ or H/K‐ATPase‐mediated recovery from an acute acid load or (ii) pHi in CCDs from CMA animals suggests that the channel does not participate in the adaptation of α‐ICs to CMA.

Abstract 92: Hvcn1 Contributes to Superoxide Production in the Medullary Thick Ascending Limb

We have previously reported that H + efflux stimulates superoxide (O 2 - ) production in the medullary thick ascending limb of the loop of Henle (mTAL) in rats by activation of an as yet unidentified H + transport pathway. As HVCN1, a voltage gated H + channel associated with NADPH oxidase in immune cells, is also expressed in mTAL, we hypothesized that HVCN1 is responsible for H + efflux induced production of O 2 - in mTAL. In order to test this hypothesis we utilized HVCN1 -/- mice and wild type litter mates obtained from KOMP. To confirm that HVCN1 was functionally knocked out in HVCN1 -/- mice we stimulated the respiratory burst using PMA (100uM) in peritoneal macrophages extracted from HVCN1 -/- and WT mice and determined maximal O 2 - production using L-012 luminescence. Maximal O 2 - production during the respiratory burst was significantly lower in macrophages from HVCN1 -/- mice (23±4AU; n=6) compared to WT mice (92±3AU; n=5; p&lt;0.05), consistent with loss of HVCN1 function. mTAL were isolated from the inner stripe of the outer-medulla by micro-dissection and loaded for 60 min with the O 2 - sensitive dye dihydroethidium (DHE). Live mTAL were then imaged on a heated chamber attached to a fluorescent microscope and the ratio of ethidium (Eth) to DHE quantified using metafluor imaging software as an index of O 2 - production. O 2 - producing H + currents were isolated in 0 Na + , 100uM BaCl 2 media and cellular H + efflux stimulated using an NH 4 Cl (20mM) prepulse. Following removal of NH 4 Cl from the bath there was a significant increase in O 2 - production in mTAL from WT mice (Delta slope Eth/DHE 15.9±3.6 AU/sec; p&lt;0.02 (n=5)) but not HVCN1 -/- mice (delta slope 4.7±9.6; P=0.64 (n=6)). The response of WT mice was significantly greater than that of HVCN1 -/- mice (P&lt;0.001). We conclude that HVCN1 is required for maximal O 2 - production in mTAL in response to H + efflux. These data suggest that HVCN1 may be a novel target to prevent renal oxidative stress.

Pyk2 regulates H+-ATPase-mediated proton secretion in the outer medullary collecting duct via an ERK1/2 signaling pathway

Acid-secreting intercalated cells respond to changes in systemic pH through regulation of apical H(+) transporters. Little is known about the mechanism by which these cells sense changes in extracellular pH (pH(o)). Pyk2 is a nonreceptor tyrosine kinase activated by autophosphorylation at Tyr402 by cell-specific stimuli, including decreased pH, and is involved in the regulation of MAPK signaling pathways and transporter activity. We examined whether the Pyk2 and MAPK signaling pathway mediates the response of transport proteins to decreased pH in outer medullary collecting duct cells. Immunoblot analysis of phosphorylated Pyk2 (Tyr402), ERK1/2 (Thr202/Tyr204), and p38 (Thr180/Tyr182) was used to assay protein activation. To examine specificity of kinase activation and its effects, we used Pyk2 small interfering RNA to knockdown Pyk2 expression levels, the Src kinase inhibitor 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP 1) to inhibit Pyk2 phosphorylation, and the MEK inhibitor U0126 to inhibit ERK1/2 phosphorylation. The pH-sensitive fluorescent probe 2'-7'-bis(carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl ester (BCECF-AM) was used to assay H(+) transporter activity. The activity of H(+) transporters was measured as the rate of intracellular pH (pH(i)) recovery after an NH(4)Cl prepulse. We show that Pyk2 is endogenously expressed and activated by acid pH in mouse-derived outer medullary collecting duct (mOMCD1) cells. Incubation of mOMCD1 cells in acid media [extracellular pH (pH(o)) 6.7] increased the phosphorylation of Pyk2, ERK1/2, and p38. Reduction in pH(i) induced by an NH(4)Cl prepulse also increased the phosphorylation of Pyk2, ERK1/2, and p38. Consistent with our previous studies, we found that mOMCD1 cells exhibit H(+)-ATPase and H(+),K(+)-ATPase activity. Pyk2 inhibition by Pyk2 siRNA and PP 1 prevented Pyk2 phosphorylation as well as H(+)-ATPase-mediated recovery in mOMCD1 cells. In addition, ERK1/2 inhibition by U0126 prevented acid-induced ERK1/2 phosphorylation and H(+)-ATPase-mediated pH(i) recovery but not phosphorylation of p38. We conclude that Pyk2 and ERK1/2 are required for increasing H(+)-ATPase, but not H(+),K(+)-ATPase, activity at decreased pH(i) in mOMCD1 cells.

Luminal flow modulates H+-ATPase activity in the cortical collecting duct (CCD).

Epithelial Na(+) channel (ENaC)-mediated Na(+) absorption and BK channel-mediated K(+) secretion in the cortical collecting duct (CCD) are modulated by flow, the latter requiring an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), microtubule integrity, and exocytic insertion of preformed channels into the apical membrane. As axial flow modulates HCO(3)(-) reabsorption in the proximal tubule due to changes in both luminal Na(+)/H(+) exchanger 3 and H(+)-ATPase activity (Du Z, Yan Q, Duan Y, Weinbaum S, Weinstein AM, Wang T. Am J Physiol Renal Physiol 290: F289-F296, 2006), we sought to test the hypothesis that flow also regulates H(+)-ATPase activity in the CCD. H(+)-ATPase activity was assayed in individually identified cells in microperfused CCDs isolated from New Zealand White rabbits, loaded with the pH-sensitive dye BCECF, and then subjected to an acute intracellular acid load (NH(4)Cl prepulse technique). H(+)-ATPase activity was defined as the initial rate of bafilomycin-inhibitable cell pH (pH(i)) recovery in the absence of luminal K(+), bilateral Na(+), and CO(2)/HCO(3)(-), from a nadir pH of ∼6.2. We found that 1) an increase in luminal flow rate from ∼1 to 5 nl·min(-1)·mm(-1) stimulated H(+)-ATPase activity, 2) flow-stimulated H(+) pumping was Ca(2+) dependent and required microtubule integrity, and 3) basal and flow-stimulated pH(i) recovery was detected in cells that labeled with the apical principal cell marker rhodamine Dolichos biflorus agglutinin as well as cells that did not. We conclude that luminal flow modulates H(+)-ATPase activity in the rabbit CCD and that H(+)-ATPases therein are present in both principal and intercalated cells.

Nedd4-1 and β-Arrestin-1 Are Key Regulators of Na+/H+ Exchanger 1 Ubiquitylation, Endocytosis, and Function

The ubiquitously expressed mammalian Na(+)/H(+) exchanger 1 (NHE1) controls cell volume and pH but is also critically involved in complex biological processes like cell adhesion, cell migration, cell proliferation, and mechanosensation. Pathways controlling NHE1 turnover at the plasma membrane, however, are currently unclear. Here, we demonstrate that NHE1 undergoes ubiquitylation at the plasma membrane by a process that is unprecedented for a mammalian ion transport protein. This process requires the adapter protein β-arrestin-1 that interacts with both the E3 ubiquitin ligase Nedd4-1 and the NHE1 C terminus. Truncation of NHE1 C terminus to amino acid 550 abolishes binding to β-arrestin-1 and NHE1 ubiquitylation. Overexpression of β-arrestin-1 or of wild type but not ligase-dead Nedd4-1 increases NHE1 ubiquitylation. siRNA-mediated knock-down of Nedd4-1 or β-arrestin-1 reduces NHE1 ubiquitylation and endocytosis leading to increased NHE1 surface levels. Fibroblasts derived from β-arrestin-1 and Nedd4-1 knock-out mice show loss of NHE1 ubiquitylation, increased plasmalemmal NHE1 levels and greatly enhanced NHE1 transport compared with wild-type fibroblasts. These findings reveal Nedd4-1 and β-arrestin-1 as key regulators of NHE1 ubiquitylation, endocytosis, and function. Our data suggest a broader role for β-arrestins in the regulation of membrane ion transport proteins than currently known.

CaSR Mediated Gastric Acid Secretion via the Vacuolar H+-ATPase - With Special Emphasis on the Effect of Extracellular Calcium and Changes in Systemic pH

Purpose: One of the most important functions of the stomach is gastric acid secretion. Since the H+, K+ATPase is the main proton pump in the production of gastric acid, little attention has been paid to the vacuolar H+-ATPase. Therefore, our goal was to evaluate whether this ion channel can be activated by calcium via the CaSR like the H+, K+ATPase. We also hypothesized that the activity of the H+-ATPase is influenced by extracellular pH disturbances such as alkalosis and acidosis, therefore leading to a change in acid secretion. Methods: Isolated gastric glands from male Sprague-Dawley rats were used to measure intracellular pH recovery rates, which were monitored using the pH sensitive dye BCECF. Recovery rates were measured after maximally alkalizing the cells using the NH4Cl prepulse technique. To make sure that the recovery rate was due to the vacuolar H+-ATPase we used sodium and potassium free flushing solutions. Calcimimetic effects were induced by adding R641 (400 nM), an allosteric modifier of the CaSR, to the Na+ and K+ free solution. For imitating pH disturbances, all solutions were titrated to pH 7.2 (for acidosis) and 7.6 (for alkalosis). Finally, we compared the two different influences (calcium and pH-change) in order to determine which factor had a greater influence on gastric acid secretion. Results: Allosteric modification of the CaSR via R641 significantly increases vacuolar H+-ATPase activity and thus gastric acid secretion (0.09815± 0.00780 versus 0.03001±0.00305, p-value <0.01). Metabolic acidosis decreases (0.02467±0.00203 versus 0.03160±0.00187, p-value=0.0179) and metabolic alkalosis increases hydrochloric acid secretion (0.04833 ±0.00256 versus 0.03160±0.00187, p-value <0.01). Combining the two influences (Ca2+ and pH change) we discovered that acid-induced decreased acid secretion can be stimulated again through addition of R641. In contrast, metabolic alkalosis-induced acid secretion cannot be further increased by adding a calcimimetic. Conclusion: The stomach has a regulatory function on the acid-base homeostasis of the body through its secretion of hydrochloric acid. Additionally, extracellular-induced pH changes influence gastric acid secretion via the H+-ATPase. Hypercalcemic conditions, such as in patients with hyperparathyroidism or end stage renal disease, significantly increase gastric acid secretion through stimulation of the H+-ATPase via the CaSR. This fact might be able to explain the comparatively high numbers of coexisting gastric ulcers in those patients.

A Novel Amiloride-Sensitive H + Transport Pathway Mediates Enhanced Superoxide Production in Thick Ascending Limb of Salt-Sensitive Rats, Not Na + /H + Exchange

It has been reported previously that H(+) efflux via the Na(+)/H(+) exchange stimulates NAD(P)H oxidase-dependent superoxide (O(2)(.-)) production in medullary thick ascending limb. We have demonstrated recently that N-methyl-amiloride-sensitive O(2)(.-) production is enhanced in the thick ascending limb of Dahl salt-sensitive (SS) rats, suggesting that H(+) efflux through Na(+)/H(+) exchangers may promote renal oxidative stress and the development of hypertension in these animals. In the current study we demonstrate, using selective and potent inhibitors, that inhibition of Na(+)/H(+) exchange does not mediate the ability of N-methyl-amiloride to inhibit thick ascending limb O(2)(.-) production. To determine the mechanism of action of N-methyl-amiloride, we examined H(+) efflux and O(2)(.-) production in SS and SS.13(BN) thick ascending limbs of prehypertensive, 0.4% NaCl-fed rats. Tissue strips containing the medullary thick ascending limb were isolated from male SS and salt-resistant consomic SS.13(BN) rats, loaded with either dihydroethedium or 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester, and imaged in a heated tissue bath. In Na(+)-replete media, activation of Na(+)/H(+) exchange using an NH(4)Cl prepulse did not stimulate thick ascending limb O(2)(.-) production. In Na(+)-free media containing BaCl(2) in which Na(+)/H(+) activity was inhibited, an NH(4)Cl prepulse stimulated O(2)(.-) production in medullary thick ascending limb renal tubular segments. This response was enhanced in medullary thick ascending limb of SS rats (slope Deltaethidium/Deltadihydroethedium=0.029+/-0.004) compared with SS.13(BN) rats (slope=0.010+/-0.004; P<0.04) and could be inhibited by N-methyl-amiloride (slope=0.005+/-0.002 and 0.006+/-0.002 for SS and SS.13(BN), respectively). We concluded that only H(+) efflux through a specific, as-yet-unidentified, amiloride-sensitive H(+) channel promotes O(2)(.-) production in the medullary thick ascending limb and that this channel is upregulated in SS rats.

T1977 Rand Appropriateness Study with Development of Recommendations for NSAID Treatment

NH4Cl prepulse followed by a Na+-free solution. Alkalinization in Na+-free solution represents intracellular acid production and eliminates the possibility of contribution by the Na+/ H+ proteins. Studies were done in the presence or absence of indomethacin (100μM) or acetylsalicylic acid (ASA) (100-500μM). Results: In the presence of indomethacin, a rapid decrease in pHi is seen upon the removal of Na+, followed by an immediate rise in pHi, approaching pH of 7.0. In the absence of indomethacin, no alkalinization was observed until Na+ was added back to the solution. In a separate series of experiments, a similar result was observed with ASA. In the presence of ASA, a rapid alkalinization was observed in the Na+-free solution, representing intracellular acid production. This effect was not seen in the absence of drug. Conclusions: This study suggests that indomethacin and ASA have a direct effect on activation of acid secretion in the parietal cell. This is a novel mechanism in which NSAID use may increase risk of PUD. The direct stimulatory effect on acid secretion provides evidence for a rapid onset of PUD via the decreased barrier function coupled with enhanced acid secretion that occurs in the absence of secretagogues.

T1979 Pepsinogen II As Marker of Inflammation in H.P Infection and in NSAIDs Damage

NH4Cl prepulse followed by a Na+-free solution. Alkalinization in Na+-free solution represents intracellular acid production and eliminates the possibility of contribution by the Na+/ H+ proteins. Studies were done in the presence or absence of indomethacin (100μM) or acetylsalicylic acid (ASA) (100-500μM). Results: In the presence of indomethacin, a rapid decrease in pHi is seen upon the removal of Na+, followed by an immediate rise in pHi, approaching pH of 7.0. In the absence of indomethacin, no alkalinization was observed until Na+ was added back to the solution. In a separate series of experiments, a similar result was observed with ASA. In the presence of ASA, a rapid alkalinization was observed in the Na+-free solution, representing intracellular acid production. This effect was not seen in the absence of drug. Conclusions: This study suggests that indomethacin and ASA have a direct effect on activation of acid secretion in the parietal cell. This is a novel mechanism in which NSAID use may increase risk of PUD. The direct stimulatory effect on acid secretion provides evidence for a rapid onset of PUD via the decreased barrier function coupled with enhanced acid secretion that occurs in the absence of secretagogues.