Intracellular pH Recovery Research Articles

New advances in the protective mechanisms of acidic pH after ischemia: Participation of NO

BackgroundIt has been previously demonstrated that the maintenance of ischemic acidic pH or the delay of intracellular pH recovery at the onset of reperfusion decreases ischemic-induced cardiomyocyte death. ObjectiveTo examine the role played by nitric oxide synthase (NOS)/NO-dependent pathways in the effects of acidic reperfusion in a regional ischemia model. MethodsIsolated rat hearts perfused by Langendorff technique were submitted to 40 min of left coronary artery occlusion followed by 60 min of reperfusion (IC). A group of hearts received an acid solution (pH = 6.4) during the first 2 min of reperfusion (AR) in absence or in presence of l-NAME (NOS inhibitor). Infarct size (IS) and myocardial function were determined. In cardiac homogenates, the expression of P-Akt, P-endothelial and inducible isoforms of NOS (P-eNOS and iNOS) and the level of 3-nitrotyrosine were measured. In isolated cardiomyocytes, the intracellular NO production was assessed by confocal microscopy, under control and acidic conditions. Mitochondrial swelling after Ca2+ addition and mitochondrial membrane potential (Δψ) were also determined under control and acidosis. ResultsAR decreased IS, improved postischemic myocardial function recovery, increased P-Akt and P-eNOS, and decreased iNOS and 3-nitrotyrosine. NO production increased while mitochondrial swelling and Δψ decreased in acidic conditions. l-NAME prevented the beneficial effects of AR. ConclusionsOur data strongly supports that a brief acidic reperfusion protects the myocardium against the ischemia-reperfusion injury through eNOS/NO-dependent pathways.

β3-Adrenoceptor as a new player in the sympathetic regulation of the renal acid-base homeostasis.

Efferent sympathetic nerve fibers regulate several renal functions activating norepinephrine receptors on tubular epithelial cells. Of the beta-adrenoceptors (β-ARs), we previously demonstrated the renal expression of β3-AR in the thick ascending limb (TAL), the distal convoluted tubule (DCT), and the collecting duct (CD), where it participates in salt and water reabsorption. Here, for the first time, we reported β3-AR expression in the CD intercalated cells (ICCs), where it regulates acid-base homeostasis. Co-localization of β3-AR with either proton pump H+-ATPase or Cl-/HCO3 - exchanger pendrin revealed β3-AR expression in type A, type B, non-A, and non-B ICCs in the mouse kidney. We aimed to unveil the possible regulatory role of β3-AR in renal acid-base homeostasis, in particular in modulating the expression, subcellular localization, and activity of the renal H+-ATPase, a key player in this process. The abundance of H+-ATPase was significantly decreased in the kidneys of β3-AR-/- compared with those of β3-AR+/+ mice. In particular, H+-ATPase reduction was observed not only in the CD but also in the TAL and DCT, which contribute to acid-base transport in the kidney. Interestingly, we found that in in vivo, the absence of β3-AR reduced the kidneys' ability to excrete excess proton in the urine during an acid challenge. Using ex vivo stimulation of mouse kidney slices, we proved that the β3-AR activation promoted H+-ATPase apical expression in the epithelial cells of β3-AR-expressing nephron segments, and this was prevented by β3-AR antagonism or PKA inhibition. Moreover, we assessed the effect of β3-AR stimulation on H+-ATPase activity by measuring the intracellular pH recovery after an acid load in β3-AR-expressing mouse renal cells. Importantly, β3-AR agonism induced a 2.5-fold increase in H+-ATPase activity, and this effect was effectively prevented by β3-AR antagonism or by inhibiting either H+-ATPase or PKA. Of note, in urine samples from patients treated with a β3-AR agonist, we found that β3-AR stimulation increased the urinary excretion of H+-ATPase, likely indicating its apical accumulation in tubular cells. These findings demonstrate that β3-AR activity positively regulates the expression, plasma membrane localization, and activity of H+-ATPase, elucidating a novel physiological role of β3-AR in the sympathetic control of renal acid-base homeostasis.

Hydrocortisone cardioprotection in ischaemia/reperfusion injury involves antioxidant mechanisms.

Glucocorticoid (GR) and mineralocorticoid (MR) receptors are highly expressed in cardiac tissue, and both can be activated by corticosteroids. MR activation, in acute myocardial infarction (AMI), worsens cardiac function, and increase NHE activity contributing to the deleterious process. In contrast, effects of GR activation are not fully understood, probably because of the controversial scenario generated by using different doses or potencies of corticosteroids. We tested the hypothesis that an acute dose of hydrocortisone (HC), a low-potency glucocorticoid, in a murine model of AMI could be cardioprotective by regulating NHE1 activity, leading to a decrease in oxidative stress. Isolated hearts from Wistar rats were subjected to regional ischemic protocol. HC (10 nmol/L) was added to the perfusate during early reperfusion. Infarct size and oxidative stress were determined. Isolated papillary muscles from non-infarcted hearts were used to evaluate HC effect on sodium-proton exchanger 1 (NHE1) by analysing intracellular pH recovery from acute transient acidosis. HC treatment decreased infarct size, improved cardiac mechanics, reduced oxidative stress after AMI, while restoring the decreased level of the pro-fusion mitochondrial protein MFN-2. Co-treatment with the GR-blocker Mifepristone avoided these effects. HC reduced NHE1 activity by increasing the NHE1 pro-inhibiting Ser648 phosphorylation site and its upstream kinase AKT. HC restored the decreased AKT phosphorylation and anti-apoptotic BCL-2 protein expression detected after AMI. Our results provide the first evidence that acute HC treatment during early reperfusion induces cardioprotection against AMI, associated with a non-genomic HC-triggered NHE1 inhibition by AKT and antioxidant action that might involves mitochondrial dynamics improvement.

TRPV4 expression in the renal tubule is necessary for maintaining whole body K+ homeostasis.

The Ca2+-permeable transient receptor potential vanilloid type 4 (TRPV4) channel serves as the sensor of tubular flow, thus being well suited to govern mechanosensitive K+ transport in the distal renal tubule. Here, we directly tested whether the TRPV4 function is significant in affecting K+ balance. We used balance metabolic cage experiments and systemic measurements with different K+ feeding regimens [high (5% K+), regular (0.9% K+), and low (<0.01% K+)] in newly created transgenic mice with selective TRPV4 deletion in the renal tubule (TRPV4fl/fl-Pax8Cre) and their littermate controls (TRPV4fl/fl). Deletion was verified by the absence of TRPV4 protein expression and lack of TRPV4-dependent Ca2+ influx. There were no differences in plasma electrolytes, urinary volume, and K+ levels at baseline. In contrast, plasma K+ levels were significantly elevated in TRPV4fl/fl-Pax8Cre mice on high K+ intake. K+-loaded knockout mice exhibited lower urinary K+ levels than TRPV4fl/fl mice, which was accompanied by higher aldosterone levels by day 7. Moreover, TRPV4fl/fl-Pax8Cre mice had more efficient renal K+ conservation and higher plasma K+ levels in the state of dietary K+ deficiency. H+-K+-ATPase levels were significantly increased in TRPV4fl/fl-Pax8Cre mice on a regular diet and especially on a low-K+ diet, pointing to augmented K+ reabsorption in the collecting duct. Consistently, we found a significantly faster intracellular pH recovery after intracellular acidification, as an index of H+-K+-ATPase activity, in split-opened collecting ducts from TRPV4fl/fl-Pax8Cre mice. In summary, our results demonstrate an indispensable prokaliuretic role of TRPV4 in the renal tubule in controlling K+ balance and urinary K+ excretion during variations in dietary K+ intake. NEW & NOTEWORTHY The mechanoactivated transient receptor potential vanilloid type 4 (TRPV4) channel is expressed in distal tubule segments, where it controls flow-dependent K+ transport. Global TRPV4 deficiency causes impaired adaptation to variations in dietary K+ intake. Here, we demonstrate that renal tubule-specific TRPV4 deletion is sufficient to recapitulate the phenotype by causing antikaliuresis and higher plasma K+ levels in both states of K+ load and deficiency.

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.

Crosstalk between Yeast Cell Plasma Membrane Ergosterol Content and Cell Wall Stiffness under Acetic Acid Stress Involving Pdr18.

Acetic acid is a major inhibitory compound in several industrial bioprocesses, in particular in lignocellulosic yeast biorefineries. Cell envelope remodeling, involving cell wall and plasma membrane composition, structure and function, is among the mechanisms behind yeast adaptation and tolerance to stress. Pdr18 is a plasma membrane ABC transporter of the pleiotropic drug resistance family and a reported determinant of acetic acid tolerance mediating ergosterol transport. This study provides evidence for the impact of Pdr18 expression in yeast cell wall during adaptation to acetic acid stress. The time-course of acetic-acid-induced transcriptional activation of cell wall biosynthetic genes (FKS1, BGL2, CHS3, GAS1) and of increased cell wall stiffness and cell wall polysaccharide content in cells with the PDR18 deleted, compared to parental cells, is reported. Despite the robust and more intense adaptive response of the pdr18Δ population, the stress-induced increase of cell wall resistance to lyticase activity was below parental strain levels, and the duration of the period required for intracellular pH recovery from acidification and growth resumption was higher in the less tolerant pdr18Δ population. The ergosterol content, critical for plasma membrane stabilization, suffered a drastic reduction in the first hour of cultivation under acetic acid stress, especially in pdr18Δ cells. Results revealed a crosstalk between plasma membrane ergosterol content and cell wall biophysical properties, suggesting a coordinated response to counteract the deleterious effects of acetic acid.

Regulation of electrogenic Na+ /HCO3- cotransporter 1 (NBCe1) function and its dependence on m-TOR mediated phosphorylation of Ser245.

Astrocytes are pivotal responders to alterations of extracellular pH, primarily by regulation of their principal acid-base transporter, the membrane-bound electrogenic Na+ /bicarbonate cotransporter 1 (NBCe1). Here, we describe amammalian target of rapamycin (mTOR)-dependent and NBCe1-mediated astroglial response to extracellular acidosis. Using primary mouse cortical astrocytes, we investigated the effect of long-term extracellular metabolic acidosis on regulation of NBCe1 and elucidated the underlying molecular mechanisms by immunoblotting, biotinylation of surface proteins, intracellular H+ recording using the H+ -sensitive dye 2',7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein, and phosphoproteomic analysis. The results showed significant increase of NBCe1-mediated recovery of intracellular pH from acidification in WT astrocytes, but not in cortical astrocytes from NBCe1-deficient mice. Acidosis-induced upregulation of NBCe1 activity was prevented following inhibition of mTOR signaling by rapamycin. Yet, during acidosis or following exposure of astrocytes to rapamycin, surface protein abundance of NBCe1 remained -unchanged. Mutational analysis in HeLa cells suggested that NBCe1 activity was dependent on phosphorylation state of Ser245 , a residue conserved in all NBCe1 variants. Moreover, phosphorylation state of Ser245 is regulated by mTOR and is inversely correlated with NBCe1 transport activity. Our results identify pSer245 as a novel regulator of NBCe1 functional expression. We propose that context-dependent and mTOR-mediated multisite phosphorylation of serine residues of NBCe1 is likely to be a potent mechanism contributing to the response of astrocytes to acid/base challenges during pathophysiological conditions.

Cardiac Na/Ca Exchange Suppression: A Late‐Breaking Knockout Story Showing That There Is No Free Lunch

Cardiac Na/Ca Exchange Suppression: A Late‐Breaking Knockout Story Showing That There Is No Free Lunch

Deficient Active Transport Activity in Healing Mucosa After Mild Gastric Epithelial Damage.

Peptic ulcers recur, suggesting that ulcer healing may leave tissue predisposed to subsequent damage. In mice, we have identified that the regenerated epithelium found after ulcer healing will remain abnormal for months after healing. To determine whether healed gastric mucosa has altered epithelial function, as measured by electrophysiologic parameters. Ulcers were induced in mouse gastric corpus by serosal local application of acetic acid. Thirtydays or 8months after ulcer induction, tissue was mounted in an Ussing chamber. Transepithelial electrophysiologic parameters (short-circuit current, Isc. resistance, R) were compared between the regenerated healed ulcer region and the non-ulcerated contralateral region, in response to luminal hyperosmolar NaCl challenge (0.5M). In unperturbed stomach, luminal application of hyperosmolar NaCl transiently dropped Isc followed by gradual recovery over 2h. Compared to the starting baseline Isc, percent Isc recovery was reduced in 30-day healing mucosa, but not at 8months. Prior to NaCl challenge, a lower baseline Isc was observed in trefoil factor 2 (TFF2) knockout (KO) versus wild type (WT), with no Isc recovery in either non-ulcerated or healing mucosa of KO. Inhibiting Na/H exchanger (NHE) transport in WT mucosa inhibited Isc recovery in response to luminal challenge. NHE2-KO baseline Isc was reduced versus NHE2-WT. In murine gastric organoids, NHE inhibition slowed recovery of intracellular pH and delayed the repair of photic induced damage. Healing gastric mucosa has deficient electrophysiological recovery in response to hypertonic NaCl. TFF2 and NHE2 contribute to Isc regulation, and the recovery and healing of transepithelial function.

The CO-releasing molecule CORM-3 protects adult cardiomyocytes against hypoxia-reoxygenation by modulating pH restoration

Several studies have reported that CORM-3, a water-soluble carbon monoxide releasing molecule, elicits cardioprotection against myocardial infarction but the mechanism remains to be investigated. Numerous reports indicate that inhibition of pH regulators, the Na+/H+ exchanger (NHE) and Na+/HCO3− symporter (NBC), protect cardiomyocytes from hypoxia/reoxygenation injury by delaying the intracellular pH (pHi) recovery at reperfusion. Our goal was to explore whether CORM-3-mediated cytoprotection involves the modulation of pH regulation. When added at reoxygenation, CORM-3 (50 μM) reduced the mortality of cardiomyocytes exposed to 3 h of hypoxia and 2 h of reoxygenation in HCO3−-buffered solution. This effect was lost when using inactive iCORM-3, which is depleted of CO and used as control, thus implicating CO as the mediator of this cardioprotection. Interestingly, the cardioprotective effect of CORM-3 was abolished by switching to a bicarbonate-free medium. This effect of CORM-3 was also inhibited by 5-hydroxydecanoate, a mitochondrial ATP-dependent K+ (mKATP) channel inhibitor (500 μM) or PD098059, a MEK1/2 inhibitor (10 μM). In additional experiments and in the absence of hypoxia-reoxygenation, intracellular pH was monitored in cardiomyocytes exposed to cariporide to block NHE activity. CORM-3 inhibited alkalinisation and this effect was blocked by PD098059 and 5-HD. In conclusion, CORM-3 protects the cardiomyocyte against hypoxia-reoxygenation injury by inhibiting a bicarbonate transporter at reoxygenation, probably the Na+/HCO3− symporter. This cardioprotective effect of CORM-3 requires the activation of mKATP channels and the activation of MEK1/2.

ER Stress Induced Apoptosis of Smooth Muscle in Pulmonary Hypertension is Regulated by the Sodium‐Hydrogen Exchanger

ObjectiveAltering the apoptosis‐resistant phenotype of pulmonary arterial smooth muscle cells (PASMCs) may be key to reversing pathologic pulmonary vascular remodeling in pulmonary hypertension (PH). Endoplasmic reticulum (ER) stress and sodium‐hydrogen exchanger (NHE) activity have each been implicated in vascular remodeling in PH. We studied the effect of NHE activity upon ER stress‐induced apoptosis in PASMCs isolated from the SU5416‐hypoxia (SuHx) rat model of PH.MethodsPH was induced in male Wistar rats via exposure to the vascular endothelial growth factor receptor inhibitor SU5416 plus 3 wk of 10% oxygen followed by 2 wk of normoxia (SuHx model), while control rats were exposed to vehicle and 5 wk of normoxia. PASMCs were isolated from SuHx and control rats and expanded in culture. Apoptosis was measured using Hoechst stain. Thioflavin fluorescence was quantified as a measure of ER stress. Protein levels of the ER stress‐induced transcription factor, CCAAT/enhancer‐binding protein homologous protein (CHOP), were measured via immunoblot. To measure NHE activity, PASMCs were incubated with pH‐sensitive fluorescent dye and the Na+‐dependent recovery of intracellular pH following acidification by ammonium pulse challenge was measured via fluorescence microscopy. Cell stress was induced with 250 μM H2O2. NHE was pharmacologically inhibited with 10 μM ethyl‐isopropyl amiloride.ResultsER stress, a known stimulus of apoptosis, was increased by H2O2 in control PASMCs and was elevated at baseline in SuHx PASMCs. H2O2 exposure increased apoptosis in control PASMCs. Despite increased ER stress, SuHx PASMCs exhibited low levels of apoptosis both at baseline and in response to H2O2. CHOP protein expression, which is induced by ER stress and triggers the apoptotic cascade, was increased by H2O2 in control cells, but remained low in H2O2‐treated SuHx PASMCs. NHE activity was increased in PASMCs from SuHx rats, and pharmacologic NHE inhibition in SuHx PASMCs increased both basal and H2O2‐induced CHOP expression, and also increased apoptosis.ConclusionsPH PASMCs are resistant to ER‐stress induced apoptosis due to decreased CHOP expression. NHE inhibition increases CHOP expression in PH PASMCs and restores ER‐stress induced apoptosis, suggesting a novel mechanism to control vascular remodeling in PH.Support or Funding InformationNIH K08HL133475 &amp; R01HL073859This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

P38-MAP Kinase Negatively Regulates the Slow Force Response to Stretch in Rat Myocardium through the Up-Regulation of Dual Specificity Phosphatase 6 (DUSP6).

Myocardial stretch increases cardiac force in two consecutive phases: The first one due to Frank-Starling mechanism, followed by the gradually developed slow force response (SFR). The latter is the mechanical counterpart of an autocrine/paracrine mechanism involving the release of angiotensin II (Ang II) and endothelin (ET) leading to Na⁺/H⁺ exchanger 1 (NHE-1) phosphorylation and activation. Since previous evidence indicates that p38-MAP kinase (p38-MAPK) negatively regulates the Ang II-induced NHE1 activation in vascular smooth muscle and the positive inotropic effect of ET in the heart, we hypothesized that this kinase might modulate the magnitude of the SFR to stretch. Experiments were performed in isolated rat papillary muscles subjected to sudden stretch from 92 to 98% of its maximal length, in the absence or presence of the p38-MAPK inhibitor SB202190, or its inactive analogous SB202474. Western blot technique was used to determine phosphorylation level of p38-MAPK, ERK1/2, p90RSK and NHE-1 (previously immunoprecipitated with NHE-1 polyclonal antibody). Dual specificity phosphatase 6 (DUSP6) expression was evaluated by RT-PCR and western blot. Additionally, the Na⁺-dependent intracellular pH recovery from an ammonium prepulse-induced acid load was used to asses NHE-1 activity. The SFR was larger under p38-MAPK inhibition (SB202190), effect that was not observed in the presence of an inactive analogous (SB202474). Myocardial stretch activated p38-MAPK, while pre-treatment with SB202190 precluded this effect. Inhibition of p38-MAPK increased stretched-induced NHE-1 phosphorylation and activity, key event in the SFR development. Consistently, p38-MAPK inhibition promoted a greater increase in ERK1/2-p90RSK phosphorylation/activation after myocardial stretch, effect that may certainly be responsible for the observed increase in NHE-1 phosphorylation under this condition. Myocardial stretch induced up-regulation of the DUSP6, which specifically dephosphorylates ERK1/2, effect that was blunted by SB202190. Taken together, our data support the notion that p38-MAPK activation after myocardial stretch restricts the SFR by limiting ERK1/2-p90RSK phosphorylation, and consequently NHE-1 phosphorylation/activity, through a mechanism that involves DUSP6 up-regulation.

P38 mitogen activated protein kinase mediates cardiac Na+/H+ exchanger inhibition induced by Sildenafil

Since the original description as potent antianginal compounds, phosphodiesterase 5A inhibitors have continuously increased their possible therapeutic applications. In the heart, Sildenafil was shown to protect against an ischemic insult by decreasing cardiac Na+/H+ exchanger (NHE1) activity, action that was mediated by protein kinase G. p38 mitogen activated protein kinase (p38MAPK) activation was described in cardiac ischemia, but its precise role remains elusive. It has been shown that p38MAPK is activated by protein kinase G (PKG) in certain non-cardiac tissues, while in others modulates NHE1 activity. Current study was aimed to seek the role of p38MAPK in the Sildenafil-triggered pathway leading to NHE1 inhibition in myocardium. Rat isolated papillary muscles were used to evaluate NHE1 activity during intracellular pH recovery from an acidic load. Protein kinases phosphorylation (activation) was determined by western blot. Sustained acidosis promoted NHE1 hyperactivity by enhancing Ser703 phosphorylation, effect that was blunted by Sildenafil. p38MAPK inhibition reversed the effect of Sildenafil on NHE1. Activation of p38MAPK, by Sodium Arsenite or Anisomycin, mimicked the inhibitory effect of Sildenafil on the exchanger. Consistently, Sildenafil induced p38MAPK phosphorylation/activation during acidosis. Neither Sildenafil nor p38MAPK inhibition affected extracellular signal–regulated kinases 1/2 phosphorylation, kinases upstream NHE1. Furthermore, inhibition of NHE1 after p38MAPK activation was precluded by preventing the activation of protein phosphatase 2A with Okadaic Acid. Taken together, these results suggest that activation of p38MAPK is a necessary step to trigger the inhibitory effect of Sildenafil on cardiac NHE1 activity, thorough a mechanism that involves protein phosphatase 2A-mediated exchanger dephosphorylation.

Elevated arterial lactate delays recovery of intracellular muscle pH after exercise.

We evaluated muscle proton elimination following similar exercise in the same muscle group following two exercise modalities. Seven rowers performed handgrip or rowing exercise for ~ 5min. The intracellular response of the wrist flexor muscles was evaluated by 31P nuclear magnetic resonance spectroscopy, while arterial and venous forearm blood was collected. Rowing and handgrip reduced intracellular pH to 6.3 ± 0.2 and 6.5 ± 0.1, arterial pH to 7.09 ± 0.03 and 7.40 ± 0.03 and venous pH to 6.95 ± 0.06 and 7.20 ± 0.04 (P < 0.05), respectively. Arterial and venous lactate increased to 17.5 ± 1.6 and 20.0 ± 1.6mM after rowing while only to 2.6 ± 0.8 and 6.8 ± 0.8mM after handgrip exercise. Arterio-venous concentration difference of bicarbonate and phosphocreatine recovery kinetics (T50% rowing 1.5 ± 0.7min; handgrip 1.4 ± 1.0min) was similar following the two exercise modalities. Yet, intramuscular pH recovery in the forearm flexor muscles was 3.5-fold slower after rowing than after handgrip exercise (T50% rowing of 2 ± 0.1 vs. 7 ± 0.3min for handgrip). Rowing delays intracellular-pH recovery compared with handgrip exercise most likely because rowing, as opposed to handgrip exercise, increases systemic lactate concentration. Thus the intra-to-extra-cellular lactate gradient is small after rowing. Since this lactate gradient is the main driving force for intracellular lactate removal in muscle and, since pHi normalization is closely related to intracellular lactate removal, rowing results in a slower pHi recovery compared to handgrip exercise.

Signaling pathways involved in the rapid biphasic effect of aldosterone on Na+/H+ exchanger in rat proximal tubule cells

The receptors and signaling pathways for nongenomic effects of aldosterone (Aldo) on the proximal Na+/H+ exchanger are still unknown; therefore, the aim of this study was to investigate the mineralocorticoid receptor (MR) and/or glucocorticoid receptor (GR) participation in rapid Aldo effects on NHE1 (basolateral Na+/H+ exchanger isoform) and cytosolic calcium concentration ([Ca2+]i). In addition, phospholipase C (PLC), protein kinase C (PKC), and mitogen-activated protein kinase kinase (MEK) involvement in signaling pathways of such effects was evaluated, using immortalized proximal tubule cells of rat (IRPTC) as an experimental model. MR and GR expression was investigated using reverse transcription polymerase chain reaction and immunoblotting. The intracellular pH recovery rate (after acid loading) and [Ca2+]i were determined by the probes BCECF-AM and FURA 2-AM, respectively. Aldo (10−12 M) promoted a moderate increase in [Ca2+]i and stimulation of NHE1, whereas Aldo (10−6 M) greatly increased the [Ca2+]i, but inhibited the NHE1. BAPTA-AM (a calcium chelator), GR antagonism and inhibition of PLC, PKC and MEK pathway abolished the biphasic and dose-dependent effect of Aldo on NHE1 and decreased the [Ca2+]i; whereas MR do not appear to participate in this rapid signaling in IRPTC cells. The reduction of GR content, by gene silencing, abolished the Aldo effect on NHE1, in low concentration, confirming the importance of this receptor in the rapid modulation of proximal sodium and hydrogen transports.

Time course of red blood cell intracellular pH recovery following short-circuiting in relation to venous transit times in rainbow trout,Oncorhynchus mykiss

Accumulating evidence is highlighting the importance of a system of enhanced hemoglobin-oxygen (Hb-O2) unloading for cardiovascular O2 transport in teleosts. Adrenergically stimulated sodium-proton exchangers (β-NHE) create H+ gradients across the red blood cell (RBC) membrane that are short-circuited in the presence of plasma-accessible carbonic anhydrase (paCA) at the tissues; the result is a large arterial-venous pH shift that greatly enhances O2 unloading from pH-sensitive Hb. However, RBC intracellular pH (pHi) must recover during venous transit (31-90 s) to enable O2 loading at the gills. The halftimes ( t1/2) and magnitudes of RBC β-adrenergic stimulation, short-circuiting with paCA and recovery of RBC pHi, were assessed in vitro, on rainbow trout whole blood, and using changes in closed-system partial pressure of O2 as a sensitive indicator for changes in RBC pHi. In addition, the recovery rate of RBC pHi was assessed in a continuous-flow apparatus that more closely mimics RBC transit through the circulation. Results indicate that: 1) the t1/2 of β-NHE short-circuiting is likely within the residence time of blood in the capillaries, 2) the t1/2 of RBC pHi recovery is 17 s and within the time of RBC venous transit, and 3) after short-circuiting, RBCs reestablish the initial H+ gradient across the membrane and can potentially undergo repeated cycles of short-circuiting and recovery. Thus, teleosts have evolved a system that greatly enhances O2 unloading from pH-sensitive Hb at the tissues, while protecting O2 loading at the gills; the resulting increase in O2 transport per unit of blood flow may enable the tremendous athletic ability of salmonids.

Rho Kinase and Na+/H+ Exchanger Mediate Endothelin‐1 Induced Pulmonary Arterial Smooth Muscle Cell Proliferation and Migration

ObjectiveRemodeling of the pulmonary vasculature, involving increased proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), is a key component of the pathology underlying pulmonary hypertension (PH). Endothelin‐1 (ET‐1) is known to be an important contributor to both pulmonary vasoconstriction and remodeling in PH. We aimed to determine whether ET‐1 modulates PASMC function via a pathway involving Rho kinase (ROCK) and the Na+/H+ exchanger (NHE).MethodsPASMCs were isolated from distal pulmonary arteries of male C57/B6 mice as well as male Wistar rats and expanded in culture. To measure NHE activity, cells were incubated with pH‐sensitive fluorescent dye and the Na+‐dependent recovery of intracellular pH (pHi) following acidification by ammonium pulse challenge was measured via fluorescence microscopy. NHE1 mRNA expression was measured via qRT‐PCR and protein expression was measured via immunoblot. Proliferation was assessed via ELISA for BrdU incorporation over 24 hr. Migration was measured over 24 hr using Transwell filters with 8 μm pores. Cells were treated with 10−8 M ET‐1 versus vehicle for 24–48 hr. NHE was pharmacologically inhibited with 10 μM ethyl‐isopropyl amiloride. ROCK was inhibited with 10 μM Y‐27632.ResultsET‐1 treatment, compared to vehicle, resulted in increased resting pHi and NHE activity in PASMCs. ET‐1 did not result in increased NHE1 mRNA or protein expression. ROCK inhibition prevented ET‐1‐induced NHE activation. PASMC proliferation and migration were both increased by ET‐1 treatment, compared to vehicle. ROCK inhibition attenuated PASMC proliferation and migration in the presence of ET‐1. Similarly, NHE inhibition also attenuated ET‐1‐induced PASMC proliferation and migration.ConclusionsET‐1 results in increased PASMC proliferation and migration via a pathway involving ROCK and NHE activation. This suggests a novel pathway which may contribute to vascular remodeling in PH.Support or Funding InformationNIH K08HL133475 &amp; R01HL073859This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The H+/K+ ATPase Inhibitor SCH-28080 Inhibits Insulin Secretion and Induces Cell Death in INS-1E Rat Insulinoma Cells

Background/Aims: Glucose-stimulated insulin secretion (GSIS) of pancreatic β-cells involves glucose uptake and metabolism, closure of K_ATP channels and depolarization of the cell membrane potential (V_mem), activation of voltage-activated Ca²⁺ currents (ICa_v) and influx of Ca²⁺, which eventually triggers hormone exocytosis. Beside this classical pathway, K_ATP-independent mechanisms such as changes in intracellular pH (pH_i) or cell volume, which also affect β-cell viability, can elicit or modify insulin release. In β-cells the regulation of pH_i is mainly accomplished by Na⁺/H⁺ exchangers (NHEs). To investigate if other proton extrusion mechanisms than NHEs are involved in pH regulation, we tested for the presence of the non-gastric H⁺/K⁺ ATPase in rat insulinoma cells and assessed effects of the H⁺/K⁺ ATPase inhibitor SCH-28080 on insulin secretion, cell viability and apoptosis. Methods: In INS-1E cell cultures, H⁺/K⁺ ATPase gene and protein expression was analyzed by reverse transcription PCR and Western blotting. Intracellular pH (pH_i) recovery after acute acidic load was measured by NH₄Cl prepulsing using BCECF. Insulin secretion was determined by ELISA from the cell culture supernatant. V_mem, K⁺ and Ca²⁺ currents were recorded using patch clamp. Overall cell responses were determined using resazurin (viability) and cytotoxicity assays. The mean cell volume (MCV), cell granularity (side-scatter; SSC), phosphatidylserine (PS) exposure, cell membrane integrity, caspase activity and the mitochondrial membrane potential (ΔΨ_m) were measured by flow cytometry. Results: We found that the α-subunit of the non-gastric H⁺/K⁺ ATPase (HKα2) is expressed on mRNA and protein level. However, compared to rat colon tissue, in INS-1E cells mRNA abundance was very low. In NH₄Cl prepulsing experiments no K⁺-dependent pH_i recovery was observed under Na⁺-free extracellular conditions. Nonetheless within 1 h, 20 µM SCH-28080 inhibited GSIS by ∼50%, while basal release was unaffected. The L-type ICa_v blocker nifedipine caused a full inhibition of GSIS at 10 and 20 µM. At 20 µM, SCH-28080 inhibited ICa_v comparable to 20 µM nifedipine and in addition augmented IK_ATP recorded at -60 mV and hyperpolarized V_mem by ∼15 mV. Cell viability 2 and 24 h post treatment with SCH-28080 was dose-dependently inhibited with IC₅₀ values of 22.9 µM and 15.3 µM, respectively. At 20 µM the percentages of Annexin-V+, caspase+ and propidium iodide+ cells were significantly increased after 24 and 48 h. Concurrently, the MCV was significantly decreased (apoptotic volume decrease, AVD) and the SSC signal was increased. At concentrations >40–50 µM, SCH-28080 became progressively cytotoxic causing a steep increase in necrotic cells already 2 h post treatment and a breakdown of ΔΨ_m within 4 h under 50 and 100 µM while 10 and 20 µM had no effect on ΔΨ_m within 24 h. Conclusion: We demonstrate expression of HKα2 in rat INS-1E cells. However, the pump is apparently non-functional under the given conditions. Nonetheless the H⁺/K⁺ ATPase blocker SCH-28080 inhibits insulin secretion and induces cell death. Importantly, we show that SCH-28080 inhibits ICa_v - and activates K_ATP channels identifying them as novel “off-targets” of the inhibitor, causing hyperpolarization of V_mem and inhibition of insulin secretion.

The V-ATPase is expressed in the choroid plexus and mediates cAMP-induced intracellular pH alterations.

The cerebrospinal fluid (CSF) pH influences brain interstitial pH and, therefore, brain function. We hypothesized that the choroid plexus epithelium (CPE) expresses the vacuolar H+‐ATPase (V‐ATPase) as an acid extrusion mechanism in the luminal membrane to counteract detrimental elevations in CSF pH. The expression of mRNA corresponding to several V‐ATPase subunits was demonstrated by RT‐PCR analysis of CPE cells (CPECs) isolated by fluorescence‐activated cell sorting. Immunofluorescence and electron microscopy localized the V‐ATPase primarily in intracellular vesicles with only a minor fraction in the luminal microvillus area. The vesicles did not translocate to the luminal membrane in two in vivo models of hypocapnia‐induced alkalosis. The Na+‐independent intracellular pH (pHi) recovery from acidification was studied in freshly isolated clusters of CPECs. At extracellular pH (pHo) 7.4, the cells failed to display significant concanamycin A‐sensitive pHi recovery (i.e., V‐ATPase activity). The recovery rate in the absence of Na+ amounted to <10% of the pHi recovery rate observed in the presence of Na+. Recovery of pHi was faster at pHo 7.8 and was abolished at pHo 7.0. The concanamycin A‐sensitive pHi recovery was stimulated by cAMP at pH 7.4 in vitro, but intraventricular infusion of the membrane‐permeant cAMP analog 8‐CPT‐cAMP did not result in trafficking of the V‐ATPase. In conclusion, we find evidence for the expression of a minor fraction of V‐ATPase in the luminal membrane of CPECs. This fraction does not contribute to enhanced acid extrusion at high extracellular pH, but seems to be activated by cAMP in a trafficking‐independent manner.

Acute Effects of Vitamin C Exposure On Colonic Crypts: Direct Modulation of pH Regulation

Background/Aim: Colorectal cancer is still considered a leading cause of death in the United States and worldwide. One potential way to improve survival besides detection is to look to new therapeutic agents that can be taken prophylactically to reduce the risk of tumor formation. For cancer cells to grow and invade, a higher (more alkaline) intracellular pH must occur. We chose to examine a specific nutraceutical agent, which is Vitamin C. The acute effect of Vitamin C exposure on normal colonic crypts has been studied, providing some insight into how Vitamin C achieve its effect. Methods: Distal colon was excised from rats. Following enzymatic digestion single colonic crypts were isolated. Colonic crypts were loaded with pH sensitive dye to measure the intracellular pH changes. Crypts were exposed to solutions +/- Vitamin C. Results: 10 mM Vitamin C decreased Na⁺-dependent intracellular pH recovery. Vitamin C modulates SVCT leading to changes in proton extrusion. Vitamin C entry occurs via either SVCT2 on the basolateral membrane or by transcellular passive diffusion through tight junctions to the apical membrane and then active transport via SVCT1. Conclusion: Acute addition of Vitamin C to the basolateral membrane maintains low intracellular pH for a longer period which could halt and/or prevent tumor formation.