Acid-base Transport Research Articles

Differential regulation of vacuolar H+ -ATPase subunits by transforming growth factor-β1 in salivary ducts.

Bicarbonate concentration in saliva is controlled by the action of acid-base transporters in salivary duct cells. We show for the first time expression of ATP6V1B1 in submandibular gland and introduce transforming growth factor-beta (TGF-β) as a novel regulator of V-ATPase subunits. Using QRT-PCR, immunoblotting, biotinylation of surface proteins, immunofluorescence, chromatin immunoprecipitation, and intracellular H(+ ) recording with H(+ )-sensitive dye 2',7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein we show that in the human submandibular gland (HSG) cell line, activation of TGF-β signaling upregulates ATP6V1E1 and ATP6V1B2, downregulates ATP6V1B1, and has no effect on ATP6V1A. TGF-β1 effects on ATP6V1B1 are mediated through the canonical, the soluble adenylate cyclase, and ERK signaling. A CREB binding sequence was identified in the ATP6V1B1 promoter and CREB binding decreased after TGF-β1 treatment. Following acidosis, a bafilomycin-sensitive and Na+ -independent cell pH recovery was observed in HSG cells, an effect that was not influenced after disruption of acidic lysosomes. Moreover, neutralization of TGF-βs, inhibition of TGF-β receptor, or inhibition of the canonical pathway decreased membrane expression of ATP6V1A and prevented the acidosis-induced increased V-ATPase activity. The results suggest multiple modes of action of TGF-β1 on V-ATPase subunits in HSG cells: TGF-β1 may regulate transcription or protein synthesis of certain subunits and trafficking of other subunits in a context-dependent manner. Moreover, surface V-ATPase is active in salivary duct cells and involved in intracellular pH regulation following acidosis.

A new coupling of an acid-base transporter to PKD and cyst formation.

A new coupling of an acid-base transporter to PKD and cyst formation.

The acid-base transport proteins NHE1 and NBCn1 regulate cell cycle progression in human breast cancer cells

ABSTRACTPrecise acid-base homeostasis is essential for maintaining normal cell proliferation and growth. Conversely, dysregulated acid-base homeostasis, with increased acid extrusion and marked extracellular acidification, is an enabling feature of solid tumors, yet the mechanisms through which intra- and extracellular pH (pHi, pHe) impact proliferation and growth are incompletely understood. The aim of this study was to determine the impact of pH, and specifically of the Na+/H+ exchanger NHE1 and Na+, HCO3− transporter NBCn1, on cell cycle progression and its regulators in human breast cancer cells. Reduction of pHe to 6.5, a common condition in tumors, significantly delayed cell cycle progression in MCF-7 human breast cancer cells. The NHE1 protein level peaked in S phase and that of NBCn1 in G2/M. Steady state pHi changed through the cell cycle, from 7.1 in early S phase to 6.8 in G2, recovering again in M phase. This pattern, as well as net acid extrusion capacity, was dependent on NHE1 and NBCn1. Accordingly, knockdown of either NHE1 or NBCn1 reduced proliferation, prolonged cell cycle progression in a manner involving S phase prolongation and delayed G2/M transition, and altered the expression pattern and phosphorylation of cell cycle regulatory proteins. Our work demonstrates, for the first time, that both NHE1 and NBCn1 regulate cell cycle progression in breast cancer cells, and we propose that this involves cell cycle phase-specific pHi regulation by the two transporters.

CryoEM structure of the human SLC4A4 sodium-coupled acid-base transporter NBCe1

Na+-coupled acid–base transporters play essential roles in human biology. Their dysfunction has been linked to cancer, heart, and brain disease. High-resolution structures of mammalian Na+-coupled acid–base transporters are not available. The sodium-bicarbonate cotransporter NBCe1 functions in multiple organs and its mutations cause blindness, abnormal growth and blood chemistry, migraines, and impaired cognitive function. Here, we have determined the structure of the membrane domain dimer of human NBCe1 at 3.9 Å resolution by cryo electron microscopy. Our atomic model and functional mutagenesis revealed the ion accessibility pathway and the ion coordination site, the latter containing residues involved in human disease-causing mutations. We identified a small number of residues within the ion coordination site whose modification transformed NBCe1 into an anion exchanger. Our data suggest that symporters and exchangers utilize comparable transport machinery and that subtle differences in their substrate-binding regions have very significant effects on their transport mode.

Downregulation of the Cl-/HCO3-Exchanger Pendrin in Kidneys of Mice with Cystic Fibrosis: Role in the Pathogenesis of Metabolic Alkalosis

Background/Aims: Patients with cystic fibrosis (CF) are prone to the development of metabolic alkalosis; however, the pathogenesis of this life threatening derangement remains unknown. We hypothesized that altered acid base transport machinery in the kidney collecting duct underlies the mechanism of impaired bicarbonate elimination in the CF kidney. Methods: Balance studies in metabolic cages were performed in WT and CFTR knockout (CF) mice with the intestinal rescue in response to bicarbonate loading or salt restriction, and the expression levels and cellular distribution of acid base and electrolyte transporters in the proximal tubule, collecting duct and small intestine were examined by western blots, northern blots and/or immunofluorescence labeling. Results: Baseline parameters, including acid-base and systemic vascular volume status were comparable in WT and CF mice, as determined by blood gas, kidney renin expression and urine chloride excretion. Compared with WT animals, CF mice demonstrated a significantly higher serum HCO₃^- concentration (22.63 in WT vs. 26.83 mEq/l in CF mice; n=4, p=0.013) and serum pH (7.33 in WT vs. 7.42 in CF mice; n=4, p=0.00792) and exhibited impaired kidney HCO₃^- excretion (urine pH 8.10 in WT vs. 7.35 in CF mice; n=7, p=0.00990) following a 3-day oral bicarbonate load. When subjected to salt restriction, CF mice developed a significantly higher serum HCO₃^- concentration vs. WT animals (29.26 mEq/L in CF mice vs. 26.72 in WT; n=5, p=0.0291). Immunofluorescence labeling demonstrated a profound reduction in the apical expression of the Cl^-/HCO₃^- exchanger pendrin in cortical collecting duct cells and western and northern blots indicated diminished plasma membrane abundance and mRNA expression of pendrin in CF kidneys. Conclusions: We propose that patients with cystic fibrosis are prone to the development of metabolic alkalosis secondary to the inactivation of the bicarbonate secreting transporter pendrin, specifically during volume depletion, which is a common occurrence in CF patients.

Haploinsufficiency of the Mouse Atp6v1b1 Gene Leads to a Mild Acid-Base Disturbance with Implications for Kidney Stone Disease

Background/Aims: Homozygous mutations or deletion of the ATP6V1B1 gene encoding for the B1 subunit of the vacuolar H⁺-ATPase leads to distal renal tubular acidosis in man and mice. In humans, heterozygous carriers of B1 mutations can develop incomplete dRTA with nephroclacinosis. Here, we investigated whether Atp6v1b1^+/- mice also develop acid-base disturbances during an HCl acid load. Methods: We subjected Atp6v1b1^+/+, Atp6v1b1^+/-, Atp6v1b1^-/- to an HCl-load for 7 days and investigated acid-base status, kidney function, and expression of renal acid-base transport proteins. Results: Atp6v1b1^-/- mice had more alkaline urine and low ammoniuria, whereas Atp6v1b1^+/- mice showed no difference in their urine parameters but higher blood chloride and lower blood pCO₂ compared to controls. Subcellular localization of a4 and B2 subunits of H⁺-ATPase were unchanged within the 3 genotypes and Atp6v1b1^+/+ and Atp6v1b1^+/- mice exhibited a similar luminal localization of B1 subunit in intercalated cells. However, B1, B2 and a4 expression were decreased in renal membrane fractions from Atp6v1b1^+/- mice compared to Atp6v1b1^+/+ while B2 and a4 were unchanged and B1 protein was reduced in Atp6v1b+^-/- kidneys. Compensatory mechanisms of B1 ablation were found only in the collecting duct with a down-regulation of pendrin in Atp6v1b1^-/- mice. Conclusions: In conclusion, 1) Atp6v1b1^+/- mice developed a mild incomplete dRTA. dRTA is partly compensated by respiration. 2) Compensatory mechanisms for the absence of B1 take place only in the collecting duct of Atp6v1b1^-/- kidneys.

The murine choroid plexus epithelium expresses the 2Cl-/H+ exchanger ClC-7 and Na+/H+ exchanger NHE6 in the luminal membrane domain.

The choroid plexus epithelium within the brain ventricles secretes the majority of the cerebrospinal fluid (CSF). The luminal Na+-K+-ATPase acts in concert with a host of other transport proteins to mediate efficient fluid secretion across the epithelium. The CSF contains little protein buffer, but the pH value seems nonetheless maintained within narrow limits, even when faced with acid-base challenges. The involvement of choroid plexus acid-base transporters in CSF pH regulation is highlighted by the expression of several acid-base transporters in the epithelium. The aim of the present study was to identify novel acid-base transporters expressed in the luminal membrane of the choroid plexus epithelium to pave the way for systematic investigations of each candidate transporter in the regulation of CSF pH. Mass spectrometry analysis of proteins from epithelial cells isolated by fluorescence-activated cell sorting identified the Cl-/H+ exchangers ClC-3, -4, -5, and -7 in addition to known choroid plexus acid-base transporters. RT-PCR on FACS isolated epithelial cells confirmed the expression of the corresponding mRNAs, as well as Na+/H+ exchanger NHE6 mRNA. Both NHE6 and ClC-7 were immunolocalized to the luminal plasma membrane domain of the choroid plexus epithelial cells. Dynamic imaging of intracellular pH and membrane potential changes in isolated choroid plexus epithelial cells demonstrated Cl- gradient-driven changes in intracellular pH and membrane potential that are consistent with Cl-/H+ exchange. In conclusion, we have detected for the first time NHE6 and ClC-7 in the choroid plexus, which are potentially involved in pH regulation of the CSF.

The ins and outs of acid-base transport in skeletal muscle.

Aalkjær and Nielsen discuss new data revealing the basis of acid–base transport in t-tubules of skeletal muscle.

Versatility of NaCl transport mechanisms in the cortical collecting duct.

The cortical collecting duct (CCD) forms part of the aldosterone-sensitive distal nephron and plays an essential role in maintaining the NaCl balance and acid-base status. The CCD epithelium comprises principal cells as well as different types of intercalated cells. Until recently, transcellular Na+ transport was thought to be restricted to principal cells, whereas (acid-secreting) type A and (bicarbonate-secreting) type B intercalated cells were associated with the regulation of acid-base homeostasis. This review describes how this traditional view has been upended by several discoveries in the past decade. A series of studies has shown that type B intercalated cells can mediate electroneutral NaCl reabsorption by a mechanism involving Na+-dependent and Na+-independent Cl-/[Formula: see text] exchange, and that is energetically driven by basolateral vacuolar H+-ATPase pumps. Other research indicates that type A intercalated cells can mediate NaCl secretion, through a bumetanide-sensitive pathway that is energized by apical H+,K+-ATPase type 2 pumps operating as Na+/K+ exchangers. We also review recent findings on the contribution of the paracellular route to NaCl transport in the CCD. Last, we describe cross-talk processes, by which one CCD cell type impacts Na+/Cl- transport in another cell type. The mechanisms that have been identified to date demonstrate clearly the interdependence of NaCl and acid-base transport systems in the CCD. They also highlight the remarkable versatility of this nephron segment.

Inherited Proximal and Distal Renal Tubular Acidosis

Acid-base homeostasis by the kidney is maintained through proximal tubular reclamation of filtered bicarbonate and the excretion of the daily acid load by collecting duct type A intercalated cells. The impairment of either process results in renal tubular acidosis (RTA), a group of disorders characterized by a reduced net acid excretion and a persistent hyperchloremic, non–anion gap metabolic acidosis. The primary or hereditary forms of proximal (pRTA) and distal renal tubular acidosis (dRTA) have received increased attention because of advances in the understanding of the molecular mechanism, whereby mutations in the main proteins involved in acid-base transport result in either reduced bicarbonate reabsorption or reduced H+ secretion and impaired acid excretion. dRTA is characterized by reduced net acid excretion and an inability to lower urine pH despite severe acidemia (but minimal HCO3– wastage). pRTA (type 2), by contrast, is characterized by marked HCO3– wastage but preserved ability to lower urine pH when plasma HCO3– (and therefore filtered HCO3–) is below a certain threshold. In children with dRTA, growth retardation caused by chronic metabolic acidosis is the key manifestation but is fully reversible with appropriate alkali therapy if initiated early in life. A striking manifestation of many patients with dRTA is the development of severe hypokalemia that may cause muscle paralysis. In this review, we discuss these types of hereditary RTA and the mechanisms involved in the genesis of these inherited tubular disorders. This review contains 5 figures, 1 table, and 103 references. Key words: Proximal renal tubular acidosis (pRTA), Distal renal tubular acidosis (dRTA), Hyperchloremic, non–anion gap metabolic acidosis, Hypokalemia, Fractional HCO3– excretion, Urinary gap, Fanconi Syndrome.ATP6V1B1 and ATP6V0A4 gene mutations . Intercalated cells ,

Carbonic anhydrase inhibitors modify intracellular pH transients and contractions of rat middle cerebral arteries during CO2/HCO3– fluctuations

The CO2/HCO3– buffer minimizes pH changes in response to acid–base loads, HCO3– provides substrate for Na+,HCO3–-cotransporters and Cl–/HCO3–-exchangers, and H+ and HCO3– modify vasomotor responses during acid–base disturbances. We show here that rat middle cerebral arteries express cytosolic, mitochondrial, extracellular, and secreted carbonic anhydrase isoforms that catalyze equilibration of the CO2/HCO3– buffer. Switching from CO2/HCO3–-free to CO2/HCO3–-containing extracellular solution results in initial intracellular acidification due to hydration of CO2 followed by gradual alkalinization due to cellular HCO3– uptake. Carbonic anhydrase inhibition decelerates the initial acidification and attenuates the associated transient vasoconstriction without affecting intracellular pH or artery tone at steady-state. Na+,HCO3–-cotransport and Na+/H+-exchange activity after NH4+-prepulse-induced intracellular acidification are unaffected by carbonic anhydrase inhibition. Extracellular surface pH transients induced by transmembrane NH3 flux are evident under CO2/HCO3–-free conditions but absent when the buffer capacity and apparent H+ mobility increase in the presence of CO2/HCO3– even after the inhibition of carbonic anhydrases. We conclude that (a) intracellular carbonic anhydrase activity accentuates pH transients and vasoconstriction in response to acute elevations of pCO2, (b) CO2/HCO3– minimizes extracellular surface pH transients without requiring carbonic anhydrase activity, and (c) carbonic anhydrases are not rate limiting for acid–base transport across cell membranes during recovery from intracellular acidification.

Regulation of glomerulotubular balance: flow-activated proximal tubule function.

The purpose of this review is to summarize our knowledge and understanding of the physiological importance and the mechanisms underlying flow-activated proximal tubule transport. Since the earliest micropuncture studies of mammalian proximal tubule, it has been recognized that tubular flow is an important regulator of sodium, potassium, and acid-base transport in the kidney. Increased fluid flow stimulates Na+ and HCO3- absorption in the proximal tubule via stimulation of Na/H-exchanger isoform 3 (NHE3) and H+-ATPase. In the proximal tubule, brush border microvilli are the major flow sensors, which experience changes in hydrodynamic drag and bending moment as luminal flow velocity changes and which transmit the force of altered flow to cytoskeletal structures within the cell. The signal to NHE3 depends upon the integrity of the actin cytoskeleton; the signal to the H+-ATPase depends upon microtubules. We have demonstrated that alterations in fluid drag impact tubule function by modulating ion transporter availability within the brush border membrane of the proximal tubule. Beyond that, there is evidence that transporter activity within the peritubular membrane is also modulated by luminal flow. Secondary messengers that regulate the flow-mediated tubule function have also been delineated. Dopamine blunts the responsiveness of proximal tubule transporters to changes in luminal flow velocity, while a DA1 antagonist increases flow sensitivity of solute reabsorption. IP3 receptor-mediated intracellular Ca2+ signaling is critical to transduction of microvillus drag. In this review, we summarize our findings of the regulatory mechanism of flow-mediated Na+ and HCO3- transport in the proximal tubule and review available information about flow sensing and regulatory mechanism of glomerulotubular balance.

Influence of training intensity on adaptations in acid/base transport proteins, muscle buffer capacity, and repeated-sprint ability in active men.

McGinley C, Bishop DJ. Influence of training intensity on adaptations in acid/base transport proteins, muscle buffer capacity, and repeated-sprint ability in active men. J Appl Physiol 121: 1290-1305, 2016. First published October 14, 2016; doi:10.1152/japplphysiol.00630.2016-This study measured the adaptive response to exercise training for each of the acid-base transport protein families, including providing isoform-specific evidence for the monocarboxylate transporter (MCT)1/4 chaperone protein basigin and for the electrogenic sodium-bicarbonate cotransporter (NBCe)1. We investigated whether 4 wk of work-matched, high-intensity interval training (HIIT), performed either just above the lactate threshold (HIITΔ20; n = 8), or close to peak aerobic power (HIITΔ90; n = 8), influenced adaptations in acid-base transport protein abundance, nonbicarbonate muscle buffer capacity (βmin vitro), and exercise capacity in active men. Training intensity did not discriminate between adaptations for most proteins measured, with abundance of MCT1, sodium/hydrogen exchanger (NHE) 1, NBCe1, carbonic anhydrase (CA) II, and CAXIV increasing after 4 wk, whereas there was little change in CAIII and CAIV abundance. βmin vitro also did not change. However, MCT4 protein content only increased for HIITΔ20 [effect size (ES): 1.06, 90% confidence limits × / ÷ 0.77], whereas basigin protein content only increased for HIITΔ90 (ES: 1.49, × / ÷ 1.42). Repeated-sprint ability (5 × 6-s sprints; 24 s passive rest) improved similarly for both groups. Power at the lactate threshold only improved for HIITΔ20 (ES: 0.49; 90% confidence limits ± 0.38), whereas peak O2 uptake did not change for either group. Detraining was characterized by the loss of adaptations for all of the proteins measured and for repeated-sprint ability 6 wk after removing the stimulus of HIIT. In conclusion, 4 wk of HIIT induced improvements in each of the acid-base transport protein families, but, remarkably, a 40% difference in training intensity did not discriminate between most adaptations.

The Na+/HCO3− co-transporter is protective during ischemia in astrocytes

The sodium bicarbonate co-transporter (NBC) is the major bicarbonate-dependent acid–base transporter in mammalian astrocytes and has been implicated in ischemic brain injury. A malfunction of astrocytes could have great impact on the outcome of stroke due to their participation in the formation of blood-brain barrier, synaptic transmission, and electrolyte balance in the human brain. Nevertheless, the role of NBC in the ischemic astrocyte death has not been well understood. In this work, we obtained skin biopsies from healthy human subjects and had their fibroblasts grown in culture and reprogrammed into human-induced pluripotent stem cells (hiPSCs). These hiPSCs were further differentiated into neuroprogenitor cells (NPCs) and then into human astrocytes. These astrocytes express GFAP and S100β and readily propagate calcium waves upon mechanical stimulation. Using pH-sensitive dye BCECF [2′,7′-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein] and qPCR technique, we have confirmed that these astrocytes express functional NBC including electrogenic NBC (NBCe). In addition, astrocytes exposed to an ischemic solution (IS) that mimics the ischemic penumbral environment enhanced both mRNA and protein expression level of NBCe1 in astrocytes. Using IS and a generic NBC blocker S0859, we have studied the involvement of NBC in IS-induced human astrocytes death. Our results show that a 30μM S0859 induced a 97.5±1.6% (n=10) cell death in IS-treated astrocytes, which is significantly higher than 43.6±4.5%, (n=10) in the control group treated with IS alone. In summary, a NBC blocker exaggerates IS-induced cell death, suggesting that NBC activity is essential for astrocyte survival when exposed to ischemic penumbral environment.

Both monensin and plant extract alter ruminal fermentation in sheep but only monensin affects the expression of genes involved in acid-base transport of the ruminal epithelium

The effects of a plant extract (broadleaf plantain, peppermint) or monensin on dry matter intake (DMI), growth performance, ruminal fermentation products, as well as morphology and the expression of genes involved in acid-base regulation of the ruminal epithelium were determined. Thirty two male Afshari finishing lambs on a high-concentrate diet were subjected to four consecutive 14-d periods on one of four treatments with supplementation of (1) no additive (control, CON), (2) 2g blend of plant extract/(d×lamb) (PE), or (3) 30mg monensin/(d×lamb) (MON) over all four treatment periods. The fourth group received periodical inclusion of 30mg monensin/(d×lamb) in periods 1 and 3 with no additive in periods 2 and 4 (MON-p). Despite no significant overall effects on DMI, ruminal pH, average daily gain (ADG), ADG:DMI ratio and plasma albumin, the interactions of treatment×period were significant for these measures (P<0.01). Compared with CON, periods with decreased DMI occurred selectively in groups MON (period 1 and 2) and MON-p (period 2 and 3), a period with decreased ruminal pH occurred in PE (period 1), and periods with decreased plasma albumin occurred in groups MON (period 1) and MON-p (period 2) (all P<0.05 or lower). In period 2, MON sheep had higher ADG than PE and MON-p (P<0.05) and a higher ADG:DMI ratio than all other groups (P<0.05). While total concentrations of ruminal short chain fatty acids were unchanged, increased propionate proportions and decreased acetate proportions were observed in PE (P<0.05) and, as a trend, in MON, resulting in decreased acetate:propionate ratios in PE and MON compared to CON (P<0.05). The proportion of butyrate was decreased by MON only (P<0.05). Compared with CON, the relative mRNA abundance of Na+/H+ exchanger isoforms NHE1 and NHE3 were lower and that of monocarboxylate transporter isoform MCT1 was greater for MON (P<0.05). In MON-p animals, the mRNA abundance of NHE1 was lower and that of MCT4 was greater compared to CON (P<0.05). Morphometry results did not differ between treatments. It is concluded that monensin and the used plant extract were equally effective to increase ruminal propionate fermentation. Moreover, the study elucidated for the first time that the ruminal epithelium itself is affected by the ionophore properties of monensin and needs to adapt epithelial acid-base transporter expression.

Na+, HCO3--cotransporter NBCn1 increases pHi gradients, filopodia, and migration of smooth muscle cells and promotes arterial remodelling.

Arterial remodelling can cause luminal narrowing and obstruct blood flow. We tested the hypothesis that cellular acid-base transport facilitates proliferation and migration of vascular smooth muscle cells (VSMCs) and enhances remodelling of conduit arteries. [Formula: see text]-cotransport via NBCn1 (Slc4a7) mediates net acid extrusion and controls steady-state intracellular pH (pHi) in VSMCs of mouse carotid arteries and primary aortic explants. Carotid arteries undergo hypertrophic inward remodelling in response to partial or complete ligation in vivo, but the increase in media area and thickness and reduction in lumen diameter are attenuated in arteries from NBCn1 knock-out compared with wild-type mice. With [Formula: see text] present, gradients for pHi (∼0.2 units magnitude) exist along the axis of VSMC migration in primary explants from wild-type but not NBCn1 knock-out mice. Knock-out or pharmacological inhibition of NBCn1 also reduces filopodia and lowers initial rates of VSMC migration after scratch-wound infliction. Interventions to reduce H(+)-buffer mobility (omission of [Formula: see text] or inhibition of carbonic anhydrases) re-establish axial pHi gradients, filopodia, and migration rates in explants from NBCn1 knock-out mice. The omission of [Formula: see text] also lowers global pHi and inhibits proliferation in primary explants. Under physiological conditions (i.e. with [Formula: see text] present), NBCn1-mediated [Formula: see text] uptake raises VSMC pHi and promotes filopodia, VSMC migration, and hypertrophic inward remodelling. We propose that axial pHi gradients enhance VSMC migration whereas global acidification inhibits VSMC proliferation and media hypertrophy after carotid artery ligation. These findings support a key role of acid-base transport, particularly via NBCn1, for development of occlusive artery disease.

Acidic Eye‐Drops De‐Swell the Edematous Corneas of Slc4a11‐null Mice; Evidence for Functional Coupling between Slc4a11 and the H+/lactate Cotransporter Mct1

SLC4A11 is the most divergent member of the SLC4 family of acid‐base transporters. Mutations in SLC4A11 are associated with congenital hereditary endothelial dystrophy (CHED): a clouding of the cornea caused by the accumulation of fluid in the corneal stroma. Slc4a11‐null mice have edematous corneas. Slc4a11 is expressed in the basolateral membrane of corneal endothelial cells; these cells normally promote fluid removal from the stroma by absorbing stromal osmolytes such as Na+, HCO3− (via the basolateral Na+/HCO3− cotransporter NBCe1) and lactate (via the basolateral H+/lactate cotransporter MCT1). The contribution of Slc4a11 to endothelial function is not fully understood. In a separate study in Xenopus oocytes, we have observed that mouse Slc4a11 mediates H+ conduction when extracellular pH is raised. At the basolateral membrane of corneal‐endothelial cells, Mct1‐mediated H+/lactate import would tend to raise surface pH (pHs) in the vicinity of Slc4a11. The near‐zero membrane potential reported for actively‐transporting corneal endothelia (Hodson and Wigham, J. Physiol., 1989), predicts that Slc4a11 should mediate H+ efflux in response to elevations in pHs. Thus we hypothesize that Mct1 action promotes Slc4a11‐mediated H+ efflux, which in turn supports Mct1 action and thereby the maintenance of corneal clarity. To investigate coupling between Mct1 and Slc4a11, we expressed Mct1 ± Slc4a11 in Xenopus oocytes. Under voltage clamp at 0 mV, we simultaneously monitored pHs and the Slc4a11‐dependent H+‐current as we applied lactate to the cells. Lactate application caused a sharp rise in pHs in all cells due to Mct action. In cells co‐expressing Mct1+Slc4a11, lactate application also caused a sustained increase in current due to Slc4a11‐mediated H+‐efflux (n=3), consistent with functional coupling between Mct1 and Slc4a11. Our preliminary data suggest that even in the absence of lactate, Slc4a11‐mediated H+‐conduction is significantly enhanced by Mct1 co‐expression (P=0.01, n=3), as if Mct1 also physically enhances the functional expression of Slc4a11. We performed two further assays to investigate the effect of Slc4a11 loss on H+ and lactate handling by mouse corneas. [1] We extracted tissue samples from the anterior section of wild‐type and Slc4a11‐null mouse eyes; Slc4a11‐null samples contained more lactate than wild‐type equivalents (P=0.02, n=4). [2] We measured corneal thickness in wild‐type and Slc4a11‐null mice by pachymetry. The application of acidic eye‐drops to the edematous corneas of Slc4a11‐null mice resulted in their de‐swelling (n=6, P&lt;0.01). The thickness of de‐swollen corneas was not different from the thickness of wild‐type corneas. Together these data indicate that Slc4a11 plays an important role in support of Mct1 action, and thereby endothelial function.Support or Funding InformationSupported by Start‐up Funds from UB:SUNY (to MDP), an Unrestricted Grant from Research to Prevent Blindness (to SPP), and NIH R21‐EY021646 (to MLJ and MDP).

Hypoxia and Its Acid-Base Consequences: From Mountains to Malignancy.

Hypoxia, depending upon its magnitude and circumstances, evokes a spectrum of mild to severe acid-base changes ranging from alkalosis to acidosis, which can alter many responses to hypoxia at both non-genomic and genomic levels, in part via altered hypoxia-inducible factor (HIF) metabolism. Healthy people at high altitude and persons hyperventilating to non-hypoxic stimuli can become alkalotic and alkalemic with arterial pH acutely rising as high as 7.7. Hypoxia-mediated respiratory alkalosis reduces sympathetic tone, blunts hypoxic pulmonary vasoconstriction and hypoxic cerebral vasodilation, and increases hemoglobin oxygen affinity. These effects and others can be salutary or counterproductive to tissue oxygen delivery and utilization, based upon magnitude of each effect and summation. With severe hypoxia either in the setting of profound arterial hemoglobin desaturation and reduced O2 content or poor perfusion (ischemia) at the global or local level, metabolic and hypercapnic acidosis develop along with considerable lactate formation and pH falling to below 6.8. Although conventionally considered to be injurious and deleterious to cell function and survival, both acidoses may be cytoprotective by various anti-inflammatory, antioxidant, and anti-apoptotic mechanisms which limit total hypoxic or ischemic-reperfusion injury. Attempts to correct acidosis by giving bicarbonate or other alkaline agents under these circumstances ahead of or concurrent with reoxygenation efforts may be ill advised. Better understanding of this so-called "pH paradox" or permissive acidosis may offer therapeutic possibilities. Rapidly growing cancers often outstrip their vascular supply compromising both oxygen and nutrient delivery and metabolic waste disposal, thus limiting their growth and metastatic potential. However, their excessive glycolysis and lactate formation may not necessarily represent oxygen insufficiency, but rather the Warburg effect-an attempt to provide a large amount of small carbon intermediates to supply the many synthetic pathways of proliferative cell growth. In either case, there is expression and upregulation of many genes involved in acid-base homeostasis, in part by HIF-1 signaling. These include a unique isoform of carbonic anhydrase (CA-IX) and numerous membrane acid-base transporters engaged to maintain an optimal intracellular and extracellular pH for maximal growth. Inhibition of these proteins or gene suppression may have important therapeutic application in cancer chemotherapy.

A novel, protective role of ursodeoxycholate in bile-induced pancreatic ductal injury.

We have previously shown that chenodeoxycholic acid (CDCA) strongly inhibits pancreatic ductal HCO3 (-) secretion through the destruction of mitochondrial function, which may have significance in the pathomechanism of acute pancreatitis (AP). Ursodeoxycholic acid (UDCA) is known to protect the mitochondria against hydrophobic bile acids and has an ameliorating effect on cell death. Therefore, our aim was to investigate the effect of UDCA pretreatment on CDCA-induced pancreatic ductal injury. Guinea pig intrainterlobular pancreatic ducts were isolated by collagenase digestion. Ducts were treated with UDCA for 5 and 24 h, and the effect of CDCA on intracellular Ca(2+) concentration ([Ca(2+)]i), intracellular pH (pHi), morphological and functional changes of mitochondria, and the rate of apoptosis were investigated. AP was induced in rat by retrograde intraductal injection of CDCA (0.5%), and the disease severity of pancreatitis was assessed by measuring standard laboratory and histological parameters. Twenty-four-hour pretreatment of pancreatic ducts with 0.5 mM UDCA significantly reduced the rate of ATP depletion, mitochondrial injury, and cell death induced by 1 mM CDCA and completely prevented the inhibitory effect of CDCA on acid-base transporters. UDCA pretreatment had no effect on CDCA-induced Ca(2+) signaling. Oral administration of UDCA (250 mg/kg) markedly reduced the severity of CDCA-induced AP. Our results clearly demonstrate that UDCA 1) suppresses the CDCA-induced pancreatic ductal injury by reducing apoptosis and mitochondrial damage and 2) reduces the severity of CDCA-induced AP. The protective effect of UDCA against hydrophobic bile acids may represent a novel therapeutic target in the treatment of biliary AP.

Acid-Base Homeostasis.

Acid-base homeostasis and pH regulation are critical for both normal physiology and cell metabolism and function. The importance of this regulation is evidenced by a variety of physiologic derangements that occur when plasma pH is either high or low. The kidneys have the predominant role in regulating the systemic bicarbonate concentration and hence, the metabolic component of acid-base balance. This function of the kidneys has two components: reabsorption of virtually all of the filtered HCO3(-) and production of new bicarbonate to replace that consumed by normal or pathologic acids. This production or generation of new HCO3(-) is done by net acid excretion. Under normal conditions, approximately one-third to one-half of net acid excretion by the kidneys is in the form of titratable acid. The other one-half to two-thirds is the excretion of ammonium. The capacity to excrete ammonium under conditions of acid loads is quantitatively much greater than the capacity to increase titratable acid. Multiple, often redundant pathways and processes exist to regulate these renal functions. Derangements in acid-base homeostasis, however, are common in clinical medicine and can often be related to the systems involved in acid-base transport in the kidneys.