Ethanol potentiates hypoxic liver injury: role of hepatocyte Na(+) overload

Centrilobular hypoxia has been suggested to contribute to hepatic damage caused by alcohol intoxication. However, the mechanisms involved are still poorly understood. We have investigated whether alterations of Na(+) homeostasis might account for ethanol-mediated increase in hepatocyte sensitivity to hypoxia. Addition of ethanol (100 mmol/l) to isolated rat hepatocytes incubated under nitrogen atmosphere greatly stimulated cell death. An increase in intracellular Na(+) levels preceded cell killing and Na(+) levels in hepatocytes exposed to the combination of ethanol and hypoxia were almost twice those in hypoxic cells without ethanol. Na(+) increase was also observed in hepatocytes incubated with ethanol in oxygenated buffer. Ethanol addition significantly lowered hepatocyte pH. Inhibiting ethanol and acetaldehyde oxidation with, respectively, 4-methylpyrazole and cyanamide prevented this effect. 4-methylpyrazole, cyanamide as well as hepatocyte incubation in a HCO(3)(-)-free buffer or in the presence of Na(+)/H(+) exchanger blocker 5-(N,N-dimethyl)-amiloride also reduced Na(+) influx in ethanol-treated hepatocytes. 4-methylpyrazole and cyanamide similarly prevented ethanol-stimulated Na(+) accumulation and hepatocyte killing during hypoxia. Moreover, ethanol-induced Na(+) influx caused cytotoxicity in hepatocytes pre-treated with Na(+), K(+)-ATPase inhibitor ouabain. Also in this condition 4-methylpyrazole and 5-(N,N-dimethyl)-amiloride decreased cell killing. These results indicate that ethanol can promotes cytotoxicity in hypoxic hepatocytes by enhancing Na(+) accumulation.     

Contrasting role of Na(+) ions in modulating Cu(+2) or Cd(+2) induced hepatocyte toxicity

Previously we showed that hepatocyte lysis induced by Cu(+2)/Cd(+2) could be partly attributed to membrane lipid peroxidation induced by Cu(+2) or mitochondrial toxicity induced by Cd(+2) [5]. Changes in Na(+) and Ca(+2) homeostasis induced when Cu(+2) was incubated with hepatocytes markedly differed from that induced by Cd(+2). Na(+) omission from the media or addition of the Na(+)/H(+) exchange inhibitor 5-(N,N-dimethyl)-amiloride markedly increased Cu(+2) cytotoxicity even though Cu(+2) did not increase hepatocyte Na(+) when the media contained Na(+). Intracellular Ca(+2) levels however were markedly increased when the hepatocytes were incubated with Cu(+2) in a Na(+) free media and removing media Ca(+2) with EGTA also prevented Cu(+2) induced hepatocyte cytotoxicity. This suggests that intracellular Ca(+2) accumulation contributes to Cu(+2) induced cytotoxicity and a Na(+)-dependent Ca(+2) transporter is involved in controlling excessive Ca(+2) accumulation caused by Cu(+2). The omission of Cl(-) from the media or addition of glycine, a Cl(-) channel blocker also enhanced Cu induced cytotoxicity. By contrast Cd(+2) induced cytotoxicity was prevented by Na(+) omission from the media or by the addition of 5-(N,N-dimethyl)-amiloride. Furthermore the omission of Cl(-) from the media or addition of glycine also prevented Cd(+2) induced hepatocyte toxicity. A hypotonic media also increased Cd(+2) but not Cu(+2) induced hepatocyte cytotoxicity. This suggests that Cd(+2) but not Cu(+2) cytotoxicity could be partly attributed to disruption of cell volume regulation mechanisms. The increased osmotic load caused by the uncontrolled accumulation of intracellular Na(+) in Cd(+2) treated hepatocytes likely resulted from the activation of Na(+)/H(+) exchanger and the Na(+)/HCO(3)(-) cotransporter by the acidosis and ATP depletion caused by mitochondrial toxicity.     

Overexpression of the Na+/H+ exchanger and ischemia-reperfusion injury in the myocardium

In the myocardium, the Na(+)/H(+) exchanger isoform-1 (NHE1) activity is detrimental during ischemia-reperfusion (I/R) injury, causing increased intracellular Na(+) (Na(i)(+)) accumulation that results in subsequent Ca(2+) overload. We tested the hypothesis that increased expression of NHE1 would accentuate myocardial I/R injury. Transgenic mice were created that increased the Na(+)/H(+) exchanger activity specifically in the myocardium. Intact hearts from transgenic mice at 10-15 wk of age showed no change in heart performance, resting intracellular pH (pH(i)) or phosphocreatine/ATP levels. Transgenic and wild-type (WT) hearts were subjected to 20 min of ischemia followed by 40 min of reperfusion. Surprisingly, the percent recovery of rate-pressure product (%RPP) after I/R improved in NHE1-overexpressing hearts (64 +/- 5% vs. 41 +/- 5% in WT; P < 0.05). In addition, NMR spectroscopy revealed that NHE1 overexpressor hearts contained higher ATP during early reperfusion (levels P < 0.05), and there was no difference in Na(+) accumulation during I/R between transgenic and WT hearts. HOE642 (cariporide), an NHE1 inhibitor, equivalently protected both WT and NHE1-overexpressing hearts. When hearts were perfused with bicarbonate-free HEPES buffer to eliminate the contribution of HCO(3)(-) transporters to pH(i) regulation, there was no difference in contractile recovery after reperfusion between controls and transgenics, but NHE1-overexpressing hearts showed a greater decrease in ATP during ischemia. These results indicate that the basal activity of NHE1 is not rate limiting in causing damage during I/R, therefore, increasing the level of NHE1 does not enhance injury and can have some small protective effects.     

Restitution of the bullfrog gastric mucosa is dependent on a DIDS-inhibitable pathway not related to HCO3- ion transport

This study was conducted to determine the contribution of ion transport to restitution after injury in the gastric mucosa. For this, intact sheets of stomach from the bullfrog, Rana catesbeiana, were mounted in Ussing chambers. Restitution was evaluated in the presence or absence of ion transport inhibitors amiloride, DIDS, and bumetanide to block Na(+)/H(+) exchange, Cl(-)/HCO(3)(-) exchange and Na(+)/HCO(3)(-) co-transport, and Na(+)-K(+)-2Cl(-) cotransport, respectively. Ion substitution experiments with Na(+)-free, Cl(-)-free, and HCO(3)(-)-free solutions were also performed. Injury to the mucosa was produced with 1 M NaCl, and restitution was evaluated by recovery of transepithelial resistance (TER), mannitol flux, and morphology. Amiloride, bumetanide, Cl(-)-free, or HCO(3)(-)-free solutions did not affect restitution. In Na(+)-free solutions, recovery of TER and mannitol flux did not occur because surface cells did not attach to the underlying basement membrane. In contrast, all aspects of restitution were inhibited by DIDS, a compound that inhibits Na(+)-dependent HCO(3)(-) transport. Because HCO(3)(-)-free solutions did not inhibit restitution, it was concluded that DIDS must block a yet undefined pathway not involved in HCO(3)(-) ion transport but essential for cell migration after injury and restitution in the gastric mucosa.     

Relative contributions of Na+/H+ exchange and Na+/HCO3- cotransport to ischemic Nai+ overload in isolated rat hearts

The Na(+)/H(+) exchanger (NHE) and/or the Na(+)/HCO(3)(-) cotransporter (NBC) were blocked during ischemia in isolated rat hearts. Intracellular Na(+) concentration ([Na(+)](i)), intracellular pH (pH(i)), and energy-related phosphates were measured by using simultaneous (23)Na and (31)P NMR spectroscopy. Hearts were subjected to 30 min of global ischemia and 30 min of reperfusion. Cariporide (3 microM) or HCO(3)(-)-free HEPES buffer was used, respectively, to block NHE, NBC, or both. End-ischemic [Na(+)](i) was 320 +/- 18% of baseline in HCO(3)(-)-perfused, untreated hearts, 184 +/- 6% of baseline when NHE was blocked, 253 +/- 19% of baseline when NBC was blocked, and 154 +/- 6% of baseline when both NHE and NBC were blocked. End-ischemic pH(i) was 6.09 +/- 0.06 in HCO(3)(-)-perfused, untreated hearts, 5.85 +/- 0.02 when NHE was blocked, 5.81 +/- 0.05 when NBC was blocked, and 5.70 +/- 0.01 when both NHE and NBC were blocked. NHE blockade was cardioprotective, but NBC blockade and combined blockade were not, the latter likely due to a reduction in coronary flow, because omission of HCO(3)(-) under conditions of NHE blockade severely impaired coronary flow. Combined blockade of NHE and NBC conserved intracellular H(+) load during reperfusion and led to massive Na(+) influx when blockades were lifted. Without blockade, both NHE and NBC mediate acid-equivalent efflux in exchange for Na(+) influx during ischemia, NHE much more than NBC. Blockade of either one does not affect the other.     

Inhibition of NHE protects reoxygenated cardiomyocytes independently of anoxic Ca(2+) overload and acidosis

We investigated the question of whether inhibition of the Na(+)/H(+) exchanger (NHE) during ischemia is protective due to reduction of cytosolic Ca(2+) accumulation or enhanced acidosis in cardiomyocytes. Additionally, the role of the Na(+)-HCO(3)(-) symporter (NBS) was investigated. Adult rat cardiomyocytes were exposed to simulated ischemia and reoxygenation. Cytosolic pH [2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)], Ca(2+) (fura 2), Na(+) [sodium-binding benzolfuran isophthatlate (SBFI)], and cell length were measured. NHE was inhibited with 3 micromol/l HOE 642 or 1 micromol/l 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), and NBS was inhibited with HEPES buffer. During anoxia in bicarbonate buffer, cells developed acidosis and intracellular Na and Ca (Na(i) and Ca(i), respectively) overload. During reoxygenation cells underwent hypercontracture (44.0 +/- 4.1% of the preanoxic length). During anoxia in bicarbonate buffer, inhibition of NHE had no effect on changes in intracellular pH (pH(i)), Na(i), and Ca(i), but it significantly reduced the reoxygenation-induced hypercontracture (HOE: 61.0 +/- 1.4%, EIPA: 68.2 +/- 1.8%). The sole inhibition of NBS during anoxia was not protective. We conclude that inhibition of NHE during anoxia protects cardiomyocytes against reoxygenation injury independently of cytosolic acidification and Ca(i) overload.     

Intracellular pH regulation in colonocytes of rat proximal colon

The regulation of intracellular pH (pH(i)) in colonocytes of the rat proximal colon has been investigated using the pH-sensitive dye BCECF and compared with the regulation of pH(i) in the colonocytes of the distal colon. The proximal colonocytes in a HEPES-buffered solution had pH(i)=7.24+/-0.04 and removal of extracellular Na(+) lowered pH(i) by 0.24 pH units. Acid-loaded colonocytes by an NH(3)/NH(4)(+) prepulse exhibited a spontaneous recovery that was partially Na(+)-dependent and could be inhibited by ethylisopropylamiloride (EIPA). The Na(+)-dependent recovery rate was enhanced by increasing the extracellular Na(+) concentration and was further stimulated by aldosterone. In an Na(+)- and K(+)-free HEPES-buffered solution, the recovery rate from the acid load was significantly stimulated by addition of K(+) and this K(+)-dependent recovery was partially blocked by ouabain. The intrinsic buffer capacity of proximal colonocytes at physiological pH(i) exhibited a nearly 2-fold higher value than in distal colonocytes. Butyrate induced immediate colonocyte acidification that was smaller in proximal than in distal colonocytes. This acidification was followed by a recovery phase that was both EIPA-sensitive and -insensitive and was similar in both groups of colonocytes. In a HCO(3)(-)/CO(2)-containing solution, pH(i) of the proximal colonocytes was 7.20+/-0.04. Removal of external Cl(-) caused alkalinization that was inhibited by DIDS. The recovery from an alkaline load induced by removal of HCO(3)(-)/CO(2) from the medium was Cl(-)-dependent, Na(+)-independent and blocked by DIDS. Recovery from an acid load in EIPA-containing Na(+)-free HCO(3)(-)/CO(2)-containing solution was accelerated by addition of Na(+). Removal of Cl(-) inhibited the effect of Na(+). In summary, the freshly isolated proximal colonocytes of rats express Na(+)/H(+) exchanger, H(+)/K(+) exchanger ((H(+)-K(+))-ATPase) and Na(+)-dependent Cl(-)/HCO(3)(-) exchanger that contribute to acid extrusion and Na(+)-independent Cl(-)/HCO(3)(-) exchanger contributing to alkali extrusion. All of these are likely involved in the regulation of pH(i) in vivo. Proximal colonocytes are able to maintain a more stable pH(i) than distal cells, which seems to be facilitated by their higher intrinsic buffer capacity.     

High sodium intake increases HCO(3)- absorption in medullary thick ascending limb through adaptations in basolateral and apical Na+/H+ exchangers

A high sodium intake increases the capacity of the medullary thick ascending limb (MTAL) to absorb HCO(3)(-). Here, we examined the role of the apical NHE3 and basolateral NHE1 Na(+)/H(+) exchangers in this adaptation. MTALs from rats drinking H(2)O or 0.28 M NaCl for 5-7 days were perfused in vitro. High sodium intake increased HCO(3)(-) absorption rate by 60%. The increased HCO(3)(-) absorptive capacity was mediated by an increase in apical NHE3 activity. Inhibiting basolateral NHE1 with bath amiloride eliminated 60% of the adaptive increase in HCO(3)(-) absorption. Thus the majority of the increase in NHE3 activity was dependent on NHE1. A high sodium intake increased basolateral Na(+)/H(+) exchange activity by 89% in association with an increase in NHE1 expression. High sodium intake increased apical Na(+)/H(+) exchange activity by 30% under conditions in which basolateral Na(+)/H(+) exchange was inhibited but did not change NHE3 abundance. These results suggest that high sodium intake increases HCO(3)(-) absorptive capacity in the MTAL through 1) an adaptive increase in basolateral NHE1 activity that results secondarily in an increase in apical NHE3 activity; and 2) an adaptive increase in NHE3 activity, independent of NHE1 activity. These studies support a role for NHE1 in the long-term regulation of renal tubule function and suggest that the regulatory interaction whereby NHE1 enhances the activity of NHE3 in the MTAL plays a role in the chronic regulation of HCO(3)(-) absorption. The adaptive increases in Na(+)/H(+) exchange activity and HCO(3)(-) absorption in the MTAL may play a role in enabling the kidneys to regulate acid-base balance during changes in sodium and volume balance.     

Euryhaline pufferfish NBCe1 differs from nonmarine species NBCe1 physiology

Marine fish drink seawater and eliminate excess salt by active salt transport across gill and gut epithelia. Euryhaline pufferfish (Takifugu obscurus, mefugu) forms a CaCO(3) precipitate on the luminal gut surface after transitioning to seawater. NBCe1 (Slc4a4) at the basolateral membrane of intestinal epithelial cell plays a major role in transepithelial intestinal HCO(3)(-) secretion and is critical for mefugu acclimation to seawater. We assayed fugu-NBCe1 (fNBCe1) activity in the Xenopus oocyte expression system. Similar to NBCe1 found in other species, fNBCe1 is an electrogenic Na(+)/HCO(3)(-) cotransporter and sensitive to the stilbene inhibitor DIDS. However, our experiments revealed several unique and distinguishable fNBCe1 transport characteristics not found in mammalian or other teleost NBCe1-orthologs: electrogenic Li(+)/nHCO(3)(-) cotransport; HCO(3)(-) independent, DIDS-insensitive transport; and increased basal intracellular Na(+) accumulation. fNBCe1 is a voltage-dependent Na(+)/nHCO(3)(-) cotransporter that rectifies, independently from the extracellular Na(+) or HCO(3)(-) concentration, around -60 mV. Na(+) removal (0Na(+) prepulse) is necessary to produce the true HCO(3)(-)-elicited current. HCO(3)(-) addition results in huge outward currents with quick current decay. Kinetic analysis of HCO(3)(-) currents reveals that fNBCe1 has a much higher transport capacity (higher maximum current) and lower affinity (higher K(m)) than human kidney NBCe1 (hkNBCe1) does in the physiological range (membrane potential = -80 mV; [HCO(3)(-)] = 10 mM). In this state, fNBCe1 is in favor of operating as transepithelial HCO(3)(-) secretion, opposite of hkNBCe1, from blood to the luminal side. Thus, fugu-NBCe1 represents the first ortholog-based tool to study amino acid substitutions in NBCe1 and how those change ion and voltage dependence.     

Plasmalemmal V-H(+)-ATPases regulate intracellular pH in human lung microvascular endothelial cells

The lung endothelium layer is exposed to continuous CO(2) transit which exposes the endothelium to a substantial acid load that could be detrimental to cell function. The Na(+)/H(+) exchanger and HCO(3)(-)-dependent H(+)-transporting mechanisms regulate intracellular pH (pH(cyt)) in most cells. Cells that cope with high acid loads might require additional primary energy-dependent mechanisms. V-H(+)-ATPases localized at the plasma membranes (pmV-ATPases) have emerged as a novel pH regulatory system. We hypothesized that human lung microvascular endothelial (HLMVE) cells use pmV-ATPases, in addition to Na(+)/H(+) exchanger and HCO(3)(-)-based H(+)-transporting mechanisms, to maintain pH(cyt) homeostasis. Immunocytochemical studies revealed V-H(+)-ATPase at the plasma membrane, in addition to the predicted distribution in vacuolar compartments. Acid-loaded HLMVE cells exhibited proton fluxes in the absence of Na(+) and HCO(3)(-) that were similar to those observed in the presence of either Na(+), or Na(+) and HCO(3)(-). The Na(+)- and HCO(3)(-)-independent pH(cyt) recovery was inhibited by bafilomycin A(1), a V-H(+)-ATPase inhibitor. These studies show a Na(+)- and HCO(3)(-)-independent pH(cyt) regulatory mechanism in HLMVE cells that is mediated by pmV-ATPases.     

Mechanism of glucocorticoid-mediated reversal of inhibition of Cl(-)/HCO(-)(3) exchange during chronic ileitis

In the normal ileum, coupled NaCl absorption occurs via the dual operation of Na(+)/H(+) and Cl(-)/HCO(-)(3) exchange on the brush-border membrane (BBM) of villus cells. In a rabbit model of chronic small intestinal inflammation we determined the cellular mechanism of inhibition of NaCl absorption and the effect of steroids on this inhibition. Cl(-)/HCO(-)(3) but not Na(+)/H(+) exchange was reduced in the BBM of villus cells during chronic ileitis. Cl(-)/HCO(-)(3) exchange was inhibited secondary to a decrease in the affinity for Cl(-) rather than an alteration in the maximal rate of uptake of Cl(-) (V(max)). Methylprednisolone (MP) stimulated Cl(-)/HCO(-)(3) exchange in the normal ileum by increasing the V(max) of Cl(-) uptake rather than altering affinity for Cl(-). MP reversed the inhibition of Cl(-)/HCO(-)(3) exchange in rabbits with chronic ileitis. However, MP alleviated the Cl(-)/HCO(-)(3) exchange inhibition by restoring the affinity for Cl(-) rather than altering the V(max) of Cl(-) uptake. These data suggest that glucocorticoids mediate the alleviation of Cl(-)/HCO(-)(3) exchange inhibition in chronically inflamed ileum by reversing the same mechanism that was responsible for inhibition of this transporter rather than exerting a direct effect on the transporter itself, as was the case in normal ileum.     

Muscarinic activation of Na+-dependent ion transporters and modulation by bicarbonate in rat submandibular gland acinus

To investigate the interaction between the ion channels and transporters in the salivary fluid secretion, we measured the membrane voltage (V(m)) and intracellular concentrations of Ca(2+), Na(+) ([Na(+)](c)), Cl(-), and H(+) (pH(i)) in rat submandibular gland acini (RSMGA). After a transient depolarization induced by a short application of acetylcholine (ACh; 5 muM, 20 s), RSMGA showed strong delayed hyperpolarization (V(h,ACh); -95 +/- 1.8 mV) that was abolished by ouabain. In the HCO(3)(-)-free condition, the V(h,ACh) was also blocked by bumetanide, a blocker of Na(+)-K(+)-2Cl(-) cotransporter (NKCC). In the presence of HCO(3)(-) (24 meq, bubbled with 5% CO(2)), however, the V(h,ACh) was not blocked by bumetanide, but it was suppressed by ethylisopropylamiloride (EIPA), a Na(+)/H(+) exchanger (NHE) inhibitor. Similarly, the ACh-induced increase in [Na(+)](c) was totally blocked by bumetanide in the absence of HCO(3)(-), but only by one-half in the presence of HCO(3)(-). ACh induced a prominent acidification of pH(i) in the presence of HCO(3)(-), and the acidification was further increased by EIPA treatment. Without HCO(3)(-), an application of ACh strongly accelerated the NKCC activity that was measured from the decay of pH(i) during the application of NH(4)(+) (20 mM). Notably, the ACh-induced activation of NKCC was largely suppressed in the presence of HCO(3)(-). In summary, the ACh-induced anion secretion in RSMGA is followed by the activation of NKCC and NHE, resulting an increase in [Na(+)](c). The intracellular Na(+)-induced activation of electrogenic Na(+)/K(+)-ATPase causes V(h,ACh). The regulation of NKCC and NHE by ACh is strongly affected by the physiological level of HCO(3)(-).     

Cloning and characterization of a Na+-driven anion exchanger (NDAE1). A new bicarbonate transporter

Regulation of intra- and extracellular ion activities (e.g. H(+), Cl(-), Na(+)) is key to normal function of the central nervous system, digestive tract, respiratory tract, and urinary system. With our cloning of an electrogenic Na(+)/HCO(3)(-) cotransporter (NBC), we found that NBC and the anion exchangers form a bicarbonate transporter superfamily. Functionally three other HCO(3)(-) transporters are known: a neutral Na(+)/ HCO(3)(-) cotransporter, a K(+)/ HCO(3)(-) cotransporter, and a Na(+)-dependent Cl(-)-HCO(3)(-) exchanger. We report the cloning and characterization of a Na(+)-coupled Cl(-)-HCO(3)(-) exchanger and a physiologically unique bicarbonate transporter superfamily member. This Drosophila cDNA encodes a 1030-amino acid membrane protein with both sequence homology and predicted topology similar to the anion exchangers and NBCs. The mRNA is expressed throughout Drosophila development and is prominent in the central nervous system. When expressed in Xenopus oocytes, this membrane protein mediates the transport of Cl(-), Na(+), H(+), and HCO(3)(-) but does not require HCO(3)(-). Transport is blocked by the stilbene 4,4'-diisothiocyanodihydrostilbene- 2, 2'-disulfonates and may not be strictly electroneutral. Our functional data suggest this Na(+) driven anion exchanger (NDAE1) is responsible for the Na(+)-dependent Cl(-)-HCO(3)(-) exchange activity characterized in neurons, kidney, and fibroblasts. NDAE1 may be generally important for fly development, because disruption of this gene is apparently lethal to the Drosophila larva.     

Acclimation of ion regulatory capacities in gills of marine fish under environmental hypercapnia

The preservation of ion balance and pH despite environmental fluctuations is essential for the maintenance of vital cellular functions. While several ion transporters contribute to acid-base regulation in fish, the involvement and expression of key transporters under hypercapnia remain to be established. Here, two members of the HCO(3)(-) transporter family (Na(+)/HCO(3)(-) cotransporter NBC1 and Cl(-)/HCO(3)(-) exchanger AE1) were described for the first time in gills of marine fish. Benthic eelpout Zoarces viviparus were acclimated to 10,000 ppm CO(2). Hypercapnia did not affect whole animal oxygen consumption over a period of 4 days. During a time series of 6 wk NBC1 mRNA levels first decreased by about 40% (8 to 24 h) but finally increased about threefold over control. mRNA expression of AE1 decreased transiently by 50% at day 4 but recovered to control levels only. Reduced mRNA levels were also found for two Na(+)/H(+) exchangers (NHE1A, NHE1B) during the first days (by 50-60% at days 1 and 2), followed by restoration of control levels. This pattern was mirrored in a slight decrease of NHE1 protein contents and its subsequent recovery. In contrast, Na(+)-K(+)-ATPase mRNA and protein contents, as well as maximum activity, rose steadily from the onset of hypercapnia, and reached up to twofold control levels at the end. These results indicate shifting acclimation patterns between short- and long-term CO(2) exposures. Overall, ion gradient-dependent transporter mRNA levels were transiently downregulated in the beginning of the disturbance. Upregulation of NBC1 on long timescales stresses the importance of this transporter in the hypercapnia response of marine teleosts. Long-term rearrangements include Na(+)-K(+)-ATPase at higher densities and capacities, indicating a shift to elevated rates of ion and acid-base regulation under environmental hypercapnia.     

KCl reabsorption by the lower malpighian tubule of rhodnius prolixus: inhibition by Cl(-) channel blockers and acetazolamide

Iono- and osmoregulation by the blood-feeding hemipteran Rhodnius prolixus involves co-ordinated actions of the upper and lower Malpighian tubules. The upper tubule secretes ions (Na(+), K(+), Cl(-)) and water, whereas the lower tubule reabsorbs K(+) and Cl(-) but not water. The extent of KCl reabsorption by the lower tubule in vitro was monitored by ion-selective microelectrode measurement of Cl(-) and/or K(+) concentration in droplets of fluid secreted by Malpighian tubules isolated under oil. An earlier study proposed that K(+) reabsorption involves an omeprazole-sensitive apical K(+)/H(+) ATPase and Ba(2+)-sensitive basolateral K(+) channels. This paper examines the effects acetazolamide and of compounds that inhibit chloride channels, Cl(-)/HCO(3)(-) exchangers and Na(+)/K(+)/2Cl(-) or K(+)/Cl(-) co-transporters. The results suggest that Cl(-) reabsorption is inhibited by acetazolamide and by Cl(-) channel blockers, including diphenylamine-2-carboxylate(DPC) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), but not by compounds that block Na(+)/K(+)/Cl(-) and K(+)/Cl(-) co-transporters. Measurements of transepithelial potential and basolateral membrane potential during changes in bathing saline chloride concentration indicate the presence of DPC- and NPPB-sensitive chloride channels in the basolateral membrane. A working hypothesis of ion movements during KCl reabsorption proposes that Cl(-) moves from lumen to cell through a stilbene-insensitive Cl(-)/HCO(3)(-) exchanger and then exits the cell through basolateral Cl(-) channels.     

Contribution of anion transporters to the acidosis-induced swelling and intracellular acidification of glial cells

This study examines the contribution of anion transporters to the swelling and intracellular acidification of glial cells from an extracellular lactacidosis, a condition well-known to accompany cerebral ischemia and traumatic brain injury. Suspended C6 glioma cells were exposed to lactacidosis in physiological or anion-depleted media, and different anion transport inhibitors were applied. Changes in cell volume and intracellular pH (pH(i)) were simultaneously quantified by flow cytometry. Extracellular lactacidosis (pH 6.2) led to an increase in cell volume to 125.1 +/- 2.5% of baseline within 60 min, whereas the pH(i) dropped from the physiological value of 7.13 +/- 0.05 to 6.32 +/- 0.03. Suspension in Cl(-)-free or HCO(3)(-)/CO(2)-free media or application of anion transport inhibitors [0.1 mM bumetanide or 0.5 mM 4, 4'-diisothio-cyanatostilbene-2,2'-disulfonic acid (DIDS)] did not affect cell volume during baseline conditions but significantly reduced cell swelling from lactacidosis. In addition, the Cl(-)-free or HCO(3)(-)/CO(2)-free media and DIDS attenuated intracellular acidosis on extracellular acidification. From these findings it is concluded that besides the known activation of the Na(+)/H(+) exchanger, activation of the Na(+)-independent Cl(-)/HCO(3)(-) exchanger and the Na(+)-K(+)-Cl(-) cotransporter contributes to acidosis-induced glial swelling and the intracellular acidification. Inhibition of these processes may be of interest for future strategies in the treatment of cytotoxic brain edema from cerebral ischemia or traumatic brain injury.     

Mechanism of acid adaptation of a fish living in a pH 3.5 lake

Despite unfavorable conditions, a single species of fish, Osorezan dace, lives in an extremely acidic lake (pH 3.5) in Osorezan, Aomori, Japan. Physiological studies have established that this fish is able to prevent acidification of its plasma and loss of Na(+). Here we show that these abilities are mainly attributable to the chloride cells of the gill, which are arranged in a follicular structure and contain high concentrations of Na(+)-K(+)-ATPase, carbonic anhydrase II, type 3 Na(+)/H(+) exchanger (NHE3), type 1 Na(+)-HCO(3)(-) cotransporter, and aquaporin-3, all of which are upregulated on acidification. Immunohistochemistry established their chloride cell localization, with NHE3 at the apical surface and the others localized to the basolateral membrane. These results suggest a mechanism by which Osorezan dace adapts to its acidic environment. Most likely, NHE3 on the apical side excretes H(+) in exchange for Na(+), whereas the electrogenic type 1 Na(+)-HCO(3)(-) cotransporter in the basolateral membrane provides HCO(3)(-) for neutralization of plasma using the driving force generated by Na(+)-K(+)-ATPase and carbonic anhydrase II. Increased expression of glutamate dehydrogenase was also observed in various tissues of acid-adapted dace, suggesting a significant role of ammonia and bicarbonate generated by glutamine catabolism.     

Responses of gill mitochondria-rich cells in Mozambique tilapia exposed to acidic environments (pH 4.0) in combination with different salinities

On exposure to hyposmotic acidic water, teleost fish suffer from decreases in blood osmolality and pH, and consequently activate osmoregulatory and acid-base regulatory mechanisms to restore disturbed ion and acid-base balances. In Mozambique tilapia Oreochromis mossambicus exposed to acidic (pH 4.0) or neutral (pH 7.4-7.7) freshwater in combination with 0mM or 50mM NaCl, we examined functional and morphological changes in gill mitochondria-rich (MR) cells. We assessed gene expression of Na(+)/H(+) exchanger-3 (NHE3), Na(+)/Cl(-) cotransporter (NCC), vacuolar-type H(+)-ATPase (V-ATPase) and Na(+)/HCO(3)(-) cotransporter-1 (NBC1) in the gills. The mRNA expression of NHE3 and NCC in tilapia gills were higher in acidic freshwater than in that supplemented with 50mM NaCl, while there was no significant difference in mRNA levels of V-ATPase and NBC1. In addition, immunocytochemical observations showed that apical-NHE3 MR cells were enlarged, and frequently formed multicellular complexes with developed deep apical openings in acidic freshwater with 0mM and 50mM NaCl. These findings suggest that gill MR cells respond to external salinity and pH treatments, by parallel manipulation of osmoregulatory and acid-base regulatory mechanisms.     

Dual role of CO2/HCO3(-) buffer in the regulation of intracellular pH of three-dimensional tumor growths

Intracellular pH (pH(i)), a major modulator of cell function, is regulated by acid/base transport across membranes. Excess intracellular H(+) ions (e.g. produced by respiration) are extruded by transporters such as Na(+)/H(+) exchange, or neutralized by HCO(3)(-) taken up by carriers such as Na(+)-HCO(3)(-) cotransport. Using fluorescence pH(i) imaging, we show that cancer-derived cell lines (colorectal HCT116 and HT29, breast MDA-MB-468, pancreatic MiaPaca2, and cervical HeLa) extrude acid by H(+) efflux and HCO(3)(-) influx, largely sensitive to dimethylamiloride and 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS), respectively. The magnitude of HCO(3)(-) influx was comparable among the cell lines and may represent a constitutive element of tumor pH(i) regulation. In contrast, H(+) efflux varied considerably (MDA-MB-468 > HCT116 > HT29 > MiaPaca2 > HeLa). When HCO(3)(-) flux was pharmacologically inhibited, acid extrusion in multicellular HT29 and HCT116 spheroids (～10,000 cells) was highly non-uniform and produced low pH(i) at the core. With depth, acid extrusion became relatively more DIDS-sensitive because the low extracellular pH at the spheroid core inhibits H(+) flux more than HCO(3)(-) flux. HCO(3)(-) flux inhibition also decelerated HCT116 spheroid growth. In the absence of CO(2)/HCO(3)(-), acid extrusion by H(+) flux in HCT116 and MDA-MB-468 spheroids became highly non-uniform and inadequate at the core. This is because H(+) transporters require extracellular mobile pH buffers, such as CO(2)/HCO(3)(-), to overcome low H(+) ion mobility and chaperone H(+) ions away from cells. CO(2)/HCO(3)(-) exerts a dual effect: as substrate for membrane-bound HCO(3)(-) transporters and as a mobile buffer for facilitating extracellular diffusion of H(+) ions extruded from cells. These processes can be augmented by carbonic anhydrase activity. We conclude that CO(2)/HCO(3)(-) is important for maintaining uniformly alkaline pH(i) in small, non-vascularized tumor growths and may be important for cancer disease progression.     

Mechanisms contributing to intracellular pH homeostasis in an immortalised human chondrocyte cell line

The maintenance of chondrocyte pH is an important parameter controlling cartilage matrix turnover rates. Previous studies have shown that, to varying degrees, chondrocytes rely on Na(+)/H(+) exchange to regulate pH. HCO(3)(-)-dependent buffering and HCO(3)(-)-dependent acid-extrusion systems seem to play relatively minor roles. This situation may reflect minimal carbonic anhydrase activity in cartilage cells. In the present study, the pH regulation of the human chondrocyte cell line, C-20/A4 has been characterised. Intracellular pH (pH(i)) was measured using the H(+)-sensitive fluoroprobe BCECF. In solutions lacking HCO(3)(-)/CO(2), pH(i) was approximately 7.5, and the recovery from intracellular acidification was predominantly mediated by a Na(+)-dependent, amiloride- and HOE 694-sensitive process. A small additional component which was sensitive to chloro-7-nitrobenz-2-oxa-1,3-diazole, an inhibitor of the V-type H(+)-ATPase, was also apparent. In solutions containing HCO(3)(-)/CO(2), pH(i) was approximately 7.2. Comparison of buffering capacity in the two conditions showed that this variable was not significantly augmented in HCO(3)(-)/CO(2)-containing media. The recovery from intracellular acidification was more rapid in the presence of HCO(3)(-)/CO(2), although under these conditions it was again largely dependent on Na(+) ions and inhibited by amiloride and HOE 694. A small component was inhibited by SITS, although this effect did not reach the level of statistical significance. These findings indicate that HCO(3)(-)-dependent processes play only a minimal role in pH regulation in C-20/A4 chondrocytes. pH regulation instead relies heavily on the Na(+)/H(+) exchanger together with a H(+)-ATPase. The absence of extrinsic (HCO(3)(-)/CO(2)) buffering is likely to reflect the low levels of carbonic anhydrase in these cells. In addition to providing fundamental information about a widely-used cell line, these findings support the contention that the unusual nature of pH regulation in chondrocytes reflects the paucity of carbonic anhydrase activity in these cells.