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We have studied [Cl.sup.-] transport mechanisms in freshwater rainbow trout gill mitochondrion-rich (MR) cells using intracellular pH ([pH.sub.i]) imaging. Scanning electron microscopy demonstrated maintenance of cellular polarity in isolated MR cells. MR cell subtypes were identified by [Na.sup.+] introduction to the bath, and [Cl.sup.-] transport mechanisms were subsequently examined. [Cl.sup.-]-free exposure resulted in an alkalinization of [pH.sub.i] in both MR cell subtypes, which was dependent on HC[O.sup.-.sub.3] in the bath and inhibited by 1 mM DIDS. Recovery of [pH.sub.i] from an acidified state in [Na.sup.+]-free conditions was also DIDS sensitive. These results are the first functional evidence for [Cl.sup.-]/HC[O.sup.-.sub.3] exchangers in fish gill MR cells. A direct switch from NaCl to [Cl.sup.-]-free conditions caused a [pH.sub.i] acidification in a subset of MR cells, which was enhanced in the absence of HC[O.sup.-.sub.3]. The acidification was replaced by an alkalinization when [Cl.sup.-] removal was performed in the presence of NPPB (500 [micro]M) or EIPA (500 [micro]M). Finally, we found that the [Na.sup.+]-induced alkalinization of [pH.sub.i] found in a previous study is inhibited by EIPA. This inhibitor profile's results suggest the presence of a [Cl.sup.-]-dependent [Na.sup.+]/[H.sup.+] exchange mechanism. [pH.sub.i] imaging; NHE; acid/base regulation; anion exchange

Posterior isolated gills of Neohelice (Chasmagnathus) granulatus were symmetrically perfused with hemolymph-like saline of varying [HC[O.sup.-.sub.3]] and pH. Elevating [HC[O.sup.-.sub.3]] in the saline from 2.5 to 12.5 mmol/l (pH 7.75 in both cases) induced a significant increase in the transepithelial potential difference ([V.sub.te]), a measure of ion transport. The elevation in [HC[O.sup.-.sub.3]] also induced a switch from acid secretion (-43.7 [+ or -] 22.5 [micro]equiv x [kg.sup.-1] x [h.sup.-1]) in controls to base secretion (84.7 [+ or -] 14.4 [micro]equiv x [kg.sup.-1] x [h.sup.-1]). The [HCO.sup.-.sub.3]-induced [V.sub.te] increase was inhibited by basolateral acetazolamide (200 [micro]mol/l), amiloride (1 mmol/l), and ouabain (5 mmol/l) but not by bafilomycin (100 nmol/l). The [V.sub.te] response to HC[O.sup.-.sub.3] did not take place in [Cl.sup.-1]-free conditions; however, it was unaffected by apical SITS (2 mmol/l) or DIDS (1 mmol/l). A decrease in pH from 7.75 to 7.45 pH units in the perfusate also induced a significant increase in [V.sub.te], which was matched by a net increase in acid secretion of 67.8 [+ or -] 18.4 [micro]equiv [kg.sup.-1] [h.sup.-1]. This stimulation was sensitive to basolateral acetazolamide, bafilomycin, DIDS, and [Na.sup.+]-free conditions, but it still took place in [Cl.sup.-]-free saline. Therefore, the cellular response to low pH is different from the HC[O.sup.-.sub.3]-stimulated response. We also report V-[H.sup.+]-[ATPase.sup.-] and [Na.sup.+-] [K.sup.+]-ATPase-like immunoreactivity in gill sections for the first time in this crab. Our results suggest that carbonic anhydrase (CA), basolateral [Na.sup.+]/[H.sup.+] exchangers and [Na.sup.+]-[K.sup.+]-ATPase and apical anion exchangers participate in the HC[O.sup.-.sub.3]-stimulated response, while CA, apical V-[H.sup.+]-ATPase and basolateral HC[O.sup.-.sub.3]-dependent cotransporters mediate the response to low pH. acid/base regulation; [HCO.sup.-.sub.3] secretion; V-[H.sup.+]-ATPase; carbonic anhydrase; electrogenic [Na.sup.+]/[H.sup.+] exchanger; amiloride; acetazolamide; bafilomycin

We investigated the involvement of carbonic anhydrase (CA) in mediating V-[H.sup.+]-ATPase translocation into the basolateral membrane in gills of alkalotic Squalus acanthias. Immunolabeling revealed that CA is localized in the same cells as V-[H.sup.+]-ATPase. Blood plasma from dogfish injected with acetazolamide [30 mg/kg at time (t) = 0 and 6 h] and infused with NaHC[O.sub.3] for 12 h (1,000 [micro]eq x [kg.sup.-1] x [h.sup.-1]) had significantly higher plasma HC[O.sup.-.sub.3] concentration than fish that were infused with NaHC[O.sub.3] alone (28.72 [+ or -] 0.41 vs. 6.57 [+ or -] 2.47 mmol/l, n = 3), whereas blood pH was similar in both treatments (8.03 [+ or -] 0.11 vs. 8.04 [+ or -] 0.11 pH units at t = 12 h). CA inhibition impaired V-[H.sup.+]-ATPase translocation into the basolateral membrane, as estimated from immunolabeled gill sections and Western blotting on gill cell membranes (0.24 [+ or -] 0.08 vs. 1.00 [+ or -] 0.28 arbitrary units, n = 3; P < 0.05). We investigated V-[H.sup.+]-ATPase translocation during a postfeeding alkalosis ('alkaline tide'). Gill samples were taken 24-26 h after dogfish were fed to satiety in a natural-like feeding regime. Immunolabeled gill sections revealed that V-[H.sup.+]-ATPase translocated to the basolateral membrane in the postfed fish. Confirming this result, V-[H.sup.+]-ATPase abundance was twofold higher in gill cell membranes of the postfed fish than in fasted fish (n = 4-5; P < 0.05). These results indicate that 1) intracellular [H.sup.+] or HC[O.sub.3] produced by CA (and not blood pH or HC[O.sup.-.sub.3]) is likely the stimulus that triggers the V-[H.sup.+]-ATPase translocation into the basolateral membrane in alkalotic fish and 2) V-[H.sup.+]-ATPase translocation is important for enhanced HC[O.sup.-.sub.3] secretion during a naturally occurring postfeeding alkalosis. Squalus acanthias; bicarbonate secretion; proton reabsorption; acid-base regulation; acetazolamide; ion transport; feeding doi:10.1152/ajpregu.00814.2006

Isolated mitochondria-rich (MR) cells from the rainbow trout gill epithelium were subjected to intracellular pH ([pH.sub.i]) imaging with the pH-sensitive dye BCECF-AM. MR cells were categorized into two distinct functional subtypes based on their ability to recover [pH.sub.i] from an N[H.sup.4]Cl-induced acidification in the absence of [Na.sup.+]. An apparent link between resting [pH.sub.i] and [Na.sup.+]-independent [pH.sub.i] recovery was made. We observed a unique [pH.sub.i] acidification event that was induced by extracellular [Na.sup.+] addition. This further classified the mixed MR cell population into two functional subtypes: the majority of cells (77%) demonstrated the [Na.sup.+]-induced [pH.sub.i] acidification, whereas the minority (23%) demonstrated an alkalinization of [pH.sub.i] under the same circumstances. The focus of this study was placed on the [Na.sup.+]-induced acidification and pharmacological analysis via the use of amiloride and phenamil, which revealed that [Na.sup.+] uptake was responsible for the intracellular acidification. Further experiments revealed that [pH.sub.i] acidification could be abolished when [Na.sup.+] was allowed entry into the cell, but the activity of an electrogenic [Na.sup.+]-HC[O.sup.-.sub.3] cotransporter (NBC) was inhibited by DIDS. The electrogenic NBC activity was supported by a DIDS-sensitive, [Na.sup.+]-induced membrane potential depolarization as observed via imaging of the voltage-sensitive dye bis-oxonol. We also demonstrated NBC immunoreactivity via Western blotting and immunohistochemistry in gill tissue. We propose a model for transepithelial [Na.sup.+] uptake occurring via an apical [Na.sup.+] channel linked to a basolateral, electrogenic NBC in one subpopulation of MR cells. epithelial sodium channels; sodium-induced acidification; amiloride; phenamil; DIDS; acid-base; peanut lectin agglutinin binding; membrane potential; BCECF-AM; bis-oxonol; ion uptake

The hypoxic and acidic tumor environment necessitates intracellular pH (pHi) regulation for tumor progression. Carbonic anhydrase IX (CA IX; hypoxia-induced) is known to facilitate CO2 export and generate HCO3− in the extracellular tumor space. It has been proposed that HCO3− is re-captured by the cell to maintain an alkaline pHi. A diverse range of HCO3− transporters, coupled with a lack of a clear over-expression in cancers have limited molecular identification of this cellular process. Here, we report that hypoxia induces the Na+/HCO3− co-transporter (NBCe1) SLC4A4 mRNA expression exclusively in the LS174 colon adenocarcinoma cell line in a HIF1α dependent manner. HCO3− dependent pHi recovery observations revealed the predominant use of an NBC mechanism suggesting that reversal of a Cl−/HCO3− exchanger is not utilized for tumor cell pHi regulation. Knockdown of SLC4A4 via shRNA reduced cell proliferation and increased mortality during external acidosis and spheroid growth. pHi recovery from acidosis was partially reduced with knockdown of SLC4A4. In MDA-MB-231 breast cancer cells expressing high levels of SLC4A4 compared to LS174 cells, SLC4A4 knockdown had a strong impact on cell proliferation, migration, and invasion. SLC4A4 knockdown also altered expression of other proteins including CA IX. Furthermore the Na+/HCO3− dependent pHi recovery from acidosis was reduced with SLC4A4 knockdown in MDA-MB-231 cells. Combined our results indicate that SLC4A4 contributes to the HCO3− transport and tumor cell phenotype. This study complements the on-going molecular characterization of the HCO3− re-uptake mechanism in other tumor cells for future strategies targeting these potentially important drug targets. J. Cell. Physiol. 230: 1954-1963, 2015. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]