Intestinal carbonic anhydrase, bicarbonate, and proton carriers play a role in the acclimation of rainbow trout to seawater

Abrupt transfer of rainbow trout from freshwater to 65% seawater caused transient disturbances in extracellular fluid ionic composition, but homeostasis was reestablished 48 h posttransfer. Intestinal fluid chemistry revealed early onset of drinking and slightly delayed intestinal water absorption that coincided with initiation of NaCl absorption and HCO(3)(-) secretion. Suggestive of involvement in osmoregulation, relative mRNA levels for vacuolar H(+)-ATPase (V-ATPase), Na(+)-K(+)-ATPase, Na(+)/H(+) exchanger 3 (NHE3), Na(+)-HCO(3)(-) cotransporter 1, and two carbonic anhydrase (CA) isoforms [a general cytosolic isoform trout cytoplasmic CA (tCAc) and an extracellular isoform trout membrane-bound CA type IV (tCAIV)], were increased transiently in the intestine following exposure to 65% seawater. Both tCAc and tCAIV proteins were localized to apical regions of the intestinal epithelium and exhibited elevated enzymatic activity after acclimation to 65% seawater. The V-ATPase was localized to both basolateral and apical regions and exhibited a 10-fold increase in enzymatic activity in fish acclimated to 65% seawater, suggesting a role in marine osmoregulation. The intestinal epithelium of rainbow trout acclimated to 65% seawater appears to be capable of both basolateral and apical H(+) extrusion, likely depending on osmoregulatory status and intestinal fluid chemistry.     

Upregulation of apical NHE3 in renal OK cells overexpressing the rodent alpha(1)-subunit of the Na(+) pump

Vectorial Na(+) reabsorption across the proximal tubule is mediated by apical entry of Na(+), primarily via Na(+)/H(+) exchanger isoform 3 (NHE3), and basolateral extrusion via the Na(+) pump (Na(+)-K(+)-ATPase). We hypothesized that regulation of Na(+) reabsorption should involve not only the activity of the basolateral Na(+)-K(+)-ATPase, but also the apical NHE3, in a concerted manner. To generate a cell line that overexpresses Na(+)-K(+)-ATPase, opossum kidney (OK) cells were transfected with the rodent Na(+)-K(+)-ATPase alpha(1)-subunit (pCMV ouabain vector), and native cells were used as a control. The existence of distinct functional classes of Na(+)-K(+)-ATPase in wild-type and transfected cells was confirmed by the inhibition profile of Na(+)-K(+)-ATPase activity by ouabain. In contrast to wild-type cells, transfected cells exhibited two IC(50) values for ouabain: the first value was similar to the IC(50) of control cells, and the second value was 2 log units greater than the first, consistent with the presence of rat and opossum alpha(1)-isozymes. It is shown that transfection of OK cells with Na(+)-K(+)-ATPase increased Na(+)-K(+)-ATPase and NHE3 activities. This was associated with overexpression of the Na(+)-K(+)-ATPase alpha(1)-subunit and NHE3 in transfected OK cells. The abundance of the Na(+)-K(+)-ATPase beta(1)-subunit was slightly lower in transfected OK cells. In conclusion, the increase in expression and function of Na(+)-K(+)-ATPase in cells transfected with the rodent Na(+) pump alpha(1)-subunit cDNA is expected to stimulate apical Na(+) influx into the cells, thereby accounting for the observed stimulation of the apical NHE3 activity.     

Aldosterone regulation of intestinal Na absorption involves SGK-mediated changes in NHE3 and Na+ pump activity

Aldosterone-induced intestinal Na(+) absorption is mediated by increased activities of apical membrane Na(+)/H(+) exchange (aNHE3) and basolateral membrane Na(+)-K(+)-ATPase (BLM-Na(+)-K(+)-ATPase) activities. Because the processes coordinating these events were not well understood, we investigated human intestinal Caco-2BBE cells where aldosterone increases within 2-4 h of aNHE3 and alpha-subunit of BLM-Na(+)-K(+)-ATPase, but not total abundance of these proteins. Although aldosterone activated Akt2 and serum glucorticoid kinase-1 (SGK-1), the latter through stimulation of phosphatidylinositol 3-kinase (PI3K), only the SGK-1 pathway mediated its effects on Na(+)-K(+)-ATPase. Ouabain inhibition of the early increase in aldosterone-induced Na(+)-K(+)-ATPase activation blocked most of the apical NHE3 insertion, possibly by inhibiting Na(+)-K(+)-ATPase-induced changes in intracellular sodium concentration ([Na](i)). Over the next 6-48 h, further increases in aNHE3 and BLM-Na(+)-K(+)-ATPase activity and total protein expression were observed to be largely mediated by aldosterone-activated SGK-1 pathway. Aldosterone-induced increases in NHE3 mRNA, for instance, could be inhibited by RNA silencing of SGK-1, but not Akt2. Additionally, aldosterone-induced increases in NHE3 promoter activity were blocked by silencing SGK-1 as well as pharmacological inhibition of PI3K. In conclusion, aldosterone-stimulated intestinal Na(+) absorption involves two phases. The first phase involves stimulation of PI3K, which increases SGK-dependent insertion and function of BLM-Na(+)-K(+)-ATPase and subsequent increased membrane insertion of aNHE3. The latter may be caused by Na(+)-K(+)-ATPase-induced changes in [Na] or transcellular Na flux. The second phase involves SGK-dependent increases in total NHE3 and Na(+)-K(+)-ATPase protein expression and activities. The coordination of apical and BLM transporters after aldosterone stimulation is therefore a complex process that requires multiple time- and interdependent cellular processes.     

Gramicidin-perforated patch recording revealed the oscillatory nature of secretory Cl- movements in salivary acinar cells

Elevations of cytoplasmic free calcium concentrations ([Ca(2+)](i)) evoked by cholinergic agonists stimulate isotonic fluid secretion in salivary acinar cells. This process is driven by the apical exit of Cl(-) through Ca(2+)-activated Cl(-) channels, while Cl(-) enters the cytoplasm against its electrochemical gradient via a loop diuretic-sensitive Na(+)-K(+)-2Cl(-) cotransporter (NKCC) and/or parallel operations of Cl(-)-HCO(3)(-) and Na(+)-H(+) exchangers, located in the basolateral membrane. To characterize the contributions of those activities to net Cl(-) secretion, we analyzed carbachol (CCh)-activated Cl(-) currents in submandibular acinar cells using the "gramicidin-perforated patch recording configuration." Since the linear polypeptide antibiotic gramicidin creates monovalent cation-selective pores, CCh-activated Cl(-) currents in the gramicidin-perforated patch recording were carried by Cl(-) efflux via Cl(-) channels, dependent upon Cl(-) entry through Cl(-) transporters expressed in the acinar cells. CCh-evoked oscillatory Cl(-) currents were associated with oscillations of membrane potential. Bumetanide, a loop diuretic, decreased the CCh-activated Cl(-) currents and hyperpolarized the membrane potential. In contrast, neither methazolamide, a carbonic anhydrase inhibitor, nor elimination of external HCO(3)(-) had significant effects, suggesting that the cotransporter rather than parallel operations of Cl(-)-HCO(3)(-) and Na(+)-H(+) exchangers is the primary Cl(-) uptake pathway. Pharmacological manipulation of the activities of the Ca(2+)-activated Cl(-) channel and the NKCC revealed that the NKCC plays a substantial role in determining the amplitude of oscillatory Cl(-) currents, while adjusting to the rate imposed by the Ca(2+)-activated Cl(-) channel, in the gramicidin-perforated patch configuration. By concerting with and being controlled by the cation steps, the oscillatory form of secretory Cl(-) movements may effectively provide a driving force for fluid secretion in intact acinar cells.     

Immunocytochemical localization of Na+-HCO3- cotransporters and carbonic anhydrase dependence of fluid transport in corneal endothelial cells

In corneal endothelium, there is evidence for basolateral entry of HCO(3)(-) into corneal endothelial cells via Na(+)-HCO(3)(-) cotransporter (NBC) proteins and for net HCO(3)(-) flux from the basolateral to the apical side. However, how HCO(3)(-) exits the cells through the apical membrane is unclear. We determined that cultured corneal endothelial cells transport HCO(3)(-) similarly to fresh tissue. In addition, Cl(-) channel inhibitors decreased fluid transport by at most 16%, and inhibition of membrane-bound carbonic anhydrase IV by benzolamide or dextran-bound sulfonamide decreased fluid transport by at most 29%. Therefore, more than half of the fluid transport cannot be accounted for by anion transport through apical Cl(-) channels, CO(2) diffusion across the apical membrane, or a combination of these two mechanisms. However, immunocytochemistry using optical sectioning by confocal microscopy and cryosections revealed the presence of NBC transporters in both the basolateral and apical cell membranes of cultured bovine corneal endothelial cells and freshly isolated rabbit endothelia. This newly detected presence of an apical NBC transporter is consistent with its being the missing mechanism sought. We discuss discrepancies with other reports and provide a model that accounts for the experimental observations by assuming different stoichiometries of the NBC transport proteins at the basolateral and apical sides of the cells. Such functional differences might arise either from the expression of different isoforms or from regulatory factors affecting the stoichiometry of a single isoform.     

pH microdomains in oligodendrocytes

Oligodendrocytes (OLs) are cells that produce myelin in the central nervous system. Here we use ratiometric pH indicator dye to analyze intracellular pH in OLs in culture. The results reveal alkaline microdomains, which predominate in the perikaryon and proximal dendrites, and acidic microdomains, which predominate in distal dendrites. Spatial nonuniformity of pH is generated by differential subcellular distribution of Na(+)/H(+) exchanger (NHE), which is localized in a punctate distribution in the perikaryon and proximal processes, Na(+)/HCO(3)(-) cotransporter (NBC), which is localized in a punctate distribution in distal dendrites, and carbonic anhydrase isotype II (CAII), which is colocalized with either NHE or NBC. Inhibition of NHE activity by amiloride inhibits regeneration of alkaline microdomains after cytoplasmic acidification, whereas the inhibition of CAII activity with ethoxyzolamide inhibits acidification of dendrites. Fluorescence correlation spectroscopy analysis of CAII microinjected into OLs reveals freely diffusing protein throughout the cell as well as protein associated predominantly with NHE in the perikaryon and predominantly with NBC in the dendrites. Alkaline and acidic microdomains could be generated by transport metabolons consisting of CAII associated with NHE or NBC, respectively. This study provides the first evidence for pH microdomains in cells and describes a mechanism for how they are generated.     

Gill Na(+)-K(+)-2Cl(-) cotransporter abundance and location in Atlantic salmon: effects of seawater and smolting

Na(+)-K(+)-2Cl(-) cotransporter abundance and location was examined in the gills of Atlantic salmon (Salmo salar) during seawater acclimation and smolting. Western blots revealed three bands centered at 285, 160, and 120 kDa. The Na(+)-K(+)-2Cl(-) cotransporter was colocalized with Na(+)-K(+)-ATPase to chloride cells on both the primary filament and secondary lamellae. Parr acclimated to 30 parts per thousand seawater had increased gill Na(+)-K(+)-2Cl(-) cotransporter abundance, large and numerous Na(+)-K(+)-2Cl(-) cotransporter immunoreactive chloride cells on the primary filament, and reduced numbers on the secondary lamellae. Gill Na(+)-K(+)-2Cl(-) cotransporter levels were low in presmolts (February) and increased 3.3-fold in smolts (May), coincident with elevated seawater tolerance. Cotransporter levels decreased below presmolt values in postsmolts in freshwater (June). The size and number of immunoreactive chloride cells on the primary filament increased threefold during smolting and decreased in postsmolts. Gill Na(+)-K(+)-ATPase activity and Na(+)-K(+)-2Cl(-) cotransporter abundance increased in parallel during both seawater acclimation and smolting. These data indicate a direct role of the Na(+)-K(+)-2Cl(-) cotransporter in salt secretion by gill chloride cells of teleost fish.     

Time course analysis of the mechanism by which silver inhibits active Na+ and Cl- uptake in gills of rainbow trout

A time course analysis using (110m)Ag, (24)Na(+), and (36)Cl(-) examined gill silver accumulation and the mechanism by which waterborne silver (4.0 x 10(-8) M; 4.3 microg/l) inhibits Na(+) and Cl(-) uptake in gills of freshwater rainbow trout. Analyses of gill and body fluxes allowed calculation of apical uptake and basolateral export rates for silver, Na(+), and Cl(-). To avoid changes in silver bioavailability, flow-through conditions were used to limit the buildup of organic matter in the exposure water. For both Na(+) and Cl(-) uptake, apical entry, rather than basolateral export, was the rate-limiting step; Na(+) and Cl(-) uptake declined simultaneously and equally initially, with both uptakes reduced by approximately 500 nmol.g(-1).h(-1) over the 1st h of silver exposure. There was a further progressive decline in Na(+) uptake until 24 h. Carbonic anhydrase activity was inhibited by 1 h, whereas Na(+)-K(+)-ATPase activity was not significantly inhibited until 24 h of exposure. These results indicate that carbonic anhydrase inhibition can explain the early decline in Na(+) and Cl(-) uptake, whereas the later decline is probably related to Na(+)-K(+)-ATPase blockade. Contrary to previous reports, gill silver accumulation increased steadily to a plateau. Despite the rapid inhibition of apical Na(+) and Cl(-) uptake, apical silver uptake (and basolateral export) increased until 10 h, before decreasing thereafter. Thus silver did not inhibit its own apical uptake in the short term. These results suggest that reduced silver bioavailability is the mechanism behind the pattern of peak and decline in gill silver accumulation previously reported for static exposures to silver.     

Brush border Mg2+-HCO-3-ATPase, supernatant carbonic anhydrase and other enzyme activities isolated from rat intestinal mucosa: effect of adrenalectomy and aldosterone administration

The possible role of Mg2+-HCO3-ATPase, carbonic anhydrase and several other enzymes in rat intestinal mucosa as mediators of the action of aldosterone has been examined. The small-intestinal tract was cut into seven segments, 15 cm each in length and the mucosa was scraped off, homogenized in 50 mM D-mannitol-2 mM Tris-HCl buffer (pH 7.1), differentially fractionated and a crude brush border was obtained. The mucosa from the colon and rectum was combined and used as the large-intestinal sample. Five days after the adrenalectomy, activities of brush border Mg2+-HCO3-ATPase and supernatant carbonic anhydrase from the upper small intestine decreased to about 60 and 40% of normal values, respectively. Activities of Na+-K+-ATPase, beta-glycerophosphatase and succinate dehydrogenase were all decreased. Two and 4 h after i.p. injection of aldosterone (40 micrograms/kg) to adrenalectomized rats, all enzyme activities increased except for Na+-K+-ATPase in the upper small intestine. In contrast, Mg2+-HCO-3-ATPase and carbonic anhydrase activities were unchanged 3 h after i.p. injection of dexamethasone (200 micrograms and 1 mg/kg). The activation of both Mg2+-HCO3-ATPase and carbonic anhydrase by a single injection of aldosterone was blocked by pretreatment with cycloheximide (1 mg/kg). These results suggest that aldosterone may induce the synthesis of enzyme proteins in the intestinal mucosa.     

Gene expression of Na+/H+ exchanger in zebrafish H+ -ATPase-rich cells during acclimation to low-Na+ and acidic environments

In mammalian nephrons, most of the Na(+) and HCO(3)(-) is reabsorbed by proximal tubular cells in which the Na(+)/H(+) exchanger 3 (NHE3) is the major player. The roles of NHEs in Na(+) uptake/acid-base regulation in freshwater (FW) fish gills are still being debated. In the present study, functional genomic approaches were used to clone and sequence the full-length cDNAs of the nhe family from zebrafish (Danio rerio). A phylogenetic tree analysis of the deduced amino acid sequences showed that zNHE1-8 are homologous to their mammalian counterparts. By RT-PCR analysis and double/triple in situ hybridization/immunocytochemistry, only zebrafish NHE3b was expressed in zebrafish gills and was colocalized with V-H(+)-ATPase but not with Na(+)-K(+)-ATPase, indicating that H(+)-ATPase-rich (HR) cells specifically express NHE3b. A subsequent quantitative RT-PCR analysis demonstrated that acclimation to low-Na(+) FW caused upregulation and downregulation of the expressions of znhe3b and zatp6v0c (H(+)-ATPase C-subunit), respectively, in gill HR cells, whereas acclimation to acidic FW showed reversed effects on the expressions of these two genes. In conclusion, both NHE3b and H(+)-ATPase are probably involved in Na(+) uptake/acid-base regulation in zebrafish gills, like mammalian kidneys, but the partitioning of these two transporters may be differentially regulated depending on the environmental situation in which fish are acclimatized.     

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.     

Theoretical considerations underlying Na(+) uptake mechanisms in freshwater fishes

Ion and acid-base regulating mechanisms have been studied at the fish gill for almost a century. Original models proposed for Na(+) and Cl(-) uptake, and their linkage with H(+) and HCO(3)(-) secretion have changed substantially with the development of more sophisticated physiological techniques. At the freshwater fish gill, two dominant mechanisms for Na(+) uptake from dilute environments have persisted in the literature. The use of an apical Na(+)/H(+) exchanger driven by a basolateral Na(+)/K(+)-ATPase versus an apical Na(+) channel electrogenically coupled to an apical H(+)-ATPase have been the source of debate for a number of years. Advances in molecular biology have greatly enhanced our understanding of the basic ion transport mechanisms at the fish gill. However, it is imperative to ensure that thermodynamic principles are followed in the development of new models for gill ion transport. This review will focus on the recent molecular advances for Na(+) uptake in freshwater fish. Emphasis will be placed on thermodynamic constraints that prevent electroneutral apical NHE function in most freshwater environments. By combining recent advances in molecular and functional physiology of fish gills with thermodynamic considerations of ion transport, our knowledge in the field should continue to grow in a logical manner.     

The putative mechanism of Na(+) absorption in euryhaline elasmobranchs exists in the gills of a stenohaline marine elasmobranch, Squalus acanthias

We recently cloned an NHE3 orthologue from the gills of the euryhaline Atlantic stingray (Dasyatis sabina), and generated a stingray NHE3 antibody to unequivocally localize the exchanger to the apical side of epithelial cells that are rich with Na(+)/K(+)-ATPase (A MRC). We also demonstrated an increase in NHE3 expression when stingrays are in fresh water, suggesting that NHE3 is responsible for active Na(+) absorption. However, the vast majority of elasmobranchs are only found in marine environments. In the current study, immunohistochemistry with the stingray NHE3 antibody was used to localize the exchanger in the gills of the stenohaline marine spiny dogfish shark (Squalus acanthias). NHE3 immunoreactivity was confined to the apical side of cells with basolateral Na(+)/K(+)-ATPase and was excluded from cells with high levels of vacuolar H(+)-ATPase. Western blots detected a single protein of 88 kDa in dogfish gills, the same size as NHE3 in stingrays and mammals. These immunological data demonstrate that the putative cell type responsible for active Na(+) absorption in euryhaline elasmobranchs is also present in stenohaline marine elasmobranchs, and suggest that the inability of most elasmobranchs to survive in fresh water is not due to a lack of the gill ion transporters for Na(+) absorption.     

Interactions between Na+ channels and Na+-HCO3- cotransporters in the freshwater fish gill MR cell: a model for transepithelial Na+ uptake

Isolated mitochondria-rich (MR) cells from the rainbow trout gill epithelium were subjected to intracellular pH (pH(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(i) from an NH(4)Cl-induced acidification in the absence of Na(+). An apparent link between resting pH(i) and Na(+)-independent pH(i) recovery was made. We observed a unique pH(i) acidification event that was induced by extracellular Na(+) addition. This further classified the mixed MR cell population into two functional subtypes: the majority of cells (77%) demonstrated the Na(+)-induced pH(i) acidification, whereas the minority (23%) demonstrated an alkalinization of pH(i) under the same circumstances. The focus of this study was placed on the Na(+)-induced acidification and pharmacological analysis via the use of amiloride and phenamil, which revealed that Na(+) uptake was responsible for the intracellular acidification. Further experiments revealed that pH(i) acidification could be abolished when Na(+) was allowed entry into the cell, but the activity of an electrogenic Na(+)-HCO(3)(-) cotransporter (NBC) was inhibited by DIDS. The electrogenic NBC activity was supported by a DIDS-sensitive, Na(+)-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(+) uptake occurring via an apical Na(+) channel linked to a basolateral, electrogenic NBC in one subpopulation of MR cells.     

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.     

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.     

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)(-).