Bicarbonate secretion plays a role in chloride and water absorption of the European flounder intestine

Experiments performed on isolated intestinal segments from the marine teleost fish, the European flounder (Platichthys flesus), revealed that the intestinal epithelium is capable of secondary active HCO3(-) secretion in the order of 0.2-0.3 micromol x cm(-2) x h(-1) against apparent electrochemical gradient. The HCO3(-) secretion occurs via anion exchange, is dependent on mucosal Cl(-), results in very high mucosal HCO3(-) concentrations, and contributes significantly to Cl(-) and fluid absorption. This present study was conducted under in vivo-like conditions, with mucosal saline resembling intestinal fluids in vivo. These conditions result in a transepithelial potential of -16.2 mV (serosal side negative), which is very different from the -2.2 mV observed under symmetrical conditions. Under these conditions, we found a significant part of the HCO3(-) secretion is fueled by endogenous epithelial CO2 hydration mediated by carbonic anhydrase because acetazolamide (10(-4) M) was found to inhibit HCO3(-) secretion and removal of serosal CO(2) was found not to influence HCO3(-) secretion. Reversal of the epithelial electrochemical gradient for Cl(-) (removal of serosal Cl(-)) and elevation of serosal HCO3(-) resulted in enhanced HCO3(-) secretion and enhanced Cl(-) and fluid absorption. Cl(-) absorption via an anion exchange system appears to partly drive fluid absorption across the intestine in the absence of net Na(+) absorption.     

HCO3-secretion by bullfrog duodenum: dependence on nutrient Na+ during secretory stimulation

HCO3(-) secretion across in vitro duodenal mucosa of Rana catesbeiana was investigated under baseline conditions and during secretory stimulation. Baseline secretion was abolished by removal of CO2-HCO3(-)and reduced approximately 60% by removal of nutrient Na+, but was not sensitive to changes in Cl- or K+. Baseline secretion was not directly altered by exposure to 10(-3) M amiloride or 10(-3) M H2DIDS (dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) in the nutrient solution and only mildly reduced by acetazolamide. Following removal and restoration of Na+, recovery of secretion was impaired by exposure to acetazolamide (5 x 10(-4) M) or H2DIDS (5 x 10(-4) M) in the nutrient solution. Secretion stimulated by glucagon (10(-6) M) or 16,16-dimethyl prostaglandin E2 (10 microg.mL(-1)) was markedly attenuated by removal of Na+ or by exposure to H2DIDS, but secretion was not altered by acetazolamide (5 x 10(-4) M) or nutrient amiloride (1 mM). Thus, the HCO3(-) that is secreted under nonstimulated conditions derives partly from basolateral Na(+)-dependent uptake and partly from cellular CO2 hydration. Secretagogue-stimulated secretion by duodenal surface epithelium depends on stilbene-sensitive Na+(HCO3(-))n uptake across the basolateral membrane.     

Regulation of apical H⁺-ATPase activity and intestinal HCO₃⁻ secretion in marine fish osmoregulation

The absorption of Cl(-) and water from ingested seawater in the marine fish intestine is accomplished partly through Cl(-)/HCO(3)(-) exchange. Recently, a H(+) pump (vacuolar-type H(+)-ATPase) was found to secrete acid into the intestinal lumen, and it may serve to titrate luminal HCO(3)(-) and facilitate further Cl(-)/HCO(3)(-) exchange, especially in the posterior intestine, where adverse concentration gradients could limit Cl(-)/HCO(3)(-) exchange. The H(+) pump is expressed in all intestinal segments and in gill tissue of gulf toadfish (Opsanus beta) maintained in natural seawater. After acute transfer of toadfish to 60 ppt salinity, H(+) pump expression increased 20-fold in the posterior intestine. In agreement with these observations was a fourfold-increased H(+)-ATPase activity in the posterior intestine of animals acclimated to 60 ppt salinity. Interestingly, Na(+)-K(+)-ATPase activity was elevated in the anterior intestine and gill, but not in the posterior intestine. Apical acid secretion by isolated intestinal tissue mounted in Ussing chambers fitted with pH-stat titration systems increased after acclimation to hypersalinity in the anterior and posterior intestine, titrating >20% of secreted bicarbonate. In addition, net base secretion increased in hypersalinity-acclimated fish and was ～70% dependent on serosal HCO(3)(-). Protein localization by immunohistochemistry confirmed the presence of the vacuolar-type H(+)-ATPase in the apical region of intestinal enterocytes. These results show that the H(+) pump, especially in the posterior intestine, plays an important role in hypersaline osmoregulation and that it likely has significant effects on HCO(3)(-) accumulation in the intestinal lumen and, therefore, the continued absorption of Cl(-) and water.     

Independence of apical Cl-/HCO3- exchange and anion conductance in duodenal HCO3- secretion

Reduced gastrointestinal HCO3- secretion contributes to malabsorption and obstructive syndromes in cystic fibrosis. The apical HCO3- transport pathways in these organs have not been defined. We therefore assessed the involvement of apical Cl-/HCO3- exchangers and anion conductances in basal and cAMP-stimulated duodenal HCO3- secretion. Muscle-stripped rat and rabbit proximal duodena were mounted in Ussing chambers, and electrical parameters, HCO3- secretion rates, and 36Cl-, 22Na+, and 3H+ mannitol fluxes were assessed. mRNA expression levels were measured by a quantitative PCR technique. Removal of Cl- from or addition of 1 mM DIDS to the luminal perfusate markedly decreased basal HCO3- secretion but did not influence the HCO3- secretory response to 8-bromo-cAMP, which was inhibited by luminal 5-nitro-2-(3-phenylpropylamino)-benzoate. Bidirectional 22Na+ and 36Cl- flux measurements demonstrated an inhibition rather than a stimulation of apical anion exchange during cAMP-stimulated HCO3- secretion. The ratio of Cl- to HCO3- in the anion secretory response was compatible with both Cl- and HCO3- being secreted via the CFTR anion channel. CFTR expression was very high in the duodenal mucosa of both species. We conclude that in rat and rabbit duodena, an apical Cl-/HCO3- exchanger mediates a significant part of basal HCO3- secretion but is not involved in the HCO3- secretory response to cAMP analogs. The inhibitor profile, the strong predominance of Cl- over HCO3- in the anion secretory response, and the high duodenal CFTR expression levels suggest that a major portion of cAMP-stimulated duodenal HCO3- secretion is directly mediated by CFTR.     

Transport of cations and anions across forestomach epithelia: conclusions from in vitro studies

Secretion of saliva as well as absorptive and secretory processes across forestomach epithelia ensures an optimal environment for microbial digestion in the forestomachs. Daily salivary secretion of sodium (Na+) exceeds the amount found in plasma by a factor of 2 to 3, while the secretion of bicarbonate (HCO3-) is 6 to 8 times higher than the amount of HCO3- in the total extracellular space. This implies a need for efficient absorptive mechanisms across forestomach epithelia to allow for an early recycling. While Na+ is absorbed from all forestomachs via Na+/H+ exchange and a non-selective cation channel that shows increased conductance at low concentrations of Mg2+, Ca2+ or H+ in the luminal microclima and at low intracellular Mg2+, HCO3- is secreted by the rumen for the buffering of ingesta but absorbed by the omasum to prevent liberation of CO2 in the abomasum. Fermentation provides short chain fatty acids and ammonia (NH3) that have to be absorbed both to meet nutrient requirements and maintain ruminal homeostasis of pH and osmolarity. The rumen is an important location for the absorption of essential minerals such as Mg2+ from the diet. Other ions can be absorbed, if delivered in sufficient amounts (Ca2+, Pi, K+, Cl- and NH4+). Although the presence of transport mechanisms for these electrolytes has been described earlier, our knowledge about their nature, regulation and crosstalk has increased greatly in the last years. New transport pathways have recently been added to our picture of epithelial transport across rumen and omasum, including an apical non-selective cation conductance, a basolateral anion conductance, an apical H+-ATPase, differently expressed anion exchangers and monocarboxylate transporters.     

Membrane localization of H+ and HCO3- transporters in the rat pancreatic duct

The pancreatic duct secretes alkaline fluid that is rich in HCO3- and poor in Cl-. The molecular mechanisms that mediate ductal secretion and are responsible for the axial gradients of Cl- and HCO3- along the ductal tree are not well understood because H+ and HCO3- transport by duct cells have not been characterized or localized. To address these questions, we microdissected the intralobular, main, and common segments of the rat pancreatic duct. H+ and HCO3- transporters were characterized and localized by following intracellular pH while perfusing the bath and the lumen of the ducts. In intralobular ducts, Na(+)-dependent and amiloride-sensitive recovery from acid load in the absence of HCO3- was used to localize a Na+/H+ exchanger to the basolateral membrane (BLM). Modification of Cl- gradients across the luminal (LM) and BLM in the presence of HCO3- showed the presence of Cl- /HCO3- exchangers on both membranes of intralobular duct cells. Measurement of the effect of Cl- on one side of the membrane on the rate and extent of pHi changes caused by removal and addition of Cl- to the opposite side suggested that both exchangers are present in the same cell. In the presence of HCO3-, intralobular duct cells used three separate mechanisms to extrude H+: (a) BLM-located Na+/H+ exchange, (b) Na(+)-independent vacuolar-type H+ pump, and (c) BLM-located, Na(+)- dependent, amiloride-insensitive, and 4',4'-diisothiocyanatostilbene- 2,2'-disulfonic acid sensitive mechanism, possibly a Na(+)-dependent HCO3- transporter. The main and common segments of the duct displayed similar mechanisms and localization of H+ and HCO3- transporters to the extent studied in the present work. In addition to the transporters found in intralobular ducts, the main and common ducts showed Na+/H+ exchange activity in the LM. Three tests were used to exclude a significant luminal to basolateral Na+ leak as the cause for an apparent luminal Na+/H+ exchange in an HCO3- secreting cells: (a) addition of amiloride and removal of Na+ from the LM had a profound effect on Na+/H+ exchange activity on the BLM and vice versa; (b) inhibition of all transporters in the BLM by bathing the duct in the inert hydrocarbon Fluorinert FC-75 did not prevent cytosolic acidification caused by removal of luminal Na+; and (c) luminal Na+ did not activate the basolateral Na(+)-dependent HCO3- transporter. An Na(+)-independent, bafilomycin-sensitive H+ pumping activity was marginal in the absence of HCO3-.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evolutionary aspects of intestinal bicarbonate secretion in fish

Experiments compared intestinal HCO3- secretion in the intestine of marine teleost Gulf toadfish, Opsanus beta, to representatives of early chondrostean and chondrichthyan fishes, the Siberian sturgeon, Acipenser baerii, and white-spotted bamboo shark, Chiloscyllium plagiosum, respectively. As seen in marine teleosts, luminal HCO3- concentrations were 10-fold plasma levels in all species when exposed to hyperosmotic conditions. While intestinal water absorption left Mg2+ and SO4(2-) concentrated in intestinal fluids up to four-fold ambient seawater concentrations, HCO3- was concentrated up to 50 times ambient levels as a result of intestinal HCO3- secretion. Reduced luminal Cl- concentrations in the intestine of all species suggest that HCO3- secretion also occurs via Cl-/HCO3- exchange in chondrostean and chondrichthyan fishes. Sturgeon began precipitating carbonates from the gut after only 3 days at 14 per thousand, a mechanism utilized by marine teleosts to reduce intestinal fluid osmolality and maintain calcium homeostasis. Analysis of published intestinal fluid composition in the cyclostome Lampetra fluviatilis reveals that this species likely also utilize intestinal HCO3- secretion for osmoregulation. Analysis of existing cyclostome data and our results indicate that intestinal Cl-/HCO3- exchange plays an integral role in maintaining hydromineral balance not only in teleosts, but in all fish (and perhaps other animals) with a need to drink seawater.     

Mechanism of augmented duodenal HCO(3)(-) secretion after elevation of luminal CO(2)

The proximal duodenum is exposed to extreme elevations of P(CO(2)) because of the continuous mixture of secreted HCO(3)(-) with gastric acid. These elevations (up to 80 kPa) are likely to place the mucosal cells under severe acid stress. Furthermore, we hypothesized that, unlike most other cells, the principal source of CO(2) for duodenal epithelial cells is from the lumen. We hence examined the effect of elevated luminal P(CO(2)) on duodenal HCO(3)(-) secretion (DBS) in the rat. DBS was measured by the pH-stat method. For CO(2) challenge, the duodenum was superfused with a high Pco(2) solution. Intracellular pH (pH(i)) of duodenal epithelial cells was measured by ratio microfluorometry. CO(2) challenge, but not isohydric solutions, strongly increased DBS to approximately two times basal for up to 1 h. Preperfusion of the membrane-permeant carbonic anhydrase inhibitor methazolamide, or continuous exposure with indomethacin, fully inhibited CO(2)-augmented DBS. Dimethyl amiloride (0.1 mM), an inhibitor of the basolateral sodium-hydrogen exchanger 1, also inhibited CO(2)-augumented DBS, although S-3226, a specific inhibitor of apical sodium-hydrogen exchanger 3, did not. DIDS, an inhibitor of basolateral sodium-HCO(3)(-) cotransporter, also inhibited CO(2)-augemented DBS, as did the anion channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid. CO(2) decreased epithelial cell pH(i), followed by an overshoot after removal of the CO(2) solution. We conclude that luminal CO(2) diffused in the duodenal epithelial cells and was converted to H(+) and HCO(3)(-) by carbonic anhydrase. H(+) initially exited the cell, followed by secretion of HCO(3)(-). Secretion was dependent on a functioning basolateral sodium/proton exchanger, a functioning basolateral HCO(3)(-) uptake mechanism, and submucosal prostaglandin generation and facilitated hydration of CO(2) into HCO(3)(-) and H(+).     

Cation specificity and modes of the Na+:CO3(2-):HCO3- cotransporter in renal basolateral membrane vesicles

The cation specificity and possible exchange modes of the Na+:CO3(2-):HCO3- cotransporter were evaluated by use of basolateral membrane vesicles isolated from rabbit renal cortex. External Li+ inhibited HCO3- gradient-stimulated 22Na uptake, indicating that Li+ interacts with the Na+:CO3(2-):HCO3- cotransporter. No interaction with K+, choline, Rb+, Cs+, or NH4+ could be similarly detected. Imposing an outward Li+ gradient caused quenching of acridine orange fluorescence in the presence but not in the absence of HCO3-, suggesting that Li+:base cotransport takes place via the Na+:CO3(2-):HCO3- cotransporter. Imposing an outward gradient of unlabeled Na+ stimulated the initial rate of 22Na uptake and induced its transient uphill accumulation, indicating Na(+)-Na+ exchange. Na(+)-Na+ exchange was observed in the presence but not in the absence of HCO3- and was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), suggesting that it occurs via the Na+:CO3(2-):HCO3- cotransporter. Similarly, an outward Li+ gradient stimulated uphill 22Na accumulation, indicating Na(+)-Li+ exchange. Na(+)-Li+ exchange was observed in the presence but not in the absence of HCO3-, and was inhibited by DIDS, suggesting that it also occurs via the Na+:CO3(2-):HCO3- cotransporter. Both Na(+)-Na+ and Li(+)-Na+ exchange modes were sensitive to inhibition by harmaline but not by amiloride. We conclude that Li+ is an alternative substrate for the renal Na+:CO3(2-):HCO3- cotransporter. Transport modes of the system include cation:base cotransport and HCO3-dependent cation-cation exchange.     

Oxalate transport via the sulfate/HCO3 exchanger in rabbit renal basolateral membrane vesicles

We evaluated the mechanism of oxalate transport in basolateral membrane vesicles isolated from the rabbit renal cortex. An outward HCO3- gradient induced the transient uphill accumulation of oxalate and sulfate, indicating the presence of oxalate/HCO3- exchange and sulfate/HCO3- exchange. For oxalate, sulfate, or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, the K1/2 value for oxalate/HCO3- exchange was nearly identical to that for sulfate/HCO3- exchange, suggesting that both exchange processes occur via the same transport system. This was further supported by the finding of sulfate/oxalate exchange. Thiosulfate/sulfate exchange and thiosulfate/oxalate exchange were also demonstrated, but a variety of other tested anions including Cl-, p-aminohippurate, and lactate did not exchange for sulfate or oxalate. Na+ did not affect sulfate or oxalate transport, indicating that neither anion undergoes Na+ co-transport or Na+-dependent anion exchange in these membrane vesicles. Finally, we found that the stoichiometry of exchange is 1 sulfate or oxalate per 2 HCO3-, or a thermodynamically equivalent process. We conclude that oxalate, but not other organic or inorganic anions of physiologic importance, can share the sulfate/HCO3- exchanger in renal basolateral membrane vesicles. In series with luminal membrane oxalate/Cl- (formate) exchange, exchange of oxalate for HCO3- or sulfate across the basolateral membrane provides a possible transcellular route for oxalate transport in the proximal tubule.     

pH sensitivity of the Na+:HCO3- cotransporter in basolateral membrane vesicles isolated from rabbit kidney cortex.

HCO3- exit across the basolateral membrane of the kidney proximal tubule cell is mediated via an electrogenic Na+:HCO3- cotransporter. We have studied the effect of pH on the activity of this cotransport system in basolateral membrane vesicles isolated from rabbit renal cortex. At constant internal pH 6.0, increasing the external pH and [HCO3-] increased the rate of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive 22Na+ influx into the vesicles. To determine the role of internal pH on the activity of the Na+:HCO3- cotransport system, the influx of 22Na+ via HCO3-dependent Na(+)-Na+ exchange was measured in the absence of an initial pH and [HCO3-] gradient (pH(i) = pH(o), 5% CO2). Increasing the pH from 6.8 to 7.2 increased whereas, increasing the pH from 7.4 to 8.0 decreased the rate of 22Na+ influx via this exchange. Increasing pH at constant [HCO3-] (pH(i) = pH(o) = 8.0, 1.5% CO2 versus pH(i) = pH(o) = 7.2, 10% CO2) reduced the influx of 22Na+ via HCO3-dependent Na(+)-Na+ exchange. Increasing pH at constant [CO3(2-)](pH(i) = pH(o) = 8.0, 1.5% CO2 versus pH(i) = pH(o) = 7.2, 60% CO2) was associated with reduced 22Na+ uptake. Decreasing the pH (pH(i) = pH(o) = 6.3, 60% CO2 versus pH(i) = pH(o) = 7.2, 5% CO2) was associated with a reduced rate of HCO3(-)-dependent Na(+)-Na+ exchange. We conclude that the Na+:HCO3- cotransporter displays a significant pH sensitivity profile with the cotransporter being more functional at pH 7.0-7.4 and less active at more acid or alkaline pH. In addition, the results suggest that the pH sensitivity arises at the inner surface of the basolateral membrane.     

Nerve growth factor inhibits HCO3- absorption in renal thick ascending limb through inhibition of basolateral membrane Na+/H+ exchange

Nerve growth factor (NGF) inhibits transepithelial HCO3- absorption in the rat medullary thick ascending limb (MTAL). To investigate the mechanism of this inhibition, MTALs were perfused in vitro in Na+-free solutions, and apical and basolateral membrane Na+/H+ exchange activities were determined from rates of pHi recovery after lumen or bath Na+ addition. NGF (0.7 nM in the bath) had no effect on apical Na+/H+ exchange activity, but inhibited basolateral Na+/H+ exchange activity by 50%. Inhibition of basolateral Na+/H+ exchange activity with ethylisopropyl amiloride (EIPA) secondarily reduces apical Na+/H+ exchange activity and HCO3- absorption in the MTAL (Good, D. W., George, T., and Watts, B. A., III (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 12525-12529). To determine whether a similar mechanism could explain inhibition of HCO3- absorption by NGF, apical Na+/H+ exchange activity was assessed in physiological solutions (146 mM Na+) by measurement of the initial rate of cell acidification after lumen EIPA addition. Under these conditions, in which basolateral Na+/H+ exchange activity is present, NGF inhibited apical Na+/H+ exchange activity. Inhibition of HCO3- absorption by NGF was eliminated in the presence of bath EIPA or in the absence of bath Na+. Also, NGF blocked inhibition of HCO3- absorption by bath EIPA. We conclude that NGF inhibits basolateral Na+/H+ exchange activity in the MTAL, an effect opposite from the stimulation of Na+/H+ exchange by growth factors in other systems. NGF inhibits transepithelial HCO3- absorption through inhibition of basolateral Na+/H+ exchange, most likely as the result of functional coupling in which primary inhibition of basolateral Na+/H+ exchange activity results secondarily in inhibition of apical Na+/H+ exchange activity. These findings establish a role for basolateral Na+/H+ exchange in the regulation of renal tubule HCO3- absorption.     

Differential effects of superoxide on luminal and basolateral Na+/H+ exchange in the thick ascending limb

Superoxide (O2-) increases Na+ reabsorption in the thick ascending limb (THAL) by enhancing Na/K/2Cl cotransport. However, the effects of O2- on other THAL transporters, such as Na(+)/H+ exchangers, are unknown. We hypothesized that O2- stimulates Na(+)/H+ exchange in the THAL. We assessed total Na(+)/H+ exchange activity by measuring recovery of intracellular pH (pH(i)) after acid loading in isolated perfused THALs before and after adding xanthine oxidase (XO) and hypoxanthine (HX). We found that XO and HX decreased total pH(i) recovery rate from 0.26 +/- 0.05 to 0.21 +/- 0.04 pH units/min (P < 0.05), and this net inhibition decreased steady-state pH(i) from 7.52 to 7.37. Because THALs have different Na(+)/H+ exchanger isoforms on the luminal and basolateral membrane, we tested the effects of xanthine oxidase and hypoxanthine on luminal and basolateral Na(+)/H+ exchange by adding dimethylamiloride to either the bath or lumen. Xanthine oxidase and hypoxanthine increased luminal Na(+)/H+ exchange from 3.5 +/- 0.8 to 6.7 +/- 1.4 pmol.min(-1).mm(-1) (P < 0.01) but decreased basolateral Na(+)/H+ exchange from 10.8 +/- 1.8 to 6.8 +/- 1.1 pmol.min(-1).mm(-1) (P < 0.007). To ascertain whether these effects were caused by O2- or H2O2, we examined the ability of tempol, a superoxide dismutase mimetic, to block these effects. In the presence of tempol, xanthine oxidase and hypoxanthine had no effect on luminal or basolateral Na(+)/H+ exchange. We conclude that O2- inhibits basolateral and stimulates luminal Na(+)/H+ exchangers, perhaps because different isoforms are expressed on each membrane. Inhibition of basolateral Na(+)/H+ exchange may enhance stimulation of luminal Na(+)/H+ exchange by providing additional protons to be extruded across the luminal membrane. Together, the effects of O2- on Na(+)/H+ exchange may increase net HCO3- reabsorption by the THAL.     

Ca2+-activated Cl- channels can substitute for CFTR in stimulation of pancreatic duct bicarbonate secretion.

This study addresses the mechanisms by which a defect in CFTR impairs pancreatic duct bicarbonate secretion in cystic fibrosis. We used control (PANC-1) and CFTR-deficient (CFPAC-1; DeltaF508 mutation) cell lines and measured HCO3- extrusion by the rate of recovery of intracellular pH after an alkaline load and recorded whole cell membrane currents using patch clamp techniques. 1) In PANC-1 cells, cAMP causes parallel activation of Cl- channels and of HCO3- extrusion by DIDS-sensitive and Na+-independent Cl-/HCO3- exchange, both effects being inhibited by Cl- channel blockers NPPB and glibenclamide. 2) In CFPAC-1 cells, cAMP fails to stimulate Cl-/HCO3- exchange and Cl- channels, except after promoting surface expression of DeltaF508-CFTR by glycerol treatment. Instead, raising intracellular Ca2+ concentration to 1 micromol/l or stimulating purinergic receptors with ATP (10 and 100 micromol/l) leads to parallel activation of Cl- channels and HCO3- extrusion. 3) K+ channel function is required for coupling cAMP- and Ca2+-dependent Cl- channel activation to effective stimulation of Cl-/HCO3- exchange in control and CF cells, respectively. It is concluded that stimulation of pancreatic duct bicarbonate secretion via Cl-/HCO3- exchange is directly correlated to activation of apical membrane Cl- channels. Reduced bicarbonate secretion in cystic fibrosis results from defective cAMP-activated Cl- channels. This defect is partially compensated for by an increased sensitivity of CF cells to purinergic stimulation and by alternative activation of Ca2+-dependent Cl- channels, mechanisms of interest with respect to possible treatment of cystic fibrosis and of related chronic pancreatic diseases.     

Ionic mechanism of Na+-HCO3- cotransport in rabbit renal basolateral membrane vesicles

The exit of HCO3- across the basolateral membrane of the proximal tubule cell occurs via the electrogenic cotransport of 3 eq of base per Na+. We have used basolateral membrane vesicles isolated from rabbit renal cortex to identify the ionic species transported via this pathway. Media of varying pH and pCO2 were employed to evaluate the independent effects of HCO3- and CO3(2-) on 22Na transport. Na+ uptake was stimulated when [CO3(2-)] was increased at constant [HCO3-], indicating the existence of a transport site for CO3(2-). In the presence of HCO3-, Na+ influx was stimulated more than 3-fold by an inward SO3(2-) gradient. SO3(2-)-stimulated Na+ influx was stilbene-sensitive, confirming that it occurs via the Na+-HCO3- cotransport system. Na+-SO3(2-) cotransport was demonstrated and found to have a 1:1 stoichiometry. Increasing [CO3(2-)] at constant [HCO3-] reduced the stimulation of Na+ influx by SO3(2-), suggesting competition between SO3(2-) and CO3(2-) at a common divalent anion site. Additional divalent anions that were tested, such as SO4(2-), oxalate2-, and HPO4(2-), did not interact at this site. SO3(2-) stimulation of Na+ influx was absolutely HCO3-(-)dependent and was increased as a function of [HCO3-], indicating the presence of a separate HCO3- site. Lastly, we tested whether Na+ interacts via ion pair formation with CO3(2-) or binds to a distinct site. Na+, which has lower affinity than Li+ for ion pair formation with CO3(2-), was found to have greater than 5-fold higher affinity than Li+ for the Na+-HCO3- cotransport system. Moreover, when its inhibition was studied as a function of [Na+], harmaline was found to be a competitive inhibitor of Na+ influx, indicating the existence of a distinct cation site. Our data are compatible with a model in which base transport across the basolateral membrane of the proximal tubule cell takes place via 1:1:1 cotransport of CO3(2-), HCO3-, and Na+ on distinct sites.     

Membrane potential and bicarbonate secretion in isolated interlobular ducts from guinea-pig pancreas

The interlobular duct cells of the guinea-pig pancreas secrete HCO(3)(-) across their luminal membrane into a HCO(3)(-)-rich (125 mM) luminal fluid against a sixfold concentration gradient. Since HCO(3)(-) transport cannot be achieved by luminal Cl-/HCO(3)(-) exchange under these conditions, we have investigated the possibility that it is mediated by an anion conductance. To determine whether the electrochemical potential gradient across the luminal membrane would favor HCO(3)(-) efflux, we have measured the intracellular potential (V(m)) in microperfused, interlobular duct segments under various physiological conditions. When the lumen was perfused with a 124 mM Cl- -25 mM HCO(3)(-) solution, a condition similar to the basal state, the resting potential was approximately -60 mV. Stimulation with dbcAMP or secretin caused a transient hyperpolarization (approximately 5 mV) due to activation of electrogenic Na+-HCO(3)(-) cotransport at the basolateral membrane. This was followed by depolarization to a steady-state value of approximately -50 mV as a result of anion efflux across the luminal membrane. Raising the luminal HCO(3)(-) concentration to 125 mM caused a hyperpolarization (approximately 10 mV) in both stimulated and unstimulated ducts. These results can be explained by a model in which the depolarizing effect of Cl- efflux across the luminal membrane is minimized by the depletion of intracellular Cl- and offset by the hyperpolarizing effects of Na+-HCO(3)(-) cotransport at the basolateral membrane. The net effect is a luminally directed electrochemical potential gradient for HCO(3)(-) that is sustained during maximal stimulation. Our calculations indicate that the electrodiffusive efflux of HCO(3)(-) to the lumen via CFTR, driven by this gradient, would be sufficient to fully account for the observed secretory flux of HCO(3)(-).     

Regulation of ion transport by pH and [HCO3-] in isolated gills of the crab Neohelice (Chasmagnathus) granulata

Posterior isolated gills of Neohelice (Chasmagnathus) granulatus were symmetrically perfused with hemolymph-like saline of varying [HCO3-] and pH. Elevating [HCO3-] 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 (Vte), a measure of ion transport. The elevation in [HCO3-] also induced a switch from acid secretion (-43.7 +/- 22.5 microequiv.kg(-1).h(-1)) in controls to base secretion (84.7 +/- 14.4 microequiv.kg(-1).h(-1)). The HCO3(-)-induced Vte increase was inhibited by basolateral acetazolamide (200 micromol/l), amiloride (1 mmol/l), and ouabain (5 mmol/l) but not by bafilomycin (100 nmol/l). The Vte response to HCO3(-) did not take place in Cl(-)-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 Vte, which was matched by a net increase in acid secretion of 67.8 +/- 18.4 microequiv kg(-1) h(-1). This stimulation was sensitive to basolateral acetazolamide, bafilomycin, DIDS, and Na+-free conditions, but it still took place in Cl(-)-free saline. Therefore, the cellular response to low pH is different from the HCO3(-)-stimulated response. We also report V-H+-ATPase- and Na+-K+-ATPase-like immunoreactivity in gill sections for the first time in this crab. Our results suggest that carbonic anhydrase (CA), basolateral Na+/H+ exchangers and Na+-K+-ATPase and apical anion exchangers participate in the HCO3(-)-stimulated response, while CA, apical V-H+-ATPase and basolateral HCO3(-)-dependent cotransporters mediate the response to low pH.     

HCO3- transport in basolateral membrane vesicles isolated from rat renal cortex

The mechanism of HCO3- translocation across the proximal tubule basolateral membrane was investigated by testing for Na+-HCO3- cotransport using isolated membrane vesicles purified from rat renal cortex. As indicated by 22Na+ uptake, imposing an inwardly directed HCO3- concentration gradient induced the transient concentrative accumulation of intravesicular Na+. The stimulation of basolateral membrane vesicle Na+ uptake was specifically HCO3(-)-dependent as only basolateral membrane-independent Na+ uptake was stimulated by an imposed hydroxyl gradient in the absence of HCO3-. No evidence for Na+-HCO3- cotransport was detected in brush border membrane vesicles. Charging the vesicle interior positive stimulated net intravesicular Na+ accumulation in the absence of other driving forces via a HCO3(-)-dependent pathway indicating the flow of negative charge accompanies the Na+-HCO3- cotransport event. Among the anion transport inhibitors tested, 4-4'-diisothiocyanostilbene-2,2'-disulfonic acid demonstrated the strongest inhibitor potency at 1 mM. The Na+-coupled transport inhibitor harmaline also markedly inhibited HCO3- gradient-driven Na+ influx. A role for carbonic anhydrase in the mechanism of Na+-HCO3- cotransport is suggested by the modest inhibition of HCO3- gradient driven Na+ influx caused by acetazolamide. The imposition of Cl- concentration gradients had a marked effect on HCO3- gradient-driven Na+ influx which was furosemide-sensitive and consistent with the operation of a Na+-HCO3- for Cl- exchange mechanism. The results of this study provide evidence for an electrogenic Na+-HCO3- cotransporter in basolateral but not microvillar membrane vesicles isolated from rat kidney cortex. The possible existence of an additional basolateral membrane HCO3(-)-translocating pathway mediating Na+-HCO3- for Cl- exchange is suggested.     

Na+/HCO3-co-transport in basolateral membrane vesicles isolated from rabbit renal cortex

Recent studies suggest that the major pathway for exit of HCO3- across the basolateral membrane of the proximal tubule cell is electrogenic Na+/HCO3- co-transport. We therefore evaluated the possible presence of Na+/HCO3- co-transport in basolateral membrane vesicles isolated from the rabbit renal cortex. Imposing an inward HCO3- gradient induced the transient uphill accumulation of Na+, and imposing an outward Na+ gradient caused HCO3- -dependent generation of an inside-acid pH gradient as monitored by quenching of acridine orange fluorescence, findings consistent with the presence of Na+/HCO3- co-transport. In the absence of other driving forces, generating an inside-positive membrane potential by imposing an inward K+ gradient in the presence of valinomycin caused net Na+ uptake via a HCO3- -dependent pathway, indicating that Na+/HCO3- co-transport is electrogenic and associated with a flow of negative charge. Imposing transmembrane Cl- gradients did not appreciably affect HCO3- gradient-stimulated Na+ influx, suggesting that Na+/HCO3- co-transport is not Cl- -dependent. The rate of HCO3- gradient-stimulated Na+ influx was a simple, saturable function of the Na+ concentration (Km = 9.7 mM, Vmax = 160 nmol/min/mg of protein), was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (I50 = 100 microM), but was inhibited less than 10% by up to 1 mM amiloride. We could not demonstrate a HCO3- -dependent or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive component of Na+ influx in microvillus membrane vesicles. This study thus indicates the presence of a transport system mediating electrogenic Na+/HCO3- co-transport in basolateral, but not luminal, membrane vesicles isolated from the rabbit renal cortex. Analogous to the use of renal microvillus membrane vesicles to study Na+/H+ exchange, renal basolateral membrane vesicles may be a useful model system for examining the kinetics and possible regulation of Na+/HCO3- co-transport.     

CFTR inhibition augments NHE3 activity during luminal high CO2 exposure in rat duodenal mucosa

We hypothesized that the function of duodenocyte apical membrane acid-base transporters are essential for H(+) absorption from the lumen. We thus examined the effect of inhibition of Na(+)/H(+) exchanger-3 (NHE3), cystic fibrosis transmembrane regulator (CFTR), or apical anion exchangers on transmucosal CO(2) diffusion and HCO(3)(-) secretion in rat duodenum. Duodena were perfused with a pH 6.4 high CO(2) solution or pH 2.2 low CO(2) solution with the NHE3 inhibitor, S3226, the anion transport inhibitor, DIDS, or pretreatment with the potent CFTR inhibitor, CFTR(inh)-172, with simultaneous measurements of luminal and portal venous (PV) pH and carbon dioxide concentration ([CO(2)]). Luminal high CO(2) solution increased CO(2) absorption and HCO(3)(-) secretion, accompanied by PV acidification and PV Pco(2) increase. During CO(2) challenge, CFTR(inh)-172 induced HCO(3)(-) absorption, while inhibiting PV acidification. S3226 reversed CFTR(inh)-associated HCO(3)(-) absorption. Luminal pH 2.2 challenge increased H(+) and CO(2) absorption and acidified the PV, inhibited by CFTR(inh)-172 and DIDS, but not by S3226. CFTR inhibition and DIDS reversed HCO(3)(-) secretion to absorption and inhibited PV acidification during CO(2) challenge, suggesting that HCO(3)(-) secretion helps facilitate CO(2)/H(+) absorption. Furthermore, CFTR inhibition prevented CO(2)-induced cellular acidification reversed by S3226. Reversal of increased HCO(3)(-) loss by NHE3 inhibition and reduced intracellular acidification during CFTR inhibition is consistent with activation or unmasking of NHE3 activity by CFTR inhibition, increasing cell surface H(+) available to neutralize luminal HCO(3)(-) with consequent CO(2) absorption. NHE3, by secreting H(+) into the luminal microclimate, facilitates net transmucosal HCO(3)(-) absorption with a mechanism similar to proximal tubular HCO(3)(-) absorption.