Adaptation to respiratory acidosis in the turtle bladder

The effect of in vivo respiratory acidosis for 4 and 48 hr was examined in the turtle bladder by placing turtles in hypercapnic chambers. Blood pH was significantly lowered and pCO2 was significantly elevated over control values both 4 and 48 hr, while blood bicarbonate was only increased after 48 hr. In vitro rates for H+ secretion determined by the reverse short-circuit current were significantly greater in bladders from 48 hr of respiratory acidosis than those of controls (27.3 +/- 2.7 vs 20.6 +/- 1.7 microA, P less than 0.05). In vitro rates for HCO3- secretion determined by pH stat were not altered. Fluorescence microscopy was used to study cell morphology. The number of carbonic anhydrase cells (corrected for the total number of cells) as determined by four different fluorescence stains (6-carboxyfluorescein, rhodamine 123, acridine orange, and 3,3'-diethyloxacarbocyaninine iodide) was increased both after 4 and 48 hr of respiratory acidosis. However, the number of HCO3(-)-secreting (beta subtype) carbonic anhydrase cells, determined by a probe for the anion exchanger, NBD-taurine, was not increased. In vitro 1% CO2 for 4 hr also resulted in an increase in H+ secretion and in the number of 6-carboxyfluorescein-positive cells, both of which could be blocked with SITS pretreatment. We conclude that CO2 changes the mucosal cells more toward the carbonic anhydrase phenotype, and that if NBD-taurine accurately identifies the beta cells, that the adaptation produces or recruits more alpha-carbonic anhydrase cells.     

Effects of acetazolamide on cerebrospinal fluid ions in metabolic alkalosis in dogs

We hypothesized that inhibition of carbonic anhydrase in the central nervous system by acetazolamide should limit the rise in cisternal cerebrospinal fluid (CSF) [HCO3-] observed in metabolic alkalosis. To test this hypothesis, isosmotic isonatremic metabolic alkalosis was produced in two groups of anesthetized, paralyzed, and mechanically ventilated dogs (8 in each group). Group II animals received 50 mg/kg of acetazolamide intravenously 1 h before induction of metabolic alkalosis of 5-h duration. Renal effects of acetazolamide were eliminated by ligation of renal pedicles. In both groups cisternal CSF [Na+] remained relatively constant during metabolic alkalosis. In group I CSF [Cl-] decreased 3.6 and 8.2 meq/l, respectively, 2.5 and 5 h after induction of metabolic alkalosis. Respective increments in CSF [HCO3-] were 3.4 and 6.0 meq/l. In acetazolamide-treated dogs, during metabolic alkalosis, increments in CSF [HCO3-] (4.8 and 7.2 meq/l, respectively, at 2.5 and 5 h) and decrements in CSF [Cl-] (9.1 and 13.3 meq/l) were greater than those observed in group I. We conclude that, in dogs with metabolic alkalosis and bilateral ligation of renal pedicles, acetazolamide impairs CSF regulation of HCO3- and Cl- ions; acetazolamide not only failed to impede HCO3- rise but actually appeared to increase it. The mechanisms for these observations are discussed.     

V-H+ -ATPase translocation during blood alkalosis in dogfish gills: interaction with carbonic anhydrase and involvement in the postfeeding alkaline tide

We investigated the involvement of carbonic anhydrase (CA) in mediating V-H(+)-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(+)-ATPase. Blood plasma from dogfish injected with acetazolamide [30 mg/kg at time (t) = 0 and 6 h] and infused with NaHCO(3) for 12 h (1,000 microeq.kg(-1).h(-1)) had significantly higher plasma HCO(3)(-) concentration than fish that were infused with NaHCO(3) alone (28.72 +/- 0.41 vs. 6.57 +/- 2.47 mmol/l, n = 3), whereas blood pH was similar in both treatments (8.03 +/- 0.11 vs. 8.04 +/- 0.11 pH units at t = 12 h). CA inhibition impaired V-H(+)-ATPase translocation into the basolateral membrane, as estimated from immunolabeled gill sections and Western blotting on gill cell membranes (0.24 +/- 0.08 vs. 1.00 +/- 0.28 arbitrary units, n = 3; P < 0.05). We investigated V-H(+)-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(+)-ATPase translocated to the basolateral membrane in the postfed fish. Confirming this result, V-H(+)-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(+) or HCO(3)(-) produced by CA (and not blood pH or HCO(3)(-)) is likely the stimulus that triggers the V-H(+)-ATPase translocation into the basolateral membrane in alkalotic fish and 2) V-H(+)-ATPase translocation is important for enhanced HCO(3)(-) secretion during a naturally occurring postfeeding alkalosis.     

Functional and molecular adaptation of Cl/HCO3- exchanger to chronic alkaline media in renal cells.

The Cl(-)/HCO3- exchanger (AE) is one of the mechanisms that cells have developed to adjust pH Despite its importance, the role of AE isoforms in controlling steady-state pH during alkalosis has not been widely investigated. In the present study, we have evaluated whether conditions simulating acute and chronic metabolic alkalosis affected the transport activity and protein levels of Cl-/HCO3- exchangers in a rat cortical collecting duct cell line (RCCD1). pH(i) was monitored using the fluorescent dye BCECF in monolayers grown on permeable supports. Anion exchanger function was assessed by the response of pH(i) to acute chloride removal. RT-PCR and immunoblot assays were also performed. Our results showed that RCCD1 cells express two members of the anion exchanger gene family: AE2 and AE4. Functional studies demonstrated that while in acute alkalosis pH(i) became alkaline and was not regulated, after 48 h adaptation; steady-state pH(i) reached a value similar to the physiological one. Chronic treated cells also resulted in a 3-fold rise in Cl(-)/HCO3- exchange activity together with a 2.2-fold increase in AE2, but not AE4, protein abundance. We conclude that RCCD1 cells can adapt to chronic extracellular alkalosis reestablishing its steady-state pH(i) and that AE2 would play a key role in cell homeostasis.     

Compensation of respiratory alkalosis induced after acclimation to simulated altitude

Conscious intact rats previously acclimated for 3 wk to barometric pressure of 370-380 Torr (3WHx) were made alkalotic for 3 h by a decrease in inspired O2 fraction from 0.10 to 0.075 at ambient barometric pressure (730-740 Torr). Controls were normoxic littermates (Nx) in which inspired O2 fraction was lowered from approximately 0.21 to 0.10 for 3 h. Arterial PCO2 decreased progressively and similarly in both groups (65-70% of control at 15 min). Initially, arterial pH increased less in 3WHx (0.09 +/- 0.004 vs. 0.15 +/- 0.008). As hypocapnia continued, delta[HCO3-]/delta pH (mmol.l-1.pH) became more negative in Nx, from -15.2 +/- 2.5 at 15 min to -37.0 +/- 2.9 at 3 h, indicating nonrespiratory compensation of alkalosis. In 3WHx, delta[HCO3-]/delta pH did not change during alkalosis. Cumulative renal excretion of base (mueq/100 g) during alkalosis increased by 73.2 +/- 11.1 in Nx and 25.4 +/- 7.3 in 3WHx. This difference was mainly due to a larger increase in HCO3- excretion in Nx. The data suggest that the smaller compensation of hypocapnic alkalosis in 3WHx is partly due to the smaller increase in renal base excretion. Because base availability limits renal base excretion, the smaller renal response of 3WHx may be secondary to the low plasma HCO3- concentration that accompanies altitude acclimation.     

Cellular remodeling of HCO3(-)-secreting cells in rabbit renal collecting duct in response to an acidic environment

The renal cortical collecting duct (CCD) consists of principal and intercalated cells. Two forms of intercalated cells, those cells involved in H+/HCO3- transport, have recently been described. H+-secreting cells are capable of apical endocytosis and have H+ATPase on the apical membrane and a basolateral Cl-/HCO3- exchanger. HCO3(-)-secreting cells bind peanut agglutinin (PNA) to apical membrane receptors and have diffuse or basolateral distribution of H+ATPase; their Cl-/HCO3- exchanger is on the apical membrane. We found that 20 h after acid feeding of rabbits, there was a fourfold increase in number of cells showing apical endocytosis and a numerically similar reduction of cells binding PNA. Incubation of CCDs at pH 7.1 for 3-5 h in vitro led to similar, albeit less pronounced, changes. Evidence to suggest internalization and degradation of the PNA binding sites included a reduction in apical binding of PNA, decrease in pH in the environment of PNA binding, and incorporation of electron-dense PNA into cytoplasmic vesicles. Such remodeling was dependent on protein synthesis. There was also functional evidence for loss of apical Cl-/HCO3- exchange on PNA-labeled cells. Finally, net HCO3- flux converted from secretion to absorption after incubation at low pH. Thus, exposure of CCDs to low pH stimulates the removal/inactivation of apical Cl-/HCO3- exchangers and the internalization of other apical membrane components. Remodeling of PNA-labeled cells may mediate the change in polarity of HCO3- flux observed in response to acid treatment.     

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.     

Dual contribution theory of regulation of CSF HCO3 in respiratory acidosis.

Regulation of CSF HCO3-in respiratory acidosis was studied in light of the "dual contribution theory," which proposed that there were two sources for the CSF HCO3-increase: 1) HCO3-by diffusion from plasma and 2) HCO3-generated in the CNS and catalyzed by the local carbonic anhydrase (J. Appl. Physiol. 38: 504-512, 1975). In anesthetized dogs with an increase in Paco2 of 30 mmHg for 4 h the plasma HCO3 increased 2 meq/1 and CSF 6 meq/1. In combined respiratory and metabolic acidosis, plasma HCO3-did not increase but CSF HCO3-increased 6 meq/1. In combined acidosis and intraventricular injections of acetazolamide no increase in plasma or CSF HCO3-occurred. In combined respiratory acidosis and metabolic alkalosis and intraventricular acetazolamide, plasma HCO3-increased 15 meq/1 but CSF HCO3-increased 6 meq/1. Brain and CSF ammonia increased linearly and selectively with the increase in the relative contribution of CNS HCO3-increase. Therefore regulation of CSF HCO3-in respiratory acidosis depends on both components of the dual contribution theory, where each component can provide the total CSF HCO3-increase under appropriate experimental conditions. The control mechanism may be sensitive to changes in [H+] on the brain side of the blood-brain barrier.     

HCO3- secretion in the esophageal submucosal glands

The mammalian esophagus has the capacity to secrete a HCO(3)(-) and mucin-rich fluid in the esophageal lumen. These secretions originate from the submucosal glands (SMG) and can contribute to esophageal protection against refluxed gastric acid. The cellular mechanisms by which glandular cells achieve these secretions are largely unknown. To study this phenomenon, we used the pH-stat technique to measure luminal alkali secretion in an isolated, perfused pig esophagus preparation. Immunohistochemistry was used to localize receptors and transporters involved in HCO(3)(-) transport. The SMG-bearing esophagus was found to have significant basal alkali secretion, predominantly HCO(3)(-), which averaged 0.21 +/- 0.04 microeq.h(-1).cm(-2). This basal secretion was doubled when stimulated by carbachol but abolished by HCO(3)(-) or Cl(-) removal. Basal- and carbachol-stimulated secretions were also blocked by serosal application of atropine, pirenzipine, DIDS, methazolamide, and ethoxzolamide. The membrane-impermeable carbonic anhydrase inhibitor benzolamide, applied to the serosal bath, partially inhibited basal HCO(3)(-) secretion and blocked the stimulation by carbachol. Immunohistochemistry using antibodies to M(1) cholinergic receptor or carbonic anhydrase-II enzyme showed intense labeling of duct cells and serous demilunes but no labeling of mucous cells. Labeling with an antibody to Na(+)-(HCO(3)(-))(n) (rat kidney NBC) was positive in ducts and serous cells, whereas labeling for Cl(-)/HCO(3)(-) exchanger (AE2) was positive in duct cells but less pronounced in serous cells. These data indicate that duct cells and serous demilunes of SMG play a role in HCO(3)(-) secretion, a process that involves M(1) cholinergic receptor stimulation. HCO(3)(-) transport in these cells is dependent on cytosolic and serosal membrane-bound carbonic anhydrase. HCO(3)(-) secretion is also dependent on serosal Cl(-) and is mediated by DIDS-sensitive transporters, possibly NBC and AE2.     

Effects of SITS, an anion transport blocker, on CSF ionic composition in metabolic alkalosis

Disulfonic stilbenes combine with the carrier protein involved in anion transport and inhibit the exchange of Cl- for HCO3- in a variety of biomembranes. Our aim was to determine whether such a mechanism is operative in the regulation of cerebrospinal fluid (CSF) [HCO3-] in metabolic alkalosis. In anesthetized, curarized, and artificially ventilated dogs either mock CSF (group I, 9 dogs) or mock CSF containing SITS, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (group II, 7 dogs) was periodically injected into both lateral cerebral ventricles. During 6 h of isocapnic metabolic alkalosis, produced by intravenous infusion of Na2CO3 solution, plasma [HCO3-] was increased by approximately 14 meq/l in both groups. In SITS-treated animals the mean cisternal CSF [HCO3-] increased by 7.7 meq/l after 6 h, and this was significantly higher than the respective increment, 3.5 meq/l, noted in the control group. Increments in CSF [HCO3-] in both groups were reciprocated by decrements in CSF [Cl-] with CSF [Na+] remaining unchanged. Cisternal CSF PCO2 and lactate concentrations showed similar increments in both groups. It is hypothesized that in metabolic alkalosis a carrier transports HCO3- out of cerebral fluid in exchange for Cl- and that SITS inhibits this mechanism. The efflux of HCO3- out of CSF in metabolic alkalosis would minimize the rise in CSF [HCO3-] brought about by HCO3-] influx from blood into CSF and therefore contributes to the CSF [H+] homeostasis.     

Colonic anion secretory defects and metabolic acidosis in mice lacking the NBC1 Na+/HCO3- cotransporter

The NBC1 Na+/HCO3- cotransporter is expressed in many tissues, including kidney and intestinal epithelia. NBC1 mutations cause proximal renal tubular acidosis in humans, consistent with its role in HCO3- absorption in the kidney. In intestinal and colonic epithelia, NBC1 localizes to basolateral membranes and is thought to function in anion secretion. To test the hypothesis that NBC1 plays a role in transepithelial HCO3- secretion in the intestinal tract, null mutant (NBC1-/-) mice were prepared by targeted disruption of its gene (Slc4a4). NBC1-/- mice exhibited severe metabolic acidosis, growth retardation, reduced plasma Na+, hyperal-dosteronism, splenomegaly, abnormal dentition, intestinal obstructions, and death before weaning. Intracellular pH (pH(i)) was not altered in cAMP-stimulated epithelial cells of NBC1-/- cecum, but pH(i) regulation during sodium removal and readdition was impaired. Bioelectric measurements of NBC1-/- colons revealed increased amiloride-sensitive Na+ absorption. In Ringer solution containing both Cl- and HCO3-, the magnitude of cAMP-stimulated anion secretion was normal in NBC1-/- distal colon but increased in proximal colon, with the increase largely supported by enhanced activity of the basolateral NKCC1 Na+-K+-2Cl- cotransporter. Anion substitution studies in which carbonic anhydrase was inhibited and transepithelial anion conductance was limited to HCO3- revealed a sharp decrease in both cAMP-stimulated HCO3- secretion and SITS-sensitive current in NBC1-/- proximal colon. These results are consistent with the known function of NBC1 in HCO3- absorption in the kidney and demonstrate that NBC1 activity is a component of the basolateral mechanisms for HCO3- uptake during cAMP-stimulated anion secretion in the proximal colon.     

Effect of short-chain fatty acids on cyclic 3',5'-guanosine monophosphate-mediated colonic secretion

Short chain fatty acids (SCFA) prevent and reverse cyclic 3',5'-adenosine monophosphate (cAMP) but not Ca(2+)-mediated Cl- secretion. Mucosal [HCO3-]i has an opposite effect on these secretagogues. We examined whether SCFA and [HCO3-]i affect cyclic 3',5'-guanosine monophosphate (cGMP)-induced secretion. Stripped segments of male Sprague-Dawley rat (Rattus norvegicus) proximal and distal colon, and cultured T84 cells were studied in Using chambers, and pHi and [HCO3-]i were determined. Mucosal [cGMP] was measured in proximal colon. In T84 cells, the increase in Cl- secretion (measured as Isc) induced by mucosal 0.25 microM Escherichia coli heat-stable enterotoxin (STa) was prevented/reversed by bilateral 50 mM Na+ butyrate (71%/73%), acetate (58%/76%), propionate (68%/73%) and (poorly metabolized) isobutyrate (80%/79%). In proximal colon in HCO3- Ringer, basal Cl- secretion was not affected by [HCO3-]i or 25 mM butyrate. Mucosal 0.25 microM STa decreased net Na+ and Cl- absorption. Bilateral but not mucosal 25 mM SCFA reversed STa-induced effects on Na+ absorption and Cl- secretion. Bilateral and mucosal 25 mM SCFA but not [HCO3-]i prevented STa-induced Cl- secretion and increases in mucosal [cGMP]. STa did not produce Cl- secretion in distal colon. It was concluded that SCFA but not [HCO3-]i can prevent and reverse cGMP-induced colonic Cl- secretion.     

Chloride-induced increment in short-circuiting current of the turtle bladder. Effects of in-vivo acid-base state

Evidence for the participation of conductive and non-conductive (exchange) transmembrane anion pathways in the luminal acidification, alkalinization, and chloride-reabsorptive functions of the turtle bladder is provided from the pattern of Cl- -induced changes in transepithelial electrical parameters of isolated urinary bladders from three groups of donor turtles: control or post-absorptive turtles (those killed 5 days after feeding); acidotic turtles (NH4Cl-loaded); and alkalotic turtles (NaHCO3-loaded). The predominance of each of the three aforementioned transport functions as well as the response to Cl- -addition is altered by the in-vivo electrolyte balance of the turtle. In post-absorptive bladders, which are poised for acidification and Cl- reabsorption, the mucosal and serosal addition of Cl- to Na+-free, (HCO3- + CO2)-containing media increases the negative short-circuiting current (Isc). In acidotic bladders, which are poised for acidification but not Cl- reabsorption, mucosal Cl- addition has no effect on this Isc whereas serosal Cl- addition increases the negative Isc in a manner identical to that observed in the post-absorptive bladders. Alkalotic bladders do not possess an acidification function but instead are poised for Cl- reabsorption and cAMP-dependent electrogenic alkali secretion (positive Isc). In these bladders, serosal Cl- addition is without effect while mucosal Cl- addition produces transient changes in this positive Isc. It is found that these results can be replicated by a model of the turtle bladder in which transmembrane Cl- and HCO3- conductive and exchange paths mediate transepithelial acidification, alkalinization and Cl- reabsorption.     

Effects of feeding on metabolism, gas transport, and acid-base balance in the bullfrog Rana catesbeiana

Massive feeding in ectothermic vertebrates causes changes in metabolism and acid-base and respiratory parameters. Most investigations have focused on only one aspect of these complex changes, and different species have been used, making comparison among studies difficult. The purpose of the present study was, therefore, to provide an integrative study of the multiple physiological changes taking place after feeding. Bullfrogs (Rana catesbeiana) partly submerged in water were fed meals (mice or rats) amounting to approximately (1)/(10) of their body weight. Oxygen consumption increased and peaked at a value three times the predigestive level 72-96 h after feeding. Arterial PO(2) decreased slightly during digestion, whereas hemoglobin-bound oxygen saturation was unaffected. Yet, arterial blood oxygen content was pronouncedly elevated because of a 60% increase in hematocrit, which appeared mediated via release of red blood cells from the spleen. Gastric acid secretion was associated with a 60% increase in plasma HCO3(-) concentration ([HCO3(-)]) 48 h after feeding. Arterial pH only increased from 7.86 to 7.94, because the metabolic alkalosis was countered by an increase in PCO(2) from 10.8 to 13.7 mm Hg. Feeding also induced a small intracellular alkalosis in the sartorius muscle. Arterial pH and HCO3(-) returned to control values 96-120 h after feeding. There was no sign of anaerobic energy production during digestion as plasma and tissue lactate levels remained low and intracellular ATP concentration stayed high. However, phosphocreatine was reduced in the sartorius muscle and ventricle 48 h after feeding.     

Carbonic anhydrase inhibitors: antisecretory and cytoprotective properties

Experimental studies located carbonic anhydrase (CA) in the parietal cells close to secretory canaliculi, in superficial epithelial cells and gastric microvasculature. The role of CA is CO2 hydration resulting H+ for acid secretion and conversion of OH into HCO3-. Our studies showed that the physiological secretagogue histamine, acetylcholine and gastrin are all CA activators, achieving potentiating interactions. Catecholamines are also strong enzymatic activators. Beside sulfonamides, other CA inhibitors are anticholinergics, PGE and PGI2, some calcium channel blockers, alpha 2- and beta 1-adrenoceptor blockers and Zn2+. Cytoprotective properties of CA inhibitors gained experimental evidence in the past years. These effects could be based on increase of gastric mucosal blood flow, proved experimentally, which might be mediated by increase of endogenous prostaglandin synthesis and sulfhydryls and, respectively, motility changes. The unique combination of strong antisecretory effect with the cytoprotective action explain the outstanding clinical efficacy of CA inhibitors in the healing of gastric and duodenal ulcers.     

Effects of endothelin-1 on duodenal bicarbonate secretion and mucosal integrity in rats

Effects of endothelin-1 on gastric acid secretion, duodenal HCO3- secretion, and duodenal mucosal integrity were investigated in anesthetized rats, in comparison with those of TY-10957, a stable analogue of prostacyclin. A rat stomach mounted on an ex-vivo chamber or a proximal duodenal loop was perfused with saline, and gastric acid or duodenal HCO3- secretion was measured using a pH-stat method and by adding 100 mM NaOH or 10 mM HCl, respectively. Duodenal lesions were induced by mepirizole (200 mg/kg) given subcutaneously. Intravenous administration of endothelin-1 (0.6 and 1 nmol/kg) caused an increase of duodenal HCO3- secretion with concomitant elevation of blood pressure; this effect was antagonized by co-administrahon of BQ-123 (ET(A) antagonist; 3 mg/kg, i.v.) and significantly mitigated by vagotomy. Likewise, endothelin-1 caused a significant decrease in histamine-stimulated acid secretion, and this effect was also significantly antagonized by BQ-123. Although TY-10957 (10 and 30 mg/kg, i.v.) produced a temporal decrease of blood pressure, this agent caused not only an increase of duodenal HCO3- secretion, independent of vagal nerves, but also a decrease of acid secretion as well. In addition, both endothelin-1 and TY-10957 significantly prevented mepirizole-induced duodenal lesions at the doses that caused an increase of duodenal HCO3- secretion and a decrease of gastric acid secretion. These results suggest that endothelin-1 affects the duodenal mucosal integrity by modifying both gastric acid and duodenal HCO3- secretions, the effects being mediated by ET(A) receptors.     

Novel role for pendrin in orchestrating bicarbonate secretion in cystic fibrosis transmembrane conductance regulator (CFTR)-expressing airway serous cells

In most HCO(3)(-)-secreting epithelial tissues, SLC26 Cl(-)/HCO(3)(-) transporters work in concert with the cystic fibrosis transmembrane conductance regulator (CFTR) to regulate the magnitude and composition of the secreted fluid, a process that is vital for normal tissue function. By contrast, CFTR is regarded as the only exit pathway for HCO(3)(-) in the airways. Here we show that Cl(-)/HCO(3)(-) anion exchange makes a major contribution to transcellular HCO(3)(-) transport in airway serous cells. Real-time measurement of intracellular pH from polarized cultures of human Calu-3 cells demonstrated cAMP/PKA-activated Cl(-)-dependent HCO(3)(-) transport across the luminal membrane via CFTR-dependent coupled Cl(-)/HCO(3)(-) anion exchange. The pharmacological and functional profile of the luminal anion exchanger was consistent with SLC26A4 (pendrin), which was shown to be expressed by quantitative RT-PCR, Western blot, and immunofluorescence. Pendrin-mediated anion exchange activity was confirmed by shRNA pendrin knockdown (KD), which markedly reduced cAMP-activated Cl(-)/HCO(3)(-) exchange. To establish the relative roles of CFTR and pendrin in net HCO(3)(-) secretion, transepithelial liquid secretion rate and liquid pH were measured in wild type, pendrin KD, and CFTR KD cells. cAMP/PKA increased the rate and pH of the secreted fluid. Inhibiting CFTR reduced the rate of liquid secretion but not the pH, whereas decreasing pendrin activity lowered pH with little effect on volume. These results establish that CFTR predominately controls the rate of liquid secretion, whereas pendrin regulates the composition of the secreted fluid and identifies a critical role for this anion exchanger in transcellular HCO(3)(-) secretion in airway serous cells.     

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(+).     

Structural adaptation of intercalated cells in rat renal cortex to acute metabolic acidosis and alkalosis

The structural responses of cells in the distal convoluted, connecting, and collecting tubule to acute acid/base changes were investigated by electron microscopy. Acute metabolic acidosis was induced by administration of ammonium chloride, and acute metabolic alkalosis by potassium or sodium bicarbonate. Morphometric analyses were performed on micrographs of randomly selected distal nephron cells. No structural responses were found in distal convoluted tubule cells, connecting tubule cells, or principal cells but prominent changes were observed in intercalated cells (I cells). Thus, the surface density of the luminal membrane in I cells was significantly higher in acidotic animals and lower in KHCO3 alkalotic animals than in controls. On the contrary, the surface density of the membrane that bounds apical vesicles was higher in KHCO3 alkalotic and lower in acidotic animals than in controls. These results suggest that the luminal membrane is internalized during alkalosis and that the membrane that bounds apical vesicles is transferred to the luminal membrane during acidosis. Since a proton translocating ATPase may be present in the luminal membrane the observations are consistent with the possibility that cortical I cells participate in the maintenance of acid/base homeostasis.