HCO3 Secretion Research Articles

HCO3− Secretion by Murine Nasal Submucosal Gland Serous Acinar Cells during Ca2+-stimulated Fluid Secretion

Airway submucosal glands contribute to airway surface liquid (ASL) composition and volume, both important for lung mucociliary clearance. Serous acini generate most of the fluid secreted by glands, but the molecular mechanisms remain poorly characterized. We previously described cholinergic-regulated fluid secretion driven by Ca2+-activated Cl− secretion in primary murine serous acinar cells revealed by simultaneous differential interference contrast (DIC) and fluorescence microscopy. Here, we evaluated whether Ca2+-activated Cl− secretion was accompanied by secretion of HCO3−, possibly a critical ASL component, by simultaneous measurements of intracellular pH (pHi) and cell volume. Resting pHi was 7.17 ± 0.01 in physiological medium (5% CO2–25 mM HCO3−). During carbachol (CCh) stimulation, pHi fell transiently by 0.08 ± 0.01 U concomitantly with a fall in Cl− content revealed by cell shrinkage, reflecting Cl− secretion. A subsequent alkalinization elevated pHi to above resting levels until agonist removal, whereupon it returned to prestimulation values. In nominally CO2–HCO3−-free media, the CCh-induced acidification was reduced, whereas the alkalinization remained intact. Elimination of driving forces for conductive HCO3− efflux by ion substitution or exposure to the Cl− channel inhibitor niflumic acid (100 μM) strongly inhibited agonist-induced acidification by >80% and >70%, respectively. The Na+/H+ exchanger (NHE) inhibitor dimethylamiloride (DMA) increased the magnitude (greater than twofold) and duration of the CCh-induced acidification. Gene expression profiling suggested that serous cells express NHE isoforms 1–4 and 6–9, but pharmacological sensitivities demonstrated that alkalinization observed during both CCh stimulation and pHi recovery from agonist-induced acidification was primarily due to NHE1, localized to the basolateral membrane. These results suggest that serous acinar cells secrete HCO3− during Ca2+-evoked fluid secretion by a mechanism that involves the apical membrane secretory Cl− channel, with HCO3− secretion sustained by activation of NHE1 in the basolateral membrane. In addition, other Na+-dependent pHi regulatory mechanisms exist, as evidenced by stronger inhibition of alkalinization in Na+-free media.

Phosphodiesterase Isozymes Involved in Regulation of Secretion in Isolated Mouse Stomach in Vitro

(+/-)-(E)-4-Ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide] (NOR-3), a nitric-oxide (NO) donor, is known to increase HCO(3)(-) secretion in rat stomachs, intracellularly mediated by cGMP; yet, there is no information about the phosphodiesterase (PDE) isozyme involved in this process. We examined the effects of various isozyme-selective PDE inhibitors on the secretion of HCO(3)(-) in the mouse stomach in vitro and the type(s) of PDE isozymes involved in the response to NO. The gastric mucosa of DDY mice was stripped of the muscle layer and mounted on an Ussing chamber. HCO(3)(-) secretion was measured at pH 7.0 using a pH-stat method and by adding 2 mM HCl. NOR-3, 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP), and various PDE inhibitors were added to the serosal side. Vinpocetine (PDE1 inhibitor) or zaprinast (PDE5 inhibitor) was also added serosally 30 min before NOR-3 or 8-Br-cGMP. Both NOR-3 and 8-Br-cGMP stimulated HCO(3)(-) secretion in a dose-dependent manner, and the response to NOR-3 was significantly inhibited by methylene blue. Likewise, the secretion induced by NOR-3 or 8-Br-cGMP was significantly attenuated by 6-((2S,3S)-3-(4-chloro-2-methylphenylsulfonylaminomethyl)-bicyclo(2.2.2)octan-2-yl)-5Z-hexenoic acid (ONO-8711), the PGE receptor (EP)1 antagonist, as well as indomethacin and potentiated by both vinpocetine and zaprinast at doses that had no effect by themselves on the basal secretion, whereas other subtype-selective PDE inhibitors had no effect. NOR-3 increased the mucosal PGE(2) content in a methylene blue-inhibitable manner. These results suggest that NO stimulates gastric HCO(3)(-) secretion mediated intracellularly by cGMP and modified by both PDE1 and PDE5, and this response is finally mediated by endogenous PGE(2) via the activation of EP1 receptors.

Hormonal and purinergic stimulation of bicarbonate secretion in oviducts of rhesus monkey

Because an increase in the HCO(3)(-) concentration of oviductal liquid at midcycle is believed to markedly enhance fertility, we have studied active secretion of HCO(3)(-) across highly differentiated cultures of monkey oviductal epithelium. Cultured cell sheets were mounted in Ussing chambers and bathed in medium containing 25 mM HCO(3)(-). Purinergic agents potently stimulated short-circuit current (I(sc)) with an initial transient response declining within approximately 2 min to a sustained response. The potency sequence of ATP approximately UTP > ADP >> AMP suggested that the I(sc) response was mediated mainly by P2Y(2) receptors. Acetazolamide, an inhibitor of carbonic anhydrase, had little or no effect on baseline I(sc) or the transient response to ATP but abolished the sustained response to ATP. Similar results were obtained on sheets of native epithelium. In pH-stat experiments, the abluminal medium of cell cultures was bathed in HCO(3)(-)-CO(2) medium, and the pH of the unbuffered luminal medium was maintained at approximately 7.4 by addition of strong acid or base. ATP stimulated base secretion, and this was inhibited by acetazolamide. Furthermore, these changes in secretion of base were in good quantitative agreement with the I(sc) responses. When phenol red (an estrogen) was removed from the culture medium, ATP-dependent HCO(3)(-) secretion was markedly reduced but could be restored by treatment with estradiol. Estrogens also markedly increased ciliation of the cultures. These results suggest that the midcycle increase in the HCO(3)(-) concentration of oviductal liquid may be mediated by the effects of estradiol on purinergic pathways or on ATP secretion.

Identification of intestinal bicarbonate transporters involved in formation of carbonate precipitates to stimulate water absorption in marine teleost fish

Marine teleost fish precipitate divalent cations as carbonate deposits in their intestine to minimize the potential for excessive calcium entry and to stimulate water absorption by removing osmolyte ions. This carbonate deposit formation therefore plays an important role in osmoregulation in the seawater environment and also requires controlled secretion of HCO3− to match the amount of calcium entering the intestinal lumen. Despite this physiological importance, the process of carbonate precipitate formation has not been characterized at the molecular level. We first isolated 24 candidate cDNA clones representing all possible membrane proteins capable of transporting HCO3− from the euryhaline pufferfish, mefugu Takifugu obscurus based on the DNA sequence information on the T. rubripes genome, then performed Northern blot analyses to select clones highly expressed in the intestine of seawater mefugu, and finally determined the cellular locations by immunohistochemistry. Based on these observations, we proposed a model for HCO3− excretion across the intestinal epithelium of marine teleost fish that includes a basolateral NBCe1 (Na+/bicarbonate cotransporter) and apical Slc26a6 (Cl−/HCO3− exchangers).

Transport activities involved in intracellular pH recovery following acid and alkali challenges in rat brain microvascular endothelial cells

Transport activities involved in intracellular pH (pH(i)) recovery after acid or alkali challenge were investigated in cultured rat brain microvascular endothelial cells by monitoring pH(i) using a pH-sensitive dye. Following relatively small acid loads with pH(i) approximately 6.5, HCO(-)(3) influx accounted for most of the acid extrusion from the cell with both Cl(-)-independent and Cl(-)-dependent, Na(+)-dependent transporters involved. The Cl(-)-independent component has the same properties as the NBC-like transporter previously shown to account for most of the acid extrusion near the resting pH(i). Following large acid loads with pH(i) < 6.5, most of the acid extrusion was mediated by Na(+)/H(+) exchange, the rate of which was steeply dependent on pH(i). Concanamycin A, an inhibitor of V-type ATPase, had no effect on the rates of acid extrusion. Following an alkali challenge, the major component of the acid loading leading to recovery of pH(i) occurred by Cl(-)/HCO(-)(3) exchange. This exchange had the same properties as the AE-like transporter previously identified as a major acid loader near resting pH(i). These acid-loading and acid-extruding transport mechanisms together with the Na(+), K(+), ATPase may be sufficient to account not only for pH(i) regulation in brain endothelial cells but also for the net secretion of HCO(-)(3) across the blood-brain barrier.

Identification of intestinal bicarbonate transporters involved in formation of carbonate precipitates to stimulate water absorption in marine teleost fish

Marine teleost fish precipitate divalent cations as carbonate deposits in the intestine to minimize the potential for excessive Ca2+ entry and to stimulate water absorption by reducing luminal osmotic pressure. This carbonate deposit formation, therefore, helps maintain osmoregulation in the seawater (SW) environment and requires controlled secretion of HCO3(-) to match the amount of Ca2+ entering the intestinal lumen. Despite its physiological importance, the process of HCO3(-) secretion has not been characterized at the molecular level. We analyzed the expression of two families of HCO3(-) transporters, Slc4 and Slc26, in fresh-water- and SW-acclimated euryhaline pufferfish, mefugu (Takifugu obscurus), and obtained the following candidate clones: NBCe1 (an Na+-HCO3(-) cotransporter) and Slc26a6A and Slc26a6B (putative Cl(-)/HCO3(-) exchangers). Heterologous expression in Xenopus oocytes showed that Slc26a6A and Slc26a6B have potent HCO3(-)-transporting activity as electrogenic Cl(-)/nHCO3(-) exchangers, whereas mefugu NBCe1 functions as an electrogenic Na+-nHCO3(-) cotransporter. Expression of NBCe1 and Slc26a6A was highly induced in the intestine in SW and expression of Slc26a6B was high in the intestine in SW and fresh water, suggesting their involvement in HCO3(-) secretion and carbonate precipitate formation. Immunohistochemistry showed staining on the apical (Slc26a6A and Slc26a6B) and basolateral (NBCe1) membranes of the intestinal epithelial cells in SW. We therefore propose a mechanism for HCO3(-) transport across the intestinal epithelial cells of marine fish that includes basolateral HCO3(-) uptake (NBCe1) and apical HCO3(-) secretion (Slc26a6A and Slc26a6B).

A critical analysis of carbonic anhydrase function, respiratory gas exchange, and the acid-base control of secretion in the rectal gland of Squalus acanthias

We compared in vivo responses of rectal gland secretion to carbonic anhydrase (CA) inhibition (10(-4) mol l(-1) acetazolamide) in volume-loaded dogfish with in vitro responses in an isolated-perfused gland stimulated with 5 x 10(-6) mol l(-1) forskolin and removed from systemic influences. We also measured respiratory gas exchange in the perfused gland, described the acid-base status of the secreted fluid, and determined the relative importance of various extracellular and intracellular acid-base parameters in controlling rectal gland secretion in vitro. In vivo, acetazolamide inhibited Cl(-) secretion and decreased pHi in the rectal gland, but interpretation was confounded by an accompanying systemic respiratory acidosis, which would also have contributed to the inhibition. In the perfused gland, M(CO(2)) and M(O(2)) increased in linear relation to increases in Cl(-) secretion rate. CA inhibition (10(-4) mol l(-1) acetazolamide) had no effect on Cl(-) secretion rate or pHi in the perfused gland, in contrast to in vivo, but caused a transitory 30% inhibition of M(CO(2)) (relative to stable M(O(2))) and elevation in secretion P(CO(2)) effects, which peaked at 2 h and attenuated by 3.5-4 h. Secretion was inhibited by acidosis and stimulated by alkalosis; the relationship between relative Cl(-) secretion rate and pHe was almost identical to that seen in vivo. Experimental manipulations of perfusate pH, P(CO(2)) and HCO(3)(-) concentration, together with measurements of pHi, demonstrated that these responses were most strongly correlated with changes in pHe, and were not related to changes in P(CO(2)), extracellular HCO(3)(-), or intracellular HCO(3)(-) levels, though changes in pHi may also have played a role. The acid-base status of the secreted fluid varied with that of the perfusate, secretion pH remaining about 0.3-0.5 units lower, and changing in concert with pHe rather than pHi; secretion HCO(3)(-) concentrations remained low, even in the face of greatly elevated perfusate HCO(3)(-) concentrations. We conclude that pH effects on rectal gland secretion rate are adaptive, that CA functions to catalyze the hydration of CO(2), thereby maintaining a gradient for diffusive efflux of CO(2) from the working cells, and that differences in response to CA inhibition likely reflect the higher perfusion-to-secretion ratio in vitro than in vivo.

5‐Hydroxytryptamine contributes significantly to a reflex pathway by which the duodenal mucosa protects itself from gastric acid injury

Although duodenal mucosal bicarbonate secretion (DMBS) is currently accepted as an important defense mechanism against acid-induced duodenal injury, the mechanism and the regulation of DMBS are largely unknown. 5-HT may regulate DMBS, but little is known about its physiological relevance in DMBS and the underlying mechanism(s). Thus, the aims of the present study were to demonstrate the role of 5-HT in acid-stimulated DMBS and to further elucidate the precise mechanisms involved in this process. Luminal acid stimulation significantly increased 5-HT release from the duodenal mucosa (P<0.01). SB204070, a selective 5-HT4 receptor antagonist, dose-dependently reduced luminal acid-stimulated HCO3(-) secretion of mice in vivo. In Ussing chamber studies, 5-HT-induced I(SC) and DMBS were abolished by removal of extracellular Ca2+, and significantly attenuated by pharmacological blockade of the Na+/Ca2+ exchanger (NCX), intermediate Ca2+-activated K+ channels (IK(Ca)), or cystic fibrosis transmembrane conductance regulator (CFTR). 5-HT increased cytoplasmic free calcium ([Ca2+]cyt) in SCBN cells, a duodenal epithelial cell line, and knockdown of NCX1 proteins with a specific siRNA greatly decreased this 5-HT-mediated Ca2+ signaling. Taken together, our data suggest that 5-HT plays a physiological role in acid-stimulated DMBS via a Ca2+ signaling pathway, in which the plasma membrane NCX transporter as well as IK(Ca) and CFTR channels may be involved.

Transporters involved in regulation of intracellular pH in primary cultured rat brain endothelial cells

Fluid secretion across the blood-brain barrier, critical for maintaining the correct fluid balance in the brain, entails net secretion of HCO(3)(-), which is brought about by the combined activities of ion transporters situated in brain microvessels. These same transporters will concomitantly influence intracellular pH (pH(i)). To analyse the transporters that may be involved in the maintenance of pH(i) and hence secretion of HCO(3)(-), we have loaded primary cultured endothelial cells derived from rat brain microvessels with the pH indicator BCECF and suspended them in standard NaCl solutions buffered with Hepes or Hepes plus 5% CO(2)/HCO(3)(-). pH(i) in the standard solutions showed a slow acidification over at least 30 min, the rate being less in the presence of HCO(3)(-) than in its absence. However, after accounting for the difference in buffering, the net rates of acid loading with and without HCO(3)(-) were similar. In the nominal absence of HCO(3)(-) the rate of acid loading was increased equally by removal of external Na(+) or by inhibition of Na(+)/H(+) exchange by ethylisopropylamiloride (EIPA). By contrast, in the presence of HCO(3)(-) the increase in the rate of acid loading when Na(+) was removed was much larger and the rate was then also significantly greater than the rate observed in the absence of both Na(+) and HCO(3)(-). Removal of Cl(-) in the presence of HCO(3)(-) produced an alkalinization followed by a resumption of the slow acid gain. Removal of Na(+) following removal of Cl(-) increased the rate of acid gain. In the presence of HCO(3)(-) and initial presence of Na(+) and Cl(-), DIDS inhibited the changes in pH(i) produced by removal of either Na(+) or Cl(-). These are the expected results if these cells possess an AE-like Cl(-)/HCO(3)(-) exchanger, a 'channel-like' permeability allowing slow influx of acid (or efflux of HCO(3)(-)), a NBC-like Cl(-)-independent Na(+)-HCO(3)(-) cotransporter, and a NHE-like Na(+)/H(+) exchanger. The in vitro rates of HCO(3)(-) loading via the Na(+)-HCO(3)(-) cotransporter could, if the transporter is located on the apical, blood-facing side of the cells, account for the net secretion of HCO(3)(-) into the brain.

Chloride Uptake and Base Secretion in Freshwater Fish: A Transepithelial Ion‐Transport Metabolon?

Despite all the efforts and technological advances during the last few decades, the cellular mechanisms for branchial chloride uptake in freshwater (FW) fish are still unclear. Although a tight 1 : 1 link with HCO-3 secretion has been established, not much is known about the identity of the ion-transporting proteins involved or the energizing steps that allow for the inward transport of Cl- against the concentration gradient. We propose a new model for Cl- uptake in FW fish whereby the combined action of an apical anion exchanger, cytoplasmic carbonic anhydrase, and basolateral V-type H+ -ATPase creates a local [HCO-3] high enough to energize Cl- uptake. Our model is based on analyses of structure-function relationships, reinterpretation of previous results, and novel observations about gill cell subtypes and immunolocalization of the V-H+ -ATPase.

Epithelial carbonic anhydrases facilitate PCO2 and pH regulation in rat duodenal mucosa

The duodenum is the site of mixing of massive amounts of gastric H+ with secreted HCO3-, generating CO2 and H2O accompanied by the neutralization of H+. We examined the role of membrane-bound and soluble carbonic anhydrases (CA) by which H+ is neutralized, CO2 is absorbed, and HCO3- is secreted. Rat duodena were perfused with solutions of different pH and PCO2 with or without a cell-permeant CA inhibitor methazolamide (MTZ) or impermeant CA inhibitors. Flow-through pH and PCO2 electrodes simultaneously measured perfusate and effluent pH and PCO2. High CO2 (34.7 kPa) perfusion increased net CO2 loss from the perfusate compared with controls (pH 6.4 saline, PCO2 approximately 0) accompanied by portal venous (PV) acidification and PCO2 increase. Impermeant CA inhibitors abolished net perfusate CO2 loss and increased net HCO3- gain, whereas all CA inhibitors inhibited PV acidification and PCO2 increase. The changes in luminal and PV pH and [CO2] were also inhibited by the Na+-H+ exchanger-1 (NHE1) inhibitor dimethylamiloride, but not by the NHE3 inhibitor S3226. Luminal acid decreased total CO2 output and increased H+ loss with PV acidification and PCO2 increase, all inhibited by all CA inhibitors. During perfusion of a 30% CO2 buffer, loss of CO2 from the lumen was CA dependent as was transepithelial transport of perfused 13CO2. H+ and CO2 loss from the perfusate were accompanied by increases of PV H+ and tracer CO2, but unchanged PV total CO2, consistent with CA-dependent transmucosal H+ and CO2 movement. Inhibition of membrane-bound CAs augments the apparent rate of net basal HCO3- secretion. Luminal H+ traverses the apical membrane as CO2, is converted back to cytosolic H+, which is extruded via NHE1. Membrane-bound and cytosolic CAs cooperatively facilitate secretion of HCO3- into the lumen and CO2 diffusion into duodenal mucosa, serving as important acid-base regulators.

Distinct mechanisms of acid-induced HCO3−secretion in normal and slightly permeable stomachs

We investigated the regulatory mechanism of acid-induced HCO(3)(-) secretion in the slightly permeable rat stomach after an exposure to hyperosmolar NaCl. Under urethane anesthesia, a rat stomach was mounted on a chamber and perfused with saline, and the secretion of HCO(3)(-) was measured at pH 7.0 using a pH-stat method and by adding 2 mM HCl. Acidification of the normal stomach with 100 mM HCl increased HCO(3)(-) secretion, and this response was totally inhibited by pretreatment with indomethacin but not N(G)-nitro-l-arginine methyl ester (l-NAME) or chemical ablation of capsaicin-sensitive afferent neurons. Exposure of the stomach to 0.5 M NaCl deranged the unstirred mucus gel layer without damaging the surface epithelial cells. The stomach responded to 0.5 M NaCl by secreting slightly more HCO(3)(-), in an indomethacin-inhibitable manner, and responded to even 10 mM HCl with a marked rise in HCO(3)(-) secretion, although 10 mM HCl did not have an effect in the normal stomach. The acid-induced HCO(3)(-) response in the NaCl-treated stomach was significantly but partially attenuated by indomethacin, l-NAME, or sensory deafferentation and was totally abolished when these treatments were combined. These results suggest that gastric HCO(3)(-) secretion in response to acid is regulated by two independent mechanisms, one mediated by prostaglandins (PGs) and the other by sensory neurons and nitric oxide (NO). The acid-induced HCO(3)(-) secretion in the normal stomach is totally mediated by endogenous PGs, but, when the stomach is made slightly permeable to acid, the response is markedly facilitated by sensory neurons and NO.

Ouabain-sensitive bicarbonate secretion and acid absorption by the marine teleost fish intestine play a role in osmoregulation

The gulf toadfish (Opsanus beta) intestine secretes base mainly in the form of HCO3- via apical anion exchange to serve Cl- and water absorption for osmoregulatory purposes. Luminal HCO3- secretion rates measured by pH-stat techniques in Ussing chambers rely on oxidative energy metabolism and are highly temperature sensitive. At 25 degrees C under in vivo-like conditions, secretion rates averaged 0.45 micromol x cm(-2) x h(-1), of which 0.25 micromol x cm(-2) x h(-1) can be accounted for by hydration of endogenous CO2 partly catalyzed by carbonic anhydrase. Complete polarity of secretion of HCO3- and H+ arising from the CO2 hydration reaction is evident from equal rates of luminal HCO3- secretion via anion exchange and basolateral H+ extrusion. When basolateral H+ extrusion is partly inhibited by reduction of serosal pH, luminal HCO3- secretion is reduced. Basolateral H+ secretion occurs in exchange for Na+ via an ethylisopropylamiloride-insensitive mechanism and is ultimately fueled by the activity of the basolateral Na+-K+-ATPase. Fluid absorption by the toadfish intestine to oppose diffusive water loss to the concentrated marine environment is accompanied by a substantial basolateral H+ extrusion, intimately linking osmoregulation and acid-base balance.

Chloride and bicarbonate have similar affinities to the intestinal anion exchanger DRA (down regulated in adenoma)

DRA (down regulated in adenoma, SLC26A3) is an anion exchanger that mediates electroneutral NaCl absorption in the ileum and proximal colon together with NHE3 (Na/H exchanger isoform 3), and that is involved in duodenal and possibly pancreatic bicarbonate secretion. Thus, its chloride and bicarbonate affinities are important for both processes. [Cl]i and pHi transients were measured using MQAE and BCECF. HEK293 cells stably expressing DRA were exposed to 0 mM Cl at various [HCO3] (9 to 51 mM, at 5% CO2 or 15 to 57 mM, at pH 7.5) to determine the HCO3 affinity. After intracellular Cl depletion, 10, 30, and 90 mM Cl were readded at various [HCO3]s to determine the relative Cl and HCO3 affinities. The k0.5 for extracellular HCO3 is between 18.5 and 32.8 mM. Cl and HCO3 compete with similar affinities for transport by DRA. DRA activity is independent of pHo between 7.0 and 7.75. DRA is activated by alkaline pHi. Competition of Cl and HCO3 does not significantly impair NaCl absorption, because in the ileum and colon, luminal Cl is comparably high. Activation at alkaline pHi supports functional coupling of DRA and NHE3 by the subapical pHi. In the distal pancreatic ductal system, luminal HCO3 is high compared to luminal Cl. Under these conditions, competition of Cl and HCO3 is difficult to reconcile with a role of DRA in Cl reabsorption in exchange for HCO3. Our data, thus, provide indirect evidence against a role of DRA in pancreatic HCO3 secretion.

95 Bicarbonate-rich choleresis induced by secretin in normal rat is taurocholate dependent and involves AE2 anion exchanger

Canalicular bile is modified along bile ducts through reabsorptive and secretory processes regulated by nerves, bile salts, and hormones such as secretin. Secretin stimulates ductular cystic fibrosis transmembrane conductance regulator (CFTR)–dependent Cl efflux and subsequent biliary HCO3 secretion, possibly via Cl /HCO3 anion exchange (AE). However, the contribution of secretin to bile regulation in the normal rat, the significance of choleretic bile salts in secretin effects, and the role of Cl /HCO3 exchange in secretin-stimulatedHCO3 secretion all remainunclear.Here, secretinwas administered tonormal ratswithmaintainedbile acidpool via continuous taurocholate infusion. Bile flow and biliary HCO3 and Cl excretion were monitored following intrabiliary retrograde fluxes of saline solutions with and without the Cl channel inhibitor 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) or the Cl /HCO3 exchange inhibitor 4,4 -diisothiocyanatostilbene-2,2 -disulfonic acid (DIDS). Secretin increased bile flow and biliary excretion ofHCO3 andCl . Interestingly, secretin effects were not observed in the absence of taurocholate.Whereas secretin effects were all blocked by intrabiliary NPPB, DIDS only inhibited secretin-induced increases in bile flow and HCO3 excretion but not the increased Cl excretion, revealing a role of biliary Cl /HCO3 exchange in secretininduced, bicarbonate-rich choleresis in normal rats. Finally, small hairpin RNA adenoviral constructs were used to demonstrate the involvement of the Na -independent anion exchanger 2 (AE2) through gene silencing in normal rat cholangiocytes. AE2 gene silencing caused amarked inhibition of unstimulated and secretin-stimulated Cl /HCO3 exchange. In conclusion, maintenance of the bile acid pool is crucial for secretin to induce bicarbonate-rich choleresis in the normal rat and that this occurs via a chloride–bicarbonate exchange process consistent with AE2 function. (HEPATOLOGY 2006;43:266-275.)

Disruption of the SLC26A3-mediated anion transport is associated with male subfertility

Male subfertility in congenital chloride diarrhea (CLD) was possible after identification of expression of an epithelial Cl-/HCO3- exchanger SLC26A3 in the male reproductive tract and by the observation that adult men with CLD had very few children. A prospective clinical and laboratory study among eight adult Finnish men with CLD revealed constant oligoasthenoteratozoospermia but normal spermatogenesis, high chloride and low pH in seminal plasma, and three spermatoceles, suggesting that male subfertility is a clinical manifestation of CLD and could be caused by an analogous defect in the epithelial Cl-/HCO3- and water transport, as described for the CLD intestine.

Melatonin Modulates Acid/Base Transport in Human Pancreatic Carcinoma Cells

Melatonin was found to improve pancreatic organfunction in diseased animals. To study whetherpancreatic bicarbonate secretion is stimulated bymelatonin, investigations were done in two humanductal pancreatic adenocarcinoma cell lines MIAPaCa-2 (MIA) and PANC-1 (PANC). Using thefluorescence pH-sensor BCECF-AM, we monitoredmelatonin effects on basal intracellular pH (pH_i), andon pH_i recovery after intracellular alkalinizationproduced by the removal of extracellular HCO₃^-/CO₂.Exposure to 1µM melatonin for 24hrs and presenceof the indoleamine during the experiment increasesthe basal pH_i. Moreover, pHi recovery and HCO₃^-secretion are facilitated after the alkaline load. Thesefindings are in line with the observed increase in mRNAexpression of the Na⁺/HCO₃^--cotransporter SLC4A4bfor the uptake and the Cl^-/HCO₃^--exchanger SLC26A6for the secretion of HCO₃^-. The reduction in Na⁺/H⁺-exchanger SLC9A1 mRNA would favor pH_i recoveryafter alkalinization, but it does not explain the initialincrease in pHi. This controversial effect and therequirement for continuous presence of melatoninthroughout the experiment suggest that nontranscriptionalsignalling may contribute to the effectsof melatonin on acid/base movements. In summary,we show a stimulatory effect of melatonin onbicarbonate secretion in the pancreatic cancer celllines which may help to prevent duodenal damage.

Serotonin increases protective duodenal bicarbonate secretion via enteric ganglia and a 5-HT4-dependent pathway

Objective. Serotonin (5-HT) is present in much larger amounts in the gut than in the central nervous system and is predominantly synthesized and stored in mucosal enterochromaffin cells. Bicarbonate secretion by the duodenal mucosa is the major mechanism in maintaining mucosal integrity, neutralizing invading protons within the surface mucus gel. In this study the role of local 5-HT in the control of the protective secretion was investigated. Material and methods. A segment of proximal duodenum was perfused in situ in anaesthetized rats and the alkaline secretion was continuously recorded by pH-stat. Intracellular calcium signalling was measured in clusters of human and rat duodenal enterocytes devoid of neural tissue. After loading with the fluorescent probe, fura-2, the clusters were attached to the bottom of a temperature-controlled perfusion chamber. Results. Close intra-arterial infusion to the duodenal segment of 5-HT (20–200 nmol kg−1 h−1) dose-dependently increased duodenal mucosal HCO3 secretion. A higher dose (2000 nmol kg−1 h−1) did not further increase secretion. Responses were inhibited by the ganglionic blocker and nicotinic receptor antagonist hexamethonium, and were abolished by the 5-HT4 receptor antagonist SB 204070. The 5-HT3 antagonist tropisetron, in contrast, caused only slight inhibition. Viable human and rat duodenal enterocytes responded to 5-HT (100–500 nM) with an increase in intracellular calcium concentration. Pretreatment with SB 204070 or removal of external calcium abolished the response. Conclusions. Stimulation of the duodenal protective secretion by 5-HT thus involves receptors of the 5-HT4 subtype as well as nicotinic transmission, the myenteric plexus being a likely location. In addition, serotonin acts on enterocyte membrane receptors, inducing intracellular calcium signalling.

Gastric HCO3− secretion induced by mucosal acidification: Different mechanisms depending on acid concentration

We compared the HCO3- secretory responses induced by mucosal acidification at different HCl concentrations (100 and 200 mM HCl) in the rat stomach. Under urethane anesthesia, the stomach was mounted on an ex vivo chamber and perfused with saline under inhibition of acid secretion by omeprazole (60 mg/kg, i.p.). TheHCO3- secretion was measured at pH 7.0 using a pH-stat method and by adding 2 mM HCl. The acidification was performed by exposure of the mucosa to 100 mMor 200 mM HCl for 10 min. The secretion of HCO3- was increased by acidification of the mucosa at both 100 and 200 mM of HCl, and the maximal HCO3- response was 1.5-times greater at the latter concentration. The HCO3- responses induced by 100 and 200 mM HCl were both totally inhibited by prior administration of indomethacin, an inhibitor of prostaglandin (PG) production. The HCO3- stimulatory effect of 200 mM HCl was also significantly attenuated by pre-treatment with N(G)-nitro L-arginine methyl ester (L-NAME), the inhibitor of nitric oxide (NO) synthase, as well as chemical ablation of capsaicin-sensitive afferent neurons, whereas that of 100 mM HCl was affected by neither of these treatments. We conclude that the mucosal acidification stimulates gastric HCO3- secretion in different mechanisms, depending on the concentration of acid; the response caused by 100 mM HCl is mediated only by PGs, while that caused by 200 mM HCl is mediated by both capsaicin-sensitive afferent neurons and NO, in addition to PGs.

Mechanisms Underlying Capsaicin-Stimulated Secretion in the Stomach: Comparison with Mucosal Acidification

The effects of capsaicin and mucosal acidification on gastric HCO3(-) secretion were compared in wild-type and prostacyclin (PGI2) IP receptor or prostaglandin E receptor EP1 or EP3 knockout C57BL/6 mice as well as rats. Under urethane anesthesia, the stomach was mounted on an ex vivo chamber, perfused with saline, and the secretion of HCO3(-) was measured at pH 7.0 using the pH-stat method. Capsaicin or 200 mM HCl was applied to the chamber for 10 min. Capsaicin increased the secretion of HCO3(-) in rats and wild-type mice, the response at 0.3 mg/ml being equivalent to that induced by acidification. This effect of capsaicin in rats was abolished by ablation of capsaicin-sensitive afferent neurons and attenuated by indomethacin, N(G)-nitro-L-arginine methylester (L-NAME), and capsazepine [transient receptor potential vanilloid type 1 (TRPV1) antagonist] but not FR172357 [3-bromo-8-[2,6-dichloro-3-[N[(E)-4-(N,N-dimethylcarbamoyl) cinnamidoacetyl]-N-methylamino]benzyloxy]-2-metylimidazo[1,2-a]pyridine; bradykinin B2 antagonist] or the EP1 antagonist. The acid-induced HCO3(-) secretion was attenuated by indomethacin, L-NAME, the EP1 antagonist, and sensory deafferentation, but not affected by capsazepine or FR172357. Prostaglandin E2 (PGE2), NOR-3 [(+/-)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamine] (NO donor), and bradykinin stimulated the secretion of HCO3(-), and the effect of bradykinin was blocked by indomethacin and L-NAME as well as FR172357. The stimulatory effect of capsaicin disappeared in IP (-/-) mice, whereas that of acidification disappeared in EP1 (-/-) mice. Intragastric application of capsaicin increased mucosal PGI2 but not PGE2 levels in the rat stomach. These results suggested that both capsaicin and acid increase gastric HCO3(-) secretion via a common pathway, involving PG and NO as well as capsaicin-sensitive afferent neurons, yet their responses differ concerning TRPV1 or prostanoid receptor dependence.