HCO3 Secretion Research Articles

Role of Prostaglandin E Receptor Subtypes in Gastroduodenal HCO3 - Secretion

Gastroduodenal HCO3- secretion is a key process that aids in preventing acid-peptic injury. Endogenous prostaglandins (PGs) play a particularly important role in the local control of this secretion. The secretion of HCO3- in both the stomach and duodenum was increased in response to PGE2 as well as mucosal acidification, the latter occurring with concomitant enhancement of mucosal PG generation. These HCO3- responses in the duodenum were markedly reduced by prior administration of the EP4 antagonist in rats, and profoundly decreased in the animals lacking EP3 receptors but not EP1 receptors. In contrast, gastric HCO3- responses induced by PGE2 and mucosal acidification were prevented by the EP1 antagonist and disappeared in EP1, but not EP3-knockout mice. Consistent with these findings, duodenal HCO3- secretion was stimulated by both EP3 and EP4 agonists but not EP1 or EP2 agonists, while gastric HCO3- secretion was increased by the EP1 agonist but not EP2, EP3 or EP4 agonists. In addition, the HCO3- stimulatory action of sulprostone (EP1/EP3 agonist) in the stomach was inhibited by the Ca2+ antagonist verapamil but not affected by IBMX, the inhibitor of phosphodiesterase, while that in the duodenum was inhibited by verapamil and enhanced by IBMX. Forskolin, the stimulator of adenylate cyclase, increased HCO3- secretion in the duodenum but not the stomach. Thus, the HCO3- stimulatory action of PGE2 in the duodenum is mediated by both EP3 and EP4 receptors being coupled intracellularly with both Ca2+ and cAMP, while that in the stomach is mediated by EP1 receptors, coupled with Ca2+.

Dapsone impairs the bile salt-independent fraction of bile flow in rats: Possible involvement of its N-hydroxylated metabolite

The effects of dapsone (DDS) treatment (30 mg/kg body wt, twice a day, for 4 days) on biliary secretory function, with special emphasis on bile salt independent bile flow (BSIF), were investigated in male and in female Wistar rats. Because DDS is metabolized to its N-hydroxylated parent compound only in male rats, any gender difference in DDS effect can be causally attributed to this metabolite. The two main driving forces for BSIF, the biliary secretion of HCO 3 − and glutathione species, were assessed. BSIF was decreased by about 20% in male but not in female rats after DDS treatment. Basal biliary HCO 3 − secretion was decreased also by 20% in males. This was associated with a diminished (−37%) expression of the HCO 3 − canalicular transporter, anion exchanger 2 (AE2), detected by western blotting. Biliary output of reduced glutathione (GSH) was not modified by DDS irrespective of gender, whereas excretion of oxidized glutathione (GSSG) was increased by 830% in males. This latter finding confirmed a gender-dependent oxidative stress associated with formation of the N-hydroxylated metabolite of DDS. The expression of multidrug resistance-associated protein 2 (Mrp2), a putative transporter of glutathione species, was decreased by 38% as detected by western blotting, clearly dissociating from preserved or increased biliary excretion of GSH and GSSG. In conclusion, our results show an impairment of BSIF by DDS mainly due to a decreased AE2-mediated biliary excretion of HCO 3 −, formation of the N-hydroxylated metabolite of DDS being a likely mediator. The clinical relevance of these findings is discussed.

Bicarbonate Secretion

Understanding of the mechanism of colonic electrolyte transport has markedly increased over the past three decades. This article provides a brief summary of the critical features of Na, Cl, and K transport in the large intestine and how these processes may be altered in diarrhea. Less understood is the mechanism of colonic HCO3 secretion. Recent progress in the regulation of HCO3 secretion in the distal colon is summarized with emphasis on the interrelationship between Cl-dependent, short-chain fatty acid (SCFA)-dependent, and cAMP-induced HCO3 secretion. cAMP down-regulates Cl-dependent HCO3 secretion, while SCFA stimulates HCO3 secretion but also inhibits both Cl-dependent and cAMP-induced HCO3 secretion. As SCFAs are the primary anions in stool, it is likely that SCFA-dependent HCO3 secretion is the primary mechanism of HCO3 secretion in the mammalian colon. Future studies will undoubtedly provide increased understanding of the mechanism of HCO3 secretion in health and disease.

MECHANISMS OF BICARBONATE SECRETION IN THE PANCREATIC DUCT

In many species the pancreatic duct epithelium secretes HCO3- ions at a concentration of around 140 mM by a mechanism that is only partially understood. We know that HCO3- uptake at the basolateral membrane is achieved by Na+-HCO3- cotransport and also by a H+-ATPase and Na+/H+ exchanger operating together with carbonic anhydrase. At the apical membrane, the secretion of moderate concentrations of HCO3- can be explained by the parallel activity of a Cl-/HCO3- exchanger and a Cl- conductance, either the cystic fibrosis transmembrane conductance regulator (CFTR) or a Ca2+-activated Cl- channel (CaCC). However, the sustained secretion of HCO3- into a HCO- -rich luminal fluid cannot be explained by conventional Cl-/HCO3- exchange. HCO3- efflux across the apical membrane is an electrogenic process that is facilitated by the depletion of intracellular Cl-, but it remains to be seen whether it is mediated predominantly by CFTR or by an electrogenic SLC26 anion exchanger.

Mechanism of augmented duodenal HCO3−secretion after elevation of luminal CO2

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(+).

Acid and base secretion in the Calu-3 model of human serous cells.

Submucosal glands are the primary source of airway mucus, a critical component of lung innate defenses. Airway glands are defective in cystic fibrosis (CF), showing a complete absence of secretion to vasoactive intestinal peptide or forskolin, which increase intracellular cAMP concentration. This defect is attributed to gland serous cells, which express the cystic fibrosis transmembrane conductance regulator. Calu-3 cells, which mimic many features of serous cells, secrete Cl(-) and HCO(3)(-), with HCO(3)(-) secretion predominating for forskolin stimulation and Cl(-) secretion predominating for stimuli that open basolateral K(+) channels to hyperpolarize the cells. We used pH stat and ion substitution experiments to clarify the mechanisms and consequences of these two modes of secretion. We confirm that Calu-3 cells secrete primarily HCO(3)(-) in response to forskolin. Unexpectedly, HCO(3)(-) secretion continued in response to K(+) channel openers, with Cl(-) secretion being added to it. Secretion of HCO(3)(-) from hyperpolarized cells occurs via the conversion of CO(2) to HCO(3)(-) and is reduced by approximately 50% with acetazolamide. A gap between the base equivalent current and short-circuit current was observed in all experiments and was traced to secretion of H(+) via a ouabain-sensitive, K(+)-dependent process (possibly H(+)-K(+)-ATPase), which partially neutralized the secreted HCO(3)(-). The conjoint secretion of HCO(3)(-) and H(+) may help explain the puzzling finding that mucus secreted from normal and CF glands has the same acidic pH as does mucus from glands stimulated with forskolin or ACh. It may also help explain how human airway glands produce mucus that is hypotonic.

Participation of prostaglandin E receptor EP4 subtype in duodenal bicarbonate secretion in rats.

We examined, by using a specific PGE receptor subtype EP4 agonist and antagonist, the involvement of EP4 receptors in duodenal HCO(3)(-) secretion induced by PGE(2) and mucosal acidification in rats. Mucosal acidification was achieved by exposing a duodenal loop to 10 mM HCl for 10 min, and various EP agonists were given intravenously 10 min before the acidification. Secretion of HCO(3)(-) was dose-dependently stimulated by AE1-329 (EP4 agonist), the maximal response being equivalent to that induced by sulprostone (EP1/EP3 agonist) or PGE(2). The stimulatory action of AE1-329 and PGE(2) but not sulprostone was attenuated by AE3-208, a specific EP4 antagonist. This antagonist also significantly mitigated the acid-induced HCO(3)(-) secretion. Coadministration of sulprostone and AE1-329 caused a greater secretory response than either agent alone. IBMX potentiated the stimulatory action of both sulprostone and AE1-329, whereas verapamil mitigated the effect of sulprostone but not AE1-329. Chemical ablation of capsaicin-sensitive afferent neurons did not affect the response to any of the EP agonists used. We conclude that EP4 receptors are involved in the duodenal HCO(3)(-) response induced by PGE(2) or acidification in addition to EP3 receptors. The process by which HCO(3)(-) is secreted through these receptors differs regarding second-messenger coupling. Stimulation through EP4 receptors is mediated by cAMP, whereas that through EP3 receptors is regulated by both cAMP and Ca(2+); yet there is cooperation between the actions mediated by these two receptors. The neuronal reflex pathway is not involved in stimulatory actions of these prostanoids.

A not-so-basic case of metabolic alkalosis: an analysis featuring Leendert Paul and emphasizing Occam’s razor

Abstract A patient had the onset of a severe degree of metabolic alkalosis associated with a very large daily loss of diarrheal fluid. Because the expected acid-base disorder in this setting is metabolic acidosis, factors that could result in the markedly increased concentration of bicarbonate (HCO 3 − ) in plasma (Phco 3 ) were sought. In our approach, there is an emphasis on electroneutrality, mass balance, a quantitative analysis, and the application of basic concepts in physiology at the bedside. We suggest that there were 3 major factors that could explain why the Phco 3 was so high. First, a severe degree of extracellular fluid volume contraction was probably the most important factor that raised her Phco 3 . Second, there may have been an unusual way to have an electroneutral addition of HCO 3 − and removal of chloride ions from her extracellular fluid compartment. Third, the diarrheal fluid could have contained little HCO 3 − because of a down-regulated intestinal secretion of HCO 3 − . This analysis challenges the traditional pathophysiology of metabolic alkalosis.

Central nervous alpha1-adrenoceptor stimulation induces duodenal luminal release of melatonin.

Intracerebroventricular (i.c.v.) infusion of the alpha1-adrenoceptor agonist phenylephrine elicits vagal and sympathetic neural stimulation of the bicarbonate secretion by the duodenal mucosa. Melatonin originating from mucosal enterochromaffin cells (EC cells) has been proposed to mediate this centrally elicited stimulation. However, the release of intestinal melatonin has not been studied. Rats were anesthetized with thiobarbiturate, a 12-mm segment of duodenum with intact blood supply was cannulated in situ and bicarbonate secretion titrated by pH-stat. The mean arterial blood pressure was continuously recorded. Melatonin in the duodenal luminal perfusate was determined by high-performance liquid chromatography with electrochemical detection. Intracerebroventricular infusion of phenylephrine (12.2 microM/kg/hr) induced more than 10-fold increase in release of melatonin into the duodenal lumen and an increase in HCO secretion from 7.6 +/- 0.5 to 18.6 +/- 2.1 microEq/cm/hr. The melatonin receptor (MT2 > MT1) antagonist luzindole (600 nM/kg, i.v.) almost abolished the marked rise in bicarbonate secretion induced by i.c.v. phenylephrine but, in contrast, did not affect the release of melatonin. These results strongly suggest that release of melatonin from the mucosa mediates the duodenal secretory response to centrally elicited neural stimulation.

Targeted disruption of cftr increases the resistance of duodenal epithelial cells to luminal acid challenge in mice

indomethacin-sensitive manner. In lP (-/-) mice, however, the HCOf secretion was similarly increased in response to acid but not capsaicin. By contrast, the acid-induced duodenal HCO3 secretion was profoundly mitigated in EP3 (4-) mice while capsaicin increased the HCO3 secretion in these knockout mice. In addition, cicaprost, the PG12 agonlst, increased HCO3 secretion in both WT ( + / + ) and EP3 (4-) mice, but this response disappeared in IP (4-) animals. Conclusion: The presence of EP3 but not IP receptors is essential for acidinduced duodenal HCOf secretion in mice, although activation of 1P receptors results in stimulation of the HCOf secretion. In contrast, the capsaicin-induced HCO3 secretion is dependent on endogenous PGs and requires the presence of IP but not EP3 receptors. It is assumed that both acidand capsaicin-induced HCOf secretion depend on the afferent neurons, yet the mode of their interaction with the afferem neurons may be different regarding the PGI2/IP receptor dependency.

Relation of hypoxia inducible factor-1A to vasoactive factors during healing of gastric ulcers

agoinst and antagonist, In the present study, we examined, using specific EP4 agonist and antagonist, the involvement of EP4 ~ceptors in the HCO~ secretory response in rat duodenums and investigated the intracellular signaling coupled with EP4 receptors, in comparison with that of EP3 receptors. Methods: Male SD rats were used aider 18 h fasting. Under urethane anesthesia, a proximal duodenal loop was perfused with saline, and the HCO3 secretion was measured at pH 7.0 using a pH-stat method and adding 10 mM HCI. PGE2 (1 ragg stimulatory action of ONOAE1-329 but not Sul was significantly attenuated ONO-AE3-208. Likewise, ONO-AE3208, the EP4 antagonist, partially but sign~l~cantly mitigated the HCO3 secretion in response to PGE2 as well as luminal acidification. [BMX potentiated the HCOf stimulatoty action of both Sul and ONO-AF1-329, while verapamil s~ginficantly mitigated the effect of Sul but not ONO-AE1-329 Sensory deatterentadon did not af|ect the H C Q secretion induced either Snl or ONO-AE1-329. Conclusion: These results suggest that 1) EP4 receptors are invob:,ed in the HCO~ stitmdamry action of PGE2 m rat duodenums, in addition to EP3 receptors, 2) the process of HCO~ secretion difffrs in second messenger coupling; the stinmlation through EP4 receptors is mediated cAMP, while that by EP3 receptors is mediated both cAMP and Ca ~ and 3) Sensory neurons have no interaction with the HCO~ stimulatory action of PGE2

Melatonin in the duodenal lumen is a potent stimulant of mucosal bicarbonate secretion.

Melatonin, originating from intestinal enterochromaffin cells, mediates vagal and sympathetic neural stimulation of the HCO secretion by the duodenal mucosa. This alkaline secretion is considered the first line of mucosal defense against hydrochloric acid discharged from the stomach. We have studied whether luminally applied melatonin stimulates the protective secretion and whether a melatonin pathway is involved in acid-induced stimulation of the secretion. Rats were anaesthetized (Inactin) and a 12-mm segment of proximal duodenum with an intact blood supply was cannulated in situ. Mucosal HCO secretion (pH-stat) and the mean arterial blood pressure were continuously recorded. Luminal melatonin at a concentration of 1.0 micro m increased (P < 0.05) the secretion from 7.20 +/- 1.35 to 13.20 +/- 1.51 micro Eq/cm/hr. The MT2 selective antagonist luzindole (600 nmol/kg, i.v.) had no effect on basal HCO secretion, but inhibited (P < 0.05) secretion stimulated by luminal melatonin. Hexamethonium (10 mg/kg i.v. followed by continuous i.v. infusion at a rate of 10 mg/kg/hr), abolishes neurally mediated rises in secretion and also inhibited (P < 0.05) the stimulation by luminal melatonin. Exposure of the lumen to acid containing perfusate (pH 2.0) for 5 min increased (P < 0.05) the HCO secretion from 5.85 +/- 0.82 to 12.35 +/- 1.51 micro Eq/cm/hr, and luzindole significantly inhibited (P < 0.05) this rise in secretion. The study thus demonstrates that luminal melatonin is a potent stimulant of duodenal HCO secretion and, furthermore, strongly suggests melatonin as an important mediator of acid-induced secretion.

CFTR and bicarbonate secretion by [correction of to] epithelial cells.

Defective HCO(3)(-) and fluid secretion are hallmarks of the pathophysiology of the pancreas of cystic fibrosis patients. Recently, impaired HCO(3)(-) secretion has been shown in most tissues known to express the cystic fibrosis transmembrane conductance regulator (CFTR). New results suggest that CFTR plays an important role in the transcellular secretion of HCO(3)(-).

Secretion of acid and base equivalents by intact distal airways.

Secretion of HCO(3)(-) by airway submucosal glands is essential for normal liquid and mucus secretion. Because the liquid bathing the airway surface (ASL) is acidic, it has been proposed that the surface epithelium may acidify HCO(3)(-)-rich glandular fluid. The aim of this study was to investigate the mechanisms by which intact distal bronchi, which contain both surface and glandular epithelium, modify pH of luminal fluid. Distal bronchi were isolated from pig lungs, cannulated in a bath containing HCO(3)(-)-buffered solution, and perfused continually with an aliquot of similar, lightly buffered solution (LBS) in which NaCl replaced NaHCO(3)(-) (pH 7 with NaOH). The pH of this circulating LBS initially acidified (by 0.053 +/- 0.0053 pH units) and transepithelial potential difference (PD) depolarized. The magnitude of acidification was increased when pH(LBS) was higher. This acidification was unaffected by luminal dimethylamiloride (DMA, 100 microM) but was inhibited by 100 nM bafilomycin A(1) (by 76 +/- 13%), suggesting involvement of vacuolar-H(+) ATPase. Addition of ACh (10 microM) evoked alkalinization of luminal LBS and hyperpolarization of transepithelial PD. The alkalinization was inhibited in HCO(3)(-)-free solutions containing acetazolamide (1 mM) and by DMA and was enhanced by bumetanide (100 microM), an inhibitor of Cl(-) secretion. The hyperpolarization was unaffected by these maneuvers. The anion channel blocker 5-nitro-2-(3-phenylpropylamino)benzoate (300 microM) and combined treatment with DMA and bumetanide blocked both the alkalinization and hyperpolarization responses to ACh. These results are consistent with earlier studies showing that ACh evokes glandular secretion of HCO(3)(-) and Cl(-). Isolated distal airways thus secrete both acid and base equivalents.

Quantitative Assessment of Motility-Associated Changes in Gastric and Duodenal Luminal pH in Humans

Background: Interdigestive pain relieved by food is a common feature of ulcer disease. We tested the hypothesis that the duodenal bulb is intermittently acidified in association with phase III of the interdigestive motility cycle, and tried to quantify the balance between acid and duodenal bicarbonate secretion during this particular period. Methods: The experiments were performed in Helicobacter- negative healthy volunteers. Gastric and duodenal luminal pH was measured with a triple antimon electrode before, during, and after phase III of the migrating motor complex. Gastric acid secretion rate was measured in real time with a perfusion system and duodenal bicarbonate secretion was estimated from a continuous recording of the transmucosal potential difference (PD) in the duodenal bulb. Results: No significant changes in bulb pH occurred before, during, or after phase III. During the studied time window, the stomach produced 2.24 ± 0.55 mmol of acid at a peak pH of 1.74 ± 0.10. Basal HCO3 secretion calculated from bulb PD was 0.82 ± 0.12 mmol × 30 min−1 to which was added 0.47 ± 0.07 mmol of HCO3− during duodenal phase III. The contribution of retroperistalsis-driven HCO3− reflux was small (0.08 ± 0.02 mmol). Conclusions: Both the pH recording and the quantitative assessment of secretion rates show that in healthy subjects, fasting gastric acid production and duodenal bicarbonate secretion are of similar magnitude and dynamically coordinated. The mechanism behind the linkage may be reflex activation by motor activity, or a luminal PCO2 rise during phase III activity.

Immunolocalization of electroneutral Na(+)-HCO cotransporters in human and rat salivary glands.

Patterns of salivary HCO secretion vary widely among species and among individual glands. In particular, virtually nothing is known about the molecular identity of the HCO transporters involved in human salivary secretion. We have therefore examined the distribution of several known members of the Na(+)-HCO cotransporter (NBC) family in the parotid and submandibular glands. By use of a combination of RT-PCR and immunoblotting analyses, the electroneutral cotransporters NBC3 and NBCn1 mRNA and protein expression were detected in both human and rat tissues. Immunohistochemistry demonstrated that NBC3 was present at the apical membranes of acinar and duct cells in both human and rat parotid and submandibular glands. NBCn1 was strongly expressed at the basolateral membrane of striated duct cells but not in the acinar cells in the human salivary glands, whereas little or no NBCn1 labeling was observed in the rat salivary glands. The presence of NBCn1 at the basolateral membrane of human striated duct cells suggests that it may contribute to ductal HCO secretion. In contrast, the expression of NBC3 at the apical membranes of acinar and duct cells in both human and rat salivary glands indicates a possible role of this isoform in HCO salvage under resting conditions.

Identification of an apical Cl(-)/HCO3(-) exchanger in the small intestine.

HCO3(-) secretion is the most important defense mechanism against acid injury in the duodenum. However, the identity of the transporter(s) mediating apical HCO3(-) secretion in the duodenum remains unknown. A family of anion exchangers, which include downregulated in adenoma (DRA or SLC26A3), pendrin (PDS or SLC26A4), and the putative anion transporter (PAT1 or SLC26A6) has recently been identified. DRA and pendrin mediate Cl(-)/base exchange; however, the functional identity and distribution of PAT1 (SLC26A6) is not known. In these studies, we investigated the functional identity, tissue distribution, and membrane localization of PAT1. Expression studies in Xenopus oocytes demonstrated that PAT1 functions in Cl(-)/HCO3(-) exchange mode. Tissue distribution studies indicated that the expression of PAT1 is highly abundant in the small intestine but is low in the colon, a pattern opposite that of DRA. PAT1 was also abundantly detected in stomach and heart. Immunoblot analysis studies identified PAT1 as a approximately 90 kDa protein in the duodenum. Immunohistochemical studies localized PAT1 to the brush border membranes of the villus cells of the duodenum. We propose that PAT1 is an apical Cl(-)/HCO3(-) exchanger in the small intestine.

Correction of CFTR malfunction and stimulation of Ca 2+-activated Cl − channels restore HCO secretion in cystic fibrosis bile ductular cells

In view of the occurrence of hepatobiliary disorders in cystic fibrosis (CF) this study addresses the role of the cystic fibrosis transmembrane conductance regulator (CFTR) and of Ca 2+-activated Cl − channels in promoting HCO secretion in bile ductular cells. Human cholangiocytes were isolated from control livers and from 1 patient with CF (ΔF508/G542X mutations). Single channel and whole cell currents were analyzed by patch clamp techniques, and HCOsecretion was determined by fluorometric analysis of the rate of recovery of intracellular pH following alkaline loading. In control cholangiocytes, both cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) catalytic subunit, activated CFTR Cl − channels that exhibited a nonrectifying conductance of 8 pS and appeared in clusters. Activation of Cl − current by cAMP was associated with an increase in the rate of HCO secretion. The basal rate of HCO secretion was lower in CF than in control cholangiocytes. In both control and CF cholangiocytes, raising intracellular Ca 2+ concentrations with ionomycin led to a parallel activation of Cl − current and HCO secretion. Consistent with reports that premature stop codon mutations (class I; e.g., G542X) can be read over by treatment with aminoglycoside antibiotics, exposure of CF cholangiocytes to gentamicin restored activation by cAMP of Cl − current and HCO secretion. The observation that activation of Ca 2+-dependent Cl − channels can substitute for cystic fibrosis transmembrane conductance regulator (CFTR) in supporting HCO secretion and the efficacy of gentamicin in restoring CFTR function and HCO secretion in class I mutations are of potential clinical interest. (H EPATOLOGY 2002;35:95-104.)

Transepithelial measurements of bicarbonate secretion in Calu-3 cells.

AbstractThe importance of impaired HCO3 secretion in the pathophysiology of the pancreas of cystic fibrosis (CF) patients has been well documented and known for many years (1). Studies in 1990s from the Welsh (2) and Boucher (3) laboratories suggested that HCO3 secretion may also be impaired in the airways of CF patients. Perhaps because Cl- secretion has been assumed to be of higher importance and thus received the greater attention, the transcellular mechanisms of HCO3 secretion remain poorly understood and underinvestigated. Studies from our own laboratory (4) and the laboratories of Wine and Widdicombe (5) have now established that the human airway serous cell line, Calu-3 cells, secrete HCO3 and not Cl-, in response to a cAMP-mediated secretory agonist. Serous cells are the most abundant cell type of the submucosal glands (6) and are the predominate site of CFTR expression in the airways (7,8). Thus, a better understanding of the transport mechanisms of serous cells is of critical importance in establishing how mutations in CFTR lead to submucosal gland and airways pathophysiology. The focus of this chapter is to provide a description of the methods used to study Calu-3 cell monolayers grown on permeable supports by the short-circuit current (ISC) technique. In addition, methods for ion flux studies and ion substitution studies of short-circuited monolayers are described along with background information necessary to interpret the experimental results specifically as they relate to the net secretion of HCO3 . To introduce the subject of HCO3 secretion, we provide a brief description of the mechanisms of transepithelial HCO3 secretion as they are presently understood. KeywordsBasolateral MembraneVoltage ClampCold SideUssing ChamberSubmucosal GlandThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Structure and function of the heat-stable enterotoxin receptor/guanylyl cyclase C.

Guanylyl cyclase C (GC-C) was found to function as the principal receptor for heat-stable enterotoxins (STa), major causative factors in E. coli-induced secretory diarrhea. GC-C is enriched in intestinal epithelium, but was also detected in other epithelial tissues. The enzyme belongs to the family of receptor guanylyl cyclases, and consists of an extracellular receptor domain, a single transmembrane domain, a kinase homology domain, and a catalytic domain. GC-C is modified by N-linked glycosylation and, at least in the small intestine, by proteolysis, resulting in a STa receptor that is coupled non-covalently to the intracellular domain. So far two endogenous ligands of mammalian GC-C have been identified i.e. the small cysteine-rich peptides guanylin and uroguanylin. The guanylins are released in an auto- or paracrine fashion into the intestinal lumen but may also function as endocrine hormones in gut-kidney communication and as regulators of ion transport in extra-intestinal epithelia. They are thought to activate GC-C by inducing a conformational change in the extracellular portion of the homotrimeric GC-C complex, which allows two of the three intracellular catalytic domains to dimerize and form two active catalytic clefts. In the intestine, activation of GC-C results in a dual action: stimulation of Cl and HCO3 secretion, through the opening of apical CFTR Cl channels; and inhibition of Na absorption, through blockade of an apical Na/H exchanger. The principal effector of the GC-C effect on ion transport is cGMP dependent protein kinase type II, which together with GC-C and the ion transporters, may form a supramolecular complex at the apical border of epithelial cells.