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Ion Transport Across the Gallbladder Epithelium
Abstract
The function of the gallbladder is not only to store bile, but also to concentrate it during the interdigestive phase by means of salt-dependent water reabsorption. On the contrary, secretions of water and salt take place during the digestive phase. Dysregulation of ion absorption or secretion are common in many gallbladder diseases, such as colelithiasis. Transepithelial absorptions are determined by the Na+/K+ pump on the basolateral membrane, and by several apical membrane Na(+)-coupled transporters. Among these, some isoforms of Na+/H+ and Cl-/HCO3(-) exchangers have been studied. The presence of a Na(+)-Cl(-) simport has been molecularly and functionally characterized in some animal species. The ion transepithelial secretion is mainly dependent on an apical chloride transport attributable to a CFTR-like cAMP-activated channel with high permeability to HCO3(-). The apical membrane electrical potential is one of the factors influencing anion secretion and is maintained by the activity of cAMP-dependent K+ channels. The regulation of the activity of these channels is complex, because of their sensitivity to voltage, and to intracellular calcium and pH. The coordinated interplay underlying the regulation of transporters and channels needs to be clarified yet, as well as the interactions between transporters, channels and aquaporins.
... However, in studies on human and primate gallbladders, it was discovered that these electrically silent absorptive organs can secrete, under the influence of secretin, HCO 3 À -containing fluid after meals (Igimi et al. 1992;Svanvik et al. 1984). This secretion seems to depend on CFTR-like cAMP-activated channels that have relatively high permeability to HCO 3 À (Meyer et al. 2005). The importance of CFTR is highlighted in pig models of cystic fibrosis, where the disruption of CFTR leads to gallbladder and bile duct abnormalities (Rogers et al. 2008). ...
... This preparation is often used as an experimental model of human cholelithiasis, due to its unique propensity for developing gallstones on high-cholesterol chow. The basolateral HCO 3 À transport in gallbladder epithelium is carried out by pNBC1, and the driving force for secretion is maintained by cAMP-stimulated K + channels that are also sensitive to Ca 2+ and pH, but their identity is not clear (Moser et al. 2007;Meyer et al. 2005). ...
Certain epithelia secrete HCO3 − to buffer luminal fluids and protect cells against acids. Dysregulation of HCO3 − secretion can lead to conditions such as malabsorption, acid/base disturbances, cystic fibrosis, biliary cirrhosis and peptic and duodenal ulcers. In addition to transport of HCO3 − across the epithelium, epithelial cells also need to maintain intracellular pH, despite significant HCO3 − extrusion and sometimes even despite exposure to external acid. In this chapter, we will introduce the main plasma membrane acid/base transporters and describe their role in general cellular homeostasis. The same transporters are also used in building the molecular machinery for vectorial HCO3 − transport, i.e. bicarbonate secretion. We will highlight HCO3 −-secreting epithelia by examples from the digestive system (pancreas, salivary glands, hepatobiliary system and duodenum), the renal collecting duct type-B intercalated cell, and the choroid plexus epithelium of the brain. We seek an integrative approach to understand the HCO3 − secretion processes by combining historical perspectives with molecular and genetic studies as well as studies of selected regulatory systems.
... Furthermore, intestinal malabsorption might affect the enterohepatic circulation of bile acids and enhance urinary oxalate level, thereby playing a role in the pathogenesis of cholesterol gallstones and renal stones [66]. Meanwhile, water or ion channels act as mediators in processing bile and urine, and the transport alteration of water or ion could further promote bile and urinary concentration, leading to stone formation in the gallbladder and kidney [13,67]. Hyperuricemia is a prominent risk factor for UA stones, and UA is also found to induce fat accumulation in hepatocytes [68]. ...
BACKGROUND
A body of evidence has suggested bidirectional relationships among gallstone disease (GSD), non-alcoholic fatty liver disease (NAFLD), and kidney stone disease (KSD). However, the results are inconsistent, and studies on this topic in China are relatively few. Our goal is to explore the bidirectional associations among these three diseases through a multicenter study, systematic review, and meta-analysis.
AIM
To explore the bidirectional associations among these three diseases through a multicenter study, systematic review, and meta-analysis. The results may help to investigate the etiology of these diseases and shed light on the individualized prevention of these three diseases.
METHODS
Subjects who participated in physical examinations in Beijing, Tianjin, Chongqing in China were recruited. Multivariable logistic regression was employed to explore the bidirectional relationships among GSD, KSD, and NAFLD. Systematic review and meta-analysis were initiated to confirm the epidemiologic evidence from previous observational studies. Furthermore, trial sequential analysis (TSA) was conducted to evaluate whether the evidence was sufficient and conclusive.
RESULTS
Significant bidirectional associations were detected among the three diseases, independent of potential confounding factors. The pooled results of the systematic review and meta-analysis also corroborated the aforementioned results. The combined evidence from the multicenter study and meta-analysis was significant [pooled odds ratio (OR) = 1.42, 95%CI: 1.16-1.75, KSD → GSD; pooled OR = 1.48, 95%CI: 1.31-1.67, GSD → KSD; pooled OR = 1.31, 95%CI: 1.17-1.47, GSD → NAFLD; pooled OR = 1.37, 95%CI: 1.26-1.50, NAFLD → GSD; pooled OR = 1.28, 95%CI: 1.08-1.51, NAFLD → KSD; pooled OR = 1.21, 95%CI: 1.16-1.25, KSD → NAFLD]. TSA indicated that the evidence was sufficient and conclusive.
CONCLUSION
The present study presents relatively sufficient evidence for the positive bidirectional associations among GSD, KSD, and NAFLD. The results may provide clues for investigating the etiology of these three diseases and offer a guideline for identifying high-risk patients.
... It is commonly believed that bile stasis is the prime factor for gallstone formation. The function of the gallbladder is to store bile and to concentrate it during the inter-digestive phase [2]. Gallbladder and biliary tract epithelium excrete high concentrations of potentially harmful compounds [3]. ...
... The function of the gallbladder is not only to store bile, but also to concentrate it during the interdigestive phase by means of salt-dependent water reabsorption. (2) Gallstones are common, affecting about one fourth of women and 10% to 15% of men over the age of 50 (3) . Documented risk factors for gallstone disease include age , sex , obesity, rapid weight loss, high dietary intake of fat, multiple deliveries and congenital hemolytic anemias, and some medications (4,5,6) . ...
Cholelithiasis is defined as the presence of stones within the lumen of the gall bladder or in the extrahepatic biliary tree. The goal of this study was to identify the ultrastructural ultrations of gall bladder epithelium in cholelithiesis. Gallbladder specimens were collected from patients who underwent cholectstectomy. Minute specimens were also fixed and processed to evaluate the fine structures of the gall bladder epithelium. The histological changes in semithin sections of methylene blue stain showed disruption of gall bladder epithelium and discontinuity of this epithelium with appearance of Rokitansky-Aschoff sinuses, there is hyperplasia of epithelial cells, all these changes associated with mucous gland metaplasia in the lamina properia of cholecystitic gall bladder. At the ultrastructural level, abraded and altered microvilli accompanied by mitochondrial damages, dilitation of intercellular spaces was revealed by thin-section electron microscopy associated with intracellular vacuoles and irregularity and herniation of cell outlines. The epithelial cells contain mucous droplet, some of these droplets shedded to the exterior of the cells. Gallstones are accompanied by major changes in the gallbladder epithelium, as shown by both light and electron microscopy.
... Third, intestinal malabsorption can result in decreased bile acid resorption and increased urinary oxalate 27 . Finally, water and ion channels in the gallbladder might affect urine composition 28 . ...
The present study evaluated the associations between gallstones and renal stones using a national sample cohort of the Korean population. The Korean National Health Insurance Service-National Sample Cohort was collected from 2002 to 2013. We designed two different longitudinal follow-up studies. In study I, we extracted gallstone patients (n = 20,711) and 1:4-matched control I subjects (n = 82,844) and analyzed the occurrence of renal stones. In study II, we extracted renal stone patients (n = 23,615) and 1:4-matched control II subjects (n = 94,460) and analyzed the occurrence of gallstones. Matching was performed for age, sex, income, region of residence, and history of hypertension, diabetes mellitus, and dyslipidemia. Crude and adjusted hazard ratios (HRs) were calculated using a Cox proportional hazards model, and the 95% confidence intervals (CIs) were calculated. Subgroup analyses were performed according to age and sex. The adjusted HR of renal stones was 1.93 (95% CI = 1.75–2.14) in the gallstone group (P < 0.001). The adjusted HR of gallstones was 1.97 (95% CI = 1.81–2.15) in the renal stone group (P < 0.001). The results were consistent in all subgroup analyses. Gallstones increased the risk of renal stones, and renal stones increased the risk of gallstones.
... The concentrating process involves a number of epithelial ion and water transport systems that lead to net apical NaCl transport and passive osmotic water absorption across the GB epithelial cells. 11,12 As a result, the hepatic bile can be concentrated up to 10-fold by GB mucosa, so that the BA concentration in the GB may reach as high as 275 mmol/L. 13 Meanwhile, the concentrations of cholesterol and phosphatidylcholine in GB bile also increase significantly, though not to a similar extent. ...
It is commonly held that the gallbladder (GB) is not indispensable for life. However, recent studies strongly suggest that GB removal can lead to the development of metabolic syndrome (MetS). With the recent recognition of the role of bile acids (BAs) in systemic metabolic regulation, it is worthwhile to re‐examine the function of the GB, which can be regarded as the physiological ‘pacemaker’ of BA flow. Thus in the present study, we review the role of the GB in BA flow regulation, describe the epidemiologic evidence that associates cholecystectomy with various components of MetS, and discuss the possible mechanism behind these connections, in order to demonstrate the pivotal role that GB plays in metabolic regulation.
... The function of the gallbladder is not only to store bile, but also to concentrate it during the interdigestive phase by means of salt-dependent water reabsorption. (2) Gallstones are common, affecting about one fourth of women and 10% to 15% of men over the age of 50 (3) . Documented risk factors for gallstone disease include age , sex , obesity, rapid weight loss, high dietary intake of fat, multiple deliveries and congenital hemolytic anemias, and some medications (4,5,6) . ...
... allstone disease is a common health problem worldwide forming about 95% of the biliary tract disorders. The function of gallbladder is not only to store bile, but also to concentrate it during the interdigestive phase by means of salt-dependent water reabsorption [1] .There are three main types of gallstones, about 80% are cholesterol stones containing crystalline cholesterol monohydrate; the remainder are either pigment stones composed predominantly of bilirubin calcium salts or mixed stones which are mixture of the components of the both other stones. Cholesterol stones are usually yellow-green while pigment stones are small dark stones made of bilirubin [2] . ...
Abstract:
Background & Objective: The aim of this study is to investigate the histological changes of gall bladder mucosa in relation
to different gallstones whether it was cholesterol, pigment or mixed stone.
Patients and Methods: Thirty specimens of gallbladders collected from patients who underwent cholecystectomy, Paraffin
sections were stained with haematoxylin and eosin to demonstrate the general histology while Periodic acid-Schiff
and Alcian blue were utilized to evaluate the intraepithelial mucin content.
Results: The histological changes were found to be more obvious in gall bladders with cholesterol stones, except the
muscular hypertrophy which was more prominent in gallbladder with pigment stones. The mucosal hyperplasia in
gall bladders with cholesterol stones was found in 26.6% of cases, Roktansky-Aschoff sinuses in 13.3% of cases,
distended columnar cells were found in 23.3%, mucous gland metaplasia was found in 10% of cases while lymphatic
cells infiltration and haemorrhage with congestion both were found in 26.6% of cases. The neutral
mucopolysaccharides and sulfate acid mucopolysaccharides were increased in the basement membrane and found to
be more prominent in gall bladders with cholesterol stones than in pigment and mixed stones.
Conclusion: Gallstones are accompanied by major changes in the gallbladder epithelium. These changes were clearer in
gallbladder mucosa with cholesterol stones may be due to the large size stones leading to more irritation to the
mucosa in addition to the toxic effect of the lithogenic bile which produce chemical injury to the mucosa.
Key Words: Gallbladders, Cholesterol stone, Roktansky-Aschoff sinuses.
... The gallbladder functions not only to store the bile, but also to concentrate it during the inter-digestive phase by means of salt-dependent water reabsorption. 2 Gallbladder epithelium cells and biliary tract is exposed to high concentrations of potentially dangerous exogenous and endogenous compounds excreted into pri- mary bile. 3 All columnar epithelial cells are lined by a blan- ket of mucus, a native physiological gel-like secretion which separates the host mucosal cells from the external milieu. ...
Introduction: The widespread use of routine ultrasound (USG) of abdomen has led to increased detection of asymptomatic gallstone disease. The cholecystectomy is the gold standard treatment for symptomatic gallstones. However, there is always a controversy regarding management of silent gallstones. Aim: Present study designed to analyze the incidence of diverse histological changes found in gallbladder mucosa in silent gallstone diseases. Material and methods: We evaluated the mucosal changes in 135 specimens collected from patients undergoing routine cholecystectomy for silent gallstone disease for a period of 2 years. After gross examination, the specimens were formalin fixed. Sections were taken from different areas of formalin fixed gallbladder, stained with hematoxylin and eosin and examined under light microscope. Results: Abnormal mucosa was found in 86 (63,70%) cases of gallbladder grossly. On microscopy, chronic cholecystitis was found in 121 (89,63%) cases and xanthogranulomatous cholecystitis found in 4 cases (2,96%). Follicular cholecystitis was found in 5 (3,70%) cases. Cholesterolosis was found in 25 (18.52%) cases. Epithelial adenomatoid hyperplasia and papillary hyperplasia were found in 1 case each. Eighteen (13.33%) cases of Gastric metaplasia, 6 (4.44%) cases of intestinal metaplasia, 1 (0.74%) case of dysplasia and 2 cases (1.48%) adenocarcinoma were found. Discussion: Silent gallstone diseases, despite being asymptomatic, always show series of mucosal changes. Data revealed metaplastic changes especially intestinal metaplasia are associated significantly with dysplastic changes. Conclusion: Cholecystectomy should be done in all surgically fit silent gallstone cases and histopathological evaluation is important in every case to exclude metaplasia, dysplasia and carcinoma.
... Gallstones are classified into cholesterol stones containing more than 80 %crystalline cholesterol monohydrate, pigment stones composed predominantly of bilirubin calcium salts and mixed stones with components of both types. [2] Cholesterol stones are more common in the West whereas pigment stone is the predominant type in non-western population. Hypomotility of gallbladder,accumulation of lipids in bile and mucus hypersecretion contribute to the formation of cholesterol stones. ...
Background: Gallstones are a common cause of morbidity world wide. Presence of gallstones in gallbladder results
in diverse histological changes. Some of them could be precursor lesions for malignancy. This study was aimed to
correlate the various histological changes in the gallbladder to the chemical composition of gallstones whether it was
cholesterol, mixed or pigment variety.
Method: We analysed gallbladders of 100 patients who underwent cholecystectomy for gall stones.The age ,sex
distribution and the incidence of different types of gallstones were studied.The histological changes in the gallbladders
were observed and correlation with the type of gallstones was evaluated.
Result: Gallstones were more common in the 40 -49 age group with increased incidence in females. Many histological
changes including hyperplasia ,lymphoid follicles ,prominent RokitanskyAschoff sinuses, muscular hypertrophy,
metaplasia and carcinoma were observed. Most of these histological changes were seen in the gallbladders with
cholesterol stones. Hyperplasia was observed in 31.5%,lymphoid follicles in 31.5% ,RokitanskyAshoff sinus in 36.8%,
muscular hypertrophy in 47.3%,pyloric metaplasia in 26% of gallbladders with cholesterol stones. Intestinal metaplasia
was commonly associated with pigment stones(11%) and carcinoma was seen in gallbladders with mixed stones (6.7%).
Conclusion: Gallstones are common in the adult population with a female predominance. Mixed stones were the
common stones encountered . Correlation of histological changes with the chemical composition of gallstones showed
increased incidence of changes in gallbladders with cholesterol stones.This could be due to the larger size of the
cholesterol stones leading to more irritation and chemical injury produced by lithogenic bile.
... Endogenous EFs was initially discovered more than 150 years ago from a cut in his own finger by Du Bois-Reymond. Polarized epithelia transport ions directionally and maintain trans-epithelial potentials (TEP).The TEPs are present in many types of epithelia, including respiratory, gastrointestinal, urinary, and bile duct systems, and in prostate, breast, cerebral cavities, retina, and ocular lens [4][5][6][7][8][9][10][11][12][13][14] .When a wound occurs, it breaks the barrier and instantly short-circuits the TEP, and then generates wound EFs. This wound-induced electrical signal was as large as 42-150 mV/mm, detected by micro needle arrays and Bio-Electric Imager® [15,16] . ...
... The concentrating function of gallbladder, which is abnormally enhanced during the early stage of CGD, acts to continuously increase the lithogenicity of the gallbladder bile [2]. The removal of water from the gallbladder bile involves different epithelial ion transport systems that lead to net NaCl apical absorption [3]. Previous investigations in a variety of species demonstrate that gallbladder Na + and Clabsorption is predominantly mediated by the parallel Na + /H + exchanger (NHE) and Cl -/HCO 3 anion exchanger (AE) on the apical membrane of gallbladder epithelial cells (GBECs). ...
Enhanced gallbladder concentrating function is an important factor for the pathogenesis of cholesterol gallstone disease (CGD), but the mechanism is unknown. Potential candidates for regulation of gallbladder ion absorption are suggested to be Na(+)/H(+) exchanger isoform 3 (NHE3). In this study, we investigated the expression and subcellular localization of NHE3 in both acalculous and calculous human gallbladders.
Adult human gallbladder tissue was obtained from 23 patients (7 men, 16 women) who had undergone cholecystectomy. The patients were divided into two groups: Group A (acalculous group) and Group B (calculous group). Gene expression of NHE3 was quantitatively estimated by real-time PCR. Protein expression was studied by Western blotting assays. Furthermore, expression of immunoreactive NHE3 was investigated by immunohistochemistry.
There was no significant difference in the NHE3 mRNA expression between calculous and acalculous human gallbladders. NHE3 protein expression in gallbladders from patients with cholelithiasis is increased compared to those without gallstones. Immunohistochemistry studies prove that NHE3 is located both on the apical plasma membrane and in the intracellular pool in human GBECs.
NHE3 may play a role in the pathogenesis of human CGD. Additional studies are required to further delineate the underlying mechanisms.
... Some details in the regulation of gallbladder ion transport have been explored and provide further information regarding the cellular mechanisms behind the enhanced absorption efficiency of gallbladder epithelium. The removal of water from gallbladder bile involves different epithelial ion transport systems that lead to net NaCl apical absorption [43][44][45][46] (Figure 1). Previous investigations in a variety of species demonstrate that gallbladder Na þ and Cl À absorption is predominantly mediated by the parallel Na þ /H þ exchanger (NHE) and Cl À /HCO 3 À anion exchanger (AE) on the apical membrane of gallbladder epithelial cells (GBECs). ...
Gallstone disease (GSD) is one of the most common biliary tract diseases worldwide in which both genetic and environmental factors have roles in its pathogenesis. Biliary cholesterol supersaturation from metabolic defects in the liver is traditionally seen as the main pathogenic factor. Recently, there have been renewed investigative interests in the downstream events that occur in gallbladder lithogenesis. This article focuses on the role of the gallbladder in the pathogenesis of cholesterol GSD (CGD). Various conditions affecting the crystallization process are discussed, such as gallbladder motility, concentrating function, lipid transport, and an imbalance between pro-nucleating and nucleation inhibiting proteins.Laboratory Investigation advance online publication, 15 December 2014; doi:10.1038/labinvest.2014.140.
... The function of the gallbladder is not only to store bile, but also to concentrate it during the interdigestive phase by means of salt-dependent water reabsorption. 2 Epithelium of the gallbladder and biliary tract is exposed to high concentrations of potentially harmful exogenous and endogenous compounds excreted into primary bile. 3 All columnar epithelial cells are lined by a blanket of mucus, a native physiological gel-like secretion which separates the host mucosal cells from the external milieu. ...
Objectives: Pathological changes related to gallstone formation are still the focus of intensive research. The hypothesis most widely accepted is the stasis of bile caused by gallbladder dyskinesia, while dyskinesia may be the result of pathologic changes in the gallbladder wall. The aim of this study is to investigate the relation between gallstones and light as well as electron microscopic changes in the gallbladder epithelium. Methods: Gallbladder specimens were collected from patients who underwent cholectstectomy. Paraffin sections were stained with haematoxylin and eosin to demonstrate the general histology. Periodic acid-Schiff and alcian blue were utilized to evaluate the intraepithelial mucin content. Minute specimens were also fixed and processed to evaluate the fine structures of the gall bladder epithelium. Results: PAS positive materials were increased in the basement membrane. Additionally, an increase in the intensity of alcian blue reaction was observed. At the ultrastructural level, abraded and altered microvilli accompanied by mitochondrial damages,
... The anti-TGR5 antiserum detected a broad band around 48 kDa in all 19 gallbladders, which corresponds to the glycosylated form of the TGR5 protein. The unglycosylated form of the TGR5 protein was visible just below 37 kDa in some gallbladders (2,4,8,5,19). Detection of GAPDH served as loading control for densitometric analysis. ...
Unlabelled:
TGR5 (Gpbar-1) is a plasma membrane-bound, G protein-coupled receptor for bile acids. TGR5 messenger RNA (mRNA) has been detected in many tissues, including rat cholangiocytes and mouse gallbladder. A role for TGR5 in gallstone formation has been suggested, because TGR5 knockout mice did not develop gallstones when fed a lithogenic diet. In this study, expression and localization of TGR5 was studied in human gallbladders. TGR5 mRNA and protein were detected in all 19 gallbladders. Although TGR5 mRNA was significantly elevated in the presence of gallstones, no such relation was found for TGR5 protein levels. In order to study the localization of TGR5 in human gallbladders, a novel antibody was generated. The receptor was localized in the apical membrane and the rab11-positive recycling endosome of gallbladder epithelial cells. Furthermore, the TGR5 staining colocalized with the cyclic adenosine monophosphate-regulated chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) and the apical sodium-dependent bile salt uptake transporter, suggesting a functional coupling of TGR5 to bile acid uptake and chloride secretion. Stimulation with bile acids significantly increased cyclic adenosine monophosphate concentration in human gallbladder tissue. Incubation of gallbladder epithelial cells with a TGR5 agonist led to a rise of N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE)-fluorescence, suggestive of a decrease in intracellular chloride concentration. The TGR5 agonist-dependent increase in MQAE-fluorescence was absent in TGR5 knockout mice or in the presence of a CFTR inhibitor, indicating that TGR5 mediates chloride secretion via activation of CFTR. The presence of the receptor in both the plasma membrane and the recycling endosome indicate that TGR5 can be regulated by translocation.
Conclusion:
The data suggest a role for TGR5 in bile acid-induced fluid secretion in biliary epithelial cells.
... Cyclic AMP (cAMP) signaling is of central importance in the regulation of cell growth, ionic composition and volume, as well as in specialized tissue functions such as transepithelial fluid secretion and absorption, and muscle contraction (Meyer et al., 2005;Murthy, 2006;van Staveren et al., 2006). Pathological elevation in cAMP as a result of toxins, hormonal imbalance or G-protein mutations is involved in the pathogenesis of enterotoxin-mediated secretory diarrheas (Sack et al., 2004), polycystic kidney disease (Sweeney and Avner, 2006), and certain adenomas and other tumors (Murakami et al., 1999). ...
We carried out a "pathway" screen of 50,000 small molecules to identify novel modulators of cAMP signaling. One class of compounds, the 2-(acylamino)-3-thiophenecarboxylates, strongly suppressed cAMP and cGMP in multiple cell lines in response to different agonists acting on G-protein-coupled receptors, adenylyl cyclase, and guanylyl cyclase. The best compounds from structure-activity analysis of 124 analogs, including several synthesized chiral analogs, had and IC(50) of <5 microM for suppression of agonist-induced cAMP and cGMP elevation. Measurements of cAMP, cGMP, and downstream signaling in response to various activators/inhibitors suggested that the 2-(acylamino)-3-thiophenecarboxylates function as nonselective phosphodiesterase activators, although it was not determined whether their action on phosphodiesterases is direct or indirect. The 2-(acylamino)-3-thiophenecarboxylates suppressed CFTR-mediated Cl(-) current in T84 colonic cells in response to cholera and Escherichia coli (STa) toxins, and prevented intestinal fluid accumulation in a closed-loop mouse model of secretory diarrhea. They also prevented cyst growth in an in vitro renal epithelial cell model of polycystic kidney disease. The 2-(acylamino)-3-thiophenecarboxylates represent the first small-molecule cyclic nucleotide suppressors, whose potential therapeutic indications include secretory diarrheas, polycystic kidney disease, and growth inhibition of cAMP-dependent tumors.
... Furthermore, since the magnitude of the decline in I sc with luminal SITS (38%) was almost comparable to that of Cl-free solution (Fig. 3A), and both Cl and HCO 3 may share the same pathway (see discussion above), this apical Cl channel appears capable of transporting both Cl and, to a lesser extent, HCO 3 . Interestingly, the presence in BSCE of a prominent apical membrane, SITS-sensitive Cl channel makes it, from the perspective of the secreting digestive tract, more like colonic epithelium than that of small intestine (or gallbladder), whose most prominent apical Cl channel is the SITS-insensitive cystic fibrosis transmembrane regulator (3,7,16). ...
The human esophagus is lined by stratified squamous epithelium (ESSE), and in some subjects with reflux disease the distal esophagus becomes lined by Barrett's specialized columnar epithelium (BSCE). ESSE and BSCE differ both histologically and functionally, the latter evident by differences in their in vivo transmural electrical potential difference (PD), ESSE averaging -15 mV and BSCE being greater than -25 mV. In this report we examine the basis for this difference in PD. This is done by mounting endoscopic biopsies of ESSE from 25 subjects without esophageal disease and BSCE from 19 with Barrett's esophagus in mini-Ussing chambers for electrical recordings basally and after bathing solution ion replacement. The results show that the PD of human ESSE reflects a low level of active ion transport (5.1 +/- 0.8 muA/cm(2)) combined with a high level of tissue (electrical) resistance (344 +/- 34 Omega.cm(2)) and that of BSCE reflects a high level of active transport (43.6 +/- 11.6 muA/cm(2)) combined with a low level of resistance (69 +/- 8 Omega.cm(2)). Furthermore, active transport in ESSE was principally due to sodium absorption whereas in BSCE it was equally divided between sodium absorption and anion (chloride/bicarbonate) secretion, the latter through an apical membrane, 4-acetamido4'-isothiocyano-2,2'-stilbenedisulfonic acid-sensitive anion channel. As an anion-secreting tissue with bicarbonate secretory capacity more than fivefold greater than ESSE, BSCE is better suited than ESSE for defense of the esophagus against reflux disease.
Mucocele formation in dogs is a unique and enigmatic muco-obstructive disease of the gallbladder caused by amassment of abnormal mucus that bears striking pathological similarity to cystic fibrosis. We investigated the role of CFTR in the pathogenesis of this disease. The location and frequency of disease-associated variants in the coding region of CFTR was compared using whole genome sequence data from 2,642 dogs representing breeds at low-risk, high-risk, or with confirmed disease. Expression, localization, and ion transport activity of CFTR was quantified in control and mucocele gallbladders by NanoString, Western blotting, immunofluorescence imaging, and studies in Ussing chambers. Our results establish significant loss of CFTR-dependent anion secretion by mucocele gallbladder mucosa. A significantly lower quantity of CFTR protein was demonstrated relative to E-cadherin in mucocele compared to control gallbladder mucosa. Immunofluorescence identified CFTR along the apical membrane of epithelial cells in control gallbladders but not in mucocele gallbladder epithelium. Decreases in mRNA copy number for CFTR was accompanied by decreases in mRNA for the Cl ⁻ /HCO 3 ⁻ exchanger SLC26A3, K ⁺ channels ( KCNQ1, KCNN4), and vasoactive intestinal polypeptide receptor ( VIPR1) which suggest a driving force for change in secretory function of gallbladder epithelial cells in the pathogenesis of mucocele formation. There were no significant differences in CFTR gene variant frequency, type, or predicted impact comparing low risk, high risk, and definitively diagnosed groups of dogs. This study describes a unique, naturally occurring muco-obstructive disease of the canine gallbladder, with uncanny similarity to cystic fibrosis, and driven by underlying failure of CFTR function.
Cholelithiasis is the presence of gallstones, which are concretions that form in the biliary tract, usually in the gallbladder. Gallstones are hardened deposits of the digestive fluid “bile” that form within the gallbladder, varying in size and shape, ranging from as small as a grain of sand to as large as a golf ball. Gallstones occur when there is an imbalance in the chemical constituents of bile that results in the precipitation of one or more components. Gallstone disease is often thought to be a major affliction in modern society; however, it has its origins dating back to 1000 BCE. Gallstones are increasingly seen commonly in all age groups, but the incidence increases with age. It is more prevalent in females than males. The scientific data pertaining to males shows that stones are 50% less frequent. The factors leading to gall formation include both environmental cues and genetic factors. Evidence suggests that gallstones are related to age, gender, female physiological status, obesity, cardiovascular disease, the microbiome, sugar metabolism, and various environmental exposures. The mechanisms underlying these factors that lead to gallstone formation are poorly understood. Gallstone disease may not be symptomatic until there are complications caused by inflammation, infection, ductal obstruction, etc. A detailed account of gallstone disease with current information about risk factors, pathophysiology, and factors responsible for gallstone formation has been provided.
Objective: The most widely accepted hypothesis is bile stasis caused by gall bladder dyskinesia. The aim of this study is to investigate about relationship between gall stones and different gall bladder pathologies. Specimens for study was sent by surgery department forMethods: histopathology after cholecystectomy. After grossing, sections were submitted for tissue processing. Grossly, size of gall bladder wasResults: normal in 91 (60.66%) but brotic in 15 (10%) and enlarged in 44 (29.34%) patients. Wall thickness was normal (3 mm) in 71 patients (47.33%). In microscopy 110 (73.34%) were of chronic cholecystitis, cholesterolosis 15 (10%) cases, xathogranulomatous and acute on chronic cholecystitis 3 cases (2%) each, 1 case (0.67%) each of mucocele and follicular cholecystitis. Hyperplasia was observed in 8 (5.33%) cases. Hyperplasia subclassied into adenomatous and adenomyomatous. Five (3.33%) cases of cholecystitis with metaplasia were seen. There were 4 cases (2.66%) of carcinoma, predominant histologic type being adenocarcinoma. Presence of stones may lead to different mucosal changes fromConclusion: chronic inammation to carcinoma. Therefore histopathological examination of all cholecystectomy specimens with or without cholelithiasis is essential to diagnose benign as well as malignant pathology.
Certain epithelia secrete HCO3⁻ to drive fluid secretion, to modify luminal pH and properties of secreted mucus, and to fulfill other functions of a given epithelium. Dysregulation of HCO3⁻ secretion can lead to conditions such as malabsorption, acid/base disturbances, cystic fibrosis, biliary cirrhosis, peptic, and duodenal ulcers. In addition to the transport of HCO3⁻ across the epithelium, epithelial cells also need to maintain intracellular pH, despite significant HCO3⁻ extrusion and sometimes even despite exposure to external acid. In this chapter, we will introduce the main plasma membrane acid/base transporters and describe their role in general cellular homeostasis. The same transporters are also used in building the molecular machinery for vectorial HCO3⁻ transport, i.e., bicarbonate secretion. We will highlight HCO3⁻ secreting epithelia by examples from the digestive system (pancreas, salivary glands, hepatobiliary system, and duodenum), the renal collecting duct B-intercalated cell, as well as the choroid plexus epithelium of the brain. We seek an integrative approach to understand the HCO3⁻ secretion processes by combining historical perspectives with molecular and genetic studies as well as studies of selected regulatory systems.
Dysfunction of the Na+/H+ exchanger 3 (NHE3) contributes to the formation of cholesterol gallstones. We aimed to investigate whether NHE3 dysfunction is associated with abnormalities in NHE3 complex formation. We fed C57BL/6 mice with control or lithogenic diet and study the expression of NHE3, ezrin, and Na+/H+ exchanger regulatory factor 1 (NHERF1) in the gallbladder (GB) using RT–PCR and western blot. Immunofluorescence and immunoprecipitation were performed to investigate the interactions of NHE3 with ezrin or NHERF1. To explore the initiating factor that leads to NHE3 dysfunction, we stimulated cholangiocarcinoma cells with taurochenodeoxycholate (TCDC) and/or forskolin. The effects of TCDC on the expression of NHE3 regulatory proteins, as well as their bindings to NHE3, were detected by western blot and immunoprecipitation. Enzyme-linked immunosorbent assay was used to study the regulation of cAMP production by TCDC. The expression of NHERF1 and ezrin phosphorylation level were increased in the gallbladder epithelial cells (GBECs) of C57BL/6 mice with cholesterol gallstones. Immunofluorescence studies demonstrated that the subcellular localization of ezrin and NHERF1 were similar to that of NHE3 in GBECs. Immunoprecipitation revealed that ezrin formed macrocomplex with NHE3, which were enhanced after gallstone formation. TCDC increased forskolin-induced cAMP accumulation, and NHERF1 and PKCα expression in cholangiocarcinoma cells. Under the synergistic effect of forskolin, TCDC stimulated ezrin phosphorylation, with enhanced interaction between ezrin and NHE3. The formation of cholesterol gallstones is associated with abnormal formation of NHE3 complexes. Decreased biliary TCDC may be an initiating factor that leads to abnormal GB absorption.
The pathophysiology of canine gallbladder diseases, including biliary sludge, gallbladder mucoceles, and gallstones, is poorly understood. This study aimed to evaluate the composition of gallbladder contents and bacterial infection of the gallbladder in order to elucidate the pathophysiology of biliary sludge and gallbladder mucoceles. A total of 43 samples of canine gallbladder contents (biliary sludge, 21 and gallbladder mucoceles, 22) were subjected to component analysis by infrared spectroscopy, and the resultant infrared spectra were compared with that of swine mucin. Of the 43 samples, 41 were also evaluated by aerobic and anaerobic bacterial culture. The contents of 20 (95.2%) biliary sludge and 22 (100%) gallbladder mucocele samples exhibited similar infrared spectra as swine mucin. Although biliary sludge and gallbladder mucocele contents exhibited similar infrared spectra, one sample of biliary sludge (4.8%) was determined to be composed of proteins. The rate of bacterial infection of the gallbladder was 10.0% for biliary sludge and 14.3% for gallbladder mucoceles. Almost all of the identified bacterial species were intestinal flora. These results indicate that the principal components of gallbladder contents in both gallbladder mucoceles and biliary sludge are mucins and that both pathophysiologies exhibit low rates of bacterial infection of the gallbladder. Therefore, it is possible that gallbladder mucoceles and biliary sludge have the same pathophysiology, and, rather than being independent diseases, they could possibly represent a continuous disease. Thus, biliary sludge could be considered as the stage preceding the appearance of gallbladder mucoceles.
Na(+)/H(+) exchanger isoform 3 (NHE3) dysfunction is thought to contribute to the altered gallbladder absorption that occurs in cholesterol gallstone disease, but the mechanism is unknown. The current study was undertaken to examine the expression, phosphorylation, and subcellular localization of NHE3 in gallbladder epithelium cells (GBECs) of male C57BL/6 mice on a control or lithogenic diet. Thirty-six 8-week-old male C57BL/6 mice were randomly assigned to receive a high cholesterol diet or a regular diet for 8 weeks. Gallstone formation was recorded. Gallbladder bile cholesterol, phospholipid, and total bile acids were examined. RT-PCR was used to measure NHE3 mRNA expression. NHE3 protein expression and subcellular localization were examined by Western blotting and immunofluorescence microscopy, respectively. Gallstones were formed in all mice fed the lithogenic diet. Despite higher NHE3 mRNA expression in gallbladders of the mice on the lithogenic diet than in those on the control diet, there was no significant difference in expression of total NHE3 protein. However, a higher level of NHE3 phosphorylated at serine-552 (P-NHE3) was seen on the lithogenic diet. In immunofluorescence studies, NHE3 protein was expressed both on the apical membrane and in the cytoplasm of mouse GBEC. This pattern of subcellular distribution of NHE3 strongly corroborates an exchanger trafficking mechanism in NHE3 activity regulation in mouse GBEC. We conclude that increased phosphorylation of NHE3 following gallstone formation leads to turnover of the exchanger, resulting in decreased gallbladder concentrating function.
The biliary epithelium of the liver is a system of interconnecting ducts that transport bile out of the liver to the duodenum. The bile ducts are lined by epithelial cells (cholangiocytes) which actively modify bile by secreting or absorbing solutes and water using transporters, exchangers, channels, and receptors, and by processes such as endocytosis, exocytosis, and pinocytosis. Gastrointestinal hormones, neurotransmitters, growth factors, bile acids, osmosensors, and mechanoreceptors regulate cholangiocyte function through multiple, complex pathways and mechanisms. Cholangiocyte pathophysiology is studied in vivo using bile duct cannulated animals and cholestatic models of primary biliary cirrhosis and primary sclerosing cholangitis and in vitro using freshly isolated intrahepatic and extrahepatic cholangiocytes, cholangiocyte monolayers, cell lines, and isolated intrahepatic bile duct units. Cholangiocytes express phase I and II enzymes as well as cholangiocyte-specific proteins. Additionally, receptors and transport proteins are differentially expressed by cholangiocytes of different sizes lining small and large ducts, respectively.
All cells recognize chemicals in their immediate environment. They detect and respond specifically to substances as simple and ubiquitous as sodium ions to substances as complex as protein antigens. In many cases the response involves the translocation of the ligand or the activation of an ion channel.
The biliary tree of the liver is a system of ducts that transport bile out of the liver to the duodenum. The ducts are lined by epithelial cells (cholangiocytes) which actively modify bile by secreting or absorbing solutes and water using transporters, exchangers, channels, and receptors, and by processes such as endocytosis, exocytosis, and pinocytosis. Cholangiocyte function is regulated by hormones, neurotransmitters, growth factors, bile acids, osmosensors, and mechanoreceptors. In man, bile is stored and concentrated in the gallbladder for secretion during digestion. In numerous vertebrates, however, a gallbladder is lacking. Thus, storage/concentration of biliary constituents is not a requirement for normal biliary function. Cholangiocyte physiology is studied in vivo using bile-duct cannulated animals and in vitro using freshly isolated cholangiocytes, cholangiocyte monolayers, cell lines, and isolated bile duct units. Cholangiocytes express phase I and II enzymes plus cholangiocyte-specific proteins. Additionally, receptors and transport proteins are differentially expressed by cholangiocytes from large versus small ducts.Liver architecture and volume is restored following moderate injury or resection via a cascade of cytokines and growth factors that induces proliferation of all liver cell types. In contrast, a stem cell compartment (oval or progenitor cells) begins to proliferate following massive or chronic liver injury, which induces a variable 'ductular reaction' correlated with the degree of inflammation and fibrosis. Diseases of the biliary tree (cholangiopathies) are collectively termed 'vanishing bile duct syndromes,' due to progressive destruction of intra- and extrahepatic bile ducts. Primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) are the most prevalent immune-mediated cholangiopathies. Proposed etiology of PBC is formation of 'neo-antigens' (chemically modified lipoyl domains of mitochondrial matrix proteins) that elicit immunogenic autoepitopes via chronic low-level protein turnover (i.e., breakdown of tolerance). New knockout mouse models suggest that PSC is caused by elevations of cytotoxic bile acids in ductular bile and/or leaky tight junctions (TJ) leading to cholangiocyte injury, portal tract inflammation, and peribiliary fibrosis. Proposed etiologies for drug-induced cholangiocyte injury are direct cytotoxicity by drugs or their reactive metabolites, or initiation of an immune-mediated (drug hypersensitivity) response that targets cholangiocytes. Genetic and environmental risk factors identified for PBC and PSC include certain human leukocyte antigen (HLA) alleles, urban living, and proximity to superfund toxic waste sites (i.e., environmental toxicants); risk factors for drug-induced cholangiopathies include HLA alleles, gender, age, dose, number of courses of therapy, and concomitant viral infection. Although knowledge of cholangiocyte physiology and function has been considerably improved in the last 10 years, critical insight into the mechanisms by which cholangiopathies occur continues to elude investigators.
A series of 4-benzofuranyloxynicotinamide derivatives were identified to be novel, potent, and orally available TGR5 agonists. Among them, compound 9r had the highest potency in vitro (hTGR5 EC50 = 0.28 nM, mTGR5 EC50 = 0.92 nM). Further in vivo studies disclosed that 9r could effectively lower the blood glucose, but meantime caused an increase in the gallbladder volume of mice. Subsequent research toward eliminating the gallbladder toxicity resulted in compound 19 with low permeability. Although the EC50 of mTGR5 of 19 was larger one order than that of 9r, it still had good glucose-lowing activity. Nevertheless, 19 also caused the adverse effects to the gallbladder. The drug levels detection disclosed that the concentration of 19 was only lower than that of 9r in plasma but was higher in bile and gallbladder tissue. This result indicated that low exposure in plasma could not guarantee low exposure in bile and gallbladder tissue, and thus resulting in the gallbladder toxicity of 19.
Classical physiology regards the gallbladder as an absorptive epithelium. The gallbladder epithelium has one of the highest rates and capacities to absorb water among all gastrointestinal epithelia studied (1). This is effected through active sodium transport coupled with passive water absorption. Chloride is exchanged with bicarbonate. The movements of electrolytes and the effects of Donnan equilibrium forces have been extensively studied (2). There is increasing evidence that the gallbladder mucosa may also modify bile by its secretory function. For example, the secretion of mucin, immunoglobulin, as well as hydrogen ion by the gallbladder epithelium (3± 5) has been documented. We have recently suggested that the postprandial human gallbladder can secrete a color
Summary Basolateral membrane vesicles isolated from rat jejunal enterocyte and well purified from brush border contamination were tested to examine Cl and HCO3 movements. Uptake experiments provided no evidence for a coupling between Na and HCO3 fluxes; K-HCO3 and K-Cl cotransports also could be excluded. Transport studies revealed the presence of a Cl/HCO3 exchanger accepting other anions and inhibitable by the disulfonic stilbenes SITS and DIDS. We can exclude that the evidenced HCO3-dependent Cl uptake is due to brush border contamination, since in jejunal brush border membranes this mechanism, if present, has a very low transport rate. Besides the Cl/HCO3 antiporter, a Cl-conductive pathway seems to exist in jejunal basolateral membranes.
Intracellular microelectrode techniques and extracellular pH mea- surements were used to study the dependence of apical Na+/H § exchange on mucosal and intracellular pH and on mucosal solution Na § concentration ((Na+)o). When mucosal solution pH (pHo) was decreased in gallbladders bathed in Na § containing solutions, aNai fell. The effect of pHo is consistent with titration of a single site with an apparent pK of 6.29. In Na+-depleted tissues, increasing (Na+)o from 0 to values ranging from 2.5 to 110 mM increased aNal; the relationship was well described by Michaelis-Menten kinetics. The apparent Km was 15 mM at pHo 7.5 and increased to 134 mM at pHo 6.5, without change in Vm~. In Na+-depleted gallbladders, elevating (Na+)o from 0 to 25 mM increased aNai and pHi and caused acidification of a poorly buffered mucosal solution upon stopping the superfusion; lowering pHo inhibited both apical Na + entry and mucosal solution acidification. Both effects can be ascribed to titration of a single site; the apparent pK's were 7.2 and 7.4, respectively. Diethylpyrocarbo nate (DEPC), a histidine-spe- cific reagent, reduced mucosal acidification by 58 _+ 4 or 39 -+ 6% when exposure to the drug was at pHo 7.5 or 6.5, respectively. Amiloride (1 raM) did not protect against the DEPC inhibition, but reduced both apical Na § entry and mucosal acid- ification by 63 _+ 5 and 65 -+ 9%, respectively. In the Na+-depleted tissues mean pHi was 6.7. Cells were alkalinized by exposure to mucosal solutions containing high concentrations of nicotine or methylamine. Estimates of apical Na § entry at varying pHi, upon increasing (Na+)o from 0 to 25 mM, indicate that Na+/H + exchange is active at pH~ 7.4. Intracellular H + stimulated apical Na + entry by titra- tion of more than one site (apparent pK 7.1, Hill coefficient 1.7). The results sug- gest that external Na + and H + interact with one site of the Na+/H + exchanger and that cytoplasmic H § acts on at least two sites. The external titratable group seems to be an imidazolium, which is apparently different from the amiloride-bindin g site. The dependence of Na + entry on phi supports the notion that the Na+/H + exchanger is operational under normal transport conditions.
Existing data on the relation between gallstones and kidney stones are provocative but limited. Therefore, we determined whether symptomatic radiographically confirmed gallstones (and/or cholecystectomy) and symptomatic kidney stone disease are independently associated.
We conducted cross-sectional and prospective analyses in the Nurses' Health Studies I and II (older and younger women, respectively) and the Health Professionals Follow-Up Study (men) that included more than 240,000 participants followed for 14 to 24 years. Regression models adjusted for age, body size, thiazide use, diet and other factors.
At baseline the multivariate odds ratio of kidney stone history in individuals with gallstone history compared to those without was 1.65 (95% CI 1.46-1.86) in older women, 1.85 (95% CI 1.65-2.07) in younger women and 1.61 (95% CI 1.41-1.85) in men. Prospectively, the multivariate relative risk of incident kidney stones in participants with gallstone history compared to those without was 1.26 (95% CI 1.09-1.44) in older women, 1.32 (95% CI 1.14-1.52) in younger women and 1.28 (95% CI 1.03-1.57) in men. The multivariate relative risk of incident gallstones in participants with kidney stone history compared to those without was 1.17 (95% CI 1.06-1.29) in older women, 1.31 (95% CI 1.19-1.45) in younger women and 1.51 (95% CI 1.35-1.68) in men. Prospective lag analyses instituting a delay of 4 years between the diagnoses of gallstones and kidney stones yielded similar results.
Gallstones and kidney stones are independently associated. Additional studies are needed to identify shared mechanisms underlying both diseases.
Experiments were performed using in vitro perfused medullary thick ascending limbs of Henle (MTAL) and in suspensions of MTAL tubules isolated from mouse kidney to evaluate the effects of arginine vasopressin (AVP) on the K+ dependence of the apical, furosemide-sensitive Na+:Cl- cotransporter and on transport-related oxygen consumption (QO2). In isolated perfused MTAL segments, the rate of cell swelling induced by removing K+ from, and adding one mM ouabain to, the basolateral solution [ouabain(zero-K+)] provided an index to apical cotransporter activity and was used to evaluate the ionic requirements of the apical cotransporter in the presence and absence of AVP. In the absence of AVP cotransporter activity required Na+ and Cl-, but not K+, while the presence of AVP the apical cotransporter required all three ions. 86Rb+ uptake into MTAL tubules in suspension was significant only after exposure of tubules to AVP. Moreover, 22Na+ uptake was unaffected by extracellular K+ in the absence of AVP while after AVP exposure 22Na+ uptake was strictly K(+)-dependent. The AVP-induced coupling of K+ to the Na+:Cl- cotransporter resulted in a doubling in the rate of NaCl absorption without a parallel increase in the rate of cellular 22Na+ uptake or transport-related oxygen consumption. These results indicate that arginine vasopressin alters the mode of a loop diuretic-sensitive transporter from Na+: Cl- cotransport to Na+: K+: 2Cl- cotransport in the mouse MTAL with the latter providing a distinct metabolic advantage for sodium transport. A model for AVP action on NaCl absorption by the MTAL is presented and the physiological significance of the coupling of K+ to the apical Na+: Cl- cotransporter in the MTAL and of the enhanced metabolic efficiency are discussed.
In order to investigate the regulation of intracellular pH (pHi) in freshly isolated human colonocytes, we have used a newly developed technique for the rapid isolation and covalent attachment of these cells to glass surfaces and microspectrofluorimetric measurement of the pH-sensitive fluorescence of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-loaded specimens in a perfusion chamber (37 degrees C). In N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic-acid-(HEPES)-buffered Ringer solution (HBS) a baseline pHi of 7.35 +/- 0.03 (mean +/- SD; n = 42) was found for human colonocytes and in HBS, NH4Cl-prepulse-induced intracellular acidification in colonocytes is reversed rapidly by the ubiquitous amiloride-sensitive (1 mmol/l) Na+/H+ exchanger. Switching from HBS to HCO(3-)-buffered solution (BBS) led to a transient intracellular acification (7.29 +/- 0.09), followed by a recovery to a final resting pHi of 7.43 +/- 0.03. One-third of the acid extrusion in BBS is amiloridesensitive; the remaining two-thirds are caused by the dihydroderivative of 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (H2DIDS)-sensitive HCO(3-)-dependent mechanisms. The functional activity of an acid-extruding Na+/HCO3- cotransporter in human colonocytes was observed in response to the reintroduction of Na+ into amiloride-containing Na+/Cl(-)-free BBS. In addition, the mechanism leading to alkalinization (7.56 +/- 0.05) in Cl(-)-free BBS was identified as Na(+)-dependent Cl-/HCO3- exchange, by its H2DIDS sensitivity and the specific requirement for Cl- and Na+. The intrinsic buffering capacity (beta i) of the human colonocytes was calculated from pH changes induced by sequential NH4Cl-loading steps during blockage of acid/base transporters.(ABSTRACT TRUNCATED AT 250 WORDS)
Inhibition of the metabolism of arachidonic acid by the epoxygenase (cytochrome P-450) pathway with the inhibitor ketoconazole results in excessive cell swelling upon exposure to hyposmolality instead of the rapid and complete regulatory volume decrease (RVD) normally observed. NaCl entry from bathing solutions to cell interior was shown to cause this swelling, with Na influx occurring across the basolateral membrane and electrically silent Cl influx across the apical membrane. Ion substitution experiments show that the KCl efflux mediating RVD was unimpaired by ketoconazole, but was overwhelmed by the NaCl influx. Measurements of transepithelial fluid flux, Cl concentration, osmolality and pH showed that gallbladders treated with ketoconazole transiently secreted fluid rather than the normal absorption. We conclude that inhibition of arachidonic acid metabolism does not directly affect RVD by Necturus gallbladder, but that blockade of the epoxygenase pathway can have a profound influence on NaCl entry into gallbladder epithelial cells.
In the rabbit gallbladder epithelium, hydrochlorothiazide (HCTZ) was shown to inhibit the transepithelial NaCl transport and the apical Na+-Cl− symport, to depolarize the apical membrane potential and to enhance the cell-to-lumen Cl− backflux (radiochemically measured), this increase being SITS-sensitive. To better investigate the causes of the depolarization and the Cl− backflux increase, cells were punctured with conventional microelectrodes on the luminal side (incubation in bicarbonate-free saline at 27°C) and the apical membrane potential (V
m) was studied either with prolonged single impalements or with a set of short multiple impalements. The maximal depolarization was of 3–4 mV and was reached with 2.5 × 10−4m HCTZ. It was significantly enhanced by reducing luminal Cl− concentration to 30 mm; it was abolished by SCN−, furosemide, SITS; it was insensitive to DPC. SITS converted the depolarization into a hyperpolarization of about 4 mV; this latter was apamin, nifedipine and verapamil sensitive. It was concluded that HCTZ concomitantly opens apical Cl− and (probably) Ca2+ conductances and, indirectly, a Ca2+-sensitive, apamin inhibitable K+ conductance: since the intracellular Cl− activity is maintained above the value predicted at the electrochemical equilibrium, the opening of the apical Cl− conductance depolarizes V
mand enhances Cl− backflux. In the presence of apamin or verapamil, to avoid the hyperpolarizing effects due to HCTZ, the depolarization elicited by this drug was fully developed (7–10 mV) and proved to be Ca2+ insensitive. On this basis and measuring the transepithelial resistance and the apical/basolateral resistance ratio, the Cl− conductance opened by HCTZ has been estimated and the Cl− backflux increase calculated: it proved to be in the order of that observed radiochemically. The importance of this Cl− leak to the lumen in the overall inhibition of the transepithelial NaCl transport by HCTZ has been evaluated.
The absorption of water and electrolytes by the gallbladder seems to be largely dependent upon a Na+/H+ exchange at the apical membrane of the gallbladder epithelium. To find out if the exchanger involved is the NHE3 isoform, as in other absorbing epithelia, two studies were performed using the rabbit gallbladder. First, we studied 22Na absorption in Ussing chambers with Krebs buffer as a control solution, and in the presence of amiloride (100, 200 or 1000 microM), ethyl-isopropyl-amiloride (EIPA, 1 or 5 microM), or the phorbol ester, phorbol 12-myristate 13-acetate (PMA, 1 microM). A net mucosal-to-serosal Na+ flux was observed with control buffer. No inhibition of this net flux was observed with 5 microM EIPA, and the IC50 for amiloride was found to be 200 microM. PMA induced a reduction of absorption by 30% that was prevented by incubation with calphostin C. Resistance to amiloride and EIPA, and inhibition by PMA are consistent with the involvement of the NHE3 isoform. The second study involved reverse-transcriptase polymerase chain reaction (RT-PCR) of total gallbladder RNA, with two primers designed to amplify a 645-base-pair fragment from NHE3 mRNA. A cDNA fragment of the expected size was actually obtained from gallbladder RNA, while RT-PCR of RNA from the liver, which does not contain NHE3, gave negative results. A sequence of 492 nucleotides of the amplified product was determined, which was almost superimposable onto the known sequence of the corresponding fragment of rabbit NHE3. It is concluded that, in rabbit gallbladder, neutral NaCl absorption is, at least in part, dependent on the NHE3 isoform of the Na+/H+ exchanger.
Electrogenic cation transport across the caecal epithelium of the leech Hirudo medicinalis was investigated using modified Ussing chambers. Transepithelial resistance (RT) and potential difference (VT) were 61.0 +/- 3.5 omega.cm2 and -1.1 +/- 0.2 mV (n = 149), respectively, indicating that leech caecal epithelium is a "leaky" epithelium. Under control conditions short circuit current (ISC) and transepithelial Na+ transport rate (INa) averaged at 22.1 +/- 1.5 microA.cm-2 and 49.7 +/- 2.6 microA.cm-2, respectively. Mucosal application of amiloride (100 mumol.l-1) or benzamil (50 mumol.l-1) influenced neither ISC nor INa. The transport system in the apical membrane showed no pronounced cation selectivity and a linear dependence on mucosal Na+ concentration. Removal of mucosal Ca2+ increased ISC by about 50% due to an increase of transepithelial Na+ transport. Trivalent cations (La3+ and Tb3+, 1 mmol.l-1 both) added to the mucosal Ringer solution reduced INa by more than 40%. Serosal ouabain (1 mmol.l-1) almost halved ISC and INa while 0.1% (= 5.4 mmol.l-1) DNP decreased INa to 11.8 +/- 5.1% of initial values. Serosal addition of cAMP increased both ISC and INa whereas the neurotransmitters. FMRFamide, acetylcholine, GABA, L-dopa, serotonin and dopamine failed to show any effects; octopamine, glycine and L-glutamate reduced INa markedly. On the basis of these results we conclude that in leech caecal epithelium apical uptake of monovalent cations is mediated by non-selective cation conductances which are sensitive to extracellular Ca2+ but insensitive to amiloride. Basolaterally Na+ is extruded via ouabain-sensitive and -insensitive ATPases. cAMP activates Na+ transport across leech caecal epithelium, although the physiological stimulus for cAMP-production remains unknown.
1. The explanted blastoderm of the Japanese quail was used to explore the role of ions and carbon dioxide in determining the rate of sub-embryonic fluid (SEF) production between 54 and 72 h of incubation.
2. Amiloride, an inhibitor of Na+/H+ exchange, at concentrations of 10-3 to 10-6 M substantially decreased the rate of SEF production when added to the albumen culture medium. N-ethylmaleimide, an inhibitor of V type H+ ATPase, also decreased this rate but only to a small extent at the highest dose applied, 10-3 M. Both inhibitors had no effect on SEF production when added to the SEF. 3. The inhibitors of cellular bicarbonate and chloride exchange, 4-acetamido-4-'isothiocyano-2, 2-'disulphonic acid (SITS) and 4,4'diisothiocyanostilbene-2,2-'disulphonic acid (DIDS), had no effect upon SEF production.
4. Ouabain, an inhibitor of Na+/K+ ATPase, decreased SEF production substantially at all concentrations added to the SEF (10-3 to 10-6 M). Three sulphonamide inhibitors of carbonic anhydrase, acetazolamide, ethoxzolamide and benzolamide, decreased SEF production when added to the SEF at concentrations of 10-3 to 10-6 M. Benzolamide was by far the most potent. Neither ouabain nor the sulphonamides altered SEF production when added to the albumen culture medium.
5. Using a cobalt precipitation method, carbonic anhydrase activity was localised to the endodermal cells of the area vasculosa. The carbonic anhydrase activity was primarily associated with the lateral plasma membranes, which together with the potent inhibitory effect of benzolamide, suggests the carbonic anhydrase of these cells is the membrane-associated form, CA IV.
6. The changes in SEF composition produced by inhibitors were consistent with the production of SEF by local osmotic gradients.
7. It is concluded that a Na+/K+ ATPase is located on the basolateral membranes of the endodermal cells of the area vasculosa , and that a sodium ion/hydrogen ion exchanger is located on their apical surfaces. Protons for this exchanger would be provided by the hydration of CO2 catalysed by the membrane-associated carbonic anhydrase. Furthermore, it is proposed that the prime function of the endodermal cells of the area vasculosa is the production of SEF.
Isolated hepatocytes release 2-3 nmol Mg2+/mg protein or approximately 10% of the total cellular Mg2+ content within 2 minutes from the addition of agonists that increase cellular cAMP, for example, isoproterenol (ISO). During Mg2+ release, a quantitatively similar amount of Ca2+ enters the hepatocyte, thus suggesting a stoichiometric exchange ratio of 1 Mg2+:1Ca2+. Calcium induced Mg2+ extrusion is also observed in apical liver plasma membranes (aLPM), in which the process presents the same 1 Mg2+:1Ca2+ exchange ratio. The uptake of Ca2+ for the release of Mg2+ occurs in the absence of significant changes in Deltapsi as evidenced by electroneutral exchange measurements with a tetraphenylphosphonium (TPP+) electrode or 3H-TPP+. Collapsing the Deltapsi by high concentrations of TPP+ or protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) does not inhibit the Ca2+-induced Mg2+ extrusion in cells or aLPM. Further, the process is strictly unidirectional, serving only in Ca2+ uptake and Mg2+ release. These data demonstrate the operation of an electroneutral Ca2+/Mg2+ exchanger which represents a novel pathway for Ca2+ accumulation in liver cells following adrenergic receptor stimulation.
Dysregulation of gallbladder ion and water absorption and/or secretion has been linked to cholesterol crystal and gallstone formation. We have recently demonstrated that obese, leptin-deficient (Lep(ob)) mice have enlarged gallbladder volumes and decreased gallbladder contractility and that leptin administration to these mice normalizes gallbladder function. However, the effect of leptin on gallbladder absorption/secretion is not known. Therefore, we sought to determine whether leptin would alter the expression of genes involved in water and ion transport across the gallbladder epithelium. Affymetrix oligonucleotide microarrays representing 39,000 transcripts were used to compare gallbladder gene-expression profiles from 12-wk-old control saline-treated Lep(ob) and from leptin-treated Lep(ob) female mice. Leptin administration to Lep(ob) mice decreased gallbladder volume, bile sodium concentration, and pH. Leptin repletion upregulated the expression of aquaporin 1 water channel by 1.3-fold and downregulated aquaporin 4 by 2.3-fold. A number of genes involved in sodium transport were also influenced by leptin replacement. Epithelial sodium channel-alpha and sodium hydrogen exchangers 1 and 3 were moderately downregulated by 2.0-, 1.6-, and 1.3-fold, respectively. Carbonic anhydrase-IV, which plays a role in the acidification of bile, was upregulated 3.7-fold. In addition, a number of inflammatory cytokines that are known to influence gallbladder epithelial cell absorption and secretion were upregulated. Thus leptin, an adipocyte-derived cytokine involved with satiety and energy balance, influences gallbladder bile volume, sodium, and pH as well as multiple inflammatory cytokine genes and genes related to water, sodium, chloride, and bicarbonate transport.
Cl– influx at the luminal border of the epithelium of rabbit gallbladder was measured by 45-sec exposures to36Cl– and3H-sucrose (as extracellular marker). Its paracellular component was evaluated by the use of 25mm SCN– which immediately and completely inhibits Cl– entry into the cell. Cellular influx was equal to 16.7eq cm–2 hr–1 and decreased to 8.5eq cm–2 hr–1 upon removal of HCO
3
–
from the bathing media and by bubbling 100% O2 for 45 min. When HCO
3
–
was present, cellular influx was again about halved by the action of 10–4
m acetazolamide, 10–5 to 10–4
m furosemide, 10–5 to 10–4
m 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate (SITS), 10–3
m amiloride. The effects of furosemide and SITS were tested at different concentrations of the inhibitor and with different exposure times: they were maximal at the concentrations reported above and nonadditive. In turn, the effects of amiloride and SITS were not additive. Acetazolamide reached its maximal action after an exposure of about 2 min. When exogenous HCO
3
–
was absent, the residual cellular influx was insensitive to acetazolamide, furosemide and SITS. When exogenous HCO
3
–
was present in the salines, Na+ removal from the mucosal side caused a slow decline of cellular Cl– influx; conversely, it immediately abolished cellular Cl– influx in the absence of HCO
3
–
. In conclusion, about 50% of cellular influx is sensitive to HCO
3
–
, inhibitable by SCN–, acetazolamide, furosemide, SITS and amiloride and furthermore slowly dependent on Na+. The residual cellular influx is insensitive to bicarbonate, inhibitable by SCN–, resistant to acetazolamide, furosemide, SITS and amiloride, and immediately dependent on Na+. Thus, about 50% of apical membrane NaCl influx appears to result from a Na+/H+ and Cl–/HCO
3
–
exchange, whereas the residual influx seems to be due to Na+–Cl– contranport on a single carrier. Whether both components are simultaneously present or the latter represents a cellular homeostatic counterreaction to the inhibition of the former is not clear.
By incorporating plasma membrane vesicles into planar lipid bilayers, we previously characterized a family of four types of Ca(2+)-activated K+ channels from rat brain (Reinhart et al., 1989). Two of these are "large-conductance" or "maxi"-K+ channels, which differ in their gating kinetics and toxin sensitivity and are henceforth referred to as "type 1" and "type 2" channels. Here we show that the gating of these two channel types can be modulated by phosphorylation and dephosphorylation. The effects of cAMP-dependent protein kinase catalytic subunit (PK-A) on type 1 maxi-K+ channels are complex in that, while half of these channels are upregulated by the kinase, about one out of seven channels is downregulated. Thus, there may be several distinct channels within the type 1 category. Type 2 maxi-K+ channels are consistently downregulated by PK-A. The effects of PK-A on both channel types are reversed by the catalytic subunit of protein phosphatase 2A (PP-2A), but not by protein phosphatase 1 (PP-1). Furthermore, some of the type 1 maxi-K+ channels can be modulated by PP-2A, even without any prior PK-A treatment, indicating they are in a phosphorylated state when they are incorporated into the bilayer. The results demonstrate that (1) type 1 and type 2 maxi-K+ channels are substrates for PK-A; (2) phosphorylation can shift the open probability of channels in either direction, by a mechanism involving multiple phosphorylation sites; (3) phosphorylation alters the Ca2+/voltage sensitivity of these channels; and (4) dephosphorylation of type 1 and type 2 channels is catalyzed by specific phosphatases.
We have used a well-differentiated human colonic cell line, the T84 cell line, as a model system to study the pathways of cellular ion transport involved in vasoactive intestinal polypeptide (VIP)-induced chloride secretion. A modified Ussing chamber was used to study transepithelial Na+ and Cl- fluxes across confluent monolayer cultures of the T84 cells grown on permeable supports. In a manner analogous to isolated intestine, the addition of VIP caused an increase of net Cl- secretion which accounted for the increase in short circuit current (Isc). The effect of VIP on Isc was dose dependent with a threshold stimulation at 10(-10) M VIP, and a maximal effect at 10(-8) M. Bumetanide prevented or reversed the response to VIP. Inhibition by bumetanide occurred promptly when it was added to the serosal, but not to the mucosal bathing media. Ion replacement studies demonstrated that the response to VIP required the simultaneous presence of Na+, K+, and Cl- in the serosal media. Utilizing cellular ion uptake techniques, we describe an interdependence of bumetanide-sensitive 22Na+, 86Rb+, and 36Cl- uptake, which is indicative of a Na+,K+,Cl- cotransport system in this cell line. This transport pathway was localized to the basolateral membrane. Extrapolated initial velocities of uptake for each of the three ions was consistent with the electroneutral cotransport of 1 Na+:1 K+ (Rb+):2 Cl-. Our findings indicate that VIP-induced Cl- secretion intimately involves a bumetanide-sensitive Na+,K+,Cl- cotransport system which is functionally localized to the basolateral membrane.
Intracellular microelectrode techniques were employed to study the effect of cyclic AMP on apical membrane Cl-/HCO3- exchange and electrodiffusive HCO3- transport in Necturus gallbladder epithelium. Intracellular cAMP levels were raised by addition of either the phosphodiesterase inhibitor theophylline (3 X 10(-3) M) or the adenylate cyclase activator forskolin (10(-5) M) to the serosal bathing solution. Measurements of pH in a poorly buffered control mucosal solution upon stopping superfusion show acidification, owing to secretion of both H+ and HCO3-. When the same experiment is performed after addition of amiloride or removal of Na+ from the mucosal bathing medium, alkalinization is observed since H+ transport is either inhibited or reversed, whereas HCO3- secretion persists. The changes in pH in both amiloride or Na-free medium were significantly decreased in theophylline-treated tissues. Theophylline had no effect on the initial rates of fall of intracellular Cl- activity (aCli) upon reducing mucosal solution [Cl-] to either 10 or 0 mM, although membrane voltage and resistance measurements were consistent with stimulation of apical membrane electrodiffusive Cl- permeability. Estimates of the conductive flux, obtained by either reducing simultaneously mucosal [Cl-] and [HCO3-] or lowering [Cl-] alone in the presence of a blocker of anion exchange (diphenylamine-2-carboxylate), indicate that elevation of intracellular cAMP inhibited the anion exchanger by approximately 50%. Measurements of net Cl- uptake upon increasing mucosal Cl- from nominally zero to levels ranging from 2.5 to 100 mM suggest that the mechanism of inhibition is a decrease in Vmax. Consistent with these results, the rate of intracellular alkalinization upon reducing external Cl- was also inhibited significantly by theophylline. Reducing mucosal solution [HCO3-] from 10 to 1 mM under control conditions caused intracellular acidification and an increase in aCli. Theophylline inhibited both changes, by 62 and 32%, respectively. These data indicate that elevation of intracellular cAMP inhibits apical membrane anion (Cl-/HCO3-) exchange. Studies of the effects of rapid changes in mucosal [HCO3-] on membrane voltages and the apparent ratio of membrane resistances, both in the presence and in the absence of theophylline, with or without Cl- in the mucosal solution, do not support the hypothesis that cAMP produces a sizable increase in apical membrane electrodiffusive HCO3- permeability.
Electrically silent Na(+)-(K+)-Cl- transporter systems are present in a wide variety of cells and serve diverse physiological functions. In chloride secretory and absorbing epithelia, these cotransporters provide the chloride entry mechanism crucial for transcellular chloride transport. We have isolated cDNAs encoding the two major electroneutral sodium-chloride transporters present in the mammalian kidney, the bumetanide-sensitive Na(+)-K(+)-Cl- symporter and thiazide-sensitive Na(+)-Cl- cotransporter, and have characterized their functional activity in Xenopus laevis oocytes. Despite their differing sensitivities to bumetanide and thiazides and their different requirements for potassium, these approximately 115-kDa proteins share significant sequence similarity (approximately 60%) and exhibit a topology featuring 12 potential membrane-spanning helices flanked by long non-hydrophobic domains at the NH2 and COOH termini. Northern blot analysis and in situ hybridization indicate that these transporters are expressed predominantly in kidney with an intrarenal distribution consistent with their recognized functional localization. These proteins establish a new family of Na(+)-(K+)-Cl- cotransporters.
Ion secretion in guinea pig gall-bladder epithelium can be electrogenic. Therefore ion channels were studied utilizing the patch-clamp technique. Using the cell-attached configuration on dissociated cells, K+ channels with a single-channel conductance of about 160 pS, for negative voltage values, were identified. An inward rectification was usually observed. The channel open probabilities at physiological voltage values were low (less than 0.1) and increased 3- to 4-fold with depolarization of 20–30 mV. Using inside-out configuration and symmetrical 145 mM KCI, the curve was linear and was not modified by the presence of Mg2+ and ATP on the cytoplasmic side. The channels were selective for K+ over Na+ (PNa+/PK+ = 0.05). They were activated by increased levels of [Ca2+] or pH on the cytoplasmic side. Data suggest that the activity of these channels is not only influenced by voltage, [Ca2+], pH but also by unidentified cytoplasmic factors responsible for the inward rectification.
The patch-clamp technique was used to characterize K+ channel activity in the basolateral membrane of isolated crypts from rat distal colon. In cell-attached patches with KCl in the pipette, channels with conductances ranging from 6 pS to 80 pS appeared. With NaCl in the pipette and KCl in the bath in excised inside-out membrane patches a small-conductance channel with a mean conductance of 126 pS (n=18) was observed. The channel has been identified as K+ channel by its selectivity for K+ over Na+ and by its sensitivity to conventional K+ channel blockers, Ba2+ and tetraethylammonium (TEA+). Changes of cytosolic pH did not attenuate channel activity. Activity of the 12-pS channel was increased by membrane depolarization and elevated cytosolic Ca2+ concentration. In addition, a maxi K+ channel with a mean conductance of 18715 pS (n=4) in symmetrical KCl solutions was only occasionally recorded. The maxi K+ channel could be blocked by Ba2+ (5 mmol/l) on the cytosolic side. Using the slow-whole cell recording technique under control conditions, a cell membrane potential of –7010mV (n=18) was measured. By application of various K+ channel blockers such as glibenclamide, charybdotoxin, apamin, risotilide, Ba2+ and TEA+ in the bath, only Ba2+ and TEA+ depolarized the cell membrane. The present data suggest that the small K+ channel (12 pS) is involved in the maintenance of the cell membrane resting potential.
In the epithelium of rabbit gallbladder, in the nominal absence of bicarbonate, intracellular Cl– activity is about 25mm, about 4 times higher than intracellular Cl– activity at the electrochemical equilibrium. It is essentially not affected by 10–4
m acetazolamide and 10–4
m 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate (SITS) even during prolonged exposures; it falls to the equilibrium value by removal of Na+ from the lumen without significant changes of the apical membrane potential difference. Both intracellular Cl– and Na+ activities are decreased by luminal treatment with 25mm SCN–; the initial rates of change are not significantly different. In addition, the initial rates of change of intracellular Cl– activity are not significantly different upon Na+ or Cl– entry block by the appropriate reduction of the concentration of either ion in the luminal solution. Luminal K+ removal or 10–5
m bumetanide do not affect intracellular Cl– and Na+ activities or Cl– influx through the apical membrane. It is concluded that in the absence of bicarbonate NaCl entry is entirely due to a Na+–Cl– symport on a single carrier which, at least under the conditions tested, does not cotransport K+.
In guinea pig gallbladder epithelium, a secretion of fluid, secondary to an electrogenic secretion of Cl− and HCO−3, is elicited in the presence of a high intracellular concentration of adenosine 3′-5′-cyclic monophosphate (cAMP). The aim of this study was to analyze the effects of secretagogues on the activity of anionic channels in isolated epithelial cells using the patch-clamp technique and measuring the electrical potential difference of the cellular membrane (pdcm). In cell-attached configuration, with the microelectrode filled with a solution of N-methylglucamine–Cl, or in inside-out configuration (symmetrical solution), it was possible to demonstrate the presence of an 18-pS Cl− channel with linear current/voltage (I/V) relationship and voltage independence; this channel is not activated by cAMP (cell-attached configuration). In inside-out configuration (symmetrical solution), another anionic channel with a conductance of 2.8 pS, voltage independence, and a linear I/V relationship was also identified. This channel was stimulated by cAMP (cell-attached configuration) and by PKA + ATP + cAMP (inside-out configuration). The channel was inhibited by NPPB (10−5 M), but not by other anionic inhibitors. Measurements of the pdcm value suggested that in isolated cells, as in whole tissue, cAMP activates conductance for both Cl− and HCO−3. The selectivity of the channel was gluconate < SO2−4 < Cl− < Br− < I− < HCO−3 < SCN− and the PHCO3/PCl was 2.6. Some features of the channel resemble those of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and RT-PCR performed on mRNA from isolated epithelial cells detected the presence of a CFTR homologue mRNA. The results obtained indicate that this channel is responsible for the HCO−3 conductance activated by cAMP.
The effects of cytosolic ATP on Ca2+-dependent K+ (KCa) channel activation in cultured bovine adrenal chromaffin cells were investigated by using single-channel recording patch-clamp techniques. Application of ATP to the intracellular surface of excised inside-out patches activated KCa channels in a dose-dependent manner at 30 μM to 10 mM. The KCa channels also were activated by 3 mM of adenosine 5′-O-(3′-thiotriphosphate) (ATPγS), a non-hydrolyzable analogue of ATP, but not by 5′-adenylylimidodiphosphate (AMP-PNP) (from 300 μM to 3 mM). Furthermore, other nucleotides also activated KCa channels in inside-out patches. This modulation took place without addition of exogenous protein kinase and was dependent on the presence of Mg2+ in the bathing solution. Staurosporine, a non-specific kinase inhibitor, or H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide), a cAMP-dependent protein kinase inhibitor, was unable to alter ATP-mediated KCa channel activation. Following complete removal of Mg2+, a higher concentration of ATP (10 mM) and other nucleotides was required to activate KCa channels; however, Mg2+ was ineffective in altering the activation of KCa channels by itself. It is concluded that intracellular ATP and other nucleotides activate KCa channels directly. These nucleotides may regulate catecholamine release by changing the cell membrane potential in adrenal chromaffin cells.
Obligatory, coupled cotransport of Na(+), K(+), and Cl(-) by cell membranes has been reported in nearly every animal cell type. This review examines the current status of our knowledge about this ion transport mechanism. Two isoforms of the Na(+)-K(+)-Cl(-) cotransporter (NKCC) protein (approximately 120-130 kDa, unglycosylated) are currently known. One isoform (NKCC2) has at least three alternatively spliced variants and is found exclusively in the kidney. The other (NKCC1) is found in nearly all cell types. The NKCC maintains intracellular Cl(-) concentration ([Cl(-)](i)) at levels above the predicted electrochemical equilibrium. The high [Cl(-)](i) is used by epithelial tissues to promote net salt transport and by neural cells to set synaptic potentials; its function in other cells is unknown. There is substantial evidence in some cells that the NKCC functions to offset osmotically induced cell shrinkage by mediating the net influx of osmotically active ions. Whether it serves to maintain cell volume under euvolemic conditons is less clear. The NKCC may play an important role in the cell cycle. Evidence that each cotransport cycle of the NKCC is electrically silent is discussed along with evidence for the electrically neutral stoichiometries of 1 Na(+):1 K(+):2 Cl- (for most cells) and 2 Na(+):1 K(+):3 Cl(-) (in squid axon). Evidence that the absolute dependence on ATP of the NKCC is the result of regulatory phosphorylation/dephosphorylation mechanisms is decribed. Interestingly, the presumed protein kinase(s) responsible has not been identified. An unusual form of NKCC regulation is by [Cl(-)](i). [Cl(-)](i) in the physiological range and above strongly inhibits the NKCC. This effect may be mediated by a decrease of protein phosphorylation. Although the NKCC has been studied for approximately 20 years, we are only beginning to frame the broad outlines of the structure, function, and regulation of this ubiquitous ion transport mechanism.
Acute pancreatitis is associated with passage of gallstones, although the mechanism(s) linking the two processes remains undefined. Bile reflux into the pancreatic duct could play a role but the experimental conditions often employed to induce pancreatitis rarely develop clinically. Here we examined whether low concentrations of bile affect ductal electrophysiology as an indirect measure of ductal epithelial integrity and function in vitro.
The main duct was dissected out of freshly harvested bovine pancreata, cut into 1- x 2-cm sections, placed in tissue culture for 48-72 h, then placed in Ussing chambers. Changes in tissue resistance (Rt) and short-circuit current (Isc) were monitored. The responses to forskolin and bile (taurodeoxycholic acid, TDCA) were examined separately and together.
Forskolin (10 microM) produced a decrease in the Isc without a significant change in Rt, suggesting a secretory response, followed by a return to baseline. TDCA caused a similarly reversible decrease in the Isc at low doses, but a persistent drop at higher concentrations. A concurrent drop in Rt was noted at all TDCA concentrations, the duration of which correlated with dosage and degree of histological damage. Prior exposure to low (0.5 mM) doses of TDCA significantly blunted the response to subsequent forskolin challenge.
Acute exposure to TDCA in vitro causes epithelial damage at levels lower than those normally used to induce experimental pancreatitis. At the lower concentrations, Rt returns to baseline rapidly, suggesting recovery (restitution) from epithelial damage but with a persistent loss of the response to forskolin. Reflux of minute amounts of bile into the pancreatic duct could play a significant role in the pathogenesis of gallstone pancreatitis by uncoupling the normal stimulus-secretion apparatus of the ductal system and breaking down the epithelial barrier.
Fluid transport and net fluxes of Na, K, Cl and HCO3 by guinea pig gallbladder were investigated in vitro. A perfused gallbladder preparation was devised to simultaneously study unidirectional fluxes of 22Na and 36Cl. The net Cl flux exceeded the net Na flux during fluid absorption in the presence of HCO3. This Cl excess was counterbalanced by a net HCO3 secretion: a HCO3-Cl exchange. PGE1 reversed the direction of fluid transport and abolished the net Cl flux. The magnitude of the HCO3 secretion remained unchanged, but shifted from a HCO3-Cl exchange to a net secretion of NaHCO3 and KHCO3. Furosemide inhibited both the HCO3-Cl exchange and HCO3 secretion after PGE1 without influencing fluid absorption. Ouabain inhibited the HCO3-Cl exchange as well as fluid absorption; only the effect on the HCO3 secretion was entirely reversible. Secreted HCO3 appeared not to be derived from metabolic sources since HCO3 secretion was abolished in a HCO3-free bathing medium. HCO3 secretion was also dependent on the Na concentration of the bathing fluid. Three lines of evidence are presented in favor of an active HCO3 secretion in guinea pig gallbladder. HCO3 is secreted against: (i) a chemical gradient, (ii) an electrical gradient and (iii) the direction of fluid movement under control conditions.
Bicarbonate presence in the bathing media doubles Na+ and fluid transepithelial transport and in parallel significantly increases Na+ and Cl− intracellular concentrations and contents, decreases K+ cell concentration without changing its amount, and causes a large cell swelling. Na+ and Cl− lumen-to-cell influxes are significantly enhanced, Na+ more so than Cl−. The stimulation does not raise any immediate change in luminal membrane potential and cannot be due to a HCO
3−-ATPase in the brush border. The stimulation goes together with a large increase in a Na+-dependent H+ secretion into the lumen. All of these data suggests that HCO
3− both activates Na+−Cl− cotransport and H+−Na+ countertransport at the luminal barrier.
Thiocyanate inhibits Na+ and fluid transepithelial transport without affecting H+ secretion and HCO
3−-dependent Na+ influx. It reduces Na+ and Cl− concentrations and contents, increases the same parameters for K+, causes a cell shrinking, and abolishes the lumen-to-cell Cl− influx. It enters the cell and is accumulated in the cytoplasm with a process which is Na+-dependent and HCO
3−-activated. Thus, SCN− is likely to compete for the Cl− site on the cotransport carrier and to be slowly transferred by the cotransport system itself.
Loperamide, a new antidiarrhoeal compound, effectively antagonised prostaglandin induced diarrhoea in adult healthy male volunteers and in patients undergoing pregnancy termination with prostaglandins. This compound is effective in inhibiting prostaglandin induced fluid accumulation in the small intestine in rats. Loperamide also blocked smooth muscle stimulating action of prostaglandins, acetylcholine and histamine on gastrointestinal smooth muscle preparations from several laboratory animals.
Prostaglandins (PGs) F2alpha, E1 and E2 exerted a triphasic influence on the fluid transport of isolated guinea-pig gall-bladders, when applied to the serosal side. PGE1 and PGE2 produced these effects in lower concentrations than F2alpha. Directly after PG addition to the serosal side a short stimulation of fluid transport to between 200 and 400% was observed. The stimulatory effect of PGs was most distinct in gall-bladders from female guinea-pigs, less pronounced in male and nearly absent in pregnant animals. Since PGs increased intraluminal hydrostatic pressure in gall-bladders by contraction of the smooth muscle, experiments were performed in which hydrostatic pressure was increased by different procedures. These included the addition of imidazole (10- minus 2 M), raising of K+ in the bathing solution and an increase in intraluminal pressure by addition of Ringer's solution into the lumen. All three procedures stimulated fluid reabsorption temporarily in the same way as PGs, hence increase of intraluminal pressure is thought to be the reason for the observed temporary stimulation of fluid transport. Direct evidence for this thesis was obtained when the gall-bladder was mounted as a flat sheet over a chamber; in this preparation no stimulation of fluid transport was obtained. The second phase of the PG influence was characterized by a concentration-related inhibition of fluid reabsorption followed by a significant but small reverse of fluid transport (secretion of fluid). When PGs were applied to the mucosal side, only an inhibition of fluid transport was observed, which was much weaker compared to the addition to the serosal side.
Effects of adenosine 3',5'-cyclic monophosphate (cAMP) on Ca(2+)-dependent K+ channel and Cl- conductance in the plasma membrane of isolated canine pancreatic acinar cells were studied by patch-clamp methods. In whole-cell current recordings on isolated cells dialyzed with K(+)-rich solution containing 0.5 mM EGTA, addition of 0.5 mM dibutyryl cAMP (dbcAMP), or 50 microM forskolin to the bath increased outward K+ and inward Cl- currents associated with depolarizing and hyperpolarizing voltage jumps, respectively. In intact cells (cell-attached configurations), addition of 0.5 mM dbcAMP or 50 microM forskolin to the bath increased the opening of single K+ channel. In Ca(2+)-free external solution (bath and pipette) 50 microM forskolin or 0.5 mM dbcAMP application evoked an increase in the opening of single K+ channel in intact cells. Addition of 0.5 mM dbcAMP to the bath solution containing 10 mM EGTA without Ca2+ increased the currents of whole-cell dialyzed with K(+)-rich solution containing 10 mM EGTA. When cell was dialyzed with 20 mM EGTA, dbcAMP, or forskolin application did not increase the whole-cell currents. In excised inside-out patches, addition of the catalytic subunit of cAMP-dependent protein kinase (16 U/ml) in the presence of 0.3 mM ATP to the cytoplasmic face of membrane activated the K+ channel, but 0.1 mM cAMP did not. These results suggest that cAMP-dependent phosphorylation can activate Ca(2+)-dependent K+ channels without increase in intracellular free Ca2+ and cAMP-dependent mechanism can activate Ca(2+)-dependent Cl- conductances without the increase in Ca2+ in canine pancreatic acinar cells.
Retrovirus-mediated transfection of cDNA for the cystic fibrosis (CF) gene into the CF pancreatic cell line, CFPAC-1, confers adenosine 3',5'-cyclic monophosphate (cAMP)-dependent regulation of Cl conductance. We used patch-clamp techniques to identify the single-channel basis of this conductance pathway and to study its properties. Forskolin or cAMP activated Cl channels with a conductance of 9 +/- 1 pS in 26 of 62 cell-attached patches of cystic fibrosis transmembrane conductance regulator (CFTR)-transfected CFPAC-1 cells. The current-voltage (I-V) relation showed slight outward rectification (chord conductance of 10 +/- 2 pS at +80 mV vs. 7 +/- 1 pS at -80mV) with high Cl concentrations (170 mM) in the pipette solution. Channel kinetics were voltage sensitive, with longer openings at positive clamp voltages. Channel properties were unaffected by the substitution of N-methyl-D-glucamine for pipette Na or by the addition of disulfonic stilbenes (100 microM DNDS or DIDS) to the pipette. The channels usually inactivated within seconds of patch excision, but in three of nine patches, activity could be maintained by addition of the catalytic subunit of protein kinase A and ATP. With equal Cl concentrations on both membrane surfaces, the single-channel I-V relation was linear, suggesting that the outward rectification of the cell-attached channel is due to a pipette-to-cell Cl gradient. Anion substitution on the extracellular side of the membrane indicates a halide permselectivity of Br approximately Cl greater than I.(ABSTRACT TRUNCATED AT 250 WORDS)
Patch clamp recordings on the apical membrane of marginal cells of the stria vascularis of the gerbil were made in the cell-attached and excised configuration. Marginal cells are thought to secrete K+ into and absorb Na+ from endolymph. Four types of channel were identified; the most frequently observed channel was a small, nonselective cation channel which was highly similar to that found in the apical membrane of vestibular dark cells (Marcus et al., (1992) Am. J. Physiol. 262, C1423-C1429). The small nonselective cation channel was equally conductive (26.7 +/- 0.3 pS; N = 49) for K+, Na+, Rb+, Li+ and Cs+, 1.6 times more permeable to NH4+, but not permeable to Cl-, Ca2+, Ba2+ or N-methyl-D-glucamine. This channel yielded linear current-voltage relations which passed nearly through the origin (intercept: -2.2 +/- 0.4 mV, N = 49) when conductive monovalent cations were present on both sides of the membrane in equal concentrations. Channel activity required the presence of Ca2+ at the cytosolic face but not the extracellular (endolymphatic) face; there was essentially no activity for cytosolic Ca2+ less than or equal to 10(-7) M Ca2+ and full activity for greater than or equal to 10(-5) M. Cell-attached recordings had a conductance of 28.6 +/- 2.2 pS (N = 6) and a reversal voltage of -2.2 +/- 5.2 mV (N = 3) which was interpreted to reflect the intracellular potential of marginal cells under the present conditions. The three other types of channel were a Cl- channel (approximately 50 pS; N = 2), a maxi-K+ channel (approximately 230 pS; N = 1), and another large channel, probably cation nonselective (approximately 170 pS; N = 1). The 27 pS nonselective cation channel may be involved in K+ secretion and Na+ absorption under stimulated conditions which produce an elevated intracellular Ca2+; however, consideration of the apparent channel density in relation to the total transepithelial K+ flux suggests that these channels are not sufficient to account for K+ secretion.
Recordings were made on excised apical membrane patches from vestibular dark cells from the semicircular canal of gerbils to determine if ion channels could be involved in the process of K+ secretion. Both nonselective cation channels [Am. J. Physiol. 262 (Cell Physiol. 31): C1430-C1436, 1992] and K(+)-selective channels were found. The K+ channels occurred in only 0.7% of the patches. In symmetrical 145 mM KCl solutions, the current-voltage (I-V) relation of the K(+)-selective channel was linear, indicating the absence of rectification, and the conductance was 240 +/- 8 pS (n = 8). The Goldman-Hodgkin-Katz equation for current carried solely by K+ could be fitted to the I-V relation in asymmetrical K+ and Na+ solutions and yielded a K+ permeability of 5.78 x 10(-13) cm3/s (n = 12). The channel was shown to be impermeable to Li+, NH4+, N-methyl-D-glucamine, and Cl-. Channel activity increased with depolarization and with increasing free [Ca2+]; for voltages between +40 and -60 mV, the strongest regulation occurred in the range 10(-6) to 10(-5) M Ca2+. Tetraethylammonium (2 x 10(-2) M) had from the cytosolic side no effect on the open probability (Po) but completely inhibited activity from the extracellular side. Po was reduced by Ba2+ (5 x 10(-3) M), verapamil (10(-4) M), quinine (10(-4) M), and quinidine (10(-4) and 10(-3) M), while lidocaine (5 x 10(-3) M) had no measurable effect on Po but decreased the amplitude. Rb+ and Cs+ were either poorly permeable or partially blocked the channel in a voltage-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)
Using the patch-clamp technique we have identified a Ca2(+)-sensitive, voltage-dependent, maxi-K+ channel on the basolateral surface of rat pancreatic duct cells. The channel had a conductance of approximately 200 pS in excised patches bathed in symmetrical 150 mM K+, and was blocked by 1 mM Ba2+. Channel open-state probability (Po) on unstimulated cells was very low, but was markedly increased by exposing the cells to secretin, dibutyryl cyclic AMP, forskolin or isobutylmethylxanthine. Stimulation also shifted the Po/voltage relationship towards hyperpolarizing potentials, but channel conductance was unchanged. If patches were excised from stimulated cells into the inside-out configuration, Po remained high, and was not markedly reduced by lowering bath (cytoplasmic) Ca2+ concentration from 2 mM to 0.1 microM. However, activated channels were still blocked by 1 mM Ba2+. Channel Po was also increased by exposing the cytoplasmic face of excised patches to the purified catalytic subunit of cyclic AMP-dependent protein kinase. We conclude that cyclic AMP-dependent phosphorylation can activate maxi-K+ channels on pancreatic duct cells via a stable modification of the channel protein itself, or a closely associated regulatory subunit, and that phosphorylation alters the responsiveness of the channels to Ca2+. Physiologically, these K+ channels may contribute to the basolateral K+ conductance of the duct cell and, by providing a pathway for current flow across the basolateral membrane, play an important role in pancreatic bicarbonate secretion.
Recent studies suggest that altered gallbladder absorptive function may be an important and previously unrecognized factor in the pathogenesis of experimentally induced gallstones. The present study was designed to define the specific changes in gallbladder epithelial ion transport that occur during mixed gallstone formation. Fifteen prairie dogs were fed either control or corn-alfalfa chow for six months. No control animals developed gallstones or crystals. Three of eight corn-alfalfa-fed animals had large black stones, and the remaining five had crystals ("pregallstone" group). Corn-alfalfa-fed animals had significant increases in gallbladder bile cholesterol, phospholipids, and calcium as compared to controls. Gallbladders were removed and mounted in a Ussing chamber for electrophysiologic and ion flux studies. Gallbladders from animals fed corn-alfalfa demonstrated significant decreases in short-circuit current and potential difference as compared to controls (P less than 0.05). 22Na and 36Cl were used to determine unidirectional ion fluxes. While net ion fluxes were similar in pregallstone animals and controls, stone-forming animals exhibited a significant decrease in net Na+ flux and a significant reversal in the direction of net Cl- flux (from secretion to absorption) as compared to controls (P less than 0.05). These data indicate that mixed gallstone formation is associated with alterations in gallbladder ion transport. The role of these changes in the pathogenesis of mixed gallstones remains to be determined.
The marked sensitivity of Cl and fluid secretion in mammalian airways to basolateral application of loop diuretics has led to postulates that electrically neutral Na-Cl entry plays a critical role during secretion. Electrically neutral Na-Cl(K) cotransport was investigated by determining the initial rate of 22Na and 36Cl efflux in epithelial cells isolated from rabbit trachea and preequilibrated with radioactive tracer at 25 degrees C. Tracer transport was initiated by 10-fold dilution of an aliquot of cells in radioisotope-free medium. The initial rate of radiolabeled ion transport was calculated from the linear portion of efflux curves. Base-line Na and Cl transport rates were not affected by furosemide or bumetanide. l-Epinephrine stimulated Na and Cl transport rates 1.8-fold each in Ca2(+)-replete and 2.6- and 2.3-fold, respectively, in Ca2(+)-deficient transport medium. Loop diuretics and yohimbine, an alpha 2-adrenergic antagonist, blocked the effects of l-epinephrine, and, clonidine, an alpha 2-adrenergic agonist, stimulated yohimbine- and furosemide-sensitive Cl transport. The beta-adrenergic agonist l-isoproterenol alone did not affect tracer transport and, in combination with clonidine, did not affect the response to clonidine. Elevation of intracellular Ca2+ with ionomycin stimulated tracer transport, and buffering of intracellular Ca2+ with 1,2-bis(aminophenoxy)ethane- N,N,N',N'-tetraacetic acid blocked the stimulatory effects of alpha-adrenergic agents. These results indicate an alpha 2-adrenergic stimulation of loop diuretic-sensitive Na and Cl transport that requires elevated intracellular Ca2+ as the second messenger. The transport mechanism is probably a Na-Cl or Na-K-2Cl cotransport located in the basolateral membrane.
Eicosanoids have been shown to be important modulators of intestinal secretion. In cholera, cAMP is often regarded as the sole mediator, but recent data suggest that 5-hydroxytryptamine (5-HT) and prostaglandin (PG) E2 also play important roles. Thus cholera toxin (CT) increases their release from the rat jejunum in vivo, and human cholera is associated with an increased luminal 'overflow' of PGE2. In vitro evidence of secretion can be obtained with PG concentrations 100- to 1000-fold lower than those required for activation of the adenylate cyclase. Furthermore, 5-HT induces secretion associated with increased 'overflow' of PGE2, but without a change in mucosal cAMP. CT-induced release of PGE2 and fluid secretion can be decreased by indomethacin or by the 5-HT2-receptor antagonist, ketanserin, whereas the release of 5-HT and cAMP is not affected by either substance. Secretion caused by vasoactive intestinal polypeptide (VIP) is associated with increased mucosal cAMP levels, without a change in PGE2 release, and is unaffected by indomethacin and ketanserin. These results suggest that CT stimulates the release of 5-HT, which in turn causes the release of PGE2. The latter substances probably act via a local intramural reflex and contribute to secretion by mechanisms that are independent of cAMP.
Single channels in the apical cell membrane of primary cultured chick kidney cells were studied using the patch clamp technique. Cell-attached recordings revealed the presence of a 107 +/- 6 pS channel that increased fractional open time upon depolarization. Experiments with inside-out excised patches indicated that the channel is K+ selective, Ca2+ activated, and inhibited by Ba2+. The addition of forskolin or antidiuretic hormone (ADH) to the bath during cell-attached recordings caused an increase in the fractional open time of the channel. The activation of a K+ channel by increases in cAMP may be one way in which K+ secretion in the kidney is stimulated by ADH in vivo.
Guinea pig gallbladder epithelium secretes HCO3- by electroneutral mechanisms, resulting in transepithelial Cl- -HCO3- exchange. Adenosine 3',5'-cyclic monophosphate (cAMP) converts HCO3- secretion into an electrogenic process. This transformation was examined using voltage-clamp, pH-stat, and microelectrode techniques. Prostaglandin E1 (PGE1; 10(-6) M) was used to raise intracellular cAMP levels. It increased short-circuit current (Isc) by approximately 1.8 mumol.cm-2.h-1, an effect dependent on serosal HCO3- and, partly, on mucosal Cl-. Mucosal 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS; 10(-3) M) halved Isc, but only in Cl- containing solutions. PGE1 increased the secretory HCO3- flux from approximately 2.0 to approximately 2.7 mumol.cm-2.h-1 and reduced the absorptive HCO3- flux from approximately 1.1 to approximately 0.5 mumol.cm-2.h-1, with net HCO3- secretion accounting for the increase in Isc. During single-cell impalements, PGE1 depolarized the apical membrane by greater than 10 mV (transiently in the absence of HCO3-) and decreased the apparent ratio of membrane resistances (Ra/Rb) from 5-8 to a value close to zero. These effects were largely reduced in magnitude and rapidity by removing Cl- and HCO3- from both sides of the epithelium. Ion substitutions in the luminal perfusate revealed substantial Cl- and HCO3- permeabilities at the apical membrane under PGE1 conditions. Our results indicate that, in the presence of PGE1 (cAMP), HCO3- crosses the apical membrane by two different routes. A SITS-sensitive fraction leaves the cell in exchange for luminal Cl-, which, in turn, recycles into the lumen by electrodiffusion. The remaining HCO3- exits through a HCO3- conductive pathway.
Loperamide inhibits PGE1-induced electrogenic HCO3 secretion in guinea-pig gallbladder. Underlying changes in epithelial cell membrane properties were investgated using intracellular microelectrode techniques in vitro. In the absence of PGE1, mucosal loperamide (10−4 mol/l) reversibly depolarized both cell membranes by ∼ 6 mV. The apparent ratio of membrane resistances (R
a/R
b) remained unchanged and so did voltage responses to luminal Cl removal and Na reduction. The depolarizing response to elevation of luminal K concentration from 5 to 76 mmol/l was decreased from 13 to 8 mV. In the presence of 1 PGE1, the apical membrane is mainly permeable to Cl and HCO3. Under these conditions, loperamide reduced membrane potentials by ∼ 10 mV,R
a/R
b remaining constant at ∼ 0.4. Effects on voltage responses to changes in luminal Na or K concentration were unchanged. Responses to luminal Cl removal (transient depolarization) were greatly enhanced (from 22 to 42 mV) as predictable from the fall in K permeability that hinders Cl efflux from cell into lumen. Less marked but significant effects were obtained with 10−5 mol/l (mucosal side) and serosal loperamide (10−4 mol/l). We suggest that loperamide inhibits electrogenic HCO3 secretion by reducing apical membrane K permeability. The resulting depolarization diminishes the driving force for conductive anion efflux from cell into lumen. This conclusion is supported by the ability of luminal K elevation to mimick loperamide inhibition of the secretory flux of HCO3 (pH-stat experiments).
Effects of adenosine 3',5'-cyclic monophosphate (cAMP) on single Ca-activated K current (IK(Ca)) in cultured smooth muscle cells of the rat aorta were investigated with the patch-clamp technique. In cell-attached patch configurations, extracellular application of isoproterenol (10(-5) M) increased the Ca-activated K currents. The increase in the currents was due to an increase in the probability of channel openings (Po). Neither unit conductance nor the maximum number of the channel in the patch was affected by the drug. The effects were inhibited by adding propranolol (10(-6) M). The extracellular application of forskolin (10(-5) M) or dibutyryl cAMP (10(-4) M) mimicked the effects of isoproterenol. In inside-out patch configurations, activated cAMP-dependent protein kinase (A kinase) in the bathing solution increased the sensitivity of the Ca-activated K channels to intracellular free calcium concentration ([Ca]i) and enhanced Po. Kinetic analyses of the IK(Ca) showed that cAMP-dependent phosphorylation of the Ca-activated K channels significantly reduced the mean closed time between bursting openings. We conclude from these observations that the Ca-activated K channels in aortic cells may increase Po through cAMP-dependent phosphorylation.
Recent studies indicate that gallbladder absorption increases during the early stages of experimentally-induced cholesterol gallstone formation. The purpose of the present study was to ascertain whether pharmacologic inhibition of gallbladder ion transport and absorption reduces the incidence of experimentally-induced cholesterol gallstones. Prairie dogs were fed either a control chow or a 1.2% cholesterol-enriched chow for 15 days. One group of cholesterol-fed animals received saline via an orogastric tube; another group received amiloride, a drug known to inhibit in vitro ion transport in the prairie dog gallbladder. The incidence of gallstones in cholesterol-fed animals was reduced from 83% to 13% (p less than 0.025) when the animals were treated with amiloride; this occurred despite a cholesterol-saturation index comparable to that observed in gallstone animals. Additionally, although biliary calcium decreased in the gallbladder, hepatic bile did not in the amiloride-treated animals. These data provide further evidence that altered gallbladder absorption and increased biliary calcium are important factors in the pathogenesis of cholesterol gallstones.
The properties and, in particular, the cAMP-sensitivity of apical Na/H exchange in guinea-pig gallbladder epithelium were investigated using gravimetric, pH-stat, and microelectrode techniques. Proton secretion was Na-dependent, inhibited by ouabain and amiloride, and insensitive to changes in apical membrane potential. It was markedly reduced by 8-Br-cAMP and PGE1. PGE1 also attenuated the changes in intracellular Na activity produced by luminal Na removal and restoration. Our results suggest inhibition of Na/H exchange by cAMP. In conjunction with the cAMP-induced rise in apical membrane Cl permeability shown previously, this effect can account for inhibition of NaCl absorption by cAMP.
The effects of bathing solution HCO3-/CO2 concentrations on baseline cell membrane voltages and resistances were measured in Necturus gallbladder epithelium with conventional intracellular microelectrode techniques. Gallbladders were bathed in either low HCO3-/CO2 Ringer's solutions (2.4 mM HCO3-/air or 1 mM HEPES/air) or a high HCO3-/CO2 Ringer's (10 mM HCO3-/1% CO2). The principal finding of these studies was that the apical membrane fractional resistance (fRa) was higher in tissues bathed in the 10 mM HCO3-/CO2 Ringer's, averaging 0.87 +/- 0.06, whereas fRa averaged 0.63 +/- 0.07 and 0.48 +/- 0.08 in 2.4 mM HCO3- and 1 mM HEPES, respectively. Intraepithelial cable analysis was employed to obtain estimates of the individual apical (Ra) and basolateral membrane (Rb) resistances in tissues bathed in 10 mM HCO3-/1% CO2 Ringer's. Compared to previous resistance measurements obtained in tissues bathed in a low HCO3-/CO2 Ringer's, the higher value of fRa was found to be due to both an increase in Ra and a decrease in Rb. The higher values of fRa and lower values of Rb confirm the recent observations of others. To ascertain the pathways responsible for these effects, cell membrane voltages were measured during serosal solution K+ and Cl- substitutions. The results of these studies suggest that an electrodiffusive Cl- transport mechanism exists at the basolateral membrane of tissues bathed in a 10 mM HCO3-/1% CO2 Ringer's, which can explain in part the fall in Rb. The above observations are discussed in terms of a stimulatory effect of solution [HCO3-]/PCO2 on transepithelial fluid transport, which results in adaptive changes in the conductive properties of the apical and basolateral membranes.
In this review, I focus on how airway epithelia transport electrolytes and what regulates this transport. This is a relatively new topic in physiology. Researchers have recognized only recently that airways are more than conduits for airflow to the alveoli, the first work on airway transport properties having been published in 1975 (118). I primarily review the literature but also emphasize uncertainties and gaps in our knowledge. This approach may help focus important but unanswered questions. I occasionally refer to other epithelia, when a comparison or analog may help our understanding of airway epithelia. At the end, I briefly address the pathophysiology of airway epithelia because abnormal electrolyte transport is part of several airway diseases. I do not cover mucus secretion.
1. Sodium ion (Na+) transport, a principal function of the gallbladder epithelium, was studied by measuring the flux of 22Na across isolated, inflamed human gallbladder mucosa maintained in a modified ‘Ussing’ flux chamber. Tissue was obtained from cholecystectomy specimens in symptomatic patients with cholelithiasis.
2. In 30 gallbladders studied, 57% had a net Na+ flux from mucosa to serosa (Na+ absorption), while 23% had a net Na+ flux from serosa to mucosa (Na+ secretion). The remaining 20% showed no overall net Na+ flux.
3. Indomethacin added to the serosal fluid reversed the direction of net Na+ flux in secreting gallbladders and caused an absorption of Na+. In Na+-absorbing gallbladders, indomethacin caused a slight reduction in Na+ absorption. No change in Na+ flux was induced in gallbladders with no initial net Na+ flux.
4. These results demonstrate that instead of absorbing Na+, some inflamed human gallbladders may secrete Na+. As this secretion can be reversed to the more usual absorption by indomethacin, it is likely that this secretion is mediated by prostaglandins.
The hypothesis that the presence of cholelithogenic bile during the early stages of cholesterol gallstone formation promotes gallbladder absorption of water and electrolytes was tested in a prairie dog gallstone model. An increase in gallbladder transport of water and sodium was observed in cholesterol-fed prairie dogs at a time when cholesterol crystals were present, but before gallstone formation. These data suggest that in the presence of cholesterol-saturated bile, in vivo gallbladder absorption is increased during the early stages of cholesterol gallstone formation. The resulting increase in the solute concentration may promote nucleation and, therefore, be an important etiologic factor in cholesterol gallstone formation.
Transepithelial HCO3- movement in guinea pig gallbladder was investigated in vitro. Absorptive (JHCO3ms) and secretory (JHCO3sm) HCO3- fluxes, determined by use of the pH-stat method were approximately 1.0 and 2.1 mumol X cm-2 X h-1, respectively. The resultant net secretion equaled in magnitude, and balanced electrically, the excess in net absorption of Cl- over that of Na+ X JHCO3sm was dependent on luminal Cl- and serosal Na+; it was inhibited by mucosal 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS; 10(-3) M) and serosal ouabain (3 X 10(-5) M) but not by serosal amiloride (10(-3) M) and scarcely by bilateral methazolamide (10(-4) M). JHCO3ms was reduced by mucosal Cl- but enhanced by serosal Cl-; it was dependent on mucosal Na+ and inhibited by mucosal amiloride and bilateral methazolamide. Our findings are consistent with a model in which 1) serosal HCO3- enters the cell in cotransport with Na+ and is then extruded into the lumen by Cl-(-)HCO3- exchange at the apical membrane; 2) mucosal HCO3- enters the cell secondary to apical membrane Na+-H+ exchange and recycles into the lumen via Cl-(-)HCO3- exchange or is, to a lesser extent, absorbed across the basolateral membrane.
In the presence of indomethacin serosal application of PGE1 and PGE2 caused significant inhibition of net isosmotic fluid transport by the gall bladder with maximum effect (50% inhibition) at 0.5 μg PGE/ml. Mucosal application was much less effective. PGF2α was about 100 times less potent than PGE. In the absence of indomethacin the sensitivity to low concentrations of PGE1 was about 10 times less than in the presence of this drug (p < 0.001). Maximum effective doses of PGE1 depressed the unidirectional Na+ efflux about 35% and the Na+ influx about 20%. Transepithelial potential difference and ohmic resistance remained unchanged. It is concluded that PGE inhibits isosmotic fluid transport by an effect mainly on the active component of the transfer process. In the absence of indomethacin endogenous PGE2 (but not PGE1) was released to both serosal and mucosal medium. Net release to mucosal medium was 35-600 pg/hr-1 mg dry weight-1; release was higher to the serosal medium. Mucosal concentrations of 0.2-4.5 ng PGE2/ml were independent of the serosal concentration, which did not exceed 0.8 ng/ml under the present conditions. The data suggest that mucosal PGE2 release originates, in part at least, from the epithelial cells. Indomethacin (10 μg/ml) blocked almost completely the in vitro PGE biosynthesis.
1. The effects of low amiloride concentrations (less than 2.5 x 10(-5) M) on the apical membrane (Vm) and transepithelial (Vms) potential differences, transepithelial resistance (Rep) and apical/basolateral membrane resistance ratio (Rm/Rs) have been determined in the gall-bladders of the rabbit and guinea-pig. 2. Vm was hyperpolarized, Vms decreased towards negative values, Rm/Rs increased, but Rep remained unchanged. 3. K+ selectivity of the apical membrane was also checked by increasing luminal K+ concentration from 5.9 to 29.4 mM; the corresponding change in the apical electromotive force was much higher in the rabbit than in the guinea-pig. 4. Pre-incubations with Cl--and HCO3--free salines did not modify K+ selectivity in the rabbit, but nearly doubled it in the guinea-pig. 5. Pre-incubations with control solutions containing 10(-5) M-amiloride in the lumen converted the apical membrane of rabbit gall-bladder into a perfectly K+-perm-selective membrane; the same results were obtained in the guinea-pig only on bathing the tissue with Cl-- and HCO3--free salines together with 10(-5) M-amiloride. 6. It is suggested that in the apical membrane of rabbit gall-bladder K+ and Na+ conductances exist, whereas in guinea-pig gall-bladder both K+, Na+ and anion (Cl- and/or HCO3-) conductances are present; in both species conductive Na+ pathways are inhibited by amiloride. .
Nervous or hormonal stimulation of many exocrine glands evokes release of cellular K+ (ref. 1), as originally demonstrated in mammalian salivary glands2,3, and is associated with a marked increase in membrane conductance1,4,5. We now demonstrate directly, by using the patch-clamp technique6, the existence of a K+ channel with a large conductance localized in the baso-lateral plasma membranes of mouse and rat salivary gland acinar cells. The K+ channel has a conductance of approximately 250 pS in the presence of high K+ solutions on both sides of the membrane. Although mammalian exocrine glands are believed not to possess voltage-activated channels1,7, the probability of opening the salivary gland K+ channel was increased by membrane depolarization. The frequency of channel opening, particularly at higher membrane potentials, was increased markedly by elevating the internal ionized Ca2+ concentration, as previously shown for high-conductance K+ channels from cells of neural origin8-10. The Ca2+ and voltage-activated K+ channel explains the marked cellular K+ release that is characteristically observed when salivary glands are stimulated to secrete.
In NaCl-absorbing epithelia such as proximal renal tubule, small intestine and gallbladder, Na+-dependent Cl- entry across the luminal membrane is an electroneutral transport process that has been attributed to Na-Cl symport, Na-K-Cl symport, or a double (Na-H, Cl-HCO3) antiport. At the basolateral (antiluminal) membrane, Na+ extrusion can be attributed to the Na+-K+ pump, and Cl- transport could be explained in principle by electrodiffusion since the intracellular Cl- activity is higher than predicted for equilibrium distribution. However, in Necturus gallbladder, as in other epithelia, the electrodiffusional Cl- permeability of the membrane (PCl) is too low to account for the transepithelial Cl- transport rate. Because K+ is at a higher chemical potential in the cell than in the extracellular fluid, and because serosal Cl- substitutions have only small effects on membrane potential, the hypothesis of carrier-mediated electroneutral KCl co-transport was proposed. The experiments reported here were designed to test this hypothesis in Necturus gallbladder epithelium. Intracellular Cl- and K+ activities (aCli, aKi) were measured with ion-sensitive intracellular microelectrodes before, during and after ionic substitutions of the serosal (basolateral) bathing medium. The results demonstrate a Na+-independent basolateral membrane KCl symport.
The stimulating effect of external HCO3- on Na+ salt transport has been examined in rabbit and guinea-pig gall-bladder by electrophysiological methods, as a sequel to a previous study carried out by radiochemical techniques. At steady state, cell K+ activity was found to be significantly reduced in the presence of HCO3-, whereas cell Na+ activity significantly increased; in parallel the apical membrane p.d. was depolarized; K+ equilibrium potential was higher than membrane p.d. in every case. The apical p.d. dependence on K+ was unaffected by HCO3-, but in the guinea-pig it was affected by Cl-. Rapid increases in HCO3- concentration on the luminal side caused a depolarization of the apical p.d. of the guinea-pig within about 30 sec, an effect that did not occur if the tissue was pre-treated with 10(-4) M-acetazolamide; the epithelial resistance and apical/basolateral resistance ratio were unchanged in all cases. The primary action of HCO3- is confirmed to be on the apical membrane; an HCO3- conductance does not seem to be present at this level, either in the rabbit or guinea-pig, nor does HCO3- affect Na+ influx through the apical conductive pathway, so that all the stimulating effects of the anion are confirmed to be on the neutral transports of Na+ salts; in spite of this, the apical electromotive force is modified due to the changed cell K+ activity. The rapid depolarization caused by the anion in the guinea-pig is in agreement with an HCO3- electrogenic secretion and/or a basolateral conductance for the anion. Polyelectrolyte dissociation from protons increases in the absence of external HCO3-: the negative charges are mainly counterbalanced by bound Na+ in the rabbit and by free K+ in the guinea-pig. K+ leakage from the cell into the lumen is calculated to be minimal in the rabbit and all K+ lost could be reabsorbed through the paracellular pathways; K+ efflux to the subepithelial layer via conductive routes is insufficient to account for the over-all K+ efflux.
The intracellular pH in animal cells in generally maintained at a higher level than would be expected if H+ were passively distributed across the plasma membrane. In a wide variety of cells including sea urchin eggs, skeletal muscle, renal and intestinal epithelial cells, and neuroblastoma cells, plasma membrane Na+-H+ exchangers mediate the uphill extrusion of H+ coupled to, and thus energized by, the downhill entry of Na+. Plasma membrane vesicles isolated from the luminal (microvillus, brush border) surface of renal proximal tubular cells possess a Na+-H+ exchanger that seems to be representative of the Na+-H+ exchangers found in other tissues. For example, the renal microvillus membrane Na+-H+ exchanger, like other Na+-H+ exchangers, mediates electroneutral cation exchange, is sensitive to inhibition by the diuretic drug amiloride, and has affinity for Li+ in addition to Na+ and H+ (refs 5, 9). Here we have examined the effect of internal H+ on the activity of the Na+-H+ exchanger in renal microvillus membrane vesicles. Our results suggest that internal H+, independent of its role as a substrate for exchange with external independent of its role as a substrate for exchange with external independent of its role as a substrate for exchange with external Na+, has an important modifier role as an allosteric activator of the Na+-H+ exchanger. Allosteric behaviour with respect to internal H+ is a property that would enhance the ability of plasma membrane Na+-H+ exchangers to extrude intracellular acid loads and thereby contribute to the regulation of intracellular pH.
Cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels appear to be regulated by hydrolysis of ATP and are inhibited by a product of hydrolysis, ADP. We assessed the effect of the other product of hydrolysis, inorganic phosphate (P(i)), on CFTR Cl- channel activity using the excised inside-out configuration of the patch-clamp technique. Millimolar concentrations of P(i) caused a dose-dependent stimulation of CFTR Cl- channel activity. Single-channel analysis demonstrated that the increase in macroscopic current was due to an increase in single-channel open-state probability (po) and not single-channel conductance. Kinetic modeling of the effect of P(i) using a linear three-state model indicated that the effect on po was predominantly the result of an increase in the rate at which the channel passed from the long closed state to the bursting state. P(i) also potentiated activity of channels studied in the presence of 10 mM ATP and stimulated Cl- currents in CFTR mutants lacking much of the R domain. Binding studies with a photoactivatable ATP analog indicated that Pi decreased the amount of bound nucleotide. These results suggest that P(i) increased CFTR Cl- channel activity by stimulating a rate-limiting step in channel opening that may occur by an interaction of P(i) at one or both nucleotide-binding domains.
In a voltage clamp setting (Ussing chamber), the antidiarrhoeal drug, loperamide (Lop) slightly augmented prostaglandin E 1 (PGE 1 ) plus theophylline‐stimulated net chloride secretion above control values at low concentrations (10 ⁻¹⁰ and 10 ⁻⁹ m ) but inhibited it at higher concentrations (10 ⁻⁶ and 10 ⁻⁵ m ). The apparently weak prosecretory action component of Lop was turned into a clear cut antisecretory effect by pretreatment with 2 times 10 ⁻⁷ m naloxonazine plus 10 ⁻⁷ m CTOP‐NH 2 , two selective μ opioid receptor antagonists. This indicates a prosecretory effect of loperamide at μ opioid receptors. The antisecretory effect of low Lop concentrations, uncovered by μ opioid receptor blockade, was prevented by additional blockade of κ opioid receptors by 5 times 10 ⁻⁹ m nor‐binaltorphmiine (nor‐BNI).
The nonselective opioid antagonist, naloxone, at 10 ⁻⁶ m did not significantly reduce either PGE 1 plus theophylline‐stimulated net chloride secretion in Lop‐free controls or the antisecretory action of Lop. By contrast, the partial agonist ethylketocyclazocine (EKC), which activates κ but blocks μ opioid receptors, concentration‐dependently inhibited PGE 1 plus theophylline‐stimulated net chloride secretion without any consistent prosecretory action component. Nor‐BNI at 5 times 10 ⁻⁸ m significantly blocked the antisecretory action of EKC.
It is concluded that, in the guinea‐pig colonic mucosa under the present conditions, μ opioid receptors mediate enhancement and κ opioid receptors inhibition of PGE 1 ‐stimulated net chloride secretion by low Lop concentrations. The two opposite actions are largely masked by superimposition. An opioid receptor‐independent mechanism of action contributes to the antisecretory effect of Lop at high concentrations.
Mucin is the main secretory product of gallbladder epithelial cells. Increased gallbladder mucus secretion has been implicated in gallstone formation in humans. The mechanisms underlying control of mucin synthesis and secretion by the gallbladder are not known. This study aimed to elucidate the efficacy of a panel of secretagogues to stimulate mucin secretion and to determine the intracellular second messengers involved. Studies were carried out on normal well-differentiated epithelial cells from dog gallbladder grown in monolayer culture. Intracellular adenosine 3',5'-cyclic monophosphate (cAMP) as measured by radioimmunoassay increased in response to prostaglandin (PG) E2, PGE1, vasoactive intestinal peptide, epinephrine, and isoproterenol. The greatest effect, a 37-fold increase in cAMP level, was noted with PGE2 at 1.0 microM concentration. In contrast, three breakdown products of phosphatidylinositol (inositol triphosphate, inositol bisphosphate and inositol monophosphate) were not detected with any of the secretagogues tested. Assay of mucin secretion using tritiated N-acetyl-D-glucosamine, a mucin precursor, showed that the same secretagogues noted to increase intracellular cAMP led to an increase in mucin secretion. No correlation was noted, however, between the magnitude of the intracellular cAMP rise and the amount of mucin secreted. A membrane-permeable form of cAMP, dibutyryl cAMP, mimicked PGE2-induced mucin secretion. The results unequivocally show that secretagogue-stimulated mucin secretion in these normal gallbladder epithelial cells can proceed via a cAMP signal transduction pathway.
1. Whole-cell currents were investigated in the model salt-secreting epithelium, human T84 cell line, by means of the perforated patch-clamp technique. In the control extracellular medium containing Cl-, depolarizing voltage ramps evoked current responses which peaked at 5.43 +/- 0.81 pA pF-1 at +60 mV and had a reversal potential (Erev) of -38.4 +/- 2.5 mV (n = 23). 2. Activation of the cAMP pathway with forskolin increased the current at +60 mV from 3.81 +/- 0.61 to 20.79 +/- 5.08 pA pF-1 (n = 18). In thirteen cells, Erev was initially shifted towards positive potentials (Erev of the cAMP-activated initial current was -18.2 +/- 1.2 mV) and subsequently shifted towards more negative potentials, consistent with the activation of both Cl- and K+ currents during cAMP stimulation. 3. Increasing the intracellular Ca2+ concentration, [Ca2+]i, with ionomycin (1 microM) or with acetylcholine (1 microM), increased the current at +60 mV from 7.79 +/- 1.57 to 57.50 +/- 12.10 pA pF-1 (n = 6) and from 6.36 to 34.13 pA pF-1 (n = 4), respectively. With both agonists, Erev was shifted either towards the reversal potential for potassium, EK, or towards the reversal potential for chloride, ECl, depending on the cell. 4. In the absence of chloride ions (gluconate substituted), stimulation of the Ca2+ pathway activated a time-independent outward current of large amplitude. This current exhibited inward rectification at positive voltages, reverted at -89.5 +/- 0.2 mV and was markedly reduced by charybdotoxin (10 nM), a specific blocker of Ca(2+)-activated K+ channels. When a voltage step protocol was used, increased [Ca2+]i also activated an outward current at potentials more positive than -40 mV which slowly relaxed during depolarizing steps. 5. The activation of both (i) a time-dependent inwardly rectifying charybdotoxin-sensitive K+ current, and (ii) a time-dependent slowly inactivating current was also produced by cAMP stimulation. 6. We concluded that (i) in the T84 epithelial cells, both Cl- and K+ currents are concomitantly increased by secretagogue stimulation, and (ii) two different types of K+ conductances are activated by either the cAMP or the intracellular Ca2+ secreting pathways.
Hepatobiliary complications in cystic fibrosis result predominantly from lesions of the biliary epithelium. These abnormalities affect the intrahepatic as well as extrahepatic bile ducts and the gallbladder. The protein cystic fibrosis transmembrane conductance regulator (CFTR), the gene product defective in cystic fibrosis, functions as a cAMP-activated chloride channel in the plasma membrane. As such, it may represent an important driving force for fluid transport across the epithelium.
The purpose of this study was to investigate the expression of CFTR in human gallbladder epithelial cells and to examine the chloride ion transport properties of these cells. Immunolocalization was performed on tissue sections. The reverse transcription-PCR was used to analyze the expression of CFTR mRNA in freshly isolated and cultured gallbladder epithelial cells. The CFTR protein was detected by Western blotting and immunoprecipitation. The chloride ion transport properties of the cells were determined by 36Cl efflux studies.
The CFTR protein was immunodetected in human gallbladder in situ and localized predominantly to the apical membrane of epithelial cells. High levels of CFTR mRNA and protein were maintained in gallbladder epithelial cells in primary cultured. Glycosylated forms of CFTR were present as confirmed by treatment with N-glycanase. Chloride efflux was stimulated by Ca(++)-dependent pathways but more intensely by cAMP-dependent pathways. Stimulation of chloride efflux by agonist of the cAMP-pathway was inhibited by diphenylamine carboxylic acid, a chloride channel blocker. Two physiologically active peptides--acting via cAMP, vasoactive intestinal peptide, and secretin--also stimulated chloride efflux in vitro.
Our results are consistent with a high expression of endogenous functional CFTR protein in human gallbladder epithelial cells. Physiologically active peptides, vasoactive intestinal peptide and secretin, stimulate chloride conductance in these cells. These findings indicate that CFTR play an important role in the pathophysiology of the biliary epithelium, including the gallbladder epithelium.