Prevalence of Staphylococcus aureus in wild hedgehogs (Erinaceus europaeus) and first report of mecC-MRSA in Hungary.

In 2011 mecC, a new mecA gene homologue, was described in a bovine isolate in the UK. Since then, mecC-positive methicillin-resistant Staphylococcus aureus (mecC-MRSA) has also been found in wild animals. An especially high prevalence of mecC-MRSA has been reported among hedgehogs in Sweden (64%) and Denmark (61%). Based on these findings we aimed to survey the hedgehog population for mecC-MRSA in Hungary. Altogether 200 hedgehogs were screened for Staphylococcus aureus using a culture-based method. The antibiotic susceptibility of the isolates to nine drugs was determined, their genetic relatedness was established by PFGE and spa-typing, and virulence genes were identified by PCR. Whole genome sequencing was performed for the single mecC-MRSA isolate found. Of the 200 animals, 13 were carriers of S. aureus (6.5%). Among these, one isolate was mecA positive and one was mecC positive. The isolates were susceptible to non-beta-lactam antibiotics. Toxin genes were not found, but the majority carried genes responsible for adhesion and biofilm production. The mecC-MRSA isolate was a single-locus variant of ST130, had a new spa type (t19701) and belonged to SCCmec type XI. It carried a recently described, novel exfoliative toxin (etE). This is the first report of mecC-MRSA in Hungary and the first survey of staphylococcus carriage among wild animals in the country. The mecC prevalence was much lower than in Northern European countries and rather similar to other countries in our region. MecC-MRSA could potentially emerge as a novel human pathogen, especially where close contact occurs between humans and animals.

Bicarbonate Evokes Reciprocal Changes in Intracellular Cyclic di-GMP and Cyclic AMP Levels in Pseudomonas aeruginosa.

The formation of Pseudomonas aeruginosa biofilms in cystic fibrosis (CF) is one of the most common causes of morbidity and mortality in CF patients. Cyclic di-GMP and cyclic AMP are second messengers regulating the bacterial lifestyle transition in response to environmental signals. We aimed to investigate the effects of extracellular pH and bicarbonate on intracellular c-di-GMP and cAMP levels, and on biofilm formation. P. aeruginosa was inoculated in a brain−heart infusion medium supplemented with 25 and 50 mM NaCl in ambient air (pH adjusted to 7.4 and 7.7 respectively), or with 25 and 50 mM NaHCO3 in 5% CO2 (pH 7.4 and 7.7). After 16 h incubation, c-di-GMP and cAMP were extracted and their concentrations determined. Biofilm formation was investigated using an xCelligence real-time cell analyzer and by crystal violet assay. Our results show that HCO3− exposure decreased c-di-GMP and increased cAMP levels in a dose-dependent manner. Biofilm formation was also reduced after 48 h exposure to HCO3−. The reciprocal changes in second messenger concentrations were not influenced by changes in medium pH or osmolality. These findings indicate that HCO3− per se modulates the levels of c-di-GMP and cAMP, thereby inhibiting biofilm formation and promoting the planktonic lifestyle of the bacteria.

Three-Dimensional Culture of Ameloblast-Originated HAT-7 Cells for Functional Modeling of Defective Tooth Enamel Formation.

Background: Amelogenesis, the formation of dental enamel, is well understood at the histomorphological level but the underlying molecular mechanisms are poorly characterized. Ameloblasts secrete enamel matrix proteins and Ca2+, and also regulate extracellular pH as the formation of hydroxyapatite crystals generates large quantities of protons. Genetic or environmental impairment of transport and regulatory processes (e.g. dental fluorosis) leads to the development of enamel defects such as hypomineralization. Aims: Our aims were to optimize the culture conditions for the three-dimensional growth of ameloblast-derived HAT-7 cells and to test the effects of fluoride exposure on HAT-7 spheroid formation. Methods: To generate 3D HAT-7 structures, cells were dispersed and plated within a Matrigel extracellular matrix scaffold and incubated in three different culture media. Spheroid formation was then monitored over a two-week period. Ion transporter and tight-junction protein expression was investigated by RT-qPCR. Intracellular Ca2+ and pH changes were measured by microfluorometry using the fluorescent dyes fura-2 and BCECF. Results: A combination of Hepato-STIM epithelial cell differentiation medium and Matrigel induced the expansion and formation of 3D HAT-7 spheroids. The cells retained their epithelial cell morphology and continued to express both ameloblast-specific and ion transport-specific marker genes. Furthermore, like two-dimensional HAT-7 monolayers, the HAT-7 spheroids were able to regulate their intracellular pH and to show intracellular calcium responses to extracellular stimulation. Finally, we demonstrated that HAT-7 spheroids may serve as a disease model for studying the effects of fluoride exposure during amelogenesis. Conclusion: In conclusion, HAT-7 cells cultivated within a Matrigel extracellular matrix form three-dimensional, multi-cellular, spheroidal structures that retain their functional capacity for pH regulation and intracellular Ca2+ signaling. This new 3D model will allow us to gain a better understanding of the molecular mechanisms involved in amelogenesis, not only in health but also in disorders of enamel formation, such as those resulting from fluoride exposure.

TRPM7-Mediated Calcium Transport in HAT-7 Ameloblasts.

TRPM7 plays an important role in cellular Ca2+, Zn2+ and Mg2+ homeostasis. TRPM7 channels are abundantly expressed in ameloblasts and, in the absence of TRPM7, dental enamel is hypomineralized. The potential role of TRPM7 channels in Ca2+ transport during amelogenesis was investigated in the HAT-7 rat ameloblast cell line. The cells showed strong TRPM7 mRNA and protein expression. Characteristic TRPM7 transmembrane currents were observed, which increased in the absence of intracellular Mg2+ ([Mg2+]i), were reduced by elevated [Mg2+]i, and were inhibited by the TRPM7 inhibitors NS8593 and FTY720. Mibefradil evoked similar currents, which were suppressed by elevated [Mg2+]i, reducing extracellular pH stimulated transmembrane currents, which were inhibited by FTY720. Naltriben and mibefradil both evoked Ca2+ influx, which was further enhanced by the acidic intracellular conditions. The SOCE inhibitor BTP2 blocked Ca2+ entry induced by naltriben but not by mibefradil. Thus, in HAT-7 cells, TRPM7 may serves both as a potential modulator of Orai-dependent Ca2+ uptake and as an independent Ca2+ entry pathway sensitive to pH. Therefore, TRPM7 may contribute directly to transepithelial Ca2+ transport in amelogenesis.

STRO-1 positive cell expansion during osteogenic differentiation: A comparative study of three mesenchymal stem cell types of dental origin.

OBJECTIVE: Although the osteogenic differentiation potential of mesenchymal stem cells of dental origin is well established, the roles of different marker proteins in this process remain to be clarified. Our aim was to compare the cellular and molecular changes, focusing in particular on mesenchymal stem cell markers, during in vitro osteogenesis in three dental stem cell types: dental follicle stem cells (DFSCs), periodontal ligament stem cells (PDLSCs) and dental pulp stem cells (DPSCs). DESIGN: Human DFSCs, PDLSCs and DPSCs were isolated, cultured and their osteogenic differentiation was induced for 3 weeks. Mineralization was assessed by von Kossa staining and calcium concentration measurements. The expression of mesenchymal and osteogenic markers was studied by immunocytochemistry and qPCR techniques. Alkaline phosphatase (ALP) activity and the frequency of STRO-1 positive cells were also quantified. RESULTS: The three cultures all showed abundant mineralization, with high calcium content by day 21. The expression of vimentin and nestin was sustained after osteogenic induction. The osteogenic medium induced a considerable elevation of STRO-1 positive cells. By day 7, the ALP mRNA level had increased more than 100-fold in DFSCs, PDLSCs, and DPSCs. Quantitative PCR results indicated dissimilarities of osteoblastic marker levels in the three dental stem cell cultures. CONCLUSIONS: DFSCs, PDLSCs and DPSCs have similar functional osteogenic differentiation capacities although their expressional profiles of key osteogenic markers show considerable variations. The STRO-1 positive cell fraction expands during osteogenic differentiation while vimentin and nestin expression remain high. For identification of stemness, functional studies rather than marker expressions are needed.

Saliva as a Candidate for COVID-19 Diagnostic Testing: A Meta-Analysis.

Background: COVID-19 is a serious and potentially deadly disease. Early diagnosis of infected individuals will play an important role in stopping its further escalation. The present gold standard for sampling is the nasopharyngeal swab method. However, several recent papers suggested that saliva-based testing is a promising alternative that could simplify and accelerate COVID-19 diagnosis. Objectives: Our aim was to conduct a meta-analysis on the reliability and consistency of SARS-CoV-2 viral RNA detection in saliva specimens. Methods: We have reported our meta-analysis according to the Cochrane Handbook. We searched the Cochrane Library, Embase, Pubmed, Scopus, Web of Science and clinical trial registries for eligible studies published between 1 January and 25 April 2020. The number of positive tests and the total number of tests conducted were collected as raw data. The proportion of positive tests in the pooled data were calculated by score confidence-interval estimation with the Freeman-Tukey transformation. Heterogeneity was assessed using the I 2 measure and the χ2-test. Results: The systematic search revealed 96 records after removal of duplicates. Twenty-six records were included for qualitative analysis and 5 records for quantitative synthesis. We found 91% (CI 80-99%) sensitivity for saliva tests and 98% (CI 89-100%) sensitivity for nasopharyngeal swab (NPS) tests in previously confirmed COVID-19 patients, with moderate heterogeneity among the studies. Additionally, we identified 18 registered, ongoing clinical trials of saliva-based tests for detection of the virus. Conclusion: Saliva tests offer a promising alternative to NPS for COVID-19 diagnosis. However, further diagnostic accuracy studies are needed to improve their specificity and sensitivity.

Author Correction: Organic osmolytes preserve the function of the developing tight junction in ultraviolet B-irradiated rat epidermal keratinocytes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Organic osmolytes preserve the function of the developing tight junction in ultraviolet B-irradiated rat epidermal keratinocytes.

Epidermal barrier function is provided by the highly keratinised stratum corneum and also by tight junctions (TJs) in the granular layer of skin. The development of the TJ barrier significantly deteriorates in response to ultraviolet B radiation (UVB). Following exposure to UVB, keratinocytes accumulate organic osmolytes, which are known to preserve cell volume during water stress. Since TJs are intimately associated with control of water homeostasis in skin, we hypothesised that there may be a direct influence of osmolytes on TJ development. Exposure of rat epidermal keratinocytes (REKs) to a single dose of UVB reduced the function of developing TJs. This was concomitant with dislocalisation of claudin-1 and claudin-4 from the keratinocyte plasma membrane, phosphorylation of occludin and elevation of reactive oxygen species (ROS). In the presence of organic osmolytes, these effects were negated but were independent of the effects of these molecules on cell volume, elevation of ROS or the gene expression of TJ proteins. These data suggest that organic osmolytes affect TJs via post-translational mechanism(s) possibly involving protection of the native conformation of TJ proteins.

Bicarbonate-rich fluid secretion predicted by a computational model of guinea-pig pancreatic duct epithelium.

KEY POINTS: The ductal system of the pancreas secretes large volumes of alkaline fluid containing HCO3- concentrations as high as 140 mm during hormonal stimulation. A computational model has been constructed to explore the underlying ion transport mechanisms. Parameters were estimated by fitting the model to experimental data from guinea-pig pancreatic ducts. The model was readily able to secrete 140 mm HCO3- . Its capacity to do so was not dependent upon special properties of the cystic fibrosis transmembrane conductance regulator (CFTR) anion channels and solute carrier family 26 member A6 (SLC26A6) anion exchangers. We conclude that the main requirement for secreting high HCO3- concentrations is to minimize the secretion of Cl- ions. These findings help to clarify the mechanism responsible for pancreatic HCO3- secretion, a vital process that prevents the formation of protein plugs and viscous mucus in the ducts, which could otherwise lead to pancreatic disease. ABSTRACT: A computational model of guinea-pig pancreatic duct epithelium was developed to determine the transport mechanism by which HCO3- ions are secreted at concentrations in excess of 140 mm. Parameters defining the contributions of the individual ion channels and transporters were estimated by least-squares fitting of the model predictions to experimental data obtained from isolated ducts and intact pancreas under a range of experimental conditions. The effects of cAMP-stimulated secretion were well replicated by increasing the activities of the basolateral Na+ -HCO3- cotransporter (NBC1) and apical Cl- /HCO3- exchanger (solute carrier family 26 member A6; SLC26A6), increasing the basolateral K+ permeability and apical Cl- and HCO3- permeabilities (CFTR), and reducing the activity of the basolateral Cl- /HCO3- exchanger (anion exchanger 2; AE2). Under these conditions, the model secreted ∼140 mm HCO3- at a rate of ∼3 nl min-1  mm-2 , which is consistent with experimental observations. Alternative 1:2 and 1:1 stoichiometries for Cl- /HCO3- exchange via SLC26A6 at the apical membrane were able to support a HCO3- -rich secretion. Raising the HCO3- /Cl- permeability ratio of CFTR from 0.4 to 1.0 had little impact upon either the secreted HCO3- concentration or the volume flow. However, modelling showed that a reduction in basolateral AE2 activity by ∼80% was essential in minimizing the intracellular Cl- concentration following cAMP stimulation and thereby maximizing the secreted HCO3- concentration. The addition of a basolateral Na+ -K+ -2Cl- cotransporter (NKCC1), assumed to be present in rat and mouse ducts, raised intracellular Cl- and resulted in a lower secreted HCO3- concentration, as is characteristic of those species. We conclude therefore that minimizing the driving force for Cl- secretion is the main requirement for secreting 140 mm HCO3- .

No Change in Bicarbonate Transport but Tight-Junction Formation Is Delayed by Fluoride in a Novel Ameloblast Model.

We have recently developed a novel in vitro model using HAT-7 rat ameloblast cells to functionally study epithelial ion transport during amelogenesis. Our present aims were to identify key transporters of bicarbonate in HAT-7 cells and also to examine the effects of fluoride exposure on vectorial bicarbonate transport, cell viability, and the development of transepithelial resistance. To obtain monolayers, the HAT-7 cells were cultured on Transwell permeable filters. We monitored transepithelial resistance (TER) as an indicator of tight junction formation and polarization. We evaluated intracellular pH changes by microfluorometry using the fluorescent indicator BCECF. Activities of ion transporters were tested by withdrawal of various ions from the bathing medium, by using transporter specific inhibitors, and by activation of transporters with forskolin and ATP. Cell survival was estimated by alamarBlue assay. Changes in gene expression were monitored by qPCR. We identified the activity of several ion transporters, NBCe1, NHE1, NKCC1, and AE2, which are involved in intracellular pH regulation and vectorial bicarbonate and chloride transport. Bicarbonate secretion by HAT-7 cells was not affected by acute fluoride exposure over a wide range of concentrations. However, tight-junction formation was inhibited by 1 mM fluoride, a concentration which did not substantially reduce cell viability, suggesting an effect of fluoride on paracellular permeability and tight-junction formation. Cell viability was only reduced by prolonged exposure to fluoride concentrations greater than 1 mM. In conclusion, cultured HAT-7 cells are functionally polarized and are able to transport bicarbonate ions from the basolateral to the apical fluid spaces. Exposure to 1 mM fluoride has little effect on bicarbonate secretion or cell viability but delays tight-junction formation, suggesting a novel mechanism that may contribute to dental fluorosis.

Evidence for Active Electrolyte Transport by Two-Dimensional Monolayers of Human Salivary Epithelial Cells.

Functional reconstruction of lost tissue by regenerative therapy of salivary glands would be of immense benefit following radiotherapy or in the treatment of Sjogren's syndrome. The purpose of this study was to develop primary cultures of human salivary gland cells as potential regenerative resources and to characterize their acinar/ductal phenotype using electrophysiological measurements of ion transport. Human salivary gland cultures were prepared either from adherent submandibular gland cells (huSMG) or from mixed adherent and nonadherent cells (PTHSG) and were cultivated in Hepato-STIM or minimum essential medium (MEM). Expression of key epithelial marker proteins was determined by quantitative reverse transcription polymerase chain reaction (RT-PCR). Transepithelial electrical resistance (TER) was monitored following seeding the cells on Transwell membranes. Transepithelial ion transport was estimated by short-circuit current (Isc) measurements in an Ussing chamber. Both huSMG and PTHSG cells showed epithelial characteristics when cultivated in Hepato-STIM, while fibroblast-like elements dominated in MEM. Compared to intact tissue, cultivation of the cells resulted in substantial decreases in AQP5 and NKCC1 expression and moderate increases in claudin-1 and ENaC expression. Both cultures achieved high TER and transepithelial electrolyte movement in Hepato-STIM, but not in MEM. The Isc was substantially reduced by basolateral Cl(-) and bicarbonate withdrawal, indicating the involvement of basolateral-to-apical anion transport, and by the blockade of apical ENaC by amiloride, indicating the involvement of apical-to-basolateral Na(+) transport. An almost complete inhibition was observed following simultaneous ENaC block and withdrawal of the two anions. Isc was enhanced by either apical adenosine triphosphate (ATP) or basolateral carbachol application, but not by forskolin, confirming the expected role of Ca(2+)-activated regulatory pathways in electrolyte secretion. Inhibition of basolateral NKCC1 by bumetanide reduced the response to ATP, indicating the active involvement of this transporter in Cl(-) secretion. In conclusion, we have demonstrated that both PTHSG and huSMG primary cultures cultivated in Hepato-STIM form two-dimensional monolayers in vitro on permeable supports and achieve active vectorial transepithelial electrolyte transport. The presence of both basolateral-to-apical anion fluxes and an apical-to-basolateral Na(+) flux indicates both acinar and ductal characteristics. With further refinement, this model should provide a firm basis for new interventions to correct salivary gland dysfunction.

Pathophysiologic Changes in Extracellular pH Modulate Parathyroid Calcium-Sensing Receptor Activity and Secretion via a Histidine-Independent Mechanism.

The calcium-sensing receptor (CaR) modulates renal calcium reabsorption and parathyroid hormone (PTH) secretion and is involved in the etiology of secondary hyperparathyroidism in CKD. Supraphysiologic changes in extracellular pH (pHo) modulate CaR responsiveness in HEK-293 (CaR-HEK) cells. Therefore, because acidosis and alkalosis are associated with altered PTH secretion in vivo, we examined whether pathophysiologic changes in pHo can significantly alter CaR responsiveness in both heterologous and endogenous expression systems and whether this affects PTH secretion. In both CaR-HEK and isolated bovine parathyroid cells, decreasing pHo from 7.4 to 7.2 rapidly inhibited CaR-induced intracellular calcium (Ca(2+)i) mobilization, whereas raising pHo to 7.6 potentiated responsiveness to extracellular calcium (Ca(2+)o). Similar pHo effects were observed for Ca(2+)o-induced extracellular signal-regulated kinase phosphorylation and actin polymerization and for L-Phe-induced Ca(2+)i mobilization. Intracellular pH was unaffected by acute 0.4-unit pHo changes, and the presence of physiologic albumin concentrations failed to attenuate the pHo-mediated effects. None of the individual point mutations created at histidine or cysteine residues in the extracellular domain of CaR attenuated pHo sensitivity. Finally, pathophysiologic pHo elevation reversibly suppressed PTH secretion from perifused human parathyroid cells, and acidosis transiently increased PTH secretion. Therefore, pathophysiologic pHo changes can modulate CaR responsiveness in HEK-293 and parathyroid cells independently of extracellular histidine residues. Specifically, pathophysiologic acidification inhibits CaR activity, thus permitting PTH secretion, whereas alkalinization potentiates CaR activity to suppress PTH secretion. These findings suggest that acid-base disturbances may affect the CaR-mediated control of parathyroid function and calcium metabolism in vivo.

Role of organic osmolytes in water homoeostasis in skin.

The ability to conserve water is fundamental to terrestrial life. A number of organs such as the kidney and the bladder have important roles in the regulation of body water balance. The epidermis of skin is also fundamental to this process, and it is in a constant battle to prevent loss of water to the external, dry environment. Given this important role of the epidermis as a barrier to water loss, it is perhaps surprising that many of the cellular mechanisms by which human keratinocytes achieve cell volume homoeostasis, maintain epidermal hydration and adapt to biological effects from environmental stressors such as ultraviolet radiation are poorly understood. This article reviews what is known thus far and speculates about other potential mechanisms through which skin conducts water homoeostasis, with a particular emphasis on the putative role of organic osmolytes.

Role of anion exchangers in Cl- and HCO3- secretion by the human airway epithelial cell line Calu-3.

Despite the importance of airway surface liquid pH in the lung's defenses against infection, the mechanism of airway HCO3- secretion remains unclear. Our aim was to assess the contribution of apical and basolateral Cl-/HCO3- exchangers to Cl- and HCO3- transport in the Calu-3 cell line, derived from human airway submucosal glands. Changes in intracellular pH (pHi) were measured following substitution of Cl- with gluconate. Apical Cl- substitution led to an alkalinization in forskolin-stimulated cells, indicative of Cl-/HCO3- exchange. This was unaffected by the anion exchange inhibitor DIDS but inhibited by the CFTR blocker CFTRinh-172, suggesting that the HCO3- influx might occur via CFTR, rather than a solute carrier family 26 (SLC26) exchanger, as recently proposed. The anion selectivity of the recovery process more closely resembled that of CFTR than an SLC26 exchanger, and quantitative RT-PCR showed only low levels of SLC26 exchanger transcripts relative to CFTR and anion exchanger 2 (AE2). For pHi to rise to observed values (∼7.8) through HCO3- entry via CFTR, the apical membrane potential must reverse to at least +20 mV following Cl- substitution; this was confirmed by perforated-patch recordings. Substitution of basolateral Cl- evoked a DIDS-sensitive alkalinization, attributed to Cl-/HCO3- exchange via AE2. This appeared to be abolished in forskolin-stimulated cells but was unmasked by blocking apical efflux of HCO3- via CFTR. We conclude that Calu-3 cells secrete HCO3- predominantly via CFTR, and, contrary to previous reports, the basolateral anion exchanger AE2 remains active during stimulation, providing an important pathway for basolateral Cl- uptake.

Deletion of Slc26a6 alters the stoichiometry of apical Cl-/HCO-3 exchange in mouse pancreatic duct.

To define the stoichiometry and molecular identity of the Cl(-)/HCO(3)(-) exchanger in the apical membrane of pancreatic duct cells, changes in luminal pH and volume were measured simultaneously in interlobular pancreatic ducts isolated from wild-type and Slc26a6-null mice. Transepithelial fluxes of HCO(3)(-) and Cl(-) were measured in the presence of anion gradients favoring rapid exchange of intracellular HCO(3)(-) with luminal Cl(-) in cAMP-stimulated ducts. The flux ratio of Cl(-) absorption/HCO(3)(-) secretion was ∼0.7 in wild-type ducts and ∼1.4 in Slc26a6(-/-) ducts where a different Cl(-)/HCO(3)(-) exchanger, most likely SLC26A3, was found to be active. Interactions between Cl(-)/HCO(3)(-) exchange and cystic fibrosis transmembrane conductance regulator (CFTR) in cAMP-stimulated ducts were examined by measuring the recovery of intracellular pH after alkali-loading by acetate prepulse. Hyperpolarization induced by luminal application of CFTRinh-172 enhanced HCO(3)(-) efflux across the apical membrane via SLC26A6 in wild-type ducts but significantly reduced HCO(3)(-) efflux in Slc26a6(-/-) ducts. In microperfused wild-type ducts, removal of luminal Cl(-), or luminal application of dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid to inhibit SLC26A6, caused membrane hyperpolarization, which was abolished in Slc26a6(-/-) ducts. In conclusion, we have demonstrated that deletion of Slc26a6 alters the apparent stoichiometry of apical Cl(-)/HCO(3)(-) exchange in native pancreatic duct. Our results are consistent with SLC26A6 mediating 1:2 Cl(-)/HCO(3)(-) exchange, and the exchanger upregulated in its absence, most probably SLC26A3, mediating 2:1 exchange.

High glucose inhibits HCO3(-) and fluid secretion in rat pancreatic ducts.

Cellular mechanisms underlying the impairment of pancreatic fluid and electrolyte secretion in diabetes were examined using interlobular ducts isolated from rat pancreas. Fluid secretion was assessed by monitoring changes in luminal volume. HCO3(-) uptake across the basolateral membrane was estimated from the recovery of intracellular pH following an acid load. Exposure to high glucose concentrations inhibited fluid secretion and reduced the rate of basolateral HCO3(-) uptake in secretin-stimulated ducts isolated from normal rats. In ducts isolated from streptozotocin-treated diabetic rats, fluid secretion and basolateral HCO3(-) uptake were also severely impaired but could be largely reversed by incubation in normal-glucose solutions. Sodium-dependent glucose cotransporter 1 (SGLT1), glucose transporter (GLUT)1, GLUT2, and GLUT8 transcripts were detected by reverse transcriptase polymerase chain reaction in isolated ducts. Raising the luminal glucose concentration in microperfused ducts caused a depolarization of the membrane potential, consistent with the presence of SGLT1 at the apical membrane. Unstimulated ducts filled with high-glucose solutions lost luminal fluid by a phlorizin-sensitive mechanism, indicating that pancreatic ducts are capable of active glucose reabsorption from the lumen via SGLT1. In ducts exposed to high glucose concentrations, continuous glucose diffusion to the lumen and active reabsorption via SGLT1 would lead to elevation of intracellular Na+ concentration and sustained depolarization of the apical membrane. These two factors would tend to inhibit the basolateral uptake and apical efflux of Cl(-) and HCO3(-) and could therefore account for the impaired fluid and electrolyte secretion that is observed in diabetes.

Bicarbonate transport by the human pancreatic ductal cell line HPAF.

OBJECTIVES: The human pancreatic duct cell line, HPAF, has been shown previously to secrete Cl(-) in response to Ca(2+)-mobilizing stimuli. Our aim was to assess the capacity of HPAF cells to transport and secrete HCO3(-). METHODS: HPAF cells were grown as confluent monolayers on permeable supports. Short-circuit current was measured by voltage clamp. Intracellular pH (pHi) was measured by microfluorometry in cells loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). RESULTS: In HCO3(-)-free solutions, ATP-evoked changes in short-circuit current were inhibited by bumetanide, and the recovery of pHi from acid loading was abolished by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA). In the presence of HCO3(-), ATP-evoked secretion was no longer inhibited by bumetanide, and there was a strong EIPA-insensitive recovery from acid loading, which was inhibited by 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate (H2DIDS). ATP, but not forskolin, stimulated HCO3(-) efflux from the cells. CONCLUSIONS: In the absence of HCO3(-), ATP-evoked Cl(-) secretion is driven by a basolateral Na(+)-K(+)-2Cl(-) cotransporter, and pH(i) is regulated by apical and basolateral Na(+)/H(+) exchangers. In the presence of HCO3(-), ATP-evoked secretion is sustained in the absence of Na(+)-K(+)-2Cl(-) cotransporter activity and is probably driven by basolateral Na(+)-HCO3(-) cotransport.

Ducts isolated from the pancreas of CFTR-null mice secrete fluid.

The pancreatic pathology in cystic fibrosis (CF) is normally attributed to the failure of ductal fluid secretion resulting from the lack of functional CF transmembrane conductance regulator (CFTR). However, murine models of CF show little or no pancreatic pathology. To resolve this dichotomy we analysed the transport mechanisms involved in fluid and electrolyte secretion by pancreatic ducts isolated from CFTR-null mice. Experiments were performed on cultured interlobular duct segments isolated from the pancreas of the Cftr(tm1Cam) strain of CFTR-null mouse. Fluid secretion to the closed luminal space was measured by video microscopy. The secretory response of ducts isolated from CF mice to cAMP-elevating agonists forskolin and secretin was significantly reduced compared with wild type but not abolished. The Cl(-)- and HCO(3) (-) -dependent components of the ductal secretion were affected equally by the absence of CFTR. The secretory response to carbachol stimulation was unaltered in CF ducts. Loading the ductal cells with the Ca2+ chelator BAPTA completely abolished carbachol-evoked secretion, but did not affect forskolin-evoked secretion in CF or wild-type ducts. We conclude that pancreatic duct cells from CF mice can secrete a significant amount of water and electrolytes by a cAMP-stimulated mechanism that is independent of CFTR and cannot be ascribed to the activation of calcium-activated chloride channels.

Molecular and cellular regulation of pancreatic duct cell function.

PURPOSE OF REVIEW: The pancreatic duct epithelium is remarkable for its capacity to secrete HCO(3)(-) ions at concentrations as high as 140 mmol/l. The properties of the key transporters involved in this process and the central role played by cystic fibrosis transmembrane conductance regulator (CFTR) are the main focus of this review. RECENT FINDINGS: The Cl(-)/HCO(3)(-) exchanger at the apical membrane of pancreatic duct cells is now known to be SLC26A6. The 1: 2 stoichiometry and electrogenicity of this exchanger enable it to contribute to the secretion of HCO(3)(-) at high concentrations. The apical CFTR channels also appear to have sufficient HCO(3)(-) permeability to contribute directly to HCO(3)(-) secretion. There is a strong possibility that the Ca(2+)-activated Cl(-) channels at the apical membrane are members of the bestrophin family which, like CFTR, are also permeable to HCO(3)(-). More has been learned about the complex interactions between CFTR and other transporters within macromolecular complexes coordinated at the apical membrane by scaffolding proteins. Further details are also emerging of the protective paracrine roles of nucleotides, nucleosides, bile acids and trypsin in the regulation of ductal secretion. SUMMARY: Most of the key transporters involved in Cl(-) and HCO(3)(-) secretion have now been identified and characterized. Current research focuses on the molecular interactions between these transporters and the ways in which they are regulated by extracellular signals.

CFTR functions as a bicarbonate channel in pancreatic duct cells.

Pancreatic duct epithelium secretes a HCO(3)(-)-rich fluid by a mechanism dependent on cystic fibrosis transmembrane conductance regulator (CFTR) in the apical membrane. However, the exact role of CFTR remains unclear. One possibility is that the HCO(3)(-) permeability of CFTR provides a pathway for apical HCO(3)(-) efflux during maximal secretion. We have therefore attempted to measure electrodiffusive fluxes of HCO(3)(-) induced by changes in membrane potential across the apical membrane of interlobular ducts isolated from the guinea pig pancreas. This was done by recording the changes in intracellular pH (pH(i)) that occurred in luminally perfused ducts when membrane potential was altered by manipulation of bath K(+) concentration. Apical HCO(3)(-) fluxes activated by cyclic AMP were independent of Cl(-) and luminal Na(+), and substantially inhibited by the CFTR blocker, CFTR(inh)-172. Furthermore, comparable HCO(3)(-) fluxes observed in ducts isolated from wild-type mice were absent in ducts from cystic fibrosis (Delta F) mice. To estimate the HCO(3)(-) permeability of the apical membrane under physiological conditions, guinea pig ducts were luminally perfused with a solution containing 125 mM HCO(3)(-) and 24 mM Cl(-) in the presence of 5% CO(2). From the changes in pH(i), membrane potential, and buffering capacity, the flux and electrochemical gradient of HCO(3)(-) across the apical membrane were determined and used to calculate the HCO(3)(-) permeability. Our estimate of approximately 0.1 microm sec(-1) for the apical HCO(3)(-) permeability of guinea pig duct cells under these conditions is close to the value required to account for observed rates of HCO(3)(-) secretion. This suggests that CFTR functions as a HCO(3)(-) channel in pancreatic duct cells, and that it provides a significant pathway for HCO(3)(-) transport across the apical membrane.