Targeting the bicarbonate transporter SLC4A4 overcomes immunosuppression and immunotherapy resistance in pancreatic cancer.

Solid tumors are generally characterized by an acidic tumor microenvironment (TME) that favors cancer progression, therapy resistance and immune evasion. By single-cell RNA-sequencing analysis in individuals with pancreatic ductal adenocarcinoma (PDAC), we reveal solute carrier family 4 member 4 (SLC4A4) as the most abundant bicarbonate transporter, predominantly expressed by epithelial ductal cells. Functionally, SLC4A4 inhibition in PDAC cancer cells mitigates the acidosis of the TME due to bicarbonate accumulation in the extracellular space and a decrease in lactate production by cancer cells as the result of reduced glycolysis. In PDAC-bearing mice, genetic or pharmacological SLC4A4 targeting improves T cell-mediated immune response and breaches macrophage-mediated immunosuppression, thus inhibiting tumor growth and metastases. In addition, Slc4a4 targeting in combination with immune checkpoint blockade is able to overcome immunotherapy resistance and prolong survival. Overall, our data propose SLC4A4 as a therapeutic target to unleash an antitumor immune response in PDAC.

Aquaporin-1 Facilitates Transmesothelial Water Permeability: In Vitro and Ex Vivo Evidence and Possible Implications in Peritoneal Dialysis.

We previously showed that mesothelial cells in human peritoneum express the water channel aquaporin 1 (AQP1) at the plasma membrane, suggesting that, although in a non-physiological context, it may facilitate osmotic water exchange during peritoneal dialysis (PD). According to the three-pore model that predicts the transport of water during PD, the endothelium of peritoneal capillaries is the major limiting barrier to water transport across peritoneum, assuming the functional role of the mesothelium, as a semipermeable barrier, to be negligible. We hypothesized that an intact mesothelial layer is poorly permeable to water unless AQP1 is expressed at the plasma membrane. To demonstrate that, we characterized an immortalized cell line of human mesothelium (HMC) and measured the osmotically-driven transmesothelial water flux in the absence or in the presence of AQP1. The presence of tight junctions between HMC was investigated by immunofluorescence. Bioelectrical parameters of HMC monolayers were studied by Ussing Chambers and transepithelial water transport was investigated by an electrophysiological approach based on measurements of TEA+ dilution in the apical bathing solution, through TEA+-sensitive microelectrodes. HMCs express Zo-1 and occludin at the tight junctions and a transepithelial vectorial Na+ transport. Real-time transmesothelial water flux, in response to an increase of osmolarity in the apical solution, indicated that, in the presence of AQP1, the rate of TEA+ dilution was up to four-fold higher than in its absence. Of note, we confirmed our data in isolated mouse mesentery patches, where we measured an AQP1-dependent transmesothelial osmotic water transport. These results suggest that the mesothelium may represent an additional selective barrier regulating water transport in PD through functional expression of the water channel AQP1.

Anisakis Allergy: Is Aquacultured Fish a Safe and Alternative Food to Wild-Capture Fisheries for Anisakis simplex-Sensitized Patients?

BACKGROUND: Anisakis simplex (A. simplex) infection, in humans, causes a series of clinical manifestations affecting the gastro-intestinal tract known as Anisakiasis/Anisakidosis. Patients may also present allergic manifestations such as hives and/or angioedema and even anaphylactic shock. The aim of this study was to investigate whether aquacultured fish could be considered A.simplex-free food and constitute a safe, alternative, wild-capture fish food for Gastro-Allergic Anisakiasis (GAA)-sensitized subjects. METHODS: Protein extracts from A. simplex larvae in the third stage (L3) and from edible part of heavily infected horse mackerel (Trachurus trachurus) and aquacultured sea bream, have been tested for A. simplex allergens presence by immunological analysis. Western blot analysis using, as source of specific Anisakis allergens antibodies, serum samples from subjects referring allergic symptoms after raw fish ingestion, was performed. These subjects showed high levels of specific IgE anti A.simplex allergens determined by clinical laboratory tests (ISAC test). RESULTS: Our data demonstrate the presence of Ani s4 allergen in both infected and aquacultured fish extracts, providing a possible interpretation for the allergic manifestations reported by subjects, already sensitized to A. simplex, who ate frozen or well-cooked or, even, aquacultured fish. CONCLUSIONS: The present data stimulate more accurate prophylaxis suggestions for Anisakis allergy and more specific controls of fishmeal used in aquaculture.

Soy Metabolism by Gut Microbiota from Patients with Precancerous Intestinal Lesions.

BACKGROUND: Colorectal cancer (CRC) requires the presence of a variety of factors predisposing a tumorigenic milieu. Excluding familial clustering and hereditary CRC syndromes, the development of sporadic CRC from precancerous lesions is influenced by tissue inflammation, modulation of intestinal immunity, hormones, dietary habits and gut microbiota composition. As concerning the last two aspects, the intestinal presence of equol, the most biologically active metabolite of the soy isoflavone daidzein and the presence of a genetic determinant of gut microbiota able to metabolize daidzein, seem to lower the CRC risk. It has been hypothesized that the anaerobic microorganisms of the Bacteroides genus play a role in equol production. AIM: To evaluate the presence of (i) anaerobic gut microbiota and (ii) the urinary levels of soy isoflavones (daidzein, genistein and equol) in patients with and without precancerous lesions, challenged with a daidzein-rich soy extract. METHODS: Consecutive subjects undergoing colonoscopy participated to the study. Feces were collected from all patients one week before colonoscopy for gut microbiota studies. After the endoscopy examination and the histological evaluation, 40 subjects, 20 with sporadic colorectal adenomas (SCA/P group) and 20 without proliferative lesions (control group) were enrolled for the study. Urine levels of soy isoflavones daidzein, genistein and their metabolite equol, were determined by high performance liquid chromatographic (HPLC) analysis and gut microbiota analysis was performed by Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) procedure. RESULTS: Seventeen different bacterial species were identified in the fecal samples of the forty subjects participating to the study. Ten bacterial species resulted anaerobic Gram-negative bacteria, all belonging to the Bacteroides genus. A significant difference of bacteria species was evidenced in the fecal samples of the two groups of subjects. Particularly important was the evidence of Parabacteroides distasonis, Clostridium clostridioforme and Pediococcus pentasaceus only in control fecal samples, such as the presence of Bacteroides fragilis and Prevotella melaningenica only in SCA/P fecal samples. Concerning the soy isoflavones levels, no statistically significant differences were revealed in the genistein and daidzein urinary levels between the two groups of subjects. On the contrary, urinary equol levels were undetectable in ten SCA/P subjects and in two controls; moreover, when present, the levels of urinary equol were significantly lower in SCA/P subjects compared to controls (0.24 ± 0.27 mg/24 hrs vs. 21.25 ± 4.3 mg/24 hrs, respectively, p = 1.12 × 10-6). CONCLUSIONS: Our results suggest that the presence of anaerobic Bacteroides in the colon, and the production of equol from soy, could determine a milieu able to contrast the development of colonic mucosa proliferative lesions.

Cardiofaciocutaneous syndrome with rare structural variant in DOCK8 gene associated with neurodevelopmental disorders.

We describe a girl with clinical signs of cardiofaciocutaneous syndrome who simultaneously presents a mutation in the BRAF gene and a 9p24.3 microduplication. This genetic condition has never been described in the literature and could explain the phenotypic variability observed in the girl.

Interleukin-13 increases pendrin abundance to the cell surface in bronchial NCI-H292 cells via Rho/actin signaling.

Interleukin-13 (IL13) is a major player in the development of airway hyperresponsiveness in several respiratory disorders. Emerging data suggest that an increased expression of pendrin in airway epithelia is associated with elevated airway hyperreactivity in asthma. Here, we investigate the effect of IL13 on pendrin localization and function using bronchiolar NCI-H292 cells. The data obtained revealed that IL13 increases the cell surface expression of pendrin. This effect was paralleled by a significant increase in the intracellular pH, possibly via indirect stimulation of NHE. IL13 effect on pendrin localization and intracellular pH was reversed by theophylline, a bronchodilator compound used to treat asthma. IL13 upregulated RhoA activity, a crucial protein controlling actin dynamics, via G-alpha-13. Specifically, IL13 stabilized actin cytoskeleton and promoted co-localization and a direct molecular interaction between pendrin and F-actin in the plasma membrane region. These effects were reversed following exposure of cells to theophylline. Selective inhibition of Rho kinase, a downstream effector of Rho, reduced the IL13-dependent cell surface expression of pendrin. Together, these data indicate that IL13 increases pendrin abundance to the cell surface via Rho/actin signaling, an effect reversed by theophylline.

NKCC2 activity is inhibited by the Bartter's syndrome type 5 gain-of-function CaR-A843E mutant in renal cells.

BACKGROUND INFORMATION: The gain-of-function A843E mutation of the calcium sensing receptor (CaR) causes Bartter syndrome type 5. Patients carrying this CaR variant show a remarkably reduced renal NaCl reabsorption in the thick ascending limb (TAL) of Henle's loop resulting in renal loss of NaCl in the absence of mutations in renal Na(+) and Cl(-) ion transporters. The molecular mechanisms underlying this clinical phenotype are incompletely understood. We investigated, in human embryonic kidney 293 (HEK 293) cells and porcine kidney epithelial (LLC-PK1) cells, the functional cross-talk of CaR-A843E with the Na(+):K(+):2Cl(-) co-transporter, NKCC2, which provides NaCl reabsorption in the TAL. RESULTS: The expression of the CaR mutant did not alter the apical localisation of NKCC2 in LLC-PK1 cells. However, the steady-state NKCC2 phosphorylation and activity were decreased in cells transfected with CaR-A843E compared with the control wild-type CaR (CaR WT)-transfected cells. Of note, low-Cl(-)-dependent NKCC2 activation was also strongly inhibited upon the expression of CaR-A843E mutant. The use of either P450 ω-hydroxylase (CYP4)- or phospholipase A2 (PLA2)-blockers suggests that this effect is likely mediated by arachidonic acid (AA) metabolites. CONCLUSIONS: The data suggested that the activated CaR affects intracellular pathways modulating NKCC2 activity rather than NKCC2 intracellular trafficking in renal cells, and throw further light on the pathological role played by active CaR mutants in Bartter syndrome type 5.

Trimethylangelicin promotes the functional rescue of mutant F508del CFTR protein in cystic fibrosis airway cells.

Cystic fibrosis transmembrane conductance regulator (CFTR) carrying the F508del mutation is retained in endoplasmic reticulum and fails to traffic to the cell surface where it functions as a protein kinase A (PKA)-activated chloride channel. Pharmacological correctors that rescue the trafficking of F508del CFTR may overcome this defect; however, the rescued F508del CFTR still displays reduced chloride permeability. Therefore, a combined administration of correctors and potentiators of the gating defect is ideal. We recently found that 4,6,4'-trimethylangelicin (TMA), besides inhibiting the expression of the IL-8 gene in airway cells in which the inflammatory response was challenged with Pseudomonas aeruginosa, also potentiates the cAMP/PKA-dependent activation of wild-type CFTR or F508del CFTR that has been restored to the plasma membrane. Here, we demonstrate that long preincubation with nanomolar concentrations of TMA is able to effectively rescue both F508del CFTR-dependent chloride secretion and F508del CFTR cell surface expression in both primary or secondary airway cell monolayers homozygous for F508del mutation. The correction effect of TMA seems to be selective for CFTR and persisted for 24 h after washout. Altogether, the results suggest that TMA, besides its anti-inflammatory and potentiator activities, also displays corrector properties.

Toxic mechanisms induced by fumonisin b1 mycotoxin on human intestinal cell line.

The gastrointestinal tract is the main target of exposure to mycotoxin fumonisin B1 (FB1), common natural contaminant in food. Previous studies reported that proliferating cells are more sensitive than confluent cells to the toxic effect of FB1. This study aims to investigate, by dose- and time-dependent experiments on human colon proliferating intestinal cell line (HT-29), the modifications induced by FB1 at concentrations ranging from 0.25 to 69 µM. The choice of highest FB1 concentration considered the low toxicity previously reported on intestinal cell lines, whereas the lowest one corresponded to the lower FBs levels permitted by European Commission Regulation. Different functional parameters were tested such as cell proliferation, oxidative status, immunomodulatory effect and changes in membrane microviscosity. In addition FB1-FITC localization in this cell line was assessed by using confocal laser scanning microscopy. Lipid peroxidation induction was the main and early (12 h) effect induced by FB1 at concentrations ranging from 0.5 to 69 µM, followed by inhibition of cell proliferation (up to 8.6 µM), the immunomodulatory effect (up to 17.2 µM), by assessing IL-8 secretion, and increase in membrane microviscosity (up to 34.5 µM). The toxic effects observed in different functional parameters were not dose-dependent and could be the consequence of the FB1 intracytoplasmatic localization as confirmed by confocal microscopy results. The different timescales and concentrations active of different functional parameters could suggest different cellular targets of FB1.

Bovine pericardium patch wrapping intestinal anastomosis improves healing process and prevents leakage in a pig model.

Failure of intestinal anastomosis is a major complication following abdominal surgery. Biological materials have been introduced as reinforcement of abdominal wall hernia in contaminated setting. An innovative application of biological patch is its use as reinforcement of gastrointestinal anastomosis. The aim of study was to verify whether the bovine pericardium patch improves the healing of anastomosis, when in vivo wrapping the suture line of pig intestinal anastomosis, avoiding leakage in the event of deliberately incomplete suture. Forty-three pigs were randomly divided: Group 1 (control, n = 14): hand-sewn ileo-ileal and colo-colic anastomosis; Group 2 (n = 14): standard anastomosis wrapped by pericardium bovine patch; Group 3 (n = 1) and 4 (n = 14): one suture was deliberately incomplete and also wrapped by patch in the last one. Intraoperative evaluation, histological, biochemical, tensiometric and electrophysiological studies of intestinal specimens were performed at 48 h, 7 and 90 days after. In groups 2 and 4, no leak, stenosis, abscess, peritonitis, mesh displacement or shrinkage were found and adhesion rate decreased compared to control. Biochemical studies showed mitochondrial function improvement in colic wrapped anastomosis. Tensiometric evaluations suggested that the patch preserves the colic contractility similar to the controls. Electrophysiological results demonstrated that the patch also improves the mucosal function restoring almost normal transport properties. Use of pericardium bovine patch as reinforcement of intestinal anastomosis is safe and effective, significantly improving the healing process. Data of prevention of acute peritonitis and leakage in cases of iatrogenic perforation of anastomoses, covered with patch, is unpublished.

Glucose increases extracellular [Ca2+] in rat insulinoma (INS-1E) pseudoislets as measured with Ca2+-sensitive microelectrodes.

Secretory granules of pancreatic ß-cells contain high concentrations of Ca2+ ions that are co-released with insulin in the extracellular milieu upon activation of exocytosis. As a consequence, an increase in the extracellular Ca2+ concentration ([Ca2+]ext) in the microenvironment immediately surrounding ß-cells should be expected following the exocytotic event. Using Ca2+-selective microelectrodes we show here that both high glucose and non-nutrient insulinotropic agents elicit a reversible increase of [Ca2+]ext within rat insulinoma (INS-1E) ß-cells pseudoislets. The glucose-induced increases in [Ca2+]ext are blocked by pretreatment with different Ca2+ channel blockers. Physiological agonists acting as positive or negative modulators of the insulin secretion and drugs known to intersect the secretory machinery at different levels also induce [Ca2+]ext changes as predicted on the basis of their described action on insulin secretion. Finally, the glucose-induced [Ca2+]ext increase is strongly inhibited after disruption of the actin web, indicating that the dynamic [Ca2+]ext changes recorded in INS-1E pseudoislets by Ca2+-selective microelectrodes occur mainly as a consequence of exocytosis of Ca2+-rich granules. In conclusion, our data directly demonstrate that the extracellular spaces surrounding ß-cells constitute a restricted domain where Ca2+ is co-released during insulin exocytosis, creating the basis for an autocrine/paracrine cell-to-cell communication system via extracellular Ca2+ sensors.

Cadmium inhibits acid secretion in stimulated frog gastric mucosa.

Cadmium, a toxic environmental pollutant, affects the function of different organs such as lungs, liver and kidney. Less is known about its toxic effects on the gastric mucosa. The aim of this study was to investigate the mechanisms by which cadmium impacts on the physiology of gastric mucosa. To this end, intact amphibian mucosae were mounted in Ussing chambers and the rate of acid secretion, short circuit current (I(sc)), transepithelial potential (V(t)) and resistance (R(t)) were recorded in the continuous presence of cadmium. Addition of cadmium (20 microM to 1mM) on the serosal but not luminal side of the mucosae resulted in inhibition of acid secretion and increase in NPPB-sensitive, chloride-dependent short circuit current. Remarkably, cadmium exerted its effects only on histamine-stimulated tissues. Experiments with TPEN, a cell-permeant chelator for heavy metals, showed that cadmium acts from the intracellular side of the acid secreting cells. Furthermore, cadmium-induced inhibition of acid secretion and increase in I(sc) cannot be explained by an action on: 1) H(2) histamine receptor, 2) Ca(2+) signalling 3) adenylyl cyclase or 4) carbonic anhydrase. Conversely, cadmium was ineffective in the presence of the H(+)/K(+)-ATPase blocker omeprazole suggesting that the two compounds likely act on the same target. Our findings suggest that cadmium affects the functionality of histamine-stimulated gastric mucosa by inhibiting the H(+)/K(+)-ATPase from the intracellular side. These data shed new light on the toxic effect of this dangerous environmental pollutant and may result in new avenues for therapeutic intervention in acute and chronic intoxication.

The Vibrio cholerae cytolysin promotes chloride secretion from intact human intestinal mucosa.

BACKGROUND: The pathogenicity of the Vibrio cholerae strains belonging to serogroup O1 and O139 is due to the production of virulence factors such as cholera toxin (CT) and the toxin-coregulated pilus (TCP). The remaining serogroups, which mostly lack CT and TCP, are more frequently isolated from aquatic environmental sources than from clinical samples; nevertheless, these strains have been reported to cause human disease, such as sporadic outbreaks of watery diarrhoea and inflammatory enterocolitis. This evidence suggested the possibility that other virulence factor(s) than cholera toxin might be crucial in the pathogenesis of Vibrio cholerae-induced diarrhoea, but their nature remains unknown. VCC, the hemolysin produced by virtually all Vibrio cholerae strains, has been proposed as a possible candidate, though a clear-cut demonstration attesting VCC as crucial in the pathogenesis of Vibrio cholerae-induced diarrhoea is still lacking. METHODOLOGY/PRINCIPAL FINDINGS: Electrophysiological parameters and paracellular permeability of stripped human healthy colon tissues, obtained at subtotal colectomy, mounted in Ussing chamber were studied in the presence or absence of VCC purified from culture supernatants of V. cholerae O1 El Tor strain. Short circuit current (I(SC)) and transepithelial resistance (R(T)) were measured by a computerized voltage clamp system. The exposure of sigmoid colon specimens to 1 nM VCC resulted in an increase of I(SC) by 20.7%, with respect to the basal values, while R(T) was reduced by 12.3%. Moreover, increase in I(SC) was abolished by bilateral Cl(-) reduction. CONCLUSION/SIGNIFICANCE: Our results demonstrate that VCC, by forming anion channels on the apical membrane of enterocytes, triggers an outward transcellular flux of chloride. Such an ion movement, associated with the outward movement of Na(+) and water, might be responsible for the diarrhoea caused by the non-toxigenic strains of Vibrio cholerae.

Ca2+-dependent K+ efflux regulates deoxycholate-induced apoptosis of BHK-21 and Caco-2 cells.

BACKGROUND & AIMS: Deoxycholate (DC) has proapoptotic and tumorigenic effects in different cell types of the gastrointestinal tract. Exposure of BHK-21 (stromal) cells to DC induces Ca(2+) entry at the plasma membrane, which affects intracellular Ca(2+) signaling. We assessed whether DC-induced increases in [Ca(2+)] can impinge on plasma membrane properties (eg, ionic conductances) involved in cell apoptosis. METHODS: Single- and double-barreled microelectrodes were used to measure membrane potential (V(m)) and extracellular [K(+)] in BHK-21 fibroblasts and Caco-2 colon carcinoma cells. Apoptosis was assessed by Hoechst labeling, propidium iodide staining, and caspase-3 and caspase-7 assays. RESULTS: DC-induced cell membrane hyperpolarization was directly measured with intracellular microelectrodes in both cell lines. Diverse Ca(2+) mobilizing agents, such as membrane receptor agonists, an inhibitor of the sarco/endoplasmic reticulum Ca(2+) adenosine triphosphatase and a Ca(2+) ionophore, also induced increases in V(m). Removal of extracellular Ca(2+) reduced the agonist- and DC-induced membrane hyperpolarization by approximately 15% and 60%, respectively. These findings indicate a prominent role for Ca(2+) entry at the plasma membrane in the action of this bile salt. Blockade of Ca(2+)-activated K(+) conductances by charybdotoxin and apamin reduced DC-induced hyperpolarization by 75% and 64% in BHK-21 and Caco-2 cells, respectively. These inhibitors also reduced the DC-induced increase in extracellular [K(+)] by 75% and cell apoptosis by approximately 50% in both cell lines. CONCLUSIONS: Ca(2+)-dependent K(+) conductance is an important regulator of DC-induced apoptosis in stromal and colon cancer cells.

Taurodeoxycholate-induced intestinal injury is modulated by oxidative stress-dependent pre-conditioning like mechanisms.

Mechanisms by which hydrophobic bile salts cause tissue changes below their critical micellar concentration (CMC, 1-2mM) and above (4-8mM) remain poorly understood. In this study, rat colonic mucosa was exposed to different concentrations of taurodeoxycholate (TDC), t-butyl-hydroperoxide (t-BH) or glutathione ester with or without pre-incubation with 2mM TDC. Exposure to 2mM TDC was associated with 10% higher tissue levels of total glutathione (GSH, basal values: 33.7+/-3.3 nmol/mg prot). With TDC 8mM, GSH decreased to 16.4+/-2.3 nmol/mg prot (P<0.05), oxidized glutathione (GSSG) increased by 60% (P<0.05), glutathione peroxidase (GSH-Px) and reductase activities were threefold increased, protein carbonyls fourfold increased, protein sulfhydrils decreased by 78%, lactate dehydrogenase (LDH) and GSSG release in the incubation medium were sixfold higher. In 2mM TDC pre-treated tissues, the subsequent incubation with 8mM TDC induced a lower loss of tissue GSH, and a lower release of LDH and GSSG. Pre-incubation with 2mM TDC partly protected against t-BH toxicity, while glutathione ester protected against 8mM TDC toxicity. In conclusion, TDC exposure causes opposite effects depending on CMC: induction of antioxidant protective systems including glutathione system (pre-conditioning effect) was observed with TDC below CMC, oxidative damages pointing to decreased mucosal detoxification potential with above CMC.

Real time measurements of water flow in amphibian gastric glands: modulation via the extracellular Ca2+-sensing receptor.

The mechanisms for the formation of the osmotic gradient driving water movements in the gastric gland and its modulation via the extracellular Ca(2+)-sensing receptor (CaR) were investigated. Real time measurements of net water flux in the lumen of single gastric glands of the intact amphibian stomach were performed using ion-selective double-barreled microelectrodes. Water movement was measured by recording changes in the concentration of impermeant TEA(+) ions ([TEA(+)](gl)) with TEA(+)-sensitive microelectrodes inserted in the lumen of individual gastric glands. Glandular K(+) (K(+)(gl)) and H(+) (pH(gl)) were also measured by using K(+)- and H(+)-sensitive microelectrodes, respectively. Stimulation with histamine significantly decreased [TEA](gl), indicating net water flow toward the gland lumen. This response was inhibited by the H(+)/K(+)-ATPase inhibitor, SCH 28080. Histamine also elicited a significant and reversible increase in [K(+)](gl) that was blocked by chromanol 293B, a blocker of KCQN1 K(+) channels. Histamine failed to induce net water flow in the presence of chromanol 293B. In the "resting state," stimulation of CaR with diverse agonists resulted in significant increase in [TEA](gl). CaR activation also significantly reduced histamine-induced water secretion and apical K(+) transport. Our data validate the strong link between histamine-stimulated acid secretion and water transport. We also show that cAMP-dependent [K(+)](gl) elevation prior to the onset of acid secretion generates the osmotic gradient initially driving water into the gastric glands and that CaR activation inhibits this process, probably through reduction of intracellular cAMP levels.

Extracellular calcium acts as a "third messenger" to regulate enzyme and alkaline secretion.

It is generally assumed that the functional consequences of stimulation with Ca2+ -mobilizing agonists are derived exclusively from the second messenger action of intracellular Ca2+, acting on targets inside the cells. However, during Ca2+ signaling events, Ca2+ moves in and out of the cell, causing changes not only in intracellular Ca2+, but also in local extracellular Ca2+. The fact that numerous cell types possess an extracellular Ca2+ "sensor" raises the question of whether these dynamic changes in external [Ca2+] may serve some sort of messenger function. We found that in intact gastric mucosa, the changes in extracellular [Ca2+] secondary to carbachol-induced increases in intracellular [Ca2+] were sufficient and necessary to elicit alkaline secretion and pepsinogen secretion, independent of intracellular [Ca2+] changes. These findings suggest that extracellular Ca2+ can act as a "third messenger" via Ca2+ sensor(s) to regulate specific subsets of tissue function previously assumed to be under the direct control of intracellular Ca2+.

A reassessment of the effects of luminal [Ca2+] on inositol 1,4,5-trisphosphate-induced Ca2+ release from internal stores.

Inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ release from intracellular stores displays complex kinetic behavior. While it well established that cytosolic [Ca2+] can modulate release by acting on the InsP3 receptor directly, the role of the filling state of internal Ca2+stores in modulating Ca2+ release remains unclear. Here we have reevaluated this topic using a technique that permits rapid and reversible changes in free [Ca2+] in internal stores of living intact cells without altering cytoplasmic [Ca2+], InsP3 receptors, or sarcoendoplasmic reticulum Ca2+ ATPases (SERCAs). N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylene diamine (TPEN), a membrane-permeant, low affinity Ca2+ chelator was used to manipulate [Ca2+] in intracellular stores, while [Ca2+] changes within the store were monitored directly with the low-affinity Ca2+ indicator, mag-fura-2, in intact BHK-21 cells. 200 microM TPEN caused a rapid drop in luminal free [Ca2+] and significantly reduced the extent of the response to stimulation with 100 nm bradykinin, a calcium-mobilizing agonist. The same effect was observed when intact cells were pretreated with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid(acetoxymethyl ester) (BAPTA-AM) to buffer cytoplasmic [Ca2+] changes. Although inhibition of Ca2+ uptake using the SERCA inhibitor tBHQ permitted significantly larger release of Ca2+ from stores, TPEN still attenuated the release in the presence of tBHQ in BAPTA-AM-loaded cells. These results demonstrate that the filling state of stores modulates the magnitude of InsP3-induced Ca2+release by additional mechanism(s) that are independent of regulation by cytoplasmic [Ca2+] or effects on SERCA pumps.

Basolateral Ca2+-dependent K+-channels play a key role in Cl- secretion induced by taurodeoxycholate from colon mucosa.

The diarrhea associated with malabsorption of bile salts such as the secondary hydrophobic taurodeoxycholate (TDC) may be partly explained by the TDC-induced increase in colon Cl(-) secretion. We, therefore, investigated the effects of TDC (0.5-8 mM) on electrical parameters and electrolyte transport of rat proximal colon mucosa mounted in Ussing chambers. Colonic secretion, measured as short circuit current (I(SC)), progressively increased on mucosal incubation with TDC ranging 0.5-2 mM; up to TDC 2 mM, a spontaneous recovery toward control values with no changes in epithelial resistance (Rt), and lactate dehydrogenase (LDH) release was observed. In contrast, for TDC > 2 mM, I(SC) increased further and the effect was progressive and associated with a significant decrease in the Rt and increased LDH release, implying a cytolytic effect. Mucosal preincubation with the Cl(-) channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), fully prevented the precytolytic effect of TDC on I(SC). Serosal preincubation with furosemide, a Na(+)/K(+)/2Cl(-) cotransporter inhibitor, significantly reduced TDC-induced increase in I(SC). Inhibition of the basolateral Ca(2+)-dependent K(+) channel-rSK4-with serosal clotrimazole or incubation with mucosal Ca(2+)-free (EGTA) buffer completely prevented precytolytic TDC-induced increase in I(SC). In conclusion, Cl(-) secretion is activated in colon mucosa by TDC low concentrations; while at higher concentrations, a detergent cytotoxic effect intervenes. Activation of the Ca(2+)-dependent basolateral K(+) pathway, through TDC-induced apical Ca(2+) influx, provides the Na(+)/K(+)/2Cl(-) basolateral activation, thereby the driving force for the apical exit of Cl(-) ions. These findings further enhance the knowledge of the pathogenic mechanisms of diarrhea associated with bile salt malabsorption.