Half-Life Extension and Biodistribution Modulation of Biotherapeutics via Red Blood Cell Hitch-Hiking with Novel Anti-Band 3 Single-Domain Antibodies.

Small therapeutic proteins are receiving increased interest as therapeutic drugs; however, their clinical success has been limited due to their rapid elimination. Here, we report a half-life extension strategy via strategy via red blood cell red blood cell (RBC) hitch-hiking. This manuscript details the development and characterization of novel anti-RBC single-domain antibodies (sdAbs), their genetic fusion to therapeutic antibody fragments (TAF) as bispecific fusion constructs, and their influence on TAF pharmacokinetics and biodistribution. Several sdAbs specific to the band 3 antigen were generated via phage-display technology. Binding affinity to RBCs was assessed via flow cytometry. Affinity maturation via random mutagenesis was carried out to improve the binding affinity of the sdAbs. Bi-specific constructs were generated by fusing the anti-RBC sdAbs with anti-tissue necrosis factor alpha (TNF-α) TAF via the use of a glycine-serine flexible linker, and assessments for binding were performed via enzyme-linked immunosorbent assay and flow cytometry. Pharmacokinetics of anti-RBC sdAbs and fusion constructs were evaluated following intravenous bolus dosing in mice at a 1 mg/kg dose. Two RBC-binding sdAbs, RB12 and RE8, were developed. These two clones showed high binding affinity to human RBC with an estimated KD of 17.7 nM and 23.6 nM and low binding affinity to mouse RBC with an estimated KD of 335 nM and 528 nM for RB12 and RE8, respectively. Two derivative sdAbs, RMA1, and RMC1, with higher affinities against mouse RBC, were generated via affinity maturation (KD of 66.9 nM and 30.3 nM, respectively). Pharmacokinetic investigations in mice demonstrated prolonged circulation half-life of an anti-RBC-TNF-α bispecific construct (75 h) compared to a non-RBC binding control (1.3 h). In summary, the developed anti-RBC sdAbs and fusion constructs have demonstrated high affinity in vitro, and sufficient half-life extension in vivo.

Role of the Second Extracellular Loop of the Adenosine A1 Receptor on Allosteric Modulator Binding, Signaling, and Cooperativity.

Allosteric modulation of adenosine A1 receptors (A1ARs) offers a novel therapeutic approach for the treatment of numerous central and peripheral disorders; however, despite decades of research, there is a relative paucity of structural information regarding the A1AR allosteric site and mechanisms governing cooperativity with orthosteric ligands. We combined alanine-scanning mutagenesis of the A1AR second extracellular loop (ECL2) with radioligand binding and functional interaction assays to quantify effects on allosteric ligand affinity, cooperativity, and efficacy. Docking and molecular dynamics (MD) simulations were performed using an A1AR homology model based on an agonist-bound A2AAR structure. Substitution of E172ECL2 for alanine reduced the affinity of the allosteric modulators PD81723 and VCP171 for the unoccupied A1AR. Residues involved in cooperativity with the orthosteric agonist NECA were different in PD81723 and VCP171; positive cooperativity between PD81723 and NECA was reduced on alanine substitution of a number of ECL2 residues, including E170ECL2 and K173ECL2, whereas mutation of W146ECL2 and W156ECL2 decreased VCP171 cooperativity with NECA. Molecular modeling localized a likely allosteric pocket for both modulators to an extracellular vestibule that overlaps with a region used by orthosteric ligands as they transit into the canonical A1AR orthosteric site. MD simulations confirmed a key interaction between E172ECL2 and both modulators. Bound PD81723 is flanked by another residue, E170ECL2, which forms hydrogen bonds with adjacent K168ECL2 and K173ECL2. Collectively, our data suggest E172ECL2 is a key allosteric ligand-binding determinant, whereas hydrogen-bonding networks within the extracellular vestibule may facilitate the transmission of cooperativity between orthosteric and allosteric sites.

Extracellular Loop 2 of the Adenosine A1 Receptor Has a Key Role in Orthosteric Ligand Affinity and Agonist Efficacy.

The adenosine A1 G protein-coupled receptor (A1AR) is an important therapeutic target implicated in a wide range of cardiovascular and neuronal disorders. Although it is well established that the A1AR orthosteric site is located within the receptor's transmembrane (TM) bundle, prior studies have implicated extracellular loop 2 (ECL2) as having a significant role in contributing to orthosteric ligand affinity and signaling for various G protein-coupled receptors (GPCRs). We thus performed extensive alanine scanning mutagenesis of A1AR-ECL2 to explore the role of this domain on A1AR orthosteric ligand pharmacology. Using quantitative analytical approaches and molecular modeling, we identified ECL2 residues that interact either directly or indirectly with orthosteric agonists and antagonists. Discrete mutations proximal to a conserved ECL2-TM3 disulfide bond selectively affected orthosteric ligand affinity, whereas a cluster of five residues near the TM4-ECL2 juncture influenced orthosteric agonist efficacy. A combination of ligand docking, molecular dynamics simulations, and mutagenesis results suggested that the orthosteric agonist 5'-N-ethylcarboxamidoadenosine binds transiently to an extracellular vestibule formed by ECL2 and the top of TM5 and TM7, prior to entry into the canonical TM bundle orthosteric site. Collectively, this study highlights a key role for ECL2 in A1AR orthosteric ligand binding and receptor activation.

Mechanisms of ADC Toxicity and Strategies to Increase ADC Tolerability.

Anti-cancer antibody-drug conjugates (ADCs) aim to expand the therapeutic index of traditional chemotherapy by employing the targeting specificity of monoclonal antibodies (mAbs) to increase the efficiency of the delivery of potent cytotoxic agents to malignant cells. In the past three years, the number of ADCs approved by the Food and Drug Administration (FDA) has tripled. Although several ADCs have demonstrated sufficient efficacy and safety to warrant FDA approval, the clinical use of all ADCs leads to substantial toxicity in treated patients, and many ADCs have failed during clinical development due to their unacceptable toxicity profiles. Analysis of the clinical data has demonstrated that dose-limiting toxicities (DLTs) are often shared by different ADCs that deliver the same cytotoxic payload, independent of the antigen that is targeted and/or the type of cancer that is treated. DLTs are commonly associated with cells and tissues that do not express the targeted antigen (i.e., off-target toxicity), and often limit ADC dosage to levels below those required for optimal anti-cancer effects. In this manuscript, we review the fundamental mechanisms contributing to ADC toxicity, we summarize common ADC treatment-related adverse events, and we discuss several approaches to mitigating ADC toxicity.

Use of Payload Binding Selectivity Enhancers to Improve Therapeutic Index of Maytansinoid-Antibody-Drug Conjugates.

Systemic exposure to released cytotoxic payload contributes to the dose-limiting off-target toxicities of anticancer antibody-drug conjugates (ADC). In this work, we present an "inverse targeting" strategy to optimize the therapeutic selectivity of maytansinoid-conjugated ADCs. Several anti-maytansinoid sdAbs were generated via phage-display technology with binding IC50 values between 10 and 60 nmol/L. Co-incubation of DM4 with the anti-maytansinoid sdAbs shifted the IC50 value of DM4 up to 250-fold. Tolerability and efficacy of 7E7-DM4 ADC, an anti-CD123 DM4-conjugated ADC, were assessed in healthy and in tumor-bearing mice, with and without co-administration of an anti-DM4 sdAb. Co-administration with anti-DM4 sdAb reduced 7E7-DM4-induced weight loss, where the mean values of percentage weight loss at nadir for mice receiving ADC+saline and ADC+sdAb were 7.9% ± 3% and 3.8% ± 1.3% (P < 0.05). In tumor-bearing mice, co-administration of the anti-maytansinoid sdAb did not negatively affect the efficacy of 7E7-DM4 on tumor growth or survival following dosing of the ADC at 1 mg/kg (P = 0.49) or at 10 mg/kg (P = 0.9). Administration of 7E7-DM4 at 100 mg/kg led to dramatic weight loss, with 80% of treated mice succumbing to toxicity before the appearance of mortality relating to tumor growth in control mice. However, all mice receiving co-dosing of 100 mg/kg 7E7-DM4 with anti-DM4 sdAb were able to tolerate the treatment, which enabled reduction in tumor volume to undetectable levels and to dramatic improvements in survival. In summary, we have demonstrated the utility and feasibility of the application of anti-payload antibody fragments for inverse targeting to improve the selectivity and efficacy of anticancer ADC therapy.

Development and Evaluation of Competitive Inhibitors of Trastuzumab-HER2 Binding to Bypass the Binding-Site Barrier.

Our group has developed and experimentally validated a strategy to increase antibody penetration in solid tumors through transient inhibition of antibody-antigen binding. In prior work, we demonstrated that 1HE, an anti-trastuzumab single domain antibody that transiently inhibits trastuzumab binding to HER2, increased the penetration of trastuzumab and increased the efficacy of ado-trastuzumab emtansine (T-DM1) in HER2+ xenograft bearing mice. In the present work, 1HE variants were developed using random mutagenesis and phage display to enable optimization of tumor penetration and efficacy of trastuzumab-based therapeutics. To guide the rational selection of a particular 1HE mutant for a specific trastuzumab-therapy, we developed a mechanistic pharmacokinetic (PK) model to predict within-tumor exposure of trastuzumab/T-DM1. A pharmacodynamic (PD) component was added to the model to predict the relationship between intratumor exposure to T-DM1 and the corresponding therapeutic effect in HER2+ xenografts. To demonstrate the utility of the competitive inhibition approach for immunotoxins, PK parameters specific for a recombinant immunotoxin were incorporated into the model structure. Dissociation half-lives for variants ranged from 1.1 h (for variant LG11) to 107.9 h (for variant HE10). Simulations predicted that 1HE co-administration can increase the tumor penetration of T-DM1, with inhibitors with longer trastuzumab binding half-lives relative to 1HE (15.5 h) further increasing T-DM1 penetration at the expense of total tumor uptake of T-DM1. The PK/PD model accurately predicted the response of NCI-N87 xenografts to treatment with T-DM1 or T-DM1 co-administered with 1HE. Model predictions indicate that the 1HE mutant HF9, with a trastuzumab binding half-life of 51.1 h, would be the optimal inhibitor for increasing T-DM1 efficacy with a modest extension in the median survival time relative to T-DM1 with 1HE. Model simulations predict that LG11 co-administration will dramatically increase immunotoxin penetration within all tumor regions. We expect that the mechanistic model structure and the wide range of inhibitors developed in this work will enable optimization of trastuzumab-cytotoxin penetration and efficacy in solid tumors.

Early Melanoma Diagnosis With Sequential Dermoscopic Images.

Dermatologists often diagnose or rule out early melanoma by evaluating the follow-up dermoscopic images of skin lesions. However, existing algorithms for early melanoma diagnosis are developed using single time-point images of lesions. Ignoring the temporal, morphological changes of lesions can lead to misdiagnosis in borderline cases. In this study, we propose a framework for automated early melanoma diagnosis using sequential dermoscopic images. To this end, we construct our method in three steps. First, we align sequential dermoscopic images of skin lesions using estimated Euclidean transformations, extract the lesion growth region by computing image differences among the consecutive images, and then propose a spatio-temporal network to capture the dermoscopic changes from aligned lesion images and the corresponding difference images. Finally, we develop an early diagnosis module to compute probability scores of malignancy for lesion images over time. We collected 179 serial dermoscopic imaging data from 122 patients to verify our method. Extensive experiments show that the proposed model outperforms other commonly used sequence models. We also compared the diagnostic results of our model with those of seven experienced dermatologists and five registrars. Our model achieved higher diagnostic accuracy than clinicians (63.69% vs. 54.33%, respectively) and provided an earlier diagnosis of melanoma (60.7% vs. 32.7% of melanoma correctly diagnosed on the first follow-up images). These results demonstrate that our model can be used to identify melanocytic lesions that are at high-risk of malignant transformation earlier in the disease process and thereby redefine what is possible in the early detection of melanoma.

Intestinal helminth infections among reproductive age women in Vietnam: prevalence, co-infection and risk factors.

Intestinal helminth infections are a significant public health problem for Vietnamese women, but prevalence and risk factor data are scarce. The objectives of this paper were to (1) determine the prevalence of helminth infections among women; (2) investigate interactions among intestinal helminth species in individuals and (3) identify risk factors that contribute to intestinal helminth infections. In a nationwide survey conducted in 1995, 9550 households in 53 provinces were covered using a stratified two-stage cluster survey. Stool specimens were examined by Kato-Katz technique. Of 5,127 women, 76% were infected with one or more helminth species, 36% with hookworm, 59% with Ascaris lumbricoides and 28% with Trichuris trichiura. A. lumbricoides and T. trichiura were more likely to be concurrent than expected by chance. There was significant interaction between prevalence and intensity of infection in all three species. All three helminth species were more common in certain ecologic zones than others. Hookworm infection was associated with farming [Odd ratio (OR) = 2.1] and lack of a closed latrine (OR = 2.0), A. lumbricoides with use of untreated feces as fertilizer (OR = 1.2) and coinfection with T. trichiura (OR = 2.1) and T trichiura with A. lumbricoides co-infection (OR = 2.1). Our findings suggest that reproductive-age women, especially rural farmers, should be included among the high priority groups for helminth control programs through mass chemotherapy and improving sanitation.

Design, implementation and operation of a multimodality research imaging informatics repository.

BACKGROUND: Biomedical imaging research increasingly involves acquiring, managing and processing large amounts of distributed imaging data. Integrated systems that combine data, meta-data and workflows are crucial for realising the opportunities presented by advances in imaging facilities. METHODS: This paper describes the design, implementation and operation of a multi-modality research imaging data management system that manages imaging data obtained from biomedical imaging scanners operated at Monash Biomedical Imaging (MBI), Monash University in Melbourne, Australia. In addition to Digital Imaging and Communications in Medicine (DICOM) images, raw data and non-DICOM biomedical data can be archived and distributed by the system. Imaging data are annotated with meta-data according to a study-centric data model and, therefore, scientific users can find, download and process data easily. RESULTS: The research imaging data management system ensures long-term usability, integrity inter-operability and integration of large imaging data. Research users can securely browse and download stored images and data, and upload processed data via subject-oriented informatics frameworks including the Distributed and Reflective Informatics System (DaRIS), and the Extensible Neuroimaging Archive Toolkit (XNAT).

Fecal occult blood testing in a general medical clinic: comparison between guaiac-based and immunochemical-based tests.

PURPOSE: Guaiac-based fecal occult blood tests are limited by poor patient compliance, and low sensitivity, specificity, and positive predictive value. Newer immunochemical-based tests are designed to improve accuracy and patient compliance. We compared patient compliance and the test characteristics of these two types of tests. METHODS: The laboratory outcomes associated with use of different fecal occult blood tests were examined in a Veterans Affairs-based general medicine clinic that was divided into two firms with similar patient and provider characteristics. Tests were ordered for colorectal cancer screening or for symptom evaluation. Patients were given one of the two tests depending on their firm. The completion and positivity rates, time to test completion, completion of diagnostic follow-up, and positive predictive values were compared. RESULTS: The percentage of returned test cards was similar between the two groups (47% [1369/2964] for guaiac-based tests vs. 48% [1410/2965] for immunochemical-based tests) as was the positivity rate (9.0% [122/1396] and [128/1410] for both groups). In patients with positive tests who underwent further colon evaluation, the proportion with adenomas was similar between groups (59% [38/64] vs. 58% [40/69]). However, 17% (12/69) with a positive immunochemical-based test had an adenoma >1 cm or a colorectal malignancy, versus 30% (19/64) for guaiac-based tests (P = 0.09). CONCLUSION: Overall, immunochemical-based and guaiac-based fecal occult blood tests had comparable performance. However, although immunochemical-based testing is reported to be easier for patients than guaiac-based testing, we found that patients were no more likely to return cards for analysis. The similar positive predictive value and additional cost of immunochemical-based tests call into question their utility in general practice.

Cyclic AMP potentiates Ca2+-dependent exocytosis in pancreatic duct epithelial cells.

Exocytosis is evoked by intracellular signals, including Ca(2+) and protein kinases. We determined how such signals interact to promote exocytosis in exocrine pancreatic duct epithelial cells (PDECs). Exocytosis, detected using carbon-fiber microamperometry, was stimulated by [Ca(2+)](i) increases induced either through Ca(2+) influx using ionomycin or by activation of P2Y2 or protease-activated receptor 2 receptors. In each case, the exocytosis was strongly potentiated when cyclic AMP (cAMP) was elevated either by activating adenylyl cyclase with forskolin or by activating the endogenous vasoactive intestinal peptide receptor. This potentiation was completely inhibited by H-89 and partially blocked by Rp-8-Br-cAMPS, inhibitors of protein kinase A. Optical monitoring of fluorescently labeled secretory granules showed slow migration toward the plasma membrane during Ca(2+) elevations. Neither this Ca(2+)-dependent granule movement nor the number of granules found near the plasma membrane were detectably changed by raising cAMP, suggesting that cAMP potentiates Ca(2+)-dependent exocytosis at a later stage. A kinetic model was made of the exocytosis stimulated by UTP, trypsin, and Ca(2+) ionophores with and without cAMP increase. In the model, without a cAMP rise, receptor activation stimulates exocytosis both by Ca(2+) elevation and by the action of another messenger(s). With cAMP elevation the docking/priming step for secretory granules was accelerated, augmenting the releasable granule pool size, and the Ca(2+) sensitivity of the final fusion step was increased, augmenting the rate of exocytosis. Presumably both cAMP actions require cAMP-dependent phosphorylation of target proteins. cAMP-dependent potentiation of Ca(2+)-induced exocytosis has physiological implications for mucin secretion and, possibly, for membrane protein insertion in the pancreatic duct. In addition, mechanisms underlying this potentiation of slow exocytosis may also exist in other cell systems.

Protease-activated receptor-2 increases exocytosis via multiple signal transduction pathways in pancreatic duct epithelial cells.

Protease-activated receptor-2 (PAR-2) is activated when trypsin cleaves its NH(2) terminus to expose a tethered ligand. We previously demonstrated that PAR-2 activates ion channels in pancreatic duct epithelial cells (PDEC). Using real-time optical fluorescent probes, cyan fluorescence protein-Epac1-yellow fluorescence protein for cAMP, PH(PLC-delta1)-enhanced green fluorescent protein for phosphatidylinositol 4,5-bisphosphate, and protein kinase Cgamma (PKCgamma)-C1-yellow fluorescence protein for diacylglycerol, we now define the signaling pathways mediating PAR-2 effect in dog PDEC. Although PAR-2 activation does not stimulate a cAMP increase, it induces phospholipase C to hydrolyze phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate and diacylglycerol. Intracellular Ca(2+) mobilization from inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores and a subsequent Ca(2+) influx through store-operated Ca(2+) channels cause a biphasic increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), measured with Indo-1 dye. Single-cell amperometry demonstrated that this increase in [Ca(2+)](i) in turn causes a biphasic increase in exocytosis. A protein kinase assay revealed that trypsin also activates PKC isozymes to stimulate additional exocytosis. Paralleling the increased exocytosis, mucin secretion from PDEC was also induced by trypsin or the PAR-2 activating peptide. Consistent with the serosal localization of PAR-2, 1 microm luminal trypsin did not induce exocytosis in polarized PDEC monolayers; on the other hand, 10 microm trypsin at 37 degrees C damaged the epithelial barrier sufficiently so that it could reach and activate the serosal PAR-2 to stimulate exocytosis. Thus, in PDEC, PAR-2 activation increases [Ca(2+)](i) and activates PKC to stimulate exocytosis and mucin secretion. These functions may mediate the reported protective role of PAR-2 in different models of pancreatitis.

Novel short chain fatty acids restore chloride secretion in cystic fibrosis.

Phenylalanine deletion at position 508 of the cystic fibrosis transmembrane conductance regulator (DeltaF508-CFTR), the most common mutation in cystic fibrosis (CF), causes a misfolded protein exhibiting partial chloride conductance and impaired trafficking to the plasma membrane. 4-Phenylbutyrate corrects defective DeltaF508-CFTR trafficking in vitro, but is not clinically efficacious. From a panel of short chain fatty acid derivatives, we showed that 2,2-dimethyl-butyrate (ST20) and alpha-methylhydrocinnamic acid (ST7), exhibiting high oral bioavailability and sustained plasma levels, correct the DeltaF508-CFTR defect. Pre-incubation (>or=6h) of CF IB3-1 airway cells with >or=1mM ST7 or ST20 restored the ability of 100microM forskolin to stimulate an (125)I(-) efflux. This efflux was fully inhibited by NPPB, DPC, or glibenclamide, suggesting mediation through CFTR. Partial inhibition by DIDS suggests possible contribution from an additional Cl(-) channel regulated by CFTR. Thus, ST7 and ST20 offer treatment potential for CF caused by the DeltaF508 mutation.

Pattern of Ca2+ increase determines the type of secretory mechanism activated in dog pancreatic duct epithelial cells.

Intracellular calcium concentration ([Ca(2+)](i)) is a key factor controlling secretion from various cell types. We investigated how different patterns of [Ca(2+)](i) signals evoke salt secretion via ion transport mechanisms and mucin secretion via exocytosis in dog pancreatic duct epithelial cells (PDEC). Activation of epithelial P2Y(2) receptors by UTP generated two patterns of [Ca(2+)](i) change: 2-10 microm UTP induced [Ca(2+)](i) oscillations, whereas 100 microm UTP induced a sustained [Ca(2+)](i) increase, both in the micromolar range. As monitored by carbon-fibre amperometry, the sustained [Ca(2+)](i) increase stimulated a larger increase in exocytosis than [Ca(2+)](i) oscillations, despite their similar amplitude. In contrast, patch-clamp recordings revealed that [Ca(2+)](i) oscillations synchronously activated a K(+) current as efficiently as the sustained [Ca(2+)](i) increase. This K(+) current was mediated by intermediate-conductance Ca(2+)-activated K(+) channels (32 pS at -100 mV) which were sensitive to charybdotoxin and resistant to TEA. Activation of these Ca(2+)-dependent K(+) channels hyperpolarized the plasma membrane from a resting potential of -40 mV to -90 mV, as monitored in perforated whole-cell configuration, in turn enhancing Na(+)-independent, Cl(-)-dependent and DIDS-sensitive HCO(3)(-) secretion, as monitored through changes in intracellular pH. PDEC therefore encode concentrations of purinergic agonists as different patterns of [Ca(2+)](i) changes, which differentially stimulate K(+) channels, the Cl(-)-HCO(3)(-) exchanger, and exocytosis. Thus, in addition to amplitude, the temporal pattern of [Ca(2+)](i) increases is an important mechanism for transducing extracellular stimuli into different physiological effects.

Regulation of exocytosis by purinergic receptors in pancreatic duct epithelial cells.

In epithelial cells, several intracellular signals regulate the secretion of large molecules such as mucin via exocytosis and the transport of ions through channels and transporters. Using carbon fiber amperometry, we previously reported that exocytosis of secretory granules in dog pancreatic duct epithelial cells (PDEC) can be stimulated by pharmacological activation of cAMP-dependent protein kinase (PKA) or protein kinase C (PKC), as well as by an increase of intracellular free Ca2+ concentration ([Ca2+]i). In this study, we examined whether exocytosis in these cells is modulated by activation of endogenous P2Y receptors, which increase cAMP and [Ca2+]i. Low concentrations of ATP (<10 microM) induced intracellular Ca2+ oscillation but no significant exocytosis. In contrast, 100 microM ATP induced a sustained [Ca2+]i rise and increased the exocytosis rate sevenfold. The contribution of Ca2+ or cAMP pathways to exocytosis was tested by using the Ca2+ chelator BAPTA or the PKA inhibitors H-89 or Rp-8-bromoadenosine 3',5'-cyclic monophosphorothioate. Removal of [Ca2+]i rise or inhibition of PKA each partially reduced exocytosis; when combined, they abolished exocytosis. In conclusion, ATP at concentrations >10 microM stimulates exocytosis from PDEC through both Ca2+ and cAMP pathways.

Effects of bile acids on dog pancreatic duct epithelial cell secretion and monolayer resistance.

Pancreatic duct epithelial cells (PDEC) mediate the secretion of fluid and electrolytes and are exposed to refluxed bile. In nontransformed cultured dog PDEC, which express many ion transport pathways of PDEC, 1 mM taurodeoxycholic acid (TDCA) stimulated an (125)I(-) efflux inhibited by DIDS and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and a (86)Rb(+) efflux inhibited by charybdotoxin. Inhibition by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM suggests mediation via increased intracellular Ca(2+) concentration, whereas the absence of lactate dehydrogenase release excludes cellular toxicity. At 1 mM, TDCA stimulated a larger (125)I(-) efflux than glycodeoxycholate; two dihydroxy bile acids, taurochenodeoxycholate and TDCA, were similarly effective, whereas a trihydroxy bile acid, taurocholate, was ineffective. In Ussing chambers, 1 mM serosal or 2 mM luminal TDCA stimulated an I(sc) increase from confluent PDEC monolayers. TDCA also stimulated 1) a short-circuit current (I(sc)) increase from basolaterally permeabilized PDEC subject to a serosal-to-luminal Cl(-) gradient that was inhibited by BAPTA-AM, DIDS, and NPPB and 2) an I(sc) increase from apically permeabilized PDEC subject to a luminal-to-serosal K(+) gradient inhibited by BAPTA-AM and charybdotoxin. Along with the efflux studies, these findings suggest that TDCA interacts directly with PDEC to stimulate Ca(2+)-activated apical Cl(-) channels and basolateral K(+) channels. Monolayer transepithelial resistance was only minimally affected by 1 mM serosal and 2 mM luminal TDCA but decreased after exposure to higher TDCA concentrations (2 mM serosal and 4 mM luminal). A secretory role for bile acids should be considered in pancreatic diseases associated with bile reflux.

Angiotensin II evokes calcium-mediated signaling events in isolated dog pancreatic epithelial cells.

INTRODUCTION: Calcium-activated chloride conductance has been identified in normal pancreatic duct cells. Recent in vitro evidence suggests that angiotensin II (AngII) stimulates pancreatic secretion in both cystic fibrosis (CFPAC) and transformed pancreatic cells. AIMS: To investigate calcium-mediated stimulatory effects of AngII in both nontransformed dog pancreatic duct epithelial (DPDE) and CFPAC cells. METHODS: Western blots were performed in both cells seeking AngII receptors. In additional studies, DPDE and CFPAC cells were grown on vitrogen-coated glass cover slips and loaded with Indo-1-AM dye. Cells were placed in a confocal microscope's perfusion chamber and perfused with 100 microM AngII or ATP (control). Cells were excited with UV light, and intracellular calcium ([Ca+2]i) was read using fluorescence emission at 405 and 530 nm. Finally, single channels in the DPDE cells were examined using cell-attached patch clamps. Current amplitude histograms provided estimates of the conductance and open probability of channels. RESULTS: Western blots demonstrated presence of both AT and AT AngII receptors in DPDE and CFPAC cells; the density of AT receptors appeared lower than that of AT receptors. Basal intracellular calcium concentrations did not differ between DPDE (109 +/- 11 nM) and CFPAC (103 +/- 8 nM) cells. AngII significantly increased measured intracellular calcium concentrations in both DPDE (909 +/- 98 nM) and CFPAC (879 +/- 207 nM) cells, as did ATP (DPDE = 1722 +/- 228 nM; CFPAC = 1522 +/- 245 nM). In the patch clamp studies, a variety of different channels were observed; they appeared to be an 11pS nonselective cation (NSC) channel, a 4.6pS Na+ channel, a 3pS anion channel, and an 8pS chloride channel. The latter channel had characteristics similar to cystic fibrosis transmembrane conductance regulator (CFTR). Apical or basolateral application of AngII activated both the 11pS NSC and the 3pS channels. CONCLUSION: In nontransformed DPDE and CFPAC cells, specific AngII receptors mediate increases in [Ca ]. The latter effect of AngII may elicit activation of calcium-mediated chloride channels, suggesting a role for AngII as an alternative mediator of pancreatic ductal secretion.