Toan D. Nguyen

Trypsin activates pancreatic duct epithelial cell ion channels through proteinase-activated receptor-2

Proteinase-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved by trypsin within the NH2-terminus, exposing a tethered ligand that binds and activates the receptor. We examined the secretory effects of trypsin, mediated through PAR-2, on well-differentiated nontransformed dog pancreatic duct epithelial cells (PDEC). Trypsin and activating peptide (AP or SLIGRL-NH2, corresponding to the PAR-2 tethered ligand) stimulated both an 125I- efflux inhibited by Ca2+-activated Cl- channel inhibitors and a 86Rb+ efflux inhibited by a Ca2+-activated K+ channel inhibitor. The reverse peptide (LRGILS-NH2) and inhibited trypsin were inactive. Thrombin had no effect, suggesting absence of PAR-1, PAR-3, or PAR-4. In Ussing chambers, trypsin and AP stimulated a short-circuit current from the basolateral, but not apical, surface of PDEC monolayers. In monolayers permeabilized basolaterally or apically with nystatin, AP activated apical Cl- and basolateral K+ conductances. PAR-2 agonists increased [Ca2+]i in PDEC, and the calcium chelator BAPTA inhibited the secretory effects of AP. PAR-2 expression on dog pancreatic ducts and PDEC was verified by immunofluorescence. Thus, trypsin interacts with basolateral PAR-2 to increase [Ca2+]i and activate ion channels in PDEC. In pancreatitis, when trypsinogen is prematurely activated, PAR-2-mediated ductal secretion may promote clearance of toxins and debris.

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 NH2 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, PHPLC-δ1-enhanced green fluorescent protein for phosphatidylinositol 4,5-bisphosphate, and protein kinase Cγ (PKCγ)-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 Ca2+ mobilization from inositol 1,4,5-trisphosphate-sensitive Ca2+ stores and a subsequent Ca2+ influx through store-operated Ca2+ channels cause a biphasic increase in intracellular Ca2+ concentration ([Ca2+]i), measured with Indo-1 dye. Single-cell amperometry demonstrated that this increase in [Ca2+]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 μm luminal trypsin did not induce exocytosis in polarized PDEC monolayers; on the other hand, 10 μm trypsin at 37 °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 [Ca2+]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.

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

Intracellular calcium concentration ([Ca2+]i) is a key factor controlling secretion from various cell types. We investigated how different patterns of [Ca2+]i signals evoke salt secretion via ion transport mechanisms and mucin secretion via exocytosis in dog pancreatic duct epithelial cells (PDEC). Activation of epithelial P2Y2 receptors by UTP generated two patterns of [Ca2+]i change: 2–10 μm UTP induced [Ca2+]i oscillations, whereas 100 μm UTP induced a sustained [Ca2+]i increase, both in the micromolar range. As monitored by carbon‐fibre amperometry, the sustained [Ca2+]i increase stimulated a larger increase in exocytosis than [Ca2+]i oscillations, despite their similar amplitude. In contrast, patch‐clamp recordings revealed that [Ca2+]i oscillations synchronously activated a K+ current as efficiently as the sustained [Ca2+]i increase. This K+ current was mediated by intermediate‐conductance Ca2+‐activated K+ channels (32 pS at −100 mV) which were sensitive to charybdotoxin and resistant to TEA. Activation of these Ca2+‐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 HCO3− secretion, as monitored through changes in intracellular pH. PDEC therefore encode concentrations of purinergic agonists as different patterns of [Ca2+]i changes, which differentially stimulate K+ channels, the Cl−–HCO3− exchanger, and exocytosis. Thus, in addition to amplitude, the temporal pattern of [Ca2+]i increases is an important mechanism for transducing extracellular stimuli into different physiological effects.

Isolation and long-term culture of gallbladder epithelial cells from wild-type and CF mice

SummaryMice with targeted disruption of the cftr gene show pathophysiologic changes in the gallbladder, which correlate with hepatobiliary disease seen in cystic fibrosis patients. As gallbladder epithelium secretes mucin, and as this epithelium consists of a relatively homogenous cell type, study of CFTR function in these cells would be beneficial to delineate the complex cellular functions of this protein. The size and anatomic location of the murine gallbladder makes such studies difficult in vivo. Therefore, the need exists for in vitro models of gallbladder epithelium. We describe a method to isolate and culture murine gallbladder epithelium from wild-type and CF mice. Cells were grown in a monolayer on porous inserts over a feeder layer of fibroblasts. These nontransformed cells can be successively passaged and maintain a well-differentiated epithelial cell phenotype as shown by morphologic criteria, characterized by polarized columnar epithelial cells with prominent microvilli and intercellular junctions. Organotypic cultures showed columnar cells simulating in vivo morphology. This culture system should be valuable in delineating cellular processes relating to CFTR in gallbladder epithelium.

Calcium-activated potassium conductances on cultured nontransformed dog Pancreatic duct epithelial cells

Pancreatic duct epithelial cells (PDECs) mediate the pancreatic secretion of fluid and electrolytes. Membrane K+ channels on these cells regulate intracellular K+ concentration; in combination with the Na+/H+ antiport and Na+, K+-adenosine triphosphatase (ATPase), they may also mediate serosal H+ secretion, balancing luminal HCO−3 secretion. We describe the K+ conductances on well-differentiated and functional nontransformed cultured dog PDECs. Through 86Rb+ efflux studies, we demonstrated Ca2+-activated K+ channels that were stimulated by A23187, thapsigargin, and 1-ethyl-2-benzimidazolinone, but not forskolin. These conductances also were localized on the basolateral membrane because 86Rb+ efflux was directed toward the serosal compartment. Of the K+ channel blockers, BaCl2, charybdotoxin, clotrimazole, and quinidine, but not 4-aminopyridine, apamin, tetraethylammonium, or iberiotoxin, inhibited 86Rb+ efflux. This efflux was not inhibited by amiloride, ouabain, and bumetanide, inhibitors of the Na+/H+ antiport, the Na+, K+-ATPase pump, and the Na+, K+, 2Cl− cotransporter, respectively. When apically permeabilized PDEC monolayers were mounted in Ussing chambers with a luminal-to-serosal K+ gradient, A23187 and 1-ethyl-2- benzimidazolinone stimulated a charybdotoxin-sensitive shortcircuit current (Isc) increase. Characterization of K+ channels on these cultured PDECs, along with previous identification of Cl− channels (1), further supports the importance of these cells as models for pancreatic duct secretion.

Culture of human main pancreatic duct epithelial cells

SummaryAttempts to grow human pancreatic duct epithelial cells in long-term culture have proven difficult. We have developed a system of growing these cells for several passages by adapting methods used to culture dog pancreatic duct cells. Epithelial cells were enzymatically dissociated from the main pancreatic duct and plated onto collagen-coated culture inserts suspended above a human fibroblast feeder layer. After primary culture, the cells were either passaged onto new inserts or plastic tissue culture plates in the absence of collagen. Cells grown on the latter plates were maintained in a serum-free medium. Primary pancreatic duct epithelial cells grow steadily to confluence as a monolayer in the feeder layer system. After primary culture, cells passaged onto new inserts with fresh feeder layer or plastic plates and fed with serum-free medium continued to develop into confluent monolayers for up to four passages. The cells were columnar with prominent apical microvilli, sub-apical secretory vesicles, and lateral intercellular junctions resembling the morphology of normal in vivo epithelial cells. These cells were also positive for cytokeratin 19, 7, and 8 and carbonic anhydrase II, as measured by immunohistochemistry. Metabolically, these cells synthesized and secreted mucin, as measured by incorporation of tritiated N-acetyl-d-glucosamine. In conclusion, we demonstrated that human pancreatic epithelial cells from the main duct can be successfully grown in culture and repeatedly passaged using a feeder layer system, with serum-free medium, and in organotypic cultures.

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 microscopes perfusion chamber and perfused with 100 &mgr;M 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 AT1 and AT2 AngII receptors in DPDE and CFPAC cells; the density of AT1 receptors appeared lower than that of AT2 receptors. Basal intracellular calcium concentrations did not differ between DPDE (109 ± 11 n M) and CFPAC (103 ± 8 n M) cells. AngII significantly increased measured intracellular calcium concentrations in both DPDE (909 ± 98 n M) and CFPAC (879 ± 207 n M) cells, as did ATP (DPDE = 1722 ± 228 n M; CFPAC = 1522 ± 245 n M). 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+2]i. 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.

Growth and function of isolated canine pancreatic ductal cells

These studies investigated the growth characteristics and functional properties of isolated canine pancreatic ductal epithelial cells. Cells were isolated from the accessory pancreatic duct and cultured by using three conditions: on vitrogen-coated petri dishes with fibroblast conditioned medium (nonpolarized); in vitrogen-coated Transwells above a fibroblast feeder layer (polarized); or as organotypic rafts above a fibroblast-embedded collagen layer (polarized). Growth characteristics, transepithelial resistances, and carbonic anhydrase and cyclic adenosine monophosphate (AMP) responses were evaluated. Under polarized conditions, the cells grew as monolayers with columnar epithelial characteristics. The monolayers developed high transepithelial resistance and became impervious to the passage of horseradish peroxidase. Epithelial growth factor (EGF) (2 ng/ml) stimulated ductal cell growth and accelerated the formation of a high-resistance monolayer. Forskolin (10 microM) rapidly decreased transepithelial resistance. Carbonic anhydrase activity, which was lower in nonpolarized compared with polarized conditions, was stimulated by carbachol (175 microM). Secretin, however, did not stimulate carbonic anhydrase activity in these cells. Although secretin stimulated adenylyl cyclase activity in early-passage cells, this response was lost in later-passage cells. Both vasoactive intestinal polypeptide (VIP; 1 microM) and forskolin (10 microM) consistently increased adenylyl cyclase activity. Isolated canine pancreatic ductal epithelial cells proliferate in vitro, develop high-resistance epithelial monolayers, and respond to stimuli that activate adenylyl cyclase. These cells should provide a useful model for regulatory studies of ductal cell functions.

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

Exocytosis is evoked by intracellular signals, including Ca2+ 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 [Ca2+]i increases induced either through Ca2+ 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 Ca2+ elevations. Neither this Ca2+-dependent granule movement nor the number of granules found near the plasma membrane were detectably changed by raising cAMP, suggesting that cAMP potentiates Ca2+-dependent exocytosis at a later stage. A kinetic model was made of the exocytosis stimulated by UTP, trypsin, and Ca2+ ionophores with and without cAMP increase. In the model, without a cAMP rise, receptor activation stimulates exocytosis both by Ca2+ 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 Ca2+ 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 Ca2+-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.