Michael A. Gray

Secretin-regulated chloride channel on the apical plasma membrane of pancreatic duct cells.

SummaryUsing the patch clamp technique we have identified a small conductance ion channel that typically occurs in clusters on the apical plasma membrane of pancreatic duct cells. The cell-attached current/voltage (I/V) relationship was linear and gave a single channel conductance of about 4 pS. Since the reversal potential was close to the resting membrane potential of the cell, and unaffected by changing from Na+-rich to K+-rich pipette solutions, the channel selects for anions over cations in cell-attached patches. The open state probability was not voltagedependent. Adding 25mm-bicarbonate to the bath solution caused a slight outward rectification of theI/V relationship, but otherwise, the characteristics of the channel were unaffected. In excised, inside-out, patches theI/V relationship was linear and gave a single channel conductance of about 4 pS. A threefold chloride concentration gradient across the patch (sulphate replacement) shifted the single channel current reversal potential by −26 mV, indicating that the channel is chloride selective. Stimulation of duct cells with secretin (10nm), dibutyryl cyclic AMP (1mm) and forskolin (1 μm) increased channel open state probability and also increased the number of channels, and/or caused disaggregation of channel clusters, in the apical plasma membrane. Coupling of this channel to a chloride/bicarbonate exchanger would provide a mechanism for electrogenic bicarbonate secretion by pancreatic duct cells.

Dynamic Regulation of CFTR Bicarbonate Permeability by [Cl−]i and Its Role in Pancreatic Bicarbonate Secretion

BACKGROUND & AIMS Pancreatic bicarbonate (HCO3-) secretion is important for a healthy pancreas as well as digestive physiology. However, how human pancreatic duct cells secrete copious amounts of HCO3- has long been a puzzle. Here, we report that a dynamic increase in the cystic fibrosis transmembrane conductance regulator (CFTR) HCO3- permeability by intracellular Cl- concentration ([Cl-]i)-sensitive mechanisms plays a pivotal role in pancreatic HCO3- secretion. METHODS The role of [Cl-]i-sensitive kinases in CFTR-mediated HCO3- transport was examined in heterologous expression systems, PANC1 human pancreatic duct cells, and human and guinea pig pancreatic tissues using an integrated molecular and physiologic approach. RESULTS In human pancreatic tissues, CFTR-positive duct cells abundantly expressed with-no-lysine (WNK1) kinase, oxidative stress-responsive kinase 1 (OSR1), and sterile 20/SPS1-related proline/alanine-rich kinase (SPAK), which are known to be activated by low [Cl-]i. Interestingly, CFTR activation rapidly decreased [Cl-]i in response to luminal Cl- depletion in polarized PANC1 human pancreatic duct cells. Notably, the WNK1-mediated OSR1 and SPAK activation by low [Cl-]i strongly increased CFTR HCO3- permeability in CFTR-transfected HEK 293T, PANC1, and guinea pig pancreatic duct cells, making CFTR primarily an HCO3- channel, which is essential for the secretion of pancreatic juice containing HCO3- at a concentration greater than 140 mmol/L. In contrast, OSR1 and SPAK activation inhibited CFTR-dependent Cl-/HCO3- exchange activity that may reabsorb HCO3- from the high HCO3--containing pancreatic juice. CONCLUSIONS These results indicate that the [Cl-]i-sensitive activation of the WNK1-OSR1/SPAK pathway is the molecular switch to generate HCO3--rich fluid in the human pancreatic duct.

Development of substituted Benzo[c]quinolizinium compounds as novel activators of the cystic fibrosis chloride channel.

Chloride channels play an important role in the physiology and pathophysiology of epithelia, but their pharmacology is still poorly developed. We have chemically synthesized a series of substituted benzo[c]quinolizinium (MPB) compounds. Among them, 6-hydroxy-7-chlorobenzo[c]quinolizinium (MPB-27) and 6-hydroxy-10-chlorobenzo[c]quinolizinium (MPB-07), which we show to be potent and selective activators of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. We examined the effect of MPB compounds on the activity of CFTR channels in a variety of established epithelial and nonepithelial cell systems. Using the iodide efflux technique, we show that MPB compounds activate CFTR chloride channels in Chinese hamster ovary (CHO) cells stably expressing CFTR but not in CHO cells lacking CFTR. Single and whole cell patch clamp recordings from CHO cells confirm that CFTR is the only channel activated by the drugs. Ussing chamber experiments reveal that the apical addition of MPB to human nasal epithelial cells produces a large increase of the short circuit current. This current can be totally inhibited by glibenclamide. Whole cell experiments performed on native respiratory cells isolated from wild type and CF null mice also show that MPB compounds specifically activate CFTR channels. The activation of CFTR by MPB compounds was glibenclamide-sensitive and 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid-insensitive. In the human tracheal gland cell line MM39, MPB drugs activate CFTR channels and stimulate the secretion of the antibacterial secretory leukoproteinase inhibitor. In submandibular acinar cells, MPB compounds slightly stimulate CFTR-mediated submandibular mucin secretion without changing intracellular cAMP and ATP levels. Similarly, in CHO cells MPB compounds have no effect on the intracellular levels of cAMP and ATP or on the activity of various protein phosphatases (PP1, PP2A, PP2C, or alkaline phosphatase). Our results provide evidence that substituted benzo[c]quinolizinium compounds are a novel family of activators of CFTR and of CFTR-mediated protein secretion and therefore represent a new tool to study CFTR-mediated chloride and secretory functions in epithelial tissues.

Effects of bile acids on pancreatic ductal bicarbonate secretion in guinea pig

Background and aims: Acute pancreatitis is associated with significant morbidity and mortality. Bile reflux into the pancreas is a common cause of acute pancreatitis and, although the bile can reach both acinar and ductal cells, most research to date has focused on the acinar cells. The aim of the present study was to investigate the effects of bile acids on HCO3− secretion from the ductal epithelium. Methods: Isolated guinea pig intralobular/interlobular pancreatic ducts were microperfused and the effects of unconjugated chenodeoxycholate (CDC) and conjugated glycochenodeoxycholate (GCDC) on intracellular calcium concentration ([Ca2+]i) and pH (pHi) were measured using fluorescent dyes. Changes of pHi were used to calculate the rates of acid/base transport across the duct cell membranes. Results: Luminal administration of a low dose of CDC (0.1 mM) stimulated ductal HCO3− secretion, which was blocked by luminal H2DIDS (dihydro-4,4′-diisothiocyanostilbene-2,2′-disulfonic acid). In contrast, both luminal and basolateral administration of a high dose of CDC (1 mM) strongly inhibited HCO3− secretion. Both CDC and GCDC elevated [Ca2+]i, and this effect was blocked by BAPTA-AM (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid), caffeine, xestospongin C and the phospholipase C inhibitor U73122. BAPTA-AM also inhibited the stimulatory effect of low doses of CDC on HCO3− secretion, but did not modulate the inhibitory effect of high doses of CDC. Conclusions: It is concluded that the HCO3− secretion stimulated by low concentrations of bile acids acts to protect the pancreas against toxic bile, whereas inhibition of HCO3− secretion by high concentrations of bile acids may contribute to the progression of acute pancreatitis.

Regulation of maxi-K+ channels on pancreatic duct cells by cyclic AMP-dependent phosphorylation.

SummaryUsing the patch-clamp technique we have identified a Ca2+-sensitive, voltage-dependent, maxi-K+ channel on the basolateral surface of rat pancreatic duct cells. The channel had a conductance of ∼200 pS in excised patches bathed in symmetrical 150mm K+, and was blocked by 1mm Ba2+. Channel openstate probability (Po) on unstimulated cells was very low, but was markedly increased by exposing the cells to secretin, dibutyryl cyclic AMP, forskolin or isobutylmethylxanthine. Stimulation also shifted thePo/voltage relationship towards hyperpolarizing potentials, but channel conductance was unchanged. If patches were excised from stimulated cells into the inside-out configuration,Po remained high, and was not markedly reduced by lowering bath (cytoplasmic) Ca2+ concentration from 2mm to 0.1 μm. However, activated channels were still blocked by 1mm Ba2+. ChannelPo was also increased by exposing the cytoplasmic face of excised patches to the purified catalytic subunit of cyclic AMP-dependent protein kinase., We conclude that cyclic AMP-dependent phosphorylation can activate maxi-K+ channels on pancreatic duct cells via a stable modification of the channel protein itself, or a closely associated regulatory subunit, and that phosphorylation alters the responsiveness of the channels to Ca2+. Physiologically, these K+ channels may contribute to the basolateral K+ conductance of the duct cell and, by providing a pathway for current flow across the basolateral membrane, play an important role in pancreatic bicarbonate secretion.

Novel Role for Pendrin in Orchestrating Bicarbonate Secretion in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-expressing Airway Serous Cells

In most HCO3−-secreting epithelial tissues, SLC26 Cl−/HCO3− transporters work in concert with the cystic fibrosis transmembrane conductance regulator (CFTR) to regulate the magnitude and composition of the secreted fluid, a process that is vital for normal tissue function. By contrast, CFTR is regarded as the only exit pathway for HCO3− in the airways. Here we show that Cl−/HCO3− anion exchange makes a major contribution to transcellular HCO3− transport in airway serous cells. Real-time measurement of intracellular pH from polarized cultures of human Calu-3 cells demonstrated cAMP/PKA-activated Cl−-dependent HCO3− transport across the luminal membrane via CFTR-dependent coupled Cl−/HCO3− anion exchange. The pharmacological and functional profile of the luminal anion exchanger was consistent with SLC26A4 (pendrin), which was shown to be expressed by quantitative RT-PCR, Western blot, and immunofluorescence. Pendrin-mediated anion exchange activity was confirmed by shRNA pendrin knockdown (KD), which markedly reduced cAMP-activated Cl−/HCO3− exchange. To establish the relative roles of CFTR and pendrin in net HCO3− secretion, transepithelial liquid secretion rate and liquid pH were measured in wild type, pendrin KD, and CFTR KD cells. cAMP/PKA increased the rate and pH of the secreted fluid. Inhibiting CFTR reduced the rate of liquid secretion but not the pH, whereas decreasing pendrin activity lowered pH with little effect on volume. These results establish that CFTR predominately controls the rate of liquid secretion, whereas pendrin regulates the composition of the secreted fluid and identifies a critical role for this anion exchanger in transcellular HCO3− secretion in airway serous cells.

Volume-activated chloride currents in pancreatic duct cells.

We have used the patch clamp technique to study volume-activated Cl− currents in the bicarbonatesecreting pancreatic duct cell. These currents could be elicited by a hypertonic pipette solution (osmotic gradient 20 mOsm/l), developed over about 8 min to a peak value of 91 ± 5.8 pA/pF at 60 mV (n = 123), and were inhibited by a hypertonic bath solution. The proportion of cells which developed currents increased from 15% in freshly isolated ducts to 93% if the ducts were cultured for 2 days. The currents were ATP-dependent, had an outwardly rectifying current/voltage (I-V) plot, and displayed time-dependent inactivation at depolarizing potentials. The anion selectivity sequence was: ClO4 = I = SCN > Br = NO3 > Cl > F > HCO3 > gluconate, and the currents were inhibited to a variable extent by DIDS, NPPB, dideoxyforskolin, tamoxifen, verapamil and quinine. Increasing the intracellular Ca2+ buffering capacity, or lowering the extracellular Ca2+ concentration, reduced the proportion of duct cells which developed currents. However, removal of extracellular Ca2+ once the currents had developed was without effect. Inhibiting protein kinase C (PKC) with either the pseudosubstrate PKC (19–36), calphostin C or staurosporine completely blocked development of the currents. We speculate that cell swelling causes Ca2+ influx which activates PKC which in turn either phosphorylates the Cl− channel or a regulatory protein leading to channel activation.

Non-selective cation channel on pancreatic duct cells

We have identified a non-selective cation channel on pancreatic duct cells. These epithelial cells secrete the bicarbonate ions found in pancreatic juice; a process controlled by the hormone secretin, which uses cyclic AMP as an intracellular messenger. The non-selective channel is located on both apical and basolateral plasma membranes of the duct cell, is equally permeable to sodium and potassium, and has a linear I/V relationship with a single-channel conductance of about 25 pS. Channel opening requires the presence of 1 microM Ca2+ on the cytoplasmic face of the membrane, and is also increased by depolarization. Intracellular ATP, ADP, magnesium, and a rise in pH all decreased channel activity. The channel was not affected by 10 mM TEA, 1 mM Ba2+ or 0.5 mM decamethonium applied to the cytoplasmic face of the membrane, but 0.5 mM quinine caused a flickering block which was more pronounced at depolarizing potentials. We observed the channel only rarely in cell-attached patches on unstimulated duct cells, and acute exposure to stimulants did not cause channel activation. However, after prolonged stimulation, the proportion of cell-attached patches containing active channels was increased 9-fold. The role of this channel in pancreatic duct cell function remains to be elucidated.

Sendai virus-mediated CFTR gene transfer to the airway epithelium

The potential for gene therapy to be an effective treatment for cystic fibrosis has been hampered by the limited gene transfer efficiency of current vectors. We have shown that recombinant Sendai virus (SeV) is highly efficient in mediating gene transfer to differentiated airway epithelial cells, because of its capacity to overcome the intra- and extracellular barriers known to limit gene delivery. Here, we have identified a novel method to allow the cystic fibrosis transmembrane conductance regulator (CFTR) cDNA sequence to be inserted within SeV (SeV-CFTR). Following in vitro transduction with SeV-CFTR, a chloride-selective current was observed using whole-cell and single-channel patch-clamp techniques. SeV-CFTR administration to the nasal epithelium of cystic fibrosis (CF) mice (CftrG551D and Cftrtm1UncTgN(FABPCFTR)#Jaw mice) led to partial correction of the CF chloride transport defect. In addition, when compared to a SeV control vector, a higher degree of inflammation and epithelial damage was found in the nasal epithelium of mice treated with SeV-CFTR. Second-generation transmission-incompetent F-deleted SeV-CFTR led to similar correction of the CF chloride transport defect in vivo as first-generation transmission-competent vectors. Further modifications to the vector or the host may make it easier to translate these studies into clinical trials of cystic fibrosis.

Ca2+ and cAMP-activated Cl− conductances mediate Cl− secretion in a mouse renal inner medullary collecting duct cell line

1 The nature of Cl− conductance(s) participating in transepithelial anion secretion by renal inner medullary collecting duct (IMCD, mIMCD‐K2 cell line) was investigated. 2 Extracellular ATP (100 μM) stimulated a transient increase in both whole‐cell Cl− conductance and intracellular free Ca2+. In contrast, ionomycin (10–100 nM) caused a sustained increase in whole‐cell Cl− conductance. Pre‐loading cells with the Ca2+ buffer BAPTA abolished the ATP‐dependent responses and delayed the onset of the increase observed with ionomycin. 3 The Ca2+‐activated whole‐cell Cl− current stimulated by ATP (peak) and ionomycin (maximal) displayed (i) a linear steady‐state current‐voltage relationship and (ii) time and voltage dependence with slow activation at +80 mV and slow inactivation at −80 mV. In BAPTA‐loaded cells, ionomycin‐elicited whole‐cell currents exhibited pronounced outward rectification with time‐dependent activation/inactivation. 4 Ca2+‐activated and forskolin‐activated Cl− conductances co‐exist since ATP activation of whole‐cell current occurred during a maximal stimulation by forskolin in single cell recordings. 5 In IMCD epithelial layers, ATP and ionomycin stimulated an inward short circuit current (Isc) dependent upon basal medium Na+ and Cl−/HCO3− but independent of the presence of apical bathing medium Na+ and Cl−/HCO3−. This was identical to forskolin stimulation and consistent with transepithelial anion secretion. 6 PCR amplification of reverse‐transcribed mRNA using gene‐specific primers demonstrated expression of both cystic fibrosis transmembrane conductance regulator (CFTR) mRNA and Ca2+‐activated Cl− channel (mCLCA1) mRNA in mIMCD‐K2 cells. 7 Ca2+ and forskolin‐activated Cl− conductances participate in anion secretion by IMCD.