Viktória Venglovecz

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.

The acinar-ductal tango in the pathogenesis of acute pancreatitis

There is an unacceptably high mortality in acute pancreatitis, which is due to the lack of specific treatments for the disease. A major reason stated to account for the inability to develop effective treatments is that there are multiple pathobiologic pathways activated in the acinar cell mediating pancreatitis making it difficult to choose molecular targets for therapeutic strategies. However, this reasoning limits opportunities for therapeutic development because it does include another important participant in pancreatitis - the pancreatic duct cells. The most recent advance in pancreatitis research is that depletion of both glycolytic and oxidative ATP synthesis is a common event in both acinar and ductal cells. Although ATP has a very short half-life in the blood and is hydrolysed to ADP, there is clear evidence that encapsulating ATP into liposomes can effectively drive ATP into the cells which can be effective in protecting them from necrosis. In this review, we will examine the effects of different insults associated with pancreatitis on both the acinar and ductal components of the exocrine pancreas pointing out the role of the ductal epithelial responses in both attenuating and increasing the severity of pancreatitis. In addition, we propose that exogenous ATP administration may restore ductal and acinar function providing therapeutic benefit.

New therapeutic targets in ulcerative colitis: The importance of ion transporters in the human colon

Background: The absorption of water and ions (especially Na+ and Cl−) is an important function of colonic epithelial cells in both physiological and pathophysiological conditions. Despite the comprehensive animal studies, there are only scarce available data on the ion transporter activities of the normal and inflamed human colon. Methods: In this study, 128 healthy controls and 69 patients suffering from ulcerative colitis (UC) were involved. We investigated the expressional and functional characteristics of the Na+/H+ exchangers (NHE) 1–3, the epithelial sodium channel (ENaC), and the SLC26A3 Cl−/HCOSymbol exchanger downregulated in adenoma (DRA) in primary colonic crypts isolated from human biopsy and surgical samples using microfluorometry, patch clamp, and real‐time reverse‐transcription polymerase chain reaction (RT‐PCR) techniques. Symbol. No caption available. Results: Data collected from colonic crypts showed that the activities of electroneutral (via NHE3) and the electrogenic Na+ absorption (via ENaC) are in inverse ratio to each other in the proximal and distal colon. We found no significant differences in the activity of NHE2 in different segments of the colon. Surface cell Cl−/HCOSymbol exchange is more active in the distal part of the colon. Importantly, both sodium and chloride absorptions are damaged in UC, whereas NHE1, which has been shown to promote immune response, is upregulated by 6‐fold. Symbol. No caption available. Conclusions: These results open up new therapeutic targets in UC. (Inflamm Bowel Dis 2011;)

Non-conjugated chenodeoxycholate induces severe mitochondrial damage and inhibits bicarbonate transport in pancreatic duct cells

We read the manuscripts by Lee and Muallem1 and Venglovecz et al 2 recently published in Gut with great interest. In both articles the authors highlighted the role of pancreatic ducts in maintaining the integrity of the pancreas. Venglovecz et al showed that a high concentration of the non-conjugated chenodeoxycholate (CDC) inhibits pancreatic ductal bicarbonate secretion; however, the mechanisms of the inhibition were not clarified. This is a follow-up study in which we show that this reduction of ductal bicarbonate secretion by CDC is evoked by inhibition of glycolytic and oxidative (caused by severe mitochondrial damage) metabolism with a consequent depletion of intracellular ATP levels. Physiologically, pancreatic ductal fluid and HCO3− secretion are necessary to wash out the digestive enzymes from the acinar cells into the duodenum. Under pathophysiological conditions toxic factors (such as bile acids and ethanol) involved in the pathogenesis of acute pancreatitis have dual effects on ductal HCO3− secretion.1 Low doses of CDC and ethanol were found to stimulate fluid and HCO3− secretion. However, these toxic agents in high concentrations inhibit the secretion. These data suggest that an elevation in pancreatic ductal fluid and HCO3− secretion may have protective roles. However, since under physiological conditions the pressure in the main pancreatic duct is higher than in the bile ducts, it is still controversial as to whether bile acids enter the pancreatic ductal tree. We have recently shown that a high concentration (1 mM) of the non-conjugated bile acid CDC has strong inhibitory effects on the activities of acid/base transporters (Na+/H+ exchanger (NHE), Na+/HCO3− cotransporter (NBC) …

Trypsin Reduces Pancreatic Ductal Bicarbonate Secretion by Inhibiting CFTR Cl− Channels and Luminal Anion Exchangers

BACKGROUND & AIMS The effects of trypsin on pancreatic ductal epithelial cells (PDECs) vary among species and depend on the localization of proteinase-activated receptor 2 (PAR-2). We compared PAR-2 localization in human and guinea-pig PDECs, and used isolated guinea pig ducts to study the effects of trypsin and a PAR-2 agonist on bicarbonate secretion. METHODS PAR-2 localization was analyzed by immunohistochemistry in guinea pig and human pancreatic tissue samples (from 15 patients with chronic pancreatitis and 15 without pancreatic disease). Functionally, guinea pig PDECs were studied by microperfusion of isolated ducts, measurements of intracellular pH and intracellular Ca(2+) concentration, and patch clamp analysis. The effect of pH on trypsinogen autoactivation was assessed using recombinant human cationic trypsinogen. RESULTS PAR-2 localized to the apical membrane of human and guinea pig PDECs. Trypsin increased intracellular Ca(2+) concentration and intracellular pH and inhibited secretion of bicarbonate by the luminal anion exchanger and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. Autoactivation of human cationic trypsinogen accelerated when the pH was reduced from 8.5 to 6.0. PAR-2 expression was strongly down-regulated, at transcriptional and protein levels, in the ducts of patients with chronic pancreatitis, consistent with increased activity of intraductal trypsin. Importantly, in PAR-2 knockout mice, the effects of trypsin were markedly reduced. CONCLUSIONS Trypsin reduces pancreatic ductal bicarbonate secretion via PAR-2-dependent inhibition of the apical anion exchanger and the CFTR Cl(-) channel. This could contribute to the development of chronic pancreatitis by decreasing luminal pH and promoting premature activation of trypsinogen in the pancreatic ducts.

Pathophysiological relevance of apical large-conductance Ca 2+ -activated potassium channels in pancreatic duct epithelial cells

Background Acute pancreatitis is among the few inflammatory diseases for which no specific pharmacological treatment is available. It has previously been shown that bile acids alter pancreatic ductal secretion and these effects are probably involved in the pathogenesis of bile-induced pancreatitis. Objective To understand the mechanism responsible for bile-induced hypersecretion and, in particular, to identify the molecular target for bile acids in native pancreatic duct epithelial cells (PDECs). Methods Patch clamp recordings and spectrofluorimetry were used to measure whole cell currents and rates of HCO3− secretion, respectively, from isolated guinea pig pancreatic ducts. Expression of ion channels and receptors was investigated by immunohistochemistry/immunofluorescence of intact pancreatic tissue. Results Exposing PDECs to chenodeoxycholate (CDC, 100 μM) reversibly increased whole cell K+ currents and hyperpolarised cell membrane potential. Bile acid-stimulated K+ currents were inhibited by Ba2+ (2 mM), iberiotoxin (100 nM), and suppressed by strong intracellular Ca2+ buffering. Luminally applied iberiotoxin also blocked CDC-stimulated HCO3− secretion from microperfused ducts; however, the inhibitor did not influence the stimulatory effect of secretin, carbachol or luminally applied ATP. The specific large-conductance Ca2+-activated potassium (BK) channel activator, NS11021, induced a similar increase in HCO3− secretion to CDC. Immunohistochemical analysis showed strong BK channel protein expression on the apical membrane of PDECs, while the G-protein-coupled bile acid receptor-1 was not detected in PDECs, but was present in acinar cells. Conclusion It was shown for the first time that BK channels (i) are expressed at the apical membrane of guinea pig PDECs; (ii) have a crucial role in regulating HCO3− secretion and (iii) are also essential for the bile acid-induced hypersecretion and, therefore, underlie the response of the pancreas to this noxious agent.

Central role of mitochondrial injury in the pathogenesis of acute pancreatitis

Acute pancreatitis is an inflammatory disease with no specific treatment. One of the main reasons behind the lack of specific therapy is that the pathogenesis of acute pancreatitis is poorly understood. During the development of acute pancreatitis, the disease‐inducing factors can damage both cell types of the exocrine pancreas, namely the acinar and ductal cells. Because damage of either of the cell types can contribute to the inflammation, it is crucial to find common intracellular mechanisms that can be targeted by pharmacological therapies. Despite the many differences, recent studies revealed that the most common factors that induce pancreatitis cause mitochondrial damage with the consequent breakdown of bioenergetics, that is, ATP depletion in both cell types. In this review, we summarize our knowledge of mitochondrial function and damage within both pancreatic acinar and ductal cells. We also suggest that colloidal ATP delivery systems for pancreatic energy supply may be able to protect acinar and ductal cells from cellular damage in the early phase of the disease. An effective energy delivery system combined with the prevention of further mitochondrial damage may, for the first time, open up the possibility of pharmacological therapy for acute pancreatitis, leading to reduced disease severity and mortality.

The Crucial Role of Early Mitochondrial Injury in L-Lysine-Induced Acute Pancreatitis

AIMS Large doses of intraperitoneally injected basic amino acids, L-arginine, or L-ornithine, induce acute pancreatitis in rodents, although the mechanisms mediating pancreatic toxicity remain unknown. Another basic amino acid, L-lysine, was also shown to cause pancreatic acinar cell injury. The aim of the study was to get insight into the mechanisms through which L-lysine damages the rat exocrine pancreas, in particular to characterize the kinetics of L-lysine-induced mitochondrial injury, as well as the pathologic responses (including alteration of antioxidant systems) characteristic of acute pancreatitis. RESULTS We showed that intraperitoneal administration of 2 g/kg L-lysine induced severe acute necrotizing pancreatitis. L-lysine administration caused early pancreatic mitochondrial damage that preceded the activation of trypsinogen and the proinflammatory transcription factor nuclear factor-κB (NF-κB), which are commonly thought to play an important role in the development of acute pancreatitis. Our data demonstrate that L-lysine impairs adenosine triphosphate synthase activity of isolated pancreatic, but not liver, mitochondria. INNOVATION AND CONCLUSION Taken together, early mitochondrial injury caused by large doses of L-lysine may lead to the development of acute pancreatitis independently of pancreatic trypsinogen and NF-κB activation.

CFTR Expression But Not Cl- Transport Is Involved in the Stimulatory Effect of Bile Acids on Apical Cl-/HCO3- Exchange Activity in Human Pancreatic Duct Cells

Objectives: Low doses of chenodeoxycholate (CDC) stimulate apical anion exchange and HCO3− secretion in guinea pig pancreatic duct cells (Gut. 2008;57:1102-1112). We examined the effects of CDC on intracellular pH (pHi), intracellular Ca2+ concentration ([Ca2+]i), and apical Cl−/HCO3− exchange activity in human pancreatic duct cells and determined whether any effects were dependent on cystic fibrosis transmembrane conductance regulator (CFTR) expression and Cl− channel activity. Methods: Polarized CFPAC-1 cells (expressing F508del CFTR) were transduced with Sendai virus constructs containing complementary DNAs for either wild-type CFTR or &bgr;-galactosidase. Microfluorimetry was used to record pHi and [Ca2+]i and apical Cl−/HCO3− exchange activity. Patch clamp experiments were performed on isolated guinea pig duct cells. Results: Chenodeoxycholate induced a dose-dependent intracellular acidification and a marked increase in [Ca2+]i in CFPAC-1 cells. CFTR expression slightly reduced the rate of acidification but did not affect the [Ca2+]i changes. Luminal administration of 0.1 mmol/L of CDC significantly elevated apical Cl−/HCO3− exchange activity but only in cells that expressed CFTR. However, CDC did not activate CFTR Cl− conductance. Conclusions: Bile salts modulate pHi, [Ca2+]i, and apical anion exchange activity in human pancreatic duct cells. The stimulatory effect of CDC on anion exchangers requires CFTR expression but not CFTR channel activity.

NHE1 activity contributes to migration and is necessary for proliferation of human gastric myofibroblasts

Myofibroblasts play central roles in wound healing, deposition of the extracellular matrix and epithelial function. Their functions depend on migration and proliferation within the subepithelial matrix, which results in accelerated cellular metabolism. Upregulated metabolic pathways generate protons which need to be excreted to maintain intracellular pH (pHi). We isolated human gastric myofibroblasts (HGMs) from surgical specimens of five patients. Then we characterized, for the first time, the expression and functional activities of the Na+/H+ exchanger (NHE) isoforms 1, 2 and 3, and the functional activities of the Na+/HCO3− cotransporter (NBC) and the anion exchanger (AE) in cultured HGMs using microfluorimetry, immunocytochemistry, reverse transcription polymerase chain reaction and immunoblot analysis. We showed that NHE1–3, NBC and AE activities are present in HGMs and that NHE1 is the most active of the NHEs. In scratch wound assays we also demonstrated (using the selective NHE inhibitor HOE-642) that carbachol and insulin like growth factor II (IGF-II) partly stimulate migration of HGMs in a NHE1-dependent manner. EdU incorporation assays revealed that IGF-II induces proliferation of HGMs which is inhibited by HOE-642. The results indicate that NHE1 is necessary for IGF-II-induced proliferation response of HGMs. Overall, we have characterized the pHi regulatory mechanisms of HGMs. In addition, we demonstrated that NHE1 activity contributes to both IGF-II- and carbachol-stimulated migration and that it is obligatory for IGF-II-induced proliferation of HGMs.