Regulation of pancreatic excretory function by ion channels Viktoria Venglovecz

Morphology of the pancreas exocrine/endocrine gland acinar and ductal cells acinar cells: rough ER smooth ER nucleus Golgi apparatus secretory granules acinar lobules terminates with the centroacinar cells centroacinar cells intercalated ducts intra/interlobular ducts Main duct shares a duodenal openinig with the common bile duct at the Ampulla of Vater

Composition of pancreatic juice 1–2 liters of pancreatic juice per day acini secrete isotonic, plasma-like fluid pancreatic duct absorbs most of the Cl- and secrete HCO3- Human, 140 mM HCO3- Mouse and rat, 50–70mM HCO3- The cation composition of the juice is nearly constant Also contains Mg2, Zn2, PO43- and SO42-

Fluid and electrolyte secretion by acinar cells Two-step process Acinar cells secrete a small amount of volume Most fluid is secreted by the ductal cells Duct modify the electrolyte composition to generate the final fluid

Na+/K+ ATPase Fluid and electrolyte secretion is fueled by the cellular Na gradient hydrolyzes ATP to exchange 3 Na+ in for 2 K+ out generate the transcellular Na+ and K+ gradients provides the electrochemical gradient determines the membrane potential  

K+ channels Acinar cell membrane potential is set by the K+ channels Ca2+ -activated K+ channel BK channels (voltage-activated) IK channels (time- and voltage independent) Gene deletion revealed that both channels are required to sustain acinar function

Na+/K+/2Cl- cotransporter (NKCC) NKCC1 is ubiquitous activated by cell shrinkage mediate regulatory volume increase to restore cell volume expressed at the basolateral membrane inhibited by the diuretics, furosemide and bumetanide mediates 70% of the Cl- uptake provide most of the Na+ necessary to fuel the Na+/K+ pump

Na+/H+ (NHE) and Cl-/HCO3- (AE) exchangers NHE1 and AE2 NHE1 and AE2 are ubiquitous housekeepers of cytoplasmic pH activated by small changes in intracellular pH NHE1 is activated by acidic pHi AE2 is activated by alkaline pHi NHE1 and AE2 are involved in many cellular functions including mediation of acinar cell fluid and electrolyte secretion

TMEM16A Cl- exit in acinar cells is mediated by apical Ca2+ -activated Cl- channel extensively characterized by biophysically molecular identity of the channel is still unknown TMEM16A/Anoctamin 1 has been identify in salivary and pancreas acini knock down of TMEM16A reduces salivary secretion .

AQUAPORINS (AQPs) AQP family consists of 13 members water channels glyceroporins AQPs functions: cell adhesion, proliferation, migration, and cell survival Diseases: Sjögren’s syndrome and pancreatitis The established AQP in acinar cells is AQP5 (luminal membrane of salivary gland acini) Regulate both trans- and paracellular water transport Deletion of AQP5 disrupt the integrity of tight junctions and reduce water permeability

Fluid and electrolyte secretion by acinar cells Secretion is fueled by the basolateral Na+/K+ ATPase pump intracell. Na+ = 20 mM, intracell. K+ =140 mM NKCC1 and NHE1 are the main routes of Na+ influx Ca2+ -activated K+ channels set the membrane potential at 50 to 60 mV NKCC1 and AE2 are the major route of Cl- influx. Cl- = 60 mV NHE1 and AE2 set cytoplasmic pH at 7.2 Tight junctions are permeable to Na+ and is the main route for transcellular Na+ flux

Fluid and electrolyte secretion by ductal cells Studies on ductal function lagged behind studies on acinar function Advanced techniques over the past 25 years changed this

TRANSPORTERS AT THE BASOLATERAL MEMBRANE

Na+/H+ exchanger 1 (NHE1) electroneutral 1Na+/1H+ exchangers NHE1 ubiquitous pHi homeostasis localized at the basolateral membrane provide a small portion of HCO3- influx NHE2 and NHE3 localized at the luminal membrane role of NHE2 is not clear deletion of NHE2 in mice has no obvious phenotype NHE3 plays role in HCO3- salvage

TRANSPORTERS AT THE BASOLATERAL MEMBRANE

Na+/HCO3+ cotransporter (NBC) Acid-base homeostasis, Regulation of cell volume and intracellular pH NBC1 NBC3 expressed at the BASOLATERAL membrane of most epithelia electroGENIC transporter 1 Na+-2 HCO3- stoichiometry NBC uses Na+ gradient to accumulate cytosolic HCO3- mediates the bulk of basolateral HCO3+ entry expressed at the LUMINAL membrane electroNEUTRAL transporter 1 Na+-1 HCO3+ stoichiometry Regulated by CFTR inhibition With NHE3 are part of the HCO3- regulatory complex

TRANSPORTERS AT THE BASOLATERAL MEMBRANE

TRANSPORTERS AT THE BASOLATERAL MEMBRANE

TRANSPORTERS AT THE LUMINAL MEMBRANE

CFTR CFTR is the protein mutated in cystic fibrosis belongs to the ATP-binding cassette (ABC) transporters superfamily ABC transporters functions as a membrane pumps and transport their substrate against the ec. gradient anion channel activity functions as a small-conductance (5–10 pS) Cl_ channel activated by the cAMP/PKA pathway Cl- channel with limited permeability to HCO3+ Central role in ductal fluid secretion

CFTR exists as a macromolecular complex composed of two, 6 span membrane bound regions each connected to a nuclear binding domain (NBD) R-domain – comprised of many charged amino acids mutations identified in CF occur in the first nucleotide binding domain (NBD1) Activation of CFTR: phosphorylation of the R domain binding of ATP to NBD R domain is highly conserved between species

TRANSPORTERS AT THE LUMINAL MEMBRANE

ANION EXCHANGERS conserved family of ion transporters 10 members, Slc26a1-11 genes encode proteins that transport mono- and divalent ions (Cl-, HCO3- , I-, oxalate, formate, hydroxyl) each SLC26 paralog has different anion specificities Group 1 selective for SO4- (i.e. SLC26A1, -2), Group 2 as Cl-/HCO3- exchangers (i.e. SLC26A3, -4, -6) Group 3 as ion channels (i.e. SLC26A7, -9) transporters in Group 2 have potent Cl- / HCO3- exchange activity SLC26a3 – 2 Cl- and 1 HCO3-, SLC26a6 – 1 Cl- and 2 HCO3- SLC26a4 - 1 Cl- and 1 HCO3-

ANION EXCHANGERS Physiological role: SCL26 are expressed in the kidney (except 3) Kidney: Cl- homeostasis, oxalate excretion, kidney stone formation, vascular volume and blood pressure regulation acid/base balance Pancreas: secretion of juice skeletal development  thyroid hormone synthesis Genetic disorders: SLC26A2 - chondrodysplasias, SLC26A3 - chloride-losing diarrhea, SLC26A4 - Pendred syndrome and hereditary deafness

SLC26A6 SLC26A3 Gene name Aliases Locus Substrates Distribution Related diseases SLC26A1 Sat-1 4p16.3   Liver, kidney SLC26A2 DTDST 5q31-34 SO42-, Cl- Widespread Chondrodysplasia SLC26A3 DRA, CLD 7q31 SO42-, Cl-,HCO3-, OH-, oxalate Intestine, sweat gland, pancreas, prostate Congenital chloride-losing diarrhea SLC26A4 Pendrin Cl-, HCO3-, I-, formate Inner ear, kidney, thyroid Pendred syndrome, DFNB4 SLC26A5 Prestin 7q22 Inner ear Non-syndromic hearing loss SLC26A6 CFEX, PAT-1 3p21.3 SO42-, Cl-,HCO3-, OH-, oxalate, formate SLC26A7 8q22.2 SO42-, Cl-, oxalate Kidney, GI tract SLC26A8 Tat1 6p21.3 Sperm, brain SLC26A9 1q31-32 Lung, Kidney, GI tract SLC26A11 17q25 SO42-

MECHANISM OF HCO3- SECRETION Proposed model: HCO3- is derived from CO2 Additional Na+ dependent HCO3- uptake mechanism has been identified, namely the NBC CA catalyses the formation of carbonic acid and the dissipation into HCO3- and H+ H+ are extruded through the NHE Driving force for the NHE is provided by the pump K + channels allow the recirculation of K + and maintain m.pot. Na + enter the secretion via paracellualr pathway HCO3- leaves the cell on the apical membrane via the exchanger in exchange for Cl- and Cl- recycles via CFTR

RELATIONSHIP BETWEEN ACINAR AND DUCTAL CELLS

Physiology Pathophysiology STRESS enzymes Alcohol 45% Bile 45% Others 10% activating enzymes And now I would like to talk about the pathophysiology of pancreatic ion transport processes. As I mentioned under physiological conditions, acinar cells secrete the inactive form of digestive enzymes which will be transported through the ductal tree to the duodenum where it become active and take part int he digestion. The ductal cells secrete the bicarbonate rich alkaline solution which help to wash out the enzymes from the pancreas. However, int he case of any stress, such as excessive alcohol consuption, the present of gallstones or other factors such as ercp, viral infection this will cause premature activation of digestive enzymes, which leads to autodigestion and inflammation of the pancreas. fluid and HCO3- AUTODIGESTION INFLAMMATION DIGESTION CELL DEATH

Development of Acute Pancreatitis Inducing Factor Intracinar Events Immun Response - Bile acid - Ethanol Other factors -Pathological Ca2+ signal -Colocalization of lysosomal enzymes and zymogens Leukocyte activation Cytocines (IL-6, TNFα, etc.) ROS -Intrapancreatic trypsinogen activation This schematic figure shows the development of AP. The process starts with an inducing factor like alcohol or bile acids, this will induce intraacinar events, like … and it will swith the immune response which involves. Although the mortality and morbidity of pancreatitis is unacceptedly high no specif therapy is currently available for the disease. -Autodigestion, Inflammation NO SPECIFIC THERAPY ACUTE PANCREATITIS

Insufficient electrolyte and fluid secretion by ductal cells in CF Acinar cells Insufficient electrolyte and fluid secretion by ductal cells in CF Destruction of acinar cells Primary defect in membrane trafficking of zymogens Correction of the luminal pH reverses the membrane trafficking defects and largely restores the membrane dynamics Pancreatic duct obstruction Freedman SD, Gastroenterology, 2001 c Model of post-ERCP pancreatitis in rats pH 6.9 pH 7.3 pH 6.0 Pancreatic damage Noble MD, Gut, 2008 Lower extracellular pH enhances secretagogue-induced zymogen activation Bhoomagoud M , Gastroenterology, 2009 It has been shown that insufficient electrolyte and fluid secretion by pancreatic ductal cells in cystic fibrosis leads to destruction of acinar cells. In addition, this insufficient fluid secretion leads to a primary defect in membrane trafficking at the apical plasma membrane of acinar cells. Importantly, correction of the luminal pH reverses the membrane trafficking defects in pancreatic acinar cells and restores the membrane dynamics required for exocytosis of zymogen granules. The importance of luminal pH is also suggested in a model of post ERCP pancreatitis in rats. Contrast solution at pH 6.0 injected into the pancreatic duct caused histological damage, increase in serum amylase and pancreatic odema. Whereas contrast solution at pH7.3 caused only a little damage. It has been recently shown by Smuel Mualem workgroup that corticosteroid treatment repairs pancreatic damage and improves ductal HCO3 secretion in patient with AIP. The decreased fluid secretion in AIP is probably due to the mislocalization of CFTR to the cytosol, whereas corticosteroids treatment corrected its targeting to the apical membrane. Furthermore, recent studies have shown that lowering extracellular pH, which can occur during the deficit of luminal bicarbonate concentration enhances secretagogue-induced zymogen activation in acinar cells. Mislocalization of CFTR in AIP Decreased pancreatic ductal function Reversal by corticosteroid treatment Ko SB, Gastroenterology, 2010

alterations in pancreatic ductal fluid Ductal cells Acinar cells alterations in pancreatic ductal fluid and bicarbonate secretion can increase patients’ risk to pancreatitis restoration of pancreatic ductal bicarbonate and fluid secretion may have therapeutic benefits

ETIOLOGICAL FACTORS IN ACUTE PANCREATITIS Gallstone (bile acid) Alcohol Trypsinogen → Trypsin ?

Venglovecz V et al. Gut 2008;57:1102-12. Using isolated inra/interlobular pancreatic ducts we have characterized the effect of bile acids on pancreatic ducts. We have shown that low concentration of the non-conjugated bile acid, CDC induce physiological calcium signalling in the ductal cell. The increased intracellular calcium activates BK channels at the apical membrane which in turn increases the electrochemical driving force for ion secretion. We also investigated the inhibitory effect of high dose of CDC. We found, that contrast to low conc. of CDC, high conc induces toxic calcium signalling, and mitochondrial damage and consequently depletion of ATPi. In the absence of ATP the iontransporters do not work properly, which probably leads to the impaired bicarbonate secretion. Most probably when the ductal defence mechanism is damaged, bile acids can reach the acinar cells at high concentrations. The key point in the toxic effects of bile acid on acinar cells is the generation of global sustained Ca2+ waves. Bile acids elevate the Ca2+i by stimulating Ca2+ efflux from the (i) endoplasmic reticulum (ER) via IP3R and ryanodine receptors, from the (ii) acidic Ca2+ stores (AS) and by (iii) stimulating indirectly the opening of store-operated Ca2+ channels (SOCC). In addition, bile acids inhibit Ca2+ restoration to the basal level by blocking both the sarco/ER Ca2+ ATPase (SERCA)-dependent Ca2+ reloading into intracellular pools and the plasma membrane Ca2+ ATPase (PMCA)-dependent Ca2+ excretion. Venglovecz V et al. Gut 2008;57:1102-12. Ignath I. et al. Pancreas 2009;38:921-9. Venglovecz V, et al. Gut 2011;60:361-9. Maleth J et al. Gut 2011;60:136-8.

Maléth J. et al.unpiblished Administration of ethanol in low concentration (10mM) induced short lasting, repetitive Ca2+ spikes in ductal cells, which released from the ER via the IP3 receptor. In addition ethanol, at low concentration was able to stimulated bicarbonate secretion. The stimulatory effect of ethanol is depend on an elevation in intracellular calcium concentration. High concentration of ethanol (100mM) induced a small sustained [Ca2+]i elevation, whereas high concentration of POA which is the non-oxidative metabolite of EtOH induced toxic sustained two-phases [Ca2+]i rise. We have also shown that high conc of etoh and POA strongly inhibited bicarbonate secretion. Using patch clamp technique we showed that both ethanol and POA at high conc. strongly inhibited forskolin stimulated cftr currents as well. High concentration of ethanol and POA also inhibited mitochondrial function and induced (ATP)i depletion. Taken together high concentration of POA inhibit both bicarbonate efflux and CFTR, induce sustained intracellular calcium signals in PDEC, (iv) inhibit mitochondrial function and decrease (ATP)i level Similar mechanisms occur in the acinar cells which finally leads to secretory block and pancreatitis. Hegyi P. et al. Gut 2011;60:544-52. Maléth J. et al.unpiblished

The trypsin vicious cycle Several studies indicated that early intra-acinar or luminal activation of trypsin is a key event int he development of acute pancreatitis. The effect of trypsin on pancreatic ductal cells differs among species. In dog trypsin activates iontransporters and bicarbonate secretion whereas in bovine inhibits. In addition the effect of trypsin also depends ont he localization of PAR-2 receptors In guinea pig, we characterized the effect of trypsin ont he ductal cells. We showed that PAR-2 is highly expressed int he luminal membrane of small intra/interlobular ducts. Luminal administration of the PAR-2 activation peptide or trypsin caused a dose-dependent calcium signalling and evoked alkalosis. We have also shown that trypsin inhibited the forskolin-stimulated CFTR currents and bicarbonate secretion. Since the anion exchanger not work properly the HCO3 concentration in the pancreatic juice decreases, which leads to the acidification of the juice. Since autoactivation of trypsinogen is pH-dependent process, at acidic pH the autoactivation of trypsinogen markedly increased which can leads to the autodigestion of the gland. Pallagi et al, Gastroenterology 2011 Dec;141(6):2228-2239 35

HCO3- What are the roles of bicarbonate secretion? To neutralize the acid content secreted by acinar cells To curtail trypsinogen autoactivation within the pancreatic ductal system To neutralise the acid chyme entering the duodenum from the stomach To defend the pancreas by washing out the toxic agents HCO3- HCO3- HCO3- The main pancreatitis-inducing factors (bile acids and ethanol) are strong inhibitors of pancreatic ductal bicarbonate secretion HCO3- HCO3- HCO3-