Regulation of pancreatic excretory function by ion channels. Viktoria Venglovecz

Transcription

1 Regulation of pancreatic excretory function by ion channels Viktoria Venglovecz

2 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

3 Composition of pancreatic juice 1 2 liters of pancreatic juice per day acini secrete isotonic, plasmalike fluid pancreatic duct absorbs most of the Cl and secrete HCO 3 Human, 140 mm HCO 3 Mouse and rat, 50 70mM HCO 3 The cation composition of the juice is nearly constant Also contains Mg 2, Zn 2, PO 4 3 and SO 4 2

4 Fluid and electrolyte secretion by acinar cells Twostep 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

5 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

6 K + channels Acinar cell membrane potential is set by the K + channels Ca 2+ activated K + channel BK channels (voltageactivated) IK channels (time and voltage independent) Gene deletion revealed that both channels are required to sustain acinar function

7 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

8 Na + /H + (NHE) and Cl /HCO 3 (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

9 TMEM16A Cl exit in acinar cells is mediated by apical Ca 2+ 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

10 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

11 Fluid and electrolyte secretion by acinar cells NHE1 and AE2 set cytoplasmic ph at 7.2 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 Ca 2+ 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 Tight junctions are permeable to Na + and is the main route for transcellular Na + flux

12 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

13 TRANSPORTERS AT THE BASOLATERAL MEMBRANE

14 Na + /H + exchanger 1 (NHE1) electroneutral 1Na + /1H + exchangers NHE1 ubiquitous phi homeostasis localized at the basolateral membrane provide a small portion of HCO 3 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 HCO 3 salvage

15 TRANSPORTERS AT THE BASOLATERAL MEMBRANE

16 Na + /HCO 3 + cotransporter (NBC) Acidbase homeostasis, Regulation of cell volume and intracellular ph NBC1 expressed at the BASOLATERAL membrane of most epithelia electrogenic transporter 1 Na + 2 HCO 3 stoichiometry NBC uses Na + gradient to accumulate cytosolic HCO 3 mediates the bulk of basolateral HCO 3 + entry NBC3 expressed at the LUMINAL membrane electroneutral transporter 1 Na + 1 HCO 3 + stoichiometry Regulated by CFTR inhibition With NHE3 are part of the HCO 3 regulatory complex

17 TRANSPORTERS AT THE BASOLATERAL MEMBRANE

18 TRANSPORTERS AT THE BASOLATERAL MEMBRANE

19 TRANSPORTERS AT THE LUMINAL MEMBRANE

20 CFTR CFTR is the protein mutated in cystic fibrosis belongs to the ATPbinding cassette (ABC) transporters superfamily ABC transporters functions as a membrane pumps and transport their substrate against the ec. gradient activated by the camp/pka pathway Cl channel with limited permeability to HCO 3 + anion channel activity functions as a smallconductance (5 10 ps) Cl _ channel Central role in ductal fluid secretion

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

22 TRANSPORTERS AT THE LUMINAL MEMBRANE

23 ANION EXCHANGERS conserved family of ion transporters 10 members, Slc26a111 genes encode proteins that transport mono and divalent ions (Cl, HCO 3, I, oxalate, formate, hydroxyl) each SLC26 paralog has different anion specificities Group 1 selective for SO 4 (i.e. SLC26A1, 2), Group 2 as Cl /HCO 3 exchangers (i.e. SLC26A3, 4, 6) Group 3 as ion channels (i.e. SLC26A7, 9) transporters in Group 2 have potent Cl / HCO 3 exchange activity SLC26a3 2 Cl and 1 HCO 3, SLC26a6 1 Cl and 2 HCO 3 SLC26a4 1 Cl and 1 HCO 3

24 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 chloridelosing diarrhea, SLC26A4 Pendred syndrome and hereditary deafness

25 Gene name Aliases Locus Substrates Distribution Related diseases SLC26A1 Sat1 4p16.3 Liver, kidney SLC26A2 DTDST 5q3134 SO 4 2, Cl Widespread Chondrodysplasia SLC26A3 DRA, CLD 7q31 SO 4 2, Cl,HCO 3, OH, oxalate Intestine, sweat gland, pancreas, prostate Congenital chloridelosing diarrhea SLC26A4 Pendrin 7q31 Cl, HCO 3, I, formate Inner ear, kidney, thyroid Pendred syndrome, DFNB4 SLC26A5 Prestin 7q22 Inner ear Nonsyndromic hearing loss SLC26A6 CFEX, PAT1 3p21.3 SO 4 2, Cl,HCO 3, OH, oxalate, formate Widespread SLC26A7 8q22.2 SO 2 4, Cl, oxalate Kidney, GI tract SLC26A8 Tat1 6p21.3 SO 2 4, Cl, oxalate Sperm, brain SLC26A9 1q3132 SO 2 4, Cl, oxalate Lung, Kidney, GI tract SLC26A11 17q25 SO 2 4 Widespread

26 MECHANISM OF HCO 3 SECRETION Proposed model: HCO 3 is derived from CO 2 Additional Na + dependent HCO 3 uptake mechanism has been identified, namely the NBC CA catalyses the formation of carbonic acid and the dissipation into HCO 3 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 HCO 3 leaves the cell on the apical membrane via the exchanger in exchange for Cl and Cl recycles via CFTR

27 RELATIONSHIP BETWEEN ACINAR AND DUCTAL CELLS

28 Physiology Pathophysiology STRESS activating enzymes enzymes Alcohol 45% Bile 45% Others 10% fluid and HCO 3 AUTODIGESTION DIGESTION INFLAMMATION CELL DEATH

29 Development of Acute Pancreatitis Inducing Factor Intracinar Events Immun Response Bile acid Ethanol Other factors NO SPECIFIC THERAPY Pathological Ca 2+ signal Colocalization of lysosomal enzymes and zymogens Intrapancreatic trypsinogen activation Autodigestion, Inflammation ACUTE PANCREATITIS Leukocyte activation Cytocines (IL6, TNFα, etc.) ROS

30 Ductal cells Acinar cells Insufficient electrolyte and fluid secretion by ductal cells in CF Destruction of acinar cells Primary defect Pancreatic duct in membrane trafficking obstruction of zymogens Correction of the luminal ph reverses the membrane trafficking defects and largely restores the membrane dynamics Freedman SD, Gastroenterology, 2001 Model of postercp pancreatitis in rats ph 6.0 ph 6.9 ph 7.3 Pancreatic damage c Noble MD, Gut, 2008 Lower extracellular ph Mislocalization of CFTR in AIP Bhoomagoud M, Gastroenterology, 2009 enhances secretagogueinduced zymogen activation Decreased pancreatic ductal function Reversal by corticosteroid treatment Ko SB, Gastroenterology, 2010

31 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

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

33 Venglovecz V et al. Gut 2008;57: Ignath I. et al. Pancreas 2009;38:9219. Venglovecz V, et al. Gut 2011;60:3619. Maleth J et al. Gut 2011;60:1368.

34 Hegyi P. et al. Gut 2011;60: Maléth J. et al.unpiblished

35 The trypsin vicious cycle Pallagi et al, Gastroenterology 2011 Dec;141(6):

36 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 HCO 3 HCO 3 HCO 3 HCO 3 HCO 3 The main pancreatitisinducing factors HCO (bile acids and ethanol) are strong 3 inhibitors of pancreatic ductal bicarbonate secretion