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, 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-

4. 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

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
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

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-/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

9. 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 .

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
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

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 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

15. TRANSPORTERS AT THE BASOLATERAL MEMBRANE

16. 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

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 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

21. 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

22. TRANSPORTERS AT THE LUMINAL MEMBRANE

23. 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-

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 - chloride-losing diarrhea,
SLC26A4 - Pendred syndrome and hereditary deafness

25. 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-

26. 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

27. RELATIONSHIP BETWEEN ACINAR AND DUCTAL CELLS

28. Physiology Pathophysiology
STRESS
enzymes
Alcohol 45%
Bile 45%
Others %
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

29. 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

30. 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

31. 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

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

33. 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:
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.

34. 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:
Maléth J. et al.unpiblished

35. 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): 35

36. 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-