Coordination of Pancreatic HCO3

Min Goo Lee
, Wooin Ahn
, Jin Ah Lee
, Joo Young Kim
, Joo Young Choi
, Orson W Moe
Sharon L Milgram
, Shmuel Muallem
, Kyung Hwan Kim
Department of Pharmacology, Yonsei University College of Medicine. Seoul, Korea.
Department of
Physiology and
Department of Internal Medicine, University of Texas, Southwestern Medical Center.
Dallas, Texas (USA).
Department of Cell and MolecularPhysiology, University of North Carolina.
Chapel Hill, North Carolina (USA)
Increasing evidence suggests that protein-
protein interaction is essential in many
biological processes including epithelial
transport. In this report, we discuss the
significance of protein interactions to HCO3
secretion in pancreatic duct cells. In pancreatic
ducts HCO3
secretion is mediated by cystic
fibrosis transmembrane conductance regulator
(CFTR) activated luminal Cl
activity and HCO3
absorption is achieved by
-dependent mechanisms including Na
exchanger 3 (NHE3). We found biochemical
and functional association between CFTR and
NHE3. In addition, protein binding through
PDZ modules is needed for this regulatory
interaction. CFTR affected NHE3 activities in
two ways. Acutely, CFTR augmented the
cAMP-dependent inhibition of NHE3. In a
chronic mechanism, CFTR increases the
luminal expression of Na
exchange in
pancreatic duct cells. These findings reveal that
protein complexes in the plasma membrane of
pancreatic duct cells are highly organized for
efficient HCO3
Fluid secretion is required for proper
functioning of essential organs such as the lung
and pancreas. HCO3
, an important component
of the secreted fluids, is the subject of increased
attention since it governs the luminal pH and
solubility of protein in the secreted fluids. We
have previously reported that cystic fibrosis
transmembrane conductance regulator (CFTR)
participates in HCO3
secretion by stimulating a
- dependent HCO3
transport, in the form of
exchange activity [1, 2]. Another
important mechanism in HCO3
homeostasis is
a HCO3
-absorbing processes in the resting
state. In the pancreatic duct 50% of HCO3
absorption is mediated by Na
exchanger 3
(NHE3) and 50% by a novel, yet unidentified,
-dependent mechanism [3]. An interesting
feature of HCO3
homeostasis is the possibility
that the activity of multiple mechanisms is
regulated by interaction between the
transporters mediated by scaffolding proteins
such as ezrin-binding phosphoprotein 50
(EBP50) [4]. Both PDZ (PSD95, Dlg1, ZO-1)
domains of EBP50 bind the C-terminus of
CFTR to dimerize it and regulate its activity as
a Cl
channel [5]. NHE3 interacts with EBP50
via the second PDZ domain [6]. In a recent
work, we observed regulatory interaction
between CFTR and NHE3, possibly through
EBP50, in a heterologous expression system of
PS120 cells and in the native pancreatic duct
[7]. Here, we discuss the significance of protein

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JOP. Journal of the Pancreas – – Vol.2, No.4 Suppl – July 2001
interactions to HCO3
secretion in pancreatic
duct cells.
Initially, we examined whether CFTR and
NHE3 exist in the same protein complexes.
NHE3 was found in the anti-CFTR
immunoprecipitates when CFTR and NHE3
were co-expressed in PS120 cells,
demonstrating that exogenously expressed
CFTR and NHE3 may associate in a stable
complex. To determine whether CFTR and
NHE3 also associate in native cells, we
performed the same experiments using
pancreata from wild type (WT) and CFTR-
impaired homozygote F508 (F) mice. NHE3
was detected in anti-CFTR immunoprecipitates
from the pancreas of WT mouse. In contrast,
only a very small amount of NHE3 was found
in CFTR immunoprecipitates from the pancreas
of F mouse.
Next we studied the effect of CFTR on NHE3
activity. Treatment of PS120/NHE3 cells with
forskolin inhibited NHE3 activity dose-
dependently, which was maximal at 10 µM.
Forskolin also inhibited NHE3 activity in
CFTR co-expressing cells. However, the
inhibition of NHE3 activity was significantly
higher at any given forskolin concentrations
when compared to control cells and nearly
maximum at 0.1 µM of forskolin. Thuswe
concluded that activation of CFTR augments
cAMP-mediated inhibition of NHE3 in PS120
In an immunolocalization study, we observed
the co-localization of CFTR, NHE3, and
EBP50 in the luminal area of mouse pancreatic
duct cells. Therefore we determined whether
CFTR expression affects the Na
activity in the luminal membrane of the
perfused pancreatic duct. When the luminal
NHE3 activity was measured in pancreatic
ducts from F mice, it was evident that the
basal activity was significantly lower than that
from WT mice. The reduced activity in F
mice was independent of age. Similar degree of
reduction in NHE3 activity was found in as
early as 2-week-old mice, suggesting that an
innate mechanism is responsible for the
decreased activity rather than an adaptive
process necessary for survival (Table 1).
Subsequent quantitative confocal microscopy
revealed 53% reduced luminal expression of
NHE3 in ducts from F mice. In another set of
experiment using mice ages from 3 to 6
months, we found that 10 µM forskolin
inhibited the luminal NHE3 activity by 40% in
WT mice, similar to the findings in PS120 cells.
However, the same concentration of forskolin
failed to show significant inhibition on the
residual NHE3 activity in F mice.
The present findings may have importance in
understanding the overall role of CFTR in
epithelial physiology and in cystic fibrosis.
Notably, co-expression of CFTR increased the
basal activity and expression levels of NHE3 in
the luminal membrane of pancreatic duct cells.
By forming a protein complex, CFTR may
enhance the stability of the expressed NHE3 or
its delivery to the luminal membrane of the
pancreatic duct. Alternatively, CFTR may
increase the transcription of NHE3 mRNA or
its half-life. In an acute mechanism, CFTR
augmented the cAMP-dependent inhibition of
Table 1. Luminal NHE3 activity in pancreatic ducts from WT and F mice.
Age of mice
Luminal Na+/Hexchange activity (∆∆pH/min)
2 weeks
1.05 ± 0.15
0.29 ± 0.13**
2 months
1.09 ± 0.09
0.25 ± 0.13**
6 months
0.92 ± 0.10
0.38 ± 0.12**
Pancreatic ducts were microdissected from WT and F mice, cannulated and used to measure the luminal Na+/Hexchange
activity [3].

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JOP. J. Pancreas (Online) 2001; 2(4 Suppl):203-206.
JOP. Journal of the Pancreas – – Vol.2, No.4 Suppl – July 2001
NHE3 in both PS120 cells and pancreatic ducts.
Pancreatic ductal fluid and HCO3
secretion is
stimulated by the Gs-coupled secretin or
vasoactive intestinal polypeptide (VIP)
receptors. Upon cell stimulation, cellular cAMP
is increased and the CFTR-EBP50-NHE3
complex either is formed or may undergo a
conformational change to allow regulatory
inhibition of Na
-dependent H
fluxes by
CFTR. This inhibits HCO3
absorption by the
duct cells. At the same time, CFTR stimulates
secretion by activating a Cl
exchange process in the luminal membrane of
the pancreatic duct [1, 2]. The overall result is
production of an alkaline pancreatic juice.
These findings demonstrate a coordinated
regulation of HCO3
secretion mediated by the
CFTR-NHE3 protein complex. In this respect,
it is of particular interests that many of the G
protein-coupled membrane receptors and
transporters related to HCO3
secretion in
pancreatic duct cells have a PDZ-binding motif
on their C-terminus (Figure 1). In addition,
most are associated with cAMP-dependent
processes. It has been shown that the scaffolds
EBP50 and E3KARP can recruit possible A-
kinase anchoring proteins (AKAP) such as
ezrin to the protein complex, hence increasing
the signaling efficiency of cAMP [8]. Such an
arrangement allows for precise and tight control
of HCO3
homeostasis by CFTR.
Key words Bicarbonates; Cystic Fibrosis
Transmembrane Conductance Regulator;
Pancreas; Protein Binding; Sodium-Hydrogen
Abbreviations AKAP: A-kinase anchoring
proteins; CFTR: cystic fibrosis transmembrane
conductance regulator; EBP50: ezrin-binding
phosphoprotein 50; NHE: Na
PDZ: PSD95, Dlg1, ZO-1; VIP: vasoactive
intestinal polypeptide; WT: wild type; F:
CFTR-impaired homozygote F508
Acknowledgments This work was supported
by the Brain Korea 21 Project for Medical
Sciences, Yonsei University (K.H.K.) and the
Korean Medical Association in the program
year of 2000 (W.A.).
Min Goo Lee
Department of Pharmacology
Yonsei University College of Medicine
134 Sinchon-dong
Seoul 120-752
Phone: +82-2-361.5221
Fax: +82-2-313.1894
E-mail address:
1. Lee MG, Wigley WC, Zeng W, Noel LE, Marino
CR, Thomas PJ, Muallem S. Regulation of Cl-/ HCO3-
Figure 1. Possible multiple protein interactions
controlling HCO3
secretion in pancreatic ducts. Several
membrane receptors and transporters participating in
homeostasis in pancreatic duct cells have a PDZ-
binding motif (-X-T/S-X-hydrophobic amino acid) on
their C-terminus. The C-terminal sequences are based on
the human clones. Except for several purinergic
receptors, most of the proteins are associated with
cAMP-dependent processes.
The abbreviations used are: DRA, down-regulated in
adenoma; NBC, Na+-HCO3
co-transporter; P2,
purinergic receptor; RII, regulatory subunit of protein
kinase A type II.

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JOP. J. Pancreas (Online) 2001; 2(4 Suppl):203-206.
JOP. Journal of the Pancreas – – Vol.2, No.4 Suppl – July 2001
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