Cell Parietal Fisiol Annulas Rew 2003

14 Jan 2003 13:55 AR AR177-PH65-05.tex AR177-PH65-05.
SGM LaTeX2e(2002/01/18) P1: fhd

10.1146/annurev.physiol.65.072302.114200
Annu. Rev. Physiol. 2003. 65:10331

doi: 10.1146/annurev.physiol.65.072302.114200
Copyright c 2003 by Annual Reviews. All rights reserved
First published online as a Review in Advance on December 9, 2002
CELL BIOLOGY OF ACID SECRETION BY

THE PARIETAL CELL
Xuebiao Yao1,2 and John G. Forte1

1
Department of Molecular and Cell Biology University of California, Berkeley, California
94720, and 2Laboratory of Cell Dynamics, University of Science and Technology of China,
Annu. Rev. Physiol. 2003.65:103-131. Downloaded from arjournals.annualreviews.org
Hefei, China 230027; email: xbyao@uclink.berkeley.edu; jforte@uclink.berkeley.edu
Key Words membrane recruitment, membrane fusion, gastric secretion,

cytoskeleton, SNARE, HCl secretion
by HINARI on 05/16/07. For personal use only.
Abstract Acid secretion by the gastric parietal cell is regulated by paracrine,

endocrine, and neural pathways. The physiological stimuli include histamine, acetyl-
choline, and gastrin via their receptors located on the basolateral plasma membranes.
Stimulation of acid secretion typically involves an initial elevation of intracellular
calcium and/or cAMP followed by activation of a cAMP-dependent protein kinase
cascade that triggers the translocation and insertion of the proton pump enzyme,
H,K-ATPase, into the apical plasma membrane of parietal cells. Whereas the
H,K-ATPase contains a plasma membrane targeting motif, the stimulation-mediated
relocation of the H,K-ATPase from the cytoplasmic membrane compartment to the
apical plasma membrane is mediated by a SNARE protein complex and its regulatory
proteins. This review summarizes the progress made toward an understanding of the
cell biology of gastric acid secretion. In particular we have reviewed the early signaling
events following histaminergic and cholinergic activation, the identification of multi-
ple factors participating in the trafficking and recycling of the proton pump, and the
role of the cytoskeleton in supporting the apical pole remodeling, which appears to be
necessary for active acid secretion by the parietal cell. Emphasis is placed on identify-
ing protein factors that serve as effectors for the mechanistic changes associated with
cellular activation and the secretory response.
INTRODUCTION
Secretion of HCl into the stomach is mediated by the oxyntic (acid-secreting)

cell, one of several epithelial cell types within gastric glands. In mammalian gas-
tric mucosa, oxyntic cells project peripherally, onto the walls of the gland and
thus are commonly called parietal cells, which is the designation we use in this
review. Gastric acid secretion is a tightly regulated process triggered by ligand-
receptor binding at the basolateral plasma membrane with ultimate output of H+,
Cl and H2O across the apical plasma membrane of the parietal cell. The phys-
iological stimuli include acetylcholine, gastrin, and especially, histamine, which
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104 YAO FORTE
operate via basolateral membrane receptors. Stimulation of acid secretion typically

involves an initial elevation of intracellular calcium and cAMP, followed by acti-
vation of protein kinase cascades, which trigger the translocation and insertion of
H,K-ATPasethe proton pump enzymeinto the apical plasma membrane of the
parietal cell. This review primarily focuses on research over the past 10 years that
has sought to identify and describe specific roles for proteins involved in the activa-
tion, membrane translocation, and recycling processes that underlie acid secretion.
Table 1 provides a summary of the various proteins that have been implicated in the
functional activity of parietal cell secretion. In the course of this review, relevant
earlier studies are briefly discussed and references given to related review articles.
AN ELABORATE STRUCTURAL BASIS

FOR ACID SECRETION
The parietal cell is a highly specialized epithelial cell with several distinctive
morphological characteristics that directly bear on its functional activities (1).
The apical plasma membrane has the unusual structural form of a series of small
canals (canaliculi) that invaginate from the surface and project throughout the
entire cell interior, with frequent interconnections among canaliculi. In the non-
secreting or resting parietal cell, these apical canalicular surfaces are lined with
short, stubby microvilli that are supported by prominent actin microfilaments in-
cluding accessory actin-binding proteins. Throughout the cytoplasmic space there
is an abundance of membranous structures, rich in H,K-ATPase, that take the mor-
phological form of vesicles, tubules, and cisternal sacs; these are commonly called
H,K-ATPase-rich tubulovesicles.
Parietal cells undergo a profound morphological transition upon activation of
acid secretion. Although limited by the optics of light microscopy, careful histo-
logical examination by Golgi in the late 19th century noted the enlargement of
canaliculi that occurred in secreting parietal cells. Electron micrographs of maxi-
mally stimulated cells have since revealed dilated canalicular spaces, a dramatically
expanded apical membrane surface with elongated microvilli, and diminution of
cytoplasmic tubulovesicles (24). Mounting evidence has contributed to a general
consensus for the membrane recycling hypothesis which holds that activation of
acid secretion results in a fusion-based recruitment of H,K-ATPase-rich cytoplas-
mic membranes into the apical plasma membrane (5). However, an alternate view
has argued that the H,K-ATPase-rich membranes are contiguous with the apical
plasma membrane in both resting and stimulated states, thus negating the need
for a fusion-based recruitment process (68). Recent evidence has clarified the
morphological argument through the use of high-pressure, rapid-freeze fixation to
prepare gastric parietal cells for electron microscopy (EM) and analysis of three-
dimensional structure (9). Models constructed from ultra-thin serial sections, as
well as tomographic reconstruction of thick tissue sections using high voltage EM,
clearly demonstrate the separation of tubulovesicular membranes from the plasma
CELL BIOLOGY OF ACID SECRETION 105
TABLE 1 Summary of parietal cell proteins implicated in the cell activation process
Mass Cellular Presumed
Name (kDa) location function Reference
Proton pump
H,K-ATPase = 96 Tubulovesicle (resting) H+ pumping (86)
-, -subunits = 6080 Apical PM (secreting) stabilize -subunit (115)
Trafficking and membrane recycling proteins
Syntaxin 1 34 Apical PM (?) Docking protein (92, 93)
Syntaxin 3 34 Cytoplasm (resting) H,K-ATPase translocation (92, 94)
Apical PM (secreting) (94)
SNAP-25 25 Apical PM SNARE pairing (92)
VAMP-2 18 Cytoplasm (resting) H,K-ATPase translocation (92, 93)

Apical PM (secreting)
Rab11 23 Cytoplasm (resting) H,K-ATPase translocation (94, 101)
Apical PM (secreting) (94, 101)
Rab25 23 ? H,K-ATPase translocation (100)
Rab11-interacting Tubulovesicle (resting) Rab11-myosin Vb binding (103)
proteins (FIP1 and FIP2)
pp75/Rip11 75 Tubulovesicle (resting) Rab11 binding (103)

SCAMPs 37 Tubulovesicle (resting) Vesicle trafficking (93)
Clathrin (heavy and 160; 35 Apical PM, tubulovesicles Endocytosis (104, 105)
light chains)
Dynamin 110 Apical PM Endocytosis (105)
-, -, -adaptins Tubulovesicle (resting) Endocytosis (104, 105)
Regulatory kinases
PKA cat. = 43 Cytoplasm Activation (17)
reg. = 52 Basolateral PM Anchoring
PKC (multiple isozymes) 80 Cytosol; membranes Protein phosphorylation (52, 53)
PI3 kinase cat. = 110 ? PI phosphorylation
reg. = 85
CAM kinase 50 Cytosol Protein phosphorylation (54)
MAP kinase 34 Cytosol Protein phosphorylation (59, 60)
MLCK 110 Cytosol Myosin lc phosphorylation (146)
Cytoskeleton
Actin
-cytoplasmic 43 Apical microvilli Microvillar formation (134)
-cytoplasmic 43 Basolateral PM Polarity formation (?) (134)
Tubulins 55 Cytoplasm TV trafficking ? (150)
Ezrin 80 Apical microvilli PKA-regulated membrane- (22, 24)
actin-filament linker
Coroninse 66 Apical microvilli PKC-dependent membrane- (28)
actin linker
Myosin V 110 Endosome Rab11a-binding; recycling (113)
Lasp-1 28 Apical PM Membrane-cytoskeleton (39, 41)
linking protein
Ankyrin 110 Apical PM H,K-ATPase binding (125)
linking protein
Spectrin 220/240 Apical PM Membrane-cytoskeleton (125)
Other proteins
AKAP120 120 ? PKA anchoring (146)
Calpain I cat. = 80 Cytosol Hydrolysis of ezrin (147)
reg. = 30
Parchorin 65 Cytosol (resting) Cl channel regulation (26, 42)
PM (stimulated)
PM = plasma membrane; TV = tubulovesicles.

106 YAO FORTE
membrane and generally confirm early studies using chemical fixation techniques.
This revisiting of parietal cell ultrastructure, together with the requirement for
recruitment/fusion-based proteins in parietal cell activation (see below) reinforces
the recruitment-recycling model of parietal cell activation. In addition, this new
fixation method has revealed that the abundant parietal cell mitochondria form an
extensive reticular network throughout the cytoplasm, as has been reported for
several other cell types. Because of the close coupling between acid secretion and
oxidative metabolism, it will be of interest to evaluate whether, and to what extent,
dynamics of mitochondria are related to the cell activation.
SIGNAL TRANSDUCTION UNDERLYING GASTRIC

ACID SECRETION
Activation of acid secretion by parietal cells is triggered by paracrine, endocrine,
and neural stimuli in vivo. Study of the cell biology of acid secretion has been
greatly facilitated by the technique of gastric gland isolation, developed by
Berglindh & Obrink, along with the method to monitor acid secretion in vitro
by accumulation of the weak base, aminopyrine (10).
From the pioneering work of Chew and her colleagues (11, 12), primary cul-
tures of parietal cells have also been of special value in localizing functionally
relevant proteins and defining events in the secretory cascade. Many intracellular
signaling pathways have now been identified with their contributing roles in pari-
etal cell activation, including protein kinase A (PKA), protein kinase C (PKC),
Ca2+-calmodulin (CaM) kinase II, phosphatidylinositol 3-kinase (PI3 kinase), and
several other downstream kinases. Histaminergic stimulation is by far the most
potent activation pathway observed for the stimulation of gastric acid secretion
in vitro. Although cholinergic and gastrinergic stimulation can be seen in vitro,
the magnitude of stimulation for many species is much reduced compared with
stimulation in vivo, most likely because parietal cells are in close contact with
histamine-releasing enterochromaffin-like (ECL) cells in vivo (13). Isolated ca-
nine parietal cells appear to be an exception, tending to be more responsive to
cholinergic stimuli than to histamine (1). In this review we first focus on signaling
events underlying cAMP-mediated stimulation and the PKA stimulation pathway,
and then move to a discussion of cholinergic pathways and an overview of other
kinase pathways.
cAMP-Mediated PKA Activation and Protein Phosphorylation

Evidence implicating cAMP as an intracellular messenger mediating HCl secre-
tion was presented by Roth & Ivy more than 50 years ago (14). Using isolated
gastric glands, Chew et al. (15) directly showed that histamine elevates intracel-
lular levels of cAMP leading to activation of PKA (16), and specifically type I
cAMP-dependent protein kinase (17). Activation of PKA initiates a cascade of
phosphorylation events through the activation of other downstream effectors. Col-

lectively, these events trigger membrane and cytoskeletal rearrangements within
the parietal cell, as well as increase electrical conductance across the gastric epithe-
lium (1). In the 1980s, several groups reported on stimulus-associated phosphory-
lation of parietal cell proteins, most of which were simply identified by molecular
size (1821). By tracing the phosphorylation of proteins that occurred concomi-
tant with stimulation of gastric glands by histamine, Urushidani and his colleagues
(19, 22) identified an 80-kDa peripheral membrane protein, later characterized as
the cytoskeletal-associated protein ezrin (23, 24), and a 120-kDa protein, now
called parchorin and recently implicated in Cl and water transport (25, 26). In
addition, several stimulation-related phosphoproteins earlier identified by Chew

and her colleagues on the basis of molecular size (18, 27) are now recognized as
40-kDa lasp-1, 66-kDa coroninse (28), and CSPP-28, the latter being a calcium-
sensitive phosphoprotein of 28-kDa (29). Possible functional roles for each of
these phosphoproteins are discussed below.
The early studies of signaling processes related to acid secretion were carried
out on intact gastric glands. Because the plasma membrane provides a barrier
to separate intracellular environment from extracellular bath, the development of
permeable parietal cell models was important for a more direct manipulation of
cytosol. Several permeabilized models, in which electroporation (30) and digitonin
(3033) were used to render gastric glandular cells permeable, have provided useful
information. However, none of these earlier permeabilized preparations could be
transformed from the resting to secreting state by the addition of secretagogues or
cAMP. This problem was eliminated by the use of -toxin, isolated from Staphylo-
coccus aureus, as a permeabilizing agent. The -toxin permeabilized gastric gland
model can be triggered by cAMP to effect the resting-to-secreting transition that
is correlated with the phosphorylation, from 32P-ATP, of a dozen phosphoproteins
(34), several of which are similar to those mentioned above from the intact gland
studies.
The narrow pore size generated by -toxin (3 nm in diameter) allows passage
of only small molecules such as nucleotides. Thus the -toxin model has been
useful in studies of metabolism and protein phosphorylation associated with stim-
ulation (34, 35), but access to large macromolecules is required for biochemical
reconstitution of parietal cell activation. To this end, several permeabilized sys-
tems have been developed to allow the introduction of large peptides and proteins
while retaining the resting-to-secreting transition in response to the addition of
cAMP. One system employing the detergent -escin tested a variety of inhibitory
peptides that block the activities of several protein kinases, including PKA, PKC,
myosin light chain kinase (MLCK), and CaM kinase (36). These peptide inhibi-
tion experiments confirmed the roles of PKA and MLCK, but not CaM kinase and
PKC, in parietal cell secretion. In addition, the inclusion of peptides that inhibit
Arf (adenosine ribosylation factor) into -escin-permeabilized glands was shown
to attenuate the cAMP-stimulated parietal cell activation, suggesting a functional
role for Arf protein in parietal cell secretion.
108 YAO FORTE
An alternative model system has used the Streptococcal toxin streptolysin O

(SLO) to permeabilize gastric glands. With this model, Ammar et al. (37) were
able to show the incorporation of fluorescent-labeled actin into the pool of endoge-
nous F-actin in SLO-permeabilized glands. They also tested the action of several
syntaxin isoforms on the cAMP-activated secretory response (as discussed below
in the section on SNARE proteins).
Yet another approach for permeabilized models is to use a depleted system,
such as the digitonin model, and probe for cytoplasmic constituents that restore
functional activity. Searching for players underlying the cAMP-mediated parietal
cell activation, Akagi et al. (38) carried out a reconstitution experiment in which
they added brain cytosol and purified fractions into digitonin-permeabilized gas-
tric glands. They demonstrated a stimulatory activity by brain cytosol as judged by
aminopyrine uptake. The stimulatory activity was further characterized as phos-
phatidylinositol transfer protein (PITP). Although PITP is a stimulatory factor for
acid secretion, it is not associated with the cAMP-dependent pathway. This re-
constitution model promises to be of importance for the further identification and
function of critical components necessary for parietal cell activation, including

other PKA effectors; it will also address questions of how known proteins such as
ezrin and lasp integrate the cAMP signal to membrane cytoskeleton remodeling.
Lasp-1 was initially identified in the parietal cell as a 40-kDa phosphoprotein
related to cAMP-mediated activation of acid secretion (18). Lasp-1 has been iden-
tified as a signaling molecule that is phosphorylated upon elevation of [cAMP]i in
pancreas and intestine, as well as gastric mucosa, and is selectively expressed in
ion-transporting cells within epithelial tissues, such as cortical regions of pancreatic
and salivary duct cells and in certain F-actin-rich cells in the distal tubule/collecting
duct (34, 35). Lasp-1 has been characterized as a new LIM protein subfamily mem-
ber containing an N-terminal LIM domain, a C-terminal SH3 domain, and two
internal nebulin repeats, with speculation that lasp-1 may function as an adaptor
molecule to relay the upstream signaling to downstream effectors (39). Recent
biochemical evidence revealed that lasp-1 binds to nonmuscle filamentous (F)
actin, but not monomeric (G) actin, in a phosphorylation-dependent manner (40).
The apparent Kd of bacterially expressed his-tagged lasp-1 binding to F-actin was
2 M, with a saturation stoichiometry of 1:7. Phosphorylation of recombinant
lasp-1 with PKA increased the Kd for lasp-1 binding to F-actin and decreased the
Bmax. The major PKA-dependent phosphorylation sites were localized in rabbit
lasp-1 to S99 and S146 (40).
Immunostaining of gastric glands stimulated by the cAMP-dependent pathway
showed that lasp-1 was redistributed from the basolateral membrane to the api-
cal canalicular membrane of parietal cells upon stimulation (41). Alternatively,
exposure of gastric mucosal fibroblasts to the protein kinase C activator, PMA, in-
duced the formation of lamellipodial extensions and nascent focal complexes that
contained both lasp-1 and F-actin (40). The selective phosphorylation-dependent
regulation of lasp-1 in F-actin-rich epithelial cells and the recruitment of lasp-1 to
cellular regions associated with dynamic actin turnover suggest that this protein
may play an integral and specific role in the regulation of cytoskeletal/membrane-

based cellular activities.
Parchorin was first identified by SDS-PAGE as a 120-kDa phosphoprotein as-
sociated with histamine/cAMP stimulation of acid secretion (19); the cDNA se-
quence of parchorin has recently been cloned, having an actual molecular mass of
65 kDa (26). Its anomalous migration on gels is due to an unusually high content
of acidic amino acids. Parchorin is a novel protein that has significant homology
to the family of chloride intracellular channels (CLIC) and has been localized to
a variety of epithelial tissues that are active in the transport of salt and water,
especially choroid plexus, salivary glands, and lacrimal glands, in addition to the
parietal cell (25, 26). Endogenous parietal cell parchorin is present in the cytosol
and relocated to the apical plasma membrane upon histamine stimulation, very
reminiscent of the translocation of H,K-ATPase in response to stimulation. When
parchorin, linked to green fluorescent protein (GFP), was exogenously expressed
in kidney cells, GFP-parchorin also appeared in the cytosol and was relocated to
the plasma membrane when Clefflux from the cells was triggered. Interestingly,
parchorin was co-purified with a new type of kinase activity (42), suggesting that
there may be some functional link between that kinase and the phosphorylated
form of parchorin.
Cholinergic Pathways of Activation

Activation of parietal cells by cholinergic agents acting on muscarinic M3 receptors
clearly involves an elevation of intracellular Ca2+ ([Ca2+]i). Studies on isolated
gastric glands with Ca2+-sensitive fluorescent probes revealed a rapid spike in
parietal cell [Ca2+]i, up to 0.150.5 M within 46 s after addition of the M3
agonist carbachol, followed by a sustained lower-level elevation of [Ca2+]i, that
persisted during the resulting stimulation of acid secretion by the glands (43, 44).
The secretagogue-dependent changes in [Ca2+]i occur from an intracellular store
of bound Ca2+ and are independent of extracellular Ca2+, unless the stores are first
depleted (45, 46). Buffering of the rise in [Ca2+]i by the chelating agent BAPTA
prevents the acid secretory response to carbachol but permits subsequent stimula-
tion by histamine or forskolin (47). Intracellular Ca2+ stores are relatively easily
depleted by cholinergic stimulation in a Ca2+-free medium, although the stores can
be replenished from extracellular sources through a La3+-sensitive pathway (45).
The carbachol-induced rise in [Ca2+]i has been correlated with a rise in parietal
cell inositol trisphosphate (IP3) levels, thus implicating IP3 as the messenger from
the M3 receptor to the internal Ca2+ stores (43). The role of PKC in cholinergic
activation has been more controversial, particularly through the use of PKC activa-
tors such as phorbol esters (e.g., TPA), which have been shown to inhibit or activate
both carbachol- and histamine-stimulated acid secretion (4851). Chew et al. (52)
measured the PKC expression in parietal cells, as well as the response of gastric
glands to a variety of PKC inhibitors. Their data showed several isozymes of PKC
present with some, particularly PKC-, being localized by immunocytochemistry
110 YAO FORTE
to a parietal cell compartment similar to filamentous actin (52). Inhibitors of

PKC activity, such as Ro 31-8220, were found to potentiate both carbachol- and
histamine-mediated acid secretion, also causing a dose-dependent decrease in the
phosphorylation of the cytoskeletal-related proteins ezrin and pp66, later charac-
terized as coroninse (28, 52). These data suggest a negative modulatory role for
PKC in acid secretion and were confirmed in principle by an independent study
(53), although there was some disagreement regarding responsiveness of specific
PKC isozymes.
A role for CaM kinase in parietal cell activation stimulated by the cholinergic
pathway was suggested by Tsunoda et al. (54) who found that KN-62, a pharma-
cological inhibitor of CaM kinase II, inhibited carbachol-mediated acid secretion,

but did not alter the rise in cytosolic Ca2+ caused by carbachol. KN-62 was without
effect on histamine- or forskolin-mediated responses (55). The presence of CaM
kinase II in gastric cells was verified by enzymatic assay (54).
The calcium-sensitive phosphoprotein of 28 kDa known as CSPP28 was orig-
inally discovered based on its phosphorylation associated with cholinergic stim-
ulation (27). CSPP28 is rapidly phosphorylated in intact parietal cells in re-

sponse to both the cholinergic agonist, carbachol, and the calcium ionophore,
ionomycin (29). Subsequent studies showed that recombinant CSPP28 was phos-
phorylated by both crude parietal cell homogenate and purified CaM kinase II in
a calcium/calmodulin-dependent manner (29). Whereas CSPP28 is closely tied to
a calcium-mediated phosphorylation, it would be of great interest to ascertain the
nature of CaM kinase-mediated phosphorylation on CSPP28 and whether such
modification regulates the function of CSPP28 in the activation process.
Carbachol has been shown to induce IB kinase activity in canine parietal
cells via Ca2+- and PKC-dependent pathways (56). IB kinase appears to be a
key element in the signaling cascade that activates NF-B (57, 58), which is an
important transcription factor and regulator of numerous genes modulating the
inflammatory response. Carbachol is also a potent inducer of several families of
protein kinases known as mitogen-activated protein kinases (MAPKs) or extra-
cellular signal-regulated protein kinases (ERKs) and the c-Jun N-terminal protein
kinases (JNKs) in the canine parietal cell system (5961). Generally, these kinases
are known to be fundamental signaling elements in cellular functions of growth,
differentiation, and secretion (6265). However, it is uncertain as to what specific
role these kinases play in the acid secretory activation pathway because presumed
specific inhibitors of the various kinases reportedly produce both inhibitory and
enhancing effects. For example, a pronounced up-regulation of p38 kinase, another
member of the MAPK family of protein kinases, occurred in response to carba-
chol and could be blocked by the p38 kinase-specific inhibitor SB-203580 (66).
Interestingly, carbachol-induced aminopyrine uptake by intact canine parietal cells
was potentiated several-fold by SB-203580, suggesting that active p38 kinase was
inhibitory to acid secretion (66). Collectively these data demonstrate the extensive
interrelationship of signaling elements that are activated via M3-muscarinic stim-
ulation of gastric parietal cells. It remains to be established which elements are
directly involved in the activation of parietal cell secretion, which are modulatory
or inhibitory to the activation, and which are true downstream effector proteins
rather than secondary and tertiary reporters of changes in metabolic load.
Modulation by EGF and TGF-

To reveal the mechanisms underlying the actions of EGF and TGF- on acid se-
cretion, Chew et al. (67) studied the effects of these agents on isolated parietal cells
and primary cultures. They categorized two apparently opposed responses: (a) an
acute inhibition of histamine- and carbachol-mediated acid secretion and (b) an en-
hancement of acid secretory function with chronic exposure to the growth factors.
Because of the observed differential effects of the acute and chronic responses to
tyrosine kinase inhibitors, they proposed that EGF and TGF- modulate parietal
cell function by multiple signaling pathways. They reasoned that a soluble tyrosine
kinase was involved in mediating the chronic effects of EGF, whereas the acute
potentiation of histamine-stimulated secretion by certain tyrosine kinase inhibitors
was probably not mediated by receptor-associated tyrosine kinases. In a further

study of these growth factors on parietal cell activation, these authors showed that
EGF biphasically activated extracellular signal-regulated protein kinases, known
as ERK-1 and ERK-2, with a peak response occurring at approximately 5 min,
followed by a sustained activation for at least 2 h (68). In contrast to EGF, the phor-
bol ester PKC activator TPA induced a sustained activation of ERK-1 and ERK-2
for at least 2 h. Carbachol also activated ERK-1 and ERK-2, but the response was
weaker and monophasic. However, the ERK-signaling cascade was not modulated
by elevated intracellular free Ca2+ or cAMP.
Takeuchi et al. (59, 60) confirmed the biphasic effects of EGF on isolated canine
parietal cells and tested the role of ERKs as possible signal modulators for gastric
secretion. In their studies, carbachol caused a pronounced increase in the kinase
activity of ERK2 in extracts prepared from canine parietal cells (gastrin and EGF
had modest effects, and histamine had no effect), but unlike Chews group, these
authors found that the stimulatory response to carbachol appeared to be dependent
on elevated [Ca2+]i and that a specific inhibitor of ERK, PD-98059, abolished the
stimulatory response. Addition of the ERK inhibitor to intact parietal cells led to a
modest elevation, at best, of aminopyrine uptakes stimulated by carbachol or other
secretagogues, thus the ERK pathway has a minimal role in the direct activation
of gastric secretion. This conclusion is supported by later work suggesting that
the increased ERK activity stimulated by EGF appears coupled to H,K-ATPase
-subunit expression (69) which may be the reason for the slightly enhanced
secretory activity associated with ERK activation and may even form the basis for
the long-term stimulatory effects of EGF. The serine-threonine protein kinase Akt
appears to be another element in the EGF activation cascade leading to increased
H,K-ATPase expression in canine parietal cells (70).
Tsunoda et al. (71) employed chemical inhibitors of tyrosine kinases, genis-
tein and erbstatin, to test for their ability to release the parietal cell from the
112 YAO FORTE
inhibitory action of EGF/TGF- on acid secretion. Whereas these two inhibitors

totally abolished the TGF--mediated inhibition, genistein but not erbstatin, also
potentiated histamine- and forskolin-triggered acid secretion. Because genistein
is a relatively selective inhibitor for cytosolic tyrosine kinases such as c-src, it is
likely that some cytosolic tyrosine kinase negatively regulates parietal cell secre-
tion. It has been reported that activation of tyrosine kinase such as c-src stimulates
the dynamin-dependent endocytosis of renal outer medullary potassium channels
(72). It is possible that EGF/TGF- exert their inhibition on histamine-stimulated
acid secretion by promoting the retrieval of H,K-ATPase from the apical plasma
membrane. Although a tyrosine residue in the cytoplasmic tail of the -subunit
of H,K-ATPase has been implicated in proton pump recycling (73), it would be

of great interest to test whether tyrosine kinases such as c-src accelerate the re-
cycling of H,K-ATPase and to ascertain the molecular mechanism underlying the
regulation of H,K-ATPase recycling by tyrosine phosphorylation.
Modulation by Duodenal and Neural Peptides

Physiological modulation of gastric secretion by a variety of peptides, including
the duodenal hormone cholecystokinin (CCK) and the neuropeptide vasoactive
intestinal peptide (VIP), has been well established. Of prime importance for the
action of CCK and VIP is the paracrine release of somatostatin by D cells in the
antrum and fundus (74, 75). Some of the effects of somatostatin are peripheral to
parietal cells, e.g., inhibit release of gastrin from antral G cells (75, 76) and inhibit
release of histamine from fundic ECL cells (77, 78), but somatostatin also has
direct inhibitory effects on parietal cells (79, 80). Somatostatin receptors on the
parietal cell have been characterized as sstR2-type receptors (84), which operate
via an inhibitory guanine nucleotide-binding protein (Gi) to attenuate the activity
of adenylate cyclase. Inhibition of Gi by pertussis toxin reversed the ability of
somatostatin to inhibit acid secretion and restored the adenylate cyclase-mediated
production of cAMP by isolated canine parietal cells stimulated with histamine and
forskolin, but did not alter the stimulatory effects of dbcAMP or Ca2+ pathway
(79, 82). In addition somatostatin may have long-term effects on parietal cells
because it has been shown to inhibit expression of early response genes, such as
c-fos, via a pertussis toxin-sensitive inhibitory pathway in isolated canine parietal
cells (83, 84).
TRAFFICKING OF H,K-ATPASE ASSOCIATED WITH

ACID SECRETION
In the resting parietal cell the proton pump resides in cytoplasmic tubulovesicles in
an inactive form, presumably because of low permeability of these membranes to
K+ (85). Upon stimulation by secretagogues, H,K-ATPase translocates and inserts
into the apical plasma membrane which has, or acquires, K+ and Cl conductance
pathways that lead to active proton pumping. H,K-ATPase is a P-type ATPase

composed of a catalytic subunit (-subunit) and an accessory subunit (-subunit).
The H,K-ATPase catalyzes the electroneutral exchange of intracellular protons
for extracellular potassium ions, thus generating the enormous proton gradients
associated with gastric HCl secretion (1, 86). Early EM studies support a model
in which H,K-ATPase-containing vesicles fuse with the apical plasma membrane
upon stimulation, thus recruiting the pump enzyme into the plasma membrane
(87). In the following section we discuss those protein elements that have been
identified in parietal cells and have been shown (or implicated) to have a role in
directing the trafficking and recycling of H,K-ATPase-rich membranes underlying
HCl secretion. Figure 1 provides a cartoon summary of the membrane recycling

scheme, indicating the location of putative participating proteins.
SNARE Proteins
It is widely accepted that the fundamental components of the machinery required

for membrane fusion are highly conserved in both constitutive and regulated mem-
brane trafficking pathways (88, 89). These components include N-ethylmaleimide-
sensitive factor (NSF), the soluble NSF attachment proteins (-, -, and -SNAPs),
and SNAP receptors (SNAREs). Membrane-associated SNAREs were first identi-
fied in detergent extracts of brain membranes (90). In these extracts, the SNAREs
formed a tightly bound complex consisting of SNAP-25, syntaxin, and a protein
known as vesicle-associated membrane protein (VAMP). The specific cleavage
of each of these proteins by specific neurotoxins causes a blockade in neuro-
transmitter release, indicating that SNAP-25, syntaxin, and VAMP are essential
for exocytosis in synaptic termini (91). Peng et al. (92) showed that a syntaxin
isoform known as syntaxin 3 and the VAMP-2 isoform (also known as synapto-
brevin) are specifically associated with H,K-ATPase-containing tubulovesicles in
gastric parietal cells, suggesting their possible involvement in secretory activation.
Calhoun & Goldenring (93) and Calhoun et al. (94) confirmed the localization of
syntaxin 3, VAMP-2, and another secretory carrier membrane protein known as
SCAMP to the tubulovesicle fraction, and more importantly demonstrated their re-
location to the apical plasma membrane after stimulation. Because these data were
consistent with a role for syntaxin 3 in apical trafficking of H,K-ATPase, Ammar
et al. (37) carried out experiments in which recombinant syntaxin 3 was added
into SLO-permeabilized gastric glands as a potential competitor for endogenous
SNARE complex formation. Significantly, exogenous syntaxin 3, but not the syn-
taxin 5 isoform, blocked cAMP/ATP-mediated HCl secretion in a dose-dependent
manner. The authors reasoned that the soluble exogenous syntaxin 3 reacted with
endogenous cognate SNAREs to prevent the recruitment of secretory pumps, thus
providing direct evidence for the involvement of the syntaxin 3 isoform in parietal
cell activation.
Primary cultures of parietal cells have been particularly useful in defining the
trafficking and recycling of membrane components associated with stimulation of
114 YAO FORTE
acid secretion (95). Karvar et al. (96) used an adenovirus-based GFP reporter to
show that GFP-VAMP-2 is co-localized with H,K-ATPase to cytoplasmic mem-
branes of resting parietal cell cultures and that GFP-VAMP-2 is translocated along
with the pump enzyme to the apical plasma membrane after stimulation by his-
tamine. To test the involvement of VAMP-2 in parietal cell activation, they treated
SLO-permeabilized glands with tetanus toxin, which is a highly specific pro-
tease for VAMP proteins. They demonstrated a correlation between the cleavage
of VAMP-2 and the inhibition of glandular acid secretion, confirming an earlier
suggestion that the proteolytic cleavage of VAMP-2 inhibited acid secretion in
isolated parietal cells that were permeabilized by SLO and stimulated with cAMP
(97). Further support for the participation of SNARE proteins in the recruitment of
H,K-ATPase is offered by recent studies of Karvar et al., who doubly infected pari-
etal cell cultures with genes constructed with different fluorescent proteins linked
to VAMP-2 and SNAP-25 (98). In resting cells, the two proteins were localized
to different membrane compartments: VAMP-2 to H,K-ATPase-rich cytoplasmic
vesicles, and SNAP-25 to apical membrane vacuoles. Upon stimulation with his-
tamine, the two signals became coincident in the expanded plasma membrane
consistent with SNARE complex formation associated with membrane recruit-
ment. Furthermore, introduction of a SNAP-25 construct lacking the functional C
terminus inhibited acid secretion by the isolated cells.
Rab Proteins
In addition to SNARE proteins, small GTPase proteins have long been implicated
in vesicular membrane trafficking (99). Studies in several systems, primarily in
neuronal synapses, have demonstrated that Rab3 family members redistribute from
a membrane-bound location to the soluble cytosolic fraction upon fusion of secre-
tory granules with target plasma membranes. Rab proteins are then recycled back
onto mature secretory vesicles after reinternalization of the membrane. Although
this cycle is well established for Rab3, far less is known about the functional activ-
ity of other Rab proteins during vesicle fusion and recycling. In the gastric parietal
cell, there are several Rab proteins, including Rab11 and Rab25 (93, 100, 101). In
nonsecreting parietal cells, the Rab11a isoform is associated with H,K-ATPase-
containing tubulovesicles that fuse with the apical plasma membrane in response
to secretory agonists such as histamine (94).
Using matrix-assisted laser desorption mass spectrometry, Duman et al. (102)
confirmed that Rab11 is associated with H,K-ATPase-enriched gastric micro-
somes, and their data provided a stoichiometric estimate of one Rab11 per six
copies of H,K-ATPase. Furthermore, Rab11 exists in at least three forms on rabbit
gastric microsomes: The two most prominent resemble Rab11a, whereas the third
resembles Rab11b. Using an adenoviral expression system, Duman et al. expressed
the dominant-negative mutant Rab11a N124I, in primary cultures of rabbit pari-
etal cells. Rab11a N124I is a mutant of Rab11a in which aspargine (N) at position
124 has been replaced with isoleucine (I). The mutant was well expressed with a
distribution similar to that of H,K-ATPase in resting parietal cells; however, mutant

Rab11a arrested the stimulus-dependent morphological transition of the parietal
cells and acid secretion measured by aminopyrine uptake. Specificity was verified
by addition of tetracycline, which blocked mutant Rab expression and restored the
morphological transition and acid secretory function. These studies demonstrate
that Rab11a is a prominent GTPase associated with gastric tubulovesicles and
suggest that it plays a direct role in regulating parietal cell activation.
In a search for downstream effectors, a number of Rab11-interacting proteins
have been identified as co-enriched with Rab11a and H,K-ATPase on parietal cell
tubulovesicle membranes: rab11-family interacting protein 1 (rab11-FIP1), rab11-
family interacting protein 2 (rab11-FIP2), and pp75/rip11 (103). The binding of

rab11-FIP1 and rab11-FIP2 to Rab11a was dependent upon a conserved C-terminal
amphipathic -helix. Moreover, these Rab11-interacting proteins were found to
translocate with Rab11a and the H,K-ATPase upon stimulation of parietal cells with
histamine. These results suggest that the function of Rab11a in plasma membrane
recycling is dependent upon a number of protein effectors.
Clathrin Coat and Other Adaptor Proteins

An important, though somewhat ignored, aspect of regulating gastric secretion
has to do with the uptake and internalization of the extensive apical membrane
when the stimulus is withdrawn and the parietal cell returns to the resting state.
Given the similarities of regulated H,K-ATPase trafficking with recycling of cargo
through the apical recycling endosomes of many epithelial cells, it seems reason-
able to speculate that clathrin and its adaptor proteins regulate some part of an
apical recycling pathway as in other epithelial cells. Using immunological probes,
Okamoto et al. (104) identified -adaptin and clathrin heavy chain on tubulovesi-
cles isolated from gastric parietal cells. Further biochemical characterization of
the tubulovesicular clathrin adaptor complex suggested a subunit composition in-
cluding -, - and -adaptins, as well as other subunits with molecular masses
of 50 and 19 kDa, all of which could polymerize in vitro into basket-like struc-
tures of 120 nm diameter (105). For parietal cells in the nonsecreting state,
immuno-electron microscopy revealed that clathrin is relatively abundant at or
near the apical canalicular membrane specifically in association with coated pits
and coated vesicles, as well as accumulated at the ends of tubulovesicular pro-
files where the membranes are acutely curved. These results indicate that, even in
resting cells, active membrane trafficking between the apical membrane and the
tubulovesicular compartment is an ongoing process. They are also consistent with
an hypothesis that the secretory cycle is regulated with respect to the relative rates
of exocytic (stimulation) and endocytic (recycling) processes, rather than a binary
on or off system. It must be pointed out that, although clathrin and its adaptors are
well represented and appear actively involved in some aspect of apical membrane
recycling in the parietal cell, there is only presumptive evidence that H,K-ATPase
is the cargo.
116 YAO FORTE
Dynamin
Another component of clathrin-dependent trafficking pathways is the large
GTPase dynamin, which is essential for receptor-mediated endocytosis and is
clearly known to interact with clathrin and coat-forming proteins, but its precise
function in vesicle formation remains controversial (106, 107). Given the distri-
bution of clathrin and clathrin adaptors in the parietal cell, it is not surprising that
a dynamin-like molecule should be associated with the apical membrane. In fact,
dynamin II has been immunolocalized to the apical membrane of both resting
and stimulated parietal cells (94, 105). Interestingly, among the various cell types
in gastric glands, dynamin appears to be expressed predominantly, if not exclu-
sively, in the parietal cell (105). Although it has been proposed that dynamin acts
as a pinchase for constriction of endocytic membrane separation from plasma
membrane, this remains to be established (107).
Using a GST-dynamin II fusion protein as an affinity matrix, Okamoto et al.
identified two proteins that bind to dynamin II, the nonreceptor tyrosine kinase
c-src and lasp-1 (108). Moreover, the binding of c-src and lasp-1 was specific for the
proline-rich domain of dynamin II. Their study also showed the presence of c-src
on tubulovesicular membranes, with an active tyrosine kinase that phosphorylated
tubulovesicular proteins in vitro, one of which may be the 100-kDa H,K-ATPase.
A potential trafficking role for c-src on tubulovesicular membranes is of inter-
est because c-src has been found on endosomal membranes (109), as a regulator
of other apical membrane trafficking pathways (110, 111), and shown to regu-
late membrane transporters directly by phosphorylation (112). The interaction of
lasp-1 with dynamin II is also of interest, suggesting the possibility of some regula-
tory role for lasp-1 in coordinating the actin cytoskeleton and vesicular trafficking
machinery via dynamin (108). As with c-src, the functional consequences of lasp-1
dynamin II interaction remain to be characterized.
Although these interactive data provide interesting possibilities for connection
between signaling molecules such as c-src and the downstream effectors, such
as dynamin, lasp-1 and even H,K-ATPase, the role of the in vitro protein-protein
interactions in parietal cell physiology and their regulation during cell activation
remain to be characterized.
Myosin Vb
Given the importance of Rab11a in tubulovesicle trafficking, Lapierre et al. (113)
set up a yeast two-hybrid screen for proteins interacting with active Rab11a.
Among three Rab11-interacting proteins discovered, one is myosin Vb. These
authors further characterized myosin Vb in polarized MDCK cells. They found
that myosin Vb is associated with apical endosomes. Furthermore, this associa-
tion depends on the integrity of microtubules, providing the interesting suggestion
of a role for myosin Vb in plasma membrane recycling in connection with the
microtubule cytoskeleton. Expression of the C-terminal tail of myosin Vb dis-
persed the distribution profile of transferrin receptor and retarded recycling of the
receptor back to the plasma membrane. By analogy with a proposed function of

myosin Va in melanosome trafficking (114), it is also possible that the mutant
disrupted the association of the myosin Vb-endosomal complex with the actin-
based cytoskeleton, which resulted in an inhibition of apical plasma membrane
recycling.
Structure and Function of H,K-ATPase

H,K-ATPase shares several structural homologies with another P-type ATPase,
Na,K-ATPase (86, 115). They are both composed of an -subunit, predicted to
span the membrane 10 times, and a glycosylated -subunit of type II integral

membrane protein. Early in the biosynthetic pathway, the -subunits assemble with
-subunits in the endoplasmic reticulum. Assembly of the holoenzyme appears to
involve interactions between cytoplasmic, extracytoplasmic and transmembrane
domains of both subunits (116, 117) and requires insertion of the / complex
into the plasma membrane. Although cross-assembly of H,K-ATPase and Na,K-

ATPase subunits can occur under contrived in vitro conditions, the -subunit of
each ATPase preferentially assembles with its respective -subunit (118).
Whereas the amino acid sequence between the H,K-ATPase and Na,K-ATPase is
highly conserved, these enzymes differ in several important attributes, including the
cations they respectively transport and their targeted locations. The Na,K-ATPase
is concentrated at the basolateral membrane of most epithelial cells, whereas, the
gastric H,K-ATPase is ultimately targeted to the apical membrane of secreting
parietal cells. Also, the H,K-ATPase represents the major cargo protein for the
regulated recycling associated with acid secretion.
The recruitment of vesicular coat proteins has been shown to depend upon both
the presence of membrane protein cargo and the lipid composition of the target
membranes (119). Thus cargo can regulate the formation of vesicular carriers.
Complete deletion of either the -subunit (120) or the -subunit (121) in knockout
mice was shown to have predictably profound effects on parietal cell structure and
function. In both cases, the mice were achlorhydric. Although parietal cells were
histologically identifiable, both knockout mice exhibited a dramatic diminution
of tubulovesicular membranes. The residual amount of -subunit expressed in the
-subunit knockout mice was not sufficient to produce a tubulovesicular network
(120), suggesting that the assembled holoenzyme is required as cargo for the
biogenesis of the tubulovesicular compartment.
Expression studies in heterologous cell lines have shown that the -subunit
of the gastric H,K-ATPase encodes sorting and targeting information responsible
for the apical distribution of the pumps. By analyzing the sorting behavior of a
number of chimeric pumps composed of complementary portions of -subunits
from the H,K-ATPase and Na,K-ATPase, Dunbar et al. (122) identified a portion
of the gastric H,K-ATPase, which is sufficient to redirect the normally basolateral
Na,K-ATPase to the apical surface in transfected LLC-PK cells. This motif resides
within the fourth of the ten predicted transmembrane domains of the -subunit.
118 YAO FORTE
There are seven N-glycosylation sites on the -subunit of the H,K-ATPase

(123). To examine the role of the carbohydrate chains on holoenzyme activity and
stability, Asano et al. (124) carried out site-directed mutagenesis on the -subunit
in which one, several, or all of the asparagine residues in the N-glycosylation sites
were replaced by glutamine. Removing any one of seven carbohydrate chains from
the -subunit did not have a significant effect on the activity of H,K-ATPase or
its delivery to the apical surface. However, removal of all the carbohydrate chains
prevented assembly of the holoenzyme and all associated functional activity. In
contrast, enzyme activity and /-subunit assembly were retained when three
carbohydrate chains were removed, but surface delivery of the -subunit and its
associated -subunit was arrested, indicating that the surface delivery mechanism
is more dependent on the carbohydrate chains than is the expression of H,K-ATPase

activity and holoenzyme assembly.
In addition to stabilizing its complementary -subunit, the -subunit of
H,K-ATPase may contain a tyrosine-based endocytotic motif that is essential for
recycling of the pump after withdrawal of stimulation. Courtois-Coutry et al. (73)
made transgenic mice that expressed a mutant H,K-ATPase -subunit, in which a

critical tyrosine residue in the cytoplasmic tail was mutated to alanine. These mice
developed a pathology resembling that in idiopathic hypersecretion of acid into
the stomach. Immuno-electron microscopy data suggest that the -subunit, and
presumably the -subunit, in these transgenic mice are constitutively expressed at
the apical membrane and fail to re-internalize. The authors concluded that that the
mutated -subunit, and therefore the pump enzyme, could not interact with the
endocytotic machinery to allow re-sequestration of the stimulated apical surface
in reversion to the resting state.
Biochemical studies suggest that the cytoskeletal proteins ankyrin and spectrin
interact with the H,K-ATPase in the microsomal fraction of resting parietal cells
and appear to relocate with H,K-ATPase to the apical plasma membrane of the
secreting parietal cells (125). Whereas these authors speculated that ankyrin and
spectrin form a functional complex to stabilize H,K-ATPase at the apical membrane
upon the stimulation, it would be of great interest to ascertain whether ankyrin and
spectrin directly bind to H,K-ATPase and how such interactions are regulated.
ACTIN-BASED CYTOSKELETON IS ESSENTIAL

FOR PARIETAL CELL ACTIVATION
Actin
Interactions between the plasma membrane and the cytoskeleton play a central
role in a variety of physiological processes including mobilization of membrane
receptors, endocytosis, exocytosis (e.g., hormone secretion and transmitter re-
lease), cell polarity, and morphology (126, 127). An essential role for the cortical
actin cytoskeleton in parietal cell secretion has been implied from morphological
analyses (13) and from studies with microfilament disrupters such as cytocha-
lasin (128, 129). Highly organized microfilaments are a characteristic feature of
microvilli within the canaliculus of the gastric parietal cell. However, the radial
arrangement of the actin filaments in proximity to the microvillar membrane in
parietal cells (3) is distinctly different from the central core localization of actin
filaments of intestinal microvilli (130).
The actin gene family encodes a number of structurally related, but functionally
distinct, protein isoforms that modulate contraction in muscle tissues and control
shape and motility of nonmuscle cells (131). In mammals, there are at least six
different actin isoforms, each encoded by a separate gene, and they differ by
<10% of the amino acid sequence, primarily in the N-terminal region (132).
The development of cytoplasmic - and -actin isoform-specific antibodies has

provided powerful tools for localizing actin isoforms in situ (133).
Using actin isoform-specific antibodies and isolated gastric glands as an ep-
ithelial model, Yao et al. (134) demonstrated that actin isoforms, in particular
the cytoplasmic - and -actin nonmuscle isoforms, are differentially polarized
in epithelial cells. They found that -actin was predominantly localized to the
apical plasma membrane of all glandular cells, including the tortuous microvilli-
enriched canalicular surface of parietal cells, as well as the entire gland lumen,
whereas -actin was predominantly localized to the basolateral membrane.
The canaliculi of resting parietal cells are replete with short microvilli that
include radially organized actin microfilament bundles projecting along the mi-
crovillus and extending one or more micrometers into the cytoplasm (3). The
radical membrane transformations of cell activation result in elongation of mi-
crovilli, which suggests a commensurate shift in the ratio of monomeric G-actin
to filamentous F-actin. However, measurements on the state of actin in gastric
glands revealed the following: (a) Parietal cell actin is primarily organized in the
F-actin form (90%); (b) the microfilament disrupter cytochalasin D destabilizes
the F-actin and inhibits acid secretion; and (c) phalloidin, which stabilizes F-actin
filaments, does not inhibit acid secretion (135). Surprisingly, the authors could find
no significant change in the ratio of F- to G-actin when the cells were transformed
from rest to maximal secretion: F-actin remained about 90% of the total. These
data are consistent with an hypothesis that microfilamentous actin is necessary for
membrane recruitment underlying parietal cell secretion; however, they suggest
that rapid exchange between G- and F-actin is not essential for the secretory pro-
cess and that the parietal cell maintains actin in a highly polymerized state. This
conclusion was underscored by the work of Ammar et al. (136) who tested the actin
monomer-sequestering agent latrunculin B (Lat B) on parietal cell structure and
function. Lat B inhibited acid secretion and increased the extractable monomeric
actin but only at relatively high doses (1070 M). Because the authors observed
high sensitivity of other parietal cell functions to Lat B (e.g., formation of lamel-
lipodia was inhibited at 0.1 M), they reasoned that there were distinct pools of
exchangeable actin in parietal cells, with microvillar microfilaments being partic-
ularly stable owing to the presence of stabilizing, capping, and bundling proteins.
120 YAO FORTE
They proposed that the resistance of acid secretory function to Lat B is the result
of stable actin filament-turnover pathways that minimize the accessibility of the
actin monomer to Lat B.
The actin cytoskeleton has been linked to the mechanism of potentiation of
cAMP-mediated acid secretion by carbachol in rabbit gastric glands (137). The
observed potentiation was dependent upon release of Ca2+ from intracellular stores
regulated by the type 3 IP3 receptor (the major subtype in the parietal cell), but
it was unaffected by changes in extracellular Ca2+ or inhibitors of either PKC or
CAM kinase II. On the other hand, the disrupter of actin filaments, cytochala-
sin D, preferentially blocked the secretory effect of carbachol and its synergism
with cAMP, as well as the release of Ca2+ from stores. Treatment of glands
with cytochalasin D also caused redistribution of the IP3 receptor from a plasma
membrane-like fraction to the microsomal fraction, suggesting a dissociation of
the stores from the plasma membrane and a functional coupling between actin
filaments and the receptor-mediated Ca2+ store.
Ezrin-Actin Interactions
An actin-binding/regulatory protein that may have a primary role in parietal cell
function was mentioned above as the stimulation-dependent 80-kDa phospho-
protein called ezrin. Ezrin was independently identified by various investigators
for their special interests (138140) and implicated as a cytoskeleton-membrane
linker protein. Ezrin is the best studied member of the ERM (ezrin-radixin-moesin)
family (141). These proteins share approximately 75% primary sequence identity
and contain an N-terminal globular domain followed by an -helical region and
a C-terminal tail domain (142). Although ezrin was originally purified with the
microfilament bundles from intestinal microvilli, the native, full-length protein
possessed no convincing F-actin binding activity in tests that used -actin from
skeletal muscle, a commonly used rich source of pure actin. However, C-terminal
fragments of ezrin were shown to bind filamentous -actin (143); consequently,
Bretscher et al. (142) developed a model of intra- and intermolecular folding of
full-length ezrin to account for the discrepancy.
Ezrin was first identified in parietal cells on the basis of PKA-dependent phos-
phorylation concomitant with stimulation of gastric glands (19, 22). Double im-
munostaining for ezrin and for -actin revealed that these two proteins are abundant
and primarily co-localized to the same regions within parietal cells, characteris-
tic of the tortuous apical canalicular surface wending through most of the cell
(23, 24, 134). Interestingly, ezrin is almost exclusively localized to parietal cells,
whereas staining for the -actin isoform is intense along the apical borders of all
cell types lining the gland lumen as well as within apical canaliculi of parietal
cells. Because the -actin isoform is primarily distributed near the basolateral
membrane of parietal cells (134), it appears that ezrin is co-localized with -actin,
but not -actin, as a subset of the F-actin microfilaments.
On the basis of actin-ezrin cyto-localization studies and the knowledge that
-actin is not expressed in parietal cells, Yao et al. (144) hypothesized that ezrin
preferentially binds to nonmuscle actin isoforms rather than to skeletal muscle

-actin. Using a co-sedimentation assay that compared purified -actin with skele-
tal muscle -actin, ezrin appeared in the pellet only when -actin was polymer-
ized, whereas a known actin-binding protein, the S1 tryptic fragment of myosin II,
showed no selectivity between these actin isoforms. The calculated data suggest a
stoichiometry of 1 ezrin bound per 12 actin molecules. These studies indicate that
gastric ezrin possesses F-actin binding in an isoform-specific manner. In fact, the
binding of full-length ezrin to actin was lately demonstrated using a novel solid
phase actin-binding assay (145).
In their search for a novel class of pharmacological antiulcer agents, Urushidani
et al. (146) reported that a compound designated ME3407, a potent acid secretion
inhibitor, arrested the morphological transition of cells stimulated by histamine.
Because ME3407 was found to liberate the phosphoprotein ezrin from the api-
cal plasma membrane, as well as to inhibit histamine-triggered translocation of
H,K-ATPase, these authors hypothesized that ME3407 may exert its inhibitory
action on the modulation of protein phosphorylation required for recruitment of
H,K-ATPase. Dephosphorylation of ezrin has been correlated with its dissociation

from the plasma membrane-cytoskeleton (147). ME3407 was also observed to in-
hibit MLCK and PI3 kinase activities measured in vitro (44). Because wortmannin
is a well known inhibitor of these latter kinases, it was tested and found to inhibit
glandular acid secretion, similar to the effect of ME3407. Recent studies, however,
have differentiated the effects of wortmannin and ME3407 (37). Both compounds
were found to inhibit acid secretion by intact gastric glands, but only ME3407
e inhibited acid secretion in SLO-permeabilized glands and liberated ezrin from
its membrane-cytoskeletal locus at the canalicular surface. Thus it appears that
the targets of these two inhibitors are not identical. Future studies may attempt to
define whether and to what extent phosphorylation of ezrin might be altered by
ME3407 and relate it to the modulation of parietal cell secretion.
Ezrin Interacts with Other Cellular Proteins

Using an overlay assay, Dransfield et al. (148) showed that ezrin binds to the
regulatory RII subunit of PKA in parietal cells, suggesting it might be involved in
the functional localization of PKA as an anchoring protein. In addition, the authors
showed that the RII subunit of PKA is capable of binding to full-length ezrin in
cell extracts, arguing that inter- or intramolecular interaction of ezrin masks ezrin
activity in binding to other cellular proteins. It was also reported that the N-terminal
domain of ezrin binds to EBP50, which mediates ezrin association with the plasma
membrane, although the physiological relevance of such interaction remains to be
characterized (144).
In a search for Ca2+-mediated signaling events in parietal cell activation, Yao
et al. (149) discovered the presence of calpain I (-calpain) in gastric parietal cells
and its interaction with ezrin. Further studies showed that parietal cell calpain
I can be activated by an ionomycin-mediated elevation of intracellular calcium;
furthermore, the activation of calpain triggers the release and hydrolysis of ezrin
122 YAO FORTE
(149), followed by the collapse of the actin cytoskeleton in parietal cells. These
studies suggest the importance of ezrin in the integrity of the actin cytoskeleton of
parietal cells. Significantly, the activation of calpain, as evidenced by the hydrolysis
of ezrin, was correlated with the inhibition of parietal cell secretion, presumably
through the loss of ezrin. This ezrin-calpain interaction is unique because the
calpain cleavage site is located to the C-terminal 200 amino acids of ezrin where
sequences of the ERM family are divergent. To explore the possible regulation
by calcium and calpain I of the interaction between -actin and ezrin, Herman
and his associates overexpressed calpastatin, an endogenous inhibitor of calpain,
in fibroblast cells. The overexpression resulted in a diminished calpain activity
and increased ezrin protein level, which was experimentally correlated with an
inhibition of filopodial formation (150), suggesting that spatial activation of calpain
may be necessary for actin dynamics associated with spreading at the leading edge.
It will be of interest to evaluate whether the hydrolysis of ezrin, or the liberation of
ezrin from actin cytoskeleton, is essential for formation of cytoplasmic extensions
such as filopodia or microvilli.
A NOTE ON THE FUSION REACTION

Thus far we have discussed the signal transduction events of parietal cell activation
in terms of (a) the structural elements (cytoskeleton) that support and promote the
translocation of H,K-ATPase-rich membranes from their cytoplasmic location to
the apical membrane and (b) the cognitive elements (SNAREs) that assure mem-
branes are directed to and associate with highly specific docking sites. (c) A third
and equally important element has generally been given less attention, the fusion
mechanism itself, i.e, the means by which two distinct apposing lipid bilayers
interact and flow into one. A schematic view of these three cooperative processes
contributing to membrane recruitment is shown in Figure 2. The proposal that
phospholipid surface chemistry and fusion processes play a role in membrane
recruitment and secretion extends back to 1963, when morphological transforma-
tions of acid-secreting cells were correlated with phospholipid turnover in bullfrog
gastric mucosa (87). Although this was an important step in formulating the mem-
brane recycling hypothesis, little work was done on the fusion process per se until
recently. Using separate assays of fluorescence dequenching and intervesicular
protein transfer, Duman et al. (151) have demonstrated that gastric tubulovesicular
membranes will fuse with each other (homotypic fusion) or with liposomes of
the appropriate composition. The fusion reaction is highly specific for a trigger-
ing system that must include either Ca2+ or ATP (EC50 is 0.15 M and 1 mM,
respectively) and is dependent on the presence of protein in at least one of the
two vesicular pools, although the specific protein has not been identified. Whereas
these in vitro fusion data are preliminary, they do offer a system by which the
interfacial reactions of fusion might be studied, with the potential to identify new
classes of proteins that facilitate the lipid mixing processes.
PERSPECTIVES
Although great progress has been made over the past 10 years in characterizing
the mechanisms and participants in parietal cell secretion, the challenge ahead is
to define the precise function and the physiological regulation of the implicated
proteins and identify new players (152). By analyzing both the cell biology and
physiology of phenotypic changes in cultured parietal cells and even transgenic
animals expressing mutant regulatory proteins, it will be possible to determine
whether and how a particular molecule operates upon the signaling cascade and
downstream effector reactions of parietal cell activation. Molecular engineering
of fluorescent reporters on a desired molecule has emerged as an exciting way to

correlate the cytological changes to molecular function of individual regulatory
proteins in real-time imaging of live parietal cells. Such studies will consolidate
protein-protein interactions that have been inferred from indirect techniques into
a physiological model for parietal cell activation and relate these data to molec-
ular medicine of associated disorders such as peptic ulcer disease and gastric
carcinoma.
The Annual Review of Physiology is online at http://physiol.annualreviews.org
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isoforms in gastric parietal cells. Mol. 145. Roy C, Martin M, Mangeat P. 1997. A
Biol. Cell 6:54157 dual involvement of the amino-terminal
135. Forte JG, Ly B, Rong Q, Ogihara S, domain of ezrin in F- and G-actin bind-
Ramilo M, et al. 1998. State of actin in ing. J. Biol. Chem. 272:2008895
gastric parietal cells. Am. J. Physiol. Cell 146. Urushidani T, Muto Y, Nagao T, Yao
Physiol. 274:C97C104 X, Forte JG. 1997. ME-3407, a new an-
136. Ammar DA, Nguyen PN, Forte JG. 2001. tiulcer agent, inhibits acid secretion by
interfering with redistribution of H+-K+- 150. Potter DA, Tirnauer JS, Janssen R, Croall
ATPase. Am. J. Physiol. Gastrointest. DE, Hughes CN, et al. 1998. Calpain regu-
Liver Physiol. 272:G1122G34 lates actin remodeling during cell spread-
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Dephosphorylation of ezrin as an early 151. Duman JG, Singh G, Lee GY, Machen TE,
event in renal microvillar breakdown and Forte JG. 2002. Ca2+ and Mg2+/ATP in-
anoxic injury. Proc. Natl. Acad. Sci. USA dependently trigger homotypic membrane
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148. Dransfield DT, Bradford AJ, Smith J, Traffic 3:20317
Martin M, Roy C, et al. 1997. Ezrin is a 152. Soroka CJ, Chew CS, Hanzel DK, Smolka
cyclic AMP-dependent protein kinase an- A, Modlin IM, Goldenring JR. 1993.
choring protein. EMBO J. 16:3543 Characterization of membrane and cy-

149. Yao X, Thibodeau A, Forte JG. 1993. toskeletal compartments in cultured pari-
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265:C36C46 Cell Biol. 60:7687
22 Jan 2003 20:43 AR AR177-05-COLOR.tex AR177-05-COLOR.SGM LaTeX2e(2002/01/18) P1: GDL

Figure 1 Schematic model for regulated tubulovesicle cycling in the parietal cell.
In resting cells, the proton pump H,K-ATPase is sequestered in inactive cytoplasmic
tubulovesicles (TVs). Secretagogue stimulation triggers the translocation of TVs ulti-
mately leading to activation of proton pumping (solid arrows). Once the stimulus has
decayed, the proton pump is internalized to terminate acid secretion (dotted arrows).
The regulated TV cycle can be divided into following steps: (a) Docking. TVs that
contains H,K-ATPase and intrinsic factors dock at the active zone of the target mem-
brane. H,K-ATPase is inactive owing to low permeability to K+. Docking is defined as
the initial contact/interaction between vesicle membrane and target membrane medi-
ated by specific proteins, such as VAMP-2, syntaxins, SNAP-25, and exocyst protein
complex SEC6/8. Docking can occur between vesicles and apical plasma membrane
(heterotypic) and among vesicle membranes themselves (homotypic). (b) Priming.
After docking, TVs go through a maturation process that enables competency for
membrane fusion, possibly mediated by PKA-dependent phosphorylation and Ca2+-
dependent activation. (c) Fusion/proton pumping. Lipids of primed TVs are competent
to mix with those of the apical membrane allowing H,K-ATPase to partition into the
membrane. The proton pump begins active H+ transport while TV contents such as
intrinsic factor exocytose into the gland lumen. (d ) Endocytosis. After the stimulus
removal, the H,K-ATPase-rich regions of apical membrane are retrieved into the cy-
toplasm. The initial internalization is facilitated via clathrin-coated pits and dynamin.
(e) Endosome fusion. Coated TVs fuse with apical early endosome. This may be facil-
itated by small GTPases and their accessory proteins. ( f ) Budding. TVs are reformed
chiefly by budding from recycling endosomes. The newly formed TVs are either com-
petent for translocation and subsequent docking, or committed to a homotypic fusion
to gain competence. (g) Translocation. H,K-ATPase-containing TVs translocate back
to the active zone either by diffusion or by motor proteins.
22 Jan 2003 20:43 AR AR177-05-COLOR.tex AR177-05-COLOR.SGM LaTeX2e(2002/01/18) P1: GDL

Figure 2 Schematic model representing three stages in the membrane recruitment

process. (a) The trafficking and migration of cytoplasmic membranes containing cargo
transport proteins is facilitated by the cytoskeleton (microfilaments and microtubules)
possibly through resident motor proteins. (b) Effective recruitment can occur only
where cargo vesicles can recognize and dock on target plasma membranes via highly
specific interactive proteins (e.g., SNARE proteins), possibly regulated by small GT-
Pases such as the Rab proteins. (c) After the membranes are tightly docked, fusion can
occur only when microscopic regions of the two highly distinctive membrane interfaces
flow together to form the fusion pore. For biological membranes, the reaction probably
involves activation of hydrophobic protein(s) that spans the hydrophilic interface and
permits phospholipids to flow into a fusion pore. Phospholipases may also participate.
All of these processes are carefully regulated by receptor-mediated signaling events
involving protein kinases and phosphatases.
P1: FDS
January 17, 2003 11:23 Annual Reviews AR177-FM
Annual Review of Physiology,

Volume 65, 2003
CONTENTS
FrontispieceJean D. Wilson xiv
PERSPECTIVES, Joseph F. Hoffman, Editor
A Double Life: Academic Physician and Androgen Physiologist,

Jean D. Wilson 1
CARDIOVASCULAR PHYSIOLOGY, Jeffrey Robbins, Section Editor
Lipid Receptors in Cardiovascular Development, Nick Osborne
and Didier Y.R. Stainier 23

Cardiac Hypertrophy: The Good, the Bad, and the Ugly, N. Frey
and E.N. Olson 45
Stress-Activated Cytokines and the Heart: From Adaptation to
Maladaptation, Douglas L. Mann 81
CELL PHYSIOLOGY, Paul De Weer, Section Editor
Cell Biology of Acid Secretion by the Parietal Cell, Xuebiao Yao
and John G. Forte 103
Permeation and Selectivity in Calcium Channels, William A. Sather
and Edwin W. McCleskey 133
Processive and Nonprocessive Models of Kinesin Movement,
Sharyn A. Endow and Douglas S. Barker 161
COMPARATIVE PHYSIOLOGY, George N. Somero, Section Editor
Origin and Consequences of Mitochondrial Variation in Vertebrate
Muscle, Christopher D. Moyes and David A. Hood 177
Functional Genomics and the Comparative Physiology of Hypoxia,
Frank L. Powell 203
Application of Microarray Technology in Environmental
and Comparative Physiology, Andrew Y. Gracey and
Andrew R. Cossins 231
ENDOCRINOLOGY, Bert W. OMalley, Section Editor
Nuclear Receptors and the Control of Metabolism,
Gordon A. Francis, Elisabeth Fayard, Frederic Picard, and
Johan Auwerx 261
vii
P1: FDS
viii CONTENTS
Insulin Receptor Knockout Mice, Tadahiro Kitamura, C. Ronald Kahn,

and Domenico Accili 313
The Physiology of Cellular Liporegulation, Roger H. Unger 333
GASTROINTESTINAL PHYSIOLOGY, John Williams, Section Editor
The Gastric Biology of Helicobacter pylori, George Sachs,
David L. Weeks, Klaus Melchers, and David R. Scott 349
Physiology of Gastric Enterochromaffin-Like Cells, Christian Prinz,
Robert Zanner, and Manfred Gratzl 371
Insights into the Regulation of Gastric Acid Secretion Through Analysis of
Genetically Engineered Mice, Linda C. Samuelson and Karen L. Hinkle 383

NEUROPHYSIOLOGY, Richard Aldrich, Section Editor
In Vivo NMR Studies of the Glutamate Neurotransmitter Flux
and Neuroenergetics: Implications for Brain Function,
Douglas L. Rothman, Kevin L. Behar, Fahmeed Hyder,

and Robert G. Shulman 401
Transducing Touch in Caenorhabditis elegans, Miriam B. Goodman
and Erich M. Schwarz 429
Hyperpolarization-Activated Cation Currents: From Molecules
to Physiological Function, Richard B. Robinson and
Steven A. Siegelbaum 453
RENAL AND ELECTROLYTE PHYSIOLOGY, Steven C. Hebert, Section Editor
Macula Densa Cell Signaling, P. Darwin Bell, Jean Yves Lapointe,
and Janos Peti-Peterdi 481
Paracrine Factors in Tubuloglomerular Feedback: Adenosine, ATP,
and Nitric Oxide, Jurgen Schnermann and David Z. Levine 501
Regulation of Na/Pi Transporter in the Proximal Tubule,
Heini Murer, Nati Hernando, Ian Forster, and Jurg Biber 531
Mammalian Urea Transporters, Jeff M. Sands 543
Terminal Differentiation of Intercalated Cells: The Role of Hensin,
Qais Al-Awqati 567
RESPIRATORY PHYSIOLOGY, Carole R. Mendelson, Section Editor
Current Status of Gene Therapy for Inherited Lung Diseases,
Ryan R. Driskell and John F. Engelhardt 585
The Role of Exogenous Surfactant in the Treatment of Acute Lung
Injury, James F. Lewis and Ruud Veldhuizen 613
Second Messenger Pathways in Pulmonary Host Defense,
Martha M. Monick and Gary W. Hunninghake 643
P1: FDS
CONTENTS ix
Alveolar Type I Cells: Molecular Phenotype and Development,

Mary C. Williams 669
SPECIAL TOPIC: LIPID RECEPTOR PROCESSES, Donald W. Hilgemann,
Special Topic Editor
Getting Ready for the Decade of the Lipids, Donald W. Hilgemann 697
Aminophospholipid Asymmetry: A Matter of Life and Death,
Krishnakumar Balasubramanian and Alan J. Schroit 701
Regulation of TRP Channels Via Lipid Second Messengers,
Roger C. Hardie 735
Phosphoinositide Regulation of the Actin Cytoskeleton,

Helen L. Yin and Paul A. Janmey 761
Dynamics of Phosphoinositides in Membrane Retrieval and
Insertion, Michael P. Czech 791
SPECIAL TOPIC: MEMBRANE PROTEIN STRUCTURE, H. Ronald Kaback,

Special Topic Editor
Structure and Mechanism of Na,K-ATPase: Functional Sites
and Their Interactions, Peter L. Jorgensen, Kjell O. Hakansson,
and Steven J. Karlish 817
G Protein-Coupled Receptor Rhodopsin: A Prospectus,
Slawomir Filipek, Ronald E. Stenkamp, David C. Teller, and
Krzysztof Palczewski 851
INDEXES
Subject Index 881
Cumulative Index of Contributing Authors, Volumes 6165 921
Cumulative Index of Chapter Titles, Volumes 6165 925
ERRATA
An online log of corrections to Annual Review of Physiology chapters
may be found at http://physiol.annualreviews.org/errata.shtml

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Annu. Rev. Physiol. 2003. 65:10331

CELL BIOLOGY OF ACID SECRETION BY

Xuebiao Yao1,2 and John G. Forte1

Hefei, China 230027; email: xbyao@uclink.berkeley.edu; jforte@uclink.berkeley.edu

Key Words membrane recruitment, membrane fusion, gastric secretion,

Abstract Acid secretion by the gastric parietal cell is regulated by paracrine,

Secretion of HCl into the stomach is mediated by the oxyntic (acid-secreting)

104 YAO FORTE

operate via basolateral membrane receptors. Stimulation of acid secretion typically

AN ELABORATE STRUCTURAL BASIS

CELL BIOLOGY OF ACID SECRETION 105

VAMP-2 18 Cytoplasm (resting) H,K-ATPase translocation (92, 93)

pp75/Rip11 75 Tubulovesicle (resting) Rab11 binding (103)

PM = plasma membrane; TV = tubulovesicles.

106 YAO FORTE

SIGNAL TRANSDUCTION UNDERLYING GASTRIC

cAMP-Mediated PKA Activation and Protein Phosphorylation

CELL BIOLOGY OF ACID SECRETION 107

phosphorylation events through the activation of other downstream effectors. Col-

addition, several stimulation-related phosphoproteins earlier identified by Chew

108 YAO FORTE

An alternative model system has used the Streptococcal toxin streptolysin O

function of critical components necessary for parietal cell activation, including

CELL BIOLOGY OF ACID SECRETION 109

may play an integral and specific role in the regulation of cytoskeletal/membrane-

Cholinergic Pathways of Activation

110 YAO FORTE

to a parietal cell compartment similar to filamentous actin (52). Inhibitors of

cological inhibitor of CaM kinase II, inhibited carbachol-mediated acid secretion,

ulation (27). CSPP28 is rapidly phosphorylated in intact parietal cells in re-

CELL BIOLOGY OF ACID SECRETION 111

Modulation by EGF and TGF-

was probably not mediated by receptor-associated tyrosine kinases. In a further

112 YAO FORTE

inhibitory action of EGF/TGF- on acid secretion. Whereas these two inhibitors

of H,K-ATPase has been implicated in proton pump recycling (73), it would be

Modulation by Duodenal and Neural Peptides

TRAFFICKING OF H,K-ATPASE ASSOCIATED WITH

CELL BIOLOGY OF ACID SECRETION 113

pathways that lead to active proton pumping. H,K-ATPase is a P-type ATPase

HCl secretion. Figure 1 provides a cartoon summary of the membrane recycling

It is widely accepted that the fundamental components of the machinery required

114 YAO FORTE

CELL BIOLOGY OF ACID SECRETION 115

distribution similar to that of H,K-ATPase in resting parietal cells; however, mutant

family interacting protein 2 (rab11-FIP2), and pp75/rip11 (103). The binding of

Clathrin Coat and Other Adaptor Proteins

116 YAO FORTE

CELL BIOLOGY OF ACID SECRETION 117

receptor back to the plasma membrane. By analogy with a proposed function of

Structure and Function of H,K-ATPase

span the membrane 10 times, and a glycosylated -subunit of type II integral

into the plasma membrane. Although cross-assembly of H,K-ATPase and Na,K-

118 YAO FORTE

There are seven N-glycosylation sites on the -subunit of the H,K-ATPase

is more dependent on the carbohydrate chains than is the expression of H,K-ATPase

made transgenic mice that expressed a mutant H,K-ATPase -subunit, in which a

ACTIN-BASED CYTOSKELETON IS ESSENTIAL

CELL BIOLOGY OF ACID SECRETION 119

The development of cytoplasmic - and -actin isoform-specific antibodies has

120 YAO FORTE

CELL BIOLOGY OF ACID SECRETION 121