Nucleotide metabolism by gastric glands and
H+-K+-ATPase-enriched
membranes
Qinfen
Rong,
Olga
Utevskaya,
Marlon
Ramilo,
Dar C.
Chow, and
John G.
Forte
Department of Molecular and Cell Biology, University of
California, Berkeley, California 94720
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ABSTRACT |
-Toxin-permeabilized gastric glands represent
a functional model in which acid secretion can be elicited by either
adenosine 3',5'-cyclic monophosphate (cAMP) or ATP, with
proven morphological and functional transition between resting and
secretory states [X. Yao, S. M. Karam, M. Ramilo, Q. Rong, A. Thibodeau, and J. G. Forte. Am. J. Physiol. 271 (Cell
Physiol. 40): C61-C73, 1996.] In this study
we use
-toxin-permeabilized rabbit gastric glands to study energy
metabolism and the interplay between nucleotides to support acid
secretion, as indicated by the accumulation of aminopyrine (AP). When
permeabilized glands were treated with a phosphodiesterase inhibitor,
the secretory response to cAMP was inhibited, whereas the secretory
response to ATP was potentiated. This implied that
1) ATP provided support not only as
an energy source but also as substrate for adenylate cyclase,
2) activation of acid secretion by
cAMP needed ATP, and 3) ATP and cAMP
exchanged rapidly inside parietal cells. To address these issues, we
tested the action of adenine nucleotides in the presence and absence of
oxidizable substrates. All adenine nucleotides, including AMP, ADP,
ATP, and cAMP, could individually enhance the glandular AP accumulation
in the presence of substrates, whereas only a high concentration of ATP
(5 mM) was able to support secretory activity in substrate-free buffer.
Moreover, ATP could maintain 75-80% of maximal secretory activity
in phosphate-free buffer; cAMP alone could not support secretion in
phosphate-free buffer. In glands and in
H+-K+-adenosinetriphosphatase-rich
gastric microsomes, we showed the operation of adenylate kinase,
creatine kinase, and ATP/ADP exchange activities. These enzymes,
together with endogenous adenylate cyclase and phosphodiesterase,
provide the recycling of nucleotides essential for the viability of
-toxin-permeabilized gastric glands and imply the importance of
nucleotide recycling for energy metabolism in intact parietal cells.
adenylate kinase; creatine kinase; nucleotide exchange;
-toxin
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INTRODUCTION |
THE SECRETION OF 150 mM HCl places an extraordinary
energy demand on the acid-secreting parietal cell. Parietal cells are well adapted to meet these energetic requirements by virtue of their
large, abundant mitochondria and functionally adapted enzymes to
support high oxidative potential and nucleotide metabolism. For
example, parietal cells have a high capacity to metabolize fatty acids
(5, 11), their lactic dehydrogenase is predominantly of the
H4 isotype, and creatine kinase
activity has been localized to the same tubulovesicles that contain the
primary proton pump, the
H+-K+-adenosinetriphosphatase
(ATPase) (32).
Isolated gastric glands have been widely used as a model of parietal
cell function, typically using the uptake of the weak base aminopyrine
(AP) as an index of the secretory response to secretagogues (2). For
gastric glands freshly isolated from rabbit stomach, the most potent
secretagogues are those that evoke secretion via the protein kinase A
(PKA) pathway, e.g., dibutyryl-adenosine 3',5'-cyclic
monophosphate (cAMP), forskolin, or histamine, operating through a
Gs-coupled
H2 receptor, plus
phosphodiesterase inhibitors (6, 7). Earlier studies from our
laboratory demonstrated the utility of the
-toxin-permeabilized
gastric gland model to evaluate second messenger pathways in parietal
cell secretion (35, 38), and these have essentially been confirmed by
the recent report of Miller and Hersey (24). When bathed in high K+ buffer containing a supply of
oxidizable substrates, the
-toxin-permeabilized glands were
responsive to the addition of 0.1 mM cAMP, and this was potentiated by
a background of 0.1 mM ATP, the latter presumably as a supporting
energy substrate. Our studies also showed that
-toxin-permeabilized
glands responded to ATP alone at concentrations of 0.1 mM or higher. We
interpreted these data to generally support the cAMP-PKA pathway of
parietal cell activation. In the absence of cAMP, when ATP
concentration was sufficient, we presumed that endogenous adenylate
cyclase activity generated the cAMP required for the
activation. Thus we were surprised by our more recent observation that cAMP activation of
-toxin-permeabilized gastric glands was blocked by the addition of phosphodiesterase inhibitors, such as 3-isobutyl-1-methylxanthine (IBMX). The present experiments were undertaken to more fully evaluate cAMP involvement in parietal cell activation and provide a more complete picture of nucleotide metabolism in the system.
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MATERIALS AND METHODS |
Preparation of Gastric Glands
Gastric glands were prepared from New Zealand White rabbits as
described by Berglindh and Obrink (2). After isolation, gastric glands
were subjected to washing three times with minimum essential medium
(GIBCO) and maintained in the resting condition in the same buffer
containing 10 µM cimetidine.
Permeabilization of Glands
Freshly isolated glands were washed once in a permeabilization buffer
rich in K+ (K medium), including
(in mM) 100 KCl, 20 NaCl, 1.2 MgSO4, 1 NaH2PO4,
40 mannitol, 20 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and 10 tris(hydroxymethyl)aminomethane (Tris), pH 7.4. Glands were then resuspended in K medium at 10% cytocrit and incubated with
Staphylococcus
-toxin at the
concentration of 0.11 mg/ml for 45 min at 37°C.
Staphylococcus
-toxin was a
generous gift from Dr. S. Bhakdi, purified from S. aureus (4, 25). After permeabilization, the gland
suspension was diluted to a 5% cytocrit in K medium in which 10 mM
succinate and 1 mM pyruvate were added as oxidative substrates (35).
Functional activity of glands was characterized by AP uptake assay. AP
accumulation is reported either as the AP accumulation ratio, as
described by Berglindh and Obrink (2), or as the AP index, which is the
percentage of maximum AP accumulation for a given preparation (35).
Preparation of
H+-K+-ATPase-Enriched
Subfraction of Microsomes
H+-K+-ATPase-containing
microsomes were prepared from rabbit stomach as previously reported (8,
28). New Zealand White rabbits (2-3 kg) were injected with
cimetidine (20 mg/kg body wt) 1 h before the animals were euthanized
with pentobarbital sodium (Nembutal). The stomach was then removed and
washed with ice-cold isotonic saline. The mucosa from the fundus and
body region was scraped, minced, and homogenized in a Potter-Elvehjem homogenizer with 15 passes in a hypotonic solution containing ice-cold
(in mM) 113 mannitol, 37 sucrose, 0.4 EDTA, and 5 piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), pH 6.7. The homogenate was centrifuged at 14,500 g for 10 min at 4°C. The
supernatant was further centrifuged at 100,000 g for 1 h. The resulting pellet was
resuspended in a suspending medium (in mM; 300 sucrose, 5 Tris, and 0.2 EDTA, pH 7.4). The crude microsomal suspension was brought up to 43%
sucrose and overlaid with successive gradient layers of 30 and 10%
sucrose (wt/vol) in 5 mM Tris and 0.2 mM EDTA, pH 7.4, and centrifuged for 4 h at 90,000 g using a Beckman
SW27 rotor.
H+-K+-ATPase-enriched
rabbit gastric microsomes were collected as the flotation layer at the
interface between the 10 and 30% layers and stored in aliquots at
20°C until use.
Probing Nucleotide Interchange by TLC
Permeabilized glands were bathed in K medium containing 0.2-0.5
µCi of
[8-14C]adenosine di-
or triphosphate and additional additives, as specified by the
conditions in each experiment. The reaction was stopped by 0.6 N
perchloric acid (PCA), and nucleotides were extracted by adsorption
onto charcoal (14) and finally eluted from the charcoal with 2%
NH3 in 50% ethanol. Samples were
assayed for individual nucleotides by thin-layer chromatography (TLC).
Enzymatic characterization of
H+-K+-ATPase-enriched
microsomes was assessed by a modified method of Reenstra et al. (29). Isolated gastric microsomes (10 µg protein) were incubated with various substrates at room temperature (23-24°C) in buffer
containing 1 mM MgSO4, 20 mM NaCl,
15 mM PIPES (pH 6.8), and 0.1 µCi/100 µl
[8-14C]ADP with 0.1 mM
ADP. Aliquots were taken at assigned time intervals, and the reaction
was stopped by 20 mM EDTA. Protein was precipitated with 0.6 N PCA
followed by neutralization with ice-cold KOH. Samples were clarified by
centrifugation, and the supernatant was saved for assay by TLC.
TLC was carried out on 20-cm2
polyethyleneimine-cellulose plastic plates (Selecto Scientific,
Norcross, GA). Aliquots of samples were applied 3 cm from the bottom of
the plate. Unlabeled nucleotides were applied as a standard for
identification. Chromatograms were developed in different solvent
systems, depending on the purpose of the specific nucleotide separation
(14, 17, 27). To separate ATP, ADP, and AMP the chromatogram was first
developed with 2 M sodium formate (pH 3.4) up to 6 cm from the bottom
and then with 4 M sodium formate (pH 3.4) to the top of the plate (14). To separate cAMP from other nucleotides the chromatogram was developed in distilled water to the top of the plate, followed by 0.25 M LiCl
after air drying (adapted from Ref. 17). The position of nucleotides
was identified under ultraviolet light. Radioactivity of nucleotides
was evaluated by radioautography and liquid scintillation counting of
eluted spots.
Statistics
Results from different experiments were averaged and presented as the
means ± SE. Differences between experiments were evaluated by
Student's t-test, with
P < 0.05 being considered
significant.
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RESULTS |
Support of AP Accumulation in
-Toxin-Permeabilized
Gastric Glands
Phosphodiesterase inhibitors block stimulatory effects of cAMP in
permeabilized gastric glands.
The effects of a phosphodiesterase inhibitor on the AP accumulation
ratio of permeabilized gastric glands are shown in Table 1. Without IBMX the glands responded
briskly to the addition of 0.1 mM cAMP, and in this case there was no
measurable potentiation when 0.1 mM ATP was included. Similar to our
previous results (35, 38), even without added cAMP AP accumulation was
also increased if 1 mM ADP (Table 1) or ATP (cf. Fig.
1) was added. When 50 µM IBMX was
included, the cAMP-elicited secretory activity was inhibited by about
50%, whereas no significant difference was found in AP ratios mediated
by either 1 mM ADP or 0.1 mM cAMP-0.1 mM ATP. The inhibitory effect of
IBMX and its analog is just the opposite of what has been seen in
intact glands (1, 33). IBMX blocks the degradation of cAMP to AMP, and
in normal intact gastric glands with a constitutively active adenylate
cyclase, IBMX results in the elevation of intracellular cAMP,
stimulating acid secretion itself and/or potentiating the
action of secretagogues like histamine. These latter effects of IBMX in
intact tissue and glands have long been used to support the cAMP-PKA
pathway for parietal cell activation. However, the inhibition by IBMX of cAMP-elicited acid secretion in the
-toxin model was unexpected and required some alternative explanation.
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Table 1.
cAMP-elicited acid secretion in
-toxin-permeabilized glands is inhibited by
inhibiting phosphodiesterase activity
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Fig. 1.
Phosphodiesterase inhibitors potentiate acid secretory index in
-toxin-permeabilized gastric glands. Gastric glands were isolated,
permeabilized with -toxin, and placed in K medium, including
indicated concentrations of ATP, and in absence ( ) or presence ( )
of 50 µM 3-isobutyl-1-methylxanthine (IBMX). Aminopyrine (AP)
accumulation was measured as AP accumulation index as described in
MATERIALS AND METHODS. Values are
means ± SE, n = 3.
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ATP supports AP accumulation as an energy source and as a source of
cAMP.
The dose response to ATP for AP accumulation by
-toxin-permeabilized
glands is shown in Fig. 1 in the absence and presence of IBMX. AP
accumulation increased gradually as ATP concentration increased, and
over the entire range the accumulation of AP was enhanced in the
presence of 50 µM IBMX, with the potentiating effect of IBMX being
significant (P < 0.05) at 0.2 mM ATP
and higher. These data are consistent with our previous explanation that ATP may serve both as an energy source and as a precursor for cAMP
through endogenous adenylate cyclase activity (35, 38). Accordingly, as
predicted from results on intact preparations, IBMX would presumably
operate to preserve the generated cAMP. The plausibility of this latter
explanation is supported by our experiments measuring the distribution
of 14C among nucleotides in
-toxin-permeabilized glands treated with [14C]ATP. Figure
2 shows that there is a low level of
[14C]cAMP produced in
conditions where glands were treated with 0.1 mM forskolin and 0.1 mM
[14C]ATP, and that the
[14C]cAMP spot became
more intense in preparations treated with IBMX, consistent with IBMX
blocking the degradation of cAMP. It is also apparent from the
autoradiographs that the level of
[14C]AMP was quite
high in both conditions, with or without IBMX, suggesting a high flow
path from ATP to AMP. (With this solvent system to specifically
separate AMP and cAMP, ATP could not be separated from ADP.)

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Fig. 2.
Permeabilized glands produce cAMP from ATP. -Toxin-permeabilized
gastric glands were incubated for 20 min at 37°C in K medium
supplemented with 0.1 mM ATP containing 0.5 µCi
[14C]ATP, 0.1 mM
forskolin, and in presence (+) or absence (-) of 5 × 10 5 M IBMX, as indicated.
The reaction was stopped with 0.6 N perchloric acid (PCA), and
nucleotides were extracted using charcoal separation and spotted onto
polyethyleneimine (PEI)-cellulose plates.
14C-labeled nucleotides were
resolved by thin-layer chromatography (TLC) developed first with
distilled water to the front line followed by 0.25 M LiCl after air
drying.
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Functional oxidative metabolism is essential for AMP and cAMP to
support nucleotide interchange and AP accumulation.
To test the ability of AMP to serve as a source of nucleotide in
-toxin-permeabilized glands, AP accumulation was measured in
response to exogenous AMP. Figure 3 shows
that AP accumulation was progressively increased as AMP was raised from
0.01 to 1 mM AMP. The AP ratios produced by 1 mM AMP were comparable to
those produced by 1 mM ATP or ADP. Figure 3 also shows that the
addition of 50 µM IBMX greatly diminishes the stimulatory effect of
cAMP but does not appreciably alter the responses to AMP or ADP. In earlier studies we demonstrated that AP accumulation by
-toxin-permeabilized glands could be supported by either ATP or ADP
(35), but that in the case of ADP, oxidative substrates and functional
mitochondria were necessary for optimal secretory activity. Without
functional mitochondrial oxidation, stimulation of AP accumulation by
either cAMP or ADP diminished and, as shown in Fig. 3, only relatively high concentrations of ATP could recover the activation. In the case of
activation by AMP, the AP ratio was totally abolished (from 60.5 ± 4.0 to 5.9 ± 0.5) in the absence of oxidative substrates (Fig. 3).
Thus, with intact oxidative phosphorylation, ATP, ADP, AMP, and even
cAMP, will all serve as a source of adenine nucleotide to support
energy metabolism and the H+ pump,
suggesting that phosphate interchange among the adenine nucleotides is
very efficient and is essential to maintain parietal cell function.

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Fig. 3.
AMP, cAMP, or ADP will each serve as a nucleoside triphosphate source
to support AP accumulation by -toxin-permeabilized glands when
oxidative metabolism is functional. Gastric glands were permeabilized
with -toxin and placed into K buffer, including the various
nucleotides as indicated (values, nucleotide concentration in mM; cim,
10 4 M cimetidine with no
nucleotide added). Except where indicated all incubation media
contained 10 mM succinate and 1 mM pyruvate as oxidative substrates. In
1 set of experiments 50 µM IBMX was included to block
phosphodiesterase. Values are means ± SE,
n = 3.
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Pi is essential for nucleotide exchange.
To further address nucleotide interchange, we investigated the effect
of Pi on AP accumulation. In
Pi-free buffer, ATP (both 0.1 and
1.0 mM) retained about 80% of the AP accumulation observed in
phosphate buffer (Fig. 4). The stimulation
elicited solely by cAMP was completely abolished, and the large
stimulation ordinarily produced by 0.1 mM ATP plus 0.1 mM cAMP was
greatly attenuated (60-70%) in
Pi-free buffer. Synthesis of ATP
from the mono- or diphosphate requires a source of
Pi in the buffer. The addition of
sufficient ATP provides a direct energy source for proton transport, as
well as a source of phosphate for nucleotide exchange and recycling. Nevertheless, in Pi-free buffer,
ATP could not support the maximum AP accumulation, which is probably
due to the trapping of phosphate in other stores, such as protein
phosphorylation.

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Fig. 4.
Influence of Pi on AP accumulation
supported by cAMP or ATP in -toxin-permeabilized gastric glands.
Glands were isolated, permeabilized with -toxin, and incubated in K
buffer containing oxidative substrates, with or without
Pi. Medium was also supplemented
with nucleotides (in mM) as indicated. Values are means ± SE,
n = 3.
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The conclusion we draw thus far is that there is a rapid flow of
adenine nucleotide from AMP or cAMP to ATP, sufficiently rapid to keep
up with the demand set by proton transport associated with AP
accumulation. In each of the cases shown above, AP accumulation was
completely inhibited by the
H+-K+-ATPase
inhibitor omeprazole (data not shown). The purpose of the next group of
experiments was to test for the operation of a variety of enzymes
associated with nucleotide metabolism and exchange, using both the
permeabilized cell model and isolated H+-K+-ATPase-rich
microsomal vesicles.
Nucleotide Metabolism and Interchange in Permeabilized Glands
In addition to functional oxidative metabolism, several enzymatic
pathways are involved in the cycling of nucleotides, and we used
[14C]ADP as a
precursor to assess the operation of these enzymes in the permeabilized
gastric gland model. Figure 5 shows the
results of experiments in which glands were incubated with 0.1 mM ADP containing tracer
[14C]ADP. These
experiments were performed in the absence of oxidative substrates to
eliminate the formation of labeled ATP from ADP through oxidative
phosphorylation. When the incubation included 0.1 mM ADP alone there
was a great loss of
[14C]ADP with a large
increase in [14C]AMP,
suggesting the net degradation of ADP via ADP phosphohydrolase reactions, which we generically indicate as ADP
AMP + Pi. On the basis of data shown
below, the modest production of ATP was most likely the result of
adenylate kinase activity, 2ADP
ATP + AMP. When the incubation
included 1 mM phosphocreatine (PCr) there was a large production of ATP
and much of the
[14C]ADP was
preserved, indicating the operation of creatine kinase activity, PCr + ADP
ATP + creatine. The inclusion of several nucleoside
triphosphate substrates (NTP), such as ATP (not shown), ITP, or GTP,
produced qualitatively similar results as PCr, i.e., the synthesis of
14C-labeled ATP and the
preservation of ADP. These latter results are consistent with a
nucleoside phosphate exchange reaction, NTP + ADP
ATP + NDP (26, 29).

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Fig. 5.
Nucleotide metabolism and interchange in -toxin-permeabilized
gastric glands. Permeabilized glands were incubated in K buffer in
absence of oxidative substrates and with indicated concentrations of
nucleotides or phosphocreatine (PCr), as well as 0.5 µCi
[14C]ADP. After 5 min
incubation at 37°C reaction was stopped by 0.6 N PCA and
nucleotides were extracted by charcoal adsorption, spotted onto PEI-TLC
plates and developed successively in 2 M and 4 M sodium formate (pH
3.4) to separate ATP, ADP, and AMP. With no glands present
([14C]ADP blank),
label is present only as ADP.
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Nucleotide Metabolism and Interchange by Isolated Gastric Microsomes
Adenylate kinase activity.
We next undertook a study of nucleotide reactions within purified
gastric microsomal membranes that are rich in
H+-K+-ATPase.
In many of the subsequent experiments a potent inhibitor of
H+-K+-ATPase,
Sch-28080, was used to prevent this enzyme from dominating the data.
For example, one experiment tested the ability of the microsomes to
metabolize [14C]ADP,
similar to what we did with the permeabilized glands above. Figure
6A shows a
representative time course of the relative amounts of label from
[14C]ADP that appear
in AMP and ATP. After the reaction was initiated, there was an
exponential decrease in
[14C]ADP. The initial
rate of [14C]ADP
decrease was accompanied by a rise in
14C-label appearing as ATP and an
equivalent increase appearing as AMP; the sum of increased
[14C]ATP and
[14C]AMP was
stoichiometrically equivalent to the loss of
[14C]ADP. These data
are consistent with the presence of adenylate kinase activity in the
microsomal fraction. The feasibility of this conclusion was tested by
running the same conditions of reaction but in the presence of an
inhibitor of adenylate kinase, P1,
P5-Di(adenosine-5')pentaphosphate
(Ap5A; Ref. 21). As demonstrated in Fig. 6B, no ATP is formed from
[14C]ADP, and only the
slow appearance of
[14C]AMP from
dephosphorylating
[14C]ADP is seen when
Ap5A is included in the medium.
Table 2 provides summary data for adenylate
kinase activity measured as the initial rate of
[14C]ATP formation on
three microsomal preparations.

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Fig. 6.
Adenylate kinase activity is expressed in
H+-K+-ATPase-enriched
gastric microsomes. Gastric microsomes were incubated in 0.2 ml buffer
containing 0.1 mM ADP, 0.2 µCi
[14C]ADP, and 10 µM
Sch-28080 as an inhibitor of
H+-K+-ATPase
activity. Samples were taken at times indicated for nucleotide
extraction and analysis by TLC, similar to Fig. 5. Radioactivity was
measured in spots corresponding to ATP ( ), ADP ( ), and AMP ( ).
A: in absence of adenylate kinase
inhibitor P1,
P5-Di(adenosine-5')pentaphosphate
(Ap5A).
B: in presence of 200 µM
Ap5A.
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Creatine kinase activity.
Sistermans et al. (32) reported the operation of efficient creatine
kinase activity in hog gastric microsomes. We tested for microsomal
creatine kinase activity in much the same manner as for adenylate
kinase. Figure 7 shows the results of
incubating microsomes with 1.25 mM phosphocreatine and 0.1 mM ADP (plus
tracer [14C]ADP). In
the absence of the adenylate kinase inhibitor
Ap5A (Fig.
7A), the synthesis of ATP occurs
both by creatine kinase and adenylate kinase, the latter occurring with
the greatest velocity. When adenylate kinase was inhibited by
Ap5A (Fig.
7B) there was a steady production of
[14C]ATP from
[14C]ADP, presumably
driven by phosphocreatine. The initial rate of
[14C]ATP production
was taken as creatine kinase activity. A summary of creatine kinase
activity measured on several preparations is given in Table 2.

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Fig. 7.
Creatine kinase activity is expressed in
H+-K+-ATPase-enriched
gastric microsomes. Gastric microsomes were incubated in 0.2 ml buffer
containing 1.25 mM PCr, 0.1 mM ADP, 0.2 µCi
[14C]ADP, 10 µM
Sch-28080 as an inhibitor of
H+-K+-ATPase
activity and in absence (A) and
presence (B) of 200 µM adenylate
kinase inhibitor Ap5A. Samples
were taken at times indicated for nucleotide extraction and analysis by
TLC. Radioactivity was measured in spots corresponding to ATP ( ),
ADP ( ), and AMP ( ).
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NTP/NDP exchange activity.
Gastric microsomes also display potent ATP/ADP exchange activity, and
this has been a proposed partial reaction of the
H+-K+-ATPase
(26), analogous to what has been seen for the
Na+-K+-ATPase
(10). Accordingly, we have measured active ATP/ADP exchange activity in
gastric microsomes. As shown in Fig.
8A, when
1.25 mM ATP was included in the medium along with 0.1 mM ADP containing tracer [14C]ADP there
was a relatively prompt exchange of
14C-labeled nucleotide from ADP to
ATP, i.e., ATP + [14C]ADP
[14C]ATP + ADP. Figure
8B shows the CTP/ADP exchange reaction
in gastric microsomes, i.e., CTP + [14C]ADP
[14C]ATP + CDP. It is
important to account for, or inhibit, adenylate kinase activity for an
accurate measurement of nucleotide exchange rate. For the experiments
shown here we included 0.2 mM Ap5A
as an inhibitor of adenylate kinase, and it is clear that rather little
[14C]AMP is formed
over the time course of reaction. We also included the
H+-K+-ATPase
inhibitor Sch-28080 (10 µM), which is especially important for
ascertaining the initial rate of
[14C]ATP production
from nucleoside triphosphates other than ATP. Control experiments
established that NTP/ADP exchange activity was not significantly
altered by Sch-28080 (data not shown). Sch-28080 is known to prevent
the turnover of the
H+-K+-ATPase
by inhibiting K+-stimulated
dephosphorylation of the phosphoenzyme intermediate (37), and thus it
is not surprising to find that the
K+-independent formation of
phosphoenzyme intermediate from ATP and the catalyzed transfer of
-phosphate to ADP remains unaltered, as has been shown for another
K+ site inhibitor of
H+-K+-ATPase,
AHR-9294 (29) Furthermore, our data summarized in Table 2 suggest that
the exchange reaction is in fact a general nucleoside triphosphate/diphosphate (NTP/NDP) exchange activity, catalyzing the
transfer of
-phosphate from a variety of nucleotides to ADP. The
specificity of nucleotides for the rate of
-phosphate exchange was
ATP
ITP > CTP > GTP. The relative lack of specificity among nucleotides for the triphosphate/diphosphate exchange reaction is in
contrast to the high degree of specificity that has been reported for
H+ transport by the
H+-K+-ATPase
(30).

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Fig. 8.
Nucleoside triphosphate/diphosphate exchange reaction is a prominent
activity of
H+-K+-ATPase-enriched
gastric microsomes. Gastric microsomes were incubated in 0.2 ml buffer
containing 0.1 mM ADP, 0.2 µCi
[14C]ADP, 10 µM
Sch-28080 as an inhibitor of
H+-K+-ATPase
activity, 200 µM Ap5A as an
adenylate kinase inhibitor, and either 1.25 mM ATP
(A) or 1.25 mM CTP
(B). Samples were taken at times
indicated for nucleotide extraction and analysis by TLC. Radioactivity
was measured in spots corresponding to ATP ( ), ADP ( ), and AMP
( ).
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DISCUSSION |
Gastric parietal cells are highly active metabolizing systems,
requiring intense oxidative activity to sustain the energy demands for
the secretory product of ~0.16 N HCl. To meet the energy requirements
the parietal cell has many large mitochondria, occupying a larger
portion of the parietal cell volume (25-45%) than any other
vertebrate cell type (18, 39). The requirement for a fluid supply of
ATP as the primary energy source for the proton pump, the
H+-K+-ATPase,
has been known for many years (12, 13, 30). Thus it has been no
surprise that various permeabilized parietal cell models have a
requirement for supplementary ATP (16, 23, 35).
Our earlier studies with the
-toxin-permeabilized parietal cell
model demonstrated that either cAMP or ATP was capable of supporting
histamine-stimulated AP accumulation, although maximal stimulation was
achieved with combined cAMP plus ATP (35, 38). We reasoned that the
"ATP effect" was mediated by endogenous adenylate cyclase
activity to provide an activating level of cAMP. However, no clear view
was formulated as to why cAMP alone would be an effective stimulant,
except to postulate some residual level of ATP. The present data
support the former conclusion and provide a clear explanation for the
latter effect. In
-toxin-permeabilized glands, AP accumulation
stimulated by increasing levels of ATP is potentiated by adding low
levels of cAMP or by inhibiting glandular phosphodiesterase activity.
This action of phosphodiesterase inhibitors supports the role of cAMP
in the activation pathway along with the present demonstration of cAMP
synthesis through endogenous adenylate cyclase activity. Furthermore,
the previously observed inhibition of AP accumulation and
cAMP-stimulated protein phosphorylation by H-89, a PKA inhibitor (35,
38), reinforce the cAMP-PKA pathway for activating HCl secretion,
although other signals (e.g., Ca2+) may have a potentiating
role. The addition of cAMP to permeabilized glands, without exogenous
ATP, provides an adequate stimulus of AP accumulation as long as
phosphodiesterase remains active, suggesting that the product AMP
participates in the reaction. It is now clear that the parietal cell
can effectively utilize AMP as a nucleotide source to synthesize
nucleoside triphosphate as long as oxidative metabolism is intact.
Adenylate kinase is the principal reaction for recycling AMP, and
relatively high levels of adenylate kinase activity were demonstrated
in isolated gastric glands as well as in
H+-K+-ATPase-enriched
microsomes derived therefrom. Thus the adenylate kinase reaction, AMP + ATP
2ADP, coupled with functioning mitochondria, i.e.,
ADP + Pi
ATP, produces a
redistribution of added nucleoside monophosphate to nucleoside
triphosphate. Because of the virtual autocatalytic production of ATP by
these reactions, the initial level of ATP can be extremely low.
Several other reactions associated with nucleotide metabolism and
phosphate metabolism were evident in parietal cells, including the
reaction promoting phosphate exchange between NTP/NDP exchange and
creatine kinase (Cr + ATP
PCr + ADP). Sistermans et al. (32)
demonstrated the colocalization of creatine kinase in isolated gastric
tubulovesicles, and that PCr + ADP were capable of supporting proton transport by the pump in those vesicles. Our experiments show
that NTP/NDP exchange colocalizes with
H+-K+-ATPase-rich
tubulovesicles, most likely the ATP/ADP exchange that is a recognized
partial reaction of P-type ATPases (10, 26). As with the creatine
kinase, we have found that NTP/NDP exchange activity is capable of
providing sufficient ATP, e.g., GTP + ADP
ATP + GDP, to power
the proton pump in isolated tubulovesicles (unpublished observation).
In the skeletal and cardiac muscle literature a large body of evidence
has demonstrated that distinctive isoenzymes of creatine kinase (36)
and adenylate kinase (40) operate at sites of ATP utilization in
addition to those mitochondrial isoenzyme forms that function within
sites of ATP generation. It has been proposed that these enzymes
function to transfer or shuttle high-energy phosphoryl groups from
sites of production to sites of energy consumption, serving as spatial
and temporal energy buffers in cells that consume large amounts of ATP
(3). Moreover, functional interactions between creatine kinase and
adenylate kinase systems have been suggested (9, 31). Apart from the
studies on muscle, a functional coupling has been shown to occur
between the creatine kinase system and various P-type ATPases,
including
Na+-K+-ATPase
(5), Ca2+-ATPase (20), and
H+-K+-ATPase
(32). The present results, as well as those of Sistermans et al. (32), show that several of the enzymes associated with nucleotide and phosphate metabolism are present at membrane sites in
the periphery of parietal cells, at or near the site of ATP utilization. Because the parietal cell has a regulated secretory cycle
with high-energy demand, it is reasonable to propose that creatine
kinase and adenylate kinase in the vicinity of energy consumption may
help to buffer the ATP level and ATP-to-ADP ratio under differing
functional secretory states.
We suggest at least one additional function for high activity adenylate
kinase, which is to prevent a depletion of the cellular nucleotide
pool. Accumulation of AMP leads to deamination of AMP and degradation
to hypoxanthine derivatives (19), with a potential loss to the adenine
nucleotide pool. It is clear from this and earlier studies that
parietal cells maintain relatively high activity phosphodiesterase,
even in a vegetative state, e.g., inhibitors of phosphodiesterase
produce 50-100% activation of parietal cells (1, 33). Thus the
regulated flow of nucleotide from ATP
cAMP
AMP may be
considerable and represents a potential loss from the ATP/ADP energy
pool, in addition to other leakage pathways that produce AMP. Results
here show that parietal cell adenylate kinase can effectively recycle
AMP to the ATP/ADP pool. The equilibrium constant for the adenylate
kinase reaction is close to 1.0 (34) and therefore as cellular
metabolism normally maintains a high ATP-to-ADP ratio, AMP will be
driven toward ATP (AMP + ATP
2ADP and ADP + Pi
ATP). When cells are
maintained in an anaerobic or metabolically stressful environment,
there can be a serious depletion of the nucleotides, and in many
tissues such treatment can appear irreversible as the resynthesis of
adenine nucleotides from IMP and hypoxanthine derivatives requires
large amounts of energy expenditure (22).
Data presented in this study provide a comprehensive view of nucleotide
metabolism in the parietal cell that is schematically represented in
Fig. 9. The pool of ATP is modulated by
balancing the productive pathways (principally mitochondrial oxidative
phosphorylation) and the major
(H+-K+-ATPase)
and minor (protein phosphorylation) consumptive pathways. The
high-energy demand of acid secretion is met by a steady flow of oxygen
and substrates through the large mitochondrial pool. Extramitochondrial
sources of creatine kinase and adenylate kinase help to buffer ATP
level and the ATP-to-ADP ratio throughout the cytoplasm. ATP also
serves an essential role in the process of activation, both in the
production of the intracellular message and as a substrate for protein
phosphorylation. Adenylate cyclase and phosphodiesterase modulate the
level of cAMP, hence cell activation, through receptor-regulated
pathways. The resulting AMP is recycled back into the high-energy
nucleotide pool via adenylate kinase.

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|
Fig. 9.
Representation of nucleotide metabolism in parietal cell. Bold arrows,
major pathways; dashed arrows, interchangeable nucleotide pool.
Enzymatic reactions include protein kinase A (PKA, subscripts i and a
indicate inactive and active forms, respectively),
H+-K+-ATPase
(H/K), adenylate cyclase (AC), phosphodiesterase (PDE), adenylate
kinase (AK), and creatine kinase (CK). Transformation of
H+-K+-ATPase
from functionally inactive vesicular form to apical plasma membrane
presumably occurs by protein phosphorylations (Prot-P) mediated via
PKAa. Channels for
K+ and
Cl at apical plasma
membrane promote, respectively, recycling of
K+ through
H+/K+
exchange pump and net flow of
Cl to follow
H+ as HCl.
|
|
 |
ACKNOWLEDGEMENTS |
This work was supported in part by the National Institute of
Diabetes and Digestive and Kidney Diseases Grant DK-10141.
 |
FOOTNOTES |
Address for reprint requests: J. G. Forte, Dept. of Molecular and Cell
Biology, 241 LSA, Univ. of California, Berkeley, CA 94720-3200.
Received 11 August 1997; accepted in final form 6 October 1997.
 |
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