Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan
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ABSTRACT |
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Gastric vesicles purified from acid-secreting rabbit stomach display K+ permeability manifested by the valinomycin-independent proton pumping of H+-K+-ATPase as monitored by acridine orange quenching. This apparent K+ permeability is attenuated by the treatment of the membrane with 5 mM Mg2+, and this phenomenon has been attributed to membrane-bound phosphoprotein phosphatase. However, with the exception of the nonspecific inhibitor pyrophosphate, protein phosphatase inhibitors failed to inhibit the loss of K+ permeability. Preincubation of the membrane with neomycin, a phospholipase C inhibitor, surrogated the effect of Mg2+, whereas another inhibitor, U-73122, did not. Phosphatidylinositol 4,5-bisphosphate (PIP2) restored the attenuated K+ permeability by treatment with either Mg2+ or neomycin. Furthermore, either phosphatidylinositol bound to phosphatidylinositol transfer protein or phosphatidylinositol 4,5,6-trisphosphate (PIP3) surrogated the effect of PIP2. Mg2+ and neomycin reduced K+ permeability in the membrane as determined by Rb+ influx and K+-dependent H+ diffusion. Treatment with Mg2+ reduced the contents of PIP2 and PIP3 in the membrane. These results suggest that PIP2 and/or PIP3 maintain K+ permeability, which is essential for proton pumping in the apical membrane of the secreting parietal cell.
hydrogen; potassium; adenosinetriphosphatase; neomycin
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INTRODUCTION |
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GASTRIC ACID
SECRETION IS conducted by the proton pump
H+-K+-ATPase, which exchanges H+ on
the cytoplasmic side with K+ on the opposite side using
energy supplied by ATP hydrolysis. When the parietal cell is in its
resting state, H+-K+-ATPase exists mainly on
the intracellular membranous structure called the tubulovesicles.
Because the tubulovesicle membrane has poor K+ and
Cl conductance, the exchange cycle cannot continue even
though enough ATP is present in the vicinity (for review, see Ref.
29). This has been manifested by vesicular proton
transport monitored by the acridine orange quenching technique
(15). When the microsomes obtained from resting stomach
were used, little proton pumping occurred in the presence of KCl and
ATP. For the transport to operate at full activity, valinomycin, a
K+ ionophore, is required (7). When the apical
membrane fraction purified from the acid-secreting stomach was used,
the vesicular proton transport was no longer dependent on valinomycin,
indicating that the apical membrane of the stimulated parietal cell
acquired K+ conductance (32). Stimulation of
acid secretion consists of two steps: the tubulovesicle fuses with the
apical membrane of the parietal cell and the secretory membrane
acquires K+ and Cl
conductance. Recent
studies (1, 6, 20) on the membrane fusion process in the
parietal cell have shown that several proteins are involved in membrane
recruitment and recycling. However, little is known about the
regulation of K+ and Cl
conductance, which is
thought to be the direct trigger for the activation of proton pumping.
As for the Cl
permeability involved in gastric acid
secretion, a candidate channel has been cloned (18) from
the cDNA library of gastric parietal cells, activated by cAMP-dependent
protein kinase A (PKA). In contrast, the molecular entity required for
K+ permeability in acid secretion has not yet been
identified. Although secretagogues such as histamine, gastrin, and ACh
have been shown to stimulate several intracellular signaling pathways
including cAMP, Ca2+, and inositol
1,4,5-trisphosphate/diacylglycerol in the parietal cell
(29), PKA is considered to be the essential component for the activation (1, 3). Therefore, it would be
conceivable that the putative K+ channel or transporter is
expected to be phosphorylated and activated by PKA. This assumption has
been supported by the early work of Im et al. (13). They
(13) showed that the K+ permeability of
gastric vesicles obtained from stimulated rat gastric mucosa was
reduced when the vesicles were treated with a high concentration of
Mg2+. Im et al. (13) attributed this
phenomenon to the putative membrane-bound Mg2+-activated
phosphoprotein phosphatase, because the reduction of K+
permeability was prevented by pyrophosphate, a nonspecific phosphatase inhibitor. However, further examination of this phenomenon has not been
performed. We examined the results of Im et al. (13) and
found that the decrease of K+ permeability by
Mg2+ treatment was not due to the dephosphorylation of the
putative K+ channel. Here we report that the phenomenon is
related to the metabolism of inositol polyphosphate, which appears to
be essential for K+ conductance in gastric proton pumping.
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MATERIALS AND METHODS |
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Membrane preparations and ATPase assay.
Gastric vesicles enriched in the apical membranes of stimulated
parietal cells were purified from the gastric mucosa of rabbit that had
been stimulated in vivo according to the method previously described
(32) with slight modifications. Japanese White rabbits (Shiraishi, Tokyo, Japan) were allowed to feed on staple food for 20 min followed by a subcutaneous injection of chlorpheniramine maleate (1 mg/kg body wt) and two histamine injections (0.1 mmol/kg each) 10 and
20 min after chlorpheniramine injection. The stomach was taken 5 min
after the last injection under anesthesia. The oxyntic glandular region
of the mucosa was homogenized in 30 vol of ice-cold homogenizing buffer
(125 mM mannitol, 40 mM sucrose, 1 mM EDTA, and 5 mM PIPES, pH 6.7) by
15 passes at 600 rpm through a Teflon piston homogenizer
(Potter-Elveheim). The homogenate was centrifuged at 100 g
for 5 min, and the supernatant was centrifuged at 6,000 g
for 10 min. The pellet was suspended with 18% Ficoll in the suspending
medium (300 mM sucrose, 5 mM Tris, and 0.2 mM EDTA, pH 7.4), layered
underneath the suspension medium, and then centrifuged at 100,000 g for 2 h. The material on the 18% Ficoll layer was
harvested, diluted in 20 vol of the suspending medium, and
recentrifuged at 100,000 g for 45 min. The final material was suspended in the suspending medium and stored at 80°C until use.
Measurement of proton accumulation rates by acridine orange quenching. Proton transport into the intravesicular space was monitored by the acridine orange quenching technique as described previously (15). An aliquot of membrane suspension (20 µl of ~2.5 mg protein/ml; 50 µg/ml final) was added to 1 ml of the uptake medium (40 mM KCl, 110 mM choline chloride, 10 mM PIPES-Tris, pH 6.8, 1 µM acridine orange, 0.5 mM MgATP, and 2 mM phosphocreatine). Accordingly, when the membrane had been pretreated with ions or drugs, the final concentration of the ions or drugs in the assay medium was one-fiftieth of the pretreated concentration. The mixture was excited with the wavelength of 493 nm under continuous stirring at 37°C, and the emission through a 540 ± 6-nm bandpass filter was recorded by a spectrophotofluorometer (CAF-110; JASCO, Tokyo, Japan).
Acridine orange is a weak base and membrane permeable when it is in the uncharged form in neutral solution. When the intravesicular pH is decreased by the action of H+-K+-ATPase, acridine orange is protonated and accumulated within the vesicles, and subsequently the fluorescent intensity decreases because of the red shift of the emission (15). This quenching reflects the continuous revolving of the H+-K+-exchanging pump. This means the membrane has some permeability to K+, because there is a continuous supply of K+ to the intravesicular site (34). In contrast to the apical membranes from stimulated parietal cells, the tubulovesicle membranes from resting parietal cells show a different feature. Addition of the vesicles to the reaction mixture does not induce acridine orange quenching, because the membrane has little K+ permeability, and thus the revolution of the pump does not occur. To continue the pumping by supplying K+ to the intravesicular site, the addition of valinomycin, a K+-specific ionophore (see Fig. 1A; 7, 32) is necessary. K+ permeability of the membrane was therefore estimated by the valinomycin dependency of the acridine orange quenching, that is the relative fluorescence before and after addition of valinomycin (1 µl of 10 mM stock solution in ethanol was added to make a final concentration of 10 µM). To ensure membrane integrity, membranes once frozen and then thawed were used up in a series of experiments and freezing-thawing was never repeated.
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Measurement of passive diffusion of H+ from the gastric vesicle. Passive diffusion of H+ across the vesicular membrane was measured as previously described (24) with slight modifications. The membrane preparation was incubated for 15 min at room temperature in a medium containing 1.5 µM acridine orange, 10 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP), 10 mM succinate, 300 mM sucrose, and 5.6 mM Tris base (pH 4). The extravesicular pH was promptly increased to 8 by addition of 25 mM Tris base (25 µl of 1 M solution) with continuous monitoring of the fluorescence as described above. By extravesicular alkalization, the fluorescence of acridine orange abruptly quenched according to the pH gradient made. Thereafter, intravesicular H+ ions passively diffused to the external medium as manifested by the recovery of fluorescence. This diffusion was restricted and reached an equilibrium even in the presence of the protonophore CCCP, because of the electrochemical potential created in the absence of any other movable ions. The addition of K+ gluconate caused some recovery of fluorescence. This is attributed to the cancellation of the electrical potential by K+ influx and thus the recovery rate of fluorescence is taken to be a reflection of the K+ influx rate. Inclusion of the proton pump inhibitor, omeprazole or SCH-28080 (10 µM each), showed no effect on K+-dependent recovery of fluorescence, suggesting that the apparent K+ influx was not through the reversed revolution of the pump but via the putative K+ channel or transporter(s).
Preparation of recombinant phosphatidylinositol transfer
protein-.
Rattus cloned phosphatidylinositol transfer protein-
(PITP
) in
pET-21a-d(+) vector was kindly given by Dr. H. Arai (Graduate School of
Pharmaceutical Sciences, University of Tokyo). The cDNA was cloned into
pGEX-4T-1 and transformed XL1-blue. Expression of the PITP
glutathione-S-transferase fusion protein was induced by
isopropyl-
-thiogalactopyranoside (0.1 mM) for 2 h at 25°C, and the bacterial cells were collected and resuspended in 150 mM NaCl,
3 mM K2HPO4, 0.64 mM
NaH2PO4, and 10 mM EDTA (pH 7.0). After
freeze-thawing, the sample was centrifuged at 40,000 g for 30 min at 4°C. Recombinant protein was purified from the supernatant using glutathione-Sepharose 4B resin. Purified fusion protein was
cleaved by thrombin treatment and incubated with 10 mg/ml phosphatidylinositol at 4°C overnight.
86Rb+ influx across membrane vesicles. Measurement of 86Rb+ influx across the vesicular membrane was performed as previously described (26) with slight modifications. Vesicles (1 mg/ml protein) were loaded with 150 mM RbCl, 125 mM sucrose, 2 mM PIPES-Tris (pH 7.4), and 86Rb+ (6 µCi/ml) at room temperature. The influx reaction was stopped at 15, 60, 300, and 600 s by a 20-fold dilution of the mixture with ice-cold stop solution (357 mM sucrose plus 2 mM PIPES-Tris; pH = 7.4) and promptly filtered through a Millipore filter (HAWP, 0.45 µm) that had previously been wetted with ice-cold stop solution. The filter was washed twice with 5 ml of ice-cold stop solution, transferred to a scintillation vial, and solubilized with 3 ml liquid scintillation cocktail. 86Rb+ inside the vesicles was analyzed by a liquid scintillation counter. This series of experiments was carried out in the Isotope Center of the University of Tokyo.
Measurement of phosphoinositides in the membrane preparation.
Labeling of phosphatidylinositol in the membrane preparation was
basically carried out as previously described (19, 28). The sample was added to 0.6 mM MgSO4 and 1 µCi/ml
[-32P]ATP. The mixture was incubated for 1 or 5 min at
30°C, and then the reaction was stopped by the addition of 0.5 mM
EDTA, and centrifuged at 16,000 rpm for 10 min. The pellet was
suspended in the suspending medium.
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RESULTS |
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Phosphoprotein phosphatase inhibitors did not prevent reduction of K+ permeability by treatment with high Mg2+. We first examined whether the results reported by Im et al. (13) were reproducible. The apical membrane-rich fraction obtained from acid-secreting rabbit parietal cell was added to a solution containing acridine orange, and quenching of fluorescence due to the accumulation of protons within the vesicles was consistently observed. This was completely independent of addition of valinomycin as shown in Fig. 1A, confirming that this membrane had already acquired enough K+ permeability. The membranes were then treated with 5 mM MgSO4 at 37°C for 10 min and added to the quenching medium. As shown in Fig. 1A, quenching was markedly reduced by this treatment and subsequent addition of valinomycin clearly caused further quenching. This indicates that the reduction of the quenching is not due to the decrease in the pumping activity per se but to the decrease of K+ permeability of the membrane. This effect of Mg2+ appeared to require some metabolic step(s), because the membrane needed to be pretreated with Mg2+ at 37°C for 10 min or more and incubation on ice did not develop the valinomycin dependency. Inclusion of 20 mM sodium pyrophosphate, a nonspecific phosphatase inhibitor, during the preincubation time, prevented the development of valinomycin dependency, showing exactly the same trace as the control membrane (Fig. 1A). These results were consistent with observations reported by Im et al. (13) using stimulated rat gastric membranes.
We started an examination of the work by Im et al. (13) using specific inhibitors. Calyculin A was expected to abolish any activities of phosphoprotein phosphatases I, IIA, and IIC at 1 µM. However, this drug failed to prevent Mg2+-induced development of valinomycin dependency (data not shown). To confirm this, we used the following protocol. The stimulated apical membranes were incubated with 5 mM MgSO4 and 1 mM ATP (Mg2+ treatment), with 5 mM Mg2+, 1 mM ATP, and 1 µM calyculin A (with protein phosphatase inhibitor), and with 5 mM Mg2+, 1 mM ATP, 1 µM calyculin A, and 20 U/ml PKA catalytic subunit (with PKA plus phosphatase inhibitor). As shown in Fig. 1B, all these showed the same degree of reduction in K+ permeability. These observations clearly indicate that involvement of protein phosphatases I, IIA, and IIC is excluded and that the putative phosphoprotein does not appear to be the substrate for PKA. Because there remained the possibility that protein phosphatase IIB (calcineurin) was involved in this process, we examined the effects of 50 nM calcineurin autoinhibitory peptide or 10 µM deltamethrin, which was reported (10) to be sufficient to achieve complete inhibition of calcineurin activity. It was found that neither inhibitor could prevent the effect of Mg2+ (Fig. 1C). Based on the results shown in Fig. 1, we excluded the possibility of involvement of protein phosphatases and PKA from the present system. We then moved on to the other candidates related to phosphorylation.Effects of phospholipase C inhibitors and aminoglycoside
antibiotics on acridine orange quenching.
As the involvement of phosphoprotein phosphatases appeared to be of
minor effect in the decrease of K+ permeability by
Mg2+ treatment, we examined another dephosphorylation
enzyme, phospholipase. Neither PLA2 inhibitor (AA-861, 10 µM) nor phospholipase D inhibitor (propranolol, 100 µM) was
effective (data not shown). We found that neomycin, a phospholipase C
(PLC) inhibitor, showed an interesting effect. As shown in Fig.
2A, preincubation of the
stimulated membrane vesicles with neomycin at 37°C for 10 min reduced
the acridine orange quenching in a dose-dependent manner. This effect
of neomycin was considered to be not due to the direct inhibition of
proton pumping but due to reduced K+ permeability, because
the reduced quenching was recovered by the addition of valinomycin, as
shown in Fig. 2A. Estimating the K+
permeability by the degree of valinomycin-independent acridine orange
quenching, we found that the effect of 150 µM neomycin appeared to be
equivalent to that of 5 mM Mg2+ (Fig. 2B). The
mode of action of neomycin seemed to be similar to that of
Mg2+, because it was necessary for neomycin to be
preincubated with membrane at 37°C for 10 min or more. The effect of
neomycin was evident at concentrations higher than 50 µM and reached
a maximum as high as 1 mM in the preincubation. The concentration of
neomycin in the cuvette during acridine orange quenching assay was
reduced to one-fiftieth of that in preincubation, i.e., 1 mM to 20 µM. Neomycin added just before the assay had little effect on the K+ permeability in this concentration range. When the
concentration of neomycin was increased to the millimolar range in the
assay condition, the acridine orange quenching was reduced and was not recovered by the addition of valinomycin (data not shown). This might
be due to its direct inhibition of the pumping machinery, as we
previously reported (24). It was therefore demonstrated that preincubation with 50 to 1,000 µM neomycin specifically reduced the K+ permeability in stimulated apical membranes.
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PIP2 restored attenuated acridine orange quenching by
neomycin and Mg2+.
It was reported (9) that the inhibition of neomycin on PLC
activity was due to its binding to the substrate PIP2. We
thought that the action of neomycin might be on the
phosphoinositide metabolism in the membrane by trapping
PIP2. We thus added PIP2 to the
membrane during preincubation with neomycin and found that
PIP2 protected the membrane from neomycin (data not shown).
Furthermore, the membrane with reduced K+ permeability from
neomycin pretreatment restored acridine orange quenching with the
addition of 2 µM PIP2 into the cuvette (Fig. 3A). Although the curve was
steep, the effect of PIP2 was found to be dose dependent in
the range of 1-10 µM (Fig. 3B). We also checked the
dose-dependent effect of PIP2 on Mg2+-treated
vesicles and found a similar recovery of K+ permeability
(Fig. 3, C and D). To elucidate the mode of
action of PIP2, we purified the microsomal fraction
containing resting tubulovesicles from cimetidine-treated rabbit
stomach. As shown in Fig. 3E, addition of PIP2
never caused acridine orange quenching in resting tubulovesicles,
whereas valinomycin added subsequently induced a marked quenching,
demonstrating that the resting tubulovesicles have little
K+ permeability. From this experiment, it was demonstrated
that PIP2 neither works as a K+ ionophore nor
activates the putative endogenous K+ channel/transporter in
this membrane preparation.
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Mg2+ and neomycin decreased
K+ permeability of membrane vesicles.
In the above experiments, we estimated the K+ permeability
by an indirect measure, i.e., valinomycin dependency of the
H+-K+-ATPase-operated proton pumping as
monitored by acridine orange quenching. As shown in Fig.
5, we measured
86Rb+ uptake by the vesicle and tested whether
the observed effects were related to the K+ permeability.
Although there was no difference in the 86Rb+
uptake at 5 min or later, it was significantly inhibited by
preincubation with 500 µM neomycin at the early time point (15 s
after the addition of the ion). This result indicates that inhibition
of K+ permeability by neomycin was only detectable when the
concentration gradient was large. As the K+ movement
appeared to be too fast for the filter method, we employed another
technique to measure the K+ permeability.
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Measurement of PIP2 content in gastric membranes.
To elucidate the mechanism of action of Mg2+ treatment, we
tried to measure the contents of phosphoinositides. It was
suggested that Mg2+ could deplete PIP2 from the
membrane (12) or absorb PIP2
(27), although the actual data have not been presented. We
pulse labeled the phospholipids in apical membrane vesicles with
32P, and the vesicles were incubated with 10 mM
Mg2+ for 20 min at 37°C. As the optimal condition for the
detection differed between PIP2 and PIP3,
slight modifications were made in the labeling time and the solvent
system. Figure 7A shows the autoradiography of the chromatogram suitable for
PIP2. It is clearly shown that Mg2+
treatment decreased 32P label in PIP2.
Consistent with the results of acridine orange quenching, addition of
20 mM pyrophosphate together with Mg2+ prevented the
decrease in 32P label in PIP2. In contrast,
treatment with neomycin did not decrease but rather increased the
amount of labeled PIP2. With TLC, we could not detect any
labeling in the place corresponding to PIP under the present condition.
In this system, however, the retardation factor of PIP3 was
too small to detect. Figure 7B shows the
autoradiography of the chromatogram suitable for PIP3 by
extending the labeling time and changing the solvent. It is clearly
shown that Mg2+ treatment decreased 32P label
also in PIP3. Although there were several radioactive spots
other than these authentic phosphoinositides, and some of them
even showed changes in radioactivity by these treatments, we could not
identify any of them so far.
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DISCUSSION |
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When H+-K+-ATPase was first discovered, the mechanism of its activation was an enigma, i.e., the enzyme was activated by K+ but existed on the intracellular membranes where the concentration of K+ was much higher than the Michaelis constant of the enzyme. This mystery was partially solved by the observation that H+-K+-ATPase required the K+ ionophore valinomycin for its maximal activity (7). Namely, the proton pumping activity was thought to be latent when the membrane had little K+ permeability and thus the K+ site facing the luminal side is not accessible for the cation. Since then, much effort has been made to find out the nature of that K+-transporting activity. Although some candidates have been suggested (23), no conclusion has been obtained yet.
It should be reasonable to postulate that activation of the putative K+ transporter or channel is due to phosphorylation via PKA, because PKA activation has been shown to be essential for acid secretion, at least in rabbit parietal cells (1, 3). As evidence, Im et al. (13) showed that 1) gastric heavy microsomes obtained from acid-secreting rat stomach showed ATP-dependent, valinomycin-independent proton transport; 2) incubation of the membrane with Mg2+ and high protein concentration made the vesicle valinomycin dependent; 3) the effect of Mg2+ was prevented by a nonspecific phosphatase inhibitor, pyrophosphate; and 4) the microsomal membrane preparation actually showed phosphoprotein phosphatase activity. From these findings, Im et al. (13) concluded that the gastric membranes contained membrane-bound, Mg2+-dependent phosphoprotein phosphatase, which desphosphorylated the putative K+ carrier. The role of Mg2+ was postulated to activate the enzyme and to facilitate the membrane-membrane contact for the dephosphorylation reaction. However, these results (13) only indirectly suggested the possible involvement of protein phosphorylation in activation of K+-transporting activity. One disadvantage for the researchers at that time was that no specific inhibitors of phosphoprotein phosphatase were available. As the reaction of H+-K+-ATPase contains a dephosphorylation step, the usual phosphatase inhibitors also blocked the revolution of the pump and thus analysis utilizing the pump activity (like acridine orange quenching) became difficult. In recent years, calyculin A and okadaic acid have been available as specific inhibitors of phosphoprotein phosphatase type I, IIA, and IIC. Of these, we previously reported (30) that calyculin A stimulated acid secretion in isolated rabbit gastric glands, suggesting that protein phosphorylation plays an important role in acid secretory response.
For the present study, we examined previous reports using rabbits instead of rats and found that similar results were obtained, i.e., the K+ permeability manifested by valinomycin-independent proton transport in vesicles from stimulated gastric mucosa was attenuated by treatment with Mg2+, and this effect was prevented by pyrophosphate, a nonspecific phosphatase inhibitor. However, calyculin A at 1 µM, which was expected to completely suppress the phosphoprotein phosphatases I, IIA, and IIC, failed to prevent the attenuation of K+ permeability induced by Mg2+. Moreover, K+ permeability was reduced by treatment with Mg2+ even in the presence of ATP, PKA, and calyculin A. These results clearly indicated that the effect of Mg2+ did not involve dephosphorylation by phosphoprotein phosphatases I, IIA, and IIC and that the dephosphorylation, if any, did not occur on the substrate(s) for PKA. The possible involvement of protein phosphatase IIB, or calcineurin, was excluded by using calcineurin inhibitory peptide and deltamethrin. These results suggest that there was little possibility for the involvement of phosphoprotein phosphatases in the phenomenon. We then decided to search for other possibilities.
Even though protein phosphatases were excluded from the process, it was reasonable to postulate that other phosphorylation events might be involved, because the effects of Mg2+ treatment were effectively prevented by pyrophosphate. During screening with phospholipase inhibitors, we found that Mg2+ treatment was surrogated by the treatment with neomycin, a PLC inhibitor. Based on published data, we postulated the following possibilities at that time: 1) PLC plays a pivotal role for K+ permeability, because neomycin was reported to block PIP2 metabolism and it modulated the open probability of the Ca2+-sensitive K+ channel (31); 2) neomycin has a direct blocking effect on K+ permeability, because aminoglycoside antibiotics were shown to block the P/Q-type Ca2+ channel (11); and 3) neomycin binds to PIP2 and changes the functions of some proteins regulated by PIP2 (9). The first possibility was excluded by using another PLC inhibitor, U-73122, which failed to surrogate the effect of neomycin. The second possibility was denied by the observation that other aminoglycoside antibiotics, gentamycin and kanamycin, did not mimic neomycin.
Considering the mechanism of neomycin, we postulated that its effect was similar to Mg2+ and related to the metabolism of PIP2, because the effects of both neomycin and Mg2+ were 1) prevented by pyrophosphate, 2) not prompt and required at least 10 min of pretreatment, and 3) restored by addition of PIP2. We speculate that the phosphorylation/dephosphorylation cycle is working in the membrane and neomycin traps PIP2 to prevent its function, whereas Mg2+ affects the cycle by reducing the membrane contents of PIP2. This assumption is partially supported by the observation that the amount of PIP2 was reduced by Mg2+ but increased by neomycin.
To identify the key molecule among the phosphoinositides, we
checked several compounds and found that PIP3 also
recovered K+ permeability, whereas neither PIP nor
phosphatidylinositol did. This result was somewhat surprising because
biomembranes usually possess an enzyme complex for phosphoinositide
metabolism (8) and the supply of phosphatidylinositol is
the rate-limiting step. We thought that the lack of effect of the
latter two was due to the difficulty of their incorporation into the
membranes. It has been suggested that the supply of PIP or
phosphatidylinositol was accelerated in the living cell by the protein
PITP (4). We then prepared a recombinant PITP and found
that it restored the reduced K+ permeability by
Mg2+ or neomycin treatment after the protein had been
preadsorbed with phosphatidylinositol. These results suggest that
treatment by Mg2+ or neomycin caused a shortage of some
phosphoinositide(s) by affecting the phosphatidylinositol metabolism.
The direct measurement of phosphoinositides in the membrane showed that treatment with Mg2+ actually reduced the labeled PIP2 as well as PIP3, and the reduction was prevented by pyrophosphate. A possible explanation is that Mg2+ might activate a phosphatase or inhibit a kinase for the component(s) downstream of PIP2, and pyrophosphate antagonizes that reaction. It was reported (2) that pyrophosphate affected the contents of phosphoinositides in the neutrophil membranes, although it increased the contents of PIP2 in this case. However, it is possible that pyrophosphate acts differently in another type of cell with a different phosphoinositide metabolism.
In contrast to Mg2+, neomycin treatment did not decrease but rather increased the phosphate label in PIP2. Considering the fact that neomycin binds to PIP2 and inhibits its metabolism (9), we postulate that neomycin intercepted the sequence of phosphorylation/dephosphorylation at the level of PIP2, and subsequently the labeled compound was accumulated in the membrane. We have not identified the molecules by which the label is decreased from treatment with neomycin.
Wolosin and Forte (33) reported that various cations,
including Mg, Ni2+, and Zn2+, potently
inhibited Cl and K+ conductances in the
stimulated apical membrane of rabbit parietal cells. They
(33) suggested that these cations directly affected the
putative channels. Direct effects of Mg2+ on the putative
channels could be excluded because that demanded some metabolic process
and the Mg2+ concentration during the assay was reduced to
one-fiftieth of that in preincubation. On the other hand, the
observation of Wolosin and Forte (33) that
Ni2+ and Zn2+ inhibited K+
conductance at submillimolar concentration might have been due to the
reduction of phosphoinositides in the membrane. However, we did not
observe any changes in the contents of PIP2 and
PIP3 in the membrane preparation by treatment with these cations.
Of the phosphoinositides, PIP2 has been reported to modulate the activity of various channels or transporters, including inward rectifier K+ channels (ROMK, ROMK2 /Kir6.2, GIRK1/4, GIRK2, and IRK1; 12, 16), Na+-gated nonselective cation channel (34), and Na+/Ca2+ exchanger (22). It is reasonable to postulate that the putative K+ channel or transporter essential for gastric proton pumping demands PIP2 for its activity. This information should be quite useful to identify the molecular entity of the putative K+ channel or transporter in a future experiment. However, considering the fact that PIP3 surrogated PIP2, the metabolite(s) of PIP2 would be the key molecule(s). Alternatively, PIP3 might have been metabolized to be PIP2 within the membrane. More work is necessary to identify the molecule responsible for K+ permeability by phosphoinositides in the gastric membrane.
In the present study, the K+ permeability of the membrane
was mainly estimated by indirect measurements, i.e.,
valinomycin-dependent proton transport operated by
H+-K+-ATPase and H+ passive
diffusion where K+ alone exists as the permeable ion
population. The direct measurement using 86Rb uptake
revealed that the K+ (Rb+) permeability of the
membrane was relatively high even though the
H+-K+-ATPase-operated acridine orange quenching
became highly valinomycin dependent from treatment with neomycin. We
consider that the intrinsic permeability of the membrane to
K+ or Rb+ is relatively high even in the
resting state. This possibility has been repeatedly pointed out, and
there used to be a hypothesis that K+ permeability is not
necessarily accelerated during the activation of acid secretion
(5, 26). However, even though the membrane containing
H+-K+-ATPase has some K+
permeability, H+ cannot accumulate within the vesicle when
permeability to H+ in the membrane is higher than that to
K+. The observed acridine orange quenching was the function
of the intrinsic pump activity, and K+, Cl,
and H+ permeability. In this system, we can observe the
steady-state level based on these factors and thus the sensitivity to
detect the reduction of K+ permeability is quite high. In
the case of Rb+ uptake, the observed measurement is the
result of uniflux and is thus much less sensitive. Looking at the
traces of the H+ passive diffusion experiments, the
difference in the influx rate manifested as the proton counterflow,
with or without treatment, was only evident within a minute and
disappeared when the K+ concentration inside the vesicle
approached that of the outside, which was consistent with the data of
Rb+ uptake.
It is noteworthy that the addition of PIP2 did not induce K+ permeability in resting tubulovesicles. This indicates that the molecular entity of the putative K+ channels or transporters does not exist on the tubulovesicular membrane but on the other intracellular membrane that fuses with the apical membrane under stimulation (14), or it resides on the apical membrane. Alternatively, the putative K+ channels or transporters exist on the tubulovesicles in the resting state and are insensitive to PIP2 but become sensitive to it when the cell is stimulated.
In conclusion, we found in the present study that phosphatidylinositol (possibly PIP2 and/or PIP3) is an essential determinant for the K+ permeability involved in gastric proton pumping. In spite of many efforts to identify the molecular entity of the K+ transporter that is the direct switch of gastric acid secretion, no conclusive results have been obtained. For example, a study by Supplisson et al. (25) that used a patch-clamp method described K+ channels as being possibly located on the apical membrane of the parietal cell. However, it is very difficult to conclude that the observed channel is coupled with the gastric proton pump by electrophysiology alone. To this end, the present study has supplied a powerful tool, phosphoinositides and the drugs affecting their function and metabolism, in the identification of the molecular entity responsible for K+ permeability. It is also an interesting question how phosphoinositides participate in the regulation of gastric acid secretion. If it is a direct switch of acid secretion, we have to search for a missing link between PKA and phosphoinositides. If it is indirect, there should be some mechanism that allows the system to be sensitive to this lipid. In any event, we are sure that the present work represents a breakthrough in understanding the molecular basis of gastric proton pumping.
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ACKNOWLEDGEMENTS |
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We thank John H. Jennings for editing the manuscript.
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FOOTNOTES |
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This study was supported in part by the Japanese Ministry of Education, Science, Sports, and Culture.
Address for reprint requests and other correspondence: T. Urushidani, Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Univ. of Tokyo, Tokyo 113-0033, Japan (E-mail: urushi{at}mol.f.u-tokyo.ac.jp).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 29 December 2000; accepted in final form 14 May 2001.
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