Biochimica et Biophysica Acta, 728 (1983) 31-38
`Elsevier Biomedical Press
`
`BBA 71532
`
`31
`
`INHIBITION OF GASTRIC (H + + K + )-ATPase BY THE SUBSTITUTED BENZIMIDAZOLE,
`PICOPRAZOLE
`
`B. WALLMARK •, G. SACHS ••, S. MARDH ***and E. FELLENIUS
`
`AB Hassle, Research Laboratories, S-431 83 Molnda/ (Sweden)
`
`(Received August 13th, 1982)
`
`Key words: ( H + + K +)-A TPase inhibition; Proton transport; Picoprazole; Acid secretion; (Porcine stomach)
`
`The substituted benzimidazole, picoprazole, inhibited the gastric (H + + K + )-A TPase in a concentration- and
`time-dependent manner. Half-maximal inhibition of the (H + + K +)-A TPase activity was obtained at about
`2·10 - 6 M under standard conditions. In addition to the inhibition of ATPase activity, parallel inhibition of
`phosphoenzyme formation and the proton transport activity were achieved. Radiolabelled picoprazole was
`found to bind to 100 kDa peptide; this peptide was shown by phosphorylation experiments to contain the
`catalytic centre of the (H + + K +)·A TPase. Studies on the (Na + + K + )-ATPase indicated that this enzyme
`was unaffected by picoprazole. From the data presented and from other pharmacological studies, it is
`proposed that this compound inhibits acid secretion at the level of the parietal cell by its ability to inhibit the
`gastric proton pump, the (H + + K + )·A TPase.
`
`Introduction
`
`The substituted benzimidazoles inhibit gastric
`acid secretion both in vivo and in vitro [1-3].
`Furthermore, in isolated gastric gland prepara(cid:173)
`tions [4] these inhibitors have been shown to in(cid:173)
`hibit acid production monitored as accumulation
`of a weak base, aminopyrine [5,6]. In this glandu(cid:173)
`lar system and in isolated guinea pig mucosa, the
`
`* To whom correspondence should be addressed.
`** Institute of Medical and Physiological Chemistry Biomedi(cid:173)
`cal Centre, Box 575, Uppsala University, S-751 23 Upp(cid:173)
`sala, Sweden.
`*** Laboratory of Membrane Biology, University of Alabama
`in Birmingham, University Station, Birmingham, AL
`35294, U.S.A.
`Abbreviations: (H + + K + )-A TPase, Proton plus potassium(cid:173)
`stimulated A TP phosphohydrolase; (N a+ K + )-A TPase, Sodium
`plus potassium ion transport A TP phosphohydrolase (EC
`3.6.1.3); Pipes, piperazine-N, N' -bis-(2-ethanesulfonic acid);
`Tris,
`tris(hydroxymethyl)aminornethane; dibutyryl-cAMP,
`N 6,0 21 -dibutyryladenosine 3': 5'-cyclic monophosphate.
`
`substituted benzimidazoles inhibited not only basal
`secretion, but also secretion induced by
`the
`secretatogues histamine and dibutyryl-cAMP. In(cid:173)
`hibition was found to be noncompetitive [1,6].
`Furthermore, aminopyrine accumulation stimu(cid:173)
`lated by high K + concentration was also
`antagonized by the compounds [6]. Based on these
`data, a mechanism of action peripheral to cell
`receptor sites and probably located within the
`parietal cell seemed likely [5,6]. During recent
`years, an (H + + K +)-A TPase has been purified
`from the gastric mucosa of various species [7-9]
`and has been localized to the secretory surface of
`the parietal cell [1 OJ. The addition of A TP to
`isolated vesicles containing the enzyme results in
`H+ uptake into the intravesicular space in ex(cid:173)
`change for intra vesicular K + [ 11, 12]. The trans(cid:173)
`port process is apparently electroneutral [ 13]. En(cid:173)
`try of KCl into the vesicle is rate-limiting for
`proton translocation. The (H + + K +)-A TPase thus
`plays an important role in H+ secretion across the
`membranes of the secretory canaliculus of the
`
`0005-2736/83/0000-0000j$03.00 ® 1983 Elsevier Biomedical Press
`
`

`
`32
`
`parietal cell [13,14]. It seemed possible, therefore,
`that the substituted benzimidazoles affect acid
`secretion by acting directly on the (H + + K + )(cid:173)
`ATPase. This paper presents data for one sub(cid:173)
`stituted benzimidazole, picoprazole, and describes
`its inhibitory action on the (H + + K +)-A TPase in
`more detail.
`
`Material and Methods
`
`Materials. Gastric (H + + K +)-A TPase was pre(cid:173)
`pared from hog stomachs as described by Sac(cid:173)
`comani et al. [15]. In brief, membrane vesicles
`containing the (H+ + K+)-ATPase were prepared
`by differential and zonal density gradient centrifu(cid:173)
`gation. The microsomal fraction was centrifuged
`through a 0.25 M sucrosej0.25 M sucrose-8%
`(w jw) Ficoll ® step gradient in a Kontron TZT 48
`zonal rotor (Basel, Switzerland) for 4 h at 47 000
`rpm. The material retained at the interface was
`used for the experiments.
`For transport experiments, the vesicles were
`equilibrated in a mixture of 150 mM KCl, 2 mM
`MgC1 2 and 2 mM Pipes adjusted to pH 6.7 with
`Tris base. After equilibration, the vesicles were
`stored at 4°C until use on the same day. All
`subsequent mixing of the sample solutions was
`done near isotonicity of the equilibrated sample.
`For experiments in which free access of ions to
`either face of the enzyme was required, the vesicle
`preparation was lyophilized and stored at - 80°C
`until resuspension. The (Na + + K + )-ATPase pre(cid:173)
`paration was obtained as described by J0rgensen
`[ 16].
`Reagents. [ y- 32 P]A TP was obtained from the
`Radiochemical Centre, Amersham, U.K. ATP was
`obtained from Sigma, U.S.A. Acridine orange
`was obtained from Merck, Darmstadt, F.R.G. Pi(cid:173)
`coprazole, (H 149 /94), methyl 6-methyl-2-[[(3-
`methyl-2-pyridinyl)methyl]sulfinyl]-1 H -benzimid(cid:173)
`azole-5-carboxylate and [ 14C]picoprazole were
`synthetized by the Chemical Department, AB
`Hassle, Molndal, Sweden (Fig. 1). Picoprazole was
`dissolved in methanol. The final methanol con(cid:173)
`centration did not exceed 1% which alone had no
`effect on any of the activities measured.
`Enzyme assays. K +-stimulated A TPase activity
`of the (H + + K +)-A TPase is designated K +(cid:173)
`A TPase activity. (N a+ + K +)-stimulated A TPase
`
`Picoprazole
`
`( H 149/94)
`
`Fig. I. Structural formula of picoprazole. • indicates the posi(cid:173)
`tion of the 14 C label.
`
`activity of the (Na + + K + )-ATPase is designated
`(Na + + K + )-ATPase activity. K +-stimulated p(cid:173)
`nitrophenylphosphatase activity of the (H+ +
`K+)-ATPase and (Na+ + K+)-ATPase are desig(cid:173)
`nated p-nitrophenylphosphatase ((H+ + K + )(cid:173)
`ATPase) and p-nitrophenylphosphatase ((Na+ +
`K + )-ATPase), respectively. It should be noted that
`p-nitrophenylphosphatase ((N a+ + K +)-A TPase)
`was assayed in the absence of Na +.
`A TPase activity. This was measured by the
`release of inorganic phosphate, which was assayed
`according to the method of Fiske and SubbaRow
`[I 7].
`K + -ATPase activity. The assay medium con(cid:173)
`sisted of 2 mM MgC1 2 , 75 mM Pipes-Tris buffer
`(pH 7.4), 2 mM Na 2 ATP, with or without 10 mM
`KCl in a total volume of 2 mi. The assay was
`incubated for 15 min at 37°C. The reaction was
`then stopped by the addition of I ml 10% trichlo(cid:173)
`roacetic acid. A maximum of 10% of the substrate
`was hydrolyzed over the incubation period, giving
`linear reaction rates. In lyophilized material and in
`the absence of K + , reaction rates were about 10
`ttmol PJmg protein per h. In the presence of both
`Mg 2 + and K +, reaction rates were about 100
`,umol PJmg protein per h. For all inhibition ex(cid:173)
`periments. reaction rates in the presence of Mg 2 +
`and K + but in the absence of inhibitor were set to
`100%.
`(Na + + K + )-ATPase activity was assayed in a
`medium consisting of 5 mM Pipes-Tris buffer (pH
`7.4), 2 mM MgC1 2 , 2 mM Na 2 ATP with or without
`10 mM KCI plus 100 mM N aCl in a total volume
`of 2 ml, and incubated for 3 min at 37°C. The
`reaction was stopped by the addition of l ml 10%
`trichloroacetic acid. In the absence of K + and
`Na +, reaction rates were about 20 ttmol PJmg
`protein per h. In the presence of N a+ and K + ,
`
`

`
`reaction rates were about 400 p.mol Pjmg protein
`per h. For the inhibition experiments reaction
`rates in the presence of Mg2+, Na+ and K+ but
`in the absence of inhibitor were set to 100%.
`p-Nitrophenylphosphatase activity. This was
`measured by the release of p-nitrophenol from
`p-nitrophenyl phosphate [18].
`((H + + K + )(cid:173)
`p-N itrophenylphosphatase
`ATPase) activity was assayed in a medium consist(cid:173)
`ing of 20 mM imidazole-acetic acid buffer (pH
`7.5), 3 mM MgC1 2 , 3 mM Tris-p-nitrophenyl
`phosphate with or without 10 mM KCI in a total
`volume of 0.5 ml. The assay was incubated for 3
`min at 37°C. The reaction was stopped by the
`addition of 25 p.l 50% trichloroacetic acid. In the
`absence of K +, reaction rates were about 4 p.mol
`p-nitrophenol/mg protein per h. In the presence
`of both Mg+ and K +, reaction rates were about
`75 p.mol p-nitrophenol/mg protein per h. This
`reaction rate was given a value of 100%.
`((Na+ + K + )(cid:173)
`p-Nitrophenylphosphatase
`ATPase) activity. This was assayed as described
`for p-nitrophenylphosphatase (H+ + K + -ATPase).
`In the absence of K + , reaction rates were about 7
`p.mol p-nitrophenol/mg protein per h. In the pres(cid:173)
`ence of both Mg + and K + , reaction rates were
`about 160 p.mol p-nitrophenol/mg protein per h,
`which was taken as the 100% value.
`Phosphorylation of the gastric membranes. (H +
`+ K +)-A TPase was preincubated for 30 min at
`37°C. The medium contained 10- 6 , 10- 5 and
`10- 4 M picoprazole, 40 mM Tris-HCl (pH 7.4)
`and 20 p.g of membrane protein. The phosphory(cid:173)
`lation reaction was then measured by the addition
`of 2 mM MgC1 2 and 5 p.M [y- 32 P]ATP for 15 s, at
`22°C. The reaction was stopped by the addition of
`1 ml ice-cold 10% HC104 containing 5 mM ATP
`and 40 mM Na 2 P04 • The precipitated protein was
`collected on a 4 p.m Millipore ® type SSWP filter.
`The filter was washed with 70 ml of 5% HC104
`containing 10 mM Na 2 P04 • The Cerenkov radia(cid:173)
`tion of the filter was measured in a scintillation
`counter.
`SDS-polyacrylamide gel-electrophoresis. Mem(cid:173)
`branes undergoing SDS-polyacrylarnide gel elec(cid:173)
`trophoresis were either first phosphorylated by
`means of [ y- 32 P]A TP or incubated with C4 C]pico(cid:173)
`prazole. The phosphorylation procedure was car(cid:173)
`ried out in a medium consisting of 200 p.gjml
`
`33
`
`gastric membranes, 2 mM MgC1 2 and 5 p.M [y-
`32 P]A TP in the presence or absence of 0.1 M KCI.
`The final volume was 200 p.l. The phosphorylation
`reaction was stopped with 1% SDS to solubilize
`the membranes. About 40 p.g protein was applied
`to a density gradient polyacrylamide gel with a
`starting density of 4% and a final density of 30%.
`Gels were run in a 0.1 M Tris-borate buffer (pH
`6.0) in 0.1% SDS for 2.5 h. Protein was stained
`with Coomassie blue and scanned
`in a gel
`densitometer at 550 nm. Gels run with 32 P-labelled
`membranes were sectioned manually immediately
`after electrophoresis and each slice was counted.
`14 C-picoprazole,
`For binding studies with
`gastric membranes (10 1-'g/ml) were incubated at
`37°C in 2 mM Pipes-Tris (pH 7.4) with 10- 4 M
`14 C]picoprazole in a total volume of 20 ml for 2 h
`[
`and then centrifuged for 1 h at 100 000 X g. The
`resulting pellet was resuspended in 1% SDS and
`about 40 1-'g protein applied to each gel. Gels were
`then treated as described for the phosphorylation
`experiments.
`Proton transport experiments. H + transport rates
`and the extent of pH gradient formation were
`monitored in an Aminco DW2 spectrophotometer
`(America} Instrument Company, U.S.A.) in the
`dual beam model set at 490 and 535 nm [19].
`Vesicles were preequilibrated at a concentration of
`40 p.gjml in 2 mM Pipes-Tris, 150 mM KCI and 2
`mM MgC1 2 at pH 6.7 for 3 h at 22°C. The equi(cid:173)
`librated sample was mixed with 10- 4 M pico(cid:173)
`prazole and after various incubation times, a sam(cid:173)
`ple was withdrawn and placed in a cuvette. 20 p.M
`Acridine orange was added and a baseline re(cid:173)
`corded, after which 0.6 mM Na 2 ATP was added
`to the incubation and the proton transport rate
`assayed.
`Protein determination. Protein was measured by
`the method of Lowry et al. [20].
`
`Results
`
`Kinetics of the inactivation of K + -A TPase activity
`The time course for the inhibition of K +(cid:173)
`ATPase activity is shown in Fig. 2A. The inhibi(cid:173)
`tion was found to be both time- and concentra(cid:173)
`tion-dependent, since the reduction in enzyme ac(cid:173)
`tivity
`increased progressively with
`increased
`
`

`
`~
`
`>
`.;=
`:;;_
`
`~
`
`1.5
`
`f
`g'
`c
`~
`cr
`~
`F 11
`
`34
`
`~
`
`>
`.;=
`:;;_
`~
`.g 50
`[?'
`c
`~
`&'
`-:.
`
`A
`
`1~M
`
`30
`
`60
`90
`30
`T1me M1nutes
`Time , M1nutes
`Fig. 2. A. Time course for inactivation of (H + + K + )-A TPase.
`Membrane suspensions (10 p.g of protein;ml) were incubated
`in a medium consisting of 5 mM Pipes-Tris (pH 7.4) at
`concentrations of picoprazole as indicated. After incubation
`times, indicated at the abscissa, samples were assayed for
`K +-A TPase activity. The K +-stimulated ATPase activity in the
`absence of picoprazole was given a value of 100%. Each time
`point is the mean of four separate determinations. The bars
`indicate the standard error of the mean (S.E.) for each time
`point. B. Semilogarithmic point of the data in Fig. 2A.
`
`inhibitor concentrations.
`time and
`incubation
`When the data from Fig. 2A were replotted semi(cid:173)
`logarithmically (Fig. 2B), the reaction of pico(cid:173)
`prazole with the enzyme was linear over the first
`75% of the total reaction. However, for the remain(cid:173)
`ing 25% a deviation from a straight-line relation(cid:173)
`ship between the logarithm of the remaining en(cid:173)
`zyme activity and time was obtained. From the
`linear portions of the curves in Fig. 2B, half-lives,
`
`2.0
`
`Stope= -1.1
`
`c
`0
`~
`
`~
`lll
`.s:
`r... .e
`~ -+-
`
`C'l
`.2
`
`1.0
`
`-6
`
`-7
`
`tog [PicoprazoleJ
`Fig. 3. Double logarithmic plot of the t 112 of inactivation
`against picoprazole concentrations. The t 112 was calculated
`from the linear part of Fig. 2B.
`
`.....
`>o
`·s:
`~
`~
`QJ
`E
`>o so
`N c:
`UJ
`Cl c:
`·c:
`'iii
`E
`QJ
`0::
`~ 0
`
`-6
`
`-4
`
`-5
`log [Picoprazolel
`Fig. 4. Concentration dependence for inhibition of (H + + K + )(cid:173)
`I' g of
`A TPase by picoprazole. Gastric membranes ( 10
`protein/ml) were incubated for 30 min in 5 mM Pipes-Tris (pH
`7.4) with picoprazole at concentrations indicated. After incuba(cid:173)
`tion, samples were assayed for K +-A TPase activity. The en(cid:173)
`zyme activity in the absence of picoprazole was given a value of
`100%. Each time point is the mean of two separate determina(cid:173)
`tions.
`
`t 112 , were calculated for the inhibition reaction at
`each inhibitor concentration. These values were
`then plotted in a double logarithmic plot against
`the picoprazole concentrations (Fig. 3). This plot
`was linear with a slope close to one. This allows
`the conclusion that inhibition follows pseudo(cid:173)
`first-order kinetics [21], at least over the first 75%.
`The concentration-effect relationship is shown in
`Fig. 4. Increasing concentrations of picoprazole
`resulted in progressive inhibition of the K +(cid:173)
`ATPase activity. Half-maximal inhibition occurred
`at about 2 ·_10- 6 M.
`
`pH dependence of inhibition
`From the results in Fig. 5, it was found that
`enzyme inhibition increased progressively as the
`proton concentration was increased. Despite the
`greater inhibition at lower pH, however, a pH of
`7.4 was taken as standard for most subsequent
`experiments, since this was the optimal pH for the
`enzyme activity [22].
`
`Binding of picoprazo/e to ( H + + K +)-A TPase
`The binding of picoprazole to (H + + K + )(cid:173)
`A TPase was analyzed by SDS-polyacrylarnide gel
`electrophoresis. A major peak was evident at about
`
`

`
`0
`
`0
`
`100
`
`>-
`......
`·:;::
`~ u
`d
`QJ
`E
`>-
`N c: so
`Cl c:
`:~
`E
`QJ
`0:
`~ 0
`
`QJ
`
`6.0
`
`7.0
`
`8.0
`
`pH
`Fig. 5. Effects of pH on the inactivation of (H + + K + )-ATPase.
`Membrane suspension (10 p.g of proteinjml) was incubated in
`2 mM Pipes-Tris buffer at pH 5.7, 6.3, 6.8, 7.4 and 7.8 for 30
`min, with picoprazole at concentrations of I p.M (0--0);
`5 p.M (0--0) and 10 p.M (L>.--L>.). After incubation,
`samples were adjusted to pH 7.4 by the addition of 75 mM
`Pipes-Tris buffer and K +-A TPase activity was assayed. K +(cid:173)
`A TPase activity in the absence of picoprazole was given a value
`of 100%.
`
`1.7
`
`E
`c:
`0 ...,
`...,
`~ 0.95
`~
`"'
`.Q
`~
`.Q
`<(
`
`Qj
`
`A
`
`100K-
`
`[3Zp)
`dpm
`
`20000
`
`10000
`
`8 9
`
`10
`
`11 12
`
`35
`
`100 kDa (Fig. 6B). This peak was also found to
`contain large amounts of radioactivity. However,
`large quantities of radioactivity that did not corre(cid:173)
`spond to any protein peak were retained at the end
`of the gel. This was probably due to binding of
`14C]picoprazole to lipid components, as the low
`[
`molecular weight region of the gel stained with
`Sudan black, a marker for lipids (data not shown).
`In order to identify the protein containing the
`catalytic peptide of the (H+ + K + )-ATPase, we
`studied the incorporation of 32 P from AT 33 P [23].
`The distribution of 32 P after phosphorylation of
`the membrane preparation is shown in Fig. 6A.
`The 100 kDa peptide was found to contain the
`32 P-label (open bars) and, furthermore, when the
`experiment was performed in the presence of K + ,
`the label of the 100 kDa peptide was lost (shaded
`bars). It can therefore be concluded that both the
`14C- and 32 P-labels are confined to the same
`peptide.
`
`r -
`
`B
`
`r -
`
`[ 14c I
`dpm
`
`6000
`
`100 K ....
`
`r--
`
`.--1)
`
`~
`
`.--
`
`4000
`
`.....-
`
`2000
`
`~ v f-v ~
`~ ~ V1
`v
`v
`
`jA.
`
`'""' h Jli\
`
`1 2 3 4 5 6 7 8 9 10 11 12 13
`Gel slice no.
`
`Fig. 6. A. Binding of [y-nP]ATP to (H+ + K + )-ATPase. Gastric membranes (200 p.g of proteinjml) were incubated and
`phosphorylated as described in Methods. Open bars show the distribution of the 32 P-label in the presence of MgAT 32 P and shaded
`bars in the presence of MgAT 32 P plus 100 mM KCI. Gels were prepared and run as detailed in the Methods section. 100 K indicates
`the position of the 100 kDa peptide. B. Binding of [ 14 C]picoprazole to (H+ + K + )-ATPase. Gastric membranes (10 p.g of proteinjml)
`were incubated with w- 4 M [ 14 C]picoprazole, after which the incubate was centrifuged at 100000 X g for 60 min and solubilized in
`1% sodium dodecyl sulphate. Open bars show the distribution of [14 C]picoprazole. 100 K indicates the position of the 100 kDa
`peptide.
`
`

`
`36
`
`Effect of picoprazole on phosphoenzyme formation
`the (H + + K + )(cid:173)
`During the catalytic cycle,
`ATPase forms a phosphoenzyme intermediate
`[24-26]. Since picoprazole was found to bind to
`the I 00 kDa peptide and also inhibited the A TPase
`activity (Figs. 4 and 6), its effect on steady-state
`phosphoenzyme concentration was studied. Pico(cid:173)
`prazole inhibited the phosphoenzyme levels in a
`concentration-related manner. At w- 6 M, 95% of
`the control level was obtained; at w-s M and
`10- 4 M 61% and 35%, respectively, of the control
`level was reached. The control level was 720 pmol
`of phosphoenzyme per milligram of membrane
`protein. Thus, inhibition of K + -ATPase activity
`and phosphoenzyme formation were found to oc(cid:173)
`cur in parallel.
`
`Inhibition of proton transport by picoprazole
`When the (H + + K +)-A TPase is isolated in
`vesicular form, proton transport can be measured
`[12, 19]. Addition of A TP to vesicles preequi(cid:173)
`librated in a medium containing KCl results in
`vesicles energization, and proton transport rates
`can be monitored. In this experiment, gastric
`vesicles were incubated with picoprazole. After the
`times indicated under each curve in Fig. 7 A, a
`sample was withdrawn and the transport signal
`measured. From Fig. 7 A, three parameters were
`calculated for each curve: the inital rate of proton
`transport, taken as the initial slope; the magnitude
`of the signal, and the time at which the maximal
`magnitude was reached. These parameters are
`shown in Fig. 7B, from which it can be seen that
`both the intial rate and maximal magnitude of the
`transport signal were inhibited in parallel. In addi(cid:173)
`tion, the time required to reach the maximal am(cid:173)
`plitude of the transport curve increased progres(cid:173)
`sively with incubation time.
`
`Effects on p-nitrophenylphosphatase (( H + + K + )(cid:173)
`ATPase)
`As stated earlier, proton transport in the iso(cid:173)
`lated vesicle system required extra vesicular MgA TP
`and intra vesicular K +. However, the (H + + K + )(cid:173)
`A TPase can hydrolyze substrates other
`than
`MgA TP. In the presence of p-nitrophenyl phos(cid:173)
`phate and Mg 2 + , K + stimulates the splitting of
`p-nitrophenyl phosphate into p-nitrophenol and
`inorganic phosphate. Two differences are evident
`
`A
`
`ATP
`I
`
`-
`
`0
`
`j -004
`E
`::0
`
`-OOB
`
`-012
`
`0
`
`B
`
`100
`
`.. c...,
`~"g
`Oi '2 50
`:Eg'
`.s~
`
`Il
`
`Trme,sec
`
`200 "'
`]
`'E
`Cl
`~
`i
`150 .f
`~
`;::
`
`'-/KJID-------:6:'::-0------..,±-___::s...~ 100
`0
`
`Time, min
`
`Fig. 7. A. Inhibition of proton by picoprazole. Gastric mem(cid:173)
`brane vesicles (40 JLg of protein/ml were equilibrated in 150
`mM KCl, 2 mM MgC1 2 and 2 mM Pipes-Tris (pH 6.7) at room
`temperature for 3 h, after which 10- 4 M picoprazole was added
`to the equilibration mixture. At the times indicated under the
`curves (0, 2, 13, 24, 35, 57, 68 and 143 min), a sample was
`withdrawn and the proton transport signal assayed. At time
`zero, indicated on the time axis in the figure, the sample and 20
`JLM Acridine orange were added to the cuvette (volume= I ml)
`and a base-line was recorded for about 25 s after which 0.6 mM
`ATP was added to initiate proton transport. B. Three parame(cid:173)
`ters were calculated from the date in Fig. 7A: Initial slope
`(e--e); maximal magnitude taken as the lowest value of
`each curve (.A--.6) and the time from the addition of ATP
`at which the signal reached a maximum ( 0 - - 0 ). All data
`are given as percent of the control signal, which was taken as
`the transport signal in the absence of inhibitor (lowest curve in
`Fig. 7A).
`
`when p-nitrophenyl phosphate is used as substrate
`instead of MgA TP. First, in order to stimulate
`hydrolysis of p-nitrophenyl phosphate, K + is re(cid:173)
`quired on the outside of the vesicle whereas in(cid:173)
`travesicular K + is required for the hydrolysis of
`
`

`
`37
`
`found that even at concentrations as high as
`w- 4 M, the (Na+ + K+)-ATPase activity was un(cid:173)
`affected by the inhibitor (Fig. 8). Furthermore, the
`p-nitrophenylphosphatase ((Na + + K + )-ATPase)
`activity was not inhibited, even at high picoprazole
`concentrations (Fig. 8).
`
`Discussion
`
`The ability to inhibit acid secretion in mam(cid:173)
`malian gastric mucosa has hitherto depended on
`the availability of H 2-receptor antagonists, such as
`cimetidine, or of anticholinergic agents [28,29].
`The substituted benzimidazoles represent a novel
`group of gastric acid secretion inhibitors. Their
`mechanism of action appears to be distinct from
`those of the H 2-blockers or anticholinergics, since
`pharmacological data for the benzimidazole de(cid:173)
`rivative, picoprazole, indicate that its antisecretory
`activity is exerted at a site distal to the production
`of cAMP in the events leading to acid formation
`[I ,6].
`It has been established that the gastric (H + +
`K +)-A TPase plays an essential role in the secre(cid:173)
`tion of acid by the parietal cells [13]. The data
`presented here show that picoprazole, a known
`inhibitor of gastric acid secretion [1-3], inhibits
`the (H + + K +)-A TPase. In addition, the phos(cid:173)
`phorylation and K +-dependent phosphatase reac(cid:173)
`tions were also inhibited. Moreover, SDS-poly(cid:173)
`acrylamide gel electrophoresis showed that the in(cid:173)
`hibitor was bound to the ATPase peptide. The
`inhibition of the proton transport reaction by pi(cid:173)
`coprazole can thus be attributed to inhibition of
`the A TPase activity.
`Since all partial reactions of the (H + + K + )(cid:173)
`A TPase were inhibited by picoprazole it was not
`possible to define the exact point at which the
`reaction pathway was inhibited. A puzzling feature
`of the inhibition of enzyme activity and of H+
`transport by the A TPase is the long preincubation
`requirement. Picoprazole is a hydrophobic com(cid:173)
`pound, which in the unprotonated form, distrib(cid:173)
`utes as a weak base into acidic compartments [6].
`Accordingly, limited penetration of picoprazole is
`unlikely to account for the time dependence of
`inhibition.
`Acidic pH facilitated the inhibition, but it was
`not determined whether this was due to a change
`
`10"6
`[ Picoprazole J
`
`Fig. 8. Effects of picoprazole on p-nitrophenylphosphatase
`((H + + K + )-A TPase),
`(N a+ + K + )-ATPase and p(cid:173)
`nitrophenylphosphatase ((Na + + K + )-ATPase) activity.
`Gastric membranes (10 1-1g of proteinjml) were incubated with
`picoprazole for 30 min in 5 mM Pipes-Tris buffer, pH 7.4
`<•--•). Samples were assayed as detailed in Methods.
`K + -p-nitrophenylphosphatase ((H + + K + )-ATPase) activity in
`the absence of inhibitor was taken as 100%. Kidney membranes
`(10 1-'g of proteinjml) were incubated with picoprazole in 5
`mM Pipes-Tris buffer (pH 7.4) for 30 min. Samples were
`assayed either for (Na + + K + )-ATPase activity (e--e) or
`for p-nitrophenylphosphatase ((Na + + K + )-ATPase) activity
`(N a+ + K + )-ATPase activity and p(cid:173)
`(& - - &),
`nitrophenylphosphatase ((Na + + K + )-ATPase) activity in the
`absence of picoprazole were taken as 100%.
`
`MgA TP. Secondly, hydrolysis of p-nitrophenyl
`phosphate cannot promote proton transport [23].
`From the experiment presented in Fig. 8, it is
`the p(cid:173)
`evident
`that picoprazole
`inhibited
`nitrophenylphosphatase ((H + + K + )-ATPase) ac(cid:173)
`tivity in a concentration-dependent manner. Half(cid:173)
`maximal inhibition was obtained at a concentra(cid:173)
`tion of 5 · 10- 6 M, which is in agreement with the
`value found for the inhibition of K +-A TPase.
`
`Effects on ( N a+ + K +)-A TPase
`Picoprazole was not able to discriminate be(cid:173)
`tween K + -A TPase, p-nitrophenylphosphatase
`((H + + K +)-A TPase) phosphoenzyme formation
`or proton transport. This finding led us to examine
`whether this lack of discrimination was only true
`of the various (H + + K +)-A TPase reactions. We
`therefore also studied the effect of picoprazole on
`a related enzyme system, namely the (Na + + K + )(cid:173)
`A TPase which was chosen because of its close
`similarities to the (H + + K +)-A TPase [27]. We
`
`

`
`38
`
`in reactivity of the inhibitor or to the protein.
`However, both the enhanced reactivity at low pH
`and the trapping of picoprazole in the secretory
`canaliculus [6] would be expected to enhance the
`activity of the compound in the intact cell.
`From the data presented, it can be concluded
`that picoprazole cannot discriminate between K + -
`ATPase, p-nitrophenylphosphatase ((H+ + K + )(cid:173)
`ATPase), phosphoenzyme levels or transport reac(cid:173)
`tions of the enzyme. However, a closely related
`enzyme, (N a+ + K + )-A TPase, was not affected by
`picoprazole. It therefore appears that local acidic
`compartments within the gastric gland are not the
`only factor governing
`the selectivity of pico(cid:173)
`prazole; when tested on the isolated enzymes, pi(cid:173)
`coprazole inhibited the (H + + K +)-A TPase more
`readily than the (N a+ + K +)-A TPase. This com(cid:173)
`pound thus appears to act selectively on the parietal
`cell, by virtue of its inhibition of the gastric proton
`pump.
`
`Acknowledgement
`
`The authors thank Miss Britt-Marie Jaresten
`for excellent technical assistance, Mrs. Greta Tebr(cid:173)
`ing for typing the manuscript and Mrs. Berit
`Elander for drawing the graphs.
`
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