Defects in TCIRG1 subunit of the vacuolar proton pump are responsible for a subset of human autosomal recessive osteopetrosis.

Osteopetrosis includes a group of inherited diseases in which inadequate bone resorption is caused by osteoclast dysfunction. Although molecular defects have been described for many animal models of osteopetrosis, the gene responsible for most cases of the severe human form of the disease (infantile malignant osteopetrosis) is unknown. Infantile malignant autosomal recessive osteopetrosis (MIM 259700) is a severe bone disease with a fatal outcome, generally within the first decade of life. Osteoclasts are present in normal or elevated numbers in individuals affected by autosomal recessive osteopetrosis, suggesting that the defect is not in osteoclast differentiation, but in a gene involved in the functional capacity of mature osteoclasts. Some of the mouse mutants have a decreased number of osteoclasts, which suggests that the defect directly interferes with osteoclast differentiation. In other mutants, it is the function of the osteoclast that seems to be affected, as they show normal or elevated numbers of non-functioning osteoclasts. Here we show that TCIRG1, encoding the osteoclast-specific 116-kD subunit of the vacuolar proton pump, is mutated in five of nine patients with a diagnosis of infantile malignant osteopetrosis. Our data indicate that mutations in TCIRG1 are a frequent cause of autosomal recessive osteopetrosis in humans.

Faster evolution of highly conserved DNA in tropical plants.

A faster rate of nuclear DNA evolution has recently been found for plants occupying warmer low latitudes relative to those in cooler high latitudes. That earlier study by our research group compared substitution rates within the variable internal transcribed spacer (ITS) region of the ribosomal gene complex amongst 45 congeneric species pairs, each member of which differed in their latitudinal distributions. To determine whether this rate differential might also occur within highly conserved DNA, we sequenced the 18S ribosomal gene in the same 45 pairs of plants. We found that the rate of evolution in 18S was 51% faster in the tropical plant species relative to their temperate sisters and that the substitution rate in 18S correlated positively with that in the more variable ITS. This result, with a gene coding for ribosomal structure, suggests that climatic influences on evolution extend to functionally important regions of the genome.

Biosystematics and conservation: a case study with two enigmatic and uncommon species of Crassula from New Zealand.

BACKGROUND AND AIMS: Crassula hunua and C. ruamahanga have been taxonomically controversial. Here their distinctiveness is assessed so that their taxonomic and conservation status can be clarified. METHODS: Populations of these two species were analysed using morphological, chromosomal and DNA sequence data. KEY RESULTS: It proved impossible to differentiate between these two species using 12 key morphological characters. Populations were found to be chromosomally variable with 11 different chromosome numbers ranging from 2n = 42 to 2n = 100. Meiotic behaviour and levels of pollen stainability were both variable. Phylogenetic analyses showed that differences exist in both nuclear and plastid DNA sequences between individual plants, sometimes from the same population. CONCLUSIONS: The results suggest that these plants are a species complex that has evolved through interspecific hybridization and polyploidy. Their high levels of chromosomal and DNA sequence variation present a problem for their conservation.

[3H]Bafilomycin as a probe for the transmembrane proton channel of the osteoclast vacuolar H(+)-ATPase.

Bone resorption by the osteoclast is dependent on acidification of the bone surface by a vacuolar type H+-ATPase (V-ATPase) present in the ruffled membrane of the actively resorbing cell. V-ATPases are a highly conserved family of proton pumps consisting of two functional complexes: a cytoplasmic catalytic sector (VC) and a membrane bound proton channel (VB). Bafilomycin A1, a macrolide antibiotic, is a highly potent inhibitor of V-ATPases, and inhibits bone resorption in vitro in isolated rat calvariae. In order to investigate the binding of bafilomycin to the osteoclast V-ATPase, we used a tritiated bafilomycin which had been prepared by acetylating the 21-hydroxyl group of bafilomycin A1. Osteoclast ruffled membrane vesicles were prepared from purified chicken osteoclasts by differential centrifugation and proton transport in these vesicles was shown to be inhibited by [3H]bafilomycin (IC50 approximately 2 nM). Control membrane vesicles or membrane vesicles partially inhibited with [3H]bafilomycin were solubilized and separated by centrifugation on 15-30% glycerol gradients. V-ATPase activity and reconstitutable proton transport activity could be recovered in high density fractions of the gradient. However, the peak of [3H]bafilomycin radioactivity (>70% of total radioactivity in the gradient) was present in a single peak at lower density. Antibodies against subunits of VC (70, 56 and 40 kDa) reacted only in fractions containing the peak V-ATPase activity whereas an antibody to the 39 kDa subunit of VB reacted both with fractions containing the peak V-ATPase activity but also, and more strongly, in fractions containing the peak [3H]bafilomycin. The fractions in the control gradient corresponding to the peak of [3H]bafilomycin were reconstituted into liposomes and shown to mediate passive bafilomycin A1-inhibitable proton conductance. SDS-PAGE followed by autoradiography, indicated that the bafilomycin was not covalently bound to the V-ATPase or the proton channel. Quantification of VB by [3H]bafilomycin binding or by antibody staining suggested an excess of the free proton channel to that of the intact holoenzyme. A corresponding amount of free catalytic sector could not be found in any fraction throughout the isolation procedure of the V-ATPase from the initial homogenate. Thus, in conclusion, bafilomycin inhibits the V-ATPase by binding tightly but non-covalently to the proton channel region of the V-ATPase which appears to be present in excess over the intact holoenzyme in the osteoclast. The possible role of an excess of the proton channel subcomplex in the osteoclast is discussed.

Effects of NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid) on chloride transport in intestinal tissues and the T84 cell line.

NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid) has been reported to block Cl- channels in isolated rabbit nephrons with high potency (IC50 = 80 nM). The effects of this compound on Cl(-)-mediated transport processes in intestinal tissues have been studied using agonist-stimulated short-circuit current (T84) in Ussing chamber experiments and 36Cl- fluxes in monolayers of a colonic cell line (T84). NPPB inhibited PGE1-stimulated Isc in rabbit distal colon and ileum at concentrations in the range 20 to 100 microM. However, NPPB at the same concentrations also inhibited glucose-stimulated Isc in rabbit ileum, suggesting that its effects were not restricted to those on Cl- transport. Consistent with this, exposure of rabbit distal colon to 100 microM NPPB was found to reduce endogenous ATP levels by 69%, implying that, at these concentrations, NPPB could impair active transport processes by an effect on cellular energy metabolism. Clear evidence for a direct effect of NPPB on epithelial chloride channels was found in studies on Cl- fluxes in T84 cell monolayers. NPPB inhibited VIP-stimulated Cl- uptake into T84 cells with an IC50 of 414 microM. NPPB (1 mM) also inhibited Cl- efflux from pre-loaded cells confirming its effect as a weak Cl- channel blocker in this system.

Characterisation of the effects of anthranilic and (indanyloxy) acetic acid derivatives on chloride transport in membrane vesicles.

The effects of the Cl- channel blockers, NPPB, IAA94/95 and a number of related compounds on 36Cl- transport in membrane vesicles from bovine kidney cortex and rabbit ileum mucosa brush borders have been studied. These vesicles have been previously shown to be enriched in Cl- channel and Cl-/anion cotransport activity, respectively. Chloride transport was assayed in both types of vesicles by measuring the uptake of 36Cl- in response to an outwardly-directed Cl- concentration gradient. In kidney microsomes, a large proportion of the observed 36Cl- uptake was mediated by an electrogenic uniport and could be substantially reduced by clamping the membrane potential at zero mV using K+ and valinomycin. Chloride uptake was inhibited by both NPPB and IAA94/95 with apparent IC50 values of around 10 microM under optimal conditions (i.e., 4 min uptake at 4 degrees C). Under other conditions (e.g., 10 min uptake at 25 degrees C), where uptake had reached a steady-state level, much higher concentrations of inhibitor were required to cause inhibition. Therefore, previous differences in the reported potency of these compounds may, in part, have been due to the conditions under which Cl- uptake was measured. In addition, both NPPB and, to a lesser extent, IAA94/95 were found to have other effects on the vesicles, in that, when added at a concentration of 100 microM, they induced a leakage of pre-accumulated 36Cl-. This was probably caused by either dissipation of membrane potential or damage to the vesicle membranes. The sulphonic acid derivatives of NPPB and IAA94/95 (NPPB-S and ISA94/95, respectively) blocked 36Cl- uptake with around the same potency as NPPB and IAA94/95, but did not cause any non-specific Cl- leakage, when added at concentrations up to 100 microM. Inhibition of 36Cl- uptake by all four compounds was almost completely reversible. However, when vesicles were incubated with the inhibitors in the presence of an outward Cl- concentration gradient, or if vesicles were freeze/thawed in the presence of the compounds, inhibition could be only partially reversed. In rabbit brush border membrane vesicles, 36Cl- uptake was not reduced when the vesicles were voltage clamped using valinomycin and K+, and was therefore probably mediated by Cl-/Cl- exchange. However, despite the lack of effect of valinomycin, 36Cl- uptake was inhibited by both NPPB (approx. 80% inhibition at 100 microM) and, to a lesser extent, by IAA94/95 (approx. 30% inhibition at 100 microM).(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of proton transport in bone-derived membrane vesicles.

ATP-dependent proton transport in membrane vesicles prepared from the medullary bone of egg-laying hens, a source rich in osteoclasts, was characterized. Proton transport was abolished by bafilomycin A1 (10 nM) and N-ethylmalemide (50 microM), but not by oligomycin (15 micrograms/ml), vanadate (100 microM) or SCH 28080 (100 microM), thereby differentiating this H(+)-ATPase from the F1F0- and phosphorylated-type of ATPases. Preincubation of the membrane vesicles at 0 degrees C for 1 h in the presence of KCl (0.3 M) and Mg-ATP (5 mM) resulted in almost complete loss of H(+)-transport activity (cold-inactivation). Preventing the formation of a membrane potential by voltage clamp (Kin+ = Kout+ + valinomycin) increased both the rate of H(+)-transport and the equilibrium delta pH, suggesting an electronic proton transport mechanism. Thus, the H(+)-ATPase in this bone-derived membrane vesicle preparation shows the characteristics of a vacuolar H(+)-ATPase in its inhibitor- and cold-sensitivity and its electrogenic mechanism. The anion sensitivity of the H(+)-ATPase was investigated by varying the intra- and/or extra-vesicular salt composition. The H(+)-ATPase had no absolute requirement for any specific anion, but membrane permeable anions were found to stimulate proton transport activity, presumably by acting as charge compensators for the electrogenic hydrogen ion transport. However, some anions, such as sulfate, acetate and nitrate were directly inhibitory to the ATPase. The results are in agreement with the recently proposed mechanism of osteoclast acidification: a vacuolar H(+)-ATPase working in parallel with a Cl(-)-channel resulting in electroneutral HCl secretion.

Fourier transform infrared spectroscopic studies on gastric H+/K+-ATPase.

Suspensions of membrane-bound H+/K+-ATPase in both H2O and 2H2O were investigated using Fourier transform infrared (FT-IR) spectroscopy. Second-derivative techniques were used to reveal the overlapping bands in the 1800-1500 cm-1 region. Analysis of the amide I band shows that the protein component contains substantial amounts of both alpha-helical and beta-sheet structures. Addition of 10 mM KCl to a suspension in 2H2O does not significantly affect the amide I band, indicating that the E1-E2 conformational transition of the enzyme, induced by K+, does not involve a gross change in protein secondary structure. Analysis of the amide II band in the spectra of suspensions in 2H2O shows that inhibition of the enzyme with omeprazole increases the rate of 1H-2H exchange, indicating an increase in conformational flexibility. Furthermore, an additional feature at 1628 cm-1 in the spectra of the inhibited samples in 2H2O could either support a conformational change or arise from a vibrational mode of omeprazole in its enzyme-bound form. The frequency of the band due to the symmetric stretching vibrations of the methylene groups of the lipid acyl chains increases steadily with increasing temperature indicating that there is no co-operative melting process in the lipid component of the membrane over the temperature range 9-50 degrees C. For comparison, FT-IR studies on aqueous suspensions of Na+/K+-ATPase were also carried out. These show that the protein components in the Na+/K+- and H+/K+-ATPases have similar secondary structures.

Characterization and cellular distribution of the osteoclast ruffled membrane vacuolar H+-ATPase B-subunit using isoform-specific antibodies.

Acidification of the bone surface, leading to bone resorption, is accomplished by a vacuolar-type H+-ATPase present in a specialized domain of the plasma membrane of the osteoclast known as the ruffled membrane. Structure and function appears to be highly conserved within this class of multisubunit enzymes. However, cloning and sequencing of complementary DNA has shown that one of the subunits in the catalytic domain, the B-subunit, exists in at least two forms, B1 and B2. B1 messenger RNA has been found almost exclusively in the kidney, whereas messenger RNA for B2 has been found in all tissues studied, including the kidney. It has been speculated that the B1 isoform might be involved in targeting to the plasma membrane. In the present study, we have characterized the B-subunit of the chicken osteoclast H+-ATPase using antibodies directed against peptides with isoform-specific or conserved sequences of the B-subunit. Western analysis was performed on chicken osteoclast membrane vesicles and on partially purified chicken osteoclast H+-ATPase and was compared with similar analysis of H+-ATPase isolated from bovine kidney and brain. The B1-specific antibody reacted with a polypeptide of approximately 56 kD on immunoblots of the renal H+-ATPase, whereas no reaction could be detected against the osteoclast H+-ATPase or the osteoclast membrane vesicle preparation. In contrast, the antibody against a B2-specific sequence reacted with a peptide of approximately 56 kD on immunoblots of the osteoclast H+-ATPase, the renal H+-ATPase, and the clathrin-coated vesicle H+-ATPase. The antibody against a conserved region of the B-subunit did not generate any evidence for the presence of isoforms other than B2 in the osteoclast. Immunocytochemistry of rat osteoclasts on bovine bone slices using the B2 antibody showed intense polarized staining along the plasma membrane facing the bone surface in actively resorbing osteoclasts whereas nonresorbing osteoclasts were diffusely stained throughout the cytoplasm. By confocal microscopy, the B2 staining was located to the level of the ruffled membrane and appeared to be concentrated to the peripheral areas of the membrane adjacent to the sealing zone. We conclude that the osteoclast vacuolar H+-ATPase contains the B2 isoform and suggest that upon initiation of resorption the pump is translocated from the cell interior to a special domain of the ruffled membrane close to the sealing zone.

Reversible inhibitors of the gastric (H+/K+)-ATPase. 2. 1-Arylpyrrolo[3,2-c]quinolines: effect of the 4-substituent.

Further work on compounds 1 has identified the 4-position as a site where substantial modifications are tolerated, leading to analogues which are more potent and less toxic than those described previously. The best compound in the series is 13a (SK&F 96356), which is a potent inhibitor of gastric acid secretion in both the pentagastrin-stimulated rat and the histamine-stimulated dog. This compound shows reversible, K(+)-competitive binding to the enzyme. Because of its fluorescent properties, it is also proving useful in vitro as a probe of the structure and function of the (H+/K+)-ATPase.

Reversible inhibitors of the gastric (H+/K+)-ATPase. 4. Identification of an inhibitor with an intermediate duration of action.

3-Acyl-4-(arylamino)quinolines were previously identified as gastric (H+/K+)-ATPase inhibitors, and clinical efficacy has been demonstrated for compound 3 (SK&F 96067). In the present study the further structure-activity relationship of this series is developed. Only a limited range of substituents are tolerated on the N-aryl ring or the 6- and 7-positions of the quinoline, and although hydroxylated derivatives were identified possessing markedly greater affinity for the enzyme, none of these proved to have adequate potency after oral dosing. In contrast, the 8-position of the quinoline ring proved suitable for a wide variety of substituents, allowing modification of physicochemical properties while retaining primary activity. This led to the identification of compound 4 (SK&F 97574), which combines good oral potency with a somewhat longer duration of action than 3 (though much shorter than covalent inhibitors such as omeprazole). This compound was selected for further development and evaluation in man.

Reversible inhibitors of the gastric (H+/K+)-ATPase. 5. Substituted 2,4-diaminoquinazolines and thienopyrimidines.

Quinazolines bearing a secondary 4-(arylamino) substituent demonstrate an SAR for inhibition of the gastric (H+/K+)-ATPase different from the previously described 3-acylquinolines, suggesting that, although these compounds are also K(+)-competitive, they probably bind to the enzyme in a different orientation. Compounds bearing a tertiary 4-(arylamino) substituent, however, in particular 4-(N-methylarylamino), appear to possess an SAR quite similar to the 3-acylquinolines. We show that this arises from the effect of the N-methylation, which is to orientate the 4-(arylamino) substituent syn to C5, analogous to the 3-acylquinolines. Compounds possessing both a 4-(N-methylarylamino) substituent and a 2-(arylamino) substituent proved to be very potent, K(+)-competitive inhibitors of K(+)-stimulated ATPase activity with Ki values down to 12 nM. Some compounds also proved to be effective inhibitors of stimulated acid secretion in both the rat and dog when dosed intravenously. However, although a number of these demonstrated activity after oral administration in the dog, the level and variability precluded further evaluation.

Reversible inhibitors of the gastric (H+/K+)-ATPase. 3. 3-substituted-4-(phenylamino)quinolines.

Previously, gastric (H+/K+)-ATPase inhibitors such as 2 have been prepared as analogues of 1a on the presumption that the 3-carbethoxy substituent plays a key role in establishing the orientation of the 4-arylamino group. In this paper we explore further the contribution made to activity by the quinoline 3-substituent. We show that, for compounds bearing such a substituent, only a particular combination of properties provides high activity, both in vitro and as inhibitors of gastric acid secretion in vivo. The ability of the substituent to affect activity by restricting rotation about the Cquin-N bond through a combination of both a pi-electron withdrawal and hydrogen bonding is supported by the current study. However, high activity is only achieved if the effect of this group on the quinoline pK(a) is kept to a minimum. 3-Acyl substituents provide an optimum combination of electronic properties. From this series, compound 17c (SK&F 96067) was shown to be a potent inhibitor of histamine-stimulated gastric acid secretion after oral dosing in the Heidenhain pouch dog and was selected for further development and evaluation in man.

Reversible inhibitors of the gastric (H+/K+)-ATPase. 1. 1-Aryl-4-methylpyrrolo[3,2-c]quinolines as conformationally restrained analogues of 4-(arylamino)quinolines.

The 4-(arylamino)quinoline 4, previously described as an antiulcer compound, is shown to be an inhibitor of the gastric (H+/K+)-ATPase. It is postulated that 1-arylpyrrolo[3,2-c]quinolines 6 act as conformationally restrained analogues of 4. A series of derivatives of 6 has been prepared and shown to be potent inhibitors of the target enzyme in vitro. Substitution in the ortho position of the aryl ring is important for activity. Unsaturation in the 5-membered ring makes little difference, but introduction of heteroatoms into the same ring markedly reduces activity. In more detailed kinetic experiments, 15c and 4 both show reversible, K(+)-competitive binding to the enzyme, with submicromolar Ki values. The compounds appear to act at the lumenal face of the enzyme and to require protonation for activity. Several compounds in the series are shown to be potent inhibitors of pentagastrin-stimulated acid secretion in the rat.

2-[[(4-Amino-2-pyridyl)methyl]sulfinyl]benzimidazole H+/K+-ATPase inhibitors. The relationship between pyridine basicity, stability, and activity.

The benzimidazole sulfoxide class of antisecretory H+/K+-ATPase inhibitors need to possess high stability under neutral physiological conditions yet rearrange rapidly at low pH to the active sulfenamide 2. Since the initial reaction involves internal nucleophilic attack by the pyridine nitrogen, control of the pyridine pKa is critical. In this paper we show that by utilizing the powerful electron-donating effect of a 4-amino substituent on the pyridine, moderated by the electron-withdrawing effect of a 3- or 5-halogen substituent, a combination of high potency (as inhibitors of histamine-stimulated gastric acid secretion) and good stability under physiological conditions can be obtained. Furthermore, the role of the steric interaction between the 3/5-substituents and the 4-substituent in modifying the electron-donating ability of the 4-amino group is exemplified, and additional factors affecting stability are identified. One compound, in particular, 2-[[(3-chloro-4-morpholino-2- pyridyl)methyl]sulfinyl]-5-methoxy-(1H)-benzimidazole (3a, SK&F 95601), was chosen for further development and evaluation in man.

The specificity of omeprazole as an (H+ + K+)-ATPase inhibitor depends upon the means of its activation.

Omeprazole (OME) is a novel acid secretion inhibitor, believed to act directly on the gastric proton pump, the (H+ + K+)-ATPase. Inhibition of ATPase activity is associated with an incorporation of [14C]OME into gastric vesicles containing the (H+ + K+)-ATPase, and both processes are greatly enhanced if the OME is exposed to acidic pH. This, and other evidence, suggests that the acidic environment of the (H+ + K+)-ATPase generates from OME a reactive intermediate which covalently inhibits the pump. We have compared the means by which the OME was acid-activated with the specificity of inhibition (amount of incorporation of omeprazole required to produce 100% inhibition of K+-stimulated ATPase activity). The stoichiometry of incorporation has been related to the number of detectable catalytic phosphorylation sites in each preparation (an index of the number of functional pumps). In lyophilised gastric vesicles, where the membrane barriers separating the cytoplasmic and luminal faces of the enzyme are substantially destroyed, incubation with OME at pH 6.1 produced a progressive inhibition and incorporation over 120 min. Complete inhibition of K+-ATPase required 13 +/- 3 (SEM; N = 4) moles of OME incorporation per phosphorylation site. In intact gastric vesicles, under conditions shown independently to result in proton pumping and the acidification of the vesicle interior (150 mM KCl, 9 microM valinomycin, 2 mM Mg-ATP pH 7.0), inhibition and incorporation occurred more rapidly (15 min). Complete inhibition of K+-ATPase required only 1.8 +/- 0.15 (SEM; N = 3) moles of OME per phosphorylation site.(ABSTRACT TRUNCATED AT 250 WORDS)

SCH 28080 is a lumenally acting, K+-site inhibitor of the gastric (H+ + K+)-ATPase.

SCH 28080 (2-methyl-8-(phenylmethoxy)imidazo[1,2-a] pyridine-3-acetonitrile) is an effective inhibitor of acid secretion in vivo and is a reversible, K+-competitive inhibitor of the gastric (H+ + K+)-ATPase in vitro. The actions of SCH 28080 have been studied on gastric vesicle preparations containing the (H+ + K+)-ATPase. At pH 7, inhibition was competitive with respect to K+ for both ATPase (Ki = 24 nM) and pNPPase (Ki = 275 nM) activities. A close analogue of SCH 28080 (methylated in the 1-N position), that was not expected to cross membranes freely, inhibited ATPase and pNPPase activity less effectively in intact vesicle preparations, where the lumenal (extracellular) face of the membrane was not directly accessible. This suggested that SCH 28080 inhibited both enzyme activities at a lumenal site on the enzyme. Being a protonatable weak base (pKa = 5.6), SCH 28080 would be expected to accumulate on the lumenal, acidic side of the parietal cell membrane in its protonated form. The potency of SCH 28080, relative to that of the "non-protonatable" analogue, increased at low pH, commensurate with the proportion of SCH 28080 in the protonated form. Thus the accumulating protonated form was the active inhibitory species. SCH 28080 (50 nM) blocked the rapid, K+-stimulated dephosphorylation of the catalytic phosphoenzyme intermediate of the (H+ + K+)-ATPase at room temperature. At 4 degrees, higher concentrations of the inhibitor were required, suggesting that the rate of inhibitor binding was slow at low temperatures.

By 1023/SK&F 96022: biochemistry of a novel (H+ + K+)-ATPase inhibitor.

The mechanism by which the substituted benzimidazole sulphoxide BY 1023/SK&F 96022 inhibited the (H+ + K+)-ATPase, the enzyme responsible for hydrogen ion secretion in the stomach, was studied in a variety of in vitro preparations. In gastric preparations that were capable of active hydrogen ion transport with consequent lumenal acidification, BY 1023/SK&F 96022 inhibited with high potency and in a time-dependent manner consistent with the acid-induced conversion of the parent benzimidazole sulphoxide to a covalent inhibitor (cyclic sulphenamide). The following IC50 values were obtained for the inhibition of aminopyrine accumulation: intact gastric glands stimulated with 1 mM dibutyryl cAMP, 1.0 microM; permeabilized gastric glands stimulated with 5 mM ATP, 0.42 microM; intact gastric vesicles stimulated with 150 mM KCl, 9 microM valinomycin and 2 mM MgATP, 3.5 microM. In a preparation that could not generate pH gradients, lyophilized gastric vesicles at pH 7.4, BY 1023/SK&F 96022 inhibited K(+)-stimulated ATPase activity with relatively low potency, 70 microM, indicating its good chemical stability at neutral pH. As assessed by ATPase inhibition, this stability was three times greater than that of omeprazole. Inhibition by BY 1023/SK&F 96022 was not reversed by dilution in either permeabilized gastric glands or intact gastric vesicles. Inhibition could, however, be completely reversed by subsequent incubation with 20 mM beta-mercaptoethanol (intact gastric glands) or 100 mM dithiothreitol (intact gastric vesicles) suggesting a disulphide link between inhibitor and enzyme. The concentration of glutathione needed to protect against inhibition by BY 1023/SK&F 96022 was 10,000 times higher in intact, compared with lyophilized, gastric vesicles indicating an interaction with the lumenal (extra-cellular) face of the (H+ + K+)-ATPase. BY 1023/SK&F 96022 and omeprazole were also found to inhibit acidification in purified kidney lysosomes with IC50 values of 194 and 75 microM, respectively. Protection by 10 microM glutathione suggested that this did not result from intralysosomal activation of these inhibitors. Thus, BY 1023/SK&F 96022 has the combined properties of good chemical stability at neutral pH and effective conversion to the cyclic sulphenamide at acidic pH. In this way the activation to the cyclic sulphenamide may be optimally restricted to the parietal cell canaliculus.

SK&F 96067 is a reversible, lumenally acting inhibitor of the gastric (H+ + K+)-ATPase.

SK&F 96067 [3-butyryl-4-(2-methylphenylamino)-8-methoxyquinoline] has been identified, from a novel class of 4-aminoquinolines, as a reversible inhibitor of the gastric (H+ + K+)-ATPase. This compound has been studied in gastric membrane vesicle preparations enriched in the (H+ + K+)-ATPase. At pH 7.0, SK&F 96067 inhibited K(+)-stimulated ATPase activity competitively with respect to the activating cation K+, with a Ki value of 0.39 +/- 0.05 microM. Under comparable conditions, SK&F 96067 was 32 times more potent as an inhibitor of the gastric (H+ + K+)-ATPase relative to the closely related (Na+ + K+)-ATPase. Studies in intact gastric vesicles showed that SK&F 96067 also inhibited hydrogen ion transport. Using the initial rate of acridine orange quenching as the index of acidification, an IC50 of 0.84 +/- 0.24 microM was observed. Steady state acidification, as measured by aminopyrine accumulation, was inhibited with greater potency (IC50 = 0.06 +/- 0.01 microM) consistent with the accumulation of this inhibitor into the intravesicular acidic space to a site of action on the inside (lumenal) face of the enzyme. Inhibition of ATPase activity in the presence of both SK&F 96067 and the K(+)-competitive (H+ + K+)-ATPase inhibitor, SCH 28080, indicated that their binding was mutually exclusive, consistent with SK&F 96067 acting at the same lumenal binding site as does SCH 28080. The steady-state inhibition kinetics of SK&F 96067 against K(+)-stimulated ATPase activity were followed as a function of pH. At pH 6.6 and 7.0 the inhibition was competitive with respect to the activating cation K+. At pH 7.5 and 8.1 a mixed pattern of inhibition was detected. Thus, at alkaline pH values, the binding of SK&F 96067 and K+ were no longer mutually exclusive. The potency of SK&F 96067 decreased as pH rose, consistent with the protonated form of the inhibitor being the preferred inhibitory species. A kinetic model is discussed, in which, at acidic pH, the protonated form of SK&F 96067 binds to the enzyme competitively with respect to K+, whereas, at alkaline pH, the neutral form of SK&F 96067 can bind simultaneously with K+.

Studies on the mechanism of action of omeprazole.

The effects of omeprazole on preparations of pig gastric (H+ + K+)-ATPase have been studied. Omeprazole was found to inhibit the (H+ + K+)-ATPase activity in a time-dependent manner. Inhibition was more pronounced at pH 6.1 compared with pH 7.4 and decreased as the concentration of (H+ + K+)-ATPase preparation increased. The potency of omeprazole was therefore highly dependent upon the conditions used. When pre- incubated with (H+ + K+)-ATPase preparation (30 micrograms protein/ml) for 30 min at 37 degrees and pH 6.1, omeprazole inhibited the (H+ + K+)-ATPase activity with an IC50 of 3.9 microM. This inhibition was shown to be irreversible in nature. Whilst omeprazole itself was not very potent as an inhibitor of the (H+ + K+)-ATPase activity at pH 7.4 (IC50 = 36 microM), transient acidification of omeprazole resulted in the formation of a compound(s) which produced marked inhibition at this pH (IC50 = 5.2 microM). The effects of omeprazole in the absence of acidification may have resulted from the rate-limiting formation of this compound. Radiolabelled omeprazole was shown to incorporate into the (H+ + K+)-ATPase preparation in a time-dependent and pH-dependent manner. Omeprazole, radiolabelled in three separate positions (the sulphur atom and the two adjacent carbon atoms), incorporated with equivalent time courses suggesting that the incorporation did not involve a fragmentation of the omeprazole molecule. Under conditions shown to produce a 50% inhibition of (H+ + K+)-ATPase activity, [14C] omeprazole had incorporated to a level of 4-5 nmoles/mg protein. Incorporation continued beyond the point required to produce 100% inhibition of (H+ + K+)-ATPase activity and reached 30 nmoles/mg protein after 5 hr. Prior acidification of the omeprazole resulted in a more rapid initial rate of incorporation although the final level of incorporation was lower than for omeprazole. Omeprazole was also shown to interact with the (Na+ + K+)-ATPase from dog kidney. Omeprazole inhibited the (Na+ + K+)-ATPase activity (IC50 = 186 microM). Acid-degraded omeprazole inhibited the (Na+ + K+)-ATPase activity with greater potency (IC50 = 19 microM) and was also shown to incorporate into this enzyme preparation.