Search

Pantoprazole is a pyridinyl-2-methylenesulfinyl-2-benzimidazole derivative. This compound inhibits the vesicular gastric H+/K(+)-ATPase (cytoplasmic side out) under acid transporting conditions by accumulating in the acid space generated by the pump. Pantoprazole is then converted in an acid-catalysed reaction to a cationic sulfenamide and reacts with cysteines available in or from the acidic extracytoplasmic space. This compound binds to the hog gastric H+/K(+)-ATPase with a stoichiometry of 3 nmol per mg protein, resulting in 94% inhibition of ATPase activity. Tryptic cleavage of the intact vesicles which had been reacted with [14C]pantoprazole at a 1 to 4 trypsin to protein ratio removed most of the cytoplasmic domain leaving the pairs of membrane spanning segments and their connecting extracytoplasmic loops intact. The peptides remaining in the membrane were dissolved in SDS and available cysteine residues labelled with fluorescein-5-maleimide. The peptides were separated on Tricine gradient gels, transferred to PVDF membranes and identified by fluorescence and radioactivity. From N-terminal sequence, fluorescence and molecular mass, it is concluded that pantoprazole is able to label both Cys-813 and Cys-822. These cysteines are predicted to be located in the extracytoplasmic loop connecting membrane segments 5 and 6 and in membrane segment 6. The major cytoplasmic tryptic cleavage site at this location moved from position 776 in unmodified enzyme to positions 784 and 792 following pantoprazole labelling, showing that the configuration of this region changed with pantoprazole labelling. A similar result was obtained by reduction of the enzyme with dithiothreitol. Covalent binding of the cationic sulfenamide to this region of the enzyme is able to block the conformation necessary for phosphorylation of the enzyme by ATP, accounting for its inhibitory effect on acid secretion.

Initiation of acid secretion in the gastric mucosa is accompanied by a morphological transformation in which the acid pump, the H+/K(+)-ATPase, translocates from a cytoplasmic vesicular location to the secretory surface lining the canaliculi. Associated with the morphological changes, activation of K+ and Cl- pathways are necessary to supply K+ to the extracytoplasmic face of the pump. Although the pump in the secretory membrane is known to secrete acid, it is not known whether activation of the KCl pathway occurs in the tubulovesicular membrane prior to the formation of the canaliculus, or when the pump is in the secretory membrane. The cellular site of activation of acid secretion in the rabbit gastric parietal cell was investigated using the covalent binding of [3H]omeprazole as a probe of acid secretion in rabbit gastric glands that were undergoing stimulation in vitro. This compound depends on an acidic environment for activation and covalent binding to the H+/K(+)-ATPase. Electron microscopic autoradiography showed that activation of the enzyme occurred only when it was present in the canalicular membrane and not when it was present in the cytoplasmic tubulovesicular membrane. Hence there is likely to be a physical separation of K+ and/or Cl- pathways from the ATPase in the resting cell, and stimulation of acid secretion is dependent on colocalization of these pathways in the canalicular membrane.

It cannot be predicted from hydropathy analysis whether the C-terminal end of the alpha subunit of the gastric H,K-ATPase is cytoplasmic or extracytoplasmic. The sideness of the C-terminal amino acids was determined by taking advantage of the two C-terminal tyrosines in the primary sequence of the enzyme. Intact, cytoplasmic side out vesicles derived from hog gastric mucosa or detergent solubilized vesicles were iodinated by the lactoperoxidase method and then the C-terminal amino acids hydrolyzed by carboxypeptidase Y. The alpha and beta subunits were separated by SDS gel electrophoresis. The level of iodination of the alpha subunit following solubilization was about three fold greater than when intact vesicles were iodinated, and the beta subunit was iodinated only when solubilized enzyme was used. Carboxypeptidase Y removed 28 +/- 4% of the radioactivity from the alpha subunit iodinated in intact vesicles. These data are consistent with a cytoplasmic location of the C-terminal amino acids of the alpha subunit and with a mostly extracytoplasmic location of the amino acids of the beta subunit.

Omeprazole was found to inhibit the (H + + K + )-ATPase activity in isolated gastric vesicles only when acid was accumulated in the vesicle lumen. The ATPase activity was time- and dose-dependently inhibited in the presence of K + and valinomycin. Under conditions in which no pH-gradient was generated, i.e., in the presence of K + alone or NH 4 + , no effect of omeprazole was found. The degree of inhibition was directly correlated to the amount of inhibitor bound to the preparation. A stoichiometry of 2 mol radiolabelled inhibitor bound per mol phosphoenzyme was found on total inhibition of the K + plus valinomycin-stimulated activity. This inhibitory action of omeprazole on the ATPase activity could be fully reversed by addition of β-mercaptoethanol. The inhibition of the proton transport in the (H + + K + )-ATPase-containing vesicles by omeprazole was also strictly correlated to the amount of bound inhibitor. The stoichiometry of binding at total inhibition of this reaction was found to be 1.4 mol per mol phosphoenzyme. The K + -stimulated p -nitrophenylphosphatase activity was inhibited in parallel with the ATPase activity, whereas the phosphoenzyme levels were affected to a lesser extent by omeprazole. Gel electrophoresis of an omeprazole-inhibited vesicle preparation showed that the radiolabel was mainly found at 94 kDa, the molecular weight of the (H + + K + )-ATPase catalytic subunit(s).

The absorbance change of the weak base dye probe, Acridine orange, was used to monitor alterations of pH gradients across renal brush border membrane vesicles. The presence of Na+/H+ or Li+/H+ exchange was demonstrated by diluting Na2SO4 or Li2SO4 loaded vesicles into Na+- or Li+-free solutions, which caused dye uptake. About 20% of the uptake was abolished by lipid permeable cations such as valinomycin-K+ or tetraphenylphosphonium, indicating perhaps the presence of a finite Na+ conductance smaller than electroneutral Na+/H+ exchange. The protonophore tetrachlorosalicylanilide raised the rate of dye uptake under these conditions, hence the presence of an Na+ conductance greater than the H+ conductance was suggested. K+ gradients also induced changes of pH, at about 10% of the Na+ or Li+ rate. Partial inhibition (21%) was seen with 0.1 mM amiloride indicating that K+ was a low affinity substrate for the Na+/H+ exchange. Acceleration both by tetrachlorosalicylanilide (2-fold) and valinomycin (4-fold) suggested the presence of 2 classes of vesicles, those with high and those with low K+ conductance. The larger magnitude of the valinomycin dependent signal suggested that 75% of the vesicles had a low K+ conductance. Inward Cl− gradients also induced acidification, partially inhibited by the presence of tetraphenylphosphonium, and accelerated by tetrachlorosalicylanilide. Thus both a Cl− conductance greater than the H+ conductance and a Cl−/OH− exchange were present. The rate of Na+/H+ exchange was amiloride sensitive with a pH optimum of 6.5 and an apparent Km for Na+ or Li+ of about 10 mM and an EA of 14.3 kcal per mol. A 61-fold Na2SO4 gradient resulted in a pH gradient of 1.64 units which increased to 1.8 with gramicidin. An equivalent NaCl gradient gave a much lower ΔpH even in the presence of gramicidin showing that the H+ and Cl− pathways could alter the effects of the Na+/H+ exchange.

ATP-induced transport by fractions of frog gastric microsomes prepared either by density gradient centrifugation or by free flow electrophoresis were K+ dependent and hence considered due to a K+-activated ATPase. Significant activity of this enzyme was, however, only found in the anodic peak of the free flow electrophoretic separation, which in addition to separating transporting from non-transporting particles, also separated membranes containing a phosphorylatable peptide ( M r = 105 000 ) region as the major peptide on SDS-polyacrylamide gel electrophoresis from those containing a peptide ( M r = 44 000 ) on SDS-polyacrylamide gel electrophoresis. H+ uptake, measured either by acridine orange or 3,3′-diethyloxadicarbocyanine + tetrachlorosalicylanilide absorbance changes was dependent on K+ intravesicularly. Using 86Rb+, active extrusion of the cation followed ATP addition. SCN−, an inhibitor of acid secretion did not affect the latter, but blocked signals due to H+ uptake, in contrast to mammalian preparations.

Highly purified gastric membranes were extracted by ionic or non-ionic detergents. Further fractionation was carried out on acrylamide gel electrophoresis. Some defined bands, incorporated into artificial bilayer membranes, produced discrete conductance changes characteristics of channel activity. A low molecular weight peak, purified through two cycles of electrophoresis was characterized as an anion-selective, positively voltage dependent channel, and a model to account for the action of the transport ATPase incoporating this channel concept is suggested.

A highly purified membrane fraction was derived from hog gastric mucosa by a combination of differential and density gradient centrifugation and free flow electrophoresis. This final fraction was 35-fold enriched with respect to cation activated ouabain-insensitive ATPase. Antibody against this fraction was shown to be bound to the luminal surface of the gastric glands. The addition of ATP to this fraction or the density gradient fraction resulted in H + uptake into an osmotically sensitive space. The apparent K m for ATP was 1.7 · 10 −4 M in the absence of a K + gradient similar to that found for ATPase activity. The reaction is specific for ATP and requires cation in the sequence K + > Rb + > Cs + > Na + > Li + and is inhibited by ATPase inhibitors such as N , N ′-dicylclohexylcarbodiimide. Maximal H + uptake occurs with an outward K + gradient but the minimal apparent K A is found in the absence of a K + gradient. The pH optimum for H + uptake is between 5.8 and 6.2 which corresponds to the pH range for phosphorylation of the enzyme, but is considerably less than the pH maximum of the K + dependent dephosphorylation. In the presence of an inward K − gradient, protonophores such as tetrachlorsalicylanilide only partially abolish the H + gradient but valinomycin dissipates 75% of the gradient, and nigericin abolishes the gradient. The vesicles therefore have a low K + conductance but a measurable H + conductance, hence a K + gradient can produce an H + gradient in the presence of valinomycin. The uptake and spontaneous leak of H + are temperature sensitive skin with a similar transition temperature. Ultraviolet irradiation inactivates ATPase and proton transport at the same rate, approximately at twice the rate of p -nitrophenylphosphatase inactivation. It is concluded that H + uptake by these vesicles is probably due to a dimeric (H + + K + )-ATPase and is probably non-electrogenic.

Transport activity of the hog gastric (H+ + K+)-ATPase system was measured either as the formation of a proton gradient using the dye probe acridine orange or as the formation of a proton diffusion potential using the cyanine dye 3,3′-diethyloxdicarbocyanine iodide in the presence of the protonophore tetrachlorosalicylanilide. The development of these gradients has been compared in K+ media in the presence of either Cl− or SO42− as the anionic species. This comparison of proton diffusion potential formation to proton gradient formation has been used to demonstrate that a Cl− conductance in this vesicular system results from limited enzymic digestion with either trypsin or α-chymotrypsin and from the ageing process itself. The possible significance of this finding is discussed.

The mechanism of gastric antisecretory action for trifluoperazine, verapamil and 8-(N,N- diethylamino)octyl -3,4,5- trimethoxybenzoate (TMB-8) has been studied utilizing isolated hog gastric membranes enriched with ( H + + K + )- ATPase . The drugs inhibited the gastric ATPase due to their apparent competition with K+ for the luminal high-affinity K+-site of the ATPase. The dose to inhibit 50% (ID50) of the ATPase in the membranes rendered freely permeable to K+ (20mM) was 50 μM for trifluoperazine and 1.5 mM for verapamil and TMB-8. In intact hog gastric membranes which develop a pH gradient in the presence of valinomycin, ATP and KCl, however, trifuoperazine at 4 μM, verapamil and TMB-8 at 15 μM inhibited 40 and 30% of the valinomycin-stimulated ATPase activity, respectively, and also blocked the ionophore-dependent intravesicular acidification as measured by aminopyrine accumulation. The enhanced potency of the drugs to inhibit the ATPase in the intact membrane vesicles may be attributed to the accumulation of the drugs as a weak base within the vesicles, where the luminal K+-site of the ATPase is accessible. Calmodulin and Ca2+ had no effect on the extent of H+-accumulation as measured by aminopyrine accumulation in the membrane vesicles which were prepared in the presence of 1 mM EGTA. Since the drugs showed similar potency in interfering with H+ movements either in the membrane vesicles or isolated rabbit gastric glands stimulated by dibutylryl cAMP, it is reasonable to suggest the inhibitory effect of the drugs on ( H + + K + )- ATPase as a primary cause for such interferences in both cases. A trifluoperazine analog and other lipophilic amine drugs similarly inhibited ( H + + K + - ATPase and H+ accumulation in the membrane vesicles or in the glands. We have concluded that a tertiary amine, the only common functional group among these drugs, is primarily responsible for their ability to interact with the high-affinity K+ site of the gastric ATPase.

A micropuncture technique is described for isolated surface epithelial cells and oxyntic cells of Necturus gastric mucosa, and for intact isolated tubules from the same tissue. The potential obtained is similar to that found for the cells in the intact tissue, whereas the measured membrane resistance is about 100 times higher. This is explained by cell-cell coupling in the intact mucosa, and is confirmed by direct two-electrode experiments in the intact tissue or isolated tubule. The calculated tissue resistance is about twice as high as the measured resistance after allowance is made for glandular infolding, hence the possibility remains that there is a low resistance shunt across the tissue.

A vesicular preparation derived from acid-secreting cells of dog gastric mucosa was studied in a Cary 60 modified for simultaneous CD and absorbance measurements. Significant changes in the spectrum were obtained on addition of MgATP, but not MgAMP, which were only partly due to changes in scattering, etc . Thus ATP may induce conformational changes in the protein constituents of the vesicle membrane.

An SCN−-inhibited, HCO3−-stimulated ATPase (ATP phosphohydrolase, EC 3.1.6.3) has been shown to be localized in the oxyntic cells of Necturus gastric mucosa. Carbonic anhydrase is also associated with this cell type. High activity of the ATPase was found in a pure smooth microsomal fraction of a homogenate of these oxyntic cells. A method is described for solubilization of the enzyme resulting in a marked increase in the sensitivity to HCO3− and SCN−. High activity of the enzyme is found in tissues of Necturus which transport H+ and/or C1− and which have a high carbonic anhydrase content.

Thiocyanate in addition to its well-known action in inhibiting HCl secretion by the amphibian gastric mucosa has been shown to inhibit 42K flux and the potential difference change due to K+ or Rb+ concentration changes in the bathing solutions. Cs+ does not substitute for K+ in the stomach. Other secretory inhibitors have similar effects. These data may be interpreted by regarding the mucosa as consisting of two parallel circuits residing in the parietal and surface cells, with the parietal cell being responsible for the major fraction of the 42K movement through the mucosa, which is associated with HCl secretion, and the surface cell possessing the K+ perm-selective membrane.