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Disulphonic stilbenes are effective inhibitors of an anion exchanger which is present in the plasma membranes of many cells (Cabantchik et al. 1978). In the present study, the effects of 4,4′‐diisothiocyano‐2,2′‐disulphonic stilbene acid (DIDS) on the transport activity of the hydrochloric acid pump isolated from pig stomach (H,K‐ATPase, EC 3.6.1.36) were tested. Half‐maximal inhibition of proton transport carried out by the H,K‐ATPase in the isolated vesicles was observed at micromolar concentrations of DIDS. The effects of DIDS on the adenosine‐triphosphatase and p‐nitrophenyl‐phosphatase activities of isolated H,K‐ATPase were also studied and compared with those of the kinetically and structurally related Na,K‐ATPase (EC 3.6.1.37). Half‐maximal inhibition of the enzymatic activities of both enzymes were observed in the micromolar range of DIDS. The lipid bilayer of the gastric vesicle membrane is highly asymmetric and the original cytosolic side is facing the outside of the vesicle. Since DIDS does not readily cross the membrane, it is most likely that DIDS exerts its inhibitory effects by modifying the transport ATPases on their cytosolic sides.

Hydrolysis of adenosine 5'-triphosphate (ATP) and p-nitrophenyl phosphate by the hydrogen ion-transporting potassium-stimulated adenosine triphosphatase (H,K-ATPase) was investigated. Hydrolysis of ATP was studied at pH 7.4 in vesicles treated with the ionophore nigericin. The kinetic analysis showed negative cooperativity with one high affinity (Km1 = 3 microM) and one low affinity (Km2 = 208 microM) site for ATP. The rate of hydrolysis decreased at 2000 microM ATP indicating a third site for ATP. When the pH was decreased to 6.5 the experimental results followed Michaelis-Menten enzyme kinetics with one low affinity site (Km = 116 microM). Higher concentrations than 750 microM ATP were inhibitory. Proton transport was measured as accumulation of acridine orange in vesicles equilibrated with 150 mM KCl. The transport at various concentrations of ATP in the pH interval from 6.0 to 8.0 correlated well with the Hill equation with a Hill coefficient between 1.5-1.9. The concentration of ATP resulting in half-maximal transport rate (S0.5) increased from 5 microM at pH 6.0 to 420 microM at pH 8.0. At acidic pH the rate of proton transport decreased at 1000 microM ATP. The K+-stimulated p-nitrophenylphosphatase (pNPPase) activity resulted in a Hill coefficient close to 2 indicating cooperative binding of substrate. The pNPPase was noncompetitively inhibited by ATP and ADP; half-maximal inhibition was obtained at 2 and 100 microM, respectively. Phospholipase C-treated vesicles lost 80% of the pNPPase activity, but the Hill coefficient did not change. These kinetic results are used for a further development of the reaction scheme of the H,K-ATPase.

The transverse distribution of the phospholipids in vesicular H+-translocating membranes prepared from pig gastric mucosa was investigated with the aid of phospholipase C, sphingomyelinase, and trinitrobenzenesulfonic acid. The major part (80-90%) of the phosphatidylcholine and the phosphatidylethanolamine, 60% of the phosphatidylserine, and 45% of the sphingomyelin was located on the external, cytoplasmic side of the vesicle membranes. After treatment with phospholipase C the vesicles still behaved as osmometers and appeared as closed vesicles on the electron micrographs. 31P NMR indicated that the phospholipids in untreated vesicles as well as the unhydrolyzed phospholipids in phospholipase C-treated vesicles were arranged in lamellar structures. The 31P NMR spectrum of untreated vesicles to which Pr3+ ions had been added supported the conclusion that the major part of the membrane phospholipids was located on the external surface of the vesicles. A small fraction of the lipids, 3.6 mol %, was found to consist of glycosphingolipids which occurred at a concentration of 52 nmol/mg of protein.

The effects of some inhibitors of acid secretion were tested on isolated, purified pig parietal cells. The cells were stimulated with 10-4 M histamine, 10-5 M carbachol, or 10-7 M pentagastrin. The H, K-ATPase inhibitors SCH 28080 and omeprazole inhibited both the basal and secretagogue-stimulated acid production, as measured by aminopyrine accumulation, irrespective of the type of stimulator used. The IC50 value was 3-5 ± 10-9 M for SCH 28080 and 1-3 ± 10-8 M for omeprazole. Ranitidine inhibited the histamine-stimulated but not the basal acid production. The IC50 value was 2 ± 10-5 M. Stimulation of acid production with carbachol was blocked by pirenzepine, with an IC50 of 6 ± 10-7 M. Pirenzepine (10-5 M) specifically blocked the carbachol-stimulated increase in cytosolic free Ca2+ in fura-2-loaded cells but not the increase in cytosolic free Ca2+ induced by histamine or pentagastrin. Ranitidine (10-4 M) prevented the histamine-induced increase in Ca2+ and was also the only one of the four inhibitors which prevented the histamine-stimulated cAMP formation. SCH 28080 (10-5 M) significantly potentiated the histamine-stimulated increase in cytosolic free Ca2+ and the formation of cAMP, whereas omeprazole (10-5 M) was without effect.