Abstract
When gastric microsomes were purified from resting and stimulated rabbit mucosae, they were found to be generally similar in (H+ + K+)-ATPase activity, peptide composition in single-dimension sodium dodecyl sulfate-gel electrophoresis, and in size. In the stimulated vesicles, optimal proton transport activity was found at pH 7.4, 20-50 mM KCl, and 1 mM ATP-Mg. However, in the case of resting vesicles, the presence of valinomycin and an inward Cl-gradient was also necessary for Mg-ATP-dependent proton transport. Measurement of K+ and Cl-diffusion potentials using 3,3-dipropylthiadicarboxocyanine iodide as a potential sensitive dye showed that both resting and stimulated vesicles developed K+ gradient-dependent potentials in the presence of an impermeant anion, but that Cl- gradient-dependent potentials were observed only in the stimulated preparation. 86Rb+ self-exchange was found in both types of vesicles, but Cl- self-exchange was confined to vesicles derived from stimulated mucosae. Putative inhibitors of anion conductance such as furosemide and anthracene 9-carboxylic acid blocked proton transport, Cl- conductance, 36Cl- uptake, and Cl- exchange. The inhibition of proton transport was overcome by valinomycin. ATPase activity in the presence of nigericin, an H+:K+ exchanger, was unaffected by these inhibitors. K+ conductance, Rb+ uptake, and Rb+ exchange were insensitive to these inhibitors. Thus, activation of acid secretion by the stimulated parietal cell appears to involve at least the appearance of a discrete Cl- conductance in the pump-associated membrane.
Highlights
From the Center for Ulcer Research and Education, Veterans Administration WeLstos Angeles Hospital Center, Wadsworth Division, and Department of Medicine, Universityof California, Los Angeles, Los Angeles, California90073
Inhibitors of the (H' K')-ATPase such as vanadate and omeprazole inhibstimulated gastricmucosae were isolated at the same density ited H' transport in this fraction
There is no obvious difference in the morphology of the vesicles derived from resting as opposed to stimulateadnimalEs.lectron micrographs were kindly prepared by Dr.Herbert Helander
Summary
Activation of proton transport by CO, ad- the pump This limitation could be overcome in part by the dition to turtle bladder in uitro appears to depend on fusion addition of the ionophore valinomycin or transiently by preof vesicles containing a n ATP-driven H+pump to the luminalequilibration in either KC1 or K,SO, [11, 13]. The assaywas performed in a 1-ml reaction mixture activate the pump The elements constituting this permeabcoiln-taining 0.3 M sucrose, 8 mM Tris, 4 mM Pipes, pH 7.4, and 20 mM ity pathway couldbeacoupled electroneutralsymportor KCI. The reaction was terminated by ulated mucosa seems to be the appearance of a furosemide- adding a charcoal slurry in 1% (v/v) perchloric acid (0 "C) toremove inhibitable C1- conductance, absent in membranes from rest- ATP and any added inhibitors. The homogenates were centrifuged a t 13,000 X g for 15 min
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