Direct measurement of extracellular proton flux from isolated gastric glands.
We used the microphysiometer, a sensitive extracellular pH sensor, to resolve luminal (or apical) H+ secretion and basolateral release of OH- as well as liberation of acidic metabolites in rabbit gastric glands. Stimulation of glands via the adenosine 3',5'-cyclic monophosphate pathway produced a biphasic change in the extracellular acidification rate (EAR): after an initial transient decrease below the unstimulated baseline (-40.9 +/- 3.4%), the EAR increased to a steady-state maximal plateau (+98.1 +/- 5.3%) within 30 min (n = 37). We interpret the biphasic EAR profile as an initial excess of basolaterally released OH- followed by delayed luminal efflux of simultaneously produced H+. The elevated EAR at steady state reflected liberation of metabolic acid attributed to H(+)-K(+)-ATPase enzymatic activity. The presence of H2-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid prevented OH- release and reduced steady-state EAR. Basolateral OH- release and steady-state EAR were also inhibited by the H(+)-K(+)-ATPase inactivators omeprazole and SCH-28080. Inhibition of Na+/H+ exchange did not reduce steady-state EAR and did not affect apical H+ production, as judged by the accumulation of the weak base aminopyrine. Sodium thiocyanate (1 mM), which short circuits intraluminal H+ accumulation, blocked OH- release, demonstrating its dependence on H(+)-OH- separation at the apical membrane. A computerized model was developed to illustrate how the observed biphasic EAR profile would result from a delayed luminal efflux of H+ due to transitory intraluminal compartmentalization.
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
A
A
Thibodeau
R C
RC
Kuo
J M
JM
Crothers
X
X
Yao
J C
JC
Owicki
J G
JG
Forte
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D017920:Antiporters / Q000037:antagonists & inhibitors; D027963:Chloride-Bicarbonate Antiporters; D005110:Extracellular Space / Q000378:metabolism*; D005260:Female; D005744:Gastric Acid / Q000378:metabolism; D005753:Gastric Mucosa / Q000378:metabolism*; D017506:H(+)-K(+)-Exchanging ATPase / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D011522:Protons; D011817:Rabbits; D013268:Stimulation, Chemical; D013861:Thiocyanates / Q000494:pharmacology
D017920:Antiporters; D027963:Chloride-Bicarbonate Antiporters; D011522:Protons; D013861:Thiocyanates; C024553:sodium thiocyanate; D017506:H(+)-K(+)-Exchanging ATPase; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid