Proc. NatL Acad. Sci. USA Vol. 78, No. 9, pp. 5908-5912, September 1981 Physiological Sciences Properties of the gastric proton pump in unstimulated permeable gastric glands (permeabilization/digitonin/parietal cell/ATP/ionophore effects) D. H. MALINOWSKA*, H. R. KOELZ*t, S. J. HERSEYt, AND G. SACHS*§ *Laboratory of Membrane Biology, University of Alabama in Birmingham, Birmingham, Alabama 35294; and tDepartment of Physiology, Emory University, Atlanta, Georgia 30322 Communicated byJared M. Diamond, June 15, 1981 ABSTRACT Itwas found that digitonin-permeabilized resting and endoplasmic reticulum are relatively unaffected (15) be- gastric glands retained considerable acid-secretory ability. Ofi- cause they contain little cholesterol compared to the plasma gomycin abolished this, and ATP was able to bypass this inhibition membrane (16). In the present work, digitonin has been suc- and restore acid secretion. Moreover, the effect ofanoxia was also cessfully used to permeabilize parietal cells of isolated resting bypassed by ATP in these preparations. As in intact glands, acid gastric glands of rabbits without affecting the acid-secretory secretion was K+ dependent, and the concentration for half-max- membrane as in shocked glands. Moreover, mitochondrial dam- imal effect was 18.5 ± 1.76 mM in Na+-free solutions, a value age was minimal by morphological and functional criteria. The similar to that found for resting intact glands. The slight inhibition of acid secretion were investi- ofATP-dependent secretion by either valinomycin or 2,4-dinitro- ATP, 02, and K+ dependence phenol, but total inhibition by a combination of the ionophores, gated, and it has been possible to examine the electrogenicity is interpreted to mean that, in resting gastric glands, the in situ of the H' pump and the nature ofthe associated KC1 pathway. proton pump is electroneutral and the KCI pathway supplying K+ The results obtained establish digitonin permeabilized gastric to the luminal face of the pump is probably electroneutral. glands as a useful model for studying some of the properties of acid secretion by the parietal cell. Isolation ofpurified gastric membrane vesicles made it possible to investigate and model the mechanism of H+ transport by the MATERIALS AND METHODS parietal cell. Historically, the K+ dependence ofacid secretion Rabbit gastric glands were prepared as described (17), and the was recognized in the frog gastric mucosa in vitro (1), and the accumulation ratio of the weak base [14C]aminopyrine was used presence of a K+-activated ATPase was demonstrated (2). The to monitor acid-secretory capacity (18). The glands were washed ability of the ATPase to transport H+ was shown in dog micro- three times at room temperature in Ca2+-free, high-K+ medium somes (3), and the mechanism was revealed to involve H+-for- containing: K+, 100 mM; Na+, 31 mM; Mg2e, 1.2 mM; Cl-, 120 K+ exchange in purified hog gastric vesicles (4). The H+-pump mM; SO2-, 1.2 mM; HPO2-, 5.0 mM; H2PO , 1.0 mM; Hepes, K+-leak hypothesis was put forward to explain these observa- 20 mM; Tris, 9.7 mM; phenol red, 10 ,ug/ml; glucose, 2 mg/ tions and has been discussed in several articles (4-6). However, ml; rabbit serum albumin, 2 mg/ml. The glands were then in- it has proven difficult to explain some of the properties of acid cubated in this medium in a shaking water bath at 37°C. 02 secretion observed in the intact tissue by using the vesicle sys- consumption of the glands was measured by using a Warburg tem. Among these are: the primary energy source for acid se- respirometer (17). cretion (7), the absolute 02 dependence of acid secretion (8), Permeable gastric glands were prepared by treating a sus- the apparent electrogenicity of the pump in vitro (9), and the pension of glands [50 mg (wet weight)/ml] with 20 ,ug of digi- increase of resistance with SCNG inhibition (10). Therefore it tonin per ml of gland suspension at 37°C. Digitonin was dis- was necessary to develop a model intermediate in complexity solved in methanol and added in a minimal volume. Methanol between the intact cell and isolated vesicles in order to inves- alone had no effect. Leakiness of the cells was monitored by tigate these properties. trypan blue uptake (11) and by the appearance of lactate de- One attempt to develop such a model involved permeabili- hydrogenase (LDH) activity in the medium (19). zation ofparietal cell membranes ofisolated rabbit gastric glands To determine the K+ dependence ofthe secretory response, by using high-voltage shocks (11). Although evidence was ob- the solutions were made Na+ free and the concentration of K+ tained in support of the. view that ATP is the primary energy was varied. Tetramethylammonium was used as the substitut- source for acid secretion, electron microscopy of the shocked ing cation to maintain constant osmolarity. Previous experi- cells revealed massive mitochondrial damage, indicated by ments showed that tetramethylammonium is less toxic than cho- swelling and vacuolation (H. F. Helander, personal commu- line (20). Ouabain (1 mM) was included in all solutions. K+ and nication). This finding raised questions about the interpretation Na+ concentrations were checked by flame photometry, using ofsome ofthe experiments with shocked glands. Methods were a 443 flame photometer (Instrumentation Laboratory) with ex- therefore sought that would render the parietal cell membrane ternal lithium standards for calibration. permeable without causing gross mitochondrial damage. To investigate the effects of anoxia, gastric glands were sus- The detergent digitonin increases the permeability ofvarious pended in the glass chamber ofa Gilson 02 electrode apparatus cell types to inorganic ions, metabolites, and enzymes (12, 13). and allowed to consume all the oxygen present in the solution This effect is thought to be due to the interaction of digitonin prior to the addition of ATP. The 02 tension was continuously with cholesterol (14) in the plasma membrane. Mitochondria Abbreviations: LDH, lactate dehydrogenase; DNP, 2,4-dinitrophenol. The publication costs ofthis article were defrayed in part by page charge t Present address: Abteilung fur Gastroenterologie, Inselspital, CH- payment. This article must therefore be hereby marked "advertise- 3010 Bern, Switzerland. ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. § To whom reprint requests should be addressed. 5908 Downloaded by guest on September 28, 2021 Physiological Sciences: Malinowska etd Proc. NatL Acad. Sci. USA 78 (1981) 5909 monitored to ensure that the glands remained anoxic through- out the experiment. Digitonin-treated glands were fixed for electron microscopy in 2% (vol/vol) glutaraldehyde in medium for 30-45 min and postfixed for 15 min in 1% osmium tetroxide. After dehydration, the glands were embedded in Spurr embedding medium. Thin -0 sections were prepared and examined in a Phillips 400 S.3 microscope. The metachromatic fluorescence shift ofacridine orange was 8 403 used as described (21) for visual assessment ofthe cells respond- ing to ATP. The glands were observed with a Zeiss Photomi- croscope III. Oligomycin was dissolved in ethanol, and the ionophores valinomycin and 2,4-dinitrophenol (DNP) were dissolved in methanol. These compounds were always added in minimal volumes and the solvents alone had no effect. All chemicals were of reagent grade. Collagenase (type I), 0 0.1 1.0 10 100 rabbit albumin, digitonin, oligomycin, valinomycin, ouabain, and ATP, as the disodium salt or Tris salt (for experiments in Oligomycin, ,ug/ml Na'-free conditions) were obtainedfrom Sigma; ['4C]aminopyrine FIG. 2. Effect ofoligomycin on the aminopyrine ratio of intact gas- (84 mCi/mmol; 1 Ci = 3.7 x 1010 becquerels) was purchased tric glands incubated for 30 min. Inhibition of aminopyrine accumu- from New England Nuclear, and DNP was from Fisher. lation was close to maximum at an oligomycin concentration of 1 «ig/ ml. RESULTS Effect of Digitonin. When gastric glands were incubated 50%, and higherconcentrations ofoligomycin caused no further with digitonin at 20 Ag/ml, more than 80% ofthe parietal cells inhibition. Therefore, oligomycin, which is known not to affect took up trypan blue. The effect of digitonin on aminopyrine the gastric H+,K+-ATPase (22) was used at 10 ug/ml to pretreat accumulation by the glands and on LDH release from the glands the glands for 30 min. As shown in Fig. 1, the addition ofATP was then followed simultaneously. and digitonin then restored the aminopyrine ratio to control Gastric glands incubated in high-K+ medium in the absence levels, an increase of over 10-fold. The effect was transitory, of stimuli accumulated aminopyrine to a ratio of about 60 as with the maximum increase occurring at 10 min after ATP shown in Fig. 1 (control). The addition of digitonin at 20 ,g/ addition; ml slowly reduced the aminopyrine accumulation by 50%. The effect of digitonin on LDH release from the gastric However, addition of 5 mM ATP with digitonin to the glands glands is shown in Fig. 3. Release of 50-60% of total cellular resulted in a transient elevation of the aminopyrine ratio to about 100. ATP alone had little effect, strongly suggesting that digitonin was permeabilizingparietal cells. Although a response to ATP was found, a lower initial aminopyrine ratio was desirable. 60 F Fig. 2 illustrates the effect of oligomycin on aminopyrine accumulation in intact gastric glands incubated for 30 min. At 1 Ag/ml, oligomycin lowered the aminopyrine ratio by over 90%. 02 consumption of the glands was rapidly inhibited by a4-4 40 [ 100 go CD 4) 94 Dig + ATP 20 - 0- ---- ., .0 50 ,_ .Oligo I o 0 15 30 45 60 1Dig +ATP Time, min FIG. 3. Effect of digitonin (Dig) on the release ofLDHfrom gastric glands in the same experiment as shown in Fig. 1. Oligomycin (Oligo), 15 30 45 60 when present, was added at time 0; digitonin alone ordigitonin + ATP Time, min (as indicated) were added at 30 min.