Proc. NatI. Acad. Sci. USA Vol. 76, No. 12, pp. 6689-6693, December 1979 Physiological Sciences Cellular site of gastric acid secretion (H+ transport/membrane transformation/H+,K+-adenosinetriphosphatase/parietal cell/canaliculus) D. R. DIBONA*, S. ITOt, T. BERGLINDHW, AND G. SACHS* *Department of Physiology and Biophysics, Nephrology Research and Training Center, and *Laboratory of Membrane Biology, University of Alabama in Birmingham, University Station, Birmingham, Alabama 35294; and tDepartment of Anatomy, Harvard Medical School, Boston, Massachusetts 02115 Communicated by Alex B. Novikoff, August 30,1979 ABSTRACT Isolated gastric glands of the rabbit were ex- emission spectrum shifts in an analogous way as the local dye amined both with differential interference-ontrast microscopy concentration increases, either in free solution or when the dye and with electron microscopy to describe the morphologic is bound to an anionic polymer such as RNA or correlates of acid secretion. Stimulation of the glands with heparin. histamine resulted in the development of intracellular spaces In this report, we applied these approaches in discrete within the parietal cells. A similar transformation was produced combinations in an attempt to specifically identify the site of by addition of 1 mM aminopyrine, whether the weak base was gastric acid secretion. added in the presence of normal-K+ (5.4 mM) or high-K+ (108 mM) solutions. The intracellular space was compatible with the METHODS expanded canaliculus described in stimulated parietal cells. Confirmation that the space produced by histamine is the site Rabbit gastric glands were prepared by described methods (4). of acid secretion was gained by combining fluorescence and These involved high-pressure perfusion of the stomach through interference-contrast methods in the presence of the dye acri- the gastric artery with phosphate-buffered saline at 370C, dine orange, which displays a pH-dependent metachromasia mincing the separated epithelial layer with scissors, and di- in its emission spectrum. Human gastrin I resulted in an ob- gestion with collagenase. The isolated glands were incubated servable discharge of peptic granules. at 370C in a stirred vessel in a medium containing: Na+, 143.4 Although it is generally assumed that the parietal cell is re- mM; Cl-, 139.8 mM; K+, 5.4 mM; PO42-, 6.0 mM; Ca2+, 1.0 sponsible for acid secretion by the stomach, exact localization mM; Mg2+, 1.2 mM; So42-, 1.2 mM; glucose, 2 mg/ml, and of the process not rabbit albumin, 2 mg/ml. In some experiments, the K+ con- has been previously demonstrated in intact centration was increased to 108 mM at the expense of Na+; in tissue (1). It is clear that there is a dramatic trapsformation of some experiments, 15 mM NaSCN was also present. Uptake of parietal cell morphology accompanying stimulation of acid amino[14C]pyrine was monitored as described (6). Acridine secretion (2), in which a complex intracellular array of mem- orange was added at 100 ,M to the incubation medium. branes (the tubulovesicles) is replaced by an extensive micro- Tetraphenylphosphonium bromide was added at 100 ,uM. villous infolding of the luminal plasma membrane to form an Accumulation of 36C1- was measured by addition of 1 MCi (3.7 expanded canaliculus. A direct investigation of the significance X 104 becquerels) of -`Cl- to 1 ml of gland suspension, with or of this structural change in terms of its relationship to acid se- without the addition of secretagogue. cretion seemed possible, given recent improvements in cell and For differential interference-contrast (DIC) and fluorescence gland isolation techniques (3, 4), methods for assessment of acid microscopy, a Zeiss inverted microscope (1M-35) was used. secretion (5, 6), and the practical application of microscopic Glands were sampled from the incubation mixture at 370C at examination of living epithelial tissues (7, 8). prescribed times after treatment; for each sample, photographs Acid secretion levels can be readily measured by the con- were taken of glands as they were randomly encountered within centration uptake by the glands of the weak base aminopyrine 3 min after sampling. DIC microscopy was performed by (pK, 5.0) (6). Under maximal histamine stimulation the transillumination with a tungsten/halogen source while fluo- gland-to-medium ratio may be as high as 200. Knowing both rescence was studied with an epi-illumination system and an the size of this compartment and the gland-to-medium distri- HBO-50 light source. Excitation was with Hg lines 404.7 and bution ratio of aminopyrine would allow a direct calculation 435 nm; emission was observed and photographed through a of the pH of the compartment contents. In this respect, it is also high-pass reflecting filter (cut off at 510 nm; Zeiss component of interest to examine the consequences of increased medium pair no. 48-77-07). For electron microscopy, the glands were K+ concentration. The role of K+ in acid secretion by the fixed in a solution containing 2% (wt/wt) formaldehyde, 2.5% stomach (9) has been specified by the transport characteristics (wt/wt) glutaraldehyde, and 0.02% 2,3,6-trinitrophenol in pH of the gastric H+,K+-ATPase (10) and it has been demonstrated 7.2 sodium phosphate buffer for 2 hr, postfixed in 1% OS04 for that increased medium K+ results in an aminopyrine accu- 1 hr. dehydrated, and embedded in Epon for thin sectioning. mulation nearly equivalent to that found in the secretagogue- stimulated state (6). RESULTS Characterization of an identified compartment can be fa- cilitated by the use of acridine orange, a fluorescent dye that Stimulation of Acid Secretion by Histamine. The time has been used as an indicator of energization in many mem- course of histamine stimulation of acid secretion has been brane systems and notably for everted submitochondrial par- documented with measurements of the rate of amino['4C]- ticles (11). In gastric vesicles, acridine orange is distributed pyrine accumulation (6). After addition of the secretagogue at across and bound on membranes as a function of imposed pH 100 AM, there is a rapid increase in weak base accumulation, gradients (12). At low pH the green fluorescence at 530 nm is which reaches a maximum at 25-30 min. The morphologic quenched and a red fluorescence at 624 nm appears. The transformation of histamine-stimulated glands that we visual- ized by DIC microscopy follows a similar course. Resting glands, The publication costs of this article were defrayed in part by page as in Fig. la, allow identification of both parietal and peptic charge payment. This article must therefore be hereby marked "ad- (chief) cells. The parietal cells are conical, showing triangular vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: DIC, differential interference contrast. 6689 Downloaded by guest on September 30, 2021 6690 Physiological Sciences: DiBona et al. Proc. Natl. Acad. Sci. USA 76 (19* FIG. 1. Gastric gland. (DIC optics; X650.) (a) Resting gland suspended in normal Ringer's solution. Characteristic features: P, parietal cell; Z, peptic cell; M, mitochondria; G, pepsinogen granules. (b) Gland 30 min after histamine Addition. Note the appearance of vacuoles (V). profiles, and contain characteristic 1-am particles-mito- so that by 30 min some cells were observed to contain one or chondria-densely packed toward the basal end of the cell. The more intracellular fluid compartments that involved as much peptic cells are recognized as those containing granules of about as half of the parietal cell volume. Typically, maximally stim- 2 ,um diameter, concentrated toward the luminal surface. No ulated glands appeared as in Fig. lb. Electron micrographs of morphologic changes in the peptic cells were evident after glands fixed under similar conditions showed a comparable histamine addition, while a progressive development of intra- transformation with a loss of the tubulovesicles of the resting cellular vacuoles was noted in the parietal cells. Individual cell and formation of canalicular profiles lined with microvilli vacuoles became prominentin many parietal cells by 10 min much as has been previously described (2). These results suggest to and coalesce that the enlarging vacuolar space may be'the site of aminopy- after histamine addition; these appeared enlarge rine accumulation. b JPP";; ^; FIG. 2. Gastric gland. (DIC optics; X650.) (a) Gland incubated in 5.4 mM K+, recorded after the addition of 1 mM aminopyrine. Formed vacuoles (V) were restricted to a small fraction of the parietal cells. (b) Gland incubated in 108 mM K+ Ringer's solution, recorded 5 min after addition of 1 mM aminopyrine. Vacuolation extends to all observed parietal cells. Downloaded by guest on September 30, 2021 Physiological Sciences: DiBona et al. Proc. Natl. Acad. Sci. USA 76 (1979) 6691 2.;. *te,s = FIG. 3. (a) Electron micrograph of a glandular parietal cell incubated in 5.4 mM K+ Ringer's solution and 1 mM aminopyrine. Stages of development of osmotically generated canaliculi are seen. Large canaliculi have few microvilli, conceivably due to stretching of the membrane. (x2300.) (b and c) Glandular parietal cells incubated in 108 mM K+ Ringer's solution and 1 mM aminopyrine, showing large expanded intracellular canaliculi with few microvilli (b) as well as canaliculi with multiple microvilli (c). (b, X2300; c, X3600.) Effects of Addition of Aminopyrine. In unstimulated Use of Acridine Orange. The pH-dependent distribution glands, aminopyrine accumulation increases with increasing and fluorescence spectrum of the dye acridine orange offered K+ concentration of the incubation medium (6). The amino- an exceptional opportunity to further characterize the parietal pyrine accumulation ratio (when the base is added at 1 MM) is cell vacuolization noted above. In unstimulated resting glands about 20 in normal medium, and it rises to 75 when K+ con- (not shown here) addition of acridine orange resulted in a rather centration is increased from 5.4 mM to 108 mM at the expense uniform green fluorescence of all of the glandular components.