Site Of Acid Secretion Research Articles

A microinjection technique using a pH-sensitive dye to determine the gut pH of Calanus helgolandicus

By applying a microinjection technique previously used in cell biology, a pH-sensitive dye, 2′,7′-bis(2-carboxyethyl)-5,6-carboxyfluorescein) (BCECF), was injected into the gut of the copepod Calanus helgolandicus to determine pH under a range of feeding conditions. The median gut pH of the fore- and hindguts of starved individuals was 6.86 and 7.19, respectively. This was a consistently lower pH than that observed in copepods feeding on either the diatom Thalassiosira weissflogii, the dinoflagellate Prorocentrum micans, or the coccolithophorid Emiliania huxleyi (Strain 92D), all of which had a median gut pH>7.0. In all treatments, the median pH of the foregut was more acidic than the median of the hindgut, implying that the foregut is the site of acid secretion. The results, which demonstrate pH values down to 6.11, are discussed in terms of coccolith dissolution, pigment degradation and digestive enzyme activity.

The site of acid secretion in the mammalian parietal cell

Initiation of acid secretion in the gastric mucosa is accompanied by a morphological transformation in which the acid pump, the H +/K +-ATPase, translocates from a cytoplasmic vesicular location to the secretory surface lining the canaliculi. Associated with the morphological changes, activation of K + and Cl — pathways are necessary to supply K + to the extracytoplasmic face of the pump. Although the pump in the secretory membrane is known to secrete acid, it is not known whether activation of the KCl pathway occurs in the tubulovesicular membrane prior to the formation of the canaliculus, or when the pump is in the secretory membrane. The cellular site of activation of acid secretion in the rabbit gastric parietal cell was investigated using the covalent binding of [ 3H]omeprazole as a probe of acid secretion in rabbit gastric glands that were undergoing stimulation in vitro. This compound depends on an acidic environment for activation and covalent binding to the H +/K +-ATPase. Electron microscopic autoradiography showed that activation of the enzyme occurred only when it was present in the canalicular membrane and not when it was present in the cytoplasmic tubulovesicular membrane. Hence there is likely to be a physical separation of K + and/or Cl — pathways from the ATPase in the resting cell, and stimulation of acid secretion is dependent on colocalization of these pathways in the canalicular membrane.

Acid secretion and membrane reorganization in single gastric parietal cell in primary culture

A digitally-enhanced videomicroscopy study of rabbit gastric parietal cells in primary culture was performed using alternate observations with differential interference contrast and fluorescence optics of cells mounted and perfused on a temperature-controlled microscope stage. The effect of histamine, a physiological effector of acid secretion, was followed. Isolated parietal cells possess an internal apical vacuole, which kept the cell in a pseudopolarized state. This apical vacuole is a site of acid secretion. This was demonstrated by the direct visualization of the uptake of the fluorescent weak base 9-amino acridine and of the concomitant enormous swelling of the acid vacuole which reached an estimated size of 3–7 times the normal cell volume. This morphological change of shape and acidification of apical vacuoles was fully reversible and cells could respond to successive stimulations. A quantitative study of these events provided a value of the acid accumulation index for each single cell in response to histamine. Individual cell response varied within a factor of 7. The cellular localization of the proton pump complex responsible for acid secretion and of the major components of the secretory microvilli, actin and ezrin, a histamine-dependent phosphorylation target of protein kinase A, were detected by indirect immunofluorescence microscopy in resting and stimulated cells. Both actin and ezrin colocalized at the apical vacuole membrane in resting and stimulated cells, whereas the proton pump shifted from an intracytoplasmic pool to the apical vacuole membrane upon stimulation.

Sites of carbonic anhydrase in avian gastric mucosa identified by electron microscope autoradiography.

Carbonic anhydrase was localized at the ultrastructural level by autoradiography of [3H]acetazolamide in quail gastric mucosa. Grains were associated predominantly with mucin granules at the apical end of surface mucosal cells. Secreted mucin was also labeled. Oxynticopeptic cells of the multilobular glands were not labeled. The pH of fluid secreted by was 7.0-7.4 compared with a pH of 1.3-1.8 of freshly secreted fluid in the gastric lumen. The results of this study suggest that surface mucosal cells, which are strikingly similar in birds, amphibians, and mammals, are the site of acid secretion in the vertebrate stomach.

Probes of parietal cell function

The site of acid secretion in the gastric mucosa has been inferred, but never proven. Using differential interference-contrast (Nomarski) microscopy an expansion of intracellular vacuoles was observed in the parietal cells of living rabbit gastric glands following histamine stimulation. A similar vacuolization occurring only in part of a parietal cell population could be induced by high concentrations of accumulated weak base, aminopyrine, in the absence of secretagogue. In high-K+ medium, 10(-3) M aminopyrine induced massive vacuolization in all parietal cells, consistent with the strong effect of high K+ in stimulating aminopyrine uptake by isolated glands. Electron micrographs showed that the apparent vacuoles correspond to the secretory canaliculi in various stages of swelling. Acridine orange, a fluorescent dye which is distributed across natural membranes as a function of a pH gradient and binds in a multimolecular fashion (stacking) to negative sites, was accumulated by gastric glands as a function of acid secretion. Visualization of such glands by fluorescence or a combination of Nomarski and fluorescence microscopy showed a red fluorescence in the expanding secretory canaliculi that was in sharp contrast to the green fluorescence in the rest of the cell. From these data it is concluded that the site of acid secretion is indeed the secretory canaliculus of the parietal cell. It is also possible that the formation of secretory canaliculi may be induced osmotically and that the peripheral "parietal" position and triangular shape of the parietal cell is necessary to allow expansion and oriented apical flow of HCl.

Cellular site of gastric acid secretion.

Isolated gastric glands of the rabbit were examined both with differential interference-contrast microscopy and with electron microscopy to describe the morphologic correlates of acid secretion. Stimulation of the glands with histamine resulted in the development of intracellular spaces within the parietal cells. A similar transformation was produced by addition of 1 mM aminopyrine, whether the weak base was added in the presence of normal-K+ (5.4 mM) or high-K+ (108 mM) solutions. The intracellular space was compatible with the expanded canaliculus described in stimulated parietal cells. Confirmation that the space produced by histamine is the site of acid secretion was gained by combining fluorescence and interference-contrast methods in the presence of the dye acridine orange, which displays a pH-dependent metachromasia in its emission spectrum. Human gastrin I resulted in an observable discharge of peptic granules.

Characterization of gastric mucosal membranes. X. Immunological studies of gastric (H+ + K+)-ATPase.

Gastric mucosal homogenates from hog were fractionated by differential and density gradient centrifugation and free-flow electrophoresis. The two major membrane fractions (FI and FII) thus obtained are distinct both enzymically and in terms of transport reactivity. This heterogenicity extends to their antigenic activity. Purified antibodies which were raised against the K+-ATPase-containing H+ transport fraction FI were of two types: inhibitory and non-inhibitory. Inhibitory antibodies reduced the K+-ATPase activity by approximately 80% and the K+-p-nitro-phenylphosphatase activity by approximately 40% in a concentration-dependent manner, while the small Mg++-dependent component of the enzyme activity was unaffected. Antibodies inhibiting the K+-ATPase also inhibited H+ transport. These antibodies did not cross-react with the other major membrane fraction isolated by free-flow electrophoresis, FII, and gave a single band on rocket immunoelectrophoresis. Antibodies against this FII fraction also did not react with the K+-ATPase and were heterogeneous, giving at least four bands with rocket immunoelectrophoresis and inhibiting both the 5'-nucleotidase and Mg++-ATPase of this fraction. Immunofluorescent staining of tissue sections showed that the FI was derived from the parietal cell of gastric tissue and was localized to the supranuclear area of the cell. Staining of isolated rat gastric cell suspensions by FI antibodies confirmed the selectivity of the antibody and showed a polar, plasma membrane localization. FII antibodies also largely stained the parietal cells in tissue sections. In the 16 hog tissues tested, FI antibodies cross-reacted only with gastric fundus, thyroid and weakly with thymus. Immunoelectronmicroscopy showed that FI antibodies reacted strongly with the secretory membrane at the apical cell surface of the parietal cells and at the secretory canaliculi, weakly with the apical surface of the zymogen cell, and not with the basal-lateral surface of the cells. Thus, the protontranslocating ATPase is localized in the parietal cells and in the region postulated to be the site of acid secretion.