Reconstitution of acid secretion in digitonin-permeabilized rabbit gastric glands. Identification of cytosolic regulatory factors

When isolated rabbit gastric glands were permeabilized with digitonin, they lost their ability to secrete acid, as monitored by [14C]aminopyrine accumulation, and they never recovered by supplement with cytosol prepared from gastric mucosa. However, the permeabilized glands elicited acid secretion when brain cytosol was supplemented. Fractionation of gastric cytosol by gel filtration revealed that the fraction at 30 kDa stimulated permeabilized glands by itself, whereas the 200-kDa fraction potently inhibited brain cytosol-stimulated acid secretion. Brain cytosol contained only the former stimulatory factor. With further gel filtration, the 30-kDa activator was separated into two components, 20 kDa (peak 1) and 1.8 kDa (peak 2), both of which are necessary for full activity. We purified peak 1 from bovine brain, and phosphatidylinositol transfer protein (PITP) was identified as the main component of the activity. The stimulating activity in brain and gastric mucosa correlated with the contents of PITP, and recombinant PITP mimicked the effect of peak 1, suggesting that PITP is one of the essential components in gastric acid secretion. When gastric glands were stimulated, the inhibitory activity, but not stimulatory activity, in the cytosol was increased. This suggests a regulatory mechanism such as stimulation translocates the inhibitory component from the secretory site on the membrane to cytosol. These results demonstrate a high degree of usefulness for our present model, the reconstituted digitonin-permeabilized gastric glands.     

Purification and characterization of a fatty-acid-binding protein from the gastric mucosa of rats. Possible identity with heart fatty-acid-binding protein and its parietal cell localization

Fatty acid-binding protein (FABP) was purified from rat gastric mucosa by successive Sephadex G-75 chromatography, DEAE-cellulose chromatography and HPLC on an RP-2 (Merck) reversed-phase column. The purified stomach FABP migrated as a single band corresponding to an apparent molecular mass of 15 kDa on SDS/PAGE. Stomach FABP appeared to be identical with rat heart FABP, as judged from its electrophoretic mobility, amino acid composition and tryptic peptide map. In addition, the amino acid sequences of two selected tryptic peptides coincided completely with the rat heart FABP sequence deduced from that of cDNA. Stomach FABP showed immunochemical identity with rat heart FABP when tested with an antiserum against rat heart FABP. Immunohistochemically, stomach FABP was specifically stained with anti-(rat heart FABP) serum in parietal cells of the gastric mucosa. The results suggested that the primary structure of stomach FABP is identical with that of rat heart FABP, and showed that stomach FABP is localized in parietal cells of the gastric mucosa.     

Partial purification of a novel stimulant of gastric acid secretion from canine non-antral mucosa

A novel stimulant of gastric acid secretion was extracted and purified from the non-antral gastric mucosa of the canine stomach and some of its biological properties were examined. Tissue was boiled in water and extracted in 2% trifluoroacetic acid. The stimulatory activity was purified by a combination of reverse-phase high pressure liquid chromatography (HPLC) and gel filtration. Fractions were assayed for a stimulation of basal, pentagastrin- and histamine-stimulated gastric acid secretion in the anaesthetized rat. Stimulatory activity was eluted from reverse-phase HPLC columns with acetonitrile and its elution from Sephadex G-10 and G-50 columns suggested a molecular weight of 1,000 to 3,000. The highly purified extracts enhanced basal, pentagastrin- and histamine-stimulated acid secretion in the rat. A stimulatory fraction was purified which was devoid of immunoreactive gastrin and gastrin-releasing peptide and contained only small amounts of histamine. Its chromatographic properties differed from those of histamine and acetylcholine. On two occasions the stimulant was purified to homogeneity and found to contain amino acids. Insufficient pure material was obtained for full characterization. The stimulant has been tentatively called oxyntin.     

One-step purification of the serotonin transporter located at the human platelet plasma membrane.

A 68-kDa glycoprotein bearing the biological activity of the plasma membrane serotonin (5-hydroxytryptamine, 5-HT) transporter has been purified from human blood platelets, a classical cell model for the study of 5-HT uptake. After treatment of the whole platelet population or its plasma membrane fraction by sulfhydryl-dependent bacterial protein toxins or by digitonin, purification was reproducibly obtained by a one-step affinity chromatography using two different columns with 5-HT or 6-fluorotryptamine as ligands and elution by 5-HT or Na(+)-free buffer. The purified fraction migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band with an apparent molecular mass of 68 kDa and exhibited an apparent isoelectric point of 5.6-6.2. Two sialic acid residues were detected in the purified material. The purified glycoprotein bound the 5-HT uptake blocker [3H]paroxetine with a Kd (0.25 nM) similar to the one observed for intact human platelets. It also bound [3H] 5-HT but neither [3H]hydroxytetrabenazine nor [3H] ouabain, the respective markers of the granular monoamine transporter and of the Na+,K(+)-ATPase associated to the plasma membrane 5-HT transporter. 5-HT derivatives and 5-HT uptake inhibitors exhibited similar Ki values for 5-HT uptake and paroxetine binding in intact human platelets and in the purified glycoprotein. Under laser UV irradiation, 40% of this purified glycoprotein could be labeled by either [3H]paroxetine or [3H]cyanoimipramine. No labeling was detected with either [3H] gamma-aminobutyric acid or [3H]GBR 12783, the respective markers of gamma-aminobutyric acid and dopamine carriers. The purified 68-kDa protein is therefore likely to correspond at least to the binding domain of the 5-HT transporter located at the human platelet plasma membrane.     

Purification of substrate proteins of casein kinases from the cytosol fraction of AH-66 hepatoma cells

We have attempted to purify endogenous substrate proteins for casein kinases I and II from the cytosol of AH-66 hepatoma cells. Utilizing the fact that only a few substrates are concentrated in the fraction eluted from DEAE-cellulose between 0.3 and 0.6 M NaCl, two substrates were purified from this fraction by DEAE-cellulose chromatography, hydroxyapatite chromatography, and HPLC on a DEAE-5PW column. The purified substrate proteins had molecular masses of 30.5 kDa and 31 kDa. The 31-kDa protein substrate was markedly phosphorylated by casein kinase II, but only slightly by casein kinase I. The radioactive phosphate incorporated into 31-kDa substrate by casein kinase II was 0.2 mol/mol of the protein and phosphorylation occurred on both threonine and serine residues. The 30.5 kDa protein was only slightly phosphorylated by casein kinase II, but not at all by casein kinase I.     

Purification of substrate proteins of casein kinases from the cytosol fraction of AH-66 hepatoma cells

We have attempted to purify endogenous substrate proteins for casein kinases I and II from the cytosol of AH-66 hepatoma cells. Utilizing the fact that only a few substrates are concentrated in the fraction eluted from DEAE-cellulose between 0.3 and 0.6 M NaCl, two substrates were purified from this fraction by DEAE-cellulose chromatography, hydroxyapatite chromatography, and HPLC on a DEAE-5PW column. The purified substrate proteins had molecular masses of 30.5 kDa and 31 kDa. The 31-kDa protein substrate was markedly phosphorylated by casein kinase II, but only slightly by casein kinase I. The radioactive phosphate incorporated into 31-kDa substrate by casein kinase II was 0.2 mol/mol of the protein and phosphorylation occurred on both threonine and serine residues. The 30.5 kDa protein was only slightly phosphorylated by casein kinase II, but not at all by casein kinase I.     

Purification and characterization of bile acid-CoA:amino acid N-acyltransferase from human liver

The bile acid-conjugating enzyme, bile acid-CoA: amino acid N-acyltransferase, was purified 480-fold from the soluble fraction of homogenized frozen human liver. Purification was accomplished by a combination of anion exchange chromatography, chromatofocusing, glycocholate-AH-Sepharose affinity chromatography, and high performance liquid chromatography (HPLC) gel filtration. Following purification, the reduced, denatured enzyme migrated as a single 50-kDa protein band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A similar molecular mass was obtained for the native enzyme by HPLC gel filtration. Elution from the chromatofocusing column suggested an apparent isoelectric point of 6.0 (+/- 0.2). Using a rabbit polyclonal antibody raised against the purified enzyme, Western blot analysis using 100,000 x g human liver supernatant confirmed that the affinity-purified polyclonal antibody was specific for human liver bile acid-CoA:amino acid N-acyltransferase. The purified enzyme utilized glycine, taurine, and 2-fluoro-beta-alanine (a 5-fluorouracil catabolite), but not beta-alanine, as substrates. Kinetic studies revealed apparent Km values for taurine, 2-fluoro-beta-alanine, and glycine of 1.1, 2.2, and 5.8 mM, respectively, with corresponding Vmax values of 0.33, 0.19, and 0.77 mumol/min/mg protein. These data demonstrate that a single monomeric enzyme is responsible for the conjugation of bile acids with glycine or taurine in human liver.     

Platelet plasma membrane: characterization of the serotonin transporter]

After treatment of human platelets by a sulfhydryl-dependent bacterial protein cytolysin, a glycoprotein was reproducibly purified by a one-step affinity chromatography using 6-fluorotryptamine as ligand and elution by serotonin (5-HT), cyanoimipramine, citalopram, or a Na(+)-free buffer. The purified fraction migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band with an apparent molecular mass of 68 kDa. The purified glycoprotein bound the 5-HT uptake blockers 3H-paroxetine, 3H-cyanoimipramine, and 3H-citalopram with Kds similar to the ones observed for intact human platelets. No binding was detected with 3H-hydroxytetrabenazine, 3H-ouabain, 3H-gamma aminobutyric acid or 3H-BTCP, the respective markers of the granular monoamine transporter, the plasma membrane Na+, K(+)-ATPase, the gamma aminobutyric acid and dopamine carriers. The purified 68-kDa glycoprotein is therefore likely to correspond at least to the paroxetine and imipramine binding domains of the 5-HT transporter located at the human platelet plasma membrane. Finally a 68-kDa protein was purified in the same conditions from the human megakaryocytic cell line Dami and to a lesser extent from the human megakaryoblastic cell line MEG-01 but not from the human erythroleukaemic cell line HEL.     

Purification of an AlF4- and G-protein beta gamma-subunit-regulated phospholipase C-activating protein.

A 150-kDa phospholipase C has previously been purified from turkey erythrocytes and has been shown by reconstitution with turkey erythrocyte membranes to be a receptor- and G-protein-regulated enzyme (Morris, A. J., Waldo, G. L., Downes, C.P., and Harden, T. K. (1990) J. Biol. Chem. 265, 13501-13507; Morris, A.J., Waldo, G.L., Downes, C.P., and Harden, T.K. (1990) J. Biol. Chem. 265, 13508-13514). Combination of this 150-kDa protein with phosphoinositide substrate-containing phospholipid vesicles prepared with a cholate extract from purified turkey erythrocyte plasma membranes resulted in conferrence of AlF4- sensitivity to the purified phospholipase C. Guanosine 5'-3-O-(thio)triphosphate also activated the reconstituted phospholipase C in a manner that was inhibited by guanosine 5'-2-O-(thio)-diphosphate. The magnitude of the AlF4- stimulation was increased with increasing amounts of plasma membrane extract, and was also dependent on the concentration of purified phospholipase C. Using reconstitution of AlF4- sensitivity as an assay, the putative G-protein conferring regulation to the 150-kDa phospholipase C was purified to near homogeneity by sequential chromatography over Q-Sepharose, Sephacryl S-300, octyl-Sepharose, hydroxylapatite, and Mono-Q. Reconstituting activity co-purified with an approximately 43-kDa protein identified by silver staining; lesser amounts of a 35-kDa protein was present in the final purified fractions, as was a minor 40-kDa protein. The 43-kDa protein strongly reacted with antiserum against a 12-amino acid sequence found at the carboxyl terminus of Gq and G11, the 35-kDa protein strongly reacted with G-protein beta-subunit antiserum, and the 40-kDa protein reacted with antiserum that recognizes Gi3. Immunoprecipitation of the 43-kDa protein resulted in loss of phospholipase C-stimulating activity of the purified fraction. The idea that this is a phospholipase C-regulating G-protein is further supported by the observation that co-reconstitution of G-protein beta gamma-subunit with the purified phospholipase C-activating fraction resulted in a beta gamma-subunit-dependent inhibition of AlF(4-)-stimulated phospholipase C activity in the reconstituted preparation.     

Isolation and partial characterization of an amphiphilic 56-kDa fatty acid binding protein from rat renal basolateral membrane

We have identified a 56-kDa fatty acid binding protein in rat renal basolateral membrane and purified it by extraction in nonionic detergent (Triton X-100), followed by gel filtration, DEAE-cellulose chromatography, and affinity chromatography. The purified protein was homogeneous on polyacrylamide gel electrophoresis in the presence of Triton X-100 or SDS. It showed amphiphilic properties on gel filtration, polyacrylamide gel electrophoresis, and oleate-Sepharose 4B chromatography. Its molecular mass was estimated to be 56 kDa by SDS-polyacrylamide gel electrophoresis. The protein showed optimal binding activity at pH 7.5 and 37 degrees C. The apparent Kd for palmitic acid was 0.79 microM. It was immunologically clearly distinct from renal cytosolic fatty acid binding protein.     

Gastric parietal cell antigens of 60-90, 92, and 100-120 kDa associated with autoimmune gastritis and pernicious anemia. Role of N-glycans in the structure and antigenicity of the 60-90-kDa component

Thirty-four human sera containing parietal cell autoantibodies (PCA) specifically immunoprecipitated two antigens, with apparent molecular masses of 60-90 kDa and 100-120 kDa under nonreducing conditions and 60-90 kDa and 120-150 kDa under reducing conditions, from porcine gastric membrane extracts. A third antigen of 92 kDa was only observed in immunoprecipitates analyzed under reducing conditions. By immunoblotting, 24 of the 34 PCA-positive sera reacted with only the 60-90-kDa antigen, three reacted with a broad 60-120-kDa smear, one reacted only with a 92-kDa antigen and six did not react. Reactivity with the 60-90-kDa antigen was observed with gastric membranes from dog, pig, rat, and rabbit. Twenty PCA-negative sera did not react with these components by immunoprecipitation or immunoblotting. PCA reactivity with the 60-90-kDa antigen was abolished when the gastric membranes were (a) digested with Pronase, (b) reduced with 100 mM dithiothreitol, (c) treated with sodium periodate, or (d) digested with N-glycanase. The 60-90-kDa and 100-120-kDa components were insensitive to neuraminidase treatment. N-glycanase digestion of 125I-labeled antigens purified by immunoprecipitation and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis collapsed the 60-90-kDa antigen to a sharp 34-kDa band; the 100-120-kDa component was unaffected. These observations suggest that (i) parietal cell antigens comprise three components of 60-90, 92, and 100-120 kDa; (ii) the epitopes differ in conformational sensitivity; (iii) the 60-90-kDa antigen is a conserved molecule comprising a 34-kDa core protein extensively glycosylated with N-linked oligosaccharides; (iv) sialic acid residues are not present in the 60-90- and 100-120-kDa molecules, and (v) the carbohydrate and protein moieties of the 60-90-kDa molecule are required for antibody binding.     

Primary structure of a 15-kDa protein from rat intestinal epithelium. Sequence similarity to fatty-acid-binding proteins

An abundant and novel cytosolic protein was purified from the rat intestinal epithelium by gel filtration, ion-exchange and hydroxylapatite chromatography. The protein was eluted into two different positions (fractions 1 and 2) on DEAE-cellulose chromatography. We have completed the primary structure of the protein of fraction 1 by Edman degradation. The protein (144565 Da) contains 127 amino acid residues and has an acetylated alanine at its NH2-terminus. Comparison of the primary structure of the protein with porcine gastrotropin [Walz, A. D., Wider, M. D., Snow, J. W., Dass, C. & Desiderio, D. M. (1988) J. Biol. Chem. 263, 14189-14195] and rat hepatic fatty-acid-binding protein revealed that identical residues within these proteins are found in 90 and 54 out of a total of 127 positions, respectively. Bioactivity studies demonstrated that neither the protein nor liver and intestinal fatty-acid-binding proteins influence gastric acid secretory activity in rats with gastric fistulas compared to pentagastrin. The protein showed very low affinity for palmitic-acid-binding in vitro assay system and only trace amounts of endogenous fatty acids were detected from the protein. The protein, rat intestinal 15-kDa protein is considered to be a new member of the fatty-acid-binding protein family based on its structural features.     

Ribonuclease inhibitors in Malus x domestica (common apple): isolation and partial characterization

A ribonuclease inhibitory activity was detected in the fruits of common apple, Malus x domestica, cv. Fuji, and purified by affinity chromatography on ribonuclease A-Sepharose. It inhibited hydrolysis of cyclic-2':3'-CMP by bovine pancreatic ribonuclease A with an apparent inhibition constant of about 5 x 10(-8) M. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the purified protein gave two peaks corresponding to the mass numbers of 55,658 and 62,839, while three bands of 43-, 34-, and 21-kDa were detected by SDS-PAGE. These results suggested that the inhibitor preparation was a mixture of two proteins comprised of 43- and 21-kDa subunits or of 34- and 21-kDa subunits. Attempts to separate these two proteins were unsuccessful. Amino acid composition and N-terminal amino acid sequence of these subunits were also identified and N-terminal sequences showed some similarity to that of cottonseed storage globulin. The significance of the presence of ribonuclease inhibitors in apple fruits is not clear, but it might allow some speculation about their possible involvement in the control of the self-incompatibility ribonuclease of Rosaceae plants.     

Target molecules of molecular chaperone (HSP70 family) in injured gastric mucosa in vivo

AimsSeveral recent studies, including ours, have indicated the importance of heat shock proteins (HSPs) in cytoprotection against cytotoxic agents and environmental stresses mediated by the chaperone function of HSPs (molecular chaperones). However, the target molecule that is recognized by HSPs in damaged cells currently remains unknown. As HSPs rapidly recognize and bind to degenerated protein in cells, target molecules of HSPs might be key molecules for the initiation and pathogenesis of cellular damage. In the present study, gastric mucosal proteins that specifically bind to the HSP70 family (HSC70) were analyzed using HSC70-affinity chromatography.Main methodsThe gastric mucosa was removed from Sprague–Dawley rats after exposure to water immersion-stress for 0, 1, 3 or 5 h. Soluble fractions of each gastric mucosa were applied to the HSC70-affinity column separately. After washing off non-specific binding proteins, specific binding proteins were eluted by ATP-containing buffer. Binding proteins were analyzed by SDS-polyacrylamide gel electrophoresis. In addition, the amino acid sequence of purified proteins was also analyzed.Key findingsSpecific HSC70-binding proteins with a molecular weight of 200-kDa and 45-kDa were eluted from an affinity column when gastric mucosal homogenate of 1-h stress exposure was applied. The amino acid sequencing showed that these binding proteins were cytoskeletal myosin (heavy chain) and actin, respectively.SignificanceDuring the pathogenesis of stress-induced gastric mucosal damage, structurally degenerated cytoskeletal myosin (heavy chain) and actin may be key or initiation molecules which structural changes were firstly recognized by molecular chaperone.     

Identification of a high-affinity binding protein for a hepta-beta-glucoside phytoalexin elicitor in soybean.

A putative receptor protein for a hepta-beta-glucoside phytoalexin elicitor was identified by photoaffinity labeling of detergent-solubilized proteins from soybean root membranes. Incubation of partially purified beta-glucan-binding proteins with a photolabile 125I-labeled 2-(4-azidophenyl)ethyl-amino conjugate of the heptaglucoside elicitor, followed by irradiation with ultraviolet light (366 nm) resulted in specific labeling of a 70-kDa band in SDS/PAGE. Half-maximal inhibition of the 125I-labeling of the protein band by underivatized hepta-beta-glucoside was achieved by 15 nM heptaglucoside. Analysis of the affinity of radiolabel incorporation into the protein by ligand-saturation experiments, gave an apparent Kd value of 3 nM, in full agreement with the results from radioligand-binding studies. Good correlation was also observed between the amount of radiolabel incorporated into the protein and the binding activity of the fractions obtained at different stages in the purification of heptaglucoside-binding activity. Photoaffinity labeling of proteins purified by glucan-affinity chromatography showed the 70-kDa band as the main component along with weak 125I-labeling of a 100-kDa band. The 70-kDa band was also the major protein visualized by silver staining after SDS/PAGE of this fraction, suggesting that it is the predominant form of the heptaglucoside-binding proteins in detergent-solubilized soybean membranes.     

Monoclonal antibodies specific for the core protein of the beta-subunit of the gastric proton pump (H+/K+ ATPase). An autoantigen targetted in pernicious anaemia.

The gastric H+/K(+)-transporting adenosine triphosphatase (H+/K+ ATPase) (proton pump) consists of a catalytic alpha-subunit and a recently proposed 60-90-kDa glycoprotein beta-subunit. Using dog gastric membranes as the antigen, we have produced two murine monoclonal antibodies, 4F11 (IgG1) and 3A6 (IgA), which are specific for the 60-90-kDa glycoprotein. The monoclonal antibodies (1) specifically stained the cytoplasm of unfixed and formalin-fixed dog gastric parietal cells; (2) specifically reacted by ELISA with gastric tubulovesicular membranes; (3) recognised epitopes located on the luminal face of parietal cell tubulovesicular membranes, the site of the proton pump, by immunogold electron microscopy; (4) immunoblotted a 60-90-kDa molecule from tubulovesicular membranes and a 35-kDa component from peptide N-glycosidase-F-treated membrane extracts; (5) immunoblotted the 60-90-kDa parietal cell autoantigen associated with autoimmune gastritis and pernicious anemia, purified by chromatography on parietal cell autoantibody- or tomato-lectin-Sepharose 4B affinity columns, and the 35-kDa protein core of this autoantigen; this autoantigen has amino acid sequence similarity to the beta-subunit of the related Na+/K(+)-transporting adenosine triphosphatase (Na+/K+ ATPase) [Toh et al. (1990) Proc. Natl Acad. Sci. 87, 6418-6422]; (6) co-precipitated a molecule of 95 kDa with the 60-90-kDa molecule from 125I-labelled detergent extracts of dog tubulovesicular membranes; and (7) co-purified the catalytic alpha-subunit of the H+/K+ ATPase with the 60-90-kDa molecule by immunoaffinity chromatography of tubulovesicular membrane extracts on a monoclonal antibody 3A6-Sepharose 4B column, indicating a physical association between the two molecules. These results provide further evidence that the 60-90-kDa glycoprotein is the beta-subunit of the gastric H+/K+ ATPase. We conclude that the monoclonal antibodies specifically recognise luminal epitopes on the 35-kDa core protein of the 60-90-kDa beta-subunit of the gastric proton pump, a major target molecule in autoimmune gastritis and pernicious anaemia. These monoclonal antibodies will be valuable probes to study the structure and function of this associated beta-subunit, as well as the ontogeny of the gastric proton pump.     

Purification and characterization of a protein tyrosine kinase from bovine spleen

Protein tyrosine kinase was purified extensively from a 30,000 X g particulate fraction of bovine spleen by a procedure involving four column chromatographies: DEAE-Sepharose, polyamino acids affinity, hydroxylapatite, and Sephacryl S-200 molecular sieving. The purification resulted in more than 3,000-fold enrichment in [Val5]angiotensin II phosphorylation activity (specific activity 202 nmol/min/mg). All column chromatography profiles showed single protein tyrosine kinase activity peaks with the exception of that of affinity chromatography, where about 50% of the enzyme activity appeared with the breakthrough fraction; only the bound enzyme was further purified. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of a purified sample phosphorylated in the presence of [gamma-32P]ATP revealed the presence of a single phosphorylated polypeptide of molecular weight 50,000 which represents about 40% of total protein. Analysis by polyacrylamide gel electrophoresis under nondenaturing conditions showed that protein tyrosine kinase activity co-migrated with the phosphoprotein. Stoichiometry of the phosphorylation of the 50-kDa polypeptide was found to be 1.0 mol/mol. The purified sample did not appear to contain phosphotyrosine protein phosphatase activity. Both casein and histone could be phosphorylated by the purified sample, and the phosphorylation occurred only at tyrosine residue, suggesting that there was no protein serine and threonine kinase contamination.     

Functional characterization and expression of PBR in rat gastric mucosa: stimulation of chloride secretion by PBR ligands

Previous studies have demonstrated that gastric mucosa contained high levels of the polypeptide diazepam binding inhibitor, the endogenous ligand of the peripheral-type benzodiazepine receptor (PBR). However, the expression and function of this receptor protein in these tissues have not been investigated. Immunohistochemistry identified an intense PBR immunoreactivity in the mucous and parietal cells of rat gastric fundus and in the mucous cells of antrum. Immunoelectron microscopy revealed the mitochondrial localization of PBR in these cells. Binding of isoquinoline PK 11195 and benzodiazepine Ro5-4864 to gastric membranes showed that fundus had more PBR-binding sites than antrum, displaying higher affinity for PK 11195 than Ro5-4864. In a Ussing chamber, PK 11195 and Ro5-4864 increased short-circuit current (I(sc)) in fundic and antral mucosa in a concentration-dependent manner in the presence of GABA(A) and central benzodiazepine receptor (CBR) blockers. This increase in I(sc) was abolished after external Cl(-) substitution and was sensitive to chloride channels or transporter inhibitors. PK 11195-induced chloride secretion was also 1) sensitive to verapamil and extracellular calcium depletion, 2) blocked by thapsigargin and intracellular calcium depletion, and 3) abolished by the mitochondrial pore transition complex inhibitor cyclosporine A. PK 11195 had no direct effect on H(+) secretion, indicating that it stimulates a component of Cl(-) secretion independent of acid secretion in fundic mucosa. These data demonstrate that mucous and parietal cells of the gastric mucosa express mitochondrial PBR functionally coupled to Ca(2+)-dependent Cl(-) secretion, possibly involved in the gastric mucosa protection.