Volume 117, Issue 3, Pages 605-618 (September 1999) Gastric H+, K+-adenosine triphosphatase β subunit is required for normal function, development, and membrane structure of mouse parietal cells  Katrina L. Scarff, Louise M. Judd, Ban–Hock Toh, Paul A. Gleeson, Ian R. van Driel  Gastroenterology  Volume 117, Issue 3, Pages 605-618 (September 1999) DOI: 10.1016/S0016-5085(99)70453-1 Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 Targeting of the mouse gastric H+,K+-ATPase β-subunit gene by homologous recombination. (A) Wild-type gastric H+,K+-ATPase β-subunit locus, (B) targeting vector pPNTβ.PH5.6, and (C) mutated β-subunit allele. The targeting vector was constructed as described in Materials and Methods. A homologous recombination event results in the replacement of 35 bp of exon 1 with the phosphoglycerate kinase I (PGK)-neomycin gene. The internal (int) and external (ext) probes were used to detect homologous recombination events. The PGK-thymidine kinase (tk) gene is also shown. Restriction sites shown are BamHI (B), PstI (P), EcoRI (E), and HindIII (H). (D) DNA from H+,K+-ATPase β subunit–deficient mice was digested with BamHI, electrophoresed on an agarose gel, and transferred to a nylon membrane. The membrane was hybridized with the 400-bp external probe (ext) shown in A. The wild-type allele (+/+) generates a 6.7-kb fragment, whereas the mutated allele generates a 1.7-kb fragment (−/−). Heterozygous mice show both fragments (+/−). (E) Analysis of gastric H+,K+-ATPase β-subunit mRNA expression in the stomach of H+,K+-ATPase β subunit–deficient mice. RNA was extracted from stomachs of wild-type (+/+), heterozygous (+/−), and H+,K+-ATPase β subunit–deficient (−/−) mice and incubated with oligonucleotides corresponding to sequences of the H+,K+-ATPase α or β subunits in the presence (+) or absence (−) of reverse transcriptase, as indicated. The resulting cDNA was amplified as described in Materials and Methods. The PCR products were electrophoresed on a DNA gel, transferred to nylon membrane, and hybridized to 32P-labeled α- or β-subunit cDNAs. The α-subunit primers generated a product of 536 bp, and the β-subunit primers generated a product of 650 bp. Amplification was also performed with oligonucleotides corresponding to β-actin sequences, and a similar amount of product was generated with all samples (not shown). Gastroenterology 1999 117, 605-618DOI: (10.1016/S0016-5085(99)70453-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 Targeting of the mouse gastric H+,K+-ATPase β-subunit gene by homologous recombination. (A) Wild-type gastric H+,K+-ATPase β-subunit locus, (B) targeting vector pPNTβ.PH5.6, and (C) mutated β-subunit allele. The targeting vector was constructed as described in Materials and Methods. A homologous recombination event results in the replacement of 35 bp of exon 1 with the phosphoglycerate kinase I (PGK)-neomycin gene. The internal (int) and external (ext) probes were used to detect homologous recombination events. The PGK-thymidine kinase (tk) gene is also shown. Restriction sites shown are BamHI (B), PstI (P), EcoRI (E), and HindIII (H). (D) DNA from H+,K+-ATPase β subunit–deficient mice was digested with BamHI, electrophoresed on an agarose gel, and transferred to a nylon membrane. The membrane was hybridized with the 400-bp external probe (ext) shown in A. The wild-type allele (+/+) generates a 6.7-kb fragment, whereas the mutated allele generates a 1.7-kb fragment (−/−). Heterozygous mice show both fragments (+/−). (E) Analysis of gastric H+,K+-ATPase β-subunit mRNA expression in the stomach of H+,K+-ATPase β subunit–deficient mice. RNA was extracted from stomachs of wild-type (+/+), heterozygous (+/−), and H+,K+-ATPase β subunit–deficient (−/−) mice and incubated with oligonucleotides corresponding to sequences of the H+,K+-ATPase α or β subunits in the presence (+) or absence (−) of reverse transcriptase, as indicated. The resulting cDNA was amplified as described in Materials and Methods. The PCR products were electrophoresed on a DNA gel, transferred to nylon membrane, and hybridized to 32P-labeled α- or β-subunit cDNAs. The α-subunit primers generated a product of 536 bp, and the β-subunit primers generated a product of 650 bp. Amplification was also performed with oligonucleotides corresponding to β-actin sequences, and a similar amount of product was generated with all samples (not shown). Gastroenterology 1999 117, 605-618DOI: (10.1016/S0016-5085(99)70453-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 Targeting of the mouse gastric H+,K+-ATPase β-subunit gene by homologous recombination. (A) Wild-type gastric H+,K+-ATPase β-subunit locus, (B) targeting vector pPNTβ.PH5.6, and (C) mutated β-subunit allele. The targeting vector was constructed as described in Materials and Methods. A homologous recombination event results in the replacement of 35 bp of exon 1 with the phosphoglycerate kinase I (PGK)-neomycin gene. The internal (int) and external (ext) probes were used to detect homologous recombination events. The PGK-thymidine kinase (tk) gene is also shown. Restriction sites shown are BamHI (B), PstI (P), EcoRI (E), and HindIII (H). (D) DNA from H+,K+-ATPase β subunit–deficient mice was digested with BamHI, electrophoresed on an agarose gel, and transferred to a nylon membrane. The membrane was hybridized with the 400-bp external probe (ext) shown in A. The wild-type allele (+/+) generates a 6.7-kb fragment, whereas the mutated allele generates a 1.7-kb fragment (−/−). Heterozygous mice show both fragments (+/−). (E) Analysis of gastric H+,K+-ATPase β-subunit mRNA expression in the stomach of H+,K+-ATPase β subunit–deficient mice. RNA was extracted from stomachs of wild-type (+/+), heterozygous (+/−), and H+,K+-ATPase β subunit–deficient (−/−) mice and incubated with oligonucleotides corresponding to sequences of the H+,K+-ATPase α or β subunits in the presence (+) or absence (−) of reverse transcriptase, as indicated. The resulting cDNA was amplified as described in Materials and Methods. The PCR products were electrophoresed on a DNA gel, transferred to nylon membrane, and hybridized to 32P-labeled α- or β-subunit cDNAs. The α-subunit primers generated a product of 536 bp, and the β-subunit primers generated a product of 650 bp. Amplification was also performed with oligonucleotides corresponding to β-actin sequences, and a similar amount of product was generated with all samples (not shown). Gastroenterology 1999 117, 605-618DOI: (10.1016/S0016-5085(99)70453-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 Immunoblot analysis of gastric membranes from H+,K+-ATPase β subunit–deficient mice. Gastric membranes were prepared from the stomachs of wild-type (+/+), heterozygous (+/−), and H+,K+-ATPase β subunit–deficient (−/−) mice. Approximately 1 μg/lane of membrane proteins was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose. (A) The membrane was probed with monoclonal antibodies specific for the α or β subunits of gastric H+,K+-ATPase or an isotype-matched negative control (C). Binding of anti–mouse immunoglobulin–horseradish peroxidase conjugate was detected by enhanced chemiluminescence. (B) Alternatively, the membrane was probed with a polyclonal rabbit serum specific for a carboxyl-terminal peptide of the α subunit (αC2) or normal rabbit serum (N), followed by anti-rabbit immunoglobulin–horseradish peroxidase. Films were exposed for (A) 10 seconds or (B) 60 seconds. *Prominent breakdown product. Gastroenterology 1999 117, 605-618DOI: (10.1016/S0016-5085(99)70453-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 Reactivity of gastric H+,K+-ATPase–specific monoclonal antibodies to frozen stomach sections from H+,K+-ATPase β subunit–deficient mice. Frozen stomach sections of (A–C) wild-type and (D–F) H+,K+-ATPase β subunit–deficient mice were incubated with monoclonal antibodies specific for the (A and D) β or (B and E) α subunits of gastric H+,K+-ATPase or (C and F) with an isotype-matched control. The sections were incubated with anti-mouse immunoglobulin–FITC and examined by using confocal microscopy. Arrowheads indicate some cells that contain large vacuole-like structures. (Bar in F = 25 μm and applies to all panels.) Gastroenterology 1999 117, 605-618DOI: (10.1016/S0016-5085(99)70453-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 Analysis of gastric pH and serum gastrin levels in H+,K+-ATPase β subunit–deficient mice. The pH of gastric contents and levels of plasma gastrin of fasted wild-type (+/+, ●), heterozygous (+/−, ▴), and H+,K+-ATPase β subunit–deficient (−/−, ■) mice was determined as described in Materials and Methods. Each point represents the datum from 1 animal. Horizontal bars in each graph indicate the mean value of each set of data from that genotype. Gastroenterology 1999 117, 605-618DOI: (10.1016/S0016-5085(99)70453-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 5 Morphological analysis of gastric mucosa in H+,K+-ATPase β subunit–deficient mice. Sections of stomach from (A–D) 15-day-old, (E–H) 17-day-old, and (I–L) 35-day-old wild-type (A, B, E, F, I, and J) and H+,K+-ATPase β subunit–deficient (C, D, G, H, K, and L) mice were stained with H&E. +, Cells with typical parietal cell structure. *Vacuole-like structures. (Bar in A = 50 μm and applies to A, C, E, and G; bar in B = 25 μm and applies to B, D, F, H, J, and L; bar in I = 100 μm and applies to I and K.) Gastroenterology 1999 117, 605-618DOI: (10.1016/S0016-5085(99)70453-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 6 Reactivity of H+,K+-ATPase α subunit–specific antiserum to frozen stomach sections from H+,K+-ATPase β subunit–deficient mice. Sections of frozen stomach from (A and D) 15-day-old, (B and E) 17-day-old, and (C and F) 35-day-old wild-type (A–C) and H+,K+-ATPase β subunit–deficient (D–F) mice were incubated with a polyclonal rabbit serum specific for a carboxyl-terminal peptide of the α subunit (HKαC2) followed by anti–rabbit immunoglobulin–FITC and examined by using confocal microscopy. Arrowheads indicate some cells that contain large vacuole-like structures. No staining was observed when normal rabbit serum was used in place of HKαC2 serum (not shown). (Bar in A = 50 μm and applies to all panels.) Gastroenterology 1999 117, 605-618DOI: (10.1016/S0016-5085(99)70453-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 7 Ultrastructure of parietal cells of H+,K+-ATPase β subunit–deficient mice prepared by aldehyde fixation. Gastric mucosae of (A) 17-day-old wild-type and (B) H+/K+-ATPase β subunit–deficient mice as well as (C and H) 35-day-old wild-type and (D–G and I) H+,K+-ATPase β subunit–deficient were fixed in aldehydes before imaging by transmission electron microscopy. In 17-day-old normal parietal cells (A), a typical secretory canaliculus is present (C), although we did not observe tubulovesicular membranes in parietal cells of this age. In parietal cells of H+,K+-ATPase β subunit–deficient mice (B), an abnormal dilated canaliculus was present (asterisk) that had shorter microvilli at a lower density. Vesicles (v) are also present in the cytoplasm. In parietal cells of 35-day-old wild-type mice (C and H), the secretory canaliculus (c) and tubulovesicular membranes (t) were observed. In H+,K+-ATPase β subunit–deficient mice (D–G and I), parietal cells with the abnormal canaliculi (asterisk) were the dominant morphological feature. Some cells also had large vesicles (v). The difference between the typical tubulovesicular structures in normal parietal cells and the large vesicles in H+,K+-ATPase β subunit–deficient parietal cells can be clearly seen by comparing H and I. (Bars in A–F = 2.5 μm; bars in H and I = 1 μm.) Gastroenterology 1999 117, 605-618DOI: (10.1016/S0016-5085(99)70453-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 8 Ultrastructure of parietal cells of 35-day-old H+,K+-ATPase β subunit–deficient mice prepared by fast freeze-fixation/freeze-substitution. Gastric mucosae of (A and B) wild-type and (C and D) H+,K+-ATPase β subunit–deficient mice were fixed by rapid freezing and cryosubstituted before imaging by transmission electron microscopy. In parietal cells (p) from wild-type animals, the tubular membranes (t) and secretory canaliculus (c) were present. In H+,K+-ATPase β subunit–deficient parietal cells, the vesicular structures obvious in many of the aldehyde-fixed cells (see Figure 7) were rare. The structure of the abnormal canaliculus is, on the other hand, similar to that found in aldehyde-fixed cells. (Bars in A and C = 2.5 μm; bars in B and D = 1 μm.) Gastroenterology 1999 117, 605-618DOI: (10.1016/S0016-5085(99)70453-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions