1 Departments of Medicine, Medical College of Virginia-Virginia Commonwealth University and McGuire Department of Veterans Affairs Medical Center, Richmond, Virginia 23249; 2 Department of Asthma/Allergy, Novartis, CH-4002 Basel, Switzerland; 3 Department of Anesthesiology, Johns Hopkins University College of Medicine, Baltimore, Maryland 21287; 4 Second Medical Department, Bogenhausen Hospital, 81675 Munich, Germany; and 5 Department of Pathology, McGuire Department of Veterans Affairs Medical Center, Richmond, Virginia 23249
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ABSTRACT |
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Nitric oxide synthases (NOS) are enzymes that catalyze the generation of nitric oxide (NO) from L-arginine and require nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. At least three isoforms of NOS have been identified: neuronal NOS (nNOS or NOS I), inducible NOS (iNOS or NOS II), and endothelial NOS (eNOS or NOS II). Recent studies implicate NO in the regulation of gastric acid secretion. The aim of the present study was to localize the cellular distribution and characterize the isoform of NOS present in oxyntic mucosa. Oxyntic mucosal segments from rat stomach were stained by the NADPH-diaphorase reaction and with isoform-specific NOS antibodies. The expression of NOS in isolated, highly enriched (>98%) rat parietal cells was examined by immunohistochemistry, Western blot analysis, and RT-PCR. In oxyntic mucosa, histochemical staining revealed NADPH-diaphorase and nNOS immunoreactivity in cells in the midportion of the glands, which were identified as parietal cells in hematoxylin and eosin-stained step sections. In isolated parietal cells, decisive evidence for nNOS expression was obtained by specific immunohistochemistry, Western blotting, and RT-PCR. Cloning and sequence analysis of the PCR product confirmed it to be nNOS (100% identity). Expression of nNOS in parietal cells suggests that endogenous NO, acting as an intracellular signaling molecule, may participate in the regulation of gastric acid secretion.
nitric oxide; brain nitric oxide synthase; nicotinamide adenine dinucleotide phosphate-diaphorase; acid; immunohistochemistry; stomach
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INTRODUCTION |
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NITRIC OXIDE (NO) is an important regulator of a variety of physiological functions (27). Depending on the situation, NO may function as a neurotransmitter, intracellular signal messenger, or paracrine agent (46). In the stomach, NO may mediate receptive relaxation and participate in the regulation of mucosal blood flow as well as mucus and acid secretion (2, 7, 10, 20, 23, 45).
NO is synthesized from L-arginine via the catalytic action of a group of enzymes, the NO synthases (NOS). NOS contains two distinct domains, an NH2-terminal domain that contains the L-arginine binding site and a COOH-terminal reductase domain that transfers electrons to the NH2-terminal domain and contains the binding site for nicotinamide adenine dinucleotide phosphate (NADPH) (19). Three isoforms of NOS have been identified using biochemical, immunohistochemical, and molecular biological techniques (19, 28). Constitutive calcium/calmodulin-dependent isoforms were initially localized to neurons in brain (nNOS, bNOS, or NOS I) (4) and vascular endothelial cells (eNOS or NOS III) (31) but are now known to be more widely distributed (1, 21, 22, 29, 38, 44). A cytokine-inducible calcium-independent isoform (iNOS, mNOS, or NOS II), first identified in immunocytes (43), can also be induced in other cell types (15, 30). All NOS isoforms require NADPH as a cofactor and have highly conserved consensus sequences for NADPH binding sites. These sites exhibit NADPH diaphoretic reductase activity and can be identified by NADPH-diaphorase histochemistry, reflecting NOS-catalyzed reduction of nitroblue tetrazolium (11, 17). Each of the isoforms can be distinguished with specific antibodies (11, 48).
In the gastrointestinal tract, NO may influence muscle tone as well as endocrine and exocrine secretion. In isolated mouse stomach, NO donors inhibit and NOS blockers stimulate acid secretion induced by distension (23), implying that endogenous NO participates in the regulation of acid secretion. Although calcium-dependent NOS activity (36) as well as nNOS expression by Western blotting (32, 33) have been detected in gastric mucosa, the precise cellular location of the isoform that may influence acid secretion is not known. In the present study, we have used the NADPH-diaphorase reaction, NOS immunohistochemistry, Western blotting, and RT-PCR to identify the expression of nNOS in rat parietal cells.
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METHODS |
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Animals. Male Sprague-Dawley rats weighing 150-200 g were deprived of solid food overnight but were allowed to drink water containing 10% dextrose. The animals were anesthetized with 20% urethan (5 ml/kg body wt ip). The protocols were approved by the Virginia Commonwealth University Institutional Animal Care and Use Committee.
Preparation of oxyntic mucosal sections. Rat stomachs were excised and opened along the greater curvature. The muscle and serosal layers were removed by dissection. Oxyntic mucosal sections were fixed in 4% (wt/vol) paraformaldehyde in PBS (pH 7.4) for 90-120 min and then dehydrated in an increasing gradient of sucrose in PBS (5-20%, in 5% steps, 45 min each). The tissue samples were snap-frozen in liquid nitrogen and embedded in a 1:2 solution containing OCT compound (Miles, Elkhart, IN) and 20% sucrose in PBS. Sections of 2-4 µm were thaw-mounted onto precleaned slides (Superfrost Plus; Fisher Scientific, Springfield, NJ).
Preparation of parietal cells. Rat parietal cells were isolated as previously described (41). Briefly, rat mucosal cells were released by enzymatic digestion (Pronase E), separated according to size and density by sequential use of counterflow elutriation and density gradient centrifugation, and then cultured. After 48 h in primary culture, the purity of parietal cells was 98-100% as determined by immunohistochemistry using an antibody directed against H+-K+-ATPase. The parietal cells were fixed in Bouin's solution for 2-4 h, centrifuged at 6,000 g for 10 min, and stored in 70% ethanol at 4°C until use. For immunohistochemistry, the cells were embedded in paraffin, cut into 4-µm-thick sections, and mounted onto slides that had been coated with CellTak (Collaborative Biomedical Products, Bedford, MA).
NADPH-diaphorase staining.
After being washed with PBS, cryostat oxyntic mucosal tissue sections
were stained for NADPH-diaphorase activity by incubation in 50 mM
Tris · HCl buffer (pH 8.0) containing 1.2 mM -NADPH and 0.3 mM nitroblue tetrazolium (Sigma Chemical, St. Louis, MO) for 40 min at
37°C as previously described (34). After being washed in
PBS, sections were covered with a glass coverslip and examined using
bright-field microscopy. In negative control slides,
-NADPH
substrate was omitted from the incubation medium.
Immunohistochemistry.
Immunohistochemistry on oxyntic mucosal sections and isolated parietal
cells was performed as previously described (48, 49), with
minor modification. Before staining, parietal cell sections were
deparaffinized, rehydrated, and incubated in a steamer with Tissue
Revival Solution (Cell Marque, Austin, TX) for 15 min for antigen
enhancement. Tissues were permeabilized with 0.2% Triton X-100 in 0.1 M PBS for 10 min (Sigma Chemical) and then incubated with 3% hydrogen
peroxide in 0.1 M PBS at pH 7.3 for 10 min to block endogenous
peroxidase activity. After blocking with 4% normal goat serum (Sigma
Chemical) or Cas Block (Zymed, San Francisco, CA), sections were washed
twice in PBS for 10 min and incubated overnight at 4°C with a
polyclonal antibody raised in rabbits against rat brain NOS [a
well-characterized antibody directed against amino acids 1-181
produced by T. M. Dawson and S. H. Snyder (1:500 dilution),
Johns Hopkins University (3, 11); antibody no. 24312 directed against amino acids 724-739 (1:800 dilution), Oxis Health
Products (Portland, OR); or antibody no. N-198 directed against amino
acids 1409-1429 (1:500), Research Biologicals (Natick, MA)], a
polyclonal antibody raised against human eNOS (1:100 dilution;
Transduction Laboratories, Lexington, KY), a polyclonal antibody raised
against mouse iNOS (1:500 dilution; Oxis Health Products), or murine
monoclonal antibody HK 12.18 directed against the
H+-K+-ATPase -subunit (1:500 dilution;
kindly supplied by A. Smolka, Medical University of South Carolina;
Ref. 42). Negative control slides were incubated with
normal rabbit serum, rabbit IgG, normal mouse serum, or mouse IgG,
according to the primary antibodies used, or by omitting the primary
antibody from the incubation buffer. Sections of rat cerebellum served
as positive controls for nNOS immunostaining (12). After
unbound primary antibody was washed off with PBS, the sections were
stained using the Dako LSAB2 peroxidase-based kit (Dako, Carpinteria,
CA) specific for rat tissue, according to the manufacturer's
instructions followed by the Dako Liquid DAB (diaminobenzidine)
Substrate-Chromagen System kit. The kits contain biotinylated
anti-rabbit and anti-mouse IgG antibody that has been absorbed to
abolish cross-reactivity, streptavidin peroxidase conjugated to
horseradish peroxidase (HRP), and DAB.
Hematoxylin and eosin staining. Parallel slides with step sections adjacent to the immunostained or NADPH-diaphorase-stained sections were doubly stained with eosin in 95% ethanol (30 s) and hematoxylin in water (3 min) for identification of mucosal cells.
Western blotting. The parietal cells were lysed on ice in a solution containing 10 mM HEPES (pH 7.5), 1.5 mM MgCl2, 10 mM KCl, 0.5% tergitol, 100 µg/ml phenylmethylsulfonyl fluoride, and 2 µg/ml leupeptin (Sigma Chemical), transferred into microfuge tubes, and spun at 14,000 g for 5 min at 4°C. The protein concentration in the supernatant was measured using the Pierce BCA Protein Assay kit, according to the manufacturer's instructions (Pierce, Rockford, IL). Equal amounts of parietal cell protein (maximum load 30 µg/well) were loaded onto small-format 10% SDS-PAGE gels (Bio-Rad, Hercules, CA). After running, protein was transferred to nitrocellulose (Intermountain Scientific, Kaysville, UT) or polyvinylidene fluoride (Immobilon-P, Millipore, Bedford, MA) membranes using the Mini Trans-Blot electrophoretic transfer cell kit (Bio-Rad). The blots were blocked with a buffer consisting of 3% bovine serum albumin (fraction V; Fischer Scientific, Fair Lawn, NJ) and 0.1% Tween-20 (Amersham Life Sciences, Arlington Heights, IL) in PBS for 1 h at room temperature. The transferred proteins were probed with the same panel of nNOS antibodies described above for immunostaining [Dawson and Snyder nNOS antibody (1:500), Oxis Health Products nNOS antibody no. 24312 (1:1,000), and Research Biochemicals nNOS antibody no. N-198 (1:500)]. The blots were incubated for 1 h at room temperature with goat anti-rabbit secondary antibody conjugated with HRP (1:1,000 dilution; Amersham Life Sciences). The bands were identified by enhanced chemiluminescence reagents (ECL Plus kit, Amersham Pharmacia Biotech, Piscataway, NJ) and visualized in a luminescent image analyzer (LAS-1000, Fujifilm, Tokyo, Japan). Specificity was revealed by the presence of a signal to recombinant rat nNOS (Alexis Biochemicals, San Diego, CA) and absence of any signal to eNOS protein (Cayman Chemical, Ann Arbor, MI) or iNOS protein (Alexis Biochemicals) after preabsorption of the nNOS antibody with nNOS protein.
RT-PCR. Total cellular RNA was extracted from isolated, enriched parietal cells using oligo-dT linked paramagnetic beads according to the protocol provided in the Dynabeads mRNA Direct kit (Dynal, Lake Success, NY). Single-strand cDNA was synthesized using a RT reaction solution containing (in mM) 50 Tris · HCl, 75 KCl, 3 MgCl2, 10 dithiothreitol, and 0.5 dNTP with 0.5 µg of oligo-dT primers (Boehringer-Mannheim, Indianapolis, IN) and 200 units of Superscript II RT (Gibco BRL, Gaithersburg, MD). The cDNA product was amplified by PCR for 30 cycles (94°C for 1 min, 51°C at 1 min, and 72°C at 2 min) followed by a final extension cycle (72°C at 10 min). Optimal primers were chosen using Vector NIT (Informax, Bethesda, MD). The upstream primer was 5'-GAACCCCCAAGACCATCC-3', and the downstream primer was 5'-GGCTTTGCTCCCACTGTT-3' (IDT Technologies, Coralville, IA). The amplified products were analyzed by ethidium bromide-stained agarose gel electrophoresis, and the DNA was extracted using the Gene Clean Spin Kit (Bio101, Vista, CA). The PCR product was cloned into TOPO vector (Invitrogen, Carlsbad, CA) and subjected to blue/white colony selection. The extracted DNA was sequenced by Commonwealth Biotechnologies (Richmond, VA) and identified using the Gene Blast program. In negative control studies, cDNA was omitted to control for amplification of contaminating RNA or DNA.
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RESULTS |
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Identification of nNOS in parietal cells by histochemistry. In rat fundic mucosa, NADPH-diaphorase enzyme activity, a marker for rat nNOS in paraformaldehyde-fixed tissues (11, 12, 24, 26, 47), was observed within cells in the midportion of the oxyntic glands. In adjacent step sections stained with hematoxylin and eosin, the cells were large and rounded or triangular, with centrally located nuclei and intensely acidophilic cytoplasm. The location, morphology, and staining characteristics of the cells were consistent with those of parietal cells.
Because NADPH-diaphorase activity cannot distinguish among the different isoforms of NOS and it is possible that other NADPH-diaphorases may exist in gastric mucosa, the results of NADPH-diaphorase histochemistry were confirmed by NOS immunohistochemistry using antibodies directed against nNOS, eNOS, and iNOS. All three nNOS antibodies immunostained cells located in the midportion of the oxyntic glands, which, on hematoxylin and eosin staining of step sections, were identified as parietal cells. In control sections in which rabbit serum or IgG was substituted for the nNOS antibody or the nNOS antibody was simply omitted, no immunoreactivity was detected. In rat cerebellum, a positive control, nNOS-immunoreactive neurons were robustly labeled by all three nNOS antibodies. The eNOS antibody stained vascular epithelium only, and no immunostaining was observed with the iNOS antibody. To verify that the nNOS immunostaining occurred in parietal cells, the immunohistochemical studies were repeated in isolated rat parietal cells, in which the purity was determined to be >98%, by immunohistochemistry using a murine monoclonal antibody directed against the H+-K+-ATPase
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Identification of nNOS in parietal cells by Western
blot analysis.
Western blotting was performed to confirm the specificity of the
antibodies and the presence of specific polypeptides corresponding to
nNOS in parietal cells. All three nNOS antibodies detected a distinct
band of ~155 kDa in crude protein lysates prepared from isolated rat
parietal cells (Fig. 2). The molecular
mass of the detected signal was similar in size to the signal detected using recombinant rat nNOS protein (Fig. 2). The antibodies were specific for nNOS protein and did not cross-react with eNOS and iNOS
proteins, because there were no proteins at 135-kDa or 130-kDa mass
(the masses of eNOS and iNOS) (Fig. 2).
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Identification of nNOS expression in parietal cells
by RT-PCR, cloning, and sequence analysis.
With nNOS-specific primers, a distinct RT-PCR product of the predicted
size (309 bp) was obtained from isolated rat parietal cells (Fig.
3). The nNOS-specific product was cloned
into TOPO vector and sequenced in both directions, yielding a 309-bp
sequence that was 100% identical with rat nNOS. Control experiments
without cDNA did not yield PCR products.
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DISCUSSION |
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The major finding of this study is that nNOS, the enzyme responsible for synthesis of NO, is expressed in rat parietal cells. This was demonstrated in 1) oxyntic mucosa tissue sections by NADPH-diaphorase histochemistry, a stain that colocalizes with nNOS (11, 17, 24, 39) and by nNOS immunostaining and 2) isolated parietal cells by nNOS immunostaining, Western blotting, and RT-PCR.
In oxyntic mucosa, both methods, i.e., NADPH-diaphorase histochemistry and nNOS immunostaining, stained cells in the midportion of the glands that were subsequently identified as parietal cells in hematoxylin and eosin-stained step sections. Thus NADPH-diaphorase histochemical reactivity is also a marker of nNOS in parietal cells.
Decisive evidence for the expression of nNOS in parietal cells was obtained by performing immunostaining and Western blotting on highly enriched (>98%), isolated rat parietal cells using three different nNOS antibodies, each directed at different regions of the protein, and by RT-PCR. The nNOS immunostaining in parietal cells was specific because 1) it was observed with each of the nNOS antibodies but was absent with omission of the antibody, preabsorption of the antibody with nNOS, or substitution of the antibody with eNOS antibody, iNOS antibody, rabbit IgG, or normal rabbit serum; 2) cerebellar neurons, known to contain nNOS, were positively stained; and (3) by Western analysis, the nNOS antibodies detected pure nNOS protein but did not cross-react with eNOS or iNOS proteins. Western blotting experiments confirmed the expression of immunoreactive nNOS in parietal cells and showed that its apparent molecular mass is similar to that of recombinant rat nNOS (155 kDa). Not only was nNOS protein present in the isolated parietal cells, but RT-PCR with nNOS-specific primers detected mRNA transcripts of the predicted size for rat nNOS. Cloning and subsequent sequence analysis of the PCR product revealed it to be 100% identical to that of rat nNOS.
Although nNOS, named for the tissue from which it was first cloned, was initially isolated from brain, this isoform has since been localized to a variety of nonneuronal tissues including smooth and skeletal muscle, respiratory epithelium, white blood cells, and pancreas (5, 9, 22, 29, 39, 40). With Western blotting, a protein tentatively identified as nNOS has been detected in full-thickness samples of rat stomach and in crude preparations of human fundic mucosa (14). With immunohistochemistry, nNOS has been localized to chief cells in guinea pig stomach (13), surface epithelial cells in rat stomach (18, 32), and somatostatin cells in rat and human stomach (8). In contrast to the findings of Fiorucci et al. (13), who reported nNOS immunoreactivity in ~70% of guinea pig chief cells, we did not detect nNOS immunoreactivity or NADPH-diaphorase staining in rat chief cells or, in preliminary studies, in rabbit chief cells.
In rat oxyntic mucosa tissue sections, nNOS immunoreactivity has been reported in surface epithelial cells, in brush or caveolated cells by Kugler et al. (25), and in mucus cells by Price et al. (32). Consistent with these findings, we also observed moderate nNOS immunoreactivity and NADPH-diaphorase activity in surface epithelial cells (data not shown). Although Price et al. (32) did not detect nNOS immunoreactivity in the midportion of the glands where parietal cells reside, it should be noted that, in agreement with our results, they did detect NADPH-diaphorase activity, a marker for nNOS (11, 17, 39), in this region. The disparity may be caused by differences in technique. First, our mucosal staining was performed on cryosections, which better preserve tissue immunogenicity (12, 39). It has been reported that NADPH-diaphorase histochemistry may be a more sensitive and reliable method of detecting nNOS than nNOS immunohistochemistry in fixed, paraffin-embedded tissues (12). Second, our isolated parietal cells were pretreated to optimize antigen retrieval. Antigen retrieval is often required to reveal the nNOS epitope (12). Third, we used antibodies directed against the rat nNOS sequence, whereas their antibody was directed against the human sequence.
Using a double immunolabeling method, Burrell et al. (8) reported colocalization of nNOS and somatostatin immunoreactivity in endocrine cells of rat and human fundus. Because endocrine cells account for only 1-3% of all epithelial cells in rats, of which only 10% are somatostatin cells (16), and we did not use specific antiserum to identify somatostatin cells, our studies do not exclude the possibility that nNOS may also be present in somatostatin cells.
Recent studies suggest that endogenous NO participates in the regulation of parietal cell function. In isolated rabbit parietal cells, Sakai et al. (35) reported that NG-monomethyl-L-arginine, an inhibitor of NOS, attenuates and sodium nitroprusside, a NO donor, augments prostaglandin E2-induced activation of a basolateral chloride channel. In anesthetized rats, Saperas et al. (37) reported that central vagal activation by intracisternal injection of thyrotropin-releasing hormone stimulates gastric NO release and acid secretion. The NOS inhibitor NG-nitro-L-arginine attenuates secretagogue- and meal-stimulated acid secretion in dogs (2) and distension-induced acid secretion in the isolated mouse stomach (23), implying that endogenous NO acts to stimulate acid secretion. In contrast to these findings, Kato et al. (20) reported that the NOS inhibitor NG-nitro-L-arginine methyl ester slightly augments pentagastrin-stimulated acid secretion in the anesthetized rat and Brown et al. (6) reported that the NO donor S-nitroso-N-acetyl-penicillamine inhibits secretory activity in isolated rat parietal cells.
Although NO released by various stimuli, including vagal activation and distension, can influence acid secretion, it should be noted that the source of NO and the mechanism by which NO regulates acid secretion are not known. The present study demonstrates for the first time, using immunohistochemical and molecular biological techniques, that nNOS is expressed in parietal cells. The precise contribution of NO derived from parietal cells, neurons, and possibly other cell types in the regulation of acid secretion is not known. The findings of the present study raise the possibility that NO may influence parietal cell secretion directly, acting as an intracellular signaling molecule, and/or indirectly by diffusing to and acting on adjacent neurons and/or endocrine cells, e.g., somatostatin cells or histamine-containing enterochromaffin-like cells.
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ACKNOWLEDGEMENTS |
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This work was supported by the Department of Veterans Affairs Medical Research Fund.
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. L. Schubert, McGuire VAMC, Code 111N, Division of Gastroenterology, 1201 Broad Rock Blvd., Richmond, VA 23249 (E-Mail: mitchell.schubert{at}med.va.gov).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 16 March 2000; accepted in final form 8 September 2000.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Balligand, JL,
Kobzik L,
Xan X-Q,
Kaye DM,
Belhassen L,
O'Hara DS,
Kelly RA,
Smith TW,
and
Michel T.
Nitric oxide-dependent parasympathetic signaling is due to activation of constitutive endothelial (type III) nitric oxide synthase in cardiac myocytes.
J Biol Chem
270:
14582-14586,
1995
2.
Bilski, J,
Konturek PC,
Konturek SJ,
Cieszkowski M,
and
Czarnobilski K.
Role of endogenous nitric oxide in the control of gastric acid secretion, blood flow and gastrin release in conscious dogs.
Regul Pept
53:
175-184,
1994[ISI][Medline].
3.
Bredt, DS,
Glatt CE,
Hwang PM,
Fotuhi M,
Dawson TM,
and
Snyder SH.
Nitric oxide synthase protein and mRNA are discretely localized in neuronal populations of the mammalian CNS together with NADPH diaphorase.
Neuron
7:
615-624,
1991[ISI][Medline].
4.
Bredt, DS,
and
Snyder SH.
Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme.
Proc Natl Acad Sci USA
87:
682-685,
1990[Abstract].
5.
Brenman, JE,
Chao DS,
Xia HH,
Aldape K,
and
Bredt DS.
Nitric oxide synthase is complexed with dystrophin and absent from skeletal muscle sarcolemma in Duchenne muscular dystrophy.
Cell
82:
743-752,
1995[ISI][Medline].
6.
Brown, JF,
Hanson PJ,
and
Whittle BJR
The nitric oxide donor, S-nitroso-N-acetyl-penicillamine, inhibits secretory activity in rat isolated parietal cells.
Biochem Biophys Res Commun
195:
1354-1359,
1993[ISI][Medline].
7.
Brown, JF,
Keates AC,
Hanson PJ,
and
Whittle BJR
Nitric oxide generators and cGMP stimulate mucus secretion by rat gastric mucosal cells.
Am J Physiol Gastrointest Liver Physiol
265:
G418-G422,
1993
8.
Burrell, MA,
Montuenga LM,
García M,
and
Villaro AC.
Detection of nitric oxide synthase (NOS) in somatostatin-producing cells of human and murine stomach and pancreas.
J Histochem Cytochem
44:
339-346,
1996
9.
Chakder, S,
Bandyopadhyay A,
and
Rattan S.
Neuronal NOS gene expression in gastrointestinal myenteric neurons and smooth muscle cells.
Am J Physiol Cell Physiol
273:
C1868-C1875,
1997
10.
Currò, D,
Volpe AR,
and
Preziosi P.
Nitric oxide synthase activity and non-adrenergic non-cholinergic relaxation in the rat gastric fundus.
Br J Pharmacol
117:
717-723,
1996[Abstract].
11.
Dawson, TM,
Bredt DS,
Fotuhi M,
Hwang PM,
and
Snyder SH.
Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues.
Proc Natl Acad Sci USA
88:
7797-7801,
1991[Abstract].
12.
Downen, M,
Zhao ML,
Lee P,
Weidenheim KM,
Dickson DW,
and
Lee SC.
Neuronal nitric oxide synthase expression in developing and adult human CNS.
J Neuropathol Exp Neurol
58:
12-21,
1999[ISI][Medline].
13.
Fiorucci, S,
Distrutti E,
Chiorean M,
Santucci L,
Belia S,
Fano G,
De Giorgio R,
Stanghellini V,
Corinaldesi R,
and
Morelli A.
Nitric oxide modulates pepsinogen secretion induced by calcium-mediated agonist in guinea pig gastric chief cells.
Gastroenterology
109:
1214-1223,
1995[ISI][Medline].
14.
Fischer, H,
Becker JC,
Boknik P,
Huber V,
Lüss H,
Neumann J,
Schmitz W,
Domschke W,
Stachura J,
and
Konturek JW.
Expression of constitutive nitric oxide synthase in rat and human gastrointestinal tract.
Biochim Biophys Acta
1450:
414-422,
1999[ISI][Medline].
15.
Geller, DAC,
Lowenstein CJ,
Shapiro RA,
Nussler AK,
Silvio MD,
Wang SC,
Nakayama DK,
Simmons RL,
Snyder SH,
and
Billiar TR.
Molecular cloning and expression of inducible nitric oxide synthase in hepatocytes.
Proc Natl Acad Sci USA
90:
522-526,
1993[Abstract].
16.
Häkanson, R,
Ekelund M,
and
Sundler F.
Evolution and Tumour Pathology of the Neuroendocrine System. Amsterdam: Elsevier, 1984, p. 371-398.
17.
Hope, BT,
Michael GJ,
Knigge KM,
and
Vincent SR.
Neuronal NADPH diaphorase is a nitric oxide synthase.
Proc Natl Acad Sci USA
88:
2811-2814,
1991[Abstract].
18.
Ichikawa, T,
Ishihara K,
Kusakabe T,
Kurihara M,
Kawakami T,
Takenaka T,
Saigenji K,
and
Hotta K.
Distinct effects of tetragastrin, histamine, and CCh on rat gastric mucin synthesis and contribution of NO.
Am J Physiol Gastrointest Liver Physiol
274:
G138-G146,
1998
19.
Jaffrey, SR,
and
Snyder SH.
Nitric oxide: a neural messenger.
Annu Rev Cell Biol
11:
417-440,
1995[ISI][Medline].
20.
Kato, S,
Kitamura M,
Korolkiewicz RP,
and
Takeuchi K.
Role of nitric oxide in regulation of gastric acid secretion in rats: effects of NO donors and NO synthase inhibitor.
Br J Pharmacol
123:
839-846,
1998[Abstract].
21.
Kirsch, EA,
Yuhanna IS,
Chen Z,
German Z,
Sherman TS,
and
Shaul PW.
Estrogen acutely stimulates endothelial nitric oxide synthase in H441 human airway epithelial cells.
Am J Respir Cell Mol Biol
20:
658-666,
1999
22.
Kobzik, L,
Bredt DS,
Lowenstein CJ,
Drazen J,
Gastor B,
Sugarbaker D,
and
Stamler JS.
Nitric oxide synthase in human and rat lung: immunocytochemical and histochemical localization.
Am J Respir Cell Mol Biol
9:
371-377,
1993[ISI][Medline].
23.
Koduru, S,
Vuyyuru L,
and
Schubert ML.
Nitric oxide mediates the stimulation of acid secretion induced by distension of the gastric fundus (Abstract).
Gastroenterology
108:
A132,
1995[ISI].
24.
Koike S, Hisa Y, Uno T, Murakami Y, Tamada Y, and Ibata Y. Nitric
oxide synthase and NADPH-diaphorase in neurons of the rat, dog and
guinea pig nodose ganglia. Acta Otolaryngol (Stockh)
110-112, 1998.
25.
Kugler, P,
Höfer D,
Mayer B,
and
Drenckhahn D.
Nitric oxide synthase and NADP-linked glucose-6-phosphate dehydrogenase are co-localized in brush cells of rat stomach and pancreas.
J Histochem Cytochem
42:
1317-1321,
1994
26.
Matsumoto, T,
Nakane M,
Pollock JS,
Kuk JE,
and
Förstermann U.
A correlation between soluble brain nitric oxide synthase and NADPH-diaphorase activity is only seen after exposure of the tissue fixative.
Neurosci Lett
155:
61-64,
1993[ISI][Medline].
27.
Moncada, S.
Nitric oxide gas: mediator, modulator, and pathophysiologic entity.
J Lab Clin Med
120:
187-191,
1992[ISI][Medline].
28.
Nadaud, S,
and
Soubrier F.
Molecular biology and molecular genetics of nitric oxide synthase genes.
Clin Exp Hypertens
18:
113-143,
1996[ISI][Medline].
29.
Nakane, M,
Schmidt HHHW,
Pollock JS,
Forstermann U,
and
Murad F.
Cloned human brain nitric oxide synthase is highly expressed in skeletal muscle.
FEBS Lett
316:
175-180,
1993[ISI][Medline].
30.
Nunokawa, Y,
Ishida N,
and
Tanaka S.
Cloning of inducible nitric oxide synthase in rat vascular smooth muscle cells.
Biochem Biophys Res Commun
191:
89-94,
1993[ISI][Medline].
31.
Palmer, RMJ,
and
Moncada S.
A novel citrulline-forming enzyme implicated in the formation of nitric oxide by vascular endothelial cells.
Biochem Biophys Res Commun
158:
348-352,
1989[ISI][Medline].
32.
Price, KJ,
Hanson PJ,
and
Whittle BJR
Localization of constitutive isoforms of nitric oxide synthase in the gastric glandular mucosa of the rat.
Cell Tissue Res
285:
157-163,
1996[ISI][Medline].
33.
Rajnakova, A,
Goh PM,
Chan ST,
Ngoi SS,
Alponat A,
and
Moochhala S.
Expression of differential nitric oxide synthase isoforms in human normal gastric mucosa and gastric cancer tissue.
Carcinogenesis
18:
1841-1845,
1997[Abstract].
34.
Rengasamy, A,
Xue C,
and
Johns RA.
Immunohistochemical demonstration of a paracrine role of nitric oxide in bronchial function.
Am J Physiol Lung Cell Mol Physiol
267:
L704-L711,
1994
35.
Sakai, H,
Kumano E,
Ikari A,
and
Takeguchi N.
A gastric housekeeping Cl channel activated via prostaglandin EP3 receptor-mediated Ca2+/nitric oxide/cGMP pathway.
J Biol Chem
270:
18781-18785,
1995
36.
Salter, M,
Knowles RG,
and
Moncada S.
Widespread tissue distribution, species distribution, and changes in activity of Ca2+-dependent and Ca2+-independent nitric oxide synthases.
FEBS Lett
291:
145-149,
1991[ISI][Medline].
37.
Saperas, E,
Mourelle M,
Santos J,
Moncada S,
and
Malagelada J-R.
Central vagal activation by an analogue of TRH stimulates gastric nitric oxide release in rats.
Am J Physiol Gastrointest Liver Physiol
268:
G895-G899,
1995
38.
Sase, K,
and
Michel T.
Expression of constitutive endothelial nitric oxide synthase in human blood platelets.
Life Sci
57:
2049-2055,
1995[ISI][Medline].
39.
Schmidt, HH,
Gagne GD,
Nakane M,
Pollock JS,
Miller MF,
and
Murad F.
Mapping of neural nitric oxide synthase in the rat suggests frequent co-localization with NADPH diaphorase but not with soluble guanylyl cyclase, and novel paraneural functions for nitrinergic signal transduction.
J Histochem Cytochem
40:
1439-1456,
1992
40.
Schmidt, HH,
Warner TD,
Ishii K,
Sheng H,
and
Murad F.
Insulin secretion from pancreatic B cells caused by L-arginine-derived nitrogen oxides.
Science
255:
721-723,
1992[ISI][Medline].
41.
Schmidtler, J,
Dehne K,
Allescher H-D,
Schusdziarra V,
Classen M,
Holst JJ,
Polack A,
and
Schepp W.
Rat parietal cell receptors for GLP-1-(7-36) amide: Northern blot, cross-linking, and radioligand binding.
Am J Physiol Gastrointest Liver Physiol
267:
G423-G432,
1994
42.
Smolka, A,
Alverson L,
Fritz R,
Swiger K,
and
Swiger R.
Gastric H,K-ATPase topography: amino acids 888-907 are cytoplasmic.
Biochem Biophys Res Commun
180:
1356-1364,
1991[ISI][Medline].
43.
Stuehr, DJ,
and
Marletta MA.
Mammalian nitrate biosynthesis: mouse macrophages produce nitrite and nitrate in response to Escherichia coli lipopolysaccharide.
Proc Natl Acad Sci USA
82:
7738-7742,
1985[Abstract].
44.
Teng, BQ,
Murthy KS,
Kuemmerle JF,
Grider JR,
Sase K,
Michel T,
and
Makhlouf GM.
Expression of endothelial nitric oxide synthase in human and rabbit gastrointestinal smooth muscle cells.
Am J Physiol Gastrointest Liver Physiol
275:
G342-G351,
1998
45.
Whittle, BJR,
Lopez-Belmonte J,
and
Moncada S.
Nitric oxide mediates rat mucosal vasodilatation induced by intragastric capsaicin.
Eur J Pharmacol
218:
339-341,
1992[ISI][Medline].
46.
Wood, J,
and
Garthwaite J.
Models for diffusional spread of nitric oxide: implications for neural nitric oxide signaling and its pharmacological properties.
Neuropharmacology
33:
1235-1244,
1994[ISI][Medline].
47.
Wörl, J,
Wiesand M,
Mayer B,
Greskötter K-R,
and
Neuhuber WL.
Neuronal and endothelial nitric oxide synthase immunoreactivity and NADPH-diaphorase staining in rat and human pancreas: influence of fixation.
Histochemistry
102:
353-364,
1994[ISI][Medline].
48.
Xue, C,
and
Johns RA.
Histochemical evidence of endothelial nitric oxide synthase in vascular endothelium of patients with abnormally thickened pulmonary arteries.
N Engl J Med
333:
1642-1644,
1995
49.
Xue, C,
Rengasamy A,
LeCras TD,
Koberna PA,
Dailey GC,
and
Johns RA.
Distribution of NOS in normoxic vs hypoxic rat lung: upregulation of NOS by chronic hypoxia.
Am J Physiol Lung Cell Mol Physiol
267:
L667-L678,
1994
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