Neuronal nitric oxide synthase: expression in rat parietal cells

Shyamal Premaratne1, Chun Xue2, John M. McCarty1, Muhammad Zaki1, Robert W. McCuen1, Roger A. Johns3, Wolfgang Schepp4, Bruno Neu4, Robert Lippman5, P. D. Melone1, and Mitchell L. Schubert1

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


    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


    INTRODUCTION
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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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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 beta -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, beta -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 alpha -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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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 alpha -subunit (42) (Fig. 1, C and D). All three nNOS antibodies strongly stained the parietal cells (Fig. 1A). No immunostaining was observed in control slides in which nNOS antibody was omitted or eNOS antibody, iNOS antibody, normal rabbit serum, or rabbit IgG was substituted for the nNOS antibody (Fig. 1B).


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Fig. 1.   Immunostaining of isolated rat parietal cells with neuronal nitric oxide synthase (nNOS) antibody and H+-K+-ATPase antibody. A: immunostaining with nNOS antibody (×200). B: control staining with rabbit IgG substituted for nNOS antibody (×200). C: immunostaining with H+-K+-ATPase antibody (×200). D: control staining with mouse IgG substituted for H+-K+-ATPase antibody (×200).

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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Fig. 2.   Western blot analysis of nNOS from isolated rat parietal cells. Cells were lysed, and the lysates were subjected to acrylamide gel electrophoresis. The separated proteins were transferred onto nitrocellulose sheets, and the blots were probed with nNOS antibodies. The nNOS antibodies recognized a single band of molecular mass ~155 kDa that corresponded to that obtained with pure nNOS protein. The nNOS antibodies did not crossreact with endothelial (eNOS) or inducible (iNOS) NOS proteins.

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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Fig. 3.   Expression of nNOS in isolated rat parietal cells determined by RT-PCR using rat nNOS-specific primers. The arrow (right lane) indicates a band at the expected size for rat nNOS (309 bp). The PCR product was cloned, and sequence analysis revealed complete homology with the known rat nNOS sequence.


    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.


    ACKNOWLEDGEMENTS

This work was supported by the Department of Veterans Affairs Medical Research Fund.


    FOOTNOTES

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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Am J Physiol Gastrointest Liver Physiol 280(2):G308-G313




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