Gastroenterology

Gastroenterology

Volume 123, Issue 2, August 2002, Pages 566-576
Gastroenterology

Basic–Alimentary Tract
Nitric oxide regulates the release of somatostatin from cultured gastric rabbit primary D-cells,☆☆

https://doi.org/10.1053/gast.2002.34749Get rights and content

Abstract

Background & Aims: Neuronal nitric oxide synthase (nNOS) is present in gastric D-cells. Mucosal somatostatin is diminished in H. pylori gastritis, where production of nitric oxide (NO) is increased. Therefore, we investigated the role of NO in D-cell function and the effects of prolonged exposure of D-cells to NO. Methods: Rabbit gastric D-cells were cultured. Somatostatin-14 was measured after 2 hours to examine the effects of arginine, nitric oxide sythase (NOS) inhibitors, and NO donors. Some cells were preincubated with a slow releasing NO donor for 12 hours. Results are expressed as percentage of total cell content. Nitrate content was measured by chemiluminescent assay. Results:L-arginine increased somatostatin-14 release in the presence of CCK8 from 4.4% ± 0.5% to 6.4% ± 0.4% (P < 0.02), and this was accompanied by NO release from 27 ± 7 μmol/L to 86 ± 12 μmol/L (P = 0.001). D-arginine and L-lysine had no effect. NOS inhibitors LNNA, SMT, and 7NI significantly attenuated the stimulatory response to L-arginine. NO donors sodium nitroprusside (SNP), 1 mmol/L, and S-nitroso-N-acetyl-D-L-penicillamine, 0.1 mmol/L, significantly increased basal and cholecystokinin-8 (CCK8) stimulated somatostatin release. Oxyhemoglobin attenuated the effect of SNP but not of L-arginine. Neither cyclic guanosine monophosphate nor guanylate cyclase were involved in the response to NO. However, inhibition of adenosine diphosphate (ADP) ribosyltransferase significantly decreased the response to L-arginine. Preincubation for 12 hours with 150 μmol/L (Z)-1-[(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate; IP3, inositol triphosphate decreased the 2-hour cellular response to CCK8 and SNP. Conclusions: NO regulates rabbit D-cells. Acute exposure stimulates somatostatin mediated by ADP ribosylation, whereas long-term exposure reduces cellular responses to stimuli. The latter pathway may be responsible for the suppression of somatostatin in H. pylori gastritis.

GASTROENTEROLOGY 2002;123:566-576

Section snippets

Chemicals

Collagenase (type 1 and type H), ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), deoxyribonuclease (DNAse), bovine serum albumin (BSA), glutamine, HEPES, streptomycin sulphate, gentamicin sulphate, penicillin, hydrocortisone, bovine insulin, fetal calf serum (FCS), gelatin, CCK8, sodium nitroprusside (SNP), sodium nitrite, L-arginine, L-lysine, epinephrine, 3-aminobenzamide and lactate dehydrogenase (LDH) diagnostic kit were all purchased from Sigma Chemicals (Poole, Dorset,

Immunocytochemistry

At the time of experimentation, 45% of cells in the preparation were D-cells (Figure 1).

. (A) Cells stained with monoclonal rat antisomatostatin antibody (arrows) at a dilution of 1:200 (×40). (B) Control cells without monoclonal antibody (×40).

The rest of the cells were mostly mucous cells: Thirty percent of the cells in the preparation were PAS-positive.

Effects of amino acids

L-arginine, 1 mmol/L, significantly stimulated the release of somatostatin in the D-cell preparation in the presence of CCK8. Hormone release

Discussion

The present studies show that L-arginine-stimulated somatostatin release is prevented by NOS inhibitors and that NO donors release somatostatin. We found evidence that ADP ribosylation is involved in the effect of NO on D-cells but no evidence that cGMP is involved. The stimulatory effect of NO on D-cells occurred over 2 hours, but preincubation with a slow-releasing NO donor for 12 hours suppressed the subsequent 2-hour response of D-cells to stimuli.

Although major advances have been made in

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    Address requests for reprints to: Naila Arebi, M.D., Gastroenterology, Hammersmith Hospital, London W12 0NN, United Kingdom. e-mail: [email protected]; fax: (440) 20-8749-3436.

    ☆☆

    Supported by a grant from the Astra Foundation (to Z.H.) and from the Digestive Diseases Foundation (to P.B.).

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