ARTICLE |
Correspondence to: Lars-Inge Larsson, Div. of Cell Biology, Dept. of Anatomy and Physiology, Royal Veterinary and Agricultural University, Gronnegaardsvej 7, DK-1870 Frederiksberg C, Denmark.
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Summary |
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The turnover of the epithelium of the gastrointestinal tract is regulated by a balance between cell multiplication and cell loss. We examined the effects of starvation on apoptosis in endocrine and other epithelial cells of rat antropyloric mucosa. Apoptosis was determined by the TUNEL reaction combined with immunocytochemical staining for gastrin and somatostatin. Apoptotic cell morphology was determined by bisbenzimide staining for DNA. Both gastrin and somatostatin cells showed a significantly lower apoptotic index than the general epithelium. This agrees with the longer turnover kinetics of gastric endocrine cells. On starvation, the apoptotic index of the general epithelium and of the gastrin but not of the somatostatin, cells increased significantly. This was prevented by the nitric oxide synthase (NOS) inhibitor L-NAME but not by its inactive stereoisomer D-NAME. Immunoreactive neuronal NOS was present in somatostatin cells, in nonendocrine cells predominating in the surface and pit epithelium, and in rare nerve fibers. Endothelial cell NOS was present in vessels, whereas the inducible isoform was barely detectable. Thus, endogenous NOS isoforms participate in regulating antropyloric epithelial apoptosis during starvation. The close paracrine relation between somatostatin cells and gastrin cells suggests that the former regulates apoptosis of the latter through release of NO. (J Histochem Cytochem 48:123131, 2000)
Key Words: gastrin, somatostatin, apoptosis, nitric oxide synthase, L-NAME, stomach, NO
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Introduction |
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The epithelium of the gastrointestinal tract is continuously renewed through a balance between cell multiplication and programmed cell death (apoptosis) (
In this study we developed a method for studying the apoptotic index in immunocytochemically identified endocrine cells, assessed the apoptotic index of endocrine gastrin and somatostatin cells and of the general epithelium in freely fed and starved rats, studied the distribution and expression of different NOS isoforms in antropyloric mucosa, and investigated the role of NO formation in apoptosis of gastrin and somatostatin cells and of the general epithelium.
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Materials and Methods |
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Tissue Material
Groups of six adult female Wistar rats each (body weight 200 g) were fasted for 18 hr in wire-bottomed cages and were then left untreated or given either L- or D-NAME (1.125 mg/kg) (Sigma; St Louis, MO) by gavage, and were decapitated 3 hr later. A fourth group of six female rats were freely fed. Preliminary experiments established that administration of L-NAME by gavage to freely fed rats produced unacceptably large variations in results that were ascribed to the widely variable amounts of food remaining in the stomachs of freely fed rats. The rats were fasted overnight because earlier studies have established that this period of fasting results in significant decreases in gastrin cell numbers (
Immunocytochemistry
Three-µm paraformaldehyde-fixed paraffin sections were microwaved (Polar Patent PP-780; Axlab, Copenhagen, Denmark) three times for 5 min at 780 W in 10 mM citrate buffer, pH 6.0. Cryostat sections were hydrated in Tris-buffered saline (TBS). All sections were reacted overnight with rabbit antisera to synthetic human gastrin I and synthetic somatostatin-14 (Dako; Glostrup, Denmark) or to synthetic peptides derived from the middle [728743] region (Chemicon International; Temecula, CA) or C-terminal [14111433] region of the human nNOS sequence (Research & Diagnostic Antibodies; Berkeley, CA). The two regions of nNOS are highly conserved. Therefore, the human nNOS [728743] region is identical to the [724739] region of rat nNOS and the human [14111433] region differs from the rat [14071429] region by only a single amino acid substitution (Ala/Thr) (
Detection and Quantitation of Apoptosis In Situ
Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end-labeling (TUNEL) was performed essentially as described (
For quantitations, alkaline phosphatase-stained TUNEL sections were immunocytochemically stained with rabbit anti-gastrin or rabbit anti-somatostatin antisera, followed by peroxidase-labeled swine anti-rabbit Ig (Dako) and development in 3-amino-9-ethylcarbazoleH2O2 medium (
Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)
Oligonucleotide primers were synthesized on an Applied Biosystems 392 DNA/RNA synthesizer (Applied BioSystems; Foster City, CA). A sense (5'-CCGGAATTCGAATACCAGCCTGATCCATGGAA) and antisense (5'-GCCGGATCCTCCAGGAGGGTGTCCACCGCATG) primer set was used to detect nNOS mRNA. Total RNA was isolated from scraped antropyloric mucosa by the TRizol reagent (Life Technologies; Grand Island, NY). Reverse transcription was carried out using the GeneAmp RNA PCR kit (PerkinElmer; Roche Molecular Systems, Branchburg, NJ). The cDNA obtained was amplified using optimized buffers containing 1.5 mM MgCl2. All reactions used 2.5 U/100 µl AmpliTaq DNA polymerase. PCR was carried out with the GeneAmp PCR System 2400 (PerkinElmer) using 40 cycles (94C for 45 sec, 59C for 60 sec, and 72C for 90 sec). Controls were made by omitting template cDNA and by amplifying rat ß-actin using primers placed in different exons of the gene. With genomic DNA, the amplicon size using this primer set would be 713 bp instead of the 503 bp generated from cDNA. In addition, reverse transcriptase was deleted in some experiments. Amplicon size was checked by 2% agarose gel electrophoresis.
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Results |
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Distribution and Quantitation of Apoptotic Cells
By the TUNEL method, apoptotic cells were detected in the surface epithelium, the pits, and the glands. In freely fed rats, 0.35 ± 0.02% (n = 6) of all epithelial cells were apoptotic (Figure 1). The frequencies of apoptotic G- and D-cells were determined by first reacting sections using the TUNEL method, and then restaining them for gastrin and somatostatin, respectively (Figure 2). Apoptotic G- and D-cells predominated in the bottom half of the glands. In freely fed rats, 0.23 ± 0.06% (n = 6) of all G-cells and 0.13 ± 0.05% (n = 6) of all D-cells were apoptotic. This difference was significant (p=0.02). In addition, the AI of the general epithelium (0.35 ± 0.02%) was significantly higher than that of the G- (p<0.01) and the D-cells (p<0.01) (Figure 1). After fasting for 18 hr, the AI of G-cells increased to 0.34 ± 0.06% (n = 6) (p<0.02 compared to freely fed animals) and the AI of the general epithelium increased to 0.61 ± 0.08% (n = 6) (p<0.02 compared to freely fed animals). The AI of the D-cells did not change significantly on fasting [0.16 ± 0.06 (n = 6); p=0.4].
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Controls and Validation of the TUNEL Method in Rat Antropyloric Mucosa
Omission of either TdT or digoxigenindUTP from the TUNEL reaction produced no staining. To correlate the morphology of the cells to TUNEL positivity, some sections were first stained by the TUNEL method (using immunofluorescent detection) and then restained for DNA using bisbenzimide. Such sections revealed that all TUNEL-positive cells of the rat antropyloric mucosa also displayed the typical morphology of apoptotic cells. This morphology included early stages displaying condensed chromatin and later stages displaying fragmentation of condensed chromatin and eventual formation of apoptotic bodies. By triple staining we could confirm that this also applied to endocrine cells (Figure 2).
Effects of Inhibition of NOS Activity on Antropyloric Apoptosis
Female rats fasted for 18 hr received either L-NAME or its inactive stereoisomer D-NAME by gavage and were sacrificed 3 hr later. Comparisons between apoptotic indices of untreated, fasted rats (n = 6) with D-NAME-treated, fasted rats (n = 6) showed no differences with respect to general epithelium (0.61 ± 0.08 vs 0.59 ± 0.07; p=0.6), G-cells (0.34 ± 0.06 vs 0.33 ± 0.04; p=0.9), or D-cells (0.16 ± 0.06 vs 0.13 ± 0.05; p=0.4) (Figure 1). In contrast, L-NAME treatment (n = 6) significantly reduced the AI of the general epithelium (0.35 ± 0.04; p<0.01) and of the G-cells (0.24 ± 0.05; p<0.02) while having no effect on the AI of the D-cells (0.13 ± 0.05; p=0.9).
Distribution of NOS-positive Cells
Staining of rat antropyloric mucosa by the polyclonal nNOS antisera demonstrated scattered strongly stained endocrine-like cells, which by double staining were found to correspond to somatostatin cells (Figure 2). In addition, the mid-region nNOS antibody also produced weaker staining of many epithelial cells that were mainly present in the pits and surface epithelium (Figure 3). Only very sparse nerve terminals were detected in the mucosal layer. Preabsorption of both polyclonal antisera against the corresponding synthetic peptide eliminated all staining, whereas absorption against somatostatin or poly-L-lysine did not affect the staining. Staining with the monoclonal nNOS antibody was difficult to interpret because of heavy nuclear background staining. A monoclonal antibody against iNOS stained only extremely sparse immunopositive cells of the lamina propria. The ecNOS monoclonal antibody produced staining of endothelial cells present in many blood vessels (not shown).
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RT-PCR
By use of nNOS-specific primers, one strong band corresponding in size to that expected from nNOS transcripts was obtained from rat antropyloric RNA (Figure 4). Simultaneous amplification using an intron-spanning ß-actin primer set produced only the band expected from RNA (503 bp) and excluded the presence of genomic contamination. Moreover, no band could be detected in controls from which reverse transcriptase was excluded.
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Discussion |
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Our results demonstrate that the frequency of apoptosis in the antropyloric region of the stomach varies with the prandial state and that NO synthesis is required for the increased apoptosis seen after fasting. Because all three known isoforms of NOS can be inhibited by L- but not by D-NAME, they could conceivably all contribute to the fasting-induced apoptosis observed. Our present data and data from others show that the two Ca2+calmodulin-dependent enzyme forms are present in the gastric mucosa (
Our data leave room for the hypothesis that D-cells regulate apoptosis in G-cells by paracrine release of NO. Thus, local release of NO can induce apoptosis (
Very recently,
In addition to the effects on G-cells, starvation and NOS inhibitor treatment also influenced the AI of the general antropyloric epithelium. Apoptotic general epithelial cells were detected also in the surface and pit epithelium, where D-cells are virtually absent. Possibly, changes in gastric blood flow caused by L-NAME treatment could affect the degree of apoptosis. However, this mechanism would be expected to be rather unspecific and to affect all gastric epithelial cell types, including the D-cells. A more likely explanation is that the other nNOS-positive epithelial cells, which were found to predominate in the surface and pit regions, could be important for regulating adaptive apoptosis during starvation. Alternatively, the expression of other cellular survival factors, including gastrin itself or neuronal factors, may be regulated by NO-dependent mechanisms during starvation.
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Footnotes |
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1 Present address: Musculoskeletal Research Center, Department of Orthopaedic Surgery, Pittsburgh, PA.
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Acknowledgments |
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Supported by grants from the Danish Medical Research Council and the Danish Cancer Foundation. Dr Cao was supported by a scholarship from the Danish National Research Foundation.
Received for publication May 13, 1999; accepted August 10, 1999.
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