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Keywords:

  • gastrointestinal tract;
  • stomach;
  • survival

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Abstract  The factors underlying the survival and maintenance of interstitial cells of Cajal (ICC) are not well understood. Loss of ICC is often associated with loss of neuronal nitric oxide synthase (nNOS) in humans, suggesting a possible link. The aim of this study was to determine the effect of neuronal NO on ICC in the mouse gastric body. The volumes of ICC were determined in nNOS−/− and control mice in the gastric body and in organotypic cultures using immunohistochemistry, laser scanning confocal microscopy and three-dimensional reconstruction. ICC numbers were determined in primary cell cultures after treatment with an NO donor or an NOS inhibitor. The volumes of myenteric c-Kit-immunoreactive networks of ICC from nNOS−/− mice were significantly reduced compared with control mice. No significant differences in the volumes of c-Kit-positive ICC were observed in the longitudinal muscle layers. ICC volumes were either decreased or unaltered in the circular muscle layer after normalization for the volume of circular smooth muscle. The number of ICC was increased after incubation with S-nitroso-N-acetylpenicillamine and decreased by N(G)-nitro-l-arginine. Neuronally derived NO modulates ICC numbers and network volume in the mouse gastric body. NO appears to be a survival factor for ICC.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Coordinated gastrointestinal motility requires a normal complement of interstitial cells of Cajal (ICC).1 ICC have diverse functions in the gastrointestinal (GI) tract. ICC generate and propagate electrical slow waves functioning as pacemaker cells,2,3 release carbon monoxide to set the membrane potential and the membrane potential gradient across the circular muscle layer,4 act as mechanosensors5,6 and mediate enteric neurotransmission.7 Decreased numbers or disrupted networks of ICC are associated with a number of human GI motility disorders, including slow transit constipation,8,9 pseudo-obstruction,10–12 and diabetic enteropathy.13

The factors that modulate survival of ICC in the GI tract are incompletely understood. ICC express c-Kit, a receptor tyrosine kinase, that is a well-established marker for ICC.3,14 Stem cell factor, also known as steel factor is the ligand for c-Kit. Mouse models have shown that a normal steel factor/c-Kit signalling pathway is necessary for the development and maintenance of ICC.2,3,15,16 However, steel factor appears to be not the only factor required for ICC survival. Injection of newborn mice with a neutralizing antibody for c-Kit is only effective in depleting ICC in one particular strain and is not effective in adult mice.16,17 Moreover, ICC survive in culture without steel factor, albeit in reduced numbers.15

Enteric nerves and ICC are often found in close proximity and their interactions seem to be required to maintain normal function of the GI tract.18 In nearly all human motility disorders associated with loss of ICC, there is a concomitant loss of enteric neurones.13,19–21 When the subtypes of enteric neurones that were decreased in association with ICC loss were examined, a decrease in neuronal nitric oxide synthase (nNOS), the synthetic enzyme for nitric oxide (NO), was found.13 This relationship between nNOS and ICC is also seen in mouse models of diabetes. In non-obese diabetic (NOD) mice, diabetes is associated with both a loss of nNOS and a decreased number of ICC.22,23

Together with its well-established role as an inhibitory nonadrenergic/noncholinergic (NANC) neurotransmitter24 in the GI tract, NO has several other functions. NO is not only a cytotoxic free radical but it is also cytoprotective depending on the cellular expression and local concentration.25 NO can act as a cytoprotective molecule through several mechanisms, including through a cGMP-independent antioxidative process by S-nitrosylation or S-nitrosoglutathione-scavenging free radicals and inhibition of caspase-3, or a cGMP-dependent mechanism such as upregulation of anti-apoptotic bcl-2.26,27

The concomitant loss of enteric neurones and ICC in several motility disorders, the human and mouse studies showing loss of nNOS expression in enteric neurones in parallel with loss of ICC, coupled with the cytoprotective functions of NO, suggest the possibility that NO generated from nNOS is a survival factor for ICC. To explore this hypothesis we used nNOS−/− mice, lacking the gene encoding nNOSα28 and ICC-enriched cell cultures. (Parts of this study have previously been published in abstract form.)29

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Animals

Eight- to 12-week-old homozygous mutant female mice, B6;129S-Nos1tm1Plh/J, obtained from Jackson Laboratories (Bar Harbor, ME, USA) with a targeted disruption of the nNOSα gene (nNOS−/−)28 were used to determine the effect of loss of nNOS on ICC. All protocols were approved by the Mayo Foundation Institutional Animal Care and Use Committee (IACUC). Female B6129SF2/J 101045 mice (Jackson Laboratories) were used as controls.

Dissection of tissue

The gastric body (corpus) was chosen as the region to be studied based on previously published reports that described a decrease in nNOS expression and ICC in this region.22,23 The intensive computing and analysis required for these studies precluded investigation of more than one region of the stomach. A rectangular section of the gastric body was removed by making a cut at the oesophago-gastric junction and continuing along the white cap (denoted by the dotted line in Fig. 1A) to the greater curvature and then extending posteriorly back up to the junction. A second cut was made parallel to the first, along a line equidistant from the junction between the squamous and glandular epithelium similar to the first cut. The squamous–glandular epithelial junction is indicated by an asterisk in Fig. 1A. This second cut was also extended posteriorly. The tissue resected was rectangular in shape. This method, rather than using fixed dimensions, was used to take into account differences in gastric size between control mice and nNOS−/− mice that have enlarged stomachs.30 The size of the resected tissue was 0.4 ± 0.08 cm ×1.0 ± 0.2 cm in control mice and 0.68 ± 0.16 cm ×1.51 ± 0.2 cm in nNOS−/− mice. Eight areas were studied within this piece of tissue. Areas 1–4 were from the proximal body and 5–8 from the distal body. Areas 2, 3, 6 and 7 were closer to the greater curvature and 1, 4, 5 and 8 closer to the lesser curvature (Fig. 1A). The tissue pieces were then pinned out (mucosa side up), in Hanks balanced salt solution (Mediatech, Inc., Herndon, PA, USA) without calcium or magnesium, onto blocks of Sylgard and the mucosa removed.

image

Figure 1.  Distribution of c-Kit-positive ICC in the wall of the gastric body in control mice and in nNOS−/− mice. (A) Schematic diagram of the mouse stomach and the areas studied. The cartoon represents anterior and posterior views of the stomach and the rectangular tissue section studied. The indicated size of the rectangular section is for control mice. The edge of the white cap is denoted by the dotted line. The junction between the squamous and glandular epithelial portion is indicated by an asterisk. (B) Volumes of c-Kit-positive ICC in control (open bars) and nNOS−/− mice (solid bars) in each of the eight areas studied are shown for the total muscle wall (ICC-Total), circular muscle layer (ICC-CM), myenteric plexus region (ICC-MY) and the longitudinal muscle layer (ICC-LM). *P < 0.05, mean ± SEM, n = 6 animals. (C) Volume rendered images of myenteric ICC from control mice (upper) and nNOS−/− mice (lower panels) for each of the eight areas. Note the lower volume of c-Kit-positive ICC-MY in the nNOS−/− mice. The scale bar (upper panel left) is 50 μm.

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Primary cell cultures of ICC

Primary cultures of ICC were prepared from the small intestine of neonatal control mice (3–6 days postnatum) as previously published.15,31 Briefly, the muscle layers were peeled off and dissociated using a cocktail containing 2400 U collagenase, 20 mg bovine serum albumin, 20 mg trypsin inhibitor, 5.5 mg adenosine triphosphate and 10 mL of Hank's balanced salt solution at 32 °C for 17 min. The freshly dispersed cells were cultured on 22-mm glass coverslips at a density of 3 × 105 cells/coverslip. The coverslips were precoated with irradiated fibroblasts, genetically modified to express the steel factor. This procedure results in a significantly greater number of ICC than if the steel factor-producing fibroblasts are omitted.15,31S-nitroso-N-acetylpenicillamine (SNAP) was used as an NO donor at a concentration of 100 μmol L−1 twice daily32 and N(G)-nitro-l-arginine (l-NNA) at a concentration of 200 μmol L−1 daily as an nNOS inhibitor33,34 daily.

Organotypic cultures

Tissues of nNOS−/− mice were used in these experiments. Tissue from region 7 was removed and pinned flat on Sylgard-coated dishes. The mucosa was removed with sharp dissection scissors in sterile Hanks balanced salt solution (Mediatech, Inc., Herndon, PA, USA) without calcium or magnesium. After removal of the mucosa, the tissue was rinsed several times with Hanks. The tissue was then placed in Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10% characterized fetal bovine serum (Hyclone, Logan, UT, USA) and 1% antibiotic–antimycotic (Invitrogen) and rinsed five times with media before being placed into an incubator at 37 °C with 5% CO2. The cultures were treated twice a day for 6 days by replacing half the media with media alone for control or media with 200 μmol L−1 SNAP for a final concentration of 100 μmol L−1. After 7 days in culture, the tissues were rinsed six times for 2 min with 1x phosphate-buffered saline (PBS) and then fixed in acetone for 15 min at 4 °C on a shaker. Immunohistochemistry, confocal microscopy and three-dimensional volume reconstruction for the myenteric plexus region were completed as described below.

Immunohistochemistry

Interstitial cells of Cajal were identified in whole-mount preparations using fluorescent immunolabelling for c-Kit as previously described.35 Briefly, the tissue pieces were fixed in cold acetone for 15 min at 4 °C. Following fixation, the preparation was rinsed for 30 min in 1x PBS containing 10 mmol L−1 Na2HPO4, pH 7.4 and 150 mmol L−1 NaCl. For immunostaining, non-specific labelling was blocked by incubation in PBS containing 10% normal donkey serum (NDS; Jackson ImmunoResearch Lab, Inc., West Grove, PA, USA) and 0.3% Triton X-100 (Sigma, St Louis, MO, USA). The tissue was next incubated for 2 days at 4 °C with the primary antibody against c-Kit (Table 1, ACK2; eBioscience, San Diego, CA, USA) diluted in PBS containing 5% NDS and 0.3% Triton X-100. After washing with PBS, specific labelling was detected by incubation with a Cy3-conjugated donkey anti-rat secondary antibody (Table 1; Jackson ImmunoResearch Lab, Inc.) diluted in PBS containing 2.5% NDS and 0.3% Triton X-100. After rinsing in PBS, nuclei in the tissue were stained by incubating the tissue for 30 min at 4 °C with 0.3 μmol L−1 4′,6-diamidino-2-phenylindole,dihydrochloride (DAPI; Molecular Probes, Inc., Eugene, OR, USA) in 1x PBS.

Table 1.   Antibodies used for immunohistochemistry
Purpose of IHCAntibodyFinal concentration or titerFixative
PrimarySecondaryPrimarySecondary
  1. PFA, paraformaldehyde; FITC, flourescein isothiocyanate; IHC, immunohistochemistry; nNOS, neuronal nitric oxide synthase.

c-Kit in whole mounts and cell cultureACK-2Donkey anti-rat CY31.7 μg ml−1μg ml−1Acetone (15 min, 4 °C)
Enteric neurones in whole mountsHu antiserumDonkey anti-human FITC1 : 50010 μg ml−14% PFA (>2 h, 4 °C)
nNOS in cell culturesRabbit anti-NOS1Donkey anti-rabbit CY31 : 4000μg ml−14% PFA (10 min, 4 °C)
iNOS in cell culturesRabbit anti-NOS2Donkey anti-rabbit CY31 : 250μg ml−14% PFA (10 min, 4 °C)
eNOS in cell culturesRabbit anti-NOS3Donkey anti-rabbit CY31 : 250μg ml−14% PFA (10 min, 4 °C)

Neurones were identified using human anti-Hu antiserum (gift from Dr Vanda Lennon, Mayo Clinic, Rochester, MN, USA) in whole-mount preparations as previously published.36 Tissue was prepared as described above. The tissue was fixed in 4% paraformaldehyde (PFA) for 2 h. After a wash in PBS, the tissue was incubated overnight in a blocking solution consisting of PBS, 4% NDS and 0.5% Triton X-100. The anti-Hu antiserum (Table 1) was pre-absorbed in guinea-pig liver powder for 2 h at room temperature. The tissue was then incubated in pre-absorbed human anti-Hu antibody diluted in PBS containing 4% NDS and 0.5% Triton X-100 for 24 h at 4 °C. After washing in PBS the tissue was incubated overnight with donkey anti-human flourescein isothiocyanate (FITC) (Table 1) diluted in PBS. The number of myenteric ganglia was counted in an area with a relatively less severe loss of c-Kit-positive ICC (area 1) and in an area with the most severe loss of c-Kit-positive ICC (area 7) in the myenteric plexus region of nNOS−/− mice. Ganglia were counted using low-power objectives (10×) from the same fields used to collect confocal images for c-Kit-positive ICC. The number of ganglia was calculated per millimetre.2 The number of neurones was counted using a higher power objective (20×) from 45 randomly selected ganglia in the same areas.

Interstitial cells of Cajal in primary cell culture were identified by immunolabelling for c-Kit as previously reported.31 NOS-positive cells in the primary cultures were identified by using antibodies selective for subtypes of NOS (see Table 1). ICC-enriched cultures were immunolabelled for NOS subtypes using the antibodies shown in Table 1 by a modification of the standard procedure for c-Kit immunolabelling.15,31 Cells on 25-mm glass coverslips were washed and fixed in the appropriate fixative (see Table 1), washed again, incubated with blocking solution for 2 h and then in primary antibody at the indicated titre overnight at 4 °C. After washing with PBS, the secondary antibody was added at the indicated titre (Table 1) for 2 h at room temperature followed by washing with PBS and mounting the coverslips on microscope slides to view. For double labelling, coverslips were fixed in acetone and immunolabelled for c-Kit with ACK-2 as described above, then washed and fixed again in 2% paraformaldehyde for 10 min in order to immunolabel for the other markers including nNOS (Chemicon, Temecula, CA, USA), inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) (Santa Cruz, Santa Cruz, CA, USA). For a positive control, iNOS was induced in cultured fibroblasts with 50 ng ml−1 human interferon-gamma (IFN-γ; R&D System, Inc., Minneapolis, MN, USA) and 5 ng mL−1 interleukin-1-beta (IL-1-β; R&D System, Inc.) and the induced fibroblasts then immunolabelled with an antibody raised against iNOS (NOS2; Table 1). The endothelial lining of blood vessels in mouse jejunal tissue sections provided a positive control for labelling with an antibody raised against eNOS (NOS3; Table 1). In all experiments, serial dilutions were carried out to determine the optimal antibody concentration, and negative controls omitting the primary antibody were also carried out.

ICC volume analysis

The volume of c-Kit-positive immunoreactivity in each of the eight areas of the gastric body whole mount was determined as previously described.35 Briefly, confocal image stacks were collected from each area using a laser scanning confocal microscope (Zeiss LSM510; Carl Zeiss, Inc., Oberkochen, Germany) equipped with a water immersion 40× objective (NA 1.3). The sample from each area consisted of six image stacks collected from each of the eight areas. The average number of confocal slices per stack was 239 ± 107. The voxel size was 0.45 μm ×0.45 μm × 0.60 μm. The dimensions of each confocal plane were 512 × 512 pixels, i.e. 230.4 × 230.4 μm. The images in the stack were divided into four categories: total muscle layer, circular muscle layer, myenteric plexus region and longitudinal muscle layer. The volume of c-Kit immunoreactivity was determined and reconstructed using AnalyzeTM (Mayo Foundation, Rochester, MN, USA) by an analyst blind to the source of each data set. Mast cells were not reconstructed. The thickness of the smooth muscle layers was determined by using the DAPI-labelled nuclei to mark the beginning and end of each layer in each image stack.

Western blots

Homogenates of gastric body tissue pieces were lysed in a solution consisting of 50 mmol L−1 Tris-HCl, 1% NP-40, 0.25% Na-deoxycholate, 150 mmol L−1 NaCl, 1 mmol L−1 ethylenediaminetetraacetic acid (EDTA), 1 mmol L−1 activated Na3VO4, 1 mmol L−1 NaF and protease inhibitors including 1 μg mL−1 of aprotinin, leupeptin, pepstatin and 1 mmol L−1 phenylmethylsulphonyl fluoride. From the tissue lysates, 30 μg of protein was resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% gel. Western blots were obtained after SDS-PAGE and transferred to immuno-blot polyvinylidene flouride (PVDF) membranes (BioRad Laboratories, Hercules, CA, USA). Either rabbit polyclonal c-Kit or anti-nNOS (NOS1) antibodies (Stressgen Biotechnologies Corporation, Victoria, BC, Canada and Santa Cruz Biotech. Inc., respectively) were used as primary antibodies. Bands were scanned and quantified against local background using the Bio-Rad Gel Doc system (Bio-Rad).

Statistical analysis

Data are presented as mean ± SEM. Statistical significance was determined using paired Student's t-test. A P-value of <0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

c-Kit protein expression is decreased in the gastric body of nNOS−/− mice

To determine whether loss of nNOS results in changes in ICC in the gastric body, the amount of c-Kit receptor protein expressed in the muscle layers of the mouse gastric body was determined by Western blot analysis. As expected, nNOS protein was not detected in protein lysates from the gastric body of nNOS−/− mice, although a faint non-specific band was observed in blots of nNOS−/− tissues, as previously reported.30 c-Kit protein was present at much lower levels in preparations from nNOS−/− mice compared with preparations from control mice (Fig. 2A). The difference in c-Kit expression between control (1.94 ± 0.70) and nNOS−/− (0.65 ±0.32) mice was significant (t-test, P < 0.05) and reproducible as determined by densitometry of the bands from independent experiments using tissue from four control and four nNOS−/− mice (Fig. 2B). The same amount of protein was loaded in each lane as determined by blotting for the housekeeping genes β-tubulin and glyceraldehyde-3-phosphate dehydrogenase (Fig. 2A).

image

Figure 2.  c-Kit expression is decreased in the gastric body of nNOS−/− mice. (A) Western blot using polyclonal antibodies against either nNOS or c-Kit. β-tubulin and GAPDH were used to assess protein loading. (B) Optical density (OD) of the bands for c-Kit was measured from four controls and four nNOS−/− mice. *P < 0.05.

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ICC are distributed in a heterogeneous fashion in the gastric body of mice

Under low-power magnification, it was apparent that there was a considerable variation in the number and distribution of ICC in the eight areas of the mouse gastric body studied. Therefore, the total volume of c-Kit immunoreactivity was determined in the total tissue, circular muscle layer, myenteric plexus region and longitudinal muscle layer in the eight defined areas in control tissue (see Fig. 1A). The overall quantitative results obtained for the C57BL/6J mice used as controls for the nNOS−/− mice were similar to those previously reported for other strains of mice.37–39 Specifically, for all types of ICC, higher volumes of c-Kit-positive ICC were detected in the areas along the greater curvature (open bars of areas 2, 3, 6 and 7; Fig. 1B) when compared with volumes of c-Kit-positive ICC along the lesser curvature (open bars of areas 1, 4, 5 and 8; Fig. 1B). Segmentation of the total volumes into circular (ICC-CM), myenteric (ICC-MY) and longitudinal regions (ICC-LM) showed that this difference was mainly due to the higher volume of c-Kit-positive ICC-MY in these regions (Fig. 1B). No c-Kit-positive ICC-MY were detected in tissues from regions near the lesser curvature on the posterior aspect of the gastric body (areas 4 and 5). The distribution of ICC-MY was inverse to the distribution of ICC in the longitudinal muscle layer (Fig. 1B). The volume of c-Kit-positive ICC in the longitudinal muscle layer was low in all areas apart from areas 4 and 5 (near the lesser curvature on the posterior aspect), the areas where ICC-MY were not present. The volume of c-Kit-positive ICC in the circular muscle layer was larger in the proximal gastric body compared with the volume of c-Kit-positive ICC in the distal gastric body (areas 1, 2, 3 and 4 compared with areas 5, 6, 7 and 8; P < 0.05).

Distribution and volume of ICC in the gastric body of mice is altered in nNOS−/− mice

The volume of c-Kit-positive ICC in the gastric body of nNOS−/− mice was quantified in the same eight regions that were studied in control tissue (Fig. 1). There were significant differences between control (open bar) and nNOS−/− mice (solid bar) in the total volume of c-Kit-positive ICC. In areas 1, 2 and 3, a significant decrease in total c-Kit-positive ICC volume was detected compared with controls (a decrease of 31 ± 2.1%, 43 ± 4.3% and 23 ± 7.1%, respectively; Fig. 1B). In areas 4, 5, 7 and 8 there was no difference in the total volume of c-Kit-positive ICC compared with controls whereas in area 6 the total volume of c-Kit-positive ICC was increased (137 ± 3.3%; Fig. 1B). In contrast to the total volumes, the volumes of c-Kit-positive ICC in the myenteric plexus region from nNOS−/− mice were significantly lower in all the areas where ICC-MY were present when compared with the volumes for the same regions from control mice (53 ± 11.4%, 71 ± 5.8%, 43 ± 1.1%, 49 ± 6.7%, 71 ± 8.0%, 58 ± 8.4% lower for areas 1, 2, 3, 6, 7 and 8 respectively; P < 0.05, Fig. 1B). The volumes of c-Kit-positive ICC-IM in the circular muscle layer were significantly higher in areas 6 (430 ± 43%), 7 (339 ± 36%) and 8 (240 ± 13.9%) (distal gastric body, P < 0.05; Fig. 1B). There were no significant differences in c-Kit-positive ICC volumes in the longitudinal muscle layer between nNOS−/− and control mice. Representative volume rendered images of c-Kit-positive ICC-MY from control mice and nNOS−/− mice are shown in Fig. 1C.

To determine if there was disruption of the remaining ICC networks, images obtained from areas 2 and 7, the areas where the decrease in c-Kit-positive ICC-MY in the nNOS−/− mice was most severe (Fig. 3), were examined more closely. c-Kit-positive ICC from control mice showed well-developed ICC with intact processes and, in the myenteric plexus region, intact networks with multiple processes from the cell bodies. In nNOS−/− mice, the remaining ICC cell bodies were often less well formed (arrows) and the processes blunted and decreased resulting, in the myenteric plexus region, in disrupted networks.

image

Figure 3.  High magnification images of c-Kit-positive ICC in control and nNOS−/− mice. Confocal image stacks were collected from either the circular muscle layer (CM) or myenteric plexus region (MY) of areas 2 and 7 using a laser scanning confocal microscope (40×). Arrows show that the remaining ICC cell bodies in the areas from nNOS−/− mice were less well formed (arrows) and the processes blunted and decreased resulting in disrupted networks in the myenteric plexus region. The scale bar (left upper panel) is 50 μm and applies to all images.

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Muscle layer thickness is increased in the mouse gastric body of nNOS−/− mice

In nNOS−/− animals, the uniform decrease in c-Kit-positive ICC volumes in the myenteric plexus region contrasted with the changes observed in the circular muscle layer. The stomach of the nNOS−/− mice is enlarged when compared with controls, with the muscle wall reported to be 60% thicker than that of controls.30 Therefore, we measured the thickness of the circular muscle layer of nNOS−/− mice to determine whether the increased volume of c-Kit-positive ICC in the circular smooth muscle layer in specific areas simply reflected an increase in the thickness of the muscle layer. In all regions examined, the circular smooth muscle layer was two- to threefold thicker compared with controls (Table 2). When the volume of c-Kit-positive ICC in each area studied was normalized to the volume of the circular smooth muscle layer in the same area, the density of c-Kit-positive ICC in the circular muscle layer of the nNOS−/− mouse was significantly lower in the proximal gastric body compared with controls (areas 1–4; Fig. 4). There was no difference in the density of c-Kit-positive ICC in the circular muscle layer in the distal gastric body (areas 6–8; Fig. 4) with the exception of area 5.

Table 2.   Thickness of the circular smooth muscle layer in the gastric body
AreaControl (μm)nNOS−/− (μm)
  1. *P < 0.05. Data are expressed as mean ± SEM, n = 6.

156.9 ± 6.395.4 ± 15.4*
254.4 ± 6.8111.9 ± 20.7*
354.8 ± 10.7118.7 ± 22.9*
436.1 ± 4.488.4 ± 11.4*
549.4 ± 6.896.9 ± 23.1*
643.1 ± 6.3155.9 ± 38.1*
741.0 ± 3.6125.5 ± 35.8*
845.8 ± 6.3107.2 ± 29.6*
image

Figure 4.  Percentage of total circular muscle volume positive for c-Kit-positive ICC. The volume of c-Kit-positive ICC in the circular muscle layer was normalized by the volume of circular muscle wall in each of the eight areas examined. The percentage of total circular muscle volume containing c-Kit-positive ICC was less in areas 1–5 and not different in areas 6–8. *P < 0.05, mean ± SEM, n = 6 animals.

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As the whole stomach in the nNOS−/− mice is enlarged compared with controls,22,30 the tissue pieces from the nNOS−/− mice were larger. It was therefore possible that the ICC-MY were simply spread over a larger area resulting in a lower apparent density of cells. This possibility was tested by staining myenteric neurones with an antibody to Hu and determining the number of myenteric ganglia and the number of neurones in controls and nNOS−/− mice in areas 1 and 7. No significant differences in numbers of ganglia between control and nNOS−/− mice were observed in either area (11.4 ± 0.8/mm2 in control and 10.7 ± 0.7/mm2 in nNOS−/− mice in area 1, 10.1 ± 0.9/mm2 in control and 10.1 ± 1.2/mm2 in nNOS−/− mice in area 7, n = 4, P > 0.05). There were also no significant differences in numbers of neuronal cell bodies in either area (905 ± 67 per 45 ganglia in control and 896 ± 49 per 45 ganglia in nNOS−/− mice in area 1, 1010 ± 46 per 45 ganglia in control and 979 ± 33 per 45 ganglia in nNOS−/− mice in area 7, n = 4, P > 0.05) between control and nNOS−/− mice.

Number of ICC in primary culture and organotypic culture is increased by an NO donor

Neuronal NOS is absent throughout development in nNOS−/− mice. Therefore, changes in ICC may reflect an indirect response to changes in other cell types in the muscle wall that may also be altered by the decrease in nNOS availability. To determine if NO has a direct effect on ICC, ICC cultures were treated for 2 days with either the NO donor SNAP (100 μmol L−1) twice a day or the nNOS inhibitor l-NNA (200 μmol L−1) daily. Treatment with the NO donor SNAP increased the number of c-Kit-positive ICC (37 ± 8% increase, P < 0.05, n = 4; Fig. 5A) and treatment with the NO inhibitor l-NNA decreased the number of c-Kit-positive ICC (56 ± 27% decrease, P < 0.05, n = 4; Fig. 5B) in culture.

image

Figure 5.  The NO donor SNAP and the NOS inhibitor l-NNA alter ICC number in primary cell culture. Cells were treated with either (A) 100 μm SNAP (cross-hatched bar) or (B) 200 μmol L−1l-NNA (solid bar). Control (open bar) numbers were normalized to 100%. The panels above the bar graphs show representative images from control ICC enriched cultures (left panels) and cultures exposed to SNAP or l-NNA (right panels). SNAP increased c-Kit-positive ICC numbers by 37 ± 8% (P < 0.05, n = 4), whereas l-NNA decreased the number of c-Kit-positive ICC by 56 ± 27% (P < 0.05, n = 4). ICC-enriched primary cultures were stained for nNOS (C) iNOS (D), and eNOS (E). Low power (10×, left panel) and higher power (40×, right panel) images of the nNOS staining showed the presence of NOS expressing cells with the characteristic morphology of neurones in the cultures. There was no staining for iNOS (left panel, cultures, right panel, fibroblasts induced by IFN-γ and IL-1-β as a positive control) or eNOS (left panel, cultures, right panel, tissue section showing a blood vessel as a positive control) in the cultures. The scale bars are 100 μm.

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The effect of l-NNA on c-Kit-positive ICC indicated that the cultures contain endogenous sources of NO from cells expressing one or more of the subtypes of NOS.40 Therefore, we used antibodies specific for nNOS, eNOS and iNOS to identify cells within the cultures that were generating NO. nNOS-positive cells that had the characteristic morphology of neurones were labelled in the cultures (Fig. 5C). eNOS or iNOS immunoreactivity was detected in positive controls (right panels of Fig. 5D,E) but not in the cultures (left panels of Fig. 5D,E).

The effect of the NO donor was also tested in organotypic cultures from nNOS−/− mice. Tissue was cultured from area 7, an area with marked decrease in c-Kit-positive ICC-MY in nNOS−/− mice compared with controls. The volume of c-Kit-positive ICC was increased 149 ± 26% (n = 4, P < 0.05) when the tissue was treated with 100 μmol L−1 SNAP for 6 days compared with untreated nNOS−/− controls (Fig. 6).

image

Figure 6.  The volume of c-Kit-positive ICC-MY was increased in nNOS−/− mouse organotypic cultures treated with SNAP. (A) Representative rendered images of ICC-MY from organotypic cultures obtained from nNOS−/− mice (untreated, left panel, SNAP 100 μmol L−1 for 6 days, right panel). Scale bar (upper panel left) is 25 μm and applies to both images. (B) Volume of c-Kit-positive ICC-MY in the organotypic cultures from untreated nNOS−/− mouse tissues (open bar) and SNAP treated (solid bar). *P < 0.05, unpaired t-test, mean ± SEM, n = 4 animals.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This study shows that NO plays a role in the survival and maintenance of ICC networks in the mouse gastric body. We observed that in the absence of nNOS-derived NO in NOS−/− mice, the c-Kit-positive ICC volumes in the gastric body were lower compared with tissues from control animals and that this difference was most pronounced in ICC from the myenteric plexus region. The remaining ICC did not appear to be normal with blunted processes and disrupted networks. Furthermore, we determined that administration of an NO donor to primary cultures and organotypic cultures resulted in increased numbers and volumes of ICC and that inhibition of NOS activity resulted in decreased numbers of ICC in primary culture, suggesting a direct effect of NO on ICC numbers. nNOS but not iNOS or eNOS expressing cells were present in the cultures. These complementary experimental approaches suggest that NO derived from nNOS plays a direct role in survival of ICC. Increased survival may be due to decreased cell death or increased proliferation, as both would result in increased numbers of ICC. Our experiments do not allow us to differentiate between the two, although the rapid increase in c-Kit-positive ICC volume in organotypic cultures in response to the NO donor suggests a proliferative effect. This effect of NO on ICC has not been previously reported. Indeed, the only well-accepted regulator of ICC survival and development is the steel factor, the ligand for the Kit tyrosine kinase receptor.15,41,42 Insulin and IGF-1 also modulate ICC numbers, but their effect also appears to be mediated through the steel factor.43 There are however several lines of evidence that raise the possibility that NO may act as a cytoprotective molecule for ICC. In particular, there is an established relationship between nitrergic neurotransmission and ICC. ICC and nNOS-positive nerve endings are closely associated physically,44,45 and depletion of ICC is associated with impaired nitrergic inhibitory neurotransmission.7 In humans, infantile hypertrophic pyloric stenosis is associated with low expression of nNOS and depleted numbers of ICC, changes that are reversible.21 Loss of nNOS and depletion of ICC are seen in diabetic enteropathy.13 A recent paper looking at the effect of diabetes on the human antrum also showed loss of c-Kit and nNOS expression. There was also a loss of substance P expression. However in this study, loss of kit and nNOS expression was limited to the circular muscle layer and not found in the myenteric plexus46 and the possible relation between loss of c-Kit and nNOS expression was not explored. These studies suggest that both changes of expression in c-Kit and in nNOS be examined in patients before ascribing a documented change in gut function to a particular cell type. In mouse models of diabetes, slowed gastric emptying, loss of nNOS and depletion of ICC are observed.22,23 However, it is not clear from these studies whether the initial injury was depletion of ICC or loss of nNOS and whether the concomitant loss of nNOS and ICC were causally linked or unrelated. The current study suggests that there may, in fact, be a causal link between the observed loss of nNOS and ICC and that NO may act as a cytoprotective molecule on mature ICC.

A neuronal source for a cytoprotective molecule for ICC was not considered likely as a study on GDNF−/− mice, which lack enteric neurones, suggested that ICC were qualitatively unaffected.42 Subsequent studies47,48 suggested that myenteric-like ICC were lacking in adult ls/ls and newborn c-ret−/− mice, and the morphology of the ICC in these animals resembled only intramuscular ICC. Furthermore, ICC with myenteric-like ICC morphology were present in culture experiments only when enteric neurones were present.47 The data in the present study are in agreement with these findings as loss of c-Kit-positive ICC volume was most pronounced in ICC from the myenteric plexus region and suggest that neuronally derived NO is particularly required for maintenance of ICC.

The functional changes in the stomach of nNOS−/− mice are both an obstruction due to impaired pyloric relaxation and a generalized impairment of gastric motility.30 nNOS−/− mice have an increase in smooth muscle thickness throughout the stomach unlike the localized thickening of only the pyloric muscle layer in infantile pyloric stenosis.30 This finding was confirmed in the present study which showed a two- to threefold increase in the thickness of the circular muscle layer compared with controls. The increase in size of the stomach in nNOS−/− mice and the increase in thickness of the muscle walls raised the possibility that the decrease in ICC in nNOS−/− mice was simply a dilutional effect of the increased tissue mass. This did not appear to be the case as changes in c-Kit-positive ICC volume were greater for ICC-MY than ICC-IM and it would be expected that a dilutional effect would be more noticeable in the circular muscle layer. Moreover, the myenteric plexus region was not significantly affected in nNOS−/− mice as documented by a similar number of enteric neurones in nNOS−/− mice compared with controls. These data also suggest that, in contrast to the effect of loss of nNOS on ICC, loss of nNOS does not lead to decreased neuronal numbers, as first demonstrated by Watkins et al.22

The delay in gastric emptying in nNOS−/− mice also raised the possibility that a functional obstruction could account for the decrease in ICC numbers. Partial small bowel obstruction in mice has been shown to decrease ICC numbers.49 It appears unlikely that functional obstruction could account for the decrease in ICC numbers. In the mouse model of intestinal obstruction, the depletion of ICC was highest just proximal to the obstruction and all sub-types of ICC were lost.49 In contrast, in the stomach of nNOS−/− mice, myenteric ICC were significantly more affected than other classes of ICC and the loss of c-Kit-positive ICC was more marked in the proximal gastric body further from the pylorus compared with the distal gastric body closer to the pylorus. Furthermore, the ICC culture experiments in the present study strongly suggest that NO can directly affect ICC numbers.

The cellular mechanisms by which loss of neuronally derived NO results in lower c-Kit-positive volumes of ICC-MY in the intact animal and fewer ICC in culture were not determined in the present study. NO is a well-established regulator of cell proliferation,50 cell death51,52 and cell migration.53 There are therefore several possible mechanisms by which loss of NO could alter the volume and distribution of ICC networks. NO has been shown to increase cellular proliferation through several mechanisms including S-nitrosylation or S-nitrosoglutathione-scavenging free radicals and inhibition of caspase-3, upregulation of anti-apoptotic bcl-2 and upregulation of PI-3 kinase and through Raf/MEK/ERK signalling pathways that are cGMP-independent.25–27,54 NO has been shown to be cytoprotective through a cGMP/protein kinase G-dependent pathway and also through multiple kinase pathway including PI-3 kinase, MEK, PKA and CaMK.55 The thickening of the muscle layer in the stomach is similar to the proliferation of smooth muscle cells that occurs when NO availability is reduced in vascular tissue.56 Thus, given that ICC and smooth muscle cells are derived from the same progenitor cells,17 it is possible that the hyperplasia and hypertrophy of gastric smooth muscle cells in the nNOS−/− mouse may occur at the expense of ICC development. However, it is more likely that the effects on smooth muscle hyperplasia and decreased c-Kit-positive ICC volume are independent because of the results of the ICC culture experiments and because it appears that after ICC reach a mature phenotype, neuronal factors are more important for ICC development47 than other factors.

In summary, using different approaches in intact animals and ICC cultures, we have shown that nNOS derived NO modulates c-Kit-positive ICC volumes and ICC numbers. Our findings suggest a novel role for NO as a cytoprotective molecule for ICC and add to our current understanding of the survival factors that regulate ICC numbers in the GI tract. Understanding the mechanisms that underlie this effect of NO on ICC numbers may lead to strategies to prevent or reverse ICC loss in human motility disorders.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We thank Gary Stoltz for tissue dissection and cell dissociation, Cheryl Bernard for assistance with immunohistochemistry and Kristy Zodrow for secretarial assistance. Grant support: NIH DK 68055 and DK 57061.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  • 1
    Der-Silaphet T, Malysz J, Hagel S, Larry Arsenault A, Huizinga JD. Interstitial cells of cajal direct normal propulsive contractile activity in the mouse small intestine. Gastroenterology 1998; 114: 72436.
  • 2
    Ward SM, Burns AJ, Torihashi S, Sanders KM. Mutation of the proto-oncogene c-kit blocks development of interstitial cells and electrical rhythmicity in murine intestine. J Physiol 1994; 480 (Pt 1): 917.
  • 3
    Huizinga JD, Thuneberg L, Kluppel M, Malysz J, Mikkelsen HB, Bernstein A. W/kit gene required for interstitial cells of Cajal and for intestinal pacemaker activity. Nature 1995; 373: 3479.
  • 4
    Farrugia G, Lei S, Lin X et al. A major role for carbon monoxide as an endogenous hyperpolarizing factor in the gastrointestinal tract. Proc Natl Acad Sci U S A 2003; 100: 856770.
  • 5
    Strege PR, Ou Y, Sha L et al. Sodium current in human intestinal interstitial cells of Cajal. Am J Physiol Gastrointest Liver Physiol 2003; 285: G111121.
  • 6
    Won KJ, Sanders KM, Ward SM. Interstitial cells of Cajal mediate mechanosensitive responses in the stomach. Proc Natl Acad Sci U S A 2005; 102: 149138.
  • 7
    Burns AJ, Lomax AE, Torihashi S, Sanders KM, Ward SM. Interstitial cells of Cajal mediate inhibitory neurotransmission in the stomach. Proc Natl Acad Sci U S A 1996; 93: 1200813.
  • 8
    He CL, Burgart L, Wang L et al. Decreased interstitial cell of cajal volume in patients with slow-transit constipation. Gastroenterology 2000; 118: 1421.
  • 9
    Lyford GL, He CL, Soffer E et al. Pan-colonic decrease in interstitial cells of Cajal in patients with slow transit constipation. Gut 2002; 51: 496501.
  • 10
    Kenny SE, Vanderwinden JM, Rintala RJ et al. Delayed maturation of the interstitial cells of Cajal: a new diagnosis for transient neonatal pseudoobstruction. Report of two cases. J Pediatr Surg 1998; 33: 948.
  • 11
    Vanderwinden JM, Rumessen JJ. Interstitial cells of Cajal in human gut and gastrointestinal disease. Microsc Res Tech 1999; 47: 34460.
  • 12
    Feldstein AE, Miller SM, El-Youssef M et al. Chronic intestinal pseudoobstruction associated with altered interstitial cells of cajal networks. J Pediatr Gastroenterol Nutr 2003; 36: 4927.
  • 13
    He CL, Soffer EE, Ferris CD, Walsh RM, Szurszewski JH, Farrugia G. Loss of interstitial cells of cajal and inhibitory innervation in insulin-dependent diabetes. Gastroenterology 2001; 121: 42734.
  • 14
    Burns AJ, Herbert TM, Ward SM, Sanders KM. Interstitial cells of Cajal in the guinea-pig gastrointestinal tract as revealed by c-Kit immunohistochemistry. Cell Tissue Res 1997; 290: 1120.
  • 15
    Rich A, Miller SM, Gibbons SJ, Malysz J, Szurszewski JH, Farrugia G. Local presentation of Steel factor increases expression of c-kit immunoreactive interstitial cells of Cajal in culture. Am J Physiol Gastrointest Liver Physiol 2003; 284: G31320.
  • 16
    Maeda H, Yamagata A, Nishikawa S, Yoshinaga K, Kobayashi S, Nishi K. Requirement of c-kit for development of intestinal pacemaker system. Development 1992; 116: 36975.
  • 17
    Torihashi S, Nishi K, Tokutomi Y, Nishi T, Ward S, Sanders KM. Blockade of kit signaling induces transdifferentiation of interstitial cells of cajal to a smooth muscle phenotype. Gastroenterology 1999; 117: 1408.
  • 18
    Daniel EE, Posey-Daniel V. Neuromuscular structures in opossum esophagus: role of interstitial cells of Cajal. Am J Physiol 1984; 246: G30515.
  • 19
    Horisawa M, Watanabe Y, Torihashi S. Distribution of c-Kit immunopositive cells in normal human colon and in Hirschsprung's disease. J Pediatr Surg 1998; 33: 120914.
  • 20
    Wedel T, Spiegler J, Soellner S et al. Enteric nerves and interstitial cells of Cajal are altered in patients with slow-transit constipation and megacolon. Gastroenterology 2002; 123: 145967.
  • 21
    Vanderwinden JM, Liu H, Menu R, Conreur JL, De Laet MH, Vanderhaeghen JJ. The pathology of infantile hypertrophic pyloric stenosis after healing. J Pediatr Surg 1996; 31: 15304.
  • 22
    Watkins CC, Sawa A, Jaffrey S et al. Insulin restores neuronal nitric oxide synthase expression and function that is lost in diabetic gastropathy. J Clin Invest 2000; 106: 37384.
  • 23
    Ordog T, Takayama I, Cheung WK, Ward SM, Sanders KM. Remodeling of networks of interstitial cells of Cajal in a murine model of diabetic gastroparesis. Diabetes 2000; 49: 17319.
  • 24
    Stark ME, Szurszewski JH. Role of nitric oxide in gastrointestinal and hepatic function and disease. Gastroenterology 1992; 103: 192849.
  • 25
    Kroncke KD, Fehsel K, Kolb-Bachofen V. Nitric oxide: cytotoxicity versus cytoprotection – how, why, when, and where? Nitric Oxide 1997; 1: 10720.
  • 26
    Stamler JS. Redox signaling: nitrosylation and related target interactions of nitric oxide. Cell 1994; 78: 9316.
  • 27
    Andoh T, Lee SY, Chiueh CC. Preconditioning regulation of bcl-2 and p66shc by human NOS1 enhances tolerance to oxidative stress. FASEB J 2000; 14: 21446.
  • 28
    Huang PL, Dawson TM, Bredt DS, Snyder SH, Fishman MC. Targeted disruption of the neuronal nitric oxide synthase gene. Cell 1993; 75: 127386.
  • 29
    Choi KM, Gibbons SJ, Lurken MS et al. Loss of neuronal nitric oxide synthase results in altered volume and distribution of interstitial cells of Cajal in mouse stomach. Neurogastroenterol Motil 2005; 17: 612, A33.
  • 30
    Mashimo H, Kjellin A, Goyal RK. Gastric stasis in neuronal nitric oxide synthase-deficient knockout mice. Gastroenterology 2000; 119: 76673.
  • 31
    Gibbons SJ, Rich A, Distad MA et al. Kit/stem cell factor receptor-induced phosphatidylinositol 3′-kinase signalling is not required for normal development and function of interstitial cells of Cajal in mouse gastrointestinal tract. Neurogastroenterol Motil 2003; 15: 64353.
  • 32
    Sasaki S, Siragy HM, Gildea JJ, Felder RA, Carey RM. Production and role of extracellular guanosine cyclic-3′,5′-monophosphate in sodium uptake in human proximal tubule cells. Hypertension 2004; 43: 28691.
  • 33
    Matsuda NM, Miller SM, Sha L, Farrugia G, Szurszewski JH. Mediators of non-adrenergic non-cholinergic inhibitory neurotransmission in porcine jejunum. Neurogastroenterol Motil 2004; 16: 60512.
  • 34
    Zyromski NJ, Duenes JA, Kendrick ML, Balsiger BM, Farrugia G, Sarr MG. Mechanism mediating nitric oxide-induced inhibition in human jejunal longitudinal smooth muscle. Surgery 2001; 130: 48996.
  • 35
    Miller SM, Farrugia G, Schmalz PF, Ermilov LG, Maines MD, Szurszewski JH. Heme oxygenase 2 is present in interstitial cell networks of the mouse small intestine. Gastroenterology 1998; 114: 23944.
  • 36
    Linden DR, Couvrette JM, Ciolino A et al. Indiscriminate loss of myenteric neurones in the TNBS-inflamed guinea-pig distal colon. Neurogastroenterol Motil 2005; 17: 75160.
  • 37
    Seki K, Komuro T. Distribution of interstitial cells of Cajal and gap junction protein, Cx 43 in the stomach of wild-type and W/Wv mutant mice. Anat Embryol (Berl) 2002; 206: 5765.
  • 38
    Ordog T, Baldo M, Danko R, Sanders KM. Plasticity of electrical pacemaking by interstitial cells of Cajal and gastric dysrhythmias in W/W mutant mice. Gastroenterology 2002; 123: 202840.
  • 39
    Song G, David G, Hirst S, Sanders KM, Ward SM. Regional variation in ICC distribution, pacemaking activity and neural responses in the longitudinal muscle of the murine stomach. J Physiol 2005; 564: 52340.
  • 40
    Mashimo H, Goyal RK. Lessons from genetically engineered animal models. IV. Nitric oxide synthase gene knockout mice. Am J Physiol 1999; 277: G74550.
  • 41
    Bernex F, De Sepulveda P, Kress C, Elbaz C, Delouis C, Panthier JJ. Spatial and temporal patterns of c-kit-expressing cells in WlacZ/+ and WlacZ/WlacZ mouse embryos. Development 1996; 122: 302333.
  • 42
    Ward SM, Ordog T, Bayguinov JR et al. Development of interstitial cells of Cajal and pacemaking in mice lacking enteric nerves. Gastroenterology 1999; 117: 58494.
  • 43
    Horvath VJ, Vittal H, Ordog T. Reduced insulin and IGF-I signaling, not hyperglycemia, underlies the diabetes-associated depletion of interstitial cells of Cajal in the murine stomach. Diabetes 2005; 54: 152833.
  • 44
    Nemeth L, Puri P. Three-dimensional morphology of c-Kit-positive cellular network and nitrergic innervation in the human gut. Arch Pathol Lab Med 2001; 125: 899904.
  • 45
    Wang XY, Sanders KM, Ward SM. Intimate relationship between interstitial cells of cajal and enteric nerves in the guinea-pig small intestine. Cell Tissue Res 1999; 295: 24756.
  • 46
    Iwasaki H, Kajimura M, Osawa S et al. A deficiency of gastric interstitial cells of Cajal accompanied by decreased expression of neuronal nitric oxide synthase and substance P in patients with type 2 diabetes mellitus. J Gastroenterol 2006; 41: 107687.
  • 47
    Wu JJ, Rothman TP, Gershon MD. Development of the interstitial cell of Cajal: origin, kit dependence and neuronal and nonneuronal sources of kit ligand. J Neurosci Res 2000; 59: 384401.
  • 48
    Hagl CI, Holland-Cunz S, Schafer KH. What do knockout models teach us about the enteric nervous system? Eur J Pediatr Surg 2003; 13: 1705.
  • 49
    Chang IY, Glasgow NJ, Takayama I, Horiguchi K, Sanders KM, Ward SM. Loss of interstitial cells of Cajal and development of electrical dysfunction in murine small bowel obstruction. J Physiol 2001; 536: 55568.
  • 50
    Papapetropoulos A, Garcia-Cardena G, Madri JA, Sessa WC. Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells. J Clin Invest 1997; 100: 31319.
  • 51
    Contestabile A, Ciani E. Role of nitric oxide in the regulation of neuronal proliferation, survival and differentiation. Neurochem Int 2004; 45: 90314.
  • 52
    Tuteja N, Chandra M, Tuteja R, Misra MK. Nitric oxide as a unique bioactive signaling messenger in physiology and pathophysiology. J Biomed Biotechnol 2004; 2004: 22737.
  • 53
    Boerth NJ, Dey NB, Cornwell TL, Lincoln TM. Cyclic GMP-dependent protein kinase regulates vascular smooth muscle cell phenotype. J Vasc Res 1997; 34: 24559.
  • 54
    Pervin S, Singh R, Hernandez E, Wu G, Chaudhuri G. Nitric oxide in physiologic concentrations targets the translational machinery to increase the proliferation of human breast cancer cells: involvement of mammalian target of rapamycin/eIF4E pathway. Cancer Res 2007; 67: 28999.
  • 55
    Mejia-Garcia TA, Paes-de-Carvalho R. Nitric oxide regulates cell survival in purified cultures of avian retinal neurons: involvement of multiple transduction pathways. J Neurochem 2007; 100: 38294.
  • 56
    Cooke JP. The pivotal role of nitric oxide for vascular health. Can J Cardiol 2004; 20 (Suppl B): 7B15B.