Distribution of constitutive nitric oxide synthase in the jejunum of adult rat

Abstract

AIM: To study the distribution of the constitutive nitric oxide synthase (NOS) in the jejunum of adult rat.

METHODS: The distribution of endothelial NOS (eNOS) was detected by immunohistochemistry. Immunofluorescence histochemical dual staining technique were used for studying the distribution of neuronal NOS (nNOS) and eNOS. The dual stained slides were observed under a confocal laser scanning microscope.

RESULTS: Positive neuronal NOS (nNOS) and endothelial NOS (eNOS) cells were found to be distributed in lamina propria of villi, and the epithelial cell was not stained. eNOS was mainly located in submucosal vascular endothelia, while nNOS was mainly situated in myenteric plexus. Some cells in the villi had both nNOS and eNOS. More than 80% of the cells were positive for both nNOS and eNOS, the rest cells were positive either for nNOS or for eNOS.

CONCLUSION: The two constitutive nitric oxide synthases are distributed differently in the jejunum of rat. nNOS distributed in myenteric plexus is a neurotransmitter in the non-adrenergic non-cholinergic (NANC) inhibitory nerves. eNOS distributed in endothelial and smooth muscle cells of blood vessels plays vasodilator role. eNOS and nNOS are coexpressed in some cells of lamina propria of villi. NO generated by those NOS is very important in the physiological and pathological process of small intestine.

Key Words: $[Keywords]

INTRODUCTION

Nitric oxide (NO) is an intercellular and endocellular signal molecule, and has an important role in the physiological process of intestine. For example, NO can regulate muscular contraction and blood circulation of the intestine[1,2]. Nitric oxide synthase (NOS) is widely distributed in the intestine, and has several isoforms, such as constitutive nitric oxide synthase (nNOS and eNOS) and inducible NOS (iNOS)[3]. In previous studies, NOS was mostly located in small intestine and could be shown by enzyme cytochemistry, but different isoforms of NOS[4-8] coud not be distinguished. In order to study the characteristics and distribution of NOS, immunohistochemistry and immunofluorescence histochemical duel staining technique were used to investigate the distribution of the constitutive nitric oxide synthase (NOS) in the jejunum of adult rat, to provide morphological basis of digestive physiology.

MATERIALS AND METHODS

Specimens

Segments (1-2 cm) of the jejunum were removed from decapitated male Sprague-Dawley rats (250-300 g) and placed immediately in a fixative consisting of 4% paraformaldehyde and 0.1 M phosphate buffer (PB, pH7.4). The fixed jejunum segments were rinsed for at least 12 h at 4 °C in 0.1 M PB (pH7.2) containing 30% sucrose, and then cryostat sections were made at 6 μm thickness and mounted onto glass slides.

Reagents

Rabbit anti-rat eNOS antibody and SP kit were purchased from Beijing Zhongshan Biotechinical Company. Mouse anti-rat nNOS antibody, FITC-conjugated anti-mouse IgG and PE-conjugated anti-rabbit IgG were purchased from Wuhan Boster Biological Technology Company.

Immunohistochemistry

Immunohistochemical staining for eNOS was performed using SP technique with the following procedure. (1) The slides were washed in 0.01 M phosphate-buffered saline (PBS). Endogenous peroxidase was blocked by 0.3% H₂O in methanol for 25 minutes, followed by incubation in normal goat serum for 30 minutes at room temperature. (2) The slides were the incubated with a 1:75 dilution of the primary rabbit anti-rat eNOS antibody for 12 hour at 4 °C. A biotin-streptavidin detection system was employed with diaminobenzidine as the chromogen. (3) Then the slides were washed with PBS and incubated with a reagent (biotinylated anti-immunoglobulin) for 60 minutes at 37 °C. After rinsing in PBS, the slides were incubated with the peroxidase-conjugated streptavidin label for 60 minutes at 37 °C, and incubated with diaminobenzidine and H₂O₂ for 5 minutes. Finally the sections were counterstained with hematoxylin.

Immunofluorescence histochemical dual-staining technique

The slides were incubated with normal goat serum for 30 minutes, followed by incubation with rabbiu anti-rat eNOS antibody and mouse anti-rat nNOS antidody for 48 hour at 4 °C. Then, These were washed with PBS and incubated with FITC-conjugated anti-mouse IgG and PE-conjugated anti-rabbit IgG for 24 hour at 4 °C. After rinsing in PBS, the slides were observed under a confocal laser scanning microscope (MR/A₂, Nikon). Excitation of FITC and PE were 488 and 495 nm respectively, emission of FITC and PE were 525 and 578 nm. 0.01 M PBS was used as a substitute for primary antibody for negative control groups.

RESULTS

Immunohistochemistry showed that eNOS was lealid in the cytoplasm solely. eNOS was distributed mainly in the endothelia of submucosa vessels. Part of the smooth muscle of submucosa vessels was also positive for eNOS (Figure 1). There were strongly positive substances in the cells of lamina propria of the villi, and in the cells near the striated border of villous epithelia. The epithelial cells were unstained (Figure 2).

Figure 1 Immunohistochemical stain of eNOS in jejunal submucosa, showing the positive endothelium (↑) and microvascular smooth muscle (▲). × 170

Figure 2 Immunohistochemical stain of eNOS in jejunal villi, showing the positive cell in the proper layer (↑). × 170

Under the confocal laser scanning microscope, the positive substances of nNOS labeled by FITC were green, and those of eNOS labeled by PE were red. The positive substances of nNOS were distributed mainly in myenteric plexus, rarely in the submucosal plexus (Figure 3). In the lamina propria of the villi, more than 80% of the cells were positive for both nNOS and eNOS, the rest of them were positive either for nNOS or for eNOS (Figure 3 b and c).

Figure 3 Immunofluorescence histochemical double-stain in jejunum observed under a confocal laser scanning microscope, (a) showing the positive substances of nNOS were distributed mainly in myenteric plexus. × 350 (b) and (c) showing the nNOS-positive cell (▲), the eNOS-positive cell (★) and double-stained cell (↑) in the proper layer of villi. × 250

DISCUSSION

The two constitutively expressed, Ca²⁺-dependent NOS isoforms previously identified in neurons (nNOS) and endothelial cells (eNOS) are now known to be distributed more widely[9-11]. eNOS is found in cardiac myocytes[12,13], epithelial cells[14-16], human platelets[17] and various neurons, particularly the pyramidal neurons of the hippocampus, where it is coexpressed with nNOS[18]. nNOS is found in the cytoskeleton of fast-contracting skeletal muscle fibers[19]. In our study, we found nNOS and eNOS were coexpressed in some cells of lamina propria of the villi. NO generated by those cells plays an important role in absorption and protection of microvasculature. Inhibition of endogenous NOS by N_G-nitro-L-auginine methyl ester (L-NAME) caused secretion of water and ions, and this secretion was reversed by administration of the NOS substrate L-arginine[20]. Previous studies indicated that norepinephrine[21,22], somatostatin[23], and neuropeptide Y[24] increased ileal water and ion absorption at a similar magnitude to that observed with L-arginine. It is consistent with the hypothesis that endogenous NO has a proabsorptive influence in the intestine in the basal state. Furthermore, endogenous NO can reduce the vascular albumin leakage provoked by lipopolysaccharide (LPS)[25] and maintain microvascular integrity[26-29].

Our study showed that nNOS was distributed mainly in the myenteric plexus, rarely in submucosal plexus. Recent pharmacological and physiological studies demonstrated that NO is a neurotransmitter in the non-adrenergic non-cholinergic (NANC) inhibitory nerves of the gut[30-34]. During nerve stimulation, NO generated by nNOS in nerve terminals regulates the release of vasoactive intestinal polypeptide (VIP) when diffuses to muscle cells to participate in muscle relaxation[35-38]. Teng et al[39] found eNOS was selectively expressed in rabbit gastric and human intestinal smooth muscle cells. In turn, VIP acts on smooth muscle cells to generate NO. The NO formed in the muscle cells constitutes the predominant component (60%-80%) of NO formed during nerve stimulation.

Using NOS histochemistry and endothelial cell immunohistochemisty, Nichols et al[40] provided the first anatomic evidence of NOS in both endothelial and smooth muscle cells of submucosal blood vessels in the intestines of rat and human, but he could not distinguish the isoform. We found eNOS was distributed in the endothelial and smooth muscle cells of submucosal blood vessels. This particular localization of eNOS was unexpected since only the inducible isoform of NOS had been reported in the vascular smooth muscle cells[41-44]. These anatomical data strongly supported the proposed vasodilator role of NO in the mammalian gastrointestinal tract. There is both basal and stimulated release of NO from the endothelium. Stimulated NO release is affected by certain antagonists (acetylcholine, ATP, or bradykinin) or by physical stimuli such as fluid shear stress[45-49] or low arterial PO₂[50]. Vascular smooth muscle-derived NO behaves as an autocrine factor that plays a role in modulation of vasodilator tone and represents a reserve pool of NOS, which may be required when the tissue is under a local stress. The source of NO within the vascular wall, either intimal or medial, should be a consideration in future studies in terms of the relative contribution of these sources to vasodilator tone in the gut wall.