Effect of age on NADPH-diaphorase-containing myenteric neurones of rat ileum and proximal colon

The effect of age on the distribution of NADPH-diaphorase-containing neurones was investigated in the myenteric plexus of ileum and proximal colon of embryonic day-19 rats, as well as in rats at postnatal day 4, 6 months and 26 months. The mean percentage of NADPH-diaphorase-stained neurones per ganglion was established using protein gene product 9.5 (protein found in most if not all neurones)-immunostained neurones as 100%. The results revealed that there was a significant relative increase in NADPH-diaphorase-positive neurones with increasing age in the myenteric plexus of proximal colon with nearly all protein gene product 9.5-immunoreactive neurones staining for NADPH-diaphorase in 26-month-old rats. This was in marked contrast with the ileum, where no significant relative increase in NADPH-diaphorase-positive neurones was seen in aged rats. The implications of these findings in relation to programmed cell survival and cell death are discussed.     

Comparative studies of quinacrine-positive neurones in the myenteric plexus of stomach and intestine of guinea-pig,rabbit and rat

Summary The number of quinacrine-fluorescent nerve cell bodies and the percentage of the ganglion area occupied by this fluorescence within stretch preparations of the myenteric plexus of the stomach and ileum of the guineapig, rabbit and rat were assessed. The number of quinacrine-positive cell bodies per cm² of plexus varied between 1045 in the rabbit ileum to 2633 in the rat stomach, whilst the percentage of the ganglionic area occupied by fluorescence was approximately 10 %. The distribution of quinacrine-fluorescent nerve fibres and cell bodies in the myenteric plexus was compared to the distribution of nerves revealed by catecholamine fluorescence and by staining for acetylcholinesterase in the stomach and ileum of all three species. Quinacrine fluorescence appears to be selective for non-adrenergic, non-cholinergic nerves; the possibility that it binds to high levels of ATP is discussed.     

Neural and endothelial nitric oxide synthase activity in rat penile erectile tissue

NADPH-diaphorase (NADPH-D) activity and immunoreactivity for neural and endothelial nitric oxide synthase (nNOS and eNOS, respectively) were used to investigate nitric oxide (NO) regulation of penile vasculature. Both the histochemical and immunohistochemical techniques for NOS showed that all smooth muscles regions of the penis (dorsal penile artery and vein, deep penile vessels, and cavernosal muscles) were richly innervated. The endothelium of penile arteries, deep dorsal penile vein, and select veins in the crura and shaft were also stained for NADPH-D and eNOS. However, the endothelium of cavernous sinuses was unstained by both techniques. Fewer fibers were seen in the glans penis, those present being associated with small blood vessels and large nerve bundles near the trabecular walls. All penile neurons in the pelvic plexus, located by retrograde transport of a dye placed in the corpora cavernosa penis, were stained by the NADPH-D method. Essentially similar results were obtained with an antibody to nNOS. These data suggest that penile parasympathetic neurons comprise a uniform population, as all seem capable of forming nitric oxide. However, in contrast to the endothelium of penile vessels, the endothelium lining the cavernosal spaces may not be capable of nitric oxide synthesis.     

A large proportion of pelvic neurons innervating the corpora cavernosa of the rat penis exhibit NADPH-diaphorase activity

Nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry, which indicates the presence of neural nitric oxide synthase, the enzyme responsible for the generation of nitric oxide, was used in combination with retrograde labelling methods to determine, in whole-mounts and sections of rat major pelvic ganglia, whether neurons destined for the penile corpora cavernosa were able to produce nitric oxide. In whole-mount preparations of pelvic ganglia, among the 607±106 retrogradely labelled neurons innervating the penile corpora cavernosa, 84±7% were NADPH-diaphorase-positive, 30±7% of which were intensely histochemically stained. In serial sections of pelvic ganglia, out of a mean count of 451 retrogradely labelled neurons, 65% stained positively for NADPH-diaphorase. An average of 1879±363 NADPH-diaphorase positive cell bodies was counted in the pelvic ganglion. In the major pelvic ganglion, neurons both fluorescent for Fluorogold or Fast Blue and intensely stained for NADPH-diaphorase were consistently observed in the dorso-caudal part of the ganglia in the area close to the exit of the cavernous nerve and within this nerve. This co-existence was much less constant in other parts of the ganglion. In the rat penis, many NADPH-diaphorase-positive fibres and varicose terminals were observed surrounding the penile arteries and running within the wall of the cavernous spaces. This distribution of NADPH-diaphorase-positive nerve cells and terminals is consistent with the idea that the relaxation of the smooth muscles of the corpora cavernosa and the dilation of the penile arterial bed mediated by postganglionic parasympathetic neurons is attributable to the release of nitric oxide and that nitric oxide plays a crucial role in penile erection. Moreover, the existence in the pelvic ganglion of a large number of NADPH-diaphorase-positive neurons that are not destined for the corpora cavernosa suggests that nitric oxide is probably also involved in the function of other pelvic tissues.     

Colocalization of neuropeptides and NADPH-diaphorase in the intra-adrenal neuronal cell bodies and fibres of the rat

Colocalization of vasoactive intestinal peptide, neuropeptide Y, calcitonin gene-related peptide, substance P, and tyrosine hydroxylase, respectively, with NADPH-diaphorase staining in rat adrenal gland was investigated using the double labelling technique. All vasoactive intestinal peptide- and some neuropeptide Y-immunoreactive intrinsic neuronal cell bodies seen in the gland were double stained with NADPH-diaphorase. Double labelling also occurred in some nerve fibres immunoreactive to vasoactive intestinal peptide and neuropeptide Y in the medulla and cortex. No colocalization of calcitonin gene-related peptide, substance P or tyrosine hydroxylase immunoreactivity with NADPH-diaphorase staining was observed. However, nerve fibres with varicosities immunoreactive for all the neuropeptides examined were closely associated with some of the NADPH-diaphorase-stained neuronal cell bodies. Thus, in rat adrenal gland, nitric oxide is synthesized in all ganglion cells containing vasoactive intestinal peptide and in some containing neuropeptide Y, but not in those containing calcitonin gene-related peptide, substance P or tyrosine hydroxylase.     

Culture of rat mesenteric arteriolar smooth muscle cells: effects of platelet-derived growth factor, angiotensin, and nitric oxide on growth

We cultured smooth muscle cells as explants from rat mesenteric arterioles (40–200m in diameter) obtained by injecting a suspension of iron oxide intraarterially and magnetically separating the arterioles after collagenase digestion of adventitial tissue. In third-passaged cells we ascertained smooth muscle purity of >98% by characteristic morphology, contraction responses, and specific immunofluorescence staining. Treatment of growth-arrested (in 0.4% fetal calf serum) cells with platelet-derived growth factor (0.3–7.5 nM) or angiotensin II (0.001–1000 nM) induced ³H-thymidine incorporation and cell proliferation in a dose-dependent manner (P<0.01). S-nitroso-N acetylpencillamine (0.05–0.5 mM), a nitric oxide-generating compound, inhibited 10% fetal calf serum-induced ³H-thymidine incorporation (P<0.05) and cell proliferation (P<0.01). The antimitogenic effect of S-nitroso-N-acetylpencillamine was significantly reduced by hemoglobin and potentiated by superoxide dismutase (P<0.01). In addition to a new technique for culturing mesenteric arteriolar smooth muscle cells, these findings provide evidence that platelet-derived growth factor, angiotensin II, and nitric oxide may be involved in their growth control.     

Distribution and colocalization of nitric oxide synthase and NADPH-diaphorase in adrenal gland of developing,adult and aging Sprague-Dawley rats

The distribution and colocalization of nitric oxide synthase and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-diaphorase) was investigated in the adrenal gland of developing, adult and aging rats with the use of immunohistochemical and histochemical techniques. Nitric oxide synthase-immunoreactive neurons within the adrenal gland were found from the 20th day of gestation onwards. During early development the neurons were found as small clusters of smaller-size cells compared to those observed in the adult gland. Their number reached that of adult level by the 4th day after birth, and in the glands from aging rats a 28.6% increase was observed. Whilst no immunofluorescence was seen in chromaffin cells during early development, some cells from glands of aging rats showed nitric oxide synthase-immunoreactivity with varying intensity. The immunoreactive neurons from postnatal rat adrenals were also positive for NADPH-diaphorase, whilst those in prenatal rats were negative or lightly stained. Nitric oxide synthase-immunoreactive nerve fibres were present in all adrenal glands examined from the 16th day of gestation onwards. A considerable degree of variation in the distribution of immunoreactive fibres both in medulla and outer region of cortex at the different age groups was observed and described. Most, but not all, nitric oxide synthase-immunoreactive nerve fibres also showed NADPH-diaphorase staining.     

Nitric oxide nerves in the uterus are parasympathetic,sensory, and contain neuropeptides

Nitric oxide (NO) is synthesized in neurons and is a potent relaxor of vascular and nonvascular smooth muscle. The uterus contains abundant NO-synthesizing nerves which could be autonomic and/or sensory. This study was undertaken to determine: 1) the source(s) of NO-synthesizing nerves in the rat uterus and 2) what other neuropeptides or transmitter markers might coexist with NO in these nerves. Retrograde axonal tracing, utilizing Fluorogold injected into the uterine cervix, was employed for identifying sources of uterine-projecting neurons. NO-synthesizing nerves were visualized by staining for nicotinamide adenine dinucleotide phosphate (reduced)-diaphorase (NADPH-d) and immunostaining with an antibody against neuronal/type I NO synthase (NOS). NADPH-d-positive perikarya and terminal fibers were NOS-immunoreactive (-I). Some NOS-I/NADPH-d-positive nerves in the uterus are parasympathetic and originate from neurons in the pelvic paracervical ganglia (PG) and some are sensory and originate from neurons in thoracic, lumbar, and sacral dorsal root ganglia. No evidence for NOS-I/NADPH-d-positive sympathetic nerves in the uterus was obtained. Furthermore, double immunostaining revealed that in parasympathetic neurons, NO-I/NADPH-d-reactivity coexists with vasoactive intestinal polypeptide, neuropeptide Y, and acetylcholinesterase and in sensory nerves, NOS-I/NADPH-d-reactivity coexists with calcitonin generelated peptide and substance P. In addition, tyrosine hydroxylase(TH)-I neurons of the PG do not contain NOS-I/NADPH-d-reactivity, but some TH-I neurons are apposed by NOS-I varicosities. These results suggest NO-synthesizing nerves in the uterus are autonomic and sensory, and could play significant roles, possibly in conjunction with other putative transmitter agents, in the control of uterine myometrium and vasculature.     

Distribution of intracardiac neurones and nerve terminals that contain a marker for nitric oxide,NADPH-diaphorase,in the guinea-pig heart

There is strong evidence that NADPH-diaphorase can be used as a marker for neurones that employ nitric oxide as a messenger molecule. In the present study, the NADPH-diaphorase activity of intracardiac neurones and nerve terminals in whole-mount stretch preparations and sections of the newborn and adult guinea-pig atria and interatrial septum has been examined histochemically. Together with epicardial, endothelial and endocardial cells, which displayed some NADPH-diaphorase staining, a subpopulation of intracardiac neurones exhibited moderate-heavy labelling for NADPH-diaphorase, while the majority of neurones were only lightly stained or negative. Intracardiac ganglia containing positive neuronal cell bodies were located between the epicardial cells and atrial myocytes in four main regions: in association with the superior and inferior vena cavae, the points of entry of the pulmonary veins, and within the interatrial septum. Nerve terminals exhibiting NADPH-diaphorase activity were seen throughout the atrial tissue, forming basket-like endings around intracardiac neuronal cell bodies; varicose terminals were also observed on atrial myocytes and other non-neuronal structures. A proportion of the nerve fibres was clearly of intrinsic origin, other terminals may well have originated from neuronal cell bodies present outside the heart.     

Ultrastructural investigation of nitric oxide synthase-immunoreactive nerves associated with coronary blood vessels of rat and guinea-pig

Ultrastructural investigation of nitrix oxide synthase-immunoreactive nerves closely associated with blood vessels in rat and guinea-pig hearts revealed many labelled nerve fibres in the walls of the main branches of the coronary arteries, and in arterioles, capillaries and post-capillary venules. The number of nitric oxide synthase-containing nerve fibres associated with different vessels, even those of the same calibre, varied. Terminal regions of nitric oxide synthase-immunoreactive fibres were observed in the endocardium and myocardium. Nitric oxide synthase-labelled fibres displayed electrondense immunoproduct in both varicose and intervaricose regions. Immunoreactive axonal varicosities contained both small and large synaptic vesicles. The characteristics of the nitric oxide synthase-immunoreactive nerve fibres observed in the heart and the possibility that these fibres represent the processes of intracardiac neurones and/or sensory neurones of extrinsic origin are discussed.     

The expression of the gene coding for parathyroid hormone-related protein (PTHrP) during tooth development in the rat

By means of in situ hybridisation studies, it is shown that parathyroid hormone-related protein (PTHrP) mRNA is strongly expressed in the developing enamel organs of rat teeth. In particular, the cervical loop hybridises strongly with the PTHrP probe and expression is maintained at this site throughout life in the permanently erupting incisor teeth. In mature molar teeth, expression is downregulated to low levels and confined to the epithelial cell rests of Malassez and/or cementoblasts which may derive from these. The gene is also expressed at low levels in the tissue overlying the erupting molars and, thereafter, in the junctional epithelia and connective tissue cells of the epithelial attachment on all tooth surfaces. The premise that PTHrP may undergo post-translational processing and that the resultant products could act in different ways raises the possibility of its exerting multiple paracrine actions during tooth development. These could include the control of cell division and local vascular dilation during development.     

Nitric oxide synthase in the rat carotid body and carotid sinus

The participation of nitric oxide synthase (NOS) in the innervation of the rat carotid body and carotid sinus was investigated by means of NADPH-diaphorase histochemistry and NOS immunohistochemistry using antisera raised against purified neuronal NOS and a synthetic tridecapeptide. NOS was detected in 23% of neurons at the periphery of the carotid bodies. Some negative neurons were surrounded by NOS-positive terminals. NOS-containing varicose nerve fibres innervated the arterial vascular bed and, to a lesser extent, the islands of glomus cells. These fibres persisted after transection of the carotid sinus nerve and are probably derived from intrinsic neurons. Large NOS-positive axonal swellings in the wall of the carotid sinus were absent after transection of the sinus nerve, indicating their sensory origin. The results suggest a neuronal nitrergic control of blood flow, neuronal activity and chemoreception in the carotid body, and an intrinsic role of NO in the process of arterial baroreception.     

Nitric oxide involvement in the effect of acute lithium administration on the nonadrenergic noncholinergic-mediated relaxation of rat gastric fundus

INTRODUCTION: Lithium has largely met its initial promise as the first drug to be discovered in the modern era of psychopharmacology. However, the mechanism for its action remains an enigma. The aim of the present study was to verify the effect of acute lithium administration on the nonadrenergic noncholinergic (NANC)-mediated relaxation of rat isolated gastric fundus and to evaluate the role of nitric oxide pathway in this manner. MATERIALS AND METHODS: The isolated rat gastric fundus strips were precontracted with 0.5 microM serotonin and electrical field stimulation (EFS) was applied at 5 Hz frequency to obtain NANC-mediated relaxation in the presence or absence of lithium (0.1, 0.5, 1 and 5 mM). Also, effects of combining lithium (0.1 mM) with the NO synthase (NOS) inhibitor L-NAME (0.03 microM) or the guanylyl cyclase inhibitor ODQ (1 microM) on relaxant responses to EFS was investigated. Moreover, effects of combining lithium (1 mM) with 0.1 mM L-arginine (a precursor of NO) on neurogenic relaxation were assessed. Also, the effect of lithium (1 mM) on relaxation to sodium nitroprusside (SNP; 1 nM-0.1 mM) and glyceryltrinitrate (GTN; 0.1-10 microM) was investigated. RESULTS: The NANC-mediated relaxation was significantly (P<0.001) reduced by lithium in a dose- and time-dependent manner. Combination of lithium (0.1 mM) with L-NAME (0.03 microM), which separately had partial inhibitory effect on relaxations, significantly (P<0.001) reduced the NANC-mediated relaxation of gastric fundus. ODQ (1 microM) significantly inhibited the neurogenic relaxations in the presence or absence of lithium (0.1 and 1 mM). Although L-arginine at 0.1 mM had no effect on relaxation to EFS, it prevented the inhibition by lithium (1 mM) of relaxant responses to EFS. Also, SNP and GTN produced concentration-dependent relaxation in precontracted rat gastric fundus which was not altered by lithium incubation (1 mM). DISCUSSION: Our experiments indicated that lithium likely by interfering with L-arginine/NO pathway in nitrergic nerve can result in impairment of NANC-mediated relaxation of rat gastric fundus.     

Immunolocalisation of P2X and P2Y nucleotide receptors in the rat nasal mucosa

Purinoceptor subtypes were localised to various tissue types present within the nasal cavity of the rat, using immunohistochemical methods. P2X₃ receptor immunoreactivity was localised in the primary olfactory neurones located both in the olfactory epithelium and vomeronasal organs (VNO) and also on subepithelial nerve fibres in the respiratory region. P2X₅ receptor immunoreactivity was found in the squamous, respiratory and olfactory epithelial cells of the rat nasal mucosa. P2X₇ receptor immunoreactivity was also expressed in epithelial cells and colocalised with caspase 9 (an apoptotic marker), suggesting an association with apoptosis and epithelial turnover. P2Y₁ receptor immunoreactivity was found within the respiratory epithelium and submucosal glandular tissue. P2Y₂ receptor immunoreactivity was localised to the mucus-secreting cells within the VNO. The possible functional roles of these receptors are discussed.     

Glucose stimulates the expression and activities of nitric oxide synthases in incubated rat islets: an effect counteracted by GLP-1 through the cyclic AMP/PKA pathway

We have examined the expression and activity of inducible nitric oxide synthase (iNOS) and the activity of neuronal constitutive NOS (ncNOS) in isolated rat pancreatic islets, stimulated by a hyperglycaemic concentration of glucose, and whether the NOS activities could be modulated by activation of the cyclic AMP/protein kinase A (cyclic AMP/PKA) system in relation to the insulin secretory process. Here, we show that glucose stimulation (20 mmol/l) induces iNOS and increases ncNOS activity. No iNOS is detectable at basal glucose levels (3.3 mmol/l). The addition of glucagon-like-peptide 1 (GLP-1) or dibutyryl-cAMP to islets incubated with 20 mmol/l glucose results in a marked suppression of iNOS expression and activity, a reduction in ncNOS activity and increased insulin release. The GLP-1-induced suppression of glucose-stimulated iNOS activity and expression and its stimulation of insulin release is, at least in part, PKA dependent, since the PKA inhibitor H-89 reverses the effects of GLP-1. These observations have been confirmed by confocal microscopy showing the glucose-stimulated expression of iNOS, its suppression by GLP-1 and its reversion by H-89 in -cells. We have also found that the NO scavenger cPTIO and the NOS inhibitor L-NAME potentiate the insulin response to glucose, again suggesting that NO is a negative modulator of glucose-stimulated insulin release. We conclude that the induction of iNOS and the increase in ncNOS activity caused by glucose in rat islets is suppressed by the cyclic AMP/PKA system. The inhibition of iNOS expression by the GLP-1/cyclic AMP/PKA pathway might possibly be of therapeutic potential in NO-mediated -cell dysfunction and destruction.     

Comparison of rat mesenchymal stem cells derived from bone marrow,synovium, periosteum,adipose tissue,and muscle

Mesenchymal stem cells (MSCs) are increasingly being reported as occurring in a variety of tissues. Although MSCs from human bone marrow are relatively easy to harvest, the isolation of rodent MSCs is more difficult, thereby limiting the number of experiments in vivo. To determine a suitable cell source, we isolated rat MSCs from bone marrow, synovium, periosteum, adipose, and muscle and compared their properties for yield, expansion, and multipotentiality. After two passages, the cells in each population were CD11b (−), CD45 (−), and CD90 (+). The colony number per nucleated cells derived from synovium was 100-fold higher than that for cells derived from bone marrow. With regard to expansion potential, synovium-derived cells were the highest in colony-forming efficiency, fold increase, and growth kinetics. An in vitro chondrogenesis assay demonstrated that the pellets derived from synovium were heavier, because of their greater production of cartilage matrix, than those from other tissues, indicating their superiority in chondrogenesis. Synovium-derived cells retained their chondrogenic potential after a few passages. The Oil Red-O positive colony-rate assay demonstrated higher adipogenic potential in synovium- and adipose-derived cells. Alkaline phosphatase activity was greater in periosteum- and muscle-derived cells during calcification. The yield and proliferation potential of rat MSCs from solid tissues was much better than those from bone marrow. In particular, synovium-derived cells had the greatest potential for both proliferation and chondrogenesis, indicating their usefulness for cartilage study in a rat model. This study was supported in part by grants from the Japan Latest Osteoarthritis Society and from the Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone in Tokyo Medical and Dental University (to T.M.), and by the Japan Society for the Promotion of Science (grant no. 18591657 to I.S.). Recombinant human bone morphogenetic protein-2 was kindly provided by Astellas Pharma.     

Differential binding patterns of three antibodies (VOBM1, VOBM2, and VOM2) in the rat vomeronasal organ and accessory olfactory bulb

Immunohistochemical properties of monoclonal antibodies raised against the rat vomeronasal epithelium were examined in adult rats. Three monoclonal antibodies, VOBM1, VOBM2, and VOM2, reacted specifically to the luminal surface of the sensory epithelium of the vomeronasal organ. In addition, the reactivities of VOBM1 and VOBM2 were detected in the vomeronasal nerve layer and the glomerular layer of the accessory olfactory bulb. Electron-microscopic study revealed differential patterns of the immunoreactivity of the three antibodies to the microvilli of vomeronasal sensory epithelium. VOBM1 immunoreactivity was found on the microvilli of the supporting cells, whereas VOBM2 immunoreactivity was found on those of the sensory cells. VOM2 immunoreactivity was observed on the microvilli of both the sensory and supporting cells. These results suggest that the three antibodies recognize different antigens on the vomeronasal sensory epithelium. In particular, VOBM2 antibody appears to react to an antigen specific to the microvilli of the vomeronasal sensory cells.     

Met-enkephalin receptor-mediated increase of membrane fluidity modulates nitric oxide (NO) and cGMP production in rat brain synaptosomes

The association of [³H]-Met-enkephalin with synaptosomes isolated from rat brain cortex, when incubated for 30 min at 25°C follows a sigmoid path with a Hill coefficient h=1.25±0.04. Binding of Met-enkephalin into synaptosomes was saturable, with an apparent binding constant of 8.33±0.48 nM. At saturation, Met-enkephalin specific receptors corresponded to 65.5±7.2 nmol/mg synaptosomal protein. The Hill plot in combination with the biphasic nature of the curve to obtain the equilibrium constant, showed a moderate degree of positive cooperativity in the binding of Met-enkephalin into synaptosomes of at least one class of high affinity specific receptors. Met-enkephalin increased the lipid fluidity of synaptosomal membranes labelled with 1,6-diphenyl-1,3,5-hexatriene (DPH), as indicated by the steady-state fluorescence anisotropy [(r_o/r)–1]^–1. Arthenius-type plots of [(r_o/r)–1]^–1 indicated that the lipid separation of the synaptosomal membranes at 23.4±1.2°C was perturbed by Met-enkephalin such that the temperature was reduced to 15.8±0.8°C. Naloxone reversed the fluidizing effect of Met-enkephalin, consistent with the receptor-mediated modulation of membrane fluidity. Naloxone alone had no effect on membrane fluidity. NO release and cGMP production by NO-synthase (NOS) and soluble guanylate cyclase (sGC), both located in the soluble fraction of synaptosomes (synaptosol) were decreased by 82% and 80% respectively, after treatment of synaptosomes with Met-enkephalin (10^–10–10^–4 M). These effects were reversed by naloxone (10^–4 M) which alone was ineffective in changing NO and cGMP production. We propose that Met-enkephalin achieved these effects through receptor mediated perturbations of membrane lipid structure and that inhibition of the L-Arg/NO/cGMP pathway in the brain may result in the antinociceptive effects of Met-enkephalin.     

Role of endogenous nitric oxide (NO) and NO synthases in healing of indomethacin-induced intestinal ulcers in rats

We examined the roles of nitric oxide (NO) and NO synthase (NOS) isozymes in the healing of indomethacin-induced small intestinal ulcers in rats. Animals were given indomethacin (10 mg/kg, s.c.) and killed 1, 4 and 7 days after the administration. Indomethacin (2 mg/kg), N(G)-nitro-L-arginine methyl ester (L-NAME: a nonselective NOS inhibitor: 10 mg/kg) and aminoguanine (a relatively selective iNOS inhibitor: 20 mg/kg) were given s.c. once daily for 6 days, the first 3 days or the last 3 days during a 7-day experimental period. Both indomethacin and L-NAME significantly impaired healing of these lesions, irrespective of whether they were given for 6 days, first 3 days or last 3 days. The healing was also impaired by aminoguanine given for the first 3 days but not for the last 3 days. Expression of iNOS mRNA in the intestine was up-regulated after ulceration, persisting for 2 days thereafter, and the Ca(2+)-independent iNOS activity also markedly increased with a peak response during 1-2 days after ulceration. Vascular content in the ulcerated mucosa as measured by carmine incorporation was decreased when the healing was impaired by indomethacin and L-NAME given for either the first or last 3 days as well as aminoguanidine given for the first 3 days. These results suggest that endogenous NO plays a role in healing of intestinal lesions, in addition to prostaglandins, yet the NOS isozyme mainly responsible for NO production differs depending on the stage of healing: iNOS in the early stage and cNOS in the late stage.