Interleukin-1 beta and tumor necrosis factor-alpha stimulate the cat-2 gene of the L-arginine transporter in cultured vascular smooth muscle cells

The production of nitric oxide (NO) from L-arginine by nitric oxide synthase (NOS) in cytokine-stimulated vascular smooth muscle cells (VSMC) is thought to play an important role in the pathophysiology of several vascular disease states including septic shock. This study examines the relationship between cytokine-stimulated NO production and L-arginine transport in cultured VSMC. Cultured VSMC from rat aorta were stimulated with interleukin-1 beta, tumor necrosis factor-alpha, and/or angiotensin II (Ang II); and the accumulation of nitrite, a stable product of NO metabolism, in the culture media and the rates of net L-arginine uptake were measured. Interleukin-1 beta and tumor necrosis factor-alpha, alone or in combination, stimulated both the uptake of L-arginine and the accumulation of nitrite in the culture media in a dose-dependent manner. Inhibition of NOS activity by substituted analogues of L-arginine had no effect on cytokine-stimulated L-arginine transport. Ang II in the presence of cytokines up-regulated L-arginine transport while inhibiting nitrite accumulation. Two forms of the L-arginine transporter, cat-1b and cat-2, are expressed in VSMC. Northern analysis revealed that the cytokine-stimulated increase in L-arginine transport coincided with increased levels of cat-2 mRNA. In contrast, cat-1b does not appear to be regulated by cytokines at the mRNA level, although significant increases in response to Ang II were observed. These results show that, while cytokines can stimulate both NOS activity and L-arginine uptake, NO production is not required to signal the increase in L-arginine transport. Furthermore, Ang II and cytokine stimulation of L-arginine uptake involves the differential regulation of the cationic amino acid transporter (cat) genes.     

Corneal penetration of diclofenac from a fixed combination of diclofenac-gentamicin eyedrops

Recent studies suggest a dual role for nitric oxide (NO) in tumour biology. High concentrations of NO can mediate tumouricidal activity, whereas lower concentrations have been shown to promote tumour growth. In this study, NO synthase (NOS) activity was investigated in cells that were prepared from tissue from primary and metastatic sites and from malignant effusions in 41 cases of suspected ovarian cancer. NO biosynthesis, determined by nitrite + nitrate (NOx) accumulation in medium from cultured cells prepared from disaggregated tumours or effusions and indicative of the inducible NO synthase isoform, was detected in 37% of the cases investigated (range 10.2-114 microM). There was a significant relationship between NOx and tumour differentiation (P = 0.014), with NOx being significantly higher for the more differentiated tumours. NOS activity, determined by the conversion of radiolabelled L-arginine to citrulline by tissue or cell extracts, was detected in 29% of cases (range 0.9-6.9 pmol/min per mg of protein), with all samples tested being moderately or poorly differentiated. Seventy percent of this activity was calcium dependent, indicative of constitutive NOS isoforms. Morphological and immunohistochemical assessment of tumour samples indicated a significant relationship between high macrophage content and NOS activity (as NOx biosynthesis) (rs = 0.726, N = 16, P < 0.01). The relationship between NOS expression, immune response, and disease progression is complex and not simply dependent on the differentiation status of ovarian cancer.     

Effect of endogenous L-arginine on the measurement of nitric oxide synthase activity in the rat kidney

The endogenous level of L-arginine in renal cortex, outer medulla, and inner medulla and its effect on the measurement of nitric oxide synthase (NOS) activity in these regions was determined. L-Arginine was measured with an amino acid analyzer and total NOS activity was measured as the rate of formation of radiolabeled L-citrulline from L-[14C]arginine. Total NOS activity was that activity inhibited by NG-nitromonomethyl-L-arginine (300 microM). The endogenous L-arginine concentration was 452 +/- 45 (mean +/- SEM), 313 +/- 25, and 95 +/- 8 pmol/mg wet weight in the cortex, outer medulla, and inner medulla, respectively (n = 12). Total NOS activity was 61 +/- 19, 709 +/- 94, and 1347 +/- 76 pmol.h-1.mg protein-1 in the cortex, outer medulla, and inner medulla, respectively, when endogenous L-arginine was not considered in the calculation. Correcting for endogenous L-arginine gave values of 185 +/- 61, 1714 +/- 239, and 1707 +/- 104 pmol.h-1.mg protein-1, respectively. Dowex extraction to remove endogenous L-arginine from samples also increased NOS activity in cortex and medulla. The data indicate that there is a differential distribution of endogenous L-arginine in the kidney and that these levels must be taken into account when measuring NOS activity. NOS activity is also distributed differentially, with activity in the medulla being nearly 10-fold higher than in the cortex.     

Nitric oxide synthase activity in human lung cancer

Nitric oxide synthases (NOS) exist in human tumor cell lines and solid tumor tissues, and it has been suggested that NO may play important roles in growth, progression or metastasis of tumors. We investigated the activity and distribution of NOS in a series of human cancer and normal lung tissues. Seventy-two primary lung cancer samples (44 cases of adenocarcinoma, 18 of squamous cell carcinoma, 4 of large cell carcinoma, 2 of small cell carcinoma, 2 of adenosquamous carcinoma, and 2 of carcinoids) and corresponding normal lung samples were obtained from surgically treated patients. In normal lung tissues, little NOS activity was observed with no correlation between the patient's age and NOS activity. The total NOS activities in lung adenocarcinoma samples were significantly higher than those in other types of lung cancers of normal lung samples (P < 0.05). Analysis by tumor grade of the adenocarcinoma samples revealed no significant difference of NOS activity between grades. TNM classification showed that, although T stage did not correlate with NOS activity, cancer tissues from patients with N2 disease tended to have lower activity than those from patients with NO or N1 disease. Immunohistochemical studies revealed that the intensity of NOS immunoreactivity correlated with NOS activity. These results suggest that NO may play an important role in the metabolism and behavior of lung cancers, especially adenocarcinoma.     

Physiological and physiopathological aspects of nitric acid in mammalian tissues

In recent years, nitric oxide (NO), a single but highly reactive molecule has become known as the central point of many researchs. NO is synthesized by the enzyme nitric oxide synthase (NOS) in mammals from the amino-acid L-arginine. The products of L-arginine oxidation by NOS are L-citrulline and NO. Nitric oxide has a very short half life, is lipid soluble, reacts easily with several enzymatic systems, and is produced by a wide amount of cells. At least, three kinds of enzymes NOS have been described: two of them are calcium-dependent and continuously present in select cells (constitutive NOS, cNOS). One cNOS isoform is present in the cytosol of neuronal cells, while the other isoform is present in membrane-bound form, in endothelial cells. cNOS produces small quantities of NO, following stimulation by specific agonist. NO produced by cNOS frequently mediates cellular communications and cellular signaling. A third isoform is calcium-independent, is not present in unstimulated cells, and produces large quantities of NO following stimulation of the appropriate cell with cytokines or LPS (inducible NOS, iNOS). NO is a mediator of both physiological and pathological process. It acts directly on its targets, one of them, maybe the most important, is the soluble guanylate cyclase, and produces a variety of biological effects, ranged from cytoprotection to cytotoxicity. An analysis of the biochemistry and physiology of NO is the focus of this review, together with its biological action and potential therapeutical implications.     

Identification of nuclear matrix protein alterations associated with renal cell carcinoma

PURPOSE: Neoplastic transformation, including renal cell carcinoma (RCC), is always accompanied by changes in nuclear morphology. Nuclear grading of RCC is based on characteristic alterations in nuclear shape, size, area and other morphologic parameters. The nuclear matrix, which forms the skeleton of the nucleus, determines nuclear morphology. Alterations in nuclear matrix protein (NMP) composition specific to tissue and cancer type have been described in a variety of human cancers. We conducted a study to analyze the nuclear matrix protein composition of renal cell carcinoma and compare it to that of normal renal tissue and renal cell carcinoma cells grown in culture. MATERIALS AND METHODS: We analyzed the nuclear matrix protein composition of RCC tumor tissue and that of normal kidney tissue obtained from seventeen patients undergoing radical nephrectomy for RCC. We also analyzed the NMP composition of two renal cancer cell lines (A-498 and 769-P). RESULTS: We were able to identify five different and unique NMPs which were present only in the human RCC tumor samples and were absent in all normal kidney tissue. One NMP was found specifically in the normal kidney tissue. All five RCC specific NMPs were also identified in the nuclear matrix of the two cell lines analyzed. CONCLUSIONS: Five nuclear matrix proteins specific and unique to RCC were identified. These NMPs are different from those previously identified in other tissues and neoplasms. The RCC specific NMPs identified in this study can potentially be used as diagnostic markers for renal cell carcinoma and for therapeutic tumor targeting.     

L-arginine treatment in ischemia/reperfusion injury

We tested whether treatment with exogenous L-arginine, the precursor of nitric oxide (NO), could protect the skeletal muscle from ischemia/reperfusion (I/R) injury. A rabbit hindlimb I/R model (2.5 h ischemia/2 h reperfusion) was used. Morphological changes were elucidated by morphometry. Plasma concentrations of malondialdehyde (pMDA), as well as L-arginine and L-citrulline content in the plasma and skeletal muscle were measured. I/R injury in the skeletal muscle was manifested by development of prominent interstitial edema (fraction of interfiber area was 26.23% vs 15.09% in sham operated control, p < .005) and severe microvascular constriction (capillary area was 11.41 microns2 vs 16.92 in control, p <.005). These changes were accompanied by increased pMDA levels, indicating a process of lipid peroxidation in the cell membranes. L-arginine treatment (4 mg/kg/min intravenously, for 1 h, infusion initiated 30 min before reperfusion) caused an intracellular accumulation of this amino acid in the SM. Intracellular concentrations of L-citrulline increased (201.0 mumol/dm3 after reperfusion vs 176.0 before ischemia onset, p < .005), suggesting stimulated endogenous NO synthesis. L-arginine treatment protected capillary constriction (capillary area was 17.64 microns2 vs 11.41 in the untreated animals, p < .0005) and reduced interstitial edema after reperfusion (fraction of interfiber area was 17.80% vs 26.23 in untreated animals, p < 0.005). The protective effect of L-arginine treatment on I/R injury of SM may be related to its ability to prevent microvascular constriction and reduce permeability disorders by the stimulation of endogenous NO production.     

Cancer-associated expression of glycolipid sulfotransferase gene in human renal cell carcinoma cells

Human renal cell carcinoma (RCC) tissue and a cell line derived therefrom, SMKT-R3, showed markedly increased glycolipid sulfotransferase [cerebroside sulfotransferase (CST); EC 2.8.2.11] activity and accumulated sulfoglycolipids. Recently, we cloned a human CST cDNA from a SMKT-R3 cDNA library (K. Honke et al., J. Biol. Chem., 272: 4864-4868, 1997). In this study, we investigated the expression of the CST gene in seven human RCC lines (SMKT-R1, SMKT-R2, SMKT-R3, SMKT-R4, TOS-1, TOS-2, and ACHN) and their normal counterpart, human renal proximal tubular cells. On Northern blot analysis, a marked increase of CST mRNA was observed in every RCC line, except for ACHN, as compared with normal cells. ACHN cells showed a slightly increased level of CST mRNA. CST activity was correlated with the amount of mRNA. Sulfoglycolipid analysis revealed that expression of lactosylceramide sulfate was correlated with the CST level. Furthermore, we examined the effects of epidermal growth factor (EGF), tetradecanoylphorbol-13-acetate, and genistein, which are known to regulate CST activity in SMKT-R3 cells, on CST-gene expression in various RCC cells. On treatment with EGF, CST mRNA time-dependently increased in accord with its activity in SMKT-R3 cells. Yet, augmentation by EGF was only observed in SMKT-R3. In contrast, a reduction of CST mRNA and activity by tetradecanoylphorbol-13-acetate and genistein was observed in all of the lines examined. Taken together, these findings indicate that in human RCC cells, the CST gene is generally overexpressed via a signaling pathway involving protein kinase-C and tyrosine kinases.     

Purification and characterization of a nitric-oxide synthase from rat liver mitochondria

The biosynthesis of nitric oxide (NO.) in different cell types occurs concomitantly with the conversion of L-arginine to L-citrulline by the enzyme nitric-oxide synthase (NOS). NO. has been identified as a major participant in a number of basic physiological functions such as neurotransmission, vasodilation, and immune response. At the subcellular level, mitochondria have been identified as targets for NO.; however, to date, no unambiguous evidence has been presented to identify these organelles as sources of NO.. In this study, a NOS was isolated to homogeneity from Percoll-purified rat liver mitochondria. Kinetic parameters, molecular weight, requirement of cofactors, and cross-reactivity to monoclonal antibodies against macrophage NOS suggest similarities to the inducible form. However, the constitutive expression of the mitochondrial enzyme and its main membrane localization indicate the presence of either a distinctive isoform or a macrophage isoform containing posttranslational modifications that lead to different subcellular compartments. The detection of NADPH-oxidizing activities and a production of superoxide anion catalyzed by mtNOS and recombinant cytochrome P450 reductase were consistent with the sequence homology reported for these two proteins. Given the role of NO. as cellular transmitter, messenger, or regulator, the presence of a functionally active mitochondrial NOS may have important implications for the intermediary metabolism.     

The versatile and complex enzymology of nitric oxide synthase

The biogenesis of nitric oxide is catalyzed by nitric oxide synthase (NOS) which forms L-citrulline and NO from L-arginine. Here we review the enzymology of NOS. We discuss its modular structure, its prosthetic groups and cofactors, and we provide a brief account of present knowledge regarding cellular targeting and regulation of the different isoforms. The various reactions which are catalyzed by NOS are reviewed, and an inventory of different inhibitor types is given. Special attention is paid to the role of the cofactor tetrahydrobiopterin (BH4) and of the dimeric structure, and to the possibility that the main product of NOS catalysis under some conditions may not be NO. Based on a number of recent observations, we postulate that neuronal NOS with one equivalent of BH4 per dimer (a state which may be physiologically relevant) catalyzes the concerted formation of peroxynitrite.     

Substrate binding and calmodulin binding to endothelial nitric oxide synthase coregulate its enzymatic activity

Endothelial nitric oxide synthase (NOS) is a constitutively expressed flavin-containing heme protein that catalyzes the formation of NO from L-arginine, NADPH, and molecular oxygen. We purified bovine endothelial NOS from transfected embryonic kidney cells by conventional chromatographic techniques and characterized the activity of the detergent-solubilized enzyme. Endothelial NOS displays a much lower specific activity of NO synthesis (143 +/- 11 nmol NO/min/mg enzyme) than the constitutive neuronal NOS or inducible NOS isoforms. Like the neuronal isoform, endothelial NOS requires binding of Ca2+/calmodulin to achieve Vmax NO synthase activity; however, we observed a basal level of NO synthesis even when Ca2+/calmodulin was omitted and 0.5 mM EDTA was present in the assay solution. Moreover, endothelial NOS demonstrates a high-affinity bonding interaction with calmodulin such that the enzyme as purified has a NO synthase activity at about 80% of Vmax. We also observed a more than twofold increase in NADPH consumption by endothelial NOS when it was coupled to arginine oxygenation as opposed to when oxygen is activated in the absence of substrate. Substrate binding was also shown to stimulate heme reduction in the absence of added calmodulin. Thus, the enzymatic synthesis of NO from L-arginine by endothelial NOS appears to be partially regulated by binding of both calmodulin and substrate. These findings for endothelial NOS represent a significant departure from the enzymatic properties of the other constitutive NOS isoform, neuronal NOS, and we interpret this result in terms of the physiological implications.     

Effects of intracerebroventricular leptin administration on food intake, body weight gain and diencephalic nitric oxide synthase activity in the mouse

1. Intracranial administration of leptin reduces both food intake and body weight gain in the mouse. Inhibitors of nitric oxide (NO) synthase produce similar effects. 2. To investigate the role of the brain L-arginine/NO pathway in mediating this effect of leptin, we have evaluated food intake and body weight gain after daily (5 days) intracerebroventricular (i.c.v.) administration of leptin (0.5-2 microg) alone or in association with L-arginine (10 microg). Moreover, we measured diencephalic nitric oxide synthase (NOS) activity after a single i.c.v. leptin (0.25-2 microg) injection and after consecutive doses of leptin (0.25-2 microg) over 5 days. The time course of the effect of leptin on NOS activity was also evaluated. 3. I.c.v. injected leptin (1 and 2 microg) significantly and dose-dependently reduced food intake and body weight gain with respect to vehicle (food intake: 5.97+/-0.16 g 24 h(-1) and 4.27+/-0.18 g 24 h(-1), respectively, vs 8.05+/-0.34 g 24 h(-1), P<0.001, n=6 for each group; body weight gain: -10.7+/-0.46% and -15.7+/-0.65%, respectively, vs 5.14+/-0.38%, P<0.001, n=6 for each group). This effect was antagonized by L-arginine (food intake: 7.90+/-0.37 g 24 h; body weight gain: 5.11+/-0.31%, n=6). Diencephalic NOS activity was significantly reduced by the highest doses of leptin with respect to vehicle (vehicle: 0.90+/-0.04 nmol citrulline min(-1) g(-1) tissue; leptin 1 microg: 0.62+/-0.03 nmol citrulline min(-1) g(-1) tissue, P<0.001; leptin 2 microg: 0.44+/-0.03 nmol citrulline min(-1) g(-1) tissue, P<0.001, n=6 for each group). Similar results were obtained in animals treated with daily consecutive doses of leptin. The inhibitory effect appeared rapidly (within 30 min) and was long lasting (up to 12 h). 4. Our results suggest that the brain L-arginine/NO pathway may be involved in the central effect of leptin on feeding behaviour and body weight gain in mice.     

Oxygen radicals and nitric oxide in rat mesenteric ischaemia-reperfusion: modulation by L-arginine and NG-nitro-L-arginine methyl ester

1. The aims of the present study were to detect changes in superoxide anion (O2.-), nitric oxide (NO) and other reactive oxygen species (ROS) directly by measurement of chemiluminescence (CL) and to investigate the role of L-arginine, a nitric oxide synthase (NOS) substrate, and NG-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor, together with their molecular enantiomers D-arginine and D-NAME, in a rat mesenteric ischaemia-reperfusion (I/R) model. 2. Seventy-nine female Wistar albino rats were divided into eight groups. The first three groups underwent sham operation; group 1 was the control group, group 2 received L-arginine and group 3 received L-NAME. Ischaemia was produced in the remaining five groups by ligation of the superior mesenteric artery for 30 min followed by 60 min reperfusion. Group 4 rats were control I/R rats and groups 5-8 received either L-arginine, L-NAME, D-arginine or D-NAME, respectively. 3. Both luminol and lucigenin CL was significantly increased in I/R groups compared with sham-operated groups. L-Arginine significantly reduced CL measurements. D-Arginine was also protective, but not as much as L-arginine. Both L- and D-arginine had in vitro O2.- (-)scavenging potential, as tested by the xanthine-xanthine oxidase system. NG-Nitro-L-arginine methyl ester decreased lipid peroxidation values in addition to reducing CL measurements. Nitric oxide concentrations were significantly increased in I/R groups in comparison with sham-operated groups. Peroxynitrite formation was increased by I/R. Treatment with L-NAME was beneficial by reducing NO concentrations in the reperfused ileum. 4. In our I/R model, O2.-, NO and other ROS were increased. Although NOS inhibitors were effective in reducing oxidative damage, increasing NO concentrations with L-arginine was also beneficial, presumably due to the ability of L-arginine to inhibit phagocyte adherence and its radical scavenging potential. In fact, NO may have different effects in terms of tissue injury or protection depending on the concentration of oxygen and the haemodynamic state of the tissue.     

Modulation by protein kinase C of nitric oxide and cyclic GMP poffation in cultured cerebellar granule cells

The possible modulation of nitric oxide (NO) synthase (NOS) activity by protein kinase C (PKC) was investigated in primary cultures of rat cerebellar neurons. Incubation of the cells with L-arginine and nicotinamide-adenine dinucleotide phosphate (NADPH) produced detectable levels of NO, as quantified by photometric assay [0.14 +/- 0.03 nmol/h/dish (2.5 x 10(6) cells)]. The NO producing activity was paralleled by concomitant accumulation of cyclic GMP (cGMP) (0.12 +/- 0.02 pmol/dish). Downregulation of PKC by prolonged treatment with phorbol esters or inhibition of the kinase by treatment with 4taurosporine raised the basal levels of NO and cGMP five fold. When granule cells were incubated in the absence of extracellular Mg2+, N-methyl-D-aspartate and to a lesser extent, glutamate became effective in enhancing NO formation and cGMP accumulation with respect to the control. The NO and cGMP increases induced by the two agonists were almost doubled by treatment of the cells with staurosporine or depletion of PKC. Calphostin C. an inhibitor of the regulatory domain of PKC, was as effective as staurosporine in increasing the formation of NO in both resting and excited cells. These results indicate that downregulation or inhibition of PKC increase NOS activity in cerebellar neurons, and suggest that phosphorylation of NOS by PKC negatively modulates the catalytic activity of the enzyme in these cells.     

Memory-related changes of nitric oxide synthase activity and nitrite level in rat brain

The changes of nitric oxide synthase (NOS) activity and nitrite level in rat brain regions after spatial learning were investigated. NOS activity was assayed by conversion of [3H]L-arginine to [3H]L-citrulline, and a sensitive fluorometric assay for quantification of nitrite was used. Compared with sham-trained rats, NOS activity and nitrite level in hippocampus and cortex, and also the nitrite level in cerebellum, was elevated significantly one day after rats had learnt a water-rewarded spatial alteration task. These results suggest a spatial memory-related changes of endogenous NO in rat brain, and support the idea that NO participates in learning and memory processes.     

Sperm nitric oxide and motility: the effects of nitric oxide synthase stimulation and inhibition

Nitric oxide (NO) is synthesized from L-arginine by a family of enzymes known as the nitric oxide synthases (NOS). We have recently shown a NOS similar to constitutive brain NOS (bNOS) and endothelial NOS (ecNOS) to be present in spermatozoa. The aim of this study is to investigate NO production by human spermatozoa and the effects of stimulation and inhibition of NOS. This was carried out using the Iso-NO, an isolated NO meter and sensor, which provides rapid, accurate and direct measurements of NO. Semen samples with normozoospermic and asthenozoospermic profiles were prepared using a direct swim-up technique. Basal concentrations of NO and stimulated NO production were measured after exposure to the calcium ionophore (A23187; 0.01-10 microM) a potent activator of constitutive NOS. NO production in human spermatozoa was significantly increased by the addition of A23187 30 seconds after stimulation. Furthermore, this response was greatly diminished by pre-incubating the samples with competitive inhibitors of L-arginine, the substrate for NOS, before treatment with calcium ionophore. In the presence of N(G)-nitro-L-arginine methyl ester (L-NAME), N(G)-nitro-L-arginine (L-NA) or N(G)-methyl-L-arginine (L-NMMA; all at 10 microM), NO production was inhibited with a rank order of potency L-NAME > L-NMMA > L-NA which is in accordance with the inhibition of an endothelial type of constitutive NOS.     

Objective evaluation of Papanicolaou staining

Altered endothelium-dependent vasodilation has been observed in congestive heart failure (CHF), a disease characterized by a sustained adrenergic activation. The purpose of our study was to test the hypothesis that chronically elevated catecholamines influence the nitric oxide (NO) pathway in the human endothelium. Human umbilical vein endothelial cells (HUVEC) were exposed for 7 days to a concentration of noradrenaline (NA, 1 ng/mL) similar to that found in the blood of patients with CHF. Kinetics of endothelial constitutive NO synthase (ecNOS) and inducible NO synthase (iNOS) activity, measured by [3H]L-arginine to [3H]L-citrulline conversion, and protein expression of ecNOS and iNOS, assessed by Western blot analysis, were unaffected by chronic NA treatment. Furthermore, no changes in subcellular fraction-associated ecNOS were found; this indirectly shows that chronic NA did not cause phosphorylation of the enzyme. Moreover, [3H]L-arginine transport through the plasma membrane was conserved in chronically NA-treated cells. The data demonstrate that prolonged in vitro exposure to pathologic CHF-like NA does not affect the L-arginine: NO pathway in human endothelial cells.     

Regional differences in the nitrergic innervation between the proximal and the distal colon in rats

BACKGROUND & AIMS: Functional differences in the inhibitory neural pathway between the proximal and the distal colon are unknown. METHODS: We investigated the nonadrenergic, noncholinergic (NANC) relaxation, nitric oxide synthase (NOS) synthesis, and NOS messenger RNA (mRNA) expression of the myenteric plexus in the proximal and the distal colon in rats. RESULTS: Transmural nerve stimulation of the neuromuscular preparations from the proximal colon showed greater NANC relaxations than those from the distal colon. NANC relaxations were abolished by the NO biosynthesis inhibitor (NG-nitro-L-arginine methyl ester) in the proximal and the distal colon, suggesting mediation by NO released from the myenteric plexus. The average number of NOS-immunoreactive cells was significantly higher in the tissue from the proximal colon than in the tissue from the distal colon. Western and Northern blot analyses showed a higher density of the immunoreactive NOS band and the NOS mRNA band in the tissue from the proximal colon than in that from the distal colon. CONCLUSIONS: These observations indicate that the number of NOS-containing neurons and the NOS activity are increased in the myenteric plexus of the proximal colon compared with the distal colon, resulting in greater NANC relaxation in the proximal colon. These findings may explain the physiological role of the proximal colon as an organ for fecal storage and absorption of excess fluid.