Clinical features of a new disease concept, IgG4-related thyroiditis.

OBJECTIVES: Immunoglobulin (Ig)G4-related disease is a recently proposed systemic disorder that includes autoimmune pancreatitis (AIP), Mikulicz's disease, and various other organ lesions. In the present retrospective study, we examined whether thyroid lesions should also be included in IgG4-related disease (Ig4-RD) under the new term IgG4-related thyroiditis. METHOD: We enrolled 114 patients with Ig4-RD, including 92 patients with AIP, 15 patients with Mikulicz's disease, and seven patients with IgG4-related cholangitis, and analysed clinical findings, function, serum values of activity markers, computed tomography (CT) images, and histology of the thyroid gland. RESULTS: Among the 22 patients (19%) in our cohort who were found to have hypothyroidism [thyroid stimulating hormone (TSH) > 4 mIU/L], 11 patients had clinical hypothyroidism [free thyroxine (FT4) < 1 ng/dL] and 11 patients had subclinical hypothyroidism (FT4 ≥ 1 ng/dL). Serum concentrations of IgG, IgG4, circulating immune complex (CIC), and ß2-microglobulin (ß2-MG) were significantly higher in the hypothyroidism group compared with the remaining 92 euthyroid patients, and serum C3 concentration was significantly lower. After prednisolone treatment, TSH values had decreased significantly (p = 0.005) in this group and FT4 values had increased significantly (p = 0.047). CT images showed that the thyroid glands of patients with clinical hypothyroidism had a significantly greater volume than those of the euthyroid and other groups. Pathological analysis of one resected thyroid gland disclosed a focused lesion with infiltration of lymphocytes and IgG4-bearing plasma cells and loss of thyroid follicles. CONCLUSIONS: Thyroid lesions associated with hypothyroidism can be considered as a new disease termed IgG4-related thyroiditis. Awareness of this condition should lead to appropriate corticosteroid treatment that may prevent progression to a fibrous state.

Inhibitory effects of cyclic AMP elevating agents on lipopolysaccharide (LPS)-induced microvascular permeability change in mouse skin.

Anti-inflammatory effects of cyclic AMP elevating agents were examined in a mouse model of lipopolysaccharide (LPS)-induced microvascular permeability change. Vascular permeability on the back skin was measured by the local accumulation of Pontamine sky blue (PSB) after subcutaneous injection of LPS (400 microg site-1) from Salmonella typhimurium. Dye leakage in the skin was significantly increased 2 h after injection of LPS. This LPS-induced dye leakage was suppressed by phosphodiesterase inhibitors, including pentoxifylline (160 mg kg-1), milrinone (5 - 10 mg kg-1), rolipram (0.5 - 10 mg kg-1) and zaprinast (5 - 10 mg kg-1). The dye leakage was also inhibited by beta-adrenoceptor agonists, including isoproterenol (0.5 - 5 mg kg-1) and salbutamol (0.05 - 5 mg kg-1), an adenylate cyclase activator, forskolin (5 mg kg-1), and a cell permeable cyclic AMP analogue, 8-bromo-cyclic AMP (8-Br-cAMP, 10 mg kg-1). LPS caused a transient increase in serum TNF-alpha level peaking at 1 h after the injection. This increase in serum TNF-alpha was completely blocked by a pretreatment with pentoxifylline (160 mg kg-1), milrinone (5 mg kg-1), rolipram (1 mg kg-1), zaprinast (10 mg kg-1), salbutamol (0.5 mg kg-1), forskolin (1 mg kg-1) and 8-Br-cAMP (10 mg kg-1). LPS caused an increase in serum IL-1alpha level peaking at 3 h after injection. This increase in serum IL-1alpha was not significantly suppressed by the cyclic AMP elevating agents. Our study suggests that cyclic AMP elevating agents attenuate LPS-induced microvascular permeability change by suppressing TNF-alpha up regulation.

Molecular and functional characterization of HSC54, a novel variant of human heat-shock cognate protein 70.

A novel variant of human heat-shock cognate protein 70 (HSC70) transcript, named heat-shock cognate protein 54 (HSC54), was identified and characterized. The transcript encodes the protein lacking 153 amino acid residues of HSC70 in a part of the protein-binding and variable domains, resulting in a calculated molecular mass of 53.5 kDa. HSC54 mRNA was detected in all human cells and tissues examined. The protein was also detected in peripheral mononuclear cells and U937 human histiocytic lymphoma cells. Heat treatment of U937 cells up-regulated the expression of HSC54. The chaperoning activity of HSC54 was examined by luciferase renaturation assay. HSC70 recovered the luciferase activity in the presence of reticulocyte lysate as a source of cochaperones. However, HSC54 did not facilitate the recovery of denatured luciferase; besides, HSC54 significantly inhibited the HSC70-mediated chaperoning activity. In pull-down experiments, HSC54 interacted with cochaperones, p60, HSP40, and p48, as HSC70 did. The resonant mirror detection analysis showed that p60 binds to HSC54 with a higher association rate constant than HSC70 with a similar affinity constant. These results suggest that HSC54 is constitutively expressed and also inducible by stress and may function as an endogenous inhibitory regulator of HSC70 by competing the cochaperones.

Role of inflammatory mediators in lipid A analogue (ONO-4007)-induced vascular permeability change in mouse skin.

1. Endotoxin shock is accompanied by an increase in peripheral vascular permeability. It has been postulated that most biological activities of LPS are derived from lipid A moiety. Here we examined the effect of lipid A analogue ONO-4007 in increasing vascular permeability and the possible mediators in mouse skin by a dye leakage method. 2. Subcutaneous injection of ONO-4007 (1 - 2 mg site(-1)) induced a dose-dependent increase in vascular permeability which was evident after 120 min. 3. ONO-4007-induced dye leakage was significantly attenuated by pretreatments with anti-tumour necrosis factor-alpha (TNF-alpha) and anti-interleukin-1alpha (IL-1alpha) antibodies, but not with indomethacin (5 mg kg(-1)) or diphenhydramine (10 mg kg(-1)). ONO-4007-induced dye leakage was significantly inhibited by a pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME) (10 mg kg(-1)) but not with aminoguanidine (50 mg kg(-1)). In inducible nitric oxide synthase (iNOS)-deficient mice, ONO-4007 significantly increased the dye leakage, while ONO-4007 dilated rat thoracic aortic rings pre-contracted with phenylephrine, and the L-NAME pretreatment inhibited the dilation. 4. Thus, TNF-alpha, IL-1alpha and constitutive NOSs-derived nitric oxide but not prostaglandins or histamine play a role in ONO-4007-induced increase in vascular permeability. Although ONO-4007 mimics LPS in increasing vascular permeability, mechanisms of permeability change elicited by ONO-4007 were not identical to those of LPS.

Effect of lipoteichoic acid on dermal vascular permeability in mice.

Lipoteichoic acid (LTA), the cell wall component of Gram-positive bacteria, has been shown to cause inflammatory responses comparable to lipopolysaccharide (LPS) of Gram-negative bacteria. This study examined the activity of LTA to induce dermal microvascular permeability changes in mice. Vascular permeability was assessed by extravasation of Pontamine sky blue. Subcutaneous injection of LTA (200-400 microg/site) in mice that were preinjected i.v. with the dye increased local dye leakage in the skin at 1 to 3 h. The LTA-induced dye leakage was inhibited by indomethacin, valeryl salicylate, diphenhydramine, and a platelet-activating factor antagonist but not by inhibitors of nitric-oxide synthase, cyclooxygenase-2, or guanylate cyclase or by antibodies against tumor necrosis factor-alpha or interleukin-1alpha. LTA induced comparable increases in dye leakage in inducible nitric-oxide synthase-deficient mice and wild-type controls. Pretreatment of normal mice with i.v. LTA did not confer tolerance to LTA- or LPS-induced dye leakage. In contrast, systemic LPS administration induced tolerance against subsequent challenge with LPS but not LTA. Serum corticosterone levels, which were suggested to induce tolerance, were not increased by LTA pretreatment but were increased by LPS. Thus, LTA increases dermal microvascular permeability in mice. Among the inflammatory mediators, eicosanoids, platelet-activating factor, and histamine mediate the effect of both LTA and LPS, whereas nitric oxide, tumor necrosis factor-alpha, and interleukin-1alpha may not play a major role in LTA-induced dye leakage. The difference between LTA and LPS to stimulate corticosterone may partially explain the failure of LTA to induce tolerance against vascular dye leakage.

Infant leukemia suggestive of natural killer cell precursor origin followed an unusual clinical course.

We report a patient with infant leukemia showing the rare phenotypes of CD2+, CD4+, CD7+, CD56+, CD3-, CD5- who followed an unusual clinical course. The patient was a 3-month-old girl who was admitted because of unusual purpura looking like a black ring. On admission WBC count was 85.8 x 10(9)/l and bone marrow aspiration revealed a nucleated cell count of 112.0 x 10(9)/l with 70.8% atypical lymphocytes. On the 3rd hospital day, the WBC count decreased by about 1/5 without chemotherapy and partial remission was obtained. But about 3 weeks later, the WBC count increased again and she died. Based on surface marker analysis, genotypic analysis and autopsy, we diagnosed infant leukemia suggestive of natural killer (NK) cell precursor origin.

Protein kinase C mediates tumor necrosis factor-alpha-induced inhibition of obese gene expression and leptin secretion in brown adipocytes.

Previously we showed that tumor necrosis factor-alpha (TNF-alpha) inhibits lipoprotein lipase (LPL) activity and its gene expression, an early marker of adipocyte differentiation, in cultured brown adipocytes. To know whether TNF-alpha also affects late events in brown adipocyte maturation, we examined the effect of TNF-alpha on obese gene expression and leptin secretion in mouse brown adipocytes differentiated in culture. TNF-alpha caused a concentration-dependent decrease in leptin accumulation in culture medium and leptin mRNA amount in brown adipocytes which constitutively express the ob gene. Time-course study showed that TNF-alpha significantly suppressed leptin secretion during incubation for 16, 24 and 48 h. Since some effect of TNF-alpha is mediated by activation of protein kinase C (PKC), the role of PKC in TNF-alpha-induced downregulation of ob gene expression and leptin secretion was studied. The suppressive effect of TNF-alpha on both ob gene expression and leptin secretion was blocked by PKC inhibitors such as bisindolylmaleimide I (BIM) and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7). Incubation of brown adipocytes with TNF-alpha (20 ng/ml, 15 min) caused a rapid shift of PKC activity from the cytosolic to the membrane fraction, suggesting an activation of PKC by TNF-alpha in brown adipocytes. This effect of TNF-alpha was blocked by a selective PKC inhibitor, BIM. These results suggest that TNF-alpha promotes dedifferentiation of the brown adipocytes as evidenced by a downregulation in ob gene expression and leptin secretion via PKC-dependent mechanisms.

Evidence for modulation of osteocalcin containing gamma-carboxyglutamic acid residues synthesis by insulin-like growth factor-I and vitamin K2 in human osteosarcoma cell line MG-63.

The effect of insulin-like growth factor-I (IGF-I) and 2-methyl-3-all-trans-tetraphenyl-1,4-naphtoquinone (vitamin K2) on the synthesis of osteocalcin containing gamma-carboxyglutamic acid (Gla) residues which is the physiologically relevant form in bone metabolism was studied in cultured human osteoblast-like (MG-63) cells. Both IGF-I and vitamin K2 stimulated 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-induced osteocalcin containing Gla secretion in a concentration-dependent manner. This stimulatory effect of IGF-I and vitamin K2 was additive. Vitamin K2-enhanced osteocalcin containing Gla secretion was selectively suppressed by 3-(alpha-acetonyl-benzyl)-4-hydroxy-coumarin (warfarin). The stimulatory effect of IGF-I was completely abolished by the presence of cycloheximide; in contrast the effect of vitamin K2 was still observed in the presence of cycloheximide. Treatment of MG-63 cells with IGF-I caused an approximately 2.2-fold increase in osteocalcin mRNA levels (determined by reverse transcription-polymerase chain reaction). Vitamin K2 had no effect on either the stimulation of mRNA level by IGF-I or the basal level. IGF-I-stimulated osteocalcin containing Gla secretion was inhibited by one of its binding proteins (insulin-like growth factor binding protein-4) in a concentration-dependent manner. These findings suggest that the modes of action of IGF-I and vitamin K2 on 1.25(OH)2D3-induced osteocalcin containing Gla secretion in MG-63 cells are different.

Nitric oxide mediates down regulation of lipoprotein lipase activity induced by tumor necrosis factor-alpha in brown adipocytes.

We previously reported that tumor necrosis factor-alpha (TNF-alpha)/cachectin suppresses lipoprotein lipase activity and its gene expression in brown adipocytes differentiated in culture. Recent evidence suggests that the effect of TNF-alpha over various cells is related to the enhanced production of nitric oxide (NO). The present study examined whether the suppressive effect of TNF-alpha on lipoprotein lipase activity is mediated by production of NO in the brown adipocytes. A reverse transcription-polymerase chain reaction (RT-PCR) assay revealed that TNF-alpha caused a concentration- and time-dependent expression of inducible NO synthase in brown adipocytes. Increasing concentrations of TNF-alpha (0.5-50 ng/ml) for 24 h resulted in a concentration-dependent decrease in lipoprotein lipase activity with reciprocal increase in nitrite production in the medium. The suppressive effect of TNF-alpha on lipoprotein lipase activity was significantly prevented by NO synthase inhibitors, NG-nitro-L-arginine methyl ester (L-NAME) and aminoguanidine, but not by D-NAME, an inactive isomer. Furthermore, 8-bromoguanosine 3',5'-cyclic monophosphate, cell permeant cGMP, suppressed lipoprotein lipase activity and 1 H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one, a selective inhibitor for soluble guanylate cyclase, restored the TNF-alpha-suppressed lipoprotein lipase activity. These results suggest that TNF-alpha stimulates brown adipocytes to express inducible NO synthase, followed by production of NO, which in turn mediates the suppressive effect of TNF-alpha on lipoprotein lipase activity. The effect of NO is mediated, at least partly, through production of cGMP.

Estrogen and parathyroid hormone regulate insulin-like growth factor binding protein-4 in SaOS-2 cells.

The effect of 17beta-estradiol and parathyroid hormone (PTH) on the expression of insulin-like growth factor-binding protein-4 (IGFBP-4) messenger RNA (mRNA) was studied in the cultured human osteoblast-like SaOS-2 cells. Treatment of SaOS-2 cells with PTH for 3 h caused 3.3-fold increase in IGFBP-4 mRNA levels which was determined by reverse transcription-polymerase chain reaction. 17beta-Estradiol had no effect on either the stimulation of mRNA level by PTH or the basal level. Together with our previous report that 17beta-estradiol inhibits the PTH-induced reduction of IGFBP-4 proteolysis in these cells, the results obtained may help to explain the mechanisms of determining IGFBP-4 availability by systemic hormones in osteoblast cells.

Role of nitric oxide, prostaglandins and tyrosine kinase in vascular endothelial growth factor-induced increase in vascular permeability in mouse skin.

We investigated role of nitric oxide (NO), prostaglandins (PG) and tyrosine kinase in vascular endothelial growth factor (VEGF)-induced increase in vascular permeability in mouse skin. Subcutaneous injection of VEGF (0.5-2.0 ng/site) induced dose- and time-dependent increase in vascular permeability at the injection site determined by a leakage of Pontamine sky blue. VEGF (1 ng/site)-induced dye leakage was partially inhibited by N(G)-nitro-L-arginine methyl ester (an inhibitor for both constitutive and inducible NO synthase) (5 and 10 mg/kg, i.v.) and by aminoguanidine (a selective inducible NO synthase inhibitor) (5-20 mg/kg, i.v.), but not by an inactive enantiomer, N(G)-nitro-D-arginine methyl ester (10 mg/kg, i.v.). Pretreatment with an intraperitoneal injection of indomethacin (a nonselective cyclooxygenase inhibitor) (5 mg/kg) or N-(2-cyclohexyloxy-4-nitrophenyl) methanesulphonamide (a cyclooxygenase-2 selective inhibitor) (1-100 microg/kg) almost completely inhibited the effect of VEGF (1 ng/site). Coadministration of PGE2 (3 and 30 nmol/site) with VEGF did not restore the inhibitory effect of indomethacin on VEGF (1 ng/site)-induced increase in vascular permeability. Lavendustin A (a selective tyrosine kinase inhibitor) (10 and 50 microg/kg, s.c.) dose-relatedly inhibited the VEGF (1 ng/site)-induced increase in dye leakage, whereas its negative control, lavendustin B (10 microg/kg, s.c.) had no effect. Another tyrosine kinase inhibitor, genistein (2.5 mg/kg, s.c.) also inhibited the response. Cycloheximide (a protein biosynthesis inhibitor) (35 mg/kg, s.c.) suppressed the response of VEGF (1 ng/site). Histologically, no cellular infiltration was observed in the area of VEGF injection. These results suggest that increased vascular permeability induced by VEGF is mediated by local production of NO and arachidonic acid metabolites other than PGE2, which are most probably produced by inducible NO synthase and cyclooxygenase-2, respectively. Protein tyrosine kinase-mediated phosphorylation and synthesis of any new proteins are likely to be required in this effect of VEGF in mouse skin.

Cationic amino acid transporter-2 mRNA induction by tumor necrosis factor-alpha in vascular endothelial cells.

Nitric oxide (NO) synthesis may be coupled to the activity of the cellular L-arginine transporter, namely the cationic amino acid transporter. The present study examined tumor necrosis factor (TNF)-alpha-induced alterations in the gene expression of the cationic amino acid transporter (CAT) and NO production in human umbilical vein endothelial cells. In quiescent endothelial cells, CAT-1 mRNA expression, determined by reverse transcription-polymerase chain reaction, was dominant to that of CAT-2. TNF-alpha (10 ng/ml for 1-24 h) induced a time-dependent increase in CAT-2 but not CAT-1 expression. Moreover, TNF-alpha (1-30 ng/ml) treatment for 6 h induced a concentration-dependent increase in CAT-2 mRNA expression. The upregulation of CAT-2 expression by TNF-alpha was associated with enhanced nitrite accumulation in the culture medium (70% increase compared with vehicle-treated cells at 24 h). Thus, induction of the cationic amino acid transporter may constitute one mechanism for the TNF-alpha-induced NO production in human umbilical vein endothelial cells.

Tolerance to lipopolysaccharide-induced increase in vascular permeability in mouse skin.

We investigated whether tolerance develops to the lipopolysaccharide-induced increase in vascular permeability of mouse skin on pretreatment with Salmonella typhimurium lipopolysaccharide. Lipopolysaccharide-induced plasma extravasation was assessed by determining Pontamine sky blue dye accumulation in the skin where lipopolysaccharide was injected s.c. 2 h previously. When mice were pretreated with lipopolysaccharide (0.15 mg/kg i.p.), the dye leakage induced by s.c. challenge with lipopolysaccharide (400 micrograms/site) was significantly, inhibited for 2-24 h after pretreatment, indicating the development of lipopolysaccharide tolerance. At 4 h after lipopolysaccharide (0.15 mg/kg i.p.), the dose-response curve of dye leakage against the challenge dose of lipopolysaccharide shifted about 2-fold to the higher dose. The dye leakage induced by lipopolysaccharide was inhibited by pretreatment with lipopolysaccharide in a dose-dependent manner (0.05-0.15 mg/kg i.p.). Lipopolysaccharide tolerance was not seen in adrenalectomized mice. When mice were pretreated with lipopolysaccharide and NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide (NO) synthase inhibitor, at the same time, the hyporesponsiveness to lipopolysaccharide challenge disappeared. However, L-NAME was ineffective to inhibit the development of lipopolysaccharide tolerance when administered 24 h after lipopolysaccharide pretreatment or just before the lipopolysaccharide challenge. Tumor necrosis factor-alpha and interleukin-1 alpha but not interleukin-6 induced a similar hyporesponsiveness to lipopolysaccharide. These results suggest that tolerance develops to the lipopolysaccharide-induced increase in vascular permeability in mouse skin after a single lipopolysaccharide administration and that endogenous glucocorticoids and NO are necessary for induction of lipopolysaccharide tolerance. Hyporesponsiveness induced by lipopolysaccharide pretreatment may be mediated by production of some cytokines such as tumor necrosis factor-alpha or interleukin-1 alpha.

Histochemical staining of cadmium with 2-(8-quinolylazo)-4,5-diphenylimidazole.

In order to develop a stain with increased sensitivity and selectivity for cadmium (Cd), we synthesized and characterized 2-(8-quinolylazo)-4,5-diphenylimidazole (QAI). This chelating agent was more than twice as sensitive for Cd than the best conventional staining agents, including benzothiazolylazo-beta-naphthol. Differentiation between Cd and zinc (Zn) was achieved by immersing tissue sections in TRIS(2-aminoethyl)amine before they were stained with QAI. This pretreatment made it possible to selectively stain for Cd by blocking Zn.

Developmental changes in the response of rat isolated duodenum to nicotine.

Developmental changes in the response to ganglionic stimulants, nicotine and dimethylphenylpiperazinium, were investigated in rat isolated duodenum by recording isotonic mechanical activity. The duodenal response to nicotine/dimethylphenylpiperazinium (3 x 10(-7) to 10(-3) M) in neonatal rats was contraction, which was blocked by hexamethonium, tetrodotoxin and hyoscine. The response to nicotine/dimethylphenylpiperazinium (10(-6) to 10(-4) M) in the adult duodenum was relaxation, which was blocked by tetrodotoxin and hexamethonium, but by neither guanethidine nor hyoscine. The transition of the response to nicotine/dimethylphenylpiperazinium from contraction to relaxation occurred at around the 3rd postnatal week. Nicotine-induced relaxation of adult duodenum was significantly inhibited by preincubation with alpha-chymotrypsin, a proteolytic enzyme, and a combination of nucleotide pyrophosphatase and 8-phenyltheophylline, a P1 purinoceptor antagonist. Nicotine-induced relaxation was desensitized by alpha, beta-methylene ATP, a stable P2x purinoceptor agonist. These results suggest that the contractile response of isolated duodenum to nicotine is mediated through cholinergic transmission in neonatal rats and the relaxant response is mediated through non-adrenergic, non-cholinergic transmission, which involves both peptidergic and purinergic transmission, in adult rats.

Crohn's disease monocytes are primed for accentuated release of toxic oxygen metabolites.

BACKGROUND: Inflammatory bowel disease occurs in regions of the intestine characterized by a bowel content high in bacteria. Intestinal bacteria synthesize cell wall products such as lipopolysaccharide; when normal monocytes or macrophages come in contact with these products, they can be primed to release a number of inflammatory mediators. Mediators such as toxic oxygen metabolites released as part of the respiratory burst may contribute to inflammatory tissue damage. The aim of this study was to determine if monocytes from patients with Crohn's disease are primed by lipopolysaccharide for a greater respiratory burst. METHODS: The generation of superoxide anion was measured by superoxide dismutase inhibitable reduction of ferricytochrome c. RESULTS: Freshly isolated monocytes from active untreated Crohn's disease patients (n = 8) showed enhanced stimulated release of superoxide anion when compared with normal monocytes (n = 15; 3.80 +/- 0.12 vs. 1.02 +/- 0.06 nmol/5 min; P < 0.001). We tested the hypothesis that the monocyte priming factor in Crohn's disease serum may be lipopolysaccharide by showing that Crohn's disease serum lost its ability to prime normal monocytes after lipopolysaccharide was removed (0.25 +/- 0.25 nmol/5 min, P < 0.001). CONCLUSIONS: These studies indicate that bacterial cell wall products may be important proinflammatory molecules involved in the initiation and/or perpetuation of Crohn's disease.

Effect of caerulein on expression of the immediate-early genes c-fos and zif/268 in the rat brain.

The effect of caerulein, an analog of cholecystokinin-8, on expression of the immediate-early genes c-fos and zif/268 was studied in the rat brain using Northern blot analysis and an in situ hybridization technique. Intraperitoneal injection of caerulein did not change the basal c-fos and zif/268 expression. Administration of the convulsant, pentylenetetrazole (PTZ), caused a dramatic increase of c-fos and zif/268 mRNAs in the hippocampus and dentate gyrus. Pretreatment with caerulein suppressed the PTZ-induced c-fos and zif/268 expression. It is considered that systemically administrated caerulein modifies neuronal activities by exerting a suppressed effect on induction of the immediate-early genes.

L-NG-nitro-arginine inhibits nicotine-induced relaxation of isolated rat duodenum.

We studied the effects of L-NG-nitro-arginine (L-NOARG), which inhibits nitric oxide (NO) biosynthesis from L-arginine, on the non-adrenergic, non-cholinergic (NANC)-mediated relaxation induced by nicotine in isolated rat duodenum. L-NOARG reduced nicotine-induced relaxation, and L-arginine prevented the inhibitory effect of L-NOARG. However, L-NOARG did not inhibit the tetrodotoxin-insensitive relaxation induced by adenosine 5'-triphosphate, alpha, beta-methyleneadenosine 5'-triphosphate, thyrotropin-releasing hormone or the calcitonin gene-related peptide. Endogenous NO thus could possibly be involved in the NANC-mediated relaxation of rat duodenum induced by nicotine.