Nitric oxide is overproduced by peritoneal macrophages in rat taurocholate pancreatitis: the mechanism of inducible nitric oxide synthase expression

To investigate the pathobiology of severe acute pancreatitis, we studied the expression of inducible nitric oxide synthase (iNOS) in peritoneal macrophages of experimental pancreatitis. Taurocholate (TCA) pancreatitis and cerulein (CE) pancreatitis were used as models of lethal and self-limited pancreatitis, respectively, and the mechanism of iNOS expression in peritoneal macrophages was studied. Serum nitrate and nitrite (NOx) concentrations increased during the course of TCA pancreatitis, and iNOS-immunoreactivity was detected in the peritoneal macrophages 12 h after the induction of TCA pancreatitis, but these phenomena were not observed in CE pancreatitis. Despite the difference in the iNOS expression, the iNOS messenger RNA (mRNA) and the activation of nuclear factor-kappa B (NF-kappa B) were detected in the peritoneal macrophages of both pancreatitis models. The supernatant of TCA pancreatitis ascites could induce iNOS in the peritoneal macrophages of normal rats in vitro, but the peritoneal lavage fluid of CE pancreatitis rats could not. The results indicated that there may be qualitative or quantitative differences in the macrophage activation between the two types of experimental pancreatitis and suggested that the ascites of rats with lethal acute pancreatitis contains some soluble factors that activate the macrophage/monocyte system and cause an overproduction of NO by the iNOS expression.     

Nitric oxide synthase in the vestibulocochlear system of mice

PURPOSE: To evaluate the use of helical computed tomography (CT) without contrast material enhancement for prediction of a favorable outcome in ureterolithiasis. MATERIALS AND METHODS: CT studies were reviewed in 69 patients with a single ureteral stone not located at the ureteropelvic junction. CT findings (tissue rim sign, hydronephrosis, perinephric fat stranding, perinephric fluid collections, and thickening of renal fascia) were graded on a scale of 0-3. Stone diameter and renal parenchymal enlargement were also measured. RESULTS: Twenty-two patients had spontaneous passage, 12 did not respond to conservative treatment, and 35 were lost to follow-up. When the latter 35 patients were excluded, perinephric fat stranding (P = .044) and perinephric fluid collections (P = .021) were graded significantly higher in patients with spontaneous stone passage. Mean stone diameter was significantly larger (P < .001) in patients in whom conservative treatment failed (mean, 7.8 mm) than in patients with spontaneous stone passage (mean, 2.9 mm). The presence of a tissue rim sign and the grade of hydronephrosis, renal fascial thickening, and renal parenchymal enlargement were not significantly different between the two groups. CONCLUSION: In addition to stone size, the degree of perinephric fat stranding and the presence of perinephric fluid collections are useful ancillary signs for help in predicting the likelihood of stone passage.     

Nitric oxide synthase in neuroepithelial brain tumors

Immunohistochemical investigation of NO-synthase in brain astrocytic tumors revealed intense reaction in many tumor cells as well as direct correlation in the intensity of reaction and the degree of tumor anaplasia. Grade I astrocytomas did not show immunoreactivity in contrast to high anaplastic tumors where many cells had positive reaction with a different degree of intensity. Positive immunoreaction was shown in many giant cells. Small cell glioblastomas and oligodendrogliomas were immunonegative. There was a direct correlation between NO-synthase expression and glial fibrillar acidic protein.     

Nitric oxide synthase in cardiac sarcoplasmic reticulum

NO. is a free radical that modulates heart function and metabolism. We report that a neuronal-type NO synthase (NOS) is located on cardiac sarcoplasmic reticulum (SR) membrane vesicles and that endogenous NO. produced by SR-associated NOS inhibits SR Ca2+ uptake. Ca2+-dependent biochemical conversion of L-arginine to L-citrulline was observed from isolated rabbit cardiac SR vesicles in the presence of NOS substrates and cofactors. Endogenous NO. was generated from the vesicles and detected by electron paramagnetic resonance spin-trapping measurements. Immunoelectron microscopy demonstrated labeling of cardiac SR vesicles by using anti-neuronal NOS (nNOS), but not anti-endothelial NOS (eNOS) or anti-inducible NOS (iNOS) antibodies, whereas skeletal muscle SR vesicles had no nNOS immunoreactivity. The nNOS immunoreactivity also displayed a pattern consistent with SR localization in confocal micrographs of sections of human myocardium. Western blotting demonstrated that cardiac SR NOS is larger than brain NOS (160 vs. 155 kDa). No immunodetection was observed in cardiac SR vesicles from nNOS knockout mice or with an anti-nNOS mu antibody, suggesting the possibility of a new nNOS-type isoform. 45Ca uptake by cardiac SR vesicles, catalyzed by Ca2+-ATPase, was inhibited by NO. produced endogenously from cardiac SR NOS, and 7-nitroindazole, a selective nNOS inhibitor, completely prevented this inhibition. These results suggest that a cardiac muscle nNOS isoform is located on SR of cardiac myocytes, where it may respond to intracellular Ca2+ concentration and modulate SR Ca2+ ion active transport in the heart.     

Nitric oxide production in mouse peritoneal macrophages enhanced with glycyrrhizin

The enhancement of nitric oxide (NO) production in glycyrrhizin (GL)-induced macrophages (M phi) in response to lipopolysaccharide (LPS) was investigated. No production in GL-induced macrophage culture supernatants was stimulated in response to LPS (10 micrograms/ml) for 24- or 48- h cultures, and these levels were compared three times with the levels in saline-induced peritoneal exudate cell cultures. Furthermore, M phi induced with proteose peptone (PP) containing GL could generate greater NO production than M phi induced with PP alone. However, no stimulation of NO production was observed by addition of GL in the cultures of M phi induced with thioglycollate or Bacillus Calmette Guerin. Moreover, GL-induced M phi showed cytostasis against such tumor target cells as L 1210 and P 388 lymphoma cell lines. These observations indicate that GL can activate the M phi in vivo system and stimulate NO production in response to LPS.     

Induction of apoptosis in myocardial infarction and its possible relationship to nitric oxide synthase in macrophages

Activated macrophages produce nitric oxide through the inducible form of nitric oxide synthase (iNOS). Previously, a significant increase of iNOS activity in macrophages in infarcted rabbit heart tissue was observed. The present study is concerned with the induction of apoptosis in macrophages and cardiomyocytes in infarcted rabbit heart tissue. The left anterior descending artery of rabbits was ligated. The heart was excised five hours, one, two, three, ten and twenty days later, and DNA was extracted from infarcted and non-infarcted region and subjected to electrophoresis. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) was carried out, and iNOS activity was measured by conversion of L-[14C]-arginine to L-[14C]-citrulline. Positive staining by TUNEL was seen in some cardiomyocytes five hours after coronary ligation and on postoperative day (POD) 1; internucleosomal DNA fragmentation was not noted. On POD 2 and 3, many infiltrating cells, immunohistochemically identified as macrophages, were positively stained by TUNEL; DNA fragmentation was also present. Apoptosis was not found on POD 10 and 20. The peak activity of iNOS was noted on POD 3, which corresponded with the induction of apoptosis. It is tempting to speculate that a causal relationship exists between increased iNOS formation and induction of apoptosis in macrophages in infarcted rabbit heart tissue.     

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.     

Nitric oxide production by murine macrophages is inhibited by prolonged elevation of cyclic AMP

Murine macrophages produce large amounts of nitric oxide (NO) on stimulation by interferon (IFN)-gamma and lipopolysaccharide (LPS) or a high concentration of LPS alone. Agents which increase intracellular cAMP levels inhibit cytokine production by macrophages. The effect of increased intracellular cAMP levels on NO production was investigated, using a murine macrophage cell line, J774. NO production was reduced by prolonged elevation of cAMP, but not by a transient increase.     

Neuropathic pain in rats is associated with altered nitric oxide synthase activity in neural tissue

Peripheral nerve injury may lead to a chronic neuropathic pain state that results from an increase in excitability of central neurons. This central sensitization is mediated via an N-methyl-D-aspartic acid (NMDA) receptor and may involve the production of nitric oxide (NO). As NO is suggested to play a role in nociceptive transmission following nerve injury, we examined for altered NO synthase activity at multiple levels of peripheral and spinal neural tissue in a rat model of neuropathic pain. Peripheral neuropathy was induced in rats (N = 12) by ligation of the left L5 and L6 nerve roots. Six other rats had sham surgery. An ipsilateral decrease in paw withdrawal threshold to mechanical stimuli confirmed the presence of a neuropathic pain state. Samples of the lumbar and thoracic spinal cords, L4, L5, and L6 dorsal root ganglia (DRGs), and the sciatic nerves were obtained from the lesioned and contralateral sides at 2 and 4 weeks after neuropathic surgery (N = 6 per group). In the lumbar spinal cord, a bilateral decrease in nitric oxide synthase (NOS) activity was observed 2 and 4 weeks after neuropathic surgery. NOS activity was increased in the ipsilateral L5 and 6 DRGs 2 weeks following neuropathic surgery. An increase in NOS activity in the DRG may be an early mechanism for inducing more central changes. The bilaterally decreased NOS activity in the lumbar spinal cord may be secondary to a negative feedback mechanism resulting from increased NO production in the spinal dorsal root ganglia. Multiple alterations in expression of NOS activity that occur in both peripheral and central processing may play a role in the pain behavior resulting from peripheral nerve injury. (Preliminary results of these studies have been presented in abstract form at the annual meetings of the Society for Neuroscience, 1994, and the American Society of Anesthesiologists, 1994).     

Nitric oxide synthase is co-localized with vasoactive intestinal polypeptide in postganglionic parasympathetic nerves innervating the rat vas deferens

Cross-sections of the vas deferens taken from control adult male rats showed positive histochemical reactivity to acetylcholinesterase and immunoreactivity for antibodies to protein gene product 9.5, tyrosine hydroxylase, neuropeptide Y, vasoactive intestinal polypeptide, nitric oxide synthase and calcitonin gene-related peptide. Immunoreactivity to substance P was very sparse. Histochemical reactivity to acetylcholinesterase and immunoreactivity to vasoactive intestinal polypeptide and nitric oxide synthase was concentrated in the subepithelial lamina propria and inner smooth muscle layers. Complete surgical denervation resulting from transection of the nerve arising from the pelvic ganglion which supplies the vas deferens totally abolished the immunoreactivity to all of the antibodies tested as well as the histochemical reactivity to acetylcholinesterase. In sections of the prostatic end of the vas deferens taken from rats neonatally pretreated with capsaicin, immunoreactivity to calcitonin gene-related peptide and substance P was reduced by 75 and 83%, respectively. Immunoreactivity to neuropeptide Y, vasoactive intestinal polypeptide and nitric oxide synthase was similar in tissue sections taken from capsaicin-treated rats and those taken from control tissues. Pretreatment of rats with guanethidine or 6-hydroxydopamine decreased immunoreactivity to tyrosine hydroxylase and neuropeptide Y by 60-70%, but immunoreactivity to substance P, vasoactive intestinal polypeptide and nitric oxide synthase was unchanged, while immunoreactivity to calcitonin gene-related peptide and acetylcholinesterase staining was increased by guanethidine but not by 6-hydroxydopamine treatment. Triple labelling experiments showed nitric oxide synthase, vasoactive intestinal polypeptide and acetylcholinesterase all to be co-localized in some nerve fibres. These results indicate that the nitric oxide synthase contained in the nerve fibres innervating the rat vas deferens is unaffected by pretreatment of rats with capsaicin, 6-hydroxydopamine or guanethidine but is abolished by surgical denervation, of postganglionic parasympathetic, sympathetic and sensory nerves. Therefore it appears that nitric oxide synthase is co-localized with vasoactive intestinal polypeptide in the postganglionic parasympathetic nerves which innervate the rat vas deferens.     

Nitric oxide synthase in developing retinas and after optic tract section

Protein kinase C (PKC) is a key enzyme in the intracellular signaling network. Upon activation by 12-O-tetradecanoylphorbol 13-acetate, the alpha-isoform of PKC translocates to the detergent-soluble and the detergent-insoluble fractions. Besides cofactors, the activity and stability of this protein is critically regulated by multisite phosphorylations. At least three distinct sites, Thr497, Thr638 and Ser657, are involved. We have previously shown that the replacement of Ser657 by alanine leads to a premature down-regulation in the detergent soluble compartment of LLC-PK1 cells [Gysin, S. & Imber, R. (1996) Eur. J. Biochem. 240, 747-750]. More detailed analysis revealed that, in contrast to the wild-type molecule, the down-regulation of the mutant protein is in vivo preceded by a rapid dephosphorylation after phorbol-ester-induced translocation to both the detergent-soluble and insoluble compartments. The [Ala657]PKC-alpha mutant protein molecule showed in vitro a strongly increased sensitivity towards protein phosphatase 2A whereas its overall proteolytic sensitivity remained unchanged when compared to wild type. The in vitro studies led to the suggestion that further dephosphorylation of the mutant protein is a prerequisite in order to become proteolytically down-regulated. Therefore phosphorylation of Ser657 controls the duration of activation of this PKC isozyme upon agonist-induced translocation by preventing premature proteolytic down-regulation via protecting the protein from dephosphorylation.     

Inducible nitric oxide synthase expression in smooth muscle cells and macrophages of human transplant coronary artery disease

BACKGROUND: The inducible isoform of the nitric oxide synthase (iNOS) produces large amounts of nitric oxide in response to cytokine stimulation. Previous investigations have demonstrated iNOS expression in the setting of acute and chronic rejection in experimental cardiac transplant models. The goal of this study was to investigate whether iNOS is upregulated in human transplant coronary artery disease (TCAD), a major cause of late mortality after cardiac transplantation. METHODS AND RESULTS: We studied 15 patients with TCAD and 10 with normal coronary arteries. In situ hybridization and immunohistochemistry were used in tissue sections to localize iNOS mRNA and protein, respectively. The presence of peroxynitrite was indirectly assessed by immunostaining with an anti-nitrotyrosine antibody. Normal coronary arteries had no evidence of iNOS expression. In contrast, 30 of 36 coronary artery segments with TCAD (83%) were immunostained by the iNOS antibody. The presence of iNOS mRNA was demonstrated in these vessels by in situ hybridization. Specific cell markers identified iNOS-positive cells as neointimal macrophages and smooth muscle cells. Nitrotyrosine immunoreactivity colocalized with iNOS expression in arteries with TCAD, distributed in macrophages and smooth muscle cells. CONCLUSIONS: iNOS mRNA and protein are expressed in human arteries with TCAD, where they are associated with extensive nitration of protein tyrosines. These findings indicate that the high-output nitric oxide pathway and possibly the oxidant peroxynitrite might be involved in the process leading to the development of TCAD.     

Nitric oxide synthase inhibitors alter ventilation in isoflurane anesthetized rats

BACKGROUND: Nitric oxide (NO) is present in medullary structures and can modulate respiratory rhythm. The authors determined if spontaneous ventilation at rest and in response to increased carbon dioxide is altered by selective neuronal NO synthase (NOS; 7-nitro-indazole, 7-NI) or nonselective (neuronal plus endothelial) NOS (NG-L-arginine methyl ester [L-NAME] and NG-monomethyl L-arginine [L-NMMA]) inhibitors in rats anesthetized with isoflurane. METHODS: Fifty-four rats received either L-NAME or L-NMMA (1, 10, and 30 mg/kg) or 7-NI (20, 80, and 400 mg/kg) and were compared with time controls (isoflurane = 1.4%), with isoflurane concentrations (1.6%, 1.8%, and 2%) increased consistent with the increased anesthetic depth caused by NOS inhibitors, or with L-arginine (300 mg/kg). Tidal volume (VT), respiratory frequency (f), minute ventilation (VE), and ventilatory responses to increasing carbon dioxide were determined. RESULTS: L-NAME and L-NMMA decreased resting VT and VE, whereas 7-NI had no effect. Increasing concentrations of isoflurane decreased resting f, VT, and VE. L-NAME and L-NMMA decreased VT and VE, whereas 7-NI had no effect at 8%, 9%, and 10% end-tidal carbon dioxide (ETCO2). Increasing concentrations of isoflurane decreased f, VT, and VE at 8%, 9%, and 10% ETCO2. The slope of VE versus ETCO2 was decreased by isoflurane but was unaffected by L-NAME, L-NMMA, or 7-NI. L-arginine alone had no effect on ventilation. CONCLUSIONS: Nonselective NOS inhibitors decreased VT and VE at rest and at increased carbon dioxide levels but did not alter the slope of the carbon dioxide response. Selective neuronal NOS inhibition had no effect, suggesting that endothelial NOS may be the isoform responsible for altering ventilation. Finally, the cause of the decreased ventilation is not a result of the enhanced anesthetic depth caused by NOS inhibitors.