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Background: Nitric oxide (NO) mediates different cellular functions by activating soluble guanylate cyclase (sGC) that converts guanosine-5'-triphosphate (GTP) to cyclic guanosine-3',5'-monophosphate (cGMP). Membrane-bound GCs produce cGMP in response to natriuretic peptides in osteoblasts, but neither the NO-target enzyme sGC, nor the phosphorylation sites of NOS III, nor their regulation by extracellular signal-regulated kinases 1 and 2 (ERK1/2) and Akt/protein kinase B (Akt/PKB) in osteoclasts have been established., Methods: Rat molars with periodontium were perfusion- and post-fixed, decalcified, and frozen-sectioned. Free-floating sections were stained using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) and tartrate-resistant acid phosphatase (TRAP) histochemical techniques and immunoreacted with antisera against NO-synthase (NOS) I-III, NOS III phoshorylated at Thr495, NOS III phoshorylated at Serine1177 (Ser1177), ERK1/2, phosphorylated ERK1/2, Akt/PKB, phosphorylated Akt/PKB, sGC (alpha2/beta1), and cGMP., Results: NADPH-d staining and immunostaining of NOS I-III, NOS III phosphorylated at Ser1177, ERK1/2, Akt/PKB, phosphorylated Akt/PKB, sGC (alpha2 and beta1-subunits), and cGMP were detected in osteoclasts. Immunohistochemical reaction products for NOS III phosphorylated at threonine495 (Thr495) and phosphorylated ERK1/2 could not be identified in osteoclasts. Comparison of TRAP activity and immunostaining for sGC beta1-subunit revealed that sGC beta1-subunit is only expressed in a sub-population of osteoclasts., Conclusions: NO is likely to be generated by NOS I and NOS III in osteoclasts. The inconstant expression of NOS II in some osteoclasts may be explained with inducible expression of NOS II upon physiological cell activation. Localization of the sGC alpha2- and beta1-subunits and cGMP in osteoclasts is compatible with an involvement of NO-sGC signaling in the homeostasis of osteoclasts. The phosphorylation site of NOS III at Ser1177 and phosphorylated Akt/PKB are involved in regulation of NO production by NOS III in osteoclasts under basal conditions.

Nitric oxide (NO) is thought to play an important role as a signaling molecule in embryonic and adult cardiomyocytes; however, its involvement in muscarinic signaling is still unclear. The aim of the present work was to analyze the muscarinic modulation of the L-type Ca2+ current (ICa) in early- and late-stage embryonic ventricular cardiomyocytes. Muscarinic stimulation depressed basal ICa by 30.1 +/- 3.2% (n=27) in early-stage cardiomyocytes. Pharmacological evidence suggested that the muscarinic modulation was mediated through generation of NO, activation of cGMP-dependent phosphodiesterase (PDE) 2, and ensuing lowering of cyclic AMP/protein kinase A (cAMP/PKA) levels. Conversely, in late-stage cardiomyocytes, muscarinic regulation of ICa occurred in a NO-independent manner via inhibition of prestimulated adenylyl cyclase (AC). To unequivocally prove the involvement of NO and to identify the nitric oxide synthase (NOS) isoform(s), we analyzed muscarinic signaling in embryonic ventricular cardiomyocytes of NOS2 (-/-) and NOS3 (-/-) mice. The early-stage NOS3 (-/-) cardiomyocytes lacked muscarinic modulation, whereas it was preserved in NOS2 (-/-) cells. Moreover, at the late embryonic stage, muscarinic modulation of ICa was intact in both strains. Thus, NO is the key regulator of muscarinic signaling in the early embryonic ventricle, whereas at later stages, signaling occurs through a NO-independent pathway.

Background: Nitric oxide (NO) is produced by a group of synthase enzymes (NOS). By means of different pathways, NO exerts several functions in benign and malignant human bladder tissues. The current paper describes the NO/guanylate cyclase (sGC)/cyclic guanosine monophosphate (cGMP) and the NO/oxidative pathways in human bladder tissues., Methods: Bladder carcinoma tissues were collected from 18 patients by transurethral resection procedures. Normal benign vesical tissue specimens from a further eight patients with benign diseases served as controls. Immunohistochemistry was conducted for localization of sGC, cGMP, and nitrotyrosine in benign and malignant vesical tissues, evaluating two-three tissue sections per patient., Results: Positive immunolabeling for sGC and cGMP was detected in vascular endothelial cells of normal and malignant vesical tissues. Those signals were most intense in bladder carcinoma tissues. Immunolabeling for sGC and cGMP was also detected in normal urothelial cells. In bladder carcinoma cells, a heterogeneous immunolabeling for sGC and cGMP was seen, with a wide spectrum of signal intensity. Positive immunostaining for sGC and cGMP was also observed in stromal round cells in benign and malignant bladder tissues. Immunolabeling for nitrotyrosine was mainly observed in endothelial cells, with a much stronger immunolabeling in bladder carcinoma tissues compared to normal benign controls. A weak immunolabeling for nitrotyrosine was also observed in bladder carcinoma cells. Normal urothelial cells did not show such nitrotyrosine expression., Conclusions: The current report provides evidences that NO play several roles through different pathways in benign and malignant vesical tissues. The influences generated by NO molecules can be divided into cGMP-mediated effects (those resulting from the NO/sGC/cGMP pathway) and non-cGMP-mediated effects (those resulting from the NO/oxidative pathway). Increased angiogenesis is a cGMP-mediated effect, while nitrotyrosine production is a non cGMP-mediated oxidative effect. Such an NO/oxidative pathway is observed more often in bladder carcinoma., (Copyright 2002 American Cancer Society.DOI 10.1002/cncr.10942)

Abstract: Multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) have long been regarded as primarily T helper cell type 1-mediated diseases. However, recent evidence suggests that TH17 cells, a mostly unexplored subset of T helper cells, may be even more pathogenic than TH1 cells. In the EAE model, this cell type is crucial for the recruitment of leukocytes into the CNS and for triggering parenchymal inflammation. In humans, TH17 cells are found in acutely active and on the borders of chronically active lesions. Overall, CD4+ T cells only recognize antigens presented on MHC class II complexes, and these are seldom found in the CNS. MHC class I, in contrast, can be induced on neurons and myelin. This also makes CD8+ T cells promising candidates as effector cell types. Indeed, CD8+ T cells outnumber CD4+ T cells in the lesions of MS patients, and can induce axonal pathology. New data on B cells have likewise stimulated unconventional paths of reasoning about the disease. B cells can contribute to the pathogenesis by secreting autoantibodies and presenting antigens to T cells. By the formation of ectopic B cell aggregates in the CNS, B cell differentiation and response can take place remote from the periphery, thus autonomously fueling pathology. In addition, cells of the innate immune system including macrophages, dendritic cells and mast cells are present in the inflamed CNS. On the one hand, these cells can recognize pathogen-associated molecular patterns via Toll-like receptors (TLRs), generating proinflammatory signals that trigger adaptive immune responses. On the other hand, these cells support the autoimmune process by the secretion of effector molecules such as nitric oxide (NO). Apart from a solely pathogenic autoimmune role, regulatory T cells, NK cells and NKT cells can suppress autoreactive cells. In this paper, we review data on how a complex network of immune mechanisms is involved in the pathogenesis of MS and EAE. We also critically reevaluate the traditional CD4/TH1 paradigm. [Copyright &y& Elsevier]

Background: Previous investigations suggested apoptosis as a contributing factor to early failure of allograft heart valves. As myocardial apoptosis may be induced by nitric oxide (NO) release, this study investigated NO synthase [NOS-III] activation and apoptotis induction in human cryopreserved allografts during the thawing process. Methods: Frozen myocardial tissue from ten human allograft heart valves, unsuitable for implantation, was submitted to the following conditions: (1) thawing in paraformaldehyde (Control), thawing according to the standard clinical protocol (Standard), standard-thawing with addition of the NOS-inhibitor N-omega-nitro-l-arginine (L-NA; Standard-LNA), and standard thawing with the NOS-stimulator angiotensin II (Standard-AT-II). Cryo-thin sections were investigated by immunostaining for activated NOS-III, cyclic guanosine monophosphate (cGMP), activated caspase-3, and poly-ADP-ribose polymerase (PARP). Quantitative analyses was performed by television densitometry. Results: For activated NOS-III, gray unit values were significantly higher in the Standard and Standard-AT-II group than in the Control and Standard-LNA groups (p < 0.001). Gray unit values for cGMP, a downstream NO-signal-pathway molecule, showed results grossly corresponding to NOS-III activation. Activated caspase-3 and PARP showed high levels of expression in all groups with no significant differences. Conclusions: Significant activation of NOS-III and subsequent NO-cGMP signal pathway occurs in human cryopreserved allografts during the thawing process and can be significantly reduced by a NOS-III inhibitor administered during thawing. Activation of the apoptosis pathway is also present after thawing, which was not correlated to NOS-III activation. Further experimental investigation focused on the time course and mechanisms of apoptosis and NOS-III activation are required to evaluate NOS and(or) apoptosis inhibitors as therapeutic strategies for improved allograft preservation. [Copyright &y& Elsevier]

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Background: The epithelial rests of Malassez (ERM) are derived from the Hertwig's epithelial root sheath during tooth development. The ERM contain endothelial nitric oxide synthase (eNOS), but the existence of phosphorylation site/s of eNOS in the ERM is unclear. Methods: Rat molars with periodontium were perfusion- and post-fixed, decalcified, and frozen-sectioned. Free-floating sections were incubated using antisera against total eNOS, eNOS phosphorylated at serine (Ser)1177 Ser116 and threonine (Thr)495. The signal intensities of t-eNOS, p-eNOS at Ser1177 and Ser116 in the ERM were measured by densitometry and statistically analyzed. Results: In the ERM, localization of total eNOS and the phosphorylation sites of eNOS at Ser1177 and Ser116 were detected, while a basal localization of eNOS phosphorylated at Thr495 in the ERM was undetectable. For p-eNOS at Ser116 regional differences in phosphorylation were detected. Conclusions: The basal production of NO by eNOS in the ERM is modulated by phosphorylation of eNOS at Ser1177 and Ser116 residues, while the basal activity of the eNOS is not influenced by phosphorylation of eNOS at Thr495 residue. This provides evidence that phosphorylation plays a key role for regulation of the catalytic activity of eNOS. [ABSTRACT FROM AUTHOR]

It is known that total endothelial nitric oxide synthase (eNOS) is localized in peripheral nerve fibers, but the existence of the phosphorylation site/s of eNOS in peripheral nerve fibers is unknown. In the perfusion-fixed and decalcified sections of rat palates, eNOS and eNOS phosphorylated at Ser116 were identified in the nerve fibers of the glandulae palatinae. The localization of eNOS phosphorylated at Ser1177 and Thr495 in nerve fibers was undetectable. It is concluded that eNOS is phosphorylated at Ser116 in nerve fibers of the glandulae palatinae under basal conditions. The basal Ser1177 and Thr495 phosphorylation of eNOS are missing in nerve fibers of the glandulae palatinae. [Copyright &y& Elsevier]