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Background: Nitric oxide (NO) is believed to participate in the regulation of airway clearance and non-specific cellular immunity. Recent studies have suggested that airway epithelial cells of allergic and non-allergic individuals may differ in their ability to produce this molecule., Objective: The aim of this study was to detect the difference in NO production in human nasal epithelial cells between normal subjects and patients with perennial allergic rhinitis (AR), and to assess the relationship between the expression of nitric oxide synthase (NOS) isoforms and the severity of the disease., Methods: Nasal epithelial cells were obtained from the inferior turbinate. The expression of mRNAs encoding constitutive endothelial NOS (eNOS) and inducible NOS (iNOS) was studied by reverse transcription-polymerase chain reaction (RT-PCR). Direct NO production in living cells was visualized and quantified by a fluorescent indicator, DAF-2 DA., Results: RT-PCR analysis demonstrated that AR patients with a RAST score of 5 or 6 showed significant increases in the levels of iNOS mRNA and slight reductions in those of eNOS mRNA. Patients with a RAST score of 2-4 also revealed the same tendency however, the difference was not significant. DAF-2 DA imaging demonstrated that epithelial cells, especially the ciliated cells, produced a larger amount of NO than non-epithelial inflammatory cells. Preincubation with L-NAME resulted in an approximate 40% decrease in both groups., Conclusion: These results directly indicate that nasal epithelial cells of AR patients overall produce higher levels of NO through the concomitant expression of different NOS isoforms. Continuous NO production by the epithelial cells in normal subjects further support the hypothesis that NO derived from epithelium may play dual roles in the regulation of nasal airway clearance and in the host defense. In addition, the use of DAF-2 DA provides a reliable method to visualize and quantify the direct NO production of living cells.

Study Design: This in vitro study clarifies the role of nitric oxide (NO) in human lumbar intervertebral disc metabolism., Objective: To investigate the effects of NO on proteoglycan synthesis in human lumbar discs and to test the hypothesis that NO is a mediator of the changes in proteoglycan synthesis in response to hydrostatic pressure., Summary of Background Data: The authors have clarified that hydrostatic pressure has an apparent effect on proteoglycan synthesis as well as matrix metalloproteinase production in the intervertebral disc. The cellular mechanisms underlying the response of disc cells to hydrostatic pressure remain to be clarified. Herniated lumbar discs produce NO in response to interleukin (IL)-1 beta. In articular cartilage, NO mediates the change of proteoglycan synthesis by IL-1 or shear stress., Methods: Fifty-eight lumbar intervertebral disc specimens were obtained from patients who had undergone posterior discectomy. The specimens were chopped into 1-2-mm cubes and were incubated in a plastic syringe with 1 mL Dulbecco's modified Eagle's medium (DMEM). The syringes were placed in a water-filled pressure vessel kept at 37 C. Hydrostatic pressures of 1 (control), 3, and 30 atmospheres (atm) were applied. Proteoglycan synthesis was determined from (35)S-sulfate incorporation rates. Nitrite (the stable oxidation product of NO) concentration in DMEM was determined by a spectrophotometric method based on the Griess reaction. As a competitive inhibitor of NO synthases, N(G)-methyl-l-arginine (l-NMA, 10-1000 micromol) and as an organic donor of NO, S-nitroso-N-acetylpenicillamine (SNAP, 1-200 micromol) were used., Results: Addition of l-NMA suppressed NO production and increased proteoglycan synthesis rates in the intervertebral disc specimens in a dose-dependent fashion. Addition of SNAP increased exogenous NO content in the medium significantly and suppressed proteoglycan synthesis rates in a dose-dependent fashion. Three-atmosphere hydrostatic pressure stimulated the proteoglycan synthesis rates. Rates were approximately 1.3-fold greater than at 1 atm, whereas 30-atm pressure inhibited proteoglycan synthesis rates. However, the hydrostaticpressure had inverse effect on NO production. At 3 atm, NO production decreased slightly relative to 1 atm, whereas at a pressure of 30 atm, NO production was increased and was approximately 1.32-fold greater than at 1 atm. L-NMA enhanced the 3-atm pressure-induced increase in proteoglycan synthesis and also relieved the suppression of proteoglycan synthesis at a pressure of 30 atm., Conclusion: The current study confirmed the previous finding that human herniated lumbar disc cultures spontaneously produce NO. Endogenously generated and exogenously supplied NO inhibited proteoglycan synthesis in the intervertebral disc. Hydrostatic pressure influenced NO production by disc cells, and NO is one of the mediators that changes proteoglycan synthesis in response to hydrostatic pressure. These results may show that autocrine and paracrine mechanisms of NO play an important role in the regulation of disc cell metabolism under mechanical stress and in the pathophysiology of intervertebral disc degeneration.

The primary function of well-differentiated ciliated epithelium in the paranasal sinus is to eliminate harmful agents through the beating action of cilia. Respiratory epithelium also contributes to local inflammatory processes through the release of various proinflammatory cytokines. Recently, considerable attention has been focused on the intimate relationship between the cytokine-dependent regulation of the ciliary beat frequency (CBF) and intra-cellular production of nitric oxide (NO) in ciliated epithelial cells. The aims of this study are to examine the effect of tumor necrosis factor-alpha (TNF-alpha), one of the major proinflammatory cytokines, on the ciliary activity of human sinus epithelial cells and to assess the hypothesis that NO is involved in this regulatory mechanism. Human maxillary or ethmoidal sinus mucosa (n = 23) were cultured by the explant-outgrowth method. CBF of the outgrowth ciliated cells was measured by the photoelectrical method before and after being treated with TNF-alpha (0.1, 1 and 10 ng/ml) or dexamethasone (10(-6) M and 10(-7) M). We also investigated the expression of nitric oxide synthase (NOS) isoforms, enzymes responsible for NO synthesis, by fluorescent immunohistochemistry. TNF-alpha increased CBF at relatively low concentrations (0.1 and 1 ng/ml) and decreased CBF at a high concentration (10 ng/ml). Dexamethasone decreased CBF at a concentration of 10(-6) M. Fluorescent immunohistochemistry demonstrated that the expression of inducible NOS was augmented by TNF-alpha and attenuated by dexamethasone, whereas that of endothelial NOS remained unchanged. We conclude that human sinus epithelial cells potentially contribute to the inflammatory process by regulating their ciliary motility through an NO-dependent pathway. Proinflammatory cytokines and steroids are able to modulate this mechanism by the induction or inhibition of expression of different NOS isoforms.