Shawn A. Milligan

Nitric oxide synthase in circulating vs. extravasated polymorphonuclear leukocytes

It is becoming increasingly apparent that certain forms of acute and chronic inflammation are associated with enhanced production of nitric oxide (NO). Although substantial information has been obtained describing the regulation of NO synthase (NOS) in macrophages, little information is available regarding the biochemistry and molecular biology of NOS in circulating vs. extravasated polymorphonuclear leukocytes (PMNs). The objective of this study was to characterize the molecular and biochemical properties of the inducible NO synthase (iNOS) in circulating vs. extravasated rat and human PMNs. Circulating rat and human PMNs were purified from peripheral blood and extravasated PMNs were elicited in rats by intraperitoneal injection of 1% oyster glycogen or in humans by peritoneal dialysis of patients with peritonitis. Inducible NOS mRNA from circulating and elicited PMNs was quantified using slot blot hybridization analysis with a cDNA probe specific for iNOS. iNOS protein was identified using Western immunoblot analysis, and NOS activity was quantified by measuring the NG‐monomethyl‐L‐arginine (L‐NMMA)‐inhibitable conversion of 14C‐labeled L‐arginine to L‐[14C]citrulline. In a separate series of experiments, circulating or extravasated PMNs were cultured for 4 h and the accumulation of L‐NMMA‐inhibitable nitrite (NO2−) in the supernatant was determined and used as a measure of NO production in vitro. We found that circulating PMNs (rat or human) contained no iNOS mRNA, protein, or enzymatic activity. Furthermore, circulating rat or human PMNs (2 × 106 cells/well) were unable to generate significant amounts of NO2− when cultured for 4 h in vitro. In contrast, iNOS mRNA levels in 4‐ and 6‐h elicited rat PMNs increased 21‐ and 42‐fold, respectively, when compared with circulating cells. Western blot analysis revealed the presence of iNOS protein in the elicited rat PMNs and iNOS enzymatic activity increased from normally undetectable levels in circulating rat PMNs to 81 and 285 pmol/min/mg for the 4‐ and 6‐h elicited rat PMNs, respectively. Approximately 20–30% of the total iNOS activity was Ca2+‐dependent. Nitrite formation by elicited rat PMNs in the absence of any exogenous stimuli increased from normally undetectable amounts for circulating PMNs to approximately 8 and 11 μM/106 cells for the 4‐ and 6‐h elicited PMNs, respectively. Highly enriched preparations of extravasated human PMNs contained neither message, protein nor iNOS enzymatic activity. Taken together our data demonstrate that inflammation‐induced extravasation of rat PMNs upregulates the transcription and translation of iNOS in a time‐dependent fashion and that 20–30% of the total inducible NOS is Ca2+‐dependent. In contrast, neither circulating nor extravasated human PMNs contained iNOS message, protein, or enzymatic activity. These data suggest that the human PMN iNOS gene is under very different regulation than is the rat gene.

Superoxide released from neutrophils causes a reduction in nitric oxide gas

Exhaled nitric oxide (NO) is increased in some inflammatory airway disorders but not in others such as cystic fibrosis and acute respiratory distress syndrome. NO can combine with superoxide ([Formula: see text]) to form peroxynitrite, which can decompose into nitrate. Activated polymorphonuclear neutrophils (PMNs) releasing[Formula: see text] could account for a reduction in exhaled NO in disorders such as cystic fibrosis. To test this hypothesis in vitro, we stimulated confluent cultures of LA-4 cells, a murine lung epithelial cell line, to produce NO. Subsequently, human PMNs stimulated to produce [Formula: see text] were added to the LA-4 cells. A gradual increase in NO in the headspace above the cultures was observed and was markedly reduced by the addition of PMNs. An increase in nitrate in the culture supernatant fluids was measured, but no increase in nitrite was detected. Superoxide dismutase attenuated the PMN effect, and xanthine/xanthine oxidase reproduced the effect. No changes in epithelial cell inducible NO synthase protein or mRNA were observed. These data demonstrate that [Formula: see text]released from PMNs can decrease NO by conversion to nitrate and suggest a potential mechanism for modulation of NO levels in vivo.

Restless legs syndrome in the older adult: Diagnosis and management

Restless legs syndrome (RLS) is common in the elderly, with an estimated prevalence of 10 to 35% in individuals over 65 years of age. RLS is characterised by paraesthesias and dysaesthesias of the legs, typically occurring in the evening. The symptoms occur at rest and result in motor restlessness; movement often temporarily relieves the symptoms. Patients with poorly controlled RLS may develop related problems including insomnia (due to sleep-onset restlessness or periodic limb movements or related sleep fragmentation) and depression. RLS can be a primary disorder that develops in the young and includes familial cases. Secondary RLS occurs in association with iron-deficiency anaemia, uraemia and polyneuropathies. Typically, RLS is misdiagnosed or undiagnosed for years. In the elderly, both primary and secondary types of the disorder are common.It is thought that RLS represents lower CNS levels of, or reduced responsiveness to, dopamine. The symptoms improve with dopaminergic therapy. Ergotamine dopamine-receptor agonists such as pergolide, and the non-ergotamine dopamine-receptor agonists pramipexole and ropinirole, are becoming more commonly used to treat RLS. The dopamine precursor levodopa, in combination with carbidopa, is another effective therapeutic agent. An advantage of levodopa is lower cost than non-ergotamine and ergotamine dopamine-receptor agonists. However, the adverse effect of symptom augmentation appears to develop more frequently with levodopa than dopamine-receptor agonists; therefore, levodopa may currently be used somewhat less often as first-line therapy. Patients with painful symptoms may respond favourably to the anticonvulsants gabapentin and carbamazepine. Opioids and hypnosedatives are helpful in selected patients; however, these agents may have troubling adverse effects in the elderly. Correction of iron deficiency improves symptoms in patients with low ferritin levels. Lifestyle modification may also be helpful. Therapy is directed at symptoms, and most symptomatic patients benefit from treatment. It is important to consider RLS in the differential diagnosis of any patient with paraesthesias of the limbs.

Inhibition of pleural mesothelial cell collagen synthesis by nitric oxide

The pleural mesothelial cell has a critical role in repairing the mesothelium after injury via its ability to produce connective tissue macromolecules. We have recently shown that proinflammatory cytokines and lipopolysaccharide induce pleural mesothelial cells to produce nitric oxide. The present study examined the effect of nitric oxide on pleural mesothelial cell protein synthesis. Rat pleural mesothelial cells were exposed to various combinations of tumor necrosis factor, interleukin-1, interferon-gamma, and lipopolysaccharide or to the nitric oxide donors: 6-morpholino-sydnonimine, S-nitroso-N-acetyl-D,L-penicillamine, sodium nitroprusside, and spermine-NO adduct for 24-48 h. Nitrate and nitrite (an index of nitric oxide production) and not collagen and noncollagen protein production (uptake of 3H-proline into collagenase-sensitive protein) were then determined. Net collagen production was significantly inhibited by the cytokine-lipopolysaccharide combinations tested. Collagen inhibition paralleled the time course of increased nitric oxide production. The inhibition of collagen production was also significantly reversed by the addition of NG-nitro-L-arginine methyl ester, and was reproduced by the addition of a 5:1 molar excess of L-arginine to NG-nitro-L-arginine methyl ester. Additionally, nitric oxide-generating compounds significantly inhibited collagen production in a dose-dependent manner compared to unexposed control cells. Net collagen production was inhibited to a greater degree than noncollagen protein synthesis. These results suggest that nitric oxide may be a significant mediator of PMC collagen production during conditions of significant pleural inflammation.

Growth factor modulation of rat pleural mesothelial cell mitogenesis and collagen synthesis

This study examined the effects of an epithelial and a mesenchymal growth factor on pleural mesothelial cell proliferation and collagen synthesis, functions that may be important in the response of the pleura to injury. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) added singly caused significant increases relative to control in both the uptake of [3H]thymidine into the cellular DNA of subconfluent monolayers and of [3H]proline into collagenase-sensitive protein. Combinations of EGF and PDGF resulted in more than additive increases in proliferation and additive increases in collagen production relative to each factor alone. Media from control and growth factor-stimulated PMC demonstrated no gelatinase or collagenase activity, suggesting that the increase in net collagen production was secondary to enhanced synthesis. These data demonstrate that both epithelial and mesenchymal growth factors can stimulate PMC proliferation and collagen synthesis and that these growth factors have even greater effects when combined, particularly in regard to cellular proliferation. Increases in PMC proliferation and collagen synthesis in response to these growth factors may be important in healing the pleura after injury by a variety of disease processes.

Nitric Oxide Synthesis by Rat Pleural Mesothelial Cells: Induction by Growth Factors and Lipopolysaccharide

The purpose of this study was to determine if certain growth factors and bacterial products induce pleural mesothelial cells (PMC) to produce nitric oxide (NO). Confluent monolayers of rat PMC were exposed to epidermal growth factor (EGF), platelet-derived growth factor (PDGF), or lipopolysaccharide (LPS) individually and in various combinations for 24-72 h. Concentrations of nitrite and nitrate were quantified and used as an indirect measure of NO production. LPS stimulation resulted in a significant increase in nitrite/nitrate concentration, but neither EGF nor PDGF alone or combined had any significant effect relative to control. However, LPS combined with either EGF or PDGF caused a significant increase in nitrite/nitrate concentration relative to LPS alone and growth factor alone. The highest level level of nitrite/nitrate concentration was observed with the triple combination of LPS, EGF, and PDGF. Nitrite/nitrate accumulation was significantly increased at 24 h by all combinations, and continued to increase, with the highest concentration observed after 72 h of exposure. Nitrite/nitrate production was significantly inhibited by NG-nitro-L-arginine methyl ester and this inhibition was reversed by the addition of L-arginine, suggesting that nitrite and nitrate were derived from the L-arginine-dependent formation of NO. These data indicate that PMC can be induced to produce relatively large amounts of NO in response to growth factors combined with LPS.

Inhibition of rat pleural mesothelial cell nitric oxide synthesis by transforming growth factor-beta 1.

Pleuritis is a common initial clinical manifestation of tuberculosis. It is associated with an accumulation of a variety of cytokines in the pleura and pleural fluid. We have recently shown that these proinflammatory cytokines induce the pleural mesothelial cell to produce large amounts of nitric oxide, a nitrogen intermediate that has been shown to have a tuberculocidal effect. TGF-β has also been found in situ in tuberculous effusions and pleural tissues and is thought to suppress the immune response and promote tissue repair. This study examined the effects of TGF-β on cytokine-induced NO synthesis by rat pleural mesothelial cells in vitro. Results demonstrated that TGF-β significantly inhibited NO synthesis and that this inhibition was associated with a proportionate decrease in iNOS mRNA and iNOS protein. Suppression of pleural mesothelial cell NO synthesis by TGF-β may be important in the pathogenesis of tuberculous pleuritis.

Nitric Oxide-Dependent N-Nitrosating Activity of Rat Pleural Mesothelial Cells

Recent studies have demonstrated that nitric oxide (NO)-derived N-nitrosating agents may promote mutagenesis and carcinogenesis from the nitrosative deamination of DNA bases via the formation of nitrosamine intermediates. The objective of this study was to determine if pleural mesothelial cells (PMC) stimulated with proinflammatory cytokines could promote the N-nitrosation of a primary aromatic amine via the L-arginine-dependent formation of NO-derived N-nitrosation agents. N-nitrosating activity was determined by measuring the N-nitrosation of a model amine, 2,3-diaminonapthalene, to yield its fluorescent triazole (1-naptho-2,3-triazole) derivative. Results show that specific combinations of TNF, IL-1, interferon gamma, and LPS significantly increased N-nitrosating activity. There was a significant positive correlation between nitrite plus nitrate and triazole production. Triazole formation was inhibited by NG-nitro-L-arginine methyl ester, suggesting that triazole was derived from L-arginine-dependent formation of NO. These data indicate that PMC have the capacity to promote the N-nitrosation of primary aromatic amines via the formation of NO.

Characterization of proteoglycans produced by rat pleural mesothelial cells in vitro

The mesothelial cell envelopes the surface of the parietal and visceral pleura. These cells are known to synthesize most of the protein constituents of the pleural basement membrane and interstitium. This study examined the ability of a rat pleural mesothelial cell line to synthesize proteoglycans in vitro. Cells were labeled with inorganic 35SO4 to label the glycosaminoglycan moiety of proteoglycans. The medium and combined cell membrane/extracellular matrix fractions contained 73 and 25% of the proteoglycan radioactivity, respectively. The medium contained a single chondroitin/dermatan sulfate proteoglycan of approximately 190 kDa, consistent with biglycan. As determined by Northern analysis of steady-state levels of messenger RNA, the cells contained message for biglycan. Stimulation of the cells with epidermal growth factor resulted in the appearance of a second chondroitin/dermatan sulfate proteoglycan of approximately 97 kDa, characteristic of decorin. The cell membrane/matrix contained a biglycan-like chondroitin/dermatan proteoglycan and several heparan sulfate proteoglycans. Pleural mesothelial cells in vitro are capable of synthesizing a variety of interstitial and basement membrane proteoglycans.