Phagocytosis is regulated by nitric oxide in murine microglia.

Nitric oxide (NO) is produced by inducible nitric oxide synthase (iNOS) in activated microglia and has been shown to participate in host defense mechanisms. However, the role of NO produced by constitutive nitric oxide synthase (cNOS) in microglia is poorly understood. In this report, NO was found to regulate phagocytosis in murine BV-2 microglial cells as quantified by flow cytometry. Addition of NO-generating compounds caused impaired phagocytosis as compared to untreated microglia. The addition of nitric oxide synthase (NOS) inhibitors to microglial cells resulted in potentiation of phagocytosis, suggesting that constitutive NO was participating in the regulation of phagocytosis. The inverse correlation between NO production and phagocytosis was also observed when Alzheimer's beta-amyloid peptide was added. With beta-amyloid treatment, constitutive NO production decreased while phagocytosis increased. Cell extracts prepared from untreated microglia were found to contain both neuronal and endothelial NOS isoforms, but not the inducible form. The correlation of spontaneous NO production with attenuated phagocytosis suggests that constitutive NOS enzymes participate in microglial regulation.

4-Hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol) inhibits peroxynitrite-mediated phenol nitration.

Peroxynitrite (PN), a very reactive oxidant formed by the combination of superoxide and nitric oxide, appears to play a role in producing tissue damage in a number of inflammatory conditions. Pharmacological scavenging and decomposition of PN within these areas has therapeutic value in several tissue injury models. Recently, we have been interested in nitroxide free radical-containing compounds as possible scavengers of PN decomposition products. Nitroxides can undergo redox reactions to the corresponding hydroxylamine anion or oxoammonium cation in biological systems as shown by its ability to react with superoxide, leading to the formation of hydrogen peroxide and molecular oxygen. We found that 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol) inhibits PN-mediated nitration of phenolic compounds in the presence of a large molar excess of PN, suggesting a catalytic-like mechanism. In these experiments, Tempol inhibited PN-mediated nitration over the pH range of 6.5-8.5. This inhibition was specific for nitration and had no effect on hydroxylation. After the inhibition of PN-mediated nitration, Tempol was recovered from the reaction mixtures unmodified. In addition, Tempol was effective in protecting PC-12 cells from death induced by SIN-1, a PN-generating compound. The exact mechanism of Tempol's interaction with PN is not clear; however, we propose that an intermediate in this reaction may be a nitrogen dioxide radical-Tempol complex. This complex could react with water to form either nitrite or nitrate, or with a phenol radical to produce nitrophenol or nitrosophenol products and regenerate the nitroxide.

Alzheimer's beta-amyloid peptide 1-42 induces a phagocytic response in murine microglia.

Beta-amyloid (A beta) peptides are a key component of the senile plaques that characterize Alzheimer's disease. Cytokine-producing microglia have been shown to be intimately associated with amyloid deposits and have also been implicated as scavengers responsible for clearing A beta deposits. However, little is known about the initial activation of these microglia or the effect of A beta on phagocytosis. Murine BV-2 microglia were used to assess the effect of synthetic A beta 1-42 on phagocytosis by quantifying uptake of fluorescent microspheres, acetylated low-density lipoproteins, and zymosan particles by flow cytometry. A beta 1-42 stimulated microglial phagocytosis in a time- and dose-dependent manner. A beta fibrils produced the greatest potentiation, and once activated, phagocytosis remained elevated after removal of A beta from the cultures. A beta-stimulated phagocytosis could be blocked if proteoglycans were first complexed to A beta fibrils. These data suggest that A beta fibrils act as an immune signal to stimulate microglial phagocytosis and that extracellular matrix molecules may modify A beta function.

Effect of Alzheimer's brain extracts on dynein immunoreactivity in PC12 cells.

The neurodegenerative process in Alzheimer's disease (AD) has been suggested to occur as a consequence of microtubule disruption and subsequent loss of intracellular transport. Structural microtubule-associated proteins (MAPs) have been investigated for their role in the etiology of AD, but dynein, a force-producing MAP which mediates intracellular transport, has not been examined. In this report, dynein (MAP1C) immunoreactivity in AD brain tissue homogenates was observed increased 3.7-fold compared with control brain homogenate preparations. Similarly, NGF-differentiated PC12 cells cultured in the presence of soluble extracts prepared from AD brain tissue homogenates, exhibited an approximate 15-fold increase in dynein immunoreactivity compared to that of control brain tissue extracts. In contrast, AD clarified extracts had little effect upon "kinesin-like" protein immunoreactivity increased (approximately 2-fold); whereas, tau immunoreactivity was observed to be moderately increased (5-fold) over that of control brain extract treated PC12 cells. Chemical dephosphorylation and alkaline phosphatase treatment of AD extract-treated PC12 cell lysate prior to Western blotting resulted in complete loss of immunoreactivity, suggesting the dynein being monitored is a phosphorylated isoform. Furthermore, treatment of clarified brain tissue extracts with trypsin and (NH4)2SO4 suggests the endogenous elements giving rise to increased PC12 cell dynein intermediate chain immunoreactivity to be proteinaceous in nature. The observed increase in dynein intermediate-chain dynein immunoreactivity following exposure of neuronal cells to endogenous elements of AD brain may be reflective of dynein-microtubular array differences. Such an approach may be useful in assessing the effect of endogenous biomolecules on retrograde axonal transport in neuronal culture models.

Usefulness of dobutamine echocardiography in the hemodynamic assessment of mechanical prostheses in the aortic valve position.

Dobutamine stress echocardiography is a simple, safe, and readily available method to evaluate prosthetic valve hemodynamics under various flow conditions. A normal response of aortic valve prostheses to dobutamine infusion is a 100% increment in transprosthetic pressure gradients with an unchanged valve area.

Use of dobutamine stress echocardiography in assessing mechanical aortic prostheses: comparison with exercise echocardiography.

BACKGROUND AND AIMS OF THE STUDY: Relatively high resting flow velocities are occasionally seen in asymptomatic patients with apparently normal mechanical aortic prostheses. Doppler echocardiography in conjunction with bicycle exercise or dobutamine have been used to 'unmask' symptoms or abnormal hemodynamics in these patients. The aim of this study was to compare the Doppler-derived hemodynamic response to exercise and to dobutamine in patients with normally functioning mechanical aortic prostheses. METHODS: Bicycle ergometry (from 200-400 kg x m/min) and dobutamine (from 5-40 micrograms/kg/min) in conjunction with Doppler echocardiography were performed in 25 asymptomatic patients (21 men, four women; mean age 61 years) with mechanical aortic prostheses (range: 19 mm to 27 mm) who had normal resting hemodynamics and normal left ventricular function. Aortic valve area (continuity equation) and maximal instantaneous and mean gradients were estimated at rest and at peak stress. RESULTS: Target heart rate was achieved in 10/25 (40%) patients with exercise, and in 21/25 (84%) with dobutamine (p < 0.005). At peak stress, exercise induced a 25% increase in peak flow velocity, a 55% increase in valve gradients, and no significant change in valve area. In comparison, dobutamine induced a 48% increase in peak flow velocity, a 105% increase in valve gradients, and also no significant change in valve area. The flow velocity and pressure gradients at peak stress were significantly higher with dobutamine than with exercise. CONCLUSIONS: In normally functioning aortic valve prostheses, the target heart rate can be reached more often with dobutamine than with supine bicycle exercise. Despite significant increase in the transvalvular gradients, the valve area remained unchanged. The clinical significance of exercise or dobutamine in symptomatic patients with mechanical prostheses is yet to be proven.