A novel assay to study autophagy: regulation of autophagosome vacuole size by amino acid deprivation.

Autophagy is a normal degradative pathway that involves the sequestration of cytoplasmic portions and intracellular organelles in a membrane vacuole called the autophagosome. These vesicles fuse with lysosomes and the sequestered material is degraded. Owing to the complexity of the autophagic pathway and to its inaccessibility to external probes, little is known about the molecular mechanisms that regulate autophagy in higher eukaryotic cells. We used the autofluorescent drug monodansylcadaverine (MDC), a specific autophagolysosome marker to analyze at the molecular level the machinery involved in the autophagic process. We have developed a morphological and biochemical assay to study authophagy in living cells based on the incorporation of MDC. With this assay we observed that the accumulation of MDC was specifically induced by amino acid deprivation and was inhibited by 3-methlyadenine, a classical inhibitor of the autophagic pathway. Additionally, wortmannin, an inhibitor of PI3-kinases that blocks autophagy at an early stage, inhibited the accumulation of MDC in autophagic vacuoles. We also found that treatment of the cells with N-ethylmaleimide (NEM), an agent known to inhibit several vesicular transport events, completely blocked the incorporation of MDC, suggesting that an NEM-sensitive protein is required for the formation of autophagic vacuoles. Conversely, vinblastine, a microtubule depolymerizing agent that induces the accumulation of autophagic vacuoles by preventing their degradation, increased the accumulation of MDC and altered the distribution and size of the autophagic vacuoles. Our results indicate that in the presence of vinblastine very large MDC-vacuoles accumulated mainly under starvation conditions, indicating that the expansion of autophagosomes is upregulated by amino acid deprivation. Furthermore, these MDC-vacuoles were labeled with LC3, one of the mammalian homologues of the yeast protein Apg8/Aut7 that plays an important role in autophagosome formation.

Human monocytes lose 5-lipoxygenase and FLAP as they mature into monocyte-derived macrophages in vitro.

Previous studies in mononuclear phagocytes have shown that macrophages have substantially greater 5-lipoxygenase activity than monocytes and that this is associated with greater amounts of 5-lipoxygenase and its activating protein (FLAP). The aim of this study was to examine the effect of mononuclear phagocyte maturation in vitro on 5-lipoxygenase expression. At baseline, monocytes had significant 5-lipoxygenase activity, but then lost all detectable 5-lipoxygenase activity over 7 days. Immunoblot and Northern blot analysis revealed that immunoreactive protein and mRNA for both 5-lipoxygenase and FLAP were significantly decreased over time. These studies demonstrate that in vitro differentiation of monocytes into a macrophage phenotype is not accompanied by the enhanced expression of 5-lipoxygenase and FLAP seen in macrophages derived from in vivo sources. In fact, baseline expression of 5-lipoxygenase and FLAP by monocytes is lost in vitro. These studies have clear implications for the use of cultured monocytes as a model of macrophages, and they also further our understanding of the regulation of the 5-lipoxygenase pathway.

Lymphocytes stimulate expression of 5-lipoxygenase and its activating protein in monocytes in vitro via granulocyte macrophage colony-stimulating factor and interleukin 3.

The aim of this study was to examine the role of lymphocytes in regulating expression of the 5-lipoxygenase pathway in monocytes. When monocytes were cultured over a period of days with lymphocytes, calcium ionophore-stimulated 5-lipoxygenase activity was enhanced. If lymphocytes alone were activated with lectins and their supernatants added to monocytes, stimulated 5-lipoxygenase activity was increased, whereas supernatants from lymphocytes cultured without lectins had no effect. Increased immunoreactive protein and mRNA for 5-lipoxygenase and 5-lipoxygenase activating protein were present in cells conditioned with lectin-activated lymphocyte supernatants. The effect of activated-lymphocyte supernatants could be mimicked by either GM-CSF or IL-3, but there was no additive effect with both cytokines. Both GM-CSF and IL-3 were present in the supernatant from lectin-activated lymphocytes at concentrations above their ED50, but were undetectable in the supernatant from nonactivated lymphocytes. The effect of lectin-activated lymphocyte supernatant could be inhibited by neutralizing antibodies to both cytokines, but not to either cytokine alone. We conclude that lymphocytes can regulate the expression of 5-lipoxygenase in monocytes, over a period of days, via the release of soluble factors, primarily GM-CSF and IL-3.

Captopril inhibits neutrophil synthesis of leukotriene B4 in vitro and in vivo.

The aim of this investigation was to determine the effects of metalloproteinase inhibitors on leukotriene (LT) A4 hydrolase in human neutrophil cytosol and to examine the effects of captopril on intact neutrophils in vitro and in vivo. Cytosolic fractions were assayed for LTA4 hydrolase and 5-lipoxygenase activity in the presence or absence of inhibitors. Only bestatin, 1,10-O-phenanthroline, captopril and fosinoprilat demonstrated significant effects. The IC50 of captopril and fosinoprilat for LTA4 hydrolase activity were 500 microM and 1 mM, respectively. No inhibition of 5-lipoxygenase activity in cytosolic fractions was detected. The effect of captopril was only minimally reversed by ZnSO4. The IC50 of captopril for inhibition of LTB4 synthesis in intact neutrophils was 63 microM. Furthermore, 5-HETE production in intact cells was diminished 25.3 +/- 8.5% in the presence of 1 mM captopril. Oral captopril inhibited stimulated LTB4 release by subsequently isolated neutrophils by 48.1 +/- 5.6% and 5-HETE release by 43.2 +/- 5.5%. Thus, captopril is an inhibitor of LTB4 synthesis in neutrophils in vitro and in vivo. However, there are differences between the potency of this drug as assessed in cytosol and intact cell studies. This study significantly extends previous reports in that it demonstrates that captopril is a more potent inhibitor of LTB4 synthesis in intact neutrophils than in cytosol and in that it demonstrates an inhibitory effect of captopril on synthesis of LTB4 by neutrophils exposed to captopril in vivo.

Leukotriene A4 hydrolase in human bronchoalveolar lavage fluid.

We examined cell-free human bronchoalveolar lavage fluid (BALF) for enzymes of the 5-lipoxygenase pathway. BALF was obtained from six patients who were active smokers and six nonsmokers. Enzymatic activity in cell-free BALF was assessed by specific assays for leukotriene (LT) A4 hydrolase, 5-lipoxygenase, and LTC4 synthase using HPLC. Only LTA4 hydrolase enzymatic activity was found. This activity ranged from 101 to 667 when expressed as picomoles of LTB4 produced per milliliter BALF. Enzymatic activity in smokers vs nonsmokers was 484 +/- 120 vs 129 +/- 32 pmol LTB4/ml BALF (mean +/- SD, P < 0.0001). There were no leukotrienes found in BALF before assay. Immunoblot analysis revealed an immunoreactive band at a relative molecular mass of 69,000 D in all samples, consistent with LTA4 hydrolase, but no evidence of 5-lipoxygenase. BALF had greater LTA4 hydrolase activity per milligram of protein than neutrophil cytosol, epithelial cell cytosol, plasma, or serum. The synthesis of LTB4 was significantly increased when neutrophils were stimulated in BALF. These data indicate the selective presence of LTA4 hydrolase in BALF which is significantly increased in smokers. This enzyme in BALF may contribute to the inflammatory response in tobacco-related lung disease.

Rate of oxyhemoglobin desaturation in obstructive versus nonobstructive apnea.

Preapneic thoracic gas volume (Vtg), arterial saturation (SaO2), and mixed venous oxygen saturation (SvO2), have been shown to influence the rate of SaO2 fall (dSaO2/dt) during apnea. We asked the following question: does tissue oxygen consumption (tVO2) affect the dSaO2/dt during apnea? We attempted to answer this question by comparing dSaO2/dt during obstructive apneas (high tVO2) with dSaO2/dt during nonobstructive apneas (low tVO2) in six adult baboons. Fiberoptic central venous and arterial catheters were used for continuous monitoring of SvO2, SaO2, and cardiac output. A sapphire-bearing turbine monitored minute ventilation and airflow cessation. A Respitrace and esophageal pressures were used to assess relative differences in Vtg. Obstructive apneas (30, 45, and 60-s) were created by clamping an indwelling cuffed endotracheal tube at end-expiration. Nonobstructive apneas were created by paralyzing the animals with atracurium and interrupting ventilation for periods equivalent to those of the obstructed apneas. The ventilator was adjusted to duplicate the respiratory rate, tidal volume, and relative Vtg of the spontaneously breathing animal. Mean tVO2 during spontaneous breathing was 110 ml/min (Fick method) and decreased to 90 ml/min during paralysis (p less than 0.05). The dSaO2/dt for the three apnea durations (mean, all animals), obstructive versus nonobstructed were: 0.85 and 0.74%/s (n = 6), 0.87 and 0.75%/s (n = 6), and 0.60 and 0.48%/s (n = 4), respectively. The dSaO2/dt was significantly lower during the nonobstructive apneas. We conclude that differences in VO2 during apnea may affect the dSaO2/dt and that for the same duration apnea, central apneas may show less desaturation than obstructive apneas where vigorous muscular efforts at overcoming obstruction are common.

Effect of cardiac output reduction on rate of desaturation in obstructive apnea.

The nadir of SaO2 during an obstructive apnea is dependent upon the apnea's duration and the rate of fall of saturation (dSaO2/dt). We postulated that a low Q, such as in patients with congestive heart failure with sleep apnea, or a reduction in Q, as seen in some humans during obstructive sleep apnea, might steepen dSaO2/dt. The mechanism postulated was lowering of SvO2 with increased pulmonary capillary blood oxygen uptake and faster depletion of alveolar oxygen. This study examines dSaO2/dt following the onset of apnea in eight spontaneously breathing adult baboons. Nonrepetitive obstructive apneas (30, 45, and 60 seconds) were created by clamping an indwelling cuffed endotracheal tube at the end of expiration. Following baseline measurements, the animals were given a bolus of a rapid-acting beta-adrenergic blocker followed by continuous infusion to reduce cardiac output and to limit the cardiovascular response to obstructive asphyxia. Fiberoptic catheters were used for continuous monitoring of SaO2, SvO2, and cardiac output. Esophageal pressure and relative thoracic gas volume (Respitrace) were monitored to insure equivalence of lung volume at the onset of apnea. Beta-adrenergic blockade reduced resting Q by a mean of 25 percent. The blocked vs unblocked dSaO2/dt was 0.73 vs 0.72 percent/s, 0.76 vs 0.73 percent/s, and 0.70 vs 0.71 percent/s for 30-second, 45-second, and 60-second apneas, respectively. Thus, mean dSaO2/dt for all durations of apneas was unaffected by beta-adrenergic blockade. We concluded that dSaO2/dt is not influenced by limited Q preceding or induced by obstructive asphyxia.

Atelectasis affects the rate of arterial desaturation during obstructive apnea.

Chronic hemodynamic disturbances are more profound in patients with obstructive sleep apnea when underlying lung disease with abnormal gas exchange (low arterial PO2) is present. Previous studies suggest that pulmonary gas exchange could influence the rate of fall of arterial oxygen saturation (dSaO2/dt) in obstructive sleep apnea. We postulated that abnormal gas exchange in the form of atelectasis would steepen dSaO2/dt and thereby lower nadir arterial oxyhemoglobin saturation (SaO2) for the same duration of apnea. Apneas were created by clamping an indwelling cuffed endotracheal tube at end expiration in eight spontaneously breathing adult baboons. Apneas of the same duration were then repeated during temporary endobronchial occlusion of one lobe of the lung. SaO2 and mixed venous O2 saturation were continuously monitored, and cardiac output was calculated. Worsening of pulmonary gas exchange during atelectasis was documented by an increase in calculated venous admixture from 10.5 +/- 0.8 to 25.0 +/- 0.7% (P less than 0.001). The dSaO2/dt was independent of apnea duration at 30, 45, and 60 s. During endobronchial occlusion, apnea dSaO2/dt increased 20%, and nadir SaO2 was significantly lower. Possible mechanisms for steepening of dSaO2/dt during atelectasis are discussed.