Michel Nasser

Apoptosis and the activity of ceramide, Bax and Bcl-2 in the lungs of neonatal rats exposed to limited and prolonged hyperoxia

BackgroundThe aim of the study is to examine the effect of limited and prolonged hyperoxia on neonatal rat lung. This is done by examining the morphologic changes of apoptosis, the expression of ceramide, an important mediator of apoptosis, the expression of inflammatory mediators represented by IL-1β and the expression of 2 proto-oncogenes that appear to modulate apoptosis (Bax and Bcl-2).MethodsNewborn rats were placed in chambers containing room air or oxygen above 90% for 7 days. The rats were sacrificed at 3, 7 or 14 days and their lungs removed. Sections were fixed, subjected to TUNEL, Hoechst, and E-Cadherin Staining. Sections were also incubated with anti-Bcl-2 and anti-Bax antisera. Bcl-2 and Bax were quantitated by immunohistochemistry. Lipids were extracted, and ceramide measured through a modified diacylglycerol kinase assay. RT-PCR was utilized to assess IL-1β expression.ResultsTUNEL staining showed significant apoptosis in the hyperoxia-exposed lungs at 3 days only. Co-staining of the apoptotic cells with Hoechst, and E-Cadherin indicated that apoptotic cells were mainly epithelial cells. The expression of Bax and ceramide was significantly higher in the hyperoxia-exposed lungs at 3 and 14 days of age, but not at 7 days. Bcl-2 was significantly elevated in the hyperoxia-exposed lungs at 3 and 14 days. IL-1β expression was significantly increased at 14 days.ConclusionExposure of neonatal rat lung to hyperoxia results in early apoptosis documented by TUNEL assay. The early rise in Bax and ceramide appears to overcome the anti-apoptotic activity of Bcl-2. Further exposure did not result in late apoptotic changes. This suggests that apoptotic response to hyperoxia is time sensitive. Prolonged hyperoxia results in acute lung injury and the shifting balance of ceramide, Bax and Bcl-2 may be related to the evolution of the inflammatory process.

Modulation of total ceramide and constituent ceramide species in the acutely and chronically hypoxic mouse heart at different ages

Ceramide has been implicated in regulatory processes vital for cell survival under different stressors, most notably hypoxia. Little has been done to investigate the contributions of the different ceramide species to the regulation of cell survival. This study aims to highlight the patterns of variation in total ceramide and its species in the growing and hypoxic mouse heart. Mus musculus mice were placed in a hypoxic environment at birth. Control animals remained in room air. The hearts were extracted at different time points: 1 day, 1 week, 4 weeks, and 8 weeks. The total ceramide content and the amounts of component species were assayed by a modified diacylglycerol kinase assay and high-performance liquid chromatography-tandem mass spectroscopy, respectively. Data was collected from both ventricles in hypoxic and control conditions. There was significant polycythemia in the hypoxic versus control animals with a nearly twofold increase in hematocrit levels. Hypoxic right ventricle (RV) mass significantly increased over that of controls at different age groups. When ceramide content was compared in the hypoxic versus control animals, there was a significant increase at day 1 and a significant decrease at week 4 in the left ventricle, whereas a significant decrease was found in the RV at 1 week, 4 weeks, and 8 weeks. There was also a differential involvement of the RV with regard to levels of N-palmitoyl-D-erythro-sphingosine (C16-Cer) and its synthetic precursor dihydro-N-palmitoyl-D-erythro-sphinganine (DHC-16-Cer). The decrease in C16-Cer observed in both hypoxic and control RVs over time was paralleled by a significant increase in DHC-16-Cer in hypoxic (142.1+/-15.0 pmol; p<0.05) but not control (52.8+/-4.0 pmol) RVs suggesting a role for DHC-16-Cer in the RV adaptive response to hypoxia. Another species, N-arachidoyl-D-erythro-sphingosine (C20-Cer), was specifically and significantly decreased in the hypoxic RV. These studies support the presence of distinct roles for different ceramide species and their precursors. A better assessment of cyanotic congenital heart disease in light of the mechanism and timing of cardiomyocyte death, will lead to punctual interventions and even novel cardioprotective strategies.

Endocrine Changes in a Rat Model of Chronic Hypoxia Mimicking Cyanotic Heart Disease

Objective. The endocrine system plays an important role in the adaptation to hypoxia. The aim of this study is to assess the effect of chronic hypoxia on endocrine changes in a neonatal animal model mimicking cyanotic heart disease. Methods. Sprague–Dawley rats were placed in a normobaric hypoxic environment at birth and oxygen levels were maintained at 10% in an airtight Plexiglas chamber. Controls remained in room air. Animals were sacrificed at 4 and 8 weeks of life. Hematocrit, Free T4 (FT4), Thyrotropin (TSH), corticosterone, and Growth hormone (GH) were measured. Results. Significant polycythemia developed in the hypoxic rats. Free T4 levels were significantly lower in the hypoxic (H) group compared to the control (C) group at 4 and 8 weeks with FT4 of 2.44 ± 1.11 ng/dL (H) and 4.35 ± 1.62 (C) at 4 weeks with a p value <0.005 and FT4 of 2.01 ± 0.36 (H) and 3.25 ± 0.54 (C) ng/dL at 8 weeks with p < 0.01. At 8 weeks TSH levels were significantly lower in the hypoxic group (1.84 ± 0.9 ng/mL (H) vs. 3.11 ± 1.1 (C)) with p < 0.05. Corticosterone levels were higher in the hypoxic group with values of 126 ± 14.8 ng/mL (H) and 114.1 ± 12.6 (H) at 4 and 8 weeks respectively, when compared to the control group with values of 82.9 ± 18.1 (C) and 92.7 ± 10.3 (C) and 4 and 8 weeks with p < 0.0005 and <0.05 respectively. Growth hormone levels were significantly lower in the hypoxic group at 4 and 8 weeks with p < 0.05 and p < 0.001, respectively. Conclusion. Chronic hypoxia in our neonatal rat model was associated with a decrease in growth hormone levels and an increase in corticosterone levels. Furthermore, hypoxia resulted in thyroid hormone axis suppression. This effect seems to be centrally mediated.

Lack of apoptosis in the hypoxic brain of a rat model mimicking cyanotic heart disease

Objective : To assess the effect of chronic hypoxia on brain neuronal apoptosis, an animal model mimicking cyanotic heart disease was utilized. Methods : Rats were placed in an hypoxic environment at birth and oxygen levels were maintained at 10% in an air-tight Plexiglass chamber. Controls remained in room air. Animals were sacrificed and the brains were harvested at 1 and 4 weeks, respectively. Results : Significant polycythemia developed in the hypoxic rats at 1 and 4 weeks. Indexed brain mass to body weight was significantly increased in the hypoxic groups by 18% ( p < 0.01) and 38% ( p < 0.01) as compared to controls at 1 and 4 weeks, respectively. There was no difference in the number of apoptotic neurons between the chronically hypoxic rats and controls, as assayed by TUNEL labelling and Hoechst staining. The role of the sphingolipid ceramide was then examined because of its reported role in stress response, growth suppression and apoptosis. It was found that the brain ceramide accumulation was not significantly different in the hypoxic and control groups at 1 and 4 weeks. Conclusion : A protective adaptive response to chronic hypoxia in the neonatal brain may exist.

Activated protein C attenuates acute lung injury and apoptosis in a hyperoxic animal model.

Evidence suggests that activated protein C (APC) attenuates acute lung injury (ALI) through antithrombotic and anti-inflammatory mechanisms. The aim of this study was to determine the effects of APC on ALI in adult rats exposed to hyperoxic environment. Rats were divided into control, hyperoxia, hyperoxia + APC, and APC. Hyperoxia and hyperoxia + APC were exposed to 1, 3, and 5 days of hyperoxia. Hyperoxia + APC and APC were injected with APC (5 mg/kg, i.p.) every 12 h. Control and hyperoxia received isotonic sodium chloride solution injection. Measurement of wet to dry ratio and albumin leak demonstrated significant improvement in hyperoxia + APC when compared with hyperoxia. Apoptosis, as measured by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, was significantly reduced in hyperoxia + APC when compared with hyperoxia. Histological evaluation of lung sections showed significant reduction in inflammation, edema, and in the number of marginating neutrophils in hyperoxia + APC as compared with hyperoxia. Transcriptional expression of lung inflammatory mediators demonstrated a time-dependent surge in the levels TNF-&agr;, IL-1&bgr;, and IL-6 in response to hyperoxia that was attenuated with APC administration in the presence of hyperoxia. In this rat model, APC attenuates lung injury and the expression of inflammatory mediators in ALI secondary to hyperoxia.

Dopamine receptors in normal and diabetic liver plasma membrane

Dopamine binding to liver plasma membrane isolated from diabetic livers was significantly reduced (P less than 0.01) as compared to dopamine binding to the normal membrane. Diabetic membranes exhibited a significant decrease (P less than 0.01) in specific dopamine binding as compared to the normal membranes; whereas no significant change (P less than 0.5) was noticed in the nonspecific binding patterns. The dopamine binding capacity of both membranes is temperature dependent. Procaine at different concentrations inhibited significantly (P less than 0.01) dopamine binding to normal and diabetic membranes. Treatment of the normal and diabetic membranes with insulin showed a 33% decrease in the binding capacity of the normal and 42% decrease in the binding capacity of the diabetic membrane.

Receptors of prostaglandin E1 in the plasma membrane of normal liver. Effect of membrane fluidity on PGE1 binding

Prostaglandin E1 binding to isolated liver plasma membrane as a function of PGE1 concentration showed saturability of the binding sites at PGE1 concentration of 2.5 X 10(-4) M. Scatchard analysis revealed heterogeneous population of binding sites with a binding capacity of 470 and 990 nmol/mg protein for the higher and lower affinity binding sites respectively. PGE1 binding to liver plasma membrane was progressively and significantly decreased (P less than 0.01) as the incubation temperature was reduced to 22 degrees and 4 degrees C. Procaine at 1 X 10(-3) M concentration showed a significant decrease (P less than 0.01) in the binding capacity of the liver plasma membrane. Colchicine plus cytochalasin-B inhibited PGE1 binding significantly (P less than 0.01) but their inhibition is not equivalent to that of procaine.

Cytoskeletal control of calcium absorption across the rat small intestine

1. The effect of colchicine, cytochalasin-B and procaine on calcium transport across the rat small intestine was investigated. The results obtained show the following: 2. Colchicine and cytochalasin-B at different concentrations inhibited significantly (P less than 0.001) calcium accumulation in rat intestinal cells, whereas procaine at different concentrations increased significantly (P less than 0.001) calcium accumulation in the rat small intestine. 3. Unidirectional influx of calcium across the rat small intestine was significantly inhibited (P less than 0.01) in the presence of colchicine and cytochalasin-B in the preincubation medium. Procaine, on the other hand, caused a significant increase (P less than 0.01) in the unidirectional influx of calcium across the rat intestinal cells. 4. The cell water content was not altered in the presence of the different drugs indicating that the changes in calcium transport across the rat intestinal cells are not due to alterations in the structure of the cell membrane.

Effect of enzymatic digestion on phenylalanine transport across the small intestine

Phenylalanine accumulation in mucosal strips isolated from rat small intestine was significantly inhibited (P less than 0.01) after preincubation with trypsin, chymotrypsin, phospholipase D and neuraminidase. Unidirectional phenylalanine influx across the small intestine was significantly reduced (P less than 0.01) when the mucosal strips were preincubated with the above mentioned enzymes. Intestinal cell water and volume were not significantly changed (P greater than 0.6) when the intestinal tissues were preincubated with these enzymes.

Prostacyclin-enhanced calcium sequestration by microsomes isolated from rat left ventricle

The objective of these experiments was to investigate the direct effect of prostacyclin on calcium binding and uptake by microsomal vesicles isolated from rat left ventricle. The protein content of the preparation was found to be 2.73 +/- 0.18 mg microsomal protein/g of left ventricular wet weight. Steady-state calcium binding and uptake by microsomal vesicles was reached at 6 minutes in control animals and 20 sec in prostacyclin-treated experiments. Prostacyclin increased steady-state calcium binding and uptake from a control of 52.48 +/- 4.16 up to 109.31 +/- 3.06 nmol/mg protein (p less than 0.05) and from 238.07 +/- 12.37 up to 314.85 +/- 1.23 nmol/mg protein (p less than 0.05) respectively. Doubling the concentration of prostacyclin from initial dose of 1.2 x 10(-8) M did not alter calcium binding and uptake further.