Mechanisms and regulation of transferrin and iron transport in a model blood-brain barrier system.

For peripheral iron to reach the brain, it must transverse the blood-brain barrier. In order for the brain to obtain iron, transferrin receptors are present in the vascular endothelial cell to facilitate movement of transferrin bound iron into the brain parenchyma. However, a number of significant voids exist in our knowledge about transport of iron into the brain. These gaps in our knowledge are significant not only because iron is an essential neurotrophic factor but also because the system for delivery of iron into the brain is being viewed as an opportunity to circumvent the blood-brain barrier for delivery of neurotoxins to tumors or trophic factors in neurodegenerative diseases. In this study, we have used fluorescein-transferrin-59Fe in a bovine retinal endothelial cell culture system to determine the mechanism of transferrin-iron transport and to test the hypothesis that the iron status of the endothelial cells would influence iron transport. Our results indicated that iron is transported across endothelial cells both bound to and not bound to transferrin. The ratio of non-transferrin-bound iron to transferrin-bound iron transported is dependent upon the iron status of the cells. Blocking acidification of endosomes led to a significant decrease in transport of non-transferrin-bound iron but not transferrin-bound iron. Blocking pinocytosis had no effect on either transferrin or iron transcytosis. These results indicate that there is both transferrin-mediated and non-transferrin-mediated transcytosis of iron and that the process is influenced by the iron status of the cells. These data have considerable implications for common neurodegenerative diseases that are associated with excess brain iron accumulation and the numerous neurological complications associated with brain iron deficiency.

Insulin rescues retinal neurons from apoptosis by a phosphatidylinositol 3-kinase/Akt-mediated mechanism that reduces the activation of caspase-3.

The ability of insulin to protect neurons from apoptosis was examined in differentiated R28 cells, a neural cell line derived from the neonatal rat retina. Apoptosis was induced by serum deprivation, and the number of pyknotic cells was counted. p53 and Akt were examined by immunoblotting after serum deprivation and insulin treatment, and caspase-3 activation was examined by immunocytochemistry. Serum deprivation for 24 h caused approximately 20% of R28 cells to undergo apoptosis, detected by both pyknosis and activation of caspase-3. 10 nm insulin maximally reduced the amount of apoptosis with a similar potency as 1.3 nm (10 ng/ml) insulin-like growth factor 1, which acted as a positive control. Insulin induced serine phosphorylation of Akt, through the phosphatidylinositol (PI) 3-kinase pathway. Inhibition of PI 3-kinase with wortmannin or LY294002 blocked the ability of insulin to rescue the cells from apoptosis. SN50, a peptide inhibitor of NF-kappaB nuclear translocation, blocked the rescue effect of insulin, but neither insulin or serum deprivation induced phosphorylation of IkappaB. These results suggest that insulin is a survival factor for retinal neurons by activating the PI 3-kinase/Akt pathway and by reducing caspase-3 activation. The rescue effect of insulin does not appear to be mediated by NF-kappaB or p53. These data suggest that insulin provides trophic support for retinal neurons through a PI 3-kinase/Akt-dependent pathway.

Vascular endothelial growth factor induces rapid phosphorylation of tight junction proteins occludin and zonula occluden 1. A potential mechanism for vascular permeability in diabetic retinopathy and tumors.

Vascular endothelial growth factor (VEGF) may have a physiologic role in regulating vessel permeability and contributes to the pathophysiology of diabetic retinopathy as well as tumor development. We set out to ascertain the mechanism by which VEGF regulates paracellular permeability in rats. Intra-ocular injection of VEGF caused a post-translational modification of occludin as determined by a gel shift from 60 to 62 kDa. This event began by 15 min post-injection and was maximal by 45 min. Alkaline phosphatase treatment revealed this modification was caused by a change in occludin phosphorylation. In addition, the quantity of extracted occludin increased 2-fold in the same time frame. The phosphorylation and increased extraction of occludin was recapitulated in retinal endothelial cells in culture after VEGF stimulation. The data presented herein are the first demonstration of a change in the phosphorylation of this transmembrane protein under conditions of increased endothelial permeability. In addition, intra-ocular injection of VEGF also caused tyrosine phosphorylation of ZO-1 as early as 15 min and increased phosphorylation 4-fold after 90 min. In conclusion, VEGF rapidly increases occludin phosphorylation as well as the tyrosine phosphorylation of ZO-1. Phosphorylation of occludin and ZO-1 likely contribute to regulated endothelial paracellular permeability.

The testis isoform of the phosphorylase kinase catalytic subunit (PhK-gammaT) plays a critical role in regulation of glycogen mobilization in developing lung.

In order to identify the form of phosphorylase kinase catalytic subunit expressed in developing lung, degenerate polymerase chain reaction primers were designed based on conserved domains of the two known catalytic subunits, expressed primarily in muscle and testis. Amplification of cDNA from day 19 fetal rat lung followed by cloning and sequence analyses indicated that only the testis isoform of phosphorylase kinase (PhK-gammaT) was detectable in fetal lung. In situ hybridization analyses indicated that expression of PhK-gammaT RNA in developing lung tissue was widespread and not restricted to Type II epithelial cells; PhK-gammaT protein expression was temporally and spatially correlated with expression of PhK-gammaT RNA. PhK-gammaT RNA and protein expression was also characterized in the PhK-deficient glycogen storage disease (gsd) rat. PhK-gammaT RNA levels were similar in Type II cells isolated from wild type and gsd/gsd fetuses; in contrast, PhK-gammaT protein was virtually undetectable in gsd/ gsd Type II cells and enzyme activity was very low. These results suggest that PhK-gammaT plays a critical role in mobilization of glycogen during fetal lung development and that failure to catabolize glycogen in the gsd/gsd rat is related to an untranslatable PhK-gammaT RNA or unstable protein.

Matrix Gla protein mRNA expression in cultured type II pneumocytes.

Matrix Gla protein (MGP) was first isolated from the matrix fraction of bone. This highly conserved vitamin K-dependent protein of 14 kDa has been identified in numerous tissues and cells, and its mRNA was recently found to be abundant in rat lung. Relatively low MGP protein levels in many soft tissues where its mRNA is high suggests an important secretory function for this protein. We have found a high specific activity of vitamin K-dependent carboxylase in microsomes of rat pulmonary type II cells and the presence of numerous endogenous substrates, including one of 13-15 kDa. To investigate the possibility that MGP and its mRNA could be localized in type II cells, rat MGP and actin cDNA probes were hybridized to total RNA obtained from freshly isolated type II cells and from cells cultured for up to 6 days. MGP mRNA increased 5- to 6-fold relative to beta-actin mRNA from days 3 to 6 in primary culture and MGP secretion increased nearly 60-fold during that interval. MGP mRNA and MGP secretion decreased 25-75% if cultures were supplemented with vitamin K quinone. Vitamin K deficiency, caused by carbon stripping the serum or treatment of cell cultures with warfarin, resulted in an induction of carboxylase activity and elevated MGP mRNA. In parallel experiments, carboxylase specific activity also increased during culture in the presence or absence of vitamin K. Retinoic acid further increased steady-state mRNA levels and MGP secretion at later culture intervals, an effect which was serum dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Gamma-carboxyglutamic acid excretion into rat amniotic fluid during late gestation.

The amino acid gamma-carboxyglutamate is the product of post-translational vitamin K-dependent carboxylation of peptide bound glutamic acid residues. Activity of the microsomal vitamin K-dependent carboxylase which catalyzes gamma-carboxyglutamate formation has been studied in numerous tissues, including liver and lung. Catabolism of gamma-carboxyglutamate containing proteins leads to gamma-carboxyglutamate excretion into the urine, thus quantitation of urinary gamma-carboxyglutamate can be used to assess vitamin K status, as well as the turnover of gamma-carboxyglutamate containing proteins. Since fetal urine is a major component of amniotic fluid, samples were obtained during late gestation in the rat (days 18-20) and analyzed for gamma-carboxyglutamate by reversed phase liquid chromatography to better define gestational changes in fetal vitamin K-dependent carboxylation. Relative to gestational age 18 days, amniotic fluid gamma-carboxyglutamate concentrations increased by 25% at 19 days (P less than 0.02) and by 105% at 20 days (P less than 0.001). When expressed per unit creatinine to correct for change in body mass and/or amniotic fluid volume, these differences are 15% (NS) at 19 days and 70% (P less than 0.02) at 20 days. These increases are prevented by maternal treatment with sodium warfarin. Amniotic fluid gamma-carboxyglutamate concentrations are 7-12 times greater than those in adult rat urine. During the same developmental interval (18-20 days), both lung and liver carboxylase activities increase by more than two-fold. These studies suggest that gestational age associated increases in carboxylase activity measured in vitro are associated with increased turnover of gamma-carboxyglutamate containing proteins in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Minimizing perioperative hypoxemia does not affect postpneumonectomy lung growth.

The effects of preventing the acute hypoxemia common during lung resection on postpneumonectomy lung growth were investigated. Rats that had undergone translaryngeal tracheal intubation and were supported with intermittent positive-pressure ventilation during pneumonectomy (IPPV) were compared with those allowed to breathe room air spontaneously via the natural airway (SV). A pulse oximeter was used to document intraoperative and postoperative oxygen saturation (SaO2). Almost all SV animals became acutely hypoxemic during thoracotomy [SaO2 less than 50% for 2.5 +/- 0.5 min (8/9), less than 30% for 1.7 +/- 0.4 (8/9)], whereas IPPV animals largely maintained oxygenation [SaO2 less than 50% for 0.3 +/- 0.2 min (8/13), less than 30% for 0.05 +/- 0.05 min (1/13)]. Direct measurements of oxygen saturation correlated well with the pulse oximeter (slope of regression line = 0.90, correlation 0.91), and arterial blood gases showed the SV group to be hypercapneic and acidotic as well as hypoxemic during lung removal. These abnormalities resolved soon after chest closure. Intubated animals had mild postextubation hypoxemia that normalized within 3 h of surgery. Two weeks postoperative, there were no differences in lung mass or content of water, RNA, DNA, and protein between the two groups.