Rita Ball

Angiotensin II‐induced fluid phase endocytosis in human cerebromicrovascular endothelial cells is regulated by the inositol‐phosphate signaling pathway

The involvement of the early signaling messengers, inositol tris‐phosphate (IP3), intracellular calcium, [Ca2+]i, and protein kinase C (PKC), in angiotensin II (AII)‐induced fluid phase endocytosis was investigated in human brain capillary and microvascular endothelial cells (HCEC). AII (0.01–10 μM) stimulated the uptake of Lucifer yellow CH, an inert dye used as a marker for fluid phase endocytosis, in HCEC by 50–230%. AII also triggered a fast accumulation of IP3 and a rapid increase in [Ca2+]i in cells loaded with the Ca2+‐responsive fluorescent dye fura‐2. The prompt AII‐induced [Ca2+]i spike was not affected by incubating HCEC in Ca2+‐free medium containing 2 mM EGTA or by pretreating the cultures with the Ca2+ channel blockers, methoxyverapamil (D600; 50 μM), nickel (1 mM), or lanthanum (1 mM), suggesting that the activation of AII receptors on HCEC triggers the release of Ca2+ from intracellular stores. The AII‐triggered increases in IP3, [Ca2+]i, and Lucifer yellow uptake were inhibited by the nonselective AII receptor antagonist, Sar1, Val5, Ala8‐AII (SVA‐AII), and by the phospholipase C (PLC) inhibitors, neomycin and U‐73122. By contrast, the protein kinase C (PKC) inhibitors, staurosporine and calphostin C, failed to affect any of these AII‐induced events. This study demonstrates that increased fluid phase endocytotosis induced by AII in human brain capillary endothelium, an event thought to be linked to the observed increases in blood‐brain barrier permeability in acute hypertension, is likely dependent on PLC‐mediated changes in [Ca2+]i and independent of PKC.

Developmental regulation of glutamate transporters and glutamine synthetase activity in astrocyte cultures differentiated in vitro.

Glutamate plays an important role in brain development, physiological function, and neurodegeneration. Astrocytes control synaptic concentration of glutamate via the high affinity glutamate transporters, GLT‐1 and GLAST, and the glutamate catabolizing enzyme, glutamine synthetase. In this study we show that astrocytes cultured from rat brain in various stages of development including embryonic (E18), postnatal (P1–P21) and mature (P50), show distinct patterns of GLT‐1 and GLAST expression, glutamine synthetase activity, and phenotypic changes induced by dibutyryl‐cyclic adenosine monophosphate. The transcripts for GLT‐1 message were detectable in embryonic astrocytes only, whereas the GLAST message was highly expressed in E18 and P1–P4 astrocyte cultures, declined in P10–P21, and was undetectable in P50 astrocytes. Uptake of 3H‐glutamate correlated well with GLAST expression in astrocyte cultures of all developmental stages. Glutamine synthetase activity significantly declined from high embryonic levels in P4 astrocytes and remained low throughout postnatal maturation. Exposure of astrocyte cultures to the differentiating agent, db‐cAMP (250–500 μM; 6 days), resulted in a pronounced stellation, up‐regulation of GLT‐1 and GLAST in E18, and GLAST in P4 cultures, while it was ineffective in P10 astrocytes. By contrast, db‐cAMP induced a more pronounced stimulation of glutamine synthetase activity (up to 10‐fold above basal) in P10 than in E18 cultures (up to 2 times above basal). The differences in expression/inducibility of glutamate transporters and glutamine synthetase observed in astrocyte cultures derived from various stages of fetal and postnatal development suggest that astrocytes in vivo might also respond differently to environmental or injurious stimuli during development and maturation.

Stimulation of glutamate uptake and Na, K‐ATPase activity in rat astrocytes exposed to ischemia‐like insults

The postsynaptic actions of glutamate are rapidly terminated by high affinity glutamate uptake into glial cells. In this study we demonstrate the stimulation of both glutamate uptake and Na,K‐ATPase activity in rat astrocyte cultures in response to sublethal ischemia‐like insults. Primary cultures of neonatal rat cortical astrocytes were subjected to hypoxia, or to serum‐ and glucose‐free medium, or to both conditions (ischemia). Cell death was assessed by propidium iodide staining of cell nuclei. To measure sodium pump activity and glutamate uptake, 3H‐glutamate and 86Rb were both simultaneously added to the cell culture in the presence or absence of 2 mM ouabain. Na,K‐ATPase activity was defined as ouabain‐sensitive 86Rb uptake. Concomitant transient increases (2–3 times above control levels) of both Na,K‐ATPase and glutamate transporter activities were observed in astrocytes after 4–24 h of hypoxia, 4 h of glucose deprivation, and 2–4 h of ischemia. A 24 h ischemia caused a profound loss of both activities in parallel with significant cell death. The addition of 5 mM glucose to the cells after 4 h ischemia prevented the loss of both sodium pump activity and glutamate uptake and rescued astrocytes from death observed at the end of 24 h ischemia. Reoxygenation after the 4 h ischemic event caused the selective inhibition of Na,K‐ATPase activity. The observed increases in Na,K‐ATPase activity and glutamate uptake in cultured astrocytes subjected to sublethal ischemia‐like insults may model an important functional response of astrocytes in vivo by which they attempt to maintain ion and glutamate homeostasis under restricted energy and oxygen supply.

Free radical-induced endothelial membrane dysfunction at the site of blood-brain barrier: relationship between lipid peroxidation, Na,K-ATPase activity, and 51Cr release.

Na,K-ATPase activity, membrane lipid peroxidation (TBARM), and membrane ‘leakiness’ for small molecules were examined in rat cerebromicrovascular endothelial cells (RCEC) following exposure to hydrogen peroxide and xanthine/xanthine oxidase. Whereas short-term (15–30 min) exposure to either oxidant decreased ouabain-sensitive86Rb uptake and increased TBARM in a concentration-dependent fashion, significant release of51Cr (30–40%) from cells was observed only after one hour exposure to the oxidants. By comparison, much longer exposure times (i.e., 4 hours) were needed to induce significant lactate dehydrogenase release from oxidant-treated cells. The oxidant-evoked decrease in Na,K-ATPase activity and increases in TBARM and RCEC ‘permeability’ were abolished in the presence of the steroid antioxidants U-74500A and U-74389G (5–20 μM). Reduced glutathione (4 mM) partially attenuated oxidant-induced changes, whereas ascorbic acid (2 mM) and the disulfide bond-protecting agent, dithiothreitol (1 mM), were ineffective. These results suggest that the oxidant-induced loss of Na,K-ATPase activity in RCEC results primarily from changes in membrane lipids, and implicate both the inhibition of Na,K-ATPase and membrane lipid peroxidation in the mechanism responsible for the delayed free radical-induced increase in RCEC membrane ‘permeability’.

Evidence for the role of protein kinase C in astrocyte-induced proliferation of rat cerebromicrovascular endothelial cells.

The proliferation of cerebral endothelial cells is a crucial step in neural angiogenesis and is a process responsive to changes in the surrounding environment. Serum-free medium conditioned by rat cortical astrocytes was found to accelerate DNA synthesis, induce transient activation of protein kinase C (PKC), and increase the endogenous phosphorylation of the PKC-specific substrate, the 85 kDa MARCKS protein, in rat cerebromicrovascular endothelial cells (RCEC). The stimulatory factor(s) in astrocyte conditioned media (ACM) were heat- and trypsin-sensitive and found to have an apparent molecular weight greater than 10 kDa. The potent PKC activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA), also stimulated RCEC proliferation, whereas the inhibition of PKC by staurosporine caused a concomitant loss in ACM-induced PKC translocation, MARCKS protein phosphorylation and DNA synthesis. These findings implicate PKC activation as a critical early event in cerebral endothelial cell proliferation triggered by astrocyte-derived mitogen(s).

Glutamate Uptake and Na,K-ATPase Activity in Rat Astrocyte Cultures Exposed to Ischemia

In this study we demonstrate the stimulation of both glutamate uptake and Na,K-ATPase activity in rat astrocyte cultures in response to a sublethal ischemic insult in vitro. To measure sodium pump activity and glutamate uptake, 3H-glutamate and 86Rb were simultaneously added to the cultures in the presence or absence of 2 mM ouabain. Na,K-ATPase activity was defined as ouabain-sensitive 86Rb uptake. Cell death was assessed by exclusion of the vital dye, calcein-AM from cells. Concomitant transient increases (2-3 fold above control levels) in both Na,K-ATPase and glutamate transporter activities were observed in astrocytes after 2-4 hours of ischemia. By contrast, 24 hours of ischemia caused a profound loss of both activities which paralleled significant cell death. The addition of 5 mM glucose to the cells after 4 hours of ischemia prevented the loss of sodium pump activity and glutamate uptake, and rescued astrocytes from the lethal effects of 24 hours of ischemia.

Formulation of glycerolipidic prodrugs into PEGylated liposomes for brain delivery

Although numerous drugs are used to treat HIV infection with increasing efficacy, the patient’s brain is often infected by the virus and acts as a sanctuary where drugs cannot penetrate due to their low passage through the blood brain barrier. Therefore, the design of new medicine able to reach the brain is extremely challenging. An approach based on prodrug synthesis and encapsulation into PEGylated nanocarriers was proposed and applied to didanosine, a nucleosidic analogue used to treat HIV-1 associated dementia. In this study, appropriate formulations of PEGylated liposomes were designed to incorporate two glycerolipidic prodrugs of didanosine. Preparation methods based on Bangham’s or emulsion/evaporation techniques were optimized for each prodrug formulation according to the influence of critical parameters on vesicle size distribution. The obtained formulations exhibited particle size under 300 nm with high incorporation of prodrugs as shown by light scattering, optical microscopy experiments and differential scanning calorimetry. Finally the uptake of fluorescently labeled PEGylated formulations by rat brain immortalized endothelial cells modeling the BBB was evidenced by confocal laser scanning microscopy. All the results suggest that the encapsulation of didanosine prodrugs into PEGylated liposomes is a promising approach in the goal of increasing didanosine concentration in the brain and treating HIV-1-associated dementia.

Calcium and Protein Kinase C Signaling in Response to Vasoactive Peptides in Human Cerebromicrovascular Endothelial Cells

Vasoactive peptides endothelin-1 (ET-1) and bradykinin (BK) were shown to induce immediate increases in intracellular calcium concentrations, [Ca2+]i, and endogenous phosphorylation of the protein kinase C (PKC)-specific substrate, 85 kD MARCKS protein, in human cerebromicrovascular endothelial cells (HBEC). The peptides-induced [Ca2+]i surges were not affected by incubating the cells in Ca2+-free medium or by pretreating them with Ca2+-channel blocker D600 (50 µ;M). BK-stimulated [Ca2+]i increases were completely inhibited, whereas ET-1-induced [Ca2+]i, increases were insensitive to ADP-ribosylation of G-proteins by pertussis toxin. Both peptides-triggered [Ca2+]i, surges and MARCKS protein phosphorylation were abolished by the inhibitor of inositol phospholipid hydrolysis, U73122 (2.5–5 µM).

The Role of Protein Kinase C and Marcks Protein Phosphorylation in Rat Cerebromicrovascular Endothelial Cell Proliferation Induced by Astrocyte-Derived Factors

Serum-free medium conditioned by rat cortical astrocytes was found to prevent apoptosis induced by growth factor-deprivation, accelerate DNA synthesis, induce transient activation of protein kinase C (PKC), and increase the endogenous phosphorylation of the PKC-specific substrate, the 85 kD MARCKS protein, in rat cerebromicrovascular endothelial cells (RCEC). The trophic and stimulatory factor(s) in astrocyte conditioned media (ACM) were heat-and trypsin-sensitive and found to have an apparent molecular weight greater than 10 kD. The potent PKC activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA), also stimulated RCEC proliferation, whereas the inhibition of PKC by staurosporine caused a concomitant loss in ACM-induced PKC translocation, MARCKS protein phosphorylation and DNA synthesis. These findings implicate PKC activation as a critical early event in cerebral endothelial cell proliferation triggered by astrocyte-derived mitogen(s).