Hypoxia Dramatically Increases the Nonspecific Transport of Blood-Borne Proteins to the Brain

Abstract: Increased cerebrovascular permeability is an important factor for the development of cerebral edema. To investigate the effect of hypoxia on the transport of blood-borne proteins to the brain, we used a cell culture model of the blood-brain barrier (BBB) consisting of a coculture of brain capillary endothelial cells and astrocytes that closely mimics the in vivo situation. The permeability of albumin, a marker of the nonspecific transcellular route, is extremely low in this in vitro model of the BBB. After hypoxia, a huge increase in the permeability of albumin is detected. Despite the opening of the tight junctions already demonstrated after hypoxia, the increase in the permeability of albumin is mainly attributed to an increase of the nonspecific vesicular transport in the cell, attested by the temperature dependence of the phenomenon and the visualization of labeled apotransferrin in the cytoplasm. The increase of this pathway could participate in the development of brain edema during hypoxia.     

N-Acetylaspartylglutamate Stimulates Metabotropic Glutamate Receptor 3 to Regulate Expression of the GABAAα6 Subunit in Cerebellar Granule Cells

Abstract: We have shown that the vertebrate neuropeptide N-acetylaspartylglutamate (NAAG) meets the criteria for a neurotransmitter, including function as a selective metabotropic glutamate receptor (mGluR) 3 agonist. Short-term treatment of cerebellar granule cells with NAAG (30 µM) results in the transient increase in content of GABA_Aα6 subunit mRNA. Using quantitative PCR, this increase was determined to be up to 170% of control values. Similar effects are seen following treatment with trans-1-aminocyclopentane-1,3-dicarboxylate and glutamate and are blocked by the mGluR antagonists (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine and (2S)-α-ethylglutamic acid. The effect is pertussis toxin-sensitive. The increase in α6 subunit mRNA level can be simulated by activation of other receptors negatively linked to adenylate cyclase activity, such as adenosine A1, α2-adrenergic, muscarinic, and GABA_B receptors. Forskolin stimulation of cyclic AMP (cAMP) levels abolished the effect of NAAG. The change in α6 levels induced by 30 µM NAAG can be inhibited in a dose-dependent manner by simultaneous application of increasing doses of the β-adrenergic receptor agonist isoproterenol. The increase in α6 mRNA content is followed by a fourfold increase in α6 protein level 6 h posttreatment. Under voltage-clamped conditions, NAAG-treated granule cells demonstrate an increase in the furosemide-induced inhibition of GABA-gated currents in a concentration-dependent manner, indicating an increase in functional α6-containing GABA_A receptors. These data support the hypothesis that NAAG, acting through mGluR3, regulates expression of the GABA_Aα6 subunit via a cAMP-mediated pathway and that cAMP-coupled receptors for other neurotransmitters may similarly influence GABA_A receptor subunit composition.     

Ethanol Specifically Inhibits NMDA Receptors with Affinity for Ifenprodil in the Low Micromolar Range in Cultured Cerebellar Granule Cells

Abstract: The effect of ethanol on the intracellular Ca²⁺ concentration response to NMDA in rat cerebellar granule cells grown in low or high KCI concentrations has been studied using image analysis. The cells grown in low KCI displayed high sensitivity for glycine. The subtype-selective antagonist ifenprodil inhibited the response with high (in the low micromolar range) and low (in the high micromolar range) potency. Ethanol affected the high-potency component in these cultures. In cells grown in high KCI the glycine sensitivity was lower, and a low potency for ifenprodil (high micromolar) dominated. These cells were not significantly sensitive to ethanol. The results indicate that the component displaying potency for ifenprodil in the low micromolar range with properties of the NR2B subunit is the target for ethanol action on the NMDA receptor.     

Rapid Degeneration of Cultured Human Brain Pericytes by Amyloid β Protein

Abstract: Amyloid β protein (Aβ) deposition in the cerebral arterial and capillary walls is one of the major characteristics of brains from patients with Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D). Vascular Aβ deposition is accompanied by degeneration of smooth muscle cells and pericytes. In this study we found that Aβ_1–40 carrying the "Dutch" mutation (HCHWA-D Aβ_1–40) as well as wild-type Aβ_1–42 induced degeneration of cultured human brain pericytes and human leptomeningeal smooth muscle cells, whereas wild-type Aβ_1–40 and HCHWA-D Aβ_1–42 were inactive. Cultured brain pericytes appeared to be much more vulnerable to Aβ-induced degeneration than leptomeningeal smooth muscle cells, because in brain pericyte cultures cell viability already decreased after 2 days of exposure to HCHWA-D Aβ_1–40, whereas in leptomeningeal smooth muscle cell cultures cell death was prominent only after 4–5 days. Moreover, leptomeningeal smooth muscle cell cultures were better able to recover than brain pericyte cultures after short-term treatment with HCHWA-D Aβ_1–40. Degeneration of either cell type was preceded by an increased production of cellular amyloid precursor protein. Both cell death and amyloid precursor protein production could be inhibited by the amyloid-binding dye Congo red, suggesting that fibril assembly of Aβ is crucial for initiating its destructive effects. These data imply an important role for Aβ in inducing perivascular cell pathology as observed in the cerebral vasculature of patients with Alzheimer's disease or HCHWA-D.     

Oleic Acid Inhibits Gap Junction Permeability and Increases Glucose Uptake in Cultured Rat Astrocytes

Abstract: The role of oleic acid in the modulation of gap junction permeability was studied in cultured rat astrocytes by the scrape-loading/Lucifer yellow transfer technique. Incubation with oleic acid caused a dose-dependent inhibition of gap junction permeability by 79.5% at 50 µ M , and no further inhibition was observed by increasing the oleic acid concentration to 100 µ M . The oleic acid-mediated inhibition of gap junction permeability was reversible and was prevented by bovine serum albumin. The potency of oleic acid-related compounds in inhibiting gap junction permeability was arachidonic acid > oleic acid > oleyl alcohol > palmitoleic acid > stearic acid > octanol > caprylic acid > palmitic acid > methyloleyl ester. Oleic acid and arachidonic acid, but not methyloleyl ester, increased glucose uptake by astrocytes. Neither oleic acid nor arachidonic acid increased glucose uptake in the poorly coupled glioma C6 cells. These results support that the inhibition of gap junction permeability is associated with the increase in glucose uptake. We suggest that oleic acid may be a physiological mediator of the transduction pathway leading to the inhibition of intercellular communication.     

BCL-2-Related Protein Expression in Apoptosis: Oxidative Stress Versus Serum Deprivation in PC12 Cells

Abstract: Expression of the BCL-2 protein family members, BAX, BAK, BAD, BCL-xL, BCL-xS, and BCL-2, was measured (by western blotting using specific antibodies) in PC12 cells before and during apoptosis induced by either H₂O₂ treatment or by serum deprivation and during rescue from apoptosis by nerve growth factor (NGF). H₂O₂-induced apoptosis, as measured by DNA fragmentation, caused: (a) a dose-dependent increase in BAX, (b) a dose-independent increase in BAK, and (c) a dose-dependent inhibition of BAD expression. By comparison, apoptosis induced by serum deprivation resulted in a time-dependent decrease in both BAX and BAK, along with a dramatic and sudden decrease in BAD expression. However, when PC12 cells were incubated in an apoptosis-sparing medium (i.e., NGF-supplemented serum-free medium), both BAX and BAK were increased significantly, whereas BAD expression remained inhibited. BCL-xL expression was increased by H₂O₂ but unaffected by serum deprivation or long-term NGF treatment. Neither BCL-2 nor BCL-xS expression could be detected in PC12 cells under the experimental conditions tested. Our results show that the expression of BAX, BAK, BAD, and BCL-xL is altered in a stimulus-dependent manner but cannot be used to define whether a cell will undergo or survive apoptosis. The similarity between changes in expression of BCL-2-related proteins induced by H₂O₂ exposure and NGF rescue could reflect activation in part of a common antioxidant pathway.     

Roles for Protein Kinase C and Mitogen-Activated Protein Kinase in Nicotine-Induced Secretion from Bovine Adrenal Chromaffin Cells

Abstract: Both the Ca²⁺/phospholipid-dependent protein kinases (protein kinases C, PKCs) and mitogen-activated protein kinases (MAPKs) have been implicated as participants in the secretory response of bovine adrenomedullary chromaffin cells. To investigate a possible role for these kinases in exocytosis and the relationship of these kinases to one another, intact chromaffin cells were treated with agents that inhibited each of the kinases and analyzed for catecholamine release and MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)/MAPK activation after stimulation with secretagogues of differential efficacy. Of the three secretagogues tested, inactivation of PKCs by long-term phorbol 12-myristate 13-acetate (PMA) treatment or incubation with GF109203X had the greatest inhibitory effect on nicotine-induced catecholamine release and MEK/MAPK activation, a moderate effect on KCl-induced events, and little, if any, effect on Ca²⁺ ionophore-elicited exocytosis and MEK/MAPK activation. These results indicate that PKC plays a significant role in events induced by the optimal secretagogue nicotine and a lesser role in exocytosis elicited by the suboptimal secretagogues KCl and Ca²⁺ ionophore. Treatment of cells with the MEK-activation inhibitor PD098059 completely inhibited MEK/MAPK activation (IC₅₀ 1–5 µM) and partially inhibited catecholamine release induced by all secretagogues. However, PD098059 was more effective at inhibiting exocytosis induced by suboptimal secretagogues (IC₅₀～10 µM) than that induced by nicotine (IC₅₀～30 µM). These results suggest a more prominent role for MEK/MAPK in basic secretory events activated by suboptimal secretagogues than in those activated by the optimal secretagogue nicotine. However, PD098059 also partially blocked secretion potentiated by short-term PMA treatment, suggesting that PKC can function in part by signaling through MEK/MAPK to enhance secretion. Taken together, these results provide evidence for the preferential involvement of MEK/MAPK in basic secretory events activated by the suboptimal secretagogues KCl and Ca²⁺ ionophore and the participation of both PKC and MEK/MAPK in optimal secretion induced by nicotine.     

Interactions between diurnal temperature fluctuations and salinity on expansion growth and water status of young tomato plants

Expansion growth is limited if the difference between day and night temperature (DIF) is negative. Growth is also limited high salinity. Expansion growth of tomato seedlings was studied under day/night temperatures of 16/24°C and 24/16°C, and nutrient solution salinities of 3 and 15 mS cm^-1 to ascertain whether interactions exist between the two stress forms. Water status was also studied in order to assess possible mechanisms of growth retardation. A significant interaction between DIF and salinity was found for all recorded growth variables. Hypocotyl length, plant height, leaf area and fresh and dry weight were lower at negative DIF than at positive, the reduction being greater at low salinity than at high. Increased salinity also reduced growth, more so at positive DIF than at negative. Growth reduction at negative DIF was accompanied increased shoot water and osmotic potentials. Pressure potential was unaffected DIF. Growth reduction at high salinity was accompanied reduced water and osmotic potentials. Pre-dawn pressure potential was increased at high salinity, whereas no effect of salinity on pressure potential at midday was found. The differences in effects on water status between the two stress forms may suggest differing mechanisms of growth retardation.     

Three Antigenic Regions in p17 of Human Immunodeficiency Virus Type 1 (HIV-1) Revealed by Mouse Monoclonal Antibodies and Human Antibodies in HIV-1 Carrier Sera

We investigated the murine antibody response to recombinant p17 (rp17) of human immunodeficiency virus type 1 (HIV-1) and the human antibody response directed to p17 in HIV-1 infection. Three large peptides covering residues 12-29, 53-87 and 87-115 of p17 were synthesized. The cysteine residues 57 and 87 of peptide 53-87 were reoxidized to form a disulfide bridge. Eighteen out of 19 murine monoclonal anti-rp17 antibodies had relatively high affinities (K_A = 1.9 × 10⁵?1.4 × 10⁸ M^?1) with one of the 3 p17 peptides in the liquid phase. Each monoclonal antibody reacted only with one particular peptide and had no reactivity with the other 2 p17 peptides. All the monoclonal antibodies reacted with rp17 in the liquid phase with a reasonable degree of affinity (K_A = 2.0 × 10⁵?1.8 × 10⁷ M^?1). Four HIV-1 carrier sera, which were positive in ELISA using rp17 as the antigen, reacted positively in an ELISA using 3 p17 peptides which were used to titrate murine monoclonal antibodies. Murine monoclonal antibodies having specificity for the 3 p17 peptides stained live HIV-1-infected cells by means of indirect membrane immunofluorescence, irrespective of their specificity. This suggests that the various portions of p17 (at least 3 regions of p17) were exposed on the surface of live infected cells, probably as short polypeptide chains.