Extracellular superoxide was detected in cultures of monkey and human arterial smooth muscle cells as indicated by superoxide dismutase inhibitable reduction of cytochrome c. Superoxide production by these cells in the presence of Fe or Cu resulted in modification of low density lipoprotein (LDL). The degree of LDL modification was directly proportional to the rate of superoxide production by cells. Superoxide dismutase (100 &g/ ml), and the general free radical scavengers butylated hydroxytoluene and butylated hydroxyanisole (50 p&M), inhibited Feand Cu-mediated modification of LDL by monkey smooth muscle cells, while catalase (100 pg/ml) and mannitol (25 mM) had no effect.

The effect of ammonia on water space of astrocytes in culture was determined as a means of studying the neurotoxicity of ammonia in fulminant hepatic failure (FHF). Treatment of primary astrocyte cultures obtained from neonatal rat cortices with 10 mM NH4Cl for 4 days resulted in a 29% increase in astrocytic water space, as measured by an isotopic method utilizing 3-O-methyl-[3H]-glucose. This effect was time- and dose-dependent. The ammonia-induced swelling was reversible as the water space in cultures treated with 10 mH NH4Cl for 3 days, and then returned to normal culture media for 1 day, was similar to control cultures. These findings suggest that elevated levels of ammonia lead to astrocyte swelling and may contribute to the brain edema in FHF.

We have examined the oxidative activities of inverted cytoplasmic membrane preparations from Escherichia coli bearing proline dehydrogenase. Our measurements include both direct substrate: 2,6-dichloroindophenol and substrate : 0, oxidoreductase assays and the 9-aminoacridine fluorescence assay for proton translocation, employing succinate and NADH dehydrogenases as comparative standards. Our data show the following. (a) Membranes prepared in a new buffer system bear proline dehydrogenase that is stable in both activity and membrane association. This membrane-associated enzyme shows an apparent K , for proline 20-fold lower than that estimated from the solubilized and purified enzyme. Studies employing whole bacteria or cytoplasmic membrane vesicles have shown that proline oxidation is cryptic in intact cells, it is associated with a particulate cell fraction, and it employs molecular oxygen as physiological electron acceptor [6 -81. Purified preparations of soluble proline dehydrogenase have been obtained by detergent extraction from the cytoplasmic membranes of both E. coli and S. typhimurium [6, 81. Proline oxidation by the'soluble enzyme requires an exogenous electron acceptor.The data cited above suggest that proline dehydrogenase is associated with the cytoplasmic membrane, interacting with the respiratory chain. Menzel and Roth have suggested that saturation of specific membrane sites by the enzyme leads to its accumulation in soluble form, interaction with a specific DNA control sequence, and repression of the put genes [5]. The enzyme-membrane interaction is clearly fundamental to both Ahhreviutions. Amytal, 5-ethyl-5-isopentyl barbituric acid; CIPhzC-(CN),, carbonylcyanide m-chlorophenylhydrazone; (cHxN),C, N,N'-dicyclohexylcarbodiimide; DCIP, 2,6-dichloroindophenol; HpHOQnO, 2-heptyl-4-hydroxyquinoline-N-oxide; iodonitrotetrazolium, 3-@-IOdOphenyl)-2-@-nitrophenyl)-5-phenyltetrazoiium chloride; ApH, magnitude of the transmembrane pH gradient, pHin-pH,,,, in inverted membrane vesicles.Enzyme. Proline dehydrogenase (EC 1.5.99.8).

A number of factors appear to be involved in the proliferative and hypertrophic processes which characterize reactive astrocytosis. We have investigated the possibility that ATP, an agent that is released by injured cells following tissue destruction, may be one such factor. For this purpose, we utilized primary cultures of astrocytes derived from cerebral cortices of neonatal rats to study the effect of extracellular ATP on properties associated with astrogliosis. Light microscopic studies disclosed marked stellation of astrocytes after 30-60 min of exposure to 100 microM-1 mM ATP. In addition, the content of the astrocyte-specific intermediate filament, glial fibrillary acidic protein (GFAP), was increased 35-40% following 60-min exposure to ATP; this effect persisted for 1-3 days of exposure to 100 microM ATP. [3H]Thymidine incorporation increased progressively from 1-3 days; a 3.6-fold increase in DNA synthesis was observed following 3 days of exposure to 1 mM ATP, suggesting stimulation of cellular proliferation. These findings show that high micromolar to low millimolar concentrations of extracellular ATP reproduce several features associated with reactive gliosis and suggest that extracellular ATP may be involved in the activation of astrocytes following CNS injury.

In mammalian brain peripheral benzodiazepine (PBZD) receptors are predominantly localized on astroglial cells. Previous studies utilizing whole membrane preparations from brain and peripheral organs of various species have indicated several distinctions between the drug-receptor interactions of the two prototypic PBZD receptor ligands, PK 11195 and Ro5-4864. The present study was undertaken to determine whether putative differences in the binding of PBZD receptor ligands in homogenates of primary astrocyte cultures can be interpreted as the labeling of PBZD receptor subtypes. Equilibrium competition and saturation binding experiments in homogenate preparations of primary astrocytes from cerebral cortex of new born rats revealed that [3H]PK 11195 labels twice the number of [3H]Ro5-4864 binding sites. Unlabeled Ro5-4864 competes for [3H]PK 11195 binding in a manner suggesting the existence of multiple PK 11195 binding sites. The competition binding experiments, using various benzodiazepines, indicate that one binding component of PK 11195 corresponds to Ro5-4864 binding sites, whereas the second is different. The latter binding site does not correspond to the central BZD receptor but displays the pharmacological properties of the PBZD receptor. Further differences between the binding of PK 11195 and Ro5-4864 in astrocytes were detected in the presence of ethanol which was more effective in inhibiting the binding of the latter. Subcellular distribution studies indicated, however, that the binding of both [3H]PK 11195 and [3H]Ro5-4864 is associated primarily with the mitochondrial fraction of astrocytes. Taken together, the present study indicates the existence of non-overlapping PBZD binding sites in astrocytes and thus suggests the existence of PBZD receptor subtypes.(ABSTRACT TRUNCATED AT 250 WORDS)