Effects of lactic acid on astrocytes in primary culture.

Excessive tissue lactic acidosis is considered to be detrimental to the central nervous system (CNS) and may adversely affect recovery from anoxia, ischemia, trauma and epilepsy. Since astrocytes are believed to play a role in pH regulation in the CNS, we studied the effect of this acid on primary astrocyte cultures. Cells exposed to lactic acid showed chromatin clumping, an increase of lipid and dense bodies, a loss of polyribosomal clusters, slightly increased cytoplasmic lucency, swollen mitochondria and tangled intermediate filaments. These alterations progressed with lower pH and longer exposure. Irreversible changes occurred one to two hours after exposure at pH 6; after 30 to 60 minutes (min) at pH 5.5 and after ten to 30 min at pH 5. Comparable results were obtained with the use of other weak acids indicating that the observed changes were due to increased hydrogen ion concentration rather than secondary to lactate per se. Additionally, various concentrations of lactic acid adjusted to identical pH produced similar morphologic alterations. Thus, while lactic acid caused marked and at times irreversible alterations in astrocytes, severe and prolonged acidosis was required to produce such injurious effects. This relative resistance of astrocytes to acidosis is in keeping with their potential role in pH regulation in brain.

Effect of cyclic AMP on ammonia-induced alterations in primary astrocyte cultures.

Current evidence suggests that astrocytes may be the target of ammonia toxicity. Consistent with this view are recent investigations which have shown morphologic alterations in primary astrocyte cultures following exposure to ammonia. In the present study, these alterations became severely aggravated when the cultures were not grown or maintained in dibutyryl cyclic adenosine monophosphate (AMP). Cyclic AMP analogues and agents that increase intracellular cyclic AMP levels significantly inhibited the toxic effects of ammonia. The exact mechanism responsible for this apparent protective effect of cyclic AMP on ammonia-treated astrocytes is not known. The possible means by which cyclic AMP may serve to ameliorate ammonia-induced toxicity are discussed.

Morphologic effects of ammonia on primary astrocyte cultures. II. Electron microscopic studies.

Light microscopic studies of primary astrocyte cultures following exposure to ammonia have shown several alterations. To determine the nature and significance of these changes, electron microscopic studies were performed. Ultrastructural changes consisted of proliferation, pleomorphism and swelling of mitochondria, condensation of the mitochondrial matrix, cytoplasmic lucency and vacuolization, disaggregation of polyribosomal clusters, an initial increase followed by degranulation of rough endoplasmic reticulum, proliferation of smooth endoplasmic reticulum, an accumulation of dense bodies and a loss of intermediate glial filaments. The early alterations appeared reactive and perhaps reflected ammonia detoxification. Some changes were degenerative and support the view that ammonia exerts a direct toxic effect on astrocytes. It is postulated that these changes may interfere with critical astroglial functions and thereby play a key role in the neurologic dysfunction seen in hyperammonemia.

Morphologic effects of ammonia on primary astrocyte cultures. I. Light microscopic studies.

To evaluate the astrocytic alterations commonly seen in hepatic encephalopathy and other hyperammonemic states, primary astrocyte cultures derived from neonatal rats were exposed to varying concentrations of ammonia for one to ten days. Ammonia-treated cultures initially showed an increase in basophilia, prominent cytoplasmic processes and increased cytoplasmic granularity and vacuolization. Nucleoli were increased in size and there was an increase in nucleolar/nuclear ratio. Later, fragmentation and loss of cytoplasmic processes, formation of dense bodies and frank cellular disintegration were noted. The changes were proportional to the concentration and duration of ammonia treatment. Our studies show that ammonia is capable of directly causing morphologic alterations in astrocytes. We believe that the use of primary cultures provides a means of exploring the precise role of astrocytes in hyperammonemic states.

Increase of cellular murine leukemia virus reverse transcriptase in interferon-treated cells.

The addition of mouse interferon (IFN) to AKR murine leukemia virus (MuLV)-producing NIH3T3 cells inhibited the extracellular appearance of AKR MuLV when assayed for reverse transcriptase activity or infectious virus production. IFN treatment had no detectable effect on proviral DNA formation during infection nor on the level of viral RNA in virus-producing cells. However, addition of IFN did alter the level of cellular viral reverse transcriptase activity. Chromatography of extracts from virus-producing cells on poly(G)-Sepharose columns revealed two peaks of reverse transcriptase activity. Peaks I and II eluted at 0.45 M and 0.65 M NaCl, respectively, while the cellular DNA polymerase beta eluted earlier at 0.3 M NaCl. IFN treatment of these chronic virus producer cells resulted in a 5-fold increase in peak I whereas peak II and polymerase beta remained essentially unchanged. When reverse transcriptase from purified virions was similarly chromatographed on poly(G)-Sepharose, all of the enzymatic activity eluted as peak I. Thus, the reverse transcriptase in peak I from cell extracts appears to be the form which is present in mature virions. Contrary to the results with chronic virus-producing cells, IFN treatment prior to exogenous infection with MuLV did not alter levels of reverse transcriptase peaks I and II or polymerase beta. These results provide further evidence that the major effect of IFN occurs at the level of MuLV maturation and assembly.

Rescue of SV40 from interferon-treated heterokaryons.

Simian virus 40 (SV40)-transformed nonpermissive cells express only the early products of SV40. Heterokaryons formed by fusion of these transformed cells with uninfected permissive cells support the activation of the resident viral genome leading to subsequent viral DNA replication, late protein synthesis and release of progeny virus. Pretreatment of heterokaryon cultures with either mouse or monkey interferon (IFN) before fusion with polyethylene glycol (PEG) produced a dose-dependent inhibition in the appearance of free viral DNA as well as production of infectious virus. The decreased yield of SV40 in these cultures was similar to the inhibition which was observed in mouse or monkey cells incubated with homologous IFN prior to exogenous infection with SV40. When IFN was added to the cultures at progressively later times after fusion with PEG, there was less inhibition of virus production. Although there was a comparable decrease in the production of virus by pretreatment with either mouse or monkey IFN, monkey IFN exerted the inhibition for a longer period of time when added after heterokaryon formation. These results demonstrate that IFN treatment applied even after initiation of SV40 replication can still inhibit virus multiplication.

Increased interferon production in human cells irradiated with ultraviolet light.

Polyinosinate-polycytidylate [poly(I).poly(C)]-induced interferon production in cultures of human foreskin fibroblast strains was increased by ultraviolet irradiation of cells at the time of exposure to inducer or at 2 h after induction. Incubation of cells with interferon prior to induction (priming) and ultraviolet irradiation exerted a cooperative enhancing effect on interferon production. The resulting interferon yields were generally somewhat higher than the yields from cultures subjected to sequential treatment with cycloheximide and actinomycin D.

Interferon induction in rabbit cells irradiated with UV light.

UV irradiation of a continuous line of rabbit kidney cells (RK13) was used as a tool for the study of the mechanism of interferon induction. Irradiation of cells prior to their exposure to Newcastle disease virus (NDV) resulted in a dose-dependent decrease in interferon production. The inhibition of total cellular RNA synthesis by UV irradiation in uninduced cultures was similar to the inactivation curve of interferon production in NDV-induced cultures. In contrast, the production of interferon with polyinosinate-polycytidylate (poly[I].poly [C]) paradoxically was enhanced in cells irradiated with a wide range of doses of UV. However, in cells stimulated with poly(I).poly(C) and "superinduced" by the sequential addition of cycloheximide and actinomycin D, the rate of inactivation of interferon production by UV light was similar to that observed with NDV. These results are not inconsistent with the idea that both poly(I).poly(C) and NDV stimulate the same interferon gene(s), but indicate that the mechanism controlling its expression may be different for each inducer.