L. O.B. Norenberg

Ammonia-induced astrocyte swelling in primary culture

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.

The benzodiazepine receptor in cultured astrocytes from genetically epilepsy-prone rats.

Peripheral-type benzodiazepine (BZD) receptors were studied in cultured astrocytes derived from genetically epilepsy-prone and control rats. Scatchard analysis of the binding of [3H]Ro 5-4864 to astrocyte homogenates from epilepsy-prone rats showed 38% fewer BZD receptors (Bmax) as compared to controls. No significant change in affinity (Kd) was observed. These findings suggest that the astrocyte peripheral-type BZD receptor may be involved in some forms of epilepsy.

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.

Effect of ammonia on calcium homeostasis in primary astrocyte cultures

Calcium influx, accumulation and efflux were studied in primary cultures of rat astrocytes treated with ammonium chloride. Treatment of the cells for 3 days with 10 mMN4Cl resulted in a 35% reduction in 45Ca influx. The decrease in calcium influx was dose-dependent between 2 and 10 mM NH4Cl. Short-term (30 min) exposure to ammonia had no effect on calcium influx. Calcium accumulation, as measured by 20-min exposure to 45Ca, decreased after treating cultures with 10 mM NH4Cl for one or 3 days; a greater effect was observed after the 3-day treatment. Studies with lanthanum, an inhibitor of calcium transport, indicated that the effect of ammonia was not due to non-specific leakage of calcium. Calcium efflux was not affected by exposure of the cultures to ammonium chloride. Purinergic-evoked calcium influx and mobilization was not altered by ammonia. While the mechanism(s) of calcium homeostasis affected by long-term hyperammonemia remain to be defined, these results suggest that reduced astrocytic calcium may be related to the pathogenesis of ammonia-related disorders such as hepatic encephalopathy.