Michael D. Norenberg

The role of astrocytes in hepatic encephalopathy

The Alzheimer type II astrocyte change is the distinctive morphologic alteration in brain of humans and experimental animals succumbing to hepatic encephalopathy (HE). Whether this change is a primary event in the pathogenesis of HE or whether it is secondary to injury of some other component(s) of the CNS has not been clarified. Studies in a rat model of HE have revealed early reactive changes in astrocytes characterized by cytoplasmic hypertrophy. During the later phases, degenerative changes ensue corresponding to the Alzheimer type II change observed by light microscopy. In view of the role of astrocytes in ammonia detoxification and the importance of ammonia in the pathogenesis of HE, we have suggested that the initial astrocytic changes are the morphological correlates of ammonia detoxification. We have speculated that the later degenerative alterations could lead to failure by astrocytes to carry out key functions (e.g., neurotransmitter uptake, ion regulation, and the like) and contribute the development of the encephalopathy. Recently, the potential involvement of astrocytes in HE has been further investigated, using primary astrocyte cultures. Exposure of cultures to ammonia at clinically relevant concentrations has shown morphologic changes closely resembling those observed in experimental HE in vivo. These deleterious effects can partly be prevented by raising cyclic AMP levels in cells. Other potential toxins (octanoic acid, phenol) have shown pathologic changes as well. Although some alterations were common to all three, they each possessed distinctive pathological effects. A synergistic interaction has also been demonstrated with these toxins. Functional studies of ammonia-treated astrocytes have shown the following: With low doses or short-term exposure, the uptakes of K+, glutamate, and GABA remained unchanged or slightly increased, whereas with higher doses or longer treatment, those activities diminished. A fall in ATP values occurred with prolonged ammonia treatment. Preliminary findings have shown no significant derangements in the beta-adrenergic receptor, except for a slight decrease in receptor affinity. However, cyclic AMP production was diminished following stimulation with isoproterenol. A slight rise in the number of benzodiazepine receptors was found. These studies indicate that profound changes occur in astrocytes following exposure to ammonia and other putative toxins. It is proposed that toxins and factors involved in the precipitation of HE do so by affecting astroglial properties. Derangements in such properties may lead to glial dysfunction (primary gliopathy), resulting in an encephalopathic state.

Protein Kinase C in Primary Astrocyte Cultures: Cytoplasmic Localization and Translocation by a Phorbol Ester

Abstract: The distribution of calcium‐activated, phospholipid‐dependent protein kinase (protein kinase C) in supernatant and paniculate fractions of primary cultures of rat astrocytes and its translocation by a phorbol ester were studied. We observed that 91% of protein kinase C activity in astrocytes was in the supernatant fraction, as measured by lysine‐rich histone phosphorylation assay. Attempts to uncover latent activity in the particulate fraction were unsuccessful. Approximately 75% of the supernatant protein kinase C activity could be translocated to the particulate fraction by prior treatment (30–60 min) of the cultures with 100 nM 12‐O‐tetradecanoyl‐phorbol 13‐acetate (TPA), but not with 4α‐phorbol, an inactive phorbol ester. Investigation of endogenous substrates for protein kinase C showed that TPA treatment brought about an increase in phosphor ylation in membrane proteins and a decrease in phosphorylation of supernatant proteins. These findings indicate that the distribution of protein kinase C in astrocytes differs substantially from that in whole brain tissue, where approximately two‐thirds of the protein kinase C activity is associated with the particulate fraction. Because protein kinase C is concentrated in the cytosol of astrocytes and most of this activity can be translocated to membranes, astrocytes may be particularly well‐suited to respond to signals that activate phosphoinositide‐linked receptors in brain.

Ammonia induced decrease in glial fibrillary acidic protein in cultured astrocytes.

Previous studies of human hepatic encephalopathy (HE) have shown decreased levels of glial fibrillary acidic protein (GFAP) in Alzheimer type II astrocytes. In view of the important role of ammonia in the pathogenesis of HE, we carried out immunocytochemical and enzyme-linked immunosorbent assay (ELISA) studies on the effect of ammonium chloride (10 mM) on GFAP content in primary astrocyte cultures. There was a 39% loss of GFAP after a four day treatment. There was no fall in total cell protein. Potential mechanisms for this apparent selective loss of GFAP are discussed.

Calcium-activated, phospholipid-dependent protein kinase and protein substrates in primary cultures of astrocytes.

Phosphoinositide-linked transmembrane signaling in the brain involves calcium-activated, phospholipid-dependent protein kinase (protein kinase C), but little is known about the glial contribution to this system. We observed that phosphorylation of several proteins in a cytosol fraction of rat astrocytes in primary culture was increased by the addition of calcium and phosphatidylserine. These agents also stimulated phosphate incorporation into lysine-rich histone, a substrate for protein kinase C. Addition of diacylglycerol, an activator of protein kinase C, further increased histone phosphorylation, whereas polymyxin B, an inhibitor of protein kinase C, blocked the stimulatory effect of calcium and phosphatidylserine. Based on enzyme units per mg protein, the activity of protein kinase C in astrocytes appears similar to that in whole brain cytosol. These results indicate that astrocytes display protein kinase C activity and suggest that the glial enzyme may be an important component of the receptor-linked phosphoinositide response system in the brain.

Effect of ammonium chloride on the astrocyte benzodiazepine receptor

We have carried out studies on the effect of ammonium chloride on the astrocyte benzodiazepine receptor. Scatchard analysis of the binding of [3H]Ro-5-4864 to homogenates prepared from primary astrocyte cultures showed a significant decrease in Kd (27% with 2 mM NH4Cl; 32% with 5 mM NH4Cl; 25% with 10 mM NH4Cl) and Bmax (14% with 10 mM NH4Cl). These findings indicate that ammonium chloride can affect the astrocyte benzodiazepine receptor, and that such receptor changes may contribute to ammonia-induced encephalopathy.

Hyperammonemia causes altered protein phosphorylation in astrocytes

Treatment of primary astrocyte cultures with ammonium chloride for one day prior to phosphoprotein labeling resulted in a reduction in phosphate incorporation in a 66-kDa protein. Increasing ammonium chloride concentrations (2, 5, and 10 mM) led to greater reductions in phosphate incorporation in this band. The specificity of the effect was indicated by the lack of change in phosphate incorporation in 7 other protein bands. These results indicate that protein phosphorylation can be affected by pathophysiological concentrations of ammonia and suggest that altered protein phosphorylation may be related to the pathogenesis of disorders such as hepatic encephalopathy and Reyes syndrome where ammonia has been implicated as an important etiological factor.

Protein phosphorylation in primary astrocyte cultures treated with and without dibutyryl cyclic AMP

Protein phosphorylation was investigated in primary rat astrocyte cultures treated with and without dibutyryl cyclic AMP. Astrocytes maintained in dibutyryl cyclic AMP for several weeks displayed increased phosphate incorporation in 5 protein bands (55, 52, 45, 43 and 28 kDa) while incorporation in one band (42 kDa) was decreased. Phosphate incorporation in several other protein bands was unchanged. Calcium-dependent phosphate incorporation was also altered by prior exposure of the cells to dibutyryl cyclic AMP: addition of calcium to broken cell preparations resulted in increased incorporation in 75, 53 and 52 kDa while decreased incorporation occurred in 100 kDa. These differences in protein phosphorylation may be related to the previously reported biochemical and morphological changes brought about by dibutyryl cyclic AMP and may provide insights into the mechanisms of reactive gliosis.

Effect of ammonia on cyclic AMP production in primary astrocyte cultures

Exposure of primary astrocyte cultures to ammonia caused a dose- and time-dependent reduction of isoproterenol-stimulated cyclic AMP (cAMP) production. This treatment did not affect basal cAMP levels. This defect in receptor-linked cAMP production may contribute to the pathogenesis of hepatic and ammonia encephalopathies.

Effect of methotrexate on glial fibrillary acidic protein content of astrocytes in primary culture

We have recently reported that methotrexate (MTX) causes degenerative as well as reactive-like astroglial changes and alters the cell cycle kinetics of astrocytes in vitro. To further characterize the nature of the reactive-like changes that were noted by light and electron microscopy following MTX exposure, the glial fibrillary acidic protein (GFAP) content of astrocytes in culture was investigated by enzyme-linked immunosorbent assay, flow cytometry and double-immunofluorescent staining. An increase in GFAP content which did not correlate with drug dosage or DNA synthesis was noted in the MTX-treated cultures. It is postulated that this increase in GFAP content of astrocytes reflects an adaptive response to MTX-induced injury and partly explains the gliosis that is seen in methotrexate encephalopathy.

Effect of phenol and sodium octanoate on the astrocyte benzodiazepine receptor

Alterations in the benzodiazepine (BZD) receptor system have been proposed as key factors in the pathogenesis of hepatic encephalopathy (HE). To date, the focus of research has been exclusively on the central-type neuronal receptor. However, astrocytes also possess BZD receptors which are of the peripheral-type. In recent studies we found an increased affinity of the astrocyte BZD receptor, using [3H]Ro5-4864 as the ligand, after treatment of cell cultures with ammonia, an agent strongly implicated in HE. The present study was undertaken to determine whether other suspected toxins in HE (phenol and octanoic acid) produce comparable effects. Scatchard analysis of the binding of [3H]Ro5-4864 to astrocyte homogenates showed a significant decrease in Bmax in cells that had been treated with 0.5, 1.0 and 3.0 mM phenol (46%, 58% and 68%, respectively). The same homogenates also showed a significant decrease in Kd after treatment with 0.5 mM phenol. No change in either affinity or receptor number was seen with 0.5, 1.0 and 3.0 mM sodium octanoate. Our results indicate that phenol, but not sodium octanoate, has an effect on the astrocyte BZD receptor. Thus, different agents that have been implicated in HE produce varying effects on the astrocytic BZD receptor. These findings suggest that the astrocyte benzodiazepine receptor may be involved in the pathogenesis of HE.