The mitochondrial permeability transition in neurologic disease

Mitochondria, being the principal source of cellular energy, are vital for cell life. Yet, ironically, they are also major mediators of cell death, either by necrosis or apoptosis. One means by which these adverse effects occur is through the mitochondrial permeability transition (mPT) whereby the inner mitochondrial membrane suddenly becomes excessively permeable to ions and other solutes, resulting in a collapse of the inner membrane potential, ultimately leading to energy failure and cell necrosis. The mPT may also bring about the release of various factors known to cause apoptotic cell death. The principal factors leading to the mPT are elevated levels of intracellular Ca 2+ and oxidative stress. Characteristically, the mPT is inhibited by cyclosporin A. This article will briefly discuss the concept of the mPT, its molecular composition, its inducers and regulators, agents that influence its activity and describe the consequences of its induction. Lastly, we will review its potential contribution to acute neurological disorders, including ischemia, trauma, and toxic-metabolic conditions, as well as its role in chronic neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis

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 mM NH 4Cl resulted in a 35% reduction in 45Ca influx. The decrease in calcium influx was dose-dependent between 2 and 10 mM NH 4Cl. 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 NH 4Cl 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 encephalography

ATP stimulates calcium influx in primary astrocyte cultures

The effect of ATP and other purines on 45Ca uptake was studied in primary cultures of rat astrocytes. Treatment of the cells with ATP for 1 to 30 min brought about an increase in cellular 45Ca. Stimulation of calcium influx by ATP was investigated using a 90 sec exposure to 45Ca and over a concentration range of 0.1 nM to 3 mM; a biphasic dose-response curve was obtained with EC 50 values of 0.3 nM and 9 uM, indicating the presence of low and high affinity purinergic binding sites. Similar levels of 45Ca influx at 90 sec were observed with ATP, ADP and adenosine (all at 100 uM). Prior treatment of the cultures with LaCl 3 blocked the purine-induced 45Ca influx. These findings indicate that one pathway for calcium entry in astrocytes involves purinergic receptor-operated, calcium channels

Characterization of cystine uptake in cultured astrocytes

Glutathione is involved in the maintenance of the structural and functional integrity of membrane proteins, in protection against free radicals and oxidative stress, and in the detoxification of xenobiotics. The cellular uptake of cystine is the rate limiting step in the biosynthesis of glutathione. The precise mechanism for such uptake is not clear as some reports indicate that the uptake occurs through a glutamate–cystine antiporter (system X c – ), whereas, others suggest that it is taken up by the glutamate transporter (system X AG ). Our studies in cultured astrocytes derived from neonatal rats showed that glutamate, D- and L-aspartate inhibited cystine uptake; that factors that increased intracellular glutamate levels, which would have enhanced the activity of the antiporter, did not stimulate cystine uptake; that the uptake was sodium dependent and partially chloride dependent; that the b o,+ and ASC systems, which have been shown to carry cystine in some cells, did not mediate cystine uptake in astrocytes; that glutamate uptake blockers such as L-aspartate-β-hydroxamate (AβH) and L- trans-pyrrolidine-2,4-dicarboxylate (PDC), as well as cystine uptake inhibitor L-α-aminoadipate (AAA) potently reduced cystine uptake. Additionally, deferoxamine (100 μM) as well as ammonium chloride (5 mM), both of which inhibit glutamate uptake, also inhibited cystine uptake. Taken together, our findings indicate that astrocytes take up cystine through a similar, if not identical, system used to take up glutamate. Interference of cystine uptake by astrocytes through the glutamate transport system may have profound effects on the redox state and the structural and functional integrity of the CNS

Glutamine synthetase and glutamate metabolism in the guinea pig cochlea

Glutamate is thought to act as a neurotransmitter of the sensory hair cells of the organ of Corti. Glutamine synthetase could be involved in a type of glutamate-glutamine cycle in the cochlea which could clear glutamate off the synaptic cleft and replenish the hair cell glutamate neurotransmitter store. Using both light and electron microscopic immunocytochemistry to localize this enzyme in the guinea pig cochlea, we have observed immunoreactive satellite glial cells surrounding parvalbumin-immunoreactive primary auditory neurons in the spiral ganglion. Glutamine synthetase was also detected in Schwann cells of the osseous spiral lamina which form the myelin sheath of nerve fibers. On the contrary, no immunoreactivity could be observed in the cochlear nerve and in the organ of Corti, although this organ contains structures able to take up glutamate. Although they confirm earlier works involving glutamine synthetase in the conversion of l-[ 3H]glutamate taken up by glial cells, our results suggest that the cochlear glutamate-glutamine cycle is not primarily involved in the recycling and replenishment of hair cell neurotransmitter glutamate. Alternatively, it is proposed that glutamine synthetase functions to limit the perilymphatic glutamate concentrations

Role of oxidative stress in the ammonia-induced mitochondrial permeability transition in cultured astrocytes

Ammonia is a neurotoxin that has been strongly implicated in the pathogenesis of hepatic encephalopathy (HE) and other neurological disorders, and astrocytes are thought to be the principal target of ammonia toxicity. While the precise mechanisms of ammonia neurotoxicity remain to be more clearly defined, altered bioenergetics and oxidative stress appear to be critical factors in its pathogenesis. It has recently been demonstrated that pathophysiological concentrations of ammonia induce the mitochondrial permeability transition (MPT) in cultured astrocytes, a process associated with mitochondrial dysfunction, and frequently caused by oxidative stress. This study investigated the potential role of oxidative stress in the induction of the MPT by ammonia. Accordingly, the effect of various antioxidants on the induction of the MPT by ammonia in cultured astrocytes was examined. Astrocytes were subjected to NH 4Cl (5 mM) treatment for 2 days with or without various antioxidants. The MPT was assessed by quantitative fluorescence imaging for the mitochondrial membrane potential (Δ Ψ m), employing the potentiometric dye TMRE; by changes in mitochondrial calcein fluorescence and by 2-deoxyglucose-6-phosphate (2-DG-6-P) changes in mitochondrial permeability. Astrocytes treated with ammonia significantly dissipated the Δ Ψ m, which was blocked by the MPT inhibitor, cyclosporin A, caused a decrease in mitochondrial calcein fluorescence and increased 2-DG-6-P permeability into mitochondria. All of these findings are consistent with induction of the MPT. Pretreatment with SOD, catalase, desferroxamine, Vitamin E, PBN and the nitric oxide synthase inhibitor, N G-nitro- l-arginine methyl ester ( l-NAME), completely blocked the ammonia-induced MPT. These data provide strong evidence that oxidative stress is involved in the induction of the MPT by ammonia, and suggest that oxidative stress and the subsequent induction of the MPT contribute to the pathogenesis of HE and other hyperammonemic disorders