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Notes: Abstract Cultures of bovine adrenomedullary chromaffin cells accumulated l-[methyl-3H]methyl-4-phenylpyridinium ([3H]MPP+) in a time- and concentration-dependent manner with an apparent Km of 0.7 μM and a Vmax of 3 pmol/min/106 cells. The uptake was sodium dependent and sensitive to inhibitors of the cell-surface catecholamine transporter. At low concentrations of MPP+, the subcellular distribution was identical to that of endogenous catecholamines in the catecholamine-containing chromaffin vesicles. However, at a higher concentration of MPP+, a larger proportion of the toxicant was recovered in the cytosolic fraction, with less in the chromaffin vesicle fractions. When cells were prelabeled with [3H]MPP+, at 1 and 300 μM, and then permeabilized with digitonin in the absence of Ca2+, there was a proportionally greater release of MPP+ from the cells labeled at the higher concentration of the toxicant. In the presence of Ca2+, cell permeabilization induced a time-dependent secretion of catecholamines and a parallel secretion of MPP+. Under these conditions, the secretion of endogenous catecholamines was unaffected by the presence of MPP+. When the permeabilization studies were carried out in the presence of tetrabenazine, a massive release of MPP+ was observed in the absence of Ca2+ and was not further increased by Ca2+. In intact cells prelabeled with 300 μM [3H]MPP+, the secretagogues nicotine and veratridine elicited a Ca2+-dependent secretion of catecholamines and MPP+ from the cells in similar proportions to their cellular contents. Barium-induced release of both species was independent of external Ca2+. Collectively, these data reveal that MPP+ is a novel substrate for both the cellular and vesicular catecholamine transporters and that, in intact cells, vesicular transport may become limiting relative to the cell-surface transporter, leading to the cytosolic accumulation of the toxicant.

Notes: Abstract: Bovine adrenomedullary chromaffin (BAMC) cells, cultured in a defined medium, were used to study the mechanisms of toxicity and cellular resistance to the catecholamine neuron toxicants 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium (MPP+). The viability of the cells was assessed biochemically [cellular catecholamine content and the catalytic activities of tyrosine hydroxylase (TH) and lactate dehydrogenase (LDH)] and anatomically (by electron microscopy). When cultures of BAMC cells were exposed to MPTP or MPP+ for 3 days, a marked loss of cellular catecholamines and TH activity was observed. The addition of an inhibitor of monoamine oxidase (MAO) B (Ro 19–6327), but not MAO A (clorgyline), prevented the toxicity of MPTP but not that of MPP+. In addition, the cellular toxicity of MPP+, but not MPTP, was antagonized by desmethylimipramine, an inhibitor of cellular catecholamine uptake. The toxicity of MPP+ was time dependent, with losses of TH and the release of cellular LDH occurring after 48 h in culture. Catecholamine depletion occurred somewhat sooner, being evident after 24 h of exposure to MPP+. The cellular toxicity of MPP+ was concentration dependent and significantly enhanced by inhibitors of catecholamine vesicular uptake (reserpine, tetrabenazine, or Ro 4–1284). Electron microscopic examination of cells treated with either MPP+, tetrabenazine, or their combination revealed that MPP+ damaged BAMC cells and that this damage was markedly potentiated by the inhibition of vesicular uptake by tetrabenazine. The concentration of glucose in the culture media of untreated cells slowly decreased as a function of time. The rate of glucose consumption was markedly accelerated by MPP+ treatment and the losses in cell TH and the release of LDH into the media were preceded by a 99% depletion of glucose from the media. In cultures not treated with MPP+, lactate accumulated in the media as a function of time. Addition of MPP+ to the media increased the formation of lactate, in a concentration-dependent manner. Reserpine pretreatment further enhanced the production of lactate in response to MPP+. Culturing cells in glucose-free medium greatly potentiated the effects of MPP+ on cellular TH and catecholamines. The toxicity observed after 3 days’exposure of BAMC cells to MPP+ could be prevented when the medium was replaced with fresh medium every 24 h. The effects of glucose deprivation and reserpine were observed to be additive. The ability of MPP+ to affect mitochondrial function is determined by the capacity of the storage vesicle to sequester the pyridinium, acting as a cytosolic “buffer.”Furthermore, under conditions where MPP+ impairs mitochondrial respiration, cells survive by increasing their reliance on glycolytic metabolism.

Notes: Abstract: Experimental allergic encephalomyelitis (EAE) is an autoimmune, animal model of multiple sclerosis (MS) in which demyelination and paralysis are evident. Quinolinic acid (QUIN) is a neurotoxin and endogenous N-methyl-d-aspartate receptor agonist formed from tryptophan. The role of neurotoxins in general and QUIN in particular in EAE or MS is unknown. Lewis rats inoculated with myelin basic protein developed signs of EAE by day 12, were killed, and their tissues assayed for QUIN by gas chromatography with mass spectrometry. QUIN levels were significantly elevated in the more caudal regions of the spinal cords of animals with EAE. Brain, serum, and liver levels of QUIN were not altered. In a similar manner, QUIN in mylin basic protein-injected, asymptomatic animals was not different from control animals. The time course for QUIN was similar to the neurological signs of the disorder; however, the initial elevation in QUIN occurred before the appearance of behavioral signs. Last, treatment with the glucocorticoid dexamethasone prevented both the signs of EAE and the elevation in spinal cord QUIN. It is not known whether QUIN contributes to the paralysis in EAE. However, if QUIN is pathogenic in EAE, this finding could have therapeutic implications for MS.

Notes: Diethyldithiocarbamic acid (DDC) potentiates in vivo neurotoxicity of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) and in vitro neurotoxicity of 1-methyl-4-phenylpyridinium (MPP+). Male C57B1/6 mice were given two or five injections of MPTP (30 rag/kg i.p.) preceded 0.5 h by DDC (400 mg/kg i.p.). The mice were tested for catalepsy, akinesia, or motor activity during and after the period of dosing. Striatal and hippocampal tissues were obtained at 2 and 7 days following the last injection and evaluated for dopamine and norepinephrine levels, respectively. These same tissues were also analyzed for the levels of glial fibrillary acidic protein (GFAP), an astrocyte-localized protein known to increase in response to neural injury. Pretreatment with DDC potentiated the effect of MPTP in striatum and resulted in substantially greater dopamine depletion, as well as a more pronounced elevation in GFAP. In hippocampus, the levels of norepinephrine and GFAP were not different from controls in mice receiving only MPTP, but pretreatment with DDC resulted in a sustained depletion of norepinephrine and an elevation of GFAP, suggesting that damage was extended to this brain area by the combined treatment. Mice receiving MPTP preceded by DDC also demonstrated a more profound, but reversible, catalepsy and akinesia compared to those receiving MPTP alone. Systemically administered MPP+ decreased heart norepinephrine, but did not alter the striatal levels of dopamine or GFAP, and pretreatment with DDC did not alter these effects, but did increase lethality. DDC is known to increase brain levels of MPP+ after MPTP, but our data indicate that this is not due to a movement of peripherally generated MPP+ into CNS. In cultured bovine adrenal medullary cells, MPP+ (300 μM) slightly decreased catecholamine levels, but had no effect on tyrosine hydroxylase activity or cellular protein. However, the incubation of these cells with both MPP+ and DDC (1.5 or 3.0 mM) caused large decreases in all indicators of cell viability. The enhancement of MPP+ neurotoxicity by DDC in an in vitro system, where distributional factors are limited, raises die possibility that mechanisms in addition to altered kinetics may account for DDC-induced potentiation of MPTP neurotoxicity in vivo.