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Dysregulation of dopamine homeostasis and elevation of the cytosolic level of the transmitter have been suggested to underlie the vulnerability of catecholaminergic neurons in Parkinson's disease. Because several known mutations in alpha-synuclein or overexpression of the wild-type (WT) protein causes familial forms of Parkinson's disease, we investigated possible links between alpha-synuclein pathogenesis and dopamine homeostasis. Chromaffin cells isolated from transgenic mice that overexpress A30P alpha-synuclein displayed significantly increased cytosolic catecholamine levels as measured by intracellular patch electrochemistry, whereas cells overexpressing the WT protein and those from knock-out animals were not different from controls. Likewise, catechol concentrations were higher in L-DOPA-treated PC12 cells overexpressing A30P or A53T compared with those expressing WT alpha-synuclein, although the ability of cells to maintain a low cytosolic dopamine level after L-DOPA challenge was markedly inhibited by either protein. We also found that incubation with low-micromolar concentrations of WT, A30P, or A53T alpha-synuclein inhibited ATP-dependent maintenance of pH gradients in isolated chromaffin vesicles and that the WT protein was significantly less potent in inducing the proton leakage. In summary, we demonstrate that overexpression of different types of alpha-synuclein disrupts vesicular pH and leads to a marked increase in the levels of cytosolic catechol species, an effect that may in turn trigger cellular oxyradical damage. Although multiple molecular mechanisms may be responsible for the perturbation of cytosolic catecholamine homeostasis, this study provides critical evidence about how alpha-synuclein might exert its cytotoxicity and selectively damage catecholaminergic cells.

Amperometry is widely used to study exocytosis of neurotransmitters and hormones in various cell types. Analysis of the shape of the amperometric spikes that originate from the oxidation of monoamine molecules released during the fusion of individual secretory vesicles provides information about molecular steps involved in stimulation-dependent transmitter release. Here we present an overview of the methodology of amperometric signal processing, including (i) amperometric signal acquisition and filtering, (ii) detection of exocytotic events and determining spike shape characteristics, and (iii) data manipulation and statistical analysis. The purpose of this review is to provide practical guidelines for performing amperometric recordings of exocytotic activity and interpreting the results based on shape characteristics of individual release events.

Using a cell fusion assay, we show here that in addition to complete fusion SNAREs also promote hemifusion as an alternative outcome. Approximately 65% of events resulted in full fusion, and the remaining 35% in hemifusion; of those, approximately two thirds were permanent and approximately one third were reversible. We predict that this relatively close balance among outcomes could be tipped by binding of regulatory proteins to the SNAREs, allowing for dynamic physiological regulation between full fusion and reversible kiss-and-run-like events.

A key question in understanding mechanisms of neurotransmitter release is whether the fusion pore of a synaptic vesicle regulates the amount of transmitter released during exocytosis. We measured dopamine release from small synaptic vesicles of rat cultured ventral midbrain neurons using carbon fiber amperometry. Our data indicate that small synaptic vesicle fusion pores flicker either once or multiple times in rapid succession, with each flicker releasing approximately 25-30% of vesicular dopamine. The incidence of events with multiple flickers was reciprocally regulated by phorbol esters and staurosporine. Thus, dopamine neurons regulate the amount of neurotransmitter released by small synaptic vesicles by controlling the number of fusion pore flickers per exocytotic event. This mode of exocytosis is a potential mechanism whereby neurons can rapidly reuse vesicles without undergoing the comparatively slow process of recycling.

Alterations in the cytosolic pool directly affect neurotransmitter synthesis and release and are suggested to be key factors in various neurodegenerative disorders. Although this cytosolic pool is the most metabolically active, it is miniscule compared with the amount of vesicular transmitter and has never been quantified separately. Here, we introduce intracellular patch electrochemistry (IPE), a technique that for the first time provides direct measurements of cytosolic oxidizable molecules in single mammalian cells. In amperometric mode, IPE detects total catechols, whereas in cyclic voltammetric mode, it preferentially measures catecholamines. In cultured chromaffin cells, the total cytosolic catechol concentration was 50-500 microm, of which approximately 10% were catecholamines. Reserpine, a vesicular monoamine transporter inhibitor, had no effect on the catecholamine pool but increased total catechols by fourfold to fivefold. Combined with pargyline, a monoamine oxidase inhibitor, reserpine increased catecholamine levels in the cytosol by approximately sixfold. Amphetamine induced a transient approximately fivefold accumulation of cytosolic catecholamines and a slow increase of total catechols. In cells incubated with 3,4-dihydroxy-L-phenylalanine (L-DOPA), catecholamines increased by approximately 2.5-fold and total catechols increased by approximately fourfold. Cytosolic catecholamines returned to control levels

We propose a simple mathematical model of liver S -adenosylmethionine (AdoMet) metabolism. Analysis of the model has shown that AdoMet metabolism can operate under two different modes. The first, with low metabolic rate and low AdoMet concentration, serves predominantly to supply the cell with AdoMet, the substrate for various cellular methylation reactions. The second, with high metabolic rate and high AdoMet concentration, provides an avenue for cleavage of excess methionine and can serve as a source of cysteine when its increased synthesis is necessary. The switch that triggers interconversion between the "low" and "high" modes is methionine concentration. Under a certain set of parameters both modes may coexist. This behavior results from the kinetic properties of (i) the two isoenzymes of AdoMet synthetase, MATI and MATIII, that catalyse AdoMet production; one is inhibited by AdoMet, whereas the other is activated by it, and (ii) glycine- N -methyltransferase that displays highly cooperative kinetics that is different from that of other AdoMet-dependent methyltransferases. Thus, the model provides an explanation for how different cellular needs are met by regulation of this pathway. The model also correctly identifies a critical role for glycine N -methyltransferase in depleting excess methionine in the high mode, thus avoiding the toxicity associated with elevated levels of this essential amino acid., (Copyright 2000 Academic Press.)

In suspensions of washed human erythrocytes, adenosine added in a single dose to concentrations of 0.1-10.0 mmol/l suspension was deaminated at rates ranging from 10 to 50 mmol/l cells h. The sum of adenosine, inosine, and hypoxanthine concentrations in the suspension, as well as the intracellular concentration of ATP, remained constant. In the presence of 25-50 mmol/l orthophosphate, addition of a single dose of adenosine into erythrocyte suspension increased the ATP concentration by up to 280% of the initial level. If the initial adenosine concentrations were greater than 5 mmol/l suspension, ATP increased independently of adenosine concentration to the level determined only by the concentration of orthophosphate. After orthophosphate was returned to its initial level, ATP in erythrocytes began to decrease. In the presence of coformycin, erythrocytes utilised adenosine at a rate of 0.2-0.3 mmol/l cells h. Their adenylate pool increased at a rate of 0.10-0.16 mmol/l cells h for several hours, but intracellular ATP increased only slightly. The energy charge of cells decreased significantly from 0.86 +/- 0.05 (control) to 0.82 +/- 0.06. Adenosine continuously pumped into erythrocyte suspensions at rates of 0.02-5.0 mmol/l cells h for several hours caused the adenylate pool of erythrocytes and intracellular ATP to increase synchronously at a rate of 0.02-0.35 mmol/l cells h. The energy charge of these erythrocytes increased significantly up to 0.91 +/- 0.03. After pumping of adenosine was stopped, the intracellular ATP and the adenylate pool began to decrease, returning sometimes to the initial level in 2-3 h.

IMP was found to activate AMP deaminase in crude glucose-depleted human erythrocyte lysates. Activation of the enzyme by IMP is due to prevention of the inhibitory effect of inorganic phosphate. At 1 mM AMP and 2-3 mM phosphate the addition of 2-5 mM IMP accelerates the AMP deamination two to three times.

Amphotericin B (1-3 mg/l) decreases the ATP content in erythrocytes by 11-26% and stimulates the K+ efflux but has no effect on the adenylate pool. Adenosine added to the erythrocyte suspension increases the adenylate pool, maintains a high intracellular ATP level for 6-8 hours of incubation at 37 degrees C and diminishes the amphotericin B-induced leakage of K+. Incubation of erythrocytes without glucose for 4-5 hours leads to a 20-50% loss of ATP accompanied by a significant reduction of the adenylate pool. Further addition of glucose partly restores the ATP level. In the presence of adenosine the ATP concentration is restored far more pronounced reaching nearly the original level due to the increase of the adenylate pool.