Intracellular Patch Electrochemistry: Regulation of Cytosolic Catecholamines in Chromaffin Cells

Abstract

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 M, of which ϳ10% 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 ϳ2.5-fold and total catechols increased by approximately fourfold. Cytosolic catecholamines returned to control levels Յ10 min after L-DOPA withdrawal, whereas total catechols remained approximately twofold elevated even after a 1.5 hr incubation in L-DOPA-free media. Our data indicate that cytosolic catecholamines are strictly maintained at a defined level, and drug-induced increases in their concentrations lead to the accumulation of other catecholamine derivatives, such as DOPAC and 3,4-dihydroxyphenylethyleneglycol. These derivatives reside in the cytosol for hours after treatment and may be an underlying cause of drug-related cytotoxicity.

Amphetamine induces dopamine efflux through a dopamine transporter channel

“…In addition, in chromaffin cells, basal concentrations of intracellular catecholamine have been found to be as high as 50 M, and AMPH increases these basal levels 6-fold. The 2 mM DA in the patch pipette to facilitate measurement of DA efflux by the amperometric electrode is in the range of the K m for DA and is in the same order of magnitude as the physiological concentration measured in chromaffin cells (23). We also performed recordings of DA efflux mediated by DAT channel-like activity with 100 M DA in the pipette, and the results were qualitatively similar.…”

Regulation of the Development of Mesencephalic Dopaminergic Systems by the Selective Expression of Glial Cell Line-Derived Neurotrophic Factor in Their Targets

“…Background-subtracted cyclic voltammograms served to identify the released substance. The DA oxidation current was converted to concentration on the basis of a calibration of 5 M DA in ACSF after the experiment (Mosharov et al, 2003).…”

Vesicular monoamine transporter 2 regulates the sensitivity of rat dopaminergic neurons to disturbed cytosolic dopamine levels

“…Reduced expression levels of VMAT2 (Mooslehner et al, 2001) or pharmacological inhibition of the transporter (Mosharov et al, 2003) has previously been demonstrated to result in higher dopamine turnover thereby exposing neurons to dopamine-dependent oxidative stress and dopamine-quinone formation. We set up a method to identify dopaminergic neurites, based on the staining of these structures with an antibody against tyrosine hydroxylase (TH) and subtraction of the cell body area using a specialized algorithm (see Materials and Methods).…”