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The Journal of Neuroscience, July 2, 2003, 23(13):5835-5845

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Cellular/Molecular
Intracellular Patch Electrochemistry: Regulation of Cytosolic Catecholamines in Chromaffin Cells

Eugene V. Mosharov,¹ Liang-Wei Gong,² Bhavanna Khanna,² David Sulzer,¹ and Manfred Lindau²

¹Departments of Neurology and Psychiatry, Columbia University, New York, New York 10032, Department of Neuroscience, New York State Psychiatric Institute, New York, New York 10032, and ²School of Applied and Engineering Physics, Cornell University, Ithaca, New York 14853

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 µM, of which 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 2.5-fold and total catechols increased by approximately fourfold. Cytosolic catecholamines returned to control levels 10 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.

Key words: patch-amperometry; cyclic voltammetry; cytosolic catecholamine; chromaffin cells; reserpine; pargyline; amphetamine; L-DOPA

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Received Feb 11, 2003; revised April 17, 2003; accepted April 21, 2003.

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