Intracellular patch electrochemistry: regulation of cytosolic catecholamines in chromaffin cells

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