Vacuolar ATPase-mediated sequestration of local anesthetics in swollen macroautophagosomes

Abstract

Purpose: Local anesthetics in their therapeutic concentration range cause a vacuolar cytopathology that has been observed in vivo and in various types of mammalian cells. We examined whether active concentration ranges of drugs and the kinetics of the vacuolar response are clinically relevant and whether this phenomenon is associated with cytotoxicity, autophagy, and cell stress signalling.

Methods: We compared procaine and lidocaine for morphological, functional, and signalling responses in a previously exploited non-neuronal system, primary smooth muscle cells. Several markers conjugated to fluorescent proteins allowed morphological and functional analysis of vacuolar cells. Signalling related to autophagy and cell stress was addressed (immunoblotting of cell lysates).

Results: Within 2-4 hr, lidocaine and procaine (> or = 1 mM) induced massive cell vacuolization, a response abated by the V-ATPase inhibitor, bafilomycin A1, and activated macroautophagic signalling (LC3 II formation) but not other stress signalling (p38, ERK1/2, p53, no influence on serum-controlled Akt phosphorylation). Novel aspects of the morphological analysis include reduced LC3 labelling of the large vacuoles in cells treated with 3-methyl-adenine, inhibition of CD63 labelling of these vacuoles by co-expression of dominant negative Rab7, retention of secretory green fluorescent protein (GFP) possessing a signal sequence in vacuolar cells, and partial vacuole labelling with lysosomal-associated membrane protein 1 (LAMP1). Lidocaine (2.5-5 mM) was not overtly cytotoxic but arrested cell division over 48 hr.

Conclusions: V-ATPase-mediated sequestration of clinically relevant concentrations of local anesthetics sequentially involves vacuolization, macroautophagic signalling, and lysosome fusion to large vacuoles. Disruption of the secretory pathway and mitotic arrest were also observed over several hours without major cytotoxicity.