title: Lidocaine induces ROCK-dependent membrane blebbing and subsequent cell death in rabbit articular chondrocytes.
abstract: Local anesthetics are administered intraarticularly for pain control in orthopedic clinics and surgeries. Although previous studies have shown that local anesthetics can be toxic to chondrocytes, the underlying cellular mechanisms remain unclear. The present study investigates acute cellular responses associated with lidocaine-induced toxicity to articular chondrocytes. Rabbit articular chondrocytes were exposed to lidocaine and their morphological changes were monitored with live cell microscopy. The viability of chondrocytes was evaluated using a fluorescence based LIVE/DEAD assay. Acute treatment of chondrocytes with lidocaine (3-30 mM) induced spherical protrusions on the cell surface (so called "membrane blebbing") in a time- and concentration-dependent manner. The concentration-response relationship for the lidocaine effect was shifted leftward by elevating extracellular pH, as expected for the non-ionized lidocaine being involved in the bleb formation. ROCK (Rho-kinase) inhibitors Y-27632 and fasudil completely prevented the lidocaine-induced membrane blebbing, suggesting that ROCK activation is required for bleb formation. Caspase-3 levels were unchanged by 10 mM lidocaine (p = 0.325) and a caspase inhibitor z-VAD-fmk did not affect the lidocaine-induced blebbing (p = 0.964). GTP-RhoA levels were significantly increased (p < 0.001), but Rho inhibitor-1 failed to suppress the membrane blebbing (p = 0.875). Lidocaine (30 mM) reduced the cell viability of isolated chondrocytes (p < 0.001) and in situ chondrocytes (p < 0.001). The chondrotoxicity was attenuated by pretreatment of cells with ROCK inhibitors or a myosin-II inhibitor blebbistatin (p < 0.001). These findings suggest that lidocaine induces ROCK-dependent membrane blebbing and thereby produces a cytotoxic effect on chondrocytes. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:754-762, 2016.
authors.affiliation: Department of Orthopedic Surgery, Shiga University of Medical Science, Otsu, Shiga, Japan.|Department of Physiology, Shiga University of Medical Science, Otsu, Shiga, Japan.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Maeda
authors.affiliation: Department of Physiology, Shiga University of Medical Science, Otsu, Shiga, Japan.
authors.forename: Futoshi
authors.initials: F
authors.lastname: Toyoda
authors.affiliation: Department of Orthopedic Surgery, Shiga University of Medical Science, Otsu, Shiga, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Imai
authors.affiliation: Department of Orthopedic Surgery, Shiga University of Medical Science, Otsu, Shiga, Japan.|Department of Physiology, Shiga University of Medical Science, Otsu, Shiga, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Tanigawa
authors.affiliation: Department of Orthopedic Surgery, Shiga University of Medical Science, Otsu, Shiga, Japan.
authors.forename: Kousuke
authors.initials: K
authors.lastname: Kumagai
authors.affiliation: Department of Physiology, Shiga University of Medical Science, Otsu, Shiga, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Matsuura
authors.affiliation: Department of Orthopedic Surgery, Shiga University of Medical Science, Otsu, Shiga, Japan.
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Matsusue
mesh_terms: D000779:Anesthetics, Local / Q000009:adverse effects*; D000818:Animals; D002358:Cartilage, Articular / Q000166:cytology / Q000187:drug effects; D053148:Caspase 3 / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects*; D019902:Chondrocytes / Q000187:drug effects*; D006576:Heterocyclic Compounds, 4 or More Rings; D008012:Lidocaine / Q000009:adverse effects*; D008297:Male; D011817:Rabbits; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D000779:Anesthetics, Local; D006576:Heterocyclic Compounds, 4 or More Rings; C472645:blebbistatin; D008012:Lidocaine; D054460:rho-Associated Kinases; D053148:Caspase 3
keywords: Rho-kinase (ROCK); chondrocyte; local anesthetics; membrane blebbing; toxicity