- Email: [email protected]
Reversible effect of lidocaine on raft formation
International Congress Series 1283 (2005) 281 – 282
www.ics-elsevier.com
Reversible effect of lidocaine on raft formation Kotoe Kamata a,b, Sumie Manno b, Yuichi Takakuwa b, Makoto Ozaki a,* a
Department of Anesthesiology, Tokyo Women’s Medical University, Tokyo, Japan b Department of Biochemistry, Tokyo Women’s Medical University, Tokyo, Japan
Abstract. Lipid rafts have been implicated in the signaling processes. Anesthesia has been postulated to represent a transient, drug-induced interruption of signal transduction. In examining the effects of lidocaine on lipid rafts, we found that the agent abolished raft formation in a reversible manner in an erythrocyte membrane model. Our results suggested that raft-related signal transduction is involved in the anesthetic mechanism. D 2005 Elsevier B.V. All rights reserved. Keywords: Erythrocyte; Raft; Lidocaine hydrochloride; Flotillin-1
1. Introduction The lipid raft, a kind of detergent-resistant microdomain enriched with cholesterol and sphingolipids, is biochemically isolated by its property of insolubility in cold nonionic detergents. Earlier reports have proposed that the lipid rafts may play various roles in cellular processes such as signal transduction and synaptic transmission. Lidocaine hydrochloride, a local anesthetic now in wide clinical use, is characterized by its rapid onset and short duration as an amphipathic agent with disarrhythmic effects, sedative effects, and activity as a neural blockade elicited via the suppression of sodium channel conductance. Although many theories have been discussed, there is no consensus on the anesthetic mechanism. In this study, we focused on the annular transition theory of protein–lipid interactions by examining the effects of lidocaine on the raft formation in an erythrocyte membrane model. 2. Materials and methods Human erythrocytes were treated with lidocaine at concentrations of up to 18.4 mM for 20 min at 37 8C and washed with Tris-buffered saline (TBS) with or without 3% bovine * Corresponding author. Tel./fax: +81 3 5269 7336. E-mail address: [email protected] (M. Ozaki). 0531-5131/ D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2005.06.077
282
K. Kamata et al. / International Congress Series 1283 (2005) 281–282
Fig. 1. (A) Flotillin-1 distribution. (B) Cholesterol/protein ratio.
serum albumin (BSA). Erythrocyte ghosts were prepared by hypotonic lysis and incubated on ice in TBS containing 1% Triton X-100, followed by ultracentrifugation in sucrose density gradient. Six fractions were collected as equal volumes from top to bottom. Each fraction was subjected to immunoblot and probed with anti-flotillin-1 antibody as a raft marker protein. Cholesterol and protein estimation were carried out on chloroform– methanol extracts of aliquots of sucrose density gradient fractions. Morphological change and osmotic fragility were also measured. 3. Results Lipid rafts were collected in fractions 3 and 4, the fractions which were reacted to anti-flotillin-1 antibody (see Fig. 1A) and high cholesterol/protein ratio (see Fig. 1B). Following lidocaine treatment depleted cholesterol content and abolished flotillin-1 in these fractions, but flotillin-1 was recovered after removing lidocaine from the erythrocyte membranes by washing with TBS containing BSA. Methyl-h-cyclodextrin (MBCD) depleted cholesterol in the erythrocyte membrane by about 30%, whereas lidocaine left the total cholesterol content unchanged (see Table 1).
4. Discussion Lidocaine prevented the raft formation in the erythrocyte membrane in which cholesterol content is maintained. Its removal from the membrane restores raft formation. This reversible lidocaine effect should be involved in its effects on raft-related signal transduction in various cell membranes.
Table 1 Cholesterol contents
Control Lidocaine Lidocaine/BSA MBCD
Depleted cholesterol (Ag/100 Al RBC)
Estimated remained-cholesterol (Ag/100 Al RBC)
Cholesterol depletion rate (%)
3.3 4.7 0 32.3
95.3 95.7 92.0 69.7
3.3 4.7 0 31.7
www.ics-elsevier.com
Reversible effect of lidocaine on raft formation Kotoe Kamata a,b, Sumie Manno b, Yuichi Takakuwa b, Makoto Ozaki a,* a
Department of Anesthesiology, Tokyo Women’s Medical University, Tokyo, Japan b Department of Biochemistry, Tokyo Women’s Medical University, Tokyo, Japan
Abstract. Lipid rafts have been implicated in the signaling processes. Anesthesia has been postulated to represent a transient, drug-induced interruption of signal transduction. In examining the effects of lidocaine on lipid rafts, we found that the agent abolished raft formation in a reversible manner in an erythrocyte membrane model. Our results suggested that raft-related signal transduction is involved in the anesthetic mechanism. D 2005 Elsevier B.V. All rights reserved. Keywords: Erythrocyte; Raft; Lidocaine hydrochloride; Flotillin-1
1. Introduction The lipid raft, a kind of detergent-resistant microdomain enriched with cholesterol and sphingolipids, is biochemically isolated by its property of insolubility in cold nonionic detergents. Earlier reports have proposed that the lipid rafts may play various roles in cellular processes such as signal transduction and synaptic transmission. Lidocaine hydrochloride, a local anesthetic now in wide clinical use, is characterized by its rapid onset and short duration as an amphipathic agent with disarrhythmic effects, sedative effects, and activity as a neural blockade elicited via the suppression of sodium channel conductance. Although many theories have been discussed, there is no consensus on the anesthetic mechanism. In this study, we focused on the annular transition theory of protein–lipid interactions by examining the effects of lidocaine on the raft formation in an erythrocyte membrane model. 2. Materials and methods Human erythrocytes were treated with lidocaine at concentrations of up to 18.4 mM for 20 min at 37 8C and washed with Tris-buffered saline (TBS) with or without 3% bovine * Corresponding author. Tel./fax: +81 3 5269 7336. E-mail address: [email protected] (M. Ozaki). 0531-5131/ D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2005.06.077
282
K. Kamata et al. / International Congress Series 1283 (2005) 281–282
Fig. 1. (A) Flotillin-1 distribution. (B) Cholesterol/protein ratio.
serum albumin (BSA). Erythrocyte ghosts were prepared by hypotonic lysis and incubated on ice in TBS containing 1% Triton X-100, followed by ultracentrifugation in sucrose density gradient. Six fractions were collected as equal volumes from top to bottom. Each fraction was subjected to immunoblot and probed with anti-flotillin-1 antibody as a raft marker protein. Cholesterol and protein estimation were carried out on chloroform– methanol extracts of aliquots of sucrose density gradient fractions. Morphological change and osmotic fragility were also measured. 3. Results Lipid rafts were collected in fractions 3 and 4, the fractions which were reacted to anti-flotillin-1 antibody (see Fig. 1A) and high cholesterol/protein ratio (see Fig. 1B). Following lidocaine treatment depleted cholesterol content and abolished flotillin-1 in these fractions, but flotillin-1 was recovered after removing lidocaine from the erythrocyte membranes by washing with TBS containing BSA. Methyl-h-cyclodextrin (MBCD) depleted cholesterol in the erythrocyte membrane by about 30%, whereas lidocaine left the total cholesterol content unchanged (see Table 1).
4. Discussion Lidocaine prevented the raft formation in the erythrocyte membrane in which cholesterol content is maintained. Its removal from the membrane restores raft formation. This reversible lidocaine effect should be involved in its effects on raft-related signal transduction in various cell membranes.
Table 1 Cholesterol contents
Control Lidocaine Lidocaine/BSA MBCD
Depleted cholesterol (Ag/100 Al RBC)
Estimated remained-cholesterol (Ag/100 Al RBC)
Cholesterol depletion rate (%)
3.3 4.7 0 32.3
95.3 95.7 92.0 69.7
3.3 4.7 0 31.7