Korean J Physiol Pharmacol.  2012 Dec;16(6):413-422. 10.4196/kjpp.2012.16.6.413.

The Effect of Lidocaine.HCl on the Fluidity of Native and Model Membrane Lipid Bilayers

Affiliations
  • 1Department of Dental Pharmacology and Biophysics, College of Pharmacy, Chung-Ang University, Seoul 156-756, Korea. iyun@pusan.ac.kr, jho9612@pusan.ac.kr
  • 2Department of Oral and Maxillofacial Surgery and Clinical Pharmacology, College of Pharmacy, Chung-Ang University, Seoul 156-756, Korea.
  • 3Department of Pharmacology, College of Pharmacy, Chung-Ang University, Seoul 156-756, Korea.
  • 4Department of Oral Physiology and Molecular Biology, School of Dentistry and Research Institute for Oral Biotechnology, Yangsan Campus of Pusan National University, Yangsan 626-870, Korea. mkbae@pusan.ac.kr

Abstract

The purpose of this study is to investigated the mechanism of pharmacological action of local anesthetic and provide the basic information about the development of new effective local anesthetics. Fluorescent probe techniques were used to evaluate the effect of lidocaine.HCl on the physical properties (transbilayer asymmetric lateral and rotational mobility, annular lipid fluidity and protein distribution) of synaptosomal plasma membrane vesicles (SPMV) isolated from bovine cerebral cortex, and liposomes of total lipids (SPMVTL) and phospholipids (SPMVPL) extracted from the SPMV. An experimental procedure was used based on selective quenching of 1,3-di(1-pyrenyl)propane (Py-3-Py) and 1,6-diphenyl-1,3,5-hexatriene (DPH) by trinitrophenyl groups, and radiationless energy transfer from the tryptophans of membrane proteins to Py-3-Py. Lidocaine.HCl increased the bulk lateral and rotational mobility of neuronal and model membrane lipid bilayes, and had a greater fluidizing effect on the inner monolayer than the outer monolayer. Lidocaine.HCl increased annular lipid fluidity in SPMV lipid bilayers. It also caused membrane proteins to cluster. The most important finding of this study is that there is far greater increase in annular lipid fluidity than that in lateral and rotational mobilities by lidocaine.HCl. Lidocaine.HCl alters the stereo or dynamics of the proteins in the lipid bilayers by combining with lipids, especially with the annular lipids. In conclusion, the present data suggest that lidocaine, in addition to its direct interaction with proteins, concurrently interacts with membrane lipids, fluidizing the membrane, and thus inducing conformational changes of proteins known to be intimately associated with membrane lipid.

Keyword

Annular lipid fluidity; Lidocaine.HCl; Membrane protein clustering; Neuronal and model membranes; Transbilayer lateral and rotational mobility

MeSH Terms

Anesthetics, Local
Cell Membrane
Cerebral Cortex
Diphenylhexatriene
Energy Transfer
Lidocaine
Lipid Bilayers
Liposomes
Membrane Lipids
Membrane Proteins
Membranes
Neurons
Phospholipids
Proteins
Tryptophan
Anesthetics, Local
Diphenylhexatriene
Lidocaine
Lipid Bilayers
Liposomes
Membrane Lipids
Membrane Proteins
Phospholipids
Proteins
Tryptophan

Figure

  • Fig. 1 The effect of lidocaine·HCl on excimer to monomer fluorescence intensity ratio (I'/I) of Py-3-Py in SPMV (A), SPMVTL (B) and SPMVPL (C). The excitation wavelength of Py-3-Py was 330 nm and the I'/I values were calculated from the 480 nm to 379 nm signal ratio. SPMV was treated±4mM TNBS, pH 8.5, at 4℃ for 80 min. SPMVTL and SPMVPL were treated±0.5 mM TNBS, pH 8.5, at 4℃ for 20 min. Py-3-Py was incorporated into SPMV, SPMVTL and SPMVPL and fluorescence measurements were performed at 37℃ (pH 7.4). Untreated (inner and outer monolayers, ▪); TNBS treated (inner monolayer, ▴); calculated for outer monolayer (•) by eq. 3 as described in Materials and Methods. Each point represents the mean±SEM of 5 determinations. An asterisk and double asterisks signify p<0.05 and p<0.01, respectively, compared to control by Student's t-test.

  • Fig. 2 The effect of lidocaine·HCl on annular lipid fluidity in SPMV. Py-3-Py was excited through RET from tryptophan (excitation wavelength, 286 nm) and the excimer to monomer fluorescence intensity ratio (I'/I) was calculated from the 480 nm to 379 nm signal ratio. Fluorescence measurements were performed at 37℃ (pH 7.4). Each point represents the mean±SEM of 5 determinations. An asterisk and double asterisks signify p<0.05 and p<0.01, respectively, compared to control by Student's t-test.

  • Fig. 3 The effect of lidocaine·HCl on protein distribution in SPMV. Efficiency of RET from tryptophan to Py-3-Py was taken as a measure of protein clustering and calculated by eq. 4. Fluorescence measurements were performed at 37℃ (pH 7.4). Each point represents the mean±SEM of 5 determinations. An asterisk and double asterisks signify p<0.05 and p<0.01, respectively, compared to control by Student's t-test.

  • Fig. 4 The effect of lidocaine·HCl on the anisotropy (r) of DPH in SPMV (A), SPMVTL (B) and SPMVPL (C). The excitation wavelength for DPH was 362 nm and fluorescence emission was read at 424 nm. SPMV was treated±2 mM TNBS, pH 8.5, at 4℃ for 40 min. SPMVTL and SPMVPL were treated±0.5 mM TNBS, pH 8.5, at 4℃ for 20 min. DPH was incorporated into SPMV, SPMVTL and SPMVPL and fluorescence measurements were performed at 37℃ (pH 7.4). Untreated (inner and outer monolayers, ▪); TNBS treated (inner monolayer, ▴); calculated for outer monolayer (•) by eq. 7 as described in Materials and Methods. Each point represents the mean±SEM of 5 determinations. An asterisk and double asterisks signify p<0.05 and p<0.01, respectively, compared to control by Student's t-test.


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