QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Raucher, D. Articles by Sheetz, M. P. Search for Related Content PubMed PubMed Citation Articles by Raucher, D. Articles by Sheetz, M. P. Social Bookmarking                         What's this?

Phospholipase C activation by anesthetics decreases membrane-cytoskeleton adhesion

¹ Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA
² Department of Biological Sciences, Box 2408, Columbia University, 1212 Amsterdam Ave., New York, NY 10027, USA

View larger version (48K): [in a new window]   Fig. 1. Tether force measurements. (a) DIC image of a tether force measurement using optical tweezers. Polystyrene beads (1 µm diameter) coated with IgG were attached to the plasma membrane of NIH-3T3 fibroblasts using laser optical tweezers (left panel) and a membrane tether was formed by pulling out the bead (right panel). At a constant length, tether force (Ftether) is pulling the bead back towards the cell opposing the force of the trap (Ftrap). (b) Schematic view of the optical tweezers force measurement that defines the local adhesion energy term. At a constant length, the force on the bead was measured by its displacement (r) from the center of the laser trap. (c) Typical displacement trace (r), showing how far the center of the bead has been moved away from the center of the optical tweezers. This tether force is a measure of the apparent membrane tension or the energy required to move membrane from the plasma membrane into the tether.

View larger version (42K): [in a new window]   Fig. 2. Effects of different local anesthetics on membrane tension and the endocytosis rate. (a) Chemical structure of met-chlorpromazine, a membrane-impermeable local anesthetic, which intercalates mainly into the lipid in the exterior half of the bilayer and expands that layer relative to the cytoplasmic half, and chlorpromazine, its membrane-permeable analog, which expands the cytoplasmic half. (b) Tether force (left panel) and endocytosis rate, measured using fluorescence (right panel), in the presence of 30 µM chlorpromazine or met-chlorpromazine. (c) Tether force (left panel) and endocytosis rate (right panel) in the presence of 30 µM lidocaine or membrane-impermeable analog bromo-lidocaine. CPZ, chlorpromazine; M-CPZ, met-chlorpromazine.

View larger version (24K): [in a new window]   Fig. 3. PLC PH domain-GFP relocalization in chlorpromazine. (a) Confocal fluorescent image of membrane localization of PLC PH domain-GFP fusion protein in transfected NIH-3T3 cells. The fluorescent intensities along the white lines were plotted as line intensity histograms, shown in the right panels. Calculation of Ipm/Icyt was used to quantify the extent of membrane localization. (b) Localization of PLC PH domain-GFP (left panel) and fluorescence intensity changes (right panel) after incubation with 30 µM chlorpromazine.

View larger version (14K): [in a new window]   Fig. 4. Time course of PIP2 relocalization and membrane tension in the presence of local anesthetics. (a) Fluorescence ratios calculated from line intensity plots (see legend to Fig. 3) were plotted against time. Chlorpromazine or met-chlorpromazine was added at 0 seconds, and images were taken at 5 minute intervals. (b) Tether force was measured as described in Fig. 1 and plotted against time. Chlorpromazine or met-chlorpromazine was added at 0 seconds, and measurements were made at 5 minute intervals. CPZ, chlorpromazine; M-CPZ, met-chlorpromazine.

View larger version (22K): [in a new window]   Fig. 5. Phospholipase C inhibitor, U73122, blocked the chlorpromazine (CPZ)-induced reduction in tether force. Incubation of cells with 30 µM chlorpromazine induces a decrease in tether force. Tether force was measured 10-15 minutes after chlorpromazine addition. Chlorpromazine-induced decrease in adhesion energy is likely mediated by activation of PLC, as 1 µM U73122, a PLC inhibitor, blocked the chlorpromazine-induced decrease in tether force.

View larger version (13K): [in a new window]   Fig. 6. Calcium response induced by (a) chlorpromazine (CPZ) alone and (b) chlorpromazine combined with the PLC inhibitor U73122. Fluorescence images were acquired using confocal time-lapse imaging of NIH-3T3 fibroblasts loaded with the Ca(2+)-sensitive dye fluo-3. Fluorescence intensity of each image was quantified as the sum of all pixel intensities.

View larger version (18K): [in a new window]   Fig. 7. The effect of 30 µM chlorpromazine on phospholipid metabolism, as measured by HPLC. Phosphatidylinositol-lipids in NIH-3T3 fibroblasts were radiolabeled with [3H]inositol during a 24 hour period. Incubation and lipids were extracted from control cells and cells treated with chlorpromazine. (a) Typical HPLC radio-trace of separated phosphatidylinositols from control cells. (b) HPLC trace of phosphatidylinositol-lipids separated from cells after 30 minutes exposure to 30 µM chlorpromazine. (c) Bar chart representing summary of HPLC data. Integrated intensity of the PI4 or PIP2 peak after chlorpromazine treatment was calculated for each sample, normalized to control value and plotted as relative count per minute (CPM).

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter