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
*Author for correspondence (e-mail: drazen{at}duke.edu)
Accepted July 3, 2001
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SUMMARY |
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Key words: Local anesthetic, Phospholipase C, Membrane-cytoskeleton adhesion, Calcium release
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INTRODUCTION |
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In previous studies, an increase in endocytosis rate correlates with a decrease in the force on membrane tethers. Tethers can be formed by pulling on membrane-attached beads with laser tweezers and the displacement of the beads in the laser tweezers gives a readout of the tether force. The tether force has components of both cytoskeleton-membrane adhesion and a tension in the membrane but several different observations indicate that the cytoskeleton-membrane adhesion accounts for 60-90% of the tether force. Recent findings suggest that cytoskeleton-membrane adhesion can be regulated by plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2) (Raucher et al., 2000). A pleckstrin homology (PH) domain of phospholipase C (PLC) fused with green fluorescent protein (GFP), which bound tightly to PIP2, and a PIP2 5' phosphatase, which hydrolyzed PIP2, both decreased membrane-cytoskeleton adhesion and tether force dramatically. It is possible that the mechanism of amphiphilic compound action could be through altering the activity of one or more of the enzymes involved in PIP2 metabolism, particularly as those enzymes are active at the surface of the plasma membrane. One criterion for the compound acting in this way is that the compounds should act at the cytoplasmic surface of the plasma membrane, as that is where the PIP2 metabolism is controlled. Local anesthetics, as other amphiphilic molecules, interact with the membrane by inserting into the bilayer-water interface and causing alterations of membrane properties such as curvature and resistance to hypotonic lysis (Roth and Seeman, 1971; Seeman, 1972). Most local anesthetics at physiological pH are present in both the cationic (charged) and the base (uncharged) forms. Anionic drugs preferentially intercalate mainly into the lipid in the exterior half of the bilayer, expand that layer relative to the cytoplasmic half, and thereby induce the cell to crenate, whereas permeable cationic drugs do the opposite and cause erythrocytes to form cup-shapes (Sheetz and Singer, 1974). Impermeable amphiphilic drugs intercalate only into the exterior half of the bilayer, and therefore cause crenation. Pairs of permeable and impermeable analogs of anesthetics have been used to probe the inner versus outer surfaces of plasma membranes, respectively (Sheetz and Singer, 1974). Thus, if the site of action of the anesthetics on endocytosis rate and membrane-cytoskeleton adhesion is at the plasma membrane, an impermeable cationic anesthetic may not be effective.
The antipsychotic amine, chlorpromazine, which has local anesthetic effects, increases the turnover of phosphoinositides and elevates the steady-state level of phosphatidylinositol-4-phosphate (PIP) in human platelets (Frolich et al., 1992). However, biochemical mechanisms underlying this increase are poorly understood. Another effect of local anesthetics is to elevate cytoplasmic Ca2+ levels (Jaimovich and Rojas, 1994). Cytoplasmic Ca2+ elevation has been linked to the release of inositol 1,4,5 trisphosphate (IP3) by the action of PLC on PIP2. It is possible that the local anesthetics activate PLC and thereby increase both lipid turnover and cytoplasmic Ca2+. To address this question we designed experiments to examine the effects of permeable and impermeable anesthetics on the PIP2 hydrolyzing enzyme, PLC. Our results indicate that PLC stimulation may be an important factor in the modulation of PIP2 concentration and cytoskeleton-membrane adhesion energy by anesthetics. These findings indicate that PLC in the plasma membrane is particularly sensitive to the effects of amphiphilic compounds.
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MATERIALS AND METHODS |
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Endocytosis rate measurement
Endocytosis was determined by FACS® analysis. Cells were incubated for 10 minutes with 5 mg/ml fluorescein-dextran (average molecular weight 4000 kDa), gently rinsed and fixed for 5 minutes in 2% formaldehyde. The data are expressed as the relative fluorescence index (RFI), relative to control cells, and represent the mean of triplicate determinations±s.d. (for at least 10,000 cells in each measurement).
Transfection of cells for confocal microscopy
PH domain of PLC was fused to the NH2 terminus of the GFP protein and this construct was transiently expressed in NIH-3T3 cells as previously described (Raucher et al., 2000).
Cells expressing PH-PLCGFP were visualized by confocal microscopy before and 10-15 minutes after incubation with local anesthetics. The extent of PH-PLC
GFP membrane localization was calculated from Ipm/Icyt, where Ipm is fluorescence intensity of the plasma membrane and Icyt is fluorescence intensity of the cytosol.
Labeling of PI lipids with [3H]inositol and analysis of inositol phosphates
NIH-3T3 cells were plated at 5-10% confluency in DMEM without inositol and 10% dialyzed FBS. [3H]inositol was added at a final concentration of 10 µCi/ml. The cells were allowed to grow for 3 days at 37°C and 5% CO2 before transfection. Cells were harvested 10-15 minutes after incubation with local anesthetics, and cellular lipids were extracted and deacylated (Guo et al., 1999). Briefly, cells were treated with 0.5 N HCl, harvested and their lipids isolated using a chloroform/methanol extraction. Lipids were treated with methamine as described and the resulting glycerophosphoinositols (gPIs) were stored at 80°C until use. The gPIs were separated by HPLC using conditions previously described (Guo et al., 1999) and the radioactivity of individual derivatives that co-eluted with gPI, gPI(3)P, gPI(4)P, gPI(3,4)P2 and gPI(4,5)P2 standards were quantified.
Quantification of PIP2 distribution in presence of local anesthetics
Cellular expression of a PLC domain-GFP fusion construct and analysis of confocal images gives a measure of plasma membrane PIP2 in living cells (Stauffer et al., 1998). The confocal images show that at low levels of expression the PLC
-PH domain-GFP fusion protein is localized to the plasma membrane in transfected NIH-3T3 cells. The fluorescent intensities along the selected cross-sections of the cells were plotted as line intensity histograms. Dividing Icyt by Ipm yields a ratio that can be used as an index of membrane localization. Therefore, the ratio Ipm/Icyt was used to quantify plasma membrane binding before and after treatment with chlorpromazine.
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RESULTS |
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Visualization of PIP2 distribution by PH domain in the presence of local anesthetics
One possible mechanism to decrease membrane-cytoskeleton adhesion is to decrease the membrane concentration of PIP2 (Raucher et al., 2000). Plasma membrane PIP2 pools can be measured by the binding of PH domains to the plasma membrane. Among the known PH domains reported to interact with PIP2, the PH domain of PLC has the highest affinity (Ferguson et al., 1995). Cellular expression of a PLC
domain-GFP fusion construct and analysis of confocal images gives a measure of plasma membrane PIP2 in living cells (Stauffer et al., 1998). The confocal image in Fig. 3a (left panel) shows that at low levels of expression the PLC
PH domain-GFP fusion protein is localized to the plasma membrane in transfected NIH-3T3 cells. Treatment of cells with chlorpromazine causes translocation of PLC
PH domain-GFP fusion protein from the plasma membrane to the cytosol, as indicated by an increase in the amount of fluorescence in the cytosol (Fig. 3b). The redistribution of fluorescence was clearly demonstrated by comparing line intensity histograms calculated at selected cross sections of the cells. Dividing Icyt by Ipm yielded a ratio that was used as an index of plasma membrane localization (Fig. 3a,b; right panel). There was about a twofold increase in the cytosol fluorescence relative to the membrane fluorescence after chlorpromazine addition (Fig. 3b).
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DISCUSSION |
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Previous studies have shown that local anesthetics have a dramatic effect on phosphoinositide metabolism in human platelets. Frolich et al. (Frolich et al., 1992) demonstrated that chlorpromazine increased the level of PIP and PIP2 in platelets pre-labeled with [32P]Pi, but the biochemical mechanism underlying this increase was poorly understood. In the present study, we have confirmed those findings in NIH-3T3 fibroblast cell line, i.e. chlorpromazine induced an almost twofold increase in PIP and a 20% increase in PIP2 concentration. Although chlorpromazine induced an increase in the total pool of PIP2, simultaneously it induced a reduction in the plasma membrane pool of PIP2, as observed by relocalization of the PIP2-specific PLC-PH domain. The time course of the chlorpromazine effects argues against the explanation that chlorpromazine competes with PH domains for PIP2 binding sites in the plasma membrane or directly binds to PH domains, thereby inhibiting their binding to PIP2. Direct binding events should occur within seconds, whereas it took 5-10 minutes for chlorpromazines effect, which is indicative of an effect on lipid metabolism. Phospholipase C is an interesting candidate enzyme as it can directly hydrolyze PIP2, thereby reducing the concentration in the plasma membrane, and it is activated in a number of signaling pathways. We find that the reduction in cytoskeleton-membrane adhesion, caused by chlorpromazine, is prevented by U73122, an inhibitor of PLC. Furthermore, chlorpromazine induces Ca2+ release from internal stores (Fig. 6), which is completely blocked by U73122. These results indicate that IP3 is released by PLC cleavage of PIP2 in the plasma membrane to cause Ca2+ release as well as a decrease in cytoskeleton-membrane adhesion energy. Previous studies (Goodman et al., 1996) have shown an increase in PLC activity by local anesthetics that modify the membrane bilayer surface. Similarly, it has also been shown that local anesthetics potentiate fMLP-stimulated PLC activity in human promyelocytic leukemic HL-60 cells (Tan et al., 1999). We favor the hypothesis that plasma membrane PIP2 is actually decreased by amphiphilic amine-dependent activation of PLC.
It is well documented that local anesthetics are capable of changing the activity of lipid-modifying membrane enzymes (Goodman et al., 1996; Tan et al., 1999). In addition, local anesthetics may affect the membrane-bound lipase activities by their direct interaction with the protein, through their effect on the physical state of the lipid micro-environment of the lipase or by a combination of those effects. For example, the rate of PIP2 hydrolysis by PLC depends exponentially on the lipid monolayer surface pressure (Rebecchi et al., 1992). Thus, the physical chemical changes at the membrane surface that are expected from chlorpromazine binding could cause the observed changes in lipid metabolism.
The changes in lipid metabolism may be responsible for the observed changes in tether force. Several recent studies have shown that the tether force provides a rapid and reliable measure of membrane-cytoskeleton adhesion (Togo et al., 2000; Dai and Sheetz, 1999; Raucher et al., 2000), and a decrease in PIP2 level in the plasma membrane is accompanied by a decrease in tether force and a marked decrease in cytoskeleton-membrane adhesion (Raucher et al., 2000). The overall interaction between the membrane and the cytoskeleton appears to be complex, given that many cytoskeletal proteins that bind to integral membrane proteins as well as to membrane phospholipids have been identified. Decreases in cytoskeleton-membrane adhesion correlate with decreases in membrane binding of GFP-PH domains, which bind PIP2 with high affinity and specificity.
Actin filament assembly is dependent on the level of PIP2 and some of the effects of anesthetics may be the result of decreased actin filament density. For example, Rabinovitch and DeStefano (Rabinovitch and DeStefano, 1975) have shown that lidocaine and chlorpromazine affect the cytoskeleton; they also induce cell rounding and inhibit motility in cultivated macrophages. Similar effects of local anesthetics are observed on BALB/3T3 cells (Nicolson et al., 1976). High concentrations of the anesthetics cause cell contraction, rounding and bleb formation, which is similar to overexpressed MARCKS (myristolylated alanine-rich C kinase substrate) mutant protein (which sequesters acidic phospholipids including PIP2) (Myat et al., 1997). Furthermore, the selective reduction of PIP2 level in plasma membrane causes NIH-3T3 fibroblasts to become round, lose their substrate attachments and form membrane blebs (Raucher et al., 2000). The tether force in blebs is very small, consistent with the absence of a membrane-cytoskeleton adhesion term (Keller and Eggli, 1998; Dai and Sheetz, 1999) and the appearance of the blebs after longer times or with higher concentrations of chlorpromazine indicates loss of cytoskeleton-membrane adhesion. Thus, the mechanism of local anesthetic action in disrupting cytoskeletal systems may be PIP2 mediated by causing stimulation of phosphatidyl inositol turnover and a decrease in plasma membrane PIP2.
An additional factor that may alter the endocytosis rate is the effect of bilayer couple asymmetry (Sheetz and Singer, 1974). In recent studies Farge et al. (Farge et al., 1999) tested the possibility that the phospholipid bilayer itself could generate the budding force for endocytosis in living cells. When asymmetry was generated by specific translocation to the inner layer by an endogenous flippase, bulk flow internalization was increased as the inner layer area was relatively increased. Similarly, Zha et al. (Zha et al., 1998) have found that hydrolysis of sphingomyelin in the outer half of the plasma membrane by sphingomyelinase treatment causes inward curvature of the plasma membrane and induces ATP-independent endocytosis. This is in qualitative agreement with the model, suggesting that an asymmetric expansion of the plasma membrane cytoplasmic surface by chlorpromazine or lidocaine may contribute a driving force for endocytosis; however, the drop in membrane-cytoskeleton adhesion is not explained. Thus, we suggest that the major factor causing the increase in endocytosis rate is the drop in membrane-cytoskeleton adhesion.
Several important cellular activities, including endocytosis, membrane extension and membrane resealing rates are controlled by membrane-cytoskeleton adhesion (Sheetz, 2001). Because of its importance, cells normally control the level of membrane-cytoskeleton adhesion quite tightly (the standard variance in the measurements of tether force is small) and the level varies in characteristic ways during mitosis, during stimulated secretion and in response to hormones or other treatments (Raucher and Sheetz, 1999; Dai et al., 1997; Raucher et al., 2000; Togo et al., 2000). Levels of phosphorylated inositol lipids play a major role in cytoskeleton assembly and dynamics as well as the adhesion between the membrane and the cytoskeleton. Many of the changes must occur at the cytoplasmic surface of the plasma membrane and it is expected that agents that alter that surface can have profound effects on cell functions through changes in the activities of lipid-modifying enzymes.
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