Proc. Nat. Acad. Sci. USA Vol. 72, No. 11, pp. 4430-4434, November 1975
Cell Biology
Local anesthetics affect transmembrane cytoskeletal control of mobility and distribution of cell surface receptors (lectins/concanavalin A/immunoglobulins/microtubules/microfilaments)
GEORGE POSTE*, DIMITRI PAPAHADJOPOULOS*, AND GARTH L. NICOLSONt * Department of Experimental Pathology, Roswell Park Memorial Institute, Buffalo, New York 14263; t Department of Cancer Biology, The Salk Institute for Biological Studies, San Diego, California 92112; and t Department of Developmental and Cell Biology, The University of California, Irvine, Irvine, Calif. 92664
Communicated by Harden M. McConnell, July 17,1975
ABSTRACT Tertiary amine local anesthetics facilitated concanavalin A-induced redistribution of lectin receptors on murine BALB/3T3 cells and enhanced the susceptibility of these cells to agglutination by concanavalin A. In contrast, these drugs at similar concentrations inhibited ligand-induced capping of immunoglobulin receptors on mouse lymphocytes. We propose that these differing effects of local anesthetics on membrane receptor mobility in fibroblasts and lymphocytes result from the action of anesthetics on membrane-associated microtubules and microfilaments involved in the transmembrane control of receptor mobility. We present electron microscopic evidence of structural alterations in microtubule and microfilament organization in anesthetic-treated cells, together with data on changes in the responsiveness of anesthetic-treated cells to drugs that act on microtubules and/or microfilaments. This evidence supports the proposal that anesthetics affect the organization of cytoskeletal components or their plasma membrane attachment points. The effects of local anesthetics on ligand-induced redistribution of membrane receptors in both 3T3 cells and lymphocytes can be duplicated by treating cells with colchicine (or Vinca alkaloids) together with cytochalasin B. We propose that the participation of membrane-associated microtubules and microfilaments in the transmembrane control of receptor mobility is such that microtubules and microfilaments play opposing roles in regulating the mobility and topograpy of cell surface receptors. Binding of multivalent ligands such as lectins or antibodies to the cell surface is known to be accompanied by changes in the topographical distribution of the appropriate receptors (1, 2). Ligand-induced changes in receptor distribution offer a potential mechanism whereby events occurring on the outer surface of the plasma membrane could be translated to the cytoplasmic side and this might provide a structural basis for transducing information between the external environment and the intracellular metabolic machinery in such phenomena as mitogenesis, cell recognition and communication, and cellular immune responses. The mobility and distribution of certain lectin and immunoglobulin receptors on the surfaces of mammalian cells may be under transmembrane control by cytoplasmic structural elements associated with the plasma membrane that are sensitive to microtubule (MT) disruptive drugs such as colchicine (Ch) and the Vinca alkaloids (2-8). These membrane-associated proteins may act as "anchors" to restrict receptor mobility. Dislocation of the linkage between surface receptors and the Ch-sensitive structures allows increased receptor mobility and facilitates receptor redistribution by external multivalent ligands (2-8). The mobility and distribution of lectin and immunoglobulin receptors is also influenced by cytochalasin B (CB), a drug that disrupts microAbbreviations: CB, cytochalasin B; Ch, colchicine; Con A, concanavalin A; MF, microfilaments; MT, microtubules.
filaments (MF) (2, 9-11). Tertiary amine local anesthetics have recently been shown to alter ligand-induced redistribution of surface receptors, causing enhancement of lectin-induced redistribution of concanavalin A (Con A) -receptors on 3T3 cells (8, 12), but inhibition of antibody-induced capping of immunoglobulin receptors on lymphocytes (13). We report here further observations on the effect of local anesthetics on the mobility and distribution of surface receptors which provide an explanation for the apparently conflicting action of these drugs on receptor mobility in 3T3 cells and lymphocytes. Our evidence indicates that local anesthetics affect the activity of both Ch- and CB-sensitive membrane-associated elements involved in the transmembrane control of receptor mobility and topography.
MATERIALS AND METHODS Chemicals. Dibucaine HC1, tetracaine HC1, and procaine HC1 were obtained from Mann Research Laboratories (Orangeburg, N.Y.); lidocaine HC1 from Astra Pharmaceuticals (Worcester, Mass.); mepivacaine from Sterling-Winthrop Research (Rensselaer, N.Y.); dimethylsulfoxide, glutaraldehyde, and colchicine from Sigma (St. Louis, Mo.); cytochalasin B from the Aldrich Chemical Co. (Milwaukee, Wisc.); vinblastine sulfate from Eli Lilly and Co. (Indianapolis, Ind.), and fluorescein isothiocyanate from BLL Division of Bioquest (Cockeysville, Md.). [3H]Colchicine (specific activity 5 mCi/mmol) was purchased from New England Nuclear of Boston, Mass. Cells. Mouse BALB/c 3T3 and their simian virus 40 (SV40) transformants (SV3T3) cells were grown in Dulbecco-modified Eagle's medium with 10 calf serum (12, 14). Spleen cells were harvested from 12-week-old BALB/c mice and purified on a Ficoll-Hypaque gradient (15) and maintained in RPMI 1640 medium supplemented with 0.5% bovine serum albumin. Lectins. Con A was purchased as a twice-crystallized preparation (Miles; Elkhart, Ind.) and purified further by affinity chromatography (16). [3H]Con A (specific activity 3.8 X 106 cpm/mg) was prepared from affinity-purified Con A (16) and specific binding of [3H]Con A to cells measured at 0° (16). Fluorescein-conjugated Con A and fluorescein-conjugated rabbit anti-mouse immunoglobulin (F1-RAMG) were prepared (16, 17) and cell agglutination by Con A was measured (14, 18). Labeling of Cell Surface Receptors in Mouse Lymphocytes and Effect of Drugs on Receptor Distribution. Suspension cultures of mouse splenic lymphocytes (1 X 107 cells per ml) were incubated in 1.0 ml of medium for 15 min at 370 with or without drugs, and then in 50 ,Ag/ml of FlRAMG. The cells were then centrifuged onto glass coverslips 4430
Proc. Nat. Acad. Sci. USA 72 (1975)
Cell Biology: Poste et al. cytocentrifuge (Shandon Instruments; Camberley, England), fixed with 2.5% glutaraldehyde for 15 min at '20°, and the attached cells examined for specific immunofluorescence (16, 17). At least 100 stained nonaggregated cells at lOOX magnification were examined for capping of immunoglobulin receptors (detectable over no more than one half of each cell) irrespective of whether the label was on the surface or had been interiorized by endocytosis. To assess the reversibility of drug-induced changes in immunoglobulin receptor distribution, we incubated similar lymphocyte populations in drug-containing medium and then washed twice with pre-warmed phosphate-buffered saline before incubation with fluorescein-conjugated rabbit anti-mouse immunoglobulin in fresh drug-free medium. Electron Microscopy. BALB/ST3 cells were grown to confluence and then fresh medium (370) containing dibucaine (0.2 mM) or tetracaine (0.6 mM) was added. Cells were incubated for 15-30 min at 370, fixed in glutaraldehyde (final concentration 0.5%) for 15 min at 370, washed in phosphate-buffered saline and post-fixed in buffered 1% osmium tetroxide for 1 hr at 200. The fixed cells were carefully removed from the substrate and oriented onto soft agar pellets by centrifugation. The agar pellets were dehydrated, stained in block with 1% uranyl acetate, and embedded in Spurr resin (Electron Microscopy Sciences, Warren, Pa.). in
Table 1. The effect of colchicine and Vinca alkaloids on Con A-mediated agglutination of mouse 3T3 and SV3T3 cells pretreated with dibucaine
a
RESULTS
Effect of Microtubule Disruptive Drugs on Lectin-Induced Agglutination of Cells Pretreated with Local Anesthetics. Incubation of 3T3 cells with local anesthetics increases their susceptibility to agglutination by Con A so that the concentration of Con A required to produce maximum cell agglutination is reduced from 1200 ug/ml to 350 ug/ml (12). Studies in this (8) and other laboratories (6) have shown that lectin-induced agglutination of 3T3 and SV3T3 cells is inhibited by pretreating cells with MT-disruptive drugs. However, agglutination of anesthetic-treated 3T3 and SV3T3 cells by Con A was unaffected by MT-disruptive drugs (Table 1). The concentrations of MT-disruptive drugs shown in Table 1 are identical to those causing inhibition of Con A-mediated agglutination of 3T3 or SVv3T3 cells (6, 8). However, incubation of anesthetic-treated 3T3 or SV3T3 cells with Ch or Vinca alkaloids at concentrations as high as 10-4 M for 1 hr did not produce significant inhibition of cell agglutination (results not shown). If as proposed (3, 4, 8), the Ch-sensitive MT act as "anchors" to restrict the mobility of lectin receptors, then dislocation of the functional linkage between these structures and the membrane receptors would be expected to enhance receptor mobility and facilitate their redistribution by lectins. Indeed, ultrastructural evidence of enhanced lectin-induced redistribution of lectin receptors has been documented in cells treated with Ch (19) and local anesthetics (8). However, in Ch-treated cells Con A redistributed receptors into a single "cap-like" aggregate (19) whereas in anesthetic-treated cells binding of Con A resulted in multiple clusters or patches (8). These differences may explain the respective actions of Ch and local anesthetics in inhibiting and enhancing cell agglutination by Con A. Formation of caps of receptors on Ch-treated cells would reduce the probability of contact between aggregated Con A receptors on adjacent cells, a probable requirement for agglutination (17, 19). In contrast, Con A-induced redistribution of receptors on anesthetic-treated cells into multiple patches would increase the likelihood of
4431
Drug concentration Treatment
(mM)
Untreated Dibucainee Dibucaine + colchicinefg Dibucaine + vinblastinef Dibucaine +
Control 0.2 0.2 0.001 0.2
vincristinef Dibucaine + colchicine + vinblastinef
0.001 0.2 0.01 0.2 0.001 0.001
Cell agglutinations by Con Ab SV3T3d 3T3C 0 ++++
++++ ++++
++++
++++
++++
++++
++++
++++
++++
++++
Mean values derived from three separate experiments. Agglutination by the indicated concentration of Con A was measured as described in Materials and Methods and scored as 0, +, ++, +++, or ++++ for 0, 25, 50, 75, or >90% cell agglutination, respectively. c Agglutination by Con A (350 gg/ml) at room temperature after a
b
20 min.
d Agglutination
by Con A (150 gg/ml) at room temperature after 20
min.
Cells incubated in suspension with dibucaine at 370 for 30 min in serum-free medium. f Cells incubated in suspension with dibucaine at 370 for 30 min after which the cells were harvested, washed twice in phosphatebuffered saline and returned to fresh medium supplemented with dibucaine plus the indicated drugs for 30 min at 37°. g Dibucaine did not alter cellular binding of colchicine as determined by the binding of [3H]colchicine to cell extracts [method of Wilson (50)]. e
interaction between groups of aggregated receptors on adjacent cells and thus favor agglutination (1, 8). Since cap formation involves a MF system sensitive to CB (2, 10, 11), formation of caps in Ch-treated cells indicates that the MF system is still intact. In contrast, the lack of capping of Con A receptors on anesthetic-treated 3T3 cells (8) suggests that local anesthetics are acting not only on the Ch-sensitive MT system, but also on the CB-sensitive MF system. This proposed action of local anesthetics on both MT and MF would mean that anesthetics would be expected to enhance ligandinduced receptor redistribution as a result of functional impairment of the MT "anchors," but redistribution of receptors could not progress to cap formation (due to simultaneous impairment of the MF system), and would instead result in formation of multiple small clusters and patches as re-
ported previously (8). If local anesthetics affect both MT and MF, or their attachment to plasma membranes, it should be possible to duplicate the action of local anesthetics with Ch plus CB. As shown in Table 2, incubation of 3T3 cells with Ch (or Vinca alkaloids) plus CB enhanced Con A agglutination without altering lectin binding to cells. Although the increase in Con A agglutinability with Ch plus CB was not as great as with local anesthetics alone (Table 2), when cells were incubated with Ch, CB and local anesthetics, the anesthetics did not produce an additive increase in cell agglutinability (Table 2). The small non-additive increase in agglutinability caused by anesthetics might result from their ability to increase the fluidity of phospholipids (20, 21); this should also contribute to enhanced receptor mobility within the membrane. The results in Table 2 therefore support the above proposal that
4432
Cell Biology: Poste et al.
Proc. Nat. Acad. Sci. USA 72 (1975)
Table 2. The effect of dibucaine, colchicine, Vinca alkaloids, and cytochalasin B on the agglutination of mouse 3T3 cells by Con A
,
., ..,:,.."I'm
.: t. '!
..."
.., t,,;-
4.' .. IT.i. ',
I
l -Pb %
.t.
sf;
Concentration of Con A
(jlg/ml) causing maxiTreatment
Drug concentration
Untreated Dibucaine Colchicine + cytochalasin Bb Vinblastine + cytochalasin Bb Vincristine + cytochalasin Bb Colchicine + cytochalasin B + dibucaineC Dilnethylsulfoxided
Control 0.2 mM 0.001 mM 10 gg/ml 0.01 mM 10 ,ug/ml 0.01 mM 10 jig/ml 0.001 mM 10 ,ug/ml 0.2 mM 0.1% (vol/vol)
2b
mum cell
agglutinationa 1400 350 450 475 475
350 1400
Cell agglutination (>90%) by the indicated Con A concentration after incubation at room temperature for 20 min. b Cells incubated with the indicated drugs in serum-free medium for 1 hr at 370 before assay for agglutination by Con A. c Cells incubated in serum-free medium containing colchicine and cytochalasin B for 1 hr at 370 after which dibucaine containing medium was added and the sample incubated for 30 min at 370 before assay for Con A agglutination. dDimethylsulfoxide was used as a solvent for cytochalasin B in the other experimental series.
a
local anesthetics affect both Ch-sensitive and CB-sensitive structures. Microtubules and Microfilament Organization in Cells Treated with Local Anesthetics. Further evidence that local anesthetics affect the organization of MT and MF was obtained by electron microscopic examination of anesthetictreated cells. Confluent 3T3 cells appear in cross-section as polygonal, flat, endothelialoid cells (Fig. la). At higher magnification, a dense plasma membrane-associated network of MF can be seen with numerous MT deeper in the cell cytoplasm (Fig. lb). Treatment of 3T3 cells with tetracaine resulted in a dramatic alteration in cell morphology. The normally flattened cells rounded up and contracted, leading to the formation of numerous surface "blebs" (Fig. 2a) that resembled the zeiotic protrusions produced by cytochalasin B (22) or cytochalasin D (23). The cytoplasmic distribution of cytoskeletal elements was also altered by tetracaine treatment. Bundles of MF were no longer found beneath the plasma membrane and few MT could be identified in the cytoplasm (Fig. 2b). Some filaments were still present deep in the cytoplasm and these may be responsible for the apparent contraction of some cellular organelles to the nuclear area. It was impossible to determine whether tetracaine treatment produced dissolution of cytoskeletal elements, disorganization, or both. Inhibition of Ligand-Induced Capping of Lymphocyte Membrane Receptors by Local Anesthetics. Both the formation of caps of surface receptors (2, 10, 11, 24) and the maintenance of caps (11) are inhibited by CB, suggesting the involvement of MF. If, as proposed above, local anesthetics impair the function of a CB-sensitive MF system, these drugs should alter ligand-induced capping. As shown in Table 3, local anesthetics produced significant inhibition
FIG. 1. Untreated 3T3 cells. (a) low magnification field of cells oriented parallel to the initial growth surface, X910; (b) subplasma membrane cytoskeletal organization: MF, microfilaments; MT, microtubules; X13,900; bar equals 0.2 ,m. FIG. 2. 3T3 cells treated 30 min with 0.6 mM tetracaine at 370. (a) low magnification demonstrating anesthetic-induced cell rounding and surface "blebs", X910; (b) subplasma membrane cytoskeletal organization disrupted; X13,900; bar equals 0.2 Mm.
of antibody-induced immunoglobulin capping on mouse lymphocytes. Indeed, these drugs produced significantly greater inhibition than CB (Table 3). However, incubation of lymphocytes with CB together with Ch or Vinca alkaloids caused more marked inhibition of capping (Table 3), similar to that produced by local anesthetics alone. This further suggested that anesthetics act on both Ch-sensitive and CB-sensitive structures or their membrane attachment points. These results confirm the observations of Ryan et al. (13) on inhibition of antibody-induced lymphocyte capping by local anesthetics. Further evidence that local anesthetics act on CB-sensitive structures was provided by the finding that local anesthetics caused breakdown and redistribution of preformed caps of receptors (Table 4), similar to that caused by CB (Table 4), or CB plus Ch or vinblastine (Table 4). DISCUSSION
The present results provide new information to support the concept that cytoskeletal structures associated with the plasma membrane can influence the mobility and distribution of membrane receptors (2-8, 19, 25-27). Our data indicate that local anesthetics cause impairment of both Ch-sensitive MT and CB-sensitive MF systems involved in transmembrane control of receptor mobility. In addition to ultrastructural evidence for anesthetic-induced alteration in MT and MF organization (28), the finding that the effects of local anesthetics can be duplicated by exposing cells simultaneously to both Ch and CB lends support to our proposal that anesthetics act on both MT and MF. The inhibitory action of local anesthetics on both MT and MF offers an explanation for the previously unexplained findings that these drugs enhance lectin agglutination of certain cells (8, 12), a process that requires increased receptor mobility redistribution (1, 16, 25), yet at similar concentrations they inhibit capping of antibody receptors on lymphocytes (13). These effects, as
Cell Biology: Poste et al.
Proc. Nat. Acad. Sci. USA 72 (1975)
Table 3. The effect of local anesthetics, cytochalasin B, colchicine, and vinblastine on antibody-induced capping of immunoglobin receptors on mouse spleen cellsa
Treatment
1. Untreated 2. Dibucaine 3. Tetracaine 4. Lidocaine 5. Procaine 6. Mepivacaine 7. Cytochalasin B 8. Cytochalasin B 9. Dimethylsulfoxide 10. Colchicine 11. Vinblastine 12. Cytochalasin B + colchicine 13. Cytochalasin B + vinblastine 14. Vinblastine + colchicine
Concentration
Control 0.2 mM 0.5 mM 5.0 mM 10.0 mM 10.0 mM 10 /ig/ml 20 Mg/ml 0.25% (vol/vol) 0.01 mM 0.01 mM 10 pg/ml 0.01 mM 10 pg/ml 0.01 mM 0.01 mM 0.01 mM
Table 4. Reversal of preformed caps of receptors on mouse spleen cells by local anesthetics, cytochalasin B,
colchicine,
and
vinblastinea
Incubation % time (min) Capsb 30 30 30 30 30 60 60
79 15 18 25 38 44 62 54
60 60 60
81 74 80
60
12
Incubation Immuno-
Treatment
Untreated Dibucaine Tetracaine
B +
colchicine
Cytochalasin
B +
60
87
Spleen cells were preincubated in serum-free RPMI 1640 medium with or without the various drug supplements for the indicated times and then incubated with fluorescein-conjugated rabbit anti-mouse immunoglobulin for 30 min at 370 after which the percentage of stained cells with caps of receptors was measured as described in Materials and Methods. The results represent mean values from three separate experiments for experimental series nos. 1-3 and 8-13 and from two separate experiments for series nos. 4, 5, 7, and 14. b Percentage of caps in stained immunoglobulin-positive cells. The latter represent 38-47% of the total lymphocyte population. c Dimethylsulfoxide at the indicated concentration was used as solvent for cytochalasin in experiments 7, 8, and 12-14.
duced receptor redistribution, but simultaneous inhibition of
0
19
0.5 mM
30
25
mM
,ug/ml
1.25% (vol/vol) 0.01 mM 0.01 mM 10 gg/ml 0.01 mM
10 ,ug/ml 0.01 mM
85
30
31
60
45
60
90
60
92
60
88
60
33
60
28
Capping of immunoglobulin receptors was induced by fluorescein-conjugated rabbit anti-mouse immunoglobulin as in Table 3. We then incubated cell populations in medium with or without various drug supplements for the indicated times at room temperature to reduce endocytosis of capped receptors which occurred rapidly at 370 and complicated observations on the ability of drugs to reverse the capping process.
a
well as previous findings on the effect of Ch and CB on receptor mobility (2-4, 7, 11, 19, 29, 30), can be explained if membrane receptors are linked to MT and MF systems which play opposing roles in controlling receptor distribution and mobility. As proposed by Edelman and Yahara (3-5), we suggest that Ch-sensitive MT serve as cytoskeletal elements that "anchor" receptors and limit their mobility. In addition, we envisage that a MF system is linked to and operates in opposition to MT via a contractile activity that can redistribute receptors into large caps. Evidence that MF possess actomyosin components and can function as a contractile system has been reviewed elsewhere (31, 46). In this scheme the topography of membrane receptors at any time would reflect the interplay between these two opposing systems. Functional dislocation of receptors from "anchoring" MT by such drugs as Ch or vinblastine would favor ligand-induced receptor redistribution, but so long as the receptors remained linked to MF the ligands would redistribute the aggregated receptors into caps, as found in cells exposed to Ch (19). Conversely, selective inhibition of contractile MF by CB would prevent capping of receptors, as reported in several laboratories (2, 10, 11, 24), but since receptors remain linked to MT "anchors," they would remain randomly distributed. Finally, in situations where both MT and MF are impaired, as in cells treated with local anesthetics or a combination of Ch and CB, loss of the MT "anchors" would facilitate ligand-in-
capsb
30
10
Cytochalasin
globulin
Control
10
Colchicine Vinblastine
time
(min)
0.2 mM
Cytochalasin B Dimethylsulfoxide
a
9
Concentration
Procaine
vinblastine
60
4433
b
Percentage of stained cells showing caps.
the contractile MF system would dictate that capping of receptors could not occur and ligands would instead produce multiple clusters or patches of receptors (8). The mechanism by which local anesthetics alter the functional activity of MT and MF and their interaction with membrane receptors is unknown. Our results indicate that these drugs produce structural or organizational alterations in both MT and MF. Similar ultrastructural alterations in MT in anesthetic-treated tissues have been reported (32, 33) and lidocaine inhibits in vitro polymerization of MT (33). One aspect of the pharmacological activity of local anesthetics that may be pertinent to their action on MT and MF concerns their ability to compete for Ca2+ and to displace Ca2+ bound to membranes (34, 35). Structural reorganization of membranes after anesthetic-induced modification of Ca2+ binding could alter the linkage between integral membrane receptors and a membrane-associated MT-MF system. This possibility has been discussed elsewhere (8, 12, 28, 36). The role of Ca2+ in regulating the depolymerization and polymerization of MT subunits both in vivo and in ittro (37-39) also introduces the possibility that structural breakdown of MT in anesthetic-treated cells might result from competitive inhibition by anesthetics of Ca2+-sensitive functions necessary for MT integrity (39, 40). Finally, the extreme sensitivity of MT polymerization to Ca2+ concentration, the dissolution of MT at [Ca2+] > 10-5 M, and the inverse correlation between cytoplasmic [Ca2+] and MT integrity (38, 39, 45) offers yet another mechanism by which displacement of Ca2+ from membranes by anesthetics could affect MT in-
tegrity. Displacement of membrane-bound Ca2+ might raise cytoplasmic [Ca2+] to levels sufficient to cause MT depolymerization. In this respect, it is interesting to note that experimentally-induced increase in intracellular [Ca2+] produced by exposure of 3T3 cells and mouse lymphocytes to the calcium ionophore A23187 has been found to produce effects
on
receptor mobility identical to those produced by
Ch and vinblastine (36).
4434
Cell Biology: Poste et al.
Interpretation of the diverse effects of local anesthetics on membranes has previously been considered primarily in terms of their action on membrane lipids (35, 41). The present results indicate, however, that these drugs also influence membrane organization via an interaction with membrane-associated MT and MF. The inhibitory effect of local anesthetics on the adhesion, spreading and locomotion of cells on solid substrates (42, 43), cell aggregation (44), axonal transport (45), endocytosis (46), and exocytosis (47), might now be reevaluated as possibly resulting from the action of these drugs on cytoskeletal elements rather than their single action on membrane lipids particularly since these various processes are also inhibited by drugs acting on MT and MF systems (48-50). This work was supported by NIH Grants CA-13393 (G.P.), GM18921 (D.P.); NIH-NCI Tumor Immunology Program Contract CB-33879 (G.L.N.), and NSF Grant GB-34178 (G.L.N.). We thank J. Smith, P. Newhouse, and A. MacKearnin for excellent technical assistance and A. Brodginski for help in preparing the manuscript. 1. Nicolson, G. L. (1974) Int. Rev. Cytol. 39,89-190. 2. de Petris, S. (1975) J. Cell Biol. 65, 123-146. 3. Edelman, G. M., Yahara, I. & Wang, J. L. (1973) Proc. Nat. Acad. Sci. USA 70, 1442-1446. 4. Yahara, I. & Edelman, G. M. (1973) Nature 236, 152-155. 5. Yahara, I. & Edelman, G. M. (1975) Exp. Cell Res. 91, 125142. 6. Yin, H. H., Ukena, T. H. & Berlin, R. D. (1972) Science 178, 177-179. 7. Berlin, R. D., Oliver, J. M., Ukena, T. E. & Yin, H. H. (1974) Nature 247,45-46. 8. Poste, G., Papahadjopoulos, D., Jacobson, K. & Vail, W. J. (1975) Biochim. Biophys. Acta 394,504-519. 9. Wessells, N. K., Spooner, B. S., Ash, J. F., Bradley, M. O., Luduena, M. A., Taylor, E. L., Wrenn, J. T. & Yamada, K. M. (1971) Science 171, 135-143. 10. Taylor, R. B., Duffus, W. P. H., Raff, M. C. & de Petris, S. (1971) Nature New Biol. 233,225-229. 11. de Petris, S. (1974) Nature 250, 54-56. 12. Poste, G., Papahadjopoulos, D., Jacobson, K. & Vail, W. J. (1975) Nature 253,552-554. 13. Ryan, G. B., Unanue, E. R. & Karnovsky, M. J. (1974) Nature
250,56-57. 14. Nicolson, G. L. (1973) J. Nat. Cancer Inst. 50, 1443-1451. 15. Perper, R. J., Zee, T. W. & Michelson, M. M. (1968) J. Lab.
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Proc. Nat. Acad. Sci. USA 72 (1975) 21. Papahadjopoulos, D., Jacobson, K., Poste, G. & Sheperd, G. (1975) Biochim. Biophys. Acta 394,520-59. 22. Butcher, F. & Perdue, J. (1973) J. Cell Biol. 56,857-861. 23. Godman, G; C., Miranda, A. F., Deitch, A. D. & Tanenbaum, S. W. (1975) J. Cell Biol. 64, 644-667. 24. Loor, F., Forni, L. & Pernis, B. (1974) Eur. J. Immunol. 2, 203-211. 25. Nicolson, G. L. (1975) in Concanavalin A, ed. Chowdhury, T. K. & Weiss, A. K. (Plenum Press, New York), pp. 153-172. 26. Nicolson, G. L. & Painter, R. G. (1973) J. Cell Biol. 59, 395406. 27. Ji, T. H. & Nicolson, G. L. (1974) Proc. Nat. Acad. Sci. USA 71,2212-2216. 28. Nicolson, G. L., Smith, J. & Poste, G. (1975) J. Cell Biol., in press. 29. Ryan, G. B., Borysenko, J. Z. & Karnovsky, M. J. (1974) J. Cell Biol. 62,351-365. 30. Unanue, E. R. & Karnovsky, M. J. (1974) J. Exp. Med. 140, 1207-1220. 31. Durham, A.C.H. (1974) Cell 2, 123-136. 32. Byers, M. R., Kendrickson, A. E., Fink, B. R., Kennedy, R. D. & Middaugh, M. E. (1973) J. Neuroblol. 4,125-143. 33. Haschke, R. H., Byers, M. R. & Fink, B. R. (1974) J. Neurochem. 22,837-843. 34. Papahadjopoulos, D. (1972) Biochim. Blophys. Acta 265, 169-186. 35. Seeman, P. (1974) Pharmacol. Rev. 24,583-655. 36. Poste, G. & Nicolson, G. L. (1975) Btochtm. Btophys. Acta, in press. 37. Weisenberg, R. C. (1972) Science 177, 1104-1105. 38. Kirschner, M. W. & Williams, R. C. (1974) J. Supramol. Struct. 2, 412-428. 39. Olmstead, J. B., Marcum, J. M., Johnson, K. A., Allen, C. & Borisy, G. G. (1974) J. Supramol. Struct. 2,449-450. 40. Wilson, L., Bryan, J., Ruby, A. & Mazia, D. (1970) Proc. Nat. Acad. Sct. USA 68,807-814. 41. Sheetz, M. P. & Singer, S. J. (1974) Proc. Nat. Acad. Sci. USA 71, 4457-4461. 42. Rabinovitch, M. & DeStefano, M. J. (1974) Exp. Cell Res. 88, 153-162. 43. Gail, M. H. & Boone, C. W. (1972) Exp. Cell Res. 73, 252258. 44. Ungar, G. (1975) in Progress in Anesthesiology, ed. Fink, B. R. (Raven Press, New York), Vol. 1, pp. 569-574. 45. Byers, M. R., Fink, B. R., Kish, S. J. & Aasheim, G. M. (1975) in Progress in Anesthesiology, ed. Fink, B. R. (Raven Press, New York), Vol. 1, pp. 25-38. 46. Allison, A. C. (1973) in CIBA Foundation Symposium 14 (Elsevier, Amsterdam), pp. 109-146. 47. Poste, G. & Allison, A. C. (1973) Btochtm. Btophys. Acta 300, 421-465. 48. McClure, W. 0. (1972) Adv. Pharmacol. Chemotherap. 10, 185-220. 49. Douglas, W. W. (1975) in Secretory Mechanisms of Exocrine Glands, eds. Thorn, N. A. & Peterson, 0. H. (Academic Press, New York), pp. 116-136. 50. Wilson, L. (1970) Biochemistry 9,4999-5007.
Cell Biology
Local anesthetics affect transmembrane cytoskeletal control of mobility and distribution of cell surface receptors (lectins/concanavalin A/immunoglobulins/microtubules/microfilaments)
GEORGE POSTE*, DIMITRI PAPAHADJOPOULOS*, AND GARTH L. NICOLSONt * Department of Experimental Pathology, Roswell Park Memorial Institute, Buffalo, New York 14263; t Department of Cancer Biology, The Salk Institute for Biological Studies, San Diego, California 92112; and t Department of Developmental and Cell Biology, The University of California, Irvine, Irvine, Calif. 92664
Communicated by Harden M. McConnell, July 17,1975
ABSTRACT Tertiary amine local anesthetics facilitated concanavalin A-induced redistribution of lectin receptors on murine BALB/3T3 cells and enhanced the susceptibility of these cells to agglutination by concanavalin A. In contrast, these drugs at similar concentrations inhibited ligand-induced capping of immunoglobulin receptors on mouse lymphocytes. We propose that these differing effects of local anesthetics on membrane receptor mobility in fibroblasts and lymphocytes result from the action of anesthetics on membrane-associated microtubules and microfilaments involved in the transmembrane control of receptor mobility. We present electron microscopic evidence of structural alterations in microtubule and microfilament organization in anesthetic-treated cells, together with data on changes in the responsiveness of anesthetic-treated cells to drugs that act on microtubules and/or microfilaments. This evidence supports the proposal that anesthetics affect the organization of cytoskeletal components or their plasma membrane attachment points. The effects of local anesthetics on ligand-induced redistribution of membrane receptors in both 3T3 cells and lymphocytes can be duplicated by treating cells with colchicine (or Vinca alkaloids) together with cytochalasin B. We propose that the participation of membrane-associated microtubules and microfilaments in the transmembrane control of receptor mobility is such that microtubules and microfilaments play opposing roles in regulating the mobility and topograpy of cell surface receptors. Binding of multivalent ligands such as lectins or antibodies to the cell surface is known to be accompanied by changes in the topographical distribution of the appropriate receptors (1, 2). Ligand-induced changes in receptor distribution offer a potential mechanism whereby events occurring on the outer surface of the plasma membrane could be translated to the cytoplasmic side and this might provide a structural basis for transducing information between the external environment and the intracellular metabolic machinery in such phenomena as mitogenesis, cell recognition and communication, and cellular immune responses. The mobility and distribution of certain lectin and immunoglobulin receptors on the surfaces of mammalian cells may be under transmembrane control by cytoplasmic structural elements associated with the plasma membrane that are sensitive to microtubule (MT) disruptive drugs such as colchicine (Ch) and the Vinca alkaloids (2-8). These membrane-associated proteins may act as "anchors" to restrict receptor mobility. Dislocation of the linkage between surface receptors and the Ch-sensitive structures allows increased receptor mobility and facilitates receptor redistribution by external multivalent ligands (2-8). The mobility and distribution of lectin and immunoglobulin receptors is also influenced by cytochalasin B (CB), a drug that disrupts microAbbreviations: CB, cytochalasin B; Ch, colchicine; Con A, concanavalin A; MF, microfilaments; MT, microtubules.
filaments (MF) (2, 9-11). Tertiary amine local anesthetics have recently been shown to alter ligand-induced redistribution of surface receptors, causing enhancement of lectin-induced redistribution of concanavalin A (Con A) -receptors on 3T3 cells (8, 12), but inhibition of antibody-induced capping of immunoglobulin receptors on lymphocytes (13). We report here further observations on the effect of local anesthetics on the mobility and distribution of surface receptors which provide an explanation for the apparently conflicting action of these drugs on receptor mobility in 3T3 cells and lymphocytes. Our evidence indicates that local anesthetics affect the activity of both Ch- and CB-sensitive membrane-associated elements involved in the transmembrane control of receptor mobility and topography.
MATERIALS AND METHODS Chemicals. Dibucaine HC1, tetracaine HC1, and procaine HC1 were obtained from Mann Research Laboratories (Orangeburg, N.Y.); lidocaine HC1 from Astra Pharmaceuticals (Worcester, Mass.); mepivacaine from Sterling-Winthrop Research (Rensselaer, N.Y.); dimethylsulfoxide, glutaraldehyde, and colchicine from Sigma (St. Louis, Mo.); cytochalasin B from the Aldrich Chemical Co. (Milwaukee, Wisc.); vinblastine sulfate from Eli Lilly and Co. (Indianapolis, Ind.), and fluorescein isothiocyanate from BLL Division of Bioquest (Cockeysville, Md.). [3H]Colchicine (specific activity 5 mCi/mmol) was purchased from New England Nuclear of Boston, Mass. Cells. Mouse BALB/c 3T3 and their simian virus 40 (SV40) transformants (SV3T3) cells were grown in Dulbecco-modified Eagle's medium with 10 calf serum (12, 14). Spleen cells were harvested from 12-week-old BALB/c mice and purified on a Ficoll-Hypaque gradient (15) and maintained in RPMI 1640 medium supplemented with 0.5% bovine serum albumin. Lectins. Con A was purchased as a twice-crystallized preparation (Miles; Elkhart, Ind.) and purified further by affinity chromatography (16). [3H]Con A (specific activity 3.8 X 106 cpm/mg) was prepared from affinity-purified Con A (16) and specific binding of [3H]Con A to cells measured at 0° (16). Fluorescein-conjugated Con A and fluorescein-conjugated rabbit anti-mouse immunoglobulin (F1-RAMG) were prepared (16, 17) and cell agglutination by Con A was measured (14, 18). Labeling of Cell Surface Receptors in Mouse Lymphocytes and Effect of Drugs on Receptor Distribution. Suspension cultures of mouse splenic lymphocytes (1 X 107 cells per ml) were incubated in 1.0 ml of medium for 15 min at 370 with or without drugs, and then in 50 ,Ag/ml of FlRAMG. The cells were then centrifuged onto glass coverslips 4430
Proc. Nat. Acad. Sci. USA 72 (1975)
Cell Biology: Poste et al. cytocentrifuge (Shandon Instruments; Camberley, England), fixed with 2.5% glutaraldehyde for 15 min at '20°, and the attached cells examined for specific immunofluorescence (16, 17). At least 100 stained nonaggregated cells at lOOX magnification were examined for capping of immunoglobulin receptors (detectable over no more than one half of each cell) irrespective of whether the label was on the surface or had been interiorized by endocytosis. To assess the reversibility of drug-induced changes in immunoglobulin receptor distribution, we incubated similar lymphocyte populations in drug-containing medium and then washed twice with pre-warmed phosphate-buffered saline before incubation with fluorescein-conjugated rabbit anti-mouse immunoglobulin in fresh drug-free medium. Electron Microscopy. BALB/ST3 cells were grown to confluence and then fresh medium (370) containing dibucaine (0.2 mM) or tetracaine (0.6 mM) was added. Cells were incubated for 15-30 min at 370, fixed in glutaraldehyde (final concentration 0.5%) for 15 min at 370, washed in phosphate-buffered saline and post-fixed in buffered 1% osmium tetroxide for 1 hr at 200. The fixed cells were carefully removed from the substrate and oriented onto soft agar pellets by centrifugation. The agar pellets were dehydrated, stained in block with 1% uranyl acetate, and embedded in Spurr resin (Electron Microscopy Sciences, Warren, Pa.). in
Table 1. The effect of colchicine and Vinca alkaloids on Con A-mediated agglutination of mouse 3T3 and SV3T3 cells pretreated with dibucaine
a
RESULTS
Effect of Microtubule Disruptive Drugs on Lectin-Induced Agglutination of Cells Pretreated with Local Anesthetics. Incubation of 3T3 cells with local anesthetics increases their susceptibility to agglutination by Con A so that the concentration of Con A required to produce maximum cell agglutination is reduced from 1200 ug/ml to 350 ug/ml (12). Studies in this (8) and other laboratories (6) have shown that lectin-induced agglutination of 3T3 and SV3T3 cells is inhibited by pretreating cells with MT-disruptive drugs. However, agglutination of anesthetic-treated 3T3 and SV3T3 cells by Con A was unaffected by MT-disruptive drugs (Table 1). The concentrations of MT-disruptive drugs shown in Table 1 are identical to those causing inhibition of Con A-mediated agglutination of 3T3 or SVv3T3 cells (6, 8). However, incubation of anesthetic-treated 3T3 or SV3T3 cells with Ch or Vinca alkaloids at concentrations as high as 10-4 M for 1 hr did not produce significant inhibition of cell agglutination (results not shown). If as proposed (3, 4, 8), the Ch-sensitive MT act as "anchors" to restrict the mobility of lectin receptors, then dislocation of the functional linkage between these structures and the membrane receptors would be expected to enhance receptor mobility and facilitate their redistribution by lectins. Indeed, ultrastructural evidence of enhanced lectin-induced redistribution of lectin receptors has been documented in cells treated with Ch (19) and local anesthetics (8). However, in Ch-treated cells Con A redistributed receptors into a single "cap-like" aggregate (19) whereas in anesthetic-treated cells binding of Con A resulted in multiple clusters or patches (8). These differences may explain the respective actions of Ch and local anesthetics in inhibiting and enhancing cell agglutination by Con A. Formation of caps of receptors on Ch-treated cells would reduce the probability of contact between aggregated Con A receptors on adjacent cells, a probable requirement for agglutination (17, 19). In contrast, Con A-induced redistribution of receptors on anesthetic-treated cells into multiple patches would increase the likelihood of
4431
Drug concentration Treatment
(mM)
Untreated Dibucainee Dibucaine + colchicinefg Dibucaine + vinblastinef Dibucaine +
Control 0.2 0.2 0.001 0.2
vincristinef Dibucaine + colchicine + vinblastinef
0.001 0.2 0.01 0.2 0.001 0.001
Cell agglutinations by Con Ab SV3T3d 3T3C 0 ++++
++++ ++++
++++
++++
++++
++++
++++
++++
++++
++++
Mean values derived from three separate experiments. Agglutination by the indicated concentration of Con A was measured as described in Materials and Methods and scored as 0, +, ++, +++, or ++++ for 0, 25, 50, 75, or >90% cell agglutination, respectively. c Agglutination by Con A (350 gg/ml) at room temperature after a
b
20 min.
d Agglutination
by Con A (150 gg/ml) at room temperature after 20
min.
Cells incubated in suspension with dibucaine at 370 for 30 min in serum-free medium. f Cells incubated in suspension with dibucaine at 370 for 30 min after which the cells were harvested, washed twice in phosphatebuffered saline and returned to fresh medium supplemented with dibucaine plus the indicated drugs for 30 min at 37°. g Dibucaine did not alter cellular binding of colchicine as determined by the binding of [3H]colchicine to cell extracts [method of Wilson (50)]. e
interaction between groups of aggregated receptors on adjacent cells and thus favor agglutination (1, 8). Since cap formation involves a MF system sensitive to CB (2, 10, 11), formation of caps in Ch-treated cells indicates that the MF system is still intact. In contrast, the lack of capping of Con A receptors on anesthetic-treated 3T3 cells (8) suggests that local anesthetics are acting not only on the Ch-sensitive MT system, but also on the CB-sensitive MF system. This proposed action of local anesthetics on both MT and MF would mean that anesthetics would be expected to enhance ligandinduced receptor redistribution as a result of functional impairment of the MT "anchors," but redistribution of receptors could not progress to cap formation (due to simultaneous impairment of the MF system), and would instead result in formation of multiple small clusters and patches as re-
ported previously (8). If local anesthetics affect both MT and MF, or their attachment to plasma membranes, it should be possible to duplicate the action of local anesthetics with Ch plus CB. As shown in Table 2, incubation of 3T3 cells with Ch (or Vinca alkaloids) plus CB enhanced Con A agglutination without altering lectin binding to cells. Although the increase in Con A agglutinability with Ch plus CB was not as great as with local anesthetics alone (Table 2), when cells were incubated with Ch, CB and local anesthetics, the anesthetics did not produce an additive increase in cell agglutinability (Table 2). The small non-additive increase in agglutinability caused by anesthetics might result from their ability to increase the fluidity of phospholipids (20, 21); this should also contribute to enhanced receptor mobility within the membrane. The results in Table 2 therefore support the above proposal that
4432
Cell Biology: Poste et al.
Proc. Nat. Acad. Sci. USA 72 (1975)
Table 2. The effect of dibucaine, colchicine, Vinca alkaloids, and cytochalasin B on the agglutination of mouse 3T3 cells by Con A
,
., ..,:,.."I'm
.: t. '!
..."
.., t,,;-
4.' .. IT.i. ',
I
l -Pb %
.t.
sf;
Concentration of Con A
(jlg/ml) causing maxiTreatment
Drug concentration
Untreated Dibucaine Colchicine + cytochalasin Bb Vinblastine + cytochalasin Bb Vincristine + cytochalasin Bb Colchicine + cytochalasin B + dibucaineC Dilnethylsulfoxided
Control 0.2 mM 0.001 mM 10 gg/ml 0.01 mM 10 ,ug/ml 0.01 mM 10 jig/ml 0.001 mM 10 ,ug/ml 0.2 mM 0.1% (vol/vol)
2b
mum cell
agglutinationa 1400 350 450 475 475
350 1400
Cell agglutination (>90%) by the indicated Con A concentration after incubation at room temperature for 20 min. b Cells incubated with the indicated drugs in serum-free medium for 1 hr at 370 before assay for agglutination by Con A. c Cells incubated in serum-free medium containing colchicine and cytochalasin B for 1 hr at 370 after which dibucaine containing medium was added and the sample incubated for 30 min at 370 before assay for Con A agglutination. dDimethylsulfoxide was used as a solvent for cytochalasin B in the other experimental series.
a
local anesthetics affect both Ch-sensitive and CB-sensitive structures. Microtubules and Microfilament Organization in Cells Treated with Local Anesthetics. Further evidence that local anesthetics affect the organization of MT and MF was obtained by electron microscopic examination of anesthetictreated cells. Confluent 3T3 cells appear in cross-section as polygonal, flat, endothelialoid cells (Fig. la). At higher magnification, a dense plasma membrane-associated network of MF can be seen with numerous MT deeper in the cell cytoplasm (Fig. lb). Treatment of 3T3 cells with tetracaine resulted in a dramatic alteration in cell morphology. The normally flattened cells rounded up and contracted, leading to the formation of numerous surface "blebs" (Fig. 2a) that resembled the zeiotic protrusions produced by cytochalasin B (22) or cytochalasin D (23). The cytoplasmic distribution of cytoskeletal elements was also altered by tetracaine treatment. Bundles of MF were no longer found beneath the plasma membrane and few MT could be identified in the cytoplasm (Fig. 2b). Some filaments were still present deep in the cytoplasm and these may be responsible for the apparent contraction of some cellular organelles to the nuclear area. It was impossible to determine whether tetracaine treatment produced dissolution of cytoskeletal elements, disorganization, or both. Inhibition of Ligand-Induced Capping of Lymphocyte Membrane Receptors by Local Anesthetics. Both the formation of caps of surface receptors (2, 10, 11, 24) and the maintenance of caps (11) are inhibited by CB, suggesting the involvement of MF. If, as proposed above, local anesthetics impair the function of a CB-sensitive MF system, these drugs should alter ligand-induced capping. As shown in Table 3, local anesthetics produced significant inhibition
FIG. 1. Untreated 3T3 cells. (a) low magnification field of cells oriented parallel to the initial growth surface, X910; (b) subplasma membrane cytoskeletal organization: MF, microfilaments; MT, microtubules; X13,900; bar equals 0.2 ,m. FIG. 2. 3T3 cells treated 30 min with 0.6 mM tetracaine at 370. (a) low magnification demonstrating anesthetic-induced cell rounding and surface "blebs", X910; (b) subplasma membrane cytoskeletal organization disrupted; X13,900; bar equals 0.2 Mm.
of antibody-induced immunoglobulin capping on mouse lymphocytes. Indeed, these drugs produced significantly greater inhibition than CB (Table 3). However, incubation of lymphocytes with CB together with Ch or Vinca alkaloids caused more marked inhibition of capping (Table 3), similar to that produced by local anesthetics alone. This further suggested that anesthetics act on both Ch-sensitive and CB-sensitive structures or their membrane attachment points. These results confirm the observations of Ryan et al. (13) on inhibition of antibody-induced lymphocyte capping by local anesthetics. Further evidence that local anesthetics act on CB-sensitive structures was provided by the finding that local anesthetics caused breakdown and redistribution of preformed caps of receptors (Table 4), similar to that caused by CB (Table 4), or CB plus Ch or vinblastine (Table 4). DISCUSSION
The present results provide new information to support the concept that cytoskeletal structures associated with the plasma membrane can influence the mobility and distribution of membrane receptors (2-8, 19, 25-27). Our data indicate that local anesthetics cause impairment of both Ch-sensitive MT and CB-sensitive MF systems involved in transmembrane control of receptor mobility. In addition to ultrastructural evidence for anesthetic-induced alteration in MT and MF organization (28), the finding that the effects of local anesthetics can be duplicated by exposing cells simultaneously to both Ch and CB lends support to our proposal that anesthetics act on both MT and MF. The inhibitory action of local anesthetics on both MT and MF offers an explanation for the previously unexplained findings that these drugs enhance lectin agglutination of certain cells (8, 12), a process that requires increased receptor mobility redistribution (1, 16, 25), yet at similar concentrations they inhibit capping of antibody receptors on lymphocytes (13). These effects, as
Cell Biology: Poste et al.
Proc. Nat. Acad. Sci. USA 72 (1975)
Table 3. The effect of local anesthetics, cytochalasin B, colchicine, and vinblastine on antibody-induced capping of immunoglobin receptors on mouse spleen cellsa
Treatment
1. Untreated 2. Dibucaine 3. Tetracaine 4. Lidocaine 5. Procaine 6. Mepivacaine 7. Cytochalasin B 8. Cytochalasin B 9. Dimethylsulfoxide 10. Colchicine 11. Vinblastine 12. Cytochalasin B + colchicine 13. Cytochalasin B + vinblastine 14. Vinblastine + colchicine
Concentration
Control 0.2 mM 0.5 mM 5.0 mM 10.0 mM 10.0 mM 10 /ig/ml 20 Mg/ml 0.25% (vol/vol) 0.01 mM 0.01 mM 10 pg/ml 0.01 mM 10 pg/ml 0.01 mM 0.01 mM 0.01 mM
Table 4. Reversal of preformed caps of receptors on mouse spleen cells by local anesthetics, cytochalasin B,
colchicine,
and
vinblastinea
Incubation % time (min) Capsb 30 30 30 30 30 60 60
79 15 18 25 38 44 62 54
60 60 60
81 74 80
60
12
Incubation Immuno-
Treatment
Untreated Dibucaine Tetracaine
B +
colchicine
Cytochalasin
B +
60
87
Spleen cells were preincubated in serum-free RPMI 1640 medium with or without the various drug supplements for the indicated times and then incubated with fluorescein-conjugated rabbit anti-mouse immunoglobulin for 30 min at 370 after which the percentage of stained cells with caps of receptors was measured as described in Materials and Methods. The results represent mean values from three separate experiments for experimental series nos. 1-3 and 8-13 and from two separate experiments for series nos. 4, 5, 7, and 14. b Percentage of caps in stained immunoglobulin-positive cells. The latter represent 38-47% of the total lymphocyte population. c Dimethylsulfoxide at the indicated concentration was used as solvent for cytochalasin in experiments 7, 8, and 12-14.
duced receptor redistribution, but simultaneous inhibition of
0
19
0.5 mM
30
25
mM
,ug/ml
1.25% (vol/vol) 0.01 mM 0.01 mM 10 gg/ml 0.01 mM
10 ,ug/ml 0.01 mM
85
30
31
60
45
60
90
60
92
60
88
60
33
60
28
Capping of immunoglobulin receptors was induced by fluorescein-conjugated rabbit anti-mouse immunoglobulin as in Table 3. We then incubated cell populations in medium with or without various drug supplements for the indicated times at room temperature to reduce endocytosis of capped receptors which occurred rapidly at 370 and complicated observations on the ability of drugs to reverse the capping process.
a
well as previous findings on the effect of Ch and CB on receptor mobility (2-4, 7, 11, 19, 29, 30), can be explained if membrane receptors are linked to MT and MF systems which play opposing roles in controlling receptor distribution and mobility. As proposed by Edelman and Yahara (3-5), we suggest that Ch-sensitive MT serve as cytoskeletal elements that "anchor" receptors and limit their mobility. In addition, we envisage that a MF system is linked to and operates in opposition to MT via a contractile activity that can redistribute receptors into large caps. Evidence that MF possess actomyosin components and can function as a contractile system has been reviewed elsewhere (31, 46). In this scheme the topography of membrane receptors at any time would reflect the interplay between these two opposing systems. Functional dislocation of receptors from "anchoring" MT by such drugs as Ch or vinblastine would favor ligand-induced receptor redistribution, but so long as the receptors remained linked to MF the ligands would redistribute the aggregated receptors into caps, as found in cells exposed to Ch (19). Conversely, selective inhibition of contractile MF by CB would prevent capping of receptors, as reported in several laboratories (2, 10, 11, 24), but since receptors remain linked to MT "anchors," they would remain randomly distributed. Finally, in situations where both MT and MF are impaired, as in cells treated with local anesthetics or a combination of Ch and CB, loss of the MT "anchors" would facilitate ligand-in-
capsb
30
10
Cytochalasin
globulin
Control
10
Colchicine Vinblastine
time
(min)
0.2 mM
Cytochalasin B Dimethylsulfoxide
a
9
Concentration
Procaine
vinblastine
60
4433
b
Percentage of stained cells showing caps.
the contractile MF system would dictate that capping of receptors could not occur and ligands would instead produce multiple clusters or patches of receptors (8). The mechanism by which local anesthetics alter the functional activity of MT and MF and their interaction with membrane receptors is unknown. Our results indicate that these drugs produce structural or organizational alterations in both MT and MF. Similar ultrastructural alterations in MT in anesthetic-treated tissues have been reported (32, 33) and lidocaine inhibits in vitro polymerization of MT (33). One aspect of the pharmacological activity of local anesthetics that may be pertinent to their action on MT and MF concerns their ability to compete for Ca2+ and to displace Ca2+ bound to membranes (34, 35). Structural reorganization of membranes after anesthetic-induced modification of Ca2+ binding could alter the linkage between integral membrane receptors and a membrane-associated MT-MF system. This possibility has been discussed elsewhere (8, 12, 28, 36). The role of Ca2+ in regulating the depolymerization and polymerization of MT subunits both in vivo and in ittro (37-39) also introduces the possibility that structural breakdown of MT in anesthetic-treated cells might result from competitive inhibition by anesthetics of Ca2+-sensitive functions necessary for MT integrity (39, 40). Finally, the extreme sensitivity of MT polymerization to Ca2+ concentration, the dissolution of MT at [Ca2+] > 10-5 M, and the inverse correlation between cytoplasmic [Ca2+] and MT integrity (38, 39, 45) offers yet another mechanism by which displacement of Ca2+ from membranes by anesthetics could affect MT in-
tegrity. Displacement of membrane-bound Ca2+ might raise cytoplasmic [Ca2+] to levels sufficient to cause MT depolymerization. In this respect, it is interesting to note that experimentally-induced increase in intracellular [Ca2+] produced by exposure of 3T3 cells and mouse lymphocytes to the calcium ionophore A23187 has been found to produce effects
on
receptor mobility identical to those produced by
Ch and vinblastine (36).
4434
Cell Biology: Poste et al.
Interpretation of the diverse effects of local anesthetics on membranes has previously been considered primarily in terms of their action on membrane lipids (35, 41). The present results indicate, however, that these drugs also influence membrane organization via an interaction with membrane-associated MT and MF. The inhibitory effect of local anesthetics on the adhesion, spreading and locomotion of cells on solid substrates (42, 43), cell aggregation (44), axonal transport (45), endocytosis (46), and exocytosis (47), might now be reevaluated as possibly resulting from the action of these drugs on cytoskeletal elements rather than their single action on membrane lipids particularly since these various processes are also inhibited by drugs acting on MT and MF systems (48-50). This work was supported by NIH Grants CA-13393 (G.P.), GM18921 (D.P.); NIH-NCI Tumor Immunology Program Contract CB-33879 (G.L.N.), and NSF Grant GB-34178 (G.L.N.). We thank J. Smith, P. Newhouse, and A. MacKearnin for excellent technical assistance and A. Brodginski for help in preparing the manuscript. 1. Nicolson, G. L. (1974) Int. Rev. Cytol. 39,89-190. 2. de Petris, S. (1975) J. Cell Biol. 65, 123-146. 3. Edelman, G. M., Yahara, I. & Wang, J. L. (1973) Proc. Nat. Acad. Sci. USA 70, 1442-1446. 4. Yahara, I. & Edelman, G. M. (1973) Nature 236, 152-155. 5. Yahara, I. & Edelman, G. M. (1975) Exp. Cell Res. 91, 125142. 6. Yin, H. H., Ukena, T. H. & Berlin, R. D. (1972) Science 178, 177-179. 7. Berlin, R. D., Oliver, J. M., Ukena, T. E. & Yin, H. H. (1974) Nature 247,45-46. 8. Poste, G., Papahadjopoulos, D., Jacobson, K. & Vail, W. J. (1975) Biochim. Biophys. Acta 394,504-519. 9. Wessells, N. K., Spooner, B. S., Ash, J. F., Bradley, M. O., Luduena, M. A., Taylor, E. L., Wrenn, J. T. & Yamada, K. M. (1971) Science 171, 135-143. 10. Taylor, R. B., Duffus, W. P. H., Raff, M. C. & de Petris, S. (1971) Nature New Biol. 233,225-229. 11. de Petris, S. (1974) Nature 250, 54-56. 12. Poste, G., Papahadjopoulos, D., Jacobson, K. & Vail, W. J. (1975) Nature 253,552-554. 13. Ryan, G. B., Unanue, E. R. & Karnovsky, M. J. (1974) Nature
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