Planta
Planta (1984) 162:415 421
9 Springer-Verlag 1984
Anesthetics alter the lipid composition of barley-root membranes Patricia C. Jackson and Judith B. St. John U.S. D e p a r t m e n t o f Agriculture, Agricultural Research Service, Beltsville, M D 20705, U S A
Abstract. The question of whether membrane expansion, which is caused by anesthetics in animal systems, alters the lipid composition of plant cell membranes was investigated. We have measured the effects of several anesthetics on the relative amounts of the principal fatty acids from the polar lipids of barley (Hordeum vulgare L.) root membranes. Procaine, dibucaine, tetracaine, chloroform and, to a lesser degree, methanol increased the proportions of palmitic, stearic and oleic acids and decreased the proportions of linoleic and linolenic acids. Ethanol had no significant effect. Total amounts of the fatty acids from the polar lipids of roots in procaine solution decreased markedly so that all of the acids decreased in amount. The anesthetic was effective as soon as the roots were introduced to the solution and the changes progressed at constant rates for 6 h. Only the polar membrane lipids were altered; other lipids were not affected. Increased hydrostatic pressure of about 1.0 MPa largely prevented the anesthetic effects, including the decrease in the total amounts of the fatty acids. Hydrostatic pressure as high as 2 MPa had no effect per se on the membrane lipid composition. These results indicate that anesthetics cause expansion of the root membranes which results in the lipid changes. That a compositional change in the membrane lipids involves a conformational change such as expansion is an indication of the nature of the link between changes in the membrane lipids and changes in function of areas where hydrophilic ions permeate.
Introduction
Changes in the membrane lipid composition of barley roots produced by undissociated organic acids and phenols (Jackson and St. John 1980, 1982) have led us to suggest that membrane expansion or swelling is involved. Involvement of swelling was suggested because 2,4-dinitrophenol (DNP) causes swelling of mitochondria and phospholipid vesicles (Bakker et al. 1973). We have tested this hypothesis in the present work by the use of anesthetics. Effects of anesthetics in animals have been widely studied and the associated changes in cell membranes have been observed (for review, see Fink 1975). Release of seed dormancy by anesthetics also has been attributed to action at the cell membranes (Hendricks and Taylorson 1980). Anesthetic effects generally can be reversed by increased pressure and this finding has led to the view that the observed membrane expansion is implicated in the anesthetic action. This and the apparent necessity for adequate lipid solubility associated with anesthetic activity and release from seed dormancy prompted us to investigate whether such expansion is accompanied by alteration of the lipid composition of membranes. Accordingly, we have measured the effects of several water-soluble anesthetics on the fatty-acid composition of polar membrane lipids of barley roots, and have studied whether these effects can be prevented by increased hydrostatic pressure.
Key words: Anesthetic - Hordeum (root membranes) - Membrane.
Material and methods
Abbreviations." 16 : 0 = palmitic acid; 18 : 0 = steanc acid; 18 : 1 =
The roots used were from 6-d-old seedlings o f barley (Hordeum vulgare L. cv. Trebi, U.S. D e p a r t m e n t o f Agiculture Branch Experiment Station, Aberdeen, Id. ; or cv. C o m p a n a , M o n t a n a
oleic acid; 18: 2 = linoleic acid ; 18 : 3 = linolenic acid
416
P.C. Jackson and J.B. St. John: Anesthetics alter barley-root membrane lipids
Table 1. Effects of several anesthetics on fatty acids from polar (membrane) lipids of barley roots a Treatment
% of total fatty acids
S/U
18 : 2 18 : 3
Total amount (btmol/g DW)
16:0
18:0
18:1
18:2
18:3
25.5 26.5 27.9 49.1 30.6
1.2 1.2 1.5 4.5 9.2
3.0 3.4 3.9 8.2 13.4
48.5 49.7 49.6 32.7 38.7
21.8 19.2 17.1 5.5 8.1
0.364 0.384 0.417 1.153 0.667
2.22 2.59 2.90 5.97 4.78
51.8 34.9 23.3 13.1 7.4
Untreated
25.3
0.9
3.5
49.2
21.4
0.356
2.28
67.1
5 m M tetracaine pH 5 pH 7
31.0 29.0
1.4 1.5
4.1 4.2
47.4 46.3
16.2 19.0
0.479 0.439
2.93 2.42
57.6 60.6
cv. Trebi
Untreated 0.2 M C2HsOH 0.2 M CH3OH 0.08 M CHC13 0.2 M procaine cv. C o m p a n a
a Treated roots were maintained in aerated solutions of the anesthetic + 0A m M CaSO4 for 6 h. S/U is the ratio of the saturated acids (16 : 0 + 18 : 0) to the unsaturated acids (18 : 1 + 18 : 2 + 18 : 3). Standard deviations are within • 3 % of the values for 16 : 0, 18:2, 18:3 and the ratios, within + 1 0 % of the values for 18:0 and 18:1, and average _+15% for total amounts. Total amount is the total fatty acids from the polar lipids (16: 0 + 18 : 0 + 18 : 2 + 18 : 3)
Table 2. Effects of dibucaine on fatty acids from the polar (membrane) lipids of roots of barley (cv. Compana)" Treatment
[D ~
% of total fatty acids 16:0
18:0
18:1
18:2
18:3
S/U
18:2 18:3
Total amount (gmol/g DW)
I pH 5.4
Control
-
28.5
0.6
2.8
44.9
23.2
0.410
1.93
30.2
Dibucaine 0.50 m M 1.0 m M
0.49 0.98
35.6 43.7
0.4 1.2
3.0 4.6
40.2 39.2
20.8 11.3
0.563 0.815
1.93 3.46
26.1 27.9
-
27.4
0.2
3.8
48.7
19.8
0.382
2.46
46.9
0.95 1.52 2.53
32.3 40.9 45.8
0.3 0.6 1.4
3.2 4.8 5.4
43.8 39.5 37.7
20.3 14.3 9.7
0.485 0.709 0.896
2.16 2.77 3.87
32.6 29.5 20.3
-
25.3
0.9
3.5
49.2
21.4
0.356
2.28
67.1
0.32 30.9
38.1 42.4
1.8 2.4
8.1 6.4
41.5 47.4
10.5 9.0
0.664 0.810
3.94 4.44
30.1 26.8
II pH 6.0 Control Dibucaine 0.3 m M 0.6 m M 1.0 m M III Control Dibucaine, 1 m M pH 5 pH 7
a Treated roots were maintained in aerated solutions of dibucaine +0.1 m M C a S O 4 for 3 h. [D ~ is the moIar concentration of the neutral species of dibucaine. For S/U and Total amount, see footnote to Table 1 State University, Bozeman, USA) which had been grown in aerated 0.1 m M CaSO 4 in the dark at 25 ~ C. The pH of the solutions was 5.6. Characteristics of growth and response of the two cultivars were quantitatively similar. The roots were excised and rinsed several times with demineralized H 2 0 just before use. Conditions for the experiments and details of the procedures are the same as described in Jackson and Stieff (1965). Briefly, roots were maintained in aerated solutions of 0.1 m M CaSO 4 with or without an anesthetic at 23~ for 15 min to 6 h. The pH was adjusted and maintained at 5.4-5.6 during treatment by periodic titration with an acid or base. Then,
the roots were removed, rinsed, and freeze-dried. The anesthetics used came from Sigma Chemical Co., St. Louis, Mo., USA. Experiments with increased hydrostatic pressure were performed by placing approx. 1 g (fresh weight) of roots in 18 ml of procaine at pH 5.8 in a 35-ml glass test tube which was placed in a pressure chamber. The chamber was an adaptation of the chamber used by Hendricks and Taylorson (1980, their Fig. 1 b). A requisite pressure of air ("Breathing A i r " ; Air Products Co., Allentown, Pa., USA) was then applied as quickly as possible. The treatment was applied for 6 h. During this time, the roots were aerated continuously by passage of air under pressure through the solution by means of a glass
P.C_ Jackson and J.B. St. John: Anesthetics alter barley-root membrane lipids
417 1 mM D i b u c a i n e
0.2 M P r o c a i n e
50 5O 40
pmol/g
Total 4(
O~ o 3c
/
v'/~ c,.,_ 1:~ 30 J" ~'t 20 4
16:0
y_ 20'
'4 30
0.6 0.5
~- 2e
10 0.4 0.3
Time of Treatment - hours
1C
Cf
18:0
o
~
~
I
0
I
2
I
4
9
6
Fig. 2. Effects of I mM dibucaine on proportions of fatty acids from polar (membrane) lipids of barley roots as a function of time of treatment. Larger circles are used for Total }~mol/g because the standard deviations of the measurements are greater. For definitions of S/U and Total gmol/g, see legend of Fig. l
Hours of treatment
Fig. 1. Effects of 0.2 M procaine on proportions of fatty acids from polar (membrane) lipids of barley roots as a function of time of treatment. S/U is the ratio of saturated to unsaturated acids. The scale of the left-hand ordinate applies to the right hand ordinate as well. Total gM/g refers to the total of the fatty acids from the polar lipids (16:0+ 18:0+ 18:1 + 18:2 +18:3)
be calculated is less precise. However, quantities of the total fatty acids are given as gmol g 1 freeze-dried roots. The dry weights are about 7% of the fresh weights. Standard deviations of the distributional data are generally within 3% of the distributional values of the three major fatty acids whereas standard deviations of total amounts are about 15% of the amounts.
Results aerator. The flow rate was adjusted by a regulated release of air from the chamber so that adequate aeration and requisite pressure were maintained. Application and removal of the increased pressure took appreciable time such that the roots were exposed to procaine at ambient pressure for 10-20 rain. This may have influenced the results to some degree. Lipids from ground, freeze-dried roots were extracted according to the procedures of Folch et al. (1957), Polar lipids (membrane lipids) were separated from non-polar lipids by rubber-membrane dialysis (Bottcher et al. 1959) and then were saponified with alcoholic K O H according to Burchfield and Storrs (t962). Methyl esters of the resulting fatty acids were prepared for gas chromatography (St. John 1976) with boron trifluoride in methanol as described by Metcalfe and Schmitz (1961). Heptadecanoic acid was added to all samples as an internal standard. The polar lipids of the roots in these studies are predominately phospholipids ( > 95%). The results are expressed as the percent by chromatographic area of the total fatty acids from the polar lipids (C 1 6 : 0 + C 1 8 : 0 + C 1 8 : 1 + C 1 8 : 2 + C 18:3) and as ratios of saturated to unsaturated or less-saturated acids. This was done because the gas-chromatographic data are obtained as relative values. The combination of weighing the samples and quantitative introduction of an internal standard so that quantities can
Procaine (diethylaminoethyl-p-aminobenzoate), tetracaine (2-dimethylaminoethyl 4-n butylaminobenzoate), dibucaine (2-butoxy-N-(2-diethylaminoethyl)cinchoninamide) and chloroform at various concentrations have marked effects on the fatty acids from polar membrane lipids of barley roots (Tables 1, 2). They greatly decreased the proportions of linolenic (18:3) and linoleic (18:2) acids and increased the proportions of oleic (18:1), stearic (18 : 0) and palmitic (16: 0) acids, thereby increasing the degree of saturation of the acids (S/ U) and the ratio, 18:2/18:3. The total amounts of the acids decreased also, so that the amounts of each of the individual acids decreased although the proportions of three of the acids increased. Methanol had much less effect than chloroform, procaine and dibucaine, and ethanol was without significant effect on the distribution. Thus, the effects of methanol on 16:0 and 18:3, and effects
418
P.C. Jackson and J.B. St. John: Anesthetics alter barley-root membrane lipids
Table 3. Effect of procaine on fatty acids from all lipid fractions of cv. Cornpana barley roots" Treatment
% of total fatty acids
S/U
Total amount (gmol/g DW)
16:0
18:0
18:1
18:2
18:3
25.5 28.8
1.2 5.2
3.9 9.6
47.8 42.9
21.7 13.6
0.363 0.515
44.0 8.2
17.7 16.2
21.4 20.8
19,9 19.4
33.9 37.1
7.2 6.6
0.641 0.585
40.5 29.5
9.9 10.9
7.6 7.9
30,0 30.7
50.9 49.3
1.7 1.2
0.211 0.232
43.4 21.1
Polar lipids Untreated Procaine
Non-polar Lipids Untreated Procaine
Free fatty acids Untreated Procaine
a Treated roots were maintained in aerated solutions of 0.1 M procaine +0.1 m M CaSO4 for 6 h. For S/U and Total amount, see footnote to Table I Table 4. Effects of procaine on the polar lipid composition of barley roots (cv. Compana) under increased hydrostatic pressure a Treatment
% of total fatty acids
S/U
18:2 18:3
Total amount (gmol/g DW)
16:0
18:0
18:1
18:2
18:3
I Untreated 0.2 M Procaine +0.87 M P a
25.9 29.8 26.2
1.4 5.8 1.8
4.2 13.6 5.2
47.5 38.0 47.2
21.0 12.7 19.7
0.375 0.554 0.387
2.27 2.99 2.40
44.3 7.7 32.5
I1 Untreated 0.01 M Procaine 0.1 M Procaine + 1.02 MPa
27.6 30.5 31.3 28.2
0.6 1.0 2.4 0.5
3.0 3.8 5.9 2.9
48.0 46.0 43.4 47.6
20.8 18.6 16.6 20.8
0.393 0.460 0.507 0.404
2.30 2.38 2.65 2.29
62.3 45.6 18.9 62.3
" Treated roots were maintained in aerated solutions of 0.1 m M CaSO4 with procaine for 6 h. For S/U and Total amount, see footnote to Table 1
of procaine, tetracaine, dibucaine and chloroform on the proportions of all of the acids are significant at the 1% level of confidence. The anesthetics decreased the total amounts significantly as well. Concentrations of the anesthetics used in these experiments were close to those used by Hendricks and Taylorson (1980) for the enhancement of seed germination, except that the concentration of chloroform was lower because of limited solubility in water. Tables 1 and 2 show that a shift in pH of the anesthetic solution from 5 to 7 has little or no effect on the fatty-acid changes produced by tetracaine (Table 1) or dibucaine (Table 2). The concentration of the positively charged anesthetic amine is essentially constant (both pK,'s = 8.5) as the concentration of the uncharged species increases 100-fold from p H 5 to 7. Thus, the major contribution to the lipid changes appears to be from the protonated species, whereas, the neutral species has only a minor effect, if any.
Effects of procaine and dibucaine on the fattyacid distribution appear to begin as soon as the roots are introduced into the solution and thereafter progress at constant rates for 6 h (Figs. 1, 2). The linear curves all pass through zero with correlation coefficients between 0.95 and 1.00. Fatty acids from roots maintained for 6 h in aerated 0.1 m M CaSO 4 or water without procaine do not change in distribution or total amount as shown by comparison of untreated with control roots in previous studies (Jackson and St. John 1980). Total amounts of the fatty acids from roots in anesthetic solutions decreased so greatly that effects of procaine on lipids other than the membrane (polar) lipids were determined to assess whether the anesthetics have a general effect on all lipids and whether fatty acids lost from the polar lipids appear with the free fatty acids. Typical results are shown for cv. Compana roots in Table 3. Procaine has no effect on the distribution of fatty acids from either the non-polar lipids or the
P.C. Jackson and J.B. St_ John: Anesthetics alter barley-root membrane lipids
50
0.2 M P r o c a i n e
4q
Total rJmol/g
"~ 3C "o
419
ever, the effects of procaine were largely prevented by increased hydrostatic pressure of about i MPa in Table 4. This iricludes the large loss of the total amounts of fatty acids as well as the distributional changes. Pressure curves (Fig. 3) dramatize the prevention and show that pressures greater than 1 MPa are required for maximum effectiveness. The half-maximum effective pressure is about 0.6 MPa for the distributional values. Pressures lower than 0.6 MPa were proportionately less effective (data not shown).
,...,
Discussion m2a
18:3
]
"6
ae
J
Hydrostatic
Pressure
18:2/18:3I 3 6 9 12 x 10
- MPa
Fig. 3. Prevention of the effects of 0.2 M procaine on fatty acids from polar membrane lipids of barley roots by increased hydrostatic pressure. S/U is the ratio of saturated to unsaturated acids. The dotted lines connect to data points of untreated control roots. The scale for the left-hand ordinate applies to the right hand ordinate as well. Total gM/g refers to the total fatty acids from the polar lipids ( 1 6 : 0 + 1 8 : 0 + 1 8 : 0 + 1 8 : 1 +18:2+18:3)
free fatty acids. Although the total amounts decrease (to 73% and 49%, respectively), these losses are not nearly as great as the loss of fatty acids from polar lipids (a decrease to 19%). Effects of procaine on cv. Trebi roots are similar with decreases in total amounts to 58% for the non-polar fraction and to 61% for the free-fatty-acid fraction. No gain is apparent in the free fatty acids so that the large losses of fatty acids from the polar lipids appear to be to the external solution. Studies were undertaken to assess the interaction of increased hydrostatic pressure with the effects of procaine on the polar lipids. Hydrostatic pressure increased to about 2 MPa (20 atmospheres) had no significant effect per se on either the distribution or the total amount of the fatty acids of roots which had not been treated with an anesthetic as long as the roots were aerated during the time under pressure (data not shown). Procaine at ambient pressure under aeration had marked effects on the fatty acids (Table 4). How-
Our results present evidence that anesthetics perturb barley-root membranes sufficiently to alter the composition of the polar membrane lipids. The changes are in the distribution of the major fatty acids from the polar lipids and often are accompanied by loss in the total amount of fatty acids (Table 1). We view the distributional changes and the losses as two effects because distributional changes are not always accompanied by fatty-acid losses (Jackson and St. John 1980, 1982). Tetracaine at 5 m M (Table 1) produced distributional changes but no significant loss in the total amount of fatty acids. Distributional changes produced by dibucaine (Table 2) are as great as those produced by procaine (Table 1 or Fig. 1), but fatty-acid losses from roots are much less in dibucaine (33%) than in procaine (86%). In our previous work, such losses were a feature of marked increase in permeability of the roots to ions (Jackson and Taylor 1970; Jackson 1982). This seems to apply to the action of anesthetics as well (data not shown). The anesthetics alter the membrane-lipid composition apparently as soon as the roots are introduced into the solution containing the anesthetic. This is indicated in Figs. 1, 2 where the effects of procaine and dibucaine, starting from zero time, progress at constant rates for 6 h. Such kinetics parallel those of the effects of undissociated organic acids and phenols on membrane lipids (Jackson and St. John 1980, 1982). These compounds were inferred to act directly on the root membranes. Thus, the anesthetics also are inferred to act primarily on the root membranes. Plasma membranes would be expected to be the most involved initially because of their proximity to the external solution. Further indication that the anesthetics act directly on the membranes comes from the absence of distributional changes in lipids other than the polar membrane lipids (Table 3). This is also true of the effects of undissociated organic acids and phenols.
420
P.C. Jackson and J.B. St. John: Anesthetics alter barley-root membrane lipids
The possibility of anesthetic action via potential changes has been considered, but seems unlikely. This comes from the close parallelism of the anesthetic effects with those of DNP in particular. The kinetics of potential changes produced by DNP vary greatly from the kinetics of DNP effects on the polar lipids and ion permeability, as discussed at length previously (Jackson 1982). Reversal of anesthetic effects by increased hydrostatic pressure is well-documented in animal systems (e.g. Johnson and Flagler 195i; Johnson and Miller 1970) and has been demonstrated in plants by Hendricks and Taylorson (1980) in studies of seed dormancy. The reversal in animal systems has led to the hypothesis that anesthetic action involves membrane expansion (for review, Seeman 1972). Kamaya et al. (1981) have shown that anesthetics expand polymer membranes and that this is counteracted by pressures of about 10 MPa, pressures which are comparable to those used in our experiments. All anesthetics disorder phospholipid bilayers and pressure restores the order (Trudell et al. 1973). Consequently, we interpret the effects of several anesthetics on membrane lipids of roots and the prevention of such effects by increased hydrostatic pressure (Table 4, Fig. 2) as indicating that the anesthetics induce expansion of the root membranes. Substantiation of this comes from the LeChatelier-Braun Principle that an equilibrium displaced by a force can be maintained by application of an opposing force (LeChatelier 1885). Cell wall compression does not preclude membrane expansion. The membrane may become more convoluted or swollen. Barley roots in solutions of anesthetics, phenols or organic acids do not appear to lose turgor for the first hours of treatment, although they generally lose turgor eventually. The increase in ion permeability begins immediately as do the lipid changes. Comparative effects of the various anesthetics used in our studies show that the order of effectiveness is not according to lipid solubility, e.g. methanol is more effective than ethanol which has little or no effect. Chloroform, the most lipid-soluble of the anesthetics used, although more effective than methanol is less effective than the tertiary amines which are predominantly cationic in the pH range used in our experiments (pKa's=8.59.0). The relative efficacy of the various anesthetics which we have used is in the order dibucaine> tetracaine>procaine, an order of effectiveness shown by anesthetic action in animal and synthetic membrane systems. Concentrations of the anesthetics used in our studies also compare closely with those used for pharmacologic activity (e.g.
Badger and Helmkamp 1982; Surewicz and Leyko 1982). We have not used the more lipid-soluble volatile anesthetics because of technical imprecision caused by the volatility and insolubility in water. Nevertheless, the comparisons here, although limited, indicate involvement of factors other than lipid solubility per se. The nature of the changes produced by the anesthetics is closely similar to the nature of the changes produced by undissociated organic acids (Jackson and St. John 1980) and undissociated phenols (Jackson and St. John 1982). All of these diverse compounds increase the relative saturation of the fatty acids which is seen principally as a marked decrease in 18:3 balanced by increase in the proportions of the more saturated acids. The rapidity with which these changes are produced is indicative of an inhibition or repression of a physiological process. Such an increase in saturation, which is a deoxidation or reduction, is indicative of a desaturase inhibition. That desaturation (i.e. oxidation) can be rate-limiting to the maintenance of 18:3 is not surprising when one considers the large reducing capacity of the roots. Inhibition of lipid synthesis is unlikely because of the changes in distribution without significant change in the total amounts of the fatty acids (Table 1; Jackson and St. John 1980, 1982). The most effective molecular species of the tertiary-amine anesthetics evidently is the positively charged protonated amine, the concentration of which changes little with a pH shift from 5 to 7. This comes from lack of a large interaction of pH with the effectiveness of tetracaine (Table 1) and dibucaine (Table 2). Experiments I and II of Table 2 show that increasing the concentrations of dibucaine from 0.3 or 0.5 mM to 1 mM greatly increases the intensity of the effect. However, the effect of 1 mM dibucaine is nearly the same in the three experiments whatever the pH and despite the ]00-fold range of the concentration of neutral dibucaine. This indicates that most, if not all, of the effect is attributable to the protonated species of dibucaine. The effectiveness of the protonated species is consistent with the action of anesthetics in other biological systems (Badger and Helmkamp 1982) and on phospholipid membranes (Boulanger et al. 1981 ; Surewicz and Leyko 1982) and the actions of organic acids (Jackson and St. John 1980) and phenols (Jackson and St. John 1982) on root membrane lipids and permeability to ions (Jackson and Taylor 1970; Jackson 1982). In every case, whether the compounds are amines, phenols or aliphatic acids, the effective species is that which is protonated; i.e., the species which is capable of
P.C. Jackson and J.B. St. John : Anesthetics alter barley-root membrane Iipids
hydrogen bonding. Carboxyl, hydroxyl and amine groups are known to bind to proteins (for review, Van Sumere et al. 1975) and head groups of polar lipids (Boulanger et al. 1981), displacing water by hydrogen bonding of the undissociated or protonated species. Presumably this action results in membrane expansion which alters both the membrane lipids and the areas where hydrophilic ions permeate. An interesting feature of our results is that a physiological process of barley roots (namely, ion uptake) and the polar membrane lipids are influenced by a physical event (swelling or expansion) resulting from interaction between effective chemicals (anesthetics, undissociated organic acids and phenols) and the membranes. References Badger, C.R., Helmkamp, G.M., Jr. (1982) Modulation of phospholipid transfer protein activity: inhibition by local anesthetics. Biochim. Biophys. Acta 692, 32-40 Bakker, E.P., Van den Heuvel, E.J., Wiechmann, A.H.C.A., Van Dam, K. (1973) A comparison between effectiveness of uncouplers of oxidative phosphorylation in mitochondria and in artificial membrane systems. Biochim. Biophys. Acta 292, 78 Bottcher, C.J., Woodford, F.P., Borlsma-Van Houte, E., Gent, C.M. (1959) Methods for the analysis of lipids extracted from human arteries and other tissues. Recl. Tray. Chim. 78, 794-814 Boulanger, Y., Schreier, S., Smith, I.C.P. (1981) Molecular details of anesthetic lipid interaction as seen by deuterium and phosphorus 31 nuclear magnetic resonance. Biochemistry 20, 6824-6830 Burchfield, H.P., Storrs, E.E. (1962) Biochemical applications of gas chromatography. Academic Press, New York London Fink, B.R., ed. (1975) Molecular mechanisms of anesthesia. Raven Press, New York Folch, J., Lees, M., Stanley, G.H. (1957) A simple method for the isolation and purification of total lipids from animal tissue. J. Biol. Chem. 226, 497-509 Hendricks, S.B., Taylorson, R.B, (1980) Reversal by pressure of seed germination promoted by anesthetics. Planta 149, 108-111
421
Jackson, P.C. (1982) Differences between effects of undissociated and anionic 2,4-dinitrophenol on permeability of roots to ions. Plant Physiol. 70, 1373-1379 Jackson, P.C., Stieff, K.J. (1965) Equilibrium and ion exchange properties of potassium and sodium accumulation by barley roots. J. Gem Physiol. 48, 601-616 Jackson, P.C., St. John, J.B. (1980) Changes in membrane lipids of roots associated with changes in permeability. I. Effects of undissociated acids. Plant Physiol. 66, 801-804 Jackson, P.C., St. John, J.B. (1982) Effects of 2,4-dinitrophenol on membrane lipids of roots. Plant Physiol. 70, 858 862 Jackson, P.C., Taylor, J.M. (1970) Effects of organic acids on ion uptake and retention in barley roots. Plant Physiol. 46, 538 542 Johnson, F.H., Flagler, E.A. (1951) Hydrostatic reversal of narcosis in tadpoles. Science 112, 91-92 Johnson, S.M., Miller, K.W. (1970) The antagonism of pressure and anesthesia. Nature (London) 228, 75-76 Kamaya, H., Yukio, S., Ueda, I., Eyring, H. (1981) Anesthetics and high pressure interaction upon elastic properties of a polymer membrane. Proc. Natl. Acad. Sci. USA 78, 3572-3575 LeChatelier, HTL. (1885) Sur les lois de la dissolution. C.R. Acad. Sci. (Paris) 100, 441444 Metcatfe, L.D., Schmitz, A.A. (1961) The rapid preparation of fatty acid esters for gas chromatographic analysis. Anal. Chem. 33, 363-364 Seeman, P. (1972) The membrane actions of anesthetics and tranquilizers. Pharmacol. Rev. 24, 583-655 St. John, J.B, (1976) Manipulation of galactolipid fatty acid composition with substituted pyridazinones. Plant Physiol. 57, 38-40 Surewicz, W.K., Leyko, W. (1982) Interaction of local anesthetics with model phospholipid membranes. The effects of pH and phospholipid composition studied by quenching of an intra membrane fluorescent probe. J. Pharm. Pharmacol. 34, 359-363 Trudell, J.R., Hubbell, W.L., Cohen, E.N. (1973) The effect of two inhalation anesthetics on the order of spin-labeled phospholipid vesicles. Biochim. Biophys. Acta 291,321-327 Van Sumere, C.F., Albrecht, J., Dedonger, A., de Pooter, H., Pc, I. (1975) Plant proteins and phenolics in the chemistry and biochemistry of plant proteins. Proc. Phytochem. Soc., Univ. of Ghent, Belgium, Sept. 1973, pp. 211-247, Harbone, J.B., Van Sumere, C.F., eds. Academic Press, London New York Received 12 December 1983; accepted 16 May 1984
Planta (1984) 162:415 421
9 Springer-Verlag 1984
Anesthetics alter the lipid composition of barley-root membranes Patricia C. Jackson and Judith B. St. John U.S. D e p a r t m e n t o f Agriculture, Agricultural Research Service, Beltsville, M D 20705, U S A
Abstract. The question of whether membrane expansion, which is caused by anesthetics in animal systems, alters the lipid composition of plant cell membranes was investigated. We have measured the effects of several anesthetics on the relative amounts of the principal fatty acids from the polar lipids of barley (Hordeum vulgare L.) root membranes. Procaine, dibucaine, tetracaine, chloroform and, to a lesser degree, methanol increased the proportions of palmitic, stearic and oleic acids and decreased the proportions of linoleic and linolenic acids. Ethanol had no significant effect. Total amounts of the fatty acids from the polar lipids of roots in procaine solution decreased markedly so that all of the acids decreased in amount. The anesthetic was effective as soon as the roots were introduced to the solution and the changes progressed at constant rates for 6 h. Only the polar membrane lipids were altered; other lipids were not affected. Increased hydrostatic pressure of about 1.0 MPa largely prevented the anesthetic effects, including the decrease in the total amounts of the fatty acids. Hydrostatic pressure as high as 2 MPa had no effect per se on the membrane lipid composition. These results indicate that anesthetics cause expansion of the root membranes which results in the lipid changes. That a compositional change in the membrane lipids involves a conformational change such as expansion is an indication of the nature of the link between changes in the membrane lipids and changes in function of areas where hydrophilic ions permeate.
Introduction
Changes in the membrane lipid composition of barley roots produced by undissociated organic acids and phenols (Jackson and St. John 1980, 1982) have led us to suggest that membrane expansion or swelling is involved. Involvement of swelling was suggested because 2,4-dinitrophenol (DNP) causes swelling of mitochondria and phospholipid vesicles (Bakker et al. 1973). We have tested this hypothesis in the present work by the use of anesthetics. Effects of anesthetics in animals have been widely studied and the associated changes in cell membranes have been observed (for review, see Fink 1975). Release of seed dormancy by anesthetics also has been attributed to action at the cell membranes (Hendricks and Taylorson 1980). Anesthetic effects generally can be reversed by increased pressure and this finding has led to the view that the observed membrane expansion is implicated in the anesthetic action. This and the apparent necessity for adequate lipid solubility associated with anesthetic activity and release from seed dormancy prompted us to investigate whether such expansion is accompanied by alteration of the lipid composition of membranes. Accordingly, we have measured the effects of several water-soluble anesthetics on the fatty-acid composition of polar membrane lipids of barley roots, and have studied whether these effects can be prevented by increased hydrostatic pressure.
Key words: Anesthetic - Hordeum (root membranes) - Membrane.
Material and methods
Abbreviations." 16 : 0 = palmitic acid; 18 : 0 = steanc acid; 18 : 1 =
The roots used were from 6-d-old seedlings o f barley (Hordeum vulgare L. cv. Trebi, U.S. D e p a r t m e n t o f Agiculture Branch Experiment Station, Aberdeen, Id. ; or cv. C o m p a n a , M o n t a n a
oleic acid; 18: 2 = linoleic acid ; 18 : 3 = linolenic acid
416
P.C. Jackson and J.B. St. John: Anesthetics alter barley-root membrane lipids
Table 1. Effects of several anesthetics on fatty acids from polar (membrane) lipids of barley roots a Treatment
% of total fatty acids
S/U
18 : 2 18 : 3
Total amount (btmol/g DW)
16:0
18:0
18:1
18:2
18:3
25.5 26.5 27.9 49.1 30.6
1.2 1.2 1.5 4.5 9.2
3.0 3.4 3.9 8.2 13.4
48.5 49.7 49.6 32.7 38.7
21.8 19.2 17.1 5.5 8.1
0.364 0.384 0.417 1.153 0.667
2.22 2.59 2.90 5.97 4.78
51.8 34.9 23.3 13.1 7.4
Untreated
25.3
0.9
3.5
49.2
21.4
0.356
2.28
67.1
5 m M tetracaine pH 5 pH 7
31.0 29.0
1.4 1.5
4.1 4.2
47.4 46.3
16.2 19.0
0.479 0.439
2.93 2.42
57.6 60.6
cv. Trebi
Untreated 0.2 M C2HsOH 0.2 M CH3OH 0.08 M CHC13 0.2 M procaine cv. C o m p a n a
a Treated roots were maintained in aerated solutions of the anesthetic + 0A m M CaSO4 for 6 h. S/U is the ratio of the saturated acids (16 : 0 + 18 : 0) to the unsaturated acids (18 : 1 + 18 : 2 + 18 : 3). Standard deviations are within • 3 % of the values for 16 : 0, 18:2, 18:3 and the ratios, within + 1 0 % of the values for 18:0 and 18:1, and average _+15% for total amounts. Total amount is the total fatty acids from the polar lipids (16: 0 + 18 : 0 + 18 : 2 + 18 : 3)
Table 2. Effects of dibucaine on fatty acids from the polar (membrane) lipids of roots of barley (cv. Compana)" Treatment
[D ~
% of total fatty acids 16:0
18:0
18:1
18:2
18:3
S/U
18:2 18:3
Total amount (gmol/g DW)
I pH 5.4
Control
-
28.5
0.6
2.8
44.9
23.2
0.410
1.93
30.2
Dibucaine 0.50 m M 1.0 m M
0.49 0.98
35.6 43.7
0.4 1.2
3.0 4.6
40.2 39.2
20.8 11.3
0.563 0.815
1.93 3.46
26.1 27.9
-
27.4
0.2
3.8
48.7
19.8
0.382
2.46
46.9
0.95 1.52 2.53
32.3 40.9 45.8
0.3 0.6 1.4
3.2 4.8 5.4
43.8 39.5 37.7
20.3 14.3 9.7
0.485 0.709 0.896
2.16 2.77 3.87
32.6 29.5 20.3
-
25.3
0.9
3.5
49.2
21.4
0.356
2.28
67.1
0.32 30.9
38.1 42.4
1.8 2.4
8.1 6.4
41.5 47.4
10.5 9.0
0.664 0.810
3.94 4.44
30.1 26.8
II pH 6.0 Control Dibucaine 0.3 m M 0.6 m M 1.0 m M III Control Dibucaine, 1 m M pH 5 pH 7
a Treated roots were maintained in aerated solutions of dibucaine +0.1 m M C a S O 4 for 3 h. [D ~ is the moIar concentration of the neutral species of dibucaine. For S/U and Total amount, see footnote to Table 1 State University, Bozeman, USA) which had been grown in aerated 0.1 m M CaSO 4 in the dark at 25 ~ C. The pH of the solutions was 5.6. Characteristics of growth and response of the two cultivars were quantitatively similar. The roots were excised and rinsed several times with demineralized H 2 0 just before use. Conditions for the experiments and details of the procedures are the same as described in Jackson and Stieff (1965). Briefly, roots were maintained in aerated solutions of 0.1 m M CaSO 4 with or without an anesthetic at 23~ for 15 min to 6 h. The pH was adjusted and maintained at 5.4-5.6 during treatment by periodic titration with an acid or base. Then,
the roots were removed, rinsed, and freeze-dried. The anesthetics used came from Sigma Chemical Co., St. Louis, Mo., USA. Experiments with increased hydrostatic pressure were performed by placing approx. 1 g (fresh weight) of roots in 18 ml of procaine at pH 5.8 in a 35-ml glass test tube which was placed in a pressure chamber. The chamber was an adaptation of the chamber used by Hendricks and Taylorson (1980, their Fig. 1 b). A requisite pressure of air ("Breathing A i r " ; Air Products Co., Allentown, Pa., USA) was then applied as quickly as possible. The treatment was applied for 6 h. During this time, the roots were aerated continuously by passage of air under pressure through the solution by means of a glass
P.C_ Jackson and J.B. St. John: Anesthetics alter barley-root membrane lipids
417 1 mM D i b u c a i n e
0.2 M P r o c a i n e
50 5O 40
pmol/g
Total 4(
O~ o 3c
/
v'/~ c,.,_ 1:~ 30 J" ~'t 20 4
16:0
y_ 20'
'4 30
0.6 0.5
~- 2e
10 0.4 0.3
Time of Treatment - hours
1C
Cf
18:0
o
~
~
I
0
I
2
I
4
9
6
Fig. 2. Effects of I mM dibucaine on proportions of fatty acids from polar (membrane) lipids of barley roots as a function of time of treatment. Larger circles are used for Total }~mol/g because the standard deviations of the measurements are greater. For definitions of S/U and Total gmol/g, see legend of Fig. l
Hours of treatment
Fig. 1. Effects of 0.2 M procaine on proportions of fatty acids from polar (membrane) lipids of barley roots as a function of time of treatment. S/U is the ratio of saturated to unsaturated acids. The scale of the left-hand ordinate applies to the right hand ordinate as well. Total gM/g refers to the total of the fatty acids from the polar lipids (16:0+ 18:0+ 18:1 + 18:2 +18:3)
be calculated is less precise. However, quantities of the total fatty acids are given as gmol g 1 freeze-dried roots. The dry weights are about 7% of the fresh weights. Standard deviations of the distributional data are generally within 3% of the distributional values of the three major fatty acids whereas standard deviations of total amounts are about 15% of the amounts.
Results aerator. The flow rate was adjusted by a regulated release of air from the chamber so that adequate aeration and requisite pressure were maintained. Application and removal of the increased pressure took appreciable time such that the roots were exposed to procaine at ambient pressure for 10-20 rain. This may have influenced the results to some degree. Lipids from ground, freeze-dried roots were extracted according to the procedures of Folch et al. (1957), Polar lipids (membrane lipids) were separated from non-polar lipids by rubber-membrane dialysis (Bottcher et al. 1959) and then were saponified with alcoholic K O H according to Burchfield and Storrs (t962). Methyl esters of the resulting fatty acids were prepared for gas chromatography (St. John 1976) with boron trifluoride in methanol as described by Metcalfe and Schmitz (1961). Heptadecanoic acid was added to all samples as an internal standard. The polar lipids of the roots in these studies are predominately phospholipids ( > 95%). The results are expressed as the percent by chromatographic area of the total fatty acids from the polar lipids (C 1 6 : 0 + C 1 8 : 0 + C 1 8 : 1 + C 1 8 : 2 + C 18:3) and as ratios of saturated to unsaturated or less-saturated acids. This was done because the gas-chromatographic data are obtained as relative values. The combination of weighing the samples and quantitative introduction of an internal standard so that quantities can
Procaine (diethylaminoethyl-p-aminobenzoate), tetracaine (2-dimethylaminoethyl 4-n butylaminobenzoate), dibucaine (2-butoxy-N-(2-diethylaminoethyl)cinchoninamide) and chloroform at various concentrations have marked effects on the fatty acids from polar membrane lipids of barley roots (Tables 1, 2). They greatly decreased the proportions of linolenic (18:3) and linoleic (18:2) acids and increased the proportions of oleic (18:1), stearic (18 : 0) and palmitic (16: 0) acids, thereby increasing the degree of saturation of the acids (S/ U) and the ratio, 18:2/18:3. The total amounts of the acids decreased also, so that the amounts of each of the individual acids decreased although the proportions of three of the acids increased. Methanol had much less effect than chloroform, procaine and dibucaine, and ethanol was without significant effect on the distribution. Thus, the effects of methanol on 16:0 and 18:3, and effects
418
P.C. Jackson and J.B. St. John: Anesthetics alter barley-root membrane lipids
Table 3. Effect of procaine on fatty acids from all lipid fractions of cv. Cornpana barley roots" Treatment
% of total fatty acids
S/U
Total amount (gmol/g DW)
16:0
18:0
18:1
18:2
18:3
25.5 28.8
1.2 5.2
3.9 9.6
47.8 42.9
21.7 13.6
0.363 0.515
44.0 8.2
17.7 16.2
21.4 20.8
19,9 19.4
33.9 37.1
7.2 6.6
0.641 0.585
40.5 29.5
9.9 10.9
7.6 7.9
30,0 30.7
50.9 49.3
1.7 1.2
0.211 0.232
43.4 21.1
Polar lipids Untreated Procaine
Non-polar Lipids Untreated Procaine
Free fatty acids Untreated Procaine
a Treated roots were maintained in aerated solutions of 0.1 M procaine +0.1 m M CaSO4 for 6 h. For S/U and Total amount, see footnote to Table I Table 4. Effects of procaine on the polar lipid composition of barley roots (cv. Compana) under increased hydrostatic pressure a Treatment
% of total fatty acids
S/U
18:2 18:3
Total amount (gmol/g DW)
16:0
18:0
18:1
18:2
18:3
I Untreated 0.2 M Procaine +0.87 M P a
25.9 29.8 26.2
1.4 5.8 1.8
4.2 13.6 5.2
47.5 38.0 47.2
21.0 12.7 19.7
0.375 0.554 0.387
2.27 2.99 2.40
44.3 7.7 32.5
I1 Untreated 0.01 M Procaine 0.1 M Procaine + 1.02 MPa
27.6 30.5 31.3 28.2
0.6 1.0 2.4 0.5
3.0 3.8 5.9 2.9
48.0 46.0 43.4 47.6
20.8 18.6 16.6 20.8
0.393 0.460 0.507 0.404
2.30 2.38 2.65 2.29
62.3 45.6 18.9 62.3
" Treated roots were maintained in aerated solutions of 0.1 m M CaSO4 with procaine for 6 h. For S/U and Total amount, see footnote to Table 1
of procaine, tetracaine, dibucaine and chloroform on the proportions of all of the acids are significant at the 1% level of confidence. The anesthetics decreased the total amounts significantly as well. Concentrations of the anesthetics used in these experiments were close to those used by Hendricks and Taylorson (1980) for the enhancement of seed germination, except that the concentration of chloroform was lower because of limited solubility in water. Tables 1 and 2 show that a shift in pH of the anesthetic solution from 5 to 7 has little or no effect on the fatty-acid changes produced by tetracaine (Table 1) or dibucaine (Table 2). The concentration of the positively charged anesthetic amine is essentially constant (both pK,'s = 8.5) as the concentration of the uncharged species increases 100-fold from p H 5 to 7. Thus, the major contribution to the lipid changes appears to be from the protonated species, whereas, the neutral species has only a minor effect, if any.
Effects of procaine and dibucaine on the fattyacid distribution appear to begin as soon as the roots are introduced into the solution and thereafter progress at constant rates for 6 h (Figs. 1, 2). The linear curves all pass through zero with correlation coefficients between 0.95 and 1.00. Fatty acids from roots maintained for 6 h in aerated 0.1 m M CaSO 4 or water without procaine do not change in distribution or total amount as shown by comparison of untreated with control roots in previous studies (Jackson and St. John 1980). Total amounts of the fatty acids from roots in anesthetic solutions decreased so greatly that effects of procaine on lipids other than the membrane (polar) lipids were determined to assess whether the anesthetics have a general effect on all lipids and whether fatty acids lost from the polar lipids appear with the free fatty acids. Typical results are shown for cv. Compana roots in Table 3. Procaine has no effect on the distribution of fatty acids from either the non-polar lipids or the
P.C. Jackson and J.B. St_ John: Anesthetics alter barley-root membrane lipids
50
0.2 M P r o c a i n e
4q
Total rJmol/g
"~ 3C "o
419
ever, the effects of procaine were largely prevented by increased hydrostatic pressure of about i MPa in Table 4. This iricludes the large loss of the total amounts of fatty acids as well as the distributional changes. Pressure curves (Fig. 3) dramatize the prevention and show that pressures greater than 1 MPa are required for maximum effectiveness. The half-maximum effective pressure is about 0.6 MPa for the distributional values. Pressures lower than 0.6 MPa were proportionately less effective (data not shown).
,...,
Discussion m2a
18:3
]
"6
ae
J
Hydrostatic
Pressure
18:2/18:3I 3 6 9 12 x 10
- MPa
Fig. 3. Prevention of the effects of 0.2 M procaine on fatty acids from polar membrane lipids of barley roots by increased hydrostatic pressure. S/U is the ratio of saturated to unsaturated acids. The dotted lines connect to data points of untreated control roots. The scale for the left-hand ordinate applies to the right hand ordinate as well. Total gM/g refers to the total fatty acids from the polar lipids ( 1 6 : 0 + 1 8 : 0 + 1 8 : 0 + 1 8 : 1 +18:2+18:3)
free fatty acids. Although the total amounts decrease (to 73% and 49%, respectively), these losses are not nearly as great as the loss of fatty acids from polar lipids (a decrease to 19%). Effects of procaine on cv. Trebi roots are similar with decreases in total amounts to 58% for the non-polar fraction and to 61% for the free-fatty-acid fraction. No gain is apparent in the free fatty acids so that the large losses of fatty acids from the polar lipids appear to be to the external solution. Studies were undertaken to assess the interaction of increased hydrostatic pressure with the effects of procaine on the polar lipids. Hydrostatic pressure increased to about 2 MPa (20 atmospheres) had no significant effect per se on either the distribution or the total amount of the fatty acids of roots which had not been treated with an anesthetic as long as the roots were aerated during the time under pressure (data not shown). Procaine at ambient pressure under aeration had marked effects on the fatty acids (Table 4). How-
Our results present evidence that anesthetics perturb barley-root membranes sufficiently to alter the composition of the polar membrane lipids. The changes are in the distribution of the major fatty acids from the polar lipids and often are accompanied by loss in the total amount of fatty acids (Table 1). We view the distributional changes and the losses as two effects because distributional changes are not always accompanied by fatty-acid losses (Jackson and St. John 1980, 1982). Tetracaine at 5 m M (Table 1) produced distributional changes but no significant loss in the total amount of fatty acids. Distributional changes produced by dibucaine (Table 2) are as great as those produced by procaine (Table 1 or Fig. 1), but fatty-acid losses from roots are much less in dibucaine (33%) than in procaine (86%). In our previous work, such losses were a feature of marked increase in permeability of the roots to ions (Jackson and Taylor 1970; Jackson 1982). This seems to apply to the action of anesthetics as well (data not shown). The anesthetics alter the membrane-lipid composition apparently as soon as the roots are introduced into the solution containing the anesthetic. This is indicated in Figs. 1, 2 where the effects of procaine and dibucaine, starting from zero time, progress at constant rates for 6 h. Such kinetics parallel those of the effects of undissociated organic acids and phenols on membrane lipids (Jackson and St. John 1980, 1982). These compounds were inferred to act directly on the root membranes. Thus, the anesthetics also are inferred to act primarily on the root membranes. Plasma membranes would be expected to be the most involved initially because of their proximity to the external solution. Further indication that the anesthetics act directly on the membranes comes from the absence of distributional changes in lipids other than the polar membrane lipids (Table 3). This is also true of the effects of undissociated organic acids and phenols.
420
P.C. Jackson and J.B. St. John: Anesthetics alter barley-root membrane lipids
The possibility of anesthetic action via potential changes has been considered, but seems unlikely. This comes from the close parallelism of the anesthetic effects with those of DNP in particular. The kinetics of potential changes produced by DNP vary greatly from the kinetics of DNP effects on the polar lipids and ion permeability, as discussed at length previously (Jackson 1982). Reversal of anesthetic effects by increased hydrostatic pressure is well-documented in animal systems (e.g. Johnson and Flagler 195i; Johnson and Miller 1970) and has been demonstrated in plants by Hendricks and Taylorson (1980) in studies of seed dormancy. The reversal in animal systems has led to the hypothesis that anesthetic action involves membrane expansion (for review, Seeman 1972). Kamaya et al. (1981) have shown that anesthetics expand polymer membranes and that this is counteracted by pressures of about 10 MPa, pressures which are comparable to those used in our experiments. All anesthetics disorder phospholipid bilayers and pressure restores the order (Trudell et al. 1973). Consequently, we interpret the effects of several anesthetics on membrane lipids of roots and the prevention of such effects by increased hydrostatic pressure (Table 4, Fig. 2) as indicating that the anesthetics induce expansion of the root membranes. Substantiation of this comes from the LeChatelier-Braun Principle that an equilibrium displaced by a force can be maintained by application of an opposing force (LeChatelier 1885). Cell wall compression does not preclude membrane expansion. The membrane may become more convoluted or swollen. Barley roots in solutions of anesthetics, phenols or organic acids do not appear to lose turgor for the first hours of treatment, although they generally lose turgor eventually. The increase in ion permeability begins immediately as do the lipid changes. Comparative effects of the various anesthetics used in our studies show that the order of effectiveness is not according to lipid solubility, e.g. methanol is more effective than ethanol which has little or no effect. Chloroform, the most lipid-soluble of the anesthetics used, although more effective than methanol is less effective than the tertiary amines which are predominantly cationic in the pH range used in our experiments (pKa's=8.59.0). The relative efficacy of the various anesthetics which we have used is in the order dibucaine> tetracaine>procaine, an order of effectiveness shown by anesthetic action in animal and synthetic membrane systems. Concentrations of the anesthetics used in our studies also compare closely with those used for pharmacologic activity (e.g.
Badger and Helmkamp 1982; Surewicz and Leyko 1982). We have not used the more lipid-soluble volatile anesthetics because of technical imprecision caused by the volatility and insolubility in water. Nevertheless, the comparisons here, although limited, indicate involvement of factors other than lipid solubility per se. The nature of the changes produced by the anesthetics is closely similar to the nature of the changes produced by undissociated organic acids (Jackson and St. John 1980) and undissociated phenols (Jackson and St. John 1982). All of these diverse compounds increase the relative saturation of the fatty acids which is seen principally as a marked decrease in 18:3 balanced by increase in the proportions of the more saturated acids. The rapidity with which these changes are produced is indicative of an inhibition or repression of a physiological process. Such an increase in saturation, which is a deoxidation or reduction, is indicative of a desaturase inhibition. That desaturation (i.e. oxidation) can be rate-limiting to the maintenance of 18:3 is not surprising when one considers the large reducing capacity of the roots. Inhibition of lipid synthesis is unlikely because of the changes in distribution without significant change in the total amounts of the fatty acids (Table 1; Jackson and St. John 1980, 1982). The most effective molecular species of the tertiary-amine anesthetics evidently is the positively charged protonated amine, the concentration of which changes little with a pH shift from 5 to 7. This comes from lack of a large interaction of pH with the effectiveness of tetracaine (Table 1) and dibucaine (Table 2). Experiments I and II of Table 2 show that increasing the concentrations of dibucaine from 0.3 or 0.5 mM to 1 mM greatly increases the intensity of the effect. However, the effect of 1 mM dibucaine is nearly the same in the three experiments whatever the pH and despite the ]00-fold range of the concentration of neutral dibucaine. This indicates that most, if not all, of the effect is attributable to the protonated species of dibucaine. The effectiveness of the protonated species is consistent with the action of anesthetics in other biological systems (Badger and Helmkamp 1982) and on phospholipid membranes (Boulanger et al. 1981 ; Surewicz and Leyko 1982) and the actions of organic acids (Jackson and St. John 1980) and phenols (Jackson and St. John 1982) on root membrane lipids and permeability to ions (Jackson and Taylor 1970; Jackson 1982). In every case, whether the compounds are amines, phenols or aliphatic acids, the effective species is that which is protonated; i.e., the species which is capable of
P.C. Jackson and J.B. St. John : Anesthetics alter barley-root membrane Iipids
hydrogen bonding. Carboxyl, hydroxyl and amine groups are known to bind to proteins (for review, Van Sumere et al. 1975) and head groups of polar lipids (Boulanger et al. 1981), displacing water by hydrogen bonding of the undissociated or protonated species. Presumably this action results in membrane expansion which alters both the membrane lipids and the areas where hydrophilic ions permeate. An interesting feature of our results is that a physiological process of barley roots (namely, ion uptake) and the polar membrane lipids are influenced by a physical event (swelling or expansion) resulting from interaction between effective chemicals (anesthetics, undissociated organic acids and phenols) and the membranes. References Badger, C.R., Helmkamp, G.M., Jr. (1982) Modulation of phospholipid transfer protein activity: inhibition by local anesthetics. Biochim. Biophys. Acta 692, 32-40 Bakker, E.P., Van den Heuvel, E.J., Wiechmann, A.H.C.A., Van Dam, K. (1973) A comparison between effectiveness of uncouplers of oxidative phosphorylation in mitochondria and in artificial membrane systems. Biochim. Biophys. Acta 292, 78 Bottcher, C.J., Woodford, F.P., Borlsma-Van Houte, E., Gent, C.M. (1959) Methods for the analysis of lipids extracted from human arteries and other tissues. Recl. Tray. Chim. 78, 794-814 Boulanger, Y., Schreier, S., Smith, I.C.P. (1981) Molecular details of anesthetic lipid interaction as seen by deuterium and phosphorus 31 nuclear magnetic resonance. Biochemistry 20, 6824-6830 Burchfield, H.P., Storrs, E.E. (1962) Biochemical applications of gas chromatography. Academic Press, New York London Fink, B.R., ed. (1975) Molecular mechanisms of anesthesia. Raven Press, New York Folch, J., Lees, M., Stanley, G.H. (1957) A simple method for the isolation and purification of total lipids from animal tissue. J. Biol. Chem. 226, 497-509 Hendricks, S.B., Taylorson, R.B, (1980) Reversal by pressure of seed germination promoted by anesthetics. Planta 149, 108-111
421
Jackson, P.C. (1982) Differences between effects of undissociated and anionic 2,4-dinitrophenol on permeability of roots to ions. Plant Physiol. 70, 1373-1379 Jackson, P.C., Stieff, K.J. (1965) Equilibrium and ion exchange properties of potassium and sodium accumulation by barley roots. J. Gem Physiol. 48, 601-616 Jackson, P.C., St. John, J.B. (1980) Changes in membrane lipids of roots associated with changes in permeability. I. Effects of undissociated acids. Plant Physiol. 66, 801-804 Jackson, P.C., St. John, J.B. (1982) Effects of 2,4-dinitrophenol on membrane lipids of roots. Plant Physiol. 70, 858 862 Jackson, P.C., Taylor, J.M. (1970) Effects of organic acids on ion uptake and retention in barley roots. Plant Physiol. 46, 538 542 Johnson, F.H., Flagler, E.A. (1951) Hydrostatic reversal of narcosis in tadpoles. Science 112, 91-92 Johnson, S.M., Miller, K.W. (1970) The antagonism of pressure and anesthesia. Nature (London) 228, 75-76 Kamaya, H., Yukio, S., Ueda, I., Eyring, H. (1981) Anesthetics and high pressure interaction upon elastic properties of a polymer membrane. Proc. Natl. Acad. Sci. USA 78, 3572-3575 LeChatelier, HTL. (1885) Sur les lois de la dissolution. C.R. Acad. Sci. (Paris) 100, 441444 Metcatfe, L.D., Schmitz, A.A. (1961) The rapid preparation of fatty acid esters for gas chromatographic analysis. Anal. Chem. 33, 363-364 Seeman, P. (1972) The membrane actions of anesthetics and tranquilizers. Pharmacol. Rev. 24, 583-655 St. John, J.B, (1976) Manipulation of galactolipid fatty acid composition with substituted pyridazinones. Plant Physiol. 57, 38-40 Surewicz, W.K., Leyko, W. (1982) Interaction of local anesthetics with model phospholipid membranes. The effects of pH and phospholipid composition studied by quenching of an intra membrane fluorescent probe. J. Pharm. Pharmacol. 34, 359-363 Trudell, J.R., Hubbell, W.L., Cohen, E.N. (1973) The effect of two inhalation anesthetics on the order of spin-labeled phospholipid vesicles. Biochim. Biophys. Acta 291,321-327 Van Sumere, C.F., Albrecht, J., Dedonger, A., de Pooter, H., Pc, I. (1975) Plant proteins and phenolics in the chemistry and biochemistry of plant proteins. Proc. Phytochem. Soc., Univ. of Ghent, Belgium, Sept. 1973, pp. 211-247, Harbone, J.B., Van Sumere, C.F., eds. Academic Press, London New York Received 12 December 1983; accepted 16 May 1984
