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The osmotic behavior and permeability to non-electrolytes of mitochondria
The Osmotic Behavior and Permeability to Non-Electrolytes of Mitochondria’* ’ Henry Tedesch? and Daniel L. Harris From
the Department
of Physiology, Received
University January
of Chicago,
Chicago,
Illinois
14, 1955
The recent literature of biochemistry and cytochemistry leaves little room for doubt that each of the observable constituents of the cell (e.g., mitochondria, cytoplasmic granules, nuclei, etc.) is characterized by a unique chemical composition and functional activity. That such an organization must have for the economy of the cell as a whole both advantages and limitations is obvious enough. It is by no means easy, however, either to define or to evaluate these attributes. Part of the difficulty arises from the fact that we know, as yet, relatively little of the barriers which separate the contents of the inclusions from the surrounding protoplasm, or of the mechanism of interchange of substances across these barriers. To fill this gap in our knowledge, forthright investigations of the permeability of these inclusions are needed; these are beginning to appear (l-3). These investigations are based on the growing belief [see review by Schneider (4)] that mitochondria are surrounded by semipermeable membranes. Recent electron micrographs (5-7) dispel earlier doubts as to the existence of a discrete membrane. However, the evidence that the membrane is semipermeable, while persuasive, is not truly compelling; most is qualitative, much is indirect. There have been repeated observations that mitochondria and other cytoplasmic granules undergo volume changes when subjected in situ or in vitro to anisotonic media [see, for example, Refs. (3, S-lo)]. The only quantitative study of this 1 A dissertation submitted by one of us (H. T.) to the Faculty of the Division of the Biological Sciences of the University of Chicago, in partial fulfillment of the requirements for the Ph.D. degree. 2 Supported in part by the Wallace C. and Clara M. Abbott Memorial Fund. 3 Arno B. Luckhardt Fellow.
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aspect of the problem is that of Harris (lo) who was able to show that pigment granules of the sea-urchin egg obey osmotic laws and that these particles have a permeability to water approximately equal to that of the cell as a whole. Less direct evidence for the existence of a semipermeable membrane comesfrom biochemical studies [see, for example, Lehninger (ll)] which show that exposure of mitochondria to dilute solutions inactivates certain enzymes and activates others. Such treatment, if sufficiently prolonged, may also result in the release of somebut not all the proteins of mitochondria (1, 4). Of the many methods which might be used for obtaining unequivocal evidence for a semipermeablemembrane and a quantitative characterization of its properties, the classicalosmotic method seemsmost promising. Although this method is indirect, the underlying theory is well understood and, used with discretion, it can yield significant data from relatively simple experiments. More importantly, it permits the application of photometric techniques (12, 13) rapid and precise enough to cope with the rapid rates which one may expect to encounter when dealing with particles as small as mitochondria. Indeed, these methods have already been successfully applied to the study of cytoplasmic granules of one sort or another (10, 2, 14). The data which follow show that mitochondria of rat liver obey osmotic laws rather exactly, and that they can withstand, reversibly, at least for a short period of time, rather large increases in volume, seemingly without significant damage or leakage of internal solute. They further show that the optical density of a mitochondrial suspensioncan be interpreted directly and simply in terms of the volume of the mitochondria themselves. Investigations of the permeability of mitochondria to dissolved non-electrolytes prove unequivocally that there is a true semipermeable barrier which is characterized by a high degree of permeability to lipide-soluble substances.In accord with the interpretation of similar data for the erythrocyte and other cells [see, for example, Davson and Danielli (15)], these findings may be taken to mean that the barrier is essentially lipide-like. METHODS Mitochondria were isolated from rat liver by a method based on those of Schneider (16) and Cleland and Slater (17). Female rats of the Sprague-Dawley strain, weighing approximately 150-250 g., were starved for 15-20 hr. and then killed by a blow on the head followed by cervical fracture of the spinal cord. The
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TEDESCHI
AND
DANIEL
L.
HARRIS
liver was excised and placed in about 30 ml. of chilled suspension medium. The rest of the procedure was conducted at 2-5°C. The suspension medium consisted of 0.25 M sucrose, 0.02 M potassium phosphate, and 0.02 M sodium Versenate, adjusted to pH 7.5, and was similar to that of Cleland and Slater (17). Such a medium obviates the agglutinating effects of ions (IS) as well as other poorly understood effects of the calcium ion on oytoplasmic inclusions (10, 3, 19). The tissues were minced with scissors and homogenized with a glass, or Teflon and glass, homogenizer in 3-5 vol. of sucrose-Versenate medium. The homogenate was then diluted about fourfold and centrifuged for 15 min. at 900 X g in an International, size 1, type SB centrifuge to remove cellular debris and nuclei. The supernatant was decanted and centrifuged at 8500 X g for 10 min. in a Servall angle centrifuge. After removal of the “fluffy” layer (20), the pellet was suspended in a small volume of buffered sucrose-Versenate medium and kept at 0” until use. Microscopic examination gave no evidence of agglutination; the mitochondria appeared to be spherical and free from visible contaminants. In view of the removal of the “fluffy” layer, submicroscopic particles were probably not a major contaminant [see (21)]. To measure the volume of mitochondria in anisotonic media, the particles were exposed to various concentrations of chilled, buffered sucrose (pH 7.5). Typically this was accomplished by dilution of the initial suspension with suitable solutions. In these cases there was a small residue (7 X 10-z M) of Versene. For very hypotonic solutions (less than 0.18 osmolal)4 the sedimented mitochondria were suspended directly in hypotonic media. They were then photographed6 as promptly as possible. A conventional compound microscope giving a total magnification on the photographic plate of 4000 times was used. This procedure permits us to measure the mitochondrial diameters at leisure and thus avoid the deterioration attendant on prolonged treatment of the particles with hypotonic solutions. An exposure time of only l/50 sec., made possible by the use of flashbulbs, was utilized to minimize Brownian motion of the suspended particles. This is of some importance in obtaining good photographs of a random sample since the larger particles tend to become fixed to the glass slide more frequently than do the smaller ones. Measurements of mitochondrial diameter were usually made directly from the negative; in a few cases, the positive was employed. Three independent experiments were carried out; several photographs were made at each concentration. From this battery of photographs, 2-7 samples of 100-300 mitochondria were measured at each concentration. Optical density was measured with a Coleman, Junior spectrophotometer, model 6A, set at 520 nut This instrument can be used directly for making “equilibrium” measurements or for following the optical density changes with time produced by slowly penetrating substances. For experiments with rapid penetrants, however, the galvanometer of this instrument is hopelessly slow (period approximately 3.5 sec.) ; in these cases, the output of the photocell was connected directly to a Cambridge Flik galvanometer which has a period of about 0.1 sec. All meas‘Osmolal is defined as total molality of all osmotically active particles and includes both ions and nondissociated molecules. 6 The photographic work was done in conjunction with Mr. J. W. Crunelle, Photographic Department, University of Chicago Clinics.
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urements were made in a constant-temperature room at 20 f 1°C. The temperature of the suspension was held within these or closer limits. In these experiments, a small volume (either 0.15 or 0.5 ml.) of the cold, concentrated suspension of mitochondria was placed in a vial (1.5 or 2.0 cm. diameter, respectively) held in a suitable adapter in the spectrophotometer. At zero time, 20 vol. of 0.3 M non-electrolyte solution buffered with 0.02 M potassium phosphate (pH 7.5) was added rapidly to the suspension with the aid of a syringe and rubber tube. In the case of slow penetrants, optical density was read at frequent intervals, thus permitting full reconstruction of the optical density-time curve. For fast penetrants it was possible to ascertain the time corresponding to but a single point in the curve. The end point chosen was necessarily arbitrary and will be defined more explicitly in the text below. RESULTS
The E$ect of Anisotonic Media on Mitochondria If a suspension of mitochondria is exposed to a hypotonic solution, there is a fall in optical density. Results of a typical experiment are given in curve 2, Fig. 1. (This graph also contains additional data obtained with the samepreparation which will be discussedlater.) It will be seen that there is a very rapid initial fall (which we know to be complete within less than 1 sec., but cannot reproduced on this time scale) followed by a slower decay which lasts for several hours. These changes in optical density might reflect any of a number of phenomena known to occur in mitochondria: (a) an increase in mitochondrial volume [see review of Schneider (4)] resulting in a decrease,by dilution, of the refractive index [seeBarer et al. (22)] of the particles; (b) a hemolysis-like leakage of high molecular weight substances (1, 4) with consequent decreasein refractive index; or (c) a fragmentation of the mitochondria (23). Of these possibilities, only the first should be fully reversible. To test this, mitochondria exposed for various lengths of time to a hypotonic solution (as in curve 2, Fig. 1) were restored to isotonic media by addition of calculated amounts of sucrose.The results (curve 4, Fig. 1) show there is an irreversible change which progressively increases in magnitude..However, its time course is so slow, that while it can account for the slow decline in optical density it cannot account for the initial, rapid fall, which must be interpreted as resulting from volume changes, per se. The irreversible change is properly to be regarded as a secondary phenomenon, dependent on and subsequent to the volume changes induced by the hypotonic solution. Figure 2A shows the optical density of a mitochondrial suspension exposed to a broad range of anisotonic solutions both before (open circles)
56
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TEDESCHI
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DANIEL
L.
HARRIS
FIG. 1. Change in optical density of a suspension of mitochondria exposed to a hypotonic solution or to a solution of a penetrating substance and its reversal by osmotic means. Aliquots (0.5 ml.) of a suspension were placed in a battery of calorimeter tubes. At zero time, a controlled amount of buffered (0.02 M potassium phosphate, pH 7.5) solution was added. At times specified by the circles, either of two procedures was followed: (a) (curves 1, 2, 5) the optical density was read; (5) (curves 3,4) buffered, hypertonic sucrose was added; optical density read l-5 min. later. 1. Effect of penetrating substance. At zero time, 10 ml. of buffered 0.3 M propylene glycol. 2. Effect of hypotonic solution. At zero time, 10 ml. of buffer alone. 3. Reversal of curve 1. At zero time, 5 ml. of buffered 0.3 84 propylene glycol; at specified times, 5 ml. of buffered 0.6 M sucrose. 4. Reversal of curve 2. At zero time, 5 ml. of buffer alone; at specified times, 5 ml. of buffered 0.6 M sucrose. 5. Control. At zero time, 10 ml. of buffered 0.3 M sucrose.
and after (closed circles) restoration of the mitochondria to the original medium. By methods explicitly discussed below, mitochondrial volume can be calculated from optical density of the suspension. This is plotted in Fig. 2B as a function of reciprocal of concentration. It will be noted that the optical density (volume) changes are largely, but not fully, reversible. However, in contrast with changes of mitochondrial volume of the magnitude indicated, the differences in reversibility with concentration are seen to be of real but minor significance. Indeed, it would appear that any volume increase, however modest, induces secondary, irreversible changes which are essentially independent of the volume. The nature of these secondary changes has not been ascertained. Presumably they include in varying degree (a) irreversible swelling, (5) loss of intramitochondrial proteins, i.e., lysis, and (c) fragmentation.
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FIG. 2. Effect of concentration of nonpenetrating substance on (A) the optical density of a suspension and (B) the volume of the mitochondria. Open circles: optical density read 5 sec. after exposure of 0.5 ml. suspension to 10 ml. of buffered (0.02 M potassium phosphate, pH 7.5) sucrose solutions of various concentrations. Closed circles: 5 sec. after exposure of 0.5 ml. suspension to 5 ml. of buffered sucrose solutions, the concentration of the medium was made 0.33 osmolal by addition of 5 ml. buffered sucrose of calculated concentrations; optical density read 30 sec. later. The volume of mitochondria was calculated from the optical density by equations described in the text. Results qualitatively indistinguishable from these have also been obtained when exposure time was 1 min.
Once initiated they seem to be progressive; following restoration to isotonic media, we have found that mitochondria exposed temporarily to hppotonic media deteriorate more rapidly than controls not so exposed. Since the optical-density changes seen during the initial phases (Fig. 1 and 2) are reflections of volume changes per se, it should be possible to calibrate them in terms of mitochondrial volume. This proves to be a relatively simple matter. We first need to establish the relation of mitochondrial volume to the osmotic pressure of the surrounding medium. To this end, photomicrographs of mitochondria exposed to various concentrations of buffered sucrose were measured (see Methods for detail). Although the results (Fig. 3) lack precision, they give little cause to doubt v=KI;+b
(1)
that the mitochondria behave osmotically in accord with the Boyle-van’t Hoff law [Eq. (l)] where C is the osmotic pressure (measured as concentration) ; V, the measured volume; 6, the volume which does not undergo change (osmotic dead space); and KI , a constant. Depending on the concentration one assumes to be isoosmotic with mitochondria, the average normal volume is (from Fig. 3) 0.175-0.190 $; the osmotic dead space is 0.085 $, i.e., of the order of 40-50 % of the normal volume. This figure, which falls in the range of those reported (24) for the erythrocyte, seems
58
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TEDESCHI
AND
DANIEL
L.
HARRIS
FIG. 3. The dependence of mitochondrial volume on concentration of medium. Closed circles: average of mean of 2-7 samples of 100-300 mitochondria each. Perpendicular lines indicate standard diviations (four or more samples). Other points are averagesof two samples. See text for further detail.
not unreasonable in view of the high concentration of proteins and lipides [seereview of Schneider (4)] within the mitochondria. It may be further seenin Fig. 3 that if the osmotic pressure of the medium is reduced by half, the effective volume (V - b) is doubled. We may tentatively infer that, at least during brief exposures to hypotonic solutions, there is little leakage of osmotically active solute from the mitochondria. Were there leakage: (a) we would not expect linearity in Fig. 3; (b) the mitochondria would swell lessthan predicted; (c) on return to isotonic media, the mitochondria would shrink to less rather than more (Fig. 2) than their original volume. This inference is particularly surprising in view of the fact that the mitochondria sustain such a large increase in volume (more than threefold) and apparent surface area (more than twofold). Finally it should be noted that photomicrographs of mitochondria exposed to extremely hypotonic solutions reveal considerable numbers of small particles. As the open circles of Fig. 3 show, this phenomenon is highly variable, possibly because of inevitable variations in exposure time. The empirical relation of optical density to the concentration of mitochondria and to the osmotic pressureof the medium proves to be absurdly simple. Analysis of Fig. 4 shows that the relation of these variables can be expressed by Eq. (2) where 4 is reciprocal of optical density, N is the concentration of mitochondria, C is the concentration of nonpenetrating
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FIO. 4. The dependence of optical density of a mitochondrial suspension on (a) the concentration of mitochondria and (5) the concentration of the medium. The lines are described by Eq. (2) with KS = 33, Kz = 0.045. K, can be evaluated graphically; for Eq. (2) when + = 0, K) = -l/C. Since at constant osmotic pressure, the optical density of a suspension of mitochondria is a function of the refractive index of the external medium (see Fig. 7), the magnitude of KS and Ka will depend on the composition of the external medium. The form of the equation be be the same.
4~=~
[
Kz 1 ,+Ks
1
(2);
solute in the medium, and KZ and KS are constants. If C is held constant, Eq. (2) reduces to Eq. (2a), and optical density is solely a function of N, as one would expect from Beer’s law. If, on the other hand, N is held constant, Eq. (2) becomes Eq. (2b), a relation formally identical with Eq. (1) (see also Fig. 2B). It follows from Eqs. (1) and (2b) that 4 is a linear function of mitochondrial volume. Although these empirical equations would be hard to predict from theoretical considerations, and may be only approximately correct, they seem accurate enough in actual practice. Similar empirical relations also hold for erythrocytes of various species (D. L. Harris, unpublished).
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TEDESCHI
AND
E$ect of Non-Electrolytes
DANIEL
L. HARRIS
on Mitochondria
If we now turn to experiments in which the mitochondria are exposed to isoosmotic solutions of various non-electrolytes, we find that the optical density of the suspension (curve 1, Fig. 1; and Fig. 5) changes in a manner qualitatively identical to that described above for mitochondria exposed to a hypotonic solution. This observation is consistent with the view that the non-electrolyte penetrates, increases the intramitochondrial osmotic pressure, and thus causes simple osmotic swelling. If this interpretation is correct, the swelling should be reversed by addition of suitable amounts of a nonpenetrating substance to the medium; if, on the other hand, it were due to some damage to the mitochondria, comparable to the so-called colloid osmotic hemolysis produced in erythrocytes by soaps, alcohols, and saponins, it should prove to be irreversible. The experiment needed to carry out this classical test, takes the same form as that described above and is plotted in curve 3, Fig. 1. It will be noted that the two sets of data (curves 1 and 2, curves 3 and 4, and Fig. 1) are qualitatively indistinguishable. It may be concluded that the same factors are operative in both experiments, and thus that the optical density changes observed in curve 1 are due to the rapid osmotic-volume changes and the slow secondary changes induced by this primary process. If now we examine the effect of a variety of non-electrolytes, we find results which are entirely comparable to those of Figs. 1 and 5 except
FIG. 5. Penetration description, see text. the horizontal line.
of a non-electrolyte The end point used
(malonamide) into in kinetic experiments
mitochondria. is indicated
For by
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I
)O
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mm
FIG. 6. Reversibility of the volume changes produced by exposure to buffered solutions of penetrating substances. At zero time, 0.5 ml. of mitochondria were exposed to 5 ml. of buffered, 0.3 M non-electrolyte solution. At end point indicated in Fig. 5,5 ml. of buffered 0.6 M sucrose added. Optical density readings made at equilibrium. Controls consisted of buffered 0.3 M sucrose, 0.15 M non-electrolyte. The optical density of mitochondria in this solution differed but slightly from that of mitochondria in buffered 0.3 M sucrose. 1. Relative optical density at the end 4. Urea. 5. Glycerol. 6. Malonamide. point. 2. Acetamide. 3. Methylurea. 7. Erythritol.
that the rate of the initial drop varies from substance to substance. As expected, the secondary decay appears to be essentially independent of the nature of the substance. Control experiments in which the swelling is reversed by addition of nonpenetrating substances at arbitrary end points indicate that all these substancesact as simple penetrants rather than as lytic agents. Representative experiments are given in Fig. 6. As would be expected from Fig. 1, the reversibility decreaseswith length of exposure. Quantitative evaluation of the rate of swelling is beset with practical difficulties. Ideally we would like to have a theoretical or empirical expression which would enable us to transform the optical-density time curves (Fig. 5) into linear functions. This has so far not proved possible. The best we can do is to ascertain the time required to reach some arbitrary end point. The one chosen is that x of the distance between the initial and final optical density. It is difficult enough to ascertain this point in the case of slow penetrants where we can, at least, follow the entire time course of swelling; the troubles are compounded in the caseof rapid penetrants where we can get but a single point on the curve. Some of the more serious difficulties are the following.
62
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HARRIS
FIG. 7. Dependence of optical density of mitochondrial suspension on refractive index of medium. Ordinate: optical density. Abscissa: difference in refractive indices of buffered, 0.38 osmolal solution and distilled water. Vertical lines indicate standsrd deviations, wherever greater than that indicated by diameter of circles. 1. KCl. 2. Malonamide. 3. Erythritol. 4. Sodium citrate. 5. Mannitol. 6. Sucrose.
(a) The optical density of a suspensionof mitochondria seemsto be primarily due to a difference between the refractive indices of mitochondria and medium. It is thus sensitive to the magnitude of the refractive index of the medium, a value which varies with the non-electrolyte being studied. This is illustrated in Fig. 7 (see also curves 3 and 4, Fig. 1). Similar results have also been obtained when the refractive index of the medium is varied by use of various concentrations of polyvinyl pyrrolidone6 in solutions of constant osmotic pressure. For slowly penetrating substances the initial optical density can be obtained by extrapolation of the optical density-time curve back to zero time. For rapid penetrants this method cannot be used, and we have to measure the refractive index of the medium and then interpolate from a graph similar to Fig. 7. Such a graph has to be prepared for each experiment. However, as the substance penetrates, the refractive index of the mitochondria tends to approach that of the medium and at “equilibrium” the optical density of the suspensionis nearly, but not entirely, independent of the nature of the penetrant (seecurves 1 and 2, Fig. 1). (b) The hnal optical density is also uncertain. In addition to the general difficulties of measuring asymptotes, the secondary, irreversible decline 6 Courtesy
of Schenley Laboratories,
Inc.
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in optical density greatly complicates a precise evaluation of the equilibrium optical density, particularly in the case of slow penetrants. As a convenient, if empirical, value we have chosen the optical density produced by a lOO-sec. exposure to buffered 0.3 M propylene glycol. This time period is sufhciently great’to allow almost complete penetration of this substance, but is not so great that it includes much of the secondary process. This procedure automatically corrects for variation in concentration of mitochondria from preparation to preparation. The times required to reach this end point for various non-electrolytes are stated in the legend of Fig. 8. The values given are the means and standard deviations; each is based on at least ten experiments. Using Eq. (3) (25), we can calculate from these data the corresponding perme-
FIN. 8. Permeability of mitochondria to various non-electrolytes as a function of olive oil-water partition coefficient. Closed circles : mitochondria; open circles : Chara (data of Collander and Barlund). Substituting in Eq. (3) (see text) the values: 2.1 X 19* sq. cm. as average surface area, A; 0.35 X 10-r* cc. as the osmotically effective volume, v, at time t; 0.09 X lO-‘a cc. as the corresponding initial volume, vo ; and the factor 3600 to convert the observed times in seconds to hours, we obtain P t = 0.1, where P has the dimensions of cm./hr. and t is given in seconds. The substances used, the observed times in seconds, and the standard deviations follow: 1. Erythritol, 258 f 20.8; 2. Malonamide, 82 f 18.0; 3. Glycerol, 39.8 f 3.7; 4. Urea, 15.6 f 1.5; 5. Thiourea; 8.0 f 3.0; 6. Methylurea, 7.5 & 2.6; 7. Propionamide, 3.5 i 0.9; 8. Acetamide, 3.5 f 1.3; 9. Butyramide, 2.0 f 0.9; 10. Formamide, 2.0 f 0.4; 11. Ethylene glycol, 2.0 f 0.5; 12. Dimethylurea, 1.6 f 0.2; 13. Propylene glycol, 1.6 f 0.6; 14. Valeramide 1.4 hO.3.
64
HENRY
TEDESCHI
AND
DANIEL
pt=14I_O ““-1 2A
L.
[ 1 vf
HARRIS
(3)
ability constants (see legend of Fig. 8). For reasons discussed below, these constants must be regarded as provisional and approximate; they are certainly correct as to order of magnitude. They are plotted in Fig. 8 as a function of the partition coefficient (olive oil/water) of the various substances as determined by Collander and Barlund (26). For comparison the results of these authors on Chara are also plotted. The agreement, both qualitative and quantitative, between the two sets of data is surprisingly great. We may properly conclude from these data that mitochondria possess a semipermeable membrane which is characterized by a high degree of permeability to lipide-soluble substances. In accord with the interpretation of this kind of data for plant and animal cells by many students of permeability [see, for example (15, ZS)], we may infer that the semipermeable membrane of mitochondria contains a high proportion of lipide. DISCUSSION
It has been shown above that on exposure to anisotonic solutions, mitochondria of rat liver can undergo very large volume changes of a magnitude predictable by osmotic laws. These changes are reversible and appear to occur without substantial damage or loss of internal solute, provided exposure to hypotonic media is not too prolonged. It has been further shown that various non-electrolytes penetrate the mitochondria at rates which are correlated with their lipide-solubility. These results provide quantitative and unequivocal evidence that mitochondria possess a semipermeable membrane. They suggest strongly that the membrane is characterized by a high content of lipide. For several reasons, the authors do not claim a high degree of accuracy for either the penetration times reported in Fig. 8 or the permeability constants calculated from them: (a) A more critical evaluation of the equilibrium position is essential to precise interpretation of the end point. (b) The media in which the mitochondria are suspended and to which they are exposed is far from a natural one, being very high in nonelectrolytes and low in the salts and proteins which normally bathe these granules in the cytoplasm. (c) Certain of the assumptions made by Jacobs (25) in the derivation of Eq. (3) are of doubtful validity for the present case. Neither the
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assumption that the surface area remains constant nor the assumption that water penetrates infinitely rapidly in comparison with the solutes, is highly probable. We do know that water penetrates rapidly; indeed, so rapidly does it penetrate that we have not yet succeeded in measuring its rate. This failure prevents us from using the more precise expressions available in the literature. However, the less exact equation probably does not introduce serious mathematical error. Its use is justified, at the present time, by the fact that approximate values of the permeability constants are better than none at all. They are certainly correct in order of magnitude. (d) The surface area of the mitochondria may be very much greater than that which appears from casual inspection. Recent electron micrographs (5-7) indicate the presence of a delicate membrane at the outer surface of the mitochondrion, as well as of thick folds which extend into the interior of the mitochondrial body. This thick layer appears to be bounded on the inside by a second delicate membrane. One possible interpretation of these electron micrographs is that the folds represent extensive convolutions of the surface membrane. If this were the case, the surface area could be, in fact, much larger than appears at first sight. Such an interpretation could account for the fact that mitochondria can withstand reversibly, for a short period of time, very large increases in volume without immediate damage, leakage, or lysis. Sjostrand and Rhodin (6) believe that the space between the two membranes contains a high proportion of lipide. This would be in accord with the interpretations reached in the classical work of Cowdry (27), Bourne (28), and Bensley (29) that mitochondria contain a peripheral lipide or lipoprotein layer. If this is the case our results would suggest that this lipide layer is the primary barrier to passage of non-electrolytes. SUMMARY
The volume changes of rat liver mitochondria exposed to hypotonic solutions or to solutions of penetrating non-electrolytes were studied by direct estimation of mitochondrial volume from photomicrographs and by changes in optical density of mitochondrial suspensions. The data permit the following conclusions about the osmotic behavior and permeability of these particles. 1. Mitochondria undergo reversible volume changes in anisotonic solutions. 2. Minor secondary changes of undetermined origin also occur.
66
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3. Mitochondria obey osmotic (Boyle-van’t Hoff) law. 4. They contain a substantial amount (40-50%) of material which does not participate in volume changes. 5. There appears to be little leakage of osmotically active solute from mitochondria during short exposure to hypotonic solutions. 6. A simple, empirical relation between the optical density of a mitochondrial suspension and mitochondrial volume has been established. 7. Non-electrolytes permeate the mitochondria and thereby induce reversible osmotic volume changes. 8. The rate of penetration is correlated with the olive oil/water partition coefficient of the compound. 9. The permeability constants of the substances studied are quantitatively similar to those found for typical cells. 10. Mitochondria possess a semipermeable membrane which probably contains a high proportion of lipide. REFERENCES
J., BERTHET, L., APPELMANS, F., AND DE DUVE, C., Biochem. J. 60, 182 (1951). CLELAND, K. W., Nature 170,497 (1952). CLELAND, K. W., AND SLATER, E. C., Quart. J. Microscop. Sci. 94, 329 (1963). SCHNEIDER, W. C., J. Histochem. and Cytochem. 1, 212 (1953). PALADE, G. E., J. Histochem. and Cytochem. 1, 188 (1953). SJ~STRAND, F. S., AND RHODIN, J., Exptl. Cell. Research 4, 426 (1953). BRADFIELD, F. R. G., Quart. J. Microscop. Sci. 94, 351 (1963). LEWIS, M. R., AND LEWIS, W. H., Am. J. Anat. 17, 339 (1915). COSTELLO, D. P., Physiol. 2001. 12, 13 (1939). HARRIS, D. L., Biol. Bull. 86, 179 (1943). LEHNINQER, A. L., Phosphorus Metabolism. Symposium on the Role of Phosphorus in the Metabolism of Plants and Animals 1, 344 (1951). $~RSKOV, S. L., Biochem. 2. 279, 241 (1935). PARPART, A. K., J. Cellular Comp. Physiol. 7, 153 (1935).
1. BERTHET, 2.
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
14. RAAFLAUB, J., Helv. Physiol. et Pharmacol. Acta 11, 142 (1953). 16. DAVSON, H., AND DANIELLI, J. F., “The Permeability of Natural Membranee.‘? Cambridge Univ. Press, London, 1943. 16. SCHNEIDER, W. C., J. Biol. Chem. 176, 259 (1948). 17. CLELAND, K. W., AND SLATER, E. C., Biochem. J. 63,547 (1953). 18. LEHNINQER, A. L., in “Enzymes and Enzyme Systems” (J. T. Edsall, ea.). Harvard Univ. Press, Cambridge, Mass., 1951. 19. SLATER, E. C., AND CLELAND, K. W., Biochem. J. 66, 666 (1963). 20. MUNTWYLER, E., SEIFTER, S., AND HARKNESS, D. M., J. Biol. Chem. 184, 181 (1950). 21. SCHNEIDER, W. C., AND HOGEBOOM, G. H., Cancer Research 11.1 (1961). 22. BARER, R., Ross, K. F. A., AND TRACZYK, S., Nature 171,720 (1953).
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23. 24. 25. 26. 27. 28. 29.
OF
MITOCHONDBIA
CLAUDE, A., J. Exptl. Med. 64, 51 (1946). LUCK$, B., AND MCCUTCHEON, M., Physiol. Revs. 12, 68 (1932). JACOBS, M. H., Harvey Lectures 22, 146 (1927). COLLANDER, R., AND BHRLUND, H., Acta Botanica Fennica 11, 5 (1933) COWDRY, E. V., Am. Naturalist 60,157 (1926). BOURNE, G., Austr. J. Expt. Biol. and Med. Sci. 13,239 (1935). BENSLEY, R. R., Anat. Record 69, 341 (1937).
67
the Department
of Physiology, Received
University January
of Chicago,
Chicago,
Illinois
14, 1955
The recent literature of biochemistry and cytochemistry leaves little room for doubt that each of the observable constituents of the cell (e.g., mitochondria, cytoplasmic granules, nuclei, etc.) is characterized by a unique chemical composition and functional activity. That such an organization must have for the economy of the cell as a whole both advantages and limitations is obvious enough. It is by no means easy, however, either to define or to evaluate these attributes. Part of the difficulty arises from the fact that we know, as yet, relatively little of the barriers which separate the contents of the inclusions from the surrounding protoplasm, or of the mechanism of interchange of substances across these barriers. To fill this gap in our knowledge, forthright investigations of the permeability of these inclusions are needed; these are beginning to appear (l-3). These investigations are based on the growing belief [see review by Schneider (4)] that mitochondria are surrounded by semipermeable membranes. Recent electron micrographs (5-7) dispel earlier doubts as to the existence of a discrete membrane. However, the evidence that the membrane is semipermeable, while persuasive, is not truly compelling; most is qualitative, much is indirect. There have been repeated observations that mitochondria and other cytoplasmic granules undergo volume changes when subjected in situ or in vitro to anisotonic media [see, for example, Refs. (3, S-lo)]. The only quantitative study of this 1 A dissertation submitted by one of us (H. T.) to the Faculty of the Division of the Biological Sciences of the University of Chicago, in partial fulfillment of the requirements for the Ph.D. degree. 2 Supported in part by the Wallace C. and Clara M. Abbott Memorial Fund. 3 Arno B. Luckhardt Fellow.
52
PERMEABILITY
OF MITOCHONDRIA
53
aspect of the problem is that of Harris (lo) who was able to show that pigment granules of the sea-urchin egg obey osmotic laws and that these particles have a permeability to water approximately equal to that of the cell as a whole. Less direct evidence for the existence of a semipermeable membrane comesfrom biochemical studies [see, for example, Lehninger (ll)] which show that exposure of mitochondria to dilute solutions inactivates certain enzymes and activates others. Such treatment, if sufficiently prolonged, may also result in the release of somebut not all the proteins of mitochondria (1, 4). Of the many methods which might be used for obtaining unequivocal evidence for a semipermeablemembrane and a quantitative characterization of its properties, the classicalosmotic method seemsmost promising. Although this method is indirect, the underlying theory is well understood and, used with discretion, it can yield significant data from relatively simple experiments. More importantly, it permits the application of photometric techniques (12, 13) rapid and precise enough to cope with the rapid rates which one may expect to encounter when dealing with particles as small as mitochondria. Indeed, these methods have already been successfully applied to the study of cytoplasmic granules of one sort or another (10, 2, 14). The data which follow show that mitochondria of rat liver obey osmotic laws rather exactly, and that they can withstand, reversibly, at least for a short period of time, rather large increases in volume, seemingly without significant damage or leakage of internal solute. They further show that the optical density of a mitochondrial suspensioncan be interpreted directly and simply in terms of the volume of the mitochondria themselves. Investigations of the permeability of mitochondria to dissolved non-electrolytes prove unequivocally that there is a true semipermeable barrier which is characterized by a high degree of permeability to lipide-soluble substances.In accord with the interpretation of similar data for the erythrocyte and other cells [see, for example, Davson and Danielli (15)], these findings may be taken to mean that the barrier is essentially lipide-like. METHODS Mitochondria were isolated from rat liver by a method based on those of Schneider (16) and Cleland and Slater (17). Female rats of the Sprague-Dawley strain, weighing approximately 150-250 g., were starved for 15-20 hr. and then killed by a blow on the head followed by cervical fracture of the spinal cord. The
54
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TEDESCHI
AND
DANIEL
L.
HARRIS
liver was excised and placed in about 30 ml. of chilled suspension medium. The rest of the procedure was conducted at 2-5°C. The suspension medium consisted of 0.25 M sucrose, 0.02 M potassium phosphate, and 0.02 M sodium Versenate, adjusted to pH 7.5, and was similar to that of Cleland and Slater (17). Such a medium obviates the agglutinating effects of ions (IS) as well as other poorly understood effects of the calcium ion on oytoplasmic inclusions (10, 3, 19). The tissues were minced with scissors and homogenized with a glass, or Teflon and glass, homogenizer in 3-5 vol. of sucrose-Versenate medium. The homogenate was then diluted about fourfold and centrifuged for 15 min. at 900 X g in an International, size 1, type SB centrifuge to remove cellular debris and nuclei. The supernatant was decanted and centrifuged at 8500 X g for 10 min. in a Servall angle centrifuge. After removal of the “fluffy” layer (20), the pellet was suspended in a small volume of buffered sucrose-Versenate medium and kept at 0” until use. Microscopic examination gave no evidence of agglutination; the mitochondria appeared to be spherical and free from visible contaminants. In view of the removal of the “fluffy” layer, submicroscopic particles were probably not a major contaminant [see (21)]. To measure the volume of mitochondria in anisotonic media, the particles were exposed to various concentrations of chilled, buffered sucrose (pH 7.5). Typically this was accomplished by dilution of the initial suspension with suitable solutions. In these cases there was a small residue (7 X 10-z M) of Versene. For very hypotonic solutions (less than 0.18 osmolal)4 the sedimented mitochondria were suspended directly in hypotonic media. They were then photographed6 as promptly as possible. A conventional compound microscope giving a total magnification on the photographic plate of 4000 times was used. This procedure permits us to measure the mitochondrial diameters at leisure and thus avoid the deterioration attendant on prolonged treatment of the particles with hypotonic solutions. An exposure time of only l/50 sec., made possible by the use of flashbulbs, was utilized to minimize Brownian motion of the suspended particles. This is of some importance in obtaining good photographs of a random sample since the larger particles tend to become fixed to the glass slide more frequently than do the smaller ones. Measurements of mitochondrial diameter were usually made directly from the negative; in a few cases, the positive was employed. Three independent experiments were carried out; several photographs were made at each concentration. From this battery of photographs, 2-7 samples of 100-300 mitochondria were measured at each concentration. Optical density was measured with a Coleman, Junior spectrophotometer, model 6A, set at 520 nut This instrument can be used directly for making “equilibrium” measurements or for following the optical density changes with time produced by slowly penetrating substances. For experiments with rapid penetrants, however, the galvanometer of this instrument is hopelessly slow (period approximately 3.5 sec.) ; in these cases, the output of the photocell was connected directly to a Cambridge Flik galvanometer which has a period of about 0.1 sec. All meas‘Osmolal is defined as total molality of all osmotically active particles and includes both ions and nondissociated molecules. 6 The photographic work was done in conjunction with Mr. J. W. Crunelle, Photographic Department, University of Chicago Clinics.
PERMEABILITY
OF MITOCHONDRIA
55
urements were made in a constant-temperature room at 20 f 1°C. The temperature of the suspension was held within these or closer limits. In these experiments, a small volume (either 0.15 or 0.5 ml.) of the cold, concentrated suspension of mitochondria was placed in a vial (1.5 or 2.0 cm. diameter, respectively) held in a suitable adapter in the spectrophotometer. At zero time, 20 vol. of 0.3 M non-electrolyte solution buffered with 0.02 M potassium phosphate (pH 7.5) was added rapidly to the suspension with the aid of a syringe and rubber tube. In the case of slow penetrants, optical density was read at frequent intervals, thus permitting full reconstruction of the optical density-time curve. For fast penetrants it was possible to ascertain the time corresponding to but a single point in the curve. The end point chosen was necessarily arbitrary and will be defined more explicitly in the text below. RESULTS
The E$ect of Anisotonic Media on Mitochondria If a suspension of mitochondria is exposed to a hypotonic solution, there is a fall in optical density. Results of a typical experiment are given in curve 2, Fig. 1. (This graph also contains additional data obtained with the samepreparation which will be discussedlater.) It will be seen that there is a very rapid initial fall (which we know to be complete within less than 1 sec., but cannot reproduced on this time scale) followed by a slower decay which lasts for several hours. These changes in optical density might reflect any of a number of phenomena known to occur in mitochondria: (a) an increase in mitochondrial volume [see review of Schneider (4)] resulting in a decrease,by dilution, of the refractive index [seeBarer et al. (22)] of the particles; (b) a hemolysis-like leakage of high molecular weight substances (1, 4) with consequent decreasein refractive index; or (c) a fragmentation of the mitochondria (23). Of these possibilities, only the first should be fully reversible. To test this, mitochondria exposed for various lengths of time to a hypotonic solution (as in curve 2, Fig. 1) were restored to isotonic media by addition of calculated amounts of sucrose.The results (curve 4, Fig. 1) show there is an irreversible change which progressively increases in magnitude..However, its time course is so slow, that while it can account for the slow decline in optical density it cannot account for the initial, rapid fall, which must be interpreted as resulting from volume changes, per se. The irreversible change is properly to be regarded as a secondary phenomenon, dependent on and subsequent to the volume changes induced by the hypotonic solution. Figure 2A shows the optical density of a mitochondrial suspension exposed to a broad range of anisotonic solutions both before (open circles)
56
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TEDESCHI
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DANIEL
L.
HARRIS
FIG. 1. Change in optical density of a suspension of mitochondria exposed to a hypotonic solution or to a solution of a penetrating substance and its reversal by osmotic means. Aliquots (0.5 ml.) of a suspension were placed in a battery of calorimeter tubes. At zero time, a controlled amount of buffered (0.02 M potassium phosphate, pH 7.5) solution was added. At times specified by the circles, either of two procedures was followed: (a) (curves 1, 2, 5) the optical density was read; (5) (curves 3,4) buffered, hypertonic sucrose was added; optical density read l-5 min. later. 1. Effect of penetrating substance. At zero time, 10 ml. of buffered 0.3 M propylene glycol. 2. Effect of hypotonic solution. At zero time, 10 ml. of buffer alone. 3. Reversal of curve 1. At zero time, 5 ml. of buffered 0.3 84 propylene glycol; at specified times, 5 ml. of buffered 0.6 M sucrose. 4. Reversal of curve 2. At zero time, 5 ml. of buffer alone; at specified times, 5 ml. of buffered 0.6 M sucrose. 5. Control. At zero time, 10 ml. of buffered 0.3 M sucrose.
and after (closed circles) restoration of the mitochondria to the original medium. By methods explicitly discussed below, mitochondrial volume can be calculated from optical density of the suspension. This is plotted in Fig. 2B as a function of reciprocal of concentration. It will be noted that the optical density (volume) changes are largely, but not fully, reversible. However, in contrast with changes of mitochondrial volume of the magnitude indicated, the differences in reversibility with concentration are seen to be of real but minor significance. Indeed, it would appear that any volume increase, however modest, induces secondary, irreversible changes which are essentially independent of the volume. The nature of these secondary changes has not been ascertained. Presumably they include in varying degree (a) irreversible swelling, (5) loss of intramitochondrial proteins, i.e., lysis, and (c) fragmentation.
PERMEABILITY
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MITOCHONDRIA
57
FIG. 2. Effect of concentration of nonpenetrating substance on (A) the optical density of a suspension and (B) the volume of the mitochondria. Open circles: optical density read 5 sec. after exposure of 0.5 ml. suspension to 10 ml. of buffered (0.02 M potassium phosphate, pH 7.5) sucrose solutions of various concentrations. Closed circles: 5 sec. after exposure of 0.5 ml. suspension to 5 ml. of buffered sucrose solutions, the concentration of the medium was made 0.33 osmolal by addition of 5 ml. buffered sucrose of calculated concentrations; optical density read 30 sec. later. The volume of mitochondria was calculated from the optical density by equations described in the text. Results qualitatively indistinguishable from these have also been obtained when exposure time was 1 min.
Once initiated they seem to be progressive; following restoration to isotonic media, we have found that mitochondria exposed temporarily to hppotonic media deteriorate more rapidly than controls not so exposed. Since the optical-density changes seen during the initial phases (Fig. 1 and 2) are reflections of volume changes per se, it should be possible to calibrate them in terms of mitochondrial volume. This proves to be a relatively simple matter. We first need to establish the relation of mitochondrial volume to the osmotic pressure of the surrounding medium. To this end, photomicrographs of mitochondria exposed to various concentrations of buffered sucrose were measured (see Methods for detail). Although the results (Fig. 3) lack precision, they give little cause to doubt v=KI;+b
(1)
that the mitochondria behave osmotically in accord with the Boyle-van’t Hoff law [Eq. (l)] where C is the osmotic pressure (measured as concentration) ; V, the measured volume; 6, the volume which does not undergo change (osmotic dead space); and KI , a constant. Depending on the concentration one assumes to be isoosmotic with mitochondria, the average normal volume is (from Fig. 3) 0.175-0.190 $; the osmotic dead space is 0.085 $, i.e., of the order of 40-50 % of the normal volume. This figure, which falls in the range of those reported (24) for the erythrocyte, seems
58
HENRY
TEDESCHI
AND
DANIEL
L.
HARRIS
FIG. 3. The dependence of mitochondrial volume on concentration of medium. Closed circles: average of mean of 2-7 samples of 100-300 mitochondria each. Perpendicular lines indicate standard diviations (four or more samples). Other points are averagesof two samples. See text for further detail.
not unreasonable in view of the high concentration of proteins and lipides [seereview of Schneider (4)] within the mitochondria. It may be further seenin Fig. 3 that if the osmotic pressure of the medium is reduced by half, the effective volume (V - b) is doubled. We may tentatively infer that, at least during brief exposures to hypotonic solutions, there is little leakage of osmotically active solute from the mitochondria. Were there leakage: (a) we would not expect linearity in Fig. 3; (b) the mitochondria would swell lessthan predicted; (c) on return to isotonic media, the mitochondria would shrink to less rather than more (Fig. 2) than their original volume. This inference is particularly surprising in view of the fact that the mitochondria sustain such a large increase in volume (more than threefold) and apparent surface area (more than twofold). Finally it should be noted that photomicrographs of mitochondria exposed to extremely hypotonic solutions reveal considerable numbers of small particles. As the open circles of Fig. 3 show, this phenomenon is highly variable, possibly because of inevitable variations in exposure time. The empirical relation of optical density to the concentration of mitochondria and to the osmotic pressureof the medium proves to be absurdly simple. Analysis of Fig. 4 shows that the relation of these variables can be expressed by Eq. (2) where 4 is reciprocal of optical density, N is the concentration of mitochondria, C is the concentration of nonpenetrating
PERMEABILITY
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59
FIO. 4. The dependence of optical density of a mitochondrial suspension on (a) the concentration of mitochondria and (5) the concentration of the medium. The lines are described by Eq. (2) with KS = 33, Kz = 0.045. K, can be evaluated graphically; for Eq. (2) when + = 0, K) = -l/C. Since at constant osmotic pressure, the optical density of a suspension of mitochondria is a function of the refractive index of the external medium (see Fig. 7), the magnitude of KS and Ka will depend on the composition of the external medium. The form of the equation be be the same.
4~=~
[
Kz 1 ,+Ks
1
(2);
solute in the medium, and KZ and KS are constants. If C is held constant, Eq. (2) reduces to Eq. (2a), and optical density is solely a function of N, as one would expect from Beer’s law. If, on the other hand, N is held constant, Eq. (2) becomes Eq. (2b), a relation formally identical with Eq. (1) (see also Fig. 2B). It follows from Eqs. (1) and (2b) that 4 is a linear function of mitochondrial volume. Although these empirical equations would be hard to predict from theoretical considerations, and may be only approximately correct, they seem accurate enough in actual practice. Similar empirical relations also hold for erythrocytes of various species (D. L. Harris, unpublished).
60
HENRY
TEDESCHI
AND
E$ect of Non-Electrolytes
DANIEL
L. HARRIS
on Mitochondria
If we now turn to experiments in which the mitochondria are exposed to isoosmotic solutions of various non-electrolytes, we find that the optical density of the suspension (curve 1, Fig. 1; and Fig. 5) changes in a manner qualitatively identical to that described above for mitochondria exposed to a hypotonic solution. This observation is consistent with the view that the non-electrolyte penetrates, increases the intramitochondrial osmotic pressure, and thus causes simple osmotic swelling. If this interpretation is correct, the swelling should be reversed by addition of suitable amounts of a nonpenetrating substance to the medium; if, on the other hand, it were due to some damage to the mitochondria, comparable to the so-called colloid osmotic hemolysis produced in erythrocytes by soaps, alcohols, and saponins, it should prove to be irreversible. The experiment needed to carry out this classical test, takes the same form as that described above and is plotted in curve 3, Fig. 1. It will be noted that the two sets of data (curves 1 and 2, curves 3 and 4, and Fig. 1) are qualitatively indistinguishable. It may be concluded that the same factors are operative in both experiments, and thus that the optical density changes observed in curve 1 are due to the rapid osmotic-volume changes and the slow secondary changes induced by this primary process. If now we examine the effect of a variety of non-electrolytes, we find results which are entirely comparable to those of Figs. 1 and 5 except
FIG. 5. Penetration description, see text. the horizontal line.
of a non-electrolyte The end point used
(malonamide) into in kinetic experiments
mitochondria. is indicated
For by
PERMEABILITY
-
OF
z-
-
MITOCHONDRIA
-
I
)O
7
61
mm
FIG. 6. Reversibility of the volume changes produced by exposure to buffered solutions of penetrating substances. At zero time, 0.5 ml. of mitochondria were exposed to 5 ml. of buffered, 0.3 M non-electrolyte solution. At end point indicated in Fig. 5,5 ml. of buffered 0.6 M sucrose added. Optical density readings made at equilibrium. Controls consisted of buffered 0.3 M sucrose, 0.15 M non-electrolyte. The optical density of mitochondria in this solution differed but slightly from that of mitochondria in buffered 0.3 M sucrose. 1. Relative optical density at the end 4. Urea. 5. Glycerol. 6. Malonamide. point. 2. Acetamide. 3. Methylurea. 7. Erythritol.
that the rate of the initial drop varies from substance to substance. As expected, the secondary decay appears to be essentially independent of the nature of the substance. Control experiments in which the swelling is reversed by addition of nonpenetrating substances at arbitrary end points indicate that all these substancesact as simple penetrants rather than as lytic agents. Representative experiments are given in Fig. 6. As would be expected from Fig. 1, the reversibility decreaseswith length of exposure. Quantitative evaluation of the rate of swelling is beset with practical difficulties. Ideally we would like to have a theoretical or empirical expression which would enable us to transform the optical-density time curves (Fig. 5) into linear functions. This has so far not proved possible. The best we can do is to ascertain the time required to reach some arbitrary end point. The one chosen is that x of the distance between the initial and final optical density. It is difficult enough to ascertain this point in the case of slow penetrants where we can, at least, follow the entire time course of swelling; the troubles are compounded in the caseof rapid penetrants where we can get but a single point on the curve. Some of the more serious difficulties are the following.
62
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HARRIS
FIG. 7. Dependence of optical density of mitochondrial suspension on refractive index of medium. Ordinate: optical density. Abscissa: difference in refractive indices of buffered, 0.38 osmolal solution and distilled water. Vertical lines indicate standsrd deviations, wherever greater than that indicated by diameter of circles. 1. KCl. 2. Malonamide. 3. Erythritol. 4. Sodium citrate. 5. Mannitol. 6. Sucrose.
(a) The optical density of a suspensionof mitochondria seemsto be primarily due to a difference between the refractive indices of mitochondria and medium. It is thus sensitive to the magnitude of the refractive index of the medium, a value which varies with the non-electrolyte being studied. This is illustrated in Fig. 7 (see also curves 3 and 4, Fig. 1). Similar results have also been obtained when the refractive index of the medium is varied by use of various concentrations of polyvinyl pyrrolidone6 in solutions of constant osmotic pressure. For slowly penetrating substances the initial optical density can be obtained by extrapolation of the optical density-time curve back to zero time. For rapid penetrants this method cannot be used, and we have to measure the refractive index of the medium and then interpolate from a graph similar to Fig. 7. Such a graph has to be prepared for each experiment. However, as the substance penetrates, the refractive index of the mitochondria tends to approach that of the medium and at “equilibrium” the optical density of the suspensionis nearly, but not entirely, independent of the nature of the penetrant (seecurves 1 and 2, Fig. 1). (b) The hnal optical density is also uncertain. In addition to the general difficulties of measuring asymptotes, the secondary, irreversible decline 6 Courtesy
of Schenley Laboratories,
Inc.
PERMEABILITY
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MITOCHONDRIA
63
in optical density greatly complicates a precise evaluation of the equilibrium optical density, particularly in the case of slow penetrants. As a convenient, if empirical, value we have chosen the optical density produced by a lOO-sec. exposure to buffered 0.3 M propylene glycol. This time period is sufhciently great’to allow almost complete penetration of this substance, but is not so great that it includes much of the secondary process. This procedure automatically corrects for variation in concentration of mitochondria from preparation to preparation. The times required to reach this end point for various non-electrolytes are stated in the legend of Fig. 8. The values given are the means and standard deviations; each is based on at least ten experiments. Using Eq. (3) (25), we can calculate from these data the corresponding perme-
FIN. 8. Permeability of mitochondria to various non-electrolytes as a function of olive oil-water partition coefficient. Closed circles : mitochondria; open circles : Chara (data of Collander and Barlund). Substituting in Eq. (3) (see text) the values: 2.1 X 19* sq. cm. as average surface area, A; 0.35 X 10-r* cc. as the osmotically effective volume, v, at time t; 0.09 X lO-‘a cc. as the corresponding initial volume, vo ; and the factor 3600 to convert the observed times in seconds to hours, we obtain P t = 0.1, where P has the dimensions of cm./hr. and t is given in seconds. The substances used, the observed times in seconds, and the standard deviations follow: 1. Erythritol, 258 f 20.8; 2. Malonamide, 82 f 18.0; 3. Glycerol, 39.8 f 3.7; 4. Urea, 15.6 f 1.5; 5. Thiourea; 8.0 f 3.0; 6. Methylurea, 7.5 & 2.6; 7. Propionamide, 3.5 i 0.9; 8. Acetamide, 3.5 f 1.3; 9. Butyramide, 2.0 f 0.9; 10. Formamide, 2.0 f 0.4; 11. Ethylene glycol, 2.0 f 0.5; 12. Dimethylurea, 1.6 f 0.2; 13. Propylene glycol, 1.6 f 0.6; 14. Valeramide 1.4 hO.3.
64
HENRY
TEDESCHI
AND
DANIEL
pt=14I_O ““-1 2A
L.
[ 1 vf
HARRIS
(3)
ability constants (see legend of Fig. 8). For reasons discussed below, these constants must be regarded as provisional and approximate; they are certainly correct as to order of magnitude. They are plotted in Fig. 8 as a function of the partition coefficient (olive oil/water) of the various substances as determined by Collander and Barlund (26). For comparison the results of these authors on Chara are also plotted. The agreement, both qualitative and quantitative, between the two sets of data is surprisingly great. We may properly conclude from these data that mitochondria possess a semipermeable membrane which is characterized by a high degree of permeability to lipide-soluble substances. In accord with the interpretation of this kind of data for plant and animal cells by many students of permeability [see, for example (15, ZS)], we may infer that the semipermeable membrane of mitochondria contains a high proportion of lipide. DISCUSSION
It has been shown above that on exposure to anisotonic solutions, mitochondria of rat liver can undergo very large volume changes of a magnitude predictable by osmotic laws. These changes are reversible and appear to occur without substantial damage or loss of internal solute, provided exposure to hypotonic media is not too prolonged. It has been further shown that various non-electrolytes penetrate the mitochondria at rates which are correlated with their lipide-solubility. These results provide quantitative and unequivocal evidence that mitochondria possess a semipermeable membrane. They suggest strongly that the membrane is characterized by a high content of lipide. For several reasons, the authors do not claim a high degree of accuracy for either the penetration times reported in Fig. 8 or the permeability constants calculated from them: (a) A more critical evaluation of the equilibrium position is essential to precise interpretation of the end point. (b) The media in which the mitochondria are suspended and to which they are exposed is far from a natural one, being very high in nonelectrolytes and low in the salts and proteins which normally bathe these granules in the cytoplasm. (c) Certain of the assumptions made by Jacobs (25) in the derivation of Eq. (3) are of doubtful validity for the present case. Neither the
PERMEABILITY
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65
assumption that the surface area remains constant nor the assumption that water penetrates infinitely rapidly in comparison with the solutes, is highly probable. We do know that water penetrates rapidly; indeed, so rapidly does it penetrate that we have not yet succeeded in measuring its rate. This failure prevents us from using the more precise expressions available in the literature. However, the less exact equation probably does not introduce serious mathematical error. Its use is justified, at the present time, by the fact that approximate values of the permeability constants are better than none at all. They are certainly correct in order of magnitude. (d) The surface area of the mitochondria may be very much greater than that which appears from casual inspection. Recent electron micrographs (5-7) indicate the presence of a delicate membrane at the outer surface of the mitochondrion, as well as of thick folds which extend into the interior of the mitochondrial body. This thick layer appears to be bounded on the inside by a second delicate membrane. One possible interpretation of these electron micrographs is that the folds represent extensive convolutions of the surface membrane. If this were the case, the surface area could be, in fact, much larger than appears at first sight. Such an interpretation could account for the fact that mitochondria can withstand reversibly, for a short period of time, very large increases in volume without immediate damage, leakage, or lysis. Sjostrand and Rhodin (6) believe that the space between the two membranes contains a high proportion of lipide. This would be in accord with the interpretations reached in the classical work of Cowdry (27), Bourne (28), and Bensley (29) that mitochondria contain a peripheral lipide or lipoprotein layer. If this is the case our results would suggest that this lipide layer is the primary barrier to passage of non-electrolytes. SUMMARY
The volume changes of rat liver mitochondria exposed to hypotonic solutions or to solutions of penetrating non-electrolytes were studied by direct estimation of mitochondrial volume from photomicrographs and by changes in optical density of mitochondrial suspensions. The data permit the following conclusions about the osmotic behavior and permeability of these particles. 1. Mitochondria undergo reversible volume changes in anisotonic solutions. 2. Minor secondary changes of undetermined origin also occur.
66
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3. Mitochondria obey osmotic (Boyle-van’t Hoff) law. 4. They contain a substantial amount (40-50%) of material which does not participate in volume changes. 5. There appears to be little leakage of osmotically active solute from mitochondria during short exposure to hypotonic solutions. 6. A simple, empirical relation between the optical density of a mitochondrial suspension and mitochondrial volume has been established. 7. Non-electrolytes permeate the mitochondria and thereby induce reversible osmotic volume changes. 8. The rate of penetration is correlated with the olive oil/water partition coefficient of the compound. 9. The permeability constants of the substances studied are quantitatively similar to those found for typical cells. 10. Mitochondria possess a semipermeable membrane which probably contains a high proportion of lipide. REFERENCES
J., BERTHET, L., APPELMANS, F., AND DE DUVE, C., Biochem. J. 60, 182 (1951). CLELAND, K. W., Nature 170,497 (1952). CLELAND, K. W., AND SLATER, E. C., Quart. J. Microscop. Sci. 94, 329 (1963). SCHNEIDER, W. C., J. Histochem. and Cytochem. 1, 212 (1953). PALADE, G. E., J. Histochem. and Cytochem. 1, 188 (1953). SJ~STRAND, F. S., AND RHODIN, J., Exptl. Cell. Research 4, 426 (1953). BRADFIELD, F. R. G., Quart. J. Microscop. Sci. 94, 351 (1963). LEWIS, M. R., AND LEWIS, W. H., Am. J. Anat. 17, 339 (1915). COSTELLO, D. P., Physiol. 2001. 12, 13 (1939). HARRIS, D. L., Biol. Bull. 86, 179 (1943). LEHNINQER, A. L., Phosphorus Metabolism. Symposium on the Role of Phosphorus in the Metabolism of Plants and Animals 1, 344 (1951). $~RSKOV, S. L., Biochem. 2. 279, 241 (1935). PARPART, A. K., J. Cellular Comp. Physiol. 7, 153 (1935).
1. BERTHET, 2.
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
14. RAAFLAUB, J., Helv. Physiol. et Pharmacol. Acta 11, 142 (1953). 16. DAVSON, H., AND DANIELLI, J. F., “The Permeability of Natural Membranee.‘? Cambridge Univ. Press, London, 1943. 16. SCHNEIDER, W. C., J. Biol. Chem. 176, 259 (1948). 17. CLELAND, K. W., AND SLATER, E. C., Biochem. J. 63,547 (1953). 18. LEHNINQER, A. L., in “Enzymes and Enzyme Systems” (J. T. Edsall, ea.). Harvard Univ. Press, Cambridge, Mass., 1951. 19. SLATER, E. C., AND CLELAND, K. W., Biochem. J. 66, 666 (1963). 20. MUNTWYLER, E., SEIFTER, S., AND HARKNESS, D. M., J. Biol. Chem. 184, 181 (1950). 21. SCHNEIDER, W. C., AND HOGEBOOM, G. H., Cancer Research 11.1 (1961). 22. BARER, R., Ross, K. F. A., AND TRACZYK, S., Nature 171,720 (1953).
PERMEABILITY
23. 24. 25. 26. 27. 28. 29.
OF
MITOCHONDBIA
CLAUDE, A., J. Exptl. Med. 64, 51 (1946). LUCK$, B., AND MCCUTCHEON, M., Physiol. Revs. 12, 68 (1932). JACOBS, M. H., Harvey Lectures 22, 146 (1927). COLLANDER, R., AND BHRLUND, H., Acta Botanica Fennica 11, 5 (1933) COWDRY, E. V., Am. Naturalist 60,157 (1926). BOURNE, G., Austr. J. Expt. Biol. and Med. Sci. 13,239 (1935). BENSLEY, R. R., Anat. Record 69, 341 (1937).
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