Ion Movements in Cell Injury - Europe PMC

Ion

Movements in Cell Injury Effects of Inhibition of Respiration and Glycolysis on the Ultastucture and Function of the Epithelial Cells of the Toad Bladder Andrew J. Saladino, M.D., and Benjamin F. Trump, M.D.

EA uLTRAsmucruRAL sruDms done in our laboratory have contributed evidence in support of the hypothesis that movements and redistributions of ions and water form the basis of many of the commonly observed ultrasctural changes which follow injury.' Since exchange of ions and water is primarily the function of plasma membrane permeability and energy-dependent ion transport, it would follow that alterations of these factors would result in altered ultrastuctural patterns. We have been exploring the hypothesis that such pattems are similar to those which occur folloNving cellular injury. Accordingly, we have attempted in the present study to investigate the relationship between alterations of ion transport resulting from the inhibition of energy metabolism and alterations of subcellular organelles. The isolated toad bladder was selected for these studies because of the simplicity of its structure, the wealth of physiologic and morphologic information concerning it, and the many similarities that it bears to the distal part of the nephron in mammals. Materials and Methods A total of 50 male and female toads (Bufo marinus) were obtained between January and July and kept in a dark, moist atmosphere until used. Before manipulation of the bladder, the cloaca was clamped, the toad pithed, and the abdominal wall incised. For in-vivo fixations, a small incision was made in the dome of the left hemibladder, a catheter inserted and secured with a silk suture, and residual urine removed; fixative was then infused rapidly. For experiments utilizing transport chambers, the bladder was removed and immersed in a Ringer's solution, approximately 23°C., of the following composition in milliequivalents per liter: Na+, 114; K+, 2.4; Ca++, 2.4; Mg++, 2.4; Cl-, 117; and PO4=, 2.4 The bladder was hemisected, each alf was placed between the 2 compartments of a Ussing transport chamber,2 and each compartment was filled with 25 mL of the Ringer's solution; the area of exposed bladder in the chamber was 7 sq. cmL From the Departnent of Pathology, Duke University, Durham, N. C. Supported by Grants AM-10698 and GM00726-07. Accepted for publication Jan. 2, 1968. Address for reprint requests: Dr. Trmp, Department of Pathology, Duke University Medical Center, Durham, N. C. 27706.

737

738

Vol. 52, No. 4

SALADINO AND TRUMP

NP or

*OX

ITrT

K ..

~

~

~ ~ ~

~~~~

TExT-EcG. 1. Diagram of the Ussing transport chamber and electrical apparatus. Tissue under study (A) separates 2 conical compartments filled with appropriate fluid. Reservoirs (B, B') communicate with conical compartments. Gas inlets (C, C') facilitate mixing and control of oxygen saturation of the fluid. Spontaneous PD is measured across the tissue with an electrometer (D) through agar bridges (E, E') and reference electrodes (F, F') immersed in a saturated KC1 solution. SCC is measured by adjusting neutralizing voltage from external battery (G) through a variable resistor (H) and reading the current on the microammeter (I). Silver spiral (J, J') and agar bridges (KR, K') facilitate passage of current through tissue.

The essential features of the Ussing transport chamber are illustrated in Text-fig. 1. The bladder wall separates 2 compartments, each of which is in communication with a reservoir. Bubbling of oxygen or nitrogen into sidearm inlets facilitates rotary mixng and saturation of the bathing medium with oxygen or replacement of oxygen with nitrogen. A potential difference (PD) develops across the normally functioning toad bladder, due principally to a metabolically dependent tansport of sodium from the mucosal to the serosal surface.3 When a neutraling voltage is added from an external battery source across a variable resistor, the current in the circuit measured on the microammeter is referred to as the "short circuit current" (SCC); this has been shown to be an accurate measure of the net influx of sodium ion.2,3 The PD's were measured continuously wvith either a Keithely 610 B or 601 electrometer or a Coring Mlodel 12 pH meter, and recorded on a Leeds-Northrup Speedomax WV or Beckman NModel 10 recorder. The SCC was measured once each hour using Simpson 2% microammeters. The tissue resistance was calculated according to Ohm's law by averaging the ratios obtained between PD and SCC when the voltage was increased or decreased in four 10-mv steps.

April 1968

ION MOVEMENTS IN CELL INJURY

739

Sodium efflux from the serosal to mucosal medium was measured by adding, to the serosal medium, a sufficient quantity of 24Na to yield a specific activity of 3 X 10 7to 6 x 107 cpm/mL This increased the original sodium concentrations by less than 0.3%. Samples of mucosal solution taken at serial time intervals were counted on a Packard Model 403 automatic gamma counter. In most cases, tissues were fixed for electron microscopy by injecting a concentrated foxm of the fixative solution into both chamber compartments in order to give the final desired concentration without removal of the original medium. The following fixatives (final concentrations) were used: (1) 1% osmium tetroxide; (2) 2.5% glutaraldehyde; (3) 1% osmium tetroxide and 2.5% glutaraldehyde mixture; and (4) 1% potassium permanganate. Tissues were fixed for 20 min. at 23°C., and those fixed initially in glutaraldehyde alone were postfixed for 30 min. in s-collidine-buffered 1% OSO4. Blocks were dehydrated and embedded in epoxy resin. Some tissues were stained en bloc with uranium acetate prior to dehydrtion.4 Sections were cut with a diamond knife in a Porter-Blum microtome and stained with uranyl magnesium acetate and lead citrate; micrographs were taken on Hitachi HU-11 or HS-7 electron microscopes. Three groups of experiments were performed: 1. Control: Bladders were maintained for 1-31 hr. in the chamber and fixed after the following time intervals: 1 hr. after mounting in the chamber; after 95-99% loss of PD and SCC; and 5 hr. after an increase in sodium efflux. 2. Anoxia: After equilibration, gassing with oxygen was discontinued and the medium was gassed with commercial nitrogen (99.9% oxygen free) which was first passed through a vanadyl sulfate oxygen trap, followed by sodium hydroxide solution and distilled water.5 Tissues were fixed 5 hr. after the increase of sodium efflulx 3. Inhibition of respiration and glycolysis: After equilibration in the chamber, potassium cyanide (final concentration 10-3 I/L.) and iodoacetic acid (final concentration 10-4 M/L.) were added to the medium. Tissues were fixed 5 hr. after the increase of sodium efflux.

Results Eectrophyskilogic Measurements

Electrophysiologic measurements on control and experimental bladders are illustrated in Text-fig. 2. Vlalues for PD, SCC, resistance, and efflux are plotted with respect to time. Control. The PD and SCC rose to a peak value, then decreased slowly toward zero. The peak occurred after 1-2 hr., and this is arbitrarily referred to and represented on the graph as "zero time." The peak values ranged from 30 to 120 mv in various experiments. This variation might relate to such factors as seasonal variation or vagaries of manipulation. (Leaf 6 has measured values as high as 120 mv with most values in the range of 20-50 mv.) Decline in PD followed decline in SCC; a loss of 95-99% of PD and SCC occurred after 15±5 hr. in the chamber. Resistance bore an inverse relationship to SCC and PD in the early hours of the experiment, and to sodium efflux in the latter hours of the experiment. The linearity of the voltage-current plot is shown in Text-fig. 3. As PD and SCC increased, resistance decreased, usually to a level of 3370±750

740

Vol. 52, No. 4

SALADINO AND TRUMP I*N-.

*~~

*EffA,r

60 M.

hei

t000

-I2ooo

0

~~~~20.000 _ 15,000 _ 10,000 a

91"k

*N.

%.

.

OAt

-h

10 _J_ e &

TIME (Hours)

TExTr-vI. 2. Comparison of electrophysiologic properties and sodium efflux

m

the

toad bladder under various conditions. See text for eplanaion.

ohm sq. cm. This is wthin the range reported by other authors.7'8 As PD and SCC decreased, resistance increased up to a value of approximately 24,500 ohm sq. cm. and remained constant for 8-10 hr.; after this time resistance again decreased. Sodium efflux, usualy 8-10 Eq. cm.2 sec.-' during presence of PD and SCC, decreased to less than 4 Eq. cm.-2 sec.-' while high tissue resistance was maintained, then increased rapidly to more than 30 ,'Eq. cm.-2 sec.-' after the decrease in resistance. Experimental. Gassing with nitrogen or addition of potassium cyanide and iodoacetate were initiated 2 hr. after placement in the chamber. This was arbitrarily referred to and represented on the graph as zero time. After zero time the PD rapidly fell to zero in the KCN+IAA tissue and less rapidly in the anoxic tissue, (although more rapidly than in the control tissue). Resistance increased only slightly in the KCN+IAA-treated tissue, then, after a short delay, fell with a subsequent increase of efflux to


April 1968

741

more than 10,000 iqtEq. cm.-2 sec.-l In anoxic tissue, resistance attained a higher value comparable to the control (24,500 ohm sq. cm.) but again fell rapidly after a delay which was shorter than the control but longer than that found in the KCN+IAA tissue (11,600 ohm sq. cm.). High efflux rates occurred slightly sooner in anoxic tissue tn i KCN+IAA tissue.

Control. Although there were marked changes in SCC and PD, the morphology after 1 hr. in the chamber, as well as after loss of 95-99% of PD and SCC, showed few changes from tissue fixed in vivo. TISSU FIXD i vivo. The fine structure of the normal toad bladder fixed in vivo 9 or immediately after removal from the toad 10 has been described and, hence, will only be summarized here. The bladder wall was 15-100 , thick depending upon degree of distention and location of section with respect to collagen and muscle bundles. The epithelial layer was composed of 4 cell types: granulated cells, basal cells, mitochondria-rich cells, and goblet cells (in decreasing order of frequency). By far the most common was the granulated cell which comprised approximately 85% of the surface cell population. The mitochondria-rich cell accounted for 11% of the population and the goblet cell, 6% or less.'1 In general, granulated cells were ovoid (Fig. 1), and mitochondriarich cells were flask-shaped (Fig. 2). The apical plasma membrane was studded with microvilli covered with a nap of filamentous material or mV 90s r

Tr-FI. 3. Linear reLibonship between SCC and PD in 1 anoxia experiment at time aft repla t of oxygen in the bathing media.

zero and 7 hr.

742

SALADINO AND TRUMP

Vol. 52, No. 4

"fuzz." The lateral membranes of adjacent cells were fused by a junctional complex in the apical region, but below were separated by narrow, slitlike spaces containing compressed and interdigitated projections (Fig. 2 and 3). The apical cytoplasm in the mitochondria-rich cell contained numerous smooth-surfaced membranous sacs, while the apical cytoplasm in granulated cells contained numerous granules of varied appearance (Fig. 3). Some of these granules were apparently in the process of discharging their contents into the lumen (Fig. 3). Rough-surfaced endoplasmic reticulum was sparse but the Golgi apparatus was well developed. Mitochondrial profiles were slender rods and often differed in size within the same cell (Fig. 4 and 5). Additional details concerning the normal fine structure appear in the figure legends. rSSUE FXuE AFTER 1 ii. IN cAMER. The appearance of this tissue is illustrated in Fig. 6-10. Epithelial cells were cuboidal or ovoid with basally situated nuclei (Fig. 6). Junctional complexes were tightly closed, but in contrast to material fixed in vivo, there was some widening of the lateral and basilar spaces with separation of the interdigitated cell processes (Fig. 6 and 7). The spaces between the surface cells and between surface and basal cells communicated as a continuous channel to the mucosal basement membrane except in regions of desmosomes. In granulated cells, the apical cytoplasm contained granules of varying shape and density (Fig. 6 and 8). Pale, oval granules fused with the apical membrane and again appeared to be discharging their contents into the mucosal bath (Fig. 8). In mitochondria-rich cells, the apical cytoplasm contained numerous round or elongated smooth-surfaced vesicles. Throughout the cytoplasm of granulated mitochondria-rich and basal cells were other bodies. Some were uniformly dense, while others contained eccentric densities, concentric proffles, or debris (Fig. 6). Multivesicular bodies were commonly found in the supranuclear cytoplasm. A few lipid droplets were present Mitochondrial proffles were concentrated in the supranuclear cytoplasm of granulated cells (Fig. 6) and were distributed throughout mitochondria-rich and basal cells. They varied in size and shape and were not usually as slender as in cells fixed in vivo; cristae were stfill parallel to each other. The Golgi apparatus was well developed in granulated cells, usually occupying a lateral or supranuclear position and sharing it with long parallel stacks of rough-surfaced endoplasmic reticulum (Fig. 6). In some granulated cells there existed a nonbranhing system of tubular profiles which were characterized by a large and relatively uniform caliber and adelicate limiting membrane (Fig. 6, 9, and 10). A fine filamen-


April 1968

743

tous and particulate material occurred within the sac. This system bore no definite spatial relationship to granules or other organelles but is thought to represent a form of smooth-surfaced endoplasmic reticulum; this system is referred to as the macrotubular system. Coursing through the cytoplasm between organelles were numerous long parallel filaments. Widely scattered ribonucleoprotein (RNP) particles and glycogen were present. Microtubules extended from the nuclear area toward the periphery and were a constant feature in glutaraldehydefixed tissue. TISSUES FiCxD AFTE 95-99% LOSS OF PD A-N) SCC

(10-20 HR. i cm,xamaE).

The ultrastructural appearance of all cells was very similar to that observed in cells fixed after 1 hr. (Fig. 11-16). The nucleus appeared to show the greatest change (Fig. 11). There was progressive scalloping of the nuclear outline, sometimes with deep cytoplasmic invaginations, and progressive clumping of nuclear chromatin. The number of cytoplasmic lipid droplets and autophagic granules were increased. Some proffles of rough-surfaced endoplasmic reticulum were swollen (Fig. 14). Fuzzcoated microvilli again occurred at regular intervals (Fig. 11) and were bound by a trilaminar apical plasma membrane (Fig. 12) .When peak-topeak measurements (made by ocular micrometer) on the plasma membranes were compared, the broad apical plasmalemma exceeded the lateral plasmalemma by approximately 30%. Microvilli occurred at regular intervals along the free surface and were covered by a coat of delicate branching filamentous material. A representative junctional complex is seen at high magnification in Fig. 11, 15, and 16. Inner and outer leaflets of the plasma membrane of 2 adjacent cells were well defined; the outer leaflet fused at the lateral border and appeared to form a single leaflet and a "tight" junction. The ratio of apical membrane thickness to tight junction thickness was 1.9 ± 0.2, indicating a fusion of the membrane outer leaflets.12 Below the tight junction, the plasma membrane separated for a short distance to form the intermediate junction. Below the intermediate junction, the plasma membranes became modified to form a desmosome which was characterized by a dense thickening of the plasma membrane. In the space between these dense thickenings, alternating pale and dense areas were seen. TIssuiE: FIXE

5

HR. AFTER INCREASED SODrum

ux

(27-33

m. IN

cHAMBER). The appearance of tissues fixed 5 hr. after increased sodium efflux is illustrated in Fig. 17-23. The epithelial layer remained a relatively flat, uniform sheet of cells; however, all cell types showed degenerative changes. In a few cells degeneration was advanced (Fig. 19, 20, and 22). These

744

SALADINO AND TRUMP

Vol. 52, No. 4

cells were characterized by pale or clear cytoplasm and large increase in volume. Multiple large vacuoles, some of which probably represented plasmalemmal invaginations, were present at lateraL basal, and apical borders. The nuclei were round to ovaL and chromatin became packed along the inner nuclear membrane. The mitochondria showed swelling of the matix compartment, some debris, very few cristae, and a few irregular densities within the matrix. The rough endoplasmic reticulum was swollen and fragmented and ribonucleoprotein particles has become detached. Cytoplasmic fibrils were fragmented and granules were not usually seen in the granulated cells. In the majority of the cells, however, degeneration was less advanced (Fig. 17,18, and 23). Increase in volume was difficult to detect. Peripheral vacuole formation was infrequent, and microvilli were infrequent, and when they did appear were long and slender. Most tight and intermediate junctions and desmosomes were still intact. Nuclei were ovoid and showed a less severe margination of chromatin. While most mitochondria showed clearing of the matrix and an apparent increase in volume, distorted tubular and branching cristae were almost always found. A few mitochondria with condensed matrix and intracristal swelling were present (Fig. 19 and 21). The Golgi apparatus was not recognizable as such, but was likely represented by perinuclear swollen vacuoles. Lipid droplets were frequent, and autophagic vacuoles were more numerous than in tissues fixed in vivo. Normal granules were small or absent. Experimental ANoxiA. The appearance of tissue fixed 5 hr. after the increase in sodium efflux (8-12 hr. in the chamber) is illustrated in Fig. 24-26. Widespread changes were present at this time. The epithelial layer was a sheet of cells whose irregular surface was characterized by numerous swelling and desquamating cells. Desquamating cells showed the most advanced changes. The basal cells, however, showed few changes. Desquamating cells varied greatly in appearance (Fig. 24 and 26). Some possessed irregular shapes with long microvilli, multiple large and small peripheral vacuoles, invaginations, and occasional outpouchings containing material of low density. Others had become round and smooth surfaced except for a few clear outpouchings. No structures resembling junctional complexes were recognizable. Nuclei were usually regular in outline with separation of chromatin material and, occasionally, disintegration of nucleoli. Granules and large, rounded homogeneous areas were often seen. The latter were surrounded by a single membrane and resembled enlarged pale granules. Mitochondria differed in appearance, often witin the same cell (Fig. 26). Some were large with numerous

April 1968


745

slightly swollen cristae; most showed condensation of matrix with swelling of intracristal spaces. Swelling of profiles of endoplasmic reticulum occurred, but the number of profiles appeared to be decreased. Cells which had maintained continuity in the epithelial layer (Fig. 25) showed fewer changes, although some were rounded with loss of microvilli and compression of lateral spaces. Nuclei possessed a very regular outline. Separation of chromatin elements was seen but was not as marked as that observed in desquamating cells. Most mitochondria were condensed, but a few had become swollen and ruptured. Basal cells showed a dramatic change from the control. Many long, slender sacs of rough-surfaced endoplasmic reticulum enveloped mitochondria, which were themselves slightly swollen. CYANIDE AIND IODOACETATE. The appearance of the tissue fixed 5 hr. after an increase in sodium efflux (14 hr. in the chamber) is illustrated in Fig. 27-31. The epithelial layer was an irregularly broken sheet of necrotic cells. Again the basal cells showed less severe change (Fig. 28). The appearance of surface epithelial cells was uniform (Fig. 27 and 28). All cells were greatly increased in volume and there was clearing of cytoplasmic ground substance. Many desmosomes (Fig. 28) and junctional complexes (Fig. 31) had come apart and microvilli were replaced by short, mound-like structures. Remnants of the extracellular coat were, however, stiRl present. Nuclei were round with central clear areas and very dense, marginated chromatin. Large dense particles were adherent to both the outer envelope and to the swollen, fragmented, rough-surfaced endoplasmic reticulum. Dense particles were also seen within greatly altered mitochondria (Fig. 28 and 30). These mitochondria were spheroidaL clear, and contained an occasional cristal remnant; expansion or loss of the outer membrane often occurred. Other membrane systems were unrecognizable. In contrast to the surface epithelium, the majority of basal cells were characterized by the absence of advanced change (Fig. 28). Cells were flattened, and cytoplasmic ground substance was condensed. Nuclei showed margination but little densification of chromatin. Mitochondria were contracted with dense matrices and an occasional swollen intracristal space. Lipid droplets were often present. The endoplasmic reticulum did not appear to be increased. Transition forms between basal cells and granulated cells were sometimes seen. Infrequently, a basal cell could be found with some cytoplasmic clearing and mitochondrial swelling and disruption. In summary, the granulated cells and mitochondria-rich cells reacted similarly. The basal cells, which normally formed an incomplete second

746

Vol. 52, No. 4

SALADINO AND TRUMP

layer, were characterized by an apparent lack of sensitivity to the interruption of glycolysis and respiration. It has been felt that these latter cells are young, undifferentiated epithelial cells 9 which may replace aged or desquamated surface cells. Epithelial desquamation, which occurs slowly in the control, is greatly accelerated after tissues were made anoxic. Presumably, neighboring cells and basal cells filled in the gaps left behind. Discussion Relatohip Beween Sodium Flux and Ult

u

I Aherations Related to Cell Injury

The results of the present study serve to underline the importance of ion and water movements in the pathogenesis of subcellular changes following cell injury. In the normal toad bladder epithelium, it can be assumed that sodium may enter the cell through the plasmalemmas on all sides of the cell; however, the bulk of evidence favors the hypothesis that in the toad bladder the epithelial layer itself represents the major resistance barrier to sodium ions, and that the predominant vectorial sodium transport is confined to the basilar part of the latter.'3 This results in the development of a potential difference between the mucosal and the serosal side, with the mucosal side negatively charged. Although our interpretations are based on this model, it is important to emphasize that this assumption has not as yet been completely established. Thus, alternative pathways of current (i.e., predominantly between the cells) may exist. Since the SCC can be considered as a measure of the net sodium transport from the mucosal to the serosal side,2'3 it can be inferred that when the SCC reaches zero, the net outward sodium transport by the epithelium has ceased. This is based on the assumption that additional sodium pumps do not exist on other sides of the celL In the present experiment, this decrease in sodium "pumping" activity resulted, presumably, from decreased concentration of substrate in control preparations and from decreased levels of high-energy phosphate, after addition of inhibitors. Two types of interference with high-energy phosphate synthesis were studied: (1) anoxia, and (2) inhibition of respiration and glycolysis. It is generally agreed that in cells which are isotonic to their environment and which have sodium as the principal extracellular cation, cell volume regulation is accomplished by the active extrusion of sodiUm.14 It is clear that the activity of the sodium pump exhibits an inverse relationship to the rate of influx of sodium, i.e., in cells that are more permeable to sodium, the sodium extrusion mechanism must be even more active. This is so chiefly because of the presence of nondiffusible intracellular protein anions which result in a tendency toward Gibbs-Donnan equilibrium if ionic permeability is not controlled.

April 1968

I


747

I

TExTr-G. 4. Proposed relationship between ion movement and ultrastructural alterations following inhibition of energy metabolism. While sufficient quantity of the energy-rich compound (X,-P) is available to drive the sodium pump, cell vohlme is maintaned by extrsion of inwardly diffusing sodium ions. After substantial decrease in quantity of X-Z.P, cell volume is maintained by alteration in permeability of plasma membrane to sodium and perhaps to other ions. Ultimately, after prolonged lack of XP, the plasma membrane becomes leaky to sodium ions and water. Entrance of sodium and water into the cell is followed by expansion of endoplasmic reticulum, and expansion of first the outer and then the inner mitochondrial compartment.

Accordingly, it would be predicted that if sodium extrusion is inhibited, the cells would begin increasing in volume immediately. In general, this seems to be the case as evidenced by physiologic experiments of other investigators 15,16 and by correlative morphologic and physiologic experiments in our laboratory.' In isolated flounder kidney tubules incubated in the presence of cyanide, for example, the cells undergo an approximately twofold increase in volume after 30 min. Similarly, measurements of water uptake in rat kidney slices cooled to 0-40 C. indicate a rapid increase in volume. In the present system, however, it was evident that a marked increase of cell volume did not immediately follow cessation of sodium pumping systems. This delay period, during which no changes in cell volume or ultrastructure occurred, was associated with an elevated tissue resistance and a decreased sodium efflux. The decreased sodium efflux might be related to the decreased potential, since the presence of a potential with the mucosal side negative tends to favor sodium efflux. On the other hand, it

748

SALADINO AND TRUMP

Vol. 5Z No. 4

could mean that the membrane had become less permeable to sodium or perhaps to other ions. Since sodium is the major extracellular cation in the present system, and since the cell did not swell for a number of hours, it is tempting to suggest that the permeability to sodium is, in fact, decreased. Further studies, however, will be needed to clarifv this point. Such an idea is, however, supported by our studies on the response of this system to surface active agents. In this case, the plasmalemma is presumably damaged rapidly by the surfactant; swelling occurs immediately and increased resistance does not occur.'1 Our concepts of the relations between morphology and ion movements are summarized in Text-fig. 4. Similar changes involving increased resistance have been observed in plant cells such as Gracilaria folifora 18 and Halocystis ovalis.'9 In the latter alga, the potential difference across the protoplasm following anoxia showed an inverse relationship to resistance as was observed here. The highest resistances measured were as much as 30 times the values in aerated preparations. There is also suggestive evidence that the cell membranes of hibernating animals may become less permeable to sodium ions at low temperatures. In the hamster, for example, water content and sodium concentration of kidney cortex slices exposed to metabolic inhibitors were not significantly greater than control slices incubated at 37C It is interesting to surmise that the existence of such mechanisms in the toad bladder might be correlated with the poikilothermic characteristics of amphibians. In our experiments, however, this change was a transient one and was followed after a few hours by a pronounced decrease in resistance and an increase in sodium permeability as measured by sodium efflux. In this case, the movement of sodium is not down an electrical gradient and, hence, is indicative of increased permeability of cells or intercellular junctions. Ultrastructural considerations indicate that there are at least 3 possible barriers to ion movement in the toad bladder epithelium: the mucosal plasmalemma; the junctional complex, probably most importantly the tight junction; and the serosal plasma membrane. With the present data it is impossible to make a firm statement regarding the mechanism of the observed increased permeability. Increased permeability of cell membranes to sodium is, however, suggested by the marked morphologic changes at the light- and electron-microscopic level that occurred in association with the increased sodium efflux and the decreased resistance. At this time, the cells underwent dramatic increases in volume and equally dramatic configurational changes in subcellular organelles. This correlation between decreased resistance and morphologic change was also observed in studies of human meningioma cells following treatment

April 1968


749

with antiserum plus complement.20 In this system, membrane depolarization and decreased resistance rapidly followed application of antibody and complement. These changes took place 10-0 sec. before changes in morphology could be seen by light microscopy. Although Prieto, Kornblith, and Pollen " reported only light-microscopic changes, the blebbing and other alterations in cell membrane were similar to those described by electron microscopy in the present system. It was noteworthy that, in the present system, expansion of cell volume did not affect all intracellular compartments equally. Indeed, there was good evidence of differential compartment expansion with respect to time. A prominent example of this, discussed in more detail below, was the differential expansion of outer and inner mitochondrial compartments. These morphologic and functional observations, coupled with the temporal resolution between decreased sodium tanLsport activity and morphologic change, are consistent with the notion that many of the subcellular changes resulting from cell injury may be a direct result of ion and water movement and redistributions. It should be noted that the toad bladder, along with certain other systems, may be an exception to the situation observed in mammals, in that morphologic changes following inhibition of mitochondrial energy production are retarded, possibly owing to membrane permeability changes. In other systems these events may be compressed into a much shorter time (e.g. the treated meningioma cells mentioned above) and hence the separation between loss of potentia, increased permeability, and morphologic change may be of the order of seconds or minutes instead of hours. Finally, since our understanding of cellular fine structure is dependent on observations of membrane-limited subcellular compartments, it seems probable that many of the changes that have been or will be discovered utilizing ultrastructural techniques may be a direct result of such modulation in membrane activities. Mitocho-drial Changes I -njued arssue

The present experiments provide an opportunity to observe changes in mitochondrial structure under 3 conditions which are known to grossly alter mitochondrial function: ( 1) inhibition of respiration and glycolysis, (2) anoxia, and (3) deprivation of substrate. The chanages observed are diagrammatically illustrated in Fig. 32. Mitochondria within epithelial cells fixed in vivo were slender, sinuous structures which contained a dense matrix compartment traversed by numerous parallel cristae (A), the leaves of which were slightly sepa-

750

SALADINO AND TRUMP

Vol. 52, No. 4

rated. This appearance was maintained for many hours in the transport chamber. In bladders possessing only negligible potentials, some mitochondria tended to be wider and less sinuous, and there were deviations from the previous parallel patterns of the cristae. Some expansion of the intracristal space occurred (B). Strildng changes took place after loss of resistance and rise of efflux in the control tissue. All mitochondria were swollen, pale, and contained distorted, deranged cristae (F-H). Some mitochondria appeared as empty spheres (I) or irregular collections of membranes (K). A wide variety of structural changes was also observed in anoxic bladders. After loss of resistance and rise of efflux, there were distinct mitochondrial configurations: stout rods, spheres, and permutations of each (A-E). Mfost of the cells within the mucosal layer contained mitochondria similar to control tissues with negligible potential. These mitochondria were less sinuous and showed slight widening and derangement of cristae (A and B). Cells in the basal layer possessed swollen rod-shaped or spherical cristae (D and E). Numerous cells were in the process of desquamation. These possessed mitochondria which were rods or spheres and their cristae were either short, buiblike, or long and thin (B-E). In general, while there was great variety in the appearance of mitochondria, they did not appear as deranged as the cyanide-iodoacetate-treated tissues. The foregoing observations, plus the evident existence of transitional forms between these proposed stages, suggest that tochondria in this system probably undergo both reversible and irreversible transformations. In the figure, it appears that transformations A-F are reversible and may involve only changes in shape. Thus, it seems possible that transformation from a rod (A) to a sphere (D or E) could occur without significant change in volume. Such transformations are, in fact, quite evident as early stages in the beaufiful time-lapse cinematography accomplished by Ch6vremont and Frederc.21 Furthermore, it appears likely that the transitions G-K are terminal, irreversible changes which occur in the stage of necrosis. Such conclusions are consonant with studies of mitochondria in vitro following exposure to swelling agents, or to mitochondna from normal as compared with injured cells.23 The differential expansion of inner and outer mitochondrial compartments, with respect to time, is strongly indicative of differences in the permeability characteristics of these membranes. Thus, the early increase in the volume of the outer compartments is probably a reflection of permeability to sodium and water of this membrane. The inner membrane, on the other hand, seems to maintain impermeability to sodium until a much later stage. The notion that these membranes differ in permeability is strongly supported by evidence that these membranes differ in protein composition,4 as well as

April 1968


751

genetic control.25 Thus, strong and dramatic differences between outer and inner mitochondrial membranes are now accepted, and recent evidence supports the contention that the outer membrane probably "belongs" to the cell and is under the genetic control of the nucleus, while the inner membrane may well be under control of mitochondrial DNA itself. Thus, we conclude that the inner membrane is probably the critical one for the maintenance of the mitochondrial internal environment. The appearance of pronounced mitochondrial swelling in anonc as well as cyanide-treated tissues represents substantial disagreement, with results of investigators studying mitochondrial "swelling" as measured by optical density change in isolated mitochondria.'6 27 In the latter systems, it has been found that both cyanide and anoxia inhibit the "swelling" induced by agents such as calcium or phosphate. It seems probable, however, that in this latter case other parameters such as membrane configuration may result in optical density changes. Significantly, in a correlative morphologic and optical density study of swollen mitochondria in vitro, Malamed28 observed that in contrast to the optical density changes, treatment with cyanide did not in any sense prevent the swelling of mitochondria that could be observed at the ultrastructural level. Summary and Conclusions 1. The ultrastructure and function of the isolated toad bladder maintained for long periods of time in Ussing chambers was studied. 2. Incubation of bladders in control oxygenated media for as long as 20 hr. revealed no significant ultrastructural change. 3. When toad bladders were incubated in media containing cyanide and iodoacetate or in media bubbled with nitrogen rather than oxygen, pronounced ultrastructural changes occurred. These were similar to the changes that characterize irreversible cell injury in other systems. 4. In all cases, an mintial response consisted of increasing resistance, decreasing potential difference and short-circuit current, and decreased efflux of sodium. At this stage, minimal ultrastructural change was observed. Following this, however, there was a decrease in resistance and an increase in sodium efflux, which was correlated with rapid onset of ultrastructural change. 5. These observations are discussed in relation to the hypothesis that ion and water movements are important determining factors in the pathogenesis of subeellular changes following injury. References 1. TRumP, B. F., and GINN, F. L. "he Ultrastructural Basis of Ceular Swelling" (Abst) In Proceedings of the Electron Microscopic Society of America,

752

2.

3. 4. 5. 6.

7. 8.

9. 10. 11.

SALADINO AND TRUMP

Vol. 52, No. 4

A.RcENEAux, C. J., Ed. Claxton's Book Store, Baton Rouge, La., 1967, pp. 206-207. UsswG, H. H., and ZERAN, K. Active transport of sodium as the source of electric current in the short-circuited isolated frog skin. Acta Physiol Scand 23:110-127, 1951. L-AF, A., ANDERSON, J., and PkGE, L. B. Active sodium transport by isolated toad bladder. I Gen Physiol 41:657-668, 1958. FARQuHAR, NM. G., and PAADE, G. E. Cell junctions in amphibian slin. J CeU Biol 26:268-291, 1965. Mris, L., and MrrEs, T. Removal of oxygen from gas streams. Anal Chem 20:984-985, 1948. LEAF, A. Transepithelial transport and its hormonal control in toad bladder. Ergebn Physiol 56:216-263, 1965. LEB, D. E., HosHmo, T., and LrL-DLEY, B. D. Effects of alkali metal cations on the potential across toad and bullfrog urinary bladder. I Gen Physiol 48: 527-540, 1965. BENTLEY, P. J. The effects of contraction of the frog bladder on sodium transport and the responses to oxytocin. Gen Comp Endoct 3:281-285, 1963. CHOI, J. K. The fine structure of the urinarv bladder of the toad, Bufo marinus. I Cell Biol 16:53-72, 1963. PEAcHEY, L. D., and RAsmussEN, H. Structure of the toad's urinary bladder as related to its physiology. J Biophys Biochem Cytol 10:529-553, 1961. KELLER, A. R. A histochemical study of the toad urinary bladder. Anat Rec

147:367-377, 1963. 12. KARNOvSKY, M. J. The ultrastructural basis of capillary permeability studied with peroxidase as a tracer. J CeU Biol 35:213-236, 1967. 13. FR4zaER, H. S. The electrical potential proffle of the isolated toad bladder. I Gen Physiol 45:515-528, 1962. 14. TOSTESON, D. C. "Regulation of Cell Volume by Sodium and Potassium Transport." In The Cellular Functions of Membrane Transport, HoFFmAN, J. F., Ed. Prentice-Hall Englewood Cliffs, N. J., 1964, pp. 1-22. 15. MIUDGE, G. H. Electrolyte and water metabolism of rabbit kidney slides: Effect of metabolic inhibitors. Amer J Physiol 167:206-223, 1951. 16. ToSTEsoN, D. C., and HoFFMAN, J. F. Regulation of ceR volume by active cation transport in high and low potassium sheep red cells. I Gen Physiol 44:169-194, 1960. 17. SALIkDNo, A. J., and TRUMP, B. F. Observations on the role of ion and water movements in the pathogenesis of subcellular reactions to injury. (Abstr). Amer J Path In press. 18. GuTKNEcwr, J. Ion distribution and tansport in the red marine alga, Gracilaria folffera. Biol Bull 129:495-509, 1965. 19. BLNxs, L. R., DAmsI, M. L., and SKow, R. K Bioelectric potentials in Halicystis: VII. The effects of low oxygen tension. J Gen Physiol 22:255-279, 1938. 20. PwE-ro, A., KoRlNJ3Lri, P. L., and PoiwEN, D. A. Electrical recordings from meningioma cells during cytolytic action of antibody and complement. Science 157:1185-1187, 1967. 21. FRiDiEiUc, J., and CHEIREMONT, M. Recherches sur les chondriosomes de cellules vivantes par la microscopie et microcinematographie en contraste de phase. Arch Biol (Liege) 63:109-131, 1952. 22. CiuEmo-r, M., and FPEDEiMCc, J. "Mitochondria" Part I and Part II. Medical and Scientific Motion Pictures. Wymne S. Eastman Productions, Houston, Tex.

April 1968


753

23. TRUMP, B. F., CALDER, J., and SMm!, M. A. Correlation between structure and fimction in mitochondria from injured cells. (abst). Fed Proc 26:514, 1967. 24. GRE, D. E., and PERDUE, J. F. Cerrelation of mitochondrial structure and function. Ann NY Acad Sci 137:667-684, 1966. 25. DAVDsON, R. G., and CowrNER, J. A. Mitochondrial malate dehydrogenase: A new genetic polymorphism in man. Science 157:1569-1571, 1967. 26. INGER, A. L. Water uptake and extrusion by mitochondria in relation to oxidative phosphorylation. Physiol Rev 42:467-517, 1967. 27. HAcKENBRocK, C. R. Ultrastructural bases for metabolically linked mechanical activity in mitochondria. J CeU Biol 30:269-297, 1966. 28. MALAME, S. The effects of cyanide plus g-hydroxybutyrate on changes in mitochondrial absorbancy and ultrastructure induced by hypotonicity and inorganic phosphate. Z Zellforsch 75:272-280, 1966. 29. TRumP, B. F., and BuILGER, R. E. Studies of cellular injury in isolated flounder tubules: Ill. Light microscopic and functional observations in tubules treated with cyanide. Lab Invest In press. 30. ROBEISON, J. R. Osmoregulation in surviving slices from the livers of guinea pigs. Proc Roy Soc (Ser. B) 140:135-144, 1953. 31. WrITIS, J. S. Characteristics of ion transport in kidney cortix of mammalian hibernators. I Gen Physiol 49:1221-1239, 1966. The authors wish to express their smcere appreciation to Dr. John Gutknecht and Dr. Peter J. Bentley, Department of Physiology, Duke University, for mny invaluable discussions, and to Mrs. Jessie Calder, Mrs. Patsy Thacker, frs. Donna Shumaker, Mr. Sumter Brawley III, and Mr. Bernard Bell for their invaluable technical assistance.

[ Illusrations follow ]

754

Vol. 52, No. 4

SALADINO AND TRUMP

Legends for IFigures Ker basal cell B C D E F g G gr H

G/OsO. G +OsO4 OsO, + PF

intercellular space junctional complex lipid mitochondria multivesicular body microtubules macrotubular system clear projections irregular invaginations filamentous cellular coat osmium tetroxide, 1% (final concentration in Ringers solution) glutaraldehyde, 2.5%-osmium tetroxide 1% mixture (final concentration in Ringer's solution) glutaraldehyde, 2.5% (final concentration in Ringers solution), postfixed in s-collidine-buffered 1% osmium tetroxide osmium tetroxide, 1% (final concentration in Ringers solution), postfixed in cacodylate-buffered 4% paraformaldehyde coated vesicles desmosomes endoplasmic reticulum cytoplasmic fibrils glycogen Golgi apparatus granules large homogeneous masses

I J L M mvb Tm TM P V Z

Fig 1. Granulated epithelial cell fixed in vivo. Apical plasma membrane is covered by nap of filamentous material. Nucleus is ovoid; perinuclear cytoplasm contains mitochondria, flattened sacs of rough-surfaced endoplasmic reticulum, well-developed Golgi apparatus, and glycogen particles Apical cytoplasm contains numerous granules. Bundles of filaments course throughout cytoplasm. Collagen fibrils and portion of a basal cell are located below surface epithelial cell. Fixation: OsO, X 16,600. Fig 2. Mitochondria-rich cell fixed in vivo. These cells are typically flask-shaped and often possess numerous blunt microvilli. Below junctional complexes, cells are separated from each other by narrow intercellular spaces containing compressed cellular projections. Mitochondria are dispersed throughout cytoplasm except for apical area which contains numerous vesicles, multivesicular bodies, smooth membrane sacs, and small and large granules. Glycogen is present throughout the cell. Fixation: OsO; x 15,400.


April 1968

z

755 _q

0/,. \-

v. 1551

.:, nos..."'Ma -6.A. IN - -..It

1IPtii.k

-

-0.0'

1.

k04, 't

VI

:-

.

46

IC

In an

t

&

.kV

I

s

;m

2

fir -9 -4.-i

Ar -

la

:-

I

-

.

Af ..*

.tp

'%

A

1.

I

'fi

.w

It

I -;.v

756

SALADINO AND TRUMP

Vol. 52, No. 4

Fig. 3. Apical cytoplasm of granulated cell fixed in vivo. This portion of cell is characterized by filamentous coated microvilli, cytoplasmic densification immediately subjacent to plasma membrane, and granules of varying shapes and densities. Mitochondria are slender sinuous rods and aggregations of gtycogen are present. Cells are attached by a junctional complex and separated by narrow intercellular spaces. Fixation: Os04; x 26,600. Fwg 4. Mitochondria-rich cell fixed in vivo. Matrix is dense, matrix granules contain pale centers, and parallel cristae are angulated (arrows). Fixation: G/Os04; X 55,800. Fig. 5. Mitochondria-rich cell fixed in vivo. Mitochondria are slender rods containing numerous parallel, angulated cristae and a few matrix granules. The Golgi region contained numerous sacs and vesicles. Fixation: Os04; x 39,100.


April 1968

757

z i

It6 txv

*

X

1 3

e .

-

41

j

.

a-

>4

A?

I.

J'

*..

--4;

...

U

(.Y.

t

,

.4

6

-.p

IW4- -,* 114

1l

.

ff.

._v 7,IF

.4 :4

-I

-._ b _l

"

*I

. .

'.

C E

4

,-Io

Vqkt is

='%

X

;o -S-

-

5

.v .

758

SALADINO AND TRUMP

Vol. 52, No. 4

Fig. 6. Control: epithelial cell fixed after 1 hr. in transport chamber. Many different types of granules are present in cytoplasm. Near cell apex, biconcave dense (1) and pale (2) granules are sometimes adjacent to round pale granules (3). Near the Golgi apparatus, smaller, dense, round granules (4) are seen. Granules with lamellar substructure (5) are distributed throughout cytoplasm. Also present in cytoplasm are numerous coated vesicles and sacs of the macrotubular system and of rough-surfaced endoplasmic reticulum. Lateral intercellular spaces are slightly widened. Fixation: G/Os04; x 28,500.


April 1968

-

-,

-

Allpw ..x:Mmk,

759

;4

--

4

427-lii APSMPT-71,

IitIA.

-

a

.

.,

'Ir

v -

it

6

..,..-

4

.,.

I

M. LO.,7

'-p .%

4

.:

IWIL

lw .

%- .,

I&

I..'

1.

ow.

--i.

760

SALADINO AND TRUMP

Vol. 52, No. 4

Fig. 7. Control: junctional complex between 2 granulated cells is composed of a tight junction (1-2), intermediate junction (2-3), and desmosome (3-4). Below this cell attachment is a widened intercellular space containing numerous lateral projections. Fixation: G/Os04; x 55,100. Fig. 8. Conetrol: apical cytoplasm of a granulated cell fixed after 1 hr. in transport chamber. Contents of a pale, round granule appears to be in the process of secretion into the mucosal medium (compare Fig. 3). Fixation: G/Os04, X 20,700. Fig 9. Control: are of cytoplasm in a granulated cell, fixed after 1 hr. in transport chamber, contains a slender profile of the macrotubular system. Fixation: G/Os04; X 41,400. Fig. 10. Control: area of cytoplasm in granulated cell, fixed after 1 hr. in transport chamber, containing terminal portion of a macrotubule whose blind end is covered by a coat of fuzzy material (arrow). Fixation: G/Os04; x 55,200. Fig. 11. Control: surface epithelial cell fixed after 95-99% loss of PD and SCC. Organelles are normally distributed throughout cytoplasm. Sacs of rough-surfaced endoplasmic reticulum are slightly swollen and there is some loss of parallel arrangement of mitochondrial cristae. Nuclear outline is irregular, and chromatin is marginated. Intercellular spaces are widened. Fixation: G+ Os04; x 29,900.

April 1968


/D,6

".

_

761

r .

7

10

11

762

SALADINO AND TRUMP

Vol. 52, No. 4

Fig. 12. Control: apical region of granulated cell, fixed after 95-99% loss of PD and SCC, reveals thick plasma membrane covered by filamentous material. Fixation: G + Os04; x 75,000. Fig. 13. Control: cytoplasmic granules containing dense central area and surrounded by trilaminar membrane. Fixed after 95-99% loss of PD and SCC. Fixation: G + Os04; x 75,000. Fig. 14. Control: perinuclear cytoplasm of a granulated cell, fixed after 95-99% loss of PD and SCC, still contains slender mitochondria and microtubules, although sacs of rough endoplasmic reticulum and mitochondrial cristae (free arrows) are slightly swollen. Fixation: G/Os04; X 75,000. Fig. 15. Control: junctional complex between 2 granulated cells fixed after 95-99% loss of PD and SCC. The tight junction (3-4) contains an intermediate line (free arrows) and is followed by an intermediate junction (2-3) and a desmosome (1-2). The appearance is identical to junctional complexes fixed after 1 hr. in chamber (compare Fig. 7). Fixation: OS04 + PF; x 88,000.

763


April IM

' .- 'Yk Mr

12

12

13S 13

1.

.o jIL4, -$ --

,

I

I. "W

I.,

'IF.

14

s . r

jr_*

-

.,

--Onr 0. -,

-,

II

v

VL

.1,

.

f

W

-.0

*

I

.

0

-t

-W

4"

T

'..1

-

L 15

-

.

r.

i~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ I *A 'C Jrz , 'A'f. 74

i4. s

. , *, uF

~~~~' r

mp*

*

I I

~~0-

-

.1.

t

,

.41. 'K "

I

[-.[.

_

b.

W.

A:P-

53 45v`-2

1160

,0

I

-. I2,

.,.

-

-i

lo

_.. -

'

't

-

;

of

!..

J

, r

.

t

#;Jj

,V---

764

SALADINO AND TRUMP

Vol. 52, No. 4

Fig 16. Control: higher magnification of the tight junction of similar area to that in Fig. 15, again showing the intermediate line (free arrows). Fixation: G + OSO4; x 126,000. Fig 17. Control: epithelial cell fixed after increased efflux of sodium. Degenerative changes are present. Nuclei are ovoid and regular in outline (compare Fig. 6 and 11). Rough-surfaced endoplasmic reticulum and mitochondria are swollen. The latter contains only tubular remnants of cristae. Large vesicular profiles occur near lateral and basal cell margins. Fixation: Os04; x 17,200. Fig 1& Control: swollen irregular mitochondria containing swollen or tubular branching cristae in mitochondria-rich cell fixed after increased effiux of sodium. Fixation: Os04; x 27,600.

April 1968


765

',

16

a:I

2~~~~~~~~~~~

17

18

766

SALADINO AND TRUMP

Vol. 52, No. 4

Fig. 19. Control: desquamating epithelial cell fixed after increased efflux of sodium. Degenerative changes are marked. Mitochondria are swollen and spheroidal, rough-surfaced endoplasmic reticulum is swollen, and lipid droplets are present. Irregular projections and invaginations are present at cell periphery. Margination of chrom3tin is marked. Fixation: G + Os04; X 13,800. Fig 20. Control: smudging and cleavage of desmosomes in granulated cell fixed after increased effiux of sodium. Fixation G + Os04; x 55,000. Fig 21. Control: swollen spherical mitochondrion containing swollen cristal space and condensed matrix in granulated cell fixed after increased efflux of sodium. Fixation:

Os04; x 46,000. Fig 22. Control: 2 mitochondria in granulated cell, fixed after increased efflux of sodium, show severe degenerative changes with interruptions of inner and outer membranes. One is almost completely unrecognizable (M'). Fixation: Os04; x 23,000. Fig 23. Control: large autophagic vacuole in granulated cell, fixed after increased efflux of sodium, contains multiple vesicles and lamellar profiles of membranes. Fixation: Os04; x 69,000.


April 1968

767

0~~~~~

X 1%

~ ~

~

~

20 t~~~~~~~~~~~- s~-

' - ' S-

;t

-s

j J**4

;,

9

9 , .* 4 4-' _ ~ s -,_.,.

4'\ S

'

19

voi~~~~~~~~~~~~~~~~~~~~~~~

21

--

23

EP~~~~~~ .K

22X t.,, .. .. t . t. 5 ~a .

e

768

SALADINO AND TRUMP

Vol. 52, No. 4

Fig. 24. Anoxia: mitochondria-rich cell fixed after increase in sodium effiux. A few granules and homogeneous masses are in cytoplasm. Mitochondria are still rods with parallel cristae. Little change from the cells fixed in vivo and after 1 hr. is evident. Fixation: Os04; x 16,100. Fig 25. Anoxia: desquamating epithelial cell fixed after increase in sodium efflux. Chro-

matin is clumped. Pale homogeneous masses are in cytoplasm together with a number of mitochondrial profiles: (1) slender mitochondria in cross section, (2) spheroidal mitochondria with parallel cristae, (3) swollen spheroidal mitochondria, and (4) swollen mitochondria with swollen or distorted cristae. Fixation: Os04; X 16,100.

April 1968

769


1-4PAO w".1-

11

*W..:,

-,;..

t

9. ..

7A.0 vv %.% -,. do

1.

.

-.,

-14"

.q "'

-.

""e":

-

v

.

px.' -, "-'-A*".

.1

!.

-

nlli.

.

io.,

-,i

24

1.

AF .

*A,,

1.

-v

;j.

.1'

A.

7

Ail

I

...,

AL

'd

-tN

k,

W- -"

.t~

., .,-, .

",

15L

t, I

,j.94NA.

V

A,

;r

V

_i

25

,A,

I

-

.16§w 1,tI

770

SALADINO AND TRUMP

Vol. 52, No. 4

Fig. 26. Anoxia: desquamating mitochondria-rich cell, fixed after increased efflux of sodium, has become round, and projections containing low-density material are present at cell surface. Rough-surfaced endoplasmic reticulum is swollen and RNP particles are detached. Mitochondria assume bizarre shapes and contain numerous swollen cristae. Nucleus is round and contains marginated chromatin. Fixation: Os04; X 18,400.

April 1968


p

5.'S

A

9. A,

f

k P

771

772

SALADINO AND TRUMP

Vol. 52, NMo. 4

Fig 27. KCN+IAA: marked degenerative changes in cells, fixed after increased sodium spheroidal. Cytoplasm is clear. Large, dense particles (free arrows) are adherent to outer nuclear envelope and to a few swollen sacs of rough-surfaced endoplasmic reticulum. Chromatin clumping is marked. Fixation: G/Os04; X 12,800. Fig 28. KCN+IAA: swelling and fragmentation of endoplasmic reticulum and distortion of mitochondrial profiles is present in surface epithelial cells fixed after increased sodium efflux. In contrast, a basal cell contains condensed cytoplasm. Fixation: G/Os04; x 10, 500.

effiux, include massive increases in cytoplasmic volume. Mitochondria are small and

April 1968


773

tN~~~~~~~~~~~~~~~~~~~~~~~~~~4 27~~~~~~~~~~~~~~~~2

'*a~~~~~~~~~* 'A;

h

-14

28~~~~~~~~~~~~~~i

AP~~~~~~~~~~~~~~

774

SALADINO AND TRUMP

Vol. 52, No. 4

Fig 29. KCN+IAA mitochondria-rich cell, fixed after increased effiux of sodium, contains swollen, empty mitochondria. Some mitochondria contain bizarre tubular profiles and densities (M', M"). Fixation: G/Os04; x 25,300. Fig 30. KCN+IAA: fixed after increased sodium effiux, mitochondrial profiles vary in size and possess distorted and interrupted membranes. Intramembranous density is present (arrow). Fixation: G + Os04; x 52,500. Fg 31. KCN+IMA cleavage of tight, intermediate junctions (free arrows) and desmosomes between adjacent cells. Fixation: G + Os04; x 22,400.


Apnil 1968

775

29

.

..t...

30

*

.-

. -.:

~~~~~~~~~~3

776

32

Fw

SALADINO AND TRUMP

Vol. 52, No. 4

W

32. conditions. Diagrammatic representation of mitochondrial profiles observed under 3 expenmental Changes take place in external shape, as well as in spatial orientation and shape of cristae. Appearance in control is represented by A. In anoxic tissue profiles change to stout rods or spheres with minor disorganization and cristal swelling, A-F were all observed. After prolonged lack of new substrate, more marked cristal disorganization and shape changes take place: B, F-I, and K. After potassium cyanide and iodoacetate, interruption of the inner and outer envelope occur J and K. The presence of intermnediate forms and overlap suggest that mitochondrial change proceeds along the lines indicated in the diagram.