An analysis of physiological states responsible for antero-posterior disintegration in Planaria dorotocephala

AN ANALYSIS OI = PHYSIOLOGICAL STATES RESPONSIBLE FOR ANTERO-POSTERIOR DISINTEGRATION

IN PLANARIA DOROTOCEPHALA

by J. WILLIAM BUCHANAN (Northwestern University)

With 5 Text-figures

Received for publication July 13, 1934

INTRODUCTION

The fact has been established by substantial evidence tha t under suitable conditions the lethal effects of a variety of agents first occur in those regions of embryos and of adults of some of the more simple organisms tha t are most active in oxidative, growth, differentiative, and other metabolic processes. This fact imposes the necessity of determining more accurately and in less general terms the fundanlental nature of the physical and physiological differences tha t are responsible for differences in lethal susceptibility within the organism and between individuals within the species. The problem of the nature of differential susceptibility is the more important for in many cases it has been shown tha t dominant regions in form determination during development and in re- constitution are characterized by relatively high susceptibility to lethal agents. The mechanism of dominance and subordination appears to be associated with these quantitat ive physiological differences.

T H E PROBLEM

The term "susceptibility" has been used in a var iety of ways. In order to simplify a t tack on the problem a distinction will be emphasized here between the death of cells resulting from the effects of an externally applied agent, and cellular and tissue disintegration, for death is not always accompanied by disintegration. Under appropriate conditions and in certain animals, e. g., Planaria, death is followed or accompanied by a disorganization of the tissues, evidenced by the outflow of white cloudy masses, the product of cytolysis. The cell exudate is amorphic and includes numbers of bringht spherules. For purposes of simplifying and concentrating the at tack on one phase of the nature of antero-posterior disintegration, attention will be largely confined to the conditions associated with the occurrence of cytolysis of this character. I t is not assumed, however, t ha t all cytolytic agents which induce cytolysis act in the same waY. In fact, the evidence that cytolysis of this type may be induced in different but related ways constitutes an important contribution incorporated into the present study.

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The work of which the present paper is a par t has been undertaken with the conviction tha t the importance of the concept of the physiological gradient demands an analysis of the factors underlying differential rate of cytolysis. Differential sensitivity to cytolytic agents suggests possible differences in lipoid distribution, s tate and distribution of calcium, or water relations along the axis of the animal, and other concommitants. The experiments reported in the present paper were designed to test further the hypothesis suggested in earlier communications, namely, tha t the gradient in cytolytic disintegration in Planaria is a consequence of axial differences in the ability of the tissues to appropriate water and tha t the cytolytic phenomena are induced by lipoid soluble agents and osmotic unbalance favoring water uptake, but are inhibited by hypertonie agents (BucItA~AI~, 1923, 1926, 1929, 1930a, 1930b, 1931).

I f the conclusion is correct tha t cytolytic disintegration is a consequence of disturbance of water relations in the organism, then the addition of the calcium ion should exert a protective effect against disintegration by distilled water and by lipoid solvents, for the stabilizing action of calcium on permeability and on tissue hydration is well established. Accordingly, in the work about to be reported, Planaria were subjected to various concentrations of calcium chloride; for purposes of comparison the characteristics of the effects of various solutions of chlorides of univalent and bivalent cations, potassium oxalate solutions, hypertonic solutions made up with and without calcium, and solutions of a lipoid solvent with and without added calcium were also studied.

MATERIALS AND METHODS

In all experiments the rapidity of the disintegrative action of distilled water was used as the basis of comparison and the relative effects of the various solutions in accelerating or retarding cytolytic disintegration in a hypotonic medium were determined.

The animals, Planaria dorotocephala, were carefully selected from con- trolled laboratory stocks and thus standardized as accurately as possible with respect to outward appearance, physiological age, and state of nutrition. Small animals, not longer than 15 ram. and consisting of only one zooid, were used. The distilled water employed was taken in seasoned containers from a block tin still after single distillation. Previous analysis showed it to be remarkably free from salts and organic materials (BucKACqA~, 1930a). Before use and before solutions were made it was brought nearly to neutrali ty by bubbling carbon aioxide free air through the stock of water for many hours. Hydrion concentrations ds determined by colorometric methods were between p H 6.8 and p H 7.2 in all experiments except those in which buffered RINGER'S solution was employed. The RII~GER'S solutions were made up in the salt proportions described as isotonic for the frog's heart and were buffered at pH 7.8. For purposes tha t will be apparent farther on, in some experiments the calcium salt was omitted from the RINGElZ formula. Differences in results arc not to be referred to differences in the H ion concentration, for the rapidity of cytolysis is not affected materially within the range of hydrion concentration employed (CHILD, 1930; BUCttAI~AIq, 1930a).

An analysis of physiological states responsible for antero-posterior disintegration &c. 499

Because of the conditioning action of Planaria on distilled water (B~cHA~AN 1930a: CmI~D, 1930), in each test only ten animals were placed in 500 ee. of distilled water or other hypotonie solution, after appropriate washings to remove traces of tap water from the animals and flasks. Temperatures in each set of experiments were room temperatures that varied during the course of the experiment but did not differ materially between the several flasks employed in the set.

Records were made of the time required for the first appearance of disintegration among the animals in each flask, the time for complete disintegration of all aninmls within the flasks, and of the order and rate of disintegration along the long axes of the animals. In order that the effects of the several environments may be compared quantitatively, it is necessary to ascribe certain numerical values to the various stages of disintegration (CHILD, 1915; B[rCHA-'~AN, 1923).

Fig. 1. Five stages in the | eytolytie disintegration of Planaria dorotocephala ar- bitrarily selected and assigned numerical values. For comparing rates of distintegration Stage I is assigned the value 10; Stage II, 8; Stage III, 6; Stage IV, 4; and Stage V, 0. /

Thus the progress of disintegration may be divided into five arbitrary stages (Fig. 1), each stage being assigned a numerical value. In Stage I there is no visible change in the animal and is assigned the value 10. Since at the beginning of each experiment all of the animals in each flask are in Stage I the value for .each flask is 100 (plotted as ordinates in the figures: time intervals between observations are plotted as abscissae). Stage I I is assigned the value 8, and the combined value within a flask is thus reduced when some of the animals show the initiation of eytolysis in the region of the head. Stage I I I represents further disintegration of the anterior end associated frequently with the initiation of disintegration of the extreme posterior end and is assigned the value 6. Stage IV indicates that disintegration is not wholly complete, the region immediately posterior to the pharynx still contains intact and living tissue. The value 4 is assigned to this stage. Complete disintegration is assigned the value 0, for no living tissues of the animal may be observed. Thus as disintegration progresses among the animals within a flask the total value of living material decreases although some animals disintegrate more slowly than others. This device makes

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it possible to distinguish sharply even slight effects of agents on the rate of disintegration.

This method of analysis of relative rates of cytolytic disintegration is useful but by no means ideal. Some of the defects will be apparent if reference is made to Figure 2. From the nature of the graphs it would appear tha t cytolysis begins only after a long delay and then continues with great abruptness, only to slow again as the process nears completion. From many sources, including results given elsewhere (BUCHANAI~, 1929) it is known tha t the cytolytie effect of distilled water, for instance, is prompt. But direct observation employed here determines only effects visible under low magnification which do not become evident immediately on exposure to the cytolytic agent. As disintegration approaches its conclusion the delay is only apparent, except in some concentrations of cMcium, for the arbi t rary values for surviving tissues, s tated above, are imperfectly established. Moreover, except in rapidly eytolyzing animals, observations are more accurate if the t ime intervals are several hours rather than shorter periods, for the method includes a subjective element not possible to eliminate.

The rate of disintegration of the animMs in distilled water varies between different lots of animals, depending upon several conditions. Consequently, a distilled water control lot was carried in each group of experiments and the effects of the agents are to be compared with tha t of distilled water only in experiments conducted simultaneously. Under ordinary laboratory conditions the animals disintegrate completely in distilled water in from ten to twenty hours.

DATA

T H E R E L A T I V E RATES OF D I S I N T E G R A T I O N OF PLANARIA I N M/500 MOLAL SOLUTIONS OF AMMONIUM, POTASSIUM, SODIUM, MA- GNESIUM, AND CALCIUM CHLORIDES AND I N D I S T I L L E D WATER

Figure 2 was plotted as indicated above and is derived from a typical experimentM series taken from a group of five which show essentially the same results. The extreme effectiveness of calcium is remarkable; its Mmost specific protective action is further shown in Figure 3. In dilutions of this character differences in effect due to differences in degree of ionization and in osmotic pressure may be considered relatively minor and are probably little concerned in the very marked effect of the Ca ion. Since the hydrion concentration was approximately the same in all solutions it seems probable the results form a true HOFFMEISTER series showing specific ionic effects. Two facts stand out as significant: Calcium is extremely effective in protecting against hypotonie disintegration; this fact accords with the view tha t the cytolytic disintegration of Planaria is an expression of water appropriation l)y the tissues. Cytolytic disintegration is from anterior toward posterior in all solutions, except that in solutions containing calcium no disintegration occurs; in the calcium solutions the protective effect operates in all regions.

An analysis of physiological states responsible for antero-posterior disintegration &e. 501

THE PROTECTIVE ACTION OF CALCIUM IN RELATION TO CONCEN- TRATION

In Figure 3 it is shown that the Ca ion continues to be effective in protecting against cytolytic disintegration, even when highly dilute. Obviously its action is highly specific in character and not due to its modification of the hypotonicity of distilled water. I t has been held by some that the lethal effects of hypotonic agents are a consequence of loss of essential salts. I t is clear that hypotonicity alone is insufficient to cause death, for the Ca solutions, but little less hypotonic than distilled water do not bring about death. Loss of salts in hypotonic solutions may be regarded as concomitant rather than causal.

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L ~

Fig. 2. Relative rates of cytolytic disintegration of Planaria dorotocephala in M/500 solutions: of: A, KC1; B, NI-I4C1; D, NaC1; E, MgCI~; F, CaC12. C is for distilled water.

B

5 [~, 20

Fig. 3. Relative rates of cytolytic disintegration of Planaria in dilute cal- einm chloride solutions and in distilled water. A, distilled water; B, M/I,000,000 CaC12; C, M/500,000 CaCl~; D, M/100,000

CaC12. In M/50,000 Ca CI~ one or two animals usually disintegrate within forty-eight hours; the others are not affected for periods up to four days. Ordinates and abscissae as

in Figure 2.

Evidently salt loss is prevented by the presence of an extremely dilute concentration of a salt in the environment, or else the Ca ion acts specifically to stabilize boundaries against permeability changes that occur in hypotonic media. Ob- viously the latter is the more probable. In natural waters it is certainly true that Planaria do not cytolyze, yet it is equally certain that these waters are not saturated salt solutions but that they do contain calcium.

In solutions of the order of M/40,000 to M/50,000 calcium chloride the region of the animal posterior to the pharynx does not disintegrate for a long

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time. I t appears that the protective action of this concentration approximately balances the cytolytic effect of the hypotonic medium in this region of the animal, but not elsewhere. Some preliminary experiments with pieces indicate tha t as a consequence of the injury produced by cutting, the posterior pieces are more susceptible to the disintegrative action of the hypotonic medium, including hypotonic calcium solutions than are anterior pieces. This is the reverse of the order of disintegration in intact animals and suggests tha t the differences in response to hypotonic agents along the axis are due to differences in physiological

state rather than differences in di-

#t

20

I0

0

\ \ \

[

Fig. 4. Relative rates of eytolytic disintegration of Planaria in potassium oxalate solutions and in distilled water. A, M/500 potassium oxalate; B, M/1000 potassium oxalate; C, M/l,500 potassium oxalate. Ordinates and

abscissae as in t~igure 2.

stribution of the calcium ion in the several regions of the animal.

T H E EFFECT OF POTASSIUM OXALATE SOLUTIONS AS

COMPARED W I T H D I S T I L L E D W A T E R

Since calcium added to a hypotonic medium retards or prevents cytolytic disintegration, it was suggested tha t the calcium present in the animal may be important in maintaining organization and that its removal might accelerate cytolysis. Figure 4 shows the relative effectiveness in causing cytolysis of several concentrations of potassium oxalate as compared with distilled water. The results demonstrating that this calcium precipitant accelerates cytolysis accord completely with those which show that calcium solutions protect against cytolysis; moreover, they in-

dicate with great disinctness the importance of the Ca ion in maintaining protoplasmic organization. As in distilled water, in oxalate solutions disintegration is initiated in the anterior region of the animal and proceeds posteriorly.

T H E EFFECT OF H Y P E R T O N I C SOLUTIONS CONTAINING CALCIUM, LACKING CALCIUM, AND CONTAINING POTASSIUM OXALATE

The question next arises as to whether the absence of clacium will bring about cytolysis if at the same time water is withdrawn from the organism, or at least prevented from entering; i. e., if the environment is hypertonic. Ob- servations having a bearing on this question are given in the form of a protocol of a typical experiment:


4/25/31. Placed fifty 15 millimeter animals in each of the following solutions: Unmodified Ringer, calcium-free Ringer, and calcium-free Ringer made up in M/500 potassium oxalate. 500 cc. flasks. Room temperature, 17 0. p H 7.8.

After ten hours : No disintegrations. Removed ten from each flask to tap water. All animals from the unmodified Ringer recovered without disintegration. Four from the Ca-free Ringer and five from the Ca-free Ringer in potassium oxalate disintegrated to Stage I I , then recovered.

After thirteen hours: No disintegration. After removal of ten from each lot to tap water all from the unmodified Ringer recovered. Three from the Ca-free Ringer a n d three from the oxalated Ca-free Ringer disintegrated to Stage I I and beyond and then recovered.

After fifteen hours: No evidence of disintegration in the solutions. On removal to tap water the animals from the unmodified Ringer completely recovered. Of the animMs removed from the Ca-free Ringer four reached Stage I I and three Stage I I I , then all recovered. Among those from the Ca-free oxalated Ringer five reached Stage I I and two Stage I I I and recovered without fur ther disintegration.

After twenty hours: No disintegration in the solutions. When ten were removed from each flask and placed in tap water, all from the unmodified Ringer recovered without disintegration. All of the animals from the other two solutions disintegrated to Stage V, disintegration proceeding from anterior toward posterior.

The fact tha t death occurred in the Ca-free Ringer and in the Ca-free oxalated Ringer showed tha t hypertonic solutions afforded no protection against death due to absence of calcium and to the presence of potassium oxalate. On the other hand, hypertonicity completely protected dead and moribund tissue against cytolysis, for on return to natural water those tissues disintegrated.

Incidental to these observations was the fact apparent in other experiments of this series tha t after prolonged exposure to Ringer's solution (two days) erosion of the body wall occurs, areas sloughing off, particularly in the region posterior to the pharynx. I t was noted tha t when these eroded animMs were removed to tap water disintegration of a cytolytic character was initiated a t the anterior end, not in the eroded regions, although the opened areas should afford a place of entrance for the water. In some preliminary experiments with pieces, mentioned above (p. 502), it was found tha t posterior pieces shortly after section are first to disintegrate in distilled water, the reverse of the order of disintegration in the intact animal. Pieces from all regions have two cut ends where the integument is broken and the parenchyma exposed so tha t free entry for the distilled water is afforded, yet the posterior pieces are first to disintegrate. These facts effectively dispose of the argument of WIr~soN (1925) tha t disintegration in Planaria is a consequence of the breakdown of the integument allowing the entrance of the cytolytie agent, and of others variously ex- pressed to the effect tha t the differential susceptibility of an organism may be at t r ibuted to the entrance of injurious substances by mouth (PARKER, 1929).

When these results are compared with those shown in Figures 2 and 3 it is clear tha t cytolysis depends not only on the absence of calcium but also

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on the presence of appropriate osmotic conditions. Depriving the tissues of water by hypertonic Ringer solutions prevents cytolysis but does not prevent death, regardless of whether or not calcium is present. Moreover, in hypertonic environments the disintegration gradient is obliterated whether or not calcium is present, but the order of death described an antero-posterior gradient, as revealed by the axial cytolysis of dead and moribund tissues when returned to tap water.

THE RELATIVE RATES OF DISINTEGRATION IN DISTILLED WATER, IN ETHYL ALCOHOL SOLUTION, AND IN ETHYL ALCOHOL SOLUTION

WITH CALCIUM CHLORIDE

Figure 5 shows that the addition of one per cent ethyl alcohol to distilled water accelerates disintegration, presumably because of its lipoid solvent property.

If calcium chloride is added the re- 190

\

Fig. 5. Relative rates of cytolytic disintegration of Planaria in distilled water, 1 ~ ethyl alcohol, and in 1 ~ ethyl alcohol made up in M/500 calcium chloride. A, alcohol plus calcium; B, distilled water; C, alcohol. Ordinates

and abscissae as in Figure 2.

sulting solution is less effective than alcohol alone. Thus the presence of calcium in some way interferes with the cytolytic action both of distilled water and of ethyl alcohol.

THE EFFECTS OF VARIOUS SOLUTIONS OF SALTS MADE UP

IN TAP WATER

The disintegrative action of all the salts except potassium oxalate employed in the preceding reported experiments was tested in tap water solutions. With no exception the animals lived without visible effect of the agent for more than three days, at which time the experiments were terminated and the animals recovered. In previous work it was shown that Planaria will tolerate one per cent ethyl alcohol in tap water for ex- tended periods (BucRAI~A~, 1923). The results reported here of the action

of these substances in distilled water solutions are true cases of the additive and antagonistic effects of hypotonic and of chemical agents.

GENERAL CONSIDERATIONS

The present work forms a part of a series of studies by the author. Other papers in the series have appeared over several years; a summary of the results is necessary in considering the significance of the present report.


The evidence of a parallelism between relative rate of oxidative metabolism and relative rate of disintegration in the presence of lethal agents is substantial. I t is of more importance here to dall attention to some apparent exceptions. For example, LYoN (1904) found that dividing sea urchin eggs show a rhy thm in susceptibility to KNC, while J. GRAY (1925) failed to find a corresponding rhythm in their rate of oxygen consumption. LYoN used the disintegration of the eggs as the criterion of death: no s ta tement is made as to whether death may precede or accompany the processes of disintegration. LUI~D (1921) found that a deficiency in oxygen supply decreases the rate of oxygen consumption of Planaria, while CHILD (1919a) showed tha t in low oxygen concentrations the disintegration of the animals in KNC is accelerated. Significant also in this connection is the work of ]=IENsttAW and FRANCIS (1934) who observed that in seedlings of Triticum vulgare radiosensitivity increases with increase in growth rate but when growth is experimentally inhibited the sensitivity remains high. From these and other evidences one is not justified in making the generali- zation tha t differentials in rate of disintegration may be in all cases directly at tr ibuted to corresponding differentials in rate of metabolism.

A conspicuous feature of antero-posterior disintegration gradients in Planaria dorotocephala is the cytolysis of the cells of the anterior region first, followed by an orderly sequence of cytolysis of tissues rfiore and more posterior. We have, therefore, to consider first the essential character of cytolysis as protoplasmic response and, secondly, the axial conditions of the animal which determine that this response is first elicited in the anterior region. The essential character of certain types of cytolysis is the swelling of the cell accompanied by an outflow of its contents. This has been studied by yon KNAFF~-LE~z (1908) who concluded that all cytolysis is due primarily to liquefaction of the cell lipoids and tha t thereupon the lipoid free proteins swell or are dissolved by taking up water. Cytolysis is also produced by the dissolving of water soluble phosphatides as well as. those soluble in non-polar solvents such as ether and other common fat solvents. HANSTEE~c-C~ANNF~ (1919, 1922) showed that distilled water dissolves or at least leaches lipoids in plant cells. RU~STRSM (1923) found tha t the lipoids in sea urchin eggs are removed in hypotonic sea water. Evidently the dissolving out or leaching of lipoids is quite commonly a par t of the cytolytic process and HEILBRUNlV (1928, p. 249) states: "Apparent ly most cytolytic agents act first by causing a solution of fats". While evidence adduced by these writers is convincing, STEWARD (1929) failed to confirm the presence of lipoids in the living protoplast and found no phosphatides leached from certain plant cells by distilled water. This is not regarded as invalidating the conclusions of the above writers, for distilled water could not be regarded as affecting more than the water soluble phosphatides and minor redistribhtion of lipoids. An~ a t tempt has been made to evaluate the 15hysiologieal conditions responsible for the ~ntero-posterior differential in rate of cytolysis and to throw some light on the question as to whether this differential is the result of differences in rate of oxidative metabolism, or more directly related to other physiological conditions. The interpretation of certain types of cytolysis outlined above suggested one approach to the problem.

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KNC is a convenient and effective disintegrating agent, inducing the effusion of cell substance in many organisms when employed under appropriate conditions. The radial NC is almost specific as a depressant of biological oxi- dations (EMERSON and BUCHANAN, 1927). Ether is described as a general anti- catalyst of biological oxidatio~as by reason of its adsorption by an enzyme concerned in the process. The effect of combinations of ether and KNC on the oxygen consumption of Planaria was tested (BIrcHANAN, 1926). The results failed to show any additive effects. In fact, KNC reduced oxygen consumption slightly less in the presence of ether than when acting alone.

The comparative effectiveness of KNC as a eytolytic agent when made up in tap water and in tap water solutions containing ether was therefore tested (BucHANAN, 1930b). I t was found that the presence of ether greatly accelerates the disintegrative action of KNC, even when the ether present is so dilute that in the absence of the cyanide the animals tolerate it without evidence of injurious effect.

From these results it seemed reasonable to infer (1) tha t since ether accelerates KNC cytolysis and inhibits its action as a depressant, the cytolytic action of KNC and its depressant action are distinct and highly independent effects; (2) if ether cytolyses by permitting protoplasmic swelling (von KNAFFL-Ln~z, 1908), then KNC disintegration may also involve a swelling effect. In previous work (BucHANAN, 1923) it had been shown tha t ethyl alcohol, another lipoid solvent, also accelerates KNC disintegration.

Direct evidence of a possible effect of KNC on cell swelling was then sought. For this purpose the eggs and early embryos of Amblystoma punctatum were used and the effects of various hypertonic and hypotonic solutions with and without KNC, and of KNC solutions in tap water, on cell vohune were determined by measuring the changes in the length of a column of eggs or of spherical embryos in a vertical tube. The relative effectiveness of KNC as a cytolytie agent in the presence of hyper- and hypotonic solutions was noted (BucHANAN, 1929). I t was found that KNC in all cases caused swelling of the embryos or distinctly lessened the shrinkage in hypertonic solutions. Thus t h e embryo volume in distilled water plus KNC and in Ringer plus KNC solutions was the algebraic sum of the KNC effect and that of the osmotic properties of the solutions. BAaRON (1931) has confirmed this swelling action of KNC and regards it as due to ad- sorbed water.

Thus it is apparent tha t among other possible effects accompanying KNC disintegration is the swelling of the cells. Osmotic conditions which decrease the volume of the embryos protect against the cytolytic action of KNC, while those which increase the cell volume accelerate the cytolytic processes. The results indicate tha t KNC, in par t at least, induces cytolysis by reason of its positive effect on protoplasmic swelling and suggest tha t its additive action with ether and with alcohol may be at tr ibuted to this common action on cell swelling.

The visible lethal effect of the KNC in iso- and hypotonic solutions was first indicated by the whitening of the region of the animal pole in eggs and of the blastoporic lip in early embryos, followed by a progressive involvement


of the organism. In hypertonic solutions no differential rate of cytolytic effect could be noted. Since abstracting water from the embryo by a hypertonic solution obliterates a differential sensitivity to the cytolytic action of KNC, it may be concluded that this differential sensitivity is in some way associated with a differential in the ability of the protoplasm to appropriate water.

Although the order of visible effect was distinct in A~tblysto~yta embryos it was not possible to determine the order of death along the long axis with any accuracy, for recoveries were uncertain. There was nothing in these observations oa the visible effects of KNC under these experimental conditions that indicated whether or not death occurred progressively as did cytolysis, or whether or not a hypertonic agent disturbed the time or order of lethal effect.

Assuming that distilled water acts as stated by ]~ANSTEE:N-CRANNER (1922), namely, by extraction of the lipoids and allowing protoplasmic swelling by reason of osmotic unbalance and adsorption, in the next group of experiments with Planaria it was employed as the cytolytic agent. KNC was abandoned to avoid the action of an ionized salt and its undoubtedly complicated effects on permeability. In distilled water the cytolytic effects might be more reasonably assumed to be due to changes in water distribution and the modification of lipoids. The effect of distilled water on the water content of Planaria was determined by weighing before and after an exposure that was just insufficient to cause disintegration. Certain important difficulties of technique were encountered which were not completely overcome. Therefore the weight determinations were not accurate indices of the amount of water uptake. The method described by Pa~1"I~ (1931) was not tried; perhaps it has some advantages over the weighing method as employed here. The data and observations (BUCHANAN, 1930a) permit the statement that after four hours exposure to distilled water the weight of the animals increases, and with or before the onset of disintegration the volume increases distinctly. I t was found that distilled water is without appreciable effect on the rate of oxygen consumption during the first few hours of exposure, but that it causes a decrease as the exposure continues. The process of disintegration in distilled water describes a perfectly clear antero-posterior gradient, thus indicating that the conditions which render the animal sensitive are more pro- nounced in the anterior than in the posterior region. Again it may be suggested that these results are to be referred to water appropriation, probably the consequence of the lipoid leaching action of hypotonic agents. KEPNER and YOE (1933) were unable to detect an antero-posterior gradient in the disintegration of Stenostomu~n oesophagium in distilled water, but in Planaria dorotocephala it occurs in almost every case when fresh stocks of animals are used and the writer does not hesitate to re-affirm his conclusions stated elsewhere (B~:c~A~A~-, 1930a). Ordinarily the stocks are kept in water drawn from a well sunk along the shore of Lake Michigan. I t was noted that when animals are kept in the Evanston city water after a few weeks they acquire a poor condition, abnormal in color, feed poorly, and occasionally head degeneration occurs. Such animals disintegrate rapidly and quite irregularly in distilled water.

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I-~EILBRUNN (1924) has shown that with temperatures of the order of 35 0 there is death and cytolysis of the eggs of Arbacia and of Cummingia and that this is accompanied by and probably due to the dissolving of lipoids. The action of high temperature as a disintegrating influence on Planaria was studied (BucHANAN, 1930b). The lethal action of high temperature also afforded an opportunity to determine by recoveries something of the relation between order of death due to heat and order of disintegration and the role played by water appropriation in the latter. With data at hand on the action of distilled water on rate of oxygen utilization it was also possible to arrive at a conclusion as to the relation between distilled water disintegration and its effect on oxidative metabolism.

Temperatures higher than 42 0 were unsatisfactory because of some fixation of the tissues. Between 33 o and 37.50 typical eytolytic disintegration occurs in tap water, describing a characteristic antero-posterior gradient. In distilled water the time required for this disintegration is very much less than in tap water. Furthermore, if the animals are pro-treated with distilled water for so short a time as half an hour before being placed in high temperature in tap water they disintegrate much more rapidly than if transferred directly in tap water. This discloses the additive nature of the cytolytic effects of distilled water and high temperature. Since the brief exposure to distilled water induces no appreciable change in rate of oxidative metabolism (BuCHA/~AN, 1931) nor any appreciable increase in water content (BucHANAN, 1930a), the additive action of distilled water and high temperature must be referred to a common effect, presumably on lipoids. Moreover, it is clear that the accelerative action of distilled water on cytolysis is not to be ascribed to an accelerative effect on oxidative metabolism.

If the difference in the ability of the several regions of the animal to appropriate water is an important factor in determining difference in rate of cytolysis along the axis, it ought to be possible to obliterate the disintegration gradient shown in high temperature, in distilled water, in KNC, and in common lipoid solvents, and to prevent cytolysis by depriving the organism of water, thereby reducing the free water content, decreasing the dispersion of lipoids, and lessening the mechanical action of distention which is concerned in the cellular breakdown. Planar~:a were therefore subjected to high temperature in Ringer's solution (BucHANAN, 1930b). In accord with expectations from the work with Amblystoma embryos (BucHANAN, 1929) and with Planaria in KNC disintegration (CHILD, 1930), no cytolysis occurred. The animals were killed but there was no disintegration of any sort if the containers were undisturbed. On agitation the animals broke up into masses. These results afford strong support for the conclusion that the differential in cytelytic disintegration along the axis represents a differential in the ability of the cells to appropriate water. That water was actually abstracted from the animals was shown by their loss of weight. After four hours in Ringer's the weight of the animals was reduced appreciably; their water content was reduced about twenty per cent.


Tests of the effect of Ringer's solution on the rate of oxygen consumption of Planaria showed that with oxygen concentration of the solution approximately at saturation the rate of oxygen consumption is accelerated during long exposures (BuchANAN, 1931). Since Ringer's prevents disintegration and accelerates respiration, the conclusion is clear tha t high disintegration rate is not a necessary association with high rate of oxidative metabolism.

But Planaria were killed by the temperatures employed, whether or not they disintegrated. This fact afforded an opportunity to discover if the hypertonie character of Ringer's alteres the antero-posterior order of death due to heat. A series of animals was subjected to high temperature in Ringer's and from time to t ime individuals were removed to tap water at room temperature (BucgA~A~, 1930b). Living portions recovered while dead or moribund portions disintegrated. The recoveries showed very plainly that the order of death is from anterior toward posterior and thus describes a perfectly clear gradient in sensitivity to the killing action of heat. Gradients in lethal susceptibility to high temperatures have been described by others. For example, BELEHRADEK and ~r (1930) state tha t in the plant Helodea in supra opt imum temperatures the cells at the base are first to die and those of the apex last; at lower temperatures the order of lethal effect is reversed.

In the experiments with Ringer's on Planaria in high temperatures obviously the hypertonic agent obliterates the disintegration gradient but does not protect from heat death nor affect the death gradient. Similar results concerning the protective action of hypertonic solutions against KNC cytolysis are reported by CmLD (1930). The evidence seems clear tha t the disintegration gradient is incidental rather than a necessary corollary of death along the long exis and is not directly conditioned upon differences in rate of metabolism. This does not contradict the s tatement of CHILD (1915) tha t the order of death in such agents is an index of the order of differences in rate of metabolism. The work reviewed here draws a sharp distinction between the lethal effect and the cytolytic action of an agent, a distinction that has not always been made clear.

I t seems improbable that the water relations in the living organism are completely independent of the oxidative metabolism. In fact, the depressive effect of long exposures to distilled water and the accelerating effect of Ringer's solution on respiration point to some sort of relationship (See also DURYEE, 1932). Are these effects evidence of impairment of the oxidative enzymes or of inter- ference with the normal distribution of enzyme and substrate by addition or withdrawal of Water ? Evidence on this question was obtained by testing the effect of varying osmotic pressures on the rate of oxygen utilization in different oxygen tensions (BucI~ANAN, 1931). I t was found tha t after four hours exposure to distilled water or to Ringer's solution the rate of oxygen Consumption varies with the oxygen tension of the environment, although under normal conditions in tap water the rate of oxygen consumption is highly independent of environ- mental supply over wide ranges (HYMA~, 1929; BUCI~ANA~ ~, 1931). In detail, it was found tha t the rate of oxygen consumption of Planaria in distilled water.

510 Buchanan

with oxygen tensions up to 9 cc. per liter is distinctly lower than normM but tha t it increases as t h e oxygen tension is increased beyond 9 cc. per liter. In Ringer's solution after four hours exposure the rate is distinctly higher than normal and also increases as the oxygen tension is increased. I t was also found tha t while the rate of oxygen consumption in distilled water with oxygen tension approximately 15 cc. per liter is distinctly higher than in oxygen tensions around the saturation point for distilled water, cytolytic disintegration is appre- cuably less rapid. These results are interpreted to mean tha t the oxidative enzymes are not impaired by distilled water and Ringer's solution and that the disintegrative action of distilled water and the protective action of Ringer's solution are not closely associated with their effects on oxidative metabolism.

I t is generally accepted tha t the Ca ion decreases permeability to toxic ions and also prevents water uptake by cells. Calcium salts precipitate water soluble phosphatides ; hence distilled water and calcium act in opposite directions. (See discussion by BEUTNER, 1933, p. 72 et seq.). PAGE, for example (1929), found tha t a phase of high resistance to cytolysis by hypotonic sea water follows fertilization in the Arbacia egg and tha t cMcium chloride prolongs this phase. In the light of the work reviewed above and of this action of the Ca ion, the data given in the present communication afford clear support for the conclusion tha t disintegration in Planaria involving cytolysis is associated with the ability of the tissues to appropriate water. Water uptake appears to be the common consequence of the action of lipoid solvents, KNC, hypotonic solutions, absence of calcium, and of high temperature. I t may be prevented by hypertonic agents ttnd the presence of the Ca ion. Thus the cytolytic gradient in Planaria represents a gradient in the order of disturbance of the lipoid-cMcium-water relation in the organism. As to why cytolytic disintegration begins at the anterior end of the animal and proceeds posteriorly before the posterior end shows similar effects there are at least two possibilities: Either the lipoid-calcium-water relation differs progressively along the long axis, or else the anterior region cytolyzes and disintegrates first because it is first to die. The former possibility receives support from the fact that in distilled water, where death results from cytolysis, the disintegrative process is initiated at the anterior and proceeds posteriorly. Also, the addition of potassium oxalate to distilled water accelerates cytolysis and lessens the interval between the time of application and the initiation of disintegration. Then, too, suitable concentrations of calcium protect posterior regions against cytolytic disintegration for a long time. Moreover, absence of calcium and the presence of potassium oxalate both result in death in salt solutions (possibly by sodium poisoning) even if the environment is hypertonie: death in these cases first occurs in the anterior end and proceeds posteriorly.

The evidence from the sources reviewed here may be said to show that in Planaria dorotocephala there is evidence of a cytolytic gradient as well as the death gradient recorded in many papers by CHILD. In general, the death gradient parallels the metabolic gradient of the normal animal, but this is not necessarily the case with the cytolytic gradient; its visible appearance, in this animal at least, is conditioned upon water appropriation by the tissues.


The effect of cytolvtic agents acting under suitable osmotic conditions is an antero-posterior order of cytolysis. The facts constitute clear evidence of a gradient in sensitivity to cytolytie agents; in the experiments reported here these agents act on some sort of lipoid-calcium-water relation. Consequently, the evidence is that this relation varies quanti tat ively from anterior toward posterior in Planaria dorotocephala. During dessication over sulphurie acid, anterior thirds of the first zooid of Planaria lose water slightly more rapidly than do posterior thirds (Bb'cHA~AN, 1930b, p. 465). Such demonstrations of differences in water relations along the axis of an organism are not unusual. HATAI (1924) reports that in a Japanese earthworm the water content is least in the anterior and posterior ends and suggests tha t low water content and high susceptibility are associated. BUCHANAN and KOPENHAVE~ (1934) report tha t in AUobophora caliginosus there is a significant difference in the water content of the body wall tissues between pieces of the anterior and posterior ends and tha t in distilled water anterior tissues gain weight.

SUMMARY

Planaria were treated with equi-molal solutions of ammonium, potassium, sodium, magnesium, and calcium chlorides, made up in distilled water and the rates of cytolysis compared with cytolysis in distilled water. Potassium and ammonium accelerate cytolysis; some protection is afforded by sodium; still more by magnesium, and complete protection by calcium in the concentrations employed.

In distilled water solutions of calcium chloride no cytolysis occurs in concentrations from M/500 to M/40,000 ; cytolysis is distinctly delayed in M/100,000. The protective action of M/I,000,000 is detectable.

Potassium oxalate accelerates disintegration in hypotonic solutions. One per cent ethyl alcohol in distilled water causes cytolysis more rapidly

than does distilled water alone, but in M/500 molal calcium chloride the alcohol solution is much less effective.

Ringer's solution minus calcium affords no protection against death due to absence of calcium and death due to potassium oxalate but completely protects against cytolysis. Death in Ringer's solution minus calcium and in Ringer's solution with potassium oxalate occurs first in the anterior region ~nd describes an antero-posterior gradient.

Cytolysis in distilled water, in potassium oxalate solutions, in alcohol solutions, and in hypotonic calcium solutions of extreme dilution is initiated in the anterior end and describes an antero-posterior gradient within a zooid.

Earlier work of the writer on the disintegrative action of lipoid solvents~ heat, KNC, hyper- and hypotonie solutions is discussed. I t is concluded tha t in Planaria dorotocephala the antero-posterior gradient in cytolytic disintegration represents an antero:posterior differential in sensitivity to disturbance of the calcium-lipoid-water relation in the organism.

512 B u c h a n a n , An analysis of physiological states responsible &c.

BIBLIOGRAPHY

BARRON, D. If., 1931, Proc. Soc. Exper. Biol. and IVied., Vol. 28, 1019. B~LEHR~DEK, J. and MELI0~A~, J., 1930, Biol. Generalis., Bd. 6, 109. B]~UT~ER, R., 1933. Physical Chemistry of Living Tissues and Life Processes. Baltimore. B~CHA~AN, J. W., 1923, Jour. Exper. Zool., Vol. 38, 331. - - , 1926, Jour. Exper. Zool., Vol. 44, 307. - - , 1929, Physiol. Zool., Vol. 2, 1925. - - , 1930a, Jour. Exper. Zool., Vol. 57, 307. - - , 1930b, Jour. Exper. Zool., Vol. 57, 455. - - , 1931, Biol. Bull., Vol. 60, 309. :Bvc~A~AN, J. W. and KOI'E~HAV]~R, M., 1932, Anat. Ree., Voh 54, 53. CHILD, C. l~V[., 1915, Senescence and Rejuvenescence. Chicago. - - , 1919a, Amer. Jour. Physiol., Vol. 48, 372. - - , 1919b, Amer. Jour. Physiol., Vol. 49, 403. - - , 1930, Physiol. Zool., Vol. 3, 90. DURYE]~, W. R., 1932, Anat. Rec., Vol. 54~ 54. E~ERSO~, O. H. and B~C~A~A~, J. W., 1927, Jour. Pharm. and Exper. Ther., Vol. 31, 387. GRAY, J., 1925, Proc. C~mb. Phil. Soc., Biol. See., No. 1, 225. I-[ANSTEEN-CRANNER, G., 1919, Ber. d. Deutsch. Bot. Ges., Bd. 37, 380. - - , 1922, Meldinger fra Norges Landbrukshoiskole, Vol. 2, 1. ( Quoted from HEIL~R~N~, L. V.

Colloid Chemistry of Protoplasm, Berlin 1928). ttATAI, S., 1924, Sci. Rep. Tohoku Imp. Univ., Ser. 4, 3. HEILBRUNN, L. V., 1924, Amer. Jour. Physiol., Vol. 69, 190. - - , 1928, The Colloid Chemistry of Protoplasm. Berlin. I-IENsItAW, P. S. and FRANCIS, D. S., 1934, Jour. Cell and Comp. Physiol., Vol. 4, 1. HYMAN, L. tL, 1929, Physiol. Zool., Vol. 2, 505. KEr~ER, W~. A., and u J. H., 1933, Jour. Exper. Zool., Vol. 66, 455. KNAFFL~LENz, E. yon, 1908, Arch. f. ges. Physiol., Bd. 123, 279. LUI~D, E. J., 1921, Biol. Bull., Vol. 41, 203. Lyo~, E. P., 1904, Amer. Jour. Physiol., Vol. l l , 52. PAGE, I. I:[., 1929, Brit. Jour. Exper. Biol., Vol. 6, 219. PA~TIN, C. F. A., 1931, Jour. Exper. Biol., Vol. 8, 63. PA~K~, G. It., 1929, Brit. Jour. Exper. Biol., Vol. 6, 412. RU~ST~5~, J., 1923, Aeta. Zoologiea, Bd. 4, 285. ST]~W~D, F. C., 1929, Brit. Jour. Exper. Biol., Voh 6, 32. WILSOn, J. W., 1925, Anat. Rec., Vol. 31, 336.

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An analysis of physiological states responsible for antero-posterior disintegration in Planaria dorotocephala