612.26: osmo-regulatory mechanism. salinities, of Balanus crenatus. Kreps found that cessation of motility

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1 612.26: RESPIRATION OF ISOLATED GILL TISSUE OF THE EEL. BY J. B. BATEMAN1 AND ANCEL KEYS2. (From the Physiological Laboratory, Cambridge.) INTRODUCTION. VARIOUS attempts have been made to show that osmotic regulation in marine and fresh-water animals is accompanied by increased respiratory exchange. The evidence is almost entirely presumptive; the respiration of a tissue is measured under conditions in which activity is assumed to occur without any proof of its occurrence; still further, from a direct correlation, the respiration of an entire animal is measured under two sets of conditions involving respectively quiescence and activity of the osmo-regulatory mechanism. The results are open to some obvious objections. The animals may be more active, mechanically, under one set of conditions [B eadle, 1931; A. V. Hill, 1931, p. 69]-a criticism whose force is shown clearly by the experiments of Kreps [1929] on the respiration, in water of different salinities, of Balanus crenatus. Kreps found that cessation of motility was accompanied by a decrease of respiration to 1/3 or 1/4 of its normal value, while changes which could be attributed directly to changing salinity were of a much smaller order. Again, total respiration of an entire animal may be subject to complex regulatory influences which may mask the behaviour of any one organ. Schlieper has upheld the view that osmo-regulatory activity is accompanied by increased respiration of the animal and he has supported it by observations on the shore crab, Carcinus maenas [1929,1930], and on Gammarus locusta [1931], and by the experiments of Tarussov [1927] on Nereis diversicolor3. In the first case the conditions for osmo-regulatory activity are well defined, but the question of muscular activity is a doubtful one; in the case of Gammarus there is no information concerning the behaviour of the body Working for the Medical Research Council. 2 Fellow of the National Research Council of America. 8 Dr Schlieper informs us that he has recently obtained similar evidence in the case of some other forms, particularly Eriocheir inen8i0. PH. LXxvII. 18

2 272 J. B. BATEMAN AND A. KEYS. fluids when the animal is in different environments, and therefore no experimental evidence in favour of Schlieper's interpretation. The evidence in the case of Nereis diversicolor derives some validity from B eadle's [1931] experiments, in which it was found that osmotic regulation occurs although it is absent in the closely related form, Nereis cultrifera. Other experiments of this kind are those of Beadle [1931] on Gunda ulvew, the osmotic properties of which had been studied by Pantin [1931] and Weil and Pantin [1931], and the observations of Hayes [1930] on Paramaecium. Where the osmo-regulatory process is probably more complex, as in the fishes, observations on the intact animals are still more difficult to interpret. R a f fy and Fontaine [1930] reported that "civelles" adapted to fresh water showed a greater oxygen consumption than the same animals adapted to sea water, although a prolonged sojourn in fresh water presumably causes cessation of gill secretory activity. K eys [1931 a], on the other hand, found that the respiration of Fundulus parvipinnis is depressed in fresh water. Schlieper [1929] measured the respiration of the isolated gill of the mussel, Mytilus edulis, and found that the gas exchange is greater in diluted media than in pure sea water. Here again, however, the observations are for the present of little significance, because there is no evidence that the gills of Mytilus play any role in osmotic regulation; it is not even known that osmotic regulation occurs in this animal. The present experiments were undertaken in view of the possibility that the secretion of chloride, which is manifested by eel gills under certain conditions [Keys, 1931 c], might also occur in the extirpated gills under similar conditions. It was shown in Keys's experiments that the amount of secretory activity of the gills is determined by the internal concentration of chloride, so that no secretion is observed when the internal medium has a freezing-point depression of less than C.; as the internal concentration is increased above this point the secretion is initiated and further small increases in the internal concentration provoke progressively larger increases in the secretion activity. These results have been confirmed by Krogh and Schlieper (unpublished experiments), who showed that the external concentration is not of itself a major factor in the phenomenon. The conditions of internal concentration which maintain the gills active or passive as regards secretion can be brought about almost as readily with the gills extirpated as in situ. The perfusion technique worked out by Keys [1931 b] made it possible to fill the gills with fluid of any

3 RESPIRATION OF ISOLATED GILL TISSUE OF EEL. 273 desired concentration. When these gills are extirpated and suspended in an appropriate solution, diffusion and passive osmotic interchange between the internal and, external solutions will tend to keep the internal concentration high or low, depending on the concentration of the external solution. If the gills on one side of an eel are filled with Ringer's solution of A = C., extirpated and suspended in a balanced medium of high concentration, it is not too much to expect that they will be more active in secretion than the gills from the other side of the same animal, which are similarly perfused with a Ringer's solution of A = 0 50 C. and suspended in a medium of low concentration. With preparations of this kind transferred to microrespirometers, it should be possible to obtain presumptive evidence for or against the utilization of oxygen in the branchial secretory process. It should be noted that the great exposure of surface in the gills makes them peculiarly suitable for respiration measurements. EXPERIMENTAL. The following aqcount deals with the general procedure adopted in most of the experiments. The eels (Anguilla vulgaris) used were kept in the laboratory in a tank of running tap water; some of these were transferred to sea water and allowed a few days for acclimatization before being used. The gills were perfused from the ventral aorta [B at em an and Keys, 1932], the cannula being connected by a two-way tap to two reservoirs containing respectively "concentrated" and "dilute" Ringer's solutions [prepared according to Keys, 1931 b] or different dilutions of sea water. The "concentrated" sea water had a freezing-point depression of 0.70 C. and the "concentrated" Ringer's solution a A of 0.72 C. The "dilute" sea water and the "dilute" Ringer's solution had freezing-point depressions of C. and 0.55 C. respectively. In a few cases the animals were anoesthetized with amytal [Keys and Wells, 1930], but the amytal was washed out rapidly by the subsequent perfusion. After perfusion with a given solution for some minutes, the gills on one side were exposed by cutting open the operculum and the afferent and efferent ends of the gill bars on this side were clamped off. At this time the reservoir tap was turned so as to perfuse the opposite side with the second solution. The first set of gills was excised by cutting away from the clamps and was suspended in the appropriate medium. After minutes the second set of gills was excised similarly. The individual 18-2

4 274 J. B. BATEMAN AND A. KEYS. gill bars were carefully separated and weighed in Ringer's solution after gentle blotting with clean filter paper. The fourth (posterior) bar on each side was discarded on account of its small size. It should be mentioned that in all these operations the gill filaments themselves were touched only in the process of removing excess liquid incidental to weighing, and, save at the extreme ends of the bar, the gill filaments suffered no manipulative damage whatever. The gills perfused with the dilute and concentrated Ringer's solutions were now transferred to B arcroft manometers containing 3 c.c. 1/9 and 5/6 sea water respectively, and 0 5 c.c. 40 p.c. KOH. After a period of 20 minutes for temperature and gas equilibration, the taps were closed to the air and manometer readings taken at 15-minute intervals for several hours, bath temperature and barometric pressure being recorded. When the measurements were completed the gills were weighed after gentle blotting, re-weighed after being heavily blotted to remove all adherent moisture, and finally the gill filaments were dissected away from the cartilaginous bar and the weight of the latter obtained. This dissection could be made quite cleanly and the proportion of relatively inert cartilage and non-filament tissue in the gill was obtained sufficiently accurately for an adequate correction to be made in computing the respiration of the gill-filament tissue proper. The respiration of the "cartilage" alone was determined in several experiments, and a mean value used in the application of this correction. The constants for the B a r croft manometers were corrected in each experiment for the different volumes of liquid and tissue used. PROPORTION OF GILL RESPIRATION DUE TO "CARTILAGE." The figures for "cartilage" respiration are given in Table I. The values for each hour of experiment are the means of the four 15-minute measurements. The mean values, 54-3 c.mm. 02 per g. per hour for the first hour and 49-4 for the second, were used in all calculations. The TABLE I. Respiration of gill cartilage at 150 C. Mean respiration in c.mm. 2 at N.T.P. per g. per hour Exp. First hour Second hour C C *3 C C Means 54*3 49.4

5 RESPIRATION OF ISOLATED GILL TISSUE OF EEL. 275 rather wide variation in the individual values is probably due to incomplete removal of filament tissue, making the lower figures more probably correct. But the corrections involved are small enough for the mean value of all experiments to be used without serious error. The proportion of the measured weight of gill bars due to " cartilage" was found usually in each individual case, but, as this determination was sometimes neglected, a mean value had to be obtained from all the figures available. These seem to be worth presenting, since they illustrate the cleanness with which the dissection of cartilage could be done (Table II). The mean value, 48 p.c., was used in calculation. TABLE II. Proportion of cartilage in gill bar. Mean weight of cartilage as p.c. No. of of total blotted Series No. determinations dry weight P P P P P P P P P The experimental procedure and method of calculating results may now be made clearer by a detailed typical protocol. The remaining data will be presented more briefly by graphs and summarizing tables. Protocol of Exp iii Eel from fresh water after 2 days in laboratory. Operation begun p.m., perfusion begun p.m. Operation complete p.m. Internal perfusion media: 1/3 sea water from start till p.m., 1/4 sea water till 1.5 p.m. External medium: tap water. Gill from right side dissected out at p.m. and transferred temporarily to 1/3 sea water. Left side out at 1.5 p.m. and transferred to 1/4 sea water. Placed in respiration chambers at 1.15 p.m., each apparatus containing 3 0 c.c. external medium and 0-5 c.c. 20 p.c. KOH. The manometer readings were continued for 4-5 hours, then the tissue was weighed. The details of external and internal media, weights of tissues, and manometers are given in Table III, while Table IV shows the detailed figures for one manometer, together with the essential stages in the calculation. The constant k for manometer 75, determined at C. and 763 mm. and containing 0-1 c.c. liquid, was 3-08, or 2-90 at N.T.P. For this experiment, with cups containing 3-5 c.c. liquid g. tissue at and 760 mm., k becomes Then the rate of 02 uptake is kx (see Table IV). This is due partly to respiration of gill filaments and partly to cartilage. Correction for the latter is applied by subtracting from kx the product (weight of cartilage) x (mean respiration rate of cartilage) = B, or 0085 x 51-8 = 4-6 (see Tables I and III). The true respiration of gill tissue is then this difference divided by the weight of gill tissue (M). In Table V the results of the calculation are given for all six manometers.

6 276 J. B. BATEMAN AND A. KEYS. TABLE III. Weights of tissue, etc., in Exp Apparatus No ' Internal medium Dilute Ringer's solution Conc. Ringer's solution 3. External medium 1/9 sea water 5/6 sea water 4. Weight of gills at end of exp.: g. 5. Weights of gill bars calc. as 0-48 x (4) 6. Weights of gill fila ment tissue (M) Time (min.) TABLE IV. Calculation of respiration rates in Exp. 2. Manometer 75. k = Weight of gill tissue g. Cartilage correction, B=4-6 throughout. Rate of move- kx ment of mano- c.mm. meter liquid: 02 per g. =x mm./hour per hour kx-b kx -B M TABLE V. Final results for Exp. 2. Respiration rates for gill tissue in c.mm. 02 per g. per hour at N.T.P. Temperature = C. Internal medium "dilute" Internal medium "concentrated" Ringer's solution. Ringer's solution. External medium 1/9 s.w. External medium 5/6 s.w. 1- A6 1 Manometer No ' 116 Time (min.) s.w. =sea water

7 RESPIRATION OF ISOLATED GILL TISSUE OF EEL. 277 RESULTS. The figures obtained in the detailed protocol, which are plotted in Fig. 1, are typical. In all other experiments of the same kind the respiration was definitely greater when the liquid bathing the gills was sea water or 5/6 sea water than when it was tap water or 1/9 sea water. Individual manometers very occasionally, as in the experiment quoted, gave irregular results, which could as a rule be traced to an imperfectly greased tap. In Fig. 2 the results of a similar experiment are plotted, the ordinate here being total 02 uptake per g. gill tissue instead of the rate which is plotted in Fig. 1. This shows the main effect rather more strikigly. TABLE VI. Respiration of isolated gills in 1/9 s.w. and in 5/6 s.w. Incre- Increment, ment Mean respiration rate in rate in as p.c. c.mm. 02 per g. per hour 5/6 s.w. of Experi- minus values mental Mean of Mean of rate in in series Internal media 1/9 s.w. series 5/6 s.w. series 1/9 s.w. 1/9 s.w. 2 "Dilute " R in 1/9 s.w "Conc. " R in 5/6 s.w /4 s.w. in 1/9 s.w /3 s.w. in 5/6 s.w Same as series Same as series Same as series 2, plus glucose and urea Same as series Mean of all 46 p.c. Each value given is the mean of from 6 to 12 determinations; a total of 234 determinations is summarized in this table. R = Ringer's solution. s.w. = sea water. The remaining experiments are summarized in Table VI, in which the mean rates of respiration are given for each manometer used, over a period of 2 hours. In column 7 the differences between the mean rates in the two media are given for each experiment, this difference being expressed in column 8 as a percentage of the "normal" respiration in 1/9 sea water. It is clear that there is a consistent, though rather variable, positive difference, amounting on the average to about 46 p.c. of the respiration in 1/9 sea water. The variability is to be expected in a

8 0 278 J. B. BATEMAN AND A. KEYS. C) p4 P.4 C 0 p4 4) P4 C5 C3 100 % Time in minutes Fig. 1. Effect of salt concentration on the respiration of extirpated gill tissue. Rates of respiration of six gills from a single eel. Solid circles, gills perfused with Ringer's solution A =0.70' and suspended in 5/6 sea water. Open circles, perfused with Ringer's solution A =0.50 and suspended in 1/9 sea water. Temp *9.*4 0'8 0 bd p.4 10X Time in minutes Fig. 2. Effect of salt concentration on the respiration of extirpated gill tissue. Total respiration of five gills from a single eel. Solid and open circles as in Fig. 1. Temp

9 RESPIRATION OF ISOLATED GILL TISSUE OF EEL. 279 tissue subjected to such drastic manipulation before the performance of the experiment. There was generally a tendency for the respiration to decrease somewhat during the course of the experiment; this may be seen in Fig. 1, which is typical of the behaviour observed. The data are not, however, sufficiently extensive to indicate any tendency of the increment due to salt concentration to decrease more rapidly than the total respiration. The existence of the effect under slightly more physiological conditions was confirmed in a series of experiments on the blood-filled gills. The procedure was as follows: the eel was pithed and the gills exposed with care to avoid serious bleeding. The afferent and efferent ends of the gills were clamped with heemostats, as in the previous case, and the gills excised with the clamps still in position. Then the individual gill bars were ligatured at both ends, the clamps removed, and the gill bars cut apart. The respiration experiments were carried out as in the other experiments and the results are summarized in Table VII. In Fig. 3 TABLE VII. Respiration of blood-filled gills in dilute and concentrated media. Pre- Incre- Increvious ment, Exment, habi- Ex- conc. as p.c. peri- tat ternal minus of mental of solu- Resp. Mean External Resp. Mean dilute dilute series eel tion rate rate solution rate rate rate rate 5 s.w. f.w S.W f.w.,, ,, ,,,, , *3 41 9,, *0, ,, 1/9 s.w /6 s.w '7 15 s.w.,, 190* ,, * *7 241*5 Mean of all 79 P.C. s.w. = sea water; f.w. = fresh water. the data of experiments 7, 8 and 9 are plotted. In each of these three experiments a small eel was used, and the whole gill from one side put into one respiration chamber. It appears that, although in a single experiment the respiration was more constant under these conditions than in the perfused gills, the actual 02 consumption was somewhat smaller and the increase of respiration in sea water even more marked, having a mean value 79 p.c. more than that of the respiration rate in fresh water. For gills from eels acclimatized to sea water the mean is 76 p.c. and for those from fresh water it is 82 p.c.; considering the wide variation in the quantities observed this agreement may be fortuitous.

10 280 J. B. BATEMAN AND A. KEYS. The most rigid proof of the reality of the phenomenon under discussion would be to show the effects of salt concentration on the respiration of the same portion of tissue. Such an experiment would put a severe strain on the capacity of the extirpated tissue to adjust to the changing concentration of its environment, and it could hardly be expected that O's 1 L5T7 p * Time in minutes Fig. 3. Effect of salt concentration on the respiration of extirpated blood-filled gills. Three experiments, eels from fresh water. Solid circles, gills suspended in sea water. Open circles, gills suspended in tap water. Temp the secretory mechanism would respond perfectly under these circumstances. Experiments, however, showed that under these conditions there is an effect of salt concentration on the oxygen consumption which, although small, is in agreement with the earlier experiments; i.e. the oxygen consumption tends to be higher in the more concentrated environment. Table VIII summarizes the results of these experiments in which the respiration of individual gills was measured first in an environment of one concentration and then in an environment of a very different concentration.

11 RESPIRATION OF ISOLATED GILL TISSUE OF EEL. 281 TABLE VIII. Effect of change of salt concentration on the respiration of individual gills. Group A, internal concentration initially A =0.700 C. First observations, Second observations, relative resp. rate relative resp. rate in environment in environment GillNo. A=1*70C. A=0-30C Means *0 Group B, internal concentration initially A =0.480 C. Second observations, First observations, relative resp. rate relative resp. rate in environment in environment Gill No. A = 1-7 C. A=0 3 C Means DISCUSSION. The increase in respiration with increase in the salt content of the environment has been observed regularly under different experimental conditions, and its reality seems certain. Its interpretation in terms of the performance of secretory work by the gills is simple and attractive, but it is necessary to consider the possibility that some other effect of changing salinity may be involved. The effects observed in the various experiments cannot be ascribed to any difference in the ph of the solutions. The initial ph of the internal solutions was always between 7-5 and 7 7, and the ph of the external solutions was between 7'8 and 8-0; the shift to greater alkalinity due to abstraction of C02 in the respiration chambers was practically the same in the various solutions. As to the normality of the tissue respiration observed, we may mention that the effect of cyanide on the gill respiration was quite normal. In some cases it appears that tissue respiration, even where no secretory activity is known to occur, may be a function of the salt concentration of the environment, but there is no general ground upon which pure osmotic effects on respiration can be predicted. The effect of

12 282 J. B. BATEMAN AND A. KEYS. hypertonic NaCl solutions in increasing the respiration of fertilized seaurchin eggs [Warburg, 1909; Meyerhof, 1911], for example, was supposed by Warburg to be a direct effect of NaCl on the respiration, while Schlieper [1931] attributes it to an attempt to prevent, by an active osmotic regulation, the entry of the poisonous NaCl. With plant cells increase of internal osmotic concentration produces no predictable general effect [Smith, ; Palladin and Sheloumova, 1918; Inman, 1921]. Hayes [1930] found that the respiration of Paramcecium is increased both in diluted and in concentrated media. Clearly, in these cases S c h lie p er's explanation of the W a rb u r g sea-urchin eggs experiment cannot be applied. In the present instance, calculation shows it to be quite possible that the decreased respiration in dilute sea water may be to some extent an injury effect due to salt loss to the surroundings; if we reason thus we must conclude that the tissue in 5/6 sea water suffers to an even greater extent from dehydration. The dilute sea water (A = about C.) is much nearer the normal internal osmotic pressure of the gills (A = about C.) than the concentrated sea water (A = about C.). Moreover, the observed respiration of gill tissue immersed in 1/3 sea water is rather less than in 5/6 sea water, as is shown in Table IX. TABLE IX. Respiration of gills in 1/3 s.w. (A =0.67 C.). Respiration rates in c.mm. 02 per g. per hour. Mean Exp. series respiration _% & - AMean rate of gills A B C D rate in 5/6 s.w s.w. =sea water. In the attempt to discover whether there is a general effect of salt concentration on the respiration of eel tissues, we have studied the respiration of the thin membranous fins, which, like the gills, are well suited for respiration measurements and are subjected to great variation in the external concentration in the normal life cycle of the animal. The technique was similar to that used in the gill experiments; the fins were extirpated and the respiration of the fin from one side of the eel was measured in 1/9 sea water, while the respiration of the corresponding fin from the other side was measured in 5/6 sea water. In each experiment eight determinations were made over a period of 2 hours. The results of the six experiments are given in Table X; there is no trace of a consistent effect of the salt concentration.

13 RESPIRATION OF ISOLATED GILL TISSUE OF EEL. 283 TABLE X. Respiration of eel fins in 1/9 and in 5/6 s.w. Relative respiratory rates per g. per hour Exp. No. In 1/9 s.w. In 5/6 s.w. F F F F F F Means Eight determinations were made in each experiment. s.w. =sea water. The data presented in this paper have clearly the defect of some of the earlier experiments on the respiration of secretory tissue; they do not show, unequivocally, that the performance of work is causally associated with increased oxygen consumption. One can only point out that the tissue has a proven secretory capacity, and that the respiration does increase under conditions which are known to be associated with the initiation and maintenance of the secretion. Assuming, for the moment, that the secretion hypothesis is the correct one, we can make some more detailed observations on the experiments reported. If the osmotic pressure of the internal medium really provides the stimulus for the secretion, then we must be able to show that the necessary change in the internal environment in the case of the bloodfilled gills can occur early in the experiment in which the external medium is 5/6 sea water. We may use data for the permeability of the perfused gills when fresh water is the external medium [Keys, 1931 b]. These data indicate a water entry of about 5 c.c. per kg. eel per hour. In eels of the size used in the present experiments this means an entry of roughly 0-15 c.c. of water per hour per gill. The gill volume may be put at 0.5 c.c. (probably less) of which 20 p.c. is osmotically inert substance. It is clear that the normal permeability will effect in 1 hour a change of at least 35 p.c. in the internal concentration. Where the concentration gradient is greater, as in the case of gills suspended in 5/6 sea water, the effect of passive diffusion will be proportionally greater. The permeability of the extirpated gills, moreover, may be taken to be considerably greater than that of the gills in situ, in the same way that any tissue subjected to manipulation shows an increased permeability. It is obvious that sufficient exchange will take place in the early stages of the experiment to stimulate or stop the secretory activity as the case may be. The

14 284 J. B. BATEMAN AND A. KEYS. results, then, are consistent with the opinion that the internal osmotic pressure is the stimulus for the secretion. This conclusion has been reached also from the recent experiments by Krogh and Schlieper [1932]. The respiration increment due to, or associated with, higher salt concentration is an excess of oxidation over the "resting" value in the lower salt concentration, and the excess energy yielded in this way may be calculated and compared with the medium energy cost of the gill secretion observed in the perfusion experiments. If it could be assumed that the salt concentration stimulus evoked the same degree of secretion activity in the extirpated gills as in the perfused gills, such a calculation would provide an answer to the question as to the efficiency of the secretory mechanism. Actually, of course, it is highly probable that the secretory activity in the extirpated gills is smaller, perhaps much smaller, than in the perfused gills, and the result of our calculation will be of interest only as a limiting value. The mean value for the "secretion" increment in all experiments is c.mm. 02 per g. per hour. Assuming that carbohydrate is burnt, the combustion of this amount of oxygen would yield 0-51 cal. The secretion observed in the perfusion experiments involves a performance of thermodynamic work amounting to cal. [Bateman and Keys, 1932], with the mean value not far from 015 cal. The first approximation to the maximum efficiency is therefore 29 p.c. Organs capable of specialized activity frequently have a high resting metabolism. It is of interest, therefore, that the respiration of eel gill tissue in a dilute medium-about 210 c.mm. 02 per g. per hour at 150 C.-is proportionally considerably greater than that of the mammalian lung. Lovatt Evans [1912] gives 5001 c.mm. 02 per g. per hour at 36 C. for the respiration of the lung tissue in the heart-lung preparation, and this works out to be about 1-5 times the rate for the body as a whole. The respiration rate of the "resting" gills is about seven times that of the resting intact eel, as measured by Krogh [1904], while the respiratory rate of the gill in the concentrated medium is about ten times that for the entire animal. Moreover, if allowance is made for the temperature difference by the application of the usual temperature coefficient-qlo about 2-0-the respiratory rates of "resting" and "active" gills become respectively 100 p.c. and 190 p.c. greater than the rate of the mammalian lung tissue. For purposes of comparison, we have 1 The value obtained from Evans's paper by Bayliss [1924, p. 612] appears to be incorrect.

15 RESPIRATION OF ISOLATED GILL TISSUE OF EEL. 285 assembled data on the respiratory rates of various mammalian and eel tissues in Table XI. TABLE XI. Respiration of various tissues. Respiration rates in c.mm. 02 per g. per hour of wet tissue. Mean respira. tory rate Approx Neumann, Evans, Meyerhof, Fenn, Original 61,, 67,,1 63,, 167,,1 273,,J 210 Tissue Mammalian kidney Mammalian kidney Mammalian suprarenal gland Mammalian submaxillary gland Mammalian submaxillary gland Mammalian lung Mouse and rat diaphragm Frog, small muscles at C. Eel, pectoral fin at 150 C. dorsal fin (strips) at C. central fin (strips) at 15-1 C. mid gut (strips) at C. blood-filled gills in 1/9 s.w. at 14*8' C. blood-filled gills in 5/6 s.w. at C. gills after perfusion, in 1/9 s.w. at C. same calculated for 370 C.* gills after perfusion, in 5/6 s.w. at C. same calculated for 370 C.* * Assui muing Qlo is 2-0. Observer Neumann, 1912 Barcroft and Brodie, 1905 Verzir, 1912 Barcroft and Piper, 1912 SUMMARY. 1. The respiration of excised eel gills, filled with Ringer's solution and blood, has been measured in dilute and concentrated external environments. 2. The percentage of gill filament tissue in the gills has been determined and the respiration of the bars minus the filaments measured. 3. The mean oxygen uptake of the gills filled with Ringer's solution, corrected for the respiration of the relatively inert cartilaginous bars, was 210 c.mm. 02 per g. per hour with 1/9 sea water as the external medium and 300 c.mm. with 5/6 sea water. In all experiments the respiration was consistently greater in the more concentrated medium. 4. With the blood-filled gills an even greater difference was found between the respiration in 1/9 and 5/6 sea water, the respiration in the latter averaging 80 p.c. greater than in the former. 5. Possible causes of this concentration effect are discussed and reasons are given for the belief that it is related to secretory activity in the gill. The possible efficiency of this activity is discussed. 6. The fins of the eel do not show any effect of osmotic concentration of the environment on the respiratory rate.,,

16 286 J. B. BATEMAN AND A. KEYS. ACKNOWLEDGMENTS. We are grateful to Prof. J. B arcroft for his kindness and interest in these experiments. We are indebted to Dr M. Dixon for the loan of some of the manometers used in the experiments. One of us (J. B. B.) also wishes to thank the Physiological Laboratory, Cambridge, for a research grant, during the tenure of which part of this work was done. REFERENCES. Barcroft, J. and Brodie, T. G. (1905). J. Physiol. 33, 52. Barcroft, J. and Piper, H. (1912). Ibid. 44, 359. Bateman, J. B. and Keys, A. (1932). Ibid. 75, 226. Bayliss, W. M. (1924). Principles of General Physiology. London. Beadle, L. C. (1931). J. exp. Biol. 8, 211. Evans, C. L. (1912). J. Physiol. 45, 213. Fenn, W. 0. (1927). Amer. J. Physiol. 83, 309. Hayes, F. R. (1930). Z. vergl. Physiol. 13, 214. Hill, A. V. (1931). Adventures in Biophysics. Oxford. Inman, 0. L. (1921). J. gen. Physiol. 3, 535. Keys, A. (1931 a). Bull. Scripps Inst. Oceanog. 2, 417. Keys, A. (1931 b). Z. vergl. Physiol. 15, 352. Keys, A. (1931 c). Ibid. 15, 364. Keys, A. and Wells, N. A. (1930). J. Pharmacol. 40, 115. Kreps, E. (1929). Pflfigers Arch. 222, 215. Krogh, A. (1904). Skand. Arch. Physiol. 16, 348. Krogh, A. and Schlieper, C. (1932). Private communication. Meyerhof, 0. (1911). Biochem. Z. 33, 29. Meyerhof, 0. (1930). Die chemischen Vorgdnge im Muskel. Berlin. Neumann, K. 0. (1912). J. Physiol. 45, 188. Palladin, V. I. and Sheloumova, A. M. (1918). See Chem. Abstr. 12 (1889). Pantin, C. F. A. (1931). J. exp. Biol. 8, 82. Raffy, A. and Fontaine, M. (1930). C. R. Soc. Biol., Paris, 104, 287. Schlieper, C. (1929). Z. vergl. Physiol. 9, 478. Schlieper, C. (1930). Biol. Rev. 5, 309. Schlieper, C. (1931). Biol. Zbl. 51, 401. Smith, I. M. ( ). Rep. Brit. Ass. 85, 725. Tarussov, B. (1927). Zurnal eksp. biol. i med. 6, 229. See Schlieper (1930). VerzAr, F. (1912). J. Physiot. 45, 39. Warburg, 0. (1909). Z. physiot. Chem. 60, 443. Weil, E. and Pantin, C. F. A. (1931). J. exp. Biol. 8, 73.