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Effect of freezing and thawing on ionic properties of the gills of Artemia salina (L.)
Comp. Biochem. Physiol., 1970, Vol. 35, pp. 959 to 963. Pergamon Press. Printed in Great Britain
SHORT COMMUNICATION EFFECT OF FREEZING AND THAWING ON IONIC P R O P E R T I E S O F T H E G I L L S O F A R T E M I A S A L I N A (L.) G. K. R E N N I E * and P. G. S M I T H t School of Biological Sciences, University of East Anglia, Norwich (Received 19 February 1970)
Abstract--1. In vivo electrical and permeability measurements have been made on the gill epithelium of the euryhaline Arternia salina before and after freezing and thawing. 2. Although the animal did not survive freezing, little change in epithelial properties occurred. 3. This suggests that tissues normally encountering widely varying osmolarities are resistant to freezing damage. INTRODUCTION
TH~ mGH ionic concentrations which occur when biological fluids are frozen cause damage to cell membranes, and it has been suggested that freezing injury is due to this effect (Lovelock, 1953). A r t e m i a salina, the brine shrimp, tolerates very high ionic concentrations in its environment (Croghan, 1958a); it therefore appeared that this animal might be resistant to freezing injury. Over the hundredfold range of external salinities in which the animal survives, the blood concentration varies only fourfold (Croghan, 1958b). T h e internal tissues do not therefore encounter a wide range of ionic concentrations; in fact the only tissue to experience the wide salinity variation of the external medium is the gill epithelium (Croghan, 1958c), and it is in this tissue that a resistance to the effects of freezing and thawing might be expected. Accordingly experiments were carried out to investigate the effects of freezing and thawing on the electrical and permeability properties of the gill epithelium of Artemia.
MATERIALS AND METHODS The gill epithelium of Artemia is far more permeable to ions than is any other part of the external cuticle and is also an order of magnitude more permeable than the gut (Croghan, 1958c; Smith, 1969a). In vivo measurements of electrical potential difference and resistance between blood and external medium are therefore largely controlled by the properties of the gill epithelium; measurements of this type were carried out in order to investigate the * Present address: Unilever Research Laboratories, Port Sunlight, Cheshire. t Present address : Department of Zoology, University of Liverpool, Liverpool L69 3BX. Please address reprint requests to P. G. Smith. 959
960
G . K . RENNIE AND P. G. SMITH
epithelial properties. The methods used have been described previously (Smith, 1969b). Briefly, an animal was ligatured at neck and anus, thus closing off the gut from the external solution, and immobilized in a small Perspex cell. Electrical potential difference (p.d.) between the blood and the external sea-water medium was measured by means of glass micropipette electrodes, using an electrometer of high input impedance. Current was passed through the epithelium via a second internal microelectrode, allowing epithelial resistance to be measured. The relative permeabilities of Na +, K + and C1- were also determined, as transport numbers; measurements were made of the change in p.d. which occurred when the external concentration of each of these ions was reduced by a factor 2. The inorganic ions were replaced by large organic ions (choline and benzenesulphonate) which were assumed to be impermeable. For a p.d. change of AVmV, the transport number T is given by T - - A V/17.8 at 25°C. The sign of AV is positive for the anion and negative for the cations. Placed immediately beside the animal was a calibrated copper-constantan thermocouple which measured the approximate temperature of the animal. Before freezing, epithelial p.d., resistance and transport numbers were measured. With the electrodes remaining in position the animal was frozen using either a Frigistor cooling device (final temperature -18°C) or a block of solid carbon dioxide (final temperature below -32°C) placed on the Perspex cell. The animal was then allowed to thaw and the parameters were remeasured. RESULTS AND D I S C U S S I O N I n unfrozen animals, the epithelial p.d. was 24 m V (blood positive) and only the sodium transport n u m b e r was significantly different from zero (Table 1). T h e s e results have been discussed in m o r e detail previously (Smith, 1969b). As cooling progressed, the p.d. fell rapidly to about 2 m V (Fig. 1) and remained at this level until thawing. Concurrently, beating of the limbs became slower and ceased completely at about - 4 ° C . T h i s was reversible, provided that no ice crystals f o r m e d within the animal. Freezing of the animal occurred at about - 10°C, when ice crystals were seen to f o r m ; thawing took place at 0°C. On thawing, the limbs did not resume beating, suggesting that damage to excitable tissues had occurred. However, there was little change in any of the measured parameters; the only significant changes were in the pooled results for epithelial p.d. and in the chloride transport n u m b e r after freezing to the lower t e m p e r a t u r e (Table 1). I f the epithelial m e m b r a n e s had ruptured, the resistance would have dropped considerably and the p.d. would have become a liquid junction potential decaying rapidly with time. T h e value of a liquid junction potential between the blood and sea water can be calculated f r o m the Henderson equation (MacInnes, 1961, p. 232) to be - 6 mV, substantially different f r o m the measured p.d. ; f u r t h e r m o r e the rate of fall of the p.d. (about 0-2 m V / m i n ) was small. It is therefore concluded that little damage to the epithelial m e m b r a n e s occurred. T o check that no gross damage to the epithelium had taken place, the permeability of the animals to the dye phenol red (molecular diameter about 10 A) was studied. Several animals were put into a sea-water solution of the dye for periods between ½ and 3 hr and were washed in sea water for a few minutes before examination. Before freezing, dye was accumulated in the gut only, with a little
p.d.,
* P < 0'02.
+0.08+0'04(12) +0.006+0.006(12) +0.04+-0"04(12)
- 2 - 2 + 0"7 * * (12) - 8 8 0 + 550 (5)
Total
+0'11 _+0'08 (5) +0.017+_0.011 (5) +0"01 +_0'01 (5)
- 0 " 5 + 1 "0 (5) + 100 + 200 (2)
Frigistor ( - 18°C)
+ 0.06 + 0.04 (7) + 0.006 + 0.006 (7) + 0.08 +_0-02" (7)
- 2"8 + 1"2 (7) - 1230 + 850 (3)
Solid CO2 ( < -32°C)
G I L L E P I T H E L I U M , AND C H A N G E S AFTER F R E E Z I N G AND
Changes after freezing and thawing
Artemia
Results are given as mean + S . E . M . (No. of readings).
0-65 +0'03 (13) -0.007+_0.004(13) 0 . 0 4 + 0 " 0 2 (13)
+ 23"7 +_0-8 (15) 1740 +_ 280 (12)
Initially
THAWING
RESISTANCE AND I O N I C TRANSPORT N U M B E R S OF
* * P < 0.01 (t-test).
Epithelial p.d. (mV) Epithelial resistance ( ~ ) Transport numbers of Na K C1
TABLE 1--EPITHELIAL
,-]
bl
0
962
G. K. RY~NIEANDP. G. SMITH
adsorbed onto the cuticle. After freezing and thawing, dye in the gut was released into the bathing medium, but even after the animal had been returned to the phenol red solution for several hours no dye was visible in the blood. The coloration intensity of phenol red is reduced by low pH; to check that the apparent absence of dye from the blood was not caused by this effect a few drops of ammonia solution were added, to raise the pH. No dye could be seen in the blood, showing that the epithelial membranes remained sufficiently intact to exclude the dye. 25 30
20
15
P IO
0
--
Time, -I
-20
OiL
rain
"~" LLJ
kTemperoture
~-
Fro. 1. Time course of temperature and epithelial p.d. on freezing Artemia, using the Frigistor unit for freezing. Temperature was measured by means of a thermocouple placed beside the animal and does not therefore correspond exactly to the temperature of the animal. The eutectic point of sodium chloride, which makes up most of the solute of both blood and sea water, is -22°C. There was therefore still a liquid phase present at -18°C, but not at the lower temperature (below -32°C) which produced more significant changes in the electrical parameters. Thus it appears that complete dehydration may have had more effect than the increase of ionic concentrations during freezing. The absence of any marked effect of freezing and thawing on the electrical and permeability properties of the Artemia gill epithelium is in contrast to the large effects on the ionic properties of other tissues. In the isolated frog skin, which normally maintains a p.d. of the order of 100 mV when bathed with Ringer solution on both sides, both p.d. and resistance fall to zero after freezing with solid carbon
EFFECT OF FREEZING AND T H A W I N G ON GILLS OF A R T E M I A S A L 1 N A
963
dioxide and subsequent thawing (B. Andersen & K. Zerahn, personal communication). Nervous tissue of the rat fails to conduct impulses (Pascoe, 1957); presumably the failure of the limbs of A r t e m i a to resume beating after freezing and thawing is caused by a similar effect. None of these tissues which show sensitivity to freezing damage is subject to high osmolarities, or to rapid changes in osmolarity, in its normal environment. It therefore appears that the resistance of tissues to freezing and thawing may be related to the range of osmolarities which the tissue encounters, and tolerates, under normal circumstances. Acknowledgements--We thank Drs. P. C. Croghan and R. T. Joy for advice, and the S.R.C. for the award of research studentships. REFERENCES
CROGHANP. C. (1958a) The survival of Artemia salina (L.) in various media, ft. exp. Biol. 35, 213-218. CROGX-IANP. C. (1958b) Osmotic and ionic regulation of Artemia salina (L.) .7. exp. Biol. 35, 219-233. CROGHANP. C. (1958c) The mechanism of osmotic regulation in Artemia salina (L.). The physiology of the branchiae, aT. exp. Biol. 35, 234--242. LOWLOCK J. E. (1953) The haemolysis of human red blood cells by freezing and thawing. Biochim. biophys. Acta 10, 414 426. MACINNES D. A. (1961) Principles of Electrochemistry. Dover, New York. PASCOE J. E. (1957) The survival of the rat's superior cervical ganglion after cooling to -76°C. Proc. R. Soc. B 147, 510-519. SMITH P. G. (1969a) The ionic relations of Artemia salina (L.)--II. Fluxes of sodium, chloride and water. ~. exp. Biol. 51,739-757. SMITH P. G. (1969b) The ionic relations of Artemia salina (L.)--I. Measurements of electrical potential difference and resistance. ~t. exp. Biol. 51, 727-738. Key Word Index--Artemia salina; brine shrimp ; freezing; cryobiology.
SHORT COMMUNICATION EFFECT OF FREEZING AND THAWING ON IONIC P R O P E R T I E S O F T H E G I L L S O F A R T E M I A S A L I N A (L.) G. K. R E N N I E * and P. G. S M I T H t School of Biological Sciences, University of East Anglia, Norwich (Received 19 February 1970)
Abstract--1. In vivo electrical and permeability measurements have been made on the gill epithelium of the euryhaline Arternia salina before and after freezing and thawing. 2. Although the animal did not survive freezing, little change in epithelial properties occurred. 3. This suggests that tissues normally encountering widely varying osmolarities are resistant to freezing damage. INTRODUCTION
TH~ mGH ionic concentrations which occur when biological fluids are frozen cause damage to cell membranes, and it has been suggested that freezing injury is due to this effect (Lovelock, 1953). A r t e m i a salina, the brine shrimp, tolerates very high ionic concentrations in its environment (Croghan, 1958a); it therefore appeared that this animal might be resistant to freezing injury. Over the hundredfold range of external salinities in which the animal survives, the blood concentration varies only fourfold (Croghan, 1958b). T h e internal tissues do not therefore encounter a wide range of ionic concentrations; in fact the only tissue to experience the wide salinity variation of the external medium is the gill epithelium (Croghan, 1958c), and it is in this tissue that a resistance to the effects of freezing and thawing might be expected. Accordingly experiments were carried out to investigate the effects of freezing and thawing on the electrical and permeability properties of the gill epithelium of Artemia.
MATERIALS AND METHODS The gill epithelium of Artemia is far more permeable to ions than is any other part of the external cuticle and is also an order of magnitude more permeable than the gut (Croghan, 1958c; Smith, 1969a). In vivo measurements of electrical potential difference and resistance between blood and external medium are therefore largely controlled by the properties of the gill epithelium; measurements of this type were carried out in order to investigate the * Present address: Unilever Research Laboratories, Port Sunlight, Cheshire. t Present address : Department of Zoology, University of Liverpool, Liverpool L69 3BX. Please address reprint requests to P. G. Smith. 959
960
G . K . RENNIE AND P. G. SMITH
epithelial properties. The methods used have been described previously (Smith, 1969b). Briefly, an animal was ligatured at neck and anus, thus closing off the gut from the external solution, and immobilized in a small Perspex cell. Electrical potential difference (p.d.) between the blood and the external sea-water medium was measured by means of glass micropipette electrodes, using an electrometer of high input impedance. Current was passed through the epithelium via a second internal microelectrode, allowing epithelial resistance to be measured. The relative permeabilities of Na +, K + and C1- were also determined, as transport numbers; measurements were made of the change in p.d. which occurred when the external concentration of each of these ions was reduced by a factor 2. The inorganic ions were replaced by large organic ions (choline and benzenesulphonate) which were assumed to be impermeable. For a p.d. change of AVmV, the transport number T is given by T - - A V/17.8 at 25°C. The sign of AV is positive for the anion and negative for the cations. Placed immediately beside the animal was a calibrated copper-constantan thermocouple which measured the approximate temperature of the animal. Before freezing, epithelial p.d., resistance and transport numbers were measured. With the electrodes remaining in position the animal was frozen using either a Frigistor cooling device (final temperature -18°C) or a block of solid carbon dioxide (final temperature below -32°C) placed on the Perspex cell. The animal was then allowed to thaw and the parameters were remeasured. RESULTS AND D I S C U S S I O N I n unfrozen animals, the epithelial p.d. was 24 m V (blood positive) and only the sodium transport n u m b e r was significantly different from zero (Table 1). T h e s e results have been discussed in m o r e detail previously (Smith, 1969b). As cooling progressed, the p.d. fell rapidly to about 2 m V (Fig. 1) and remained at this level until thawing. Concurrently, beating of the limbs became slower and ceased completely at about - 4 ° C . T h i s was reversible, provided that no ice crystals f o r m e d within the animal. Freezing of the animal occurred at about - 10°C, when ice crystals were seen to f o r m ; thawing took place at 0°C. On thawing, the limbs did not resume beating, suggesting that damage to excitable tissues had occurred. However, there was little change in any of the measured parameters; the only significant changes were in the pooled results for epithelial p.d. and in the chloride transport n u m b e r after freezing to the lower t e m p e r a t u r e (Table 1). I f the epithelial m e m b r a n e s had ruptured, the resistance would have dropped considerably and the p.d. would have become a liquid junction potential decaying rapidly with time. T h e value of a liquid junction potential between the blood and sea water can be calculated f r o m the Henderson equation (MacInnes, 1961, p. 232) to be - 6 mV, substantially different f r o m the measured p.d. ; f u r t h e r m o r e the rate of fall of the p.d. (about 0-2 m V / m i n ) was small. It is therefore concluded that little damage to the epithelial m e m b r a n e s occurred. T o check that no gross damage to the epithelium had taken place, the permeability of the animals to the dye phenol red (molecular diameter about 10 A) was studied. Several animals were put into a sea-water solution of the dye for periods between ½ and 3 hr and were washed in sea water for a few minutes before examination. Before freezing, dye was accumulated in the gut only, with a little
p.d.,
* P < 0'02.
+0.08+0'04(12) +0.006+0.006(12) +0.04+-0"04(12)
- 2 - 2 + 0"7 * * (12) - 8 8 0 + 550 (5)
Total
+0'11 _+0'08 (5) +0.017+_0.011 (5) +0"01 +_0'01 (5)
- 0 " 5 + 1 "0 (5) + 100 + 200 (2)
Frigistor ( - 18°C)
+ 0.06 + 0.04 (7) + 0.006 + 0.006 (7) + 0.08 +_0-02" (7)
- 2"8 + 1"2 (7) - 1230 + 850 (3)
Solid CO2 ( < -32°C)
G I L L E P I T H E L I U M , AND C H A N G E S AFTER F R E E Z I N G AND
Changes after freezing and thawing
Artemia
Results are given as mean + S . E . M . (No. of readings).
0-65 +0'03 (13) -0.007+_0.004(13) 0 . 0 4 + 0 " 0 2 (13)
+ 23"7 +_0-8 (15) 1740 +_ 280 (12)
Initially
THAWING
RESISTANCE AND I O N I C TRANSPORT N U M B E R S OF
* * P < 0.01 (t-test).
Epithelial p.d. (mV) Epithelial resistance ( ~ ) Transport numbers of Na K C1
TABLE 1--EPITHELIAL
,-]
bl
0
962
G. K. RY~NIEANDP. G. SMITH
adsorbed onto the cuticle. After freezing and thawing, dye in the gut was released into the bathing medium, but even after the animal had been returned to the phenol red solution for several hours no dye was visible in the blood. The coloration intensity of phenol red is reduced by low pH; to check that the apparent absence of dye from the blood was not caused by this effect a few drops of ammonia solution were added, to raise the pH. No dye could be seen in the blood, showing that the epithelial membranes remained sufficiently intact to exclude the dye. 25 30
20
15
P IO
0
--
Time, -I
-20
OiL
rain
"~" LLJ
kTemperoture
~-
Fro. 1. Time course of temperature and epithelial p.d. on freezing Artemia, using the Frigistor unit for freezing. Temperature was measured by means of a thermocouple placed beside the animal and does not therefore correspond exactly to the temperature of the animal. The eutectic point of sodium chloride, which makes up most of the solute of both blood and sea water, is -22°C. There was therefore still a liquid phase present at -18°C, but not at the lower temperature (below -32°C) which produced more significant changes in the electrical parameters. Thus it appears that complete dehydration may have had more effect than the increase of ionic concentrations during freezing. The absence of any marked effect of freezing and thawing on the electrical and permeability properties of the Artemia gill epithelium is in contrast to the large effects on the ionic properties of other tissues. In the isolated frog skin, which normally maintains a p.d. of the order of 100 mV when bathed with Ringer solution on both sides, both p.d. and resistance fall to zero after freezing with solid carbon
EFFECT OF FREEZING AND T H A W I N G ON GILLS OF A R T E M I A S A L 1 N A
963
dioxide and subsequent thawing (B. Andersen & K. Zerahn, personal communication). Nervous tissue of the rat fails to conduct impulses (Pascoe, 1957); presumably the failure of the limbs of A r t e m i a to resume beating after freezing and thawing is caused by a similar effect. None of these tissues which show sensitivity to freezing damage is subject to high osmolarities, or to rapid changes in osmolarity, in its normal environment. It therefore appears that the resistance of tissues to freezing and thawing may be related to the range of osmolarities which the tissue encounters, and tolerates, under normal circumstances. Acknowledgements--We thank Drs. P. C. Croghan and R. T. Joy for advice, and the S.R.C. for the award of research studentships. REFERENCES
CROGHANP. C. (1958a) The survival of Artemia salina (L.) in various media, ft. exp. Biol. 35, 213-218. CROGX-IANP. C. (1958b) Osmotic and ionic regulation of Artemia salina (L.) .7. exp. Biol. 35, 219-233. CROGHANP. C. (1958c) The mechanism of osmotic regulation in Artemia salina (L.). The physiology of the branchiae, aT. exp. Biol. 35, 234--242. LOWLOCK J. E. (1953) The haemolysis of human red blood cells by freezing and thawing. Biochim. biophys. Acta 10, 414 426. MACINNES D. A. (1961) Principles of Electrochemistry. Dover, New York. PASCOE J. E. (1957) The survival of the rat's superior cervical ganglion after cooling to -76°C. Proc. R. Soc. B 147, 510-519. SMITH P. G. (1969a) The ionic relations of Artemia salina (L.)--II. Fluxes of sodium, chloride and water. ~. exp. Biol. 51,739-757. SMITH P. G. (1969b) The ionic relations of Artemia salina (L.)--I. Measurements of electrical potential difference and resistance. ~t. exp. Biol. 51, 727-738. Key Word Index--Artemia salina; brine shrimp ; freezing; cryobiology.