Camp. Biochem. Physiol., 1975, Vol. 5OA,pp. 383 to 385. Pergamon Press. Printed in Great Britain
EFFECT
OF TEMPERATURE ON FLUID ABSORPTION BY TELEOST GALL BLADDER W.C.
Department
MACKAY
of Zoology, University of Alberta, Edmonton,
Alberta, Canada
(Received 30 October 1973) Ahstraet-1. Gall bladders from goldfish held for 2 weeks at 5, 10 and 30°C did not show temperature acclimation in the rate of water absorption measured in vitro. 2. No differences were found in uiuo in the volume of bile stored or the composition of bile from goldfish held at 10 and 30°C for 1 month. 3. Gall bladders from brook trout which had been held at 5 and 15°C showed temperature acclimation in the in vitro rate of water absorption. 4. The rate of water absorption measured in vitro by gall bladders from both species increased as the incubation temperature was raised. Temperature coefficients for water absorption were greater between 5 and 15°C (2~774.98) than between 15 and 30°C (1.40-2.73).
INTRODUCTION THE GALL bladder of most vertebrates stores and concentrates bile produced by the liver. Bile salts are concentrated as a result of the active absorption of an essentially isotonic solution of NaCl. The mechanism of this absorptive process has been extensively investigated in both fish and mammals by Diamond and his colleagues (Diamond, 1962a, b,c; Diamond & Tormey, 1966; Diamond & Bossert, 1967; Machen & Diamond, 1969). However, the extent to which an organism can modulate gall bladder reabsorption has received only scant attention. Himno & Bern (1972) found that the rate of water absorption in vitro by gall bladders from a variety of teleosts was not affected by adaptation to salt or fresh water or by in vivo treatments of cortisol or prolactin, but absorption was inhibited by the in vitro administration of oxytocin and arginine vasotocin. Active transport processes in both the intestine (Smith, 1966a, b) and the kidney (Mackay, 1973) of goldfish show temperature acclimation. Since the gall bladder arises embryologically from the gut it might also be expected to show temperature acclimation in the transport it performs. The purpose of the present study was to determine the extent, it any, of temperature acclimation in the rate of water absorption measured in vitro by gall bladders from the goldfish, Carassius auratus (Linnaeus), and the brook trout, Salvelinus fontinalis (Mitchill). MATERIALS AND METHODS Yearling brook trout were obtained from the National Parks of Canada Fish Hatchery at Jasper, Alberta, and
goldfish weighing from 40 to 100 g from a commercial supplier. Both species were held in the laboratory under a natural photoperiod at 15°C for several weeks before beginning temperature acclimation. Goldfish were acclimated to 5, 10 and 30°C and brook trout to 5 and 15°C for at least 2 weeks. Fish acclimated to 5°C were fasted for 2 days and all other fish were fasted for 1 day before an experiment. Gall bladders were prepared and the rate of water absorption measured in vitro using the methods described by Diamond (1962a). The physiological saline in which the gall bladders were incubated contained NaCl 125 mM, KC1 4.8 mM, CaCl, 0.8 mM, NaHCOs 3.7 mM, NaH,PO, 2.9 mM, Na,HPO, 6sOmM and glucose 5 mM. Air was constantly bubbled through the incubation medium. The temperature of each incubation bath was regulated to f 0.5”C. Rate of water absorption was generally constant at each experimental temperature (Fig. 1). The rate of water absorption was expressed as pg of weight lost per cma of bladder surface area per hour. Surface area was calculated from the weight of the bladder at the beginning of each incubation period. This calculation assumed that the gall bladder was a sphere and that the density of the bladder fluid was unity. To determine whether temperature acclimation OCcurred in vivo in the volume of bile stored or in bile composition goldfish were acclimated to 10 and 30°C for 1 month. Before samnlina the 30 and 10”Cacclimated fish were fasted for 1 and 3-days respectively. The fish were killed by a blow on the head; the gall bladder and cystic duct were freed from the surrounding tissue and weighed. The bile was removed from the bladder and stored at - 20°C. Osmolality was determined by freezing point depression on a sample of bile which had been diluted 3 : 1 with demineralized water. Sodium and calcium concentrations were measured by emission flame photometry. Chloride concentration was measured by amperometric titration. 383
384
W. C. MACKAY
0.45
I-
0.40 E 07
-
-P c
0.35
-
*g
0.30
-
3 0.25
0
I 100
I 200
I 300
I 400
J 500
Time (min) Fig. 1. Effect of temperature change on the rate of weight loss by brook trout gall bladder incubated at 30 and 5°C. The arrow indicates the time at which the incubation temperature was changed. Student’s t-test for unpaired samples was used to test for the statistical significance of differences between treatments. RESULTS The rate of fluid absorption by goldfish gall bladders measured in vitro did not show temperature acclimation. When measured at 15°C the mean rate of water absorption was not significantly different (P> 0.05) between gall bladders from 5 and 30°C acclimated goldfish (Table 1). Although water absorption by 5 and 30°C acclimated groups
which were examined in goldfish (Table 2). Bile volume, osmolality, Na, Cl and Ca concentrations of the bile were not significantly different in fasted goldfish acclimated to 10 and 30°C. Temperature acclimation seemed to occur for the rate of water absorption by brook trout gall bladders in vitro. The rate of fluid absorption by gall bladders from 5°C acclimated fish measured at 5°C was comparable to that of gall bladders from 15°C acclimated fish measured at 15°C (Table 3). When measured at 5°C the rate of water absorption was significantly greater (P x 0.05) in gall bladders from 5°C than in gall bladders from 15°C
Table 1. Effect of acclimation temperature and incubation temperature tion by goldfish gall bladder in vitro
Acclimation temperature (“C) Incubation temperature (“C) Rate of water absorption (pg cm-a hr-3
5
on the rate of water absorp-
30
10
5
15
7.2* 1.5* (11)
35.9+ 9.4 (9)
5 8.8k3.0 (8)
* Mean + 1 S.E.M., number of determinations
was significantly different (E-0.05) at 5°C this difference was in the wrong direction to be of adaptive value for the fish. Water absorption by goldfish gall bladders increased as the incubation temperature was raised. Temperature coefficients for the rate of water absorption were higher over the lower temperature interval (5.0, 4.9, 2.8) than over the higher interval (1.4, 2.1). An inverse relationship existed between acclimation temperature and the temperature coefficients over both temperature intervals. Temperature acclimation did not occur in any of the parameters of in uivo gall bladder function
10 19.5k2.9 (11)
30 84.2421.3 (9)
5 12.4k1.8 (5)
15 34.3k7.1 (12)
30 56.8k9.9 (6)
in brackets.
acclimated trout. The two acclimation groups were also significantly different (PcO.05) when measurements were made at 15°C. DISCUSSION The present data extended the observations of Hirano & Bern (1972) to another experimental condition, namely temperature acclimation, which markedly affects many other physiological parameters. No evidence was obtained for temperature acclimation in the rate of water absorption in vitro by goldfish gall bladders. Brook trout gall
Effect of temperature of fluid absorption by teleost gall bladder
385
Table 2. Effect of temperature acclimation on in vivo volume of stored bile and on bile composition of gold&h Acclimation temperature (“C)
Volume of bile @g/g body wt.)
N
mOsm
Na
Cl
Ca
10 30
2.58 t 0.53 2.28 + 0.42
6 6
293.0& 2.8 291.8+8.0
289.9 + 4.2 283.8 f 6.4
l-6+0.2 3.3kO.9
13.351.7 11*5+0.9
Concentration (mM/l.)
Table 3. Effect of acclimation temperature and incubation temperature on the rate of water absorption by brook trout gall bladder in vitro Acclimation temperature (“C) Incubation temperature (“C) Rate of water absorption (pg cm-2 hr-I)
5 5 3357 (10)
bladders appeared to show temperature acclimation but lack of fish prevented further investigation with this species. The temperature coefficients between 15 and 30°C for fluid absorption by the goldfish gall bladder (1.4) was similar to the value of 1.7 obtained by Diamond (1962a) for roach gall bladders over a similar temperature range. In the present study differences were not detected in the in vivo gall bladder function of goldfish acclimated to 10 and 30°C because both acclimation groups were fasted long enough to allow them to Iill their gall bladders with fully concentrated bile. Differences presumably would have been found it both groups had been fasted for only 1 day. Temperature exerts a marked effect on salt and water transport by teleost gall bladder, hence in vivo the bile concentrating mechanism must proceed more slowly in 10°C than in 30°C acclimated fish. However, feeding and digestion are also much slower in fish at low temperatures. If temperature acclimation did not occur in the digestive enzymes themselves there would be little selective advantage favoring temperature acclimation of gall bladder function. The gall bladder is a rather inflexible organ in terms of modulating salt and water absorption. The low chloride concentration in the bile of fasted fish indicates that in vivo low chloride concentrations may act to limit the extent of fluid transport by the gall bladder and hence limit the extent to which bile can be concentrated.
15 85+_22 (9)
15 5 15 11&5 27513 (5) (5)
30 122k37 (4)
REFERENCES DIAMONDJ. M. (1962a) The reabsorptive function of the gall bladder. J. Physiol., Lond. 161, 442-473. DIAMONDJ. M. (1962b) The mechanism of solute transport by the gall bladder. J. Physiol., Lond. 161, 474502.
DIAMOND J. M. (1962~) The mechanism of water transport by the gall bladder. J. Physiol., Lond. 161, 503-527.
DIAMONDJ. M. & BOSSERTW. H. (1967) Standinggradient osmotic flow. A mechanism for coupling of water and solute transport in epithelia. J. gen. Physiol. 50, 2061-2083.
DIAMONDJ. M. & TORMEYM. McD. (1966) Role of long extracellular channels in fluid transport across epithelia. Nature, Lond. 210, 817-820. HIRANOT. & BERNH. A. (1972) The teleost gall bladder as an osmoregulatory organ. Endocr. Jap. 19, 41-46. HOLMESW. N. & S~0r-r G. H. (1960) Studies of the respiration rates of excretory tissue in the cut-throat trout (Sulmo clarki clarki)-II. Effect of transfer to sea water. Physiol. Zool. 33, 15-20. MACHENT. E. & DIAMOND J. M. (1969) An estimate of the salt concentration in the lateral intercellular spaces of rabbit gall bladder during maximal fluid transport. J. Mem. Biol. 1, 194-213. MACKAYW. C. (1974) Effects of temperature on osmotic and ionic regulation of goldfish. J. Comp. Physiol. 88, 1-19. SMITH M. W. (1966a) Influence of temperature ac-
climatization on sodium-glucose interactions in the goldfish intestine. J. Physiol., Land. 182, 574-590. SMITHM. W. (1966b) Time course and nature of temperature-induced changes in sodium-glucose interactions of the goldfish intestine. J. Physiol., Lomi. 183, 649-657.
Acknowledgement.s-I would like to thank Mr. Bert Kooyman of the National Parks Branch for providing the brook trout and Mr. Ken Tollefson for his technical assistance. This study was supported by operating grant number A-6587 from the National Research Council of Canada.
Key Word Index-Carassius aura&s; Salvelinus fontinalis; goldfish; brook trout; gall bladder; tempera-
ture; bile; temperature acclimation.
EFFECT
OF TEMPERATURE ON FLUID ABSORPTION BY TELEOST GALL BLADDER W.C.
Department
MACKAY
of Zoology, University of Alberta, Edmonton,
Alberta, Canada
(Received 30 October 1973) Ahstraet-1. Gall bladders from goldfish held for 2 weeks at 5, 10 and 30°C did not show temperature acclimation in the rate of water absorption measured in vitro. 2. No differences were found in uiuo in the volume of bile stored or the composition of bile from goldfish held at 10 and 30°C for 1 month. 3. Gall bladders from brook trout which had been held at 5 and 15°C showed temperature acclimation in the in vitro rate of water absorption. 4. The rate of water absorption measured in vitro by gall bladders from both species increased as the incubation temperature was raised. Temperature coefficients for water absorption were greater between 5 and 15°C (2~774.98) than between 15 and 30°C (1.40-2.73).
INTRODUCTION THE GALL bladder of most vertebrates stores and concentrates bile produced by the liver. Bile salts are concentrated as a result of the active absorption of an essentially isotonic solution of NaCl. The mechanism of this absorptive process has been extensively investigated in both fish and mammals by Diamond and his colleagues (Diamond, 1962a, b,c; Diamond & Tormey, 1966; Diamond & Bossert, 1967; Machen & Diamond, 1969). However, the extent to which an organism can modulate gall bladder reabsorption has received only scant attention. Himno & Bern (1972) found that the rate of water absorption in vitro by gall bladders from a variety of teleosts was not affected by adaptation to salt or fresh water or by in vivo treatments of cortisol or prolactin, but absorption was inhibited by the in vitro administration of oxytocin and arginine vasotocin. Active transport processes in both the intestine (Smith, 1966a, b) and the kidney (Mackay, 1973) of goldfish show temperature acclimation. Since the gall bladder arises embryologically from the gut it might also be expected to show temperature acclimation in the transport it performs. The purpose of the present study was to determine the extent, it any, of temperature acclimation in the rate of water absorption measured in vitro by gall bladders from the goldfish, Carassius auratus (Linnaeus), and the brook trout, Salvelinus fontinalis (Mitchill). MATERIALS AND METHODS Yearling brook trout were obtained from the National Parks of Canada Fish Hatchery at Jasper, Alberta, and
goldfish weighing from 40 to 100 g from a commercial supplier. Both species were held in the laboratory under a natural photoperiod at 15°C for several weeks before beginning temperature acclimation. Goldfish were acclimated to 5, 10 and 30°C and brook trout to 5 and 15°C for at least 2 weeks. Fish acclimated to 5°C were fasted for 2 days and all other fish were fasted for 1 day before an experiment. Gall bladders were prepared and the rate of water absorption measured in vitro using the methods described by Diamond (1962a). The physiological saline in which the gall bladders were incubated contained NaCl 125 mM, KC1 4.8 mM, CaCl, 0.8 mM, NaHCOs 3.7 mM, NaH,PO, 2.9 mM, Na,HPO, 6sOmM and glucose 5 mM. Air was constantly bubbled through the incubation medium. The temperature of each incubation bath was regulated to f 0.5”C. Rate of water absorption was generally constant at each experimental temperature (Fig. 1). The rate of water absorption was expressed as pg of weight lost per cma of bladder surface area per hour. Surface area was calculated from the weight of the bladder at the beginning of each incubation period. This calculation assumed that the gall bladder was a sphere and that the density of the bladder fluid was unity. To determine whether temperature acclimation OCcurred in vivo in the volume of bile stored or in bile composition goldfish were acclimated to 10 and 30°C for 1 month. Before samnlina the 30 and 10”Cacclimated fish were fasted for 1 and 3-days respectively. The fish were killed by a blow on the head; the gall bladder and cystic duct were freed from the surrounding tissue and weighed. The bile was removed from the bladder and stored at - 20°C. Osmolality was determined by freezing point depression on a sample of bile which had been diluted 3 : 1 with demineralized water. Sodium and calcium concentrations were measured by emission flame photometry. Chloride concentration was measured by amperometric titration. 383
384
W. C. MACKAY
0.45
I-
0.40 E 07
-
-P c
0.35
-
*g
0.30
-
3 0.25
0
I 100
I 200
I 300
I 400
J 500
Time (min) Fig. 1. Effect of temperature change on the rate of weight loss by brook trout gall bladder incubated at 30 and 5°C. The arrow indicates the time at which the incubation temperature was changed. Student’s t-test for unpaired samples was used to test for the statistical significance of differences between treatments. RESULTS The rate of fluid absorption by goldfish gall bladders measured in vitro did not show temperature acclimation. When measured at 15°C the mean rate of water absorption was not significantly different (P> 0.05) between gall bladders from 5 and 30°C acclimated goldfish (Table 1). Although water absorption by 5 and 30°C acclimated groups
which were examined in goldfish (Table 2). Bile volume, osmolality, Na, Cl and Ca concentrations of the bile were not significantly different in fasted goldfish acclimated to 10 and 30°C. Temperature acclimation seemed to occur for the rate of water absorption by brook trout gall bladders in vitro. The rate of fluid absorption by gall bladders from 5°C acclimated fish measured at 5°C was comparable to that of gall bladders from 15°C acclimated fish measured at 15°C (Table 3). When measured at 5°C the rate of water absorption was significantly greater (P x 0.05) in gall bladders from 5°C than in gall bladders from 15°C
Table 1. Effect of acclimation temperature and incubation temperature tion by goldfish gall bladder in vitro
Acclimation temperature (“C) Incubation temperature (“C) Rate of water absorption (pg cm-a hr-3
5
on the rate of water absorp-
30
10
5
15
7.2* 1.5* (11)
35.9+ 9.4 (9)
5 8.8k3.0 (8)
* Mean + 1 S.E.M., number of determinations
was significantly different (E-0.05) at 5°C this difference was in the wrong direction to be of adaptive value for the fish. Water absorption by goldfish gall bladders increased as the incubation temperature was raised. Temperature coefficients for the rate of water absorption were higher over the lower temperature interval (5.0, 4.9, 2.8) than over the higher interval (1.4, 2.1). An inverse relationship existed between acclimation temperature and the temperature coefficients over both temperature intervals. Temperature acclimation did not occur in any of the parameters of in uivo gall bladder function
10 19.5k2.9 (11)
30 84.2421.3 (9)
5 12.4k1.8 (5)
15 34.3k7.1 (12)
30 56.8k9.9 (6)
in brackets.
acclimated trout. The two acclimation groups were also significantly different (PcO.05) when measurements were made at 15°C. DISCUSSION The present data extended the observations of Hirano & Bern (1972) to another experimental condition, namely temperature acclimation, which markedly affects many other physiological parameters. No evidence was obtained for temperature acclimation in the rate of water absorption in vitro by goldfish gall bladders. Brook trout gall
Effect of temperature of fluid absorption by teleost gall bladder
385
Table 2. Effect of temperature acclimation on in vivo volume of stored bile and on bile composition of gold&h Acclimation temperature (“C)
Volume of bile @g/g body wt.)
N
mOsm
Na
Cl
Ca
10 30
2.58 t 0.53 2.28 + 0.42
6 6
293.0& 2.8 291.8+8.0
289.9 + 4.2 283.8 f 6.4
l-6+0.2 3.3kO.9
13.351.7 11*5+0.9
Concentration (mM/l.)
Table 3. Effect of acclimation temperature and incubation temperature on the rate of water absorption by brook trout gall bladder in vitro Acclimation temperature (“C) Incubation temperature (“C) Rate of water absorption (pg cm-2 hr-I)
5 5 3357 (10)
bladders appeared to show temperature acclimation but lack of fish prevented further investigation with this species. The temperature coefficients between 15 and 30°C for fluid absorption by the goldfish gall bladder (1.4) was similar to the value of 1.7 obtained by Diamond (1962a) for roach gall bladders over a similar temperature range. In the present study differences were not detected in the in vivo gall bladder function of goldfish acclimated to 10 and 30°C because both acclimation groups were fasted long enough to allow them to Iill their gall bladders with fully concentrated bile. Differences presumably would have been found it both groups had been fasted for only 1 day. Temperature exerts a marked effect on salt and water transport by teleost gall bladder, hence in vivo the bile concentrating mechanism must proceed more slowly in 10°C than in 30°C acclimated fish. However, feeding and digestion are also much slower in fish at low temperatures. If temperature acclimation did not occur in the digestive enzymes themselves there would be little selective advantage favoring temperature acclimation of gall bladder function. The gall bladder is a rather inflexible organ in terms of modulating salt and water absorption. The low chloride concentration in the bile of fasted fish indicates that in vivo low chloride concentrations may act to limit the extent of fluid transport by the gall bladder and hence limit the extent to which bile can be concentrated.
15 85+_22 (9)
15 5 15 11&5 27513 (5) (5)
30 122k37 (4)
REFERENCES DIAMONDJ. M. (1962a) The reabsorptive function of the gall bladder. J. Physiol., Lond. 161, 442-473. DIAMONDJ. M. (1962b) The mechanism of solute transport by the gall bladder. J. Physiol., Lond. 161, 474502.
DIAMOND J. M. (1962~) The mechanism of water transport by the gall bladder. J. Physiol., Lond. 161, 503-527.
DIAMONDJ. M. & BOSSERTW. H. (1967) Standinggradient osmotic flow. A mechanism for coupling of water and solute transport in epithelia. J. gen. Physiol. 50, 2061-2083.
DIAMONDJ. M. & TORMEYM. McD. (1966) Role of long extracellular channels in fluid transport across epithelia. Nature, Lond. 210, 817-820. HIRANOT. & BERNH. A. (1972) The teleost gall bladder as an osmoregulatory organ. Endocr. Jap. 19, 41-46. HOLMESW. N. & S~0r-r G. H. (1960) Studies of the respiration rates of excretory tissue in the cut-throat trout (Sulmo clarki clarki)-II. Effect of transfer to sea water. Physiol. Zool. 33, 15-20. MACHENT. E. & DIAMOND J. M. (1969) An estimate of the salt concentration in the lateral intercellular spaces of rabbit gall bladder during maximal fluid transport. J. Mem. Biol. 1, 194-213. MACKAYW. C. (1974) Effects of temperature on osmotic and ionic regulation of goldfish. J. Comp. Physiol. 88, 1-19. SMITH M. W. (1966a) Influence of temperature ac-
climatization on sodium-glucose interactions in the goldfish intestine. J. Physiol., Land. 182, 574-590. SMITHM. W. (1966b) Time course and nature of temperature-induced changes in sodium-glucose interactions of the goldfish intestine. J. Physiol., Lomi. 183, 649-657.
Acknowledgement.s-I would like to thank Mr. Bert Kooyman of the National Parks Branch for providing the brook trout and Mr. Ken Tollefson for his technical assistance. This study was supported by operating grant number A-6587 from the National Research Council of Canada.
Key Word Index-Carassius aura&s; Salvelinus fontinalis; goldfish; brook trout; gall bladder; tempera-
ture; bile; temperature acclimation.
