Volume Regulation of Muscle Fibres in the Killifish, Fundulus heteroclitus BODIL SCHMIDT-NIELSEN Mount Desert Island Biological Laboratory, Salsburg Coue, Maine 04672
ABSTRACT Cell volume regulation has been observed in vitro in many tissues. In the killifish it was studied in vivo during transfer from SW to FW and from FW to SW. It was found that the cell water content in acclimated fish varies inversely with the osmolality of the medium. During transfer from SW to FW the plasma osmolality decreased. Cell swelling was prevented through net loss of solutes from the cells. The solutes lost were not potassium or sodium and are presumed to be organic solutes. During transfer from FW to SW the plasma osmolality increased, cell shrinkage occurred and no significant increase in cell solute content could be found during eight hours. During prolonged acclimation cell water content returns to a higher value.
When animal cells are exposed to osmotic changes in the extracellular fluid, their volumes change according to the Boyle-Van't Hoff law, which states that osmotically active water content (V) times the osmotic pressure ( w ) is constant: PV = k (House, '74). Thus, if the cell acts as a simple osmometer, the osmotically active water content varies inversely with the osmotic pressure of the medium. Many cells studied in vitro have been shown to have the capacity to regulate their volume (reviewed by SchmidtNielsen, '75). Following acute osmotic dilution of the suspension medium, the cell volume increases abruptly and then gradually decreases toward the original volume. This type of regulation (regulatory volume decrease, RVD) has been seen in nucleated red cells of ducks (Kregenow, '71a) and of flounders (Cala, '76), in mammalian renal tubule cells (Dellasega and Grantham, '73), in mammalian lymphoblasts (Rosenberg et al., '72) and in the muscle fibres of the blue crab (Lang and Gainer, '69). Volume regulation was not found in the frog satorius muscle studied by Blinks ('65). Following shrinkage in a hyperosmotic medium, volume regulation (regulatory volume increase, RVI) was seen in the flounder red cells (Cala, '76). Kregenow ('71b) noticed that RVI in duck red cells took place only when the potassium concentration in the medium was elevated above normal levels. Lang and Gainer ('69) found no RVI in the muscle fibre of the blue crab. J. EXP. ZOOL.,199; 411-418.
The present study deals with the regulations in cell water and ion content that take place in the muscle fibres of the euryhaline fish, the killifish Fundulus heteroclitus, following acute transfer from freshwater (FW) to seawater (SW) and vice versa. In contrast to the studies mentioned above, the present measurements were made in vivo. This precludes the manipulation of the extracellular medium other than that which occurs naturally during acclimation to different salinities. It does show, however, the normal changes which take place in plasma concentrations during the acclimation period and the reaction of the cells to these changes. It was found that the water content of the cells is maintained at a higher level in FW vs. SW acclimated fish. Cell volume regulation (RVD) follows transfer of the fish from SW to FW. It was further seen that in the killifish RVD is primarily due to loss of nonelectrolyte solutes. RVI was not apparent during eight hours following transfer from FW to SW but was apparent over a period of days. METHODS
The killifish were caught at the mouth of Northeast Creek on Mount Desert Island. They were maintained in the laboratory for at least a week prior to the experi1 This work was supported by National Institutes of Health grant AM 15972 to Bodil Schmidt-Nielsen and National Science Foundation grant GB 7851 to the Mount Desert Island Biological Laboratory.
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Fig. 1 Plasma concentrations and intracellular amounts in muscle fibres of killifish following transfer from SW to FW. Each data point represents five fish, except the last which represents three fish. Two or three tissue samples were taken from each fish. The values for plasma are expressed i n mM. Values on the left represent Mean t S.E. (n = 6). The values for amounts are expressed in pmo1eslg.d.c.s. (the product of the intracellular concentrations times the water content of the cells). Mean S.E. For values of n see table 2.
*
TABLE 1
Fundulus heteroclitus acclimated
sw
Osm Na K c1
FW
Plasma concentration mM n=6 n=6 409516 346f4 194c9 157f4 5.00 f0.23 4.54 rt 0.58 156f17 143513
P value
ABSTRACT Cell volume regulation has been observed in vitro in many tissues. In the killifish it was studied in vivo during transfer from SW to FW and from FW to SW. It was found that the cell water content in acclimated fish varies inversely with the osmolality of the medium. During transfer from SW to FW the plasma osmolality decreased. Cell swelling was prevented through net loss of solutes from the cells. The solutes lost were not potassium or sodium and are presumed to be organic solutes. During transfer from FW to SW the plasma osmolality increased, cell shrinkage occurred and no significant increase in cell solute content could be found during eight hours. During prolonged acclimation cell water content returns to a higher value.
When animal cells are exposed to osmotic changes in the extracellular fluid, their volumes change according to the Boyle-Van't Hoff law, which states that osmotically active water content (V) times the osmotic pressure ( w ) is constant: PV = k (House, '74). Thus, if the cell acts as a simple osmometer, the osmotically active water content varies inversely with the osmotic pressure of the medium. Many cells studied in vitro have been shown to have the capacity to regulate their volume (reviewed by SchmidtNielsen, '75). Following acute osmotic dilution of the suspension medium, the cell volume increases abruptly and then gradually decreases toward the original volume. This type of regulation (regulatory volume decrease, RVD) has been seen in nucleated red cells of ducks (Kregenow, '71a) and of flounders (Cala, '76), in mammalian renal tubule cells (Dellasega and Grantham, '73), in mammalian lymphoblasts (Rosenberg et al., '72) and in the muscle fibres of the blue crab (Lang and Gainer, '69). Volume regulation was not found in the frog satorius muscle studied by Blinks ('65). Following shrinkage in a hyperosmotic medium, volume regulation (regulatory volume increase, RVI) was seen in the flounder red cells (Cala, '76). Kregenow ('71b) noticed that RVI in duck red cells took place only when the potassium concentration in the medium was elevated above normal levels. Lang and Gainer ('69) found no RVI in the muscle fibre of the blue crab. J. EXP. ZOOL.,199; 411-418.
The present study deals with the regulations in cell water and ion content that take place in the muscle fibres of the euryhaline fish, the killifish Fundulus heteroclitus, following acute transfer from freshwater (FW) to seawater (SW) and vice versa. In contrast to the studies mentioned above, the present measurements were made in vivo. This precludes the manipulation of the extracellular medium other than that which occurs naturally during acclimation to different salinities. It does show, however, the normal changes which take place in plasma concentrations during the acclimation period and the reaction of the cells to these changes. It was found that the water content of the cells is maintained at a higher level in FW vs. SW acclimated fish. Cell volume regulation (RVD) follows transfer of the fish from SW to FW. It was further seen that in the killifish RVD is primarily due to loss of nonelectrolyte solutes. RVI was not apparent during eight hours following transfer from FW to SW but was apparent over a period of days. METHODS
The killifish were caught at the mouth of Northeast Creek on Mount Desert Island. They were maintained in the laboratory for at least a week prior to the experi1 This work was supported by National Institutes of Health grant AM 15972 to Bodil Schmidt-Nielsen and National Science Foundation grant GB 7851 to the Mount Desert Island Biological Laboratory.
411
412
400
z 300
BODIL SCHMIDT-NIELSEN
- 9 Osm -
\$/'\gL$
i4.0Y 33.5I
1
I
I
1
_F
' H2°1
-P I
I
I
I
I
Fig. 1 Plasma concentrations and intracellular amounts in muscle fibres of killifish following transfer from SW to FW. Each data point represents five fish, except the last which represents three fish. Two or three tissue samples were taken from each fish. The values for plasma are expressed i n mM. Values on the left represent Mean t S.E. (n = 6). The values for amounts are expressed in pmo1eslg.d.c.s. (the product of the intracellular concentrations times the water content of the cells). Mean S.E. For values of n see table 2.
*
TABLE 1
Fundulus heteroclitus acclimated
sw
Osm Na K c1
FW
Plasma concentration mM n=6 n=6 409516 346f4 194c9 157f4 5.00 f0.23 4.54 rt 0.58 156f17 143513
P value
