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Sodium influx and efflux in the spiny dogfish Squalus acanthias
Comp. Biochem. Physzol ,
1966, Vol. 19, pp. 649 to 653. _Pergamon Press Ltd
Prtnted ~n Great Britain
SODIUM INFLUX AND EFFLUX IN THE SPINY DOGFISH
SQUALUS ACANTHIAS J. W E N D E L L B U R G E R and D. C. T O S T E S O N Trinity College, Hartford, Connecticut, and Department of Physiology and PhalTnacology, Duke University School of Medicine, Durham, North Carolina, U.S.A. (Received
26
April
1966)
Abstract--1. With the use of Na 22, measurements were made of the influx of
Na via the anterior end and both the total and renal efflux of Na in the spmy dogfish. 2. In two experiments with unfed animals, anterior end influxes were 1'0 and 0"89 mMoles/kg-hr while total effluxes were 0.92 and 0"86 mMoles/ kg-hr respectively, 3. Renal effluxes in these two fish were 0"10 and 0"20 mMoles/kg-hr. 4. These results show that Na is taken up by the anterior end of the dogfish at a rate about equal to the total body efflux of this ion. 5. Since only 10-30 per cent of the total Na efflux is mediated by the kidney, substantial extra-renal loss of Na must occur. 6. The implications of these results for salt and water metabolism in elasmobranchs is discussed. INTRODUCTION ANALYSES of elasmobranch body fluids (e.g. Smith, 1931) show the plasma to be markedly hypertonic to the external sea water medium. It has been assumed that this situation provides for the osmotic uptake of water necessary to the fish. More recently, it was pointed out (Burger, 1962) that the unfed dogfish whose total urine and rectal gland (salt gland) fluids were collected i n t o t o for long periods gave a daily average output of chloride at 374 mMoles/1 (1 ml/kg/hr) versus a plasma level of about 240 mMoles/1. This strongly suggests that the dogfish takes up not only water but also salt. This paper reports experiments designed to explore this problem directly by measuring the Na influx into the head end and both the total and renal Na efflux. MATERIALS AND METHODS Two dogfish (Fish 1, 2) captured 3 and 7 days before use, and maintained unfed, were studied. Fish 2 was completely free from wounds. Fish 1 had a hook-wound carefully sutured 1 day before use. Both fish were kept in a small laboratory aquarium in running sea water (13.5°-14°C), except as noted below. One hour prior to experimentation, an indwelling polyethylene catheter was inserted into the caudal artery. T h r o u g h this catheter samples of blood were taken and injections made. Care was taken to negate the dead space of the catheter in all manipulations. After determination of the sodium space (see below), the catheter was removed, 649
650
J. WENDELLBURGERANDD. C. TOSTESON
the hole sutured, and further blood samples taken by arterial puncture. After initial efflux data were obtained, the urinary papilla was catheterized for urine collection. For the influx study, a box with lid in which a dogfish could lie unrestrained was tilted so that only the head and gills of the fish were under a measured amount of sea water aerated by an aquarium bubbler and cooled by ice in plastic bags. For Fish 2, the bath temperature was kept at 9°C for the whole period. For Fish 1, the temperature was 14--10°C for the first 30 min, falling abruptly to 5°C for 5 rain, and returned to 8°C for 31 rain. Both fish lay quietly and showed strong, even respiratory movements. Once the fish were set up in the box, a measured amount of Na 22was introduced to the bath. Fish 1 spent 66 rain and Fish 2 spent 2 hr in the Na 22 labelled bath. At the end of this time, samples of bath and blood were taken for analysis and the fish placed in running sea water. Additional blood samples were taken 30 rain and 1 hr later for Fish 1, and 1 hr later for Fish 2. At this time, a measured amount of Na e2 was injected into the caudal artery in order to measure the Na space. The amount of Na 22 injected was measured accurately by using the same syringe pipette to make the injection and to transfer aliquots of the injected labelled solution into volumetric flasks for counting. For Fish 1, blood samples were taken 0.5, 5, 23, 47 hr after injection. For Fish 2, blood samples were taken 1, 5, 22, 46 hr after injection. The sodium space was estimated from the 30-min plasma sample of Fish 1, and the hour sample of Fish 2. Blood samples were centrifuged and the Na 2~ concentrations measured in the separated plasma. All measurements of Na 22 were made with a well-type, crystal scintillation counter. Influx via the head end (iMHa, mMoles/kg-hr) was calculated as follows: iMH a
= ANat'/At ~XI~a
(1)
tc/ t
where ANal2 = change in Na 22 content of fish ~ - g j At --- time (hr) of exposure to Na 22 labelled bathing solution
I
1
SXl~a = specific activity of Na 22 in ambient sea water \mMoles]
ANa~2 was computed by multiplying the concentration of Na 22 in the blood plasma (Na~2)p, ( ~ - ~ ) b y the NA space (ml). The Na space was estimated from the relation Na ~2 injected Na space - (Na22)~._ (Na~2)~,°
(2)
651
SODIUM INFLUX AND EFFLUX I N THE S P I N Y DOGFISH
where (Na22),T' = concentration of Na 22 in plasma at the time T~ (0.5 hr in Fish 1 and 1"0 hr in Fish 2), while (Na22)pro = concentration of Na ~2 in plasma prior to injection of the isotope. Total Na efflux (°Mr~a) was estimated from the equation OM~a = [(NaZ2)~ ° - (Na22)~ E] x N a space
(3)
where (Na2~)~0 and (Na22)~E are the concentrations of Na ~2 in blood plasma at the start and end °f the efflux peri°d respectively (c-~), and PYr°~Naand P X ~ [ c/m 1 are the plasma specific activities of Na z2 ~m---M-0q~oles/at the start and end of the efflux period which was of duration te (hr). The renal efflux of Na (°M~a)was estimated from the relation (NaZZ)uT.x V
(4)
where (Na2~)~° is the concentration of Na 22 (c_~) in the urine at the end of the efltux period (re, hr) and V is the volume of urine produced. In making all of the above calculations, it was assumed that the concentration of Na in sea water was 440 mMoles/l and in blood plasma 250 mMoles/l. RESULTS AND DISCUSSION The results of the measurements of head end influx and total as well as renal effluxes of Na are shown in Table 1. The values for Na influx given in the table are quite reliable since the estimates of Na space used in the calculation (1) were made from measurements of the Na 22 concentration in samples of plasma taken at the same interval after injection as the time allowed for distribution of Na ~2 taken TABLE 1 - - N a
FLUXES I N DOGFISH
Fmh
Wt. (kg)
Na space (ml)
Na influx head end (mMoles/kg-hr)
1 2
4"0 3"8
898 678
1"0 0'89
Na effiuxes (mMoles/kg-hr) Total
Renal
Extra-renal
0"92 0"86
0"10 0"20
0"82 0"66
652
J. WENDELLBURGERANDD. C. TOSTESON
up during exposure of the anterior end of the fish to labelled sea water (0.5 hr and 1.0 hr for Fishes 1 and 2 respectively). These measurements of Na influx establish unequivocally that Na ions enter the head end of the unfed dogfish. The values for total Na effiux shown in Table 2 are somewhat less reliable. At least two sources of error should be noted. First, the calculation (3) assumes that the only sink for Na 22 disappearing from plasma during the efflux period is loss to the external running non-labelled sea water. Some of the loss of tracer from the plasma may be to slowly exchanging pools of Na in the fish, e.g. muscle, cartilage, etc. Furthermore, the calculation also assumes that the Na space during the effiux period does not change from the 0.5 hr (Fish 1) or 1.0 hr (Fish 2) value. That this assumption is not valid is suggested by the fact that the Na effiux calculated by this method tended to decrease with time. Obviously, these difficulties can be overcome by measuring the Na 22 lost to a known volume of non-labelled sea water directly rather than attempting to estimate Na 2~ loss from analyses of plasma Na 2~ concentration and Na space as was the case in these experiments. This will be done in future experiments. Despite these difficulties, the total Na effiux data shown in Table 1 do permit certain conclusions. They show that uptake of Na via the anterior end of the fish is more than enough to balance the total Na effiux. This suggests that the animals are approximately in Na balance and that all of the Na absorbed enters through the anterior end. The measurements of renal Na effiux are reliable since the loss of Na 22 by this route was measured directly. Note that the ratio of renal to total Na effiux was small and variable (0.15-0.25). Variations in urinary Na excretion are common in dogfish in which concentrations of Na in the urine may vary from 165 to 367 mMoles/1. (Burger, 1965). It is obvious from these measurements of renal Na efflux that most of the Na which enters the head end of dogfish is lost by some extra-renal route. Some is undoubtedly excreted by the rectal gland, but it is uncertain whether this will account for all or even most of the head end Na influx (Burger, 1962). It is possible that considerable Na efflux also occurs in the anterior end of the dogfish. In conclusion, it is perhaps appropriate to speculate briefly on the locus and possible mechanism of Na uptake by the anterior end of dogfish. Since the fish studied in these experiments were unfed, and since dogfish probably do not drink sea water (Smith, 1931), it is unlikely that Na is taken up via the gastro-intestinal tracts. In view of their salt-transporting role in marine teleosts, the gills seem a much more probable locus for uptake. It is unlikely that active Na transport is required to perform the uptake. This conclusion is supported by an examination of the ratio of head end influx to extra-renal efltux in e.g. Fish 2. The flux ratio is 1.3 which agrees quite well with what would be predicted, 1-8, from the flux ratio (Ussing, 1949) assuming that the electrical potential difference across the head end transport site ( ? gills) is small and can be neglected. Thus passive Na movement across the gill epithelium could produce a net uptake of Na more than sufficient to balance urinary loss of the ion. Clearly, further experiments are necessary to define this problem more adequately.
SODIUM INFLUXAND EFFLUXIN THE SPINY DOGFISH
653
REFERENCES BURGERJ. W. (1962) Further studles on the function of the recta] gland in the spiny dogfish.
Physiol. Zool. 35, 205-217. BURGER J. W. (1962) Roles of the rectal gland and the kidneys in salt and water excretion m the spiny dogfish. Physiol. Zool. 38, 191-196. SMITH H. W. (1931) The absorption and excretion of water and salts by elasmobranch fishes--II. Marine elasmobranchs. Am. J. Physiol. 98, 296-310. USSING H. H. (1949) Transport of ions across cellular membranes. Physiol. Rev. 29, 127-155.
1966, Vol. 19, pp. 649 to 653. _Pergamon Press Ltd
Prtnted ~n Great Britain
SODIUM INFLUX AND EFFLUX IN THE SPINY DOGFISH
SQUALUS ACANTHIAS J. W E N D E L L B U R G E R and D. C. T O S T E S O N Trinity College, Hartford, Connecticut, and Department of Physiology and PhalTnacology, Duke University School of Medicine, Durham, North Carolina, U.S.A. (Received
26
April
1966)
Abstract--1. With the use of Na 22, measurements were made of the influx of
Na via the anterior end and both the total and renal efflux of Na in the spmy dogfish. 2. In two experiments with unfed animals, anterior end influxes were 1'0 and 0"89 mMoles/kg-hr while total effluxes were 0.92 and 0"86 mMoles/ kg-hr respectively, 3. Renal effluxes in these two fish were 0"10 and 0"20 mMoles/kg-hr. 4. These results show that Na is taken up by the anterior end of the dogfish at a rate about equal to the total body efflux of this ion. 5. Since only 10-30 per cent of the total Na efflux is mediated by the kidney, substantial extra-renal loss of Na must occur. 6. The implications of these results for salt and water metabolism in elasmobranchs is discussed. INTRODUCTION ANALYSES of elasmobranch body fluids (e.g. Smith, 1931) show the plasma to be markedly hypertonic to the external sea water medium. It has been assumed that this situation provides for the osmotic uptake of water necessary to the fish. More recently, it was pointed out (Burger, 1962) that the unfed dogfish whose total urine and rectal gland (salt gland) fluids were collected i n t o t o for long periods gave a daily average output of chloride at 374 mMoles/1 (1 ml/kg/hr) versus a plasma level of about 240 mMoles/1. This strongly suggests that the dogfish takes up not only water but also salt. This paper reports experiments designed to explore this problem directly by measuring the Na influx into the head end and both the total and renal Na efflux. MATERIALS AND METHODS Two dogfish (Fish 1, 2) captured 3 and 7 days before use, and maintained unfed, were studied. Fish 2 was completely free from wounds. Fish 1 had a hook-wound carefully sutured 1 day before use. Both fish were kept in a small laboratory aquarium in running sea water (13.5°-14°C), except as noted below. One hour prior to experimentation, an indwelling polyethylene catheter was inserted into the caudal artery. T h r o u g h this catheter samples of blood were taken and injections made. Care was taken to negate the dead space of the catheter in all manipulations. After determination of the sodium space (see below), the catheter was removed, 649
650
J. WENDELLBURGERANDD. C. TOSTESON
the hole sutured, and further blood samples taken by arterial puncture. After initial efflux data were obtained, the urinary papilla was catheterized for urine collection. For the influx study, a box with lid in which a dogfish could lie unrestrained was tilted so that only the head and gills of the fish were under a measured amount of sea water aerated by an aquarium bubbler and cooled by ice in plastic bags. For Fish 2, the bath temperature was kept at 9°C for the whole period. For Fish 1, the temperature was 14--10°C for the first 30 min, falling abruptly to 5°C for 5 rain, and returned to 8°C for 31 rain. Both fish lay quietly and showed strong, even respiratory movements. Once the fish were set up in the box, a measured amount of Na 22was introduced to the bath. Fish 1 spent 66 rain and Fish 2 spent 2 hr in the Na 22 labelled bath. At the end of this time, samples of bath and blood were taken for analysis and the fish placed in running sea water. Additional blood samples were taken 30 rain and 1 hr later for Fish 1, and 1 hr later for Fish 2. At this time, a measured amount of Na e2 was injected into the caudal artery in order to measure the Na space. The amount of Na 22 injected was measured accurately by using the same syringe pipette to make the injection and to transfer aliquots of the injected labelled solution into volumetric flasks for counting. For Fish 1, blood samples were taken 0.5, 5, 23, 47 hr after injection. For Fish 2, blood samples were taken 1, 5, 22, 46 hr after injection. The sodium space was estimated from the 30-min plasma sample of Fish 1, and the hour sample of Fish 2. Blood samples were centrifuged and the Na 2~ concentrations measured in the separated plasma. All measurements of Na 22 were made with a well-type, crystal scintillation counter. Influx via the head end (iMHa, mMoles/kg-hr) was calculated as follows: iMH a
= ANat'/At ~XI~a
(1)
tc/ t
where ANal2 = change in Na 22 content of fish ~ - g j At --- time (hr) of exposure to Na 22 labelled bathing solution
I
1
SXl~a = specific activity of Na 22 in ambient sea water \mMoles]
ANa~2 was computed by multiplying the concentration of Na 22 in the blood plasma (Na~2)p, ( ~ - ~ ) b y the NA space (ml). The Na space was estimated from the relation Na ~2 injected Na space - (Na22)~._ (Na~2)~,°
(2)
651
SODIUM INFLUX AND EFFLUX I N THE S P I N Y DOGFISH
where (Na22),T' = concentration of Na 22 in plasma at the time T~ (0.5 hr in Fish 1 and 1"0 hr in Fish 2), while (Na22)pro = concentration of Na ~2 in plasma prior to injection of the isotope. Total Na efflux (°Mr~a) was estimated from the equation OM~a = [(NaZ2)~ ° - (Na22)~ E] x N a space
(3)
where (Na2~)~0 and (Na22)~E are the concentrations of Na ~2 in blood plasma at the start and end °f the efflux peri°d respectively (c-~), and PYr°~Naand P X ~ [ c/m 1 are the plasma specific activities of Na z2 ~m---M-0q~oles/at the start and end of the efflux period which was of duration te (hr). The renal efflux of Na (°M~a)was estimated from the relation (NaZZ)uT.x V
(4)
where (Na2~)~° is the concentration of Na 22 (c_~) in the urine at the end of the efltux period (re, hr) and V is the volume of urine produced. In making all of the above calculations, it was assumed that the concentration of Na in sea water was 440 mMoles/l and in blood plasma 250 mMoles/l. RESULTS AND DISCUSSION The results of the measurements of head end influx and total as well as renal effluxes of Na are shown in Table 1. The values for Na influx given in the table are quite reliable since the estimates of Na space used in the calculation (1) were made from measurements of the Na 22 concentration in samples of plasma taken at the same interval after injection as the time allowed for distribution of Na ~2 taken TABLE 1 - - N a
FLUXES I N DOGFISH
Fmh
Wt. (kg)
Na space (ml)
Na influx head end (mMoles/kg-hr)
1 2
4"0 3"8
898 678
1"0 0'89
Na effiuxes (mMoles/kg-hr) Total
Renal
Extra-renal
0"92 0"86
0"10 0"20
0"82 0"66
652
J. WENDELLBURGERANDD. C. TOSTESON
up during exposure of the anterior end of the fish to labelled sea water (0.5 hr and 1.0 hr for Fishes 1 and 2 respectively). These measurements of Na influx establish unequivocally that Na ions enter the head end of the unfed dogfish. The values for total Na effiux shown in Table 2 are somewhat less reliable. At least two sources of error should be noted. First, the calculation (3) assumes that the only sink for Na 22 disappearing from plasma during the efflux period is loss to the external running non-labelled sea water. Some of the loss of tracer from the plasma may be to slowly exchanging pools of Na in the fish, e.g. muscle, cartilage, etc. Furthermore, the calculation also assumes that the Na space during the effiux period does not change from the 0.5 hr (Fish 1) or 1.0 hr (Fish 2) value. That this assumption is not valid is suggested by the fact that the Na effiux calculated by this method tended to decrease with time. Obviously, these difficulties can be overcome by measuring the Na 22 lost to a known volume of non-labelled sea water directly rather than attempting to estimate Na 2~ loss from analyses of plasma Na 2~ concentration and Na space as was the case in these experiments. This will be done in future experiments. Despite these difficulties, the total Na effiux data shown in Table 1 do permit certain conclusions. They show that uptake of Na via the anterior end of the fish is more than enough to balance the total Na effiux. This suggests that the animals are approximately in Na balance and that all of the Na absorbed enters through the anterior end. The measurements of renal Na effiux are reliable since the loss of Na 22 by this route was measured directly. Note that the ratio of renal to total Na effiux was small and variable (0.15-0.25). Variations in urinary Na excretion are common in dogfish in which concentrations of Na in the urine may vary from 165 to 367 mMoles/1. (Burger, 1965). It is obvious from these measurements of renal Na efflux that most of the Na which enters the head end of dogfish is lost by some extra-renal route. Some is undoubtedly excreted by the rectal gland, but it is uncertain whether this will account for all or even most of the head end Na influx (Burger, 1962). It is possible that considerable Na efflux also occurs in the anterior end of the dogfish. In conclusion, it is perhaps appropriate to speculate briefly on the locus and possible mechanism of Na uptake by the anterior end of dogfish. Since the fish studied in these experiments were unfed, and since dogfish probably do not drink sea water (Smith, 1931), it is unlikely that Na is taken up via the gastro-intestinal tracts. In view of their salt-transporting role in marine teleosts, the gills seem a much more probable locus for uptake. It is unlikely that active Na transport is required to perform the uptake. This conclusion is supported by an examination of the ratio of head end influx to extra-renal efltux in e.g. Fish 2. The flux ratio is 1.3 which agrees quite well with what would be predicted, 1-8, from the flux ratio (Ussing, 1949) assuming that the electrical potential difference across the head end transport site ( ? gills) is small and can be neglected. Thus passive Na movement across the gill epithelium could produce a net uptake of Na more than sufficient to balance urinary loss of the ion. Clearly, further experiments are necessary to define this problem more adequately.
SODIUM INFLUXAND EFFLUXIN THE SPINY DOGFISH
653
REFERENCES BURGERJ. W. (1962) Further studles on the function of the recta] gland in the spiny dogfish.
Physiol. Zool. 35, 205-217. BURGER J. W. (1962) Roles of the rectal gland and the kidneys in salt and water excretion m the spiny dogfish. Physiol. Zool. 38, 191-196. SMITH H. W. (1931) The absorption and excretion of water and salts by elasmobranch fishes--II. Marine elasmobranchs. Am. J. Physiol. 98, 296-310. USSING H. H. (1949) Transport of ions across cellular membranes. Physiol. Rev. 29, 127-155.