Intracellular and extracellular osmoregulation of temperature acclimated goldfish: Carassius auratus L.

~NTRAC~LLU~A~ AND EXTRACELLULA~ OSMOREGULATION OF TEMPERATURE ACCLIMATED GOLDFISH: CARASSIUS AURAlrUS L. ROBERT

H.CATLE~

AND DAVID

R. MILLICH*

Department of Biology, University of Colorado, Colorado Springs, CO 80907 U.S.A.

Abstract-t. CoIdfM were acclimated to temperatures ranging from 21.5 to 1°C. 2. Acclimation to near freezing temperatures caused a reduction in plasma osmolality due to decreased organic solutes as well as reduced plasma inorganic ions. 3. Plasma Na’ cvncn decreased in lower temperatures; plasma Cl- did not. 4. RBC response to decreased plasma osmolality included phases: osmotic swelling followed by cellular shrinkage 5. Cellular shrinkage resulted from efflux of KC1 and water. 6. The over-all response is an example of “isosmotic intracellular regulation” achieved by adjusting osmotically active particles.

iNTRODUCTlON

The study of vertebrate osmoregulatio~ has been primarily devoted to the extracellular system. It is assumed that in teleosts osmoregulation is exclusively extracellular because their excretory organs are capable of ma~ntainiug osmolality at a relatively constant level. Variations in the osmolality of these fishes do occur. One cause of variation is temperature. Umminger (1969) demonstrated that when the acclimation temp was lowered from 20 to - lS”C serum osmolality increased by 200/;;in the salt water adapted killifish, Fz~ndzfl~s~~~t~roel~tus.Serum electrolytes, sodium, chloride, calcium, magnesium and bicarbonate, increased in the subzero cold by 12, 17, 30, 33 and 11% respectively. Umminger (1969) suggested that these changes in serum electrolytes did not indicate osmoregulatory failure, since the newly established levels were maintained constant. Many marine fish survive quite well in the cold, and serum osmolality increases in most of these fold-acclimated fish (Scholander rt al., 1957; Pearcy, 1961; Gordon er u/., 1962; Rao, 1969). Similar studies have been made for freshwater fish. Parvatheswararao (1967) found a lowered sodium and chloride level in the tissue Auids of the Indian fish. Etr#plus ~c~~ut~.~. Umminger (1971) illustrated several patterns of osmoregulation demonstrated by freshwater fish exposed to the cold. These patterns showed a decrease in serum inorg~jc ion concentration. The response of serum osmolality varied from decreases commensurate with serum electrolytes to unchanged serum osmofality. In the latter case, organic substances are added to the serum in quantities sufficient to fully compensate for the reduction in electrolytes. To understand fully the osmotic adjustments made by freshwater fish to low temperature, serum osmolality as well as serum electrolyte levels should be studied. * Present address: Department of Veterinary Pathology. University of California. Davis. U.S.A.

There are 2 theories to explain why serum osmolality of marine fish increases and serum osmol~ity of freshwater fish decreases in response to cold acclimation. One states that the change is due to a reduced ability of these fish to osmoregnlate in the cold. The other suggests that this response is adaptive, since the amount of energy the fish requires to maintain osmotic gradients is less if the gradient is reduced (Prosser et al., 1970). Umminger (1971) proposes that both are probably oversimplifications of a much more complex osmoregulatory response. This phenomenon presents a unique situation to study, in cite, a mechanism of intracellular osmoregulation. There exists in these fish a naturally occurring, inducible alteration of serum osmolality to which the erythrocytes must conform. Lange (1964) working with the echinoderm. Stro~~loe~~tr~tl~~ drueboc&errs&c,suggests a model for intracellular osmoregulation. The term “isomotic intracellular regulation” is used to describe a form of volume regulation common to euryhaline invertebrates. The mechanism functions by adjusting the number of effective intracellular osmotic particles. In vitro studies, using duck erythrocytes exposed to nonhemolytic hypotonic media, also indicate some type of erythrocyte volumecontrolling me~h~ism (Kregenow, 1971). Potassium, chloride and water were implicated in the volume regulation of the erythrocyte. Lange & Fugelli (1965) have done in z&m studies of flounder and threespined stickleback erythroeytes exposed to hypotonic solutions. They found a vol~e”~ntrolling mechanism implicating ninhydrin-positive substances (NPS) and trimethylamine oxide (TMAO) as possible osmoregulatory intracellular particles as well as inorganic ions. This investigation was concerned with the goldfish, Carassius auratus and the effect of temperature on plasma osmolality and the concomitant effects on the erythrocytes. Aspects of goldfish acclimation to cold temperatures relative to osmoregulation have been examined. Prosser et al. (1970) found reduced serum sodium and chloride concentrations in 5°C accli-

261

ROBERT H. CATLETT A~YDDAVID

262

mated fish; however, no serum osmolalities were reported. There are conflicting views on water balance in cold acclimated goldfish. Meyer et al. (1956) reported an increase in water content of the entire body in the cold. Blood water content was found to increase in the cold in goldfish (Platner, 1950). The testes, liver (Clemens & Grant 1964) and muscle, (Hoar & Cottle, 1952) of Cu~uss~u~ u~rat~s showed dehydration in the cold. No change in water content at low temperatures were reported for the liver (Murphy, I961; Das, 1967) muscle and gill (Das, 1967) of goldfish. A more comprehensive study was necessary to gain an understanding of this phenomenon in the goldfish. An examination of selected erythrocyte constituents and indices and plasma constituents was indicated. It is apparent that the total response must be ascribed to the interaction of 2 different regulatory systems, the extracellular system and the intracellular system (Lange & Fugeili, 1965). In order to gain a fuller understanding of the nature of this entire system, the following plasma constituents were examined: osmolality, sodium, potassium, chloride, glucose, total protein and plasma water. With respect to the erythrocyte the following were examined: erythrocyte count, hematocrit, mean corpuscular volume (MCV), osmo-

tic fragility, sodium, potassium, water. MATERIALS

chloride

and cell

AND METHODS

All fish were obtained from a local supplier. The fish were inspected for external parasites and maintained in the laboratory for 2 weeks at room temp before acciimation to specific temp began. Mean wt of the fish was 14.2 g. Room temp was 215°C. Four groups of fish, 10 per group, were acclimated to 4 temps: 215°C lo”C, 5°C and 1%. The acclimation process consisted of lowering the temp 1’C per day until the appropriate temp was reached. The acclimation time was 6 weeks. The fish were kept in glass, 5 gallon aquaria equipped with bottom filters and a constant flow of atmospheric air. The aquaria were placed in constant temp baths adjusted to the appropriate temperature. Except for temperature, all fish received the same treatment. Naturally occurring day-night light cycles and approximate nutritional states were attained by feeding the 1°C and 5°C fish on alternate days, the WC fish daily and the 215°C twice daily (Prosser et ui., 1970). The fish were not fed for 24hr prior to the time of autopsy. To avoid erratic levels of plasma constituents, “training’” the fish for 2 weeks prior to autopsy was necessary (Pickford er ul.. 1969). The training procedure consisted of catching the fish each day and allowing them to swim in 3 I of aerated water for 20 min. The fish were then returned to their home tanks and fed. Standard metabolic rates were determined for each acclimation temperature using “trained” fish. Fish were anaesthetized with a 0.13, solution of tricaine methonesulfonate. The tail w-as severed, and free-flowing blood was collected from the caudal artery with a 180 ~1 amonium heparin blood collecting pipette. Five ELIand IO ~1 samples were taken for blood cell count and hematocrit. Thirty ~1 samples of whole blood were removed for osmotic fragility and then the pipette was sealed and the blood centrifuged at 3800 rev/min for 40 min. All centrifu-

R.

MILLICN

gations and blood collections were performed at the acclimation temperatures. After centrifugation the pipettes were broken at the white huffy layer and sealed immediately for storage at 5°C until the chemical determinations were made. All samples were transferred with 5 ,~l and 10 ~1 disposable pipettes, Plasma osmolality was measured by a melting point apparatus using 5 ~1 samples compared to reference standards. Melting times were recorded and plotted against osmolality. Plasma sodium and potassium determinations were made with a Beckman Model B flame spectrophotometer using sodium and potassium standard solutions for reference. Sodium determinations were made on 5 ~1 samples with a dilution factor of 1:lOO using distilled water. Potassium determinations were performed on 10 ~1 samples with a dilution factor of 1:40. Plasma chloride was measured with an Aminco-Cotlove chloride titrator using 5 ~1 samples. Plasma glucose was determined with an ultramicro adaptation of the Glucostat enzymatic method, following the procedures of Cawley et al. (1959). Plasma proteins were determined by the biuret method using an adaptation for small sample size (Bausch & Lomb, 1965). Twenty ~1 samples were employed and a Bausch & Lomb Spectronic 20 spectrophotometer was used. The standard solution was bovine albumin. Percent plasma water was obtained by determining wet and dry wts of 5 ,uI samples of plasma. The dry wt was determined using a Thelco Model 18 laboratory oven set at 100°C for 24 hr. Erythrocyte count was made using 5 pt samples of whole, unheparinized blood. A standard hemocytometer was used. Hematocrit determinations were made on 15 ~1 samples of blood drawn directly from the fish. Centrifugation was performed at the same speed and time as the larger samples, 3800 rev/mm for 40 min. The hematocrit values were corrected for percent trapped plasma. Osmotic fragility was measured by a micro method specified by Ezell ef al. (f969). Concentrations of NaCl in “tris” buffer ranging from 0.57: to 0.20;;,were used. The pH of this solution was 7.4. The mean corpuscular fragility (MCF) is defined as the concn of NaCl in which SOS
Intracellular and extracellular osmoregulation - 1 pt, PPO -50 mg/l, POPOP -4 g/l, total vol 15 ml. Samples were added directly to the flur mix and counted. Standard solutions were made in water, and 1 ,~l of inulin-“‘C was introduced into each sample. Duplicate readings on 5 replications were performed. The CPM were converted to DPM and normal standard corrections made to produce a value of 3.0% trapped plasma. (Valberg et al., 1965). RBC chloride was determined using an Aminco-Cotlove chloride titrator using 5 ~1 samples. The samples were diluted in 0.5 ~1 distilled water to produce a hemolysate soluble in the blank solution. A correction for percent trapped plasma was made. Percent water was obtained by determining wet and dry wts of 5 ~1 samples of packed cells. A Thelco Model 18 laboratory oven set at 100°C for 24 hr was used. A correction for percent trapped plasma was made. Standard metabolic rate (SMR) was determined using a YSI oxygen analyzer and reported as cm3 O&/kg. The fish were sealed in a 2 1, air tight container, the water acclimation temperature was maintained and the oxygen content of the water was measured at 5 min intervals. A magnetic stirring apparatus was used to ensure constant flow through the electrode membrane. Five determinations were performed at each acclimation temperature. All statistics presented in this paper are means +S.E. calculated for small sample numbers. Sample size for all mean values was 10 unless otherwise specified. Statistical significance of the difference was determined using the student’s t test.

RESULTS

Plasma osmolality, sodium chloride and potassium In Carassius auratus there is a statistically significant decrease in plasma osmolality upon acclimation to cold temperature (Fig. 1, Table 1). At 21.5”C the mean plasma osmolality was 294.6 + 2.06 mOsm/l. At 10°C the mean plasma osmolality was 288.4 f 2.32 mOsm/l, which is a significant reduction (P < 0.05). The largest difference in reduction of plasma osmolality occurred between 10 and YC, the mean value was 275.6 & 4.06 mOsm/l at 5°C (P < 0.01) and at 1°C it was 270.0 + 2.73 mOsm/l (P < 0.01). This represents a decrease of 8.5% in plasma osmolality over the entire temperature range. Table 1 summarizes the mean values of plasma electrolytes. Sodium concentrations were significantly lower than the control value for acclimation temperatures of 10, 5, and 1°C: -4.8 mM/l, -8.9 mM/l, and - 14.3 mM/l respectively. Chloride concentrations remained unchanged over the temperature range of 21.5-5°C. At 1°C the plasma chloride concentration increased slightly + 1.59 mM/l (P < 0.05). The plasma potassium level increased significantly (P < 0.01) at lO”C, was unchanged at 5°C and decreased slightly at 1’C (p < 0.05). At lO”C, reduction in plasma electrolytes can account for 92.6% of the reduction in plasma osmolality. At 5°C reduction in electrolyte concentrations can account for only 51.3% and at 1°C 54.1% of the reduction in plasma osmolality. Plasma organic constituents Table 2 summarizes the mean values of the organic constituents of the plasma. Plasma glucose was relatively unchanged over the temperature range of 21.5-10°C. However, at 5 and 1°C the glucose level

263

nearly doubled and represented a highly significant increase (P < 0.01). Total plasma protein at 21.5”C was 4.39 + 0.23 g/lOOml. This value remained unchanged over the temperature range of 21.5-10°C. At 5°C the total protein concentration was significantly decreased (P < 0.01) to 3.44 + 20 g/lOOml which represented a 21.7% decrease in plasma protein. At 1°C the value was also significantly lower and represented a 23.7% decrease. Erythrocyte indices The mean RBC count at 21.5”C was 1.88 & 0.05 x lo6 (Table 3). At 10 and 5°C the values were not significantly different from the control (P = 0.394 and P = 0.182 respectively). At 1°C the RBC count was 1.49 f 0.90 x 106. This was a highly significant decrease in RBC/mm3. The mean hematocrit at 21.5”C was 35.18% _t 0.51. The values at 10 and 5°C were not statistically different from the control value (P = 0.505 and 0.154 respectively). The mean hematocrit at 1°C of 28% k 1.96 was highly significantly different from the control value (P < 0.01). The mean corpuscular vol (MCV) at 21.5”C was 185.42 f 4.02~~. The value at 10°C was 203.53 + 7.08 p3 and was significantly higher (P < 0.05). The MCV at 5 and 1°C were not statistically different from the control value. The mean corpuscular fragility (MCF) values at 21.5 and 10°C were 0.388% NaCl and 0.382% respectively. There was a marked decrease in osmotic fragility at 5 and l”C, 0.335% and 0.337% respectively. These values were determined from percent hemolysis curves (Fig. 2). Erythrocyte electrolytes and intracellular water Table 4 summarizes mean electrolyte and water cones at the 4 acclimation temps. The control value for potassium, 110.43 f 2.06 mM/l was lowered at each acclimation temp. At 21.5”C the intracellular chloride concn was 107.3 f 1.08 mM/L The chloride concn was reduced at each acclimation temp. Sodium concns were reduced slightly from the control values at each temp. Intracellular water increased significantly (P < 0.01) at 10 and 5°C. At 1°C the percent water increased, but this value was not statistically significant (P = 0.176).

290-

260’

, 10

50

Acclimation

loo temperature

(“Cl

210

Fig. 1. Plasma osmolality determined at acclimation temperatures. Circles represent mean values; vertical bars S.E.M.

ROBERTH. CATLETTAND DAVIDR. MILLICH

264

Table I. Plasma osmolality and electrolyte concentration of Curussius aurutusacclimated to various temperatures. All values are means + standard error of the mean Parameter and units

215’C

Sodium mM,/l Chloride mM/l Potassium mM/l Osmolality mOsm/l

148.20 + 111.85 f 3.86 + 294.60 k

1crC 0.61 1.65 0.06 2.06

143.40 * 110.57 k 4.20 * 28X.40 f

2.21* 1.61 0.07** 2.32*

5c 139.30 * I1 1.25 * 3.64 * 275.60 +

IC 1.90** 1.02 0. I4 4.06**

133.YO+ 1.21** 113.44 * 0.72* 3.27 & 0.20* 270,OOk 2.73**

* Significantly different from values of controls at 21.5’C (P < 0.05). ** Highly significantly different from values of controls at 21.5’C (P < 0.01). Table 2. Plasma organic constituents

of Carassius auratus acclimated to various temperatures. + standard error of the mean

Parameter and Units

21.5”C

IO’C

42.29 + 6.97 4.39 -i_0.23

41.34 + 6.12 4.35 * 0.52

x4.19 * 4.59** 3.44 * 0.20**

75.43 f 6.10** 3.35 * 0.1x**

92.37 & 0.72

94.1 I * 0.7x

94.37 & 0.55*

94.6X k 0.72*

Glucose mg/lOO ml Total protein g/100 ml Water y,;,weight

All values are means

1°C

5c

* Significantly different from values of controls at 21.5’C (P < 0.05). ** Highly significantly different from values of controls at 2 I .5 ‘C (P < 0.01). Table 3. Erythrocyte physical indices. All values are means f standard error of the mean Parameter and units

21.5’C

Erythrocyte count cells/mm” ( x IO’) Hematocrit ?,Acell volume Mean corpuscular volume ($) Mean corpuscular fragility* (“(, NaCl)

IO’c

5C

I’C

1.X8f 0.05

I .X0 & 0.06

35.18 & 0.51

36.56 & 1.96

33.13 + 1.x

2X.00 k 1.96

185.42 k 4.02

203.53 * 7.0x**

3X7.36+ 7.68

187.92 f 8.16

0.3X81,,

0.382””

0.335”,,

0.337:;,

1.7x + 0.05

1.49 f 0.09t

* Determined graphically from mean values of “:, hemolysis. ** Significantly different from mean value of control at 21.5’C (P < 0.05). -i-Highly signiticantly different from mean values of control at 21.5’C (P < 0.01).

Table 4 represents concns of electrolytes in mmoles/l of packed cells. Since the volume of these cells varies (Table 3). it cannot be determined from these data if the alterations in electrolytes are relative, due to water influx, or absolute. MCV values are known at each temp and it is possible to determine the absolute electrolyte concn per cell (Table 5). The absolute concns of intracellular potassium, chloride and sodium do not significantly vary between the temp range of 21.51O’C. At 5 and I “C these values are significantly lower. Figure 3 summarizes electrolyte and water balance at each acclimation temp.

temp (Table 1; Fig. 1). This decrease, 294.6-270.0 mOsm/l, is a moderate reduction in comparison to other species studied. In the brown bullhead, 1ctaluru.s nebulosus, the decrease for a temp range of 2&1’C was 286.G201.0 mOsm/l and in Fundulus heteroclitus.

SMR values are shown in Fig. 4. At a temp of 21.5”C the mean SMR was 292.7 + 15.51 cm3 Oz,Jl,lhrikg. At 1°C the SMR was reduced to 76.2 f 8.23 cm3 O,/l/hr/kg. This represents a 73.87: decrease in SMR over the entire temp range. DiSCUSSlON Extracellular Curassius

plasma

osmoregulation auratus

osmolality

showed a significant reduction in upon acclimation to near freezing

Fig. 2. Hemolysis determmed at acclimation temperatures n = 21YC, 0 = 1O’C. 0 = 5°C. a = 1’C.

Intracellular Table

4. Erythrocyte

Parameter

electrolyte

concentrations

and units

and extracellular and water

content.

21.5”c

Sodium mM/l Chloride mM/l Potassium mM/l Water Y{,weight

13.97 107.30 110.43 66.89

f * k k

All values are means f standard

10°C 0.27 1.08 2.06 1.68

11.90 98.89 100.65 72.5

f 0.24* f 0.59** f 1.16** + 1.18**

5. Erythrocytes

Parameter Sodium mM/cell Chloride mM/cell Potassium mM,kell MCV ($)

electrolyte

and water values calculated are means k standard

error

11.76 102.18 94.70 70.80

+ k * rt

of the mean 1°C

5°C 0.30* 0.69** 0.75** 0.51**

II.09 94.48 91.25 70.00

per mean cell volume error of the mean

at various

temperatures.

5°C

* Significantly different from values of controls at 21.5”C (P -c0.05). ** Highly significantly different from values of controls at 21.5”C (P<

Table

265

osmoregulation

+ + f rf:

0.27** 1.19** 0.85** 1.42

0.01). All values

and units

21.5”C

10°C

(x IO-‘)

0.258 f 0.005

0.242 k 0.049

0.220 * 0.010**

0.208 * 0.007**

( x lo- “)

1.990 k 0.023

2.010 f 0.014

1.900 * 0.014**

1.790 + 0.022**

( x lo- ‘)

2.048 k 0.038 185.420 + 4.020

2.050 + 0.024 203.530 & 7.08*

1.760 f 0.014** 187.360 + 7.680

1.730 * 0.017** 187.920 k 8.160

1°C

* Significantly different from values of controls at 21.5”C (P < 0.05). ** Highly significantly different from values of controls at 21.5”C (P< 0.01).

adapted to freshwater, the decrease was 372G316.0 mOsm/l (Umminger, 1971). The nature of the reduction in plasma osmolality in the goldfish appears to be unique. Plasma sodium and potassium concns decreased, but the plasma chloride concn actually increased slightly in the 1°C fish. A decrease in plasma chloride has been reported in Etroplus macularus (Parvatheswaratae, 1967). Prosser et al., (1970) also found a reduction in plasma chloride concn in the goldfish when acclimated to 5°C. In this study plasma chloride decreased slightly at 5°C. but then rose above the control value at 1°C. It is apparent from these data that Na+, Cl- and K+ concns alone cannot account for the total decrease in plasma osmolality. It is highly unlikely that other plasma electrolytes; Mg”, Ca2+, HCO;, could account for this reduction since these electrolytes are present in such low concns. Table 2 summarizes the plasma organic constituents determined at each temp. Plasma glucose increased lOOoi;,over the control value at 5°C and slightly less at 1°C. Hyperglycemia is a well-known

response to stress in teleosts (Chavin, 1964). Nate rt al. (1964) showed that environmental temp exerts some influence on blood sugar level. By keeping toadfish at low temps, blood sugar levels, higher than normal, were produced. Similar results were reported for brown bullheads and the killifish by Umminger (1971). This increase in plasma glucose is quite marked, but osmotically it represents <2 mOsm/l over the temp range of 21.551°C. This decrease probably accounts for a portion of the reduced plasma osmolality. Since the exact nature of the plasma proteins are not known, a quantitative value representing colloid osmotic pressure is not possible. Considering the rather large disparity in osmotic pressure over the entire temp range, the slight reduction in colloid osmotic pressure is assumed here to be negligible. Percent plasma water increased consistently over the temp range of 21.5l”C. This is consistent with the lowered sodium and potassium concns as well

0 f

Fig. 3. Comparative

I

electrolyte and water mation temperatures.

levels at accli-

Fig. 4. Standard metabolic rate at acclimation temperatures. Circles represent mean values and vertical bars S.E.M.

266

ROBERT H. CATLETT AND DAVID

as lowered total protein. Umminger (1969) states that marine fishes lose water in the cold and that freshwater fishes gain water in the cold if data for whole body water content are compared. The literature shows no consistent trends in water content of tissues as affected by temp. All possible responses, increase, decrease, no change, have been suggested for the goldfish at low temps. While it is generally agreed that freshwater fish lower plasma osmolality in the cold, it is certainly not agreed as to how this process takes place. In studies where non-acclimated fish are immersed in near freezing water, hemodilution occurs as a result of water swallowing (Platner, 1950). This is no indication that acclimated fish swallow water. One hypothesis is that renal failure and reduced urinary water output may occur, resulting in dilution of body electrolytes by water taken up osmotically. This is contraindicated by the study of Mackay & Beatly (1968), who found that water reabsorption in the kidney was not significantly affected by environmental temp over the range of 142°C. Another hypothesis is generalized dilution of extracellular electrolytes as a result of mass movement of water from the cellular to the extracellular compartment. This implies that exposure to cold causes a decrease in the cellular concn of osmotically active materials resulting in a transfer of fluid to the extracellular compartment (Houston, 1962). This seems unlikely in the goldfish since percent cell water and cell vol increased at 10°C (Table 3 & 4). Another possibility is that the low temp depresses the ion transport mechanism (Krogh, 1939) to the extent that NaCl cannot be absorbed by gill epithelium rapidly enough to balance the water osmotically (Meyer et al., 1956). Wikgreen (1953) found that ion loss in both the lamprey and the crucion carp was greater at low temp than at high temp. Histological studies by Fedrov (1967) and Woodhead & Woodhead (1965) showed temp dependence of the activity of the chloride-secreting cells of the gill in the cold. A recent review of the literature (Maetz, 1971) gives an excellent account of the current knowledge concerning these “chloride cells.” Histologically these cells are larger than the flat epithelial cells specialized for respiratory gas exchange. They contain numerous mitochondria and an extraordinary development of cytoplasmic microtubules resembling a smooth endoplasmic reticulum. It has been suggested that passive electrolyte transfer occurs across the respiratory cells (Kirschner, 1970) and that active transfer occurs in the “chloride cells” because of the abundance of energyproviding mitochondria and the presence of rate limiting enzymes related to Na+ and Cl- transport, carbonic anhydrase and Na-K activated ATPase. The activities of both enzymes increase during adaptation to seawater in parallel with the increase in “chloride cell” number (Utida et al., 1971). Maetz, (1971) strongly suggests the existence of 2 independent active pumps probably located on the mucosal surface of the epithelial cells of the gill concerned with transport of Na+ and Cl-. He states that sodium and chloride exchanges are frequently of very different intensities and sometimes in different directions Differences in the absorption of ions of different charges can only he cuplained by exchange with endo-

R.

MILLICH

genous ions of the same charge. Maetz & Shaw (1960) proposed a model of the “chloride cell” in freshwater which states that ionized or free ammonia is normally exchanged against Na‘ and at the higher rates of Na+ absorption, H+ ion excretion supplements the ammonium excretion to account for the Na+ exchanged. The model also suggests that HCO; ions are the endogenous ions exchanged against Cl-. Referring to this model, possible mechanisms can be proposed to explain the electrolyte concns obtained in this study. The account of free ammonia available for exchange with Na+ may be limited and Na+ absorption could be inhibited. There is evidence that ammonium concns do affect Na+ absorption and can inhibit it (Maetz, 1971). Temperature may also affect the Na-K pump located on the serosal surface of the “chloride cell” and inhibit Na+ absorption. The fact that the Cl- concn did not parallel Na+ concn is not surprising since the model depicts 2 independent exchange mechanisms for Na+ and Cll transport. Since Cl- concn remained fairly constant over the temp range of 21.55°C and rose at 1°C it is possible that in Carassius auratus the Clexchange for HCO; mechanism is not inhibited by low temp. This would imply sufficient COZ production as well as normal carbonic anhydrase activity over the temp range of 21.551C. There is another possibility of a compartmental shift of Cl- from the intracellular to extracellular phase, since the intracellular Cl- concn decreased at low temp (Table 4). It can be demonstrated that the large decrease in cell chloride concn in the temp range of 5-1°C coincides with the increased plasma chloride concn. One or a combination of these factors result in hemodilution in Carassius uuratus when subjected to near freezing temps. This response of freshwater fish is considered to be adaptive. The amount of energy the fish needs to maintain osmotic gradients is less if the gradient is reduced. Since these cold-acclimated fish are metabolically sluggish, their limited energy need not be wasted on osmotic work if lowered osmolality can be tolerated (Prosser et al., 1970). Unfortunately, lowered osmolality also predisposes these fish to freeze, which can in no way be termed adaptive. In many cases the compensatory processes invoked because of the lowered osmolality are extremely expensive in terms of energy. In the freshwater adapted killifish, Fundulus heteroclitus, the serum NaCl concn is reduced 103.2 mM/l over a temp range of 11-1°C. The serum osmolality is reduced only 56 mOsm/l. This is accomplished by an increase of serum glucose from 2.8-59.2 mM/l (Umminger, 1970). The energy demand necessary to build up 56.4 mM/I of glucose must be extreme for a metabolically sluggish fish. This investigator is of the opinion that reduction in plasma osmolality is indicative of at least partial reduction in the ability to osmoregulate accompanied by osmotic tolerance and/or compensatory processes. Due to the variation in response (Umminger, 1971) it appears that the degree of tolerance or compensatory processes are species specific. Carassius auratus is able to regulate plasma osmolality relatively efficiently over this temp range and appears able to tolerate the moderate reduction with

Intracellular and extracellular osmoregulation

little stress. However, the subsequent intracellular osmoregulatory processes necessitated within the erythrocyte must be examined. RBC intracellular

osmoregulation

The erythrocyte count decreased slightly over the temp range of 21.555°C however, a significant decrease (P < 0.01) occurred at 1°C (Table 3). It would appear that in carassius auratus erythropoiesis is not significantly affected until ambient temps reach extremely low levels, i.e., 4-1°C. This lowered RBC count probably reflects decreased tissue oxygen demand as well as increased O2 water saturation at low temps. The hematocrit increased slightly at 10°C but was not statistically significant. At 1°C the hematocrit was reduced significantly (Table 3). Umminger (1969) reported that in all fish studied the hematocrit was definitely lowered between 20 and 10°C but at temps < 10°C the hematocrit no longer changed. The mean corpuscular vol data (Table 3) indicate that the erythrocytes swell at 10°C and return to the original vols at 5 and 1°C. This would indicate a volume regulatory process. Examination of cell electrolytes and cell water (Table 4) are necessary to explain this phenomenon. Cell water increased dramatically at 10°C and at 1°C percent cell water was not statistically different from the control value. Cell sodium, potassium and chloride concns all decreased over the temp range. Whether the decreases are due to cellular hydration or an absolute decrease in these electrolytes can be determined because the cell vol at each temp are known. Table 5 represents the absolute electrolyte values/cell. It can be noted that the absolute electrolyte values at 10°C do not differ statistically from the control values. The decrease in concn of these electrolytes was due to cellular hydration. The values at 5 and 1°C represent an absolute decrease in cellular electrolytes. Figure 3 illustrates these absolute variations in electrolyte and water balance for each temp. The reduction in intracellular electrolytes is further indicated by the osmotic fragility study (Table 3, Fig. 2). The decrease in osmotic fragility over the temp range is consistent with the fact that the cells have been diluted and therefore will resist hemolysis at lower osmotic concns. It is apparent that the erythrocyte response to reduction in plasma osmolality follows a pattern consisting of 2 phases: an initial phase of osmotic swelling due to an influx of water and a second phase consisting of vol regulation in which the cells shrink until they approach their initial isotonic vol. Such a system has been described by Kregenow (1971) for duck erythrocytes incubated in nonhemolytic hypotonic media and Lange & Fugelli (1965) for flounder erythrocytes incubated in hypotonic media. At 10°C there is a water influx and no actual electrolyte efflux. According to Davson (1937) there is a critical vol to which the RBC must swell before

it becomes permeable to potassium. Because of the relatively slight reduction in plasma osmolality, 6.2 mOsm/l at 10°C this critical vol is not reached and no regulatory phase follows the osmotic swelling phase. In the 5 and 1°C fish the reduction in plasma osmolality is sufficient to cause the RBC vol to reach the critical vol and initiate the secondary regulating

267

phase. This phase is characterized by a transient increase in the efflux of potassium, chloride, sodium to a slight extent and water. Potassium is the primary cation lost from the cell. Chloride is the primary anion lost while sodium remains comparatively impermeable. Upon loss of these electrolytes water leaves the cell until the initial cell vol is reached. The magnitude of this reaction depends on the hypotonicity of the plasma. Note the differences in intracellular K+ and Cl- concns at 5 and 1°C (Table 4). The loss of chloride, if accompanied by an additional expected passive loss of HCO;, is sufficient to maintain electroneutrality within the cell (Kregenow, 1971). The secondary process, the volume regulating phase, is responsible for the final volume adjustment. A necessary part of this mechanism is a means of producing the controlled loss of osmotic particles from the cell (effector) as well as a device sensitive to some parameter associated with cell vol which in turn can control the effector (Kregenow, 1971). The potassium loss is in the same direction as the electrochemical gradient for potassium. The increase in K+ efflux could result from the active transport of K+ down this gradient; however, it would be more economical for the cell to execute the cation loss by increasing the K+ leak. This would be consistent with the fact that at 1°C energy-dependent ion transport would be metabolically costly. Kregenow (1971) believes that one parameter, membrane elasticity, is the possible volume sensitive indicator, although as the cell contents are diluted there are a number of intracellular substances that could function in this capacity. Membrane elasticity is a possibility since it is theoretically capable of large relative changes as ‘the cell swells. This possibility is increased by the finding of a fibrous system in the human erythrocyte membrane which appears to have some relationship to the elastic properties of the membrane and when isolated is associated with the Ca2+-sensitive adenosine triphosphatase activity (Kregenow & Rosenthal, in prep). Changes in membrane elasticity could develop in areas of the membrane and somehow cause increased Kf leak permeability. Since the essential cellular change appears to be a modification in the cation content of the cell, the possibility of the Na-K exchange pump being involved was also investigated by Kregenow (1971). He found that 10m4 M ouabain does not affect the change in cell volume as duck erythrocytes regulate their volume in hypotonic media. This is consistent with the fact that intracellular Na+ did not increase during volume regulation (Table 4). Since the cells return to their original hydration state and volume, the total response can be considered an example of “isosmotic intracellular regulation.” This term defines the cellular mechanism whereby a cell’s hydration and volume are controlled by adjusting the number of intracellular osmotic particles when alterations occur in the osmolality of the external medium (Lange, 1964). It is considered a primitive adaptation and has been demonstrated in euryhaline invertebrates (Potts & Perry, 1964). In invertebrates the organic constituents of the cell account for the majority of the altered intracellular osmotic particles. The major changes occur in the low mol. wt non-essential amino acids and in some in-

268

ROBERTH. CATLETTAND

stances in taurine and trimethylamine oxide (Florkin & Schoffeniels, 1969). Lange & Fugelli (1965); Fugelli (1967) attempted to account for the volume adjustment of vertebrate erythrocytcs in terms of a reduction in the intracellular concn of free ninhydrinpositive substances (NPS). However, only l/8 of the expected decrease in the intracellular osmotically active solutes could be attributed to the decrease in cellular NPS if the latter are unionized. This study has demonstrated an in viva intracellular osmoregulatory mechanism similar to that described for euryhaline invertebrates and described in in vitro studies with several species of vertebrate erythrocytes. As a result of this mechanism the erythrocytes are in osmotic equilibrium with the plasma. The diluted erythrocytes are less prone to hemolysis in dilute plasma because of the newly established solute concentrations. The limiting factor would seem to be how much variation in dilution can the cell tolerate and still be physiologically efficient. This would be extremely critical in cells with excitable membranes. Goldfish acclimated to 5°C have significantly lower muscle potassium than goldfish acclimated to 15 and 25°C (Prosser et al., 1970). This would suggest further study into the mechanism of “isosmotic intracellular regulation” in freshwater fish that exhibit extreme variation in dilution of plasma concentration, i.e. the brown bullhead 1ctaluru.s n~bulosus.

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