Cation transport and energy metabolism in the nucleated erythrocyte of the dogfish shark, Squalus acanthias

Comp. Biochem. Phhysiol., 1972, Vol. 42A, pp. 639 to 654. Pergamon Press. Printed in Great Britain

CATION TRANSPORT AND ENERGY METABOLISM IN THE NUCLEATED ERYTHROCYTE OF THE DOGFISH SHARK, SQUALUS ACANTHIAS” JAMES H. VICTOR

THEODORE,

MURDAUGH,

EUGENE JR.t

D. ROBIN,

and CARROLL

E. CROSS

Departments of Medicine and Physiology, Stanford University School of Medicine, Stanford, California; + Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; and the Mount Desert Island Biological Laboratory, Salisbury Cove, Maine (Received 9 October 1971) Abstract-l. Electrolyte distribution in the nucleated erythrocyte of the dogfish shark, Squalus acunthius, resembles that found in low Na+-high Kf mammalian erythrocytes. 2. Na+ efllux (20°C) averaged 14.9 + 2.7 m-equiv x kg RBC HaO-’ x hr-i, being reduced significantly by monoiodoacetate (lOea M), Na exposure, and ouabain (low4 M). 3. K+ influx (20°C) averaged 9*1 f 4.0 m-equiv x kg RBC H,O-’ x hr-l, being reduced significantly by exposure to 12°C and ouabain (10e4 M). 4. Total 0, consumption (30°C) averaged 3.14 f O-76 mM x kg RBC H,O-’ x hr-‘. Sixty-five per cent of 00, is related to ATP generation. 5. Lactate production (30°C) averaged 1.52 mM x kg RBC H@i x hr-l. Inhibition of oxidative phosphorylation produced a Pasteur effect. 6. Total energy availability exceeds that required for cation transport, suggesting that other energy-consuming processes are present. A substantial amount of energy required for cation transport is presumably derived from oxidative phosphorylation. INTRODUCTION

between energy provision and energy utilization is a problem of general biological interest. The mature non-nucleated mammalian erythrocyte has been an important model in considering this relationship. In the mammalian erythrocyte, energy is supplied by a single metabolic pathway, anaerobic glycolysis (Whittam, 1964). This permits an accurate estimate of energy supply by measurements of glycolytic rate. A major energy consuming process involves the active transport of cations. The minimal work of cation transport can be calculated from simple thermodynamic considerations (Koefoed-Johnson & Ussing, 1960). Assessment of these factors has permitted approximations of the energy requirements for cation THE

RELATIONSHIP

* This work was supported by Grants HE-12123 Institutes of Health. 639

and HE-13784

from the National

640

J. THEODORE, E. D. ROBIN, H. V. MURDAUCH, JR. AND C. E. CROSS

transport and its relationship to energy supply (Bernstein, 1953 ; Whittam, 1964; Robin et al., 1971b). Comparable data involving energy supply and electrolyte metabolism for the various nucleated erythrocytes of non-mammalian vertebrates are scanty. In contrast to mammalian erythrocytes, nucleated non-mammalian erythrocytes generally possess O,-consuming metabolic pathways. A portion of this 0, consumption is involved in the generation of ATP by oxidative phosphorylation. The relationship between ATP generated by glycolysis and that generated by oxidative phosphorylation is of obvious interest. The electrolyte composition of extracellular fluid of non-mammalian vertebrates may differ substantially from that present in mammals. The effect of this difference on electrolyte composition and electrolyte transport in the erythrocyte is likewise of interest. In the present studies some of these problems have been investigated in the nucleated erythrocyte of the elasmobranch, Squuh acanthius (spiny dogfish). This cell is nucleated, manifests substantial 0, consumption, and exists in vim in a plasma containing approximately 250 m-equiv/l. of Na+ and 250 m-equiv/l. of Cl-, values substantially higher than the corresponding values in human plasma. Moreover, plasma osmolality is approximately three times as great as compared to human plasma (Smith, 1936). This study will include data concerning steady-state plasma-erythrocyte gradients of Na f, K+ and Cl-; the magnitude of Naf efflux and K+ influx; a quantitative estimate of oxidative phosphorylation and glycolysis; and will consider the relationship between energy supply and the energy cost of cation transport. MATERIALS

AND METHODS

Over 100 North American female dogfish (S’qualus acunthias) were employed. Animals were maintained in live cars immersed in the ocean (12-15°C) for no longer than l-2 days prior to study. Steady

state electrolyte composition of erythrocytes

and plasma

The methods used in estimating [Na+], [K+] and [Cl-] have been described previously (Robin et al., 1964). Preparation

in plasma and red cell water

of erythrocytes for transport and metabolic studies

Fresh heparinized blood was obtained by direct puncture of the caudal artery and centrifuged at 1200 g at 10°C for 5 min. The huffy coat and plasma were discarded, the erythrocytes resuspended and washed three times in elasmobranch Ringers Solution (ER) composed of Naf 255 m-equiv/l., K+ 5.5 m-equiv/l., Caa+ 5 m-equiv/l., Mg*+ 3 m-equiv/l., 0.5 m-equiv/l., glucose 5 mM, urea Cl- 270 m-equiv/l., HCOa- 5 m-equiv/l., H,PO*350 mM with the pH adjusted to 7.50. Each wash suspension contained 1 vol. of cells to 4 vol. of ER. The final supernatant was discarded and the cells quickly utilized. Each experiment was performed on 20-30 ml of erythrocytes obtained from a single animal. Mixing of cells from different sharks was found to produce hemolysis presumably because of the existence of different blood groups. Care was required in handling the cells as substantial hemolysis occurred if the cells were excessively agitated during manipulations.

CATION

TRANSPORT

AND

FNERGETICS-SHARK

641

ERYTHROCYTE

Magnitude of trap. A limitation of the accuracy of measurements of intracellular electrolyte concentrations is the magnitude of trapped plasma. This is of great quantitative importance in this species because of the relatively high plasma [Na+] and [Cl-]. Small differences in trapped plasma could lead to large differences in estimates of intracellular electrolyte concentrations. Samples of erythrocytes were washed three times in ER and processed as described above. Approximately 10 PC of 14C-labeled Dextran (mol. wt. 70,000) was added to each sample of erythrocytes suspended in ER (approximate hematocrit = 25 per cent). The suspensions were incubated at 12°C for 60 min. Radioactivity was counted in aliquots of cells and extracellular fluid using a liquid scintillation counter with appropriate corrections for quench. The percentage trap was calculated as the ratio of counts/mm per g of red cell water to counts/min per g of extracellular water x 100. The percentage trap in seventeen determinations averaged 3.6 kO.71 (SD.). In view of the reproducibility of trap measurements, we used a value of 3 per cent for trapped fluid corrections in all studies. Red cell Na+, Kf, Cl- and water contents were corrected for trapped Na+, K+, Cl- and H,O by application of the following formula: Corrected RBC content = [observed RBC content - 0.03 x plasma content]/0*97. Trapped extracellular fluid was not independently measured in subsequent studies. Nat and K+ transport (a) Na e@ux. For measurements of Na+ efflux, approximately 25 ml of washed and packed erythrocytes were resuspended in 100 ml of ER with glucose (5 mM/l.) to which penicillin (100,000 units), streptomycin (O-05 g) and 25 PC *%Na+ had been added. This suspension was incubated for approximately 12 hr at 5°C with the mixture being frequently stirred. After labeling, the cells were washed five times with unlabeled ER (cells comprised 10 per cent of wash volume) to remove the extracellular *rNa+. Following the removal of ER after the last wash, the final aaNa+ labeled cell pack had a hematocrit of greater than 98 per cent. Approximately 2-g aliquots of the labeled cells were suspended in 10 ml of ER with glucose (5 mM/l.) and the mixtures were incubated at 20 + 2°C using a Dubinoff metabolic shaker. Approximately 4-ml samples were obtained at 1, 2, 3 and 4 hr for determinations of red cell radioactivity and “sNa+ concentration measurements. The samples were processed in the following fashion. The cells were separated by rapid centrifugation and radioactivity determined on weighed aliquots of the separated cells and O-5-ml aliquots of the supernatant using a gamma spectrometer. The weighed cells were then dried to a constant weight at lOO”C, and [Na+] was determined on nitric acid digests of the dried cells (Robin et cd., 1964). Cell water was determined by the difference in weights of the wet and dried cells. Erythrocyte [Na+] and radioactivity were calculated per kg of erythrocyte water. [r3Na+] was also determined on the supematants. All erythrocyte *aNa+ and 23Na+ measurements were corrected for an assumed 3 per cent trap of extracellular fluid in the separated cell fraction. Naf efflux was calculated from measurements of the decrease in intracellular azNa+ concentrations per unit time (Robin et al., 1971b). In brief, Na+ efflux was calculated from the following formula: (m-equiv

x

Naf efllux kg RBC H,O-i

x

hr-l) =

ARBC radioactivity x kg RBC H,O-’ RBC radioactivity/[88Na+]

x

hr-l

’

In this equation the numerator represents the measured aaNa+ efflux rates and the denominator represents the mean specific activity of intracellular Na+ in the erythrocyte during the period of study. The decrease in intracellular aaNa+ changed in a first-order fashion after the first hour, whereas the intracellular Na+ concentration remained essentially unchanged. No corrections were made for back diffusion. Na+ efflux was measured under aerobic (air) and anaerobic (100% N,) conditions, and as a function of the addition of ouabain (10e4 M) and monoiodoacetate (MIA) (10-a M). (b) K+ influx. Approximately 2 g of erythrocytes were added to an incubation medium consisting of 10 ml ER and 4aK+. Erythrocyte 4eK+ influx was determined by measuring

642

J. THEODORE, E. D. ROBIN, H. V. MURDAUGH, JR. AND C. E. CROSS

the rate at which the raK+ added to the supernatant at the beginning of the incubation period entered the cells. Since the supernatant PnK+ radioactivity was high, a greater degree of accuracy was obtained by washing the separated cells three times with ER containing no radioactivity prior to counting. The method of calculating K+ influx involved the serial measurements of 48K+ radioactivity and K+ concentration in the ER and erythrocyte PaK+ radioactivity at 30 and 150 min of the incubation. As the cells were washed substantially free of extracellular fluid 4aK+ prior to determining their radioactivity, it was unnecessary to correct cell 4aK+ radioactivity for trapped 4aK+. Knowing the incubation media (ER) specific activities, and the change in cell 4sK+ radioactivity, the K+ influx is calculated from the following equation : K+ influx (m-equiv x kg RBC H,O-l

x hr-‘)

= AraK+ radioactivity x kg RBC H,O-l x hr-’ Ringer’s 4rK+ radioactivity/Ringer’s [40K+] *

No corrections were made for back diffusion. K+ influx was measured at 12+2”C 20 + 2”C, under aerobic (air) conditions, and during exposure to ouabain (lop4 M).

and

Metabolic studies (a) Oxygen consumption. Preparation of the erythrocytes was performed as described above. Approximately 2 ml of packed erythrocytes were placed in respirometer flasks containing 4 ml of ER, with or without metabolic inhibitors. All flasks contained penicillin (100,000 units) and streptomycin (0.05 g). The flasks were weighed before and after the addition of erythrocytes to determine the weight of the added cells. The center wells contained 10% KOH in which filter paper wicks were placed. The flasks were then gassed with 100% 0, and incubated at 12 or 30°C in a modified Gilson Differential Respirometer with all glass tubing.* After gassing with O,, measurements of Or consumption were performed at 15-min intervals. Following the initial 15 to 30 min, a steady state was achieved. The subsequent 15 min readings were used to calculate 0, consumption. 0, consumption values were corrected to STPD and expressed as mM x kg RBC HeO-’ x hr-l. Studies were performed under control conditions (no inhibitors) and following exposure to NaCN (10e3 M), antimycin A (50 pg/flask), Dinitrophenol (DNP) (lOes M) and ouabain (1O-4 M). (b) Measurement of lactate production. Two ml of packed erythrocytes were added to 4 ml ER with glucose (5 mM), penicillin (100,000 units) and streptomycin (0.05 g). The weight of the erythrocytes was obtained by the difference in the weight of the flasks before and after the addition of cells. After the initial mixing of cells and media for several minutes, l-ml aliquots of the mixture were obtained to determine the time zero (To) lactate concentration. The mixtures were then incubated with or without metabolic inhibitors under aerobic conditions at 30°C. Those with inhibitors included cyanide (lOes M), antimycin A (50 pg/flask), dinitrophenol (1O-6 M) and ouabain (10e4 M). After incubation for 2 hr, lactate concentration was measured in the mixture. Lactate production was calculated from the difference between the To and TB hour samples in mM x kg red cell HsO-’ x hr-‘. Lactate was determined enzymatically using a modification of the Bucher technique (Bucher et al., 1959). RESULTS

Steady-state

electrolyte

Steady-state

concentrations

concentrations

of Na f, K+, Cl- in plasma and erythrocyte water

* The plastic tubes were replaced by glass tubes to prevent alterations of gas composition produced by diffusion of gases through the plastic tubing.

CATION

TRANSPORT

AND

RNRRGETICS-SHARK

643

ERYTHROCYFE

are shown in Table 1. * We have previously presented evidence for the rapid thermodynamic equilibrium of Cl- between plasma and erythrocyte water (Robin et al., 1964). Assuming this, the transmembrane potential at 12°C has been calculated by means of the Nernst equation. The calculated transmembrane potential TABLE

I-STEADY-STATE

ELECTROLYTE

Plasma (m-equiv x kg plasma H,O-‘)

CONCENTRATIONS

(m-equiv

x

IN DOGFISH

ERYTHROCYTRS

RBC kg red cell HsO-‘)

Wa+li = N+l,

0,14 f 0.04

240 f 36

lK+l

3.6 k 0.6

218+43

-lK+li = 63.9 + 21

[Cl-l

259 + 20

137+15

-[Cl -1i = 0.53 f 0.04

Transmembrane potential: 16 f 2 (SD.) Mean f 1 SD. from ten animals. i = Intracellular concentration. o = Outside concentration.

33 f 12

-

[Na +I

[K+l,

El-10

mV.

averaged 16 + 2 mV (S.D.). Na+ and K+ are not distributed in accordance with thermodynamic equilibrium, suggesting active transport of these ions. Table 2 compares these values with typical values in the human and the seal. Also shown in this table are the intracellular concentrations of Na+ and K+ which would be present if electrochemical equilibrium was obtained. It is obvious that despite the high [Cl-] in shark plasma, the ratio of intracellular to extracellular Cl- concentrations is similar to that present in non-nucleated mammalian erythrocytes. As a result, the calculated transmembrane potential is similar in all three erythrocytes. Na+ and K+ in all three cells are not in thermodynamic equilibrium. The extensive departure from equilibrium in the shark red cell is obvious. Despite the high Naf concentration in shark plasma, the ratio of intracellular Na+ to extracellular Na+ is similar to that in the human erythrocyte (low Na+ cell) and not like that in the high Naf cell (seal). Likewise, the ratio of intracellular Kf to extracellular K+ resembles that found in human erythrocytes (high K+) and not that found in seal erythrocytes (low K+). Naf eflux These data are summarized in Table 3. Na+ efflux averaged 14.9 + 2.7 mequivx kg red cell HsO-l x hr-l at 20°C. No data were obtained at 12°C (the * These data have been reported previously (Robin et aZ., 1964), but are presented here because of their relevance to the present study. 23

J. THEODORE,E. D. ROBIN, H. V. MURDAUGH,JR. ANDC. E. CROSS

644 TABLE 2-A

COMPARISON OF STEADY-STATE ELECTROLYTECONCENTRATIONS IN HUMAN,SEAL AND DOGFISH ERYTHROCYTES

Temperature

in viva:

Wa+ld Wa+l,

P. vitulina 40°C

S. acanthias 12°C

10

147 161 0.91 258

33 240 0.14 456

150 0.07 255

RN*+ Equilibrium concentration

W+li lK+l, RK+

Equilibrium concentration [Cl -14 [Cl-l, RCIT.M.P.* * Trans-membrane R, = blih4,.

H. sapiens 37°C

150 4 37.5 6.8

8 4 2.0 6.4

218 4 54.5 7.6

65 110 0.59 14

70 116 0.60 13

135 260 0.52 16

potential in mV.

physiological temperature of this animal). It is clear that the efflux is substantially higher at 20°C than efflux in low Na+ mammalian erythrocytes (such as man) despite the higher temperature in mammals. Ouabain (lo-*M) produced a significant reduction in Na + efflux to 2.4 + 1.7 m-equiv x kg cell H,O-1 x hr-l (P < 0.001). The “active” Na+ efflux (total flux- ouabain flux) averaged 11.5 & 3.4 m-equiv x kg cell H,O-l x hr-r(77 per cent of total flux). This value is approximately fivefold that found in human erythrocytes. TABLE

3-SODIUMEFFLUX

INDOGFISHERYTHROCYTJBAT

20°C

Na+ flux (m-equiv x RBC HsO-l x hr-‘) Total Na+ flux Ouabain (10e4 M) Na+ flux “Active” Na+ flux (total flux - ouabain flux) Twenty-four

14.9 k 2.7 2.4 rf:I.7 II.5 f 3.4

P

< O*OOl

studies.

Table 4 summarizes data concerning the effect of various inhibitors on Na+ efflux. Inhibition of glycolysis by MIA (10-s M), which interferes with 3 phosphoglyceraldehyde dehydrogenase, produced a significant reduction in Na+ efflux to 7.4 f 2.3 m-equiv x kg RBC H,O-l (PC 0.001). This is similar to the effect of

CATION TRANSPORT

TABLE ~-THE

AND ENERGETICS-SHARK

645

ERYTHROCYTE

EFFECTSOF ANAEROBIOSIS AND MONOIODOACETATE ON SODIUMEFFLUX IN DOGFISH ERYTHROCYTESAT

20°C

Na+ efflux (m-equiv x kg RBC H,O-’ Control MIA (1O-2 M) Nitrogen Nitrogen + MIA(10-2

M)

14.9 7.4 9.7 6.7

x hr-‘)

+ 2.7 (24) f 2.3 (14) k4.0 (11) + 3.0 (8)

P < 0.001 < 0.001 < 0.001

Number in parentheses indicates number of studies.

MIA on Na+ transport in low Na+ non-nucleated erythrocytes. This could occur as a result of reduced ATP generation by glycolysis. Alternatively this could occur because of limitation of pyruvate supply to the Krebs cycle with an ultimate limitation of ATP generation by oxidative phosphorylation. Exposure to approximately 100% N, resulted in a significant reduction of Na+ efflux to 9.7 f 4-O m-equiv x kg RBC H,O-l x hr-1 (PC 0.001). This finding suggests that some of the energy for Na+ transport is derived from oxidative phosphorylation. This finding differs from that noted in non-nucleated erythrocytes in which exposure to N, produces no change in Naf transport (Whittam, 1964). Exposure of erythrocytes simultaneously to MIA and N, does not further significantly reduce Na+ efflux as compared to N, alone (P> 05) or MIA alone (P> O-5). This suggests that active transport may continue on the basis of energy provided by a preformed ATP pool.

K+ injlux The data concerning K+ transport are summarized in Table 5. K+ influx measured at 20°C averaged 9-l+ 4-O m-equiv x kg red cell H,O-1 x hr-1. Exposure to ouabain ( 10m4M) significantly reduced this value to 4.2 m-equiv x kg red cell H,O-l (P < 0.001) so that calculated “active” K+ influx (total flux - ouabain flux) TABLE

~-POTASSIUM

INFLUX INDOGFISH

ERYTHROCYTES

K+ flux (m-equiv x kg RBC H20-’ x hr-‘) Temperature : 20°C 12°C Total K+ flux Ouabain (1O-4 M) K+ flux “Active” Kf flux (total flux - ouabain flux)

9.1 f 4.0 (6) 4.2 + 1.2 (6) 4.9 + 2.8 (6)

2.3 jlO.7 (6) 1.4 + 0.3 (6) 0.9 + 0.7 (6)

Number in parentheses indicates number of studies.

P < 0*005 < 0.001

646

J. THEODORE,E. D. ROBIN, H. V. MURDAUGH,JR. ANDC. E. CROSS

averaged 4.9 f 2.8 m-equiv x kg red cell HsO-l x hr-l. The total K+ influx and ouabain sensitive influx at 20°C are substantially greater in these cells than in human erythrocytes despite the higher temperature (37°C) in the latter species. Metabolic studies 0, consumption. The data concerning 0, consumption (00,) are summarized in Table 6. 40, at 30°C averaged 3.145 0.76 mM x kg cell HsO-l x hr-l. Exposure of shark erythrocytes to both CN- (lOes M) (inhibits the terminal oxidase of the electron transport chain) and antimycin A (SOrJ,g/flask) (relatively specific inhibitor of cytochrome b of the electron transport chain) produces significant reductions of 00,. Exposure to DNP (10e6 M) which uncouples oxidative

TABLE ~--AEROBIC METABOLISM IN DOGFISHERYTHROCYTES

(mM x kg RBC HeO-’ x hr-i) Temperature Control

30°C

CN- (1O-8 M) Antimycin A (50 pg) DNP (1O-5 M) Ouabain (low4 M) Temperature Control

3.14 + 0.76 (22) 1.08 1.11 4.34 2.98

P -

f 0.24 (8) kO.23 (11) +_1.48 (16) + 0.54 (11)

< 0.001 < 0.001 < 0.025 N.S.

0.97 + 0.35 (11)

< 0.001

12°C

Number in parentheses indicates number of studies.

phosphorylation produces a significant increase in 00, to 4.34 f 1.48 mMxkg cell H,O-l x hr-l (P< 0.025). These findings constitute strong biochemical evidence for oxidative phosphorylation in these cells despite the apparent absence of mitochondria (Doyle, 1966). Approximately 65 per cent of total 00, is inhibited by CN- and antimycin A. Assuming that this fraction of total 0, consumption (approximately 2 mM x kg cell H,O-l x hr-l) is devoted to ATP generation at 3O”C, the energy yield by this pathway amounts to approximately 500 cal x kg cell H,O-l x hr-l.* Exposure to ouabain does not produce a significant change in 00,. At 12°C there is a significant reduction in 00, to 0.97 + 0.35 mM x kg cell HsO-’ x hr-l (PC 0.001). Assuming that 65 per cent of this value represents ATP generation by oxidative phosphorylation, the energy yield is approximately 150 cal x kg cell H,O-l x hr- 1. It should be noted that the Q1s of Qs consumption is 1.8, a value which is similar to that found in most Qs consuming processes. * It has been assumed that the free energy of the hydrolysis of ATP to ADP is 7000 Cal/M-’ (Lehninger, 1964).

647

CATION TRANSPORT AND ENERGETICS-SHARK ERYTHROCYTE

Anaerobic glycolysis Table 7 summarises data concerning anaerobic glycolysis. Under aerobic conditions lactate production averaged 1.52 mM x kg cell H,O-l x hr-l at 30°C. This compares to a value of approximately 2-O in the human erythrocyte (Whittam, 1964). The rate of aerobic glycolysis in this cell when corrected for temperature is similar to that seen in mammalian erythrocytes (Robin et al., 1971b). Interference with oxidative phosphorylation by CN- (1O-s M), DNP (1O-5 M) or antimycin A (50 pg/flask) produces significant increases in lactate production TABLE 7----ANAEROBIC GLYCOLYSIS IN DOGFISH ERYTHROCYTES AT

& lactate (mM x kg RBC H,O-’ Control

1.52 f0.93

CN- (1O-s M)

2.88 + 1.77 (16)

Antimycin A (50 pg) DNP (1O-5 M) Ouabain (lo-* M)

5.43 ? 1.25 (9) 2.30 + 1.13 (12) 1.25 + 0.83 (9)

Number

in parentheses

indicates

number

x hr-‘)

30°C

P

(16) < 0.025 < 0.001 < 0.025 N.S.

of studies.

(Pasteur effect). These studies show that: (a) under aerobic conditions at 30°C approximately 10 cal x kg cell H,O-l x hr-l in ATP equivalents is made available by glycolysis; (b) the shark erythrocyte shows a brisk Pasteur effect which is strong evidence for the presence of oxidative phosphorylation; (c) maximal lactate production (evoked by antimycin A) yields as much as 38 cal x kg cell H,O-1 x hr-l. Unlike human erythrocytes (Minakami et al., 1964; Minakami & Yoshikawa, 1966), ouabain exposure does not lead to a decrease in lactate production. DISCUSSION

An important aspect of these studies concerns the relationship between nonmammalian nucleated erythrocytes and mature mammalian erythrocytes. Since most studies of erythrocyte function have focused on the latter cell types, any approach to defining function in the shark erythrocyte must be based on studies in the latter. This is not entirely appropriate. The mature mammalian erythrocyte represents a senescent (or degenerated) cell. As the cell develops from erythroblast through the normoblast and reticulocyte states, there is loss of subcellular organelles including the nucleus, mitochondria, RNA granules, microsomes, etc. Biochemically the mammalian erythrocyte loses ability to synthesize DNA, RNA, proteins and heme; and no longer possesses a Krebs tricarboxylic acid cycle or enzymes to carry out oxidative phosphorylation through the electron transport chain (Harris, 1965). The shark erythrocyte more closely resembles the early nucleated forms of mammalian erythrocytes (see below). However, there are a number of cellular functions which may properly be compared.

648

J. THEODORE,E. D. ROBIN, H. V. MURDAUGH,

JR. ANDC. E. CROSS

One of these concerns the nature of intracellular electrolyte composition. Mammalian erythrocytes are characterized by high permeability for small anions (Cl-, HCO,-, OH-) as well as H+, which are in thermodynamic equilibrium across the erythrocyte membrane. These are asymmetrically distributed because of the Gibbs-Donnan effect leading to transmembrane potentials which are more or less similar. High anion and H+ permeability would appear to be necessary for adequate respiratory function. In the tissues metabolically generated CO, is converted to HCOs- by OH- furnished through the isohydric shift of hemoglobin intracellularly. HCO,- is the major transport form of CO, in the blood. In the lung, H+ furnished by the isohydric shift converts HCOa- to CO, for pulmonary excretion. During these processes there are appropriate shifts of small anions like Cl- in or out of the erythrocyte which maintain electroneutrality. These rapid shifts require high permeability and it is not surprising that this is present in all mammalian erythrocytes. There are, however, two different types of erythrocytes with respect to cation distribution, the high electrochemical gradient type (HECG) and the low electrochemical gradient type (LECG). The differences between these two types of HECG cells show wide electrochemical erythrocytes are outlined in Table 8.

HECG cell type

LECG

cell type

Permeability to small anions

High

High

Transmembrane

potential

5-20

S-20

Electrochemical Na+

gradient

Wide

Narrow but [Na+li less than

for

N+l,

Electrochemical gradient for Kf

Wide

Exceedingly narrow

Ratio-Na+

Relatively low, approximately 3 Na+ to 2 K+ in a number of species

Very high because of high Na+ flux

Marked

Absent

Present

Low to absent

Influence of metabolic inhibitors

Marked decrease in Na+ and Kf flux

Na+ and K+ flux not affected

Ethanol 5 x 10-i M

Does not transport

Inhibits Naf transport

Representative species

Man, rabbit

flux : K+ flux

Sensitivity of Naf-K+ cardiac glycosides Naf-Kf activity

activated

flux to ATPase

affect

Na+

Cat, dog, seal

CATION

TRANSPORT

AND

ENERGETICS-SHARK

ERYTHROCYTR

649

gradients for both Na+ and K +; the ratio of Na+ flux to K+ flux is fairly small (approximately 3 Na+ transported for 2 K+), both Na+ flux and K+ flux are sensitive to the inhibition of Na+-K+ activated ATPase by cardiac glycosides. Depression of ATP generation by inhibition of anaerobic glycolysis depresses Na+ and K+ flux. LECG cells show intracellular Na+ concentrations fairly close to plasma Na+ concentrations and intracellular K+ concentrations very close to electrochemical equilibrium. Cardiac glycosides produce little or no reduction in Na+ and Kf flux. Short-term inhibition of metabolism produces little change in the flux rate of both ions. Ethanol depresses Na+ flux in this cell type (Davson & Reiner, 1942; Robin et al., 1971b). There are no substantial data regarding non-mammalian vertebrate erythrocytes with respect to these properties. As predicted, Cl- distribution is similar in the shark erythrocyte to mammalian erythrocytes. The cation pattern in the shark ery-throcyte is that of the HECG type. The wide electrochemical gradients for both Na+ and K+ closely resemble those seen in mammalian HECG cells. Despite substantial differences in absolute Na+ and K+ flux, the ratio of Na+ efflux to K+ influx closely resembles that seen in HECG cells. There is substantial depression of Na+ flux by ouabain. Metabolic inhibition leads to a decrease in Na+ flux. Previous studies of other vertebrate nucleated erythrocytes (chicken, turtle and duck) suggest that these cells likewise are HECG cells (Maizels, 1954; Clarkson & Maizels, 1955; Tosteson & Robertson, 1956). It is possible that all nucleated erythrocytes (mammalian pre-erythrocytes as well as non-mammalian) are HECG cells. LECG cells may represent a characteristic which develops late in the developmental cycle of the cell. Alternatively, LECG characteristics may represent a feature of certain special erythrocytes occurring at early states of red cell development. The resolution of this issue will await a systematic survey of non-mammalian vertebrate erythrocytes to determine the possible occurrence of LECG types and studies of cation distribution patterns in early nucleated forms of mammalian erythrocytes. In addition to these general aspects of electrolyte composition, there are specific features related to the special properties of extracellular fluid in this species. Intracellular Cl- concentrations are substantially higher than those found in other vertebrate red cells. This reflects the high [Cl-] of elasmobranch plasma and the existence of high Cl- permeability across the erythrocyte membrane. Intracellular Na+ concentrations are considerably higher than in other vertebrate erythrocytes, presumably reflecting the increased concentration of Na+ in elasmobranch plasma. Intracellular K+ concentration is moderately higher than in most mammalian high K+ erythrocytes. Although the ratio of Na+ flux to K+ flux is comparable to that seen in other HECG cell types, the absolute fluxes are substantially higher. These increased flux rates involve both total flux as well as “active” flux (total flux - ouabain flux). The maintenance of these high active fluxes obviously requires high energy expenditures as compared to human erythrocytes.

650

J. THEODORE, E. D. ROBIN, H. V. MURDAUGH, JR. AND C. E. CROSS

There are other differences in erythrocyte composition. Serum osmolality amounts to approximately 1 Osmole/l. (Rodnan et al., 1962), a value which is three times as high as the corresponding value in mammalian plasma. Intraerythrocytic osmolality must be correspondingly greater than mammalian intraerythrocytic osmolality. Despite this difference in red cell osmolality, the percentage of water in shark red cells amounts to 72 per cent (Robin et al., 1964), a value approximately the same as that in mammalian erythrocytes. Approximately 38 per cent of total osmolality of shark plasma is accounted for by urea (Smith, 1936; Maren, 1967). A special mechanism (facilitated diffusion) exists for urea transport in shark erythrocytes (Murdaugh et al., 1964). Urea concentrations in shark erythrocyte water are in equilibrium with urea concentrations in plasma water (Robin & Murdaugh, unpublished data). Schmidt-Nielsen has recently presented evidence for linked urea-Na+ transport in the elasmobranch kidney (Schmidt-Nielsen, 1971). The relationship between urea transport and cation transport in the shark erythrocyte has not been specifically studied. Turning to energy metabolism, it may be noted that electron microscopy fails to reveal definite evidence of mitochondria (Doyle, 1966). However, the evidence that shark erythrocytes utilize molecular 0, for ATP generation mediated by electron transport chain metabolism seems incontrovertible. The decrease in 0, consumption produced by CN- and antimycin A; the increase in 0, consumption produced by DNP ; and the increased lactate generation in cells exposed to N, (Bricker et al., 1968), to CN-, antimycin A and DNP (Pasteur effect), establishes the existence of functional oxidative phosphorylation. Assuming that the antimycin A sensitive fraction of 0, consumption represents the fraction of 0, consumption devoted to ATP generation, it may be estimated that at 20°C 288 cal x kg cell H,O-l x hr-r are available through this pathway. In addition, this cell shows a fairly brisk rate of aerobic glycolysis, which provides an additional energy source amounting to 8 cal x kg RBC H,O-i at 20°C. The total energy availability at 20°C amounts to 296 cal x kg RBC H,O-l x hr-l. The brisk Pasteur effect present in these cells is capable of providing as much as ATP generation. 30 cal x kg HsO-l x hr-i, even in the absence of 0, dependent The relationship between the energy required for cation transport and energy The minimal work involved in cation transport may be supply is of interest. estimated on the basis of relatively simple thermodynamic considerations (KoefoedJohnsen & Ussing, 1960) from the relationship: W=Nj

RTln@

&zF(#,-IG,)+RTlnB], a

&

0

where W = work in calories, Nj = moles of cation transported, R = gas content, T = absolute temperature, uy$ = activity of cation in erythrocyte

H,O,

CATION

TRANSPORT

AND

ENERGETICS-SHARK

ERYTHROCYTE

= activity of cation in plasma HsO, z = cation charge, equal to + 1, F = Faraday, (&--+,) = potential difference across the erythrocyte ,FL~ = magnitude of flux in, p0 = magnitude of flux out.

651

aJo

membrane,

resistance related to In the (normal) steady state, pi = pO, and the frictional bulk flow is eliminated. Substituting experimentally determined values for the various variables and assuming that the moles of Na+ and K+ actively transported can be approximated flux of each ion, it can be calculated that the minimal work by the “active” amounts to 26 cal x kg red cell H,O-l x hr-l in the dogfish erythrocyte. The corresponding value in human erythrocytes is 8 cal x kg RBC HsO-l x hr-l. The biological and technical problems inherent in applying this equation to erythrocytes have previously been discussed (Robin et al., 1971b). One major problem is the difficulty of determining an absolute flux in vitro which corresponds to the true in vivo flux. In this respect, it is useful to discuss previously published observations by Bricker and his colleagues. These workers in a series of careful studies characterized some aspects of Naf transport and glycolysis in the red cells of Squalus acanthias (Bricker et al., 1968). Although their findings are similar to those reported here, there were three important quantitative differences : (1) The absolute efflux rate noted by these workers averaged 5.81 m-equiv x kg red cells-l x hr-l, a value which, converted to red cell water, is approximately 60 per cent of the value found in the present studies. There are a number of methodologic differences between the two studies, the major difference being that the trap problem was met by washing the red cells in isotonic MgCI,.* Although this is an acceptable technique in systems with low Na+ permeability, it is less so in systems with high Na+ permeability. In alveolar macrophages (which possess high Naf permeability) exposure to Na + free media leads to loss of 50-70 per cent of the internal Na+ within l-2 min (Robin et al., 1971a). There is loss of intracellular Na+ from human and chicken erythrocytes when washed in Naf free media (Clarkson & Maizels, 1966). This also occurs in the elasmobranch erythrocyte (Cross et al., 1966). Thus, intracellular Na+ concentrations determined by Bricker et al. (1968) were lower than those found by us leading to a high specific activity and a lower calculated flux. In general, an exact separation between extracellular and intracellular phases in the erythrocyte is difficult and the problem of absorbed (membrane-bound) Na+ has not been clearly resolved. Assuming that absolute Na+ flux amounts to approximately 6-9 m-equiv x kg cell H,O-l x hr-l as noted by Bricker et al. (1968) such a value would result in a Na+ * Na+ free Ringer solution produces alterations in cell structure which may also influence the suitability of this technique for reducing trapped Na+ in elasmobranch erythrocytes (Cross et al., 1966).

652

J.THEODORE,E.

D. ROBIN, H.V.

MURDAUGH,JR.AND C. E. CROSS

to K+ flux ratio that was less than 1, a finding which has not been noted in any red cell system. The ratio of Na+ flux to K+ flux noted by us is approximately 3 : 2 which is the value found in most HECG cell types. In any case, both the studies by Bricker et al. and our group show Na+ efflux considerably higher than in most HECG cell types even at a substantially lower temperature. (2) Bricker et al. originally noted that anaerobiosis reduced efflux rates to approximately 50 per cent of those noted under aerobic conditions (Bricker et al., 1965). In a subsequent study no difference in efflux between anaerobiosis and aerobiosis was detected (Bricker et al., 1968). In the present studies a significant decrease in Na+ transport was found during anaerobiosis. The basis of these differences is not clear. (3) The rates of lactate generation noted by Bricker under control conditions were approximately one-fifth of the values found by us. Even with N, exposure in these studies, the amount of lactate generated at 20°C was substantially less than that found during aerobic conditions in the present studies. The basis of this difference is not clear. In the dogfish erythrocyte, given an energy availability of 296 cal x kg HsO-1 x hr-l and an energy cost of cation transport of 26 cal x kg H,O-1 x hr-r, it is obvious that energy supply substantially exceeds that required for cation transport. Unlike the non-nucleated mammalian erythrocyte, it is probable that nucleated erythrocytes engage in a number of energy requiring processes other than cation transport (Harris, 1965). A more difficult question is whether the energy for cation transport is primarily derived from glycolysis. The calculations of energy derived from aerobic glycolysis as compared to the energy required for cation transport would suggest that glycolysis by itself would be inadequate to support transport. This conclusion is supported by the decrease in Na + efflux produced by exposure to lOOoh N,. On the other hand, the energy made available by maximal glycolytic rate following exposure to antimycin A would appear adequate to maintain Naf and K+ transport for at least a period of time. In a purely glycolytic system like the mammalian erythrocyte, glycolysis is sufficient to maintain transport indefinitely given adequate substrate. Whether this is also true of the shark red cell is not clear. Major uncertainties concerning the exact energy provided by ATP hydrolysis in &JO (Burton & Krebs, 1953; Podolfsky & Morales, 1956; Lehninger, 1964) are present. As pointed out above, the exact in aivo fluxes are difficult to estimate. Studies using inhibitors must be interpreted with caution. Few inhibitors have a single and precise action. * ATP stores may be sufficient to maintain cellular processes for substantial periods, despite inhibition of a given metabolic pathway. For these reasons an exact determination of the role of glycolysis in cation transport in these cells is not possible. * In this context one may quote Davenport’s law (H. Davenport, personal communication) that the specificity of an inhibitor is inversely proportional to the experience of the investigator.

CATIONTRANSPORT ANDENBRGETICS-SHARK ERYTHROCYTE

653

Aside from the specific application of these studies to cation transport, it is suggested that a more general application is involved. In an evolutionary sense it may be considered that the transition from young nucleated erythrocyte precursors to adult non-nucleated red cells represents a recapitulation of “phylogeny” during the “ontogeny” of these cells. Vertebrate nucleated red cells represent a useful model for defining a number of cellular physiological processes in young mammalian pre-erythrocytes. REFERENCES BERNSTEINR. E. (1953) Rates of glycolysis in human red cells in relation to energy requirements for cation transport. Nature, Lond. 172, 911-912. BRICKER N. S., GUERRA L., KLAHR S., BEAUMANW. & MARCHENAC. (1968) Sodium transport and metabolism by erythrocytes of the dogfish shark. Am. J. Physiol. 215, 383-388. BRICKERN. S., KLAHR S., HOLLE L. & MAYER S. (1965) Sodium transport by the red blood cells of the dogfish (Squalus acanthias). Bull. Mt Desert Isl. biol. Lab. 5,4 (Abstr.). BUCHER T. et al. (1959) Described in technical bulletin issued by the Boehringer Biochemical, Manneheim, Germany. BURTONK. & KREBS H. A. (1953) The free-energy changes associated with individual steps of the tricarboxylic acid cycle, glycolysis, and alcoholic fermentation, and with the hydrolysis of the pyro-phosphate groups of adenosine-triphosphate. Biochem. J. 54, 94-107. CLARKSONE. M. & MAIZELS M. (1955) S o dium transfer in human and chicken erythrocytes. r. Physiol., Lond. 129, 476-503. CROSS C. E., THEODOREJ., MURDAUGHH. V., JR. & ROBIN E. D. (1966) Methodologic problems in the study of cation transport in erythrocytes of the dogfish. Bull. Mt Desert Isl. biol. Lab. 6, 13 (Abstr.). DAVSONH. & REINER J. M. (1942) Ionic permeability: an enzyme-like factor concerned in the migration of sodium through the cat erythrocyte membrane. r. cell. camp. Physiol. 20, 325-342. DOYLE W. L. (1966) The occurrence of mitochondria in mature erythrocytes of myxine glutinosa. Bull. Mt Desert Isl. biol. Lab. 6, 17 (Abstr.). HARRISJ. W. (1965) The Red Cell. Harvard University Press, Cambridge, Mass. KOEFOED-JOHNSENV. & USSING H. H. (1960) Ion transport. In Mineral Metabolism (Edited by COMARC. L. & BRONNERF.), pp. 169-203. Academic Press, New York. LEHNINGERA. L. (1964) The Mitochondrion: Molecular Basis of Structure and Function. W. A. Benjamin, New York. MAIZELSM. (1954) Cation transport in chicken erythrocytes. r. Physiol., Lond. 125, 263277, MARENT. H. (1967) Special body fluids of the elasmobranch. In Sharks, Skates and Rays (Edited by PERRY W. G., MATHEWSONR. F. & RALL D. P.), pp. 287-292. The Johns Hopkins Press, Baltimore, Maryland. MINAKAMIS., KAKINUMAK. & YOSHIKAWAH. (1964) The control of erythrocyte glycolysis by active cation transport. Biochim. biophys. Acta 90, 434-436. MINAKAMIS. & YOSHIKAWA H. (1966) Studies in erythrocyte glycolysis-III. The effects of active cation transport, pH and inorganic phosphate concentration on erythrocyte glycolysis. J. Biochem. (Tokyo) 59, 145-150. MURDAUGHH. V., ROBIN E. D. & HEARN C. D. (1964) Urea: apparent carrier-mediated transport by facilitated diffusion in dogfish erythrocytes. Science, N.Y. 144, 52-53. PODOLFSKYR. J. & MORALESM. F. (1956) The enthalpy change of adenosine-triphosphate hydrolysis. J. biol. Chem. 218, 945-959.

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ROBIN E. D., MURDAUCHH. V., JR., CROSS C. E., SMITH J. & THEODOREJ. (1971b) Cation transport and energy metabolism in the high Na f, low K+ erythrocyte of the harbor seal, Phoca vitulina. Comp. Biochem. Physiol. 39A, 807-821. ROBIN E. D., MURDAUGHH. V., JR. & WEISS E. (1964) Acid-base, fluid, and electrolyte metabolism in the elasmobranch-I. Ionic composition of erythrocytes, muscle, and brain. J. cell. camp. Physiol. 64, 409-418. ROBIN E. D., SMITH J. D., TANSER A. R., ADAMSONJ. S., MILLEN J. E. & PACKERB. (1971a) Ion and macromolecular transport in the alveolar macrophage. Biochim. biophys. Acta 241, 117-128. RODNANG. P., ROBIN E. D. & ANDRUS M. H. (1962) Dogfish coelomic fluid-I. Chemical anatomy. Bull. Mt Desert Isl. biol. Lab. 4, 69-70 (Abstr.). SCHMIDT-NIELSEN B. (1971) Renal volume and osmoregulation in the dogfish. Paper presented at Elasmobranch Biology Symposium, June 20-23, 1971, Bar Harbor, Maine. Proceedings to be published in Comp. Biochem. Physiol. SMITH H. W. (1936) The retention and physiological role of urea in the elasmobranchii. Biol. Rev. 11, 49-82. TOSTE~OND. C. & ROBERTSONJ. S. (1956) Potassium transport in duck red cells. J. cell. camp. Physiol. 47, 147-166. WHITTAM R. (1964) Transport and D@‘usion in Red Blood Cells. Monograph No. 13 of the Physiological Society. Williams & Wilkins, Baltimore, Maryland. Key Word Index-Cation transport; energetics; low Na+ vertebrate erythrocytes; electrolyte distribution; 0, consumption; oxidative phosphorylation; glycolysis; electrochemical gradient; energy cost; cyanide; dinitrophenol; antimycin A; nitrogen; monoiodoacetate ; ouabain; Squalus acanthias.