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Metabolic and functional characteristics of erythrocytes from Glycera and Noetia
Camp. Biochem. Physiol. Vol. SSA, No. 3, pp. 397403, Printed in Great Britain
1987 0
0300-9629/87 $3.00 + 0.00 1987 Pergamon Journals Ltd
METABOLIC AND FUNCTIONAL CHARACTERISTICS ERYTHROCYTES FROM GLYCERA AND NOETIA N. A. MAURO Research
OF
and R. E. ISAACKS
Laboratories of the Veterans Administration Medical Center, and the Department University of Miami School of Medicine, Miami, FL 33125, USA
of Medicine,
(Received 11 February 1987) Abstract-l. Annelid and molluscan red blood cells (RBC) may de differentiated metabolically from vertebrate RBC by their increased permeability to substrate, their magnitude of amino acid catabolism and their higher aerobic metabolism. 2. At 22’C, Glycera and Noetiu RBC oxidize glucose and glutamate to CO, without accumulation of either D- or L-lactate. By comparison, the oxidation of glutamate by rat and chicken RBC is negligible at this temperature despite its incorporation into the cells. 3. At 37”C, chicken RBC oxidize glutamate at a rate 4 times greater than at 22”C, with oxygen uptake still lower than that in Noetiu RBC at 32°C. At 37”C, rat RBC do not increase their oxidation of glutamate above that at 22°C. but oxygen uptake increases to slightly more than half that of chicken RBC. 4. Our findings indicate that RBC of these two invertebrate species have both a higher aerobic metabolism and lower anerobic capacity than vertebrate RBC. 5. Moreover, the annelid and molluscan RBC have a relatively lower activity of the pentose phosphate (PPO, ) pathway than vertebrate RBC, as evidenced by their higher thermal sensitivity of oxygen uptake and their higher *C,02/*C,0, isotope ratio.
INTRODUCTION
The information available on metabolism of invertebrate erthrocytes (RBC) is scant despite the perception that these red cells represent the early ancestral condition of all RBC. These cells are not merely carbon copies of nucleated vertebrate RBC, much less anucleated mammalian RBC. Although our knowledge of invertebrate RBC is largely restricted to their morphology and their contents of OS-carrying proteins (Mangum, 1977; Terwilliger et al., 1985; Mangum and Mauro, 1985) limited data are available on oxygen uptake and their responses to osmotic stress (reviewed by Pierce and Maugel, 1985; reviewed by Mangum and Mauro. 1985). In addition, some information has been collected on carbonic anhydrase activity, non-metabolic enzymes, and protein and RNA synthesis (Hoffman and Mangum, 1970; Shafie et al., 1976; R. P. Henry, unpublished data). Moreover, no intensive investigation has been made on the site of formation and destruction of these cells or their life histories, or the biosynthesis of their most important constituent, the oxygen carrier (Mangum and Mauro, 1985). The purpose of this study was to take two representative species, the annelid bloodworm Glycera dibrachiata and the blood clam Noetia ponderosa and to elucidate some of the basic metabolic and physiological characteristics of their red cells in comparison with those of higher vertebrates.
MATERIALS AND METHODS Coktion
and preparation of RBC
Rat and chicken blood were collected in heparinized syringes to prevent clotting, transferred to heparinized tubes. centrifuged. the plasma and buffy coat removed by
aspiration and the RBC washed twice by resuspension in 0.9% NaCl and centrifugation. Noetia and Glycera bloods do not clot, thus Noetia blood was obtained by gently scraping the mantle tissue and collecting the pooled blood; Glycera blood was obtained by slitting the body wall and collecting the coelomic fluid into Petri dishes. After collection the blood cells were washed in physiological salines which in the case of invertebrate species, was sea-water (33X+; pH adjusted with HCI to 7.2 for Noetia or 7.4 for Glycera). Standard hematological measurement of hemoglobin concentration and hematocrit were made on aliquots of blood from all organisms. Flux measurements and CO, production from radio-labelled substrates Glutamate and glucose uptake were determined on washed RBC suspended at an approximate hematocrit of 10% according to Kim and Isaacks (1978). In this procedure, aliquots (0.4 ml) of the RBC suspension were taken at frequent intervals and layered onto dibutyl phthalate (0.8 ml). These mixtures are quickly centrifuged and the radioactive counts of the cell pellets and supernatant fluid determined. Radio-labelled *CO, production from radioactive substrates was measured according to procedures described by Mauro and Mangum (1982). Red cell suspensions of 1.5 ml per flask and approximate hematocrit of 10% were incubated with 1 mM of unlabelled substrate at 22 or 37°C. The labelled CO, produced was collected into 0.3 ml hyamine hydroxide and the experiments terminated by acidification of the cell suspension with OSml of 1 N H,SO,. The radio-labelled *CO, collected into base was added to scintillation vials containing 10 ml of scintillation fluid and the radioactivity determined with a Packard Tri-Carb liquid scintillation spectrometer (Model 3380). Erythrocyte density and osmotic fragility determinations Blood cell densities were determined by methods of Danon and Marikovsky (1964). Methyl phthalate and di-nbutyl phthalate were mixed in different proportions to yield a series of solutions in which the specific gravity differed by 397
398
N. A. MAURO
increments of 0.004. Blood was introduced onto the series of liquids of different densities placed in microhematocrit capillaries. After centrifugation, denser cells were separated from less dense cells by a transparent layer of the non-water miscible phthalate esters. The percentage of RBCs of a given density was calculated from the proportion of packed cells below the separation fluid. In the determination of osmotic fragility, whole blood from Noeria and Glycera was centrifuged and the red cells initially washed two times in 100% sea-water (SW 33%0, pH adjusted with HCI to 7.4). A series of sea-water concentrations ranging from distilled water (0% SW) to 100% SW were mixed. One millilitre from each concentration was added to 50 ul of washed red cells and gently mixed. After I5 min at 22°C the RBCs in each concentration were gently resuspended and the solutions centrifuged. From the supernatant fluid, 200 pl aliquots were taken, added to Drabkins reagent and then read spectrophotometrically. A comparable aliquot of whole cell suspension was directly added to Drabkins reagent for total hemoglobin determination (Drabkin, 1945). The per cent hemolysis was thus deter-
Fig.
1. Electron
micrograph
and R. E. ISAACKS mined by calculating the amount relative to the total hemoglobin Oxygen
of hemoglobin in solution in the red cell suspension,
uptake
Oxygen uptake of cell suspensions was measured by monitoring the depletion of oxygen with a Yellow Springs Oxygen probe (Model 53) and recorder (BRC Model) in the PO, range of llO~l59Torr. Lactate analysis After being washed in physiological salines, the hematocrit of invertebrate blood cell samples was determined and adjusted to 25%. D- and L-lactate levels were obtained before and after incubation at 22 and 37’C with Sigma lactate test kits. Since the standard Sigma lactate kit measures only L-lactate, a separate o-lactate test kit was also used. Phosphate analysis The water-soluble erythrocytes washed
of a single Noetia ponderosa red blood mithochondria (M), and vacuoles (V).
phosphates were with trichloroacetic
cell showing
extracted from acid (TCA) as
its nucleus
(N),
Erythrocytes
399
from Glycera and Noetia
previously described (Isaacks ef al., 1976). The phosphates of each extract were separated on a Dowex l-X8 formate column with a linear gradient (O-5 N ammonium formate buffer, pH 3.45), identified, and quantitated as previously described (Isaacks et al., 1976).
Electron microscopy RBCs were fixed in 3% glutaradehyde in sea-water with a post-fix of 1% OsO,. After dehydration through an ethanol series, the RBCs were embedded in epoxy resin. Thin sections (0.1 PM) were cut with glass knives, stained with uranyl acetate (2.5%) and lead citrate and viewed with a Phillips-300 Electron Microscope. DENSITY
RESULTS
General hematological characteristics
Fig. 3. Density distribution of Noeria, Glycera and rat RBC. Noetiu (a), Glyceru (m), and rat (A).
The blood of Noetia and Glycera have at least two cell types: amoeboid white cells and hemoglobin containing erythrocytes. RBC of these species are nucleated and range in size from 15-17 pm in Noetia to 17-27 p M in Glycera. Noetia blood has a packed cell volume of 611% as compared to 1542% in Glycera with similar hemoglobin concentration (Hoffman and Mangum, 1970; Freadman and Mangum, 1976). The concentrations of hemoglobin in Noetia range between 2.4 and 5.6g/dl blood as compared to a hemoglobin concentration between 2.3 and 5.7 g/d1 blood in Glycera. Our values for hematocrit and for hemoglobin concentration in Noetia and Glvcera blood fell within these reported ranges. GIycera and Noetia RBC are easily differentiated on the basis of ultrastructure. (Reviewed by Mangum and Mauro, 1985.) Unlike Noetia RBC, Glycera RBC lack marginal bands and possess smaller vacuoles (Figs 1 and 2). Glycera RBC also have more
distinct mitochondria which contain prominent cristae (Fig. 2). The comparative densities of these cells (Fig. 3) reveal that Noetia RBC are less dense than GIycera or rat RBC with a DSO(density point) for half the cell population of 1.099 as compared to 1.108 and 1.104 for G&era and rat RBC, respectively. Although Noetia RBC are less dense than Glycera RBC, they have a lower osmotic fragility (Fig. 4). These findings are similar to those reported by Mangum (1980). A more detailed account of the osmotic fragility of these and other RBC can be found in Demanche (1980). Total phosphate per cm’ RBC (TP,) measures 33.7pmol in Noetia and 55.6pmol in Glycera (Figs 5 and 6, respectively). ATP and ADP are the predominant organic phosphates in the RBC of these
Fig. 2 Electron
micrograph
of a Glycera dibranchiafared blood cell showing vacuoles (V).
its mitochondria
04 a.nd
N. A. MAURO and R. E. ISAACKS
400
0
I 0
I
1YL 20
40
% SEA
60
80
100
WATER
33~100)
(lco%SW
Fig. 4. Red blood cell fragility of Noetia and Gl~era RBC after a 15-min exposure to a given osmotic pressure. Noetia (a), and Glycera (m).
species. The concentration of ATP in erythrocytes of Noetia and Glycera is 5.4 and 5.9 mM, representing 42.9 and 30.5% of their cell phosphate, respectively. Inorganic phosphate (P,) constitutes 6.4 and 22.7 PM cm3 RBC or 19.0 and 40.8% of the total P, in Noetiu and Glycera RBC. respectively. Glucose uptake and metabolism Plasma glucose levels for Noetia and Glycera are similar at approx. 1.4mM (28 mg/dl). By comparison. the plasma glucose levels of the rat and chicken are higher and range between 3.1 and 4.2 mM and 11 .I and 14.4 mM for the rat and chicken. respectively. Figure 7 shows the results of the net glucose uptake into the RBC obtained from Noetia, Gll,cera, and vertebrate RBC. Glucose uptake is fastest in Glycera
RBC followed by Noetiu. The sluggish uptake of glucose by the chicken and rat RBC is probably the result of a lower glucose permeability and a low concentration of glucose in medium as compared to normal serum levels; the lower incubation temperature of 22’C relative to the physiological norms of 394O’C for the chicken and 37’C for the rat may also be a factor. The Glwera RBC, which demonstrate the highest permeability to glucose are only about half as permeable to ribose with an uptake of 0.32 f 0.01 (SE: N = 4) pmol/ml RBC/hr (22’C). Uptake of ribose into Noetia RBC is similar to that of glucose: 0.42 f 0.01 (SE: N = 4) pmol/ml RBC per hr (22 C). The metabolic fate of glucose was also compared. Mammalian RBC utilize the Emden-Meyerhof and pentose PO, pathway for energy production with L-lactate produced as the primary end product. We observed no measurable D- or-L lactate accumulation by Noetia or G&era RBC, after a I-2-hr incubation at either 22 or 37’C. The limitations of the anaerobic capacity of Glycera RBC are further demonstrated by the 25% hemolysis after a 15-min exposure to KCN (lo-‘M). Rapid hemolysis of Glycera RBC also occurs when either whole sera or washed cells are exposed to a PO, of zero (Mangum and Carhart, 1972). A similar exposure of Noetia to KCN (10m3 M) did not produce lysis. Noetia RBC membranes thus appear to be stronger and are osmotically less fragile than Glvceru. Besides glycolysis, glucose may alternatively be oxidized by RBC to CO, via the PPO, pathway, or in the case of nucleated RBCs, by means of the TCA cycle. Since the production of CO? is greater when glucose is completely oxidized by the TCA cycle as compared to partial oxidiation by the PPO, pathway, the relative level of CO? production gives indirect
umoles Pi/ml
ATP
i
-10
0
20
40
80 100 FRACTIONS l5ml)
Fig. 5. Column chromatogram of a trichloroacetic acid extract of 1.35 cm3 Noefia RBC. The chromatogram was developed with a linear gradient O-5 N ammonium formate buffer. pH 3.45. as described by Isaacks et al. (1976). The heavy solid line represents pmol P,/ml of fraction; the narrow solid line respresents absorbance at 260 nM.
Erythrocytes
from Gl.vcera and Noetia
401
bmoles Pi/ml 1.0-
1.6-
1.4-
g 1.2@I > l.O2 $ a % 2
.6-
.6 -
FRACTIONS I5 ml)
Fig. 6. Column chromatogram of a trichloroacetic acid extract of 4.40cm3 Glycera RBC. The chromatogram was developed with a linear gradient O-5 N ammonium formate buffer, pH 3.45, as described by Isaacks et al. (1976). The heavy solid line represents pmol P,/ml of fraction; the narrow solid line represents absorbance at 260 nM.
evidence of the level of activity of the PPOl pathway and the TCA cycle. Compared to the rat RBC, whose primary route of CO, production is by the PPO, pathway, all of the nucleated RBCs examined here have a considerably higher level of *CO2 production from labelled glucose (Table 1). The decreased importance of the PPOl pathway in Glycera and Noetia RBC compared to the rat was further substantiated by the production of labelled CO, after incubation with glucose labelled in either the C6 or C, position. Because CO, is made preferentially in the PPO, 0.8
0.6
F
t
/
.-
=-m
/
pathway from the first C and triose PO, from the 6th, the lower the ratio of C60z/C,02 the greater the relative activity of the PPO, pathway. Rat, Noetiu, and Glycera RBC have ratios of 0.02 and 0.06 and 0.13, respectively. Therefore, the rat has a higher relative activity of this pathway than Noetia or Glycera. Moreover, the relative activity of the PPO, pathway is twice as great in Noetia as in Glycera. This observation may explain in part the higher uptake of ribose by Noetia RBC compared with Glycera and, conversely the reduced uptake of ribose compared to glucose in Glycera RBC. Still further substantiation of the decreased importance of the PPO, pathway in invertebrate RBC is indicated by the increased thermal sensitivity of their oxygen uptake. Cells which show a predominance of the PPO, pathway have a lower temperature dependence of their oxygen consumption (Hochachka and Hayes, 1962; Mauro and Mangum, 1982). The lower thermal sensitivity of the vertebrate RBC, as evidenced by a lower Q,,, , indicates a greater dependence on the PPO, pathway than Noetia RBC (Table 2). Glutamate uptake and metabohm
The uptake and oxidation of glutamate by RBC gives a better evaluation of the activity of the TCA Table
I.
Ratio of *CO2 production
from labelled glucose by RBC relative to *CO2 production from uniformly labelled glucose by rat RBC TIME (minsl
Fig. 7. Glucose accumulation at 22°C by Noeria (a), Glycera (m), rat (A), and chicken (0) RBC. Flux media containing 1 mM cold glucose.
Species
Ratio
Rat Chicken
2.64
Noeria Gl.vcera
6.25 5.91
I .oo
N. A. MAURO and R. E.
402 Table
2
RBC
oxygen uptake (pl,‘ml Mean + SE. .N = 6-X
RBC,‘hr)
TWIpetXtUre 32 c
37 C Human Rat Chxken NWfiU G/wru
199.*4 276 i_ 2 444 i_ I 658 t 2
22 c
Q,n
122i5 160+ I l79i2 337 * 3 434 k 6
I .4 I .4 1.X 2.0
Table 3. Glutamate accumulation by RBC at 22 C after I hr incubatmn (PM x IO ‘/ml RBC). Mean & SE. N = 6-R Accumulation 5 * 0.4 9 z 0.4 39 i 7.0 lh+O.l
Table 4. Radio-labelled carbon dioxide (‘CO,) production from umformly labelled glutamate by RBC after I hr at 22 and 37’C (PM x IO ‘/ml RBC). Mean i SE. N = f%g Amount of glutamate Oxidzed Species
22 C
Noetiu GlKera Rat Chicken
39 54 87il3 1+0.1 I + 0.2
*CO Produced
37 c
22 c
37 c
I kO.1 4kO.l
195 435 5 5
5 20
cycle than glucose since this substrate enters directly into the TCA cycle after deamination. Of the RBC examined, glutamate accumulation is greatest in Noetia RBC and smallest in the mammalian RBC (Table 3). Regardless of the magnitude of glutamate oxidation, glutamate is apparently permeable to all RBC examined. Glutamate oxidation is considerably greater in Noetia and Glycera RBC than in the vertebrate RBC (Table 4). Vertebrate RBC, even avian RBC, which possess a TCA cycle, are limited in their ability to oxidize exogenous glutamate. This limitation occurs at both 22°C and the more physiological temperature of 37 C. At 37’C, the rat RBC are still unable to oxidize more than trace amounts of glutamate and the values for chicken RBC are more than an order of magnitude lower than observed for Gll’cera or Noetia.
DISCUSSION
The metabolism of the invertebrate RBC studied here is clearly different from vertebrate RBC. Vertebrate RBC are lower in permeability to exogenous substrates than annehd or molluscan RBC. Many fish RBC, in addition to the rat and chicken RBC, are either impermeable or low in permeability to glucose (Bolis and Luly, 1972; Kim and Isaacks, 1978). Even mammalian RBC. which lack mitochrondria and must rely upon glycolysis for energy production, may be glucose impermeable (Kim and McManus, 1971). The adaptive advantage of this phenomenon is unknown. although it is known that glucose imper-
ISAACKS
meability is apparently confined to RBC taken from adult pigs and that RBC taken from the fetal pigs are permeable to glucose (Zeidler, 1976). In other words, annelid and molluscan RBC resemble embryonic mammalian RBC more closely than adult mammalian RBC in terms of this physiological feature as well as their nucleated condition. After entering the RBC the oxidation of exogenous substrate is regulated by the size of endogenous substrate pools and the activity and/or existence of the oxidizing pathways, The low oxidation of glutamate by chicken RBC compared to the invertebrate RBC is surprising in view of the fact that the endogenous pool of glucose in chicken RBC is small, measuring 0.8 PM/ml RBC (Mauro and Isaacks, unpublished data). These observations suggest that the TCA cycle of avian RBC may either play a less significant role in energy production and/or may indicate a specialization of avian RBC for carbohydrate oxidation. It is not known whether these low levels of amino acid oxidation are unique to avian RBCs or are characteric of nucleated RBC from other groups of vertebrates. This area is worthy of further examination. Annelid and molluscan RBC can be differentiated further from avian and mammalian RBC by their level of ATP. ATP levels in GIycera and Noetia are higher than those found in birds or mammals although similar to those found in some fish and reptiles (Bartlett, 1980; Isaacks and Harkness, 1980; Johansen et al.. 1978; Kim et al., 1984). Unlike vertebrates the organic phosphates of invertebrates apparently play no role in the modulation of hemoglobin oxygen affinity (Mangum and Mauro, 1985). The question thus becomes, what are some of the possible roles of ATP within invertebrate RBC? One of the primary functions of ATP in RBC is to sustain cell membrane integrity. Reduction in the level of intracellular ATP results in hemolysis. In Glycera RBC the presence of the electron transport inhibitor. KCN. causes a rapid hemolysis. A similar reduction in ATP levels in mammalian RBC results in a weakening and breakdown of the cell membranes (Clader, 1976). Another potential role of ATP in RBC may be in the active transport of substrates and ions. This need for ATP may be seen in the distribution of K+ and Na’ ions across the Glycera RBC membranes. In a medium of 500 mOsm Na+ and 16mOsm K’, the intracellular concentration of K+ and Nat ions measures 100 mOsm for each ion (Mauro and Isaacks. upublished data). Although intraceilular Na’ ion concentrations of cells have been shown to vary greatly, intracellular K+ ion concentration remains more constant, ranging between 100 and 125 mOsm (Presser, 1973). In summary. annelid and molluscan RBC can be metabolically characterized as aerobic cells sharing much in common with other aerobic cells. They have a limited anerobic tolerance; they are highly permeable to exogenous substrates; they oxidize exogenous substrates readily; and they lack substrate specificity. By using this metabolic profile as archetypal for the ancestral condition of all RBC. the metabolic specialization and evolution of the vertebrate RBC can be more easily interpreted.
Erythrocytes
from G&era
Acknowledgements-The authors are grateful to Phyllis Goldman and Chang Kim for their technical help, to Tommie Stapleton, Linda Numan. and Linda Schoomaker for typing the manuscript, and to Dr Charlotte P. Mangum for her suggestions and review of the manuscript.
REFERENCES Bartlett G. R. (1980) Phosphate compounds in vertebrate red blood cells. Am. Zool. 20, 103-l 14. Bolis L. and Luly P. (1972) Monosaccharide permeability in brown trout Salmo !ratfa L. erythrocytes. In Role o/ Membranes in Secretory Processes (Edited by Bolis L.. Keynes R. D. and Wilbrandt W). pp. 215-221. NorthHolland, London and Amsterdam. Dannon D. and Marikovsky Y. (1964) Determination of density distribution of red cell population. J. Lab. clin. Med. 64, 6688674. Demanche R. (1980) The osmotic fragility of red blood cells of marine animals: a comparative-study. M. A. Thesis, Colleee of William & Marv._ Williamsbure. Vireinia. Drabkin D. R. (1945) Crystallographic and optical properties of human hemoglobin. A proposal for the standardization of hemoglobin. Am. J. Med. Sci. 209, 2688370. Freadman M. A. and Mangum C. P. (1976) The function of hemoglobin in the arcid clam Noe/ia ponderosa, I. Oxgenation in vitro and in riro. Camp. Biochem. Physiol. 53A, 173-179. Glader B. F. (1976) Salicylate-induced injury of pyruvatekinase deficient erythrocytes. N. Engl. J. Med. 249, 916918. Hochachka P. W. and Hayes F. R. (1962) The effect of temperature acclimation on pathways of glucose metabolism in the trout. Can J. Zool. 40, 261-270. Hoffman R. J. and Mangum C. P. (1970) The function of coelomic cell hemoglobin in the polychaete Glvcera dilbranchiata. Comp.Biochem. Physiol36, 21 l-228: Isaacks R. E. and Harkness D. R. (1980) Ervthrocvte organic phosphate and hemoglobin function -in birds, reptiles and fishes. Am. Zool. 20, 115-129. Isaacks R. E., Harkness D. R.. Froeman G. A. and Sussman S. A. (1976) Studies on avian erythrocyte metabolism. 1. Procedure for separation and quantitaton of the major phosphorylated metabolic intermediates by anion exchange chromatography. Camp. Biochem. Physiol. 53A, 95-99. Johansen K.. Mangum C. P. and Lykkeboe G. (1978) Respiratory properties of the blood of Amazon fishes. Can. J. Zool. 56, 8988906. Kim H. D. and lsaacks R. E. (1978) The membrane w.
_
and Noeria
403
permeability of nonelectrolytes and carbohydrate metabolism of Amazon fish red cells. Can. J. Zool. 56, 8633869. Kim H. D. and McManus T. J. (1971) Studies on the energy metabolism of pig red cells. I. The limiting role of membrane permeability in glycolysis. B&him. biophys. Acta 230, 1-I I. Kim H. D., Zeidler R. B.. Sallis J.. Nicol S. and Isaacks R. E. (I 984) Metabolic properties of low .4TP erthrocytes of the monotremes. FEBS-167, 83-87. Mangum C. P. (1977) The annelid hemoglobins: a dichotomy in structure and function. In Essays in Memory of Dr. Olga Hartman (Edited by Reish D. J. and Fauchald K.), p. 407. Allan Hancock Foundation, Special Publication, University of Southern California Press, Los Angeles. Mangum C. P. (1980) Distribution of respiratory pigments and the role of anaerobic metabolism in the lamellibranch molluscs. In Animals and Enaironment Fitness (Edited by Gilles R.). pp. 171.-184. Pergamon Press, Oxford and New York. Mangum C. P. and Carhart J. A. (1972) Oxygen equilibrium of coelomic cell hemoglobin from the bloodworm G&era dibranchiata. Camp. Biochem. Physiol. 43A, 949-957. Mangum C. P. and Mauro N. A. (1985) Metabolism of invertebrate red cells: a vacuum in our knowledge. In C’h-culalion. Respiration, and Metabolism-Current Corn pararire Approaches (Edited by Giles R.), pp. 28@-289. Springer, New York. Mauro N. A. and Mangum C. P. (1982) The role of the blood in the temperature dependence of oxidative metabolism in decapod crustaceans. I. Intraspecific responses to seasonal differences in temperature. J. e.vp. Zool. 219, 1799188. Pierce S. K. and Maugel T. K. (1985) A comparison of the water regulating responses of bivalves and polychoete red cells to osmotic stresses. In Blood Cells of Marine Invertebrates (Edited by Cohen W. D.), pp. 1677193. Allen R. Liss. New York. Prosser C. L. (1973) Comparafit~e Animal Physiology. W. B. Saunders. Philadelphia, London, Toronto. Shafie S. M., Vinogradov S. N., Larson L. and McCormick J. J. (1976) RNA and protein synthesis in the nucleated erythrocytes of Glycera dibranchiata. Camp. Biochem. Physiol. 53A, 85-88. Terwilliger N. B.. Terwilliger R. C. and Schabtach E. (1985) Intracellular respiratory proteins of Sipuncula Echiura. and Annelids. In Blood Cells of Marine Inoertebrates (Edited by Cohen W. D.). pp. 193-227. Allen R. Liss. New York. Zeidler R. B., Lee P. and Kim H. D. (1976) Kinetics of 3-O-methyl glucose transport in red cells of newborn pigs. J. gen. Physiol. 67, 67.-80.
1987 0
0300-9629/87 $3.00 + 0.00 1987 Pergamon Journals Ltd
METABOLIC AND FUNCTIONAL CHARACTERISTICS ERYTHROCYTES FROM GLYCERA AND NOETIA N. A. MAURO Research
OF
and R. E. ISAACKS
Laboratories of the Veterans Administration Medical Center, and the Department University of Miami School of Medicine, Miami, FL 33125, USA
of Medicine,
(Received 11 February 1987) Abstract-l. Annelid and molluscan red blood cells (RBC) may de differentiated metabolically from vertebrate RBC by their increased permeability to substrate, their magnitude of amino acid catabolism and their higher aerobic metabolism. 2. At 22’C, Glycera and Noetiu RBC oxidize glucose and glutamate to CO, without accumulation of either D- or L-lactate. By comparison, the oxidation of glutamate by rat and chicken RBC is negligible at this temperature despite its incorporation into the cells. 3. At 37”C, chicken RBC oxidize glutamate at a rate 4 times greater than at 22”C, with oxygen uptake still lower than that in Noetiu RBC at 32°C. At 37”C, rat RBC do not increase their oxidation of glutamate above that at 22°C. but oxygen uptake increases to slightly more than half that of chicken RBC. 4. Our findings indicate that RBC of these two invertebrate species have both a higher aerobic metabolism and lower anerobic capacity than vertebrate RBC. 5. Moreover, the annelid and molluscan RBC have a relatively lower activity of the pentose phosphate (PPO, ) pathway than vertebrate RBC, as evidenced by their higher thermal sensitivity of oxygen uptake and their higher *C,02/*C,0, isotope ratio.
INTRODUCTION
The information available on metabolism of invertebrate erthrocytes (RBC) is scant despite the perception that these red cells represent the early ancestral condition of all RBC. These cells are not merely carbon copies of nucleated vertebrate RBC, much less anucleated mammalian RBC. Although our knowledge of invertebrate RBC is largely restricted to their morphology and their contents of OS-carrying proteins (Mangum, 1977; Terwilliger et al., 1985; Mangum and Mauro, 1985) limited data are available on oxygen uptake and their responses to osmotic stress (reviewed by Pierce and Maugel, 1985; reviewed by Mangum and Mauro. 1985). In addition, some information has been collected on carbonic anhydrase activity, non-metabolic enzymes, and protein and RNA synthesis (Hoffman and Mangum, 1970; Shafie et al., 1976; R. P. Henry, unpublished data). Moreover, no intensive investigation has been made on the site of formation and destruction of these cells or their life histories, or the biosynthesis of their most important constituent, the oxygen carrier (Mangum and Mauro, 1985). The purpose of this study was to take two representative species, the annelid bloodworm Glycera dibrachiata and the blood clam Noetia ponderosa and to elucidate some of the basic metabolic and physiological characteristics of their red cells in comparison with those of higher vertebrates.
MATERIALS AND METHODS Coktion
and preparation of RBC
Rat and chicken blood were collected in heparinized syringes to prevent clotting, transferred to heparinized tubes. centrifuged. the plasma and buffy coat removed by
aspiration and the RBC washed twice by resuspension in 0.9% NaCl and centrifugation. Noetia and Glycera bloods do not clot, thus Noetia blood was obtained by gently scraping the mantle tissue and collecting the pooled blood; Glycera blood was obtained by slitting the body wall and collecting the coelomic fluid into Petri dishes. After collection the blood cells were washed in physiological salines which in the case of invertebrate species, was sea-water (33X+; pH adjusted with HCI to 7.2 for Noetia or 7.4 for Glycera). Standard hematological measurement of hemoglobin concentration and hematocrit were made on aliquots of blood from all organisms. Flux measurements and CO, production from radio-labelled substrates Glutamate and glucose uptake were determined on washed RBC suspended at an approximate hematocrit of 10% according to Kim and Isaacks (1978). In this procedure, aliquots (0.4 ml) of the RBC suspension were taken at frequent intervals and layered onto dibutyl phthalate (0.8 ml). These mixtures are quickly centrifuged and the radioactive counts of the cell pellets and supernatant fluid determined. Radio-labelled *CO, production from radioactive substrates was measured according to procedures described by Mauro and Mangum (1982). Red cell suspensions of 1.5 ml per flask and approximate hematocrit of 10% were incubated with 1 mM of unlabelled substrate at 22 or 37°C. The labelled CO, produced was collected into 0.3 ml hyamine hydroxide and the experiments terminated by acidification of the cell suspension with OSml of 1 N H,SO,. The radio-labelled *CO, collected into base was added to scintillation vials containing 10 ml of scintillation fluid and the radioactivity determined with a Packard Tri-Carb liquid scintillation spectrometer (Model 3380). Erythrocyte density and osmotic fragility determinations Blood cell densities were determined by methods of Danon and Marikovsky (1964). Methyl phthalate and di-nbutyl phthalate were mixed in different proportions to yield a series of solutions in which the specific gravity differed by 397
398
N. A. MAURO
increments of 0.004. Blood was introduced onto the series of liquids of different densities placed in microhematocrit capillaries. After centrifugation, denser cells were separated from less dense cells by a transparent layer of the non-water miscible phthalate esters. The percentage of RBCs of a given density was calculated from the proportion of packed cells below the separation fluid. In the determination of osmotic fragility, whole blood from Noeria and Glycera was centrifuged and the red cells initially washed two times in 100% sea-water (SW 33%0, pH adjusted with HCI to 7.4). A series of sea-water concentrations ranging from distilled water (0% SW) to 100% SW were mixed. One millilitre from each concentration was added to 50 ul of washed red cells and gently mixed. After I5 min at 22°C the RBCs in each concentration were gently resuspended and the solutions centrifuged. From the supernatant fluid, 200 pl aliquots were taken, added to Drabkins reagent and then read spectrophotometrically. A comparable aliquot of whole cell suspension was directly added to Drabkins reagent for total hemoglobin determination (Drabkin, 1945). The per cent hemolysis was thus deter-
Fig.
1. Electron
micrograph
and R. E. ISAACKS mined by calculating the amount relative to the total hemoglobin Oxygen
of hemoglobin in solution in the red cell suspension,
uptake
Oxygen uptake of cell suspensions was measured by monitoring the depletion of oxygen with a Yellow Springs Oxygen probe (Model 53) and recorder (BRC Model) in the PO, range of llO~l59Torr. Lactate analysis After being washed in physiological salines, the hematocrit of invertebrate blood cell samples was determined and adjusted to 25%. D- and L-lactate levels were obtained before and after incubation at 22 and 37’C with Sigma lactate test kits. Since the standard Sigma lactate kit measures only L-lactate, a separate o-lactate test kit was also used. Phosphate analysis The water-soluble erythrocytes washed
of a single Noetia ponderosa red blood mithochondria (M), and vacuoles (V).
phosphates were with trichloroacetic
cell showing
extracted from acid (TCA) as
its nucleus
(N),
Erythrocytes
399
from Glycera and Noetia
previously described (Isaacks ef al., 1976). The phosphates of each extract were separated on a Dowex l-X8 formate column with a linear gradient (O-5 N ammonium formate buffer, pH 3.45), identified, and quantitated as previously described (Isaacks et al., 1976).
Electron microscopy RBCs were fixed in 3% glutaradehyde in sea-water with a post-fix of 1% OsO,. After dehydration through an ethanol series, the RBCs were embedded in epoxy resin. Thin sections (0.1 PM) were cut with glass knives, stained with uranyl acetate (2.5%) and lead citrate and viewed with a Phillips-300 Electron Microscope. DENSITY
RESULTS
General hematological characteristics
Fig. 3. Density distribution of Noeria, Glycera and rat RBC. Noetiu (a), Glyceru (m), and rat (A).
The blood of Noetia and Glycera have at least two cell types: amoeboid white cells and hemoglobin containing erythrocytes. RBC of these species are nucleated and range in size from 15-17 pm in Noetia to 17-27 p M in Glycera. Noetia blood has a packed cell volume of 611% as compared to 1542% in Glycera with similar hemoglobin concentration (Hoffman and Mangum, 1970; Freadman and Mangum, 1976). The concentrations of hemoglobin in Noetia range between 2.4 and 5.6g/dl blood as compared to a hemoglobin concentration between 2.3 and 5.7 g/d1 blood in Glycera. Our values for hematocrit and for hemoglobin concentration in Noetia and Glvcera blood fell within these reported ranges. GIycera and Noetia RBC are easily differentiated on the basis of ultrastructure. (Reviewed by Mangum and Mauro, 1985.) Unlike Noetia RBC, Glycera RBC lack marginal bands and possess smaller vacuoles (Figs 1 and 2). Glycera RBC also have more
distinct mitochondria which contain prominent cristae (Fig. 2). The comparative densities of these cells (Fig. 3) reveal that Noetia RBC are less dense than GIycera or rat RBC with a DSO(density point) for half the cell population of 1.099 as compared to 1.108 and 1.104 for G&era and rat RBC, respectively. Although Noetia RBC are less dense than Glycera RBC, they have a lower osmotic fragility (Fig. 4). These findings are similar to those reported by Mangum (1980). A more detailed account of the osmotic fragility of these and other RBC can be found in Demanche (1980). Total phosphate per cm’ RBC (TP,) measures 33.7pmol in Noetia and 55.6pmol in Glycera (Figs 5 and 6, respectively). ATP and ADP are the predominant organic phosphates in the RBC of these
Fig. 2 Electron
micrograph
of a Glycera dibranchiafared blood cell showing vacuoles (V).
its mitochondria
04 a.nd
N. A. MAURO and R. E. ISAACKS
400
0
I 0
I
1YL 20
40
% SEA
60
80
100
WATER
33~100)
(lco%SW
Fig. 4. Red blood cell fragility of Noetia and Gl~era RBC after a 15-min exposure to a given osmotic pressure. Noetia (a), and Glycera (m).
species. The concentration of ATP in erythrocytes of Noetia and Glycera is 5.4 and 5.9 mM, representing 42.9 and 30.5% of their cell phosphate, respectively. Inorganic phosphate (P,) constitutes 6.4 and 22.7 PM cm3 RBC or 19.0 and 40.8% of the total P, in Noetiu and Glycera RBC. respectively. Glucose uptake and metabolism Plasma glucose levels for Noetia and Glycera are similar at approx. 1.4mM (28 mg/dl). By comparison. the plasma glucose levels of the rat and chicken are higher and range between 3.1 and 4.2 mM and 11 .I and 14.4 mM for the rat and chicken. respectively. Figure 7 shows the results of the net glucose uptake into the RBC obtained from Noetia, Gll,cera, and vertebrate RBC. Glucose uptake is fastest in Glycera
RBC followed by Noetiu. The sluggish uptake of glucose by the chicken and rat RBC is probably the result of a lower glucose permeability and a low concentration of glucose in medium as compared to normal serum levels; the lower incubation temperature of 22’C relative to the physiological norms of 394O’C for the chicken and 37’C for the rat may also be a factor. The Glwera RBC, which demonstrate the highest permeability to glucose are only about half as permeable to ribose with an uptake of 0.32 f 0.01 (SE: N = 4) pmol/ml RBC/hr (22’C). Uptake of ribose into Noetia RBC is similar to that of glucose: 0.42 f 0.01 (SE: N = 4) pmol/ml RBC per hr (22 C). The metabolic fate of glucose was also compared. Mammalian RBC utilize the Emden-Meyerhof and pentose PO, pathway for energy production with L-lactate produced as the primary end product. We observed no measurable D- or-L lactate accumulation by Noetia or G&era RBC, after a I-2-hr incubation at either 22 or 37’C. The limitations of the anaerobic capacity of Glycera RBC are further demonstrated by the 25% hemolysis after a 15-min exposure to KCN (lo-‘M). Rapid hemolysis of Glycera RBC also occurs when either whole sera or washed cells are exposed to a PO, of zero (Mangum and Carhart, 1972). A similar exposure of Noetia to KCN (10m3 M) did not produce lysis. Noetia RBC membranes thus appear to be stronger and are osmotically less fragile than Glvceru. Besides glycolysis, glucose may alternatively be oxidized by RBC to CO, via the PPO, pathway, or in the case of nucleated RBCs, by means of the TCA cycle. Since the production of CO? is greater when glucose is completely oxidized by the TCA cycle as compared to partial oxidiation by the PPO, pathway, the relative level of CO? production gives indirect
umoles Pi/ml
ATP
i
-10
0
20
40
80 100 FRACTIONS l5ml)
Fig. 5. Column chromatogram of a trichloroacetic acid extract of 1.35 cm3 Noefia RBC. The chromatogram was developed with a linear gradient O-5 N ammonium formate buffer. pH 3.45. as described by Isaacks et al. (1976). The heavy solid line represents pmol P,/ml of fraction; the narrow solid line respresents absorbance at 260 nM.
Erythrocytes
from Gl.vcera and Noetia
401
bmoles Pi/ml 1.0-
1.6-
1.4-
g 1.2@I > l.O2 $ a % 2
.6-
.6 -
FRACTIONS I5 ml)
Fig. 6. Column chromatogram of a trichloroacetic acid extract of 4.40cm3 Glycera RBC. The chromatogram was developed with a linear gradient O-5 N ammonium formate buffer, pH 3.45, as described by Isaacks et al. (1976). The heavy solid line represents pmol P,/ml of fraction; the narrow solid line represents absorbance at 260 nM.
evidence of the level of activity of the PPOl pathway and the TCA cycle. Compared to the rat RBC, whose primary route of CO, production is by the PPO, pathway, all of the nucleated RBCs examined here have a considerably higher level of *CO2 production from labelled glucose (Table 1). The decreased importance of the PPOl pathway in Glycera and Noetia RBC compared to the rat was further substantiated by the production of labelled CO, after incubation with glucose labelled in either the C6 or C, position. Because CO, is made preferentially in the PPO, 0.8
0.6
F
t
/
.-
=-m
/
pathway from the first C and triose PO, from the 6th, the lower the ratio of C60z/C,02 the greater the relative activity of the PPO, pathway. Rat, Noetiu, and Glycera RBC have ratios of 0.02 and 0.06 and 0.13, respectively. Therefore, the rat has a higher relative activity of this pathway than Noetia or Glycera. Moreover, the relative activity of the PPO, pathway is twice as great in Noetia as in Glycera. This observation may explain in part the higher uptake of ribose by Noetia RBC compared with Glycera and, conversely the reduced uptake of ribose compared to glucose in Glycera RBC. Still further substantiation of the decreased importance of the PPO, pathway in invertebrate RBC is indicated by the increased thermal sensitivity of their oxygen uptake. Cells which show a predominance of the PPO, pathway have a lower temperature dependence of their oxygen consumption (Hochachka and Hayes, 1962; Mauro and Mangum, 1982). The lower thermal sensitivity of the vertebrate RBC, as evidenced by a lower Q,,, , indicates a greater dependence on the PPO, pathway than Noetia RBC (Table 2). Glutamate uptake and metabohm
The uptake and oxidation of glutamate by RBC gives a better evaluation of the activity of the TCA Table
I.
Ratio of *CO2 production
from labelled glucose by RBC relative to *CO2 production from uniformly labelled glucose by rat RBC TIME (minsl
Fig. 7. Glucose accumulation at 22°C by Noeria (a), Glycera (m), rat (A), and chicken (0) RBC. Flux media containing 1 mM cold glucose.
Species
Ratio
Rat Chicken
2.64
Noeria Gl.vcera
6.25 5.91
I .oo
N. A. MAURO and R. E.
402 Table
2
RBC
oxygen uptake (pl,‘ml Mean + SE. .N = 6-X
RBC,‘hr)
TWIpetXtUre 32 c
37 C Human Rat Chxken NWfiU G/wru
199.*4 276 i_ 2 444 i_ I 658 t 2
22 c
Q,n
122i5 160+ I l79i2 337 * 3 434 k 6
I .4 I .4 1.X 2.0
Table 3. Glutamate accumulation by RBC at 22 C after I hr incubatmn (PM x IO ‘/ml RBC). Mean & SE. N = 6-R Accumulation 5 * 0.4 9 z 0.4 39 i 7.0 lh+O.l
Table 4. Radio-labelled carbon dioxide (‘CO,) production from umformly labelled glutamate by RBC after I hr at 22 and 37’C (PM x IO ‘/ml RBC). Mean i SE. N = f%g Amount of glutamate Oxidzed Species
22 C
Noetiu GlKera Rat Chicken
39 54 87il3 1+0.1 I + 0.2
*CO Produced
37 c
22 c
37 c
I kO.1 4kO.l
195 435 5 5
5 20
cycle than glucose since this substrate enters directly into the TCA cycle after deamination. Of the RBC examined, glutamate accumulation is greatest in Noetia RBC and smallest in the mammalian RBC (Table 3). Regardless of the magnitude of glutamate oxidation, glutamate is apparently permeable to all RBC examined. Glutamate oxidation is considerably greater in Noetia and Glycera RBC than in the vertebrate RBC (Table 4). Vertebrate RBC, even avian RBC, which possess a TCA cycle, are limited in their ability to oxidize exogenous glutamate. This limitation occurs at both 22°C and the more physiological temperature of 37 C. At 37’C, the rat RBC are still unable to oxidize more than trace amounts of glutamate and the values for chicken RBC are more than an order of magnitude lower than observed for Gll’cera or Noetia.
DISCUSSION
The metabolism of the invertebrate RBC studied here is clearly different from vertebrate RBC. Vertebrate RBC are lower in permeability to exogenous substrates than annehd or molluscan RBC. Many fish RBC, in addition to the rat and chicken RBC, are either impermeable or low in permeability to glucose (Bolis and Luly, 1972; Kim and Isaacks, 1978). Even mammalian RBC. which lack mitochrondria and must rely upon glycolysis for energy production, may be glucose impermeable (Kim and McManus, 1971). The adaptive advantage of this phenomenon is unknown. although it is known that glucose imper-
ISAACKS
meability is apparently confined to RBC taken from adult pigs and that RBC taken from the fetal pigs are permeable to glucose (Zeidler, 1976). In other words, annelid and molluscan RBC resemble embryonic mammalian RBC more closely than adult mammalian RBC in terms of this physiological feature as well as their nucleated condition. After entering the RBC the oxidation of exogenous substrate is regulated by the size of endogenous substrate pools and the activity and/or existence of the oxidizing pathways, The low oxidation of glutamate by chicken RBC compared to the invertebrate RBC is surprising in view of the fact that the endogenous pool of glucose in chicken RBC is small, measuring 0.8 PM/ml RBC (Mauro and Isaacks, unpublished data). These observations suggest that the TCA cycle of avian RBC may either play a less significant role in energy production and/or may indicate a specialization of avian RBC for carbohydrate oxidation. It is not known whether these low levels of amino acid oxidation are unique to avian RBCs or are characteric of nucleated RBC from other groups of vertebrates. This area is worthy of further examination. Annelid and molluscan RBC can be differentiated further from avian and mammalian RBC by their level of ATP. ATP levels in GIycera and Noetia are higher than those found in birds or mammals although similar to those found in some fish and reptiles (Bartlett, 1980; Isaacks and Harkness, 1980; Johansen et al.. 1978; Kim et al., 1984). Unlike vertebrates the organic phosphates of invertebrates apparently play no role in the modulation of hemoglobin oxygen affinity (Mangum and Mauro, 1985). The question thus becomes, what are some of the possible roles of ATP within invertebrate RBC? One of the primary functions of ATP in RBC is to sustain cell membrane integrity. Reduction in the level of intracellular ATP results in hemolysis. In Glycera RBC the presence of the electron transport inhibitor. KCN. causes a rapid hemolysis. A similar reduction in ATP levels in mammalian RBC results in a weakening and breakdown of the cell membranes (Clader, 1976). Another potential role of ATP in RBC may be in the active transport of substrates and ions. This need for ATP may be seen in the distribution of K+ and Na’ ions across the Glycera RBC membranes. In a medium of 500 mOsm Na+ and 16mOsm K’, the intracellular concentration of K+ and Nat ions measures 100 mOsm for each ion (Mauro and Isaacks. upublished data). Although intraceilular Na’ ion concentrations of cells have been shown to vary greatly, intracellular K+ ion concentration remains more constant, ranging between 100 and 125 mOsm (Presser, 1973). In summary. annelid and molluscan RBC can be metabolically characterized as aerobic cells sharing much in common with other aerobic cells. They have a limited anerobic tolerance; they are highly permeable to exogenous substrates; they oxidize exogenous substrates readily; and they lack substrate specificity. By using this metabolic profile as archetypal for the ancestral condition of all RBC. the metabolic specialization and evolution of the vertebrate RBC can be more easily interpreted.
Erythrocytes
from G&era
Acknowledgements-The authors are grateful to Phyllis Goldman and Chang Kim for their technical help, to Tommie Stapleton, Linda Numan. and Linda Schoomaker for typing the manuscript, and to Dr Charlotte P. Mangum for her suggestions and review of the manuscript.
REFERENCES Bartlett G. R. (1980) Phosphate compounds in vertebrate red blood cells. Am. Zool. 20, 103-l 14. Bolis L. and Luly P. (1972) Monosaccharide permeability in brown trout Salmo !ratfa L. erythrocytes. In Role o/ Membranes in Secretory Processes (Edited by Bolis L.. Keynes R. D. and Wilbrandt W). pp. 215-221. NorthHolland, London and Amsterdam. Dannon D. and Marikovsky Y. (1964) Determination of density distribution of red cell population. J. Lab. clin. Med. 64, 6688674. Demanche R. (1980) The osmotic fragility of red blood cells of marine animals: a comparative-study. M. A. Thesis, Colleee of William & Marv._ Williamsbure. Vireinia. Drabkin D. R. (1945) Crystallographic and optical properties of human hemoglobin. A proposal for the standardization of hemoglobin. Am. J. Med. Sci. 209, 2688370. Freadman M. A. and Mangum C. P. (1976) The function of hemoglobin in the arcid clam Noe/ia ponderosa, I. Oxgenation in vitro and in riro. Camp. Biochem. Physiol. 53A, 173-179. Glader B. F. (1976) Salicylate-induced injury of pyruvatekinase deficient erythrocytes. N. Engl. J. Med. 249, 916918. Hochachka P. W. and Hayes F. R. (1962) The effect of temperature acclimation on pathways of glucose metabolism in the trout. Can J. Zool. 40, 261-270. Hoffman R. J. and Mangum C. P. (1970) The function of coelomic cell hemoglobin in the polychaete Glvcera dilbranchiata. Comp.Biochem. Physiol36, 21 l-228: Isaacks R. E. and Harkness D. R. (1980) Ervthrocvte organic phosphate and hemoglobin function -in birds, reptiles and fishes. Am. Zool. 20, 115-129. Isaacks R. E., Harkness D. R.. Froeman G. A. and Sussman S. A. (1976) Studies on avian erythrocyte metabolism. 1. Procedure for separation and quantitaton of the major phosphorylated metabolic intermediates by anion exchange chromatography. Camp. Biochem. Physiol. 53A, 95-99. Johansen K.. Mangum C. P. and Lykkeboe G. (1978) Respiratory properties of the blood of Amazon fishes. Can. J. Zool. 56, 8988906. Kim H. D. and lsaacks R. E. (1978) The membrane w.
_
and Noeria
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permeability of nonelectrolytes and carbohydrate metabolism of Amazon fish red cells. Can. J. Zool. 56, 8633869. Kim H. D. and McManus T. J. (1971) Studies on the energy metabolism of pig red cells. I. The limiting role of membrane permeability in glycolysis. B&him. biophys. Acta 230, 1-I I. Kim H. D., Zeidler R. B.. Sallis J.. Nicol S. and Isaacks R. E. (I 984) Metabolic properties of low .4TP erthrocytes of the monotremes. FEBS-167, 83-87. Mangum C. P. (1977) The annelid hemoglobins: a dichotomy in structure and function. In Essays in Memory of Dr. Olga Hartman (Edited by Reish D. J. and Fauchald K.), p. 407. Allan Hancock Foundation, Special Publication, University of Southern California Press, Los Angeles. Mangum C. P. (1980) Distribution of respiratory pigments and the role of anaerobic metabolism in the lamellibranch molluscs. In Animals and Enaironment Fitness (Edited by Gilles R.). pp. 171.-184. Pergamon Press, Oxford and New York. Mangum C. P. and Carhart J. A. (1972) Oxygen equilibrium of coelomic cell hemoglobin from the bloodworm G&era dibranchiata. Camp. Biochem. Physiol. 43A, 949-957. Mangum C. P. and Mauro N. A. (1985) Metabolism of invertebrate red cells: a vacuum in our knowledge. In C’h-culalion. Respiration, and Metabolism-Current Corn pararire Approaches (Edited by Giles R.), pp. 28@-289. Springer, New York. Mauro N. A. and Mangum C. P. (1982) The role of the blood in the temperature dependence of oxidative metabolism in decapod crustaceans. I. Intraspecific responses to seasonal differences in temperature. J. e.vp. Zool. 219, 1799188. Pierce S. K. and Maugel T. K. (1985) A comparison of the water regulating responses of bivalves and polychoete red cells to osmotic stresses. In Blood Cells of Marine Invertebrates (Edited by Cohen W. D.), pp. 1677193. Allen R. Liss. New York. Prosser C. L. (1973) Comparafit~e Animal Physiology. W. B. Saunders. Philadelphia, London, Toronto. Shafie S. M., Vinogradov S. N., Larson L. and McCormick J. J. (1976) RNA and protein synthesis in the nucleated erythrocytes of Glycera dibranchiata. Camp. Biochem. Physiol. 53A, 85-88. Terwilliger N. B.. Terwilliger R. C. and Schabtach E. (1985) Intracellular respiratory proteins of Sipuncula Echiura. and Annelids. In Blood Cells of Marine Inoertebrates (Edited by Cohen W. D.). pp. 193-227. Allen R. Liss. New York. Zeidler R. B., Lee P. and Kim H. D. (1976) Kinetics of 3-O-methyl glucose transport in red cells of newborn pigs. J. gen. Physiol. 67, 67.-80.