Cow red blood cells. II. Stimulation of bovine red cell glycolysis by plasma MICHAEL Department
J. SEIDER AND HYUN uf Physiology, University
DJU
KIM
of Arizona,
SEIDER, MICHAEL J., AND HYUN DJU KIM. Cow red blood cells. II. Stimulation of bovine red cell glycolysis by plasma. Am. J. Physiol. 236(5): C262-C267,1979 or Am. J. Physiol.: Cell Physiol. 5(3): C262-C267, 1979.-Cow red cell glycolysis, which can be stimulated by a variety of purines and pyrimidines, was also found to be elevated by its own plasma. Dialyzed or charcoal-treated plasma could no longer stimulate glycolysis, suggesting that the stimulating factors may be purines or pyrimidines. Determination of purines or pyrimidines in plasma revealed the presence of xanthine (0.31 FM), hypoxanthine (0.60 PM), and adenosine (0.05 PM), as well as unknown compounds. A physiologic level of hypoxanthine, with or without xanthine and adenosine approximating their concentrations in plasma, resulted in the stimulation of cow red cell glycolytic rate by 16% (P < 0.01). These findings suggest that plasma-borne purines may act on cow red cells in concert with as yet unidentified factors. Moreover, exchanging calf and cow plasmas produced no stimulatory effect on either calf or cow red cell glycolysis, suggesting that a) calf red cells lack some of the cellular components that respond to this stimulator and, b) only cow plasma contains this specific stimulator. In other species, including dog, cat, rabbit, rat, guinea pig, and human, stimulation of glycolysis by plasma was not observed. purines; pyrimidines; chromatography
ion-exchange
chromatography;
thin-layer
EFFECTS ofcertainpurine andpyrimidine compounds on cow red cell glycolysis were described in the companion paper (20). The site of this enhancement could possibly be at the level of hexokinase, which can be stimulated by these agents. Inasmuch as the newborn calf red cells, endowed with more than threefold greater glycolytic capacity than cow cells, displayed no discernible reponse to these agents, this phenomenon represents a unique paradigm of postnatal adaptation of energy metabolism in cow cells. In the course of this investigation, we also found that glycolysis of the cow, but not the calf, red cells can be equally augmented by their own, but not alien, plasma kept at a pH 7.4. In this communication, results of experiments designed to determine the plasma-borne agents responsible for an enhancement of cow red cell glycolysis are presented.
THE STIMULATORY
MATERIALS
AND METHODS
Sources and preparation of blued and plasma. Blood was obtained from animaIs housed at the University of Arizona farm or the Arizona Health Sciences Center, as C262
Health
Sciences
Center,
Tucson, Arizona
85724
described in the companion paper (20). Red blood cells were spun down in a Sorvall GLC centrifuge at 4OC and the upper two-thirds of the plasma was saved. The white buffy coat, along with the remaining plasma, was aspirated and discarded. Red cells were thoroughly washed in isotonic NaCl solution prior to use. MetaboEic studies. Prior to addition of red blood cells, plasma was allowed to equilibrate with air for a minimum of 2 h at 37OC. For all studies, pH was checked every half-hour during incubation with a Radiometer waterjacketed blood electrode and maintained within 0.1 pH units of 7.4 with isotonic NaOH or HCl. Cell extraction procedures, as well as determination of glucose and lactate, were the same as described in the previous paper. Composition of balanced salt solution (BSS) is the same as described in the previous paper (20). Ion-exchange and thin-layer chromatography of nucleosides and purines in plasma. Plasma purines and nucleosides were determined with ion-exchange chromatography according to the methods of Bartlett (2). At least 400 ml of blood were obtained at the local slaughterhouse and rapidly chilled in small aliquots; the plasma was separated from cells by centrifugation. After deproteinating the plasma with 10% trichloracetic acid (TCA), the resultant supernatant was extracted four times with 4 volumes of ether to remove TCA. The extract was loaded onto a Dowex 50 x 8 resin (ZOO-400 mesh), 1 x 20 cm column. The column was eluted with 1,000 ml of linear gradient of O-4 N HCl. The column eluant was read at 260 nm, and those fractions showing appreciable absorbance were further analyzed by scanning over the entire untraviolet range (240-320 nm) at pH’s of 2, 7, and 12 in order to identify the eluted compound. The absorption profile was compared with those of known standards run in an identical manner. Plasma levels of nucleosides and purines were also measured by thin-layer chromatography as described by Akaoka, Nishizawa, and Nishida (1) or by Bockman, Berne, and Rubio (6). In the method described by Akaoka et al. (1), blood was rapidly cooled and centrifuged, and the plasma was separated from the erythrocytes. The plasma was deproteinized with 5% perchloric acid (PCA) and the supernatant neutralized with 5% KOH. After centrifugation to remove the precipitated salt, the supernatant was lyophilized to dryness, and the dry powder was resuspended in 0.6 ml distilled water. All the supernatant fluid was spotted on the bottom of a cellulose thin-layer chromatography (TLC) plate (Kodak), and the plates were developed in ascending fashion
0363”6143/79/0000-UOOO$O1.25
Copyright
0 1979 the American
Physiological
Society
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PLASMA
STIMULATION
OF
C263
GLYCOLYSIS
in a closed tank with a solvent consisting of 95% ethanol and 1 M ammonium acetate, pH 7.5 (75:30 vol/vol). The elution position of standards of hypoxanthine, xanthine, and uric acid were identified by visualization under ultraviolet light. In the method described by Bockman et al. (6), at least 100 ml blood were collected in heparinized tubes and then immediately poured into equal volumes of ice-cold saline. This mixture was spun down at 1,000 g within 5 min, and the plasma-saline mixture was separated from the red cells. The plasma proteins were precipitated with enough 5 N PCA to give a final concentration of 0.5 N PCA. The samples were then centrifuged at 14,000 g in an International refrigerated centrifuge (model B-20) and the supernatant fractions were neutralized with 5.63 N K&03. The resulting precipitate was spun down, and the neutralized supernatant was saved. Adenosine, inosine, hypoxanthine, and xanthine in this neutralized extract were adsorbed onto Norit charcoal (5 mg for each ml of plasma), After a brief centrifugation, the supernatant was discarded and the charcoal was then eluted with two successive 15-ml volumes of 10% pyridine in 50% aqueous ethanol. The eluants were air dried, suspended in 0.4 ml water, and spotted onto TLC plates. The plates were developed in 15% ethanol at 4”C, with the necessary standards spotted on one side. In both methods, the plasma compounds were identified under ultraviolet light by their position on the TLC plate, which was compared with standards. To quantitate the amount of compounds in the plasma, areas on the plates corresponding to the known standards were scraped off and eluted with three successive lo-ml volumes of 50% ethanol. The eluants were air dried and resuspended in water. The nucleosides and bases were assayed by conversion to uric acid according to the method of Kalckar (8). Standards were run to test for recovery in all experiments, and recoveries ranged from 80-90%, similar to previous reports (1, 6). In all cases, no other noticeable UV-absorbing material was found on TLC plates spotted with various standards. Sources of materials. All materials were obtained from the same suppliers as in the companion paper. Purity of nucleosides and purines was examined using both thinlayer and column chromatographic methods described above. We found no UV-absorbing impurities with any of these methods. RESULTS
Rapoport (18) reported that mammalian red cells suspended in a balanced salt solution (BSS) decreased the medium pH by 0.2 to 0.5 pH units per hour at 38°C. When calf or cow red cells were suspended in BSS containing glucose, the pH slowly decreased as a function of time, due to the increasing concentration of lactic acid produced as an end-product of anaerobic metabolism (Fig. 1). This decrease was about 0.5 and 0.3 pH units per hour in calf and cow red cells, respectively, with the most rapid decline in calf cells, due to their higher glycolytic rate. In contrast, when washed calf or cow red cells were added to their own plasma, the plasma pH rose to 8.0 for calf red cells, and 8.2 for cow cells (Fig. l), presumably due to the equilibration of plasma CO2 with
cow plasm
cow BSS cdf BSS
FIG. 1. Comparison of the pH change in plasma solution (BSS) containing calf or cow red cells. Red either to balanced salt solution or to plasma fortified glucose to give a hematocrit of 1045%. Calf cells in (a-----+); calf cells in balanced salt solution (A-A); own plasma (o------O); cow cells in balanced salt solution 4 h incubation in calf plasma, the calf cells began to alent results were seen in 2 other experiments.
vs. balanced salt cells were ad .ded with additional their own plasma cow cells in their (A-A). After hemolyze. Equiv-
air as reported in other cells (3). The calf plasma pH rose no further, probably due to extensive hemolysis that occurred by this time. Zeidler and Kim (21) have shown that elevation of the internal pH of calf red cells, but not cow cells, produces rapid hemolysis. Figure 2 shows the effect of pH on cow red cell glycolysis. The desired pH was maintained within 0.1 pH units by titrating with isotonic acid or base during a 7-h incubation. Results are expressed as percent of control at pH 7.4, and numerical values for initial rates at pH 7.4 are given in Table 1. At pH 8.2, the glucose consumption rate is nearly 55% greater than at 7.4, but this elevation is less than in several other species, including rats and humans, which increase their glycolytic rate 82% and 164%, respectively (18). Table 1 shows the effects of plasma on calf and cow red cell glycolysis. Although calf and cow plasma have a glucose concentration between 1 and 3.5 mM, additional glucose was added to equalize plasma and BSS glucose concentrations. As in other species (7, 10, II), red cells obtained from newborn calf had a much higher glycolytic rate than did cow red cells (Table 1). In contrast to calf red cells, which displayed identical glycolytic rates in both BSS or plasma, cow red cells significantly (P < 0.02) increased their glycolytic rate nearly 50% when suspended in their own plasma. To discover the chemical composition of the plasma activator, approximately 20 ml of cow plasma was dialyzed in 15 in. of no, 8 tubing for 4 and 24 h against 3 liters of isotonic, buffered BSS at 4°C and the dialyzed plasma was fortified with 5 mM glucose to replace lost glucose. Figure 3 shows the effect of dialyzed plasma on glycolysis. The levels of lactate in undialyzed cow plasma were quite variable from animal to animal and exceeded the intracellular lactate concentration. Thus, for the purpose of comparison, lactate levels were normalized against initial lactate levels. Figure 3 shows that plasma
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C264
M.
80
80
60
60 z F 40 2
t
-60 616
7.-O
7: 4
7:8
a2
6:6
f0
7:4
718
ti
PH
PH FIG. 2. Effect of pH on cow red cell glycolysis. E1xperimental conditions are the same as in Fig. 1. Similar results were seen in 2 other experiments. TABLE
1. Calf and cow red cell glucose
consumption and lactate formation in plasma us. BSS Calf HSS
Glucose consumption Lactate formation Ratio: lactate/ glucose
2.05 -+ 0.49 (61 3.28 k 0.70 (6) 1.60
COW
PlZiSfIM
BSS
pm0 I/ (ml cells . h) 2.17 k 0.51 0.60 k 0.04 6) 03 3.39 k 0.51 0.87 t 0.05 03 (6) 1.56 1.45
PhIMa
J. SEIDER
AND
H. 11. KIM
To determine the plasma levels of purines and nucleosides, both ion-exchange and thin-layer chromatography procedures were employed. With the Bartlett method (2), cow plasma elution profiles showed peaks corresponding to xanthine and hypoxanthine and two unknown peaks that had ultraviolet absorption patterns exhibiting a maxima of 251.5 nm, but did not have any characteristics of known purines or nucleosides (Fig. 4). Adenosine was found in very low concentrations with this method. TLC procedures of Akaoka et al. (1) allowed us to quantitate the amount of purines on smaller quantities of calf, cow, and human plasma. Human plasma was found to have concentrations of uric acid, xanthine, and hypoxanthine that were higher than either calf or cow plasma (data not shown). As in the case of ionexchange chromatography, TLC of cow plasma showed a rapidly migrating ultraviolet-absorbing peak that was absent in both calf and human plasmas. In dialyzed plasma, all of the ultraviolet-absorbing areas were lost, implying that the rapidly migrating compound was not an artifact of the TLC preparative method. When compared with either human plasma (16) or dog plasma (19), the concentration of purines and nucleosides in cow and calf plasma were extremely low (Table 4). Data in Table 5 summarizes the effect of hypoxanthine approximating the plasma concentration on cow red cell Plasma + J Hypoxanthine
0.89 t 0.06* 03 1.32 t 0.11”
PI osma \ Dialyzed Plastrtu + H ypoxanthine
03 1.55
Dialyzed /Plasma
Values are means k SE, with number of experiments in parentheses. Balanced salt solution (BSS): 5 mM KCl, 150 mM NaCl, 10 mM Na phusphate buffer, pH 7.4, and 5 mM glucose. Hematocrit, 10-15s; temperature 37 OC. * P < 0.02 comparing cow BSS to cow plasma.
elevated the lactate formation rate when compared with BSS, but that plasma dialyzed for 24 h lost its stimulatory capability. The 4-h-dialyzed plasma (not shown) gave rates that were intermediate to the two extremes shown in Fig. 3. The results implied that the stimulatory agent was a small molecule. If the stimulating factor(s) in plasma were purines, then elevated glycolytic rates should be restored by adding this in vitro stimulator to dialyzed plasma. Addition of 1 mM hypoxanthine to dialyzed plasma restored the glycolytic rate to that found in untreated plasma (Fig. 3). Further elevation of the glycolytic rate was not seen when hypoxanthine was added to nondialyzed plasma, suggesting that plasma stimulates red cell glycolysis maximally. Several methods were used in attempting to identify and isolate the plasma stimulator(s). Charcoal-treated plasma failed to stimulate cow red cell glycolysis (Table 2). Plasma, boiled for 30 min to denature the proteins, was tested for its stimulatory effect. Boiled plasma had a lower lactate formation rate than t.hat seen in normal plasma, but still elevated gycolysis 33% over a BSS control (Table 3). The reason for the drop from the plasma control may have been heat destruction or loss of activator in the protein precipitate.
bSS
0/
234567 Hours FIG. 3. Effect of plasma on cow cell glycolysis. Plasma was dialyzed for 24 h against buffered balanced salt solution. After dialysis, 5 mM glucose or I mM hypoxanthine was added, depending upon experiment. In 24-h dialysis experiment, fresh blood was drawn from same animal that provided plasma the day earlier. Dialyzed plasma (A-A); undialyzed plasma (c-1); balanced salt solution (PF----A); dialyzed plasma + I mM hypoxanthine (m); undialyzed plasma + 1 mM hypoxanthine { nj. Equivalent results were seen in 4 other experiments. 0
I
TABLE 2. Effect of charcoal-extracted on cow red cell glycolysis Lactate
_-
BSS control Plasma control Charcoal-extracted
plasma
plasma kite, pmol/(ml cells-h)
0.79 1.21 0.79
53 0
Typical results from I of 3 experiments is shown. Norit charcoal, 5 mg, was added to each ml plasma; the mixture was stirred for 3 h at 4°C and the charcoal was spun out. The plasma supernatant was glucose fortified, and then washed red cells were added to make a final hematocrit of 1045%. Controls consisted of red cells suspended in glucose-fortified BSS, or untreated plasma. Rates were calculated using least-squares regression analysis, with correlation coefficient r > 0.95 in all cases. RSS, balanced salt solution.
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PLASMA
STIMULATION
OF
C265
GLYCOLYSIS
3. Effect of boiled plasma cow red cell glycolysis
on
TABLE
Lactate
Procedure
BSS control Plasma control Boiled plasma
Rate, pmol/( cells. h)
ml
%
0.66 0.99
50
0.88
Cow plasma was boiled removed by centrifugation,
33
for 30 min and the resulting BSS, balanced salt solution.
precipitate
was
cells can be “turned on” only by their own plasma. Plasma other than cow and calf hemolyzed cow red cells within minutes. When cat, rat, guinea pig, and dog red cells were added to cow plasma, they rapidly hemolyzed within 10 min. When compared to cow plasma, calf plasma was not effective in elevating cow red celI glycolysis, implying that calf plasma lacked all the cow plasma stimulator(s). When calf red cells were suspended in cow plasma, their glycolytic rate was unaffected, implying that calf cells also lacked the internal machinery necessary for producing stimulation of glycolysis. To determine whether in vitro nucleosides and purinestimulated glycolysis were unique to cow red cells, several other animal cells were also tested with adenine. Figure 7 summarized the results. It is apparent that adenine is not as effective in other species as it is in cow red cells. DISCUSSION
In several early RBC glycolytic studies utilizing plasma, the elevated glycolytic rate appeared to be accounted for by the increased plasma pH (3,lZ). In strictly pH-controlled plasma, glycolytic rates of human red cells were the same as cells suspended in balanced salt solution
.l
I
I
T
I
1
.2
.4
.6
.8
1.0
Liters
4. Levels of nucleosides in animal plasma
TABLE
Xanthine
cow Dog"
Human
(4)
and bases ----~--~
Hypoxanthine
pmoL/ml Calf (6)
0.40 0.31
* 0.07 k 0.06
3.16
t
1.45+
0.60 0.55 0,44 3.60
t t Al k
0.07 0.05 0.12 1.9Ot
5. Effect of 1 pM hypoxanthine cow red cell glycolysis -.
on
TABLE
4. Ion-exchange chromatography of cow plasma. 100 ml of cow plasma were extracted with trichloroacetic acid (TCA), which in turn was removed by ether. Resulting extract was loaded onto a Dowex 50 x 8 resin-filed column (1 x 20 cm), Purines were eluted from column using 1,000 ml of a linear gradient of O-4 N HCl. Absorbance at 260 nm is plotted against liters through the column. Each compound was identified on the basis of its elution position and its absorption profile in three different pH’s over the entire ultraviolet range (200-320 nm). HX, hypoxanthine; X, xanthine. Results from 1 of 6 experiments, FIG.
Inosine
Adenosine
plasma 0.14
* 0*04 ND 0.12 f 0.03
0.09 0.05 0.11 0.31
* * t t
Values are means & SE, with number of animals in parentheses. not detected. * From Bockman et al. (6). t From Akaoka $ From Mills et al. (16). (1).
0.01 0.02 0.04 0.29$
ND, et al.
glycolysis. There was a small but significant 16% (P < 0.02) elevation of glycolysis, and addition of xanthine or adenosine at concentrations found in plasma produced no further increase in the glycolytic rate. These findings showed that physiologic levels of purines may stimulate cow red cell glycolysis to some extent. When dog, cat, rabbit, rat, guinea pig, and human red cells were suspended in either glucose-fortified balanced salt solution or plasma, the rates of glycolysis were the same, suggesting that other animal plasmas lacked stimulators of glycolysis (Fig. 5). On the other hand, stimulators may have been present in all plasmas, but the cells were not responsive to these compounds. To test this possibility, cross-plasma experiments were performed, with the results shown in Fig. 6. Apparently, cow red
Lactate
Formation,
pmol/(ml
Cow No. Glucose
1
Glucose
alone
0.52
Cow cells were pH 7.4, at 37°C. cat
t
% Difference
+ 1 pM hypoxanthine
0.6 0.65 0.50 0.57 0.55 0.34 0.50 0.55 0.4 0.57 0.49
2 3 4 5 6 7 8 9 10 11
------ - ---
cells. h)
0.84 0.67 0.52 0.59 0.79 0.41 0.56 0.63 0.46 0.67 0.56 0.03
suspended * Using ml
0.61
k 0.04
at Hct 15-20% paired t test. rabbit
36.8 4.7 2.6 4.6 45.2 21.1 12.5 14.7 7.0 17.1 15.4 16.5 k 04.09 P < 0.01*
in balanced
guinea
rat
salt solution,
human
Pi&l
Bss
PL
E3ss PL
Bss
PL
5. Comparison of glycolysis fied balanced salt solution (BSS) animals. FIG.
Bss
PL
Bs
PL
Rss
PL
of red blood cells in glucose-fortivs. plasma (PL) from a variety
of
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(2266
M.
2.6
- 2.6
-
i cow
ii cat
rat
cow
cow
guinea Pig cow
MiI rabbit
human
cow
cow
cow
calf
**’ 2 P 00 3 sg cm 3g p: ’ 1.0 $2 qg 3 -
/ calf calf
6. Calf and cow red cell glycolysis in plasma obtained from several different species. Rapid hemolysis of red cells occurred when dog, cat, rat, rabbit, or guinea pig red cells were suspended in cow plasma. FIG.
dog
rabbit
tat
sheep
human
6 5k
z
0
4s
c, 3F
3s
k
-
g
v, zx
z
II o-
G G+A G G+A G G+A G G+A 7. Effect of adenine on glycolysis of red blood cells obtained from a variety of animals. G, glucose-fortified balanced salt solution (BSS); G + A, BSS with I mM adenine + glucose. FIG.
(17). In contrast, cow red cell glycolysis was found to be stimulated by its own plasma (Table 1). This stimulatory effect of plasma on cow cells was somewhat unusual, inasmuch as other species including the dog, cat, rabbit, rat, guinea pig, and human did not respond to their own plasma. Dialysis of cow red cells for 6 h against balanced salt solution still produced the same stimulation upon exposure to plasma, suggesting that no unusual intracellular factors were required. In view of the findings reported in the companion paper, in which cow red cell glycolysis was shown to be augmented by a variety of purines and nucleosides, it seemed reasonable to suspect those purines and nucleosides in the plasma as agents responsible for the plasma stimulation. Indeed, dialysis or charcoal treatment of plasma resulted in the inactivation of this stimulation. However, actual quantitation of the purines and nucleosides in the plasma revealed that these compounds were present in l/loo the amount needed for maximal stimulation of cow red cell glycolysis in vitro. Unidentified compounds were found by both TLC and liquid-chromatographic methods in cow, but not in calf or human plasmas. Isolation of these compounds was attempted to determine whether they were responsible for stimulation of cow red cell glycolysis. The highly acidic eluant from the first peak of the column was neutralized with a strong base, diluted to isotonic strength, and then added to incubating cow red cells.
J. SEIDER
AND
H. D. KIM
This peak failed to stimulate the red cell glycolytic rate. However, the possibility for acid inactivation of this compound cannot be ruled out. The second unknown peak was not tested due to excessive acidity. In yet another attempt, the rapidly migrating UV-absorbing spot on the TLC plate was scraped off, extracted, and added to BSS. It was found that cow red cells responded to a small extent to this extract, but control extracts from unspotted, developed TLC plates also elevated cow red. cell glycolysis, thereby obscuring our initial results. It has been shown that nucleosides and purines formed in tissues, including the heart (9), brain (5), and liver (13), may be transported by red cells to other regions of the body (13). Adenosine, which is a potent vasodilator, has been found in the coronary venous blood as well (4) and is released into the circulation during hypoxia (4, 14). If adenosine and other purine bases indeed participate in the stimulatory effect in vivo, one potential metabolic benefit could be the augmented production of ATP by stimulation of glycolysis. It should be recalled that cow red cells undergo an unusually rapid breakdown of ATP when incubated without metabolizable substrate. Factors responsible for this rapid breakdown in cells with a low Naf-K’and Ca2+-ATPase activity are not known (15)
Calf red cells, which have higher glycolytic rates, did not respond to exogenously added purines or their own plasma. Both the glycolytic rate and intracellular ATP levels fall rapidly in the growing animal. It is not known when the need for the plasma stimulator arises during this neonatal period. The exchange of plasmas from calf and cow failed to markedly stimulate glycolysis in either of these cells, suggesting that a) calf red cells lacked the internal machinery necessary for reacting to the plasma stimulator and b) calf plasma lacked the plasma stimulator. Other ruminants have low rates of glycolysis and intracellular ATP concentrations. It is presently unknown whether all ruminants have this particular type of glycoly-tic stimulation, although sheep are known to be unaffected by exogenously added purines ( Fig. 6). Our data show that plasma concentrations of purines and nucleosides in BSS cannot produce the same magnitude of stimulation that plasma itself can. This implies that compounds in plasma other than purines also act to stimulate cow red cell glycolysis. The expert technical assistance of Mr. Paul Cook and Mr. Y. S, Bak is gratefully acknowledged. We thank Mr. Robert Schonberg at the University of Arizona experimental farm for providing blood samples used in this study. Special thanks are due to Dr. R. Zeidler for his stimulating discussions. This work was supported in part by National Institutes of Health Grant AM-17723 and Training Grants HL-05884 and HL-07249. A part of this work represents partial fulfillment of the requirements for the PhD by Michiel Seider, University of Arizona, 1977. H. D. Kim is the recipient of a National Institutes of Health Research Career Development Award AM-00316. A preliminary report was presented at the Federation meetings, Chicago, IL, April 1977. Present address of M. J. Seider is Dept, of Physiology, The University of Texas Medical School, Health Science Center, Houston, TX 77025.
Received
28 Nov
1977; accepted
in final
form
12 Dee 1978.
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PLASMA REFERENCES
STIMULATION
OF
GLYCOLYSIS
C267
,
1. AKAOKA, I., T. NICHIZAWA, AND Y. NISHIDA. Determination of hypoxanthine and xanthine in plasma separated by thin-layer chromatography. B&hem. Med. 14: 285-289, 1975. 2. BARTLETT, G. R. Adenine: chemistry, analytical methods, sources, purity, specification. Transfusion 17: 333-338, 1977. 3. BARLETT, G. R., AND A. A. MARLOW, Comparative effects of the different formed elements on normal human blood glycolysis. J. AppZ. Physiol. 6: 335-347, 1953. 4. BERNE, R. M. Cardiac nucleotides in hypoxia: possible role in regulation of coronary blood flow. Am. J, PhysioZ. 204: 317-322, 1963. 5. BERNE, R. M., AND R. R. CURNISH. Adenosine formation in brain tissue (Abstract). Federation Proc, 31: 402, 1972, 6. BOCKMAN, E. L., R. M. BERNE, AND R. RUBIO. Adenosine and active hyperemia in dog skeletal muscle. Am. J. PhysioZ. 230: 15311537, 1976. 7. GROSS, R. T., AND R. E. HURWITZ. The pentose phosphate pathway in human erythrocytes. Relationship between age of the subject and enzyme activity. Pediatrics 22: 453-460, 1958. 8. KALCKAR, H. M. Differential spectroscopy of purine compounds by means of specific enzymes. J BioZ. Chem. 167: 429-459, 1947. 9, KATORI, M., AND R. M, BERNE. Release of adenosine from anoxic hearts. Circ. Res. 19: 420-425, 1966. 10. KIM, H. D., T. J. MCMANUS, AND G. BARTLETT. In: Erythrocytes, Thrombocytes, L/eukocytes, edited by E. Gerlach, K. Moser, E. Deutsch, and W. Wilmans. Stuttgart: George Thiene, 1972, p. 146148. 11. LACHHEIN, L., E. GRUBE, C. JOHNIGH, AND H. MATTHIES. Der Verbraught an Glucose, Galaktose, Ribose, und Inosine von Erwachsenene und Nabelschnur Erythocyten, Klin. Wochenschr, 39:
875, 1961. 12. LARIS, P, C. Permeability and utilization of glucose in mammalian erythrocytes, J. CeZZ. Camp. PhysioZ. 51: 273-307, 1958. 13. LERNER, M, H., AND B. A. LOWY. The formation of adenosine in rabbit liver and its possible role as a direct precursor of adenosine nucleotides. J. Biol. Chem. 249: 959-966, 1974. 14. LIU, M. S., AND H. FEINBERG, Incorporation of adenosine-8-14C and inosine-8-14C into rabbit heart adenine nucleotides. Am. J. PhysioZ. 220: 1242-1247, 1971. 15. LUTHRA, M. G., G. R. HILDENBRANDT, H. D, KIM, AND D. J. HANAHAN. Observations on the (Ca’+ + Mg”)-ATPase activator found in various mammahan erythrocytes. Biochim. Biophys. Acta 419: 180-186, 1976. 16. MILLS, G. C., F. C. SCHMALSTIEG, K. B. TRIMMER, A. S. GOLDMAN, AND R. M. GOLDBLUM, Purine metabolism in adenosine deaminase deficiency. Proc. NatZ. Acad. Sci. USA 73: 2867-2871, 1976. 17. MURPHY, J. R. Erythrocyte metabolism. II. Glucose metabolism and pathways. J. Lab, Clin. Med. 55: 286-302, 1960, 18. RAPOPORT, S. The regulation of glycolysis in mammalian erythrocytes. Essays Biochem. 4: 69-103, 1968. 19. RUBIO, R., R. M. BERNE, AND M. KATORI. Release of adenosine in reactive hyperemia of the dog heart. Am. J. Physiol. 216: 56-62, 1969. 20. SEIDER, M. J., AND H. D. KIM. Cow red blood cells. I. Effect of purines, pyrimidines, and nucleosides in bovine red cell glycolysis. Am. J. Physiol. 236: C255-C261, 1979 or Am. J. Physiol.: CeZZ physiol. 5: C255-C261, 1979. 21. ZEIDLER, R., AND H. D. KIM. Preferential hemolysis of postnatal calf red cells induced by internal alkalinization. J. Gen. Physiol. 70: 385-401, 1977.
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 11, 2019.
J. SEIDER AND HYUN uf Physiology, University
DJU
KIM
of Arizona,
SEIDER, MICHAEL J., AND HYUN DJU KIM. Cow red blood cells. II. Stimulation of bovine red cell glycolysis by plasma. Am. J. Physiol. 236(5): C262-C267,1979 or Am. J. Physiol.: Cell Physiol. 5(3): C262-C267, 1979.-Cow red cell glycolysis, which can be stimulated by a variety of purines and pyrimidines, was also found to be elevated by its own plasma. Dialyzed or charcoal-treated plasma could no longer stimulate glycolysis, suggesting that the stimulating factors may be purines or pyrimidines. Determination of purines or pyrimidines in plasma revealed the presence of xanthine (0.31 FM), hypoxanthine (0.60 PM), and adenosine (0.05 PM), as well as unknown compounds. A physiologic level of hypoxanthine, with or without xanthine and adenosine approximating their concentrations in plasma, resulted in the stimulation of cow red cell glycolytic rate by 16% (P < 0.01). These findings suggest that plasma-borne purines may act on cow red cells in concert with as yet unidentified factors. Moreover, exchanging calf and cow plasmas produced no stimulatory effect on either calf or cow red cell glycolysis, suggesting that a) calf red cells lack some of the cellular components that respond to this stimulator and, b) only cow plasma contains this specific stimulator. In other species, including dog, cat, rabbit, rat, guinea pig, and human, stimulation of glycolysis by plasma was not observed. purines; pyrimidines; chromatography
ion-exchange
chromatography;
thin-layer
EFFECTS ofcertainpurine andpyrimidine compounds on cow red cell glycolysis were described in the companion paper (20). The site of this enhancement could possibly be at the level of hexokinase, which can be stimulated by these agents. Inasmuch as the newborn calf red cells, endowed with more than threefold greater glycolytic capacity than cow cells, displayed no discernible reponse to these agents, this phenomenon represents a unique paradigm of postnatal adaptation of energy metabolism in cow cells. In the course of this investigation, we also found that glycolysis of the cow, but not the calf, red cells can be equally augmented by their own, but not alien, plasma kept at a pH 7.4. In this communication, results of experiments designed to determine the plasma-borne agents responsible for an enhancement of cow red cell glycolysis are presented.
THE STIMULATORY
MATERIALS
AND METHODS
Sources and preparation of blued and plasma. Blood was obtained from animaIs housed at the University of Arizona farm or the Arizona Health Sciences Center, as C262
Health
Sciences
Center,
Tucson, Arizona
85724
described in the companion paper (20). Red blood cells were spun down in a Sorvall GLC centrifuge at 4OC and the upper two-thirds of the plasma was saved. The white buffy coat, along with the remaining plasma, was aspirated and discarded. Red cells were thoroughly washed in isotonic NaCl solution prior to use. MetaboEic studies. Prior to addition of red blood cells, plasma was allowed to equilibrate with air for a minimum of 2 h at 37OC. For all studies, pH was checked every half-hour during incubation with a Radiometer waterjacketed blood electrode and maintained within 0.1 pH units of 7.4 with isotonic NaOH or HCl. Cell extraction procedures, as well as determination of glucose and lactate, were the same as described in the previous paper. Composition of balanced salt solution (BSS) is the same as described in the previous paper (20). Ion-exchange and thin-layer chromatography of nucleosides and purines in plasma. Plasma purines and nucleosides were determined with ion-exchange chromatography according to the methods of Bartlett (2). At least 400 ml of blood were obtained at the local slaughterhouse and rapidly chilled in small aliquots; the plasma was separated from cells by centrifugation. After deproteinating the plasma with 10% trichloracetic acid (TCA), the resultant supernatant was extracted four times with 4 volumes of ether to remove TCA. The extract was loaded onto a Dowex 50 x 8 resin (ZOO-400 mesh), 1 x 20 cm column. The column was eluted with 1,000 ml of linear gradient of O-4 N HCl. The column eluant was read at 260 nm, and those fractions showing appreciable absorbance were further analyzed by scanning over the entire untraviolet range (240-320 nm) at pH’s of 2, 7, and 12 in order to identify the eluted compound. The absorption profile was compared with those of known standards run in an identical manner. Plasma levels of nucleosides and purines were also measured by thin-layer chromatography as described by Akaoka, Nishizawa, and Nishida (1) or by Bockman, Berne, and Rubio (6). In the method described by Akaoka et al. (1), blood was rapidly cooled and centrifuged, and the plasma was separated from the erythrocytes. The plasma was deproteinized with 5% perchloric acid (PCA) and the supernatant neutralized with 5% KOH. After centrifugation to remove the precipitated salt, the supernatant was lyophilized to dryness, and the dry powder was resuspended in 0.6 ml distilled water. All the supernatant fluid was spotted on the bottom of a cellulose thin-layer chromatography (TLC) plate (Kodak), and the plates were developed in ascending fashion
0363”6143/79/0000-UOOO$O1.25
Copyright
0 1979 the American
Physiological
Society
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PLASMA
STIMULATION
OF
C263
GLYCOLYSIS
in a closed tank with a solvent consisting of 95% ethanol and 1 M ammonium acetate, pH 7.5 (75:30 vol/vol). The elution position of standards of hypoxanthine, xanthine, and uric acid were identified by visualization under ultraviolet light. In the method described by Bockman et al. (6), at least 100 ml blood were collected in heparinized tubes and then immediately poured into equal volumes of ice-cold saline. This mixture was spun down at 1,000 g within 5 min, and the plasma-saline mixture was separated from the red cells. The plasma proteins were precipitated with enough 5 N PCA to give a final concentration of 0.5 N PCA. The samples were then centrifuged at 14,000 g in an International refrigerated centrifuge (model B-20) and the supernatant fractions were neutralized with 5.63 N K&03. The resulting precipitate was spun down, and the neutralized supernatant was saved. Adenosine, inosine, hypoxanthine, and xanthine in this neutralized extract were adsorbed onto Norit charcoal (5 mg for each ml of plasma), After a brief centrifugation, the supernatant was discarded and the charcoal was then eluted with two successive 15-ml volumes of 10% pyridine in 50% aqueous ethanol. The eluants were air dried, suspended in 0.4 ml water, and spotted onto TLC plates. The plates were developed in 15% ethanol at 4”C, with the necessary standards spotted on one side. In both methods, the plasma compounds were identified under ultraviolet light by their position on the TLC plate, which was compared with standards. To quantitate the amount of compounds in the plasma, areas on the plates corresponding to the known standards were scraped off and eluted with three successive lo-ml volumes of 50% ethanol. The eluants were air dried and resuspended in water. The nucleosides and bases were assayed by conversion to uric acid according to the method of Kalckar (8). Standards were run to test for recovery in all experiments, and recoveries ranged from 80-90%, similar to previous reports (1, 6). In all cases, no other noticeable UV-absorbing material was found on TLC plates spotted with various standards. Sources of materials. All materials were obtained from the same suppliers as in the companion paper. Purity of nucleosides and purines was examined using both thinlayer and column chromatographic methods described above. We found no UV-absorbing impurities with any of these methods. RESULTS
Rapoport (18) reported that mammalian red cells suspended in a balanced salt solution (BSS) decreased the medium pH by 0.2 to 0.5 pH units per hour at 38°C. When calf or cow red cells were suspended in BSS containing glucose, the pH slowly decreased as a function of time, due to the increasing concentration of lactic acid produced as an end-product of anaerobic metabolism (Fig. 1). This decrease was about 0.5 and 0.3 pH units per hour in calf and cow red cells, respectively, with the most rapid decline in calf cells, due to their higher glycolytic rate. In contrast, when washed calf or cow red cells were added to their own plasma, the plasma pH rose to 8.0 for calf red cells, and 8.2 for cow cells (Fig. l), presumably due to the equilibration of plasma CO2 with
cow plasm
cow BSS cdf BSS
FIG. 1. Comparison of the pH change in plasma solution (BSS) containing calf or cow red cells. Red either to balanced salt solution or to plasma fortified glucose to give a hematocrit of 1045%. Calf cells in (a-----+); calf cells in balanced salt solution (A-A); own plasma (o------O); cow cells in balanced salt solution 4 h incubation in calf plasma, the calf cells began to alent results were seen in 2 other experiments.
vs. balanced salt cells were ad .ded with additional their own plasma cow cells in their (A-A). After hemolyze. Equiv-
air as reported in other cells (3). The calf plasma pH rose no further, probably due to extensive hemolysis that occurred by this time. Zeidler and Kim (21) have shown that elevation of the internal pH of calf red cells, but not cow cells, produces rapid hemolysis. Figure 2 shows the effect of pH on cow red cell glycolysis. The desired pH was maintained within 0.1 pH units by titrating with isotonic acid or base during a 7-h incubation. Results are expressed as percent of control at pH 7.4, and numerical values for initial rates at pH 7.4 are given in Table 1. At pH 8.2, the glucose consumption rate is nearly 55% greater than at 7.4, but this elevation is less than in several other species, including rats and humans, which increase their glycolytic rate 82% and 164%, respectively (18). Table 1 shows the effects of plasma on calf and cow red cell glycolysis. Although calf and cow plasma have a glucose concentration between 1 and 3.5 mM, additional glucose was added to equalize plasma and BSS glucose concentrations. As in other species (7, 10, II), red cells obtained from newborn calf had a much higher glycolytic rate than did cow red cells (Table 1). In contrast to calf red cells, which displayed identical glycolytic rates in both BSS or plasma, cow red cells significantly (P < 0.02) increased their glycolytic rate nearly 50% when suspended in their own plasma. To discover the chemical composition of the plasma activator, approximately 20 ml of cow plasma was dialyzed in 15 in. of no, 8 tubing for 4 and 24 h against 3 liters of isotonic, buffered BSS at 4°C and the dialyzed plasma was fortified with 5 mM glucose to replace lost glucose. Figure 3 shows the effect of dialyzed plasma on glycolysis. The levels of lactate in undialyzed cow plasma were quite variable from animal to animal and exceeded the intracellular lactate concentration. Thus, for the purpose of comparison, lactate levels were normalized against initial lactate levels. Figure 3 shows that plasma
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C264
M.
80
80
60
60 z F 40 2
t
-60 616
7.-O
7: 4
7:8
a2
6:6
f0
7:4
718
ti
PH
PH FIG. 2. Effect of pH on cow red cell glycolysis. E1xperimental conditions are the same as in Fig. 1. Similar results were seen in 2 other experiments. TABLE
1. Calf and cow red cell glucose
consumption and lactate formation in plasma us. BSS Calf HSS
Glucose consumption Lactate formation Ratio: lactate/ glucose
2.05 -+ 0.49 (61 3.28 k 0.70 (6) 1.60
COW
PlZiSfIM
BSS
pm0 I/ (ml cells . h) 2.17 k 0.51 0.60 k 0.04 6) 03 3.39 k 0.51 0.87 t 0.05 03 (6) 1.56 1.45
PhIMa
J. SEIDER
AND
H. 11. KIM
To determine the plasma levels of purines and nucleosides, both ion-exchange and thin-layer chromatography procedures were employed. With the Bartlett method (2), cow plasma elution profiles showed peaks corresponding to xanthine and hypoxanthine and two unknown peaks that had ultraviolet absorption patterns exhibiting a maxima of 251.5 nm, but did not have any characteristics of known purines or nucleosides (Fig. 4). Adenosine was found in very low concentrations with this method. TLC procedures of Akaoka et al. (1) allowed us to quantitate the amount of purines on smaller quantities of calf, cow, and human plasma. Human plasma was found to have concentrations of uric acid, xanthine, and hypoxanthine that were higher than either calf or cow plasma (data not shown). As in the case of ionexchange chromatography, TLC of cow plasma showed a rapidly migrating ultraviolet-absorbing peak that was absent in both calf and human plasmas. In dialyzed plasma, all of the ultraviolet-absorbing areas were lost, implying that the rapidly migrating compound was not an artifact of the TLC preparative method. When compared with either human plasma (16) or dog plasma (19), the concentration of purines and nucleosides in cow and calf plasma were extremely low (Table 4). Data in Table 5 summarizes the effect of hypoxanthine approximating the plasma concentration on cow red cell Plasma + J Hypoxanthine
0.89 t 0.06* 03 1.32 t 0.11”
PI osma \ Dialyzed Plastrtu + H ypoxanthine
03 1.55
Dialyzed /Plasma
Values are means k SE, with number of experiments in parentheses. Balanced salt solution (BSS): 5 mM KCl, 150 mM NaCl, 10 mM Na phusphate buffer, pH 7.4, and 5 mM glucose. Hematocrit, 10-15s; temperature 37 OC. * P < 0.02 comparing cow BSS to cow plasma.
elevated the lactate formation rate when compared with BSS, but that plasma dialyzed for 24 h lost its stimulatory capability. The 4-h-dialyzed plasma (not shown) gave rates that were intermediate to the two extremes shown in Fig. 3. The results implied that the stimulatory agent was a small molecule. If the stimulating factor(s) in plasma were purines, then elevated glycolytic rates should be restored by adding this in vitro stimulator to dialyzed plasma. Addition of 1 mM hypoxanthine to dialyzed plasma restored the glycolytic rate to that found in untreated plasma (Fig. 3). Further elevation of the glycolytic rate was not seen when hypoxanthine was added to nondialyzed plasma, suggesting that plasma stimulates red cell glycolysis maximally. Several methods were used in attempting to identify and isolate the plasma stimulator(s). Charcoal-treated plasma failed to stimulate cow red cell glycolysis (Table 2). Plasma, boiled for 30 min to denature the proteins, was tested for its stimulatory effect. Boiled plasma had a lower lactate formation rate than t.hat seen in normal plasma, but still elevated gycolysis 33% over a BSS control (Table 3). The reason for the drop from the plasma control may have been heat destruction or loss of activator in the protein precipitate.
bSS
0/
234567 Hours FIG. 3. Effect of plasma on cow cell glycolysis. Plasma was dialyzed for 24 h against buffered balanced salt solution. After dialysis, 5 mM glucose or I mM hypoxanthine was added, depending upon experiment. In 24-h dialysis experiment, fresh blood was drawn from same animal that provided plasma the day earlier. Dialyzed plasma (A-A); undialyzed plasma (c-1); balanced salt solution (PF----A); dialyzed plasma + I mM hypoxanthine (m); undialyzed plasma + 1 mM hypoxanthine { nj. Equivalent results were seen in 4 other experiments. 0
I
TABLE 2. Effect of charcoal-extracted on cow red cell glycolysis Lactate
_-
BSS control Plasma control Charcoal-extracted
plasma
plasma kite, pmol/(ml cells-h)
0.79 1.21 0.79
53 0
Typical results from I of 3 experiments is shown. Norit charcoal, 5 mg, was added to each ml plasma; the mixture was stirred for 3 h at 4°C and the charcoal was spun out. The plasma supernatant was glucose fortified, and then washed red cells were added to make a final hematocrit of 1045%. Controls consisted of red cells suspended in glucose-fortified BSS, or untreated plasma. Rates were calculated using least-squares regression analysis, with correlation coefficient r > 0.95 in all cases. RSS, balanced salt solution.
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PLASMA
STIMULATION
OF
C265
GLYCOLYSIS
3. Effect of boiled plasma cow red cell glycolysis
on
TABLE
Lactate
Procedure
BSS control Plasma control Boiled plasma
Rate, pmol/( cells. h)
ml
%
0.66 0.99
50
0.88
Cow plasma was boiled removed by centrifugation,
33
for 30 min and the resulting BSS, balanced salt solution.
precipitate
was
cells can be “turned on” only by their own plasma. Plasma other than cow and calf hemolyzed cow red cells within minutes. When cat, rat, guinea pig, and dog red cells were added to cow plasma, they rapidly hemolyzed within 10 min. When compared to cow plasma, calf plasma was not effective in elevating cow red celI glycolysis, implying that calf plasma lacked all the cow plasma stimulator(s). When calf red cells were suspended in cow plasma, their glycolytic rate was unaffected, implying that calf cells also lacked the internal machinery necessary for producing stimulation of glycolysis. To determine whether in vitro nucleosides and purinestimulated glycolysis were unique to cow red cells, several other animal cells were also tested with adenine. Figure 7 summarized the results. It is apparent that adenine is not as effective in other species as it is in cow red cells. DISCUSSION
In several early RBC glycolytic studies utilizing plasma, the elevated glycolytic rate appeared to be accounted for by the increased plasma pH (3,lZ). In strictly pH-controlled plasma, glycolytic rates of human red cells were the same as cells suspended in balanced salt solution
.l
I
I
T
I
1
.2
.4
.6
.8
1.0
Liters
4. Levels of nucleosides in animal plasma
TABLE
Xanthine
cow Dog"
Human
(4)
and bases ----~--~
Hypoxanthine
pmoL/ml Calf (6)
0.40 0.31
* 0.07 k 0.06
3.16
t
1.45+
0.60 0.55 0,44 3.60
t t Al k
0.07 0.05 0.12 1.9Ot
5. Effect of 1 pM hypoxanthine cow red cell glycolysis -.
on
TABLE
4. Ion-exchange chromatography of cow plasma. 100 ml of cow plasma were extracted with trichloroacetic acid (TCA), which in turn was removed by ether. Resulting extract was loaded onto a Dowex 50 x 8 resin-filed column (1 x 20 cm), Purines were eluted from column using 1,000 ml of a linear gradient of O-4 N HCl. Absorbance at 260 nm is plotted against liters through the column. Each compound was identified on the basis of its elution position and its absorption profile in three different pH’s over the entire ultraviolet range (200-320 nm). HX, hypoxanthine; X, xanthine. Results from 1 of 6 experiments, FIG.
Inosine
Adenosine
plasma 0.14
* 0*04 ND 0.12 f 0.03
0.09 0.05 0.11 0.31
* * t t
Values are means & SE, with number of animals in parentheses. not detected. * From Bockman et al. (6). t From Akaoka $ From Mills et al. (16). (1).
0.01 0.02 0.04 0.29$
ND, et al.
glycolysis. There was a small but significant 16% (P < 0.02) elevation of glycolysis, and addition of xanthine or adenosine at concentrations found in plasma produced no further increase in the glycolytic rate. These findings showed that physiologic levels of purines may stimulate cow red cell glycolysis to some extent. When dog, cat, rabbit, rat, guinea pig, and human red cells were suspended in either glucose-fortified balanced salt solution or plasma, the rates of glycolysis were the same, suggesting that other animal plasmas lacked stimulators of glycolysis (Fig. 5). On the other hand, stimulators may have been present in all plasmas, but the cells were not responsive to these compounds. To test this possibility, cross-plasma experiments were performed, with the results shown in Fig. 6. Apparently, cow red
Lactate
Formation,
pmol/(ml
Cow No. Glucose
1
Glucose
alone
0.52
Cow cells were pH 7.4, at 37°C. cat
t
% Difference
+ 1 pM hypoxanthine
0.6 0.65 0.50 0.57 0.55 0.34 0.50 0.55 0.4 0.57 0.49
2 3 4 5 6 7 8 9 10 11
------ - ---
cells. h)
0.84 0.67 0.52 0.59 0.79 0.41 0.56 0.63 0.46 0.67 0.56 0.03
suspended * Using ml
0.61
k 0.04
at Hct 15-20% paired t test. rabbit
36.8 4.7 2.6 4.6 45.2 21.1 12.5 14.7 7.0 17.1 15.4 16.5 k 04.09 P < 0.01*
in balanced
guinea
rat
salt solution,
human
Pi&l
Bss
PL
E3ss PL
Bss
PL
5. Comparison of glycolysis fied balanced salt solution (BSS) animals. FIG.
Bss
PL
Bs
PL
Rss
PL
of red blood cells in glucose-fortivs. plasma (PL) from a variety
of
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(2266
M.
2.6
- 2.6
-
i cow
ii cat
rat
cow
cow
guinea Pig cow
MiI rabbit
human
cow
cow
cow
calf
**’ 2 P 00 3 sg cm 3g p: ’ 1.0 $2 qg 3 -
/ calf calf
6. Calf and cow red cell glycolysis in plasma obtained from several different species. Rapid hemolysis of red cells occurred when dog, cat, rat, rabbit, or guinea pig red cells were suspended in cow plasma. FIG.
dog
rabbit
tat
sheep
human
6 5k
z
0
4s
c, 3F
3s
k
-
g
v, zx
z
II o-
G G+A G G+A G G+A G G+A 7. Effect of adenine on glycolysis of red blood cells obtained from a variety of animals. G, glucose-fortified balanced salt solution (BSS); G + A, BSS with I mM adenine + glucose. FIG.
(17). In contrast, cow red cell glycolysis was found to be stimulated by its own plasma (Table 1). This stimulatory effect of plasma on cow cells was somewhat unusual, inasmuch as other species including the dog, cat, rabbit, rat, guinea pig, and human did not respond to their own plasma. Dialysis of cow red cells for 6 h against balanced salt solution still produced the same stimulation upon exposure to plasma, suggesting that no unusual intracellular factors were required. In view of the findings reported in the companion paper, in which cow red cell glycolysis was shown to be augmented by a variety of purines and nucleosides, it seemed reasonable to suspect those purines and nucleosides in the plasma as agents responsible for the plasma stimulation. Indeed, dialysis or charcoal treatment of plasma resulted in the inactivation of this stimulation. However, actual quantitation of the purines and nucleosides in the plasma revealed that these compounds were present in l/loo the amount needed for maximal stimulation of cow red cell glycolysis in vitro. Unidentified compounds were found by both TLC and liquid-chromatographic methods in cow, but not in calf or human plasmas. Isolation of these compounds was attempted to determine whether they were responsible for stimulation of cow red cell glycolysis. The highly acidic eluant from the first peak of the column was neutralized with a strong base, diluted to isotonic strength, and then added to incubating cow red cells.
J. SEIDER
AND
H. D. KIM
This peak failed to stimulate the red cell glycolytic rate. However, the possibility for acid inactivation of this compound cannot be ruled out. The second unknown peak was not tested due to excessive acidity. In yet another attempt, the rapidly migrating UV-absorbing spot on the TLC plate was scraped off, extracted, and added to BSS. It was found that cow red cells responded to a small extent to this extract, but control extracts from unspotted, developed TLC plates also elevated cow red. cell glycolysis, thereby obscuring our initial results. It has been shown that nucleosides and purines formed in tissues, including the heart (9), brain (5), and liver (13), may be transported by red cells to other regions of the body (13). Adenosine, which is a potent vasodilator, has been found in the coronary venous blood as well (4) and is released into the circulation during hypoxia (4, 14). If adenosine and other purine bases indeed participate in the stimulatory effect in vivo, one potential metabolic benefit could be the augmented production of ATP by stimulation of glycolysis. It should be recalled that cow red cells undergo an unusually rapid breakdown of ATP when incubated without metabolizable substrate. Factors responsible for this rapid breakdown in cells with a low Naf-K’and Ca2+-ATPase activity are not known (15)
Calf red cells, which have higher glycolytic rates, did not respond to exogenously added purines or their own plasma. Both the glycolytic rate and intracellular ATP levels fall rapidly in the growing animal. It is not known when the need for the plasma stimulator arises during this neonatal period. The exchange of plasmas from calf and cow failed to markedly stimulate glycolysis in either of these cells, suggesting that a) calf red cells lacked the internal machinery necessary for reacting to the plasma stimulator and b) calf plasma lacked the plasma stimulator. Other ruminants have low rates of glycolysis and intracellular ATP concentrations. It is presently unknown whether all ruminants have this particular type of glycoly-tic stimulation, although sheep are known to be unaffected by exogenously added purines ( Fig. 6). Our data show that plasma concentrations of purines and nucleosides in BSS cannot produce the same magnitude of stimulation that plasma itself can. This implies that compounds in plasma other than purines also act to stimulate cow red cell glycolysis. The expert technical assistance of Mr. Paul Cook and Mr. Y. S, Bak is gratefully acknowledged. We thank Mr. Robert Schonberg at the University of Arizona experimental farm for providing blood samples used in this study. Special thanks are due to Dr. R. Zeidler for his stimulating discussions. This work was supported in part by National Institutes of Health Grant AM-17723 and Training Grants HL-05884 and HL-07249. A part of this work represents partial fulfillment of the requirements for the PhD by Michiel Seider, University of Arizona, 1977. H. D. Kim is the recipient of a National Institutes of Health Research Career Development Award AM-00316. A preliminary report was presented at the Federation meetings, Chicago, IL, April 1977. Present address of M. J. Seider is Dept, of Physiology, The University of Texas Medical School, Health Science Center, Houston, TX 77025.
Received
28 Nov
1977; accepted
in final
form
12 Dee 1978.
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PLASMA REFERENCES
STIMULATION
OF
GLYCOLYSIS
C267
,
1. AKAOKA, I., T. NICHIZAWA, AND Y. NISHIDA. Determination of hypoxanthine and xanthine in plasma separated by thin-layer chromatography. B&hem. Med. 14: 285-289, 1975. 2. BARTLETT, G. R. Adenine: chemistry, analytical methods, sources, purity, specification. Transfusion 17: 333-338, 1977. 3. BARLETT, G. R., AND A. A. MARLOW, Comparative effects of the different formed elements on normal human blood glycolysis. J. AppZ. Physiol. 6: 335-347, 1953. 4. BERNE, R. M. Cardiac nucleotides in hypoxia: possible role in regulation of coronary blood flow. Am. J, PhysioZ. 204: 317-322, 1963. 5. BERNE, R. M., AND R. R. CURNISH. Adenosine formation in brain tissue (Abstract). Federation Proc, 31: 402, 1972, 6. BOCKMAN, E. L., R. M. BERNE, AND R. RUBIO. Adenosine and active hyperemia in dog skeletal muscle. Am. J. PhysioZ. 230: 15311537, 1976. 7. GROSS, R. T., AND R. E. HURWITZ. The pentose phosphate pathway in human erythrocytes. Relationship between age of the subject and enzyme activity. Pediatrics 22: 453-460, 1958. 8. KALCKAR, H. M. Differential spectroscopy of purine compounds by means of specific enzymes. J BioZ. Chem. 167: 429-459, 1947. 9, KATORI, M., AND R. M, BERNE. Release of adenosine from anoxic hearts. Circ. Res. 19: 420-425, 1966. 10. KIM, H. D., T. J. MCMANUS, AND G. BARTLETT. In: Erythrocytes, Thrombocytes, L/eukocytes, edited by E. Gerlach, K. Moser, E. Deutsch, and W. Wilmans. Stuttgart: George Thiene, 1972, p. 146148. 11. LACHHEIN, L., E. GRUBE, C. JOHNIGH, AND H. MATTHIES. Der Verbraught an Glucose, Galaktose, Ribose, und Inosine von Erwachsenene und Nabelschnur Erythocyten, Klin. Wochenschr, 39:
875, 1961. 12. LARIS, P, C. Permeability and utilization of glucose in mammalian erythrocytes, J. CeZZ. Camp. PhysioZ. 51: 273-307, 1958. 13. LERNER, M, H., AND B. A. LOWY. The formation of adenosine in rabbit liver and its possible role as a direct precursor of adenosine nucleotides. J. Biol. Chem. 249: 959-966, 1974. 14. LIU, M. S., AND H. FEINBERG, Incorporation of adenosine-8-14C and inosine-8-14C into rabbit heart adenine nucleotides. Am. J. PhysioZ. 220: 1242-1247, 1971. 15. LUTHRA, M. G., G. R. HILDENBRANDT, H. D, KIM, AND D. J. HANAHAN. Observations on the (Ca’+ + Mg”)-ATPase activator found in various mammahan erythrocytes. Biochim. Biophys. Acta 419: 180-186, 1976. 16. MILLS, G. C., F. C. SCHMALSTIEG, K. B. TRIMMER, A. S. GOLDMAN, AND R. M. GOLDBLUM, Purine metabolism in adenosine deaminase deficiency. Proc. NatZ. Acad. Sci. USA 73: 2867-2871, 1976. 17. MURPHY, J. R. Erythrocyte metabolism. II. Glucose metabolism and pathways. J. Lab, Clin. Med. 55: 286-302, 1960, 18. RAPOPORT, S. The regulation of glycolysis in mammalian erythrocytes. Essays Biochem. 4: 69-103, 1968. 19. RUBIO, R., R. M. BERNE, AND M. KATORI. Release of adenosine in reactive hyperemia of the dog heart. Am. J. Physiol. 216: 56-62, 1969. 20. SEIDER, M. J., AND H. D. KIM. Cow red blood cells. I. Effect of purines, pyrimidines, and nucleosides in bovine red cell glycolysis. Am. J. Physiol. 236: C255-C261, 1979 or Am. J. Physiol.: CeZZ physiol. 5: C255-C261, 1979. 21. ZEIDLER, R., AND H. D. KIM. Preferential hemolysis of postnatal calf red cells induced by internal alkalinization. J. Gen. Physiol. 70: 385-401, 1977.
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