Influence of cyclic 3′, 5′-AMP on glycolysis in human erythrocytes

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BBA Report BBA 21344 I n f l u e n c e o f cyclic 3 ' , 5 ' - A M P o n glycolysis in h u m a n e r y t h r o c y t e s

DAVID L. FORD and AKIRA OMACHI Department of Physiology., University o f lllinois at the Medical Center, Chicago, Ill. 60612 (U.S.A.)

(Received August 29th, 1972)

SUMMARY Small increases in lactate production and in glucose utilization were observed when human red cells were incubated in Tris-Ringer's medium with 5 mM adenosine 3',5'-monophosphate (cyclic 3',5'-AMP) or its dibutyryl analogue. 5 mM caffeine facilitated this response whereas 5'-AMP, cyclic 2',3'-AMP, and guanosine 3',5'-AMP had no effect.

The importance of adenosine 3' ,5'-monophosphate (cyclic 3',5'-AMP) in mediating hormonal effects in a wide variety of different tissues has been amply demonstrated I . It was thought at one time that mammalian erythrocytes might be an exception to the general rule 2, but recent studies have shown that the cyclic nucleotide can be produced in five species of mammalian ghosts 3. The possible effects of this agent on erythrocyte physiology, however, remain unknown. The present study was conducted in order to test whether a change in erythrocyte glycolysis might be produced when cyclic 3',5'-AMP is added to a suspension of human erythrocytes incubated in vitro. Blood from young adult males was collected in acid-citrate-dextrose medium (N.I.H. Formula B) or in citrate-phosphate-dextrose medium 4. Samples were removed after different periods of storage up to 16 days and centrifuged for 5 rain at 800 × g and 5 °C. The plasma, buffy coat and top layer of erythrocytes were removed. The erythrocytes were washed with an equal volume of a salt medium containing, in mM: NaC1, 120; KC1, 5.6; MgC12, 2; Na2HPO4, 1 ; Tris, 24; and glucose, 10. The pH of this solution was 7.40 at 37 °C. After two additional washes, the cells were washed a final time with the same medium except that it now contained 2 mM CaC12 (Tris-Ringer's medium). 1 ml of washed erythrocytes was added to 2 ml of Tris-Ringer's medium which gave a hematocrit of 25%, and the suspension was incubated for 2.5 h at 37 °C in a Dubnoff shaker cycled Biochim. Biophys. Acta, 279 (1972) 587-592

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at 100/min. No notable differences in pH or in hematocrit were recorded between control and experimental flasks in any of the cases described below. The mean leukocyte and platelet levels were 0.016% and 0.13%, respectively, of the erythrocyte count which averaged 2.8- 106 cells/mm 3 in the final suspension (N = 7). Perchloric acid extracts were prepared in duplicate from aliquots of the erythrocyte suspension and lactic acid was analyzed with lactate dehydrogenase s. Glucose was determined with glucose oxidase 6 in zinc hydroxide extracts of the whole suspension. The various agents used in this study had no effect on these enzymatic assay procedures. The different nucleotides used in this study as well as caffeine were obtained from Sigma Chemical Company. In preliminary experiments, lactate production was found to be linear up to 2.5 h, both in the presence and absence of 5 mM cyclic 3',5'-AMP. Lactate production was therefore calculated routinely from values measured at time zero and at 2.5 h. Similar changes were elicited by cyclic 3',5'-AMP in erythrocytes that had been stored for 7 - 1 4 days as in cells stored for 0 - 2 days ( N = 7). Cells of any storage age up to 16 days were therefore assumed to be the same for the purposes of this study. Erythrocytes incubated with 5 mM cyclic AMP produced approximately 15% more lactate than control erythrocytes (Tables I, II and III). With a 10-mM concentration, lactate production was 35% greater than the control ( N = 6) whereas no significant change was recorded with a 1-mM concentration (N = 9). As this nucleotide is generally regarded not to be freely permeable, it has been estimated that exogenous levels that are 10 000 times the intracellular level may be required to produce an effect 7. The differences noted between control and experimental series in this study were generally small, but the use of the paired t-test of Student which is recommended with paired observations 8 indicated that the differences were statistically significant. Since ptlosphodiesterase which degrades cyclic 3',5'-AMP to 5'-AMP has been reported to be present in human erythrocytes 9, it was thought that the inhibition of this TABLE I INCREASE IN LACTATE PRODUCTION IN THE PRESENCE OF CYCLIC 3',5'-AMP, CAFFEINE AND BOTH AGENTS Means -+S.E. from 7 experiments are shown. The figures in parentheses refer to % change from control. All added agents were present at 5 mM final concentration. The paired t-test was used to evaluate statistical significance. *P < 0.05; **P < 0.01; ***P < 0.001. Agent added

Non~ Cyclic 3',5'-AMP (A) Caffeine (B) Cyclic 3',5'-AMP + caffeine (C)

Lactate production (#moles/ml of cells per h) Observed rate

Difference from control

2.72 +-0.28 3.09 -+0.31 3.12 -+0.32 3.68 -+0.37

0.37 -+0.06** (+ 14) 0.40 -+0.06*** (+ 15) 0.96 -+0.11"** (+ 35)

Biochim. Biophys. Acta, 279 (1972) 587-592

C-{A + B)

0.19 -+0.07* (+ 7)

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T A B L E II INFLUENCE OF VARIOUS ANALOGUES OF CYCLIC 3',5'-AMP ON LACTATE PRODUCTION IN THE PRESENCE AND ABSENCE OF 5 mM CAFFEINE Means ± S.E. are shown. The figures in parentheses refer to % change from control. All added agents were present at 5 mM final concentration. The paired t-test was used to evaluate statistical significance. The caffeine effect was statiStically significant (P < 0.05) in all of the comparisons in Expt A but not in Expt B, presumably due to the smaller number of experiments. *P < 0.05; ***P < 0.001.

Nucleotide added

Lactate production #tmoles/ml o f cells per h) Without caffeine

With caffeine

A. None 5'-AMP Cyclic 3',5'-AMP

2.67 t 0.28 2.76 ± 0.35 (+ 3) 3.13 +- 0.34*** (+ 17)

2.87 +- 0.33 2.97 -+ 0.37 (+ 3) 3.57 -+ 0.37*** (+ 24)

8 8 8

B. None Cyclic 2',3'-AMP Dibutyryl cyclic 3',5'-AMP

2.19 ± 0.48 2.30 ± 0.55 3.00 ± 0.64*

2.30 -+ 0.47 2.41 -+ 0.57 3.32 -+ 0.74*

4 4 4

(+ 6) (+ 37)

No. of expts

(+ 5) (+ 44)

T A B L E III GLUCOSE UTILIZATION AND THE RESPONSE OF WASHED ERYTHROCYTES TO CYCLIC 3',5'-AMP FOLLOWING REDUCTION OF LEUKOCYTE COUNT Means ± S.E. from 4 experiments are shown. The figures in parentheses refer to % change from control. Blood stored in citrate-phosphate-dextrose medium for less than 6 days was used. Erythrocytes passed through cotton I s appeared better oxygenated and the suspension pH was more alkaline by 0.1 pH unit. The paired t-test was used to evaluate statistical significance. *P < 0,05; **P < 0.01. Lactate production with dibutyryl cyclic 3',5'-AMP was significantly greater than with cyclic 3',5'AMP in both Series I (P < 0.001) and II (P < 0.05). Glucose utilization with dibutyryl 3',5'-AMP was not significantly different from the control in both Series I (P < 0.08) and II (P < 0.07); however, P was less than 0.05 when the number of comparisons was increased to six.

II) Regular procedure

(II) Preliminary passage through cotton

Leukocyte count, per mm 3 of suspension

4 5 2 ± 78

Lactate production (vmoles/ml of ceils per h) Control 5 mM cyclic 3',5'-AMP 5 mM dibutyryl cyclic 3',5 '-AMP

3.65 ± 0.31 4.27 -+ 0.25* (+ 19) 4.84 +_0.27 (+ 35)**

3.85 ± 0.24 4.67 + 0.07* (+ 15) 5.10 + 0.44* (+ 32)

Glucose utilization (umoles/ml of cells per h) Control 5 mM cyclic 3',5'-AMP 5 mM dibutyryl cyclic 3',5'-AMP

1.45 ± 0.10 2.05 -+ 0.14" (+ 43) 1.95 ± 0.23 (+ 36)

1.89 ± 0.16 2.42 + 0.31" (+ 28) 2.39 ± 0.32 (+ 25)

41 +

18

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enzyme might increase the cyclic 3',5'-AMP effect. Methylxanthines, such as caffeine, are generally regarded to be potent inhibitors of this enzyme 1°, and a 5-mM concentration of caffeine has been shown to sustain high intracellular cyclic AMP levels in suspensions of epinephrine-stimulated pigeon erythrocytes2. In our experiments, caffeine alone produced a significant increase when at least seven experiments were conducted (Tables I and IIA). In addition, cyclic 3',5'-AMP caused greater lactate output when 5 mM caffeine was also present (Tables I and IIA). In fact, the combined influence of both agents could be greater than the sum of the individual responses (Table I). These results indicate that caffeine may exert a protective action against the degradation of cyclic 3',5'-AMP in intact human erythrocytes. Dibutyryl cyclic 3',5'-AMP is believed to penetrate cell membranes more readily and to be broken down less easily than cyclic 3',5'-AMP 11 so that a greater effect is generally observed with this analogue. In Tables liB and III, lactate production was approximately twice as great in the presence of this agent as with cyclic 3',5'-AMP. Our results appear, therefore, to be in accord with previous observations. On the other hand, 5'-AMP caused no change in lactate production even in the presence of caffeine (Table IIA). Since this nucleotide is the product of cyclic 3',5'-AMP degradation by phosphodiesterase, it would appear that the action of the cyclic nucleotide is not mediated through formation of this compound. It is possible, however, that 5'-AMP does not penetrate the cell membrane because a stimulatory effect on lactate production might have been expected had this compound entered the cell and affected certain enzymes such as phosphofructokinase 12°a. With cyclic 2',3'-AMP, which differs from cyclic 3',5'-phosphate only in one phosphate-ribose linkage, the results were also negative (Table liB). Finally, in three experiments with guanosine 3',5'-AMP, no change in lactate production was detected. These results support the view that the action of cyclic 3',5'-AMP is a specific effect that cannot be duplicated by several other structurally similar nucleotides. In a final group of experiments, glucose utilization was found to be increased by 5 mM cyclic 3',5'-AMP and the percent change was twice that seen for lactate production (Table III). With dibutyryl cyclic 3',5'-AMP, glucose utilization was raised to the same extent but lactate production was greater than with cyclic 3',5'-AMP. These effects may be explained by the relatively higher internal cyclic 3',5'-AMP concentration that could be attained when dibutyryl cyclic 3',5'-AMP is used, due to its greater penetrability. Thus, it seems possible that glucose utilization may be stimulated to a greater extent than lactate production at one cyclic 3',5'-AMP concentration whereas, at a higher internal concentration (i.e. with dibutyryl cyclic 3',5'-AMP), lactate production may be activated to a greater extent, approaching the percent change in glucose utilization. These results suggest, therefore, that cyclic 3',5'-AMP could be acting at two separate sites which differ in their responsiveness to this agent. The glucose data appear also to rule out an alternate explanation for the cyclic 3',5'-AMP effect, viz. that the increased lactate production may be due simply to the cyclic nucleotide serving as an added substrate for glycolysis. Substantial lactate production could result if adenosine, for example, were formed by the removal of a Biochim. Biophys. Acta, 279 (1972) 587-592

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phosphate group from cyclic 3',5'-AMP by the successive action of phosphodiesterase and phosphatase. Under these conditions, however, glucose utilization would tend to be depressed due to the sparing action of an alternate substrate 14 rather than to be accelerated. Included in the final set of experiments was a series in which leukocyte count was reduced to low values by preliminary filtering of the blood through cotton ~s before washing the cells (Table III). The changes seen following cyclic 3',5'-AMP addition were essentially the same in erythrocyte suspensions with different leukocyte concentrations (compare Columns I and II). Thus, the presence of leukocytes did not appear to complicate our findings. Platelets also do not seem to be significantly involved because lactate production by these elements is negligible compared to lactate formation by leukocytes 16. In summary, addition of cyclic 3',5'-AMP to suspensions of intact human erythrocytes resulted in small but significant increases in glycolysis. This is believed to be a specific effect because (1) a similar response was not elicited by several other structurally related nucleotides, (2) a greater response was produced by dibutyryl cyclic 3',5'-AMP, and (3) the effect was augmented by caffeine. Stimulation of glycolytic enzymes appears to be a reasonable explanation for this effect since certain of these enzymes have been reported to be activated by cyclic 3',5'-AMP in other systems. For example, phosphofructokinase in heart muscle ~2 and in primate sperm ~3 have been reported to be stimulated by this agent. Also, the activities of glyceraldehyde-3-phosphate dehydrogenase in yeast 17 and pyruvate kinase in loach embryos ~8 are augmented by this cyclic nucleotide. On the other hand, an action on phosphorylase seems doubtful since the human red cell ordinarily contains little glycogen~9. It appears that further studies on the metabolic localization of cyclic 3',5'-AMP action as well as on the identities of the primary messengers can be effectively pursued with human erythrocyte suspensions in vitro because of their metabolic simplicity. This investigation was supported in part by U.S. Public Health Service Grants GM-738 and HL-13567.

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