Effects of adenosine deaminase inhibition on active uptake and metabolism of adenosine in astrocytes in primary cultures

168

Brain Research, 515 (1990) 168-172 Elsevier

BRES 15415

Effects of adenosine deaminase inhibition on active uptake and metabolism of adenosine in astrocytes in primary cultures H. Matz and L. Hertz Department of Pharmacology, Universityof Saskatchewan, Saskatoon, Saskatchewan (Canada) (Accepted 3 October 1989) Key words: Adenosine; Active transport, Astrocyte; Primary cultures; Salvage pathway; 2"-Deoxycoformycln; Purine metabolism

The use of a relatively specific adenosine deaminase inhibitor, 2"-deoxycoformycm (1.0/tM), has revealed an active transport of adenosine into astrocytes in primary cultures. The abolishment of part of the metabolic degradation and of a concentration gradient, which may favour influx, did not lead to a decreased total uptake. The concentration of labelled, i e exchangeable adenosine rose to become several fold higher than m the medium. Thus, as previously shown in neurons, the uptake of adenosine into astrocytes occurs by an active and concentrativeprocess As a result of the increase in the adenosine concentration when the inhibitor was present, ewdence for an increased phosphorylation to the nucleotides (i.e. ATP, ADP, AMP) was obtained. This is in contrast to previous findings in neurons where the incorporation of labelled adenosine into these compounds was decreased in the presence of 2"-deoxycoformycin. This difference may suggest that the salvage pathway from inosine to adenine nucleotides ~s crucaal for nucleotide synthes~s m neurons, but not in astrocytes. INTRODUCTION Glial cells, particularly astrocytes, have been implicated in the regulation of the neuronal micro-environment. A m a j o r function of these cells is to remove neuroactive c o m p o u n d s from the extracellular cleft in o r d e r to t e r m i n a t e their actions 1°'13A6. This also applies to adenosine, a substance whose n e u r o m o d u l a t o r y role is t e r m i n a t e d by u p t a k e into brain cells and/or deamination to the inactive c o m p o u n d , inosine 29. A n intense u p t a k e for adenosine has been demonstrated in both astrocytes and neurons 5"12. We have o b s e r v e d 2° p r o n o u n c e d differences in adenosine metabolism b e t w e e n cerebral cortical neurons (considerable d e a m i n a t i o n ) and cerebral cortical astrocytes (mainly p h o s p h o r y l a t i o n and little deamination). In a previous study, we have r e p o r t e d is that the uptake of adenosine into neurons in p r i m a r y cultures occurs by a rapid, concentrative, i.e., active transport. This was done by d e m o n s t r a t i n g that the u p t a k e was not decreased in the presence of the adenosine deaminase inhibitor, 2"deoxycoformycin ( D C F ) , although it occurred against a steep concentration gradient of adenosine itself (not including metabolites). A similar uptake mechanism may not necessarily exist in o t h e r brain cell types. It was the p u r p o s e of this investigation to d e t e r m i n e if astrocytes possess such a transport mechanism. In addition, the extent to which inhibition of adenosine deamination

affects the m a j o r p a t h w a y for adenosine metabolism in astrocytes (i.e. adenosine p h o s p h o r y l a t i o n ) was investigated. A g a i n , we used DCF. This c o m p o u n d easily p e n e t r a t e s cell m e m b r a n e s and is a highly p o t e n t ( K 1 = 10-11 to 10 -12 M) inhibitor of adenosine d e a m i n a s e ( E C 3.5.4.4) activity 1. High concentrations of D C F are known to inhibit not only d e a m i n a t i o n of adenosine, but also other metabolic pathways (i.e. purine ribonucleotide synthesis; adenylate d e a m i n a t i o n ; conversion of inosinate to adenine and guanine nucleotides via the purine salvage pathway). H o w e v e r , the action by D C F is quite specific for adenosine d e a m i n a t i o n at lower concentrations H, e.g. 1.0 btM. We have therefore e x a m i n e d u p t a k e and metabolism of 14C-labelled adenosine u n d e r control conditions and in the presence of 1.0 ~ M 2"-deoxycoformycin. MATERIALS AND METHODS Preparation of cell cultures Primary cultures of astrocytes were prepared as described by Hertz et al 14,16from the neopallium of the cerebral hemispheres of newborn Swiss mice They were maintained in cultures for 4-5 weeks, and from the age of 2 weeks exposed to 0 25 mM dibutyryl cyclic AMP (dBcAMP), a procedure which reduces a pronounced morphological and functional differentiation Such cultures are known to constitute good models of their in VlVOcounterparts TM Uptake studies For the uptake studies the cells were exposed for 5 s and 15, 30 and 60 mln at 37 °C to 10/~M [8-14C]adenoslne with and without the

Correspondence L. Hertz, Department of Pharmacology, Umverslty of Saskatchewan, Saskatoon, Saskatchewan, Canada, S7N 0W0

169 adenosine deaminase inhibitor, 2"-deoxycoformycin (1.0/zM). At the end of the incubation penod the cultures were washed 3 times with ice-cold phosphate-buffered saline (pH 7.3). Subsequently, 0.6 mi of 0.3 M ice-cold perchioric acid (PCA) was added and the cells were kept on ice for at least 15 min. They were then scraped off the dishes, and the cell extracts with the precipitated proteins were centrifuged for 10 min at 18,700 g. Protein content per culture was determined by the method of Lowry et al. 19. Special care was taken to expose all cultures to PCA for a similar total period of time (45 min) since some breakdown of adenosine and its metabolites inevitably occurs during this period. Recovery studies showed that the deaminated products were only slightly affected by PCA (80-85% recovery), whereas ATP and ADP were affected to a larger extent (recovery 60 and 50%, respectively). These recovenes are consistent with previous results by Webster et al. 2s, and appropriate corrections were used in the calculation of cellular contents. Immediately after centnfugation, the PCA extract was neutralized with Alamine 336/Freon TM,and another centrifugation followed (10 min; 4600 g). Samples and standards were stored for at most 2 weeks at -20 °C before HPLC analysis. The aqueous phase, which contained adenosine and its metabohtes, was injected (100/zl) into a Waters HPLC system. Separatzon of nucleotides, nucleosides and bases was achieved using a reverse phase/zBondapak column and a concave gradient elution (program No. 10 on Waters 680 automated gradient controller with 0.05 M potassium phosphate (pH 6 0) and 10% methanol in phosphate buffer as mobde phase A and B, respectively). Thas is a modification of the procedure originally described by Schweinsberg and LoozS. Adenosine and its metabohtes were detected by UV absorption at a wavelength of 254 nm. Radioactivity was monitored in 1.0-min fractions of the effluent to ensure determination of total radioactivity of each peak and to separate closely adjacent peaks as well as possible. However, even with this technique, peaks for ATP and ADP were not completely resolved so these two metabolztes were combined. This is of no consequence for the present studzes or thezr interpretation.

Statisucal analysis Pool sizes are indicated + S.E.M. whereas no S.E.M. values are indicated for the labelled contents which were calculated from the product of pool sizes and specific activities. Statistical sigmficances between means in the absence and presence of DCF were calculated by the aid of the Student's t-test 27.

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sured by HPLC) and the protein content of the culture, and total concentrations in the intracellular water phase were calculated on the basis of an assumed water content of 10/zl/mg protein. This is the intracellular water content in brain 17 and it is somewhat higher than that actually measured in our cultures (7.0/A/mg protein; H.S. White, L. Hertz, S.Y. Chow, and D.M. Woodbury, unpublished results). Thus, the concentration in the cells, if anything, was underestimated. The labelled pool sizes and concentrations were calculated in a similar manner based upon the radioactivity incorporated into each compound and the specific activity of the medium (100,000 d.p.m./ nmol2°). Such a calculation is appropriate for adenosine

Materials [8-14C]Adenosine (specific activity of 43 mCi/mmol) in ethanolwater was obtained from New England Nuclear, Lachine, Quebec, Canada, and its purity (especially with respect to possible contamination with labeled inosine) checked, 2"-deoxycoformycin (Pentostatm) and chemicals for the preparatzon of standards and of tissue culture medium were purchased from Sigma Chemical Company, St. Louis, MO, U.S.A.; methanol was obtained from Burdlck & Jackson, Muskegon, MI, U.S.A.; and all water used for media or mobile phases was from BDH, Toronto. The latter point is important since other sources of water were contaminated w~th UV-absorblng impurities. RESULTS

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The intracellular concentrations ~mol/ml intracellular water) and contents (nmol/mg protein) of the total and labelled pools of adenosine (Fig. 1), inosine (Fig. 2), AMP (Fig. 3), ATP + ADP (Fig. 4), and hypoxanthine (Fig. 5) with and without 2"-deoxycoformycin (DCF) are shown as a function of the length of incubation with 10 /zM adenosine. The total contents (pools) are calculated from the amounts of each compound per culture (mea-

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itself, but probably not for its metabolites since intracellular adenosine (the precursor) had a specific activity which was low compared to the specific activity of adenosine in the medium. The latter value can also not be used since intracellular adenosine must be compartmentalized. From Fig. 1, it can be seen that the total concentration of adenosine in the cells (100-250 #M) was considerably higher than the extracellular concentration (10 pM), and that it was approximately doubled in the presence of DCF at 15 rain. The concentration of labelled adenosine rose above that in the medium, both in the absence and presence of DCF, but most markedly in the presence of the drug, where all values from 15 min and onwards were

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significantly (P < 0.02 or better) higher than the adenosine concentration in the medium (10pM). Both in the absence and presence of DCF, the specific activity, indicated by the ratio between labelled and total adenosine contents, was shghtly below 50%. Since the pool size of adenosine was small and its uptake rate high (see below), the absence of equilibration (i.e. achieving the same specific activity in the cells and in the medium), even after 60 min, indicates that the intracellular adenosine must be compartmentalized into more and less easily exchangeable pools. Since DCF is an inhibitor of adenosine deaminase activity, one would expect a lower intracellular concentration and content of both total and labelled inosine, the product of the enzymatic reaction, in the presence of this drug. Fig. 2 shows that there was indeed a strong tendency towards a decreased mosine content in the presence of DCF. However, due to the low amount of inosine under all conditions, the statistical variation was large and the differences between results with and without DCF varied at different time periods. The decrease in labelled inosine after 60 min of incubation was significantly (P < 0.02) decreased in the presence of DCF. Fig. 3 shows the contents of total and radiolabelled ATP + ADP. The total contents were identical in the presence and absence of DCF at the start of the incubation, followed by a slow, gradual increase of the content in the presence, but not in the absence of DCF, and after 60 rain the difference was statistically significant (P < 0.05). The incorporation of radioactivity into ATP + ADP showed a tendency in the same direction, but no significant differences were found. In agreement with the

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F=g 5. Total mtracellular concentrations ~ M ) and total contents (nmol/mg protein) of the total + S.E M. (closed symbols) and rad]olabelled (open symbols) pools of adenosine plus all its measured metabohtes (ATP + ADP, AMP, mosme, and hypoxanthine) as a function of the length of incubation w~th 10 /~M adenosine in the absence of 2"-deoxycoformycin ( 0 , 0 ) and in the presence of 2"-deoxycoformycm (O,II) Results are from the same experiments as m Fig 1

171 large pool size, the specific activity remained low throughout the incubation. DCF had also little effect (results not shown) on AMP content. At longer incubation times, the total and radiolabelled pools of hypoxanthine tended to show an increase when exposed to DCF (Fig. 4). The total pool sizes were quite variable, but the increase in hypoxanthine after incubation with DCF for 60 min was highly significant (P < 0.001). The apparent difference after 5 s was not statistically significant. Fig. 5 shows the total intracellular concentration and total content of adenosine plus all its measured metabolites (i.e. ATP + ADP, AMP, inosine, and hypoxanthine) and the total incorporation of radioactivity as a function of the length of incubation with 10 pM adenosine in the presence and absence of DCF. As in the case of ATP + ADP, the DCF-treated cultures showed no differences during the initial part of the incubation, but after 60 min there was a significant (P < 0.05) increase above the control. From the incorporation of label, the accumulation of radioactivity during the first 15 min can be calculated as 0.31 nmol/min per mg protein in the absence of DCF and at least as much in its presence. DISCUSSION We have previously demonstrated that adenosine uptake into neurons in primary cultures to a large extent occurs by an active and concentrative uptake. This was done by inhibiting adenosine deaminase (EC 3.5.4.4) activity by the relatively specific inhibitor, 2"-deoxycoformycin (DCF). Under these conditions, the total uptake was unaltered or was increased (at 60 min) although approx, one half of the metabolism was abolished. Moreover, the concentration of labelled adenosine itself (i.e. not including any of its metabolites), within 15 min rose to ~0.5 nmol/mg protein, or at least 50 pM in the intracellular water phase, i.e., a 5 times higher concentration than in the medium 2°. Adenosine is accumulated not only into neurons, but also into astrocytes. The K m values are almost identical in the two cell types, but the Vm~x was ~50% higher in astrocytes 5. It is therefore of considerable interest to study whether adenosine uptake into astrocytes also occurred by an active and concentrative uptake. In contrast to neurons, most of the accumulated adenosine in astrocytes is phosphorylated, i.e., metabolized via AMP to ADP and ATP. This means on the one hand that the effect of inhibiting deaminase activity will be less dramatic and on the other hand it raises the question of what happens to the formation of the nucleotides when deamination is inhibited. The present study showed that already after 5 s of

incubation, the concentration of labelled adenosine and its metabolites was ~15 /~M in the intracellular water phase (i.e. one and a half times the extracellular concentration). This value rose to about 40/~M in the absence of the inhibitor and to about 80 /~M in the presence of DCF in spite of this disruption of the concentration gradient. The total uptake rate was high under control conditions, i.e., 0.31 nmol/min per mg protein during the first 15 min. This value is identical to that (0.33 nmol/min per protein) previously determined in similar cultures 12. The uptake was not decreased, but possibly enhanced in the presence of the inhibitor (Fig. 5). Thus, as in neurons, abolishment of part of the metabolic degradation did not lead to a decreased total uptake. This means that active uptake of adenosine is not limited to neurons, but that also astrocytes accumulate adenosine by an active and concentrative uptake. The concept that adenosine transport into astrocytes and into neurons occurs by an active, energy dependent, and concentrative transport process is supported by previous findings in brain slices and in synaptosomes of a concentrative, partially sodium- and calcium-dependent, cyanide and 2,4-dinitrophenol-inhibited uptake of adenosine 3'6'7"22'24. It is also consistent with temperature effects on adenosine uptake in cultured astrocytes5, brain cortex slices3 and synaptosomes 4'6. Moreover, conventional nucleoside transport inhibitors (like papaverine and dipyridamole) inhibit long-term uptake of radioactivity from labelled adenosine in both astrocytes and neurons in primary cultures 5. In the neurons, the incorporation into ATP + ADP was unaltered for the first 15 min in the presence of DCF, but thereafter showed a distinct inhibition is. This means that phosphorylation became decreased m spite of a pronounced increase in adenosine concentration. In contrast, in the present study, the incorporation of radioactivity into ATP + ADP was not decreased after 60 min of incubation. The increased pool sizes of these nucleotides after longer incubation could be a direct result of the doubling of the adenosine concentration. Three enzymes, adenosine kinase (EC 2.7.1.20), adenosine deaminase (adenosine utilizing enzymes) and 5"nucleotidase (EC 3.1.3.5; adenosine synthesizing enzyme), are simultaneously active and probably involved in the maintenance of the steady state adenosine concentration 2. In the presence of DCF, metabolism of adenosine to inosine was blocked by inhibition of adenosine deaminase, creating an increase in the adenosine concentration. It is not likely that this increase causes a stimulation of adenosine phosphorylation since the K m value (~1 pM) of the adenosine kinase is low 2'26. However, degradation of nucleotides to adenosine is at least under certain conditions inhibited by the end

172 p r o d u c t , adenosine 2, and a block of nucleotide degradation to a d e n o s i n e explains the elevated contents of A D P + ATP, especially at later time periods. Utilization of a direct p a t h w a y from A M P via inosine 5"-monophosphate ( I M P ) to inosine and hypoxanthine might explain the increase of hypoxanthine d e m o n s t r a t e d in Fig. 4. A ' t r a p p i n g ' of A T P + A D P 21 by D C F would also explain the increase in the sum of adenosine and all its metabolites (Fig. 5). T h e absence of a corresponding increase in A T P + A D P in neurons, where D C F caused a decrease of A T P + A D P after 15 min 15, suggests that the salvage p a t h w a y (e.g. Fox 9) from inosine to adenine nucleotides is quantitatively crucial for nucleotide synthesis in neurons, but not in astrocytes. The very

m o d e r a t e rate of inosme f o r m a t i o n in astrocytes under normal conditions is c o m p a t i b l e with this concept. Both in the p r e s e n t study and in the previous study of neurons, a d e a m i n a s e inhibitor was used. This raises the question of what would h a p p e n to adenosine u p t a k e in the presence of a p h o s p h o r y l a t i o n inhibitor. Such compounds have been studied in brain slices s'26 and all were found to inhibit p h o s p h o r y l a t i o n and u p t a k e in parallel. H o w e v e r , this does not necessarily indicate that the phosphorylation process p e r se is essential for adenosine uptake since the contents of A T P and thus the ability to maintain energy-utilizing processes might be rapidly impaired when synthesis o f A T P is inhibited.

REFERENCES

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