adenosine metabolism in kidney slices under normoxic conditions

JOURNAL OF CELLULAR PHYSIOLOGY 143:344-351 (1990)

Adenosine Metabolism in Kidney Slices Under Normoxic Conditions

JULIA BLANCO, JOSEFA MALLOL, CARMEN LLUIS, ENRIC I. CANELA, AND R. FRANCO* Departament de Bioquimica i Fisiologia, Facultat de Quimica, Universitat de Barcelona,

Diagonal 647, 0807 I Barcelona, Catalunya, Esparia

The effect of adenosine in rat kidney under normoxic conditions has been studied. It is demonstrated that adenosine modulates cell nucleotide levels. HPLC analysis of the purine compounds inside the cell indicates that adenosine improves the ATP/ADP ratio, whereas it diminishes the adenine content. This behaviour is not due to mediation by specific receptors, as agonists at P, purinoceptors did not have any effect. Further evidence using inosine as well as dipiridamole and deoxycoformycin indicates that all effects are dependent on the previous uptake of adenosine. The origin of free adenine in the kidney has been investigated, and it appears to come from the phosphorolysis of S’-methylthioadenosine. This report is the first to describe the activity of methylthioadenosine phosphorylase (E.C. 2.4.2.28) in the kidney. It is concluded that 1 ) extracellular adenosine improves guinea pig renal function by increasing the ATP level and the ATP/ADP ratio; and 2) there exists a functional pathway in the kidney that produces adenine and AMP coming from methionine and ATP. This latter pathway probably produces spermine and spermidine, which are likely to be important for renal function.

Cell adenine nucleotides play a critical role in the regulation of numerous intracellular processes in nor- mal kidney cells and in the pathogenesis of cell injury in disease states, particularly those involving oxygen deprivation (Siegel et al., 1980; Weinberg, 1985; Jones, 1986; Weinberg and Humes, 1986; Soltoff, 1986). After 1 minute of ischaemia, whole kidney ATP content is decreased by 70% (Hems and Brosnan, 1970). With longer durations (10 minutes), ATP decreases to less than 10% of the control levels (Siegel et al., 1983). Is- chaemia is accompanied by the loss of ion transport functions, as well as structural damage to membranes, which is characterized in the early stages of ischEmia by the loss of brush border in the proximal convoluted tubule (Venkatachalan et al., 1978; Siegel et al., 1980; Johnston et al., 1984).

Recovery from hypoxia requires the restoration of intracellular ATP levels. It has been proved that extracellular adenine nucleotides, mainly ATP, recover ATP levels in rabbit renal tubules after anoxia (Wein- berg and Humes, 1986; Mandel et al., 1988). The effect of extracellular adenine nucleotides is even evident in normoxic rabbit kidney (Weinberg and Humes, 1986; Weinberg et al., 1988). In these studies extracellular adenosine is less effective than extracellular ATP for maintaining or restoring intracellular nucleotide levels notwithstanding that, in ischEmic conditions in vivo, adenosine is more likely to be present in intersti- tial fluid than ATP. In fact, it is well established that extracellular adenosine produced in conditions of cellular damage is part of a general mechanism by which the energetic state of the cell is improved (as a consequence of adenosine transport and ulterior conversion into ATP) and appropriate systemic changes are pro-

duced, mediated by specific receptors (PI ,purinoceptors) (Burnstock and Kennedy, 1986; Phillis et al., 1987; Stefanovich, 1988; Shryock et al., 1988; Meghji et al., 1988). As an example of these responses mediated by receptors, infusion of adenosine in vivo produces the following: renal constriction (Hedqvist and Fredholm, 1976; Spielman et al., 1980; Tagawa and Vander, 1980); inhibition of renal adenylate cyclase (Mechenzie and Baer, 1973); neurotransmitter release (Hedqvist et al., 1978); renin release (Osswald et al., 1980; Spielman and Thompson, 1983); and reduction of glomerular filtration (Arend et al., 1985) and sodium excretion (Tagawa and Vander, 1980). The study of the metabolic effects produced by adenosine has been carried out ex- tensively for some types of cells, for instance, in erythrocytes (Plageman, 1986).

This study investigates the incorporation of adenosine into cellular adenine nucleotides and purine de- rivatives in the presence and absence of adenosine agonists, and adenosine transport inhibitors using nor- mal kidney slices. Because adenine is found in renal cells and its level changes after incubation with adenosine, the mechanism of production of the purine base was investigated; 5‘-methylthioadenosine phosphory-

Received August 29, 1989; accepted December 11, 1989. *To whom reprint requestdcorrespondence should be addressed. Abbreviations used: ADP, adenosine diphosphate; AMP, adenosine monophosphate; ATP, adenosine triphosphate; HPLC, high-performance liquid chromatography; NECA, N-ethylcarbox- amidoadenosine; PIA, phenylisopropyladenosine; PRPP, phospho- ribosy Ipyrophosphate.

@> 1990 WILEY-LISS, INC.

ADENOSINE METABOLISM IN KIDNEY

lase activity, which could account for the adenine produced, has been detected. Furthermore, as i t was demonstrated that free adenine can come only from 5'- methylthioadenosine, it is concluded that a relatively important metabolic flux in the pathway leading to the synthesis of polyamines (spermine and spermidine) exists in kidney.

MATERIALS AND METHODS The following products were used: adenosine, ino-

sine, deoxycoformycin, 5'-methylthioadenosine (Sig- ma), dipyridamole (Boehringer Ingelheim); NAD + , NECA, R-PIA, PRPP, Tris (Boehringer Mannheim); and ATP, ADP, AMP, adenine, inosine, Freon, and uric acid (Merck). The remaining compounds were reactive- grade quality. Female albino guinea pigs weighing 300-350 g were used for these studies.

Isolation of cortical slices Freshly isolated kidneys were cut into two halves

and the medulla removed. Slices of cortical tissue ap- proximately 1 mm wide were taken by using a razor blade. Dissection was performed on an ice-cold tile. Pieces of tissue were blotted on absorbent paper moist- ened with modified Krebs' solution (solution A) containing (mM) 133 NaC1, 5 KC1, 1.8 CaCI,, 0.6 MgSO,, 16.3 NaHCO,, 1.3 NaH,PO,, and 8 glucose (pH 7.4). The wet weight of these segments was obtained by placing them into preweighed drops of buffer and re- weighing. Care was taken to minimize tissue exposure to air. After weighing, they were used immediately.

Incubation procedure Incubations were performed by pretreating kidney

slices (50-70 mg wet weight) with 25 ml of a bath solution obtained by bubbling carbogen (95% 0, and 5% C02) to solution A and adjusting the pH to 7.4. Carbogen was continuously bubbled and, after 1 hour of standing, the pH of the aerated solution varied by less than 0.3 units. Fragments were subsequently placed in 5 ml of fresh and aerated solution A (pH 7.4) containing the appropriate modulator compound. Car- bogen was continuously bubbled, and after 5 minutes the pH of the aerated incubation mixture varied by less than 0.15 units. When two successive compounds (or a mixture of compounds) were used, the timing was 5 minutes of incubation with the first compound (or mixture of compounds) and 5 minutes with the second compound (or mixture of compounds). All incubations were performed at 30 +_ 0.1"C. The stability of the preparations was assessed by measuring the ATP content at different incubation time intervals with aerated A solution. After l hour of standing with aerated solution A, the ATP levels did not change appreciably during the subsequent incubation times (from 1 to 1.5 hours). Incubations with R-PIA or NECA were carried out in the presence of commercial adenosine deaminase (Boeh- ringer Mannheim, 1 UIlml of incubation mixture) to avoid the competition between endogenous adenosine and R-PIA for adenosine receptors.

Anoxic conditions were obtained by previously bubbling the incubation solutions with nitrogen; pH was adjusted to 7.4, and the incubation mixtures were performed in sealed tubs to avoid the presence of atmo- spheric oxygen.

7

1 4 6

/

345

8 9

@

Fig. 1. Typical elution pattern of a mixture of standards by HPLC. The exact chromatographic procedure is given in Methods. 1: ATP; 2: ADP; 3: hypoxanthine; 4 xanthine; 5 A M P 6 inosine; 7: NAD * , 8: adenine: 9: adenosine.

Homogenization of tissue and measurement of purine levels

At the end of the incubation process, cortical slices were washed by immersion in a continuous flow of cold (0-4°C) fresh A solution and immediately homogenized a t 0-4°C with a Polytron (Kinematical in 2 ml of an ice-cold 0.6 N perchloric acid solution. After standing for 10 minutes, suspensions were filtered through 0.45 pm Millipore filters, and the filtrate was extracted with one volume of heptane-Freon 1:4 (vlv). These two latter steps were carried out at 0-4°C during a short interval (less than 30 seconds). The aqueous phase was neutralized with a 1.2 N NaOH solution and either immediately analyzed or frozen (-20°C) until use. Nu- cleotides, nucleosides, and purine bases were quanti- fied on 20 pl aliquots of samples by HPLC (Waters HPLC equipment) at 254 nm with a LiChrospher 100 RP-18 (5 pm) (Merck) reverse-phase column using the following elution pattern: 1) 7 minutes of isocratic elution with 50 mM potassium phosphate buffer, pH 5.6; 2) 14 minutes of linear gradient with the same buffer and methanol to a final concentration of the alcohol of 10% (v/v); 3) 4 minutes of isocratic elution with 10% methanol. Reequilibration with the phosphate buffer lasted 10-15 minutes. The flow rate was 1 ml/minute. A typical elution profile is given in Figure 1. Resolu- tion of mixtures containing 5'-methylthioadenosine and adenine was performed with the same column and

346 BLANCO ET AL.

TABLE 1. Effect of adenosine on the intracellular level of purine compounds'

Comoound

Adenosine Control (n = 8) Incubation with (100 pM) (7)

(10 min) (5 mink (10 min) ATP 1.43 i 0.08 ADP 0.71 ? 0.09 AMP 0.41 i 0.09 NAD+ 0.35 ? 0.07 Adenosine 0.008 rc_ 0.002 Inosine u.d. Methylthioadenosine u.d. Hypoxanthine 0.11 2 0.03

Adenine 0.018 2 0.007 Xanthine 0.04 ? 0.02

1.8 2 O. l* :L* 0.69 t 0.1 0.40 i 0.06 0.41 2 0.08

0.008 ? 0.004 u.d. u.d.

0.09 2 0.02 0.03 -t 0.01

0.009 2 0.004"

2.4 f 0.1*** 0.77 f 0.04 0.42 2 0.05 0.42 f 0.05

0.008 rc_ 0.002 u.d. u.d.

0.08 2 0.01* 0.03 t 0.01 0.006 t 0.02**

ATPiADP ratio 2.0 2.7 3.1 2: (adenine nucleotides) 2.6 3.0 3.6

'Kidney slices were incubated with aerated Krebs' solution in the absence (control) or presence of adenosine. Values ( ~ m o l i g wet weight) are the mean ? SD of the experiments given in parentheses. Values for controls made at 5 minutes were fully analogous to those corresponding to 10 minutes. u.d. = undetectable. P values (paired student's t-test) are with respect to control. *P < 0.1. **P e. 0.05. ***P -< 0.01.

according to the following procedure: 15 minutes of a linear gradient from 20% to 40% of methanol in a 50 mM potassium phosphate buffer, pH 5.6. The flow rate was 1 ml/minute. In these conditions adenine had a retention time of 5 minutes, and methylthioadenosine had a retention time of 9.5 minutes.

Enzymatic studies Fresh renal cortices were homogenized by means of a

Polytron in four volumes of a medium that, unless oth- erwise stated, contained (mM) 250 sucrose and 50 tris, adjusted to pH 7.4 with HC1. The suspension was cen- trifuged 105,OOOg (Beckman L5-75 ultracentrifuge) for 90 minutes a t 4°C. The supernatant was considered to be the cytosolic fraction and contained 10-12 mg protein/ml. Pellet was washed twice with 50 mM tris-HC1 buffer (pH 7.4) and suspended in the same medium. For measuring the activities of purine nucleoside phosphorylase and methylthioadenosine phosphorylase, ho- mogenates were carried out in 50 mM phosphate buffer, pH 7.4, containing 1 mM dithiothreitol and cen- trifuged as above.

Adenosine deaminase activity was measured according to the method of Kalckar (1947) as described else- where (Franc0 et al., 1986). All other enzyme activities, measured a t 30 k O.l"C, of either cytosolic fraction or incubation media were performed by HPLC analysis. After incubating the enzyme solution with the appropriate substrate, an acidic deproteinization was carried out as described above. Quantification of the corresponding reaction substrate and product was accom- plished by HPLC after the heptane-Freon extraction (see above). Incubation mixture for the assay of adenine phosphoribosyltransferase in the forward reaction (production of AMP) contained (mM in 50 mM tris-HC1 buffer, pH 7.4) 2 Mg2+, 0.5 adenine and 0.5 PRPP. The incubation mixture used in the measurement of the reverse reaction contained (mM in 50 mM tris-HC1 buffer, pH 7.4) 0.1-1 AMP and 1-10 potassium pyro- phosphate. Incubation mixture for the assay of purine nucleoside phosphorylase contained (mM, in 50 mM potassium phosphate buffer, pH 7.4) 1 adenosine. Incu-

bation mixture for the assay of methylthioadenosine phosphorylase contained (mM, in 50 mM potassium phosphate buffer, pH 7.4) 0.05 methylthioadenosine and 1 dithiothreitol. Protein was measured according to the method of Lowry et al. (1951) employing bovine serum albumin as standard.

RESULTS Effects of adenosine

As indicated in Table 1, after 5 or 10 minutes of incubation with kidney slices, adenosine significantly increased the ATP/ADP ratio as well as the adenine nucleotide content. The remaining purines analyzed did not suffer any significant variation, with the sole exception of adenine, the level of which was diminished by the action of adenosine. These effects were progres- sive with time (Table 1). Kidney slices maintained for 5 minutes in anoxic conditions (see Methods) showed a high increase in the adenine content (0.031 pmol/g wet weight), whereas the ATP/ADP ratio was severely diminished (close to 1). In such conditions of hypoxia, treatment with 100 p M adenosine (5 minutes) produced an improvement of the ATPiADP ratio, similar to that obtained under normoxic conditions (see above).

Effects of adenosine transport inhibitors Dipyridamole is a potent inhibitor of the carrier-me-

diated transport of adenosine. In assays performed with dipyridamole, it has been demonstrated that the increase in the ATP/ADP ratio produced by adenosine is prevented when the nucleoside cannot enter the cell. Thus, in the presence of dipyridamole (alone or in combination with adenosine), purine levels did not change significantly with respect to control values (Table 2).

Effects of deoxycoformycin Deoxycoformycin is a tight-binding inhibitor of ecto-

solic and cytosolic-adenosine deaminase. This compound alone had no effect (Table 21, but when assayed with adenosine it further improved the ATP/ADP ratio and diminished the cellular adenine level. To assess the extent of the exogenous catabolism of adenosine, which could also account for the potentiation of aden-

ADENOSINE METABOLISM IN KIDNEY 347 TABLE 2. Modulation of adenosine effect by some compounds or mixture of compounds'

Dipyridamole Deoxycoformycin DCF + DCF 10 pM 0.2 p M dipyridamole R-PIA + dipyridamole NECA

100 pM 100 pM 100 pM Adenosine Adenosine Adenosine

~

Adensine Adenosine Adenosine 1 PM + R-PIA 1 r*M

Compound ( - ) [31 ( + ) I61 ( - ) 131 ( + ) 161 ( + ) [61 ( - ) [6l ( - 1 L6l ( - 1 141 ATP 1.4 ? 0.1 1.46 f 0.08 1.48 F 0.05 2.0 2 0.1**:* 1.5 i 0.1 1.39? 0.9 1.49 2 0.08 1.4 f 0.1 ADP 0.73 t 0.05 0.67 2 0.09 0.69 2 0.05 0.68 5 0.07 0.72 f 0.08 0.71 ? 0.09 0.71 5 0.08 0.7 f 0.1 AMP 0.40 ? 0.03 0.40 f 0.05 0.40 2 0.05 0.39 2 0.03 0.38 i 0.05 0.37 2 0.06 0.38 2 0.06 0.3 2 0.1 NAD 0.39 t 0.08 0.43 f 0.07 0.39 2 0.04 0.43 2 0.06 0.37 f 0.09 0.32 ? 0.09 0.34 2 0.08 0.4 2 0.1 Adenosine 0.008 2 0.002 0.007 5 0.002 0.009 t 0.002 0.007 f 0.002 0.008 -t 0.002 0.008 ? 0.002 0.007 2 0.002 0.008 2 0.004 Inosine u.d. u.d. u.d. u.d. u.d. u.d. u.d. u.d. Hypoxanthine 0.10 t 0.03 0.10 t 0.03 0.11 5 0.02 0.09 5 0.02 0.11 f 0.01 0.10 i- 0.02 0.10 2 0.02 0.11 2 0.05 Xanthine 0.05 ? 0.01 0.03 f 0.01 0.03 F 0.01 0.024 5 0.006 0.05 f 0.02 0.04 2 0.02 0.04 2 0.02 0.05 f 0.02 Adenine 0.019 2 0.005 0.018 2 0.005 0.016 t 0.005 0.010 2 0.002" 0.016 2 0.004 0.016 ? 0.003 0.017 2 0.003 0.017 f 0.08 ATPiADP ratio 2.0 2.2 2.1 2.9 2.1 2.0 2.1 2.0 E (adenine 2.5 2.5 2.6 3.0 2.6 2.5 2.6 2.4

nucleotides)

'Kidney slices were incubated for 5 minutes in aerated Krebs' solution with the indicated compound (or mixture of compounds) before adding adenosine (adenosine I i I); total incubation time was 10 minutes. Adenosine (-i indicates that no addition was carried out after the first 5 minutes. Values are the mean * SD of the experiments given in brackets. u.d. = undetectable. P values (paired student's t-test) are with respect to the control values as in Table 1. fP < 0.1. ***P < 0.01

osine effects exerted by the inhibitor of the deaminase, chromatographic analyses of the incubation media have been performed. The typical profiles shown in Figure 2 indicate that the catabolism of adenosine (in the incubation medium) during the first 5 minutes of incubation goes farther to inosine (2.05 pmolsig wet weight) and hypoxanthine (0.5 pmols/g wet weight) and is completely abolished in the presence of deoxycoformycin. For comparison, Figure 2 shows that dipyridamole had no effect on the catabolism of adenosine (in the incubation mixture). The activity of the adenosine deaminase in the washed total membranes fraction (see Methods) was 210 IT 34 pmollminute g wet weight (mean 2 SD for five separate renal cortices). This activity is relatively high if compared with that of the homogenate: 675 IT 46 pmollminute g wet weight (mean 5 SD for six separate renal cortices).

Effects of adenosine analogues in kidney slices

When added alone, R-PIA, which is an adenosine agonist at P, receptors, did not produce any significant change with respect to nontreated cortices (Table 2). To prevent uptake of the compound, incubations were performed together with both R-PIA and a nucleoside transport inhibitor (dipyridamole); the same results were obtained, which indicates that R-PIA does not exert any direct (through coupling to receptors) or indirect (through uptake) effect. In similar experiments performed with NECA, another adenosine agonist a t A, and A, receptors, the results have been identical to those obtained with R-PIA. Thus, it is concluded that adenosine increment of the ATPiADP ratio in kidney slices is not mediated via P, purinergic receptors.

Effects of inosine Inosine is a poor inhibitor of adenosine deaminase

and is inactive as ligand for purinergic receptors; however, controversy exists concerning its role in inhibiting adenosine transport. Inosine alone (Table 3) or in combination with dipyridamole (data not shown) did not exert any effect on adenine nucleotide levels or

on the ATP/ADP ratio, but it did produce significant increases in hypoxanthine probably due to its intracellular catabolism. On the other hand, the changes produced by adenosine were impaired when the cortices were incubated with inosine before the addition of adenosine. Nonetheless, when slices were previously incubated with adenosine and inosine was added afterwards, the increase in ATP levels was evident (Table 3). These results suggest that, when both compounds are present, adenosine and inosine compete for the transporter. This has been proved in brush-border membranes from rat kidney (LeHir and Dubach 1985; Franco et al., 1989b). The catabolism of inosine in the incubation mixture gives the appare- ance of only hypoxantine (1.6 pmolsig wet weight). The study of the extracellular catabolism of both adenosine and inosine displayed, as expected, high levels of adenosine and/or inosine as well as variable amounts of hypoxanthine (Fig. 2); this confirms the presence in the medium or in the cell surface of adenosine deaminase and purine nucleoside phosphorylase activities.

Adenine metabolism In many tissues, adenosine uptake goes forward to

the synthesis of adenine nucleotides by means of the adenosine kinase activity. In kidney, where as demonstrated here, it is possible to find measurable levels of adenine, another mechanism is possible through the enzyme adenine phosphoribosyltransferase (E.C. 2.4.2.7). We have been able to detect the forward reaction (production of AMP) in cytosolic fraction of kidney (Fig. 3). Adenine phosphoribosyltransferase activity was 345 nmollminute g wet weight. A putative reverse reaction (adenine production) has been impossible to detect with concentrations in the range of 0.05-1 mM of AMP and 1-10 mM of PPi. Another alternative pathway of adenine formation from adenosine by means purine nucleoside phosphorylase activity was tested in a 50 mM potassium phosphate buffer, pH 7.4, with 1 mM adenosine. Adenine was not found in the HPLC analysis of the incubate. The most likely pathway

348 BLANCO ET AL.

Inosine idenos ine

1 no:

Fig. 2. Extracellular catabolism of adenosine. Purine analysis of the incubation media was performed by HPLC. A Incubation mixture without any addition; B Kidney slices incubated 10 minutes with adenosine 100 JLM. C: Kidney slices incubated 5 minutes with 1 pM dipyridamole and 5 minutes more with 1 pM dipyridamole and 100

pM adenosine. D Kidney slices incubated 5 minutes with 10 p M dipyridamole and 1 p M deoxycoformycin and then 5 minutes more with 10 pM dipyridamole and 1 pM deoxycoformycin and 100 pM adenosine.

TABLE 3. Effect of inosine on intracellular levels of purine compounds'

Inosine 100 (LM Inosine 100 pM (10 min) Adenosine 100 pM

Compound [61 ( + 1 [61 ATP 1.45 -t 0.09 1.53 t 0.08 ADP 0.7 ? 0.1 0.69 -t 0.09 AMP 0.38 -t 0.06 0.43 t 0.07 NAD + 0.33 2 0.06 0.35 i- 0.07 Adenosine 0.008 ? 0.003 0.008 t 0.004 Inosine ud ud Hypoxanthine 0.15 t 0.02* 0.25 ? 0.03*** Xanthine 0.06 ? 0.02* 0.06 t 0.03 Adenine 0.016 2 0.005 0.016 t 0.007 ATPiADP ratio 2.1 2.2 X (adenine nucleotides) 2.5 2.6

Adenosine 100 uM Inosine 100 pM

( + ) 151 . . 1.92 5 0.08***

0.39 -t 0.07 0.71 -t 0.07

0.39 i 0.07 0.009 i- 0.003

ud 0.17 t 0.03" 0.05 ? 0.02

0.009 f 0.003% 2.7 x n

-

'Kidney slices were incubated with aerated Krebs' solution containing inosine. When both adenosine and inosine were assayed, a preincubation of 5 minutes with one of the nucleosides was carried out before adding the second one (total time = 10 minutes). Values (pmoUg wet weight) are the mean t SD of the experiments given in brackets. P (paired Student's t-test) are with respect to the control values of Table 1. Incubation with inosine for 5 minutes led to the same results as for 10 minutes. u.d. - undetectable. 'P <: 0.1. **iP .c 0.01.

of adenine production in mammals is from 5'-methylthioadenosine, a by-product of polyamine (spermine and spermidine) synthesis coming from S-adenosylme- thionine. This thioderivative of adenosine is metabo- lized to adenine by means of a specific protein display- ing phosphorolytic activity (5'-deoxy-5'-methylthioadenosine: ortophosphate methylthioribosyltransferase, E.C. 2.4.2.28). By incubating an aliquot of kidney cytosolic fraction with 5'-methylthioadenosine in 50 mM potassium phosphate buffer, pH 7.4, containing 1 mM dithiothreitol, variable amounts of adenine were formed when the reaction was stopped at

different times, as shown in Figure 4 (1.2 nmoll minute mg protein o 4.8 nmoliminute g wet weight). As expected, the recovery of the purine ring of methylthioadenosine as adenine is nearly stoichiometric (Fig. 4).

DISCUSSION The results presented in this report indicate that, in

kidney, adenosine produces an increase of ATP levels and ATP/ADP ratios in normoxic conditions (Table 1). These effects are mediated by the uptake of adenosine by cells, as they are completely abolished in the pres-

ADENOSINE METABOLISM IN KIDNEY 349

1600

:400

i 2 0 0

: 3 i O 0 G

u BOO

Y

I

2 6.30 i 8 1 100 "

2 00 P

000 7 0.00 5.00 10.00 15.00 SO 00 25.00 30.00

TIME (mi")

Fig. 3. Anabolism of adenine. AMP production by the reaction cat- alyzed by adenine phosphoribosyltransferase was detected by HPLC; 50 F l h l of kidney cytosolic fraction (see Methods) were incubated in a media containing (mM in 50 mM tris-HC1 buffer, pH 7.4) 2 MgCl,, 0.5 adenine, and 0.5 PRPP.

ence of dipyridamole, an inhibitor of mediated adenosine transport. Although in some cells there exists dipyridamole-resistant active nucleoside transporter, this varies from species to species, and within a given species it depends on the type of cell (Plageman et al., 1988). Thus, with guinea pig cerebral cortical synaptosomes, Lee and Jarvis (1988) find a 50-60% of sensi- tive transport, whereas Heaton and Clanachan (1987) demonstrate, in guinea pig myocytes, a total inhibition of the transport by dipyridamole. Our results are in accordance with the latter and indicate that in kidney slices the percentage of insensitive transport is very low. On the other hand, there is no involvement of PI purinoceptors, as the agonists R-PIA and NECA did not exert any effect.

In kidney the effects of adenine nucleotides added exogenously have been studied in normoxic conditions (Weinberg et al., 1988) as well as during recovery from hypoxia (Weinberg and Humes, 1986; Sumpio et al. 1987; Mandel et al., 1988). In these studies it is demonstrated that, irrespective of the oxygenation conditions, the addition of adenine nucleotides causes an increase of intracellular ATP levels. These metabolic events are dependent on extracellular conversion of ATP, ADP, or AMP into adenosine, which is subsequently taken up by the cell. However, adenosine itself is less effective than adenine nucleotides for recovering intracellular ATP levels. This may be explained by as- suming a substrate inhibition of adenosine kinase by moderate concentrations of adenosine (Mandel et al., 1988; Weinberg et al., 1988). Another possibility that may be considered is that stressed by Mandel et al. (1988) and based on a previous finding in the perfused rat heart by Frick and Lowenstein (1978), i.e., that adenosine produced from AMP by an ecto-5'-nu-

40 00

35.00

30.00 G

P d

F 2500

5 20.00

\ v1 n -

15 00

10.00

5 00 I I 5.00 10.00 15.00 20.00 25.00 30.00

TIME (min)

Fig. 4. Conversion of methylthioadenosine into adenine. Adenine (A) and methylthioadenosine (0) were analyzed by HPLC; 200 pliml of kidney cytosolic fraction (see Methods) were incubated in a medium containing (mM in 50 mM potassium phosphate buffer, pH 7.4) 1 dithiothreitol and 0.05 methylthioadenosine.

cleotidase may be transported preferentially into the cells.

Extracellular metabolism of adenosine goes forward to hypoxanthine, indicating that adenosine deaminase and purine nucleoside phosphorylase are leaking into the incubation medium. There is a possibility that in- stead of enzymes being leaked into the medium, they are true ectoenzymes. Furthermore, xanthine oxidase, which has a relatively important activity in guinea pig kidney and produces intracellular xanthine (Table 1) or uric acid (data not shown), did not act upon exogenous hypoxanthine. The ectoenzyme behaviour of adenosine deaminase has been demonstrated recently by both indirect (Franco et al., 1986, 1988) and direct methods (Meghji et al., 1988; Franco et al., 1989a).

Inosine, in our kidney preparation, did not affect the cellular levels of ATP; but it did prevent the increase of adenine nucleotide levels induced by extracellular adenosine (Table 3). Thus, when adenosine is incubated in the presence of inosine, apart from the logical increase of cellular hypoxanthine, it is observed that the ATP level does not reach the peak obtained when incubation is performed with adenosine alone. Yet it seems that a competition between adenosine and inosine is produced. An explanation would be that inosine and adenosine are, at least to some extent, transported by the same protein molecule. Franco et al. (1989131, working with brush-border membranes of the proximal

350 BLANCO ET AL

A ATP-

Adenosine I \

l 1 Iu 1 3 I 1 7

Inosine I

k-- \ \ Hyuoxanthine

f l

Methionine

Adenosine Methylation react ions

Inosine Putrescine (spermidine)

Spermidine (spermine)

P. 12

Nucleotide Catabolism pool to uric acid

Fig. 5. Scheme of the metabolic events concerning adenosine and adenine in kidney. 1: Ectoadenosine deaminase; 2: Ectopurine nucleoside phosphorylase: 3 Adenosine deaminase (E.C. 3.5.4.4); 4 Purine nucleoside phosphorylase (E.C.2.4.2.1); 5 Nucleoside transporter; 6 Nucleobase transporter; 7: Adenosine kinase (E.C. 2.7.1.20); 8 5'- nucleotidase (E.C. 3.1.3.5); 9 Methionine adenosyltransferase (E.C. 2.5.1.6); 10: S-adenosyl-L-methionine decarboxylase (E.C. 4.1.1.50);

tubule from rat kidney, have demonstrated that influx of adenosine is enhanced by the intravesicular presence of inosine, thus suggesting that both nucleosides can be partially transported by the same carrier molecule (or complex). In contrast, working with isolated tubular cells from rabbit, Weinberg et al. (1988) sug- gested that other purines, such as hypoxanthine and uridine, that may use the same transporter did not modify the effects of adenosine in increasing ATP levels.

In this work we have observed a significant level of adenine in our kidney preparations. We observed that adenine phosphoribosyltransferase functions only in the direction of production of AMP. Our results also confirm the well-known fact that adenosine is a poor substrate of purine nucleoside phosphorylase. Thus, for kidney cells, the production of adenine is possible only by means of the enzyme 5'-methylthioadenosine phosphorylase. In kidney this enzymatic reaction functions in the direction of adenine production because of the following: 1) Km for methylthioadenosine is very low (0.47 pM; Ferro et al., 1979); 2) methylthioadenosine in kidney is either undetectable (Table 1) or very low (2.2 nmolig wet weight in rat kidney; Della Ragione et al., 1981); 3) adenine level is measurable in kidney (Table 1); and 4) the cellular concentration of Pi is probably very much higher than that of methylthioribose- l-phosphate.

The fact that adenosine diminishes adenine content (Table 1) is quite surprising. The only possible explanation lies in the displacement of the reaction cata- lyzed by adenine phosphoribosyltransferase toward the formation of AMP. Because ATP is needed for the synthesis of PRPP (Fig. 5), i t is likely that PRPP levels

1 (Decarbdxylated S- ." \

11: Propylamine transferase (E.C. 2.5.1.16); 12: 5'-methylthioadenosine phosphorylase (E.C. 2.4.2.28); 13: Adenine phosphori- boxyltransferase (E.C. 2.4.2.7); 14 Pathway demonstrated in mammalians by Backlund and Smith (1981); 1 5 Phospho- ribosylpyrophosphate synthetase. Some trivial reaction products and cofactors have been omitted for clarity.

increase upon recovery of ATP levels. In these conditions, the purine ring of adenine is incorporated di- rectly into the pool of adenine nucleotides. This incorporation is quantitatively small, but it produces a displacement of the reactionb) toward ATP formation due to a favourable mass-action ratio. This hypothesis is consistent with a higher concentration of adenine obtained in anoxic conditions (see Results); in oxygen- deprivation conditions, PRPP level, as a consequence of (ATP) reduction, is probably reduced, resulting in an impairment of the conversion of adenine to AMP.

The existence of free adenine in kidney cells, as well as the functional activity of methylthioadenosine phosphorylase, provides evidence that polyamine synthesis is relatively important in kidney. This is supported by the moderately high levels of spermine (1.11 nmol/mg protein) and spermidine (3.3 nmol/mg protein) found in kidney (Piacentini e t al., 1986; values from rat). A summary of reactions concerning adenine and adenosine metabolism in kidney is given in Figure 5.

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ADENOSINE METABOLISM IN KIDNEY 351

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