Characterization of nitrobenzylthioinosine binding sites in the mitochondrial fraction of rat testis

Life Sciences,Vol. 58,No.9,p~.?53-?59,19% CoWright* 19% Elsevier science Inc. PrintedintheUSA. Allrightsreserved W?4-3205/% $15.00 t .oo 0024-3205(95)02353-4

ELSEVIER

CHARACTERIZATION

OF NITROBENZYLTHIOINOSINE

BINDING

SITES

IN THE

MITOCHONDRIAL FRACTION OF RAT TESTIS A. Camins, A. Jimenez, F.X. Sureda, M. Pall&, E. Escubedo and J. Camarasa"' Laboratory of Pharmacology and Pharmacognosy. Faculty of Pharmacy. University of Barcelona. 08028 Barcelona. SPAIN. (Received in final form December 12, 1995)

Summarv The presence of L3Hl NBMPR binding sites in the mitochondrial fraction of rat testis is described. The disssociation constant (K,) from saturation studies was 0.16 + 0.04 104. The association and dissociation rate constants (k, and k.,) were 3.95 f 0.57 x 1Oa Mm' min.' and 0.025 + 0.002 min.', respectively. The number of binding sites was 2,100 + 163 fmols/mg protein. L3Hl NBMPR binding was inhibited, in a nanomolar range, by NBMPR (K,= 0.23 f 0.02 nM), OHNBMPR (K,= 2.30 + 0.55 nM) and HNBTG (K,= 2.58 + 0.33 nM). In the micromol& range, adenosine receptor lygands such as PIA (3.46 k 1.36 PM), 2-chloroadenosine (18.81 ? 3.36 PM) and NECA (8.26 + 3.90 PM) , and mitochondrial benzodiazepine receptor ligands such as RO 5-4864 (5.15 f 1.82 PM) and PK 11195 inhibited the specific binding of [3H] NBMPR. These results suggest the existence of a nucleoside transport system in the mitochondrial fraction of rat testis. KQJ Wordr: nitrobenzylthioinosine binding sites, mitochondrial

fraction, testis

Adenosine is an important neuromodulator with sedative, anticonvulsant, antiaggregant and vasodilatative properties (1). These physiological functions are mediated through the binding to different specific receptors, A, and 4. subdivided on the basis of their pharmacological profile, inhibiting or stimulating the adenylate cyclase, respectively. Recently a new receptor subtype, &, has been characterized in rat testis (2). Furthermore, it has been demonstrated that rat (3) and bovine testis (4) have A, receptors, and a role of adenosine in reproduction has been suggested, perhaps regulating the energetic equilibrium (5). Nitrobenzylthioinosine (NBMPR) is a potent inhibitor of the nucleoside transport across the cytoplasmatic membrane, and it has been used as a definitive probe to identify this nucleoside transport (6). NBMPR-resistant transporters have been described (7). It has been proposed that in brain tissue, phosphorylation of adenosine to ATP could be the regulator of adenosine uptake (8). In myocardial ischaemia, one of the protective effects of NBMPR is due to an intracellular increase of adenosine levels and ATP regeneration (9). In the mitochondrial fraction from guinea pig cortex the presence of high affinity [Q-NBMPR b'Ind ing sites has been demonstrated (6), and also high levels of adenosine has been found in rat kidney mitochondria (10). In previous studies with the mitochondrial fraction of several tissues, using peripheral-type benzodiazepine ligands, as Ro 5-4864 and PK 11195 (11, 12), we have described a possible relationship between the inhibitors of the adenosine uptake and mitochondrial-benzodiazepine receptor (MBR). The aim of the present study was to demonstrate the presence and characterize high affinity L3Hl NBMPR binding sites in the mitochondrial

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fraction of rat testis using various compounds (see Fig. 1) with different affinity profiles. To sum up, specific agonist of adenosine receptors (PIA, NECA and 2-chloroadenosine) and specific markers of cytoplasmatic membrane adenosine transport (NBMPR derivates) has been used.

RO 5-4854

PK 11195

Fig. 1 Structures of different compounds used in the study. Materials and Methods Materials. [?Hl NBMPR (27.3 Ci/mmol) was purchased from New England Nuclear (Boston, MA, U.S.A). Ro 5-4864 was purchased from Fluka Chemie AG (Buchs, Switzerland). PK 11195 was a gift of Pharmuka Laboratories (Asnieres, France). S- (p-nitrobenzylj-6-thioinosine (NBMPR), S-(2-hydroxy5nitrobenzyl)-6thioguanosine (HNBTG),S-(2-hydroxy-5-nitrobenzyl)-6-thioinosine (OH-NBMPR),5'(N-Ethylcarboxamido) -adenosine (NECA), N6-phenylisopropyladenosine (PIA), 6amino-2-chloropurine riboside (2-chloroadenosine) and Sigma Diagnostics 5'Nucleotidase were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.). All other chemicals were purchased from commercial sources. Drug solutions were prepared in ethanol and diluted with the buffer solution. The final concentration of ethanol was maintained at < 1% and produced no effect on binding. Tissue ureuaration. Adult male Sprague Dawley rats, weighing 250-275 g, were obtained from CERJ (Le G&rest, France). They were maintained on a 12h light/dark cycle and fasted 24h before the experiments. Animals were killed by decapitation, and testis were removed, washed, and placed in ice cold homogenization buffer (0.32 M sucrose and O.OlM Hepes, pH 7.4). The organs were then homogenized with a Kinematica Polytron. The homogenate was centrifuged at 800 x g for 10 min; the supernatant was centrifuged at 7,000 x g for 10 min. The pellet was resuspended in buffer and incubated for 5 min at 37°C in order to

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eliminate, as possible, the endogenous adenosine content. The homogenate was centrifuged and washed twice at 7,000 x g for 10 min. The pellet was resuspended in the assay buffer ( 50 mM Tris-HCl, pH 7.4) with a Potter. Protein content was determined by the method of Bradford (13) using bovine serum albumin as the standard. The enrichment and purity of the mitochondrial fraction was assessed by enzymatic assays and electron microscopy. In the final pellet, succinate dehydrogenase activity, a mitochondrial marker, was performed by the method of Earl and Korner (14). This activity (101.95 f 8.05 nmol/min/mg protein) was enriched about 4 fold relative to that of the postnuclear supernatant. In the same pellet, 5'-nucleotidase activity (203 + 32 pg P,/h), a cytoplasmic membrane marker, was only 1.5% of that of the postnuclear supernatant. Electron microscopy ( 20,000X ) of the final pellet was performed according to the method of Hayat (1972) and showed a mitochondrial purity greater than 95% and confirmed the absence of functional cytoplasmic membrane. All results are expressed as mean + SEM (standard error of the mean) from 3-6 different experiments. Radiolisand bindins assays. Equilibrium binding assays were performed at 25°C using L3H1 NBMPR. Assays were done in a final volume of 1 ml (pH 7.4) including 100 ~1 ['HI NBMPR in a concentration range of 0.025-5 nM (saturation studies) or 0.5 nM (kinetic and competition studies) and 100~1 of mitochondrial preparation (200 pg of protein per assay). Specific binding was defined as the difference between the radioactivity bound in the absence (total binding) and in the presence (non-specific binding) of 10 PM of unlabeled NBMPR. The specific binding was usually 95%. After incubation for 30 min, samples were filtered under vacuum over Whatman GF/B glass fiber filters soaked with 0.5% polyethyleneimine. Filters were rapidly washed with three aliquots of 4 ml of ice-cold Tris-HCl and placed in vials containing 10 ml of Cocktail Biogreen 1 l(Scharlau). Radioactivity was measured by liquid scintillation spectroscopy in a Beckman LS-1800 with an efficiency of 55%. K, and B,,,in saturation studies and K, in competition studies were determined by the LIGAND program. Kinetic parameters were determined by linear regression. The association (k,) and dissociation (k.,) rate constants were calculated from the slopes of plots of In (B/B-Bt) versus time (B=the amount of radioligand specifically bound at equilibrium; Bt= the amount of radioligand specifically bound at time t) in the case of association experiments, or ln(Bt/Bo) versus time (Bo is the concentration of radioligand-receptor complex at time 0) in the dissociation studies. Results Fig 2 shows the time course of ['HI NBMPR binding to rat mitochondrial testis. Binding appeared to reach equilibrium within 15-25 min with a kobs0.34 * 0.04 min.I. Calculated association rate constant (k,) was 3.95 ? 0.57 x 10' M-' min.'. The time course of dissociation of L3Hl NBMPR from binding sites of rat mitochondrial testis, after the addition of 10 pM of unlabeled NBMPR, is presented also in Fig 2. The semilogarithmic plot of dissociation data yields a k~, of 0.025 ? 0.002 min.'. The dissociation constant (KD= k-,/k,)for ['HI NBMPR computed from kinetic data was 0.06 * 0.01 nM. Equilibrium saturation experiments were performed with 9 different concentrations of L3Hl NBMPR ranging from 0.025 to 5 nM. The specific binding was saturated by increasing concentrations of ligand. The linear Scatchard plot indicated one population of binding sites (Fig 3). This was confirmed by nonlinear regression analysis, which yielded a K, of 0.16 ? 0.04 nM and a number of binding sites (B,,,)of 2,100 f 163 fmols/mg of protein. Hill coefficient was 0.94 2 0.06 and was consistent with the presence of a single class of binding sites.

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Fig. 2 Association (right pannel) and dissociation (left pannel) time course of 13H1 NBMPR bindinq to rat mitochondrial testis. The association was measured by -addition of 2 nM ['HI NBMPR to the incubation mixture. Preparations were incubated with L3Hl NBMPR for 30 min before initiation of dissociation by addition of 10 PM NBMPR at the indicated times. Data are the mean of three different experiments. Inset: Semilogarithmic plots of the same data. 2500

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Fig. 3 Saturation of ['HI NBMPR binding. Mitochondrial preparations of rat testis were incubated as described in Materials and Methods with increasing concentrations of radioligand ranging from 0.025 to 5 nM. Data are the mean of four experiments carried out in triplicate. Inset: Scatchard plot of the same data.

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To characterize further the pharmacological profile of L3H] NBMPR binding sites in rat testis mitochondria, we compared the potencies of an array of compounds as inhibitors of ['HI NBMPR binding (Figs. 4 and 5).

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g m

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0 -11

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[drug]

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(Ml

Fig. 4 Inhibition of c3H] NBMPR binding by NBMPR (H), OH-NBMPR (O), HNBTG (A), and PK 11195 (7). Data are the mean of four different experiments carried out in triplicate.

100

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a0 -

-0 ii g

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E z

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z F

20 -

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-7

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(Ml

Fig. 5 Inhibition of ['HI NBMPR binding by PIA (W), NECA (O), RO 5-4864 (A] and 2-Chloroadenosine (v). Data are the mean of three to six different experiments carried out in triplicate. As expected, NBMPR competitively displaced L3H] NBMPR from its binding sites (slope factor 1.04 f 0.12), with a K, of 0.23 + 0.02 nM which is in agreement with the K, value derived from equilibrium saturation studies.

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The congeners of NBMPR and potent inhibitors of nucleoside transport, OHNBMPR and HNBTG also inhibited with high affinity (K, value of 2.30 k 0.55 no and 2.58 k 0.33 nM, respectively) and competitively (slope factor 0.98 + 0.02 and 0.95 + 0.05, respectively) the binding of ['HI NBMPR. PIA and 2-chloroadenosine, two compounds that bind with high affinity to A, adenosine receptors, displaced the specific binding of L3HJ NBMPR with a K, of 3.46 + 1.36 PM and 18.81 i-3.36 FM, respectively. NECA, a non selective A,/% agonist, was also able to displace competitively ['HI NBMPR bound only in a micromolar range (K, value of 8.26 + 3.90 PM). Because in previous studies we have described a possible relationship between the mitochondrial benzodiazepine receptor (MBR) and adenosine uptake system inhibitors (11). we tested the effect of Ro 5-4864 (a MBR agonist) and PK 11195 (a putative antagonist of MBR) on ['HI NBMPR binding. Ro 5-4864, competitively inhibited the binding of ['HI NBMPR with a K, of 5.15 + 1.82 PM, meanwhile PK 11195 only was able to produce an inhibition of 50% at a concentration of 75 PM. Discussion Although adenosine receptors have been characterized in rat (5) and bovine (4) testis, and important amounts of adenosine has been detected into mitochondria of some tissues (lo), the presence of a specific and saturable mitochondrial adenosine transporter has not as yet been characterized. The present results demonstrate the existence of specific, saturable, time-dependent and displaceable L3Hl NBMPR binding sites in mitochondrial membrane of rat testicular tissue. The characterization of [?Hl NBMPR binding was assessed by the competitive displacement curves performed with specific inhibitors such as OH-NBMPR, NBMPR, HNBTG, adenosine analogs and peripheral-type benzodiazepines. As expected, only specific inhibitors of the cytoplasmic nucleoside transporter, displaced [?Hl NBMPR from its binding sites with high affinity. This results provided conclusive evidences of the existence of an specific adenosine transport system in mitochondrial membrane of rat testis. Recognition sites for L3Hl NBMPR are distinct from membrane adenosine receptors, as the specific A, agonist PIA and 2-chloroadenosine, showed an affinity significantly lower than that reported for their binding to adenosine receptors in rat testis (5). NECA (a non specific A,/A, agonist) showed also a high K, value. This experiments allows us reject that the binding sites studied are adenosine receptors. In our study RO 5-4864 displaced ['HI NBMPR from its binding sites in a micromolar range. This result is in agreement with similar K, values obtained using [?H] NBMPR in cytoplasmatic membrane preparations from human erythrocytes (16) and guinea pig brain (6). On the other hand, PK 11195 only displaced 50% of [?H] NBMPR bound. The different interaction between RO 5-4864 and PK 11195 for ['HI NBMPR binding sites could be explained by their structure differences as RO 5-4864 is a benzodiazepine compound and PK 11195 is an isoquinoline derivative. Also, it has been hypothetised that both compounds interact with different domains of the MBR (17), involving that only the domain marked by Ro 5-4864 overlap with the adenosine transport system. Thus, at high concentrations, Ro 5-4864 could modulate the mitochondrial 13Hl NBMPR binding as occurs in the cytoplasmic membrane adenosine uptake system (18). It has been described that in mitochondria of rat brain ATP can be formed from adenosine (19). As it is known, adenosine plays a modulatory role in the motility of sperm (3), this action can be probably due to an increase in ATP levels. Perhaps, the knowledge of the role of adenosine in the energetic balance in rat testis, could be a key step in the function of this nucleoside in reproduction. The characterization of an adenosine transporter in mitochondrial membranes could explain the role of adenosine in the energetic function into the mitochondria. Finally, it is the first time that the existence of a putative nucleoside transport system is described in the mitochondrial fraction of rat testis.

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