Ammonia-induced upregulation of peripheral-type benzodiazepine receptors in cultured astrocytes labeled with [3H]PK 11195

ELSEVIER

Neuroscience Letters 177 (1994) 35-38

N[UROSCI[HC[ LETTERS

Ammonia-induced upregulation of peripheral-type benzodiazepine receptors in cultured astrocytes labeled with [3H]PK 11195 Yossef Itzhak a'*, Michael D. Norenberg a-~ "Department of Biochemistry and Molecular Biology and hPathology, University of Miami School of Medicine, Miami, FL, USA ' Veterans Administration Medical Center, Miami. FL, USA

Received 18 April 1994; Revised version received6 June 1994; Accepted 13 June 1994

Abstract

Evidence suggests that peripheral-type benzodiazepine receptors (PBRs) may play a role in hepatic encephalopathy (HE), a condition associated with increased levels of ammonia in brain. In the present study, the regulation of [3H]PK 11195-binding to PBRs in cultured rat astrocytes that had been previously exposed to NH4C1 was investigated. 24 h treatment of 21-28-day-old cultures with 2, 5 or 10 mM NHaC1 resulted in 25 + 3, 48 + 3 and 42 + 4% increase in the number of [3H]PK 11195-binding sites, respectively. No further change in [3H]PK 11195-binding was observed after exposure of astrocytes to 5 mM NHaC1 for 48 or 72 h. Ammonia treatment did not cause any significant alteration in the affinity of [3H]PK 11195 for PBRs. The present study demonstrates the susceptibility of the PK 11195-binding site of PBRs in cultured astrocytes to ammonia and suggests that increase in brain ammonia concentration causes a supersensitivity of PBRs. Key words: Astrocyte; Ammonia; Peripheral benzodiazepine receptor; Hepatic encephalopathy

Ammonia is considered to be one of the major toxic agents in hepatic encephalopathy (HE) and animals with hyperammonemia develop many of the clinical and morphological features that mimic this condition [4,14,19]. However, the mechanism by which ammonia contributes to H E is unclear. Recent studies suggest the possible involvement of the peripheral-type benzodiazepine receptors (PBRs) in HE. Postmortem studies have shown increase in the number of PBRs labeled with [3H]PK 11195 in brain from patients with H E [10] and, after portacaval shunting in rats [6], an animal model of chronic HE. Additionally, an increase in the affinity of [3H]Ro5-4864-binding to PBRs in primary cultured astrocytes from rat cortex exposed to ammonia was noted [5].

*Corresponding author. Address: Department of Biochemistryand Molecular Biology(R-629), PO Box 012629, University of Miami School of Medicine Miami, FL 33101, USA. Fax: (1) (305) 547 3955. 0304-3940/94/$7.00© 1994 Elsevier ScienceIreland Ltd. All rights reserved SSD1 0304-3940(94)00488-V

In brain, PBRs are primarily localized in astroglial cells [2,21,22]. The existence of two PBR subtypes designated 'isoquinoline' and 'benzodiazepine' sites is supported by ligand receptor-binding experiments and cloning of the isoquinoline-binding protein [16,17,20]. The two selective PBR ligands, P K 11195 and Ro5-4864, preferentially label the isoquinoline and benzodiazepine sites, respectively [17]. Because our recent studies indicate marked differences in the properties of Ro5-4864and P K 11195-binding to PBRs in astrocytes [8,9], the present study was undertaken to determine the effect of ammonia on the binding parameters of [3H]PK 11195 in cultured astrocytes as compared with the modulation of [3H]Ro5-4864-binding that we previously reported [5]. Primary astrocyte cultures were prepared as previously described [3] with some modifications. Briefly, cerebral cortices obtained from 1-2-day-old Fischer rat pups were dissected free of meninges, minced, dissociated by trituration and vortexing, passed through sterile nylon sieves and then placed in Dulbecco's modified Eagle's medium ( D M E M ) containing penicillin (100

36

g Bzhak, M.D. Norenberg/ Neuroscience Letters 177 /1994) 3 5 ~ 8

U/ml), streptomycin (110 pg/ml), fungizone (2.5/.tg/ml) and 15% fetal calf serum (FCS). -1 x 10 6 Trypan blueexcluding cells were seeded in each 60-ram tissue culture plate (Falcon) and incubated at 37°C in a humidified chamber provided with 5% CO~-95% air. The culture media were changed twice weekly. The first medium change contained 15% FCS, the second 10% FCS. At confluency (after -11 12 days), cells were changed over from 10% FCS to 10% horse serum. Culture plates were maintained in the absence or presence of 0.5 mM dibutyryl cAMP (dBcAMP). This agent causes morphological and biochemical differentiation [7] and has a distinct effect on the binding of [3H]Ro5-4864 and [3H]PK 1t 195 to PBRs [9]. Based on the immunohistochemical identification of glial fibrillary acidic protein and glutamine synthetase, the cultures were composed of at least 9598% astrocytes. To determine the effect of various concentrations of ammonia on the binding of [3H]PK 11195 to PBRs, 2128-day-old cultures were maintained for 24 h in serumcontaining DMEM (pH 7.4) in the absence or presence of 2, 5 or 10 mM NHaCI. These concentrations of NH4C1 did not change the medium pH. To investigate the time course of ammonia effect on [3H]PK 11195-binding, cells were maintained in the presence of 5 mM NH4C1 for 24, 48 or 72 h. Levels up to 5 mM ammonia have been observed in animal models of HE [24]. After treatment, cells were rinsed 3-4 x with 0.32 M sucrose, scraped and homogenized in 0.32 M sucrose with a Teflon-glass homogenizer (25 strokes, 0°C). Aliquots of the homogenate in 6 vols. of 0.32 M sucrose (-2.5 mg protein/ml) were kept at -80°C until used for the binding experiments. The homogenate was thawed at room temperature and diluted in 10 vols. of ice-cold Tris-HC1 (50 mM, pH 7.4, 25°C). For saturation-binding assays, samples containing -0.2 mg proteirdml were incubated in a final volume of 0.5 ml with various concentrations of [3H]PK 11195 (0.2-8 nM). Nonspecific binding was determined in the presence of 1 pM unlabeled PK 11195. The reaction mixture was incubated at 4°C for 120 min, a time period that allowed equilibrium. Reaction was stopped by a rapid vacuum filtration (Brandel M12) through Whatman GF/B filters that were subsequently washed twice with 4 ml Tris-HC1 buffer. Radioactivity remaining on filters was determined by scintillation spectroscopy. Protein concentration was determined by method of Lowry et al. [11]. Results indicated that ammonia treatment did not cause any significant change in cells' protein concentration. The binding data were analysed using the ligand curve-fitting and receptor-binding analysis program [13], version 2.3.10, to calculate affinity constants (Kd) and maximal number of receptor sites (Bma0. Statistical differences between the binding parameters of [3H]PK 11195 in control and ammonia-treated cells were determined by two-tailed Student's t test. Saturation-binding experiments of [3H]PK 11195 to

homogenate of cultured astrocytes maintained in the absence or presence of dBcAMP (0.5 mM) indicated that this reagent does not affect the binding parameters of [3H]PK 11195 (Fig. 1, [9]). In the absence of dBcAMR the Kd was 2.21 + 0.15 nM and the Bma x w a s 1.72 + 0.053 pmol/mg protein while, in the presence of dBcAMR Kd was 2.42 + 0.21 nM; Bma x w a s 1.78 + 0.065 pmol/mg protein. As shown in Fig. 1, treatment of cells for 24 h with various concentrations of NH4C1 had a similar effect on the binding parameters of [3H]PK 11195 to astrocytes maintained in the absence or presence of dBcAMR At the lowest concentration tested, NH4C1 (2 mM) resulted in 25-28% increase in the number of [3H]PK 11195-binding sites (P < 0.05 as compared with control, 0 mM ammonia) while 5 mM ammonia caused an increase of 44-48% in the Bm~x(P < 0.05 compared with control). Raising ammonia concentration to 10 mM resulted in 42-44% increase in the number of [3H]PK-binding sites (P < 0.05 compared with control; Fig. 1). These changes in Bma x w e r e not accompanied by any significant alteration in the affinity (K~) of the ligand for the receptor site (Fig. 1). Time-course studies indicated that treatment with 5 mM ammonia for 24, 48 and 72 h resulted in very similar effect. No significant changes in the binding parameters of [3H]PK 11195 were observed in cells treated for 48 and 72 h with ammonia as compared with the binding of [3H]PK 11195 in cells treated with 5 mM ammonia for 24 h (data not shown). These findings suggest that the optimal upregulation of [3H]PK 11195-binding sites is obtained within 24 h of exposure to ammonia. The present study indicates that exposure of rat cortical astrocytes to ammonia induces a significant upregulation of PK 11195-binding sites. Since the elevation of brain ammonia concentration is considered as a major factor in HE, the ammonia-induced increase in PK 11195-binding sites suggests an important role for these receptors in HE. The present findings are consistent with a previous study demonstrating a 25-48% increase in the number of PK 11195-binding sites in brain autopsy samples from patients with HE [10]. In addition, upregulation of PK 11195-binding sites was observed in several rat brain regions after portacaval shunting [6], an animal model of chronic HE. We previously reported that a similar treatment of cultured astrocytes with ammonia resulted in an increase in the affinity of [3H]Ro5-4864 but no significant change in the number of receptors sites was observed [5]. The differential effect of ammonia on [3H]PK 11195- and [3H]Ro5-4864-binding further supports the distinction between the isoquinoline/PK 11195- and the benzodiazepine/Ro5-4864-binding sites [17]. Several recent findings from our laboratory indicate the divergence between these binding sites in cultured astrocytes. (1) PK 11195 labels nearly 50% more binding sites than [3H]Ro5-4864 [8]. (2) Ro5-4864-binding sites are downregulated after treatment with dBcAMP but PK 11195-binding sites are

E Itzhak, M.D. Norenberg/Neuroscience Letters 177 (1994) 35-38

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receptors [23]. Ammonia-induced increase in PBRs may, therefore, stimulate the biosynthesis of neurosteroids, a process that can contribute to altered states of CNS excitability. The major histopathological change associated with HE is the development of Alzheimer type II astrocytes [14]. Since PBRs are primarily localized in mitochondria [1,8], the increase in PK 11195-binding sites may be a result of the increase in size and number of mitochondria which is found in Alzheimer type II astrocytes. The present results not only support the role of astrocytes in the pathogenesis of HE but also provide a possible mechanism by which ammonia may contribute to the encephalopathy. In summary, the present study indicates that PK 11195-binding to PBRs in cultured astrocytes is susceptible to ammonia, suggesting that increase in brain ammonia levels causes supersensitivity of PBRs. Blockade of PBRs by specific ligands may provide a novel mechanism for attenuating ammonia-induced toxicity.

[NH4CI] (raM) Fig. 1. Effect of NH4CI on binding parameters of [3H]PK 11195 to astrocyte homogenates maintained in absence (A) or presence (B) of dBcAMP (0.5 mM). Saturation assays were carried out in homogenate from control (0 mM ammonia) and ammonia-treated cells as described in text. Ammonia treatment resulted in a significant increase (*P < 0.0.5) in number of [3H]PK l l195-binding sites (Bmax) as compared with control. No significant change in binding affinity (Kd) was observed. Results represent mean + S.E.M. values of three or four experiments.

not [9]. (3) Monoamines, such as dopamine, serotonin and norepinephrine, have distinctive effect on Ro54864- and PK l l195-binding sites (Itzhak and Norenberg, in prep.). Thus, the differential effect of ammonia on [3H]Ro5~864- and [3H]PK 11195-binding sites ([5], present study) may be due to the distinct properties of these receptor sites. Recently, we reported that the upregulation of [3H]PK 11195- and [3H]Ro5-4864-binding sites in cultured astrocytes by hypoosmotic stress in dependent on the presence ofdBcAMP [9]. Therefore, in the present study, we investigated the regulation of [3H]PK 11195-binding sites by ammonia in the absence and presence of dBcAMR The data presented in Fig. 1 indicate that ammonia had a similar effect on [3H]PK 11195-binding sites in the absence or presence of dBcAMR These findings suggest that different mechanisms are associated with the regulation of PK 11195-binding sites by ammonia and hypoosmotic stress. The precise role of PBRs in the pathogenesis of HE remains to be determined. Several studies suggest the involvement of PBRs in the biosynthesis of neurosteroids [12,15] which are thought to affect the function of neuronal GABAA [18] and N-methyl-D-aspartate (NMDA)

Supported by National Institute of Health DK 38153 and NS 30291, Department of Veterans Affairs and GRECC. We thank A. Vaishnav for excellent technical assistance. [1] Anholt, R.R.H., Pedersen, EL., DeSouza, E.B. and Snyder, S.H., The peripheral-type benzodiazepine receptor: localization to the mitochondrial outer membrane, J. Biol. Chem., 261 (1986) 5 7 6 583. [2] Bender, A.S. and Hertz, L., Flunitrazepam binding to intact and homogenized astrocytes and neurones in primary cultures, J. Neurochem., 43 (1984) 1319-1327. [3] Booher, J. and Sensenbrenner, M., Growth and cultivation of dissociated neurons and glial cells from embryonic chic, rat and human brain in flask cultures, Neurobiology, 2 (1972) 97-105. [4] Cooper, A.J.L. and Plum, F., Biochemistry and physiology of brain ammonia, Physiol. Rev., 67 (1987) 440 519. [5] Ducis, I., Norenberg, L.-O.B. and Norenberg, M.D., Effect of ammonium chloride on astrocyte benzodiazepine receptor, Brain Res., 493 (1989) 362-365. [6] Giguere, J.-F., Hamel, E. and Butterworth, R.F., Increased densities of binding sites for the 'peripheral-type' benzodiazepine receptor ligand [3H]PK 11195 in rat brain following portacaval anastomosis, Brain Res., 585 (1992) 295-298. [7] Hertz, L., Dibutyryl cyclic AMP treatment of astrocytes in primary cultures as a substitute for normal morphogenic and 'functiogenic' transmitter signal. In J.M. Lauder et al. (Eds.), Molecular Aspects of Development and Aging of the Nervous System, Adv. Exp. Med. Biol., Vol. 265, Plenum, New York, NY, 1990, pp. 227-243.

[8] ltzhak, Y., Baker, L.L. and Norenberg, M.D., Characterization of the peripheral-type benzodiazepine receptors in cultured astrocytes: evidence for multiplicity, Glia, 9 (1993) 211 218. [9] Itzhak, Y., Bender, A.S. and Norenberg, M.D., Effect of hypoosmotic stress on peripheral-type benzodiazepine receptors in cultured astrocytes, Brain Res., in press. [10] Lavoie, J., Layragues, G.P. and Butterworth, R.F., Increased densities of peripheral-type benzodiazepine receptors in brain autopsy samples from cirrhotic patients with hepatic encephalopathy, Hepatology, 11 (1990) 874-878. [11] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J.,

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Y Itzhak, M.D. Norenberg/ Neuroscience Letters 177 (1994) 35-38

Protein measurement with Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. [12] Mukhin, A.G., Papadopoulos, V., Costa., E. and Krueger, K.E., Mitochondrial benzodiazepine receptors regulate steroid biosynthesis, Proc. Natl. Acad. Sci. USA, 86 (1989) 9813-9816. [13] Munson, P.J. and Rodbard, D., LIGAND: a versatile computerized approach for the characterization of ligand binding systems, Anal. Biochem., 107 (1980) 220-239. [14] Norenberg, M.D., The astrocyte in liver disease, In S. Fedoroff et al. (Eds.), Advances in Cellular Neurobiology, Vol. 2, Academic Press, New York, NY, 1981, pp. 303-352. [15] Papadopoulos, V., Berkovich, A., Krueger, K.E., Costa, E. and Guidotti, A., Diazepam binding inhibitor and its processing products stimulate mitochondrial steroid biosynthesis via an interaction with mitochondrial benzodiazepine receptors, Endocrinology, 129 (1991) 1481-1488. [16] Parola, A.L., Stump, D.G., Peperel, D.J., Kueger, K.E., Regan, J.W. and Larid, H.E., II. Cloning and expression of a pharmacologically unique bovine peripheral-type benzodiazepine receptor isoquinoline binding protein, J. Biol. Chem., 266 (1991) 1408214087. [17] Parola, A.L. and Yamamura, H.I., Molecular Properties of mitochondrial benzodiazepine receptors. In E. Giesen-Crouse (Ed.), Peripheral Benzodiazepine Receptors, Academic Press, London, UK, 1993, pp. 3-26.

[18] Paul, S.M. and Purdy, R.H., Neuroactive steroids, FASEB J., (1992) 2311-2322. [19] Record, C.O., Neurochemistry of hepatic encephalopathy, Gut. 32 (1991) 1261-1263. [20] Riond, J., Mattei, M.G., Kaghad, M., Dumont, X., Guillemot, J.C., LeFur, G., Caput, D. and Ferrara, P., Molecular cloning and chromosomal localization of a human peripheral-type benzodiazepine receptor, Eur. J. Biochem., 195 (1991) 305-311. [21] Schoemaker, H., Morelli, M., Deshmukh, P. and Yamamura, H.I., [3H]Ro5-4864 benzodiazepine binding in the kainate lesioned striatum, and Huntington's disease basal ganglia, Brain Res., 248 (1982) 296-401. [22] Sher, P.K. and Machen, V.L., Properties of [3H]diazepam binding sites on cultured murine glia and neurons, Brain Res., 316 (1984) 16. [23] Smith, S.S., The effects of estrogen and progesterone on GABA and glutamate responses at extrahypothalamic sites. In E. Costa and S.M. Paul (Eds.), Neurosteroids and Brain Function, Fidia Research Foundation Symposium Series, Vol. 8, Thiem Medical Publisher, New York, NY, 1991, pp. 95-102. [24] Swaine, M., Butterworth, R.F. and Blei, A.T., Ammonia and related amino acids in the pathogenesis of brain edema in acute ischemic liver failure in rats, Hepatology, 15 (1992) 449~,53.