SYNAPSE 7:141-150 (1991)

The Contribution of Endogenous BenzodiazeDine Recethor Ligands to the Pathogenesis of Hepahe EnGphalopathy ANTHONY S. BASILE Section on Neurobiology, Laboratory of Neuroscience, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

KEY WORDS

Benzodiazepine receptors, Fulminant hepatic failure, Diazepam, Mass spectroscopy

ABSTRACT The involvement of the y-aminobutyric acid A (GABAA)receptor complex in the pathogenesis of hepatic encephalopathy (HE) was examined in galactosaminetreated rabbits with HE caused by fulminant hepatic failure. Radioligand binding to the constituent components of the GABAAreceptor complex was unchanged in rabbits with HE. However, partially purified extracts from encephalopathic rabbit brain were approximately three times more potent in inhibiting L3H1Ro 15-1788binding t o benzodiazepine (BZ)receptors than extracts from control rabbits. The inhibition of radioligand binding to the BZ receptor produced by these extracts was competitive and reversible and was significantly enhanced by GABA. Further purification of these extracts by high-performance liquid chromatography (HPLC) indicated that the inhibitory activity was localized in several peaks, some of which had retention times corresponding to 1,4-benzodiazepine standards. The presence of diazepam in these extracts was confirmed using mass spectroscopy.Both mass spectroscopic and radiometric techniques demonstrated that the concentration of diazepam in brain extracts from encephalopathic rabbits was approximately 4 times greater than control extracts. These findings link the presence of BZ receptor agonists to the development of a neuropathologcal condition, thereby providing a rational basis for the use of BZ receptor antagonists in the management of HE in man.

INTRODUCTION He atic encephalopathy (HE) is a neuropsychiatric disor er arising from the complex metabolic abnormalities that accompany liver failure (Sherlock, 1989). The underlying liver failure may result from alcoholic cirrhosis, hepatitis or drug overdose. The encephalopathy is initially characterized by personality disorders and hypersomnia, with a gradual deterioration of cognitive and neuromuscular function leading to coma and death. The mechanism of the pathogenesis of HE is unclear. It has been proposed that an increase in the activity of y-aminobutyric acid (GABA)-containingpathways contributes to the manifestations of this syndrome (Basile and Gammal, 1988; Schafer and Jones, 1982). Investigations of this hypothesis have been aided by the develo ment of an animal model of HE, the galactosamine a1N)-treated rabbit model of fulminant hepatic failure (FHF)(Blitzer et al., 1978). Studies of visual evoked responses in this model indicate that GABAergictone is elevated (Bassett et al., 1987; Schafer et al., 1984), but the mechanism(s) responsible for this increase have not been resolved. Several mechanisms that could explain the augmentation of GABAergicneurotransmission observed in HE include increased density or affinity of GABA receptors, andlor increased GABA potency mediated through an allosteric modulatory site, such as the benzodiazepine (BZ) receptor (Basile and Gammal, 1988).

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Subse uent electrophysiological studies of single neurons ave served to clarify further the mechanism involved in the increased activity of GABAergic systems observed in HE. Cerebellar Purkinje neurons from encephalo athic rabbits were hypersensitive to depression by t e GABA receptor agonist muscimol and the BZ receptor agonist flunitrazepam, but not to the rw-adrenoceptor agonist phenylephrine (Basile et al., 1988). In contrast, BZ receptor antagonists increased the s ontaneous firing rate of these neurons and reverse their hypersensitivity to muscimol at concentrations that did not affect the firin rate of control rabbit neurons (Basile et al., 1988). hese observations indicated that the increased GABAergic activity observed in HE may be mediated by the BZ receptor, possibly as a consequence of increases in the concentration or availability of a BZ receptor ligandk). The following study further investigates the mechanism(s) underlyin the role of GABAergicsystems in the pathogenesis of E. Neither the e uilibrium binding constants of radioligands at the G AA receptor complex nor the couplin between the constituents of the GAEiAreceptor comp ex is altered in the brain of rabbits with HE. However, significant elevations in the levels of

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Received April 23,1990; accepted in revised form July 12,1990. Address reprint requests to Dr. Anthony S Basile, Laboratory of Neuroscience Building 8, Room 111, NIDDK, National Institutes of Health, Bethesda, M 6 20892.

142

A.S. BASILE

BZ receptor ligands are observed in extracts from the brain and peripheral tissues of this animal model. These extracts contain several substances that bind to the BZ receptor, the neurochemical properties of which are consistent with those of BZ receptor agonists. The content of one of the active fractions is structurally identified as the 1,4-substituted benzodiazepine, diazepam. These results indicate that increased concentrations of BZ receptor agonists may be causally related to the enhanced GABAergic tone that contributes to the electrophysiological and behavioral manifestations of HE due to FHF. MATERIALS AND METHODS Animals Male New Zealand (NZ) white rabbits (=2 kg, Small Animal Section, VRB, NIH, Bethesda, MD) were maintained under a 12-h daylnight cycle with free access to food and water. FHF was induced using a modification of the method of Blitzer et al. (1978). Animals were infused with galactosamine HCl(4.0 mmol/kg dissolved in 8 ml of 0.9% saline pH 6.8) over a 2-min period through an ear vein. Control animals received an e ual volume of the vehicle. Neurologc fkatures of encep alopathy were manifest within 12-18 h after the administration of galactosamine. Clinical stages of encephalopathy were stage I: lethargy; stage 11: mild ataxia, poor1 maintained head posture; stage 111: severe ataxia, hin limb extension, loss of righting reflex, nystagmus; and sta e IV: coma (Jones et al., 1987; Bassett et al., 1987). On rabbits in stages 11-111 of encephalopathy were stuJed. Radioligand binding assays Radioligand binding to the central BZ receptor was assessed using previously described techniques (Squires and Braestrup, 1978; Mohler and Richards, 1981). Rabbits were decapitated according to AAALAC guidelines. The brains were rapidly removed and placed into 320 mM sucrose ( 0 4 C ) before dissection, weighing, and homogenization. For measurements of [3HlRo 15-1788 binding, the cortex and cerebellum were homogenized in 50 v of 50 mM Tris-citrate buffer, pH 7.4, using a Polytron (Brinkman Instruments, setting 6.5, 15 s). The homogenate was centrifuged at 20,OOOg (0-4"C) for 20 min and the pellet resuspended in an equal volume of Tris-citrate buffer. This washin procedure was repeated five times. After the last wasff, the pellet was resuspended in 20 vol of buffer and stored at -80°C for ~ 2 days 1 before use. Before assa , the tissue preparation was thawed, and a 50-pl a5quot (containing -0.12 mg protein) was gidded to each assay, which also contained 50 p1 of [clHIRo15-1788 (final concentration, 0.25-10 nM), 50 pl of Ro 14-7437 (final concentration, 10 pM), or buffer. Sufficient Tris-citrate buffer was used t o bring the final assay volume to 0.5 ml. Assays of total and nonspecific binding at each concentration were done in triplicate and duplicate, respectively. The assay was terminated after incubation (60 min at 25°C)by rapid filtration over Whatman GFB filter strips using a Brandel M-24R filtering manifold. Samples were washed with 2 x 5 ml aliquots of cold buffer. A similar procedure was used to assay the enhancement of [3H]flunitrazepam binding by NaCl(10400 mM) and GABA (0.1-200 phl), using a l-nM final

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Concentration of [3H]flunitrazepam and incubating the assa at 04"C for 60 min. F3 2SJt-Butylbicyclophosphorothionate(TBPS) binding to the GABA-gated chloride ionophore was assayed using a modification of the methods of Havoundjian et al. (1986). Tissue homogenates were used immediately after the fifth wash. A 300-pl ali uot of tissue homogenate (-0.4 mg protein) was adde to 50 pl of [:35S]TBPS (final concentration, 3.5 nM), 100 pl NaCl (final concentration, 200 mM), and either 50 p1 of cold TRPS (final concentrations, 5-160 nM), picrotoxin (final concentration, 20 pM) or Tris-citrate buffer. Assays were performed in triplicate and incubated at 25°C for 90 min. [3HlMuscimol binding to the GABAA receptor was assayed in washed P2 fractions re ared as previously described (Beaumont et al., 19787.TKe pellet was resuspended in 10 vol of Tris-citrate buffer; 25 pl (-0.5 m protein) was added to 50 pl of 2.5-100 nM [3Hlmuscimo and 50 pl muscimol (50-1,000 nM for total binding, yielding a final concentration range of 2.5-1100 nM),50 pl muscimol (100 pM) for nonspecific binding, and sufficient buffer to achieve a final volume of 0.5 ml. The assays were performed in tri licate, incubated at 0-4"C for 1hr, and terminated by fi tering under high vacuum (>20 Hg) through the Brandel manifold using Whatman GF/B filter disks pretreated with 0.5% polyethyleneimine, followed by washing with 2 x 5 ml aliquots of 50 mM Tris-citrate (0-4"C). The radioactivity retained by the filters was measured in a Beckman LS 5802 liquid scintillation spectrometer. Protein was determined using the BCA method (Pierce Chemicals, Rockford, IL). Equilibrium bindin constants for all ligands were determined using a non-%near regression analysis program (Accufit 1, Lundon Inc., Chagrin Falls, OH). Extraction The brains and eripheral tissues of control rabbits and rabbits with E were extracted using a modification of previous techniques (Foerster et al., 1978, Osselton, 1977). Tissues were homogenized in 4 vol (wlv)of 1 M Tris base (pH 10.9) with 1 mg/g tissue of alkaline rotease using a Brinkmann Polytron (setting6.5,30 s). The homogenate was then incubated in a water bath with continuous shaking for 1 h at 5540°C. At the end of the incubation, the saturated Na2C03. T stirring with 8 vol centrifuged (15,00Og, 10 min, 0-4"C) and the organic layer removed. The l-chlorobutane was then extracted by stirring with 4 vol of 1M HC1 for 20 min, the aqueous layer was retained, and the pH ad'usted to 9 using concentrated ammonium hydroxide. he a ueous la er was then extracted against 8 vol of l-chlorautane. '$he organic layer was retained and evaporated under vacuum (Buchi Rotavap, Switzerland) at 50°C. Seven to 10 g of tissue was routinely extracted for uantitation by radioreceptor assay, while 17-25 g of rain were extracted for mass spectral analysis. All glassware was siliconized with dimethyldichlorosilane and either discarded or washed in chromic acid after each use. Chromatography Crude extracts were purified using reverse-phase HPLC (RP-HPLC) techniques. Samples were applied

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143

BENZODIAZEPINES IN HEPATIC ENCEPHALOPATHY

(U6K injector, Waters Corp., Milford, MA) t o a Bondapak CI8 (7.8 x 300 mm) RP column (Waters/ billipore). Absorbance was monitored at 229 nm (Waters model 490 E detector). The FBondapak column was eluted with a gradient of 90% trifluoroacetic acid (TFA, 0.143% v/v)/lO%acetonitrile (solution A) and 10% TFAI 90% acetonitrile (solution B), developed at a rate of 0 4 0 % solution B over 120 min at a flow rate of 1 mumin. Fractions were collected every minute. At the end of each run, the column was washed with 120 ml of 10% TFA/9O% acetonitrile and re-equilibrated with 60 ml of 90% TFA/10% acetonitrile. Maxima 710 software (Waters) was used for data acquisition and pump control. All fractions were dried under vacuum (Speedvac, Savant Instruments, Farmingdale, NY) a t 45°C and stored a t -20°C until assayed. Quantitative analysis The pharmacological characteristics and semi uantitative anal sis of crude extracts were determine using radioligan binding assays only. Quantitative analysis of HPLC-purified extracts was performed usin either radiometric or mass spectroscopic techniques. $he following internal standards were used to monitor the extraction efficiency of BZs from the initial brain homogenate (see under Extraction); prazepam (100 ng) was the internal standard for the quantitation of materials by HPLC/radiometric assay; d5-diazepam (50 ng) was used for quantitation by HPLC/mass spectroscopy. Radiometric analysis was performed using [3H]-Ro 151788 (final concentration, 1 nM) using previously described techniques (Basile et al., 1989, and this section), except that 5 times washed membranes prepared from rat cerebral cortex (containing ~ 0 . 1 m 2 rotein, Basile receptors. et al., 1989) were used as the source of The retention times of fractions that inhibited radioligand binding by 10-90% under these assay conditions were compared with 1,Cbenzodiazepine standards. The amount of inhibitory material in each fraction was quantitated by constructing com etition curves using known concentrations of unlabele 1,4-benzodiazepines assayed under identical conditions. The minimum quantity of diazepam reliably detected by this radioreceptor assay was 0.3 nglassay tube, respective1 The recovery was determined using the ratio of the nown extraction efficiency of the particular BZ to the observed efficiency of the prazepam internal standard. The concentration of material determined by the radioreceptor assay was then corrected for this recovery. The mean extraction efficiency for 10-100 n of diazepam and prazepam from 1-2 g of normal rat rain was 68 ? 7.2 (n = 13) and 16 5.3% (n = ll), respectively. When the total quantity of inhibitory activity was expressed in diazepam equivalents, the normalization of the quantity of BZ based on extraction efficiency was not used. Mass spectroscopic analysis was performed using a LKB-2091 spectrometer in the electron-impact mode with 70-eV ionizing voltage and 5O-kA ionizing current. The source was maintained at 290°C. The sample was admitted by a direct insertion probe and rapidly heated to 300°C. Materials [3H]Ro 15-1788 (sp. act. 78 Cilmmol), E3H1flunitrazepam (sp. act. 87 Cilmmol), [35SlTBPS(sp. act. 69

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Cilmmol), and [3H]muscimol (sp. act. 30.3 Cilmmol) were obtained from New En land Nuclear (Boston, MA). Ro 14-7437, Ro-7-1986, eschlorodiazepam, desand N-desmethyldichlorolorazepam, diazeEm, r. Peter Sorter (Hoffmannazepam were gifts from LaRoche, Nutle ,NJ). Oxaze am was a gift from Ayerst Pharmaceutica s (Chicago, I ). Prazepam, lorazepam, d5-diazepam, and alkaline protease were purchased from Sigma Chemicals (St. Louis, MO). Galactosamine HCl was urchased from ICN Biochemicals (Cleveland, OH). I-ehlorobutane was purchased from Fluka (Buchs, Switzerland). HPLC-grade water, methanol, acetonitrile (Fisher, Fairlawn, NJ), and TFA (Pierce Chemicals, Rockford, IL) were used for mobile phases. RESULTS The & and B,, for the binding of [3Hlmuscimol to the GABAAreceptor, r3H1Ro 15-1788t o the BZ receptor, and [35S]TBPSto the chloride ionophore in cerebrocortical or cerebellar membrane preparations from GalNtreated rabbits with HE were not si ificantly different from the vehicle-treated rabbits (Ta le I). Furthermore, neither the potency nor efficacy of NaCl andfor GABA in enhancing C3Hlflunitrazepam binding to membranes from either the cerebral cortex or cerebellum from rabbits with HE were significantly altered compared with control rabbits (Table 11). Extracts of GalN-treated rabbit brains significantly inhibited [3H]Ro 15-1788 binding to the BZ receptor (Fig. 1). These extracts (ICs0 = 490 i 76 pl) were approximately threefold more potent than identically preared extracts from vehicle-treated rabbits (extrapofated ICso = 1573 i 817 yl). Brain extracts from rabbits with HE inhibited [ HIRo 15-1788 binding in a competitive fashion (Fig. 21, since statistically significant reductions in the apparent affinity of this radioligand were observed (&:HE = 4.0 t 0.58 nM, n = 8; control = 1.5 F 0.14 nM, n = 5; buffer = 1.9 k 0.26 nM, n = 6 , P = 0.005, HE vs. control), with no change in B,, (HE = 2.4 t 0.28 pmollmg; control = 2.6 5 0.15 pmollmf. buffer = 2.3 ? 0.28 pmollm 1. Furthermore, the inhi itory effects of brain extracts rom rabbits with HE were reversible (Fig. 3) and were not significantly affected by heating to 100°C for 15 min (inhibitory activity decreased 14.4 2 9.6% after heating). Extracts of heart, liver, plasma, and kidney (but not small intestine or skeletal muscle) from rabbits with HE inhibited significantly more L3H]Ro 15-1788 binding than did equal volumes of correspondin extract prepared from vehicle-treated rabbit tissues ( able 111). Subsequent urification of the crude extracts from control rabbit rains usin RP-HPLC revealed 4-10 fractions that inhibited [3fi]R0 15-1788 binding (Fig. 4B). Two of these fractions had retention times (Rt: 77.8 0.7;83.5 i 0.3,mean tSEM,n = 9)corresponding to diazepam (R, = 78.1 i 0.7 min, n = 12) and oxazepam (R, = 84.9 ? 0.9 min, n = 10) standards, res ectively (Fig. 4B,C). The prazepam internal standard e uted with a retention time of 107 0.1 min. Fractions from buffer extracts (no tissue) and mobile hase residues had no effect on the radioligand bin ing assay. Brain extracts from rabbits with HE contained a larger number of fractions with significant inhibitory activity than did control extracts (Fig. 4A). These additional fractions had retention times of 79.5 -+ 0.6,82.6 5 0.34,

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A S . BASILE TABLE I. Radioligand binding to the GABAa receptor complex in control and GalN-treated rabbits'-3

(nM)

Bmax 1 (pmol/mg prot)

(nM)

(pmol/mg prot)

3.5 f 0.29

1.2 F 0.02

71 k 12

2.6 k 0.20

3.2 F 0.60

0.97 f 0.16

81 F 24

4.1 k 1.7

I .9 i 0.11

2.2 i 0.28

29 i 8.8

3.1 I0.59

1.8 f 0.28

2.0 F 0.22

28 F 5.4

3.9 10.68

48 f 4.4

2.6 i 0.22

-

Kd

Ligand/Region

[3H] Muscimol CTRL CTX HE CTX CTRL CB HE CB ["'S]-TBPS CTRL CTX

1

Kd

2

Bmax

HE CTX

45

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2.2 f 0.17

-

-

CTRL CB

43 I4.3

1.7 f 0.09

-

-

HE CB

49 k 6.0

1.5 f 0.14

-

-

1.3 k 0.04

3.1 i 0.22

-

-

13H]-Ro 15-1788 CTRL CTX HE CTX CTRL CB

1.3 k 0.06

3.2 i 0.04

-

-

1.1 I0.06

0.9 f 0.04

-

-

HE CB

1.2

* 0.03

1.1 F 0.02

-

-

2

'Theresults from thesebinding assays representthe mean k SEMof4 observationsconsisting of 1-Zrabbits per observation for L3H] muscimol; 6-8 obseruations for [35S]TBPS;and 4 observations for [JH]Ro- 15-1788.N o significant differences were observed between respective tissue groups from control rabbits and rabbits with HE, regardless of the radioligand used. :Results determined using nonlinear regression analysis (Accufit 1, Lundon Software). .'HE, rabbits with HE caused by GalN-induced FHF; CTRL, vehicle injected rabbits; CTX, cerebral cortex; CB, cerebellum; R,,, maximum binding density; Kd, apparent equilibrium dissociation constant.

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85.2 2 0.4, and 91.7 0.6 min (n -= ll),correspondin to deschlorolorazepam (R, = 82.4 2 0.9 min, n = 9) an$ lorazepam (R,= 90.6 t 0.9 min, n = 9)' as well as diazepam and oxazepam (Fig. 4A,C). All active fractions with these retention times were not found in every extract from encephalopathic rabbits. In addition, the retention times of several other active fractions did not correspond to those of any of the BZ standards available for analysis. The identity of these fractions as BZ was further sup orted by adding 100-ng quantities of N-desmethy diazepam, diaze am, and oxazepam to control brain homogenates, fol owed by extraction, chromatography, and assay (Fig. 5). The ability of the fractions from HE rabbit brain to inhibit [3H]Ro 15-1788 binding was increased by 63% in the presence of 200 pM GABA [from 18.5 2 4.2 ng/g diazepam equivalents (without GABA) t o 30.1 k 5 ng/g (with GABA), n = 6, P = 0.0396, paired Student's t-test, l-tailed (Fig. 611. This indicates that the contents of the extracts have pro erties consistent with that of BZ rece tor a onists. d i n g mass spectroscopic techniques, t e su stance eluting at 78-81 min was determined to be diazepam (Fig. 6). The concentration of diazepam in HE extracts as determined by mass spectroscopy was threefold

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eater than in control rabbit extracts (Table IV). SimiEr results were observed using radiometric quantitation techniques, which determined that the concentration of diazepam in extracts from rabbits with HE was 4 times higher than in control extracts (Table IV). DISCUSSION He atic encephalopathy is a neuropsychiatric disorder t at occurs as a result of acute or chronic liver failure (cf. Basile and Gammal, 1988, for review). Electrophysiological findings (Basile et al., 1988; Bassett et al., 1987; Schafer et al., 1984) in the GalN-treated rabbit model ofHE attributable to FHF stron ly suggest that an increase in GABAergic tone is invo ved in the atho enesis of this syndrome. Thus, in rabbits with HE, l3% receptor antagonists were found to excite Purkinje cell neurons, reverse their hy ersensitivity to depression by a GABAmimetic (Basi e et al., 19881, and improve their neurologic status (Bassett et al., 1987). These findings are most readily ex lained by an increase in the concentration or avai ability of a substance(s) with agonist properties at the BZ receptor. A subsequent autoradiographic investi ation provided additional support for the presence of Z receptor

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BENZODIAZEPINES IN HEPATIC ENCEPHALOPATHY TABLE 11. Enhancement of [3H]flunitrazepambinding by C1- and GABA in cortex and cerebellum of HE and control rabbits'-3 Region CTRL CTX HE CTX CTRL CB HE CB CTRL CTX HE CTX CTRL CB HE CB CTRL CTX HE CTX CTRL CB HE CB

nH

Emax (Wof baseline)

160 f 5.7 mM

3.0 i 0.8

18 f 3.8

160 f 1.3

2.1 i 1.4

17 i 8.0

122 I 16

1.4 i 0 . 3

23 i 1.3

144 k 16

0.8 i 0.2

30 i 8.2

* 0.2

42 f 2.1

Treatment

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2.5

* 0.6 pM

1.1

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6.0

* 1.4

2.3 f 0.3

1.2 f 0.1

38

0.7 f 0.6

0.9 rt 0.1

51 f 3.2

1.1 i 0.6

0.8 f 0.1

43 f 2.6

1.3 i 0.2 pM

0.7 t 0.1

85 i 3.2

1.6 2 0 . 3

0.8 rt 0.1

77 i 4.9

0.6 f 0.6

0.5 f 0.1

110 f 3.4

0.6 f 1.3

0.6 rt 0.1

96 f 10

'["H]Flunitrazepam binding (1 nM) to well-washed cerebral cortical and cerebellar membrane preparationswas measured in the presenceofNaCl(l0-400 mM), GABA (0.1-100 pM),and NaCl + GABA (200 mM NaCl 0.1-100 pM GABA). Values represent the mean f SEM of 4-5 observations. No significant differences in the binding potency or efficacy of GABA and/or NaCl in enhan~ing[~Hjflunitrazepam to respective brain regions were observed betweencontrol and GalN-treated rabbits. 2Results determined using nonlinear regression analysis (Graph-Pad 3.0, GraphPad Software). :%HE,rabbits with HE due to to GalN-induced FHF; CTRL, vehicle injected rabbits; CTX, cerebral cortex; CR, cerebellum; nfj, Hill coefficient, Emax,maximum enhancement.

145

0.5 1.0 1.5 2.0 2.5 [%]&I 15-1788 Bound (pmol/q protein)

3.0

Fig. 2. Representative Scatchard plots of the inhibition of i3H1Ro 15-1788 binding to normal rat cerebrocortical membranes by brain extracts from rabbits with HE (m)and control rabbits ( 0 )compared with buffer alone (0).Assays were performed using 50 pl of 13HJ-Ro15-1788 (0.5,1,2,4, 8, and 10 nM, final concentrations), 350 p1 of crude brain extract from HE or control rabbits, or buffer. For nonspecific binding, 50 p1 of Ro 14-7437 (10 pM final volume) was added. Total assay volume was 0.5 ml. Note the decrease in the apparent affinity (increased K,) of I3H1-Ro 15-1788 for the BZ receptor in the presence of brain extract from GalN-treated rabbits (3.1 nM) compared with control rabbit extracts (1.4 nM) or Tris-citrate buffer (1.9nM), characteristic of competitive inhibition processes. No significant change in the B,, (2.2, 2.6, and 2.4 pmoVm protein, HE, control, and buffer, respectively) was observed. Bound/kee values are in units of (pmoVmg of protein bound)/(pmoVassay).

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Extract Volume (ml) Fig. 1. Inhibition of [3H]Ro 15-1788 (1nM) binding to rat cerebral cortex membranes by extracts of brains from rabbits with HE (m) and control rabbits (0). The crude isolates were prepared by extracting protease-treated brain homogenates with chlorobutane. The IC,, value for the inhibition of [3H]Ro 15-1788 binding by brain extracts from rabbits with HE is 490 ? 76 p1. Because of the low level of inhibitory activity in the control rabbit extracts, the IC,, for the inhibition of [3HJ-Ro15-1788 binding was estimated by extrapolation from the competition curve (- - -) to be 1,573 k 817 pl. Sigmoidal competition curves were fitted to the data points, and IC,, values determined using Graph-Pad 3.0. Values represent the mean ?SEM of 3-20 determinations.

Fig. 3. Reversibility of the inhibition of 13H]Ro 15-1788 binding to cerebrocortical membranes by brain extracts from rabbits with HE. AIiquots of Tris-citrate buffer (400 p1) (m)or brain extracts from rabbits with HE ( 0 )were added to tissue samples and incubated for 1h a t 25°C. Sufficient extract from rabbits with HE was added to inhibit approximately 70% of [3HIRo 15-1788 binding. For 12 assay tubes, 4.8 ml of extract was combined with 0.6 ml of rat cortex membrane preparation. This mixture was incubated for 1 h at W " C . After incubation, three 450-p1 samples were taken for the 0 wash point of the assay. The remaining mixture was centrifuged, the supernatant removed, and the pellet resuspended in 4.05 ml offresh buffer; three 450-pl samples were takenfor the first wash point ofthe assay. This procedure was repeated for a total ofthree washes, using decreasing volumes ofwash buffer. All tubes were maintained on ice during the wash procedures. After all the washing steps were completed, the binding assay was initiated by the addition of 1 nM.f3H]Ro 15-1788, incubated for 1 h at 0 4 ° C and terminated according t o techniques described under Methods. Values represent the mean tSEM of 6-8 observations. ** = P < 0.01, * = P < 0.05.

146

AS. BASILE 15.0

TABLE III. Inhibition of [SHIRo 15-1788 binding to benzodiarepine receptors by extracts of tissues from HE and control Tissue

HE3

Control

Heart Liver Piasma Kidney Rrain

60 f 10* 65 i 6.7** 55 i 7.6t 35 i 9.91 52 i 146

16 i.4.5 20 i 9.8 21 -f 6.3 15 i 5.8 17

2.0

‘Each value is the mean i S E M of 4-1 1 observationsof the percentage inhibition of [“HI-Ro 15-1788binding (1 nM) to well-washed rat cerebrocortical membranes by 350.~1aliquots of extract (-350 mg of tissue, wet weight) from GalN-treated and control rabbits. %” significant differences were found in the inhibition mediated by extracts of small intestine or skeletal muscle from control and HE rabbits. 3Inhihition of [3H]-Ro 151788 binding by extracts of HE rabbit tissue was significantly greater than control group extracts using the 1-tailedStudent’s t-test. *P = 0.004. **P= 0.003. tP = 0.014. t P = 0.047. §P = 0.05.

20

0

0.0207 1

40

60

80

106

- 2.0

B. CTRL

-

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0.015 -

1.5

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agonists in HE (Basile et al., 1990a). A significant decrease in the level of [3Hl-Ro 15-1788 binding to BZ receptors was recorded in the cerebral cortex, cerebellum, and midbrain nuclei of thin brain sections from rabbits with HE. Simple washing of the sections before incubating them with the radioligand raised the bindin density to control levels. This observation suggeste that a ligand was present in HE that bound reversibly to the BZ receptor. Further analyses indicated that this ligand had pharmacologic properties consistent with that of a BZ receptor agonist. However, no definitive studies of the ossible role of alterations in the GABA receptor comp ex in HE were performed, nor was the chemical structure of the BZ receptor ligand(s) present in HE determined in these investigations. The resent study provides evidence that alterations in the 8ABA or BZ receptors or in the chloride ionophore do not play a role in the enhancement of GABAergictone observed in this animal model of HE. This is indicated by the lack of change in the e uilibrium binding constants for radioligands s ecific or the constituent components of the GABA-B!2 receptor com lex in the cortex and cerebellum from rabbits with HE. urthermore, no alterations were found in the ability of C1- and/or GABA to enhance [3H]flunitraze am binding to BZ receptors in the cortex and cerebe lum from rabbits with HE. These observations are consistent with previous reports indicating the lack of change in GABA and BZ receptors in other animal models of HE (Maddison et al., 1987a,b; Roy et al., 1988)or in brain samples from patients with HE (La1et al., 1987). This effectively rules out changes in the density or affinity of GABA and/or BZ receptors as a factor contributing to the enhanced GABAergicneurotransmission reported in HE. By contrast, elevated levels of a substance(s) that inhibits radioligand binding to the BZ receptor were observed in the brain and in several peripheral tissues from GalN-treated rabbits. This heat-stable, reversible, competitive inhibitor of the BZ receptor has the properties of a BZ receptor a onist, as indicated by the increased inhibition of [#HI-Ro 15-1788 binding by extracts of encephalopathic rabbit brain in the presence of GABA (Karobath et al., 1983). Subsequent urification of crude brain extracts from rabbits with E and con-

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Fig. 4. Chromatograms of extracts from 9.7 g of brain from a rabbit with HE (A), 8.7 g of a control rabbit brain (B), and LOO ng each of 1,4-benzodiazepinestandards (C) purified using HPLC techniques (see under Methods). The 1,4-benzodiazepine standards include deschlorodiazepam (DCD, R, = 55.8 min), desmethyldiazepam (DMD, R, = 66.3 min), Ro 7-1986 (R, R, = 73.6) diazepam (D, R, = 78.5), deschlorolorazepam (DCL, R = 83.0), oxazepam (0, R, = 85.5) and absorption monitored a t 229 nm. A,B lorazepam (L, R, = 91.3). Chromatograms overlie bar graphs of the equivalent quantity of diazepam (ng/g tissue wet weight) found in each fraction usin radio metric uantitation. The diazepam concentrations determine$ radio: metrically for the HE (fractions 78-80) and control (fractions 78, 79) rabbit pre arations were 3.8 and 1.3 ng/g, respectively. Total BZ receptor iniibitory activity in the HE and control rabbit preparations were 24.5 and 7.77 ng/g, respectively.

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trols using HPLC resolved several fractions that inhibit radioligand binding to BZ receptors. Many, but not all, of these fractions were found to comigrate with known BZs. Identical profiles of fractions containing inhibitory substances were not detected in every extract, but substances comigrating with the diazepam standard were consistently present. Mass s ectroscopic anal sis confirmed the identity of one oft ese unknown su stances as diazepam. While the presence of 1,4-benzodiazepines

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Fig. 5. Comigration of inhibitory fractions from an HE rabbit brain extract (9 g,+) with control rabbit brain extract (18.5g, ---m--- bars), spiked with 100 ng of N-desmethyldiazepam (R, = 6 M 7 m i d , diazepam (R, = 77-79 min), andoxazepam (R, = 85 mid. Fraction activity is represented as the percentage inhibition of f3HIRo 15-1788binding to cerebral BZ receptors by 100-pl aliquots of control brain fractions, and 1ml of HE brain fractions.

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Fig. 6. Enhanced inhibition of L3HJ-Ro15-1788binding to BZ receptors by an HE rabbit brain preparation in the presence of 200 p M GABA. Fraction activity is represented as the percentage inhibition of [3H]Ro 15-1788 binding to BZ rece tors Each bar represents the activity of 500-pl aliquots of HPLC corumnfractions from an extract of 7.1 g of HE rabbit brain. Total BZ receptor inhibitory activity in these HE rabbit fractions was 5.8 n lg without GABA, and 17.7 ngig in the presence of GABA. The total BE receptor inhibitory activity in a control rabbit extract prepared in parallel with the HE rabbit extract was 1.19 nglg. The retention time of a diazepam standard for this run was 80.6 min, with the TFA concentration in the mobile phase = 0.1%.

in mammalian brain has been previously reported (Manning et al., 1986; Medina et al., 1988; Sangameswaran et al., 1986;Unseld and Klotz, 1989; Wildmann et al., 1987), this is the first report of an elevated concentration of a BZ receptor ligand being associated with a pathophysiologic state. The diazepam concentration in extracts of control rabbit brains (1 ng/g) are consistent with levels previously reported in normal rat, bovine, and human brains (Medina et al., 1988; Sangameswaran et al., 1986; Wildmann et al., 1987). In

Fig. 7. Mass s ectra of fractions with inhibitory activity comigrating with diazepam Kom an extract of 17.6 g of HE rabbit brain (A), an extract of 19.4g of control rabbit brain (B), and pure standards (C). C: Standards containin 100ng of diazepam and d5-diaze am dissolvedin ethanol and appliefdirectly to the sample cup. A,!%: 50 ng of d5diazepam was used as the internal standard. The peak ratios used for quantitative analysis are 2561261 and 2831289 d z .

contrast, the diazepam concentration was increased three- to fourfold in brain extracts from GalN-treated rabbits. Furthermore, the total concentration of HPLCurified substances inhibiting radioligand binding to [Z rece tors (expressed in diazepam equivalents) was elevateifourfold (Table IV, Fig. 4). This involvement of BZ receptor agonists in the pathogenesis of HE is supported by recent findin s of elevated concentrations of BZ-like substances in t e brain and eripheral tissues of rats with thioacetamide-induced HF (Basile et al., 1989,1990b) and in the cerebrospinal fluid and plasma of pat.ients with HE (Mullen et al., 1989; Olaasma et al., 1989), indicating that this is neither a species- nor model-dependent phenomenon.

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'Brains were extracted using techniques described under Methods. Extracts from 7-10 g of rabbit brain were applied to a C18 reverse-phase column and eluted with a TFAIacetonitrile gradient over 120 min. Fractions were taken every minute, dried, and assayed for their ability to inhibit [3H]-Ro 15-1788binding to BZ receptors a s described under Methods. Mean retention times for the active fractions were correlated with retention times for diazepam and prazepam standards.Therecovery of diazepam from the extractis based on the ratio of themean extraction efficiency of diazepam and the observed extraction efficiency of the prazepam standard in the preparation. 2Total inhibitory activity was determined for ail active fractions in the extract and expressed a s diazepam equivalents (ng/g tissue wet weight) without normalization for extraction efficiency. When massspectroscopywas used for quantitation, 16-25g of tissue was extracted and purified by the gradient HPLC technique described. 3Fractions corresponding to the retention time of the d5-diazepam standard were dried into sample cups for mass spectroscopy. 4Values are the mean fSEM of 4-11 observations; HE, significantly different from control values, as determined by Student's t-test (I-tailed). *P< 0.05. t P 0.01.

The presence of BZ receptor ligands in HE is compatible with the observation that the constituent components of the GABA-BZ receptor complex are unchanged. Most studies of the pharmacologc regulation of BZ receptors report little (Rosenberg and Chiu, 1981; Crawley et al., 1982) or no (Mohler et al., 1978; Braestrup et al., 1979) chan e in the density and no change in the affinity of radioyigand binding t o BZ receptors after the administration of moderate doses (2.5-5 mgl kg) of BZ receptor agonists (diazepam, flurazepam, clonazepam) daily for periods of 5-21 days. Similarly, a moderate decrease in the level of GABA-enhanced [3H]flunitrazepam and [3H]lormetazepam binding was observed after 9-21 days of benzodiazepine administration (Stephens and Schneider, 1985; Gallager et al., 1984). Given the requirement for the long-term presence of BZ receptor agonists to elicit modest degrees of desensitization, there may be neither sufficient time nor a stimulus of sufficient intensity for compensatory changes in the GABA-BZ receptor complex to occur in the GalN-treated rabbit model of HE before the animal succumbs to liver failure. Whether the whole-brain concentrations of the BZ receptor agonists observed in rabbits with HE are elevated sufficiently to account for the neuropsychiatric manifestations of HE are unclear. After the administration of 1.3 m /kg diazepam (a behaviorally active but nonhypnotic ose) to normal rats, brain diazepam levels were 35-fold higher than the concentrations reported here (Mennini and Garattini, 1983). Nonetheless, autoof brains from rabbits with HE regional heterogeneity in the agonists, such that radioliand binding to BZ receptors in the cortex and cerebelk m was reduced by 22% and 42%, respectively (Basile et al., 1990a).These values are similar to the levels ofBZ receptor occupation observed after the administration of 4-18 mgkg diazepam (anticonvulsant and hypnotic dose, respectively) to normal rats (Lippa et al., 1979;

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Paul et al., 1979).In addition, the ability of BZ rece tor antagonists to correct the electrophysiological an behavioral manifestations of HE in animal models (Basile et al., 1988; Gammal et al., 1990) and man (Bansky et al., 1989; Ferenci et al., 1989; Grimm et al., 1988; Scollo-Lavizzariand Steinmann, 1985)indicate that the regionally high concentrations of BZ receptor agonists, in HE may be sufficient to produce some of the behavioral manifestations of the syndrome. These effects may be further enhanced by the other central nervous system changes accompanying HE, such as increased GABA concentrations (Bassett et al., 19901, and/or altered glutamate metabolism (Schenker and Brady, 1988). The origin of the diazepam detected in normal and encephalopathic rabbit brains is presently unknown. However, dietary sources may play an important role, since 1,4-BZsand other BZ receptor ligands have been reported in a number of plant and animal foodstuffs (Medina et al., 1988; Wildmann et al., 19881, including animal chow (unpublished observations). Whether these dietary sources directly provide all the substances observed in this study or only their precursors is currently under investigation. However, in view of the ability of fungi (Luckner, 1984) and cultured cell lines (DeBlas et al., 1987) to synthesize BZs, the possible contributions of gut flora, adrenal, and/or neuronal tissue to the production of "endogenous" BZs in mammals must be considered. Together, dietary sources and the endogenous de novo synthesis of benzodiazepines may be responsible for the BZs present in animal brains. Thus, naturally occurring 1,4-benzodiazepines and other BZ receptor agonists may play significant roles not only in the pathogenesis of HE, but in other pathologcal states, such as anxiety (Lippa et al., 1979, Medina et al., 1983; Rag0 et al., 1988) and sleep disorders (Lavie, 1989). SUMMARY The presence of increased levels of BZ rece tor agonists in this and other animal models of HE (gasile et al., 1988, 1989, 1990a,b; Gammal et al., 1990), and in humans with HE (Mullen et al., 1989; Olasmaa et al., 1989) suggests that therapeutic modalities directed at decreasing the effective concentrations of these compounds at the BZ receptor, such as the administration of BZ receptor antagonists (Bansky et al., 1989; Ferenci et al., 1989; Grimm et al., 1988; Scollo-Lavizzari and Steinmann, 1985) may be useful in the management of this important neuropsychiatric syndrome in man. ACKNOWLEDGMENTS I would like to acknowled e the contributions of E.A. Jones, S.H. Gammal, K.D. lkfullen, L. Pannell, H. Fales, T. Jaouni, and P. Skolnick, without whose help this research would not have been possible. REFERENCES

2

TABLE IV. Levels of diazepam and total diazepam equivalents in brain extracts fromcontrol rabbits and rabbits with HE'-4

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8.

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Wildmann, J.,Vetter, W., Ranalder, U.B., Schmidt, K., Maurer, R., and Mohler, H. (1988)Occurrence of pharmacologically active benzodiazepines in trace amounts in wheat and potato. Biochem. Pharmacol., 37:3549-3559. Unseld, E., and Klotz, U. (1989) Benzodiazepines: Are they of natural orign? Pharmaceut. Res., 6:l-3.