A study of 3H-PK 11 195 binding to “peripheral-type” benzodiazepine receptors on human lymphocytes

Journal q[' the Neurok)gical Sciences, 102 ( 1991 ) 209-219 Elsevier

209

JNS 03508

A study of 3H-PK 11 195 binding to "peripheral-type" benzodiazepine receptors on human lymphocytes E v i d e n c e of decreased binding in hepatic e n c e p h a l o p a t h y P. Ferrero ~, P. Rocca 2, A. Gualerzi 2, P. Benna ~, F. Enrichens 3, G. Olivero s, P, Mao s, L, Ravizza 2 and B. Bergamasco ~ Departments ~f ~Neurologv, 2psychiatry and 3Emergency Surgery, University of Turin, Turin (ltalvj (Received 8 July, 1990) (Revised, received 26 October, 1990) (Accepted 30 October, 1990)

K
Summary In an attempt to assess the involvement of the "peripheral-type" benzodiazepine receptors (pBDZR) in hepatic cncephalopathy (HE), we examined the binding of the isoquinoline carboxamide derivative 3H-PK 11 195 to lymphocyte membranes from a group of patients with liver cirrhosis with or without clinical signs of HE and normal controls. Lymphocyte 3H-PK 11 195 binding is saturable, with high affinity and presents the pharmacological specificity corresponding to pBDZR. A significant 40 °,0 decrease in the number of~H-PK 11 195 binding sites, without a concomitant change in the apparent affinity, is observed in the group with HE as compared to the controls, but not in that with liver diseases without HE. The decrease in binding capacity correlates significantly with the clinical grading of HE, but not with age, sex, aetiology of cirrhosis or presence of surgical shunt. In contrast to the reduction of pBDZR, 3H-N-methylscopolamine binding to lymphocyte muscarinic receptors is not affected in HE. These findings are consistent with a role for pBDZR in HE and may stimulate studies of endogenous modulators and pharmacological agents for these receptors in the disease.

Introduction Hepatic encephalopathy (HE) is a complex neuropsychiatric syndrome that occurs as a complication of liver failure in patients with acute or chronic hepatocellular diseases. Despite extensive investigations, the pathogenesis of HE is unknown although it probably reflects the neuronal exposure to toxic substances not cleared by the liver (Jones and Gammal 1988). Since the early 1980s, theories on the neurochemical basis of H E have focussed on activation of inhibitory gamma-aminobutyric acid systems, but the mechanisms involved are still unclear (see Basile and Gammal (1988) and Butterworth and Layrargues (1990) for review). Neurotransmission effected by GABA is predominantly mediated by a gated chloride channel intrinsic to the GABA A receptor. The GABAA receptor is a large supramolecular complex that includes a recognition site for Corre.spondetwe to. Patrizia Ferrero, M.D., Neurological Clinic, Via Cherasco 15, 10126 Torino, Italy. Tel./Tclefax: (01 I)6963487.

G A B A and an allosteric modulatory center with binding sites for several compounds, including benzodiazepines (BDZ) and neuropeptides derived from diazepam binding inhibitor (DBI) (Guidotti et al. 1983; Ferrero et al. 1986; Barbaccia et al. 1988; Barnard et al. 1989). The enhancement of GABA-ergic tone in HE could occur as a result of an increased availability of GABA at its receptors, a higher density of GABA or BDZ receptors or an allosteric augmentation of the potency or efficacy of GABA mediated by the molecular events at the BDZ receptors. Although investigations of the first two possibilities have yielded inconsistent results, recent electrophysiological and biochemical findings strongly suggest that the increased GABA-ergic tone in H E is attributable to the presence of increased concentrations of an endogenous ligand for the BDZ recognition sites (Bassett et al. 1987: Basile and Gammal 1988; Mullen et al. 1988). Several reports have indeed demonstrated that changes in the content of endogenous compounds with BDZ-like structure do occur in H E and, in recent studies, the levels of DBI and its processing product octadecaneuropeptide were also significantly

0022-510X/91 '$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)

2 ll) increased in the cerebrospinal fluid and brain of clinical and experimental H E (Basile et al. 1989: Olasmaa et al. 1989: Mullen 1989; Rothstein et al. 1989: Lavoie et al. 1989). Moreover, a number of recent clinical studies report of beneficial effects of flumazenil, a selective BDZ antagonist (Moheler and Richards 1981), in ameliorating symptoms of H E in the patients (Scollo-Lavizzari and Steinmann 1985: Grimm et al. 1988; Bansky et al. 1989: Ferenci et al. 1989). In addition to BDZ binding sites associated with GABA A receptors, molecular biological and radio/receptors studies have revealed the presence of another class of BDZ binding sites, highly concentrated in the membranes of mitochondria isolated from certain peripheral tissues. In the central nervous system, these BDZ binding sites. referred to as "peripheral-type" BDZ receptors (pBDZR) are mainly located in glial cells (see Squires (1984) Anholt (1986) and Saano et al. (1989) for review). Despite their abundance and widespread distribution, the physiological and pharmacological relevance of p B D Z R remains to be established. Several endogenous substances have been identified that could physiologically subserve these pBDZ binding sites, including porphyrins and peptides derived from DBI (Verma et al. 1987; Slobodyansky 1989). There are relatively few reports on the status of p B D Z R in human pathology. An increase in their number was reported in the brain during carcinogenesis. Huntington's chorea and Parkinson's disease, which was associated with events of glial proliferation (Benavides etal. 1987: Starosta-Rubinstein et al. 1987: Ferrarese et al, 1989). By contrast, a reduced density of p B D Z R was demonstrated in platelets from patients with anxiety disorders, nonabstinent alcoholics and patients chronically treated with neuroleptics, but the reasons for such changes are unclear (Gavish et al. 1986b; Weizman et al. 1986, 1987: SuranyiCadotte et al. 1988). In the current study, we have attempted to investigate a possible involvement o f p B D Z R in H E and we have studied the binding of PK 11 195, an isoquinoline carboxamide derivative that binds selectively p B D Z R (Benavides et al. 1983), in lymphocyte membranes from a population of patients with advanced chronic liver diseases with or without clinical symptoms of H E and in a comparable group of controls. If an altered metabolism of an endogenous benzodiazepine receptor ligand may represent a fundamental pathogenetic factor in HE. a disorder of p B D Z R might be theoretically expected.

Materials and methods

Subjects In a preliminary study, lymphocyte pBDZR were assayed in a population of 35 healthy controls (age range

20-75 years) in order to examine the binding properties and possible relationships to physiological and clinical variables. Forty patients with liver cirrhosis, who were both in- or outpatients at the Departments of Emergency and Surgery and Neurology, University of Turin. were selected for the study. Twenty-one out of the patients did not exhibit clinical signs of H E (group liver diseases. LD) whereas 19 were encephalopathics and had an impaired mental status of different degrees (group HE). Thirty healthy volunteers with normal hepatic function served as a control group. No member of either group had taken BDZ or other psychotropic drugs for at least 3 months or had a history of neuropsychiatrlc or immunologic diseases, as verified by patient, family and physician interviews and scrutiny of all medication records. Informed consent was obtained from all participants, or from their relatives whenever appropriate. "after the procedure was fully explained Demographic and clinical details of the studied population are summarized in Table 1. Patients with hepatic diseases were diagnosed on the basis of biochemical, scintigraphic, altrasonographic and endoscopy findings and by liver histological examinations as well. None of the patients was suffering from other non hepatic diseases that might cause deterioration in mental state. All patients received a standard treatment with a low protein diet: lactulose was administered orally or by enema to the obtunded patients. Patients evaluation

The clinicians were informed about the planned study and instructed not to administer BDZ or other sedative drugs before the start of the study. Routine toxicologic screenings for BDZ or barbiturates were performed in all patients on blood and urine samples, collected on the morning of the blood lymphocyte purification. All analyses were performed in the general clinical laboratory, Ospedale S. Giovanni Battista, Turin, using semiquantitativeenzyme immunoassays (Emit tox serum and DAU. Syva Co.. detection limits: secobarbital, 3/~g/ml serum and 0.3 #g/ml urine: diazepam 0.3/ag/ml serum and oxazepam 0.3 gg/ml urine). No samples contained traces of these drugs. Each patient underwent two serial neurologic and psychiatric examinations. A computerized tomography (CT) scan of the head was performed on each case within 2-7 days of the patient's testing. All scans were performed without contrast medium and without sedation. Quantitative CT scan indexes for the measures of cerebral atrophy were not performed. In 36 patients CT scans revealed no evidence of organic brain lesions. However. in the other 5 patients, the presence of mild diffuse sulcus widening and or ventricular enlargment was noted (onecase withLD and 4 with HE). Clinical neurological status was assessed

211 TABLE 1 CHARACTERISTICS OF THE PATIENTS WITH LIVER DISEASES AND CONTROLS HE indicates hepatic encephalopathy, LD liver diseases without HE, MMSE Mini Mental State Examination, DS Digit span, ND not determined. Values arc mean + SD. Parameters

Number of subjects Agc (years) Sex (males/females)

Group HE Stage l-II

Stage Ill-IV

12 53.3 + 12.8 5/7

7 60.3 _+9.2 6il

Group LD

Controls

21 49.6 _+ 12.3 12/9

30 47.3 + 19.1 12/18 -

Aetiolugy (cirrhosis) HBV-related Alcoholic Mixed Carcinoma

2 5 3 2

3 3 1 -

8 13 -

Esophageal varices Sclerotherapy Warren shunt Port() caval shunt

i2 10 5 2

7 7 4 -

15 15 3 3

0/7

21,,'0

30/0

18.0 _+5.2

ND

28.1 _+ 1.0

28.5 _+ 1.2

4.1 _+ 1.9 2.3 _+ 1.5

ND

6.5 + 1.2 4.6 _+ 1.4

6.7 _+ 1.2 4.7 + 1.1

EEG ~, normal/abnormal MMSE Digit span forward backward

4,,'8

-

" Normal EEG include minimal transient theta rhythm. on each patient in combination with a conventional E E G recording on the day of blood sampling. E E G s were analyzed independently by the neurological staff. Normal E E G s were considered those with predominant parietooccipital alpha rhythm and minimal transient theta activity. A standardized clinical protocol was adopted. Besides a complete neurologic examination with attention to mental function, this protocol included a semi-structured psychiatric interview using the Brief Psychiatric Rating Scale, the Mini Mental State Examination ( M M S E ) to detect diffuse cognitive dysfunction and the Digit Span, a subtest of the Wechsler Adult Intelligent Scale, to assess attention and concentration (Folstein et al. 1975). Other psychometric tests such as Trailmaking and Digit Symbol, which seems particularly sensitive to detecting the presence of subclinical H E (Rikkers et al. 1978; Tarter et al. 1984) were not performed. Only patients who could cooperate were psychometrically tested. Normal range for each psychometric test was defined as the mean _+ 2 SD determined in the control group. Clinically, the degree o f H E was semiquantifled to stage I - I V , essentially on the criteria proposed by Pappas and Jones (1983); stage 1, mild confusion but preserved reaction to simple verbal commands, ability to recall past events, shortened attention span, mild impairment of addition and subtraction, irritability, agitation,

tremulousness, slowed motor coordination; stage 2, drowsiness, lethargy, gross personality changes, disorientation to time, poor recall, asterixis, paratonia, ataxia; stage 3, delirium or profound confusion, somnolence with arousability, disorientation to time and place, incomprensible speech; stage 4, coma. According, patients with H E were subdivided in mild-moderate H E (stage I and II, n = 5 and 7, respectively) and severe H E (stage III and IV, n = 5 and 2). All patients accepted into the group with L D showed normal neurological examination and E E G recordings, except for 3 subjects with fine postural tremor. Their mental status was considered normal as indicated by the clinical evaluation and psychometric testing. Conversely, the encephalopathic subjects were variably affected on these parameters. The results of psychometric tests and E E G recordings in patients and controls groups are indicated in Table 1. Routine blood test examinations were performed in all patients and controls. Blood T-lymphocyte subpopulations were also analysed in each participant by direct immunofluorescence on an automated laser flow cytometry system (Ortho-Spectrum). Monoclonal antibodies of the O K T series were used (Ortho-Immune): OKT3 defines mature T cells, O K T 4 marks the helper/inducer subsets and O K T 8 the cytotoxic/suppressor. In our laboratory, the normal

212 FABLI! 2 LABORATORY

DATA OF PATIENTS

WITH LIVER DISEASES

AND CONTROLS

H E i n d i c a t e s h e p a t i c e n c e p h a l o p a t h y . L D liver d i s e a s e s w i t h o u t H E . B U N b l o o d u r e a n i t r o g e n . Values a r e m e a n _~ SD. Parameters

Group HE S t a g c 1-11

Group LD

Controls

Stage Ill-IV

A l b u m i n (g/dl)

3,1 -

0.7

2.8 z

0.5

3.3 +

0,8

4.4 z

Bilirubin (total, m g / d l )

3.1 -

1.4

4.0 z

1.5

2.0 +

13

0.8 +_ ().3

17,5 z

4.2

20.6 z

5.8

18.0 ±

7.1

P r o t h r o m b i n t i m e (sec) A m m o n i a (/tg/dl) BUN (mg'dll W h i t e cells Itotal. 1 × 103 )

109.3 + 42.8 49.9-

18.6

13.2 ±

0.4

2.2

107.8 _+ 39.1

84.4 + 27 7

65,0 ~_ 8.6

38.4 z 10.5

46.3 - 14.4

40.0 + 11,5

5.2 z 1.8

4.9 z 0.7

5.1 - 1 2

0.0 + 1.5

O K T 3 ~mean % )

73.9 z 7.f)

76.2 _+ 4.9

74.1 ,_- 4.8

73.1 ~ 6.8

OKT4 (mean %)

47.2 _+ 4.8

49.2 z 7.2

45.4 + 4.2

43.6 .t 6.1

O K T 8 ( m e a n ~,)

27.6 _, 5,6

28.6 z 5.0

29.5 - 5,(,

30,0 * 6.4

percentages (mean _+ SD) for OKT3. OKT4 and OKT8 were respectively 73 z 7.43 + 7 and 33 z 7%. The profile of some biochemical parameters in the patients and control groups are shown in Table 2.

L ymphocytes preparation Lymphocytes were purified from blood (50-70ml) according to the method of Boyum (1968), with minor modifications. Briefly, heparinized blood diluted with 1 volume phosphate-buffered saline, pH 7.4. was centrifuged at 1000 × g for 20 min at 24 °C, over a density gradient of Lymphoprep (Nycomed). Lymphocytes were harvested and washed twice in Hank's balanced salt solution (GIBCO). Ten ml blood yielded approximately 1-2 x 107 mononuclear cells consisting of approximately 80-85°% lymphocytes, 10-15% monocytes over 985o cell viability, as assessed by trypan blue exclusion. The fresh lymphocyte pellet was resuspended in 10 ml ice cold 10 mM Tris-HCl buffer, pH 7.4, containing 10 mM NaC1 3 mM MgC12, and cells lysated using a Brinkmann Polytron homogenizer (setting 6) for 20 sec. Lymphocyte membranes were sedimentated at 48 000 × g for 20 min at 5 ° C, washed once in the same buffer and then stored at - 4 0 °C until used (5-10 days later). On the day of assay, lymphocyte membranes were resuspended in the respectwe assays buffers, washed twice by centrifugation (48 000 x g for 10 min) and then used immediately. In selected experiments sheep erythrocyte rosette forming cells (E-RFC) and non E-RFC were separated from the total blood lymphocyte preparation, deprived o f adherent cells. E-RFC plus T cell preparations (purity higher than 95~o as checked by specific anti-CD3 monoclonal antibody, kindly provided by the Laboratory of Cell Biology, University of Turin l were resuspended in RPMI 1640 medium containing glutamine (Gibco) at a concentration of 5 × 105cells'0.5ml and then processed for 3H-PK 11 195 binding assay.

~H-PK ll 195 binding assay Binding assays for 3H-PK 11 195 (New England Nuclear, specific activity 82.3 Ci/mmol) to lymphocyte membranes were conducted in 50 mM Tris-HC1, pH 7.4 at 4 ° C. The binding mixture, in a final volume of 500/~1, contained 400/~1 membranes (50 #g proteins), 50/~t 3H-PK 11 t95 at the desired concentrations and 50 #1 buffer or t ~M PK 11 195 to assess the extent of non specific binding. Following a 30-min incubation time. the reaction was terminated by the addition of 3 ml cold buffer and vacuum filtration through GF~ C filters that were washed 3 times with 3 ml buffer. The radioactivity retained by the filters was counted 12 h later in a conventional scintillauon counter. For each person studied, saturation isotherms of 3H-PK 11 t95 binding were determined using at least 6 radiotigand concentrations (ranging from 0.5 to 50 nM). H-N-Methylscopolamine l ~H-NMS) binding assay Lymphocyte muscarinic receptors were assayed in a restricted population of patients with or without H E and comparable controls by studying the binding of the specific antagonist 3H -N-methylscopolamine (3H-N MS). ~H-NM S binding was performed as previously described (Eva et at. 1989). Analysis of data Binding was quantified per milligram of protein as assayed using the method of Lowry etal. (t95I), with bovine serum albumine as internal standard. Binding indexes, maximum number of binding sites (B~a x) and the apparent dissociation constant (Ka) were calculated from the respective saturation binding isotherms by Scatchard analysis. Binding parameters for the various patients groups and controls were compared by statistical analysis usmg Student's two-tailed t test. Correlations among the various biochemical and clinical parameters were determined by linear regression analysis.

213 Results

Properties of 3H-PK ll 195 binding to human lymphocyte membranes Radioligand binding experiments performed with lymphocyte membranes prepared from fresh pooled blood of healthy donnors revealed the existence of specific and saturable binding sites for 3H-PK 11 195. Representative isotherms of 3H-PK 11 195 specific and non specific binding to this membrane preparation are shown in Fig. 1. The specific binding increased in a hyperbolic fashion with increasing concentrations of 3H-PK l 1 195. The non-specific binding was less than 10°'o of the total binding and increased linearly with increasing concentrations of 3H-PK 11 195. Scatchard analysis of the data (inset), together with a Hill number of 1.04, suggested a homogeneous population of binding sites. In these membranes, a K d of 9.3 _+ 0.3 nM and a B .... of 10.9 + 0.4 pmol/mg protein were calculated. Kinetics of3H-PK 11 195 binding at 4 °C to lymphocyte membranes revealed that specific binding of this ligand was rapid, reaching steady-state conditions after 15 rain. Furthermore, after the equilibrium was reached, the binding was almost completely' reversible by 1 #M PK 11 195. The binding was linear with respect to protein content (up to 250 #g proteins) (data not shown). Increasing the incubation temperature from 4 : C to 24 °C and treatment of lymphocyte membranes with 0.01 ",, Triton X-100 failed to

change the binding kinetics (results not shown). In other experiments (n = 3), the effect of chloride ions on 3H-PK 11 195 binding was studied by saturation analysis in the absence of CI (Na-K phosphate buffer) and in the presence of 50 mM CI (Tris-HCl buffer). Chloride was found not to affect significantly 3H-PK 11 195 binding. A binding site density of 11.8 + 0.9 and 12.8 + 1.0 pmol/mg protein and an affinity (Kd) of 8.5 + 0.9 and 8.0 _+ 0.6 nM was found in the absence and presence of chloride, respectively. Sonication of lymphocytes probably denaturates the receptors, since virtually no specific binding was detectable in these cell membrane preparations.

Spec([iciO' q/ 3H-PK 11 195 bindin~ to human lymphocyte membranes The rank potency order of displacing agents suggests that 3H-PK 11 195 binds pBDZR on lymphocyte (Table 3). Unlabelled PK 11 195 was the most potent compound tested. Of the BDZ, Ro5-4864 also inhibited the binding with high affinity followed by diazepam and flunitrazepam. N-Desmethyl-2'-chlorodiazepam and N-desmethyldiazepare were much less effective whereas clonazepam and flumazenil were both inactive. The binding was inhibited by high nmol concentrations of protoporphyrin IX and mesoporphyrin IX. Human DBI at 5 #M concentration inhibited approximately 60°','o of the binding. 3H-PK 11 195 binding to human lymphocyte membranes TABLE 3 DISPLACEMENT OF 3H-PK 11 195 SPECIFICALLY BOUND TO H U M A N LYMPHOCYTE MEMBRANES

10

m/o.s

-~.

"-.

/ m

GABA, muscimol, bicuculline, picrotoxin, ethyl (BCCE) and methyl (BCCM)-fl-carboline-3-carboxylate and FG 7142 (methylamide-fl-carboline-3-carboxylate) were ineffective up to 10 ~M concentration. K~ values were calculated from the equation K~ = IC,,/( 1 + L/K~,), where L is the concentration of radioligand in the incubation medium. K d value was taken from Fig. 1. IC5o values were obtained from a semilogaritm plot of the dose-response curves derived from at least 5 concentrations of each drug tested in 3 different experiments, except for human DBI where results refer to a single experiment. DBI (diazepam binding inhibitor) was prepared according to Ferrero et al, (1988). n u indicates Hill coefficient. Values are mean _+ SEM.

°"

/

Displacer

K I (riM)

;qj

PK 11 195 Ro 5-4865 Protoporphyrin IX Diazepam Flunitrazepam Mesoporphyrin IX N-Desmethyl-2' -chlorodiazepam Desmethyldiazepam DBI human Clonazepam Flumazenil

8.9 + 0.6 42.9 + 4.5 165.3 + 7.3 236.1 , 11.8 302.3 + 36.3 340.0 _+ 25.0 2 800 5 900 3 800 > 10 000 > I 000fl

(I.98 0.86 0.97 (I.87 0.90 0.89 -

BOUND

___.___,___----Non S p e c i f i c ~ t

10

~ I

30

3H-PK

I

'~0

11195 ( n M )

Fig. 1. Specific and non specific binding of3H-PK 11 195 to lymphocyte membranes as a function of3H-PK 11 195 concentration. Data shown are representative of five experiments. Inset: Scatchard plot of the specific binding of 3H-PK 11 195 calculated from the saturation isotherms, B ..... = 10.9 _+ 0.4 pmol/mg protein, K d = 9.3 + 0.3 nM.

214 was not affected by GABA or other compounds specifically active at the GABA A receptor complex. Furthermore, in other experiments, Scatchard analyses of equilibrium saturation isotherms obtained in the presence and absence of 100/~M GABA or muscimol indicated that neither the binding affinity nor the density was significantly affected (data not shown).

B max

2o

"IES

O.

i

E

TABLE 4

Values are mean ± SEM. Statistical comparison between groups was by two-tailed Student's t-test. No.

3H-PK 11 195 specific binding Bm~ (pmol/mg prot.)

Controls Hepatic encephalopathy: Total Clinical stage Stage I - I I Stage I l l - I V Aetiology/surgery Alcoholic Non alcoholic Surgical shunt No surgery Liver diseases: Total Aetiology/surgery Alcoholic Non-alcoholic Surgical shunt No surgery

e•

0~0

£

10

!

•

1

30

10.0 _+ 0.5

Kd (nM) 9.8 _+ 0.6

19

5.2 + 0.8*

10.1 + 1.1

12 7

6.4 + 1.1 ~ 3.3 -2-_0.3 #

9.9 _+ 1.0 10.4 +_ 0.8

8 7 11 8

4.7 5.9 5.6 4.7

9.3 10.8 9.9 10.2

21

8.9 + 0.7

10.3 ± 0.6

13 8 6 15

8.8 8.9 9.4 8.7

9.7 10.9 9.9 10.3

_+ 0.4 + 1.9 ± 1.3 ± 0.5

± ± # ±

0.7 0.6 0.8 0.6

± 0.5 _+ 1.5 +_ 0.8 ± 0.8

+ ± ± ±

0.8 0.5 0.7 0.7

* P < 0.01 compared with controls (t = 5.597) and patients with liver diseases (t = 4.007). P < 0.01 compared with controls (t = 3.565) and P < 0.025 compared with liver diseases. # P < 0 . 0 1 compared with controls (t = 6.225) and liver diseases (t = 6.450) and P < 0.025 compared with stage I - I I HE. Age was not correlated significantly with Bm~~ in any group studied (controls: r = 0.263; HE: r = 0.03; LD: r = 0.23).

•

" --

o_

x

o. t

3:

.!

Controls

SPECIFIC B I N D I N G O F 3H-PK 11 195 TO L Y M P H O C Y T E MEMB R A N E S OF C O N T R O L S A N D PATIENTS W I T H LIVER DISEASES

Subjects

•

"3 O.

3H-PK 11195 lymphocyte binding in healthy controls and patients with liver diseases Clinical and biochemical details of the population included in the study are shown in Tables 1 and 2. Scatchard analyses of lymphocyte 3H-PK 11195 binding were linear and showed a single non interacting class of binding sites in each case (data not shown). The binding parameters (mean B m a x and Kd) for the cirrhotic patients with or without clinical signs of H E and the healthy controls are presented in Table 4, while scatter-

oo

Liver Diseases

_ Hepatic ~elflml~ty

Fig. 2. Scattergram of 3H-PK 11 195 maximal binding capacity(Bma x j to lymphocyte membranes from controls, patients with liver diseases without mental status changes a n d patients with hepatic encephalopathy. Each point represents an individual subject. Solid line-represents the mean. dotted lines represent SEM. The difference in the Bm~~ values is statistically significant (P < 0.0I ) (see TaMe 4 for mearL values).

grams of individual B m a x values are in Fig. 2. The apparent affinity (Kd) did not change significantly between the patients and controls. However. the density of lymphocyte 3H-PK 11 195 binding sites differed significantly among the groups. The average Bm,x was significantly lower in patients with H E than in normal controls (40%) or non encephalopathic patients (35°,,). In contrast, the B .... for the hepatic patients without H E was not significantly different than that for the controls. In order to examine whether the change in binding density was specifically related to the presence of HE. we studied the effect of other variables on Bm~~ values. Some of the results are given in Table 4. There was no significant correlation between the maximal binding capacity and age, neither in controls nor in the group with LD or with HE. Similarly, there was no difference in B..... values between males and females in any group examined. Among the patients most of them were suffering from alcoholic cirrhosis: the average Bma× did not change significantly between the alcoholics and non-alcoholics, neither in the encephalopathic group nor in that without HE. There was also no difference in binding density between the patients who had a surgical shunt and who had not in both the diagnostic groups. To evaluate the relationship between decrease in binding capacity and clinical grading of HE. Bm,~ values have been

215 compared in mild-moderate (stage l - l I ) and severe (stage Ill-IV) HE patients. The average number of ~H-PK 11 195 binding sites was 25 ~'0 less in the group of severe HE than in that of mild-moderate, and the difference was significant. Moreover, there was also a significant negative correlation between the number of lymphocyte pBDZR and clinical grading of HE in the patients (Fig. 3). Changes in serum ammonia did not correlate reliably with the B .... values, in either HE patients or LD only. Nevertheless, no correlation was observed between serum ammonia levels and clinical HE stages. We could examine three patients during and after the recovery from HE. One patient was not encephalopathic at the time of first sampling and had a normal (mean + 2 SD) lymphocyte pBDZR density (8.90 pmol/mg protein). Following porto-caval shunt surgery, she developed a severe encephalopathy and had a B ...... values of 3.11 pmol/mg protein. Conversely, the other two patients were encephalopatic at the first assay and both showed Bm~~ levels significantly less than normal (5.82 and 4.72 pmol/mg protein, respectively). After improvement in their mental status, the receptors number tended to increase in both subjects (6.92 and 6.34 pmol/mg protein), but it remained still lower than in controls. Binding to peripheral blood lymphocytes represents mainly the contribution of T cells. Thus, the change in pBDZR associated with HE could reflect modifications occurring directly on T cells. To verify this hypothesis in a small group of HE patients and controls (n = 5, respectively), ~H-PK 11 195 binding was measured directly in purified preparations of T lymphocytes. The binding level, measured at 5 nM -~H-PK 11 195, was significantly lower in

O. ~a

m o

E ¢,n

t'~

10

Z

°

0

~.

°

4o

I

II

IN HE

IV

Clinical S c o r e

Fig. 3. Correlation between individual *H-PK 11 195 maximal binding capacity (B ...... ) and clinical ratings of hepatic encephalopathy in patients with stages l - I V hepatic encephalopathy. Linear regression analysis indicates a significant negative correlation (r = -0.51, P < 0.05).

-FABLE 5 SPECIFIC B I N D I N G OF 3H-N-METHYLSCOPOLAMINE (3HNMS) TO L Y M P H O C Y T E M E M B R A N E S OF C O N T R O L S A N D PATIENTS WITH LIVER DISEASES Values are mean _+ SEM. Subjects

Controls Hepatic encephalopathy Liver diseases

No.

20 15 15

~H-NMS specific binding B,~,:,x (fmol.'mg prot.)

Kd (nM)

156.6 + 16.3 186.0 + 15.I 172.7 ± 18.0

18.9 ± 1.1 19.9 ± 0.9 18.1 ± 1.0

T cells from HE patients than in the controls (Specific binding, fmol/1 x 106, mean _+ SEM' 50.5 + 10.8 vs. 145.8 _+ 19.0, P < 0.01).

L ymphoo,te muscarinic receptors In order to establish whether the alteration in HE was in some way specific for pBDZR, we examined the binding of 3H-NMS to lymphocyte muscarinic receptors in a restricted population of patients with or without HE and controls. As shown in Table 5, there were no significant differences in the affinity or B ....... of 3H-N MS for its binding sites on lymphocyte membranes between the 3 groups.

Discussion Our results indicate that (1)peripheral circulating lymphocytes possess pBDZR, that are readily demonstrated by the binding of the selective ligand -~H-PK 11 195, (2)the patients with HE exhibit a significant reduction in the number of pBDZR in their lymphocytes that differentiate them not only from the healthy controls but also from the patients with liver diseases without clinical signs of HE, (3)the abnormality of the lymphocyte receptors associated with HE is in some way specific for pBDZR, since preliminary experiments with 3H-NMS binding to lymphocyte muscarinic receptors show no changes associated with HE. Benzodiazepines (BDZ) are among the most widely prescribed drugs, mainly due to their therapeutic action in relieving anxiety. Their anxiolytic effect has been attributed to a modulation of GABA-operated chloride channels (Costa and Guidotti 1979; Tallmann et al. 1980; Pritchett etal. 1989). In addition to binding to this class of neurotransmitter receptors, BDZ have also been reported to bind with high affinity to several other proteins (Taft and De Lorenzo 1984; Kirkness and Turner 1988} of which the peripheral-type BDZ recognition sites have been studied most extensively (Anholt 1986). Despite the fact that pBDZR were demonstrated over ten years ago, their cellu-

216 lar functions as well as the potential side effects of BDZ at these sites remain to be elucidated. These sites are virtually present in all mammalian tissues and could be responsible for the BDZ actions in steroidogenesis, cardiovascular function, and immune response (Mestre et al. 1985; Ruff et al. 1985; Ritta et al. 1987; French and Matlib 1988; Papadopoulos et al. 1990). In addition, they may also play a role in anion transport, cell proliferation, mitochondrial respiration, convulsive threshold and stress (Lueddens and Skolnick 1987; Wang et al. 1984; Weissman et al. 1985; Drugan et at. 1986; Hirsch et al, 1989). The present study was designated to examine the status of p B D Z R in lymphocyte membranes from patients with hepatic encephalopathy (H E). Lymphocytes that are readily accessible cells bear p B D Z R (Moingeon et al. 1983, 1984) and may represent a reliable experimental model for studying the pathological conditions that affect their function. The suggestion was of special interest in the case of HE, since recent observations strongly suggest that the pathogenesis of this disorder is related to accumulation of endogenous compounds with BDZ-like structure and properties, possibly as a result of the altered liver metabolism (Basile et al. 1989; Mullen et al. 1988; Olasmaa et aL 1989, 1990). The identity, as well as the origin, of these endogenous BDZ is at present uncertain although it seems that the most likely candidates are compounds with diazepam-related structure, that naturally occur in traces in mammalian tissues and body fluids (Deckert et al. 1984); Sangameswaran et al. 1986; Wildmann et al. 1986; De Bias et al. 1987; Medina et al. 1988, 1989; Unseld et al. 1989, 1990).

Characteristics of lymphocyte 3H-PK 11 195 binding sites In order to characterize p B D Z R in lymphocytes we studied the binding of the specific ligand 3H-PK 11 195 to preparations of lymphocyte membranes (Benavides et at. 1983). In previous investigations that report the presence of p B D Z R in human lymphocytes, the receptors have been characterized using 3H-diazepam and Ro5-4865 as ligands (Moingeon et al. 1983, 1984). The number of p B D Z R that could be demonstrated in lymphocytes by both the radioligands was low and the binding measurements were always complicated by a large extent of non-specific binding. The results of our study show that these methodological problems that could make it difficult to detect discrete changes of lymphocyte p B D Z R in patients, are avoided by the selection of 3H-PK 11 195 as a ligand. We found that the binding of 3H-PK 11 195 to lymphocyte membranes is specific, saturable and occurs with relatively high capacity and affinity. The non specific binding was always less than 10<°~ of the total. The sites labelled by 3H-PK 11 195 in lymphocyte membranes have the pharmacological characteristics of

pBDZR because they possess high affinity for the peripheral ligands PK 11 195 and Ro5-4865 and are unaffected at nM concentrations by the central ligands flumazenil and ¢lonazepam as well as by several other compounds active at the GABAA receptor complex. This conclusion is further supported by the relatively high affinity displacement of 3H-PK 1 t 195 binding by porphyrins that are endogenous ligands for the p B D Z R located in the outer mitochondrial membrane (Verma et al. 1987). tn addition. our data indicate that human DBI is also effectivein inhibiting the binding, at a concentration close to that reported for rat DBI in other tissues (Guidotti et al. 1988). The ratios of affinities of BDZ related drugs to displace 3H-PK 11 195 binding from lymphocyte membranes are m close agreement with those reported in human platetets (Benavides et al. 1984). It is of interest that also in lymphocytes, Ro5-4865 is significantly tess potent (K~ 6-fold less) in displacing the tracer than PK 11 195. This observation suggests that the site labelled by PK 11 195 and Ro5-4865 in lymphocyte membranes may not he identical. Differences in binding sites for BDZ and isoquinoline compounds have been demonstrated in various tissues and species and may reflect heterogeneity in pBDZR structure (Paul et at. 1981; Doble et al. 1985; Skowronski et al. 1987: Snvder et al. 1987: Lueddens and Skolnick 1987: Antkiewicz-Michaluk et al. 1988).

Cbmpar~on of lymphocyte 3H-PK 11 195 binding sites in controls and patients with liver diseases Although the role of p B D Z R in the pathogenesis of H E remains to be elucidated, our study clearly demonstrates that a profound reduction of the number of these receptors occurs in lymphocytes from patients with HE. Moreover. p B D Z R were not decreased in lymphocytes from patients with hepatic diseases without neurological abnormalities. suggesting that the receptors response is in some way specific for the presence of HE. This conclusion is further supported by the fact that the receptor density correlates with clinical degrees of H E and tends to return t o normal in those few patients who could be reexamined after the recovery from HE. The reduction in lymphocyte pBDZR measured here in H E subjects is apparently not a result of age, sex. tissues preparation or storage. Furthermore. total white cells blood counts as well as percentages of total T lymphocytes or T lymphocytes subsets, in the HE patients were comparable to non-encephalopathic patients and controls, suggesting that the reduction in lymphocyte 3H-PK 1l t95 binding sites may not be secondary to changes in the number or relative proporuon of peripheral circulating lymphocytes. Since it appears that the binding sites for 3H-PK 1 t 195 exist m all the various populations of circulating mononuclear cells (Ferrero et al. 1989). an important issue concerns

217 the contribution of the various cell types to the alteration in binding observed in HE. From our results, it seems possible that the reduction in p B D Z R in H E reflects a change occurring directly in T lymphocytes, since we found that the binding to these cells was significantly less in HE patients than in controls. Whether the reported decrease in binding, could also originate from differences in B cells and monocytes will be the subject of further studies. Although data on the effect of acute and chronic ethanol administration on lymphocyte p B D Z R are not available to date. the observed changes are not likely to be related to a vulnerability for alcoholism, since there was no difference in the binding between alcoholic and non-alcoholic cirrhoscs neither in the group with HE, nor in that without neurological complications. To explain the alteration in lymphocyte p B D Z R in HE, two major possibilities can be considered. Potentially, this could be related to some non specific effect occurring in the lymphocyte menlbranes. However, the fact that lymphocytes from encephalopathic patients exhibit a normal muscarinic cholinergic binding might argue against this suggestion. Alternatively, the change in density of p B D Z R rnay reflect a compensatory response due to prolonged exposure to an endogenous c o m p o u n d with agonistic property for the receptors, but this possibility remains highly speculative. A down-regulatory effect on p B D Z R has been previously reported to occur "in vitro" after exposure to certain B D Z and "'in vivo" in certain forms of stress, hormonal changes and alcohol abuse (Gavish et al. 1986a; Johnson et al. 1986; Drugan et al. 1986; Weizman et al. 1987 ; Suranyi-Cadotte et al. 1988 ; Karp et al. 1989). Moreover, recent observations describe increased levels of endogenous BDZ-Iike activity also in sertnn of patients with HE (Olasmaa et al. 1989, 1990; Mullen et al. 1990). Sonte characteristics of this B D Z activity have been already determined and it may contain variable amounts of N-desmcthyldiazepam, diazepanl and other two unidentified BDZ. Plasma levels of the endogenous BDZ-like material in encephalopatic patients correspond to concentrations between 10 v and 10 5 that are in the range of those effective for diazepam to bind to lymphocyte p B D Z R . However, until the substance is purified to serum extracts its affinity l\)r B D Z receptors remains unknown and the level of concentration uncertain. If so, any implications of these findings as an explanation for the alteration of pB DZ R in lyre phocytes of encephalopatic patients cannot be established. Otherwise, it is important to point out that as no single agent is likely to be completely responsible for HE, other endogenous inhibitors of radioligand binding to p B D Z R including porphyrins and DBI and/or DB1 peptide processing products, that have been also implicated in HE, may be responsible for the decrease in lymphocyte p B D Z R (Verma et al. 1987: Rothstein et al. 1989; Lavoie et al.

1990). Measurements of these c o m p o u n d s in serum of patients with H E in conjunction with the binding of p B D Z R in lymphocytes, would be important in clarifying this issue. As a final point of discussion, it is noteworthy that recent observations suggest that alterations of p B D Z R occur also in the brain of patients and animal models with H E (Giguere et al. 1989, Lavoie et al. 19901. However in contrast to our findings in lymphocytes, the receptors density in the brain was found to increase following HE, possibly as a consequence of astroglial proliferation. Differences in the regulation of p B D Z R among the various tissues have been previously reported and might be secondary to changes in p B D Z R conformational structure (Schoemaker et al. 1983, Tamborska and Marangos 1986: Awad and Gavish 1987: Gavish and Weizman 1989). In conclusion, the present results suggest that the binding of-~H-PK 11 195 to lymphocyte p B D Z R represents a useful addition to the few biochemical markers available in HE. However, additional studies are still necessary to establish the validity of our proposal and the role of p B D Z R in HE. In addition, based on our data, it would be of interest to investigate whether agents active at p B D Z R could prevent certain symptoms or complications associated with HE.

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