COULD AN ENDOGENOUS BENZODIAZEPINE LIGAND CONTRIBUTE TO HEPATIC ENCEPHALOPATHY?

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hepatic encephalopathy by promoting GABA-ergic

to

Occasional

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neurotransmission. INTRODUCTION

COULD AN ENDOGENOUS BENZODIAZEPINE LIGAND CONTRIBUTE TO HEPATIC ENCEPHALOPATHY? KEVIN D. MULLEN JOSEPH V. MARTIN MARK L. BASSETT WALLACE B. MENDELSON E. ANTHONY JONES

Department of Internal Medicine, Gastroenterology Division, Cleveland Metropolitan General Hospital, Case Western Reserve University, Cleveland, Ohio, USA; Department of Psychiatry and Behavioral Science, State University of New York, Stonybrook, New York; and Liver Diseases Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland.

Summary

butyric acid

High affinity recognition sites for benzodiazepines are part of the &ggr;-amino(GABA) supramolecular complex on the

plasma membrane of neurons in the mammalian brain. Synthetic agonist benzodiazepines promote GABA-ergic neurotransmission, and hence the hypnotic and anxiolytic effects of this class of drugs, by binding to these sites. A normal physiological role for these binding sites is unknown, and an endogenous ligand for benzodiazepine receptors has not been definitely identified in normal animals. In animals and human beings with hepatic encephalopathy, however, benzodiazepine receptor antagonists have induced amelioration of the encephalopathy, and an endogenous substance that competitively binds to benzodiazepine receptors has been found in cerebrospinal fluid. These findings suggest that an endogenous ligand for the benzodiazepine receptor with agonist properties contributes 12. Hoxie JA, Matthews DM, Callahan KJ, et al. Transient modulation and internalization of T4 antigen induced by phorbol esters. J Immunol 1986; 137: 1194-201 13 Sodroski J, Goh WC, Rosen C, et al. Role of the HTLV-III/LAV envelope in syncitium formation and cytopathicity. Nature 1986; 322: 470-74. 14 Walker CM, Moody DJ, Stites DP, Levy JA. CD8- lymphocytes can control HIV infection in vitro by suppressing virus replication. Science 1986; 234: 1563-66 15. Harper ME, Marselle LM, Gallo R, Wong-Stall F Detection of lymphocytes expressing human T-lymphotropic virus type III in lymph nodes and peripheral blood from infected individuals by in situ hybridization. Proc Natl Acad Sci USA

1986; 83: 772-76. a network theory of the immune system. Ann Immonol 1974; 125C: 373-85. 17. Moller G, ed Idiotypic networks. Immunol Rev 1984, 79. 18. Cosenza H, Kohler H. Specific suppression of the antibody response by antibodies to receptors. Proc Natl Acad Sci USA 1972; 69: 2701-05 19. Koprowski H, Melchers F, eds. Images of biologically active structures in the immune system. Current Topics Microbiol Immunol 1984; 119. 20. Dreesman GR, Kanda P, Chang TC, et al Definition of HIV determinants with vaccine potential. J Cell Biochem 1987; 262 (suppl 11D): 34. 21 Wofsy DDC, Mayes J, Woodrock J, Seaman WE. Inhibition of humoral immunity in vivo by monoclonal antibody to L3T4 J Immunol 1985; 135: 1698-701. 22. Goronzy J, Weyand CM, Fathman CG. Long-term humoral unresponsiveness in vivo induced by treatment with monoclonal antibody against L3T4. J Exp Med 1986;

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Jeme NK. Towards

164: 911-25. 23. Lundin K, Karlsson

al Certain human gp 120-HIV antibodies react with anti-CD4 antibodies. Scand J Immunol 1988; 27: 113-17. 24 Beretta A, et al HIV env glycoprotem shares a cross-reacting epitope with a surface

25

26 27

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et

protein present on activated human monocytes and involved in antigen presentation Eur J Immunol 1987; 17: 1793-98. Afrasiabi R, Mitsuyasu RT, Nishanian P, et al. Characterization of a distinct subgroup of high risk persons with Kaposi’s sarcoma and good prognosis who present with normal T4 cell number and T4:T8 ratio and negative HT1V II I/LAV serologic test results Am J Med 1986; 81: 969-81. Salahudin SZ, Markham PD, Redfield RR, et al. HTLV-III m symptom-free seroneganve persons. Lancet 1984, ii: 1418-19 Mayer KH, Stoddard AM, McCusker J, et al. Human T-lymphotropic virus type III in high-risk, antibody negative homosexual men Ann Intern Med 1986; 104: 194-201 Moller G, ed Acquisition of T-cell repertoires. Immunol Rev 1986; 90.

hypothesis implicating the y-aminobutyric acid (GABA) neurotransmitter system in the mediation of hepatic encephalopathy was originally based on a series of experimental findings in rabbits with hepatic encephalopathy due to galactosamine-induced fulminant hepatic failure. One of the original findings was a change in the status of benzodiazepine binding sites on the GABA/ benzodiazepine receptor complex.2 The initial hypothesis, however, proposed that increased GABA-ergic neurotransmission occurred in hepatic encephalopathy due to The

increased interaction between GABA and its receptors, rather than to potentiation of the action of GABA caused by events at the benzodiazepine binding site.12 More recent experimental findings indicate that both mechanisms should be considered. During the past decade, endogenous ligands for the benzodiazepine receptor have been sought in normal animals without success.3,4 In contrast, during the past three years, persuasive evidence has been found in animals and in human beings with hepatic encephalopathy which directly,S,6 and indirectly,7-14 suggests the presence of an endogenous substance with agonist properties at the benzodiazepine receptor. GABA-ergic tone may, therefore, be increased in hepatic encephalopathy because of the interaction of an endogenous benzodiazepine agonist with benzodiazepine receptors. NEUROPHARMACOLOGY OF BENZODIAZEPINES

Since the original reports of the existence of high affinity,

saturable, and stereospecific benzodiazepine receptors in the mammalian central nervous system,"," a great deal of

Jerne NK, Rolland J, Cazenave PA Recurrent idiotypes and internal images. EMBO J 1982; 1: 243-47 30 Martinez-A C, Bernabe RR, de la Hera A. idiotypic helper T-cell repertoires are established early in life Nature 1985, 317: 721-23. 31 Klinman NR, Press JL The characterization of the B-cell repertoire specific for the 2,4-dinitrophenyl and 2,4-6-trinitrophenyl determinants in neonatal Balb/c mice. J Exp Med 1975; 141: 1133-46 32. Sigal NH, Press PJ, Klinman NR. Late acquisition of a germ line specificity. Nature 1976; 259: 51-53. 33. Martinez-A C, Pereira P, et al. Internal complementarities in the immune system: regulation of the expression of hepler T-cell isiotypes Proc Natl Acad Sci USA 1987, 81: 4520-24. 34 Holmberg D, Freitas AA, Portnoi D, et al. Antibody repertoires in normal Balb/c mice: B lymphocyte subpopulations defined by the state of activation. Immunol Rev 1986; 93: 147-69 35. Coutinho A, Marquez C, Aranjo PMF, et al. Antibody dependent T-cell idiotypes are exclusively expressed by natural activated lymphocytes in non-immunized mice. Eur JImmunol 1987, 17: 821-26. 36. Bernabe RR, Coutinho A, Cazenave PA, Forni L Suppressionn of a recurrent idiotype results in profound alteration of the whole B-cell compartment Proc Natl Acad Sci USA 1981; 78: 6416-20 37. Martinez-A C, Toribio ML, de la Hera A, et al. Maternal transmission of idiotypic network interactions selecting available T-cell repertoires. Eur J Immunol 1986; 16: 29.

1445-48 38 Martinez-A C, Marcos MAR, Pereira P,

39.

et al Turning (Ir gene) low responders into high responders by idiotypic manipulation of the developing immune system. Proc Natl Acad Sci USA 1987, 84: 3812-16. Mitsuda H, Broder S. Strategies for antiviral therapy m AIDS Nature 1987; 325:

773-78.

R, Broader S. Development of anti-retroviral therapy for the acquired immunodeficiency syndrome and related disorders N Engl J Med 1987; 316:

40. Yarchoan

557-64

BD, Levine AM, Mildvan D, et al. Suramin therapy in AIDS and related disorders Report of the U.S Suramin Working group JAMA 1987, 258: 1347-51. 42. Sultan Y, Kazatchkine MD, Maisonneuse P, Nydegger UE Antiidiotype suppression of autoantibodies to factor VIII (anti-haemophilic factor) by high doses of intravenous gammaglobulin Lancet 1984; ii: 765. 43 Intravenous immune globulin and the compromised host Am J Med 1984; 76: 1-213.

41. Cheson

458 be altered, but the significance of these changes remains uncertain. During the past decade, an endogenous ligand for benzodiazepine receptors has been intensively sought in normal animals, but no such substance has been definitely identified. Suggestions have included P-carboline seems to

derivatives,2223 inosine, hypoxanthine 21 nicotinamide,25 diazepam binding inhibitor, 26 endozepines,2’ nepenthin,2s

desmethyldiazepam,29 and various unidentified low and high molecular weight proteins.34All of these substances are capable of binding to the benzodiazepine receptor, but none of them has yet been shown to be an active endogenous benzodiazepine receptor ligand in vivo. Potent and specific benzodiazepine receptor antagonists (eg, CGS 8216, Ro 15-1788) mediate few obvious neurobehavioural effects when administered to normal animals .30,31 This suggests either that very little, if any, of an endogenous benzodiazepine ligand is present under normal physiological conditions, or that its function is too subtle for reversal of its effect to be detected. BENZODIAZEPINE RECEPTOR LIGANDS AND HEPATIC ENCEPHALOPATHY

Diagrammatic representation of the GABA/benzodiazepine receptor/chloride ionophore complex in the surface membrane of a postsynaptic neuron. Receptors are depicted for: (a) GABA, GABA receptor agonists (eg, muscimol), and GABA receptor antagonists (eg, bicuculline); (b) picrotoxin (the barbiturate binding site); and (c) benzodiazepine receptor agonists (eg, diazepam), benzodiazepine receptor antagonists (eg, CGS 8216, Ro 151788), and benzodiazepine receptor inverse agonists (eg, beta-carbolines). A shows the receptor complex in the unactivated state with the Cl- channel closed. B shows the receptor complex in the activated state with the Clchannel open. Activation is induced by GABA or GABA agonists binding to GABA receptors, or by barbiturates or benzodiazepine agonists interacting with their specific receptors, in the presence of GABA. Activation of the receptor complex is associated with conformational changes and opening of the Cl- channel. These phenomena promote Cl- conductance across the cell membrane from synaptic cleft to the cytoplasm, and transform the resting cell membrane potential to a hyperpolarised level. This mechanism mediates GABA-ergic inhibitory neurotransmission. (Reproduced with modifications from Biological Ps3chtatrv.’’)

evidence has shown that these receptors mediate the principal pharmacological effects of synthetic benzodiazepines.16,17 Benzodiazepine receptors are one component of an oligomeric membrane complex, on which there are also recognition sites for GABA, and multiple allosteric sites at, or near to, a GABA-gated chloride ionophore for cage convulsants and barbiturates (fig). Occupation of any of these sites can alter neuronal excitability by modulating transmembrane chloride flux. The chloride ionophore can, therefore, be considered the effector unit of this membrane receptor complex.1s The main action of benzodiazepine agonist compounds, such as diazepam, appears to be augmentation of inhibitory postsynaptic neurotransmission, mediated by GABA. Benzodiazepine receptors in the central nervous system were considered to be of a single type, but recently it has been shown that they are heterogeneous: at least two subclasses have been identified.19 In normal animals, the physiological function of benzodiazepine receptors is unknown. Complex alterations in the coupling of the chloride channel and benzodiazepine binding sites have been reported in rats with acute stress,20 and probable increases in the densities of both benzodiazepine and GABA receptors have been reported in the brains of some animals with hepatic encephalopathy.2,7-21 Thus, in certain pathological states, the receptor complex

Benzodiazepine Receptor Agozzist Induced Encephalopathy The effects of administration of a benzodiazepine receptor agonist should mimic hepatic encephalopathy if increased GABA-ergic tone, promoted by events at benzodiazepine receptors, is implicated in the mediation of hepatic encephalopathy. Animals with hepatic encephalopathy have been compared to animals with diazepam-induced encephalopathy.8.9,32 Visual evoked been to monitor recorded are to reflect the and considered electrophysiological status, of the brain in the pattern of post-synaptic potentials regions being monitored .33 3’ Rabbits with hepatic encephalopathy due to galactosamine-induced fulminant hepatic failure, and rabbits with diazepam-induced encephalopathy, showed similar clinical features of encephalopathy and similar abnormalities of visual evoked responses .11,32 The clinical features of encephalopathy and abnormalities of the visual evoked responses were also similar in rats with hepatic encephalopathy due to thioacetamide-induced fulminant hepatic failure, and in rats with diazepam-induced encephalopathy.9 Other agents that induce encephalopathy in rabbits and rats by mechanisms that do not appear to involve activation of the GABA neurotransmitter system, do not produce similar changes in visual evoked responses to those seen in hepatic encephalopathy,323538 Diazepam induces neural inhibition as a consequence of its interaction with benzodiazepine receptors promoting GABA-ergic neurotransmission.1617 These findings, therefore, are compatible with an analogous mechanism contributing to the neural inhibition of hepatic encephalopathy. responses

have

Bnzzodiazepine Receptor Antagonist Induced Amelioration of Encephalopathy The administration of a benzodiazepine receptor antagonist should ameliorate hepatic encephalopathy if increased GABA-ergic tone, due to events at benzodiazepine receptors, is implicated in causing the syndrome. CGS 8216, a potent benzodiazepine receptor antagonist, induces clinical and electrophysiological improvement of encephalopathy in galactosamine-treated rats.’ Ro 15-1788, another benzodiazepine receptor antagonist, induces transient but unequivocal clinical and electrophysiological remission of hepatic encephalopathy in

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rabbits8 and in rats9 with fulminant hepatic failure. Ro 15-1788 has been reported to induce clinical and remissions of hepatic encephalopathy beings with fulminant hepatic failure’0,17 or cirrhosis,11.3 and to reverse chronic intractable portalsystemic encephalopathy.14 These observations suggest that benzodiazepine receptor antagonists induce an improvement in hepatic encephalopathy by displacing an endogenous ligand with benzodiazepine agonist-like properties from benzodiazepine receptors. The improvement of hepatic encephalopathy in rats which was induced by the partial inverse benzodiazepine agonist, Ro 15-4513, was more complete than that induced by Ro

electrophysiological in human

15-1788.’ This suggests that a component of the neural inhibition involved in the mediation of hepatic encephalopathy is additional to that attributable to occupation of the benzodiazepine receptor by an agonist ligand alone. The superior potency of a partial inverse benzodiazepine agonist in reversing hepatic encephalopathy is most probably due to the intrinsic activity of the drug

decreasing GABA-ergic tone.

Benzodiazepine Binding Activity in Cerebrospinal Fluid Cerebrospinal fluid from rabbitss and human beings6 with hepatic encephalopathy contains a substance which binds avidly to benzodiazepine receptors. This has been shown by the ability of cerebrospinal fluid to displace from neural membranes [3H]-Ro 15-1788 or pH]-p-carboline-3carboxylate, both of which are ligands with a high affmity for benzodiazepine receptors. This substance, found in cerebrospinal fluid in hepatic encephalopathy, competitively inhibits benzodiazepine binding, is heat stable, and has a molecular weight of less than 10 000 daltons. Benzodiazepine receptor binding activity has not yet been detected in serum from rabbits or human beings with hepatic encephalopathy. CONCLUSIONS

Hepatic encephalopathy and benzodiazepine receptor agonist induced encephalopathy are similar in terms of clinical and behavioural features, and electrophysiological changes, as assessed by visual evoked responses. Benzodiazepine receptor antagonists can mediate clinical and electrophysiological improvements in hepatic encephalopathy. Appreciable benzodiazepine binding activity has been detected in cerebrospinal fluid from rabbits and human beings with hepatic encephalopathy. We suggest that an endogenous substance, with benzodiazepine agonist-like properties, contributes to the syndrome of hepatic encephalopathy by potentiating GABA-ergic neurotransmission. This work was supported in part by a grant from the Cuyahoga Hospital Foundation and Case Western Reserve University biomedical research support grant # RR-05410-26.

Correspondence should be addressed to E. A. J., Liver Diseases Section, Building 10, Room 4D52, National Institutes of Health, Bethesda, Maryland 20892. REFERENCES

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