Klaus J. Fehske

Benzodiazepine antagonism by harmane and other β-carbolines in vitro and in vivo

Abstract Harmane and other related β-carbolines are putative endogenous ligands of the benzodiazepine receptor. Since the compounds are potent convulsants they may have agonist activities at the benzodiazepine receptor while the benzodiazepines may be antagonists. This hypothesis was proved by comparing the in vivo and in vitro antagonism of benzodiazepines by harmane and other β_carboliness. Harmane is clearly a competitive inhibitor of benzodiazepine receptor binding in vitro. Moreover, harmane-induced convulsions can be inhibited reversibly by diazepam in a manner which is consistent with the assumption of competitive antagonism in vivo. For some β-carboline derivatives a correlation was found between the affinity for the benzodiazepine receptor in vitro and the convulsive potency in vivo. Thus, the data reported suggest that harmane or other related β-carbolines are putative endogenous agonists of the benzodiazepine receptor. This suggestion is further supported by the observation that diazepam is equally potent in inhibiting harmane- or picrotoxin-induced convulsions, indicating a convulsive mechanism within the GABA receptor-benzodiazepine receptor system.

1-Methyl-β-carboline (Harmane), a potent endogenous inhibitor of benzodiazepine receptor binding

SummaryThe interaction of several β-carbolines with specific [3H]-flunitrazepam binding to benzodiazepine receptors in rat brain membranes was investigated. Out of the investigated compounds, harmane and norharmane were the most potent inhibitors of specific [3H]-flunitrazepam binding, with IC50-values in the micromolar range. All other derivatives, including harmine, harmaline, and several tetrahydroderivatives were at least ten times less potent. Harmane has been previously found in rat brain and human urine, so it is the most potent endogenous inhibitor of specific [3H]-flunitrazepam binding known so far, with a several hundred fold higher affinity for the benodiazepine receptor than inosine and hypoxanthine. Thus, we suggest that harmane or other related β-carbolines could be potential candidates as endogenous ligands of the benzodiazepine receptor.

On the neuropharmacology of Harmane and other β-carbolines

β-Carbolines have been recently proposed as candidates for the unknown endogenous ligand of the benzodiazepine receptor. Out of the β-carboline derivatives already found in the mammalian CNS, harmane is clearly the most potent inhibitor of benzodiazepine receptor binding. Therefore, it has been considered as possible endogenous ligand for this new receptor system. However, a certain degree of specificity might be a basic condition to accept the hypothesis of harmane as the endogenous ligand. Thus, the effects of harmane as well as other β-carbolines on several neurotransmitter receptor binding systems in vitro and on some neuropharmacological tests in vivo were investigated. Harmane developed the highest affinity towards the benzodiazepine binding site among all systems investigated. Its IC50-values for inhibiting opiate and muscarinic cholinergic receptor binding were about four times lower than those for dopamine and serotonin receptor binding but were about four times higher than that found for the benzodiazepine receptor binding. Norharmane exerted a remarkable displacing activity only at the benzodiazepine binding site. Harmine affected mainly the opiate and cholinergic muscarinic system, whereas tetrahydronorharmane turned out to be a potent inhibitor of serotonin and dopamine receptor binding. Doses of harmane needed to produce convulsions as indication of its possible benzodiazepine receptor agonistic properties are also sufficient to diminish nociception and decrease body temperature whereas the apomorphine-induced licking rate was affected at higher doses. The data demonstrate that harmane affects not only the benzodiazepine binding site but also other neuronal mechanisms. Furthermore, only minor changes of the β-carboline structure lead to substantially different effects. Therefore, the search for other β-carbolines with higher affinity for the benzodiazepine binding site as harmane seems to be promising.

A highly reactive tyrosine residue as part of the indole and benzodiazepine binding site of human serum albumin

The interaction of L-tryptophan and four benzodiazepine derivatives with tyrosine-modified human serum albumin was investigated by equilibrium dialysis and circular dichroism measurements. Out of the 18 tyrosine residues of the human serum albumin molecule, only 9 could be modified with tetranitromethane. At least up to a degree of modification of 5, the conformation of human serum albumin was not changed and no crosslinking and fractionation has been found, as revealed from circular dichroism measurements in the far ultraviolet range and from SDS polyacrylamide electrophoresis. The modification of only 2 out of the 9 accessible tyrosine residues of human serum albumin strongly affects the binding of L-tryptophan and diazepam to their common, stereospecific bindining site. This was evidently shown by a reduction of the association constants by more than 90% and by a large reduction of the extrinsic Cotton effects of four benzodiazepines bound to human serum albumin. The numbers of binding sites remained unchanged. Strong evidence was presented that only one tyrosine residue, which reacts faster with tetranitromethane than all others, is mainly involved in the specific indole and benzodiazepine binding site of human serum albumin. The location of this highly reactive tyrosine residue and that of the specific indole and benzodiazepine binding site within the human serum albumin primary structure is discussed.

Direct demonstration of the highly reactive tyrosine residue of human serum albumin located in fragment 299–585

Abstract The reactivity of tyrosine residues in human serum albumin (HSA) was investigated by the nitration of the albumin with tetranitromethane. It could clearly be demonstrated that one tyrosine residue reacts faster and with about 20-fold higher reactivity than all others. This highly reactive tyrosine residue is located in fragment 299–585 of HSA and is supposed to be Tyr 411. This tyrosine residue is specifically involved in the indole and benzodiazepine binding site of HSA. Since some other amino acid residues, located in fragment 124–298, are also involved in the indole and benzodiazepine binding site, it is concluded that this important binding site of HSA depends on the tertiary structure of the albumin.

β-Carboline binding indicates the presence of benzodiazepine receptor subclasses in the bovine central nervous system

SummaryReceptor binding studies were performed with tritiated propyl β-carboline-3-carboxylate ([3H]PrCC), tritiated ethyl β-carboline-3-carboxylate ([3H]ECC), and tritiated flunitrazepam ([3H]FNT) in membrane preparations from different regions of the bovine brain and retina. Specific binding in all regions investigated was associated with benzodiazepine receptor sites. However, not all benzodiazepine receptor sites. However, not all benzodiazepine receptors in the regions investigated as determined by the specific binding of tritiated flunitrazepam ([3H]FNT) are available for [3H]PrCC suggesting that specific [3H]PrCC binding labels only one subclass or subpopulation of the benzodiazepine receptor. This benzodiazepine receptor subclass is sensitive to GABAergic modulation and amounts for about 60% of the benzodiazepine receptors in bovine cortex, hippocampus, and retina but for about 80% of the benzodiazepine receptors in the bovine cerebellum. By contrast, specific [3H]ECC binding in the cerebellum and the hippocampus labeled the same number of benzodiazepine receptors as [3H]FNT, giving no evidence for a benzodiazepine receptor subclass specificity of this compound in the bovine CNS.

Azapropazone binding to human serum albumin

SummaryAzapropazone, a new non-steroidal antiinflammatory drug, is strongly bound to human serum albumin. As revealed by Scatchard analysis, one high-affinity binding site with an association constant of about 1.2×106 M−1 and two low-affinity binding sites with association constants of about 0.05×106 M−1 were found. While the high-affinity binding site of azapropazone is clearly not identical with the diazepam or digitoxin binding sites of human serum albumin, contradictory evidence was found by optical measurements and displacement studies for the similarity of the azapropazone and the warfarin binding site of human serum albumin. At present, it is suggested that both drugs bind to different areas of the same binding site. Therefore, the pronounced effects of various disease states on the plasma protein binding of azapropazone can not be explained by a binding to an unusual binding site, but seem to be due to an extreme sensitivity of the azapropazone binding area to the putative endogenous binding inhibitors, present in the blood during those disease states.

β-Carboline inhibition of benzodiazepine receptor binding in vivo

The in vivo potencies of 4 beta-carboline derivatives as inhibitors of benzodiazepine receptor binding were investigated. For all 4 derivatives maximal inhibition was seen within 2 min after i.v. application but decreased continuously for the next 20 min and after this time accounted for less than one-third of maximal inhibition. The in vivo potencies of the beta-carbolines as inhibitors of benzodiazepine receptor binding are much smaller than one would expect from their affinities measured in vitro. Thus, it is assumed than only a small portion of the i.v. dose reaches the brain.

High-Affinity Binding of Ethacrynic Acid Is Mediated by the Two Most Important Drug Binding Sites of Human Serum Albumin

The binding of ethacrynic acid to human serum albumin was investigated by means of circular dichroism and equilibrium dialysis measurements, using native human serum albumin and albumin derivatives with chemical modifications impairing specifically drug binding to the indole and benzodiazepine binding site or the azapropazone-warfarin binding area, respectively. The data presented indicate that the high-affinity binding of ethacrynic acid to human serum albumin is mediated by these two important drug binding sites. Accordingly, even at relatively low concentrations ethacrynic acid displaces other drugs from both binding sites.

The Modification of the Lone Tryptophan Residue in Human Serum Albumin by 2-Hydroxy-5-nitrobenzyl Bromide

The possible function of the lone tryptophan residue of human serum albumin in the stereospecific binding site for indole and benzodiazepine compounds was investigated by chemical modification. This residue can be selectively modified with 2-hydroxy-5-nitrobenzyl bromide. The modification alters the conformation of the albumin only slightly, as revealed by circular dichroism, fluorescence, and ultraviolet absorption measurements. A decrease in the association constants of L-tryptophan and diazepam of about 30 - 50% and a decrease in the extrinsic Cotton effects of four benzodiazepine derivatives of about 10 - 15% were found as specific effects of the tryptophan modification. The tryptophan modification itself did not change the number of binding sites of diazepam and L-tryptophan. It is suggested that the lone tryptophan residue of human serum albumin is not directly involved in the specific binding site for indole and benzodiazepine compounds. However, the modification alters the properties of the binding site either by an incomplete refolding of the albumin after urea treatment, or a more selective allosteric effect of the modified tryptophan residue.