Urinary and brain beta-carboline-3-carboxylates as potent inhibitors of brain benzodiazepine receptors.

Abstract

Benzodiazepines probably exert their anxiolytic, hypnotic, and anticonvulsant effects by interacting with brain-specific high-affinity benzodiazepine receptors. In searching for possible endogenous ligands for these receptors, we have purified a compound 107-fold from human urine by extractions, treatment with hot ethanol, and column chromatography. The compound was identified as ,-carboline-3-carboxylic acid ethyl ester (IIc) by mass spectrometry, NMR spectrometry, and synthesis; HIc was also isolated from brain tissues (20 ng/g) by similar procedures. Very small concentrations of TIc displaced [3H]diazepam completely from specific cerebral receptors, but not from liver andkidney binding sites; the concentration causing 50% inhibition of specific [3H]diazepam binding (IC50) was 4-7 nM compared to ca. 5 nM for the potent benzodiazepine lorazepam. Specific binding sites for quinuclidinyl benzilate, naloxone, spiroperidol, serotonin, muscimol, and WB 4101 were not affected by IIc. In contrast to benzodiazepines, I1c exhibits "mixed type" competitive inhibition of forebrain benzodiazepine receptors (negative cooperativity). We surmise that an endogenous ligand for benzodiazepine receptors may be a derivative of P-carboline-3-carboxylic acid.[3H]Diazepam and [3H]flunitrazepam bind specifically and with high affinity to brain membranes from all higher vertebrates, including man (1-5). Binding is brain specific (6), correlated to pharmacological potency (1, 7-10), located on neurons (11-17), distributed unevenly in the brain (6), and slightly sensitive to seizures and stressful conditions (18,19 5.5. Further impurities were removed by washing twice with 0.05 vol of borate buffer (100 mM, pH 9) and three times with 0.05 vol of aqueous NH3 (100 mM). All aqueous phases were washed twice in counter current fashion with ethyl acetate to decrease losses. The combined ethyl acetate phase was evaporated to dryness and the residue was dissolved in 300 ml of 0.18-0.25 M HCI and washed with 500 ml of diethyl ether, which removed large amounts of impurities. The active fraction was then extracted twice with 500 ml of diethyl ether at pH 5-5.5. After evaporation the brown-orange waxy residue was dissolved in 3 ml of 50% ethanol and chromatographed on a Sephadex LH-20 column [1.5 X 84 cm, elution volume (Ve) = 144 ml] in 50% ethanol. The active fraction (y-fraction) was eluted in a volume of [15][16][17][18][19][20][21][22][23][24][25]

Effects of Melatonin on Sleep and Neurochemistry in the Rat

“…Not only do the benzodiazepines and melatonin both promote sleep but melatonin, at higher doses, also shares the muscle relaxant and anticonvulsant properties of the benzodiazepines (Sugden, 1980), suggesting that melatonin, or a metabolite, could act as an agonist at the receptor. Furthermore there are structural similarities between AMK and the I-carbolines, one of which may be an endogenous ligand for the benzodiazepine receptor (Braestrup, Nielsen & Olsen, 1980). Although the brain concentrations of melatonin and its pyrollase metabolites are probably too low for any to be considered an endogenous ligand at the benzodiazepine receptor (Koslow, 1974;Pang, et al, 1977), it is possible that the psychopharmacological effects of melatonin administration may be explained by this mechanism.…”

The anti‐epileptic drug levetiracetam reverses the inhibition by negative allosteric modulators of neuronal GABA‐ and glycine‐gated currents

“…Even if some of them have been suggested as endogenous (Braestrup et al, 1980;Medina et al, 1989;PenÄ a et al, 1986), b-carbolines are unlikely to be important pathogenic agents causing epilepsy. In contrast, the data on zinc are remarkable when confronted to the possibility that the hyperexcitability of the epileptic hippocampus may be associated with circuit and cellular alterations involving changes both in the subunit expression pattern of GABA A receptors in dentate granule cells (DGC) and in sprouting of zinc-containing DGC axons back onto the inner molecular layer of the dentate gyrus.…”

Benzodiazepines modulate the A2 adenosine binding sites on 108CC15 neuroblastoma × glioma hybrid cells

“…A variety of endogenous ligands for the benzodiazepine binding sites have been proposed (Skolnick et al, 1978;Marangos et al, 1979;Braestrup et al, 1980;Massotti & Guidotti, 1980;Davies & Cohen 1980;Woolf & Nixon 1981) ranging in nature from purines to high molecular weight proteins. An involvement of purines in the actions of benzodiazepines has been suggested and Phillis and his coworkers (Bender et al, 1980b;Phillis et al, 1980;Wu et al, 1981); have proposed that inhibition of adenosine uptake could be involved in some of the pharmacological effects of benzodiazepines.…”