CA1 pyramidal cells were voltage clamped, and GABA was applied to individual cells with a modified U-tube, rapid drug application system. With Vh = -50 mV, inward currents elicited by 10 microM GABA were inhibited by GABAA receptor (GABAR) antagonists and were baclofen insensitive, suggesting that GABA actions on isolated CA1 pyramidal cells were GABAR mediated. GABA concentration-response curves averaged from all cells were fitted best with a two-site equation, indicating the presence of at least two GABA binding sites, a higher-affinity site (EC50-1 = 11.0 microM) and a lower-affinity site (EC50-2 = 334.2 microM), on two or more populations of cells. The effects of GABAR allosteric modulators on peak concentration-dependent GABAR currents were complex and included monophasic (loreclezole) or multiphasic (diazepam) enhancement, mixed enhancement/inhibition (DMCM, zolpidem) or multiphasic inhibition (zinc). Monophasic (70% of cells) or biphasic (30% of cells) enhancement of GABAR currents by diazepam suggested three different sites on GABARs (EC50-1 =1.8 nM; EC50-2 = 75.8 nM; EC50-3 = 275.9 nM) revealing GABAR heterogeneity. The imidazopyridine zolpidem enhanced GABAR currents in 70% of cells with an EC50 = 222.5 nM, suggesting a predominance of moderate affinity alpha2 (or alpha3-) subtype-containing BZ Type IIA receptors. A small fraction of cells (10%) had a high affinity for zolpidem, something that is suggestive of alpha1 subtype-containing BZ Type I receptors. The remaining 30% of cells were insensitive to or inhibited by zolpidem, suggesting the presence of alpha5 subtype-containing BZ Type IIB receptors. Whether BZ Type I and Type II receptors coexist could not be determined. The beta-carboline methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) inhibited GABAR currents in all cells at midnanomolar concentrations, but in addition, potentiated GABAR currents in some cells at low nanomolar concentrations, characterizing two groups of cells, the latter likely due to functional assembly of alpha5betaxgamma2GABARs. In all cells, GABAR currents were moderately sensitive (EC50 = 9 microM) to loreclezole, consistent with a relatively greater beta3 subtype, than beta1 subtype, subunit mRNA expression. Two populations of cells were identified based on their sensitivities to zinc(IC50 = 28 and 182 microM), suggesting the presence of at least two GABAR isoforms including alpha5beta3gamma2 GABARs. Consistent with the heterogeneity of expression of GABAR subunit mRNA and protein in the hippocampus and based on their differential responses to GABA and to allosteric modulators, distinct populations of CA1 pyramidal cells likely express multiple, functional GABAR isoforms. (+info)
Occurrence of stereoisomers of 1-(2'-pyrrolidinethione-3'-yl)- 1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid in fermented radish roots and their different mutagenic properties.
Stereoisomers of the tetrahydro-beta-carboline derivative, 1-(2-pyrrolidinethione)-3-yl)-1,2,3,4-tetrahydro-beta-carboline- 3-carboxylic acid (PTCC), were formed from L-tryptophan with 4-methylthio-3-butenyl isothiocyanate, and their mutagenic properties and contents in different types of the radish products were studied. The isomers were identified as (1S*, 3S*, 3R*)- and (1R*, 3S*, 3R*)-PTCCs; the former was found as the major compound but had no mutagenic activity, while the latter was mutagenic toward Salmonella typhimurium TA 98 in the presence of a rat microsomal fraction. Both (1S*, 3S*, 3R*)- and (1R*, 3S*, 3R*)-PTCC were detected in a ratio of about 4:1 in a product fermented for 8 months, but only a trace was apparent in products manufactured within a few weeks. (+info)
Heterocyclic aromatic amines induce DNA strand breaks and cell transformation.
Heterocyclic aromatic amines (HAAs), formed during the cooking of foods, are known to induce tumours in rodent bioassays and may thus contribute to human cancer risk. We tested six HAAs in a morphological transformation assay and in three in vitro genotoxicity assays. The morphological transforming abilities of HAAs were tested, in the presence of rat-liver S9, in the C3H/M2 fibroblast cell line. Concentration levels of 50 microM 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), 100 microM 2-amino-3,4,8-trimethylimidazo-[4,5-f]quinoxaline (4,8-DiMeIQx), 50 microM 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 100 microM 2-amino-9H-pyrido[2,3-b]indole (AalphaC), 100 microM 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAalphaC) and 15 microM 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induced maximum transformation potencies of 5.5, 6.6, 6.3, 5.2, 7.3 and 9.2 transformed foci per 10(4) surviving cells, respectively. Bacterial mutagenic activity was determined in the presence of rat-liver S9 using the Salmonella typhimurium reverse-mutation assay employing strain YG1019. Mutagenic potencies of 3800 revertants (revs)/ng with 8-MeIQx, 2900 revs/ng with 4,8-DiMeIQx, 3480 revs/ng with IQ, 1.6 revs/ng with AalphaC, 2.9 revs/ng with MeAalphaC and 5 revs/ng with PhIP were observed. Clastogenic activity in vitro was analysed by the micronucleus assay in metabolically competent MCL-5 cells. Dose-dependent induction of micronuclei was observed for all HAAs tested with 1-5.4% of cells containing micronuclei at 10 ng/ml. Micronucleus induction was in the order 4,8-DiMeIQx > 8-MeIQx > IQ > MeAalphaC > PhIP > AalphaC. DNA strand-breaking activity in MCL-5 cells was measured by the alkaline single cell-gel (comet) assay. The lowest effect doses for significant increases (P < or = 0.0007, Mann-Whitney test) in comet tail length (microm) were 45.5 microg/ml (200 microM) for PhIP, 90.9 microg/ml (410-510 microM) for 4,8-DiMeIQx, IQ, MeAalphaC and AalphaC, and 454.5 microg/ml (2130 microM) for 8-MeIQx. It is not yet clear which of these assays most accurately reflects the genotoxic potential to humans of compounds of this class of environmental carcinogens. (+info)
Mapping quantitative trait loci for seizure response to a GABAA receptor inverse agonist in mice.
To define the genetic contributions affecting individual differences in seizure threshold, a beta carboline [methyl-beta-carboline-3-carboxylate (beta-CCM)]-induced model of generalized seizures was genetically dissected in mice. beta-CCM is a GABAA receptor inverse agonist and convulsant. By measuring the latency to generalized seizures after beta-CCM administration to A/J and C57BL6/J mice and their progeny, we estimated a heritability of 0.28 +/- 0.10. A genome wide screen in an F2 population of these parental strains (n = 273) mapped quantitative trait loci (QTLs) on proximal chromosome 7 [logarithm of the likelihood for linkage (LOD) = 3.71] and distal chromosome 10 (LOD = 4.29) for seizure susceptibility, explaining approximately 22 and 25%, respectively, of the genetic variance for this seizure trait. The best fitting logistic regression model suggests that the A/J allele at each locus increases the likelihood of seizures approximately threefold. In a subsequent backcross population (n = 223), we mapped QTLs on distal chromosome 4 (LOD = 2.88) and confirmed the distal chromosome 10 QTLs (LOD = 4.36). In the backcross, the C57BL/6J allele of the chromosome 10 QTL decreases the risk of seizures approximately twofold. These QTLs may ultimately lead to the identification of genes influencing individual differences in seizure threshold in mice and the discovery of novel anticonvulsant agents. The colocalization on distal chromosome 10 of a beta-CCM susceptibility QTL and a QTL for open field ambulation and vertical movement suggests the existence of a single, pleiotropic locus, which we have named Exq1. (+info)
The fluorescence of scorpions and cataractogenesis.
BACKGROUND: Protein cross-linking and fluorescence are widely recognized markers of oxidative aging in human proteins. Oxidative protein aging is a combinatorial process in which diversity arises from the heterogeneity of the targets and is amplified by the nonselective nature of the reactants. The cross-links themselves defy analysis because they are generally embedded in a covalent matrix. Arthropods rely upon oxidative cross-linking in the hardening of the cuticle - a process known as sclerotization. Among arthropods, scorpions are noteworthy in that the process of sclerotization is accompanied by the buildup of strong visible fluorescence. To date, the nature of the fluorescent species has remained a mystery. RESULTS: We have identified one of the soluble fluorescent components of the scorpions Centuroides vittatus and Pandinus imperator as beta-carboline - a tryptophan derivative that has previously been identified by hydrolysis and oxidation of lens protein. We have also shown that beta-carboline-3-carboxylic acid is released from both scorpion exuvia (the shed cuticle) and human cataracts upon hydrolysis, suggesting that the protein-bound beta-carboline and free beta-carboline have common chemical origins. CONCLUSIONS: Cataractogenesis and cuticular sclerotization are disparate oxidative processes - the former is collateral and the latter is constitutive. The common formation of beta-carbolines shows that similar patterns of reactivity are operative. These fundamental mechanisms provide predictive insight into the consequences of human protein aging. (+info)
Apparent pA2 values of benzodiazepine antagonists and partial agonists in monkeys.
Drugs that bind to benzodiazepine recognition sites of gamma-aminobutyric acid type A receptor complexes may function as agonists in some behavioral assays and as antagonists in other behavioral assays. The present studies compared the effects of the benzodiazepines midazolam, flumazenil, bretazenil, Ro 41-7812, and Ro 42-8773 and the beta-carboline, beta-carboline-3-carboxylate-t-butyl ester (beta-CCt) under two different types of schedule-controlled responding in squirrel monkeys. One group of monkeys responded under a fixed-ratio schedule of stimulus-shock termination, and a second group of monkeys responded under a multiple fixed-ratio schedule of food presentation involving suppressed and nonsuppressed behavior. Under the schedule of stimulus-shock termination, midazolam produced dose-related decreases in response rate, and these effects were surmountably antagonized by flumazenil, bretazenil, Ro 41-7812, Ro 42-8773, and beta-CCt. Schild plot analysis of these data revealed the following mean pA(2) values: flumazenil, 7.18; bretazenil, 7.62; Ro 41-7812, 7. 06; Ro 42-8773, 6.95. Apparent pA(2) values were not calculated for beta-CCt because the CL of the slope of the Schild plot included positive values. Under the multiple schedule, midazolam, bretazenil, and Ro 42-8773 dose-dependently increased rates of suppressed responding, whereas flumazenil, Ro 41-7812, and beta-CCt had no significant rate-altering effects. Flumazenil antagonized the antisuppressant effects of midazolam and bretazenil; however, individual variability in these effects prohibited the determination of apparent pA(2) values. These results indicate that in vivo pA(2) values may be determined for benzodiazepine-site ligands. These results further demonstrate that some benzodiazepine-site ligands, e. g., bretazenil and Ro 42-8773, may function as both agonists and as competitive antagonists in vivo. (+info)
Dual mode of stimulation by the beta-carboline ZK 91085 of recombinant GABA(A) receptor currents: molecular determinants affecting its action.
In electrophysiological measurements the beta-carboline ethyl 6-benzyloxy-beta-carboline-3-carboxylate (ZK 91085) acts as a positive allosteric modulator on rat recombinant alpha1beta2gamma2 GABA(A) receptors and binds with high affinity (IC50-1.5 nM) to the [3H]-flunitrazepam site. Flumazenil was able to partially counteract the current modulation. These observations indicate an action of ZK 91085 at the benzodiazepine binding site. At the dual subunit combination alpha1beta2, which lacks the gamma subunit required for benzodiazepine modulation, we still observed a potentiation of GABA currents. Thus ZK 91085 acts via an additional site on the channel. At the subunit combination alpha1beta1, ZK 91085 potentiation is strongly reduced as compared to alpha1beta2. In binding studies, ZK 91085 was able to decrease [35S]-TBPS binding in alpha1beta2gamma2 and alpha1beta2 but not in alpha1beta1. This selectivity of ZK 91085 for receptors containing the beta2 isoform over those containing the beta1 isoform is reminiscent of the action of loreclezole. To identify amino acid residues important for the second type of modulation, we functionally compared wild type alpha1beta2 and mutant receptors for stimulation by ZK 91085. The mutation beta2N265S, that abolishes loreclezole effects, also abolishes ZK 91085 stimulation. The mutation beta2Y62L increased stimulation by ZK 91085 3-4 fold, locating an influencing entity of the second type of action of ZK 91085 at an alpha/beta subunit interface. Structural intermediates of ZK 91085 and the beta-carboline abecarnil, the latter of which only slightly potentiated GABA currents in alpha1/beta2, were analysed to determine structural requirements for modulation. ZK 91085 thus allosterically stimulates the GABA(A) receptor through two sites of action: the benzodiazepine site and the loreclezole site in contrast to classical beta-carbolines, that confer negative allosteric modulation through the benzodiazepine site. (+info)
Improvement in pain and bowel function in female irritable bowel patients with alosetron, a 5-HT3 receptor antagonist.
BACKGROUND: No currently available treatment provides consistent relief of irritable bowel syndrome. Colonic sensory and motor function are modulated partly through 5HT3-receptors. AIM: To evaluate effects of the 5HT3-receptor antagonist, alosetron, in irritable bowel syndrome. METHODS: Randomized, double-blind, placebo-controlled, dose-ranging (1, 2, 4, 8 mg b.d. alosetron), 12-week trial in 370 patients with diarrhoea-predominant or alternating constipation and diarrhoea irritable bowel syndrome. Weekly measurement of adequate relief was the key end-point; other irritable bowel syndrome symptoms were collected daily using an electronic phone system. RESULTS: Alosetron (1 mg or 2 mg b.d.) significantly (P < 0.05 vs. placebo) increased the proportion of females, but not males, reporting adequate relief. Stool consistency, frequency and percentage days with urgency improved over placebo (P < 0.05) within the first month with all doses of alosetron, and persisted throughout the trial with all doses in female patients. With 1 mg b.d. alosetron, females had improved stool consistency and urgency within the first week, and adequate relief and improved stool frequency within the first 2 weeks. There was no consistent improvement in bowel function among male patients. CONCLUSION: In female irritable bowel syndrome patients with predominant diarrhoea or alternating constipation and diarrhoea, alosetron is effective in treatment of abdominal pain and discomfort and bowel-related symptoms. (+info)