Hui Zheng

Anxiolytic Effect of Wogonin, a Benzodiazepine Receptor Ligand Isolated from Scutellaria Baicalensis Georgi

The search for novel anxiolytics devoid of undesirable side-effects typical of classical benzodiazepines (BDZs) has been intense, and flavonoids, as a relative new class of ligands, have been shown to possess anxiolytic effects in vivo. The present study evaluated the pharmacological properties of a naturally occurring monoflavonoid, 5,7-dihydroxy-8-methoxyflavone or wogonin. The affinity (K(i)) of wogonin for the benzodiazepine site (BZD-S) on the gamma-aminobutyric acid(A) (GABA(A)) receptor complex was 0.92 microM. Using electrophysiological techniques, we showed that wogonin enhanced the GABA-activated current in rat dorsal root ganglion neurons, and in Xenopus laevis oocytes expressing recombinant rat GABA(A) receptors, the enhancement was partially reversed by the co-application of a 1 microM concentration of the BZD-S antagonist anexate (Ro15-1788). Acute toxicity and behavioral effects were examined in mice. Acute lethal activity was low, with an LD(50) of 3.9 g/kg. Oral administration of wogonin (7.5 to 30 mg/kg) elicited an anxiolytic response that was similar to that elicited by diazepam in the elevated plus-maze; a dose-dependent increase in open arm entries and time spent in open arms was observed. More importantly, its anxiolytic effect was blocked by the co-administration of Ro15-1788. In the holeboard test, not only did wogonin-treated mice experience an increased number of head-dips but they also spent more time at it, showing no signs of sedation. Furthermore, wogonin did not cause myorelaxant effects in the horizontal wire test. Taken together, these data suggest that wogonin exerts its anxiolytic effect through positive allosteric modulation of the GABA(A) receptor complex via interaction at the BZD-S. Its anxiolytic effect was not accompanied by sedative and myorelaxant side-effects typical of BDZs.

Anxiolytic-like action of orally administered dl-tetrahydropalmatine in elevated plus-maze

dl-Tetrahydropalmatine (dl-THP), a naturally occurring alkaloid, has been intensively studied for its sedative and hypnotic effects. Putative explanation for its mechanism and target of action involves the dopaminergic neurotransmission system. In view of the close interactions between the dopaminergic and the GABAergic neurons in the amygdala, pharmacological effects of dl-THP were tested for activity at the GABAA receptor benzodiazepine site (BDS). Effects of dl-THP were examined in mice employing the elevated plus-maze, the holeboard and the horizontal-wire tests. In the elevated plus-maze, mice treated with low doses of dl-THP (0.5-10 mg/kg) exhibited significant increase in the percentage of entries and time spent in open arms without altering the number of closed-arm entries when compared to the control group, indicative of its selective anxiolytic effect. In the holeboard and horizontal wire tests, where exploratory behavior and potential muscle relaxant effect were assessed, respectively, only mice treated with as much as 50 mg/kg dl-THP manifested sedation and myorelaxation, as observed in the significant decrease in the number of head dips and the decrease in the percentage of mice grasping wire in comparison to control. Notably, coadministration of the BDS antagonist flumazenil abolished the dl-THP-induced anxiolysis as seen in the reversal of the increase of both the number of entries and time spent in open arms back to basal levels in the elevated plus-maze test. The results suggest that dl-THP at defined low dosages acts as anxiolytics in mice, and the BDS mediates, at least in part, such anxiolytic effect of dl-THP.

Topology characterization of a benzodiazepine‐binding β‐rich domain of the GABAA receptor α1 subunit

Structural investigation of GABAA receptors has been limited by difficulties imposed by its trans‐membrane‐complex nature. In the present study, the topology of a membrane‐proximal β‐rich (MPB) domain in the C139–L269 segment of the receptor α1 subunit was probed by mapping the benzodiazepine (BZ)‐binding and epitopic sites, as well as fluorescence resonance energy transfer (FRET) analysis. Ala‐scanning and semiconservative substitutions within this segment revealed the contribution of the phenyl rings of Y160 and Y210, the hydroxy group of S186 and the positive charge on R187 to BZ‐binding. FRET with the bound BZ ligand indicated the proximity of Y160, S186, R187, and S206 to the BZ‐binding site. On the other hand, epitope‐mapping using the monoclonal antibodies (mAbs) against the MPB domain established a clustering of T172, R173, E174, Q196, and T197. Based on the lack of FRET between Trp substitutionally placed at R173 or V198 and bound BZ, this epitope‐mapped cluster is located on a separate end of the folded protein from the BZ‐binding site. Mutations of the five conserved Cys and Trp residues in the MPB domain gave rise to synergistic and rescuing effects on protein secondary structures and unfolding stability that point to a CCWCW‐pentad, reminiscent to the CWC‐triad “pin” of immunoglobulin (Ig)‐like domains, important for the structural maintenance. These findings, together with secondary structure and fold predictions suggest an anti‐parallel β‐strand topology with resemblance to Ig‐like fold, having the BZ‐binding and the epitopic residues being clustered at two different ends of the fold.

Two β‐rich structural domains in GABAA receptor α1 subunit with different physical properties: Evidence for multidomain nature of the receptor

The type A γ‐aminobutyric acid (GABAA) receptor is a major inhibitory neurotransmitter‐gated ion channel. Previously, we identified a membrane‐proximal β‐rich (MPBR) domain in fragment C166‐L296 of GABAA receptor α1 subunit, forming nativelike pentamers. In the present study, another structural domain, the amino‐terminal domain, was shown to exist in the fragment Q28‐E165. The secondary structures of both fragments were β‐rich as measured using FTIR spectroscopy and estimated from the CD spectra to be 42% and 51% β‐strand for Q28‐E165 and C166‐L296, respectively. The CD spectrum of the combined fragment Q28‐L296 was additive of the spectra of the two fragments. In addition, denaturation curves of both fragments were characteristic of cooperative transitions, supporting their domainlike nature. C166‐L296 required 6.5 M of guanidine chloride for total denaturation, therefore it is extraordinarily stable, more so than Q28‐E165. Moreover, effects of detergent on the molecular masses of Q28‐E165 and C166‐L296, as monitored using laser‐scattering spectroscopy, indicated that intermolecular interactions were much more significant in C166‐L296 than in Q28‐E165. Effects of pH on their molecular masses suggested that ionic forces were involved in these interactions. Together the results show that the two adjacent fragments form independent folding units, MPBR and amino‐terminal domains, different in secondary structure content, denaturation profile, and polymerization status, and suggest that the former may play a more important role in receptor assembly and that the extraordinary stability may underlie its intrinsic tendency to form oligomers. More significantly, the present study has provided direct evidence for the long‐postulated multidomain nature of this family of receptors.

Preparation of Highly Purified Hemoglobin by Affinity Elution

The large scale production of recombinant hemoglobin (Hb) from microorganism or transgenic hosts for Hb-based blood substitutes places utmost emphasis on purity. In the present study, a high-resolution, convenient and inexpensive purification method is developed for purification of Hb from mixtures containing E. coli extract and bovine serum. This method is based on affinity elution by pyrophosphate (PPi) of Hb adsorbed on an FPLC column of the anion exchanger Toyopearl DEAE-650M. Compared to pH elution or NaCl elution, PPi elution makes possible the preparation of Hb of much higher purity. A procedure combining pH elution and PPi elution sequentially using a single column proves particularly valuable. The purification method is also applicable to the purification of cyanomet-Hb (CNHb+).