Inhibitory actions of bisabolol on α7-nicotinic acetylcholine receptors.

Bisabolol is a plant-derived monocyclic sesquiterpene alcohol with antinociceptive and antiinflammatory actions. However, molecular targets mediating these effects of bisabolol are poorly understood. In this study, using a two-electrode voltage-clamp and patch-clamp techniques and live cellular calcium imaging, we have investigated the effect of bisabolol on the function of human α7 subunit of nicotinic acetylcholine receptor (nAChR) in Xenopus oocytes, interneurons of rat hippocampal slices. We have found that bisabolol reversibly and concentration dependently (IC50 = 3.1 µM) inhibits acetylcholine (ACh)-induced α7 receptor-mediated currents. The effect of bisabolol was not dependent on the membrane potential. Bisabolol inhibition was not changed by intracellular injection of the Ca(2+) chelator BAPTA and perfusion with Ca(2+)-free solution containing Ba(2+), suggesting that endogenous Ca(2+)-dependent Cl(-) channels are not involved in bisabolol actions. Increasing the concentrations of ACh did not reverse bisabolol inhibition. Furthermore, the specific binding of [(125)I] α-bungarotoxin was not attenuated by bisabolol. Choline-induced currents in CA1 interneurons of rat hippocampal slices were also inhibited with IC50 of 4.6 µM. Collectively, our results suggest that bisabolol directly inhibits α7-nAChRs via a binding site on the receptor channel.

Dopamine D2 and D3 receptors are linked to the actin cytoskeleton via interaction with filamin A.

We have used a yeast two-hybrid approach to uncover protein interactions involving the D2-like subfamily of dopamine receptors. Using the third intracellular loop of the D2S and D3 dopamine receptors as bait to screen a human brain cDNA library, we identified filamin A (FLN-A) as a protein that interacts with both the D2 and D3 subtypes. The interaction with FLN-A was specific for the D2 and D3 receptors and was independently confirmed in pull-down and coimmunoprecipitation experiments. Deletion mapping localized the dopamine receptor-FLN-A interaction to the N-terminal segment of the D2 and D3 dopamine receptors and to repeat 19 of FLN-A. In cultures of dissociated rat striatum, FLN-A and D2 receptors colocalized throughout neuronal somata and processes as well as in astrocytes. Expression of D2 dopamine receptors in FLN-A-deficient M2 melanoma cells resulted in predominant intracellular localization of the D2 receptors, whereas in FLN-A-reconstituted cells, the D2 receptor was predominantly localized at the plasma membrane. These results suggest that FLN-A may be required for proper cell surface expression of the D2 dopamine receptors. Association of D2 and D3 dopamine receptors with FLN-A provides a mechanism whereby specific dopamine receptor subtypes may be functionally linked to downstream signaling components via the actin cytoskeleton.

Enhanced proliferation, survival, and dopaminergic differentiation of CNS precursors in lowered oxygen.

Standard cell culture systems impose environmental oxygen (O(2)) levels of 20%, whereas actual tissue O(2) levels in both developing and adult brain are an order of magnitude lower. To address whether proliferation and differentiation of CNS precursors in vitro are influenced by the O(2) environment, we analyzed embryonic day 12 rat mesencephalic precursor cells in traditional cultures with 20% O(2) and in lowered O(2) (3 +/- 2%). Proliferation was promoted and apoptosis was reduced when cells were grown in lowered O(2), yielding greater numbers of precursors. The differentiation of precursor cells into neurons with specific neurotransmitter phenotypes was also significantly altered. The percentage of neurons of dopaminergic phenotype increased to 56% in lowered O(2) compared with 18% in 20% O(2). Together, the increases in total cell number and percentage of dopaminergic neurons resulted in a ninefold net increase in dopamine neuron yield. Differential gene expression analysis revealed more abundant messages for FGF8, engrailed-1, and erythropoietin in lowered O(2). Erythropoietin supplementation of 20% O(2) cultures partially mimicked increased dopaminergic differentiation characteristic of CNS precursors cultured in lowered O(2). These data demonstrate increased proliferation, reduced cell death, and enhanced dopamine neuron generation in lowered O(2), making this method an important advance in the ex vivo generation of specific neurons for brain repair.

The dopamine D3 receptor interacts with itself and the truncated D3 splice variant d3nf: D3-D3nf interaction causes mislocalization of D3 receptors.

We have generated a stable cell line expressing FLAG epitope-tagged D3 dopamine receptors and used this cell line to study D3 receptor-protein interactions. To analyze protein interactions, we separately introduced into the stable cell line either D3 receptors carrying an hemagglutinin (HA) epitope tag, or an HA-tagged version of the D3 receptor splice variant D3nf. A combination of confocal laser microscopy and coimmunoprecipitation was used to assay the formation and expression pattern of D3-D3 homodimers or D3-D3nf heterodimers. When coexpressed in HEK 293 cells, FLAG- and HA-tagged D3 receptors exhibited a similar plasma membrane distribution. Using an HA epitope tag-specific antibody, we coimmunoprecipitated HA- and FLAG-tagged D3 receptors, suggesting that D3 receptors are capable of forming homodimers. Epitope-tagged D3nf polypeptides exhibited a markedly different cellular distribution than D3 receptors. When expressed in HEK 293 cells, either alone or in combination with FLAG-tagged D3 receptors, D3nf exhibited a punctate perinuclear distribution. When D3nf was introduced into the stable D3-expressing cell line, D3 receptors were no longer visualized at the plasma membrane. Instead, D3 and D3nf showed a similar, predominantly cytosolic, localization. Using the HA-specific antibody, we were able to coimmunoprecipitate D3 and D3nf polypeptides from transfected cells. These data suggest the existence of physical interaction between D3 and D3nf. This interaction appears to result in the mislocalization of D3 receptors from the plasma membrane to an intracellular compartment, a finding that could be of significance in the etiology of schizophrenia.