TSPO ligand etifoxine attenuates LPS-induced cognitive dysfunction in mice.

The translocator protein (TSPO), once known as peripheral-type benzodiazepine receptor, was reported to be related with several physiological functions. Etifoxine is a clinically available anxiolytic drug, and has recently shown neuroprotective effects as a TSPO ligand. The aim of the present study was to investigate the influence of etifoxine on LPS-induced neuroinflammation and cognitive dysfunction. C57/BL6 male mice were injected with etifoxine (50 mg/kg, i.p.) three days before lipopolysaccharide (LPS, 500 µg/kg, i.p.) administration. Etifoxine pretreatment alleviated hippocampal inflammation, increased brain levels of progesterone, allopregnanolone and attenuated cognitive dysfunction in LPS-injected mice. While LPS increased expression of caspase-3 and decreased p-Akt/Akt, etifoxine returned caspase-3 and p-Akt/Akt to control levels. Finasteride, a 5α-reductase inhibitor that blocked allopregnanolone production, partially reversed the effects of etifoxine. We concluded that etifoxine exerted neuroprotective effects in LPS-induced neuroinflammation and the neuroprotection may be related with increase of neurosteroids synthesis and decrease of apoptosis.

Beta-adrenergic signaling accelerates and synchronizes cardiac ryanodine receptor response to a single L-type Ca2+ channel.

As the most prototypical G protein-coupled receptor, beta-adrenergic receptor (betaAR) regulates the pace and strength of heart beating by enhancing and synchronizing L-type channel (LCC) Ca(2+) influx, which in turn elicits greater sarcoplasmic reticulum (SR) Ca(2+) release flux via ryanodine receptors (RyRs). However, whether and how betaAR-protein kinase A (PKA) signaling directly modulates RyR function remains elusive and highly controversial. By using unique single-channel Ca(2+) imaging technology, we measured the response of a single RyR Ca(2+) release unit, in the form of a Ca(2+) spark, to its native trigger, the Ca(2+) sparklet from a single LCC. We found that acute application of the selective betaAR agonist isoproterenol (1 microM, < or = 20 min) increased triggered spark amplitude in an LCC unitary current-independent manner. The increased ratio of Ca(2+) release flux underlying a Ca(2+) spark to SR Ca(2+) content indicated that betaAR stimulation helps to recruit additional RyRs in synchrony. Quantification of sparklet-spark kinetics showed that betaAR stimulation synchronized the stochastic latency and increased the fidelity (i.e., chance of hit) of LCC-RyR intermolecular signaling. The RyR modulation was independent of the increased SR Ca(2+) content. The PKA antagonists Rp-8-CPT-cAMP (100 microM) and H89 (10 microM) both eliminated these effects, indicating that betaAR acutely modulates RyR activation via the PKA pathway. These results demonstrate unequivocally that RyR activation by a single LCC is accelerated and synchronized during betaAR stimulation. This molecular mechanism of sympathetic regulation will permit more fundamental studies of altered betaAR effects in cardiovascular diseases.

[Electrophysiology of two human hyperpolarization activated cyclic nucleotide-gated potassium channels expressed in HEK293 cells].

OBJECTIVE: To express the human HCN2 and HCN4 genes in HEK293 cells and investigate the electrophysiology of the expressed channel protein. METHODS: cDNA encoding human HCN2 or HCN4 gene was ligated into a shuttle vector pAdTrack-CMV. Homologous recombination was performed in Escherichia coli of the line BJ5183. Human embryonic kidney cells of the line 293 (HEK293 cells) were cultured and transfected with the positive recombinant adenovirus plasmid. Then the HEK293 cells were infected by AdhHCN2 or AdhHCN4 and the whole cell hyperpolarization-activated currents were recorded in HEK293 cells transfected with hHCN2 and hHCN4. RESULTS: If-like currents could be found in the HEK293 cells transfected with hHCN2 and hHCN4. The channels were activated by hyperpolarized potentials. Boltzmann equation showed that the half-activation voltage of the hHCN2 and hHCN4 channels were -114.8 mV +/- 3.3 mV and -125.9 mV +/- 2.9 mV respectively (P = 0.024). The reversal slope factors of the hHCN2 and hHCN4 channels were 11.1 mV +/- 1.2 mV and 13.7 mV +/- 1.3 mV respectively (P = 0.22). The activation kinetics was faster in hHCN2 than in hHCN4, with the activation constants at -110 mV being 0.99 s +/- 0.21 s and 8.47 s +/- 2.85 s respectively. The relative permeation ratio for sodium and potassium were 0.40 and 0.34 respectively in these two channels. Caesium chloride of the concentration of 2 mmol/L prominently inhibited both currents. CONCLUSION: The target genes hHCN2 and hHCN4 are successfully expressed in HEK293 cells, and the expressed functional channels have profoundly different activation kinetics.