Reversible inhibition of intracellular calcium influx through NMDA receptors by imidazoline I(2) receptor antagonists.

Intracellular calcium ([Ca(2+)]i) influx through N-methyl-d-aspartic acid (NMDA) receptors in cortical neurons is central to NMDA receptor-mediated excitotoxicity. Drugs that uncompetitively modulate NMDA receptor-mediated [Ca(2+)]i influx are potential leads for development to treat NMDA receptor-mediated neuronal damage since these drugs spare NMDA receptor normal functions. Ligands to alpha(2)-adrenoceptors and imidazoline I(2) receptors confer neuroprotection possibility through modulating NMDA receptor-mediated [Ca(2+)]i influx. Here, we investigated the characteristics of several ligands to alpha(2)-adrenoceptors and imidazoline I(2) receptor, in inhibiting NMDA receptor-mediated [Ca(2+)]i influx in cultured cortical neurons using a ratiometric calcium imaging technique. In contrast to MK801, which non-reversibly blocks NMDA receptor-mediated [Ca(2+)]i influx, imidazoline I(2) receptor antagonists, Idazoxan, and 2-(2-benzofuranyl)-2-imidazoline (2-BFI)-mediated inhibition of [Ca(2+)]i influx can be rapidly reversed when removed, in a manner similar to that of memantine, an uncompetitive antagonist to NMDA receptors. Interestingly, ligands to alpha(2)-adrenoceptors, including agmatine sulfate and yohimbine, and a ligand to the nicotinic receptor, levamisol, neither inhibited NMDA receptor-mediated [Ca(2+)]i influx, nor provided neuroprotection against glutamate toxicity, suggesting selective inhibition of NMDA receptor activities. The inhibition of NMDA receptor by Idazoxan and 2-BFI also led to the suppression of NMDA receptor-mediated calpain activity as a result of blocking NMDA receptor activity, rather than through direct inhibition of calpain activity. Collectively, these studies demonstrated that imidazoline I(2) receptor antagonists transiently and reversibly block NMDA receptor-mediated [Ca(2+)]i influx. These compounds are leads for further development as uncompetitive antagonists to NMDA receptor-mediated excitotoxicity.

bFGF expression mediated by a hypoxia-regulated adenoviral vector protects PC12 cell death induced by serum deprivation.

Basic fibroblast growth factor (bFGF) is a known neuroprotectant against a number of brain injury conditions such as cerebral ischemia. However, bFGF also regulates a plethora of brain developmental processes and functions as a strong mitogen. Therefore, unregulated long-term expression of bFGF in brain may potentially be tumorigenic, limiting its utility in brain therapy. Here, we report the successful construction of an adenoviral vector (Ad-5HRE-bFGF) expressing bFGF under the regulation of five hypoxia-responsive elements (5HRE) and a minimal cytomegalovirus promoter (CMVmp). Following hypoxia treatment in a hypoxic chamber with less than 1% of oxygen, Ad-5HRE-bFGF induced a significant and time-dependent expression of bFGF protein and the fluorescent tag, humanized GFP (hrGFP) protein, in infected PC12 cells. In contrast, normoxia treatment evoked extremely low level of bFGF and hrGFP expression, demonstrating that the 5HRE-CMVmp cassette was effective in regulating the expression of bFGF gene in response to hypoxia. More importantly, bFGF expressed by the Ad-5HRE-bFGF viral vector under the regulation of hypoxia was significantly neuroprotective against PC12 cell death evoked by serum deprivation. Taken together, these studies demonstrated the feasibility to express bFGF in a hypoxia-regulated fashion to provide neuroprotection. The Ad-5HRE-bFGF can be further developed as an effective tool to provide neuroprotection against hypoxia-induced brain diseases, such as cerebral ischemia.

Electrostatic charge activates inflammatory vanilloid (VR1) receptors.

The pathophysiology of neurogenic inflammation culminates in the overt symptoms of tissue inflammation through a series of events which are initiated by the activation of vanilloid receptors (VR1). This study was designed to test the hypothesis that a sufficiently negative, electrostatic charge carried on a particulate matter (PM) particle, could acquire a cloud of protons sufficient to activate proton-sensitive VR1 receptors and acid-sensitive ionic channels (ASICs) pathways. For this, nanometer-sized, synthetic polystyrene micells (SPM) or those charged with chemical groups (e.g. diamino, carboxyl) were used. These chemical groups imparted either a net positive (i.e. diamino) or negative (i.e. carboxyl) charge on the SPM when suspended in a neutral ionic medium. The zeta potential, a measure of the SPM's electronegativity, was taken in both cell culture nutrient medium and in ultraviolet light-distilled water (UV-DW). In both vehicles, the rank order of electronegativity (most to least negative) was carboxyl > polystyrene > diamino-SPM. Individual types of SPM were exposed to human, immortalized bronchial-tracheal epithelial cells (i.e. BEAS-2B) and endpoints of biological activation (i.e. membrane depolarization, increases in intracellular calcium (i.e. [Ca(2+)](i)) levels, IL-6 release) were measured. Cells loaded with a fluorescent probe for membrane depolarization (3,3'-dihexyloxacarbocyanine iodide, DiOC-6-3) showed a positive reaction when exposed to carboxyl-SPM but not to diamino-SPM. BEAS-2B cells exposed to carboxyl-SPM responded with significant increases in [Ca(2+)](i), and IL-6 release relative to uncharged SPM or diamino-SPM. This IL-6 release could be reduced by pretreatment with antagonists to the VR1 receptor (i.e. capsazepine) or to acid-sensitive ionc channels (i.e. amiloride). Although both diamino and carboxyl-SPM groups stimulated increases in IL-6 transcript, only the more electronegatively charged carboxyl-SPM stimulated mRNA-VR1 receptor. These data suggest that measurable inflammatory changes can be stimulated in human epithelial target cells by the electrostatic charge carried on an inert particle. Further, these changes appear to be mediated through acid-sensitive VR1 receptors and ASICs.