Abstract
Ethyl 5-amino-6,7,8,9-tetrahydro-2-methyl-4-phenylbenzol[1,8] naphthyridine-3-carboxylate (ITH4012) is a novel tacrine derivative that can reduce cell death induced by various compounds with different mechanisms of action, such as thapsigargin (reticular stress), H2O2 (free radicals), and veratridine (calcium overload), in bovine chromaffin cell. Cell viability, quantified as lactic dehydrogenase release, was significantly reduced by ITH4012 at concentrations ranging from 0.01 to 3 μM. In the human neuroblastoma cell line SH-SY5Y, ITH4012 also reduced amyloid β25-35-induced apoptosis, determined by flow cytometry. ITH4012 caused a slight elevation in the cytosolic concentration of Ca2+ in fura 2-loaded bovine chromaffin cells, which could be related to the induction of protein synthesis relevant for cell survival. Blockade of protein synthesis by cycloheximide or blockade of Bcl-2's active site with HA14-1 (ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate) reversed the cytoprotective action of ITH4012. Furthermore, exposure of bovine chromaffin cells for 24 or 48 h to neuroprotective concentrations of this compound enhanced, nearly 3-fold, the expression of the antiapoptotic protein Bcl-2. In conclusion, ITH4012 is a tacrine derivative that maintains acetylcholinesterase-inhibiting activity (IC50 = 0.8 μM) but has the additional property of acting as a calcium promotor, a property leading to neuroprotection through the induction of antiapoptotic proteins.
Footnotes
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This work was supported in part by the Dirección General de Investigación Científica y Técnica Grants BFI2003-02722 (to A.G.G.) and SAF2003-04596 (to M.G.L.), Red CIEN, Instituto Carlos III, and Fundación La Caixa (Barcelona).
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Parts of these data have been presented previously. C.O., C.d.l.R., J.L.M., M.G.L., and M.V. TK 19: a new Ca2+ promotor with neuroprotective properties. XXV Congress of the Spanish Society of Pharmacology. October 8 to 11, 2003, Cadiz, Spain.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.104.068189.
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ABBREVIATIONS: AD, Alzheimer's disease; Aβ, amyloid β; [Ca2+]c, cytosolic concentration of Ca2+; AChE, acetylcholinesterase; ITH4012, ethyl 5-amino-6,7,8,9-tetrahydro-2-methyl-4-phenylbenzol[1,8]naphthyridine-3-carboxylate; Bay K8644, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl]phenyl)-pyridine-3-carboxylic acid methyl ester; DMEM, Dulbecco's modified Eagle's medium; LDH, lactic dehydrogenase; PI, propidium iodide; PBS, phosphate buffer solution; HA14-1, ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate.
- Received March 11, 2004.
- Accepted April 22, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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