Abstract
Microglial cells play a critical role in the neuroinflammatory response that accompanies various diseases of the central nervous system, such as ischemic stroke, and ATP is a major signaling molecule regulating the response of these cells to these pathophysiological conditions. Experiments were carried out to determine the effects of afobazole on microglial function and to identify the molecular mechanisms by which afobazole affects microglial cells. Afobazole was found to inhibit migration of microglial cells in response to ATP and UTP chemoattraction in a concentration-dependent manner. Inhibition of either σ-1 or σ-2 receptors decreased the effects of afobazole on microglia. In addition to inhibiting microglial cell migration, activation of σ receptors by afobazole decreased intracellular calcium elevation produced by focal application of ATP and UTP in isolated microglial cells. Furthermore, afobazole blocked membrane currents elicited by rapid application of ATP in microglial cells. Taken together, our data indicate that afobazole inhibits microglial response to P2Y and P2X purinergic receptor activation by functioning as a pan-selective σ-receptor agonist. In addition to modulating response to purinergic receptor activation, the effects of afobazole on microglial survival during in vitro ischemia were assessed. Application of afobazole during in vitro ischemia decreased microglial cell death during the ischemic episode and after a 24-h recovery period. Moreover, when afobazole was only applied after the ischemic episode, a significant enhancement in cell survival was still observed. Thus, afobazole acts via σ receptors to decrease microglial response to ATP and provides cytoprotection during and after ischemia.
Footnotes
This study was supported by grants from RF Pharmaceuticals Sarl and MDR Pharmaceutical Limited (to J.C.).
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.111.182816.
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ABBREVIATIONS:
- DTG
- 1,3-di-o-tolyl-guanidine
- Afob
- afobazole
- AM
- acetoxymethyl ester
- ASIC
- acid-sensing ion channels
- SM-21
- (±)-tropanyl 2-(4-chlorophenoxy)butanoate maleate], rimcazole [9-[3-(cis-3,5-dimethyl-1-piperazinyl)propyl]-9H-carbazole dihydrochloride
- BD 1063
- 1-[2-(3,4-dichlorophenyl)ethyl]-4-methylpiperazine dihydrochloride
- BD 1047
- N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine dihydrobromide
- DMEM
- Dulbecco's modified Eagle's medium
- DMSO
- dimethyl sulfoxide
- MET
- metaphit
- PBS
- phosphate-buffered saline
- PSS
- physiological saline solution
- [Ca2+]i
- intracellular calcium concentration
- Δ[Ca2+]i
- change in intracellular calcium concentration
- LDH
- lactate dehydrogenase
- TNF-α
- tissue necrosis factor-α
- DAPI
- 4′-6-diamidino-2-phenylindole
- NE-100
- 4-methoxy-3-(2-phenylethoxy)-N,N-dipropylbenzeneethanamine hydrochloride.
- Received April 8, 2011.
- Accepted June 28, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
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