NMDA receptor upregulation occurring after prolonged alcohol administration in rodent models has been implicated in neural cytotoxicity, via an increase in calcium-flux resulting from l-glutamate activation of these calcium channels. Calpains are a class of calcium-activated cysteine proteases that are known to mediate calcium-mediated injury in neural cells. The focus of this project is to a) examine the mechanism of l-glutamate mediated cell injury using a PC12 cell model; b) determine whether inhibition of calpain activity would result in significant cytoprotection; c) test a variety of calpain inhibitors as cytoprotectants in an l-glutamate model of cytotoxicity; d) extend these observations to a rodent model of neural cell injury. It was previously shown in this laboratory that ritonavir, an HIV protease inhibitor, is also a competitive inhibitor of calpain activity, with a Ki of ~10 microM, well within range found during clinical dosing of the drug in humans when used as an anti-retroviral. Using fura-2, it was found that l-glutamate singificantly increased intracellular concentrations of free calcium. The increase in intracellular free calcium induced by l-glutamate was also shown to result in a signficant increase in calpain protease activity. Calpain activation is followed by degradation of a variety of cytoskeletal components, and in this model it was found that l-glutamate exposure resulted in significant degradation of actin, tau, and NF68, which was blocked by MK801, calpain inhibitor I and ritonavir. In cytotoxicity tests, it was found that MK801, calpain inhibitor I, and ritonavir also all inhibited l-glutamate mediated cell death. Neither a caspase inhibitor (Z-DEVD-FMK) nor DNQX (AMPA inhibitor) had any protectant effects, nor did they prevent l-glutamate induced breakdown of cytoskeletal proteins. We conclude from the initial phases of this work that l-glutamate mediated cytotoxicity in PC12 cells is a calpain-dependent process. In future work, the neuroprotective effects of these agents will be examined in a rodent model. If this is successful, this would raise the possibility that ritonavir or its analogues might be useful as neuroprotective agents when cellular injury is thought to occur via a calcium-mediated process.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Intramural Research (Z01)
Project #
1Z01AA000203-01
Application #
6413411
Study Section
(LCS)
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
2000
Total Cost
Indirect Cost
Name
Alcohol Abuse and Alcoholism
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Wan, Wenshuai; DePetrillo, Paolo B (2002) Ritonavir inhibition of calcium-activated neutral proteases. Biochem Pharmacol 63:1481-4
Rackoff, J; Yang, Q; DePetrillo, P B (2001) Inhibition of rat PC12 cell calpain activity by glutathione, oxidized glutathione and nitric oxide. Neurosci Lett 311:129-32
DePetrillo, P B; Yang, Q; Rackoff, J et al. (2000) Surface fractal computation and its application to immunofluorescent histochemical studies of calpain and calpastatin in PC12 cells. J Neurosci Methods 103:191-7