Certain types of cellular cysteine proteases may play an important role in modulating the activity of G-protein coupled receptors. The activity of this class of enzymes is closely regulated by cytoplasmic and nuclear inhibitors. The interaction of these inhibitors with the target proteases is, in part, mediated by strong hydrophobic interactions. The effects of ethyl alcohol exposure on cysteine protease activity has been studied in cell culture utilizing a PC12 cell line. Activity of these proteases appears to be strongly affected by alcohol exposure. We have established that exposure of PC12 cells to ethyl alcohol for 96 hours results in: a) a decrease in calcium-stimulated protease activity, which is evident at exposure concentrations of 20 mM and, b) a decrease in both isoforms of calpain, which is evident at 40 mM and 80 mM. The results of this work were published (J Neurochem 1997;68:1863-9). An abstract is available at www.well.com/user/ pdeep/abstracts/pub12ab.html. We are working towards defining a mechanism for this alcohol-induced inhibition of calcium-activated protease activity. We have generated a polyclonal antibody to Domain IV of calpastatin, a cellular inhibitor of protease activity, and have determined that alcohol exposure increases the levels of calpastatin in PC12 cells. The increased levels of the inhibitor following ethanol exposure may explain the observed decrease in calcium-stimulated calpain activities. Both calpastatin and the 5HT-3 receptor are known to be up-regulated by treatment of PC-12 cells with nerve growth factor, suggesting that a common pathway involved in message expression might be operating. Using quantitative RT-PCR, we are determining whether ethanol exposure increases the expression of calpastatin message. We are also examining whether message for the 5HT-3 receptor, which functions as a calcium-ion gate, is affected by ethanol exposure. A polyclonal antibody for the 5HT-3 receptor is being raised so that we can determine if protein levels for the receptor are also modulated by ethanol exposure.
| 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 |
