A Secreted Caspase-3-Substrate-Cleaving Activity at Low pH Belongs to Cathepsin B: a Study on Primary Brain Cell Cultures

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M. V. Onufriev¹, A. A. Yakovlev^1,2, A. A. Lyzhin³, M. Yu. Stepanichev¹, L. G. Khaspekov³, and N. V. Gulyaeva^1*

¹Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, ul. Butlerova 5a, 117485 Moscow, Russia; fax: (495) 952-4007; E-mail: nata_gul@pisem.net

²Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, ul. Institutskaya 3, 142290 Pushchino, Moscow Region, Russia

³Neurology Research Center, Russian Academy of Medical Sciences, Volokolamskoe Shosse 80, 125367 Moscow, Russia

^* To whom correspondence should be addressed.

Received June 11, 2008; Revision received August 5, 2008

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The cysteine proteases caspase-3 and cathepsins are involved in both neuronal plasticity and neuropathology. Using primary neuroglial and glial cerebellar cultures, the pH dependence of cleavage of a synthetic caspase-3 substrate, Ac-DEVD-AMC, was studied. At acidic pH, cathepsin B cleaved Ac-DEVD, this activity being significantly higher than that of caspase-3 at pH 7.4. This activity is blocked by peptide inhibitors of both caspase-3 and cathepsin B. Substitution of culture medium for balanced salt solution stimulated cathepsin B secretion in both types of cultures. Ischemia (oxygen–glucose deprivation) significantly decreased secretion of cathepsin B activities into the culture medium.

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KEY WORDS: cathepsin B, caspase-3, ischemia/reoxygenation, neurons, glia

DOI: 10.134/S0006297909030067

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