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Inhibition of calpain expression by E-64d in the rat retina subjected to ischemia/reperfusion injury

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Abstract

To investigate the effect of E-64d, a selective inhibitor of calpain, on the expression of calpain and calpastatin in rat retina was subjected to ischemia/reperfusion injury (IRI). An animal model of retinal IRI was set up by increasing the intraocular pressure (110 mm Hg) of a rat eye for 1 h. The retinal thickness and morphologic changes were detected by histology. The protein expression of m-calpain (a calpain isoform) in the retina was assessed by immunohistochemistry and Western blot assay. The mRNA of m-calpain, as well as calpastatin (an endogenous protein inhibitor of calpain), in the retina was assessed by RT-PCR, and the ratio of m-calpain/calpastatin was then calculated. To evaluate the effect of E-64d on the expression of calpain, the drug (5 μl of 100 μM) was injected intravitreously immediately after IRI. There were retinal edematous changes, particularly in the inner plexiform layer after IRI. The protein expression of m-calpain in the retina was increased 24 h after IRI, an effect that was inhibited by E-64d (P < 0.05). The mRNA expression of m-calpain and calpastatin was also increased 24 h and 3 h after IRI, respectively. Neither m-calpain nor calpastatin mRNA expression was influenced by E-64d (P > 0.05). The mRNA ratio of m-calpain to calpastatin was increased at the 6 h, 24 h and 72 h after IRI, and only at 24 h the increase of the ratio of m-calpain to calpastatin was inhibited by E-64d (P < 0.05). In the rat retina of IRI, E-64d inhibits the increase of m-calpain protein expression, as well as the mRNA ratio increase of m-calpain to calpastatin. E-64d also inhibited the retinal damage induced by IRI, suggesting a role for E-64d in the protection of the retinal apoptosis induced by IRI.

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References

  1. Osborne N.N., Casson R.J., Wood J.P., Chidlow G., Graham M., Melena J. 2004. Retinal ischemia: Mechanisms of damage and potential therapeutic strategies. Progr. Retin. Eye. Res. 23, 91–147.

    Article  CAS  Google Scholar 

  2. Buchi E.R., Suivaizdis I., Fu J. 1991. Pressure-induced retinal ischemia in rats: An experimental model for quantitative study. Ophthalmologica. 203, 138–147.

    Article  PubMed  CAS  Google Scholar 

  3. Reed J.C. 1994. Bcl-2 and regulation of programmed cell death. J. Cell. Biol. 124, 1–6.

    Article  PubMed  CAS  Google Scholar 

  4. Takahashi A., Masuda A., Sun M., Centonze V.E., Herman B. 2004. Oxidative stress-induced apoptosis is associated with alterations in mitochondrial caspase activity and Bcl-2-dependent alterations in mitochondrial pH (pHm). Brain Res. Bull. 62, 497–504.

    Article  PubMed  CAS  Google Scholar 

  5. Katai N., Yoshimura N. 1999. Apoptotic retinal neuronal death by ischemia-reperfusion is executed by two distinct caspase family proteases. Invest. Ophthalmol. Vis. Sci. 40, 2697–2705.

    PubMed  CAS  Google Scholar 

  6. Sakamoto Y.R., Nakajima T.R., Fukiage C.R., Sakai O.R., Yoshida Y.R., Azuma M.R., Shearer T.R. 2000. Involvement of calpain isoforms in ischemia-reperfusion injury in rat retina. Curr. Eye Res. 21, 571–580.

    Article  PubMed  CAS  Google Scholar 

  7. Tamada Y., Nakajima E., Nakajima T., Shearer T.R., Azuma M. 2005. Proteolysis of neuronal cytoskeletal proteins by calpain contributes to rat retinal cell death induced by hypoxia. Brain Res. 1050, 148–155.

    Article  PubMed  CAS  Google Scholar 

  8. Pontremoli S., Salamino F., Sparatore B., De Tullio R., Pontremoli R., Melloni E. 1988. Characterization of the calpastatin defect in erythrocytes from patients with essential hypertension. Biochem. Biophys. Res. Commun. 157, 867–874.

    Article  PubMed  CAS  Google Scholar 

  9. Pontremoli S., Melloni E., Viotti P.L., Michetti M., Salamino F., Horecker B.L. 1991. Identification of two calpastatin forms in rat skeletal muscle and their susceptibility to digestion by homologous calpains. Arch. Biochem. Biophys. 288, 646–652.

    Article  PubMed  CAS  Google Scholar 

  10. Tamai M., Matsumoto K., Omura S., Koyama I., Ozawa Y., Hanada K. 1986. In vitro and in vivo inhibition of cysteine proteases by EST, a new analog of E-64. J. Pharmacobiodyn. 9, 672–677.

    PubMed  CAS  Google Scholar 

  11. Tamai M., Omura S., Kimura M., Hanada K., Sugita H. 1987. Prolongation of life span of dystrophic hamster by cysteine proteinase inhibitor, loxistation (EST). J. Pharmacobiodyn. 10, 678–681.

    PubMed  CAS  Google Scholar 

  12. Ray S.K., Matzelle D.C., Wilford G.G., Hogan E.L., Banik N.L. 2000. E-64-d prevents both calpiain upregulation and apoptosis in the lesion and penumbra following spinal cord injury in rats. Brain Research. 867, 80–89.

    Article  PubMed  CAS  Google Scholar 

  13. Ray S.K., Matzelle D.D., Sribnick E.A., Guyton M.K., Wingrave J.M., Banik N.L. 2003. Calpain inhibitor prevented apoptosis and maintained transcription of proteolipid protein and myelin basic protein genes in rat spinal cord injury. J. Chem. Neuroanat. 26, 119–124.

    Article  PubMed  CAS  Google Scholar 

  14. Muller A., Pietri S., Villain M., Frejaville C., Bonne C., Culcas M.. Free radicals in rabbit retina under ocular hyperpressure and functional consequences. Exp. Eye Res. 64, 637–643.

  15. Lafuente M.P., Villegas-Perez M.P., Selles-Navarro I., Mayor-Torroglosa S., Miralles de Imperial J., Vidal-Sanz M. 2002. Retinal ganglion cell death after acute retinal ischemia is an ongoing process whose severity and duration depends on the duration of the insult. Neuroscience. 109, 157–168.

    Article  PubMed  CAS  Google Scholar 

  16. Joo C.K., Choi J.S., Ko H.W., Park K.Y., Sohn S., Chun M.H., Oh Y.J., Gwag B.J. 1999. Necrosis and apoptosis after retinal ischemia: Involvement of NMDA-mediated excitotoxicity and p53. Invest. Ophthalmol. Vis. Sci. 40, 713–720.

    PubMed  CAS  Google Scholar 

  17. Kuriyama H., Nakagawa M., Tsuda M. 2001. Intracellular Ca2+ changes induced by in vitro ischemia in rat retinal slices. Exp. Eye Res. 73, 365–374.

    Article  PubMed  CAS  Google Scholar 

  18. Kuriyama H., Waki M., Nakagawa M., Tsuda M. 2001. Involvement of oxygen free radicals in experimental retinal ischemia and the selective vulnerability of retinal damage. Ophthalmic. Res. 33, 196–202.

    Article  PubMed  CAS  Google Scholar 

  19. Adachi K., Kashii S., Masai H., Ueda M., Morizane C., Kaneda K., Kume T., Akaike A., Honda Y. 1998. Mechanism of the pathogenesis of glutamate neurotoxicity in retinal ischemia. Graefes. Arch. Clin. Exp. Ophthalmol. 236, 766–774.

    Article  PubMed  CAS  Google Scholar 

  20. Bull N.D., Barnett N.L. 2004. Retinal glutamate transporter activity persists under simulated ischemic conditions. J. Neurosci. Res. 78, 590–599.

    Article  PubMed  CAS  Google Scholar 

  21. Tamada Y., Fukiage C., Daibo S., Yoshida Y., Azuma M., Shearer T.R. 2002. Involvement of calpain in hypoxia-induced damage in rat retina in vitro. Comp. Biochem. Physiol. Biochem. Mol. Biol. 131, 221–225.

    Article  Google Scholar 

  22. Singh M., Savitz S.I., Hoque R., Gupta G., Roth S., Rosenbaum P.S., Rosenbaum D.M. 2001. Cell-specific caspase expression by different neuronal phenotypes in transient retinal ischemia. J. Neurochem. 77, 466–475.

    Article  PubMed  CAS  Google Scholar 

  23. Molinari M., Carafoli E. 1997. Calpain: A cytosolic proteinase active at the membrane. J. Membr. Biol. 156, 1–8.

    Article  PubMed  CAS  Google Scholar 

  24. Carafoli E., Molinari M. 1998. Calpain: A proteinase in search of a function? Boichem. Biophys. Res. Comm. 247, 193–203.

    Article  CAS  Google Scholar 

  25. Sorimachi H., Ishiura S., Suzuki K. 1997. Structure and physiological function of calpain. Biochem. J. 328, 721–732.

    PubMed  CAS  Google Scholar 

  26. Saido T.C., Sorimachi H., Suzuki K. 1994. Calpain: New perspectives in molecular diversity and physiological-pathological involvement. FASB J. 8, 814–822.

    CAS  Google Scholar 

  27. Markgraf C.G., Velayo N.L., Johnson M.P., McCarty D.R., Medhi S., Koehl J.R., Chmielewski P.A., Linnik M.D. 1998. Six-hour window of opportunity for calpain inhibition in focal cerebral ischemia in rats. Stroke. 29, 152–158.

    PubMed  CAS  Google Scholar 

  28. Komatsu K., Inazuki K., Hosoya J., Satoh S. 1986. Beneficial effect of new thiol protease inhibitors, epoxide derivatives on dystrophic mice. Exp. Neurol. 9, 23–29.

    Article  Google Scholar 

  29. Ray S.K., Matzelle D.D., Wilford G.G., Hogan E.L., Banik N.L. 2000. Increased calpain expression is associated with apoptosis in rat spinal cord injury: Calpain inhibitor provides neuroprotection. Neuro. Chem. Res. 25, 1191–1198.

    CAS  Google Scholar 

  30. Ray S.K., Hogan E.L., Banik N.L. 2003. Calpain in the pathophysiology of spinal cord injury: Neruoprotection with calpain inhibitors. Brain Res. Rev. 42, 169–185.

    Article  PubMed  CAS  Google Scholar 

  31. Toriu N., Akaike A., Yasuyoshi H., Zhang S., Kashii S., Honda Y., Shimazawa M., Hara H. 2000. Lomerizine, a Ca2+ channel blocker, reduces glutamate-induced neurotoxicity and ischemia/reperfusion damage in rat retina. Exp. Eye Res. 70, 475–484.

    Article  PubMed  CAS  Google Scholar 

  32. Bartus R.T., Hayward N.J., Elliott P.J., Sawyer S.D., Baker K.L., Dean R.L., Akiyama A., Straub J.A., Harbeson S.L, Li Z., Powers J. 1994. Calpain inhibitor AK295 protects neurons from focal brain ischemia. Effects of post occlusion intra-arterial administration. Stroke. 25, 2265–2270.

    PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Ophthalmology, the Second Affiliated Hospital of the Medical College, Zhejiang University, Hangzhou, China

    Zhiqing Chen, Ke Yao, Wen Xu & Renyi Wu

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  1. Zhiqing Chen
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  2. Ke Yao
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  3. Wen Xu
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  4. Renyi Wu
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Correspondence to Zhiqing Chen.

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Published in Russian in Molekulyarnaya Biologiya, 2008, Vol. 42, No. 2, pp. 258–264.

The text was submitted by the authors in English.

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Chen, Z., Yao, K., Xu, W. et al. Inhibition of calpain expression by E-64d in the rat retina subjected to ischemia/reperfusion injury. Mol Biol 42, 227–233 (2008). https://doi.org/10.1134/S0026893308020088

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  • DOI: https://doi.org/10.1134/S0026893308020088

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