DOI: 10.1007/s00441-010-1063-8

Hypothermia translocates nitric oxide synthase from cytosol
to membrane in snail neurons

Supplementary Information

Tamás Rőszer1CA*, Éva Kiss-Tóth1, Dávid Rózsa1, Tamás Józsa2,

A. József Szentmiklósi3, Gáspár Bánfalvi1

1 Department of Microbial Biotechnology and Cell Biology (formerly Animal Anatomy and Physiology), Faculty of Science and Technology, University of Debrecen, Hungary

2 Department of Pediatrics, Medical and Health Science Center, University of Debrecen, Hungary

3 Department of Pharmacology and Pharmacotherapy, Medical and Health Science Center, University of Debrecen, Hungary

Short title: NOS in hibernating neurons


Immunocytochemsitry of HelixNOS

Specific antibody reacting with Helix pomatia NOS is still not available, therefore we purchased from Transduction Laboratories the same antibody which was used in the first biochemical characterization of Helix pomatia NOS by Huang et al (1997). Distribution pattern for NADPHd showed a strong overlap with immunostained neurons (S1), although many NADPHd positive neurons did not show NOS immunoreactivity. We also tested a rabbit antiserum raised against mammalian inducible NOS (Santa Cruz), which did not label NADPHd stained neurons, in accordance with a previous description in snail ganglia (Peruzzi et al. 2004). We used 5 specimens (whole CNS) for immunostainings. Further information and discussion on the use of mammalian antibodies in snail neurons is available in works of Cooke et al (1994), Vignola et al (1995), Huang et al (1997) and Pisu et al (1999).

Determination of tested ligand concentrations

Ligand concentrations used in the core study were choosen based on existing literature data (Morishita et al. 1998) or determined on cultured neurons kept at 24 °C. NOS activity was assayed as it has been described in the Materials and Methods. The known NOS inhibitor L-w-nitro-L-arginine (NOARG) dose dependently reduced NOS activity (S2). The PKC inhibitor Ro-31-8220 (50, 500mM, S3), FrK (1-100 mM, S4), DXM (100, 500 nM, S5) and QRC (1-50 mM, S6) did not affect NOS activity. However, higher doses of QRC (100, 500 mM) reduced the amount of nitrite in the culture supernatant, which can be explained by its NO-scavenging capacity or proapoptotic effect (Rzymowska et al. 1999; López-López et al. 2004). In the core studies we used 50 mM QRC, therefore possible proapoptotic effect of QRC was tested with MTT assay (Berridge et al. 2005), showing that this amount of QRC did not affect cell viability (S7). Measurements were carried out three times.

Recovery of NOS activity after hypothermia

To recover NOS activity after hypothermia, cell cultures were warmed up to 24 ºC, incubated for 1-3 hours and their NOS activity was assayed. To reach the original NOS activity (considered as 100%) neuron cultures required ~3 hours, while in neuron-glia co-cultures NOS activity was restored within 1 hour. Administration of tested ligands did not affect the recovery from hypothermia (S8). Tests were repeated three times.

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