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Catalase activity during postnatal cardiac development correlates with recovery following reperfusion injury
ABSTRACTS / Journal of Molecular and Cellular Cardiology 44 (2008) 711–825
possibility that the commonly used anti-dual phosphorylation antibody (Cell Signalling, product #9211) recognises a monophospho-form. Western blot analysis using the dual phospho-specific antibody confirmed as expected, augmentation of apparent p38-MAPK dual phosphorylation in plasmid-mediated overexpression of WTp38-MAPK and TAB1 in HEK293 cells. In comparison, overexpression of TAB1 with T180A p38 reduced the dual phospho signal, as expected, whereas co overexpression of Y182Fp38, yielded a strong dual phospho signal. We demonstrated with the use of an antibody recognising only phospho Tyr182, that TAB1-mediated phosphorylation of p38-MAPK occurs on Thr180 alone. The MKK3-dependent p38 dual phoshorylation signal was also abolished by Y182F mutation but T180A was phosphorylated indicating dual phosphorylation of T180 and Y182 within the activation lip. In conclusion, we have shown that TAB1 mediated phosphorylation of p38-MAPK is by phosphorylation of Thr180, rather than dual phosphorylation.
771
This work was supported by a research grant, 2001-08-09-067, from the Ankara University Research Foundation. Keywords: Nitric oxide; Phosphodiesterases; Heart doi:10.1016/j.yjmcc.2008.02.143
Abstract No. 143 Catalase activity during postnatal cardiac development correlates with recovery following reperfusion injury Sarah Martin, Nilima Shukla, Hua Lin, Jamie Jeremy, M.-Saadeh Suleiman ⁎. Bristol Heart Institute, Faculty of Medicine and Dentistry, University of Bristol, Bristol, UK ⁎ Corresponding author. Tel.: +44 117 9283519; fax: +44 117 9252736. E-mail address: [email protected]
Keywords: p38-MAPK; Ischaemia doi:10.1016/j.yjmcc.2008.02.142
Abstract No. 142 Nitric oxide effects depend on different phosphodiesterases activity in the rat heart Emine Demirel-Yilmaz ⁎, Basar Cenik. University of Ankara, Faculty of Medicine, Department of Pharmacology and Clinical Pharmacology, Sihhiye, Ankara 06100, Turkey ⁎ Corresponding author. Tel.: +90 312 310 6268; fax: +90 312 310 6268. E-mail address: [email protected] Cardiac functions are modulated by nitric oxide (NO) however the signaling pathways responsible for the cardiac effects of NO are poorly identified. NO regulates cardiac activity through activation of cGMP-dependent protein kinase (PKG) and by alteration of phosphodiesterases (PDEs) activity. This study examined the effects of NO donor diethylamine NONOate (DEA) on the activity of the isolated right atrium and left papillary muscle from the rat heart in the presence of different PDE inhibitors. DEA (0.1–100 μM) decreased contractions and the sinus rate of right atrium but had no effect on the papillary muscle. In the presence of vinpocetine (PDE1 inhibitor) (1–10 μM) or milrinone (PDE3 inhibitor) (1–10 μM), DEA decreased papillary muscle contraction. EHNA (PDE2 inhibitor) (1–10 μM) or sildenafil (PDE5 inhibitor) (1 μM) reversed only DEA-induced decrease in resting force of the right atrium. The effect of DEA on right atrium contractions and the sinus rate was blocked by rolipram (PDE4 inhibitor) (1–10 μM). The present study has indicated that the effect of NO depends on a variety of PDE activities in different regions of the rat heart.
The growing myocardium undergoes different stages of development during which the heart responds differently to cardiac insults. The vulnerability of the rat heart to ischaemia and reperfusion is at a minimum at 14 days and maximal at 4 days and in adult heart (60 days old) (Modi and Suleiman, Amino Acids 2004, 26:65–70). An important trigger of reperfusion injury is the generation of reactive oxygen species (ROS). Therefore the ability of heart cells to generate and scavenge ROS will determine the extent of their vulnerability to the damaging effects of cardiac insults. The aim of this study was to investigate postnatal developmental changes in the activity of the myocardial antioxidant enzymes superoxide dismutase (SOD) and catalase. SOD catalyses the reaction 2O2U− + 2H+ → H2O2 + O2 whereas catalase catalyses H2O2 → 2H2O + O2. Rat hearts were extracted from 4, 7, 14, 21, and 28 days postnatally and from adult rats aged 60 days. Ventricular tissue was homogenised and enzymes extracted and assayed as described by the manufacturer's protocol (CALBIOCHEM®). Catalase activity (U/mg protein) was found to follow a bell shaped curve reaching a maximum at the age of 14 days and a minimum at 60 days old. The age-dependent changes in catalase activity closely correlate with functional recovery after ischaemia and reperfusion. This is in contrast to superoxide dismutase activity where the changes were small and did not correlate with vulnerability to ischaemia and reperfusion. These data suggest that an increase in cardiac catalase but not SOD activity may be responsible for the observed increase in resistance to ischaemia and reperfusion during postnatal development. This work is supported by the British Heart Foundation. Keywords: Heart; Development; Oxidative stress doi:10.1016/j.yjmcc.2008.02.144
possibility that the commonly used anti-dual phosphorylation antibody (Cell Signalling, product #9211) recognises a monophospho-form. Western blot analysis using the dual phospho-specific antibody confirmed as expected, augmentation of apparent p38-MAPK dual phosphorylation in plasmid-mediated overexpression of WTp38-MAPK and TAB1 in HEK293 cells. In comparison, overexpression of TAB1 with T180A p38 reduced the dual phospho signal, as expected, whereas co overexpression of Y182Fp38, yielded a strong dual phospho signal. We demonstrated with the use of an antibody recognising only phospho Tyr182, that TAB1-mediated phosphorylation of p38-MAPK occurs on Thr180 alone. The MKK3-dependent p38 dual phoshorylation signal was also abolished by Y182F mutation but T180A was phosphorylated indicating dual phosphorylation of T180 and Y182 within the activation lip. In conclusion, we have shown that TAB1 mediated phosphorylation of p38-MAPK is by phosphorylation of Thr180, rather than dual phosphorylation.
771
This work was supported by a research grant, 2001-08-09-067, from the Ankara University Research Foundation. Keywords: Nitric oxide; Phosphodiesterases; Heart doi:10.1016/j.yjmcc.2008.02.143
Abstract No. 143 Catalase activity during postnatal cardiac development correlates with recovery following reperfusion injury Sarah Martin, Nilima Shukla, Hua Lin, Jamie Jeremy, M.-Saadeh Suleiman ⁎. Bristol Heart Institute, Faculty of Medicine and Dentistry, University of Bristol, Bristol, UK ⁎ Corresponding author. Tel.: +44 117 9283519; fax: +44 117 9252736. E-mail address: [email protected]
Keywords: p38-MAPK; Ischaemia doi:10.1016/j.yjmcc.2008.02.142
Abstract No. 142 Nitric oxide effects depend on different phosphodiesterases activity in the rat heart Emine Demirel-Yilmaz ⁎, Basar Cenik. University of Ankara, Faculty of Medicine, Department of Pharmacology and Clinical Pharmacology, Sihhiye, Ankara 06100, Turkey ⁎ Corresponding author. Tel.: +90 312 310 6268; fax: +90 312 310 6268. E-mail address: [email protected] Cardiac functions are modulated by nitric oxide (NO) however the signaling pathways responsible for the cardiac effects of NO are poorly identified. NO regulates cardiac activity through activation of cGMP-dependent protein kinase (PKG) and by alteration of phosphodiesterases (PDEs) activity. This study examined the effects of NO donor diethylamine NONOate (DEA) on the activity of the isolated right atrium and left papillary muscle from the rat heart in the presence of different PDE inhibitors. DEA (0.1–100 μM) decreased contractions and the sinus rate of right atrium but had no effect on the papillary muscle. In the presence of vinpocetine (PDE1 inhibitor) (1–10 μM) or milrinone (PDE3 inhibitor) (1–10 μM), DEA decreased papillary muscle contraction. EHNA (PDE2 inhibitor) (1–10 μM) or sildenafil (PDE5 inhibitor) (1 μM) reversed only DEA-induced decrease in resting force of the right atrium. The effect of DEA on right atrium contractions and the sinus rate was blocked by rolipram (PDE4 inhibitor) (1–10 μM). The present study has indicated that the effect of NO depends on a variety of PDE activities in different regions of the rat heart.
The growing myocardium undergoes different stages of development during which the heart responds differently to cardiac insults. The vulnerability of the rat heart to ischaemia and reperfusion is at a minimum at 14 days and maximal at 4 days and in adult heart (60 days old) (Modi and Suleiman, Amino Acids 2004, 26:65–70). An important trigger of reperfusion injury is the generation of reactive oxygen species (ROS). Therefore the ability of heart cells to generate and scavenge ROS will determine the extent of their vulnerability to the damaging effects of cardiac insults. The aim of this study was to investigate postnatal developmental changes in the activity of the myocardial antioxidant enzymes superoxide dismutase (SOD) and catalase. SOD catalyses the reaction 2O2U− + 2H+ → H2O2 + O2 whereas catalase catalyses H2O2 → 2H2O + O2. Rat hearts were extracted from 4, 7, 14, 21, and 28 days postnatally and from adult rats aged 60 days. Ventricular tissue was homogenised and enzymes extracted and assayed as described by the manufacturer's protocol (CALBIOCHEM®). Catalase activity (U/mg protein) was found to follow a bell shaped curve reaching a maximum at the age of 14 days and a minimum at 60 days old. The age-dependent changes in catalase activity closely correlate with functional recovery after ischaemia and reperfusion. This is in contrast to superoxide dismutase activity where the changes were small and did not correlate with vulnerability to ischaemia and reperfusion. These data suggest that an increase in cardiac catalase but not SOD activity may be responsible for the observed increase in resistance to ischaemia and reperfusion during postnatal development. This work is supported by the British Heart Foundation. Keywords: Heart; Development; Oxidative stress doi:10.1016/j.yjmcc.2008.02.144