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Probucol induced antioxidants confers protection against I-R injury
CULTURED ENDOTHELIAL INDUCE STRONG GROWTH RESPONSES IN CARDIAC MYOCYTES BUT NOT IN SMOOTH MUSCLE CELLS Thomas Kubin, Jutta Webel, Dimitri Scholz, Wolfgang Schaper, Dietmar v.d. Ahe 81 Ren6 Zimmermann. Vascular-Genomics, Kerckhoff Klinik, Bad Nauheim, Germany We investigated the effect of endothelial cells (EC) on aortic smooth muscle cells (SMC) and neonatal cardiac m ocytes (NEO) by using serum-free su matant (CM) oP porcine microvascular and aortic EI?. Cell culture
were positive for a-smooth muscle actin, desmin and ne ative for EC-markers. SMC and NE0 were exposef to 5% fetal calf serum (FCS), CM or 10 rig/ml PDGF for 2d and are shown as x-fold increaseover pure medium treated controls. CM (FCS) induced a 6-fold (3-fold) increase in protein synthesis (PROT , a 4-fold increase in surface area (3-5716)and a 3-fol cl (IO-fol increase in DNA-s nthesis (DNA) in NEO. In SM3 CM, FCS and PDG8. induced a 2x increasein PROT. In contrast to a 2-fold increase in DNA in CM-treated SMC, FCS or PDGF induced >150-fold increase in DNA. SMC number doubled and significant morphological than es were visible after FCS or PDGF stimulation but no ca anges in number and morphology of CM-treated SMC were detectable. SMC did not show signs of deterioration such as DNA laddering, ATP loss or necrotic morphology excluding c totoxic effects in cultures. In conclusion, EC might p Yay a major role in heart muscle growth while key regulators in SMC growth might originate from blood.
Role of cGMP-dependent Protein Kinase in the Regulation of L-type Calcium Current in Human Atrial Cells. Rajiv Kmuar, Manjari Mishra & Ronald W. Joyner. Dept. of Pediatrics, Emory University, Atlanta, GA 30322 We have shown that cGMP-dependent protein kinase (PKG) mediates stimulation of L-type calcium current (13 by cGMP in rabbit atria1 cells. We found that human atrial cells have similar PKG dependent regulation of ka. To elucidate the significance of PKG in cardiac function, we have isolated human cardiac PKG type Ia cDNA, determined the sequence, and analyzed expression of PKG in human atrium. The coding region of human cardiac PKG Ia cDNA showed 99.9% homology to previously published human PKG Ia except for the base number 1983 where G was substituted for T and this resulted in an amino acid substitution from Leua9 to Phe”‘. The cloned PKG Ia cDNA was expressed in COS cells and the expressed PKG showed cGMP stimulated PKG activity and immunoreactivity. We determined the specific expression of PKG Ia mRNA by ribonuclease protection assay using a biotinylated riboprobe from N-terminal of Ia specific region and found that PKG Ia is highly expressed in human atrium. Western blot analysis showed the presence of high levels of PKG (I 5557 ng PKGimg protein, n=4) at 79kDa. PKG enzyme activity in homogenates from human atrium was also high (7.4k1.2 mnoles Pi/min/mp protein, n=7) and was completely inhibited by PKG inhibitor KT-5823. Immunofluorescence staining of isolated human atria1 cells with PKG antibody forther confbmed that PKG is highly expressed in human atrial cells. PKG stimulation by intracellular application of 8BrcGMP caused an increase in I,, in human atrial cells and KT5823 blocked the stimulatory effect of PKG. These findings suggest that PKG is highly expressed in human atrium and PKGdependent phosphorylation may be important in regulation of calcium channel activity in human atria1 cells.
A62
PROBUCOL INDUCED ANTIOXIDANTS CONFERS PROTECTION AGAINST I-R INJURY Dlnender Kumar, Vlnce Palace, lgor Danellsen, Bodh JugdutP 8 Pawan K.Slngal. lnst of Cardlovasc Sci, Dept of Physiology, University of Manitoba, Winnipeg, Canada; l Dept of Cardiology, University of Alberta, Edmonton, Alberta, Canada.
I.
We have examined the effects of probucol on myocardial antioxidants as well as global &hernia-repetfusion (I-R) injury. Rats were treated with probucol (10 mglkglbody wt., i. p.) for 4 weeks on alternate days. Isolated hearts from control (C), and probucol (P) groups were subjected to either 80 min of normal perfusion (non I-R) or to 60 min of global ischemia and 20 min of reperfusion (I-R). In non I-R hearts, myocardial glutathione peroxidase (GSHPx) was higher in the P group in the right ventricular wall (RWV) and the left ventricular wall (LVW) as compared to the C group, while catalase (CAT) activity was not different in two groups. After I-R, recovery of the contractile function in ischemic hearts was 74% in the P group as compared to 36% in the C group. Upon I-R, C had a significant decrease in GSHPx in the RVW and in the LVW, and in the P group, GSHPx decreased in the RVW whereas LVW and S showed no significant change. A significant decline in CAT activity in C was observed in the RVW and S after I-R, whereas no change was observed in the P group. In the LVW, an increase in CAT in C and P was seen after I-R. Lipid peroxidation, afler I-R, was significantly reduced in the RVW and S in the P group as compared to the C group, whereas LVW did not show any change. These data indicate that probucol treatment improves endogenous antioxidants in different regions of the heart, and offer better recovery after, I-R. (Supported by Canadian Institutes of Health Research.)
EFFECT OF CAPTOPRIL ON VASOACTIVE INTESTINAL PEPTIDE LEVELS IN THE HYPERTHYROID RAT HEART ATRIA Jltka Kuncova & Jana SlavikovB. Dept. of Physiology, Charles University, PlzeiI, Czech Republic. Vasoactive intestinal peptide (VIP), neurotransmitter detected in the cardiac innervation, is at least partly degraded by angiotensin I converting enzyme (ACE). Hyperthyroidism is known to be associated with the increased activity of ACE in the Plasma and heart and decreased VIP levels in the heart atria of adult rats (1). In our study, we investigated the effect of ACE inhibition by captopril (C) on VIP levels in the atria of intact and hyperthyroid‘ rais. dais were given thyroxine (T4), C and T4+C for 10 days. The table shows body weights (Bw), serum T4 levels, heart rates (HR), and VIP concentrations, determined in the right and lefl atria (RA. LA) by radioimmunoassay and expresses in pg pe g protein. BW ISerum T4 1 HR 1 VIP&! 1 VIP-LA 1
C administration led to a sliaht increase in DeDtide levels in both atria. T4 administration decreased &ii1 VIP levels significantly compared to control animals (P < 0.01). Combined treatment by T4 and C resulted in VIP concentrations not dieting from control values. In conclusion, increased activity of ACE that contributes to the degradation of VIP may play a role in the effect of hyperthyroidism on VIP levels in the rat heart atria. 1. KuncoJ J, Slavikoti J: Physiol. Res., 2000, 49, 427-434. Supported by grant GACR No. 305VMJ263
were positive for a-smooth muscle actin, desmin and ne ative for EC-markers. SMC and NE0 were exposef to 5% fetal calf serum (FCS), CM or 10 rig/ml PDGF for 2d and are shown as x-fold increaseover pure medium treated controls. CM (FCS) induced a 6-fold (3-fold) increase in protein synthesis (PROT , a 4-fold increase in surface area (3-5716)and a 3-fol cl (IO-fol increase in DNA-s nthesis (DNA) in NEO. In SM3 CM, FCS and PDG8. induced a 2x increasein PROT. In contrast to a 2-fold increase in DNA in CM-treated SMC, FCS or PDGF induced >150-fold increase in DNA. SMC number doubled and significant morphological than es were visible after FCS or PDGF stimulation but no ca anges in number and morphology of CM-treated SMC were detectable. SMC did not show signs of deterioration such as DNA laddering, ATP loss or necrotic morphology excluding c totoxic effects in cultures. In conclusion, EC might p Yay a major role in heart muscle growth while key regulators in SMC growth might originate from blood.
Role of cGMP-dependent Protein Kinase in the Regulation of L-type Calcium Current in Human Atrial Cells. Rajiv Kmuar, Manjari Mishra & Ronald W. Joyner. Dept. of Pediatrics, Emory University, Atlanta, GA 30322 We have shown that cGMP-dependent protein kinase (PKG) mediates stimulation of L-type calcium current (13 by cGMP in rabbit atria1 cells. We found that human atrial cells have similar PKG dependent regulation of ka. To elucidate the significance of PKG in cardiac function, we have isolated human cardiac PKG type Ia cDNA, determined the sequence, and analyzed expression of PKG in human atrium. The coding region of human cardiac PKG Ia cDNA showed 99.9% homology to previously published human PKG Ia except for the base number 1983 where G was substituted for T and this resulted in an amino acid substitution from Leua9 to Phe”‘. The cloned PKG Ia cDNA was expressed in COS cells and the expressed PKG showed cGMP stimulated PKG activity and immunoreactivity. We determined the specific expression of PKG Ia mRNA by ribonuclease protection assay using a biotinylated riboprobe from N-terminal of Ia specific region and found that PKG Ia is highly expressed in human atrium. Western blot analysis showed the presence of high levels of PKG (I 5557 ng PKGimg protein, n=4) at 79kDa. PKG enzyme activity in homogenates from human atrium was also high (7.4k1.2 mnoles Pi/min/mp protein, n=7) and was completely inhibited by PKG inhibitor KT-5823. Immunofluorescence staining of isolated human atria1 cells with PKG antibody forther confbmed that PKG is highly expressed in human atrial cells. PKG stimulation by intracellular application of 8BrcGMP caused an increase in I,, in human atrial cells and KT5823 blocked the stimulatory effect of PKG. These findings suggest that PKG is highly expressed in human atrium and PKGdependent phosphorylation may be important in regulation of calcium channel activity in human atria1 cells.
A62
PROBUCOL INDUCED ANTIOXIDANTS CONFERS PROTECTION AGAINST I-R INJURY Dlnender Kumar, Vlnce Palace, lgor Danellsen, Bodh JugdutP 8 Pawan K.Slngal. lnst of Cardlovasc Sci, Dept of Physiology, University of Manitoba, Winnipeg, Canada; l Dept of Cardiology, University of Alberta, Edmonton, Alberta, Canada.
I.
We have examined the effects of probucol on myocardial antioxidants as well as global &hernia-repetfusion (I-R) injury. Rats were treated with probucol (10 mglkglbody wt., i. p.) for 4 weeks on alternate days. Isolated hearts from control (C), and probucol (P) groups were subjected to either 80 min of normal perfusion (non I-R) or to 60 min of global ischemia and 20 min of reperfusion (I-R). In non I-R hearts, myocardial glutathione peroxidase (GSHPx) was higher in the P group in the right ventricular wall (RWV) and the left ventricular wall (LVW) as compared to the C group, while catalase (CAT) activity was not different in two groups. After I-R, recovery of the contractile function in ischemic hearts was 74% in the P group as compared to 36% in the C group. Upon I-R, C had a significant decrease in GSHPx in the RVW and in the LVW, and in the P group, GSHPx decreased in the RVW whereas LVW and S showed no significant change. A significant decline in CAT activity in C was observed in the RVW and S after I-R, whereas no change was observed in the P group. In the LVW, an increase in CAT in C and P was seen after I-R. Lipid peroxidation, afler I-R, was significantly reduced in the RVW and S in the P group as compared to the C group, whereas LVW did not show any change. These data indicate that probucol treatment improves endogenous antioxidants in different regions of the heart, and offer better recovery after, I-R. (Supported by Canadian Institutes of Health Research.)
EFFECT OF CAPTOPRIL ON VASOACTIVE INTESTINAL PEPTIDE LEVELS IN THE HYPERTHYROID RAT HEART ATRIA Jltka Kuncova & Jana SlavikovB. Dept. of Physiology, Charles University, PlzeiI, Czech Republic. Vasoactive intestinal peptide (VIP), neurotransmitter detected in the cardiac innervation, is at least partly degraded by angiotensin I converting enzyme (ACE). Hyperthyroidism is known to be associated with the increased activity of ACE in the Plasma and heart and decreased VIP levels in the heart atria of adult rats (1). In our study, we investigated the effect of ACE inhibition by captopril (C) on VIP levels in the atria of intact and hyperthyroid‘ rais. dais were given thyroxine (T4), C and T4+C for 10 days. The table shows body weights (Bw), serum T4 levels, heart rates (HR), and VIP concentrations, determined in the right and lefl atria (RA. LA) by radioimmunoassay and expresses in pg pe g protein. BW ISerum T4 1 HR 1 VIP&! 1 VIP-LA 1
C administration led to a sliaht increase in DeDtide levels in both atria. T4 administration decreased &ii1 VIP levels significantly compared to control animals (P < 0.01). Combined treatment by T4 and C resulted in VIP concentrations not dieting from control values. In conclusion, increased activity of ACE that contributes to the degradation of VIP may play a role in the effect of hyperthyroidism on VIP levels in the rat heart atria. 1. KuncoJ J, Slavikoti J: Physiol. Res., 2000, 49, 427-434. Supported by grant GACR No. 305VMJ263