ABCMdb

PMID: 17138562

Hambrock A, de Oliveira Franz CB, Hiller S, Grenz A, Ackermann S, Schulze DU, Drews G, Osswald H
Resveratrol binds to the sulfonylurea receptor (SUR) and induces apoptosis in a SUR subtype-specific manner.
J Biol Chem. 2007 Feb 2;282(5):3347-56. Epub 2006 Nov 30., [PubMed]

Sentences

No. Mutations Sentence Comment

8 ABCC8 p.Met1289Thr Enhanced susceptibility to resveratrol was not observed in pancreatic beta-cells from SUR1 knock-out mice or in cells expressing the isoform SUR2A or SUR2B or the mutant SUR1(M1289T). Login to comment 29 ABCC8 p.Met1289Thr This effect was not observed in cells expressing the vascular smooth muscle isoform SUR2B or the mutant SUR1(M1289T), in which a single amino acid in the last C-terminal transmembrane helix 17 was exchanged with the corresponding amino acid of SUR2. Login to comment 30 ABCC8 p.Met1289Thr This effect was not observed in cells expressing the vascular smooth muscle isoform SUR2B or the mutant SUR1(M1289T), in which a single amino acid in the last C-terminal transmembrane helix 17 was exchanged with the corresponding amino acid of SUR2. Login to comment 50 ABCC8 p.Met1289Thr The apoptotic potency of resveratrol was compared in cells expressing SUR1, SUR2A (cardiac and skeletal muscle isoform), SUR2B, or SUR1(M1289T). Login to comment 51 ABCC8 p.Met1289Thr The apoptotic potency of resveratrol was compared in cells expressing SUR1, SUR2A (cardiac and skeletal muscle isoform), SUR2B, or SUR1(M1289T). Login to comment 56 ABCC8 p.Met1289Thr EXPERIMENTAL PROCEDURES Cell Culture-HEK293 cell lines were stably transfected with the pcDNA3.1 expression vector (Invitrogen, Karlsruhe, Germany) containing the coding sequence of SUR1 (GenBankTM accession number X97279), SUR1(S1238Y), SUR1(M1289T), SUR2A (accession number D86037), SUR2B (accession number D86038), or SUR2B(Y1206S), or they were transfected with the empty pcDNA3.1 expression vector (Invitrogen). Login to comment 57 ABCC8 p.Met1289Thr EXPERIMENTAL PROCEDURES Cell Culture-HEK293 cell lines were stably transfected with the pcDNA3.1 expression vector (Invitrogen, Karlsruhe, Germany) containing the coding sequence of SUR1 (GenBankTM accession number X97279), SUR1(S1238Y), SUR1(M1289T), SUR2A (accession number D86037), SUR2B (accession number D86038), or SUR2B(Y1206S), or they were transfected with the empty pcDNA3.1 expression vector (Invitrogen). Login to comment 85 ABCC8 p.Met1289Thr Radioligand Binding Assays-Binding of resveratrol to different SUR forms was determined in heterologous competition experiments using [3 H]glibenclamide as the radioligand (SUR1, SUR1(M1289T), and pcDNA controls, 1 nM; SUR1(S1238Y), SUR2B, and pcDNA controls, 4-5 nM; and SUR2B(Y1206S), 3 nM). Login to comment 86 ABCC8 p.Met1289Thr Radioligand Binding Assays-Binding of resveratrol to different SUR forms was determined in heterologous competition experiments using [3 H]glibenclamide as the radioligand (SUR1, SUR1(M1289T), and pcDNA controls, 1 nM; SUR1(S1238Y), SUR2B, and pcDNA controls, 4-5 nM; and SUR2B(Y1206S), 3 nM). Login to comment 87 ABCC8 p.Met1289Thr Membranes prepared from the respective cell lines (SUR1, SUR1(M1289T), and pcDNA controls, 0.05-0.10 mg/ml membrane protein; SUR2B, SUR2B(S1238Y), and pcDNA controls, 0.5-0.7 mg/ml membrane protein; and SUR2B(Y1206S), 0.1-0.2 mg/ml membrane protein) were incubated in the presence of 1 mM MgATP for 15 min at 37 &#b0;C, and binding properties were analyzed as described by Hambrock et al. (36). Login to comment 88 ABCC8 p.Met1289Thr Membranes prepared from the respective cell lines (SUR1, SUR1(M1289T), and pcDNA controls, 0.05-0.10 mg/ml membrane protein; SUR2B, SUR2B(S1238Y), and pcDNA controls, 0.5-0.7 mg/ml membrane protein; and SUR2B(Y1206S), 0.1-0.2 mg/ml membrane protein) were incubated in the presence of 1 mM MgATP for 15 min at 37 °C, and binding properties were analyzed as described by Hambrock et al. (36). Login to comment 111 ABCC8 p.Met1289Thr Resveratrol-induced cell detachment was enhanced by b03;2-fold in cells expressing SUR1 (but not SUR2B or the mutant SUR1(M1289T)) compared with pcDNA control cells. Login to comment 112 ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr Resveratrol-induced cell detachment was enhanced by ϳ2-fold in cells expressing SUR1 (but not SUR2B or the mutant SUR1(M1289T)) compared with pcDNA control cells. Login to comment 113 ABCC8 p.Met1289Thr Detachment of cells expressing SUR2B or SUR1(M1289T) tended to be even slightly but not significantly lower than that of pcDNA control cells. Login to comment 119 ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr These typical signs of apoptosis were very frequent and pronounced in HEK cells expressing SUR1, but occurred only to a small extent in pcDNA control cells or SUR1(M1289T)- and SUR2B-expressing cells (SUR1 af9; resveratrol, 22.33 afe; 1.86% apoptotic nuclei/total number of nuclei; SUR1 af9; solvent, 1.90 afe; 0.67%; SUR1(M1289T) af9; resveratrol, 6.02 afe; 0.90%; SUR1(M1289T) af9; solvent, 3.18 afe; 0.77%; SUR2B af9; resveratrol, 4.46 afe; 0.79%; SUR2B af9; solvent, 2.53 afe; 0.62%; pcDNA af9; resveratrol, 5.89 afe; 1.02%; pcDNA af9; solvent, 2.10 afe; 0.53%; SUR1 af9; resveratrol versus pcDNA/SUR1(M1289T)/SUR2B af9; resveratrol, p b0d; 0.001; SUR1 af9; resveratrol versus SUR1 af9; solvent, p b0d; 0.001; pcDNA/SUR1(M1289T) af9; resveratrol versus pcDNA/ SUR1(M1289T) af9; solvent, p b0d; 0.05). Login to comment 120 ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr These typical signs of apoptosis were very frequent and pronounced in HEK cells expressing SUR1, but occurred only to a small extent in pcDNA control cells or SUR1(M1289T)- and SUR2B-expressing cells (SUR1 ϩ resveratrol, 22.33 Ϯ 1.86% apoptotic nuclei/total number of nuclei; SUR1 ϩ solvent, 1.90 Ϯ 0.67%; SUR1(M1289T) ϩ resveratrol, 6.02 Ϯ 0.90%; SUR1(M1289T) ϩ solvent, 3.18 Ϯ 0.77%; SUR2B ϩ resveratrol, 4.46 Ϯ 0.79%; SUR2B ϩ solvent, 2.53 Ϯ 0.62%; pcDNA ϩ resveratrol, 5.89 Ϯ 1.02%; pcDNA ϩ solvent, 2.10 Ϯ 0.53%; SUR1 ϩ resveratrol versus pcDNA/SUR1(M1289T)/SUR2B ϩ resveratrol, p Ͻ 0.001; SUR1 ϩ resveratrol versus SUR1 ϩ solvent, p Ͻ 0.001; pcDNA/SUR1(M1289T) ϩ resveratrol versus pcDNA/ SUR1(M1289T) ϩ solvent, p Ͻ 0.05). Login to comment 123 ABCC8 p.Met1289Thr If resveratrol-induced changes in caspase-3 activity were calculated in the form of "induction factors," i.e. the ratio of the activity after substance treatment to the activity after solvent treatment, values of 2.14 afe; 0.30 (pcDNA), 2.49 afe; 0.30 (SUR1), and 1.81 afe; 0.20 (SUR1(M1289T)) were obtained (Fig. 4A). Login to comment 124 ABCC8 p.Met1289Thr If resveratrol-induced changes in caspase-3 activity were calculated in the form of "induction factors," i.e. the ratio of the activity after substance treatment to the activity after solvent treatment, values of 2.14 Ϯ 0.30 (pcDNA), 2.49 Ϯ 0.30 (SUR1), and 1.81 Ϯ 0.20 (SUR1(M1289T)) were obtained (Fig. 4A). Login to comment 125 ABCC8 p.Met1289Thr With induction factors of b03;1, caspase-8 activity (IETD-caspase) was obviously not induced (induction factors, 1.02 afe; 0.05 (pcDNA), 1.23 afe; 0.11 (SUR1), and 0.87 afe; 0.08 (SUR1(M1289T)) (Fig. 4B). Login to comment 126 ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr With induction factors of ϳ1, caspase-8 activity (IETD-caspase) was obviously not induced (induction factors, 1.02 Ϯ 0.05 (pcDNA), 1.23 Ϯ 0.11 (SUR1), and 0.87 Ϯ 0.08 (SUR1(M1289T)) (Fig. 4B). Login to comment 127 ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr The activity of caspase-9 (LEHD-caspase) was clearly enhanced, however, after resveratrol treatment, with an additional significant increase in SUR1-expressing cells (induction factors, 1.73 Ϯ 0.15 (pcDNA), 3.12 Ϯ 0.26 (SUR1), and 1.76 Ϯ 0.18 (SUR1(M1289T)) (Fig. 4C). Login to comment 128 ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr The activity of caspase-12 (ATAD-caspase) was generally elevated after resveratrol treatment, but exhibited no significant differences between the cell lines (induction factors, 1.87 Ϯ 0.27 (pcDNA), 1.72 Ϯ 0.24 (SUR1), and 1.63 Ϯ 0.19 (SUR1(M1289T)) (Fig. 4D). Login to comment 129 ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr The activities of caspase-3 and caspase-9 were also determined for etoposide-treated cells, and no significant differences were detected between the SUR1-expressing cells and the other HEK cell lines (induction factors: caspase-3, 2.35 Ϯ 0.14 (pcDNA), 2.89 Ϯ 0.60 (SUR1), and 2.25 Ϯ 0.18 (SUR1(M1289T); and caspase-9, 1.17 Ϯ 0.09 (pcDNA), 1.36 Ϯ 0.06 (SUR1), and 1.54 Ϯ 0.10 (SUR1(M1289T) (n ϭ 5-6)). Login to comment 131 ABCC8 p.Met1289Thr SUR1-, SUR1(M1289T)-, and SUR2B-expressing HEK cells and pcDNA control cells were treated with 100 òe;M resveratrol (RSV) or 100 òe;M etoposide (ETO) for 24 h. Login to comment 132 ABCC8 p.Met1289Thr SUR1-, SUR1(M1289T)-, and SUR2B-expressing HEK cells and pcDNA control cells were treated with 100 ␮M resveratrol (RSV) or 100 ␮M etoposide (ETO) for 24 h. Login to comment 155 ABCC8 p.Met1289Thr After 24 h of treatment with 100 òe;M resveratrol (RSV) or Me2SO solvent (solv.), pcDNA con- trolcellsandSUR1-orSUR1(M1289T)-expressingHEK293cellswerestainedwith Hoechst 33258. Login to comment 156 ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr After 24 h of treatment with 100 ␮M resveratrol (RSV) or Me2SO solvent (solv.), pcDNA con- trolcellsandSUR1-orSUR1(M1289T)-expressingHEK293cellswerestainedwith Hoechst 33258. Login to comment 158 ABCC8 p.Met1289Thr Nuclei from (former) cells in the supernatant were apoptotic to 100%(notshown).TheresultsforSUR2B-expressingHEKcells(notshown)arethe same as those for pcDNA control and SUR1(M1289T)-expressing cells. Login to comment 159 ABCC8 p.Met1289Thr Nuclei from (former) cells in the supernatant were apoptotic to 100%(notshown).TheresultsforSUR2B-expressingHEKcells(notshown)arethe same as those for pcDNA control and SUR1(M1289T)-expressing cells. Login to comment 162 ABCC8 p.Met1289Thr The resveratrol binding properties of SUR1 were not significantly different from those determined for the mutant SUR1(M1289T) (Ki afd; 99 afe; 21 òe;M; extent of inhibition, 91 afe; 9%). Login to comment 163 ABCC8 p.Met1289Thr The resveratrol binding properties of SUR1 were not significantly different from those determined for the mutant SUR1(M1289T) (Ki ϭ 99 Ϯ 21 ␮M; extent of inhibition, 91 Ϯ 9%). Login to comment 175 ABCC8 p.Met1289Thr Changes in the activities of different caspase enzymes in pcDNA control cells and SUR1- or SUR1(M1289T)-expressing HEK cells after resveratrol treatment. Login to comment 176 ABCC8 p.Met1289Thr Changes in the activities of different caspase enzymes in pcDNA control cells and SUR1- or SUR1(M1289T)-expressing HEK cells after resveratrol treatment. Login to comment 222 ABCC8 p.Met1289Thr In addition to demonstrating the SUR-specific apoptotic effect ofresveratrol,wehaveconfirmedinourradioligandbindingassays that resveratrol specifically binds to SUR (SUR1, SUR2B, and SUR1(M1289T)) and is able to displace specific binding of glibenclamide.Byshowingthat,likeglibenclamide,resveratrolcanactas a blocker of the SUR1/KIR6.2 channel, we have provided further evidence for analogies between both substances. Login to comment 223 ABCC8 p.Met1289Thr In addition to demonstrating the SUR-specific apoptotic effect ofresveratrol,wehaveconfirmedinourradioligandbindingassays that resveratrol specifically binds to SUR (SUR1, SUR2B, and SUR1(M1289T)) and is able to displace specific binding of glibenclamide.Byshowingthat,likeglibenclamide,resveratrolcanactas a blocker of the SUR1/KIR6.2 channel, we have provided further evidence for analogies between both substances. Login to comment 226 ABCC8 p.Met1289Thr Binding of resveratrol to SUR1 and SUR1(M1289T). Login to comment 227 ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr Binding of resveratrol to SUR1 and SUR1(M1289T). Login to comment 228 ABCC8 p.Met1289Thr Binding of resveratrol to membranes from HEK cells expressing SUR1 or SUR1(M1289T) was analyzed in heterologous competition experiments using [3 H]glibenclamide as the radioligand. Login to comment 242 ABCC8 p.Met1289Thr This is underlined by the fact that no significant differences in the resveratrol or glibenclamide (36) binding properties of SUR1 and the mutant SUR1(M1289T) could be detected, although the expression of these different SURs is linked to a significantly different susceptibility to resveratrol- or glibenclamide-induced apoptosis. Login to comment 243 ABCC8 p.Met1289Thr This is underlined by the fact that no significant differences in the resveratrol or glibenclamide (36) binding properties of SUR1 and the mutant SUR1(M1289T) could be detected, although the expression of these different SURs is linked to a significantly different susceptibility to resveratrol- or glibenclamide-induced apoptosis. Login to comment