ABCMdb

PMID: 16306272

Hambrock A, de Oliveira Franz CB, Hiller S, Osswald H
Glibenclamide-induced apoptosis is specifically enhanced by expression of the sulfonylurea receptor isoform SUR1 but not by expression of SUR2B or the mutant SUR1(M1289T).
J Pharmacol Exp Ther. 2006 Mar;316(3):1031-7. Epub 2005 Nov 23., [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCC8 p.Met1289Thr Glibenclamide-Induced Apoptosis Is Specifically Enhanced by Expression of the Sulfonylurea Receptor Isoform SUR1 but Not by Expression of SUR2B or the Mutant SUR1(M1289T) Annette Hambrock, Claudia Bernardo de Oliveira Franz, Sabrina Hiller, and Hartmut Osswald Department of Pharmacology and Toxicology, Medical Faculty, University of Tu¨ bingen, Tu¨ bingen, Germany Received October 20, 2005; accepted November 21, 2005 ABSTRACT Sulfonylurea receptor 1 (SUR1) is the regulatory subunit of the pancreatic ATP-sensitive Kϩ channel (KATP channel), which is essential for triggering insulin secretion via membrane depolarization. Login to comment 3 ABCC8 p.Met1289Thr To investigate the role of SUR in apoptosis induction, we tested the effect of glibenclamide on recombinant human embryonic kidney 293 cells expressing either SUR1, the smooth muscular isoform SUR2B, or the mutant SUR1(M1289T) at which a single amino acid in transmembrane helix 17 (TM17) was exchanged by the corresponding amino acid of SUR2. Login to comment 4 ABCC8 p.Met1289Thr By analyzing cell detachment, nuclear condensation, DNA fragmentation, and caspase-3-like activity, we observed a SUR1-specific enhancement of glibenclamide-induced apoptosis that was not seen in SUR2B, SUR1(M1289T), or control cells. Login to comment 6 ABCC8 p.Met1289Thr In conclusion, expression of SUR1, but not of SUR2B or SUR1(M1289T), renders cells more susceptible to glibenclamide-induced apoptosis. Login to comment 37 ABCC8 p.Met1289Thr Cells expressing the mutant SUR1(M1289T) at which a single amino acid in transmembrane helix 17 (TM17) was exchanged by the corresponding amino acid of SUR2 were also investigated. Login to comment 43 ABCC8 p.Met1289Thr Cells were transfected with pcDNA3.1 expression vector (Invitrogen, Karlsruhe, Germany) containing the coding sequences of SUR1 (GenBank X97279), SUR1(M1289T), or SUR2B (GenBank D86038). Login to comment 44 ABCC8 p.Met1289Thr The mutant SUR1(M1289T) had been constructed from SUR1 using the QuikChange Mutagenesis System (Stratagene, Amsterdam, The Netherlands). Login to comment 89 ABCC8 p.Met1289Thr Results Cell Morphology after Glibenclamide Treatment of Different Cell Lines. HEK293 cells stably expressing either SUR1, SUR2B, or the mutant SUR1(M1289T) were incubated with glibenclamide concentrations (5-100 ␮M) over a 7-day culture period and compared with cells that were only treated with the same amount (0.1%) of solvent. Login to comment 90 ABCC8 p.Met1289Thr Results Cell Morphology after Glibenclamide Treatment of Different Cell Lines. HEK293 cells stably expressing either SUR1, SUR2B, or the mutant SUR1(M1289T) were incubated with glibenclamide concentrations (5-100 òe;M) over a 7-day culture period and compared with cells that were only treated with the same amount (0.1%) of solvent. Login to comment 95 ABCC8 p.Met1289Thr Cells expressing the mutant SUR1(M1289T) resembled pcDNA cells after glibenclamide treatment. Login to comment 96 ABCC8 p.Met1289Thr Cells expressing the mutant SUR1(M1289T) resembled pcDNA cells after glibenclamide treatment. Login to comment 104 ABCC8 p.Met1289Thr Effect of glibenclamide treatment on the morphology of SUR1, SUR1(M1289T), SUR2B, or pcDNA cells. Login to comment 105 ABCC8 p.Met1289Thr Effect of glibenclamide treatment on the morphology of SUR1, SUR1(M1289T), SUR2B, or pcDNA cells. Login to comment 111 ABCC8 p.Met1289Thr B, pcDNA, SUR1, and SUR1(M1289T) cells were compared in parallel experiments with n ϭ 4. Login to comment 112 ABCC8 p.Met1289Thr B, pcDNA, SUR1, and SUR1(M1289T) cells were compared in parallel experiments with n afd; 4. Login to comment 114 ABCC8 p.Met1289Thr For cells expressing SUR1(M1289T), the number of detached cells was reduced compared with SUR1 cells but slightly higher than the amount of detached pcDNA cells (Fig. 2B). Login to comment 115 ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr However, these differences between SUR1(M1289T) cells and SUR1 or pcDNA cells were not significant. Login to comment 116 ABCC8 p.Met1289Thr However, these differences between SUR1(M1289T) cells and SUR1 or pcDNA cells were not significant. Login to comment 120 ABCC8 p.Met1289Thr Again, the degree of detached SUR1 cells after glibenclamide treatment (462 Ϯ 82% of solvent control) was considerably higher than that of the other cells (pcDNA, 320 Ϯ 66%; SUR1(M1289T), 262 Ϯ 71%; and SUR2B, 254 Ϯ 30%). Login to comment 121 ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr The amount of glibenclamide-induced detachment of SUR1 cells was nearly the same as that of etoposide-induced cell detachment (pcDNA, 434 Ϯ 75%; SUR1, 475 Ϯ 127%; SUR1(M1289T), 488 ee; 78%; and SUR2B, 389 Ϯ 85%). Login to comment 122 ABCC8 p.Met1289Thr The amount of glibenclamide-induced detachment of SUR1 cells was nearly the same as that of etoposide-induced cell detachment (pcDNA, 434 afe; 75%; SUR1, 475 afe; 127%; SUR1(M1289T), 488 afe; 78%; and SUR2B, 389 afe; 85%). Login to comment 132 ABCC8 p.Met1289Thr In SUR1 cells, apoptotic nuclei occurred to a much larger extent and were significantly more condensed and fragmented than in SUR2B (data not shown), SUR1(M1289T) , or pcDNA cells (Fig. 4). Login to comment 133 ABCC8 p.Met1289Thr In SUR1 cells, apoptotic nuclei occurred to a much larger extent and were significantly more condensed and fragmented than in SUR2B (data not shown), SUR1(M1289T) , or pcDNA cells (Fig. 4). Login to comment 150 ABCC8 p.Met1289Thr After 48 h, caspase-3-like activity in SUR1 and pcDNA cells was significantly higher in the glibenclamide-treated groups than in the solvent-treated groups, but this difference was not observed in SUR1(M1289T) cells (Fig. 5). Login to comment 151 ABCC8 p.Met1289Thr After 48 h, caspase-3-like activity in SUR1 and pcDNA cells was significantly higher in the glibenclamide-treated groups than in the solvent-treated groups, but this difference was not observed in SUR1(M1289T) cells (Fig. 5). Login to comment 152 ABCC8 p.Met1289Thr SUR1(M1289T) cells showed even slightly lower activity than pcDNA controls after glibenclamide treatment, but this difference was not significant. Login to comment 153 ABCC8 p.Met1289Thr SUR1(M1289T) cells showed even slightly lower activity than pcDNA controls after glibenclamide treatment, but this difference was not significant. Login to comment 154 ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr Caspase-3-like activity was a little higher in SUR1 cells than in pcDNA and SUR1(M1289T) cells in most experiments, and pcDNA cells for their part showed slightly but not significantly higher activity than SUR1(M1289T) cells. Login to comment 155 ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr Yet, if total activity after glibenclamide treatment was referred to the respective solvent-dependent activity, the increase in caspase-3-like activity due to glibenclamide was still higher in SUR1 cells (323 Ϯ 40%) compared with pcDNA cells (269 Ϯ 37%) or SUR1(M1289T) cells (200 Ϯ 41%). Login to comment 156 ABCC8 p.Met1289Thr Yet, if total activity after glibenclamide treatment was referred to the respective solvent-dependent activity, the increase in caspase-3-like activity due to glibenclamide was still higher in SUR1 cells (323 afe; 40%) compared with pcDNA cells (269 afe; 37%) or SUR1(M1289T) cells (200 afe; 41%). Login to comment 158 ABCC8 p.Met1289Thr A basal level of apoptosis was also present in sham-transfected control cells as well as in cells expressing the SUR2B isoform or the mutant SUR1(M1289T). Login to comment 159 ABCC8 p.Met1289Thr A basal level of apoptosis was also present in sham-transfected control cells as well as in cells expressing the SUR2B isoform or the mutant SUR1(M1289T). Login to comment 172 ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr Hoechst dye 33258 staining after 48 h of treatment with 100 ␮M glibenclamide (GBC) or ethanol/DMSO treatment of pcDNA, SUR1, and SUR1(M1289T) cells. Login to comment 173 ABCC8 p.Met1289Thr Hoechst dye 33258 staining after 48 h of treatment with 100 òe;M glibenclamide (GBC) or ethanol/DMSO treatment of pcDNA, SUR1, and SUR1(M1289T) cells. Login to comment 174 ABCC8 p.Met1289Thr Results for SUR2B cells (data not shown) were the same as for pcDNA or SUR1(M1289T) cells. Login to comment 175 ABCC8 p.Met1289Thr Results for SUR2B cells (data not shown) were the same as for pcDNA or SUR1(M1289T) cells. Login to comment 176 ABCC8 p.Met1289Thr Determination of caspase-3-like activity in pcDNA, SUR1, and SUR1(M1289T) cells after treatment with 100 ␮M glibenclamide (GBC) or solvent for 48 h. Login to comment 177 ABCC8 p.Met1289Thr Determination of caspase-3-like activity in pcDNA, SUR1, and SUR1(M1289T) cells after treatment with 100 òe;M glibenclamide (GBC) or solvent for 48 h. Login to comment 197 ABCC8 p.Met1289Thr It still has to be noted that caspase-3-like activity in solvent-treated controls is slightly higher in SUR1 cells compared with pcDNA cells but lower in SUR1(M1289T) cells. Login to comment 198 ABCC8 p.Met1289Thr It still has to be noted that caspase-3-like activity in solvent-treated controls is slightly higher in SUR1 cells compared with pcDNA cells but lower in SUR1(M1289T) cells. Login to comment 199 ABCC8 p.Met1289Thr Perhaps the expression of SUR1 per se [but not of SUR1(M1289T)] results in slightly higher caspase-3-like activity, which could be due to interaction of SUR1 with endogenous compounds or supplements in the culture medium, for example. Login to comment 200 ABCC8 p.Met1289Thr Perhaps the expression of SUR1 per se [but not of SUR1(M1289T)] results in slightly higher caspase-3-like activity, which could be due to interaction of SUR1 with endogenous compounds or supplements in the culture medium, for example. Login to comment 202 ABCC8 p.Met1289Thr SUR1(M1289T) is able to form functional plasmalemmal channels with Kir6.2 according to electrophysiological experiments (Moreau et al., 2000) and thus possesses essential properties of an intact SUR protein. Login to comment 203 ABCC8 p.Met1289Thr SUR1(M1289T) is able to form functional plasmalemmal channels with Kir6.2 according to electrophysiological experiments (Moreau et al., 2000) and thus possesses essential properties of an intact SUR protein. Login to comment 204 ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr However, the maximal amount of glibenclamide-binding sites is reduced at SUR1(M1289T) by approximately 30 to 50% at physiological MgATP concentrations (S. Hiller, unpublished results), which might explain the lower sensitivity of SUR1(M1289T) cells to glibenclamide-induced apoptosis. Login to comment 205 ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr However, the maximal amount of glibenclamide-binding sites is reduced at SUR1(M1289T) by approximately 30 to 50% at physiological MgATP concentrations (S. Hiller, unpublished results), which might explain the lower sensitivity of SUR1(M1289T) cells to glibenclamide-induced apoptosis. Login to comment 206 ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr In contrast to this finding, glibenclamide-induced apoptosis in SUR1(M1289T) cells is at the same level as that of control cells (or is even lower for SUR1(M1289T) cells in case of caspase-3-like activity). Login to comment 207 ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr In contrast to this finding, glibenclamide-induced apoptosis in SUR1(M1289T) cells is at the same level as that of control cells (or is even lower for SUR1(M1289T) cells in case of caspase-3-like activity). Login to comment 208 ABCC8 p.Met1289Thr In this respect, SUR1(M1289T) is probably similar to the SUR2B isoform, which is also less effective in triggering glibenclamide-induced apoptosis. Login to comment 209 ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr It has been shown previously that binding and effect of several KATP channel openers are clearly enhanced in SUR1(M1289T) cells (Moreau et al., 2000; Hambrock et al., 2004). Login to comment 210 ABCC8 p.Met1289Thr It has been shown previously that binding and effect of several KATP channel openers are clearly enhanced in SUR1(M1289T) cells (Moreau et al., 2000; Hambrock et al., 2004). Login to comment 211 ABCC8 p.Met1289Thr Therefore, it will be an interesting task to investigate whether openers exert different protective effects in cells expressing SUR1, SUR1(M1289T), or SUR2. Login to comment 212 ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr In conclusion, glibenclamide induces apoptotic processes in HEK293 cells that are markedly enhanced by expression of SUR1 but not of SUR2B or the mutant SUR1(M1289T). Login to comment 213 ABCC8 p.Met1289Thr In conclusion, glibenclamide induces apoptotic processes in HEK293 cells that are markedly enhanced by expression of SUR1 but not of SUR2B or the mutant SUR1(M1289T). Login to comment