Pharmacological and molecular characterization of ATP-sensitive K+ channels in the TE671 human medulloblastoma cell line

Miller, Thomas R.; Taber, Rachel Davis; Molinari, Eduardo J.; Whiteaker, Kristi L.; Monteggia, Lisa M.; Scott, Victoria E.; Brioni, Jorge D.; Sullivan, James P.; Gopalakrishnan, Murali

doi:10.1016/s0014-2999(99)00128-4

Cited by 29 publications

(22 citation statements)

References 25 publications

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“…Extraction and isolation of mRNA was achieved with oligo(dT) 25 -linked magnetic beads (Dynal), and first-strand cDNA was synthesized from mRNA using Ready-To-Go You-Prime FirstStrand Beads (Amersham Biosciences) according to established protocols. 14 Reverse transcription was initiated by addition of 0.5 g Oligo(dT) 15 Primer (Promega) at 37°C for 75 minutes. Protocols were performed using 1 L of the cDNA in 30 L reaction containing 0.2 mol/L of each primer specific for K ATP , 200 mol/L of each dNTP, and 0.7 U of Taq polymerase (PCR Supermix, Life Technologies).…”

Section: Reverse Transcription-polymerase Chain Reaction Analysismentioning

confidence: 99%

“…Primers were used for detection of the inwardly rectifying Kir6.1 (expected product, 737 bp) and Kir6.2 (636 bp) and the sulfonylurea receptors SUR1 (134 bp) and SUR2A/2B (451/312 bp), as described before. 15,16 The primers for SUR2A/2B were designed to identify both SUR2A and SUR2B. Cycling conditions were as follows: for Kir6.1 and 6.2, 96°C, 3 minutes, 40 cycles (96°C, 15 seconds; 55°C, 30 seconds; 72°C, 78 seconds) and 72°C, 7 minutes; and for SUR1 and SUR2A/2B, 94°C, 3 minutes, 40 cycles (94°C, 30 seconds; 58.8°C, 1 minute; 72°C, 1 minute) and 72°C, 7 minutes.…”

Section: Reverse Transcription-polymerase Chain Reaction Analysismentioning

confidence: 99%

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Diabetes Mellitus Impairs Vasodilation to Hypoxia in Human Coronary Arterioles

Miura

Wachtel

Loberiza

et al. 2003

Circulation Research

166

156

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Abstract-ATP-sensitive K ϩ channels (K ATP ) contribute to vasomotor regulation in some species. It is not fully understood the extent to which K ATP participate in regulating vasomotor tone under physiological and pathophysiological conditions in the human heart. Arterioles dissected from right atrial appendage were studied with video microscopy, membrane potential recordings, reverse transcription-polymerase chain reaction, and immunohistochemistry. Hypoxia produced endothelium-independent vasodilation and membrane hyperpolarization of vascular smooth muscle cells, both of which were attenuated by glibenclamide. Aprikalim, a selective K ATP opener, also induced a potent endothelium-independent and glibenclamide-sensitive vasodilation with membrane hyperpolarization. Reverse transcription-polymerase chain reaction detected mRNA expression for K ATP subunits, and immunohistochemistry confirmed the localization of the inwardly rectifying Kir6.1 protein in the vasculature. In patients with type 1 or type 2 diabetes mellitus (DM), vasodilation was reduced to both aprikalim (maximum dilation, DM(ϩ) 90Ϯ2% versus DM(Ϫ) 96Ϯ1%, PϽ0.05) and hypoxia (maximum dilation, DM(ϩ) 56Ϯ8% versus DM(Ϫ) 85Ϯ5%, PϽ0.01) but was not altered to sodium nitroprusside or bradykinin. Baseline myogenic tone and resting membrane potential were not affected by DM. We conclude that DM impairs human coronary arteriolar dilation to K ATP opening, leading to reduced dilation to hypoxia. This reduction in K ATP function could contribute to the greater cardiovascular mortality and morbidity in DM.

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Section: Reverse Transcription-polymerase Chain Reaction Analysismentioning

confidence: 99%

Section: Reverse Transcription-polymerase Chain Reaction Analysismentioning

confidence: 99%

Diabetes Mellitus Impairs Vasodilation to Hypoxia in Human Coronary Arterioles

Miura

Wachtel

Loberiza

et al. 2003

Circulation Research

166

156

View full text Add to dashboard Cite

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“…[19] Labeling of cells cultured in 12-well strips was continuously observed in 12-channel fluorescence detectors at 30 8C until the distribution of DiBAC 4 (3) across the plasma membrane reached equilibrium (60-90 min). Test compounds were added in a volume comprising 1/10 total buffer volume and gently mixed resulting in a time-dependent decrease in fluorescence indicating hyperpolarization of the membrane potential.…”

Section: Pharmacological Methodsmentioning

confidence: 99%

Synthesis of Benzofuran, Benzothiophene, and Benzothiazole‐Based Thioamides and their Evaluation as K_ATP Channel Openers

et al. 2010

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Several series of benzofurans, benzothiophenes, and benzothiazoles, all featuring the thioamide group, were synthesized and tested as novel K(ATP) channel openers in artificial cell systems: CHO cells transfected with SUR1/Kir6.2, and HEK 293 cells transfected with SUR2B/Kir6.1; these served as model systems for insulin-secreting pancreatic β cells and smooth muscle cells, respectively. All compounds were investigated with respect to their binding affinity for the SUR2B-type K(ATP) channels using [(3)H]P1075 as radioligand. Selected compounds were also tested as agonists in intact cells using DiBAC(4)(3) and DyeB (R7260) as membrane potential dyes. Remarkable affinity for SUR2B/Kir6.1 channels in the single-digit micromolar range was observed. In addition, benzothiazole-derived thioamides with sterically demanding, lipophilic substituents showed >100-fold selectivity in favor of SUR2B/Kir6.1. A one-carbon spacer between the heterocyclic skeleton and the thioamide moiety was observed to be crucial for affinity and selectivity. Two of the most potent and selective compounds were studied by crystal structure analyses.

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“…[31,32] Labeling of cells cultured in 12-well strips was continuously observed in 12-channel fluorescence detectors at 30 8C until the distribution of DiBAC4(3) across the plasma membrane reached equilibrium (60-90 min). Test compounds were added in a volume comprising 10 % of total buffer volume and gently mixed resulting in a time-dependent reduction of fluorescence indicating hyperpolarization of the membrane potential.…”

Section: Pharmacological Methodsmentioning

confidence: 99%

Synthesis of Modified 4H‐1,2,4‐Benzothiadiazine‐1,1‐dioxides and Determination of their Affinity and Selectivity for Different Types of K_ATP Channels

et al. 2009

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4H-1,2,4-Benzothiadiazine-1,1-dioxides with various substituents in positions 3, 5, and 7 were synthesized and tested as K(ATP) channel agonists in artificial cell systems (CHO cells transfected with SUR1/Kir6.2, and HEK 293 transfected with SUR2B/Kir6.1) as model systems for insulin-secreting pancreatic beta-cells and for smooth muscle cells, respectively. The effects of agonists were tested in intact cells using DiBAC4(3) [bis-(1,3-dibarbituric acid)trimethine oxanol] as a membrane potential dye, and the results compared with their binding affinity for the SUR2B-type K(ATP) channels using the radioligand [(3)H]P1075. Compounds with cycloalkyl and (cycloalkyl)methyl side chains in position 3 had higher affinities towards the SUR2B/Kir6.1 receptor compared with the parent compound diazoxide (1 a). Compounds with bulky, nonpolar residues in position 3 exhibited remarkable selectivity for SUR2B-type K(ATP) channels. The compound substituted with a bulky (1-adamantyl)methyl residue exhibited micromolar affinity and activity on SUR2B-type K(ATP) channels without being able to activate the SUR1-type K(ATP) channels.

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Pharmacological and molecular characterization of ATP-sensitive K+ channels in the TE671 human medulloblastoma cell line

Cited by 29 publications

References 25 publications

Diabetes Mellitus Impairs Vasodilation to Hypoxia in Human Coronary Arterioles

Diabetes Mellitus Impairs Vasodilation to Hypoxia in Human Coronary Arterioles

Synthesis of Benzofuran, Benzothiophene, and Benzothiazole‐Based Thioamides and their Evaluation as K_ATP Channel Openers

Synthesis of Modified 4H‐1,2,4‐Benzothiadiazine‐1,1‐dioxides and Determination of their Affinity and Selectivity for Different Types of K_ATP Channels

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Pharmacological and molecular characterization of ATP-sensitive K+ channels in the TE671 human medulloblastoma cell line

Cited by 29 publications

References 25 publications

Diabetes Mellitus Impairs Vasodilation to Hypoxia in Human Coronary Arterioles

Diabetes Mellitus Impairs Vasodilation to Hypoxia in Human Coronary Arterioles

Synthesis of Benzofuran, Benzothiophene, and Benzothiazole‐Based Thioamides and their Evaluation as KATP Channel Openers

Synthesis of Modified 4H‐1,2,4‐Benzothiadiazine‐1,1‐dioxides and Determination of their Affinity and Selectivity for Different Types of KATP Channels

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Product

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Synthesis of Benzofuran, Benzothiophene, and Benzothiazole‐Based Thioamides and their Evaluation as K_ATP Channel Openers

Synthesis of Modified 4H‐1,2,4‐Benzothiadiazine‐1,1‐dioxides and Determination of their Affinity and Selectivity for Different Types of K_ATP Channels