Molecular Mechanisms of the Inhibitory Effects of Bupivacaine, Levobupivacaine, and Ropivacaine on Sarcolemmal Adenosine Triphosphate–sensitive Potassium Channels in the Cardiovascular System

2004 ◽  
Vol 101 (2) ◽  
pp. 390-398 ◽  
Author(s):  
Takashi Kawano ◽  
Shuzo Oshita ◽  
Akira Takahashi ◽  
Yasuo Tsutsumi ◽  
Yoshinobu Tomiyama ◽  
...  
Keyword(s):  
Cardiovascular System ◽  
Adenosine Triphosphate ◽  
Local Anesthetics ◽  
Molecular Mechanisms ◽  
Transmembrane Domain ◽  
Katp Channels ◽  
Inhibitory Effect ◽  
Amino Acid Residues ◽  
Sulfonylurea Receptor ◽  
Inhibitory Effects

Background Sarcolemmal adenosine triphosphate-sensitive potassium (KATP) channels in the cardiovascular system may be involved in bupivacaine-induced cardiovascular toxicity. The authors investigated the effects of local anesthetics on the activity of reconstituted KATP channels encoded by inwardly rectifying potassium channel (Kir6.0) and sulfonylurea receptor (SUR) subunits. Methods The authors used an inside-out patch clamp configuration to investigate the effects of bupivacaine, levobupivacaine, and ropivacaine on the activity of reconstituted KATP channels expressed in COS-7 cells and containing wild-type, mutant, or chimeric SURs. Results Bupivacaine inhibited the activities of cardiac KATP channels (IC50 = 52 microm) stereoselectively (levobupivacaine, IC50 = 168 microm; ropivacaine, IC50 = 249 microm). Local anesthetics also inhibited the activities of channels formed by the truncated isoform of Kir6.2 (Kir6.2 delta C36) stereoselectively. Mutations in the cytosolic end of the second transmembrane domain of Kir6.2 markedly decreased both the local anesthetics' affinity and stereoselectivity. The local anesthetics blocked cardiac KATP channels with approximately eightfold higher potency than vascular KATP channels; the potency depended on the SUR subtype. The 42 amino acid residues at the C-terminal tail of SUR2A, but not SUR1 or SUR2B, enhanced the inhibitory effect of bupivacaine on the Kir6.0 subunit. Conclusions Inhibitory effects of local anesthetics on KATP channels in the cardiovascular system are (1) stereoselective: bupivacaine was more potent than levobupivacaine and ropivacaine; and (2) tissue specific: local anesthetics blocked cardiac KATP channels more potently than vascular KATP channels, via the intracellular pore mouth of the Kir6.0 subunit and the 42 amino acids at the C-terminal tail of the SUR2A subunit, respectively.

Molecular Mechanisms Underlying Ketamine-mediated Inhibition of Sarcolemmal Adenosine Triphosphate-sensitive Potassium Channels

2005 ◽  
Vol 102 (1) ◽  
pp. 93-101 ◽  
Author(s):  
Takashi Kawano ◽  
Shuzo Oshita ◽  
Akira Takahashi ◽  
Yasuo Tsutsumi ◽  
Katsuya Tanaka ◽  
...  
Keyword(s):  
Potassium Channels ◽  
Adenosine Triphosphate ◽  
Molecular Mechanisms ◽  
Transmembrane Domain ◽  
Katp Channels ◽  
Adenosine Diphosphate ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Intracellular Magnesium ◽  
Type Mutant

Background Ketamine inhibits adenosine triphosphate-sensitive potassium (KATP) channels, which results in the blocking of ischemic preconditioning in the heart and inhibition of vasorelaxation induced by KATP channel openers. In the current study, the authors investigated the molecular mechanisms of ketamine's actions on sarcolemmal KATP channels that are reassociated by expressed subunits, inwardly rectifying potassium channels (Kir6.1 or Kir6.2) and sulfonylurea receptors (SUR1, SUR2A, or SUR2B). Methods The authors used inside-out patch clamp configurations to investigate the effects of ketamine on the activities of reassociated Kir6.0/SUR channels containing wild-type, mutant, or chimeric SURs expressed in COS-7 cells. Results Ketamine racemate inhibited the activities of the reassociated KATP channels in a SUR subtype-dependent manner: SUR2A/Kir6.2 (IC50 = 83 microM), SUR2B/Kir6.1 (IC50 = 77 microM), SUR2B/Kir6.2 (IC50 = 89 microM), and SUR1/Kir6.2 (IC50 = 1487 microM). S-(+)-ketamine was significantly less potent than ketamine racemate in blocking all types of reassociated KATP channels. The ketamine racemate and S-(+)-ketamine both inhibited channel currents of the truncated isoform of Kir6.2 (Kir6.2DeltaC36) with very low affinity. Application of 100 mum magnesium adenosine diphosphate significantly enhanced the inhibitory potency of ketamine racemate. The last transmembrane domain of SUR2 was essential for the full inhibitory effect of ketamine racemate. Conclusions These results suggest that ketamine-induced inhibition of sarcolemmal KATP channels is mediated by the SUR subunit. These inhibitory effects of ketamine exhibit specificity for cardiovascular KATP channels, at least some degree of stereoselectivity, and interaction with intracellular magnesium adenosine diphosphate.

Molecular Mechanisms of the Inhibitory Effects of Propofol and Thiamylal on Sarcolemmal Adenosine Triphosphate–sensitive Potassium Channels

2004 ◽  
Vol 100 (2) ◽  
pp. 338-346 ◽  
Author(s):  
Takashi Kawano ◽  
Shuzo Oshita ◽  
Akira Takahashi ◽  
Yasuo Tsutsumi ◽  
Yoshinobu Tomiyama ◽  
...  
Keyword(s):  
Adenosine Triphosphate ◽  
Katp Channel ◽  
Molecular Mechanisms ◽  
Katp Channels ◽  
Site Directed Mutagenesis ◽  
Sulfonylurea Receptor ◽  
Inhibitory Effects ◽  
Inwardly Rectifying Potassium Channel ◽  
Mutagenesis Study ◽  
Inwardly Rectifying

Background Both propofol and thiamylal inhibit adenosine triphosphate-sensitive potassium (KATP) channels. In the current study, the authors investigated the effects of these anesthetics on the activity of recombinant sarcolemmal KATP channels encoded by inwardly rectifying potassium channel (Kir6.1 or Kir6.2) genes and sulfonylurea receptor (SUR1, SUR2A, or SUR2B) genes. Methods The authors used inside-out patch clamp configurations to investigate the effects of propofol and thiamylal on the activity of recombinant KATP channels using COS-7 cells transfected with various types of KATP channel subunits. Results Propofol inhibited the activities of the SUR1/Kir6.2 (EC50 = 77 microm), SUR2A/Kir6.2 (EC50 = 72 microm), and SUR2B/Kir6.2 (EC50 = 71 microm) channels but had no significant effects on the SUR2B/Kir6.1 channels. Propofol inhibited the truncated isoform of Kir6.2 (Kir6.2DeltaC36) channels (EC50 = 78 microm) that can form functional KATP channels in the absence of SUR molecules. Furthermore, the authors identified two distinct mutations R31E (arginine residue at position 31 to glutamic acid) and K185Q (lysine residue at position 185 to glutamine) of the Kir6.2DeltaC36 channel that significantly reduce the inhibition of propofol. In contrast, thiamylal inhibited the SUR1/Kir6.2 (EC50 = 541 microm), SUR2A/Kir6.2 (EC50 = 248 microm), SUR2B/Kir6.2 (EC50 = 183 microm), SUR2B/Kir6.1 (EC50 = 170 microm), and Kir6.2DeltaC36 channels (EC50 = 719 microm). None of the mutants significantly affects the sensitivity of thiamylal. Conclusions These results suggest that the major effects of both propofol and thiamylal on KATP channel activity are mediated via the Kir6.2 subunit. Site-directed mutagenesis study suggests that propofol and thiamylal may influence Kir6.2 activity by different molecular mechanisms; in thiamylal, the SUR subunit seems to modulate anesthetic sensitivity.

Inhibitory Effects of Etomidate and Ketamine on Adenosine Triphosphate–Sensitive Potassium Channel Relaxation in Canine Pulmonary Artery

2003 ◽  
Vol 98 (1) ◽  
pp. 104-113 ◽  
Author(s):  
Ju-Tae Sohn ◽  
Paul A. Murray
Keyword(s):  
Adenosine Triphosphate ◽  
Inhibitory Effect ◽  
Isometric Tension ◽  
Relaxation Response ◽  
Inhibitory Effects ◽  
Channel Activation ◽  
K Channels ◽  
Dependent Component ◽  
Pulmonary Arterial ◽  
Relaxation Responses

Background The authors recently demonstrated that etomidate and ketamine attenuated endothelium-dependent pulmonary vasorelaxation mediated by nitric oxide and Ca -activated K + channels. In the current study, they tested the hypothesis that these intravenous anesthetics inhibit pulmonary vasorelaxation mediated by adenosine triphosphate-sensitive potassium (K + ATP ) channel activation. Methods Endothelium intact and denuded pulmonary arterial rings were suspended in organ chambers for isometric tension recording. The effects of etomidate (5 x 10(-6) and 5 x 10(-5) m) and ketamine (5 x 10(-5) and 10(-4) m) on vasorelaxation responses to lemakalim (K + ATP channel activator), prostacyclin, and papaverine were assessed in phenylephrine-precontracted rings. The effect of cyclooxygenase inhibition with indomethacin was assessed in some protocols. Results Etomidate (5 x 10(-6) m) only inhibited the vasorelaxant response to lemakalim in endothelium intact rings, whereas a higher concentration of etomidate (5 x 10(-5) m) inhibited relaxation in both intact and endothelium-denuded rings. Pretreatment with indomethacin abolished the endothelium-dependent attenuation of lemakalim-induced relaxation caused by etomidate. Ketamine (5 x 10(-5) and 10(-5) m) inhibited the relaxation response to lemakalim to the same extent in both endothelium-intact and -denuded rings, and this effect was not prevented by indomethacin pretreatment. Etomidate and ketamine had no effect on the relaxation responses to prostacyclin or papaverine. Conclusions These results indicate that etomidate, but not ketamine, attenuates the endothelium-dependent component of lemakalim-induced pulmonary vasorelaxation an inhibitory effect on the cyclooxygenase pathway. Both anesthetics inhibit K + ATP -mediated pulmonary vasorelaxation a direct effect on pulmonary vascular smooth muscle.

Molecular Mechanisms of the Inhibitory Effects of Clonidine on Vascular Adenosine Triphosphate–Sensitive Potassium Channels

2011 ◽  
Vol 113 (6) ◽  
pp. 1374-1380 ◽  
Author(s):  
Shinji Kawahito ◽  
Takashi Kawano ◽  
Hiroshi Kitahata ◽  
Jun Oto ◽  
Akira Takahashi ◽  
...  
Keyword(s):  
Potassium Channels ◽  
Adenosine Triphosphate ◽  
Molecular Mechanisms ◽  
Inhibitory Effects

scholarly journals N-terminal transmembrane domain of SUR1 controls gating of Kir6.2 by modulating channel sensitivity to PIP2

2011 ◽  
Vol 137 (3) ◽  
pp. 299-314 ◽  
Author(s):  
Emily B. Pratt ◽  
Paul Tewson ◽  
Cathrin E. Bruederle ◽  
William R. Skach ◽  
Show-Ling Shyng
Keyword(s):  
Katp Channel ◽  
Transmembrane Domain ◽  
Katp Channels ◽  
Full Length ◽  
Regulatory Subunit ◽  
Functional Coupling ◽  
Sulfonylurea Receptor ◽  
Open Probability ◽  
Protein Levels ◽  
Channel Inactivation

Functional integrity of pancreatic adenosine triphosphate (ATP)-sensitive potassium (KATP) channels depends on the interactions between the pore-forming potassium channel subunit Kir6.2 and the regulatory subunit sulfonylurea receptor 1 (SUR1). Previous studies have shown that the N-terminal transmembrane domain of SUR1 (TMD0) interacts with Kir6.2 and is sufficient to confer high intrinsic open probability (Po) and bursting patterns of activity observed in full-length KATP channels. However, the nature of TMD0–Kir6.2 interactions that underlie gating modulation is not well understood. Using two previously described disease-causing mutations in TMD0 (R74W and E128K), we performed amino acid substitutions to study the structural roles of these residues in KATP channel function in the context of full-length SUR1 as well as TMD0. Our results revealed that although R74W and E128K in full-length SUR1 both decrease surface channel expression and reduce channel sensitivity to ATP inhibition, they arrive there via distinct mechanisms. Mutation of R74 uniformly reduced TMD0 protein levels, suggesting that R74 is necessary for stability of TMD0. In contrast, E128 mutations retained TMD0 protein levels but reduced functional coupling between TMD0 and Kir6.2 in mini-KATP channels formed by TMD0 and Kir6.2. Importantly, E128K full-length channels, despite having a greatly reduced Po, exhibit little response to phosphatidylinositol 4,5-bisphosphate (PIP2) stimulation. This is reminiscent of Kir6.2 channel behavior in the absence of SUR1 and suggests that TMD0 controls Kir6.2 gating by modulating Kir6.2 interactions with PIP2. Further supporting this notion, the E128W mutation in full-length channels resulted in channel inactivation that was prevented or reversed by exogenous PIP2. These results identify a critical determinant in TMD0 that controls Kir6.2 gating by controlling channel sensitivity to PIP2. Moreover, they uncover a novel mechanism of KATP channel inactivation involving aberrant functional coupling between SUR1 and Kir6.2.

scholarly journals Analysis of the mechanism of anagrelide-induced thrombocytopenia in humans

Blood ◽  
1992 ◽  
Vol 79 (8) ◽  
pp. 1931-1937 ◽  
Author(s):  
EM Mazur ◽  
AG Rosmarin ◽  
PA Sohl ◽  
JL Newton ◽  
A Narendran
Keyword(s):  
Colony Size ◽  
Molecular Mechanisms ◽  
Human Peripheral Blood ◽  
Inhibitory Effect ◽  
Colony Development ◽  
Maturation Stage ◽  
Inhibitory Effects ◽  
Plasma Clot ◽  
Liquid Suspension ◽  
Normal Human Peripheral Blood

Abstract Anagrelide is a new therapeutic compound recently demonstrated to have a rapid and selective thrombocytopenic effect in humans. The effects of anagrelide were evaluated in plasma clot and liquid suspension cultures of optimally stimulated normal human peripheral blood megakaryocyte progenitors in order to determine the mechanism of its thrombocytopenic activity. In plasma clot cultures, at clinically relevant, therapeutic concentrations (5 to 50 ng/mL), anagrelide exerted no significant inhibitory effect on megakaryocyte colony numbers or colony size. Only at anagrelide concentrations of 10 to 500 times therapeutic doses did anagrelide inhibit megakaryocyte colony development: an anagrelide concentration of 5 micrograms/mL reduced colony numbers by 57% and colony size by 31%. In contrast, lower, therapeutic anagrelide concentrations exerted profound effects in liquid culture on megakaryocyte cytoplasmic maturation, size, and DNA content. When present for the entire 12-day culture duration, anagrelide induced left- shifted megakaryocyte maturation and reduced both megakaryocyte ploidy and megakaryocyte diameter. Anagrelide, at concentrations of 5 to 50 ng/mL, shifted the modal cultured megakaryocyte morphologic stage from III to II, reduced the model ploidy value from 16N to 8N, and decreased the mean megakaryocyte diameter by up to 22%, from 27.6 microns to 21.6 microns. Megakaryocyte diameter was significantly reduced in most instances, even when analyzed as a function of morphologic stage. When anagrelide was added to the cultures after 6- and 9-day delays (during the final 6 and 3 days, respectively, of culture), similar inhibitory effects on megakaryocyte maturation stage and ploidy distribution were observed. However, the magnitude of the left-shift in ploidy appeared to be less as the duration of anagrelide exposure was reduced. Conversely, megakaryocyte diameter was not significantly affected by the shorter 3- and 6-day anagrelide exposures. These data indicate that therapeutic concentrations of anagrelide influence primarily the postmitotic phase of megakaryocyte development, decreasing platelet production by reducing megakaryocyte size and ploidy, as well as by disrupting full megakaryocyte maturation. Inhibition of megakaryocyte diameter appears to require more prolonged anagrelide exposure than inhibition of maturation stage and ploidy. The molecular mechanisms responsible for the inhibitory effects of anagrelide on megakaryocytopoiesis remain to be defined.

scholarly journals Application of the adenosine triphosphate sensitivity assay in infantile vascular anomalies

2019 ◽  
Author(s):  
Li Li ◽  
Bin Yang ◽  
Li Wei ◽  
Bin Zhang ◽  
Xiao-feng Han ◽  
...  
Keyword(s):  
Adenosine Triphosphate ◽  
Comprehensive Evaluation ◽  
Vascular Malformations ◽  
Inhibitory Effect ◽  
Vascular Anomalies ◽  
Vascular Tumors ◽  
Inhibitory Effects ◽  
Inhibition Effect ◽  
Multiple Drugs

Abstract BACKGROUND: The term vascular anomalies include various vascular tumors and vascular malformations, among them infantile hemangiomas and capillary malformations are the most well-known associated diseases in early ages. Multiple drugs have been introduced for intervention, but susceptibility test in vitro were scarcely reported.OBJECTIVE: To evaluate the inhibition effect of adenosine triphosphate sensitivity assay in vitro before the treatment of infantile hemangiomas and capillary malformations. METHODS: Specimens were selected from 5 cases of infantile hemangiomas and 11 cases of capillary malformations. Propranolol, rapamycin, sildenafil and itraconazole were tested for their growth inhibition effect by using the adenosine triphosphate sensitivity assay. RESULTS: Propranolol demonstrated inhibitory effects on infantile hemangiomas cells. Rapamycin and itraconazole both showed inhibitory effects on infantile hemangiomas cells and capillary malformations cells. Sildenafil has no growth inhibitory effect on infantile hemangiomas cells or capillary malformations cells. CONCLUSION: Adenosine triphosphate sensitivity assay is a sensitive and useful testing method before the management of vascular anomalies, and individualized medication suggestions for the choice of therapeutic drugs were offered based on the testing result and together with a comprehensive evaluation of each infant.

scholarly journals Noble Gas Xenon Is a Novel Adenosine Triphosphate-sensitive Potassium Channel Opener

2010 ◽  
Vol 112 (3) ◽  
pp. 623-630 ◽  
Author(s):  
Carsten Bantel ◽  
Mervyn Maze ◽  
Stefan Trapp
Keyword(s):  
Blood Brain Barrier ◽  
Adenosine Triphosphate ◽  
Katp Channel ◽  
Katp Channels ◽  
Brain Barrier ◽  
Hek293 Cells ◽  
Sulfonylurea Receptor ◽  
Channel Opener ◽  
Blood Brain ◽  
Sulfonylurea Receptor 1

Background Adenosine triphosphate-sensitive potassium (KATP) channels in brain are involved in neuroprotective mechanisms. Pharmacologic activation of these channels is seen as beneficial, but clinical exploitation by using classic K channel openers is hampered by their inability to cross the blood-brain barrier. This is different with the inhalational anesthetic xenon, which recently has been suggested to activate KATP channels; it partitions freely into the brain. Methods To evaluate the type and mechanism of interaction of xenon with neuronal-type KATP channels, these channels, consisting of Kir6.2 pore-forming subunits and sulfonylurea receptor-1 regulatory subunits, were expressed in HEK293 cells and whole cell, and excised patch-clamp recordings were performed. Results Xenon, in contrast to classic KATP channel openers, acted directly on the Kir6.2 subunit of the channel. It had no effect on the closely related, adenosine triphosphate (ATP)-regulated Kir1.1 channel and failed to activate an ATP-insensitive mutant version of Kir6.2. Furthermore, concentration-inhibition curves for ATP obtained from inside-out patches in the absence or presence of 80% xenon revealed that xenon reduced the sensitivity of the KATP channel to ATP. This was reflected in an approximately fourfold shift of the concentration causing half-maximal inhibition (IC50) from 26 +/- 4 to 96 +/- 6 microm. Conclusions Xenon represents a novel KATP channel opener that increases KATP currents independently of the sulfonylurea receptor-1 subunit by reducing ATP inhibition of the channel. Through this action and by its ability to readily partition across the blood-brain barrier, xenon has considerable potential in clinical settings of neuronal injury, including stroke.

scholarly journals Expression of Phi11 Gp07 Causes Filamentation in Escherichia coli

2018 ◽  
Vol 12 (1) ◽  
pp. 107-115 ◽  
Author(s):  
Avijit Das ◽  
Sumit Biswas ◽  
Malabika Biswas
Keyword(s):  
Growth Rate ◽  
Inhibitory Effect ◽  
Terminal Region ◽  
Amino Acid Residues ◽  
Inhibitory Effects ◽  
E Coli ◽  
Amino Terminal ◽  
Growth Inhibitory ◽  
Carboxy Terminal ◽  
Amino Terminal Domain

Background:The Gp07 protein of aureophage Phi11 exhibits growth inhibitory effects when overexpressed inE. coli.The protein harbors two domains- an amino terminal Bro-like domain and a carboxy terminal Ant superfamily like KilA domain, of which the KilA domain retains the growth inhibitory effect of Gp07.Methods:We studied the effects exerted by the overexpression of Gp07 and its separate domains upon the growth rate as well as the morphology of theE. colicells. Additionally, we generated a mutant of Gp07 (designated as ΔGp07) by deleting the first eleven amino acid residues from the amino-terminal region of Gp07, and studied its growth inhibitory effects uponE. coli.Results:Our results indicate that Gp07, ΔGp07 as well as the Carboxy-terminal region of Gp07 upon overexpression, retards the growth rate of theE. colicells and also induces filamentation in the cells. Surprisingly, our data clearly suggests that the growth inhibition and filamentation induced by the the amino-terminal domain of Gp07 is temporal in nature.Conclusion:The carboxy-terminal of domain of gp07 is essential for its activity.

Mechanism of Cardiac Sarcolemmal Adenosine Triphosphate–sensitive Potassium Channel Activation by Isoflurane in a Heterologous Expression System

2006 ◽  
Vol 105 (3) ◽  
pp. 534-540 ◽  
Author(s):  
Martin Bienengraeber ◽  
David C. Warltier ◽  
Zeljko J. Bosnjak ◽  
Anna Stadnicka
Keyword(s):  
Heterologous Expression ◽  
Adenosine Triphosphate ◽  
Katp Channel ◽  
Expression System ◽  
Katp Channels ◽  
Sulfonylurea Receptor ◽  
Channel Activity ◽  
Heterologous Expression System ◽  
Open Probability ◽  
Channel Activation

Background Activation of the cardiac sarcolemmal adenosine triphosphate-sensitive potassium (KATP) channel during metabolic stress initiates cellular events that preserve cardiac performance. Previous studies showed that halogenated anesthetics prime KATP channels under whole cell voltage clamp and act in intracellular pH (pHi)-dependent manner on KATP channels in excised membrane patches. However, it is not known how halogenated anesthetics interact with these channels. Methods The authors evaluated the effect of pHi and isoflurane on the KATP channel subunits, the pore-forming inward rectifier Kir6.2, and the regulatory sulfonylurea receptor SUR2A, using HEK293 cells as a heterologous expression system. Single channel activity was recorded in the inside-out patch configuration. Results At pHi 7.4, isoflurane had negligible effect on activity of wild-type Kir6.2/SUR2A, but at pHi 6.8, the channel open probability was increased by isoflurane (0.177 +/- 0.077 to 0.364 +/- 0.164). By contrast, the open probability of truncated Kir6.2DeltaC26, which forms a functional channel without SUR2A, was attenuated by isoflurane at both pHi 7.4 and pHi 6.8. Coexpression of Kir6.2DeltaC26 with SUR2A restored pHi sensitivity of channel activation by isoflurane. Site-directed mutagenesis within the Walker motifs of SUR2A abolished isoflurane activation of KATP channel at pHi 6.8. In addition, the pancreatic-type channels expressing sulfonylurea receptor SUR1 could not be activated by isoflurane. Conclusions The nucleotide binding domains of SUR2A play a crucial role in isoflurane facilitation of the KATP channel activity at moderately acidic pHi as would occur during early ischemia. These findings support direct and differential interaction of isoflurane with the subunits of the cardiac sarcolemmal KATP channel.

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