Local Anesthetic Interaction with Human Ether-a-go-go-related Gene (HERG) Channels: Role of Aromatic Amino Acids Y652 and F656

Background: Human ether-a-go-go-related gene (HERG) potassium channels constitute a potential target involved in cardiotoxic side effects of amino-amide local anesthetics. The molecular interaction site of these low-affinity blockers with HERG channels is currently unknown. The aim of this study was to determine the effect of the mutations Y652A and F656A in the putative drug binding region of HERG on the inhibition by bupivacaine, ropivacaine, and mepivacaine.

Methods: The authors examined the inhibition of wild-type and mutant HERG channels, transiently expressed in Chinese hamster ovary cells by bupivacaine, ropivacaine, and mepivacaine. Whole cell patch clamp recordings were performed at room temperature.

Results: Inhibition of HERG wild-type and mutant channels by the different local anesthetics was concentration dependent, stereoselective, and reversible. The sensitivity decreased in the order bupivacaine > ropivacaine > mepivacaine for wild-type and mutant channels. The mutant channels were approximately 4-30 times less sensitive to the inhibitory action of the different local anesthetics than the wild-type channel. The concentration-response data were described by Hill functions (bupivacaine: wild-type IC50 = 22 +/- 2 [mu]m, n = 38; Y652A IC50 = 95 +/- 5 [mu]m, n = 31). The mutations resulted in a change of the stereoselectivity of HERG channel block by ropivacaine. The potency of the local anesthetics to inhibit wild-type and mutant channels correlated with the lipophilicity of the drug (r > 0.9).     

Molecular interaction of droperidol with human ether-a-go-go-related gene channels: prolongation of action potential duration without inducing early afterdepolarization

BACKGROUND: The cardiac safety of droperidol given at antiemetic doses is a matter of debate. Although droperidol potently inhibits human ether-a-go-go-related gene (HERG) channels, the molecular mode of this interaction is unknown. The role of amino acid residues typically mediating high-affinity block of HERG channels is unclear. It is furthermore unresolved whether droperidol at antiemetic concentrations induces action potential prolongation and arrhythmogenic early afterdepolarizations in cardiac myocytes. METHODS: Molecular mechanisms of HERG current inhibition by droperidol were established using two-electrode voltage clamp recordings of Xenopus laevis oocytes expressing wild-type and mutant channels. The mutants T623A, S624A, V625A, Y652A, and F656A were generated by site-directed mutagenesis. The effect of droperidol on action potentials was investigated in cardiac myocytes isolated from guinea pig hearts using the patch clamp technique. RESULTS: Droperidol inhibited currents through HERG wild-type channels with a concentration of half-maximal inhibition of 0.6-0.9 microM. Droperidol shifted the channel activation and the steady state inactivation toward negative potentials while channel deactivation was not affected. Current inhibition increased with membrane potential and with increasing duration of current activation. Inhibition of HERG channels was similarly reduced by all mutations. Droperidol at concentrations between 5 and 100 nM prolonged whereas concentrations greater than 300 nm shortened action potentials. Early afterdepolarizations were not observed. CONCLUSIONS: Droperidol is a high-affinity blocker of HERG channels. Amino acid residues typically involved in high-affinity block mediate droperidol effects. Patch clamp results and computational modeling allow the hypothesis that interaction with calcium currents may explain why droperidol at antiemetic concentrations prolongs the action potential without inducing early afterdepolarizations.     

Local anaesthetic sensitivities of cloned HERG channels from human heart: comparison with HERG/MiRP1 and HERG/MiRP1 T8A

Background. Myocardial potassium channels are complexes formedby different subunits. The subunit composition may influencethe cardiotoxic action of local anaesthetics. The effects ofamide local anaesthetics on HERG channels co-expressed withthe putative subunit MiRP1 have not been established. It isalso unclear if the common polymorphism MiRP1_T8A that predisposesindividuals to drug-induced cardiac arrhythmia increases local-anaestheticsensitivity of HERG/MiRP1 channels. This may suggest the presenceof genetic risk factors for local-anaesthetic-induced cardiacarrhythmia. Methods. Whole-cell patch-clamp recordings and site-directedmutagenesis were combined to compare local anaesthetic sensitivitiesof cloned and mutated human potassium channel subunits. Theion channels were activated by a protocol that approximatedventricular action potentials. Results. The amide local anaesthetics bupivacaine, levobupivacaineand ropivacaine inhibited HERG channels at toxicologically relevantconcentrations, with IC₅₀ values of 20 (SEM 2) µM (n=29),10 (1) µM (n=40) and 20 (2) µM (n=49), respectively.Hill coefficients were close to unity. There were no indicationsof qualitative differences in channel inhibition between thethree anaesthetics. The putative subunit MiRP1 did not alterlocal anaesthetic sensitivity of HERG channels. The common singlenucleotide polymorphism producing MiRP1_T8A did not increaselocal anaesthetic sensitivity of HERG/MiRP1 channels. Conclusions. Amide local anaesthetics target HERG and HERG/MiRP1channels with identical potency. The effects on these ion currentsmay significantly contribute to local-anaesthetic-induced cardiacarrhythmia. MiRP1_T8A does not seem to confer an increased riskof severe cardiac side-effects to carriers of this common polymorphism. Br J Anaesth 2004; 92: 93–101     

Muscle and plasma amino acids after injury: the role of inactivity.

The amino acid pattern following total hip replacement is characterized by increases in muscle of the branched chain amino acids (leucine, isoleucine and valine), the aromatics (phenylalanine and tyrosine) as well as methionine. The nonessential amino acids in muscle tend to decline, glutamine having the most marked change. Plasma levels of the essential amino acids increase while the nonessentials tend to decrease. This pattern differs from that observed in other catabolic states (uremia, starvation, untreated diabetes) and is significantly different from the effects of inactivity and starvation combined. This suggests that injury can be characterized by a unique pattern of muscle and plasma amino acids.     

Cardioprotective effects of propofol and sevoflurane in ischemic and reperfused rat hearts: role of K(ATP) channels and interaction with the sodium-hydrogen exchange inhibitor HOE 642 (cariporide).

BACKGROUND: Sodium ion-hydrogen ion (Na(+)-H(+)) exchange inhibitors are effective cardioprotective agents. The N(+)-H(+) exchange inhibitor HOE 642 (cariporide) has undergone clinical trials in acute coronary syndromes, including bypass surgery. Propofol and sevoflurane are also cardioprotective via unknown mechanisms. The authors investigated the interaction between propofol and HOE 642 in the ischemic reperfused rat heart and studied the role of adenosine triphosphate-sensitive potassium (K(ATP)) channels in the myocardial protection associated with propofol and sevoflurane. METHODS: Isolated rat hearts were perfused by the Langendorff method at a constant flow rate, and left ventricular function and coronary pressures were assessed using standard methods. Energy metabolites were also determined. To assess the role of K(ATP) channels, hearts were pretreated with the K(ATP) blocker glyburide (10 microM). Hearts were then exposed to either control buffer or buffer containing HOE 642 (5 microM), propofol (35 microM), sevoflurane (2.15 vol%), the K(ATP) opener pinacidil (1 microM), or the combination of propofol and HOE 642. Each heart was then subjected to 1 h of global ischemia followed by 1 h of reperfusion. RESULTS: Hearts treated with propofol, sevoflurane, pinacidil, or HOE 642 showed significantly higher recovery of left ventricular developed pressure and reduced end-diastolic pressures compared with controls. The combination of propofol and HOE 642 provided superior protection toward the end of the reperfusion period. Propofol, sevoflurane, and HOE 642 also attenuated the onset and magnitude of ischemic contracture and preserved high-energy phosphates (HEPs) compared with controls. Glyburide attenuated the cardioprotective effects of sevoflurane and abolished the protection observed with pinacidil. In contrast, glyburide had no effect on the cardioprotection associated with propofol treatment. CONCLUSION: HOE 642, propofol, and sevoflurane provide cardioprotection via different mechanisms. These distinct mechanisms may allow for the additive and superior protection observed with the combination of these anesthetics and HOE 642.     

Percutaneous nephrostomy: assessment of renal damage associated with semi-rigid (24F) and balloon (36F) dilation

In an effort to discern the amount of renal damage incurred by antegrade percutaneous nephrostomy and nephrostomy tract dilation, we studied the long-term effect of acutely created 24F and 36F percutaneous nephrostomy tracts on 12 pig kidneys. Half of the nephrostomy tracts were dilated using a semi-rigid 24F fascial dilating system (Amplatz design) and half were dilated using a 36F balloon dilation system. Animals were killed at six weeks and the nephrostomy tract was dissected, fixed in formalin, and microscopic cross-sections were stained with Masson's trichrome stain for collagen prior to morphometry using computerized planimetry. In this study, renal damage from the nephrostomy tract averaged 0.15% of the total renal cortical surface. Balloon dilation to 36F incurred no more significant damage than fascial dilation to 24F.     

Absorption of amino acids in patients after partial gastrectomy (B II) and truncal vagotomy with pyloroplasty or antrectomy (author's transl)

After surgery for gastric or duodenal ulcer the resorption of amino acids is changed due to alteration of morphology and function of stomach, bile system, pancreas and small bowel. We used the stable isotope labelled amino acid 15N-glycine in patients after partial gastrectomy (B II), truncal vagotomy with pylorplasty as well as antrectomy and in healthy persons. It could be shown that in contrast to the control group the absorption of the amino acid in the surgical group took place more rapid and more intensive.     

Morphine enhances pharmacological preconditioning by isoflurane: role of mitochondrial K(ATP) channels and opioid receptors

BACKGROUND: Adenosine triphosphate-regulated potassium channels mediate protection against myocardial infarction produced by volatile anesthetics and opioids. We tested the hypothesis that morphine enhances the protective effect of isoflurane by activating mitochondrial adenosine triphosphate-regulated potassium channels and opioid receptors. METHODS: Barbiturate-anesthetized rats (n = 131) were instrumented for measurement of hemodynamics and subjected to a 30 min coronary artery occlusion followed by 2 h of reperfusion. Myocardial infarct size was determined using triphenyltetrazolium staining. Rats were randomly assigned to receive 0.9% saline, isoflurane (0.5 and 1.0 minimum alveolar concentration [MAC]), morphine (0.1 and 0.3 mg/kg), or morphine (0.3 mg/kg) plus isoflurane (1.0 MAC). Isoflurane was administered for 30 min and discontinued 15 min before coronary occlusion. In eight additional groups of experiments, rats received 5-hydroxydecanoic acid (5-HD; 10 mg/kg) or naloxone (6 mg/kg) in the presence or absence of isoflurane, morphine, and morphine plus isoflurane. RESULTS: Isoflurane (1.0 MAC) and morphine (0.3 mg/kg) reduced infarct size (41 +/- 3%; n = 13 and 38 +/- 2% of the area at risk; n = 10, respectively) as compared to control experiments (59 +/- 2%; n = 10). Morphine plus isoflurane further decreased infarct size to 26 +/- 3% (n = 11). 5-HD and naloxone alone did not affect infarct size, but abolished cardioprotection produced by isoflurane, morphine, and morphine plus isoflurane. CONCLUSIONS: Combined administration of isoflurane and morphine enhances the protection against myocardial infarction to a greater extent than either drug alone. This beneficial effect is mediated by mitochondrial adenosine triphosphate-regulated potassium channels and opioid receptors in vivo.     

Randomized, multicenter study of interaction between Org 9426 (rocuronium bromide) and anesthetic agents in Japanese population

BACKGROUND: The purpose of this randomized, multi-center phase III trial was to investigate the influence of sevoflurane and propofol on the neuromuscular blocking effects and pharmacokinetic parameters of Org 9426 (rocuronium bromide) in Japanese population. METHODS: Thirty-nine adult Japanese patients participated in this randomized, multi-center study. Neuromuscular function was monitored continuously with TOF-Watch SX (Organon NV, Netherlands) after anesthetic induction with propofol. These subjects randomly received either 0.6 mg x kg(-1) or 0.9 mg x kg(-1) of rocuronium for endotracheal intubation. These two groups were further divided to two anesthetic regiments : sevoflurane group and propofol group. The difference in onset and recovery of rocuronium-induced neuromuscular block was statistically analyzed with two-way ANOVA. RESULTS: Mean duration for maximal block was 76 seconds and 66 seconds, respectively. The duration between Org 9426 administration and 25% recovery of first twitch response was significantly prolonged in patients given 0.9 mg x kg(-1) of Org 9426. Sevoflurane also significantly increased this duration. However, the serum concentration of Org 9426 was not statistically different between the four study groups. CONCLUSIONS: The duration of Org 9426-induced neuromuscular blockade was significantly increased under sevoflurane anesthesia compared to propofol anesthesia. This difference may be attributed to pharmacodynamic change.     

A novel hemostatic agent: the potential role of recombinant activated factor VII (rFVIIa) in anesthetic practice.

PURPOSE: To review the role of recombinant factor VIIa in anesthetic practice. SOURCE: A review of the published literature. MAIN FINDINGS: The mechanism of action of rFVIIa suggests enhancement of hemostasis limited to the site of injury without systemic activation of the coagulation cascade. In addition to its indication for use in patients with hemophilia, use of rFVIIa for treatment of uncontrolled massive hemorrhage in various peroperative settings appears to be rational, safe, and effective. Published results suggest that in trauma patients rFVIIa may play a role as an adjunctive hemostatic measure in addition to surgical hemostatic techniques There is preliminary evidence that hemorrhagic complications (eg. epistaxis, vaginal bleeding) associated with profound thrombocytopenia can be reversed with rFVIIa even at platelet counts below 10,000 per microL. Various case reports outlining the successful treatment with recombinant factor VIIa of patents experiencing intractable bleeding after valve replacement surgery, and with severe hemorrhage during therapy with left ventricular assist device, indicate the potential therapeutic efficacy of this agent in cardiac surgical procedures. Additionally, rFVIIa has been used successfully for treatment of massive postoperative bleeding following general surgery. CONCLUSIONS: rFVIIa is a novel hemostatic agent that shows promise in non-hemophiliac patents of a significant therapeutic role in variety of coagulopathic and hemorrhagic conditions in clinical situations ranging from thrombocytopenia, disseminated intravascular coagulation and transfusion-related coagulopathy, as well as in patients experiencing massive blood loss undergoing orthotopic liver transplantation, cardiac, orthopedic and genitourinary surgery.     

Mouse seminal vesicle secretory protein of 99 amino acids (MSVSP99): characterization and hormonal and developmental regulation.

Polyclonal antibodies have been generated to investigate the localization, tissue and species distribution, androgen regulation, and ontogeny of a protein secreted by mouse seminal vesicle, designated as MSVSP99 (ie, mouse seminal vesicle secretory protein of 99 amino acids). MSVSP99 is a polymorphic compound with a molecular weight of around 14 kilodaltons and a positive immunoreactivity range of 5.23 to 5.70. Positive immunoreactivity was restricted to the epithelial cells of the seminal vesicle. Western blot analysis showed organ specificity for MSVSP99, which could not be detected in several organs in the mouse. Time course decrease of MSVSP99 after castration closely followed that of its mRNA. In contrast, the length of time required to restore control levels after testosterone treatment was higher for the protein than it was for its mRNA. Whereas the MSVSP99 gene is already active in 10-day-old males, MSVSP99 is first detected at 27 days. Then, we conclude that factors other than the accumulation of the mRNA regulate MSVSP99 expression.     

The role of mitochondrial and sarcolemmal K(ATP) channels in canine ethanol-induced preconditioning in vivo

Chronic consumption of small doses of ethanol protects myocardium from ischemic injury. We tested the hypothesis that mitochondrial and sarcolemmal adenosine triphosphate-dependent potassium (K(ATP)) channels mediate these beneficial effects. Dogs (n = 76) were fed with ethanol (1.5 g/kg) or water mixed with dry food bid for 6 or 12 wk, fasted overnight before experimentation, and instrumented for measurement of hemodynamics. Dogs received intracoronary saline (vehicle), 5-hydroxydecanoate (a mitochondrial K(ATP) channel antagonist; 6.75 mg/kg over 45 min), or HMR-1098 (a sarcolemmal K(ATP) channel antagonist; 45 microg/kg over 45 min) and were subjected to a 60 min coronary artery occlusion followed by 3 h of reperfusion. A final group of dogs was pretreated with ethanol and chow for 6 wk before occlusion and reperfusion. Myocardial infarct size and transmural coronary collateral blood flow were measured with triphenyltetrazolium chloride staining and radioactive microspheres, respectively. The area at risk of infarction was similar between groups. A 12-wk pretreatment with ethanol significantly reduced infarct size to 13% +/- 2% (mean +/- SEM; n = 8) of the area at risk compared with control experiments (25% +/- 2%; n = 8), but a 6-wk pretreatment did not (21% +/- 2%; n = 8). 5-hydroxydecanoate and HMR-1098 abolished the protective effects of 12-wk ethanol pretreatment (24% +/- 2% and 29% +/- 3%, respectively; n = 8 for each group) but had no effect in dogs that did not receive ethanol (22% +/- 2% and 23% +/- 4%, respectively; n = 8 for each group). No differences in hemodynamics or transmural coronary collateral blood flow were observed between the groups. The results indicate that mitochondrial and sarcolemmal K(ATP) channels mediate ethanol-induced preconditioning in dogs independent of alterations in systemic hemodynamics or coronary collateral blood flow. IMPLICATIONS: Mitochondrial and sarcolemmal K(ATP) channels mediate ethanol-induced preconditioning independent of alterations in systemic hemodynamics or coronary collateral perfusion in vivo.