Presence Of Quinidine Research Articles

Protein 14-3-3 Influences the Response of the Cardiac Sodium Channel Nav1.5 to Antiarrhythmic Drugs.

The cardiac sodium channel Nav1.5 is a key contributor to the cardiac action potential, and dysregulations in Nav1.5 can lead to cardiac arrhythmias. Nav1.5 is a target of numerous antiarrhythmic drugs (AADs). Previous studies identified the protein 14-3-3 as a regulator of Nav1.5 biophysical coupling. Inhibition of 14-3-3 can remove the Nav1.5 functional coupling and has been shown to inhibit the dominant-negative effect of Brugada syndrome mutations. However, it is unknown whether the coupling regulation is involved with AADs' modulation of Nav1.5. Indeed, AADs could reveal important structural and functional information about Nav1.5 coupling. Here, we investigated the modulation of Nav1.5 by four classic AADs, quinidine, lidocaine, mexiletine, and flecainide, in the presence of 14-3-3 inhibition. The experiments were carried out by high-throughput patch-clamp experiments in an HEK293 Nav1.5 stable cell line. We found that 14-3-3 inhibition can enhance acute block by quinidine, whereas the block by other drugs was not affected. We also saw changes in the use- and dose-dependency of quinidine, lidocaine, and mexiletine when inhibiting 14-3-3. Inhibiting 14-3-3 also shifted the channel activation toward hyperpolarized voltages in the presence of the four drugs studied and slowed the recovery of inactivation in the presence of quinidine. Our results demonstrated that the protein 14-3-3 and Nav1.5 coupling could impact the effects of AADs. Therefore, 14-3-3 and Nav1.5 coupling are new mechanisms to consider in the development of drugs targeting Nav1.5. SIGNIFICANCE STATEMENT: The cardiac sodium channel Nav1.5 is a target of commonly used antiarrhythmic drugs, and Nav1.5 function is regulated by the protein 14-3-3. The present study demonstrated that the regulation of Nav1.5 by 14-3-3 influences Nav1.5's response to antiarrhythmic drugs. This study provides detailed information about how 14-3-3 differentially regulated Nav1.5 functions under the influence of different drug subtypes. These findings will guide future molecular studies investigating Nav1.5 and antiarrhythmic drugs outcomes.

Comparative pharmacokinetic profile of cimicoxib in dogs and cats after IV administration

Cimicoxib is a selective COX-2 inhibitor (coxib) registered for the treatment of pain and inflammation in dogs. Pharmacokinetics of some coxibs have been described in dogs and cats. In cats, total body clearance values are lower and terminal half-lives of the coxibs are longer than those in dogs. The aim of this work was to evaluate if this is also the case for cimicoxib. For this purpose, blood pharmacokinetics and urinary excretion after IV administration were compared between these species. The in vitro metabolism of cimicoxib was also evaluated using canine and feline microsomes.In canine and feline microsomes, the formation rate of demethyl-cimicoxib, a phase 1 metabolite, was decreased in presence of quinidine, a specific human cytochrome P450 (CYP)2D6 inhibitor. IC50 values were 1.6 μM and 0.056 μM with canine and feline microsomes, respectively. As quinidine was about 30 times more potent in feline microsomes, the affinity of cimicoxib to the enzyme was considered to be about 30 times lower than that in canine microsomes. Total body clearance (ClB) of cimicoxib, was 0.50 L/h kg in dogs and 0.14 L/h kg in cats (P < 0.01) and terminal half-life, T½λz, was 1.92 and 5.25 h, respectively (P < 0.01). Dose eliminated in urine was 12.2% in dogs and 3.12% in cats (P < 0.01). Conjugated demethyl-cimicoxib represented 93.7% of this amount in dogs and 67.5% in cats. Thus cimicoxib, like other veterinary coxibs, was eliminated more slowly in cats. Both CYP2D15 (the canine ortholog of CYP2D6) and UDP-glucuronyltransferase enzyme systems have reduced ability to produce metabolites of cimicoxib in cats.

Entomopathogenic fungi Beauveria sp. and Aspergillus sclerotiorum can produce secondary metabolite quinidine

Entomopathogenic fungi have been reported to produce secondary metabolite quinolines. One of the quinoline alkaloids is quinidine, a compound that has antiarrhythmic properties, therefore it has an economic important value. The aim of the experiment reported here was to study the potency of entomopathogenic fungi Beauveria sp. IPBCC. 19.1499 and Aspergillus sclerotiorum IPBCC.19.1500 in producing quinidine. The quinidine was obtained from a broth culture of each fungus grown in Potato Dextrose Broth medium (pH 6.2, static, room temperature) for 7, 14, and 21 days incubation period. At harvest, broth of each culture was extracted using chloroform. The extract then dried using an evaporator. The presence of quinidine in the extracted was detected by HPLC. The dry weight of fungal biomass and the crude extract of broth culture were also recorded. HPLC analysis showed that quinidine was produced by both fungi tested. The species of the fungus and incubation period significantly affected quinidine production. Beauveria sp. IPBCC.19.1499, in general, produced quinidine in higher concentration than that of A. sclerotiorum IPBCC.19.1500. The quinidine concentration increased significantly with increasing incubation period. The quinidine concentration was also positively correlated with biomass dry weight, while the crude extract dry weight was negatively correlated with quinidine concentration.

Involvement of Renal Efflux Transporter MATE1 in Renal Excretion of Flecainide.

Flecainide, an anti-arrhythmic drug, undergoes renal excretion through active renal tubular secretion in addition to passive glomerular filtration. The contribution of renal uptake and efflux transporters in active renal tubular secretion of flecainide remains unclear except that flecainide is a substrate of human multidrug resistance protein 1 (MDR1). To elucidate renal efflux and uptake transporters involved with active renal tubular secretion of flecainide, we conducted in vitro interaction studies of flecainide using organic cation transporter 2 (OCT2), multidrug and toxin extrusion (MATE) 1, and MATE2-K. Uptake transporter inhibition assays using hOCT2-Chinese hamster ovary (CHO), hMATE1-CHO, and hMATE2-K-Madin Darby canine kidney strain II (MDCKII) cells revealed that flecainide (2.5 µM) inhibited hMATE1-mediated transport by 40% with an IC50 value of 6.7 µM; however, it showed no or weak inhibitory effects on hOCT2- and hMATE2-K-mediated transport. For investigating flecainide as a substrate of hMATE1, the accumulation of flecainide in hMATE1-CHO was compared with that in control cells. Uptake transporter substrate assay revealed that flecainide (1 µM) showed 1.11-fold accumulation though the hMATE1-related active transport was significantly decreased in the presence of quinidine (42.0 ± 23.9 vs. 11.8 ± 4.1 pmol/mg in transfected cells; p < 0.05). These results suggest that flecainide is a weak substrate of hMATE1, which is involved in the renal tubular secretion of cationic drugs, and hMATE1 may be less important in the pharmacokinetic drug-drug interaction for renal excretion of flecainide. However, in vivo drug-drug interaction studies of flecainide with substrates of hMATE1 may be needed because flecainide has the potential to inhibit hMATE1.

Deuterated (d6)-dextromethorphan elicits antidepressant-like effects in mice

The over-the-counter antitussive dextromethorphan (DM) may have rapid antidepressant actions based on its overlapping pharmacology with ketamine, which has shown fast antidepressant effects but whose widespread use remains limited by problematic side effects. We have previously shown that DM produces antidepressant-like effects in the forced swim test (FST) and tail suspension test (TST) that are mediated in part through α-amino-3-hydroxy-5-methyl-4-isoxazole propionic (AMPA) and sigma-1 receptors, two protein targets associated with a faster onset of antidepressant efficacy. To utilize DM clinically, however, a major challenge that must be addressed is its rapid first-pass metabolism. Two strategies to inhibit metabolism of DM and maintain stable therapeutic blood levels are 1) chemically modifying DM and 2) adding quinidine, an inhibitor of the primary metabolizer of DM, the cytochrome P450 (CYP) 2D6 enzyme. The purpose of this study was to determine if modified DM (deuterated (d6)-DM) elicits antidepressant-like effects and if AMPA and sigma-1 receptors are involved. Furthermore, d6-DM was tested in conjunction with quinidine to determine if further slowing the metabolism of d6-DM affects its antidepressant-like actions. In the FST and TST, d6-DM produced antidepressant-like effects. Upon further investigation in the FST, the most validated animal model for predicting antidepressant efficacy, d6-DM produced antidepressant-like effects both in the absence and presence of quinidine. However, pretreatment with neither an AMPA receptor antagonist (NBQX) nor sigma-1 receptor antagonists (BD1063, BD1047) significantly attenuated the antidepressant-like effects. The data suggest d6-DM has antidepressant-like effects, though it may be recruiting different molecular targets and/or acting through a different mix or ratio of metabolites from regular DM.

Development of a cyclosporin A derivative with excellent anti-hepatitis C virus potency

Synthetic modification of cyclosporin A at P3-P4 positions led to the discovery of NIM258, a next generation cyclophilin inhibitor with excellent anti-hepatitis C virus potency, with decreased transporter inhibition, and pharmacokinetics suitable for coadministration with other drugs. Herein is disclosed the evolution of the synthetic strategy to from the original medicinal chemistry route, designed for late diversification, to a convergent and robust development synthesis. The chiral centers in the P4 fragment were constructed by an asymmetric chelated Claisen rearrangement in the presence of quinidine as the chiral ligand. Identification of advanced crystalline intermediates enabled practical supply of key intermediates. Finally, macrocyclization was carried out at 10% weight concentration by a general and unconventional “slow release” concept.

PKC inhibition results in a Kv 1.5 + Kv β1.3 pharmacology closer to Kv 1.5 channels.

The Kv β1.3 subunit modifies the gating and pharmacology of Kv 1.5 channels in a PKC-dependent manner, decreasing channel sensitivity to bupivacaine- and quinidine-mediated blockade. Cardiac Kv 1.5 channels associate with receptor for activated C kinase 1 (RACK1), the Kv β1.3 subunit and different PKC isoforms, resulting in the formation of a functional channelosome. The aim of the present study was to investigate the effects of PKC inhibition on bupivacaine and quinidine block of Kv 1.5 + Kv β1.3 channels. HEK293 cells were transfected with Kv 1.5 + Kv β1.3 channels, and currents were recorded using the whole-cell configuration of the patch-clamp technique. PKC inhibition was achieved by incubating the cells with either calphostin C or bisindolylmaleimide II and the effects of bupivacaine and quinidine were analysed. The voltage-dependent inactivation of Kv 1.5 + Kv β1.3 channels and their pharmacological behaviour after PKC inhibition with calphostin C were similar to those displayed by Kv 1.5 channels alone. Indeed, the IC50 values for bupivacaine were similar in cells whose PKC was inhibited with calphostin C or bisindolylmaleimide II. Similar results were also observed in the presence of quinidine. The finding that the voltage-dependence of inactivation and the pharmacology of Kv 1.5 + Kv β1.3 channels after PKC inhibition resembled that observed in Kv 1.5 channels suggests that both processes are dependent on PKC-mediated phosphorylation. These results may have clinical relevance in diseases that are characterized by alterations in kinase activity.

Amino acid prodrug of quinidine: An approach to circumvent P-glycoprotein mediated cellular efflux

In the present study, we investigated the effect of large neutral amino acid modification in overcoming P-gp mediated cellular efflux of quinidine. L-isoleucine ester prodrug of quinidine (Ile-quinidine) was synthesized in our laboratory. [14C]-erythromycin was selected as a model substrate to study interaction of quinidine and Ile-quinidine with P-gp. Transport studies were conducted to study translocation kinetics of quinidine and Ile-quinidine in MDCK-MDR1 cells. In cellular accumulation study, uptake rate of [14C]-erythromycin elevated drastically in presence of cyclosporine A and GF 120918. This result indicates that [14C]-erythromycin is an excellent substrate of P-gp. Similarly, uptake rate of [14C]-erythromycin was enhanced significantly in presence of quinidine (25 and 50 μM). However, [14C]-erythromycin uptake rate remained fairly constant in presence of Ile-quinidine (25 μM). Apparent A-B and B-A permeability of quinidine observed in MDCK-MDR1 cells were 1.6 ± 0.2 × 10(-6) and 7.0 ± 0.4 × 10(-6)cm/s, a 4.4-fold difference. Moreover, A-B permeability of quinidine increased dramatically in the presence of cyclosporine A and GF 120918. Apparent permeability values of Ile-quinidine observed from A-B and B-A direction were 4.3 ± 0.9 × 10(-6) and 5.5 ± 0.4 × 10(-6)cm/s, a 1.3-fold difference. Importantly, A-B transport of Ile-quinidine did not change dramatically in the presence of cyclosporine and GF 120918. Based on these results, it was apparent that quinidine displayed higher substrate affinity toward P-gp relative to Ile-quinidine. Chemical or enzymatic hydrolysis of Ile-quinidine resulted in regeneration of low quantities of quinidine during transport studies. Competitive inhibition studies demonstrated that Ile-quinidine was recognized by multiple amino acid transporters such as LAT1, LAT2 and cationic amino acid transporter. In conclusion, chemical modification of quinidine with neutral amino acids results in circumvention of P-gp mediated drug efflux. Hence, amino acid transporter targeted prodrug delivery seems to be a viable strategy for improving drug accumulation in P-gp overexpressing cells.

Effects of physiological and biochemical factors based on aging, pathological conditions and drug combinations on propranolol pharmacokinetics using physiologically-based pharmacokinetic model in humans

Sensitivity of physiological and biochemical parameters based on aging, pathological conditions and drug combinations on propranolol pharmacokinetics was investigated to define factors contributing to larger inter-individual variability on propranolol pharmacokinetics after oral administration than that after intravenous (iv) administration. Using a physiologically-based pharmacokinetic model, plasma propranolol concentration was simulated with wide-ranging physiological and biochemical parameters. Then calculated AUC and half-life (t1/2) were compared with those obtained from the basic condition. It was found that the total blood flow through the body (Qtot), hepatic volume, unbound fraction in the blood and metabolic parameters affected propranolol AUC. Except for Qtot, effects were greater with after oral administration than iv administration. The propranolol AUC decreased by half with a two-fold increase in hepatic volume after oral administration possibly because of the increased first-pass metabolism. By contrast, some distribution parameters such as the skin tissue-to-blood distribution ratio and blood-to-plasma concentration ratio affected propranolol t1/2. Finally, propranolol plasma concentrations were simulated in the case that metabolizing activity decreased to one half and one fifth with an assumption of metabolic enzyme inhibition. The simulated propranolol curves correlated well with the previously reported plasma concentration in the absence and presence of quinidine, which is well known to be a potent and selective CYP2D6 inhibitor. Thus, plasma propranolol pharmacokinetics was affected by parameters which related to the hepatic metabolism and the distribution, especially after oral administration.

Variability of Bioavailability and Intestinal Absorption Characteristics of Bisoprolol

We previously reported that renal function is partly responsible for the interindividual variability of the pharmacokinetics of bisoprolol. The aim of the present study was to examine the variability of bioavailability (F) of bisoprolol in routinely treated Japanese patients and intestinal absorption characteristics of the drug. We first analyzed the plasma concentration data of bisoprolol in 52 Japanese patients using a nonlinear mixed effects model. We also investigated the cellular uptake of bisoprolol using human intestinal epithelial LS180 cells. The oral clearance (CL/F) of bisoprolol in Japanese patients was positively correlated with the apparent volume of distribution (V/F), implying variable F. The uptake of bisoprolol in LS180 cells was temperature-dependent and saturable, and was significantly decreased in the presence of quinidine and diphenhydramine. In addition, the cellular uptake of bisoprolol dissolved in an acidic buffer was markedly less than that dissolved in a neutral buffer. These findings suggest that the rate/extent of the intestinal absorption of bisoprolol is another cause of the interindividual variability of the pharmacokinetics, and that the uptake of bisoprolol in intestinal epithelial cells is highly pH-dependent and also variable.

Mitochondrial localization of P-glycoprotein and peptide transporters in corneal epithelial cells – Novel strategies for intracellular drug targeting

This study was designed to investigate functional localization of both efflux (P-glycoprotein, P-gp) and influx (peptide) transporters in the mitochondrial membrane of cultured rabbit primary corneal epithelial cells (rPCECs). Isolation and purification of mitochondria was performed by optimized cell fractionation method. Mitochondrial integrity was measured by JC-1 uptake experiment. The efflux activity of P-gp was assessed by performing in vitro uptake studies on isolated mitochondria with Rhodamine 123 (Rho-123) alone and in the presence of P-gp inhibitors (quinidine and cyclosporine A) using fluorimetry and flow cytometry analysis. Functional activity of peptide transporter was assessed by performing in vitro uptake studies of [3H] Gly-sar on isolated mitochondria in the presence or absence of peptide transporter substrate (Val–Val). Molecular characterization of P-gp and peptide transporter was assessed by western blot and confocal analysis. Enhanced JC-1 accumulation in the isolated fraction confirmed mitochondrial membrane integrity. Significantly higher uptake of Rho-123 on isolated mitochondria was observed in the presence of quinidine (75 and 100 μM) and cyclosporine A (10 μM). Significantly lower uptake of [3H] Gly-sar was observed in the presence of val–val due to competitive inhibition of peptide transporter on isolated mitochondria. Western blot and confocal analysis further confirmed the presence of P-gp and peptide transporter on the mitochondrial membrane of rPCECs. The present study demonstrates the functional and molecular characterization of P-gp and peptide transporters in the mitochondrial membranes of rPCECs. This knowledge of mitochondrial existence of P-gp and peptide transporter will aid in the development of subcellular ocular drug delivery strategies.

Multifocal Ectopic Purkinje-Related Premature Contractions: A New SCN5A-Related Cardiac Channelopathy

The aim of this study was to describe a new familial cardiac phenotype and to elucidate the electrophysiological mechanism responsible for the disease. Mutations in several genes encoding ion channels, especially SCN5A, have emerged as the basis for a variety of inherited cardiac arrhythmias. Three unrelated families comprising 21 individuals affected by multifocal ectopic Purkinje-related premature contractions (MEPPC) characterized by narrow junctional and rare sinus beats competing with numerous premature ventricular contractions with right and/or left bundle branch block patterns were identified. Dilated cardiomyopathy was identified in 6 patients, atrial arrhythmias were detected in 9 patients, and sudden death was reported in 5 individuals. Invasive electrophysiological studies demonstrated that premature ventricular complexes originated from the Purkinje tissue. Hydroquinidine treatment dramatically decreased the number of premature ventricular complexes. It normalized the contractile function in 2 patients. All the affected subjects carried the c.665G>A transition in the SCN5A gene. Patch-clamp studies of resulting p.Arg222Gln (R222Q) Nav1.5 revealed a net gain of function of the sodium channel, leading, in silico, to incomplete repolarization in Purkinje cells responsible for premature ventricular action potentials. In vitro and in silico studies recapitulated the normalization of the ventricular action potentials in the presence of quinidine. A new SCN5A-related cardiac syndrome, MEPPC, was identified. The SCN5A mutation leads to a gain of function of the sodium channel responsible for hyperexcitability of the fascicular-Purkinje system. The MEPPC syndrome is responsive to hydroquinidine.

Time-course activities of Oct1, Mrp3, and cytochrome P450s in cultures of cryopreserved rat hepatocytes

The organic cation transporter 1 (Oct1) has been shown to be one of the most abundant uptake transporters responsible for the uptake of xenobiotics from the sinusoidal blood across the basolateral membrane of hepatocytes. On the same membrane the multidrug resistance-associated protein 3 (Mrp3) mediates the efflux of xenobiotics or their metabolites from the hepatocytes to the blood allowing their systemic exposure. In the present study we investigated the expression and activity of Oct1 and Mrp3 in suspensions and in monolayer- and sandwich cultures, and activities of CYP2B1/2, 2D1, and 3A1 in monolayer- and sandwich cultures of cryopreserved rat hepatocytes. Oct1-mediated active uptake of 10 μM [ 3H]-1-methyl-4-phenylpyridinium (MPP+) into hepatocytes was assessed in the presence of quinidine (1 mM). The results showed the presence of active uptake of MPP+ in suspended hepatocytes (∼91 pmol/min/mg protein). In hepatocytes in cultures (monolayer and sandwich) a time-dependent decrease in MPP+ uptake was observed from day 0 to 4, from 80 to 90 pmol/min/mg protein at day 0 to ca. 17 pmol/min/mg protein at day 4. Mrp3 activity in suspensions and in monolayer- and sandwich cultures were investigated by measuring the efflux of [ 3H]-taurocholate from hepatocytes in the presence of the Mrp3 inhibitor taurolithocholate-3-sulfate (TLC-S) (500 μM). Cells in suspensions showed efflux of taurocholate by an active transport mechanism indicating Mrp3 activity. Experiments in monolayer- and sandwich cultures also showed Mrp3 activity at day 0 and 1 in culture whereas experiments performed at day 2–4 showed no difference in efflux of taurocholate in the presence or absence of TLC-S, suggesting an absence of Mrp3 activity. The time-dependent decrease in Oct1 activity from day 0 to day 4 in cultures was confirmed by qPCR data also showing a time-dependent decrease in mRNA expression, whereas qPCR data did not support the observed time-dependent decrease in Mrp3 activity in cultures. Time-course activities of CYP2B1/2, 2D1, and 3A1 were also investigated by using bupropion, bufuralol, and midazolam as respective substrates. Activities of CYP2D1 and 3A1 were reduced by ∼75% and ∼80%, respectively, from day 0 to day 4 in cultures, whereas activity of CYP2B1/2 was reduced by ∼50% from day 0 to day 4.

Enhancing effects of salicylate on quinidine-induced block of human wild type and LQT3 related mutant cardiac Na+ channels

It is unknown whether salicylate enhances the action of antiarrhythmic agents on human Na+ channels with state dependency and tissue specificity. We therefore investigated effects of salicylate on quinidine-induced block of human cardiac and skeletal muscle Na+ channels. Human cardiac wild-type (hH1), LQT3-related mutant (ΔKPQ), and skeletal muscle (hSkM1) Na+ channel α subunits were expressed in COS7 cells. Effects of salicylate on quinidine-induced tonic and use-dependent block of Na+ channel currents were examined by the whole-cell patch-clamp technique. Salicylate enhanced the quinidine-induced tonic and use-dependent block of both hH1 and hSkM1 currents at a holding potential (HP) of -100 mV but not at -140 mV. Salicylate decreased the IC50 value for the quinidine-induced tonic block of hH1 at an HP of -100 mV, and produced a negative shift in the steady-state inactivation curve of hH1 in the presence of quinidine. According to the modulated receptor theory, it is probable that salicylate decreases the dissociation constant for quinidine binding to inactivated-state channels. Furthermore, salicylate significantly enhanced the quinidine-induced tonic and use-dependent block of the peak and steady-state ΔKPQ channel currents. The results suggest that salicylate enhances quinidine-induced block of Na+ channels via increasing the affinity of quinidine to inactivated state channels.

The effect of acyclovir on the tubular secretion of creatinine in vitro

BackgroundWhile generally well tolerated, severe nephrotoxicity has been observed in some children receiving acyclovir. A pronounced elevation in plasma creatinine in the absence of other clinical manifestations of overt nephrotoxicity has been frequently documented. Several drugs have been shown to increase plasma creatinine by inhibiting its renal tubular secretion rather than by decreasing glomerular filtration rate (GFR). Creatinine and acyclovir may be transported by similar tubular transport mechanisms, thus, it is plausible that in some cases, the observed increase in plasma creatinine may be partially due to inhibition of tubular secretion of creatinine, and not solely due to decreased GFR. Our objective was to determine whether acyclovir inhibits the tubular secretion of creatinine.MethodsPorcine (LLC-PK1) and human (HK-2) renal proximal tubular cell monolayers cultured on microporous membrane filters were exposed to [2-14C] creatinine (5 μM) in the absence or presence of quinidine (1E+03 μM), cimetidine (1E+03 μM) or acyclovir (22 - 89 μM) in incubation medium.ResultsResults illustrated that in evident contrast to quinidine, acyclovir did not inhibit creatinine transport in LLC-PK1 and HK-2 cell monolayers.ConclusionsThe results suggest that acyclovir does not affect the renal tubular handling of creatinine, and hence, the pronounced, transient increase in plasma creatinine is due to decreased GFR, and not to a spurious increase in plasma creatinine.

Ca2+-Dependent and Na+-Dependent K+ Conductances Contribute to a Slow AHP in Thalamic Paraventricular Nucleus Neurons: A Novel Target for Orexin Receptors

Thalamic paraventricular nucleus (PVT) neurons exhibit a postburst apamin-resistant slow afterhyperpolarization (sAHP) that is unique to midline thalamus, displays activity dependence, and is abolished in tetrodotoxin. Analysis of the underlying sI(AHP) confirmed a requirement for Ca(2+) influx with contributions from P/Q-, N-, L-, and R subtype channels, a reversal potential near E(K)(+) and a significant reduction by UCL-2077, barium or TEA, consistent with a role for K(Ca) channels. sI(AHP) was significantly reduced by activation of either the cAMP or the protein kinase C (PKC) signaling pathway. Further analysis of the sAHP revealed an activity-dependent but Ca(2+)-independent component that was reduced in high [K(+)](o) and blockable after Na(+) substitution with Li(+) or in the presence of quinidine, suggesting a role for K(Na) channels. The Ca(2+)-independent sAHP component was selectively reduced by activation of the PKC signaling pathway. The sAHP contributed to spike frequency adaptation, which was sensitive to activation of either cAMP or PKC signaling pathways and, near the peak of membrane hyperpolarization, was sufficient to cause de-inactivation of low threshold T-Type Ca(2+) channels, thus promoting burst firing. PVT neurons are densely innervated by orexin-immunoreactive fibers, and depolarized by exogenously applied orexins. We now report that orexin A significantly reduced both Ca(2+)-dependent and -independent sI(AHP), and spike frequency adaptation. Furthermore orexin A-induced sI(AHP) inhibition was mediated through activation of PKC but not PKA. Collectively, these observations suggest that K(Ca) and K(Na) channels have a role in a sAHP that contributes to spike frequency adaptation and neuronal excitability in PVT neurons and that the sAHP is a novel target for modulation by the arousal- and feeding-promoting orexin neuropeptides.

Quinidine interaction with Shab K+ channels: pore block and irreversible collapse of the K+ conductance

Quinidine is a commonly used antiarrhythmic agent and a tool to study ion channels. Here it is reported that quinidine equilibrates within seconds across the Sf9 plasma membrane, blocking the open pore of Shab channels from the intracellular side of the membrane in a voltage-dependent manner with 1:1 stoichiometry. On binding to the channels, quinidine interacts with pore K(+) ions in a mutually destabilizing manner. As a result, when the channels are blocked by quinidine with the cell bathed in an external medium lacking K(+), the Shab conductance G(K) collapses irreversibly, despite the presence of a physiological [K(+)] in the intracellular solution. The quinidine-promoted collapse of Shab G(K) resembles the collapse of Shaker G(K) observed with 0 K(+) solutions on both sides of the membrane: thus the extent of G(K) drop depends on the number of activating pulses applied in the presence of quinidine, but is independent of the pulse duration. Taken together the observations indicate that, as in Shaker, the quinidine-promoted collapse of Shab G(K) occurs during deactivation of the channels, at the end of each activating pulse, with a probability of 0.1 per pulse at 80 mV. It appears that when Shab channels are open, the pore conformation able to conduct is stable in the absence of K(+), but on deactivation this conformation collapses irreversibly.

Effects of flecainide and quinidine on action potential and ventricular arrhythmogenic properties in Scn3b knockout mice

1. Flecainide and quinidine exert contrasting pro-arrhythmic and anti-arrhythmic effects in mouse hearts containing the loss-of-function, Scn5a(+/-), and the gain-of-function, Scn5a(+/DeltaKPQ), mutations in their sodium channel alpha-subunits. 2. The following properties were accordingly compared in wild-type and Scn3b(-/-) hearts modelling modifications in the beta-subunit, before and after introduction of either agent: (i) ventricular arrhythmogenecity and effective refractory periods (VERP) in response to programmed electrical stimulation (PES); (ii) monophasic action potential waveforms recorded from the left ventricular epicardium and endocardium; (iii) action potential durations (APD) obtained from the monophasic action potentials; and (iv) critical intervals derived from the APD and VERP values. 3. Ventricular tachycardia was induced by PES in 11 out of 15 Scn3b(-/-) hearts and 0 out of 17 wild-type hearts. This incidence was reduced to three out of eight Scn3b(-/-) hearts but increased to three out of eight wild-type hearts with flecainide. 4. Arrhythmogenic incidence was reduced to two out of eight Scn3b(-/-) hearts and remained at 0 out of eight wild-type hearts in the presence of quinidine. 5. Ventricular effective refractory periods were prolonged and endocardial and epicardial APD shortened, resulting in negative critical intervals in both Scn3b(-/-) and wild-type hearts treated by either flecainide or quinidine. Nevertheless, endocardial APD remained consistently longer than epicardial APD, leaving similar, positive endocardial-epicardial, differences, DeltaAPD, in treated and untreated Scn3b(-/-) and wild-type hearts. 6. It is concluded that both flecainide and quinidine exert anti-arrhythmogenic effects in Scn3b(-/-) hearts, doing so through modifying VERP rather than DeltaAPD, in contrast to their differing effects in Scn5a(+/-) and Scn5a(+/DeltaKPQ) hearts.

Hepatic Uptake and Excretion of (–)-N-{2-[(R)-3-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)piperidino]ethyl}-4-fluorobenzamide (YM758), a Novel If Channel Inhibitor, in Rats and Humans

(-)-N-{2-[(R)-3-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)piperidino]ethyl}-4-fluorobenzamide (YM758), a novel "funny" If current channel (If channel) inhibitor, is being developed as a treatment for stable angina and atrial fibrillation. The hepatic uptake/excretion of YM758 was clarified using transporter-expressing mammalian cells and hepatocytes mainly in humans and partly in rats. cDNA-expressing human embryonic kidney 293 cells were used to determine that YM758 was greatly taken up via organic anion-transporting polypeptide (OATP) 1B1 and slightly via human organic cation transporter (hOCT) 1/rat organic cation transporter 1 but not via OATP1B3. In addition, the uptake of 17beta-estradiol-d-17beta-glucuronide via OATP1B1 was inhibited in the presence of YM758, whereas that via OATP1B3 was not. In contrast, time-dependent uptake of YM758 into rat/human hepatocytes at 37 degrees C was observed, as was concentration-dependent uptake into human hepatocytes (K(m) value of 87.9 microM). This saturable uptake of YM758 into human hepatocytes was inhibited in the presence of quinidine (an inhibitor for OATP1B1) but not cimetidine (an inhibitor for the hOCT family). Moreover, the permeation clearance ratios for the transcellular transport of YM758 across multidrug resistance (MDR) 1-expressing LLC-PK1 cells were extensively higher than those across LLC-PK1 cells, which indicate that MDR1-mediated transport is one of the possible pathways through which YM758 may be excreted into the bile. These results indicate that YM758 is taken up into hepatocytes mainly via OATP1B1, but not via hOCT1, and is excreted into the bile via MDR1 in humans; however, passive diffusion or an unknown uptake/excretion mechanism could be at work in the hepatocytes. This study is the first to clarify the saturable hepatic uptake and/or the excretion mechanism by the If channel inhibitor.

Effects of flecainide and quinidine on arrhythmogenic properties of Scn5a+/Δ murine hearts modelling long QT syndrome 3

Long QT3 (LQT3) syndrome is associated with incomplete Na+ channel inactivation, abnormal repolarization kinetics and prolonged cardiac action potential duration (APD). Electrophysiological effects of flecainide and quinidine were compared in Langendorff-perfused wild-type (WT), and genetically modified (Scn5a+/Delta) murine hearts modelling LQT3. Extra stimuli (S2) following trains of pacing stimuli (S1) applied to the right ventricular epicardium triggered ventricular tachycardia (VT) in 16 out of 28 untreated Scn5a+/Delta and zero out of 12 WT hearts. Paced electrogram fractionation analysis then demonstrated increased electrogram durations (EGD), expressed as EGD ratios, in arrhythmogenic Scn5a+/Delta hearts, and prolonged ventricular effective refractory periods in initially non-arrhythmogenic Scn5a+/Delta hearts. Nevertheless, comparisons of epicardial and endocardial monophasic action potential recordings demonstrated negative transmural repolarization gradients in both groups, giving DeltaAPD(90) values at 90% repolarization of -20.88 +/- 1.93 ms (n = 11) and -16.91 +/- 1.43 ms (n = 23), respectively. Flecainide prevented initiation of VT in 13 out of 16 arrhythmogenic Scn5a+/Delta hearts, reducing EGD ratio and restoring DeltaAPD90 to + 7.55 +/- 2.24 ms (n = 9) (P < 0.05). VT occurred in four out of eight non-arrhythmogenic Scn5a+/Delta hearts in the presence of quinidine, which increased EGD ratio but left DeltaAPD90 unchanged. In contrast (P < 0.05), WT hearts had positive DeltaAPD90 values (+ 11.72 +/- 2.17 ms) (n = 20). Flecainide then increased arrhythmic tendency and EGD ratio but conserved DeltaAPD90; reduced EGD ratios and unaltered DeltaAPD90 values accompanied the lower arrhythmogenicity associated with quinidine treatment. In addition to the changes in EGD ratio shown by WT hearts, these findings attribute arrhythmogenesis and its modification by flecainide and quinidine to alterations in DeltaAPD90 in Scn5a+/Delta hearts. This is consistent with a hypothesis in which incomplete Na+ channel inactivation in Scn5a+/Delta hearts generates functional substrates dependent on altered refractoriness that cause abnormalities in activation and conduction of subsequent cardiac impulses. Any spatial heterogeneities between the epicardial and endocardial layers would thus cause fragmentation of the activation wavefront and contribute to electrogram spreading.