Photoinactivation of Leishmania donovani infantum in red cell suspensions by a flexible thiopyrylium sensitizer.

BACKGROUND AND OBJECTIVES: Leishmaniasis is a disease caused by protozoan parasites of the genus Leishmania, which are intracellular parasites of monocytes and macrophages. Transmission of the organism has been observed by transfusion of infected blood from asymptomatic donors to immunocompromised recipients, leading to clinically apparent disease. There is no licensed Leishmania screening test currently available. MATERIALS AND METHODS: This study investigated the potential for a novel DNA-intercalating photosensitizer, thiopyrylium (TP), to inactivate Leishmania donovani infantum in red cell (RBC) suspensions. RESULTS: A 5.7 TCID50 reduction of Leishmania was observed in samples treated with 12.5 micromole l(-1) TP and 1.1 J cm(-2) light. CONCLUSIONS: Leishmania is highly sensitive to photoinactivation under conditions that have been previously demonstrated to maintain RBC properties during 42 days of storage.

Pathogen inactivation of Leishmania donovani infantum in plasma and platelet concentrates using riboflavin and ultraviolet light.

BACKGROUND AND OBJECTIVES: Leishmania is transmitted by the bite of the phlebotomine sandfly or by transfusion of infected blood products. Leishmaniasis currently poses a significant problem in several parts of the world, and is an emerging problem in others. The Mirasol PRT technology is based on the use of riboflavin and ultraviolet light to generate chemical reactions in the nucleic acids of pathogens, which prevents replication and leads to inactivation. The intent of this study was to examine the ability of the Mirasol PRT System to kill the Leishmania parasite in human plasma and platelet concentrates. MATERIALS AND METHODS: In visceral Leishmaniasis, amastigotes are present in the blood and in the reticuloendothelial system within monocytes. For each unit of plasma or platelets treated, isolated mononuclear cells obtained from 100 ml of normal donor whole blood were incubated with 1.0 x 10(8) Leishmania donovani infantum promastigotes to produce amastigote-laden macrophages. The infected macrophages were added to 250 ml of human plasma or to 250 ml of platelet concentrates. Infected units were cultured pretreatment in 10-fold serial dilutions to determine the limits of detection. Thirty millilitres of 500 microM riboflavin was added to each unit, which was then illuminated with 5.9 J/cm2 of ultraviolet light (6.24 J/ml). After treatment and after 2 months of frozen storage, plasma units were cultured in 10-fold serial dilutions. Platelets were cultured on the day of treatment and on day 5 of storage post-illumination. RESULTS: A 5 log reduction of Leishmania was demonstrated in five of six units of plasma, and a 7 log reduction of Leishmania was demonstrated in one plasma unit. A 5 log reduction of Leishmania was demonstrated in five of six units of platelets, and a 6 log reduction of Leishmania was demonstrated in one unit. CONCLUSIONS: There is no donor screen for Leishmania and other pathogens constantly emerging in our blood supply. The Mirasol PRT System for Platelets and Plasma is an effective means of killing Leishmania and other emerging pathogens in these blood products.

White particulate matter found in blood collection bags consist of platelets and leukocytes.

BACKGROUND: In late January 2003, some blood centers and hospitals throughout the US voluntarily sus-pended the use of some RBC and plasma units for trans-fusion due to the presence of unknown white particulate matter (WPM) in these units. To better understand the WPM phenomena, a number of technologies were used to establish the nature of the particulates observed in Terumo Collection sets. STUDY DESIGN AND METHODS: All AS-5 nonleuko-reduced RBCs and plasma units were visually inspected for WPM by placing the bags on a flat counter, undisturbed, for approximately 10 minutes and then perform-ing a visual examination for particles. Particles were isolated and placed on microscope slides or in plastic tubes for further analysis. Electron microscopy, bright field microscopy, differential interference contrast microscopy, infrared spectroscopy, and flow cytometry procedures were performed to establish the nature of the particulate matter. In addition, leukoreduction filters and blood transfusion sets were used on RBCs units with WPM. RESULTS: The particles were mostly composed of PLTs and WBCs, and fragments of these cells. All macroscopic WPM was removed from RBCs with leukoeduction and transfusion filters. CONCLUSIONS: WPM originated from PLTs and WBCs. Foreign matter (e.g., plastic) was not observed in any of the units. Leukoreduction and transfusion filters can be used to remove macroscopic WPM.

Functional expression of L- and T-type Ca2+ channels in murine HL-1 cells.

In the search for a readily available source of native cardiac cells, we investigated the molecular and pharmacological properties of the immortalized cardiac atrial myocyte cell line, HL-1 cells. This work focused on the expression pattern of voltage-gated Ca2+ channels (VGCC). Reverse transcription-polymerase chain reaction analysis revealed that HL-1 cells have mRNA for several types of Ca2+ channels including the L-types, alpha1C and alpha1D, as well as T-types, alpha1H and alpha1G, but are lacking N-type, alpha1B and the T-type, alpha1I. Western blot analysis demonstrated significant alpha1C protein subunit expression, with less alpha1D subunit apparent, while alpha1A, alpha1B and alpha1E subunit expression was undetectable. Immunocytochemical staining showed that the alpha1C protein subunit is expressed predominantly on the cell surface, whereas the alpha1D protein is expressed mostly intracellularly. Whole-cell patch-clamp measurements demonstrated the presence of low (ICa,T) and high (ICa,L) voltage-activated Ca2+ currents, with preferential sensitivity to mibefradil and nimodipine, respectively. Addition of increasing external Ca2+ concentrations, [Ca2+]o, resulted in Ca2+ influx measured by fluorometric imaging with an EC50 of 0.8 mM [Ca2+]o. At a fixed [Ca2+]o of 0.125 mM, Ca2+ influx was also triggered by increasing the extracellular K+ concentration, [K+]o, with an EC50 of 3.7 mM [K+]o. As increasing [K+]o depolarizes the cell, this latter result is consistent with Ca2+ influx through a voltage-dependent mechanism. L-type (nimodipine and verapamil) and T-type (mibefradil and pimozide) Ca2+ channel blockers inhibited Ca2+ influx with IC50s of 1, 2, 0.4 and 0.2 microM, respectively. Antagonists of N-type (omega-conotoxins GVIA) and P/Q-type (MVIIC or omega-agatoxin IVA) did not inhibit Ca2+ influx, consistent with the lack of expression of N-, P-, or Q-type channels observed in the molecular studies. Taken together, these findings indicate that HL-1 cells express L- and T-subtypes of VGCC and are a unique in vitro model system for the study of native, mammalian cardiac Ca2+ channels.

Left ventricular hypertrophy decreases slowly but not rapidly activating delayed rectifier potassium currents of epicardial and endocardial myocytes in rabbits.

BACKGROUND: Delayed rectifier K(+) currents are critical to action potential (AP) repolarization. The present study examines the effects of left ventricular hypertrophy (LVH) on delayed rectifier K(+) currents and their contribution to AP repolarization in both epicardial (Epi) and endocardial (Endo) myocytes. METHODS AND RESULTS: VH was induced in rabbits by a 1-kidney removal, 1-kidney vascular clamping method. Slowly (I(Ks)) and rapidly (I(Kr)) activating delayed rectifier K(+) currents were recorded by the whole-cell patch-clamp technique, and APs were recorded by the microelectrode technique. In normal rabbit left ventricular myocytes, I(Ks) densities were larger in Epi than in Endo (1.1+/-0.1 versus 0.43+/-0.07 pA/pF), whereas I(Kr) density was similar between Epi and Endo (0.31+/-0.05 versus 0.36+/-0.07 pA/pF) at 20 mV. LVH reduced I(Ks) density to a similar extent (approximately 40%) in both Epi and Endo but had no significant effect on I(Kr) in either Epi or Endo. Consequently, I(Kr) was expected to contribute more to AP repolarization in LVH than in control. This was confirmed by specific I(Kr) block with dofetilide, which prolonged AP significantly more in LVH than in control (31+/-3% versus 18+/-2% in Epi; 53+/-6% versus 32+/-4% in Endo at 2 Hz). In contrast, L-768,673 (a specific I(Ks) blocker) prolonged AP less in LVH than in control. The very small I(Ks) density in Endo with LVH is consistent with the greater incidence of early afterdepolarizations induced in this region by dofetilide. CONCLUSIONS: LVH induces a decrease in I(Ks) density and increases the propensity to develop early afterdepolarizations, especially in Endo.

Synthesis and class III type antiarrhythmic activity of 4-aroyl (and aryl)-l-aralkylpiperazines.

The synthesis and in vitro Class III antiarrhythmic activity of several 4-aroyl (and aryl)-1-aralkylpiperazine and piperidine derivatives are described. Among several potent compounds identified in the series, RWJ-28810 (3), with its EC20 of 3 nM, ranks as one of the most potent (in vitro) compounds reported.

Modification of cardiac Na(+) current by RWJ 24517 and its enantiomers in guinea pig ventricular myocytes.

We examined the effects of the cardiotonic agent RWJ 24517 (Carsatrin, racemate) and its (S)- and (R)-enantiomers on action potential duration, Na(+) current (I(Na)), and delayed rectifier K(+) current (I(K)) of guinea pig ventricular myocytes. RWJ 24517 (0. 1 and 1 microM) prolongation of action potential duration could not be accounted for by suppression of either the rapid (I(Kr)) or slow (I(Ks),) component of I(K), although RWJ 24517 did reduce I(Kr) at concentrations of 1 microM. A more dramatic effect of RWJ 24517 (0.1-1 microM) and the (S)-enantiomer of RWJ 24517 (0.1-3 microM) was an increase in peak I(Na) and slowing of the rate of I(Na) decay, eliciting a large steady-state current. Neither RWJ 24517 nor the (S)-enantiomer affected the fast time constant for I(Na) decay, but both significantly increased the slow time constant, in addition to increasing the proportion of I(Na) decaying at the slow rate. Both agents elicited a use-dependent decrease of peak I(Na) (3-10 microM), which probably resulted from a slowing of both fast and slow rates of recovery from inactivation. In contrast, the (R)-enantiomer of RWJ 24517 did not induce a steady-state component I(Na) or increase peak I(Na) up to 10 microM, but it decreased peak I(Na) at 30 microM. The (R)-enantiomer displayed little use-dependent reduction of I(Na) during trains of repetitive pulses and had no effect on rates of inactivation or recovery from inactivation. These actions of the racemate and the (S)-stereoisomer to slow inactivation and to prolong both Na(+) influx and action potential duration may contribute to the positive inotropic actions of these agents because the resulting accumulation of intracellular Na(+) would increase intracellular Ca(2+) via Na(+)/Ca(2+) exchange.

A novel benzodiazepine that activates cardiac slow delayed rectifier K+ currents.

The slowly activating delayed rectifier K+ current, IKs, is an important modulator of cardiac action potential repolarization. Here, we describe a novel benzodiazepine, [L-364,373 [(3-R)-1, 3-dihydro-5-(2-fluorophenyl)-3-(1H-indol-3-ylmethyl)-1-methyl-2H- 1,4-benzodiazepin-2-one] (R-L3), that activates IKs and shortens action potentials in guinea pig cardiac myocytes. These effects were additive to isoproterenol, indicating that channel activation by R-L3 was independent of beta-adrenergic receptor stimulation. The increase of IKs by R-L3 was stereospecific; the S-enantiomer, S-L3, blocked IKs at all concentrations examined. The increase in IKs by R-L3 was greatest at voltages near the threshold for normal channel activation, caused by a shift in the voltage dependence of IKs activation. R-L3 slowed the rate of IKs deactivation and shifted the half-point of the isochronal (7.5 sec) activation curve for IKs by -16 mV at 0.1 microM and -24 mV at 1 microM. R-L3 had similar effects on cloned KvLQT1 channels expressed in Xenopus laevis oocytes but did not affect channels formed by coassembly of KvLQT1 and hminK subunits. These findings indicate that the association of minK with KvLQT1 interferes with the binding of R-L3 or prevents its action once bound to KvLQT1 subunits.

Blockade of HERG channels by the class III antiarrhythmic azimilide: mode of action.

1. The class III antiarrhythmic azimilide has previously been shown to inhibit I(Ks) and I(Kr) in guinea-pig cardiac myocytes and I(Ks) (minK) channels expressed in Xenopus oocytes. Because HERG channels underly the conductance I(Kr), in human heart, the effects of azimilide on HERG channels expressed in Xenopus oocytes were the focus of the present study. 2. In contrast to other well characterized HERG channel blockers, azimilide blockade was reverse use-dependent, i.e., the relative block and apparent affinity of azimilide decreased with an increase in channel activation frequency. Azimilide blocked HERG channels at 0.1 and 1 Hz with IC50s of 1.4 microM and 5.2 microM respectively. 3. In an envelope of tail test, HERG channel blockade increased with increasing channel activation, indicating binding of azimilide to open channels. 4. Azimilide blockade of HERG channels expressed in Xenopus oocytes and I(Kr) in mouse AT-1 cells was decreased under conditions of high [K+]e, whereas block of slowly activating I(Ks) channels was not affected by changes in [K+]e. 5. In summary, azimilide is a blocker of cardiac delayed rectifier channels, I(Ks) and HERG. Because of the distinct effects of stimulation frequency and [K+]e on azimilide block of I(Kr) and I(Ks) channels, we conclude that the relative contribution of block of each of these cardiac delayed rectifier channels depends on heart frequency. [K+]e and regulatory status of the respective channels.

Distortion-product otoacoustic emissions hearing screening in high-risk newborns.

Universal infant hearing screening has recently been recommended by the National Institutes of Health. Otoacoustic emissions have been proposed as the first-level screening technique. Although transient evoked otoacoustic emissions have shown limited applications, distortion-product otoacoustic emissions hold promise as a screening technique but have not been fully investigated. The purpose of this study was to determine the validity of distortion-product otoacoustic emissions as a hearing screening technique. A total of 208 ears of 104 infants at risk for hearing loss were tested with both automated auditory brain stem response and distortion-product otoacoustic emission screening protocols. Acoustic brain stem response results were used as the standard for normal hearing. Distortion-product otoacoustic emission results were analyzed by means of calculation of the difference between the mean of the response levels and the mean of the noise floor levels from five frequency pairs between 2000 and 4000 Hz. Pass-fail rates for response above noise floor criteria of 5, 10, and 15 dB were examined. The sensitivity of distortion-product otoacoustic emissions was 50%, 67%, and 87%, and the specificity was 94%, 68%, and 38% at the 5, 10, and 15 dB levels, respectively. The pass-fail criterion of distortion-product otoacoustic emissions should be based on instrumentation calibration, infant status, and an acceptable false-positive, false-negative yield. The ability to test rapidly the hearing of all infants with distortion-product otoacoustic emissions points to the feasibility of using this test as a first-stage screen.

Mechanism of action potential prolongation by RP 58866 and its active enantiomer, terikalant. Block of the rapidly activating delayed rectifier K+ current, IKr.

BACKGROUND: The class III antiarrhythmic agent RP 58866 and its active enantiomer, terikalant, are reported to selectively block the inward rectifier K+ current, IK1. These drugs have demonstrated efficacy in animal models of cardiac arrhythmias, suggesting that block of IK1 may be a useful antiarrhythmic mechanism. The symmetrical action potential (AP)-prolonging and bradycardic effects of these drugs, however, are inconsistent with a sole effect on IK1. METHODS AND RESULTS: We studied the effects of RP 58866 and terikalant on AP and outward K+ currents in guinea pig ventricular myocytes. RP 58866 and terikalant potently blocked the rapidly activating delayed rectifier K+ current, IKr, with IC50S of 22 and 31 nmol/L, respectively. Block of IK1 was approximately 250-fold less potent; IC50S were 8 and 6 mumol/L, respectively. No significant block of the slowly activating delayed rectifier, IK1, was observed at < or = 10 mumol/L. The phenotypical IKr currents in mouse AT-1 cells and Xenopus oocytes expressing HERG were also blocked 50% by 200 to 250 nmol/L RP 58866 or terikalant, providing further conclusive evidence for potent block of IKr. RP 58866 < or = 1 mumol/L and dofetilide increased AP duration symmetrically, consistent with selective block of IKr. Only higher concentrations (> or = 10 mumol/L) of RP 58866 slowed the rate of AP repolarization and decreased resting membrane potential, consistent with an additional but substantially less potent block of IK1. CONCLUSIONS: These data demonstrate that RP 58866 and terikalant are potent blockers of IKr and prompt a reinterpretation of previous studies that assumed specific block of IK1 by these drugs.

IK of rabbit ventricle is composed of two currents: evidence for IKs.

The delayed rectifier K+ current (IK) in rabbit heart has long been thought to consist of only a single, rapidly activating, dofetilide-sensitive current, IKr. However, we find that IK of rabbit ventricular myocytes actually consists of both rapid and slow components, IKr and IKs, respectively, that can be isolated pharmacologically. Thus, after complete blockade of IKr with dofetilide, the remaining current, IKs, is homogeneous as judged by an envelope of tails test. IKs activates and deactivates slowly, continues to activate during sustained depolarizations, has a half-activation potential of 7.0 +/- 0.8 mV and slope factor of 11.0 +/- 0.7 mV, reverses at -77.2 +/- 1.3 mV (extracellular K+ concentration = 4 mM), is increased by removing extracellular K+, and is enhanced by isoproterenol and stocked by azimilide. Northern analysis demonstrates that the minK (IsK) gene, which encodes a subunit of the channel that underlies the IKs current, is expressed in rabbit heart. Expression of the rabbit protein in Xenopus oocytes elicits a slowly activating, voltage-dependent current, IsK, similar to those expressed previously from mouse, rat, guinea pig, and human genes. The results demonstrate that IKs is present in rabbit ventricle and therefore contributes to cardiac repolarization in this species.

Water precautions in children with tympanostomy tubes.

OBJECTIVE: To compare the effectiveness of antibiotic ear drops (suspension of polymyxin B sulfate, neomycin sulfate, and hydrocortisone [Pediotic]), prefabricated ear molds, or no precautions in decreasing the incidence of posttympanostomy water-related otorrhea. DESIGN: Five-year prospective controlled study. SETTINGS: University referral center. PATIENTS: Five hundred thirty-three pediatric patients who were undergoing tympanostomy tube placement (including those who were undergoing tonsillectomy, adenoidectomy, or both) were self-selected into four groups. INTERVENTIONS: The use of antibiotic ear drops that contained polymyxin B, neomycin, and hydrocortisone and the use of prefabricated ear molds. Group 1 comprised patients who were not given any water precautions with swimming regardless of the depth or type of water; group 2 comprised patients in whom antibiotic ear drops were applied after all forms of swimming; group 3 comprised patients who used ear molds with all forms of swimming (all children were advised against diving and swimming more than 180 cm below the surface, and parents were cautioned to avoid the entrance of soapy water into their child's ears during bathing); and group 4 comprised patients who were selected not to swim at all (they served as a control group). RESULTS: No statistically significant difference was observed in the incidence of posttympanostomy swimming-related otorrhea among the three swimming groups (11%, 14%, and 20% of children in groups 1, 2, and 3, respectively, reported swimming-related otorrhea [P=.26, df-2, chi-square=2.66]). Children who did not swim at all (group 4) did not differ significantly in their overall incidence of otorrhea (59%) from the three swimming groups combined (68%) during the follow-up period (P=.11, df=1, chi-square=2.54). CONCLUSION: Young children with tympanostomy tubes who surface swim and do not dive receive no additional benefit from the taking of water precautions in the form of earplugs or antibiotic ear drops.

Use-dependent effects of the class III antiarrhythmic agent NE-10064 (azimilide) on cardiac repolarization: block of delayed rectifier potassium and L-type calcium currents.

We studied the effects of NE-10064 (azimilide), a new antiarrhythmic agent reported to be a selective blocker of the slowly activating component of the delayed rectifier, IKs. In ferret papillary muscles, NE-10064 increased effective refractory period (ERP) and decreased isometric twitch tension in a concentration-dependent manner (0.3-30 microM). Increases in ERP showed reverse use-dependence, and were greater at 1 than at 3 Hz. In contrast, changes in tension were use dependent, with larger decreases observed at 3 than at 1 Hz. In guinea pig ventricular myocytes, NE-10064 (0.3-3 microM) significantly prolonged action potential duration (APD) at 1 Hz. At 3 Hz, NE-10064 (0.3-1 microM) increased APD only slightly, and at 10 microM decreased APD and the plateau potential. NE-10064 potently blocked the rapidly activating component of the delayed rectifier, IKr (IC50 0.4 microM), and inhibited IKs (IC50 3 microM) with nearly 10-fold less potency. NE-10064 (10 microM) did not block the inward rectifier potassium current (IKl). NE-10064 (10 microM) blocked the L-type calcium current (ICa) in a use-dependent manner; block was greater at 3 than at 1 Hz. We conclude that (a) NE-10064's block of potassium currents is relatively selective for IKr over IKs, (b) NE-10064 inhibits ICa in a use-dependent fashion, and (c) NE-10064's effects on ERP and tension in papillary muscle as well as APD and action potential plateau level in myocytes may be explained by its potassium and calcium channel blocking properties.

Comparison of binding to rapidly activating delayed rectifier K+ channel, IKr, and effects on myocardial refractoriness for class III antiarrhythmic agents.

Saturation binding studies in guinea pig ventricular myocytes with 3H-dofetilide, a radioligand for the cardiac rapidly activating delayed rectifier K+ IKr channel, indicated specific high-affinity binding with a Kd of 83 nM and a Bmax of 0.18 pmol/mg cellular protein (1.36 x 10(6) sites/cell). Using displacement of high-affinity 3H-dofetilide binding as a measure of interaction with the IKr channel, potencies (Ki values) for binding to the IKr channel in guinea pig myocytes for six class III antiarrhythmic agents were characterized and compared to indices of functional electrophysiologic activity in isolated guinea pig papillary muscles [EC25 values, concentration required to increase effective refractory period (ERP) 25% above baseline]. Dofetilide, E-4031, sematilide, and d-sotalol, which have been characterized previously as selective IKr blockers, displayed good agreement between Ki values for displacement of 3H-dofetilide binding (47 +/- 7 nM, 38 +/- 8 nM, 12 +/- 5 microM, and approximately 100 microM, respectively) and EC25 values for increasing ERP in papillary muscles (45.0 nM, 76.9 nM, 20.2 microM and 63.5 microM, respectively). Ibutilide and RP58866, which have been reported to act via mechanisms other than IKr block, had Ki values for displacement of 3H-dofetilide binding (16 +/- 7 nM and 17 +/- 2 nM, respectively) that were approximately 10-fold lower than EC25 values for increasing ERP in papillary muscles (185.8 nM and 223.5 nM, respectively). The potent displacement of high-affinity 3H-dofetilide binding by ibutilide and RP58866 strongly suggest a role for interaction with IKr in their actions.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiac electrophysiological actions of the histamine H1-receptor antagonists astemizole and terfenadine compared with chlorpheniramine and pyrilamine.

We compared the cardiac electrophysiological actions of two types of H1-receptor antagonists--the piperidines, astemizole and terfenadine, and the nonpiperidines, chlorpheniramine and pyrilamine-in vitro in guinea pig ventricular myocytes and in vivo in chloralose-anesthetized dogs. Astemizole and terfenadine significantly increased action potential duration of guinea pig myocytes. This concentration-dependent prolongation of action potential duration was reverse frequency dependent and led to development of early afterdepolarizations, which occurred more frequently at higher concentrations and slower pacing frequencies. Astemizole and terfenadine potently blocked the rapidly activating component of the delayed rectifier, IKr, with IC50 values of 1.5 and 50 nmol/L, respectively. At 10 mumol/L, terfenadine but not astemizole blocked the slowly activating component of the delayed rectifier, IKs (58.4 +/- 3.1%), and the inward rectifier, IK1 (20.5 +/- 3.4%). Chlorpheniramine and pyrilamine blocked IKr relatively weakly (IC50 = 1.6 and 1.1 mumol/L, respectively) and IKs and IK1 less than 20% at 10 mumol/L. Astemizole and terfenadine (1.0 to 3.0 mg/kg IV) significantly prolonged the QTc interval and ventricular effective refractory period in vivo. Chlorpheniramine and pyrilamine (< or = 3.0 mg/kg) did not significantly affect these parameters. Block of repolarizing K+ currents, particularly IK1, by astemizole and terfenadine produces reverse rate-dependent prolongation of action potential duration and development of early afterdepolarizations, delays ventricular repolarization, and may underlie the development of torsade de pointes ventricular arrhythmias observed with the use and abuse of these agents.

Cardiac electrophysiologic and antiarrhythmic actions of two long-acting spirobenzopyran piperidine class III agents, L-702,958 and L-706,000 [MK-499].

The cardiac electrophysiologic and antiarrhythmic actions of two Class III ketone- and alcohol-containing spirobenzopyran piperidine analogs, L-702,958 and L-706,000 [MK-499], respectively, were assessed in vitro and in vivo. L-702,958 and L-706,000 [MK-499] selectively blocked the rapidly activating component of the delayed rectifier K+ current in guinea pig isolated ventricular myocytes (IC50 values, 14.6 and 43.9 nM, respectively), and prolonged effective refractory period in ferret isolated papillary muscles (EC25 values, 10.5 and 53.8 nM, respectively). In anesthetized dogs, L-702,958 and L-706,000 [MK-499] increased ventricular refractory periods (ED20 values, 3.3 and 9.2 micrograms/kg i.v., respectively) and concomitantly increased ECG QT interval and left ventricular+dP/dt. Cumulative i.v. administrations of up to 100 micrograms/kg of L-702,958 and 300 micrograms/kg L-706,000 [MK-499] in anesthetized dogs increased atrial and ventricular refractoriness and prolonged the ECG QT interval, but did not alter atrial, atrioventricular nodal, His-Purkinje or ventricular conduction indices. In anesthetized dogs studied chronically (9.2 +/- 1.1 days) after anterior myocardial infarction, the cumulative i.v. administrations of 100 micrograms/kg of L-702,958 and 300 of micrograms/kg L-706,000 [MK-499] suppressed the induction of ventricular tachyarrhythmia by programmed ventricular stimulation (suppression rates: 8 of 10, 80% and 9 of 11, 82%, respectively) and reduced the incidence of lethal ventricular arrhythmias (incidence of lethal ischemic arrhythmias: 4 of 10, 40% and 1 of 11 9%, respectively, compared to 34 of 40, 85%, in vehicle controls. L-702,958 and L-706,000 [MK-499] (cumulative 100 and 300 micrograms/kg i.v., respectively) did not facilitate the induction of arrhythmias by programmed ventricular stimulation in postinfarction dogs. After equivalently effective p.o. doses in conscious dogs, L-702,958 (10 micrograms/kg) and L-706,000 [MK-499] (30 micrograms/kg) increased ECG QT interval with long durations of action of approximately 9 and 14 hr, respectively. L-706,000 [MK-499] elicited a more consistent and sustained prolongation of the QT interval than L-702,958. These findings show that both L-702,958 and L-706,000 [MK-499] are potentially useful agents for the prevention of malignant ventricular arrhythmias in the setting of myocardial ischemic injury.

K+ currents expressed from the guinea pig cardiac IsK protein are enhanced by activators of protein kinase C.

We have isolated cardiac cDNA and genomic clones encoding the guinea pig IsK protein. The deduced amino acid sequence is approximately 78% identical to the rat, mouse, and human variants of this channel, and the structure of the gene encoding the protein is also similar to that in other species. For example, the gene is present only once in the haploid genome, the protein-coding sequence is present on a single uninterrupted exon, an intron exists in the 5' untranslated domain, and multiple alternative polyadenylation sites are used in processing the transcript. Expression of the guinea pig protein in Xenopus oocytes results in a slowly activating, voltage-dependent K+ current, IsK, similar to those expressed previously from the rat, mouse, and human genes. However, in sharp contrast to the rat and mouse currents, activation of protein kinase C with phorbol esters increases the amplitude of the guinea pig IsK current, analogous to its effects on the endogenous IKs current in guinea pig cardiac myocytes. Mutagenesis of the guinea pig cDNA to alter four cytoplasmic amino acid residues alters the phenotype of the current response to protein kinase C from enhancement to inhibition, mimicking that of rat and mouse IsK currents. This mutation is consistent with reports that phosphorylation of Ser-102 by protein kinase C decreases the current amplitude. These data explain previously reported differences in the regulatory properties between recombinant rat or mouse IsK channels and native guinea pig IKs channels and provide further evidence that the IsK protein forms the channels that underlie the IKs current in the heart.

Species variants of the IsK protein: differences in kinetics, voltage dependence, and La3+ block of the currents expressed in Xenopus oocytes.

We have compared the slowly activating K+ currents (IsK) resulting from the expression of the human, mouse, or rat IsK proteins in Xenopus oocytes, utilizing natural, species-dependent sequence variations to initiate structure-function studies of this channel. Differences were found between the human and rodent currents in their voltage dependence, kinetics, and sensitivity to external La3+. The current/voltage relationships of the human and rat IsK currents differed significantly, with greater depolarizations required for activation of the human channel. The first 30 s of activation during depolarizations to potentials between -10 and +40 mV was best described by a triexponential function for each of the three species variants. The activation rates were, however, significantly faster for the human current than for either of the rodent forms. Similarly, deactivation kinetics were best described as a biexponential decay for each of the species variants but the human currents deactivated more rapidly than the rodent currents. The human and the rodent forms of IsK were also differentially affected by external La3+. Low concentrations (10, 50 microM) rapidly and reversibly reduced the magnitude of the mouse and rat currents during a test depolarization and increased the deactivation rates of the tail currents. In contrast, the magnitude and deactivation rates of the human IsK currents were unaffected by 50 microM La3+.