Effects of the atrial antiarrhythmic drug AVE0118 on cardiac ion channels.

Previous studies in pigs and goats have demonstrated that AVE0118 prolongs atrial refractoriness without any effect on the QT-interval. The purpose of the present study was to investigate the effect of the compound on various cardiac ion channels. AVE0118 blocked the pig Kv1.5 and the human Kv1.5 expressed in Xenopus oocytes with IC(50) values of 5.4+/-0.7 microM and 6.2+/-0.4 microM respectively. In Chinese hamster ovary (CHO) cells, AVE0118 decreased the steady-state hKv1.5 current with an IC(50) of 1.1+/-0.2 microM. The hKv4.3/KChIP2.2 current in CHO cells was blocked by AVE0118 by accelerating the apparent time-constant of inactivation ( tau(inact)), and the integral current was inhibited with an IC(50) of 3.4+/-0.5 microM. At 10 microM AVE0118 tau(inact) decreased from 9.3+/-0.6 ms ( n=8, control) to 3.0+/-0.3 ms ( n=8). The K(ACh) current was investigated in isolated pig atrial myocytes by application of 10 microM carbachol. At a clamp potential of -100 mV the I(KACh) was half-maximally blocked by 4.5+/-1.6 microM AVE0118. In the absence of carbachol, AVE0118 had no effect on the inward current recorded at -100 mV. Effects on the I(Kr) current were investigated on HERG channels expressed in CHO cells. AVE0118 blocked this current half-maximally at approximately 10 microM. Comparable results were obtained in isolated guinea pig ventricular myocytes, where half-maximal inhibition of the I(Kr) tail current occurred at a similar concentration of AVE0118. Other ionic currents, like the I(Ks), I(KATP) (recorded in guinea pig ventricular myocytes), and L-type Ca(2+) (recorded in pig atrial myocytes) were blocked by 10 microM AVE0118 by 10+/-3% ( n=6), 28+/-7% ( n=4), and 22+/-13% ( n=5) respectively. In summary, AVE0118 preferentially inhibits the atrial K(+) channels I(Kur), I(to) and I(KACH). This profile may explain the selective prolongation of atrial refractoriness described previously in pigs and goats.

Development and evaluation of high throughput functional assay methods for HERG potassium channel.

Three functional hERG channel assay methods have been developed and evaluated. The methods were tested against five known hERG channel inhibitors: dofetilide, terfenadine (Seldane), sertindole (Serdolect), astemizole (Hismanal), and cisapride (Propulsid). The DiBAC4(3)-based assays were found to be the most economical but had high false-hit rates as a result of the interaction of dye with the test compounds. The membrane potential dye assay had fewer color-quenching problems but was expensive and still gave false hits. The nonradioactive Rb+ efflux assay was the most sensitive of all the assays evaluated and had the lowest false-hit rate.

Interactions of the antimalarial drug mefloquine with the human cardiac potassium channels KvLQT1/minK and HERG.

Mefloquine is a quinoline antimalarial drug that is structurally related to the antiarrhythmic agent quinidine. Mefloquine is widely used in both the treatment and prophylaxis of Plasmodium falciparum malaria. Mefloquine can prolong cardiac repolarization, especially when coadministered with halofantrine, an antagonist of the human ether-a-go-go-related gene (HERG) cardiac K+ channel. For these reasons we examined the effects of mefloquine on the slow delayed rectifier K+ channel (KvQT1/minK) and HERG, the K+ channels that underlie the slow (I(Ks)) and rapid (I(Kr)) components of repolarization in the human myocardium, respectively. Using patch-clamp electrophysiology we found that mefloquine inhibited KvLQT1/minK channel currents with an IC50 value of approximately 1 microM. Mefloquine slowed the activation rate of KvLQT1/minK and more block was evident at lower membrane potentials compared with higher ones. When channels were held in the closed state during drug application, block was immediate and complete with the first depolarizing step. HERG channel currents were about 6-fold less sensitive to block by mefloquine (IC50 = 5.6 microM). Block of HERG displayed a positive voltage dependence with maximal inhibition obtained at more depolarized potentials. In contrast to structurally related drugs such as quinidine, mefloquine is a more effective antagonist of KvLQT1/minK compared with HERG. Block of KvLQT1/minK by mefloquine may involve an interaction with the closed state of the channel. Inhibition by mefloquine of KvLQT1/minK in the human heart may in part explain the synergistic prolongation of QT interval observed when this drug is coadministered with the HERG antagonist halofantrine.

The antipsychotic drugs sertindole and pimozide block erg3, a human brain K(+) channel.

The antipsychotic drugs sertindole and pimozide are known to prolong the QT interval on the electrocardiogram via a high affinity block of the cardiac K(+) channel known as HERG (human ether-a-go-go-related gene; erg1). We wished to test whether these drugs also displayed high affinity for the related neuronal K(+) channel erg3. The cDNA encoding erg3 channel was cloned from a human brain library. Northern analysis confirmed that the channel was localized to brain relative to other tissues including heart, liver and lung. Within the brain, erg3 was expressed in higher amounts in the frontal lobe and cerebellum relative to the temporal, parietal and occipital lobes. Transient expression of erg3 in Chinese hamster ovary cells produced outwardly directed K(+) currents that activated at approximately -50 mV and produced a large transient component at positive membrane potentials. Inward tail currents measured at -100 mV were blocked in a dose-dependent fashion by sertindole resulting in an IC(50) value of 43 nM. Significant inhibition was observed at concentrations as low as 3 nM. Block of erg3 by sertindole also displayed a positive voltage-dependence. Pimozide blocked erg3 channel currents with an IC(50) of 103 nM and significant inhibition was noted at concentrations of 10 nM and higher. We conclude that erg3 can be blocked by certain antipsychotic drugs like sertindole and pimozide. Inhibition of erg3 or related K(+) channels in the brain may contribute to the efficacy/side effect profiles of some antipsychotic drugs.

Amyloid-beta peptide fragments p3 and p4 induce pro-inflammatory cytokine and chemokine production in vitro and in vivo.

Alzheimer's disease (AD) pathology is characterized by senile plaques containing amyloid-beta (A beta) peptide, a protein with neurotoxic and glial immune activating potential. In addition to the highly amyloidogenic peptides A beta(1--40/42), plaques contain amino-terminal truncated A beta peptides including the alpha secretase-generated p3 fragments A beta(17--40/42). In the present study, A beta(17--40/42), A beta(1--40/42), A beta(1--16), and A beta(25--35) aged in different solvents exhibited varying capacity to activate the murine microglia cell line MG-7 depending on solvent, peptide 'aging', and peptide sequence that did not strictly correlate with beta-sheet formation. A beta(17--40/42) or A beta(1--42) stimulated production of the pro-inflammatory cytokines interleukin (IL)-1 alpha, IL-1 beta, IL-6 and tumor necrosis factor-alpha (TNF-alpha), and the chemokine MCP-1 from differentiated human monocytes (THP-1) while little or no stimulation was observed with the other A beta fragments. MG7 cells also produced these five pro-inflammatory proteins in response to A beta(1-42) whereas A beta(17--40/42) elicited mainly TNF-alpha and MCP-1. Murine and human astrocyte cell lines (D30 and U373, respectively) were generally less responsive to A beta fragments producing mainly IL-6 and MCP-1 in response to A beta(1--42) or A beta(17--40/42) fragments. In mice, an intracerebroventricular infusion of A beta(1--42) significantly increased IL-1 alpha, IL-1 beta, IL-6 and MCP-1 while A beta(17--40/42) increased MCP-1 and A beta(17--40) increased IL-1 beta. These results demonstrate that p3 and p4 A beta fragments are pro-inflammatory glial modulators and thus may play a role in development of the immunopathology observed in AD.

Interactions of a series of fluoroquinolone antibacterial drugs with the human cardiac K+ channel HERG.

Administration of certain fluoroquinolone antibacterials has been associated with prolongation of the QT interval on the electrocardiogram and, on rare occasions, ventricular arrhythmia. Blockade of the human cardiac K+ channel HERG often underlies such clinical findings. Therefore, we examined a series of seven fluoroquinolones for their ability to interact with this channel. Using patch-clamp electrophysiology, we found that all of the drugs tested inhibited HERG channel currents, but with widely differing potencies. Sparfloxacin was the most potent compound, displaying an IC50 value of 18 microM, whereas ofloxacin was the least potent compound, with an IC50 value of 1420 microM. Other IC50 values were as follows: grepafloxacin, 50 microM; moxifloxacin, 129 microM; gatifloxacin, 130 microM; levofloxacin, 915 microM; and ciprofloxacin, 966 microM. Block of HERG by sparfloxacin displayed a positive voltage dependence. In contrast to HERG, the KvLQT1/minK K+ channel was not a target for block by the fluoroquinolones. These results provide a mechanism for the QT prolongation observed clinically with administration of sparfloxacin and certain other fluoroquinolones because free plasma levels of these drugs after therapeutic doses approximate those concentrations that inhibit HERG channel current. In the cases of levofloxacin, ciprofloxacin, and ofloxacin, inhibition of HERG occurs at concentrations much greater than those observed clinically. The data indicate that clinically relevant HERG channel inhibition is not a class effect of the fluoroquinolone antibacterials but is highly dependent upon specific substitutions within this series of compounds. HERG channel affinity should be an important criterion for the development of newer fluoroquinolones.

Interactions of the 5-hydroxytryptamine 3 antagonist class of antiemetic drugs with human cardiac ion channels.

Administration of the 5-hydroxytryptamine 3 receptor class of antiemetic agents has been associated with prolongation in the QRS, JT, and QT intervals of the ECG. To explore the mechanisms underlying these findings, we examined the effects of granisetron, ondansetron, dolasetron, and the active metabolite of dolasetron MDL 74,156 on the cloned human cardiac Na(+) channel hH1 and the human cardiac K(+) channel HERG and the slow delayed rectifier K(+) channel KvLQT1/minK. Using patch-clamp electrophysiology we found that all of the drugs blocked Na(+) channels in a frequency-dependent manner. At a frequency of 3 Hz, the IC(50) values for block of Na(+) current measured 2.6, 88.5, 38.0, and 8.5 microM for granisetron, ondansetron, dolasetron, and MDL 74,156, respectively. Block was relieved by strong hyperpolarizing potentials, suggesting a possible interaction with an inactivated channel state. Recovery from inactivation was impaired at -80 mV compared with -100 mV, and the fractional recovery was impaired by drug in a concentration-dependent manner. IC(50) values for block of the HERG cardiac K(+) channel measured 3.73, 0.81, 5.95, and 12.1 microM for granisetron, ondansetron, dolasetron, and MDL 74,156, respectively. Ondansetron (3 microM) also slowed decay of HERG tail currents. In contrast, none of these drugs (10 microM) produced greater than 30% block of the slow delayed rectifier K(+) channel KvLQT1/minK. We concluded that the antiemetic agents tested in this study block human cardiac Na(+) channels probably by interacting with the inactivated state. This may lead to clinically relevant Na(+) channel blockade, especially when high heart rates or depolarized/ischemic tissue is present. The submicromolar affinity of ondansetron for the HERG K(+) channel likely underlies the prolongation of cardiac repolarization reported for this drug.

High affinity blockade of the HERG cardiac K(+) channel by the neuroleptic pimozide.

Pimozide is an antipsychotic agent also used to treat facial tics. Pimozide can cause acquired long QT syndrome and ventricular arrhythmias. To elucidate the mechanism behind these clinical findings, we examined the effects of pimozide on the cloned human cardiac K(+) channels HERG (human ether-a-go-go-related gene; rapid component of delayed rectifier), Kv1.5 (ultra-rapid delayed rectifier) and KvLQT1/minK (slow component of delayed rectifier). Using patch clamp electrophysiology, we found that pimozide was a potent inhibitor of HERG displaying an IC(50) value of 18 nM. In contrast, pimozide (10 microM) was a weak inhibitor of KvLQT1/minK and Kv1.5. We conclude that pimozide is a specific, high affinity antagonist of HERG, and that this interaction leads to prolongation of cardiac repolarization.

Cardiovascular safety of fexofenadine HCl.

BACKGROUND: Certain first- and second-generation H1-receptor antagonists are associated with prolongation of the corrected QT interval (QTc) and, in rare instances, with ventricular dysrhythmias, including torsades de pointes ventricular tachycardia. OBJECTIVE: To assess the effect of fexofenadine HCl, a new non-sedating antihistamine, on QTc. METHODS: Dose-tolerance, safety, and drug-interaction studies with healthy volunteers; and clinical efficacy studies with seasonal allergic rhinitis patients were conducted. Twelve-lead ECG data were collected pre- and postdosing or serially throughout these studies. Outliers were defined as QTc >440 msec with a >/=10 msec increase from baseline. RESULTS: Fexofenadine HCl at single doses up to 800 mg q.d. (once daily) and multiple doses up to 690 mg b.d. for 28 days in healthy volunteers resulted in no increases in QTc (recommended dose range is 120-180 mg daily); QTc changes were similar to placebo. Compared with placebo, there were no statistically significant QTc increases in patients receiving fexofenadine HCl 80 mg b.d. for 3 months, 60 mg b. d. for 6 months, or 240 mg q.d. for 12 months. No statistically significant increases in QTc were detected when fexofenadine HCl 120 mg b.d. was administered in combination with erythromycin (500 mg t. d.) or ketoconazole (400 mg q.d.) after dosing to steady-state (6.5 days). In seasonal allergic rhinitis patients (n = 1160) treated with 40, 60, 120, or 240 mg b.d. fexofenadine HCl for 2 weeks, there were no dose-related increases in QTc and no significant increases in mean QTc compared with placebo. Frequency and magnitude of QTc outliers with fexofenadine HCl and placebo were similar in all studies. No case of fexofenadine-associated torsades de pointes has been observed in controlled trial experience with more than 6000 patients. CONCLUSION: Fexofenadine HCl has been investigated more extensively for possible electrophysiological effects than any other antihistamine. Fexofenadine HCl has no significant effect on QTc, even at doses much higher than those used in clinical practice.

The antipsychotic agent sertindole is a high affinity antagonist of the human cardiac potassium channel HERG.

Acquired long QT syndrome is a side effect seen with some pharmacological agents, including antipsychotic drugs, and is associated with the development of ventricular arrhythmias. This syndrome is often caused by the blockade of repolarizing potassium channels the human heart. A new antipsychotic agent, sertindole, has been shown to produce QT prolongation after therapeutic doses in humans. We therefore examined the effects of sertindole on two cloned human cardiac potassium channels, the human ether-a-go-go-related gene (HERG) and Kv1.5, stably transfected into mammalian cell lines. Using patch clamp electrophysiology, we found sertindole blocked HERG currents with an IC50 value of 14.0 nM when tail currents at -40 mV were measured after a 2-sec depolarization to +20 mV. When currents were measured at the end of prolonged (20 sec) depolarizing pulses, the IC50 of sertindole measured 2.99 nM. Sertindole enhanced the rate of current decay during these prolonged voltage steps and displayed a positive voltage dependence. Sertindole was approximately 1000-fold less active at blocking Kv1.5 displaying an IC50 value of 2.12 microM. By comparison, the potent class III antiarrhythmic agent dofetilde blocked HERG with an IC50 value of 9.50 nM but did not enhance HERG current decay or block Kv1. 5 channel currents. It is concluded that sertindole is a high affinity antagonist of the human cardiac potassium channel HERG and that this blockade underlies the prolongation of QT interval observed with this drug. Furthermore, the sertindole molecule may provide a useful starting point for the development of very high affinity ligands for HERG.

A mechanism for the proarrhythmic effects of cisapride (Propulsid): high affinity blockade of the human cardiac potassium channel HERG.

Cisapride (Propulsid) is a gastrointestinal prokinetic agent commonly used to treat nocturnal heartburn as well as a variety of other gastrointestinal disorders. The use of cisapride has been associated with acquired long QT syndrome and ventricular arrhythmias such as torsades de pointes which produces sudden cardiac death. These cardiotoxic effects can be due to blockade of one or more types of K+ channel currents in the human heart. For this reason we compared the effects of cisapride on two cloned human cardiac K+ channels, Kv1.5 and the human ether-a-go-go-related gene (HERG) stably transfected into mammalian cells. Using patch clamp electrophysiology, we found that cisapride was a potent inhibitor of HERG displaying an IC50 value of 44.5 nmol/l when tail currents at -40 mV were measured following a 2 s test depolarization to +20 mV. When HERG currents were measured at the end of prolonged (20 s) depolarizing steps to +20 mV, the apparent affinity of cisapride was increased and measured 6.70 nmol/l. The main effect of cisapride was to enhance the rate of HERG current decay thereby reducing current at the end of the voltage clamp pulse. Furthermore, the potency of cisapride for the HERG channel was similar to that observed for the class III antiarrhythmic agent dofetilide (IC50 = 15.3 nmol/l) and the nonsedating antihistamine terfenadine (IC50 = 56.0 nmol/l). In contrast to its effects on HERG, cisapride inhibited Kv1.5 channel currents weakly displaying an IC50 value of 21.2 micromol/l. It is concluded that cisapride displays specific, high affinity block of the human cardiac K+ channel HERG. It is likely that this interaction underlies the proarrhythmic effects of the drug observed under certain clinical settings.

Interactions of the nonsedating antihistamine loratadine with a Kv1.5-type potassium channel cloned from human heart.

The use of nonsedating antihistamines may, on rare occasions, be associated with cardiac arrhythmias. This could be due to blockade of voltage-dependent K+ channels in the heart, leading to a prolongation in repolarization in the human myocardium. For this reason, we examined the effects of the nonsedating antihistamine loratadine on a rapidly activating delayed-rectifier K+ channel (Kv1.5) cloned from human heart and stably expressed in HEK 293 cells or mouse Ltk- cells. Using patch-clamp electrophysiology, we found that loratadine blocked Kv1.5 current measured from inside-out membrane patches at concentrations of > or = 100 nM, resulting in an IC50 value of 808 nM at +50 mV. The drug enhanced the rate of Kv1.5 current decay, and block was enhanced at membrane potentials near threshold relative to higher potentials. Loratadine did not alter the kinetics of Kv1.5 current activation or deactivation. Unitary Kv1.5 currents were recorded in cell-attached patches. At the single-channel level, the main effect of loratadine was to reduce the mean probability of opening of Kv1.5. This effect of loratadine was achieved by a reduced number of openings in bursts and burst duration. Finally, loratadine (10 microM) failed to inhibit HERG K+ channel currents expressed in Xenopus laevis oocytes. It is concluded that loratadine is an effective blocker of Kv1.5 that interacts with an activated state or states of the channel. This interaction suggests a potential for loratadine to alter cardiac excitability in vivo.

Blockade of the human cardiac K+ channel Kv1.5 by the antibiotic erythromycin.

Erythromycin administration has been associated with a prolongation of cardiac repolarization in certain clinical settings. This could be due to blockade of voltage-dependent K+ channels in the human heart. For this reason we examined the effects of erythromycin on a rapidly activating delayed rectifier K+ channel (Kv1.5) cloned from human heart and stably expressed in human embryonic kidney cells. When examined using the whole-cell patch clamp technique, erythromycin (100 microM) blocked Kv1.5 current in a time-dependent manner but required prolonged exposure to do so. However, when we examined Kv1.5 current using inside-out macro-patches, erythromycin applied to the cytoplasmic surface rapidly (within 1-2 min) inhibited Kv1.5 current with an IC50 value of 2.6 x 10(-5)M (1.7 - 3.9 x 10(-5)M, 95% C.L.). The main effect of erythromycin was to accelerate the rate of Kv1.5 current decay thereby reducing the current at the end of a prolonged voltage-clamp pulse. Erythromycin also blocked Kv1.5 current in both a voltage- and frequency-dependent manner but had little effect on the activation kinetics, deactivation kinetics, or the steady-state inactivation properties of Kv1.5. These data suggest that erythromycin acts as a blocker of an activated state of the Kv1.5 channel and that it may access its binding site from the intracellular face of the channel. This study is the first to examine the effects of erythromycin on a cloned human cardiac K+ channel. It is concluded that erythromycin blocks Kv1.5 at clinically relevant concentrations. Blockade of voltage-dependent K+ channels in the heart could contribute to the alterations in cardiac repolarization that have been observed with erythromycin.

Stable expression and characterization of the human brain potassium channel Kv2.1: blockade by antipsychotic agents.

We have cloned the cDNA encoding the voltage-dependent K+ channel Kv2.1 from human brain (hKv2.1). RNase protection and RT-PCR (reverse transcriptase-PCR) experiments reveal abundant Kv2.1 transcripts in human brain with virtually no expression detectable in human heart. hKv2.1 has been stably transfected into a human glioblastoma cell line, and transformed cells display large, slowly activating outward currents. The kinetics, steady-state activation and inactivation parameters, and external tetraethylammonium sensitivity were all similar to those described previously for hKv2.1 channels transiently expressed in Xenopus oocytes or other mammalian cell lines. A number of dopamine receptor antagonist/antipsychotic agents were shown to block hKv2.1. Trifluoperizine, trifluperidol and pimozide produced time-dependent blockade of hKv2.1 with IC50 values of approx. 1-2 microM. The diphenylbutylpiperidine fluspirilene was shown to be 4-5-fold more potent than the other agents tested inhibiting hKv2.1 current with an IC50 value of 297 nM. The block produced by fluspirilene was both time- and frequency-dependent. Furthermore, fluspirilene (1 microM) shifted the midpotential of the hKv2.1 steady-state inactivation curve by approx. 15 mV in the hyperpolarizing direction. These results demonstrate the usefulness of this transfection system for the pharmacological characterization of hKv2. 1. Fluspirilene proved to be a relatively potent blocker of hKv2.1 and may provide a useful starting point for the development of more potent and selective agents active against this brain K+ channel.

Voltage- and time-dependent block by perhexiline of K+ currents in human atrium and in cells expressing a Kv1.5-type cloned channel.

Perhexiline maleate is an antianginal drug that has been shown to have antiarrhythmic effects in humans. To examine whether some of these clinical observations could be caused by block of cardiac K+ channels, we examined the effects of perhexiline on a rapidly activating delayed rectifier K+ channel (Kv1.5) cloned from human heart and stably expressed in human embryonic kidney cells as well as a corresponding K+ current (the ultra-rapid delayed rectifier, IKur) in human atrial myocytes. With the use of inside-out macropatches, we found that perhexiline inhibited Kv1.5 current in a time- and voltage-dependent manner with an IC50 value of 1.5 x 10(-6) M at +50 mV. Perhexiline reduced Kv1.5 tail current amplitude and slowed its decay relative to control. These data are consistent with blockade of open channels, probably from the intracellular surface. Perhexiline (3 microM) also blocked IKur in human atrial myocytes. The block that was observed was both time- and voltage-dependent in qualitatively similar ways to block of Kv1.5 channels. However, the time-dependent block of IKur by perhexiline was somewhat slower and its voltage-dependence steeper relative to its effects on Kv1.5. These data indicate that perhexiline blocks both cloned and native human cardiac K+ channels. Blockade of one or more types of voltage-dependent K+ channels may explain some of the electrophysiological effects of perhexiline observed in humans.

Effects of terfenadine and its metabolites on a delayed rectifier K+ channel cloned from human heart.

Use of the nonsedating antihistamine terfenadine has been associated with altered cardiac repolarization in certain clinical settings. For this reason we examined the effects of terfenadine, and its metabolites, on a rapidly activating delayed rectifier K+ channel (fHK) cloned from human heart. fHK was stably expressed in human embryonic kidney cells, and both whole-cell currents and currents from excised inside-out patches were recorded. Terfenadine (3 microM) blocked whole-cell fHK current by 72 +/- 6%. In inside-out patches, terfenadine applied to the cytoplasmic surface blocked fHK with an IC50 value of 367 nM. The main effect of terfenadine was to enhance the rate of inactivation of fHK current and thereby reduce the current at the end of a prolonged voltage-clamp pulse. The blockade displayed a weak voltage dependence, increasing at more positive potentials. The mechanism of action of terfenadine is therefore consistent with blockade of open channels. In contrast, the metabolites of terfenadine were weakly active on fHK. IC50 values for all of the metabolites tested ranged from 27-fold to 583-fold higher than that obtained for terfenadine. It is concluded that terfenadine, but not its metabolites, blocks at least one type of human cardiac K+ channel at clinically relevant concentrations and that this activity may underlie the cardiac arrhythmias that have been associated with the use of this drug.

Verapamil blocks a rapidly activating delayed rectifier K+ channel cloned from human heart.

Verapamil is an antagonist of L-type Ca2+ channels, and part of its binding site is located in the sixth transmembrane segment (S6) in the fourth repeat of the protein. Verapamil also blocks K+ channels, which are members of the same supergene family as Ca2+ channels. We examined the effects of verapamil on a rapidly activating delayed rectifier K+ channel (designated fHK) cloned from human heart. Verapamil inhibited 86Rb+ efflux from fHK-transfected human embryonic kidney cells with an EC50 of 4.5 x 10(-5) M. Whole-cell patch-clamp experiments revealed that verapamil induced a rapid component of fHK current inactivation but was without effect on activation. The effect was concentration and voltage dependent and was attributed to open channel blockade. The apparent association and dissociation rate constants measured at +50 mV were about 1.65 x 10(5) M-1 sec-1 and 3.48 sec-1, respectively. S6 of fHK has significant homology to that portion of the verapamil binding site identified in Ca2+ channels, and S6 is thought to form part of the inner mouth of K+ channel pores. The data support a role for verapamil as a blocker of the inner pore of voltage-dependent K+ channels in human myocardium.

Comparison of the in vitro and in vivo cardiovascular effects of two structurally distinct Ca++ channel activators, BAY K 8644 and FPL 64176.

We compared the cardiovascular effects of two structurally distinct L-type Ca++ channel activators, the 1,4-dihydropyridine Bay K 8644 and the benzoylpyrrole FPL 64176. Both compounds prolonged action potential duration and enhanced contractility in guinea pig papillary muscle with these responses being greater in the presence of FPL 64176 compared to (S)-Bay K 8644. (S)-Bay K 8644 (300 nM) and FPL 64176 (300 nM) increased whole-cell Ca++ channel current amplitude in neonatal rat ventricular cells by 249 +/- 14 and 484 +/- 100%, respectively. (S)-Bay K 8644 had little effect on Ca++ channel activation but significantly enhanced the rate of Ca++ channel current inactivation. FPL 64176 significantly slowed Ca++ channel current activation and inactivation. Tail current decay at -50 mV was monoexponential in the presence of (S)-Bay K 8644 and had a time constant of 4.59 +/- 0.16 msec. FPL 64176 produced biexponential tail current decays at -50 mV with fast and slow time constants of 4.30 +/- 0.30 and 44.52 +/- 4.56 msec, respectively. Intravenous administration (1-100 micrograms/kg) of Bay K 8644 and FPL 64176 produced large increases in cardiac contractile force and diastolic blood pressure in anesthetized dogs. Pretreatment with nifedipine attenuated the blood pressure response to FPL 64176 but not the effects on cardiac contractility. This study demonstrates that the benzoylpyrrole FPL 64176 defines a new and potent class of Ca++ channel agonist molecule and that this compound has pharmacological activity that differs, at least in some respects, from the 1,4-dihydropyridine group of agonists.

New synthetic ligands for L-type voltage-gated calcium channels.

The pharmacology of the L-type Ca2+ channel has been the subject of considerable basic and clinical investigation over the past two decades primarily because of the clinical activities of nifedipine, verapamil and diltiazem. However, it is quite clear that this Ca2+ channel is, in common with other pharmacologic receptors, a multiple drug receptor. There are probably as many as six or more discrete drug binding sites associated with this Ca2+ channel. Continued investigation of these sites may yield both new therapeutic agents, structural clues to ligands active at other classes of Ca2+ channel and structures active at other classes of ion channel.

[3H]PN200-110 and [3H]glibenclamide binding in normal and cardiomyopathic hamsters.

1. We examined the binding of the Ca2+ channel ligand [3H]PN200-110 and the ATP-sensitive K+ channel ligand [3H]glibenclamide to brain and heart from cardiomyopathic hamsters and compared them to controls. 2. We found that [3H]PN200-110 binding site density was elevated in the heart, but not in the brain, of 30- and 180-day old cardiomyopathic hamsters when compared to controls. 3. [3H]Glibenclamide binding site density was greatly reduced in the heart of 180-day old cardiomyopathic animals compared with all other groups. 4. Quantitative autoradiography revealed that [3H]glibenclamide binding was elevated in several brain areas of 30-day old cardiomyopathic hamsters relative to controls. 5. It is concluded that alterations in both Ca2+ and K+ channels exist in the cardiomyopathic hamster.