Suppression of alcohol and saccharin preference in rats by a novel Ca2+ channel inhibitor,Goe 5438

The effect of the novel 1,4-dihydronaphthyridine Ca²⁺ channel inhibitor Goe 5438 (CI-951) on voluntary ethanol consumption was examined in selectively bred alcohol-preferring (P) rats in a free choice two bottle preference test versus water. Intraperitoneally injected Goe 5438 dose-dependently (5, 10 or 20 µmol/kg, twice daily) inhibited ethanol and increased water intake over the 24 h period (injection day). The drug decreased ethanol preference, originally above 90%, by 6%, 19% and 45% at respective doses, on the injection day. That inhibitory effect of the highest dose of Goe 5438 on ethanol preference remained significant also on days 2 and 3 after injections (–51% and –18%, respectively). Goe 5438, in the highest dose, also tended to decrease granulated chow consumption during the injection day only. To further test whether the inhibition of ethanol preference is secondary to decrease in reinforcing properties of ethanol and not due to interference with satiety mechanisms, we compared the effect of two higher doses (10 and 20 µmol/kg, intraperitoneally, twice daily) of Goe 5438 on spontaneous preference for a non-caloric 0.04% saccharin solution in Sprague-Dawley rats. We observed a dose-dependent suppression of preference (by 44% and 58%, respectively) during the injection day, but not the subsequent 24 h period. However, Goe 5438 also significantly alleviated food pellet intake on the injection day. In conclusion, Goe 5438 produces potent and long-lasting inhibition of voluntary ethanol consumption, which may be secondary to attenuation of reinforcing properties of ethanol. Additionally, this particular Ca²⁺ channel inhibitor appears to have mild anorectic properties which may be conducive to acute suppression of alcohol intake.     

Effects of the class III antiarrhythmic drug ambasilide on outward currents in human atrial myocytes

We have studied the inhibitory influence of the class III antiarrhythmic drug ambasilide (LU 47110) on the transient outward current I _to1 and the sustained current T _so following inactivation of I _to1 in human atrial myocytes. The two currents are separated by a mathematical procedure based on the amplitudes and time constants of the biexponential inactivation of the total outward current. The frequency dependence, the recovery from inactivation and the kinetics of activation and inactivation are described. Ambasilide reversibly and concentration dependently inhibited I _to1, I _so and the sodium current I _Na. Concentration required for half maximal inhibition (IC₅₀) for the effects on I _to1 and I _so were 23.3 mol/l and 45.7 mol/l respectively, concentrations shown by others to be effective in terminating and preventing fibrillation in a dog atrial arrhythmia model. Ambasilide not only reduced the amplitude of I _to1 and I _so but also accelerated the time course of inactivation from 14.22 to 6.69 ms and from 202.3 to 87.9 ms respectively. The amplitude of I _to1 showed only a small dependence on stimulation frequency characteristic for human atrial myocytes, whereas I _so was reduced significantly at higher stimulation frequencies. Ambasilide did not change these relationships (0.1–4 Hz) and therefore did not show the reverse use-dependence known from other class III antiarrhythmic agents and which is an important property for a prospective antiarrhythmic drug. The lack of an effect of ambasilide on both steady-state activation and inactivation of I _to1, and the time constant of recovery from inactivation, suggests that ambasilide acts by changing conductance rather than by influencing the gating mechanism. The described characteristics make ambasilide an interesting substance in the group of class III antiarrhythmic drugs.     

Effects of SEA0400 and KB-R7943 on Na+/Ca2+ exchange current and L-type Ca2+ current in canine ventricular cardiomyocytes

SEA0400 and KB-R7943 are compounds synthesised to block transsarcolemmal Na⁺/Ca²⁺ exchange current (I_Na/Ca); however, they Have also been shown to inhibit L-type Ca²⁺ current (I_Ca). The potential value of these compounds depends critically on their relative selectivity for I_Na/Ca over I_Ca. In the present work, therefore, the concentration-dependent effects of SEA0400 and KB-R7943 on I_Na/Ca and I_Ca were studied and compared in canine ventricular cardiomyocytes using the whole-cell configuration of the patch clamp technique. SEA0400 and KB-R7943 decreased I_Na/Ca in a concentration-dependent manner, having EC₅₀ values of 111±43 nM and 3.35±0.82 M, when suppressing inward currents, while the respective EC₅₀ values were estimated at 108±18 nM and 4.74±0.69 M in the case of outward current block. SEA0400 and KB-R7943 also blocked I_Ca, having comparable EC₅₀ values (3.6 M and 3.2 M, respectively). At higher concentrations (10 M) both drugs accelerated inactivation of I_Ca, retarded recovery from inactivation and shifted the voltage dependence of inactivation towards more negative voltages. The voltage dependence of activation was slightly modified by SEA0400, but not by KB-R7943. Based on the relatively good selectivity of submicromolar concentrations of SEA0400—but not KB-R7943—for I_Na/Ca over I_Ca, SEA0400 appears to be a suitable tool to study the role of I_Na/Ca in Ca²⁺ handling in canine cardiac cells. At concentrations higher than 1 M, however, I_Ca is progressively suppressed by the compound.     

Stereoselective inhibition of calmodulin-dependent cAMP phosphodiesterase from bovine heart by (+)- and (−)-nimodipine

Summary The inhibitory effects of racemic (±)-nimodipine and of optically pure (+)- and (–)-nimodipine on the basal and calmodulin-dependent activity of a cAMP phosphodiesterase from bovine heart were investigated. The inhibition by (±)-nimodipine could not be overcome by an excess of calmodulin. However, increase of the cAMP concentration in the assay from 2 × 10^–4 mol/l to 2 × 10^–2 mol/l caused a shift of the IC₅₀ for the inhibition by (±)-nimodipine from 2.8 × 10^–6 mol/l to 6 × 10^–5 mol/l. Dixon-plot analysis revealed an inhibitory constant of K _i = 2.3 mol/l, Experiments with the two enantiomers showed that (+)-nimodipine is by about one order of magnitude more potent than (–)-nimodipine. This contrasts with the stereoselectivity of the Ca²⁺ channel inhibitory activity on isolated rings of the rabbit basilar artery where (–)-nimodipine is more effective than (+)-nimodipine in relaxing the smooth muscle contracted by K⁺-depolarisation. It is concluded that cAMP phosphodiesterase may be an intracellular target for nimodipine and its inhibition may contribute to the pharmacological activity of this 1,4-dihydropyridine. Send offprint requests to Ch. Schachtele at the above address     

Inhibitory Potencies of 1,4-dihydropyridine Calcium Antagonists to P-glycoprotein-Mediated Transport: Comparison with the Effects on CYP3A4

Purpose. Recently, we clarified the inhibitory effects of 13 kinds of 1,4-dihydropyridine calcium antagonists on human cytochrome P450 (CYP) 3A4. It has been reported that the substrates and/or inhibitors are overlapped between CYP3A4 and P-glycoprotein (P-gp). The purpose of this study was to investigate the inhibitory effects of 13 kinds of 1,4-dihydropyridine calcium antagonists on P-gp-mediated transport in order to evaluate the overlapping specificity of the inhibitors between P-gp and CYP3A4. Methods. The transcellular transports of [³H]daunorubicin or [³H]digoxin by monolayers of LLC-GA5-COL150 cells in which P-gp was overexpressed were measured in the presence or absence of the 1,4-dihydropyridine calcium antagonists. Results. The transport of [³H]daunorubicin was strongly inhibited by manidipine, barnidipine, benidipine, (–)-efonidipine, nicardipine, (+)-efonidipine, and amlodipine with the IC₅₀ values of 4.6, 8.6, 9.5, 17.3, 17.5, 20.6, and 22.0 M, respectively. The transport of [³H]digoxin was strongly inhibited by benidipine, nicardipine, barnidipine, and manidipine. Conclusions. It was clarified that 13 kinds of 1,4-dihydropyridine calcium antagonists have different inhibitory potencies and substrate specificities to the transport of [³H]daunorubicin or [³H]digoxin. Some compounds did not demonstrate the overlapping specificity for inhibition between P-gp and CYP3A4. It was also clarified that ni- cardipine, benidipine, manidipine, and barnidipine were strong inhibitors of P-gp as well as CYP3A4.     

Class Ia anti-arrhythmic drug ajmaline blocks HERG potassium channels: mode of action

Ajmaline is a class Ia anti-arrhythmic drug used in several European countries and Japan as first-line treatment for ventricular tachyarrhythmia. Ajmaline has been reported to induce cardiac output (QT) prolongation and to inhibit cardiac potassium currents in guinea pig cardiomyocytes. In order to elucidate the molecular basis of these effects, we examined effects of ajmaline on human ether a-go-go related gene HERG potassium channels. Electrophysiological experiments were performed with human embryonic kidney (HEK) cells (whole-cell patch clamp) and Xenopus oocytes (double-electrode voltage clamp) expressing wild-type and mutant HERG channels. Ajmaline blocked HERG currents with an IC₅₀ of 1.0 mol/l in HEK cells and 42.3 mol/l in Xenopus oocytes. The onset of block was fast and reached steady-state conditions after 180 s. The inhibitory effect was completely reversible upon wash-out. In HERG mutant channels Y652A and F656A lacking aromatic residues in the S6 domain, the inhibitory effect of ajmaline was completely abolished. Ajmaline induced a small shift in HERG current half-maximal activation voltage towards more negative potentials. Ajmaline did not markedly affect HERG inactivation. Inhibitory effects were not voltage-dependent. Ajmaline block exhibited positive frequency dependence. Ajmaline blocked HERG channels in the open, but not in the closed states. Binding of ajmaline to inactivated HERG channels may also be possible. In inactivation-deficient HERG S620T channels, the sensitivity to ajmaline was markedly reduced. The IC₅₀ of HERG channel blockade in HEK cells lies within the range of unbound therapeutic plasma concentrations of ajmaline. Therefore, inhibitory effects on HERG channels may contribute to both the high anti-arrhythmic efficacy of ajmaline and to its pro-arrhythmic potential.     

Inhibition of tyrosine hydroxylase in rabbit mesenteric artery and vas deferens by catechol oestrogens

Summary 1. In the present study we have investigated the effects of oestrogens, catechol oestrogens, and catecholamines on tyrosine hydroxylase (TH) activity derived from rabbit mesenteric artery and vas deferens. 2. Both catechol oestrogens, 2-hydroxyoestradiol (2OHE₂) and 2-hydroxyoesterone (2OHE₁), inhibited TH activity in mesenteric artery and vas deferens in a concentration-dependent manner with potencies that were higher than those for noradrenaline but lower than that for dopamine. 3. When added to the reaction medium along with increasing concentrations of a pterin cofactor (200 to 1,500 mol/l DMPH₄), the catechol oestrogens (200 mol/l) increased the apparent K _m for DMPH₄ without altering the maximum velocity (V _max) of the reaction. Similar results were obtained with the addition of noradrenaline (200 mol/l) and dopamine (120 /mol). Apparent K _i values obtained for the catecholamines and catechol oestrogens were within the same order of magnitude and varied from 30 mol/l for dopamine and 2OHE₂ to 183 mol/l for 2OHE₁. 4. Oestradiol (E₂) and 2-methoxyoestradiol (2MeOE₂), i.e., oestrogens that do not possess a catechol moiety, exhibited only weak inhibitory effects on TH activity. At the highest concentration tested (1 mmol/l), they did not reduce enzyme activity below 58% of control values. Kinetic analysis revealed that these two oestrogens did not consistently affect either the V _max of hydroxylation or the K _m for DMPH₄. 5. It is concluded that catechol oestrogens inhibit TH activity with a potency comparable to noradrenaline and dopamine. This inhibition is by competition with the pterin cofactor. Oestrogens that to not possess a catechol moiety are not effective inhibitors of TH. It is suggested that this property of the catechol oestrogens is most likely to be physiologically relevant only in tissues that either can synthesize them in situ from precursor oestrogens or can accumulate sufficient amounts of circulating catechol oestrogens such as may occur during pregnancy.Abbreviations 2OHE₁ 2-hydroxyoestrone - 2OHe₂ 2-hydroxyoestradiol - 2MeOE₂ 2-methoxyoestradiol - E₂ oestradiol Supported in part by the American Heart Association, West Virginia Affiliate and NIH grants HL30351 and K04-HLO1228A preliminary report of this study was presented to the American Society for Pharmacology and Experimental Therapeutics (Panek et al. 1985) Send offprint requests to R. E. Stitzel at the above address     

Mechanisms underlying the slow onset of action of a new dihydropyridine,NZ-105, on a cultured smooth muscle cell line

Summary The inhibitory effect of a new dihydropyridine derivative, (±)-2-[benzyl(phenyl)amino]ethyl-1,4-dihydro-2,6-dimethyl-5-(5,5-dimethyl-2-oxo-1,3,2-dioxaphosphorinan-2-yl)-4-(3-nitrophenyl)-3-pyridinecarboxylate hydrochloride (NZ-105), on whole cell Ca²⁺ current (I_Ca) in cultured vascular smooth muscle cells was investigated with the patch clamp technique. NZ-105 blocked I_Ca in a concentration-dependent manner when the command pulse ranged from +10 mV to –50 mV. The inhibitory effect of NZ-105 appeared at concentrations higher than 10 mol/l and it blocked I_Ca completely at a concentration of 1 nmol/l. The concentration which produced the half-maximal inhibitory effect was estimated to be around 20 mol/l. NZ-105 (500 pmol/l) completely blocked I_Ca elicited by depolarization to + 10 mV at a holding potential of –40 mV, whereas it blocked I_Ca by only 67% at a holding potential of –90 mV. NZ-105 (100 mol/l) shifted the steady-state inactivation curve by 40 mV to more negative potentials without affecting its slope factor. The blocking time constant of 500 mol/l NZ-105 was 57.6 + 9.9 s at a holding potential of –70 mV. These results indicate that NZ-105 has characteristics typical of dihydropyridines and binds to Ca²⁺ channels of vascular smooth muscle cells with a high affinity. They also suggested that the slow onset of its action is due to the slow binding of the drug to Ca²⁺ channels. Send offprint requests to S. Kokubun at the above address     

Risperidone-induced action potential prolongation is attenuated by increased repolarization reserve due to concomitant block of <Emphasis Type="Italic">I</Emphasis><Subscript>Ca,L</Subscript>

The neuroleptic risperidone is an effective blocker of the rapidly activating component of the delayed rectifier current (I_Kr) and hence is expected to prolong cardiac action potential duration (APD). However, unlike with other typical I_Kr blockers we failed to demonstrate a marked prolongation of late repolarization with risperidone. It is hypothesized that the APD-prolonging effect of risperidone is masked by the high repolarization reserve due to the prominent delayed rectifier currents I_Kr and I_Ks in guinea pig papillary muscle. Action potentials and force of contraction were recorded in isolated guinea pig papillary muscles. L-type calcium current I_Ca,L and I_Kr were measured using the standard patch clamp technique in single ventricular cardiomyocytes. Reduction of the repolarization reserve by the blocking of I_Ks with chromanol 239B augmented the effect of the selective I_Kr blocker E-4031, but not of risperidone, although both drugs completely blocked I_Kr. In contrast to E-4031 risperidone markedly reduced the force of contraction due to the partial blocking of I_Ca,L in the same concentration range as required for block of I_Kr. Reduction of the repolarization reserve by the blocking of I_Ks cannot exacerbate the APD-prolonging effect of risperidone. However, even incomplete concomitant blocking of I_Ca,L attenuates the APD-prolonging effect of the complete blocking of I_Kr. This behaviour may explain the small APD-prolonging effect of risperidone despite the drugs robust blocking of I_Kr.T. Christ and E. Wettwer contributed equally to this work     

Pharmacological characterization of A1 adenosine receptors in isolated rat ventricular myocytes

Summary The aim of the present study was the characterization of adenosine receptors in isolated rat ventricular myocytes. The CAMP-levels of rat ventricular myocytes in the presence of 1 mol/l isoprenaline were reduced by up to 48% by adenosine analogues; the rank order of potency was: R-N⁶-phenylisopropyladenosine (IC₅₀ 60 nmol/1), 5-N-ethylcarboxamidoadenosine (IC₅₀ 360 nmol/l) and S-N⁶-phenylisopropyladenosine (IC₅₀ 16 ol/l). The adenosine receptor antagonist XAC (xanthine amine congener) antagonized the effect of R-N⁶-phenylisopropyladenosine in a concentration-dependent manner with a K_i-value of 20 nmol/l. The A₁ receptor-selective radioligand R-N⁶-¹²⁵I-p-hydroxyphenylisopropyladenosine bound to membranes prepared from rat ventricular myocytes in a saturable manner with a B _max of 17.7 fmol/mg protein and a K _D-value of 1.1 nmol/l. Adenosine analogues competed for the binding with the same rank order of potency as for the inhibition of the isoprenaline-induced cAMP-increase. GTP inhibited radioligand binding with an IC₅₀-value of 73 ol/l. These results suggest the presence of A₁ adenosine receptors on rat ventricular myocytes, which mediate an inhibition of adenylate cyclase. The receptors may be responsible for the effects of adenosine and its analogues on the heart.Abbreviations ¹²⁵I-HPIA R-N⁶-¹²⁵I-p-hydroxyphenylisopropyladenosine - PIA N⁶-phenylisopropyladenosine - NECA 5-N-ethyl-carboxamidoadenosine - XAC 8-4-[([(2-aminoethyl)aminocarbonyl]methyl)oxy]phenyl-1,3-dipropylxanthine (xanthine amine congener) - Ro 20-1724 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone - ScAMPTME 2-O-monosuccinyladenosine-3,5-cyclic monophosphate tyrosyl methyl ester - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - GTP guanosine-5-tri-phosphate Send offprint requests to D. Martens     

Effects of AN-132, a novel antiarrhythmic lidocaine analogue,and of lidocaine on membrane ionic currents of guinea-pig ventricular myocytes

Summary We studied the effects of AN-132 (10, 30 and 100 mol/1), an analogue of lidocaine, on membrane currents and action potentials of single guinea-pig ventricular cells using whole-cell clamp techniques. The effects of lidocaine, an authentic class I antiarrhythmic agent were used for comparative purposes. (1) AN-132 decreased the Na current (I _Na) in a concentration-dependent manner, with a greater efficacy than seen with lidocaine. The concentration of the half maximal inhibition on I _Na (K _d) was (31.7 mol/1), for AN-132 and 94.9 mol/l for lidocaine. (2) AN-132 also decreased the Ca current (I _Ca), concentration-dependently, while lidocaine had only a minor effect on I _Ca. The half maximal inhibition on I _Ca (K _d) was 23.1 mol/l and 27.4 mol/l for AN-132 and lidocaine, respectively. (3) AN-132 decreased the I _K1, in a concentration-dependent manner; lidocaine was without effect. (4) AN-132 increased the unspecified steady state outward current, at positive potentials and depressed the time- and voltage-dependent outward K current (I _K). Lidocaine had no effect on either current. (5) AN-132 shortened the action potential duration (APD), in a concentration-dependent manner, without altering the resting potential. From these findings, we conclude that apart from a potent inhibitory effect on I _Na, AN-132 had a variety of effects on other currents, properties not shared by lidocaine. Such multiple blocking effects on the membrane currents may relate to the alleged potent antiarrhythmic effect of AN-132. Send offprint requests to M. Arita at the above address     

Inhibitory effects of AMP 579, a novel cardioprotective adenosine A1/A2A receptor agonist, on native I Kr and cloned HERG current

We investigated the effects of 1S-[1a,2b,3b,4a(S*)]-4-[7-[[1-[(3-chloro-2-thienyl)methylpropyl]propyl-amino]-3H-imidazo[4,5-b] pyridyl-3-yl]-N-ethyl-2,3-dihydroxycyclopentane carboxamide (AMP 579), a novel cardioprotective adenosine A₁/A_2A receptor agonist, on the rapid and slow components of the delayed rectifier K⁺ current (I_Kr and I_Ks) in guinea-pig ventricular myocytes and on the human ether-a-go-go-related gene (HERG) channel expressed in human embryonic kidney (HEK 293) cells. Whole-cell current and membrane potential were recorded using patch-clamp techniques. In guinea-pig ventricular myocytes, AMP 579 inhibited I_Kr in a concentration-dependent manner with IC₅₀ value of 15.2 M, when I_Kr was blocked by chromanol 293B. On the contrary, AMP 579 (10 M) did not affect I_Ks in the presence of the I_Kr blocker E-4031. The former effect of AMP 579 was unaffected by either the selective adenosine A₁ receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine or the non-selective adenosine A₁/A₂ receptor antagonist 8-sulphophenyltheophylline. Moreover, AMP 579-induced inhibition of I_Kr was not voltage- and frequency-dependent. In HEK 293 cells expressing HERG channels, AMP 579 (10 M) significantly blocked the HERG current at +10 mV by 34.9±7.0% (n=4, p<0.05), and the degree of inhibition was comparable with that observed in guinea-pig ventricular myocytes (36.8±6.0%, n=4). AMP 579 (10 M) significantly inhibited the L-type Ca²⁺ current (I_Ca) by 41.0±6.8% (n=5, p<0.05), which was unaffected by 8-sulphophenyl-theophylline. Consequently, despite its inhibitory actions on I_Kr or HERG current, the drug significantly shortened the action potential duration measured at 90% repolarization from 275.6±19.4 to 208.3±18.6 ms (n=4, p<0.05). Thus, AMP 579 inhibits both native I_Kr and cloned HERG channels with additional inhibitory effect of I_Ca, and such inhibitory effects may at least partially underlie the observed antifibrillatory action of the drug during myocardial ischemia/reperfusion.     

Comparison of the effects of cromakalim,a potassium conductance enhancer,and nimodipine,a calcium antagonist,on 5-hydroxytryptamine responses in a variety of vascular smooth muscle preparations

Summary We have examined the effects of the potassium conductance enhancer cromakalim (BRL 34915) and the calcium entry blocker nimodipine upon 5-hydroxytryptamine (5-HT) induced contractions in ring preparations from rabbit basilar and mesenteric arteries, and from pig coronary arteries. Cumulative concentration-response (CR) curves to 5-HT were biphasic in basilar and mesenteric arteries, and monophasic in coronary arteries. Coronary artery 5-HT CR curves and the first component of the mesenteric artery 5-HT CR curve were antagonized by ketanserin (pK _B values 8.9 and 8.8, respectively), whereas basilar artery CR curves were not. Prazosin antagonized the second component of the mesenteric 5-HT CR curve, but not that of the basilar artery. Cromakalim (0.1–10 mol/1) and nimodipine (0.001–1 mol/l) both caused reductions in resting tension in basilar and coronary arteries denuded of their endothelia, but this effect was not seen with mesenteric arteries. Procaine (5 mmol/1) abolished this vasorelaxant effect of cromakalim in basilar artery. Both agents concentration-dependently depressed the 5-HT CR curve in coronary artery, the effect of cromakalim was antagonized by lidocaine (100 mol/1). In basilar artery, only the first component was cromakalim sensitive unlike nimodipine which depressed both components of the CR curve. In mesenteric artery, 5-HT CR curves were depressed by cromakalim, but only slightly affected by nimodipine (1 mol/l). It is concluded that cromakalim, like nimodipine, possesses anti-vasospastic activity; however, differences exist in the sensitivity of the 5-HT mediated contractions of the three arterial preparations to the agents. Thus, although both cromakalim and nimodipine may have potential in the treatment of subarachnoidal haemorrhage and angina pectoris, differences in their therapeutic profiles are also likely to be found. Send offprint requests to C. R. Cain at the above address     

Calcium agonists and antagonists of the dihydropyridine type: Antinociceptive effects,interference with opiate-μ-receptor agonists and neuropharmacological actions in rodents

The calcium antagonist dihydropyridine derivative nimodipine and its enantiomers BAY N 5247, BAY N 5248, as well as BAY R 4407 (calcium antagonist (+)-enantiomer of the calcium agonist dihydropyridine BAY K 8644) do not exert antinociceptive effects in the rat as measured by the vocalization test in doses up to 100 g/kg IV, and in the mouse as measured by the hot plate test in oral doses up to 100 mg/kg. The calcium agonists BAY K 8644 and BAY R 5417 ((–)-enantiomer of BAY K 8644) are also ineffective in the rat vocalization test but BAY K 8644 increases reaction time in the hot plate test (mouse) dose-dependently (1–10 mg/kg PO). -Receptor agonist (fentanyl) antinociceptive effects are potentiated by simultaneous IV administration of the calcium antagonists, the (–)-enantiomer of nimodipine BAY N 5248 being the most potent. This applies for the rat (vocalization test) and the mouse (hot plate test). The influence on fentanyl antinociception in the rat of the calcium agonist BAY K 8644 and its (–)-enantiomer BAY R 5417 is biphasic: low doses attenuate, high doses potentiate fentanyl antinociception. In the mouse (hot plate test) antinociceptive effects of BAY K 8644 plus fentanyl are less than additive, indicating that the calcium agonist decreases fentanyl effects. The relative potency of calcium antagonists in potentiation of fentanyl antinociception correlates with their relative potency as calcium antagonists as measured by receptor binding studies, effects on vascular and cardiac muscle, and with their neuropharmacological actions (anticonvulsive effects, inhibition of balance and spontaneous motility as well as tranquilizing effects in the mouse). It is concluded that calcium antagonism potentiates -receptor agonist antinociceptive effects, whereas calcium agonism antagonizes -receptor agonist antinociception.Dedicated to Prof. Dr. H. Coper (Berlin) on the occasion of his 60th anniversary     

Drug binding to aromatic residues in the HERG channel pore cavity as possible explanation for acquired Long QT syndrome by antiparkinsonian drug budipine

Budipine is a non-dopaminergic antiparkinsonian drug causing acquired forms of Long QT syndrome (aLQTS). As a consequence, the manufacturer has restricted the use of budipine in patients who exhibit additional risk factors for the development of Torsades-de-Pointes tachycardias (TdP). The molecular basis of this serious side effect has not been elucidated yet.Human ether-a-go-go related gene (HERG) channel block being the main cause of drug induced QT prolongation, we investigated the effect of budipine on the rapid component of the delayed-rectifier potassium current (I_K(r)) in guinea pig cardiomyocytes and on HERG potassium channels heterologously expressed in Xenopus oocytes.In guinea pig cardiomyocytes, budipine (10 M) inhibited I_K(r) by 86% but was without any effect on calcium currents. In Xenopus oocytes, HERG potassium channels were blocked by budipine with an IC₅₀ of 10.2 M. Onset of block was fast and block was only slowly and incompletely reversible upon washout.Budipine blocked HERG channels in the open and inactivated state, but not in the closed states. The half-maximal activation voltage was slightly shifted towards more negative potentials. Steady-state inactivation of HERG was also influenced by budipine. Budipine block was neither voltage- nor frequency-dependent.In HERG channel mutants Y652A and F656A, drug affinity was reduced dramatically. Therefore, these two aromatic residues in the channel pore are likely to form a main part of the binding site for budipine.In summary, this is the first study that provides a molecular basis for the budipine-associated aLQTS observed in clinical practice. Furthermore, these findings underline the importance of the aromatic residues Y652 and F656 in the binding of lipophilic drugs to HERG channels.Abbreviations I_K(r) The rapid component of the delayed-rectifier potassium current - HERG Human ether-a-go-go related gene - aLQTS Acquired Long QT syndrome - TdP Torsade-de-Pointes tachycardia E.P. Scholz and E. Zitron contributed equally to this work     

The in vitro effects of alkanes,alcohols, and ketones on rat lung cytochrome P450-dependent alkoxyphenoxazone dealkylase activities

The sensitivities of cytochrome P450 (EC 1.14.14.1)-dependent benzyloxy- and ethoxyphenoxazone dealkylase (BzOPh'ase and EtOPh'ase, respectively) activities towards a series of aliphatic hydrocarbons were measured in the microsomal fraction of lung obtained from -naphthoflavone-treated rats. The unsubstituted hydrocarbons were straight-chain (n-hexane through n-undecane) and branch-chain (eight carbons). The substituted compounds were alcohols and ketones of hexane and octane. The data are expressed as I₅₀ values, i.e. the hydrocarbon concentration required to cause 50% decrease in the rate of enzymecatalyzed product (resorufin) formation. The unsubstituted aliphatic hydrocarbons exhibited I₅₀ values towards BzOPh'ase from 0.76 M (2,5-dimethylhexane) to 8.8 M (n-hexane). The lung EtOPh'ase activity was insensitive to-wards the tested unsubstituted aliphatic hydrocarbons. When the alcohols and ketones of hexane and octane were tested against lung BzOPh'ase activity, I₅₀ values ranged from 16 M (1-octanol) to 4.8 mM (2,5-hexanedione). Lung EtOPh'ase activity exhibited some sensitivity towards the alcohols and ketones, and I₅₀ values ranged from 0.52 mM (4-octanol) to 40.5 mM (2-hexanol). The data show rat lung BzOPh'ase and EtOPh'ase activites are differentially sensitive towards the selected unsubstituted aliphatic hydrocarbons and corresponding alcohols and ketones. The difference in sensitivities may reflect different requirements for an adventitious interaction between a hydrocarbon and enzyme active site.     

Characteristics of L-type calcium channel blockade by lacidipine in guinea-pig ventricular myocytes

1. The Ca²⁺-antagonistic properties of lacidipine were investigated in patch-clamp guinea-pig ventricular myocytes.
2. In basal conditions, 0.1 μM lacidipine reduced the action potential duration, associated with a decrease in the L-type calcium current (I_Ca,L) to 66±4% of the control value, without a change in the current-voltage relationship. Sodium current and background potassium currents were not affected. All the effects reached a steady state within 2 min.
3. The Ca²⁺-antagonistic effect of lacidipine was voltage-dependent: a marked negative shift (about 20 mV) of the steady-state inactivation curve was observed with long (10 s) conditioning prepulses, but not with short (350 ms) prepulses.
4. The onset of and recovery from the voltage-dependent effect caused by 0.1 μM lacidipine were significantly slower when compared to those of equiactive concentrations of nimodipine (0.5 μM) and nisoldipine (0.1 μM). I_Ca,L measured after prepulses at −40 mV lasting 500 ms or less was unchanged (95±5% of maximum current value) while it was reduced to 49±10% by nimodipine and 43±9% by nisoldipine (P<0.05 vs lacidipine for both).
5. Similarly, the recovery from block in the presence of lacidipine was slower than with nimodipine and nisoldipine. After a prepulse of 1 s at −80 mV, I_Ca,L recovered up to 54±2% of the maximum current value in the presence of lacidipine, and up to 91±3% and 93±5% in the presence of nimodipine and nisoldipine, respectively (P<0.05 vs lacidipine).
6. Blockade of I_Ca,L by lacidipine was use-dependent. After ten 200 ms long pulses (1 Hz) from −80 mV, I_Ca,L was reduced to 55±7% of the current measured at the first pulse. In the presence of nimodipine and nisoldipine, I_Ca,L elicited by the tenth pulse amounted to 93±3% and 80±6% of the first pulse value, respectively (P<0.05 vs lacidipine). Lacidipine did not cause use-dependent blockade of I_Ca,L in cells stimulated with 10 ms long pulses.
7. These results demonstrate that lacidipine selectively inhibits I_Ca,L in isolated cardiomyocytes and suggest that this effect occurs mainly through binding to the inactivated Ca²⁺ channels.

     

Transient outward current (I A) in clonal anterior pituitary cells: blockade by aminopyridine analogs

Summary Whole cell voltage-clamp recordings from GH₃ cells, a clonal cell line derived from a rat anterior pituitary tumor, demonstrated a rapidly activating and inactivating (transient) voltage-dependent outward current. This current, referred to as I _A, was elicited by step depolarization from holding potentials negative to –50 mV, showed strong outward rectification at potentials positive to –30 mV, and exhibited steady state inactivation with V _1/2 near –64 mV. The current rose to a peak within < 10–20 ms following depolarization and decayed in two exponential phases, I _Af and IA _AS with time constants of 30–50 and 500–700 ms, respectively. Both I _A components exhibited similar voltage dependencies for activation and inactivation. Aminopyridines (2 mol/l – –5 mmol/l) produced a dose dependent, reversible blockade of I _A (70% inhibition at 0.5 to 2 mmol/l) with the following rank order of potencies: 4-aminopyridine > 3,4-diaminopyridine = 3-aminopyridine > 2-aminopyridine. These drugs reduced the peak conductance of I _A, and produced complex effects on its time-dependent decay. With submaximal degrees of block, there was an increase in the inactivation rate, suggesting that open channels are preferentially blocked by the drugs. It is concluded that GH₃ pituitary cells possess an aminopyridine-sensitive transient outward current comparable to the A-current in neural cells. However, this cell line is unusual in that it expresses both rapidly and slowly decaying A-current components.Abbreviations n-AP n-aminopyridine - 3,4-DAP 3,4-diaminopyridine - TEA tetraethylammonium - EGTA ethylene glycol bis(-aminoethyl ether)N,N-tetraacetic acid - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid Send offprint requests to M. A. Rogawski at the above address     

Effect of MANT-nucleotides on L-type calcium currents in murine cardiomyocytes

Membranous adenylyl cyclases play a major role in G-protein-coupled receptor signalling and regulate various cellular responses, such as cardiac contraction. Cardiac apoptosis and development of cardiac dysfunction is prevented in mice lacking AC 5, a predominant isoform in the heart. In the search for a potent and selective AC 5 inhibitor, we recently identified 2′(3′)-methylanthraniloyl-inosine-5′-triphosphate(MANT-ITP) as the most potent AC 5 inhibitor with a K _i of 13 nM. Therefore, AC inhibition of MANT-ITP was assessed in ventricular cardiomyocytes and compared to three other MANT-nucleotides to evaluate its effect on cardiac signalling. Basal and isoproterenol-induced L-type calcium currents (I _Ca,L) in murine ventricular cardiomyocytes were recorded by whole-cell patch-clamp technique, using four different MANT-nucleotides. The effects of the MANT-nucleotides on I _Ca,L were unexpectedly complex. All MANT-nucleotides exhibited an inhibitory effect on basal I _Ca,L. Additionally, several MANT-nucleotides, i.e., MANT-ITPγS, MANT-ATP, and MANT-ITP, caused a strong initial increase in basal I _Ca,L within the first 2.5 min that appeared to be unrelated to AC 5 inhibition. However, we detected a significant reduction on isoproterenol-induced I _Ca,L with MANT-ITP, supporting the notion that AC 5 plays an important role in agonist-stimulated activation of I _Ca,L. Collectively, MANT-nucleotides are useful tools for the characterization of recombinant ACs, for fluorescence studies and crystallography, but in intact cardiomyocytes, caution must be exerted since MANT-nucleotides apparently possess additional effects than AC 5 inhibition, limiting their usefulness as tools for intact cell studies.     

Exposure to the n-3 polyunsaturated fatty acid docosahexaenoic acid impairs α1-adrenoceptor-mediated contractile responses and inositol phosphate formation in rat cardiomyocytes

The beneficial effects of n-3 polyunsaturated fatty acids of fish oil in the prevention of fatal arrhythmias in myocardial ischemia were suggested to be at least in part mediated by a modulation of dihydropyridine-sensitive L-type calcium channels. As cardiac ₁-adrenoceptor stimulation has been suggested to have no significant effect on L-type calcium channels, the aim of this study using cultured neonatal rat cardiomyocytes was to investigate whether chronic n-3 polyunsaturated fatty acid exposure may have an influence on ₁-adrenoceptor-induced positive inotropic effects and induction of arrhythmias. Pretreatment of the rat cardiomyocytes for 3 days in the presence of the n-3 polyunsaturated fish oil-derived fatty acid docosahexaenoic acid (60 mol/l) markedly decreased ₁-adrenoceptor-stimulated increase in contraction velocity and induction of arrhythmias. The increase in contraction velocity of the cardiomyocytes induced by the -adrenoceptor agonist isoprenaline was also markedly reduced by the n-3 fatty acid pretreatment. Basal contractile amplitude and spontaneous beating frequency of the cardiomyocytes were not significantly altered by the docosahexaenoic acid exposure. The pretreatment of the rat cardiomyocytes for 3 days in the presence of docosahexaenoic acid (60 mol/l) decreased ₁-adrenoceptor-stimulated formation of the calcium-mobilizing second messenger IP₃ and its metabolites IP₂ and IP₁ by 55%. The depression of IP₃ formation by docosahexaenoic acid treatment was not mediated by a decreased uptake of myo-inositol into the cardiomyocytes nor by a decreased synthesis of phosphatidylinositol bisphosphate (PIP₂), the substrate of phospholipase C. The level of glycerol-3-phosphate, an important substrate of the phosphoinositide cycle, was unaltered by the docosahexaenoic acid pretreatment. Receptor binding studies revealed that the dissociation constant and maximal binding capacity of the ₁-adrenoceptor antagonist (³H)prazosin was unchanged by the n-3 polyunsaturated fatty acid exposure. -Adrenoceptor-and forskolin-stimulated adenylyl cyclase activities were not diminished by the docosahexaenoic acid pretreatment. Chronic exposure of the cardiomyocytes to the n-6 polyunsaturated fatty acid arachidonic acid (60 mol/l) did neither significantly alter ₁-adrenoceptor-induced inositol phosphate formation nor ₁-adrenoceptor-stimulated increase in contraction velocity. The results presented show that chronic n-3 polyunsaturated fatty acid pretreatment of rat cardiomyocytes leads to a marked impairment of ₁-adrenoceptor-induced positive inotropic effects and induction of arrhythmias concomitant with a n-3 fatty acid-induced decrease in IP₃ formation. This derangement of the phosphonositide pathway by chronic n-3 fatty acid exposure may, thus, contribute to the beneficial effects of fish oil-derived fatty acids in the prevention of fatal arrhythmias in myocardial ischemia.