Effects of (+)- and (±)-sotalol on repolarizing outward currents and pacemaker current in sheep cardiac Purkinje fibres

Summary This study was aimed to differentiate the action of (+)- and (±)-sotalol (10–1000 mol/l) on membrane currents which are active during the repolarization of cardiac action potentials Effects where studied in shortened sheep cardiac Purkinje fibres with the two-microelectrode voltage-clamp technique Action potentials were activated at a frequency of 0.25 Hz and membrane currents at 0.03 Hz or 0.05 Hz in most experiments.Out of the currents investigated the transient outward current (i_to) reacted most sensitively to (+)- and (±)-sotalol. I_to-amplitude was decreased on the average to 77% of reference at 10 mol/l and to 53% at 1000 mol/l (+)- or (±)-sotalol. The maximally available ito-current was decreased but the voltage-dependent control of inactivation was left nearly unchanged. The initial inwardly rectifying current (i_Ki), which propels the last repolarization phase of the action potential and controls resting potential to a large extent was reduced on the average to 93% of reference at 10 mol/l and to 62% at 1000 mol/l (+)- or (±)-sotalol. Time-dependent (delayed) outward current (i_K) was on the average not affected by (+)- or (±)-sotalol up to 100 mol/l and was decreased to 84% of reference current under the influence of 1000 mol/l. An initial outward current, which is activated at positive membrane potentials (i_inst) was not clearly affected by (+)- or (±)-sotalol at concentrations up to 1000 mol/l Pacemaker current (i_f) was not influenced by the drugs up to 100 mol/l. Only at 1000 mol/l was the amount of available i_f-current decreased to 79% of reference. (The potential-dependent control of activation was not affected) Time constants of time-dependent currents i_to, i_K and i_f did not change in concentrations up to 1000 mol/l of the drug.Action potential duration increased at (+)- or (±)-sotalol concentrations 10 mol/l and maximal prolongation was achieved at concentrations of 100–300 mol/l Resting potential remained nearly unchanged at these concentrations, but the membranes depolarized at 1000 mol/l. According to our data action potential prolongation in sheep Purkinje fibres under the influence of (+)- and (±)-sotalol correlates to the drug-induced block to i_to-current and inwardly rectifying i_K1-current.Supported by the Deutsche Forschungsgemeinschaft SFB 242, C 1 Send offprint requests to U. Borchard at the above address     

Effects of the calcium entry blocker bepridil on repolarizing and pacemaker currents in sheep cardiac Purkinje fibres

Summary (1) Effects of bepridil (0.3–100 mol/l) on transmembrane currents which are active during the repolarization of the cardiac action potential were studied in sheep cardiac Purkinje fibres with the two-microelectrode voltage-clamp technique. Transmembrane currents were activated at a frequency of 0.03 Hz. (2) The initial inwardly rectifying current (i _K1) was reduced by 1.8 mol/l bepridil to 70% of the reference i _K1-current in the absence of the drug. (3) An initial outward current, which is activated at positive membrane potentials (i _inst) was depressed to 70% of reference by 14 mol/l bepridil. (4) The time-dependent outward current (i _K) was decreased by 1.8 mol/l bepridil to 70% of its amplitude in the absence of bepridil. The biexponential time course of i _K-current activation changed to be monoexponential with 100 mol/l bepridil. The effect of bepridil on i _K-current resulted in a shift of the activation curve of i _K-current to more positive membrane potentials (10 mol/l bepridil) and an additional decrease of driving force and/or conductance of the i _K-channels with higher bepridil concentrations (100 mol/l). (5) The transient outward current (i _to) was completely blocked by 30 mol/l bepridil. Inhibition to 70% of reference occurred with 1 mol/l bepridil. The voltage-dependent inactivation of i _to-current was affected by bepridil: the amplitude of the steady-state inactivation curve was reduced and i _to-current was inactivated faster after application of bepridil. Bepridil caused no pronounced shift of the steady-state inactivation curve along the voltage axis. (6) The pacemaker current (i _f) was slightly increased under the influence of low bepridil concentrations (0.3, 1 mol/l) while it was reduced to 70% of reference by 100 mol/l bepridil. (7) The blocking action of bepridil on outward currents in sheep cardiac Purkinje fibres will explain the action potential prolongation, which is observed in different mammalian cardiac tissues under the influence of bepridil.Supported by the Deutsche Forschungsgemeinschaft SFB 242, C 1 Send offprint requests to U. Borchard at the above address     

Inhibition of potassium outward currents and pacemaker current in sheep cardiac Purkinje fibres by the verapamil derivative YS 035

Summary The electrophysiologic mode of action and potency of the verapamil derivative YS 035 (N,N-bis-(3,4-dimethoxyphenethyl)-N-methyl amine) were investigated in sheep cardiac Purkinje fibres. Action potential duration measured at a repolarization level of –60 mV (APD-60) and membrane currents recorded with the two-microelectrode voltage-clamp technique were evaluated. At 10 mol/l YS 035 APD-60 was increased to about 115% of reference. Prolongation measured as percentage of the respective control exhibited on the average no dependence on stimulation frequency (0.17–2 Hz). At 100 mol/l membrane became depolarized to about –50 mV and action potentials could no longer be elicited. Further study was focussed on effects on outward currents, mostly activated at a frequency of 0.05 Hz. Transient outward current (i_to) was completely blocked at 100 mol/l and half-maximal inhibition occurred at about 14 mol/l. Inwardly rectifying potassium current (i_K1) was reduced to 47% of reference at 100 mol/l. An initially activating outward current at positive membrane potentials (i_inst) was reduced to 73% at 100 mol/l. Time-dependent (delayed) outward current (i_K) was on the average not affected up to 100 ol/l. Besides inhibition of repolarizing outward currents YS 035 completely blocked pacemaker current (i_f) at 100 mol/l and half-maximal reduction was achieved at 5 mol/l. YS 035 (1–100 mol/l) did not clearly affect time constants of activation at selected test potentials (I_K: +35 mV; i_f: –90 mV) or inactivation (i_to: 0 mV). Voltage-dependent control mechanisms of currents (it_to, i_f) were not influenced by YS 035 but the amount of available current was reduced.In conclusion, the verapamil derivative YS 035 inhibited pacemaker current and potassium outward currents which correlated to a prolongation of cardiac action po tentials. Electrophysiological actions of the compound favour it to be tested in vivo as an antiarrhythmic drug candidate. Send offprint requests to U. Borchard at the above address     

Effects of the bradycardic agent ZD 7288 on membrane voltage and pacemaker current in sheep cardiac Purkinje fibres

The bradycardic mechanism of ZD 7288 (4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)pyrimidinium chloride) was investigated in sheep cardiac Purkinje fibres. The pacemaker i_f-current measured with the two-microelectrode voltage-clamp technique, as well as the diastolic depolarization rate and the frequency of spontaneously active fibres were evaluated.ZD 7288 did inhibit i_f-current. The i_f-amplitude recorded with a 0.8s-lasting test pulse from about –50 mV to –100 mV was reduced to 50% of control at 0.85 mol/l and to 5% of control at 10 mol/l. The threshold potential of i_f-activation was unaffected at a concentration of 1 mol/l ZD 7288. The time constant of i_f-activation at different test potentials was not changed by 1 mol/l ZD 7288. The drug was equally effective during i_f-activation with a 0.5 s-lasting test pulse applied at 0.05 Hz or 0.5 Hz. During long lasting (5 s) hyperpolarizing test pulses (–120 mV) the inhibition of i_f-current was removed.In constantly stimulated Purkinje fibres (0.5 Hz) the slope of the early diastolic depolarization was decreased by ZD 7288. The half-maximal effect occurred at 0.92 mol/l. There was strong correlation over the concentration range of 0.01 to 10 mol/l ZD 7288 between the decrease of the slope of early diastolic depolarization and inhibition of i_f-amplitude recorded with 0.8s-lasting test pulses to –100 mV. The correlation coefficient was r = 0.97.These results will explain the decrease in frequency of spontaneously active (about 0.6 Hz) Purkinje fibres. At 0.3 mol/l ZD 7288 spontaneous activity had stopped in 8 of 11 preparations. Complete recovery of drug-induced effects on the frequency was gained after 3 h of wash-out with drug-free solution. Correspondence to: U. Borchard at the above address     

Electrophysiologic actions of aprindine in rabbit atrioventricular node

Summary Aprindine hydrochloride is a potent antiarrhythmic agent against various atrial and ventricular tachyarrhythmias. To elucidate its pharmacological actions in the atrioventricular node, electrophysiologic experiments were conducted by applying microelectrode and voltage clamp methods to small preparations of the rabbit atrioventricular node.At a concentration 1 mol/l, aprindine decreased the spontaneous firing frequency, maximal rate of depolarization, action potential amplitude, and take-off potential (P < 0.05, n = 7). The spontaneous and rate-controlled action potential durations at 50 and 100% repolarization were prolonged by aprindine. Voltage-clamp experiments using the double microelectrode method revealed that aprindine blocked the slow inward current (I_si) in a voltage-dependent manner with a dissociation constant of 10 mol/l and Hill coefficient of 0.8. The steady-state inactivation curve for I_si was shifted toward more negative potentials by 2.5 ± 0.9 mV (P < 0.05, n = 5) without a significant change in the slope factor. This finding suggests that aprindine has a higher affinity for inactivated slow inward (or Ca²⁺) channels than for resting channels. Aprindine caused use-dependent block of I_si, a result consistent with the drug's slow dissociation from inactivated Ca²⁺ channels. The delayed rectifying K⁺ current (I_K) tail obtained on repolarization from +10 mV to –60 mV was significantly decreased from 15.4 ± 2.4 to 6.8 ± 1.4 nA (P < 0.01, n = 6) and the deactivation time constant significantly increased by 20.7% (P < 0.01, n = 6). The steady-state activation curve for I_K was shifted in the hyperpolarized direction by 6.9 ± 2.9 mV, suggesting a potent voltage-dependent block of this current by aprindine. The hyperpolarization-activated inward current (1_h) was decreased from 14.4 ± 5.4 to 12.0 ± 5.5 nA (P < 0.05, n = 5). The transient outward and inward currents induced by 1 mol/l acetylstrophanthidin were almost completely suppressed after the addition of 1 mol/l aprindine.These results suggest that aprindine exerts a negative chronotropic action both by slowing deactivation of I_K and by reducing Is; and Ih, and delays atrioventricular nodal conduction by reducing Is; and IK. These blocking actions of aprindine together with its inhibition of the transient outward and inward currents may explain its antiarrhythmic effects on the atrioventricular node. Send offprint requests to Yoshio Watanabe at the above address     

Cocaine inhibits cromakalim-activated K<Superscript>+</Superscript> currents in follicle-enclosed<Emphasis Type="Italic"> Xenopus</Emphasis> oocytes

The effect of cocaine on K⁺ currents activated by the K_ATP channel opener cromakalim was investigated in follicular cells of Xenopus oocytes. The results indicate that cocaine in the concentration range of 3–500 M reversibly inhibits cromakalim-induced K⁺ currents. The IC₅₀ value for cocaine was 96 M. Inhibition of the cromakalim-activated K⁺ current by cocaine was noncompetitive and voltage independent. Pretreatment with the Ca²⁺ chelator BAPTA did not modify the cocaine-induced inhibition of cromakalim-induced K⁺ currents, suggesting that Ca²⁺-activated second messenger pathways are not involved in the actions of cocaine. Outward K⁺ currents activated by the application of 8-Br-cAMP or forskolin were also inhibited by cocaine. The EC₅₀ and slope values for the activation of K⁺ currents by cromakalim were 184±19 M and 1.14 in the absence of cocaine as compared to 191±23 M and 1.03 in the presence of cocaine (300 M). Cocaine also blocked K⁺ currents mediated through C-terminally deleted form of Kir6.2 (KirC26) in the absence of sulfonylurea receptor with an IC₅₀ value of 87 M, suggesting that cocaine interacts directly with the channel forming Kir6.2 subunit. Radioligand binding studies indicated that cocaine (100 M) did not affect the binding characteristics of the K_ATP ligand, [³H]glibenclamide. These results demonstrate that cromakalim-activated K⁺ currents in follicular cells of Xenopus oocytes are modulated by cocaine.     

Uptake,accumulation and release of ouabain by isolated rat hepatocytes

Summary We investigated uptake of ouabain into isolated rat hepatocytes and release of ouabain from preloaded hepatocytes, thus assessing separately the two membrane transport steps, involved in biliary elimination of the drug. The following results were obtained:Uptake of ouabain was saturable (K _m=263±61M, V=3.3±1.0 nmol/mg prot.×min), energy-dependent, sensitive to dinitrofluorobenzene and temperature-dependent (E=80–96 kJ/mol). It had no pH-optimum in the physiological pH-range and was independent of the extracellular cation composition.Uptake of ouabain was competitively inhibited by the cardiac glycoside digitoxin (K _i=0.3 M).Uptake was not inhibited in the presence of the glycosidic sugar l-rhamnose, but it was competitively inhibited by the steroid taurocholate (K _i=6.3 M).Ouabain was accumulated within hepatocytes 170-fold. The predominant fraction of intracellular ouabain beeing unbound.Release of ouabain from preloaded cells was energyindependent, independent of the Na⁺-concentration and not susceptible to inhibition by dinitrofluorobenzene or taurocholate.It is concluded, first that hepatocellular uptake of ouabain is mediated by a carrier for steroids and second that the pathway of release is distinct from that of uptake. We assume, that the high bile/plasma concentration-gradient of ouabain in vivo is generated during active uptake into the cell and not during release into bile.     

Influence of papaverine derivatives on phosphodiesterase activity,cyclic 3′,5′-AMP levels and relaxing effect on rabbit ileum

Summary The correlations between the relaxing effect of papaverine derivatives, inhibition of low K_m-phosphodiesterase (cAMP-PDE=EC 3.1.4.17) activity and cyclic 3,5-AMP (cAMP) levels in isolated rabbit ileum were investigated. There was a strong correlation between the relaxing effect, inhibition of PDE activity and cAMP content for eupaverine, ethylpapaverine and papaverine. Eupaverine was the most effective relaxing agent (I₅₀=7.5 M) and the most potent inhibitor of PDE activity (K_i=0.6 M), followed by ethylpapaverine (I₅₀=10 M); K_i=0.8 M) and papaverine (I₅₀=20 M; K_i=2 M). In contrast, there was a strong relaxing effect (I₅₀=6 M) but only slight inhibition of PDE activity (K_i=350 M) by tetrahydropapaveroline (THP). The adenylate cyclase stimulating effect of THP which was shown by others is most likely the reason for comparatively higher cAMP levels, which were found to be elevated about seven times over basal levels of 0.35 nmoles/g wet weight, and effective relaxation. Relaxation could be induced by exogenously added cAMP (I₅₀=45 M) and dibutyryl-cAMP (I₅₀=450 M). Our results support the assumption that smooth muscle relaxation in rabbit ileum is mediated by cAMP. Some of these observations have been published in abstract form (Schulz and Berndt, 1972).     

Effects of norfluoxetine on the action potential and transmembrane ion currents in canine ventricular cardiomyocytes

Norfluoxetine is the most important active metabolite of the widely used antidepressant compound fluoxetine. Although the cellular electrophysiological actions of fluoxetine are well characterized in cardiac cells, little is known about the effects of its metabolite. In this study, therefore, the effects of norfluoxetine on action potential (AP) configuration and transmembrane ion currents were studied in isolated canine cardiomyocytes using the whole cell configuration of patch clamp techniques. Micromolar concentrations of norfluoxetine (1–10 M) modified AP configuration: amplitude and duration of the AP and maximum velocity of depolarization were decreased in addition to depression of the plateau and elimination of the incisura of AP. Voltage clamp experiments revealed a concentration-dependent suppression of both L-type Ca²⁺ current, I_Ca (EC₅₀=1.13±0.08 M) and transient outward K⁺ current, I_to (EC₅₀=1.19±0.17 M) having Hill coefficients close to unity. The midpoint potential of the steady-state inactivation of I_Ca was shifted from –20.9±0.75 mV to –27.7±1.35 mV by 3 M norfluoxetine (P<0.05, n=7). No such shift in the steady-state inactivation curve was observed in the case of I_to. Similarly, norfluoxetine caused no change in the steady-state current–voltage relationship of the membrane or in the density of the inward rectifier K⁺ current, I_K1. All these effects of norfluoxetine developed rapidly and were fully reversible. Comparing present results with those obtained previously with fluoxetine, it can be concluded that norfluoxetine displays stronger suppression of cardiac ion channels than fluoxetine. Consequently, the majority of the cardiac side effects observed during fluoxetine treatment are likely to be attributed to its metabolite norfluoxetine.     

Stereoselective analysis of the disposition of tosufloxacin enantiomers in man

Summary The pharmacokinetics of tosufloxacin enantiomers after oral administration of racemic tosufloxacin were examined in healthy volunteers. Only small differences were observed in time to peak concentration (2.6±0.3 [mean ± SEM] h for (+)-tosufloxacin vs 2.4±0.2 h for (–)-tosufloxacin), elimination half-life (3.61±0.24 h vs 3.49±0.23 h), and area under the curve (2.78±0.19 h·g/ml vs 2.87±0.19 h·g/ml); however, peak concentration (0.40±0.03 g/ml vs 0.44±0.03 g/ml), renal clearance (226±10 ml/min vs 202±10 ml/min), and urinary recovery (35.4±2.2% vs 32.4±1.9%) differed significantly between enantiomers.     

Evidence for an A2 adenosine receptor in guinea pig lung

Summary Adenosine receptors in guinea pig lung were characterized by measurement of cyclic AMP formation and radioligand binding. 5-N-Ethylcarboxamidoadenosine (NECA) increased cyclic AMP levels in lung slices about 4-fold over basal values with an EC₅₀ of 0.32 mol/l. N⁶-R-(–)-Phenylisopropyladenosine (R-PIA) was 5-fold less potent than NECA. 5-N-Methylcarboxamidoadenosine (MECA) and 2-chloroadenosine had EC₅₀-values of 0.29 and 2.6 mol/l, whereas adenosine and inosine had no effect. The adenosine receptors in guinea pig lung can therefore be classified as A₂ receptors. Several xanthine derivatives antagonized the NECA-induced increase in cyclic AMP levels. 1,3-Diethyl-8-phenylxanthine (DPX; K _i 0.14 mol/l) was the most potent analogue, followed by 8-phenyltheophylline (K _i 0.55 mol/l), 3-isobutyl-1-methylxanthine (IBMX; K _i 2.9 mol/l) and theophylline (K _i 8.1 mol/l). In contrast, enprofylline (1 mmol/l) enhanced basal and NECA-stimulated cyclic AMP formation. In addition, we attempted to characterize these receptors in binding studies with [³H]NECA. The K _D for [³H]NECA was 0.25 mol/l and the maximal number of binding sites was 12 pmol/mg protein. In competition experiments MECA (K _i 0.14 mol/l) was the most potent inhibitor of [³H]NECA binding, followed by NECA (K _i 0.19 mol/l) and 2-chloroadenosine (K _i 1.4 mol/l). These results correlate well with the EC₅₀-values for cyclic AMP formation in lung slices. However, the K _i-values of R-PIA and theophylline were 240 and 270 mol/l, and DPX and 8-phenyltheophylline did not compete for [³H]NECA binding sites. Therefore, a complete characterization of A₂ adenosine receptors by [³H]NECA binding was not achieved. In conclusion, our results show the presence of adenylate cyclase-coupled A₂ adenosine receptors in lung tissue which are antagonized by several xanthines.     

Phenytoin partially antagonized L-type Ca2+ current in glucagon-secreting tumor cells (ITC-1)

Summary Transmembrane Ca²⁺ currents were investigated by means of a whole-cell clamp technique in a hamster glucagon-secreting tumor cell line (ITC-1). Two types of Ca²⁺ current were identified in ITC-1 cells. The low-threshold and transient (T-type) current became detectable above the potential level around –60 mV and decayed rapidly with an inactivation time constant of 95 ms (at –40 mV and 23°C), while the high-threshold and long-lasting (L-type) one was activated by depolarization more positive to –30 mV with non-inactivating kinetics. The voltage dependence and kinetics of these currents were identical to those reported in guinea-pig pancreatic ₂ cells. Both currents were augmented by equimolar substitution of Ca²⁺ with Ba²⁺ and completely abolished by adding 1 M La³⁺. Phenytoin, a well known anti-epileptic drug and a postulated T-type specific Ca²⁺ current antagonist, surprisingly blocked the L-type current without affecting the T-type current in ITC-1 cells. While phenytoin antagonized the L-type Ba²⁺ current selectively, 60% of the current remained even in supramaximal concentration range over 500 M. The residual component of the L-type current was completely abolished by adding nifedipine.     

5-Hydroxytryptamine 5-HT1D receptors mediating inhibition of cyclic AMP accumulation in Madin-Darby canine kidney (MDCK) cells

5-Hydroxytryptamine 5-HT_1B/5-HT_1D receptors are members of the same receptor subfamily, but display a different pharmacology (Hartig et al. (1992) Trends Pharmacol Set 13:152–159). Whereas several cell lines have been reported to contain 5-HT_1B receptors, none has been described, however, that endogenously expresses well-characterized 5-HT_1D receptors. The present study deals with the identification of 5-HT_1D receptors inhibiting cyclic AMP accumulation in Madin-Darby canine kidney (MDCK) cells. 5-HT (1 nM– 10 M) induced a concentration-dependent inhibition of the cyclic AMP accumulation stimulated by prostaglandin E₁ (1 M) in MDCK cells. The maximal effect of 5-HT averaged 50% inhibition and was abolished after a pre-treatment of the cells with pertussis toxin. Other agonists mimicked the effects of 5-HT, with the following rank order of potency (pEC₅₀ ± SEM, n 3): 5-carboxamidotryptamine (8.36 ± 0.48) > PAPP (p-aminophenylethyl-m-trifluoromethylphenyl piperazine, 7.89 ± 0.23) > 5-HT (7.35 ± 0.05) > sumatriptan (6.65 ± 0.27). PAPP behaved as a partial agonist. 8-OH-DPAT (8-hydroxy-2(di-n-propylamino)tetralin) was less potent, its maximal effect being not reached at 0.1 mM. Methiothepin, GR127935, (–)propranolol, rauwolscine and ketanserin were all devoid of intrinsic activity (up to 10 M or 0.1 mM). Methiothepin (10 nM, 0.1 M and 1 M) antagonized 5-HT effect (pA₂ 8.57 ± 0.44, Schild slope 1.17 ± 0.21, n = 3). GR127935 (1 nM, 10 nM and 0.1 M) shifted the curve of 5-HT to the right, but the antagonism was not fully surmountable (apparent pK_B value, 9.80 ± 0.16, n = 9). From the shifts obtained with rauwolscine (1 M) and (–)propranolol (10 M), respective pK_B values were estimated 6.68 ± 0.30 and 5.4 (n = 3 each). PAPP, when tested as an antagonist at 1 M, also shifted the curve of 5-HT to the right, with a pK_B of 8.27 ± 0.16 (n = 3). Finally, ketanserin (10 M) also antagonized the effects of 5-HT, the pK_B being 6.54 ± 0.16 (n = 9). The rank orders of agonist and antagonist potencies strongly suggest 5-HT receptors mediating inhibition of cyclic AMP accumulation in MDCK cells to be 5-HT_1D receptors. This is the first report of a cell line expressing endogenous, well-characterized, 5-HT_1D receptors. With regard to the 5-HT_1D receptor subtype involved, the relatively high potency of ketanserin would suggest it to be a 5-HT_1D subtype or a mixture of 5-HT_1D/5-HT_1D\ subtypes. However, caution must be exercised here, owing to the poor knowledge of canine 5-HT_1D receptor subtypes.     

Does the carrier of chromaffin granules transport the protonated or the uncharged species of catecholamines?

Summary By osmotic lysis in the presence of urea ghosts (60–100 nmol catecholamine/mg prot.) were prepared from chromaffin granules (4–6 mol catecholamine/mg prot.) of the bovine adrenal medulla. In the presence of 1–300 mol/l³H-catecholamine and ATP-Mg²⁺, ghosts show a net uptake of catecholamine. The net uptake is sensitive to reserpine or agents (uncouplers and ammonium) which diminish the electrochemical potential difference for protons at the granule membrane (p). The same uptake was found by³H-counting or by fluorimetric measurements. At various pH-values (pH 6.2–82.) theK _m andV _max of the ATP-stimulated rate of uptake of³H-catecholamine into ghosts was determined (at 30°C) to identify the species of catecholamine (protonated, uncharged, or anionic) which is the substrate for the granule carrier. The pH difference (pH=pH_out-pH_in) and the electrical potential difference () were determined to calculate p under conditions of³H-catecholamine uptake. When the pH_out was increased (pH 6.2, 7.4, 8.2), the apparentK _m of uptake decreased (50, 5, 1–2 mol/l), showing a linear relation between pH and logarithm ofK _m. TheK _m was calculated for the uncharged catecholamine (with pK₁=8.8 and pK₂=10.0); it was nearly pH-independent and amounted to about 0.2 mol/l. TheV _max declined only in the extreme pH-range. Between pH 6.6 and 7.8V _max and p showed a slight increase from 16 to 20 nmoles/(mg prot.·min) and from 110 to 140 mV, resp. In the same pH-range the pH_in inside ghosts increased from pH 5.2 to 5.7, whereas was constant (30 mV). At constant pH_out (=7.3) ammonium (0–30 mmol/l) caused an increase of pH_in from 5.5 to 6.6. The increase of pH_in was accompanied by an increase ofK _m from 5 to 20 mol/l³H-catecholamine and by a decrease of bothV _max and p from 20 to 5 nmoles/(mg prot.·min) and from 123 to 85mV, respectively. From the dependence of theK _m of uptake on pH_out is concluded that the uncharged species of catecholamine is transported, whereas the dependence ofK _m on pH_in suggests that the translocation of the catecholamine-carrier complex across the granule membrane is not the rate-limiting step of catecholamine uptake.A preliminary account was presented to the Deutsche Pharmakologische Gesellschaft (Kobold and Burger 1983)     

5-Hydroxytryptamine 5-HT1B receptors inhibiting cyclic AMP accumulation in rat renal mesangial cells

A clonal cell line derived from rat renal mesangial cells was shown to express endogenous 5-hydroxytryptamine (serotonin, 5-HT) receptors that mediate inhibition of cyclic AMP accumulation. These receptors were characterized as being of the 5-HT_1B receptor subtype. 5-HT₁ receptor agonists inhibited forskolin-stimulated cyclic AMP accumulation in rat renal mesangial cells (60–70% maximal inhibition) with the following rank order of potency (mean pEC₅₀ values±SEM, n 3): ergotamine (9.58±0.51)>RU 24969 (8.67±0.23)5-CT (8.42±0.06)CP 93129 (8.15±0.27)>5-HT (7.75±0.11) > sumatriptan (6.29±0.30) > 8-OH-DPAT (4.32±0.15). 5-HT₂ and 5-HT₄ receptor agonists were without effect. 5-HT-induced inhibition of cyclic AMP accumulation was abolished by a pre-treatment of the cells with pertussis toxin. (-)Propranolol was a partial agonist (27% maximal inhibition, pEC₅₀ 7.19±0.24, n = 3); when used as an antagonist at 1 M, it shifted the concentration-response curve of 5-HT to the right (pKB 7.22±0.35, n = 3). Methiothepin was a competitive antagonist of 5-HT (pA₂ 8.04±0.10, Schild slope 0.87±0.21, n = 3). Rauwolscine (10 M) had no antagonist activity. There was a significant correlation (r = 0.98, P = 0.0001) between the cyclic AMP data obtained in rat mesangial cells and 5-HT_1B binding data reported in rat brain cortex. The same pattern of responses was observed in early passages of primary cultures of rat mesangial cells. This study shows that rat mesangial cells can be used as a convenient source of functional 5-HT_1B receptors. It also constitutes further evidence for the widespread distribution of 5-HT_1B receptors outside the brain.     

Effects of lead on cloned voltage-operated neuronal potassium channels

The action of lead (Pb ²⁺) on cloned voltage-operated potassium channels of the rat brain was investigated in oocytes of Xenopus laevis. Pb²⁺ was found to decrease the potassium currents. This effect was due to a shift of the current-voltage relation in a positive direction (up to 30 mV). The Pb²⁺ effect appeared at a threshold concentration of about 0.1 mol/l and was maximal at a concentration of about 30 mol/l. At a potential of – 30 mV, the concentration needed for a 50% reduction of the potassium current was 1.0 mol/l. The depressant effect of Pb²⁺ was obtained with all potassium channels tested (Kv1.1, Kv1.2, Kv1.4, Kv2.1, Kv3.4). It was minimal for the Kv2.1 channel and maximal for the Kv1.1 channel at potentials negative to 0 mV. An effect comparable with that of Pb²⁺ could not be induced by the application of magnesium or calcium. The external application of Pb²⁺ led to a decrease of potassium currents in outside-out but not in inside-out membrane patches. Overall, Pb²⁺ had a significant effect on the potassium channels which may contribute to the mechanisms of Pb²⁺ neurotoxicity.     

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 IC₅₀ values of 5.4±0.7 M and 6.2±0.4 M respectively. In Chinese hamster ovary (CHO) cells, AVE0118 decreased the steady-state hKv1.5 current with an IC₅₀ of 1.1±0.2 M. The hKv4.3/KChIP2.2 current in CHO cells was blocked by AVE0118 by accelerating the apparent time-constant of inactivation (_inact), and the integral current was inhibited with an IC₅₀ of 3.4±0.5 M. At 10 M AVE0118 _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 M carbachol. At a clamp potential of –100 mV the I_KACh was half-maximally blocked by 4.5±1.6 M 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 M. 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 Ca²⁺ (recorded in pig atrial myocytes) were blocked by 10 M 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.     

Dicentrine,an α-adrenoceptor antagonist with sodium and potassium channel blocking activities

To elucidate the electrophysological effect of dicentrine, an alkaloid isolated from Lindera megaphylla, we examined action potential and membrane currents in single cardiac cells. The tight-seal whole cell clamp technique was used. In the current clamp condition, 3M dicentrine prolonged rat ventricular action potential duration (APD₅₀) from 38.9±(SEM)9.8 ms to 147.8 ± 19.7 ms (n = 12) and reduced its maximal rate of depolarization ( _max) from 220.5±20.3 V/s to 37.0±4.0 V/s. The same concentration of quinidine increased APD₅₀from 42.5 ± 5.2 ms to 182.8 ± 15.6 ms (n = 6) and decreased the _max from 225.4± 19.5 V/s to 32.2±3.0 V/s. Voltage clamp study revealed that dicentrine (1 to 100 M) inhibited the integral of the transient outward current (I_to - I₂₀₀) dose-dependently with a K_D value of 3.0±0.5 M. At 50 mV, the suppression of I_to by 3 M dicentrine was accompanied by shortening of its inactivation timeconstant from 41.0±4.9 ms to 18.8±2.1 ms. V _0.5 for the steady state inactivation curve of I_to was shifted from -25.5±2.8 mV to –40.6±2.1 mV. Compared to dicentrine, quinidine exerted stronger but the same mode of inhibition of I_to. 4-Aminopyridine, however, blocked I_to without modification of its inactivation time constant. In addition to the inhibition of I_to, the late outward current (I_lo) was significantly reduced byt0 M eachof dicentrine and quinidine to 60.0±13.307o and 37.5±6.3070, respectively. 4-Aminopyridine (2 mM) failed to inhibit this current. The delayed rectifier (I_k) in guinea pig ventricular cells was also inhibited by dicentrine with IC₅₀ value between 3 and 10 M. Like quinidine, dicentrine exerted equipotent inhibition of sodium inward current but without inhibition of calcium inward current. These results indicate that dicentrine exerts cardiac effect by mode of action like class I and class III antiarrhythmic agents. Correspondence to: Ming-Jai Su at the above address     

Inhibition of pacemaker current by the bradycardic agent ZD 7288 is lost use-dependently in sheep cardiac Purkinje fibres

The inhibition of the pacemaker current (i _f) in sheep cardiac Purkinje fibres by ZD 7288 [4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)pyrimidinium chloride] is lost use-dependently. This disinhibition of i _f was investigated by using the two-microelectrode voltage-clamp technique. The pulse protocol consisted of a rest period (holding potential of about -50 mV, 1–10 mol/l ZD 7288) followed by a train of test pulses (potential negative to -100 mV, stimulation frequency 0.05 Hz). At the beginning of the first test pulse there was an immediate reduction of i _f but inhibition was lost during continued stimulation. Activation of i _f is sigmoidal and the early delay in current activation was prolonged from 33 ms (no ZD 7288) to 424 ms (10 mol/l ZD 7288). Therefore hardly any disinhibition occurred during short test pulses (0.5 s). During longer test pulses (5 s, -120 mV, 10 mol/l) disinhibition developed with a time constant of about 2 s. The inhibition of i _f by ZD 7288 was lost voltage-dependently. With 10 mol/l ZD 7288 the half-maximal disinhibition occurred at -92 mV and the slope factor of the disinhibition/voltage curve (Boltzmann relation) was 4.8 mV. The voltage-dependent disinhibition could be abolished largely by extracellular application of protease (0.5 mg/ml, 7 min). After prior disinhibition, reinhibition at the holding potential (about -50 mV) followed a bi-exponential time course indicating that inhibition may be produced by a fast (=0.7 min) and a slow component (=20–30 min). Increasing ZD 7288 concentration from 1 to 10 mol/l accelerated reinhibition, mainly by an increase of the amplitude (A) of the fast component. The ratio A _fast/A _sIow was 0.399 at 1 mol/l and 2.65 at 10 mol/1 ZD 7288. The reinhibition of i _f was unchanged by shifting the holding potential from -50 mV to -20 mV Trials to wash out the effects of 10 mol/l ZD 7288 gave two results. The inhibition of i _f was slightly reversed after a wash-out of 1.5 h with drug-free solution. A second effect of the drug, the fast reinhibition, could be completely removed by washout. In summary i _f is inhibited by ZD 7288 at membrane potentials at which the virtual i _f gate is closed. Disinhibition occurs during long-lasting hyperpolarization but will hardly be operative in unclamped fibres under physiological conditions.     

Changes in the excretion of endogenous glycine conjugate as a possible artifact in toxicological experiments

Changes in the urinary excretion of hippuric acid (HIA) and phenaceturic acid (PUA) as well as their metabolic precursors, i.e. benzoic (BA) and phenylacetic acid (PAA), in rats housed in glass metabolic cages for 4 days were monitored using gas-liquid chromatography. The amount of HIA excreted was 128±63 mol/kg for female and 79±43 mol/kg for male rats in the first 24 h and decreased to 11±7 mol/kg (p< 0.01) for female and 3.2±2.4 mol/kg (p< 0.001) for male rats on the 2nd day. These values remained nearly at the same level until the end of the experiment. The amount of PUA decreased from 48±12 mol/kg on the 1st day to 22±9 mol/kg (p< 0.05) on the 2nd day by male rats, whereas by the females the decrease from 30±9 mol/kg to 21±8 mol/kg was not significant. The decrease in the excretion of glycine conjugates was compensated by a parallel increase in the level of unconjugated BA and PAA.