Different mechanisms of the inhibition of the transient outward current in rat ventricular myocytes

The mechanism of drug-induced inhibition of the transient outward current, I_to, has been investigated in rat ventricular myocytes using the whole cell patch clamp technique. I_to was activated by 300 ms depolarizing voltage clamp steps in 10 mV increments from –50 mV up to +40 mV. At +40 mV, I_to peaked after about 3 ms, and the time course of inactivation was appropriately described by two time constants, _fast = 17 ms and _slow = 203 ms. Verapamil, quinidine sulfate and nifedipine preferentially depressed I_to at the end of the 300 ms depolarizing voltage clamp step; the inactivation of I_to was accelerated by all drugs, whereas peak I_to was less affected. The time course of drug action at +40 mV was calculated by the fractional changes of I_to. Verapamil, quinidine sulfate and nifedipine exerted a block of I_to. increasing during the depolarizing voltage clamp step. The onset of block in response to verapamil, quinidine sulfate and nifedipine (30 mol/each) was appropriately described by monoexponential functions with time constants _on = 9.3, 1.7 and 1.1 ms, respectively. Relief from block by verapamil, quinidine sulfate and nifedipine at –50 mV was assessed by comparison of the recovery process of peak I_to from inactivation with or without drugs. _off amounted to 695 ms in the case of quinidine sulfate; verapamil and nifedipine did not significantly affect the recovery process so that the determination of the time course of relief from block was not possible. 4-Aminopyridine preferentially depressed peak I_to in a concentration-dependent manner, whereas I_to at the end of the 300 ms depolarizing voltage step remained unaffected. The block of I_to by 4-aminopyridine (3 mmol/l) decreased during the voltage step from –50 mV to +40 mV. Relief from block was described by _off = 30.4 ms. The efficacy of 4-aminopyridine was diminished at short and enhanced at long pulse intervals (reverse use-dependence). The time course of 4-aminopyridine-induced block of Ito was described by _on = 1561 ms. Phenylephrine (30 mol/l),papaverine (30 mol/I) and tetraethylammonium chloride (5 mmol/l) reduced I_to at the peak and at the end of the 300 ms depolarizing voltage step in a time-independent manner. It is concluded that verapamil, quinidine sulfate and nifedipine bind to the I_to channel in the open state at positive membrane potentials. In contrast, 4-aminopyridine obviously binds to the channel in the closed state at negative membrane potentials. Phenylephrine, papaverine and tetraethylammonium chloride seem to block I_to independent of the channel state. Correspondence to: H. Nawrath at the above address     

Usefulness of the Kohlrausch-Williams-Watts Stretched Exponential Function to Describe Protein Aggregation in Lyophilized Formulations and the Temperature Dependence Near the Glass Transition Temperature

Purpose. We studied the feasibility of using the Kohlrausch-Williams-Watts stretched exponential function (KWW equation) to describe protein aggregation in lyophilized formulations during storage. Parameters representing mean aggregation time (_a) and stretched exponential constant (_a) were calculated according to the KWW equation by assuming that the time required for protein molecules to aggregate () varies because of the fact that protein aggregation occurs at a rate that depends on the degree of protein deformation resulting from stresses created during freeze-drying. The temperature dependence of the parameters near the glass transition temperature was examined to discuss the possibility of predicting protein aggregation by accelerated testing. Methods. Protein aggregation in lyophilized bovine serum -globulin (BGG) formulations containing dextran or methylcellulose, at temperatures ranging from 10 to 80°C, was followed by size-exclusion chromatography. Results. Non-exponential BGG aggregation in lyophilized formulations could be described by the KWW equation. The _a and _a parameters changed abruptly around the NMR relaxation-based critical mobility temperature for formulations containing dextran and methylcellulose. In the glassy state, in contrast, the _a parameter of these formulations exhibited continuous temperature dependence. The parameter , as calculated from _a and _a, reflected differences in values between the two excipients. Conclusions. The results indicate that the parameter _a is reflective of physical changes wihtin lyophilized formulations. Within the temperature range, during which no abrupt changes in _a were observed, knowledge regarding the _aand _a parameters allows the rate of protein aggregation to be predicted. The parameter was found to be useful in comparing the protein aggregation behavior of formulations having different _a and _a values.     

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.     

Inactivation and Aggregation of β-Galactosidase in Lyophilized Formulation Described by Kohlrausch-Williams-Watts Stretched Exponential Function

Purpose. To examine whether the empirical Kohlrausch-Williams-Watts (KWW) equation is applicable not only to protein aggregation but also to protein denaturation in lyophilized formulations. Lyophilized -galactosidase (-GA) formulations containing polyvinylalcohol and methylcellulose were used as model formulations. The possibility of predicting storage stability based on the temperature dependence of the estimated parameters of inactivation/aggregation—time constant () and its distribution () is discussed. Methods. Protein aggregation in lyophilized -GA formulations at 10-70°C and 6-43% relative humidity was determined as a function of time by size exclusion chromatography. Enzyme activity was also determined using 2-nitrophenyl--D-galactopyranoside as a substrate. Results. Inactivation and aggregation of -GA were describable with the empirical KWW equation, regardless of whether the temperature was above or below the NMR relaxation-based critical mobility temperature (T_mc) or whether protein molecules with different degrees of deformation resulting from stresses during lyophilization exist in the formulation. The estimated parameter for protein aggregation decreased rapidly as temperature increased beyond T_mc because the mobility of polymer molecules increased in the initial stages of glass transition. The time required for 10% enzyme to aggregate (t₉₀) calculated from the and parameters exhibited a change in temperature dependence gradient near T_mc. In contrast, t₉₀ for protein inactivation exhibited temperature dependence patterns varying with the excipients. Conclusions. The t₉₀ calculated from the estimated and parameters was found to be a useful parameter for evaluating the stability of lyophilized -GA formulations. The prediction of t₉₀ by extrapolation was possible in the temperature range in which did not rapidly vary with temperature.     

Metabolism,Distribution, and Transdermal Permeation of a Soft Corticosteroid,Loteprednol Etabonate

The soft corticosteroid, loteprednol etabonate (chloromethyl 17-ethoxycarbonyloxy-11-hydroxy-3-oxoandrosta-1,4-diene-17-carboxylate), I, was designed based on the inactive metabolite approach. Accordingly, I should be metabolized by hydrolysis to the corresponding inactive cortienic acid derivative, II. The in vitro and in vivo metabolism of I indeed yielded mainly this inactive metabolite, which is more hydrophilic and thus readily eliminated from the body. Relatively high levels of I were found in tissues after intravenous administration of the drug in rats. The permeability of I through hairless mouse skin was comparable to what has been found for related hard steroids, without significant metabolism taking place in the skin.     

The Effect of Salts on the Stability of β-Galactosidase in Aqueous Solution,as Related to the Water Mobility

The effect of salts (KI, KBr, NaCl, KC1, KF, phosphate, and Na₂SO₄) on the stability of -galactosidase in aqueous solution was studied from the aspect of changes in water mobility. At salt concentrations up to 200 mM, the inactivation rate of -galactosidase in all the salt solutions studied increased with increasing salt concentration. At higher concentrations, those salts which had little effect on the spin-lattice relaxation time, T ₁, of water (KI, KBr, and KC1) continued to increase the inactivation rate of -galactosidase with increasing concentration, while those salts which decreased the T _l of water (KF, phosphate, and Na₂SO₄) decreased the inactivation rate. It appeared that the decrease in water mobility caused by KF, phosphate, and Na₂SO₄ resulted in stabilization of -galactosidase. The results indicate that water mobility is an important factor in the denaturation rate of proteins.     

Dehydro-digitoxosides of digitoxigenin and digoxigenin: Binding to beef heart (Na++K+)-ATPase in relation to unchanged digitoxosides

Summary Dehydro-digitoxosides are metabolites of digitalis glycosides. In order to study their possible biological activity their affinity to (Na⁺+K⁺)-activated ATPase was determined and compared with unchanged glycosides. Based on the dissociation constants of glycoside-enzyme-complexes, the affinity of the dehydro-digitoxosides ranged in the same order of magnitude as that of the native glycosides. Comparing mono-, bis-, and tris-digitoxosides of digitoxigenin (dt-1, dt-2, dt-3) and of digoxin (dg-1, dg-2, dg-3) with the corresponding dehydrodigitoxosides (3-dehydro-dt-1, 9-dehydro-dt-2, 15-dehydro-dt-3, 3-dehydro-dg-1 and 9-dehydro-dg-2, respectively) the dehydro-digitoxosides had lower affinities to the enzyme. The highest dissociation constants (K _D)were found for 3-dehydro-dt-1 and 3-dehydro-dg-1. The half maximal inhibition of (Na⁺+K⁺)-ATPase activity (I₅₀) corresponded to affinity measurements in all but two cases: dehydro-dt-3 and dehydro-dt-2 showed very low I₅₀ values.     

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.     

Quantitative Structure–Pharmacokinetics Relationships: II. A Mechanistically Based Model to Evaluate the Relationship Between Tissue Distribution Parameters and Compound Lipophilicity

The tissue-to-unbound plasma distribution coefficients (Kpus) of 14 rat tissues after iv administration of nine 5-n-alkyl-5-ethyl barbituric acids, determined in a previous study, were used to identify a model of the relationship between tissue distribution and lipophilicity of the homologs, expressed in terms of their octanol to water partition ratio, P. Based on mechanistic considerations and assumptions, the parameter model was expressed asKpu= f_W,[l + a(nP_l,)P^b], where f_W, is the tissue water content, (nP_l, ) is the binding capacity of the tissue, n is the number of the binding sites, a and b are the parameters of the relationship Ka = aP^b and Ka is the binding association constant of each tissue. The parameter model was linearized and fitted to the predetermined Kpu values, yielding correlation coefficients ranging between .940 and .997. The predictive performance of the parameter model was evaluated using a leave-one-out procedure with subsequent computation of the mean prediction error (ME = measurement of the prediction bias) and the square root of the mean squared prediction error (RMSE = measurement of the prediction accuracy). The ME varied between –22.48 and 61.14%, indicating a slight tendency for overpredicting. The RMSE was between 24.73 and 102% for the individual tissues across the different homologs; and between 28.33 and 85.2% for the individual homologs across the different tissues. The apparently high Kpu prediction errors, when translated through the low sensitivity of the barbiturate whole-body physiologically based pharmacokinetic model, established previously, leads to predicted tissue concentration–time profiles within 5 to 20% of the original ones. Therefore, it is concluded, that the identified mechanistically based model is a good predictor of the tissue-to-unbound Kpus in the rat tissues.     

Analysis of the Compression Mechanics of Pharmaceutical Agglomerates of Different Porosity and Composition Using the Adams and Kawakita Equations

Purpose. To analyze the mechanics of some pharmaceuticalagglomerates during uniaxial confined compression by using compressionparameters derived from the Heckel, Kawakita and Adams equations, and tostudy the influence of these compression parameters on the tablet-formingability of agglomerates. Methods. Force and displacement data sampled during in-diecompression of agglomerates was used to calculate compression parametersaccording to the Heckel ( _y ), Kawakita(1/b and a), and Adams (₀)equations. Mechanical strength of single agglomerates as well as the airpermeability and tensile strength of tablets prepared from them were alsodetermined. Results. _y from the Heckelequation did not differ between agglomerates of different porosity. Both1/b and ₀ varied with agglomerate porosityand composition. These two compression parameters were linearly related toeach other. No general correlation was found between 1/b and₀ and the strength of single agglomerates. The twoparameters were related to the intergranular pore structure and tensilestrength of tablets formed from the agglomerates. Conclusions. 1/b and ₀ maybe interpreted as measures of the agglomerate shear strength during uniaxialconfined compression, and as such they may be used as indicators of thetabletting performance of the agglomerates.     

Desmethylimipramine attenuates cocaine withdrawal in rats

Depression and anhedonia are two major symptoms of cocaine withdrawal in humans. Hence, pharmacological treatments effective in depression might also alleviate the symptoms of cocaine withdrawal. In the present study, the effects of acute and repeated administration of a tricyclic antidepressant, desmethylimipramine (DMI), were investigated in naive and cocaine-withdrawing rats. An animal model of cocaine withdrawal was used that employs the elevation in intracranial self-stimulation (ICSS) thresholds following the termination of prolonged periods of cocaine self-administration as a measure of an animal's anhedonic state. The influence of chronic DMI treatment on-adrenergic receptor binding and affinity was also correlated with the behavioral signs of cocaine withdrawal. Neither acute nor repeated DMI treatment influenced reward functions in rats that were not undergoing cocaine withdrawal. However, repeated DMI treatment significantly down-regulated-adrenergic receptors, and shortened the duration of the post-cocaine anhedonia (elevation in thresholds). Furthermore, the magnitude of the-adrenergic receptor down-regulation correlated significantly with the degree of effectiveness of DMI treatment in reversing the post-cocaine anhedonia. However, chronic DMI treatment did reduce the amount of cocaine self-administered by the animals. The reversal of the post-cocaine anhedonia in this animal model of cocaine withdrawal by chronic DMI treatment demonstrates the potential usefulness of the model in identifying new pharmacotherapies for cocaine withdrawal. In addition, the results indicate that tricyclic antidepressants may be able to ameliorate some of the symptoms of cocaine withdrawal.     

Effects of bucumolol,nadolol and nifenalol on maximum upstroke velocity of action potential in guinea pig papillary muscles

Summary The effects of bucumolol (BUC), nadolol (NAD) and nifenalol (NIF) on contractile forces and on action potentials (APs) were investigated in isolated guinea pig atrial and papillary muscles, respectively. Log 1/ED₄₀ values for the negative inotropic effects of these drugs were 0.097,10 and 0.74 mmol/l in this order. BUC (50 mol/l), NAD (0.5 mmol/l) and NIF (0.2 mmol/l) produced about 60,20 and 20% reduction of V _max at 1 Hz. The frequency-dependent reductions at these and higher concentrations were greatest for BUC, intermediate for NAD and least for NIF. These potencies at certain frequencies were, as a whole, consistent with log P-potency relationship established in our previous papers (Harada et al. 1981; Ban et al. 1985). The reductions of V _max in APs in response to premature stimuli during basic stimuli at the rate of 0.25 or 0.027 Hz decayed exponentially during diastolic intervals (DI). The time constants of these decay processes () estimated by linear and nonlinear regression analyses and by eye were 12.2–9.6 s for BUC (50–100 mol/l) and 2.9–4.8 s for NAD (1–2 mmol/l) and 57–87 ms for NIF (0.2–1 mmol/l). In terms of the molecular weight (MW)-log relationship (Ban et al. 1985), these values are within the 95% fiducial limit for BUC and NAD and deviated from the lower fiducial limit for NIF. The frequency-dependent reductions of V _max by these drugs were explained in terms of a function of and the intercept A_o. Based on the study mady by Cohen et al. (1984) we estimated the distortion of the time course of recovery of V _max from that of sodium channel availability in the presence of drugs. Our computation shows that the relative change of the estimated at the same level of the zero-intercept does, but that of the estimated at different levels does not, reflext exactly that of the time constants of the recovery of sodium channel availability.     

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     

A pharmacokinetic slide rule for more accurate drug treatment

Summary A slide rule has been devised which is based on the general mathematical models of pharmacokinetics. It permits calculation of exact dosage regimens for individual patients from certain basic parameters. First, from the patient's renal clearance, the proportionality constant characterizing renal excretion of a certain drug (a) and its non-renal rate constant of elimination (k _nr), the rate constant of total elimination (k _e) can be calculated. Second, fromk _e, the apparent volume of distribution (V _d) and the desired final mean concentration of a drug (c), exact values can readily be obtained for the loading dose (D*) and the dosage schedule, which consists of the maintenance dose (D), the dosing intervals () and the infusion rate for intravenous administration. In addition the slide rule provides information about the rate at which c is reached ifD alone is administered at , and the fluctuation in the concentration around c to be anticipated during . By use of this calculation, the slide rule facilitates the decision whether a loading dose should be given, and what dosage schedule is best suited to the therapeutic problem. It is possible, therefore, to calculate exact dosage regimens for individual patients, even for those with excretory dysfunction. The slide rule should also help physicians to comprehend the nature and significance of pharmacokinetic mechanisms.     

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).     

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     

Pharmacological and molecular discrimination of brain I2-imidazoline receptor subtypes

I₂-imidazoline receptors labelled with [³H]-idazoxan in the rabbit and rat brains displayed high and low affinity, respectively, for the guanidide amiloride; reinforcing the previous definition of I_2A-imidazoline receptors expressed in the rabbit brain and Its-imidazoline receptors expressed in the rat brain. Other drugs tested displayed biphasic curves in competition experiments, indicating the existence of high and low affinity sites for both subtypes of I₂-imidazoline receptors. Among the drugs studied, bromoxidine, moxonidine, (+)- and (-)-medetomidine and clorgyline were more potent on the high and/or low affinity sites of 1_2B- than on their corresponding of I_2A-imida-zoline receptors (K _iH ratios 20 to 65). No correlation was found for the potencies of the drugs tested at the low affinity sites of both I₂-imidazoline receptor subtypes. Preincubation (30 min at 25°°C) with 10^-6 M clorgyline reduced by 60% the B _max of [³H]-idazoxan binding to I₂ B-imidazoline receptors in the rat brain, but it did not affect the binding parameters of the radioligand saturation curves to I_2A-imidazoline receptors in the rabbit brain. These results indicated that I_2A- and I_2B-imidazoline receptor subtypes differ in the pharmacological profiles of their high and low affinity sites and in the ability to irreversibly bind clorgyline. In rat cortical membranes western blot detection of immunoreactive imidazoline receptor proteins revealed a double band of 29/30 kDa and two less intense bands of 45 and 66 kDa. In rabbit cortical membranes the antibody used detected proteins of 30, 57 and 66 kDa. It is suggested that different imidazoline receptor proteins (45 vs 57 kDa) may account for the different pharmacological profiles of I2-imidazoline receptor subtypes.     

Inhibition of minK protein induced K+ channels in Xenopus oocytes by estrogens

Previously it was shown that minK protein expression in uterus is regulated by estrogen. In the present study, we were interested in putative direct effects of estrogen on minK protein induced K⁺ currents (I_minK) in Xenopus oocytes. Superfusion with 17--estradiol (1 M) resulted in an inhibition of minK-induced currents, but had no appreciable effects on the delayed rectifier and inward rectifier K⁺ channels Kv1.1 and Kir2.1, respectively. The inhibition of I_minK by 17--estradiol was concentration-dependent, with an IC₅₀ of approximately 0.5 M. In the presence of 17--estradiol, the conductance-voltage relationship was shifted to more depolarized potentials. I_minK inhibition occurred also in the presence of the estrogen-receptor antagonist tamoxifen, suggesting that a mechanism independent of estrogen receptors is involved. The synthetic estrogen diethylstilbestrol (DES) also inhibited I_minK but with a lower affinity (IC₅₀ of 4.5 M), while cortisol and progesterone had only weak effects on I_minK. In summary, the results indicate that estrogens directly inhibit I_minK.     

The significance ofβ-adrenoceptor down regulation in the desipramine action in the forced swimming test

Summary The present studies were undertaken to clarify whether central-adrenoceptor down regulation is responsible for the greater effect of chronic treatment with desipramine (DMI) compared with acute treatment in the forced swimming test in rats. Repetitive administration of DMI activated the rat behaviour pattern and consequently reduced the duration of immobility. The degree of activation depended on the length of treatment, i.e. no effect when given in a single dose, moderate effect when given subchronically (3 doses) and marked activation after chronic (31 doses) treatment. Chronic treatment with DMI also produced a decrease in³H-dihydroalprenolol (³H-DHA) binding site in the cerebral cortex. Acute stimulation of brain-adrenoceptors by intracerebroventricular (i.c.v.) isoprenaline significantly, though partially, attenuated the behavioural effect of chronic DMI by ₁-adrenoceptor-related mechanisms. Similarly, chronic i.c.v. co-administration of atenolol or practolol, ₁-adrenoceptor antagonists, together with DMI attenuated both-adrenoceptor down regulation and the behavioural activation by chronic DMI. On the other hand, chronic i.c.v administration of isoprenaline, supposedly leading to down regulation of-adrenoceptors, facilitated the activating behavioural effect of DMI, as a single dose became effective. Changes, however, in³H-DHA binding parameters in the cerebral cortex were not observed after chronic isoprenaline. These results suggest that down regulation of-adrenoceptors in brain is reponsible, at least in part, for the marked activatory effect of chronic DMI in the forced swimming test, possibly by reducing an inhibitory function of ₁-adrenoceptor mediated mechanisms.     

Saluretic effect of the loop diuretic torasemide in chronic renal failure

Summary The effects of torasemide have been studied in 7 healthy controls and 9 patients with stable chronic renal failure of various degrees.After a control period of 3 days torasemide 20 mg i. v. caused a dramatic increase in diuresis and electrolyte excretion without affecting the glomerular filtration rate. The duration of action of torasemide, , averaged 6 h and was independent of the creatinine clearance, CL_CR. When related to the drug-induced excretion of Cl^–, Na⁺, K⁺, Ca²⁺ and Mg²⁺ showed strong linear dependence on CL_CR. Both the kaliuresis and the calciuresis during were tightly correlated with the natriuresis over the broad range of CL_CR. Similarly, the excretion of Mg²⁺ was dependent on the kaliuresis.The torasemide-induced kaliuresis amounted to 12% of natriuresis, as after furosemide. The kaliuretic effect of loop diuretics is smaller than that of the thiazides. After , e. g. over a 24 h period, kaliuresis was not correlated with natriuresis. The magnitude of the rebound effect was diminished with increasing renal impairment.In memory of our colleague and friend U. Wais who left us too early