Jörg W. Wegener

Activation of soluble guanylyl cyclase by YC‐1 in aortic smooth muscle but not in ventricular myocardium from rat

1 The effects of YC‐1 (3‐(5′‐hydroxymethyl‐2′‐furyl)‐1‐benzyl indazole), an activator of soluble guanylyl cyclase, on tension, levels of cyclic GMP and cyclic AMP, and cardiac L‐type Ca2+‐current (ICa(L)) were investigated in aortic smooth muscle and ventricular heart muscle from rat. 2 YC‐1 (0.1–30 μM) induced a concentration‐dependent relaxation in aortic rings precontracted with phenylephrine (3 μM). The relaxant effects of YC‐1 were reversed by 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (30 μM; ODQ), potentiated by zaprinast (10 μM) and antagonized by Rp‐8‐Br‐cGMPS (100 μM). 3 In ventricular heart muscle strips, YC‐1 (30 μM) exhibited no effects on force of contraction (Fc) in the absence or presence of either zaprinast (10 μM) or 3‐isobutyl‐1‐methylxanthine (30 μM). Fc was slightly increased by YC‐1 (30 μM) in the presence of isoprenaline (100 nM), but this effect was not influenced by ODQ (30 μM). 4 Cardiac ICa(L) was not significantly affected by YC‐1 (30 μM), either in the absence or presence of isoprenaline (30 nM). 5 In aortic rings, cyclic GMP levels were increased almost 3 fold by YC‐1 (30 μM); this effect was abolished by ODQ (30 μM). In isolated ventricular cardiomyocytes, cyclic GMP levels were not affected by YC‐1 (30 μM) but almost doubled by activation of particular guanylyl cyclase with atriopeptin II (100 nM). 6 YC‐1 (30 μM) did not increase cyclic AMP levels either in aortic rings or in ventricular cardiomyocytes. In contrast, isoprenaline (3 μM) increased cyclic AMP levels about two fold in both tissues. In cardiomyocytes, the effect of isoprenaline (3 μM) was slightly enhanced by YC‐1 (30 μM). 7 It is concluded that relaxation of smooth muscle preparations by YC‐1 is mediated mainly by activation of soluble guanylyl cyclase and subsequent increase in cyclic GMP levels. The failure of YC‐1 to affect cardiac Fc, levels of cyclic GMP, and ICa(L) suggests that soluble guanylyl cyclase is not influenced by YC‐1 in rat heart muscle or only barely present in this tissue.

Cardiac effects of isoliquiritigenin

The effects of isoliquiritigenin on force of contraction (Fc), L-type Ca2+ current (I(Ca)) and intracellular Ca2+ concentration ([Ca2+]i) were investigated in rat ventricular heart muscle. Isoliquiritigenin increased Fc and I(Ca) and, after longer exposure times, resting tension and [Ca2+]i. The effect of isoliquiritigenin (100 microM) on I(Ca) was diminished by Rp-cAMPS (30 microM). 1H-[1,2,4]oxa- diazolo[4,3-a]quinoxalin-1-one (50 microM) did not influence the effects of isoliquiritigenin on Fc and I(Ca). The positive inotropic effects of isoprenaline and forskolin, but not of 3-isobutyl-1-methylxanthine, were potentiated by isoliquiritigenin (100 microM). In the presence of milrinone (10 microM), no further effects of isoliquiritigenin (100 microM) on Fc and I(Ca) were observed. It is suggested that the increase in Fc and I(Ca) by isoliquiritigenin is due to an accumulation of cyclic AMP. These effects are probably unrelated to an effect of the drug on soluble guanylyl cyclase, as reported for smooth muscle, but rather due to a direct inhibition of phosphodiesterase III activity.

Kinetics and state-dependent effects of verapamil on cardiac L-type calcium channels

Abstract The voltage dependence and the kinetics of block by verapamil of L-type calcium current (ICa) were investigated in ventricular myocytes from rat hearts using the whole-cell patch-clamp technique. ICa was elicited repetitively in response to depolarizing voltage pulses from –80 mV to 0 mV at different pulse intervals and durations. Verapamil reduced the magnitude of ICa in a frequency-dependent manner without tonic component. The time course of ICa remained unchanged suggesting that not open but inactivated channels were affected by the drug. The interaction of verapamil with inactivated channels was investigated by the application of twin pulses. In the presence of verapamil, the duration of the first pulse significantly determined the magnitude of ICa during the second pulse. Variation of the duration of the first pulse between 12 and 3000 ms, followed by a pulse interval of 100 ms, resulted into a gradual decrease of ICa during the second pulse (180 ms), described by concentration-dependent monoexponential decay curves (t = 1060 ± 138 ms at 0.3 μM (n = 3); t = 310 ± 24 ms at 1 μM (n = 6), and t = 125 ± 7 ms at 10 μM (n = 5); means ± SEM). Under control conditions, the changes in ICa were comparably negligible. The recovery of ICa from block was analyzed by the application of a twin pulse protocol in which two depolarising voltage pulses at fixed length (1. pulse at 3 s and 2. pulse at 180 ms) were interrupted by variable pulse intervals (6 ms–60 s). Under control conditions, recovery from inactivation was fast (t = 11 ± 0.7 ms; means ± SEM; n = 3). In the presence of verapamil, recovery from block was about 500times slower than under control conditions, independent of the drug concentration (t = 5.05 ± 0.44 s at 0.3 μM (n = 3), t = 6.7 ± 0.69 s at 1 μM (n = 4), and t = 6.02 ± 0.9 s at 10 μM (n = 5); means ± SEM). Since development of block was dependent on the concentration of verapamil, whereas recovery from block was independent from the drug concentration, it is assumed that the described time constants for block and unblock reflect voltage-dependent net binding (τon) and unbinding (τoff), respectively, of verapamil at its receptor sites. A computer simulation, including the time constants of block development at 0 mV and of recovery from block at –80 mV, predicted reasonably well the observed frequency-dependent block of ICa by verapamil. The development of either measured or calculated block of ICa, using 180 ms depolarising voltage pulses from –80 mV to 0 mV, was fitted by identical monoexponential association curves (t = 7 s each at 0.2 Hz and t = 1.7 s each at 1 Hz). When Ba2+ was used as the charge carrier, which removes the calcium-dependent inactivation of the current, verapamil (3 μM) was less efficient: ICa was decreased by 57 ± 6 % (means ± SEM; n = 6), whereas IBa was decreased by 24 ± 4 % (means ± SEM; n = 5). It is proposed that verapamil binds to calcium channels in their inactivated state at more positive potentials and dissociates from the channels in the resting state at more negative potentials. In the proposed scheme of periodical drug binding and unbinding, dependent on the state of the channels, the development of frequency-dependent block of ICa by verapamil is adequately predicted by the construction of cumulative association/dissociation curves which include the experimentally determined time constants of development and recovery from block at 0 mV and –80 mV, respectively.

Extracellular site of action of phenylalkylamines on L-type calcium current in rat ventricular myocytes

The effects of the phenylalkylamines verapamil, gallopamil, and devapamil on L-type calcium currents (ICa) were studied in ventricular myocytes from rat hearts using the whole-cell patch-clamp technique. In particular, the question was addressed, whether the pharmacological binding sites for these drugs were located at the inner and/or at the outer surface of the cell membrane. Therefore, tertiary verapamil, gallopamil, and devapamil and their corresponding quaternary derivatives were applied either from the outside or the inside of the cell membrane. Extracellular application of verapamil, gallopamil and devapamil (each at 3 μM) reduced Ica to 16.1 ±8.6%, 11 ± 8.9 %, and 9.3 ± 6 % of control, respectively. Intracellular application of the same substances, via the patch pipette filled with 30 μM of either verapamil, gallopamil, or devapamil, failed to depress ICa. The quaternary derivatives of the phenylalkylamines (30 μM) were ineffective both when applied extracellularly or intracellularly. It is suggested that phenylalkylamines block ICa in ventricular myocytes by acting on a binding site of the calcium channel molecule located at the outer surface of the cell membrane.

Effects of nitric oxide donors on cardiac contractility in wild-type and myoglobin-deficient mice

The effects of the nitric oxide (NO) donors S‐nitroso‐N‐acetylpenicillamine (SNAP), sodium(Z)‐1‐(N,N‐diethylamino)diazen‐1‐ium‐1,2‐diolate (DEA‐NONOate), and (Z)‐1‐[N‐(2‐Aminoethyl)‐N‐(2‐ammonioethyl)amino]diazen‐1‐ium‐1,2‐diolate (DETA‐NONOate) on force of contraction (Fc) were studied in atrial and ventricular muscle strips obtained from wild‐type (WT) and myoglobin‐deficient (myo−/−) mice. SNAP slightly reduced Fc in preparations from WT mice at concentrations above 100 μM; this effect was more pronounced in myo−/− mice. DEA‐NONOate reduced Fc in preparations from myo−/− mice to a larger extent than those from WT mice. DETA‐NONOate reduced Fc in preparations from myo−/− but not from WT mice. Pre‐incubation with an inhibitor of the soluble guanylyl cyclase (1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one; 100 μM) prevented the effects of SNAP, DEA‐NONOate and DETA‐NONOate on Fc in myo−/− mice. It is suggested that, in physiological conditions, myoglobin acts as intracellular scavenger preventing NO from reaching its intracellular receptors in cardiomyocytes, whereas, in myoglobin‐deficient conditions, NO is able to reduce contractility via activation of the soluble guanylyl cyclase/cyclic GMP pathway.

Failure of 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ) to inhibit soluble guanylyl cyclase in rat ventricular cardiomyocytes

The effects of 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ), an inhibitor of soluble guanylyl cyclase (sGC), were investigated in aortic rings and ventricular cardiomyocytes from rats. The production of cyclic GMP was stimulated by NO•‐donors or carbachol. Additionally, the effects of ODQ were studied in cytosolic extracts from both tissues in which the cyclic GMP production was stimulated by S‐nitroso‐N‐acetylpenicillamine (SNAP). In endothelium‐intact aortic rings, SNAP (100 μM), 2,2′‐(hydroxynitrosohydrazino)bis‐ethanamine (DETA NONOate; 100 μM), or carbachol (10 μM) increased cyclic GMP levels about 4 fold. These effects were abolished by ODQ (50 μM). In cardiomyocytes, SNAP (100 μM), DETA NONOate (100 μM), or carbachol (10 μM) increased cyclic GMP levels about 2 fold. These effects were not affected by ODQ (50 μM). In cytosolic extracts from aortic rings and cardiomyocytes, SNAP (100 μM) induced about 50 fold increases in cyclic GMP levels. ODQ (50 μM) reduced these effects by about 50%. In extracts from cardiomyocytes, increases by SNAP (100 μM) of cyclic GMP levels were attenuated by myoglobin dependent on concentration: at 300 μM myoglobin, SNAP (100 μM) increased cyclic GMP levels only 3 fold. Inhibitory effects of ODQ (50 μM) were abolished by 300 μM myoglobin. It is suggested that both NO• and ODQ can bind to myoglobin which, at high concentrations, can diminish their effects on sGC. Such a scavenger function of myoglobin could explain why NO• and ODQ exert only minor effects in cardiomyocytes (with high myoglobin content), but strong effects in aortic tissue (virtually devoid of myoglobin).

Effects of quinine and quinidine on the transient outward and on the L-type Ca2+ current in rat ventricular cardiomyocytes

The effects of the enantiomers quinine and quinidine on the transient outward current (I_to) and on the L-type Ca²⁺ current (I_Ca) were investigated in rat ventricular cardiomyocytes using the patch-clamp technique. At a stimulation frequency of 2 Hz, both quinine and quinidine depressed the magnitude of I_to and I_Ca; the half-maximal effects on I_to were achieved at 11 and 15 µmol/l, respectively, and those on I_Ca at 14 and 10 µmol/l, respectively. At 0.2 Hz, both drugs depressed the magnitude of I_to, but not that of I_Ca. A change in extracellular pH from 7.3 to 8.3 did not significantly influence the effects of the drugs (which are protonated to 98% at pH 7.3) on I_to or I_Ca. It is concluded that neither the different chemical structure nor the amount of protonation of quinine and quinidine controls their effects on I_to or I_Ca.

Barnidipine block of L-type Ca2+ channel currents in rat ventricular cardiomyocytes

The effects of barnidipine and nifedipine on L‐type Ca2+ current (ICa(L)) were investigated in ventricular cardiomyocytes from rats. Both barnidipine and nifedipine reduced ICa(L) in a concentration and voltage dependent manner; the EC50 were 80 and 130 nM at a holding potential of −80 mV, respectively, and 18 and 6 nM at −40 mV, respectively. Both drugs induced a leftward shift of the steady‐state inactivation curve of ICa(L). Using a twin pulse protocol, the relationships between the amount of block of ICa(L) by either drug, seen during the second pulse, and the length of the first pulse were described by monoexponential functions reflecting onset of block, dependent on drug concentration. The onset of block by barnidipine was three times faster than that by nifedipine. With both drugs, recovery of ICa(L) was 50 times slower than under control conditions and described by monoexponential functions reflecting offset of block (independent of drug concentration). The offset of block with barnidipine was three times slower than that with nifedipine. The time constants of block and unblock of ICa(L) by both drugs were used to calculate binding and unbinding and to predict their effects at two frequencies. It is suggested that barnidipine exhibits a higher affinity to the inactivated Ca2+ channel state as compared to nifedipine.

Action of tertiary phenylalkylamines on cardiac transient outward current from outside the cell membrane

The effects of the phenylalkylamines verapamil (V), gallopamil (G), and devapamil (D) and their corresponding quaternary derivatives on the transient outward current (Ito) were examined in rat ventricular cardiomyocytes using the whole-cell patch-clamp technique. The question was addressed, whether phenylalkylamines act on Ito from the inside or the outside or from both sides of the cell membrane. To this end, the myocytes were either superfused extracellularly or perfused intracellularly with drug-containing solutions. In addition, the effects of verapamil were investigated at different pH-values. V, G, and D (30 μM each), applied extracellularly, reduced the steady state current of Ito, Ito(150 ms), to 34 ± 3.3, 33 ± 6, and 30 ± 5, respectively (% of control; means ± SEM). The effects of V (30 μM) on Ito were similar at various external pH-values (reduction of Ito(15o ms) by 69 ± 6 at pH 6.5, by 66 ± 4 at pH7.4, by 68 ± 8 at pH 8.5, and by 58 ± 0 at pH 9.5; % of control; means ± SEM). In contrast, the effect of 4-aminopyridine (300 μM) on Ito was enhanced after alkalinisation: the peak current of Ito was reduced to 49 ± 5 at pH 7.4 and to 5 ± 2 at pH 9.2 (% of control; means ± SEM). V, G, and D (300 μM) failed to produce any effect on Ito, when applied intracellularly (values of Ito(150 ms): 97 ± 6, 105 ± 4, and 94 ± 4, respectively; % of control; means ± SEM). In contrast, 4-aminopyridine (3 mM) depressed the peak current of Ito to 69 ± 6% of control (mean ± SEM), when applied intracellularly. The permanently charged quaternary derivatives of the phenylalkylamines q-V, q-G, and q-D (300 μM) did not significantly affect Ito, when applied extracellularly (values of Ito(150 ms): 94 ± 2, 90 ± 3, and 94 ± 3, respectively; % of control; means ± SEM) but diminished Ito, when applied intracellularly (reduction of Ito(150 ms) to 43 ± 5, 56 ± 7, and 63 ± 4, respectively; % of control; means ± SEM). Intracellularly applied V (300 μM) did not reduce Ito at pH 6.5 at which V is protonated to 99.4%. It is suggested that tertiary phenylalkylamines act on Ito by binding to a membrane site accessible from the outside, whereas their quaternary derivatives affect Ito by binding to a membrane site located at the inside of the cell membrane. In contrast, 4-aminopyridine is supposed to act on Ito from the inside of the cell membrane.

Alkalinisation does not modify the effect of verapamil on myocardial Ca2+ current

The effect of verapamil on L-type Ca2+ current (ICa) was compared at external pH 7.4 and 8.5 in rat ventricular myocytes. Alkalinisation increases the fraction of uncharged molecules of verapamil (pK 8.75) and thereby facilitates membrane permeation of the drug. Verapamil (1 microM) reduced the amplitude of ICa (ICa(peak)) by 36 +/- 4% at pH 7.4 and by 40 +/- 6% at pH 8.5, whereas alkalinisation from pH 7.4 to 8.5, without drug, increased ICa(peak) by 12 +/- 3%. It is suggested that the efficiency of verapamil is not influenced by the amount of protonation or membrane permeation.