Hermann Nawrath

On the mechanism underlying the action of D-600 on slow inward current and tension in mammalian myocardium.

D-600, the methoxy derivative of verapamil, is said to affect the force of cardiac contraction and the slow inward current (Isl) specifically by reducing the membrane conductance for Ca2+ (gsi). However it is apparent that many effects of D-600 cannot be adequately explained solely by an effect on gsi. We studied the effects of D-600 on membrane current and tension of cat papillary muscle, using a conventional single sucrose gap voltage clamp technique. The results indicate that D-600 not only reduces the maximal Ca conductance but also, depending on concentration and duration of exposure, alters both the kinetics of the Ca-carrying system and the amplitude of the steady state outward current. No changes in the steady state activation and inactivation variables or in the rate of Isi inactivation were found. However, a substantial increase in the time to peak Isl, as much as 7 times normal, was observed after exposure to D-600 (0.S × 10-6 to 2.0 × 10-6 M) for at least 20 minutes. Because approximately only 75% of the reduction in lsl induced by D-600 could be attributed to change in the maximum value of gsi (gsi), we conclude that the change in time to peak and about 25% of the reduction in Isi must be due to a change in the activation kinetics of the Ca-carrying system. Calculations suggest that the time to 70% activation of gsi can be prolonged to as much as 10 times normal by prolonged exposure to negatively inotropic concentrations of D-600.

Cardioprotective effects of adenosine A1 and A 3 receptor activation during hypoxia in isolated rat cardiac myocytes

Adenosine (ADO) is a well-known regulator of a variety of physiological functions in the heart. In stress conditions, like hypoxia or ischemia, the concentration of adenosine in the extracellular fluid rises dramatically, mainly through the breakdown of ATP. The degradation of adenosine in the ischemic myocytes induced damage in these cells, but it may simultaneously exert protective effects in the heart by activation of the adenosine receptors. The contribution of ADO to stimulation of protective effects was reported in human and animal hearts, but not in rat hearts. The aim of this study was to evaluate the role of adenosine A1 and A3 receptors (A1R and A3R), in protection of isolated cardiac myocytes of newborn rats from ischemic injury. The hypoxic conditions were simulated by exposure of cultured rat cardiomyocytes (4–5 days in vitro), to an atmosphere of a N2 (95%) and CO2 (5%) mixture, in glucose-free medium for 90 min. The cardiotoxic and cardioprotective effects of ADO ligands were measured by the release of lactate dehydrogenase (LDH) into the medium. Morphological investigation includes immunohistochemistry, image analysis of living and fixed cells and electron microscopy were executed. Pretreatment with the adenosine deaminase considerably increased the hypoxic damage in the cardiomyocytes indicating the importance of extracellular adenosine. Blocking adenosine receptors with selective A1 and A3 receptor antagonists abolished the protective effects of adenosine. A1R and A3R activation during the hypoxic insult delays onset of irreversible cell injury and collapse of mitochondrial membrane potential as assessed using DASPMI fluorochrom. Cardioprotection induced by the A1R agonist, CCPA, was abolished by an A1R antagonist, DPCPX, and was not affected by an A3R antagonist, MRS1523. Cardioprotection caused by the A3R agonist, Cl-IB-MECA, was antagonized completely by MRS1523 and only partially by DPCPX. Activation of both A1R and A3R together was more efficient in protection against hypoxia than by each one alone. Our study indicates that activation of either A1 or A3 adenosine receptors in the rat can attenuate myocyte injury during hypoxia. Highly selective A1R and A3R agonists may have potential as cardioprotective agents against ischemia or heart surgery.

Inotropic and electrophysiological effects of histamine on human ventricular heart muscle.

1. The effects of histamine were investigated on mechanical and electrophysiological parameters in isolated electrically driven human ventricular papillary muscles. The effects of cimetidine and propranolol on histamine responses were also investigated. 2. The effects of histamine were compared with those of noradrenaline, isoprenaline, dimaprit, a selective H2‐receptor agonist, and a cyclic AMP derivative, 8‐(4‐chlorphenylthio) cyclic AMP. 3. The effects of histamine and dimaprit and the effects of cimetidine on histamine responses were also investigated in guinea‐pig right ventricular papillary muscles in order to allow a comparison with human papillary muscles. 4. In human papillary muscles, histamine caused concentration‐dependent increases in the force of contraction and reductions in both time‐to‐peak tension and time‐to‐half‐maximal relaxation. Histamine simultaneously caused distinct changes in the action potential configuration with increases in the height and duration of the plateau phase and an increase in the over‐all action potential duration. 5. Noradrenaline and isoprenaline produced similar responses to histamine, as did 8‐(4‐chlorphenylthio) cyclic AMP, consistent with the view that the effects of histamine as well as the beta‐adrenoceptor agonists on human ventricle, were associated with cyclic AMP mediated increases in calcium‐dependent slow inward current.

Membrane currents and tension in cat ventricular muscle treated with cardiac glycosides.

The effect of cardiac glycosides on membrane currents and tension in cat ventricular muscle was studied using the single sucrose gap voltage clamp method. Complete tension-voltage and current-voltage relations were obtained in five preparations before and during treatment with dihydro-ouabain (DHO, 1.7 × 10−5m). After 1–2 minutes of DHO, the developed tension was 15% greater than control, but there was no change in either the slow inward (calcium) current (ICa) or the level of the outward current flowing at the end of a 300-msec depolarization (Iout)- After 6–8 minutes of DHO, there was a 60% increase in developed tension, a noticeable increase in resting tension, a 20% decrease in Ica, and a smaller increase in Iout. It seems possible that the reduction of ICa was due to a reduced driving force. In preparations treated with ouabain (5 × 10−7m, 3–5 minutes), developed tension was 45–150% greater than control with no change in ICa or Iout between −45 and +15 mv. We conclude that the inotropic action of these cardiac glycosides is not mediated by an increase in I(a.

Positive inotropic response to 5-HT in human atrial but not in ventricular heart muscle

SummaryThe effects of 5-hydroxytryptamine (5-HT) on force of contraction (FC), action potential (AP) and calcium current (ICa) were studied in human right atrial and left ventricular heart muscle. 5-HT exerted a concentration-dependent increase in FC in multicellular atrial preparations; the EC50 was approximately 3 × 10−7 mol/l. Maximal increases in FC (252±58% of control values; means ± SEM, n=6) were obtained at 5-HT 10−5 mol/l. At this concentration, ICa was increased four- to sevenfold in enzymatically isolated atrial myocytes. In contrast, ventricular preparations did not respond to 5-HT; FC, AP and ICa remained unaffected. In the same preparations, FC was increased by isoprenaline three- to fourfold. These results confirm the observation that 5-HT induces a positive inotropic effect in the human atrium, possibly mediated by activation of the adenylyl cyclase — cyclic AMP system. Our study demonstrates, however, the complete lack of functional 5-HT receptors, with respect to changes in FC, in the human ventricle. Since the positive inotropic effect of 5-HT in the human heart is obviously restricted to the atrium, our findings question the concept of developing 5-HT receptor agonists for the treatment of heart failure.

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, Ito, has been investigated in rat ventricular myocytes using the whole cell patch clamp technique. Ito was activated by 300 ms depolarizing voltage clamp steps in 10 mV increments from −50 mV up to +40 mV. At +40 mV, Ito peaked after about 3 ms, and the time course of inactivation was appropriately described by two time constants, τfast = 17 ms and τslow = 203 ms. Verapamil, quinidine sulfate and nifedipine preferentially depressed Ito at the end of the 300 ms depolarizing voltage clamp step; the inactivation of Ito was accelerated by all drugs, whereas peak Ito was less affected. The time course of drug action at +40 mV was calculated by the fractional changes of Ito. Verapamil, quinidine sulfate and nifedipine exerted a block of Ito. increasing during the depolarizing voltage clamp step. The onset of block in response to verapamil, quinidine sulfate and nifedipine (30 μmol/each) was appropriately described by monoexponential functions with time constants τon = 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 Ito from inactivation with or without drugs. τoff 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 Ito in a concentration-dependent manner, whereas Ito at the end of the 300 ms depolarizing voltage step remained unaffected. The block of Ito by 4-aminopyridine (3 mmol/l) decreased during the voltage step from −50 mV to +40 mV. Relief from block was described by τoff = 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 τon = 1561 ms. Phenylephrine (30 μmol/l),papaverine (30 μmol/I) and tetraethylammonium chloride (5 mmol/l) reduced Ito 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 Ito 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 Ito independent of the channel state.

Stimulatory effects of DB-c-AMP and adrenaline on myocardial contraction and 45Ca exchange. Experiments at reduced calcium concentration and low frequencies of stimulation

SummaryThe effects of adrenaline (2.2×10−6 M) and cyclic N6-2′-O-dibutyryl-adenosine-3′,5′-monophosphate (DB-c-AMP; 10−3 M) on mechanical performance, 45Ca uptake and total tissue calcium concentration were investigated in electrically stimulated left auricles isolated from female rats weighing 180–220 g. The experiments were performed at reduced [Ca]e of 0.45 mM and at various frequencies of stimulation (0–120 beats/min). In the first series of experiments 45Ca incubation time was 5 min. Under these conditions DB-c-AMP as well as adrenaline enhanced contractile force to 300–450% of the control values at all frequencies tested (Fig.1). This increase in contractile force was accompanied by a significant enhancement in 45Ca exchange (Fig.2) while the total tissue calcium concentration remained unchanged (Table 1). In resting auricles 45Ca exchange was not altered under the influence of both drugs.At long periods of 45Ca exposure (40–90 min) both drugs augmented contractile force in a way similar to that of the first series of experiments (Fig.4) but no influence of DB-c-AMP or adrenaline on 45Ca exchange could be detected (Fig.3).It is concluded that DB-c-AMP can mimic the wellknown effects of adrenaline on myocardial calcium movements. Under the assumption that DB-c-AMP is representative for c-AMP, the results thus provide experimental support for the view that the positive inotropic effect of adrenaline is mediated by primary changes in the intracellular level of c-AMP which secondarily might enhance calcium fluxes across the cardiac cell membrane.

Possible role of cyclic AMP in the relaxation process of mammalian heart: effects of dibutyryl cyclic AMP and theophylline on potassium contractures in cat papillary muscles.

SummaryThe effect of dibutyryl cyclic AMP (DB-c-AMP; 3×10−4–3×10−3 M) on electrically induced twitch and high potassium (142.4 mM KCl)-induced contracture tension was studied in papillary muscles from normal and reserpinized cats ([Ca]0 1.8 mM; 25°C; pH 7.4). In both groups of preparations, the increase in twitch tension evoked by DB-c-AMP was accompanied by an abbreviation of the time to peak force and of relaxation time. In the same preparations, the high potassium contracture was markedly depressed by DB-c-AMP in a concentration-dependent manner. Similar results were obtained with the N6-monobutyryl derivative of cyclic AMP.The relaxing effects of the cyclic nucleotides on KCl contractures did not appear to be due to possible non-cyclic breakdown products: adenosine; 5′-AMP and sodium butyrate did not attenuate contracture tension at concentrations up to 3×10−3 M. The same applies to ATP and non-cyclic N6-2′-O-3′-O-tributyryl-adenosine-monophosphate. Theophylline (10−2 M) was found to prolong the relaxation time of the twitch and to enhance the high KCl contracture.It is concluded that cyclic AMP may be capable of modulating the relaxation process of mammalian heart and that not only the positive inotropic but also the relaxant effects of catecholamines on myocardium described before may be mediated by the cyclic AMP system. The relaxant effects of cyclic AMP derivatives on intact myocardial preparations are attributed to a stimulation by cyclic AMP of the calcium transport of the sarcoplasmic reticulum (SR) and are interpreted to be a corollary to the effects of cyclic AMP previously obtained on isolated SR preparations.

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.

Influence of cyclization and acyl substitution on the inotropic effects of adenine nucleotides

SummaryThis study was designed to further elucidate relevance and mechanism of the positive inotropic action of cyclic N6-2′-O-dibutyryl-AMP (DB-c-AMP). For this purpose the effects of cyclic N6-monobutyryl-AMP (N6-MB-c-AMP), noncyclic N6-2′-O-3′-O-tributyryl-5′-AMP (TB-AMP), c-AMP, adenosine and various adenine nucleotides (ATP, ADP, AMP) on myocardial contractile force (CF) were investigated and compared to that of DB-c-AMP. The experiments were performed on isolated, electrically driven (frequency 2 Hz) rat left auricles, i.e. on a preparation in which DB-c-AMP consistently produced positive inotropic effects. The following results were obtained:1.N6-MB-c-AMP (2×10−4–5×10−3 M) produced a concentration-dependent positive inotropic effect (Fig. 2). This effect began 5.5 to 1.8 min after addition of the drug (Table 2), developed gradually (Fig.2), and was maximal within 44.5 to 9.5 min (Table 2). It was preceded by a transitory decrease in CF which was also concentration-dependent (Fig.2, Table 1).2.The inotropic effects of cyclic N6-MB-AMP and cyclic DB-AMP were qualitatively similar. Quantitatively, the positive inotropic effect of N6-MB-c-AMP developed more slowly (Table 2) and was smaller in magnitude than that of DB-c-AMP (Fig.2). The initial negative inotropic effect, however, was more pronounced (Table 1) and longer in duration (Table 2) with N6-MB-c-AMP than with DB-c-AMP.3.The time course of the reversal of the positive inotropic effects during washout in drug-free solution was practically not different with both drugs (Fig.3). Predrug levels were reached within 50–60 min.4.No positive inotropic effects were found with the non-cyclic but butyryl substituted adenine nucleotide, TB-AMP. In contrast, this compound (10−7 to 10−3 M) produced a concentration-dependent decrease in CF (Fig.4).5.c-AMP, ATP, ADP, AMP and adenosine depressed CF. No secondary positive inotropic effects could be detected within 60 min (Fig.5; Fig.6). From the failure of non-cyclic TB-AMP to increase contractile force it is concluded that the positive inotropic effects seen with cyclic DB-AMP and cyclic N6-MB-AMP are due to both acyl substitution and the intact cyclophosphate structure. In so far we probably have ruled out one of the objections to the view that the positive inotropic responses to acyl substituted derivatives of c-AMP are “representative” for c-AMP itself. Since the effects of cyclic DB-AMP and cyclic N6-MB-AMP were qualitatively similar and since the time course of the loss of the positive inotropic effects during washout were practically not differen with both drugs, one may further conclude that within the cell both drugs are likely to act via the same compound. This agent could be c-AMP. However, cyclic N6-MB-AMP as the active intracellular compound cannot be ruled out.