Dibutyryl cyclic AMP and adrenaline increase contractile force and 45Ca uptake in mammalian cardiac muscle

Summary The effects of dibutyryl cyclic AMP (DB-AMP; 10^–3M) and adrenaline (2.2×10^–6 M) on contractile force, ⁴⁵Ca uptake, and total myocardial Ca concentration were investigated in electrically driven left auricles isolated from rat hearts. The experiments were performed at an extracellular Ca concentration of 0.45 mM and at low frequency of stimulation (15 beats/min). ⁴⁵Ca exposure was 5 min. Under the conditions used, both drugs increased contractile force and enhanced ⁴⁵Ca uptake (expressed as relative specific activity) by about 30% (DB-AMP) and 40% (adrenaline), respectively. Thus, the results provide evidence that the effects of adrenaline on ⁴⁵Ca uptake in mammalian cardiac muscle can be mimicked by DB-AMP.     

Temperature dependence of the effects of isoprenaline, aminophylline and calcium ionophores on the isometric contraction of the isolated hemidiaphragm of the rat.

Both parameters of isometric contraction, Td and dT/dt max, of the isolated hemidiaphragm of the rat, as measured during the action of isoprenaline in the course of direct electrical stimulation, were significantly affected by lowering the temperature of the bath to 18°C. The action of isoprenaline was only depressed, whereas the effect of aminophylline was completely blocked by this procedure. The effect of calcium chloride in antagonizing the blocking action of di-Na-EDTA was even stronger at 18°C than at 36°C. Calcium ionophores Ro-2-2985 and A 23187 produced an increase in Td and dT/dt max when used in lower concentrations, but higher concentrations regularly produced depression or block of the isometric contraction. The increasing effect of Ro-2-2985 on Td and dT/dt max was completely reversed by lowering the temperature to 18°C. It was shown that at 18°C neither calcium nor isoprenaline antagonized the blocking action of verapamil on Td and dT/dt max, thus indicating the alteration of calcium channels at low temperatures. A 23187 potentiated the action of isoprenaline on Td and dT/dt max, whereas Ro-2-2985 blocked it. Similarly, the inrcreasing effect of db-cAMP on Td and dT/dt max was reversed by Ro-2-2985. It is concluded that both lowering the temperature of the medium and the calcium ionophores produce changes in the response to isoprenaline and aminophylline, during direct electrical stimulation, in a way which is compatible with the view that the interaction between calcium and cAMP system is essential to the isometric contraction of skeletal muscle.     

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

Summary The 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.     

Interactions of calcium,dibutyryl cyclic AMP,isoprenaline and aminophylline on the isometric contraction of the isolated hemidiaphragm of the rat

Summary Both di-Na-EDTA and verapamil depressed the tension (T) and the maximum rate of rise of tension (dT/dt _max) of twitch responses of the isolated hemidiaphragm of the rat to direct electrical stimulation. Depression was preceded by a transient facilitation. The blocking action of di-Na-EDTA was promptly reversed by calcium chloride, whereas the same procedure failed to antagonize the blocking action of verapamil. Isoprenaline and db-cAMP were found to antagonize the blocking action of verapamil.In calcium-free medium verapamil quickly produced block of isometric contractions. The depression of contraction produced by verapamil in calcium-free medium was only slightly or not restored by isoprenaline and db-cAMP. This indicates that the membrane calcium is indispensable for the action of isoprenaline and db-cAMP.The effect of aminophylline on T and dT/dt _max depends markedly on calcium in the external medium. In a calcium-free solution, as well as in the presence of verapamil, aminophylline failed to produce, any change in the isometric contraction. It is concluded that the actions of isoprenaline, db-cAMP and aminophylline on the isometric contractions of the isolated hemidiaphragm of the rat produced by direct electrical stimulation are possible only in the presence of an optimal concentration of external calcium and of functionally intact calcium channels in the membrane.     

The effects of cocaine and denervation on the sensitivity to noradrenaline,its uptake and the termination of its action in isolated venous tissue

Summary The action of staphylococcal alpha toxin (ST) on potential difference (PD) and short-circuit current (SCC) of the isolated frog skin was studied. Ringer solution bathed the corial side and 20 mM NaCl bathed the epidermal side. PD and SCC decreased to about half after the administration of ST to the corial side of the skin. Later, SCC raised considerably. The replacement of 20 mM NaCl by KCl on the epidermal side of the ST-pretreated skin did not cause any substantial decrease of PD, while in the untreated skin the same replacement caused a sharp drop of PD. No secondary increase of SCC was observed after ST administration when the Ringer solution bathing the corial side of the skin contained 1/2 S0₄ ^– instead Cl^–. In contrast to the normal skin, dilution from 120 to 2 mM of the NaCl solution on the epidermal side led to a PD increase in the toxin-treated skin.Na⁺ fluxes across the skin in both directions were measured by means of radioisotopes. The direction of net flux of Na⁺ was reversed after treatment with the toxin. The results demonstrated two phases of ST action. Na⁺ transport is damaged in the first phase; in the second phase an outflux of Cl^– is induced.The changes of water permeability of the frog urinary bladder were determined by weighing bags formed from the bladders. The addition of the toxin to the medium bathing the serosal side resulted in increased weight losses. The transport of water was increased.A preliminary account of some of these results was presented (Kadlec and apek, 1969).     

Presynaptic effects of scopolamine,oxotremorine, noradrenaline and morphine on [3H] acetylcholine release from the myenteric plexus at different stimulation frequencies and calcium concentrations

Summary The inhibition by three modulators (oxotremorine, noradrenaline, morphine) of acetylcholine release from the myenteric plexus preincubated with [³H]choline was investigated at different stimulation frequencies and calcium concentrations. Moreover, [³H]acetylcholine release evoked by a low (0.1 Hz) or a high (10 Hz) stimulation rate was investigated at different calcium concentrations either in the absence or presence of scopolamine. A reduced calcium concentration (0.6 mmol/l) inhibited acetylcholine release more at 0.1 Hz (74% ± 3%) than at 10 Hz (44% ± 8%). Scopolamine enhanced the stimulated acetylcholine release at a calcium concentration of 1.8 mmol/l. At calcium concentrations higher than 1.8 mmol/l scopolamine failed to enhance transmitter release markedly. A reduction of the calcium concentration (< 1.8 mmol/l) significantly enhanced the effect of scopolamine, when acetylcholine release was evoked at 0.1 Hz. Oxotremorine (10 mol/l) completely suppressed acetylcholine release at 1 Hz (120 pulses). When 120 pulses were applied at 10 Hz the maximal effect was only a 64% inhibition and the concentration-response curve was significantly shifted to the right. However, after a reduction of both the train length or the calcium concentration oxotremorine produced a complete inhibition of acetylcholine release evoked at 10 Hz. In contrast to the effect of oxotremorine, the concentration-response curves for morphine and noradrenaline were similar at 1 Hz and 10 Hz. Following conclusions can be drawn: 1. The present findings fit into the concept that residual calcium accumulates in the nerve terminal during 10 Hz stimulation. 2. The results obtained with scopolamine and oxotremorine are consistent with the view that muscarine autoreceptor activation triggers a reduction of the intraneuronal availability of calcium for the stimulus-secretion coupling. 3. The presynaptic effect of morphine and partly that of noradrenaline might be mediated by a different mechanism, probably by a reduction of release sites. Send offprint requests to I. Wessler at the above address     

Weak effects of local and systemic administration of the GABA uptake inhibitor,SK&F 89976, on extracellular GABA in the rat striatum

Summary The effects of local and systemic administration of the potent GABA uptake inhibitor, SK&F 89976, on GABA overflow from the striatum of conscious rats were investigated in brain dialysis experiments. Administration of the compound via the dialysis probe at concentrations of 25 or 100 gmol/l significantly increased basal GABA overflow about 2-fold. Overflow evoked by 104 mmol/l K⁺ remained unaltered at the lower and was almost doubled at the higher concentration; this increase did, however, not reach statistical significance.Given systemically at 50 mg/kg i.p., a dose which is severalfold higher than those which exhibit anticonvulsant effects, SK&F 89976 caused a significant enhancement of K⁺-stimulated GABA overflow by about a factor of 2; the lower dose of 20 mg/kg i.p. was not effective. Basal GABA overflow was not significantly increased by either dose. These results suggest that the marked effects of nipecotic acid on basal GABA overflow reported by several authors seem to be related to GABA displacement rather than uptake inhibition, and that uptake inhibition does not improve the interpretability of measurements of GABA release by brain dialysis. They neither support the idea that the relative insensitivity of extracellular GABA to low Ca²⁺ and tetrodotoxin is indirectly due to very efficient removal of GABA by neuronal and/or glial uptake, leaving only residual amounts to be measured. Send offprint requests to P. Waldmeier at the above address     

Evidence for adenosine receptor-mediated isoprenaline-antagonistic effects of the adenosine analogs PIA and NECA on force of contraction in guinea-pig atrial and ventricular cardiac preparations

Summary The effects of the adenosine agonists (–)-N⁶-phenylisopropyladenosine (PIA) and 5-N-ethylcarboxamideadenosine (NECA) on force of contraction, adenylate cyclase activity and normal as well as slow action potentials were studied in guinea-pig isolatedatrial (left auricles) andventricular preparations (papillary muscles).Inauricles PIA and NECA exerted concentration-dependent negative inotropic effects with similar potenticies (mean EC₅₀:0.05 mol l^–1 for PIA and 0.03 mol l^–1 for NECA). Similar results were obtained in the presence of isoprenaline.Inpapillary muscles PIA and NECA alone had no effect on force of contraction but produced negative inotropic effects in the presence of isoprenaline (mean EC₅₀:0.19 mol l^–1 for PIA and 0.10 mol l^–1 for NECA).In both preparations, the negative inotropic effects of PIA and NECA in the presence of isoprenaline were antagonized by the adenosine receptor antagonist 8-phenyltheophylline.In both preparations, PIA and NECA did not affect adenylate cyclase activity, both in the absence and presence of isoprenaline.Inauricles the negative inotropic effects of both nucleosides were accompanied by shortening of the action potential. This effect was also observed in the presence of isoprenaline. Inpapillary muscles the adenosine analogs did not detectably alter the shape of the normal action potential. Ca²⁺-dependent slow action potentials elicited in potassium-depolarized preparations also remained unaltered in the presence of PIA or NECA alone. However, the isoprenaline-induced enhancement of the maximal rate of depolarization of slow action potentials was attenuated by PIA or NECA.It is concluded that in guinea-pig atrial and ventricular cardiac preparations the adenosine analogs PIA and NECA exert isoprenaline-antagonistic effects on force of contraction via adenosine receptors the existence of which can thus be shown in a functional way. These receptors are not detectably coupled to the adenylate cyclase. The negative inotropic effect in theauricle is most likely due to a shortening of the action potential resulting from an activation of potassium channels, which in turn indirectly reduces the Ca²⁺ influx during the action potential. In theventricle the adenosine receptor is either not linked to these potassium channels or adenosine-sensitive potassium channels do not exist in the ventricle. Instead the activation of the receptor causes a decrease of the slow Ca²⁺ inward current but this effect is observed only when the slow Ca²⁺ inward current had previously been enhanced by a cyclic AMP-dependent mechanism.     

The effect of various cardenolides and bufadienolides with different cardiac activity on the 86Rubidium uptake of human erythrocytes

Summary The inhibitory effect of 23 cardiac glycosides, genins and derivatives on the ⁸⁶Rb-uptake of human erythrocytes was measured. Proscillaridin and its acetates, methyl ethers and -epoxide were the most active inhibitors of ⁸⁶Rb-uptake, followed by ouabain, digitoxin and digoxin. Inhibition induced by genins occurred at concentrations distinctly higher than with the glycosides. Substances with 3--configuration were less active than those with 3--configuration.The glycoside concentrations exerting half maximal inhibition of ⁸⁶Rb-uptake were correlated with the minimum lethal doses in guinea pigs (r=0.71) and cats (r=0.75).     

The effects of levosimendan and OR-1896 on isolated hearts, myocyte-sized preparations and phosphodiesterase enzymes of the guinea pig

The concentration dependences of the Ca(2+)-sensitizing and the phosphodiesterase-inhibitory effects of levosimendan (the (-) enantiomer of [[4-(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-pyridazinyl)phenyl]hydrazono]propanedinitrile) and its active metabolite, OR-1896 (the (-) enantiomer of N-[4-(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-pyridazinyl)phenyl] acetamide), were compared with their positive inotropic effects to reveal their mechanisms of action in guinea pig hearts. In Langendorff-perfused hearts, left ventricular +dP/dt(max) increased by 26+/-4% and 25+/-3% (mean+/-S.E.M.), with EC(50) values of 15+/-2 and 25+/-1 nM for levosimendan and OR-1896, respectively. In permeabilized myocyte-sized preparations, levosimendan and OR-1896 both increased isometric force production via Ca(2+) sensitization (at pCa 6.2), by 51+/-7% and 52+/-6%, with EC(50) values of 8+/-1 and 36+/-7 nM (P<0.05), respectively. Thus, the two molecules could be defined as Ca(2+) sensitizers and positive inotropes with very similar concentration dependences. However, major differences appeared when the phosphodiesterase-inhibitory effects of levosimendan and OR-1896 were probed on the two phosphodiesterase isoforms (phosphodiesterases III and IV) dominant in the left ventricular cardiac tissue. Levosimendan was a 40-fold more potent and a 3-fold more selective phosphodiesterase III inhibitor (IC(50) for phosphodiesterase III=2.5 nM, and IC(50) for phosphodiesterase IV=25 microM, selectivity factor approximately 10000) than OR-1896 (IC(50) for phosphodiesterase III=94 nM, and IC(50) for phosphodiesterase IV=286 microM, selectivity factor approximately 3000). Hence, our data support the hypothesis that levosimendan and OR-1896 both exert positive inotropy via a Ca(2+)-sensitizing mechanism and not via simultaneous inhibition of the phosphodiesterases III and IV isozymes in the myocardium at their maximal free plasma concentrations.     

Effect of the triaminopyridine flupirtine on calcium uptake,membrane potential and ATP synthesis in rat heart mitochondria

1. Flupirtine is an analgesic agent which exhibits neuronal cytoprotective activity and may have value in the treatment of conditions involving cell injury and apoptosis. Since flupirtine has no action on known receptor sites we have investigated the effect of this drug on mitochondrial membrane potential, and the changes in intramitochondrial calcium concentration in particular.
2. The findings show that flupirtine increases Ca²⁺ uptake in mitochondria in vitro. At clinically relevant flupirtine concentrations, corresponding to flupirtine levels in vitro of 0.2 to 10 nmol mg⁻¹ mitochondrial protein, there was a 2 to 3 fold increase in mitochondrial calcium levels (P<0.01). At supra-physiological flupirtine concentrations of 20 nmol mg⁻¹ mitochondrial protein and above, the mitochondrial calcium concentrations were indistinguishable from those in untreated mitochondria.
3. Mitochondrial membrane potential closely paralleled the changes in mitochondrial calcium levels showing a 20% (P<0.01) increase when the flupirtine concentration was raised from 0.2 nmol to 10 nmol mg⁻¹ mitochondrial protein and a return to control values at 20 nmol mg⁻¹ protein.
4. The increase in mitochondrial calcium uptake and membrane potential were accompanied by an increase in mitochondrial ATP synthesis (30%; P<0.05) and a similar percentage reduction in mitochondrial volume.
5. Calcium at 80 and 160 nmol mg⁻¹ mitochondrial protein decreased ATP synthesis by 20–25% (P<0.001). This decrease was prevented or diminished if flupirtine at 10 nmol mg⁻¹ protein was added before the addition of calcium.
6. Since intracellular levels of flupirtine in intact cells never exceeded 10 nmol mg⁻¹ mitochondrial protein, these findings are supportive evidence for an in vivo cytoprotective action of flupirtine at the mitochondrial level.

     

The effects of nifedipine and other calcium antagonists on the glibenclamide-sensitive K+ currents in smooth muscle cells from pig urethra

1. The effects of nifedipine on both levcromakalim-induced membrane currents and unitary currents in pig proximal urethra were investigated by use of patch-clamp techniques (conventional whole-cell configuration and cell-attached patches).
2. Nifedipine had a voltage-dependent inhibitory effect on voltage-dependent Ba²⁺ currents at −50 mV (K_i=30.6 nM).
3. In current-clamp mode, subsequent application of higher concentrations of nifedipine (⩾30 μM) caused a significant depolarization even after the membrane potential had been hyperpolarized to approximately −82 mV by application of 100 μM levcromakalim.
4. The 100 μM levcromakalim-induced inward current (symmetrical 140 mM K⁺ conditions, −50 mV) was inhibited by additional application of three different types of Ca antagonists (nifedipine, verapamil and diltiazem, all at 100 μM). In contrast, Bay K 8644 (1 μM) possessed no activating effect on the amplitude of this glibenclamide-sensitive current.
5. When 100 μM nifedipine was included in the pipette solution during conventional whole-cell recording at −50 mV, application of levcromakalim (100 μM) caused a significant inward membrane current which was suppressed by 5 μM glibenclamide. On the other hand, inclusion of 5 μM glibenclamide in the pipette solution prevented levcromakalim from inducing an inward membrane current.
6. The levcromakalim-induced K⁺ channel openings in cell-attached configuration were suppressed by subsequent application of 5 μM glibenclamide but not of 100 μM nifedipine.
7. These results suggest that in pig proximal urethra, nifedipine inhibits the glibenclamide-sensitive 43 pS K⁺ channel activity mainly through extracellular blocking actions on the K⁺ channel itself.