Notes: Summary The effects of theophylline (3×10−5 M–5×10−3 M), isoprenaline (10−6 M), excess calcium (7.2 mM) and ouabain (3×10−7 M) on force of contraction and c-AMP content were studied in electrically driven (frequency 3 Hz) left auricles isolated from guinea pigs pretreated with reserpine. 1. Theophylline (2×10−3 M) increased c-AMP consistently. The theophylline-induced increase in c-AMP proceeded very rapidly. It became significant after an incubation period of 5 s, reached its peak after 5 min and then remained stable. The positive inotropic effect of theophylline also developed very rapidly, reached its peak after 1 min and then declined. The increase in force of contraction corresponded to the increase in c-AMP, but the decline of the positive inotropic effect occurred while c-AMP remained elevated. 2. The concentration-response curves for the theophylline-induced increases in c-AMP and force of contraction were virtually parallel at concentrations up to 2×10−3 M. 3. Washout of the effects of theophylline on c-AMP and force of contraction was also similar. Complete reversal occurred within 15 min. 4. The effects of theophylline on c-AMP and force of contraction were not affected by 10−7 M propranolol added 30 min before theophylline. 5. Isoprenaline (10−6 M) increased c-AMP and force of contraction to approximately the same extent upon incubation for up to 30 min. 6. Ouabain (3×10−7 M) or the elevation of the extracellular calcium concentration to 7.2 mM increased the force of contraction without producing a detectable increase in c-AMP. 7. It is concluded that the theophylline-produced increase in force of contraction may be the result of an increase in c-AMP and a subsequent increase in slow inward current. This effect may be counteracted by a direct, c-AMP independent effect of theophylline on intracellular calcium stores which could account for the decline of the positive inotropic effect of theophylline during prolonged exposure and at high concentrations. 8. The present data also support the view that the positive inotropic effect of isoprenaline is mediated by c-AMP, whereas the effects of ouabain and excess calcium on force of contraction appear to be independent of the c-AMP system.

Notes: Summary The influence of a low concentration of 3-isobutyl-1-methylxanthine (IBMX; 5μmol l−1) on the positive inotropic effect of vanadate (NH4VO3; 10–1,000 μmol l−1) and isoprenaline (ISO; 0.1–300 nmol l−1) was studied in electrically driven (frequency 1 Hz) papillary muscles isolated from guinea pigs. Furthermore the influence of maximally effective concentrations of IBMX (100μmol l−1), ISO (100nmol l−1) and dihydro-ouabain (DHO; 70μmol l−1) on the inotropic effect of NH4VO3 was investigated. 1. IBMX (5μmol l−1) shifted the concentrationresponse curve for ISO to the left (EC50-ratio 2.4). In contrast, the positive inotropic effect of NH4VO3 (mean EC50 81.7μmol l−1) was not influenced by IBMX at this concentration. 2. In the presence of high concentrations of IBMX or ISO, NH4VO3 exerted a concentration-dependent (10–1,000μmol l−1) atropine-insensitive negative inotropic effect with mean EC50 values of 40.1 μmol l−1 and 73.0μmol l−1, respectively. 3. In the presence of the maximally effective concentration of DHO, NH4VO3 had no effect on force of contraction, i.e. it neither further increased nor reduced the DHO-induced positive inotropic effect. 4. The negative inotropic effect of NH4VO3 in the presence of IBMX or ISO in guinea-pig papillary muscles was not accompanied by a shortening of the action potential. 5. From the failure of IBMX (5μmol l−1) to enhance the positive inotropic effect of NH4VO3 it is concluded that the recently described small increase in c-AMP, which accompanies the NH4VO3-produced increase in force of contraction in intact papillary muscles, is not of major importance in mediating the agent's positive inotropic effect. 6. The mechanism of the negative inotropic effect of NH4VO3 in the presence of high concentrations of IBMX or ISO remains obscure. Apparently, it is not due to a shortening of the action potential. A stimulation of muscarinic cholinergic receptors is also unlikely although there are obvious similarities between the negative inotropic effect of NH4VO3 and that of acetylcholine described under similar conditions in ventricular heart muscle preparations. Interestingly, all drugs hitherto found capable of evoking a negative inotropic effect of vanadate in ventricular tissue are well known to increase myocardial c-AMP levels.

Notes: 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 μmol l−1 for PIA and 0.03 μmol 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 μmol l−1 for PIA and 0.10 μmol 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.

Notes: Summary Pertussis toxin, which specifically inactivates guanine nucleotide-binding proteins (N-proteins) involved in the signal transduction in various receptor systems, did not influence the positive inotropic effect of the alpha1-adrenoceptor agonist phenylephrine in rat isolated left auricles. This indicates that the alpha1-adrenoceptor-mediated positive inotropic effect does not involve a pertussis toxin-sensitive N-protein.

Notes: Summary The present study was performed to compare the effects of the new positive inotropic phosphodiesterase III inhibitors pimobendan, adibendan, and saterinone on the isometric force of contraction in electrically driven ventricular trabeculae carneae isolated from explanted failing (end-stage myocardial failure) with those from nonfailing (prospective organ donors) human hearts. In preparations from nonfailing hearts the phosphodiesterase inhibitors, as well as the aβ-adrenoceptor agonist isoprenaline, the cardiac glycoside dihydroouabain, and calcium, which were studied for comparison, revealed pronounced positive inotropic effects. The maximal effects of pimobendan, adibendan, and saterinone amounted to 56%, 36% and 45%, respectively, of the maximal effect of calcium. In contrast, in preparations from failing hearts the phosphodiesterase III inhibitors failed to significantly increase the force of contraction and the effect of isoprenaline was markedly reduced. The effects of dihydroouabain and calcium were almost unaltered. The diminished effects of isoprenaline were restored by the concomitant application of phosphodiesterase inhibitors. To elucidate the underlying mechanism of the lack of effect of the phosphodiesterase III inhibitors in the failing heart we also investigated the inhibitory effects of these compounds on the activities of the phosphodiesterase isoenzymes I–III separated by DEAE-cellulose chromatography from both kinds of myocardial tissue. Furthermore, the effects of pimobendan and isoprenaline on the content of cyclic adenosine monophosphate (determined by radioimmunoassays) of intact contracting trabeculae were studied. The lack of effect of the phosphodiesterase inhibitors in failing human hearts could not be explained by an altered phosphodiesterase inhibition, since the properties of the phosphodiesterase isoenzymes I–III and also the inhibitory effects of the phosphodiesterase inhibitors on these isoenzymes did not differ between failing and nonfailing human myocardial tissue. Instead, it may be due to a diminished formation of cyclic adenosine monophosphate in failing hearts, presumably caused mainly by a defect in receptor-adenylate cyclase coupling at least in idiopathic dilated cardiomyopathy. Both the basal and the pimobendan-stimulated or isoprenaline-stimulated contents of cyclic adenosine monophosphate of intact contracting trabeculae from failing hearts were decreased compared with the levels in nonfailing hearts. However, under the combined action of isoprenaline and pimobendan the cyclic adenosine monophosphate level reached values as high as with each compound alone in nonfailing preparations, and in addition the positive inotropic effect of isoprenaline was restored. These findings may have important clinical implications. Along with the elevated levels of circulating catecholamines the positive inotropic effects of the phosphodiesterase inhibitors may be maintained in patients with heart failure. Furthermore, the concomitant application of a β-adrenoceptor agonist and a phosphodiesterase inhibitor might be beneficial in terminal heart failure refractory to conventional therapeutic regimens.

Notes: Summary Chronic β-adrenoceptor stimulation leads to desensitization of the myocardial adenylyl cyclase signalling pathway which includes β-adrenoceptor downregulation and upregulation of Gi-protein α-subunits. However, these investigations have mainly been done in cellular preparations. In this study we report that isoprenaline infusion in vivo leads to an increase in myocardial Gia and present evidence for functional consequences of this increase. Rats were treated by a 4-day subcutaneous infusion with isoprenaline (2.4 mg/kg·d), propranolol (9.9 mg/kg·d) and triiodothyronine (T3, 0.5 mg/kg·d) for comparison. Isoprenaline treatment increased the pertussis toxin-sensitive amount of Gia by 22±6% and decreased β1- and β2-adrenoceptor density from 35±4 to 23±6 fmol/mg protein and 24±4 to 8±6 fmol/mg protein, respectively. Contraction experiments on electrically driven papillary muscles revealed that the negative inotropic potency of the M-cholinoceptor agonist carbachol in the presence of isoprenaline was increased as compared to control (mean EC50-values: 0.04 μmol/l vs. 0.28 μmol/l). All isoprenaline-induced effects were antagonized by simultaneously administered propranolol. T3 treatment had no influence on the parameters investigated. The results suggest that chronic β-adrenoceptor stimulation desensitizes myocardial adenylyl cyclase by at least two mechanisms: β-adrenoceptor downregulation leading to diminished signal transduction in the stimulatory pathway and Giα upregulation leading to sensitization of the inhibitory pathway. Such adaptation might protect the heart from chronic exposure to catecholamines in heart diseases with elevated plasma catecholamine levels.

Notes: Summary The 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.

Notes: Summary Adenosine and its analogs (-)-N6-phenylisopropyladenosine and 5′-N-ethylcarboxamideadenosine inhibit cAMP and cGMP phosphodiesterase activity in guinea-pig atrial and ventricular preparations at concentrations of 100 μmol l−1 and higher. These effects are probably unrelated to the inotropic effects of these substances. However, inhibition of cAMP breakdown may compensate for the adenosine-induced inhibition of adenylate cyclase and may thus at least partially explain why with this drug no changes in cAMP or cGMP content have previously been observed in intact cardiac tissue.