Positive Inotropic Effects Of Catecholamines Research Articles

Investigating potential modifier genes of chronic PKA activation in heart failure

The cAMP-dependent protein kinase (PKA) is a major downstream effector of β-adrenergic signaling responsible for the positive inotropic effect of catecholamines. The PKA holoenzyme is a heterotetramer composed of two catalytic (C) and two regulatory (R) subunits. Three C subunits (Cα, Cβ and Cγ) are coded by three genes and four R subunits (RIα, RIβ, RIIα and RIIβ) are encoded by four genes but the heart expresses predominantly Cα, RIα and RIIα. We have shown that conditional knock-out of RIα in cardiomyocytes from adult mice (RIα-icKO) have constitutive PKA activation and spontaneously develop heart failure with reduced ejection fraction (HFrEF) upon aging, resulting in 50% mortality after 1 year (Bedioune et al. submitted). In this work, we aimed to better characterize the HF phenotype of RIα-icKO mice. We performed echocardiography, RT-qPCR, western blot and bulk RNA sequencing in RIα-icKO mice and control littermates at 1 year of age. We show that the HF phenotype of the surviving RIα-icKO mice at 1 year is heterogeneous: among 51 mice analyzed by echocardiography and gravimetry, 23 (45%) showed clear signs of HF (EF < 50%, cardiac hypertrophy and dilatation, lung congestion, named HFKO), whereas 18 (35%) had none (named NFKO) and 10 had an intermediary phenotype (20%). RIα residual expression and PKA activity appeared similar in HFKO (n = 6–9) and NFKO (n = 6–10) Transcriptomic analysis revealed dysregulation of typical HF marker genes in NFKO that was generally exacerbated in HFKO. Gene ontology analysis also showed TOP 10 up-regulated and down-regulated pathways classically associated with HF that were similar in HFKO and NFKO. A total of 542 genes were differentially expressed (fc 1.5, P < 0.05) between HFKO and NFKO. Cross-referencing with a list of 821 genes containing genes associated with HF from NCBI and components of β-AR signaling yielded 35 differentially expressed genes, among which 9 were selected as potential modifiers of the phenotype based on the literature. Our results suggest that NFKO represent a compensated stage of HF development. Comparison with HFKO identified new genes that could modulate the impact of chronic PKA activation on HF development. Further studies are required to identify other potential candidates and validate them in neonatal cardiomyocytes with chronic PKA activation.

Treprostinil potentiates the positive inotropic effect of catecholamines in adult rat ventricular cardiomyocytes

Prostanoids have been shown to improve exercise tolerance, hemodynamics and quality of life in patients with pulmonary arterial hypertension (PAH). We investigated whether treprostinil exerts direct contractile effects on cardiomyocytes that may explain partly the beneficial effects of these drugs. Ventricular cardiomyocytes from adult rats were paced at a constant frequency of 0.5 to 2.0 Hz and cell shortening was monitored via a cell edge detection system. Twitch amplitudes, expressed as percent cell shortening of the diastolic cell length, and maximal contraction velocity, relaxation velocity, time to peak of contraction and time to reach 50% of relaxation were analyzed. Treprostinil (0.15 - 15 ng ml(-1)) slightly increased contractile dynamics of cardiomyocytes at clinically relevant concentrations. However, the drug significantly improved cell shortening of cardiomyocytes in the presence of isoprenaline, a beta-adrenoceptor agonist. Treprostinil exerted this effect at all beating frequencies under investigation. Treprostinil mimicked this potentiating effect in a Langendorff preparation as well. The potentiating effect of treprostinil on isoprenaline-dependent cell shortening was no longer seen after phosphodiesterase inhibition. Long-term cultivation of cardiomyocytes with treprostinil did not modify load free cell shortening of these cells, but reduces the duration of contraction. We conclude that the clinically used prostanoid treprostinil potentiates the positive inotropic effects of catecholamines in adult ventricular cardiomyocytes. This newly described effect may contribute to the beneficial clinical effects of prostanoids in patients with PAH.

A complex phosphodiesterase system controls β-adrenoceptor signalling in cardiomyocytes

beta-Adrenergic signalling mediates the positive inotropic effect of catecholamines on cardiomyocytes, mainly through cAMP generation and subsequent activation of PKA (protein kinase A). Given the large diversity of PKA targets within cardiac cells, a precisely regulated and confined activity of such signalling pathways is essential for the specificity of response. PDEs (phosphodiesterases) constitute the only cAMP-degrading mechanism and are expressed in the cardiomyocyte in at least 5 family variants. Each PDE family is characterized by unique functional properties and contributes to a cAMP-degrading system enabling the modulation of PKA activity in a stimulus-dependent fashion.

Compartmentalized Phosphodiesterase-2 Activity Blunts β-Adrenergic Cardiac Inotropy via an NO/cGMP-Dependent Pathway

beta-Adrenergic signaling via cAMP generation and PKA activation mediates the positive inotropic effect of catecholamines on heart cells. Given the large diversity of protein kinase A targets within cardiac cells, a precisely regulated and confined activity of such signaling pathway is essential for specificity of response. Phosphodiesterases (PDEs) are the only route for degrading cAMP and are thus poised to regulate intracellular cAMP gradients. Their spatial confinement to discrete compartments and functional coupling to individual receptors provides an efficient way to control local [cAMP]i in a stimulus-specific manner. By performing real-time imaging of cyclic nucleotides in living ventriculocytes we identify a prominent role of PDE2 in selectively shaping the cAMP response to catecholamines via a pathway involving beta3-adrenergic receptors, NO generation and cGMP production. In cardiac myocytes, PDE2, being tightly coupled to the pool of adenylyl cyclases activated by beta-adrenergic receptor stimulation, coordinates cGMP and cAMP signaling in a novel feedback control loop of the beta-adrenergic pathway. In this, activation of beta3-adrenergic receptors counteracts cAMP generation obtained via stimulation of beta1/beta2-adrenoceptors. Our study illustrates the key role of compartmentalized PDE2 in the control of catecholamine-generated cAMP and furthers our understanding of localized cAMP signaling.

Possible involvement of cyclic adenosine monophosphate-independent mechanism in the positive chronotropic effect of norepinephrine in the isolated guinea pig right atrium.

Although both positive chronotropic and inotropic effects of beta-adrenergic stimulation are thought to be mediated by cyclic adenosine 3'5'-monophosphate, phosphodiesterase III inhibitors such as amrinone and milrinone potentiate the positive inotropic effect of catecholamines with minimum influence on the heart rate in clinical setting. The aim of the current study was to compare the positive chronotropic effect of norepinephrine with that of forskolin to elucidate whether cyclic adenosine monophosphate is relevant to the chronotropic effect of norepinephrine. Concentration-response curves for the positive chronotropic effects of norepinephrine and forskolin on the spontaneously beating right atria of guinea pigs were determined in the absence and presence of phosphodiesterase inhibitors or ion channel inhibitors. In some experiments, the left atria driven electrically were used to determine the positive inotropic effect of norepinephrine. Norepinephrine and forskolin increased the beating rate in a concentration-dependent manner. The positive chronotropic effect of forskolin was potentiated by amrinone and 3-isobutyl-1-methylxanthine, whereas the positive chronotropic effect of norepinephrine was not potentiated by the phosphodiesterase inhibitors. In contrast, the positive inotropic effect of norepinephrine was potentiated by amrinone. The hyperpolarization-activated inward current inhibitor cesium chloride and L-type voltage-dependent Ca2+ current inhibitor verapamil suppressed the chronotropic effect of norepinephrine, whereas these inhibitors did not affect the chronotropic effect of forskolin. Norepinephrine increases the spontaneously beating rate by a different mechanism from that of forskolin, suggesting that cyclic adenosine monophosphate is causally unrelated to the positive chronotropic effect of norepinephrine in the guinea pig heart.

Preface

We observed that heterozygous knockout (+/-, KO) of either endothelin-A- (ET(A)) or -B- (ET(B)) receptors significantly reduced the pressor responses to systemically administered endothelin-1 (ET-1) in ET(A) or ET(B) (+/-) KO mice when compared to wild-type (WT) mice (data not shown). Also, we observed that basal mean arterial pressure (MAP) is significantly higher in ET(B) (+/-) (92.7 +/- 1.2 mmHg) (n = 53, p < 0.05) but not ET(A) (+/-) KO mice (70.6 +/- 1.8 mmHg) (n = 23) when compared to their anaesthetized WT littermates (70.1 +/- 0.7 mmHg) (n = 118). A 90 min treatment with either BQ-123 (10 mg/kg), an ET(A)-selective antagonist, or BQ-928 (10 mg/kg), a mixed ET(A)/ET(B) antagonist, administered intraperitoneally, significantly reduced basal MAP of ET(B) (+/-) KO mice almost to the level of their WT treated counterparts (94.9 +/- 4.9 mmHg) (n = 6) vs (+ BQ-123: 59.7 +/- 0.3 mmHg, n = 8); (+ BQ-928: 72.4 +/- 2.6 mmHg, n = 5). It is worthy of note that BQ-123 significantly reduced basal MAP in WT mice but to a lesser extent than in ET(B) (+/-) KO mice (69.6 +/- 2.3 mmHg, n = 8) vs (+ BQ-123: 57.3 +/- 1.4 mmHg, n = 8). In contrast, the ET(B)-selective antagonist, BQ-788 (10 mg/kg i.p.), had no significant effect on MAP even after 90 min of treatment (ET(B) (+/-) KO: (92.3 +/- 2.3 mmHg, n = 6) vs (+ BQ-788: 89.7 +/- 3.1 mmHg, n = 6); WT: (70.5 +/- 3.7 mmHg, n = 7) vs (+ BQ-788: 71.2 +/- 2.0 mmHg, n = 6). Therefore heterozygous KO of either ET(A)- or ET(B)-receptors significantly alters the phenotypic pressor properties of ET-1. We also suggest that there is less ET clearance in ET(B) (+/-) KO mice than in WT mice, which can explain the ET(A)-dependent hypertensive state of the former strain.

Inotropic responses to isoproterenol in congestive heart failure subsequent to myocardial infarction in rats

It is well known that the positive inotropic effect of catecholamines is mediated through the activation of adrenergic receptors and the formation of cyclic adenosine monophosphate (AMP) in the cardiac cell. Although attenuated responses of failing hearts to catecholamines are commonly seen in patients and experimental animals with coronary artery disease, their mechanisms are poorly understood. To examine the status of beta-adrenergic receptors and postadrenergic receptor mechanisms in congestive heart failure due to myocardial infarction, the left coronary artery in rats was ligated for 4 and 8 weeks before studying the hemodynamic and biochemical changes due to isoproterenol, a beta-adrenoceptor agonist, or forskolin, a postreceptor agonist. Isolated perfused rat heart preparations were also used for studying changes in contractile function and cyclic AMP content. The hemodynamic actions and changes in the left ventricular cyclic AMP content in the experimental animals were depressed, not only in response to isoproterenol but also to forskolin. The density of beta-adrenoceptors was also decreased in the failing myocardium. In isolated perfused hearts from the 8-week experimental animals, isoproterenol-induced as well as forskolin-induced increases in both contractile force and cyclic AMP content in the left ventricle were depressed. These data suggest that defects in both beta-adrenergic receptor and postreceptor sites may be involved in attenuating the response of infarcted hearts to catecholamines.

Contribution of PI turnover to the positive inotropic effect of catecholamines

Contribution of PI turnover to the positive inotropic effect of catecholamines

Studies on the mechanism of action of the bipyridine milrinone on the heart

Milrinone is a positive inotropic and vasodilator agent when tested in experimental animals and in human heart-failure patients. It is generally believed that milrinone acts by inhibiting phosphodiesterase IV, thus increasing cyclic AMP, [Ca++]i and cardiac contractile force and relaxation. Maximal force produced by milrinone is greater when single-dose response curves are compared to cumulative dose-response curves. In vitro, milrinone produces a tachyphylaxis, the extent of which is both dose- and time-dependent. Recovery of tachyphylaxis is both dose- and time-dependent and is not influenced by inhibitors of protein or RNA synthesis. There is a specific cross-tachyphylaxis between milrinone and amrinone, theophylline, papaverine, and Bay K8644. This tachyphylaxis may explain the low maximal contractile response of the cumulative dose-response observed in isolated tissues. Milrinone increased cyclic AMP in dog and guinea pig cardiac muscle. As previously shown by Endoh et al., milrinone in low doses produced a biphasic effect on cyclic AMP. The early increase (first 60-70 s) in cyclic AMP shows a good correlation with contractile force changes. If cyclic AMP is determined at maximal contractile force this correlation was poor. Here we also present instances where the increase in cyclic AMP after milrinone (determined at maximal effect) does not correlate with the contractile response. The cross-tachyphylaxis of milrinone with Bay K8644 suggests that milrinone has an action on the sarcolemmal Ca++ channels. Bay K8644 suppresses the positive inotropic effect of catecholamines by 50%, but not the cyclic AMP response. The inotropic effect of milrinone, in contrast to norepinephrine is highly sensitive to [Ca++]0, stimulation rate, and [K+]0. In this respect milrinone behaves more like Bay K8644. We postulate that the main inotropic action of milrinone is due to a sarcolemmal effect. The early cyclic AMP production described could be in the sarcolemmal compartment and this may explain some of the similarities of milrinone's actions with those of Bay K8644. The tachyphylaxis observed with the inotropic effect of milrinone does not extend to the decreases in relaxation time. This and other findings to be discussed suggest that the positive inotropic and reduction in relaxation time by milrinone depend on different mechanisms, possibly through differential compartmentalization of cyclic AMP.

Modification of the positive inotropic effects of catecholamines, cardiac glycosides and Ca 2+ by the orally active male contraceptive, gossypol, in isolated guinea-pig heart

Gossypol is an orally active male contraceptive with cardio-depressant side effects. To understand the mechanism of its cardiac actions, the interaction of gossypol with positive inotropic grugs was examined in isolated atrial muscle preparations obtained from guinea- pig heart. Gossypol delayed the onset of arrhythmias caused by digoxin. In the presence of gossypol, the positive inotropic effect of isoproterenol declined rapidly, and the effect of isoproterenol to increase tissue cyclic AMP concentrations was smaller. Pretreatment of atrial muscle with the combination of gossypol and isoproterenol markedly reduced effects of isoproterenol on developed tension and cyclic AMP concentrations when these effects were tested after the washout of the first dose of isoproterenol. These effects, however, were not specific to isoproterenol. The gossypol-isoproterenol pretreatment reduced the positive inotropic effect of ouabain or extracellular Ca 2+ These results indicate that gossypol has pharmacodynamic interactions with several positive inotropic agents that are known to enhance developed tension by increasing intracellular Ca 2+ transients.

Phorbol ester does not mimic, but antagonizes the alpha-adrenoceptor-mediated positive inotropic effect in the rabbit papillary muscle.

The phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) was used to examine the hypothesis that phosphoinositide turnover is involved in the regulation of myocardial contractility mediated by stimulation of alpha-adrenoceptors in the mammalian cardiac muscle. Exposure of the isolated rabbit papillary muscle electrically driven at a rate of 1 Hz at a temperature of 37 degrees C to TPA in concentrations of 10-1000 nmol/l for 30 min did not affect the basal force of contraction. The concentration-response curve for the positive inotropic effect of (-)-phenylephrine mediated by stimulation of alpha-adrenoceptors in the presence of (+/-)-bupranolol (100 nmol/l) was shifted to the right and downward by TPA in concentrations of 30-1000 nmol/l, while the effect of (-)-phenylephrine mediated by stimulation of beta-adrenoceptors in the presence of prazosin (100 nmol/l) was not decreased, but slightly enhanced by exposure of the muscle to relatively low concentrations of TPA (10-100 nmol/l). Incubation of the membrane fraction isolated from the rabbit ventricular muscle with TPA in vitro under the same condition as employed in the physiological experiments decreased the specific binding of [3H]prazosin but not that of [3H]CGP-12177, while the non-tumor promoting phorbol ester, alpha PDD, was ineffective. These results indicate that activation of protein kinase C by TPA does not mimic the positive inotropic effect of catecholamines mediated by activation of myocardial alpha-adrenoceptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship between β‐adrenoceptors and calcium channels in human ventricular myocardium

The stoichiometric relationship between adrenoceptors and saturable binding sites for 1,4-dihydropyridines in calcium channels was investigated in human ventricular myocardium. Membrane particles were prepared from heart specimens of patients undergoing open heart surgery. The patients suffered from hypertrophic obstructive cardiomyopathy (HOCM) or mitral valve disease. Using [3H]-prazosin and [125I]-2-beta-hydroxy-3-iodiphenyl-ethyl-aminoethyl tetralone ([125I]-HEAT) as labels we detected only a marginal density of alpha 1-adrenoceptors, regardless of disease. No alpha 2-adrenoceptors were detected with [3H]-rauwolscine. In HOCM patients we estimated 72 +/- 10 fmol mg-1 (n = 12) beta-adrenoceptors labelled with [3H]-(-)-dihydroalprenolol and 74 +/- 5 fmol mg-1 (n = 2) beta-adrenoceptors labelled with [125I]-(-)-iodocyanopindolol; the equilibrium dissociation constants KD, were 1.2 +/- 0.2 nmol l-1 for [3H]-(-)-dihydroalprenolol and 7 +/- 1 pmol l-1 for [125I]-(-)-iodocyanopindolol. In patients with mitral valve disease we estimated 84 +/- 11 fmol mg-1 (n = 3) labelled with [3H]-(-)-dihydroalprenolol and 66 +/- 13 fmol mg-1 (n = 2) labelled with [125I]-(-)-iodocyanopindolol. The KD values were 1.8 +/- 0.6 nmol l-1 for [3H]-(-)-dihydroalprenolol and 8 +/- 2 pmol l-1 for [125I]-(-)-iodocyanopindolol. In 14 HOCM patients we estimated 107 +/- 12 fmol mg-1 calcium channel sites labelled with [3H]-nimodipine with a KD of 280 +/- 4 pmol l-1. In 5 patients with mitral valve disease the density of calcium channel sites labelled with [3H]-nimodipine was 78 +/- 5 fmol mg-1 with a KD of 290 +/- 20 pmol l-1, In HOCM patients the density of calcium channel sites labelled with the benzoxadiazol 1,4-dihydropyridine ([3H]-(+)-PN 200-110) was 1.6 fold of that labelled with [3H]-nimodipine with a KD of 84 +/- 11 pmol l-1. In a group of 4 HOCM patients in which calcium channels were labelled with [125I]-iodipine, the density of sites was 1.37 +/- 0.07 fold the density of sites labelled by [3H]-(+)-PN 200-11-. The KD value of [125I]-iodipine was 246 +/- 16 pmol-1. (+)-PN 200-110 was approximately 100 fold more potent than (-)-PN 200-110 as a competitor of [125I]-iodipine binding. For the HOCM group a significant correlation was found between beta-adrenoceptor density and calcium channel density, whereas in the mitral valve group no such correlation was found. This does not prove that there is causal interaction leading to a relationship between the density of beta-adrenoceptors and calcium channels. However, because positive inotropic effects of catecholamines mediated by beta-adrenoceptors are associated with opening of calcium channels, this suggests that the density of both beta-adrenoceptors and calcium channels could be co-regulated.

EVIDENCE AGAINST THE ADENOSINE-CATECHOL AMINE ANTAGONISM IN THE IN SITU DOG HEART: 183

Adenosine has been reported to attenuate the positive inotropic effects of catecholamines in in vitro-heart preparations of rodents. These results were not confirmed in anesthetized dogs during intracoronary infusion of isoproterenol. However, these experiments did not study the effects of adenosine on the inotropic effects of endogenously released catecholamines. Therefore we performed cardiac sympathetic nerve stimulation (CSNS) in 5 anesthetized, vagotomized dogs. The left circumflex coronary artery was perfused at a constant pressure of 130±4 mmHg. The contractile function of the circumflex-perfused myocardium was analyzed by sonomicrometry. CSNS at 1, 2, 5, 10 and 20 Hz increased systolic segment shortening (SScx) in a frequency-dependent manner from 10.9±2.6 at control to 15.3±3.4 % at 20 Hz. During intracoronary infusion of adenosine (50 μg/ kg/min) CSNS still increased SSCX frequency-dependently from 9.9±3.5 to 16.3±4.5 %. The lack of any adenosine-catecholamine antagonism in our experiments was not due to the sequence of procedures, nor to the marked flow increase induced by adenosine, since SSCX at rest was rather reduced with adenosine infusion and since during intracoronary infusion of papaverine and sodium-nitroprusside similar results were obtained. It seems that the observation of adenosine-catecholamine antagonism in rodents is a species dependent phenomenon. Supported by SFB 30 - Kardiologie, Dusseldorf

Catecholamine effects on intracellular sodium activity and tension in dog heart.

Because catecholamines have been reported to stimulate the sodium pump and Na+-K+-ATPase in skeletal and cardiac muscle, we examined the effects of isoproterenol (0.2-1.0 X 10(-6) M) and norepinephrine (2-4 X 10(-7) M) on intracellular sodium activity (aiNa) and twitch tension in dog Purkinje strands and ventricular muscles, aiNa was measured with Na+-sensitive microelectrodes. During the initial rapid increase in tension induced by catecholamines in Purkinje strands, no changes in aiNa were found. After 5-10 min aiNa decreased by about 2 mM, coincident with a small decline in twitch tension. When the catecholamine was removed, tension declined rapidly to a level less than control. Recovery of tension to its control level less than control. Recovery of tension to its control level occurred simultaneously with recovery of aiNa. Comparable changes in aiNa occurred in ventricular muscle, but the biphasic effect of catecholamines on tension was not seen. These results are consistent with sodium pump stimulation in cardiac muscle. In Purkinje strands the resulting changes in aiNa may alter the direct positive inotropic effect of catecholamines, probably by influencing Na+-Ca2+ exchange.

Degradation of perfused adenine compounds up to uric acid in isolated rat heart

Cells are generally impermeable to nucleotides like ATP, ADP and AMP while nucleosides and bases readily cross the plasma membrane. A release of adenosine and of its catabolic derivatives by the myocardium of different animal species has been demonstrated in physiological and physiopathological conditions [2, 4, 13]. Furthermore there are many studies on the uptake of adenosine and inosine by the myocardial cells and their incorporation into tissue nucleotides [7–9, 15]. Taking into account the extracellular localization of adenosine receptors [5] and the role of this nucleoside as regulator of coronary blood flow and modulator of the positive inotropic effects of catecholamines [4] it was of interest to study a possible extracellular formation of adenosine from adenine nucleotides. Most mammalian tissues like muscle, liver and adipose tissue and cells like platelets, leukocytes and lymphocytes possess some enzyme activities associated with the cell surface membrane. Ecto-ATPase (EC 3.6.1.3/15) and ecto-5′-nucleotidase (EC 3.1.3.5) activities have been demonstrated in myocardial, smooth-muscle and endothelial cell membranes [3,4,8,12]. In the present paper we report a comparative study on the ability of the isolated rat heart to breakdown exogenous adenine nucleotides, nucleosides and bases: we have determined in the coronary perfusate all the degradation products in basal conditions and after perfusion with ATP, ADP, AMP, adenosine, inosine and hypoxanthine. All the perfused compounds were degraded up to uric acid that amounted to about 10%. Nucleotides were catabolized at a higher rate than nucleosides and so adenosine may accumulate outside the cells.

An initial characterization of human heart beta-adrenoceptors and their mediation of the positive inotropic effects of catecholamines.

The positive inotropic effects of catecholamines were studied on samples of ventricular myocardium taken from patients undergoing open heart surgery. The adenylyl cyclase and binding of 3H-(-)-bupranolol were examined in membrane particles prepared from similarly obtained samples. The equilibrium dissociation constant (KD) for (-)-bupranolol was estimated in 4 ways: blockade of the positive inotropic effects of catecholamines, blockade of the stimulation of the adenylyl cyclase by catecholamines, saturation binding of 3H-(-)-bupranolol, inhibition of the binding of 3H-(-)-bupranolol by its unlabeled stereoisomers. The estimates of KD fall in the range 0.5-1.4 nmol/l. The stereo-selectivity ratio (KD (+)-isomer/KD (-)-isomer) is 73. Both values for bupranolol are very similar in cat and man. The inotropic potency of (-)-noradrenaline is nearly 2 orders of magnitude higher in cat heart tissues than in tissues from human hearts. The difference in inotropic potencies between species is only partially accounted for by the five-fold lower potency of (-)-noradrenaline for the human heart adenylyl cyclase as compared to the cat enzyme.

Investigations on glycerinated cardiac muscle fibres in relation to the problem of regulation of cardiac contractility--effects of Ca++ and c-AMP.

Alterations in myocardial contractile force and maximum unloaded shortening velocity (Vmax) occurring in the course of isometric twitch contraction and with changes in inotropism are assumed to be mediated by changes in intracellular Ca++ and/or c-AMP concentration. In the present study, the influences of Ca++ and cyclic AMP upon the contractility of briefly glycerinated myocardial preparations are described. It is shown that Ca2++ ions affect tension and Vmax, as measured by rectangular releases in length, in different concentration ranges. This suggests that, besides the number of attached crossbridges regulated by Ca++ binding to troponin C, a Ca++-dependent phosphorylation of the P-light chain of myocardial myosin may be involved in the regulation of Vmax. Cyclic AMP, on the other hand, induces phosphorylation of troponin I, thereby reducing the sensitivity of tension to Ca++. It is concluded that the positive inotropic effect of catecholamines may be mediated by the described actions of intracellular Ca++ and c-AMP upon the contractile structures where c-AMP-dependent troponin phosphorylation could account for the acceleration of relaxation.

Frequency dependence of cyclic AMP in mammalian myocardium

ALTHOUGH there is now good evidence that cyclic AMP is a mediator of positive inotropic effects of catecholamines in the heart, the details of the metabolism of this nucleotide in the intact myocardial cells are still unknown. It has been shown that the intracellular concentration of cyclic AMP oscillates during each contraction cycle of the frog heart1,2. But little information is available for the influence of frequency of stimulation, which regulates myocardial contractility in physiological conditions3, on the intracellular level of cyclic AMP. We therefore investigated the relationship between cyclic AMP level and the strength of contraction of the ventricular myocardium during the induction of different contractile states by changing frequency of stimulation. In order to analyse the influence of frequency of stimulation on cyclic AMP level, the effects of changes in concentrations of extracellular calcium ions or of D600, which is thought to inhibit the influx of calcium ions through the myocardial cell membrane4, were also investigated. It was found that the intracellular level of cyclic AMP and the contractile force of isolated rabbit papillary muscles had a reciprocal relationship during induction of different contractile states by changing frequencies of stimulation. The influence of frequency was simulated by reduction of the extracellular calcium ions or by D600.

Relaxing and inotropic effects of cyclic AMP on skinned cardiac cells

THE positive inotropic effect of catecholamines in cardiac muscle is assumed to result from an increase in the intracellular level of cyclic AMP (adenosine 3′,5′-monophosphate)1,2. Cyclic AMP may enhance the contraction by increasing the trans-sarcolemmal flux of Ca2+ during the plateau of the action potential3,4, but the flux seems insufficient to activate directly the myofilaments and it is generally assumed that additional Ca2+ may be released from intracellular stores (ref. 5), possibly by a Ca2+-triggered release from the sarcoplasmic reticulum (SR)6–8.

Comparative study of intropic effects of catecholamines on blood-perfused canine papillary muscle.

The relative inotropic potencies of isoproterenol, norepinephrine, epinephrine and phenylephrine were compared in the blood-perfused canine papillary muscle preparation. The muscle was perfused at a constant pressure of 100mm Hg at 38-39°C with arterial blood from a donor animal. The isometric tension developed by the papillary muscle and the positive inotropic effect of catecholamines were a function of the stimulus frequency. The potencies of the inotropic effect of catecholamines were compared at various stimulus frequencies from 90 to 240/min. Isoproterenol, norepinephrine and epinephrine, 0.001 to 0.1μg, caused reproducible positive inotropic effects lasting for from 2 to 5min, when the perfused blood flow of the muscles was from 3 to 5ml/min. Phenylephrine, 0.01 to 1μg, caused no inotropie effect. The order of potencies of inotropic effect of each amine was isoproterenol>norepinephrine>epinephrine>>phenylephrine. The dose ratio required to produce 100% increase in isometric tension was IS:NE:EP=1:3:6 at stimulus frequencies of 90 and 120/min. The ratio of the percentage increase produced by 0.03μg of each agent was roughly IS:NE:EP=3:2:1 and 5:3:1 at 90 and 120/min, respectively. Norepinephrine produced a distinctly stronger positive inotropic effect than did epinephrine and the differences between these amines were statistically significant.