Augmented Contributions of Voltage-Gated Ca2 Channels to Contractile Responses in Spontaneously Hypertensive Rat Mesenteric Arteries

The observation that organic Ca²⁺ channel blockers are more effective in lowering blood pressure and peripheral resistance in hypertensive compared to normotensive subjects suggests that there is a greater contribution from voltage-gated Ca²⁺ channels (Ca_L) to vascular force maintenance in hypertensive arteries. This study tests this hypothesis by comparing the effects of Bay k 8644 and nisoldipine on basal force development, contractile responses to norepinephrine and serotonin, and Ca²⁺ currents (I_Ca) in mesenteric artery (MA) from Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). MA rings were used to record isometric contractions at L_max. Single cells were isolated by collagenase plus elastase for measurement of Ca_L properties by patch-clamp methods. Contractile responses to Bay k 8644 were larger and more sensitive in SHR than WKY, and were larger in endothelium-denuded compared to intact rings. In SHR, the addition of 10 nmol/L Bay k 8644 increased contractile sensitivity to norepinephrine (NE) and serotonin (5HT), and increased maximum response to 5HT. In WKY, 10 nmol/L Bay k 8644 produced a small increase in 5HT sensitivity with no effect on maximum response, and had no effect on NE responses. In the presence of 1 μmol/L nisoldipine, the maximum response and the sensitivity to both NE and 5HT were decreased in both WKY and SHR with the inhibitory effects of nisoldipine being larger in SHR than WKY. Peak I_Ca was larger in SHR, and current-voltage curves were shifted toward more negative voltages compared to WKY. Bay k 8644 increased I_Ca in both WKY and SHR myocytes with no apparent difference in the magnitude of its effect when expressed as a percent of control I_Ca. These results suggest that Ca_L contribute significantly to tonic force maintenance as well as to agonist responses in MA from both WKY and SHR, but with a much larger contribution in SHR. Differences in the sensitivity of Ca_L to Bay k 8644 were not responsible for the differences in contractile responses to this agonist.     

Subcellular mechanism of the positive inotropic effect of a new quinolinone derivative OPC-8490 on the dog ventricular myocardium

Summary OPC-8490 [3,4-dihydro-6-[4-(4-oxo-4-phenylbutyl)-1-piperazinylcarbonyl]-2(1H)-quinolinone citrate] elicited a positive inotropic effect in a concentration-dependent manner on the isolated dog ventricular trabeculae electrically driven at 0.5 Hz in Krebs-Henseleit solution bubbled with 95% O₂-5% CO₂ at 37°C. The-adrenoceptor antagonist, bupranolol (3×10^–7 mol/l), did not influence the effect of OPC-8490. The maximal effect of OPC-8490 was 0.19 compared with isoproterenol (1.0). The time course of the increase in contractile force coincided with that of the concomitant cyclic AMP accumulation induced by OPC-8490. The concentration-response curve for the OPC-8490-induced increase in contractile force was superimposable on that of the elevation of cyclic AMP levels. OPC-8490 (10^–5 mol/l) shifted the concentration-response curve for isoproterenol to the left and upward. These results imply that the accumulation of cyclic AMP induced by OPC-8490 through an inhibition of peak III PDE may be responsible for its positive inotropic effect. However, the OPC-8490-induced increase in the contractile force was not abolished by carbachol (3×10^–6 mol/l) when the cyclic AMP accumulation caused by the compound was completely inhibited by carbachol. In addition, OPC-8490 did cause a change in the time course of isometric contractions characteristic of cyclic AMP accumulation. These findings indicate that both the cyclic AMP-dependent (peak III PDE inhibition) and the cyclic AMP-independent mechanisms (prolongation of action potential duration by inhibition of K⁺ conductance) may be involved in the positive inotropic effect of OPC-8490 on the dog ventricular muscle.     

Mechanism of the positive inotropic effect of milrinone in cultured embryonic chick ventricular cells

Milrinone, a potent positive inotropic and vasodilating agent, has shown promise in the clinical treatment of congestive heart failure, but significant controversy about its mechanism of action exists. To approach these mechanistic problems in a non-innervated, non-diffusion-limited system, the effects of milrinone on cultured embryonic chick ventricular cells were examined. At 37 degrees C in physiologic buffer, milrinone produced a rapid, concentration-dependent increase in amplitude of contraction that was 45% of the maximum increment in contraction produced by elevated extracellular calcium; the EC50 was 8 microM. This peak response was quantitatively similar to the contractile response produced by isobutyl methylxanthine, a potent phosphodiesterase inhibitor. Milrinone inhibited 70% of total phosphodiesterase activity of cultured ventricular cells with an EC50 of 11 microM. Exposure to 1 X 10(-4) M milrinone resulted in rapid increase in cyclic AMP content to levels greater than 100% above control within 4 min. The same concentration also produced a 43% increase in the rate of transsarcolemmal 45Ca uptake. The stimulation of 45Ca uptake rate was similar to the response produced by 1 microM isoproterenol and could be completely abolished by 10 microM verapamil. Thus, in cultured embryonic chick myocardial cells, the positive inotropic effect of milrinone is largely, if not entirely, attributable to phosphodiesterase inhibition, leading to intracellular cyclic AMP accumulation and stimulation of transsarcolemmal calcium influx via the slow calcium channel.     

The force-frequency relationship is dependent on Ca2+-influx via L-type- and SR-Ca2+-channels in human heart

The present study investigated the influence of Bay K 8644 and nifedipine (Nif) on the force-frequency relationship and on tetanic tension and force of contraction of failing human myocardium (PAP, n = 12). In addition, ryanodine (Rya) was studied on the force-frequency relationship. Bay K 8644 (0.1 μM) increased, but Nif (0.01 μM) reduced isometric force of contraction significantly. However, both, Bay K 8644 (2 Hz vs. 0.5 Hz: Control: −31.6 ± 7.8 %; +Bay K 8644: +103 ± 30 % (% basal); p < 0.005) as well as Nif (2 Hz vs. 0.5 Hz: Control: −8.8 ± 9.7 %; +Nif: +90.9 ± 31.5 %) (% basal); p < 0.05), were able to restore a positive FFR in PAP. By measurement of tetanic tension and posttetanic potentiation in the presence of the 1,4-dihydropyridines, we support the hypothesis of the existence and functional relevance of a dihydropyridin-ryanodine receptor junctional complex. In skinned fiber preparations, Bay K 8644 showed no effect on Ca²⁺-sensitivity or caffeine induced Ca²⁺-release. Rya (10 μM) decreased force of contraction in PAP and was effective in restoring a postive FFR (2 Hz vs. 0.5 Hz: Control: −7.3 ± 5.1 %; +Rya: +98.0 ± 31.9 % (% basal); p < 0.05). Thus, the altered FFR and Ca²⁺-homeostasis in failing human myocardium may result from changes in sarcolemmal Ca²⁺-influx and/or from altered SR-Ca²⁺-load. Received: 27 August 1998, Returned for 1. revision: 24 September 1998, 1. Revision received: 23 October 1998, Returned for 2. revision: 20 November 1998, 2. Revision received: 18 December 1998, Accepted: 22 December 1998     

Comparison between the cardiac effects induced by muzolimine and furosemide in guinea-pig atria

Summary Muzolimine (10–500 M) induced a concentration-dependent reduction of both the contractile force and frequency in spontaneously beating atria and in electrically driven left atrium from reserpine-treated guinea pigs. This negative inotropic response was unaffected by the addition of atropine to the perfusion fluid, and it was highly sensitive to changes in external Ca²⁺ concentration. Both in spontaneously beating and in electrically driven atrium, muzolimine (50–400 M) antagonized, in an apparently competitive manner, the increase in contractile force induced by cumulative addition of CaCl₂ (0.68–9.59 mM) to the bathing fluid. Muzolimine (50–100 M) reduced the inotropic response to low (5–30 nM), but not high (50–100 nM) concentrations of Bay K 8644, a calcium-channel agonist. The inotropic effects of 8-phenyltheophylline and of ouabain were antagonized by muzolimine (10–100 M) in a noncompetitive manner, while the response to noradrenaline was not altered. Similar to muzolimine, verapamil at a concentration suitable to block calcium channels inhibited, in a noncompetitive way, the inotropic effect induced by 8-phenyltheophylline and by ouabain without altering the contractile response to noradrenaline. Furosemide (10 and 100 M) did not influence the contractile force or the frequency of spontaneously beating atria, nor the inotropic effect induced by CaCl₂, 8-phenyltheophylline, ouabain, or noradrenaline. These results indicate that the influence of muzolimine on guinea-pig atria originates from an inhibition of Ca²⁺ influx into cardiac cells and that furosemide does not mimic the effect of muzolimine at this level.     

Effects of a new 1,3-thiazole Derivative ZSY-39 on force of contraction and cyclic AMP content in canine ventricular muscle

Summary A newly synthesized 1,3-thiazole derivative ZSY-39 increased the force of contraction in a concentrationdependent manner in association with elevation of tissue cyclic AMP levels in the isolated canine ventricular trabeculae electrically driven at 0.5 Hz at 37°C. ZSY-39 shortened the duration of isometric contractions mainly by abbreviation of the relaxation time. The maximal response to and EC₅₀ of ZSY-39 were 0.7 (isoproterenol=1.0) and 4.6×10^–5 M. Bupranolol (3×10^–7 M) did not affect the positive inotropic effect of ZSY-39. The time course of increases in the force of contraction induced by ZSY-39 (10^–4 M) coincided with that of cyclic AMP accumulation. The concentration-response curve for the increase in the force of contraction produced by ZSY-39 was superimposable on that of the elevation of cyclic AMP levels. Carbachol (3×10^–6 M) shifted the concentration-response curve for the increase in force by ZSY-39 to the right and downward, and decreased the accumulation of cyclic AMP induced by ZSY-39 (10^–4 M). ZSY-39 (10^–5 M) enhanced significantly the positive inotropic effect of isoproterenol. The relationship between the force of contraction and cyclic AMP levels after the administration of ZSY-39 was not modified by the addition of carbachol or isoproterenol. These findings indicate that cyclic AMP plays an important role in the positive inotropic effect of ZSY-39 on canine ventricular muscle.     

Regulation of force and intracellular calcium transients by cyclic AMP generated by forskolin,MDL 17,043 and isoprenaline,and its modulation by muscarinic receptor agents: a novel mechanism for accentuated antagonism

Summary The relation of changes in intracellular calcium transients and force of isometric contractions in response to an elevation or reduction of cyclic AMP levels was investigated in isolated dog ventricular trabeculae and rabbit papillary muscles, in which multiple superficial cells have been microinjected with the calcium sensitive bioluminescent protein aequorin. Forskolin, MDL 17,043 and isoprenaline elevated the tissue cyclic AMP level, increased consistently the peak aequorin signals and force, and abbreviated the duration of both signals in a concentration-dependent manner. When the effect of isoprenaline was compared with that of alteration of extracellular calcium concentration ([Ca²⁺]₀), the increase in force by isoprenaline was associated with higher peak aequorin signals than that by alteration of [Ca²⁺]₀ for a given increase in force, indicating the decrease in calcium sensitivity of myofibrils by cyclic AMP generated byβ-adrenoceptor stimulation. Carbachol, which did not affect significantly the basal force and cyclic AMP levels, lowered the cyclic AMP levels elevated previously by forskolin, MDL 17,043 or isoprenaline in the isolated dog ventricular trabeculae. It antagonized the increase in peak aequorin signals and force caused by these agents in a concentration-dependent manner. When carbachol had been administered prior to isoprenaline and the concentration-response curve for isoprenaline was determined in the presence of carbachol, the relation of force peak aequorin signals was not modified by carbachol in the rabbit papillary muscle. Carbachol, when administered during induction of the positive inotropic action by forskolin, MDL 17,043 and isoprenaline, decreased the force more than peak aequorin signals in a concentration-dependent manner in the dog ventricular trabeculae. Therefore, the relation of force to peak aequorin signals was shifted downwards during the carbachol-induced inhibition, indicating a further decrease of calcium sensitivity of myofibrils by carbachol. This effect of carbachol appears to be specific to the cyclic AMP-mediated positive inotropic action, since theα-adrenoceptor-mediated (cyclic AMP-independent) action was unaffected by carbachol. This mechanism may play an important role for “accentuated antagonism” in the mammalian ventricular myocardium. This paper is supported partly by Grant-in-Aid for Scientific Research on Priority Areas No. 62624004 and No. 63641002 from the Ministry of Education, Science and Culture, Japan and National Institutes of Health grant HL-12186.     

Calcium-sensitizing inotropic agents in the treatment of heart failure: A critical view

Summary Interventions that augment the contractile state of the heart are associated with, or caused by, alterations in Ca²⁺ exchange in heart muscles. New inotropic agents have been developed that increase the sensitivity of the myofilaments to Ca²⁺. To examine the effect of calcium-sensitizing agents on force development, we measured systolic and diastolic intracellular Ca²⁺ concentration ([Ca²⁺]_i) and constructed [Ca²⁺]_i-force relationships in normal (n=6) and myopathic human hearts (n=10). Using the bioluminescent calcium indicator aequorin, we found that the diastolic [Ca²⁺]_i was 225±52 nM in normal muscles, whereas in myopathic muscles diastolic [Ca²⁺]_i was significantly higher at 361±68 nM. Calcium-sensitizing agents that shift the [Ca²⁺]_i-force relationship toward lower [Ca²⁺]_i increase the diastolic force of myopathic hearts significantly more than in normal human hearts. This leads us to the conclusion that inotropic agents that increase the sensitivity of the myofilaments to Ca²⁺ further impair relaxation in myopathic hearts, resulting in a reduced contractile reserve and diminished actice force production.     

Effects of Milrinone on Atrioventricular Conduction in the Canine Heart Under Normal Conditions,During Atrial Flutter and After Ligation and Reperfusion of the Septal Artery

Milrinone and AV Conduction. Milrinone, in addition to its vasodilatory and inotropic actions, has been shown to enhance or restore electrical activity in depolarized tissues and to improve conduction in isolated cardiac tissues depressed by exposure to an ischemic environment. These electrophysiological actions of the drug are thought to be secondary to milrinone's ability to enhance the transmembrane calcium current (I_Ca). The present study was designed to evaluate the effectiveness of milrinone (10–100 μg/kg, IV bolus) to modulate atrioventricular (AV) nodal conduction in anesthetized dogs under normal conditions, after ligation and reperfusion of the septal coronary arteries and during atrial flutter. All dosage regimens (10–100 μg/kg milrinone) produced a positive inotropic effect with no significant changes in blood pressure. Under normal conditions, 100 jig/kg of milrinone significantly reduced the PR interval, Wenckebach cycle length and AV nodal refractory period without causing a significant change in heart rate. The improvement in AV conduction and decrease in AV nodal refractoriness led to significant dose-dependent increases in ventricular rate during atrial flutter. Low doses of milrinone also accelerated the flutter rate. Ligation of the septal coronary arteries for 2 to 6 hours produced stable atrioventricular conduction disturbances in six of the 11 dogs studied: complete AV block (n = 1), second-degree AV block (n= 2) and first-degree AV block (n= 3). Milrinone (100 μg/kg) improved AV conduction in five of the six dogs. Reperfusion of the septal arteries following 6 hours of ligation produced a sustained high degree AV block in two out of five dogs. Milrinone promptly restored 1:1 conduction in both cases. Our results suggest that milrinone may be of benefit in the treatment of some cases of AV conduction disturbance and that the drug, through its effects to facilitate AV conduction and abbreviate AV refractoriness, can lead to a significant and perhaps dangerous increase in ventricular rate in patients with atrial flutter or fibrillation. (J Cardiovasc Electrophysiol, Vol 1. pp. 93–102. 1990)     

Potentiation of the inotropic effects of norepinephrine and dibutyryl cyclic AMP by theophylline

The positive inotropic response of the heart to catecholamines has been postulated to result from an increase in the intracellular level of cyclic AMP (adenosine 3′,5′-monophosphate), produced by activation of myocardial adenylate cyclase. According to this hypothesis, inhibition of phosphodiesterase, the enzyme which hydrolyzes cyclic AMP to inactive 5′-AMP, should enhance the cardiac effects of catecholamines. We therefore examined the effects of theophylline, an inhibitor of cardiac phosphodiesterase, on the isometric contractile response of cat papillary muscles to varying concentrations of norepinephrine (10⁻¹¹ to 10⁻⁵m), cyclic AMP (10⁻⁴ to 5 × 10⁻³m) and dibutyryl cyclic AMP (10⁻⁵ to 5 × 10⁻³m). The latter agent is a more lipid soluble derivative of cyclic AMP which is thought to reproduce the intracellular effects of the parent compound. Cyclic AMP produced no changes in isometric contractile function either in the presence or absence of theophylline. However, at a theophylline concentration (2.5 × 10⁻⁴m) which caused little increase in baseline contractile function (average increment in active tension less than 10%), a marked potentiation of the positive inotropic effects of norepinephrine and dibutyryl cyclic AMP was observed. The concentration of norepinephrine producing half-maximal increases in active tension and rate of tension development decreased from 10⁻⁷ to 10⁻⁸m while that of dibutyryl cyclic AMP decreased from 8 × 10⁻⁴ to 4 × 10⁻⁴m. Theophylline did not alter the positive inotropic effects of increasing extracellular calcium concentration (2.5 to 10 mm), an intervention which is not thought to directly influence intracellular cyclic AMP levels. Although indirect, these findings provide further evidence that the positive inotropic effects of catecholamines are mediated via the adenylate cyclase cyclic AMP system.     

Decreased inotropic but relatively preserved relaxation response to cyclic adenosine monophosphate-dependent agents in myopathic human myocardium

Background:

Increased intracellular concentrations of cyclic adenosine monophosphate (AMP) stimulate a positive inotropic and lusitropic response in healthy myocardial tissue. Heart failure results in an attenuated inotropic response to cyclic AMP-dependent agents. This study compares the inotropic versus relaxation response to cyclic AMP-dependent and cyclic AMP-independent agents in myocardium from patients with end-stage heart failure and control patients without heart failure.

Methods and Results:

Fifty-four control and 59 myopathic human ventricular trabeculae, 1 mm or less in diameter were placed in physiologic saline, 2.5 mM Ca²⁺, and stretched to the length at which maximal isometric force developed at 30°C, 0.33 Hz. Dose-response curves plotted as percentage change from baseline versus concentration of drug were determined for acetylstrophanthidin, isoproterenol, isobutylmethylxanthine, and milrinone. Acetylstrophanthidin, a cyclic AMP-independent agent, showed similar increases in peak tension relative to baseline over the entire dose range tested for both control and myopathic heart muscle; its effect on relaxation of control and failing cardiac muscle was equivalent over the dose range tested. In contrast, the inotropic actions of the cyclic AMP-dependent agents, isoproterenol and the phosphodiesterase inhibitors, were significantly decreased in myopathic muscle compared with control muscle, but effects on relaxation in the control and myopathic groups remained relatively preserved.

Conclusions:

A relatively preserved relaxant effect is associated with the cyclic AMP-dependentagents, despite significant diminution of their inotropic effects. Thus, in advanced heart failure, patients may continue to benefit from the lusitropic effects of the cyclic AMP-dependent agents, even when the inotropic effects of these agents are severely attenuated.     

Respiratory acid-base changes and myocardial contractility: interaction between calcium and hydrogen ions.

The mechanism whereby respiratory acid-base changes alter myocardial contractility is poorly understood. Accordingly, 46 cat right ventricular papillary muscles contracting isometrically at 30°C were studied while varying both the pH and [Ca²⁺] of the bathing medium. The pH changes were induced by varying the PCO₂ of the gassing mixture. A decrease in pH from 7.45 to 7.10 in the presence of 2.5 mm Ca²⁺ caused a rapid fall in active force and rate of force development ($\text{d}\text{F}\text{d}\text{t}$) to levels 76% of control followed by a gradual recovery to approximately 89% of control during a 60 min observation period despite continued acidosis. Increasing the [Ca²⁺] to 5.0 mm markedly attenuated the negative inotropic effects of hypercarbic acidosis. Increasing the pH from either 7.10 to 7.45 or 7.45 to 7.75 caused a rapid 15% increase in active force and $\text{d}\text{F}\text{d}\text{t}$ followed by a return to near control levels after 60 min. The biphasic contractile response to either respiratory acidosis or alkalosis was not influenced by pretreating the muscles with 1 × 10^?6m propranolol. In 10 additional muscles series elastic load-extension relations at varying pH levels were determined using standard quick-release techniques. Respiratory acid-base changes did not alter the measured series elastic properties. These findings indicate that the contractile effects produced by carbon dioxide-induced pH alterations are exerted at the level of the contractile element and that the muscle has the intrinsic capacity to attenuate these inotropic effects with time. Furthermore, our results suggest that the contractile effects produced by changing [H⁺] may involve an interaction with Ca²⁺ at a sarcolemmal or intracellular binding site.     

Sensitization of dog and guinea pig heart myofilaments to Ca2+ activation and the inotropic effect of pimobendan: comparison with milrinone

We compared the effects of the newer inotropic drugs, pimobendan (UD-CG 115 BS) and milrinone (Win 47203), on the electrical, mechanical and biochemical activity of intact and detergent-skinned preparations of cardiac muscle. Both of these agents increased contractile force of guinea pig papillary muscle preparations bathed under physiological conditions or depolarized with 25 mM K+o. The positive inotropic action was associated with potentiation of the Ca2+-dependent slow action potentials (APS). Contractile force developed in the presence of 25 mM [K]o and 1 microM isoproterenol was increased further by addition of 50 microM pimobendan with no effect on the slow action potential. Milrinone (50 microM) did not produce a further increase in the force or potentiate the slow APs. Pimobendan, in a dose-dependent manner, increased active tension developed by chemically-skinned dog heart muscle fibers at submaximally activating concentrations of Ca2+, whereas milrinone did not. At pCa 6.25, the half-maximal concentration of pimobendan for stimulation of force development was about 40 microM. At maximally activating levels of Ca2+ (pCa 4.5), pimobendan had little or no effect on force development. The effect of pimobendan on force was paralleled by changes in the Ca2+-activated Mg-ATPase activity of the isometric skinned fiber preparations. Moreover, the tension-cost (unit increase in ATPase rate/unit increase in force) was unchanged in the presence of pimobendan. Milrinone did not affect ATP hydrolysis by the skinned fiber preparations. Force-pCa and ATPase-pCa relations of skinned fiber preparations contracting isometrically were shifted to the left by 0.15-0.20 pCa units in the presence of 50 microM pimobendan. In contrast, there was no effect of pimobendan on the ATPase activity of unloaded myofibrillar preparations. The stimulation of force and ATPase activity of the skinned heart muscle fibers could be accounted for by an effect of pimobendan on the affinity of the regulatory (low affinity, Ca2+-specific) binding sites of cardiac troponin C. Ca2+ binding to the "structural" high affinity sites of troponin C was slightly inhibited. The results indicate that the positive inotropic actions of pimobendan, but not milrinone, may involve activation of the cardiac myofilaments by a direct effect involving an increased affinity of the regulatory site on troponin C for Ca2+.     

Mci-154, a Cardiac Ca2+ Sensitizer, Reverses the Depression in Maximal Ca2+-Activated Force by Inorganic Phosphate and Acidic pH in Skinned Fiber of Guinea Pig Heart

Intracellular accumulation of inorganic phosphate (Pi) and intracellular acidosis, which occur in ischemic and hypoxic hearts, reduce the force of contraction by decreasing the responsiveness of contractile system to Ca²⁺. In the present study we investigated the effects of MCI-154, a Ca²⁺ sensitizer that can enhance crossbridge interaction, on the decline in maximal Ca²⁺-activated force by Pi or acidic pH in skinned fiber bundles of guinea pig hearts. MCI-154 can concentration-dependently reverse the depression in maximal Ca²⁺-activated force (pCa 4.4) by 20mM Pi, which was not recovered by a higher concentration of Ca²⁺ ion (pCa 4.0). The effects of MCI-154 were observed even at a concentration (0.01 M) at which the drug has no effect on the pCa4.4–induced maximal force in the absence of 20 mM Pi when given alone. MCI-154 inhibited the rightward shift of the pCa–tension relationships, with a marked decrease of maximal force produced by 20 mM Pior acidic pH (decrease in pH from 7.0 to 6.6). MCI-154 also improved the decline in maximal Ca²⁺-activated force by 20 mM Pi under acidic pH, but the acidosis did not further decrease the effect of 20 mM Pi. Milrinone, a cyclic AMP–dependent phosphodiesterase inhibitor, and pimobendan, another Ca²⁺ sensitizer, did not improve the Pi-induced contractile failure. These results indicate that the Ca²⁺ sensitizer MCI-154 could reverse the contractile failure induced by Pi and/or acidic pH in a skinned fiber preparation via modulation of the strong crossbridge reaction with myosin. MCI-154 may be a promising agent for myocardial contractile failure, in which Pi and H⁺ progressively increase. This revised version was published online in July 2006 with corrections to the Cover Date.     

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

Summary Alterations in myocardial contractile force and maximum unloaded shortening velocity (V_max) 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 Ca⁺⁺ ions affect tension and V_max, 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 V_max. 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.

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Untersuchungen zur Regulation der Kontraktilität in glyzerinextrahierten Herzmuskelpräparaten-Einflüsse von Ca⁺⁺ und c-AMP

Zusammenfassung Es wird vermutet, daß Veränderungen der kontraktilen Kraft des Myokards und der maximalen lastfreien Verkürzungsgeschwindigkeit (V_max) während der isometrischen Zuckung und bei inotropen Interventionen durch Änderungen der intrazellulären Ca⁺⁺-und c-AMP-Konzentrationen mediiert werden. In der vorliegenden Arbeit werden Einflüsse von Ca⁺⁺-Ionen und c-AMP auf die Kontraktilität kurz glyzerinierter Herzmuskelpräparate beschrieben. Ca⁺⁺-Ionen beeinflussen Kraft und V_max, gemessen durch rechteckförmiges release der Länge, in unterschiedlichen Konzentrationsbereichen. Dies deutet darauf hin, daß, neben der durch Ca⁺⁺-Bindung an Troponin C regulierten Anzahl angehefteter Querbrücken, eine Ca⁺⁺-abhängige Phosphorylierung der P leichten Kette des myokardialen Myosins an der Regulation von V_max beteiligt sein könnte. c-AMP-abhängige Phosphorylierung des Troponin I dagegen erniedrigt die Ca⁺⁺-Empfindlichkeit der Kraft.Es wird postuliert, daß positiv inotrope Effekte nach Gabe von Katecholaminen durch die beschriebenen Einflüsse von intrazellulärem Ca⁺⁺ und c-AMP auf die kontraktilen Strukturen selbst mediiert werden könnten, wobei die c-AMP-abhängige Troponin-Phosphorylierung für die Beschleunigung der Relaxation bedeutsam sein könnte.

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Paper, presented at the Erwin Riesch Symposium, Tübingen, April 3–7, 1979

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With 3 figures

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Supported by the Deutsche Forschungsgemeinschaft (SFB 90)     

甲腈吡酮、黄夹次甙乙及地高辛强心作用的比较

戊巴比妥钠引起的心力衰竭犬,静注甲腈吡酮(milrinone,Mil)0.05,0.1mg/kg及地高辛(digoxin,Dig)0.05mg/kg后,左室压力上升、下降速率(±LVdp/dt max)和左室收缩力(LVCF)明显增加。这表明有明显正性肌力和正性舒张作用。静注Mil 0.1mg/kg后,动脉平均压和左室舒张末期压均降低,心率稍加快;0.05mg/kg对上述指标无影响。静注Dig 0.05mg/kg后血压升高;十二指肠注入0.2mg/kg的Mil及黄夹次甙乙(neriifolin,Ner)亦有明显的强心作用。同剂量的Dig则作用不明显。普萘洛尔(心得安)引起的心衰豚鼠在静脉恒速注射Mil、Ner及Dig后均有强心作用。Ner的治疗量最小,Mil的安全范围最宽。Dig和Ner均能引起心律失常,Mil的此种作用不明显。     

Effects of Pimobendan on the L-Type Ca2+ Current and Developed Tension in Guinea-Pig Ventricular Myocytes and Papillary Muscle: Comparison with IBMX, Milrinone, and Cilostazol

Summary. In this study, we compared the effects of pimobenda (PIM), a putative Ca²⁺-sensitizer and phosphodiesterase (PDE) inhibitor, on the L-type Ca²⁺ current (I_Ca) of guinea-pig ventricular myocytes and contractile tension of ventricular papillary muscles with those of a nonselective PDE inhibitor, isobutylmethylxanthine (IBMX), and selective PDE-III inhibitors, that is, milrinone (MIL) and cilostazol (CIL). The efficacy (maximum attainable effect) of these drugs for increasing I_Ca or developed tension (DT) ranged in the order of IBMX MIL > PIM > CIL. This finding suggests that the positive inotropic effect of each drug is roughly proportional to its increasing effect on I_Ca. The additional effect of PIM (a Ca²⁺-sensitizing effect) was not identified in intact preparations, and the potentiating effects of PIM on the DT and I_Ca were virtually the same as those observed for MIL. To isolate the Ca²⁺-sensitizing effect of PIM on the DT, we studied the effects of PIM in the presence of H89, an isoquinoline derivative possessing a selective inhibitory effect on cAMP-dependent protein kinase. In the absence of H89, 50 µM PIM increased the DT by 68 ± 11% (mean ± SE, n = 6). However, in the presence of 20 µM H89, which completely blocked the PIM-induced increase in I_Ca, PIM (50 µM) significantly increased the DT by 19 ± 6% (n = 6), thereby indicating the presence of a positive inotropic effect attributable to a mechanism other than increased intracellular cAMP, that is, a Ca²⁺-sensitizing effect. The latter notion was supported by the finding that in the presence of H89 (20 µM), the PIM-induced augmentation of DT was accompanied by a prolongation of the time to 50% relaxation of contractile tension. In contrast, MIL (50 µM) and forskolin, a direct activator of adenylate cyclase (1–10 nM), did not increase DT in the presence of 20 µM H89. These results suggest that the fraction of positive inotropic effect of PIM attributable to its Ca²⁺-sensitizing effect is masked by its potent PDE-III inhibitory effect in intact ventricular preparations.     

Regulation of sugar transport in the myocardium: effects of ouabain in resting and beating isolated guinea pig left atria.

The effect of ouabain on sugar transport in quiescent beating guinea pig left atria was studied by measuring the tissue/medium distribution of 3-methylglucose. The data were correlated with simultaneously measured changes in intracellular Na⁺ and K⁺ and in contractile force. In resting atria, as in other types of muscle, sugar transport paralleled internal Na⁺, both being decreased by low (10⁻⁹ g/ml) and increased by high (10⁻⁵ g/ml) ouabain. In atria contracting at 75 beat/min there was an additional stimulating effect of ouabain which closely followed the positive inotropic effect of the drug, with maximal increase in sugar transport and contractile force around 10⁻⁶ g/ml. Higher concentrations elicited contracture, and sugar transport was decreased to the level seen in resting tissues. Changes in Na⁺ and K⁺ were the same as in resting atria and did not reflect the inotropic effect of ouabain. As changes in Ca²⁺ distribution and fluxes result from both alterations in contractile force and in internal Na⁺ level (via Ca²⁺-Na⁺ exchange), the effects of ouabain on sugar transport are consistent with the hypothesis that regulation of sugar transport in muscle is mediated by the availability of cytoplasmic Ca²⁺ for binding to a regulatory site.     

Stimulatory and inhibitory effects of cyclic AMP on pancreatic glucagon release from monolayer cultures and the controlling role of calcium

Summary When glucagon release from monolayer cultures of newborn rat pancreas was measured over four hours in media containing 2.5 mM Ca⁺⁺, a significant cyclic AMP-related inhibition of release was observed. This was noted whether intracellular cyclic AMP levels were raised by the addition of exogenous cyclic AMP or dibutyryl cyclic AMP, by phosphodiesterase inhibition with theophylline, or by the stimulation of adenylate cyclase with cholera toxin. The inhibition was concentration dependent for cyclic AMP and could not be reproduced by the addition of AMP, ADP or ATP. Adenosine also inhibited glucagon release while ATP was stimulatory. From time course studies it appeared that the inhibitory effects of cyclic AMP and cholera toxin were progressive after two hours of incubation. With cholera toxin an early stimulation of glucagon release was observed. The effects of cyclic AMP and cholera toxin on argininestimulated glucagon release were to stimulate further the glucagon release during the first hour of the incubation. Thus, the effects of raising intracellular cyclic AMP levels were biphasic in that both an early stimulation and a late inhibition of glucagon release were observed. In examining the nature of these responses a remarkable controlling role for Ca⁺⁺ was uncovered: at Ca⁺⁺ concentrations of 0.3 mM and lower no effect of cyclic AMP on glucagon release was found. With 1 mM Ca⁺⁺ in the medium cyclic AMP stimulated glucagon release early (30 min) and thereafter had no further effect. In the presence of 2.5 mM Ca⁺⁺ cyclic AMP did not stimulate early but did cause the delayed inhibition of release. It is concluded that the effect of cyclic AMP on glucagon release can be either stimulatory or inhibitory depending upon the Ca⁺⁺ concentration of the medium and the duration of exposure to raised cyclic AMP levels.     

Physiologic assessment of the inotropic,vasodilator and afterload reducing effects of milrinone in subjects without cardiac disease

Milrinone increases left ventricular (LV) shortening. Whether these changes result from vasodilation alone or from a combination of vasodilation and a positive inotropic action is controversial. Load-independent end-systolic indexes of LV contractility were measured over a wide range of aortic pressures generated by methoxamine infusion before and during milrinone administration. Sixteen studies were performed using echocardiography and calibrated carotid pulse tracings in 11 normal subjects. Milrinone loading doses of 30, 45 or 60 μg/kg were given intravenously over 10 minutes, followed by a maintenance infusion to achieve steady-state drug levels. Milrinone induced a dose-dependent decrease in baseline (i.e., before methoxamine) total systemic resistance (p < 0.05) and afterload as measured by end-systolic wall stress (p < 0.001). The associated changes in the end-systolic pressure-dimension, stress-shortening and stressvelocity of fiber shortening relations were characteristic of a positive inotropic intervention. All end-systolic indexes of LV contractility demonstrated greater inotropic effect at the higher milrinone plasma concentrations. Thus, load-independent indexes of LV contractility show that milrinone has a dose-related positive inotropic effect separate from its vasodilator (total systemic resistance) and afterload (end-systolic stress) reducing effects.