G. Grupp

Specific binding of [3H]nitrendipine to membranes from coronary arteries and heart in relation to pharmacological effects. Paradoxical stimulation by diltiazem

Abstract High affinity binding sites for the calcium channel inhibitor [ 3 H]nitrendipine have been identified in microsomes from pig coronary arteries (K D =1.6 nM; B max =35 fmol/mg) and in purified sarcolemma from dog heart (K D =0.11 nM; B max =230 fmol/mg). [ 3 H]nitrendipine binding to coronary artery microsomes was completely inhibited by nifedipine, partially by verapamil and D600 and, surprisingly, was stimulated by d-cis-diltiazem but not by 1-cis-diltiazem, a less active isomer. Half-maximal relaxation of KCl-depolarized coronary rings occurred in a slow process at 1 nM nitrendipine or 100 nM d-cis-diltiazem. In dog trabecular strips, nitrendipine caused a negative inotropic response (ED 50 =1μM). These results suggest that there may be multiple binding sites for different “subclasses” of calcium channel inhibitors, and that drug binding sites may be different molecular entities from the putative calcium channels.

Effects of vanadate on cardiac contraction and adenylate cyclase

Abstract Vanadate produces a positive inotropic effect on ventricular muscle from rat, rabbit, guinea pig and cat; a positive inotropic effect on the atria of rat and rabbit, but a negative inotropic effect on the atria of guinea pig and cat. The effects of vanadate are completely reversible and occur in a concentration range of 10 −5 M to 10 −3 M. In this same concentration range, vanadate also causes a marked activation of cardiac adenylate cyclase suggesting that the positive inotropic action might be due in part to an elevation of cyclic AMP levels. The effects of vanadate are not influenced by alprenolol, cimetidine, or mepyramine, indicating a lack of involvement of β-adrenergic or histamine H 2 and H 1 receptors.

Effects of Bay k 8644, a dihydropyridine analog, on [3H]nitrendipine binding to canine cardiac sarcolemma and the relationship to a positive inotropic effect.

Equilibrium dissociation constants of Bay k 8644, a calcium agonist, and nitrendipine, a calcium antagonist, were determined in canine cardiac sarcolemma. The equilibrium dissociation constant for Bay k 8644 was compared to the concentration that produced a fifty percent increase, and the equilibrium dissociation constant for nitrendipine was compared to the concentration that produced a fifty percent decrease, in contractile force in canine heart trabecular muscle. Both saturation and inhibition binding data suggest that Bay k 8644 and nitrendipine bind to and compete for a high affinity dihydropyridine-binding site present in isolated cardiac sarcolemma preparations. The equilibrium dissociation constant (7–10 NM) and concentration that produced a fifty percent increase in contractile force in the canine trabecular muscle (30 ± 8 nM) of Bay k 8644 were in a similar concentration range, but the equilibrium dissociation constant (0.29 ± 0.025 nM) of nitrendipine binding was more than a thousand-fold lower than the concentration that produced a fifty percent decrease in contractile force in canine trabecular muscle (613 ± 109 n). These data suggest that binding of Bay k 8644 to high affinity binding sites is pharmacologically relevant, and is related to a positive inotropic effect.

Comparison of the effects of neuropeptide Y (NPY) and 4-norleucine-NPY on isolated perfused rat hearts; effects of NPY on atrial and ventricular strips of rat heart and on rabbit heart mitochondria.

Isolated perfused rat hearts were used to compare the effects of the synthetic neuropeptide Y (NPY) and 4-norleucine-NPY on cardiac function. Each peptide exhibited both negative inotropic and chronotropic effects, and also caused coronary vasoconstriction leading to a reduction in coronary flow. A comparison of the IC50 values from dose-response curves using 10(-14) to 10(-7) M peptides (IC50 is the peptide concentration that produced a 50% decrease of the maximal effect) indicated that NPY was more potent as inhibitor of contractility and less potently inhibited coronary flow and heart rate, whereas 4-norleucine-NPY had more inhibitory influence on coronary flow and heart rate and less on cardiac contractility. This difference in potencies suggests that the inhibitory effects of NPY on contractility, coronary flow and heart rate may be independent of each other. Since NPY also decreased the contractile force of isolated left atrial and right ventricular strips of the rat heart, the coronary flow decrease cannot be the cause of the negative inotropy of isolated heart. Pretreatment of atrial and ventricular strips with NPY did not influence the positive inotropic effect produced by the cardiac glycoside ouabain indicating that sarcolemmal Na+, K+-ATPase was not involved in the inhibitory inotropic effect of NPY. Further studies towards elucidating the mechanism of the negative inotropy of cardiac muscles using isolated heart mitochondria revealed that NPY uncoupled oxidative phosphorylation and blocked mitochondrial calcium uptake; the former event fosters negative inotropy. Since these effects on mitochondria occurred at concentrations 100-fold higher than those required for negative inotropy, the two effects of NPY may not be related.

Vasodilatory action of amlodipine on rat aorta, pig coronary artery, human coronary artery, and on isolated Langendorff rat heart preparations.

Amlodipine inhibited contractions of rat aortic rings induced by 40 mM KCl (IC50 = 7.5 x 10(-9) M). The time to attain the maximum inhibitory effect of KCl-induced contractions was long (hours) and dependent on the concentration of amlodipine. After 6 h of washing in drug-free normal Krebs-Ringer solution the contractions recovered only partially. The KCl-induced contractions appeared to be more sensitive to inhibition by amlodipine than were norepinephrine-induced contractions. CaCl2-induced contraction of KCl-depolarized aortic rings was inhibited by amlodipine in a complex manner. Amlodipine not only increased ED50 but also inhibited the maximal tension induced by CaCl2. Amlodipine also inhibited 35 mM KCl-induced contractions of pig coronary artery rings (IC50 = 2.2 x 10(-8) M) and human coronary artery rings (IC50 = 2.1 x 10(-8) M). In Langendorff rat heart preparations, low concentrations of amlodipine increased coronary flow (ED50, 10(-9) M) whereas higher concentrations (greater than 10(-7) M) decreased coronary flow. Amlodipine also decreased the rate of contraction (+ dP/dt, IC50 = 3 x 10(-7) M) and the rate of relaxation (-dP/dt, IC50 = 1.2 x 10(-7) M). Amlodipine decreased heart rate but only at high concentrations (greater than 300 nM). The results of this study indicate that amlodipine is a potent vasodilator with similar cardiovascular actions to other dihydropyridines except that its effects are slower in onset and longer lasting.

Effects of vanadate on myocardial function

SummaryThe influence of vanadate (NH4VO3, Na3VO4) and vanadyl (VOSO4) on myocardial function was studied using a variety ofin vivo andex vivo cardiac preparations. In addition, the influence of vanadate on a variety of cardiac subcellular systems was examinedin vitro.In isolated ventricular and atrial muscle of dog, cat, guinea pig, rabbit and rat, vanadate produces a positive inotropic effect. The effective concentration for positive inotropy in these tissues ranged from 10 to 500 μM. Vanadyl sulfate was less effective than either sodium ortho-vanadate or ammonium meta-vanadate. Vanadate produced negative inotropic effects in guinea pig and cat isolated left atrium, at concentrations lower than those required to produce positive inotropic effects in sensitive species and tissues.In blood perfused papillary muscle or whole heart, or in whole animal (anesthetized or conscious), vanadate generally reduced ventricular performance and produced a marked peripheral and coronary vasoconstriction. Vanadate also contracted isolated poocine coronary artery strips, an effect similar to that produced by ouabain.Vanadate caused a variety of effects on isolated subcellular systems of cardiac muscle. Low concentrations (0.1–100 μM) inhibited sarcolemmal Na+, K+-ATPase and Ca2+-ATPase, sarcoplasmic reticulum Ca2+-ATPase, Ca2+-binding to and uptake by sarcoplasmic reticulum, but stimulated hormone-sensitive adenylate cyclase. Higher concentrations of vanadate (100–1000 μM) inhibited myofibrillar ATPase, phosphorylation of troponin I, and mitochondrial ATPase.On the basis of these effects of vanadate on cardiac subcellular systems, mechanisms by which vanadate acts on intact myocardium are proposed. Furthermore, consideration of anin vivo, regulatory role of vanadate is presented with regard to myocardial function.ZusammenfassungDie Wirkung von Vanadat (NH4VO3, Na3VO4) und Vanadyl (VOSO4) auf die Myokardfunktion wurde an einer Reihe von In-vivo- und In-vitro-Präparationen des Herzens untersucht, ebenso wie der Vanadateffekt auf subzelluläre kardiale Systeme.Vanadat wirkt positiv inotrop in isolierten Ventrikeln und Vorhöfen von Hund, Katze, Meerschweinchen, Kaninchen und Ratte. Die dafür notwendigen Konzentrationen waren 10–500 μM. Vanadyl war weniger wirksam als Na3VO4 oder NH4VO3 Vanadat wirkt negativ inotrop an Meerschweinchen- und Katzenvorhofpräparaten in niedrigeren Konzentrationen als für den positiv inotropen Effekt notwendig sind.An mit Blut perfundierten Papillarmuskeln oder Herzen sowie am narkotisierten oder nicht narkotisierten Tier reduziert Vanadat die linksventrikuläre Funktion und verursacht eine deutliche periphere und koronare Vasokonstriktion. Isolierte Koronararterienstreifen (Schwein) gehen durch Vanadat in Kontraktur über ähnlich der g-Strophanthinwirkung.An isolierten subzellulären Systemen des Herzmuskels hemmen niedrige Vanadatkonzentrationen (0.1–100 μM) die sarkolemmale Na+, K+-ATPase, die Ca++-ATPase des sarkoplasmatischen Retikulums sowie dessen Ca++-Bindung und Ca++-Aufnahme. Andererseits wird die kardiale Adenylatzyklase stimuliert. Höhere Vanadatkonzentrationen (100–1000 μM) hemmen die myofibrilläre ATPase, die Phosphorylation von Troponin I und die mitochondriale ATPase.Auf der Basis der verschiedenen Wirkungen des Vanadats werden die Mechanismen zur Diskussion gestellt, über die Vanadat am intakten Herzen wirken könnte. Außerdem wird die mögliche regulatorische Rolle des Vanadats in vivo in Hinsicht auf die Myokardfunktion beurteilt.

Effects of vanadate on biochemical and contractile properties of rabbit hearts

Perfusion of working rabbit hearts with 300 μUM vanadate caused a 30% increase in left ventricular pressure (dp/dt max) and right ventricular wall force (dF/dt) with no change in negative dp/dt max or dF/dt max. Troponin I (TnI) a substrate for cyclic adenosine monophosphate dependent protein kinase, was isolated by affinity chromatography from hearts freeze-clamped at the peak of the inotropic response. The covalent phosphate content of TnI fell from 1.2 moles/mole in control hearts to 0.8 mole/mole in hearts perfused with vanadate. We also isolated myofibrils, microsomes enriched in vesicles derived from the sarcoplasmic reticulum (SR), and mitochondria, and determined the influence of vanadate on their Ca2+-ATPase and, in the case of SR vesicles, on calcium transport. While the ATPase and respiratory activity of mitochondria were unaffected by up to 100 μUM vanadate, SR ATPase and rate of calcium transport measured with 5 mM oxalate and myofibrillar ATPase were inhibited by vanadate with a similar Ki of 50 to 70 μUM. SR ATPase measured without oxalate was more sensitive to inhibition by vanadate (Ki = 5 μUM). Moreover, steady-state accumulation of Ca2+ by SR vesicles measured in the absence of oxalate was not affected by up to 60 μUM vanadate and inhibited by only 11% at 120 μUM vanadate. Our data indicate that the inotropic effects of vanadate may involve sites of action on myofibrils and SR and that vanadate may be a useful tool in the study of ATPase mechanisms.

Direct Correlation of Na+/K+-ATPase -Isoform Abundance and Myocardial Contractility in Mouse Heart

In earlier experiments on rat heart ventricles (1, 2 and 7) we found that high-and low-affinity positive inotropic effects of ouabain (PIE) were correlated with the abundance of α2 and α1 Na+/K+-ATPase isoforms respectively, whereas rat atria showed only low affinity PIE and only α1 isoforms. We also reported (3) that rat ventricular strips at high affinity concentrations (0.1 to 5 μM) accumulated aiNa significantly by 2mM. We now report the characteristics of the hearts of 2 mouse strains “FVB/N” and “129” with regard to contractile response and Na+/K+-ATPase isoform abundance. We measured myocardial contractility in atrial and ventricular strips under isometric conditions. Resting tension (final 0.5 to 1.0 g) was increased stepwise close to the point of maximally developed force (Lmax). At this level cumulative ouabain dose-response curves (10 nM to 200 μM) were obtained. The strips were suspended in oxygenated Krebs-Henseleit solution at 35° C and stimulated at 1 Hertz, pH 7.4. Relative abundance of al and α2 isoform mRNAs and enzyme activities were determined by northern blot and enzyme coupled spectrophotometric assays, respectively.

Action of Calcium Slow Channel Inhibitors on Cardiac and Vascular Smooth Muscle Membranes

Cardiac and most vascular smooth muscle cells depend on extracellular Ca2+ for contraction. The influx of Ca2+ in these cells is known to occur through Ca2+-selective slow channels during the phase 2 of the action potential when these channels are presumably “open.” A number of organic compounds classified as calcium antagonists (1) were found to antagonize the extracellular Ca2+-dependent contraction. These compounds are also referred to as “calcium slow channel inhibitors,” “calcium channel blockers,” or “calcium entry blockers.” While they differ in chemical structures (Fig. 1), they do exert at least one common effect, viz., inhibition of contraction or relaxation in cardiac and vascular smooth muscles. They are much more potent in vascular smooth muscle than in cardiac muscle, producing vasodilation at low concentrations and negative inotropy at much higher concentrations. By virtue of this differential effect, some of these calcium antagonists have been found to be useful in the treatment of angina pectoris.

Biphasic contractile response to ouabain: Species specific? Calcium dependent? Altered sensitivity?

Up to now, only the rat, an “ouabain insensitive” rodent, has shown a consistent biphasic positive inotropic response to ouabain, “low-dose” effect ED50 ~ 0.3–0.5 µM, “high dose” effect ED50 > 10 µM. The “low-dose” (high affinity?) effect was masked by high Ca++ (2 mM) and emphasized in low Ca++ (0.5 mM). The “low-dose” inotropic effect in rat ventricular strips was significantly diminished by repeated exposure to high ouabain concentrations as was 3H-ouabain binding (KD and Bmax). The decrease of the “low-dose” effect could be desensitization, tachyphylaxis (quick tolerance) or continued receptor occupancy by residual ouabain. The contractile activity of the preexposed strips did not indicate the presence of residual ouabain since their basal contractile force was decreased 12% compared to initial control instead of an expected 20% increase if ouabain occupancy had continued. The maximal response to high ouabain concentrations was identical in both exposures. In Langendorff perfused rat heart preparations the “low-dose” response was not diminished by repeated ouabain exposure and 3H-ouabain binding was not altered. The “low-dose” effect was not present in rat atrial strip preparations.