Hasso Scholz

Cardiac glycoside receptor, (Na+ + K+)ATPase activity and force of contraction in rat heart

Abstract In order to elucidate the role of (Na+ + K+)ATPase and of the ouabain binding sites in the pharmacological effects of ouabain in the digitalis insensitive species rat, specific [3H]ouabain binding and (Na+ + K+)ATPase activity were measured simultaneously in a rat heart cell membrane preparation. Specific [3H]ouabain binding, 86Rb+-uptake and force of contraction were also measured simultaneously in electrically stimulated contracting ventricular strips of rat heart. The following results were obtained: (1) Rat heart cell membranes exhibit two classes of [3H]ouabain binding sites with apparent dissociation constants (KD) of the [3H]ouabain-receptor complex 1–2.3 × 10−7 M and 2.8 × 10−5 M. (2) (Na+ + K+)ATPase activity of rat heart cell membranes is half-maximally inhibited by ouabain at a concentration of 4 × 10−5 M when assayed at the same conditions as [3H]ouabain binding. (3) Specific [3H]ouabain binding to electrically stimulated (1 Hz) contracting ventricular strips of rat heart exhibited only one class of receptors (KD = 3 × 10−7M). Force of contraction increased half-maximally at 3 × 10−7 M ouabain when measured simultaneously and 86Rb+uptake was inhibited half-maximally at 3 × 10−5 M ouabain. Thus, there is a serious discrepancy between the effect of ouabain on (Na+ + K+)ATPase activity and 86Rb+ uptake on one hand and on force of contraction on the other hand, whereas there is a good correlation between [3H]ouabain-receptor binding and increase in force of contraction. These results indicate that inhibition by ouabain of active cation transport is not a mandatory prerequisite of its positive inotropic effect, at least in the rat heart.

Effectiveness of theophylline to increase cyclic AMP levels and force of contraction in electrically paced guinea-pig auricles. Comparison with isoprenaline, calcium and ouabain.

SummaryThe 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.

The positive inotropic effect of phenylephrine in the presence of propranolol. Increase in time to peak force and in relaxation time without increase in c-AMP

SummaryThe effects of phenylephrine on the shape of the contraction curve and on the cyclic adenosine 3′,5′-monophosphate (c-AMP) content were studied in electrically driven (frequency 0.2 Hz) cat papillary muscles. All experiments were done in the presence of 1 μM propranolol in order to minimize interference from β-adrenoceptors. 1.Phenylephrine increased the force of contraction in a concentration-dependent manner. Maximal effects (about 200% of control) occurred at 30 μM phenylephrine.2.The positive inotropic effect (PIE) of phenylephrine was antagonized by phentolamine. Phentolamine, 5μM, produced a parallel shift of the concentration-response curve for the PIE of phenylephrine by about two log units to the right.3.The PIE of 30 μM phenylephrine occurred without any detectable increase in the c-AMP levels of the preparations.4.The PIE of 30μM phenylephrine developed about three times more slowly than the PIE of an equieffective concentration of isoprenaline.5.The PIE of phenylephrine was accompanied by significant, concentration-dependent increases in both time to peak force and relaxation time.6.It is concluded that the PIE of phenylephrine in the presence of propranolol is mediated mainly by a stimulation of α-adrenoceptors. It is unlikely to be related to an increase in c-AMP. With respect to time course and influence on the shape of the contraction curve it is qualitatively different from the effects of β-adrenoceptor stimulation. These data are taken to support the hypothesis that the mechanical effects of α-and β-adrenoceptor stimulating agents on the heart are produced by different mechanisms.

Stimulatory (insulin-mimetic) and inhibitory (ouabain-like) action of vanadate on potassium uptake and cellular sodium and potassium in heart cells in culture

(1) The influence of vanadate (Na3VO4) on sodium and potassium uptake as well as on cellular ion contents of sodium and potassium has been studied in heart muscle and non-muscle cells obtained from various species. An ouabain-like inhibition of potassium uptake (up to 50%), combined with a decrease of cellular potassium (up to 20%) has been observed by vanadate (10(-4)-10(-3) M) in heart non-muscle cells obtained from neonatal guinea pigs and chick embryos. In heart muscle and non-muscle cells prepared from neonatal rats, as well as in Girardi human heart cells, a vanadate-induced stimulation of potassium uptake (up to 100%), combined with a rise in cellular potassium (up to 20%) and without significant alteration of cellular sodium, has been found. A slight increase of 22Na+ influx can be measured in rat heart muscle cells and in Girardi human heart cells in the presence of vanadate (10(-4)--10(-3) M). (2) In beating rat heart muscle cells in culture, detrimental effects of serum deprivation--concerning beating properties, potassium uptake and cellular potassium--can at least in part be overcome by addition of vanadate. Furthermore, this compound prevents ouabain-induced signs of toxicity (contractures) in these cells. (3) The stimulatory effects of vanadate on potassium can be mimicked by insulin (1-10 mU/ml). Furthermore, vanadate produces an insulin-like stimulation of 2-deoxy-D-glucose uptake in rat heart muscle and non-muscle cells as well as in Girardi human heart cells. (4) The experimental data demonstrate an ouabain-like inhibition as well as an insulin-mimetic stimulation of potassium-uptake in various heart cells. The reason for this antagonistic mode of action may be due to the different capabilities of the heart cell types to reduce vanadium in the V-valence state of vanadium in the IV-valence state, thereby favouring either ouabain-like inhibition (vanadium V) or insulin-mimetic stimulation (vanadium IV) of potassium transport.

Effects of vanadate on the c-AMP system of the heart

Summary300 μM vanadate (NH4VO3) caused an about 65% increase in force of contraction in isolated electrically driven cat papillary muscles. The positive inotropic effect (PIE) was accompanied by only a small (about 20%) but significant increase in c-AMP levels. 0.3 μM isoprenaline (ISO) produced a much greater increase in c-AMP levels (about 80%) while the PIE of ISO was as great as the PIE observed with 300 μM NH4VO3. In cat right ventricular adenylate cyclase preparations NH4VO3 and ISO stimulated adenylate cyclase activity by nearly the same extent. Propranolol (1 μM and 5 μM) prevented the ISO-induced adenylate cyclase stimulation but not the stimulation due to NH4VO3. Phosphodiesterase activity was not affected by NH4VO3 (up to 1000 μM). It is concluded that the small vanadate-induced increase in c-AMP levels, which is due to adenylate cyclase stimulation, inintact papillary muscles maycontribute to its PIE, but other mechanisms may be at least equally important.Zusammenfassung300 μM Vanadat (NH4VO3) steigerte an isolierten, elektrisch gereizten Katzenpapillarmuskeln die Kontraktionskraft um ca. 65%. Der positiv inotrope Effekt (PIE) ging einher mit einem nur geringen (ca. 20%), aber signifikanten Anstieg des c-AMP-Gehaltes. 0.3 μM Isoprenalin (ISO) verursachte einen sehr viel größeren Anstieg des myokardialen c-AMP-Gehaltes (ca. 80%), während der PIE von ISO gleich groß war wie der PIE von NH4VO3. In einer Adenylatcyclasepräparation von rechten Ventrikeln des Katzenherzens stimulierten NH4VO3 und ISO die Adenylatcyclaseaktivität in nahezu gleichem Ausmaß. Propranolol (1 μM und 5 μM) verhinderte die Aktivierung der Adenylatcyclase durch ISO, aber nicht die Aktivierung durch NH4VO3. Die Phosphodiesteraseaktivität wurde durch NH4VO3 (bis zu einer Konzentration von 1000 μM) nicht beeinflußt. Aus den Ergebnissen wird geschlossen, daß der geringe vanadatinduzierte c-AMP-Anstieg, der über eine Stimulation der Adenylatcyclase zustande kommt, zwar am Zustandekommen des PIE beteiligt sein mag, daß aber andere Mechanismen von mindestens ebenso großer physiologischer Bedeutung sind.

On the mechanism of positive inotropic effects of alpha-adrenoceptor agonists.

SummaryThe positive inotropic effect of the alpha1-adrenoceptor agonist phenylephrine is accompanied by an increase in the presumed second messengers inositol 1,4,5-trisphosphate (1,4,5-IP3) and inositol 1,3,4,5-tetrakisphosphate (1,3,4,5-IP4). Both 1,4,5-IP3 and 1,3,4,5-IP4 sensitize myocardial contractile proteins in chemically skinned fibers. In addition to the Ca++ releasing effect of 1,4,5-IP3 from the sarcoplasmic reticulum the Ca++-sensitizing effect of the inositol phosphates may play a role in alpha1-adrenergic positive inotropism. In isolated heart muscle preparations from patients with endstage heart failure (due to dilated cardiomyopathy) beta-adrenergic as well as alpha1-adrenergic effects are reduced compared to preparations from healthy hearts. The reduced beta-adrenergic effects can in part be explained by an increased content of signal transducing G1-proteins. It is tempting to investigate whether other G proteins are also altered in severe congestive heart failure.

Effect of vanadate on myocardial force of contraction

SummaryAmmonium vanadate (NH4VO3; 50–1000 μM) increased the force of contraction of isolated electrically driven cat papillary muscles in a concentration-dependent manner. The positive inotropic effect (PIE) of NH4VO3 became significant at 50 μM and was maximal at 500 to 1000 μM. It was accompanied by an increase in the rate of force development, in the rate of relaxation and in relaxation time of the isometric contraction. Similar results as with NH4VO3 were obtained with NaVO3 and with Na3VO4. The effects of NH4VO3 were also observed in the presence of 1 μM propranolol, 5 μM phentolamine or after reserpine-pretreatment (5 mg/kg i.p.). These results indicate that vanadate produces a direct PIE inventricular cardiac muscle which is unlikely to be mediated by alpha-or beta-adrenoceptor stimulation. In cat left atrial strips, however, vanadate ions produced a negative inotropic effect through a hitherto unknown mechanism. Vanadate effects similar to those observed in the cat heart were obtained in ventricular and atrial preparatins from bovine hearts.ZusammenfassungDie Wirkung von Vanadat (NH4VO3, NaVO3, Na3VO4) auf die Kontraktionskraft des Herzens wurde an isolierten, elektrisch gereizten Katzenpapillarmuskeln untersucht. NH4VO3 (50–1000 μM) wirkte konzentrationsabhängig positiv inotrop. Der Effekt begann nach etwa 1 min, war nach 6–10 min maximal und blieb während der gesamten Versuchsdauer (30 min) erhalten. Die Wirkung von Vanadat wurde auch in Gegenwart von Propranolol und Phentolamin oder nach Reserpinvorbehandlung beobachtet. NH4VO3 steigerte Anstiegs- und Erschlaffungsgeschwindigkeit der isometrischen Kontraktion. Die Kontraktionsdauer wurde verlängert; dieser Effekt beruhte auf einer Verlängerung der Erschlaffungszeit. Mit NaVO3 und Na3VO4 wurden ähnliche Ergebnisse erzielt. Die Versuche sprechen dafür, daß Vanadat anventrikulären Herzmuskelpräparaten direkt positiv inotrop wirkt und daß diese Wirkung nicht durch Alpha- oder Beta-Adrenozeptoren vermittelt wird. An linken Katzenvorhofstreifen führte Vanadat zu einem negativ inotropen Effekt, dessen Ursache bisher ungeklärt ist. An isolierten Ventrikel- und Vorhofstrabekeln aus Rinderherzen hatte Vanadat ähnliche Effekte wie am Katzenherzen.

Negative inotropic effect of vanadate in ventricular myocardium in the presence of 3-isobutyl-1-methylxanthine or isoprenaline

SummaryThe 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 agents 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.

On the mechanism of action of theophylline on the heart and other organs

Evidence is summarized which indicates that the positive inotropic effect (PIE) of theophylline, which is similar to that observed with β-adrenoceptor stimulating agents, in mammalian cardiac muscle is mainly due to an increase in slow inward current, Isi . The increase in Isi and, hence, in force of contraction conceivably results from an inhibition of phosphodiesterase (PDE) activity with a subsequent increase in myocardial cAMP levels. The increase in cAMP levels is probably also responsible for the relaxant effects of theophylline in smooth muscle. Finally it is discussed that the PIE of theophylline in the heart is unlikely to result from an adenosine-antagonistic action because adenosine produces a negative inotropic effect only in atria, whereas in ventricular cardiac muscle it even increases the force of contraction.

Evidence against a role of (Na++K+-ATPase in the alpha-adrenoceptor mediated positive inotropic effect of phenylephrine

Phenylephrine (0.1–100 μM) in the presence of 1 μM propranolol increased the force of contraction in electrically driven papillary muscles from cats. This presumably alpha-adrenoceptor mediated positive inotropic effect of phenylephrine occurred without any influence on (Na++K+-ATPase activity.