Peroxynitrite is a positive inotropic agent in atrial and ventricular fibres of the frog heart.

1. We report opposite inotropic effects of NO donors in frog cardiac fibres. The negative effect, elicited by either 3-morpholino-sydnonimine (SIN-1) or S-nitroso-N-acetyl-penicillamine (SNAP), involved cyclic GMP (cGMP) production. However, SIN-1, unlike SNAP, could elicit a positive effect, in a superoxide dismutase (SOD)-sensitive manner. SIN-1, unlike SNAP, can release both NO and superoxide anion, the precursors of peroxynitrite (OONO-). The role of these messengers was examined. 2. Catalase did not reduce the positive inotropic effect of SIN-1. Thus, a conversion of superoxide anion into hydrogen peroxide was not involved in this effect. In addition, catalase did not modify the negative effects of SIN-1 plus SOD, or SNAP plus SOD. 3. LY 83583, a superoxide anion generator, elicited a positive inotropic effect, like SIN-1. The effect of LY 83583 was additive to the negative effects of SIN-1 or SNAP, and to the positive effect of SIN-1. Thus, superoxide anion generation, per se, did not account for the positive effect of SIN-1. 4. Authentic peroxynitrite (OONO-), but not mock-OONO- (negative control plus decomposed OONO-), exerted a dramatic positive inotropic effect in cardiac fibres. The effect of OONO- was larger in atrial fibres, as compared with ventricular fibres. 5. The positive effect of OONO- was not additive with that of SIN-1, suggesting a common mechanism of action. In contrast, the effects of either OONO- or SIN-1 were additive with the negative inotropic effect of SNAP. Furthermore, the effect of OONO-, like that of SIN-1, was not antagonized by 1H-[1,2,4]xidiazolo[4, 3-a]quinoxaline-1-one (ODQ; 10 microM), the guanylyl cyclase inhibitor. 6. The positive inotropic effects of SIN-1 and OONO- were not modified by hydroxyl radical scavengers, such as dimethyl-thio-urea (DMTU; 10 mM). 7. The positive inotropic effect of SIN-1 (100 microM) was abolished in sodium-free solutions, a treatment that eliminates the activity of the sodium-calcium exchanger. In contrast, the effect of SIN-1 was unchanged by a potassium channel inhibitor (tetraethyl-ammonium, 20 mM), or a sodium-potassium pump inhibitor (ouabain 10 microM). 8. We conclude that OONO- is a positive inotropic agent in frog cardiac fibres. The generation of OONO- accounts for the positive inotropic effect of SIN-1. OONO- itself was responsible for the positive inotropic effect, and appeared to modulate the activity of the sodium-calcium exchanger.

Positive and negative inotropic effects of NO donors in atrial and ventricular fibres of the frog heart.

1. The cardiac effects of the NO donors sodium nitroprusside (SNP), S-nitroso-N-acetyl-penicillamine (SNAP) and 3-morpholino-sydnonimine (SIN-1) were studied in frog fibres to evaluate the contribution of cyclic GMP-dependent mechanisms. 2. SNP and SNAP (0.1-100 microM) reduced the force of contraction in a concentration-dependent manner in atrial and ventricular fibres. This effect was associated with a reduction in the time to peak (TTP) and the time for half-relaxation of contraction (T). 3. SIN-1 (100 microM) also reduced the force of contraction in two-thirds of the atrial fibres. However, it exerted a positive inotropic effect in the remaining atrial fibres, as well as in most ventricular fibres. 4. The guanylyl cyclase inhibitor 1H-[1,2,4]oxidiazolo[4,3-a]quinoxaline-1-one (ODQ, 10 microM) antagonized the negative inotropic effects of SIN-1 (50 microM) and SNAP (25 microM) but had no effect on the positive inotropic response to SIN-1 (100 microM). 5. In the presence of SIN-1, superoxide dismutase (SOD, 50-200 U ml-1) either potentiated the negative inotropic effect or turned the positive inotropic effect of the drug into a negative effect. SOD had no effects when applied alone or in the presence of SNAP. 6. 6-Anilino-5,8-quinolinedione (LY 83583, 3-30 microM), a superoxide anion generator also known as a cyclic GMP-lowering agent, exerted a positive inotropic effect, which was antagonized by SOD (200-370 U ml-1) but not by ODQ (10 microM). 7. We conclude that SNP, SNAP and SIN-1 exert cyclic GMP-dependent negative inotropic effects, which are attributed to the generation of NO. In addition, SIN-1 and LY 83583 exert cyclic GMP-independent positive inotropic effects, which require the generation of superoxide anion.

Nitric oxide synthase does not participate in negative inotropic effect of acetylcholine in frog heart.

In the heart, the parasympathetic neurotransmitter acetylcholine (ACh) reduces the force of contraction. Although the effect of ACh can be partly explained by an inhibition of adenylyl cyclase, some of the effects of ACh may also be mediated via stimulation of nitric oxide synthase (NOS) and production of guanosine 3', 5'-cycle monophosphate (cGMP). NOS inhibitors can prevent the negative chronotropic effect of ACh on spontaneously beating cardiomyocytes and suppress the inhibition of the L-type calcium current (ICa) by ACh in sinoatrial myocytes. This pathway may be relevant not only to the chronotropic effect of ACh but also to its inotropic effect, because ACh, NO, and cGMP regulate the force of contraction and ICa in the cardiac ventricle. Here we report the effects of L-arginine (L-Arg), the substrate of NOS, and NG-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine (L-NNA), two NOS inhibitors, on muscarinic effects in the cardiac ventricle. We found that L-Arg, L-NMMA, and L-NNA have no effect on the muscarinic inhibition of ICa in isolated frog myocytes. In addition, these compounds have no significant effects on basal ICa or beta-adrenergic stimulation of ICa. L-Arg and its analogues did not change the negative inotropic effect of ACh in frog ventricular fibers. Basal active tension and the positive inotropic effect of isoproterenol, a beta-adrenergic agonist, also were unaffected. We conclude that NOS in not involved in muscarinic inhibition of ICa in isolated from ventricular myocytes or the negative inotropic effect of ACh in the frog ventricle.

Influence of Bistramide A on the twitch tension in rat atrial heart muscle.

Bistramide A, a new toxin isolated from the Urochordate Lissoclinum bistratum Sluiter, was applied to rat auricular heart muscle bundles. At a stimulation frequency of 0.2 Hz, the toxin induces a dose-dependent reduction of the stimulated twitch tension force; it decreases Vmax and shortens the duration of the plateau and the slow repolarizing phase of the action potential. In the control solution, switching from a stimulation frequency of 0.2 Hz to 1 Hz decreases the force with which a positive potentiation develops either at a maintained high frequency or after switching from 1 Hz to 0.2 Hz. Bistramide A reduces both the force evoked at 1 Hz and the potentiation. The data suggest that Bistramide A blocks Na+ conductance; inhibits Ca++ channels in a time- and frequency-dependent manner; reduces Na(+)-Ca++ exchange activity; but does not modify the ability of the sarcoplasmic reticulum to be refilled although the rate of Ca++ accumulation is decreased.

The polyether Bistramide A affects the calcium sensitivity of the contractile proteins in frog atrial heart muscle.

The effects of Bistramide A, a new toxin isolated from the Urochordate Lissoclinum bistratum Sluiter have been studied on the mechanical activity of frog heart atrial muscle preparations. The peak tension of isolated trabeculae was sensitive to nanomolar concentrations of Bistramide A. Lineweaver-Burk relationships suggest that Bistramide A competes with Ca for a common site. In voltage-clamped trabeculae, the toxin inhibited both the cadmium-sensitive Ca current and the phasic component of the tension with a dissociation constant of 3.3 microM and a stoichiometry of 2. Bistramide A decreased the isometric tension of skinned fibres in a dose-dependent manner with a dissociation constant of 400 nM and a stoichiometry of 2. The toxin reduced the maximum Ca activated force and decreased the sensitivity of the contractile proteins to Ca. The data suggest that Bistramide A decreases the Ca-sensitivity of contractile proteins prior to blocking the Ca current.

Directional variability of stimulation threshold measurements in isolated guinea pig cardiomyocytes: relationship with orthogonal sequential defibrillating pulses.

Reports on delivery of separated orthogonal pulses markedly improving cardiac defibrillation have suggested that the stimulation threshold of heart fibers varies in accordance with their orientation within the electric field. The present work was aimed at investigating the directional variability of stimulation thresholds in isolated guinea pig cardiomyocytes. This variability was measured in 48 single myocytes by rotating each one through a theta (theta) angle between two-fixed parallel electrodes 1.1 cm apart, thus making theta vary between the electric field and the myocyte axis. For theta = 0 degrees, the mean longitudinal current stimulation threshold was 16.92 +/- 4.20 mA (n = 48). When theta was increased by increments of 10 degrees up to 90 degrees, the stimulation threshold increased in an exponential way. For theta = 90 degrees, the mean transverse stimulation threshold was 63.13 +/- 13.30 mA. These results clearly demonstrate the dependence of isolated cardiomyocyte stimulation thresholds on their orientation within the electric field and may account for the improved efficacy of defibrillation previously observed after delivery of orthogonal pulses.

Reduction of energy required for defibrillation by delivering shocks in orthogonal directions in the dog.

Reduction of energy required to defibrillate (ERD) seems to represent a necessary condition for intensive development of implantable defibrillator, so as for minimization of cardiac and pulmonary damages provoked by high energy transthoracic defibrillation electric shocks. The present work describes a defibrillation method using shocks delivered in orthogonal directions and separated by a 100 ms delay. Defibrillation threshold measured with classical unidirectional shocks on 30 dogs has been found to be 286.8 +/- 22.2 joules. In the same animals, defibrillation threshold measured by use of orthogonal shocks has been found to be 101.4 +/- 14.9 joules. We conclude that this crossed shocks method leads to a substantial reduction of ERD (64%).

Intracellular pH regulation in papillary muscle cells from streptozotocin diabetic rats: an ion-sensitive microelectrode study.

Intracellular pH regulation was studied in papillary muscle from STZ-induced diabetic rat hearts. In control bicarbonate solution there was no difference between the steady-state pHi values recorded from diabetic or normal papillary muscle. The addition of insulin had no effect on the pHi of either group. The amplitude of NH4+-induced alkalinization and the time course of recovery from alkalinization were similar in both normal and diabetic muscles. In both preparations, the recovery from alkalinization was similarly delayed by the disulfonic stilbene DIDS. This suggests the participation of a Cl-/HCO3- exchange in the recovery from alkalosis in rat myocardial cells that is not changed by diabetes. On the other hand, the amplitude of the acidification induced by the withdrawal of NH4+ was markedly increased in diabetic papillary muscles as compared to normal muscles. Moreover, there was a marked slowing down of the recovery from acidosis in the diabetics. The amplitude of NH+4 withdrawal-induced acidification was increased equally by amiloride in both normal and diabetic muscles. These findings suggest that diabetes is associated with a change in the activity of the amiloride-sensitive Na+/H+ exchange.

[Discontinuous feedback amplifier: principles and application to the measurement of transmembrane potentials and currents of frog atrial fibers]. / Amplificateur à asservissement discontinu: principe et application à la mesure des potentiels et des courants transmembranaires des fibres atriales de grenouille.

The study and achievement of a discontinuous feedback amplifier to measure membrane potentials and currents in frog atrial fibres using the double sucrose gap technique was achieved. It was shown that, with the present device, the effects of the resistance in series with the membrane resistance and the membrane capacity on the measure of cardiac membrane potentials and fast currents are markedly reduced.

Staircase in frog ventricular muscle. Its dependence on membrane excitation and extracellular ionic composition.

Staircase was studied in frog ventricle strip preparations where it was possible to alter extracellular ionic composition extremely rapidly in the diastolic interval between beats. Several findings strongly indicate that staircase in this tissue is a result of progressively increasing calcium influx per beat, rather than a beat-by-beat augmentation of an intracellular calcium pool which contributes to activation. After a steady state of force development, the very next beat could be graded, from approximately zero force to the steady state value attained during the staircase progression, by grading the calcium concentration of a new Ringer's solution switched to perfuse the muscle in the diastolic period immediately before that beat. Also, action potentials, elicited during the "quiescent" period in the virtual absence of contraction (in 0.025 mm calcium Ringer's solution), markedly increase force development and accelerate the staircase seen upon return to normal Ringer's solution. Staircase is augmented and accelerated by prior exposure of the muscle, during the quiescent period, to calcium-poor media and markedly suppressed by prior exposure to sodium-poor media. Tetrodotoxin, in a dose that markedly slows the action potential upstroke, has no effect on staircase. Finally, staircase is seen to occur during a train of depolarizations (by voltage clamp) to inside positive levels greater than the equilibrium potential for sodium. It is concluded that changes in intracellular sodium concentration will alter the staircase response and may contribute to its genesis, but that this cannot be the sole cause of staircase.