Inhibition of nitric oxide synthase augments the positive inotropic effect of nitric oxide donors in the rat heart

Abstract

1. In this investigation we studied the effects of nitric oxide on contractility and heart rate in normal saline-perfused rat hearts where shear stress-induced endothelial NO synthesis substantially contributes to total cardiac NO production. In addition, we sought to estimate the concentrations of exogenous NO producing inotropic effects. 2. We investigated the effects of glyceryl trinitrate (GTN), S-nitroso-d,l-penicillamine (SNAP), sodium (Z)-1-(N, N-diethylamino)diazen-1-ium-1,2-diolat (DEA/NO), and DEA/NO in the presence of the NO synthase inhibitor Nomega-nitro-L-arginine (L-NA) in constant-flow-perfused spontaneously beating rat Langendorff hearts and in rat working hearts. 3. In Langendorff hearts, GTN (10 nM to 100 microM, n = 32) induced a positive inotropic response that plateaued at 1 microM GTN with a maximal rate of increase of left ventricular pressure during ventricular contraction (+dP/dtmax) of 6. 33 +/- 2.56 % (n = 11, P < 0.5). Similarly, both spontaneous NO donors (0.1 nM to 1 microM, corresponding to approximately 0.03-0.3 microM NO) induced a positive inotropic response of 10.6 +/- 3.1 % (SNAP; n = 15, P < 0.05) and 11.5 +/- 2.7 % (DEA/NO, n = 15, P < 0. 05). 4. The positive inotropic effect of SNAP and DEA/NO progressively declined from 1 microM to 100 microM of the NO donors (corresponding to approximately 0.3-30 microM NO). 5. In the isolated working rat heart, 0.1 microM DEA/NO induced an increase of +dP/dtmax of 7.5 +/- 2.5 % (n = 9, P < 0.05). Inhibition of NO synthase by L-NA produced a 4-fold increase in this effect of DEA/NO. 6. We suggest that physiological NO concentrations support myocardial performance. In normal rat hearts the positive inotropic effect of NO appears to be almost maximally exploited by the endogenous NO production.

Figure 1. Reversible positive inotropic effects of NO donors

In the upper panel representative original recordings of the mean effect of DEA/NO and SNAP on left ventricular pressure of isolated constant-flow-perfused Langendorff hearts from Wistar rats are shown. The time point of subjection of the hearts to the drugs is indicated by an arrow. The lower panel shows the reversibility of the effect of 0.1 μm DEA/NO. The +dP/dt_max values are from 15 individual hearts before infusion of DEA/NO (equilibration) and after a washout period of 10 min (recovery). The direction of the individual changes are indicated by the connecting lines.

Figure 2. Effect of increasing concentrations of the organic nitrate glyceryl trinitrate (GTN) on left ventricular peak pressure (LVP), +dP/dt_max and -dP/dt_max of spontaneously beating constant-volume-perfused Langendorff hearts of normal Wistar rats (n = 32)

Mean values of percentage changes related to the basal values before drug application are shown. Vertical bars indicate {"Single column legend" off}{"Single column legend" on}s.e.m. The maximal responses are significantly different from zero (P < 0.05, paired t test). Equilibration values for LVP, +dP/dt_max and -dP/dt_max of the hearts used for these experiments are 64.4 ± 4.1 mmHg, 2264 ± 243.3 mmHg s⁻¹ and 1419 ± 96 mmHg s⁻¹, respectively. The concentration-response curves are not significantly different from each other (ANOVA).

Figure 4. Effect of increasing concentrations of the spontaneous NO donor sodium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolat (DEA/NO) on left ventricular peak pressure (LVP), +dP/dt_max and -dP/dt_max of spontaneously beating constant-volume-perfused Langendorff hearts of normal Wistar rats (n = 23)

Mean values of percentage changes related to the basal values before drug application are shown. Vertical bars indicate s.e.m. The maximal responses are significantly different from zero (P < 0.05, paired t test). Equilibration values for LVP, +dP/dt_max and -dP/dt_max of the hearts used for these experiments are 56.5 ± 3.9 mmHg, 1987 ± 91.21 mmHg s⁻¹ and 1245 ± 83 mmHg s⁻¹, respectively. The concentration- response curves are not significantly different from each other (ANOVA).

Figure 5. Effect of increasing concentrations of glyceryl trinitrate (GTN, n = 32), SNAP (n = 33) and DEA/NO (n = 23), on coronary perfusion pressure (CPP) of spontaneously beating constant-volume-perfused Langendorff hearts of normal Wistar rats

Mean values of percentage changes related to the basal values before drug application are shown. Vertical bars indicate s.e.m. The maximal responses are significantly different from zero (P < 0.05, paired t test). The concentration-response curves for DEA/NO and SNAP are not significantly different from each other (ANOVA).

Figure 3. Effect of increasing concentrations of the spontaneous NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP), on left ventricular peak pressure (LVP), +dP/dt_max and -dP/dt_max of spontaneously beating constant-volume-perfused Langendorff hearts of normal Wistar rats (n = 33)

Mean values of percentage changes related to the basal values before drug application are shown. Vertical bars indicate s.e.m. The maximal responses are significantly different from zero (P < 0.05, paired t test). Equilibration values for LVP, +dP/dt_max and -dP/dt_max of the hearts used for these experiments are 62.4 ± 5.9 mmHg, 2194 ± 181.0 mmHg s⁻¹ and 1374 ± 101 mmHg s⁻¹, respectively. The concentration- response curves are not significantly different from each other (ANOVA).

Figure 6. Effect of 0.1 μm DEA/NO on +dP/dt_max, -dP/dt_max and cardiac output of spontaneously beating isolated working hearts of normal Wistar rats (n = 9)

Mean values of percentage changes related to the basal values before drug application are shown (absolute values are given in Table 3). Vertical bars indicate s.e.m. Inhibition of NO synthase by L-NA strongly increases the positive inotropic effect of DEA/NO. Significant differences were calculated by two-tailed unpaired t test: †P < 0.05 vs. baseline, *P < 0.05 vs. DEA/NO.