Proceedings of The Physiological Society
Kings College London (2005) J Physiol 565P, C172
Effects of Nitric Oxide on cardiac contractility: role of NOS isoforms and signalling pathways
Gonzalez, Daniel R; Fernandez, Ignacio ; Boric, Mauricio P;
1. P. Universidad Catolica de Chile, Santiago, Chile.
The role of NO in the cardiac function remains controversial. Several NO donors cause both positive and negative inotropic responses. Furthermore, adrenergic and cholinergic agonists induce NO production in cardiomyocytes, suggesting that NO may be important in the autonomic regulation of the heart. Cardiomyocytes express endothelial (eNOS) and neuronal (nNOS) isoforms of nitric oxide synthase, but eNOS is more abundant, while nNOS expression is restricted to the sarcoplasmic reticulum. We hypothesized that the opposite effects of NO may be related to its concentration, the NOS isoform and intracellular pathways that are activated. eNOS activation may release enough NO to trigger cGMP production to reduce contractility, whereas nNOS activation release small amounts of NO able to nitrosylate sarcoplasmic reticulum proteins. We tested this idea in the rat heart, Langendorff preparation, using S-nitroso-N-acetylpenicillamine (SNAP) as NO donor, and Isoprenaline as inotropic agent. Male Sprague-Dawley rats (290-300 g) were anesthetized with ketamine (90 mg kg-1 I.P.) and xilazine (10 mg kg-1 I.P.). Under deep anaesthesia, hearts were excised, and mounted on the perfusion system and paced at 360 beats min-1. The contractile response was measured as change in dP/dtmax (% basal). Pulses (1-min) of SNAP 0.1, 1.0 and 10 μM increased contractility (16±5%, 25±3 and 16±1%, respectively, n=6), but 100μM SNAP decreased it (−22±3%, n=10). To assess possible nitrosylation, we used TEMPOL, a superoxide dismutase mimetic, assuming superoxide is an intermediary in this reaction. TEMPOL 100 μM abolished the positive inotropic effect of 1 μM SNAP (n=3). To analyse the cGMP route, we used ODQ, a guanylyl cyclase blocker and 8-Br-cGMP. In the presence of ODQ 10 μM, the effect of SNAP 100 μM turned to a positive inotropic effect (21±5%, n=4). Pulses of 8-Br-cGMP 1, 10 and 100 μM reduced contractility (−8±3%, −24±7% and −25± %, respectively, n=4). The maximal response to Isoprenaline was reduced during nNOS inhibition with S-Methyl-L-thiocitrulline 300 nM (211±11% v/s 247±33% in control, p≤0.05, paired t test, n=5). On the contrary, during broad NOS inhibition with L-NAME 100 μM the maximal response to Isoprenaline increased (158±23% v/s 97±21% in control, p≤0.05, paired t test). In addition, the β3 adrenergic agonist, BRL-37344 (100 nM), reduced contractility (−24±3%, n=4), and this effect was abolished by ODQ 10 μM. These data suggest that positive inotropism is related to nNOS activation and low NO levels, possibly due to protein nitrosylation, while the negative inotropic effects observed at higher NO concentrations involve cGMP generation.
Where applicable, experiments conform with Society ethical requirements