Nitric oxide signalling by selective β2‐adrenoceptor stimulation prevents ach‐induced inhibition of β2‐stimulated Ca2+ current in cat atrial myocytes

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Abstract
The present study determined the effects of acetylcholine (ACh) on the L‐type Ca2+ current (ICa,l) stimulated by β1‐ or β2‐adrenergic receptor (AR) agonists in cat atrial myocytes. When isoproterenol (ISO; 0.1 μm) plus the β2‐AR antagonist ICI 118,551 (ISO‐β1‐AR stimulation) or 0.1 μm fenoterol, a β2‐AR agonist (FEN‐β2‐AR stimulation) increased ICa,l, ACh (1 μm) inhibited ICa,l by –60 ± 4 and –63 ± 6 %, respectively. When ISO plus the β1‐AR antagonist atenolol (ISO‐β2‐AR stimulation) or 1 μm zinterol (ZIN‐β2‐AR stimulation) increased ICa,l, ACh‐induced inhibition of ICa,l was significantly smaller, at –21 ± 3 and −24 ± 3 %, respectively. l‐N5‐(1‐iminoethyl)ornithine (l‐NIO, 10 μm), an inhibitor of nitric oxide (NO) synthase, enhanced ACh‐induced inhibition of ICa,l when stimulated by ZIN‐β2‐ARs, but not when stimulated by ISO‐β1‐ARs or FEN‐β2‐ARs. Haemoglobin (50 μm), a NO scavenger, also enhanced ACh‐induced inhibition when ICa,l was stimulated by ZIN‐β2‐ARs, but not when stimulated by FEN‐β2‐ARs. ACh‐induced inhibition of ICa,l stimulated by ZIN‐β2‐ARs was not affected by 10 μm 1H‐[1,2,4] oxadiazolo[4,3‐a] quinoxaline‐1‐one (ODQ) a guanylate cyclase inhibitor, but was significantly enhanced by 500 μm reduced glutathione or 100 μm dithiothreitol, agents that act as sinks for S‐nitrosylation. ACh‐induced inhibition was smaller when ICa,l was stimulated by spermine/NO, a NO donor, than by milrinone, a phosphodiesterase type III inhibitor. ISO (ISO‐β1/β2‐AR stimulation) increased ICa,l and even though ISO releases NO, ACh prominently inhibited ICa,l. This inhibitory effect of ACh was enhanced by l‐NIO. Stimulation of ZIN‐β2‐ARs increased intracellular NO, whereas ISO‐β1‐ARs or FEN‐β2‐ARs failed to increase intracellular NO. These results indicate that in atrial myocytes, NO released by selective β2‐AR stimulation prevents ACh‐induced inhibition of ICa,l stimulated by β2‐ARs. NO acts via a cGMP‐independent, S‐nitrosylation mechanism. Although FEN acts via β2‐ARs, it fails to stimulate Gi‐/NO signalling and preferentially stimulates Gs‐/adenylate cyclase signalling, similar to β1‐ARs. These findings indicate that NO signalling modulates muscarinic receptor inhibition of atrial function stimulated by β2‐ARs.
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