A comparative study of the effects of three guanylyl cyclase inhibitors on the L-type Ca²⁺ and muscarinic K⁺ currents in frog cardiac myocytes

Abstract

1. To investigate the participation of guanylyl cyclase in the muscarinic regulation of the cardiac L-type calcium current (I_Ca), we examined the effects of three guanylyl cyclase inhibitors, 1H-[1,2,4]oxidiazolo[4,3-a]quinoxaline-1-one (ODQ), 6-anilino-5,8-quinolinedione (LY 83583), and methylene blue (MBlue), on the β-adrenoceptor; muscarinic receptor and nitric oxide (NO) regulation of I_Ca and on the muscarinic activated potassium current I_K,ACh, in frog atrial and ventricular myocytes.

2. ODQ (10 μM) and LY 83583 (30 μM) antagonized the inhibitory effect of an NO-donor (S-nitroso-N-acetylpenicillamine, SNAP, 1 μM) on the isoprenaline (Iso)-stimulated I_Ca which was consistent with their inhibitory action on guanylyl cyclase. However, MBlue (30 μM) had no effect under similar conditions.

3. In the absence of SNAP, LY 83583 (30 μM) potentiated the stimulations of I_Ca by either Iso (20 nM), forskolin (0.2 μM) or intracellular cyclic AMP (5–10 μM). ODQ (10 μM) had no effect under these conditions, while MBlue (30 μM) inhibited the Iso-stimulated I_Ca.

4. LY 83583 and MBlue, but not ODQ, reduced the inhibitory effect of up to 10 μM acetylcholine (ACh) on I_Ca.

5. MBlue, but not LY 83583 and ODQ, antagonized the activation of I_K,ACh by ACh in the presence of intracellular GTP, and this inhibition was weakened when I_K,ACh was activated by intracellular GTPγS.

6. The potentiating effect of LY 83583 on Iso-stimulated I_Ca was absent in the presence of either DL-dithiothreitol (DTT, 100 μM) or a combination of superoxide dismutase (150 u ml⁻¹) and catalase (100 u ml⁻¹).

7. All together, our data demonstrate that, among the three compounds tested, only ODQ acts in a manner which is consistent with its inhibitory action on the NO-sensitive guanylyl cyclase. The two other compounds produced severe side effects which may involve superoxide anion generation in the case of LY 83583 and alteration of β-adrenoceptor and muscarinic receptor-coupling mechanisms in the case of MBlue.

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