Local response of L‐type Ca2+ current to nitric oxide in frog ventricular myocytes

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Abstract
1 The regulation of L-type Ca2+ current (ICa) by the two nitric oxide (NO) donors sodium nitroprusside (SNP, 1 μm to 1 mm) and (±)-S-nitroso-N-acetylpenicillamine (SNAP, 3 or 10 μm) was investigated in frog ventricular myocytes using double voltage clamp and double-barrelled microperfusion techniques. 2 SNP and SNAP depressed the isoprenaline (ISO, 10-100 nm)- or forskolin (FSK, 1 μm)-mediated stimulation of ICa via cGMP activation of the cGMP-stimulated phosphodiesterase (PDE2). Complete inhibition of the ISO (100 nm) response was observed at 1 mm SNP. 3 When SNP was applied locally, i.e. to one-half of the cell, and ISO to the whole cell, the response of ICa to ISO was strongly antagonized in the cell half exposed to SNP (up to 100 % inhibition at 1 mm SNP) but a relatively small depression was observed in the other half of the cell (only 20 % inhibition at 1 mm SNP). 4 The NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO, 1 mm) reversed the local effect of SNAP (3 μm) on FSK-stimulated ICa when applied to the same side as the NO donor, but had no effect when applied to the other side of the cell. 5 A local application of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA, 30 μm), a selective inhibitor of PDE2, fully reversed the local effect of SNP (100 μm) or SNAP (10 μm) on ICa but had no effect on the distant response. 6 When EHNA was applied on the distant side, with SNP (1 mm) and ISO (100 nm) applied locally, the distant effect of SNP was fully reversed. 7 Our results demonstrate that in frog ventricular myocytes stimulation of guanylyl cyclase by NO leads to a strong local depletion of cAMP near the L-type Ca2+ channels due to activation of PDE2, but only to a modest reduction of cAMP in the rest of the cell. This may be explained by the existence of a tight microdomain between L-type Ca2+ channels and PDE2.
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