Endothelial nitric oxide synthase (NOS3) regulates the functional response to β-adrenergic (β-AR) stimulation via modulation of the L-type Ca2+ current (ICa). However, the NOS3 signaling pathway modulating ICa is unknown. This study investigated the contribution of soluble guanylate cyclase (sGC) and phosphodiesterase type 5 (PDE5), a cGMP-specific PDE, in the NOS3-mediated regulation of ICa. Myocytes were isolated from NOS3 knockout (NOS3-/-) and wildtype (WT) mice. We measured ICa (whole-cell voltage-clamp), and simultaneously measured Ca2+ transients (Fluo-4 AM) and cell shortening (edge detection). Zaprinast (selective inhibitor of PDE5), decreased β-AR stimulated (isoproterenol, ISO)-ICa, and Ca2+ transient and cell shortening amplitudes in WT myocytes. However, YC-1 (NO-independent activator of sGC) only reduced ISO-stimulated ICa, but not cardiac contraction. We further investigated the NOS3/sGC/PDE5 pathway in NOS3-/- myocytes. PDE5 is mislocalized in these myocytes and we observed dissimilar effects of PDE5 inhibition and sGC activation compared to WT. That is, zaprinast had no effect on ISO-stimulated ICa, or Ca2+ transient and cell shortening amplitudes. Conversely, YC-1 significantly decreased both ISO-stimulated ICa, and cardiac contraction. Further confirming that PDE5 localizes NOS3/cGMP signaling to ICa; YC-1, in the presence of zaprinast, now significantly decreased ISO-stimulated Ca2+ transient and cell shortening amplitudes in WT myocytes. The effects of YC-1 on ICa and cardiac contraction were blocked by KT5823 (a selective inhibitor of the cGMP-dependent protein kinase, PKG). Our data suggests a novel physiological role for PDE5 in restricting the effects of NOS3/sGC/PKG signaling pathway to modulating β-AR stimulated ICa, while limiting effects on cardiac contraction.
- Cyclic GMP
- L-type Ca channel
ASJC Scopus subject areas
- Molecular Biology
- Cardiology and Cardiovascular Medicine