Phosphodiesterase 5 restricts NOS3/Soluble guanylate cyclase signaling to L-type Ca2+ current in cardiac myocytes.
Endothelial nitric oxide synthase (NOS3) regulates the functional response to beta-adrenergic (beta-AR) stimulation via modulation of the L-type Ca(2+) current (I(Ca)). However, the NOS3 signaling pathway modulating I(Ca) 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 I(Ca). Myocytes were isolated from NOS3 knockout (NOS3(-/-)) and wildtype (WT) mice. We measured I(Ca) (whole-cell voltage-clamp), and simultaneously measured Ca(2+) transients (Fluo-4 AM) and cell shortening (edge detection). Zaprinast (selective inhibitor of PDE5), decreased beta-AR stimulated (isoproterenol, ISO)-I(Ca), and Ca(2+) transient and cell shortening amplitudes in WT myocytes. However, YC-1 (NO-independent activator of sGC) only reduced ISO-stimulated I(Ca), 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 I(Ca), or Ca(2+) transient and cell shortening amplitudes. Conversely, YC-1 significantly decreased both ISO-stimulated I(Ca), and cardiac contraction. Further confirming that PDE5 localizes NOS3/cGMP signaling to I(Ca); YC-1, in the presence of zaprinast, now significantly decreased ISO-stimulated Ca(2+) transient and cell shortening amplitudes in WT myocytes. The effects of YC-1 on I(Ca) 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 beta-AR stimulated I(Ca), while limiting effects on cardiac contraction.
Department of Physiology and Cell Biology, Davis Heart and Lung Research Institute, The Ohio State University, 304 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210, USA.
Honglan
H
Wang
Mark J
MJ
Kohr
Christopher J
CJ
Traynham
Mark T
MT
Ziolo
D000818:Animals; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5 / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006162:Guanylate Cyclase / Q000378:metabolism*; D007191:Indazoles / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects; D007545:Isoproterenol / Q000494:pharmacology; D051379:Mice; D008954:Models, Biological; D009200:Myocardial Contraction / Q000187:drug effects; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000201:enzymology*; D052250:Nitric Oxide Synthase Type III / Q000172:deficiency / Q000378:metabolism*; D058986:Phosphodiesterase 5 Inhibitors; D011688:Purinones / Q000494:pharmacology; D011943:Receptors, Adrenergic, beta / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D000071756:Soluble Guanylyl Cyclase
D020746:Calcium Channels, L-Type; D007191:Indazoles; D058986:Phosphodiesterase 5 Inhibitors; D011688:Purinones; D011943:Receptors, Adrenergic, beta; D018160:Receptors, Cytoplasmic and Nuclear; C090937:3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; D017869:Cyclic GMP-Dependent Protein Kinases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; C517382:Pde5a protein, mouse; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase; C011145:zaprinast; D006152:Cyclic GMP; D007545:Isoproterenol