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.
Wang
Honglan
H
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.
Kohr
Mark J
MJ
Traynham
Christopher J
CJ
Ziolo
Mark T
MT
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