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Activation of distinct cAMP-dependent and cGMP-dependent pathways by nitric oxide in cardiac myocytes
M G Vila-Petroff1, A Younes, J Egan
1Laboratory of Cardiovascular Science, and Laboratory of Clinical Investigation, Intramural Research Program, National Institute on Aging, Gerontology Research Center, Baltimore, MD, USA.
Abstract:
Nitric oxide (NO) donors were recently shown to produce biphasic contractile effects in cardiac tissue, with augmentation at low NO levels and depression at high NO levels. We examined the subcellular mechanisms involved in the opposing effects of NO on cardiac contraction and investigated whether NO modulates contraction exclusively via guanylyl cyclase (GC) activation or whether some contribution occurs via cGMP/PKG-independent mechanisms, in indo 1-loaded adult cardiac myocytes. Whereas a high concentration of the NO donor S-nitroso-N-acetylpenicillamine (SNAP, 100 micromol/L) significantly attenuated contraction amplitude by 24.4+/-4.5% (without changing the Ca2+ transient or total cAMP), a low concentration of SNAP (1 micromol/L) significantly increased contraction amplitude (38+/-10%), Ca2+ transient (26+/-10%), and cAMP levels (from 6.2 to 8.5 pmol/mg of protein). The negative contractile response of 100 micromol/L SNAP was completely abolished in the presence of the specific blocker of PKG KT 5823 (1 micromol/L); the positive contractile response of 1 micromol/L SNAP persisted, despite the presence of the selective inhibitor of GC 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 micromol/L) alone, but was completely abolished in the presence of ODQ plus the specific inhibitory cAMP analog Rp-8-CPT-cAMPS (100 micromol/L), as well as by the NO scavenger oxyhemoglobin. Parallel experiments in cell suspensions showed significant increases in adenylyl cyclase (AC) activity at low concentrations (0.1 to 1 micromol/L) of SNAP (AC, 18% to 20% above basal activity). We conclude that NO can regulate both AC and GC in cardiac myocytes. High levels of NO induce large increases in cGMP and a negative inotropic effect mediated by a PKG-dependent reduction in myofilament responsiveness to Ca2+. Low levels of NO increase cAMP, at least in part, by a novel cGMP-independent activation of AC and induce a positive contractile response.
Insights
Nitric oxide (NO) affects cardiac contraction differently based on its concentration. Low NO levels enhance contraction via cAMP and adenylyl cyclase (AC) activation, while high NO levels depress contraction through cGMP and protein kinase G (PKG) pathways.
Area of Science:
- Cardiovascular Physiology
- Molecular Cardiology
- Cell Signaling
Background:
- Nitric oxide (NO) exhibits biphasic effects on cardiac contractility, increasing it at low concentrations and decreasing it at high concentrations.
- The precise subcellular mechanisms underlying these opposing NO effects in cardiac tissue remain incompletely understood.
- Investigation is needed to determine if NO exclusively acts via guanylyl cyclase (GC) or involves cGMP/protein kinase G (PKG)-independent pathways.
Purpose of the Study:
- To elucidate the subcellular mechanisms responsible for the dual contractile effects of nitric oxide (NO) in adult cardiac myocytes.
- To investigate whether NO modulates cardiac contraction solely through guanylyl cyclase (GC) activation or if cGMP/PKG-independent pathways are involved.
Main Methods:
- Utilized indo 1-loaded adult cardiac myocytes to measure intracellular calcium transients and contraction amplitude.
- Employed the NO donor S-nitroso-N-acetylpenicillamine (SNAP) at varying concentrations.
- Used specific inhibitors for guanylyl cyclase (ODQ) and protein kinase G (KT 5823), along with cAMP analogs and an NO scavenger (oxyhemoglobin).
- Assessed adenylyl cyclase (AC) activity in cardiac cell suspensions.
Main Results:
- High SNAP concentrations (100 µmol/L) attenuated contraction amplitude by 24.4% via a PKG-dependent mechanism.
- Low SNAP concentrations (1 µmol/L) increased contraction amplitude by 38%, augmented Ca2+ transient by 26%, and elevated cAMP levels.
- The positive contractile effect of low SNAP persisted despite GC inhibition (ODQ) but was blocked by inhibiting cAMP signaling or scavenging NO.
- Low SNAP concentrations significantly increased adenylyl cyclase (AC) activity (18-20% above basal).
Conclusions:
- Nitric oxide (NO) differentially regulates both adenylyl cyclase (AC) and guanylyl cyclase (GC) in cardiac myocytes.
- High NO levels induce a negative inotropic effect mediated by cGMP and PKG, reducing myofilament responsiveness to Ca2+.
- Low NO levels promote a positive inotropic effect through a novel cGMP-independent activation of AC, leading to increased cAMP and enhanced contractility.