The nitric oxide (NO)/cGMP signaling cascade has an established role in synaptic plasticity. However, with conventional methods, the underlying cGMP signals were barely detectable. Here, we set out to confirm the well-known NMDA-induced cGMP increases, to test the impact of AMPA on those signals, and to identify the relevant phosphodiesterases (PDEs) using a more sensitive fluorescence resonance energy transfer (FRET)-based method. Therefore, a “knock-in” mouse was generated that expresses a FRET-based cGMP indicator (cGi-500) allowing detection of cGMP concentrations between 100 nM and 3 μM. Measurements were performed in cultured hippocampal and cortical neurons as well as acute hippocampal slices. In hippocampal and cortical neurons, NMDA elicited cGMP signals half as high as the ones elicited by exogenous NO. Interestingly, AMPA increased cGMP independently of NMDA receptors and dependent on NO synthase (NOS) activation. NMDA- and AMPA-induced cGMP signals were not additive indicating that both pathways converge on the level of NOS. Accordingly, the same PDEs, PDE1 and PDE2, were responsible for degradation of NMDA- as well as AMPA-induced cGMP signals. Mechanistically, AMPAR induced calcium influx through L-type voltage-gated calcium channels leading to NOS and finally NO-sensitive guanylyl cyclase activation. Our results demonstrate that in addition to NMDA also AMPA triggers endogenous NO formation and hence cGMP production.
In the NO/cGMP signaling cascade, relevant in the cardiovascular system, two NO-sensitive guanylyl cyclase (NO-GC) isoforms are responsible for NO-dependent cGMP generation. Here, the impact of the major NO-GC isoform, NO-GC1, on fibrosis development in the cardiovascular system was studied in NO-GC1-deficient mice treated with AngiotensinII (AngII), known to induce vascular and cardiac remodeling. Morphometric analysis of NO-GC1 KO’s aortae demonstrated an enhanced increase of perivascular area after AngII treatment accompanied by a higher aortic collagen1 mRNA content. Increased perivascular fibrosis also occurred in cardiac vessels of AngII-treated NO-GC1 KO mice. In line, AngII-induced interstitial fibrosis was 32% more pronounced in NO-GC1 KO than in WT myocardia associated with a higher cardiac Col1 and other fibrotic marker protein content. In sum, increased perivascular and cardiac interstitial fibrosis together with the enhanced collagen1 mRNA content in AngII-treated NO-GC1-deficient mice represent an exciting manifestation of antifibrotic properties of cGMP formed by NO-GC1, a finding with great pharmaco-therapeutic implications.
In the central nervous system, the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signalling cascade has an established role in fine‐tuning of synaptic transmission. In the present study, we asked which isoform of NO‐sensitive guanylyl cyclase, NO‐GC1 or NO‐GC2, is responsible for generation of N‐methyl‐d‐aspartate (NMDA)‐ and AMPA (α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid)‐induced cGMP signals and which of the phosphodiesterases (PDEs) is responsible for degradation. To this end, we performed live cell fluorescence measurements of primary hippocampal neurons isolated from NO‐GC isoform‐deficient mice. Although both isoforms contributed to the NMDA‐ and AMPA‐induced cGMP signals, NO‐GC2 clearly played the predominant role. Whereas under PDE‐inhibiting conditions the cGMP levels elicited by both glutamatergic ligands were comparable, NMDA‐induced cGMP signals were clearly higher than the AMPA‐induced ones in the absence of PDE inhibitors. Thus, AMPA‐induced cGMP signals are more tightly controlled by PDE‐mediated degradation than NMDA‐induced signals. In addition, these findings are compatible with the existence of at least two different pools of cGMP in both of which PDE1 and PDE2—known to be highly expressed in the hippocampus—are mainly responsible for cGMP degradation. The finding that distinct pools of cGMP are equipped with different amounts of PDEs highlights the importance of PDEs for the shape of NO‐induced cGMP signals in the central nervous system.
The occurrence of NO/cGMP signalling in cardiac cells is a matter of debate. Recent measurements with a FRET-based cGMP indicator in isolated cardiac cells revealed NO-induced cGMP signals in cardiac fibroblasts while cardiomyocytes were devoid of these signals. In a fibroblast/myocyte co-culture model though, cGMP formed in fibroblasts in response to NO entered cardiomyocytes via gap junctions. Here, we demonstrate gap junction-mediated cGMP transfer from cardiac fibroblasts to myocytes in intact tissue. In living cardiac slices of mice with cardiomyocyte-specific expression of a FRET-based cGMP indicator (αMHC/cGi-500), NO-dependent cGMP signals were shown to occur in myocytes, to depend on gap junctions and to be degraded mainly by PDE3. Stimulation of NO-sensitive guanylyl cyclase enhanced Forskolin- and Isoproterenol-induced cAMP and phospholamban phosphorylation. Genetic inactivation of NO-GC in Tcf21-expressing cardiac fibroblasts abrogated the synergistic action of NO-GC stimulation on Iso-induced phospholamban phosphorylation, identifying fibroblasts as cGMP source and substantiating the necessity of cGMP-transfer to myocytes. In sum, NO-stimulated cGMP formed in cardiac fibroblasts enters cardiomyocytes in native tissue where it exerts an inhibitory effect on cAMP degradation by PDE3, thereby increasing cAMP and downstream effects in cardiomyocytes. Hence, enhancing β-receptor-induced contractile responses appears as one of NO/cGMP’s functions in the non-failing heart.
In isolated perfused rat hearts (medium: Krebs-Ringer solution containing about 15% bovine red cells) the following parameters were estimated: heart rate (F), left intraventricular peak pressure (P), dP/dt, oxygen consumption (VO2); myocardial tissue content of glycogen, ATP, ADP, AMP, cAMP, phosphocreatin (PC) and inorganic phosphate (Pi). Isoproterenol (ISO) was administered to the non circulating system in the concentration range of 5 x 10(-10) to 5 x 10(-5) M for 4 min and 1 hour respectively by infusion into the perfusate close to the aortic canula. After administration period of 4 min dependent on the concentration of ISO, P, dP/dt, VO2 and the content of cAMP are increased. The ratios of PC/Pi and ATP/ADP as well as glycogen content are reduced. For an administration period of 1 hour at a level of 5 x 10(-9) M the effects of isoproterenol are maintained. At ISO concentration higher than 5 x 10(-9) M the effects on mechanical parameters and VO2 fall to the level of those values which are produced at 5 x 10(-9) M ISO. In contrast, high energy phosphates and glycogen content remain reduced while that of cAMP is elevated.
Effects of Sr++ and isoproterenol were studied in rat hearts perfused with red cell containing media. Sr++ in the presence of Ca++ causes a positive inotropic effect without corresponding metabolic changes. Without Ca++ 0.5 mM Sr++ causes an immediate arrest, 2 mM Sr++ a complete contracture (14 min) and 5 mM a contracture after about 44 min. At a level of 10 mM Sr++ phasic contractions are maintained (60 min). Occurring phasic contractions are prolonged 3 to 6fold. Administration of isoproterenol (ISO) at a level of 0.5 mM Sr++ causes a delayed occurrence of cardiac arrest and incomplete contracture. At a concentration of 2 and 5 mM Sr++ positive inotropic responses proceed to a contracture (7 min, 50 min resp.). VO2 is reduced by 0.5 mM Sr++ initially by 2 mM Sr++ with delay. 5 and 10 mM Sr++ induce an initial increase. Subsequent decrease is smallest at 10 mM Sr++ ISO at all Sr++ concentrations induces an increase in VO2 initially and strong reduction finally. During 10 min administration, high energy phosphate stores (HEP) are reduced at all Sr++ concentrations, but to the smallest extent at 10 mM Sr++, ISO at levels of 0.5 and 2 mM Sr++ induces a partial recovery of HEP, but at 5 and 10 mM a further reduction. Finally, under the influence of ISO the metabolic state is similar to that without ISO. Sr++ at high concentrations in absence of Ca++ seems to be capable of substituting in principle for Ca++ also concerning metabolism. Severe metabolic disturbances at low Sr++ concentrations indicate a failure of regulation of oxidative phosphorylation.
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