The differential roles of the two NO-GC isoforms in adjusting airway reactivity.

The enzyme, nitric oxide-sensitive guanylyl cyclase (NO-GC), is activated by binding NO to its prosthetic heme group and catalyzes the formation of cGMP. The NO-GC is primarily known to mediate vascular smooth muscle relaxation in the lung, and inhaled NO has been successfully used as a selective pulmonary vasodilator. In comparison, NO-GC's impact on the regulation of airway tone is less acknowledged and, most importantly, little is known about the issue that NO-GC signaling is accomplished by two isoforms: NO-GC1 and NO-GC2, implying the existence of distinct "cGMP pools." Herein, we investigated the functional role of the NO-GC isoforms in respiration by measuring lung function parameters of isoform-specific knockout (KO) mice using noninvasive and invasive techniques. Our data revealed the participation and ongoing influence of NO-GC1-derived cGMP in the regulation of airway tone by showing that respiratory resistance was enhanced in NO-GC1-KOs and increased more pronouncedly after the challenge with the bronchoconstrictor methacholine. The tissue resistance and stiffness of NO-GC1-KOs were also higher because of narrowed airways that cause tissue distortion. Contrariwise, NO-GC2-KOs displayed reduced tissue elasticity, elastic recoil, and airway reactivity to methacholine, which did not even increase in an ovalbumin model of asthma that induced hyperresponsiveness in NO-GC1-KOs. In addition, conscious NO-GC2-KOs showed a higher breathing rate with a shorter duration of inspiration and expiration time, which remained faster even in the presence of bronchoconstrictors that slow down breathing. Thus, we provide evidence of two distinct NO/cGMP pathways in airways, accomplished by either NO-GC1 or NO-GC2, adjusting differentially the airway reactivity.

Hippocampal AMPA- and NMDA-induced cGMP signals are mainly generated by NO-GC2 and are under tight control by PDEs 1 and 2.

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.

Dysfunctional nitric oxide signalling increases risk of myocardial infarction.

Myocardial infarction, a leading cause of death in the Western world, usually occurs when the fibrous cap overlying an atherosclerotic plaque in a coronary artery ruptures. The resulting exposure of blood to the atherosclerotic material then triggers thrombus formation, which occludes the artery. The importance of genetic predisposition to coronary artery disease and myocardial infarction is best documented by the predictive value of a positive family history. Next-generation sequencing in families with several affected individuals has revolutionized mutation identification. Here we report the segregation of two private, heterozygous mutations in two functionally related genes, GUCY1A3 (p.Leu163Phefs*24) and CCT7 (p.Ser525Leu), in an extended myocardial infarction family. GUCY1A3 encodes the α1 subunit of soluble guanylyl cyclase (α1-sGC), and CCT7 encodes CCTη, a member of the tailless complex polypeptide 1 ring complex, which, among other functions, stabilizes soluble guanylyl cyclase. After stimulation with nitric oxide, soluble guanylyl cyclase generates cGMP, which induces vasodilation and inhibits platelet activation. We demonstrate in vitro that mutations in both GUCY1A3 and CCT7 severely reduce α1-sGC as well as ß1-sGC protein content, and impair soluble guanylyl cyclase activity. Moreover, platelets from digenic mutation carriers contained less soluble guanylyl cyclase protein and consequently displayed reduced nitric-oxide-induced cGMP formation. Mice deficient in α1-sGC protein displayed accelerated thrombus formation in the microcirculation after local trauma. Starting with a severely affected family, we have identified a link between impaired soluble-guanylyl-cyclase-dependent nitric oxide signalling and myocardial infarction risk, possibly through accelerated thrombus formation. Reversing this defect may provide a new therapeutic target for reducing the risk of myocardial infarction.

Angiotensin-(1-7)-induced Mas receptor activation attenuates atherosclerosis through a nitric oxide-dependent mechanism in apolipoproteinE-KO mice.

Angiotensin (Ang)-(1-7) ameliorates vascular injury by increasing nitric oxide (NO) bioavailability. Evidence that Ang-(1-7) attenuates the development of atherosclerosis through a NO-dependent mechanism is still missing. Moreover, it has been postulated that Ang-(1-7) may mediate its effects by other mechanisms than Mas receptor activation. To investigate Ang-(1-7)-dependent Mas receptor function, we treated apoE-KO and apoE/Mas-KO mice chronically with Ang-(1-7) (82 µg/kg per hour) or saline for 6 weeks. Flow-mediated dilation (FMD), a measure for NO-dependent vasodilation and the most accepted prognostic marker for the development of atherosclerosis, was measured in vivo. Chronic Ang-(1-7) treatment improved FMD and attenuated the development of atherosclerosis in apolipoproteinE (apoE)-KO but not in apoE/Mas-KO mice. These effects were accompanied by increased aortic nitrite and cGMP levels. To test whether Ang-(1-7) modulates atherosclerosis through a NO-dependent mechanism, apoE-KO mice were treated with the NO synthase inhibitor L-NAME (20 mg/kg/day) in the presence or absence of Ang-(1-7). L-NAME treatment reduced aortic nitrite content and increased blood pressure and exaggerated atherosclerosis compared to untreated apoE-KO mice. In L-NAME-treated apoE-KO mice, chronic Ang-(1-7) treatment did not increase aortic nitrite content and consequently showed no effect on blood pressure and the development of atherosclerosis. The present study proves that Ang-(1-7) mediates its protective vascular effects through Mas receptor activation. Moreover, Ang-(1-7)-mediated NO generation is essential for improving vascular function and prevents atherosclerosis in apoE-KO mice.

cGMP Imaging in Brain Slices Reveals Brain Region-Specific Activity of NO-Sensitive Guanylyl Cyclases (NO-GCs) and NO-GC Stimulators.

Impaired NO-cGMP signaling has been linked to several neurological disorders. NO-sensitive guanylyl cyclase (NO-GC), of which two isoforms-NO-GC1 and NO-GC2-are known, represents a promising drug target to increase cGMP in the brain. Drug-like small molecules have been discovered that work synergistically with NO to stimulate NO-GC activity. However, the effects of NO-GC stimulators in the brain are not well understood. In the present study, we used Förster/fluorescence resonance energy transfer (FRET)-based real-time imaging of cGMP in acute brain slices and primary neurons of cGMP sensor mice to comparatively assess the activity of two structurally different NO-GC stimulators, IWP-051 and BAY 41-2272, in the cerebellum, striatum and hippocampus. BAY 41-2272 potentiated an elevation of cGMP induced by the NO donor DEA/NO in all tested brain regions. Interestingly, IWP-051 potentiated DEA/NO-induced cGMP increases in the cerebellum and striatum, but not in the hippocampal CA1 area or primary hippocampal neurons. The brain-region-selective activity of IWP-051 suggested that it might act in a NO-GC isoform-selective manner. Results of mRNA in situ hybridization indicated that the cerebellum and striatum express NO-GC1 and NO-GC2, while the hippocampal CA1 area expresses mainly NO-GC2. IWP-051-potentiated DEA/NO-induced cGMP signals in the striatum of NO-GC2 knockout mice but was ineffective in the striatum of NO-GC1 knockout mice. These results indicate that IWP-051 preferentially stimulates NO-GC1 signaling in brain slices. Interestingly, no evidence for an isoform-specific effect of IWP-051 was observed when the cGMP-forming activity of whole brain homogenates was measured. This apparent discrepancy suggests that the method and conditions of cGMP measurement can influence results with NO-GC stimulators. Nevertheless, it is clear that NO-GC stimulators enhance cGMP signaling in the brain and should be further developed for the treatment of neurological diseases.

Impact of the NO-Sensitive Guanylyl Cyclase 1 and 2 on Renal Blood Flow and Systemic Blood Pressure in Mice.

Nitric oxide (NO) modulates renal blood flow (RBF) and kidney function and is involved in blood pressure (BP) regulation predominantly via stimulation of the NO-sensitive guanylyl cyclase (NO-GC), existing in two isoforms, NO-GC1 and NO-GC2. Here, we used isoform-specific knockout (KO) mice and investigated their contribution to renal hemodynamics under normotensive and angiotensin II-induced hypertensive conditions. Stimulation of the NO-GCs by S-nitrosoglutathione (GSNO) reduced BP in normotensive and hypertensive wildtype (WT) and NO-GC2-KO mice more efficiently than in NO-GC1-KO. NO-induced increase of RBF in normotensive mice did not differ between the genotypes, but the respective increase under hypertensive conditions was impaired in NO-GC1-KO. Similarly, inhibition of endogenous NO increased BP and reduced RBF to a lesser extent in NO-GC1-KO than in NO-GC2-KO. These findings indicate NO-GC1 as a target of NO to normalize RBF in hypertension. As these effects were not completely abolished in NO-GC1-KO and renal cyclic guanosine monophosphate (cGMP) levels were decreased in both NO-GC1-KO and NO-GC2-KO, the results suggest an additional contribution of NO-GC2. Hence, NO-GC1 plays a predominant role in the regulation of BP and RBF, especially in hypertension. However, renal NO-GC2 appears to compensate the loss of NO-GC1, and is able to regulate renal hemodynamics under physiological conditions.

NO-Sensitive Guanylate Cyclase Isoforms NO-GC1 and NO-GC2 Contribute to Noise-Induced Inner Hair Cell Synaptopathy.

Nitric oxide (NO) activates the NO-sensitive soluble guanylate cyclase (NO-GC, sGC) and triggers intracellular signaling pathways involving cGMP. For survival of cochlear hair cells and preservation of hearing, NO-mediated cascades have both protective and detrimental potential. Here we examine the cochlear function of mice lacking one of the two NO-sensitive guanylate cyclase isoforms [NO-GC1 knockout (KO) or NO-GC2 KO]. The deletion of NO-GC1 or NO-GC2 did not influence electromechanical outer hair cell (OHC) properties, as measured by distortion product otoacoustic emissions, neither before nor after noise exposure, nor were click- or noise-burst-evoked auditory brainstem response thresholds different from controls. Yet inner hair cell (IHC) ribbons and auditory nerve responses showed significantly less deterioration in NO-GC1 KO and NO-GC2 KO mice after noise exposure. Consistent with a selective role of NO-GC in IHCs, NO-GC ß1 mRNA was found in isolated IHCs but not in OHCs. Using transgenic mice expressing the fluorescence resonance energy transfer-based cGMP biosensor cGi500, NO-induced elevation of cGMP was detected in real-time in IHCs but not in OHCs. Pharmacologic long-term treatment with a NO-GC stimulator altered auditory nerve responses but did not affect OHC function and hearing thresholds. Interestingly, NO-GC stimulation exacerbated the loss of auditory nerve response in aged animals but attenuated the loss in younger animals. We propose NO-GC2 and, to some degree, NO-GC1 as targets for early pharmacologic prevention of auditory fiber loss (synaptopathy). Both isoforms provide selective benefits for hearing function by maintaining the functional integrity of auditory nerve fibers in early life rather than at old age.

Phosphodiesterase 5 inhibition ameliorates angiotensin II-dependent hypertension and renal vascular dysfunction.

Changes in renal hemodynamics have a major impact on blood pressure (BP). Angiotensin (Ang) II has been shown to induce vascular dysfunction by interacting with phosphodiesterase (PDE)1 and PDE5. The predominant PDE isoform responsible for renal vascular dysfunction in hypertension is unknown. Here, we measured the effects of PDE5 (sildenafil) or PDE1 (vinpocetine) inhibition on renal blood flow (RBF), BP, and renal vascular function in normotensive and hypertensive mice. During acute short-term Ang II infusion, sildenafil decreased BP and increased RBF in C57BL/6 (WT) mice. In contrast, vinpocetine showed no effect on RBF and BP. Additionally, renal cGMP levels were significantly increased after acute sildenafil but not after vinpocetine infusion, indicating a predominant role of PDE5 in renal vasculature. Furthermore, chronic Ang II infusion (500 ng·kg-1·min-1) increased BP and led to impaired NO-dependent vasodilation in kidneys of WT mice. Additional treatment with sildenafil (100 mg·kg-1·day-1) attenuated Ang II-dependent hypertension and improved NO-mediated vasodilation. During chronic Ang II infusion, urinary nitrite excretion, a marker for renal NO generation, was increased in WT mice, whereas renal cGMP generation was decreased and restored after sildenafil treatment, suggesting a preserved cGMP signaling after PDE5 inhibition. To investigate the dependency of PDE5 effects on NO/cGMP signaling, we next analyzed eNOS-KO mice, a mouse model characterized by low vascular NO/cGMP levels. In eNOS-KO mice, chronic Ang II infusion increased BP but did not impair NO-mediated vasodilation. Moreover, sildenafil did not influence BP or vascular function in eNOS-KO mice. These results highlight PDE5 as a key regulator of renal hemodynamics in hypertension.

Proatherosclerotic Effect of the α1-Subunit of Soluble Guanylyl Cyclase by Promoting Smooth Muscle Phenotypic Switching.

Soluble guanylate cyclase (sGC), a key enzyme of the nitric oxide signaling pathway, is formed as a heterodimer by various isoforms of its α and ß subunit. GUCY1A3, encoding the α1 subunit, was identified as a risk gene for coronary artery disease and myocardial infarction, but its specific contribution to atherosclerosis remains unclear. This study sought to decipher the role of Gucy1a3 in atherosclerosis in mice. At age 32 weeks and after 20 weeks of standard or high-fat diet, Gucy1a3(-/-)/Ldlr(-/-) mice exhibited a significant reduction of the atherosclerotic plaque size at the aortic root and the aorta for high-fat diet animals as compared with Ldlr(-/-) control mice. Collagen content in plaques in the aortic root was reduced, suggesting an alteration of smooth muscle cell function. Proliferation and migration were reduced in Gucy1a3(-/-) primary aortic smooth muscle cells (AoSMCs), and proliferation was also reduced in human AoSMCs after inhibition of sGC by 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one. Gucy1a3 deficiency in AoSMCs prevents their phenotypic switching, as indicated by the differential expression of marker proteins. The inherited Gucy1a3(-/-) loss exerts an atheroprotective effect. We suggest that sGC activity promotes the phenotypic switching of smooth muscle cells from a contractile to a synthetic state, fostering the formation of atherosclerosis. Preventing this switch by sGC inhibition may provide a novel target in atherosclerotic disease.

Angiotensin II-Induced Hypertension Is Attenuated by Reduction of Sympathetic Output in NO-Sensitive Guanylyl Cyclase 1 Knockout Mice.

In the regulation of vascular tone, the dilatory nitric oxide (NO)/cGMP pathway balances vasoconstriction induced by the renin-angiotensin and sympathetic nervous systems. NO-induced cGMP formation is catalyzed by two guanylyl cyclases (GC), NO-sensitive guanylyl cyclase 1 (NO-GC1) and NO-GC2, with indistinguishable enzymatic properties. In vascular smooth muscle cells, NO-GC1 is the major isoform and is responsible for more than 90% of cGMP formation. Despite reduced vasorelaxation, NO-GC1-deficient mice are not hypertensive. Here, the role of NO-GC1 in hypertension provoked by contractile agonists angiotensin II (Ang II) and norepinephrine (NE) was evaluated in NO-GC1-deficient mice. Hypertension induced by chronic Ang II treatment did not differ between wild-type (WT) and NO-GC1 knockout mice (KO). Also, attenuation of NO-dependent aortic relaxation induced by the Ang II treatment was similar in both genotypes and was most probably attributable to an increase of phosphodiesterase 1 expression. Analysis of plasma NE content-known to be influenced by Ang II-revealed lower NE in the NO-GC1 KO under Ang II-treated- and nontreated conditions. The finding indicates reduced sympathetic output and is underlined by the lower heart rate in the NO-GC1 KO. To find out whether the lack of higher blood pressure in the NO-GC1 KO is a result of reduced sympathetic activity counterbalancing the reduced vascular relaxation, mice were challenged with chronic NE application. As the resulting blood pressure was higher in the NO-GC1 KO than in WT, we conclude that the reduced sympathetic activity in the NO-GC1 KO prevents hypertension and postulate a possible sympatho-excitatory action of NO-GC1 counteracting NO-GC1's dilatory effect in the vasculature.

Role of Phosphodiesterase 5 and Cyclic GMP in Hypertension.

Cyclic GMP (cGMP) is a ubiquitous intracellular second messenger that mediates a wide spectrum of physiologic processes in multiple cell types within the cardiovascular and nervous systems. Synthesis of cGMP occurs either by NO-sensitive guanylyl cyclases in response to nitric oxide or by membrane-bound guanylyl cyclases in response to natriuretic peptides and has been shown to regulate blood pressure homeostasis by influencing vascular tone, sympathetic nervous system, and sodium and water handling in the kidney. Several cGMPs degrading phosphodiesterases (PDEs), including PDE1 and PDE5, play an important role in the regulation of cGMP signaling. Recent findings revealed that increased activity of cGMP-hydrolyzing PDEs contribute to the development of hypertension. In this review, we will summarize recent research findings regarding the cGMP/PDE signaling in the vasculature, the central nervous system, and the kidney which are associated with the development and maintenance of hypertension.

Postsynaptic NO/cGMP increases NMDA receptor currents via hyperpolarization-activated cyclic nucleotide-gated channels in the hippocampus.

The nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signaling cascade participates in the modulation of synaptic transmission. The effects of NO are mediated by the NO-sensitive cGMP-forming guanylyl cyclases (NO-GCs), which exist in 2 isoforms with indistinguishable regulatory properties. The lack of long-term potentiation (LTP) in knock-out (KO) mice deficient in either one of the NO-GC isoforms indicates the contribution of both NO-GCs to LTP. Recently, we showed that the NO-GC1 isoform is located presynaptically in glutamatergic neurons and increases the glutamate release via hyperpolarization-activated cyclic nucleotide (HCN)-gated channels in the hippocampus. Electrophysiological analysis of hippocampal CA1 neurons in whole-cell recordings revealed a reduction of HCN currents and a hyperpolarizing shift of the activation curve in the NO-GC2 KOs associated with reduced resting membrane potentials. These features were mimicked in wild-type (WT) neurons with an NO-GC inhibitor. Analysis of glutamate receptors revealed a cGMP-dependent reduction of NMDA receptor currents in the NO-GC2 KO mice, which was mimicked in WT by HCN channel inhibition. Lowering extracellular Mg(2+) increased NMDA receptor currents in the NO-GC2 KO and allowed the induction of LTP that was absent at physiological Mg(2+). In sum, our data indicate that postsynaptic cGMP increases the N-methyl-D-aspartate (NMDA) receptor current by gating HCN channels and thereby is required for LTP.

It takes two to tango: cardiac fibroblast-derived NO-induced cGMP enters cardiac myocytes and increases cAMP by inhibiting PDE3.

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 ß-receptor-induced contractile responses appears as one of NO/cGMP's functions in the non-failing heart.

Hypoxic erythrocytes mediate cardioprotection through activation of soluble guanylate cyclase and release of cyclic GMP.

Red blood cells (RBCs) mediate cardioprotection via nitric oxide-like bioactivity, but the signaling and the identity of any mediator released by the RBCs remains unknown. We investigated whether RBCs exposed to hypoxia release a cardioprotective mediator and explored the nature of this mediator. Perfusion of isolated hearts subjected to ischemia-reperfusion with extracellular supernatant from mouse RBCs exposed to hypoxia resulted in improved postischemic cardiac function and reduced infarct size. Hypoxia increased extracellular export of cyclic guanosine monophosphate (cGMP) from mouse RBCs, and exogenous cGMP mimicked the cardioprotection induced by the supernatant. The protection induced by hypoxic RBCs was dependent on RBC-soluble guanylate cyclase and cGMP transport and was sensitive to phosphodiesterase 5 and activated cardiomyocyte protein kinase G. Oral administration of nitrate to mice to increase nitric oxide bioactivity further enhanced the cardioprotective effect of hypoxic RBCs. In a placebo-controlled clinical trial, a clear cardioprotective, soluble guanylate cyclase-dependent effect was induced by RBCs collected from patients randomized to 5 weeks nitrate-rich diet. It is concluded that RBCs generate and export cGMP as a response to hypoxia, mediating cardioprotection via a paracrine effect. This effect can be further augmented by a simple dietary intervention, suggesting preventive and therapeutic opportunities in ischemic heart disease.

Presynaptic nitric oxide/cGMP facilitates glutamate release via hyperpolarization-activated cyclic nucleotide-gated channels in the hippocampus.

In hippocampal neurons, synaptic transmission is affected by a variety of modulators, including nitric oxide (NO), which was proposed as a retrograde messenger as long as two decades ago. NO signals via two NO-sensitive guanylyl cyclases (NO-GCs) (NO-GC1 and NO-GC2) and the subsequent increase in cGMP. Lack of long-term potentiation in mice deficient in either one of the two NO-GCs demonstrates the involvement of both NO-GCs in synaptic transmission. However, the physiological consequences of NO/cGMP and the cellular mechanisms involved are unknown. Here, we analyzed glutamatergic synaptic transmission, most likely reflecting glutamate release, in the hippocampal CA1 region of NO-GC knockout mice by single-cell recording, and found glutamate release to be reduced under basal and stimulated conditions in the NO-GC1 knockout mice, but restorable to wild-type-like levels with a cGMP analog. Conversely, an inhibitor of NO/cGMP signaling, ODQ, reduced glutamate release in wild-type mice to knockout-like levels; thus, we conclude that presynaptic cGMP formed by NO-GC1 facilitates glutamate release. In this pathway, NO is supplied by endothelial NO synthase. In search of a cGMP target, we found that two mechanistically distinct blockers of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (ZD7288 and DK-AH269) abolished the cGMP-induced increase in glutamate release, suggesting that cGMP either directly or indirectly signals via HCN channels. In summary, we unravel a presynaptic role of NO/cGMP most likely in glutamate release and propose that HCN channels act as effectors for cGMP.

Nitric Oxide/Cyclic Guanosine Monophosphate Signaling via Guanylyl Cyclase Isoform 1 Mediates Early Changes in Synaptic Transmission and Brain Edema Formation after Traumatic Brain Injury.

Traumatic brain injury (TBI) often induces structural damage, disruption of the blood-brain barrier (BBB), neurodegeneration, and dysfunctions of surviving neuronal networks. Nitric oxide (NO) signaling has been suggested to affect brain functions after TBI. The NO exhibits most of its biological effects by activation of the primary targets-guanylyl cyclases (NO-GCs), which exists in two isoforms (NO-GC1 and NO-GC2), and the subsequently produced cyclic guanosine monophosphate (cGMP). However, the specific function of the NO-NO-GCs-cGMP pathway in the context of brain injury is not fully understood. To investigate the specific role of the isoform NO-GC1 early after brain injuries, we performed an in vivo unilateral controlled cortical impact (CCI) in the somatosensory cortex of knockout mice lacking NO-GC1 and their wild-type (WT) littermates. Morphological and electrophysiological changes of cortical neurons located 500 µm distant from the lesion border were studied early (24 h) after TBI. The CCI-operated WT mice exhibited significant BBB disruption, an impairment of dendritic spine morphology, a reduced pre-synaptic glutamate release, and less neuronal activity in the ipsilateral cortical network. The impaired ipsilateral neuronal excitability was associated with increased A-type K+ currents (IA) in the WT mice early after TBI. Interestingly, NO-GC1 KO mice revealed relatively less BBB rupture and a weaker brain edema formation early after TBI. Further, lack of NO-GC1 also prevented the impaired synaptic transmission and network function that were observed in TBI-treated WT mice. These data suggest that NO-GC1 signaling mediates early brain damage and the strength of ipsilateral cortical network in the early phase after TBI.

More than a retrograde messenger: nitric oxide needs two cGMP pathways to induce hippocampal long-term potentiation.

Although nitric oxide (NO) has been implicated as a messenger molecule in hippocampal long-term potentiation (LTP) for almost 20 years, its precise function has not been elucidated because presynaptic and/or postsynaptic actions of NO have been reported. Most of the effects of NO as a signaling molecule are mediated by the NO receptor guanylyl cyclases (NO-GCs), two heme-containing enzymes with pronounced homology in which cGMP-forming activity is stimulated on NO binding. Here we report on knock-out (KO) mice in which either one of the NO-GC receptors has been genetically deleted. By measuring NO-induced cGMP levels, similar quantities of both NO-GC receptors were determined in the hippocampus. Surprisingly, hippocampal LTP was abolished in either one of the KO strains, demonstrating that both NO-GC receptors are required in the course of LTP. Expression of LTP was restored with a cGMP analog in one of the KO strains but did not recover in the other one. Moreover, single-cell recordings of paired pulse facilitation revealed a presynaptic role of one of the NO-GC isoforms in neurotransmitter release, confirming different roles of the NO-GC receptors in LTP. Because neither one of the NO/cGMP-induced responses by itself is sufficient for LTP, two divergent, possibly presynaptically and postsynaptically localized NO-stimulated cGMP pathways are apparently required for the expression of LTP. The unexpected role of cGMP at two sites of the synaptic cleft explains many of the controversial results in former NO research in LTP and demonstrates the necessity of presynaptic and postsynaptic changes for LTP expression.

Cigarette smoke exposure has region-specific effects on GDAP1 expression in mouse hippocampus.

Early detection markers for substance use disorders are urgently needed. Recently, an association between the methylation of Ganglioside-induced differentiation-associated protein 1 (GDAP1) and alcohol addiction was found in a US and German population. In this study, we investigate whether GDAP1 expression might be affected by cigarette smoke as well and thus might be a marker of substance addiction in general. 11 adult female C57BL/6 J mice (6 wildtype and 5 lacking the NO-sensitive guanylyl cyclase1 (NO-GC1 KO)) were exposed to cigarette smoke over a period of 5 weeks, their brains immunohistochemically stained and compared to 11 non exposed mice (5 WT and 6 KO). The deletion of NO-GC1 results in a complete loss of synaptic plasticity, therefore, addiction-related alterations might become more obvious. Co-staining of anti-GDAP1 and DAPI revealed protein in the stratum granulare of the hippocampus. Three randomized frames for dentate gyrus (DG) and three for Cornu Ammonis region 1 (CA1) were used to count GDAP1. Cigarette smoke exposure significantly influenced GDAP1 expression depending on the hippocampal region but was not influenced by guanyl cyclase. In conclusion, cigarette smoke exposure alone had an effect on GDAP1 amount in both regions. Therewith, GDAP1might be a biomarker for substance addiction in general.

Spare guanylyl cyclase NO receptors ensure high NO sensitivity in the vascular system.

In the vascular system, the receptor for the signaling molecule NO, guanylyl cyclase (GC), mediates smooth muscle relaxation and inhibition of platelet aggregation by increasing intracellular cyclic GMP (cGMP) concentration. The heterodimeric GC exists in 2 isoforms (alpha1-GC, alpha2-GC) with indistinguishable regulatory properties. Here, we used mice deficient in either alpha1- or alpha2-GC to dissect their biological functions. In platelets, alpha1-GC, the only isoform present, was responsible for NO-induced inhibition of aggregation. In aortic tissue, alpha1-GC, as the major isoform (94%), mediated vasodilation. Unexpectedly, alpha2-GC, representing only 6% of the total GC content in WT, also completely relaxed alpha1-deficient vessels albeit higher NO concentrations were needed. The functional impact of the low cGMP levels produced by alpha2-GC in vivo was underlined by pronounced blood pressure increases upon NO synthase inhibition. As a fractional amount of GC was sufficient to mediate vasorelaxation at higher NO concentrations, we conclude that the majority of NO-sensitive GC is not required for cGMP-forming activity but as NO receptor reserve to increase sensitivity toward the labile messenger NO in vivo.

Genetic mouse models of the NO receptor 'soluble' guanylyl cyclases.

The NO/cGMP signalling cascade has an important role in smooth muscle relaxation, inhibition of platelet aggregation and neuronal transmission. Although the function of the main NO receptor GC (NO-GC) is well established, the particular tasks of the NO receptor isoforms (NO-GC1 and NO-GC2) are unclear and NO targets other than NO-GC have been postulated. Mice deficient in either NO receptor isoform or with a complete lack of NO-GC are now available and allow new insights in NO/cGMP signalling. The first reports about the KO strains show that, outside the neuronal system, the NO-GC isoforms can substitute for each other, and that amazingly low cGMP increases are sufficient to induce smooth muscle relaxation. In the neuronal system, however, the NO-GC isoforms obviously serve distinct functions as both isoforms are required for long term potentiation. Analysis of the complete NO-GC KO provides evidence that the vasorelaxing and platelet-inhibiting effects of NO are solely mediated by NO-GC. Thus, NO-GC appears to be the only NO receptor in these two systems.