Ronald Jäger

Design of fluorescence resonance energy transfer (FRET)-based cGMP indicators: a systematic approach

The intracellular signalling molecule cGMP regulates a variety of physiological processes, and so the ability to monitor cGMP dynamics in living cells is highly desirable. Here, we report a systematic approach to create FRET (fluorescence resonance energy transfer)-based cGMP indicators from two known types of cGMP-binding domains which are found in cGMP-dependent protein kinase and phosphodiesterase 5, cNMP-BD [cyclic nucleotide monophosphate-binding domain and GAF [cGMP-specific and -stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA] respectively. Interestingly, only cGMP-binding domains arranged in tandem configuration as in their parent proteins were cGMP-responsive. However, the GAF-derived sensors were unable to be used to study cGMP dynamics because of slow response kinetics to cGMP. Out of 24 cGMP-responsive constructs derived from cNMP-BDs, three were selected to cover a range of cGMP affinities with an EC50 between 500 nM and 6 microM. These indicators possess excellent specifity for cGMP, fast binding kinetics and twice the dynamic range of existing cGMP sensors. The in vivo performance of these new indicators is demonstrated in living cells and validated by comparison with cGMP dynamics as measured by radioimmunoassays.

Activation of PDE10 and PDE11 Phosphodiesterases

Background: The cyclic nucleotide phosphodiesterases PDE10 and PDE11 contain putatively regulatory GAF domains with unknown function. Results: Synthetic GAF domain ligands can activate both PDEs. Conclusion: PDE10 is activated by cAMP, whereas the physiological ligand of the PDE11 GAF domains remains unknown. Significance: This is the first demonstration of a functional role of the PDE10 and PDE11 GAF domains. The most recently identified cyclic nucleotide phosphodiesterases, PDE10 and PDE11, contain a tandem of so-called GAF domains in their N-terminal regulatory regions. In PDE2 and PDE5, the GAF domains mediate cGMP stimulation; however, their function in PDE10 and PDE11 remains controversial. Although the GAF domains of PDE10 mediate cAMP-induced stimulation of chimeric adenylyl cyclases, cAMP binding did not stimulate the PDE10 holoenzyme. Comparable data about cGMP and the PDE11 GAF domains exist. Here, we identified synthetic ligands for the GAF domains of PDE10 and PDE11 to reduce interference of the GAF ligand with the catalytic reaction of PDE. With these ligands, GAF-mediated stimulation of the PDE10 and PDE11 holoenzymes is demonstrated for the first time. Furthermore, PDE10 is shown to be activated by cAMP, which paradoxically results in potent competitive inhibition of cGMP turnover by cAMP. PDE11, albeit susceptible to GAF-dependent stimulation, is not activated by the native cyclic nucleotides cAMP and cGMP. In summary, PDE11 can be stimulated by GAF domain ligands, but its native ligand remains to be identified, and PDE10 is the only PDE activated by cAMP.

Activation of PDE2 and PDE5 by specific GAF ligands: delayed activation of PDE5.

BACKGROUND AND PURPOSE By controlling intracellular cyclic nucleotide levels, phosphodiesterases (PDE) serve important functions within various signalling pathways. The PDE2 and PDE5 families are allosterically activated by their substrate cGMP via regulatory so‐called GAF domains. Here, we set out to identify synthetic ligands for the GAF domains of PDE2 and PDE5.

Cell-specific impact of nitric oxide-dependent guanylyl cyclase on arteriogenesis and angiogenesis in mice

Nitric oxide (NO) acts as essential regulator of vasculogenesis and angiogenesis and is critical for arteriogenesis. Whether NO’s effects in vivo are mediated through NO-sensitive guanylyl cyclase (NO-GC) and thus by cGMP-dependent mechanisms has been only poorly addressed. Mice lacking NO-GC globally or specifically in smooth muscle cells (SMC) or endothelial cells (EC) were subjected to two established models for arteriogenesis and angiogenesis, namely hindlimb ischemia and oxygen-induced retinopathy. Our data clearly show the involvement of NO-GC in the recovery of blood flow after hindlimb ischemia, and this effect could be attributed to NO-GC in SMC. In the retina, global deletion of NO-GC led to reduced oxygen-induced vessel loss and hypoxia-induced capillary regrowth, whereas pathological neovascularization was increased. These effects were also seen in mice with SMC-specific NO-GC deletion but not in animals lacking NO-GC in EC. Intriguingly, NO-GC was found to be strongly expressed in retinal pericytes. Our data prove the involvement of NO-GC in growth and plasticity of hindlimb and retinal vasculature after ischemic/hypoxic insult.

Preserved fertility despite erectile dysfunction in mice lacking the nitric oxide receptor

•  Erectile dysfunction may result from reduced or non‐functional nitric oxide (NO)/cGMP‐mediated signalling. Mice lacking NO synthases are fertile whereas mice deficient in cGMP‐dependent protein kinase I suffer from erectile dysfunction. •  To clarify this discrepancy we performed studies on the corpus cavernosum of male mice lacking the NO receptor NO‐sensitive guanylyl cyclase (NO‐GC) either globally or specifically in smooth muscle cells. •  NO released from NO donors as well as from nitrergic neurons failed to relax precontracted corpus cavernosum from mice lacking NO‐GC either globally or specifically in smooth muscle; to our surprise, males from both knockout lines were fertile. •  Our data show that deletion of the NO receptor specifically in smooth muscle cells abolishes NO‐induced corpus cavernosum relaxation but does not lead to infertility.

Radioimmunoassay for the Quantification of cGMP Levels in Cells and Tissues

Radioimmunoassay is an established method to determine the amount of a specific substance in a given cell or tissue sample. Commercially available RIA or Elisa are very cost intensive. Here, we describe the generation of radioactive cGMP tracer and the quantification of cGMP. Although working with radioactive material requires experience and care, this method is very sensitive and rather cheap, once it is established.

Activation of PDE10 and PDE11

Phosphodiesterases (PDEs) are critically involved in the determination of intracellular cyclic nucleotide levels. PDEs are heterodimers containing a conserved C-terminal catalytic domain and an N-terminal regulatory domain. Within their regulatory domain, 5 PDE families contain GAF domains, which are potential cyclic nucleotide binding domains. cGMP is known to activate PDEs 2 and 5. Also PDE10 and 11 contain GAF domains in their N termini. However, the functional role of these domains remains a matter of debate. In chimeric constructs of PDE GAF domains and cyanobacterial adenylyl cyclase catalytic domains, cAMP and cGMP activated constructs containing the PDE10 and PDE11 GAF domains, respectively. On the other hand, binding of cAMP and cGMP was claimed not to activate the PDE10 and PDE11 holoenzymes. Here we used synthetic ligands identified by fluorescence resonance energy transfer (FRET)-based analysis of isolated GAF domains to demonstrate that PDE10 and 11 holoenzymes are activated by their GAF domains. Furthermore, we show that PDE10 is activated by cAMP and that PDE11 albeit sensitive to synthetic GAF ligands is not activated by the physiological nucleotides cGMP and cAMP.

Cell type-specific knock out models to unravel NO/cGMP signaling in smooth muscle.

NO-sensitive guanylyl cyclase (NO-GC) is accepted to be the major receptor for the signaling molecule NO. Deletion of NO-GC in mice leads to disturbed NO/cGMP signaling. As a result, these mice show abolished NO-dependent relaxation of smooth muscle-containing tissues in the cardiovascular and gastrointestinal systems. Mice with general deletion suffer from increased blood pressure, reduced bleeding time, erectile dysfunction and die prematurely due to gastrointestinal dysmotility. Several of the phenotypical changes caused by NO-GC deficiency are due to increased smooth muscle tone. Therefore, our work concentrated on NO/cGMP-mediated regulation of smooth muscle contraction/relaxation in various tissues. These include smooth muscle from blood vessels, gut, corpus cavernosum and lower urinary tract. NO-induced relaxation of all NO-GC-containing smooth muscle tissues was influenced by the deletion of the enzyme. To our surprise, the degree of smooth muscle dysfunction was not homogeneous: In contrast to vascular or urethral smooth muscle, gastric fundus and other GI muscles revealed an only a partially reduced NO-induced relaxation. Detrusor muscle of the bladder was unresponsive towards NO. Closer examination of the tissues showed variable expression of NO-GC in the different smooth muscle cells. Importantly, in addition to smooth muscle cells, many tissues showed NO-GC expression in other cell types such as endothelial cells, interstitial cells of Cajal and fibroblast-like cells. We have generated various cell-specific KO strains using the inducible Cre-lox-system. In the GI tract we were able to show a dual mechanism of NO-induced relaxation via interstitial cell of Cajal and smooth muscle cells. In addition, we identified a third type of NO-GC-expressing cell, the fibroblast-like cell, whose function is still enigmatic. In penile corpus cavernosum, strong NO-GC expression was found in smooth muscle cells and, surprisingly, also in endothelial cells. The function of NO-GC in the endothelium is currently being investigated. Further studies using double or triple KO mutants will hopefully allow advising cell-specific functions of NO/cGMP signaling in murine smooth muscle.

Role of NO-sensitive guanylyl cyclase in angiogenesis and arteriogenesis

Background As the main receptor for nitric oxide (NO), NO-sensitive guanylyl cyclase (NO-GC) is involved in the regulation of different physiological processes such as the regulation of blood pressure. cGMP synthesis increases upon NO generation by the endothelial NO synthase (eNOS) and mediates vascular smooth muscle relaxation. NO synthesis can be stimulated by the vascular endothelial growth factor (VEGF) which is an important stimulator of angiogenesis. The interconnection between the VEGF and the NO/ cGMP pathways is still unclear. In this project, we investigated the role of NO/cGMP signaling in angiogenesis and arteriogenesis using NO-GC-deficient mice. To investigate VEGF-mediated angiogenesis, the aortic ring assay was employed. Endothelial sprouting was measured in aortic rings from global NO-GC knockout mice (GCKO) and WT animals (see Figure 1). We also used the oxygen-induced retinopathy model (OIR) to monitor vessel loss and regrowth in vivo dependent on the presence of NO-GC (see figure 2). To determine a

Comparative analysis of PDE10A isoforms

Phosphodiesterases (PDE) are considered to be key players in many signal transduction pathways. They degrade the second messengers cGMP and cAMP, thereby controlling their intracellular levels. So far, eleven PDE families, typically having several isoforms and splice variants are known. The PDE10 family is encoded by one gene that gives rise to at least two different isoforms in humans: the soluble PDE10A1 and the membrane associated PDE10A2. Distribution studies in human and mouse tissues showed highest expression in medium spiny neurons of the striatum and peripherally in testis. The localization of PDE10A in medium spiny neurons has led to much attention on PDE10 as a potential therapeutic target for novel antipsychotics. In neurons, membrane targeting of PDE10A2 is regulated at least in part by PKA-dependent phosphorylation of Thr16. On the other hand, little attention has been paid to PDE10A1 and the regulation of both isoforms. Here, we compared PDE10A isoforms with regard to their tissue distribution, subcellular localization and biochemical properties.