10th German Pharm-Tox Summit / 91. Jahrestagung der Deutschen Gesellschaft für Experimentelle und Klinische Pharmakologie und Toxikologie (DGPT)

Chair

Prof. Dr.

The Significance of AOC3 in Pancreatic Adaptation to Metabolic Challenges

Nuclear receptors, enzymes and other targets

BR04

Amine oxidase copper containing 3 (AOC3) is an adhesion protein involved in complications related to metabolic syndrome. This enzyme facilitates the transformation of primary amines into their corresponding aldehydes, ammonium, and hydrogen peroxide. The goal of this study is to determine if metabolic stress influences the function and expression of AOC3 and whether it plays a role in the dysfunction of pancreatic islet cells.Experiments were carried out using MIN6 cells, whole islets, or islet cells from C57BL/6N mice. The cells were cultured in various media: standard, glucotoxic (33 mM glucose), lipotoxic (500 µM BSA-bound sodium palmitate), or glucolipotoxic (33 mM glucose, 500 µM BSA-bound sodium palmitate). Apoptosis was assessed using the TUNEL method, while gene expression levels were measured with RT-qPCR (2-ΔΔCt method). Glucose-stimulated insulin secretion (1 hour, steady-state incubation) was evaluated by an ELISA kit. BES-H2O2 (5 µM) served as a fluorescent indicator for hydrogen peroxide (H2O2) production.Benzylamine (100 µM), a substrate for AOC3 and monoamine oxidases (MAO), increases H2O2 production in primary islet cells. When AOC3 is inhibited by semicarbazide (1 mM), there is a significant decrease in the conversion of benzylamine and the resulting H2O2 production. In contrast, inhibiting MAO with pargyline (10 µM) does not have any effect. When both AOC3 and MAO are inhibited simultaneously, the H2O2 production induced by benzylamine is completely blocked. In the MIN6 cell line, Aoc3 gene expression is not altered by the inhibition of the enzyme but is significantly increased after 48 hours of culture in glucolipotoxic conditions. The impact of AOC3 inhibition on cell viability was evaluated under various stress conditions. PT010 (5 µM) does not protect primary mouse islet cells exposed to 33 mM glucose for seven days from apoptosis. However, partial protection was observed in islet cells cultured in lipotoxic conditions (7 d) or glucolipotoxic medium (7 d). Importantly, AOC3 inhibition did not adversely affect insulin secretion or apoptosis rates under control conditions, making the enzyme a promising target for potential treatments of metabolic syndrome or diabetes mellitus. Inhibiting AOC3 offers protection to islet cells subjected to lipo- and glucolipotoxic stress. The elevated expression of the Aoc3 gene in a glucolipotoxic environment underscores its potential as a valuable and interesting target for diabetes research.

Basic Research

Poster Self-Study

Runcaciguat activates soluble guanylyl cyclase via the histidine essential for heme binding and nitric oxide activation.

Soluble guanylyl cyclase (sGC) is a well-established pharmacological target for nitric oxide-releasing drugs in the treatment of acute angina pectoris and sGC stimulators in pulmonary hypertension and heart failure. All of these drugs lose efficacy when sGC loses heme under conditions of oxidative stress. In contrast, sGC activators increase the enzymatic activity of oxidized and heme-free sGC. It was assumed that the heme-free sGC mutants α1/β1H105F and α1/β1H105A were valuable research tools for studying NO independent sGC activators since the same residue is also crucial for heme binding and natural enzyme activation by NO. These mutants have been used in drug screening and animal models. We confirm that the first generation of sGC activators cinaciguat and BAY 60-2770 activate the α1/β1H105F and α1/β1H105A mutants. In contrast, we show that the second generation sGC activators runcaciguat and BAY 543 only activate heme-free sGC when the β1H105 residue is present. By testing runcaciguat in β1 H105F knock-in mice, we confirm this histidine-dependency in vivo. We propose a novel classification of sGC activators, distinguishing between the histidine-dependent activators runcaciguat and BAY 543 and the histidine-independent activators cinaciguat, BAY 60-2770 and BI 703704. The histidine-dependency of some of the sGC activators provides a compelling rationale for a re-evaluation of previous research and drug development programs based on sGC histidine mutants. Whether the classification of sGC activators based on the activation mechanism also makes a therapeutic difference needs to be clarified in the future.

Testing NO-ferroheme as a novel signalling entity that activates soluble guanylyl cyclase

Soluble guanylyl cyclase (sGC) is a key enzyme in the nitric oxide (NO) pathway. It converts GTP to cyclic GMP (cGMP), which enables the relaxation of blood vessels and the modulation of neural transmission. A new theory suggests that a significant portion of sGC exists in a heme-free apo-state that cannot be activated by NO. Recent studies indicate the presence of a mobile NO-ferroheme species capable of activating this heme-free sGC [1,2]. There is even a suggestion that NO-ferroheme could be a more important signalling entity than NO, because free NO is rapidly scavenged in vivo [3]. The present study is investigating the activation of sGC by NO-ferroheme-myoglobin (Mb) and comparing its effects with those of the well-known NO donor diethylamine NONOate (DEA/NO). The results demonstrate that NO-ferroheme-Mb and DEA/NO exhibit distinct activation dynamics and that they modulate sGC activity via unique mechanisms. In particular, NO-ferroheme Mb exhibits comparable activity levels in the heme-free sGC mutant α1/β1H105A to those observed with DEA/NO. Conversely, the heme-free sGC mutant α1/β1Y135F is not activated by NO-ferroheme-Mb. This finding corroborates the hypothesis that β1Y135 is a critical site for heme binding, as previously proposed [4]. We are currently testing other sGC mutants to gain further insight into potential differences between the signalling entities NO-ferroheme-Mb and free NO.References: Kleschyov, A.L., Zhuge, Z., Schiffer, T.A. et al. NO-ferroheme is a signaling entity in the vasculature. Nat Chem Biol 19, 1267–1275 (2023)DeMartino, A.W., Poudel, L., Dent, M.R. et al. Thiol-catalyzed formation of NO-ferroheme regulates intravascular NO signaling. Nat Chem Biol 19, 1256–1266 (2023)Kleschyov, A.L. The NO-heme signaling hypothesis. Free Radic Biol Med 112, 544–552 (2017)Schmidt, P.M., Schramm, M., Schröder, H., Wunder, F. and Stasch, J.P. Identiﬁcation of residues crucially involved in the binding of the heme moiety of soluble guanylyl cyclase. J. Biol. Chem. 279, 3025–3032 (2004)

Soluble guanylyl cyclase, neurodevelopmental delay, scribble and the hippocampus

A young girl was diagnosed with severe intellectual disability, global developmental delay, and behavioural dysfunction. Genetic sequencing identified a de novo point mutation in the GUCY1A2 gene, resulting in an amino acid substitution in the α2 subunit, which is a component of the GC-2 isoform of soluble guanylyl cyclase (sGC) [1]. As a consequence of the mutation, the GC-2 enzyme exhibited elevated activity and enhanced responsiveness to NO [1]. The heterodimeric enzyme GC‑2 (α2/β1) exhibits high expression levels in the brain, particularly in the hippocampus [2]. Given the involvement of the hippocampus in learning, memory, and social behaviour, this may provide a link between sGC and the neurodevelopmental delay observed in the young girl. GC-2 has the capacity to interact with neuronal scaffolding proteins, such as PSD-95, via the carboxy-terminus of its α2 subunit. The interaction between sGC and the scaffold protein scribble has been demonstrated to be of particular significance in modulating dendritic growth [3], with disruptions in this interaction associated with alterations in neuronal morphology. Nevertheless, it was previously assumed that the interaction between sGC and scribble was mediated by the β1 subunit, which is present in GC-1 and GC-2 [3]. The present study employed co-immunoprecipitation and fluorescence microscopy to demonstrate that this interaction is specific to the GC-2 isoform of sGC. Furthermore, we have identified PDZ domains 3 and 1 of scribble as the domains interacting with the carboxy-terminus of the α2 subunit of GC-2. We intend to conduct further tests to ascertain the causal role of the developmental delay using a knock-in mouse model that mimics the constitutively active GC-2 observed in the patient.References:Wittrien T, Ziegler A, Rühle A, Stomberg S, Meyer R, Bonneau D, Rodien P, Prunier-Mirebeau D, Coutant R, Behrends S. Heterozygous gain of function variant in GUCY1A2 may cause autonomous ovarian hyperfunction. Eur J Endocrinol. 2024 190(4):266-274Mergia E, Russwurm M, Zoidl G, Koesling D. Major occurrence of the new alpha2beta1 isoform of NO-sensitive guanylyl cyclase in brain. Cell Signal. 2003 15(2):189-95Szczurkowska, J., Lee, S.I., Guo, A., Cwetsch, A.W., Khan, T., Rao, S., Walz, G., Huber, T.B., Cancedda, L., Pautot, S., Shelly, M. A Localized Scaffold for cGMP Increase Is Required for Apical Dendrite Development. Cell Reports 2020 31: 107519

Role of glucocorticoids in preventing or treating bradykinin-induced dermal extravasation in mice

Introduction: Non-allergic angioedema is caused by an increased release of bradykinin and can be life-threatening if left untreated. Bradykinin is a vasoactive peptide that acts through B2 receptors, ultimately to stimulate the production of prostaglandins, thus leading to swelling. Glucocorticoids, by stimulating the expression of annexin A1 which inhibits the activity of phospholipase A2, lead to a reduced release of arachidonic acid and thus to a reduced release of prostaglandins. However, the efficacy of glucocorticoids in the treatment of non-allergic angioedema has never been systematically studied and is generally assumed to be ineffective.Objectives: The aim of the study was to evaluate the role of glucocorticoids in preventing or treating bradykinin-induced dermal extravasation in mice.Methods: C57Bl/6J mice received dexamethasone or vehicle (i.p.) under general anaesthesia. The mice were injected with Evans blue (i.v.). After shaving the dorsal skin of the back, intradermal injections of bradykinin, labradimil and histamine (2nmol each) were administered, and phosphate-buffered saline (PBS) as a control. Intradermal injections were performed at different time points in individual experiments: 20 min after dexamethasone injection for non-genomic signalling, 90 min for a possible combined effect and 180 min for genomic signalling. The blue extravasates formed at the injection sites were punched out, extracted and Evans blue quantified photometrically.Key Results: Dexamethasone, 20 min, showed no significant effect on dermal extravasation induced by 2 nmol each of bradykinin, labradimil and histamine (each n=6, ns vs. vehicle, Fig. 1). In contrast, a decrease in both bradykinin-induced (each n=6, from 4.26±0.27 to 3.15±0.23-fold PBS, #P<0.05) and labradimil-induced extravasation (each n=6, from 4.03±0.40 to 2.89±0.24-fold PBS, #P<0.05) was observed after 90 min compared to the vehicle treatment. Bradykinin-induced (each n=6, from 3.94±0.16 to 1.66±0.10-fold PBS, ***P<0.0001) and labradimil-induced (each n=6, from 4.08±0.16 to 1.56±0.10-fold PBS, ***P <0.0001) dermal extravasation was significantly reduced with dexamethasone 180 min compared to vehicle. Furthermore, no effect of dexamethasone on histamine-induced dermal extravasation could be demonstrated (respectively, n = 6, ns vs. vehicle).Conclusion: Our results show that glucocorticoids, likely mediated by a genomic mechanism, can effectively reduce bradykinin-induced extravasation.

XIAP interacts with the pro-apoptotic transcription factor ICER in the cytoplasm of human cells

Introduction: The X-linked inhibitor of apoptosis (XIAP) is an E3 ubiquitin-protein ligase exerting anti-apoptotic functions by binding caspases. As previously demonstrated by us, XIAP specifically interacts with the inducible cAMP early repressor (ICER) in human cells, but not in rat or mouse cells. ICER acts as a short inhibitory transcription factor with pro-apoptotic properties and binds to the BIR2 domain of the human XIAP protein. The human protein sequence for the BIR2 domain differs from rat and mouse only by a glycine at position 195.Objectives: The aim of this study was to investigate whether this glycine 195 in the human XIAP protein represents the molecular basis for the observed species dependence of binding and to determine the subcellular localization of the interaction partners.Methods: HEK293 cells were co-transfected each with distinct combinations of a myc-tagged human XIAP construct, which was either mutated or deleted at glycine 195 in the BIR2 domain, and a HA-tagged ICER construct. Afterwards, coimmunoprecipitation and Western blotting were conducted. By using cell fractionation and subsequent Western blotting the subcellular localization of the interaction partners was examined.Results: A mutant XIAP construct, in which the glycine at position 195 was replaced by aspartic acid, which is conserved in the BIR2 domain of the rat and mouse proteins at this position, was still observed to interact with ICER. In contrast, ICER did not bind to a mutant XIAP construct carrying a deletion of glycine 195, thereby demonstrating the importance of the BIR2 domain for the interaction with ICER. Cell fractionation revealed that the overexpressed ICER was localized both in the nucleus and in the cytoplasm. The endogenous XIAP was identified exclusively in the cytoplasmic fraction.Conclusion: Our findings indicate that the amino acid at position 195 of human XIAP plays an important role in binding of ICER. Nevertheless, the observed species dependence cannot be attributed to this single amino acid. The interaction between ICER and endogenous XIAP occurs in the cytoplasm, with ICER binding to the BIR2 domain of the XIAP-Protein, which is involved in the binding of active caspases. Therefore, ICER may compete with caspases under apoptotic conditions for the BIR2 domain, which may represent a new mechanism in the regulation of cell survival.

Activation of PP2A by fingolimod enhances vascular relaxation in B56α knockout mice

Introduction: Protein phosphatase 2A (PP2A) is a trimeric holoenzyme, comprising a catalytic (C), a structural (A), and regulatory (B) subunits. It plays a pivotal role in the regulation of cellular functions by dephosphorylation of various target proteins. Small molecule activators of PP2A (SMAPs) can be used to study the function of PP2A. Although the exact mechanisms by which SMAPs activate PP2A are not fully understood, regulatory B subunits such as B56α are suggested to be involved.Objective: Here we investigated whether activation by SMAPs and/or inhibition of PP2A by okadaic acid (OA) aﬀects vascular contractility in the presence or absence of the PP2A regulatory subunit B56α.Material & Methods: Vascular contractility was investigated in aortic rings from wild-type (WT) and B56α-KO mice using wire myograph measurements. Aortic rings were treated with the SMAPs fingolimod (FTY720) and DT-061 and the PP2A inhibitor OA or DMSO as a control. Subsequently aortae were pre-contracted with 10-6 M phenylephrine (PE) and relaxation was investigated by application of increasing isoproterenol (ISO) concentrations.Results: The absence of B56α in aortae leads to an 15% increased contraction after 10-6 M PE stimulation. Inhibition of PP2A with 3 nM OA had no effect on aortic contraction or ISO-mediated relaxation (n=20-35). However, application of 10 µM DT-061 increased ISO-induced relaxation of aortae without differences between the genotypes (IC50: KO 1.8±1.5 µM vs. WT 0.9±0.9 µM; n=5). The additional application of 3 nM OA had no effect on the DT-061 mediated-relaxation. Interestingly, application of 5 µM FTY720 resulted in enhanced ISO-induced relaxation only in KO (IC50: 1.1±0.4 µM) vs. WT (IC50: 5.6±3.5 µM) aortic rings (n=5). This effect was reversed in KO (IC50: 7.9±0.9µM) by PP2A inhibition with 3 nM OA + 5 µM FTY720 in aortic rings (n=5-10).Summary and Conclusion: Inhibition of PP2A by OA has no effect on vascular contraction or relaxation in WT aortae and application of the putative PP2A activator DT-061 showed an increased relaxation, but in a genotype and PP2A independent manner. Loss of B56a increased PE-mediated contraction and enhanced vascular relaxation after activation of PP2A by FTY720, which was reversed by PP2A inhibition. This suggests that the B56α regulatory subunit is involved in the regulation of PP2A activation in vascular contractility and that activation of PP2A by FTY720, but not DT-061 is modulated by the B56a subunit.

Poster Walk 6
Nuclear receptors, enzymes and other targets

Termin

Chair

Programm

Poster Walk 6Nuclear receptors, enzymes and other targets

Termin

Chair

Programm

Poster Walk 6
Nuclear receptors, enzymes and other targets