Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

Characterization of domains regulating antagonist-mediated down-regulation of 5-HT7 serotonin receptors

The human 5-HT7 serotonin receptor is a G-protein-coupled receptor that activates adenylyl cyclase constitutively and upon agonist-activation. In previous studies, some inverse agonists induce both homo- and heterologous desensitization, similar to agonist-stimulation. In addition, some inverse agonists induce receptor internalization, whereas a subset of these targeted 5-HT7 receptors for lysosomal degradation. These results demonstrated that various ligands differentially activated regulatory processes governing receptor desensitization, internalization and degradation in addition to signal transduction, providing support for the concept of functional selectivity at the 5-HT7 receptor; where different ligands stabilize different receptor conformations leading to differential effects. Interestingly, the important atypical antipsychotics olanzapine and clozapine blocked G-protein activation, but induced both internalization and degradation of 5-HT7(b) receptors. Furthermore, 5-HT7(b) receptors C-terminally fused to YFP did not undergo this degradation, indicating that key regulatory proteins bind to the C-terminal tail of 5-HT7 receptors In this study, the two important and relatively novel YXXΦ motifs were identified in the C-terminus of the 5-HT7 receptor as potential sites involved in receptor internalization and recruitment of lysosomal sorting proteins such as sorting nexin 1 (SNX1) and GPCR-associated sorting protein (GASP). Mutation in either or both YXXΦ-motifs inhibited clozapine-mediated degradation of 5-HT7(b) receptors. Using radioligand binding and adenylyl cyclase assays, the YXXΦ-mutant receptors showed no change in ligand-affinity, but displayed constitutive activity and an increase in basal AC activity. Therefore, YXXΦ-mutated motif(s) may induce a conformational change in the receptor C-tail which could lead to a different capacity of the C-tail to bind and activate G-protein in addition to regulating receptor internalization and trafficking. Furthermore, mutating both YXXΦ motifs block 5-HT-stimulated AC activity, even though the receptor is present on the plasma membrane and displays functional ligand-binding and constitutive AC activity. In addition, 5-HT7(b) receptors are constitutively degradated in both lysosomes and proteasomes, but a higher proportions of receptors are constitutively targeted to lysosomes. Incubation with clozapine, olanzapine or SB269970 sort receptors to both lysosomes and proteasomes. This study shows that YXXΦ-motifs are involved in internalization and lysosomal sorting of 5-HT7(b) receptors

Regulation of cardiomyocyte T-tubular structure: opportunities for therapy

Purpose of Review Membrane invaginations called t-tubules play an integral role in triggering cardiomyocyte contraction, and their disruption during diseases such as heart failure critically impairs cardiac performance. In this review, we outline the growing understanding of the malleability of t-tubule structure and function, and highlight emerging t-tubule regulators which may be exploited for novel therapies. Recent Findings New technologies are revealing the nanometer scale organization of t-tubules, and their functional junctions with the sarcoplasmic reticulum called dyads, which generate Ca2+ sparks. Recent data have indicated that the dyadic anchoring protein junctophilin-2, and the membrane-bending protein BIN1 are key regulators of dyadic formation and maintenance. While the underlying signals which control expression and localization of these proteins remain unclear, accumulating data support an important role of myocardial workload. Summary Although t-tubule alterations are believed to be a key cause of heart failure, the plasticity of these structures also creates an opportunity for therapy. Promising recent data suggest that such therapies may specifically target junctophilin-2, BIN1, and/or mechanotransduction

The inotropic effect of the active metabolite of levosimendan, OR-1896, is mediated through inhibition of PDE3 in rat ventricular myocardium

Aims We recently published that the positive inotropic response (PIR) to levosimendan can be fully accounted for by phosphodiesterase (PDE) inhibition in both failing human heart and normal rat heart. To determine if the PIR of the active metabolite OR-1896, an important mediator of the long-term clinical effects of levosimendan, also results from PDE3 inhibition, we compared the effects of OR-1896, a representative Ca2+ sensitizer EMD57033 (EMD), levosimendan and other PDE inhibitors. Methods Contractile force was measured in rat ventricular strips. PDE assay was conducted on rat ventricular homogenate. cAMP was measured using RII_epac FRET-based sensors. Results OR-1896 evoked a maximum PIR of 33±10% above basal at 1 μM. This response was amplified in the presence of the PDE4 inhibitor rolipram (89±14%) and absent in the presence of the PDE3 inhibitors cilostamide (0.5±5.3%) or milrinone (3.2±4.4%). The PIR was accompanied by a lusitropic response, and both were reversed by muscarinic receptor stimulation with carbachol and absent in the presence of β-AR blockade with timolol. OR-1896 inhibited PDE activity and increased cAMP levels at concentrations giving PIRs. OR-1896 did not sensitize the concentration-response relationship to extracellular Ca2+. Levosimendan, OR-1896 and EMD all increased the sensitivity to β-AR stimulation. The combination of either EMD and levosimendan or EMD and OR-1896 further sensitized the response, indicating at least two different mechanisms responsible for the sensitization. Only EMD sensitized the α1-AR response. Conclusion The observed PIR to OR-1896 in rat ventricular strips is mediated through PDE3 inhibition, enhancing cAMP-mediated effects. These results further reinforce our previous finding that Ca2+ sensitization does not play a significant role in the inotropic (and lusitropic) effect of levosimendan, nor of its main metabolite OR-1896

The Ion Channels Involved in Oxidative Stress-Related Gastrointestinal Diseases

The pathogenesis of various gastrointestinal (GI) disorders, including gastritis, ulcers, inflammatory bowel disease (IBD) and cancer, can be linked to oxidative stress. It is known that reactive species carry out a crucial role in the genesis and progression of these pathologies; however, the contribution of ionic channels in their development is still under discussion. The function of ion channels in the gastrointestinal tract influences a variety of cellular processes. Acid-base balance, mucus layer, microbiota and mucosal blood flow are only some of the essential features for maintaining the mucosal integrity of the cellular barrier in the intestine, allowing for the preservation of proper permeability and ensuring tissue homeostasis. As the functional modulation of several ion channels is altered during oxidative stress conditions associated with gastrointestinal inflammation, this review focuses on contributing new insight into the roles of and the relationship between ion channels and oxidative stress in GI diseases. The association between ion channels and oxidative stress conditions could be used in diagnostics and the development of new pharmacological treatments for major gastrointestinal diseases

Identification of essential residues for binding and activation in the human 5-HT7(a) serotonin receptor by molecular modeling and site-directed mutagenesis

The human 5-HT7 receptor is expressed in both the central nervous system and peripheral tissues and is a potential drug target in behavioral and psychiatric disorders. We examined molecular determinants of ligand binding and G protein activation by the human 5-HT7(a) receptor. The role of several key residues in the 7th transmembrane domain (TMD) and helix 8 were elucidated combining in silico and experimental mutagenesis. Several single and two double point mutations of the 5-HT7(a) wild type receptor were made (W7.33V, E7.35T, E7.35R, E7.35D, E7.35A, R7.36V, Y7.43A, Y7.43F, Y7.43T, R8.52D, D8.53K; E7.35T-R7.36V, R8.52D-D8.53K), and their effects upon ligand binding were assessed by radioligand binding using a potent agonist (5-CT) and a potent antagonist (SB269970). In addition, the ability of the mutated 5-HT7(a) receptors to activate G protein after 5-HT-stimulation was determined through activation of adenylyl cyclase. In silico investigation on mutated receptors substantiated the predicted importance of TM7 and showed critical roles of residues E7.35, W7.33, R7.36 and Y7.43 in agonist and antagonist binding and conformational changes of receptor structure affecting adenylyl cyclase activation. Experimental data showed that mutants E7.35T and E7.35R were incapable of ligand binding and adenylyl cyclase activation, consistent with a requirement for a negatively charged residue at this position. The mutant R8.52D was unable to activate adenylyl cyclase, despite unaffected ligand binding, consistent with the R8.52 residue playing an important role in the receptor-G protein interface. The mutants Y7.43A and Y7.43T displayed reduced agonist binding and AC agonist potency, not seen in Y7.43F, consistent with a requirement for an aromatic residue at this position. Knowledge of the molecular interactions important in h5-HT7 receptor ligand binding and G protein activation will aid the design of selective h5-HT7 receptor ligands for potential pharmacological use

The inotropic response to OR-1896 is enhanced by PDE4 inhibition and absent in the presence of PDE3 inhibition.

<p>The figure shows representative original tracings of experiments showing the effect of increasing concentrations of OR-1896 (OR) alone and in the presence of PDE4 <b>(A)</b> and PDE3 <b>(B)</b> inhibitors. <b>A</b>) Effect of OR-1896 in increasing concentrations (OR-7: 100 nM, OR-6: 1 μM and OR-5: 10 μM, n = 6) and the effect of OR-1896 in the presence of the PDE4 inhibitor rolipram (Rol, 10 μM, n = 6). <b>B)</b> Effect of OR-1896 in the presence of a PDE3 inhibitor, either cilostamide (Cil, 1 μM, n = 6) or milrinone (Mil, 1 μM). <b>A, B)</b> EMD57033 (EMD, 3 μM) was added after OR-5. Isoprenaline (Iso, 100 μM) administered at the end gave the maximum inotropic response achievable in the strip. <b>C)</b> Bar graph showing the inotropic response to different concentrations of OR-1896 in the presence and absence (Ctr) of PDE inhibitors in rat myocardial strips (n = 6). <b>D)</b> Bar graph comparing the inotropic response of OR-1896 (1 μM) to the inotropic response of different PDE inhibitors in rat ventricular strips (n = 6). All experiments were conducted on rat ventricular muscle strips in the absence of timolol and in the presence of α₁-AR (prazosin, 100 nM) and muscarinic receptor blockade (atropine, 1 μM). Basal force values for each group (mN): OR-1896: 4.3±0.7; Rol: 3.5±0.5; Cil: 3.2±0.5; Mil: 2.6±0.3.</p