An insight into the activation mechanism of the calcatonin-gene-related peptide receptor

The calcitonin-gene- related peptide (CGRP) receptor is unique among G-protein coupled receptors (GPCRs) as it consists of at least three proteins: calcitonin receptor like receptor (CLR), receptor activity modifying protein (RAMP)1 and receptor component protein (RCP). An endogenous agonist for this curious receptor is aCGRP, which is a sensory nerve-derived peptide made up of 37 amino acids. aCGRP acts as a potent vasodilator having pronounced effects on arterioles and capillaries. Understanding the pharmacodynamics of the CGRP receptor may have pharmaceutical benefit as the receptor has been associated with the onset of migraines and implicated in Raynauds syndrome. The primary aim of this thesis was to identify functionally important residues in the extracellular face of the CGRP receptor. Three areas of interest were selected including the extreme N-terminus of the CLR, extracellular loop 1 (ECL1) of the CLR and its associated transmembrane (TM) regions, and finally extracellular loop 3 (ECL3) of the CLR and its juxtamembrane regions. A site-directed mutagenesis (SDM) strategy was used to investigate these regions, primarily substituting the innate residues of CLR with alanine and assessing the mutation on multiple criteria including a functional cAMP assay, cell-surface expression, total expression, agonist-mediated internalisation and aCGRP binding. The results are interpreted and discussed taking into consideration contemporary concepts surrounding Secretin-like GPCRs. Moreover, the thesis also contains details of RAMP purification. Overall the thesis provides novel data that furthers insight into the complex phenomenon of CGRP receptor activation. Site-directed mutants have been identified that affect aCGRP binding, receptor signal transduction, the CLR/RAMP1 interface and the integrity of the protein complex structure.

Dysregulation of hydrogen sulfide producing enzyme cystathionine γ-lyase contributes to maternal hypertension and placental abnormalities in preeclampsia

Background—The exact etiology of preeclampsia is unknown, but there is growing evidence of an imbalance in angiogenic growth factors and abnormal placentation. Hydrogen sulfide (H2S), a gaseous messenger produced mainly by cystathionine ?-lyase (CSE), is a proangiogenic vasodilator. We hypothesized that a reduction in CSE activity may alter the angiogenic balance in pregnancy and induce abnormal placentation and maternal hypertension. Methods and Results—Plasma levels of H2S were significantly decreased in women with preeclampsia (P<0.01), which was associated with reduced placental CSE expression as determined by real-time polymerase chain reaction and immunohistochemistry. Inhibition of CSE activity by DL-propargylglycine reduced placental growth factorproduction from first-trimester (8–12 weeks gestation) human placental explants and inhibited trophoblast invasion in vitro. Knockdown of CSE in human umbilical vein endothelial cells by small-interfering RNA increased the release of soluble fms-like tyrosine kinase-1 and soluble endoglin, as assessed by enzyme-linked immunosorbent assay, whereas adenoviral-mediated CSE overexpression in human umbilical vein endothelial cells inhibited their release. Administration of DL-propargylglycine to pregnant mice induced hypertension and liver damage, promoted abnormal labyrinth vascularization in the placenta, and decreased fetal growth. Finally, a slow-releasing H2S-generating compound, GYY4137, inhibited circulating soluble fms-like tyrosine kinase-1 and soluble endoglin levels and restored fetal growth in mice that was compromised by DL-propargylglycine treatment, demonstrating that the effect of CSE inhibitor was attributable to inhibition of H2S production. Conclusions—These results imply that endogenous H2S is required for healthy placental vasculature and that a decrease in CSE/H2S activity may contribute to the pathogenesis of preeclampsia. (Circulation. 2013;127:2514-2522.)

Investigation of the activation mechanism and structural characterization of the CGRP receptor

The calcitonin gene-related peptide (CGRP) receptor is an unusual G protein-coupled receptor (GPCR) in that it comprises the calcitonin receptor-like receptor (CLR), receptor activity modifying protein 1 (RAMP1) and the receptor component protein (RCP). The RAMP1 has two other homologues – RAMP2 and RAMP3. The endogenous ligand for this receptor is CGRP, a 37 amino acid neuropeptide that act as a vasodilator. This peptide has been implicated in the aetiology of health conditions such as inflammation, Reynaud’s disease and migraine. A clear understanding of the mode of activation of this receptor could be key in developing therapeutic agents for associated health conditions. Although the crystal structure of the N-terminal extracellular domain (ECD) of this receptor (in complex with an antagonist) has been published, the details of receptor-agonist interactions at this domain, and so ultimately the mechanism of receptor activation, are still unclear. Also, the C-terminus of the CLR (in the CGRP receptor), especially around the presumed helix 8 (H8) region, has not been well studied for its role in receptor signalling. This research project investigated these questions. In this study, certain residues making up the putative N-terminal ligand-binding core of the CLR (in the CGRP receptor) were mapped out and found to be crucial for receptor signalling. They included W69 and D70 of the WDG motif in family B GPCRs, as well as Y91, F92, D94 and F95 in loop 2 of CLR N-terminus. Also, F163 at the cytoplasmic end of TM1 and certain residues spanning H8 and associated C-terminal region of CLR were found to be required for CGRP receptor signalling. These residues were investigated by site-directed mutagenesis where they were mutated to alanine (or other residues in specific cases) and the effect of the mutations on receptor pharmacology assessed by evaluating cAMP production, cell surface expression, total cell expression and aCGRP-mediated receptor internalization. Moreover, the N-terminal ECDs of the CLR and RAMPs (RAMP1, RAMP2 and RAMP3) were produced in a yeast host strain (Pichia pastoris) for the purpose of structural interaction study by surface plasmon resonance (SPR). Following expression and purification, these receptor proteins were found to individually retain their secondary structures when analysed by circular dichroism (CD). Results were analysed and interpreted with the knowledge of the secretin family receptor paradigm. The research described in this thesis has produced novel data that contributes to a clearer understanding of CGRP receptor pharmacology. The study on CLR and RAMPs ECDs could be a useful tool in determining novel interacting GPCR partners of RAMPs.

Dysregulation of hydrogen sulfide producing enzyme cystathionine γ-lyase contributes to maternal hypertension and placental abnormalities in preeclampsia

Background-The exact etiology of preeclampsia is unknown, but there is growing evidence of an imbalance in angiogenic growth factors and abnormal placentation. Hydrogen sulfide (H2S), a gaseous messenger produced mainly by cystathionine γ-lyase (CSE), is a proangiogenic vasodilator. We hypothesized that a reduction in CSE activity may alter the angiogenic balance in pregnancy and induce abnormal placentation and maternal hypertension. Methods and Results-Plasma levels of H2S were significantly decreased in women with preeclampsia (P<0.01), which was associated with reduced placental CSE expression as determined by real-time polymerase chain reaction and immunohistochemistry. Inhibition of CSE activity by DL-propargylglycine reduced placental growth factorproduction from first-trimester (8-12 weeks gestation) human placental explants and inhibited trophoblast invasion in vitro. Knockdown of CSE in human umbilical vein endothelial cells by small-interfering RNA increased the release of soluble fms-like tyrosine kinase-1 and soluble endoglin, as assessed by enzyme-linked immunosorbent assay, whereas adenoviral-mediated CSE overexpression in human umbilical vein endothelial cells inhibited their release. Administration of DL-propargylglycine to pregnant mice induced hypertension and liver damage, promoted abnormal labyrinth vascularization in the placenta, and decreased fetal growth. Finally, a slow-releasing H2S-generating compound, GYY4137, inhibited circulating soluble fms-like tyrosine kinase-1 and soluble endoglin levels and restored fetal growth in mice that was compromised by DL-propargylglycine treatment, demonstrating that the effect of CSE inhibitor was attributable to inhibition of H2S production. Conclusions-These results imply that endogenous H2S is required for healthy placental vasculature and that a decrease in CSE/H2S activity may contribute to the pathogenesis of preeclampsia. © 2013 American Heart Association, Inc.

Endothelial Nox4 NADPH oxidase enhances vasodilatation and reduces blood pressure in vivo

Objective- Increased reactive oxygen species (ROS) production is involved in the pathophysiology of endothelial dysfunction. NADPH oxidase-4 (Nox4) is a ROS-generating enzyme expressed in the endothelium, levels of which increase in pathological settings. Recent studies indicate that it generates predominantly hydrogen peroxide (H O ), but its role in vivo remains unclear. Methods and Results- We generated transgenic mice with endothelium-targeted Nox4 overexpression (Tg) to study the in vivo role of Nox4. Tg demonstrated significantly greater acetylcholine- or histamine-induced vasodilatation than wild-type littermates. This resulted from increased H O production and H O -induced hyperpolarization but not altered nitric oxide bioactivity. Tg had lower systemic blood pressure than wild-type littermates, which was normalized by antioxidants. Conclusion- Endothelial Nox4 exerts potentially beneficial effects on vasodilator function and blood pressure that are attributable to H O production. These effects contrast markedly with those reported for Nox1 and Nox2, which involve superoxide-mediated inactivation of nitric oxide. Our results suggest that therapeutic strategies to modulate ROS production in vascular disease may need to separately target individual Nox isoforms. © 2011 American Heart Association, Inc.

Resveratrol stimulates hydrogen sulfide (H2S) formation to relax murine corpus cavernosum

Introduction: Resveratrol (RVT) found in red wine protects against erectile dysfunction and relaxes penile tissue (corpus cavernosum) via a nitric oxide (NO) independent pathway. However, the mechanism remains to be elucidated. Hydrogen sulfide (H2S) is a potent vasodilator and neuromodulator generated in corpus cavernosum. Aims: We investigated whether RVT caused the relaxation of mice corpus cavernosum (MCC) through H2S. Methods: H2S formation is measured by methylene blue assay and vascular reactivity experiments have been performed by DMT strip myograph in CD1 MCC strips. Main Outcome Measures: Endothelial NO synthase (eNOS) inhibitor Nω-Nitro-L-arginine (L-NNA, 0.1mM) or H2S inhibitor aminooxyacetic acid (AOAA, 2mM) which inhibits both cystathionine-β-synthase (CBS) and cystathionine-gamma-lyase (CSE) enzyme or combination of AOAA with PAG (CSE inhibitor) has been used in the presence/absence of RVT (0.1mM, 30min) to elucidate the role of NO or H2S pathways on the effects of RVT in MCC. Concentration-dependent relaxations to RVT, L-cysteine, sodium hydrogen sulfide (NaHS) and acetylcholine (ACh) were studied. Results: Exposure of murine corpus cavernosum to RVT increased both basal and L-cysteine-stimulated H2S formation. Both of these effects were reversed by AOAA but not by L-NNA. RVT caused concentration-dependent relaxation of MCC and that RVT-induced relaxation was significantly inhibited by AOAA or AOAA+PAG but not by L-NNA. L-cysteine caused concentration-dependent relaxations, which are inhibited by AOAA or AOAA+PAG significantly. Incubation of MCC with RVT significantly increased L-cysteine-induced relaxation, and this effect was inhibited by AOAA+PAG. However, RVT did not alter the effect of exogenous H2S (NaHS) or ACh-induced relaxations. Conclusions: These results demonstrate that RVT-induced relaxation is at least partly dependent on H2S formation and acts independent of eNOS pathway. In phosphodiesterase 5 inhibitor (PDE-5i) nonresponder population, combination therapy with RVT may reverse erectile dysfunction via stimulating endogenous H2S formation. Yetik-Anacak G, Dereli MV, Sevin G, Ozzayim O, Erac Y, and Ahmed A. Resveratrol stimulates hydrogen sulfide (H2S) formation to relax murine corpus cavernosum.

Can hydrogen sulfide prevent preeclampsia and fetal growth restriction?

The exact aetiology of preeclampsia is unknown, but there is a good association with an imbalance in angiogenic growth factors and abnormal placentation [1]. Hydrogen sulphide (H2S), a gaseous messenger produced mainly by cystathionine γ-lyase (CSE), is pro-angiogenic vasodilator [2] and [3]. We hypothesized that a reduction in CSE activity may alter the angiogenic balance in pregnancy and induce abnormal placentation and maternal hypertension. Plasma levels of H2S were significantly decreased in preeclamptic women (p < 0.01), which was associated with reduced CSE message and protein expression in human placenta as determined by real-time PCR and immunohistochemistry. Inhibition of CSE activity by DL-propargylglycine (PAG) in first trimester (8–12 weeks gestation) human placental explants had reduced placenta growth factor (PlGF) production as assessed by ELISA and inhibited trophoblast invasion in vitro. Endothelial CSE knockdown by siRNA transfection increased the endogenous release of soluble fms-Like tyrosine kinase-1 (sFlt-1) and soluble endoglin, (sEng) from human umbilical vein endothelial cells while adenoviral-mediated CSE overexpression inhibited their release. Administration of PAG to pregnant mice induced hypertension, liver damage, and promoted abnormal labyrinth vascularisation in the placenta and decreased fetal growth. Finally, a slow releasing, H2S-generating compound, GYY4137, inhibited circulating sFlt-1 and sEng levels and restored fetal growth that was compromised by PAG-treatment demonstrating that the effect of CSE inhibitor was due to inhibition of H2S production. These results imply that endogenous H2S is required for healthy placental vasculature and a decrease in of CSE/H2S activity may contribute to the pathogenesis of preeclampsia. References [1] S. Ahmad, A. Ahmed, Elevated placental soluble vascular endothelial growth factor receptor-1 inhibits angiogenesis in preeclampsia, Circ Res., 95 (2004), pp. 884–891. [2] G. Yang, et al., H2S as a physiologic vasorelaxant: hypertension in mice with deletion of cystathionine gamma-lyase, Science, 322 (2008), pp. 587–590. [3] A. Papapetropoulos, et al., Hydrogen sulfide is an endogenous stimulator of angiogenesis, Proc Natl Acad Sci USA, 106 (2009), pp. 21972–21977.

Role of hydrogen sulfide in the female reproductive tract

It is well established that hydrogen sulfide (H 2S) has a signaling role in the body. So far it has been shown that H 2S is produced by intra-uterine tissues in the pregnant rat and the human placenta. Two main enzymes responsible for H 2S production, cystathionine- synthase and cystathionine-lyase, have been demonstrated in the pregnant and nonpregnant uterus, fetal membranes and placenta in the rat, and in human placenta. H 2S donors have been shown to inhibit contraction of the pregnant rat uterus. H 2S could play a role in maintaining uterine quiescence during pregnancy, as an oxygen sensor and vasodilator in the placenta, or as an anti-inflammatory. More research is required in this area to elucidate the roles of H 2S in the female reproductive tract and its mechanisms of action. © 2010 Expert Reviews Ltd.

Hydrogen sulfide:a novel mechanism for the vascular protection by resveratrol under oxidative stress in mouse aorta

Reactive oxygen species (ROS) decreases bioavailability of nitric oxide (NO) and impairs NO-dependent relaxations. Like NO, hydrogen sulfide (H2S) is an antioxidant and vasodilator; however, the effect of ROS on H2S-induced relaxations is unknown. Here we investigated whether ROS altered the effect of H2S on vascular tone in mouse aorta and determined whether resveratrol (RVT) protects it via H2S. Pyrogallol induced ROS formation. It also decreased H2S formation and relaxation induced by l-cysteine and in mouse aorta. Pyrogallol did not alter sodium hydrogensulfide (NaHS)-induced relaxation suggesting that the pyrogallol effect on l-cysteine relaxations was due to endogenous H2S formation. RVT inhibited ROS formation, enhanced l-cysteine-induced relaxations and increased H2S level in aortas exposed to pyrogallol suggesting that RVT protects against "H2S-dysfunctions" by inducing H2S formation. Indeed, H2S synthesis inhibitor AOAA inhibited the protective effects of RVT. RVT had no effect on Ach-induced relaxation that is NO dependent and the stimulatory effect of RVT on H2S-dependent relaxation was also independent of NO. These results demonstrate that oxidative stress impairs endogenous H2S-induced relaxations and RVT offers protection by inducing H2S suggesting that targeting endogenous H2S pathway may prevent vascular dysfunctions associated by oxidative stress.