Potential mechanisms behind blood pressure modulation by melatonin

Melatonin, das in der Zirbeldrüse gebildet wird, ist eine sehr vielseitige Substanz. Melatonin moduliert die innere Uhr, den Appetites, den Schlafs und den Blutdruckes und ist ein starker Radikalfänger. Es wird angenommen, dass die zwei in Säugetieren vorkommenden Melatonin-Rezeptoren MT1 und MT2 auch im Herz-Kreislaufsystem exprimiert sind, allerdings sind die Daten zu Funktion und Ort dieser Rezeptoren strittig. Es gibt viele Hinweise, dass MT1 Vasokonstriktion und MT2 Vasodilatation vermitteln. Da exogenes Melatonin den Blutdruck senkt, sind die Mechanismen, die hinter der blutdruckregulierenden Wirkung durch MT1 und MT2 stehen, von großem Interesse. Im Projekt wurden mehrere Ratten-Aorten mittels RT-PCR und RT-qPCR auf MT1 und MT2 mRNA Expression untersucht. Aus den Ergebnissen kann geschlossen werden, dass MT1 mRNA in der exprimiert ist, wohingegen MT2 mRNA in der Ratten-Aorta gar nicht vorkommt. Die Expression von MT1 mRNA scheint keinem zirkadianen Rhythmus zu folgen, da kein Unterschied in der Expressionsrate zwischen Aorten von zwei 12 h auseinanderliegenden Zeitpunkten festgestellt werden konnte. Im Gegensatz dazu stellten wir eine 4-fache Erhöhung der MT1 mRNA Expressionsrate in Aorten von spontan-hypertensiven Ratten, einem Modell für primäre Hypertonie, fest. Dieser Befund unterstützt die Annahme einer Beteiligung von MT1 an den Blutdruck-modulierenden Eigenschaften von Melatonin. Mit Hilfe der Immunfluoreszenz-Mikroskopie stellten wir fest, dass MT1 Protein hauptsächlich in der Tunica Adventitia der Ratten-Aorta exprimiert ist. Diese Lokalisation und die Abwesenheit von MT2 stehen im Gegensatz zu derzeitigen Hypothesen über den Einfluss der Melatonin-Rezeptoren MT1 und MT2 und bedürfen weiterer Untersuchung.Melatonin is a versatile substance produced in the pineal gland. It influences the body’s internal clock, appetite, sleep, blood pressure and is a potent radical scavenger. The two mammalian G-protein coupled receptors for melatonin, MT1 and MT2, are believed to be expressed in the cardiovascular system; however, the function and location of these receptors in the cardiovascular system remain under debate. Various data suggested that MT1 mediates vasoconstriction, while MT2 mediates vasodilation. Since exogenous melatonin lowers blood pressure, the mechanisms involved in blood pressure regulation via MT1 and MT2 are of great interest. We investigated various rat aortas with RT-PCR and RT-qPCR for expression of MT1 and MT2 mRNA. It can be concluded that MT1 mRNA is indeed present in the rat aorta, whereas MT2 mRNA is not expressed at all. MT1 mRNA expression exhibits no circadian variations, since we observed no difference between aortas from 12 h away time points. Moreover, we discovered that MT1 mRNA expression is about 4-fold increased in spontaneously hypertensive rats, a model for essential hypertension. This finding supports a function of MT1 in the blood pressure modulating capabilities of melatonin. By use of immunofluorescence microscopy, we learned that MT1 protein is mainly expressed in the tunica adventitia of the rat aorta. This localization of MT1 and the absence of MT2, however, are in conflict with the current hypothesis on the influence of the melatonin GPCRs on blood pressure regulation by melatonin and ask for further investigation

Determining the role of the calcium sensing receptor in vascular smooth muscle cells via targeted gene deletion

The extracellular-Ca2+ sensing receptor (CaSR) is a G protein-coupled receptor which is essential for Ca2+ homeostasis in the body. The best studied function of the CaSR lies in the parathyroid gland, where hypercalcaemia activates the receptor which in consequence inhibits parathyroid hormone secretion. However, for other tissues like the vasculature the physiological and pathophysiological roles of the CaSR are a lot less well defined. The CaSR is expressed in all layers of blood vessels, the endothelium, the vascular smooth muscle cells (VSMC) and the tunica adventitia. Previous studies have suggested roles for the vascular CaSR in protection against vascular calcification and in blood pressure regulation. In the course of my thesis, I have investigated the specific roles of the vascular CaSR by characterising the phenotype of a transgenic mouse model of targeted CaSR deletion from VSMC (SM22α-Cre x LoxP-CaSR). In characterising this mouse model, I have made three principal discoveries: 1) The VSMC CaSR protects against vascular calcification induced by high Ca2+ and Pi in vitro. No calcification was discovered in mice lacking the VSMC CaSR in vivo, suggesting that this protective effect of the CaSR might only assert itself in pathological disease. 2) The VSMC CaSR contributes to blood pressure regulation. Mice lacking the VSMC CaSR exhibit hypotension which is due to impaired vascular contractility and therefore reduced total peripheral resistance. I propose a role for the VSMC CaSR as auto- / paracrine amplifier of VSMC contraction. Furthermore, knock-out mice exhibit bradycardia and sporadic cardiac remodelling. 3) Mice lacking the VSMC CaSR exhibit profound changes in mineral ion metabolism, together with severe hypercalcaemia, hypercalciuria, hyperphosphaturia and increased 1,25-(OH)2-Vitamin D3 and phosphaturic FGF23 levels, and decreased bone mineral density, consistent with a phenotype resembling primary hyperparathyroidism. The mechanisms behind this influence remain yet to be fully understood

Emerging roles of the extracellular calcium-sensing receptor in nutrient sensing: control of taste modulation and intestinal hormone secretion

The extracellular Ca-sensing receptor (CaSR) is a sensor for a number of key nutrients within the body, including Ca ions (Ca2+) and l-amino acids. The CaSR is expressed in a number of specialised cells within the gastrointestinal (GI) tract, and much work has been done to examine CaSR's role as a nutrient sensor in this system. This review article examines two emerging roles for the CaSR within the GI tract – as a mediator of kokumi taste modulation in taste cells and as a regulator of dietary hormone release in response to l-amino acids in the intestine

Stereo-Specific Modulation of the Extracellular Calcium-Sensing Receptor in Colon Cancer Cells

Pharmacological allosteric agonists (calcimimetics) of the extracellular calcium-sensing receptor (CaSR) have substantial gastro-intestinal side effects and induce the expression of inflammatory markers in colon cancer cells. Here, we compared the effects of both CaSR-specific (R enantiomers) and -unspecific (S enantiomers) enantiomers of a calcimimetic (NPS 568) and a calcilytic (allosteric CaSR antagonists; NPS 2143) to prove that these effects are indeed mediated via the CaSR, rather than via off-target effects, e.g., on β-adrenoceptors or calcium channels, of these drugs. The unspecific S enantiomer of NPS 2143 and NPS S-2143 was prepared using synthetic chemistry and characterized using crystallography. NPS S-2143 was then tested in HEK-293 cells stably transfected with the human CaSR (HEK-CaSR), where it did not inhibit CaSR-mediated intracellular Ca2+ signals, as expected. HT29 colon cancer cells transfected with the CaSR were treated with both enantiomers of NPS 568 and NPS 2143 alone or in combination, and the expression of CaSR and the pro-inflammatory cytokine interleukin 8 (IL-8) was measured by RT-qPCR and ELISA. Only the CaSR-selective enantiomers of the calcimimetic NPS 568 and NPS 2143 were able to modulate CaSR and IL-8 expression. We proved that pro-inflammatory effects in colon cancer cells are indeed mediated through CaSR activation. The non-CaSR selective enantiomer NPS S-2143 will be a valuable tool for investigations in CaSR-mediated processes

Ligand-based rational design, synthesis and evaluation of novel potential chemical chaperones for opsin

Inherited blinding diseases retinitis pigmentosa (RP) and a subset of Leber's congenital amaurosis (LCA) are caused by the misfolding and mistrafficking of rhodopsin molecules, which aggregate and accumulate in the endoplasmic reticulum (ER), leading to photoreceptor cell death. One potential therapeutic strategy to prevent the loss of photoreceptors in these conditions is to identify opsin-binding compounds that act as chemical chaperones for opsin, aiding its proper folding and trafficking to the outer cell membrane. Aiming to identify novel compounds with such effect, a rational ligand-based approach was applied to the structure of the visual pigment chromophore, 11-cis-retinal, and its locked analogue 11-cis-6mr-retinal. Following molecular docking studies on the main chromophore binding site of rhodopsin, 49 novel compounds were synthesized according to optimized one-to seven-step synthetic routes. These agents were evaluated for their ability to compete for the chromophore binding site of opsin, and their capacity to increase the trafficking of the P23H opsin mutant from the ER to the cell membrane. Different new molecules displayed an effect in at least one assay, acting either as chemical chaperones or as stabilizers of the 9-cis-retinal-rhodopsin complex. These compounds could provide the basis to develop novel therapeutics for RP and LCA

Comparative expression of the extracellular calcium sensing receptor in rat, mouse and human kidney

The calcium sensing receptor (CaSR) was cloned over 20 years ago and functionally demonstrated to regulate circulating levels of parathyroid hormone by maintaining physiological serum ionized calcium (Ca2+) concentration. The receptor is highly expressed in the kidney; however, intra-renal and intra-species distribution remains controversial. Recently, additional functions of the CaSR receptor in the kidney have emerged, including parathyroid hormone independent effects. It is therefore critical to establish unequivocally the localization of the CaSR in the kidney in order to relate this to its proposed physiological roles. In this study we determined CaSR expression in mouse, rat and human kidney using in situ hybridisation, immunohistochemistry (using eight different commercially available and custom-made antibodies) and proximity ligation assays. Both in situ hybridisation and immunohistochemistry showed CaSR expression in the thick ascending limb, distal tubule and collecting duct of all species, with the thick ascending limb showing the highest levels. Within the collecting ducts there was significant heterogeneity of expression between cell types. In the proximal tubule, lower levels of immunoreactivity were detected by immunohistochemistry and proximity ligation assays. Proximity ligation assays were the only technique to demonstrate expression within glomeruli. This study demonstrated CaSR expression throughout the kidney with minimal discrepancy between species but with significant variation in the levels of expression between cell and tubule types. These findings clarify the intra-renal distribution of the CaSR and enable elucidation of the full physiological roles of the receptor within this organ

Treatment of autosomal dominant hypocalcemia Type 1 with the calcilytic NPSP795 (SHP635)

Autosomal dominant hypocalcemia type 1 (ADH1) is a rare form of hypoparathyroidism caused by heterozygous, gain‐of‐function mutations of the calcium‐sensing receptor gene (CAR). Individuals are hypocalcemic with inappropriately low parathyroid hormone (PTH) secretion and relative hypercalciuria. Calcilytics are negative allosteric modulators of the extracellular calcium receptor (CaR) and therefore may have therapeutic benefits in ADH1. Five adults with ADH1 due to 4 distinct CAR mutations received escalating doses of the calcilytic compound NPSP795 (SHP635) on 3 consecutive days. Pharmacokinetics, pharmacodynamics, efficacy, and safety were assessed. Parallel in vitro testing with subject CaR mutations assessed the effects of NPSP795 on cytoplasmic calcium concentrations (Ca2+i), and ERK and p38MAPK phosphorylation. These effects were correlated with clinical responses to administration of NPSP795. NPSP795 increased plasma PTH levels in a concentration‐dependent manner up to 129% above baseline (p=0.013) at the highest exposure levels. Fractional excretion of calcium (FECa) trended down but not significantly so. Blood ionized calcium levels remained stable during NPSP795 infusion despite fasting, no calcitriol and little calcium supplementation. NPSP795 was generally safe and well‐tolerated. There was significant variability in response clinically across genotypes. In vitro, all mutant CaRs were half‐maximally activated (EC50) at lower concentrations of extracellular calcium (Ca2+o) compared to wild type (WT) CaR; NPSP795 exposure increased the EC50 for all CaR activity readouts. However, the in vitro responses to NPSP795 did not correlate with any clinical parameters. NPSP795 increased plasma PTH levels in subjects with ADH1 in a dose‐dependent manner, and thus, serves as proof‐of‐concept that calcilytics could be an effective treatment for ADH1. Albeit all mutations appear to be activating at the CaR, in vitro observations were not predictive of the in vivo phenotype, or the response to calcilytics, suggesting that other parameters impact the response to the drug

Computational studies towards the identification of novel rhodopsin-binding compounds as chemical chaperones for misfolded opsins

Accumulation of misfolded and mistrafficked rhodopsin on the endoplasmic reticulum of photoreceptor cells has a pivotal role in the pathogenesis of retinitis pigmentosa and a subset of Leber’s congenital amaurosis. One potential strategy to reduce rhodopsin misfolding and aggregation in these conditions is to use opsin-binding compounds as chemical chaperones for opsin. Such molecules have previously shown the ability to aid rhodopsin folding and proper trafficking to the outer cell membranes of photoreceptors. As means to identify novel chemical chaperones for rhodopsin, a structure-based virtual screening of commercially available drug-like compounds (300,000) was performed on the main binding site of the visual pigment chromophore, the 11-cis-retinal. The best 24 virtual hits were examined for their ability to compete for the chromophore-binding site of opsin. Among these, four small molecules demonstrated the ability to reduce the rate constant for the formation of the 9-cis-retinal-rhodopsin complex, while five molecules surprisingly enhanced the formation of this complex. Compound 7, 13, 20 and 23 showed a weak but detectable increase in the trafficking of the P23H mutant, widely used as a model for both retinitis pigmentosa and Leber’s congenital amaurosis, from the ER to the cell membrane. The compounds did not show any relevant cytotoxicity in two different human cell lines, with the only exception of 13. Based on the structures of these active compounds, a series of in silico studies gave important insights on the potential structural features required for a molecule to act either as chemical chaperone or as stabiliser of the 11-cis-retinal-rhodopsin complex. Thus, this study revealed a series of small molecules that represent a solid foundation for the future development of novel therapeutics against these severe inherited blinding diseases