Publications by authors named "Rafael Franco"

273 Publications

Methamphetamine Blocks Adenosine A Receptor Activation via Sigma 1 and Cannabinoid CB Receptors.

Int J Mol Sci 2021 Mar 9;22(5). Epub 2021 Mar 9.

Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas (CiberNed), Instituto de Salud Carlos III, 28031 Madrid, Spain.

Methamphetamine is, worldwide, one of the most consumed drugs of abuse. One important side effect is neurodegeneration leading to a decrease in life expectancy. The aim of this paper was to check whether the drug affects one of the receptors involved in neurodegeneration/neuroprotection events, namely the adenosine A receptor (AR). First, we noticed that methamphetamine does not affect A functionality if the receptor is expressed in a heterologous system. However, AR becomes sensitive to the drug upon complexes formation with the cannabinoid CB receptor (CBR) and the sigma 1 receptor (σR). Signaling via both adenosine AR and cannabinoid CBR was affected by methamphetamine in cells co-expressing the two receptors. In striatal primary cultures, the AR-CBR heteromer complex was detected and methamphetamine not only altered its expression but completely blocked the AR- and the CBR-mediated activation of the mitogen activated protein kinase (MAPK) pathway. In conclusion, methamphetamine, with the participation of σR, alters the expression and function of two interacting receptors, AR, which is a therapeutic target for neuroprotection, and CBR, which is the most abundant G protein-coupled receptor (GPCR) in the brain.
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http://dx.doi.org/10.3390/ijms22052743DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7963146PMC
March 2021

Cuprizone-Induced Neurotoxicity in Human Neural Cell Lines Is Mediated by a Reversible Mitochondrial Dysfunction: Relevance for Demyelination Models.

Brain Sci 2021 Feb 22;11(2). Epub 2021 Feb 22.

Department of Morphology and Cell Biology, Faculty of Medicine, University of Oviedo, 33006 Oviedo, Spain.

Suitable in vivo and in vitro models are instrumental for the development of new drugs aimed at improving symptoms or progression of multiple sclerosis (MS). The cuprizone (CPZ)-induced murine model has gained momentum in recent decades, aiming to address the demyelination component of the disease. This work aims at assessing the differential cytotoxicity of CPZ in cells of different types and from different species: human oligodendroglial (HOG), human neuroblastoma (SH-SY5Y), human glioblastoma (T-98), and mouse microglial (N-9) cell lines. Moreover, the effect of CPZ was investigated in primary rat brain cells. Cell viability was assayed by oxygen rate consumption and by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-based (MTT) method. Our results demonstrated that CPZ did not cause death in any of the assayed cell models but affected mitochondrial function and aerobic cell respiration, thus compromising cell metabolism in neural cells and neuron-glia co-cultures. In this sense, we found differential vulnerability between glial cells and neurons as is the case of the CPZ-induced mouse model of MS. In addition, our findings demonstrated that reduced viability was spontaneous reverted in a time-dependent manner by treatment discontinuation. This reversible cell-based model may help to further investigate the role of mitochondria in the disease, and study the molecular intricacies underlying the pathophysiology of the MS and other demyelinating diseases.
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http://dx.doi.org/10.3390/brainsci11020272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7926891PMC
February 2021

The Old and New Visions of Biased Agonism Through the Prism of Adenosine Receptor Signaling and Receptor/Receptor and Receptor/Protein Interactions.

Front Pharmacol 2020 29;11:628601. Epub 2021 Jan 29.

Centro de Investigación en Red, Enfermedades Neurodegenerativas (CiberNed), Instituto de Salud Carlos iii, Madrid, Spain.

Biased signaling is a concept that has arisen in the G protein-coupled receptor (GCPR) research field, and holds promise for the development of new drug development strategies. It consists of different signaling outputs depending on the agonist's chemical structure. Here we review the most accepted mechanisms for explaining biased agonism, namely the induced fit hypothesis and the key/lock hypothesis, but we also consider how bias can be produced by a given agonist. In fact, different signaling outputs may originate at a given receptor when activated by, for instance, the endogenous agonist. We take advantage of results obtained with adenosine receptors to explain how such mechanism of functional selectivity depends on the context, being receptor-receptor interactions (heteromerization) one of the most relevant and most studied mechanisms for mammalian homeostasis. Considering all the possible mechanisms underlying functional selectivity is essential to optimize the selection of biased agonists in the design of drugs targeting GPCRs.
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http://dx.doi.org/10.3389/fphar.2020.628601DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878529PMC
January 2021

Structure and function of adenosine receptor heteromers.

Cell Mol Life Sci 2021 Feb 12. Epub 2021 Feb 12.

Laboratori de Medicina Computacional, Unitat de Bioestadística, Facultat de Medicina, Universitat Autònoma de Barcelona, Campus Universitari, 08193, Bellaterra (Barcelona), Spain.

Adenosine is one of the most ancient signaling molecules and has receptors in both animals and plants. In mammals there are four specific receptors, A, A, A, and A, which belong to the superfamily of G-protein-coupled receptors (GPCRs). Evidence accumulated in the last 20 years indicates that GPCRs are often expressed as oligomeric complexes formed by a number of equal (homomers) or different (heteromers) receptors. This review presents the data showing the occurrence of heteromers formed by A and A, A and A, and A and A receptors highlighting (i) their tetrameric structural arrangements, and (ii) the functional diversity that those heteromers provide to adenosinergic signaling.
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http://dx.doi.org/10.1007/s00018-021-03761-6DOI Listing
February 2021

Discovery of a macromolecular complex mediating the hunger suppressive actions of cocaine: Structural and functional properties.

Addict Biol 2021 Feb 8:e13017. Epub 2021 Feb 8.

Department of Biochemistry and Molecular Biomedicine, School of Biology, University of Barcelona, Barcelona, Spain.

Cocaine not only increases brain dopamine levels but also activates the sigma receptor (σ R) that in turn regulates orexigenic receptor function. Identification of interactions involving dopamine D (D R), ghrelin (GHS-R ), and σ receptors have been addressed by biophysical techniques and a complementation approach using interfering peptides. The effect of cocaine on receptor functionality was assayed by measuring second messenger, cAMP and Ca , levels. The effect of acute or chronic cocaine administration on receptor complex expression was assayed by in situ proximity ligation assay. In silico procedures were used for molecular model building. σ R KO mice were used for confirming involvement of this receptor. Upon identification of protomer interaction and receptor functionality, a unique structural model for the macromolecular complex formed by σ R, D R, and GHS-R is proposed. The functionality of the complex, able to couple to both Gs and Gq proteins, is affected by cocaine binding to the σ R, as confirmed using samples from σ R mice. The expression of the macromolecular complex was differentially affected upon acute and chronic cocaine administration to rats. The constructed 3D model is consistent with biochemical, biophysical, and available structural data. The σ R, D R, and GHS-R complex constitutes a functional unit that is altered upon cocaine binding to the σ R. Remarkably, the heteromer can simultaneously couple to two G proteins, thus allowing dopamine to signal via Ca and ghrelin via cAMP. The anorexic action of cocaine is mediated by such complex whose expression is higher after acute than after chronic administration regimens.
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http://dx.doi.org/10.1111/adb.13017DOI Listing
February 2021

Geoffrey Burnstock (1929-2020): the finest pharmacologist and an inspiring scientist.

Authors:
Rafael Franco

Purinergic Signal 2021 Mar 2;17(1):135. Epub 2021 Feb 2.

Department of Biochemistry and Molecular Biomedicine, Facultat de Biologia, University of Barcelona, Diagonal 643, 08028, Barcelona, Spain.

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http://dx.doi.org/10.1007/s11302-021-09769-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7955004PMC
March 2021

Dopamine in Health and Disease: Much More Than a Neurotransmitter.

Biomedicines 2021 Jan 22;9(2). Epub 2021 Jan 22.

Neurodegenerative Diseases, CiberNed. Network Research Center, Spanish National Health Institute Carlos III, Valderrebollo 5, 28031 Madrid, Spain.

Dopamine is derived from an amino acid, phenylalanine, which must be obtained through the diet. Dopamine, known primarily to be a neurotransmitter involved in almost any higher executive action, acts through five types of G-protein-coupled receptors. Dopamine has been studied extensively for its neuronal handling, synaptic actions, and in relation to Parkinson's disease. However, dopamine receptors can be found extra-synaptically and, in addition, they are not only expressed in neurons, but in many types of mammalian cells, inside and outside the central nervous system (CNS). Recent studies show a dopamine link between the gut and the CNS; the mechanisms are unknown, but they probably require cells to act as mediators and the involvement of the immune system. In fact, dopamine receptors are expressed in almost any cell of the immune system where dopamine regulates various processes, such as antigen presentation, T-cell activation, and inflammation. This likely immune cell-mediated linkage opens up a new perspective for the use of dopamine-related drugs, i.e., agonist-antagonist-allosteric modulators of dopamine receptors, in a variety of diseases.
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http://dx.doi.org/10.3390/biomedicines9020109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7911410PMC
January 2021

Dobutamine-sparing versus dobutamine-to-all strategy in cardiac surgery: a randomized noninferiority trial.

Ann Intensive Care 2021 Jan 26;11(1):15. Epub 2021 Jan 26.

Intensive Care Unit, Cancer Institute (ICESP), University of Sao Paulo, São Paulo, Brazil.

Background: The detrimental effects of inotropes are well-known, and in many fields they are only used within a goal-directed therapy approach. Nevertheless, standard management in many centers includes administering inotropes to all patients undergoing cardiac surgery to prevent low cardiac output syndrome and its implications. Randomized evidence in favor of a patient-tailored, inotrope-sparing approach is still lacking. We designed a randomized controlled noninferiority trial in patients undergoing cardiac surgery with normal ejection fraction to assess whether an dobutamine-sparing strategy (in which the use of dobutamine was guided by hemodynamic evidence of low cardiac output associated with signs of inadequate tissue perfusion) was noninferior to an inotrope-to-all strategy (in which all patients received dobutamine).

Results: A total of 160 patients were randomized to the dobutamine-sparing strategy (80 patients) or to the dobutamine-to-all approach (80 patients). The primary composite endpoint of 30-day mortality or occurrence of major cardiovascular complications (arrhythmias, acute myocardial infarction, low cardiac output syndrome and stroke or transient ischemic attack) occurred in 25/80 (31%) patients of the dobutamine-sparing group (p = 0.74) and 27/80 (34%) of the dobutamine-to-all group. There were no significant differences between groups regarding the incidence of acute kidney injury, prolonged mechanical ventilation, intensive care unit or hospital length of stay.

Discussion: Although it is common practice in many centers to administer inotropes to all patients undergoing cardiac surgery, a dobutamine-sparing strategy did not result in an increase of mortality or occurrence of major cardiovascular events when compared to a dobutamine-to-all strategy. Further research is needed to assess if reducing the administration of inotropes can improve outcomes in cardiac surgery. Trial registration ClinicalTrials.gov, NCT02361801. Registered Feb 2nd, 2015. https://clinicaltrials.gov/ct2/show/NCT02361801.
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http://dx.doi.org/10.1186/s13613-021-00808-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7838231PMC
January 2021

Experimental data using candesartan and captopril indicate no double-edged sword effect in COVID-19.

Clin Sci (Lond) 2021 02;135(3):465-481

Research Center for Molecular Medicine and Chronic Diseases (CIMUS), IDIS, University of Santiago de Compostela, Santiago de Compostela, Spain.

The key link between renin-angiotensin system (RAS) and COVID-19 is ACE2 (angiotensin-converting enzyme 2), which acts as a double-edged sword, because ACE2 increases the tissue anti-inflammatory response but it is also the entry receptor for the virus. There is an important controversy on several drugs that regulate RAS activity and possibly ACE2, and are widely used, particularly by patients most vulnerable to severe COVID-19. In the lung of healthy rats, we observed that candesartan (an angiotensin type-1, AT1, receptor blocker; ARB) and captopril (an ACE inhibitor; ACEI) up-regulated expression of tissue ACE2 and RAS anti-inflammatory axis receptors (AT2 and Mas receptors). This effect was particularly pronounced in rats with metabolic syndrome (obesity, increased blood pressure and hyperglycemia) and aged rats. Treatment of cultures of human type-II pneumocytes with candesartan or captopril induced up-regulation of ACE2 expression in cells. Treatment with viral spike protein induced a decrease in full-length (i.e. transmembrane) ACE2, an increase in levels of a short intracellular ACE2 polypeptide and an increase in ADAM17 activity in cells, together with an increase in levels of soluble ACE2 and major proinflammatory cytokines in the culture medium. Spike protein-induced changes and levels of spike protein internalization in cells were inhibited by pretreatment with the above-mentioned drugs. The results suggest that these drugs increase ACE2 levels and promote the anti-inflammatory RAS axis in the lung. Furthermore, possible up-regulation of viral entry by the drug-induced increase in expression of transmembrane ACE2 is counteracted by additional mechanisms, particularly by drug-induced inhibition of ADAM17 activity.
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http://dx.doi.org/10.1042/CS20201511DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7851407PMC
February 2021

Novel Interactions Involving the Mas Receptor Show Potential of the Renin-Angiotensin system in the Regulation of Microglia Activation: Altered Expression in Parkinsonism and Dyskinesia.

Neurotherapeutics 2021 Jan 20. Epub 2021 Jan 20.

Department Biochemistry and Molecular Biomedicine, School of Biology, University of Barcelona, Diagonal 643, Barcelona, Catalonia, 08028, Spain.

The renin-angiotensin system (RAS) not only plays an important role in controlling blood pressure but also participates in almost every process to maintain homeostasis in mammals. Interest has recently increased because SARS viruses use one RAS component (ACE2) as a target-cell receptor. The occurrence of RAS in the basal ganglia suggests that the system may be targeted to improve the therapy of neurodegenerative diseases. RAS-related data led to the hypothesis that RAS receptors may interact with each other. The aim of this paper was to find heteromers formed by Mas and angiotensin receptors and to address their functionality in neurons and microglia. Novel interactions were discovered by using resonance energy transfer techniques. The functionality of individual and interacting receptors was assayed by measuring levels of the second messengers cAMP and Ca in transfected human embryonic kidney cells (HEK-293T) and primary cultures of striatal cells. Receptor complex expression was assayed by in situ proximity ligation assay. Functionality and expression were assayed in parallel in primary cultures of microglia treated or not with lipopolysaccharide and interferon-γ (IFN-γ). The proximity ligation assay was used to assess heteromer expression in parkinsonian and dyskinetic conditions. Complexes formed by Mas and the angiotensin AT or AT receptors were identified in both a heterologous expression system and in neural primary cultures. In the heterologous system, we showed that the three receptors-MasR, ATR, and ATR-can interact to form heterotrimers. The expression of receptor dimers (ATR-MasR or ATR-MasR) was higher in microglia than in neurons and was differentially affected upon microglial activation with lipopolysaccharide and IFN-γ. In all cases, agonist-induced signaling was reduced upon coactivation, and in some cases just by coexpression. Also, the blockade of signaling of two receptors in a complex by the action of a given (selective) receptor antagonist (cross-antagonism) was often observed. Differential expression of the complexes was observed in the striatum under parkinsonian conditions and especially in animals rendered dyskinetic by levodopa treatment. The negative modulation of calcium mobilization (mediated by ATR activation), the multiplicity of possibilities on RAS affecting the MAPK pathway, and the disbalanced expression of heteromers in dyskinesia yield new insight into the operation of the RAS system, how it becomes unbalanced, and how a disbalanced RAS can be rebalanced. Furthermore, RAS components in activated microglia warrant attention in drug-development approaches to address neurodegeneration.
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http://dx.doi.org/10.1007/s13311-020-00986-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7817140PMC
January 2021

Carnitine palmitoyltransferase 1C negatively regulates the endocannabinoid hydrolase ABHD6 in mice, depending on nutritional status.

Br J Pharmacol 2021 Apr 12;178(7):1507-1523. Epub 2021 Feb 12.

Basic Sciences Department, Faculty of Medicine and Health Sciences, Universitat Internacional de Catalunya, Sant Cugat del Vallès, Spain.

Background And Purpose: The enzyme α/β-hydrolase domain containing 6 (ABHD6), a new member of the endocannabinoid system, is a promising therapeutic target against neuronal-related diseases. However, how ABHD6 activity is regulated is not known. ABHD6 coexists in protein complexes with the brain-specific carnitine palmitoyltransferase 1C (CPT1C). CPT1C is involved in neuro-metabolic functions, depending on brain malonyl-CoA levels. Our aim was to study CPT1C-ABHD6 interaction and determine whether CPT1C is a key regulator of ABHD6 activity depending on nutritional status.

Experimental Approach: Co-immunoprecipitation and FRET assays were used to explore ABHD6 interaction with CPT1C or modified malonyl-CoA-insensitive or C-terminal truncated CPT1C forms. Cannabinoid CB receptor-mediated signalling was investigated by determining cAMP levels. A novel highly sensitive fluorescent method was optimized to measure ABHD6 activity in non-neuronal and neuronal cells and in brain tissues from wild-type (WT) and CPT1C-KO mice.

Key Results: CPT1C interacted with ABHD6 and negatively regulated its hydrolase activity, thereby regulating 2-AG downstream signalling. Accordingly, brain tissues of CPT1C-KO mice showed increased ABHD6 activity. CPT1C malonyl-CoA sensing was key to the regulatory role on ABHD6 activity and CB receptor signalling. Fasting, which attenuates brain malonyl-CoA, significantly increased ABHD6 activity in hypothalamus from WT, but not CPT1C-KO, mice.

Conclusions And Implications: Our finding that negative regulation of ABHD6 activity, particularly in the hypothalamus, is sensitive to nutritional status throws new light on the characterization and the importance of the proteins involved as potential targets against diseases affecting the CNS.
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http://dx.doi.org/10.1111/bph.15377DOI Listing
April 2021

Functional Complexes of Angiotensin-Converting Enzyme 2 and Renin-Angiotensin System Receptors: Expression in Adult but Not Fetal Lung Tissue.

Int J Mol Sci 2020 Dec 16;21(24). Epub 2020 Dec 16.

Network Center, Neurodegenerative Diseases (CiberNed), Spanish National Health Institute Carlos III, Valderrebollo 5, 28031 Madrid, Spain.

Angiotensin-converting enzyme 2 (ACE2) is a membrane peptidase and a component of the renin-angiotensin system (RAS) that has been found in cells of all organs, including the lungs. While ACE2 has been identified as the receptor for severe acute respiratory syndrome (SARS) coronaviruses, the mechanism underlying cell entry remains unknown. Human immunodeficiency virus infects target cells via CXC chemokine receptor 4 (CXCR4)-mediated endocytosis. Furthermore, CXCR4 interacts with dipeptidyl peptidase-4 (CD26/DPPIV), an enzyme that cleaves CXCL12/SDF-1, which is the chemokine that activates this receptor. By analogy, we hypothesized that ACE2 might also be capable of interactions with RAS-associated G-protein coupled receptors. Using resonance energy transfer and cAMP and mitogen-activated protein kinase signaling assays, we found that human ACE2 interacts with RAS-related receptors, namely the angiotensin II type 1 receptor (ATR), the angiotensin II type 2 receptor (ATR), and the MAS1 oncogene receptor (MasR). Although these interactions lead to minor alterations of signal transduction, ligand binding to ATR and ATR, but not to MasR, resulted in the upregulation of ACE2 cell surface expression. Proximity ligation assays performed in situ revealed macromolecular complexes containing ACE2 and ATR, ATR or MasR in adult but not fetal mouse lung tissue. These findings highlight the relevance of RAS in SARS-CoV-2 infection and the role of ACE2-containing complexes as potential therapeutic targets.
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http://dx.doi.org/10.3390/ijms21249602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7766085PMC
December 2020

Recent Advances in the Potential of Cannabinoids for Neuroprotection in Alzheimer's, Parkinson's, and Huntington's Diseases.

Adv Exp Med Biol 2021 ;1264:81-92

Molecular Neurobiology laboratory, Department of Biochemistry and Molecular Biomedicine, Universitat de Barcelona, Barcelona, Spain.

Three prevalent neurodegenerative diseases, Parkinson's, Alzheimer's, and Huntington's are in need of symptomatic relief of slowing disease progression or both. This chapter focuses on the potential of cannabinoids to afford neuroprotection, i.e. avoid or retard neuronal death. The neuroprotective potential of cannabinoids is known from the work in animal models and is mediated by the two cannabinoid receptors (CB/CB) and eventually, by their heteromers, GPR55, orphan receptors (GPR3/GPR6/GPR12/GPR18), or PPARγ. Now, there is the time to translate the findings into patients. The chapter takes primarily into account advances since 2016 and addresses the issue of proving neuroprotection in humans. One recent discovery is the existence of activated microglia with neuroprotective phenotype; cannabinoids are good candidates to skew phenotype, especially via glial CB receptors (CBR), whose targeting has, a priori, less side effects those targeting the CBs receptor (CBR), which are expressed in both neurons and glia. The fact that a cannabis extract (SativexTM) is approved for human therapy, such that cannabis use will likely be legalized in many countries and different possibilities that cannabinoid pharmacology suggests a successful route of cannabinoids (natural or synthetic) all the way to be approved and used in the treatment of neurodegeneration.
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http://dx.doi.org/10.1007/978-3-030-57369-0_6DOI Listing
February 2021

Natural Compounds as Guides for the Discovery of Drugs Targeting G-Protein-Coupled Receptors.

Molecules 2020 Oct 30;25(21). Epub 2020 Oct 30.

CiberNed: Centro de Investigación en Red Enfermedades Neurodegenerativas, Spanish National Institute of Health Carlos III, 28031 Madrid, Spain.

G protein-coupled receptors (GPCRs), which constitute the most populous family of the human proteome, are the target of 35-45% of approved therapeutic drugs. This review focuses on natural products (excluding peptides) that target GPCRs. Natural compounds identified so far as agonists, antagonists or allosteric modulators of GPCRs have been found in all groups of existing living beings according to Whittaker's Five Kingdom Classification, i.e., bacteria (monera), fungi, protoctists, plants and animals. Terpenoids, alkaloids and flavonoids are the most common chemical structures that target GPCRs whose endogenous ligands range from lipids to epinephrine, from molecules that activate taste receptors to molecules that activate smell receptors. Virtually all of the compounds whose formula is displayed in this review are pharmacophores with potential for drug discovery; furthermore, they are expected to help expand the number of GPCRs that can be considered as therapeutic targets.
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http://dx.doi.org/10.3390/molecules25215060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7663367PMC
October 2020

Experimental and computational analysis of biased agonism on full-length and a C-terminally truncated adenosine A receptor.

Comput Struct Biotechnol J 2020 24;18:2723-2732. Epub 2020 Sep 24.

Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas. Instituto de Salud Carlos III, Madrid, Spain.

Biased agonism, the ability of agonists to differentially activate downstream signaling pathways by stabilizing specific receptor conformations, is a key issue for G protein-coupled receptor (GPCR) signaling. The C-terminal domain might influence this functional selectivity of GPCRs as it engages G proteins, GPCR kinases, β-arrestins, and several other proteins. Thus, the aim of this paper is to compare the agonist-dependent selectivity for intracellular pathways in a heterologous system expressing the full-length (AR) and a C-tail truncated (AR lacking the last 40 amino acids) adenosine A receptor, a GPCR that is already targeted in Parkinson's disease using a first-in-class drug. Experimental data such as ligand binding, cAMP production, β-arrestin recruitment, ERK1/2 phosphorylation and dynamic mass redistribution assays, which correspond to different aspects of signal transduction, were measured upon the action of structurally diverse compounds (the agonists adenosine, NECA, CGS-21680, PSB-0777 and LUF-5834 and the SCH-58261 antagonist) in cells expressing AR and AR. The results show that taking cAMP levels and the endogenous adenosine agonist as references, the main difference in bias was obtained with PSB-0777 and LUF-5834. The C-terminus is dispensable for both G-protein and β-arrestin recruitment and also for MAPK activation. Unrestrained molecular dynamics simulations, at the μs timescale, were used to understand the structural arrangements of the binding cavity, triggered by these chemically different agonists, facilitating G protein binding with different efficacy.
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http://dx.doi.org/10.1016/j.csbj.2020.09.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7550916PMC
September 2020

Angiotensin AT and AT receptor heteromer expression in the hemilesioned rat model of Parkinson's disease that increases with levodopa-induced dyskinesia.

J Neuroinflammation 2020 Aug 17;17(1):243. Epub 2020 Aug 17.

Centro de Investigación en Red, enfermedades Neurodegenerativas, CiberNed, Instituto de Salud Carlos III, Madrid, Spain.

Background/aims: The renin-angiotensin system (RAS) is altered in Parkinson's disease (PD), a disease due to substantia nigra neurodegeneration and whose dopamine-replacement therapy, using the precursor levodopa, leads to dyskinesias as the main side effect. Angiotensin AT and AT receptors, mainly known for their role in regulating water homeostasis and blood pressure and able to form heterodimers (ATHets), are present in the central nervous system. We assessed the functionality and expression of ATHets in Parkinson disease (PD).

Methods: Immunocytochemistry was used to analyze the colocalization between angiotensin receptors; bioluminescence resonance energy transfer was used to detect ATHets. Calcium and cAMP determination, MAPK activation, and label-free assays were performed to characterize signaling in homologous and heterologous systems. Proximity ligation assays were used to quantify receptor expression in mouse primary cultures and in rat striatal sections.

Results: We confirmed that AT and AT receptors form ATHets that are expressed in cells of the central nervous system. ATHets are novel functional units with particular signaling properties. Importantly, the coactivation of the two receptors in the heteromer reduces the signaling output of angiotensin. Remarkably, ATHets that are expressed in both striatal neurons and microglia make possible that candesartan, the antagonist of AT, increases the effect of AT receptor agonists. In addition, the level of striatal expression increased in the unilateral 6-OH-dopamine lesioned rat PD model and was markedly higher in parkinsonian-like animals that did not become dyskinetic upon levodopa chronic administration if compared with expression in those that became dyskinetic.

Conclusion: The results indicate that boosting the action of neuroprotective AT receptors using an AT receptor antagonist constitutes a promising therapeutic strategy in PD.
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http://dx.doi.org/10.1186/s12974-020-01908-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430099PMC
August 2020

Discovery of Homobivalent Bitopic Ligands of the Cannabinoid CB Receptor*.

Chemistry 2020 Dec 9;26(68):15839-15842. Epub 2020 Nov 9.

Medicinal Chemistry Institute, Spanish Research Council, Madrid, Spain.

Single chemical entities with potential to simultaneously interact with two binding sites are emerging strategies in medicinal chemistry. We have designed, synthesized and functionally characterized the first bitopic ligands for the CB receptor. These compounds selectively target CB versus CB receptors. Their binding mode was studied by molecular dynamic simulations and site-directed mutagenesis.
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http://dx.doi.org/10.1002/chem.202003389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7756656PMC
December 2020

Adenosine A and A Receptors Are Able to Interact with Each Other. A Further Piece in the Puzzle of Adenosine Receptor-Mediated Signaling.

Int J Mol Sci 2020 Jul 17;21(14). Epub 2020 Jul 17.

Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas (CIBERNED), 28031 Madrid, Spain.

The aim of this paper was to check the possible interaction of two of the four purinergic P1 receptors, the A and the A. Discovery of the A-A receptor complex was achieved by means of immunocytochemistry and of bioluminescence resonance energy transfer. The functional properties and heteromer print identification were addressed by combining binding and signaling assays. The physiological role of the novel heteromer is to provide a differential signaling depending on the pre-coupling to signal transduction components and/or on the concentration of the endogenous agonist. The main feature was that the heteromeric context led to a marked decrease of the signaling originating at A receptors. Interestingly from a therapeutic point of view, A receptor antagonists overrode the blockade, thus allowing A receptor-mediated signaling. The A-A receptor heteromer print was detected in primary cortical neurons. These and previous results suggest that all four adenosine receptors may interact with each other. Therefore, each adenosine receptor could form heteromers with distinct properties, expanding the signaling outputs derived from the binding of adenosine to its cognate receptors.
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http://dx.doi.org/10.3390/ijms21145070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7404137PMC
July 2020

Expression of GPR55 and either cannabinoid CB or CB heteroreceptor complexes in the caudate, putamen, and accumbens nuclei of control, parkinsonian, and dyskinetic non-human primates.

Brain Struct Funct 2020 Sep 20;225(7):2153-2164. Epub 2020 Jul 20.

Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain.

Endocannabinoids are neuromodulators acting on specific cannabinoid CB and CB G-protein-coupled receptors (GPCRs), representing potential therapeutic targets for neurodegenerative diseases. Cannabinoids also regulate the activity of GPR55, a recently "deorphanized" GPCR that directly interacts with CB and with CB receptors. Our hypothesis is that these heteromers may be taken as potential targets for Parkinson's disease (PD). This work aims at assessing the expression of heteromers made of GPR55 and CB/CB receptors in the striatum of control and parkinsonian macaques (with and without levodopa-induced dyskinesia). For this purpose, double blind in situ proximity ligation assays, enabling the detection of GPCR heteromers in tissue samples, were performed in striatal sections of control, MPTP-treated and MPTP-treated animals rendered dyskinetic by chronic treatment with levodopa. Image analysis and statistical assessment were performed using dedicated software. We have previously demonstrated the formation of heteromers between GPR55 and CB receptor (CB-GPR55_Hets), which is highly expressed in the central nervous system (CNS), but also with the CB receptor (CB-GPR55_Hets). Compared to the baseline expression of CB-GPR55_Hets in control animals, our results showed increased expression levels in basal ganglia input nuclei of MPTP-treated animals. These observed increases in CB-GPR55_Hets returned back to baseline levels upon chronic treatment with levodopa in dyskinetic animals. Obtained data regarding CB-GPR55_Hets were quite similar, with somehow equivalent amounts in control and dyskinetic animals, and with increased expression levels in MPTP animals. Taken together, the detected increased expression of GPR55-endocannabinoid heteromers appoints these GPCR complexes as potential non-dopaminergic targets for PD therapy.
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http://dx.doi.org/10.1007/s00429-020-02116-4DOI Listing
September 2020

SARS-CoV-2 as a Factor to Disbalance the Renin-Angiotensin System: A Suspect in the Case of Exacerbated IL-6 Production.

J Immunol 2020 09 17;205(5):1198-1206. Epub 2020 Jul 17.

Centro de Investigación en Red, Enfermedades Neurodegenerativas, Instituto de Salud Carlos III, 28029 Madrid, Spain.

Fever in infections correlates with inflammation, macrophage infiltration into the affected organ, macrophage activation, and release of cytokines involved in immune response, hematopoiesis, and homeostatic processes. Angiotensin-converting enzyme 2 (ACE2) is the canonical cell surface receptor for SARS-CoV-2. ACE2 together with angiotensin receptor types 1 and 2 and ACE2 are components of the renin-angiotensin system (RAS). Exacerbated production of cytokines, mainly IL-6, points to macrophages as key to understand differential COVID-19 severity. SARS-CoV-2 may modulate macrophage-mediated inflammation events by altering the balance between angiotensin II, which activates angiotensin receptor types 1 and 2, and angiotensin 1-7 and alamandine, which activate proto-oncogene and -related D receptors, respectively. In addition to macrophages, lung cells express RAS components; also, some lung cells are able to produce IL-6. Addressing how SARS-CoV-2 unbalances RAS functionality via ACE2 will help design therapies to attenuate a COVID-19-related cytokine storm.
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http://dx.doi.org/10.4049/jimmunol.2000642DOI Listing
September 2020

Expression of cannabinoid CB R-GPR55 heteromers in neuronal subtypes of the Macaca fascicularis striatum.

Ann N Y Acad Sci 2020 09 27;1475(1):34-42. Epub 2020 Jun 27.

Neurosciences Division, Centre for Applied Medical Research (CIMA), the University of Navarra, Pamplona, Spain.

The cannabinoid CB receptor (CB R) is the most abundant G protein-coupled receptor in the central nervous system, consistent with the important role of endocannabinoids as neuromodulators. Cannabinoids also modulate the function of G protein-coupled receptor 55 (GPR55), which forms heteroreceptor complexes with the CB R in the striatum. The aim was to characterize cannabinoid CB R-GPR55 heteromers (CB R/GPR55Hets) in the basal ganglia input nuclei of nonhuman primates, Macaca fascicularis, both in projection neurons and interneurons, by the in situ proximity ligation assay. Striatal projecting neurons were identified by the retrograde neuroanatomical tracer, biotinylated dextran amine (BDA), injected into external or internal subdivisions of the globus pallidus. Triple immunofluorescent stains were carried out to visualize (1) BDA-labeled neurons, (2) CB R/GPR55Hets, and (3) striatal interneurons positive for choline acetyltransferase, parvalbumin, calretinin, or nitric oxide synthase. CB R/GPR55Hets were identified within both types of projection neurons as well as all interneurons except those that are cholinergic. Moreover, CB R/GPR55Hets were found specifically in the neuronal cell surface, and also in intracellular membranes. Further research efforts will be needed to confirm the intracellular occurrence of heteromers and their potential as therapeutic targets in diseases related to motor control imbalances, particularly within a parkinsonian context (with or without levodopa-induced dyskinesia).
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http://dx.doi.org/10.1111/nyas.14413DOI Listing
September 2020

Adenosine/A2B Receptor Signaling Ameliorates the Effects of Aging and Counteracts Obesity.

Cell Metab 2020 Jul 25;32(1):56-70.e7. Epub 2020 Jun 25.

Institute of Pharmacology and Toxicology, University Hospital, University of Bonn, 53127 Bonn, Germany. Electronic address:

The combination of aging populations with the obesity pandemic results in an alarming rise in non-communicable diseases. Here, we show that the enigmatic adenosine A2B receptor (A2B) is abundantly expressed in skeletal muscle (SKM) as well as brown adipose tissue (BAT) and might be targeted to counteract age-related muscle atrophy (sarcopenia) as well as obesity. Mice with SKM-specific deletion of A2B exhibited sarcopenia, diminished muscle strength, and reduced energy expenditure (EE), whereas pharmacological A2B activation counteracted these processes. Adipose tissue-specific ablation of A2B exacerbated age-related processes and reduced BAT EE, whereas A2B stimulation ameliorated obesity. In humans, A2B expression correlated with EE in SKM, BAT activity, and abundance of thermogenic adipocytes in white fat. Moreover, A2B agonist treatment increased EE from human adipocytes, myocytes, and muscle explants. Mechanistically, A2B forms heterodimers required for adenosine signaling. Overall, adenosine/A2B signaling links muscle and BAT and has both anti-aging and anti-obesity potential.
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http://dx.doi.org/10.1016/j.cmet.2020.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7437516PMC
July 2020

Pharmacological data of cannabidiol- and cannabigerol-type phytocannabinoids acting on cannabinoid CB, CB and CB/CB heteromer receptors.

Pharmacol Res 2020 09 26;159:104940. Epub 2020 May 26.

Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CiberNed), Spain; Department of Biochemistry and Molecular Biomedicine. Universitat de Barcelona, Spain. Electronic address:

Background: Recent approved medicines whose active principles are ΔTetrahidrocannabinol (Δ-THC) and/or cannabidiol (CBD) open novel perspectives for other phytocannabinoids also present in Cannabis sativa L. varieties. Furthermore, solid data on the potential benefits of acidic and varinic phytocannabinoids in a variety of diseases are already available. Mode of action of cannabigerol (CBG), cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), cannabidivarin (CBDV) and cannabigerivarin (CBGV) is, to the very least, partial.

Hypothesis/purpose: Cannabinoid CB or CB receptors, which belong to the G-protein-coupled receptor (GPCR) family, are important mediators of the action of those cannabinoids. Pure CBG, CBDA, CBGA, CBDV and CBGV from Cannabis sativa L. are differentially acting on CB or CB cannabinoid receptors.

Study Design: Determination of the affinity of phytocannabinoids for cannabinoid receptors and functional assessment of effects promoted by these compounds when interacting with cannabinoid receptors.

Methods: A heterologous system expressing the human versions of CB and/or CB receptors was used. Binding to membranes was measured using radioligands and binding to living cells using a homogenous time resolved fluorescence resonance energy transfer (HTRF) assay. Four different functional outputs were assayed: determination of cAMP levels and of extracellular-signal-related-kinase phosphorylation, label-free dynamic mass redistribution (DMR) and ß-arrestin recruitment.

Results: Affinity of cannabinoids depend on the ligand of reference and may be different in membranes and in living cells. All tested phytocannabinoids have agonist-like behavior but behaved as inverse-agonists in the presence of selective receptor agonists. CBGV displayed enhanced potency in many of the functional outputs. However, the most interesting result was a biased signaling that correlated with differential affinity, i.e. the overall results suggest that the binding mode of each ligand leads to specific receptor conformations underlying biased signaling outputs.

Conclusion: Results here reported and the recent elucidation of the three-dimensional structure of CB and CB receptors help understanding the mechanism of action that might be protective and the molecular drug-receptor interactions underlying biased signaling.
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http://dx.doi.org/10.1016/j.phrs.2020.104940DOI Listing
September 2020

Pharmacological potential of varinic-, minor-, and acidic phytocannabinoids.

Pharmacol Res 2020 May 13;158:104801. Epub 2020 May 13.

Plena Global Holdings Inc., Victoria, Canada.

While natural Δ-tetrahidrocannabinol (ΔTHC), cannabidiol (CBD), and their therapeutic potential have been extensively researched, some cannabinoids have been less extensively investigated. The present article compiles data from the literature that highlight the health benefits and therapeutic potential of lesser known phytocannabinoids, which we have divided into varinic, acidic, and "minor" (i.e., cannabinoids that are not present in high quantities in common varieties of Cannabis sativa L). A growing interest in these compounds, which are enriched in some cannabis varieties, has already resulted in enough preclinical information to show that they are promising therapeutic agents for a variety of diseases. Every phytocannabinoid has a "preferential" mechanism of action, and often targets the cannabinoid receptors, CB and/or CB. The recent resolution of the structure of cannabinoid receptors demonstrates the atypical nature of cannabinoid binding, and that different binding modes depend on the agonist or partial agonist/inverse agonist, which allows for differential signaling, even acting on the same cannabinoid receptor. In addition, other players and multiple signaling pathways may be targeted/engaged by phytocannabinoids, thereby expanding the mechanistic possibilities for therapeutic use.
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http://dx.doi.org/10.1016/j.phrs.2020.104801DOI Listing
May 2020

Adenosine A Receptor Antagonists Affects NMDA Glutamate Receptor Function. Potential to Address Neurodegeneration in Alzheimer's Disease.

Cells 2020 04 26;9(5). Epub 2020 Apr 26.

Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Instituto de Salud Carlos III, Valderrebollo, 5, 28031 Madrid, Spain.

(1) Background. -methyl d-aspartate (NMDA) ionotropic glutamate receptor (NMDAR), which is one of the main targets to combat Alzheimer's disease (AD), is expressed in both neurons and glial cells. The aim of this paper was to assess whether the adenosine A receptor (AR), which is a target in neurodegeneration, may affect NMDAR functionality. (2) Methods. Immuno-histo/cytochemical, biophysical, biochemical and signaling assays were performed in a heterologous cell expression system and in primary cultures of neurons and microglia (resting and activated) from control and the APP transgenic mice. (3) Results. On the one hand, NMDA and A receptors were able to physically interact forming complexes, mainly in microglia. Furthermore, the amount of complexes was markedly enhanced in activated microglia. On the other hand, the interaction resulted in a novel functional entity that displayed a cross-antagonism, that could be useful to prevent the exacerbation of NMDAR function by using AR antagonists. Interestingly, the amount of complexes was markedly higher in the hippocampal cells from the APP than from the control mice. In neurons, the number of complexes was lesser, probably due to NMDAR not interacting with the AR. However, the activation of the AR receptors resulted in higher NMDAR functionality in neurons, probably by indirect mechanisms. (4) Conclusions. AR antagonists such as istradefylline, which is already approved for Parkinson's disease (Nouriast in Japan and Nourianz in the US), have potential to afford neuroprotection in AD in a synergistic-like fashion. i.e., via both neurons and microglia.
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http://dx.doi.org/10.3390/cells9051075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290564PMC
April 2020

Microbiota and Other Preventive Strategies and Non-genetic Risk Factors in Parkinson's Disease.

Front Aging Neurosci 2020 12;12:12. Epub 2020 Mar 12.

Departamento de Morfología y Biología Celular, Facultad de Medicina, Universidad de Oviedo, Oviedo, Spain.

The exact cause of Parkinson's disease (PD), the second most prevalent neurodegenerative disease in modern societies, is still unknown. Many scientists point out that PD is caused by a complex interaction between different factors. Although the main risk factor is age, there are other influences, genetic and environmental, that individually or in combination may trigger neurodegenerative changes leading to PD. Nowadays, research remains focused on better understanding which environmental factors are related to the risk of developing PD and why. In line with the knowledge on evidence on exposures that prevent/delay PD onset or that impact on disease progression, the aims of this review were: (i) to comment on the non-genetic risk factors that mainly affect idiopathic PD; and (ii) to comment on seemingly reliable preventive interventions. We discuss both environmental factors that may affect the central nervous system (CNS) or the intestinal tract, and the likely mechanisms underlying noxious or protective actions. Knowledge on risk, protective factors, and mechanisms may help to envisage why nigral dopaminergic neurons are so vulnerable in PD and, eventually, to design new strategies for PD prevention and/or anti-PD therapy. This article reviews the variety of the known and suspected environmental factors, such as lifestyle, gut microbiota or pesticide exposition, and distinguishes between those that are harmful or beneficial for the PD acquisition or progression. In fact, the review covers one of the most novel players in the whole picture, and we address the role of microbiota on keeping a healthy CNS and/or on preventing the "side-effects" related to aging.
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http://dx.doi.org/10.3389/fnagi.2020.00012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7080700PMC
March 2020

DIMERBOW: exploring possible GPCR dimer interfaces.

Bioinformatics 2020 05;36(10):3271-3272

Laboratori de Medicina Computacional, Unitat de Bioestadistica, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra 08193, Spain.

Motivation: G protein-coupled receptors (GPCRs) can form homo-, heterodimers and larger order oligomers that exert different functions than monomers. The pharmacological potential of such complexes is hampered by the limited information available on the type of complex formed and its quaternary structure. Several GPCR structures in the Protein Data Bank display crystallographic interfaces potentially compatible with physiological interactions.

Results: Here, we present DIMERBOW, a database and web application aimed to visually browse the complete repertoire of potential GPCR dimers present in solved structures. The tool is suited to help finding the best possible structural template to model GPCR homomers.

Availability And Implementation: DIMERBOW is available at http://lmc.uab.es/dimerbow/.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btaa117DOI Listing
May 2020

Expression of Melatonin and Dopamine D Receptor Heteromers in Eye Ciliary Body Epithelial Cells and Negative Correlation with Ocular Hypertension.

Cells 2020 01 8;9(1). Epub 2020 Jan 8.

Centro de Investigación en Red, Enfermedades Neurodegenerativas, CiberNed, Instituto de Salud Carlos III, 28029 Madrid, Spain.

Background: Experiments in the late nineties showed an inverse relationship in the eye levels of melatonin and dopamine, thereby constituting an example of eye parameters that are prone to circadian variations. The underlying mechanisms are not known but these relevant molecules act via specific cell surface dopamine and melatonin receptors. This study investigated whether these receptors formed heteromers whose function impact on eye physiology. We performed biophysical assays to identify interactions in heterologous systems. Particular heteromer functionality was detected using Gi coupling, MAPK activation, and label-free assays. The expression of the heteroreceptor complexes was assessed using proximity ligation assays in cells producing the aqueous humor and human eye samples. Dopamine D receptors (DRs) were identified in eye ciliary body epithelial cells. We discovered heteromers formed by DR and either MT (MTR) or MT (MTR) melatonin receptors. Heteromerization led to the blockade of DR-Gi coupling and regulation of signaling to the MAPK pathway. Heteromer expression was negatively correlated with intraocular hypertension.

Conclusions: Heteromers likely mediate melatonin and dopamine actions in structures regulating intraocular pressure. Significant expression of DR-MTR and DR-MTR was associated with normotensive conditions, whereas expression diminished in a cell model of hypertension. A clear trend of expression reduction was observed in samples from glaucoma cases. The trend was marked but no statistical analysis was possible as the number of available eyes was 2.
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http://dx.doi.org/10.3390/cells9010152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7016594PMC
January 2020

Adreno-melatonin receptor complexes control ion homeostasis and intraocular pressure - their disruption contributes to hypertensive glaucoma.

Br J Pharmacol 2020 05 26;177(9):2090-2105. Epub 2020 Feb 26.

Department of Biochemistry and Molecular Biology, Faculty of Optics and Optometry, University Complutense of Madrid, Madrid, Spain.

Background And Purpose: Often, glaucoma presents with elevated eye hydrostatic pressure, which is regulated by endogenous melatonin. Phenylephrine increases cytoplasmic [Ca ], via α -adrenoceptor activation, that is detrimental in glaucoma. The aims of this study were (a) to elucidate the role of melatonin receptors in humour production and intraocular pressure (IOP) maintenance and (b) to identify glaucoma-relevant melatonin-adrenoceptor interactions.

Experimental Approach: Biophysical and proximity ligation assays were performed to identify interactions in heterologous expression systems, in cell lines and in human eyes. G /G /G signalling was investigated in these systems and in cells producing aqueous humour. IOP was determined in a mice model of glaucoma. Retinography and topically pharmacological treatment were performed in control and in glaucomatous mice.

Key Results: α -adreno- and melatonin receptors form functional complexes in which the C-terminal tail of the adrenoceptor plays a role. Remarkably, activation of α -adrenoceptors in these complexes did not lead to cytosolic Ca increases, suggesting G instead of G coupling is involved. The number of these complexes significantly decreased in models of glaucoma and, importantly, in human samples from glaucoma patients. This has led to hypothesize that melatonin, a hypotensive agent, plus blockade of α -adrenoceptors could normalize pressure in glaucoma. Remarkably, co-instillation of melatonin and prazosin, an α -adrenoceptor antagonist, resulted in long-term decreases in IOP in a well-established animal model of glaucoma.

Conclusions And Implications: The findings are instrumental to understand the physiological function of melatonin in the eye and its potential to address eye pathologies by targeting melatonin receptors and their complexes.
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http://dx.doi.org/10.1111/bph.14971DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7161553PMC
May 2020

Angiotensin type 2 receptors: Role in aging and neuroinflammation in the substantia nigra.

Brain Behav Immun 2020 07 19;87:256-271. Epub 2019 Dec 19.

Laboratory of Cellular and Molecular Neurobiology of Parkinson's Disease, Research Center for Molecular Medicine and Chronic Diseases (CIMUS), Dept. of Morphological Sciences, IDIS, University of Santiago de Compostela, Santiago de Compostela, Spain; Networking Research Center on Neurodegenerative Diseases (CIBERNED), Spain. Electronic address:

Overactivity of the angiotensin-type-1 receptor (AT1)/NADPH-oxidase axis enhances aging processes, neuroinflammation and neurodegeneration. The role of AT2 receptors in the above-mentioned AT1-related effects in the aged brain, particularly substantia nigra, was investigated in this study. In the nigra, we observed a progressive decrease in AT2 mRNA expression with aging, and AT2 deletion led to changes in spontaneous motor behavior, dopamine receptors, renin-angiotensin system, and pro-oxidative and pro-inflammatory markers similar to those observed in aged wild type (WT) mice. Both aged WT mice and young AT2 KO mice showed an increased AT1, decreased MAS receptor and increased angiotensinogen mRNA and/or protein expression, as well as upregulation of pro-oxidative and pro-inflammatory markers. In cultures of microglial cells, activation of AT2 receptors inhibited the LPS-induced increase in AT1 mRNA and protein expression and neuroinflammatory markers. Both in AT2 KO microglial cultures and microglia obtained from adult AT2 KO mice, an increase in AT1 mRNA expression was observed. In cultured dopaminergic neurons, AT2 activation down-regulated AT1 mRNA and protein, and dopaminergic neurons from adult AT2 KO mice showed upregulation of AT1 mRNA expression. Both in microglia and dopaminergic neurons the pathway AT2/nitric oxide/cyclic guanosine monophosphate mediates the regulation of the AT1 mRNA and protein expression through downregulation of the Sp1 transcription factor. MAS receptors are also involved in the regulation of AT1 mRNA and protein expression by AT2. The results suggest that an aging-related decrease in AT2 expression plays a major role in the aging-related AT1 overexpression and AT1-related pro-inflammatory pro-oxidative effects.
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http://dx.doi.org/10.1016/j.bbi.2019.12.011DOI Listing
July 2020