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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2020 Feb 26;177(9):2090–2105. doi: 10.1111/bph.14971

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

Hanan Awad Alkozi 1, Gemma Navarro 2,3, David Aguinaga 2,4, Irene Reyes‐Resina 4,6, Juan Sanchez‐Naves 5, Maria J Pérez de Lara 1, Rafael Franco 2,4,✉, Jesus Pintor 1,†,✉
PMCID: PMC7161553  PMID: 31901203

Abstract

Background and Purpose

Often, glaucoma presents with elevated eye hydrostatic pressure, which is regulated by endogenous melatonin. Phenylephrine increases cytoplasmic [Ca2+], via α1‐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. Gs/Gi/Gq 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

α1‐adreno‐ and melatonin receptors form functional complexes in which the C‐terminal tail of the adrenoceptor plays a role. Remarkably, activation of α1‐adrenoceptors in these complexes did not lead to cytosolic Ca2+ increases, suggesting Gs instead of Gq 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 α1‐adrenoceptors could normalize pressure in glaucoma. Remarkably, co‐instillation of melatonin and prazosin, an α1‐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.

Abbreviations

4PPDOT

cis‐4‐phenyl‐2‐propionamidotetralin (antagonist of MT2R)

AR

adrenoceptor

IIK7

N‐[2‐(2‐methoxy‐6H‐isoindolo[2,1‐a]indol‐11‐yl)ethyl]butanamide (MT2R selective agonist)

IOP

intraocular pressure

MT1R

melatonin MT1 receptor

MT2R

melatonin MT2 receptor

Rluc

Renilla luciferase

YFP

yellow fluorescence protein

α1A‐MT1Hets

α1A‐MT1 receptor heteromers

What is already known

  • Epinephrine, melatonin and adrenergic and melatonin receptors are relevant in the control of intraocular pressure.

What this study adds

  • Alpha‐adrenergic/melatonin receptor complexes uncouple from cognate G‐proteins. Detrimental Gq coupling appears upon complex disruption.

What is the clinical significance

  • Melatonin plus adrenoceptor antagonis prazosin serve for glaucoma therapy. Both compounds are available for human consumption.

1. INTRODUCTION

Glaucoma, a pathology characterized by visual field loss, is associated with optic nerve damage (Casson, Chidlow, Wood, Crowston, & Goldberg, 2012). The main risk factors are inter alia aging, genetic conditions and intraocular pressure (IOP; Quigley, 2011). Besides being the second cause of blindness in the world, 61 million people suffer from glaucoma and by 2020 the number may approach 80 million (Quigley & Broman, 2006).

Normotensive IOP in adults is approximately 16 mmHg. Ocular hypertension is diagnosed when the value exceeds 21 mmHg (Li, Huang, & Zhang, 2018). Persistent ocular hypertension results in damage of the optic disc, causing degeneration of ganglion cells. The relationship between ocular hypertension and glaucomatous pathology is a well‐known phenomenon (Sihota, Angmo, Ramaswamy, & Dada, 2018). However, despite the current therapeutic arsenal to decrease IOP, ocular hypertension is still the most important risk factor for optic nerve degeneration (Rossetti et al., 2015; Tamm, Braunger, & Fuchshofer, 2015).

In the healthy eye, the control of aqueous humour production is tightly controlled by hormones/neuromodulators, and aging leads to imbalance and increased hydrostatic pressure (Delamere, 2005). The role of https://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=4 expressed in the ciliary body seems to be opposite as either α‐agonists or β‐antagonists may decrease IOP (Kiuchi, Yoshitomi, & Gregory, 1992; Mittag, Tormay, Severin, & Podos, 1985; Naito, Izumi, Karita, & Tamai, 2001). Studies in different animal models indicated that https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=479, that is, the endogenous agonist, produces a reduction in IOP, which can be blocked by https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=503, a selective https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=22 antagonist (Funk, Wagner, & Rohen, 1992; Lee, 1958; Moroi, Hao, Inoue‐Matsuhisa, Pozdnyakov, & Sitaramayya, 2000). Both https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=479 and https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=503 bind the α1‐adrenoceptor, a GPCR whose cognate heterotrimeric protein is Gq (Alexander et al., 2019).

Anti‐glaucomatous compounds reduce aqueous humour formation by the ciliary body, thus leading to a decrease in hydrostatic pressure. To combat elevated IOP, parasympathomimetics, adrenoceptor antagonists, https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2597 inhibitors and/or prostaglandins (PGs) are all prescribed (Hommer, 2010; Lee & Higginbotham, 2005), although they may cause unfavourable side effects (Beckers, Schouten, Webers, van der Valk, & Hendrikse, 2008). Better and safer interventions include drug co‐administration as a way to reduce doses and the side effects of these drugs (Polo, Larrosa, Gomez, Pablo, & Honrubia, 2001).

Interest in https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=224 is emerging in ocular diseases as it modulates aqueous humour production in the ciliary body. Thus, this indoleamine has potential in the treatment of ocular hypertension, likely by acting at https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=287 and/or https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=288 receptors (Crooke, Colligris, & Pintor, 2012; Mediero, Alarma‐Estrany, & Pintor, 2009), which also belong to the superfamily of GPCRs and whose cognate protein is Gi (Alexander et al., 2019). Accordingly, activation of MT1 and MT2 receptors engages Gi that in turn inhibits AC and leads to decreasing cAMP levels and deactivation of PKA (Vanecek, 1998). Interestingly, melatonin receptors were reported to couple to different heterotrimeric G proteins, and even a third melatonin receptor was suggested. The exact reasons for pleiotropic signalling are not known and the hypothesized third receptor is not a melatonin receptor but the enzyme quinone reductase 2 (Emet et al., 2016; Mailliet et al., 2004; Pintor, Martin, Pelaez, Hoyle, & Peral, 2001; Shiu, Pang, Tam, & Yao, 2010; Tsitn, Wong, & Wong, 1996).

It is known that GPCRs form dimers/oligomers whose functionality is different from that of individual receptors. One of the most relevant discoveries related to GPCR heteromerization is their ability to couple to more than one G protein and/or a shift in G protein coupling (Franco et al., 2018). The first report on G protein coupling shift was probably that reported for dopamine https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=214/https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=215 receptors heteromers, whereas D1 couples to Gs and D2 to Gi, the heteromer couples to Gq (Hasbi et al., 2009; Rashid et al., 2007; Verma et al., 2010). Heteromers may consist of receptors for the same endogenous ligand or receptors for different endogenous ligands. Heteromerization of the two melatonin receptors has been already reported in 2013 as a mechanism to control photoreceptor function (Baba et al., 2013). In this study we wanted (a) to assess whether adrenoceptors, which are important in the control of IOP, may form heteromers with melatonin receptors and (b) to decipher whether those heteromers may be involved in the interplay between melatonin and adrenaline on IOP effects in both the healthy eye and the glaucomatous eye.

2. METHODS

To the best of our knowledge, this manuscript adheres to BJP instructions to authors and to the guidelines elsewhere detailed (Curtis et al., 2018). Studies were designed to generate groups of equal size, using randomization and blinded analysis. Immunological based assays were conducted in line with BJP guidelines detailed in Alexander et al. (2018).

2.1. Materials

The MT1 receptor antagonist https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1363, the MT2 receptor agonist N‐[2‐(2‐methoxy‐6H‐isoindolo[2,1‐a]indol‐11‐yl)ethyl]butanamide (https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1350) and cholera and pertussis toxins were purchased from Sigma‐Aldrich (St. Louis, MO, USA). The α1A receptor agonist https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=485 hydrochloride and antagonist prazosin hydrochloride, the MT1 receptor agonist melatonin, the MT2 receptor antagonist, cis‐4‐phenyl‐2‐propionamidotetralin (https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1358), GSK 1016790A, Ro‐105824 and https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5190 were purchased from Tocris Bioscience (Bristol, UK).

2.2. Human eye post‐mortem samples

Three eyes of healthy eye normotensive subjects and two of glaucoma patients came from donations managed by the Balearic Islands tissue bank foundation. After fixation with 4% paraformaldehyde, frontal sections (10 μm thick following the sagittal axis) were collected and stored at −20°C until use.

2.3. Animals and IOP measurements

Experiments of IOP measurements were performed using female C57BL/6J [RRID:IMSR_JAX:000664] (n = 12; control) and DBA/2J [RRID:IMSR_JAX:000671] (n = 12; glaucoma model) mice of different ages delivered by Charles River Lab. Institutional and regional ethic committees approved all procedures that included ARVO Statement for Ophthalmic and Vision Research. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny, Browne, Cuthill, Emerson, & Altman, 2010) and with the recommendations made by the British Journal of Pharmacology. Mice were anaesthetized by inhalation of isoflurane and IOP determined as previously described (Martínez‐Águila, Fonseca, Pérez de Lara, & Pintor, 2016).

2.4. Cells, fusion proteins, and expression vectors

The human non‐pigmented ciliary epithelial cell line, 59HCE, was kindly supplied by Dr. Coca‐Prados (Yale University) and grown in 5% heat‐inactivated FBS high‐glucose DMEM (Gibco/Invitrogen, Carlsbad, CA, USA). HEK‐293T cells [RRID:CVCL_0063] were grown in 5% FBS‐DMEM (Gibco/Invitrogen) as previously described (Martínez‐Pinilla et al., 2017; Navarro et al., 2018).

The human cDNAs for the MT1, MT2 and α1A receptors (full length or lacking its C‐terminal domain) cloned in pcDNA3.1 were amplified without their stop codons using sense and antisense primers harbouring either unique Hind III and BamH1 sites for MT1 and MT2 receptors or EcoRI and BamHI sites for α1A receptor. The fragments were then subcloned to be in frame with pRLuc‐N1 (PerkinElmer, Wellesley, MA, USA), pEYFP‐N1 (Clontech, Heidelberg, Germany) or pGFP2‐N3 (Clontech) placed on the C‐terminal end of the receptor to generate MT1YFP, MT1mutYFP, MT2YFP, MT2mutYFP, α1AGFP2, α1AYFP and α1AmutGFP2.

2.5. Transient transfection and sample preparation

Cells were transiently transfected with the cDNA encoding for each protein/fusion proteins and the polyethylenimine (PEI, Sigma‐Aldrich) method as previously described (Navarro et al., 2018). Cells were incubated for 4 hr with the corresponding cDNA mixed with PEI (5.47 mM in nitrogen residues) and 150‐mM NaCl in serum‐free medium. Finally, medium was exchanged by supplemented DMEM and maintained for 48 hr in a humid atmosphere of 5% CO2 at 37°C.

2.6. FRET assays

HEK‐293T cells were transiently co‐transfected with the plasmid cDNA corresponding to α1AGFP2 or α1AmutGFP2 (donor) and MT1YFP, MT1mutYFP, MT2YFP, or MT2mutYFP (acceptor) proteins using a ratio of donor to acceptor specified in figure legends. Cell suspension (20 μg of protein) was distributed into 96‐well microplates (black plates with a transparent bottom) and was read in a Fluostar Optima Fluorimeter (BMG Labtechnologies, Offenburg, Germany) equipped with a high‐energy xenon flash lamp, using a 10‐nm bandwidth excitation filter at 400 nm (393–403 nm) and 10‐nm bandwidth emission filters corresponding to 506‐ to 515‐nm filter (Ch 1) and 527‐ to 536‐nm filter (Ch 2). Gain settings were identical for all experiments to keep the relative contribution of the fluorophores to the detection channels constant for spectral unmixing. The contribution of the GFP variants, GFP2 and YFP alone, to the two detection channels (spectral signature) was measured in experiments with cells expressing only one of these proteins and normalized to the sum of the signal obtained in the two detection channels. FRET quantification was performed as described elsewhere (Navarro et al., 2010).

2.7. Intracellular calcium release

Cells were co‐transfected with the cDNA for the indicated receptors and 1 μg of GCaMP6 calcium sensor (Chen et al., 2013) using the PEI method. Twenty‐four hours after transfection, cells were suspended in Mg+2‐free Locke's buffer (pH 7.4; 154‐mM NaCl, 5.6‐mM KCl, 3.6‐mM NaHCO3, 2.3‐mM CaCl2, 5.6‐mM glucose, and 5‐mM HEPES) supplemented with 10‐μM https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=727; 150,000 cells per well were placed in 96‐well black, clear‐bottom microtitre plates, treated with receptor antagonists (10 min), and subsequently treated with receptor agonists. The fluorescence emission intensity of GCaMP6 was recorded 1 min after agonists addition at 515 nm upon excitation at 488 nm on the EnSpire® multimode plate reader for 150 s every 5 s and 100 flashes per well.

2.8. cAMP determinations

Two hours before initiating the experiment, transfected HEK‐293T cells or 59HCE cells medium was replaced by serum‐starved DMEM. Then cells were detached and resuspended in growing medium containing 50‐μM zardaverine. Cells were plated in 384‐well microplates (2,500 cells per well), pretreated (15 min) with the corresponding antagonists or vehicle, and stimulated with agonists (15 min) before adding 0.5 μM forskolin or vehicle (15 min). Readings were performed after 1‐hr incubation at 25°C. Homogeneous time‐resolved fluorescence energy transfer measures were performed using the Lance Ultra cAMP kit (PerkinElmer, Waltham, MA, USA). Fluorescence at 665 nm was analysed on a PHERAstar Flagship microplate reader equipped with a homogeneous time‐resolved fluorescence energy transfer optical module (BMG Labtechnologies).

2.9. Dynamic mass redistribution assays

Cell mass redistribution induced upon receptor activation was detected by illuminating with polychromatic light the underside of a biosensor and measuring the changes in the wavelength of the reflected monochromatic light that is a sensitive function of the index of refraction. The magnitude of this wavelength shift (in picometres) is directly proportional to the amount of dynamic mass redistribution (DMR). Transfected HEK‐293T cells or human 59HCE cells were seeded in 384‐well sensor microplates to obtain 70–80% confluent monolayers constituted by approximately 10,000 cells per well. Previous to the assay, cells were washed twice with assay buffer (HBSS with 20‐mM HEPES, pH 7.15) and incubated (2 hr) with assay buffer containing 0.1% DMSO (24°C, 30 μl per well). Hereafter, the sensor plate was scanned and a baseline optical signature was recorded for 10 min before adding 10 μl of the specific antagonists for 30 min followed by the addition of 10 μl of specific agonists. All test compounds were dissolved in assay buffer. Then dynamic mass redistribution responses were monitored for at least 5,000 s in an EnSpire® Multimode Plate Reader (PerkinElmer, Waltham, MA, USA). Results were analysed using EnSpire Workstation Software v 4.10.

2.10. β‐arrestin 2 recruitment assay

β‐arrestin recruitment was determined as previously described (Hinz et al., 2018; Navarro et al., 2018). Briefly, BRET experiments were performed in transfected HEK‐293T cells 48 hr after transfection with the cDNA corresponding to the indicated receptors fused to the YFP and 1‐μg cDNA corresponding to β‐arrestin 2Rluc. Forty‐eight hours after transfection, cells were adjusted to 20 μg of protein using a Bradford assay kit (Bio‐Rad, Munich, Germany) using BSA for standardization. To quantify protein‐YFP expression, fluorescence was read in a Mithras LB 940 multimode microplate reader (Berthold Technologies, Bad Wildbad, Germany) which allows the integration of the signals detected in the short‐wavelength filter at 485 nm and the long‐wavelength filter at 530 nm. Cells were distributed in 96‐well microplates (Corning 3600, white plates with white bottom, Sigma‐Aldrich). BRET readings corresponding to energy transfer between β‐arrestin 2Rluc and receptor‐YFP were collected 1 min after addition of 5‐μM coelenterazine h (Molecular Probes, Eugene, OR, USA). To quantify protein‐Rluc expression, luminescence readings were done 10 min after 5‐μM coelenterazine h addition. Net BRET is defined as [(long − wavelength emission)/(short − wavelength emission)] − Cf, where Cf corresponds to [(long − wavelength emission)/(short − wavelength emission)] for the donor construct expressed alone in the same experiment. BRET is expressed as milliBRET, mBU (net BRET × 1,000) units.

2.11. Immunofluorescence and in situ proximity ligation assays

Frozen eye sections from healthy and glaucoma subjects were rinsed in PBS 1X and permeabilized with PBS‐0.05% Tx‐100 solution for 30 min. After blocking, antibodies raised against MT1 (1:200, Santa Cruz, Dallas, TX, USA, Cat# sc‐13179) [RRID:AB_677236], MT2 (1:1,000, antibodies‐online GmbH, Aachen, Germany, Cat# ABIN122307) and α1‐adrenoceptor (1:500, Abcam, Cat# ab3462) [RRID:AB_2224924] receptors were used. The rest of the protocol was similar to that elsewhere described using ad hoc secondary reagents. Proximity ligation (PLA) allows the ex vivo detection of molecular interactions between two endogenous proteins. PLA probes were obtained by linkage of primary anti‐MT1 or MT2 receptor antibodies to PLUS oligonucleotide (DUO92009, Sigma‐Aldrich) and the α1A‐adrenoceptor antibody to MINUS oligonucleotide (DUO92010, Sigma‐Aldrich). Samples were analysed using confocal microscope (Zeiss LSM 5, Jena, Germany) at 40× magnification. The rest of the protocol was performed as described elsewhere, red spots were counted in each of the ROIs obtained in the nuclei images and data analysis was performed using specific PLA software (Navarro et al., 2018).

2.12. Immunocytochemistry

HEK‐293T cells seeded in coverslips were transfected with α1ARluc, MT1YFP, MT2YFP, α1ARluc and MT1YFP, or α1ARluc and MT2YFP (0.5‐μg cDNA each), fixed in 4% paraformaldehyde for 15 min, and washed twice with PBS containing 20‐mM glycine before permeabilization with PBS‐glycine containing 0.2% Triton X‐100 (5‐min incubation). Cells were treated for 1 hr with PBS containing 1% BSA. HEK‐293T cells were labelled with mouse anti‐Rluc antibody (1/100; Millipore, Darmstadt, Germany) and subsequently treated with Cy3 anti‐mouse (1/200; Jackson ImmunoResearch [red]) IgG (1 hr each). Nuclei were stained with Hoechst (1/100; Sigma‐Aldrich). Samples were washed several times and mounted with 30% Mowiol (Calbiochem). Samples were observed in a Leica SP2 confocal microscope (Leica Microsystems). The Immuno‐related procedures used comply with the recommendations made by the British Journal of Pharmacology.

2.13. Data analysis

With the exception of experiments with samples from human eyes (see Section 2.2), all data were obtained from at least five independent experiments and are expressed as the mean ± SEM. Two‐group comparisons were made by the unpaired Student's t‐test, and multiple comparisons were analysed by one‐way ANOVA followed by Bonferroni's post hoc test or two‐way ANOVA followed by Tukey's post hoc test. The normality of populations and homogeneity of variances were tested prior to ANOVA. Post hoc tests were run only if F achieved P < .05 and there was no significant variance inhomogeneity. Statistical analysis was undertaken only when each group size was at least n = 5, n being the number of independent variables (technical replicates were not treated as independent variables). Unequal group sizes were due to (a) different sources depending on the wide variety of experimental approaches, (b) need of increasing the n value for data reliability in some of the assays, (c) animal availability, and/or (d) economy of resources to fulfil the 3Rs (Replacement, Reduction, and Refinement) rule in experimentation with animals. Differences were considered significant when P ≤ .05. Statistical analyses were carried out with GraphPad Prism software version 5 (San Diego, CA, USA) [RRID:SCR_002798]. Outliers tests were not used; all data points (mean of replicates) were used for analysis. The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology.

2.14. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20 (Alexander et al., 2019).

3. RESULTS

3.1. Melatonin and α1A‐adrenoceptors expressed in 59HCE cells are uncoupled from their cognate G proteins

Melatonin acts via MT1 and MT2 receptors, which control Cl− efflux from ciliary body epithelial cells (Huete‐Toral, Crooke, Martínez‐Aguila, & Pintor, 2015). Interestingly, a α1A‐adrenoceptor antagonist, prazosin, blocks the effect of melatonin on reducing IOP (Dubocovich, 1995; Huete‐Toral et al., 2015; Pintor, Peláez, Hoyle, & Peral, 2003). Hence, we hypothesized that melatonin and α1A‐adrenoceptors) could interact.

Cognate heterotrimeric G proteins are Gi for melatonin and Gq/11 for α1A‐adrenoceptors (www.guidetopharacology.org; Alexander et al., 2019). However, it has also been reported that α1A‐adrenoceptors may couple to Gs (Martin et al., 2018). We then measured, in 59HCE ciliary body epithelial cells, cAMP levels and dynamic mass redistribution upon receptor activation. The effect of MT1 or MT2 agonists (melatonin and IIK7, respectively) did not result in any decrease in forskolin‐induced cytosolic [cAMP] (Figure 1a,b). By contrast, MT1 and MT2 agonists induced a significant increase in forskolin‐induced cytosolic [cAMP] (Figure 1a,b). When cells were challenged with the α1A agonist, phenylephrine, a remarkable increase in forskolin‐induced cytosolic [cAMP] was also observed. This adrenoceptor‐mediated increase in cAMP reflects a Gs coupling able to overcome the forskolin‐induced cAMP levels. The effect was specific as the α1 antagonist prazosin, abolished it. We also investigated the canonical coupling of α1A to Gq but failed to find any effect on Ca2+ levels in 59HCE cells treated with phenylephrine (Figure 1c). These results demonstrate that human ciliary epithelial cells express melatonin MT1, MT2 and α1A receptors that are not functionally coupled to their cognate G proteins. It should be noted that the concentration of forskolin chosen for these assays (500 nM) was submaximal, thus allowing detection of either Gi or Gs coupling. Also, when MT1 and MT2 agonists were analysed in 59HCE cells in the absence of forskolin, a significant increase in [cAMP] (over the basal level) was observed (data not shown).

Figure 1.

Figure 1

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Effect of melatonin receptor agonists and of phenylephrine in human 59HCE cells. (a, b, d, e) Effect of ligands (single or combined treatment) on 0.5 μM forskolin‐induced cAMP levels in the absence (a, b) or presence (d, e) of 10 ng·ml−1 pertussis (overnight) or 100 ng·ml−1 cholera (2 hr) toxins. Data are given in percentage (100% represents the forskolin effect); they are the mean ± SEM (n = 12, each in triplicates). One‐way ANOVA followed by Bonferroni's multiple comparison post hoc test was used for statistical analysis. *P < 0.05. (c) Time course of cytosolic calcium levels induced by receptor agonists or by GSK‐1016790A, an agonist of TRPV4 channels used for positive control. (f–i) Effect of ligands (single or combined treatment) on dynamic mass redistribution (DMR) in the absence (f, g) or presence (h, i) of pertussis or cholera toxins. Concentrations in the assays were 100 nM phenylephrine, 1 μM melatonin, 100 nM IIK7, 1 μM prazosin, 1 μM luzindole and 1 μM 4PPDOT

To confirm atypical G protein coupling, similar assays were performed in the presence of cholera toxin (CTX), which alters Gs‐mediated signalling, or pertussis toxin (PTX), which alters Gi‐mediated signalling. Results show that cholera toxin but not pertussis toxin inhibited the action of these agonists, unequivocally indicating Gs involvement (Figure 1d,e). Neither cholera toxin nor pertussis toxin by themselves produced any effect (data not shown). Interestingly, the presence of the α1A‐antagonist, prazosin, abolished the effect of melatonin agonists (melatonin on MT1 and IIK7 on MT2 receptors). Reciprocally, the MT1 antagonist, luzindol, and the selective MT2 antagonist, 4PPDOT, blocked the α1A‐receptor mediated signalling. Cross‐antagonism, i.e. the blockade of agonist‐mediated activation of one receptor in the heteromer by the antagonist of the partner receptor in the same complex, is an accepted heteromer print (Franco, Martínez‐Pinilla, Lanciego, & Navarro, 2016). Further evidence of cross‐antagonism was underscored using a label‐free technique consisting of detecting cell dynamic mass redistribution. Apart from the cross‐antagonism, the signal induced by any agonist was relatively high and the combination of phenylephrine and either an MT1 agonist, melatonin (Figure 1f), or an MT2 selective agonist, IIK7 (Figure 1g), caused a more robust dynamic mass redistribution response. When the experiments were carried out in the presence of cholera or pertussis toxins, responses were only abolished by cholera toxin (Figure 1h,i). Taken together, these results suggest that the crosstalk between melatonin and α1A receptors are solely due to direct interactions and Gs coupling of resulting complexes.

3.2. Atypical signalling in ciliary cells is due to α1A‐adreno/melatonin receptor complexes: Biophysical and signalling assays

To identify potential direct interactions, a FRET biophysical approach was used in HEK‐293 cells (Figure 2a). A saturable FRET curve was obtained in cells transfected with a constant [cDNA] for α1A‐GFP2 and increasing [cDNA] for MT1R‐YFP (FRETmax 49 mFU and FRET50 44; Figure 2b). Similar experiments using increasing amounts of [cDNA] of MT2R‐YFP also provided a saturable FRET curve with FRETmax and FRET50 values of, respectively, 168 mFU and 128 (Figure 2c). Thus, both melatonin receptors may form heteromers with α1A receptors (α1A‐MT1Hets and α1A‐MT2Hets). Receptor expression and function at the plasma membrane level and colocalization between α1A and melatonin receptors were confirmed by immunocytochemistry (Figure S1).

Figure 2.

Figure 2

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Dimerization of α1‐adrenoceptor and MT1 or MT2 receptors and signalling via adrenoceptors or via melatonin receptors in single‐transfected HEK‐293T cells. (a–c) Scheme of the FRET assays (a) and results of experiments performed in HEK‐293T cells transfected with a fixed amount of cDNA for the α1‐GFP2 fusion protein and increasing amounts of cDNA for either MT1‐YFP (b) or MT2‐YFP (c). Energy transfer data are given in milliFRET units (mFU). The remaining experiments were performed in HEK‐293T cells transfected with cDNAs for α1‐adrenoceptor, MT1, and MT2 receptors. (d–f) Time course of cytosolic calcium levels induced by receptor agonists (single or combined treatment; when indicated, a preincubation with antagonists was performed). (g–i) Effect of receptor ligands (single or combined treatment) on 0.5 μM forskolin‐induced cAMP levels. Data are given in percentage (100% represents the forskolin effect); they are the mean ± SEM (n = 12, each in triplicates). For negative control, a selective agonist for dopamine D4 receptor, RO‐105824 was used. One‐way ANOVA followed by Bonferroni's multiple comparison post hoc test was used for statistical analysis. *P < 0.05. (j–l) Effect of ligands (single or combined treatment) on dynamic mass redistribution (DMR). (m–o) BRET‐based measurements of the effect of receptor agonists (single or combined treatment) on β‐arrestin 2‐Rluc recruitment to every receptor‐YFP fusion protein. Data are given in milliBRET units (mBU); they are the mean ± SEM (n = 10, each in triplicates). One‐way ANOVA followed by Bonferroni's multiple comparison post hoc test was used for statistical analysis. *P < 0.05. Concentrations in the assays in panels (d) to (o) were 100 nM phenylephrine, 1 μM melatonin, 100 nM IIK7, 1 μM prazosin, 1 μM luzindole, 1 μM 4PPDOT and 100 nM RO‐105824

To know how heteromer formation is affecting signal transduction events, functionality was first addressed in single‐transfected HEK‐293T cells. Upon α1A activation, a robust increase in cytosolic [Ca2+] detected by the fluorescence due to calcium bound to an engineered GCaMP6 calmodulin sensor was obtained (Figure 2d). The agonist effect was inhibited by prazosin, but not by the melatonin antagonists, luzindole and 4PPDOT. In single‐transfected cells, either MT1 or MT2 receptor was not coupled to Gq/11 (Figure 2e,f) but to Gi, as their activation led to decreases in forskolin‐induced cAMP levels (Figure 2h,i). The results show that, indeed, when expressed individually, receptors couple to their cognate G proteins. However, α1A activation induced a significant decrease in forskolin‐induced cAMP levels. This result demonstrates that α1A can couple both Gq/11 and Gi proteins when individually expressed in a heterologous system (Figure 2g). Activation of receptors in single‐transfected cells also provided significant dynamic mass redistribution read‐outs (Figure 2j–l). Finally, BRET assays performed using β‐arrestin 2‐Rluc and receptors fused to YFP showed recruitment to α1A, MT1 and MT2 receptors when selective agonists were used (Figure 2m–o).

Remarkably, cAMP determination data in co‐transfected cells were similar to the results obtained in 59HCE cells; agonists did not decrease forskolin‐induced cAMP levels in cells expressing α1A‐MT1Hets or α1A‐MT2Hets. Also, cross‐antagonism was identified in co‐transfected cells, as melatonin receptor antagonists blocked phenylephrine‐induced signal and the α1A antagonist prazosin completely abolished the MT1 signal and slightly decreased the MT2 signal (Figure 3a,b). Oddly, there was a lack of Ca2+ responses when phenylephrine was added to cells expressing either α1A‐MT1Hets or α1A‐MT2Hets (Figure 3c,d). Analogies between 59HCE and co‐transfected HEK‐293T cells were further found in experiments with toxins; the combined effect of agonists was blocked by cholera toxin but not by pertussis toxin (Figure 3e,f). Also, matching results in 59HCE cells, a potentiation of the label‐free dynamic mass redistribution signal was found upon co‐activation, cholera toxin did block the effect and cross‐antagonism was detected (Figure 3g–j). These results indicate a lack of productive coupling of receptor heteromers with Gq/11 or Gi. Cross‐modulation was found in β‐arrestin 2 recruitment but with a particular feature; namely, both α1A‐receptor activation and activation of MT1 receptors recruited β‐arrestin to the α1A‐receptor, co‐activation resulting in a stronger signal (Figure 3k). Antagonists of the two receptors abolished recruitment induced by agonists (cross‐antagonism was, therefore, found). Similar results were obtained in cells co‐expressing α1A‐MT2 heteroreceptor complexes using ad hoc agonists/antagonists (Figure 3l).

Figure 3.

Figure 3

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Signalling via adrenoceptor/melatonin receptor functional units in a heterologous expression system. Experiments were performed in HEK‐293T cells transfected with cDNAs for full‐length α1A‐adrenoceptor and either MT1 receptor or MT2 receptor. (a, b, e, f) Effect of ligands (single or combined treatment) on 0.5 μM forskolin‐induced cAMP levels in the absence (a, b) or presence (e, f) of 10 ng·ml−1 pertussis (overnight) or 100 ng·ml−1 cholera (2 hr) toxins. Data are given in percentage (100% represents the forskolin effect); they are the mean ± SEM (n = 12, each in triplicates). One‐way ANOVA followed by Bonferroni's multiple comparison post hoc test was used for statistical analysis. *P < 0.05. (c, d) Lack of effect of receptor agonists in cytosolic calcium levels. The agonist of TRPV4 channel, GSK‐1016790A, did produce an effect (data not shown). (g–j) Effect of ligands (single or combined treatment) on dynamic mass redistribution (DMR) in the absence (g, h) or presence (i, j) of pertussis and cholera toxins. (k, l) Effect of receptor agonists (single or combined treatment) on β‐arrestin 2 recruitment to MT1‐YFP (k) or to MT2‐YFP (l). Data are given in mBU and are the mean ± SEM (n = 10, each in triplicates). One‐way ANOVA followed by Bonferroni's multiple comparison post hoc test was used for statistical analysis. *P < 0.05. Concentrations in the assays were 100‐nM phenylephrine, 1 μM melatonin, 100 nM IIK7, 1 μM prazosin, 1 μM luzindole and 1 μM 4PPDOT

3.3. Structural insights into the mechanism underlying the atypical signalling due to receptor complex formation

Since results obtained in HEK‐293T cells were virtually identical to those obtained in 59HCE cells, we assumed that heteromers functionally coupled to Gs occur in the 59HCE cell. Looking at the primary structure and membrane topological domains, we hypothesized that intracellular domains could be responsible of such differential G protein coupling. On the one hand, the area projected by a G protein on the membrane plane almost doubles that of a GPCR. On the other hand, the “clam‐shell like” opening of the small globular domain in Gα subunits, which occurs in every GTP/GDP exchange, has been structurally elucidated (Chung et al., 2011; Westfield et al., 2011). With this information and considering the structural possibilities for G protein–receptor complexes described elsewhere (Cordomí, Navarro, Aymerich, & Franco, 2015), we noticed that the long C‐terminal domain of the α1A‐adrenoceptor could lead to steric hindrance in the context of a complex formed by at least two receptors and G proteins (Rasmussen et al., 2011). It is predicted that the human α1A‐adrenoceptor C‐terminal domain contains 137 amino acids (http://www.uniprot.org/uniprot/P35348)) and that human MT1 and MT2 receptors have much shorter C‐terminal ends (http://www.uniprot.org/uniprot/P48039 and http://www.uniprot.org/uniprot/P49286, respectively). For the three receptors, cDNAs for fusion proteins lacking most of the C‐terminal cytoplasmic domain were obtained. Energy transfer assays showed interaction of α1A receptors with truncated melatonin receptors and of truncated α1A receptors with melatonin receptors (Figure 4a–d). Truncated receptors, when expressed individually, were functional (Figure 4e). Data from controls performed in cells co‐expressing combination of truncated and full‐length receptors are shown in Figure S2A–D. Functional assays provided clues on the atypical signalling mediated by heteromers. Phenylephrine did not mobilize Ca2+ in cells co‐expressing α1A receptors and any of the truncated melatonin receptors (Figure 4f,g). In contrast, activation of the truncated α1A receptors in the heteromeric contexts led to increases in cytosolic [Ca2+] (Figure 4h,i). In cAMP determination assays, we observed that truncated melatonin receptors interacting with full‐length α1A‐receptors, were still coupled to Gs (Figure 4j,k). In summary, the C‐terminal tail of the α1A‐receptor plays a relevant role in the functionality of the heteroreceptor functional units.

Figure 4.

Figure 4

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Dimerization of truncated versions of α1‐adrenoceptor, MT1 or MT2 receptor and signalling via receptor heteromers containing truncated receptors. Experiments were performed in HEK‐293T cells expressing the receptors indicated in the heading of each of the images. (a, b) FRET assays were performed in HEK‐293T cells transfected with a fixed amount of cDNA for full‐length α1‐GFP2 or the truncated α1‐GFP2 fusion proteins and increasing amounts of cDNA for, respectively, full‐length MT1‐YFP or truncated MT1‐YFP. (c, d) FRET assays were performed in HEK‐293T cells transfected with a fixed amount of cDNA for the truncated α1‐GFP2 or α1GFP2 fusion proteins and increasing amounts of cDNA for, respectively, MT2‐YFP or truncated MT2‐YFP. Energy transfer data in panels (a) to (d) are given in milliFRET units (mFU). Parameters were FRETmax = 43 ± 3 mFU; FRET50 = 25 ± 5 (a), FRETmax = 77 ± 9 mFU; FRET50 = 85 ± 11 (b), FRETmax = 100 ± 7 mFU; FRET50 = 70 ± 8 (c) and FRETmax = 85 ± 11 mFU; FRET50 = 137 ± 18 (d). (e, j, k) Effect of ligands (single or combined treatment) on 0.5 μM forskolin ‐induced cAMP levels in cells expressing truncated α1‐adrenoceptor, truncated MT1 or truncated MT2 receptors (e) and in cells co‐expressing α1‐adrenoceptor and truncated MT1 (j) or MT2 (k) receptors. Data are given in percentage (100% represents the forskolin effect); they are the mean ± SEM (n = 12, each in triplicates). One‐way ANOVA followed by Bonferroni's multiple comparison post hoc test was used for statistical analysis. *P < 0.05. (f–i) Time course of cytosolic calcium level induced by receptor agonists (single or combined treatment) in cells co‐expressing α1‐adrenoceptor and truncated MT1 (f), α1‐adrenoceptor and truncated MT2 (g), truncated α1‐adrenoceptor and MT1 (h), or truncated α1‐adrenocdeptor and MT2 (i) receptors. Concentrations in the assays were 100 nM phenylephrine, 1 μM melatonin, 100 nM IIK7, 1 μM prazosin, 1 μM luzindole and 1 μM 4PPDOT

3.4. The glaucomatous eye has reduced heteroreceptor complex expression and altered signalling

First of all, identification of α1A‐MT1Hets and α1A‐MT2Hets was achieved in healthy and glaucomatous conditions. The in situ PLA technique is instrumental to detect receptor–receptor interactions in a native system. Red clusters coming from PLA assays proved the occurrence of both α1A‐AR/MT1 receptor and α1A‐AR/MT2 receptor complexes in 59HCE cells. The analysis of the PLA labelling provided values of 65 ± 10 dots/nucleus in the case of the α1A‐MT1Hets and 73 ± 8 dots/nucleus in the case of the MT2/α1 heteromer. The percentage of cells that presented positive PLA was 56 ± 4 for α1A‐MT1Hets and 57 ± 5 for α1A‐MT2Hets (n = 150).

Does eye hypertension correlate with altered expression of melatonin–adrenoceptor complexes? We approached this question using cells subjected to stimulation of the transient receptor potential vanilloid 4 (TRPV4) channel. As previously reported (Alkozi, Franco, & Pintor, 2017; Alkozi & Pintor, 2015), activation of the channel mimics the ion fluxes that drive the increase in hydrostatic pressure that occurs in the hypertensive/glaucomatous eye. The application to 59HCE cells of the https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=510 agonist, https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4205, modified the PLA signal in a dose‐dependent manner. As shown in Figure 5a,b, the higher the concentration of GSK1016790A, the lower the PLA signal. Therefore, a reduction in the expression α1A‐MT1Hets or α1A‐MT2Hets happens when a glaucomatous condition is reproduced in a preclinical model (Figure 5c).

Figure 5.

Figure 5

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Identification of functional units in human 59HCE cells and in samples from normotensive and hypertensive human eyes. (a, b) Determination by PLA of α1‐adrenoceptor and either MT1 (a) or MT2 (b) receptor complexes in 59HCE non‐pigmented ciliary body epithelial cells treated with increasing concentrations of the TRPV4 agonist, GSK1016790A. (c) Quantification of clusters for α1‐adrenoceptor and either MT1 (blue) receptor or MT2 (red) receptor. Data are the mean ± SEM (n = 5). *P < 0.05, using unpaired Student's t‐test. Values for negative control were, respectively, 5 ± 1 and 2 ± 1 dots/nucleus. (d–i) Immunolocalization of MT1 (d), MT2 (e), or α1‐adrenoceptor (f) receptors in the ciliary body of healthy controls. Immunolocalization of MT1 (g), MT2 (h), or α1‐adrenoceptor (i) receptors in the ciliary body of glaucoma patients. Quantitation (j) of results comparing patients and controls using fluorescence values taken in identical experimental conditions. Data, given in percentage (100% given to values from controls), are the mean ± SEM (n = 5). Statistical analysis could not be performed as three was the number of healthy eyes and two was the number of glaucomatous eyes; it should be noted that the within‐groups results were similar. (k–o) Determination by PLA of α1‐adrenoceptor and either MT1 (k) or MT2 (m) receptor complexes in the ciliary body of age‐matched IOP normotensive individuals. Determination by PLA of α1‐adrenoceptor and either MT1 (l) or MT2 (n) receptor complexes in the ciliary body of glaucoma patients. Quantitation (o) of clusters comparing data in glaucoma patients and age‐matched controls using fluorescence values taken in identical experimental conditions. Data, given in percentage (100% given to values from controls), are the mean ± SEM. Statistical analysis could not be performed as three was the number of healthy eyes and two was the number of glaucomatous eyes; it should be noted that the within‐groups results were similar

Exploratory experiments on heteromer expression were performed in post‐mortem samples obtained from glaucoma patients and age‐matched controls. Due to the limited availability of these unique samples that in part reflects ethical issues, we had access to three control (healthy) and two glaucomatous eyes. Accordingly, statistical analysis could not be performed on the results that are described below. In the eye from healthy donors, the presence of melatonin MT1, MT2, and α1A receptors was confirmed by immunoreactivity across the ciliary body (Figure 5d–f). A strong labelling for the MT1 was present in non‐pigmented epithelial cells, while the labelling for the MT2 was observed in the basal membrane of the non‐pigmented epithelium (Figure 5d,e). Melatonin receptors were not found in the stroma. Concerning the α1A‐adrenoceptor, a positive labelling was observed in the pigmented and non‐pigmented epithelial cells as well as in the stroma (Figure 5f). Similar immunohistochemical studies carried out in samples obtained from glaucoma patients showed altered receptor expression (Figure 5g–i). The expression of the α1‐ adrenoceptor showed a trend to increase (Figure 5j). In sharp contrast, immunoreactivity for melatonin receptors was markedly reduced, 81% and 54% for MT1 and MT2 receptors, respectively. For MT1/α1A and MT2/α1A receptor pairs, a marked PLA positive labelling was observed in control samples (Figure 5k,m). Remarkably, when PLA was performed in samples from glaucoma patients, the reduction of heteromers was 88% in the case of the MT1/α1A and 90% in the case of the MT2/α1A heteromers (Figure 5l,n,o). These results obtained in human samples match the results obtained in 59HCE cells treated with a TRPV4 activator to mimic a glaucoma‐like condition.

3.5. A novel therapeutic approach to combat glaucoma

The above described results show that the coupling of α1A‐adrenoceptors to Gq and the subsequent Ca2+ signalling seems detrimental to eye physiology and markedly contributes to intraocular hypertension. Accordingly, an antagonist of α1A‐adrenoceptors would be beneficial as a blocker of calcium production and of Ca2+‐regulated chloride channels (Fleischhauer, Mitchell, Peterson‐Yantorno, Coca‐Prados, & Civan, 2001). To test the hypothesis, we moved to a well‐established murine model of glaucoma (Pérez de Lara et al., 2014). Three‐month‐old DBA/2J mice have normotensive eyes and physiological levels of melatonin. Retinal electrophysiology parameters were undistinguishable from those in the control mouse (C57BL/6 background; Figure 6a–c), however prazosin antagonized the hypotensive effect of melatonin (Figure 6d). Nine‐month‐old mice display a full glaucoma‐like pathology (Figure 6a–c). At 9 months of age and despite elevated levels of melatonin (Figure 6b), the glaucomatous eye of DBA/2J mice was sensitive to the hypotensive effect of exogenously added melatonin, which reduced the IOP from 16.6 ± 0.6 to 11.8 ± 0.4 mmHg. Remarkably, prazosin enhanced melatonin hypotensive action (to 9.0 ± 0.5 mmHg) instead of antagonizing it (Figure 6f). Indeed, it was consistently found that prazosin did revert the effect of melatonin in control mice, while it significantly enhanced it in the 12‐month‐old DBA/2J mouse (Figure S2E–H). Moreover, the effect of prazosin lasted more than 6 hr, thus indicating a very appropriate therapeutic time window. These results open a new and easy‐to‐implement anti‐glaucoma treatment, consisting of melatonin/prazosin co‐administration, with few expectable side effects.

Figure 6.

Figure 6

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IOP‐lowering intervention in the DBA/2J mice. (a) Intraocular pressure (IOP) values of control C57BL/6J and DBA/2J mice at 3 and 9 months of age. Data are the mean ± SEM (n = 12). *P < .05 (Wilcoxon's test for paired samples). (b) Melatonin concentrations in the aqueous humour of C57BL/6J and DBA/2J mice at 3 and 9 months of age. Data are the mean ± SEM (n = 6). *P < .05 (Wilcoxon's test for paired samples). (c) Electroretinogram in C57BL/6J (blue) and DBA/2J (red) mice. Positive scotopic threshold response (pSTR) amplitude was significantly reduced between 3 and 9 months of age (P < .05). (d, e) Time course of the effect on IOP in 3 (d) or 9 (e) month‐old DBA/2J mice after the instillation of melatonin ± prazosin. Data are the mean ± SEM (n = 5)

4. DISCUSSION

Current therapy of glaucoma addresses symptoms by interventions which are neither efficacious in all patients nor absent of adverse events. PG analogues have been approved for use by glaucoma patients because they reduce IOP but by unknown mechanisms (Lanza et al., 2018; Sanford, 2014; Weinreb et al., 2015). Antagonist of β‐adrenoceptors or drugs able to engage α‐adrenoceptors are in the portfolio; however, the rationale behind the hypotensive action of β‐blockers and α‐receptor agonists is unknown.

Melatonin has become popular and is even available online. Its main potential is in sleep induction although other properties have been suggested. Already in 1993, Serino, D'Istria, and Monteleone (1993) described the production of melatonin in the pineal gland; in the retina it was shown that it is obtained by methylation of N‐acetyl 5‐hydroxytryptamine (serotonin) (Hardeland, 2010). Interestingly, melatonin decreases IOP, but the significant decrease in the aqueous humour of glaucoma patients is not enough to normalize IOP (Alarma‐Estrany, Crooke, Peral, & Pintor, 2007; Alkozi, Sánchez‐Naves, et al., 2017). A complete characterization of melatonin receptors has not been completed due to their atypical pharmacological and unfulfilled suspicions of the existence of a third receptor. As receptor heteromers display particular properties, we reasoned that atypical data, including pleiotropic signalling, could be due to interactions with other GPCRs (Ferré et al., 2009). Here, we selected the α1A‐adreceptor because its activation leads to increases in cytosolic [Ca2+] that in turn control ion fluxes in the ciliary body. From our data, the first relevant finding is that MT1 and MT2 receptors may directly interact with the α1A‐adrenoceptor.

The role of adrenaline in the physiology of the eye has been known since 1970 (Drance & Ross, 1970; Zalta, Shock, Stone, & Petursson, 1983). Interestingly, the lack of coupling of receptors for adrenaline to Gq/11 in the ciliary body (Figure 1), both intriguing and relevant, is due to α1A‐adrenoceptors forming heteromers with melatonin receptors. Remarkably, the glaucomatous eye expresses few functional units and the signalling mediated by Gq‐coupled α1A‐adrenoceptors negatively impacts on IOP. Our results predicted that the effect of melatonin would be enhanced by blockade of Gq‐coupled α1A‐adrenoceptors by prazosin. Indeed, combination of the two compounds led to normalize IOP in a well‐established model of glaucoma and the effect was not transient but lasted for 6 hr. The long experience using prazosin as antihypertensive indicates that it is very safe and does not display some of the serious side effects of other compounds (Brogden, Heel, Speight, & Avery, 1977).

Our results provide an explanation for the pleiotropic signalling attributed to melatonin receptors and to the reported differential coupling to G proteins depending on the experimental system (Tsitn et al., 1996). It should be noted that MT1 and MT2 receptor heteromers still couple to Gi (Ayoub, Levoye, Delagrange, & Jockers, 2004) and, therefore, the effects that are mediated by this heteromer are not yet fully elucidated from a molecular point of view. In the interaction reported in this paper, melatonin alone does not increase the concentration of intracellular calcium, however melatonin via MT1 receptors is able to mobilize Ca2+ and increase PI hydrolysis in intestinal smooth muscle cells (Ahmed et al., 2013). Whether this atypical effect, seemingly direct Gq coupling, is due to heteromerization or to an alternative mechanism is, at present, unknown. From a mechanistic point of view, the C‐terminal domain of the α1A‐adrenoceptor is relevant for the interaction and for the shift of heterotrimeric G protein in the adrenoceptor–melatonin heteroreceptor complexes. In fact, the lack of the C tail favours Gq coupling, while it is likely that it intermingles with partner melatonin receptors to impede Gq coupling. One few melatonin‐receptor‐containing melatonin receptors, is that formed by https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=107, an orphan GPCR with a long C‐terminal domain and https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=34. Interestingly, in such heteromer, there is a negative crosstalk that is abolished by removing the C‐terminal end of GPR50 (Lique Levoye et al., 2006). Therefore, it seems that melatonin receptors interacting with GPCRs with long C‐terminal domains could lead to a shift in G protein coupling. To confirm this prediction more experimental work is needed.

In summary, we have discovered a trend in glaucoma consisting of the disruption of complexes formed by adrenoceptors and melatonin receptors. Such a trend found in animal models of the disease and in samples from human eye (from glaucoma patients and age‐matched controls) makes ineffective the huge 3.2‐fold increase in the concentration of melatonin in the glaucomatous eye. The physiological functional unit is coupled to a Gs protein, whereas the disassembly leads to α1A‐adrenoceptor coupled to Gq and to melatonin receptors coupled to Gi. Increases in calcium via Gq and decreases of cAMP via Gi establish a vicious circle that negatively impacts on the ion channels controlling IOP. The mechanism consisting of allosteric interaction and shift of G protein coupling is quite noteworthy and explains one of our earlier finds in the laboratory, which is included in this paper, namely, the lack of calcium ion mobilization by phenylephrine in cells expressing α1A‐adrenoceptors (Figure 1c). Together, our findings provide a better understanding of the ciliary body physiology, completing a preclinical translational research addressed to combat glaucoma. Remarkably, a therapeutic strategy resulting from combining melatonin, sold as a supplement and lacking collateral effects even at high doses in the eye (Rosenstein et al., 2010; Sánchez‐Barceló, Mediavilla, Tan, & Reiter, 2010), and prazosin, approved for the therapy of blood hypertension (Brogden et al., 1977; Mallorga, Buisson, & Sugrue, 1988; Singleton et al., 1989; Torvik & Madsbu, 1986), could readily enter into clinical trials to assay for safety and efficacy in humans with ocular hypertension.

AUTHOR CONTRIBUTIONS

H.A.A., G.N., and I.R.‐R. participated in the design and performance of many of the experiments and analysed the results; it is considered that their contribution was similar. J.S.‐N. was instrumental in obtaining and providing human samples for this study. M.J.P.d.L. performed IOP measurements. D.A. and I.R.‐R. participated in immunohistochemistry and PLA assays. R.F. and J.P. designed and supervised the work; it is considered that their contribution was similar. H.A.A., G.N., R.F., and J.P. actively participated in writing and editing. All authors have edited the paper and have received a copy of the final version.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for https://bpspubs.onlinelibrary.wiley.com/doi/abs/10.1111/bph.14207, and https://bpspubs.onlinelibrary.wiley.com/doi/abs/10.1111/bph.14206, and as recommended by funding agencies, publishers, and other organizations engaged with supporting research.

Supporting information

Figure S1 Human adrenoceptor α1A colocalize with melatonin MT1 and MT2 receptors at the plasma membrane level in a heterologous expression system. Panels A‐E: Immunocytochemistry assays were performed in HEK‐293T cells expressing α1ARluc, MT1YFP, MT2YFP, α1ARluc and MT1YFP or α1ARluc and MT2YFP (0.5 μg cDNA each). Receptors fused to YFP were detected by its own yellow fluorescence (green), and α1ARluc was detected by a mouse anti‐Rluc antibody and a Cy3 anti‐mouse secondary antibody (red). Colocalization is shown in yellow. Cell nuclei were stained with Hoechst (blue). Scale bar: 5 μm. Panel F: cAMP assays were developed in HEK‐293T cells expressing α1‐adrenoceptor, α1‐adrenoceptor‐GFP2, MT1 receptor, MT1receptor‐YFP, MT2 receptor or MT2 receptor‐YFP. Effect of agonists was assayed over 0.5 μM forskolin‐induced cAMP levels. Data are given in percentage (100% represents the forskolin effect); they are the mean ± SEM (n = 8, each in triplicates). One‐way ANOVA followed by Bonferroni's multiple comparison post‐hoc test were used for statistical analysis. *P < 0.05

Click here for additional data file. (6.4MB, tif)

Figure S2 Panels A‐D: Effect of ligands (single or combined treatment) on 0.5 μM forskolin‐induced cAMP levels in cells co‐expressing truncated α1‐adrenoceptor and either full‐length MT1 (A), truncated MT1 (B), full‐length MT2 (C) or truncated MT2 (D) receptors. Data are given in percentage (100% represents the forskolin effect); they are the mean ± SEM (n = 12, each in triplicates). One‐way ANOVA followed by Bonferroni's multiple comparison post‐hoc test were used for statistical analysis. *P < 0.05. Concentrations in the assays were: 100 nM phenylephrine, 1 μM melatonin, 100 nM IIK7, 1 μM prazosin, 1 μM luzindole and 1 μM 4PPDOT. Panels E‐H. Differential effect of receptor ligands on IOP in C57BL/6J and DBA/2J mice. Effect on IOP of ligands (single or combined treatment) distilled into the eye of C57BL/6J (E, G) or 12‐month‐old DBA/2J (F, H) mice. Data are the mean ± SEM (n = 5 followed by Tukey post‐hoc test) *P < 0.05 versus control (two‐way ANOVA with Tukey post‐test, n = 4)

Click here for additional data file. (1.6MB, tif)

ACKNOWLEDGEMENTS

In memoriam of our co‐author Prof. Jesús (Suso) Pintor, astounding as ascientist and yet a good person. This work was supported by grants from Spanish Ministerio de Economía y Competitividad (MINECO) Refs SAF2013‐44416‐R, SAF2016‐77084‐R, and BFU2015‐64405‐R and from Spanish Ministerio de Sanidad Refs RETICS RD12/0034/0003 and RD16/0008/0017. Hanan A. Alkozi is a fellowship holder of Saudi Arabia government. MINECO grants may include EU FEDER funds. The “NBM” Molecular Neurobiology Laboratory of the University of Barcelona is considered of excellence (“grup consolidat”) by the regional Catalonian Government, which neither provides funds to the consolidated laboratory nor to perform the research here reported.

Alkozi HA, Navarro G, Aguinaga D, et al. Adreno–melatonin receptor complexes control ion homeostasis and intraocular pressure ‐ their disruption contributes to hypertensive glaucoma. Br J Pharmacol. 2020;177:2090–2105. 10.1111/bph.14971

A first preprint of this paper was deposited in BioRxiv (https://doi.org/10.1101/636688).

Hanan Awad Alkozi, Gemma Navarro, Rafael Franco, and Jesus Pintor have equal contribution.

Contributor Information

Rafael Franco, Email: rfranco@ub.edu.

Jesus Pintor, Email: jpintor@ucm.es.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1 Human adrenoceptor α1A colocalize with melatonin MT1 and MT2 receptors at the plasma membrane level in a heterologous expression system. Panels A‐E: Immunocytochemistry assays were performed in HEK‐293T cells expressing α1ARluc, MT1YFP, MT2YFP, α1ARluc and MT1YFP or α1ARluc and MT2YFP (0.5 μg cDNA each). Receptors fused to YFP were detected by its own yellow fluorescence (green), and α1ARluc was detected by a mouse anti‐Rluc antibody and a Cy3 anti‐mouse secondary antibody (red). Colocalization is shown in yellow. Cell nuclei were stained with Hoechst (blue). Scale bar: 5 μm. Panel F: cAMP assays were developed in HEK‐293T cells expressing α1‐adrenoceptor, α1‐adrenoceptor‐GFP2, MT1 receptor, MT1receptor‐YFP, MT2 receptor or MT2 receptor‐YFP. Effect of agonists was assayed over 0.5 μM forskolin‐induced cAMP levels. Data are given in percentage (100% represents the forskolin effect); they are the mean ± SEM (n = 8, each in triplicates). One‐way ANOVA followed by Bonferroni's multiple comparison post‐hoc test were used for statistical analysis. *P < 0.05

Click here for additional data file. (6.4MB, tif)

Figure S2 Panels A‐D: Effect of ligands (single or combined treatment) on 0.5 μM forskolin‐induced cAMP levels in cells co‐expressing truncated α1‐adrenoceptor and either full‐length MT1 (A), truncated MT1 (B), full‐length MT2 (C) or truncated MT2 (D) receptors. Data are given in percentage (100% represents the forskolin effect); they are the mean ± SEM (n = 12, each in triplicates). One‐way ANOVA followed by Bonferroni's multiple comparison post‐hoc test were used for statistical analysis. *P < 0.05. Concentrations in the assays were: 100 nM phenylephrine, 1 μM melatonin, 100 nM IIK7, 1 μM prazosin, 1 μM luzindole and 1 μM 4PPDOT. Panels E‐H. Differential effect of receptor ligands on IOP in C57BL/6J and DBA/2J mice. Effect on IOP of ligands (single or combined treatment) distilled into the eye of C57BL/6J (E, G) or 12‐month‐old DBA/2J (F, H) mice. Data are the mean ± SEM (n = 5 followed by Tukey post‐hoc test) *P < 0.05 versus control (two‐way ANOVA with Tukey post‐test, n = 4)

Click here for additional data file. (1.6MB, tif)

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