Adrenergic-melatonin heteroreceptor complexes are key in controlling ion homeostasis and intraocular eye pressure and their disruption contributes to hypertensive glaucoma

Abstract

<jats:title>Abstract</jats:title><jats:p>Melatonin regulates intraocular pressure (IOP) whose increase leads to glaucoma and eye nerve degeneration. Aiming at elucidating the role of melatonin receptors in humour production and IOP maintenance, we here demonstrate that glaucoma correlates with disassembly of α<jats:sub>1</jats:sub>-adrenergic/melatonin receptor functional units in cells producing the aqueous humour. Remarkably, α<jats:sub>1</jats:sub>-adrenoceptor-containing complexes do not coupled to the cognate Gq protein and, hence, phenylephrine activation of these receptors does not lead to Ca<jats:sup>2+</jats:sup> mobilization. Functional complexes are significantly decreased in models of glaucoma and, more importantly, in human samples of glaucoma patients (GP). In such glaucomatous conditions phenylephrine produces, via α<jats:sub>1</jats:sub>-adrenoceptor activation, an increase in cytoplasmic [Ca<jats:sup>2+</jats:sup>] that is detrimental in glaucoma. The results led to hypothesize that using melatonin, a hypotensive agent, plus blockade of α<jats:sub>1</jats:sub>-adrenergic receptors may normalize pressure in glaucoma. Remarkably, co-instillation of melatonin and prazosin, a α<jats:sub>1</jats:sub>-adrenergic receptor antagonist, results in long-term decreases in IOP in a well-established animal model of glaucoma. 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.</jats:p>

Expression of Melatonin and Dopamine D3 Receptor Heteromers in Eye Ciliary Body Epithelial Cells and Negative Correlation with Ocular Hypertension

“…Thus, activation of these receptors leads to a decrease in cytosolic cAMP levels. We have confirmed that this is the case in single transfected cells (Appendix A Figure A1B,C; see also our recent report [52]), thereby reproducing data reported in References [34,53]. HEK-293T cells expressing D 3 R or MT 1 and incubated with the selective agonists, 7-OH-PIPAT for D 3 and melatonin for MT 1 , led to significant decreases in the forskolin-induced cytosolic cAMP levels.…”

“…When cytosolic cAMP levels were analyzed in the cells treated with 0.5 µM forskolin, the D 3 R agonist 7-OH-PIPAT, in agreement with the heteromer's print identified in the HEK-293T cells, did not produce any effect (Figure 4A,D). Interestingly, and as previously described by Alkozi et al, [34], none of the melatonin receptor agonists, melatonin or IIK7, were able to decrease cAMP levels in these cells. Next, we analyzed ERK1/2 phosphorylation, which was significantly enhanced when 7-OH-PIPAT was added ( Figure 4B,E).…”

“…We wondered whether intraocular hypertension correlated with the altered expression of dopamine and melatonin heteroreceptor complexes. First of all, we took advantage of an in vitro model of hypertension, described in Reference [54], consisting of the activation of the transient Interestingly, and as previously described by Alkozi et al, [34], none of the melatonin receptor agonists, melatonin or IIK7, were able to decrease cAMP levels in these cells. Next, we analyzed ERK1/2 phosphorylation, which was significantly enhanced when 7-OH-PIPAT was added ( Figure 4B,E).…”

“…The MT 2 R also interacts directly with 5-hydroxytryptamine 5HT 2c receptors, the heteromer leading to biased agonism for the melatonin receptor ligand, agomelatine [33]. Although little is known about melatonin receptor heteromerization in the eye, a recent report has demonstrated the heteromerization of melatonin and α 1 -adrenergic receptors that alters canonical coupling as heteromerization impedes coupling to the cognate G proteins, Gi and Gq, respectively [26,34].…”

Specificity and nanomolar potency of melatonin on G-protein coupled melatonin MT1 and MT2 receptors expressed in HEK-293T human embryo kidney cells

“…In brief the MT1 receptor is expressed in the muscle cells and melatonin acts via Gq and not via Gi; however, the involvement of the receptors in the Gq-mediated effects is dubious as the conditions of the assays are not standard from a pharmacological point of view. In this regard, activation of melatonin receptors in a heterologous expression system does not lead to immediate Ca 2+ mobilization as it occurs in the case of other GPCRs that are coupled to Gq (31).…”