Original article
Regulation of cardiac nitric oxide signaling by nuclear β-adrenergic and endothelin receptors

https://doi.org/10.1016/j.yjmcc.2013.05.003Get rights and content

Highlights

  • Isoproterenol and endothelin-1 activate NO production in isolated cardiac nuclei.

  • Nuclear b3AR, but not b1AR, activated NOS.

  • L-NAME blocked NOS activation by isoproterenol and endothelin-1.

  • Inhibition of NOS or PKG prevented isoproterenol-induced transcription initiation.

  • Caged isoproterenol and endothelin-1 analogs activated NOS in intact cardiomyocytes.

Abstract

At the cell surface, βARs and endothelin receptors can regulate nitric oxide (NO) production. β-adrenergic receptors (βARs) and type B endothelin receptors (ETB) are present in cardiac nuclear membranes and regulate transcription. The present study investigated the role of the NO pathway in the regulation of gene transcription by these nuclear G protein-coupled receptors. Nitric oxide production and transcription initiation were measured in nuclei isolated from the adult rat heart. The cell-permeable fluorescent dye 4,5-diaminofluorescein diacetate (DAF2 DA) was used to provide a direct assessment of nitric oxide release. Both isoproterenol and endothelin increased NO production in isolated nuclei. Furthermore, a β3AR-selective agonist, BRL 37344, increased NO synthesis whereas the β1AR-selective agonist xamoterol did not. Isoproterenol increased, whereas ET-1 reduced, de novo transcription. The NO synthase inhibitor l-NAME prevented isoproterenol from increasing either NO production or de novo transcription. l-NAME also blocked ET-1-induced NO-production but did not alter the suppression of transcription initiation by ET-1. Inhibition of the cGMP-dependent protein kinase (PKG) using KT5823 also blocked the ability of isoproterenol to increase transcription initiation. Furthermore, immunoblotting revealed eNOS, but not nNOS, in isolated nuclei. Finally, caged, cell-permeable isoproterenol and endothelin-1 analogs were used to selectively activate intracellular β-adrenergic and endothelin receptors in intact adult cardiomyocytes. Intracellular release of caged ET-1 or isoproterenol analogs increased NO production in intact adult cardiomyocytes. Hence, activation of the NO synthase/guanylyl cyclase/PKG pathway is necessary for nuclear β3ARs to increase de novo transcription. Furthermore, we have demonstrated the potential utility of caged receptor ligands in selectively modulating signaling via endogenous intracellular G protein-coupled receptors.

Introduction

The G protein-coupled receptor (GPCR)7 superfamily consists of a large group of seven transmembrane domain-containing receptors that signal via heterotrimeric G proteins. GPCRs modulate a wide range of downstream effectors and, as a result, regulate multiple cellular functions in cardiomyocytes. β-Adrenergic receptors (βARs) and endothelin receptors (ETRs) are two such members of the GPCR superfamily, both of which are expressed in the myocardium. Recently, potential roles for these receptors when localized to intracellular compartments such as the nuclear membrane have been identified under both physiological and pathological conditions (reviewed in [1], [2]). We have shown that ETB, β1AR and β3AR are present on the nuclear membrane in adult cardiomyocytes [3], [4]. In addition, several of their downstream effectors have also been identified at the level of the nucleus or nuclear membrane [5], [6]. These receptors have been shown to bind ligand, couple to effectors, and regulate gene expression in isolated nuclei, with the βARs having a stimulatory effect on transcription initiation, whereas ETB activation is inhibitory [7]. The precise signaling pathways involved in the regulation of transcriptional initiation by GPCRs in the nuclear membrane have not been clearly defined and require further study.

Nitric oxide (NO) is an important signaling molecule involved in many physiological processes. NO is produced from the amino acid l-arginine via the action of NO synthases (NOS, [8]). There are three NOS subtypes: endothelial (eNOS), neuronal (nNOS) and inducible (iNOS) [8]. All three NOS subtypes are expressed in cardiomyocytes and play roles in cardiac physiology and pathology [9], [10]. In fact, NO has been linked to a wide variety of effects in both the heart and vasculature, from the regulation of vascular tone and myocardial contractility to calcium handling and apoptosis [8], [11]. Regulation of NOS activity is complex, involving several mechanisms mediated by calcium, protein kinases, and NO levels [12], [13]. The exact effect exerted by NO however, appears to depend on the NOS isoform being activated, as well as its subcellular localization and mode of action [11], [14]. NO exerts its effects via modulation of guanylyl cyclase activity leading to increases in cyclic guanosine 3′,5′-monophosphate (cGMP) levels and the subsequent activation of protein kinase G (PKG), but has also been shown to signal in a guanylyl cyclase-independent manner [15]. NO has also been implicated in the regulation of gene expression through the transcriptional regulator nuclear factor κB (NF-κB) [15], [16]. In addition, recent findings have also demonstrated that the PI3K/PKB pathway, which is activated by nuclear βARs [7], is capable of activating both eNOS and iNOS, leading to the stimulation of NO production [17], [18]. Moreover, iNOS upregulation in the nucleus appears to be linked to Gαi, the protein kinase ERK1/2, and potentially eNOS as well [19], [20], [21]. Furthermore, a link has also been established between NO production and both ETA and ETB [22] as well as the βARs [10], [23] localized at the cell surface. Additionally recent evidence has also shown a role for NO in the nucleus, where it appears to modulate calcium homeostasis and is also potentially regulated by ET-1 [20], [24].

Given the involvement of NO in the regulation of cardiac function, and its established link with both the ETRs and βARs, we wished to ascertain whether the NO pathway was involved in the regulation of gene expression observed following the stimulation of nuclear ETRs and βARs, and to identify which components of NO signaling might be implicated. Toward this end, we used a pharmacologic approach to study NO production in both isolated nuclei and intact cardiomyocytes following treatment with various agonists and inhibitors. Further, we demonstrate the potential utility of caged receptor ligands in selectively modulating nuclear signaling via GPCRs.

Section snippets

Materials

Triton X-100 (TX-100), leupeptin, PMSF and DNase I were from Roche Applied Science (Laval, Quebec). Isoproterenol, BRL 37344, CGP20712, ICI118551, SR59230A, 8-bromo-cGMP, Rp-8-Br-PET-cGMPS (Rp-8-Br) and KT5823 were from Tocris (Ellisville, MO). Endothelin-1 (ET-1) was from Peninsula Labs (Torrance, CA). Pertussis toxin (PTX), xamoterol, forskolin, alprenolol, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) and l-NAME, were from Sigma-Aldrich (Mississauga, Ontario). Triciribine,

Measurement of NO production

The link between plasma membrane GPCR signaling and nitric oxide (NO) production has been well characterized for both the ETB [24], [32] and the βARs, including the β3AR [8], [10]. Hence, given the presence of these receptors on the nuclear membrane, the recapitulation of cell surface signaling pathways in the nucleus (reviewed in [1], [2]) and the demonstrated effects of certain nuclear prostaglandin E2, bradykinin, lysophosphatidic acid type-1 receptors on iNOS and eNOS expression in

Discussion

We have demonstrated that the ETB and βAR in the nuclear membrane regulate NO production in isolated cardiac nuclei and more importantly, in intact cardiomyocytes. Using a NO-sensitive fluorescent dye, DAF-2, we observed an increase in NO production following treatment with both ISO and ET-1. In addition, pre-treatment with the NOS inhibitor l-NAME blocked the increase in DAF 2 fluorescence, clearly indicating that these two agonists enhance NOS activity in the nucleus. These results implicate

Conclusions

We have shown that the nuclear β3AR and ETB regulate NO production in the cardiomyocyte, and that Gαi is implicated in this regulation. Increased NO production was required for the ISO-mediated increase in de novo transcription. Furthermore, both PKB and PKG are involved in this pathway. Moreover, we have demonstrated that nuclear receptors can regulate both rRNA and mRNA targets even in the context of the intact cell. Taken together, these results demonstrate for the first time that nuclear

Disclosure statement

The authors declare no conflicts of interest.

Acknowledgments

This work was supported by grants from the Canadian Institutes of Health Research (MOP-77791 to BGA and MOP-79354 and MOP-119530 to TEH), the Fondation des Maladies du Coeur du Québec (to BA) and the Fonds de l'Institut de Cardiologie de Montréal (FICM). BGA was a New Investigator of the Heart and Stroke Foundation of Canada and a Senior Scholar of the Fonds de la Recherche en Santé du Québec (FRSQ). TEH holds a Chercheur National award from the FRSQ. SN holds the Paul-David Chair in

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