GTPγS Assay for Measuring Agonist-Induced Desensitization of Two Human Polymorphic Alpha2B-Adrenoceptor Variants.

α2-Adrenergic receptors (ARs) mediate many cellular actions of epinephrine and norepinephrine, including inhibition of their secretion (sympathetic inhibition) from adrenal chromaffin cells. Like many other G protein-coupled receptors (GPCRs), they undergo agonist-dependent phosphorylation and desensitization by GPCR kinases (GRKs), a phenomenon recently shown to play a major role in the sympathetic overdrive that accompanies and aggravates chronic heart failure. A three-glutamic acid deletion polymorphism in the human α2B-AR subtype gene (Glu301-303) causes impaired agonist-promoted receptor phosphorylation and desensitization, resulting in enhanced signaling to inhibition of cholinergic-induced catecholamine secretion in adrenal chromaffin cells. One of the various pharmacological assays that can be used to quantify and quantitatively compare the degrees of agonist-dependent desensitization, i.e., G protein decoupling, of these two polymorphic α2B-AR variants (or of any two GPCRs for that matter) is the guanosine-5'-O-3-thiotriphosphate (GTPγS) assay that can directly quantify heterotrimeric G protein activation.

Regulator of G-Protein Signaling-4 Attenuates Cardiac Adverse Remodeling and Neuronal Norepinephrine Release-Promoting Free Fatty Acid Receptor FFAR3 Signaling.

Propionic acid is a cell nutrient but also a stimulus for cellular signaling. Free fatty acid receptor (FFAR)-3, also known as GPR41, is a Gi/o protein-coupled receptor (GPCR) that mediates some of the propionate's actions in cells, such as inflammation, fibrosis, and increased firing/norepinephrine release from peripheral sympathetic neurons. The regulator of G-protein Signaling (RGS)-4 inactivates (terminates) both Gi/o- and Gq-protein signaling and, in the heart, protects against atrial fibrillation via calcium signaling attenuation. RGS4 activity is stimulated by ß-adrenergic receptors (ARs) via protein kinase A (PKA)-dependent phosphorylation. Herein, we examined whether RGS4 modulates cardiac FFAR3 signaling/function. We report that RGS4 is essential for dampening of FFAR3 signaling in H9c2 cardiomyocytes, since siRNA-mediated RGS4 depletion significantly enhanced propionate-dependent cAMP lowering, Gi/o activation, p38 MAPK activation, pro-inflammatory interleukin (IL)-1ß and IL-6 production, and pro-fibrotic transforming growth factor (TGF)-ß synthesis. Additionally, catecholamine pretreatment blocked propionic acid/FFAR3 signaling via PKA-dependent activation of RGS4 in H9c2 cardiomyocytes. Finally, RGS4 opposes FFAR3-dependent norepinephrine release from sympathetic-like neurons (differentiated Neuro-2a cells) co-cultured with H9c2 cardiomyocytes, thereby preserving the functional ßAR number of the cardiomyocytes. In conclusion, RGS4 appears essential for propionate/FFAR3 signaling attenuation in both cardiomyocytes and sympathetic neurons, leading to cardioprotection against inflammation/adverse remodeling and to sympatholysis, respectively.

Cardiovascular angiotensin II type 1 receptor biased signaling: Focus on non-Gq-, non-ßarrestin-dependent signaling.

The physiological and pathophysiological roles of the angiotensin II type 1 (AT1) receptor, a G protein-coupled receptor ubiquitously expressed throughout the cardiovascular system, have been the focus of intense investigations for decades. The success of angiotensin converting enzyme inhibitors (ACEIs) and of angiotensin receptor blockers (ARBs), which are AT1R-selective antagonists/inverse agonists, in the treatment of heart disease is a testament to the importance of this receptor for cardiovascular homeostasis. Given the pleiotropic signaling of the cardiovascular AT1R and, in an effort to develop yet better drugs for heart disease, the concept of biased signaling has been exploited to design and develop biased AT1R ligands that selectively activate ß-arrestin transduction pathways over Gq protein-dependent pathways. However, by focusing solely on Gq or ß-arrestins, studies on AT1R "biased" signaling & agonism tend to largely ignore other non-Gq-, non ß-arrestin-dependent signaling modalities the very versatile AT1R employs in cardiovascular tissues, including two very important types of signal transducers/regulators: other G protein types (e.g., Gi/o, G12/13) & the Regulator of G protein Signaling (RGS) proteins. In this review, we provide a brief overview of the current state of cardiovascular AT1R biased signaling field with a special focus on the non-Gq-, non ß-arrestin-dependent signaling avenues of this receptor in the cardiovascular system, which usually get left out of the conversation of "biased" AT1R signal transduction.

Sympatholytic Mechanisms for the Beneficial Cardiovascular Effects of SGLT2 Inhibitors: A Research Hypothesis for Dapagliflozin's Effects in the Adrenal Gland.

Heart failure (HF) remains the leading cause of morbidity and death in the western world, and new therapeutic modalities are urgently needed to improve the lifespan and quality of life of HF patients. The sodium-glucose co-transporter-2 (SGLT2) inhibitors, originally developed and mainly indicated for diabetes mellitus treatment, have been increasingly shown to ameliorate heart disease, and specifically HF, in humans, regardless of diabetes co-existence. Indeed, dapagliflozin has been reported to reduce cardiovascular mortality and hospitalizations in patients with HF and reduced ejection fraction (HFrEF). This SGLT2 inhibitor demonstrates these benefits also in non-diabetic subjects, indicating that dapagliflozin's efficacy in HF is independent of blood glucose control. Evidence for the effectiveness of various SGLT2 inhibitors in providing cardiovascular benefits irrespective of their effects on blood glucose regulation have spurred the use of these agents in HFrEF treatment and resulted in FDA approvals for cardiovascular indications. The obvious question arising from all these studies is, of course, which molecular/pharmacological mechanisms underlie these cardiovascular benefits of the drugs in diabetics and non-diabetics alike. The fact that SGLT2 is not significantly expressed in cardiac myocytes (SGLT1 appears to be the dominant isoform) adds even greater perplexity to this answer. A variety of mechanisms have been proposed over the past few years and tested in cell and animal models and prominent among those is the potential for sympatholysis, i.e., reduction in sympathetic nervous system activity. The latter is known to be high in HF patients, contributing significantly to the morbidity and mortality of the disease. The present minireview first summarizes the current evidence in the literature supporting the notion that SGLT2 inhibitors, such as dapagliflozin and empagliflozin, exert sympatholysis, and also outlines the main putative underlying mechanisms for these sympatholytic effects. Then, we propose a novel hypothesis, centered on the adrenal medulla, for the sympatholytic effects specifically of dapagliflozin. Adrenal medulla is responsible for the production and secretion of almost the entire amount of circulating epinephrine and of a significant percentage of circulating norepinephrine in the human body. If proven true experimentally, this hypothesis, along with other emerging experimental evidence for sympatholytic effects in neurons, will shed new light on the pharmacological effects that mediate the cardiovascular benefits of SGLT2 inhibitor drugs, independently of their blood glucose-lowering effects.

Signaling and function of cardiac autonomic nervous system receptors: Insights from the GPCR signalling universe.

The two branches of the autonomic nervous system (ANS), adrenergic and cholinergic, exert a multitude of effects on the human myocardium thanks to the activation of distinct G protein-coupled receptors (GPCRs) expressed on the plasma membranes of cardiac myocytes, cardiac fibroblasts, and coronary vascular endothelial cells. Norepinephrine (NE)/epinephrine (Epi) and acetylcholine (ACh) are released from cardiac ANS terminals and mediate the biological actions of the ANS on the heart via stimulation of cardiac adrenergic or muscarinic receptors, respectively. In addition, several other neurotransmitters/hormones act as facilitators of ANS neurotransmission in the heart, taking part in the so-called nonadrenergic noncholinergic (NANC) part of the ANS's control of cardiac function. These NANC mediators also use several different cell membrane-residing GPCRs to exert their effects in the myocardium. Cardiac ANS dysfunction and an imbalance between the activities of its two branches underlie a variety of cardiovascular diseases, from heart failure and hypertension to coronary artery disease, myocardial ischemia, and arrhythmias. In this review, we present the main well-established signaling modalities used by cardiac autonomic GPCRs, including receptors for salient NANC mediators, and we also highlight the latest developments pertaining to cardiac cell type-specific signal transduction, resulting in cell type-specific cardiac effects of each of these autonomic receptors.

Adrenal angiotensin II type 1 receptor biased signaling: The case for "biased" inverse agonism for effective aldosterone suppression.

Angiotensin II (AngII) uses two distinct G protein-coupled receptor (GPCR) types, AT1R and AT2R, to exert a plethora of physiologic effects in the body and to significantly affect cardiovascular homeostasis. Although not much is known about the signaling of the AT2R, AT1R signaling is known to be quite pleiotropic, mobilizing a variety of signal transducers inside cells to produce a biological outcome. When the outcome in question is aldosterone production from the adrenal cortex, the main transducers activated specifically by the adrenocortical AT1R to signal toward that cellular effect are the Gq/11 protein alpha subunits and the ß-arrestins (also known as Arrestin-2 and -3). The existence of various downstream pathways the AT1R signal can travel down on has led to the ever-expanding filed of GPCR pharmacology termed "biased" signaling, which refers to a ligand preferentially activating one signaling pathway over others downstream of the same receptor in the same cell. However, "biased" signaling or "biased" agonism is therapeutically desirable only when the downstream pathways lead to different or opposite cellular outcomes, so the pathway promoting the beneficial effect can be selectively activated over the pathway that leads to detrimental consequences. In the case of the adrenal AT1R, both Gq/11 proteins and ß-arrestins mediate signaling to the same end-result: aldosterone synthesis and secretion. Therefore, both pathways need to remain inactive in the adrenal cortex to fully suppress the production of aldosterone, which is one of the culprit hormones elevated in chronic heart failure, hypertension, and various other cardiovascular diseases. Variations in the effectiveness of the AT1R antagonists, which constitute the angiotensin receptor blocker (ARB) class of drugs (also known as sartans), at the relative blockade of these two pathways downstream of the adrenal AT1R opens the door to the flip term "biased" inverse agonism at the AT1R. ARBs that are unbiased and equipotent inverse agonists for both G proteins and ß-arrestins at this receptor, like candesartan and valsartan, are the most preferred agents with the best efficacy at reducing circulating aldosterone, thereby ameliorating heart failure. In the present review, the biased signaling of the adrenal AT1R, particularly in relation to aldosterone production, is examined and the term "biased" inverse agonism at the AT1R is introduced and explained, as a means of pharmacological categorization of the various agents within the ARB drug class.