As a treatment for dyslipidemia, oral doses of 1-3 grams of nicotinic acid per day lower serum triglycerides, raise high density lipoprotein cholesterol, and reduce mortality from coronary heart disease ( Tavintharan

Nicotinic acid, used for atherosclerosis treatment, has an adverse effect of skin flushing. The flushing mechanism, thought to be caused by the release of prostaglandin D(2) (PGD(2)), is not well understood. We aimed to identify which cells mediate the flushing effect. Nicotinic acid receptor (GPR109A) gene expression was assessed in various tissues and cell lines. Cells expressing GPR109A mRNA were further assayed for PGD(2) release in response to nicotinic acid. Of all samples, only skin was able to release PGD(2) upon stimulation with nicotinic acid. The responsive cells were localized to the epidermis, and immunocytochemical studies revealed the presence of GPR109A on epidermal Langerhans cells. CD34+ cells isolated from human blood and differentiated into Langerhans cells (hLC-L) also showed GPR109A expression. IFNgamma treatment increased both mRNA and plasma membrane expression of GPR109A. IFNgamma-stimulated hLC-Ls released PGD(2) in response to nicotinic acid in a dose-dependant manner (effector concentration for half-maximum response=1.2 mM+/-0.7). Acifran, a structurally distinct GPR109A ligand, also increased PGD(2) release, whereas isonicotinic acid, a nicotinic acid analog with low affinity for GPR109A, had no effect. These results suggest that nicotinic acid mediates its flushing side effect by interacting with GPR109A on skin Langerhans cells, resulting in release of PGD(2).

The three ␣ 2 -adrenergic receptor (␣ 2 AR) 1 subtypes are members of the type II, biogenic amine-binding, G protein-coupled receptor family. These receptor subtypes all couple via the G i /G o family of GTP-binding proteins to the inhibition of adenylyl cyclase, inhibition of voltage-dependent calcium channels, potentiation of potassium currents via G protein-coupled, inwardly rectifying potassium channels, activation of phospholipase D, and activation of MAP kinase in native cells (1-4). In heterologous cell systems, these receptors also couple to the activation of a variety of signaling molecules, including Ras (5-7), p70 S6 kinase (8), MAP kinase (9, 10), and phospholipase D (11).Although all three ␣ 2 ARs appear to activate similar signaling pathways, differences in the cellular trafficking of these subtypes have been reported, both in naive cells and following agonist activation. Subtype-selective differences in agonistelicited ␣ 2 AR redistribution have been noted in several experimental systems (12-18). The ␣ 2B AR subtype is readily internalized following agonist activation, whereas the ␣ 2A AR subtype typically is not (14, 18). The ␣ 2C AR subtype has not been explored in as much detail with regard to agonist-elicited redistribution because of its considerable accumulation intracellularly (14). The ␣ 2 AR subtypes also manifest different trafficking itineraries in polarized Madin-Darby canine kidney II (MDCKII) cells, even in the absence of agonist treatment. The ␣ 2A AR subtype is targeted directly to the basolateral surface (19), whereas the ␣ 2B AR subtype is delivered randomly to both the apical and basolateral surfaces but is selectively retained on the basolateral surface (t1 ⁄2 ϭ 10 -12 h) in contrast to its rapid loss from the apical surface (t1 ⁄2 ϭ 5-15 min) (20). These findings suggest that there is a molecular mechanism responsible for the selective retention of the ␣ 2B AR on the basolateral sub-domain of MDCK cells, probably a retention mechanism shared by the basolaterally targeted ␣ 2A -and ␣ 2C AR subtypes (20). Although ␣ 2C ARs, like ␣ 2A ARs, are directly targeted to and retained on the basolateral subdomain, a significant proportion of these receptors is identifiable in an intracellular pool at steady state (14,18,20); the functional relevance of this intracellular ␣ 2C AR pool has yet to be clarified.Receptor retention on the lateral subdomain of MDCKII cells likely involves the third intracellular loop of the ␣ 2 AR subtypes. For example, deletion of this loop in the ␣ 2A AR subtype (⌬3i ␣ 2A AR) results in accelerated basolateral receptor turnover (t1 ⁄2 Х 4.5 h) when compared with that for the wild-type receptor or with ␣ 2A AR structures that have been mutated in the N terminus or the C-terminal tail (all possessing a t1 ⁄2 of 10 -12 h) (21). Similarly, the ⌬3i ␣ 2B AR is not enriched at the basolateral surface of MDCKII cells at steady state (22).Based on our findings that the ␣ 2B AR is rapidly removed from the apical surface following random delivery and that removal of the...

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Nicotinic acid remains the most effective therapeutic agent for the treatment and prevention of atherosclerosis resulting from low high density lipoprotein cholesterol. The therapeutic actions of nicotinic acid are mediated by GPR109A, a G i proteincoupled receptor, expressed primarily on adipocytes, Langerhans cells, and macrophage. Unfortunately, a severe, cutaneous flushing side effect limits its use and patient compliance. The mechanism of high density lipoprotein elevation is not clearly established but assumed to be influenced by an inhibition of lipolysis in the adipose. The flushing side effect appears to be mediated by the release of prostaglandin D2 from Langerhans cells in the skin. We hypothesized that the signal transduction pathways mediating the anti-lipolytic and prostaglandin D2/flushing pathways are distinct and that agonists may be identified that are capable of selectively eliciting the therapeutic, anti-lipolytic pathway while avoiding the activation of the parallel flush-inducing pathway. We have identified a number of GPR109A pyrazole agonists that are capable of fully inhibiting lipolysis in vitro and in vivo and not only fail to elicit a flushing response but can antagonize the ability of nicotinic acid to elicit a flush response in vivo. In contrast to flushing agonists, exposure of cells expressing GPR109A to the non-flushing agonists fails to induce internalization of the receptor or to activate ERK 1/2 mitogen-activated protein kinase phosphorylation.Nicotinic acid (niacin, vitamin B3, pyridine-3-carboxylic acid) is the most effective therapeutic agent to date for raising high density lipoprotein (HDL) 2 levels. It also offers protection against other cardiovascular risk factors by lowering very low density lipoprotein (VLDL), low density lipoprotein (LDL), and lipoprotein(a) plasma concentrations (1, 2). Although the mechanism by which nicotinic acid raises HDL is not clear, one hypothesis is that it is the ability of nicotinic acid to inhibit lipolysis in adipocytes that results in a decrease in the concentration of free fatty acids available for the liver to use for triglyceride synthesis and VLDL production. The attenuated synthesis of the triglyceride-rich VLDL particles in the liver leads to a decreased rate of HDL metabolism via limiting the cholesterol ester transfer protein (CETP)-mediated exchange of cholesterol from HDL to VLDL, and triglyceride from VLDL to HDL (3-6). Another hypothesis is that nicotinic acid inhibits the uptake and subsequent catabolism of Apo-AI-containing HDL particles in hepatocytes (7,8).Identification of a high affinity nicotinic acid binding site that was localized to adipose, macrophage, and spleen tissues and appeared to function in a G i protein-coupled manner (9) led to the molecular identification of the high affinity nicotinic acid receptor GPR109A (HM74A in humans and PUMA-G in mice) (10 -12). In the adipose, GPR109A mediates an anti-lipolytic response that can attenuate cAMP-stimulated lipolysis (11). A low affinity nicotinic acid receptor ...

The ␣ 2 -adrenergic receptors (␣ 2 ARs) are localized to and function on the basolateral surface in polarized renal epithelial cells via a mechanism involving the third cytoplasmic loop. 35 S]Met-14-3-3 binds to all three native ␣ 2 AR subtypes, assessed using a solid phase binding assay (␣ 2A >␣ 2B > ␣ 2C ), and this binding depends on the presence of the 3i loops. Attenuation of the ␣ 2 AR-14-3-3 interactions in the presence of a phosphorylated Raf-1 peptide corresponding to its 14-3-3 interacting domain (residues 251-266), but not by its non-phosphorylated counterpart, provides evidence for the functional specificity of these interactions and suggests one potential interface for the ␣ 2 AR and 14-3-3 interactions. These studies represent the first evidence for G protein-coupled receptor interactions with 14-3-3 proteins and may provide a mechanism for receptor localization and/or coordination of signal transduction.The three ␣ 2 -adrenergic receptor (␣ 2 AR) 1 subtypes, encoded by distinct genes (1), all couple via the G i /G o family of GTPbinding proteins to inhibition of adenylyl cyclase, suppression of voltage-sensitive calcium channels, and activation of receptor-operated potassium channels (2). These receptors also couple to activation of Ras (3, 4), the mitogen-activated protein kinase cascade (3, 5-7), and to activation of phospholipase D (8, 9).Despite the qualitatively similar signaling properties of the three ␣ 2 AR subtypes, differences in trafficking of these receptors have been reported. For example, subtype-selective differences in agonist-elicited ␣ 2 AR redistribution occur (10 -15). In addition, selective itineraries for the ␣ 2 AR subtypes are observed in polarized Madin-Darby canine kidney (MDCKII) renal epithelial cells. Thus, the ␣ 2A AR subtype is targeted directly to the basolateral surface (16), whereas the ␣ 2B AR subtype is delivered randomly to both the apical and basolateral surfaces but is rapidly lost from the apical (t1 ⁄2 ϭ 5-15 min) and selectively retained on the basolateral (t1 ⁄2 ϭ 10 -12 h) surface (17). These findings suggest that there is a molecular mechanism responsible for the selective retention of the ␣ 2B AR on the basolateral domain of MDCK cells that may be shared by all three ␣ 2 AR subtypes, as they manifest comparable halflives on that surface (17).Receptor retention on the lateral subdomain of MDCKII cells likely involves the third intracellular loop of the ␣ 2A AR, since deletion of this loop, creating the mutant ␣ 2A ⌬3iAR, results in accelerated basolateral turnover (t1 ⁄2 Х 4.5 h) when compared with that for the wild-type receptor or with ␣ 2A AR structures that have been mutated in the N terminus or the C-terminal tail (all possessing a t1 ⁄2 of 10 -12 h) (18). The accelerated turnover of the ␣ 2A ⌬3iAR when compared with the wild-type ␣ 2A AR structure suggests that the third intracellular loop interacts with proteins that either tether ␣ 2A AR to a particular surface domain or, alternatively, mask the ␣ 2A AR from interacting with endocytosis machiner...

The discovery and profiling of 3-(1H-tetrazol-5-yl)-1,4,5,6-tetrahydro-cyclopentapyrazole (5a, MK-0354), a partial agonist of GPR109a, is described. Compound 5a retained the plasma free fatty acid lowering effects in mice associated with GPR109a agonism, but did not induce vasodilation at the maximum feasible dose. Moreover, preadministration of 5a blocked the flushing effect induced by nicotinic acid but not that induced by PGD2. This profile made 5a a suitable candidate for further study for the treatment of dyslipidemia.

1-Substituted benzotriazole carboxylic acids have been identified as the first reported examples of selective small-molecule agonists of the human orphan G-protein-coupled receptor GPR109b (HM74), a low-affinity receptor for the HDL-raising drug niacin. No activity was observed at the highly homologous high-affinity niacin receptor GPR109a (HM74A). The high degree of selectivity was attributed to a difference in the amino acid sequence adjacent to a key arginine-ligand interaction allowing somewhat larger ligands to be tolerated by GPR109b.