The Skeletal Muscle Relaxer Cyclobenzaprine Is a Potent Non-Competitive Antagonist of Histamine H1 Receptors.

Cyclobenzaprine is a tricyclic dimethylpropanamine skeletal muscle relaxant, which is used clinically to decrease muscle spasm and hypercontractility, as well as acute musculoskeletal pain. Although the absolute mechanism of action of cyclobenzaprine remains elusive, it is known to mediate its effects centrally via inhibition of tonic somatic motor function, likely through modulation of noradrenergic and serotonergic systems. While cyclobenzaprine is effective as a muscle relaxant, greater than 30% of patients experience drowsiness and sedative-hypnotic effects, yet the mechanisms that cause this adverse effect are also undescribed. Based on this common adverse effect profile and the structural similarity of cyclobenzaprine to tricyclic antidepressants, as well as ethanolamine first-generation antihistamines, we hypothesized that cyclobenzaprine facilitates sedative effects via off-target antagonism of central histamine H1 receptors (H1Rs). Here, for the first time, we present data that demonstrate that cyclobenzaprine exhibits low nanomolar affinity for the cloned human H1R, as well as that expressed in both rat and mouse brain. Using saturation radioligand binding, we also demonstrate that cyclobenzaprine binds to the H1R in a noncompetitive manner. Similarly, functional assays measuring both Ca+2 influx and novel TRUPATH G-protein subunit bioluminescence resonance energy transfer biosensors reveal that cyclobenzaprine also blocks histamine-mediated H1R functional activity in a noncompetitive manner, whereas the classical H1R antagonist diphenhydramine does so competitively. Given that cyclobenzaprine readily crosses the blood-brain barrier and its muscle relaxant effects occur centrally, our data suggest that off-target central antagonism of H1R by cyclobenzaprine facilitates the significant sedative effect of this agent seen in patients. SIGNIFICANCE STATEMENT: Cyclobenzaprine, a clinically used muscle relaxant that is strongly linked to sedation, demonstrates high-affinity noncompetitive antagonism at the histamine H1 receptor. This effect likely modulates the high degree of sedation that patients experience.

Carboxy-Terminal Phosphoregulation of the Long Splice Isoform of Free-Fatty Acid Receptor-4 Mediates ß-Arrestin Recruitment and Signaling to ERK1/2.

Free-fatty acid receptor-4 (FFA4), previously termed GPR120, is a G protein-coupled receptor (GPCR) for medium and long-chained fatty acids, agonism of which can regulate a myriad of metabolic, sensory, inflammatory, and proliferatory signals. Two alternative splice isoforms of FFA4 exist that differ by the presence of an additional 16 amino acids in the longer (FFA4-L) transcript, which has been suggested to be an intrinsically ß-arrestin-biased GPCR. Although the shorter isoform (FFA4-S) has been studied more extensively, very little is known about mechanisms of regulation or signaling of the longer isoform. Because ß-arrestin recruitment is dependent on receptor phosphorylation, in the current study, we used the endogenous agonist docosahexaenoic acid (DHA) to examine the mechanisms of FFA4-L phosphorylation, as well as DHA-dependent ß-arrestin recruitment and DHA-dependent extracellular-signal regulated kinase-1/2 (ERK1/2) signaling in human embryonic kidney 293 cells. Our results reveal differences in basal phosphorylation of the two FFA4 isoforms, and we show that DHA-mediated phosphorylation of FFA4-L is primarily regulated by G protein-coupled receptor kinase 6, whereas protein kinase-C can also contribute to agonist-induced and heterologous phosphorylation. Moreover, our data demonstrate that FFA4-L phosphorylation occurs on the distal C terminus and is directly responsible for recruitment and interactions with ß-arrestin-2. Finally, using CRISPR/Cas9 genome-edited cells, our data reveal that unlike FFA4-S, the longer isoform is unable to facilitate phosphorylation of ERK1/2 in cells that are devoid of ß-arrestin-1/2. Together, these results are the first to demonstrate phosphoregulation of FFA4-L as well as the effects of loss of phosphorylation sites on ß-arrestin recruitment and ERK1/2 activation. SIGNIFICANCE STATEMENT: Free-fatty acid receptor-4 (FFA4) is a cell-surface G protein-coupled receptor for medium and long-chained fatty acids that can be expressed as distinct short (FFA4-S) or long (FFA4-L) isoforms. Although much is known about FFA4-S, the longer isoform remains virtually unstudied. Here, we reveal the mechanisms of docosahexaenoic acid-induced phosphorylation of FFA4-L and subsequent ß-arrestin-2 recruitment and extracellular-signal regulated kinase-1/2 activity.

Free-fatty acid receptor-4 (FFA4) modulates ROS generation and COX-2 expression via the C-terminal ß-arrestin phosphosensor in Raw 264.7 macrophages.

Agonism of the G protein-coupled free-fatty acid receptor-4 (FFA4) has been shown to promote numerous anti-inflammatory effects in macrophages that arise due to interaction with ß-arrestin partner proteins. Humans express functionally distinct short and long FFA4 splice variants, such that FFA4-S signals through Gαq/11 and ß-arrestin, while FFA4-L is intrinsically biased solely towards ß-arrestin signaling. Recently, we and others have shown that phosphorylation of the FFA4 C-terminal tail is responsible for ß-arrestin interactability and signaling. Given the significance of ß-arrestin in the anti-inflammatory function of FFA4, the goal of this study was to examine the role of the C-terminal ß-arrestin phosphosensor in FFA4 signaling induced by PMA and LPS in murine Raw 264.7 macrophages. Our data reveal for the first time that both FFA4 isoforms modulate PMA-induced ROS generation, and that abolishment of the FFA4-S, but not FFA4-L C-terminal phosphosensor, is detrimental to this effect. Furthermore, we show that while both isoforms reduce PMA-induced expression of COX-2, removal of the FFA4-S phosphosensor significantly decreases this response, suggesting that these effects of FFA4-S are ß-arrestin mediated. On the contrary, FFA4-S, as well as the truncated C-terminal congener lacking the ß-arrestin phosphosensor were both able to reduce LPS-induced NF-κB activity and ERK1/2 phosphorylation. However, FFA4-L and its corresponding mutant were incapable of modulating either, suggesting that these responses are mediated by G protein coupling. Taken together, our data reveal important structure-function and signaling differences between the two FFA4 isoforms, and for the first time link FFA4 to modulation of ROS in macrophages.

Fish oil and flax seed oil supplemented diets increase FFAR4 expression in the rat colon.

BACKGROUND AND OBJECTIVE: Omega-3 fatty acids, such as α-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), are polyunsaturated fatty acids (PUFA) that have long been associated with anti-inflammatory activity and general benefit toward human health. Over the last decade, the identification of a family of cell-surface G protein-coupled receptors that bind and are activated by free-fatty acids, including omega-3 fatty acids, suggest that many effects of PUFA are receptor-mediated. One such receptor, free-fatty acid receptor-4 (FFAR4), previously described as GPR120, has been shown to modulate anti-inflammatory and insulin-sensitizing effects in response to PUFA such as ALA and DHA. Additionally, FFAR4 stimulates secretion of the insulin secretagogue glucagon-like peptide-1 (GLP-1) from the GI tract and acts as a dietary sensor to regulate energy availability. The aim of the current study was to assess the effects of dietary omega-3 fatty acid supplementation on FFAR4 expression in the rat colon. METHODS: Sprague-Dawley rats were fed control soybean oil diets or alternatively, diets supplemented with either fish oil, which is enriched in DHA and EPA, or flaxseed oil, which is enriched in ALA, for 7 weeks. GLP-1 and blood glucose levels were monitored weekly and at the end of the study period, expression of FFAR4 and the inflammatory marker TNF-α was assessed. RESULTS: Our findings indicate that GLP-1 and blood glucose levels were unaffected by omega-3 fatty acid supplementation, however, animals that were fed fish or flaxseed oil-supplemented diets had significantly heightened colonic FFAR4 and actin expression, and reduced expression of the pro-inflammatory cytokine TNF-α compared to animals fed control diets. CONCLUSIONS: These results suggest that similar to ingestion of other fats, dietary-intake of omega-3 fatty acids can alter FFAR4 expression within the colon.