Free-fatty acid receptor-1 (FFA1/GPR40) promotes papillary RCC proliferation and tumor growth via Src/PI3K/AKT/NF-κB but suppresses migration by inhibition of EGFR, ERK1/2, STAT3 and EMT.

BACKGROUND: Papillary renal cell carcinoma (pRCC) is a highly metastatic genitourinary cancer and is generally irresponsive to common treatments used for the more prevalent clear-cell (ccRCC) subtype. The goal of this study was to examine the novel role of the free fatty-acid receptor-1 (FFA1/GPR40), a cell-surface expressed G protein-coupled receptor that is activated by medium-to-long chained dietary fats, in modulation of pRCC cell migration invasion, proliferation and tumor growth. METHODS: We assessed the expression of FFA1 in human pRCC and ccRCC tumor tissues compared to patient-matched non-cancerous controls, as well as in RCC cell lines. Using the selective FFA1 agonist AS2034178 and the selective FFA1 antagonist GW1100, we examined the role of FFA1 in modulating cell migration, invasion, proliferation and tumor growth and assessed the FFA1-associated intracellular signaling mechanisms via immunoblotting. RESULTS: We reveal for the first time that FFA1 is upregulated in pRCC tissue compared to patient-matched non-cancerous adjacent tissue and that its expression increases with pRCC cancer pathology, while the inverse is seen in ccRCC tissue. We also show that FFA1 is expressed in the pRCC cell line ACHN, but not in ccRCC cell lines, suggesting a unique role in pRCC pathology. Our results demonstrate that FFA1 agonism promotes tumor growth and cell proliferation via c-Src/PI3K/AKT/NF-κB and COX-2 signaling. At the same time, agonism of FFA1 strongly inhibits migration and invasion, which are mechanistically mediated via inhibition of EGFR, ERK1/2 and regulators of epithelial-mesenchymal transition. CONCLUSIONS: Our data suggest that FFA1 plays oppositional growth and migratory roles in pRCC and identifies this receptor as a potential target for modulation of pathogenesis of this aggressive cancer.

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.

Omadacycline for management of Mycobacterium abscessus infections: a review of its effectiveness, place in therapy, and considerations for use.

The Mycobacterium abscessus complex (MABC) is a group of acid-fast, rapidly dividing non-tuberculous mycobacteria (NTM) that include a number of clinically important subspecies, including M. abscessus, M. bolletii, and M. massiliense. These organisms are prevalent in the environment and are primarily associated with human pulmonary or skin and skin structure infections (SSSI) but may cause more deep-seeded disseminated infections and bacteremia in the immunocompromised. Importantly, these NTM are resistant to most first-line anti-tuberculous agents and, due to intrinsic or acquired resistance, exhibit exceedingly low, variable, and geographically distinct susceptibilities to commonly used antibacterial agents including older tetracyclines, macrolides, aminoglycosides, cephalosporins, carbapenems, and sulfamethoxazole-trimethoprim. Omadacycline is a novel third-generation member of the tetracycline family of antibacterials that has recently been demonstrated to have potent anti-NTM effects and clinical efficacy against MABC, including M. abscessus. The purpose of this review is to present a comprehensive and up-to-date assessment on the body of literature on the role of omadacycline for M. abscessus infections. Specifically, the in vitro and in vivo microbiology, mechanisms of action, mechanisms of resistance, clinical pharmacokinetics, clinical efficacy, adverse effects, dosage and administration, and place in therapy of omadacycline in management of M. abscessus infections will be detailed.

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.

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.

ROS-mediated regulation of ß2AR function: Does oxidation play a meaningful role towards ß2-agonist tachyphylaxis in airway obstructive diseases?

ß2-adrenergic receptor (ß2AR) agonists are the clinical gold standard for treatment and prophylaxis of airway constriction in pulmonary obstructive diseases such as asthma and COPD. Inhaled ß2-agonists elicit rapid bronchorelaxation of the airway smooth muscle, yet, clinical tachyphylaxis to this response can occur over repeated and chronic use, which reduces the bronchodilatory effectiveness. Several mechanisms have been proposed to impart ß2-agonist tachyphylaxis, most notably ß2AR desensitization. However, airway tissue is known to be highly oxidative, particularly in obstructive disease states where reactive oxygen species (ROS) generation is upregulated and ROS degradation is suboptimal yielding a large oxidative burden. Recent evidence demonstrates that ß2AR can regulate ROS generation and that ROS can post-translationally alter ß2AR cysteine residues via oxidation, leading to distinct functional receptor outcomes. Herein, we discuss the growing evidence for ß2AR mediated ROS generation in airway cells and the role of ROS in regulating ß2AR via cysteine-oxidation of the receptor. Given the functional consequence of the ß2AR-ROS signaling axis in the airways, we also discuss the potential role of ROS in mediating ß2-agonist tachyphylaxis.

Gut feelings in the islets: The role of the gut microbiome and the FFA2 and FFA3 receptors for short chain fatty acids on ß-cell function and metabolic regulation.

Short-chain fatty acids (SCFAs) are biosynthesized via fermentation of polysaccharides by gastrointestinal microbiota and have been shown to have wide-reaching effects on almost all tissues, including the pancreatic islets. Historically, the effects of SCFAs have been attributed to their intracellular metabolism and function as energy sources, but the discovery of free fatty acid G protein-coupled receptors (GPCRs) in the 2000s suggested that many functional outcomes of SCFAs are receptor-mediated. The SCFA receptors FFA2/GPR43 and FFA3/GPR41 are expressed on ß-cells, where they regulate glucose-dependent insulin secretion, making them attractive targets for treatment of diabetes and other metabolic disorders. Here, we provide an update on the current evidence regarding regulation of FFA2/FFA3 receptors by specific probiotic bacterial species within the gut microbiome that synthesize SCFAs. We also review the body of research regarding the FFA2- and FFA3 receptor-specific function of SCFAs on ß-cells and discuss the somewhat controversial and opposing findings within these studies.

Free-Fatty Acid Receptor-4 (FFA4/GPR120) differentially regulates migration, invasion, proliferation and tumor growth of papillary renal cell carcinoma cells.

Kidney cancer is among the 10 most common cancers, and renal cell carcinoma (RCC), which represent 90% of all kidney cancers, has the highest mortality rate of all genitourinary cancers. Papillary RCC (pRCC) is the second most frequent subtype of RCC and demonstrates distinct characteristics compared to other subtypes, including a high degree of metastasis and resistance to treatments against the more common clear cell RCC (ccRCC) subtype. Here, we demonstrate that the Free-Fatty Acid Receptor-4 (FFA4), a G protein-coupled receptor that is endogenously activated by medium-to-long chain free-fatty acids, is upregulated in pRCC compared to patient-matched normal kidney tissue, and that the expression of FFA4 increases with the degree of pathological grading of pRCC. Our data also show that FFA4 transcript is not expressed in ccRCC cell lines, but is expressed in the well-characterized metastatic pRCC cell line ACHN. Furthermore, we show that agonism of FFA4 with the selective agonist cpdA positively regulates ACHN cell migration and invasion in a manner dependent on PI3K/AKT/NF-κB signaling to COX-2 and MMP-9, with partial-dependence on EGFR transactivation. Our results also demonstrate that FFA4 agonism induces STAT-3-driven epithelial-mesenchymal transition, suggesting a significant role for FFA4 in pRCC metastasis. On the contrary, FFA4 agonism significantly reduces cell proliferation and tumor growth, suggesting that the receptor may have opposing effects on pRCC cell growth and migration. Together, our data demonstrate that FFA4 has significant functional roles in pRCC cells and may be an attractive target for study of pRCC and development of RCC pharmacotherapeutics.

Agonists and hydrogen peroxide mediate hyperoxidation of ß2-adrenergic receptor in airway epithelial cells: Implications for tachyphylaxis to ß2-agonists in constrictive airway disorders.

Asthma and other airway obstructive disorders are characterized by heightened inflammation and excessive airway epithelial cell reactive oxygen species (ROS), which give rise to a highly oxidative environment. After decades of use, ß2-adrenergic receptor (ß2AR) agonists remain at the forefront of treatment options for asthma, however, chronic use of ß2-agonists leads to tachyphylaxis to the bronchorelaxant effects, a phenomenon that remains mechanistically unexplained. We have previously demonstrated that ß2AR agonism increases ROS generation in airway epithelial cells, which upholds proper receptor function via feedback oxidation of ß2AR cysteine thiolates to Cys-S-sulfenic acids (Cys-SOH). Our previous results also demonstrate that prevention of normal redox cycling of this post-translational oxi-modification back to the thiol prevents proper receptor function. Given that Cys-S-sulfenic acids can be irreversibly overoxidized to Cys-S-sulfinic (Cys-SO2H) or S-sulfonic (Cys-SO3H) acids, which are incapable of further participation in redox reactions, we hypothesized that ß2-agonist tachyphylaxis may be explained by hyperoxidation of ß2AR to S-sulfinic acids. Here, using airway epithelial cell lines and primary small airway epithelial cells from healthy and asthma-diseased donors, we show that ß2AR agonism generates H2O2 in a receptor and NAPDH oxidase-dependent manner. We also demonstrate that acute and chronic receptor agonism can facilitate ß2AR S-sulfination, and that millimolar H2O2 concentrations are deleterious to ß2AR-mediated cAMP formation, an effect that can be rescued to a degree in the presence of the cysteine-donating antioxidant N-acetyl-L-cysteine. Our results reveal that the oxidative state of ß2AR may contribute to receptor functionality and may, at least in part, explain ß2-agonist tachyphylaxis.

Managing the "Three Cs" of Academic Literature Authorship: Contributions, Credit, and Conflict.

Dissemination of information through publications is central to academic research, as well as professional advancement. Although seemingly a straightforward endeavor, publication authorship may present challenges. Although the International Committee of Medical Journal Editors defines authorship based on 4 required criteria, contemporary interdisciplinary collaborations can complicate authorship determinations. However, communication that occurs early and frequently in the research and writing process can help to prevent or mitigate potential conflicts, while a process for defining authorship contributions can aid in awarding proper credit. The Contributor Roles Taxonomy (CRediT) defines 14 essential roles of manuscript authors that can be utilized to characterize individual author contributions toward any given publication. This information is useful for academic administrators when evaluating contributors of faculty during promotion and tenure decisions. In the era of collaborative scientific, clinical, and pedagogical scholarship, providing faculty development, including statements of credit in the published work, and developing institutional systems to capture and assess contributions are key.

Neflamapimod inhibits endothelial cell activation, adhesion molecule expression, leukocyte attachment and vascular inflammation by inhibiting p38 MAPKα and NF-κB signaling.

Neflamapimod, a selective inhibitor of the alpha isoform of p38 mitogen-activated protein kinase (MAPKα), was investigated for its potential to inhibit lipopolysaccharide (LPS)-induced activation of endothelial cells (ECs), adhesion molecule induction, and subsequent leukocyte attachment to EC monolayers. These events are known to contribute to vascular inflammation and cardiovascular dysfunction. Our results demonstrate that LPS treatment of cultured ECs and rats leads to significant upregulation of adhesion molecules, both in vitro and in vivo, which can be effectively inhibited by neflamapimod treatment. Western blotting data further reveals that neflamapimod inhibits LPS-induced phosphorylation of p38 MAPKα and the activation of NF-κB signaling in ECs. Additionally, leukocyte adhesion assays demonstrate a substantial reduction in leukocyte attachment to cultured ECs and the aorta lumen of rats treated with neflamapimod. Consistent with vascular inflammation, LPS-treated rat arteries exhibit significantly diminished vasodilation response to acetylcholine, however, arteries from rats treated with neflamapimod maintain their vasodilation capacity, demonstrating its ability to limit LPS-induced vascular inflammation. Overall, our data demonstrate that neflamapimod effectively inhibits endothelium activation, adhesion molecule expression, and leukocyte attachment, thereby reducing vascular inflammation.

Oncogenic signaling of the free-fatty acid receptors FFA1 and FFA4 in human breast carcinoma cells.

Globally, breast cancer is the most frequent type of cancer in women, and most breast cancer-associated deaths are due to metastasis and recurrence of the disease. Dietary habits, specifically dietary fat intake is a crucial risk factor involved in breast cancer development and progression. Decades of research has revealed that free-fatty acids (FFA) modulate carcinogenic processes through fatty acid metabolism and lipid peroxidation. The ground-breaking discovery of free-fatty acid receptors, which are members of the G-protein coupled receptor (GPCR) superfamily, has led to the realization that FFA can also act via these receptors to modulate carcinogenic effects. The long-chain free-fatty acid receptors FFA1 (previously termed GPR40) and FFA4 (previously termed GPR120) are activated by mono- and polyunsaturated fatty acids including ω-3, 6, and 9 fatty acids. Initial enthusiasm towards the study of these receptors focused on their insulin secretagogue and sensitization effects, and the downstream associated metabolic regulation. However, recent studies have demonstrated that abnormal expression and/or aberrant FFA1/FFA4 signaling are evident in human breast carcinomas, suggesting that FFA receptors could be a promising target in the treatment of breast cancer. The current review discusses the diverse roles of FFA1 and FFA4 in the regulation of cell proliferation, migration, invasion, and chemotherapy resistance in human breast carcinoma cells and tissue.

Agonist- and hydrogen peroxide-mediated oxidation of the ß2 adrenergic receptor: evidence of receptor s-sulfenation as detected by a modified biotin-switch assay.

Reactive oxygen species (ROS), including hydrogen peroxide (H(2)O(2)), have recently been shown to be generated upon agonism of several members of the G protein-coupled receptor (GPCR) superfamily, including ß(2)-adrenergic receptors (ß(2)ARs). Previously, we have demonstrated that inhibition of intracellular ROS generation mitigates ß(2)AR signaling, suggesting that ß(2)AR-mediated ROS generation is capable of feeding back to regulate receptor function. Given that ROS, specifically H(2)O(2), are able to post-translationally oxidize protein cysteine sulfhydryls to cysteine-sulfenic acids, the goal of the current study was to assess whether ROS are capable of S-sulfenating ß(2)AR. Using a modified biotin-switch assay that is selective for cysteine-sulfenic acids, our results demonstrate for the first time that H(2)O(2) treatment facilitates S-sulfenation of transiently overexpressed ß(2)AR in human embryonic kidney 293 cells. It is noteworthy that stimulation of cells with the ß-agonist isoproterenol produces both dose- and time-dependent S-sulfenation of ß(2)AR, an effect that is receptor-dependent, and demonstrates that receptor-generated ROS are also capable of oxidizing the ß(2)AR. Receptor-dependent S-sulfenation was inhibited by the chemoselective sulfenic acid alkylator dimedone and the cysteine antioxidant N-acetyl-l-cysteine. Moreover, our results reveal that receptor oxidation occurs in cells that endogenously express physiologically relevant levels of ß(2)AR, because treatment of human alveolar epithelial A549 cells with either H(2)O(2) or the ß(2)-selective agonist formoterol promoted receptor S-sulfenation. These findings provide the first evidence, to our knowledge, that a mammalian GPCR can be oxidized by S-sulfenation and signify an important first step toward shedding light on the overlooked role of ROS in the regulation of ß(2)AR function.

Short-chain free-fatty acid G protein-coupled receptors in colon cancer.

The dietary role of macronutrients and their metabolites in cancer has been evident for many decades. Dietary ingestion of fat, carbohydrates, protein, and fiber, as well as probiotics that influence gut microbiota, have all been linked to gastrointestinal (GI) tract health and disease, particularly in the colon, where it has long been known that fat and fiber can regulate inflammation and carcinogenesis. Short-chained fatty acids (SCFA), including acetate, propionate, and butyrate, which are biosynthesized by microbiota-mediated metabolism of dietary fiber, have previously been shown to play important roles in colorectal health, including decreasing inflammation and oxidative stress. Since the 1980s, a growing number of studies have also demonstrated a link between SCFA and colon epithelial cell carcinogenesis and prevention of colorectal cancers (CRC). While the effects of SCFA have historically been associated with their intracellular metabolism and function, the discovery of a family of G protein-coupled free-fatty acid receptors in the early 2000s suggests that many effects of SCFA are cell-surface receptor mediated. Indeed, the SCFA GPCRs FFA2 (previously termed GPR43), FFA3 (previously termed GPR41), and GPR109A are now well established to be expressed within the GI tract, where they modulate a variety of functions in response to luminal SCFA. While the role of SCFA in cancers, including CRC, has been reviewed in detail elsewhere, the goal of this report is to provide a review on the current body of evidence in regard to the effects of SCFA on FFA2, FFA3, and GPR109A in colon cancers.

Agonism with the omega-3 fatty acids alpha-linolenic acid and docosahexaenoic acid mediates phosphorylation of both the short and long isoforms of the human GPR120 receptor.

The newly discovered G protein-coupled receptor GPR120 has recently been shown to stimulate secretion of the gut hormones glucagon-like peptide-1 and cholecystokinin upon binding of free fatty acids, thrusting it to the forefront of drug discovery efforts for treatment of type 2 diabetes as well as satiety and obesity. Although sequences for two alternative splice variants of the human GPR120 receptor have been reported, there have been no studies which directly compare the signaling of these isoforms. We have identified an additional 16 amino acid gap containing four phospho-labile serine/threonine residues which is localized to the third intracellular loop of the GPR120-long (GPR120-L) isoform. Based on this finding, we hypothesized that the agonist-stimulated phosphorylation profiles of this isoform would be distinct from that of the short isoform (GPR120-S). Using a clonal HEK293 cell model, we examined agonist-mediated phosphorylation of GPR120-S and GPR120-L with the omega-3 fatty acids alpha-linolenic acid (ALA) and docosahexaenoic acid (DHA). Our results show rapid phosphorylation of both isoforms following agonism by either ALA or DHA. Moreover, we show no significant difference in the degree or rate of phosphorylation of both isoforms upon agonism with either ALA or DHA, suggesting that the additional gap in the longer variant is not phosphorylated. Importantly, our results demonstrate that the shorter variant exhibits significantly more pronounced basal phosphorylation in the absence of agonist, suggesting that the additional gap in the long variant may contribute to masking of constitutive phosphorylation sites. These are the first results which demonstrate specific phosphorylation of GPR120 isoforms upon agonism by free fatty acids and the first which distinguish the phosphorylation profiles of the two GPR120 isoforms.

Cysteine redox state regulates human ß2-adrenergic receptor binding and function.

Bronchoconstrictive airway disorders such as asthma are characterized by inflammation and increases in reactive oxygen species (ROS), which produce a highly oxidative environment. ß2-adrenergic receptor (ß2AR) agonists are a mainstay of clinical therapy for asthma and provide bronchorelaxation upon inhalation. We have previously shown that ß2AR agonism generates intracellular ROS, an effect that is required for receptor function, and which post-translationally oxidizes ß2AR cysteine thiols to Cys-S-sulfenic acids (Cys-S-OH). Furthermore, highly oxidative environments can irreversibly oxidize Cys-S-OH to Cys-S-sulfinic (Cys-SO2H) or S-sulfonic (Cys-SO3H) acids, which are incapable of further participating in homeostatic redox reactions (i.e., redox-deficient). The aim of this study was to examine the vitality of ß2AR-ROS interplay and the resultant functional consequences of ß2AR Cys-redox in the receptors native, oxidized, and redox-deficient states. Here, we show for the first time that ß2AR can be oxidized to Cys-S-OH in situ, moreover, using both clonal cells and a human airway epithelial cell line endogenously expressing ß2AR, we show that receptor redox state profoundly influences ß2AR orthosteric ligand binding and downstream function. Specifically, homeostatic ß2AR redox states are vital toward agonist-induced cAMP formation and subsequent CREB and G-protein-dependent ERK1/2 phosphorylation, in addition to ß-arrestin-2 recruitment and downstream arrestin-dependent ERK1/2 phosphorylation and internalization. On the contrary, redox-deficient ß2AR states exhibit decreased ability to signal via either Gαs or ß-arrestin. Together, our results demonstrate a ß2AR-ROS redox axis, which if disturbed, interferes with proper receptor function.

The ß2-adrenergic receptor-ROS signaling axis: An overlooked component of ß2AR function?

ß2-Adrenergic receptor (ß2AR) agonists are clinically used to elicit rapid bronchodilation for the treatment of bronchospasms in pulmonary diseases such as asthma and COPD, both of which exhibit characteristically high levels of reactive oxygen species (ROS); likely secondary to over-expression of ROS generating enzymes and chronically heightened inflammation. Interestingly, ß2AR has long-been linked to ROS, yet the involvement of ROS in ß2AR function has not been as vigorously studied as other aspects of ß2AR signaling. Herein, we discuss the existing body of evidence linking ß2AR activation to intracellular ROS generation and importantly, the role of ROS in regulating ß2AR function. The reciprocal interplay of the ß2AR and ROS appear to endow this receptor with the ability to self-regulate signaling efficacy and ligand binding, hereby unveiling a redox-axis that may be unfavorably altered in pathological states contributing to both disease progression and therapeutic drug responses.

Docosahexaenoic acid protects motor function and increases dopamine synthesis in a rat model of Parkinson's disease via mechanisms associated with increased protein kinase activity in the striatum.

Parkinson's disease (PD) is a devastating neurodegenerative disease that leads to motor deficits and selective destruction of nigrostriatal dopaminergic neurons. PD is typically treated by dopamine replacement agents; however, dopamine replacement loses effectiveness in the later stages of the disease. Here, we describe the neuroprotective effects of the omega-3 fatty acid docosahexaenoic acid (DHA) in the medial forebrain bundle 6-hydroxydopamine (6-OHDA) model of advanced-stage PD in rats. We show that daily administration of DHA protects against core symptoms of PD, including deficits in postural stability, gait integrity, and dopamine neurochemistry in motor areas of the striatum. Our results also demonstrate that DHA increases striatal dopamine synthesis via phosphorylation of the rate-limiting catecholamine synthesizing enzyme tyrosine hydroxylase, in a manner dependent on the second messenger-linked protein kinases PKA and PKC. We also show that DHA specifically reverses dopamine loss in the nigrostriatal pathway, with no effect in the mesolimbic or mesocortical pathways. This suggests that DHA is unlikely to produce pharmacotherapeutic or adverse effects that depend on dopamine pathways other than the nigrostriatal pathway. To our knowledge, previous reports have not examined the effects of DHA in such an advanced-stage model, documented that the dopamine synthesizing effects of DHA in vivo are mediated through the activation of protein kinases and regulation of TH activity, or demonstrated specificity to the nigrostriatal pathway. These novel findings corroborate the beneficial effects of omega-3 fatty acids seen in PD patients and suggest that DHA provides a novel means of protecting patients for dopamine neurodegeneration.

Reintroduction of quazepam: an update on comparative hypnotic and adverse effects.

Insomnia is a prevalent disorder that affects over one-third of the U.S. population to varying degrees and is highly disruptive towards quality of life. Pharmacological treatments for insomnia include benzodiazepines (BZs) and the non-BZ 'Z-drugs' (zolpidem, zaleplon, eszopiclone, zopiclone), which are amongst the most widely prescribed medications. Yet, these agents can produce adverse effects such as tolerance to the hypnotic effect, rebound insomnia, next-day residual drowsiness, as well as amnesia and complex behaviours such as sleep-walking, sleep-eating and sleep-driving. Quazepam, one of the five BZ approved for treatment of insomnia, was recently relaunched to the U.S. market in 2016 and is distinguished amongst hypnotic BZ by unique pharmacological characteristics including selectivity for sleep-promoting α1-subunit containing γ-aminobutyric acid (GABA-A) receptors and a significantly lower relative receptor binding affinity. These features likely drive the decreased rate of adverse events seen clinically with quazepam, such as tolerance, rebound insomnia and amnesic behaviours, compared with other BZ. Given the recent reintroduction of quazepam as a pharmacotherapeutic option, and the lack of head-to-head comparative trials against newer agents, the purpose of this review is to provide an update on distinguishing features of quazepam with regard to its pharmacology, pharmacokinetics, sleep efficacy and potential adverse effects compared to other agents used for insomnia.

Omega-3 Fatty Acids as Druggable Therapeutics for Neurodegenerative Disorders.

Neurodegenerative disorders are commonly associated with a complex pattern of pathophysiological hallmarks, including increased oxidative stress and neuroinflammation, which makes their treatment challenging. Omega-3 Fatty Acids (O3FA) are natural products with reported neuroprotective, anti-inflammatory, and antioxidant effects. These effects have been attributed to their incorporation into neuronal membranes or through the activation of intracellular or recently discovered cell-surface receptors (i.e., Free-Fatty Acid Receptors; FFAR). Molecular docking studies have investigated the roles of O3FA as agonists of FFAR and have led to the development of receptor-specific targeted agonists for therapeutic purposes. Moreover, novel formulation strategies for targeted delivery of O3FA to the brain have supported their development as therapeutics for neurodegenerative disorders. Despite the compelling evidence of the beneficial effects of O3FA for several neuroprotective functions, they are currently only available as unregulated dietary supplements, with only a single FDA-approved prescription product, indicated for triglyceride reduction. This review highlights the relative safety and efficacy of O3FA, their drug-like properties, and their capacity to be formulated in clinically viable drug delivery systems. Interestingly, the presence of cardiac conditions such as hypertriglyceridemia is associated with brain pathophysiological hallmarks of neurodegeneration, such as neuroinflammation, thereby further suggesting potential therapeutic roles of O3FA for neurodegenerative disorders. Taken together, this review article summarizes and integrates the compelling evidence regarding the feasibility of developing O3FA and their synthetic derivatives as potential drugs for neurodegenerative disorders.