Intracellular cAMP Production Research Articles

(046) THE ROLE OF ADENOSINE IN THE RELAXANT MECHANISM OF RAT DISTAL VAGINA

Abstract Introduction Adenosine (ADO) is a purinergic neuromodulator that in the male exerts a positive effect on penile erection by controlling smooth muscle (SM) relaxation. However, its role in the relaxant/contractile pathways in the vagina is yet to be investigated. Objective The aim of this study was to characterize the effect of ADO stimulation on SM activity in distal vagina, evaluating its possible effect on the regulation of nitric oxide (NO) pathway using a validated animal model of female Sprague-Dawley rats. Methods The immunolocalization of ADORA2A and ADORA2B in distal vagina tissues was detected by immunohistological staining. In vitro contractility studies on noradrenaline (NA)-precontracted vaginal strips from intact Sprague-Dawley rats were performed; rat smooth muscle cells (rvSMCs) from distal vagina of intact animals were stimulated with ADO for cyclic adenosine monophosphate (cAMP) quantification. mRNA (messenger ribonucleic acid) isolated from vaginal tissue was analyzed by semi-quantitative Reverse Transcriptase-Polymerase Chain Reaction. Results All ADORAs mRNA were expressed in the vaginal tissue, with a significant prevalence of ADORA2B; furthermore, ADORA2A and ADORA2B immunolocalization highlighted that both receptors are highly expressed in the epithelium, in blood capillaries of the vascular bed and in the smooth muscle bundles of vagina wall. In vitro ADO stimulation induced a dose-dependent relaxation (IC50=31.9±2.38 μM), which was significantly reduced by the administration of NO-synthase inhibitor L-NAME; similar effects were observed after the mechanical ablation of endothelium and in vitro administration of the soluble guanylate cyclase inhibitor ODQ, highlighting the pivotal contribution of NO signaling to the ADO relaxant activity. In vitro stimulation with increasing doses of the selective ADO receptor ADORA2A agonist CGS2-1680 induced a dose-dependent relaxation (IC50=132.6±3.47 nM); a similar but less potent relaxant effect was observed with ADORA2B selective agonist BAY60-6583. The relaxing effect induced by ADO on NA-precontracted vaginal strips was completely counteracted by the stimulation with the re-spectively selective ADORA2A and ADORA2B antagonists SHC-442416 and PSB603; a similar effect was induced by ADORA1 selective antagonist SLV320, but not by ADORA3 selective antagonists VUF5574. The mRNA relative expression of enzymes regulating the ADO turnover [adenosine deami-nase (ADA), 5′-nucleotidase (5NTD), AMP deaminase type 1 and type 2 (AMPD1, AMPD2) and adenosine kinase (ADK)] showed a higher expression of genes regulating the de novo ADO/cAMP syn-thesis (ADK, 5NTD), compared to those acting on ADO degradation (ADA, AMPD1, AMPD2). Finally, stimulation of rvSMCs with increasing doses of ADO induced a significant production of intracellular cAMP after 3 minutes (1 and 10 μM). Conclusions These data show that ADO acts as an important relaxant modulator of the vagina through the activation of its receptors, specifically ADORA2A, and the consequent cAMP-dependent adenylate cyclase re-sponse. However, in vitro studies show that ADO also acts trough the NO-dependent pathway. These results therefore demonstrate for the first time a new relaxant pathway in distal vagina, ADO signaling, that results deeply intertwined with the well-known NO pathway. This finding unravels new biological mechanisms underlying vagina physiology that need to be further studied in future research. Disclosure No.

Comparative Analysis of Orthosteric and Allosteric GLP-1R Agonists' Effects on Insulin Secretion from Healthy, Diabetic, and Recovered INS-1E Pancreatic Beta Cells.

Despite the availability of different treatments for type 2 diabetes (T2D), post-diagnosis complications remain prevalent; therefore, more effective treatments are desired. Glucagon-like peptide (GLP)-1-based drugs are currently used for T2D treatment. They act as orthosteric agonists for the GLP-1 receptor (GLP-1R). In this study, we analyzed in vitro how the GLP-1R orthosteric and allosteric agonists augment glucose-stimulated insulin secretion (GSIS) and intracellular cAMP production (GSICP) in INS-1E pancreatic beta cells under healthy, diabetic, and recovered states. The findings from this study suggest that allosteric agonists have a longer duration of action than orthosteric agonists. They also suggest that the GLP-1R agonists do not deplete intracellular insulin, indicating they can be a sustainable and safe treatment option for T2D. Importantly, this study demonstrates that the GLP-1R agonists variably augment GSIS through GSICP in healthy, diabetic, and recovered INS-1E cells. Furthermore, we find that INS-1E cells respond differentially to the GLP-1R agonists depending on both glucose concentration during and before treatment and/or whether the cells have been previously exposed to these drugs. In conclusion, the findings described in this manuscript will be useful in determining in vitro how pancreatic beta cells respond to T2D drug treatments in healthy, diabetic, and recovered states.

Steroid-Induced Ocular Hypertension in Mice Is Differentially Reduced by Selective EP2, EP3, EP4, and IP Prostanoid Receptor Agonists.

We tested five chemically and metabolically stable prostaglandin (PG) receptor agonists in a mouse model of dexamethasone-induced ocular hypertension (OHT). Whilst all compounds significantly (p < 0.05, ANOVA) lowered intraocular pressure (IOP) after twice-daily bilateral topical ocular dosing (5 µg/dose) over three weeks, the time course and magnitude of the responses varied. The onset of action of NS-304 (IP-PG receptor agonist) and rivenprost (EP4-PG receptor agonist) was slower than that of misoprostol (mixed EP2/EP3/EP4-PG receptor agonist), PF-04217329 (EP2-PG receptor agonist), and butaprost (EP2-PG receptor agonist). The rank order of IOP-lowering efficacies aligned with the onset of actions of these compounds. Peak IOP reductions relative to vehicle controls were as follows: misoprostol (74.52%) = PF-04217329 (74.32%) > butaprost (65.2%) > rivenprost (58.4%) > NS-304 (55.3%). A literature survey indicated that few previously evaluated compounds (e.g., latanoprost, timolol, pilocarpine, brimonidine, dorzolamide, cromakalim analog (CKLP1), losartan, tissue plasminogen activator, trans-resveratrol, sodium 4-phenyl acetic acid, etc.) in various animal models of steroid-induced OHT were able to match the effectiveness of misoprostol, PF-04217329 or butaprost. Since a common feature of the latter compounds is their relatively high affinity and potency at the EP2-PG receptor sub-type, which activates the production of intracellular cAMP in target cells, our studies suggest that drugs selective for the EP2-PG receptor may be suited to treat corticosteroid-induced OHT.

The polybasic region in Gαi proteins: Relevant or not? Insights from Gαi3 research

Heterotrimeric G proteins are responsible for signal transduction from G-protein-coupled receptors (GPCRs) to intracellular effectors. This process is only possible when G proteins are located on the inner side of the cell membrane due to the specific localization of GPCR receptors. The Gα subunit is directed to the cell membrane through several signals, including modification by fatty acid moieties, interaction with the Gβγ complex, and, as observed in some Gα proteins, the presence of basic amino acid residues in the N-terminal region. In this work, we focused on investigating the influence of the polybasic region on the localization and function of a representative member of the Gαi family, Gαi3. Through the use of confocal microscopy and fluorescence lifetime microscopy, we showed that, in the case of this protein, neutralizing the positive charge does not significantly affect its abundance in the cell membrane. However, it does affect its spatial arrangement concerning the dopamine D2 receptor and influences inhibitory effect of Gαi3 on intracellular cAMP production triggered by D2 receptor stimulation. Moreover, in this work, we have shown, for the first time, that nonlipidated Gαi3 binds to negatively charged lipids through electrostatic interactions, and membrane fluidity plays a significant role in this interaction.

In-depth analysis of Gαs protein activity by probing different fluorescently labeled guanine nucleotides.

G proteins are interacting partners of G protein-coupled receptors (GPCRs) in eukaryotic cells. Upon G protein activation, the ability of the Gα subunit to exchange GDP for GTP determines the intracellular signal transduction. Although various studies have successfully shown that both Gαs and Gαi have an opposite effect on the intracellular cAMP production, with the latter being commonly described as "more active", the functional analysis of Gαs is a comparably more complicated matter. Additionally, the thorough investigation of the ubiquitously expressed variants of Gαs, Gαs(short) and Gαs(long), is still pending. Since the previous experimental evaluation of the activity and function of the Gαs isoforms is not consistent, the focus was laid on structural investigations to understand the GTPase activity. Herein, we examined recombinant human Gαs by applying an established methodological setup developed for Gαi characterization. The ability for GTP binding was evaluated with fluorescence and fluorescence anisotropy assays, whereas the intrinsic hydrolytic activity of the isoforms was determined by a GTPase assay. Among different nucleotide probes, BODIPY FL GTPγS exhibited the highest binding affinity towards the Gαs subunit. This work provides a deeper understanding of the Gαs subunit and provides novel information concerning the differences between the two protein variants.

Structural Insight on GPR119 Agonist as Potential Therapy for Type II Diabetes: A Comprehensive Review.

Diabetes Mellitus (DM) is a long-term metabolic condition that is characterized by excessive blood glucose. DM is the third most death-causing disease, leading to retinopathy, nephropathy, loss of vision, stroke, and cardiac arrest. Around 90% of the total cases of diabetic patients have Type II Diabetes Mellitus (T2DM). Among various approaches for the treatment of T2DM. G proteincoupled receptors (GPCRs) 119 have been identified as a new pharmacological target. GPR119 is distributed preferentially in the pancreas β-cells and gastrointestinal tract (enteroendocrine cells) in humans. GPR119 receptor activation elevates the release of incretin hormones such as Glucagon-Like Peptide (GLP1) and Glucose Dependent Insulinotropic Polypeptide (GIP) from intestinal K and L cells. GPR119 receptor agonists stimulate intracellular cAMP production via Gαs coupling to adenylate cyclase. GPR119 has been linked to the control of insulin release by pancreatic β-cells, as well as the generation of GLP-1 by enteroendocrine cells in the gut, as per in vitro assays. The dual role of the GPR119 receptor agonist in the treatment of T2DM leads to the development of a novel prospective anti-diabetic drug and is thought to have decreased the probability of inducing hypoglycemia. GPR119 receptor agonists exert their effects in one of two ways: either by promoting glucose absorption by β-cells, or by inhibiting α-cells' ability to produce glucose. In this review, we summarized potential targets for the treatment of T2DM with special reference to GPR119 along with its pharmacological effects, several endogenous as well as exogenous agonists, and its pyrimidine nucleus containing synthetic ligands.

PS-BPB09-2: DEVELOPMENT OF LONG-ACTING HUMAN ADRENOMEDULLIN FC FUSION PROTEINS

Background: Human adrenomedullin (hAM) is a hypotensive peptide hormone with strong anti-inflammatory effects. AM consists of 52 amino acid residues with an amidated C-terminus and a ring structure formed by intramolecular disulfide bonds. Both of these structures are necessary for binding to receptors and to exert their biological functions. AM has also shown therapeutic effects in various animal disease models, including inflammatory bowel disease, ischemic heart disease, sepsis and stroke. However, the short half-life of native AM in the blood required continuous administration for treatment. To solve this, we have developed four human IgG1 and IgG4 Fc fusion proteins containing full-length human AM or AM(6–52). Methods: Recombinant Fc-AM derivatives were generated using mammalian cells. After that, we tested the biological activities of Fc-fusion AMs (Fc-AMs) to stimulate the intracellular accumulation of cAMP in HEK-293 cells stably expressing the AM1 receptor. Next, we compared IgG1-AM (6–52) and IgG4-AM (6–52) concentrations in the peripheral blood and tissues of rats after subcutaneous injection. In addition, the antihypertensive effect of Fc-AM was examined on spontaneously hypertensive rats (SHRs) which were given high salt diets. Results: We have developed four Fc-AMs, which are long-acting AM derivatives in mammalian cells. Fc-AMs produced in mammalian cells do not require refolding or amidation. Indeed, Fc-AMs could be measured by mAM assay, which recognizes the amidated form, indicating that Fc-AMs produced in mammalian cells were amidated. Fc-AMs, generated in a mammalian cell production system can therefore easily be produced in large amounts with few purification steps. The Fc-AMs stimulated intracellular cAMP production in cultured cells stably expressing the AM type I receptor in vitro. Fc-AM (6–52) induced higher cAMP levels for the receptor than Fc-AM. The pharmacokinetic study was performed using N-terminal deficient AM derivatives, which have strong receptor binding ability. Sufficient concentrations of IgG1-AM (6–52) and IgG4-AM (6–52) were observed in blood 14 days after a single subcutaneous administration. Tissue transfer to the kidney and small intestine was observed after administration of IgG1-AM (6–52) or IgG4-AM (6–52) to rats. In addition, the single subcutaneous administration of IgG4-AM (6–52) suppressed the increase in blood pressure in SHRs which were loaded with high salt diets. Conclusion: Fc-AM produced from mammalian cells is easily prepared and seems to be an effective new therapeutic agent for hypertension.

Abstract 101: Association Of Snx19 With Flotillin-1 And Caveolin-1 In Its Lipid Raft Localization And Regulation Of D 1 R Endocytosis And Blood Pressure

Sorting nexin 19 (SNX19), a member of the SNX-PXA-RGS-PXC subfamily of sorting nexins, plays a vital role in lipid raft microdomain localization, but its association with flotillin-1 and caveolin-1, two of the most important lipid raft components, and in particular its linkage to the endocytosis and trafficking of dopamine D1 receptor (D 1 R) have not been determined in the renal proximal tubule (RPT). In the present study, we found that SNX19 was localized with lipid rafts in human and mouse RPT cells (RPTCs), which regulated D 1 R endocytosis. In wild type (WT) mouse RPTCs, SNX19 co-localized with flotillin-1 and caveolin-1, which was increased by treatment with fenoldopam (Fen, 25 nM, 30 min, n=4), a D1-like receptor agonist but not by vehicle (Veh). The targeting of SNX19 in lipid rafts and its colocalization with flotillin-1 and caveolin-1 were attenuated by deletion of a flotillin binding domain ( EEGPGTETETGLPVS ) (ΔFlot1), a caveolin-1 binding domain ( YHTVNRRYREF ) (ΔCav1), or both domains (ΔFlot1/Cav) in mouse RPTCs. The increase in intracellular cAMP production caused by Fen was also impaired or eliminated by the deletion of either flotillin-1 or caveolin-1 or both binding domains (WT-Veh: 100±16.6%, n=3, WT-Fen: 195.3±10.6%, n=4; ΔFlot1-Veh: 93.1±11.8%, n=4, ΔFlot1-Fen: 144.8±9.4%, n=3; ΔCav1-Veh: 90.9±16.3%, n=3, ΔCav1-Fen: 106.9±11.7%, n=4; ΔFlot/Cav-Veh: 63.8±10.3%, n=4, ΔFlot/Cav-Fen: 86.5±8.1%, n=4). Fen (25 nM, 30 min, n=3) decreased the colocalization of SNX19 with microtubules without an effect on actin filaments, which was abrogated by nocodazole (10 μM, 1hr), a microtubule depolymerization inhibitor. Furthermore, the renal-selective silencing of SNX19 in C57BL/6 mice (n=3) by the renal subcapsular infusion of specific SNX19 siRNA decreased renal sodium excretion and increased systolic blood pressure (Mock siRNA: 96.8±0.88 mmHg, n=3; SNX19 siRNA: 117.7±5.77 mmHg, n=3). In conclusion, SNX19 contains both flotillin-1 and caveolin-1 binding domains and its binding with flotillin-1 and caveolin-1 are important in SNX19-mediated D 1 R signaling, renal sodium transport, and blood pressure regulation.

McCune–Albright syndrome: description of three clinical cases, features of diagnostics and treatment

McCune Albright syndrome (MAS) is a rare disorder caused by a sporadic postzygotic mutation in the GNAS gene, which encodes the alpha subunit of the Gs signaling protein.Permanent activation of the Gs protein leads to uncontrolled production of intracellular cAMP, and autonomic hyperfunction of target organs. Estimated prevalence of the disease – 1\100000 to 1\1000000. For the first time, MAS syndrome was described in 1936 as a triad of symptoms – fibrous dysplasia of bones (FD), spots on the skin of the color of «coffee with milk» and premature sexual development. However, it is now known, that the phenotype of the disease is much more complex. This is primarily due to the mosaic type of mutation in the GNAS gene, which determines a wide range of clinical manifestations, and presents certain difficulties in the diagnosis of this syndrome. Various endocrinopathies can be manifestations of MAS like an acromegaly, a hyperthyroidism, Cushing’s syndrome, hypophospatemic osteomalacia as well as damage to other organs and systems, namely the pathology of the gastrointestinal tract, hepato-biliary cardiovascular systems. This article presents three late diagnosed clinical cases of MAS in patients with coffee-milk spots and FD, multinodular goiter; in two cases in combination with acromegaly. We have demonstrated the possibilities of drug therapy for acromegaly in MAS, as well as the experience of using bisphosphonate for the treatment of FD.The knowledge of clinical manifestations of the MAS, early diagnostics and possibilities of drug therapy in the management of these patients have the important prognostic value for improving the quality of patient’s life.

Functional characterization of melanocortin-3 receptor in rainbow trout (Oncorhynchus mykiss).

The melanocortin-3 receptor (MC3R) is an important regulator of energy homeostasis and inflammation in mammals. However, its function in teleost fish needs to be further explored. In this study, we characterized rainbow trout MC3R (rtMC3R), which encoded a putative protein of 331 amino acids. Phylogenetic and chromosomal synteny analyses showed that rtMC3R was closely related to bony fishes. Quantitative PCR (qPCR) revealed that the transcripts of rtMC3R were highly expressed in the brain and muscle. The cellular function of rtMC3R was further verified by the signal-pathway-specific luciferase reporter assays. Four agonists such as α-MSH, β-MSH, ACTH (1-24), and NDP-MSH can active rtMC3R, increasing the production of intracellular cAMP and upregulating MAPK/ERK signals. Moreover, we found that rtMC3R stimulated with α-MSH and NDP-MSH can significantly inhibit the NF-κB signaling pathway. This research will be helpful for further studies on the function of MC3R in rainbow trout, especially the role of energy metabolism and immune regulation.

History-dependent dopamine release increases cAMP levels in most basal amygdala glutamatergic neurons to control learning.

Dopaminergic inputs to basal amygdala (BA) instruct learning of motivational salience. This learning depends on intracellular plasticity signals such as cyclic adenosine monophosphate (cAMP), which is regulated by activation of dopamine receptors. We examine the dynamics of dopamine release and downstream signaling during multiple salient events occurring within tens of seconds. We perform real-time tracking and manipulation of cAMP in BA neurons invitro and invivo. Optogenetically evoked release of dopamine drives proportional increases in cAMP in almost all BA glutamatergic neurons, suggesting widespread actions of dopamine across neurons preferring positive or negative valence. This cAMP response decreases across trials with short intertrial intervals owing to depression of dopamine release. No such depression is evident when photostimulating cAMP production directly. cAMP and protein kinase A responses to repeated appetitive or aversive stimuli also exhibit pronounced depression. Thus, history-dependent dynamics of dopamine and cAMP may regulate learning of temporally clustered, salient stimuli.

Regulation of melanocortin-5 receptor pharmacology by two isoforms of MRAP2 in ricefield eel (Monopterus albus)

The melanocortin-5 receptor (MC5R) has been implicated in the regulation of exocrine gland secretion, immune regulation, and muscle fatty acid oxidation in mammals. Melanocortin-2 receptor accessory protein 2 (MRAP2) can modulate trafficking, ligand binding, and signaling of melanocortin receptors. To explore potential interaction between ricefield eel (Monopterus albus) MC5R and MRAP2s (maMC5R, maMRAP2X1, and maMRAP2X2), herein we studied the pharmacological characteristics of maMC5R and its modulation by maMRAP2s expressed in the human embryonic kidney cells. Three agonists, α-melanocyte-stimulating hormone (α-MSH), ACTH (1–24), and [Nle4, D-Phe7]-α-MSH, could bind to maMC5R and induce intracellular cAMP production dose-dependently. Compared with human MC5R (hMC5R), maMC5R displayed decreased maximal binding but higher binding affinity to α-MSH or ACTH (1–24). When stimulated with α-MSH or ACTH (1–24), maMC5R showed significantly lower EC50 and maximal response than hMC5R. Two maMRAP2s had no effect on cell surface expression of maMC5R, whereas they significantly increased maximal binding. Only maMRAP2X2 significantly decreased the binding affinity of ACTH (1–24). Both maMRAP2X1 and maMRAP2X2 significantly reduced maMC5R efficacy but did not affect ligand sensitivity. The availability of maMC5R pharmacological characteristics and modulation by maMRAP2s will assist the investigation of its roles in regulating diverse physiological processes in ricefield eel.

The Rho kinase (ROCK) inhibitor Y-27632 reduces the β2-adrenoceptor density but enhance cAMP formation in primary equine bronchial epithelial cells

The present study addresses the effect of the Rho-kinase (ROCK) inhibitor Y-27632 on the β2-adrenoceptor density and β-agonist-stimulated intracellular second messenger cAMP formation in primary equine bronchial epithelial cells (EBEC). Y-27632 significantly decreased the β2-adrenoceptor number (Bmax) without markedly affecting the receptor affinity (dissociation constant, KD) to the radioligand [125I]-iodocyanopindolol (ICYP). In contrast, Y-27632 augmented the β-agonist-stimulated intracellular cAMP production. Herein, Y-27632 markedly increased the maximal cAMP responses (Emax) (isoproterenol > epinephrine > norepinephrine) but did not shift the β-agonist concentration-effect curves to the left. The β2-selective antagonist ICI 118.551 and the β1/β2-antagonsit propranolol but not the β1-selctive antagonist CGP 20712A reversed the isoproterenol-induced cAMP formation equally in Y-27632-treated and control EBEC, suggesting the effect was merely related to the β2-subtype. These results show that Y-27632 differentially regulates the receptor density and function. Thus, these findings provide the first evidence that the functional interaction of the β2-adrenoceptor and Rho-kinase (ROCK) signaling pathways decreases the receptor expression but enhances receptor downstream cAMP formation. This differential regulation of the receptor density and function by Y-27632 should be further reconsidered with regard to the beneficial effect of the drug in asthma therapy.

Abstract 1704: Discovery and characterization of a novel triple A1/A2a/A2b adenosine receptor antagonist for cancer immunotherapy

Abstract Introduction: The adenosine pathways via type A2a and A2b receptors are known to suppress immunity by regulating the functions of immune cells, such as macrophages, dendritic cells, MDSC, T cells and NK cells in tumor microenvironment. Also, the adenosine pathway through an A1 receptor induces tumor growth by promoting cancer cell proliferation. On the other hand, it has been recently reported that the downregulation of A1R induces expression of tumor PD-L1. Herein, we introduce a novel antagonist of adenosine receptor, HM87277, which has potent antagonistic activity against three types of adenosine receptors A1R, A2aR and A2bR, and present preclinical results that the triple antagonist HM87277 may be more effective in treating cancer patients through appropriate combination with immune checkpoint blockers (ICBs). Materials and Methods: Receptor binding affinities were assessed using human adenosine receptors of type A1, A2a, A2b and A3. Cellular functional activity tests such as cAMP production. To identify adenosine receptor downstream signaling in cancer cells, p-ERK and p-JNK were measured by western blotting in SK-BR3 cells. PD-L1 induction by adenosine receptor antagonist was determined by flow cytometry using Canto II (BD) in PD-L1 expressing SK-MEL-28 cells. For cytotoxicity assay of T cell, isolated human CD8+ T cells were co-cultured with A1R expressing cancer cells in the presence of a high concentration of adenosine. Antitumor activity of HM87277 alone and in combination with ICBs was assessed in syngeneic mouse models using murine cancer cell lines. Results: HM87277 showed strong binding affinity toward human adenosine receptors of type A1, A2a and A2b, and potently inhibited the NECA-mediated production of intracellular cAMP in HEK293-A2aR cells. HM87277 significantly increased the proliferation of effector T cells. HM87277 potently inhibited the p-ERK and p-JNK, and induced the expression of PD-L1 in A1R-expressing cancer cells. As well, the combination of HM87277 and anti-PD-L1 mAb was observed to have synergistic inhibitory effect on tumor cell growth in co-culture with SK-MEL-28 and CD8 + T cells. Furthermore, in the mouse syngeneic model using murine cancer cell lines, HM87277 effectively inhibited tumor growth alone or in combination with anti-PD-L1 mAb. Conclusion: These results indicate that a triple antagonist HM87277, which simultaneously antagonizes type A1, A2a and A2b adenosine receptors, may not only stimulate immune cells such as cytotoxic CD8+ T cell but also inhibit the growth of cancer cells themselves through inhibition of ERK and JNK pathway. In addition, it showed a strong combination effect by enhancing the sensitivity to ICBs through the increase of PD-L1 in tumor cells. Therefore, it is considered that the triple antagonist HM87277 is worth developing as a potential anticancer agent. Citation Format: Sunyoung Jang, Seunah Jun, Hosun Lee, Yongtaek Lee, Joo-Yun Byun, Junghwa Park, Yu-Yon Kim, Young Gil Ahn, YoungHoon Kim, Kwee Hyun Suh. Discovery and characterization of a novel triple A1/A2a/A2b adenosine receptor antagonist for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1704.

Molecular Cloning and Functional Characterization of Three 5-HT Receptor Genes (HTR1B, HTR1E, and HTR1F) in Chickens.

The serotonin (5-hydroxytryptamine, 5-HT) signaling system is involved in a variety of physiological functions, including the control of cognition, reward, learning, memory, and vasoconstriction in vertebrates. Contrary to the extensive studies in the mammalian system, little is known about the molecular characteristics of the avian serotonin signaling network. In this study, we cloned and characterized the full-length cDNA of three serotonin receptor genes (HTR1B, HTR1E and HTR1F) in chicken pituitaries. Synteny analyses indicated that HTR1B, HTR1E and HTR1F were highly conserved across vertebrates. Cell-based luciferase reporter assays showed that the three chicken HTRs were functional, capable of binding their natural ligands (5-HT) or selective agonists (CP94253, BRL54443, and LY344864) and inhibiting intracellular cAMP production in a dose-dependent manner. Moreover, activation of these receptors could stimulate the MAPK/ERK signaling cascade. Quantitative real-time PCR analyses revealed that HTR1B, HTR1E and HTR1F were primarily expressed in various brain regions and the pituitary. In cultured chicken pituitary cells, we found that LY344864 could significantly inhibit the secretion of PRL stimulated by vasoactive intestinal peptide (VIP) or forskolin, revealing that HTR1F might be involved in the release of prolactin in chicken. Our findings provide insights into the molecular mechanism and facilitate a better understanding of the serotonergic modulation via HTR1B, HTR1E and HTR1F in avian species.

Regulation of Melanocortin-5 Receptor Pharmacology by Two Isoforms of MRAP2 in Ricefield Eel (Monopterus albus)

The melanocortin-5 receptor (MC5R) has been implicated in the regulation of exocrine gland secretion, immune regulation, and muscle fatty acid oxidation. However, its function in fish is not well established. Melanocortin-2 receptor accessory protein 2 (MRAP2) can modulate trafficking, ligand binding, and signaling of melanocortin receptors. Ricefield eel (Monopterus albus) is an economically and evolutionarily important fish widely distributed in tropics and subtropics. To explore potential interaction between eel MC5R and MRAP2, herein we cloned ricefield eel mc5r, mrap2X1 and mrap2X2. Eel mc5r consisted of a 1056 bp open reading frame encoding a protein of 351 amino acids. Eel mrap2X1 consisted of a 708 bp open reading frame encoding a protein of 235 amino acids, while eel mrap2X2consisted of a 567 bp open reading frame encoding a protein of 188 amino acids. Multiple sequence alignment showed that maMRAP2X1 and maMRAP2X2 shared 90.43% identity. Interestingly, maMRAP2X2 lost the transmembrane domain. Phylogenetic analysis showed that maMC5R and maMRAP2s were closely related to piscine MC5Rs and MRAP2s, respectively. The maMC5R was further demonstrated to be a functional receptor and could be modulated by maMRAP2s in pharmacological studies. Three agonists, [Nle4, D-Phe7]-alpha-melanocyte stimulating hormone (NDP-MSH), alpha-MSH, and adrenocorticotropin (ACTH), could bind to maMC5R and induce intracellular cAMP production dose-dependently. Compared to human MC5R (hMC5R), maMC5R displayed a significantly decreased Bmax but higher binding affinity to alpha-MSH or ACTH. No significant difference in constitutive activity was observed between hMC5R and maMC5R. When stimulated with α-MSH and ACTH, maMC5R showed significantly lower EC50 and Rmax than that of hMC5R. Eel MRAP2s had no effect on cell surface and total expression of maMC5R, whereas they significantly increased Bmax. Only maMRAP2X2 significantly decreased the binding affinity of ACTH. Both maMRAP2X1 and maMRAP2X2 significantly reduced Rmax but did not affect EC50 in response to alpha-MSH or ACTH stimulation. The availability of maMC5R pharmacological characteristics and the modulation by maMRAP2s will facilitate the investigation of its function in regulating diverse physiological processes in ricefield eel.

TNF-α impairs EP4 signaling through the association of TRAF2-GRK2 in primary fibroblast-like synoviocytes.

Our previous study showed that chronic treatment with tumor necrosis factor-α (TNF-α) decreased cAMP concentration in fibroblast-like synoviocytes (FLSs) of collagen-induced arthritis (CIA) rats. In this study we investigated how TNF-α impairs cAMP homeostasis, particularly clarifying the potential downstream molecules of TNF-α and prostaglandin receptor 4 (EP4) signaling that would interact with each other. Using a cAMP FRET biosensor PM-ICUE3, we demonstrated that TNF-α (20 ng/mL) blocked ONO-4819-triggered EP4 signaling, but not Butaprost-triggered EP2 signaling in normal rat FLSs. We showed that TNF-α (0.02-20 ng/mL) dose-dependently reduced EP4 membrane distribution in normal rat FLS. TNF-α significantly increased TNF receptor 2 (TNFR2) expression and stimulated proliferation in human FLS (hFLS) via ecruiting TNF receptor-associated factor 2 (TRAF2) to cell membrane. More interestingly, we revealed that TRAF2 interacted with G protein-coupled receptor kinase (GRK2) in the cytoplasm of primary hFLS and helped to bring GRK2 to cell membrane in response of TNF-α stimulation, the complex of TRAF2 and GRK2 then separated on the membrane, and translocated GRK2 induced the desensitization and internalization of EP4, leading to reduced production of intracellular cAMP. Silencing of TRAF2 by siRNA substantially diminished TRAF2-GRK2 interaction, blocked the translocation of GRK2, and resulted in upregulated expression of membrane EP4 and intracellular cAMP. In CIA rats, administration of paroxetine to inhibit GRK2 effectively improved the symptoms and clinic parameters with significantly reduced joint synovium inflammation and bone destruction. These results elucidate a novel form of cross-talk between TNFR (a cytokine receptor) and EP4 (a typical G protein-coupled receptor) signaling pathways. The interaction between TRAF2 and GRK2 may become a potential new drug target for the treatment of inflammatory diseases.

Psychotropic drugs upregulate aquaporin-2 via vasopressin-2 receptor/cAMP/protein kinase A signaling in inner medullary collecting duct cells.

Psychotropic drugs may be associated with hyponatremia, but an understanding of how they induce water retention in the kidney remains elusive. Previous studies have postulated that they may increase vasopressin production in the hypothalamus without supporting evidence. In this study, we investigated the possibility of drug-induced nephrogenic syndrome of inappropriate antidiuresis using haloperidol, sertraline, and carbamazepine. Haloperidol, sertraline, or carbamazepine were treated in inner medullary collecting duct (IMCD) suspensions and primary cultured IMCD cells prepared from male Sprague-Dawley rats. The responses of intracellular cAMP production, aquaporin-2 (AQP2) protein expression and localization, vasopressin-2 receptor (V2R) and AQP2 mRNA, and cAMP-responsive element-binding protein (CREB) were tested with and without tolvaptan and the protein kinase A (PKA) inhibitors H89 and Rp-cAMPS. In IMCD suspensions, cAMP production was increased by haloperidol, sertraline, or carbamazepine and was relieved by tolvaptan cotreatment. In primary cultured IMCD cells, haloperidol, sertraline, or carbamazepine treatment increased total AQP2 and decreased phosphorylated Ser261-AQP2 protein expression. Notably, these responses were reversed by cotreatment with tolvaptan or a PKA inhibitor. AQP2 membrane trafficking was induced by haloperidol, sertraline, or carbamazepine and was also blocked by cotreatment with tolvaptan or a PKA inhibitor. Furthermore, upregulation of V2R and AQP2 mRNA and phosphorylated CREB was induced by haloperidol, sertraline, or carbamazepine and was blocked by tolvaptan cotreatment. We conclude that, in the rat IMCD, psychotropic drugs upregulate AQP2 via V2R-cAMP-PKA signaling in the absence of vasopressin stimulation. The vasopressin-like action on the kidney appears to accelerate AQP2 transcription and dephosphorylate AQP2 at Ser261.NEW & NOTEWORTHY It is unclear whether antipsychotic drugs can retain water in the kidney in the absence of vasopressin. This study demonstrates that haloperidol, sertraline, and carbamazepine can produce nephrogenic syndrome of inappropriate antidiuresis because they directly upregulate vasopressin-2 receptor and aquaporin-2 (AQP2) via cAMP/PKA signaling. We showed that, in addition to AQP2 trafficking, AQP2 protein abundance was rapidly increased by treatment with antipsychotic drugs in association with dephosphorylation of AQP2 at Ser261 and accelerated AQP2 transcription.

Direct protein delivery into intact plant cells using polyhistidine peptides.

Polyhistidine peptides (PHPs), sequences comprising only histidine residues (>His8), are effective cell-penetrating peptides for plant cells. Using PHP-fusion proteins, we aimed to deliver proteins into cultured plant cells from Nicotiana tabacum, Oryza sativa, and Cryptomeria japonica. Co-cultivation of cultured cells with fusion proteins combining maltose-binding protein (MBP), red fluorescent protein (RFP), and various PHPs (MBP-RFP-His8-His20) in one polypeptide showed the cellular uptake of fusion proteins in all plant cell lines. Maximum intracellular fluorescence was shown in MBP-RFP-His20. Further, adenylate cyclase (CyaA), a synthase of cyclic adenosine monophosphate (cAMP) activated by cytosolic calmodulin, was used as a reporter for protein delivery in living cells. A fusion protein combining MBP, RFP, CyaA, and His20 (MBP-RFP-CyaA-His20) was delivered into plant cells and increased intracellular fluorescence and cAMP production in all cell lines. The present study demonstrates that PHPs are effective carriers of proteins into the intracellular space of various cultured plant cells.

MRAP2 Interaction with Melanocortin-4 Receptor in SnakeHead (Channa argus).

The melanocortin-4 receptor (MC4R) plays an important role in the regulation of food intake and energy expenditure. Melanocortin-2 receptor accessory protein 2 (MRAP2) modulates trafficking, ligand binding, and signaling of MC4R. The Northern snakehead (Channa argus) is an economically important freshwater fish native to East Asia. To explore potential interaction between snakehead MC4R and MRAP2, herein we cloned snakehead mc4r and mrap2. The snakehead mc4r consisted of a 984 bp open reading frame encoding a protein of 327 amino acids, while snakehead mrap2 contained a 693 bp open reading frame encoding a protein of 230 amino acids. Synteny analysis indicated that mc4r was highly conserved with similar gene arrangement, while mrap2 contained two isoforms in teleost with different gene orders. Snakehead mc4r was primarily expressed in the brain, whereas mrap2 was expressed in the brain and intestine. Snakehead mc4r and mrap2 expression was modulated by fasting and refeeding. Further pharmacological experiments showed that the cloned snakehead MC4R was functional, capable of binding to peptide agonists and increasing intracellular cAMP production in a dose-dependent manner. Snakehead MC4R exhibited high constitutive activity. MRAP2 significantly decreased basal and agonist-stimulated cAMP signaling. These findings suggest that snakehead MC4R might be involved in energy balance regulation by interacting with MRAP2. Further studies are needed to elucidate MC4R in regulating diverse physiological processes in snakehead.