Cyclic Adenosine Monophosphate Accumulation Research Articles

Pharmacologic characterization of atogepant: A potent and selective calcitonin gene-related peptide receptor antagonist.

The trigeminal sensory neuropeptide calcitonin gene-related peptide (CGRP) is identified as an essential element in migraine pathogenesis. In vitro and in vivo studies evaluated pharmacologic properties of the CGRP receptor antagonist atogepant. Radioligand binding using 125I-CGRP and cyclic adenosine monophosphate (cAMP) accumulation assays were conducted in human embryonic kidney 293 cells to assess affinity, functional potency and selectivity. Atogepant in vivo potency was assessed in the rat nitroglycerine model of facial allodynia and primate capsaicin-induced dermal vasodilation (CIDV) pharmacodynamic model. Cerebrospinal fluid/brain penetration and behavioral effects of chronic dosing and upon withdrawal were evaluated in rats. Atogepant exhibited high human CGRP receptor-binding affinity and potently inhibited human α-CGRP-stimulated cAMP responses. Atogepant exhibited significant affinity for the amylin1 receptor but lacked appreciable affinities for adrenomedullin, calcitonin and other known neurotransmitter receptor targets. Atogepant dose-dependently inhibited facial allodynia in the rat nitroglycerine model and produced significant CIDV inhibition in primates. Brain penetration and behavioral/physical signs during chronic dosing and abrupt withdrawal were minimal in rats. Atogepant is a competitive antagonist with high affinity, potency and selectivity for the human CGRP receptor. Atogepant demonstrated a potent, concentration-dependent exposure/efficacy relationship between atogepant plasma concentrations and inhibition of CGRP-dependent effects.

Roflumilast 0.3% Cream: a Phosphodiesterase 4 Inhibitor for the Treatment of Chronic Plaque Psoriasis.

Plaque psoriasis is a chronic dermatologic autoimmune disease that affects adults and children. Roflumilast 0.3% cream is currently the only topical phosphodiesterase 4 inhibitor indicated for the treatment of plaque psoriasis in patients 12 years or older. Roflumilast inhibits phosphodiesterase 4 inhibitor enzyme leading to the accumulation of cyclic adenosine monophosphate, which suppresses the inflammatory mediators interferon-γ and tumor necrosis factor-α. Roflumilast, applied once daily, reaches steady state by day 15 and has a half life of approximately 4 days in adults. Roflumilast undergoes extensive hepatic metabolism by cytochrome P450 enzymes and conjugation. Roflumilast is 99% bound to plasma proteins. Roflumilast efficacy and safety were evaluated in the DERMIS-1 and DERMIS-2 clinical trials. These identically designed, double-blind, vehicle-controlled phase 3 trials randomized 881 patients to roflumilast 0.3% cream or vehicle, applied once daily for 8 weeks. In DERMIS-1, the Investigator Global Assessment success rate was 42.4% with roflumilast 0.3% cream compared with 6.1% with the vehicle (32.3%-46.9%; P <0.001). Similarly, in DERMIS-2, the Investigator Global Assessment success rate was 37.5% with roflumilast 0.3% cream compared with 6.9% with the vehicle (20.8%-36.9%; P <0.001). Of 881 participants, 1% discontinued treatment with roflumilast cream due to adverse reactions compared with 1.3% treated with vehicle. Urticaria at the application site (0.3%) was the most common adverse reaction that led to discontinuation of roflumilast. To date, topical corticosteroids are the most commonly used agents to treat mild plaque psoriasis. Sensitive areas are often challenging to treat with existing topical therapy, including corticosteroids. Topical roflumilast has shown to be effective in treating sensitive areas, including skin folds, and may be an alternative to systemic therapy for some patients. The Food and Drug Administration approved topical roflumilast for the treatment of plaque psoriasis, including intertriginous areas, for patients 12 years or older.

AaRgs1 and AaRgs2 differential regulate fungal development, stress response and appressorium-like formation in Alternaria alternata

Alternaria alternata can recognize and respond to physichemical cues from the host plant epidermis through a complex signaling network to initiate infectious structures formation. The primarily functions of RGS as a negative regulator of G protein signaling has been characterized in several model fungi, but its biological function in A. alternata remains unclear. In present study three RGS proteins were identified in A. alternata by Blast alignment and designed as AaRgs1, AaRgs2 and AaRgs3. Targeted genes deletion demonstrated that AaRgs1 and AaRgs2 play the opposite function in vegetative growth, melanin production and secretion, stress response and appressorium-like formation rate in A. alternata, while AaRgs3 had no certain regulatory effect. Differently, AaRgs1 negatively regulated tolerance to high-osmosis stress response and cell wall synthesis inhibitors by down regulating the expression level of AaHog1 and AaSlt2. AaRgs2 acts as a negative role in regulating appressorium-like formation rates through decreasing intracellular cyclic adenosine monophosphate (cAMP) accumulation. Deletion of AaRgs1 and AaRgs2 also resulted in significantly reducing penetrating ability and pathogenicity. The findings indicated that RGS proteins play multiple roles in fungal development, stress response and appressorium-like formation of A. alternata.

Computational Design of Potent and Selective d-Peptide Agonists of the Glucagon-like Peptide-2 Receptor.

Here, we designed three d-GLP-2 agonists that activated the glucagon-like peptide-2 receptor (GLP-2R) cyclic adenosine monophosphate (cAMP) accumulation without stimulating the glucagon-like peptide-1 receptor (GLP-1R). All the d-GLP-2 agonists increased the protein kinase B phosphorylated (p-AKT) expression levels in a time- and concentration-dependent manner in vitro. The most effective d-GLP-2 analogue boosted the AKT phosphorylation 2.28 times more effectively compared to the native l-GLP-2. The enhancement in the p-AKT levels induced by the d-GLP-2 analogues could be explained by GLP-2R's more prolonged activation, given that the d-GLP-2 analogues induce a lower β-arrestin recruitment. The higher stability to protease degradation of our d-GLP-2 agonists helps us envision their potential applications in enhancing intestinal absorption and treating inflammatory bowel illness while lowering the high dosage required by the current treatments.

Sweet Taste Receptor T1R3 Expressed in Leydig Cells Is Closely Related to Homeostasis of the Steroid Hormone Metabolism Profile

Taste receptor type 1 subunit 3 (T1R3) is initially expressed in mammal tongue for recognition and response of sweet/umami tastants and is critical to nutrient absorption, even endocrine. In this study, down-regulation of related steroidogenic enzymes such as StAR, 3β-HSD, CYP17A1, and 17β-HSD with the decrease of T1R3 expression was found in Leydig cells treated by a T1R3 inhibitor (lactisole). The abundances of progesterone, 17a-hydroxyprogesterone, androstenedione, testosterone, and deoxycorticosterone were down-regulated by 2.3, 3.5, 1.4, 1.6, and 2.2 times, respectively, after T1R3 inhibition. In addition, opposite results were found in saccharin sodium treatment. T1R3 activation contributed to intracellular cyclic adenosine monophosphate (cAMP) accumulation (14.41 ± 0.58 vs 20.21 ± 0.65) and increased testosterone (20.31 ± 3.49 vs 50.01 ± 7.44) and steroidogenic metabolite levels. Coadministration of human chorionic gonadotropin and saccharin sodium resulted in elevating the testosterone and cAMP levels and enhancing the expression levels of steroidogenic-related factors. Similarly, intratesticular injection of lactisole and saccharin sodium further confirmed that T1R3 inhibition/activation affected the expression of related steroidogenic enzymes and the testosterone levels in mice. The above findings suggest that T1R3 plays a role in testicular steroidogenesis.

PTHrP Modulates the Proliferation and Osteogenic Differentiation of Craniofacial Fibrous Dysplasia-Derived BMSCs.

Fibrous dysplasia (FD) is a skeletal stem cell disease caused by mutations in the guanine nucleotide-binding protein, alpha-stimulating activity polypeptide (GNAS) gene, which results in the abnormal accumulation of cyclic adenosine monophosphate (cAMP) and hyperactivation of downstream signaling pathways. Parathyroid hormone-related protein (PTHrP) is secreted by the osteoblast lineage and is involved in various physiological and pathological activities of bone. However, the association between the abnormal expression of PTHrP and FD, as well as its underlying mechanism, remains unclear. In this study, we discovered that FD patient-derived bone marrow stromal cells (FD BMSCs) expressed significantly higher levels of PTHrP during osteogenic differentiation and exhibited greater proliferation capacity but impaired osteogenic ability compared to normal control patient-derived BMSCs (NC BMSCs). Continuous exogenous PTHrP exposure on the NC BMSCs promoted the FD phenotype in both in vitro and in vivo experiments. Through the PTHrP/cAMP/PKA axis, PTHrP could partially influence the proliferation and osteogenesis capacity of FD BMSCs via the overactivation of the Wnt/β-Catenin signaling pathway. Furthermore, PTHrP not only directly modulated cAMP/PKA/CREB transduction but was also demonstrated as a transcriptional target of CREB. This study provides novel insight into the possible pathogenesis involved in the FD phenotype and enhances the understanding of its molecular signaling pathways, offering theoretical evidence for the feasibility of potential therapeutic targets for FD.

Abstract 11076: Cardiac AKAP12 Overexpression Reduces Cyclic Amp Accumulation in the Vicinity of β 2 Ar

Introduction: In Heart Failure (HF), signaling downstream the β 2- Adrenergic Receptor (β 2 AR) is critical. Sympathetic stimulation of β 2 AR triggers Protein Kinase A (PKA) dependent phosphorylation of proteins that regulate cardiac function. Cyclic adenosine monophosphate (cAMP) accumulation in the vicinity of β 2 AR is crucial for PKA stimulation. cAMP levels are regulated in part by phosphodiesterases (PDEs). A Kinase Anchoring Protein 12 (AKAP12) has been reported to directly bind β 2 AR, PKA, and PDE4. Previously, we reported that cardiac overexpression of AKAP12 (oxAKAP12) in both male and female mice reduced left ventricular ejection fraction (EF) and fractional shortening (FS) significantly after 14-days of chronic Isoproterenol treatment when compared to control groups. Hypothesis: AKAP12 overexpression progresses HF development by reducing local cAMP levels in the vicinity of β 2 AR. Methods: AC16 cells (Human ventricular cell lines) were grouped into [1] control group or [2] cells stably transfected to overexpress human AKAP12 (oxAKAP12). cAMP accumulation in real-time downstream the β 2 AR was detected for 60 minutes in live cells using the luciferase-based biosensor (GloSensor) post-treatment with 10μM Epinephrine (EPI). Additionally, cells were pre-treated for 2 hours with either 10μM Rolipram (selective PDE4 inhibitor) or 0.1mM IBMX (non-selective PDE inhibitor) followed by 10μM EPI treatment. Results: oxAKAP12 cells (expressing AKAP12 protein 3 folds higher than controls) had significantly lower maximum cAMP accumulation post-EPI treatment downstream the β 2 AR (2.06 ± 0.32) compared to controls (9.04 ± 0.73), [p=0.0001]. Rolipram pretreatment increased cAMP levels in the oxAKAP12 group to (3.75± 0.72), however, the control group was still significantly higher (9.63 ± 0.95), [p=0.0032]. IBMX pretreatment increased cAMP levels in the oxAKAP12 group to (6.94 ± 1.55) which wasn’t significantly different from the control group (9.75 ± 2.52), [p=0.4152]. Conclusions: Cardiac overexpression of AKAP12 significantly reduces cAMP levels downstream of the β 2 AR. Potentially through scaffolding other PDEs aside from PDE4.

Constitutively Activating Mutants of Equine LH/CGR Constitutively Induce Signal Transduction and Inactivating Mutations Impair Biological Activity and Cell-Surface Receptor Loss In Vitro.

The signal transduction of the equine lutropin/choriogonadotropin receptor (eLH/CGR) is unclear in naturally occurring activating/inactivating mutants of this receptor, which plays an important role in reproductive physiology. We undertook the present study to determine whether conserved structurally related mutations in eLH/CGR exhibit similar mechanisms of signal transduction. We constructed four constitutively activating mutants (M398T, L457R, D564G, and D578Y) and three inactivating mutants (D405N, R464H, and Y546F); measured cyclic adenosine monophosphate (cAMP) accumulation via homogeneous time-resolved fluorescence assays in Chinese hamster ovary cells; and investigated cell-surface receptor loss using an enzyme-linked immunosorbent assay in human embryonic kidney 293 cells. The eLH/CGR-L457R-, -D564G-, and -D578Y-expressing cells exhibited 16.9-, 16.4-, and 11.2-fold increases in basal cAMP response, respectively. The eLH/CGR-D405N- and R464H-expressing cells presented a completely impaired signal transduction, whereas the Y546F-expressing cells exhibited a small increase in cAMP response. The cell-surface receptor loss was 1.4- to 2.4-fold greater in the activating-mutant-expressing cells than in wild-type eLH/CGR-expressing cells, but was completely impaired in the D405N- and Y546F-expressing cells, despite treatment with a high concentration of agonist. In summary, the state of activation of eLH/CGR influenced agonist-induced cell-surface receptor loss, which was directly related to the signal transduction of constitutively activating mutants.

Constitutive Activating Eel Luteinizing Hormone Receptors Induce Constitutively Signal Transduction and Inactivating Mutants Impair Biological Activity.

In contrast to the human lutropin receptor (hLHR) and rat LHR (rLHR), very few naturally occurring mutants in other mammalian species have been identified. The present study aimed to delineate the mechanism of signal transduction by three constitutively activating mutants (designated M410T, L469R, and D590Y) and two inactivating mutants (D383N and Y546F) of the eel LHR, known to be naturally occurring in human LHR transmembrane domains. The mutants were constructed and measured cyclic adenosine monophosphate (cAMP) accumulation via homogeneous time-resolved fluorescence assays in Chinese hamster ovary (CHO)-K1 cells. The activating mutant cells expressing eel LHR-M410T, L469R, and D590Y exhibited a 4.0-, 19.1-, and 7.8-fold increase in basal cAMP response without agonist treatment, respectively. However, inactivating mutant cells expressing D417N and Y558F did not completely impaired signal transduction. Specifically, signal transduction in the cells expressing activating mutant L469R was not occurred with a further ligand stimulation, showing that the maximal response exhibited approximately 53% of those of wild type receptor. Our results suggested that the constitutively activating mutants of the eel LHR consistently occurred without agonist treatment. These results provide important information of LHR function in fish and regulation with regard to mutations of highly conserved amino acids in glycoprotein hormone receptors.

Ivabradine augments high-frequency dynamic gain of the heart rate response to low- and moderate-intensity vagal nerve stimulation under β-blockade.

Our previous study indicated that intravenously administered ivabradine (IVA) augmented the dynamic heart rate (HR) response to moderate-intensity vagal nerve stimulation (VNS). Considering an accentuated antagonism, the results were somewhat paradoxical; i.e., the accentuated antagonism indicates that an activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels via the accumulation of intracellular cyclic adenosine monophosphate (cAMP) augments the HR response to VNS, whereas the inhibition of HCN channels by IVA also augmented the HR response to VNS. To remove the possible influence from the accentuated antagonism, we examined the effects of IVA on the dynamic vagal control of HR under β-blockade. In anesthetized rats (n = 7), the right vagal nerve was stimulated for 10 min according to binary white noise signals between 0 and 10 Hz (V0-10), between 0 and 20 Hz (V0-20), and between 0 and 40 Hz (V0-40). The transfer function from VNS to HR was estimated. Under β-blockade (propranolol, 2 mg/kg iv), IVA (2 mg/kg iv) did not augment the asymptotic low-frequency gain but increased the asymptotic high-frequency gain in V0-10 (0.53 ± 0.10 vs. 1.74 ± 0.40 beats/min/Hz, P < 0.01) and V0-20 (0.79 ± 0.14 vs. 2.06 ± 0.47 beats/min/Hz, P < 0.001). These changes, which were observed under a minimal influence from sympathetic background tone, may reflect an increased contribution of the acetylcholine-sensitive potassium channel (IK,ACh) pathway after IVA, because the HR control via the IK,ACh pathway is faster and acts in the frequency range higher than the cAMP-mediated pathway.NEW & NOTEWORTHY Since ivabradine (IVA) inhibits hyperpolarization-activated cyclic nucleotide-gated channels, interactions among the sympathetic effect, vagal effect, and IVA can occur in the control of heart rate (HR). To remove the sympathetic effect, we estimated the transfer function from vagal nerve stimulation to HR under β-blockade in anesthetized rats. IVA augmented the high-frequency dynamic gain during low- and moderate-intensity vagal nerve stimulation. Untethering the hyperpolarizing effect of acetylcholine-sensitive potassium channels after IVA may be a possible underlying mechanism.

Inhibition of PDE4 by apremilast attenuates skin fibrosis through directly suppressing activation of M1 and T cells.

Systemic sclerosis (SSc) is a life-threatening chronic connective tissue disease with the characteristics of skin fibrosis, vascular injury, and inflammatory infiltrations. Though inhibition of phosphodiesterase 4 (PDE4) has been turned out to be an effective strategy in suppressing inflammation through promoting the accumulation of intracellular cyclic adenosine monophosphate (cAMP), little is known about the functional modes of inhibiting PDE4 by apremilast on the process of SSc. The present research aimed to investigate the therapeutic effects and underlying mechanism of apremilast on SSc. Herein, we found that apremilast could markedly ameliorate the pathological manifestations of SSc, including skin dermal thickness, deposition of collagens, and increased expression of α-SMA. Further study demonstrated that apremilast suppressed the recruitment and activation of macrophages and T cells, along with the secretion of inflammatory cytokines, which accounted for the effects of apremilast on modulating the pro-fibrotic processes. Interestingly, apremilast could dose-dependently inhibit the activation of M1 and T cells in vitro through promoting the phosphorylation of CREB. In summary, our research suggested that inhibiting PDE4 by apremilast might provide a novel therapeutic option for clinical treatment of SSc patients.

Signal Transduction Mechanisms for Glucagon-Induced Somatolactin Secretion and Gene Expression in NileTilapia (Oreochromis niloticus) Pituitary Cells.

Glucagon (GCG) plays a stimulatory role in pituitary hormone regulation, although previous studies have not defined the molecular mechanism whereby GCG affects pituitary hormone secretion. To this end, we identified two distinct proglucagons, Gcga and Gcgb, as well as GCG receptors, Gcgra and Gcgrb, in Nile tilapia (Oreochromis niloticus). Using the cAMP response element (CRE)-luciferase reporter system, tilapia GCGa and GCGb could reciprocally activate the two GCG receptors expressed in human embryonic kidney 293 (HEK293) cells. Quantitative real-time PCR analysis revealed that differential expression of the Gcga and Gcgb and their cognate receptors Gcgra and Gcgrb was found in the various tissues of tilapia. In particular, the Gcgrb is abundantly expressed in the neurointermediate lobe (NIL) of the pituitary gland. In primary cultures of tilapia NIL cells, GCGb effectively stimulated SL release, with parallel rises in the mRNA levels, and co-incubation with the GCG antagonist prevented GCGb-stimulated SL release. In parallel experiments, GCGb treatment dose-dependently enhanced intracellular cyclic adenosine monophosphate (cAMP) accumulation with increasing inositol 1,4,5-trisphosphate (IP3) concentration and the resulting in transient increases of Ca2+ signals in the primary NIL cell culture. Using selective pharmacological approaches, the adenylyl cyclase (AC)/cAMP/protein kinase A (PKA) and phospholipase C (PLC)/IP3/Ca2+/calmodulin (CaM)/CaMK-II pathways were shown to be involved in GCGb-induced SL release and mRNA expression. Together, these results provide evidence for the first time that GCGb can act at the pituitary level to stimulate SL release and gene expression via GCGRb through the activation of the AC/cAMP/PKA and PLC/IP3/Ca2+/CaM/CaMK-II cascades.

Characterization of SGLT1-mediated glucose transport in Caco-2 cell monolayers, and absence of its regulation by sugar or epinephrine

Caco-2 cells are increasingly used to study the absorption of drugs and nutrients, including D-glucose, an important nutrient that mainly gets absorbed from the intestine by the sodium/glucose cotransporter 1 (SGLT1). However, disadvantages of Caco-2 cells for such studies have been reported, e.g., D-glucose cannot elicit translocation of the intracellular pool of SGLT1 to the apical membrane, the origin of the cells affects glucose uptake, and Caco-2 cells exhibit heterogeneity. This study aimed to characterize SGLT1-mediated glucose transport across Caco-2 cell monolayers. We found that at lower glucose concentrations (5 mM) SGLT1 contributes more to total glucose transport than at higher (10 mM) glucose concentrations, suggesting contributions by another transporter at higher glucose concentrations. This contrasts with the in vivo situation, where SGLT1 dominant glucose transporter at all glucose concentrations. We also tested whether known regulators like sugars or catecholamines can stimulate glucose transport across Caco-2 cell monolayers. Neither epinephrine nor 2-deoxy-D-glucose could stimulate glucose transport. Moreover, the epinephrine could not induce accumulation of cyclic adenosine monophosphate (cAMP) in Caco-2 cells, indicating the absence of a functional β2-adrenoceptor in Caco-2 cells, which could explain the lack of epinephrine effect on glucose transport. Also, Caco-2 cells may lack some kinases required for increased SGLT1 transport. Overall, SGLT1-mediated glucose transport and its regulation in Caco-2 cells differ from that in vivo, and caution is advised when extrapolating glucose transport results obtained with this model to the in vivo situation.

Constitutive Activation and Inactivation of Mutations Inducing Cell Surface Loss of Receptor and Impairing of Signal Transduction of Agonist-Stimulated Eel Follicle-Stimulating Hormone Receptor.

In the present study, we investigated the signal transduction of mutants of the eel follicle-stimulating hormone receptor (eelFSHR). Specifically, we examined the constitutively activating mutant D540G in the third intracellular loop, and four inactivating mutants (A193V, N195I, R546C, and A548V). To directly assess functional effects, we conducted site-directed mutagenesis to generate mutant receptors. We measured cyclic adenosine monophosphate (cAMP) accumulation via homogeneous time-resolved fluorescence assays in Chinese hamster ovary (CHO-K1) cells and investigated cell surface receptor loss using an enzyme-linked immunosorbent assay in human embryonic kidney (HEK) 293 cells. The cells expressing eelFSHR-D540G exhibited a 23-fold increase in the basal cAMP response without agonist treatment. The cells expressing A193V, N195I, and A548V mutants had completely impaired signal transduction, whereas those expressing the R546C mutant exhibited little increase in cAMP responsiveness and a small increase in signal transduction. Cell surface receptor loss in the cells expressing inactivating mutants A193V, R546C, and A548V was clearly slower than in the cell expressing the wild-type eelFSHR. However, cell surface receptor loss in the cells expressing inactivating mutant N195I decreased in a similar manner to that of the cells expressing the wild-type eelFSHR or the activating mutant D540G, despite the completely impaired cAMP response. These results provide important information regarding the structure–function relationships of G protein-coupled receptors during signal transduction.

Thienorphine induces antinociception without dependence through activation of κ- and δ-, and partial activation of μ- opioid receptor

Thienorphine hydrochloride is a new anti-relapse drug for opioid abusers that is currently in phase II clinical trial. In the present study, the antinociception, dependence, and signal transduction induced by thienorphine were examined. Thienorphine showed a potent antinociception effect in acetic acid-induced writhing test and formalin test. In the hot plate test and tail-flick test, thienorphine presented the typical partial opioid agonist character with a ceiling dose-response curve in addition to a bell-shaped curve. The hot plate test revealed that thienorphine induced approximately 50% of antinociception in μ receptor knockout (μ-KO) mice compared to wild-type controls (P < 0.05). The κ, δ selective antagonist nor-binaltorphimine (nor-BNI), and naltrindole decreased approximately 50–60% of theinorphine antinociception in μ-KO mice, respectively. The ORL1 receptor-selective antagonist J113397 did not affect theinorphine antinociception in μ-KO mice. Chronic treatment with thienorphine (1.5 mg/kg) induced some tolerance that was lower compared to buprenorphine or morphine addition. In contrast to buprenorphine or morphine, thienorphine did not lead to psychological dependence by conditioned place preference (CPP). The maximum inhibition of thienorphine on protein kinase A (PKA) activity was about 36%, 100%, 100%, and 12% in CHO-μ/κ/δ/ORL1-PKAcatEGFP cells, respectively. Similar results were observed in cyclic adenosine monophosphate (cAMP) accumulation inhibited by thienorphine in cells. Thienorphine significantly increased pERK1/2 in CHO-κ/δ-PKAcatEGFP cells. These results indicated that thienorphine induced analgesia through activation of κ- and δ-, partial activation of μ- opioid receptor without a bias between G-protein- and β-arrestin-mediated pathways. Thienorphine might be used for antinociception with minimal adverse effects.

Non-Classical Effects of the cAMP Accumulation Activators In Vivo.

Purpose: Wound-healing dipyridamole- and papaverine-based aerosols (D1/D2) as activators of the accumulation of cyclic adenosine monophosphate are promising drugs that can accelerate wound healing in wound processes of various origins. Methods: 128 rats were used in the study, including 38 in a pharmacological experiment on a model of stencil wounds and 90 in an experiment that studied the effect of spray on the number of CD34 cells in the blood of rats with chemically induced immunodeficiency. Immunodeficiency was caused by the fivefold administration of cyclophosphamide and prednisone. The expression level of CD34 was determined using flow cytofluorimeter. Results: Dipyridamole- and papaverine-based aerosols of two compositions (with and without ascorbic acid) have pronounced reparative properties, significantly accelerating epithelialization and healing of stencil wounds in rats. In terms of this type of action, they are somewhat superior to dexpanthenol. Dipyridamole- and papaverine-based aerosols have the ability to produce beneficial effect on the entire body’s immune system by stimulating the division of pluripotent CD34 cells. The combined effect of papaverine and dipyridamole on tissues leads to selective stimulation of the division of pluripotent cells in the wound, and contributes to a six-fold acceleration of restoration of the animal’s immune system after induced immunodeficiency. Conclusion: Topical application of D1/D2 aerosol samples on the skin of rats contributed to a statistically significant acceleration of regeneration processes. In terms of the appearance of granulations and epithelialization of wounds, D1/D2 aerosols were superior to dexpanthenol ointment.

A Noncompetitive and Selective Prostaglandin EP2 Antagonist with Anti‐inflammatory Property

The prostaglandin E2 (PGE2) receptor subtype EP2 promotes chronic inflammation, modulates tumorigenesis, and regulates immune function. Inhibition of EP2 receptors reduces inflammation in rodent models of epilepsy, endometriosis, and arthritis, and decreases tumor cell growth. Therefore, inhibiting EP2 receptors could be a promising method for treatment of inflammatory diseases. To date all reported EP2 antagonists have a purely competitive mode of action, which can be overcome by high concentrations of agonist. Herein, we report the characterization of a novel compound belongs to substituted pyrroloquinoxaline scaffold as the first EP2 antagonist that features a noncompetitive mode of action. Cell‐based time‐resolved fluorescence resonance energy transfer (TR‐FRET) assays were developed in 384‐well format to measure the accumulation of cyclic adenosine monophosphate (cAMP) induced by PGE2, on the basis of the fact that activation of the Gas‐coupled EP2 receptor induces cAMP formation. At a low concentration (3 μM) of the pyrroloquinoxaline antagonist, a saturating concentration of PGE2 (3 μM) could reverse the inhibition of EP2 receptor activation in human EP2‐overexpressing C6 glioma (C6G‐EP2) cells. At higher antagonist concentrations (10–30 μM), the inhibition of EP2 receptors could not be fully overcome by the agonist. Meanwhile, the maximal agonist response inhibition by the antagonist increased with increasing concentration. The maximum effect (cAMP induction) by PGE2 was decreased to 81.9 ±1.44%, unlike 100% for a typical competitive antagonist. The antagonist itself had no effect on the TR‐FRET signal since there was no change in the TR‐FRET signal in the absence of C6G‐EP2 cells. The antagonist was reversible as demonstrated by washing out the antagonist three times each at 80% efficiency. After washing out the antagonist, the maximal response of C6G‐EP2 cells to PGE2 increased to 97.6 ± 0.69% from 86.4 ± 0.85% (p &lt; 0.0001, one‐way ANOVA with Bonferroni). Furthermore, the antagonist exhibited little to no effect on the EC50 or maximal response of other Gas‐coupled prostanoid receptors (EP4, IP, and DP1). The agonist EC50s for EP2, EP4, IP and DP1 receptors were shifted 13.2, 2.2, 1.1 and 0.95‐fold to the right, respectively, in 30 μM antagonist. To determine whether the noncompetitive antagonist exerts an anti‐inflammatory action we studied the expression of IL‐6 and IL‐1β in the murine BV2 microglial cell line stably expressing human EP2 receptors (BV2‐hEP2). Treatment of these cells with a &gt;EC90 concentration (30 nM) of the EP2 selective agonist, ONO‐AE1‐259, caused elevated mRNA levels of the two cytokines (measured by qRT‐PCR) that was suppressed by the antagonist (20 μM). Thus, the pyrroloquinoxaline is the first noncompetitive EP2 receptor antagonist. This compound may provide a basis for further development of novel noncompetitive EP2 antagonists.Support or Funding InformationThis work was supported by the National Institutes of Health [Grant Numbers 1R01NS097776 (RD), 1 U01 AG052460 (TG)]. C.J. is a member of the joint training program between Emory University School of Medicine and Xiangya School of Medicine, Central South University, and is supported by the China Scholarship Council. We gratefully acknowledge the gift of ONO‐AE1‐259 by Ono Pharmaceuticals.

Differential activation of G protein-mediated signaling by synthetic cannabinoid receptor agonists.

Synthetic cannabinoid receptor agonists (SCRAs) are new psychoactive substances associated with acute intoxication and even death. However, the molecular mechanisms through which SCRAs may exert their toxic effects remain unclear—including the potential differential activation of G protein subtypes by cannabinoid receptor type 1 (CB1), a major target of SCRA. We measured CB1‐mediated activation of Gαs and Gαi/o proteins by SCRAs by examining stimulation (pertussis toxin, PTX treated) as well as inhibition (non‐PTX treated) of forskolin (FSK)‐induced cyclic adenosine monophosphate (cAMP) accumulation in human embryonic kidney (HEK) cells stably expressing CB1. Real‐time measurements of stimulation and inhibition of cAMP levels were made using a BRET biosensor. We found that the maximum concentration of SCRAs tested (10 µmol L−1), increased cAMP levels 12%‐45% above that produced by FSK alone, while the phytocannabinoid THC did not significantly alter cAMP levels in PTX‐treated HEK‐CB1 cells. All SCRAs had greater potency to inhibit FSK‐induced cAMP levels than to stimulate cAMP levels. The rank order of potencies for SCRA stimulation of cAMP (Gαs) was PB‐22 > 5F‐MDMB‐PICA > JWH‐018 ≈ AB‐FUBINACA > XLR‐11. By contrast, the potency of SCRAs for inhibition of cAMP (Gαi/o) was 5F‐MDMB‐PICA > AB‐FUBINACA > PB‐22 > JWH‐018 > XLR‐11. The different rank order of potency and EMax of the SCRAs to stimulate Gαs‐like signaling compared to Gαi/o signaling suggests differences in G protein preference between SCRAs. Understanding the apparent differences among these drugs may contribute to unravelling their complex effects in humans.

A New Highly Thyrotropin Receptor-Selective Small-Molecule Antagonist with Potential for the Treatment of Graves' Orbitopathy.

The thyrotropin receptor (TSHR) is the target for autoimmune thyroid stimulating antibodies (TSAb) triggering hyperthyroidism. Whereas elevated thyroid hormone synthesis by the thyroid in Graves' disease can be treated by antithyroid agents, for the pathogenic activation of TSHR in retro-orbital fibroblasts of the eye, leading to Graves' orbitopathy (GO), no causal TSHR directed therapy is available. Due to the therapeutic gap for severe GO, TSHR inhibitors were identified by high-throughput screening in Chinese hamster ovary cells expressing the TSHR. Stereo-selective synthesis of the screening hits led to the molecule S37, which contains seven chiral centers. Enantiomeric separation of the molecule S37 resulted in the enantiopure molecule S37a-a micro-molar antagonist of thyrotropin-induced cyclic adenosine monophosphate accumulation in HEK 293 cells expressing the TSHR. The unique rigid bent shape of molecule S37a may mediate the observed high TSHR selectivity. Most importantly, the closely related follitropin and lutropin receptors were not affected by this compound. S37a not only inhibits the TSHR activation by thyrotropin itself but also activation by monoclonal TSAb M22 (human), KSAb1 (murine), and the allosteric small-molecule agonist C2. Disease-related ex vivo studies in HEK 293 cells expressing the TSHR showed that S37a also inhibits cyclic adenosine monophosphate formation by oligoclonal TSAb, which are highly enriched in GO patients' sera. Initial in vivo pharmacokinetic studies revealed no toxicity of S37a and a remarkable 53% oral bioavailability in mice. In summary, a novel highly selective inhibitor for the TSHR is presented, which has promising potential for further development for the treatment of GO.

Are the Effects of the Cholera Toxin and Isoproterenol on Human Keratinocytes' Proliferative Potential Dependent on Whether They Are Co-Cultured with Human or Murine Fibroblast Feeder Layers?

Human keratinocyte culture has provided the means to treat burns, wounds and skin pathologies. To date, to efficiently culture keratinocytes, cells are cultured on an irradiated feeder layer (iFL), either comprising human (iHFL) or murine (i3T3FL) fibroblasts, and the culture medium is supplemented with a cyclic adenosine monophosphate (cAMP) accumulation inducing agent such as isoproterenol (ISO) or cholera toxin (CT). Previous studies have characterized how the feeder layer type and the cAMP inducer type influence epithelial cells’ phenotype independently from one another, but it is still unknown if an optimal combination of feeder layer and cAMP inducer types exists. We used sophisticated statistical models to search for a synergetic effect of feeder layer and cAMP inducer types on human keratinocytes’ proliferative potential. Our data suggests that, when culturing human keratinocytes, using iHFL over i3T3FL increases population doublings and colony-forming efficiency through signaling pathways involving Ak mouse strain thymoma (Akt, also known as protein kinase B) isoforms 1 to 3, signal transducer and activator of transcription 5 (STAT5), p53, and adenosine monophosphate activated protein kinase α1 (AMPKα1). Both tested cAMP inducers ISO and CT yielded comparable outcomes. However, no significant synergy between feeder layer and cAMP inducer types was detected. We conclude that, to promote human keratinocyte growth in the early passages of culture, co-culturing them with a human feeder layer is preferable to a murine feeder layer.