cAMP Levels In Cells Research Articles

Effect of fluoxetine on cyclic adenosine monophosphate response to follicle-stimulating hormone in HEK cells

Fluoxetine is an antidepressant that acts as a selective serotonin reuptake inhibitor. Fluoxetine has exhibited different mechanisms of action on many different cell lines. In this study, the effect of fluoxetine on cAMP production in human embryonic kidney (HEK) cells under the stimulation of bovine follicle-stimulating hormone were examined. HEK cells were incubated for 60 minutes with fluoxetine at different concentrations (0, 6.25, 12.5, 25 and 50 μM), and then cAMP accumulation, ATP level, and AMPK phosphorylation and cell viability were tested. Our results showed that fluoxetine at 25 and 50 μM attenuated cAMP levels in cells after bFSH stimulation. The depletion of cAMP at this concentration is accompanied by a decrease in ATP energy and an increased capacity to phosphorylate AMPK. As such, this study showed that fluoxetine induces a reduction in cAMP accumulation and that it is associated with the AMPK pathway in HEK cells

Phosphodiesterase 7B regulates prostanoid‐ but not ß‐adrenergic‐stimulated cAMP levels in primary human airway smooth muscle cells

Human airway smooth muscle (HASM) cells contain G‐protein coupled receptor (GPCR) signal transduction pathways that are organized in the plasma membrane and form cAMP signaling compartments. One such compartment contains ß2‐adrenergic receptors (ß2AR) coupled to adenylyl cyclase (AC)6 and is principally localized in lipid rafts. Another compartment expresses E prostanoid receptors 2 and 4 (EP2/4) coupled to AC2 and is restricted to non‐raft regions of the plasma membrane. Various phosphodiesterase (PDE) isozymes modulate cyclic AMP (cAMP) levels in response to these pathways. Non‐specific or PDE‐family specific PDE inhibitors have been used to treat asthma and chronic obstructive pulmonary disease with limited success, in part due to expression of these PDE isoforms throughout the body. Drugs that inhibit specific PDE isozymes might produce therapeutic effects in airways with fewer off‐target effects. RNA‐sequencing data in HASM cells indicates that PDE7B is the third most highly expressed PDE isoform and includes expression of both the PDE7B‐201 and PDE7B‐203 splice variants. We sought to characterize the role of PDE7B in the lipid raft and non‐raft cAMP signaling compartments. We used siRNA to knock down both PDE7B splice variants, confirmed protein reduction via Western Blot analysis, and then observed live cells’ real‐time cAMP levels in response to either isoproterenol (ßAR agonist) or PGE2 (EP agonist). The cADDis‐FMP biosensor, which localizes in lipid raft regions, did not report altered cAMP dynamics in response to ß2AR or EPR agonists following PDE7B knockdown. However, the cADDis‐FS15 biosensor, which reports cAMP levels in non‐raft plasma membrane regions, reported significant increases in cAMP levels stimulated by EPR agonists but no change in cAMP levels stimulated by ß2AR agonist. PDE7B may play a unique role in regulating non‐raft associated EP2/4 signaling without altering cAMP levels in other areas of the cell, thereby presenting a novel therapeutic target for pulmonary diseases.

Crosstalk between β2- and α2-Adrenergic Receptors in the Regulation of B16F10 Melanoma Cell Proliferation.

Adrenergic receptors (AR) belong to the G protein-coupled receptor superfamily and regulate migration and proliferation in various cell types. The objective of this study was to evaluate whether β-AR stimulation affects the antiproliferative action of α2-AR agonists on B16F10 cells and, if so, to determine the relative contribution of β-AR subtypes. Using pharmacological approaches, evaluation of Ki-67 expression by flow cytometry and luciferase-based cAMP assay, we found that treatment with isoproterenol, a β-AR agonist, increased cAMP levels in B16F10 melanoma cells without affecting cell proliferation. Propranolol inhibited the cAMP response to isoproterenol. In addition, stimulation of α2-ARs with agonists such as clonidine, a well-known antihypertensive drug, decreased cancer cell proliferation. This effect on cell proliferation was suppressed by treatment with isoproterenol. In turn, the suppressive effects of isoproterenol were abolished by the treatment with either ICI 118,551, a β2-AR antagonist, or propranolol, suggesting that isoproterenol effects are mainly mediated by the β2-AR stimulation. We conclude that the crosstalk between the β2-AR and α2-AR signaling pathways regulates the proliferative activity of B16F10 cells and may therefore represent a therapeutic target for melanoma therapy.

Multifaceted Roles of cAMP Signaling in the Repair Process of Spinal Cord Injury and Related Combination Treatments.

Spinal cord injury (SCI) results in multiple pathophysiological processes, including blood–spinal cord barrier disruption, hemorrhage/ischemia, oxidative stress, neuroinflammation, scar formation, and demyelination. These responses eventually lead to severe tissue destruction and an inhibitory environment for neural regeneration.cAMP signaling is vital for neurite outgrowth and axonal guidance. Stimulating intracellular cAMP activity significantly promotes neuronal survival and axonal regrowth after SCI.However, neuronal cAMP levels in adult CNS are relatively low and will further decrease after injury. Targeting cAMP signaling has become a promising strategy for neural regeneration over the past two decades. Furthermore, studies have revealed that cAMP signaling is involved in the regulation of glial cell function in the microenvironment of SCI, including macrophages/microglia, reactive astrocytes, and oligodendrocytes. cAMP-elevating agents in the post-injury milieu increase the cAMP levels in both neurons and glial cells and facilitate injury repair through the interplay between neurons and glial cells and ultimately contribute to better morphological and functional outcomes. In recent years, combination treatments associated with cAMP signaling have been shown to exert synergistic effects on the recovery of SCI. Agents carried by nanoparticles exhibit increased water solubility and capacity to cross the blood–spinal cord barrier. Implanted bioscaffolds and injected hydrogels are potential carriers to release agents locally to avoid systemic side effects. Cell transplantation may provide permissive matrices to synergize with the cAMP-enhanced growth capacity of neurons. cAMP can also induce the oriented differentiation of transplanted neural stem/progenitor cells into neurons and increase the survival rate of cell grafts. Emerging progress focused on cAMP compartmentation provides researchers with new perspectives to understand the complexity of downstream signaling, which may facilitate the clinical translation of strategies targeting cAMP signaling for SCI repair.

Zebrafish Model as a Screen to Prevent Cyst Inflation in Autosomal Dominant Polycystic Kidney Disease.

In autosomal dominant polycystic kidney disease (ADPKD), kidney cyst growth requires the recruitment of CFTR (cystic fibrosis transmembrane conductance regulator), the chloride channel that is defective in cystic fibrosis. We have been studying cyst inflation using the zebrafish Kupffer’s vesicle (KV) as model system because we previously demonstrated that knocking down polycystin 2 (PC2) induced a CFTR-mediated enlargement of the organ. We have now quantified the PC2 knockdown by showing that it causes a 73% reduction in the number of KV cilia expressing PC2. According to the literature, this is an essential event in kidney cystogenesis in ADPKD mice. Additionally, we demonstrated that the PC2 knockdown leads to a significant accumulation of CFTR-GFP at the apical region of the KV cells. Furthermore, we determined that KV enlargement is rescued by the injection of Xenopus pkd2 mRNA and by 100 µM tolvaptan treatment, the unique and approved pharmacologic approach for ADPKD management. We expected vasopressin V2 receptor antagonist to lower the cAMP levels of KV-lining cells and, thus, to inactivate CFTR. These findings further support the use of the KV as an in vivo model for screening compounds that may prevent cyst enlargement in this ciliopathy, through CFTR inhibition.

Presence of TSHR in NK Cells and Action of TSH on NK Cells

Introduction: Thyroid-stimulating hormone receptor (TSHR) is widely expressed in human tissues and cells. TSHR is not only involved in thyroid disease but also in the neuroendocrine-immune regulatory network. However, no study has exclusively focused on the expression and function of TSHR in natural killer (NK) cells. Methods: We studied TSHR expression using reverse transcription PCR to verify TSHR mRNA transcripts in human and mouse NK cells. Human and mouse thyroid and liver tissues as well as peripheral blood mononuclear cells (PBMCs) or spleen lymphoid cells (SLCs) were used as controls. The TSHR protein levels in NK-92 cells were determined by immunofluorescence staining. The function of TSHR in NK cells was investigated by measuring the TSH-stimulated cAMP levels. Results: TSHR mRNA was detected in human and mouse NK cells as well as in NK-92 cells and had the same sequence as that of thyroid-derived, PBMC-derived, and liver-derived mRNA. The TSHR protein was also expressed in the cell membrane of NK-92 cells. Furthermore, the cAMP levels in NK-92 cells were significantly higher after adding 10² mIU/mL of bovine TSH at p < 0.05, which stimulated cAMP production in NK-92 cells. Conclusions: Our findings confirm that TSHR is present and functional in NK cells and provide key clues for the potential regulatory effects of TSH on TSHR in NK cells in the immune system.

Gs‐DREADD Regulation of ENaC

Activity of the epithelial Na+ channel (ENaC) fine-tunes renal Na+ excretion and consequently, contributes to the normal control of blood pressure. ENaC is expressed in the apical membrane of principal cells. Importantly, ENaC is activated by the hormone vasopressin via the V2 receptor, which is coupled to the heterotrimeric Gs protein. Stimulation of the V2 receptor rapidly activates adenylyl cyclase to increase intracellular cAMP levels. We have recently developed a mouse model with targeted expression of the Gs designer receptors exclusively activated by designer drugs (DREADD) in renal principal cells (GsD). Moreover, this mouse line has a luciferase reporter downstream of a cAMP response element binding protein site. As such, it allows in vivo reporting of cAMP levels. DREADD receptors are activated by clozapine N-oxide (CNO), which normally is an inert drug with no biological activity in the absence of DREADD receptors. We test here if CNO affects the activity of ENaC to influence renal salt and water excretion in GsD mice similarly to vasopressin. In these mice, we longitudinally monitored CNO-stimulated cAMP levels in principal cells using an IVIS Lumina XR. In parallel, we quantified CNO-dependent ENaC activity and renal sodium excretion. We observed that CNO increased ENaC activity in principal cells in freshly isolated tubules from GsD mice compared to control mice. Metabolic cages experiments demonstrated that CNO treatment (0.1 mg/kg) increased cAMP levels concomitantly with reducing sodium excretion similarly to dDAVP (1 µg) in GsD mice. Finally, in COS-7 cells expressing murine ENaC tagged with YFP, CPT-cAMP increased translocation of ENaC to the plasma membrane. Here we demonstrated the utility of a new principal cell-specific Gs-DREADD mouse line. This new mouse model will be an important tool for investigating the effects of abnormal cAMP signaling in principal cells possibly informing understanding of the cellular mechanism underpinning conditions like polycystic kidney disease.

Feasibility of cerium-doped LSO particles as a scintillator for x-ray induced optogenetics

Objective. Non-invasive light delivery into the brain is needed for in vivo optogenetics to avoid physical damage. An innovative strategy could employ x-ray activation of radioluminescent particles (RLPs) to emit localized light. However, modulation of neuronal or synaptic function by x-ray induced radioluminescence from RLPs has not yet been demonstrated. Approach. Molecular and electrophysiological approaches were used to determine if x-ray dependent radioluminescence emitted from RLPs can activate light sensitive proteins. RLPs composed of cerium doped lutetium oxyorthosilicate (LSO:Ce), an inorganic scintillator that emits blue light, were used as they are biocompatible with neuronal function and synaptic transmission. Main results. We show that 30 min of x-ray exposure at a rate of 0.042 Gy s−1 caused no change in the strength of basal glutamatergic transmission during extracellular field recordings in mouse hippocampal slices. Additionally, long-term potentiation, a robust measure of synaptic integrity, was induced after x-ray exposure and expressed at a magnitude not different from control conditions (absence of x-rays). We found that x-ray stimulation of RLPs elevated cAMP levels in HEK293T cells expressing OptoXR, a chimeric opsin receptor that combines the extracellular light-sensitive domain of rhodopsin with an intracellular second messenger signaling cascade. This demonstrates that x-ray radioluminescence from LSO:Ce particles can activate OptoXR. Next, we tested whether x-ray activation of the RLPs can enhance synaptic activity in whole-cell recordings from hippocampal neurons expressing channelrhodopsin-2, both in cell culture and acute hippocampal slices. Importantly, x-ray radioluminescence caused an increase in the frequency of spontaneous excitatory postsynaptic currents in both systems, indicating activation of channelrhodopsin-2 and excitation of neurons. Significance. Together, our results show that x-ray activation of LSO:Ce particles can heighten cellular and synaptic function. The combination of LSO:Ce inorganic scintillators and x-rays is therefore a viable method for optogenetics as an alternative to more invasive light delivery methods.

Anticancer activity of repurposed hemostatic agent desmopressin on AVPR2-expressing human osteosarcoma.

Osteosarcoma is the most prevalent primary bone malignancy. Due to its high aggressiveness, novel treatment strategies are urgently required to improve survival of patients with osteosarcoma, especially those with advanced disease. Desmopressin (dDAVP) is a widely used blood-saving agent that has been repurposed as an adjuvant agent for cancer management due to its antiangiogenic and antimetastatic properties. dDAVP acts as a selective agonist of the vasopressin membrane receptor type 2 (AVPR2) present in the microvascular endothelium and in some cancer cells, including breast, lung, colorectal and neuroendocrine tumor cells. Despite the fact that dDAVP has demonstrated its antitumor efficacy in a wide variety of tumor types, exploration of its potential anti-osteosarcoma activity has, to the best of our knowledge, not yet been conducted. Therefore, the aim of the present study was to evaluate the preclinical antitumor activity of dDAVP in osteosarcoma. Human MG-63 and U-2 OS osteosarcoma cell lines were used to assess in vitro and in vivo therapeutic effects of dDAVP. At low micromolar concentrations, dDAVP reduced AVPR2-expressing MG-63 cell growth in a concentration-dependent manner. In contrast, dDAVP exhibited no direct cytostatic effect on AVPR2-negative U-2 OS cells. As it would be expected for canonical AVPR2-activation, dDAVP raised intracellular cAMP levels in osteosarcoma cells, and coincubation with phosphodiesterase-inhibitor rolipram indicated synergistic antiproliferative activity. Cytostatic effects were associated with increased apoptosis, reduced mitotic index and impairment of osteosarcoma cell chemotaxis, as evaluated by TUNEL-labeling, mitotic body count in DAPI-stained cultures and Transwell migration assays. Intravenous administration of dDAVP (12 µg/kg; three times per week) to athymic mice bearing rapidly growing MG-63 xenografts, was indicated to be capable of reducing tumor progression after a 4-week treatment. No major alterations in animal weight, biochemical or hematological parameters were associated with dDAVP treatment, confirming its good tolerability and safety. Finally, AVPR2 expression was detected by immunohistochemistry in 66% of all evaluated chemotherapy-naive human conventional osteosarcoma biopsies. Taking these findings into account, repurposed agent dDAVP may represent an interesting therapeutic tool for the management of osteosarcoma. Further preclinical exploration of dDAVP activity on orthotopic or metastatic osteosarcoma models are required.

Novel Template Plasmids pCyaA'-Kan and pCyaA'-Cam for Generation of Unmarked Chromosomal cyaA' Translational Fusion to T3SS Effectors in Salmonella.

The type III secretion systems (T3SS) encoded in pathogenicity islands SPI-1 and SPI-2 are key virulence factors of Salmonella. These systems translocate proteins known as effectors into eukaryotic cells during infection. To characterize the functionality of T3SS effectors, gene fusions to the CyaA’ reporter of Bordetella pertussis are often used. CyaA’ is a calmodulin-dependent adenylate cyclase that is only active within eukaryotic cells. Thus, the translocation of an effector fused to CyaA’ can be evaluated by measuring cAMP levels in infected cells. Here, we report the construction of plasmids pCyaA’-Kan and pCyaA’-Cam, which contain the ORF encoding CyaA’ adjacent to a cassette that confers resistance to kanamycin or chloramphenicol, respectively, flanked by Flp recombinase target (FRT) sites. A PCR product from pCyaA’-Kan or pCyaA’-Cam containing these genetic elements can be introduced into the bacterial chromosome to generate gene fusions by homologous recombination using the Red recombination system from bacteriophage λ. Subsequently, the resistance cassette can be removed by recombination between the FRT sites using the Flp recombinase. As a proof of concept, the plasmids pCyaA’-Kan and pCyaA’-Cam were used to generate unmarked chromosomal fusions of 10 T3SS effectors to CyaA’ in S. Typhimurium. Each fusion protein was detected by Western blot using an anti-CyaA’ monoclonal antibody when the corresponding mutant strain was grown under conditions that induce the expression of the native gene. In addition, T3SS-1-dependent secretion of fusion protein SipA-CyaA’ during in vitro growth was verified by Western blot analysis of culture supernatants. Finally, efficient translocation of SipA-CyaA’ into HeLa cells was evidenced by increased intracellular cAMP levels at different times of infection. Therefore, the plasmids pCyaA’-Kan and pCyaA’-Cam can be used to generate unmarked chromosomal cyaA’ translational fusion to study regulated expression, secretion and translocation of Salmonella T3SS effectors into eukaryotic cells.

Inhibition of Glioma Cell Growth and Apoptosis Induction through Targeting Wnt10B Expression by Pyrazolo[4,3-c]pyridine-4-one.

BackgroundGliomas are commonly diagnosed tumors in the central nervous system that have an elevated mortality rate. The present study evaluated pyrazolo[4,3-c]pyridine-4-one (PP-4-one) as an anti-proliferative agent against glioma cells and investigated the associated mechanism.Material/MethodsThe changes in cell growth were analyzed by Cell Counting Kit-8 (CCK-8) and apoptosis by flow cytometry using Annexin V-FITC staining kit. The FACSCalibur flow cytometer was used for analysis of DNA content and western blotting for protein expression.ResultsThe PP-4-one treatment suppressed viability of U251, C6, and U87 cells significantly at a concentration of 0.25 μM. At a concentration of 16 μM, PP-4-one treatment for 72 hours suppressed viability of U251, C6, and U87 cells to 24%, 21%, and 20%, respectively. Treatment with PP-4-one suppressed cyclic 3′,5′-adenosine monophosphate (cAMP) levels in U251 and C6 cells significantly (P<0.05) depending on the concentration. The apoptotic cells were increased significantly (P<0.05) by PP-4-one treatment in U251 and C6 cell cultures. A considerable enhancement in the proportion of U251 and C6 cells in the G0/G1 phase was recorded on incubation with PP-4-one. Treatment of U251 and C6 cells with PP-4-one markedly enhanced p21 expression relative to the control. The B-cell lymphoma (Bcl-2) level in PP-4-one treated U251 and C6 cells was markedly lower relative to the control cells. The Bax, caspase-3, and caspase-9 levels were elevated markedly by PP-4-one treatment in U251 and C6 cells.ConclusionsThis study demonstrated that PP-4-one has anti-proliferative potential for glioma cells via targeting cAMP and Bcl-2 levels. It also promoted glioma cell apoptosis through caspase activation and arrest of the cell cycle. Thus, PP-4-one may be used to develop drug candidates for the glioma treatment.

Kaempferol in ameliorating diabetes-induced fibrosis and renal damage: An in vitro and in vivo study in diabetic nephropathy mice model

BackgroundKaempferol is a natural polyflavonol that has gained considerable attention as antidiabetic therapeutics. Recent reports emphasize the role of hyperglycemia and RhoA/Rho Kinase activity in the pathogenesis of diabetic nephropathy (DN). This study aims to evaluate the GLP-1 and insulin release along with RhoA/Rho Kinase inhibition pertaining to the anti-fibrotic and reno-protective effects of Kaempferol in DN. MethodsThe effect of Kaempferol on GLP-1 and insulin release along with underlying mechanisms (Ca2+ and cAMP levels) in GLUTag and MIN6 cells as well as in their co-culture has been evaluated. Further, the effect of Kaempferol on GLP-1 and insulin release was evaluated under in-vivo circumstances in the DN C57BL/6 mouse model. Histology and fibrosis specific staining was performed to study the renal injuries and fibrosis, while the expression of mRNA and protein of interest was evaluated by RT-PCR and western blot analysis. ResultsKaempferol treatment promoted the GLP-1 and insulin release, which was accompanied by increased intracellular levels of cAMP and Ca2+ in GLUTag and MIN6 cells. In agreement with in vitro studies, Kaempferol also increased the release of GLP-1 and insulin in the DN mouse model. Notably, Kaempferol showed the potential to ameliorate the histological changes as well as renal fibrosis while decreasing the expression levels of DN markers including TGF-β1, CTGF, fibronectin, collagen IV, IL-1β, RhoA, ROCK2, and p-MYPT1 in DN kidney tissues. A rise in the expression of E-cadherin and nephrin was also noted in the same study. ConclusionThis study establishes that Kaempferol ameliorates renal injury and fibrosis by enhancing the release of GLP-1, insulin, and inhibition of RhoA/Rho Kinase. This study recommends Kaempferol for further clinical trials to be developed as novel therapeutics for improving the renal function in DN patients.

Anti-glioma effect of 7-H-pyrrolo [2,3-di]pyrimidine derivative via induction of cell death

Purpose: To investigate the effect of 7-H-pyrrolo[2,3-di]pyrimidine derivative (7-HPPD) on glioma cell growth.Methods: Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, while apoptosis was assessed in Hoechst 33342 stained cells using flow cytometry. Changes in cell morphology were assessed by scanning electron microscopy (SEM).Results: Glioma cell viability showed a concentration-dependent decrease on exposure to 7-HPPD for 72 h (p &lt; 0.05). The viabilities of C6, U251 and U87 cells were reduced by 24, 28 and 31 %, respectively, following treatment with 20 μM 7-HPPD. Exposure to various concentrations of 7-HPPDresulted in a marked decrease in BrdU LI and cAMP levels in C6, U251 and U87 glioma cells (p &lt; 0.05). Moreover, 7-HPPD induced apoptosis in U251 and U87 cell cultures, as was evident in the condensation of chromatin material, presence of apoptotic bodies and intense blue fluorescence.Treatment of U251 cells with 7-HPPD for 72 h led to a significant increase in the proportion of cells in G0/G1 phase, and significant decrease in percentage of cells in G2/M and S phases. The population of rounded cells showed a significant rise with increase in 7-HPPD concentration from 10 to 20 μM (p &lt; 0.05).Conclusion: 7-HPPD inhibits growth and proliferation of glioma cells by inducing apoptosis. Therefore, it has a potential for application in glioma chemotherapy.&#x0D; Keywords: Glioma, Fluorescence, Metastasis, Apoptosis, Infiltration

Airway Relaxation Mechanisms and Structural Basis of Osthole to Improve Lung Function in Asthma

Background and objectiveOveruse of β2‐adrenoceptor agonist bronchodilators causes receptor desensitization, resulting in decreased efficacy and an increased risk of death in asthma patients. This is a critical unmet problem in asthma management, and novel drugs are urgently needed. Here, we report that osthole, a coumarin derived from a traditional Chinese medicine, induces complete relaxation of preconstricted airways in mouse precision‐cut lung slices, irrespective of β2‐adrenoceptor desensitization.MethodsMouse precision‐cut lung slices as an in vitro model were used to analyze airway relaxation. The cAMP level in human airway smooth muscle (ASM) or HEK293 cells was determined by phosphorylation of vasodilator‐stimulated phosphoprotein using Western blot or cAMP dependent reporter luciferase assay. The secretion of prostaglandin E2 (PGE2) in ASM cells or lung slices was measured by ELISA assay. The effect of osthole on airway hyperresponsiveness (AHR) was measured in both house dust mite (HDM)‐sensitized mice and naïve regulator of G‐protein signaling 2 (RGS2) KO mice. The binding sites of osthole with PDE4D5 catalytic domain were analyzed by crystallization and X‐ray diffraction.ResultsOsthole administered in vivo ameliorates AHR, a hallmark of asthma, in murine asthma models. Mechanistic studies revealed that osthole inhibits phosphodiesterase 4D (PDE4D) activity to amplify endogenous PGE2 signaling in ASM cells, which eventually triggers cAMP/protein kinase A‐dependent relaxation of airways. The crystal structure of the PDE4D complexed with osthole reveals that osthole binds to the catalytic site to prevent cAMP binding and hydrolysis. Together, our studies elucidate a specific molecular target and mechanism by which osthole induces airway relaxation.ConclusionIdentification of osthole binding sites on PDE4D will guide further development of novel therapeutics for β2‐adrenoceptor agonist‐resistant asthma.Support or Funding InformationThis work was supported in part by the National Institutes of Health (R01HL116849) and Nebraska State LB595 Research Program, USA; the National Laboratory of Biomacromolecules and the Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences (CAS); the CAS/SAFEA International Partnership Program for Creative Research Teams; the National Key R&amp;D Program of China (2017YFA0205501); the National Natural Science Foundation of China (21777192 and 31500623)

Stimulation of the β2 adrenergic receptor rapidly increases phosphorylation of the Na+‐Clcotransporter (NCC)

Adrenergic signaling is proposed to modulate activity of the thiazide‐sensitive Na+‐Clcotransporter (NCC) by direct effects on the renal distal convoluted tubule (DCT). However, there are conflicting data on whether, α1, β1 or β2‐adrenoceptors mediate these effects by activation of the with‐no‐lysine kinases (WNKs) and STE20/SPS1‐related proline alanine‐rich kinase (SPAK) kinase pathway or by other signaling pathways. Here, we determined using RT‐PCR that the β2‐adrenergic receptor was abundantly expressed in mpkDCT cells (a model of the DCT) and in DCT cells isolated from parvalbumin EGFP mice by fluorescence‐activated cell sorting. In mice administered salbutamol (β2 adrenergic receptor agonist used to treat asthma) for 30 min, phosphorylated NCC levels increased, indicating increased activity. However, mice developed hypokalemia, a condition that also increases NCC phosphorylation. To investigate direct effects of β2 adrenergic signaling on DCT cells independent of K+, the effects of salbutamol on mpkDCT cells and in kidney slices/tubule suspensions were examined. Salbutamol gradually increased NCC phosphorylation in the pharmacologically relevant concentration range of 0.01–10 μM, an effect observed after 15 min and maintained at 60 min. Changes in NCC phosphorylation were associated with increased cAMP levels in tubule suspensions, kidney slices and mpkDCT cells. A role for the NCC modulatory PKA/I‐1/PP1 pathway was suggested by attenuated effects of salbutamol on phosphorylated NCC during PKA/PP1 pathway inhibition or in slices from I‐1 KO mice. Inhibition of the WNK‐SPAK pathway with a WNK inhibitor also attenuated, but did not prevent, the salbutamol‐induced phosphorylation of NCC. In conclusion, β2 adrenergic receptor signaling modulates NCC phosphorylation in vivo and ex vivo via PKA and WNK‐dependent pathways. We speculate that this response may be a compensatory mechanism to limit electrogenic renal K+ secretion during β2 adrenergic receptor stimulation of muscle. Furthermore, frequent use of salbutamol in e.g. asthmatic patients may lead to renal NaCl retention and influence blood pressure.Support or Funding InformationThe Novo Nordisk Foundation, The Leducq Foundation and the Danish Medical Research Council.

Molecular Determinants of Signaling and Posttranslational Modifications of GPR64

Some of the adhesion G protein‐coupled receptors (aGPCRs) show constitutive activity in the absence of their large N‐terminal fragment (NTF). We previously demonstrated that GPR64/ADGRG2, an orphan aGPCR, regulates the secretion of parathyroid hormone by interacting with the calcium‐sensing receptor and elevating the cAMP levels in parathyroid adenoma cells. In HEK293 cells, we described the role of beta‐arrestins, dynamin, and GPCR kinases (GRKs) in the signaling of NTF‐truncated GPR64. Whether structural domains and motifs other than the NTF also regulate signaling, trafficking, and posttranslational modifications of GPR64 remains unknown. In the current study, we mutated a number of extracellular motifs that are conserved among zebrafish, mouse, and human GPR64. We measured cAMP production (time‐resolved FRET), surface expression (ELISA and biotinylation assays), and ubiquitination (co‐immunoprecipitation) of WT and mutant GPR64 in HEK293 cells. We discovered that several residues in the extracellular domains alter the surface expression, ubiquitination, and degradation of GPR64 at basal and tethered agonist‐stimulated conditions. The molecular mechanisms by which these residues regulate trafficking, signaling, and ubiquitination of GPR64 are currently under investigation.Support or Funding InformationThis work was supported by NIH grant 1R01GM13061701A1 (to N.B.). Student support was provided by an NIH‐NIGMS Initiative for Maximizing Student Development Grant (R25‐GM55036) (to A.A.G.).

Anti-Allergic and Anti-Inflammatory Effects of Undecane on Mast Cells and Keratinocytes.

The critical roles of keratinocytes and resident mast cells in skin allergy and inflammation have been highlighted in many studies. Cyclic adenosine monophosphate (cAMP), the intracellular second messenger, has also recently emerged as a target molecule in the immune reaction underlying inflammatory skin conditions. Here, we investigated whether undecane, a naturally occurring plant compound, has anti-allergic and anti-inflammatory activities on sensitized rat basophilic leukemia (RBL-2H3) mast cells and HaCaT keratinocytes and we further explored the potential involvement of the cAMP as a molecular target for undecane. We confirmed that undecane increased intracellular cAMP levels in mast cells and keratinocytes. In sensitized mast cells, undecane inhibited degranulation and the secretion of histamine and tumor necrosis factor α (TNF-α). In addition, in sensitized keratinocytes, undecane reversed the increased levels of p38 phosphorylation, nuclear factor kappaB (NF-κB) transcriptional activity and target cytokine/chemokine genes, including thymus and activation-regulated chemokine (TARC), macrophage-derived chemokine (MDC) and interleukin-8 (IL-8). These results suggest that undecane may be useful for the prevention or treatment of skin inflammatory disorders, such as atopic dermatitis, and other allergic diseases.

Cyclic AMP-dependent protein kinase and D1 dopamine receptors regulate diacylglycerol lipase-α and synaptic 2-arachidonoyl glycerol signaling.

Brain endocannabinoids serve as retrograde neurotransmitters, being synthesized in post-synaptic neurons "on demand" and released to bind pre-synaptic cannabinoid receptors and suppress glutamatergic or GABAergic transmission. The most abundant brain endocannabinoid, 2 arachidonoyl glycerol (2-AG), is primarily synthesized by diacylglycerol lipase-α (DGLα), which is activated by poorly understood mechanisms in response to calcium influx following post-synaptic depolarization and/or the activation of Gq -coupled group 1 metabotropic glutamate receptors. However, the impact of other neurotransmitters and their downstream signaling pathways on synaptic 2-AG signaling has not been intensively studied. Here, we found that DGLα activity in membrane fractions from transfected HEK293T cells was significantly increased by in vitro phosphorylation using cyclic AMP-dependent protein kinase (PKA). Moreover, PKA directly phosphorylated DGLα at Ser798 in vitro. Elevation of cAMP levels in HEK293 cells expressing DGLα increased Ser798 phosphorylation, as detected using a phospho-Ser798-specific antibody, and enhanced DGLα activity; this in situ enhancement of DGLα activity was prevented by mutation of Ser798 to Ala. We investigated the impact of PKA on synaptic 2-AG mobilization in mouse striatal slices by manipulating D1-dopamine receptor (D1R) signaling and assessing depolarization-induced suppression of excitation, a DGLα- and 2-AG-dependent form of short-term synaptic depression. The magnitude of depolarization-enhanced suppression of excitation in direct pathway medium spiny neurons was increased by pre-incubation with a D1R agonist, and this enhancement was blocked by post-synaptic inhibition of PKA. Taken together, these findings provide new molecular insights into the complex mechanisms regulating synaptic endocannabinoid signaling.

Cardioprotective GLP-1 metabolite prevents ischemic cardiac injury by inhibiting mitochondrial trifunctional protein-α.

Mechanisms mediating the cardioprotective actions of glucagon-like peptide 1 (GLP-1) were unknown. Here, we show in both ex vivo and in vivo models of ischemic injury that treatment with GLP-1(28-36), a neutral endopeptidase-generated (NEP-generated) metabolite of GLP-1, was as cardioprotective as GLP-1 and was abolished by scrambling its amino acid sequence. GLP-1(28-36) enters human coronary artery endothelial cells (caECs) through macropinocytosis and acts directly on mouse and human coronary artery smooth muscle cells (caSMCs) and caECs, resulting in soluble adenylyl cyclase Adcy10-dependent (sAC-dependent) increases in cAMP, activation of protein kinase A, and cytoprotection from oxidative injury. GLP-1(28-36) modulates sAC by increasing intracellular ATP levels, with accompanying cAMP accumulation lost in sAC-/- cells. We identify mitochondrial trifunctional protein-α (MTPα) as a binding partner of GLP-1(28-36) and demonstrate that the ability of GLP-1(28-36) to shift substrate utilization from oxygen-consuming fatty acid metabolism toward oxygen-sparing glycolysis and glucose oxidation and to increase cAMP levels is dependent on MTPα. NEP inhibition with sacubitril blunted the ability of GLP-1 to increase cAMP levels in coronary vascular cells in vitro. GLP-1(28-36) is a small peptide that targets novel molecular (MTPα and sAC) and cellular (caSMC and caEC) mechanisms in myocardial ischemic injury.

G Protein-Coupling of Adhesion GPCRs ADGRE2/EMR2 and ADGRE5/CD97, and Activation of G Protein Signalling by an Anti-EMR2 Antibody

The experimental evidence that Adhesion G Protein-Coupled Receptors (aGPCRs) functionally couple to heterotrimeric G proteins has been emerging in incremental steps, but attributing biological significance to their G protein signalling function still presents a major challenge. Here, utilising activated truncated forms of the receptors, we show that ADGRE2/EMR2 and ADGRE5/CD97 are G protein-coupled in a variety of recombinant systems. In a yeast-based assay, where heterologous GPCRs are coupled to chimeric G proteins, EMR2 showed broad G protein-coupling, whereas CD97 coupled more specifically to Gα12, Gα13, Gα14 and Gαz chimeras. Both receptors induced pertussis-toxin (PTX) insensitive inhibition of cyclic AMP (cAMP) levels in mammalian cells, suggesting coupling to Gαz. EMR2 was shown to signal via Gα16, and via a Gα16/Gαz chimera, to stimulate IP1 accumulation. Finally, using an NFAT reporter assay, we identified a polyclonal antibody that activates EMR2 G protein signalling in vitro. Our results highlight the potential for the development of soluble agonists to understand further the biological effects and therapeutic opportunities for ADGRE receptor-mediated G protein signalling.