Increase In cAMP Research Articles

Nitric oxide activates AMPK by modulating PDE3A in human pulmonary artery smooth muscle cells.

Phosphodiesterase 3 (PDE3), of which there are two isoforms, PDE3A and PDE3B, hydrolyzes cAMP and cGMP—cyclic nucleotides important in the regulation of pulmonary vascular tone. PDE3 has been implicated in pulmonary hypertension unresponsive to nitric oxide (NO); however, contributions of the two isoforms are not known. Furthermore, adenosine monophosphate‐activated protein kinase (AMPK), a critical regulator of cellular energy homeostasis, has been shown to be modulated by PDE3 in varying cell types. While AMPK has recently been implicated in pulmonary hypertension pathogenesis, its role and regulation in the pulmonary vasculature remain to be elucidated. Therefore, we utilized human pulmonary artery smooth muscle cells (hPASMC) to test the hypothesis that NO increases PDE3 expression in an isoform‐specific manner, thereby activating AMPK and inhibiting hPASMC proliferation. We found that in hPASMC, NO treatment increased PDE3A protein expression and PDE3 activity with a concomitant decrease in cAMP concentrations and increase in AMPK phosphorylation. Knockdown of PDE3A using siRNA transfection blunted the NO‐induced AMPK activation, indicating that PDE3A plays an important role in AMPK regulation in hPASMC. Treatment with a soluble guanylate cyclase (sGC) stimulator increased PDE3A expression and AMPK activation similar to that seen with NO treatment, whereas treatment with a sGC inhibitor blunted the NO‐induced increase in PDE3A and AMPK activation. These results suggest that NO increases PDE3A expression, decreases cAMP, and activates AMPK via the sGC‐cGMP pathway. We speculate that NO‐induced increases in PDE3A and AMPK may have implications in the pathogenesis and the response to therapies in pulmonary hypertensive disorders.

The Ca2+ channel CatSper is not activated by cAMP/PKA signaling but directly affected by chemicals used to probe the action of cAMP and PKA

The sperm-specific Ca2+ channel CatSper (cation channel of sperm) controls the influx of Ca2+ into the flagellum and, thereby, the swimming behavior of sperm. A hallmark of human CatSper is its polymodal activation by membrane voltage, intracellular pH, and oviductal hormones. Whether CatSper is also activated by signaling pathways involving an increase of cAMP and ensuing activation of PKA is, however, a matter of controversy. To shed light on this question, we used kinetic ion-sensitive fluorometry, patch-clamp recordings, and optochemistry to study transmembrane Ca2+ flux and membrane currents in human sperm from healthy donors and from patients that lack functional CatSper channels. We found that human CatSper is neither activated by intracellular cAMP directly nor indirectly by the cAMP/PKA-signaling pathway. Instead, we show that nonphysiological concentrations of cAMP and membrane-permeable cAMP analogs used to mimic the action of intracellular cAMP activate human CatSper from the outside via a hitherto-unknown extracellular binding site. Finally, we demonstrate that the effects of common PKA inhibitors on human CatSper rest predominantly, if not exclusively, on off-target drug actions on CatSper itself rather than on inhibition of PKA. We conclude that the concept of an intracellular cAMP/PKA-activation of CatSper is primarily based on unspecific effects of chemical probes used to interfere with cAMP signaling. Altogether, our findings solve several controversial issues and reveal a novel ligand-binding site controlling the activity of CatSper, which has important bearings on future studies of cAMP and Ca2+ signaling in sperm.

A dual and conflicting role for imiquimod in inflammation: A TLR7 agonist and a cAMP phosphodiesterase inhibitor

The Toll-like receptor 7 (TLR7) agonist imiquimod is an antitumor and antiviral drug used for the treatment of skin indications such as basal cell carcinoma, squamous cell carcinoma, and genital warts caused by the human papilloma virus. We show that imiquimod has TLR7-independent activity in which it directly inhibits phosphodiesterase (PDE), leading to cAMP increase, PKA-mediated CREB phosphorylation and subsequent CRE-dependent reporter transcription. The activation of the cAMP pathway by imiquimod is synergistically amplified by the β-adrenergic receptor agonist, isoproterenol. PDE inhibition is implied from cAMP measurements and CRE-reporter assays in intact RAW264.7 macrophages and HEK293T cells, and also directly demonstrated in-vitro using macrophages lysate. Moreover, molecular docking simulated the binding of imiquimod in the active site of PDE4B, enabled by the high molecular similarity between imiquimod and the adenine moiety of cAMP. As expected from the known anti-inflammatory role of cAMP inducers in stimulated macrophages, PDE inhibition by imiquimod results in reduced expression of the key pro-inflammatory cytokine TNFα, and enhanced expression of the key anti-inflammatory cytokine IL-10, compared to a different TLR7 agonist, loxoribine, as well as to the TLR4 agonist LPS. To conclude, our results indicate that the widely used inflammatory drug, imiquimod, is not only a TLR7 agonist, but also harbors a novel anti-inflammatory function as a PDE inhibitor. This off-target affects the desired therapeutic inflammatory activity of imiquimod and may be accountable for adverse side effects.

An in vitro bioassay for evaluating the effect of inhaled bronchodilators on airway smooth muscle

PurposeThe development of inhaled drug products is expensive and involves time-consuming pharmacokinetic (PK) and pharmacodynamic (PD) studies. There are few in vitro cell-based assays to evaluate the disposition and action of orally inhaled drugs to guide early product development and minimise risk. The aim of the present study was to develop a co-culture bioassay, combining an airway epithelial cell line (Calu-3) with cultured human primary airway smooth muscle cells (ASM), integrated with apparatus to deliver pharmaceutical aerosols. MethodsAn assay for measuring cyclic adenosine monophosphate (cAMP) in ASM derived from healthy donors was adapted to provide a biochemical surrogate for ASM relaxation. Concentration-response curves for cAMP were established for three drugs that elicit ASM relaxation: isoprenaline (ISO), forskolin (FOR) and salbutamol sulphate. The ASM bioassay was incorporated into a co-culture model in which air-interfaced Calu-3 cell layers, representing the permeability barrier of the airway epithelium, were grown on transwell inserts above ASM cells cultured in the well of the base-plate. The sensitivity of this bioassay to salbutamol delivered using different formulations and aerosol products was evaluated. ResultsASM responded with concentration dependent increases in cAMP when exposed to 10-9 to 10-5 M ISO, FOR or salbutamol sulphate solutions for 15 or 30 min. Salbutamol formulated with different counter ions elicited differential cAMP responses in ASM (xinafoate > base = sulphate) suggesting that this bioassay could discriminate between formulations with different potency. A similar rank order of potency was observed for the different salbutamol salts when applied as aerosols to the co-culture model. DiscussionWe have developed a novel bioassay using human ASM in co-culture with human respiratory epithelial cells to better mimic various elements that contribute to the rate and extent of local drug availability in the lungs following topical administration. The bioassay offers an opportunity to investigate the factors determining the activity of inhaled bronchodilator drugs in a more biologically relevant system than that has previously been described and with further development and validation, this novel bioassay could provide a method to guide the more efficient development of inhaled bronchodilators, reducing the current reliance on in vivo studies.

Abstract MP174: Cardiomyocyte-specific Deletion of Parathyroid Hormone Receptor 1 During Development Leads to Left Ventricular Hypoplasia

Congenital heart defects occur in approximately 1% of all births and signify the most common human birth defects. Our lab previously demonstrated that cardiomyocyte specific deletion of Sphingosine 1-Phosphate Receptor 1 (S1P 1 ), a G protein-coupled receptor (GPCR) that couples exclusively to Gαi, leads to ventricular noncompaction in mice. Gαs- and Gαi-coupled GPCRs activate and inhibit adenylate cyclase, respectively, thereby regulating cAMP production. These GPCRs play important roles in adult cardiac health and disease, but their contributions to heart development are not well understood. We hypothesized that tight regulation of cAMP via Gαs- and Gαi-coupled GPCRs is a critical determinant of cardiomyocyte maturation and cardiac development. Here, we report that Parathyroid Hormone Receptor 1 (Pth1r) which couples to Gαs, is a mediator of cardiomyocyte maturation. Using mice with a Cre allele in the myosin light chain 2a locus ( Myl7 Cre/+, also known as Mlc2a Cre/+ ), we found that cardiomyocyte-specific deletion of Pth1r during development led to reduced postnatal survival. Hearts collected from surviving Pth1r cardiomyocyte mutant mice displayed left ventricular (LV) hypoplasia, and fetal cardiac genes Nppa (natriuretic peptide A) and β myosin heavy chain (Myh7) were upregulated in the hearts of surviving mutants. At 14.5 days post coitus (dpc) and 18.5 dpc, Pth1r cardiomyocyte mutant mice also showed LV hypoplasia. Furthermore, we observed decreased cardiomyocyte proliferation in Pth1r cardiomyocyte mutant hearts during development. Isolated cardiomyocytes from C57BL/6 wild-type embryos showed increased cAMP production after stimulation of Pth1r; Pth1r-dependent cAMP increases were inhibited by activation of S1P 1 . Overall, our results indicate a critical role for Pth1r signaling during heart development and suggest that cAMP levels must be tightly controlled in cardiomyocytes for normal cardiac development. LV hypoplasia is one of the key features of hypoplastic left heart syndrome (HLHS), one of the most severe forms of congenital heart disease; these studies may provide new approaches for the study and treatment of LV hypoplasia and HLHS.

Crosstalk between EP2 and PPARα Modulates Hypoxic Signaling and Myopia Development in Guinea Pigs

PurposeCyclic adenosine monophosphate (cAMP) and peroxisome proliferator-activated receptor alpha (PPARα) levels mediate extracellular matrix (ECM) changes by altering the levels of hypoxia-inducible factor 1-alpha (HIF-1α) in various tissues. We aimed to determine, in the sclera of guinea pigs, whether a prostanoid receptor (EP2)-linked cAMP modulation affects PPARα and HIF-1α signaling during myopia.MethodsThree-week-old guinea pigs (n = 20 in each group), were monocularly injected with either an EP2 agonist (butaprost 1 µmol/L/10 µmol/L), an antagonist (AH6809 10 µmol/L/30 µmol/L) or a vehicle solution for two weeks during normal ocular growth. Separate sets of animals received these injections and underwent form deprivation (FD) simultaneously. Refraction and axial length (AL) were measured at two weeks, followed by scleral tissue isolation for quantitative PCR (qPCR) analysis (n = 10) and cAMP detection (n = 10) using a radioimmunoassay.ResultsButaprost induced myopia development during normal ocular growth, with proportional increases in AL and cAMP levels. FD did not augment the magnitude of myopia or cAMP elevations in these agonist-injected eyes. AH6809 suppressed cAMP increases and myopia progression during FD, but had no effect in a normal visual environment. Of the diverse set of 27 genes related to cAMP, PPARα and HIF-1α signaling and ECM remodeling, butaprost differentially regulated 15 of them during myopia development. AH6809 injections during FD negated such differential gene expressions.ConclusionsEP2 agonism increased cAMP and HIF-1α signaling subsequent to declines in PPARα and RXR mRNA levels, which in turn decreased scleral fibrosis and promoted myopia. EP2 antagonism instead inhibited each of these responses. Our data suggest that EP2 suppression may sustain scleral ECM structure and inhibit myopia development.

Dibutyryl cAMP down-regulates gene expression of endothelin receptor A subtype in rat astrocytoma C6 cells

In this study, we have investigated the gene expression of endothelin receptors in rat astrocytoma C6 cells regulated by the increase of intracellular cAMP. Following treatment with 1 mM dibutyryl cAMP, the specific binding of [125I]-endothelin-1 to the C6 cells was down-regulated to about 40 % of the original level. Radioligand binding assay and Northern blot analysis indicated that endothelin receptors on C6 cells, which were either treated or untreated with 1 mM dibutyryl cAMP, belong to subtype A. Analysis of RNA by Northern blotting and slot hybridization demonstrated that the presence of dibutyryl cAMP induced the down regulation of ET(A) endothelin receptor gene in C6 cells. The level of ET(A) mRNA was rapidly reduced to about 10 % of its initial level after 4 hour treatment with dibutyryl cAMP and then was slightly elevated to about 40 % after one day treatment. All the findings suggest that the down regulation of ET(A) receptor in C6 cells by the treatment of dibutyryl cAMP occurs not only at the receptor binding level but also at the mRNA level.

Cytoskeleton regulators CAPZA2 and INF2 associate with CFTR to control its plasma membrane levels under EPAC1 activation.

Cystic Fibrosis (CF), the most common lethal autosomic recessive disorder among Caucasians, is caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) protein, a cAMP-regulated chloride channel expressed at the apical surface of epithelial cells. Cyclic AMP regulates both CFTR channel gating through a protein kinase A (PKA)-dependent process and plasma membane (PM) stability through activation of the exchange protein directly activated by cAMP1 (EPAC1). This cAMP effector, when activated promotes the NHERF1:CFTR interaction leading to an increase in CFTR at the PM by decreasing its endocytosis. Here, we used protein interaction profiling and bioinformatic analysis to identify proteins that interact with CFTR under EPAC1 activation as possible regulators of this CFTR PM anchoring. We identified an enrichment in cytoskeleton related proteins among which we characterized CAPZA2 and INF2 as regulators of CFTR trafficking to the PM. We found that CAPZA2 promotes wt-CFTR trafficking under EPAC1 activation at the PM whereas reduction of INF2 levels leads to a similar trafficking promotion effect. These results suggest that CAPZA2 is a positive regulator and INF2 a negative one for the increase of CFTR at the PM after an increase of cAMP and concomitant EPAC1 activation. Identifying the specific interactions involving CFTR and elicited by EPAC1 activation provides novel insights into late CFTR trafficking, insertion and/or stabilization at the PM and highlighs new potential therapeutic targets to tackle CF disease.

Sub-Acute Treatment of Curcumin Derivative J147 Ameliorates Depression-Like Behavior Through 5-HT1A-Mediated cAMP Signaling.

BackgroundMajor depressive disorder (MDD) is a severe mental disorder related to the deficiency of monoamine neurotransmitters, particularly to abnormalities of 5-HT (5-hydroxytryptamine, serotonin) and its receptors. Our previous study suggested that acute treatment with a novel curcumin derivative J147 exhibited antidepressant-like effects by increasing brain derived neurotrophic factor (BDNF) level in the hippocampus of mice. The present study expanded upon our previous findings and investigated the antidepressant-like effects of sub-acute treatment of J147 for 3 days in male ICR mice and its possible relevancy to 5-HT1A and 5-HT1B receptors and downstream cAMP-BDNF signaling.MethodsJ147 at doses of 1, 3, and 9 mg/kg (via gavage) was administered for 3 days, and the anti-immobility time in the forced swimming and tail suspension tests (FST and TST) was recorded. The radioligand binding assay was used to determine the affinity of J147 to 5-HT1A and 5-HT1B receptor. Moreover, 5-HT1A or 5-HT1B agonist or its antagonist was used to determine which 5-HT receptor subtype is involved in the antidepressant-like effects of J147. The downstream signaling molecules such as cAMP, PKA, pCREB, and BDNF were also measured to determine the mechanism of action.ResultsThe results demonstrated that sub-acute treatment of J147 remarkably decreased the immobility time in both the FST and TST in a dose-dependent manner. J147 displayed high affinity in vitro to 5-HT1A receptor prepared from mice cortical tissue and was less potent at 5-HT1B receptor. These effects of J147 were blocked by pretreatment with a 5-HT1A antagonist NAD-299 and enhanced by a 5-HT1A agonist 8-OH-DPAT. However, 5-HT1B receptor antagonist NAS-181 did not appreciably alter the effects of J147 on depression-like behaviors. Moreover, pretreatment with NAD-299 blocked J147-induced increases in cAMP, PKA, pCREB, and BDNF expression in the hippocampus, while 8-OH-DPAT enhanced the effects of J147 on these proteins’ expression.ConclusionThe results suggest that J147 induces rapid antidepressant-like effects during a 3-day treatment period without inducing drug tolerance. These effects might be mediated by 5-HT1A-dependent cAMP/PKA/pCREB/BDNF signaling.

Cilostazol protects against cyclophosphamide-induced ovarian toxicity in female rats: role of cAMP and HO-1

Purpose: Cancer rates have been increased among women of reproductive age nowadays. Hence, many young female will be exposed to chemotherapeutic agents as cyclophosphamide (CP), carrying the hazards on female fertility. Cilostazol is a selective phosphodiesterase-3 inhibitor drug which exhibits antioxidant, anti-inflammatory, and anti-apoptotic activities. We aimed in this study to explore the possible protective effects of cilostazol against CP-induced ovarian damage in female rats.Methods: Cilostazol (10 mg/kg/day) was administered orally for 10 days in presence and absence of CP (150 mg/kg IP single dose) treatment. Serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), estrogen (E2), and anti-Müllerian hormone (AMH) levels were determined. Ovarian oxidative stress parameters along with inflammatory biomarkers were measured. 3,5-Cyclic adenosine monophosphate (cAMP) ovarian level was detected. Ovarian histopathological examination and caspase-3 immunohistochemical study were evaluated.Results: CP-treated rats showed a significant increase in serum levels of FSH and LH with decreased serum E2 and AMH levels with an increase in the ovarian inflammatory and oxidative stress biomarkers besides a significant decrease in cAMP ovarian level with an evident histopathological picture of ovarian damage and a high caspase-3 immunoexpression. Cilostazol pretreatment significantly restored the distributed hormonal levels, the oxidative stress and inflammatory biomarkers to their normal levels with marked improvement in histopathological picture of ovarian damage with a significant decrease in caspase-3 immunoexpression.Conclusions: These data suggest that cilostazol protects against CP- induced ovarian damage, which may be related to an increase in cAMP with subsequent anti-inflammatory, antioxidant, and anti-apoptotic properties.

Rational Design of a Protein Kinase A Nuclear-cytosol Translocation Reporter

Protein Kinase A (PKA) exists as a tetrameric holoenzyme which activates with increase of cAMP and plays an important role in many physiological processes including cardiac physiology, neuronal development, and adipocyte function. Although this kinase has been the subject of numerous biosensor designs, a single-fluorophore reporter that performs comparably to Förster resonance energy transfer (FRET) has not yet been reported. Here, we have used basic observations of electrostatic interactions in PKA substrate recognition mechanism and nucleus localization sequence motif to design a phosphorylation switch that shuttles between the cytosol and the nucleus, a strategy that should be generalizable to all basophilic kinases. The resulting reporter yielded comparable kinetics and dynamic range to the PKA FRET reporter, AKAR3EV. We also performed basic characterization and demonstrated its potential use in monitoring multiple signaling molecules inside cells using basic fluorescence microscopy. Due to the single-fluorophore nature of this reporter, we envision that this could find broad applications in studies involving single cell analysis of PKA activity.

Effects of stanniocalcin 1 hormone on lactate metabolism in rat kidney under fed and fasted conditions

To test the hypothesis of STC-1 participation in maintenance of glucose homeostasis in fed and fasting (48 h) rats, we investigated that this hormone may be implicated in the regulation of renal gluconeogenesis pathway from lactate and lactate oxidation in renal cortex and medulla. Our results demonstrate the hSTC-1 role on lactate metabolism in the renal cortex and medulla from fed and fasting rats. hSTC-1 increased the gluconeogenesis activity in fed state in renal cortex, and this increase was induced by raise in Pck1 gene expression. In fasting animals hSTC-1 increase the renal medulla gluconeogenesis activity, but Pck1 gene expression was not alter. The stimulatory effect of hSTC-1 on 14C-lactate oxidation occurred only in the renal cortex from fed rats. These findings show the hSTC-1 contribution to lactate homeostasis and supplies glucose to other tissues. This response may represent a strategy of action of STC-1 in response to fasting stress as postulated by different authors. On the other hand, hSTC-1 acts downstream of adenylcyclase pathway, decreasing the gluconeogenesis activity induced by cAMP intracellular increase or stimulating the phosphodiesterase activity in the renal cortex. However, no hSTC-1 effect on 14C-lactate oxidation was found after increase in the intracellular cAMP. The findings also revealed that the renal cortex and medulla respond differently to hSTC-1, possibly due to the higher level of STC-1 gene expression in inner renal medulla than in renal cortex.

Phosphodiesterase 10A Is a Mediator of Osteogenic Differentiation and Mechanotransduction in Bone Marrow-Derived Mesenchymal Stromal Cells.

Bone marrow-derived mesenchymal stromal cells (hMSCs) are capable of differentiating into the osteogenic lineage, and for osteogenic differentiation, mechanical loading is a relevant stimulus. Mechanotransduction leads to the formation of second messengers such as cAMP, cGMP, or Ca2+ influx resulting in the activation of transcription factors mediating gene regulation. The second messengers cAMP and cGMP are degraded by phosphodiesterase isoenzymes (PDE), but the role of these enzymes during osteogenic differentiation or mechanotransduction remains unclear. Here, we focused on the isoenzyme phosphodiesterase 10A (PDE10A) and its role during osteogenic commitment and mechanotransduction. We observed a time-dependent decrease of PDE10A expression in hMSC undergoing differentiation towards the osteogenic lineage. PDE10A inhibition by papaverine diminished osteogenic differentiation. While applying mechanical strain via cyclic stretching of hMSCs led to an upregulation of PDE10A gene expression, inhibition of PDE10A using the drug papaverine repressed expression of mechanoresponsive genes. We conclude that PDE10A is a modulator of osteogenic differentiation as well as mechanotransduction in hMSCs. Our data further suggests that the relative increase of cAMP, rather than the absolute cAMP level, is a key driver of the observed effects.

Possible mechanisms mediating the protective effect of cilostazol in L-arginine induced acute pancreatitis in rats: role of cGMP, cAMP, and HO-1.

Acute pancreatitis (AP) is an inflammatory disorder with a high mortality rate. Cilostazol is a selective phosphodiesterase-3 inhibitor drug that is commonly used as an antiplatelet, antithrombotic, and vasodilator drug. It exhibits antioxidant, anti-inflammatory, and anti-apoptotic activities, but its effect on AP has not been fully elucidated yet. The present study aimed to investigate the effects of cilostazol on L-arginine-induced AP and the possible protective mechanisms. A rat model of AP was established by a single i.p. injection of 3-g/kg L-arginine on day 13 of the experiment. The treated groups received a single daily oral dose of either 100 or 300 mg/kg/day for 14 consecutive days. Rats with AP showed histopathological changes of pancreatic tissue injury together with increased serum amylase enzyme activity and decreased serum insulin, pancreatic adiponectin, and cGMP levels. Moreover, AP rats showed increased pancreatic inflammatory biomarker (TNF-α, VCAM-1, and MPO) levels with decreased anti-inflammatory IL-10 levels. In addition, oxidative stress biomarkers (MDA and NO) were increased in AP with decreased antioxidant SOD activity and GSH level. Moreover, HO-1 immunostaining was increased in the AP group. Cilostazol pretreatment reversed the histopathological change; decreased the amylase activity and the levels of TNF-α, VCAM-1, and MPO; and increased the levels of insulin, adiponectin, cGMP, cAMP, and IL-10. Moreover, cilostazol decreased MDA and NO but increased SOD and GSH. Lastly, cilostazol increased the HO-1 immunostaining more than in the AP group. These data suggest that cilostazol protects against L-arginine-induced AP, which may be related to an increase in cGMP, cAMP, and upregulation of HO-1 with subsequent anti-inflammatory and antioxidant properties.

Astrocytes with TDP-43 inclusions exhibit reduced noradrenergic cAMP and Ca2+ signaling and dysregulated cell metabolism

Most cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) have cytoplasmic inclusions of TAR DNA-binding protein 43 (TDP-43) in neurons and non-neuronal cells, including astrocytes, which metabolically support neurons with nutrients. Neuronal metabolism largely depends on the activation of the noradrenergic system releasing noradrenaline. Activation of astroglial adrenergic receptors with noradrenaline triggers cAMP and Ca2+ signaling and augments aerobic glycolysis with production of lactate, an important neuronal energy fuel. Astrocytes with cytoplasmic TDP-43 inclusions can cause motor neuron death, however, whether astroglial metabolism and metabolic support of neurons is altered in astrocytes with TDP-43 inclusions, is unclear. We measured lipid droplet and glucose metabolisms in astrocytes expressing the inclusion-forming C-terminal fragment of TDP-43 or the wild-type TDP-43 using fluorescent dyes or genetically encoded nanosensors. Astrocytes with TDP-43 inclusions exhibited a 3-fold increase in the accumulation of lipid droplets versus astrocytes expressing wild-type TDP-43, indicating altered lipid droplet metabolism. In these cells the noradrenaline-triggered increases in intracellular cAMP and Ca2+ levels were reduced by 35% and 31%, respectively, likely due to the downregulation of β2-adrenergic receptors. Although noradrenaline triggered a similar increase in intracellular lactate levels in astrocytes with and without TDP-43 inclusions, the probability of activating aerobic glycolysis was facilitated by 1.6-fold in astrocytes with TDP-43 inclusions and lactate MCT1 transporters were downregulated. Thus, while in astrocytes with TDP-43 inclusions noradrenergic signaling is reduced, aerobic glycolysis and lipid droplet accumulation are facilitated, suggesting dysregulated astroglial metabolism and metabolic support of neurons in TDP-43-associated ALS and FTD.

Assessment of Morphine Tolerance Using cAMP‐GloSensor&[trade] Real‐time Detection Assay in SH‐SY5Y Human Neuroblastoma Cells

PurposeActivation of GPCRs by morphine triggers downstream effects including inhibition of adenylyl cyclase and decrease in 3′,5′‐cyclic adenosine monophosphate (cAMP). cAMP upregulation is observed in morphine tolerance and withdrawal. Endothelin‐A receptor (ETAR) antagonist, BQ123 potentiates morphine analgesia, reverses tolerance, and reduces withdrawal symptoms. However, signaling mechanisms involved in these phenomena are unknown. The objective of this study was to determine morphine‐ and BQ123‐induced changes in cAMP signaling in SH‐SY5Y neuroblastoma cells which endogenously express mu‐opioid receptors (MORs) and ETARs.MethodsSH‐SY5Y cells (ATCC®) were grown in DMEM/10% FBS and maintained in a humidified incubator at 37°C/5%CO2. Cells were seeded in 96‐well white, opaque‐bottom plates (5×104 cells/well). Transfection conditions were optimized using ViaFect™ (Promega™) and 30% transfection efficiency was achieved. Stably transfected SH‐SY5Y cell line was established using GloSensor™ −23F cAMP plasmid (Promega™) containing hygromycin‐resistance gene. Promega’s real‐time detection system in live cells, the cAMP‐GloSensor™ assay was used to measure cAMP responses. Stably transfected cells were incubated with equilibrium buffer [cAMP GloSensor™ reagent (2% (v/v)), 88% CO2 independent medium, 10% FBS]. cAMP levels were determined by changes in luminescence using an EnSpire™ multimode microplate reader (PerkinElmer). In acute studies, cells were treated for 2 h with morphine (10–10−5 M) and BQ123 (1 μM). In chronic studies, cells were treated for 24 h with morphine (1 μM) in the presence and absence of BQ123 (1 μM). After induction of tolerance for 24 h, two challenge doses of morphine (10 μM and 20 μM) were tested. Isoproterenol was used as a positive control (1–10−8 M). Each experiment was repeated in triplicate and data was analyzed in GraphPad Prism version 8.00 (GraphPad Software, San Diego, CA).ResultsA concentration‐dependent increase in cAMP was observed with isoproterenol in SH‐SY5Y cells stably transfected with −23F cAMP plasmid. Acute morphine treatment (10–10−5 M for 2 h) produced concentration‐dependent decrease in cAMP, while acute treatment with BQ123 (1–10−5 M) increased cAMP levels. Of interest, BQ123 (1 μM) appeared to enhance morphine‐induced decrease in cAMP response. Chronic morphine treatment (1 μM for 24 h) increased cAMP response, indicative of tolerance. Cells treated with combination of morphine (1 μM) and BQ123 (1 μM) for 24 h showed dose‐dependent inhibition of cAMP levels with challenge doses of morphine.ConclusionTo better understand the mechanism by which ETAR antagonists reverse morphine tolerance and reduce withdrawal symptoms, signaling mechanisms were explored in SH‐SY5Y cells endogenously expressing ETA receptors and MORs. We found that BQ123 appears to enhance morphine‐induced cAMP signaling (measured by Glo‐Sensor™ cAMP assay) through Gi‐coupled MORs in both acute and chronic studies, which support our previous work. We will further explore the involvement of beta‐arrestin and GPCR kinases (GRKs) in these phenomena.Support or Funding InformationChicago College of Pharmacy Faculty Research Grant; College of Graduate Studies, Biomedical Sciences, Midwestern University, Downers Grove IL.

Evaluation of a genetically encoded Cl− and pH sensor (ClopHensor) for monitoring intragranular Cl− in renal endocrine juxtaglomerular (JG) cells.

In most cells, intracellular cytoplasmic Cl− is maintained between 10 and 30 mM by a combination of entry and exit transport pathways. However, in endocrine cells intra‐granule Clin secretory granules is higher than cytoplasmic Cl−. This, is in part due to an intragranular positive H+ gradient driven by V‐ATPases, which generates low intra‐granular pH (4–5), and provides an electrochemical gradient for Cl− entry via CLC‐6/7. The role of V‐ATPases in exocytosis is well known, but less is known about how changes in Cl− contribute. There are very few methods available to measure intra‐granule Cl− concentration and monitor how it changes. ClopHensor is a genetically encoded protein sensor based on a Cl ‐sensitive GFP mutant (E2‐GFP) and DsRed ( Nature Methods, v7, pages 516–518, 2010). When excited by a 457 nm laser, E2‐GFP emission is sensitive to Cl− whereas at 488 nm it is sensitive to pH, whereas DsRed (insensitive to Cl− or pH) serves to control for volume changes. Renal endocrine juxtaglomerular (JG) cells, store renin, the rate‐limiting step enzyme in the production of angiotensin II, which play an essential role in blood pressure control. Renin is stored in large secretory granules and its exocytosis is stimulated primarily by increases in cAMP. We hypothesized that targeting ClopHensor to secretory granules would allow measurement of intra‐granular Cl− in JG cells and changes in its concentration during stimulation. We first tested the ability of ClopHensor to measure intracellular Cl− in M1 cells, clamped at pH 5.5 (reported for secretory granules). M1 cells transduced with adenovirus coding for ClopHensor were permeabilized to H+ with nygericin and bathed at pH 5.5, and permeabilized to Cl−. Cells were imaged by confocal microscopy under 457, 491 and 568 nm laser excitation while simultaneously acquiring emissions at 525 (E2‐GFP) and 590nm (Dsred). Under these conditions increasing Cl− from 0, to 20, 80 and 120 mM caused a logarithmic reduction in the 457/568 emission ratio (Cl‐sensitive) with a half‐max inhibition at 10.7±1.4 mM (n =6). To monitor intra‐granule Cl− we generated adenoviruses expressing NeuroPeptide Y targeting motif (NPY)‐ClopHensor. When primary mouse JG cells were transduced with NPY‐ClopHensor fluorescence was restricted to large granules. We could not permeabilize granules to Cl‐ in live JG cells Thus we measured baseline intra‐granule Cl− immediately after a daily calibration of ClopHensor in M1 cells. These results yielded an intra‐granule Cl− concentration of 77±15 mM (n=8). After measuring baseline Cl− in granules JG cells were treated with forskolin/IBMX to increase cAMP, and then 0 mM extracellular Cl− and bafilomycin. cAMP decreased intra‐granule Cl−, evidenced by a 22±5% increase (p<0.05, n= 11) in 457/568 Cl‐sensitive ratio, which was further increased by inhibiting V‐ATPse with bafilomycin by 40±6% (p<0.05, n=13). Our data show for the first time, measurements of intra‐granule Cl− in renal JG cells and suggest that NPY‐ClopHensor can be used to monitor the effect of agonists, or V‐ATPase regulators on intra‐granule Cl−. The role of intra‐granule Cl− in renin exocytosis and renin‐angiotensin system physiology remains to be studied.Support or Funding InformationHFH Research fund: Pablo A Ortiz

β2-adrenergic stimulation induces interleukin-6 by increasing Arid5a, a stabilizer of mRNA, through cAMP/PKA/CREB pathway in cardiac fibroblasts.

Background and PurposeIn cardiovascular diseases, cardiac fibroblasts (CFs) participate in the myocardial inflammation by producing pro‐inflammatory cytokines, worsening the prognosis. β2‐adrenergic receptor (AR) and β3AR are expressed in CFs, and β‐adrenergic stimulation promotes CFs to produce pro‐inflammatory cytokines. However, the mechanism of the expression of pro‐inflammatory cytokines in response to β‐adrenergic stimulation remains to be fully elucidated.Experimental ApproachCFs were isolated from adult wild‐type or AT‐rich interactive domain‐containing protein 5A (Arid5a) knockout mice. The expression of mRNA was measured by real‐time RT‐PCR. Interleukin (IL)‐6 protein was measured by ELISA. The activity of nuclear factor‐κB (NF‐κB) and cyclic AMP (cAMP) response element binding protein (CREB) was assessed by ELISA‐like assay or Western blotting.Key ResultsThe β‐adrenergic stimulation remarkably induced IL‐6 mRNA and protein through β2AR in CFs. The activation of adenylate cyclase and the enhancement of intracellular cAMP resulted in the upregulation of IL‐6 mRNA expression. The induction of IL‐6 transcript by β2AR signaling was independent of NF‐κB. Concomitant with IL‐6, the expression of Arid5a, an IL‐6 mRNA stabilizing factor, was enhanced by β2‐adrenergic stimulation and by cAMP increase. Importantly, β2AR signaling‐mediated IL‐6 induction was suppressed in Arid5a knockout CFs. Finally, β2AR stimulation phosphorylated CREB via PKA pathway, and the activation of CREB was essential for the induction of Arid5a and IL‐6 mRNA.Conclusion and Implicationsβ2‐adrenergic stimulation post‐transcriptionally upregulates the expression of IL‐6 by the induction of Arid5a through cAMP/PKA/CREB pathway in adult CFs. β2AR/Arid5a/IL‐6 axis could be a therapeutic target against cardiac inflammation.

GLP-1 receptor agonists synergize with DYRK1A inhibitors to potentiate functional human β cell regeneration.

Glucagon-like peptide-1 receptor (GLP1R) agonists and dipeptidyl peptidase 4 inhibitors are widely prescribed diabetes drugs due to their ability to stimulate insulin secretion from remaining β cells and to reduce caloric intake. Unfortunately, they fail to increase human β cell proliferation. Small-molecule inhibitors of dual-specificity tyrosine-regulated kinase 1A (DYRK1A) are able to induce adult human β cell proliferation, but rates are modest (~2%), and their specificity to β cells is limited. Here, we provide evidence that combining any member of the GLP1R agonist class with any member of the DYRK1A inhibitor class induces a synergistic increase in human β cell replication (5 to 6%) accompanied by an actual increase in numbers of human β cells. GLP1R agonist-DYRK1A inhibitor synergy required combined inhibition of DYRK1A and an increase in cAMP and did not lead to β cell dedifferentiation. These beneficial effects on proliferation were seen in both normal human β cells and β cells derived from individuals with type 2 diabetes. The ability of the GLP1R agonist-DYRK1A inhibitor combination to enhance human β cell proliferation, human insulin secretion, and blood glucose control extended in vivo to studies of human islets transplanted into euglycemic and streptozotocin-diabetic immunodeficient mice. No adverse events were observed in the mouse studies during a 1-week period. Because of the relative β cell specificity of GLP1R agonists, the combination provides an improved, although not complete, degree of human β cell specificity.

Ilex paraguariensis extracts and its polyphenols prevent oxidative damage and senescence of human retinal pigment epithelium cells

Abstract Aged-related macular degeneration (AMD), a prevalent chronic disease leading to blindness, is associated with oxidative damage of the retinal pigment epithelium (RPE). Ilex paraguariensis (yerba mate, YM), widely consumed in South America, is an excellent source of antioxidants and could prevent RPE cell impairment. Therefore, we evaluated the effects of YM extracts or caffeic (CAF) and chlorogenic (CHL) acids, two major Ilex polyphenols on cell death or premature senescence in a cell line derived from human RPE (ARPE-19). Cultures were incubated with YM, CAF, CHL or appropriate controls and then exposed to H2O2. Protection was correlated with decreases of reactive oxygen species (ROS) and DNA breaks, phosphorylation of the cAMP response element (CREB) and increases of the nuclear factor erythroid 2-related factor 2 (NRF2), BCL2, superoxide dismutase (SOD2) and sirtuin 1 (SIRT1). These results support the protective role of YM and its polyphenols against oxidative stress induced RPE injury.