Activation Of cAMP Pathway Research Articles

Integrating network pharmacology and transcriptomic omics reveals that akebia saponin D attenuates neutrophil extracellular traps-induced neuroinflammation via NTSR1/PKAc/PAD4 pathway after intracerebral hemorrhage.

Neutrophils and their production of neutrophil extracellular traps (NETs) significantly contribute to neuroinflammation and brain damage after intracerebral hemorrhage (ICH). Although Akebia saponin D (ASD) demonstrates strong anti-inflammatory activities and blood-brain barrier permeability, its role in regulating NETs formation and neuroinflammation following ICH is uncharted. Our research focused on unraveling the influence of ASD on neuroinflammation mediated by NETs and the mechanisms involved. We found that increased levels of peripheral blood neutrophils post-ICH are correlated with worse prognostic outcomes. Through network pharmacology, we identified ASD as a promising therapeutic target for ICH. ASD administration significantly improved neurobehavioral performance and decreased NETs production in neutrophils. Furthermore, ASD was shown to upregulate the membrane protein NTSR1 and activate the cAMP signaling pathway, confirmed through transcriptome sequencing, western blot, and immunofluorescence. Interestingly, the NTSR1 inhibitor SR48692 significantly nullified ASD's anti-NETs effects and dampened cAMP pathway activation. Mechanistically, suppression of PKAc via H89 negated ASD's anti-NETs effects but did not affect NTSR1. Our study suggests that ASD may reduce NETs formation and neuroinflammation, potentially involving the NTSR1/PKAc/PAD4 pathway post-ICH, underlining the potential of ASD in mitigating neuroinflammation through its anti-NETs properties.

GPCR-Gs mediates the protective effects of ginsenoside Rb1 against oxygen-glucose deprivation/re-oxygenation-induced astrocyte injury

ObjectivesTo investigate whether the protective actions of ginsenoside Rb1 (Rb1) on astrocytes are mediated through the Gs-type G-protein-coupled receptor (GPCR-Gs). MethodsPrimary astrocyte cultures derived from neonatal mouse brain were used. Astrocyte injury was induced via oxygen-glucose deprivation/re-oxygenation (OGD/R). Cell morphology, viability, lactate dehydrogenase (LDH) leakage, apoptosis, glutamate uptake, and brain-derived neurotrophic factor (BDNF) secretion were assessed to gauge cell survival and functionality. Western blot was used to investigate the cyclic adenosine monophosphate (cAMP) and protein kinase B (Akt) signaling pathways. GPCR-Gs-specific inhibitors and molecular docking were used to identify target receptors. ResultsRb1 at concentrations ranging from 0.8 to 5 μM did not significantly affect the viability, glutamate uptake, or BDNF secretion in normal astrocytes. OGD/R reduced astrocyte viability, increasing their LDH leakage and apoptosis rate. It also decreased glutamate uptake and BDNF secretion by these cells. Rb1 had protective effects of astrocytes challenged by OGD/R, by improving viability, reducing apoptosis, and enhancing glutamate uptake and BDNF secretion. Additionally, Rb1 activated the cAMP and Akt pathways in these cells. When the GPCR-Gs inhibitor NF449 was introduced, the protective effects of Rb1 completely disappeared, and its activation of cAMP and Akt signaling pathways was significantly inhibited. ConclusionRb1 protects against astrocytes from OGD/R-induced injury through GPCR-Gs mediation.

Activation of cyclic AMP signaling pathway in dopaminergic neurons rescues locomotion defects in a Drosophila larval model of Parkinson’s disease

Parkinson’s disease (PD) is a neurodegenerative disease showing uncontrollable motor symptoms that are primarily caused by the progressive loss of dopaminergic neurons in the brain. Currently no treatment exists to prevent PD progression. Therefore, discovery of new neuroprotective strategies still has great potential to benefit PD patients. A handful of studies show that activation of cAMP pathways is neuroprotective against PD progression. However, the neuroprotective role of this signaling cascade specifically in DA neurons has not been explored. In this study, fruit fly Drosophila melanogaster was used because of its sophisticated and powerful genetic approaches, especially with related to cAMP signaling pathway. We have investigated molecular mechanisms of neuroprotection in a fly larval model of PD by administering an environmental PD toxin rotenone. Increased cAMP signaling in the dunce mutant fly carrying defects in phosphodiesterase (PDE) gene, is neuroprotective against rotenone-induced locomotion deficits. Furthermore, the neuroprotective role of cAMP signaling specifically in DA neurons has been studied as it has not been explored. By using transgenic flies expressing designer receptors exclusively activated by designer drugs (DREADDs), we have shown that an increase of cAMP levels in DA neurons rescues rotenone-induced locomotion deficits. We also showed that this neuroprotection is mediated by activation of Gαs and PKA-C1 subunits. The results provide novel findings that expand our knowledge of neuroprotective mechanisms in DA neurons affecting PD progression, which could contribute to the development of new therapeutic treatments against PD. An important future study will explore downstream targets of cAMP-PKA signaling.

Cyclic adenosine monophosphate-elevating agents inhibit amyloid-beta internalization and neurotoxicity: their action in Alzheimer's disease prevention.

Cyclic adenosine monophosphate-elevating agents inhibit amyloid-beta internalization and neurotoxicity: their action in Alzheimer's disease prevention.

New Butyroside D from Argan Press Cake Possess Anti-Melanogenesis Effect via MITF Downregulation in B16F10 and HEM Cells.

Hyperpigmentation is a skin condition where patches of skin become darker in color due to excess melanin production upon UV exposure leading to melasma, which are lentigines or post inflammatory hyperpigmentation that psychologically affecting a great number of people. The present study investigates the anti-melanogenic effect of Butyroside D and the underling mechanism. After the confirmation of the non-cytotoxic effect of Butyroside D on B16F10 cells, we proceeded with analyzing the impact of the treatment at low and high concentration (i.e., 0.2 μM and 2 μM) using gene profiling analysis and examined the differentiation in gene expression. Our results identify cyclic adenosine monophosphate (cAMP), Wnt/β-catenin and Mitogen-Activated Protein Kinase (MAPK) signaling pathways to be downregulated upon treatment with Butyroside D. These pathways were targeted to further validate the effect of Butyroside D on membrane receptors melanocortin 1 receptor (MC1R) and receptor tyrosine kinase (c-Kit), related microphthalmia-associated transcription factor (MITF) and consequently tyrosinase (TYR), and tyrosine-related protein-1 (TYRP-1) that were all shown to be downregulated and, therefore, leading to the repression of melanin biosynthesis. Finally, the anti-melanogenic effect of Butyroside D was confirmed on human epidermal melanocytes (HEM) cells by inhibiting the activation of cAMP pathway generally mediated through α-melanocyte-stimulating hormone (α-MSH) and MC1R. Overall, this study suggests the potential applicability of this purified compound for the prevention of hyperpigmentation conditions.

5HTR1E receptor interacts with Neurotrophic factor‐α1 and serotonin to activate two distinct signaling pathways

Neurotrophic factor‐α1/Carboxypeptidase E (NFα1/CPE) is a prohormone processing enzyme intracellularly and as a neurotrophic factor, extracellularly. Previously we showed that extracellular treatment of hippocampal neurons with NFα1/CPE protected them from oxidative stress‐induced cell death through ERK/BCL2 signaling. Based on these results, we hypothesized that NFα1/CPE activation of the ERK/BCL2 pathway to mediate neuroprotection should involve interaction with receptors at the cell membrane. We have identified 5‐hydroxytryptamine receptor 1E (5HTR1E), a Gi coupled serotonin receptor, as an extracellular binding partner of NFα1/CPE. This interaction was confirmed by co‐IP and pull‐down experiments followed by radioligand binding studies in HEK293‐5HTR1E overexpressing cells. Molecular modeling studies also predicted that NFα1/CPE interacted with HTR1E, not at the serotonin binding pocket, but via the extracellular loop 2 (ECL2) of 5HTR1E, forming 3 stable salt bridges with specific amino acid residues in NFα1/CPE. To identify the HTR1E downstream signaling pathways, we used HEK293 cells stably expressing 5HTR1E. First, we tested the dose dependent effect of both serotonin and NFα1/CPE on ERK, and cAMP signaling and found that both can increase ERK phosphorylation while cAMP pathway activation is serotonin dependent only. To further dissect the 5HTR1E mediated functions of both serotonin and NFα1/CPE we tested Gi, Gq, PKA, and PI3K inhibitors and observed that these proteins are not involved in NFα1/CPE mediated ERK phosphorylation, while serotonin‐stimulated ERK activation was Gi‐PKA dependent. We then explored the involvement of β‐arrestin, an alternative coupling mechanism with GPCRs for ERK activation. Studies in 5HTR1E expressing‐HEK293‐β‐arrestin KO cells revealed that NFα1/CPE‐5HTR1E complex was unable to induce ERK signaling in the absence of β‐arrestin proteins. Furthermore, molecular docking studies revealed a very strong coupling of b‐arrestin to intracellular loop (ICL) 2 and ICL3 of HTR1E. Using LDH cytotoxicity assay we demonstrated that NF‐α1/CPE was able to protect HEK‐293‐5HTR1E expressing cells against H2O2‐induced cytotoxicity, via increasing pro‐survival protein BCL2 expression; however, this protective effect of NFα1/CPE was diminished in the β‐arrestin KO cells. Our study identified two ligands for HTR1E: NFα1/CPE which interacts with HTR1E to activate β‐arrestin/ERK/BCL2 signaling pathway for protecting cells against oxidative stress; and serotonin which activated a Gi‐cAMP‐dependent pathway, the physiological role is under investigation.

A Novel Mechanism Regulating Dopamine Receptor Type 2 Signal Transduction in Pituitary Tumoral Cells: The Role of cAMP/PKA-Induced Filamin A Phosphorylation.

The actin binding protein filamin A (FLNA) is required for somatostatin receptor 2 (SSTR2) and dopamine receptor 2 (DRD2) expression and signaling in GH- and PRL-secreting PitNETs, respectively, playing a role in tumor responsiveness to somatostatin receptors ligands and dopaminergic drugs. FLNA functions are regulated by several mechanisms, including phosphorylation. It has been shown that in GH-secreting PitNETs FLNA phosphorylation on Ser2152 (P-FLNA) switches FLNA function from a scaffold that allows SSTR2 signal transduction, to a signal termination protein that hampers SSTR2 antitumoral effects. Aims of the present study were to evaluate in PRL- and ACTH-secreting PitNETs cell lines MMQ and AtT-20 the effects of cAMP pathway activation and DRD2 agonist on P-FLNA and the impact of P-FLNA on DRD2 signal transduction. We found that forskolin increased (+2.2 ± 0.8-fold, p < 0.01 in MMQ; +1.9 ± 0.58-fold, p < 0.05 in AtT-20), and DRD2 agonist BIM53097 reduced (-49.4 ± 25%, p < 0.001 in MMQ; -45.8 ± 28%, p < 0.05 in AtT-20), P-FLNA on Ser2152. The overexpression of a phosphomimetic (S2152D) FLNA mutant in both cell lines prevented DRD2 antiproliferative effects, that were comparable in cells transfected with empty vector, wild-type FLNA as well as phosphodeficient FLNA mutant (S2152A) (-20.6 ± 5% cell proliferation, p < 0.001 in MMQ; -36.6 ± 12%, p < 0.01 in AtT-20). Accordingly, S2152D FLNA expression abolished the expected ability of BIM53097 to increase or decrease, in MMQ and in AtT20 respectively, ERK phosphorylation, an effect that was maintained in S2152A FLNA expressing cells (+1.8 ± 0.65-fold, p < 0.05 in MMQ; -55 ± 13%, p < 0.01 in AtT-20). In addition, the inhibitory effects of DRD2 on hormone secretion (-34.3 ± 6% PRL, p < 0.05 in MMQ; -42.8 ± 22% ACTH, p < 0.05 in AtT-20, in cells expressing S2152A FLNA) were completely lost in S2152D FLNA transfected cells. In conclusion, our data demonstrated that cAMP pathway and DRD2 agonist regulated FLNA activity by increasing or decreasing, respectively, its phosphorylation. Moreover, we found that P-FLNA prevented DRD2 signaling in PRL- and ACTH-secreting tumoral pituitary cell lines, suggesting that this FLNA modification might represent a new regulatory mechanism shared by different GPCRs. In PitNETs expressing DRD2, modulation of P-FLNA might suggest new pharmacological strategies to overcome drug resistance, and P-FLNA might represent a new biomarker for tumor responsiveness to dopaminergic agents.

Silencing of RGS2 enhances hippocampal neuron regeneration and rescues depression-like behavioral impairments through activation of cAMP pathway

Depression is one of the most common mental disorders with an increasing incidence. However, factors involved in depression are so complex, thus it is difficult to find effective strategies to reverse the impairments. This study aims to verify the role of regulator of G protein signaling 2 (RGS2) in the mouse mode of unpredictable mild stress-induced depression-like behaviors. Knockdown of RGS2 was achieved by transfection of siRNA-RGS2 in mouse hippocampal (HT-22) cells in vitro and injection of recombinant adenovirus expressing siRNA-RGS2 in mice in vivo. An aberrant high expression of RGS2 was found in mice with depression-like behaviors through immunohistochemical analysis. Silencing of RGS2 or Forskolin (activator of cAMP pathway) developed sweet water consumption, reduced inflammation and oxidative stress injury, and attenuated cognitive impairment and neuronal damage in mice with depression-like behaviors. Furthermore, regeneration was enhanced and apoptosis was repressed in mouse hippocampal neurons in the presence of RGS2 knockdown and Forskolin. Mechanistic studies indicated that silencing of RGS2 promoted the activation of cAMP pathway, thus rescuing depression-like behaviors of mice. Collectively, our study uncovered the role of RGS2-dependent cAMP pathway in regulation of cognitive impairment and hippocampal neuron regeneration in depression-like behaviors of mice, which may be a potential therapeutic target for impairments and symptoms associated with depression.

Connexin Hemichannels Contribute to the Activation of cAMP Signaling Pathway and Renin Production.

Connexin hemichannels play an important role in the control of cellular signaling and behaviors. Given that lowering extracellular Ca2+, a condition that activates hemichannels, is a well-characterized stimulator of renin in juxtaglomerular cells, we, therefore, tested a potential implication of hemichannels in the regulation of renin in As4.1 renin-secreting cells. Lowering extracellular Ca2+ induced hemichannel opening, which was associated with cAMP signaling pathway activation and increased renin production. Blockade of hemichannels with inhibitors or downregulation of Cxs with siRNAs abrogated the activation of cAMP pathway and the elevation of renin. Further analysis revealed that cAMP pathway activation was blocked by adenylyl cyclase inhibitor SQ 22536, suggesting an implication of adenyl cyclase. Furthermore, the participation of hemichannels in the activation of the cAMP signaling pathway was also observed in a renal tubular epithelial cell line NRK. Collectively, our results characterized the hemichannel opening as a presently unrecognized molecular event involved in low Ca2+-elicited activation of cAMP pathway and renin production. Our findings thus provide novel mechanistic insights into the low Ca2+-initiated cell responses. Given the importance of cAMP signaling pathway in the control of multiple cellular functions, our findings also highlight the importance of Cx-forming channels in various pathophysiological situations.

PET Imaging of Phosphodiesterase-4 Identifies Affected Dysplastic Bone in McCune–Albright Syndrome, a Genetic Mosaic Disorder

McCune-Albright syndrome (MAS) is a mosaic disorder arising from gain-of-function mutations in the GNAS gene, which encodes the 3',5'-cyclic adenosine monophosphate (cAMP) pathway-associated G-protein, Gsα. Clinical manifestations of MAS in a given individual, including fibrous dysplasia, are determined by the timing and location of the GNAS mutation during embryogenesis, the tissues involved, and the role of Gsα in the affected tissues. The Gsα mutation results in dysregulation of the cAMP signaling cascade, leading to upregulation of phosphodiesterase type 4 (PDE4), which catalyzes the hydrolysis of cAMP. Increased cAMP levels have been found in vitro in both animal models of fibrous dysplasia and in cultured cells from individuals with MAS but not in humans with fibrous dysplasia. PET imaging of PDE4 with 11C-(R)-rolipram has been used successfully to study the in vivo activity of the cAMP cascade. To date, it remains unknown whether fibrous dysplasia and other symptoms of MAS, including neuropsychiatric impairments, are associated with increased PDE4 activity in humans. Methods: 11C-(R)-rolipram whole-body and brain PET scans were performed on 6 individuals with MAS (3 for brain scans and 6 for whole-body scans) and 9 healthy controls (7 for brain scans and 6 for whole-body scans). Results: 11C-(R)-rolipram binding correlated with known locations of fibrous dysplasia in the periphery of individuals with MAS; no uptake was observed in the bones of healthy controls. In peripheral organs and the brain, no difference in 11C-(R)-rolipram uptake was noted between participants with MAS and healthy controls. Conclusion: This study is the first to find evidence for increased cAMP activity in areas of fibrous dysplasia in vivo. No differences in brain uptake between MAS participants and controls were detected-a finding that could be due to several reasons, including the limited anatomic resolution of PET. Nevertheless, the results confirm the usefulness of PET scans with 11C-(R)-rolipram to indirectly measure increased cAMP pathway activation in human disease.

CAMP/PKA-induced filamin A (FLNA) phosphorylation inhibits SST2 signal transduction in GH-secreting pituitary tumor cells

An efficient intracellular response to somatostatin analogs (SSA) in pituitary tumors requires filamin A (FLNA). Since cAMP pathway plays an important role in GH-secreting pituitary tumors pathogenesis and FLNA is phosphorylated by PKA on S2152, aim of this study was to investigate in tumoral somatotrophs the impact of cAMP pathway activation and SSA stimulation on FLNA phosphorylation and the consequences on SST2 function.We found a PKA-mediated increase (2-fold) and SST2 agonist-induced decrease (−50%) of FLNA phosphorylation in GH3, GH4C1 and primary somatotroph tumor cells. This modification regulates FLNA function. Indeed, phosphomimetic S2152D FLNA mutant, but not phosphodeficient S2152A, abolished the known SSA antitumoral effects, namely: 1) inhibition of cell proliferation, reduction of cyclin D3 and increase of p27; 2) increase of cell apoptosis; 3) inhibition of cell migration via RhoA activation and cofilin phosphorylation. Coimmunoprecipitation and immunofluorescence assays showed that S2152A FLNA was recruited to activated SST2, whereas S2152D FLNA constitutively bound SST2 on the plasma membrane, but prevented Gαi proteins recruitment to SST2.In conclusion, we demonstrated that FLNA phosphorylation, promoted by cAMP pathway activation and inhibited by SSA, prevented SST2 signaling in GH-secreting tumoral pituitary cells.

Stimulatory Effect of Intermittent Hypoxia on the Production of Corticosterone by Zona Fasciculata-Reticularis Cells in Rats

Hypoxia or intermittent hypoxia (IH) have known to alter both synthesis and secretion of hormones. However, the effect of IH on the production of adrenal cortical steroid hormones is still unclear. The aim of present study was to explore the mechanism involved in the effect of IH on the production of corticosterone by rat ZFR cells. Male rats were exposed at 12% O2 and 88% N2 (8 hours per day) for 1, 2, or 4 days. The ZFR cells were incubated at 37 °C for 1 hour with or without ACTH, 8-Br-cAMP, calcium ion channel blockers, or steroidogenic precursors. The concentration of plasma corticosterone was increased time-dependently by administration of IH hypoxia. The basal levels of corticosterone production in cells were higher in the IH groups than in normoxic group. IH resulted in a time-dependent increase of corticosterone production in response to ACTH, 8-Br-cAMP, progesterone and deoxycorticosterone. The production of pregnenolone in response to 25-OH-C and that of progesterone in response to pregnenolone in ZFR cells were enhanced by 4-day IH. These results suggest that IH in rats increases the secretion of corticosterone via a mechanism at least in part associated with the activation of cAMP pathway and steroidogenic enzymes.

PDE4D promotes FAK-mediated cell invasion in BRAF-mutated melanoma.

The cyclic AMP (cAMP) signaling pathway is critical in melanocyte biology for regulating differentiation. It is downregulated by phosphodiesterase (PDE) enzymes, which degrade cAMP itself. In melanoma evidence suggests that inhibition of the cAMP pathway by PDE type 4 (PDE4) favors tumor progression. For example, in melanomas harboring RAS mutations, the overexpression of PDE4 is crucial for MAPK pathway activation and proliferation induced by oncogenic RAS. Here we showed that PDE4D is overexpressed in BRAF-mutated melanoma cell lines, constitutively disrupting the cAMP pathway activation. PDE4D promoted melanoma invasion by interacting with focal adhesion kinase (FAK) through the scaffolding protein RACK1. Inhibition of PDE4 activity or inhibition of PDE4D interaction with FAK reduced invasion. PDE4D expression is increased in patients with advanced melanoma and PDE4D-FAK interaction is detectable in situ in metastatic melanoma. Our study establishes the role of PDE4D in BRAF-mutated melanoma as regulator of cell invasion, and suggests its potential as a target for preventing metastatic dissemination.

Neomorphic effects of recurrent somatic mutations in Yin Yang 1 in insulin-producing adenomas

Insulinomas are pancreatic islet tumors that inappropriately secrete insulin, producing hypoglycemia. Exome and targeted sequencing revealed that 14 of 43 insulinomas harbored the identical somatic mutation in the DNA-binding zinc finger of the transcription factor Yin Yang 1 (YY1). Chromatin immunoprecipitation sequencing (ChIP-Seq) showed that this T372R substitution changes the DNA motif bound by YY1. Global analysis of gene expression demonstrated distinct clustering of tumors with and without YY1(T372R) mutations. Genes showing large increases in expression in YY1(T372R) tumors included ADCY1 (an adenylyl cyclase) and CACNA2D2 (a Ca(2+) channel); both are expressed at very low levels in normal β-cells and show mutation-specific YY1 binding sites. Both gene products are involved in key pathways regulating insulin secretion. Expression of these genes in rat INS-1 cells demonstrated markedly increased insulin secretion. These findings indicate that YY1(T372R) mutations are neomorphic, resulting in constitutive activation of cAMP and Ca(2+) signaling pathways involved in insulin secretion.

Molecular and cellular characterization of a zebrafish optic pathway tumor line implicates glia-derived progenitors in tumorigenesis.

In this study we describe the molecular and cellular characterization of a zebrafish mutant that develops tumors in the optic pathway. Heterozygous Tg(flk1:RFP)is18 transgenic adults develop tumors of the retina, optic nerve and optic tract. Molecular and genetic mapping demonstrate the tumor phenotype is linked to a high copy number transgene array integrated in the lincRNA gene lincRNAis18/Zv9_00007276 on chromosome 3. TALENs were used to isolate a 147kb deletion allele that removes exons 2–5 of the lincRNAis18 gene. Deletion allele homozygotes are viable and do not develop tumors, indicating loss of function of the lincRNAis18 locus is not the trigger for tumor onset. Optic pathway tumors in the Tg(flk1:RFP)is18 mutant occur with a penetrance of 80–100% by 1 year of age. The retinal tumors are highly vascularized and composed of rosettes of various sizes embedded in a fibrous matrix. Immunohistochemical analysis showed increased expression of the glial markers GFAP and BLBP throughout retinal tumors and in dysplastic optic nerve. We performed transcriptome analysis of pre-tumorous retina and retinal tumor tissue and found changes in gene expression signatures of radial glia and astrocytes (slc1a3), activated glia (atf3, blbp, apoeb), proliferating neural progenitors (foxd3, nestin, cdh2, her9/hes1), and glioma markers (S100β, vim). The transcriptome also revealed activation of cAMP, Stat3 and Wnt signal transduction pathways. qRT-PCR confirmed >10-fold overexpression of the Wnt pathway components hbegfa, ascl1a, and insm1a. Together the data indicate Müller glia and/or astrocyte-derived progenitors could contribute to the zebrafish Tg(flk1:RFP)is18 optic pathway tumors.

SERCA2 activity is involved in the CNP‐mediated functional responses in failing rat myocardium

Myocardial C-type natriuretic peptide (CNP) levels are increased in heart failure. CNP can induce negative inotropic (NIR) and positive lusitropic responses (LR) in normal hearts, but its effects in failing hearts are not known. We studied the mechanism of CNP-induced NIR and LR in failing hearts and determined whether sarcoplasmatic reticulum Ca(2+) ATPase2 (SERCA2) activity is essential for these responses. Contractility, cGMP levels, Ca(2+) transient amplitudes and protein phosphorylation were measured in left ventricular muscle strips or ventricular cardiomyocytes from failing hearts of Wistar rats 6 weeks after myocardial infarction. CNP increased cGMP levels, evoked a NIR and LR in muscle strips, and caused phospholamban (PLB) Ser(16) and troponin I (TnI) Ser(23/24) phosphorylation in cardiomyocytes. Both the NIR and LR induced by CNP were reduced in the presence of a PKG blocker/cGMP analogue (Rp-8-Br-Pet-cGMPS) and the SERCA inhibitor thapsigargin. CNP increased the amplitude of the Ca(2+) transient and increased SERCA2 activity in cardiomyocytes. The CNP-elicited NIR and LR were not affected by the L-type Ca(2+) channel activator BAY-K8644, but were abolished in the presence of isoprenaline (induces maximal activation of cAMP pathway). This suggests that phosphorylation of PLB and TnI by CNP causes both a NIR and LR. The NIR to CNP in mouse heart was abolished 8 weeks after cardiomyocyte-specific inactivation of the SERCA2 gene. We conclude that CNP-induced PLB and TnI phosphorylation by PKG in concert mediate both a predictable LR as well as the less expected NIR in failing hearts.

Critical Role of Histone Acetylation by p300 in Human Placental 11β-HSD2 Expression

Fetal overexposure to glucocorticoids leads to growth restriction. Optimal fetal glucocorticoid level is ensured by the expression of cortisol-inactivating enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) in placental syncytiotrophoblasts. The transcription factor selective promoter factor 1 (Sp1) is known to up-regulate 11β-HSD2 expression in the presence of enhanced histone acetylation in syncytiotrophoblasts, but the mechanisms underlying histone acetylation remain unknown. The role of p300 in histone acetylation associated with 11β-HSD2 expression in syncytiotrophoblasts was investigated. Distribution of p300 in human placenta was studied with immunohistochemistry. The role of p300 in histone-3 (H3) acetylation in association with 11β-HSD2 expression was investigated in cultured primary human placental trophoblasts in the presence of small interfering RNA (siRNA)-mediated knockdown of p300, p300 inhibitor C646, or p300 overexpression. The interaction of Sp1 and p300 was studied with chromatin immunoprecipitation and coimmunoprecipitation. Intense staining of p300 was found in the nuclei of trophoblasts. Levels of p300 and acetyl H3K9 and H3K27 associated with 11β-HSD2 promoter were increased in the course of syncytialization and by cAMP pathway activation. Chromatin immunoprecipitation and coimmunoprecipitation revealed p300 and Sp1 on 11β-HSD2 promoter and in the same protein complex in the syncytiotrophoblasts. Overexpression of p300 enhanced 11β-HSD2 expression, which was attenuated by Sp1 knockdown, whereas p300 knockdown and C646 reduced both basal and cAMP-stimulated acetylation of H3K9 and H3K27 associated with 11β-HSD2 expression. Interaction of p300 with Sp1 plays a crucial role in histone acetylation associated with 11β-HSD2 expression in syncytiotrophoblasts, which may have important implications in the establishment of the placental glucocorticoid barrier in gestation.

Cyclic AMP stimulates neurite outgrowth of lamprey reticulospinal neurons without substantially altering their biophysical properties

Reticulospinal (RS) neurons are critical for initiation of locomotor behavior, and following spinal cord injury (SCI) in the lamprey, the axons of these neurons regenerate and restore locomotor behavior within a few weeks. For lamprey RS neurons in culture, experimental induction of calcium influx, either in the growth cone or cell body, is inhibitory for neurite outgrowth. Following SCI, these neurons partially downregulate calcium channel expression, which would be expected to reduce calcium influx and possibly provide supportive conditions for axonal regeneration. In the present study, it was tested whether activation of second messenger signaling pathways stimulates neurite outgrowth of lamprey RS neurons without altering their electrical properties (e.g. spike broadening) so as to possibly increase calcium influx and compromise axonal growth. First, activation of cAMP pathways with forskolin or dbcAMP stimulated neurite outgrowth of RS neurons in culture in a PKA-dependent manner, while activation of cGMP signaling pathways with dbcGMP inhibited outgrowth. Second, neurophysiological recordings from uninjured RS neurons in isolated lamprey brain–spinal cord preparations indicated that dbcAMP or dbcGMP did not significantly affect any of the measured electrical properties. In contrast, for uninjured RS neurons, forskolin increased action potential duration, which might have increased calcium influx, but did not significantly affect most other electrical properties. Importantly, for injured RS neurons during the period of axonal regeneration, forskolin did not significantly alter their electrical properties. Taken together, these results suggest that activation of cAMP signaling by dbcAMP stimulates neurite outgrowth, but does not alter the electrical properties of lamprey RS neurons in such a way that would be expected to induce calcium influx. In conclusion, our results suggest that activation of cAMP pathways alone, without compensation for possible deleterious effects on electrical properties, is an effective approach for stimulating axonal regeneration of RS neuron following SCI.

Testosterone reduces vascular relaxation by altering cyclic adenosine monophosphate pathway and potassium channel activation in male Sprague Dawley rats fed a high-salt diet

Male gender and high-salt diet are risk factors for hypertension. The effect of chronic exposure to testosterone is an increase in vascular tone but its influence upon responses induced by other vasoactive agents is not clear. We considered the possibility of interactions between testosterone and a high-salt diet in the mechanisms that are involved in the regulation of vascular tone. Therefore, we designed experiments to assess the involvement of the cyclic adenosine monophosphate (cAMP) pathway and potassium channel activation on vascular relaxation elicited by testosterone deficiency that was induced by orchidectomy in Sprague Dawley rats on a normal or high-salt diet. Weanling male rats were randomly divided into eight groups (n = 6 each) that were either orchidectomized or sham operated with or without testosterone replacement (10 mg/kg body weight of Sustanon 250 intramuscularly, Organon, Holland) and were placed on a normal or high-salt (0.3% or 8% NaCl) diet, respectively, for 6 weeks. Arterial blood pressure was determined before and weekly throughout the experiment using the tail-cuff method. Relaxation responses to forskolin and diazoxide were studied in noradrenaline (0.1 µM) precontracted aortic rings. There was an increase in the systolic blood pressure of rats placed on a high-salt diet compared with control or orchidectomized rats. Orchidectomy elicited a reduction in the systolic blood pressure while testosterone replacement restored systolic blood pressure to values seen in intact rats. A high-salt diet reduced the relaxation response to forskolin and diazoxide but not in orchidectomized rats while testosterone replacement re-established the blunted relaxation response to forskolin and diazoxide. Inhibition of potassium channel or adenylyl cyclase activation appears to contribute to the mechanisms by which a high-salt diet increases vascular tone. These effects were counteracted by orchidectomy in male Sprague Dawley rats.

Environmental Novelty Activates β2-Adrenergic Signaling to Prevent the Impairment of Hippocampal LTP by Aβ Oligomers

A central question about human brain aging is whether cognitive enrichment slows the development of Alzheimer changes. Here, we show that prolonged exposure to an enriched environment (EE) facilitated signaling in the hippocampus of wild-type mice that promoted long-term potentiation. Akey feature of the EE effect was activation of β2-adrenergic receptors and downstream cAMP/PKA signaling. This EE pathway prevented LTP inhibition by soluble oligomers of amyloid β-protein (Aβ) isolated from AD cortex. Protection by EE occurred in both young and middle-aged wild-type mice. Exposure to novelty afforded greater protection than did aerobic exercise. Mice chronically fed a β-adrenergic agonist without EE were protected from hippocampal impairment by Aβ oligomers. Thus, EE enhances hippocampal synaptic plasticity by activating β-adrenoceptor signaling and mitigating synaptotoxicity of human Aβ oligomers. These mechanistic insights support using prolonged exposure to cognitive novelty and/or oral β-adrenergic agonists to lessen the effects of Aβ accumulation during aging.