Stimulation Of Adenylate Cyclase Research Articles

L-asparaginase induces IP3R-mediated ER Ca2+ release by targeting µ-OR1 and PAR2 and kills acute lymphoblastic leukemia cells

L-asparaginase is a standard therapeutic option for acute lymphoblastic leukemia (aLL), a hematologic cancer that claims the most lives of pediatric cancer patients. Previously, we demonstrated that L-asparaginase kills aLL cells via a lethal rise in [Ca2+]i due to IP3R-mediated ER Ca2+ release followed by calpain-1-Bid-caspase-3/12 activation (Blood, 133, 2222-2232). However, upstream targets of L-asparaginase that trigger IP3R-mediated ER Ca2+ release remain elusive. Here, we show that L-asparaginase targets µ-OR1 and PAR2 and induces IP3R-mediated ER Ca2+ release in aLL cells. In doing so, µ-OR1 plays a major role while PAR2 plays a minor role. Utilizing PAR2- and µ-OR1-knockdown cells, we demonstrate that L-asparaginase stimulation of µ-OR1 and PAR2 relays its signal via Gαi and Gαq, respectively. In PAR2-knockdown cells, stimulation of adenylate cyclase with forskolin or treatment with 8-CPT-cAMP reduces L-asparaginase-induced µ-OR1-mediated ER Ca2+ release, suggesting that activation of µ-OR1 negatively regulates AC and cAMP. In addition, the PKA inhibitor 14-22 amide (myr) alone evokes ER Ca2+ release, and subsequent L-asparaginase treatment does not induce further ER Ca2+ release, indicating the involvement of PKA inhibition in L-asparaginase-induced µ-OR1-mediated ER Ca2+ release, which can bypass the L-asparaginase-µ-OR1-AC-cAMP loop. This coincides with (a) the decreases in PKA-dependent inhibitory PLCβ3 Ser1105 phosphorylation, which prompts PLCβ3 activation and ER Ca2+ release, and (b) BAD Ser118 phosphorylation, which leads to caspase activation and apoptosis. Thus, our findings offer new insights into the Ca2+-mediated mechanisms behind L-asparaginase-induced aLL cell apoptosis and suggest that PKA may be targeted for therapeutic intervention for aLL.

Acute Treatment with the Nootropic CILTEP® Does Not Improve Cognitive Performance in Healthy Middle-Aged Participants

This study investigated the acute effects of the dietary nootropic stack CILTEP®. It contains a combination of ingredients that have been individually reported to improve cognitive performance. Especially, the ingredients luteolin, which is considered a phosphodiesterase type 4 (PDE4) inhibitor, and forskolin, an adenylate cyclase stimulator, were of interest since they can increase the second messenger cAMP and thus also intracellular signaling. Numerous studies have shown that inhibition of PDE4 can improve memory in animals and humans. We examined whether acute dosing of 3 capsules of CILTEP® would improve cognitive function in healthy participants aged 30 to 40 (n = 33). We used a randomized, double-blind, placebo-controlled, two-way cross-over design. Our test battery was aimed at measuring memory performance, attention, and sensorimotor speed. The primary outcome measures were the performance on the verbal learning task and the spatial pattern separation task. Secondary outcomes included other cognitive tests, event-related potentials (ERPs), and assessment of the activity of the enzyme beta-glucuronidase and its effect on the bioavailability of luteolin, heart rate, and blood pressure. No relevant effects of acute CILTEP® treatment were found on any measure of the test battery or ERPs. Blood plasma concentrations of luteolin increased, yet about 2000 times too low to likely exert any PDE4 inhibition. CILTEP® treatment did neither affect heart rate nor blood pressure. In summary, there is no evidence that a single standardized dose of 3 capsules of CILTEP® can improve cognitive function in healthy middle-aged participants.

Vascular reactivity is altered in the placentas of fetuses with congenital diaphragmatic hernia

IntroductionInfants with congenital diaphragmatic hernia (CDH) often develop pulmonary hypertension but frequently fail to respond to vasodilator therapy, for instance because of an altered pulmonary vasoreactivity. Investigating such alterations in vivo is impossible. We hypothesised that these alterations are also present in fetoplacental vessels, since both vasculatures are exposed to the same circulating factors (e.g. endothelin-1) and respond similarly to certain stimuli (e.g. hypoxia). As proof-of-concept, we compared fetoplacental vasoreactivity between healthy and CDH-affected placentas. MethodsFetoplacental vascular function of healthy and antenatally diagnosed left-sided CDH fetuses was assessed by wire myography. Placental expression of enzymes and receptors involved in the altered vasoreactive pathways was measured using quantitative PCR. ResultsCDH arteries (n = 6) constricted more strongly to thromboxane A2 agonist U46619 (p < 0.001) and dilated less to bradykinin (p = 0.01) and nitric oxide (NO)-donor sodium nitroprusside (p = 0.04) than healthy arteries (n = 8). Vasodilation to prostacyclin analogue iloprost and adenylate cyclase stimulator forskolin, and vasoconstriction to endothelin-1 were not different between both groups. Angiotensin II did not induce vasoconstriction. Phosphodiesterase inhibitors sildenafil and milrinone did not affect responses to sodium nitroprusside, forskolin, or U46619. The mRNA expression of guanylate cyclase 1 soluble subunit alpha 1 (p = 0.003) and protein kinase cyclic guanine monophosphate (cGMP)-dependent 1 (p = 0.02) were reduced in CDH versus healthy placentas. DiscussionThe identified changes in the thromboxane and NO-cGMP pathways in the fetoplacental vasculature correspond with currently described alterations in the pulmonary vasculature in CDH. Therefore, fetoplacental arteries may provide an opportunity to predict pulmonary therapeutic responses in infants with CDH.

3-Hydroxybutyrate ameliorates insulin resistance by inhibiting PPARγ Ser273 phosphorylation in type 2 diabetic mice

3-Hydroxybutyrate (3HB) is a small ketone body molecule produced endogenously by the body in the liver. Previous studies have shown that 3HB can reduce blood glucose level in type 2 diabetic (T2D) patients. However, there is no systematic study and clear mechanism to evaluate and explain the hypoglycemic effect of 3HB. Here we demonstrate that 3HB reduces fasting blood glucose level, improves glucose tolerance, and ameliorates insulin resistance in type 2 diabetic mice through hydroxycarboxylic acid receptor 2 (HCAR2). Mechanistically, 3HB increases intracellular calcium ion (Ca2+) levels by activating HCAR2, thereby stimulating adenylate cyclase (AC) to increase cyclic adenosine monophosphate (cAMP) concentration, and then activating protein kinase A (PKA). Activated PKA inhibits Raf1 proto-oncogene serine/threonine-protein kinase (Raf1) activity, resulting in a decrease in extracellular signal-regulated kinases 1/2 (ERK1/2) activity and ultimately inhibiting peroxisome proliferator-activated receptor γ (PPARγ) Ser273 phosphorylation in adipocytes. Inhibition of PPARγ Ser273 phosphorylation by 3HB altered the expression of PPARγ regulated genes and reduced insulin resistance. Collectively, 3HB ameliorates insulin resistance in type 2 diabetic mice through a pathway of HCAR2/Ca2+/cAMP/PKA/Raf1/ERK1/2/PPARγ.

Erratum to: "Adenylate cyclase stimulation and [3H]thymidine incorporation in human thyroid tissues and thyrocyte cultures: The effect of IgG preparation from patients with different thyroid disorders".

"Erratum to: “Adenylate cyclase stimulation and [3H]thymidine incorporation in human thyroid tissues and thyrocyte cultures: The effect of IgG preparation from patients with different thyroid disorders”" published on 01 Dec 2022 by Bioscientifica Ltd.

Abstract 822: Oncogene addiction to GNAS in GNASR201 mutant tumors

Abstract Background: The GNASR201 mutation is the single most frequent cancer-causing mutation across all heterotrimeric G proteins. This gain of function mutation in GNAS drives oncogenesis in appendiceal, colorectal, and gastric adenocarcinoma, as well as Intraductal Papillary Mucinous Neoplasms (IPMN) and Small Cell Lung Cancer (SCLC). In this study, we investigated the role of GNAS in tumor growth using peritoneal models of colorectal cancer (CRC). Methods: GNAS was knocked out in multiple GNASR201C/H mutant colon cell lines (KM12, SNU175, and SKCO1) and overexpressed in GNASWT LS174T cells. Isogenic pairs of KM12 and LS174T cells were injected into the peritoneum of NSG mice to study the role of GNAS in cell line derived xenograft (CDX) models of peritoneal metastasis. Cell lines and CDX were profiled with RNAseq, reverse phase protein assay (RPPA), and immunohistochemistry (IHC) to identify potential pathways regulated by mutant GNAS. Identified mediators of GNAS signaling were then validated using chemical inhibitors of PKA (H-89) and β-catenin (LF3). Results: GNAS knockout significantly decreased 2D colony formation by KM12 (68%), SNU175 (76%), and SKCO1 (85%) cells (all p &amp;lt; 0.0001), and decreased 3D organoid area of KM12 cells by 62% (p = 0.043). There was significant increase in colony formation by LS174T cells overexpressing GNASR201C (193%, p = 0.016) and GNASR201H (170%, p =0.0037). Mice injected with KM12 GNAS-knockout tumors exhibited a significant 68% reduction in tumor growth (n = 6, p = 0.016) and were more likely to survive at 7 weeks (0% vs. 100%, p = 0.0007) relative to those with parent KM12-GNASR201H tumors. Likewise, mice injected with GNASR201H LS174T cells showed significant increase in tumor growth (934% vs. 100%, n = 3, p = 0.042). Histology of GNAS-knockout tumors showed a marked decrease in mucinous stroma and increased lytic necrosis, suggesting an interaction between oncogenic GNAS signaling and the peritoneal environment. GNAS is known to stimulate adenylate cyclase and GNAS knockout decreased levels of cyclic AMP in KM12 cells, confirming an on-target effect. RPPA and RNAseq profiling of KM12 and LS174T PDX tumors identified phosphorylation of β-catenin and activation of Wnt/β-catenin targets (NES = 1.25, 1.24) as critical downstream effects of mutant GNAS signaling, confirmed by a 4.9-fold (p = 0.001) increase in nuclear β-catenin intensity in LS174T GNASR201H tumors (19% vs. 99% nuclei stained positive, p = 0.0002). Chemical inhibition of both PKA and β-catenin reduced growth of GNAS mutant organoids by 79% (p = 0.004) and 67% (p = 0.007) respectively, supporting the involvement of the cAMP/PKA and β-catenin pathways in mutant GNAS signaling. Conclusions: Our findings demonstrate oncogene addiction to GNAS in peritoneal models of GNASR201C/H tumors, which signal through the cAMP/PKA and Wnt/β-catenin pathways. Thus, GNAS and its downstream mediators are promising therapeutic targets for GNAS mutant tumors. Citation Format: Aditya More, Valsala Haridas, Ichiaki Ito, Saikat Chowdhury, Yue Gu, Natalie W. Fowlkes, John P. Shen. Oncogene addiction to GNAS in GNASR201 mutant tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 822.

PDE1 Inhibition Modulates Cav1.2 Channel to Stimulate Cardiomyocyte Contraction.

[Figure: see text].

The Role of cAMP in Beta Cell Stimulus-Secretion and Intercellular Coupling.

Pancreatic beta cells secrete insulin in response to stimulation with glucose and other nutrients, and impaired insulin secretion plays a central role in development of diabetes mellitus. Pharmacological management of diabetes includes various antidiabetic drugs, including incretins. The incretin hormones, glucagon-like peptide-1 and gastric inhibitory polypeptide, potentiate glucose-stimulated insulin secretion by binding to G protein-coupled receptors, resulting in stimulation of adenylate cyclase and production of the secondary messenger cAMP, which exerts its intracellular effects through activation of protein kinase A or the guanine nucleotide exchange protein 2A. The molecular mechanisms behind these two downstream signaling arms are still not fully elucidated and involve many steps in the stimulus–secretion coupling cascade, ranging from the proximal regulation of ion channel activity to the central Ca2+ signal and the most distal exocytosis. In addition to modifying intracellular coupling, the effect of cAMP on insulin secretion could also be at least partly explained by the impact on intercellular coupling. In this review, we systematically describe the possible roles of cAMP at these intra- and inter-cellular signaling nodes, keeping in mind the relevance for the whole organism and translation to humans.

Early Effects of the Soluble Amyloid β25-35 Peptide in Rat Cortical Neurons: Modulation of Signal Transduction Mediated by Adenosine and Group I Metabotropic Glutamate Receptors.

The amyloid β peptide (Aβ) is a central player in the neuropathology of Alzheimer’s disease (AD). The alteration of Aβ homeostasis may impact the fine-tuning of cell signaling from the very beginning of the disease, when amyloid plaque is not deposited yet. For this reason, primary culture of rat cortical neurons was exposed to Aβ25-35, a non-oligomerizable form of Aβ. Cell viability, metabotropic glutamate receptors (mGluR) and adenosine receptors (AR) expression and signalling were assessed. Aβ25-35 increased mGluR density and affinity, mainly due to a higher gene expression and protein presence of Group I mGluR (mGluR1 and mGluR5) in the membrane of cortical neurons. Intriguingly, the main effector of group I mGluR, the phospholipase C β1 isoform, was less responsive. Also, the inhibitory action of group II and group III mGluR on adenylate cyclase (AC) activity was unaltered or increased, respectively. Interestingly, pre-treatment of cortical neurons with an antagonist of group I mGluR reduced the Aβ25-35-induced cell death. Besides, Aβ25-35 increased the density of A1R and A2AR, along with an increase in their gene expression. However, while A1R-mediated AC inhibition was increased, the A2AR-mediated stimulation of AC remained unchanged. Therefore, one of the early events that takes place after Aβ25-35 exposure is the up-regulation of adenosine A1R, A2AR, and group I mGluR, and the different impacts on their corresponding signaling pathways. These results emphasize the importance of deciphering the early events and the possible involvement of metabotropic glutamate and adenosine receptors in AD physiopathology.

CAMP-Independent Activation of the Unfolded Protein Response by Cholera Toxin.

Cholera toxin (CT) is an AB5 protein toxin that activates the stimulatory alpha subunit of the heterotrimeric G protein (Gsα) through ADP-ribosylation. Activation of Gsα produces a cytopathic effect by stimulating adenylate cyclase and the production of cAMP. To reach its cytosolic Gsα target, CT binds to the plasma membrane of a host cell and travels by vesicle carriers to the endoplasmic reticulum (ER). The catalytic CTA1 subunit then exploits the quality control mechanism of ER-associated degradation to move from the ER to the cytosol. ER-associated degradation is functionally linked to another quality control system, the unfolded protein response (UPR). However, the role of the UPR in cholera intoxication is unclear. We report here that CT triggers the UPR after 4 h of toxin exposure. A functional toxin was required to induce the UPR, but, surprisingly, activation of the adenylate cyclase signaling pathway was not sufficient to trigger the process. Toxin-induced activation of the UPR coincided with increased toxin accumulation in the cytosol. Chemical activation of the heterotrimeric G protein or the UPR also enhanced the onset of CTA1 delivery to the cytosol, thus producing a toxin-sensitive phenotype. These results indicate there is a cAMP-independent response to CT that activates the UPR and thereby enhances the efficiency of intoxication.

Influence of Phosphodiesterase Inhibition on CRE- and EGR1-Dependent Transcription in a Mouse Hippocampal Cell Line.

Signaling pathways, depending on the second messenger molecule cAMP, modulate hippocampal cell signaling via influencing transcription factors like cAMP-regulated element-binding protein (CREB) or early growth response 1 EGR1/Krox24/zif268/ZENK (EGR1). Here, we investigated two reporter cell lines derived from an immortalized hippocampal neuronal cell line stably expressing a CRE- or EGR1-luciferase reporter gene (HT22CREluc and HT22EGR1luc, respectively). The cells were subjected to phosphodiesterase inhibitors and other cAMP-modulating agents to investigate dose- and time-dependent phosphodiesterase (PDE)-mediated fine-tuning of cAMP-dependent transcriptional signaling. The non-isoform-specific cyclic nucleotide phosphodiesterase (PDE) inhibitor isobutyl-methyl-xanthine (IBMX), as well as selective inhibitors of PDE3 (milrinone) and PDE4 (rolipram), were tested for their ability to elevate CRE- and EGR1-luciferase activity. Pharmacological parameters like onset of activity, maximum activity, and offset of activity were determined. In summary, phosphodiesterase inhibition appeared similarly potent in comparison to adenylate cyclase stimulation or direct activation of protein kinase A (PKA) via specific cAMP agonists and was at least partly mediated by PKA as shown by the selective PKA inhibitor Rp-8-Br-cAMPS. Moreover, transcriptional activation by PDE inhibition was also influenced by organic anion-exchanger action and interacted with fibroblast growth factor (FGF) receptor-mediated pathways.

Interleukin-10 contributes to PGE2 signalling through upregulation of EP4 via SHIP1 and STAT3.

Macrophage cells form part of our first line defense against pathogens. Macrophages become activated by microbial products such as lipopolysaccharide (LPS) to produce inflammatory mediators, such as TNFα and other cytokines, which orchestrate the host defense against the pathogen. Once the pathogen has been eradicated, the activated macrophage must be appropriately deactivated or inflammatory diseases result. Interleukin-10 (IL10) is a key anti-inflammatory cytokine which deactivates the activated macrophage. The IL10 receptor (IL10R) signals through the Jak1/Tyk2 tyrosine kinases, STAT3 transcription factor and the SHIP1 inositol phosphatase. However, IL10 has also been described to induce the activation of the cyclic adenosine monophosphate (cAMP) regulated protein kinase A (PKA). We now report that IL10R signalling leads to STAT3/SHIP1 dependent expression of the EP4 receptor for prostaglandin E2 (PGE2). In macrophages, EP4 is a Gαs-protein coupled receptor that stimulates adenylate cyclase (AC) production of cAMP, leading to downstream activation of protein kinase A (PKA) and phosphorylation of the CREB transcription factor. IL10 induction of phospho-CREB and inhibition of LPS-induced phosphorylation of p85 PI3K and p70 S6 kinase required the presence of EP4. These data suggest that IL10R activation of STAT3/SHIP1 enhances EP4 expression, and that it is EP4 which activates cAMP-dependent signalling. The coordination between IL10R and EP4 signalling also provides an explanation for why cAMP elevating agents synergize with IL10 to elicit anti-inflammatory responses.

Forskolin Induced Syncytialization in Placental Trophoblast Induces Changes in Glycocalyx Composition

The placenta is known to function as a bidirectional selectively permeable barrier between maternal and fetal circulatory systems during pregnancy. The fetal placental villi are surrounded by a syncytiotrophoblast‐cell layer, which is the first functional layer of the placental barrier. Syncytiotrophoblasts possess an apical extracellular matrix (ECM) which functions similarly to the endothelial glycocalyx; acting as a mechanical barrier for large particles and by regulating permeability and transport of molecules. Among the most important ECM proteins are the syndecan (SDC) family. The immortalized trophoblast‐like BeWo cell line is a commonly used in vitro model of placental trophoblasts, however, they do not spontaneously syncytialize and thus do not represent an accurate model to study placental barrier function. However, they can be induced to syncytialize by addition of forskolin, an adenylate cyclase stimulator. Here we hypothesized that forskolin‐induced syncytialization of BeWos upregulates ECM protein expression and produces a morphological phenotype through which placental barrier function can be studied in the future. To test our hypothesis, we treated BeWos with 25 uM Forskolin or DMSO as a control for 48 hours and measured SDC gene expression and protein levels. After treatment, media and cells were collected for analysis. Because SDC has four non‐allelic variants in rodents, SDC‐1, ‐2, ‐3, and ‐4, we measured the expression of all four via qRT‐PCR and normalized them to β‐actin expression. SDC‐1, ‐2, and ‐3 expression were all increased in the forskolin treated group compared to controls (p&lt;0.0001 for all comparisons). Interestingly, SDC‐4 expression was decreased by treatment with forskolin (p=0.004). Next, we wished to verify our results and also to identify the predominant SDC isoforms in BeWos. In order to do this we utilized digital droplet PCR to determine absolute copy number. Again, we saw increases in SDC‐1, SDC‐2, and SDC‐3 expression in the forskolin treated group compared to controls (p&lt;0.0001 for all comparisons). We also observed decreased SDC‐4 expression in the forskolin treated group compared to controls using this method (p&lt;0.0001). Additionally, we observed that SDC‐1 (2255 ± 119 copies/ng RNA) and SDC‐4 (2881 ± 136.6 copies/ng RNA) were the predominant isoforms expressed in BeWos under control conditions, while SDC‐2 (15 ± 0.7 copies/ng RNA) and SDC‐3 (124 ± 5.4 copies/ng RNA) were expressed at very low levels. We then measured cellular and media SDC‐1 protein levels via ELISA. Compared to controls, forskolin treatment significantly increased cellular levels of SDC‐1, which were normalized to total protein (1.23 pg/ug ± 0.05 vs 2.56 pg/ug ± 0.22, respectively; p=0.002). Forskolin treatment also significantly increased media SDC‐1 compared to controls (1189 ± 10 pg/mL vs 555.5 ± 19 pg/mL, respectively; p&lt;0.0001). From this data we concluded that forskolin‐induced syncytialization of BeWos regulates the composition and quantity of the placental glycocalyx. Future studies of placental barrier function in vitro should first induce syncytialization with forskolin treatment for 48 hours before additional protocols are carried out.Support or Funding InformationP01HL51971, P20GM104357, T32HL105324, and R01HL137791

Efforts Toward GPR88 Deorphanization: Challenges and Alternative Approaches to Troublesome Cell‐Based Second Messenger Assays

G protein‐coupled receptors (GPCR) comprise a family of over 800 integral membrane proteins involved in signal transduction and serve as the primary cellular sensors for chemical stimuli. About 35% of current drugs act on GPCR targets. In addition, of the 300 agents in current clinical trials, greater than 20% target novel GPCRs for which there are no approved drugs (orphan GPCRs). Ligand discovery and deorphanization efforts seeking to assign function to understudied GPCRs are not trivial. Notoriously complex in nature, GPCR‐dependent signaling centers around receptor‐ligand interactions that serve to stabilize conformational access to intracellular signaling partners, G proteins. In GPCR signaling, receptors and heterotrimeric G proteins (made of the Gα and Gβγ subunits) work together to transmit signals via downstream effectors and distinct pathways. The measurement of GPCR mediated second messengers such as cyclic AMP (cAMP), intracellular calcium (Ca2+) mobilization, and ERK activation (MAP kinase) are commonly used as tools to detect ligand induced GPCR activation. However, experimental pathway elucidation is complicated by the tendencies for pathway crosstalk, receptor desensitization, and receptor‐G protein interaction promiscuity. Cell‐based assays that monitor second messenger responses are often used as screening tools for potential GPCR agonists and antagonists, however, commonly used cell‐lines such as HEK‐293, express many endogenous GPCRs that can complicate data analysis. Many classes of Gα subunits exist: Gαs and Gα i/o G‐proteins modulate cyclic AMP (cAMP) through stimulation or inhibition of adenylate cyclase while Gαq subunits activate phospholipase C and a subsequent rapid release of intracellular Ca2+. Specifically, the orphan receptor, GPR88, has been linked with psychiatric diseases including schizophrenia and bipolar disorder. Lacking a validated endogenous agonist, the GPR88 signaling pathway remains poorly understood, however, in vivo knockout experiments and in vitro preclinical studies with small molecule synthetic agonists indicate that GPR88 couples to Gαi/o G‐proteins to inhibit adenylate cyclase activity and reduce intracellular cAMP in cell‐based assays. In this work, we describe our challenges in the study of the GPR88 signaling pathway using second messenger cell‐based assays and explain our decision to move to a real‐time NanoLuc‐based GPCR/G protein complementation assay.Support or Funding InformationNIH grant 1 R15 MH109034

Protein Kinase A‐mediated Regulation of SLC5A8 Surface Expression

SLC5A8 (solute carrier gene family 5A, member 8) encodes the sodium monocarboxylate transporter, SMCT1. SMCT1 mediates cotransport of sodium and monocarboxylates, such as short chain fatty acids, in many tissues, including thyroid, kidney, and colon. Inspection of SMCT1’s primary amino acid sequence identifies not only a carboxyl terminus post‐synaptic density/discs large/zona occuldens 1 (PDZ) binding motif postulated to be important in macromolecular transport protein complexing, but also consensus sites for phosphorylation by multiple classes of kinases. Notably, SMCT1 contains a single protein kinase A (PKA) consensus site: RRMT (amino acids 50–53), with T53 potentially functioning as the site of phosphorylation. The present study considers the hypothesis that PKA regulates SMCT1 surface expression, as reported for the sodium‐glucose cotransporter (SGLT1, the product of the SLC5A1 gene). The following experiments test whether elimination of the phosphorylation site disrupts PKA‐mediated regulation of surface expression. Using site‐directed mutagenesis, we disrupted the consensus motif and concomitantly disabled phosphorylation by replacing T53 with an alanine residue (T53A). To facilitate downstream assays, we generated T53A on the background of a human SMCT1 expression construct containing an amino‐terminus, extracellular hemagglutinin (HA) tag. In transiently expressing COS‐7 cells, surface luminescence measurements confirmed T53A resides in the cell surface at levels similar to wild‐type SMCT1, and immunoblot measurements indicate similar total expression levels. Wild‐type SMCT1‐expressing cells displayed increased luminescence signal (~20%) subsequent to forskolin (10 μM) treatment to stimulate adenylate cyclase and activate PKA, consistent with augmented surface expression. In contrast, cells expressing T53A were indifferent to forskolin treatment. As an independent measure of surface expression, plans are underway to perform cell‐surface immunoprecipitation (CS‐IP) experiments. Taken together, these data suggest that, although constitutive plasma membrane residency does not depend on an intact PKA consensus site, forskolin‐activated, PKA‐dependent phosphorylation can increase SMCT1 levels in the plasma membrane, and thereby augment potential transport capacity.Support or Funding InformationNIH/NIGMS Award 1R15GM101674‐01A1 (to PF)

Possible effects of pituitary adenylate cyclase activating polypeptide (PACAP) on early embryo implantation marker HB-EGF in mouse

Pituitary adenylate cyclase activating polypeptide (PACAP) was originally isolated as a hypothalamic neuropeptide stimulating adenylate cyclase activity. Besides its neuroprotective effects, numerous data proved its role in reproductive processes. However, there are limited data on its role in preimplantation embryo development and implantation. Our aim was to analyse the mRNA expression of Adcyap1 (coding region of PACAP) and Hbegf [coding region of HB-EGF (Heparin-binding EGF-like growth factor)] in embryos and pregnant uterus to investigate the possible correlation between them. Eight-week-old BDF1 mice were superovulated and subsequently mated overnight or left in their cage after hCG treatment. Day4 embryos were flushed from mated females. After morphological analysis, Adcyap1 and Hbegf gene expression of embryos and uterine tissues was assessed with qPCR.Our results showed significantly higher Adcyap1 and Hbegf mRNA levels in females producing embryos compared to non-mated ones. Robust elevation of Adcyap1 and slight elevation of Hbegf were detected in females with blastocyst embryos compared with non-blastocysts. We found low rate of Hbegf mRNA expression in uncompacted embryos, whereas morulae and blastocysts expressed high amounts of Hbegf. However, we did not find detectable Adcyap1 mRNA in embryos. Strong correlation was found between uterine tissue and embryonic Hbegf levels, slight correlation between uterine Adcyap1 and Hbegf levels. Uterine tissue Adcyap1 and embryonic Hbegf showed no correlation. In summary, our present data show, for the first time, the correlation between PACAP and HB-EGF mRNA expression suggesting that PACAP might play a role during the peri-implantation period of early mouse embryo development.

Regulation of Platelet Function By Adenosine Receptors

Introduction: We have recently shown that severe trauma and hemorrhage lead to inhibition of platelet aggregation and an elevation in cyclic adenosine monophosphate (cAMP). Adenosine is one of the few humoral agents known to stimulate cAMP in platelets. Because adenosine is released from damaged tissue, it may contribute to the platelet dysfunction seen after severe trauma. Platelets have four adenosine receptors (A1, A2a, A2b and A3). These receptors are G-Protein Coupled Receptors and have been proposed to stimulate adenylyl cyclase and increase intracellular cAMP. Although studies have shown that stimulate A2a can inhibit platelet aggregation and elevate cAMP, there is little data elucidating the function of the other receptors. Objective: Define which adenosine receptors affects platelet aggregation and cAMP production. Methods: Platelet-rich plasma (PRP) was isolated from whole blood of human volunteers, and centrifuged at 200g for 10min. Light transmission aggregometry was performed using a plate reader (Synergy Neo2 Multimode Reader, BioTek) with constant agitation. PRP was stimulated with adenosine diphosphate (ADP) with or without various adenosine agonists or antagonists, including the non-metabolizable adenosine agonist 5-(N-ethyl-carboxamido) adenosine (NECA), antagonists to receptors A1 (DPCPX), A2a (Sch 58261), A2b (GS 6201) and A3 (MRS 1220), or agonists for A2a (CGS 21680) A2b (BAY 60-6583) or agonist A1 (CCPA), A2a (CGS 21680), A2b (Bay 60-6583), A3 (2-Cl-IB-Meca). Cyclic AMP was extracted from 100ul of PRP after adding 1ml of EtOH, 10mM ammonium formate, with 10ug/ml cGMP-Br as an internal control. Samples were centrifuged at 20K g for 10min, and supernatant dried. Samples were brought up in 200ul of 0.1% formic acid for analysis by Reverse Phase liquid chromatography/ Tandem Mass Spectroscopy (Quantiva, ThrermoFisher). N-8/group. Results: Adenosine diphosphate (100uM) leads to platelet aggregation (change in mAbsorbance units, Table 1). The adenosine agonist NECA inhibited aggregation to ADP and elevated cAMP in a dose dependent manner (pg/ml per 1000 plt, Table 1). Platelet aggregation was inhibited and cAMP was elevated after stimulation with agonists for adenosine receptor A2a agonist, but not A1, A2b, or A3 (Table 2). Antagonists for A2a, but not A1, A2b, A3, blocked NECA inhibition of ADP aggregation (Table 3). Agonist for adenosine receptor A2a inhibited the ADP-induced aggregation and elevated cAMP in a dose response manner (Table 4). Discussion: Adenosine inhibits platelet aggregation to ADP. The mechanism appears to be due to elevation in intracellular cAMP, and works through the A2a receptor. These data suggest that the A2a receptor could be potential target for a resuscitation strategy that could attenuate or prevent platelet dysfunction after trauma by preventing stimulation of adenylate cyclase and synthesis of cAMP. This study was funded by the US Army medical Research and Development Command. Disclosures No relevant conflicts of interest to declare.

An Overview of VPAC Receptors in Rheumatoid Arthritis: Biological Role and Clinical Significance.

The axis comprised by the Vasoactive Intestinal Peptide (VIP) and its G protein-coupled receptors (GPCRs), VPAC1, and VPAC2, belong to the B1 family and signal through Gs or Gq proteins. VPAC receptors seem to preferentially interact with Gs in inflammatory cells, rather than Gq, thereby stimulating adenylate cyclase activity. cAMP is able to trigger various downstream pathways, mainly the canonical PKA pathway and the non-canonical cAMP-activated guanine nucleotide exchange factor (EPAC) pathway. Classically, the presence of VPACs has been confined to the plasma membrane; however, VPAC1 location has been described in the nuclear membrane in several cell types such as activated Th cells, where they are also functional. VPAC receptor signaling modulates a number of biological processes by tipping the balance of inflammatory mediators in macrophages and other innate immune cells, modifying the expression of TLRs, and inhibiting MMPs and the expression of adhesion molecules. Receptor signaling also downregulates coagulation factors and acute-phase proteins, promotes Th2 over Th1, stimulates Treg abundance, and finally inhibits a pathogenic Th17 profile. Thus, the VIP axis signaling regulates both the innate and adaptive immune responses in several inflammatory/autoimmune diseases. Rheumatoid arthritis (RA) is a complex autoimmune disease that develops on a substrate of genetically susceptible individuals and under the influence of environmental factors, as well as epigenetic mechanisms. It is a heterogeneous disease with different pathogenic mechanisms and variable clinical forms between patients with the same diagnosis. The knowledge of VIP signaling generated in both animal models and human ex vivo studies can potentially be translated to clinical reality. Most recently, the beneficial effects of nanoparticles of VIP self-associated with sterically stabilized micelles have been reported in a murine model of RA. Another novel research area is beginning to define the receptors as biomarkers in RA, with their expression levels shown to be associated with the activity of the disease and patients-reported impairment. Therefore, VPAC expression together VIP genetic variants could allow patients to be stratified at the beginning of the disease with the purpose of guiding personalized treatment decisions.

Multifaceted effects of arachidonic acid and interaction with cyclic nucleotides in human platelets

IntroductionArachidonic acid induced aggregation is a generally accepted test for aspirin resistance. However, doubts have been raised that arachidonic acid stimulated aggregation can be regarded as reliable testing for aspirin resistance. Arachidonic acid, in addition to platelet activation, can induce phosphatidylserine translocation on the outer surface of platelet membrane which could be mediated by apoptosis pathways or transformation of platelets to the procoagulant state. Materials and methodsWe explored effects of arachidonic acid over a vast range of concentrations and a wide range of read-outs for human platelet activation, procoagulant activity, and platelet viability. Additionally we tested whether cAMP- or cGMP-dependent protein kinase activation can inhibit procoagulant activity or platelet viability. ResultsArachidonic acid-induced washed platelet activation was detected at low micromolar concentrations during the first 2 min of stimulation. After longer incubation and/or at higher concentrations arachidonic acid triggered platelet procoagulant activity and reduced platelet viability. At the same time, arachidonic acid stimulated adenylate cyclase mediated protein phosphorylation which correlated with reduced platelet activation. Moreover, additional stimulation of cAMP- or cGMP-dependent protein kinase inhibited only platelet activation, but did not prevent pro-coagulant activity and platelet death. ConclusionsWhile arachidonic acid induces platelet activation at low concentrations and during short incubation time, higher concentrations and lasting incubation evokes adenylate cyclase activation and subsequent protein phosphorylation corresponding to reduced platelet activation, but also enhanced pro-coagulant activity and reduced viability. Our observations provide further proof for the complex fine tuning of platelet responses in a time and agonist concentration dependent manner.

β-Arrestin1 and 2 differentially regulate PACAP-induced PAC1 receptor signaling and trafficking.

A pituitary adenylate cyclase-activating polypeptide (PACAP)-specific receptor, PAC1R, is coupled with multiple signal transduction pathways including stimulation of adenylate cyclase, phospholipase C and extracellular-signal regulated kinase (ERK)1/2. PAC1R has been shown to exert its long-lasting and potent signals via β-arrestin1 and β-arrestin2. However, the precise roles of the two β-arrestin isoforms in PACAP-PAC1R signaling remain unclear. Here we examined the interaction between the two β-arrestin isoforms and PAC1R, β-arrestin-dependent PAC1R subcellular localization and ERK1/2 activation. Upon PACAP stimulation, although PAC1R similarly interacted with β-arrestin1 and β-arrestin2 in HEK293T cells, the complex of PAC1R and β-arrestin2 was translocated from the cell surface into cytosol, but that of β-arrestin1 remained in the cell surface regions in HeLa cells and mouse primary cultured neurons. Silencing of β-arrestin2 blocked PACAP-induced PAC1R internalization and ERK1/2 phosphorylation, but silencing of β-arrestin1 increased ERK1/2 phosphorylation. These results show that β-arrestin1 and β-arrestin2 exert differential actions on PAC1R internalization and PAC1R-dependent ERK1/2 activation, and suggest that the two β-arrestin isoforms may be involved in fine and precise tuning of the PAC1R signaling pathways.