ERK-dependent induction of the immediate-early gene Egr1 and the late gene Gpr50 contribute to two distinct phases of PACAP Gs-GPCR signaling for neuritogenesis.

Gs-coupled GPCR-stimulated neuritogenesis in PC12 and NS-1 - cells depends on activation of the MAP kinase ERK. Here, we examine changes in ERK activation (phosphorylation), and the time course of ERK-dependent gene induction, to seek transcriptional determinants for this process. Quenching of ERK activation by inhibition of MEK with U0126 at any time point for at least 24 h following addition of PACAP resulted in arrest of neurite formation. Changes in the transcriptome profile throughout this time period revealed at least two phases of gene induction: an early phase dominated by induction of immediate-early genes, and a later phase of gene induction after 4-6 h of exposure to PACAP with persistent elevation of phospho-ERK levels. Genes induced by PACAP in both phases consisted in those whose induction was dependent on ERK (i.e., blocked by U0126), and some whose induction was blocked by the protein kinase A inhibitor H89. ERK-dependent "late gene" transcripts included Gpr50, implicated earlier in facilitation of NGF-induced neurite formation in NS-1 cells. Gpr50 induction by PACAP, but not NGF, was dependent on the guanine nucleotide exchange factor RapGEF2, which has been shown to be required for PACAP-induced neuritogenesis in NS-1 cells. Expression of a Gpr50-directed shRNA lowered basal levels of Gpr50 mRNA and attenuated Gpr50 mRNA and GPR50 protein induction by PACAP, with a corresponding attenuation of PACAP-induced neuritogenesis. Gs-GPCR-stimulated neuritogenesis first requires immediate-early gene induction, including that of Egr1 (Zif268/NGF1A/Krox24) as previously reported. This early phase of gene induction, however, is insufficient to maintain the neuritogenic process without ERK-dependent induction of additional late genes, including Gpr50, upon continuous exposure to neurotrophic neuropeptide. Early (Egr1) and late (Gpr50) gene induction by NGF, like that for PACAP, was inhibited by U0126, but was independent of RapGEF2, confirming distinct modes of ERK activation by Gs-coupled GPCRs and neurotrophic tyrosine receptor kinases, converging on a final common ERK-dependent signaling pathway for neuritogenesis.

Cyclic AMP-dependent activation of ERK via GLP-1 receptor signalling requires the neuroendocrine cell-specific guanine nucleotide exchanger NCS-RapGEF2.

Cyclic AMP activation of the Rap-Braf-MEK-ERK pathway after signalling initiated by the neuropeptide pituitary adenylate cyclase-activating peptide (PACAP), via the Gs -protein coupled receptor (Gs PCR) PAC1, occurs uniquely through the neuritogenic cAMP sensor Rap guanine nucleotide exchange factor 2 (NCS-RapGEF2) in Neuroscreen-1 (NS-1) neuroendocrine cells. We examined the expression of other Family B Gs PCRs in this cell line and assessed cAMP elevation and neuritogenesis after treatment with their cognate peptide ligands. Exposure of NS-1 cells to the VIPR1/2 agonist vasoactive intestinal polypeptide, or the GLP1R agonist exendin-4, did not induce neuritogenesis, or elevation of cAMP, presumably as a result of insufficient receptor protein expression. Vasoactive intestinal polypeptide and exendin-4 did induce neuritogenesis after transduction of human VIPR1, VIPR2 and GLP1R into NS-1 cells. Exendin-4/GLP1R-stimulated neuritogenesis was MEK-ERK-dependent (blocked by U0126), indicating its use of the cAMP→RapGEF2→ERK neuritogenic signalling pathway previously identified for PACAP/PAC1 signalling in NS-1 cells. NCS-RapGEF2 is expressed in the rodent insulinoma cell lines MIN6 and INS-1, as well as in human pancreatic islets. As in NS-1 cells, exendin-4 caused ERK phosphorylation in INS-1 cells. Reduction in RapGEF2 expression after RapGEF2-shRNA treatment reduced exendin-4-induced ERK phosphorylation. Transcriptome analysis of INS-1 cells after 1 hour of exposure to exendin-4 revealed an immediate early-gene response that was composed of both ERK-dependent and ERK-independent signalling targets. We propose that cAMP signalling initiated by glucagon-like peptide 1 (GLP-1) in pancreatic beta cells causes parallel activation of multiple cAMP effectors, including NCS-RapGEF2, Epac and protein kinase A, to separately control various facets of GLP-1 action, including insulin secretion and transcriptional modulation.

Masks or N95 Respirators During COVID-19 Pandemic-Which One Should I Wear?

PURPOSE: Coronavirus Disease 2019 (COVID-19) has caused suffering and death around the world. Careful selection of facial protection is paramount for preventing virus spread among healthcare workers and preserving mask and N95 respirator supplies. METHODS: This paper is a comprehensive review of literature written in English and available on Pubmed comparing the risk of viral respiratory infections when wearing masks and N95 respirators. Current international oral and maxillofacial surgery guidelines for mask and N95 respirator use, patient COVID-19 disease status, aerosol producing procedures were also collected and incorporated into a workflow for selecting appropriate facial protection for oral and maxillofacial surgery procedures during the current pandemic. RESULTS: Most studies suggest N95 respirators and masks are equally protective against respiratory viruses. Some evidence favors N95 respirators, which are preferred for high-risk procedures when aerosol production is likely or when the COVID-19 status of a patient is positive or unknown. N95 respirators may also be used for multiple patients or reused depending on the type of procedure and condition of the respirator after each patient encounter. CONCLUSION: N95 respirators are preferred over masks against viral respiratory pathogens, especially during aerosol-generating procedures or when a patient's COVID-19 status is positive or unknown.

A Novel Approach to Fiberoptic Intubation in Patients With Coronavirus Disease 2019.

PURPOSE: In an effort to protect health care workers at the beginning and end of oral and maxillofacial surgeries, we describe a negative-pressure intubation hood (NPIH) designed to reduce the risk aerosol exposure from fiberoptic intubation (FOI) and extubation. This design is especially important during the Coronavirus disease 2019 era, as it provides greater protection from Severe Acute Respiratory Syndrome-Coronavirus-2 during FOI and extubation, which are some of the most high-risk, aerosol generating procedures of oral and maxillofacial surgery cases. MATERIALS AND METHODS: This article describes the step-by-step process of assembling a NPIH for FOI using various supplies found commonly in hospitals and surrounding community retail stores, which include transparent medical dressings, equipment covers, intravenous pole clips, polyvinylchloride pipes and adaptors, copper pipe, and a Buffalo smoke evacuator. We then discuss how to create access ports for the anesthesiologist to insert their arms and FOI instrumentation and provide a demonstration of us using the hood with a manikin on an operating room table. RESULTS: This study successfully demonstrates a novel technique for performing FOI in a NIPH assembled from basic supplies found commonly among hospital and community retail stores. CONCLUSIONS: This NIPH for FOI is easily made and adaptable to operating room tables, and provides protection against aerosols generated from FOI and subsequent extubation during oral and maxillofacial surgeries.

A GABAergic cell type in the lateral habenula links hypothalamic homeostatic and midbrain motivation circuits with sex steroid signaling.

The lateral habenula (LHb) has a key role in integrating a variety of neural circuits associated with reward and aversive behaviors. There is limited information about how the different cell types and neuronal circuits within the LHb coordinate physiological and motivational states. Here, we report a cell type in the medial division of the LHb (LHbM) in male rats that is distinguished by: (1) a molecular signature for GABAergic neurotransmission (Slc32a1/VGAT) and estrogen receptor (Esr1/ERα) expression, at both mRNA and protein levels, as well as the mRNA for vesicular glutamate transporter Slc17a6/VGLUT2, which we term the GABAergic estrogen-receptive neuron (GERN); (2) its axonal projection patterns, identified by in vivo juxtacellular labeling, to both local LHb and to midbrain modulatory systems; and (3) its somatic expression of receptors for vasopressin, serotonin and dopamine, and mRNA for orexin receptor 2. This cell type is anatomically located to receive afferents from midbrain reward (dopamine and serotonin) and hypothalamic water and energy homeostasis (vasopressin and orexin) circuits. These afferents shared the expression of estrogen synthase (aromatase) and VGLUT2, both in their somata and axon terminals. We demonstrate dynamic changes in LHbM VGAT+ cell density, dependent upon gonadal functional status, that closely correlate with motivational behavior in response to predator and forced swim stressors. The findings suggest that the homeostasis and reward-related glutamatergic convergent projecting pathways to LHbMC employ a localized neurosteroid signaling mechanism via axonal expression of aromatase, to act as a switch for GERN excitation/inhibition output prevalence, influencing depressive or motivated behavior.

PACAP signaling in stress: insights from the chromaffin cell.

Pituitary adenylate cyclase-activating polypeptide (PACAP) was first identified in hypothalamus, based on its ability to elevate cyclic AMP in the anterior pituitary. PACAP has been identified as the adrenomedullary neurotransmitter in stress through a combination of ex vivo, in vivo, and in cellula experiments over the past two decades. PACAP causes catecholamine secretion, and activation of catecholamine biosynthetic enzymes, during episodes of stress in mammals. Features of PACAP signaling allowing stress transduction at the splanchnicoadrenomedullary synapse have yielded insights into the contrasting roles of acetylcholine's and PACAP's actions as first messengers at the chromaffin cell, via differential release at low and high rates of splanchnic nerve firing, and differential signaling pathway engagement leading to catecholamine secretion and chromaffin cell gene transcription. Secretion stimulated by PACAP, via calcium influx independent of action potential generation, is under active investigation in several laboratories both at the chromaffin cell and within autonomic ganglia of both the parasympathetic and sympathetic nervous systems. PACAP is a neurotransmitter important in stress transduction in the central nervous system as well, and is found at stress-transduction nuclei in brain including the paraventricular nucleus of hypothalamus, the amygdala and extended amygdalar nuclei, and the prefrontal cortex. The current status of PACAP as a master regulator of stress signaling in the nervous system derives fundamentally from the establishment of its role as the splanchnicoadrenomedullary transmitter in stress. Experimental elucidation of PACAP action at this synapse remains at the forefront of understanding PACAP's role in stress signaling throughout the nervous system.

NCS-Rapgef2, the Protein Product of the Neuronal Rapgef2 Gene, Is a Specific Activator of D1 Dopamine Receptor-Dependent ERK Phosphorylation in Mouse Brain.

The neuritogenic cAMP sensor (NCS), encoded by the Rapgef2 gene, links cAMP elevation to activation of extracellular signal-regulated kinase (ERK) in neurons and neuroendocrine cells. Transducing human embryonic kidney (HEK)293 cells, which do not express Rapgef2 protein or respond to cAMP with ERK phosphorylation, with a vector encoding a Rapgef2 cDNA reconstituted cAMP-dependent ERK activation. Mutation of a single residue in the cyclic nucleotide-binding domain (CNBD) conserved across cAMP-binding proteins abrogated cAMP-ERK coupling, while deletion of the CNBD altogether resulted in constitutive ERK activation. Two types of mRNA are transcribed from Rapgef2 in vivo. Rapgef2 protein expression was limited to tissues, i.e., neuronal and endocrine, expressing the second type of mRNA, initiated exclusively from an alternative first exon called here exon 1', and an alternative 5' protein sequence leader fused to a common remaining open reading frame, which is termed here NCS-Rapgef2. In the male mouse brain, NCS-Rapgef2 is prominently expressed in corticolimbic excitatory neurons, and striatal medium spiny neurons (MSNs). Rapgef2-dependent ERK activation by the dopamine D1 agonist SKF81297 occurred in neuroendocrine neuroscreen-1 (NS-1) cells expressing the human D1 receptor and was abolished by deletion of Rapgef2. Corticolimbic [e.g., dentate gyrus (DG), basolateral amygdala (BLA)] ERK phosphorylation induced by SKF81297 was significantly attenuated in CamK2α-Cre+/- ; Rapgef2cko/cko male mice. ERK phosphorylation in nucleus accumbens (NAc) MSNs induced by treatment with SKF81297, or the psychostimulants cocaine or amphetamine, was abolished in male Rapgef2cko/cko mice with NAc NCS-Rapgef2-depleting AAV-Synapsin-Cre injections. We conclude that D1-dependent ERK phosphorylation in mouse brain requires NCS-Rapgef2 expression.

Guanine nucleotide exchange factor Epac2-dependent activation of the GTP-binding protein Rap2A mediates cAMP-dependent growth arrest in neuroendocrine cells.

First messenger-dependent activation of MAP kinases in neuronal and endocrine cells is critical for cell differentiation and function and requires guanine nucleotide exchange factor (GEF)-mediated activation of downstream Ras family small GTPases, which ultimately lead to ERK, JNK, and p38 phosphorylation. Because there are numerous GEFs and also a host of Ras family small GTPases, it is important to know which specific GEF-small GTPase dyad functions in a given cellular process. Here we investigated the upstream activators and downstream effectors of signaling via the GEF Epac2 in the neuroendocrine NS-1 cell line. Three cAMP sensors, Epac2, PKA, and neuritogenic cAMP sensor-Rapgef2, mediate distinct cellular outputs: p38-dependent growth arrest, cAMP response element-binding protein-dependent cell survival, and ERK-dependent neuritogenesis, respectively, in these cells. Previously, we found that cAMP-induced growth arrest of PC12 and NS-1 cells requires Epac2-dependent activation of p38 MAP kinase, which posed the important question of how Epac2 engages p38 without simultaneously activating other MAP kinases in neuronal and endocrine cells. We now show that the small GTP-binding protein Rap2A is the obligate effector for, and GEF substrate of, Epac2 in mediating growth arrest through p38 activation in NS-1 cells. This new pathway is distinctly parcellated from the G protein-coupled receptor → Gs → adenylate cyclase → cAMP → PKA → cAMP response element-binding protein pathway mediating cell survival and the G protein-coupled receptor → Gs → adenylate cyclase → cAMP → neuritogenic cAMP sensor-Rapgef2 → B-Raf → MEK → ERK pathway mediating neuritogenesis in NS-1 cells.

Differential Pharmacophore Definition of the cAMP Binding Sites of Neuritogenic cAMP Sensor-Rapgef2, Protein Kinase A, and Exchange Protein Activated by cAMP in Neuroendocrine Cells Using an Adenine-Based Scaffold.

We recently reported that the adenylate cyclase (AC) inhibitor SQ22,536 (9-tetrahydrofuranyl-adenine) also has inhibitory activity against the neuroendocrine-specific neuritogenic cAMP sensor-Rapgef2 (NCS-Rapgef2), a guanine nucleotide exchanger and activator for the small effector GTPase Rap1. Cell-based assays that distinguish signaling through the three intracellular cAMP sensors NCS-Rapgef2, exchange protein activated by cAMP (Epac), and protein kinase A (PKA), as well as AC, were used. These, collectively, assess the activities of adenine (6-amino-purine) derivatives modified at several positions to enhance selectivity for NCS-Rapgef2 by decreasing affinity for adenylate cyclase (AC), without increasing affinity for PKA or Epac. Testing of each adenine derivative in whole-cell assays incorporates features of cell permeability, target selectivity, and intrinsic potency into a single EC50 or IC50, making robust extrapolation to compound activity in vivo more likely. N6-MBC-cAMP is a selective PKA activator (EC50 = 265 µM) with low efficacy at NCS-Rapgef2. 8-CPT-2'-O-Me-cAMP and ESI-09 are confirmed as Epac-selective, for stimulation and inhibition, respectively, versus both PKA and NCS-Rapgef2. The compound N6-Phe-cAMP is a full agonist of NCS-Rapgef2 (EC50 = 256 µM). It has little or no activity against Epac or PKA. The compound N6-phenyl-9-tetrahydrofuranyladenine is a novel and potent NCS-Rapgef2 inhibitor without activity at PKA, Epac, or ACs, as assayed in the neuroendocrine NS-1 cell line. This line has been engineered to allow high-content screening for activation and inhibition of AC, PKA, Epac, and NCS-Rapgef2 and the cellular activities initiated by these signaling pathway protein components.

C-terminal amidation of PACAP-38 and PACAP-27 is dispensable for biological activity at the PAC1 receptor.

PACAP-27 and PACAP-38 are the exclusive physiological ligands for the mammalian PAC1 receptor. The role of C-terminal amidation of these ligands at that receptor was examined in neuroendocrine cells expressing the PAC1 receptor endogenously and in non-neuroendocrine cells in which the human and rat PAC1 receptors were expressed from stable single-copy genes driven by the CMV promoter, providing stoichiometrically appropriate levels of this Gs-coupled GPCR in order to examine the potency and intrinsic activity of PACAP ligands and their des-amidated congeners. We found that replacement of the C-terminal glycine residues of PACAP-27 and -38 with a free acid; or extension of either peptide with the two to three amino acids normally found at these positions in PACAP processing intermediates in vivo following endoproteolytic cleavage and after exoproteolytic trimming and glycine-directed amidated, were equivalent in potency to the fully processed peptides in a variety of cell-based assays. These included real-time monitoring of cyclic AMP generation in both NS-1 neuroendocrine cells and non-neuroendocrine HEK293 cells; PKA-dependent gene activation in HEK293 cells; and neuritogenesis and cell growth arrest in NS-1 cells. The specific implications for the role of amidation in arming of secretin-related neuropeptides for biological function, and the general implications for neuropeptide-based delivery in the context of gene therapy, are discussed.

Cyclic Adenosine 3',5'-Monophosphate Elevation and Biological Signaling through a Secretin Family Gs-Coupled G Protein-Coupled Receptor Are Restricted to a Single Adenylate Cyclase Isoform.

PC12 cells express five adenylate cyclase (AC) isoforms, most abundantly AC6 and AC7. These two ACs were individually silenced using lentiviral short hairpin RNAs, which lead to a decrease (≥80%) of the protein product of each transcript. These stable PC12 sublines were then used to examine potential AC isoform preference for signaling through a family B G protein-coupled receptor (GPCR). Cells were challenged with the endogenous agonist of the pituitary adenylate cyclase-activating polypeptide type I receptor (PAC1), pituitary adenylate cyclase-activating polypeptide (PACAP)-38, or the diterpene forskolin as an AC-proximal control. Intracellular cAMP levels were elevated by forskolin about equally in wild-type, AC6, and AC7 knockdown cells. The ability of PACAP-38 and forskolin to activate three cAMP sensors downstream of AC [protein kinase A (PKA), exchange protein activated by cAMP (Epac) 2/Rapgef4, and neuritogenic cAMP sensor (NCS)/Rapgef2] was examined by monitoring the phosphorylation status of their respective targets, cAMP response element-binding protein, p38, and extracellular signal-regulated kinase. Forskolin stimulation of each downstream target of cAMP was unaffected by knockdown of either AC6 or AC7. PACAP-38 activation of all downstream targets of cAMP was unaffected by AC7 knockdown, but abolished following AC6 knockdown. Membrane cholesterol depletion with methyl-ß-cyclodextrin mimicked the effects of AC6 silencing on PACAP signaling, without attenuating forskolin signaling. These data suggest that vicinal constraint of the GPCR PAC1 and AC6 determines the exclusive requirement for this AC in PACAP signaling, but that the coupling of the cAMP sensors PKA, Epac2/Rapgef4, and NCS/Rapgef2, to their respective downstream signaling targets, determines how cAMP signaling is parcellated to physiologic responses, such as neuritogenesis, upon GPCR-Gs activation in neuroendocrine cells.

Pharmacological characterization of mGlu1 receptors in cerebellar granule cells reveals biased agonism.

The majority of existing research on the function of metabotropic glutamate (mGlu) receptor 1 focuses on G protein-mediated outcomes. However, similar to other G protein-coupled receptors (GPCR), it is becoming apparent that mGlu1 receptor signaling is multi-dimensional and does not always involve G protein activation. Previously, in transfected CHO cells, we showed that mGlu1 receptors activate a G protein-independent, ß-arrestin-dependent signal transduction mechanism and that some mGlu1 receptor ligands were incapable of stimulating this response. Here we set out to investigate the physiological relevance of these findings in a native system using primary cultures of cerebellar granule cells. We tested the ability of a panel of compounds to stimulate two mGlu1 receptor-mediated outcomes: (1) protection from decreased cell viability after withdrawal of trophic support and (2) G protein-mediated phosphoinositide (PI) hydrolysis. We report that the commonly used mGlu1 receptor ligands quisqualate, DHPG, and ACPD are completely biased towards PI hydrolysis and do not induce mGlu1 receptor-stimulated neuroprotection. On the other hand, endogenous compounds including glutamate, aspartate, cysteic acid, cysteine sulfinic acid, and homocysteic acid stimulate both responses. These results show that some commonly used mGlu1 receptor ligands are biased agonists, stimulating only a fraction of mGlu1 receptor-mediated responses in neurons. This emphasizes the importance of utilizing multiple agonists and assays when studying GPCR function.

Separate cyclic AMP sensors for neuritogenesis, growth arrest, and survival of neuroendocrine cells.

Dividing neuroendocrine cells differentiate into a neuronal-like phenotype in response to ligands activating G protein-coupled receptors, leading to the elevation of the second messenger cAMP. Growth factors that act at receptor tyrosine kinases, such as nerve growth factor, also cause differentiation. We report here that two aspects of cAMP-induced differentiation, neurite extension and growth arrest, are dissociable at the level of the sensors conveying the cAMP signal in PC12 and NS-1 cells. Following cAMP elevation, neuritogenic cyclic AMP sensor/Rapgef2 is activated for signaling to ERK to mediate neuritogenesis, whereas Epac2 is activated for signaling to the MAP kinase p38 to mediate growth arrest. Neither action of cAMP requires transactivation of TrkA, the receptor for NGF. In fact, the differentiating effects of NGF do not require activation of any of the cAMP sensors protein kinase A, Epac, or neuritogenic cyclic AMP sensor/Rapgef2 but, rather, depend on ERK and p38 activation via completely independent signaling pathways. Hence, cAMP- and NGF-dependent signaling for differentiation are also completely insulated from each other. Cyclic AMP and NGF also protect NS-1 cells from serum withdrawal-induced cell death, again by two wholly separate signaling mechanisms, PKA-dependent for cAMP and PKA-independent for NGF.

Catecholamine receptors: prototypes for GPCR-based drug discovery.

Drugs acting at G protein-coupled receptors (GPCRs) constitute ~40% of those in current clinical use. GPCR-based drug discovery remains at the forefront of drug development, especially for new treatments for psychiatric illness and neurological disease. Here, the basic framework of GPCR signaling learned through the elucidation of catecholamine receptor signaling through G proteins and ß-arrestins, and X-ray crystallographic structure determination is reviewed. In silico docking studies developed in tandem with confirmatory empirical data gathering from binding and signaling experiments have allowed this basic framework to be expanded to drug hunting through predictive in silico searching as well as high-throughput and high-content screening approaches. For efforts moving forward for the deployment of new GPCR-acting drugs, collaborative efforts between industry and government/academic research in target validation at the molecular and cellular levels have become progressively more common. Polypharmacological approaches have become increasingly available for learning more about the mechanisms of GPCR-targeted drugs, based on interaction not with a single, but with a wide range of GPCR targets. These approaches are likely to aid in drug repurposing efforts, yield valuable insight on the side effects of currently employed drugs, and allow for a clearer picture of the actual targets of "atypical" drugs used in a variety of therapeutic contexts.

Rapgef2 connects GPCR-mediated cAMP signals to ERK activation in neuronal and endocrine cells.

G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR)-mediated increases in the second messenger cyclic adenosine monophosphate (cAMP) activate the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK), and in neuroendocrine cells, this pathway leads to cAMP-dependent neuritogenesis mediated through Rap1 and B-Raf. We found that the Rap guanine nucleotide exchange factor Rapgef2 was enriched from primary bovine neuroendocrine cells by cAMP-agarose affinity chromatography and that it was specifically eluted by cAMP. With loss-of-function experiments in the rat neuronal cell line Neuroscreen-1 (NS-1) and gain-of-function experiments in human embryonic kidney 293T cells, we demonstrated that Rapgef2 connected GPCR-dependent activation of adenylate cyclase and increased cAMP concentration with the activation of ERK in neurons and endocrine cells. Furthermore, knockdown of Rapgef2 blocked cAMP- and ERK-dependent neuritogenesis. Our data are consistent with a pathway involving the cAMP-mediated activation of Rapgef2, which then stimulates Rap1, leading to increases in B-Raf, MEK, and ERK activity.

A new site and mechanism of action for the widely used adenylate cyclase inhibitor SQ22,536.

We evaluated the efficacy, potency, and selectivity of the three most commonly used adenylate cyclase (AC) inhibitors in a battery of cell lines constructed to study signaling via three discrete cAMP sensors identified in neuroendocrine cells. SQ22,536 [9-(tetrahydrofuryl)-adenine] and 2',5'-dideoxyadenosine (ddAd) are effective and potent AC inhibitors in HEK293 cells expressing a cAMP response element (CRE) reporter gene, and MDL-12,330A [cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine hydrochloride] is not. Neuroscreen-1 (NS-1) cells were used to assess the specificity of the most potent AC inhibitor, SQ22,536, to block downstream cAMP signaling to phosphorylate CREB (via PKA); to activate Rap1 (via Epac); and to activate ERK signaling leading to neuritogenesis (via the newly described neuritogenic cAMP sensor NCS). SQ22,536 failed to inhibit the effects of cAMP analogs 8-Br-cAMP and 8-CPT-2'-O-Me-cAMP on PKA-mediated CREB activation/phosphorylation and Epac-mediated Rap1 activation, indicating that it does not inhibit these cAMP pathways beyond the level of AC. On the other hand, SQ22,536, but not ddAd, inhibited the effects of cAMP analogs 8-Br-cAMP and 8-CPT-cAMP on ERK phosphorylation and neuritogenesis, indicating that it acts not only as an AC blocker, but also as an inhibitor of the NCS. The observed off-target actions of SQ22,536 are specific to cAMP signaling: SQ22,536 does not block the actions of compounds not related to cAMP signaling, including ERK induction by PMA, and ERK activation and neuritogenesis induced by NGF. These data led us to indicate a second target for SQ22,536 that should be considered when interpreting its effects in whole cell and in vivo experiments.

Ligand bias at metabotropic glutamate 1a receptors: molecular determinants that distinguish ß-arrestin-mediated from G protein-mediated signaling.

The metabotropic glutamate 1a (mGlu1a) receptor is a G protein-coupled receptor linked with phosphoinositide (PI) hydrolysis and with ß-arrestin-1-mediated sustained extracellular signal-regulated kinase (ERK) phosphorylation and cytoprotective signaling. Previously, we reported the existence of ligand bias at this receptor, inasmuch as glutamate induced both effects, whereas quisqualate induced only PI hydrolysis. In the current study, we showed that mGlu1 receptor agonists such as glutamate, aspartate, and l-cysteate were unbiased and activated both signaling pathways, whereas quisqualate and (S)-3,5-dihydroxyphenylglycine stimulated only PI hydrolysis. Competitive antagonists inhibited only PI hydrolysis and not the ß-arrestin-dependent pathway, whereas a noncompetitive mGlu1 receptor antagonist blocked both pathways. Mutational analysis of the ligand binding domain of the mGlu1a receptor revealed that Thr188 residues were essential for PI hydrolysis but not for protective signaling, whereas Arg323 and Lys409 residues were required for ß-arrestin-1-mediated sustained ERK phosphorylation and cytoprotective signaling but not for PI hydrolysis. Therefore, the mechanism of ligand bias appears to involve different modes of agonist interactions with the receptor ligand binding domain. Although some mGlu1a receptor agonists are biased toward PI hydrolysis, we identified two endogenous compounds, glutaric acid and succinic acid, as new mGlu1 receptor agonists that are fully biased toward ß-arrestin-mediated protective signaling. Pharmacological studies indicated that, in producing the two effects, glutamate interacted in two distinct ways with mGlu1 receptors, inasmuch as competitive mGlu1 receptor antagonists that blocked PI hydrolysis did not inhibit cytoprotective signaling. Quisqualate, which is biased toward PI hydrolysis, failed to inhibit glutamate-induced protection, and glutaric acid, which is biased toward protection, did not interfere with glutamate-induced PI hydrolysis. Taken together, these data indicate that ligand bias at mGlu1 receptors is attributable to different modes of receptor-glutamate interactions, which are differentially coupled to PI hydrolysis and ß-arrestin-mediated cytoprotective signaling, and they reveal the existence of new endogenous agonists acting at mGlu1 receptors.

Signaling through the neuropeptide GPCR PAC1 induces neuritogenesis via a single linear cAMP- and ERK-dependent pathway using a novel cAMP sensor.

Both cAMP and ERK are necessary for neuroendocrine cell neuritogenesis, and pituitary adenylate cyclase-activating polypeptide (PACAP) activates each. It is important to know whether cAMP and ERK are arranged in a novel, linear pathway or in two parallel pathways using known signaling mechanisms. Native cellular responses [cAMP elevation, ERK phosphorylation, cAMP responsive element binding (CREB) phosphorylation, and neuritogenesis] and promoter-reporter gene activation after treatment with forskolin, cAMP analogs, and PACAP were measured in Neuroscreen-1 (NS-1) cells, a PC12 variant enabling simultaneous morphological, molecular biological, and biochemical analysis. Forskolin (25 µM) and cAMP analogs (8-bromo-cAMP, dibutyryl-cAMP, and 8-chlorophenylthio-cAMP) stimulated ERK phosphorylation and neuritogenesis in NS-1 cells. Both ERK phosphorylation and neuritogenesis were MEK dependent (blocked by 10 µM U0126) and PKA independent (insensitive to 30 µM H-89 or 100 nM myristoylated protein kinase A inhibitor). CREB phosphorylation induced by PACAP was blocked by H-89. The exchange protein activated by cAMP (Epac)-selective 8-(4-chlorophenylthio)-2'-O-Me-cAMP (100-500 µM) activated Rap1 without affecting the other cAMP-dependent processes. Thus, PACAP-38 potently stimulated two distinct and independent cAMP pathways leading to CREB or ERK activation in NS-1 cells. Drug concentrations for appropriate effect were derived from control data for all compounds. In summary, a novel PKA- and Epac-independent signaling pathway: PACAP → adenylate cyclase → cAMP → ERK → neuritogenesis has been identified.

The protective signaling of metabotropic glutamate receptor 1 Is mediated by sustained, beta-arrestin-1-dependent ERK phosphorylation.

Metabotropic glutamate receptor 1 (mGlu1) is a G protein-coupled receptor that enhances the hydrolysis of membrane phosphoinositides. In addition to its role in synaptic transmission and plasticity, mGlu1 has been shown to be involved in neuroprotection and neurodegeneration. In this capacity, we have reported previously that in neuronal cells, mGlu1a exhibits the properties of a dependence receptor, inducing apoptosis in the absence of glutamate, while promoting neuronal survival in its presence (Pshenichkin, S., Dolinska, M., Klauzinska, M., Luchenko, V., Grajkowska, E., and Wroblewski, J. T. (2008) Neuropharmacology 55, 500-508). Here, using CHO cells expressing mGlu1a receptors, we show that the protective effect of glutamate does not rely on the classical mGlu1 signal transduction. Instead, mGlu1a protective signaling is mediated by a novel, G protein-independent, pathway which involves the activation of the MAPK pathway and a sustained phosphorylation of ERK, which is distinct from the G protein-mediated transient ERK phosphorylation. Moreover, the sustained phosphorylation of ERK and protective signaling through mGlu1a receptors require expression of beta-arrestin-1, suggesting a possible role for receptor internalization in this process. Our data reveal the existence of a novel, noncanonical signaling pathway associated with mGlu1a receptors, which mediates glutamate-induced protective signaling.

An in depth study of the effects of socio-economic conditions on household waste recycling practices.

Waste is never consistent. Different areas use different materials, which give rise to waste streams that can vary widely in composition. With the increasing pressure on landfill operators and local governments over the amount and type of waste going to landfill, moves have to be taken to reduce, divert and recover materials. To undertake this function it is necessary to identify the composition of the waste stream. This paper contains the results of a household waste analysis conducted in a typical unitary authority of South Wales, a traditional industrial community. The waste analysis was conducted to determine whether there was a connection between an area's socio-economic standing and the quantities of waste produced. A selection of three different household categories were chosen, as described by the UK's ACORN geodemographical system. The three areas chosen were terracedd, council and semi-detached properties and were selected as they were the most dominant and easily recognised types of dwellings found in the study area. The results of the full waste analysis have been compared against a number of previous household waste characterisation studies.