Traumatic brain injury (TBI) results in several pathological changes within the hippocampus that result in adverse effects on learning and memory. Therapeutic strategies to enhance learning and memory after TBI are still in the early stages of clinical development. One strategy is to target the alpha 7 nicotinic acetylcholine receptor (nAChR), which is highly expressed in the hippocampus and contributes to the formation of long-term memory. In our previous study, we found that AVL-3288, a positive allosteric modulator of the alpha 7 nAChR, improved cognitive recovery in rats after moderate fluid-percussion injury (FPI). However, whether AVL-3288 improved cognitive recovery specifically through the alpha 7 nAChR was not definitively determined. In this study we utilized Chrna7 knockout mice and compared their recovery to wild-type mice treated with AVL-3288 after TBI. We hypothesized that AVL-3288 treatment would improve learning and memory in wild-type mice, but not Chrna7(-/-) mice after TBI. Adult male C57BL/6 wild-type and Chrna7(-/-) mice received sham surgery or moderate controlled cortical impact (CCI) and recovered for 3 months. Mice were then treated with vehicle or AVL-3288 at 30 min prior to contextual fear conditioning. At 3 months after CCI, expression of alpha 7 nAChR, choline acetyltransferase (ChAT), high-affinity choline transporter (ChT), and vesicular acetylcholine transporter (VAChT) were found to be significantly decreased in the hippocampus. Treatment of wild-type mice at 3 months after CCI with AVL-3288 significantly improved cue and contextual fear conditioning, whereas no beneficial effects were observed in Chrna7(-/-) mice. Parietal cortex and hippocampal atrophy were not improved with AVL-3288 treatment in either wild-type or Chrna7(-/-) mice. Our results indicate that AVL-3288 improves cognition during the chronic recovery phase of TBI through modulation of the alpha 7 nAChR.

Traumatic brain injury (TBI) leads to changes in the neural circuitry of the hippocampus that result in chronic learning and memory deficits. However, effective therapeutic strategies to ameliorate these chronic learning and memory impairments after TBI are limited. Two pharmacological targets for enhancing cognition are nicotinic acetylcholine receptors (nAChRs) and GABAA receptors (GABAARs), both of which regulate hippocampal network activity to form declarative memories. A promising compound, 522-054, both allosterically enhances alpha 7 nAChRs and inhibits alpha 5 subunit-containing GABAARs. Administration of 522-054 enhances long-term potentiation (LTP) and cognitive functioning in non-injured animals. In this study, we assessed the effects of 522-054 on hippocampal synaptic plasticity and learning and memory deficits in the chronic post-TBI recovery period. Adult male Sprague Dawley rats received moderate parasagittal fluid-percussion brain injury or sham surgery. At 12 wk after injury, we assessed basal synaptic transmission and LTP at the Schaffer collateral-CA1 synapse of the hippocampus. Bath application of 522-054 to hippocampal slices reduced deficits in basal synaptic transmission and recovered TBI-induced impairments in LTP. Moreover, treatment of animals with 522-054 at 12 wk post-TBI improved cue and contextual fear memory and water maze acquisition and retention without a measurable effect on cortical or hippocampal atrophy. These results suggest that dual allosteric modulation of alpha 7 nAChR and alpha 5 GABAAR signaling may be a potential therapy for treating cognitive deficits during chronic recovery from TBI.

Glucocorticoids have wide therapeutic applications in inflammation and immune disorders and have traditionally been used to treat diseases such as asthma and COPD. They exert their anti‐inflammatory effects by binding to the cytosolic glucocorticoid receptor (GRα), a nuclear receptor that translocates into the nucleus to regulate gene transcription. Several studies have shown that glucocorticoids can have rapid, non‐genomic actions. For instance, short duration studies on several cell lines revealed that glucocorticoids can rapidly modulate calcium signaling, reactive oxygen species and arachidonic acid release. Moreover, studies show that a membrane GR (mGR) may interact directly with a GPCR coupled to Gαs and/or Gq/11.ObjectiveTo better understand the underlying mechanism of the non‐genomic effects of glucocorticoids, we tested the hypothesis that glucocorticoids can directly stimulate cAMP production via a Gαs coupled GPCR.ResultsThe glucocorticoids prednisone, fluticasone and budesonide stimulate cAMP rapidly in the absence of phosphodiesterase (PDE) inhibitors as measured in real time via the cADDis assay. These effects were seen in human airway smooth muscle (HASM), HEK‐293, HFL‐1, MCF‐7 and A375 cells. Glucocorticoids retained cAMP stimulating activity in HASM cells preincubated with 10 μM IBMX, a broad spectrum PDE inhibitor, implying that they do not increase cAMP via inhibition of PDEs. Cortisol‐albumin conjugate, a cell membrane impermeable glucocorticoid, stimulated cAMP suggesting that an outward facing membrane‐bound receptor mediates glucocorticoid non‐genomic signaling. GRα knockdown had no effect on glucocorticoid‐stimulation of cAMP, implying a GRα‐independent mechanism. We tested whether glucocorticoid‐stimulated cAMP depends upon the G protein Gαs by transfecting HASM with siRNA for GNAS. Immunoblot analysis confirmed a reduction in expression of both long and short forms of Gαs in the knockdown condition. Gαs knockdown lead to diminished cAMP production by fluticasone, budesonide and formoterol but not by forskolin, indicating that Gαs is an essential part of the non‐genomic signaling mechanism of glucocorticoids. The G protein estrogen receptor (GPER) is a G‐protein coupled receptor (GPCR) that couples to Gαs and binds to specific steroid ligands and is expressed in various cell types including HASM, HFL‐1 and HEK‐293. In the presence of either G15 or G36, GPER‐specific antagonists, both budesonide‐ and fluticasone‐stimulated cAMP was unaltered, suggesting that GPER does not mediate cAMP signaling by glucocorticoids.ConclusionTaken together these results reveal that glucocorticoids induce rapid, non‐genomic signaling via the Gαs‐cAMP pathway. The identity of the cell surface receptor for glucocorticoids is unknown, but given the rapid, GRα‐independent, Gαs‐dependent nature of the response we hypothesize that a GPCR or family of GPCRs is involved.Support or Funding InformationThis work was supported by NIH grant GM107094

The nongenomic mechanisms by which glucocorticoids modulate β2 agonist-induced-bronchodilation remain elusive. Our studies aimed to elucidate mechanisms mediating the beneficial effects of glucocorticoids on agonist-induced bronchodilation. Utilizing human precision-cut lung slices (hPCLS), we measured bronchodilation to formoterol, prostaglandin E2 (PGE2), cholera toxin (CTX), or forskolin in the presence and absence of budesonide. Using cultured human airway smooth muscle (HASM), intracellular cAMP was measured in live cells following exposure to formoterol, PGE2, or forskolin in the presence or absence of budesonide. We showed that simultaneous budesonide administration amplified formoterol-induced bronchodilation and attenuated agonist-induced phosphorylation of myosin light chain, a necessary signaling event mediating force generation. In parallel studies, cAMP levels were augmented by simultaneous exposure of HASM cells to formoterol and budesonide. Budesonide, fluticasone, and prednisone alone rapidly increased cAMP levels, but steroids alone had little effect on bronchodilation in hPCLS. Bronchodilation induced by PGE2, CTX, or forskolin was also augmented by simultaneous exposure to budesonide in hPCLS. Furthermore, HASM cells expressed membrane-bound glucocorticoid receptors that failed to translocate with glucocorticoid stimulation and that potentially mediated the rapid effects of steroids on β2 agonist-induced bronchodilation. Knockdown of glucocorticoid receptor-α had little effect on budesonide-induced and steroid-dependent augmentation of formoterol-induced cAMP generation in HASM. Collectively, these studies suggest that glucocorticoids amplify cAMP-dependent bronchodilation by directly increasing cAMP levels. These studies identify a molecular mechanism by which the combination of glucocorticoids and β2 agonists may augment bronchodilation in diseases such as asthma or chronic obstructive pulmonary disease.

Glucocorticoids are widely used for the suppression of inflammation, but evidence is growing that they can have rapid, non‐genomic actions that have been unappreciated. Diverse cell signaling effects have been reported for glucocorticoids, leading us to hypothesize that glucocorticoids alone can swiftly increase the 3′,5′‐cyclic adenosine monophosphate (cAMP) production. We found that prednisone, fluticasone, budesonide, and progesterone each increased cAMP levels within 3 minutes without phosphodiesterase inhibitors by measuring real‐time cAMP dynamics using the cAMP difference detector in situ assay in a variety of immortalized cell lines and primary human airway smooth muscle (HASM) cells. A membrane‐ impermeable glucocorticoid showed similarly rapid stimulation of cAMP, implying that responses are initiated at the cell surface. siRNA knockdown of Gαs virtually eliminated glucocorticoid‐stimulated cAMP responses, suggesting that these drugs activate the cAMP production via a G protein‐coupled receptor. Estradiol had small effects on cAMP levels but G protein estrogen receptor antagonists had little effect on responses to any of the glucocorticoids tested. The genomic and non‐genomic actions of budesonide were analyzed by RNA‐Seq analysis of 24 hours treated HASM, with and without knockdown of Gαs. A 140‐gene budesonide signature was identified, of which 48 genes represent a non‐genomic signature that requires Gαs signaling. Collectively, this non‐genomic cAMP signaling modality contributes to one‐third of the gene expression changes induced by glucocorticoid treatment and shifts the view of how this important class of drugs exerts its effects.

Seizures induced by organophosphorus nerve agent exposure become refractory to treatment with benzodiazepines because these drugs engage synaptic γ-aminobutyric acid-A receptors (GABAARs) that rapidly internalize during status epilepticus (SE). Extrasynaptic GABAARs, such as those containing α4β3δ subunits, are a putative pharmacological target to comprehensively manage nerve agent-induced seizures since they do not internalize during SE and are continuously available for activation. Neurosteroids related to allopregnanolone have been tested as a possible replacement for benzodiazepines because they target both synaptic and extrasynaptic GABAARs receptors. A longer effective treatment window, extended treatment efficacy, and enhanced neuroprotection represent significant advantages of neurosteroids over benzodiazepines. However, neurosteroid use is limited by poor physicochemical properties arising from the intrinsic requirement of the pregnane steroid core structure for efficacy rendering drug formulation problematic. We tested a non-steroidal enaminone GABAAR modulator that interacts with both synaptic and extrasynaptic GABAARs on a binding site distinct from neurosteroids or benzodiazepines for efficacy to control electrographic SE induced by diisopropyl fluorophosphate or soman intoxication in rats. Animals were treated with standard antidotes, and experimental therapeutic treatment was given following 1 h (diisopropyl fluorophosphate model) or 20 min (soman model) after SE onset. We found that the enaminone 2-261 had an extended duration of seizure termination (>10 h) in the diisopropyl fluorophosphate intoxication model in the presence or absence of midazolam (MDZ). 2-261 also moderately potentiated MDZ in the soman-induced seizure model but had limited efficacy as a stand-alone anticonvulsant treatment due to slow onset of action. 2-261 significantly reduced neuronal death in brain areas associated with either diisopropyl fluorophosphate- or soman-induced SE. 2-261 represents an alternate chemical template from neurosteroids for enhancing extrasynaptic α4β3δ GABAAR activity to reverse SE from organophosphorous intoxication.

Cognitive impairments are a common consequence of traumatic brain injury (TBI). The hippocampus is a subcortical structure that plays a key role in the formation of declarative memories and is highly vulnerable to TBI. The α7 nicotinic acetylcholine receptor (nAChR) is highly expressed in the hippocampus and reduced expression and function of this receptor are linked with cognitive impairments in Alzheimer’s disease and schizophrenia. Positive allosteric modulation of α7 nAChRs with AVL-3288 enhances receptor currents and improves cognitive functioning in naïve animals and healthy human subjects. Therefore, we hypothesized that targeting the α7 nAChR with the positive allosteric modulator AVL-3288 would enhance cognitive functioning in the chronic recovery period of TBI. To test this hypothesis, adult male Sprague Dawley rats received moderate parasagittal fluid-percussion brain injury or sham surgery. At 3 months after recovery, animals were treated with vehicle or AVL-3288 at 30 min prior to cue and contextual fear conditioning and the water maze task. Treatment of TBI animals with AVL-3288 rescued learning and memory deficits in water maze retention and working memory. AVL-3288 treatment also improved cue and contextual fear memory when tested at 24 hr and 1 month after training, when TBI animals were treated acutely just during fear conditioning at 3 months post-TBI. Hippocampal atrophy but not cortical atrophy was reduced with AVL-3288 treatment in the chronic recovery phase of TBI. AVL-3288 application to acute hippocampal slices from animals at 3 months after TBI rescued basal synaptic transmission deficits and long-term potentiation (LTP) in area CA1. Our results demonstrate that AVL-3288 improves hippocampal synaptic plasticity, and learning and memory performance after TBI in the chronic recovery period. Enhancing cholinergic transmission through positive allosteric modulation of the α7 nAChR may be a novel therapeutic to improve cognition after TBI.

Two cAMP signaling compartments centered on adenylyl cyclase (AC) exist in human airway smooth muscle (HASM) cells, one containing beta(2)-adrenergic receptor AC6 and another containing E prostanoid receptor AC2. We hypothesized that different PDE isozymes selectively regulate cAMP signaling in each compartment. According to RNA-sequencing data, 18 of 24 PDE genes were expressed in primary HASM cells derived from age-and sex-matched donors with and without asthma. PDE8A was the third most abundant of the cAMP-degrading PDE genes, after PDE4A and PDE1A. Knockdown of PDE8A using shRNA evoked twofold greater cAMP responses to 1 mu M forskolin in the presence of 3-isobutyl-1-methylxanthine. Overexpression of AC2 did not alter this response, but overexpression of AC6 increased cAMP responses an additional 80%. We examined cAMP dynamics in live HASM cells using a fluorescence sensor. PF-04957325, a PDE8-selective inhibitor, increased basal cAMP concentrations by itself, indicating a significant basal level of cAMP synthesis. In the presence of an AC inhibitor to reduce basal signaling, PF-04957325 accelerated cAMP production and increased the inhibition of cell proliferation induced by isoproterenol, but it had no effect on cAMP concentrations or cell proliferation regulated by prostaglandin E-2. Lipid raft fractionation of HASM cells revealed PDE8A immunoreactivity in buoyant fractions containing caveolin-1 and AC5/6 immunoreactivity. Thus, PDE8 is expressed in lipid rafts of HASM cells, where it specifically regulates beta(2)-adrenergic receptor AC6 signaling without effects on signaling by the E prostanoid receptors 2/4-AC2 complex. In airway diseases such as asthma and chronic obstructive pulmonary disease, PDE8 may represent a novel therapeutic target to modulate HASM responsiveness and airway remodeling.

Chronic neuropathic pain may be caused, in part, by loss of inhibition in spinal pain processing pathways due to attenuation of local GABAergic tone. Nociception and nocifensive behaviors are reduced after enhancement of tonically activated extrasynaptic GABAAR-mediated currents by agonist ligands for δ subunit-containing GABAARs. However, typical ligands that target δ subunit-containing GABAARs are limited due to sedative effects at higher doses. We used the spinal nerve ligation (SNL) and gp120 models of experimental neuropathic pain to evaluate compound 2-261, a nonbenzodiazepine site positive allosteric modulator of α4β3δ GABAARs optimized to be nonsedative by selective activation of β2/3-subunit-containing GABAARs over receptor subtypes incorporating β1 subunits. Similar levels of 2-261 were detected in the brain and plasma after intraperitoneal administration. Although systemic 2-261 did not alter sensory thresholds in sham-operated animals, it significantly reversed SNL-induced thermal and tactile hypersensitivity in a GABAAR-dependent fashion. Intrathecal 2-261 produced conditioned place preference and elevated dopamine levels in the nucleus accumbens of nerve-injured, but not sham-operated, rats. In addition, systemic pretreatment with 2-261 blocked conditioned place preference from spinal clonidine in SNL rats. Moreover, 2-261 reversed thermal hyperalgesia and partially reversed tactile allodynia in the gp120 model of HIV-related neuropathic pain. The effects of 2-261 likely required interaction with the α4β3δ GABAAR because 2-301, a close structural analog of 2-261 with limited extrasynaptic receptor efficacy, was not active. Thus, 2-261 may produce pain relief with diminished side effects through selective modulation of β2/3-subunit-containing extrasynaptic GABAARs.

Inhaled corticosteroids have been shown to be highly beneficial when combined with β‐adrenergic receptor (βAR) agonists in treatment of asthma. The mechanism for this synergy has never been fully clear, but corticosteroids have been shown to increase βAR expression and reverse βAR desensitization via canonical genomic actions. The current study investigated whether inhaled corticosteroids have an effect on cAMP production in both primary and immortalized human airway smooth muscle (HASM) cells. We measured real‐time cAMP dynamics in live HASM cells using the downward cAMP difference detector in situ (cADDis) cAMP sensor (Montana Molecular, Bozeman MT). Addition of various concentrations of either prednisone, fluticasone or allopregnanolone increased cAMP production over basal within 3 min without phosphodiesterase inhibitors present. We examined various passages of HASM cells and cells from different patients and found fluticasone consistently increased cAMP production at concentrations between 10 nM and 10 μM. We also examined cAMP responses to fluticasone in human embryonic kidney (HEK‐293) cells and observed no increases in cAMP production in response to fluticasone, prednisone, or progesterone. We repeated studies in the presence of glucocorticoid receptor GR‐α antagonists, RU‐486 and GSK‐9027, to understand how corticosteroids may be increasing cAMP levels. Pretreatment with either antagonist attenuated cAMP responses to all activators, including forskolin and βAR agonists, implying these drugs have considerable non‐specific effects. We also evaluated how corticosteroids would alter responses to other cAMP‐elevating agents. Fluticasone (0.1 to 1 μM) increased the potency and maximal effect of isoproterenol, PGE2 and forskolin. These findings are consistent with the idea that corticosteroids directly stimulate cAMP signaling in a non‐genomic manner and can enhance signaling by βAR and prostaglandin E receptors. Future studies will identify the molecular mechanism for this effect and determine if GR‐α is required. These findings reveal an unappreciated action of inhaled corticosteroids that may help explain how these drugs are beneficial in combination with β‐adrenergic receptor agonists for the treatment of asthma.Support or Funding InformationThis work was supported by NIH grant GM107094 (RO)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pulmonary fibroblasts play an essential role in maintaining the structure and function of the lung. In idiopathic pulmonary fibrosis, fibroblast cells persist in an activated form and produce excessive fibrous material, leading to loss of alveolar structure. PGE2, which specifically activates EP2 and EP4 receptor subtypes, is a cAMP elevating agent and thus has anti‐fibrotic potential, since cAMP has been shown to have anti‐fibrotic properties. Although PGE2 levels are increased in the bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis (and in animal models of pulmonary fibrosis), it does not prevent pulmonary fibrosis. One hypothesis is that PGE2 effects are self‐limiting because of desensitization caused by prolonged exposure. To test this hypothesis, we pretreated HFL‐1 fibroblasts with PGE2 and other cAMP‐elevating agents then measured PGE2‐stimulated cAMP levels using the downward cAMP difference detector in situ (cADDis) cAMP sensor (Montana Molecular, Bozeman MT). After 24‐hour pretreatment with 100 nM PGE2, we changed the media and measured cAMP kinetics in response to various concentrations of PGE2. Pretreatment with PGE2 shifted the concentration‐response curve of PGE2 rightward approximately 50‐fold. Neither 100 nM isoproterenol or 1μM forskolin pretreatment for 24 hours altered PGE2 response, implying that other cAMP elevating agents do not induce desensitization. We sought to uncover the underlying mechanism responsible for PGE2 desensitization. Desensitization could result from either receptor internalization through the GRK/β‐arrestin mediated pathway or via increase PDE activity. To determine if PGE2 pretreatment increases PDE activity, we used the PDE3 inhibitor, cilostazol, and the PDE4 inhibitor, rolipram after PGE2 pretreatment. Rolipram, increased both the EC50 and Emax of PGE2 in vehicle pretreated HFL‐1 fibroblasts. In cells pretreated with PGE2, rolipram partially reversed the desensitization, indicating that PDE4 may be induced by long term PGE2 exposure. Cilostazol had minimal effects on either control or desensitized PGE2 responses, implying that PDE3 is not involved. Examination of PDE isoform mRNA levels via quantitative RT‐PCR, show that PGE2 treatment upregulates PDE3A, PDE4C and PDE4D, while pretreatment with isoproterenol or forskolin only upregulated PDE4D. Taken together, these results show that long‐term exposure to PGE2 causes desensitization of EP receptor‐stimulated cAMP signaling, partly through the increased expression of a PDE4 isoform. Defining the precise PDE isoform involved in the desensitization of PGE2 responses may lead to new therapeutic strategies for treating pulmonary fibrosis.Support or Funding InformationThis work was supported by NIH grant GM107094 (RO)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

It is widely accepted that cAMP signaling is compartmentalized within cells. However, our knowledge of how receptors, cAMP signaling enzymes, effectors, and other key proteins form specific signaling complexes to regulate specific cell responses is limited. The multicomponent nature of these systems and the spatiotemporal dynamics involved as proteins interact and move within a cell make cAMP responses highly complex. Adenylyl cyclases, the enzymatic source of cAMP production, are key starting points for understanding cAMP compartments and defining the functional signaling complexes. Three basic elements are required to form a signaling compartment. First, a localized signal is generated by a G protein-coupled receptor paired to one or more of the nine different transmembrane adenylyl cyclase isoforms that generate the cAMP signal in the cytosol. The diffusion of cAMP is subsequently limited by several factors, including expression of any number of phosphodiesterases (of which there are 24 genes plus spice variants). Finally, signal response elements are differentially localized to respond to cAMP produced within each locale. A-kinase-anchoring proteins, of which there are 43 different isoforms, facilitate this by targeting protein kinase A to specific substrates. Thousands of potential combinations of these three elements are possible in any given cell type, making the characterization of cAMP signaling compartments daunting. This review will focus on what is known about how cells organize cAMP signaling components as well as identify the unknowns. We make an argument for adenylyl cyclases being central to the formation and maintenance of these signaling complexes.

OBJECTIVE:Subunit-specific positive allosteric modulators (PAMs) of gamma-aminobutyric acid-A (GABA-A) receptors are commonly used to uncover the role of GABA-A receptor isoforms in brain function. Recently, we have designed novel PAMs selective for β2/3-subunit containing GABA-A receptors (β2/3-selective PAMs) that are nonbenzodiazepine site-mediated and do not show an α-subunit isoform selectivity, yet exhibit anxiolytic efficacy with reduced potential for sedation, cognitive impairment, and tolerance. In this study, we used three novel β2/3-selective PAMs (2-261, 2-262, and 10029) with differential β2/3-subunit potency to identify the role of β2/3-selective receptor isoforms in limbic epileptogenesis.METHODS:Experimental epileptogenesis was induced in mice by daily hippocampus stimulations until each mouse showed generalized (stage 5) seizures. Patch-clamp electrophysiology was used to record GABA-gated currents. Brain levels of β2/3-selective PAMs were determined for mechanistic correlations.RESULTS:Treatment with the β2/3-selective PAMs 2-261 (30mg/kg), 2-262 (10mg/kg), and 10029 (30mg/kg), 30min prior to stimulations, significantly suppressed the rate of development of kindled seizure activity without affecting the afterdischarge (AD) signal, indicating their disease-modifying activity. The β2/3-selective agents suppressed chemical epileptogenesis in the pentylenetetrazol model. Test doses of these agents were devoid of acute antiseizure activity in the kindling model.CONCLUSION:These findings demonstrate that β2/3-selective PAMs can moderately retard experimental epileptogenesis, indicating the protective role of β2/3-subunit GABA-A receptor isoforms in the development of epilepsy.

Human airway smooth muscle (HASM) cells exhibit two distinct cAMP signaling compartments centered around adenylyl cyclase (AC), one containing β2AR‐coupled to AC6 and another configured with E prostanoid receptors (EPR) coupled to AC2. Different cell types express a unique complement of PDE isoforms that create unique spatial and kinetic properties of cAMP signals so it may be possible to selectively modulate cAMP signaling at the PDE level based on localization to cAMP signalosomes. We previously reported that PDE8A is abundantly expressed in HASM and that shRNA knockdown of its expression enhanced forskolin stimulated cAMP responses. We sought to determine if PDE8 is specifically localized such that it regulates signaling by only certain GPCR. Lipid raft fractionation of HASM cells revealed PDE8A immunoreactivity in buoyant fractions containing caveolin‐1 and AC5/6, indicating PDE8 is spatially associated with β2AR signaling complexes. We examined real‐time cAMP dynamics in live HASM cells using the downward cAMP difference detector in situ (cADDis) cAMP sensor (Montana Molecular, Bozeman MT). We observed large, rapid decreases in fluorescence of the downward cADDis sensor in response to various concentrations of forskolin without the addition of IBMX or other PDE inhibitors. PF‐04957325, a PDE8‐selective inhibitor, dose dependently decreased sensor fluorescence over 30 minutes, indicating some basal level of cAMP synthesis. We then measured cAMP responses to various concentrations of either isoproterenol or PGE2, with or without 1 μM PF‐04957325. PF‐04957325 increased 1nM isoproterenol‐stimulated cAMP responses in a concentration‐dependent manner, but PGE2‐stimulated cAMP production was not enhanced by the PDE8 inhibitor. HASM express both EP2 and EP4 receptors, so we used specific antagonists along with PGE2 stimuli to dissect the proportion of the PGE2 response could be attributed to each receptor subtype. PGE2 concentration response curves were significantly dampened by the inclusion of the EP2 receptor antagonist, PF‐04418948 (100 nM). Only small reductions in cAMP responses stimulated by PGE2 were observed with the inclusion of the EP4R antagonist, GW‐627368X (100 nM). These results are consistent with the idea that EP2 receptors predominate over EP4 receptors in HASM. Inclusion of 1 μM PF‐04957325 did not increase either EP2‐ or EP4‐mediated cAMP increases, confirming that PDE8 does not regulate signaling by either of these receptors. We conclude that β2AR signaling microdomains appear to contain functionally responsive PDE8 while EP2R microdomains do not. In airway diseases such as asthma and COPD, PDE8 may represent a novel therapeutic target to modulate HASM responsiveness and airway remodeling.Support or Funding InformationThis work was supported by NIH grant GM107094 (RO)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Type I positive allosteric modulators (PAMs) of the alpha7-nicotinic receptor enhance its cholinergic activation while preserving the spatiotemporal features of synaptic transmission and the receptor’s characteristic rapid desensitization kinetics. Alpha7-nicotinic receptor agonists have shown promise for improving cognition in schizophrenia, but longer-term trials have been disappointing. Therefore, the type I PAM AVL-3288 was evaluated for safety and preliminary evidence of neurocognitive effect in healthy human subjects. Single-dose oral administration in ascending doses was conducted in a double-blind, placebo-controlled Phase I trial in non-smokers. The trial found indication of positive but non-significant effects on neurocognition at 10 and 30 mg, two doses that produced overlapping peak levels. There was also some evidence for effects on inhibition of the P50 auditory evoked potential to repeated stimuli, a biomarker that responds to alpha7-nicotinic receptor activation. The pharmacokinetic characteristics were consistent between subjects, and there were no safety concerns. The effects and safety profile were also assessed at 3 mg in a cohort of smokers, in whom concurrent nicotine administration did not alter either effects or safety. The trial demonstrates that a type I PAM can be safely administered to humans and that it has potential positive neurocognitive effects in central nervous system (CNS) disorders.

Autism spectrum disorder (ASD) is associated with two core symptoms (social communication deficits and stereotyped repetitive behaviors) in addition to a number of comorbidities. There are no FDA-approved drugs for the core symptoms and the changes that underlie these behaviors are not fully understood. One hypothesis is an imbalance of the excitation (E)/inhibition (I) ratio with excessive E and diminished I occurring in specific neuronal circuits. Data suggests that both gamma-aminobutyric acidA (GABAA) and α7 nicotinic acetylcholine receptors (nAChRs) significantly impact E/I. BTBR T+tf/J (BTBR) mice are a model that display an autism-like phenotype with impaired social interaction and stereotyped behavior. A β2/3-subunit containing GABAA receptor (GABAAR) subtype selective positive allosteric modulator (PAM), 2-261, and an α7 nAChR subtype selective PAM, AVL-3288, were tested in social approach and repetitive self-grooming paradigms. 2-261 was active in the social approach but not the self-grooming paradigm, whereas AVL-3288 was active in both. Neither compound impaired locomotor activity. Modulating α7 nAChRs alone may be sufficient to correct these behavioral and cognitive deficits. GABAergic and nicotinic compounds are already in various stages of clinical testing for treatment of the core symptoms and comorbidities associated with ASD. Our findings and those of others suggest that compounds that have selective activities at GABAAR subtypes and the α7 nAChR may address not only the core symptoms, but many of the associated comorbidities as well and warrant further investigation in other models of ASD.