Adenylyl Cyclase Activity Research Articles

Gsα-dependent signaling is required for postnatal establishment of a functional β-cell mass

ObjectiveEarly postnatal life is a critical period for the establishment of the functional β-cell mass that will sustain whole-body glucose homeostasis during the lifetime. β cells are formed from progenitors during embryonic development but undergo significant expansion in quantity and attain functional maturity after birth. The signals and pathways involved in these processes are not fully elucidated. Cyclic adenosine monophosphate (cAMP) is an intracellular signaling molecule that is known to regulate insulin secretion, gene expression, proliferation, and survival of adult β cells. The heterotrimeric G protein Gs stimulates the cAMP-dependent pathway by activating adenylyl cyclase. In this study, we sought to explore the role of Gs-dependent signaling in postnatal β-cell development. MethodsTo study Gs-dependent signaling, we generated conditional knockout mice in which the α subunit of the Gs protein (Gsα) was ablated from β-cells using the Cre deleter line Ins1Cre. Mice were characterized in terms of glucose homeostasis, including in vivo glucose tolerance, glucose-induced insulin secretion, and insulin sensitivity. β-cell mass was studied using histomorphometric analysis and optical projection tomography. β-cell proliferation was studied by ki67 and phospho-histone H3 immunostatining, and apoptosis was assessed by TUNEL assay. Gene expression was determined in isolated islets and sorted β cells by qPCR. Intracellular cAMP was studied in isolated islets using HTRF-based technology. The activation status of the cAMP and insulin-signaling pathways was determined by immunoblot analysis of the relevant components of these pathways in isolated islets. In vitro proliferation of dissociated islet cells was assessed by BrdU incorporation. ResultsElimination of Gsα in β cells led to reduced β-cell mass, deficient insulin secretion, and severe glucose intolerance. These defects were evident by weaning and were associated with decreased proliferation and inadequate expression of key β-cell identity and maturation genes in postnatal β-cells. Additionally, loss of Gsα caused a broad multilevel disruption of the insulin transduction pathway that resulted in the specific abrogation of the islet proliferative response to insulin. ConclusionWe conclude that Gsα is required for β-cell growth and maturation in the early postnatal stage and propose that this is partly mediated via its crosstalk with insulin signaling. Our findings disclose a tight connection between these two pathways in postnatal β cells, which may have implications for using cAMP-raising agents to promote β-cell regeneration and maturation in diabetes.

279-OR: Hydroxylation Regulation of Hepatic Gluconeogenesis via Prolyl-Hydroxylase 3 in Mice

Hepatic gluconeogenesis is critical for metabolic adaption in response to fasting, and its abnormal elevation contributes to hyperglycemia and type 2 diabetes. However, whether and how hydroxylation regulates hepatic gluconeogenesis remains largely unknown. Here we identified prolyl hydroxylase 3 (PHD3) of the hydroxylase family as a key regulator for hepatic gluconeogenesis. Liver-specific knockout of PHD3 (PHD3 LKO) mice show attenuated fasting gluconeogenic genes, glycaemia and hepatic capacity to produce glucose as evidenced by a pyruvate tolerance test. The efficacies of glucagon or adenylyl cyclase activator forskolin on stimulating the expression of gluconeogenic enzymes, such as PEPCK and G6Pase, and on glucose output are abolished in PHD3-/- mouse primary hepatocytes. To determine whether the prolyl hydroxylase activity is required for PHD3’s effects on promoting gluconeogenesis, liver-specific prolyl hydroxylase-deficient knockin mice with alanine mutation in PHD3 at Pro196 (PHD3 KI) were generated. Similar to PHD3 LKO mice, PHD3 KI mice exhibit attenuated fasting gluconeogenic genes, glycaemia and hepatic capacity to produce glucose, suggesting that PHD3 regulates hepatic gluconeogenesis in a proline hydroxylase-dependent manner. Moreover, several cellular proteins that associate with PHD3 were identified by comprehensive protein analysis using LC-MS/MS followed by bioinformatic analysis including GSK3β, IRS2 and OGT, which may act as PHD3 substrates and play roles in mediating PHD3’s effects on regulating glucose metabolism. Intriguingly, expression levels of PHD3 are increased in the liver diet-induced insulin resistant mice. Together, these results demonstrate that hydroxylation plays a critical role in the regulation of hepatic gluconeogenesis under physiological and insulin resistant conditions. Hyperactivation of PHD3 may underscore the potential role of PHD3 in the development of sustained gluconeogenesis, hyperglycemia and type 2 diabetes. Disclosure Y. Xue: None. J. Gao: None. X. Dai: None. A. Cui: None. Y. Li: None. G. Cai: None. F. Ma: None. Z. Liu: None. Y. Liu: None. W. Su: None. Z. Zheng: None. D. Ding: None. Y. Jiang: None. Funding National Key Research and Development Project of China (2019YFA0802502); National Natural Science Foundation of China (81925008); Shanghai Science and Technology Commission (19140903300)

Prostaglandin E2 Enhances Gap Junctional Intercellular Communication in Clonal Epithelial Cells.

Prostaglandins are a group of lipids that produce diverse physiological and pathological effects. Among them, prostaglandin E2 (PGE2) stands out for the wide variety of functions in which it participates. To date, there is little information about the influence of PGE2 on gap junctional intercellular communication (GJIC) in any type of tissue, including epithelia. In this work, we set out to determine whether PGE2 influences GJIC in epithelial cells (MDCK cells). To this end, we performed dye (Lucifer yellow) transfer assays to compare GJIC of MDCK cells treated with PGE2 and untreated cells. Our results indicated that (1) PGE2 induces a statistically significant increase in GJIC from 100 nM and from 15 min after its addition to the medium, (2) such effect does not require the synthesis of new mRNA or proteins subunits but rather trafficking of subunits already synthesized, and (3) such effect is mediated by the E2 receptor, which, in turn, triggers a signaling pathway that includes activation of adenylyl cyclase and protein kinase A (PKA). These results widen the knowledge regarding modulation of gap junctional intercellular communication by prostaglandins.

Modulation of Cyclic AMP Levels in Fallopian Tube Cells by Natural and Environmental Estrogens.

Autocrine/paracrine factors generated in response to 17β-estradiol (E2) within the fallopian tube (FT) facilitate fertilization and early embryo development for implantation. Since cyclic AMP (cAMP) plays a key role in reproduction, regulation of its synthesis by E2 may be of biological/pathophysiological relevance. Herein, we investigated whether cAMP production in FT cells (FTCs) is regulated by E2 and environmental estrogens (EE’s; xenoestrogens and phytoestrogens). Under basal conditions, low levels of extracellular cAMP were detectable in bovine FTCs (epithelial cells and fibroblasts; 1:1 ratio). Treatment of FTCs with forskolin (AC; adenylyl cyclase activator), isoproterenol (β-adrenoceptor agonist) and IBMX (phosphodiesterase (PDE) inhibitor) dramatically (>10 fold) increased cAMP; whereas LRE1 (sAC; soluble AC inhibitor) and 2’,5’-dideoxyadenosine (DDA; transmembrane AC (tmAC)) inhibitor decreased cAMP. Comparable changes in basal and stimulated intracellular cAMP were also observed. Ro-20-1724 (PDE-IV inhibitor), but not milrinone (PDE-III inhibitor) nor mmIBMX (PDE-I inhibitor), augmented forskolin-stimulated cAMP levels, suggesting that PDE-IV dominates in FTCs. E2 increased cAMP levels and CREB phosphorylation in FTCs, and these effects were mimicked by EE’s (genistein, 4-hydroxy-2’,4’,6’-trichlorobiphenyl, 4-hydroxy-2’,4’,6’-dichlorobiphenyl). Moreover, the effects of E2 and EE were blocked by the tmAC inhibitor DDA, but not by the ERα/β antagonist ICI182780. Moreover, BAPTA-AM (intracellular-Ca2+ chelator) abrogated the effects of E2, but not genistein, on cAMP suggesting differential involvement of Ca2+. Treatment with non-permeable E2-BSA induced cAMP levels and CREB-phosphorylation; moreover, the stimulatory effects of E2 and EEs on cAMP were blocked by G15, a G protein-coupled estrogen receptor (GPER) antagonist. E2 and IBMX induced cAMP formation was inhibited by LRE1 and DDA suggesting involvement of both tmAC and sAC. Our results provide the first evidence that in FTCs, E2 and EE’s stimulate cAMP synthesis via GPER. Exposure of the FT to EE’s and PDE inhibitors may result in abnormal non-cyclic induction of cAMP levels which may induce deleterious effects on reproduction.

The isolation, purification and complete characterization of the diterpene forskolin from nutritional supplements

Forskolin (1) is a diterpene found in the Coleus forskohlii plant that has been examined for its medical properties resulting from adenylyl cyclase activation. This article describes a straightforward purification method of 1 from commercially available weight loss capsules. In addition, there has been some ambiguity with respect to the use of the name ‘forskolin’ to describe 1 and related diterpenes, which this report serves to eliminate. Herein we detail the complete spectroscopic characterization of purified 1 as well as its single crystal X-ray structure.

Antidepressants Produce Persistent Gα s-Associated Signaling Changes in Lipid Rafts after Drug Withdrawal.

Termination of antidepressant therapy often has negative consequences. Although symptoms of antidepressant withdrawal are widely recognized, the molecular processes that underlie them are not well characterized. We show that certain aspects of Gα s signaling remain suppressed after antidepressant withdrawal, even after others have reverted to baseline. Antidepressant treatment causes translocation of Gα s protein from lipid rafts to nonraft membrane regions. This results in augmented Gα s signaling, including facilitated activation of adenylyl cyclase and increased cAMP accumulation. Using CC6 or SK-N-SH cells and a lipid raft-localized cAMP sensor, we show that Gα s signaling is reduced in lipid rafts, even while signaling is enhanced elsewhere in the cell. These signaling changes mirror the changes in Gα s localization observed after antidepressant treatment. Furthermore, we show that suppression of Gα s signaling in lipid rafts persists at least 24 hours after cessation of antidepressant treatment. Gα s localization was quantified after membrane isolation and sequential detergent extraction. We show that suppression of lipid raft Gα s signaling persists for an extended time period after antidepressant withdrawal, whereas increased nonraft membrane Gα s signaling reverts partially or fully upon cessation of antidepressant treatment. Translocation of Gα s out of lipid rafts is also persistent. These events may reflect cellular adaptations to antidepressant treatment that contribute to antidepressant discontinuation syndromes and may aid in the discovery of new treatments and strategies to mitigate the symptoms of depression and antidepressant withdrawal. SIGNIFICANCE STATEMENT: This work explores, for the first time, the effects of antidepressants on Gα s signaling after drug withdrawal. This provides novel insight into the cellular and molecular processes affected by antidepressant drugs and their persistence after discontinuation of treatment.

Adenosine Signaling in Mast Cells and Allergic Diseases.

Adenosine is a nucleoside involved in the pathogenesis of allergic diseases. Its effects are mediated through its binding to G protein-coupled receptors: A1, A2a, A2b and A3. The receptors differ in the type of G protein they recruit, in the effect on adenylyl cyclase (AC) activity and the downstream signaling pathway triggered. Adenosine can produce both an enhancement and an inhibition of mast cell degranulation, indicating that adenosine effects on these receptors is controversial and remains to be clarified. Depending on the study model, A1, A2b, and A3 receptors have shown anti- or pro-inflammatory activity. However, most studies reported an anti-inflammatory activity of A2a receptor. The precise knowledge of the adenosine mechanism of action may allow to develop more efficient therapies for allergic diseases by using selective agonist and antagonist against specific receptor subtypes.

Melanocortin 4 receptor signals at the neuronal primary cilium to control food intake and body weight.

The melanocortin 4 receptor (MC4R) plays a critical role in the long-term regulation of energy homeostasis, and mutations in the MC4R are the most common cause of monogenic obesity. However, the precise molecular and cellular mechanisms underlying the maintenance of energy balance within MC4R-expressing neurons are unknown. We recently reported that the MC4R localizes to the primary cilium, a cellular organelle that allows for partitioning of incoming cellular signals, raising the question of whether the MC4R functions in this organelle. Here, using mouse genetic approaches, we found that cilia were required specifically on MC4R-expressing neurons for the control of energy homeostasis. Moreover, these cilia were critical for pharmacological activators of the MC4R to exert an anorexigenic effect. The MC4R is expressed in multiple brain regions. Using targeted deletion of primary cilia, we found that cilia in the paraventricular nucleus of the hypothalamus (PVN) were essential to restrict food intake. MC4R activation increased adenylyl cyclase (AC) activity. As with the removal of cilia, inhibition of AC activity in the cilia of MC4R-expressing neurons of the PVN caused hyperphagia and obesity. Thus, the MC4R signaled via PVN neuron cilia to control food intake and body weight. We propose that defects in ciliary localization of the MC4R cause obesity in human inherited obesity syndromes and ciliopathies.

A Case of Chronic Bone Pain in Mc-CuneAlbright Syndrome

Introduction: McCune-Albright syndrome (MAS) is a rare genetic disorder that effects the endocrine, skin and bone systems and caused by a somatic mutation in the G protein activating adenylyl cyclase resulting in abnormal osteoblast differentiation and development of fibrous dysplasia (FD). We describe a patient with extensive fibrous dysplasia and severe bone pain in the setting of MAS. Case: A 56-year-old female with a history of MAS was evaluated in the osteoporosis clinic for chronic bone pain. She was diagnosed with MAS at the age of two when she presented with precocious puberty and treated with hormonal blockade. She had multiple orthopedic issues including chronic left hip and lower back pain with impending fracture and pelvic microfractures treated with prophylactic rodding and cement osteoplasty at age 46 and 51 respectively. She was taking Tramadol as needed. The patient reported an inch of height loss, and imaging revealed T4 compression fracture. She was treated with zoledronic acid 4 mg IV on a monthly basis for 1 year without any improvement in bone density or pain symptoms She reached menarche at age 12, and is currently menopausal. Her calcium intake was limited but vitamin D intake was adequate. The patient was quite active performing intense cardio exercises several times a week. Physical exam was remarkable for café-au-lait spots on her left trunk and abdomen not crossing the mid-line. Bone density showed spine DJD, right femoral neck and forearm T scores of -1.1 and -1.6 respectively. Recent CT scan demonstrated multiple lucent lesions in the thoracic spine, bilateral ribs and sternum consistent with fibrous dysplasia. Biochemical evaluation showed high bone turnover; urine NTX of 74 nM BCE/mM creatinine (26–124 nM BCE/mM) and bone specific alkaline phosphatase 80.7 U/L (14.2–42.7) Calcium and vitamin D levels were within limits. The patient initiated denosumab 60mg every 6 months to determine if significant reduction in bone turnover and bone pain would follow. Discussion: As patients with MAS and FD have elevated RANKL levels and increased bone resorption, treatments that reduce bone turnover markers (BTMs) have been studied. Bisphosphonates have demonstrated decline in BTMs but with mixed results regarding improvement of clinical symptoms (1). In a recent study, denosumab, a monoclonal antibody to RANK-L was given at either 3- or 6-month intervals. Patients who received 3-month interval injections demonstrated a sustained suppression of BTM and improvement of pain (2). Thus, denosumab is a promising and well-tolerated agent that may be effective in FD, especially when response to bisphosphonates is inadequate.

CRISPR/Cas9‐mediated knockout of G proteins and β‐arrestins defines their specific but interdependent roles in dopamine D1 receptor signaling

The dopamine D1 receptor (D1R) is a G protein-coupled receptor which regulates key brain functions including voluntary movement, memory, attention, and reward. Selective agonist activation of the D1R may also provide novel neurotherapeutics for neurodegenerative and neuropsychiatric disorders. The D1R canonically activates the heterotrimeric G proteins Gαs, and possibly Gαolf, which activate adenylyl cyclase (AC) to increase cAMP/PKA signaling. The D1R also engages β-arrestin proteins which may transduce β-arrestin dependent signaling by scaffolding and activating kinases. However, the distinct roles of Gαs, Gαolf versus β-arrestins 1 and 2 for D1R signaling remains largely undefined. Here we utilize CRISPR/Cas9 engineered HEK293 cells in which Gαs, Gαolf, β-arrestin1 and β-arrestin2 are stably knocked out (KO) to delineate D1R pharmacology and signaling mechanisms. Western blots confirmed a complete loss of Gαs, Gαolf, β-arrestin1 or β-arrestin2 proteins in KO cells versus parental wildtype cells. Dose responses with the D1R agonist SKF-81297 or β2AR agonist isoproterenol induced robust cAMP production in wildtype cells that was eliminated in Gαs/olf KO cells. Re-expression of Gαs or Gαolf in KO cells rescued D1R-mediated cAMP signaling similar to wildtype cells. Interestingly, the D1R had significantly higher cAMP signaling when coupling via Gαs as compared to Gαolf, while the β2AR only coupled with Gαs to stimulate cAMP signaling. Western blotting indicated Gαs/olf KO completely blocked agonist induced CREB phosphorylation by both D1R and β2AR agonists, indicating a strict dependence on Gαs/olf and cAMP/PKA signaling to activate CREB. In major contrast, β-arrestin1/2 KO significantly increased the intensity and duration of cAMP signaling and CREB phosphorylation by the β2AR. β-arrestin1/2 KO also entirely blocked agonist-induced D1R internalization and impaired D1R desensitization while Gαs/olf KO had no effect on D1R internalization. Key controls of re-expressing Gαs, Gαolf, β-arrestin1 or β-arrestin2 in KO cells completely restored signaling or internalization similar to wildtype cells, indicating CRISPR/Cas9 targeting did not result in non-specific or compensatory effects in the pathways under study. Notably, β-arrestin1/2 KO also potentiated and prolonged D1R or β2AR-induced ERK1/2 phosphorylation. D1R-induced ERK1/2 phosphorylation in β-arrestin1/2 KO cells was eliminated by the SRC inhibitor SU6656 or the MEK inhibitor U0126, suggesting that D1R signaling via a Gαs/SRC/MEK pathway is essential for ERK1/2 activation. Taken together, our studies indicate that Gαs is the dominant transducer for D1R cAMP, CREB and ERK1/2 signaling and that β-arrestins provide distinctly interdependent roles with G proteins in modulating the duration and intensity of D1R and β2AR signaling cascades. Using CRISPR/Cas9 KO model cells, future studies will examine the distinct contributions of β-arrestins 1 and 2 and Gαs/olf for D1R pharmacology, which will be useful in the development of novel D1R biased agonists.

Signaling Specificity of the Gα i G Protein Subfamily8.5.5

G protein-coupled receptors, or GPCRs, are a major class of cell surface receptors that transduce extracellular stimuli to intracellular signaling events via G proteins. GPCRs catalyze nucleotide exchange on the G protein Gα subunit leading to dissociation from Gβγ subunits. The canonical role of one family of G protein alpha subunits, Gαi, is binding and inhibition of the activity of adenylyl cyclase, leading to reduction in the level of 3′,5’-cyclic adenosine monophosphate (cAMP) in cells. Using proximity labeling mass spectrometry, our laboratory has recently discovered a novel role for Gαi in the activation of PDZ-RhoGEF (PRG), a guanine nucleotide exchange factor for the small G protein RhoA. Surprisingly, we found significant differences in the ability of Gαi1, Gαi2, and Gαi3 isoforms to activate PRG in a cell-based SRE-luciferase assay, despite over 85% sequence identity and equivalent inhibition of their common effector adenylyl cyclase. Gαi1 and Gαi3 strongly activate PRG, while Gαi2 shows very weak activation. A majority of the differences in these proteins are located in the helical domain of Gαi, which supports the central hypothesis that members of the Gαi subfamily differentially bind and activate PRG with specificity determined by structural features in the helical domain. To investigate this, the amino acid sequence of Gαi1 was divided into an N-terminal region, the helical domain (HD), and a C-terminal region. Protein chimeras were then generated, substituting one of each of these regions from Gαi1 into Gαi2 to determine if any of these domains are sufficient to confer the ability of the specific Gαi2-Gαi1 chimera to activate PRG in a SRE-luciferase assay in co-transfected HEK293 cells. All chimeras were validated for proper expression in HEK293 cells using western blotting, and all equally inhibited forskolin-stimulated cAMP production. The Gαi2-Gαi1-HD chimera activated PRG to levels similar to Gαi1, while the N-terminal and C-terminal chimeras did not activate PRG. This demonstrates that the helical domain of Gαi1 is sufficient for conferring the ability to activate PRG and structural features within this domain are responsible for the specificity amongst the Gαi1, 2, and 3 subtypes. In an effort to determine the structural features within the HD that confer Gαi-dependent activation of PRG, three subdivisions of the Gαi1 HD were then substituted into Gαi2. None of these subdivisions substituted alone resulted in activation of PRG, indicating that the requirements for PRG activation likely lie in three-dimensional coordination of multiple key residues throughout the helical domain of Gαi. Current and future work will focus on clusters of residue substitutions using three-dimensional structures and sequence alignments as a guide, substituting amino acids common between Gαi1 and Gαi3 but different in Gαi2. Constructs of interest will be examined in cell-based assays as well as purified and examined for in vitro binding to PRG. The resulting data have the potential to change the existing model for signal transfer from Gαi proteins to effectors and provides some of the first evidence for Gαi subtype-effector specificity. 8.5.5

Intrathecal Knockdown of Adenylyl Cyclase 1 Attenuates Morphine Tolerance and Withdrawal in Mice

Opioid tolerance and opioid induced hyperalgesia during repeated opioid administration and chronic pain are associated with upregulation of adenylyl cyclase activity. Therefore, we investigated if expression of components of the cyclic-AMP pathway were increased within in the nervous system tissues of morphine tolerant mice, and if inhibiting any of these changes could alleviate pain and/or tolerance in mouse models. The objective of this study was to test the hypothesis that a reduction in adenylyl cyclase 1 (AC1) activity would attenuate morphine tolerance and hypersensitivity, and inflammatory pain using murine models. Short-hairpin RNA (shRNA) gene knockdown of Adcy1 in the spinal cord and dorsal root ganglia was accomplished using a lumbar injection of an associated adenovirus viral vector (AAV9-GFP-U6-m-Adcy1-shRNA) and negative controls (AAV9-GFP-U6-m-scrambl-shRNA). Behavioral testing such as open field testing, rotarod testing, burrowing, thermal and mechanical paw withdrawal latencies were tested after injection. Morphine tolerance (15mg/kg, sc, 5 days and 10-40mg/kg escalation over 4 days) and opioid-induced hypersenstivity were also assessed after inoculation. Lumbar intrathecal administration of a vector incorporating adeno-associated virus and short-hairpin RNA against Adcy1 did not affect baseline parameters such as open field testing, rotarod testing, and burrowing testing. Morphine tolerance and withdrawal were attenuated in Adcy1 shRNA mice compared to control vector mice. Chronic exposure to morphine leads to changes in mRNA expression of proteins involved in the cAMP signaling pathway in different areas of the nervous system. Gene knockdown of Adcy1 decreases morphine tolerance and opioid-induced hypersensitivity, which could form the basis for novel therapeutics in the future. This work was supported through K01 DA042902 to AHK and the Purdue University College of Pharmacy to VJW.

Gs‐DREADD Regulation of ENaC

Activity of the epithelial Na+ channel (ENaC) fine-tunes renal Na+ excretion and consequently, contributes to the normal control of blood pressure. ENaC is expressed in the apical membrane of principal cells. Importantly, ENaC is activated by the hormone vasopressin via the V2 receptor, which is coupled to the heterotrimeric Gs protein. Stimulation of the V2 receptor rapidly activates adenylyl cyclase to increase intracellular cAMP levels. We have recently developed a mouse model with targeted expression of the Gs designer receptors exclusively activated by designer drugs (DREADD) in renal principal cells (GsD). Moreover, this mouse line has a luciferase reporter downstream of a cAMP response element binding protein site. As such, it allows in vivo reporting of cAMP levels. DREADD receptors are activated by clozapine N-oxide (CNO), which normally is an inert drug with no biological activity in the absence of DREADD receptors. We test here if CNO affects the activity of ENaC to influence renal salt and water excretion in GsD mice similarly to vasopressin. In these mice, we longitudinally monitored CNO-stimulated cAMP levels in principal cells using an IVIS Lumina XR. In parallel, we quantified CNO-dependent ENaC activity and renal sodium excretion. We observed that CNO increased ENaC activity in principal cells in freshly isolated tubules from GsD mice compared to control mice. Metabolic cages experiments demonstrated that CNO treatment (0.1 mg/kg) increased cAMP levels concomitantly with reducing sodium excretion similarly to dDAVP (1 µg) in GsD mice. Finally, in COS-7 cells expressing murine ENaC tagged with YFP, CPT-cAMP increased translocation of ENaC to the plasma membrane. Here we demonstrated the utility of a new principal cell-specific Gs-DREADD mouse line. This new mouse model will be an important tool for investigating the effects of abnormal cAMP signaling in principal cells possibly informing understanding of the cellular mechanism underpinning conditions like polycystic kidney disease.

Adenylyl Cyclase (AC) Mediates the Antidepressant-Like Effects of Tropisetron on a Mouse Model of Maternal Separation Stress.

The adenylyl cyclase (AC) pathway is involved in the pathophysiology of depression. Finding new antidepressants with high medicinal properties and low side effects is warranted. Therefore, this study was designed to determine the antidepressant-like effect of tropisetron on a maternal separation (MS) model in mice, considering the possible role of AC. NMRI male mice were divided into eleven groups. The control group was treated with saline and MS groups were treated with saline, tropisetron (a 5-HT3 receptor antagonist) at doses of 1, 3, and 5 mg/kg; forskolin (an activator of AC) at doses of 5, 10, and 25 mg/kg; a subeffective dose of forskolin with a subeffective dose of tropisetron; and an effective dose of tropisetron plus an effective dose of NB001 (3 mg/kg) (an AC inhibitor). After treatment, animals were subjected to behavioral tests including the forced swimming test (FST), splash test, and open field test (OFT). We showed that MS caused depressive-like behaviors determined as an increase in the immobility time in the forced swimming test (FST) and decreased grooming time in the splash test. Our results showed that administration of tropisetron, as well as forskolin, mitigated the depressive-like behaviors in MS mice. We found that coadministration of a subeffective dose of tropisetron plus a subeffective dose of forskolin potentiated the antidepressant-like effect of tropisetron. However, coadministration of an effective dose of NB001 with an effective dose of tropisetron did not significantly affect the antidepressant-like effect of tropisetron. We concluded that the antidepressant-like effects of tropisetron on MS mice are partially mediated through the adenylyl cyclase pathway.

A Highlight on the Inhibition of Fungal Carbonic Anhydrases as Drug Targets for the Antifungal Armamentarium.

Carbon dioxide (CO2), a vital molecule of the carbon cycle, is a critical component in living organisms’ metabolism, performing functions that lead to the building of compounds fundamental for the life cycle. In all living organisms, the CO2/bicarbonate (HCO3−) balancing is governed by a superfamily of enzymes, known as carbonic anhydrases (CAs, EC 4.2.1.1). CAs catalyze the pivotal physiological reaction, consisting of the reversible hydration of the CO2 to HCO3− and protons. Opportunistic and pathogenic fungi can sense the environmental CO2 levels, which influence their virulence or environmental subsistence traits. The fungal CO2-sensing is directly stimulated by HCO3− produced in a CA-dependent manner, which directly activates adenylyl cyclase (AC) involved in the fungal spore formation. The interference with CA activity may impair fungal growth and virulence, making this approach interesting for designing antifungal drugs with a novel mechanism of action: the inhibition of CAs linked to the CO2/HCO3−/pH chemosensing and signaling. This review reports that sulfonamides and their bioisosteres as well as inorganic anions can inhibit in vitro the β- and α-CAs from the fungi, suggesting how CAs may be considered as a novel “pathogen protein” target of many opportunistic, pathogenic fungi.

Cardiac cAMP-PKA Signaling Compartmentalization in Myocardial Infarction.

Under physiological conditions, cAMP signaling plays a key role in the regulation of cardiac function. Activation of this intracellular signaling pathway mirrors cardiomyocyte adaptation to various extracellular stimuli. Extracellular ligand binding to seven-transmembrane receptors (also known as GPCRs) with G proteins and adenylyl cyclases (ACs) modulate the intracellular cAMP content. Subsequently, this second messenger triggers activation of specific intracellular downstream effectors that ensure a proper cellular response. Therefore, it is essential for the cell to keep the cAMP signaling highly regulated in space and time. The temporal regulation depends on the activity of ACs and phosphodiesterases. By scaffolding key components of the cAMP signaling machinery, A-kinase anchoring proteins (AKAPs) coordinate both the spatial and temporal regulation. Myocardial infarction is one of the major causes of death in industrialized countries and is characterized by a prolonged cardiac ischemia. This leads to irreversible cardiomyocyte death and impairs cardiac function. Regardless of its causes, a chronic activation of cardiac cAMP signaling is established to compensate this loss. While this adaptation is primarily beneficial for contractile function, it turns out, in the long run, to be deleterious. This review compiles current knowledge about cardiac cAMP compartmentalization under physiological conditions and post-myocardial infarction when it appears to be profoundly impaired.

CAMP signaling through protein kinase A and Epac2 induces substance P release in the rat spinal cord

Using neurokinin 1 receptor (NK1R) internalization to measure of substance P release in rat spinal cord slices, we found that it was induced by the adenylyl cyclase (AC) activator forskolin, by the protein kinase A (PKA) activators 6-Bnz-cAMP and 8-Br-cAMP, and by the activator of exchange protein activated by cAMP (Epac) 8-pCPT-2-O-Me-cAMP (CPTOMe-cAMP). Conversely, AC and PKA inhibitors decreased substance P release induced by electrical stimulation of the dorsal root. Therefore, the cAMP signaling pathway mediates substance P release in the dorsal horn. The effects of forskolin and 6-Bnz-cAMP were not additive with NMDA-induced substance P release and were decreased by the NMDA receptor blocker MK-801. In cultured dorsal horn neurons, forskolin increased NMDA-induced Ca2+ entry and the phosphorylation of the NR1 and NR2B subunits of the NMDA receptor. Therefore, cAMP-induced substance P release is mediated by the activating phosphorylation by PKA of NMDA receptors. Voltage-gated Ca2+ channels, but not by TRPV1 or TRPA1, also contributed to cAMP-induced substance P release. Activation of PKA was required for the effects of forskolin and the three cAMP analogs. Epac2 contributed to the effects of forskolin and CPTOMe-cAMP, signaling through a Raf - mitogen-activated protein kinase pathway to activate Ca2+ channels. Epac1 inhibitors induced NK1R internalization independently of substance P release. In rats with latent sensitization to pain, the effect of 6-Bnz-cAMP was unchanged, whereas the effect of forskolin was decreased due to the loss of the stimulatory effect of Epac2. Hence, substance P release induced by cAMP decreases during pain hypersensitivity.

Natriuretic peptide receptor-C releases and activates guanine nucleotide-exchange factor H1 in a ligand-dependent manner

Although natriuretic peptide receptor-C (NPR-C) is involved in the clearance of natriuretic peptides from plasma, it also possesses other physiological functions, such as inhibition of adenylyl cyclase activity through Gαi. However, the physiological roles and intracellular signaling pathways of NPR-C have yet been not fully elucidated. In this study, we identified a RhoA-specific guanine nucleotide-exchange factor, GEF-H1, as a novel binding protein of NPR-C. We demonstrated that endogenous NPR-C interacted with GEF-H1 in HeLa cells, and that the interaction between NPR-C and GEF-H1 was dependent on a 37-amino acid cytoplasmic region of NPR-C. In contrast, another natriuretic peptide receptor, NPR-A, which includes the kinase homology and guanylyl cyclase domains in the intracellular region, did not interact with GEF-H1. We also revealed that the ligands of NPR-C (i.e., ANP, CNP, and osteocrin) caused dissociation of GEF-H1 from NPR-C. Furthermore, osteocrin treatment induced phosphorylation of GEF-H1 at Ser-886, enhanced the interaction of GEF-H1 with 14-3-3, and increased the amount of activated GEF-H1. These findings strongly supported that NPR-C may be involved in diverse physiological roles by regulating GEF-H1 signaling.

PAC1 Receptor Internalization and Endosomal MEK/ERK Activation Is Essential for PACAP-Mediated Neuronal Excitability.

Pituitary adenylate cyclase activating polypeptide (PACAP, Adcyap1) activation of PAC1 receptors (Adcyap1r1) can significantly increase the excitability of diverse neurons through differential mechanisms. For guinea pig cardiac neurons, the modulation of excitability can be mediated in part by PAC1 receptor plasma membrane G protein-dependent activation of adenylyl cyclase and downstream signaling cascades. By contrast, PAC1 receptor-mediated excitability of hippocampal dentate gyrus granule cells appears independent of membrane-delimited AC/cAMP/PKA and PLC/PKC signaling. For both neuronal types, there is mechanistic convergence demonstrating that endosomal PAC1 receptor signaling has prominent roles. In these models, neuronal exposure to Pitstop2 to inhibit β-arrestin/clathrin-mediated PAC1 receptor internalization eliminates PACAP modulation of excitability. β-arrestin is a scaffold for a number of effectors especially MEK/ERK and notably, paradigms that inhibit PAC1 receptor endosome formation and ERK signaling also blunt the PACAP-induced increase in excitability. Detailed PAC1 receptor internalization and endosomal ERK signaling mechanisms have been confirmed in HEK PAC1R-EGFP cells and shown to be long lasting which appear to recapitulate the sustained electrophysiological responses. Thus, PAC1 receptor internalization/endosomal recruitment efficiently and efficaciously activates MEK/ERK signaling and appears to represent a singular and critical common denominator in regulating neuronal excitability by PACAP.

Orexin-A differentially modulates inhibitory and excitatory synaptic transmission in rat inner retina

In this work, modulation by orexin-A of the release of glutamate and GABA from bipolar and amacrine cells respectively was studied by examining the effects of the neuropeptide on miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) of rat retinal ganglion cells (GCs). Using RNAscope in situ hybridization in combination with immunohistochemistry, we showed positive signals for orexin receptor-1 (OX1R) mRNA in the bipolar cell terminals and those for orexin receptor-2 (OX2R) mRNA in the amacrine cell terminals. With whole-cell patch-clamp recordings in rat retinal slices, we demonstrated that application of orexin-A reduced the interevent interval of mEPSCs of GCs through OX1R. However, it increased the interevent interval of mIPSCs, mediated by GABAA receptors, through OX2R. Furthermore, orexin-A-induced reduction of mEPSC interevent interval was abolished by the application of PI-PLC inhibitors or PKC inhibitors. In contrast, orexin-A-induced increase of GABAergic mIPSC interevent interval was mimicked by 8-Br-cAMP or an adenylyl cyclase activator, but was eliminated by PKA antagonists. Finally, application of nimodipine, an L-type Ca2+ channel blocker, increased both mEPSC and mIPSC interevent interval, and co-application of orexin-A no longer changed the mEPSCs and mIPSCs. We conclude that orexin-A increases presynaptic glutamate release onto GCs by activating L-type Ca2+ channels in bipolar cells, a process that is mediated by an OX1R/PI-PLC/PKC signaling pathway. However, orexin-A decreases presynaptic GABA release onto GCs by inhibiting L-type Ca2+ channels in amacrine cells, a process that is mediated by an OX2R/cAMP-PKA signaling pathway.