RÉSUMÉ
The effect of a promising NO donor, a binuclear nitrosyl iron complex (NIC) with 3,4-dichlorothiophenolyls [Fe2(SC6H3Cl2)2(NO)4], on the adenylate cyclase and soluble guanylate cyclase enzymatic systems was studied. In in vitro experiments, this complex increased the concentration of important secondary messengers, such as cAMP and cGMP. An increase of their level by 2.4 and 4.5 times, respectively, was detected at NIC concentration of 0.1 mM. The ligand of the complex, 3,4-dichlorothiophenol, produced a less pronounced effect on adenylate cyclase. It was shown that the effect of this complex on the activity of soluble guanylate cyclase was comparable to the effect of anionic nitrosyl complex with thiosulfate ligands that exhibits vasodilating and cardioprotective properties.
Sujet(s)
AMP cyclique , GMP cyclique , GMP cyclique/métabolisme , AMP cyclique/métabolisme , Animaux , Fer/métabolisme , Fer/composition chimique , Adenylate Cyclase/métabolisme , Donneur d'oxyde nitrique/pharmacologie , Donneur d'oxyde nitrique/composition chimique , Soluble guanylyl cyclase/métabolisme , Oxydes d'azote/pharmacologie , Oxydes d'azote/métabolisme , Oxydes d'azote/composition chimique , RatsRÉSUMÉ
The pathogenesis mechanism of acute gastric mucosal lesions (AGML) is still unclear; further exploration is urgently needed to find a new therapeutic target. This study aimed to investigate whether morphine might regulate the expression and function of transient receptor potential ankyrin 1 (TRPA1) through a cyclic adenosine monophosphate/protein kinase A (cAMP/PKA)-dependent pathway, thereby alleviating gastric mucosal lesions caused by water-immersion restraint stress (WIRS). Rats were administered with intrathecal morphine, TRPA1 antagonist (HC-030031), µ-opioid receptor antagonist, or protein kinase A inhibitor (H-89), respectively, before WIRS. After 6 hours of WIRS, microscopic lesions, hematoxylin and eosin staining, and transmission electron microscopy were applied to assess the damage of the gastric mucosa. Real-time polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay were conducted to detect the levels of TRPA1 and substance P (SP) in the dorsal root ganglia (DRG) and gastric tissues. In addition, immunofluorescence was used to explore the possible co-expression of TRPA1 and µ-opioid receptors in the DRG. The results indicated that WIRS upregulated TRPA1 and SP in gastric mucosa, and HC-030031 or H-89 could alleviate gastric mucosal lesions caused by WIRS (P < .0001). Morphine was found to suppress both WIRS-induced gastric mucosal lesions (P < .0001) and the upregulation of TRPA1 (P = .0086) and SP (P = .0013). Both TRPA1 and SP play important roles in the pathogenesis of WIRS-induced AGML. Exogenous gastroprotective strategies reduce elevated levels of TRPA1 via the cAMP/PKA-dependent pathway. Inhibition of TRPA1 upregulation in the DRG is critical for intrathecal morphine preconditioning-induced gastric protection.
Sujet(s)
Ganglions sensitifs des nerfs spinaux , Muqueuse gastrique , Isoquinoléines , Morphine , Rat Sprague-Dawley , Contention physique , Membre-1 de la sous-famille A de canaux cationiques à potentiel de récepteur transitoire , Régulation positive , Animaux , Morphine/pharmacologie , Muqueuse gastrique/effets des médicaments et des substances chimiques , Muqueuse gastrique/métabolisme , Régulation positive/effets des médicaments et des substances chimiques , Membre-1 de la sous-famille A de canaux cationiques à potentiel de récepteur transitoire/métabolisme , Mâle , Ganglions sensitifs des nerfs spinaux/métabolisme , Ganglions sensitifs des nerfs spinaux/effets des médicaments et des substances chimiques , Contention physique/effets indésirables , Rats , Isoquinoléines/pharmacologie , Acétanilides/pharmacologie , Cyclic AMP-Dependent Protein Kinases/métabolisme , Purines/pharmacologie , Stress psychologique/complications , Immersion , Récepteur mu/métabolisme , AMP cyclique/métabolisme , SulfonamidesRÉSUMÉ
Collective cell migration occurs when the orientation of cell polarity is aligned with each other in a group of cells. Such collective polarization depends on a reciprocal process between cell intrinsic mechanisms such as cell-cell adhesion and extracellular guidance mechanism such as wound healing and chemotaxis. As part of its development life cycle, individual single cells of Dictyostelium discoideum exhibit chemotaxis toward cAMP, which is secreted from a certain population of cells. During the formation of multicellular body by chemotaxis-dependent cell aggregation, D. discoideum is also known to relay on multiple cell-cell adhesion mechanisms. In particular, tail-following behavior at the contact site, called contact following of locomotion (CFL), plays a pivotal role on the formation of the multicellular body. However, whether and how CFL alone can lead to a formation of collective behavior was not well understood. KI cell is a mutant of D. discoideum that lacks all chemotactic activity. Yet, it can exhibit the CFL activity and show nontrivial collective cell migration. This mutant provides an excellent model system to analyze the mechanism of the CFL and the macroscopic phenomena brought by the CFL. This chapter describes protocols for using KI cell to understand the biophysics and cell biology behind the collective cell migration induced by CFL.
Sujet(s)
Mouvement cellulaire , Chimiotaxie , Dictyostelium , Dictyostelium/génétique , Dictyostelium/physiologie , Dictyostelium/cytologie , Chimiotaxie/génétique , Mouvement cellulaire/génétique , Mutation , AMP cyclique/métabolisme , Polarité de la cellule/génétique , Adhérence cellulaire , Modèles biologiquesRÉSUMÉ
Single cilia, 100 nm in diameter and 10 µm in length, were isolated from mouse tracheae with Triton X-100 (0.02%) treatment, and the effects of pH on ciliary beating were examined by measuring the ciliary beat frequency (CBF) and the ciliary bend distance (CBD-an index of amplitude) using a high-speed video microscope (250 fps). ATP (2.5 mM) plus 8Br-cAMP (10 µM) reactivated the CBF and CBD in the isolated cilia, similar to the cilia of in vivo tracheae. In the reactivated isolated cilia, an elevation in pH from 7.0 to 8.0 increased the CBF from 3 to 15 Hz and the CBD from 0.6 to 1.5 µm. The pH elevation also increased the velocity of the effective stroke; however, it did not increase the recovery stroke, and, moreover, it decreased the intervals between beats. This indicates that H+ (pHi) directly acts on the axonemal machinery to regulate CBF and CBD. In isolated cilia priorly treated with 1 µM PKI-amide (a PKA inhibitor), 8Br-cAMP did not increase the CBF or CBD in the ATP-stimulated isolated cilia. pH modulates the PKA signal, which enhances the axonemal beating generated by the ATP-activated inner and outer dyneins.
Sujet(s)
Adénosine triphosphate , Cils vibratiles , AMP cyclique , Trachée , Animaux , Cils vibratiles/effets des médicaments et des substances chimiques , Cils vibratiles/métabolisme , Adénosine triphosphate/métabolisme , Concentration en ions d'hydrogène , Trachée/métabolisme , Trachée/effets des médicaments et des substances chimiques , Souris , AMP cyclique/métabolisme , MâleRÉSUMÉ
Cyclic nucleotide phosphodiesterases (PDEs) consist of a family of enzymes expressed in several types of cells, including inflammatory cells, that play a pivotal role in inflammation. Several studies have demonstrated that the inhibition of PDE4 results in a reduced inflammatory response via PKA and CREB signaling. Hence, PDE4 suppression improves the inflammatory feedback typical of several diseases, such as inflammatory bowel disease (IBD). In our previous studies, we have demonstrated that miR-369-3p regulates inflammatory responses, modulating different aspects of the inflammatory process. The aim of this study was to demonstrate an additional anti-inflammatory effect of miR-369-3p targeting PDE4B, one of the widely expressed isoforms in immune cells. We found that miR-369-3p was able to reduce the expression of PDE4B, elevating the intracellular levels of cAMP. This accumulation increased the expression of PKA and pCREB, mitigating the release of pro-inflammatory cytokines and promoting the release of anti-inflammatory cytokines. To prove that PDE4B is a good therapeutic target in IBD, we also demonstrate that the expression of PDE4B was increased in UC patients compared to healthy controls, affecting the immune infiltrate. PDE4B is considered an important player in inflammatory progression; hence, our results show the ability of miR-369-3p to ameliorate inflammation by targeting PDE4B, supporting its future application as a new therapeutic approach in IBD.
Sujet(s)
Cyclic Nucleotide Phosphodiesterases, Type 4 , Maladies inflammatoires intestinales , microARN , Cyclic Nucleotide Phosphodiesterases, Type 4/métabolisme , Cyclic Nucleotide Phosphodiesterases, Type 4/génétique , Humains , microARN/génétique , microARN/métabolisme , Maladies inflammatoires intestinales/génétique , Maladies inflammatoires intestinales/métabolisme , Maladies inflammatoires intestinales/anatomopathologie , Maladies inflammatoires intestinales/traitement médicamenteux , AMP cyclique/métabolisme , Inflammation/génétique , Inflammation/métabolisme , Cyclic AMP-Dependent Protein Kinases/métabolisme , Cyclic AMP-Dependent Protein Kinases/génétique , Cytokines/métabolisme , Mâle , Transduction du signal , FemelleRÉSUMÉ
Over half of spinal cord injury (SCI) patients develop opioid-resistant chronic neuropathic pain. Safer alternatives to opioids for treatment of neuropathic pain are gabapentinoids (e.g., pregabalin and gabapentin). Clinically, gabapentinoids appear to amplify opioid effects, increasing analgesia and overdose-related adverse outcomes, but in vitro proof of this amplification and its mechanism are lacking. We previously showed that after SCI, sensitivity to opioids is reduced by fourfold to sixfold in rat sensory neurons. Here, we demonstrate that after injury, gabapentinoids restore normal sensitivity of opioid inhibition of cyclic AMP (cAMP) generation, while reducing nociceptor hyperexcitability by inhibiting voltage-gated calcium channels (VGCCs). Increasing intracellular Ca2+ or activation of L-type VGCCs (L-VGCCs) suffices to mimic SCI effects on opioid sensitivity, in a manner dependent on the activity of the Raf1 proto-oncogene, serine/threonine-protein kinase C-Raf, but independent of neuronal depolarization. Together, our results provide a mechanism for potentiation of opioid effects by gabapentinoids after injury, via reduction of calcium influx through L-VGCCs, and suggest that other inhibitors targeting these channels may similarly enhance opioid treatment of neuropathic pain.
Sujet(s)
Analgésiques morphiniques , AMP cyclique , Gabapentine , Névralgie , Transduction du signal , Traumatismes de la moelle épinière , Animaux , Névralgie/traitement médicamenteux , Névralgie/métabolisme , AMP cyclique/métabolisme , Rats , Traumatismes de la moelle épinière/traitement médicamenteux , Traumatismes de la moelle épinière/métabolisme , Analgésiques morphiniques/pharmacologie , Gabapentine/pharmacologie , Transduction du signal/effets des médicaments et des substances chimiques , Rat Sprague-Dawley , Mâle , Canaux calciques de type L/métabolisme , Calcium/métabolisme , Prégabaline/pharmacologie , Prégabaline/usage thérapeutique , Synergie des médicaments , Cellules réceptrices sensorielles/métabolisme , Cellules réceptrices sensorielles/effets des médicaments et des substances chimiquesRÉSUMÉ
Introduction: Biphasic insulin secretion is an intrinsic characteristic of the pancreatic islet and has clinical relevance due to the loss of first-phase in patients with Type 2 diabetes. As it has long been shown that first-phase insulin secretion only occurs in response to rapid changes in glucose, we tested the hypothesis that islet response to an increase in glucose is a combination of metabolism plus an osmotic effect where hypertonicity is driving first-phase insulin secretion. Methods: Experiments were performed using perifusion analysis of rat, mouse, and human islets. Insulin secretion rate (ISR) and other parameters associated with its regulation were measured in response to combinations of D-glucose and membrane-impermeable carbohydrates (L-glucose or mannitol) designed to dissect the effect of hypertonicity from that of glucose metabolism. Results: Remarkably, the appearance of first-phase responses was wholly dependent on changes in tonicity: no first-phase in NAD(P)H, cytosolic calcium, cAMP secretion rate (cAMP SR), or ISR was observed when increased D-glucose concentration was counterbalanced by decreases in membrane-impermeable carbohydrates. When D-glucose was greater than 8 mM, rapid increases in L-glucose without any change in D-glucose resulted in first-phase responses in all measured parameters that were kinetically similar to D-glucose. First-phase ISR was completely abolished by H89 (a non-specific inhibitor of protein kinases) without affecting first-phase calcium response. Defining first-phase ISR as the difference between glucose-stimulated ISR with and without a change in hypertonicity, the peak of first-phase ISR occurred after second-phase ISR had reached steady state, consistent with the well-established glucose-dependency of mechanisms that potentiate glucose-stimulated ISR. Discussion: The data collected in this study suggests a new model of glucose-stimulated biphasic ISR where first-phase ISR derives from (and after) a transitory amplification of second-phase ISR and driven by hypertonicity-induced rise in H89-inhibitable kinases likely driven by first-phase responses in cAMP, calcium, or a combination of both.
Sujet(s)
Glucose , Sécrétion d'insuline , Insuline , Animaux , Sécrétion d'insuline/effets des médicaments et des substances chimiques , Glucose/métabolisme , Rats , Humains , Insuline/métabolisme , Souris , Mâle , Ilots pancréatiques/métabolisme , Ilots pancréatiques/effets des médicaments et des substances chimiques , AMP cyclique/métabolisme , Calcium/métabolismeRÉSUMÉ
Glutamate excitotoxicity is involved in retinal ganglion cell (RGC) death in various retinal degenerative diseases, including ischemia-reperfusion injury and glaucoma. Excitotoxic RGC death is caused by both direct damage to RGCs and indirect damage through neuroinflammation of retinal glial cells. Omidenepag (OMD), a novel E prostanoid receptor 2 (EP2) agonist, is a recently approved intraocular pressure-lowering drug. The second messenger of EP2 is cyclic adenosine monophosphate (cAMP), which activates protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). In this study, we investigated the neuroprotective effects of OMD on excitotoxic RGC death by focusing on differences in cAMP downstream signaling from the perspective of glia-neuron interactions. We established a glutamate excitotoxicity model in vitro and NMDA intravitreal injection model in vivo. In vitro, rat primary RGCs were used in an RGC survival rate assay. MG5 cells (mouse microglial cell line) and A1 cells (astrocyte cell line) were used for immunocytochemistry and Western blotting to evaluate the expressions of COX-1/2, PKA, Epac1/2, pCREB, cleaved caspase-3, inflammatory cytokines, and neurotrophic factors. Mouse retinal specimens underwent hematoxylin and eosin staining, flat-mounted retina examination, and immunohistochemistry. OMD significantly suppressed excitotoxic RGC death, cleaved caspase-3 expression, and activated glia both in vitro and in vivo. Moreover, it inhibited Epac1 and inflammatory cytokine expression and promoted COX-2, pCREB, and neurotrophic factor expression. OMD may have neuroprotective effects through inhibition of the Epac pathway and promotion of the COX-2-EP2-cAMP-PKA pathway by modulating glia-neuron interaction.
Sujet(s)
Cyclic AMP-Dependent Protein Kinases , AMP cyclique , Cyclooxygenase 2 , Névroglie , Neuroprotecteurs , Cellules ganglionnaires rétiniennes , Animaux , Cellules ganglionnaires rétiniennes/effets des médicaments et des substances chimiques , Cellules ganglionnaires rétiniennes/métabolisme , Neuroprotecteurs/pharmacologie , Cyclic AMP-Dependent Protein Kinases/métabolisme , Cyclooxygenase 2/métabolisme , AMP cyclique/métabolisme , Souris , Névroglie/effets des médicaments et des substances chimiques , Névroglie/métabolisme , Sous-type EP2 des récepteurs des prostaglandines E/métabolisme , Sous-type EP2 des récepteurs des prostaglandines E/antagonistes et inhibiteurs , Sous-type EP2 des récepteurs des prostaglandines E/agonistes , Mort cellulaire/effets des médicaments et des substances chimiques , Mort cellulaire/physiologie , Transduction du signal/effets des médicaments et des substances chimiques , Transduction du signal/physiologie , Facteurs d'échange de nucléotides guanyliques/métabolisme , Rat Sprague-Dawley , Rats , Acide glutamique/métabolisme , Acide glutamique/toxicité , Souris de lignée C57BL , Mâle , N-Méthyl-aspartate/pharmacologie , N-Méthyl-aspartate/toxicité , Neurones/effets des médicaments et des substances chimiques , Neurones/métabolismeRÉSUMÉ
Inherited retinopathies are devastating diseases that in most cases lack treatment options. Disease-modifying therapies that mitigate pathophysiology regardless of the underlying genetic lesion are desirable due to the diversity of mutations found in such diseases. We tested a systems pharmacology-based strategy that suppresses intracellular cAMP and Ca2+ activity via G protein-coupled receptor (GPCR) modulation using tamsulosin, metoprolol, and bromocriptine coadministration. The treatment improves cone photoreceptor function and slows degeneration in Pde6ßrd10 and RhoP23H/WT retinitis pigmentosa mice. Cone degeneration is modestly mitigated after a 7-month-long drug infusion in PDE6A-/- dogs. The treatment also improves rod pathway function in an Rpe65-/- mouse model of Leber congenital amaurosis but does not protect from cone degeneration. RNA-sequencing analyses indicate improved metabolic function in drug-treated Rpe65-/- and rd10 mice. Our data show that catecholaminergic GPCR drug combinations that modify second messenger levels via multiple receptor actions provide a potential disease-modifying therapy against retinal degeneration.
Sujet(s)
Modèles animaux de maladie humaine , Repositionnement des médicaments , Rétinite pigmentaire , Animaux , Souris , Chiens , Rétinite pigmentaire/traitement médicamenteux , Rétinite pigmentaire/génétique , Mutation , Cyclic Nucleotide Phosphodiesterases, Type 6/génétique , Cyclic Nucleotide Phosphodiesterases, Type 6/métabolisme , Récepteurs couplés aux protéines G/génétique , Récepteurs couplés aux protéines G/métabolisme , Souris knockout , Amaurose congénitale de Leber/traitement médicamenteux , Amaurose congénitale de Leber/génétique , Bromocriptine/pharmacologie , Bromocriptine/usage thérapeutique , Cis-trans-isomerases/génétique , Cis-trans-isomerases/métabolisme , Humains , Association de médicaments , Souris de lignée C57BL , Cellules photoréceptrices en cône de la rétine/effets des médicaments et des substances chimiques , Cellules photoréceptrices en cône de la rétine/métabolisme , Cellules photoréceptrices en cône de la rétine/anatomopathologie , Femelle , AMP cyclique/métabolisme , Dégénérescence de la rétine/traitement médicamenteux , Dégénérescence de la rétine/génétique , Mâle , Calcium/métabolismeRÉSUMÉ
The kidney and brain play critical roles in the regulation of blood pressure. Neuropeptide FF (NPFF), originally isolated from the bovine brain, has been suggested to contribute to the pathogenesis of hypertension. However, the roles of NPFF and its receptors, NPFF-R1 and NPFF-R2, in the regulation of blood pressure, via the kidney, are not known. In this study, we found that the transcripts and proteins of NPFF and its receptors, NPFF-R1 and NPFF-R2, were expressed in mouse and human renal proximal tubules (RPTs). In mouse RPT cells (RPTCs), NPFF, but not RF-amide-related peptide-2 (RFRP-2), decreased the forskolin-stimulated cAMP production in a concentration- and time-dependent manner. Furthermore, dopamine D1-like receptors colocalized and co-immunoprecipitated with NPFF-R1 and NPFF-R2 in human RPTCs. The increase in cAMP production in human RPTCs caused by fenoldopam, a D1-like receptor agonist, was attenuated by NPFF, indicating an antagonistic interaction between NPFF and D1-like receptors. The renal subcapsular infusion of NPFF in C57BL/6 mice decreased renal sodium excretion and increased blood pressure. The NPFF-mediated increase in blood pressure was prevented by RF-9, an antagonist of NPFF receptors. Taken together, our findings suggest that autocrine NPFF and its receptors in the kidney regulate blood pressure, but the mechanisms remain to be determined.
Sujet(s)
Communication autocrine , Pression sanguine , AMP cyclique , Oligopeptides , Transduction du signal , Animaux , Humains , Souris , AMP cyclique/métabolisme , Oligopeptides/pharmacologie , Oligopeptides/métabolisme , Récepteur aux neuropeptides/métabolisme , Tubules contournés proximaux/métabolisme , Mâle , Rein/métabolisme , Souris de lignée C57BL , Récepteur dopamine D1/métabolismeRÉSUMÉ
The epithelial to mesenchymal transition (EMT) is a multistep process involving structural and functional alterations that are required for cancer metastasis, as well as loss of epithelial markers (e.g., E-cadherin/CDH1) and gain of mesenchymal markers (e.g., N-cadherin/CDH2, vimentin/VIM). Pathological events modify cell-cell interactions, cell-matrix adhesion and extra cellular matrix integrity leading to cell migration, evasion from the primary tumor and augmented invasiveness in the metastatic niche. This transformation is modulated by multiple paracrine factors (e.g., chemokines, growth factor), as well as SLIT2-ROBO1 signaling that collectively regulate expression of RHO GTPases (e.g., RHOA) and EMT marker genes. Yet, the roles of SLIT proteins in cancer remain enigmatic. In some cancer types, SLIT2 is anti-tumorigenic, while in other cancers it contributes towards the metastatic phenotype. Here we investigated the ambivalent metastatic activity of SLIT2 by analyzing how cAMP/RHOA signal transduction modulates SLIT-ROBO controlled metastatic parameters in response to the phosphodiesterase inhibitor IBMX (3-isobutyl-1-methylxanthine) and paracrine factors (TGF-ß/TGFß1 and FGF2). Upon SLIT2 administration cell migration and proliferation increases in colon cancer cells and decreases in cervical cancer cells, while altering cell morphology and proliferation in both cancer types. These effects are reinforced by TGF-ß/TGFß1 and FGF2, but attenuated by elevation of cAMP with IBMX, depending on the cancer cell type. Our data indicate that SLIT2 represents a potential biomarker for cancer diagnosis, prognosis, and therapy.
Sujet(s)
Transition épithélio-mésenchymateuse , Facteur de croissance fibroblastique de type 2 , Protéines et peptides de signalisation intercellulaire , Métastase tumorale , Protéines de tissu nerveux , , Transduction du signal , Facteur de croissance transformant bêta-1 , Protéine G RhoA , Humains , Protéines de tissu nerveux/métabolisme , Protéines de tissu nerveux/génétique , Protéine G RhoA/métabolisme , Protéines et peptides de signalisation intercellulaire/métabolisme , Protéines et peptides de signalisation intercellulaire/génétique , Facteur de croissance transformant bêta-1/métabolisme , Facteur de croissance fibroblastique de type 2/métabolisme , AMP cyclique/métabolisme , Tumeurs/métabolisme , Tumeurs/anatomopathologie , Tumeurs/génétique , Mouvement cellulaire , Régulation de l'expression des gènes tumorauxRÉSUMÉ
The cannabinoid receptor type 1 (CB1R) is pivotal within the endocannabinoid system regulating various signaling cascades with effects in appetite regulation, pain perception, memory formation, and thermoregulation. Still, understanding of CB1R's cellular signaling, distribution, and expression dynamics is very fragmentary. Real-time visualization of CB1R is crucial for addressing these questions. Selective drug-like CB1R ligands with a defined pharmacological profile were investigated for the construction of CB1R fluorescent probes using a reverse design-approach. A modular design concept with a diethyl glycine-based building block as the centerpiece allowed for the straightforward synthesis of novel probe candidates. Validated by computational docking studies, radioligand binding, and cAMP assay, this systematic approach allowed for the identification of novel pyrrole-based CB1R fluorescent probes. Application in fluorescence-based target-engagement studies and live cell imaging exemplify the great versatility of the tailored CB1R probes for investigating CB1R localization, trafficking, pharmacology, and its pathological implications.
Sujet(s)
Colorants fluorescents , Récepteur cannabinoïde de type CB1 , Colorants fluorescents/composition chimique , Colorants fluorescents/synthèse chimique , Récepteur cannabinoïde de type CB1/métabolisme , Humains , Simulation de docking moléculaire , Cellules HEK293 , Ligands , Pyrroles/composition chimique , Pyrroles/pharmacologie , Pyrroles/synthèse chimique , Relation structure-activité , AMP cyclique/métabolismeRÉSUMÉ
Most α2-AR agonists derived from dexmedetomidine have few structural differences between them and have no selectivity for α2A/2B-AR or Gi/Gs, which can lead to side effects in drugs. To obtain novel and potent α2A-AR agonists, we performed virtual screening for human α2A-AR and α2B-AR to find α2A-AR agonists with higher selectivity. Compound P300-2342 and its three analogs significantly decreased the locomotor activity of mice (p < 0.05). Furthermore, P300-2342 and its three analogs inhibited the binding of [3H] Rauwolscine with IC50 values of 7.72 ± 0.76 and 12.23 ± 0.11 µM, respectively, to α2A-AR and α2B-AR. In α2A-AR-HEK293 cells, P300-2342 decreased forskolin-stimulated cAMP production without increasing cAMP production, which indicated that P300-2342 activated α2A-AR with coupling to the Gαi/o pathway but without Gαs coupling. P300-2342 exhibited no agonist but slight antagonist activities in α2B-AR. Similar results were obtained for the analogs of P300-2342. The docking results showed that P300-2342 formed π-hydrogen bonds with Y394, V114 in α2A-AR, and V93 in α2B-AR. Three analogs of P300-2342 formed several π-hydrogen bonds with V114, Y196, F390 in α2A-AR, and V93 in α2B-AR. We believe that these molecules can serve as leads for the further optimization of α2A-AR agonists with potentially few side effects.
Sujet(s)
Agonistes des récepteurs alpha-2 adrénergiques , Simulation de docking moléculaire , Récepteurs alpha-2 adrénergiques , Humains , Animaux , Cellules HEK293 , Récepteurs alpha-2 adrénergiques/métabolisme , Agonistes des récepteurs alpha-2 adrénergiques/pharmacologie , Souris , Sous-unités alpha Gi-Go des protéines G/métabolisme , AMP cyclique/métabolisme , Mâle , Liaison aux protéinesRÉSUMÉ
Brain computation performed by billions of nerve cells relies on a sufficient and uninterrupted nutrient and oxygen supply1,2. Astrocytes, the ubiquitous glial neighbours of neurons, govern brain glucose uptake and metabolism3,4, but the exact mechanisms of metabolic coupling between neurons and astrocytes that ensure on-demand support of neuronal energy needs are not fully understood5,6. Here we show, using experimental in vitro and in vivo animal models, that neuronal activity-dependent metabolic activation of astrocytes is mediated by neuromodulator adenosine acting on astrocytic A2B receptors. Stimulation of A2B receptors recruits the canonical cyclic adenosine 3',5'-monophosphate-protein kinase A signalling pathway, leading to rapid activation of astrocyte glucose metabolism and the release of lactate, which supplements the extracellular pool of readily available energy substrates. Experimental mouse models involving conditional deletion of the gene encoding A2B receptors in astrocytes showed that adenosine-mediated metabolic signalling is essential for maintaining synaptic function, especially under conditions of high energy demand or reduced energy supply. Knockdown of A2B receptor expression in astrocytes led to a major reprogramming of brain energy metabolism, prevented synaptic plasticity in the hippocampus, severely impaired recognition memory and disrupted sleep. These data identify the adenosine A2B receptor as an astrocytic sensor of neuronal activity and show that cAMP signalling in astrocytes tunes brain energy metabolism to support its fundamental functions such as sleep and memory.
Sujet(s)
Adénosine , Astrocytes , Encéphale , Métabolisme énergétique , Neurones , Transduction du signal , Animaux , Femelle , Mâle , Souris , Rats , Adénosine/métabolisme , Astrocytes/métabolisme , Encéphale/métabolisme , Encéphale/cytologie , AMP cyclique/métabolisme , Cyclic AMP-Dependent Protein Kinases/métabolisme , Glucose/métabolisme , Hippocampe/métabolisme , Hippocampe/cytologie , Acide lactique/métabolisme , Souris de lignée C57BL , Plasticité neuronale , Neurones/métabolisme , Récepteur A2B à l'adénosine/déficit , Récepteur A2B à l'adénosine/effets des médicaments et des substances chimiques , Récepteur A2B à l'adénosine/génétique , Récepteur A2B à l'adénosine/métabolisme , /physiologie , Sommeil/génétique , Sommeil/physiologie , Synapses/métabolismeRÉSUMÉ
Prostaglandin E2 (PGE2) is a major contributor to inflammatory pain hyperalgesia, however, the extent to which it modulates the activity of nociceptive axons is incompletely understood. We developed and characterized a microfluidic cell culture model to investigate sensitisation of the axons of dorsal root ganglia neurons. We show that application of PGE2 to fluidically isolated axons leads to sensitisation of their responses to depolarising stimuli. Interestingly the application of PGE2 to the DRG axons elicited a direct and persistent spiking activity propagated to the soma. Both the persistent activity and the membrane depolarisation in the axons are abolished by the EP4 receptor inhibitor and a blocker of cAMP synthesis. Further investigated into the mechanisms of the spiking activity showed that the PGE2 evoked depolarisation was inhibited by Nav1.8 sodium channel blockers but was refractory to the application of TTX or zatebradine. Interestingly, the depolarisation of axons was blocked by blocking ANO1 channels with T16Ainh-A01. We further show that PGE2-elicited axonal responses are altered by the changes in chloride gradient within the axons following treatment with bumetanide a Na-K-2Cl cotransporter NKCC1 inhibitor, but not by VU01240551 an inhibitor of potassium-chloride transporter KCC2. Our data demonstrate a novel role for PGE2/EP4/cAMP pathway which culminates in a sustained depolarisation of sensory axons mediated by a chloride current through ANO1 channels. Therefore, using a microfluidic culture model, we provide evidence for a potential dual function of PGE2 in inflammatory pain: it sensitises depolarisation-evoked responses in nociceptive axons and directly triggers action potentials by activating ANO1 and Nav1.8 channels.
Sujet(s)
Anoctamine-1 , Axones , Dinoprostone , Ganglions sensitifs des nerfs spinaux , Canal sodique voltage-dépendant NAV1.8 , Canal sodique voltage-dépendant NAV1.8/métabolisme , Animaux , Dinoprostone/pharmacologie , Dinoprostone/métabolisme , Axones/métabolisme , Axones/effets des médicaments et des substances chimiques , Axones/physiologie , Ganglions sensitifs des nerfs spinaux/métabolisme , Ganglions sensitifs des nerfs spinaux/effets des médicaments et des substances chimiques , Rats , Anoctamine-1/métabolisme , Cellules réceptrices sensorielles/métabolisme , Cellules réceptrices sensorielles/effets des médicaments et des substances chimiques , Potentiels d'action/effets des médicaments et des substances chimiques , Sous-type EP4 des récepteurs des prostaglandines E/métabolisme , Rat Sprague-Dawley , Cellules cultivées , Membre-2 de la famille-12 des transporteurs de solutés/métabolisme , AMP cyclique/métabolismeRÉSUMÉ
Heterologous sensitization of adenylyl cyclase (AC) results in elevated cAMP signaling transduction that contributes to drug dependence. Inhibiting cullin3-RING ligases by blocking the neddylation of cullin3 abolishes heterologous sensitization, however, the modulating mechanism remains uncharted. Here, we report an essential role of the potassium channel tetramerization domain (KCTD) protein 2, 5, and 17, especially the dominant isoform KCTD5 in regulating heterologous sensitization of AC1 and morphine dependence via working with cullin3 and the cullin-associated and neddylation-dissociated 1 (CAND1) protein. In cellular models, we observed enhanced association of KCTD5 with Gß and cullin3, along with elevated dissociation of Gß from AC1 as well as of CAND1 from cullin3 in heterologous sensitization of AC1. Given binding of CAND1 inhibits the neddylation of cullin3, we further elucidated that the enhanced interaction of KCTD5 with both Gß and cullin3 promoted the dissociation of CAND1 from cullin3, attenuated the inhibitory effect of CAND1 on cullin3 neddylation, ultimately resulted in heterologous sensitization of AC1. The paraventricular thalamic nucleus (PVT) plays an important role in mediating morphine dependence. Through pharmacological and biochemical approaches, we then demonstrated that KCTD5/cullin3 regulates morphine dependence via modulating heterologous sensitization of AC, likely AC1 in PVT in mice. In summary, the present study revealed the underlying mechanism of heterologous sensitization of AC1 mediated by cullin3 and discovered the role of KCTD proteins in regulating morphine dependence in mice.
Sujet(s)
Adenylate Cyclase , Cullines , Dépendance à la morphine , Animaux , Adenylate Cyclase/métabolisme , Adenylate Cyclase/génétique , Cullines/métabolisme , Souris , Dépendance à la morphine/métabolisme , Cellules HEK293 , Humains , Canaux potassiques/métabolisme , Canaux potassiques/génétique , Souris de lignée C57BL , Mâle , Sous-unités bêta des protéines G/métabolisme , Sous-unités bêta des protéines G/génétique , Morphine/pharmacologie , Souris knockout , Transduction du signal , AMP cyclique/métabolismeRÉSUMÉ
Neurotransmitters are key modulators in neuro-immune circuits and have been linked to tumor progression. Medullary thyroid cancer (MTC), an aggressive neuroendocrine tumor, expresses neurotransmitter calcitonin gene-related peptide (CGRP), is insensitive to chemo- and radiotherapies, and the effectiveness of immunotherapies remains unknown. Thus, a comprehensive analysis of the tumor microenvironment would facilitate effective therapies and provide evidence on CGRP's function outside the nervous system. Here, we compare the single-cell landscape of MTC and papillary thyroid cancer (PTC) and find that expression of CGRP in MTC is associated with dendritic cell (DC) abnormal development characterized by activation of cAMP related pathways and high levels of Kruppel Like Factor 2 (KLF2), correlated with an impaired activity of tumor infiltrating T cells. A CGRP receptor antagonist could offset CGRP detrimental impact on DC development in vitro. Our study provides insights of the MTC immunosuppressive microenvironment, and proposes CGRP receptor as a potential therapeutic target.
Sujet(s)
Peptide relié au gène de la calcitonine , Carcinome neuroendocrine , Cellules dendritiques , Tumeurs de la thyroïde , Microenvironnement tumoral , Microenvironnement tumoral/immunologie , Humains , Tumeurs de la thyroïde/génétique , Tumeurs de la thyroïde/métabolisme , Tumeurs de la thyroïde/immunologie , Tumeurs de la thyroïde/anatomopathologie , Peptide relié au gène de la calcitonine/métabolisme , Carcinome neuroendocrine/génétique , Carcinome neuroendocrine/métabolisme , Carcinome neuroendocrine/anatomopathologie , Carcinome neuroendocrine/immunologie , Cellules dendritiques/immunologie , Cellules dendritiques/métabolisme , Cancer papillaire de la thyroïde/métabolisme , Cancer papillaire de la thyroïde/immunologie , Cancer papillaire de la thyroïde/génétique , Cancer papillaire de la thyroïde/anatomopathologie , Récepteurs du peptide relié au gène de la calcitonine/métabolisme , AMP cyclique/métabolisme , Lymphocytes TIL/immunologie , Lymphocytes TIL/métabolisme , Agents neuromédiateurs/métabolisme , Régulation de l'expression des gènes tumoraux , Lignée cellulaire tumorale , Antagonistes du récepteur du peptide relié au gène de la calcitonine/pharmacologie , Analyse sur cellule uniqueRÉSUMÉ
Cell-to-cell communication is fundamental to the organization and functionality of multicellular organisms. Intercellular signals orchestrate a variety of cellular responses, including gene expression and protein function changes, and contribute to the integrated functions of individual tissues. Dictyostelium discoideum is a model organism for cell-to-cell interactions mediated by chemical signals and multicellular formation mechanisms. Upon starvation, D. discoideum cells exhibit coordinated cell aggregation via cyclic adenosine 3',5'-monophosphate (cAMP) gradients and chemotaxis, which facilitates the unicellular-to-multicellular transition. During this process, the calcium signaling synchronizes with the cAMP signaling. The resulting multicellular body exhibits organized collective migration and ultimately forms a fruiting body. Various signaling molecules, such as ion signals, regulate the spatiotemporal differentiation patterns within multicellular bodies. Understanding cell-to-cell and ion signaling in Dictyostelium provides insight into general multicellular formation and differentiation processes. Exploring cell-to-cell and ion signaling enhances our understanding of the fundamental biological processes related to cell communication, coordination, and differentiation, with wide-ranging implications for developmental biology, evolutionary biology, biomedical research, and synthetic biology. In this review, I discuss the role of ion signaling in cell motility and development in D. discoideum.
Sujet(s)
Mouvement cellulaire , AMP cyclique , Dictyostelium , Transduction du signal , Dictyostelium/métabolisme , Dictyostelium/croissance et développement , Dictyostelium/génétique , Dictyostelium/cytologie , AMP cyclique/métabolisme , Chimiotaxie , Communication cellulaire , Ions/métabolisme , Différenciation cellulaire , Signalisation calciqueRÉSUMÉ
The melanocortin-4 receptor (MC4R) is a key player in the hypothalamic leptin-melanocortin pathway that regulates satiety and hunger. MC4R belongs to the G protein-coupled receptors (GPCRs), which are known to form heterodimers with other membrane proteins, potentially modulating receptor function or characteristics. Like MC4R, thyroid hormones (TH) are also essential for energy homeostasis control. TH transport across membranes is facilitated by the monocarboxylate transporter 8 (MCT8), which is also known to form heterodimers with GPCRs. Based on the finding in single-cell RNA-sequencing data that both proteins are simultaneously expressed in hypothalamic neurons, we investigated a putative interplay between MC4R and MCT8. We developed a novel staining protocol utilizing a fluorophore-labeled MC4R ligand and demonstrated a co-localization of MC4R and MCT8 in human brain tissue. Using in vitro assays such as BRET, IP1, and cAMP determination, we found that MCT8 modulates MC4R-mediated phospholipase C activation but not cAMP formation via a direct interaction, an effect that does not require a functional MCT8 as it was not altered by a specific MCT8 inhibitor. This suggests an extended functional spectrum of MCT8 as a GPCR signaling modulator and argues for the investigation of further GPCR-protein interactions with hitherto underrepresented physiological functions.
Sujet(s)
Transporteurs d'acides monocarboxyliques , Récepteur de la mélanocortine de type 4 , Type C Phospholipases , Humains , Récepteur de la mélanocortine de type 4/métabolisme , Récepteur de la mélanocortine de type 4/génétique , Transporteurs d'acides monocarboxyliques/métabolisme , Transporteurs d'acides monocarboxyliques/génétique , Type C Phospholipases/métabolisme , Cellules HEK293 , Transduction du signal , AMP cyclique/métabolisme , Symporteurs/métabolisme , Symporteurs/génétique , Liaison aux protéines , AnimauxRÉSUMÉ
Protein SUMOylation is a prevalent stress-response posttranslational modification crucial for maintaining cellular homeostasis. Herein, we report that protein SUMOylation modulates cellular signaling mediated by cAMP, an ancient and universal stress-response second messenger. We identify K561 as a primary SUMOylation site in exchange protein directly activated by cAMP (EPAC1) via site-specific mapping of SUMOylation using mass spectrometry. Sequence and site-directed mutagenesis analyses reveal that a functional SUMO-interacting motif in EPAC1 is required for the binding of SUMO-conjugating enzyme UBC9, formation of EPAC1 nuclear condensate, and EPAC1 cellular SUMOylation. Heat shock-induced SUMO modification of EPAC1 promotes Rap1/2 activation in a cAMP-independent manner. Structural modeling and molecular dynamics simulation studies demonstrate that SUMO substituent on K561 of EPAC1 promotes Rap1 interaction by increasing the buried surface area between the SUMOylated receptor and its effector. Our studies identify a functional SUMOylation site in EPAC1 and unveil a novel mechanism in which SUMOylation of EPAC1 leads to its autonomous activation. The findings of SUMOylation-mediated activation of EPAC1 not only provide new insights into our understanding of cellular regulation of EPAC1 but also will open up a new field of experimentation concerning the cross-talk between cAMP/EPAC1 signaling and protein SUMOylation, two major cellular stress response pathways, during cellular homeostasis.