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1.
Int J Mol Sci ; 25(17)2024 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-39273390

RESUMO

Chronic kidney disease (CKD) is characterized by a steady decline in kidney function and affects roughly 10% of the world's population. This review focuses on the critical function of cyclic adenosine monophosphate (cAMP) signaling in CKD, specifically how it influences both protective and pathogenic processes in the kidney. cAMP, a critical secondary messenger, controls a variety of cellular functions, including transcription, metabolism, mitochondrial homeostasis, cell proliferation, and apoptosis. Its compartmentalization inside cellular microdomains ensures accurate signaling. In kidney physiology, cAMP is required for hormone-regulated activities, particularly in the collecting duct, where it promotes water reabsorption through vasopressin signaling. Several illnesses, including Fabry disease, renal cell carcinoma, nephrogenic diabetes insipidus, Bartter syndrome, Liddle syndrome, diabetic nephropathy, autosomal dominant polycystic kidney disease, and renal tubular acidosis, have been linked to dysfunction in the cAMP system. Both cAMP analogs and phosphodiesterase inhibitors have the potential to improve kidney function and reduce kidney damage. Future research should focus on developing targeted PDE inhibitors for the treatment of CKD.


Assuntos
AMP Cíclico , Insuficiência Renal Crônica , Transdução de Sinais , Humanos , AMP Cíclico/metabolismo , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/patologia , Animais , Transdução de Sinais/efeitos dos fármacos , Terapia de Alvo Molecular , Rim/metabolismo , Rim/patologia , Inibidores de Fosfodiesterase/uso terapêutico , Inibidores de Fosfodiesterase/farmacologia
2.
Elife ; 132024 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-39255004

RESUMO

In birds and insects, the female uptakes sperm for a specific duration post-copulation known as the ejaculate holding period (EHP) before expelling unused sperm and the mating plug through sperm ejection. In this study, we found that Drosophila melanogaster females shortens the EHP when incubated with males or mated females shortly after the first mating. This phenomenon, which we termed male-induced EHP shortening (MIES), requires Or47b+ olfactory and ppk23+ gustatory neurons, activated by 2-methyltetracosane and 7-tricosene, respectively. These odorants raise cAMP levels in pC1 neurons, responsible for processing male courtship cues and regulating female mating receptivity. Elevated cAMP levels in pC1 neurons reduce EHP and reinstate their responsiveness to male courtship cues, promoting re-mating with faster sperm ejection. This study established MIES as a genetically tractable model of sexual plasticity with a conserved neural mechanism.


Assuntos
Drosophila melanogaster , Feromônios , Comportamento Sexual Animal , Animais , Feminino , Masculino , Drosophila melanogaster/fisiologia , Comportamento Sexual Animal/fisiologia , Feromônios/metabolismo , Neurônios/fisiologia , Neurônios/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , AMP Cíclico/metabolismo
3.
PLoS One ; 19(9): e0305312, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39259753

RESUMO

The primate-specific chemokine CCL18 is a potent chemoattractant for T cells and is expressed at elevated levels in several inflammatory diseases. However, the cognate receptor for CCL18 remains unconfirmed. Here, we describe attempts to validate a previous report that the chemokine receptor CCR8 is the human CCL18 receptor (Islam et al. J Exp Med. 2013, 210:1889-98). Two mouse pre-B cell lines (4DE4 and L1.2) exogenously expressing CCR8 exhibited robust migration in response to the known CCR8 ligand CCL1 but not to CCL18. Similarly, CCL1 but not CCL18 induced internalization of CCR8 on 4DE4 cells. CCR8 expressed on Chinese hamster ovarian (CHO) cells mediated robust G protein activation, inhibition of cAMP synthesis and ß-arrestin2 recruitment in response to CCL1 but not CCL18. Several N- and C-terminal variants of CCL18 also failed to stimulate CCR8 activation. On the other hand, and as previously reported, CCL18 inhibited CCL11-stimulated migration of 4DE4 cells expressing the receptor CCR3. These data suggest that CCR8, at least in the absence of unidentified cofactors, does not function as a high affinity receptor for CCL18.


Assuntos
Quimiocinas CC , Cricetulus , Receptores CCR8 , Animais , Receptores CCR8/metabolismo , Receptores CCR8/genética , Humanos , Células CHO , Camundongos , Quimiocinas CC/metabolismo , Quimiocinas CC/genética , Cricetinae , Quimiocina CCL1/metabolismo , Movimento Celular , Linhagem Celular , Quimiocina CCL11/metabolismo , AMP Cíclico/metabolismo
4.
Nat Commun ; 15(1): 7822, 2024 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-39242606

RESUMO

G protein-coupled receptors' conformational landscape can be affected by their local, microscopic interactions within the cell plasma membrane. We employ here a pleiotropic stimulus, namely osmotic swelling, to alter the cortical environment within intact cells and monitor the response in terms of receptor function and downstream signaling. We observe that in osmotically swollen cells the ß2-adrenergic receptor, a prototypical GPCR, favors an active conformation, resulting in cAMP transient responses to adrenergic stimulation that have increased amplitude. The results are validated in primary cell types such as adult cardiomyocytes, a model system where swelling occurs upon ischemia-reperfusion injury. Our results suggest that receptors' function is finely modulated by their biophysical context, and specifically that osmotic swelling acts as a potentiator of downstream signaling, not only for the ß2-adrenergic receptor, but also for other receptors, hinting at a more general regulatory mechanism.


Assuntos
AMP Cíclico , Miócitos Cardíacos , Receptores Adrenérgicos beta 2 , Transdução de Sinais , Receptores Adrenérgicos beta 2/metabolismo , Miócitos Cardíacos/metabolismo , Humanos , Animais , Ligantes , AMP Cíclico/metabolismo , Membrana Celular/metabolismo , Células HEK293 , Camundongos
5.
Mol Plant Pathol ; 25(9): e70003, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39235122

RESUMO

Sugarcane smut fungus Sporisorium scitamineum produces polyamines putrescine (PUT), spermidine (SPD), and spermine (SPM) to regulate sexual mating/filamentous growth critical for pathogenicity. Besides de novo biosynthesis, intracellular levels of polyamines could also be modulated by oxidation. In this study, we identified two annotated polyamine oxidation enzymes (SsPAO and SsCuAO1) in S. scitamineum. Compared to the wild type (MAT-1), the ss1paoΔ and ss1cuao1Δ mutants were defective in sporidia growth, sexual mating/filamentation, and pathogenicity. The addition of a low concentration of cAMP (0.1 mM) could partially or fully restore filamentation of ss1paoΔ × ss2paoΔ or ss1cuao1Δ × ss2cuao1Δ. cAMP biosynthesis and hydrolysis genes were differentially expressed in the ss1paoΔ × ss2paoΔ or ss1cuao1Δ × ss2cuao1Δ cultures, further supporting that SsPAO- or SsCuAO1-based polyamine homeostasis regulates S. scitamineum filamentation by affecting the cAMP/PKA signalling pathway. During early infection, PUT promotes, while SPD inhibits, the accumulation of reactive oxygen species (ROS) in sugarcane, therefore modulating redox homeostasis at the smut fungus-sugarcane interface. Autophagy induction was found to be enhanced in the ss1paoΔ mutant and reduced in the ss1cuao1Δ mutant. Exogenous addition of cAMP, PUT, SPD, or SPM at low concentration promoted autophagy activity under a non-inductive condition (rich medium), suggesting a cross-talk between polyamines and cAMP signalling in regulating autophagy in S. scitamineum. Overall, our work proves that SsPAO- and SsCuAO1-mediated intracellular polyamines affect intracellular redox balance and thus play a role in growth, sexual mating/filamentation, and pathogenicity of S. scitamineum.


Assuntos
Oxirredução , Poliaminas , Poliaminas/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , AMP Cíclico/metabolismo , Saccharum/microbiologia , Regulação Fúngica da Expressão Gênica , Ustilaginales/patogenicidade , Autofagia
6.
Nat Commun ; 15(1): 7684, 2024 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-39227390

RESUMO

A long-held tenet in inositol-lipid signaling is that cleavage of membrane phosphoinositides by phospholipase Cß (PLCß) isozymes to increase cytosolic Ca2+ in living cells is exclusive to Gq- and Gi-sensitive G protein-coupled receptors (GPCRs). Here we extend this central tenet and show that Gs-GPCRs also partake in inositol-lipid signaling and thereby increase cytosolic Ca2+. By combining CRISPR/Cas9 genome editing to delete Gαs, the adenylyl cyclase isoforms 3 and 6, or the PLCß1-4 isozymes, with pharmacological and genetic inhibition of Gq and G11, we pin down Gs-derived Gßγ as driver of a PLCß2/3-mediated cytosolic Ca2+ release module. This module does not require but crosstalks with Gαs-dependent cAMP, demands Gαq to release PLCß3 autoinhibition, but becomes Gq-independent with mutational disruption of the PLCß3 autoinhibited state. Our findings uncover the key steps of a previously unappreciated mechanism utilized by mammalian cells to finetune their calcium signaling regulation through Gs-GPCRs.


Assuntos
Sinalização do Cálcio , Cálcio , Fosfolipase C beta , Receptores Acoplados a Proteínas G , Humanos , Fosfolipase C beta/metabolismo , Fosfolipase C beta/genética , Células HEK293 , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Cálcio/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Sistemas CRISPR-Cas , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , AMP Cíclico/metabolismo , Animais , Edição de Genes , Citosol/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/genética , Adenilil Ciclases/metabolismo , Adenilil Ciclases/genética
7.
Circ Res ; 135(7): 722-738, 2024 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-39166328

RESUMO

BACKGROUND: The KCNQ1+KCNE1 (IKs) potassium channel plays a crucial role in cardiac adaptation to stress, in which ß-adrenergic stimulation phosphorylates the IKs channel through the cyclic adenosine monophosphate (cAMP)/PKA (protein kinase A) pathway. Phosphorylation increases the channel current and accelerates repolarization to adapt to an increased heart rate. Variants in KCNQ1 can cause long-QT syndrome type 1 (LQT1), and those with defective cAMP effects predispose patients to the highest risk of cardiac arrest and sudden death. However, the molecular connection between IKs channel phosphorylation and channel function, as well as why high-risk LQT1 mutations lose cAMP sensitivity, remain unclear. METHODS: Regular patch clamp and voltage clamp fluorometry techniques were utilized to record pore opening and voltage sensor movement of wild-type and mutant KCNQ1/IKs channels. The clinical phenotypic penetrance of each LQT1 mutation was analyzed as a metric for assessing their clinical risk. The patient-specific-induced pluripotent stem-cell model was used to test mechanistic findings in physiological conditions. RESULTS: By systematically elucidating mechanisms of a series of LQT1 variants that lack cAMP sensitivity, we identified molecular determinants of IKs channel regulation by phosphorylation. These key residues are distributed across the N-terminus of KCNQ1 extending to the central pore region of IKs. We refer to this pattern as the IKs channel PKA phosphorylation axis. Next, by examining LQT1 variants from clinical databases containing 10 579 LQT1 carriers, we found that the distribution of the most high-penetrance LQT1 variants extends across the IKs channel PKA phosphorylation axis, demonstrating its clinical relevance. Furthermore, we found that a small molecule, ML277, which binds at the center of the phosphorylation axis, rescues the defective cAMP effects of multiple high-risk LQT1 variants. This finding was then tested in high-risk patient-specific induced pluripotent stem cell-derived cardiomyocytes, where ML277 remarkably alleviates the beating abnormalities. CONCLUSIONS: Our findings not only elucidate the molecular mechanism of PKA-dependent IKs channel phosphorylation but also provide an effective antiarrhythmic strategy for patients with high-risk LQT1 variants.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico , Células-Tronco Pluripotentes Induzidas , Canal de Potássio KCNQ1 , Humanos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fosforilação , Canal de Potássio KCNQ1/genética , Canal de Potássio KCNQ1/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Síndrome de Romano-Ward/genética , Síndrome de Romano-Ward/metabolismo , AMP Cíclico/metabolismo , Miócitos Cardíacos/metabolismo , Mutação , Síndrome do QT Longo/genética , Síndrome do QT Longo/metabolismo , Células HEK293 , Canais de Potássio de Abertura Dependente da Tensão da Membrana
8.
PLoS Genet ; 20(8): e1011388, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39186815

RESUMO

Most neurons are not replaced after injury and thus possess robust intrinsic mechanisms for repair after damage. Axon injury triggers a calcium wave, and calcium and cAMP can augment axon regeneration. In comparison to axon regeneration, dendrite regeneration is poorly understood. To test whether calcium and cAMP might also be involved in dendrite injury signaling, we tracked the responses of Drosophila dendritic arborization neurons to laser severing of axons and dendrites. We found that calcium and subsequently cAMP accumulate in the cell body after both dendrite and axon injury. Two voltage-gated calcium channels (VGCCs), L-Type and T-Type, are required for the calcium influx in response to dendrite injury and play a role in rapid initiation of dendrite regeneration. The AC8 family adenylyl cyclase, Ac78C, is required for cAMP production after dendrite injury and timely initiation of regeneration. Injury-induced cAMP production is sensitive to VGCC reduction, placing calcium upstream of cAMP generation. We propose that two VGCCs initiate global calcium influx in response to dendrite injury followed by production of cAMP by Ac78C. This signaling pathway promotes timely initiation of dendrite regrowth several hours after dendrite damage.


Assuntos
Adenilil Ciclases , Canais de Cálcio Tipo L , Cálcio , AMP Cíclico , Dendritos , Animais , Adenilil Ciclases/metabolismo , Adenilil Ciclases/genética , Axônios/metabolismo , Axônios/fisiologia , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo T/metabolismo , Canais de Cálcio Tipo T/genética , Sinalização do Cálcio/genética , AMP Cíclico/metabolismo , Dendritos/metabolismo , Drosophila/genética , Drosophila melanogaster/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Regeneração Nervosa/fisiologia , Regeneração Nervosa/genética , Neurônios/metabolismo , Regeneração/genética , Regeneração/fisiologia , Transdução de Sinais
9.
Bull Exp Biol Med ; 177(2): 212-216, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-39093471

RESUMO

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.


Assuntos
AMP Cíclico , GMP Cíclico , GMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Animais , Ferro/metabolismo , Ferro/química , Adenilil Ciclases/metabolismo , Doadores de Óxido Nítrico/farmacologia , Doadores de Óxido Nítrico/química , Guanilil Ciclase Solúvel/metabolismo , Óxidos de Nitrogênio/farmacologia , Óxidos de Nitrogênio/metabolismo , Óxidos de Nitrogênio/química , Ratos
10.
Int J Mol Sci ; 25(15)2024 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-39126032

RESUMO

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.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Doenças Inflamatórias Intestinais , MicroRNAs , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Doenças Inflamatórias Intestinais/tratamento farmacológico , AMP Cíclico/metabolismo , Inflamação/genética , Inflamação/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Citocinas/metabolismo , Masculino , Transdução de Sinais , Feminino
11.
Int J Mol Sci ; 25(15)2024 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-39125708

RESUMO

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.


Assuntos
Trifosfato de Adenosina , Cílios , AMP Cíclico , Traqueia , Animais , Cílios/efeitos dos fármacos , Cílios/metabolismo , Trifosfato de Adenosina/metabolismo , Concentração de Íons de Hidrogênio , Traqueia/metabolismo , Traqueia/efeitos dos fármacos , Camundongos , AMP Cíclico/metabolismo , Masculino
12.
Turk J Gastroenterol ; 35(6): 453-464, 2024 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-39114901

RESUMO

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.


Assuntos
Gânglios Espinais , Mucosa Gástrica , Isoquinolinas , Morfina , Ratos Sprague-Dawley , Restrição Física , Canal de Cátion TRPA1 , Regulação para Cima , Animais , Morfina/farmacologia , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/metabolismo , Regulação para Cima/efeitos dos fármacos , Canal de Cátion TRPA1/metabolismo , Masculino , Gânglios Espinais/metabolismo , Gânglios Espinais/efeitos dos fármacos , Restrição Física/efeitos adversos , Ratos , Isoquinolinas/farmacologia , Acetanilidas/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Purinas/farmacologia , Estresse Psicológico/complicações , Imersão , Receptores Opioides mu/metabolismo , AMP Cíclico/metabolismo , Sulfonamidas
13.
Methods Mol Biol ; 2828: 37-43, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39147968

RESUMO

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.


Assuntos
Movimento Celular , Quimiotaxia , Dictyostelium , Dictyostelium/genética , Dictyostelium/fisiologia , Dictyostelium/citologia , Quimiotaxia/genética , Movimento Celular/genética , Mutação , AMP Cíclico/metabolismo , Polaridade Celular/genética , Adesão Celular , Modelos Biológicos
14.
Phys Rev Lett ; 133(6): 068401, 2024 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-39178438

RESUMO

When cells of the social amoeba Dictyostelium discoideum are starved of nutrients they start to synthesize and secrete the chemical messenger and chemoattractant cyclic adenosine monophosphate (cAMP). This signal is relayed by other cells, resulting in the establishment of periodic waves. The cells aggregate through chemotaxis toward the center of these waves. We investigated the chemotactic response of individual cells to repeated exposure to waves of cAMP generated by a microfluidic device. For fast-moving waves (short period), the chemotactic ability of the cells was found to increase upon exposure to more waves, suggesting the development of a memory over several cycles. This effect was not significant for slow-moving waves (large period). We show that the experimental results are consistent with a local excitation global inhibition-based model, extended by including a component that rises and decays slowly and that is activated by the temporal gradient of cAMP concentration. The observed enhancement in chemotaxis is relevant to populations in the wild: once sustained, periodic waves of the chemoattractant are established, it is beneficial to cells to improve their chemotactic ability in order to reach the aggregation center sooner.


Assuntos
Quimiotaxia , AMP Cíclico , Dictyostelium , Modelos Biológicos , Quimiotaxia/fisiologia , Dictyostelium/fisiologia , AMP Cíclico/metabolismo , Fatores Quimiotáticos/farmacologia , Fatores Quimiotáticos/metabolismo
15.
Biomolecules ; 14(8)2024 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-39199373

RESUMO

Sulfonylureas (SUs) are a class of antidiabetic drugs widely used in the management of diabetes mellitus type 2. They promote insulin secretion by inhibiting the ATP-sensitive potassium channel in pancreatic ß-cells. Recently, the exchange protein directly activated by cAMP (Epac) was identified as a new class of target proteins of SUs that might contribute to their antidiabetic effect, through the activation of the Ras-like guanosine triphosphatase Rap1, which has been controversially discussed. We used human embryonic kidney (HEK) 293 cells expressing genetic constructs of various Förster resonance energy transfer (FRET)-based biosensors containing different versions of Epac1 and Epac2 isoforms, alone or fused to different phosphodiesterases (PDEs), to monitor SU-induced conformational changes in Epac or direct PDE inhibition in real time. We show that SUs can both induce conformational changes in the Epac2 protein but not in Epac1, and directly inhibit the PDE3 and PDE4 families, thereby increasing cAMP levels in the direct vicinity of these PDEs. Furthermore, we demonstrate that the binding site of SUs in Epac2 is distinct from that of cAMP and is located between the amino acids E443 and E460. Using biochemical assays, we could also show that tolbutamide can inhibit PDE activity through an allosteric mechanism. Therefore, the cAMP-elevating capacity due to allosteric PDE inhibition in addition to direct Epac activation may contribute to the therapeutic effects of SU drugs.


Assuntos
AMP Cíclico , Fatores de Troca do Nucleotídeo Guanina , Compostos de Sulfonilureia , Humanos , Compostos de Sulfonilureia/farmacologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , AMP Cíclico/metabolismo , Transferência Ressonante de Energia de Fluorescência , Inibidores de Fosfodiesterase/farmacologia , Inibidores de Fosfodiesterase/química , Hipoglicemiantes/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Sítios de Ligação , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo
16.
Eur J Med Res ; 29(1): 433, 2024 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-39192377

RESUMO

BACKGROUND: Reduction of inflammatory damage and inhibition of nucleus pulposus (NP) apoptosis are considered to be the main effective therapy idea to reverse the intervertebral disc degeneration (IDD) and alleviate the chronic low back pain. The adenosine A2A receptor (A2AR), as a member of G protein-coupled receptor families, plays an important role in the anti-inflammation and relieving pain. So far, the impact of A2AR on IDD therapy is unclear. The aim of this study was to explore the role of Adenosine A2A receptor (A2AR) in the intervertebral disc degeneration (IDD) and clarify potential mechanism. MATERIALS AND METHODS: IL-1ß and acupuncture was used to establish IDD model rats. A2AR agonist CGS-21680 and A2AR antagonist SCH442416 were used to investigate the therapeutical effects for IDD. Histological examination, western blotting analysis and RT-PCR were employed to evaluate the the association between A2AR and cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway. RESULTS: A2AR activity of the intervertebral disc tissues was up-regulated in feedback way, and cAMP, PKA and CREB expression were also increased. But in general, IL-1ß-induced IDD promoted the significant up-regulation the expression of inflammatory factors. The nucleus pulposus (NP) inflammation was exacerbated in result of MMP3 and Col-II decline through activating NF-κB signaling pathway. A2AR agonist CGS-21680 exhibited a disc protective effect through significantly increasing A2AR activity, then further activated cAMP/PKA signaling pathway with attenuating the release of TNF-α and IL-6 via down-regulating NF-κB. In contrast, SCH442416 inhibited A2AR activation, consistent with lower expression levels of cAMP and PKA, further leading to the acceleration of IDD. CONCLUSIONS: The activation of A2AR can prevent inflammatory responses and mitigates degradation of IDD thus suggest a potential novel therapeutic strategy of IDD.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico , Inflamação , Degeneração do Disco Intervertebral , NF-kappa B , Receptor A2A de Adenosina , Transdução de Sinais , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/tratamento farmacológico , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Receptor A2A de Adenosina/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ratos , Inflamação/metabolismo , Masculino , Ratos Sprague-Dawley , Fenetilaminas/farmacologia , Núcleo Pulposo/metabolismo , Núcleo Pulposo/patologia , Núcleo Pulposo/efeitos dos fármacos , AMP Cíclico/metabolismo , Agonistas do Receptor A2 de Adenosina/farmacologia , Modelos Animais de Doenças , Adenosina/análogos & derivados
17.
Toxicol Appl Pharmacol ; 491: 117070, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39151807

RESUMO

AIMS: It is well established that intracellular cAMP contributes to the relaxation of vas deferens smooth muscle. In many tissues, intracellular cAMP is actively transported to the extracellular space, where it exerts regulatory functions, via its metabolite adenosine. These actions take place through the cAMP conversion to adenosine by ectoenzymes, a process called "extracellular cAMP-adenosine pathway". Herein, we investigated whether, in addition to ATP, extracellular cAMP might be an alternative source of adenosine, influencing the contraction of vas deferens smooth muscle. MAIN METHODS: The effects of cAMP, 8-Br-cAMP and adenosine were analyzed in the isometric contractions of rat vas deferens. cAMP efflux was analyzed by measuring extracellular cAMP levels after exposure of vas deferens segments to isoproterenol and forskolin in the presence or absence of MK-571, an inhibitor of MRP/ABCC transporters. KEY FINDINGS: While 8-Br-cAMP, a cell-permeable cAMP analog, induced relaxation of KCl-precontracted vas deferens, the non-permeant cAMP increased the KCl-induced contractile response, which was mimicked by adenosine, but prevented by inhibitors of ecto-5'-nucleotidase or A1 receptors. Our results also showed that isoproterenol and forskolin increases cAMP efflux via an MRP/ABCC transporter-dependent mechanism, since it is inhibited by MK-571. SIGNIFICANCE: Our data show that activation of ß-adrenoceptors and adenylyl cyclase increases cAMP efflux from vas deferens tissue, which modulates the vas deferens contractile response via activation of adenosine A1 receptors. Assuming that inhibition of vas deferens contractility has been proposed as a strategy for male contraception, the extracellular cAMP-adenosine pathway emerges as a potential pharmacological target that should be considered in studies of male fertility.


Assuntos
5'-Nucleotidase , AMP Cíclico , Contração Muscular , Ratos Wistar , Receptor A1 de Adenosina , Ducto Deferente , Masculino , Animais , Ducto Deferente/efeitos dos fármacos , Ducto Deferente/metabolismo , AMP Cíclico/metabolismo , 5'-Nucleotidase/metabolismo , Receptor A1 de Adenosina/metabolismo , Receptor A1 de Adenosina/efeitos dos fármacos , Ratos , Contração Muscular/efeitos dos fármacos , Adenosina/farmacologia , Adenosina/análogos & derivados , Adenosina/metabolismo , Isoproterenol/farmacologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Colforsina/farmacologia
18.
Proc Natl Acad Sci U S A ; 121(34): e2405465121, 2024 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-39145932

RESUMO

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.


Assuntos
Analgésicos Opioides , AMP Cíclico , Gabapentina , Neuralgia , Transdução de Sinais , Traumatismos da Medula Espinal , Animais , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , AMP Cíclico/metabolismo , Ratos , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/metabolismo , Analgésicos Opioides/farmacologia , Gabapentina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ratos Sprague-Dawley , Masculino , Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Pregabalina/farmacologia , Pregabalina/uso terapêutico , Sinergismo Farmacológico , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/efeitos dos fármacos
19.
BMC Neurosci ; 25(1): 38, 2024 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-39179957

RESUMO

Visual perception of X-radiation is a well-documented, but poorly understood phenomenon. Scotopic rod cells and rhodopsin have been implicated in visual responses to X-rays, however, some evidence suggests that X-rays excite the retina via a different mechanism than visible light. While rhodopsin's role in X-ray perception is unclear, the possibility that it could function as an X-ray receptor has led to speculation that it could act as a transgenically expressed X-ray receptor. If so, it could be used to transduce transcranial X-ray signals and control the activity of genetically targeted populations of neurons in a less invasive version of optogenetics, X-genetics. Here we investigate whether human rhodopsin (hRho) is capable of transducing X-ray signals when expressed outside of the retinal environment. We use a live-cell cAMP GloSensor luminescence assay to measure cAMP decreases in hRho-expressing HEK293 cells in response to visible light and X-ray stimulation. We show that cAMP GloSensor luminescence decreases are not observed in hRho-expressing HEK293 cells in response to X-ray stimulation, despite the presence of robust responses to visible light. Additionally, irradiation had no significant effect on cAMP GloSensor responses to subsequent visible light stimulation. These results suggest that ectopically expressed rhodopsin does not function as an X-ray receptor and is not capable of transducing transcranial X-ray signals into neural activity for X-ray mediated, genetically targeted neuromodulation.


Assuntos
AMP Cíclico , Rodopsina , Humanos , Células HEK293 , Rodopsina/metabolismo , Rodopsina/genética , Raios X , AMP Cíclico/metabolismo , Luz , Estimulação Luminosa/métodos
20.
Molecules ; 29(16)2024 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-39202926

RESUMO

The adenosine A2A receptor (A2AR) has been identified as a therapeutic target for treating neurodegenerative diseases and cancer. In recent years, we have highlighted the 2-aminoquinazoline heterocycle as an promising scaffold for designing new A2AR antagonists, exemplified by 6-bromo-4-(furan-2-yl)quinazolin-2-amine 1 (Ki (hA2AR) = 20 nM). Here, we report the synthesis of new 2-aminoquinazoline derivatives with substitutions at the C6- and C7-positions, and the introduction of aminoalkyl chains containing tertiary amines at the C2-position to enhance antagonist activity and solubility properties. Compound 5m showed a high affinity for hA2AR with a Ki value of 5 nM and demonstrated antagonist activity with an IC50 of 6 µM in a cyclic AMP assay. Introducing aminopentylpiperidine and 4-[(piperidin-1-yl)methyl]aniline substituents maintained the binding affinities (9x, Ki = 21 nM; 10d, Ki = 15 nM) and functional antagonist activities (9x, IC50 = 9 µM; 10d, IC50 = 5 µM) of the synthesized compounds while improving solubility. This study provides insights into the future development of A2AR antagonists for therapeutic applications.


Assuntos
Antagonistas do Receptor A2 de Adenosina , Quinazolinas , Receptor A2A de Adenosina , Quinazolinas/química , Quinazolinas/farmacologia , Quinazolinas/síntese química , Antagonistas do Receptor A2 de Adenosina/química , Antagonistas do Receptor A2 de Adenosina/síntese química , Antagonistas do Receptor A2 de Adenosina/farmacologia , Receptor A2A de Adenosina/metabolismo , Receptor A2A de Adenosina/química , Humanos , Relação Estrutura-Atividade , Estrutura Molecular , AMP Cíclico/metabolismo , Solubilidade , Ligação Proteica
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