RESUMEN
Giardiasis is a parasitosis caused by Giardia lamblia with significant epidemiological and clinical importance due to its high prevalence and pathogenicity. The lack of optimal therapies for treating this parasite makes the development of new effective chemical entities an urgent need. In the search for new inhibitors of the adenylyl cyclase gNC1 obtained from G. lamblia, 14 extracts from Argentinian native plants were screened. Lepechinia floribunda and L. meyenii extracts exhibited the highest gNC1 inhibitory activity, with IC50 values of 9 and 31 µg/mL, respectively. In silico studies showed rosmarinic acid, a hydroxycinnamic acid present in both mentioned species, to be a promising anti-gNC1 compound. This result was confirmed experimentally, with rosmarinic acid showing an IC50 value of 10.1 µM. Theoretical and experimental findings elucidate the molecular-level mechanism of rosmarinic acid, pinpointing the key interactions stabilizing the compound-enzyme complex and the binding site. These results strongly support that rosmarinic acid is a promising scaffold for developing novel compounds with inhibitory activity against gNC1, which could serve as potential therapeutic agents to treat giardiasis.
RESUMEN
Polyamines are polycationic molecules which contains two or more amino groups (-NH3+) highly charged at physiological pH, and among them we found spermine, spermidine, putrescine, and cadaverine. They interact with proteins, nucleic acids, modulate Ca2+, K+, and Na+ channels, and protect sperm from oxidative stress. In this work, we evaluate the effect of spermine, spermidine, and putrescine on the total, progressive and kinematic parameters of motility, capacitation, acrosome reaction, also in presence and absence of the dbcAMP, an analogue of the cAMP, and the IBMX, a phosphodiesterase inhibitor. In addition, we evaluated the intracellular concentrations of cAMP [cAMP]i, and performed an in silico analysis between polyamines and the sAC from mouse to predict the possible interaction among them. Our results showed that all polyamines decrease drastically the total, progressive and the kinetic parameters of sperm motility, decrease the capacitation, and only spermidine and putrescine impeded the acquisition of acrosome reaction. Moreover, the effect of polyamines was attenuated but not countered by the addition of db-cAMP and IBMX, suggesting a possible inhibition of the sAC. Also, the presence of polyamines induced a decrease of the [cAMP]i, and the in silico analysis predicted a strong interaction among polyamines and the sAC. Overall, the evidence suggests that probably the polyamines interact and inhibit the activity of the sAC.
Asunto(s)
Poliaminas , Putrescina , Masculino , Animales , Ratones , Putrescina/farmacología , Espermidina/farmacología , Espermina , 1-Metil-3-Isobutilxantina , Motilidad Espermática , SemenRESUMEN
Corticotropin releasing hormone (CRH) is crucial for basal and stress-initiated reactions in the hypothalamic-pituitary-adrenal axis (HPA) and extrahypothalamic brain circuits, where it acts as a neuromodulator to organize behavioral and humoral responses to stress. We review and describe cellular components and molecular mechanisms involved in CRH system signaling through G protein-coupled receptors (GPCRs) CRHR1 and CRHR2, under the current view of GPCR signaling from the plasma membrane but also from intracellular compartments, which establish the bases of signal resolution in space and time. Focus is placed on latest studies of CRHR1 signaling in physiologically significant contexts of the neurohormone function that disclosed new mechanistic features of cAMP production and ERK1/2 activation. We also introduce in a brief overview the pathophysiological function of the CRH system, underlining the need for a complete characterization of CRHRs signaling to design new and specific therapies for stress-related disorders.
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Sistema Hipotálamo-Hipofisario , Sistema Hipófiso-Suprarrenal , Humanos , Sistema Hipotálamo-Hipofisario/metabolismo , Sistema Hipófiso-Suprarrenal/metabolismo , Hormona Liberadora de Corticotropina/metabolismo , Sistema Nervioso Central/metabolismo , EndocitosisRESUMEN
In this work, we report a derivative of N-(piperidin-4-yl)-1H-pyrrole-2-carboxamide as a new inhibitor for adenylyl cyclase of Giardia lamblia which was obtained from a study using structural data of the nucleotidyl cyclase 1 (gNC1) of this parasite. For such a study, we developed a model for this specific enzyme by using homology techniques, which is the first model reported for gNC1 of G. lamblia. Our studies show that the new inhibitor has a competitive mechanism of action against this enzyme. 2-Hydroxyestradiol was used as the reference compound for comparative studies. Results in this work are important from two points of view. on the one hand, an experimentally corroborated model for gNC1 of G. lamblia obtained by molecular modelling is presented; on the other hand, the new inhibitor obtained is an undoubtedly excellent starting structure for the development of new metabolic inhibitors for G. lamblia.
Asunto(s)
Adenilil Ciclasas/metabolismo , Inhibidores Enzimáticos/farmacología , Giardia lamblia/enzimología , Adenilil Ciclasas/química , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Modelos Moleculares , Estructura Molecular , Relación Estructura-ActividadRESUMEN
In the field of experimental pharmacology, researchers continuously investigate new relaxant agents of the airway smooth muscle cells (ASMCs), since the pathophysiology of respiratory illnesses, such as asthma, involves hyperresponsiveness and changes in ASMC homeostasis. In this scenario, labdane-type diterpenes, like forskolin (FSK), are a class of compounds known for their relaxing action on smooth muscle cells (SMCs), being this phenomenon related to the direct activation of AC-cAMP-PKA pathway. Considering the continuous effort of our group to study the mechanism of action and prospecting for compounds isolated from natural sources, in this paper, we presented how the diterpene 8(17),12E,14-labdatrien-18-oic acid (LBD) promotes relaxant effect on ASMC, performing in vitro experiments using isolated guinea pig trachea and in silico molecular docking/dynamics simulations. In vitro experiments showed that in the presence of aminophylline, FSK and LBD had their relaxant effect potentiated (EC50 from 1.4 ± 0.2 × 10-5 M to 1.5 ± 0.3 × 10-6 M for LBD and from 2.0 ± 0.2 × 10-7 M to 6.4 ± 0.4 × 10-8 M for FSK) while in the presence of Rp-cAMPS this effect was attenuated (EC50 from 1.4 ± 0.2 × 10-5 M to 3 × 10-4 M for LBD and from 2.0 ± 0.2 × 10-7 to 3.1 ± 1.0 × 10-6 M for FSK). Additionally, in silico simulations evidenced that the lipophilic character of LBD is probably responsible for its stability on AC binding site. LBD presented two preferential orientations, where the double bonds of the isoprene moiety as well as the unique polar group (carboxylic acid) in this compound form important anchoring points. In this sense, we consider that the LBD can interact stabilizing the catalytic dimmer of AC as the FSK, although less efficiently.
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Diterpenos/farmacología , Relajación Muscular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Tráquea/efectos de los fármacos , Aminofilina/farmacología , Animales , Sitios de Unión , Colforsina/farmacología , Simulación por Computador , Diterpenos/administración & dosificación , Diterpenos/química , Femenino , Cobayas , Masculino , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Miocitos del Músculo Liso/metabolismo , Tráquea/citologíaRESUMEN
The soluble adenylyl cyclase (sAC) was identified in the heart as another source of cyclic AMP (cAMP). However, its cardiac physiological function is unknown. On the other hand, the cardiac Na+/HCO3- cotransporter (NBC) promotes the cellular co-influx of HCO3- and Na+. Since sAC activity is regulated by HCO3-, our purpose was to investigate the potential functional relationship between NBC and sAC in the cardiomyocyte. Rat ventricular myocytes were loaded with Fura-2, Fluo-3, or BCECF to measure Ca2+ transient (Ca2+i) by epifluorescence, Ca2+ sparks frequency (CaSF) by confocal microscopy, or intracellular pH (pHi) by epifluorescence, respectively. Sarcomere or cell shortening was measured with a video camera as an index of contractility. The NBC blocker S0859 (10 µM), the selective inhibitor of sAC KH7 (1 µM), and the PKA inhibitor H89 (0.1 µM) induced a negative inotropic effect which was associated with a decrease in Ca2+i. Since PKA increases Ca2+ release through sarcoplasmic reticulum RyR channels, CaSF was measured as an index of RyR open probability. The generation of CaSF was prevented by KH7. Finally, we investigated the potential role of sAC activation on NBC activity. NBC-mediated recovery from acidosis was faster in the presence of KH7 or H89, suggesting that the pathway sAC-PKA is negatively regulating NBC function, consistent with a negative feedback modulation of the HCO3- influx that activates sAC. In summary, the results demonstrated that the complex NBC-sAC-PKA plays a relevant role in Ca2+ handling and basal cardiac contractility.
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Adenilil Ciclasas/metabolismo , Contracción Miocárdica , Miocitos Cardíacos/metabolismo , Simportadores de Sodio-Bicarbonato/metabolismo , Inhibidores de Adenilato Ciclasa/farmacología , Animales , Benzamidas/farmacología , Señalización del Calcio , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Ventrículos Cardíacos/citología , Isoquinolinas/farmacología , Masculino , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Ratas , Ratas Wistar , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Simportadores de Sodio-Bicarbonato/antagonistas & inhibidores , Sulfonamidas/farmacologíaRESUMEN
Trypanosomatids are a group of flagellated unicellular eukaryotes, causing serious human diseases including Chagas disease (Trypanosoma cruzi), sleeping sickness (Trypanosoma brucei spp.) and Leishmaniasis (Leishmania spp.). The second messenger cAMP is involved in numerous and fundamental processes in these parasites including differentiation between stages, proliferation, osmoregulation, oxidative stress and quorum sensing. Interestingly, its signaling pathway is quite different from that of mammals, including structurally different adenylyl cyclases, the shortage of orthologous effector proteins and the absence of G-protein-coupled-receptors, among others. These characteristics make the proteins involved in these transduction pathways good candidates for therapeutic targets. However, the identification of new unknown druggable targets involves extensive research time and is economically very expensive, making difficult the transition from basic research to the clinical phase. Trypanosomatid PDEs have characteristic binding pockets that allow for a differential inhibition from their human orthologs. Modification in the approved drugs for human to convert them into trypanocidal treatments could lead to more effective therapies, shorter lab time and lower costs. In view of the fact that kinetoplastid PDEs are highly conserved with their mammalian counterparts, and since there are already numerous drugs on the market against human PDEs, the drug repositioning approach is highly promising. The development of new technologies, higher government and industrial involvement and more scientists committed to basic investigation, are the key to ultimately find an effective treatment and cure for the neglected tropical diseases.
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Inhibidores de Fosfodiesterasa/farmacología , Transducción de Señal/efectos de los fármacos , Adenilil Ciclasas/fisiología , Calcio/fisiología , Enfermedad de Chagas/tratamiento farmacológico , Reposicionamiento de Medicamentos , Humanos , Leishmania donovani/enzimología , Leishmania donovani/fisiología , Proteínas Quinasas/fisiología , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/enzimología , Trypanosoma brucei brucei/fisiología , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/fisiologíaRESUMEN
Mycobacterium tuberculosis remains as a threat to public health around the world with 1.7 million cases of TB-associated deaths during 2016. Despite the use of Bacillus Calmette-Guerin (BCG) vaccine, control of the infection has not been successful. Because of this, several efforts have been made in order to develop new vaccines capable of boosting previous immunization or attempted for replacing current BCG. We previously showed that over expression of the M. tuberculosis adenylyl cyclase encoding gene Rv2212 in BCG bacilli (BCG-Rv2212), induced an attenuated phenotype when administered in BALB/c mice. Moreover, two-dimensional proteomic analysis showed that heat shock proteins such as GroEL2 and DnaK were overexpressed in this BCG-Rv2212. In this report, we show that immunization of mice with BCG-Rv2212 significantly increments IFN-γ+ CD4+ and CD8+ T-lymphocytes after PPD stimulation in comparison with BCG vaccinated mice. Mice vaccinated with BCG-Rv2212 significantly reduced the bacterial load in lungs after four-month post infection with M. tuberculosis H37Rv but was similar to BCG after 6 month-post-challenge. Survival experiment showed that both vaccines administered separately in mice induce similar levels of protection after 20-week post-challenge with M. tuberculosis H37Rv. Virulence experiments developed in nude mice, showed that BCG-Rv2212 and BCG bacilli were equally safe. Our results suggest that BCG-Rv2212 is capable of stimulating cellular immune response effectively and reduce bacterial burden in lungs of mice after challenge. Particularly, it seems to be more effective in controlling bacterial burden during the first steps of infection.
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Adenilil Ciclasas/administración & dosificación , Vacuna BCG/administración & dosificación , Proteínas Bacterianas/administración & dosificación , Inmunogenicidad Vacunal , Pulmón/microbiología , Mycobacterium tuberculosis/crecimiento & desarrollo , Tuberculosis Pulmonar/prevención & control , Adenilil Ciclasas/genética , Adenilil Ciclasas/inmunología , Animales , Vacuna BCG/genética , Vacuna BCG/inmunología , Carga Bacteriana , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/microbiología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/microbiología , Modelos Animales de Enfermedad , Femenino , Inmunidad Celular , Inmunización , Pulmón/inmunología , Ratones Endogámicos BALB C , Ratones Desnudos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/inmunología , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/microbiología , Vacunas de ADN/administración & dosificaciónRESUMEN
Ascorbate, the reduced form of vitamin C, is highly concentrated in the central nervous system (CNS), including the retina, where it plays important physiological functions. In the CNS, the plasma membrane transporter sodium vitamin C co-transporter 2 (SVCT2) is responsible for ascorbate transport in neurons. The neurotransmitter dopamine (DA), acting through D1- and D2-like receptor subfamilies and classically coupled to adenylyl cyclase, is known to modulate synaptic transmission in the retina. Here, we reveal that DA controls the release of ascorbate from retinal neurons. Using primary retinal cultures, we show that this DA effect is dose-dependent, occurring by the reversal of the SVCT2, and could be elicited by brief and repetitive pulses of DA. The DA effect in inducing ascorbate release occurs by the activation of D1R and is independent of PKA. Moreover, the exchange protein directly activated by cAMP type 2 (EPAC2) is present in retinal neurons and its specific knockdown using shRNAs abrogates the D1R-induced ascorbate release. Confirming the physiological relevance of this pathway, activation of D1R or EPAC2 also triggered ascorbate release ex vivo in acute preparations of the intact retina. Overall, DA plays pivotal roles in regulating ascorbate homeostasis through an unanticipated signaling pathway involving D1R/adenylyl cyclase/cAMP/EPAC2, thereby suggesting that vitamin C might fine-tune dopaminergic neurotransmission in the retina.
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Ácido Ascórbico/metabolismo , Dopamina/farmacología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Receptores de Dopamina D1/metabolismo , Neuronas Retinianas/metabolismo , Adenilil Ciclasas/metabolismo , Animales , Células Cultivadas , Embrión de Pollo , Neuronas Retinianas/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Striatal dopamine D2 receptors activate the PLCâ¯ââ¯IP3â¯ââ¯Calcineurin-signaling pathway to modulate the neural excitability of En+ Medium-sized Spiny GABAergic neurons (MSN) through the regulation of L-type Ca2+ channels. Presynaptic dopaminergic D2 receptors modulate GABA release at striatopallidal terminals through L-type Ca2+ channels as well, but their signaling pathway is still undetermined. Since D2 receptors are Gi/o-coupled and negatively modulate adenylyl cyclase (AC), we investigated whether presynaptic D2 receptors modulate GABA release through the same signaling cascade that controls excitability in the striatum or by the inhibition of AC and decreased PKA activity. Activation of D2 receptors stimulated formation of [3H]IP1 and decreased Forskolin-stimulated [3H]cAMP accumulation in synaptosomes from rat Globus Pallidus. D2 receptor activation with Quinpirole in the presence of L 745,870 decreased, in a dose-dependent manner, K+-induced [3H]GABA release in pallidal slices. The effect was prevented by the pharmacological blockade of Gi/o ßγ subunit effects with Gallein, PLC with U 73122, IP3 receptor activation with 4-APB, Calcineurin with FK506. In addition, when release was stimulated with Forskolin to activate AC, D2 receptors also decreased K+-induced [3H]GABA release, an effect occluded with the effect of the blockade of PKA with H89 or stimulation of release with the cAMP analog 8-Br-cAMP. These data indicate that D2 receptors modulate [3H]GABA release at striatopallidal terminals by activating the PLCâ¯ââ¯IP3â¯ââ¯Calcineurin-signaling cascade, the same one that modulates excitability in soma. Additionally, D2 receptors inhibit release when AC is active. Both mechanisms appear to converge to regulate the activity of presynaptic L-type Ca2+ channels.
Asunto(s)
Cuerpo Estriado/metabolismo , Globo Pálido/metabolismo , Terminales Presinápticos/metabolismo , Receptores de Dopamina D2/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Adenilil Ciclasas/metabolismo , Animales , Calcineurina/metabolismo , Cuerpo Estriado/efectos de los fármacos , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Globo Pálido/efectos de los fármacos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Fosfoinositido Fosfolipasa C/metabolismo , Potasio/metabolismo , Terminales Presinápticos/efectos de los fármacos , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Técnicas de Cultivo de Tejidos , TritioRESUMEN
Despite its importance in the regulation of growth and differentiation processes of a variety of organisms, the mechanism of synthesis and degradation of cAMP (cyclic AMP) has not yet been described in Giardia lamblia In this work, we measured significant quantities of cAMP in trophozoites of G. lamblia incubated in vitro and later detected how it increases during the first hours of encystation, and how it then returns to basal levels at 24â h. Through an analysis of the genome of G. lamblia, we found sequences of three putative enzymes - one phosphodiesterase (gPDE) and two nucleotidyl cyclases (gNC1 and gNC2) - that should be responsible for the regulation of cAMP in G. lamblia Later, an RT-PCR assay confirmed that these three genes are expressed in trophozoites. The bioinformatic analysis indicated that gPDE is a transmembrane protein of 154â kDa, with a single catalytic domain in the C-terminal end; gNC1 is predicted to be a transmembrane protein of 74â kDa, with only one class III cyclase homology domain (CHD) at the C-terminal end; and gNC2 should be a transmembrane protein of 246â kDa, with two class III CHDs. Finally, we cloned and enriched the catalytic domain of gNC1 (gNC1cd) from bacteria. After that, we confirmed that gNC1cd has adenylyl cyclase (AC) activity. This enzymatic activity depends on the presence of Mn2+ and Ca2+, but no significant activity was displayed in the presence of Mg2+ Additionally, the AC activity of gNC1cd is competitively inhibited with GTP, so it is highly possible that gNC1 has guanylyl cyclase activity as well.
Asunto(s)
Adenilil Ciclasas/química , AMP Cíclico/química , Giardia lamblia/enzimología , Guanilato Ciclasa/química , Hidrolasas Diéster Fosfóricas/química , Proteínas Protozoarias/química , Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , Secuencia de Aminoácidos , Calcio/química , Calcio/metabolismo , Dominio Catalítico , Clonación Molecular , Cristalografía por Rayos X , AMP Cíclico/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Giardia lamblia/genética , Giardia lamblia/crecimiento & desarrollo , Guanilato Ciclasa/genética , Guanilato Ciclasa/metabolismo , Cinética , Manganeso/química , Manganeso/metabolismo , Modelos Moleculares , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología Estructural de Proteína , Especificidad por Sustrato , Trofozoítos/enzimología , Trofozoítos/genética , Trofozoítos/crecimiento & desarrolloRESUMEN
Spatiotemporal regulation of cAMP within the cell is required to achieve receptor-specific responses. The mechanism through which the cell selects a specific response to newly synthesized cAMP is not fully understood. In hepatocyte plasma membranes, we identified two functional and independent cAMP-responsive signaling protein macrocomplexes that produce, use, degrade, and regulate their own nondiffusible (sequestered) cAMP pool to achieve their specific responses. Each complex responds to the stimulation of an adenosine G protein-coupled receptor (Ado-GPCR), bound to either A2A or A2B , but not simultaneously to both. Each isoprotein involved in each signaling cascade was identified by measuring changes in cAMP levels after receptor activation, and its participation was confirmed by antibody-mediated inactivation. A2A -Ado-GPCR selective stimulation activates adenylyl cyclase 6 (AC6), which is bound to AKAP79/150, to synthesize cAMP which is used by two other AKAP79/150-tethered proteins: protein kinase A (PKA) and phosphodiesterase 3A (PDE3A). In contrast, A2B -Ado-GPCR stimulation activates D-AKAP2-attached AC5 to generate cAMP, which is channeled to two other D-AKAP2-tethered proteins: guanine-nucleotide exchange factor 2 (Epac2) and PDE3B. In both cases, prior activation of PKA or Epac2 with selective cAMP analogs prevents de novo cAMP synthesis. In addition, we show that cAMP does not diffuse between these protein macrocomplexes or 'signalosomes'. Evidence of coimmunoprecipitation and colocalization of some proteins belonging to each signalosome is presented. Each signalosome constitutes a minimal functional signaling unit with its own machinery to synthesize and regulate a sequestered cAMP pool. Thus, each signalosome is devoted to ensure the transmission of a unique and unequivocal message through the cell.
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Adenilil Ciclasas/metabolismo , AMP Cíclico/biosíntesis , Hepatocitos/metabolismo , Receptor de Adenosina A2A/metabolismo , Receptor de Adenosina A2B/metabolismo , Transducción de Señal , Proteínas de Anclaje a la Quinasa A/genética , Proteínas de Anclaje a la Quinasa A/metabolismo , Adenilil Ciclasas/genética , Animales , Calcio/metabolismo , Membrana Celular/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Regulación de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Hepatocitos/citología , Masculino , Cultivo Primario de Células , Ratas , Ratas Wistar , Receptor de Adenosina A2A/genética , Receptor de Adenosina A2B/genéticaRESUMEN
The Trypanosoma cruzi adenylyl cyclase (AC) multigene family encodes different isoforms (around 15) sharing a variable large N-terminal domain, which is extracellular and receptor-like, followed by a transmembrane helix and a conserved C-terminal catalytic domain. It was proposed that these key enzymes in the cAMP signalling pathway allow the parasite to sense its changing extracellular milieu in order to rapidly adapt to its new environment, which is generally achieved through a differentiation process. One of the critical differentiation events the parasitic protozoan T. cruzi undergoes during its life cycle, known as metacyclogenesis, occurs in the digestive tract of the insect and corresponds to the differentiation from noninfective epimastigotes to infective metacyclic trypomastigote forms. By in vitro monitoring the activity of AC during metacyclogenesis, we showed that both the activity of AC and the intracellular cAMP content follow a similar pattern of transient stimulation in a two-step process, with a first activation peak occurring during the first hours of nutritional stress and a second peak between 6 and 48 h, corresponding to the cellular adhesion. During this differentiation process, a general mechanism of upregulation of AC expression of both mRNA and protein is triggered and in particular for a major subclass of these enzymes that are present in various gene copies commonly associated to the THT gene clusters. Although the scattered genome distribution of these gene copies is rather unusual in trypanosomatids and seems to be a recent acquisition in the evolution of the T. cruzi clade, their encoded product redistributed on the flagellum of the parasite upon differentiation could be important to sense the extracellular milieu.
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Adenilil Ciclasas/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/crecimiento & desarrollo , Adenilil Ciclasas/química , Adenilil Ciclasas/genética , Secuencia de Aminoácidos , Animales , Enfermedad de Chagas/parasitología , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Estadios del Ciclo de Vida , Datos de Secuencia Molecular , Familia de Multigenes , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Alineación de Secuencia , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo , Regulación hacia ArribaRESUMEN
The aim of this work was to study the participation of membrane adenylyl cyclase in heparin-induced capacitation in cryopreserved bovine spermatozoa. Sperm suspensions were incubated in Tyrode's albumin lactate pyruvate medium in the presence of heparin (10 IU ml(-1) ) or forskolin (1-75 µm), a well-known membrane adenylyl cyclase activator. The participation of membrane adenylyl cyclase was confirmed using a specific inhibitor, 2',5'-dideoxyadenosine (6-25 µm). Spermatozoa capacitated with forskolin (25 µm) were incubated with bovine follicular fluid to evaluate their ability to undergo acrosome reaction. Capacitation percentages were determined by the fluorescence technique with chlortetracycline, and true acrosome reaction was determined by trypan blue and differential interferential contrast. The forskolin concentrations employed had no effect on progressive motility or sperm viability. Capacitation values induced by 25-µm forskolin treatment (27.80 ± 2.59%) were significantly higher respect to the control (4.80 ± 1.30%). The inhibitor 2',5'-dideoxyadenosine prevented forskolin-induced capacitation and significantly diminished capacitation induced by heparin. Follicular fluid induced physiological acrosome reaction in spermatozoa previously capacitated with 25-µm forskolin (P < 0.05). Forskolin acts as a capacitation inducer and involves the participation of membrane adenylyl cyclase as part of the intracellular mechanisms that lead to capacitation in cryopreserved bovine spermatozoa.
Asunto(s)
Reacción Acrosómica/efectos de los fármacos , Adenilil Ciclasas/fisiología , Criopreservación , Fibrinolíticos/farmacología , Heparina/farmacología , Preservación de Semen , Capacitación Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Reacción Acrosómica/fisiología , Inhibidores de Adenilato Ciclasa , Animales , Antimetabolitos/farmacología , Bovinos , Supervivencia Celular , Colforsina/farmacología , Didesoxiadenosina/farmacología , Masculino , Capacitación Espermática/fisiología , Motilidad Espermática/efectos de los fármacos , Motilidad Espermática/fisiología , Espermatozoides/fisiologíaRESUMEN
Cyclic adenosine 3',5'-monophosphate (cAMP), the first second messenger to be described, plays a central role in cell signaling in a wide variety of cell types. Over the last decades, a wide body of literature addressed the different roles of cAMP in cell physiology, mainly in response to neurotransmitters and hormones. cAMP is synthesized by a wide variety of adenylyl cyclases that can generally be grouped in two types: transmembrane adenylyl cyclase and soluble adenylyl cyclases. In particular, several aspects of sperm physiology are regulated by cAMP produced by a single atypical adenylyl cyclase (Adcy10, aka sAC, SACY). The signature that identifies sAC among other ACs, is their direct stimulation by bicarbonate. The essential nature of cAMP in sperm function has been demonstrated using gain of function as well as loss of function approaches. This review unifies state of the art knowledge of the role of cAMP and those enzymes involved in cAMP signaling pathways required for the acquisition of fertilizing capacity of mammalian sperm. This article is part of a Special Issue entitled: The role of soluble adenylyl cyclase in health and disease.
Asunto(s)
Adenilil Ciclasas/fisiología , AMP Cíclico/fisiología , Espermatozoides/fisiología , Adenilil Ciclasas/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Hidrólisis , Masculino , Transducción de Señal , Espermatozoides/enzimologíaRESUMEN
Skeletal muscle contraction is triggered by acetylcholine induced release of Ca(2+) from sarcoplasmic reticulum. Although this signaling pathway is independent of extracellular Ca(2+), L-type voltage-gated calcium channel (Cav) blockers have inotropic effects on frog skeletal muscles which occur by an unknown mechanism. Taking into account that skeletal muscle fiber expresses Ca(+2)-sensitive adenylyl cyclase (AC) isoforms and that cAMP is able to increase skeletal muscle contraction force, we investigated the role of Ca(2+) influx on mouse skeletal muscle contraction and the putative crosstalk between extracellular Ca(2+) and intracellular cAMP signaling pathways. The effects of Cav blockers (verapamil and nifedipine) and extracellular Ca(2+) chelator EGTA were evaluated on isometric contractility of mouse diaphragm muscle under direct electrical stimulus (supramaximal voltage, 2 ms, 0.1 Hz). Production of cAMP was evaluated by radiometric assay while Ca(2+) transients were assessed by confocal microscopy using L6 cells loaded with fluo-4/AM. Ca(2+) channel blockers verapamil and nifedipine had positive inotropic effect, which was mimicked by removal of extracellular Ca(+2) with EGTA or Ca(2+)-free Tyrode. While phosphodiesterase inhibitor IBMX potentiates verapamil positive inotropic effect, it was abolished by AC inhibitors SQ22536 and NYK80. Finally, the inotropic effect of verapamil was associated with increased intracellular cAMP content and mobilization of intracellular Ca(2+), indicating that positive inotropic effects of Ca(2+) blockers depend on cAMP formation. Together, our results show that extracellular Ca(2+) modulates skeletal muscle contraction, through inhibition of Ca(2+)-sensitive AC. The cross-talk between extracellular calcium and cAMP-dependent signaling pathways appears to regulate the extent of skeletal muscle contraction responses.