RESUMEN
Molecular studies have revealed that many species once thought to be wide-ranging in the Indo-West Pacific contain allopatric mosaics of endemic lineages. These lineages provide compelling evidence that substantial time is needed to evolve isolating mechanisms sufficient to permit successful secondary sympatry, and that divergence is initiated in allopatry. In this context, questions arise regarding the nature, timing, and origin of isolating mechanisms that permit secondary sympatry. We present a phylogeny of the crab subfamily Chlorodiellinae which displays allopatric mosaics within species. These allopatric lineages typically do not have divergent male genitalia, while older sympatric lineages do. We tested the relationship between genetic distance (proxy for time), sympatry, and the divergence of male genitalic morphology. Our results suggest that male genitalic divergence is not involved in the initiation of speciation in chlorodielline crabs, having likely occurred only after isolation began in allopatry. However, morphological evolution of genitalia seemingly does play an important role in completing the process of speciation in these crabs.
Asunto(s)
Braquiuros , Animales , Masculino , Filogenia , Especiación Genética , Simpatría , GenitalesRESUMEN
Five species of shrimps, four carideans and one stenopodidean, are recorded for the first time from the Caribbean coast of Panama: Lysmata vittata (Stimpson, 1860) [Lysmatidae Dana, 1852], Periclimenaeus ascidiarum Holthuis, 1951, P. bredini Chace, 1972, P. maxillulidens (Schmitt, 1936) [Palaemonidae Rafinesque, 1815], and Odontozona edyli Criales Lemaitre, 2017 [Stenopodidae Claus, 1872]. Rather surprisingly, L. vittata is recorded from the Caribbean Sea for the first time. However, the taxonomic status of all western Atlantic specimens currently assigned to L. vittata (including the Panamanian material and the Brazilian L. rauli Laubenheimer Rhyne, 2010) will need a much more careful reassessment, which will only be possible after determining the taxonomic identity of L. vittata in the Indo-West Pacific. The colour patterns of P. ascidiarum, P. bredini and O. edyli, herein illustrated for the first time, appear to be species-diagnostic and may serve as additional important taxonomic characters. For O. edyli, the previously unknown thoracic sternum of the female is illustrated, as well as the variation in the rostral dentition.
Asunto(s)
Decápodos , Animales , Región del Caribe , Femenino , Palaemonidae , PanamáRESUMEN
Since the seminal reports of adenosine receptor-mediated cardioprotection in the early 1990s, there have been a multitude of such reports in various species and preparations. Original observations of the beneficial effects of A1 receptor agonists have been followed up with numerous reports also implicating A2A, A3, and most recently A2B, receptor agonists as cardioprotective agents. Although adenosine has been approved for clinical use in the United States for the treatment of supraventricular tachycardia and coronary artery imaging, and the selective A2A agonist, regadenoson, for the latter, clinical use of adenosine receptor agonists for protecting the ischemic heart has not advanced beyond early trials. An examination of the literature indicates that existing experimental studies have several limitations in terms of clinical relevance, as well as lacking incorporation of recent new insights into adenosine receptor signaling. Such deficiencies include the lack of experimental studies in models that most closely mimic human cardiovascular disease. In addition, there have been very few studies in chronic models of myocardial ischemia, where limiting myocardial remodeling and heart failure, not reduction of infarct size, are the primary endpoints. Despite an increasing number of reports of the beneficial effects of adenosine receptor antagonists, not agonists, in chronic diseases, this idea has not been well-studied in experimental myocardial ischemia. There have also been few studies examining adenosine receptor subtype interactions as well as receptor heterodimerization. The purpose of this Perspective article is to discuss these deficiencies to highlight future directions of research in the field of adenosine receptor-mediated protection of ischemic myocardium.
RESUMEN
UNLABELLED: Membrane cholesterol levels play an important factor in regulating cell function. Sarcolemmal cholesterol is concentrated in lipid rafts and caveolae, which are flask-shaped invaginations of the plasma membrane. The scaffolding protein caveolin permits the enrichment of cholesterol in caveolae, and caveolin interactions with numerous proteins regulate their function. The purpose of this study was to determine whether acute reductions in cardiomyocyte cholesterol levels alter subcellular protein kinase activation, intracellular Ca2+ and contractility. METHODS: Ventricular myocytes, isolated from adult Sprague Dawley rats, were treated with the cholesterol reducing agent methyl-ß-cyclodextrin (MßCD, 5 mM, 1 hr, room temperature). Total cellular cholesterol levels, caveolin-3 localization, subcellular, ERK and p38 mitogen activated protein kinase (MAPK) signaling, contractility, and [Ca2+]i were assessed. RESULTS: Treatment with MßCD reduced cholesterol levels by ~45 and shifted caveolin-3 from cytoskeleton and triton-insoluble fractions to the triton-soluble fraction, and increased ERK isoform phosphorylation in cytoskeletal, cytosolic, triton-soluble and triton-insoluble membrane fractions without altering their subcellular distributions. In contrast the primary effect of MßCD was on p38 subcellular distribution of p38α with little effect on p38 phosphorylation. Cholesterol depletion increased cardiomyocyte twitch amplitude and the rates of shortening and relaxation in conjunction with increased diastolic and systolic [Ca2+]i. CONCLUSIONS: These results indicate that acute reductions in membrane cholesterol levels differentially modulate basal cardiomyocyte subcellular MAPK signaling, as well as increasing [Ca2+]i and contractility.
Asunto(s)
Colesterol/metabolismo , Contracción Miocárdica/fisiología , Miocitos Cardíacos/metabolismo , Transducción de Señal , Animales , Calcio/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Espacio Intracelular/metabolismo , Masculino , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/enzimología , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Fracciones Subcelulares/metabolismo , beta-Ciclodextrinas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Although there is a growing interest in the molecular cross-talk between the endocrine and cardiovascular systems, the cardiac effects of calcium-regulating hormones (i.e., parathyroid hormone-related peptide (PTHrP)) have not been explored. In this study, we examined the effect of PTHrP on the viability of isolated adult mouse cardiomyocytes subjected to oxidative stress. Myocytes from 19 to 22 week old male 129J/C57BL6 mice were exposed to oxidative insult in the form of H2O2 which led to more than 70% loss of cell viability. Herein we demonstrate, for the first time, that pretreatment with 100 nM PTHrP prior to 100 µM H2O2 incubation prevents H2O2 -induced cell death by more than 50%. Immunoblot analysis revealed H2O2 induction of MKP-1 protein expression while PTHrP decreased MKP-1 expression. Moreover, myocytes derived from MKP1 KO mice were resistant to oxidative injury. No added benefit of PTHrP treatment was noted in MKP-1 null cardiomyocytes. Using specific pharmacological inhibitors we demonstrated that P-p38, P-ERK and P-AKT mediated PTHrP's cardioprotective action. These data provide novel evidence that: i) down-regulation of MKP1 affords profound protection against oxidative stress; and ii) PTHrP is cardioprotective, possibly via down-regulation of MKP-1 and activation of MAPK and PI3K/AKT signaling.
Asunto(s)
Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Estrés Oxidativo , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Animales , Muerte Celular , Células Cultivadas , Fosfatasa 1 de Especificidad Dual/genética , Fosfatasa 1 de Especificidad Dual/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de SeñalRESUMEN
Bone marrow-derived endothelial progenitor cells (EPCs) contribute to neovessel formation in response to growth factors, cytokines, and chemokines. Chemokine receptor CXCR2 and its cognate ligands are reported to mediate EPC recruitment and angiogenesis. CXCR2 possesses a consensus PSD-95/DlgA/ZO-1 (PDZ) motif which has been reported to modulate cellular signaling and functions. Here we examined the potential role of the PDZ motif in CXCR2-mediated EPC motility and angiogenesis. We observed that exogenous CXCR2 C-tail significantly inhibited in vitro EPC migratory responses and angiogenic activities, as well as in vivo EPC angiogenesis. However, the CXCR2 C-tail that lacks the PDZ motif (ΔTTL) did not cause any significant changes of these functions in EPCs. In addition, using biochemical assays, we demonstrated that the PDZ scaffold protein NHERF1 specifically interacted with CXCR2 and its downstream effector, PLC-ß3, in EPCs. This suggests that NHERF1 might cluster CXCR2 and its relevant signaling molecules into a macromolecular signaling complex modulating EPC cellular functions. Taken together, our data revealed a critical role of a PDZ-based CXCR2 macromolecular complex in EPC homing and angiogenesis, suggesting that targeting this complex might be a novel and effective strategy to treat angiogenesis-dependent diseases.
Asunto(s)
Células Progenitoras Endoteliales/citología , Células Progenitoras Endoteliales/metabolismo , Receptores de Interleucina-8B/metabolismo , Animales , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Células Cultivadas , Humanos , Ratones , Ratones Endogámicos C57BL , Neovascularización Fisiológica/fisiología , Dominios PDZ , Transducción de Señal , TransfecciónRESUMEN
Renin is synthesized and released from juxtaglomerular (JG) cells. Adenosine inhibits renin release via an adenosine A1 receptor (A1R) calcium-mediated pathway. How this occurs is unknown. In cardiomyocytes, adenosine increases intracellular calcium via transient receptor potential canonical (TRPC) channels. We hypothesized that adenosine inhibits renin release via A1R activation, opening TRPC channels. However, higher concentrations of adenosine may stimulate renin release through A2R activation. Using primary cultures of isolated mouse JG cells, immunolabeling demonstrated renin and A1R in JG cells, but not A2R subtypes, although RT-PCR indicated the presence of mRNA of both A2AR and A2BR. Incubating JG cells with increasing concentrations of adenosine decreased renin release. Different concentrations of the adenosine receptor agonist N-ethylcarboxamide adenosine (NECA) did not change renin. Activating A1R with 0.5 µM N6-cyclohexyladenosine (CHA) decreased basal renin release from 0.22 ± 0.05 to 0.14 ± 0.03 µg of angiotensin I generated per milliliter of sample per hour of incubation (AngI/ml/mg prot) (P < 0.03), and higher concentrations also inhibited renin. Reducing extracellular calcium with EGTA increased renin release (0.35 ± 0.08 µg AngI/ml/mg prot; P < 0.01), and blocked renin inhibition by CHA (0.28 ± 0.06 µg AngI/ml/mg prot; P < 0. 005 vs. CHA alone). The intracellular calcium chelator BAPTA-AM increased renin release by 55%, and blocked the inhibitory effect of CHA. Repeating these experiments in JG cells from A1R knockout mice using CHA or NECA demonstrated no effect on renin release. However, RT-PCR showed mRNA from TRPC isoforms 3 and 6 in isolated JG cells. Adding the TRPC blocker SKF-96365 reversed CHA-mediated inhibition of renin release. Thus A1R activation results in a calcium-dependent inhibition of renin release via TRPC-mediated calcium entry, but A2 receptors do not regulate renin release.
Asunto(s)
Adenosina/fisiología , Angiotensina II/fisiología , Aparato Yuxtaglomerular/metabolismo , Glomérulos Renales/metabolismo , Receptor de Adenosina A1/fisiología , Renina/antagonistas & inhibidores , Transducción de Señal/fisiología , Canales de Potencial de Receptor Transitorio/fisiología , Animales , Femenino , Glomérulos Renales/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Renina/metabolismoRESUMEN
G protein coupled receptors play crucial roles in mediating cellular responses to external stimuli, and increasing evidence suggests that they function as multiple units comprising homo/heterodimers and hetero-oligomers. Adenosine and ß-adrenergic receptors are co-expressed in numerous tissues and mediate important cellular responses to the autocoid adenosine and sympathetic stimulation, respectively. The present study was undertaken to examine whether adenosine A1ARs heterodimerize with ß1- and/or ß2-adrenergic receptors (ß1R and ß2R), and whether such interactions lead to functional consequences. Co-immunoprecipitation and co-localization studies with differentially epitope-tagged A1, ß1, and ß2 receptors transiently co-expressed in HEK-293 cells indicate that A1AR forms constitutive heterodimers with both ß1R and ß2R. This heterodimerization significantly influenced orthosteric ligand binding affinity of both ß1R and ß2R without altering ligand binding properties of A1AR. Receptor-mediated ERK1/2 phosphorylation significantly increased in cells expressing A1AR/ß1R and A1AR/ß2R heteromers. ß-Receptor-mediated cAMP production was not altered in A1AR/ß1R expressing cells, but was significantly reduced in the A1AR/ß2R cells. The inhibitory effect of the A1AR on cAMP production was abrogated in both A1AR/ß1R and A1AR/ß2R expressing cells in response to the A1AR agonist CCPA. Co-immunoprecipitation studies conducted with human heart tissue lysates indicate that endogenous A1AR, ß1R, and ß2R also form heterodimers. Taken together, our data suggest that heterodimerization between A1 and ß receptors leads to altered receptor pharmacology, functional coupling, and intracellular signaling pathways. Unique and differential receptor cross-talk between these two important receptor families may offer the opportunity to fine-tune crucial signaling responses and development of more specific therapeutic interventions.
Asunto(s)
Receptor de Adenosina A1/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Agonistas del Receptor de Adenosina A1/farmacología , AMP Cíclico/metabolismo , Dimerización , Células HEK293 , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Miocardio/metabolismo , Fosforilación , Unión Proteica , Receptor de Adenosina A1/química , Receptor de Adenosina A1/genética , Receptores Adrenérgicos beta 1/química , Receptores Adrenérgicos beta 1/genética , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/genética , Proteínas Recombinantes/análisis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Transducción de Señal/efectos de los fármacos , TransfecciónRESUMEN
Adenosine is a purine nucleoside, which is produced primarily through the metabolism of adenosine triphosphate (ATP), therefore its levels increase during stressful situations when ATP utilization increases. Adenosine exerts potent cardioprotective effects on the ischemic/reperfused heart, reducing reversible and irreversible myocardial injury. Adenosine receptors (ARs) are G-protein-coupled receptors, and 4 subtypes exist--A(1), A(2A), A(2B), and A(3), all of which have been shown to be cardioprotective. Adenosine receptors are expressed on multiple cardiac cells, including fibroblasts, endothelial cells, smooth muscle cells, and myocytes. Activation of both A(1) and A(3) receptors prior to ischemia has been shown in multiple experimental models to reduce ischemia/reperfusion-induced cardiac injury. Additionally, activation of the A(2A) receptor at the onset of reperfusion has been shown to reduce injury. Most recently, there is evidence that the A(2B) receptor has cardioprotective effects upon its activation. However, controversy remains regarding the precise timing of activation of these receptors required to induce cardioprotection, as well as their involvement in ischemic preconditioning and postconditioning. Adenosine receptors have been suggested to reduce cell death through actions at the mitochondrial ATP-dependent potassium (K(ATP)) channel, as well as protein kinase C and mitogen-activated protein kinase (MAPK) signaling. Additionally, the ability of ARs to interact has been documented, and several recent reports suggest that these interactions play a role in AR-mediated cardioprotection. This review summarizes the current knowledge of the cardioprotective effects of each AR subtype, as well as the proposed mechanisms of AR cardioprotection. Additionally, the role of AR interactions in cardioprotection is discussed.
Asunto(s)
Cardiotónicos/uso terapéutico , Receptor de Adenosina A1 , Receptor de Adenosina A2A , Receptor de Adenosina A2B , Receptor de Adenosina A3 , Agonistas del Receptor de Adenosina A2/farmacología , Agonistas del Receptor de Adenosina A2/uso terapéutico , Animales , Cardiotónicos/farmacología , Enfermedades Cardiovasculares/tratamiento farmacológico , Enfermedades Cardiovasculares/metabolismo , Modelos Animales de Enfermedad , Humanos , Receptor de Adenosina A1/metabolismo , Receptor de Adenosina A2A/metabolismo , Receptor de Adenosina A2B/metabolismo , Receptor de Adenosina A3/metabolismoRESUMEN
All four adenosine receptor subtypes have been shown to play a role in cardioprotection, and there is evidence that all four subtypes may be expressed in cardiomyocytes. There is also increasing evidence that optimal adenosine cardioprotection requires the activation of more than one receptor subtype. The purpose of this study was to determine whether adenosine A(2A) and/or A(2B) receptors modulate adenosine A(1) receptor-mediated cardioprotection. Isolated perfused hearts of wild-type (WT), A(2A) knockout (KO), and A(2B)KO mice, perfused at constant pressure and constant heart rate, underwent 30 min of global ischemia and 60 min of reperfusion. The adenosine A(1) receptor agonist N(6)-cyclohexyladenosine (CHA; 200 nM) was administrated 10 min before ischemia and for the first 10 min of reperfusion. Treatment with CHA significantly improved postischemic left ventricular developed pressure (74 ± 4% vs. 44 ± 4% of preischemic left ventricular developed pressure at 60 min of reperfusion) and reduced infarct size (30 ± 2% with CHA vs. 52 ± 5% in control) in WT hearts, effects that were blocked by the A(1) antagonist 8-cyclopentyl-1,3-dipropylxanthine (100 nM). Treatments with the A(2A) receptor agonist CGS-21680 (200 nM) and the A(2B) agonist BAY 60-6583 (200 nM) did not exert any beneficial effects. Deletion of adenosine A(2A) or A(2B) receptor subtypes did not alter ischemia-reperfusion injury, but CHA failed to exert a cardioprotective effect in hearts of mice from either KO group. These findings indicate that both adenosine A(2A) and A(2B) receptors are required for adenosine A(1) receptor-mediated cardioprotection, implicating a role for interactions among receptor subtypes.
Asunto(s)
Agonistas del Receptor de Adenosina A1/farmacología , Adenosina/análogos & derivados , Infarto del Miocardio/prevención & control , Daño por Reperfusión Miocárdica/prevención & control , Miocardio/metabolismo , Receptor de Adenosina A1/efectos de los fármacos , Receptor de Adenosina A2A/metabolismo , Receptor de Adenosina A2B/metabolismo , Adenosina/farmacología , Antagonistas del Receptor de Adenosina A1/farmacología , Agonistas del Receptor de Adenosina A2/farmacología , Análisis de Varianza , Animales , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infarto del Miocardio/genética , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Daño por Reperfusión Miocárdica/genética , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/fisiopatología , Miocardio/patología , Perfusión , Receptor Cross-Talk , Receptor de Adenosina A1/metabolismo , Receptor de Adenosina A2A/deficiencia , Receptor de Adenosina A2A/genética , Receptor de Adenosina A2B/deficiencia , Receptor de Adenosina A2B/genética , Factores de Tiempo , Función Ventricular Izquierda/efectos de los fármacos , Presión Ventricular/efectos de los fármacosRESUMEN
The presence of sex differences in myocardial ß-adrenergic responsiveness is controversial, and limited studies have addressed the mechanism underlying these differences. Studies were performed using isolated perfused hearts from male, intact female and ovariectomized female mice to investigate sex differences and the effects of ovarian hormone withdrawal on ß-adrenergic receptor function. Female hearts exhibited blunted contractile responses to the ß-adrenergic receptor agonist isoproterenol (ISO) compared with males but not ovariectomized females. There were no sex differences in ß(1)-adrenergic receptor gene or protein expression. To investigate the role of adenylyl cyclase, phosphodiesterase, and the cAMP-signaling cascade in generating sex differences in the ß-adrenergic contractile response, dose-response studies were performed in isolated perfused male and female hearts using forskolin, 3-isobutyl-1-methylxanthine (IBMX), and 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP). Males showed a modestly enhanced contractile response to forskolin at 300 nM and 5 µM compared with females, but there were no sex differences in the response to IBMX or CPT-cAMP. The role of the A(1) adenosine receptor (A(1)AR) in antagonizing the ß-adrenergic contractile response was investigated using both the A(1)AR agonist 2-chloro-N(6)-cyclopentyl-adenosine and A(1)AR knockout (KO) mice. Intact females showed an enhanced A(1)AR anti-adrenergic effect compared with males and ovariectomized females. The ß-adrenergic contractile response was potentiated in both male and female A(1)ARKO hearts, with sex differences no longer present above 1 nM ISO. The ß-adrenergic contractile response is greater in male hearts than females, and minor differences in the action of adenylyl cyclase or the A(1)AR may contribute to these sex differences.
Asunto(s)
Agonistas Adrenérgicos beta/farmacología , Isoproterenol/farmacología , Contracción Miocárdica/efectos de los fármacos , Miocardio/metabolismo , Ovariectomía , Receptores Adrenérgicos beta/efectos de los fármacos , Agonistas del Receptor de Adenosina A1/farmacología , Adenilil Ciclasas/metabolismo , Análisis de Varianza , Animales , Peso Corporal , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Activadores de Enzimas/farmacología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Perfusión , Inhibidores de Fosfodiesterasa/farmacología , Hidrolasas Diéster Fosfóricas/metabolismo , ARN Mensajero/metabolismo , Receptor de Adenosina A1/deficiencia , Receptor de Adenosina A1/efectos de los fármacos , Receptor de Adenosina A1/genética , Receptores Adrenérgicos beta/genética , Receptores Adrenérgicos beta/metabolismo , Factores Sexuales , Transducción de Señal/efectos de los fármacosRESUMEN
Adenosine receptors are a member of the large family of seven transmembrane spanning G protein coupled receptors. The four adenosine receptor subtypes-A(1), A(2a), A(2b), A(3)-exert their effects via the activation of one or more heterotrimeric G proteins resulting in the modulation of intracellular signaling. Numerous studies over the past decade have documented the complexity of G protein coupled receptor signaling at the level of protein-protein interactions as well as through signaling cross talk. With respect to adenosine receptors, the activation of one receptor subtype can have profound direct effects in one cell type but little or no effect in other cells. There is significant evidence that the compartmentation of subcellular signaling plays a physiological role in the fidelity of G protein coupled receptor signaling. This compartmentation is evident at the level of the plasma membrane in the form of membrane microdomains such as caveolae and lipid rafts. This review will summarize and critically assess our current understanding of the role of membrane microdomains in regulating adenosine receptor signaling.
Asunto(s)
Microdominios de Membrana , Receptores Purinérgicos P1/metabolismo , Animales , Humanos , Transducción de SeñalRESUMEN
The mammalian myocardium expresses four adenosine receptor (AR) subtypes: A(1)AR, A(2a)AR, A(2b)AR, and A(3)AR. The A(1)AR is well known for its profound antiadrenergic effects, but the roles of other AR subtypes in modulating contractility remain inconclusive. Thus, the objective of this study was to determine the direct and indirect effects of A(2a)AR and A(2b)AR on cardiac contractility. Experiments were conducted in paced, constant pressure-perfused isolated hearts from wild-type (WT), A(2a)AR knockout (KO), and A(2b)AR KO mice. The A(2a)AR agonist CGS-21680 did not alter basal contractility or ß-adrenergic receptor agonist isoproterenol (Iso)-mediated positive inotropic responses, and Iso-induced effects were unaltered in A(2a)AR KO hearts. However, A(2a)AR gene ablation resulted in a potentiation of the antiadrenergic effects mediated by the A(1)AR agonist 2-chloro-N-cyclopentyladenosine. The nonselective AR agonist 5'-N-ethylcarboxamido adenosine and the selective A(2b)AR agonist BAY 60-6583 induced coronary flow-independent increases in contractility, but BAY 60-6583 did not alter Iso-induced contractile responses. The A(1)AR antiadrenergic effect was not potentiated in A(2b)AR KO hearts. The expression of all four AR subtypes in the heart and ventricular myocytes was confirmed using real-time quantitative PCR. Taken together, these results indicate that A(2a)AR does not increase cardiac contractility directly but indirectly alters contractility by modulating the A(1)AR antiadrenergic effect, whereas A(2b)AR exerts direct contractile effects but does not alter ß-adrenergic or A(1)AR antiadrenergic effects. These results indicate that multiple ARs differentially modulate cardiac function.
Asunto(s)
Contracción Miocárdica , Miocardio/metabolismo , Receptor de Adenosina A2A/metabolismo , Receptor de Adenosina A2B/metabolismo , Agonistas del Receptor de Adenosina A2/farmacología , Agonistas Adrenérgicos beta/farmacología , Animales , Estimulación Cardíaca Artificial , Circulación Coronaria , Regulación de la Expresión Génica , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Contracción Miocárdica/efectos de los fármacos , Perfusión , ARN Mensajero/metabolismo , Receptor de Adenosina A1/genética , Receptor de Adenosina A2A/deficiencia , Receptor de Adenosina A2A/efectos de los fármacos , Receptor de Adenosina A2A/genética , Receptor de Adenosina A2B/deficiencia , Receptor de Adenosina A2B/efectos de los fármacos , Receptor de Adenosina A2B/genética , Receptor de Adenosina A3/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Adenosine, a catabolite of ATP, exerts numerous effects in the heart, including modulation of the cardiac response to stress, such as that which occurs during myocardial ischemia and reperfusion. Over the past 20 years, substantial evidence has accumulated that adenosine, administered either prior to ischemia or during reperfusion, reduces both reversible and irreversible myocardial injury. The latter effect results in a reduction of both necrosis or myocardial infarction (MI) and apoptosis. These effects appear to be mediated via the activation of one or more G-protein-coupled receptors (GPCRs), referred to as A(1), A(2A), A(2B) and A(3) adenosine receptor (AR) subtypes. Experimental studies in different species and models suggest that activation of the A(1) or A(3)ARs prior to ischemia is cardioprotective. Further experimental studies reveal that the administration of A(2A)AR agonists during reperfusion can also reduce MI, and recent reports suggest that A(2B)ARs may also play an important role in modulating myocardial reperfusion injury. Despite convincing experimental evidence for AR-mediated cardioprotection, there have been only a limited number of clinical trials examining the beneficial effects of adenosine or adenosine-based therapeutics in humans, and the results of these studies have been equivocal. This review summarizes our current knowledge of AR-mediated cardioprotection, and the roles of the four known ARs in experimental models of ischemia-reperfusion. The chapter concludes with an examination of the clinical trials to date assessing the safety and efficacy of adenosine as a cardioprotective agent during coronary thrombolysis in humans.
Asunto(s)
Daño por Reperfusión Miocárdica/prevención & control , Receptores Purinérgicos P1/fisiología , Agonistas del Receptor de Adenosina A1 , Animales , Humanos , Precondicionamiento Isquémico Miocárdico , Receptor de Adenosina A2A/fisiología , Receptor de Adenosina A2B/fisiología , Receptor de Adenosina A3/fisiologíaRESUMEN
The mechanism by which distinct stimuli activate the same mitogen-activated protein kinases (MAPKs) is unclear. We examined compartmentalized MAPK signaling and altered redox state as possible mechanisms. Adult rat cardiomyocytes were exposed to the adenosine A(1) receptor agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA; 500 nM) or H(2)O(2) (100 microM) for 15 min. Nuclear/myofilament, cytosolic, Triton-soluble membrane, and Triton-insoluble membrane fractions were generated. CCPA and H(2)O(2) activated p38 MAPK and p44/p42 ERKs in cytosolic fractions. In Triton-soluble membrane fractions, H(2)O(2) activated p38 MAPK and p42 ERK, whereas CCPA had no effect on MAPK activation in this fraction. The greatest difference between H(2)O(2) and CCPA was in the Triton-insoluble membrane fraction, where H(2)O(2) increased p38 and p42 activation and CCPA reduced MAPK activation. CCPA also increased protein phosphatase 2A activity in the Triton-insoluble membrane fraction, suggesting that the activation of this phosphatase may mediate CCPA effects in this fraction. The Triton-insoluble membrane fraction was enriched in the caveolae marker caveolin-3, and >85% of p38 MAPK and p42 ERK was bound to this scaffolding protein in these membranes, suggesting that caveolae may play a role in the divergence of MAPK signals from different stimuli. The antioxidant N-2-mercaptopropionyl glycine (300 microM) reduced H(2)O(2)-mediated MAPK activation but failed to attenuate CCPA-induced MAPK activation. H(2)O(2) but not CCPA increased reactive oxygen species (ROS). Thus the adenosine A(1) receptor and oxidative stress differentially modulate subcellular MAPKs, with the main site of divergence being the Triton-insoluble membrane fraction. However, the adenosine A(1) receptor-mediated MAPK activation does not involve ROS formation.
Asunto(s)
Agonistas del Receptor de Adenosina A1 , Adenosina/análogos & derivados , Peróxido de Hidrógeno/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Oxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Adenosina/farmacología , Animales , Antioxidantes/farmacología , Caveolina 3/metabolismo , Membrana Celular/enzimología , Membrana Celular/metabolismo , Núcleo Celular/enzimología , Núcleo Celular/metabolismo , Citosol/enzimología , Citosol/metabolismo , Detergentes , Activación Enzimática , Técnicas In Vitro , Masculino , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/metabolismo , Octoxinol , Oxidación-Reducción , Proteína Fosfatasa 2/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Receptor de Adenosina A1/metabolismo , Fracciones Subcelulares/enzimología , Fracciones Subcelulares/metabolismo , Tiopronina/farmacologíaRESUMEN
Mechanisms of adenosine (ADO) protection of reperfused myocardium are not fully understood. We tested the hypothesis that ADO (0.1 mM) alleviates ventricular stunning by ADO A(1)-receptor stimulation combined with purine metabolic enhancements. Langendorff guinea pig hearts were stunned at constant left ventricular end-diastolic pressure by low-flow ischemia. Myocardial phosphate metabolites were measured by (31)P-NMR, with phosphorylation potential {[ATP]/([ADP].[P(i)]), where brackets indicate concentration} estimated from creatine kinase equilibrium. Creatine and IMP, glycolytic intermediates, were measured enzymatically and glycolytic flux and extracellular spaces were measured by radiotracers. All treatment interventions started after a 10-min normoxic stabilization period. At 30 min reperfusion, ventricular contractility (dP/dt, left ventricular pressure) was reduced 17-26%, ventricular power (rate-pressure product) by 37%, and [ATP]/([ADP].[P(i)]) by 53%. The selective A(1) agonist 2-chloro-N(6)-cyclo-pentyladenosine marginally preserved [ATP]/([ADP].[P(i)]) and ventricular contractility but not rate-pressure product. Purine salvage precursor inosine (0.1 mM) substantially raised [ATP]/([ADP].[P(i)]) but weakly affected contractility. The ATP-sensitive potassium channel blocker glibenclamide (50 microM) abolished ADO protection of [ATP]/([ADP].[P(i)]) and contractility. ADO raised myocardial IMP and glucose-6-phosphate, demonstrating increased purine salvage and pentose phosphate pathway flux potential. Coronary hyperemia alone (papaverine) was not cardioprotective. We found that ADO protected energy metabolism and contractility in stunned myocardium more effectively than both the A(1)-receptor agonist 2-chloro-N(6)-cyclo-pentyladenosine and the purine salvage precursor inosine. Because ADO failed to stimulate glycolytic flux, the enhancement of reperfusion, [ATP]/([ADP].[P(i)]), indicates protection of mitochondrial function. Reduced ventricular dysfunction at enhanced [ATP]/([ADP].[P(i)]) argues against opening of mitochondrial ATP-sensitive potassium channel. The results establish a multifactorial mechanism of ADO antistunning, which appears to combine ADO A(1)-receptor signaling with metabolic adenylate and antioxidant enhancements.
Asunto(s)
Adenosina Trifosfato/metabolismo , Adenosina/fisiología , Contracción Miocárdica/fisiología , Vía de Pentosa Fosfato/fisiología , Función Ventricular Izquierda/fisiología , Adenosina/antagonistas & inhibidores , Animales , Circulación Coronaria/fisiología , Citosol/metabolismo , Metabolismo Energético/fisiología , Femenino , Glucosa-6-Fosfato/metabolismo , Cobayas , Frecuencia Cardíaca/fisiología , Técnicas In Vitro , Inosina Monofosfato/metabolismo , Ácido Láctico/metabolismo , Reperfusión Miocárdica , Miocardio/metabolismo , Fosfatos/metabolismo , Fosforilación , Canales de Potasio/metabolismoRESUMEN
There is increasing evidence for interactions among adenosine receptor subtypes in the brain and heart. The purpose of this study was to determine whether the adenosine A(2a) receptor modulates the infarct size-reducing effect of preischemic administration of adenosine receptor agonists in intact rat myocardium. Adult male rats were submitted to in vivo regional myocardial ischemia (25 min) and 2 h reperfusion. Vehicle-treated rats were compared with rats pretreated with the A(1) agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA, 10 mug/kg), the nonselective agonist 5'-N-ethylcarboxamidoadenosine (NECA, 10 mug/kg), or the A(2a) agonist 2-[4-(2-carboxyethyl)phenethylamino]-5'-N-methylcarboxamidoadenosine (CGS-21680, 20 mug/kg). Additional CCPA- and NECA-treated rats were pretreated with the A(1) antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 100 mug/kg), the A(2a)/A(2b) antagonist 4-(-2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-a} {1,3,5}triazin-5-yl-amino]ethyl)phenol (ZM-241385, 1.5 mg/kg) or the A(3) antagonist 3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine carboxylate (MRS-1523, 2 mg/kg). CCPA and NECA reduced myocardial infarct size by 50% and 35%, respectively, versus vehicle, but CGS-21680 had no effect. DPCPX blunted the bradycardia associated with CCPA and NECA, whereas ZM-241385 attenuated their hypotensive effects. Both DPCPX and ZM-241385 blocked the protective effects of CCPA and NECA. The A(3) antagonist did not alter the hemodynamic effects of CCPA or NECA, nor did it alter adenosine agonist cardioprotection. None of the antagonists alone altered myocardial infarct size. These findings suggest that although preischemic administration of an A(2a) receptor agonist does not induce cardioprotection, antagonism of the A(2a) and/or the A(2b) receptor blocks the cardioprotection associated with adenosine agonist pretreatment.
Asunto(s)
Antagonistas del Receptor de Adenosina A2 , Cardiotónicos/administración & dosificación , Isquemia Miocárdica/fisiopatología , Premedicación/métodos , Agonistas del Receptor Purinérgico P1 , Triazinas/administración & dosificación , Triazoles/administración & dosificación , Animales , Combinación de Medicamentos , Masculino , Isquemia Miocárdica/patología , Isquemia Miocárdica/prevención & control , Ratas , Ratas Sprague-Dawley , Resultado del TratamientoRESUMEN
Ischemia-reperfusion activates ERK and p38 MAPK in cardiac membranes, but the role of caveolae in MAPK signaling during this stress has not been studied. The purpose of this study was to determine the effect of in vivo myocardial ischemia-reperfusion on the level and distribution of caveolin-1 and -3 and cholesterol as well as MAPK activation in caveolin-enriched fractions. Adult male rats were subjected to in vivo regional myocardial ischemia induced by 25 min of coronary artery occlusion and 10 min (n = 5) or 2 h (n = 4) of reperfusion. Another group of rats served as appropriate nonischemic time controls (n = 4). A discontinuous sucrose density gradient was used to isolate caveolae/lipid rafts from ischemic and nonischemic heart tissue. Caveolin-1 and -3, as well as cholesterol, were enriched in the light fractions. A redistribution of caveolin-3 and a reduction in caveolin-1 and cholesterol levels in the light fractions occurred after 10 min of reperfusion. The ERKs were activated in ischemic zone light and heavy fractions by 10 min of reperfusion. p44 ERK was activated after 2 h of reperfusion only in the light fractions, whereas p42 ERK phosphorylation was increased in the light and heavy fractions. Although no p38 MAPK activation occurred after 10 min of reperfusion, 2 h of reperfusion caused significant activation of p38 MAPK in nonischemic zone light and heavy fractions. These results show the importance of caveolar membrane/lipid rafts in MAPK signaling and suggest that subcellular compartmentation of p44/p42 ERKs and p38 MAPK may play distinct roles in the response to myocardial ischemia-reperfusion.