RESUMEN
The circadian clock is a cellular mechanism that synchronizes various biological processes with respect to the time of the day. While much progress has been made characterizing the molecular mechanisms underlying this clock, it is less clear how external light cues influence the dynamics of the core clock mechanism and thereby entrain it with the light-dark cycle. Zebrafish-derived cell cultures possess clocks that are directly light-entrainable, thus providing an attractive laboratory model for circadian entrainment. Here, we have developed a stochastic oscillator model of the zebrafish circadian clock, which accounts for the core clock negative feedback loop, light input, and the proliferation of single-cell oscillator noise into population-level luminescence recordings. The model accurately predicts the entrainment dynamics observed in bioluminescent clock reporter assays upon exposure to a wide range of lighting conditions. Furthermore, we have applied the model to obtain refitted parameter sets for cell cultures exposed to a variety of pharmacological treatments and predict changes in single-cell oscillator parameters. Our work paves the way for model-based, large-scale screens for genetic or pharmacologically-induced modifications to the entrainment of circadian clock function.
Asunto(s)
Ritmo Circadiano/fisiología , Modelos Biológicos , Pez Cebra , Animales , Animales Modificados Genéticamente , Butadienos/farmacología , Células Cultivadas , Ritmo Circadiano/efectos de los fármacos , Colforsina/farmacología , CMP Cíclico/análogos & derivados , CMP Cíclico/farmacología , Mediciones Luminiscentes , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Nitrilos/farmacología , Reproducibilidad de los Resultados , Análisis de la Célula Individual , Procesos Estocásticos , Pez Cebra/genéticaRESUMEN
STUDY QUESTION: Can whole exome sequencing (WES) reveal a novel pathogenic variant in asthenozoospermia in a multiplex family including multiple patients? SUMMARY ANSWER: Patients were discovered to be homozygous for a rare 2-bp deletion in the ADCY10 coding region (c.1205_1206del, rs779944215). WHAT IS KNOWN ALREADY: ADCY10 encodes for soluble adenylyl cyclase (sAC), which is the predominant adenylate cyclase in sperm. It is already established that proper sAC activity and a constant supply of cAMP are crucial to sperm motility regulation, and knockout mouse models have been reported as severely asthenozoospermic. ADCY10 is a susceptibility gene for dominant absorptive hypercalciuria (OMIM#143870); however, no ADCY10 variations have been confirmed to cause human asthenozoospermia to date. STUDY DESIGN, SIZE, DURATION: This was a retrospective genetics study of a highly consanguineous pedigree of asthenozoospermia. The subject family was recruited in Iran in 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS: The two patients were diagnosed as asthenozoospermic through careful clinical investigations. Both patients, respective parents, and an unaffected brother were subjected to WES. The discovered variant was validated by Sanger sequencing and segregated with the phenotype. To confirm the pathogenicity of the variant, sperm samples from both patients, 10 normozoospermic men and 10 asthenozoospermic patients not representing the variation, were treated with a cAMP analogue dissolved in human tubal fluid medium, followed by computer-assisted sperm analysis and statistical analyses. MAIN RESULTS AND THE ROLE OF CHANCE: The discovered homozygous variant occurs at 10 amino acids upstream of the ADCY10 nucleotide binding site leading to a premature termination (p.His402Argfs*41). Treatment of the patients' sperm samples with a cell-permeable cAMP analogue resulted in a significant increase in sperm motility, indicating the pathogenic role of the variant. Moreover, absorptive hypercalciuria, segregating within the family, was also associated with the same variant following a dominant inheritance. LIMITATIONS, REASONS FOR CAUTION: Though nonsense-mediated decay is highly likely to occur in the mutated transcripts, we were not able to confirm this due to low RNA levels in mature sperm. WIDER IMPLICATIONS OF THE FINDINGS: Our finding enlarges the phenotypic spectrum associated with the ADCY10 gene, previously described as a susceptibility gene for dominant absorptive hypercalciuria. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Royan Institute, Tehran, Iran, and San Raffaele Hospital, Milan, Italy. The authors have no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.
Asunto(s)
Adenilil Ciclasas/genética , Astenozoospermia/genética , Hipercalciuria/genética , Cálculos Renales/genética , Adulto , Astenozoospermia/diagnóstico , Calcio/orina , Consanguinidad , CMP Cíclico/análogos & derivados , CMP Cíclico/farmacología , Análisis Mutacional de ADN , Mutación del Sistema de Lectura , Homocigoto , Humanos , Hipercalciuria/diagnóstico , Hipercalciuria/orina , Irán , Cariotipificación , Cálculos Renales/diagnóstico , Cálculos Renales/orina , Masculino , Linaje , Motilidad Espermática/efectos de los fármacos , Motilidad Espermática/genética , Resultado del TratamientoRESUMEN
Peptidylarginine deiminases (PADs) are posttranslational modification enzymes that citrullinate (deiminate) protein arginine residues in a calcium-dependent manner, yielding citrulline residues. Enzymatic citrullination abolishes positive charges of native protein molecules, inevitably causing significant alterations in their structure and function. Previously, we reported the abnormal accumulation of citrullinated proteins and an increase of PAD2 content in hippocampi of patients with Alzheimer disease. In this study, we investigated PAD expression by using dibutyryl cAMP (dbcAMP) in human astrocytoma U-251MG cells. Under normal culture conditions, PAD2 and PAD3 mRNA expression is detectable with quantitative PCR in U-251MG cells. The addition of dbcAMP in a dose-dependent manner significantly increased this mRNA expression and protein levels. Moreover, PAD enzyme activity also increased significantly and dose-dependently. Furthermore, the expression of PAD2 and PAD3 mRNA was inhibited by the cAMP-dependent PKA inhibitor KT5720, suggesting that such expression of dbcAMP-induced PAD2 and PAD3 mRNA is mediated by the cAMP-PKA signaling pathway in U-251MG cells. This is the first report to document the PAD2 and PAD3 mRNA expression induced by dbcAMP and to attribute the induction of these genes to mediation by the cAMP-PKA signaling pathway in U-251MG cells. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Astrocitoma/enzimología , Proteínas Quinasas Dependientes de AMP Cíclico/biosíntesis , CMP Cíclico/análogos & derivados , Desiminasas de la Arginina Proteica/biosíntesis , Transducción de Señal/fisiología , Línea Celular Tumoral , CMP Cíclico/farmacología , Relación Dosis-Respuesta a Droga , Inducción Enzimática/fisiología , Humanos , Inhibidores de Proteínas Quinasas/farmacología , Arginina Deiminasa Proteína-Tipo 2 , Arginina Deiminasa Proteína-Tipo 3 , Desiminasas de la Arginina Proteica/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacosRESUMEN
After decades of intensive research on adenosine-3',5'-cyclic monophosphate (cAMP)- and guanosine-3',5'-cyclic monophosphate (cGMP)-related second messenger systems, also the noncanonical congeners cyclic cytidine-3',5'-monophosphate (cCMP) and cyclic uridine-3',5'-monophosphate (cUMP) gained more and more interest. Until the late 1980s, only a small number of cCMP and cUMP analogs with sometimes undefined purities had been described. Moreover, most of these compounds had been rather synthesized as precursors of antitumor and antiviral nucleoside-5'-monophosphates and hence had not been tested for any second messenger activity. Along with the recurring interest in cCMP- and cUMP-related signaling in the early 2000s, it became evident that well-characterized small molecule analogs with reliable purities would serve as highly valuable tools for the evaluation of a putative second messenger role of cyclic pyrimidine nucleotides. Meanwhile, for this purpose new cCMP and cUMP derivatives have been developed, and already known analogs have been resynthesized and highly purified. This chapter summarizes early medicinal chemistry work on cCMP and cUMP and analogs thereof, followed by a description of recent synthetic developments and an outlook on potential future directions.
Asunto(s)
CMP Cíclico/síntesis química , Nucleótidos Cíclicos/síntesis química , Profármacos/síntesis química , Uridina Monofosfato/síntesis química , Animales , Cristalización , CMP Cíclico/análogos & derivados , CMP Cíclico/metabolismo , CMP Cíclico/farmacología , Humanos , Estructura Molecular , Nucleótidos Cíclicos/metabolismo , Nucleótidos Cíclicos/farmacología , Permeabilidad , Profármacos/metabolismo , Profármacos/farmacología , Uridina Monofosfato/metabolismo , Uridina Monofosfato/farmacologíaRESUMEN
STUDY QUESTION: Do peroxiredoxins (PRDXs) control reactive oxygen species (ROS) levels during human sperm capacitation? SUMMARY ANSWER: PRDXs are necessary to control the levels of ROS generated during capacitation allowing spermatozoa to achieve fertilizing ability. WHAT IS KNOWN ALREADY: Sperm capacitation is an oxidative event that requires low and controlled amounts of ROS to trigger phosphorylation events. PRDXs are antioxidant enzymes that not only act as scavengers but also control ROS action in somatic cells. Spermatozoa from infertile men have lower levels of PRDXs (particularly of PRDX6), which are thiol-oxidized and therefore inactive. STUDY DESIGN, SIZE, DURATION: Semen samples were obtained from a cohort of 20 healthy nonsmoker volunteers aged 22-30 years old over a period of 1 year. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Sperm from healthy donors was capacitated with fetal cord serum ultrafiltrate (FCSu) in the absence or presence of thiostrepton (TSP), inhibitor of 2-Cys PRDXs or 1-Hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol lithium (MJ33), inhibitor of calcium independent-phospholipase A2 (Ca2+-iPLA2) activity of PRDX6, added at different times of incubation. Capacitation was also induced by the dibutyryl cAMP+3-isobuty1-1-methylxanthine system. Sperm viability and motility were determined by the hypo-osmotic swelling test and computer-assisted semen analysis system, respectively. Capacitation was determined by the ability of spermatozoa to undergo the acrosome reaction triggered by lysophosphatidylcholine. Percentages of acrosome reaction were obtained using the FITC-conjugated Pisum sativum agglutinin assay. Phosphorylation of tyrosine residues and of protein kinase A (PKA) substrates were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblotting with specific antibodies. Actin polymerization was determined by phalloidin labeling. MAIN RESULTS AND THE ROLE OF CHANCE: TSP and MJ33 prevented sperm capacitation and its associated actin polymerization in spermatozoa incubated with 10% FCSu (capacitation inducer) compared to non-capacitated controls (P < 0.05) without altering sperm viability. PKA substrates and tyrosine phosphorylations were prevented in FCSu-treated spermatozoa in a differential fashion depending on the type and the time of addition of the inhibitor used compared to non-capacitated controls (P < 0.05). TSP and MJ33 promoted an increase of lipid peroxidation in spermatozoa (P < 0.01) and these levels were higher in those spermatozoa incubated with the inhibitors and FCSu compared to those capacitated spermatozoa incubated without the inhibitors (P < 0.0001). Inhibition of 2-Cys PRDXs by TSP generated an oxidative stress in spermatozoa, affecting their viability compared to controls (P < 0.05). This oxidative stress was prevented by nuclephile D-penicillamine (PEN). MJ33 also promoted an increase of lipid peroxidation and impaired sperm viability compared to non-treated controls (P < 0.05) but its effect was not circumvented by PEN, suggesting that not only peroxidase but also Ca2+-iPLA2 activity of PRDX6 are necessary to guarantee viability in human spermatozoa. LARGE SCALE DATA: Not applicable. LIMITATIONS REASONS FOR CAUTION: We focused on the global effect of PRDXs inhibitors on human sperm capacitation and in two of its associated phosphorylation events. Thus, other phosphorylation events and mechanisms necessary for capacitation may also be affected. WIDER IMPLICATIONS OF THE FINDINGS: PRDXs are the major antioxidant system in ejaculated spermatozoa and are necessary to allow spermatozoon to achieve fertilizing ability (capacitation and acrosome reaction). STUDY FUNDING/COMPETING INTEREST(S): This research was supported by Canadian Institutes of Health Research (MOP 133661) and the Fonds de Recherché en Santé Quebec (FRSQS #22151) to C.O. The authors have nothing to disclose.
Asunto(s)
Peroxirredoxinas/genética , Especies Reactivas de Oxígeno/metabolismo , Capacitación Espermática/genética , Espermatozoides/enzimología , 1-Metil-3-Isobutilxantina/farmacología , Reacción Acrosómica/efectos de los fármacos , Adulto , Supervivencia Celular/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , CMP Cíclico/análogos & derivados , CMP Cíclico/farmacología , Sangre Fetal/química , Regulación de la Expresión Génica , Glicerofosfatos/farmacología , Humanos , Peroxidación de Lípido/efectos de los fármacos , Lisofosfatidilcolinas/farmacología , Masculino , Estrés Oxidativo , Penicilamina/farmacología , Peroxirredoxinas/antagonistas & inhibidores , Peroxirredoxinas/metabolismo , Fosforilación/efectos de los fármacos , Cultivo Primario de Células , Capacitación Espermática/efectos de los fármacos , Motilidad Espermática/efectos de los fármacos , Espermatozoides/citología , Espermatozoides/efectos de los fármacos , Espermatozoides/metabolismo , Tioestreptona/farmacologíaRESUMEN
AIM: To investigate whether the regulation of aquaporin 3 (AQP3) and AQP9 induced by Auphen and dibutyryl cAMP (dbcAMP) inhibits hepatic tumorigenesis. METHODS: Expression of AQP3 and AQP9 was detected by Western blot, immunohistochemistry (IHC), and RT-PCR in HCC samples and paired non-cancerous liver tissue samples from 30 hepatocellular carcinoma (HCC) patients. A xenograft tumor model was used in vivo. Nine nude mice were divided into control, Auphen-treated, and dbcAMP-treated groups (n = 3 for each group). AQP3 and AQP9 protein expression after induction of xenograft tumors was detected by IHC and mRNA by RT-PCR analysis. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and histological evaluation were used to detect apoptosis of tumor cells, and the concentration of serum α-fetoprotein (AFP) was measured using RT-PCR and an ELISA kit. RESULTS: The volumes and weights of tumors decreased significantly in the Auphen- and dbcAMP-treated mice compared with the control mice (P < 0.01). The levels of AQP3 were significantly lower in the Auphen treatment group, and levels of AQP9 were significantly higher in thedbcAMP treatment mice than in the control mice (P < 0.01). The reduction of AQP3 by Auphen and increase of AQP9 by dbcAMP in nude mice suppressed tumor growth of HCC, which resulted in reduced AFP levels in serum and tissues, and apoptosis of tumor cells in the Auphen- and dbcAMP-treated mice, when compared with control mice (P < 0.01). Compared with para-carcinoma tissues, AQP3 expression increased in tumor tissues whereas the expression of AQP9 decreased. By correlating clinicopathological and expression levels, we demonstrated that the expression of AQP3 and AQP9 was correlated with clinical progression of HCC and disease outcomes. CONCLUSION: AQP3 increases in HCC while AQP9 decreases. Regulation of AQP3 and AQP9 expression by Auphen and dbcAMP inhibits the development and growth of HCC.
Asunto(s)
Antineoplásicos/farmacología , Acuaporina 3/metabolismo , Acuaporinas/metabolismo , Carcinoma Hepatocelular/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , CMP Cíclico/análogos & derivados , Neoplasias Hepáticas/tratamiento farmacológico , Compuestos Orgánicos de Oro/farmacología , Animales , Apoptosis/efectos de los fármacos , Acuaporina 3/genética , Acuaporinas/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , CMP Cíclico/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , ARN Mensajero/genética , ARN Mensajero/metabolismo , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , alfa-Fetoproteínas/metabolismoRESUMEN
In thick ascending limbs (THALs), nitric oxide (NO) decreases NaCl reabsorption via cGMP-mediated inhibition of Na-K-2Cl cotransporter (NKCC2). In angiotensin (ANG II)-induced hypertension, endothelin-1 (ET-1)-induced NO production by THALs is impaired. However, whether this alters NO's natriuretic effects and the mechanisms involved are unknown. In other cell types, ANG II augments phosphodiesterase 5 (PDE5)-mediated cGMP degradation. We hypothesized that NO-mediated inhibition of NKCC2 activity and stimulation of cGMP synthesis are blunted via PDE5 in ANG II-induced hypertension. Sprague-Dawley rats were infused with vehicle or ANG II (200 ng·kg-1·min-1) for 5 days. ET-1 reduced NKCC2 activity by 38 ± 13% (P < 0.05) in THALs from vehicle-treated rats but not from ANG II-hypertensive rats (Δ: -9 ± 13%). A NO donor yielded similar results as ET-1. In contrast, dibutyryl-cGMP significantly decreased NKCC2 activity in both vehicle-treated and ANG II-hypertensive rats (control: Δ-44 ± 15% vs. ANG II: Δ-41 ± 10%). NO increased cGMP by 2.08 ± 0.36 fmol/µg protein in THALs from vehicle-treated rats but only 1.06 ± 0.25 fmol/µg protein in ANG II-hypertensive rats (P < 0.04). Vardenafil (25 nM), a PDE5 inhibitor, restored NO's ability to inhibit NKCC2 activity in THALs from ANG II-hypertensive rats (Δ: -60 ± 9%, P < 0.003). Similarly, NO's stimulation of cGMP was also restored by vardenafil (vehicle-treated: 1.89 ± 0.71 vs. ANG II-hypertensive: 2.02 ± 0.32 fmol/µg protein). PDE5 expression did not differ between vehicle-treated and ANG II-hypertensive rats. We conclude that NO-induced inhibition of NKCC2 and increases in cGMP are blunted in ANG II-hypertensive rats due to PDE5 activation. Defects in the response of THALs to NO may enhance NaCl retention in ANG II-induced hypertension.
Asunto(s)
Angiotensina II , Endotelina-1/farmacología , Hipertensión/metabolismo , Asa de la Nefrona/metabolismo , Óxido Nítrico/metabolismo , Miembro 1 de la Familia de Transportadores de Soluto 12/metabolismo , Animales , CMP Cíclico/análogos & derivados , CMP Cíclico/farmacología , GMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Hipertensión/inducido químicamente , Asa de la Nefrona/efectos de los fármacos , Masculino , Donantes de Óxido Nítrico/farmacología , Inhibidores de Fosfodiesterasa 5/farmacología , Ratas , Ratas Sprague-Dawley , Diclorhidrato de Vardenafil/farmacologíaRESUMEN
Formylated peptides are chemotactic agents generated by pathogens. The most relevant peptide is fMLF (formyl-Met-Leu-Phe) which participates in several immune functions, such as chemotaxis, phagocytosis, cytokine release and generation of reactive oxygen species. In macrophages fMLF-dependent responses are dependent on both, an increase in intracellular calcium concentration and on a hyperpolarization of the membrane potential. However, the molecular entity underlying this hyperpolarization remains unknown and it is not clear whether changes in membrane potential are linked to the increase in intracellular Ca2+. In this study, differentiated U937 cells, as a macrophage-like cell model, was used to characterize the fMLF response using electrophysiological and Ca2+ imaging techniques. We demonstrate by means of pharmacological and molecular biology tools that fMLF induces a Ca2+-dependent hyperpolarization via activation of the K+ channel KCa3.1 and thus, enhancing fMLF-induced intracellular Ca2+ increase through an amplification of the driving force for Ca2+ entry. Consequently, enhanced Ca2+ influx would in turn lengthen the hyperpolarization, operating as a positive feedback mechanism for fMLF-induced Ca2+ signaling.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Calcio/metabolismo , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacología , Diferenciación Celular/efectos de los fármacos , CMP Cíclico/análogos & derivados , CMP Cíclico/farmacología , Retroalimentación Fisiológica/efectos de los fármacos , Humanos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/genética , Activación del Canal Iónico/fisiología , Potenciales de la Membrana/efectos de los fármacos , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Monocitos/fisiología , Técnicas de Placa-Clamp , Interferencia de ARN , Células U937RESUMEN
cCMP is a cyclic pyrimidine nucleotide which binds to and activates cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG). In S49 lymphoma cells, cAMP induces apoptosis via PKA. In our present study, we examined the effect of cCMP on apoptosis in S49 mouse lymphoma cells and in PKA-deficient S49kin(-)cells. These two cell lines also lack PKG, hyperpolarization-activated cyclic nucleotide-gated channels 2 and 4 (HCN2 and HCN4) as assessed by real-time PCR. The cell-permeable analog cCMP-AM induced PKA- and PKG-independent apoptosis in S49 cells. In contrast, exchange protein activated by cAMP (Epac) activation did not induce apoptosis. cCMP induced caspase-dependent apoptosis via the intrinsic pathway, led to cytochrome c release from mitochondria and also activated the ER stress pathway. On the contrary, the extrinsic apoptotic pathway was not involved. Autophagy was not detectable after treatment with cCMP-AM in both cell lines. cAMP-AM, cGMP-AM, cUMP-AM as well as the cyclic nucleotides lacking the acetoxymethylester (AM)-group had no effect. cCMP-AM altered gene expression of the apoptotic-relevant gene Gadd45α and the immediate early response genes cFos and Nr4A1 in S49 wild-type (wt) cells. In conclusion, cCMP induces apoptosis of S49 lymphoma cells, independently of hitherto known cCMP target proteins.
Asunto(s)
Apoptosis/efectos de los fármacos , Caspasas/metabolismo , CMP Cíclico/farmacología , Linfoma/metabolismo , Animales , Línea Celular Tumoral , CMP Cíclico/química , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
OBJECTIVE: Oxidative stress associated with cardiovascular disease can produce various oxidized lipids, including cholesterol oxides, such as 7-hydroperoxide (7-OOH), 7-hydroxide (7-OH), and 7-ketone (7=O). Unlike 7=O and 7-OH, 7-OOH is redox active, giving rise to the others via potentially toxic-free radical reactions. We tested the novel hypothesis that under oxidative stress conditions, steroidogenic acute regulatory (StAR) family proteins not only deliver cholesterol to/into mitochondria of vascular macrophages, but also 7-OOH, which induces peroxidative damage that impairs early stage reverse cholesterol transport. APPROACH AND RESULTS: Stimulation of human monocyte-derived THP-1 macrophages with dibutyryl-cAMP resulted in substantial upregulation of StarD1 and ATP-binding cassette (ABC) transporter, ABCA1. Small interfering RNA-induced StarD1 knockdown before stimulation had no effect on StarD4, but reduced ABCA1 upregulation, linking the latter to StarD1 functionality. Mitochondria in stimulated StarD1-knockdown cells internalized 7-OOH slower than nonstimulated controls and underwent less 7-OOH-induced lipid peroxidation and membrane depolarization, as probed with C11-BODIPY (4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-inda-cene-3-undecanoic acid) and JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine iodide), respectively. Major functional consequences of 7-OOH exposure were (1) loss of mitochondrial CYP27A1 activity, (2) reduced 27-hydroxycholesterol (27-OH) output, and (3) downregulation of cholesterol-exporting ABCA1 and ABCG1. Consistently, 7-OOH-challenged macrophages exported less cholesterol to apoA-I or high-density lipoprotein than did nonchallenged controls. StarD1-mediated 7-OOH transport was also found to be highly cytotoxic, whereas 7=O and 7-OH were minimally toxic. CONCLUSIONS: This study describes a previously unrecognized mechanism by which macrophage cholesterol efflux can be incapacitated under oxidative stress-linked disorders, such as chronic obesity and hypertension. Our findings provide new insights into the role of macrophage redox damage/dysfunction in atherogenesis.
Asunto(s)
Aterosclerosis/metabolismo , Colesterol/análogos & derivados , Peroxidación de Lípido/fisiología , Macrófagos/metabolismo , Estrés Oxidativo/fisiología , Transporte Biológico , Células Cultivadas , Colesterol/metabolismo , CMP Cíclico/análogos & derivados , CMP Cíclico/farmacología , Humanos , Macrófagos/citología , Mitocondrias/metabolismo , Transporte de Proteínas , Sensibilidad y EspecificidadRESUMEN
There is increasing evidence for a role of cytidine 3',5'-cyclic monophosphate (cCMP) as second messenger. In a recent study, we showed that cCMP activates both purified guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase Iα (PKG Iα) and adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) isoenzymes with the regulatory subunits RIα and RIIα. Moreover, the membrane-permeant cCMP analog dibutyryl (DB)-cCMP induces effective vasodilation and inhibition of platelet aggregation via PKG Iα, but not via PKA. These data prompted us to conduct a systematic analysis of the effects of cyclic nucleotide (cNMP) analogs on purified PKG Iα and PKA RIα and RIIα We also studied the effect of DB-cCMP on PKA-dependent phosphorylation of the transcription factor cAMP response-binding protein (CREB) in S49 wild-type lymphoma cells and S49 kin(-) cells, devoid of the catalytic subunit of PKA. The major cellular metabolite of the prodrug DB-cCMP, N(4)-monobutyryl (4-MB)-cCMP, was a partial and low-potency activator of purified PKG Iα and a full and moderate-potency activator of PKA RIα and RIIα. Sp-cCMPS and Sp-cAMPS activated PKA RIα and RIIα with much higher potency and efficacy than PKG Iα. Molecular modeling suggested that the cytidine ring interacts with PKG Iα mainly via hydrophobic interactions, while the butyryl group projects away from the kinase. In contrast to DB-cAMP, DB-cCMP did not induce PKA-dependent phosphorylation in intact cells. Taken together, our data show that N(4)-monobutyryl-cCMP (4-MB-cCMP) activates PKA RIα and PKA RIIα more potently and with higher efficacy than PKG Iα in vitro but not in vivo. cNMP phosphorothioates constitute a starting point for the development of PKA activators with high selectivity relative to PKG.
Asunto(s)
Subunidad RIIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , CMP Cíclico/análogos & derivados , CMP Cíclico/metabolismo , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/metabolismo , Animales , Línea Celular Tumoral , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , CMP Cíclico/farmacología , Interacciones Hidrofóbicas e Hidrofílicas , Linfoma/metabolismo , Ratones , Modelos Moleculares , Fosforilación/efectos de los fármacosRESUMEN
Cardiac oxidative ATP generation is finely tuned to match several-fold increases in energy demand. Calcium has been proposed to play a role in the activation of ATP production via PKA phosphorylation in response to intramitochondrial cAMP generation. We evaluated the effect of cAMP, its membrane permeable analogs (dibutyryl-cAMP, 8-bromo-cAMP), and the PKA inhibitor H89 on respiration of isolated pig heart mitochondria. cAMP analogs did not stimulate State 3 respiration of Ca2 +-depleted mitochondria (82.2 ± 3.6% of control), in contrast to the 2-fold activation induced by 0.95 µM free Ca2 +, which was unaffected by H89. Using fluorescence and integrating sphere spectroscopy, we determined that Ca2 + increased the reduction of NADH (8%), and of cytochromes bH (3%), c1 (3%), c (4%), and a (2%), together with a doubling of conductances for Complex I + III and Complex IV. None of these changes were induced by cAMP analogs nor abolished by H89. In Ca2 +-undepleted mitochondria, we observed only slight changes in State 3 respiration rates upon addition of 50 µM cAMP (85 ± 9.9%), dibutyryl-cAMP (80.1 ± 5.2%), 8-bromo-cAMP (88.6 ± 3.3%), or 1 µM H89 (89.7 ± 19.9%) with respect to controls. Similar results were obtained when measuring respiration in heart homogenates. Addition of exogenous PKA with dibutyryl-cAMP or the constitutively active catalytic subunit of PKA to isolated mitochondria decreased State 3 respiration by only 515%. These functional studies suggest that alterations in mitochondrial cAMP and PKA activity do not contribute significantly to the acute Ca2 + stimulation of oxidative phosphorylation
Asunto(s)
Calcio/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/farmacología , Mitocondrias Cardíacas/efectos de los fármacos , Fosforilación Oxidativa/efectos de los fármacos , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Adenosina Difosfato/farmacología , Animales , Dominio Catalítico/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , CMP Cíclico/análogos & derivados , CMP Cíclico/farmacología , Grupo Citocromo b/metabolismo , Transporte de Electrón/efectos de los fármacos , Complejo I de Transporte de Electrón/metabolismo , Complejo III de Transporte de Electrones/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Isoquinolinas/farmacología , Mitocondrias Cardíacas/metabolismo , NAD/metabolismo , Consumo de Oxígeno/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Espectrometría de Fluorescencia , Espectrofotometría , Sulfonamidas/farmacología , PorcinosRESUMEN
In vitro and in vivo models of Parkinson's disease were used to investigate whether TNF-α plays a major role in the enhancement of the microglial response and dopaminergic degeneration induced by brain angiotensin hyperactivity. Treatment of primary mesencephalic cultures with low doses of the neurotoxin MPP(+) induced a significant loss of dopaminergic neurons, which was enhanced by cotreatment with angiotensin II and inhibited by TNF-α inhibitors. Treatment of primary cultures with angiotensin induced a marked increase in levels of TNF-α, which was inhibited by treatment with angiotensin type-1-receptor antagonists, NADPH-oxidase inhibitors and NFK-ß inhibitors. However, TNF-α levels were not significantly affected by treatment with angiotensin in the absence of microglia. The microglial origin of the angiotensin-induced increase in TNF-α levels was confirmed using dopaminergic (MES 23.5) and microglial (N9) cell lines. Inhibition of the microglial Rho-kinase activity also blocked the AII-induced increase in TNF-α levels. Treatment of the dopaminergic cell line with TNF-α revealed that NFK-ß activation mediates the deleterious effect of microglial TNF-α on dopaminergic neurons. Treatment of mice with MPTP also induced significant increases in striatal and nigral TNF-α levels, which were inhibited by angiotensin type-1-receptor antagonists or NFK-ß inhibitors. The present results show that microglial TNF-α plays a major role in angiotensin-induced dopaminergic cell death and that the microglial release of TNF-α is mediated by activation of angiotensin type-1 receptors, NADPH-oxidase, Rho-kinase and NFK-ß.
Asunto(s)
Neuronas Dopaminérgicas/patología , Calicreínas/metabolismo , Microglía/metabolismo , Degeneración Nerviosa/fisiopatología , Extractos Pancreáticos/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Angiotensina II/farmacología , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Animales , Bencimidazoles/farmacología , Compuestos de Bifenilo , Células Cultivadas , CMP Cíclico/análogos & derivados , CMP Cíclico/farmacología , Neuronas Dopaminérgicas/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Masculino , Mesencéfalo/citología , Ratones , Ratones Endogámicos C57BL , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/metabolismo , Neuronas/efectos de los fármacos , Neuronas/fisiología , Neurotoxinas/farmacología , Tetrazoles/farmacologíaRESUMEN
BACKGROUND: Although keratinocyte-derived factors are known to promote the proliferation and differentiation of human epidermal melanocytes, it is not fully understood whether fibroblast-derived factors work in a similar way. OBJECTIVE: The aim of this study is to clarify whether fibroblast-derived factors are involved in regulating the proliferation and differentiation of human melanocytes with or without keratinocytes using serum-free culture system. METHODS: Human epidermal melanoblasts and melanocytes were cultured in a serum-free growth medium supplemented with fibroblast-derived factors such as keratinocyte growth factor (KGF) with or without keratinocytes, and the effects of KGF on the proliferation and differentiation of melanocytes were studied. RESULTS: KGF stimulated the proliferation of melanoblasts in the presence of dibutyryl cAMP (DBcAMP), basic fibroblast growth factor (bFGF), transferrin (Tf), and endothelin-1 (ET-1). Although KGF stimulated the differentiation, melanogenesis, and dendritogenesis in the presence of DBcAMP, Tf, and ET-1 without keratinocytes, KGF required the presence of keratinocytes for the stimulation of melanocyte proliferation. CONCLUSION: These results suggest that fibroblast-derived KGF stimulates the proliferation of human melanoblasts in synergy with cAMP, bFGF, Tf, and ET-1, the differentiation of melanocytes in synergy with cAMP, Tf, and ET-1, and the proliferation of melanocytes in synergy with cAMP, Tf, ET-1, and undefined keratinocyte-derived factors.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Factor 7 de Crecimiento de Fibroblastos/farmacología , Melanocitos/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , CMP Cíclico/análogos & derivados , CMP Cíclico/farmacología , Relación Dosis-Respuesta a Droga , Endotelina-1/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Factor 7 de Crecimiento de Fibroblastos/metabolismo , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Melanocitos/metabolismo , Comunicación Paracrina , Factores de Tiempo , Transferrina/farmacologíaRESUMEN
Neurofibrillary tangles (NFTs) are hallmarks of Alzheimer's disease (AD). The main component of NFTs is TAU, a highly soluble microtubule-associated protein. However, when TAU is cleaved at Asp421 by caspases it becomes prone to aggregation leading to NFTs. What triggers caspase activation resulting in TAU cleavage remains unclear. We investigated in rat cortical neurons a potential coordination between proteasome impairment and caspase activation. We demonstrate that upon proteasome inhibition, the early accumulation of detergent-soluble ubiquitinated (SUb) proteins paves the way to caspase activation and TAU pathology. This occurs with two drugs that inhibit the proteasome by different means: the product of inflammation prostaglandin J2 (PGJ2) and epoxomicin. Our results pinpoint a critical early event, that is, the buildup of SUb proteins that contributes to caspase activation, TAU cleavage, TAU/Ub-protein aggregation and neuronal death. Furthermore, to our knowledge, we are the first to demonstrate that elevating cAMP in neurons with dibutyryl-cAMP (db-cAMP) or the lipophilic peptide PACAP27 prevents/diminishes caspase activation, TAU cleavage and neuronal death induced by PGJ2, as long as these PGJ2-induced changes are moderate. db-cAMP also stimulated proteasomes, and mitigated proteasome inhibition induced by PGJ2. We propose that targeting cAMP/PKA to boost proteasome activity in a sustainable manner could offer an effective approach to avoid early accumulation of SUb proteins and later caspase activation, and TAU cleavage, possibly preventing/delaying AD neurodegeneration.
Asunto(s)
CMP Cíclico/análogos & derivados , Neuronas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas tau/metabolismo , Animales , Caspasas/metabolismo , Muerte Celular , Supervivencia Celular , CMP Cíclico/metabolismo , CMP Cíclico/farmacología , Activación Enzimática , Femenino , Masculino , Neuronas/citología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
The system x(C)- (Sx(C)-) transporter functions to mediate the exchange of extracellular cystine (L-Cys(2)) and intracellular glutamate (L-Glu). Internalized L-Cys(2) serves as a rate-limiting precursor for the biosynthesis of glutathione (GSH), while the externalized L-Glu can contribute to either excitatory signaling or excitotoxicity. In the present study the influence of culture conditions (with and without dibutyryl-cAMP) and GSH levels on the expression of Sx(C)- were investigated in primary rat astrocyte cultures. Sx(C)- activity in dbcAMP-treated cells was nearly sevenfold greater than in untreated astrocytes and increased further (â¼threefold) following the depletion of intracellular GSH with buthionine sulfoximine. This increase in Sx(C)- triggered by GSH depletion was only observed in the dbcAMP-treated phenotype and was distinct from the Nrf2-mediated response initiated by exposure to electrophiles. Changes in Sx(C)- activity correlated with increases in both protein and mRNA levels of the xCT subunit of the Sx(C)- heterodimer, an increase in the V(max) for L-Glu uptake and was linked temporally to GSH levels. This induction of Sx(C)- was not mimicked by hydrogen peroxide nor attenuated by nonspecific antioxidants but was partially prevented by the co-administration of the cell-permeant thiols GSH-ethyl ester and N-acetylcysteine. These findings demonstrate that the expression of Sx(C)- on astrocytes is dynamically regulated by intracellular GSH levels in a cell- and phenotype-dependent manner. The presence of this pathway likely reflects the inherent vulnerability of the CNS to oxidative damage and raises interesting questions as to the functional consequences of changes in Sx(C)- activity in CNS injury and disease.
Asunto(s)
Sistemas de Transporte de Aminoácidos/metabolismo , Astrocitos/metabolismo , Cistina/metabolismo , Ácido Glutámico/metabolismo , Glutatión/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Análisis de Varianza , Animales , Animales Recién Nacidos , Antimetabolitos/farmacología , Astrocitos/efectos de los fármacos , Butionina Sulfoximina/farmacología , Células Cultivadas , Corteza Cerebral/citología , CMP Cíclico/análogos & derivados , CMP Cíclico/farmacología , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , ARN Mensajero/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismo , Tritio/metabolismoRESUMEN
Activation of the LH receptor (LHR) in Leydig cells results in the phosphorylation of ERK1/2 by cAMP-dependent and cAMP-independent pathways. Here we examine the mechanisms by which cAMP stimulates ERK1/2 phosphorylation. We show that the stimulation of steroidogenesis is not necessary or sufficient to stimulate the phosphorylation of ERK1/2 but that other cAMP-dependent mitochondrial functions are involved. Using MA-10 cells as a model, we showed that cAMP analogs increase reactive oxygen species (ROS) formation and that an uncoupler of oxidative phosphorylation and a ROS scavenger prevent this increase. These two compounds also inhibit the increase in ERK1/2 phosphorylation provoked by cAMP analogs, thus suggesting that the cAMP-induced phosphorylation of ERK1/2 is mediated by mitochondrial ROS. In agreement with this hypothesis we also show that a reduction in glutathione levels, which alters the redox state of MA-10 cells, potentiates the effect of cAMP on ERK1/2 phosphorylation. Measurements of the dephosphorylation of ERK and the activation of Ras showed that the ROS scavenger prevents the cAMP-provoked activation of Ras and that cAMP, with or without a ROS scavenger, has little or no effect on the dephosphorylation of ERK. Lastly, we show that the uncoupler of oxidative phosphorylation and the ROS scavenger also prevent the ability of cAMP analogs to increase ERK1/2 phosphorylation in primary cultures of mouse Leydig cells. We conclude that, in Leydig cells, cAMP enhances the phosphorylation of ERK1/2 via a mitochondria-derived, ROS-dependent activation of Ras.
Asunto(s)
AMP Cíclico/metabolismo , Células Intersticiales del Testículo/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas ras/metabolismo , Acetilcisteína/farmacología , Animales , Antimicina A/análogos & derivados , Antimicina A/farmacología , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Línea Celular , Gonadotropina Coriónica/metabolismo , CMP Cíclico/análogos & derivados , CMP Cíclico/farmacología , Activación Enzimática , Glutatión/deficiencia , Células Intersticiales del Testículo/efectos de los fármacos , Masculino , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Oligomicinas/farmacología , Fosforilación , Progesterona/biosíntesis , Rotenona/farmacología , Desacopladores/farmacologíaRESUMEN
Pluripotent embryonic stem cells hold a great promise as an unlimited source of tissue for treatment of chronic diseases such as Type 1 diabetes. Herein, we describe a protocol using all-trans-retinoic acid, basic fibroblast growth factor and dibutyryl cAMP (DBcAMP) in the absence of embryoid body formation, for differentiation of murine embryonic stem cells into definitive endoderm that may serve as pancreatic precursors. The produced cells were analyzed by quantitative PCR, immunohistochemistry and static insulin release assay for markers of trilaminar embryo, and pancreas. Differentiated cells displayed increased Sox17 and Foxa2 expression consistent with definitive endoderm production. There was minimal production of Sox7, an extraembryonic endoderm marker, and Oct4, a marker of pluripotency. There was minimal mesoderm or neuroectoderm formation based on expression levels of the markers brachyury and Sox1, respectively. Various assays revealed that the cell clusters generated by this protocol express markers of the pancreatic lineage including insulin I, insulin II, C-peptide, PDX-1, carboxypeptidase E, pan-cytokeratin, amylase, glucagon, PAX6, Ngn3 and Nkx6.1. This protocol using all-trans-retinoic acid, DBcAMP, in the absence of embryoid bodies, generated cells that have features of definitive endoderm that may serve as pancreatic endocrine precursors.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Células Madre Embrionarias/citología , Endodermo/citología , Animales , Células Cultivadas , CMP Cíclico/análogos & derivados , CMP Cíclico/farmacología , Cuerpos Embrioides/citología , Cuerpos Embrioides/metabolismo , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Endodermo/efectos de los fármacos , Endodermo/metabolismo , Femenino , Factor 2 de Crecimiento de Fibroblastos/farmacología , Expresión Génica/efectos de los fármacos , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Factor Nuclear 3-beta del Hepatocito/genética , Factor Nuclear 3-beta del Hepatocito/metabolismo , Inmunohistoquímica , Insulina/genética , Insulina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Páncreas/citología , Páncreas/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/efectos de los fármacos , Células Madre Pluripotentes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción SOXF/genética , Factores de Transcripción SOXF/metabolismo , Factores de Tiempo , Tretinoina/farmacologíaRESUMEN
Formation of the mitochondrial membrane potential (ΔΨ) depends on flux of respiratory substrates, ATP, ADP, and Pi through voltage-dependent anion channels (VDAC). As tubulin promotes single-channel closure of VDAC, we hypothesized that tubulin is a dynamic regulator of ΔΨ, which in cultured cancer cells was assessed by confocal microscopy of the potential-indicating fluorophore tetramethylrhodamine methylester (TMRM). Microtubule destabilizers, rotenone, colchicine, and nocodazole, and the microtubule stabilizer paclitaxel increased and decreased cellular free tubulin, respectively, and in parallel decreased and increased ΔΨ. Protein kinase A (PKA) activation by cAMP analogues and glycogen synthase kinase 3ß (GSK-3ß) inhibition decreased ΔΨ, whereas PKA inhibition hyperpolarized, consistent with reports that PKA and GSK-3ß decrease and increase VDAC conductance, respectively. Plasma membrane potential assessed by DiBAC(4)(3) was not altered by any of the treatments. We propose that inhibition of VDAC by free tubulin limits mitochondrial metabolism in cancer cells.
Asunto(s)
Potencial de la Membrana Mitocondrial/fisiología , Neoplasias/fisiopatología , Tubulina (Proteína)/fisiología , Adenosina Trifosfato/metabolismo , Animales , Carcinoma Hepatocelular/fisiopatología , Línea Celular Tumoral , Membrana Celular/fisiología , Colchicina/farmacología , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , CMP Cíclico/análogos & derivados , CMP Cíclico/farmacología , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Neoplasias Hepáticas/fisiopatología , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Potenciales de la Membrana , Microtúbulos/efectos de los fármacos , Microtúbulos/fisiología , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/fisiología , Nocodazol/farmacología , Paclitaxel/farmacología , Ratas , Ratas Sprague-Dawley , Rodaminas/farmacología , Rotenona/farmacologíaRESUMEN
We analysed the function and intracellular signalling of the cyclic pyrimidinic nucleotide cCMP. The membrane-permeable cCMP analogue dibutyryl-cCMP mediated mouse aorta relaxation. cCMP activated purified cGMP-dependent protein kinase (cGK) Iα and Iß and stimulated cGK in aorta lysates. cCMP-induced relaxation was abolished in cGKI-knockout tissue. Additionally, deletion of inositol-trisphosphate receptor associated cGKI substrate (IRAG) suppressed cCMP-mediated relaxation. Signalling of cCMP via cGKI/IRAG appears to be of broader physiological importance because cCMP-mediated inhibition of platelet aggregation was absent in cGKI- and IRAG-deficient platelets. These results demonstrate that cCMP acts as intracellular messenger molecule, most unexpectedly utilizing the cGMP signal transduction pathway.