Modulating cyclic nucleotides pathways by bioactive compounds in combatting anxiety and depression disorders.

Anxiety and depression disorders are highly prevalent neurological disorders (NDs) that impact up to one in three individuals during their lifetime. Addressing these disorders requires reducing their frequency and impact, understanding molecular causes, implementing prevention strategies, and improving treatments. Cyclic nucleotide monophosphates (cNMPs) like cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), cyclic uridine monophosphate (cUMP), and cyclic cytidine monophosphate (cCMP) regulate the transcription of genes involved in neurotransmitters and neurological functions. Evidence suggests that cNMP pathways, including cAMP/cGMP, cAMP response element binding protein (CREB), and Protein kinase A (PKA), play a role in the physiopathology of anxiety and depression disorders. Plant and mushroom-based compounds have been used in traditional and modern medicine due to their beneficial properties. Bioactive compound metabolism can activate key pathways and yield pharmacological outcomes. This review focuses on the molecular mechanisms of bioactive compounds from plants and mushrooms in modulating cNMP pathways. Understanding these processes will support current treatments and aid in the development of novel approaches to reduce the prevalence of anxiety and depression disorders, contributing to improved outcomes and the prevention of associated complications.

CUTie2: The Attack of the Cyclic Nucleotide Sensor Clones.

The detection of small molecules in living cells using genetically encoded FRET sensors has revolutionized our understanding of signaling pathways at the sub-cellular level. However, engineering fluorescent proteins and specific binding domains to create new sensors remains challenging because of the difficulties associated with the large size of the polypeptides involved, and their intrinsically huge conformational variability. Indeed, FRET sensors' design still relies on vague structural notions, and trial and error combinations of linkers and protein modules. We recently designed a FRET sensor for the second messenger cAMP named CUTie (Cyclic nucleotide Universal Tag for imaging experiments), which granted sub-micrometer resolution in living cells. Here we apply a combination of sequence/structure analysis to produce a new-generation FRET sensor for the second messenger cGMP based on Protein kinase G I (PKGI), which we named CUTie2. Coarse-grained molecular dynamics simulations achieved an exhaustive sampling of the relevant spatio-temporal coordinates providing a quasi-quantitative prediction of the FRET efficiency, as confirmed by in vitro experiments. Moreover, biochemical characterization showed that the cGMP binding module maintains virtually the same affinity and selectivity for its ligand thant the full-length protein. The computational approach proposed here is easily generalizable to other allosteric protein modules, providing a cost effective-strategy for the custom design of FRET sensors.

Novel Potassium Channels in Kidney Mitochondria: The Hyperpolarization-Activated and Cyclic Nucleotide-Gated HCN Channels.

Hyperpolarization-activated cationic HCN channels comprise four members (HCN1-4) that control dendritic integration, synaptic transmission and action potential firing. In the kidney, HCN1, HCN2 and HCN3 are differentially expressed and contribute to the transport of sodium, potassium (K+) and ammonium into the nephrons. HCN3 is regulated by K+ diets in the kidney. In this work we performed a proteomic analysis of HCN3 expressed in human embryonic kidney cells (HEK293 cells). More than 50% of the interacting proteins belonged to mitochondria. Therefore, we explored the presence of HCN channels in kidney mitochondria. By immunoblotting and immunogold electron microscopy HCN3 protein expression was found in rat kidney mitochondria; it was also confirmed in human kidney. Patch-clamp recordings of renal mitochondria and mitochondria from HEK293 cells overexpressing HCN1, HCN2 and HCN3 channels, stained with MitoTracker Green FM, indicated that only HCN3 could produce inwardly K+ currents that were inhibited by ZD7288, a specific blocker of HCN channels. Furthermore, ZD7288 caused inhibition of the oxygen consumption coupled to ATP synthesis and hyperpolarization of the inner mitochondrial membrane. In conclusion, we show for the first time that pacemaker HCN channels contribute to K+ transport in mitochondria facilitating the activity of the respiratory chain and ATP synthesis by controlling the inner mitochondrial membrane potential.

Nitric oxide impacts bovine sperm capacitation in a cGMP-dependent and cGMP-independent manner.

We aimed to elucidate whether NO acts in in vitro sperm capacitation in bovine via cGMP/PKG1 pathway. For this, cryopreserved bovine sperm were capacitated in vitro with 20 µg/ml heparin (Control) plus treatments: 1 mM L-arginine (L-arg, NO precursor), 50 µM Rp-8-Bromo-ß-phenyl-1,N2 -ethenoguanosine-3',5'-cyclic monophosphorothioate (Rp-8-Br-cGMPS, selective inhibitor of the binding site for cGMP in PKG1), 1 mM 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO, NO scavenger), and the combinations of L-arg + RP-8-Br-cGMPS and L-arg + PTIO. Sperm motility and vigour were determined by phase-contrast microscopy, capacitation status by chlortetracycline staining, and the intracellular concentration of cGMP was measured by ELISA. Data were subjected to analysis of variance and means compared with SNK test at 5% probability. Motility and vigour were lower in sperm treated with PTIO when compared to Control and other treatments (p < .05). The L-arg treatment showed the highest percentage of capacitated sperm when compared to the Control and other treatments (Rp-8-Br-cGMPS, L-arg + Rp-8-Br-cGMPS and PTIO) (69.8 ± 3.4%, 51.2 ± 3.0, 51.1 ± 2.1, 51.2 ± 3.0 and 45.5 ± 2.7, respectively) (p < .05). The capacitation ratio (%) was lower in treatments with Rp-8-Br-cGMPS, L-arg + Rp-8-Br-cGMPS and PTIO, respectively (p < .05). Lastly, cGMP concentration (pmol/ml) was lower in PTIO and L-arg + PTIO (1.3 ± 0.3 and 1.6 ± 0.4) and was higher in Rp-8-Br-cGMPS and L-arg + Rp-8-Br-cGMPS (3.7 ± 0.4 and 4.0 ± 0.5) treatments. We showed that during in vitro capacitation of cattle: (a) NO influences sperm motility and vigour; (b) NO is associated with cGMP synthesis through two independent pathways and (c) the cGMP/PKG1 pathway has a partial role in sperm capacitation and does not involve the L-arg/NO.

Elevated expression of neuropeptide signaling genes in the eyestalk ganglia and Y-organ of Gecarcinus lateralis individuals that are refractory to molt induction.

Molting is induced in decapod crustaceans via multiple leg autotomy (MLA) or eyestalk ablation (ESA). MLA removes five or more walking legs, which are regenerated and become functional appendages at ecdysis. ESA eliminates the primary source of molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH), which suppress the production of molting hormones (ecdysteroids) from the molting gland or Y-organ (YO). Both MLA and ESA are effective methods for molt induction in Gecarcinus lateralis. However, some G. lateralis individuals are refractory to MLA, as they fail to complete ecdysis by 12weeks post-MLA; these animals are in the "blocked" condition. Quantitative polymerase chain reaction was used to quantify mRNA levels of neuropeptide and mechanistic target of rapamycin (mTOR) signaling genes in YO, eyestalk ganglia (ESG), thoracic ganglion (TG), and brain of intact and blocked animals. Six of the seven neuropeptide signaling genes, three of four mTOR signaling genes, and Gl-elongation factor 2 (EF2) mRNA levels were significantly higher in the ESG of blocked animals. Gl-MIH and Gl-CHH mRNA levels were higher in the TG and brain of blocked animals and levels increased in both control and blocked animals in response to ESA. By contrast, mRNA levels of Gl-EF2 and five of the 10 MIH signaling pathway genes in the YO were two to four orders of magnitude higher in blocked animals compared to controls. These data suggest that increased MIH and CHH synthesis in the ESG contributes to the prevention of molt induction by MLA in blocked animals. The up-regulation of MIH signaling genes in the YO of blocked animals suggests that the YO is more sensitive to MIH produced in the ESG, as well as MIH produced in brain and TG of ESA animals. Both the up-regulation of MIH signaling genes in the YO and of Gl-MIH and Gl-CHH in the ESG, TG, and brain appear to contribute to some G. lateralis individuals being refractory to MLA and ESA.

Daily use of phosphodiesterase type 5 inhibitors as prevention for recurrent priapism / Prevenção do priapismo recorrente com a utilização diária de inibidores da fosfodiesterase tipo 5

Summary Objective: The pathogenesis of recurrent priapism is currently being investigated based on the regulation of the phosphodiesterase 5 (PDE5) enzyme. We explored the daily use of PDE5 inhibitors to treat and prevent priapism recurrences. Method: We administered PDE5 inhibitors using a long-term therapeutic regimen in seven men with recurrent priapism, with a mean age of 29.2 years (range 21 to 35 years). Six men (85.7%) had idiopathic priapism recurrences and one man (24.3%) had sickle cell disease-associated priapism recurrences. Tadalafil 5 mg was administered daily. The mean follow-up was 6.6 months (range 3 to 12 months). Results: Daily long-term oral PDE5 inhibitor therapy alleviated priapism recurrences in all patients. Five (71.4%) had no episodes of priapism and two (28.6%) referred decrease in their episodes of priapism. All patients referred improvement in erectile function. Conclusion: These findings suggest the hypothesis that PDE5 dysregulation exerts a pathogenic role for both sickle cell disease-associated priapism and for idiopathic priapism, and that it offers a molecular target for the therapeutic management of priapism. These preliminary observations suggest that continuous long-term oral PDE5 inhibitor therapy may treat and prevent recurrent priapism.

Resumo Objetivo: Uma das teorias propostas para explicar a etiologia do priapismo recorrente está baseada no mecanismo de regulação da fosfodiesterase tipo 5. Estudamos o uso diário dos inibidores de fosfodiesterase tipo 5 no tratamento e na prevenção do priapismo recorrente. Método: Sete homens com diagnóstico de priapismo recorrente, com idade média de 29,5 anos (21 a 35 anos), utilizaram inibidor de fosfodiesterase tipo 5 em dose diária (tadalafila 5 mg/dia) por período prolongado. Seis homens (85,7%) apresentavam priapismo recorrente de etiologia idiopática, e um homem (24,3%), de etiologia associada à anemia falciforme. O seguimento médio foi de 6,6 meses (3 a 12 meses). Resultados: Todos os pacientes se beneficiaram com a utilização de inibidores de fosfodiesterase tipo 5. Cinco (71,4%) não apresentaram nenhum episódio de priapismo e dois (28,6%) relataram diminuição dos episódios. Todos os pacientes relataram melhora da função erétil. Conclusão: Estes achados sugerem que a hipótese do mecanismo de regulação da fosfodiesterase tipo 5 exerce papel importante na patogenia do priapismo recorrente. O uso contínuo e diário de inibidores da fosfodiesterase tipo 5 pode ser uma opção no tratamento do priapismo recorrente.

Expression and cellular localization of HCN channels in rat cerebellar granule neurons.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels belong to the superfamily of voltage-gated pore loop channels. In mammals, this family consists of four different subunits (HCN1-4) and their ion channels activity have been proposed to play an essential role in regulating the membrane potential of excitable cells. Here, we describe the expression and relative abundances of HCN channels in cerebellum and primary cultures of cerebellar granule neurons (CGN). Quantitative determination of mRNA expression levels demonstrated the existence of an accumulation pattern of transcripts in cerebellum that encode HCN2 > HCN3 = HCN4 > HCN1 subunits. Immunolocalization analyses of HCN channels in cerebella revealed positive staining in Purkinje and granule cell layers. The presence of the HCN subunits in the cerebellar granule cell layer was then confirmed in primary cultures of CGN by quantitative real-time PCR (qPCR), as well as western blot and immunofluorescence analysis, demonstrating the presence of all four channel proteins.

Long-term outcomes of pediatric sinus bradycardia.

OBJECTIVES: To delineate the long-term outcomes and mechanisms of pediatric sinus bradycardia. STUDY DESIGN: Participants with sinus bradycardia who were identified from a survey of 432,166 elementary and high school students, were enrolled 10 years after the survey. The clinical course, heart rate variability, and hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4) gene were assessed. RESULTS: A total of 104 (male:female was 60:44; prevalence, 0.025%) participants were observed to have sinus bradycardia at age 15.5 ± 0.2 years with a mean heart rate of 48.4 ± 0.4 beats per minute; 86 study participants (83%) responded to clinical assessment and 37 (36%) underwent laboratory assessment. Athletes composed 37.8% of the study participants. During the extended 10-year follow-up, 15 (17%) of the participants had self-limited syncopal episodes, but none had experienced life-threatening events. According to Holter recordings, none of the participants had heart rate <30 beats per minute or a pause longer than 3 seconds. Compared with 67 age- and sex-matched controls, the variables of heart rate based on the spectral and time domain analysis of the participants with sinus bradycardia were all significantly higher, indicating higher parasympathetic activity. The results of mutation analysis were negative in the HCN4 gene in all of our participants. CONCLUSIONS: The long-term outcomes of the children and adolescents with sinus bradycardia identified using school electrocardiographic survey are favorable. Parasympathetic hyperactivity, instead of HCN4 gene mutation, is responsible for the occurrence of sinus bradycardia.