Search Results (11)

Bladder urothelium and suburothelium/lamina propria (LP) have prominent sensory and transducer functions with the active participation of afferent neurons and urothelium-derived purine mediators such as adenosine 5’-triphosphate (ATP), adenosine 5’-diphosphate (ADP), and adenosine (ADO). Effective concentrations of purines at receptor targets depend significantly on the extracellular degradation of ATP by ectonucleotidases (ENTDs). We recently reported the regulated release of soluble ENTDs (s-ENTDs) in the LP and the consequent degradation of ATP to ADP, AMP, and ADO. Afferent neurons in the LP can be activated by urothelial ATP and release peptides and other transmitters that can alter the activity of cells in their vicinity. Using a murine decentralized ex vivo detrusor-free bladder model, 1,N⁶-etheno-ATP (eATP) as substrate, and sensitive HPLC-FLD methodologies, we found that exogenous neuropeptides calcitonin gene-related peptide (CGRP), substance P (Sub P), neurokinin A (NKA), and pituitary adenylate cyclase-activating polypeptide [PACAP (1-38)] all increased the degradation of eATP by s-ENTDs that were released in the LP spontaneously and/or during bladder filling. Using antagonists of neuropeptide receptors, we observed that endogenous NKA did not modify the ATP hydrolysis by s-ENTDs, whereas endogenous Sub P increased both the constitutive and distention-induced release of s-ENTDs. In contrast, endogenous CGRP and PACAP (1-38) increased the distention-induced, but not the spontaneous, release of s-ENTDs. The present study puts forward the novel idea that interactions between peptidergic and purinergic signaling mechanisms in the LP have an impact on bladder excitability and functions by regulating the effective concentrations of adenine purines at effector cells in the LP. Full article

The current view of the mitochondrial respiratory chain complexes I, III and IV foresees the occurrence of their assembly in supercomplexes, providing additional functional properties when compared with randomly colliding isolated complexes. According to the plasticity model, the two structural states of the respiratory chain may interconvert, influenced by the intracellular prevailing conditions. In previous studies, we suggested the mitochondrial membrane potential as a factor for controlling their dynamic balance. Here, we investigated if and how the cAMP/PKA-mediated signalling influences the aggregation state of the respiratory complexes. An analysis of the inhibitory titration profiles of the endogenous oxygen consumption rates in intact HepG2 cells with specific inhibitors of the respiratory complexes was performed to quantify, in the framework of the metabolic flux theory, the corresponding control coefficients. The attained results, pharmacologically inhibiting either PKA or sAC, indicated that the reversible phosphorylation of the respiratory chain complexes/supercomplexes influenced their assembly state in response to the membrane potential. This conclusion was supported by the scrutiny of the available structure of the CI/CIII₂/CIV respirasome, enabling us to map several PKA-targeted serine residues exposed to the matrix side of the complexes I, III and IV at the contact interfaces of the three complexes. Full article

The urinary bladder requires adequate concentrations of extracellular adenosine 5′-triphosphate (ATP) and other purines at receptor sites to function properly. Sequential dephosphorylation of ATP to ADP, AMP and adenosine (ADO) by membrane-bound and soluble ectonucleotidases (s-ENTDs) is essential for achieving suitable extracellular levels of purine mediators. S-ENTDs, in particular, are released in the bladder suburothelium/lamina propria (LP) in a mechanosensitive manner. Using 1,N⁶-etheno-ATP (eATP) as substrate and sensitive HPLC-FLD methodology, we evaluated the degradation of eATP to eADP, eAMP and eADO in solutions that were in contact with the LP of ex vivo mouse detrusor-free bladders during filling prior to substrate addition. The inhibition of neural activity with tetrodotoxin and ω-conotoxin GVIA, of PIEZO channels with GsMTx4 and D-GsMTx4 and of the pituitary adenylate cyclase-activating polypeptide type I receptor (PAC1) with PACAP6-38 all increased the distention-induced but not spontaneous release of s-ENTDs in LP. It is conceivable, therefore, that the activation of these mechanisms in response to distention restricts the further release of s-ENTDs and prevents excessive hydrolysis of ATP. Together, these data suggest that afferent neurons, PIEZO channels, PAC1 receptors and s-ENTDs form a system that operates a highly regulated homeostatic mechanism to maintain proper extracellular purine concentrations in the LP and ensure normal bladder excitability during bladder filling. Full article

During the last seventy years, studies on mammalian sperm cells have demonstrated the essential role of capacitation, hyperactivation and the acrosome reaction in the acquisition of fertilization ability. These studies revealed the important biochemical and physiological changes that sperm undergo in their travel throughout the female genital tract, including changes in membrane fluidity, the activation of soluble adenylate cyclase, increases in intracellular pH and Ca²⁺ and the development of motility. Sperm are highly polarized cells, with a resting membrane potential of about −40 mV, which must rapidly adapt to the ionic changes occurring through the sperm membrane. This review summarizes the current knowledge about the relationship between variations in the sperm potential membrane, including depolarization and hyperpolarization, and their correlation with changes in sperm motility and capacitation to further lead to the acrosome reaction, a calcium-dependent exocytosis process. We also review the functionality of different ion channels that are present in spermatozoa in order to understand their association with human infertility. Full article

cAMP-PKA signaling plays a pivotal role in melanin synthesis and melanosome transport by responding to the binding of the α-melanocyte-stimulating hormone (α-MSH) to melanocortin-1 receptor (MC1R). Adenylate cyclases (ADCYs) are the enzymes responsible for the synthesis of cAMP from ATP, which comprises nine transmembrane isoforms (ADCYs 1-9) and one soluble adenylate cyclase (ADCY 10) in mammals. However, little is known about which and how ADCY isoforms regulate melanocyte generation, melanin biosynthesis, and melanosome transport in vivo. In this study, we have generated a series of single and double mutants of Adcy isoforms in zebrafish. Among them, adcy3a^-/- and adcy5^-/- double mutants cause defects in melanosome dispersion but do not impair melanoblast differentiation and melanocyte regeneration during the embryonic or larval stages. Activation of PKA, the main effector of cAMP signaling, significantly ameliorates the defects in melanosome dispersion in adcy3a^-/- and adcy5^-/- double mutants. Mechanistically, Adcy3a and Adcy5 regulate melanosome dispersion by activating kinesin-1 while inhibiting cytoplasmic dynein-1. In adult zebrafish, Adcy3a and Adcy5 participate in the regulation of the expression of microphthalmia transcription factor (Mitfa) and melanin synthesis enzymes Tyr, Dct, and Trp1b. The deletion of Adcy3a and Adcy5 inhibits melanin production and reduces pigmented melanocyte numbers, causing a defect in establishing adult melanocyte stripes. Hence, our studies demonstrate that Adcy3a and Adcy5 play essential but redundant functions in mediating α-MSH-MC1R/cAMP-PKA signaling for regulating melanin synthesis and melanosome dispersion. Full article

Late-onset hypogonadism, a male age-related syndrome characterized by a decline in testosterone production in the testes, is commonly treated with testosterone replacement therapy, which has adverse side effects. Therefore, an alternative treatment is highly sought. Supplementation of a high dosage of biotin, a water-soluble vitamin that functions as a coenzyme for carboxylases involved in carbohydrate, lipid, and amino acid metabolism, has been shown to influence testis functions. However, the involvement of biotin in testis steroidogenesis has not been well clarified. In this study, we examined the effect of biotin on testosterone levels in mice and testis-derived cells. In mice, intraperitoneal treatment with biotin (1.5 mg/kg body weight) enhanced testosterone levels in the serum and testes, without elevating serum levels of pituitary luteinizing hormone. To investigate the mechanism in which biotin increased the testosterone level, mice testis-derived I-10 cells were used. The cells treated with biotin increased testosterone production in a dose- and time-dependent manner. Biotin treatment elevated intracellular cyclic adenosine monophosphate levels via adenylate cyclase activation, followed by the activation of protein kinase A and testosterone production. These results suggest that biotin may have the potential to improve age-related male syndromes associated with declining testosterone production. Full article

Adenylate Cyclase Toxin (ACT or CyaA) is one of the important virulence factors secreted by Bordetella pertussis, the bacterium causative of whooping cough. ACT debilitates host defenses by production of unregulated levels of cAMP into the cell cytosol upon delivery of its N-terminal domain with adenylate cyclase activity (AC domain) and by forming pores in the plasma membrane of macrophages. Binding of soluble toxin monomers to the plasma membrane of target cells and conversion into membrane-integrated proteins are the first and last step for these toxin activities; however, the molecular determinants in the protein or the target membrane that govern this conversion to an active toxin form are fully unknown. It was previously reported that cytotoxic and cytolytic activities of ACT depend on membrane cholesterol. Here we show that ACT specifically interacts with membrane cholesterol, and find in two membrane-interacting ACT domains, four cholesterol-binding motifs that are essential for AC domain translocation and lytic activities. We hypothesize that direct ACT interaction with membrane cholesterol through those four cholesterol-binding motifs drives insertion and stabilizes the transmembrane topology of several helical elements that ultimately build the ACT structure for AC delivery and pore-formation, thereby explaining the cholesterol-dependence of the ACT activities. The requirement for lipid-mediated stabilization of transmembrane helices appears to be a unifying mechanism to modulate toxicity in pore-forming toxins. Full article

The propensity towards platelet-rich thrombus formation increases substantially during normal ageing, and this trend is mediated by decreases in platelet responsiveness to the anti-aggregatory nitric oxide (NO) and prostacyclin (PGI₂) pathways. The impairment of soluble guanylate cyclase and adenylate cyclase-based signalling that is associated with oxidative stress represents the major mechanism of this loss of anti-aggregatory reactivity. Platelet desensitization to these autacoids represents an adverse prognostic marker in patients with ischemic heart disease and may contribute to increased thrombo-embolic risk in patients with heart failure. Patients with platelet resistance to PGI₂ also are unresponsive to ADP receptor antagonist therapy. Apart from ischemia, diabetes and aortic valve disease are also associated with impaired anti-aggregatory homeostasis. This review examines the association of impaired platelet cyclic nucleotide (i.e., cGMP and cAMP) signalling with the emerging evidence of thromboembolic risk in cardiovascular diseases, and discusses the potential therapeutic strategies targeting this abnormality. Full article

Cyclic AMP (cAMP) is a pivotal signaling molecule existing in almost all living organisms. However, the mechanism of cAMP signaling in plants remains very poorly understood. Here, we employ the engineered activity of soluble adenylate cyclase to induce cellular cAMP elevation in Arabidopsis thaliana plants and identify 427 cAMP-responsive genes (CRGs) through RNA-seq analysis. Induction of cellular cAMP elevation inhibits seed germination, disturbs phytohormone contents, promotes leaf senescence, impairs ethylene response, and compromises salt stress tolerance and pathogen resistance. A set of 62 transcription factors are among the CRGs, supporting a prominent role of cAMP in transcriptional regulation. The CRGs are significantly overrepresented in the pathways of plant hormone signal transduction, MAPK signaling, and diterpenoid biosynthesis, but they are also implicated in lipid, sugar, K⁺, nitrate signaling, and beyond. Our results provide a basic framework of cAMP signaling for the community to explore. The regulatory roles of cAMP signaling in plant plasticity are discussed. Full article

Heme oxygenase-1 (HO-1) plays a vital role in the catabolism of heme and yields equimolar amounts of biliverdin, carbon monoxide, and free iron. We report that macrophages engulfing either the low amount of heme-containing apoptotic thymocytes or the high amount of heme-containing eryptotic red blood cells (eRBCs) strongly upregulate HO-1. The induction by apoptotic thymocytes is dependent on soluble signals, which do not include adenylate cyclase activators but induce the p38 mitogen-activated protein (MAP) kinase pathway, while in the case of eRBCs, it is cell uptake-dependent. Both pathways might involve the regulation of BTB and CNC homology 1 (BACH1), which is the repressor transcription regulator factor of the HO-1 gene. Long-term continuous efferocytosis of apoptotic thymocytes is not affected by the loss of HO-1, but that of eRBCs is inhibited. This latter is related to an internal signaling pathway that prevents the efferocytosis-induced increase in Rac1 activity. While the uptake of apoptotic cells suppressed the basal pro-inflammatory cytokine production in wild-type macrophages, in the absence of HO-1, engulfing macrophages produced enhanced amounts of pro-inflammatory cytokines. Our data demonstrate that HO-1 is required for both the engulfment and the anti-inflammatory response parts of the efferocytosis program. Full article

Polyamines are ubiquitous polycationic compounds that are highly charged at physiological pH. While passing through the epididymis, sperm lose their capacity to synthesize the polyamines and, upon ejaculation, again come into contact with the polyamines contained in the seminal fluid, unleashing physiological events that improve sperm motility and capacitation. In the present work, we hypothesize about the influence of polyamines, namely, spermine, spermidine, and putrescine, on the activity of sperm channels, evaluating the intracellular concentrations of chloride [Cl⁻]i, calcium [Ca²⁺]i, sodium [Na⁺]i, potassium [K⁺]i, the membrane V_m, and pHi. The aim of this is to identify the possible regulatory mechanisms mediated by the polyamines on sperm-specific channels under capacitation and non-capacitation conditions. The results showed that the presence of polyamines did not directly influence the activity of calcium and chloride channels. However, the results suggested an interaction of polyamines with sodium and potassium channels, which may contribute to the membrane V_m during capacitation. In addition, alkalization of the pHi revealed the possible activation of sperm-specific Na⁺/H⁺ exchangers (NHEs) by the increased levels of cyclic AMP (cAMP), which were produced by soluble adenylate cyclase (sAC) and interact with the polyamines, evidence that is supported by in silico analysis. Full article