Lucas T. Parreiras-e-Silva

Participation of kallikrein–kinin system in different pathologies

The general description of kinins refers to these peptides as molecules involved in vascular tone regulation and inflammation. Nevertheless, in the last years a series of evidences has shown that local hormonal systems, such as the kallikrein-kinin system, may be differently regulated and are of pivotal importance to pathophysiological control. The combined interpretations of many recent studies allow us to conclude that the kallikrein-kinin system plays broader and richer roles than those classically described until recently. In this review, we report findings concerning the participation of the kallikrein-kinin system in inflammation, cancer, and in pathologies related to cardiovascular, renal and central nervous systems.

Testosterone represses ubiquitin ligases atrogin-1 and Murf-1 expression in an androgen-sensitive rat skeletal muscle in vivo

Skeletal muscle atrophy induced by denervation and metabolic diseases has been associated with increased ubiquitin ligase expression. In the present study, we evaluate the influence of androgens on muscle ubiquitin ligases atrogin-1/MAFbx/FBXO32 and Murf-1/Trim63 expression and its correlation with maintenance of muscle mass by using the testosterone-dependent fast-twitch levator ani muscle (LA) from normal or castrated adult male Wistar rats. Gene expression was determined by qRT-PCR and/or immunoblotting. Castration induced progressive loss of LA mass (30% of control, 90 days) and an exponential decrease of LA cytoplasm-to-nucleus ratio (nuclear domain; 22% of control after 60 days). Testosterone deprivation induced a 31-fold increase in LA atrogin-1 mRNA and an 18-fold increase in Murf-1 mRNA detected after 2 and 7 days of castration, respectively. Acute (24 h) testosterone administration fully repressed atrogin-1 and Murf-1 mRNA expression to control levels. Atrogin-1 protein was also increased by castration up to 170% after 30 days. Testosterone administration for 7 days restored atrogin-1 protein to control levels. In addition to the well known stimulus of protein synthesis, our results show that testosterone maintains muscle mass by repressing ubiquitin ligases, indicating that inhibition of ubiquitin-proteasome catabolic system is critical for trophic action of androgens in skeletal muscle. Besides, since neither castration nor androgen treatment had any effect on weight or ubiquitin ligases mRNA levels of extensor digitorum longus muscle, a fast-twitch muscle with low androgen sensitivity, our study shows that perineal muscle LA is a suitable in vivo model to evaluate regulation of muscle proteolysis, closely resembling human muscle responsiveness to androgens.

Recent updates on GPCR biased agonism

G protein-coupled receptors (GPCRs) are the most important targets for drug discovery and not surprisingly ∼40% of all drugs currently in the market act on these receptors. Currently, one of the most active areas in GPCRs signaling is biased agonism, a phenomenon that occurs when a given ligand is able to preferentially activate one (or some) of the possible signaling pathways. In this review, we highlight the most recent findings about biased agonism, including an extension of this concept to intracellular signaling, allosterism, strategies for assessment and interpretation, and perspectives of therapeutic applications for biased agonists.

A pluridimensional view of biased agonism

When studying G protein–coupled receptor (GPCR) signaling and ligand-biased agonism, at least three dimensional spaces must be considered, as follows: 1) the distinct conformations that can be stabilized by different ligands promoting the engagement of different signaling effectors and accessory regulators; 2) the distinct subcellular trafficking that can be conferred by different ligands, which results in spatially distinct signals; and 3) the differential binding kinetics that maintain the receptor in specific conformation and/or subcellular localization for different periods of time, allowing for the engagement of distinct signaling effector subsets. These three pluridimensional aspects of signaling contribute to different faces of functional selectivity and provide a complex, interconnected way to define the signaling profile of each individual ligand acting at GPCRs. In this review, we discuss how each of these aspects may contribute to the diversity of signaling, but also how they shed light on the complexity of data analyses and interpretation. The impact of phenotype variability as a source of signaling diversity, and the influence of novel and more sensitive assays in the detection and analysis of signaling pluridimensionality, is also discussed. Finally, we discuss perspectives for the use of the concept of pluridimensional signaling in drug discovery, in which we highlight future predictive tools that may facilitate the identification of compounds with optimal therapeutic and safety properties based on the signaling signatures of drug candidates.

Hydrogen peroxide production regulates the mitochondrial function in insulin resistant muscle cells: Effect of catalase overexpression

The mitochondrial redox state plays a central role in the link between mitochondrial overloading and insulin resistance. However, the mechanism by which the ROS induce insulin resistance in skeletal muscle cells is not completely understood. We examined the association between mitochondrial function and H2O2 production in insulin resistant cells. Our hypothesis is that the low mitochondrial oxygen consumption leads to elevated ROS production by a mechanism associated with reduced PGC1α transcription and low content of phosphorylated CREB. The cells were transfected with either the encoded sequence for catalase overexpression or the specific siRNA for catalase inhibition. After transfection, myotubes were incubated with palmitic acid (500μM) and the insulin response, as well as mitochondrial function and fatty acid metabolism, was determined. The low mitochondrial oxygen consumption led to elevated ROS production by a mechanism associated with β-oxidation of fatty acids. Rotenone was observed to reduce the ratio of ROS production. The elevated H2O2 production markedly decreased the PGC1α transcription, an effect that was accompanied by a reduced phosphorylation of Akt and CREB. The catalase transfection prevented the reduction in the phosphorylated level of Akt and upregulated the levels of phosphorylated CREB. The mitochondrial function was elevated and H2O2 production reduced, thus increasing the insulin sensitivity. The catalase overexpression improved mitochondrial respiration protecting the cells from fatty acid-induced, insulin resistance. This effect indicates that control of hydrogen peroxide production regulates the mitochondrial respiration preventing the insulin resistance in skeletal muscle cells by a mechanism associated with CREB phosphorylation and β-oxidation of fatty acids.

Evidences of a role for eukaryotic translation initiation factor 5A (eIF5A) in mouse embryogenesis and cell differentiation.

Eukaryotic translation initiation factor 5A (eIF5A) has a unique character: the presence of an unusual amino acid, hypusine, which is formed by post‐translational modifications. Even before the identification of hypusination in eIF5A, the correlation between hypusine formation and protein synthesis, shifting cell proliferation rates, had already been observed. Embryogenesis is a complex process in which cellular proliferation and differentiation are intense. In spite of the fact that many studies have described possible functions for eIF5A, its precise role is under investigation, and to date nothing has been reported about its participation in embryonic development. In this study we show that eIF5A is expressed at all mouse embryonic post‐implantation stages with increase in eIF5A mRNA and protein expression levels between embryonic days E10.5 and E13.5. Immunohistochemistry revealed the ubiquitous presence of eIF5A in embryonic tissues and organs at E13.5 day. Interestingly, stronger immunoreactivity to eIF5A was observed in the stomodeum, liver, ectoderm, heart, and eye, and the central nervous system; regions which are known to undergo active differentiation at this stage, suggesting a role of eIF5A in differentiation events. Expression analyses of MyoD, a myogenic transcription factor, revealed a significantly higher expression from day E12.5 on, both at the mRNA and the protein levels suggesting a possible correlation to eIF5A. Accordingly, we next evidenced that inhibiting eIF5A hypusination in mouse myoblast C2C12 cells impairs their differentiation into myotubes and decreases MyoD transcript levels. Those results point to a new functional role for eIF5A, relating it to embryogenesis, development, and cell differentiation. J. Cell. Physiol. 225: 500–505, 2010.

Phosphodiesterase-4 inhibition reduces proteolysis and atrogenes expression in rat skeletal muscles.

Phosphodiesterase (PDE) inhibition reduces skeletal muscle atrophy, but the underlying molecular mechanism remains unclear. We used microdialysis to investigate the effects of different PDE inhibitors on interstitial tyrosine concentration as well as proteolytic activity and atrogenes expression in isolated rat muscle. Rolipram, a PDE‐4–selective inhibitor, reduced the interstitial tyrosine concentration and rates of muscle protein degradation. The rolipram‐induced muscle cAMP increase was accompanied by a decrease in ubiquitin–proteasome system (UPS) activity and atrogin‐1 mRNA, a ubiquitin‐ligase involved in muscle atrophy. This effect was not associated with Akt phosphorylation but was partially blocked by a protein kinase A inhibitor. Fasting increased atrogin‐1, MuRF‐1 and LC3b expression, and these effects were markedly suppressed by rolipram. Our data suggest that activation of cAMP signaling by PDE‐4 blockade leads to inhibition of UPS activity and atrogenes expression independently of Akt. These findings are important for identifying novel approaches to attenuate muscle atrophy. Muscle Nerve 44: 371–381, 2011

Comparative analyses of downstream signal transduction targets modulated after activation of the AT1 receptor by two β-arrestin-biased agonists

G protein-coupled receptors (GPCRs) are involved in essentially all physiological processes in mammals. The classical GPCR signal transduction mechanism occurs by coupling to G protein, but it has recently been demonstrated that interaction with β-arrestins leads to activation of pathways that are independent of the G protein pathway. Also, it has been reported that some ligands can preferentially activate one of these signaling pathways; being therefore called biased agonists for G protein or β-arrestin pathways. The angiotensin II (AngII) AT1 receptor is a prototype GPCR in the study of biased agonism due to the existence of well-known β-arrestin-biased agonists, such as [Sar1, Ile4, Ile8]-AngII (SII), and [Sar1, D-Ala8]-AngII (TRV027). The aim of this study was to comparatively analyze the two above mentioned β-arrestin-biased agonists on downstream phosphorylation events and gene expression profiles. Our data reveal that activation of AT1 receptor by each ligand led to a diversity of activation profiles that is far broader than that expected from a simple dichotomy between “G protein-dependent” and “β-arrestin-dependent” signaling. We observed clusters of activation profiles common to AngII, SII, and TRV027, as well as downstream effector activation that are unique to AngII, SII, or TRV027. Analyses of β-arrestin conformational changes after AT1 receptor stimulation with SII or TRV027 suggests that the observed differences could account, at least partially, for the diversity of modulated targets observed. Our data reveal that, although the categorization “G protein-dependent” vs. “β-arrestin-dependent” signaling can be of pharmacological relevance, broader analyses of signaling pathways and downstream targets are necessary to generate an accurate activation profile for a given ligand. This may bring relevant information for drug development, as it may allow more refined comparison of drugs with similar mechanism of action and effects, but with distinct side effects.

Insights on eukaryotic translation initiation factor 5A (eIF5A) in the brain and aging

Long-term memory, a persistent form of synaptic plasticity, requires translation of a subset of mRNA present in neuronal dendrites during a short and critical period through a mechanism not yet fully elucidated. Western blotting analysis revealed a high content of eukaryotic translation initiation factor 5A (eIF5A) in the brain of neonatal rats, a period of intense neurogenesis rate, differentiation and synaptic establishment, when compared to adult rats. Immunohistochemistry analysis revealed that eIF5A is present in the whole brain of adult rats showing a variable content among the cells from different areas (e.g. cortex, hippocampus and cerebellum). A high content of eIF5A in the soma and dendrites of Purkinje cells, key neurons in the control of motor long-term memory in the cerebellum, was observed. Detection of high eIF5A content was revealed in dendritic varicosities of Purkinje cells. Evidence is presented herein that a reduction of eIF5A content is associated to brain aging.

In vitro transdentinal effect of low-level laser therapy

Low-level laser therapy (LLLT) has been used for the treatment of dentinal hypersensitivity. However, the specific LLL dose and the response mechanisms of these cells to transdentinal irradiation have not yet been demonstrated. Therefore, this study evaluated the transdentinal effects of different LLL doses on stressed odontoblast-like pulp cells MDPC-23 seeded onto the pulpal side of dentin discs obtained from human third molars. The discs were placed in devices simulating in vitro pulp chambers and the whole set was placed in 24-well plates containing plain culture medium (DMEM). After 24 h incubation, the culture medium was replaced by fresh DMEM supplemented with either 5% (simulating a nutritional stress condition) or 10% fetal bovine serum (FBS). The cells were irradiated with doses of 15 and 25 J cm−2 every 24 h, totaling three applications over three consecutive days. The cells in the control groups were removed from the incubator for the same times as used in their respective experimental groups for irradiation, though without activating the laser source (sham irradiation). After 72 h of the last active or sham irradiation, the cells were evaluated with respect to succinic dehydrogenase (SDH) enzyme production (MTT assay), total protein (TP) expression, alkaline phosphatase (ALP) synthesis, reverse transcriptase polymerase chain reaction (RT-PCR) for collagen type 1 (Col-I) and ALP, and morphology (SEM). For both tests, significantly higher values were obtained for the 25 J cm−2 dose. Regarding SDH production, supplementation of the culture medium with 5% FBS provided better results. For TP and ALP expression, the 25 J cm−2 presented higher values, especially for the 5% FBS concentration (Mann–Whitney p < 0.05). Under the tested conditions, near infrared laser irradiation at 25 J cm−2 caused transdentinal biostimulation of odontoblast-like MDPC-23 cells.