Carotid dysfunction in senescent female mice is mediated by increased α1A-adrenoceptor activity and COX-derived vasoconstrictor prostanoids.

α-Adrenergic receptors are crucial regulators of vascular hemodynamics and essential pharmacological targets for cardiovascular diseases. With aging, there is an increase in sympathetic activation, which could contribute to the progression of aging-associated cardiovascular dysfunction, including stroke. Nevertheless, there is little information directly associating adrenergic receptor dysfunction in the blood vessels of aged females. This study determined the role of a-adrenergic receptors in carotid dysfunction of senescent female mice (accelerated-senescence prone, SAMP8), compared with a nonsenescent (accelerated-senescence prone, SAMR1). Vasoconstriction to phenylephrine (Phe) was markedly increased in common carotid artery of SAMP8 [area under the curve (AUC), 527 ± 53] compared with SAMR1 (AUC, 334 ± 30, P = 0.006). There were no changes in vascular responses to the vasoconstrictor agent U46619 or the vasodilators acetylcholine (ACh) and sodium nitroprusside (NPS). Hyperactivity to Phe in female SAMP8 was reduced by cyclooxygenase-1 and cyclooxygenase-2 inhibition and associated with augmented ratio of TXA2/PGI2 release (SAMR1, 1.1 ± 0.1 vs. SAMP8, 2.1 ± 0.3, P = 0.007). However, no changes in cyclooxygenase expression were seen in SAMP8 carotids. Selective α1A-receptor antagonism markedly reduced maximal contraction, whereas α1D antagonism induced a minor shift in Phe contraction in SAMP8 carotids. Ligand binding analysis revealed a threefold increase of α-adrenergic receptor density in smooth muscle cells (VSMCs) of SAMP8 vs. SAMR1. Phe rapidly increased intracellular calcium (Cai2+) in VSMCs via the α1A-receptor, with a higher peak in VSMCs from SAMP8. In conclusion, senescence intensifies vasoconstriction mediated by α1A-adrenergic signaling in the carotid of female mice by mechanisms involving increased Cai2+ and release of cyclooxygenase-derived prostanoids.NEW & NOTEWORTHY The present study provides evidence that senescence induces hyperreactivity of α1-adrenoceptor-mediated contraction of the common carotid. Impairment of α1-adrenoceptor responses is linked to increased Ca2+ influx and release of COX-derived vasoconstrictor prostanoids, contributing to carotid dysfunction in the murine model of female senescence (SAMP8). Increased reactivity of the common carotid artery during senescence may lead to morphological and functional changes in arteries of the cerebral microcirculation and contribute to cognitive decline in females. Because the elderly population is growing, elucidating the mechanisms of aging- and sex-associated vascular dysfunction is critical to better direct pharmacological and lifestyle interventions to prevent cardiovascular risk in both sexes.

Novel potent (dihydro)benzofuranyl piperazines as human histamine receptor ligands - Functional characterization and modeling studies on H3 and H4 receptors.

Histamine acts through four different receptors (H1R-H4R), the H3R and H4R being the most explored in the last years as drug targets. The H3R is a potential target to treat narcolepsy, Parkinson's disease, epilepsy, schizophrenia and several other CNS-related conditions, while H4R blockade leads to anti-inflammatory and immunomodulatory effects. Our group has been exploring the dihydrobenzofuranyl-piperazines (LINS01 series) as human H3R/H4R ligands as potential drug candidates. In the present study, a set of 12 compounds were synthesized from adequate (dihydro)benzofuran synthons through simple reactions with corresponding piperazines, giving moderate to high yields. Four compounds (1b, 1f, 1g and 1h) showed high hH3R affinity (pKi > 7), compound 1h being the most potent (pKi 8.4), and compound 1f showed the best efficiency (pKi 8.2, LE 0.53, LLE 5.85). BRET-based assays monitoring Gαi activity indicated that the compounds are potent antagonists. Only one compound (2c, pKi 7.1) presented high affinity for hH4R. In contrast to what was observed for hH3R, it showed partial agonist activity. Docking experiments indicated that bulky substituents occupy a hydrophobic pocket in hH3R, while the N-allyl group forms favorable interactions with hydrophobic residues in the TM2, 3 and 7, increasing the selectivity towards hH3R. Additionally, the importance of the indole NH in the interaction with Glu5.46 from hH4R was confirmed by the modeling results, explaining the affinity and agonistic activity of compound 2c. The data reported in this work represent important findings for the rational design of future compounds for hH3R and hH4R.

Functional New World monkey oxytocin forms elicit an altered signaling profile and promotes parental care in rats.

The neurohormone oxytocin is a key player in the modulation of reproductive and social behavioral traits, such as parental care. Recently, a correlation between different forms of oxytocin and behavioral phenotypes has been described in the New World Monkeys (NWMs). Here, we demonstrate that, compared with the Leu8OXT found in most placental mammals, the Cebidae Pro8OXT and Saguinus Val3Pro8OXT taxon-specific variants act as equi-efficacious agonists for the Gq-dependent pathway but are weaker agonists for the ß-arrestin engagement and subsequent endocytosis toward the oxytocin receptor (OXTR). Upon interaction with the AVPR1a, Pro8OXT and the common Leu8OXT yielded similar signaling profiles, being equally efficacious on Gq and ß-arrestin, while Val3Pro8OXT showed reduced relative efficacy toward ß-arrestin. Intranasal treatment with either of the variants increased maternal behavior and also promoted unusual paternal care in rats, as measured by pup-retrieval tests. We therefore suggest that Val3Pro8OXT and Pro8OXT are functional variants, which might have been evolutionarily co-opted as an essential part of the adaptive genetic repertoire that allowed the emergence of taxon-specific complex social behaviors, such as intense parental care in the Cebidae and the genus Saguinus.

AVPR1b variation and the emergence of adaptive phenotypes in Platyrrhini primates.

Platyrrhini (New World monkeys, NWm) are a group of primates characterized by behavioral and reproductive traits that are otherwise uncommon among primates, including social monogamy, direct paternal care, and twin births. As a consequence, the study of Platyrrhine primates is an invaluable tool for the discovery of the genetic repertoire underlying these taxon-specific traits. Recently, high conservation of vasopressin (AVP) sequence, in contrast with high variability of oxytocin (OXT), has been described in NWm. AVP and OXT functions are possible due to interaction with their receptors: AVPR1a, AVPR1b, AVPR2, and OXTR; and the variability in this system is associated with the traits mentioned above. Understanding the variability in the receptors is thus fundamental to understand the function and evolution of the system as a whole. Here we describe the variability of AVPR1b coding region in 20 NWm species, which is well-known to influence behavioral traits such as aggression, anxiety, and stress control in placental mammals. Our results indicate that 4% of AVPR1b sites may be under positive selection and a significant number of sites under relaxed selective constraint. Considering the known role of AVPR1b, we suggest that some of the changes described here for the Platyrrhini may be a part of the genetic repertoire connected with the complex network of neuroendocrine mechanisms of AVP-OXT system in the modulation of the HPA axis. Thus, these changes may have promoted the emergence of social behaviors such as direct paternal care in socially monogamous species that are also characterized by small body size and twin births.

The binding of captopril to angiotensin I-converting enzyme triggers activation of signaling pathways.

Hypertension is a global health problem, and angiotensin I (ANG I)-converting enzyme (ACE) inhibitors are largely used to control this pathology. Recently, it has been shown that ACE can also act as a transducer signal molecule when its inhibitors or substrates bind to it. This new role of ACE could contribute to understanding some of the effects not explained by its catalytic activity only. In this study, we investigated signaling pathway activation in Chinese hamster ovary (CHO) cells stably expressing ACE (CHO-ACE) under different conditions. We also investigated gene modulation after 4 h and 24 h of captopril treatment. Our results demonstrated that CHO-ACE cells when stimulated with ANG I, ramipril, or captopril led to JNK and ERK1/2 phosphorylation. To verify any physiological role at the endogenous level, we made use of primary cultures of mesangial cells from spontaneously hypertensive rats (SHR) and Wistar rats. Our results showed that ERK1/2 activation occurred mainly in primary cultures of mesangial cells from SHR rats upon captopril stimulation, suggesting that this signaling pathway could be differentially regulated during hypertension. Our results also showed that captopril treatment leads to a decrease of cyclooxygenase 2, interleukin-1ß, and ß-arrestin2 and a significant increase of AP2 gene expression levels. Our findings strengthen the fact that, in addition to the blockage of enzymatic activity, ACE inhibitors also trigger signaling pathway activation, and this may contribute to their beneficial effects in the treatment of hypertension and other pathologies.

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.

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.

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 ß-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 ß-oxidation of fatty acids.

Non-canonical signalling and roles of the vasoactive peptides angiotensins and kinins.

GPCRs (G-protein-coupled receptors) are among the most important targets for drug discovery due to their ubiquitous expression and participation in cellular events under both healthy and disease conditions. These receptors can be activated by a plethora of ligands, such as ions, odorants, small ligands and peptides, including angiotensins and kinins, which are vasoactive peptides that are classically involved in the pathophysiology of cardiovascular events. These peptides and their corresponding GPCRs have been reported to play roles in other systems and under pathophysiological conditions, such as cancer, central nervous system disorders, metabolic dysfunction and bone resorption. More recently, new mechanisms have been described for the functional regulation of GPCRs, including the transactivation of other signal transduction receptors and the activation of G-protein-independent pathways. The existence of such alternative mechanisms for signal transduction and the discovery of agonists that can preferentially trigger one signalling pathway over other pathways (called biased agonists) have opened new perspectives for the discovery and development of drugs with a higher specificity of action and, therefore, fewer side effects. The present review summarizes the current knowledge on the non-canonical signalling and roles of angiotensins and kinins.

The kinin B1 receptor regulates muscle-specific E3 ligases expression and is involved in skeletal muscle mass control.

Regulation of muscle mass depends on the balance between synthesis and degradation of proteins, which is under the control of different signalling pathways regulated by hormonal, neural and nutritional stimuli. Such stimuli are altered in several pathologies, including COPD (chronic obstructive pulmonary disease), diabetes, AIDS and cancer (cachexia), as well as in some conditions such as immobilization and aging (sarcopenia), leading to muscle atrophy, which represents a significant contribution to patient morbidity. The KKS (kallikrein-kinin system) is composed of the enzymes kallikreins, which generate active peptides called kinins that activate two G-protein-coupled receptors, namely B1 and B2, which are expressed in a variety of tissues. The local modulation of the KKS may account for its participation in different diseases, such as those of the cardiovascular, renal and central nervous systems, cancer and many inflammatory processes, including pain. Owing to such pleiotropic actions of the KKS by local modulatory events and the probable fine-tuning of associated signalling cascades involved in skeletal muscle catabolic disorders [for example, NF-κB (nuclear factor κB) and PI3K (phosphoinositide 3-kinase)/Akt pathways], we hypothesized that KKS might contribute to the modulation of intracellular responses in atrophying skeletal muscle. Our results show that kinin B1 receptor activation induced a decrease in the diameter of C2C12 myotubes, activation of NF-κB, a decrease in Akt phosphorylation levels, and an increase in the mRNA levels of the ubiquitin E3 ligases atrogin-1 and MuRF-1 (muscle RING-finger protein-1). In vivo, we observed an increase in kinin B1 receptor mRNA levels in an androgen-sensitive model of muscle atrophy. In the same model, inhibition of the kinin B1 receptor with a selective antagonist resulted in an impairment of atrogin-1 and MuRF-1 expression and IκB (inhibitor of NF-κB) phosphorylation. Moreover, knockout of the kinin B1 receptor in mice led to an impairment in MuRF-1 mRNA expression after induction of LA (levator ani) muscle atrophy. In conclusion, using pharmacological and gene-ablation tools, we have obtained evidence that the kinin B1 receptor plays a significant role in the regulation of skeletal muscle proteolysis in the LA muscle atrophy model.

Shear stress-induced Ang II AT1 receptor activation: G-protein dependent and independent mechanisms.

Mechanotransduction enables cells to sense and respond to stimuli, such as strain, pressure and shear stress (SS), critical for maintenance of cardiovascular homeostasis or pathological states. The angiotensin II type 1 receptor (AT1R) was the first G protein-coupled receptor described to display stretch-induced activation in cardiomyocytes independent of its ligand Ang II. Here, we assessed whether SS (15 dynes/cm(2), 10 min), an important mechanical force present in the cardiovascular system, activates AT1R independent of its ligand. SS induced extracellular signal-regulated kinase (ERK) activation, used as a surrogate of AT1R activation, in Chinese hamster ovary cells expressing the AT1R (CHO+AT1) but not in wild type cells (CHO). AT1R dependent SS-induced ERK activation involves Ca(2+) inflow and activation of Gαq since Ca(2+) chelator EGTA or Gαq-specific inhibitor YM-254890 decreased SS-induced ERK activation. On the other hand, the activation of JAK-2 and Src, two intracellular signaling molecules independent of G protein activation, were not differently modulated in the presence of AT1R. Also, ERK activation by SS was observed in CHO cells expressing the mutated AT1R DRY/AAY, which has impaired ability to activate Gαq dependent intracellular signaling. Altogether we provided evidence that SS activates AT1R in the absence of its ligand by both a G protein-dependent and -independent pathways. The biological relevance of these observations deserves to be further investigated since the novel mechanisms described extend the knowledge of the activation of GPCRs independent of its traditional ligand.

Angiotensin-(1-7) decreases LPS-induced inflammatory response in macrophages.

It has been previously shown that besides its classical role in blood pressure control the renin-angiotensin system, mainly by action of angiotensin II on the AT(1) receptor, exerts pro-inflammatory effects such as by inducing the production of cytokines. More recently, alternative pathways to this system were described, such as binding of angiotensin-(1-7) to receptor Mas, which was shown to counteract some of the effects evoked by activation of the angiotensin II-AT(1) receptor axis. Here, by means of different molecular approaches we investigated the role of angiotensin-(1-7) in modulating inflammatory responses triggered in mouse peritoneal macrophages. Our results show that receptor Mas transcripts were up-regulated by eightfold in LPS-induced macrophages. Interestingly, macrophage stimulation with angiotensin-(1-7), following to LPS exposure, evoked an attenuation in expression of TNF-α and IL-6 pro-inflammatory cytokines; where this event was abolished when the receptor Mas selective antagonist A779 was also included. We then used heterologous expression of the receptor Mas in HEK293T cells to search for the molecular mechanisms underlying the angiotensin-(1-7)-mediated anti-inflammatory responses by a kinase array; what suggested the involvement of the Src kinase family. In LPS-induced macrophages, this finding was corroborated using the PP2 compound, a specific Src kinase inhibitor; and also by Western blotting when we observed that Ang-(1-7) attenuated the phosphorylation levels of Lyn, a member of the Src kinase family. Our findings bring evidence for an anti-inflammatory role for angiotensin-(1-7) at the cellular level, as well as show that its probable mechanism of action includes the modulation of Src kinases activities.

Exposure of luminal membranes of LLC-PK1 cells to ANG II induces dimerization of AT1/AT2 receptors to activate SERCA and to promote Ca2+ mobilization.

ANG II is secreted into the lumens of proximal tubules where it is also synthesized, thus increasing the local concentration of the peptide to levels of potential physiological relevance. In the present work, we studied the effect of ANG II via the luminal membranes of LLC-PK(1) cells on Ca(2+)-ATPase of the sarco(endo)plasmic reticulum (SERCA) and plasma membrane (PMCA). ANG II (at concentrations found in the lumen) stimulated rapid (30 s) and persistent (30 min) SERCA activity by more than 100% and increased Ca(2+) mobilization. Pretreatment with ANG II for 30 min enhanced the ANG II-induced Ca(2+) spark, demonstrating a positively self-sustained stimulus of Ca(2+) mobilization by ANG II. ANG II in the medium facing the luminal side of the cells decreased with time with no formation of metabolites, indicating peptide internalization. ANG II increased heterodimerization of AT(1) and AT(2) receptors by 140%, and either losartan or PD123319 completely blocked the stimulation of SERCA by ANG II. Using the PLC inhibitor U73122, PMA, and calphostin C, it was possible to demonstrate the involvement of a PLC→DAG(PMA)→PKC pathway in the stimulation of SERCA by ANG II with no effect on PMCA. We conclude that ANG II triggers SERCA activation via the luminal membrane, increasing the Ca(2+) stock in the reticulum to ensure a more efficient subsequent mobilization of Ca(2+). This first report on the regulation of SERCA activity by ANG II shows a new mechanism for Ca(2+) homeostasis in renal cells and also for regulation of Ca(2+)-modulated fluid reabsorption in proximal tubules.

Angiotensin II Type 1 Receptor Tachyphylaxis Is Defined by Agonist Residence Time.

Several GPCRs (G-protein-coupled receptors) have been reported to exhibit tachyphylaxis, which is an acute loss of functional receptor response after repeated stimuli with an agonist. GPCRs are important clinical targets for a wide range of disorders. Therefore, elucidation of the ligand features that contribute to receptor tachyphylaxis and signaling events underlying this phenomenon is important for drug discovery and development. In this study, we examined the role of ligand-binding kinetics in the tachyphylaxis of AT1R (angiotensin II type 1 receptor) using bioluminescence resonance energy transfer assays to monitor signaling events under both kinetic and equilibrium conditions. We investigated AT1R signal transduction and translocation promoted by the endogenous tachyphylactic agonist Ang II (angiotensin II) and its analogs, described previously for inducing reduced receptor tachyphylaxis. Estimation of binding kinetic parameters of the ligands revealed that the residence time of Ang II was higher than that of the analogs, resulting in more sustained Gq protein activation and recruitment of ß-arrestin than that promoted by the analogs. Furthermore, we observed that Ang II led to more sustained internalization of the receptor, thereby retarding its recycling to the plasma membrane and preventing further receptor responses. These results show that the apparent lack of tachyphylaxis in the studied analogs resulted from their short residence time at the AT1R. In addition, our data highlight the relevance of complete characterization of novel GPCR drug candidates, taking into account their receptor binding kinetics as well.

Oral nitrite treatment increases S-nitrosylation of vascular protein kinase C and attenuates the responses to angiotensin II.

Nitrate and nitrite supplement deficient endogenous nitric oxide (NO) formation. While these anions may generate NO, recent studies have shown that circulating nitrite levels do not necessarily correlate with the antihypertensive effect of oral nitrite administration and that formation of nitrosylated species (RXNO) in the stomach is critically involved in this effect. This study examined the possibility that RXNO formed in the stomach after oral nitrite administration promotes target protein nitrosylation in the vasculature, inhibits vasoconstriction and the hypertensive responses to angiotensin II. Our results show that oral nitrite treatment enhances circulating RXNO concentrations (measured by ozone-based chemiluminescence methods), increases aortic protein kinase C (PKC) nitrosylation (measured by resin-assisted capture SNO-RAC method), and reduces both angiotensin II-induced vasoconstriction (isolated aortic ring preparation) and hypertensive (in vivo invasive blood pressure measurements) effects implicating PKC nitrosylation as a key mechanism for the responses to oral nitrite. Treatment of rats with the nitrosylating compound S-nitrosoglutathione (GSNO) resulted in the same effects described for oral nitrite. Moreover, partial depletion of thiols with buthionine sulfoximine prevented PKC nitrosylation and the blood pressure effects of oral nitrite. Further confirming a role for PKC nitrosylation, preincubation of aortas with GSNO attenuated the responses to both angiotensin II and to a direct PKC activator, and this effect was attenuated by ascorbate (reverses GSNO-induced nitrosylation). GSNO-induced nitrosylation also inhibited the increases in Ca2+ mobilization in angiotensin II-stimulated HEK293T cells expressing angiotensin type 1 receptor. Together, these results are consistent with the idea that PKC nitrosylation in the vasculature may underlie oral nitrite treatment-induced reduction in the vascular and hypertensive responses to angiotensin II.

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.

Inhibition of the renin-angiotensin system prevents seizures in a rat model of epilepsy.

The RAS (renin-angiotensin system) is classically involved in BP (blood pressure) regulation and water-electrolyte balance, and in the central nervous system it has been mostly associated with homoeostatic processes, such as thirst, hormone secretion and thermoregulation. Epilepsies are chronic neurological disorders characterized by recurrent epileptic seizures that affect 1-3% of the world's population, and the most commonly used anticonvulsants are described to be effective in approx. 70% of the population with this neurological alteration. Using a rat model of epilepsy, we found that components of the RAS, namely ACE (angiotensin-converting enzyme) and the AT1 receptor (angiotensin II type 1 receptor) are up-regulated in the brain (2.6- and 8.2-fold respectively) following repetitive seizures. Subsequently, epileptic animals were treated with clinically used doses of enalapril, an ACE inhibitor, and losartan, an AT1 receptor blocker, leading to a significant decrease in seizure severities. These results suggest that centrally acting drugs that target the RAS deserve further investigation as possible anticonvulsant agents and may represent an additional strategy in the management of epileptic patients.

Epilepsy Seizures in Spontaneously Hypertensive Rats After Acoustic Stimulation: Role of Renin-Angiotensin System.

Hypertension is a common comorbidity observed in individuals with epilepsy. Growing evidence suggests that lower blood pressure is associated with reduced frequency and severity of seizures. In this study, we sought to investigate whether the renin-angiotensin system (RAS), which is a critical regulator of blood pressure, is involved in the pathogenesis of audiogenic epilepsy-related seizures in a hypertensive rat model. Spontaneously hypertensive rats (SHRs) were given RAS inhibitors, angiotensin-converting enzyme (ACE) inhibitor or angiotensin II type I receptor (AT1R) antagonist, for 7 days prior to inducing epileptic seizures by acoustic stimulation. After the pretreatment phase, blood pressure (BP) of SHRs normalized as expected, and there was no difference in systolic and diastolic BP between the pretreated SHRs and normotensive rat group (Wistar). Next, treated and untreated SHRs (a high BP control) were individually subjected to acoustic stimuli twice a day for 2 weeks. The severity of tonic-clonic seizures and the severity of temporal lobe epilepsy seizures (product of forebrain recruitment) were evaluated by the mesencephalic severity index (Rossetti et al. scale) and the limbic index (Racine's scale), respectively. Seizures were observed in both untreated (a high BP control) SHRs and in SHRs treated with AT1R antagonist and ACE inhibitor. There was no statistical difference in the mesencephalic severity and limbic index between these groups. Our results demonstrate that SHRs present seizure susceptibility with acoustic stimulation. Moreover, although RAS inhibitors effectively reduce blood pressure in SHR, they do not prevent developing epileptic seizures upon acoustic stimulation in SHR. In conclusion, our study shows that RAS is an unlikely link between hypertension and susceptibility to epileptic seizures induced by acoustic stimulation in SHRs, which is in contrast with the anticonvulsant effect of losartan in other animal models of epilepsy.

Signal Transduction Profiling of Angiotensin II Type 1 Receptor With Mutations Associated to Atrial Fibrillation in Humans.

The AT1 receptor (AT1R) has a major role in the Renin-Angiotensin System, being involved in several physiological events including blood pressure control and electrolyte balance. The AT1R is a member of the G protein coupled receptors (GPCR) family, classically known to couple Gαq and engage ß-arrestin recruitment. Both G protein and arrestin signaling pathways are involved in modulation of different downstream kinases. A previous study reported that mutations in the AT1R (A244S and I103T-A244S) were positively correlated with higher risk of atrial fibrillation in men. Based on that report, we aimed to investigate if these mutations, including I103T only, could affect AT1R signal transduction profile, and consequently, implicate in atrial fibrillation outcome. To address that, we engineered an AT1R carrying the above-mentioned mutations, and functionally evaluated different signaling pathways. Phosphokinase profiler array to assess the mutations downstream effects on kinases and kinase substrates phosphorylation levels was used. Our results show that the I103T-A244S mutant receptor presents decreased ß-arrestin 2 recruitment, which could lead to a harmful condition of sustained Gαq signaling. Moreover, the phosphokinase profiler array revealed that the same mutation led to downstream modulation of kinase pathways that are linked to physiological responses such as fibrous tissue formation, apoptosis and cell proliferation.