BRAG about (s)lots.

Mutations in IQSEC2/BRAG1 cause intellectual dysfunction by impairing ARF-GEF activity and long-term depression. In this issue, Bai et al. (https://doi.org/10.1083/jcb.202307117) discover how constitutive ARF-GEF activity is regulated by a closed conformation which opens in the presence of Ca2+. Two known pathogenic mutations cause "leaky" autoinhibition with reduced synaptic dynamic range and impaired cognitive performance.

Modeling human mutations to understand TRIO GEF function during development.

In a recent study, Bonnet and colleagues leveraged in silico structure prediction and human genetic data to understand the molecular regulation of the Rac1-activating guanie nucleotide exchange factor (Rac1-GEF) domain of Trio. Their work sheds new light on the role of Trio during axon guidance and explores the mechanism by which Trio GEF function is regulated in health and dysregulated in disease.

Exchange Protein Directly Activated by cAMP 2 Enhances Respiratory Syncytial Virus-Induced Pulmonary Disease in Mice.

Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection in young children. It is also a significant contributor to upper respiratory tract infections, therefore, a major cause for visits to the pediatrician. High morbidity and mortality are associated with high-risk populations including premature infants, the elderly, and the immunocompromised. However, no effective and specific treatment is available. Recently, we discovered that an exchange protein directly activated by cyclic AMP 2 (EPAC2) can serve as a potential therapeutic target for RSV. In both lower and upper epithelial cells, EPAC2 promotes RSV replication and pro-inflammatory cytokine/chemokine induction. However, the overall role of EPAC2 in the pulmonary responses to RSV has not been investigated. Herein, we found that EPAC2-deficient mice (KO) or mice treated with an EPAC2-specific inhibitor showed a significant decrease in body weight loss, airway hyperresponsiveness, and pulmonary inflammation, compared with wild-type (WT) or vehicle-treated mice. Overall, this study demonstrates the critical contribution of the EPAC2-mediated pathway to airway diseases in experimental RSV infection, suggesting the possibility to target EPAC2 as a promising treatment modality for RSV.

The Role of RASGRP2 in Vascular Endothelial Cells-A Mini Review.

RAS guanyl nucleotide-releasing proteins (RASGRPs) are important proteins that act as guanine nucleotide exchange factors, which activate small GTPases and function as molecular switches for intracellular signals. The RASGRP family is composed of RASGRP1-4 proteins and activates the small GTPases, RAS and RAP. Among them, RASGRP2 has different characteristics from other RASGRPs in that it targets small GTPases and its localizations are different. Many studies related to RASGRP2 have been reported in cells of the blood cell lineage. Furthermore, RASGRP2 has also been reported to be associated with Huntington's disease, tumors, and rheumatoid arthritis. In addition, we also recently reported RASGRP2 expression in vascular endothelial cells, and clarified the involvement of xenopus Rasgrp2 in the vasculogenesis process and multiple signaling pathways of RASGRP2 in human vascular endothelial cells with stable expression of RASGRP2. Therefore, this article outlines the existing knowledge of RASGRP2 and focuses on its expression and role in vascular endothelial cells, and suggests that RASGRP2 functions as a protective factor for maintaining healthy blood vessels.

PI3K in T Cell Adhesion and Trafficking.

PI3K signalling is required for activation, differentiation, and trafficking of T cells. PI3Kδ, the dominant PI3K isoform in T cells, has been extensively characterised using PI3Kδ mutant mouse models and PI3K inhibitors. Furthermore, characterisation of patients with Activated PI3K Delta Syndrome (APDS) and mouse models with hyperactive PI3Kδ have shed light on how increased PI3Kδ activity affects T cell functions. An important function of PI3Kδ is that it acts downstream of TCR stimulation to activate the major T cell integrin, LFA-1, which controls transendothelial migration of T cells as well as their interaction with antigen-presenting cells. PI3Kδ also suppresses the cell surface expression of CD62L and CCR7 which controls the migration of T cells across high endothelial venules in the lymph nodes and S1PR1 which controls lymph node egress. Therefore, PI3Kδ can control both entry and exit of T cells from lymph nodes as well as the recruitment to and retention of T cells within inflamed tissues. This review will focus on the regulation of adhesion receptors by PI3Kδ and how this contributes to T cell trafficking and localisation. These findings are relevant for our understanding of how PI3Kδ inhibitors may affect T cell redistribution and function.

The survival and proliferation of osteosarcoma cells are dependent on the mitochondrial BIG3-PHB2 complex formation.

Previous studies reported the critical role of the brefeldin A-inhibited guanine nucleotide exchange protein 3-prohibitin 2 (BIG3-PHB2) complex in modulating estrogen signaling activation in breast cancer cells, yet its pathophysiological roles in osteosarcoma (OS) cells remain elusive. Here, we report a novel function of BIG3-PHB2 in OS malignancy. BIG3-PHB2 complexes were localized mainly in mitochondria in OS cells, unlike in estrogen-dependent breast cancer cells. Depletion of endogenous BIG3 expression by small interfering RNA (siRNA) treatment led to significant inhibition of OS cell growth. Disruption of BIG3-PHB2 complex formation by treatment with specific peptide inhibitor also resulted in significant dose-dependent suppression of OS cell growth, migration, and invasion resulting from G2/M-phase arrest and in PARP cleavage, ultimately leading to PARP-1/apoptosis-inducing factor (AIF) pathway activation-dependent apoptosis in OS cells. Subsequent proteomic and bioinformatic pathway analyses revealed that disruption of the BIG3-PHB2 complex might lead to downregulation of inner mitochondrial membrane protein complex activity. Our findings indicate that the mitochondrial BIG3-PHB2 complex might regulate PARP-1/AIF pathway-dependent apoptosis during OS cell proliferation and progression and that disruption of this complex may be a promising therapeutic strategy for OS.

Tombusviruses Target a Major Crossroad in the Endocytic and Recycling Pathways via Co-opting Rab7 Small GTPase.

Positive-strand RNA viruses induce the biogenesis of unique membranous organelles called viral replication organelles (VROs), which perform virus replication in infected cells. Tombusviruses have been shown to rewire cellular trafficking and metabolic pathways, remodel host membranes, and recruit multiple host factors to support viral replication. In this work, we demonstrate that tomato bushy stunt virus (TBSV) and the closely related carnation Italian ringspot virus (CIRV) usurp Rab7 small GTPase to facilitate building VROs in the surrogate host yeast and in plants. Depletion of Rab7 small GTPase, which is needed for late endosome and retromer biogenesis, strongly inhibits TBSV and CIRV replication in yeast and in planta. The viral p33 replication protein interacts with Rab7 small GTPase, which results in the relocalization of Rab7 into the large VROs. Similar to the depletion of Rab7, the deletion of either MON1 or CCZ1 heterodimeric GEFs (guanine nucleotide exchange factors) of Rab7 inhibited TBSV RNA replication in yeast. This suggests that the activated Rab7 has proviral functions. We show that the proviral function of Rab7 is to facilitate the recruitment of the retromer complex and the endosomal sorting nexin-BAR proteins into VROs. We demonstrate that TBSV p33-driven retargeting of Rab7 into VROs results in the delivery of several retromer cargos with proviral functions. These proteins include lipid enzymes, such as Vps34 PI3K (phosphatidylinositol 3-kinase), PI4Kα-like Stt4 phosphatidylinositol 4-kinase, and Psd2 phosphatidylserine decarboxylase. In summary, based on these and previous findings, we propose that subversion of Rab7 into VROs allows tombusviruses to reroute endocytic and recycling trafficking to support virus replication. IMPORTANCE The replication of positive-strand RNA viruses depends on the biogenesis of viral replication organelles (VROs). However, the formation of membranous VROs is not well understood yet. Using tombusviruses and the model host yeast, we discovered that the endosomal Rab7 small GTPase is critical for the formation of VROs. Interaction between Rab7 and the TBSV p33 replication protein leads to the recruitment of Rab7 into VROs. TBSV-driven usurping of Rab7 has proviral functions through facilitating the delivery of the co-opted retromer complex, sorting nexin-BAR proteins, and lipid enzymes into VROs to create an optimal milieu for virus replication. These results open up the possibility that controlling cellular Rab7 activities in infected cells could be a target for new antiviral strategies.

Parallel Rap1>RalGEF>Ral and Ras signals sculpt the C. elegans nervous system.

Ras is the most commonly mutated oncogene in humans and uses three oncogenic effectors: Raf, PI3K, and RalGEF activation of Ral. Understanding the importance of RalGEF>Ral signaling in cancer is hampered by the paucity of knowledge about their function in animal development, particularly in cell movements. We found that mutations that disrupt function of RalGEF or Ral enhance migration phenotypes of mutants for genes with established roles in cell migration. We used as a model the migration of the canal associated neurons (CANs), and validated our results in HSN cell migration, neurite guidance, and general animal locomotion. These functions of RalGEF and Ral are specific to their control of Ral signaling output rather than other published functions of these proteins. In this capacity Ral functions cell autonomously as a permissive developmental signal. In contrast, we observed Ras, the canonical activator of RalGEF>Ral signaling in cancer, to function as an instructive signal. Furthermore, we unexpectedly identified a function for the close Ras relative, Rap1, consistent with activation of RalGEF>Ral. These studies define functions of RalGEF>Ral, Rap1 and Ras signaling in morphogenetic processes that fashion the nervous system. We have also defined a model for studying how small GTPases partner with downstream effectors. Taken together, this analysis defines novel molecules and relationships in signaling networks that control cell movements during development of the nervous system.

Long non-coding RNA FGD5-AS1 enhances osteosarcoma cell proliferation and migration by targeting miR-506-3p/RAB3D axis.

Osteosarcoma (OSA), the malignant bone tumor, predominantly affecting children and adolescents, threatens the life and life quality of the patients. An increasing number of studies have indicated the role of long non-coding RNA (lncRNA) dysregulation in cancer biology. Herein, the study was aimed to explore the role of FGD5 antisense RNA 1 (FGD5-AS1), a lncRNA, in OSA. Expression levels of FGD5-AS1, miR-506-3p and RAB3D mRNA were quantified utilizing qRT-PCR. The expression of RAB3D protein was examined employing Western blot. A series of functional experiments including CCK-8 assay, BrdU assay, wound healing assay, Transwell assay were performed for studying the effects of FGD5-AS1 on the malignancy of OSA cell lines 143B and HOS. The binding site between miR-506-3p and FGD5-AS1 was identified and validated by luciferase reporter assay and RNA immunoprecipitation assay. It was demonstrated that the expression of FGD5-AS1 was up-regulated in OSA tissues and cell lines, and its high expression is associated with higher Enneking stage and poorer histological differentiation. Gain-of-function and loss-of-function studies suggested that FGD5-AS1 facilitated OSA cells proliferation and migration. The promoting effects of FGD5-AS1 overexpression on OSA cell proliferation and migration could be counteracted by miR-506-3p. Moreover, FGD5-AS1 competitively adsorbed miR-506-3p to repress its expression so as to up-regulate the expression of RAB3D. These results indicate that FGD5-AS1 is capable of expediting OSA cell proliferation and migration via sponging miR-506-3p to up-regulate RAB3D.

DOCK7 protects against replication stress by promoting RPA stability on chromatin.

RPA is a critical factor for DNA replication and replication stress response. Surprisingly, we found that chromatin RPA stability is tightly regulated. We report that the GDP/GTP exchange factor DOCK7 acts as a critical replication stress regulator to promote RPA stability on chromatin. DOCK7 is phosphorylated by ATR and then recruited by MDC1 to the chromatin and replication fork during replication stress. DOCK7-mediated Rac1/Cdc42 activation leads to the activation of PAK1, which subsequently phosphorylates RPA1 at S135 and T180 to stabilize chromatin-loaded RPA1 and ensure proper replication stress response. Moreover, DOCK7 is overexpressed in ovarian cancer and depleting DOCK7 sensitizes cancer cells to camptothecin. Taken together, our results highlight a novel role for DOCK7 in regulation of the replication stress response and highlight potential therapeutic targets to overcome chemoresistance in cancer.

Kalirin-RAC controls nucleokinetic migration in ADRN-type neuroblastoma.

The migrational propensity of neuroblastoma is affected by cell identity, but the mechanisms behind the divergence remain unknown. Using RNAi and time-lapse imaging, we show that ADRN-type NB cells exhibit RAC1- and kalirin-dependent nucleokinetic (NUC) migration that relies on several integral components of neuronal migration. Inhibition of NUC migration by RAC1 and kalirin-GEF1 inhibitors occurs without hampering cell proliferation and ADRN identity. Using three clinically relevant expression dichotomies, we reveal that most of up-regulated mRNAs in RAC1- and kalirin-GEF1-suppressed ADRN-type NB cells are associated with low-risk characteristics. The computational analysis shows that, in a context of overall gene set poverty, the upregulomes in RAC1- and kalirin-GEF1-suppressed ADRN-type cells are a batch of AU-rich element-containing mRNAs, which suggests a link between NUC migration and mRNA stability. Gene set enrichment analysis-based search for vulnerabilities reveals prospective weak points in RAC1- and kalirin-GEF1-suppressed ADRN-type NB cells, including activities of H3K27- and DNA methyltransferases. Altogether, these data support the introduction of NUC inhibitors into cancer treatment research.

Modeling the dynamic behaviors of the COPI vesicle formation regulators, the small GTPase Arf1 and its activating Sec7 guanine nucleotide exchange factor GBF1 on Golgi membranes.

The components and subprocesses underlying the formation of COPI-coated vesicles at the Golgi are well understood. The coating cascade is initiated after the small GTPase Arf1 is activated by the Sec7 domain-containing guanine nucleotide exchange factor GBF1 (Golgi brefeldin A resistant guanine nucleotide exchange factor 1). This causes a conformational shift within Arf1 that facilitates stable association of Arf1 with the membrane, a process required for subsequent recruitment of the COPI coat. Although we have atomic-level knowledge of Arf1 activation by Sec7 domain-containing GEFs, our understanding of the biophysical processes regulating Arf1 and GBF1 dynamics is limited. We used fluorescence recovery after photobleaching data and kinetic Monte Carlo simulation to assess the behavior of Arf1 and GBF1 during COPI vesicle formation in live cells. Our analyses suggest that Arf1 and GBF1 associate with Golgi membranes independently, with an excess of GBF1 relative to Arf1. Furthermore, the GBF1-mediated Arf1 activation is much faster than GBF1 cycling on/off the membrane, suggesting that GBF1 is regulated by processes other than its interactions Arf1. Interestingly, modeling the behavior of the catalytically inactive GBF1/E794K mutant stabilized on the membrane is inconsistent with the formation of a stable complex between it and an endogenous Arf1 and suggests that GBF1/E794K is stabilized on the membrane independently of complex formation.

Candidate Genetic Modifiers for RPGR Retinal Degeneration.

Purpose: To define genetic variants associated with variable severity of X-linked progressive retinal atrophy 1 (XLPRA1) caused by a five-nucleotide deletion in canine RPGR exon ORF15. Methods: A genome-wide association study (GWAS) was performed in XLPRA1 phenotype informative pedigree. Whole genome sequencing (WGS) was used for mutational analysis of genes within the candidate genomic region. Retinas of normal and mutant dogs were used for gene expression, gene structure, and RNA duplex analyses. Results: GWAS followed by haplotype phasing identified an approximately 4.6 Mb candidate genomic interval on CFA31 containing seven protein-coding genes expressed in retina (ROBO1, ROBO2, RBM11, NRIP1, HSPA13, SAMSN1, and USP25). Furthermore, we identified and characterized two novel lncRNAs, ROBO1-AS and ROBO2-AS, that display overlapping gene organization with axon guidance pathway genes ROBO1 and ROBO2, respectively, producing sense-antisense gene pairs. Notably, ROBO1-AS and ROBO2-AS act in cis to form lncRNA/mRNA duplexes with ROBO1 and ROBO2, respectively, suggesting important roles for these lncRNAs in the ROBO regulatory network. A subsequent WGS identified candidate genes within the genomic region on CFA31 that might be implicated in modifying severity of XLPRA1. This approach led to discovery of genetic variants in ROBO1, ROBO1-AS, ROBO2-AS, and USP25 that are strongly associated with the XLPRA1 moderate phenotype. Conclusions: The study provides new insights into the genetic basis of phenotypic variation in severity of RPGRorf15-associated retinal degeneration. Our findings suggest an important role for ROBO pathways in disease progression further expanding on our previously reported changes of ROBO1 expression in XLPRA1 retinas.

Cell wall remodeling and vesicle trafficking mediate the root clock in Arabidopsis.

In Arabidopsis thaliana, lateral roots initiate in a process preceded by periodic gene expression known as the root clock. We identified the vesicle-trafficking regulator GNOM and its suppressor, ADENOSINE PHOSPHATE RIBOSYLATION FACTOR GTPase ACTIVATION PROTEIN DOMAIN3, as root clock regulators. GNOM is required for the proper distribution of pectin, a mediator of intercellular adhesion, whereas the pectin esterification state is essential for a functional root clock. In sites of lateral root primordia emergence, both esterified and de-esterified pectin variants are differentially distributed. Using a reverse-genetics approach, we show that genes controlling pectin esterification regulate the root clock and lateral root initiation. These results indicate that the balance between esterified and de-esterified pectin states is essential for proper root clock function and the subsequent initiation of lateral root primordia.

The GEF Trio controls endothelial cell size and arterial remodeling downstream of Vegf signaling in both zebrafish and cell models.

Arterial networks enlarge in response to increase in tissue metabolism to facilitate flow and nutrient delivery. Typically, the transition of a growing artery with a small diameter into a large caliber artery with a sizeable diameter occurs upon the blood flow driven change in number and shape of endothelial cells lining the arterial lumen. Here, using zebrafish embryos and endothelial cell models, we describe an alternative, flow independent model, involving enlargement of arterial endothelial cells, which results in the formation of large diameter arteries. Endothelial enlargement requires the GEF1 domain of the guanine nucleotide exchange factor Trio and activation of Rho-GTPases Rac1 and RhoG in the cell periphery, inducing F-actin cytoskeleton remodeling, myosin based tension at junction regions and focal adhesions. Activation of Trio in developing arteries in vivo involves precise titration of the Vegf signaling strength in the arterial wall, which is controlled by the soluble Vegf receptor Flt1.

ß-Adrenergic Receptors/Epac Signaling Increases the Size of the Readily Releasable Pool of Synaptic Vesicles Required for Parallel Fiber LTP.

The second messenger cAMP is an important determinant of synaptic plasticity that is associated with enhanced neurotransmitter release. Long-term potentiation (LTP) at parallel fiber (PF)-Purkinje cell (PC) synapses depends on a Ca2+-induced increase in presynaptic cAMP that is mediated by Ca2+-sensitive adenylyl cyclases. However, the upstream signaling and the downstream targets of cAMP involved in these events remain poorly understood. It is unclear whether cAMP generated by ß-adrenergic receptors (ßARs) is required for PF-PC LTP, although noradrenergic varicosities are apposed in PF-PC contacts. Guanine nucleotide exchange proteins directly activated by cAMP [Epac proteins (Epac 1-2)] are alternative cAMP targets to protein kinase A (PKA) and Epac2 is abundant in the cerebellum. However, whether Epac proteins participate in PF-PC LTP is not known. Immunoelectron microscopy demonstrated that ßARs are expressed in PF boutons. Moreover, activation of these receptors through their agonist isoproterenol potentiated synaptic transmission in cerebellar slices from mice of either sex, an effect that was insensitive to the PKA inhibitors (H-89, KT270) but that was blocked by the Epac inhibitor ESI 05. Interestingly, prior activation of these ßARs occluded PF-PC LTP, while the ß1AR antagonist metoprolol blocked PF-PC LTP, which was also absent in Epac2-/- mice. PF-PC LTP is associated with an increase in the size of the readily releasable pool (RRP) of synaptic vesicles, consistent with the isoproterenol-induced increase in vesicle docking in cerebellar slices. Thus, the ßAR-mediated modulation of the release machinery and the subsequent increase in the size of the RRP contributes to PF-PC LTP.SIGNIFICANCE STATEMENT G-protein-coupled receptors modulate the release machinery, causing long-lasting changes in synaptic transmission that influence synaptic plasticity. Nevertheless, the mechanisms underlying synaptic responses to ß-adrenergic receptor (ßAR) activation remain poorly understood. An increase in the number of synaptic vesicles primed for exocytosis accounts for the potentiation of neurotransmitter release driven by ßARs. This effect is not mediated by the canonical protein kinase A pathway but rather, through direct activation of the guanine nucleotide exchange protein Epac by cAMP. Interestingly, this ßAR signaling via Epac is involved in long term potentiation at cerebellar granule cell-to-Purkinje cell synapses. Thus, the pharmacological activation of ßARs modulates synaptic plasticity and opens therapeutic opportunities to control this phenomenon.

The Role of Epac in Cancer Progression.

Cancer continues to be a prime contributor to global mortality. Despite tremendous research efforts and major advances in cancer therapy, much remains to be learned about the underlying molecular mechanisms of this debilitating disease. A better understanding of the key signaling events driving the malignant phenotype of cancer cells may help identify new pharmaco-targets. Cyclic adenosine 3',5'-monophosphate (cAMP) modulates a plethora of biological processes, including those that are characteristic of malignant cells. Over the years, most cAMP-mediated actions were attributed to the activity of its effector protein kinase A (PKA). However, studies have revealed an important role for the exchange protein activated by cAMP (Epac) as another effector mediating the actions of cAMP. In cancer, Epac appears to have a dual role in regulating cellular processes that are essential for carcinogenesis. In addition, the development of Epac modulators offered new routes to further explore the role of this cAMP effector and its downstream pathways in cancer. In this review, the potentials of Epac as an attractive target in the fight against cancer are depicted. Additionally, the role of Epac in cancer progression, namely its effect on cancer cell proliferation, migration/metastasis, and apoptosis, with the possible interaction of reactive oxygen species (ROS) in these phenomena, is discussed with emphasis on the underlying mechanisms and pathways.

Role of Kalirin and mouse strain in retention of spatial memory training in an Alzheimer's disease model mouse line.

Nontransgenic and 3xTG transgenic mice, which express mutant transgenes encoding human amyloid precursor protein (hAPP) along with Alzheimer's disease-associated versions of hTau and a presenilin mutation, acquired the Barnes Maze escape task equivalently at 3-9 months of age. Although nontransgenics retested at 6 and 9 months acquired the escape task more quickly than naïve mice, 3xTG mice did not. Deficits in Kalirin, a multidomain protein scaffold and guanine nucleotide exchange factor that regulates dendritic spines, has been proposed as a contributor to the cognitive decline observed in Alzheimer's disease. To test whether deficits in Kalirin might amplify deficits in 3xTG mice, mice heterozygous/hemizygous for Kalirin and the 3xTG transgenes were generated. Mouse strain, age and sex affected cortical expression of key proteins. hAPP levels in 3xTG mice increased total APP levels at all ages. Kalirin expression showed strong sex-dependent expression in C57 but not B6129 mice. Decreasing Kalirin levels to half had no effect on Barnes Maze task acquisition or retraining in 3xTG hemizygous mice.

DOCK8 is essential for LFA-1-dependent positioning of T follicular helper cells in germinal centers.

T follicular helper (Tfh) cell migration into germinal centers (GCs) is essential for the generation of GC B cells and antibody responses to T cell-dependent (TD) antigens. This process requires interactions between lymphocyte function-associated antigen 1 (LFA-1) on Tfh cells and ICAMs on B cells. The mechanisms underlying defective antibody responses to TD antigens in DOCK8 deficiency are incompletely understood. We show that mice selectively lacking DOCK8 in T cells had impaired IgG antibody responses to TD antigens, decreased GC size, and reduced numbers of GC B cells. However, they developed normal numbers of Tfh cells with intact capacity for driving B cell differentiation into a GC phenotype in vitro. Notably, migration of DOCK8-deficient T cells into GCs was defective. Following T cell receptor (TCR)/CD3 ligation, DOCK8-deficient T cells had impaired LFA-1 activation and reduced binding to ICAM-1. Our results therefore indicate that DOCK8 is important for LFA-1-dependent positioning of Tfh cells in GCs, and thereby the generation of GC B cells and IgG antibody responses to TD antigen.

Epac-mediated relaxation in murine basilar arteries depends on membrane permeability of cyclic nucleotide analogues and endothelial aging.

Cerebral blood supply is finely tuned by regulatory mechanisms depending on vessel caliber the disruption of which contributes to the development of diseases such as vascular dementia, Alzheimer's and Parkinson 's diseases. This study scopes whether cAMP-mimetic-ligands relax young and aged murine cerebral arteries, whether this relates to the activation of PKA or Epac signaling pathways and is changed with advanced age. The hormone Urocortin-1 relaxed submaximally contracted young and old basilar arteries with a similar pD2 and DMAX (~ -8.5 and ~ 90% in both groups). In permeabilized arteries, PKA activation by 6-Bnz-cAMP or Epac activation by 8-pCPT-2'- O-Me-cAMP also induced relaxation with pD2 of -6.3 vs. -5.8 in old for PKA-ligands, and -4.4 and -4.0 in old for Epac-ligands. Furthermore, aging significantly increased submaximal Ca2+-induced force. The effect of 8-pCPT-2'-O-Me-cAMP on intact arteries was attenuated by aging or nitric oxide synthase inhibition. No relaxing effect in both age-groups was observed after treatment with PKAactivator, Sp-6-Phe-cAMPS. In conclusion, our results suggest that in intact basilar arteries relaxation induced by cAMP-mimetics refers only to the activation of Epac and is impaired by smooth muscle and endothelial aging. The study presents an interesting option allowing therapeutic discrimination between both pathways, possibly for the exclusive activation of Epac in brain circulatory system.