Molecular-Scale Dynamics of Long Range Retrograde Brain-Derived Neurotrophic Factor Transport Shaped by Cellular Spatial Context.

Retrograde neurotrophin (NT) transport is a specialized form of signal transduction used to conduct information from axons to the cell bodies of central and peripheral nervous system neurons. It is activated upon NT-Trk receptor binding, NT-Trk internalization into signaling endosomes, and their motion along the axon toward the cell body. Brain-derived neurotrophic factor (BDNF) is an abundant NT that modulates key brain and spinal cord functions, and defects in BDNF trafficking are associated with neuronal death, neurodegenerative diseases and in nerve injury. Decades of study have yielded impressive progress in elucidating NT retrograde transport; however, much information remains unclear. For example, while it is known that NT function is dependent on tight control of NT-receptor intracellular trafficking, data describing the precise spatiotemporal molecular dynamics of their axonal to somatic transport are lacking. In past work, we showed the use of discrete, photo-bleaching-resistant quantum dot (QD)-BNDF probes to activate and track BDNF-TrkB receptor internalization; this revealed a rich diversity of molecular motions that intracellular BDNF signaling endosomes undergo within the soma of nodose ganglia sensory neurons. Here, we used combined techniques of discrete QD-BDNF tracking with compartmented microfluidic chambers to characterize retrograde BDNF-TrkB transport over long-ranging distances of primary dorsal root ganglion sensory neuronal axons. Our new findings show that axonal retrograde motion is comprised of heterogeneous mixtures of diffusive behaviors, pauses, and variations in net molecular-motor-dependent transport speeds. Notably, specific molecular dynamic features such as NT speed were dependent on spatial context that could be categorized in distance from distal axons and proximity to the soma and were not entirely dictated by active motor transport speed. The important implication is recognition that NT-receptor retrograde transport is comprised of molecular dynamics, which change over the course of long-range trafficking to shape overall transport and possibly signaling.

A New Intravenous Immune Globulin: Novel or Not?

OBJECTIVE: To assess the clinical use and determine the place in therapy for immune globulin intravenous (IV), human-slra, a recently approved IV immune globulin for the treatment of primary immune deficiency diseases (PIDD). DATA SOURCES: A PubMed and MEDLINE search (2010 to April 2020) was conducted for relevant articles. Data were also obtained from the package insert. STUDY SELECTION AND DATA EXTRACTION: English language publications regarding the clinical efficacy and safety of immune globulin-slra were analyzed. Publications focused on use of immune globulin products were also included. DATA SYNTHESIS: Immune globulin-slra is indicated for patients with PIDD and was specifically developed to include donor plasma with high respiratory syncytial virus (RSV) antibody titers. Efficacy was demonstrated through favorable incidence of infections and infection-related complications. Patients treated with immune globulin-slra had increases in anti-RSV neutralizing antibody titers compared with baseline. Adverse events occurred at rates similar to or less than other available immune globulin products. RELEVANCE TO PATIENT CARE AND CLINICAL PRACTICE: This review describes a new immune globulin product available for use in patients with PIDD. A novel approach to managing patients at risk of serious infections may be to utilize products with formulations proven to not only boost IgG levels, but also antibodies to specific pathogens. CONCLUSIONS: The choice of which immune globulin product to select for a patient or formulary is complex. Each product is unique, and differences between products should be taken into consideration, along with cost and availability.

Involvement of CREB-regulated transcription coactivators (CRTC) in transcriptional activation of steroidogenic acute regulatory protein (Star) by ACTH.

Studies in vivo have suggested the involvement of CREB-regulated transcription coactivator (CRTC)2 on ACTH-induced transcription of the key steroidogenic protein, Steroidogenic Acute Regulatory (StAR). The present study uses two ACTH-responsive adrenocortical cell lines, to examine the role of CRTC on Star transcription. Here we show that ACTH-induced Star primary transcript, or heteronuclear RNA (hnRNA), parallels rapid increases in nuclear levels of the 3 isoforms of CRTC; CRTC1, CRTC2 and CRTC3. Furthermore, ACTH promotes recruitment of CRTC2 and CRTC3 by the Star promoter and siRNA knockdown of either CRTC3 or CRTC2 attenuates the increases in ACTH-induced Star hnRNA. Using pharmacological inhibitors of PKA, MAP kinase and calcineurin, we show that the effects of ACTH on Star transcription and CRTC nuclear translocation depend predominantly on the PKA pathway. The data provides evidence that CRTC2 and CRTC3, contribute to activation of Star transcription by ACTH, and that PKA/CRTC-dependent pathways are part of the multifactorial mechanisms regulating Star transcription.

Intranasal application of PACAP and ß-cyclodextrin before the "critical period of proestrous stage" can block ovulation.

INTRODUCTION: It was previously shown that intracerebroventricular administration of pituitary adenylate cyclase-activating polypeptide (PACAP) prior to GnRH mobilization in proestrus prevents ovulation in rats. In this study, we examined whether PACAP given intranasally could influence luteinizing hormone (LH) and prolactin (PRL) surges and ovulation. METHODS: On the day of proestrus PACAP, p-cyclodextrin (modifier of blood-brain barrier) or PACAP + p-cyclodextrin was applied intranasally between 12:30 and 13:00. Blood samples were taken at 16:00, 18:00, and 20:00 for measuring plasma hormone levels. In the next morning, the expelled ova were counted. p-Cyclodextrin was also administered to male and diestrous female rats between 12:30 and 13:00 and blood was taken at 18:00. RESULTS: PACAP prevented LH and PRL surges and ovulation in about half of the rats, p-cyclodextrin alone more effectively prevented ovulation. When PACAP and p-cyclodextrin were administered together, more rats ovulated like when PACAP given alone. p-Cyclodextrin did not influence LH and PRL levels in diestrous females; however, in males, it significantly enhanced PRL level. DISCUSSION: Not only the intracerebroventricular, but the intranasal application of PACAP prevented ovulation. p-Cyclodextrin alone is more effective than PACAP and enhances PRL levels in male rats. PACAP and p-cyclodextrin given together weaken each other's effect. p-Cyclodextrin, as excipient of various drugs, has to be used carefully in human medications.

Chemoenzymatic Dynamic Kinetic Resolution of Amines in Fully Continuous-Flow Mode.

In this study, lipase-mediated dynamic kinetic resolution (DKR) of various benzylic amines (1a-g) is presented which is realized in a so far unprecedented fully continuous-flow system. The DKR process applying sol-gel immobilized lipase B from Candida antarctica as biocatalyst, palladium on 3-aminopropyl-functionalized silica as racemization catalyst, isopropyl 2-ethoxyacetate as acylating agent, ammonium formate as hydrogen and nitrogen sources, and 2-methyl-2-butanol as solvent under regulated pressure provided the desired products in moderate to good yields with excellent enantiomeric excesses.

Differential mast cell outcomes are sensitive to FcεRI-Syk binding kinetics.

Cross-linking of immunoglobulin E-bound FcεRI triggers multiple cellular responses, including degranulation and cytokine production. Signaling is dependent on recruitment of Syk via docking of its dual SH2 domains to phosphorylated tyrosines within the FcεRI immunoreceptor tyrosine-based activation motifs. Using single-molecule imaging in live cells, we directly visualized and quantified the binding of individual mNeonGreen-tagged Syk molecules as they associated with the plasma membrane after FcεRI activation. We found that Syk colocalizes transiently to FcεRI and that Syk-FcεRI binding dynamics are independent of receptor aggregate size. Substitution of glutamic acid for tyrosine between the Syk SH2 domains (Syk-Y130E) led to an increased Syk-FcεRI off-rate, loss of site-specific Syk autophosphorylation, and impaired downstream signaling. Genome edited cells expressing only Syk-Y130E were deficient in antigen-stimulated calcium release, degranulation, and production of some cytokines (TNF-a, IL-3) but not others (MCP-1, IL-4). We propose that kinetic discrimination along the FcεRI signaling pathway occurs at the level of Syk-FcεRI interactions, with key outcomes dependent upon sufficiently long-lived Syk binding events.

Estimation of the diffusion constant from intermittent trajectories with variable position uncertainties.

The movement of a particle described by Brownian motion is quantified by a single parameter, D, the diffusion constant. The estimation of D from a discrete sequence of noisy observations is a fundamental problem in biological single-particle tracking experiments since it can provide information on the environment and/or the state of the particle itself via the hydrodynamic radius. Here, we present a method to estimate D that takes into account several effects that occur in practice, important for the correct estimation of D, and that have hitherto not been combined together for an estimation of D. These effects are motion blur from the finite integration time of the camera, intermittent trajectories, and time-dependent localization uncertainty. Our estimation procedure, a maximum-likelihood estimation with an information-based confidence interval, follows directly from the likelihood expression for a discretely observed Brownian trajectory that explicitly includes these effects. We begin with the formulation of the likelihood expression and then present three methods to find the exact solution. Each method has its own advantages in either computational robustness, theoretical insight, or the estimation of hidden variables. The Fisher information for this likelihood distribution is calculated and analyzed to show that localization uncertainties impose a lower bound on the estimation of D. Confidence intervals are established and then used to evaluate our estimator on simulated data with experimentally relevant camera effects to demonstrate the benefit of incorporating variable localization errors.

Publisher's Note: Estimation of the diffusion constant from intermittent trajectories with variable position uncertainties [Phys. Rev. E 93, 042401 (2016)].

This corrects the article DOI: 10.1103/PhysRevE.93.042401.

A secretagogin locus of the mammalian hypothalamus controls stress hormone release.

A hierarchical hormonal cascade along the hypothalamic-pituitary-adrenal axis orchestrates bodily responses to stress. Although corticotropin-releasing hormone (CRH), produced by parvocellular neurons of the hypothalamic paraventricular nucleus (PVN) and released into the portal circulation at the median eminence, is known to prime downstream hormone release, the molecular mechanism regulating phasic CRH release remains poorly understood. Here, we find a cohort of parvocellular cells interspersed with magnocellular PVN neurons expressing secretagogin. Single-cell transcriptome analysis combined with protein interactome profiling identifies secretagogin neurons as a distinct CRH-releasing neuron population reliant on secretagogin's Ca(2+) sensor properties and protein interactions with the vesicular traffic and exocytosis release machineries to liberate this key hypothalamic releasing hormone. Pharmacological tools combined with RNA interference demonstrate that secretagogin's loss of function occludes adrenocorticotropic hormone release from the pituitary and lowers peripheral corticosterone levels in response to acute stress. Cumulatively, these data define a novel secretagogin neuronal locus and molecular axis underpinning stress responsiveness.

The CB1 receptor mediates the peripheral effects of ghrelin on AMPK activity but not on growth hormone release.

This study aimed to investigate whether the growth hormone release and metabolic effects of ghrelin on AMPK activity of peripheral tissues are mediated by cannabinoid receptor type 1 (CB1) and the central nervous system. CB1-knockout (KO) and/or wild-type mice were injected peripherally or intracerebroventricularly with ghrelin and CB1 antagonist rimonabant to study tissue AMPK activity and gene expression (transcription factors SREBP1c, transmembrane protein FAS, enzyme PEPCK, and protein HSL). Growth hormone levels were studied both in vivo and in vitro. Peripherally administered ghrelin in liver, heart, and adipose tissue AMPK activity cannot be observed in CB1-KO or CB1 antagonist-treated mice. Intracerebroventricular ghrelin treatment can influence peripheral AMPK activity. This effect is abolished in CB1-KO mice and by intracerebroventricular rimonabant treatment, suggesting that central CB1 receptors also participate in the signaling pathway that mediates the effects of ghrelin on peripheral tissues. Interestingly, in vivo or in vitro growth hormone release is intact in response to ghrelin in CB1-KO animals. Our data suggest that the metabolic effects of ghrelin on AMPK in peripheral tissues are abolished by the lack of functional CB1 receptor via direct peripheral effect and partially through the central nervous system, thus supporting the existence of a possible ghrelin-cannabinoid-CB1-AMPK pathway.

Superdiffusive transport by multivalent molecular walkers moving under load.

We introduce a model for translational molecular motors to demonstrate that a multivalent catalytic walker with flexible, uncoordinated legs can transform the free energy of surface-bound substrate sites into mechanical work and undergo biased, superdiffusive motion, even in opposition to an external load force. The walker in the model lacks any inherent orientation of body or track, and its legs have no chemomechanical coupling other than the passive constraint imposed by their connection to a common body. Yet, under appropriate kinetic conditions, the walker's motion is biased in the direction of unvisited sites, which allows the walker to move nearly ballistically away from the origin as long as a local supply of unmodified substrate sites is available. The multivalent random walker model is mathematically formulated as a continuous-time Markov process and is studied numerically. We use Monte Carlo simulations to generate ensemble estimates of the mean squared displacement and mean work done for this nonergodic system. Our results show that a residence time bias between visited and unvisited sites leads to superdiffusive motion over significant times and distances. This mechanism can be used to adapt any enzyme-substrate system with appropriate kinetics for use as a functional chemical implementation of a molecular motor, without the need for structural anisotropy or conformationally mediated chemomechanical coordination.

Transcriptional regulation of episodic glucocorticoid secretion.

Circadian and ultradian variations of basal glucocorticoid secretion and transient elevations during stress are essential for homeostasis. Using intronic qRT-PCR to measure changes in primary transcript (hnRNA) we have shown that secretory events induced by stress or ACTH injection are followed by episodic increases in transcription of rate limiting steroidogenic proteins, such as steroidogenic acute regulatory protein (StAR), cytochrome P450 side chain cleavage and melanocortin receptor associated protein. These transcriptional episodes imply rapid turnover of steroidogenic proteins and the need of de novo synthesis following each secretory event. In addition to episodic ACTH secretion, it is likely that intracellular feedback mechanisms at the adrenal fasciculata level contribute to the generation of episodes of transcription. The time relationship between activation and translocation of the CREB co-activator, transducer of regulated CREB activity (TORC) to the nucleus preceding transcriptional episodes suggest the involvement of TORC in the transcriptional activation of StAR and other steroidogenic proteins.

Mechanism of diffusive transport in molecular spider models.

Recent advances in single-molecule chemistry have led to designs for artificial multipedal walkers that follow tracks of chemicals. We investigate the motion of a class of walkers, called molecular spiders, which consist of a rigid chemically inert body and several flexible enzymatic legs. The legs can reversibly bind to chemical substrates on a surface and through their enzymatic action convert them to products. The legs can also reversibly bind to products, but at a different rate. Antal and Krapivsky have proposed a model for molecular spider motion over regular one-dimensional lattices [T. Antal and P. L. Krapivsky, Phys. Rev. E 76, 021121 (2007).]. In the model the legs hop from site to site under constraints imposed by connection to a common body. The first time a leg visits a site, the site is an uncleaved substrate, and the leg hops from this site only once it has cleaved it into a product. This cleavage happens at a rate r<1, slower than dissociation from a product site, r=1. The effect of cleavage is to slow down the hopping rate for legs that visit a site for the first time. Along with the constraints imposed on the legs, this leads to an effective bias in the direction of unvisited sites that decreases the average time needed to visit n sites. The overall motion, however, remains diffusive in the long time limit. We have reformulated the Antal-Krapivsky model as a continuous-time Markov process and simulated many traces of this process using kinetic Monte Carlo techniques. Our simulations show a previously unpredicted transient behavior wherein spiders with small r values move superdiffusively over significant distances and times. We explain this transient period of superdiffusive behavior by describing the spider process as switching between two metastates: a diffusive state D wherein the spider moves in an unbiased manner over previously visited sites, and a boundary state B wherein the spider is on the boundary between regions of visited and unvisited sites and experiences a bias in the direction of unvisited sites. We show that while the spider remains in the B state it moves ballistically in the direction of unvisited sites, and while the spider is in the D state it moves diffusively. The relative amount of time the spider spends in the two states determines how superdiffusively the spider moves. We show that the B state is Markovian, but the D state is non-Markovian because the duration of a D period depends on how many sites have been visited previously. As time passes the spider spends progressively more time in the D state (moving diffusively) and less time in the B state (moving ballistically). This explains both the transient superdiffusive motion and the eventual decay to diffusive motion as t→∞.

Role of salsolinol in the regulation of pituitary prolactin and peripheral dopamine release.

(R)-Salsolinol (SAL), a dopamine (DA)-related tetrahydroisoquinoline, has been found in extracts of the neuro-intermediate lobes (NIL) of pituitary glands and in the median eminence of the hypothalamus obtained from intact male rats and from ovariectomized and lactating female rats. Moreover, analysis of SAL concentrations in NIL revealed parallel increases with plasma prolactin (PRL) in lactating rats exposed to a brief (10 min) suckling stimulus after 4-h separation. SAL is sufficiently potent in vivo to account for the massive discharge of PRL that occurs after physiological stimuli (i.e. suckling). At the same time, it was without effect on the secretion of other pituitary hormones. It has been also shown that another isoquinoline derivative, 1-methyldihydroisoquinoline (1MeDIQ), which is a structural analogue of SAL, can dose-dependently inhibit the in-vivo PRL-releasing effect of SAL. Moreover, 1MeDIQ can inhibit the elevation of plasma PRL induced by physiological stimuli, for example suckling, or in different stressful situations also. 1MeDIQ also has a psycho-stimulant action, which is fairly similar to the effect of amphetamine, i.e. it induces an increase in plasma catecholamine concentrations. It is clear from these data that this newly discovered endogenous compound could be involved in regulation of pituitary PRL secretion. It has also been observed that SAL is present in peripheral, sympathetically innervated organs, for example the atrium, spleen, liver, ovaries, vas deferens, and salivary gland. Furthermore, SAL treatment of rats results in dose-dependent and time-dependent depletion of the DA content of the organs listed above without having any effect on the concentration of norepinephrine. More importantly, this effect of SAL can be completely prevented by amphetamine and by 1MeDIQ pretreatment. It is clear there is a mutual interaction between SAL, 1MeDIQ, and amphetamine or alcohol, not only on PRL release; their interaction with catecholamine "synthesis/metabolism" of sympathetic nerve terminals is also obvious.

Dephosphorylation/inactivation of tyrosine hydroxylase at the median eminence of the hypothalamus is required for suckling-induced prolactin and adrenocorticotrop hormone responses.

We have recently found that dopamine (DA) released from terminals of the hypothalamic neuroendocrine dopamine (NEDA) neurons plays a role not only in prolactin (PRL), but also in adrenocorticotrop hormone (ACTH) secretion, without having any influence on alpha-melanocyte-stimulating hormone (alpha-MSH) release in lactating dams. The aim of our present studies was to further investigate this DAerg regulation of ACTH using consecutively applied physiological stimulation (suckling) and pharmacological inhibition of the rate-limiting enzyme of DA synthesis (tyrosine hydroxylase, TH) by alpha-methyl-p-tyrosine (alpha-MpT) that acutely affect secretion of these pituitary hormones during lactation. Following 4h separation period, two experimental groups were formed. In the first group, lactating rats were assembled with their litters for 60 min prior to alpha-MpT. In the second group, the alpha-MpT was injected first and 60 min later suckling stimulus was applied. Plasma samples were taken in every 15 min during the 90 min experimental period. Concentrations of plasma PRL, ACTH and alpha-MSH were measured by specific RIAs. Both stimuli applied in the first sequence, significantly elevated plasma PRL and ACTH levels in separated lactating dams, without having any effect on alpha-MSH secretion. Suckling applied in the first sequence was able to block the alpha-MpT-induced elevation of ACTH secretion, while PRL response was also significantly attenuated. alpha-MpT pretreatment prevented both PRL and ACTH responses to suckling stimulus. Investigating the dephosphorylation/inactivation of TH in the arcuate nucleus-ME (TIDA) regions, no pTH-immunoreactive perikarya or terminals can be found in continuously suckled dams. In contrast, after 4h separation of the mothers from their litters, pTH-immunoreactivity can be clearly visualized in the external zone of ME. In alpha-MpT pretreated mothers following 4h separation no pTH positive terminals are visible. No changes in the TH immunostaining can be observed in any of these experimental groups. In conclusion, dephosphorylation/inactivation of TH (the rate-limiting enzyme of the DA biosynthesis) in NEDA neurons is required for suckling-induced PRL and ACTH responses.

Plasma prolactin concentrations and copulatory behavior after salsolinol injection in male rats.

PURPOSE: The dopamine-derived endogenous compound, R-salsolinol (SAL), was recently identified as a putative endogenous prolactin (PRL)-releasing factor. However, how SAL influences copulatory behavior is unknown. In this study, we examined the relationship between SAL and copulatory behavior in male rats. METHODS: Male Sprague-Dawley rats administered SAL were exposed to female rats in estrus, the plasma PRL concentration was measured, and the behavioral frequency and time during copulatory behavior were noted. RESULTS: In the control and SAL groups, plasma PRL concentrations at 15 min before exposure to the female were 7.3 ± 2.0 and 8.0 ± 1.5 ng/ml, respectively. Moreover, plasma PRL concentrations in males immediately after exposure to the female were 7.4 ± 1.2 and 68.0 ± 5.9 ng/ml, respectively (P < 0.05). All (8/8) of the control animals ejaculated in the presence of the female, whereas only 33% (2/6) of the SAL group ejaculated. An increasing tendency for mount latency and intromission latency and a decreasing tendency for intromission frequency were observed in the SAL group. CONCLUSIONS: Copulatory behavior was inhibited in male rats after SAL injection, suggesting that SAL is a copulatory behavior inhibiting factor.

High-resolution cross-reactive array for alkaloids.

A high-resolution cross-reactive array capable of classifying alkaloids over a range of concentrations was generated by systematic introduction of a nitroindole analog into a hydrophobic pocket within a DNA three-way junction to match structural motifs presented by the analytes.

Dopamine-regulated adrenocorticotropic hormone secretion in lactating rats: functional plasticity of melanotropes.

Pro-opiomelanocortin (POMC) is processed to adrenocorticotropic hormone (ACTH) and beta-lipotropin in corticotropes of the anterior lobe, and to alpha-melanocyte-stimulating hormone (alpha-MSH) and beta-endorphin in melanotropes of the intermediate lobe (IL) of the pituitary gland. While ACTH secretion is predominantly under the stimulatory influence of the hypothalamic factors, hormone secretion of the IL is tonically inhibited by neuroendocrine dopamine (NEDA) neurons. Lobe-specific POMC processing is not absolute. For example, D(2) type DA receptor (D2R)-deficient mice have elevated plasma ACTH levels, although it is known that corticotropes do not express D2R(s). Moreover, observations that suckling does not influence alpha-MSH release, while it induces an increase in plasma ACTH is unexplained. The aim of the present study was to investigate the involvement of the NEDA system in the regulation of ACTH secretion and the participation of the IL in ACTH production in lactating rats. Untreated and estradiol (E(2))-substituted ovariectomized (OVX) females were also studied. The concentration of ACTH in the IL was higher in lactating rats than in OVX rats, while the opposite change in alpha-MSH level of the IL was observed. DA levels in the IL and the neural lobe were lower in lactating rats than in OVX rats. Suckling-induced ACTH response was eliminated by pretreatment with the DA receptor agonist, bromocriptine (BRC). Inhibition of DA biosynthesis by alpha-methyl-p-tyrosine (alphaMpT) and blockade of D2R by domperidone (DOM) elevated plasma ACTH levels, but did not influence plasma alpha-MSH levels in lactating rats. The same drugs had opposite effects in OVX and OVX + E(2) animals. In lactating mothers, BRC was able to block ACTH responses induced by both alphaMpT and DOM. Surgical denervation of the IL elevated basal plasma levels of ACTH. Taken together, these data indicate that melanotropes synthesize ACTH during lactation and its release from these cells is regulated by NEDA neurons.

The role of endocannabinoids in the regulation of luteinizing hormone and prolactin release. Differences between the effects of AEA and 2AG.

It has been shown that the endocannabinoids inhibit luteinizing hormone (LH) and prolactin (PRL) secretion. When the effects of the two well-known endocannabinoids arachidonoylethanolamide (AEA, anandamide) and 2-arachidonoyl-glycerol (2AG) have been compared it became evident that AEA caused inhibition was higher than that one of 2AG. AEA also diminished the two investigated hormonal levels in CB1 receptor inactivated mice. AEA, being an endogenous ligand of vanilloid type 1 (TPRV1) receptor, while activating TPRV1 receptor has an effect on both LH and PRL levels decrease because these later were abolished when capsazepin, antagonist of TPRV1 receptor was previously administered to the CB1 KO animals. We postulate that the difference between the effects of AEA and 2AG on the serum levels of LH and PRL is due to the difference in receptor activation of these two compounds, namely AEA can activate both CB1 and TRPV1 receptor but 2AG acts only on CB1 receptor.

Cyclic AMP-dependent, protein kinase A-independent activation of extracellular signal-regulated kinase 1/2 following adenosine receptor stimulation in human umbilical vein endothelial cells: role of exchange protein activated by cAMP 1 (Epac1).

A critical process in angiogenesis is endothelial cell proliferation, which requires activation of extracellular signal-regulated kinase (ERK)1/2. This study analyzed the pathway responsible for adenosine-induced ERK1/2 phosphorylation in human umbilical vein endothelial cells (HUVEC). Characterization with adenosine receptor (AR) agonists and antagonists and the AR mRNA profile demonstrated that stimulation of the A(2B)AR can mediate ERK1/2 phosphorylation in HUVEC. The lack of sensitivity of A(2B)AR-mediated ERK1/2 phosphorylation to 3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride (GF109203X) and 3-[1-[3-(amidinothio)propyl]-1H-in-dol-3-yl]-3-(1-methyl-1H-indol-3-yl) maleimide (bisindolylmaleimide IX) (Ro31-8220) indicated that protein kinase C stimulation is not required. The response did not involve transactivation of receptors for epidermal growth factor or vascular endothelial growth factor (VEGF). The A(2B)AR-mediated response required functional G(alphas) and was mimicked by forskolin and 8-bromoadenosine 3',5'-cyclic monophosphate. However, ERK1/2 phosphorylation induced by A(2B)AR stimulation and forskolin was insensitive to protein kinase A inhibitors. It was hypothesized that the A(2B)AR-mediated ERK1/2 activation may involve exchange protein activated by cAMP (Epac), a cAMP-activated guanine nucleotide exchange factor for Rap GTPases. Reverse Transcription-polymerase chain reaction analysis detected Epac1 but not Epac2 in HUVEC. 8-(p-Chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8CPT-2Me-cAMP), an Epac activator, stimulated ERK1/2 phosphorylation. Overexpression of Epac1 enhanced A(2B)AR-mediated and forskolin-induced ERK1/2 phosphorylation, whereas response to VEGF was unaffected. Inhibition of Epac1 expression with small interfering RNA substantially reduced A(2B)AR-mediated and forskolin-induced ERK1/2 phosphorylation and abolished that by 8CPT-2Me-cAMP. A(2B)AR stimulation and forskolin activated Rap1. Expression of a dominant-negative Ras protein did not affect either forskolin-induced or A(2B)AR-mediated ERK1/2 phosphorylation. In summary, Epac1 activation in HUVEC results in ERK1/2 activation, and this protein, at least in part, mediates response to the physiologically relevant event of A(2B)AR stimulation.