Smooth muscle specific Rac1 deficiency induces hypertension by preventing p116RIP3-dependent RhoA inhibition

BACKGROUND: Increasing evidence implicates overactivation of RhoA as a critical component of the pathogenesis of hypertension. Although a substantial body of work has established that Rac1 functions antagonize RhoA in a broad range of physiological processes, the role of Rac1 in the regulation of vascular tone and blood pressure is not fully elucidated. METHODS AND RESULTS: To define the role of Rac1 in vivo in vascular smooth muscle cells (vSMC), we generated smooth muscle (SM)-specific Rac1 knockout mice (SM-Rac1-KO) and performed radiotelemetric blood pressure recordings, contraction measurements in arterial rings, vSMC cultures and biochemical analyses. SM-Rac1-KO mice develop high systolic blood pressure sensitive to Rho kinase inhibition by fasudil. Arteries from SM-Rac1-KO mice are characterized by a defective NO-dependent vasodilation and an overactivation of RhoA/Rho kinase signaling. We provide evidence that Rac1 deletion-induced hypertension is due to an alteration of cGMP signaling resulting from the loss of Rac1-mediated control of type 5 PDE activity. Consequently, cGMP-dependent phosphorylation and binding of RhoA with its inhibitory partner, the phosphatase-RhoA interacting protein (p116(RIP3)), are decreased. CONCLUSIONS: Our data reveal that the depletion of Rac1 in SMC decreases cGMP-dependent p116(RIP3)/RhoA interaction and the subsequent inhibition of RhoA signaling. Thus, we unveil an in vivo role of Rac1 in arterial blood pressure regulation and a new pathway involving p116(RIP3) that contributes to the antagonistic relationship between Rac1 and RhoA in vascular smooth muscle cells and their opposite roles in arterial tone and blood pressure

Parmodulins Inhibit Thrombus Formation Without Inducing Endothelial Injury Caused by Vorapaxar

Protease-activated receptor-1 (PAR1) couples the coagulation cascade to platelet activation during myocardial infarction and to endothelial inflammation during sepsis. This receptor demonstrates marked signaling bias. Its activation by thrombin stimulates prothrombotic and proinflammatory signaling, whereas its activation by activated protein C (APC) stimulates cytoprotective and antiinflammatory signaling. A challenge in developing PAR1-targeted therapies is to inhibit detrimental signaling while sparing beneficial pathways. We now characterize a novel class of structurally unrelated small-molecule PAR1 antagonists, termed parmodulins, and compare the activity of these compounds to previously characterized compounds that act at the PAR1 ligand–binding site. We find that parmodulins target the cytoplasmic face of PAR1 without modifying the ligand-binding site, blocking signaling through Gαq but not Gα13 in vitro and thrombus formation in vivo. In endothelium, parmodulins inhibit prothrombotic and proinflammatory signaling without blocking APC-mediated pathways or inducing endothelial injury. In contrast, orthosteric PAR1 antagonists such as vorapaxar inhibit all signaling downstream of PAR1. Furthermore, exposure of endothelial cells to nanomolar concentrations of vorapaxar induces endothelial cell barrier dysfunction and apoptosis. These studies demonstrate how functionally selective antagonism can be achieved by targeting the cytoplasmic face of a G-protein–coupled receptor to selectively block pathologic signaling while preserving cytoprotective pathways

Real-time visualization of heterotrimeric G protein Gq activation in living cells

Contains fulltext : 97296.pdf (publisher's version ) (Open Access)BACKGROUND: Gq is a heterotrimeric G protein that plays an important role in numerous physiological processes. To delineate the molecular mechanisms and kinetics of signalling through this protein, its activation should be measurable in single living cells. Recently, fluorescence resonance energy transfer (FRET) sensors have been developed for this purpose. RESULTS: In this paper, we describe the development of an improved FRET-based Gq activity sensor that consists of a yellow fluorescent protein (YFP)-tagged Ggamma2 subunit and a Galphaq subunit with an inserted monomeric Turquoise (mTurquoise), the best cyan fluorescent protein variant currently available. This sensor enabled us to determine, for the first time, the kon (2/s) of Gq activation. In addition, we found that the guanine nucleotide exchange factor p63RhoGEF has a profound effect on the number of Gq proteins that become active upon stimulation of endogenous histamine H1 receptors. The sensor was also used to measure ligand-independent activation of the histamine H1 receptor (H1R) upon addition of a hypotonic stimulus. CONCLUSIONS: Our observations reveal that the application of a truncated mTurquoise as donor and a YFP-tagged Ggamma2 as acceptor in FRET-based Gq activity sensors substantially improves their dynamic range. This optimization enables the real-time single cell quantification of Gq signalling dynamics, the influence of accessory proteins and allows future drug screening applications by virtue of its sensitivity

The Rho exchange factor Arhgef1 mediates the effects of angiotensin II on vascular tone and blood pressure

Hypertension is one of the most frequent pathologies in the industrialized world. Although recognized to be dependent on a combination of genetic and environmental factors, its molecular basis remains elusive. Increased activity of the monomeric G protein RhoA in arteries is a common feature of hypertension. However, how RhoA is activated and whether it has a causative role in hypertension remains unclear. Here we provide evidence that Arhgef1 is the RhoA guanine exchange factor specifically responsible for angiotensin II-induced activation of RhoA signaling in arterial smooth muscle cells. We found that angiotensin II activates Arhgef1 through a previously undescribed mechanism in which Jak2 phosphorylates Tyr738 of Arhgef1. Arhgef1 inactivation in smooth muscle induced resistance to angiotensin II-dependent hypertension in mice, but did not affect normal blood pressure regulation. Our results show that control of RhoA signaling through Arhgef1 is central to the development of angiotensin II-dependent hypertension and identify Arhgef1 as a potential target for the treatment of hypertension

Feedback modeling of non-esterified fatty acids in rats after nicotinic acid infusions

A feedback model was developed to describe the tolerance and oscillatory rebound seen in non-esterified fatty acid (NEFA) plasma concentrations following intravenous infusions of nicotinic acid (NiAc) to male Sprague-Dawley rats. NiAc was administered as an intravenous infusion over 30 min (0, 1, 5 or 20 μmol kg−1 of body weight) or over 300 min (0, 5, 10 or 51 μmol kg−1 of body weight), to healthy rats (n = 63), and serial arterial blood samples were taken for measurement of NiAc and NEFA plasma concentrations. Data were analyzed using nonlinear mixed effects modeling (NONMEM). The disposition of NiAc was described by a two-compartment model with endogenous turnover rate and two parallel capacity-limited elimination processes. The plasma concentration of NiAc was driving NEFA (R) turnover via an inhibitory drug-mechanism function acting on the formation of NEFA. The NEFA turnover was described by a feedback model with a moderator distributed over a series of transit compartments, where the first compartment (M1) inhibited the formation of R and the last compartment (MN) stimulated the loss of R. All processes regulating plasma NEFA concentrations were assumed to be captured by the moderator function. The potency, IC50, of NiAc was 45 nmol L−1, the fractional turnover rate kout was 0.41 L mmol−1 min−1 and the turnover rate of moderator ktol was 0.027 min−1. A lower physiological limit of NEFA was modeled as a NiAc-independent release (kcap) of NEFA into plasma and was estimated to 0.032 mmol L−1 min−1. This model can be used to provide information about factors that determine the time-course of NEFA response following different modes, rates and routes of administration of NiAc. The proposed model may also serve as a preclinical tool for analyzing and simulating drug-induced changes in plasma NEFA concentrations after treatment with NiAc or NiAc analogues

Protease Activated Receptor Signaling Is Required for African Trypanosome Traversal of Human Brain Microvascular Endothelial Cells

Human African trypanosomiasis, or sleeping sickness, occurs when single-cell trypanosome protozoan parasites spread from the blood to brain over the blood-brain barrier (BBB). This barrier is composed of brain microvascular endothelial cells (BMECs) especially designed to keep pathogens out. Safe drugs for treating sleeping sickness are lacking and alternative treatments are urgently required. Using our human BMEC BBB model, we previously found that a parasite protease, brucipain, induced calcium activation signals that allowed this barrier to open up to parasite crossing. Because human BMECs express protease-activated receptors (PARs) that trigger calcium signals in BMECs, we hypothesized a functional link between parasite brucipain and BMEC PARs. Utilizing RNA interference to block the production of one type of PAR called PAR-2, we hindered the ability of trypanosomes to both open up and cross human BMECs. Using gene-profiling methods to interrogate candidate BMEC pathways specifically triggered by brucipain, several pathways that potentially link brain inflammatory processes were identified, a finding congruent with the known role of PAR-2 as a mediator of inflammation. Overall, our data support a role for brucipain and BMEC PARs in trypanosome BBB transmigration, and as potential triggers for brain inflammation associated with the disease