PEX5 translocation into and out of peroxisomes drives matrix protein import.

Peroxisomes are ubiquitous organelles whose dysfunction causes fatal human diseases. Most peroxisomal enzymes are imported from the cytosol by the receptor PEX5, which interacts with a docking complex in the peroxisomal membrane and then returns to the cytosol after monoubiquitination by a membrane-embedded ubiquitin ligase. The mechanism by which PEX5 shuttles between cytosol and peroxisomes and releases cargo inside the lumen is unclear. Here, we use Xenopus egg extract to demonstrate that PEX5 accompanies cargo completely into the lumen, utilizing WxxxF/Y motifs near its N terminus that bind a lumenal domain of the docking complex. PEX5 recycling is initiated by an amphipathic helix that binds to the lumenal side of the ubiquitin ligase. The N terminus then emerges in the cytosol for monoubiquitination. Finally, PEX5 is extracted from the lumen, resulting in the unfolding of the receptor and cargo release. Our results reveal the unique mechanism by which PEX5 ferries proteins into peroxisomes.

FXR in the dorsal vagal complex is sufficient and necessary for upper small intestinal microbiome-mediated changes of TCDCA to alter insulin action in rats.

OBJECTIVE: Conjugated bile acids are metabolised by upper small intestinal microbiota, and serum levels of taurine-conjugated bile acids are elevated and correlated with insulin resistance in people with type 2 diabetes. However, whether changes in taurine-conjugated bile acids are necessary for small intestinal microbiome to alter insulin action remain unknown. DESIGN: We evaluated circulating and specifically brain insulin action using the pancreatic-euglycaemic clamps in high-fat (HF) versus chow fed rats with or without upper small intestinal healthy microbiome transplant. Chemical and molecular gain/loss-of-function experiments targeting specific taurine-conjugated bile acid-induced changes of farnesoid X receptor (FXR) in the brain were performed in parallel. RESULTS: We found that short-term HF feeding increased the levels of taurochenodeoxycholic acid (TCDCA, an FXR ligand) in the upper small intestine, ileum, plasma and dorsal vagal complex (DVC) of the brain. Transplantation of upper small intestinal healthy microbiome into the upper small intestine of HF rats not only reversed the rise of TCDCA in all reported tissues but also enhanced the ability of either circulating hyperinsulinaemia or DVC insulin action to lower glucose production. Further, DVC infusion of TCDCA or FXR agonist negated the enhancement of insulin action, while genetic knockdown or chemical inhibition of FXR in the DVC of HF rats reversed insulin resistance. CONCLUSION: Our findings indicate that FXR in the DVC is sufficient and necessary for upper small intestinal microbiome-mediated changes of TCDCA to alter insulin action in rats, and highlight a previously unappreciated TCDCA-FXR axis linking gut microbiome and host insulin action.

Monitoring ligand-dependent assembly of receptor ternary complexes in live cells by BRETFect.

There is currently an unmet need for versatile techniques to monitor the assembly and dynamics of ternary complexes in live cells. Here we describe bioluminescence resonance energy transfer with fluorescence enhancement by combined transfer (BRETFect), a high-throughput technique that enables robust spectrometric detection of ternary protein complexes based on increased energy transfer from a luciferase to a fluorescent acceptor in the presence of a fluorescent intermediate. Its unique donor-intermediate-acceptor relay system is designed so that the acceptor can receive energy either directly from the donor or indirectly via the intermediate in a combined transfer, taking advantage of the entire luciferase emission spectrum. BRETFect was used to study the ligand-dependent cofactor interaction properties of the estrogen receptors ERα and ERß, which form homo- or heterodimers whose distinctive regulatory properties are difficult to dissect using traditional methods. BRETFect uncovered the relative capacities of hetero- vs. homodimers to recruit receptor-specific cofactors and regulatory proteins, and to interact with common cofactors in the presence of receptor-specific ligands. BRETFect was also used to follow the assembly of ternary complexes between the V2R vasopressin receptor and two different intracellular effectors, illustrating its use for dissection of ternary protein-protein interactions engaged by G protein-coupled receptors. Our results indicate that BRETFect represents a powerful and versatile technique to monitor the dynamics of ternary interactions within multimeric complexes in live cells.

An emerging link between LIM domain proteins and nuclear receptors.

Nuclear receptors are ligand-activated transcription factors that partake in several biological processes including development, reproduction and metabolism. Over the last decade, evidence has accumulated that group 2, 3 and 4 LIM domain proteins, primarily known for their roles in actin cytoskeleton organization, also partake in gene transcription regulation. They shuttle between the cytoplasm and the nucleus, amongst other as a consequence of triggering cells with ligands of nuclear receptors. LIM domain proteins act as important coregulators of nuclear receptor-mediated gene transcription, in which they can either function as coactivators or corepressors. In establishing interactions with nuclear receptors, the LIM domains are important, yet pleiotropy of LIM domain proteins and nuclear receptors frequently occurs. LIM domain protein-nuclear receptor complexes function in diverse physiological processes. Their association is, however, often linked to diseases including cancer.

Evidence for the involvement of FXR signaling in ovarian granulosa cell function.

Farnesoid X receptor (FXR) is mainly present in enterohepatic tissues and regulates cholesterol, lipid, and glucose homeostasis in coordination with target genes such as SHP and FABP6. Although FXR has been revealed to be expressed in reproductive tissues, FXR function and expression levels in the ovary remain unknown. In this study, we investigated FXR expression in mouse ovaries and its target genes in ovarian granulosa cells. In situ hybridization and immunohistochemical staining showed that FXR was mainly distributed in secondary and tertiary follicles. The agonist-induced activation of FXR in cultured granulosa cells induced the expression of SHP and FABP6, while siRNA targeting of FXR decreased CYP19a1 and HSD17b1 expression. Upon examination of the roles of SHP and FABP6 in granulosa cells, we found that SHP overexpression significantly decreased StAR, CYP11a1, and HSD3b gene expression. In addition, siRNA targeting of FABP6 decreased CYP19a1 and HSD17b1 expression, while FABP6 overexpression increased CYP19a1 expression. In conclusion, the present study demonstrates the presence of FXR signaling in the ovary and reveals that FXR signaling may have a role in function of granulosa cells.

Activated-farnesoid X receptor (FXR) expressed in human sperm alters its fertilising ability.

The farnesoid X receptor alpha (FXR) is a bile acid sensor activated by binding to endogenous bile acids including chenodeoxycholic acid (CDCA). Although, FXR is expressed in male reproductive tissue, the relevance of the receptor on reproduction is scarcely known. Here, we demonstrated the FXR presence and its action on several human sperm features. Western blot and immunofluorescence assays evidenced the FXR expression in human spermatozoa and the localisation in the middle piece. CDCA increasing concentrations and GW4064, synthetic ligand of FXR, were used to study the FXR influence on sperm motility, survival, capacitation, acrosome reaction and on glucose as well as lipid metabolism. Interestingly, our data showed that increasing concentrations of CDCA negatively affected sperm parameters, while the receptor blockage by (Z)-Guggulsterone and by the anti-FXR Ab reversed the effects. Intriguingly, elevated CDCA levels increased triglyceride content, while lipase and G6PDH activities were reduced with respect to untreated samples, thus impeding the metabolic reprogramming typical of the capacitated sperm. In conclusion, in this study, we demonstrated for the first time a novel target for FXR and that the activated receptor alters the acquisition of sperm fertilising ability. We showed that sperm itself express the FXR and it is responsive to specific ligands of the receptor; therefore, bile acids influence this cell both in male and in female genital tracts. It might be hypothesized that bile acid levels could be involved in infertility with idiopathic origin as these compounds are not systematically measured in men undergoing medically assisted procreation.

Hepatic farnesoid X receptor protein level and circulating fibroblast growth factor 19 concentration in children with NAFLD.

BACKGROUND & AIMS: Treatment with the farnesoid X receptor (FXR) agonist obeticholic acid is ineffective in some patients with non-alcoholic steatohepatitis (NASH) but the explanation is uncertain. We investigated hepatic FXR expression, and measurements of fibroblast growth factor 19 (FGF19) and bile acids (BAs) in children with NAFLD to investigate relationships with NASH. METHODS: 33 children with NAFLD who underwent diagnostic liver biopsy were studied. Hepatic FXR protein levels and circulating FGF19 concentrations were compared with those analysed in five control subjects with proven normal liver histology. NASH was defined by the Paediatric NAFLD Histological Score (PNHS). Binary logistic regression with adjustment for covariates and potential confounders was undertaken to test factors independently associated with: a) NASH and b) hepatic FXR protein levels. RESULTS: Mean ± SD age was 13.7 ± 1.9 years. Nineteen patients had NASH (PNHS ≥ 85) and 14 did not have NASH (PNHS < 85). Hepatic FXR level and plasma FGF19 concentration varied ~10-fold and 5-fold, respectively, between groups, and was highest in control subjects, intermediate in NAFLD without NASH, and lowest in NASH (between group differences P < .001 and P < .01 respectively). NASH was independently associated with both FXR protein levels (OR = 0.18, 95% CI 0.09, 0.38) and FGF19 concentration (OR = 0.55, 95% CI 0.20, 0.89). CONCLUSIONS: FXR protein levels vary markedly between normal liver, NAFLD without NASH, and NASH. Low levels of FXR are independently associated with NASH.

Minimum datasets to establish a CAR-mediated mode of action for rodent liver tumors.

Methods for investigating the Mode of Action (MoA) for rodent liver tumors via constitutive androstane receptor (CAR) activation are outlined here, based on current scientific knowledge about CAR and feedback from regulatory agencies globally. The key events (i.e., CAR activation, altered gene expression, cell proliferation, altered foci and increased adenomas/carcinomas) can be demonstrated by measuring a combination of key events and associative events that are markers for the key events. For crop protection products, a primary dataset typically should include a short-term study in the species/strain that showed the tumor response at dose levels that bracket the tumorigenic and non-tumorigenic dose levels. The dataset may vary depending on the species and the test compound. As examples, Case Studies with nitrapyrin (in mice) and metofluthrin (in rats) are described. Based on qualitative differences between the species, the key events leading to tumors in mice or rats by this MoA are not operative in humans. In the future, newer approaches such as a CAR biomarker signature approach and/or in vitro CAR3 reporter assays for mouse, rat and human CAR may eventually be used to demonstrate a CAR MoA is operative, without the need for extensive additional studies in laboratory animals.

Farnesoid X receptor agonist GW4064 ameliorates lipopolysaccharide-induced ileocolitis through TLR4/MyD88 pathway related mitochondrial dysfunction in mice.

Inflammatory bowel disease (IBD) is a complex and relapsing inflammatory condition of the gastro intestinal tract characterized by diarrhoea and abdominal pain. Farnesoid X receptor (FXR) plays an important role in enteroprotection and mucosal injury by regulating inflammatory responses and barrier function in the intestinal tract. Here we show the mechanisms of FXR agonist, GW4064, inhibits mucosal injury in ileum caused by lipopolysaccharides (LPS). Ileum injury was induced by intraperitoneal injection of LPS in Wild-type (WT) and FXR knockout (KO) mice. GW4064 alleviates LPS-mediated tight junction dysfunction as well as macrophage infiltration in WT mice, but not in FXR KO mice. Interesting, GW4064 suppresses NACHT, LRR and PYD domains-containing protein 3 (NALP3) inflammasome mediates tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6 and IL-1ß, as well as mitochondrial respiratory complexes mRNA expression in WT and FXR KO mice treated with LPS. This results demonstrated that central roles of FXR in coordinating regulation of both inflammation and mitochondrial dysfunction. We propose that GW4064 is promising therapeutic agent for treatment of ileocolitis.

Ovarian Hemangiomas Do Not Harbor EWSR1 Rearrangements: Clinicopathologic Characterization of 10 Cases.

Hemangiomas of the ovary are rare with a majority described as individual reports of unusual clinical presentations or morphologic findings. Both the expected and unexpected pathologic features of these tumors in the ovary are not well detailed. Therefore, we collected the largest series of ovarian hemangiomas to comprehensively define their clinicopathologic associations and examine the significance of hormone receptors in their pathogenesis. In addition, a novel EWSR1-NFATC1 fusion has recently been described in a case of hemangioma of bone. To our knowledge, EWSR1 rearrangement has not been evaluated in hemangiomas of other sites or in a case series. Accordingly, we used fluorescence in situ hybridization to investigate EWSR1 status in a majority of our cases. Clinical presentation was variable and dependent on tumor size. Patient age ranged 48 to 87 yr (median 63 yr). Tumors involved the right (n=6) and left (n=3) ovaries with laterality unknown in 1 case, and size ranged from 0.2 to 5.0 cm (median 1.0 cm). Three of 4 radiologic reports were either equivocal or could not exclude malignancy. Seven cases were of the cavernous type and 3 were mixed cavernous and capillary type. All lesions formed a single discrete, circumscribed mass that displaced the surrounding cortical stroma. The cavernous type showed dilated, thin-walled vessels and vascular thrombi, some of which were associated with dystrophic calcification. In addition to cavernous morphology, the mixed form exhibited features of capillary hemangioma such as lobulated growth of capillary-sized vascular spaces that lacked atypia or multilayering and were linked to a larger feeding vessel. Each tumor expressed CD31, CD34, FLI-1, ERG, but not D240. The hemangioma stromal cells, but not endothelium, expressed estrogen and progesterone receptors in every case. Stromal luteinization was seen in 2 cases. Follow-up ranged 1 to 139 mo and all patients were disease free. All cases were negative for EWSR1 rearrangement; however, 2 cases demonstrated additional intact copies of EWSR1 indicating aneusomy 22 or a structural abnormality of chromosome 22 resulting in apparent duplication of the EWSR1 gene region (at 22q12). Although an uncommon entity, awareness of ovarian hemangioma's unique and diverse clinical presentation as well as its potential to radiologically imitate malignant ovarian neoplasms are important.

Amla prevents fructose-induced hepatic steatosis in ovariectomized rats: role of liver FXR and LXRα.

OBJECTIVES: Increased fructose consumption causes dyslipidemia and fatty liver in postmenopausal women, both independent risk factors for cardiovascular disease. This study explored the potential mechanisms by which amla (Emblica officinalis) reduced hypercholesterolemia and hypertriglyceridemia and prevented fatty liver in a fructose-fed, ovariectomized rat model of menopause. METHODS: Sham-operated and ovariectomized rats were put on a chow or high fructose diet. They were further divided into groups with or without amla. After 18 weeks of treatment, livers were harvested and subjected to Western blot and histological analyses. RESULTS: In all groups, amla increased the protein expression of liver farnesoid X receptor (FXR) and liver X receptor (LXR), key proteins involved in lipid metabolism. Fructose-fed rats developed fatty liver and amla prevented this. Here amla produced an exceptional rise in LXR and insulin-induced gene-2 (Insig-2) which prevented the maturation of sterol regulatory element-binding protein-1 and steroyl CoA desaturase-1, responsible for triglyceride synthesis. Amla also increased the protein expression of ATP binding cassette transporter A1 (ABCA1), involved in high density lipoprotein (HDL) synthesis as well as low density lipoprotein receptor (LDLR) responsible for uptake of LDL cholesterol. Besides this, amla increased the protein expression of peroxisome proliferator activated receptor α (PPARα) involved in ß oxidation of fatty acids. CONCLUSIONS: Amla increased the protein expression of liver FXR, LXRα, PPARα and their downstream proteins Insig-2, ABCA1 and LDLR. This property of amla to modulate some of the key proteins involved in lipid metabolism promises its usefulness as a preventive agent for dyslipidemia and hepatic steatosis.

The nuclear receptor FXR, but not LXR, up-regulates bile acid transporter expression in non-alcoholic fatty liver disease.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease. Patients with non-alcoholic steatohepatitis (NASH) have increased plasmatic and hepatic concentrations of bile acids (BA), suggesting that they can be associated with the progression of the disease. Hepatic nuclear receptors are known to modulate genes controlling BA metabolism; thus, in this work we aimed to compare the expression of liver nuclear receptors -farnesoid X (FXR), small heterodimer partner (SHP) and liver X alpha (LXRα) receptors- and BA transporters -sodium+/taurocholate cotransporting polypeptide (NTCP) and bile salt export pump (BSEP)- in liver biopsy samples of patients with simple steatosis (SS) and NASH. MATERIAL AND METHODS: Forty patients with biopsy-proven NALFD were enrolled between 2009 and 2012; liver biopsies were classified as SS (N = 20) or NASH (N = 20) according to the NAFLD activity score. Gene expression of nuclear FXR, LXRα, SHP, NTCP and BSEP was analyzed by real-time reverse transcription polymerase chain reaction and protein level was quantified by western blot. RESULTS: Gene expression of FXR, SHP, NTCP and BSEP was significantly up-regulated in the NASH group in comparison with SS patients (P < 0.05). In contrast, protein level for FXR, SHP and NTCP was decreased in the NASH patients vs. the SS group (P < 0.05). Gene and protein profile of LXRα did not show differences between groups. CONCLUSIONS: The results suggest that liver nuclear receptors (FXR and SHP) and BA transporters (NTCP and BSEP) are associated with the progression of NAFLD.

A viable Arabidopsis pex13 missense allele confers severe peroxisomal defects and decreases PEX5 association with peroxisomes.

Peroxisomes are organelles that catabolize fatty acids and compartmentalize other oxidative metabolic processes in eukaryotes. Using a forward-genetic screen designed to recover severe peroxisome-defective mutants, we isolated a viable allele of the peroxisome biogenesis gene PEX13 with striking peroxisomal defects. The pex13-4 mutant requires an exogenous source of fixed carbon for pre-photosynthetic development and is resistant to the protoauxin indole-3-butyric acid. Delivery of peroxisome-targeted matrix proteins depends on the PEX5 receptor docking with PEX13 at the peroxisomal membrane, and we found severely reduced import of matrix proteins and less organelle-associated PEX5 in pex13-4 seedlings. Moreover, pex13-4 physiological and molecular defects were partially ameliorated when PEX5 was overexpressed, suggesting that PEX5 docking is partially compromised in this mutant and can be improved by increasing PEX5 levels. Because previously described Arabidopsis pex13 alleles either are lethal or confer only subtle defects, the pex13-4 mutant provides valuable insight into plant peroxisome receptor docking and matrix protein import.

Microporation is an efficient method for siRNA-induced knockdown of PEX5 in HepG2 cells: evaluation of the transfection efficiency, the PEX5 mRNA and protein levels and induction of peroxisomal deficiency.

The pathomechanism of peroxisomal biogenesis disorders (PBDs), a group of inherited autosomal recessive diseases with mutations of peroxin (PEX) genes, is not yet fully understood. Therefore, several knockout models, e.g., the PEX5 knockout mouse, have been generated exhibiting a complete loss of peroxisomal function. In this study, we wanted to knockdown PEX5 using the siRNA technology (1) to mimic milder forms of PBDs in which the mutated peroxin has some residual function and (2) to analyze the cellular consequences of a reduction of the PEX5 protein without adaption during the development as it is the case in a knockout animal. First, we tried to optimize the transfection of the hepatoma cell line HepG2 with PEX5 siRNA using different commercially available liposomal and non-liposomal transfection reagents (Lipofectamine(®) 2000, FuGENE 6, HiPerFect(®), INTERFERin™, RiboJuice™) as well as microporation using the Neon™ Transfection system. Microporation was found to be superior to the transfection reagents with respect to the transfection efficiency (100 vs. 0-70%), to the reduction of PEX5 mRNA (by 90 vs. 0-50%) and PEX5 protein levels (by 70 vs. 0-50%). Interestingly, we detected that a part of the cleaved PEX5 mRNA still existed as 3' fragment (15%) 24 h after microporation. Using microporation, we further analyzed whether the reduced PEX5 protein level impaired peroxisomal function. We indeed detected a reduced targeting of SKL-tagged proteins into peroxisomes as well as an increased oxidative stress as found in PBD patients and respective knockout mouse models. Knockdown of the PEX5 protein and functional consequences were at a maximum 48 h after microporation. Thereafter, the PEX5 protein was resynthesized, which may allow the temporal analysis of the loss as well as the reconstitution of peroxisomes in the future. In conclusion, we propose microporation as an efficient and reproducible method to transfect HepG2 cells with PEX5 siRNA. We succeeded to transiently knockdown PEX5 mRNA and its protein level leading to functional consequences similar as observed in peroxisome deficiencies.

Microglial NLRP3 inflammasome activation mediates IL-1ß-related inflammation in prefrontal cortex of depressive rats.

Depression is an inflammatory disorder. Pro-inflammatory cytokine interleukin-1 beta (IL-1ß) may play a pivotal role in the central nervous system (CNS) inflammation of depression. Here, we investigated IL-1ß alteration in serum, cerebrospinal fluid (CSF) and prefrontal cortex (PFC) of chronic unpredictable mild stress (CUMS)-exposed rats, a well-documented model of depression, and further explored the molecular mechanism by which CUMS procedure induced IL-1ß-related CNS inflammation. We showed that 12-week CUMS procedure remarkably increased PFC IL-1ß mRNA and protein levels in depressive-like behavior of rats, without significant alteration of serum and CSF IL-1ß levels. We found that CUMS procedure significantly caused PFC nuclear factor kappa B (NF-κB) inflammatory pathway activation in rats. The intriguing finding in this study was the induced activation of nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome with the increased IL-1ß maturation in PFC of CUMS rats, suggesting a new grade of regulatory mechanism for IL-1ß-related CNS inflammation. Moreover, microglial activation and astrocytic function impairment were observed in PFC of CUMS rats. The increased co-location of NLRP3 and ionized calcium binding adaptor molecule 1 (Iba1) protein expression supported that microglia in glial cells was the primary contributor for CUMS-induced PFC NLRP3 inflammasome activation in rats. These alterations in CUMS rats were restored by chronic treatment of the antidepressant fluoxetine, indicating that fluoxetine-mediated rat PFC IL-1ß reduction involves both transcriptional and post-transcriptional regulatory mechanisms. These findings provide in vivo evidence that microglial NLRP3 inflammasome activation is a mediator of IL-1ß-related CNS inflammation during chronic stress, and suggest a new therapeutic target for the prevention and treatment of depression.

Expression and functional regulation of the nuclear receptors AHR, PXR, and CAR, and the transcription factor Nrf2 in rat parotid gland.

Nuclear receptors and transcription factors regulate the functions of many genes involved in cellular physiology and pathology (e.g. tumorigenesis and autoimmune diseases). The present study was performed to define the expression and the regulation of aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR), constitutive androstane receptor (CAR), and nuclear factor E2-related factor 2 (Nrf2) in the rat parotid gland. Constitutive expression, as well as expression after stimulation with specific inducers for AhR [2,3,7,8-tetrachloro-dibenzylo-p-dioxin (TCDD)], Nrf2(oltipraz), PXR (dexamethasone), and CAR (phenobarbital), was evaluated using the quantitative PCR. Cellular localization of the nuclear receptors and the transcription factor was visualized by immunohistochemical staining. The study revealed constitutive expression of AhR as well as Nrf2, and their induction by TCDD andoltipraz, respectively. Immunohistochemical analysis revealed constitutive, predominantly cytoplasmic, expression of the AhR receptor, especially in interlobular striated duct cells, with nuclear shift upon exposure to TCDD. Inducible expression of Nfr2 was found mainly in the cytoplasm of intralobular striated duct cells. Constitutive expression of PXR and CAR was not found. Bearing in mind the involvement of AhR and Nrf2 in the regulation of many genes, it seems that these factors may play also a role in salivary gland physiology and pathology.

The role of soluble guanylyl cyclase in chronic obstructive pulmonary disease.

RATIONALE: Soluble guanylyl cyclase (sGC), a cyclic guanosine 5'-monophosphate-generating enzyme, regulates smooth muscle tone and exerts antiinflammatory effects in animal models of asthma and acute lung injury. In chronic obstructive pulmonary disease (COPD), primarily caused by cigarette smoke (CS), lung inflammation persists and smooth muscle tone remains elevated, despite ample amounts of nitric oxide that could activate sGC. OBJECTIVES: To determine the expression and function of sGC in patients with COPD and in a murine model of COPD. METHODS: Expression of sGCα1, α2, and ß1 subunits was examined in lungs of never-smokers, smokers without airflow limitation, and patients with COPD; and in C57BL/6 mice after 3 days, 4 weeks, and 24 weeks of CS exposure. The functional role of sGC was investigated in vivo by measuring bronchial responsiveness to serotonin in mice using genetic and pharmacologic approaches. MEASUREMENTS AND MAIN RESULTS: Pulmonary expression of sGC, both at mRNA and protein level, was decreased in smokers without airflow limitation and in patients with COPD, and correlated with disease severity (FEV1%). In mice, exposure to CS reduced sGC, cyclic guanosine 5'-monophosphate levels, and protein kinase G activity. sGCα1(-/-) mice exposed to CS exhibited bronchial hyperresponsiveness to serotonin. Activation of sGC by BAY 58-2667 restored the sGC signaling and attenuated bronchial hyperresponsiveness in CS-exposed mice. CONCLUSIONS: Down-regulation of sGC because of CS exposure might contribute to airflow limitation in COPD.

Nanoscale distribution of mitochondrial import receptor Tom20 is adjusted to cellular conditions and exhibits an inner-cellular gradient.

The translocase of the mitochondrial outer membrane (TOM) complex is the main import pore for nuclear-encoded proteins into mitochondria, yet little is known about its spatial distribution within the outer membrane. Super-resolution stimulated emission depletion microscopy was used to determine quantitatively the nanoscale distribution of Tom20, a subunit of the TOM complex, in more than 1,000 cells. We demonstrate that Tom20 is located in clusters whose nanoscale distribution is finely adjusted to the cellular growth conditions as well as to the specific position of a cell within a microcolony. The density of the clusters correlates to the mitochondrial membrane potential. The distributions of clusters of Tom20 and of Tom22 follow an inner-cellular gradient from the perinuclear to the peripheral mitochondria. We conclude that the nanoscale distribution of the TOM complex is finely adjusted to the cellular conditions, resulting in distribution gradients both within single cells and between adjacent cells.

The new NO donor Terpy induces similar relaxation in mesenteric resistance arteries of renal hypertensive and normotensive rats.

The present work aimed to investigate the cellular mechanisms involved on the vasorelaxation induced by the new nitric oxide donor [Ru(terpy)(bdq)NO](3+) (Terpy) in isolated mesenteric resistance artery and to compare the vascular responses in isolated vessels from 2K and 2K-1C hypertensive rats. We have used this artery because it is important to the control of vascular resistance and consequently to the blood pressure control. The NO donor Terpy induced relaxation in a concentration-dependent way in mesenteric resistance arteries. There were no differences between renal hypertensive (2K-1C) and normotensive (2K) in Terpy-induced relaxation neither in NO released. The relaxation induced by Terpy was inhibited by the soluble guanylyl-cyclase (sGC) inhibitor ODQ both in 2K and in 2K-1C with similar amplitude. In agreement with these data, the protein expression of the subunits α1 and ß1 of the enzyme sGC was not different between 2K-1C and 2K mesenteric bed. The relaxation induced by Terpy was inhibited by the cGMP-dependent protein kinase (G kinase) inhibitor or by the non-selective K(+) channel blocker tetraethylamonium (TEA), but with no difference between 2K-1C and 2K arteries. The relaxation induced by Terpy was also inhibited by the SERCA inhibitor thapsigargin in both groups. Taken together, these results show that the vascular relaxation induced by the NO donor [Ru(terpy)(bdq)NO](3+) involves the activation of NO/sGC/cGMP/GK pathway, activation of K(+) channels sensitive to TEA and SERCA in normotensive and renal hypertensive rat mesenteric resistance arteries. Surprisingly, Terpy-induced vasorelaxation is similar in mesenteric resistance arteries of renal hypertensive and normotensive rats.

Nitric oxide is less effective at inhibiting neointimal hyperplasia in spontaneously hypertensive rats.

Exogenous administration of nitric oxide (NO) markedly decreases neointimal hyperplasia following arterial injury in several animal models. However, the effect of NO on neointimal hyperplasia in hypertension remains unknown. Here, we employ the spontaneously hypertensive rat (SHR) strain, inbred from Wistar Kyoto (WKY) rats, and the carotid artery balloon injury model to assess the effects of NO on neointimal hyperplasia development. 2weeks after arterial injury, we showed that both rat strains developed similar levels of neointimal hyperplasia, but local administration of NO was less effective at inhibiting neointimal hyperplasia in the SHR compared to WKY rats (58% vs. 79%, P<0.001). Interestingly, local administration of NO did not affect systemic blood pressure in either rat strain. Compared to WKY, the SHR displayed more proliferation in the media and adventitia following balloon injury, as measured by BrdU incorporation. The SHR also showed more inflammation in the adventitia after injury, as well as more vasa vasorum, than WKY rats. NO treatment reduced the vasa vasorum in the SHR but not WKY rats. Finally, while NO decreased both injury-induced proliferation and inflammation in the SHR, it did not return these parameters to levels seen in WKY rats. We conclude that NO is less effective at inhibiting neointimal hyperplasia in the SHR than WKY rats. This may be due to increased scavenging of NO in the SHR, leading to diminished bioavailability of NO. These data will help to develop novel NO-based therapies that will be equally effective in both normotensive and hypertensive patient populations.