MetSCORE: a molecular metric to evaluate the risk of metabolic syndrome based on serum NMR metabolomics.

BACKGROUND: Metabolic syndrome (MetS) is a cluster of medical conditions and risk factors correlating with insulin resistance that increase the risk of developing cardiometabolic health problems. The specific criteria for diagnosing MetS vary among different medical organizations but are typically based on the evaluation of abdominal obesity, high blood pressure, hyperglycemia, and dyslipidemia. A unique, quantitative and independent estimation of the risk of MetS based only on quantitative biomarkers is highly desirable for the comparison between patients and to study the individual progression of the disease in a quantitative manner. METHODS: We used NMR-based metabolomics on a large cohort of donors (n = 21,323; 37.5% female) to investigate the diagnostic value of serum or serum combined with urine to estimate the MetS risk. Specifically, we have determined 41 circulating metabolites and 112 lipoprotein classes and subclasses in serum samples and this information has been integrated with metabolic profiles extracted from urine samples. RESULTS: We have developed MetSCORE, a metabolic model of MetS that combines serum lipoprotein and metabolite information. MetSCORE discriminate patients with MetS (independently identified using the WHO criterium) from general population, with an AUROC of 0.94 (95% CI 0.920-0.952, p < 0.001). MetSCORE is also able to discriminate the intermediate phenotypes, identifying the early risk of MetS in a quantitative way and ranking individuals according to their risk of undergoing MetS (for general population) or according to the severity of the syndrome (for MetS patients). CONCLUSIONS: We believe that MetSCORE may be an insightful tool for early intervention and lifestyle modifications, potentially preventing the aggravation of metabolic syndrome.

Identification of Urine Metabolic Markers of Stroke Risk Using Untargeted Nuclear Magnetic Resonance Analysis.

Stroke remains the second leading cause of mortality worldwide, and the third leading cause of death and morbidity combined, affecting more than 12 million people every year. Stroke pathophysiology results from complex interactions of several risk factors related to age, family history, gender, lifestyle, and the presence of cardiovascular and metabolic diseases. Despite all the evidence, it is not possible to fully prevent stroke onset. In recent years, there has been an exploration of innovative methodologies for metabolite analysis aimed at identifying novel stroke biomarkers. Utilizing Nuclear Magnetic Resonance (NMR) spectroscopy, we investigated small molecule variations in urine across different stages of stroke risk. The Framingham Stroke Risk Score was used in people over 63 years of age living in long-term care facilities (LTCFs) to calculate the probability of suffering a stroke: low stroke risk (LSR, control), moderate stroke risk (MSR), and high stroke risk (HSR). Univariate statistical analysis showed that urinary 4-hydroxyphenylacetate levels increased while glycolate levels decreased across the different stroke risk groups, from the LSR to the HSR groups. Trimethylamine N-oxide (TMAO) had average concentration values that were significantly higher in elderly people in the HSR group, while trigonelline levels were significantly lower in the MSR group. These metabolic markers can be used for early detection and to differentiate stages of stroke risk more efficiently.

Searching for Metabolic Markers of Stroke in Human Plasma via NMR Analysis.

More than 12 million people around the world suffer a stroke every year, one every 3 s. Stroke has a variety of causes and is often the result of a complex interaction of risk factors related to age, genetics, gender, lifestyle, and some cardiovascular and metabolic diseases. Despite this evidence, it is not possible to prevent the onset of stroke. The use of innovative methods for metabolite analysis has been explored in the last years to detect new stroke biomarkers. We use NMR spectroscopy to identify small molecule variations between different stages of stroke risk. The Framingham Stroke Risk Score was used in people over 63 years of age living in long-term care facilities (LTCF) to calculate the probability of suffering a stroke. Using this parameter, three study groups were formed: low stroke risk (LSR, control), moderate stroke risk (MSR) and high stroke risk (HSR). Univariate statistical analysis showed seven metabolites with increasing plasma levels across different stroke risk groups, from LSR to HSR: isoleucine, asparagine, formate, creatinine, dimethylsulfone and two unidentified molecules, which we termed "unknown-1" and "unknown-3". These metabolic markers can be used for early detection and to detect increasing stages of stroke risk more efficiently.

TGF-ß1 in Vascular Wall Pathology: Unraveling Chronic Venous Insufficiency Pathophysiology.

Chronic venous insufficiency and varicose veins occur commonly in affluent countries and are a socioeconomic burden. However, there remains a relative lack of knowledge about venous pathophysiology. Various theories have been suggested, yet the molecular sequence of events is poorly understood. Transforming growth factor-beta one (TGF-ß1) is a highly complex polypeptide with multifunctional properties that has an active role during embryonic development, in adult organ physiology and in the pathophysiology of major diseases, including cancer and various autoimmune, fibrotic and cardiovascular diseases. Therefore, an emphasis on understanding its signaling pathways (and possible disruptions) will be an essential requirement for a better comprehension and management of specific diseases. This review aims at shedding more light on venous pathophysiology by describing the TGF-ß1 structure, function, activation and signaling, and providing an overview of how this growth factor and disturbances in its signaling pathway may contribute to specific pathological processes concerning the vessel wall which, in turn, may have a role in chronic venous insufficiency.

Variability of MMP/TIMP and TGF-ß1 Receptors throughout the Clinical Progression of Chronic Venous Disease.

Chronic venous disease (CVeD) is a prevalent condition with a significant socioeconomic burden, yet the pathophysiology is only just beginning to be understood. Previous studies concerning the dysregulation of matrix metalloproteinases (MMPs) and their inhibitors (tissue inhibitors of metalloproteinases (TIMPs)) within the varicose vein wall are inconsistent and disregard clinical progression. Moreover, it is highly plausible that MMP and TIMP expression/activity is affected by transforming growth factor (TGF)-ß1 and its signaling receptors (TGFßRs) expression/activity in the vein wall. A case-control study was undertaken to analyze genetic and immunohistochemical differences between healthy (n = 13) and CVeD (early stages: n = 19; advanced stages: n = 12) great saphenous vein samples. Samples were grouped based on anatomic harvest site and subjected to quantitative polymerase chain reaction for MMP1, MMP2, MMP8, MMP9, MMP12, MMP13, TIMP1, TIMP2, TIMP3, TIMP4, TGFßR1, TGFßR2, and TGFßR3 gene expression analysis, and then to immunohistochemistry for immunolocalization of MMP2, TIMP2, and TGFßR2. Decreased gene expression of MMP12, TIMP2, TIMP3, TIMP4, and TGFßR2 was found in varicose veins when compared to controls. Regarding CVeD clinical progression, two facts arose: results across anatomical regions were uneven; decreased gene expression of MMP9 and TGFßR3 and increased gene expression of MMP2 and TIMP3 were found in advanced clinical stages. Most immunohistochemistry results for tunica intima were coherent with qPCR results. In conclusion, decreased expression of TGFßRs might suggest a reduction in TGF-ß1 participation in the MMP/TIMP imbalance throughout CVeD progression. Further studies about molecular events in the varicose vein wall are required and should take into consideration the venous anatomical region and CVeD clinical progression.

Testosterone and atrial natriuretic peptide share the same pathway to induce vasorelaxation of human umbilical artery.

We recently observed in human umbilical artery smooth muscle cells that testosterone activates protein kinase G and stimulates large-conductance Ca²âº activated (BKCa) and voltage sensitive (KV) potassium channels. In the same work, we also show that atrial natriuretic peptide (ANP), an activator of particulate guanylate cyclase (pGC), stimulates the activity of BKCa and KV channels because of protein kinase G activation. The aim of this work was to prove that the relaxant effects of testosterone are also because of the increase of cGMP because of activation of the pGC. Subsarcolemmal cGMP signals were monitored in single cells by recording the cGMP-gated current (ICNG) in human umbilical artery smooth muscle cells expressing the wild-type rat olfactory cyclic nucleotide-gated (CNG) channel. Sodium nitroprusside (10 and 100 µM), ANP (0.1 and 1 µM), or testosterone (0.1, 1, and 10 µM) induced activation of ICNG. This activation induced by testosterone and ANP is bigger than that elicited by sodium nitroprusside. In summary, our study reveals that testosterone and ANP activate the pGC and induce vasorelaxation of human umbilical artery.

Cyclic nucleotide-dependent relaxation pathways in vascular smooth muscle.

Vascular smooth muscle tone is controlled by a balance between the cellular signaling pathways that mediate the generation of force (vasoconstriction) and release of force (vasodilation). The initiation of force is associated with increases in intracellular calcium concentrations, activation of myosin light-chain kinase, increases in the phosphorylation of the regulatory myosin light chains, and actin-myosin crossbridge cycling. There are, however, several signaling pathways modulating Ca(2+) mobilization and Ca(2+) sensitivity of the contractile machinery that secondarily regulate the contractile response of vascular smooth muscle to receptor agonists. Among these regulatory mechanisms involved in the physiological regulation of vascular tone are the cyclic nucleotides (cAMP and cGMP), which are considered the main messengers that mediate vasodilation under physiological conditions. At least four distinct mechanisms are currently thought to be involved in the vasodilator effect of cyclic nucleotides and their dependent protein kinases: (1) the decrease in cytosolic calcium concentration ([Ca(2+)]c), (2) the hyperpolarization of the smooth muscle cell membrane potential, (3) the reduction in the sensitivity of the contractile machinery by decreasing the [Ca(2+)]c sensitivity of myosin light-chain phosphorylation, and (4) the reduction in the sensitivity of the contractile machinery by uncoupling contraction from myosin light-chain phosphorylation. This review focuses on each of these mechanisms involved in cyclic nucleotide-dependent relaxation of vascular smooth muscle under physiological conditions.

Long- and short-term effects of androgens in human umbilical artery smooth muscle.

The aim of the present study was to determine the effects of androgens in the regulation of human umbilical artery (HUA) contractility. The short-term effects of testosterone on the tone of the HUA were investigated, as were the long-term effects of dihydrotestosterone (DHT) on the expression of some proteins involved in the contractile process. Endothelium-denuded HUA were treated for 24 h with DHT (2 µmol/L) or the vehicle control (ethanol) to analyse the genomic effects of androgens. Twenty-four hour treatment of HUA with DHT increased the mRNA expression of the ß(1)-subunit of the large-conductance Ca(2+)-activated (BK(Ca)) channel and decreased expression of the α-subunit of L-type calcium channels. In organ bath studies, testosterone (1-100 µmol/L) produced similar relaxant responses in DHT- and vehicle-treated HUA rings precontracted with 5-HT, histamine and KCl. However, the relaxation response obtained by the combined application of testosterone (100 µmol/L) and nifedipine (10 µmol/L) was significantly greater in DHT- compared with vehicle-treated HUA. The results indicate that the rapid vasorelaxant effects of testosterone that are dependent on both BK(Ca) and voltage-sensitive potassium (K(V)) channel activity in control arteries become dependent solely on K(V) channel activity in DHT-treated HUA. Thus, the present study reveals the importance of the investigation of both the short- and long-term effects of androgens in human arteries.

Effects of bisphenol A on human umbilical arteries.

Bisphenol A (BPA) is an endocrine-disrupting chemical widely used in the plastics industry, including food container, toys, and medical equipment. We analyzed the effect of BPA in human umbilical artery contractility and expression of some proteins modulating this function, such as ionic channels and proteins involved in the cGMP pathway. Using standard organ bath technique, rings of human umbilical arteries without endothelium were contracted by 5-HT (1 µM) and histamine (10 µM) and the effect of different concentrations of BPA (1 nM-100 µM) was analyzed. The results showed that BPA is a vasodilator of these arteries in a concentration-dependent way. Besides, qPCR studies on human umbilical smooth muscle cells (HUSMC) allowed to analyze the effects of BPA on gene expression. Thus, 12-h exposition to BPA induced reduction of expression of L-type calcium channels (LTCC), alpha subunit of BKCa channels, and Kvß1 and Kvß3 from Kv channels. BPA also decreased the expression of soluble guanylate cyclase (sGC) and natriuretic peptide receptor type A (NPRA), meanwhile increasing that of PKG, proteins involved in vasodilation of human umbilical arteries (HUA) by cGMP. Further studies will be necessary to increase knowledge about the implications of these changes induced by BPA exposure.

HIF-1alpha-pathway activation in cholangiocytes of patients with biliary atresia: An immunohistochemical/molecular exploratory study.

BACKGROUND: Biliary atresia is a neonatal disease characterized by choledochal obstruction and progressive cholangiopathy requiring liver transplantation in most patients. Hypoxia-ischemia affecting the biliary epithelium may lead to biliary obstruction. We hypothesized that ischemic cholangiopathy involving disruption of the peribiliary vascular plexus could act as a triggering event in biliary atresia pathogenesis. METHODS: Liver and porta hepatis paraffin-embedded samples of patients with biliary atresia or intrahepatic neonatal cholestasis (controls) were immunohistochemically evaluated for HIF-1alpha-nuclear signals. Frozen histological samples were analyzed for gene expression in molecular profiles associated with hypoxia-ischemia. Prospective clinical-laboratory and histopathological data of biliary atresia patients and controls were reviewed. RESULTS: Immunohistochemical HIF-1alpha signals localized to cholangiocytes were detected exclusively in liver specimens from biliary atresia patients. In 37.5% of liver specimens, HIF-1alpha signals were observed in biliary structures involving progenitor cell niches and peribiliary vascular plexus. HIF-1alpha signals were also detected in biliary remnants of 81.8% of porta hepatis specimens. Increased gene expression of molecules linked to REDOX status, biliary proliferation, and angiogenesis was identified in biliary atresia liver specimens. In addition, there was a trend towards decreased GSR expression levels in the HIF-1alpha-positive group compared to the HIF-1alpha-negative group. CONCLUSION: Activation of the HIF-1alpha pathway may be associated with the pathogenesis of biliary atresia, and additional studies are necessary to confirm the significance of this finding. Ischemic cholangiopathy and REDOX status disturbance are putative explanations for HIF-1alpha activation. These findings may give rise to novel lines of clinical and therapeutic investigation in the BA field.

HLA-A23/HLA-A24 serotypes and dementia interaction in the elderly: Association with increased soluble HLA class I molecules in plasma.

MHC class I molecules regulate brain development and plasticity in mice and HLA class I molecules are associated with brain disorders in humans. We investigated the relationship between plasma-derived soluble human HLA class I molecules (sHLA class I), HLA class I serotypes and dementia. A cohort of HLA class I serotyped elderly subjects with no dementia/pre-dementia (NpD, n = 28), or with dementia (D, n = 28) was studied. Multivariate analysis was used to examine the influence of dementia and HLA class I serotype on sHLA class I levels, and to compare sHLA class I within four groups according to the presence or absence of HLA-A23/A24 and dementia. HLA-A23/A24 and dementia, but not age, significantly influenced the level of sHLA class I. Importantly, the concurrent presence of HLA-A23/A24 and dementia was associated with higher levels of sHLA class I (p < 0.001). This study has shown that the simultaneous presence of HLA-A23/HLA-A24 and dementia is associated with high levels of serum sHLA class I molecules. Thus, sHLA class I could be considered a biomarker of neurodegeneration in certain HLA class I carriers.

Non-genomic vasorelaxant effects of 17ß-estradiol and progesterone in rat aorta are mediated by L-type Ca2+ current inhibition.

AIM: The sex hormones 17ß-estradiol (ßES) and progesterone (PRG) induce rapid non-genomic vasodilator effects which could be protective for the cardiovascular system. The purpose of this study was to analyze the mechanisms underlying their vasodilator effect in rat aortic smooth muscle preparations. METHODS: Endothelium-denuded aorta artery rings were prepared from male Wistar rats and incubated in an organ bath. The contractions of the preparation were recorded through isometric transducers. The effects of the hormones on K(+) current and L-type Ca(2+) current (LTCC) were analyzed by using the whole cell voltage-clamp technique in A7r5 cells. RESULTS: Both ßES and PRG (1-100 µmol/L) concentration-dependently relaxed the endothelium-denuded aortic rings contracted by (-)-Bay K8644 (0.1 µmol/L) or by KCl (60 mmol/L). The IC(50) values of the two hormones were not statistically different. The K(V) channel blocker 4-aminopyridine (2 mmol/L), BK(Ca) channel blocker tetraethylammonium (1 mmol/L) and K(ATP) channel blocker glibenclamide (10 µmol/L) did not significantly modify the relaxant effect of the hormones. On the other hand, the blockage of the intracellular ßES and PRG receptors with estradiol receptor antagonists ICI 182,780 (1 µmol/L) and PRG receptor antagonist mifepristone (30 µmol/L), respectively, did not significantly modify the relaxant action of the hormones. In A7r5 cells, both the hormones (1-100 µmol/L) rapidly and reversibly inhibited the basal and BAY-stimulated LTCC. However, these hormones had no effect on the basal K(+) current. CONCLUSION: The vasorelaxant effects of ßES and PRG are due to the inhibition of LTCC. The K(+) channels are not involved in the effects.

CD45RA, CD8ß, and IFNγ Are Potential Immune Biomarkers of Human Cognitive Function.

There is increasing evidence that in humans the adaptive immunological system can influence cognitive functions of the brain. We have undertaken a comprehensive immunological analysis of lymphocyte and monocyte populations as well as of HLA molecules expression in a cohort of elderly volunteers (age range, 64-101) differing in their cognitive status. Hereby, we report on the identification of a novel signature in cognitively impaired elderly characterized by: (1) elevated percentages of CD8+ T effector-memory cells expressing high levels of the CD45RA phosphate receptor (Temra hi); (2) high percentages of CD8+ T cells expressing high levels of the CD8ß chain (CD8ßhi); (3) augmented production of IFNγ by in vitro activated CD4+ T cells. Noteworthy, CD3+CD8+ Temra hi and CD3+CD8ßhi cells were associated with impaired cognition. Cytomegalovirus seroprevalence showed that all volunteers studied but one were CMV positive. Finally, we show that some of these phenotypic and functional features are associated with an increased frequency of the HLA-B8 serotype, which belongs to the ancestral haplotype HLA-A1, Cw7, B8, DR3, DQ2, among cognitively impaired volunteers. To our knowledge, this is the first proof in humans linking the amount of cell surface CD45RA and CD8ß chain expressed by CD8+ Temra cells, and the amount of IFNγ produced by in vitro activated CD4+ T cells, with impaired cognitive function in the elderly.

Challenges and Opportunities for Clinical Pharmacogenetic Research Studies in Resource-limited Settings: Conclusions From the Council for International Organizations of Medical Sciences-Ibero-American Network of Pharmacogenetics and Pharmacogenomics Meeting.

PURPOSE: The symposium Health and Medicines in Indigenous Populations of America was organized by the Council for International Organizations of Medical Sciences (CIOMS) Working Group on Clinical Research in Resource-Limited Settings (RLSs) and the Ibero-American Network of Pharmacogenetics and Pharmacogenomics (RIBEF). It was aimed to share and evaluate investigators' experiences on challenges and opportunities on clinical research and pharmacogenetics. METHODS: A total of 33 members from 22 countries participated in 2 sessions: RIBEF studies on population pharmacogenetics about the relationship between ancestry with relevant drug-related genetic polymorphisms and the relationship between genotype and phenotype in Native Americans (session 1) and case examples of clinical studies in RLSs from Asia (cancer), America (diabetes and women health), and Africa (malaria) in which the participants were asked to answer in free text their experiences on challenges and opportunities to solve the problems (session 2). Later, a discourse analysis grouping common themes by affinity was conducted. FINDINGS: The main result of session 1 was that the pharmacogenetics-related ancestry of the population should be considered when designing clinical studies in RLSs. In session 2, 21 challenges and 20 opportunities were identified. The social aspects represent the largest proportion of the challenges (43%) and opportunities (55%), and some of them seem to be common. IMPLICATIONS: The main discussion points were gathered in the Declaration of Mérida/T'Hó and announced on the Parliament of Extremadura during the CIOMS-RIBEF meeting in 4 of the major Latin American autochthonous languages (Náhualth, Mayan, Miskito, and Kichwa). The declaration highlighted the following: (1) the relevance of population pharmacogenetics, (2) the sociocultural contexts (interaction with traditional medicine), and (3) the education needs of research teams for clinical research in vulnerable and autochthonous populations.

PDE4 and PDE5 regulate cyclic nucleotides relaxing effects in human umbilical arteries.

Cyclic nucleotides (cAMP and cGMP) are the main second messengers linked to vasodilatation. They are synthesized by cyclases and degraded by different types of phosphodiesterases (PDE). The effect of PDE inhibition and cyclases stimulation on 5-hydroxytryptamine (5-HT; 1 microM) and histamine (10 microM) contracted arteries was analysed. Stimulation of guanylate cyclase or adenylate cyclase relaxed the histamine- and 5-HT-induced contractions indicating that intracellular increase of cyclic nucleotides leads to vasodilatation of the human umbilical artery. We investigated the role of different PDE families in the regulation of this effect. The presence of the different PDE types in human umbilical artery smooth muscle was analysed by RT-PCR and the expression of PDE1B, PDE3A, PDE3B, PDE4C, PDE4D and PDE5A was detected. The unspecific PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX; 50 microM) relaxed histamine-contracted human umbilical artery on 47.4+/-7.2%. This effect seems to be due to PDE4 and PDE5 inhibition because among the selective PDE inhibitors used only the PDE4 inhibitor (rolipram; 1 microM) and the PDE5 inhibitors (dipyridamole and T0156; 3 microM and 1 microM respectively) induced significant relaxation (39.0+/-8.7, 30.4+/-6.0 and 36.3+/-2.8 respectively). IBMX, dipyridamole and T0156 produced similar relaxation on 5-HT-induced contraction. After forskolin, the addition of IBMX or rolipram increased the effect of the adenylate cyclase stimulator and almost completely relaxed the human umbilical artery contracted by histamine (92.5+/-4.9 and 90.9+/-4.7 respectively), suggesting a main role of PDE4. The data obtained with 5-HT contracted arteries confirmed this, because only rolipram and IBMX significantly increased the forskolin vasodilator effect. The administration of dipyridamole and T0156 after sodium nitroprusside (SNP) induced a significant increase of the SNP relaxant effect on histamine-contracted arteries, but PDE1 and PDE3 inhibition did not increase the effect of the guanylate cyclase stimulator. Similar effects were obtained in 5-HT contracted arteries, the SNP induced relaxation was increased by the PDE5 inhibition, but not by PDE1 or PDE3 inhibition. In summary, our results demonstrate that: 1) the increase of cAMP and/or cGMP levels induces relaxation of the human umbilical vascular smooth muscle; 2) four families of PDE are expressed in this smooth muscle: PDE1, PDE3, PDE4 and PDE5; 3) between these families, PDE4 and PDE5 are the key enzymes involved in the regulation of the relaxation associated to cAMP and cGMP, respectively.

Potassium channels are involved in testosterone-induced vasorelaxation of human umbilical artery.

Recent studies have shown that testosterone induces relaxation of different arteries, although the mechanism of this action is still under debate. We investigated the involvement of potassium channels in this mechanism. Using standard organ bath techniques, rings of human umbilical arteries (HUA) without endothelium were contracted by serotonin (5-HT, 1 microM), histamine (10 microM) and potassium chloride (KCl, 30 and 60 mM), and the vasorelaxant effect of testosterone was analysed. Testosterone (100 microM) relaxed human umbilical arteries contracted with 5-HT (30.1 +/- 3.2%), histamine (55.1 +/- 2.6%), KCl 30 mM (52.9 +/- 8.3%) and KCl 60 mM (54.8 +/- 6.3%). Flutamide (10 microM), an inhibitor of classical intracellular testosterone receptor, and glibenclamide, an ATP-sensitive potassium-channels (K(ATP)) inhibitor, did not influence the testosterone relaxant effect. 4-aminopyridine, a voltage-sensitive potassium-channels (Kv) inhibitor, decreased the effect of testosterone on histamine- and 5-HT-contracted arteries. Tetraethylammonium (TEA), which inhibits Kv channels and large-conductance Ca(2+)-activated potassium channels (BK(Ca)), decreased the effect of testosterone on KCl (60 mM)-contracted and 5-HT-contracted HUA. In conclusion, testosterone induces relaxation of HUA, and this effect does not appear to be mediated via a classic intracellular testosterone receptor-dependent mechanism. Our results suggest that this relaxation is partially mediated by activation of BK(Ca) and K(V) channels. The involvement of these two channels in testosterone-relaxant mechanism is dependent on the pathways activated by the contractile agent used.

Tributyltin role on the serotonin and histamine receptors in human umbilical artery.

Some studies in animals suggest that TBT may constitute a risk factor for cardiovascular diseases. Hence, the main purpose of this study was to investigate in human umbilical artery (HUA) the effect of TBT on vascular reactivity, manly in serotonin (5-HT) and histamine receptors. Using standard organ bath techniques, rings of HUA without endothelium were contracted by 5-HT and histamine. We also investigated the effect of TBT on the expression of the receptors using Real-time PCR. The results show that TBT short term effects include concentration-dependent relaxation. Moreover, at long term exposures, the arteries treated with 100 µM of TBT do not have contraction capacity when 5-HT is added, and the gene expression of 5-HT2A receptor decrease. Regarding histamine, it was demonstrated that TBT induces a concentration-dependent relaxation and the H1 gene expression levels decrease. In conclusion TBT modifies the activity and expression of 5-HT and histamine receptors.

Cyclic guanosine monophosphate compartmentation in rat cardiac myocytes.

BACKGROUND: Cyclic guanosine monophosphate (cGMP) is the common second messenger for the cardiovascular effects of nitric oxide (NO) and natriuretic peptides, such as atrial or brain natriuretic peptide, which activate the soluble and particulate forms of guanylyl cyclase, respectively. However, natriuretic peptides and NO donors exert different effects on cardiac and vascular smooth muscle function. We therefore tested whether these differences are due to an intracellular compartmentation of cGMP and evaluated the role of phosphodiesterase (PDE) subtypes in this process. METHODS AND RESULTS: Subsarcolemmal cGMP signals were monitored in adult rat cardiomyocytes by expression of the rat olfactory cyclic nucleotide-gated (CNG) channel alpha-subunit and recording of the associated cGMP-gated current (ICNG). Atrial natriuretic peptide (10 nmol/L) or brain natriuretic peptide (10 nmol/L) induced a clear activation of ICNG, whereas NO donors (S-nitroso-N-acetyl-penicillamine, diethylamine NONOate, 3-morpholinosydnonimine, and spermine NO, all at 100 micromol/L) had little effect. The ICNG current was strongly potentiated by nonselective PDE inhibition with isobutyl methylxanthine (100 micromol/L) and by the PDE2 inhibitors erythro-9-(2-hydroxy-3-nonyl)adenine (10 micromol/L) and Bay 60-7550 (50 nmol/L). Surprisingly, sildenafil, a PDE5 inhibitor, produced a dose-dependent increase of I(CNG) activated by NO donors but had no effect (at 100 nmol/L) on the current elicited by atrial natriuretic peptide. CONCLUSIONS: These results indicate that in rat cardiomyocytes (1) the particulate cGMP pool is readily accessible at the plasma membrane, whereas the soluble pool is not; and (2) PDE5 controls the soluble but not the particulate pool, whereas the latter is under the exclusive control of PDE2. Differential spatiotemporal distributions of cGMP may therefore contribute to the specific effects of natriuretic peptides and NO donors on cardiac function.

Effects of trans- and cis-resveratrol on Ca2+ handling in A7r5 vascular myocytes.

Although the natural polyphenol resveratrol posses a direct vasorelaxant effect, its effects on cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) in vascular cells remain still unclear. Here, we have investigated the effects of the isomers trans- and cis-resveratrol on agonist- and high-K(+)-induced [Ca(2+)](i) increases and on voltage-activated transmembrane Ca(2+) fluxes using imaging and patch-clamp techniques in vascular A7r5 myocytes. Arginine vasopressin (AVP) or angiotensin II caused a biphasic increase in [Ca(2+)](i) that was reduced by preincubation with trans-resveratrol and cis-resveratrol. Both isomers also reduced the agonist-induced increase in [Ca(2+)](i) in absence of extracellular Ca(2+). In high-K(+) Ca(2+)-free solution, reintroduction of Ca(2+) caused a sustained rise in [Ca(2+)](i) that was reduced by preincubation with trans-resveratrol or cis-resveratrol. When the isomers were applied during the plateau phase of the agonist- or the high-K(+)-induced response, a biphasic change in [Ca(2+)](i) was observed: a transient reduction of the plateau (<5 min) followed by an increase (>10 min). Finally, trans-resveratrol and cis-resveratrol inhibited voltage-dependent L-type Ca(2+) currents (I(Ca(L))). In conclusion, resveratrol isomers exert a dual effect on [Ca(2+)](i) handling in A7r5 myocytes: 1) a blockade of I(Ca(L)) and 2) an increase in [Ca(2+)](i) by depletion of intracellular Ca(2+) stores (which interferes with the agonist-induced release of intracellular Ca(2+)) and influx of Ca(2+), mainly due to activation of capacitative Ca(2+) entry, although other Ca(2+)-permeable channels are also involved. Taken together, these effects may explain, in part, the endothelium-independent vasorelaxant effects of resveratrol in rat aorta.