Cyclase Pathway Research Articles

Post receptor determinants of acute platelet response to clopidogrel in patients with symptomatic myocardial ischemia

BackgroundClopidogrel resistance is more common in patients with loss-of-function CYP2C19 genotypes. Since adenylate cyclase (AC) and soluble guanylate cyclase (sGC) pathways are variably impaired in patients with ischaemic heart disease, we tested the relevance of these determinants in patients undergoing acute loading with clopidogrel (600mg) prior to non-emergent coronary stenting. MethodsInhibitory effects of prostaglandin E1 (PGE1, an AC activator) and sodium nitroprusside (NP, a sGC activator) on platelet aggregation were determined at baseline and compared with platelet responses to clopidogrel (4h after administration) assessed as ∆ADP, and Platelet Reactivity Index (∆PRI). Data were analysed according to CYP2C19 genotype. ResultsIn patients without loss of function mutations (n=18), ∆ADP but not ∆PRI, was directly correlated with baseline PGE1 responsiveness (rs=0.62, p=0.005)). NP responsiveness did not predict ∆ADP. However there was no relationship between clopidogrel responses and either PGE1 or NP responsiveness in patients with loss of function mutations. Multivariate correlates of clopidogrel response were both the genotype status (β=−0.609, p<0.001) and the baseline response to PGE1 (β=0.303, p=0.03). ConclusionsWhile genetically impaired bio-activation markedly limits acute (4h) clopidogrel response, impaired AC signalling provides an additional cause for clopidogrel resistance.

Metagenomic analysis reveals microbial functional redundancies and specificities in a soil under different tillage and crop-management regimes

Information about microbial functionality in agricultural soils is still scarce, and in this study we used a shotgun metagenomic approach to compare different soil [conventional tillage (CT) with plowing and disking, and no-tillage (NT) with direct sowing into the residues of previous crops], and crop [crop succession (CS, soybean-summer/wheat-winter) or rotation (CR, soybean/maize-summer)/wheat/lupine/oat-winter)] managements in a 13-year-old field experiment in southern Brazil. Differences were detected between NT and CT in some functional subsystems, e.g., NT had more sequences associated with the metabolism of aromatic compounds, which might be related to higher capacity to degrade pesticides, more sequences of the adenylate cyclase (cAMP) pathway, which might confer stability to the microbial community, among others. On the other hand, CT showed more sequences related to carbohydrate metabolism, what could be related with a lower content of organic matter and need to metabolize a broader range of carbon sources. Also, we detected differences related to crop management, e.g., crop rotation showed more sequences in the metabolism of amino acids and derivatives and carbohydrate subsystems, what might result from higher diversity of crop-residues added to the soil. However, it was notable that the differences in the diversity of taxa previously shown in the same experiment was far greater than the functional diversity reported now, emphasizing a high level of microbial functional redundancy.

Biologically active substances-enriched diet regulates gonadotrope cell activation pathway in liver of adult and old rats.

According to the Hippocrates’ theorem “Let food be your medicine and medicine be your food”, dietary interventions may induce changes in the metabolic and inflammatory state by modulating the expression of important genes involved in the chronic disorders. The aim of the present study was to evaluate the influence of long-term (14 months) use of biologically active substances-enriched diet (BASE-diet) on transcriptomic profile of rats’ liver. The experiment was conducted on 36 Sprague–Dawley rats divided into two experimental groups (fed with control or BASE-diet, both n = 18). Control diet was a semi-synthetic diet formulated according to the nutritional requirements for laboratory animals. The BASE-diet was enriched with a mixture of polyphenolic compounds, β-carotene, probiotics, and n-3 and n-6 polyunsaturated fatty acids. In total, n = 3,017 differentially expressed (DE) genes were identified, including n = 218 DE genes between control and BASE groups after 3 months of feeding and n = 1,262 after 14 months. BASE-diet influenced the expression of genes involved particularly in the gonadotrope cell activation pathway and guanylate cyclase pathway, as well as in mast cell activation, gap junction regulation, melanogenesis and apoptosis. Especially genes involved in regulation of GnRH were strongly affected by BASE-diet. This effect was stronger with the age of animals and the length of diet use. It may suggest a link between the diet, reproductive system function and aging.

Stress response signaling and virulence: insights from entomopathogenic fungi.

The Ascomycete fungal insect pathogens, Beauveria and Metarhizium spp. have emerged as model systems with which to probe diverse aspects of fungal growth, stress response, and pathogenesis. Due to the availability of genomic resources and the development of robust methods for genetic manipulation, the last 5years have witnessed a rapid increase in the molecular characterization of genes and their pathways involved in stress response and signal transduction in these fungi. These studies have been performed mainly via characterization of gene deletion/knockout mutants and have included the targeting of general proteins involved in stress response and/or virulence, e.g. catalases, superoxide dismutases, and osmolyte balance maintenance enzymes, membrane proteins and signaling pathways including GPI anchored proteins and G-protein coupled membrane receptors, MAPK pathways, e.g. (i) the pheromone/nutrient sensing, Fus3/Kss1, (ii) the cell wall integrity, Mpk1, and (iii) the high osmolarity, Hog1, the PKA/adenyl cyclase pathway, and various downstream transcription factors, e.g. Msn2, CreA and Pac1. Here, we will discuss current research that strongly suggests extensive underlying contributions of these biochemical and signaling pathways to both abiotic stress response and virulence.

The nitric oxide pathway and possible therapeutic options in pre‐eclampsia

Pre-eclampsia is a serious multisystem disorder with diverse clinical manifestations. Although not causal, endothelial dysfunction and reduced nitric oxide bioavailability are likely to play an important role in the maternal and fetal pathophysiology of this condition. Lack of treatment modalities that can target the underlying pathophysiological changes and reverse the endothelial dysfunction frequently leads to iatrogenic preterm delivery of the fetus, causing neonatal morbidity and mortality, and the condition itself is associated with short- and longer term maternal morbidity and mortality. Drugs that target various components of the nitric oxide-soluble guanylyl cyclase pathway can help to increase NO bioavailability. The purpose of this review is to outline the current status of clinical research involving these therapeutic modalities in the context of pre-eclampsia, with the focus being on the following: nitric oxide donors, including organic nitrates and S-nitrosothiols; l-arginine, the endogenous precursor of NO; inhibitors of cyclic guanosine 3',5'-monophosphate breakdown, including sildenafil; and other novel inhibitors of NO donor metabolism. The advantages and limitations of each modality are outlined, and scope for development into established therapeutic options for pre-eclampsia is explored.

Adenosine A3 receptors negatively regulate the engulfment-dependent apoptotic cell suppression of inflammation

Timed initiation of apoptotic cell death followed by efficient removal mediated by professional macrophages is a key mechanism in maintaining tissue homeostasis. Besides phagocytosis, clearance of apoptotic cells also involves suppression of inflammatory responses by apoptotic cells mediated by both direct inhibition of pro-inflammatory cytokine production and release of soluble anti-inflammatory factors, which act in a paracrine or autocrine fashion to amplify or sustain the anti-inflammatory response. Previous work has demonstrated that during engulfment of apoptotic cells adenosine is produced in sufficient amounts to trigger both adenosine A2A receptors (A2ARs) and A3 receptors (A3Rs). Adenosine bound to A2ARs of macrophages activated the adenylate cyclase pathway to suppress the apoptotic-cell induced, NO-dependent formation of neutrophil migration factors. Here we show by using A3R null engulfing macrophages that the adenosine produced triggers the A3Rs as well, which attenuate the A2AR signaling by inhibiting adenylate cyclase. As a result, the balance in the activation of A2ARs and A3Rs determines the amounts of NO and consequently the levels of neutrophil chemoattractants formed. Since during phagocytosis of apoptotic cells the expression of A2ARs increases, while that of A3Rs decreases, on long term adenosine suppresses the proinflammatory responses in engulfing macrophages.

Three structurally similar odorants trigger distinct signaling pathways in a mouse olfactory neuron

In the mammalian olfactory system, one olfactory sensory neuron (OSN) expresses a single olfactory receptor gene. By calcium imaging of individual OSNs in intact mouse olfactory turbinates, we observed that a subset of OSNs (Ho-OSNs) located in the most ventral olfactory receptor zone can mediate distinct signaling pathways when activated by structurally similar ligands. Calcium imaging showed that Ho-OSNs were highly sensitive to 2-heptanone, heptaldehyde and cis-4-heptenal. 2-heptanone-evoked intracellular calcium elevation was mediated by cAMP signaling while heptaldehyde triggered the diacylglycerol pathway. An increase of intracellular calcium evoked by cis-4-heptenal was due to a combination of activation mediated by the adenylate cyclase pathway and suppression generated by phospholipase C signaling. Pharmacological studies demonstrated that novel mechanisms were involved in the phospholipase C-mediated intracellular calcium changes. Binary-mixture studies and cross-adaptation data indicate that three odorants acted on the same olfactory receptor. The feature that an olfactory receptor mediates multiple signaling pathways was specific for Ho-OSNs and not established in another population of OSNs characterized. Our study suggests that distinct signaling pathways triggered by ligand-induced conformational changes of an olfactory receptor constitute a complex information process mechanism in olfactory transduction. This study has important implications beyond olfaction in that it provides insights of plasticity and complexity of G-protein-coupled receptor activation and signal transduction.

Loss of follicle-stimulating hormone receptor function causes masculinization and suppression of ovarian development in genetically female medaka.

FSH, a glycoprotein hormone, is circulated from the pituitary and functions by binding to a specific FSH receptor (FSHR). FSHR is a G protein-coupled, seven-transmembrane receptor linked to the adenylyl cyclase or other pathways and is expressed in gonadal somatic cells. In some nonmammalian species, fshr expression is much higher in the ovary than in the testis during gonadal sex differentiation, suggesting that FSHR is involved in ovarian development in nonmammalian vertebrates. However, little is known of FSHR knockout phenotypes in these species. Here we screened for fshr mutations by a medaka (Oryzias latipes) target-induced local lesion in the genomes and identified one nonsense mutation located in the BXXBB motif, which is involved in G protein activation. Next, we used an in vitro reporter gene assay to demonstrate that this mutation prevents FSHR function. We then analyzed the phenotypes of fshr mutant medaka. The fshr mutant male medaka displayed normal testes and were fertile, whereas the mutant female fish displayed small ovaries and were infertile because vitellogenesis was inhibited. The mutant females also have suppressed expression of ovary-type aromatase (cyp19a1a), a steroidogenic enzyme responsible for the conversion of androgens to estrogens, resulting in decreased 17β-estradiol levels. Moreover, loss of FSHR function caused female-to-male sex reversal in some cases. In addition, the transgenic overexpression of fshr in fshr mutants rescued FSHR function. These findings strongly suggest that in the medaka, FSH regulates the ovarian development and the maintenance mainly by the elevation of estrogen levels. We present the first FSHR knockout phenotype in a nonmammalian species.

PDE5 inhibitor efficacy is estrogen dependent in female heart disease.

Inhibition of cGMP-specific phosphodiesterase 5 (PDE5) ameliorates pathological cardiac remodeling and has been gaining attention as a potential therapy for heart failure. Despite promising results in males, the efficacy of the PDE5 inhibitor sildenafil in female cardiac pathologies has not been determined and might be affected by estrogen levels, given the hormone's involvement in cGMP synthesis. Here, we determined that the heart-protective effect of sildenafil in female mice depends on the presence of estrogen via a mechanism that involves myocyte eNOS-dependent cGMP synthesis and the cGMP-dependent protein kinase Iα (PKGIα). Sildenafil treatment failed to exert antiremodeling properties in female pathological hearts from Gαq-overexpressing or pressure-overloaded mice after ovary removal; however, estrogen replacement restored the effectiveness of sildenafil in these animals. In females, sildenafil-elicited myocardial PKG activity required estrogen, which stimulated tonic cardiomyocyte cGMP synthesis via an eNOS/soluble guanylate cyclase pathway. In contrast, eNOS activation, cGMP synthesis, and sildenafil efficacy were not estrogen dependent in male hearts. Estrogen and sildenafil had no impact on pressure-overloaded hearts from animals expressing dysfunctional PKGIα, indicating that PKGIα mediates antiremodeling effects. These results support the importance of sex differences in the use of PDE5 inhibitors for treating heart disease and the critical role of estrogen status when these agents are used in females.

TGF-β and Opioid Receptor Signaling Crosstalk Results in Improvement of Endogenous and Exogenous Opioid Analgesia under Pathological Pain Conditions

Transforming growth factor-β1 (TGF-β1) protects against neuroinflammatory events underlying neuropathic pain. TGF-β signaling enhancement is a phenotypic characteristic of mice lacking the TGF-β pseudoreceptor BAMBI (BMP and activin membrane-bound inhibitor), which leads to an increased synaptic release of opioid peptides and to a naloxone-reversible hypoalgesic/antiallodynic phenotype. Herein, we investigated the following: (1) the effects of BAMBI deficiency on opioid receptor expression, functional efficacy, and analgesic responses to endogenous and exogenous opioids; and (2) the involvement of the opioid system in the antiallodynic effect of TGF-β1. BAMBI-KO mice were subjected to neuropathic pain by sciatic nerve crash injury (SNI). Gene (PCR) and protein (Western blot) expressions of μ- and δ-opioid receptors were determined in the spinal cord. The inhibitory effects of agonists on the adenylyl cyclase pathway were investigated. Two weeks after SNI, wild-type mice developed mechanical allodynia and the functionality of μ-opioid receptors was reduced. By this time, BAMBI-KO mice were protected against allodynia and exhibited increased expression and function of opioid receptors. Four weeks after SNI, when mice of both genotypes had developed neuropathic pain, the analgesic responses induced by morphine and RB101 (an inhibitor of enkephalin-degrading enzymes, which increases the synaptic levels of enkephalins) were enhanced in BAMBI-KO mice. Similar results were obtained in the formalin-induced chemical-inflammatory pain model. Subcutaneous TGF-β1 infusion prevented pain development after SNI. The antiallodynic effect of TGF-β1 was naloxone-sensitive. In conclusion, modulation of the endogenous opioid system by TGF-β signaling improves the analgesic effectiveness of exogenous and endogenous opioids under pathological pain conditions.

Hydrogen sulfide activates heterologously expressed human CFTR in Xenopus oocytes (1181.16)

Hydrogen sulfide (H2S) has recently been recognized as a cellular signaling molecule. Physiological responses to H2S are often attributed to its impact on various sodium, calcium and potassium channels. However, little is known about the influence of H2S on chloride channels. This study investigated the putative regulation of the chloride channel CFTR (cystic fibrosis transmembrane conductance regulator) by H2S.Human CFTR was heterologously expressed in Xenopus oocytes and its activity was measured by microelectrode recordings using the two‐electrode voltage‐clamp technique. CFTR expression was verified by the application of forskolin/IBMX which elicited a transmembrane current that was sensitive to the CFTR inhibitor CFTRinh‐172. The H2S‐forming salt Na2S (5‐300 µM) significantly increased transmembrane currents of CFTR‐expressing oocytes but had no effect on native oocytes. The effect of Na2S was dose dependent and blocked by CFTRinh‐172. The slow H2S‐releasing molecule GYY4137 also activated CFTR, although to a smaller extent compared to Na2S. The adenylate cyclase inhibitor MDL‐12330A significantly decreased the activation of CFTR by Na2S. These data indicate that H2S activates CFTR in Xenopus oocytes by the cAMP/adenylate cyclase pathway. In epithelia, an increased CFTR activity may correspond to a secretory response to H2S which may be endogenously produced by the epithelium or a H2S‐generating microflora.Grant Funding Source: Supported by the German Research Foundation (AL‐1453/1‐2).

Membrane progestin receptor alpha mediates progestin-induced sperm hypermotility and increased fertilization success in southern flounder (Paralichthys lethostigma)

Progestin hormones stimulate sperm motility in teleosts but their mechanisms of action remain unclear. Preliminary results suggest that progestin upregulation of sperm motility in southern flounder and several other marine species is mediated through a sperm membrane progestin receptor with the characteristics of membrane progestin receptor alpha (mPRα, also known as Paqr7b). The hypothesis that mPRα has an important role in progestin regulation of southern flounder sperm motility and fertility was tested in the present study. The specific mPRα agonist, 10-ethenyl-19-norprogesterone (Org OD 02-0, 100nM), mimicked the stimulatory actions of the endogenous progestin, 17,20β, 21-trihydroxy-4-pregnen-3-one (20β-S, 100nM) on flounder sperm motility. The concentration of the mPRα protein on sperm plasma membranes was positively correlated to sperm motility as well as the responsiveness of sperm to progestin stimulation. Acute in vitro progestin treatment of sperm with high mPRα protein levels increased both sperm motility and fertilization success in strip spawning experiments. However, in vitro progestin treatments were ineffective on sperm with low receptor abundance. A single injection of the superactive gonadotropin-releasing hormone analog (LHRHa, 100μg/kg) increased sperm motility and fertilization success in strip spawning experiments 72h post-injection which was accompanied by an increase in mPRα protein concentrations on sperm plasma membranes. These results provide clear evidence that southern flounder sperm hypermotility is mediated through mPRα. Stimulatory G proteins, but not inhibitory G proteins, were identified in flounder sperm plasma membrane fractions. The finding that treatment of flounder sperm plasma membrane fractions with either 20β-S or Org OD 02-0 increases cAMP levels suggests progestins stimulate flounder sperm motility by activating an mPRα/stimulatory G protein/membrane adenylyl cyclase pathway. A similar mechanism has been identified in Atlantic croaker, suggesting that the signaling pathway mediated by mPRα in sperm is highly conserved in advanced teleosts. Collectively, our results indicate that progestin-stimulation of flounder sperm hypermotility and fertility is dependent on a sufficient concentration of mPRα which can be upregulated by in vivo LHRHa treatments. These findings potentially have practical applications for enhancing the fertility of male flounder broodstock.

Excitability of Guanylate Cyclase Signaling Pathway Mediating Chemotaxis

Chemotaxis, the directed migration of cells in chemical gradients, is a vital function in many biological processes, for example, morphogenesis and synapse formation. In chemotaxis of many eukaryotic cells, the localization of some key signaling mediators on the cell membrane regulates cell movements.Chemotaxis of Dictyostelium cells is mediated by some parallel signaling pathways like phosphoinositide-3-kinase (PI3K) pathway, soluble guanylate cyclase(sGC) pathway and so on. Previous work demonstrated that the response to chemoattractant, cyclic adenosine monophosphate (cAMP), of PI3K pathway has some characteristics of an excitable system. The theoretical model shows that this excitable pathway can regulate cell unidirectional movements in respond to a chemical gradient efficiently. However, what kind of property sGC pathway has remains to be clarified and it also remains unknown about the interaction between the sGC pathway and the PI3K pathway.Here, we studied that the sGC pathway also is an excitable system by analyzing the spatiotemporal dynamics of sGC. We found that focal sGC-enriched domains were spontaneously generated in a no external stimulus condition. The sGC-enriched domain generated spontaneously had same property with the domains induced by cAMP stimulation. These features are characteristics of an excitable system like the PI3K pathway. We will report that the dynamics of the PI3K pathway is influenced by modulation of the sGC pathway.The chemotactic signal transduction consists of multiple excitable pathways. This structure of signaling system may increase efficiency of respond to the steep gradient of cAMP.

Calculus Bovis-Fel Uris-Moschus Pharmacopuncture's Effect on Regional Cerebral Blood Flow and Mean Arterial Blood Pressure in Rats.

Objectives:This study was designed to investigate the effects of Calculus Bovis-Fel Uris-Moschus pharmacopuncture(BUM) on the regional cerebral blood flow (rCBF) and the mean arterial blood pressure (MABP) in normal and cerebral ischemic rats and to investigate a possible pathway involved in the effects of BUM.Methods:The changes in the rCBF and the MABP following BUM into Fengfu (GV16) were determined by using a laser-Doppler flow meter and a pressure transducer, respectively.Results:BUM significantly increased the rCBF and decreased the MABP in normal rats in a dose-dependent manner. The effect on the rCBF was significantly inhibited by pretreatment with methylene blue (0.01 mg/kg, intraperitoneal), an inhibitor of guanylate cyclase, but was not affected by pretreatment with indomethacin (1 mg/kg, intraperitoneal), an inhibitor of cyclooxygenase. The BUM-induced decrease of the MABP was changed neither by methylene blue nor by indomethacin pretreatment. In the cerebral ischemic rats, the rCBF was stably increased upon cerebral reperfusion in the BUM group in contrast to the rapid and marked increase in the control group.Conclusion:This study demonstrated that BUM into Fengbu (GV16) increased the rCBF in a dose-dependent manner in the normal state; furthermore, it improved the stability of the rCBF in the ischemic state upon reperfusion. Also, the effects of BUM on the rCBF were attenuated by inhibition of guanylate cyclase, suggesting that the effects involved the guanylate cyclase pathway.

N‐nitrosomelatonin enhances photic synchronization of mammalian circadian rhythms

Most physiological processes in mammals are synchronized to the daily light:dark cycle by a circadian clock located in the hypothalamic suprachiasmatic nucleus. Signal transduction of light-induced phase advances of the clock is mediated through a neuronal nitric oxide synthase-guanilyl cyclase pathway. We have employed a novel nitric oxide-donor, N-nitrosomelatonin, to enhance the photic synchronization of circadian rhythms in hamsters. The intraperitoneal administration of this drug before a sub-saturating light pulse at circadian time 18 generated a twofold increase of locomotor rhythm phase-advances, having no effect over saturating light pulses. This potentiation was also obtained even when inhibiting suprachiasmatic nitric oxide synthase activity. However, N-nitrosomelatonin had no effect on light-induced phase delays at circadian time 14. The photic-enhancing effects were correlated with an increased suprachiasmatic immunoreactivity of FBJ murine osteosarcoma viral oncogene and period1. Moreover, in vivo nitric oxide release by N-nitrosomelatonin was verified by measuring nitrate and nitrite levels in suprachiasmatic nuclei homogenates. The compound also accelerated resynchronization to an abrupt 6-h advance in the light:dark cycle (but not resynchronization to a 6-h delay). Here, we demonstrate the chronobiotic properties of N-nitrosomelatonin, emphasizing the importance of nitric oxide-mediated transduction for circadian phase advances.

A paradigm shift for local blood flow regulation

a remarkable example of physiological regulation is the coordination between metabolic rate and local blood flow in microscopic volumes of an organ. In skeletal muscle an increase in oxygen consumption (Vo2) over a wide range evokes a proportional (∼5 to 6 × Vo2) increase in blood flow ([29][

5-HT stimulation of heart rate inDrosophiladoes not act through cAMP as revealed by pharmacogenetics

The fruit fly, Drosophila melanogaster, is a good experimental organism to study the underlying mechanism of heart rate (HR) regulation. It is already known that many neuromodulators (serotonin, dopamine, octopamine, acetylcholine) change the HR in Drosophila melanogaster larvae. In this study, we investigated the role of cAMP-PKA signaling pathway in HR regulation and 5-HT positive chronotropic action. In order to obtain insight into the 5-HT mechanism of action in larvae cardiomyocytes, genetic and pharmacological approaches were used. We used transgenic flies that expressed the hM4Di receptor [designer receptors exclusively activated by designer drugs (DREADDs)] as one tool. Our previous results showed that activation of hM4Di receptors (modified muscarinic acetylcholine receptors) decreases or arrests the heart from beating. In this study, it was hypothesized that the positive chronotropic effect of serotonin [5-hydroxytryptamine (5-HT)] are mediated by serotonin receptors coupled to the adenylyl cyclase pathway and downstream cAMP and PKA activity. Activation of hM4Di by clozapine-N-oxide (CNO) was predicted to block the effects of serotonin by inhibiting adenylyl cyclase activity through Gαi pathway activation. Interestingly, we found here that manipulation of adenylyl cyclase activity and cAMP levels had no significant effect on HR. The ability of hM4Di receptor activation to slow or stop the heart is therefore likely mediated by activation of GIRK channels to produce hyperpolarization of cardiomyocytes, and not through inhibition of adenylyl cyclase.

Antiplatelet effects of dietary nitrate in healthy volunteers: Involvement of cGMP and influence of sex

Ingestion of vegetables rich in inorganic nitrate has emerged as an effective method, via the formation of a nitrite intermediate, for acutely elevating vascular NO levels. As such a number of beneficial effects of dietary nitrate ingestion have been demonstrated including the suggestion that platelet reactivity is reduced. In this study we investigated whether inorganic nitrate supplementation might also reduce platelet reactivity in healthy volunteers and have determined the mechanisms involved in the effects seen. We conducted two randomised crossover studies each in 24 (12 of each sex) healthy subjects assessing the acute effects of dietary nitrate (250ml beetroot juice) or potassium nitrate capsules (KNO3, 8mmol) vs placebo control on platelet reactivity. Inorganic nitrate ingested either from a dietary source or via supplementation raised circulating nitrate and nitrite levels in both sexes and attenuated ex vivo platelet aggregation responses to ADP and, albeit to a lesser extent, collagen but not epinephrine in male but not female volunteers. These inhibitory effects were associated with a reduced platelet P-selectin expression and elevated platelet cGMP levels. In addition, we show that nitrite reduction to NO occurs at the level of the erythrocyte and not the platelet. In summary, our results demonstrate that inorganic nitrate ingestion, whether via the diet or through supplementation, causes a modest decrease in platelet reactivity in healthy males but not females. Our studies provide strong support for further clinical trials investigating the potential of dietary nitrate as an adjunct to current antiplatelet therapies to prevent atherothrombotic complications. Moreover, our observations highlight a previously unknown sexual dimorphism in platelet reactivity to NO and intimate a greater dependence of males on the NO-soluble guanylate cyclase pathway in limiting thrombotic potential.

Activation of MAPK by inverse agonists in six naturally occurring constitutively active mutant human melanocortin-4 receptors

The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor that plays an essential role in regulating energy homeostasis. Defects in MC4R are the most common monogenic form of obesity, with about 170 distinct mutations identified in human. In addition to the conventional Gs-stimulated adenylyl cyclase pathway, it has been recently demonstrated that MC4R also activates mitogen-activated protein kinases, extracellular signal-regulated kinases 1 and 2 (ERK1/2). Herein, we investigated the potential of four MC4R ligands that are inverse agonists at the Gs-cAMP signaling pathway, including agouti-related peptide (AgRP), MCL0020, Ipsen 5i and ML00253764, to regulate ERK1/2 activation (pERK1/2) in wild type and six naturally occurring constitutively active mutant (CAM) MC4Rs. We showed that these four inverse agonists acted as agonists for the ERK1/2 signaling cascade in wild type and CAM MC4Rs. Three mutants (P230L, L250Q and F280L) had significantly increased pERK1/2 level upon stimulation with all four inverse agonists, with maximal induction ranging from 1.6 to 4.2-fold. D146N had significantly increased pERK1/2 level upon stimulation with AgRP, MCL0020 or ML00253764, but not Ipsen 5i. The pERK1/2 levels of H76R and S127L were significantly increased only upon stimulation with AgRP or MCL0020. In summary, our studies demonstrated for the first time that MC4R inverse agonists at the Gs-cAMP pathway could serve as agonists in the MAPK pathway. These results suggested that there were multiple activation states of MC4R with ligand-specific and/or mutant-specific conformations capable of differentially coupling the MC4R to distinct signaling pathways.

The ventral hippocampus NMDA receptor/nitric oxide/guanylate cyclase pathway modulates cardiovascular responses in rats

The hippocampus is a limbic structure that is involved in the expression of defensive reactions and autonomic changes in rats. The injection of L-glutamate (L-glu) into the ventral hippocampus (VH) decreases blood pressure and heart rate in anesthetized rats. Activation of NMDA receptors in the VH increases the production of nitric oxide (NO), leading to guanylate cyclase activation. The hypothesis of the present study was that a local NMDA receptor-NO-guanylate cyclase interaction mediates the cardiovascular effects of microinjection of L-glu into the VH. Microinjection of increasing doses of L-glu (30, 60 and 200 nmol/200 nL) into the VH of conscious rats caused dose-related pressor and tachycardiac responses. The cardiovascular effects of L-glu were abolished by local pretreatment with: the glutamate receptor antagonist AP-7 (0.4 nmol); the selective neuronal NO synthase (nNOS) inhibitor N(ω)-Propyl-L-arginine (0.04 nmol); the NO scavenger C-PTIO (2 nmol) or the guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolol [4,3-a]quinoxalin-1-one (2 nmol). Moreover, these cardiovascular responses were blocked by intravenous pretreatment with: the ganglionic blocker mecamylamine (2mg/Kg); the nonselective β-adrenergic receptor antagonist propranolol (2mg/Kg); the β1-adrenergic receptor selective antagonist atenolol (1mg/kg). However, pretreatment with the selective α1-adrenergic receptor antagonist prazosin (0,5mg/kg) caused only a small reduction in the pressor response, without affecting the L-glu evoked tachycardia. In conclusion, our results suggest that cardiovascular responses caused by L-glu microinjection into the VH are mediated by NMDA glutamate receptors and involve local nNOS and guanylate cyclase activation. Moreover, these cardiovascular responses are mainly mediated by cardiac sympathetic nervous system activation, with a small involvement of the vascular sympathetic nervous system.