Endothelin-converting enzyme-1 regulates trafficking and signalling of the neurokinin 1 receptor in endosomes of myenteric neurones

Neuropeptide signalling at the plasma membrane is terminated by neuropeptide degradation by cell-surface peptidases, and by beta-arrestin-dependent receptor desensitization and endocytosis. However, receptors continue to signal from endosomes by beta-arrestin-dependent processes, and endosomal sorting mediates recycling and resensitization of plasma membrane signalling. The mechanisms that control signalling and trafficking of receptors in endosomes are poorly defined. We report a major role for endothelin-converting enzyme-1 (ECE-1) in controlling substance P (SP) and the neurokinin 1 receptor (NK(1)R) in endosomes of myenteric neurones. ECE-1 mRNA and protein were expressed by myenteric neurones of rat and mouse intestine. SP (10 nM, 10 min) induced interaction of NK(1)R and beta-arrestin at the plasma membrane, and the SP-NK(1)R-beta-arrestin signalosome complex trafficked by a dynamin-mediated mechanism to ECE-1-containing early endosomes, where ECE-1 can degrade SP. After 120 min, NK(1)R recycled from endosomes to the plasma membrane. ECE-1 inhibitors (SM-19712, PD-069185) and the vacuolar H(+)ATPase inhibitor bafilomycin A(1), which prevent endosomal SP degradation, suppressed NK(1)R recycling by >50%. Preincubation of neurones with SP (10 nM, 5 min) desensitized Ca(2+) transients to a second SP challenge after 10 min, and SP signals resensitized after 60 min. SM-19712 inhibited NK(1)R resensitization by >90%. ECE-1 inhibitors also caused sustained SP-induced activation of extracellular signal-regulated kinases, consistent with stabilization of the SP-NK(1)R-beta-arrestin signalosome. By degrading SP and destabilizing endosomal signalosomes, ECE-1 has a dual role in controlling endocytic signalling and trafficking of the NK(1)R: promoting resensitization of G protein-mediated plasma membrane signalling, and terminating beta-arrestin-mediated endosomal signalling.

Endosomal endothelin-converting enzyme-1: a regulator of beta-arrestin-dependent ERK signaling

Neuropeptide signaling at the cell surface is regulated by metalloendopeptidases, which degrade peptides in the extracellular fluid, and beta-arrestins, which interact with G protein-coupled receptors (GPCRs) to mediate desensitization. beta-Arrestins also recruit GPCRs and mitogen-activated protein kinases to endosomes to allow internalized receptors to continue signaling, but the mechanisms regulating endosomal signaling are unknown. We report that endothelin-converting enzyme-1 (ECE-1) degrades substance P (SP) in early endosomes of epithelial cells and neurons to destabilize the endosomal mitogen-activated protein kinase signalosome and terminate signaling. ECE-1 inhibition caused endosomal retention of the SP neurokinin 1 receptor, beta-arrestins, and Src, resulting in markedly sustained ERK2 activation in the cytosol and nucleus, whereas ECE-1 overexpression attenuated ERK2 activation. ECE-1 inhibition also enhanced SP-induced expression and phosphorylation of the nuclear death receptor Nur77, resulting in cell death. Thus, endosomal ECE-1 attenuates ERK2-mediated SP signaling in the nucleus to prevent cell death. We propose that agonist availability in endosomes, here regulated by ECE-1, controls beta-arrestin-dependent signaling of endocytosed GPCRs.

Syntheses, crystal structures and properties of sterically congested heteroleptic complexes of group 10 metal ions with p-tolylsulfonyl dithiocarbimate and 1,2-bis (diphenylphosphino) ethane

Three novel heteroleptic complexes of the type cis- [ML(dppe)] [M = Ni(II), Pd(II), Pt(II); L = p-tolylsulfonyl dithiocarbimate; dppe = 1,2-bis(diphenylphosphino)ethane] have been prepared and characterized. X-ray crystallography revealed the close proximity of one of the ortho phenyl protons of the dppe ligand to the metal in the Ni(II) complex showing existence of the less common C-H center dot center dot center dot Ni anagostic interactions observed for the first time in the dithio-phosphine mixed-ligand systems. The platinum complex showed a strong photoluminescence emission near visible region in CH(2)Cl(2) solution.

Heme oxygenase-1 protects against Alzheimer’s amyloid-β1-42 induced toxicity via carbon monoxide production

Heme oxygenase-1 (HO-1), an inducible enzyme up-regulated in Alzheimer‟s disease (AD), catabolises heme to biliverdin, Fe2+ and carbon monoxide (CO). CO can protect neurones from oxidative stress-induced apoptosis by inhibiting Kv2.1 channels, which mediate cellular K+ efflux as an early step in the apoptotic cascade. Since apoptosis contributes to the neuronal loss associated with amyloid β peptide (Aβ) toxicity in AD, we investigated the protective effects of HO-1 and CO against Aβ1-42 toxicity in SH-SY5Y cells, employing cells stably transfected with empty vector or expressing the cellular prion protein, PrPc, and rat primary hippocampal neurons. Aβ1-42 (containing protofibrils) caused a concentrationdependent decrease in cell viability, attributable at least in part to induction of apoptosis, with the PrPc expressing cells showing greater susceptibility to Aβ1-42 toxicity. Pharmacological induction or genetic over-expression of HO-1 significantly ameliorated the effects of Aβ1-42. The CO-donor CORM-2 protected cells against Aβ1-42 toxicity in a concentration-dependent manner. Electrophysiological studies revealed no differences in the outward current pre- and post-Aβ1-42 treatment suggesting that K+ channel activity is unaffected in these cells. Instead, Aβ toxicity was reduced by the L-type Ca2+ channel blocker nifedipine, and by the CaMKKII inhibitor, STO-609. Aβ also activated the downstream kinase, AMP-dependent protein kinase (AMPK). CO prevented this activation of AMPK. Our findings indicate that HO-1 protects against Aβ toxicity via production of CO. Protection does not arise from inhibition of apoptosis-associated K+ efflux, but rather by inhibition of AMPK activation, which has been recently implicated in the toxic effects of Aβ. These data provide a novel, beneficial effect of CO which adds to its growing potential as a therapeutic agent.

SHIMMER (1.0): a novel mathematical model for microbial and biogeochemical dynamics in glacier forefield ecosystems

SHIMMER (Soil biogeocHemIcal Model for Microbial Ecosystem Response) is a new numerical modelling framework designed to simulate microbial dynamics and biogeochemical cycling during initial ecosystem development in glacier forefield soils. However, it is also transferable to other extreme ecosystem types (such as desert soils or the surface of glaciers). The rationale for model development arises from decades of empirical observations in glacier forefields, and enables a quantitative and process focussed approach. Here, we provide a detailed description of SHIMMER, test its performance in two case study forefields: the Damma Glacier (Switzerland) and the Athabasca Glacier (Canada) and analyse sensitivity to identify the most sensitive and unconstrained model parameters. Results show that the accumulation of microbial biomass is highly dependent on variation in microbial growth and death rate constants, Q10 values, the active fraction of microbial biomass and the reactivity of organic matter. The model correctly predicts the rapid accumulation of microbial biomass observed during the initial stages of succession in the forefields of both the case study systems. Primary production is responsible for the initial build-up of labile substrate that subsequently supports heterotrophic growth. However, allochthonous contributions of organic matter, and nitrogen fixation, are important in sustaining this productivity. The development and application of SHIMMER also highlights aspects of these systems that require further empirical research: quantifying nutrient budgets and biogeochemical rates, exploring seasonality and microbial growth and cell death. This will lead to increased understanding of how glacier forefields contribute to global biogeochemical cycling and climate under future ice retreat.

Cramoll 1,4 lectin increases ROS production, calcium levels, and cytokine expression in treated spleen cells of rats

This study reports the in vivo stimulatory effects of Cramoll 1,4 on rat spleen lymphocytes as evidenced by an increase in intracellular reactive oxygen species (ROS) production, Ca(2+) levels, and interleukin (IL)-1 beta expression. Cramoll 1,4 extracted from seeds of the Leguminosae Cratylia mollis Mart., is a lectin with antitumor and lymphocyte mitogenic activities. Animals (Nine-week-old male albino Wistar rats, Rattus norvegicus) were treated with intraperitoneal injection of Cramoll 1,4 (235 mu g ml(-1) single dose) and, 7 days later, spleen lymphocytes were isolated and analyzed for intracellular ROS, cytosolic Ca(2+), and IL-6, IL-10, and IL-1 mRNAs. Cell viability was investigated by annexin V-FITC and 7-amino-actinomycin D staining. The data showed that in lymphocytes activated by Cramoll 1,4 the increase in cytosolic and mitochondrial ROS was related to higher cytosolic Ca(2+) levels. Apoptosis and necrosis were not detected in statistically significant values and thus the lectin effector activities did not induce lymphocyte death. In vivo Cramoll 1,4 treatment led to a significant increase in IL-1 beta but IL-6 and -10 levels did not change. Cramoll 1,4 had modulator activities on spleen lymphocytes and stimulated the Th2 response.

Involvement of the AT(1) receptor in the venoconstriction induced by angiotensin II in both the inferior vena cava and femoral vein

Although angiotensin II-induced venoconstriction has been demonstrated in the rat vena cava and femoral vein, the angiotensin II receptor subtypes (AT(1) or AT(2)) that mediate this phenomenon have not been precisely characterized. Therefore, the present study aimed to characterize the pharmacological receptors involved in the angiotensin II-induced constriction of rat venae cavae and femoral veins, as well as the opposing effects exerted by locally produced prostanoids and NO upon induction of these vasomotor responses. The obtained results suggest that both AT(1) and AT(2) angiotensin II receptors are expressed in both veins. Angiotensin II concentration-response curves were shifted toward the right by losartan but not by PD 123319 in both the vena cava and femoral vein. Moreover, it was observed that both 10(-5) M indomethacin and 10(-4) M L-NAME improve the angiotensin II responses in the vena cava and femoral vein. In conclusion, in the rat vena cava and femoral vein, angiotensin II stimulates AT(1) but not AT(2) to induce venoconstriction, which is blunted by vasodilator prostanoids and NO. (C) 2010 Elsevier Inc. All rights reserved.

Synthetic modified N-POMC(1-28) controls in vivo proliferation and blocks apoptosis in rat adrenal cortex

The identity of the pro-opiomelanocortin (POMC)-derived mitogen in the adrenal cortex has been historically controversial. We have used well-established in vivo models, viz., hypophysectomized (Hyp) or dexamethasone (Dex)-treated rats, to study the effect of the synthetic modified peptide N-terminal POMC (N-POMC(1-28)) on DNA synthesis in the adrenal cortex, as assessed by BrdU incorporation and compared with adrenocorticotropic hormone (ACTH). We evaluated the importance of disulfide bridges on proliferation by employing N-POMC(1-28) without disulfide bridges and with methionines replacing cysteines. Acute administration of synthetic modified N-POMC(1-28) distinctly increased DNA synthesis in the zona glomerulosa and zona fasciculata, but not in the zona reticularis in Hyp rats, whereas in Dex-treated rats, this peptide was effective in all adrenal zones. ACTH administration led to an increase of BrdU-positive cells in all adrenal zones irrespective of the depletion of Hyp or Dex-POMC peptides. The use of the ACTH antagonist, ACTH(7-38), confirmed the direct participation of ACTH in proliferation. Two different approaches to measure apoptosis revealed that both peptides similarly exerted a protective effect on all adrenocortical zones, blocking the apoptotic cell death induced by hypophysectomy. Thus, ACTH(1-39) and N-POMC(1-28) have similar actions suggesting that the disulfide bridges are important but not essential. Both peptides seem to be important factors determining adrenocortical cell survival throughout the adrenal cortex, reinforcing the idea that each zone can be renewed from within itself.

Induction of Lymphocyte Death by Short- and Long-Duration Triathlon Competitions

LEVADA-PIRES, A. C., M. F. CURY-BOAVENTURA, R. GORJAO, S. M. HIRABARA. E. F. PUGGINA, I. L. PELLEGRINOTTI, L. A. DOMINGUES FILHO, R. CURI, and T. C. PITHON-CURI. Induction of Lymphocyte Death by Short- and Long-Duration Triathlon Competitions. Med. Sci. Sporty Exerc., Vol. 4 1, No. 10, pp. 1896-1901, 2009. Purpose: The effect of triathlon competitions on death of lymphocytes from elite athletes was investigated. Material and Methods: Blood was collected from sedentary volunteers and triathletes at rest and after a short-duration triathlon (SDT) and after a long-duration triathlon (LDT-half Ironman) competitions. Results: The athletes had lowered lymphocyte proliferation capacity compared with sedentary volunteers either at rest or after the competitions. There was no difference in the parameters associated with lymphocyte death when sedentary volunteers were compared with triathletes at rest. Lymphocytes from triathletes after SDT competition showed an increase in DNA fragmentation, phosphatidylserine externalization, and mitochondrial transmembrane depolarization and did not alter membrane integrity when compared with cells from athletes at rest. In contrast, the LDT competition raised the proportion of lymphocytes with loss of membrane integrity when compared with cells from athletes at rest and did not change the apoptotic parameters. The LDT competition induced an increase of reactive oxygen species (ROS) production by lymphocytes compared with triathletes at rest. The SDT competition did not alter ROS production by lymphocytes when compared with cells from triathletes at rest. ROS production by lymphocytes after LDT competition was 60% higher than in SDT. Conclusions: Evidence is presented herein that an LDT competition caused lymphocyte death by necrosis, whereas an SDT induced lymphocyte apoptosis. The mechanism for lymphocyte death induced by the triathlon competitions may involve an increase in ROS production at different extents.

Extracellular Signal-Regulated Kinase 1/2 Activation, via Downregulation of Mitogen-Activated Protein Kinase Phosphatase 1, Mediates Sex Differences in Desoxycorticosterone Acetate-Salt Hypertension Vascular Reactivity

Extracellular signal-regulated kinase (ERK) 1/2 has been reported to play a role in vascular dysfunction associated with mineralocorticoid hypertension. We hypothesized that, compared with female rats, an upregulation of ERK1/2 signaling in the vasculature of male rats contributes to augmented contractile responses in mineralocorticoid hypertension. Uninephrectomized male and female Sprague-Dawley rats received desoxycorticosterone acetate (DOCA) pellets (200 mg per animal) and saline to drink for 3 weeks. Control uninephrectomized rats received tap water to drink. Blood pressure, measured by telemetry, was significantly higher in male DOCA rats (191 +/- 3 mm Hg) compared with female DOCA rats (172 +/- 7 mm Hg; n=5). DOCA treatment resulted in augmented contractile responses to phenylephrine in aorta (22 +/- 3 mN; n=6) and small mesenteric arteries (13 +/- 2 mN; n=6) from male DOCA rats versus uninephrectomized male rats (16 +/- 3 and 10 +/- 2 mN, respectively; P<0.05) and female DOCA rats (15 +/- 1 and 11 +/- 1 mN, respectively). ERK1/2 inhibition with PD-98059 (10 mu mol/L) abrogated increased contraction to phenylephrine in aorta (14 +/- 2 mN) and small mesenteric arteries (10 +/- 2 mN) from male DOCA rats, without any effects in arteries from male uninephrectomized or female animals. Compared with the other groups, phosphorylated ERK1/2 levels were increased in the aorta from male DOCA rats, whereas mitogen-activated protein kinase phosphatase 1 expression was decreased. Interleukin-10 plasma levels, which positively regulate mitogen-activated protein kinase phosphatase 1 activity, were reduced in male DOCA-salt rats. We speculate that augmented vascular reactivity in male hypertensive rats is mediated via activation of the ERK1/2 pathway. In addition, mitogen-activated protein kinase phosphatase 1 and interleukin 10 play regulatory roles in this process. (Hypertension. 2010; 55: 172-179.)

Interleukin-10 attenuates vascular responses to endothelin-1 via effects on ERK1/2-dependent pathway

Giachini FR, Zemse SM, Carneiro FS, Lima VV, Carneiro ZN, Callera GE, Ergul A, Webb RC, Tostes RC. Interleukin-10 attenuates vascular responses to endothelin-1 via effects on ERK1/2-dependent pathway. Am J Physiol Heart Circ Physiol 296: H489-H496, 2009. First published December 12, 2008; doi:10.1152/ajpheart.00251.2008.-Interleukin-10 (IL-10) is an anti-inflammatory cytokine with protective actions on the vasculature. On the other hand, endothelin ( ET)-1 has potent vasoconstrictor, mitogenic, and proinflammatory activities, which have been implicated in the pathophysiology of a number of cardiovascular diseases. We hypothesized that, in a condition where ET-1 expression is upregulated, i.e., on infusion of TNF-alpha, IL-10 confers vascular protection from ET-1-induced injury. Aortic rings and first-order mesenteric arteries from male C57BL/6 (WT) and IL-10-knockout (IL-10(-/-)) mice were treated with human recombinant TNF-alpha (220 ng.kg(-1).day(-1)) or vehicle (saline) for 14 days. TNF-alpha infusion significantly increased blood pressure in IL-10(-/-), but not WT, mice. TNF-alpha augmented vascular ET-1 mRNA expression in arteries from WT and IL-10(-/-) mice. ET type A (ETA) receptor expression was increased in arteries from IL-10(-/-) mice, and TNF-alpha infusion did not change vascular ETA receptor expression in control or IL-10(-/-) mice. Aorta and mesenteric arteries from TNF-alpha-infused IL-10(-/-) mice displayed increased contractile responses to ET-1, but not the ET type B receptor agonist IRL-1620. The ETA receptor antagonist atrasentan completely abolished responses to ET-1 in aorta and mesenteric vessels, whereas the ERK1/2 inhibitor PD-98059 abrogated increased contractions to ET-1 in arteries from TNF-alpha-infused IL-10(-/-) mice. Infusion of TNF-alpha, as well as knockdown of IL-10 (IL-10(-/-)), induced an increase in total and phosphorylated ERK1/2. These data demonstrate that IL-10 counteracts ET(A)-mediated vascular responses to ET-1, as well as activation of the ERK1/2 pathway.

Heterobimetallic compounds [Ru(eta(5)-Cp)(dppf)X] (X = Cl, Br, I and N(3)): synthesis, electrochemical analysis, and the crystal structure of [Ru(eta(5)-Cp)(dppf)I] [dppf=1,1`-bis(diphenylphosphino)ferrocene]

A series of new ruthenium-iron based derivatives [Ru(eta(5)-Cp)(dppf)Cl] (1), [Ru(eta(5)-Cp)(dppf)Br] (2), [Ru(eta(5)-Cp)(dppf)I] (3) and [Ru(eta(5)-Cp)(dppf)N(3)] (4) were obtained by reactions of [Ru(eta(5)-Cp)(PPh(3))(2)Cl] with 1,1`-bis(diphenylphosphino) ferrocene (dppf) and characterized by IR, NMR ((1)H, (13)C and (31)P), (57)Fe Mossbauer spectroscopy and cyclic voltammetry. Additionally, the compound (3) was structurally characterized by X-ray crystallography, and the results were as follows: orthorhombic, Pbca, a = 18.2458(10), b = 20.9192(11), c = 34.4138(19) a""<<, alpha = beta = gamma = 90A degrees, V = 13135.3(12) a""<<(3) and Z = 16.

Superoxide Dismutase 1-mediated Production of Ethanol- and DNA-derived Radicals in Yeasts Challenged with Hydrogen Peroxide MOLECULAR INSIGHTS INTO THE GENOME INSTABILITY OF PEROXIREDOXIN-NULL STRAINS

Peroxiredoxins are receiving increasing attention as defenders against oxidative damage and sensors of hydrogen peroxide-mediated signaling events. In the yeast Saccharomyces cerevisiae, deletion of one or more isoforms of the peroxiredoxins is not lethal but compromises genome stability by mechanisms that remain under scrutiny. Here, we show that cytosolic peroxiredoxin-null cells (tsa1 Delta tsa2 Delta) are more resistant to hydrogen peroxide than wildtype (WT) cells and consume it faster under fermentative conditions. Also, tsa1 Delta tsa2 Delta cells produced higher yields of the 1-hydroxyethyl radical from oxidation of the glucose metabolite ethanol, as proved by spin-trapping experiments. A major role for Fenton chemistry in radical formation was excluded by comparing WT and tsa1 Delta tsa2 Delta cells with respect to their levels of total and chelatable metal ions and of radical produced in the presence of chelators. The main route for 1-hydroxyethyl radical formation was ascribed to the peroxidase activity of Cu, Zn-superoxide dismutase (Sod1), whose expression and activity increased similar to 5- and 2-fold, respectively, in tsa1 Delta tsa2 Delta compared with WT cells. Accordingly, overexpression of human Sod1 in WT yeasts led to increased 1-hydroxyethyl radical production. Relevantly, tsa1 Delta tsa2 Delta cells challenged with hydrogen peroxide contained higher levels of DNA-derived radicals and adducts as monitored by immuno-spin trapping and incorporation of (14)C from glucose into DNA, respectively. The results indicate that part of hydrogen peroxide consumption by tsa1 Delta tsa2 Delta cells is mediated by induced Sod1, which oxidizes ethanol to the 1-hydroxyethyl radical, which, in turn, leads to increased DNA damage. Overall, our studies provide a pathway to account for the hypermutability of peroxiredoxin-null strains.

Thioredoxin-1 promotes survival in cells exposed to S-nitrosoglutathione: Correlation with reduction of intracellular levels of nitrosothiols and up-regulation of the ERK1/2 MAP Kinases

Accumulating evidence indicates that post-translational protein modifications by nitric oxide and its derived species are critical effectors of redox signaling in cells. These protein modifications are most likely controlled by intracellular reductants. Among them, the importance of the 12 kDa dithiol protein thioredoxin-1 (TRX-1) has been increasingly recognized. However, the effects of TRX-1 in cells exposed to exogenous nitrosothiols remain little understood. We investigated the levels of intracellular nitrosothiols and survival signaling in HeLa cells over-expressing TRX-1 and exposed to S-nitrosoglutahione (GSNO). A role for TRX-1 expression on GSNO catabolism and cell viability was demonstrated by the concentration-dependent effects of GSNO on decreasing TRX-1 expression, activation of capase-3, and increasing cell death. The over-expressaion of TRX-1 in HeLa cells partially attenuated caspase-3 activation and enhanced cell viability upon GSNO treatment. This was correlated with reduction of intracellular levels of nitrosothiols and increasing levels of nitrite and nitrotyrosine. The involvement of ERK, p38 and JNK pathways were investigated in parental cells treated with GSNO. Activation of ERK1/2 MAP kinases was shown to be critical for survival signaling. lit cells over-expressing TRX-1, basal phosphorylation levels of ERK1/2 MAP kinases were higher and further increased after GSNO treatment. These results indicate that the enhanced cell viability promoted by TRX-1 correlates with its capacity to regulate the levels of intracellular nitiosothiols and to up-regulate the survival signaling pathway mediated by the ERK1/2 MAP kinases.

Prion Protein and Its Ligand Stress Inducible Protein 1 Regulate Astrocyte Development

Prion protein (PrP(C)) interaction with stress inducible protein 1 (STI1) mediates neuronal survival and differentiation. However, the function of PrP(C) in astrocytes has not been approached. In this study, we show that STI1 prevents cell death in wild-type astrocytes in a protein kinase A-dependent manner, whereas PrP(C)-null astrocytes were not affected by STI1 treatment. At embryonic day 17, cultured astrocytes and brain extracts derived from PrP(C)-null mice showed a reduced expression of glial fibrillary acidic protein (GFAP) and increased vimentin and nestin expression when compared with wild-type, suggesting a slower rate of astrocyte maturation in PrP(C)-null animals. Furthermore, PrP(C)-null astrocytes treated with STI1 did not differentiate from a flat to a process-bearing morphology, as did wild-type astrocytes. Remarkably, STI1 inhibited proliferation of both wild-type and PrP(C)-null astrocytes in a protein kinase C-dependent manner. Taken together, our data show that PrP(C) and STI1 are essential to astrocyte development and act through distinct signaling pathways.(C) 2009 Wiley-Liss, Inc.