Regulation of the rat brain endothelin system by endogenous beta-endorphin

Several lines of evidence indicate that the central endogenous opioid and endothelin (ET) system regulate each other. To explore this idea further, we determined the effect of intracerebroventricular (i.c.v.) administration of anti-beta-endorphin IgG (rabbit) on the expression level of the opioid, corticotropin-releasing hormone and endothelin receptors, and tissue concentration of ET-1. Three days after implanting cannula into the lateral ventricle, male Sprague-Dawley rats were administered 10 microl (i.c.v.) of either control rabbit IgG (2.5 microg/microl) or anti-beta-endorphin IgG (2.5 microg/microl) on days 1, 3 and 5. On day 6, animals were euthanized and caudate, cortex and hippocampus collected for Western blot analysis. Anti-beta-endorphin IgG down-regulated ET-A receptor protein expression in the caudate (51%), but had no effect on the expression of mu, delta, kappa opioid, ET-B, CRH-1 and CRH-2 receptors in any brain region. Anti-beta-endorphin IgG increased tissue ET-1 levels in the caudate by 30.3%. [35S]GTP-gamma-S binding assays demonstrated that anti-beta-endorphin IgG increased the efficacy of [D-Ala2-MePhe4, Gly-ol5]enkephalin without altering its potency in caudate. Control experiments showed that there was no detectable rabbit IgG in caudate, cortex and hippocampus samples. These results suggest that beta-endorphin in the CSF coordinately regulates ET-1 levels and the ET-A receptor in rat caudate. These findings support the hypothesis that CSF neuropeptides have regulatory effects and further demonstrate a link between opioid and ET system.     

Pulmonary vasoregulation by endothelin in conscious dogs after left lung transplantation.

We tested the hypothesis that regulation of the pulmonary circulation by endogenous endothelin (ET) during normoxia and hypoxia was altered in conscious dogs 1 mo after left lung autotransplantation (LLA). Sham-operated control and post-LLA dogs were chronically instrumented to measure the left pulmonary vascular pressure-flow (LP-Q) relationship. LP-Q plots were generated on separate days during normoxia and hypoxia (arterial PO(2) approximately 50 Torr) in the intact condition, after selective ET(A)-receptor inhibition (BQ-485), and after combined ET(A+B)-receptor inhibition (bosentan). Although LLA resulted in a chronic increase in pulmonary vascular resistance, the ET-receptor antagonists had no effect on the LP-Q relationship during normoxia in either group. The magnitude of hypoxic pulmonary vasoconstriction (HPV) was flow dependent in both groups, and the HPV response was potentiated post-LLA compared with control. ET(A)-receptor inhibition attenuated the HPV response to the same extent in both groups. ET(A+B)-receptor inhibition attenuated the HPV response to a greater extent than did ET(A)-receptor inhibition alone, and this effect was greater post-LLA compared with control. Plasma ET-1 concentration only increased during hypoxia in the LLA group. These results indicate that ET does not regulate the baseline LP-Q relationship in either group. Both ET(A)- and ET(B)-receptor activation mediate a component of HPV in conscious dogs, and the vasoconstrictor influence of ET(B)-receptor activation is enhanced post-LLA.     

Purification and characterization of bovine lung endothelin receptor.

Endothelin receptor was purified from bovine lung by a rapid and simple two-step procedure: 1) solubilization with the detergent 3[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and digitonin and 2) affinity chromatography using biotinylated endothelin and avidin-agarose. Starting from 3.5 kg of bovine lung, about 200 micrograms of pure receptor were obtained. Microsequencing of tryptic fragments of the purified protein revealed a high sequence similarity with the rat endothelin ETB receptor that has very recently been cloned by expression cloning and shown to be nonselective in terms of the ligand specificity. Purification of the receptor in the presence of low (1 mM) and high (50 mM) concentrations of EDTA yielded, as a major form, 34- and 52-kDa species, respectively, indicating that the lower Mr species (34 kDa) is a proteolytic product of the 52-kDa species. Interestingly, this metal proteinase-mediated limited proteolysis did not affect the ligand binding properties of the receptor.     

Immunohistochemical mapping of endothelin in the developing and adult mouse lung.

Endothelins (ET) are a family of regulatory peptides displaying, among other abilities, potent constrictor actions. We studied the perinatal time course expression and distribution of ET in the mouse airway epithelium. In fetal mouse, ET-immunoreactivity (IR) appeared earlier (gestational Day 18) in the epithelium of upper (bronchi and large bronchioles) than in lower airways, being scarce and mainly located in the apical cytoplasm. As the lung developed, ET-IR became gradually stronger and extended throughout the cell in both bronchi and bronchioles. ET-IR was found in most airway epithelial cells. Clara cells were positive for ET, whereas ciliated and endocrine cells were not. In adult lungs, part of the myocytes and parenchymal cells also showed ET-IR. In both developing and adult mouse lungs, the cell distribution of ET-IR in the epithelium is compatible with apical and/or basal secretion. The presence of ET in mouse airway epithelium during the perinatal period may indicate a role for ET as a growth factor in lung development and its involvement in control of lung ventilation at birth.     

Regulation and intracellular trafficking pathways of the endothelin receptors

The effects of endothelin (ET) are mediated via the G protein-coupled receptors ET(A) and ET(B). However, the mechanisms of ET receptor desensitization, internalization, and intracellular trafficking are poorly understood. The aim of the present study was to investigate the molecular mechanisms of ET receptor regulation and to characterize the intracellular pathways of ET-stimulated ET(A) and ET(B) receptors. By analysis of ET(A) and ET(B) receptor internalization in transfected Chinese hamster ovary cells in the presence of overexpressed betaARK, beta-arrestin-1, beta-arrestin-2, or dynamin as well as dominant negative mutants of these regulators, we have demonstrated that both ET receptor subtypes follow an arrestin- and dynamin/clathrin-dependent mechanism of internalization. Fluorescence microscopy of Chinese hamster ovary and COS cells expressing green fluorescent protein (GFP)-tagged ET receptors revealed that the ET(A) and ET(B) subtypes were targeted to different intracellular routes after ET stimulation. While ET(A)-GFP followed a recycling pathway and colocalized with transferrin in the pericentriolar recycling compartment, ET(B)-GFP was targeted to lysosomes after ET-induced internalization. Both receptor subtypes colocalized with Rab5 in classical early endosomes, indicating that this compartment is a common early intermediate for the two ET receptors during intracellular transport. The distinct intracellular routes of ET-stimulated ET(A) and ET(B) receptors may explain the persistent signal response through the ET(A) receptor and the transient response through the ET(B) receptor. Furthermore, lysosomal targeting of the ET(B) receptor could serve as a biochemical mechanism for clearance of plasma endothelin via this subtype.     

Regulation of lung neutrophil recruitment by VE-cadherin

Lung inflammatory disease is characterized by increased polymorphonuclear leukocyte (PMN) infiltration and vascular permeability. PMN infiltration into tissue involves signaling between endothelial cells and migrating PMNs, which leads to alterations in the organization of adherens junctions (AJs). We addressed the possible role of the protein constituents of AJs, endothelium-specific vascular-endothelial (VE)-cadherin, in the migration of PMNs. Studies were made using VE-cadherin mutant constructs lacking the extracellular domain (DeltaEXD) or, additionally, lacking the COOH-terminus beta-catenin-binding domain (DeltaEXDDeltabeta). Either construct was transduced in pulmonary microvessel endothelia of mice using cationic liposome-encapuslated cDNA constructs injected intravenously. Optimal expression of constructs was seen by Western blot analysis within 24 h. Vessel wall liquid permeability measured as the lung microvessel capillary filtration coefficient increased threefold in DeltaEXD-transduced lungs, indicating patency of interendothelial junctions, whereas the control DeltaEXDDeltabeta construct was ineffective. To study lung tissue PMN recruitment, we challenged mice intraperitoneally with LPS (3 mg/kg) for 6 h and measured PMN numbers by bronchoalveolar lavage and their accumulation morphometrically in lung tissue. DeltaEXD expression markedly reduced the PMN sequestration and migration seen in nontransfected (control wild type) or DeltaEXDDeltabeta-transfected (negative control) mice challenged with LPS. In addition, DeltaEXD transfection suppressed LPS-induced activation of NF-kappaB and consequent ICAM-1 expression. These results suggest that disassembly of VE-cadherin junctions serves as a negative signal for limiting transendothelial PMN migration secondary to decreased ICAM-1 expression in the mouse model of LPS-induced sepsis.     

Regulation of fuel metabolism by preexercise muscle glycogen content and exercise intensity.

To date, the results of studies that have examined the effects of altering preexercise muscle glycogen content and exercise intensity on endogenous carbohydrate oxidation are equivocal. Differences in the training status of subjects between investigations may, in part, explain these inconsistent findings. Accordingly, we determined the relative effects of exercise intensity and carbohydrate availability on patterns of fuel utilization in the same subjects who performed a random order of four 60-min rides, two at 45% and two at 70% of peak O(2) uptake (Vo(2 peak)), after exercise-diet intervention to manipulate muscle glycogen content. Preexercise muscle glycogen content was 596 +/- 43 and 202 +/- 21 mmol/kg dry mass (P < 0.001) for high-glycogen (HG) and low-glycogen (LG) conditions, respectively. Respiratory exchange ratio was higher for HG than LG during exercise at both 45% (0.85 +/- 0.01 vs. 0.74 +/- 0.01; P < 0.001) and 70% (0.90 +/- 0.01 vs. 0.79 +/- 0.01; P < 0.001) of Vo(2 peak). The contribution of whole body muscle glycogen oxidation to energy expenditure differed between LG and HG for exercise at both 45% (5 +/- 2 vs. 45 +/- 5%; P < 0.001) and 70% (25 +/- 3 vs. 60 +/- 3%; P < 0.001) of Vo(2 peak). Yet, despite marked differences in preexercise muscle glycogen content and its subsequent utilization, rates of plasma glucose disappearance were similar under all conditions. We conclude that, in moderately trained individuals, muscle glycogen availability (low vs. high) does not influence rates of plasma glucose disposal during either low- or moderate-intensity exercise.     

Regulation of the carotenoid content and chloroplast development by levulinic acid

Chloroplast development and chlorophyll biosynthesis are co-regulated. To understand the mechanism of regulation of chloroplast biogenesis by chlorophyll, development of the photosynthetic apparatus was monitored during greening of etiolated barley leaf discs in the presence of levulinic acid, an inhibitor of chlorophyll biosynthesis. Although not a direct inhibitor of carotenoid biosynthesis, treatment by levulinic acid resulted in a linear reduction in both chlorophyll and carotenoid contents. Chlorophyll biosynthesis appeared to control that of carotenes. In the presence of levulinic acid, photosystem II (PSII) activity decreased while photosystem I (PSI) activity increased when expressed on a chlorophyll basis. However, the activities of both photosystem I and II decreased when expressed on a per plastid basis. As expected, in the presence of low amounts of chlorophyll, the light-harvesting chlorophyll-protein complex II (LHCPII) was not visible in Coomassie-stained gels in 20 m M levulinic acidtreated tissues, but was detected as a faint band by immunoblotting. This small amount of the LHCPII induced significant amounts of grana stacking, which was monitored as an increase in the ratio of variable to maximum fluorescence. When levulinic acid was washed from the leaf discs and the latter allowed to green in its absence, the chlorophyll and carotenoid contents and the photosynthetic activities approached the control values. Levulinic acid could be used to arrest the light-induced chloroplast development at a desired phase of greening and removed by washing the leaves to restore the developmental process without any apparent toxic effect. Results demonstrate that biosynthesis of carotenes is regulated by that of chlorophylls and extremely low amounts of the LHCPII can induce grana stacking.     

Nitric oxide and endothelin secretion by brain microvessel endothelial cells: Regulation by cyclic nucleotides

Endothelin (ET)-1 was originally characterized as a potent vasoconstrictor peptide secreted by vascular endothelial cells. It possesses a wide range of biological activities within the cardiovascular system and in other organs, including the brain. Also secreted by endothelial cells, nitric oxide (NO), has recently been identified as a relaxing factor, as well as a pleiotropic mediator, second messenger, immune defence molecule, and neurotransmitter. Most of the data concerning the secretion of these two agents in vitro has been collected from studies on macrovascular endothelial cells. Given the remarkable heterogeneity of endothelia in terms of morphology and function, we have analyzed the ability of brain microvessel endothelial cells in vitro to release ET-1 and NO, which, at the level of the blood-brain barrier, have perivascular astrocytes as potential targets. The present study was performed with immortalized rat brain microvessel endothelial cells, which display in culture a non transformed phenotype. Our data demonstrate that: (1) these cells release NO when induced by IFNγ and TNFα, (2) they constitutively secrete ET-1, and (3) cAMP potentiates the cytokine-induced NO release and exerts a biphasic regulation on ET-1 secretion: micromolar concentrations of 8-Br-cAMP inhibit and higher doses stimulate ET-1 secretion. This stimulation is blocked by EGTA and the calmodulin antagonist W7, but not by protein kinase C inhibitors, suggesting the involvement of the calmodulin branch of the calcium messenger system. These results suggest that cerebral microvessel endothelial cells may participate in vivo to the regulation of glial activity in the brain through the release of NO and ET-1. © 1993 Wiley-Liss, Inc.     

Regulation of lung surfactant secretion by microRNA-150

P2X7 receptor (P2X7R) is a purinergic ion-channel receptor. We have previously shown that the activation of P2X7R in alveolar type I cells stimulates surfactant secretion in alveolar type II cells. In this study, we determined whether miR-150 regulates P2X7R-mediated surfactant secretion. The miR-150 expression level in alveolar type II cells was much higher than alveolar type I cells, which was inversely correlated with the P2X7R protein level. An adenovirus expressing miR-150 significantly reduced the P2X7R protein expression in E10 cells, an alveolar type I cell line. Furthermore, pre-treatment of E10 cells with the adenovirus reduced the surfactant secretion induced by E10 cell conditioned medium. Our study demonstrates that miR-150 regulates surfactant secretion through P2X7R.     

Chromatographic characterization of immunoreactive endothelin in rat lung

Using a specific and sensitive radioimmunoassay for endothelin, combined with gel filtration and reverse phase high performance liquid chromatography, the molecular form of immunoreactive endothelin in the rat lung was investigated. On reverse phase high performance chromatography, the major immunoreactive endothelin in the rat lung emerged at a position identical to that of authentic endothelin-1. After oxidation of the immunoreactive endothelin by H2O2, the immunoreactivity migrated to a position identical to that of Met-sulfoxide endothelin-1. These data indicate that the major immunoreactive endothelin in the rat lung is not endothelin-3 (putative rat/human endothelin), but is identical or very similar to endothelin-1 (porcine/human endothelin).     

Improvement in lung diffusion by endothelin A receptor blockade at high altitude

Lung diffusing capacity has been reported variably in high-altitude newcomers and may be in relation to different pulmonary vascular resistance (PVR). Twenty-two healthy volunteers were investigated at sea level and at 5,050 m before and after random double-blind intake of the endothelin A receptor blocker sitaxsentan (100 mg/day) vs. a placebo during 1 wk. PVR was estimated by Doppler echocardiography, and exercise capacity by maximal oxygen uptake (Vo(2 max)). The diffusing capacities for nitric oxide (DL(NO)) and carbon monoxide (DL(CO)) were measured using a single-breath method before and 30 min after maximal exercise. The membrane component of DL(CO) (Dm) and capillary volume (Vc) was calculated with corrections for hemoglobin, alveolar volume, and barometric pressure. Altitude exposure was associated with unchanged DL(CO), DL(NO), and Dm but a slight decrease in Vc. Exercise at altitude decreased DL(NO) and Dm. Sitaxsentan intake improved Vo(2 max) together with an increase in resting and postexercise DL(NO) and Dm. Sitaxsentan-induced decrease in PVR was inversely correlated to DL(NO). Both DL(CO) and DL(NO) were correlated to Vo(2 max) at sea level (r = 0.41-0.42, P < 0.1) and more so at altitude (r = 0.56-0.59, P < 0.05). Pharmacological pulmonary vasodilation improves the membrane component of lung diffusion in high-altitude newcomers, which may contribute to exercise capacity.     

Characterization of endothelin converting enzyme in rat lung

An enzyme activity which converts human big endothelin (1-38) to endothelin (1-21) and a C-terminal fragment (CTF, 22-38) was identified in a plasma membrane fraction prepared from rat lung. The conversion activity was optimal at pH 4.0, was inhibited by Pepstatin-A (IC50 = 20 nM), but was not affected by TLCK, Aprotinin, PMSF, E-64, Bestatin, Phosphoramidon or Thiorphan at 40 microM. Metal ions activated the activity by 1.5 - 2.5 fold in the order of Mn+2 greater than Zn+2 = Ca+2 greater than Ba+2. These data suggest that a Pepstatin-A inhibitable, metal ion related aspartic protease may be involved in the conversion of big endothelin to endothelin in rat lung.     

Regulation of tryptase from human lung mast cells by heparin. Stabilization of the active tetramer

Tryptase was shown to be stabilized as an enzymatically active tetramer by association with heparin and dissociated to inactive monomers in the absence of heparin at 37 degrees C in physiologic buffer and in plasma. There was a 50% loss of tryptase activity at 37 degrees C by 6-8 min in both physiologic buffer and plasma. When heparin glycosaminoglycan was present, tryptase retained nearly full activity for 2 h in buffer and in plasma. Tryptase activity also decayed under standard assay conditions in the presence of synthetic ester and peptide substrates unless bound to heparin. That tryptase is bound to heparin at the pH and physiologic NaCl concentrations employed was shown by chromatography of tryptase on heparin-agarose, gel filtration, and velocity sedimentation. Elution of tryptase from heparin-agarose occurred at 0.8 M NaCl. Maximal stabilization of tryptase by heparin occurred at a weight ratio to tryptase that was equal to or greater than unity. Kcat/Km ratios for tryptase-heparin at 0.15 M NaCl and 37 degrees C were 0.9 X 10(6) s-1 M-1 for tosyl-L-Gly-Pro-Lys-p-nitroanilide and 1.7 X 10(6) s-1 M-1 for p-tosyl-L-arginine methyl ester and are among the highest reported for tryptic enzymes. The mechanism of heparin-dependent stabilization of tryptase was not due to indirect ion binding properties of heparin and was analyzed by Superose 12 high performance liquid chromatography. Active enzyme eluted with an apparent Mr of 132,000 +/- 10,000 (n = 3, +/- S.D.), whereas tryptase inactivated by incubation without heparin eluted with an apparent Mr of 34,000. The tetrameric structure of diisopropyl fluorophosphate-inhibited tryptase was also preserved after incubation with heparin at 37 degrees C but was reduced to monomeric subunits after incubation without heparin. That no appreciable degradation of tryptase occurs under conditions that cause dissociation of subunits was directly shown by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Two different subunits of 34,000 and 33,000 Mr (after reduction) present in the intact enzyme (calculated to be 134,000 Mr) were also detected unchanged after inactivation of tryptase by dissociation of its subunits. Thus, the selective localization and association of heparin and tryptase in the human mast cell secretory granule most likely plays a major role in the regulation of tryptase after secretion.