endothelin-1 and barium-chloride

The purpose of the present study was to investigate the effect of imperatorin on vasodilatation and its possible mechanisms. Isometric tension of rat mesenteric arterial rings was recorded by a myograph system in vitro. The results showed that imperatorin at more than 10 muM concentration-dependently relaxed rat mesenteric arteries pre-contracted by potassium chloride (KCl) and endothelin-1, and human omental arteries pre-contracted by noradrenaline and U46619. Removal of the endothelium did not affect imperatorin-induced relaxant responses, suggesting that the vasodilatation effect is independent of the endothelium. Co-incubation with imperatorin resulted in rightward shift of concentration-response curves of KCl, calcium chloride (CaCl(2)) and noradrenaline in a non-parallel manner; 5-hydroxytryptamine (5-HT) concentration-response curves were shifted towards right in a parallel manner by imperatorin 10 and 30 muM, but markedly suppressed by imperatorin 100 muM. These results suggest that the inhibitory effect of imperatorin is mainly via voltage dependent calcium channel and possibly receptor operated calcium channel. beta-adrenoceptor, ATP-sensitive potassium channel and inwardly rectifying potassium channel were not involved in the vasodilatation, whereas blockage of calcium-activated potassium channel with tetraethylammonium had effect. Furthermore, in Ca(2+)-free medium, imperatorin concentration-dependently depressed the vasoconstrictions derived from noradrenaline and CaCl(2), and resulted in a decreased contractile response induced by caffeine, indicating a role of inhibiting extracellular Ca(2+) influx and intracellular Ca(2+) release from Ca(2+) store. Taken together, our results suggest that imperatorin induces vasodilatation by possible mechanisms inhibiting voltage dependent calcium channel and receptor-mediated Ca(2+)influx and Ca(2+)release. Opening calcium-activated potassium channel and competitive antagonism of 5-HT receptors may also contribute to this vasodilatation effect.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Arteries; Barium Compounds; Caffeine; Calcium; Calcium Channels; Calcium Chloride; Chlorides; Dimethyl Sulfoxide; Dose-Response Relationship, Drug; Endothelin-1; Endothelium, Vascular; Epinephrine; Furocoumarins; Glyburide; In Vitro Techniques; Mesenteric Arteries; Omentum; Potassium Chloride; Propranolol; Rats; Rats, Sprague-Dawley; Serotonin; Tetraethylammonium; Vasodilation

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide that is also known to induce a wide spectrum of biological responses in nonvascular tissue. In this study, we found that ET-1 (100 nM) inhibited the glutamate uptake in cultured astrocytes expressing the glutamate/aspartate transporter (GLAST); astrocytes did not express the glutamate transporter-1 (GLT-1). The V(max) and the K(m) of the glutamate uptake were reduced by 57% and 47%, respectively. Application of the ET(A) and ET(B) receptor antagonists BQ-123 and BQ-788 partly inhibited the ET-1-evoked decrease in the glutamate uptake, whereas the nonspecific ET receptor antagonist bosentan completely inhibited this decrease. Incubation of the cultures with pertussis toxin abolished the effect of ET-1 on the uptake. The ET-1-induced decrease in the glutamate uptake was independent of extracellular free Ca(2+) concentration, whereas the intracellular Ca(2+) antagonists thapsigargin and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester abolished the effect of ET-1 on the glutamate uptake. Incubation with the protein kinase C (PKC) antagonist staurosporine, but not with the fatty acid-binding protein bovine serum albumin, prevented the ET-1-induced decrease in the glutamate uptake. These results suggest that ET-1 impairs the high-affinity glutamate uptake in cultured astrocytes through a G protein-coupled mechanism, involving PKC and changes in intracellular Ca(2+).

Topics: Amino Acid Sequence; Animals; Astrocytes; Barium Compounds; Blotting, Western; Cells, Cultured; Cerebral Cortex; Chlorides; Depression, Chemical; Endothelin-1; Glutamic Acid; Immunohistochemistry; Molecular Sequence Data; Rats; Rats, Sprague-Dawley; Transduction, Genetic

Physiological fluid shear stress regulates endothelin-1 (ET-1) gene expression in endothelial cells by inducing an early transient upregulation followed by a sustained suppression, at times greater than 2 h in duration. We evaluated the mechanism of ET-1 mRNA downregulation in confluent monolayers of bovine aortic endothelial (BAE) cells by applying a 6 h steady laminar shear stress of magnitude 20 dyn/cm2. Inhibition of tyrosine kinases using herbimycin A (875 nM) abolished the shear-induced decrease in ET-1 mRNA expression. Similarly, chelation of intracellular calcium ([Ca2+]i) with quin 2-AM (10 microM) blocked the suppression of ET-1 mRNA by shear. To examine the role of the endothelial cytoskeleton in the response to flow, cytochalasin D was used to disrupt F-actin microfilaments. This treatment induced cell retraction and detachment under flow, whereas stabilization of F-actin with phalloidin (1 microM) did not affect shear-induced ET-1 downregulation. In contrast, disruption of the microtubule network with nocodazole (10 micrograms/ml) completely prevented, while microtubule stabilization with taxol (10 microM) did not affect the suppression of ET-1 mRNA by flow. To determine the possible contributions of mechanosensitive channels, barium (1 mM BaCl2), was added to confluent BAE monolayers in a low-sulfate/low-phosphate modified medium and was noted to abrogate the downregulation of ET-1 gene expression and to attenuate the shear-induced increase in cytoplasmic free calcium concentration. Tetraethylammonium (3 mM TEA) partially inhibited the suppression of ET-1 mRNA by shear; in contrast, gadolinium (10 microM GdCl3), an inhibitor of the stretch-activated cation channel ISA, had no effect. Membrane depolarization by elevated extracellular potassium ([K+]o) also attenuated the suppression of ET-1 mRNA by flow at [K+]o = 70 mM and completely inhibited it at [K+]o = 135 mM. In summary, the steady-state downregulation of ET-1 mRNA by physiological levels of fluid shear stress shares signaling features with the morphological and cytoskeletal response to shear stress. These include requirement for intracellular calcium, tyrosine kinase activity, an intact microtubule network, and independence from a Gd(3+)-sensitive ISA. Unlike shear-induced changes in cell morphology and the actin cytoskeleton, the shear-induced decrease in ET-1 mRNA level is blocked by cell depolarization and by Ba2+, a blocker of the shear-activated IKS which also decreases shear-ind

Topics: Actins; Animals; Barium Compounds; Calcium; Cattle; Chlorides; Cytoskeleton; Down-Regulation; Endothelin-1; Gadolinium; Gene Expression Regulation; Intracellular Fluid; Microtubules; Nitric Oxide Synthase; Protein-Tyrosine Kinases; RNA, Messenger; Tetraethylammonium