1 Effects of porcine/human endothelin (endothelin-1), a novel vasoconstrictor peptide, on various smooth muscles were examined. 2 In rat aorta, endothelin (1 pM-30nM) induced contraction in a concentration-dependent manner. Removal of endothelium shifted the concentration-response curve to the left. When added during the sustained contraction induced by 0.1 FM noradrenaline, endothelin (1 nM) induced a relaxation that was inhibited by removing endothelium or by methylene blue.3 In rat aorta without endothelium, endothelin (1-30 nM) increased cytosolic Ca2" level ([Ca2"] y followed by contraction. Endothelin induced less contraction than high K' at a given [Ca2"]cyt when the concentration of endothelin was lower (1-3 nM) and/or during the early phase of the contraction (10 nM).

Abstract-G2A is a stress-inducible G protein-coupled receptor that is expressed on several cell types within atherosclerotic lesions. We demonstrated previously that G2A deficiency in mice increased aortic monocyte recruitment and increased monocyte:endothelial interactions. To investigate the impact of G2A deficiency in macrophages, we isolated peritoneal macrophages from G2Aϩ/ϩ ApoE Ϫ/Ϫ and G2A Ϫ/Ϫ ApoE Ϫ/Ϫ mice. G2A Ϫ/Ϫ ApoE Ϫ/Ϫ macrophages had significantly lower apoptosis than control macrophages. The prosurvival genes BCL-2, BCL-xL, and cFLIP were increased in G2A Ϫ/Ϫ ApoE Ϫ/Ϫ macrophages. Macrophages from G2A Ϫ/Ϫ ApoE Ϫ/Ϫ mice also had increased proinflammatory status that was indicative of a M1 macrophage phenotype. This was indicated by significantly increased nuclear translocation of nuclear factor B, as well as production of interleukin-12p40, tumor necrosis factor ␣, and interleukin-6, and reduced expression of arginase-I. Moreover, G2AϪ/Ϫ ApoE Ϫ/Ϫ macrophages had reduced ability to engulf apoptotic cells in vitro. We examined atherosclerosis in mice fed a Western diet for 10 weeks and found that G2A deficiency increased lesion size in the aortic root by 50%. Plasma lipid levels were not changed in G2A ApoEϪ/Ϫ mice. However, we found that absence of G2A increased the number of aortic macrophages and attenuated apoptosis in this cell type. Moreover, bone marrow transplantation studies indicated that deficiency of G2A in marrow-derived cells significantly contributed to atherosclerosis development. In the absence of G2A, increased macrophage activation and decreased apoptosis is associated with accumulation of macrophages in the aorta and increased atherosclerosis. Key Words: apoptosis Ⅲ macrophages Ⅲ vascular inflammation Ⅲ atherosclerosis M acrophages and T lymphocytes play critical roles in the initiation and development of atherosclerosis. 1 Activated endothelium recruits monocytes by secretion of chemoattractants, after which monocytes bind to and subsequently transmigrate though the endothelial layer. [2][3][4][5][6] In addition to macrophages, CD4ϩ lymphocytes are detected in early atherogenesis and late-stage unstable atherosclerotic lesions, consistent with a role for acquired immunity in lesion development. [7][8][9] Lesional lymphocytes secrete the inflammatory cytokine interferon-␥, which further activate macrophages and vascular cells. 10 -13 The G protein-coupled receptor G2A is a stressinducible receptor. Overexpression in fibroblasts causes cell cycle arrest at the G2 phase of mitosis, thus the name G2A for G2 Accumulation. 14 G2A expression attenuates Bcr-Abl oncogene-mediated cell proliferation, whereas mice lacking G2A have an increased mortality rate in an oncogene-induced model of leukemia. 15 The endogenous ligand for G2A is unknown, although putative ligands include lysophosphatidylcholine, 16,17 9(S)HODE, 18 and possibly other free fatty acids. 18 G2A and other receptors within the OGR1 family (TDAG8, GPR4, and OGR1) respond to changes in extracellular pH. 19 However G2A is le...

Effects of stimulants and relaxants on the cytosolic Ca2+ level [(Ca2+]cyt) and contraction were examined in isolated canine tracheal smooth muscle. High K+ and carbachol induced a sustained increase in [Ca2+]cyt and muscle tension. Cumulative addition of KCl induced a graded increase in [Ca2+]cyt and muscle tension. Cumulative addition of carbachol induced greater contraction than high K+ at a given [Ca2+]cyt 12-Deoxyphorbol 13-isobutyrate (DPB) (50 nmol/l) induced a small sustained contraction with little effect on [Ca2+]cyt. A higher concentration (1 mumol/l) of DPB induced a larger sustained contraction with a decrease in [Ca2+]cyt. DPB (50 nmol/l) potentiated the KCl-induced contraction without or with only a small additional increase in [Ca2+]cyt. By contrast, 1 mumol/l DPB potentiated the high-K(+)-induced contraction with a decrease in [Ca2+]cyt. Addition of 50 nmol/l or 1 mumol/l DPB in the presence of carbachol inhibited both [Ca2+]cyt and muscle tension. Verapamil, isoprenaline and forskolin did not change or slightly decreased [Ca2+]cyt and muscle tension in resting trachea. Verapamil inhibited the contraction and [Ca2+]cyt stimulated by high K+ and carbachol. Isoprenaline and forskolin inhibited the high-K(+)-induced contraction without changing [Ca2+]cyt, whereas these inhibitors inhibited carbachol-induced contraction with a relatively small decrease in [Ca2+]cyt. These results suggest that (a) sustained contractions induced by high K+ and carbachol are due to the sustained increase in [Ca2+]cyt, (b) carbachol increases the sensitivity of contractile elements to Ca2+, and (c) isoprenaline and forskolin inhibit the contraction by the decrease in [Ca2+]cyt and also by the decrease in the sensitivity of contractile elements to Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

1 Xestospongin-C isolated from a marine sponge, Xestospongia sp., has recently been shown to be a membrane-permeable IP 3 receptor inhibitor. In this study we examined the e ects of this compound on smooth muscle from guinea-pig ileum. 2 In guinea-pig ileum permeabilized with a-toxin, xestospongin-C (3 mM) inhibited contractions induced by Ca 2+ mobilized from sarcoplasmic reticulum (SR) with IP 3 or carbachol with GTP, but not with ca eine. 3 In intact smooth muscle tissue, xestospongin-C (3 ± 10 mM) inhibited carbachol-and high-K + -induced increases in [Ca 2+ ] i and contractions at sustained phase. 4 It also inhibited voltage-dependent inward Ba 2+ currents in a concentration-dependent manner with an IC 50 of 0.63 mM. Xestospongin-C (3 ± 10 mM) had no e ect on carbachol-induced inward Ca 2+ currents via non-selective cation channels; but it did reduce voltage-dependent K + currents in a concentration-dependent manner with an IC 50 of 0.13 mM. 5 These results suggest that xestospongin-C inhibits the IP 3 receptor but not the ryanodine receptor in smooth muscle SR membrane. In intact smooth muscle cells, however, xestospongin-C appears to inhibit voltage-dependent Ca 2+ and K + currents at a concentration range similar to that at which it inhibits the IP 3 receptor. Xestospongin-C is a selective blocker of the IP 3 receptor in permeabilised cells but not in cells with intact plasma membrane.

Dexmedetomidine, a full agonist of the α2B-adrenoceptor that is mainly involved in vascular smooth muscle contraction, is primarily used for analgesia and sedation in intensive care units. High-dose dexmedetomidine produces hypertension in children and adults. The goal of this in vitro study was to investigate the role of the calcium (Ca(2+)) sensitization mechanism involving Rho-kinase, protein kinase C (PKC), and phosphoinositide 3-kinase (PI3-K) in mediating contraction of isolated rat aortic smooth muscle in response to dexmedetomidine. The effect of dexmedetomidine on the intracellular Ca(2+) level ([Ca(2+)]i) and tension was measured simultaneously. Dexmedetomidine concentration-response curves were generated in the presence or absence of the following antagonists: rauwolscine, Y 27632, LY 294002, GF 109203X, and verapamil. Dexmedetomidine-induced phosphorylation of PKC and membrane translocation of Rho-kinase were detected with Western blotting. Rauwolscine, Y 27632, GF 109203X, LY 294002, and verapamil attenuated dexmedetomidine-induced contraction. The slope of the [Ca(2+)]i-tension curve for dexmedetomidine was higher than that for KCl. Dexmedetomidine induced phosphorylation of PKC and membrane translocation of Rho-kinase. These results suggest that dexmedetomidine-induced contraction involves a Ca(2+) sensitization mechanism mediated by Rho-kinase, PKC, and PI3-K that is secondary to α2-adrenoceptor stimulation in rat aortic smooth muscle.

This study investigated endothelium-derived vasodilators and potassium channels involved in the modulation of ropivacaine-induced contraction. In endothelium-intact rat aortae, ropivacaine concentration-response curves were generated in the presence or absence of the following inhibitors: the nonspecific nitric oxide synthase (NOS) inhibitor N ω-nitro-L-arginine methyl ester (L-NAME), the neuronal NOS inhibitor N ω-propyl-L-arginine hydrochloride, the inducible NOS inhibitor 1400W dihydrochloride, the nitric oxide-sensitive guanylyl cyclase (GC) inhibitor ODQ, the NOS and GC inhibitor methylene blue, the phosphoinositide-3 kinase inhibitor wortmannin, the cytochrome p450 epoxygenase inhibitor fluconazole, the voltage-dependent potassium channel inhibitor 4-aminopyridine (4-AP), the calcium-activated potassium channel inhibitor tetraethylammonium (TEA), the inward-rectifying potassium channel inhibitor barium chloride, and the ATP-sensitive potassium channel inhibitor glibenclamide. The effect of ropivacaine on endothelial nitric oxide synthase (eNOS) phosphorylation in human umbilical vein endothelial cells was examined by western blotting. Ropivacaine-induced contraction was weaker in endothelium-intact aortae than in endothelium-denuded aortae. L-NAME, ODQ, and methylene blue enhanced ropivacaine-induced contraction, whereas wortmannin, N ω-propyl-L-arginine hydrochloride, 1400W dihydrochloride, and fluconazole had no effect. 4-AP and TEA enhanced ropivacaine-induced contraction; however, barium chloride and glibenclamide had no effect. eNOS phosphorylation was induced by ropivacaine. These results suggest that ropivacaine-induced contraction is attenuated primarily by both endothelial nitric oxide and voltage-dependent potassium channels.

ABSTRACT. Endothelin (ET), derived from the endothelium of blood vessels, is a potent vasoactive peptide. Although it has been reported to be involved in cardiovascular diseases, such as hypertension, the mechanism by which ET evokes vasoconstriction is still unclear. On the other hand, p42/p44 mitogen-activated protein kinase (MAPK) and p38 MAPK are activated by a variety of growth factors and cellular stresses, respectively. However, the role of p42/p44 MAPK and p38 MAPK on the ET-1-induced vasoconstriction is not fully understood. This study was undertaken to determine whether p42/p44 MAPK and p38 MAPK participate in the regulation of vascular smooth muscle contraction by ET-1. The isometric vasoconstriction and intracellular Ca 2+ ([Ca 2+ ] i ) were simultaneously measured using CAF-100. Phosphorylation of myosin light chain (MLC) and p42/p44 MAPK, p38 MAPK were determined by Western blots. In rat thoracic aorta, ET-1 induced a sustained contraction. In contrast, [Ca 2+ ] i was decreased with time. Both PD98059, an inhibitor of p42/ p44 MAPK, and SB203580, an inhibitor of p38 MAPK, partially attenuated ET-1-induced contractions in concentration-dependent manners. ET-1 increased phosphorylation of both p42/p44 MAPK and p38 MAPK, and PD98059 and SB203580 completely decreased phosphorylation of p42/p44 MAPK and p38 MAPK in response to ET-1 stimulation, respectively. On the other hand, PD98059 and SB203580 did not affect MLC phosphorylation in response to ET-1 stimulation. These results indicate that p38 MAPK, as well as p42/ p44 MAPK, may partially regulate the ET-1-induced contraction through a MLC phosphorylation-independent pathway. KEY WORDS: endothelin, mitogen-activated protein kinase, vasoconstriction.J. Vet. Med. Sci. 65(2): 225-230, 2003 Endothelin (ET) is a potent vasoactive peptide particularly derived from the endothelium of blood vessels [38]. ET is composed of 21-amino acids, and is classified into ET-1, ET-2 and ET-3 according to their amino acid composition [19]. ET-1 is the major ET generated in the endothelium. ET acts via specific plasma membrane receptors. ET A and ET B receptors have distinctive characteristics for ET [1,32]. ET A and ET B receptors on smooth muscle induce contraction and stimulate proliferation and cell hypertrophy [6]. Endothelial ET B receptors stimulate the production of nitric oxide and prostacyclin [33]. ET is known to be involved in cardiovascular diseases, including hypertension, myocardial infarction, congestive heart failure, restenosis and atherosclerosis [24].Many studies have evaluated ET-induced vasoconstriction. ET-1 evokes a long-lasting contraction, slow in onset, of isolated arteries, veins and in microcirculatory vessels from experimental animals and humans [2,4,25,38]. The mechanism by which contractions are evoked by ET-1 in isolated vascular preparations is complicated. The increase in the sensitivity of the contractile apparatus to intracellular Ca 2+ ([Ca 2+ ] i ), as well as the increase of [Ca 2+ ] i concentration, may be involved in ET-1-...

Lipid emulsions are widely used for the treatment of systemic toxicity that arises from local anesthetics. The goal of this in vitro study was to examine the cellular mechanism associated with the lipid emulsion-mediated attenuation of vasodilation induced by a toxic dose of bupivacaine in isolated endothelium-denuded rat aorta. The effects of lipid emulsion on vasodilation induced by bupivacaine, mepivacaine, and verapamil were assessed in isolated aorta precontracted with phenylephrine, the Rho kinase stimulant NaF, and the protein kinase C activator phorbol 12,13-dibutyrate (PDBu). The effects of Rho kinase inhibitor Y-27632 on contraction induced by phenylephrine or NaF were assessed. The effects of bupivacaine on intracellular calcium concentrations ([Ca2+]i) and tension induced by NaF were simultaneously measured. The effects of bupivacaine alone and lipid emulsion plus bupivacaine on myosin phosphatase target subunit 1 (MYPT1) phosphorylation induced by NaF were examined in rat aortic vascular smooth muscle cells. In precontracted aorta, the lipid emulsion attenuated bupivacaine-induced vasodilation but had no effect on mepivacaine-induced vasodilation. Y-27632 attenuated contraction induced by either phenylephrine or NaF. The lipid emulsion attenuated verapamil-induced vasodilation. Compared with phenylephrine-induced precontracted aorta, bupivacaine-induced vasodilation was slightly attenuated in NaF-induced precontracted aorta. The magnitude of the bupivacaine-induced vasodilation was higher than that of a bupivacaine-induced decrease in [Ca2+]i. Bupivacaine attenuated NaF-induced MYPT1 phosphorylation, whereas lipid emulsion pretreatment attenuated the bupivacaine-induced inhibition of MYPT1 phosphorylation induced by NaF. Taken together, these results suggest that lipid emulsions attenuate bupivacaine-induced vasodilation via the attenuation of inhibition of MYPT1 phosphorylation evoked by NaF.