Endothelin-induced prostacyclin production in rat aortic endothelial cells: role of calcium.

Endothelin (ET) is a potent vasoconstrictor peptide, released from endothelial cells, which is associated with prostaglandin (PG) release. The mechanism by which ET causes the release of PG is not clearly understood. We used rat aortic endothelial cells to investigate the role of calcium (Ca2+) in ET-1-induced prostacyclin (PGI2) release. ET-1 (10(-9) M) produced a significant increase in PGI2 release. Pretreatment of rat aortic endothelial cells with different doses (10(-9) M and 10(-6) M) of diltiazem (voltage-sensitive L-type calcium channel blocker) produced significant inhibition of ET-1- and PDBu-induced PGI2 release. Inhibition was first noted at 10(-9) M and was complete at 10(-6) M. Conversely, pretreatment of rat aortic endothelial cells with different doses (10(-9) M and 10(-6) M) of calcium channel blockers (thapsigargin, an intracellular calcium channel blocker or conotoxin, a voltage-sensitive N-type calcium channel blocker) produced no changes on ET-1- or PDBu-induced PGI2 release. These results provide further support for the concept that PKC mediates ET-induced PGI2 release in rat aortic endothelial cells via an increase in intracellular calcium and this increase is due to the influx of extracellular calcium and not to the release of calcium from the sarcoplasmic reticulum.     

Endothelin-1 attenuates increases in hydraulic conductivity due to platelet-activating factor via prostacyclin release

We previously showed that endothelin-1 (ET-1) and prostacyclin (PGI(2)) similarly attenuate increases in microvascular permeability induced by platelet-activating factor (PAF). This led us to hypothesize that ET-1 attenuates trans-endothelial fluid flux during PAF through PGI(2) release. We tested this hypothesis in three phases. First, bovine pulmonary artery endothelial cells were exposed to 0.008-8 μM ET-1 and assayed for PGI(2) release. Second, to determine whether increased transmonolayer flux after PAF could be attenuated by ET-1 or PGI(2) and reversed by PGI(2) synthesis inhibition or PGI(2) receptor blockade, we measured endothelial cell transmonolayer flux after cells were exposed to 10 nM PAF plus 10 μM PGI(2) or 80 pM ET-1, with or without 500 μM tranylcypromine (PGI(2) synthase inhibitor) or 20 μM CAY-10441 (PGI(2) receptor blocker). Finally, hydraulic conductivity (L(p)) was measured in rat mesenteric venules in vivo after exposure to 10 nM PAF and 80 pM ET-1 with or without tranylcypromine (100 and 500 μM) or CAY-10441 (2 and 20 μM). We found that in vitro, ET-1 stimulated a dose-dependent increase in PGI(2) production (from 126 to 217 pg/ml, P < 0.01). Compared with PAF alone, PGI(2) plus PAF and ET-1 plus PAF decreased transmonolayer flux similarly by 52 and 46%, respectively (P < 0.01), while tranylcypromine and CAY-10441 reversed these effects by 92 and 47%, respectively (P < 0.05). In vivo, PAF increased L(p) fourfold (P < 0.01) and ET-1 attenuated this effect by 83% (P < 0.01). Tranylcypromine and CAY-10441 reversed the ET-1 attenuation in L(p) during PAF by 55 and 45%, respectively (P < 0.01). We conclude that ET-1 may stimulate endothelial cell PGI(2) release to attenuate the increases in transmonolayer flux and hydraulic conductivity secondary to PAF.     

Comparison of the release of vasoactive factors from venous and arterial bovine cultured endothelial cells.

The release of endothelium-derived relaxing factor (EDRF), prostacyclin (PGI2), and endothelin-1 (ET-1) was measured from endothelial cells (EC) cultured from either bovine vena cava (BVCEC) or bovine aorta (BAEC). EDRF release was determined by using the superfusion bioassay technique, whereas ET-1 and PGI2 were measured by specific radioimmunoassays. Bradykinin (BK) (0.05-30 pmol) given through columns of venous or arterial EC induced a dose-dependent release of EDRF. BK (0.05 pmol) evoked a release of EDRF from venous EC that was similar to the effect of a dose of 1 pmol from arterial EC. As with BAEC, infusions of NG-monomethyl-L-arginine (30 microM) caused an inhibition of EDRF release from BVCEC that was partially reversed by coinfusions of L-arginine (L-Arg; 100 microM). BK also induced a dose-dependent release of PGI2 from BVCEC. BVCEC and BAEC produced PGI2 in equivalent amounts when arachidonic acid (9.2 and 32 pmol) was added to the Krebs' solution perfusing the cells. BVCEC and BAEC released detectable amounts of ET-1 (0.4 +/- 0.1 and 0.9 +/- 0.3 ng/mL, respectively), over a 4-h period, and the release of ET-1 was increased approximately twofold by coincubations with thrombin (0.05-1 U/mL). These findings demonstrate that venous EC have a similar capacity to arterial EC to release vasoactive factors, thus supporting the hypothesis that veins have a functional endothelium that may modulate venous tone and platelet function.     

Neuropeptide Y stimulates prostacyclin production in porcine vascular endothelial cells

We investigated the effects of neuropeptide Y on the prostacyclin production of cultured porcine aortic endothelial cells by measuring the stable metabolite of prostacyclin, 6-keto-prostaglandin F1 alpha, by radioimmunoassay. Neuropeptide Y induced dose- and time-dependent stimulation of prostacyclin production by cultured porcine aortic endothelial cells. The lowest stimulatory concentration of neuropeptide Y was 10(-8) M and maximal response, a 2.8 fold rise, was obtained with 10(-6) M. The stimulation lasted at least 24 h. The effect was associated with the stimulation of arachidonic acid release. Our data suggest that neuropeptide Y may inhibit the development of atherosclerosis by stimulating prostacyclin synthesis.     

Effects of endothelins on signal transduction and proliferation in human melanocytes

We demonstrate here that human melanocytes could be regulated by endothelin (ET) derivatives, potent vasoconstrictive peptides synthesized by endothelial cells, to stimulate their proliferation and melanization via a receptor-mediated signal transduction pathway. Receptor-binding assay using [125I]ET indicated that unlabeled ET-1 or ET-2 competitively inhibited each binding of labeled ETs to melanocytes with a concentration for half-maximal inhibition (IC50) of 0.7 or 0.9 nM, respectively. The dissociation constant (Kd) and the number of sites of the specific bindings of ET-1 and those of ET-2 were almost the same (Kd: 1.81 nM, binding sites: 7.0-8.0 x 10(4) per cell). Upon incubation with cultured cells, the mass contents of inositol 1,4,5-trisphosphate and intracellular calcium level were substantially increased by 10 nM ET-1, ET-2, and ET-3, but not by big-ET with maximal response at 80-130-s postincubation. The addition of ET-1 and ET-2 at 1-50 nM concentrations caused human melanocytes to significantly stimulate DNA [( 3H]thymidine incorporation) and melanin synthesis (3H2O release and [14C] thiouracil incorporation). Furthermore, ETs exhibited an additive stimulatory effect on basic fibroblast growth factor-stimulated DNA synthesis. In a long-term serum-free culture system, the strongest stimulation of growth by 10 nM ET-1 or ET-2 was observed in the presence of 10 nM cholera toxin and 0.2% bovine pituitary extract, resulting in a 4.5-fold increase in cell number for 12 culture days. These findings strongly suggest involvement of ET in the mechanism regulating proliferation and melanization of human melanocytes.     

Enrichment of endothelial cell arachidonate by lipid transfer from high density lipoproteins: relationship to prostaglandin I2 synthesis

We have previously shown that plasma high density lipoproteins (HDL) stimulate release of prostacyclin, measured as its stable metabolite, 6-keto-PGF1 alpha, by cultured porcine aortic endothelial cells. The present experiments were designed to elucidate the contribution of HDL lipids to endothelial cellular phospholipid pools and to prostacyclin synthesis. In experiments with reconstituted HDL, both the lipid and protein moieties were required to stimulate prostacyclin release in amounts equivalent to the native HDL particle. Endothelial cells incorporated label from reconstituted HDL containing cholesteryl [1-14C]arachidonate into the cellular neutral and phospholipid pools as well as into 6-keto-PGF1 alpha and PGE2. Labeled arachidonate incorporated into endothelial cell lipids from reconstituted HDL containing cholesteryl [1-14C]arachidonate was also metabolized to prostaglandins after the cells were exposed to the calcium ionophore, A-23187. Both rat and human HDL which stimulated 6-keto-PGF1 alpha release (rat greater than human) increased the weight percentage of arachidonate in endothelial cell phospholipids; phospholipid arachidonate in the enriched cells fell after exposure to the phospholipase activator, A-23187, with release of 6-keto-PGF1 alpha which was greater than in control cells. Rat HDL that was depleted of cholesteryl arachidonate (achieved by incubation with human low density lipoproteins (LDL) in the presence of cholesteryl ester transfer protein) stimulated 6-keto-PGF1 alpha release less than native rat HDL. LDL enriched in cholesteryl arachidonate stimulated 6-keto-PGF1 alpha release more than native LDL. ApoE-depleted HDL also stimulated 6-keto-PGF1 alpha release more than apoE-rich HDL suggesting the apoE receptor was not involved in the response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endothelin-induced prostacyclin production in rat aortic endothelial cells is mediated by protein kinase C.

Endothelin (ET) is a vasoconstrictor peptide released from endothelial cells that is known to cause prostaglandin (PG) release. The mechanism remains unclear. To determine whether the protein kinase C (PKC) signaling pathway is stimulated by endothelin, we pretreated rat aortic endothelial cells with either PKC activator or inhibitors and measured the release of prostacyclin (PGI2) by radioimmunoassay. ET (10(-9) M) produced a 10-fold increase in PGI2 release. Pretreatment with 10(-9) M of three different PKC inhibitors: 1-(5-isoquinolinesulfonyl) piperazine (CL), staurosporine, and 1-(5-isoquinolinesulfonyl-methyl) piperazine (H7) blocked ET induced PGI2 release. ET induced prostacyclin release was also blocked by pretreatment with inhibitors of either phospholipase A2 (7,7,dimethyleicosadienoic acid or trifluoromethyl ketone analogue) (10(-9) M) or cyclooxygenase (indomethacin) (10(-9) M). We conclude that ET activates PKC which activates phospholipase A2 which liberates arachidonic acid which increases PGI2 production and release.     

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.     

Endothelin increases [Ca2+]i in rat pancreatic acinar cells by intracellular release but fails to increase amylase secretion.

In individual fura-2 loaded cells of rat pancreatic acini endothelin-1 (ET-1) (10-50 nM) induced sustained oscillations in [Ca2+]i. At higher concentrations a larger, but transient increase in [Ca2+]i was observed, which was largely unaffected by removal of extracellular Ca2+. ET-1 induced the release of Ca2+i from the same store as cholecystokinin (CCK), but with less potency. At concentrations of endothelin which transiently increased Ca2+, ET-1 increased the accumulation of inositol phosphates. Specific binding sites for 125I-endothelin were demonstrated on rat pancreatic acini. A single class of binding sites was identified with an apparent Kd 108 +/- 12 pM and Bmax of 171 +/- 17 fmol/mg for ET-1. The relative potency order for displacing [125I]ET was ET-1 greater than ET-2 greater than ET-3. In contrast to CCK and the non-phorbol ester tumour promoter Thapsigargin (TG) which induce both transient and sustained components of [Ca2+]i elevation, ET-1 failed to increase amylase release over the range 100 pM-1 microM.     

Vasoactive peptides upregulate mRNA expression and secretion of vascular endothelial growth factor in human airway smooth muscle cells

Airway remodeling and associated angiogenesis are documented features of asthma, of which the molecular mechanisms are not fully understood. Angiotensin (ANG)II and endothelin (ET)-1 are potent vasoconstricting circulatory hormones implicated in asthma. We investigated the effects of ANG II and ET-1 on human airway smooth muscle (ASM) cells proliferation and growth and examined the mRNA expression and release of the angiogenic peptide, vascular endothelial growth factor (VEGF). Serum deprived (48 h) human ASM cells were incubated with ANG II (100 nM) or ET-1 (10nM) for 30 min, 1, 2, 4, 8, 16, and 24 h and the endogenous synthesis of VEGF was examined in relation to control cells receiving serum free culture medium. ET-1 induced time dependent DNA biosynthesis as determined by [³H]-thymidine incorporation assay. Using northern blot hybridization, we detected two mRNA species of 3.9 and 1.7 kb encoding VEGF in the cultured smooth muscle cells. Both ANG II and ET-1 induced the mRNA expression (two-to threefold) and secretion (1.8-to 2.8-fold) of VEGF reaching maximal levels between 4–8 h of incubation. Induced expression and release of VEGF declined after 8 h of ANG II incubation while levels remained elevated in the case of ET-1. The conditioned medium derived from ET-1-treated ASM cells induced [³H]-thymidine incorporation and cell number in porcine pulmonary artery endothelial as well as human umbilical vein endothelial cells. Moreover, the VEGF tyrosine kinase receptor inhibitor blocked the conditioned medium induced mitogenesis in endothelial cells. Our results suggest a potential role for ANG II and ET-1 in ASM cell growth and upregulation of VEGF that may participate in endothelial cell proliferation via paracrine mechanisms and thus causing pathological angiogenesis and vascular remodelling seen during asthma.     

ET-1 released histamine from guinea pig pulmonary but not peritoneal mast cells.

Endothelin(ET)-1 triggered histamine release of mast cells from pulmonary tissue but not from the peritoneal cavity of guinea pigs. The observed difference in response to ET-1 was attributable to a quantitative difference in ET-1 binding sites between both cells. The concentrations of ET-1 required for half maximal release of histamine and half maximal binding of [125I]ET-1 were approximately 0.05 and 0.08 nM, respectively. The release of histamine by ET-1 was a Ca(2+)-dependent but not a cytotoxic process. These observations, taken together, suggest that ET-1 induces histamine release from mast cells in a receptor-dependent fashion.     

Endothelin-1 expression in vascular adventitial fibroblasts

Endothelial cells are a major source of endothelin (ET)-1, but the possibility that vascular adventitial fibroblasts generate ET-1 has not been explored. We hypothesized that aortic adventitial fibroblasts have the ability to produce ET-1, which may contribute to extracellular matrix synthesis. Vascular adventitial fibroblasts were isolated from mouse aorta and incubated with various concentrations of angiotensin II (ANG II). mRNA levels of preproET-1 and type I procollagen were detected with relative RT-PCR. ET-1 levels in culture medium were measured with ELISA. Protein levels of procollagen were detected with Western blotting. ANG II (10 and 100 nM, 1 microM) induced a time- and concentration-dependent increase in preproET-1 mRNA levels (P < 0.05). Induction of preproET-1 mRNA was accompanied by release of immunoreactive peptide ET-1 (P < 0.05). ANG II-evoked increases in preproET-1 mRNA expression and ET-1 release were blocked by losartan (100 microM), an AT1 receptor antagonist, but not PD-123319 (100 microM), an AT2 receptor antagonist. To further confirm our findings, we cloned and then sequenced vascular fibroblast preproET-1 bidirectionally with T7 and M13 reverse sequencing primers. Their nucleotide sequences were identical to preproET-1 cDNA from mouse vascular endothelial cells (accession no. AB081657). Moreover, ANG II-induced type I procollagen mRNA and protein expression were inhibited by BQ-123 (10 microM), an ET(A) receptor inhibitor, but not BQ-788 (10 microM), an ET(B) receptor inhibitor, suggesting a significant role of adventitial ET-1 in regulation of extracellular matrix synthesis. The results demonstrate that vascular adventitial fibroblasts are able to synthesize and release ET-1 in response to ANG II.     

The platelet cyclooxygenase metabolite 12-L-hydroxy-5, 8, 10-hepta-decatrienoic acid (HHT) may modulate primary hemostasis by stimulating prostacyclin production

Although HHT accounts for approximately one third of the arachidonic acid (AA) metabolites produced by stimulated platelets, no well defined function has been attributed to this product. We report that HHT stimulates prostacyclin production by endothelial cells, and have identified the mechanism for this effect. In human umbilical venous endothelial cells, HHT (0.5 and 1 microM) stimulated prostacyclin (RIA for 6KPGF1 alpha) by 32 +/- 22% (1SD) and 42 +/- 38% (P less than 0.05 and less than 0.01). Similar changes were observed when the effect of HHT on exogenous [1-14C] AA metabolism in fetal bovine aortic endothelial cells (FBAECs) was studied. Kinetic analyses revealed that HHT affected vascular cyclooxygenase. HHT (1 microM) increased Vmax in test microsomes (706 +/- 21 pmol/mg/min, mean +/- 1SE) when compared to controls (529 +/- 20; P less than 0.02). No concomitant effect on Km was observed. A further effect of HHT on AA release from endothelial cell membrane phospholipids was noted. Prelabeling experiments revealed that HHT (1 microM) increased the ionophore stimulated release of AA from FBAECs (20952 +/- 555 cpm/well control mean +/- 1SE vs 25848 +/- 557 for paired HHT treated cells; P less than 0.05). The effect of HHT on platelet AA metabolism was next studied. Preincubation of washed platelets with HHT (1 microM) did not enhance thrombin or arachidonic acid induced platelet TXB2 formation. In platelets prelabelled with [1-14C]AA, HHT (1 microM) had no effect on AA release post thrombin stimulation. Conversion to cyclooxygenase metabolites was also not enhanced. HHT stimulates vascular prostacyclin without a concomitant effect on platelet AA metabolism. HHT may thus be an important local modulator of platelet plug formation.     

Endothelin: a potent stimulator of intestinal ion secretion in vitro.

Effects of endothelin (ET) on electrical properties and Na+ and Cl- fluxes in stripped rabbit ileal mucosa were investigated in vitro in Ussing chambers. Results demonstrate that serosal addition of ET-1, ET-2, ET-3 or the precursor 38 amino acid 'big endothelin' produce dose-dependent increases in short-circuit current (Isc) with maximal effects at approx. 100 nM, 100 nM, 10 nM and 100 nM, respectively and half-maximal effects at 1.4 nM, 5 nM, 1.4 nM and 20 nM, respectively. Mucosal addition of ET-3 failed to elicit a response. Changes in Isc elicited by ET-3 are accompanied by decreases in net fluxes of both Na+ and Cl-. The cyclooxygenase inhibitors, indomethacin and piroxicam, inhibited the increase in Isc produced by ET-3 and indomethacin also abolished the changes in Na+ and Cl- fluxes produced by ET-3. However, no changes in the release of PGE2, thromboxane B2 or 6-keto-prostaglandin F1 alpha could be detected up to 20 min after the addition of ET-3. Preincubation of tissues with neuronal agonists or antagonists, antihistamines or an LTD4/LTE4 receptor antagonist, SKF 104353, failed to alter the response to ET-3. Furthermore, removal of serosal Ca2+ also failed to inhibit the change in Isc produced by ET-3. These results indicate that endothelin is a potent intestinal secretagogue which does not appear to elicit its response through stimulation of PGE2, thromboxane A2 or prostacyclin.     

Production of NAADP and its role in Ca2+ mobilization associated with lysosomes in coronary arterial myocytes

The present study was designed to determine the production of nicotinic acid adenine dinucleotide phosphate (NAADP) and its role associated with lysosomes in mediating endothelin-1 (ET-1)-induced vasoconstriction in coronary arteries. HPLC assay showed that NAADP was produced in coronary arterial smooth muscle cells (CASMCs) via endogenous ADP-ribosyl cyclase. Fluorescence microscopic analysis of intracellular Ca2+ concentration ([Ca2+]i) in CASMCs revealed that exogenous 100 nM NAADP increased [Ca2+]i by 711 +/- 47 nM. Lipid bilayer experiments, however, demonstrated that NAADP did not directly activate ryanodine (Rya) receptor Ca2+ release channels on the sarcoplasmic reticulum. In CASMCs pretreated with 100 nM bafilomycin A1 (Baf), an inhibitor of lysosomal Ca2+ release and vacuolar proton pump function, NAADP-induced [Ca2+]i increase was significantly abolished. Moreover, ET-1 significantly increased NAADP formation in CASMCs and resulted in the rise of [Ca2+]i in these cells with a large increase in global Ca2+ level of 1,815 +/- 84 nM. Interestingly, before this large Ca2+ increase, a small Ca2+ spike with an increase in [Ca2+]i of 529 +/- 32 nM was observed. In the presence of Baf (100 nM), this ET-1-induced two-phase [Ca2+]i response was completely abolished, whereas Rya (50 microM) only markedly blocked the ET-1-induced large global Ca2+ increase. Functional studies showed that 100 nM Baf significantly attenuated ET-1-induced maximal constriction from 82.26 +/- 4.42% to 51.80 +/- 4.36%. Our results suggest that a lysosome-mediated Ca2+ regulatory mechanism via NAADP contributes to ET-1-induced Ca2+ mobilization in CASMCs and consequent vasoconstriction of coronary arteries.     

Synergistic activation of salmon cardiac function by endothelin and beta-adrenergic stimulation

The aim was to find out the effects of endothelin-1 (ET-1) in salmon (Salmo salar) cardiac contractile and endocrine function and its possible interaction with beta-adrenergic regulation. We found that ET-1 has a positive inotropic effect in salmon heart. ET-1 (30 nM) increased the contraction amplitude 17+/-4.7% compared with the basal level. beta-Adrenergic activation (isoprenaline, 100 nM) increased contraction amplitude 30+/-13.1%, but it did not affect the contractile response to ET-1. ET-1 (10 nM) stimulated the secretion of salmon cardiac natriuretic peptide (sCP) from isolated salmon ventricle (3.3+/-0.14-fold compared with control) but did not have any effect on ventricular sCP mRNA. Isoprenaline alone (0.1-1,000 nM) did not stimulate sCP release, but ET-1 (10 nM) together with isoprenaline (0.1 nM) caused a significantly greater increase of sCP release than ET-1 alone (5.4+/-0.07 vs. 3.3+/-0.14 times increase compared with control). The effects on the contractile and secretory function could be inhibited by a selective ETA-receptor antagonist BQ-610 (1 microM), whereas ETB-receptor blockage (by 100 nM BQ-788) enhanced the secretory response. Thus ET-1 is a phylogenetically conserved regulator of cardiac function, which has synergistic action with beta-adrenergic stimulation. The modulatory effects of ET-1 may therefore be especially important in situations with high beta-adrenergic tone.     

Pituitary site of action of endothelin: selective inhibition of prolactin release in vitro

Endothelin-3 (ET-3) inhibited in a dose-dependent, significant fashion prolactin release from cultured anterior pituitary cells (ovariectomized female and intact male rat donors, ED50 = 5 X 10(-9) M). ET-3 in log doses ranging from 10(-11) to 10(-6) M did not alter significantly the release of luteinizing hormone, growth hormone or thyroid stimulating hormone. The inhibitory effect of ET-3 (rat, human) was specific for that molecule since ET-1 (porcine, human) was ineffective and was not due to an action on the dopamine receptor since the inhibitory action was still expressed in the presence of 100 nM domperidone. These data further suggest a role for neuropeptides of the posterior lobe in the control of lactotroph function.     

Differential effects of different statins on endothelin-1 gene expression and endothelial NOS phosphorylation in porcine aortic endothelial cells

It has been reported that 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase inhibitors (statins) produce a variety of cardiovascular protective effects independent of their ability to lower total and low-density lipoprotein cholesterol. Recent studies have also reported that statins produce pleiotropic effects through improved endothelial function, enhanced fibrinolysis, and antithrombotic actions. In the present study, we examined the effects of pitavastatin, pravastatin, atorvastatin, and cerivastatin on endothelin (ET)-1 production in cultured porcine aortic endothelial cells (PAECs). Treatment with cerivastatin but not pitavastatin, pravastatin, or atorvastatin decreased basal and TNF-alpha-stimulated ET-1 release from PAECs in a dose-dependent manner (1-10 microM). Northern blot analysis showed that cerivastatin markedly suppressed prepro ET-1 mRNA expression in both conditions. In addition, these inhibitory effects of cerivastatin on ET-1 release and prepro ET-1 mRNA expression were completely abolished by simultaneous treatment with 200 microM mevalonate. Furthermore, cerivastatin did not have any effects on endothelial nitric oxide synthase (eNOS) protein levels, but induced eNOS phosphorylation at Ser1177. From these findings, it is most likely that cerivastatin suppresses ET-1 production, possibly through an increase in eNOS activity and the subsequent nitric oxide production in PAECs. These findings also suggest that cerivastatin may have beneficial effects on ET-1-related diseases.     

Endothelin-1 Stimulates Monocytes in vitro to Release Chemotactic Activity Identified as Interleukin-8 and Monocyte Chemotactic Protein-1

In the present study we examined whether endothelin-1 stimulation of human monocytes causes release of chemotactic factors. It was found that monocytes released neutrophil- and monocyte-chemotactic activity in a dose- and time-dependent manner in response to ET-1. ET-1 did not show any chemotactic activity by itself. NCA was detected in monocyte supernatants in response to ET-1 (0.01-100 nM) after 1, 4, 8 and 24 h stimulation. MCA was detected only after 24 h stimulation with ET-1 (0.1-100 nM). Preincubation of the monocyte cultures with the lipoxygenase inhibitors nordihydroguaiaretic acid (10(-4) M) or diethylcarbamazine (10(-9) M) completely abolished the appearance of NCA and MCA. NCA was neutralized by > 75% using a polyclonal antibody against human interleuktn-8. The ET-1 induced release of IL-8 was confirmed by IL-8 ELISA. A monoclonal antibody against human monocyte chemotactic protein-1 neutralized MCA by > 80%. It is concluded that ET-1 stimulation of monocytes in vitro causes release of neutrophil- and monocyte-chemotactic activity identified as IL-8 and MCP-I respectively. An intact lipoxygenase pathway is crucial for this effect of ET-1 to occur.