Atrial natriuretic peptide, nitroglycerine, and nitroprusside reduce basal and stimulated endothelin production from cultured endothelial cells

Atrial natriuretic peptide (ANP) and the nitrovasodilator drugs nitroglycerine and nitroprusside were shown here to decrease both basal and thrombin stimulated production of endothelin-1 (ET-1) from cultured human endothelial cells as measured by radioimmunoassay. 8-Bromo-3',5'-cyclic guanosine monophosphate (cGMP) and papaverine also inhibited ET-1 production. The inhibitory effect of ANP and nitrovasodilators on ET-1 production thus appears to be mediated by guanylate cyclase and cGMP. Part of the vasodilatory action of ANP, nitroprusside and nitroglycerine may be due to suppression of endothelial ET-1 production. This may be an additional mechanism whereby nitrovasodilators participate in the regulation of vascular tone.     

Interleukin 1 increases the production of endothelin-1 by cultured endothelial cells

We examined the effect of human recombinant interleukin 1 (IL-1) on the production of endothelin-1 by cultured porcine endothelial cells. The induction of endothelin-1 mRNA began within 1 hr of exposure to IL-1, showed twin peaks at 4 and 24 hr, and declined thereafter. Enzyme-linked immunosorbent assay revealed that the amount of endothelin-1 peptide in conditioned media was also increased by IL-1 in a dose- and time-dependent manner. Our results suggested that IL-1, a macrophage-derived cytokine, may affect the contraction and proliferation of vascular smooth muscle cells by stimulating the production of endothelin by endothelial cells.     

Polar secretion of endothelin-1 by cultured endothelial cells.

The aim of this study was to determine the permeability of endothelial monolayers for endothelin-1 and a possible directionality of the endothelin-1 secretion process. Human umbilical vein endothelial cells were cultured on acellular amniotic membranes, dividing the tissue culture wells into an apical (luminal) and a basolateral (abluminal) compartment. Whereas in the absence of endothelial monolayers 44.9 +/- 2.3 and 43.5 +/- 2.0% of the unilaterally added endothelin-1 permeated from the apical to the basolateral side and from the basolateral to the apical side, respectively, only 6.5 +/- 0.6 and 6.6 +/- 0.4% diffused in the presence of endothelial cells. Analyzing endothelin-1 secretion, approximately 80% of the total amount of synthesized endothelin-1 was found in the basolateral compartment; thrombin (10 units/ml) stimulated the production of endothelin-1 approximately 2-fold, but did not change the relative distribution of endothelin-1 between the apical and basolateral compartments. In the presence of dexamethasone (10(-7) M), a decrease in the level of endothelin-1 was found in the apical compartment, whereas the total amount of endothelin-1 produced was not affected. Dexamethasone did not influence the permeability of human umbilical vein endothelial cell monolayers for endothelin-1. These results strongly support the hypothesis that endothelin-1 is a local paracrine regulator of vasotone.     

Human serum stimulates endothelin-1 synthesis more potently than prostacyclin production by cultured vascular endothelial cells

Human serum stimulated the synthesis of a vasoconstrictive peptide, endothelin-1 (ET-1), and a vasodilatory prostanoid, prostacyclin (PGI2), by cultured human umbilical vein endothelial cells in a concentration- and time-dependent manner. Incubation in 20% concentration of the serum for 24 h stimulated ET-1 synthesis almost six-fold while PGI2 production increased two-fold. In addition, a tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), inhibited the serum-induced ET-1 production and stimulated PGI2 synthesis in a concentration- and time-dependent manner. Our results suggest that human serum derived factor(s) stimulate the production of vasoconstrictive ET-1 more potently than the synthesis of vasodilatory PGI2 by human vascular endothelial cells and that the production of these agents is differentially regulated by PMA.     

Endothelin-1 induces prostacyclin release from bovine aortic endothelial cells

The effects of endothelin-1 (ET-1) on the release of prostacyclin from cultured bovine aortic endothelial cells were studied. ET-1 induced a time- and dose-dependent release of 6-keto PGF1 alpha, the stable metabolite of prostacyclin, with an apparent EC50 value of 3.0 +/- 0.9 nM (n = 6). ET-1 up to a concentration of 500 nM did not affect cellular integrity. Preincubation of the cells for 30 min with 10 microM indomethacin inhibited ET-1 (100 nM) - induced prostacyclin release by 90%. These findings indicate that ET-1 can directly stimulate prostacyclin release from endothelial cells probably through a receptor mediated mechanism.     

Endothelin-1 directly modulates its own secretion: studies utilising the cell immunoblot technique

Endothelin-1 is an important factor in vasoregulation and circulating levels of the peptide are increased in a number of cardiovascular disorders. However, control of endothelin-1 secretion is only sketchily understood. The possibility that endothelin-1 influences its own release was investigated. A cell immunoblot method, which can detect local secretion of peptide from individual human vascular endothelial cells, was employed. Cells were dispersed onto a protein-binding membrane. Endothelin-1 in cells or secreted and adhering to the protein-binding membrane outside the cells was detected using immunohistochemical techniques. The numbers of cells that contained endothelin-1 and secreted endothelin-1 were counted after the cells had been incubated in control conditions, or with added endothelin-1, angiotensin-II, or endothelin receptor antagonists, bosentan and BQ788. Endothelin-1 and angiotensin-II increased the numbers of cells that secreted endothelin-1. On the other hand, bosentan and BQ788 caused a reduction in the numbers of endothelin-1-secreting cells. These results indicate that human endothelial cells contain a pathway by which endothelin-1 induces its own release. The receptor antagonists, bosentan and BQ788, inhibited basal secretion of endothelin-1.     

Turnour necrosis factor stimulates endothelin-1 gene expression in cultured bovine endothelial cells

We have studied the effect of human recombinant tumour necrosis factor-alpha (TNF-alpha) on gene expression and production of endothelin-1 in cultured bovine aortic endothelial cells. TNF-alpha (10 and 100 ng ml(-1)) increased in a time dependent manner the preproendothelin-1 mRNA levels in respect to unstimulated endothelial cells. TNF-alpha induced endothelin-1 gene expression was associated with a parallel increase in the release of the corresponding peptide in the culture medium. These findings suggest that the enhanced synthesis and release of endothelin-1 occurring in conditions of increased generation of TNF, may act as a modulatory factor that counteracts the hypotensive effect and the excessive platelet aggregation and adhesion induced by TNF.     

dsRNA-mediated innate immunity of epidermal keratinocytes

MIP-1alpha, a CC chemokine, recruits monocytes, natural killer cells, lymphocytes, and neutrophils, and plays a critical role in viral infection. Since, the lesional epidermis of herpes zoster expressed MIP-1alpha, we hypothesized that keratinocytes produce MIP-1alpha in response to virus-associated dsRNA via TLR3. To investigate this, we examined cultured human keratinocytes for MIP-1alpha production induced by poly(I:C), a TLR3 ligand. Poly(I:C) treatment induced MIP-1alpha production, interestingly, poly(I:C)-induced IFN-alpha and -beta production preceded MIP-1alpha production. A neutralizing antibody for IFN-beta significantly inhibited the poly(I:C)-induced MIP-1alpha production indicating that MIP-1alpha production is via IFN-beta. IFN-alpha priming enhanced TLR3 expression and MIP-1alpha production in poly(I:C)-treated keratinocytes. This suggests that IFN-alpha enhanced the TLR3 expression and reinforced the response of keratinocytes to poly(I:C), which resulted in an increase in MIP-1alpha production. In conclusion, normal human keratinocytes produce MIP-1alpha in response to dsRNA via TLR3, and this production is regulated by IFN-alpha/beta.     

Phosphatidic acid and phospholipase D both stimulate phosphoinositide turnover in cultured human keratinocytes

Phosphatidic acid (PA) induced a rapid dose-dependent increase in production of inositol phosphates in cultured adult human keratinocytes, peaking at 30 s. Natural and dioleoyl PA were equally effective, while other phospholipid classes had no effect. Lipid A was also active. Lyso-PA also induced inositol phosphate production, but contamination of the PA preparation by lyso-PA could not account for the effect of PA. The effect of PA could not be reproduced by treatment of cells with calcium ionophore. PA-induced inositol phosphate production could be inhibited (> 50%) by pre-treatment of cells with either pertussis toxin or 12-O-tetradecanoylphorbol 13-acetate, suggesting the involvement of a GTP-binding protein and a protein kinase C-mediated negative feedback mechanism. PA also stimulated release of arachidonic acid from keratinocytes. Treatment of cells with exogenous phospholipase D similarly induced inositol phosphate production in the keratinocytes. Since PA may be formed by receptor-mediated activation of phospholipase D, or by phosphorylation of diacylglycerol, the results suggest that PA may play a significant role in signalling mechanisms of human keratinocytes.     

Cytokine-Induced Expression of Parathyroid Hormone-Related Peptide in Cultured Human Vascular Endothelial Cells

Parathyroid hormone-related peptide (PTHrP) is a potent vasodilatory peptide whose expression has been demonstrated in various tissues. The present study was undertaken to examine the regulation of PTHrP expression in cultured endothelial cells derived from human umbilical vein. Immunoradiometric assay revealed that the amount of PTHrP in the conditioned medium was increased by both tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) in both a time- and a dose-dependent manner. The induction of PTHrP mRNA was also observed, with a peak after 2 hours of incubation with both TNF-α and IL-1β. Angiotensin II, endothelin-1, and arginine vasopressin had no affect on PTHrP production. Our results suggest that PTHrP produced in vascular endothelial cells in response to cytokines may modulate vascular function as a local factor.     

Homocysteine-induced decrease in endothelin-1 production is initiated at the extracellular level and involves oxidative products.

The increased cardiovascular risk associated with hyperhomocysteinemia has been partly related to homocysteine (Hcy)-induced endothelial cell dysfunction. However, the intra or extracellular starting point of the interaction between Hcy and endothelial cells, leading to cellular dysfunction, has not yet been identified. We investigated the effects of both intracellular and extracellular Hcy accumulation on endothelin-1 (ET-1) synthesis by cultured human endothelial cells. Incubation of cultures with methionine (1.0 mmol x L(-1)) for 2 h induced a slight increase in cellular Hcy content but no change in ET-1 production. Incubation of cells with Hcy (0.2 mmol x L(-1)) led to a significant fall in ET-1 generation, accompanied by a significant increase in cellular Hcy content. Addition of the amino-acid transport system L substrate 2-amino-2-norbornane carboxylic acid had no effect on the Hcy-induced decrease in ET-1 production but significantly inhibited the Hcy-induced increase in the cellular Hcy content. Incubation of cells with a lower Hcy concentration (0.05 mmol x L(-1)) also reduced ET-1 production without increasing the cellular Hcy content. Co-incubation with extracellular free-radical inhibitors (superoxide dismutase, catalase and mannitol) markedly reduced the effect of Hcy on ET-1 production. Thus, it is extracellular Hcy accumulation that triggers the decrease in ET-1 production by endothelial cells through oxidative products.     

Cytoprotective effect of reduced glutathione in hydrogen peroxide-induced endothelial cell injury

The kinetic effects of hydrogen peroxide (H2O2) on cultured endothelial cells isolated from bovine carotid artery were studied. The cytoprotective effects of glutathione (GSH) on H2O2-induced cell injury were also investigated. H2O2-induced a dose- and time-dependent cell injury in cultured endothelial cells. H2O2-induced cell injury was blocked by simultaneous treatment by catalase, but not by superoxide dismutase. H2O2 also induced endogenous PGI2 biosynthesis, and the maximum PGI2 production was reached after 1 h treatment. Stimulation of PGI2 production was parallel with arachidonate release from H2O2-treated cells. However the prostaglandin biosynthesis enzyme activity in cells was inhibited by H2O2 treatment. When the cells were treated with GSH, the intracellular GSH reached a plateau after 3 h treatment. Both H2O2-induced cell injury and PGI2 production were significantly inhibited by the 3 h pretreatment with GSH. The cytoprotective effect of GSH was completely inhibited by buthionine sulfoximine which is a specific inhibitor of gamma-glutamylcysteine synthetase. The results indicate that the cytoprotective effect of GSH on H2O2-induced cell injury in cultured bovine carotid artery endothelial cells depends on the increase in intracellular GSH content.     

Identification of G protein-coupled endothelin receptors in cultured bovine endothelial cells.

Autocrine and paracrine regulation of vascular endothelial cells by endothelins has been postulated and has been the target of many recent investigations. In the present study, we demonstrated, by affinity labeling, the presence of endothelin-1- and endothelin-3-specific receptors on cultured bovine endothelial cells that secrete endothelin; the endothelin-3 receptor was the major form. Analysis using the GTP analogue guanosine 5'-O- (thiotriphosphate) indicated that these receptors are coupled to G proteins.     

Positive and negative regulation of insulin action by genistein in the endothelium

Genistein is an isoflavone phytoestrogen with biological activities in management of metabolic disorders. This study aims to evaluate the regulation of insulin action by genistein in the endothelium. Genistein inhibited insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and attenuated downstream Akt and endothelial nitric oxide synthase (eNOS) phosphorylation, leading to a decreased nitric oxide (NO) production in endothelial cells. These results demonstrated its negative regulation of insulin action in the endothelium. Palmitate (PA) stimulation evoked inflammation and induced insulin resistance in endothelial cells. Genistein inhibited IKKβ and nuclear factor-кB (NF-кB) activation with down-regulation of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) production and expression. Genistein inhibited inflammation-stimulated IRS-1 serine phosphorylation and restored insulin-mediated tyrosine phosphorylation. Genistein restored insulin-mediated Akt and eNOS phosphorylation, and then led to an increased NO production from endothelial cells, well demonstrating its positive regulation of insulin action under insulin-resistant conditions. Meanwhile, genistein effectively inhibited inflammation-enhanced mitogenic actions of insulin by down-regulation of endothelin-1 and vascular cell adhesion protein-1 overexpression. PA stimulation impaired insulin-mediated vessel dilation in rat aorta, while genistein effectively restored the lost vasodilation in a concentration-dependent manner (0.1, 1 and 10 μM). These results suggested that genistein inhibited inflammation and ameliorated endothelial dysfunction implicated in insulin resistance. Better understanding of genistein action in regulation of insulin sensitivity in the endothelium could be beneficial for its possible applications in controlling endothelial dysfunction associated with diabetes and insulin resistance.     

Regulation of endothelin-1 release from human endothelial cells by sex steroids and angiotensin-II

It is well documented that there are gender differences in the incidence and patterns of cardiovascular disease; males have a higher incidence of cardiovascular disease than premenopausal women. We have therefore investigated whether the sex hormones, estradiol and testosterone, could directly influence the secretion of vascular peptides from human aortic endothelial cells (HAEC). Previously we have shown that testosterone can increase the number of HAECs that secrete adrenomedullin. In this study we investigated sex hormone regulation of endothelin-1 in HAEC. Several studies have observed a reduction in endothelin-1 secretion from endothelial cells in the presence of estradiol, the effect being more marked for stimulated cells. Studies on the actions of testosterone are much fewer and inconclusive. In this study we observed that estradiol did not change the number of cells secreting endothelin-1 during 4 h incubation under basal conditions but decreased the number of secreting cells stimulated with angiotensin-II. Testosterone induced an increase in the number of cells secreting endothelin-1 (p = 0.03). Complementary incubations revealed that testosterone up-regulated endothelin-1 mRNA at 1–3 h (p < 0.05). These results, together with our previous observations, indicate that angiotensin-II, testosterone and estradiol have parallel effects on the production of endothelin-1 as on adrenomedullin in HAEC. We conclude that there is potential for coordinated modulation by sex steroids and angiotensin-II of vasoactive peptide production in human endothelial cells.     

Actions of endothelin-1 on prostaglandin production by gestational tissues

Endothelin-1 (10(-11)M-10(-7)M) was incubated with human umbilical vein endothelial cells and cells derived from amnion and decidua and prostaglandin production was determined. The rates of biosynthesis of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and prostaglandin E2 (PGE2) by endothelial cells were increased significantly by treatment with endothelin-1. Amnion cell PGE2 production was reduced significantly by endothelin-1 treatment whereas decidual PGE2 and prostaglandin F2 alpha production was unaffected by this treatment. Thus, it is possible that endothelins may play a part in the regulation of uteroplacental hemodynamics and the mechanisms of parturition.