Regulation Of Endothelin-1 Production Research Articles

Activation of spleen tyrosine kinase is required for TNF-α-induced endothelin-1 upregulation in human aortic endothelial cells

Endothelin-1 (ET-1) promotes atherosclerosis. We tested whether spleen tyrosine kinase (Syk) mediates tumor necrosis factor-α (TNF-α)-induced ET-1 upregulation in human aortic endothelial cells (HAECs) and sought to identify the signal pathways involved. TNF-α-induced reactive oxygen species (ROS) activated Syk and phosphatidylinositol 3-kinase (PI3K), which was required for the activation of AP-1 and subsequent ET-1 gene transcription. ROS mediated c-Jun NH2-terminal kinase (JNK) is also required for AP-1 activation, but Syk and PI3K regulated AP-1 activation independently of JNK. Through regulation of ET-1 production, Syk could be implicated in atherosclerosis.

Regulation of Endothelin-1 Production in Deoxycorticosterone Acetate- Salt-Treated Endothelial Cells

The aim of this study was to investigate the direct effect of deoxycorticosterone acetate (DOCA) on the expression of endothelin-1 (ET-1) mRNA and production of ET-1 peptides in cultured bovine carotid endothelial cells (BCEC) and human umbilical vein endothelial cells (HUVEC). The mineralocorticoid, DOCA, was administered to BCEC and HUVEC with or without additional salt (200 mmol/l). The ET-1 mRNA was elevated to 150% and 180% after a 24-hour incubation with DOCA (5 µmol/l) in BCEC and HUVEC, respectively. Intracellular content of ET-1 peptides of BCEC and HUVEC was increased from 21.66 ± 0.3 to 23.64 ± 0.19 fmol/10⁵ cells and from 8.38 ± 0.82 to 11.26 ± 0.91 fmol/10⁵ cells, respectively, in the DOCA-conditioned medium. Also, DOCA treatment for 24 h increased the secretion of ET-1 peptide from 1.62 ± 0.17 to 5.33 ± 0.67 fmol/10⁵ cells in BCEC and from 0.95 ± 0.08 to 3.56 ± 0.36 fmol/10⁵ cells in HUVEC. In DOCA-salt-treated endothelium, regulation of ET-1 gene was similar to that with DOCA alone. The DOCA-induced increase in expression of ET-1 mRNA and the ET-1 peptide level were both diminished in dexamethasone (DEX, 10 nmol/l)- or mifepristone (RU486, 0.5 µmol/l)-treated endothelium. These results suggest that DOCA directly increased the ET-1 mRNA expression in endothelium and could be mediated in part by a glucocorticoid receptor pathway which was independent of salt and hyperosmolarity.

Endothelin-1 production by prostate cancer cell lines is up-regulated by factors involved in cancer progression and down-regulated by androgens.

Recent data demonstrate that endothelin-1 (ET-1) concentration increases in plasma of men with advanced, hormone-refractory prostate adenocarcinoma. In addition, ET-1 is involved in osteblastic remodelling and new bone formation, suggesting a role for this vasoactive peptide in the metastatic progression of prostate cancer to the bone. We investigated the regulation of ET-1 expression in androgen-sensitive and insensitive prostate cancer cell lines by androgens and several factors involved in progression of prostate cancer (EGF) and bone remodelling (TGFbeta-1, IL1-alpha and IGF-1). Northern analysis and radio immunoassay demonstrated that all the ET-1 pathways are tuned off in the androgen-sensitive LNCaP cell line when compared to the androgen-insensitive PC-3 and DU145. In PC-3 cells transfected with a full-length androgen receptor expression vector (PC-3-AR), treatment with androgens reduced gene expression and secretion of ET-1 without affecting the gene expression of ET-3. Collectively, these data support a role for androgens in the regulation of ET-1 production by prostate adenocarcinoma cells. In PC-3 and DU145 cells, ET-1 gene expression and secretion were up-regulated by TGFbeta-1, EGF and IL1-alpha, whereas IGF-1 was ineffective. Conversely, none of the treatments affected ECE-1 or ET-3 gene expression. In conclusion, ET-1 production by prostate adenocarcinoma cells is down-regulated by androgens and up-regulated by factors involved in tumour progression indicating a role for this peptide in the biology of prostate cancer. In view of the role exerted by ET-1 in the process of bone metastasis, our data suggest the use of ET-1 receptor antagonists in the treatment of advanced prostate cancer.

Regulation of Endothelin-1 Production in Cultured Rat Vascular Smooth Muscle Cells

Regulation of Endothelin-1 Production in Cultured Rat Vascular Smooth Muscle Cells

Regulation of endothelin-1 production in cultured rat vascular smooth muscle cells.

Endothelin-1 (ET-1) is secreted from all rat vascular smooth muscle cells (VSMCs) examined, in addition to endothelial cells (ECs). An average secretion rate of ET-1 from rat VSMCs was determined to be 10% that excreted from ECs. We examined the effects of 22 substances on ET-1 secretion from VSMCs and compared them with those from ECs. Transforming growth factor-beta1 (TGF-beta), acidic and basic fibroblast growth factors, epidermal growth factor, angiotensin II, and adrenaline stimulated ET-1 secretion from VSMCs, whereas forskolin, thrombin, and platelet-derived growth factor-BB reduced it. Only TGF-beta and phorbol ester elicited consistent effects on ET-1 secretion from VSMCs and ECs. Regulation of ET-1 and adrenomedullin secretion from VSMCs was distinctly different. These data suggest that ET- 1 production in VSMCs is regulated by a mechanism separate from that in ECs and from adrenomedullin production in VSMCs. Chromatographic analysis showed immunoreactive ET-1 secreted from VSMCs was mainly composed of big ET- 1, whereas approximately 90% of that from ECs was ET-1. By TGF-beta stimulation of VSMCs, the ratio of big ET-1 to ET-1 was further increased. Because big ET-1 is converted into ET-1 only on the surface of the ECs in the culture system, big ET-1 secreted from the VSMCs may function as a mediator transmitting a signal from VSMCs to ECs in vivo.

CHRONIC NITRIC OXIDE INHIBITION AGGRAVATES HYPERTENSION IN ERYTHROPOIETIN-TREATED RENAL FAILURE RATS

Alterations in nitric oxide (NO) and endothelin-1 (ET-1) production have recently been reported in erythropoietin (r-HuEPO)-induced hypertension in renal failure rats. The present study was designed to evaluate the effect of NO synthase inhibition with the L-arginine analog NG-nitro-L-arginine methyl ester (L-NAME) on blood pressure (BP) and ET-1 production in control and in uremic rats treated or not treated with r-HuEPO. Renal failure was induced by a two-stage 5/6 nephrectomy. Control and uremic rats were studied separately and subdivided into four groups: vehicle, r-HuEPO, L-NAME + vehicle and L-NAME + r-HuEPO. L-NAME (100 mg/kg/day), r-HuEPO (100 U/kg, subcutaneously, three times per week), the vehicle or both were administered during 4 weeks in control rats and during 2 weeks in uremic rats. Systolic BP was recorded before and after the onset of treatment at weeks 2 and 4 in control rats and at weeks 1 and 2 in uremic rats. Hematocrit, serum creatinine, plasma, blood vessel (thoracic aorta and mesenteric artery bed) and renal cortex immunoreactive (ir) ET-1 concentrations were measured at the end of the protocol. L-NAME enhanced BP in control and uremic rats and the increase was significantly higher in uremic rats under r-HuEPO therapy (222 ± 7 mmHg vs 198 ± 6 mmHg, p<0.05). L-NAME induced an increase in thoracic aorta ir-ET-1 concentrations in control and uremic rats. In contrast, ir-ET-1 concentrations were unchanged in the mesenteric arterial bed and the renal cortex of control and uremic animals. R-HuEPO increased thoracic aorta ir-ET-1 contents in L-NAME treated control and uremic rats. These results underline the important role of NO release in opposing the action of vasopressors on blood vessel tone which appears more important in uremic rats treated with r-HuEPO. L-NAME treatment increased large vessel, but not small resistance artery ir-ET-1 concentrations, suggesting differential regulation of ET-1 production in different vascular beds under chronic NO synthase inhibition.

Regulation of ET-1 biosynthesis in cerebral microvascular endothelial cells by vasoactive agents and PKC.

Endothelin-1 (ET-1) is the most potent vasoconstrictor agent known. ET-1 is elevated in the cerebrospinal fluid following hemorrhage and brain injury and can compromise cerebral microvascular homeostasis. The modulation of ET-1 production by cerebral microvascular endothelial cells and the mechanism by which such changes take place are very important in our understanding of the pathological roles of ET-1. In the present study, we investigated the effects of vasoconstrictor agents that can be released from hemolyzed blood, cAMP-dependent dilators, and the role of protein kinase C (PKC) in the regulation of ET-1 production by piglet cerebral microvascular endothelial cells in culture. ET-1 was measured by RIA. 1) Cerebral microvascular endothelial cells synthesize and release ET-1 into the media; 2) 5-hydroxytryptamine (5-HT), lysophosphatidic acid (LPA), thromboxane analog U-46619, fetal bovine serum (20%), and phorbol 12-myristate 13-acetate significantly increase ET-1 production; 3) basal and vasoconstrictor agent-induced increases in ET-1 production by endothelial cells may be mediated via PKC; 4) cAMP-dependent vasodilators attenuate the basal production of ET-1 by cerebral microvessels; and 5) pretreatment of endothelial cells with a higher concentration of LPA, U-46619, or 5-HT counterbalances the cAMP-dependent dilator agent-induced reduction in basal ET-1 production. Therefore, by-products of hemolyzed blood can stimulate the production of ET-1 by a PKC-mediated mechanism. cAMP-dependent dilators can attenuate the vasoconstrictor agent-induced elevation in ET-1 production. These results suggest that cerebral microvascular homeostasis could be compromised by effects of interactions among vasoactive agents released during conditions injurious to the brain and they may further the understanding of potential contributions of hemolyzed blood clots to subarachnoid hemorrhage-induced vasospasm.

Effects of protein kinase C activation and inhibition on endothelin-1 release from human aortic and pulmonary artery endothelial cells: comparison with effects on bovine endothelin-1 and human prostaglandin I2 release.

Protein kinase C (PKC) has been implicated in the regulation of endothelin-1 production by bovine and porcine endothelial cells. The role of PKC in the synthesis and release of endothelin-1 has not been demonstrated previously in human arterial endothelial cells. The effects of activators and an inhibitor of PKC on endothelin-1 release from cultured human arterial endothelial cells have now been examined. Endothelial monolayers were incubated with the PKC activators phorbol 12-myristate 13-acetate (PMA) or 1-oleoyl 2-acetyl glycerol (OAG) for 30 min to 8 h and the amount of endothelin-1 released into the medium was determined. PMA (0.1 micromol/L) and OAG (0.1 mmol/L) stimulated endothelin-1 release, with maximal increases of 85% and 90%, respectively, apparent at 30 min in aortic endothelial cells, and of 81% and 75%, respectively, apparent at 1 h in pulmonary artery endothelial cells. Endothelin-1 release had returned to control values by 4 h and was approximately one-third to one-half of control values after 8 h of continuous exposure of both cell types to PMA or OAG. Long-term exposure of both types of cells to the PKC inhibitor staurosporine (0.1 micromol/L) reduced both basal endothelin-1 release and the enhancement of release by PMA or OAG. The PKC agonists also stimulated endothelin-1 release by bovine aortic endothelial cells. The maximal effects of PMA and OAG in these cells were apparent at 4 h, at which time endothelin-1 release was increased by 154% and 125%, respectively; after 4 h, endothelin-1 release had decreased, but was still 132% and 120%, respectively, of the control value at 8 h. The PKC activators markedly stimulated prostaglandin I2 (PGI2) release by human pulmonary artery endothelial cells. The enhancement of PGI2 release continued for up to 8 h, at which time 109-fold and threefold increases in PGI2 release were apparent with PMA and OAG, respectively. Therefore, activation of PKC results in a rapid increase in endothelin-1 release by human arterial endothelial cells. The subsequent decrease in endothelin-1 release in the presence of PMA or OAG is presumably attributable to activator-induced downregulation of PKC, which was apparent earlier in human than in nonhuman endothelial cells. The downregulation of endothelin-1 release also occurred much earlier than that of PGI2 release in human pulmonary artery endothelial cells.

Culturing of human vascular endothelial cells strongly affects their endothelin-1 and prostacyclin production.

Cultured human umbilical vein endothelial cells (HUVECs) are a widely used model to study the regulation of endothelial production of vasoactive substances such as endothelin-1 (ET-1) and prostacyclin (PGI2) in human. As even short term culturing is known to affect the function of many cell types, we studied whether there are differences in the production of ET-1 and PGI2 between freshly isolated HUVECs and HUVECs cultured for two passages, and whether variation in cell density affects the production of ET-1 and PGI2 by these cells. At confluency, freshly isolated HUVECs produced only from one-tenth to one-fifth of ET-1, but 46-86 times more PGI2 (p < 0.001), when compared to respective productions by similar amounts of cultured HUVECs. When the cell density of freshly isolated HUVECs was lowered either by diluting the cell suspension or by plating the same amount of cells on different size wells, the production of ET-1 increased: lowering cell density to one-tenth led to 18 fold increase in ET-1 production (p < 0.001). PGI2 production was not affected by cell density. Thus our data imply that the production of both ET-1 and PGI2 are differently regulated in freshly isolated and cultured HUVECs, and that cell density is an important determinant in the regulation of ET-1 production.

Regulation of endothelin-1 production by a thromboxane A 2 mimetic in rat heart smooth muscle cells

Thromboxane A 2 (TXA 2) and ET-1 have been known to play important roles in modulating vascular contraction and growth. The present study was undertaken to examine the effect of TXA 2 on the induction of endothelin-1 (ET-1) mRNA and protein levels in smooth muscle cells derived from rat heart. U-46619, a stable TXA 2 mimetic, superinduced preproET-1 mRNA in the presence of cycloheximide in these cells. This effect could be blocked by SQ-29548, a TXA 2/prostaglandin H 2 receptor antagonist and by actinomycin D, an RNA synthesis inhibitor. In addition, H7, a protein kinase C inhibitor, could abolish the induction. Transient transfection experiment revealed that the elevated ET-1 mRNA level after U-46619 treatment was a result of the activation of ET-1 gene activity. The elevated ET-1 message level was accompanied by increased ET-1 release into the cultured medium. These results show that the short-lived TXA 2 can induce potent and long-lived ET-1. These findings support a potential role for ET-1 in the pathogenesis of coronary atherosclerosis and hypertension evoked by TXA 2.

Insulin increases the release of endothelin in endothelial cell cultures in vitro but not in vivo

Endothelin-1 (ET-1), a potent vasoconstrictor and mitogenic peptide for vascular smooth muscle cells, may be a marker for development of vascular disorders in diabetic patients. The aim of this study was to elucidate the possible role of insulin in the regulation of ET-1 production. The effect of hyperinsulinemia (with and without concomitant hyperglycemia) on the release of ET-1 was studied in 23 healthy men in vivo, as well as in human umbilical cord vein endothelial cell (HUVEC) cultures in vitro. Plasma glucose and insulin were maintained at four desired levels (from 5 to 22 mmol/L and 0.065 to 12.9 nmol/L, respectively) during the in vivo studies. The mean (SEM) plasma ET-1 during normoglycemia and a fasting insulin concentration in healthy men was 3.8 (0.4) pg/mL, and ET-1 levels did not change in response to changes in the concentration of glucose (from 5.0 to 22 mmol/L) or insulin (from 0.065 to 12.9 nmol/L). The ET-1 concentration in HUVEC culture medium increased linearly during 24 hours, and insulin further enhanced the release of ET-1 dose-dependently. ET-1 release was stimulated by angiotensin II, thrombin, and transforming growth factor-β (TGF-β), whereas treatment with glucose and insulin-like growth factor-1 (IGF-1) was not associated with changed ET-1 levels in culture medium. Our results show that although high insulin concentrations stimulate ET-1 release in vitro, hyperinsulinemia is not associated with increased plasma ET-1 levels in healthy men in vivo. The role of insulin in the regulation of ET-1 production in vivo, if any, remains unsettled.

Role of cyclic nucleotides in the regulation of endothelin-1 production by human endothelial cells.

The regulation of endothelin-1 (ET-1) production by endothelial cells is likely of crucial physiological importance in the maintenance of vascular homeostasis. The aim of the present study was to explore the possible role of cyclic nucleotides in the control of ET-1 production in human umbilical vein endothelial cells (HUVEC). ET-1 release was determined by measuring levels of immunoreactive ET-1 in HUVEC-conditioned media after 6-h incubations. In the presence of 10% fetal calf serum (FCS) there was a threefold increase in ET-1 release compared with serum-free conditions (1.96 +/- 0.17 vs. 0.56 +/- 0.06 pg/micrograms protein), respectively. Inhibition of protein kinase (PK) C using staurosporine (10 nM) reduced basal ET-1 release by approximately 50% and completely prevented the response to FCS. In contrast, the addition of other PK inhibitors had little effect on basal or serum-stimulated ET-1 release at the concentrations used. N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP) produced significant alterations in ET-1 release depending on the basal level of production. Under serum-free conditions of low basal ET-1 production, DBcAMP increased ET-1 release by 68 +/- 22% but only at the highest concentration studied (1 mM). The dose-response relationship for DBcAMP was potentiated by KT-5720 (0.1 microM), an inhibitor of PKA, with a significant shift to 10-fold lower concentrations, whereas it was blocked by KT-5823 (4 microM), which can inhibit PKG.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitric oxide regulates the expression of vasoconstrictors and growth factors by vascular endothelium under both normoxia and hypoxia.

The mechanisms by which hypoxia causes vasoconstriction in vivo are not known. Accumulating evidence implicates the endothelium as a key regulator of vascular tone. Hypoxia induces the expression and secretion of endothelin-1 (ET-1), a potent vasoconstrictor in cultured human endothelial cells. We report here that nitric oxide (NO), an endothelial-derived relaxing factor, modifies this induction of ET-1. Whereas low oxygen tension (PO2 = 20-30 Torr) increases ET-1 expression four- to eightfold above that seen at normal oxygen tension (PO2 = 150 Torr), sodium nitroprusside, which releases NO, suppresses this effect. This inhibition of hypoxia-induced ET-1 expression occurs within the first hour of exposure of cells to sodium nitroprusside. Moreover, when the endogenous constitutive levels of NO made by endothelial cells are suppressed using N-omega-nitro-L-arginine, a potent competitive inhibitor of NO synthase, the baseline levels of ET-1 produced in normoxic environments are increased three- to fourfold. The effects of hypoxia and the NO synthase inhibitor on ET-1 expression are additive. The regulation of ET-1 production by NO appears to be at the level of transcription. Similar effects of NO were observed on the expression of the PDGF-B chain gene. PDGF-B expression was suppressed by NO in a hypoxic environment and induced by N-omega-nitro-L-arginine in both normoxic and hypoxic environments. These findings suggest that in addition to its role as a vasodilator, NO may also influence vascular tone via the regulated reciprocal production of ET-1 and PDGF-B in the vasculature.

Osmolar regulation of endothelin-1 production by rat inner medullary collecting duct.

Recent evidence has implicated endothelin-1 (ET-1) as an autocrine inhibitor of inner medullary collecting duct (IMCD) sodium and water transport. The regulators of IMCD ET-1 production are, however, largely unknown. Because of the unique hypertonic environment of the IMCD, the effect of varying extracellular tonicity on IMCD ET-1 production was evaluated. Increasing media osmolality from 300 to 450 mosmol with NaCl or mannitol but not urea caused a marked dose- and time-dependent reduction in ET-1 release by and ET-1 mRNA in cultured rat IMCD cells. In contrast, increasing osmolality had no effect on ET-1 production by rat endothelial or mesangial cells. To see if ET-1 varies in a similar manner in vivo, ET-1 production was assessed in volume expanded (lower medullary tonicity) or volume depleted (high medullary tonicity) rats. Urinary ET-1 excretion and inner medulla ET-1 mRNA were significantly reduced in volume depleted as compared to volume expanded animals. These results indicate that extracellular sodium concentration inhibits ET-1 production specifically in IMCD cells. We speculate that extracellular sodium concentration, via regulation of ET-1 production, provides a link between volume status and IMCD sodium and water reabsorption.

Regulation of endothelin-1 production in cultured rat mesangial cells.

We investigated the regulatory mechanisms of endothelin (ET)-1 production in cultured rat mesangial cells (MC), with a special focus on the roles of protein kinase A (PKA)- and protein kinase C (PKC)-mediated signaling systems. Vasoactive agents and growth promoting factors, including platelet-derived growth factor, vasopressin and thrombin, which act through receptors coupled to the phospholipase C-mediated signaling system, as well as phorbol ester and fetal calf serum stimulated ET-1 production. This effect was attenuated in PKC-depleted or H-7 (a PKC inhibitor) treated MC. On the other hand, an increase in intracellular cyclic AMP by forskolin or beta-adrenergic agonist, isoproterenol, which act as anti-mitogenic agents, inhibited serum-stimulated ET-1 production. In addition this effect was mimicked by the addition of 8-bromo-cyclic AMP to the medium. The effect of isoproterenol was abolished by propranolol. H-8, a PKA inhibitor, attenuated the inhibitory effect of forskolin. These findings suggest that ET-1 production in MC is regulated by interaction of both positive and negative signals mediated by PKC- and PKA-dependent mechanisms.