Increase In Endothelin-1 Levels Research Articles

CONSTITUTIVE AND INDUCIBLE EXPRESSION OF ENDOTHELIN-1 IN PRIMARY RAT ARTICULAR CHONDROCYTE CULTURE

Endothelin-1 (ET-1) mRNA expression and protein production were examined in primary rat articular chondrocyte (AC) cultures by RT-PCR and radioimmunoassay, respectively. We found that serum-starved rat AC express ET-1 mRNA and produce the peptide constitutively. Treatment of cells with 10% FCS resulted in a marked increase in ET-1 levels with a peak at 48 h (5.6-fold). A similar concentration-dependent effect was also obtained in the presence of interleukin 1β (3.1-fold), tumour necrosis factor α (3.5-fold), lipopolysaccharide (2.7-fold), transforming growth factor β1 (3.5-fold), epidermal growth factor (5.0-fold) and insulin-like growth factor-I (4.4-fold). In addition, ET-1 was found to induce, over a period of 24 h, a potent concentration-dependent stimulation of DNA synthesis in rat AC. These findings demonstrate for the first time the constitutive expression and production of ET-1 by rat AC which could be modulated by several cytokines and growth factors, suggesting a possible role for ET-1 in autocrine regulation of chondrocyte function.

Source and cause of endothelin-1 release into cerebrospinal fluid after subarachnoid hemorrhage.

Despite years of research, delayed cerebral vasospasm remains a serious complication of subarachnoid hemorrhage (SAH). Recently, it has been proposed that endothelin-1 (ET-1) mediates vasospasm. The authors examined this hypothesis in a series of experiments. In a primate model of SAH, serial ET-1 levels were measured in samples from the perivascular space by using a microdialysis technique and in cerebrospinal fluid (CSF) and plasma during the development and resolution of delayed vasospasm. To determine whether elevated ET-1 production was a direct cause of vasospasm or acted secondary to ischemia, the authors also measured ET-1 levels in plasma and CSF after transient cerebral ischemia. To elucidate the source of ET-1, they measured its production in cultures of endothelial cells and astrocytes exposed to oxyhemoglobin (10 microM), methemoglobin (10 microM), or hypoxia (11% oxygen). There was no correlation between the perivascular levels of ET-1 and the development of vasospasm or its resolution. Cerebrospinal fluid and plasma levels of ET-1 were not affected by vasospasm (CSF ET-1 levels were 9.3 +/- 2.2 pg/ml and ET-1 plasma levels were 1.2 +/- 0.6 pg/ml) before SAH and remained unchanged when vasospasm developed (7.1 +/- 1.7 pg/ml in CSF and 2.7 +/- 1.5 pg/ml in plasma). Transient cerebral ischemia evoked an increase of ET-1 levels in CSF (1 +/- 0.4 pg/ml at the occlusion vs. 3.1 +/- 0.6 pg/ml 4 hours after reperfusion; p < 0.05), which returned to normal (0.7 +/- 0.3 pg/ml) after 24 hours. Endothelial cells and astrocytes in culture showed inhibition of ET-1 production 6 hours after exposure to hemoglobins. Hypoxia inhibited ET-1 release by endothelial cells at 24 hours (6.4 +/- 0.8 pg/ml vs. 0.1 +/- 0.1 pg/ml, control vs. hypoxic endothelial cells; p < 0.05) and at 48 hours (6.4 +/- 0.6 pg/ml vs. 0 +/- 0.1 pg/ml, control vs. hypoxic endothelial cells; p < 0.05), but in astrocytes hypoxia induced an increase of ET-1 at 6 hours (1.5 +/- 0.6 vs. 6.4 +/- 1.1 pg/ml, control vs. hypoxic astrocytes; p < 0.05). Endothelin-1 is released from astrocytes, but not endothelial cells, during hypoxia and is released from the brain after transient ischemia. There is no relationship between ET-1 and vasospasm in vivo or between ET-1 and oxyhemoglobin, a putative agent of vasospasm, in vitro. The increase in ET-1 levels in CSF after SAH from a ruptured intracranial aneurysm appears to be the result of cerebral ischemia rather than reflecting the cause of cerebral vasospasm.

Elevated blood pressure in spontaneously hypertensive rats consuming a high sucrose diet is associated with elevated angiotensin II and is reversed by vanadium.

To determine the changes in serum angiotensin II (Ang II) and endothelin-1 levels induced by vanadium treatment of sugar-fed rats in order to investigate the relationship between changes in blood pressure and Ang II and endothelin-1 levels. Male spontaneously hypertensive rats (SHR) were fed starch (control), sucrose, and sucrose plus vanadium compounds at various concentrations. The systolic blood pressure of the rats was estimated by tail-cuff plethysmography. Serum Ang II and endothelin-1 levels were measured by radioimmunoassay. There were increases in systolic blood pressure (by 8%) and in serum Ang II (by 20%) in sucrose-fed SHR compared with control. In sucrose plus vanadium-fed SHR, the lowering of the systolic blood pressure (by 11-16% of the sucrose-fed value) was accompanied by a significant decrease in Ang II levels (by 25-60% of the sucrose-fed value) and an increase in endothelin-1 level (by 61-76% of the sucrose-fed value). That Ang II levels are elevated in sucrose-induced hypertension and decreased after vanadium therapy suggests that the renin-angiotensin system plays a role in the induction of hypertension in this model. On the other hand, the elevation of endothelin-1 levels associated with a decreased systolic blood pressure might be secondary to vanadium stimulation of endothelial cells. The data suggest that endothelin-1 is not involved in sugar-induced elevations of the blood pressure.

The effect of cyclosporine on endothelin levels after orthotopic liver transplantation in rats

Abstract. To assess the effects of cyclosporin (CyA) on endothelin-1 (ET-1) in rat liver allograft rejection, we evaluated ET-1 expression in samples obtained from BN(RTln)-to-BN (group 1) rats, DA(RTla)-to-BN (group 2) rats, and DA-to-BN rats treated with 5 mg/kg per day of CyA (group 3). Serum and hepatic ET-1 levels, determined by a radioimmunoassay, remained unchanged in group 1. In group 2, the ET-1 levels peaked on postoperative day (POD) 5 in the liver at 344 ± 31.6 pg/ g wet, and on POD 7 in the serum at 38.7 ± 13.1 pg/ml. In group 3, hepatic and renal ET-1 levels showed a progressive increase until POD 10, while serum ET-1 levels remained unchanged. In conclusion, acute rejection caused a temporary increase in the ET-1 level in both the serum and the liver in the early postoperative period what might have been caused by endothelial damage due to ongoing, acute rejection. CyA caused a time-dependent increase in the ET-1 level in both the liver and the kidney without an increase in the serum ET-1 level. The serum ET-1 level might have been affected by the clearance of ET from the liver or kidney.

The effect of cyclosporin on endothelin levels after orthotopic liver transplantation in rats.

To assess the effects of cyclosporin (CyA) on endothelin-1 (ET-1) in rat liver allograft rejection, we evaluated ET-1 expression in samples obtained from BN(RT1n)-to-BN (group 1) rats, DA(RT1a)-to-BN (group 2) rats, and DA-to-BN rats treated with 5 mg/kg per day of CyA (group 3). Serum and hepatic ET-1 levels, determined by a radioimmunoassay, remained unchanged in group 1. In group 2, the ET-1 levels peaked on postoperative day (POD) 5 in the liver at 344 +/- 31.6 pg/g wet, and on POD 7 in the serum at 38.7 +/- 13.1 pg/ml. In group 3, hepatic and renal ET-1 levels showed a progressive increase until POD 10, while serum ET-1 levels remained unchanged. In conclusion, acute rejection caused a temporary increase in the ET-1 level in both the serum and the liver in the early postoperative period what might have been caused by endothelial damage due to ongoing, acute rejection. CyA caused a time-dependent increase in the ET-1 level in both the liver and the kidney without an increase in the serum ET-1 level. The serum ET-1 level might have been affected by the clearance of ET from the liver or kidney.

Endothelin and nitric oxide release modulate aortic contraction to selected thrombin receptor agonists.

The contribution of endothelin-1 (ET-1) and nitric oxide (NO) release to vascular contraction induced by synthetic peptide agonists of the alpha-thrombin receptor, TRAP-14 and TRAP-6, was evaluated with the use of rings of rat aorta. TRAP-6 induced fourfold greater contraction than that induced by addition of TRAP-14. TRAP-14, but not TRAP-6, stimulation of aortic rings resulted in a rapid (in seconds) and dose-dependent increase in ET-1 levels detected in the vessel perfusate. Release of ET-1 in vessels denuded of endothelium was indistinguishable from that of intact vessels, suggesting that endothelial cells are not required for the observed ET-1 release. The contractile potency of TRAP-14 was reduced in the presence of BQ-123, a specific antagonist of the endothelin A subtype of ET receptors, whereas TRAP-14 potency was increased significantly by pretreatment with the NO synthetase inhibitor NG-nitro-L-arginine (L-NNA). The contractile potency of TRAP-6 was not altered in the presence of BQ-123 or L-NNA, suggesting that TRAP-14 but not TRAP-6 potency is modulated by ET-1 and NO release. These data indicate that TRAP-6 has limited function relative to TRAP-14 and that thrombin receptor activation is not sufficient to induce ET-1 and NO release from rat aorta.

Dose-Dependent Effect of Endothelin-1 on Blood Flow to Normal and Collateral-Dependent Myocardium

Plasma levels of endothelin-1 (ET-1) increase during ischemia and could potentially contribute to impairment of myocardial blood flow (MBF). Because collateral vessels demonstrate enhanced responsiveness to certain vasoconstrictors, blood flow to collateral-dependent myocardium could be particularly sensitive to increases in ET-1 levels. Studies were performed in 13 dogs in which collateral vessel development was produced by fluoroscopic embolization of the midleft anterior descending coronary artery with a hollow plug 4 to 6 weeks before the study. MBF was measured with radioactive microspheres at baseline and during 30-minute infusions of ET-1 (1, 10, and 100 ng/min) into the left main coronary artery. Because ET-1 stimulates endothelial prostacyclin release, aortic and coronary sinus levels of ET-1 and 6-keto-prostaglandin F1 alpha were measured at the end of each infusion. ET-1 increased MBF from 0.82 mL.min-1.g-1 at baseline to 0.92 mL.min-1.g-1 at 10 ng/min (P < .05), which corresponded to a coronary plasma concentration of 73 +/- 16 pg/mL. Blood flow in the collateral zone was less (0.74 mL.min-1.g-1) than in the normal zone (P < .05) and did not increase at an ET-1 dose of 10 ng/min. MBF in the normal and collateral zones significantly decreased when ET-1 was increased to 100 ng/min, corresponding to a coronary sinus concentration of 175 +/- 45 pg/mL (P < .05). ET-1 produced dose-related increases in aortic and coronary sinus 6-keto-prostaglandin F1 alpha and the transcoronary difference (P < .05). To assess the importance of prostacyclin in opposing the vasoconstriction produced by ET-1, additional studies were performed after cyclooxygenase blockade with indomethacin. After indomethacin administration, ET-1 (10 ng/min) caused a 120 +/- 23% increase in collateral vascular resistance (P < .05) and abolished the vasodilation that this dose produced in the normal zone. Blood flow to normal myocardium is increased at moderate plasma elevations of ET-1, whereas collateral blood flow is unchanged. Only at significantly elevated plasma concentrations of ET-1 is blood flow to normal and collateral-dependent myocardium impaired. Coronary endothelial production of prostacyclin in response to increasing concentrations of ET-1 represents an important means of blunting the vasoconstrictor properties of ET-1 in the canine coronary circulation. Coronary collateral vessels demonstrate a much greater dependence on prostacyclin production in blunting the vasoconstrictor properties of ET-1.

Endothelin-1 levels in ischaemia, reperfusion, and haemorrhagic shock in the canine infrarenal aortic Revascularisation model

Endothelin-1 (ET-1) is a potent vasoconstrictive polypeptide produced from vascular endothelial cells. The effects of ischaemia, reperfusion, and exsanguination on plasma ET-1 levels were studied and compared in the mongrel dog after infrarenal aortic cross clamping. Ischaemia produced a trend toward increased ET-1 serum levels (p < 0.07 with Bonferroni correction) that did not reach significance. Plasma ET-1 levels were significantly increased during reperfusion and even further elevations were found following exsanguination. We found a 2-3 fold increase in ET-1 levels following reperfusion (Initial 3.19 +/- 0.27 pg/ml vs. Reperfusion maximum 6.32 +/- 0.72 pg/ml, Bonferroni p < 0.01). Haemorrhagic shock was associated with a 3-4 fold increase in ET-1 levels (Initial 3.19 +/- 0.27 pg/ml vs. Exsanguination maximum 8.37 +/- 0.97 pg/ml Bonferroni p < 0.001). These data reveal that ET-1 is released during reperfusion and exsanguination and may mediate remote vascular events associated with infrarenal aortic cross clamping and acute blood loss.

Phosphoramidon does not inhibit endogenous endothelin-1 release stimulated by hemorrhage, cytokines and hypoxia in rats

The role of phosphoramidon-sensitive endothelin converting enzyme in the release of endogenous endothelin-1 was investigated in anesthetized rats. Intravenous infusion of phosphoramidon 0.3 mg/kg/min did not suppress the release of endothelin-1 stimulated by hemorrhage or cytokines. Elevation of endothelin-1 in rats subjected to hypoxia was not modified by phosphoramidon (0.1 or 0.3 mg/kg/min for 2 h). A high dose of phosphoramidon (10 mg/kg i.v. +0.1 mg/kg/min) significantly potentiated the hypoxia-induced increases in plasma endothelin-1 levels. Increases in endothelin-1 release caused by bilateral nephrectomy were further enhanced by hypoxia. It is concluded that the release of endogenous endothelin-1 release stimulated by hemorrhage, cytokines and hypoxia is resistant to inhibition by phosphoramidon, and at high doses, phosphoramidon potentiates hemorrhage- and hypoxia-induced increases in endothelin-1 levels, most likely by preventing its degradation.

Elevated endothelin-1 levels after cigarette smoking

The effect of short-term nicotine consumption on endothelin-1 (ET-1) levels was studied in 10 male healthy smokers. Volunteers smoked in random order on 3 separate days a low-tar cigarette or a high-tar cigarette, or were studied without having smoked (no-cigarette experiment). ET-1, corticotropin, and cortisol levels, heart rate, and blood pressure were determined before and 1, 3, 5, 10, 20, and 30 minutes after smoking. In contrast to results obtained after smoking a low-tar cigarette or not smoking, smoking a high-tar cigarette resulted in a significant increase in ET-1 levels within 10 minutes, followed by an increase in corticotropin levels within 20 minutes after smoking. Thirty minutes after smoking, cortisol levels were higher after a high-tar cigarette compared with a low-tar cigarette or no smoking. Increases in heart rate and systolic blood pressure were likewise higher after smoking a high-tar cigarette than after smoking a low-tar cigarette. In conclusion, it is tempting to speculate that ET-1 may indeed act as the long-searched-for link between vasopressin and corticotropin-releasing hormone (CRH) and thus play an essential role in the stimulation of the hypothalamic-pituitary-adrenal axis. In addition, these results suggest that the increase in the level of ET-1, a powerful vasoconstrictor and mitogen, may play an important part in the disease mechanisms of atherosclerosis arising from smoking.

Insulin modulates circulating endothelin-1 levels in humans

Recent data indicate that insulin stimulates synthesis of the vasoconstrictor peptide endothelin-1 (ET-1) by cultured vascular endothelial cells in vitro. To determine whether insulin modulates ET levels in vivo and whether this effect is important in the pathogenesis of obesity-associated hypertension, we measured circulating immunoreactive ET-1 levels during euglycemic hyperinsulinemic clamps (20 mU/m 2 · min −1 for 120 minutes) in eight obese women (body mass index, 36 ± 1 kg/m 2) before and after 10 weeks on an 800-kcal/d protein-sparing liquid diet. During the clamp that preceded weight loss, insulin levels were increased from 17 ± 2 to 51 ± 3 mU/L and this was associated with an increment in ET-1 level from 28 ± 3 to 33 ± 3 pg/mL ( P < .05). After weight loss, insulin levels were increased from 10 ± 2 to 47 ± 3 mU/L during the clamp, and there was a corresponding increase in ET-1 levels from 24 ± 3 to 30 ± 3 pg/mL ( P < .025). The reduction in basal ET-1 level (from 28 ± 3 to 24 ± 3 pg/mL) with weight loss correlated strongly with the reduction in fasting immunoreactive insulin level (from 17 ± 2 to 10 ± 2 mU/L; r = .92, P < .01). The decrease in blood pressure with weight loss from 130 ± 6 73 ± 3 to 118 ± 4 72 ± 3 mm Hg) did not correlate with the corresponding reduction in circulating ET-1 levels. These results indicate that insulin modulates ET-1 levels in vivo. The importance of hyperinsulinemia-induced secretion of ET in the pathogenesis of hypertension remains to be established.

Endothelin-1 and endotoxemia.

Sepsis leads to changes in blood volume and distribution. In the experiments reported here we monitored circulatory changes during endotoxemia and their relation to portal and systemic levels of endothelin-1 (ET-1). Piglets were monitored cardiovascularly under ketamine anesthesia for 6 h after an endotoxin infusion (saline controls). ET-1 levels in plasma were analyzed by RIA. In both portal and systemic blood a twofold increase in ET-1 levels was found after 1 h. This level remained significantly elevated for a further 3 h. This increase was concurrent with a drop in cardiac output, systemic vascular resistance, and blood pressure. In the portal circulation there was increased portal pressure and vascular resistance. There was no significant difference between the systemic and portal levels of ET-1.

Endothelin-1 mediates regional blood flow during and after pulmonary operations

To investigate the role of endothelin-1, a potent vasoconstrictor peptide produced by vascular endothelial cells, in the physiologic response to surgical stress, we measured plasma endothelin-1 concentrations by a sandwich-enzyme immunoassay in patients with lung cancer undergoing pulmonary operations (n = 12). In the first group (n = 6), we measured plasma endothelin-1 concentrations at multiple sampling sites (median cubital vein, pulmonary artery, and left atrium). Plasma endothelin-1 levels were significantly increased at all sampling sites at the end of the operation. Although there was no difference between the increased plasma levels of endothelin-1 in the pulmonary artery and in the left atrium, the increased level in the median cubital vein (peripheral venous blood) was significantly higher than that in the pulmonary artery (mixed venous blood). This result suggests that the production of endothelin-1 might differ between various organs under surgical stress. In the second group (n = 6), we measured plasma endothelin-1 concentrations both in the median cubital vein and in the ipsilateral radial artery, and also measured cardiac output and forearm blood flow. The increase in endothelin-1 levels in the median cubital vein was significantly higher than that in the radial artery after the operation. The endothelin-1 output from the forearm, calculated by the forearm blood flow and the arteriovenous difference of endothelin-1 concentrations, significantly increased after the operation. Although the cardiac output significantly increased after the operation, the forearm blood flow significantly decreased. The present findings provide a novel hypothesis that the preferential release of endothelin-1 from the peripheral vasculature of the forearm may partly contribute to a compensatory response to surgical stress, for example, the reduction of local blood flow in nonvital organs so as to increase the blood flow in vital organs.