J Mol Cell Cardiol23,





of Endothelin







(Received 7 Nouember 1990, accepted II January 1991) Recently discovered endothelin is a powerful vasoconstrictor of vascular smooth muscle in vitro, which also profoundly affects the mechanical function of the heart [Z, 2]. An increased level of plasma endothelin in patients with acute myocardial infarction suggests the enhanced release of endothelin from vascular endothelium under pathophysiological conditions of the heart [3’j. The reperfnsion of ischemic myocardium is associated with the activation of polymorphonuclear leukocytes (PMN) generating oxygen-derived free radicals (OFR), as well as, enhanced responsiveness of myocardial a,-adrenergic receptors [4,5’J. Roth OFR and catecholamines are implicated in the modulation of release of endothelium-derived peptides [SJ.

in this communication, we have examined the abilities of OFR and IX- and j?-adrenergic agonists to enhance the release of endothelin from cultured bovine pulmonary artery endothelial cells. Among superoxide anion (O;), hydroxyl radical (.OH), and hydrogen peroxide (H,O,) , only .OH caused increases of 2.8-fold and 2.4-fold in the release of immunoreactive endothelin and lactate dehydrogenase (LDH), respectively. These enhanced releases were markedly inhibited by deferoxamine (as iron chelator) or catalase, which blocked the formation of .OH from H,O,. Phenylephrine, a specific a,-adrenergic agonist, and epinephrine, a non-selective adrenergic agonist, were also able to enhance the release of endothelin. These induced releases were blocked by prazosin, an a,-adrenergic antagonist. On the other hand, epinephrineenhanced release of endothelin was not blocked by yohimbine, an a,-adrenergic antagonist. Furthermore, isoproterenol, a fladrenergic agonist, also failed to cause any enhanced release of endothelin from endothelial cells. These results suggest that free 0022-2828/91/060655



radicals and catecholamines through a,-adrenergic receptors may enhance the release of endothelin from vascular cndothelium during the reperfusion of‘ ischemic myocardium. Bovine pulmonary arterial endothelial cells were grown to confluence, as described previously [7]. After removal of culture medium in T-25 flasks, the cell monolayer iabout 5 x lo6 cells) was washed three times with serum free MEM medium to remove unattached cells and serum. After 30min incubation of cells with 3ml of serum free MEM medium under 959, 0, and 5’:,, CO,, cornponents of the free radical generating system or H,O, (500 PM) or phenylephrine ( 25 pM I or epinephrine ( 100 PM) or isoproterenol i 25 PM i were added to the flasks. Thr indicated amounts of adrenergic antagonists were added 10 min prior to the addition of the agonists. The incubation times for f‘rer radicals and adrenergic agonists were 1 h ‘md 5 h. respectively. For the generation of 0;) 100 PM hypoxanthine, 24 mU xanthine oxidase, and 1 pM C 1991 Academic

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EDTA were added [S]. Generation of0; was confirmed in the culture medium alone by following the reduction of cytochrome C spectrophotometrically [a]. .OH was generated by the addition of 500~~ H,O, and 100,~~ FeS04 (or 100 PM FeCl, plus 100 PM ascorbic acid) [9]. Generation of #OH was confirmed in the culture medium alone by examining the hydroxylated products of salicylic acid by HPLC [S]. LDH activity of the cultured medium was measured by employing the LDH kit from Sigma Chemical Co., St. Louis, MO, USA. To measure endothelin, 100 ~1 of culture medium (5 x lo6 cells/3 ml medium) was freeze-dried and the residue resuspended in 100 ~1 of RIA buffer. The RIA for endothelin using ‘251-endothelin-l (human, porcine) and rabbit anti-endothelin-I (human, porcine) was performed essentially according to the procedure supplied by Peninsula Laboratories, Inc., Belmont, CA, USA. All measurements were performed on four different culture flasks. Values are expressed as mean+s.E.M. For statistical analysis, student’s l-test was employed, and P values of co.05 were considered significant. During 1 h of incubation, the xanthine oxidase and the .OH generating systems generated as much as 50~~ 0; (plus 100 PM H,O, by spontaneous dismutation) and about 5pM .OH, respectively (data not shown). Figure 1 (a) shows that endothelial cells released as much as 60 pg endothelin/106 cells/h. The release of endothelin from endothelial cells exposed to chemically generated 6; or 500~~ H,O, alone was not significantly different from that of controls. On the other hand, endothelial cells exposed to .OH released 2.8-fold higher amounts of immunoreactive endothelin as compared to unexposed cells. This enhanced release of endothelin was significantly blocked by 1 mM deferoxamine (an iron chelator) or catalase (H20, hydrolytic enzyme), which inhibited formation of .OH from H,O, in the following equation: H,O,

+ Fe’+ -Fe3 + .OH

’ + *OH (Fenton reaction),

These results suggest that .OH radical is the agent which caused the enhanced release of endothelin from endothelial cells.


R. Prasad

et al.




* Jlr *


‘OH + Catalase

FIGURE 1. Effect of free radicals on the secretion of endothelin (a) and lactate dehydrogenase (LDH) (b) from the cultured endothelial cells. The incubation of endothelial cells with free radical generating systems was performed as described in the text. The concentrations of deforoxaminc (DEF) and catalase were 1 rnM and 50 U/ml, respectively. Values represent mean *s.e.M. of four independent determinations. *P

Release of endothelin from cultured bovine endothelial cells.

Recently discovered endothelin is a powerful vasoconstrictor of vascular smooth muscle in vitro, which also profoundly affects the mechanical function...
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