Article date: February 1995
By: Francisco Pérez‐Vizcaíno, Andrea C. Cooper, Roger Corder, Alain Fournier, Timothy D. Warner, in Volume 114, Issue 4, pages 867-871
In vivo the effects of endothelin‐1 (ET‐1) are limited by its rapid removal from the circulation and possibly by its metabolism by enzymes such as neutral endopeptidase 24.11, deamidase or carboxypeptidase A. Here, using as a model the isolated perfused mesenteric arterial bed of the rat, we have examined the involvements of these enzymatic activities in the vascular responses to ET‐1.
Samples of Krebs buffer which had been recirculated through the mesenteric arterial bed for 30 min rapidly destroyed the activity of ET‐1 as assessed either by bioassay on rings of rat thoracic aorta or by high performance liquid chromatography (h.p.l.c). For instance, after 15 min incubation with the recirculated‐Krebs solution (recirc‐K) the contraction induced by 3 times 10−9m ET‐1 was reduced by more than 90%. Contractions induced by sarafotoxin 6b (3 times 10−9m) were similarly suppressed by preincubation with recirc‐K whereas those to Arg‐vasopressin (3 times 10−9m) were unaffected.
The degradation of ET‐1 by recirc‐K was prevented by 1,10‐phenanthroline (10−3m), abolished by heating the recirc‐K solution to 90°C for 15 min, and reduced by EGTA (5 times 10−3m) or ET‐1(16–21) (10−5m). For instance, in the presence of ET‐1(16–21) (n = 6) the contraction induced by ET‐1 was reduced by only 40% after 15 min incubation with recirc‐K buffer. Leupeptin (3 times 10−4m), dichloroisocoumarin (5 times 10−5m), phenylmethyl‐sulphonyl fluoride (10−3m), a combination of bacitracin (300 mg ml−1), bestatin (10−5m), captopril (10−5m), phosphoramidon (10−4m) and thiorphan (10−4m) or Polypep (a proprietary protein digest) did not inhibit the degradation of ET‐1 by recirc‐K.
In experiments examining directly the vascular responses of the isolated perfused mesentery of the rat, the addition of cumulative concentrations of ET‐1 to the recirculating Krebs solution caused small concentration‐dependent increases in perfusion pressure. The inclusion of ET‐1(16–21), ET‐1(17–21), or ET‐1(18–21) (10−5m) greatly potentiated these responses, but not those to Arg‐vasopressin or methoxamine. The effects of 1, 10‐phenanthroline or EGTA could not be examined in this system because these agents both depressed non‐specifically the vasoconstrictor responses of the mesenteric vascular bed.
Thus, the rat mesentery releases an enzyme that very rapidly destroys ET‐1 or the very closely related peptide, sarafotoxin 6b but not Arg‐vasopressin. This enzyme is most probably a metallo‐peptidase because of its sensitivity to inhibition by 1,10‐phenanthroline or EGTA. It is particularly interesting that a simple vascular bed such as the mesentery produces such a powerful endothelin metabolising enzyme. It is tempting, therefore, to speculate that the endothelin degrading enzyme active at neutral pH that we have found is important in the metabolism of ET‐1 throughout the vasculature.
In vivo the effects of endothelin‐1 (ET‐1) are limited by its rapid removal from the circulation and possibly by its metabolism by enzymes such as neutral endopeptidase 24.11, deamidase or carboxypeptidase A. Here, using as a model the isolated perfused mesenteric arterial bed of the rat, we have examined the involvements of these enzymatic activities in the vascular responses to ET‐1.
Samples of Krebs buffer which had been recirculated through the mesenteric arterial bed for 30 min rapidly destroyed the activity of ET‐1 as assessed either by bioassay on rings of rat thoracic aorta or by high performance liquid chromatography (h.p.l.c). For instance, after 15 min incubation with the recirculated‐Krebs solution (recirc‐K) the contraction induced by 3 times 10−9m ET‐1 was reduced by more than 90%. Contractions induced by sarafotoxin 6b (3 times 10−9m) were similarly suppressed by preincubation with recirc‐K whereas those to Arg‐vasopressin (3 times 10−9m) were unaffected.
The degradation of ET‐1 by recirc‐K was prevented by 1,10‐phenanthroline (10−3m), abolished by heating the recirc‐K solution to 90°C for 15 min, and reduced by EGTA (5 times 10−3m) or ET‐1(16–21) (10−5m). For instance, in the presence of ET‐1(16–21) (n = 6) the contraction induced by ET‐1 was reduced by only 40% after 15 min incubation with recirc‐K buffer. Leupeptin (3 times 10−4m), dichloroisocoumarin (5 times 10−5m), phenylmethyl‐sulphonyl fluoride (10−3m), a combination of bacitracin (300 mg ml−1), bestatin (10−5m), captopril (10−5m), phosphoramidon (10−4m) and thiorphan (10−4m) or Polypep (a proprietary protein digest) did not inhibit the degradation of ET‐1 by recirc‐K.
In experiments examining directly the vascular responses of the isolated perfused mesentery of the rat, the addition of cumulative concentrations of ET‐1 to the recirculating Krebs solution caused small concentration‐dependent increases in perfusion pressure. The inclusion of ET‐1(16–21), ET‐1(17–21), or ET‐1(18–21) (10−5m) greatly potentiated these responses, but not those to Arg‐vasopressin or methoxamine. The effects of 1, 10‐phenanthroline or EGTA could not be examined in this system because these agents both depressed non‐specifically the vasoconstrictor responses of the mesenteric vascular bed.
Thus, the rat mesentery releases an enzyme that very rapidly destroys ET‐1 or the very closely related peptide, sarafotoxin 6b but not Arg‐vasopressin. This enzyme is most probably a metallo‐peptidase because of its sensitivity to inhibition by 1,10‐phenanthroline or EGTA. It is particularly interesting that a simple vascular bed such as the mesentery produces such a powerful endothelin metabolising enzyme. It is tempting, therefore, to speculate that the endothelin degrading enzyme active at neutral pH that we have found is important in the metabolism of ET‐1 throughout the vasculature.
DOI: 10.1111/j.1476-5381.1995.tb13284.x
View this article