endothelin-1 and chymostatin

Endothelin-1 (ET-1) is a 21-amino-acid peptide, derived from vascular endothelial cells, with potent vasoconstrictor activity. ET-1 has been implicated in diverse physiological or pathological processes, including the vascular changes associated with sepsis. However, the factors that regulate ET-1-associated toxicity during bacterial infections, or in other settings, are not fully understood. Both the pathology associated with certain allergic and autoimmune disorders, and optimal host defence against bacterial and parasitic infections are mediated by mast cells. In vitro, mast cells can produce ET-1 (ref. 11), undergo ET-1-dependent and endothelin-A receptor (ET(A))-dependent activation, and release proteases that degrade ET-1 (ref. 14). Although the potential relationships between mast cells and the ET-1 system thus may be complex, the importance of interactions between ET-1 and mast cells in vivo is obscure. Here we show that ET(A)-dependent mast-cell activation can diminish both ET-1 levels and ET-1-induced pathology in vivo, and also can contribute to optimal survival during acute bacterial peritonitis. These findings identify a new biological function for mast cells: promotion of homeostasis by limiting the toxicity associated with an endogenous mediator.

Topics: Animals; Body Temperature; Body Weight; Cell Degranulation; Cell Survival; Chymases; Diarrhea; Drug-Related Side Effects and Adverse Reactions; Egtazic Acid; Endothelin-1; Female; Homeostasis; Injections, Intraperitoneal; Mast Cells; Mice; Mice, Inbred C57BL; Mice, Knockout; Mutation; Oligopeptides; Peptides, Cyclic; Peritonitis; Proto-Oncogene Proteins c-kit; Serine Endopeptidases; Stem Cells; Survival Rate

We have investigated whether a chymostatin-sensitive enzyme, such as chymase, contributes to the production of vasoconstrictor endothelin peptides from exogenous big endothelin- 1 in endothelium-denuded human umbilical vein. Big endothelin-1 contracted veins with a pD2 = 7.75 +/- 0.08 (n = 23) and was five times less potent than endothelin-1 (pD2 = 8.43 +/- 0.11, n = 10). The selective endothelin-converting enzyme inhibitor, PD159790, (30 microM, n = 17) produced a significant (P < 0.005), threefold rightward shift of the big endothelin-1 concentration-response curve with no reduction in maximum response. A further shift was obtained with 100 microM PD159790 (n = 6), with a significant decrease in the maximum response from 84.0 +/- 5.3%KCl to 34.2 +/- 6.7%KCl (P < 0.005). Chymostatin (30 microM) had no effect on the big endothelin-1 response, however, the inhibitory effect of 100 microM chymostatin was significant (P < 0.005). Unlike PD159790, chymostatin did not significantly modify the maximum response to big endothelin-1. The combination of 30 microM PD159790 and 100 microM chymostatin was more effective than either inhibitor alone. Our data suggest a role for both endothelin-converting enzyme and a chymostatin-sensitive enzyme, such as chymase, in the conversion of big endothelin-1 to constrictor endothelin peptides in human umbilical vein smooth muscle. In cardiovascular diseases in which endothelin-converting enzyme activity is increased, inhibition of both enzymes may be required for effective therapeutic intervention.

Topics: Aspartic Acid Endopeptidases; Chymases; Endothelin-1; Endothelin-Converting Enzymes; Humans; In Vitro Techniques; Metalloendopeptidases; Oligopeptides; Potassium Chloride; Serine Proteinase Inhibitors; Umbilical Veins; Vasoconstriction; Vasoconstrictor Agents

The intratracheal (i.t.) instillation of Sephadex beads into rat induced inflammation and a 30-fold increase in the endothelin-1-like immunoreactivity (ET-1-LI) of broncho-alveolar lavage fluid. The levels were highest 24 h after the instillation and had declined significantly after 48 h. At a dose of 1 mg kg-1 i.t., the glucocorticosteroid budesonide almost abolished this response. Phosphoramidon, which inhibits neutral endopeptidase, an enzyme reported to degrade ET-1 and also to inhibit the endothelin-converting enzyme, potentiated the Sephadex-induced rise in ET-1-LI. Chymostatin and heparin, which are reported to reduce the formation of ET-1, did not affect the increase in ET-1-LI. The present model represents a very reactive system for analyzing the changes in ET-1 levels during inflammation.

Topics: Animals; Bronchoalveolar Lavage Fluid; Budesonide; Chromatography, High Pressure Liquid; Dextrans; Endothelin-1; Enzyme Inhibitors; Glycopeptides; Heparin; Hydrogen Peroxide; Male; Oligopeptides; Pneumonia; Pregnenediones; Rats; Rats, Sprague-Dawley

We examined conversion of Big endothelin-1 (ET-1) to mature ET-1 and pressor action during perfusion of the isolated perfused rat lung with Big ET-1. Big ET-1 caused a concentration-related increase in perfusion pressure and the pressor molar potency of the peptide was fivefold less than that of ET-1. Pressor responses to Big ET-1 were accompanied by an increase in immunoreactive-ET (IR-ET) levels in the perfusate and in the lung tissues. Pretreatment with phosphoramidon (10(-4) M), a metalloproteinase inhibitor, markedly suppressed the pressor action and increment in IR-ET in the tissues. Unexpectedly, the amount of IR-ET in the perfusate during perfusion of Big ET-1 was not influenced by phosphoramidon treatment. On the other hand, chymostatin, an inhibitor of chymotrypsin-like enzymes, effectively suppressed IR-ET levels in the perfusate; however, this enzyme inhibitor was without effect on the pressor action of Big ET-1 or on the increase in IR-ET levels in lung tissues. We tentatively conclude that the phosphoramidon-sensitive conversion of Big ET-T to ET-1 is linked to the pressor action of Big ET-1 in the isolated perfused rat lung. In addition, it seems likely that chymostatin-sensitive conversion of Big ET-1 to ET-1 does not play a major role in the conversion of the precursor to the mature form. We propose that IR-ET present in the tissues rather than that in the perfusate is a better indicator of the functional conversion of Big ET-1 in the rat lung.

Topics: Animals; Aspartic Acid Endopeptidases; Chymotrypsin; Dose-Response Relationship, Drug; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Glycopeptides; In Vitro Techniques; Kinetics; Lung; Male; Metalloendopeptidases; Oligopeptides; Perfusion; Protein Precursors; Pulmonary Circulation; Rats; Rats, Sprague-Dawley