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Abstract: Up-regulation of vascular endothelin type A (ETA) and type B (ETB) receptors are implicated in the pathogenesis of cardiovascular disease. Culture of arteries has been shown to induce similar receptor alterations and has therefore been suggested as a suitable method for in detail delineation of the regulation of endothelin receptors. We hypothesize that protein kinase C (PKC) and mitogen-activated kinases (MAPK) are involved in the regulation of endothelin receptors. Porcine coronary arteries were studied before and after 24 h of culture, using in vitro pharmacology, real-time PCR and immunofluorescence techniques. Sarafotoxin 6c and endothelin ET-1 were used to examine the endothelin ETA and ETB receptor effects. The involvement of PKC and MAPK in the receptor regulation was examined by culture in the presence of antagonists. Organ culture resulted in increased sarafotoxin 6c and endothelin-1 contractions, endothelin ETA and ETB receptor immunofluorescence staining intensities and endothelin ETB, but not ETA, receptor mRNA levels. The general PKC inhibitors, bisindolylmaleimide I (10 μM) or Ro-32-0432 (10 μM), inhibited these effects. Also, the increase in sarafotoxin 6c contraction, endothelin ETB receptor and mRNA levels and endothelin ETA and ETB immunofluorescence staining intensities were inhibited by MAPK inhibitors for extracellular signal related kinases 1 and 2 (ERK1/2), PD98059 (10 μM), C-jun terminal kinase (JNK), SP600125 (10 μM), but not by p38 MAPK, SB203580 (10 μM). In conclusion, PKC and MAPK seem to be involved in the regulation of endothelin receptor expression in porcine coronary arteries. Inhibiting these intracellular signal transduction pathways may provide a future therapeutic target for hindering the development of vascular endothelin receptor changes in cardiovascular disease. [Copyright &y& Elsevier]

Abstract: Culture of intact arteries is a frequently employed experimental model for investigating the mechanisms governing the regulation of vascular endothelin receptors. Endothelin type A (ETA) and type B (ETB) receptors on vascular smooth muscle cells are up-regulated in organ culture and the enhanced vasoconstriction mimics the changes that occur in cardiovascular disease. The effect of organ culture on endothelial dilatory endothelin ETB receptors is not known. We hypothesize that organ culture decreases the endothelin receptor-mediated dilatation and that this is one possible mechanism by which the effects of the endothelin in blood vessels are altered during culture. Porcine coronary arteries were studied before and after 24 h of culture, using in vitro pharmacology and immunofluorescence. Sarafotoxin 6c and endothelin-1 were used to examine the endothelin ETA and ETB receptor effects, and the antagonists, Nω-nitro-l-arginine (l-NOARG) for nitric oxide (NO), indomethacin for prostaglandins and charybdotoxin in combination with apamin for endothelium-derived hyperpolarizing factor (EDHF), were used to study the endothelium-derived dilatory mediators. Organ culture induced up-regulation of the sarafotoxin 6c (ETB receptor agonist) and endothelin-1 (ETA receptor agonist) elicited vasoconstriction. The sarafotoxin 6c contraction was stronger after endothelium denudation, suggesting endothelium-dependent dilatation. The endothelin-1 contraction was not affected by endothelium denudation. The increase in sarafotoxin 6c contraction after removal of the endothelium was more pronounced before than after organ culture, suggesting down-regulated endothelial endothelin ETB receptors. Also, the immunofluorescence staining intensities for endothelial endothelin ETB receptors were higher before than after organ culture. Pre-incubation with inhibitors for dilatory mediators suggested that both NO and EDHF play a vasodilatory role, while prostaglandins are not involved. In conclusion, endothelial endothelin ETB receptors induce NO and EDHF mediated vasodilatation in porcine coronary arteries. In organ culture, endothelial endothelin ETB receptors are down-regulated, mimicking the changes that occur in cardiovascular disease. Down-regulation of endothelial endothelin ETB receptors may in part explain the increased endothelin ETB receptor-mediated vasoconstriction frequently studied in organ culture. [Copyright &y& Elsevier]