Stimulation of endothelin mRNA and secretion in rat vascular smooth muscle cells: a novel autocrine function.

Abstract

Endothelin (ET), a peptide originally isolated from the supernatants of cultured endothelial cells, exerts a wide variety of biological effects in different tissues. Endothelial-cell-synthesized ET-1 has been proposed to act in a paracrine manner on adjacent smooth muscle cells (SMC) in vivo, with effects that include both vascular reactivity (vasodilation/vasoconstriction) and mitogenesis. This study, by the use of immunocytochemically characterized SMC (rVSMC) isolated from the aortas of spontaneously hypertensive rats, has investigated a possible autocrine role for ET in regulation of the vasculature. Although quiescent cultures of rVSMC apparently did not constitutively express prepro ET-1mRNA, ET-specific transcripts could be induced by a variety of growth factors (transforming growth factor beta [TGF-beta]; platelet-derived growth factor-AA homodimer [PDGF-A chain]) and vasoactive hormones (angiotensin II [Ang II], arginine-vasopressin, and ET-1 itself). The kinetics for prepro ET-1mRNA induction in rVSMC were characteristically rapid in onset and transient. Down-regulation of protein kinase C by 48 h pretreatment of rVSMC with phorbol ester markedly reduced the subsequent ability of rVSMC to express ET-1 transcripts and secrete ET-1 peptide in response to Ang II. Inducible prepro ET-1mRNA expression was accompanied by a cycloheximide-inhibitable release of ET-1 peptide into the medium of rVSMC. ET-1 peptide was determined by both radioreceptor- and radioimmunoassay. Stimulated rVSMC accumulated ET-1 (approximately 200 pg.10(6) cells-1 x 4 h-1) at levels that attained biological relevance (approximately 10(-10) M). Sep-pak C18 extracts of medium from stimulated rVSMC elicited contraction of isolated endothelium-denuded rat mesenteric resistance vessels, and this response was characteristically protracted and difficult to "wash out." Synthetic (porcine) ET-1 promoted the expression of transcripts for PDGF-A chain, TGF-beta, and thrombospondin in quiescent rVSMC. Such effects of ET-1 on gene expression may be relevant to the mitogenic potential of ET-1 on VSMC. Our findings imply a role for ET-1 in the control of vascular function via both paracrine and autocrine regulatory mechanisms. The expression of prepro ET-1mRNA and peptide biosynthesis by rVSMC may have both short-term (e.g., vasoconstriction) and long-term (e.g., structural remodeling) consequences. A sustained loop of autocrine stimulation by ET-1 in SMC could contribute toward the pathogenesis of vasospasm and/or atherosclerosis.

Increased endothelin-1 content in blood vessels of deoxycorticosterone acetate-salt hypertensive but not in spontaneously hypertensive rats.

“…3 Thus, it is conceivable that a certain proportion of the increased vascular content of ET-1 originates in smooth muscle cells. However, no significant ir-ET-1 staining was found in the media of blood vessels from DOCA-salt hypertensive or uninephrectomized rats.…”

“…ET-1 is a 21-amino acid peptide produced primarily in endothelial cells' but also in vascular smooth muscle cells. 3 Locally produced ET-1 exerts its effects in an autocrine and paracrine fashion to constrict blood vessels by acting on smooth muscle. Through its effect on endothelial cells, it induces the production of endothelium-derived relaxing factor and prostacyclin, which relax underlying smooth muscle cells.…”

Endothelin stimulated by angiotensin II augments contractility of spontaneously hypertensive rat resistance arteries.

“…28 Purified, RNA containing lysates were slot-blotted directly onto Hybond N membrane (Amersham, UK), air-dried, and irradiated with ultraviolet light for 3 minutes at 302 nm to cross-link the RNA to the membrane. Hybridization of membranes to random-primed, radioactive probes (endothelin, 27 major histocompatibility complex [MHC] 29 occurred overnight at 65°C. 27 The endothelin specific probe used was a 1.1 kb, partial cDNA probe, cloned in the £coRI sites of the vector pUC 18.…”

“…Hybridization of membranes to random-primed, radioactive probes (endothelin, 27 major histocompatibility complex [MHC] 29 occurred overnight at 65°C. 27 The endothelin specific probe used was a 1.1 kb, partial cDNA probe, cloned in the £coRI sites of the vector pUC 18. Using this enzyme, the insert was recovered and isolated.…”

Bosentan reduces neuronal apoptosis following spinal cord ischemic reperfusion injury

“…A previous study confirmed that ET-1 exerts its biological effect by autocrine and paracrine action, resulting in induction of self-expression. 22 Differences in animal models, species and tissue specificity may also play a role in these various findings.…”