[Ala1,3,11,15]-Endothelin 1

Two new endothelin receptor radioligands, [125I]-BQ3020 and [125I]-[Ala1,3,11,15]ET-1, were characterized in tissue sections of human right atrium and left ventricle. Both radioligands had high affinity ([125I]-BQ3020 right atrium: KD = 0.145 +/- 0.037 nM, left ventricle: KD = 0.107 +/- 0.004 nM; [125I]-[Ala1,3,11,15]ET-1 right atrium: KD = 0.239 +/- 0.036 nM, left ventricle: KD = 0.199 +/- 0.027 nM). Competition binding experiments were performed in the left ventricle. The selective ETA receptor compound BQ123 competed with low affinity against [125I]-BQ3020 (KD = 28.7 +/- 2.7 microM) and [125I]-[Ala1,3,11,15]ET-1 (KD = 28.5 +/- 4.2 microM). The selective ETB receptor compound BQ3020 competed with high affinity against [125I]-BQ3020 (KD = 40.8 +/- 6.6 pM) and [125I]-[Ala1,3,11,15]ET-1 (KD = 0.276 +/- 0.099 nM). Another selective ETB receptor compound, [Ala1,3,11,15]ET-1 also competed with high affinity against [125I]-BQ3020 (KD = 0.663 +/- 0.120 nM) and [125I]-[Ala1,3,11,15]ET-1 (KD = 0.643 +/- 0.124 nM). These results indicate that [125I]-BQ3020 and [125I]-[Ala1,3,11,15]ET-1 are selective ETB receptor radioligands. [Ala1,3,11,15]ET-1 competed with the non-selective radioligand [125I]-ET-1 in left ventricle and revealed the presence of ETA and ETB receptors in the proportions of 76:24% respectively in the human left ventricle.

Production of tissue inhibitors of matrix metalloproteinases (TIMPs), a family of secreted proteins with inhibitory actions on matrix metalloproteinases (MMPs), is up-regulated following nerve injuries and is suggested to have protective effects against MMP-mediated tissue damages. To clarify the extracellular signals involved in TIMP production in the brain, the effects of endothelins (ETs), a family of vasoconstricting peptides, were examined. I.c.v. administration of 500 pmol/day Ala(1,3,11,15)-ET-1, an ET(B) receptor agonist, increased the level of TIMP-1 mRNA in rat hippocampus, caudate-putamen and cerebrum. Ala(1,3,11,15)-ET-1 increased the level of TIMP-3 mRNA in the cerebrum, but not in the hippocampus or caudate-putamen. TIMP-2 mRNA was not affected in these brain regions. Ala(1,3,11,15)-ET-1 also stimulated the production of TIMP-1 and TIMP-3 proteins in the cerebrum. Immunohistochemical observations in the hippocampi of Ala(1,3,11,15)-ET-1-infused rats showed that NeuN-positive neurons and glial fibrillary acidic protein-positive astrocytes were immunoreactive for TIMP-1. In the cerebrum, astrocytes had TIMP-1 and TIMP3 reactivity, but neurons did not. In rat cultured astrocytes, both 100 nM Ala(1,3,11,15)-ET-1 and ET-1 increased the mRNA levels and protein release of TIMP-1 and TIMP-3 mRNAs. The effects of ET-1 on astrocytic TIMP-1 and TIMP-3 mRNAs were inhibited by BQ788, an ET(B) antagonist. These findings indicate that activation of brain ET(B) receptors causes production of TIMP-1 and TIMP-3, and suggest the involvement of astrocytes in ET-induced TIMP production.

A linear peptide analog of endothelin (ET)-1, [Ala1,3,11,15]ET-1 (4AlaET-1), and its truncated peptide analogs were synthesized to study the structural requirements of ET-1 for the recognition of ETs-nonselective ETB receptors. ET-1 exhibited sub-nanomolar binding to two distinct ET receptor subtypes (ETA and ETB), but 4AlaET-1 bound to ETB with an affinity 1,700 times higher than that seen during binding to ETA. The truncated linear peptides 4AlaET-1(6-21), 4AlaET-1(8-21) and N-acetyl-4AlaET-1(10-21) still had high affinity for ETB, whereas 4AlaET-1(6-20) and 4AlaET-1(11-21) displayed remarkably reduced affinity for ETB. Therefore, ET-1 requires the Glu10-Trp21 sequence for ETB binding, but not the disulfide bridges. These ETB-binding peptides elicit endothelium-dependent vasorelaxation of porcine pulmonary arteries in parallel with the binding affinity for ETB, suggesting that they are ETB agonists.