Search

Human Girardi heart cells expressing endothelin ETB receptors (GHB cells) were transfected with human ETA cDNA, and coexpression of ETA and ETB in the ratio of 4:6 was demonstrated by Scatchard analysis. [125I]Endothelin (ET)-1 binding to ETA-transfected GH cells (GHAB cells) was displaced by an ETA antagonist, BQ-123, in a biphasic manner. An ETB agonist, BQ-3020, and an ETB antagonist, BQ-788, inhibited [125I]ET-1 binding to GHAB cells in a monophasic manner with low affinities (IC50=2,800 and 890 nM, respectively); IC50 values for ETB receptors seemed to be as weak as those for ETA receptors. However, BQ-3020 and BQ-788 had a high affinity for ETB receptors in a binding experiment using [125I]ET-1 in the presence of 1 μM BQ-123, where ETA receptors are masked (IC50 =0.49 and 0.89 nM, respectively). The ETB-mediated increase in intracellular calcium concentrations in GHAB cells was not affected by 0.1 μM BQ-788 alone but was inhibited significantly by the same concentration of BQ-788 in combination with 10 μM BQ-123. ET-1 suppressed forskolin-stimulated accumulation of cAMP through the activation of ETA and ETB in GHAB cells; 1 μM BQ-123 or BQ-788 inhibited the suppression by only 20%, whereas a mixture of BQ-123 and BQ-788 (1 μM each) completely inhibited the cAMP decrease. These findings suggest that the stimulation of ETA receptors with ET-1 results in a lowering of the affinity of BQ-3020 and BQ-788 for ETB receptors in GHAB cells. We conclude that there is intracellular cross-talk between ETA and ETB receptors in GHAB cells. [ABSTRACT FROM AUTHOR]

Phosphoramidon-sensitve endothelin-converting enzyme of bovine endothelial cells showed substrate selectivity for big endothelin-1 (ET-1) when compared to big ET-1(1–380, big ET-2(1–37), big ET-2(1–38) and big ET-3(1–41). To investigate the big ET-1 structure important for specific conversion by the endothelin-converting enzyme, we synthesized a series of truncated analogues of big ET-1, measured the hydrolysis of their Trp21-Val22 bonds, and found that a 16-residue peptide, big ET-1(19–34), is the minimal peptide sequence. This suggests that an unusually long carboxy-terminal sequence is required for big ET-1 conversion. Alanine substitution for individual amino acids in the carboxy-terminal region of big ET-1(19–34) demonstrated that Hiss27, Val29, Pro30, Tyr31, Gly32, Leu33 and Gly34 are more important than Asn23, Thr24, Pro25, Glu26 and Val28 for eliciting efficient hydrolysis of the Trp21-Val22 bond, even though the former residues are located at more distant positions from the cleavage sites than are the latter. These results, together with the fact that big ET-2 and big ET-3 show heterogeneity in the big ET-1 residues His27, Val28, Val29 and Gly34, suggest that the His27-Val-Val-Pro-Tyr-Gly-Leu-Gly34 sequence in the carboxy-terminal region of big ET-1 plays the most important role in selective conversion by endothelin converting enzyme. [ABSTRACT FROM AUTHOR]

A plasminogen activator inhibitor (PAI) was purified from bovine endothelial cell conditioned medium by a simple procedure in the absence of protein denaturant. The yield was 2.2 mg from 1.61 conditioned medium in a typical experiment. The purified inhibitor showed a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis and reverse fibrin autography with an apparent molecular mass of 45 Wa. The amino-terminal 40-amino-acid sequence was determined and found to be 70% similar to the reported corresponding sequence of human PAI-1. The amino acid composition also revealed a close relationship between bovine PAI and human PAI-1. The purified PAI was substantially inactive (570 U/mg) but it could be activated by treatment with protein denaturants such as 1% SDS (1.8 × 105 U/mg) and 4 M guanidine-HCl (1.5 × 105 U/mg). A more effective activation of this latent PAI was achieved by heat treatment at 100°C for 2.5 min, generating the specific activity of 1.0 × 106 U/mg. The heat-activated PAI lost its activity during incubation at 56°C for 30 min, but repeated heat at 100°C for 2.5 min could regenerate about 70% of the initial activity. Treatment at 37°C, 56°C and 80°C. however, failed to activate the latent PAI at all. These findings suggest that the buried reactive site of the latent PAI is exposed as a result of a heat-induced, specific conformational change, but tends to be masked again during renaturation under mild conditions, i.e. the PAI protein takes on preferentially a latent form. [ABSTRACT FROM AUTHOR]