Cloning and sequencing of a human endothelin converting enzyme in renal adenocarcinoma (ACHN) cells producing endothelin-2.

Endothelin (ET)-2 is a 21 residue vasoactive peptide which is biosynthesized from big ET-2(1-38) by a specific cleavage at Trp21-Val22 with an ET converting enzyme (ECE). To identify an ECE in ACHN (human renal adenocarcinoma) cells which produce ET-2, we have cloned and sequenced a novel cDNA encoding a human ECE in ACHN (hAECE). It encodes a 770 amino acid protein with a zinc-binding motif and a single membrane spanning region. The sequences of nucleic acids and amino acids from Leu45 to Trp770 of hAECE are identical to those from Leu33 to Trp758 of a human ECE in HUVEC (hHECE). The sequences in the amino-terminal moiety are divergent between hAECE and hHECE. Based on the difference of the amino-terminal amino acid sequences, ECEs reported so far, can be classified into two isoforms. These results strongly suggest that an alternative splicing might occur in the 5'-terminal region of the ECE pre-mRNA.

Expression of intercellular adhesion molecule-1 on cardiac myocytes for myocarditis before and during immunosuppressive therapy.

Right ventricular endomyocardial biopsies were performed in 15 patients with unexplained cardiac dysfunction, and the expression of intercellular adhesion molecule-1 (ICAM-1) on the myocardium was examined using the streptavidin-biotin complex method. Three of 15 patients (20%) had histologic evidence of myocarditis, and 2 of 15 patients (13%) had borderline myocarditis. The patients with biopsy-proven myocarditis received immunosuppressive therapy (prednisolone 60 mg/day). Two of the 3 patients demonstrated a substantial increase (> 10%) in percent fractional shortening and left ventricular ejection fraction during therapy. Subsequent biopsies revealed ongoing myocarditis in 1 patient, and resolved myocarditis in the other. The remaining patient had persistent cardiac dysfunction, but the subsequent biopsy revealed resolving myocarditis. ICAM-1 immunoreactivity was observed on cardiac myocytes, vascular endothelial cells, and interstitial cells of the patients with myocarditis. However, in patients without myocarditis, ICAM-1 immunoreactivity was observed only on vascular endothelial cells and interstitial cells. ICAM-1 immunoreactivity was still observed on cardiac myocytes in the patients with ongoing or resolving myocarditis during immunosuppressive therapy, but was not detected in the patient with resolved myocarditis. This study demonstrates that ICAM-1 is expressed on cardiac myocytes of patients with myocarditis and persistent cardiac dysfunction despite immunosuppressive therapy. The persistent expression of ICAM-1 may cause chronic inflammation of the myocardium.

Immunohistochemical localization of plasminogen activator inhibitor-1 in human coronary atherosclerotic lesions involved in acute myocardial infarction.

Immunohistochemical localization of plasminogen activator inhibitor-1 (PAI-1) was studied using the streptavidin-biotin method in human atherosclerotic coronary arteries. The patients (one male and two females), whose ages ranged from 61 to 78 years, died of anteroseptal acute myocardial infarction without having received any thrombolytic therapy. PAI-1 immunoreactivities (IRs) were mainly detected in the endothelial cells, smooth muscle cells, and collagen fibers of the coronary arterial intima and not in thrombi. Remarkable immunohistochemical staining was seen in intimal collagen fibers. In one patient, intimal PAI-1 IRs partly bordered a thrombus and surrounded a large atheroma rich in cholesterol crystals. Our results suggest that PAI-1 is present in both cellular and extracellular components of human coronary atherosclerotic lesions.

[A case of primary cardiac amyloidosis with amyloid A protein].

A case of cardiac amyloidosis in a 46-year-old male is reported. He was admitted for dyspnea. Physical examination revealed third and forth heart sound and hepatomegaly. Radiographic heart-thoracic ratio was 53%. Electrocardiogram showed first degree A-V block, rS pattern in V1-V4 leads, and ambulatory electrocardiogram showed ventricular tachycardia. Echocardiogram revealed hypertrophy and highly refractile echoes of the left ventricular wall. Endomyocardial biopsy was performed and it demonstrated amyloid fibrils, which were characterized immunohistochemically as Amyloid A (AA) protein, which is generally a constituent in secondary amyloidosis. Urine protein electrophoresis showed lambda type Bence-Jones protein, but bone marrow biopsy was normal. There was no evidence of malignancy, chronic inflammatory disease, or collagen disease. This case was diagnosed as primary amyloidosis with AA protein. It is rare that, in spite of its being a case of primary amyloidosis, its constituent protein is AA protein.

Endothelin-2-converting enzyme from human renal adenocarcinoma cells is a phosphoramidon-sensitive, membrane-bound metalloprotease.

From the membrane fraction of cultured human renal adenocarcinoma (ACHN) cells, two endothelin-2-converting enzymes (ECE-2A and ECE-2B) were solubilized with detergent Lubrol PX and separated by hydrophobic butyl fast-performance liquid chromatography. The pH range of the converting activity of ECE-2B for big endothelin-1 (big ET-1), big ET-2, or big ET-3, was very narrow, and the optimal pH for each substrate was significantly different; the pH optimum for big ET-1 was 6.8 and that for big ET-2 or big ET-3 was 6.4. The ET-converting activity was abolished by phosphoramidon, 1,10-phenanthroline and EDTA but was not inhibited by thiorphan, E-64, leupeptin, PCMS, p-APMSF, or pepstatin A. The conversion efficiency for big ET-2 or big ET-3 by ECE-2B was approximately one-eighth of that for big ET-1. The molecular weight of ECE-2B was estimated to be 400 kDa by gel filtration. Because these characteristics of ECE-2B are very similar to those of ET-1-converting enzyme (ECE-1) in endothelial cells, these results raise the possibility that ECE-2B is identical to ECE-1 and that a single ECE physiologically converts all big ETs to the corresponding ETs in ET-producing cells.

Effect of phosphoramidon on big endothelin-2 conversion into endothelin-2 in human renal adenocarcinoma (ACHN) cells. Analysis of endothelin-2 biosynthetic pathway.

The biosynthetic pathway of endothelin (ET)-2 was analyzed in cultured ACHN cells. In the supernatant, we detected three ET-2-related peptides, ET-2, big ET-2(1-38) and big ET-2(22-38). Phosphoramidon decreased the amount of ET-2 and increased that of big ET-2(1-38) dose-dependently. The amount of big ET-2(1-37) did not significantly change. These results suggest that big ET-2 is composed of 38 and not 37 amino acid residues, and that a putative ET-2-converting enzyme (ECE-2) should be classified as a phosphoramidon-sensitive neutral metalloprotease, bearing a resemblance to the putative ET-1-converting enzyme (ECE-1) in endothelial cells.

[A case of malignant hemangioendothelioma effectively treated with intra-arterial continuous infusion of interleukin-2].

An 82-year-old female presented with a tumor in the right-frontal region and was diagnosed as MHE, based on the clinical and pathological findings. Increased LAK (lymphokine-activated killer cell) activity was observed during treatment with intraarterial continuous infusion of rIL-2. In addition, the decrease in tumor size was started when LAK activity became high. Treatments for MHE and mechanism of these therapies were discussed using data from this case and other authors' reports.