Secretion of endothelin and related peptides from renal epithelial cell lines

Using specific radioimmunoassays (RIAs) for endothelin (ET) and big ET, we have studied whether ET and related peptides are secreted from renal epithelial cell lines (LLCPK1 and MDCK) of non-endothelial origin. Dilution curves of extracts of conditioned media from both LLCPK1 and MDCK cell lines were parallel to those of standard porcine (p) ET and big pET in each RIA. Both cell lines incubated in serum-free medium secreted ET- and C-terminal fragment (CTF)-like immunoreactivity (LI) of big ET as a function of time. Reverse-phase HPLC coupled with both RIAs of the extracted media from both cell lines revealed a single component with ET-LI coeluting with pET(1-21) and several components with CTF-LI, one corresponding to the elution position of big pET(1-39), one to its CTF(22-39), and the others eluting earlier than CTF. These data indicate that endothelin and related peptides are synthesized by and secreted from cells other than endothelial cells.     

Characterization of endothelin converting enzyme in rat lung

An enzyme activity which converts human big endothelin (1-38) to endothelin (1-21) and a C-terminal fragment (CTF, 22-38) was identified in a plasma membrane fraction prepared from rat lung. The conversion activity was optimal at pH 4.0, was inhibited by Pepstatin-A (IC50 = 20 nM), but was not affected by TLCK, Aprotinin, PMSF, E-64, Bestatin, Phosphoramidon or Thiorphan at 40 microM. Metal ions activated the activity by 1.5 - 2.5 fold in the order of Mn+2 greater than Zn+2 = Ca+2 greater than Ba+2. These data suggest that a Pepstatin-A inhibitable, metal ion related aspartic protease may be involved in the conversion of big endothelin to endothelin in rat lung.     

A simple method for measurement of phosphoramidon-sensitive endothelin converting enzyme activity.

We have developed a rapid and convenient assay for measurement of the action of endothelin (ET) converting enzyme (ECE) using the scintillation proximity assay (SPA) principle. On incubation of [125I]big ET-1 at 37 degrees C for 0.5-6 hr with an enzyme preparation, the reaction was terminated by the addition of an ET-1-specific antibody formulated in a buffer designed to shift the pH to alkaline. The antibody was allowed to come to equilibrium for 1 hr at room temperature and the amount of ET-1 produced, detected in a single step by the addition of protein A SPA beads. Using this assay, ECE activities of enzyme preparations obtained from porcine cultured endothelial cells and rat lung were clearly detected. These activities were inhibited by phosphoramidon in a concentration-dependent manner. The SPA based assay is homogeneous requiring no separation steps and takes a half day to complete. This method is therefore suitable for the high throughput screening of potential ECE inhibitors.     

Intestinal intraepithelial lymphocyte derived angiotensin converting enzyme modulates epithelial cell apoptosis

Background & Aims: Intestinal adaptation in short bowel syndrome (SBS) consists of increased epithelial cell (EC) proliferation as well as apoptosis. Previous microarray analyses of intraepithelial lymphocytes (IEL) gene expression after SBS showed an increased expression of angiotensin converting enzyme (ACE). Because ACE has been shown to promote alveolar EC apoptosis, we examined if IEL-derived ACE plays a role in intestinal EC apoptosis. Methods: Mice underwent either a 70% mid-intestinal resection (SBS group) or a transection (Sham group) and were studied at 7 days. ACE expression was measured, and ACE inhibition (ACE-I, enalaprilat) was used to assess ACE function. Results: IEL-derived ACE was significantly elevated in SBS mice. The addition of an ACE-I to SBS mice resulted in a significant decline in EC apoptosis. To address a possible mechanism, tumor necrosis factor alpha (TNF-α) mRNA expression was measured. TNF-α was significantly increased in SBS mice, and decreased with ACE-I. Interestingly, ACE-I was not able to decrease EC apoptosis in TNF-α knockout mice. Conclusions: This study shows a previously undescribed expression of ACE by IEL. SBS was associated with an increase in IEL-derived ACE. ACE appears to be associated with an up-regulation of intestinal EC apoptosis. ACE-I significantly decreased EC apoptosis.     

Synthesis of a phostone glycomimetic of the endothelin converting enzyme inhibitor phosphoramidon.

The phostone analog of phosphoramidon, an inhibitor of endothelin converting enzyme, was synthesized from L-rhamnose. Coupling of the cyclic phosphonic acid with the dipeptide H-Leu-Trp-OMe gave, after deprotection and purification by reverse-phase HPLC, the desired phostone which exhibited an IC50 of 5.05+/-2.7 microM.     

Identification of endothelin converting enzyme in bovine lung membranes using a new fluorogenic substrate.

An enzyme partially purified from bovine lung membranes appears to be endothelin converting enzyme (ECE). This enzyme specifically cleaves big endothelin-1 (big ET-1) at the proper site, between Trp21 and Val22, with maximum activity at pH 7.5 and with a Km of roughly 3 microM, to produce endothelin-1 (ET-1) and C-terminal peptide (CTP). This same enzyme hydrolyzes the fluorogenic substrate succinyl-Ile-Ile-Trp-methylcoumarinamide to release the highly fluorescent 7-amino-4-methylcoumarin. The peptide derivative has the same amino acid sequence as big ET-1 and is a good substrate with a Km of about 27 microM. This enzyme is a metalloproteinase. It is not inhibited by five common proteinase inhibitors (pepstatin A, PMSF, NEM, E-64 and thiorphan) but it is inhibited by phosphoramidon and chelating compounds. The apoenzyme is restored to nearly full activity by a zinc-EDTA buffer with pZn = 13.     

Differential regulation of junctional complex assembly in renal epithelial cell lines

Several signaling pathways that regulate tight junction and adherens junction assembly are being characterized. Calpeptin activates stress fiber assembly in fibroblasts by inhibiting SH2-containing phosphatase-2 (SHP-2), thereby activating Rho-GTPase signaling. Here, we have examined the effects of calpeptin on stress fiber and junctional complex assembly in Madin-Darby canine kidney (MDCK) and LLC-PK epithelial cells. Calpeptin induced disassembly of stress fibers and inhibition of Rho GTPase activity in MDCK cells. Interestingly, calpeptin augmented stress fiber formation in LLC-PK epithelial cells. Calpeptin treatment of MDCK cells resulted in a displacement of zonula occludens-1 (ZO-1) and occludin from cell-cell junctions and a loss of phosphotyrosine on ZO-1 and ZO-2, without any detectable effect on tight junction permeability. Surprisingly, calpeptin increased paracellular permeability in LLC-PK cells even though it did not affect tight junction assembly. Calpeptin also modulated adherens junction assembly in MDCK cells but not in LLC-PK cells. Calpeptin treatment of MDCK cells induced redistribution of E-cadherin and -catenin from intercellular junctions and reduced the association of p120ctn with the E-cadherin/catenin complex. Together, our studies demonstrate that calpeptin differentially regulates stress fiber and junctional complex assembly in MDCK and LLC-PK epithelial cells, indicating that these pathways may be regulated in a cell line-specific manner. calpeptin; tight junctions; adherens junctions; Rho; cadherin; p120ctn     

Degradation of the Alzheimer's amyloid beta peptide by endothelin converting enzyme

The deposition of β-amyloid (Aβ) peptides in the brain is an early and invariant feature of all forms of Alzheimer's disease (AD). As such, a major focus of AD research has been the elucidation of the mechanisms responsible for the generation of Aβ. As with any peptide, however, the degree of Aβ accumulation is dependent not only on its production, but also on the mechanisms responsible for its removal. In cell-based and in vitro assays we have identified endothelin-converting enzymes (ECEs) as novel Aβ-degrading enzymes that appear to cleave predominately in an intracellular compartment. Overexpression of ECE-1 in cells that lack endogenous ECE activity reduces Aβ accumulation by up to 90%, and this effect is completely reversed by treatment of the cells with phosphoramidon. Additionally, we have shown that recombinant soluble ECE-1 is capable of hydrolyzing synthetic Aβ40 and Aβ42 in vitro at multiple sites, with a favorable kinetic profile. While several enzymes have been identified that can degrade Aβ in vitro , only neprilysin has thus far been reported to influence Aβ accumulation in the brains of knock-out mice. To examine the physiological role of ECE activity on Aβ accumulation in the brain we compared the amount of Aβ in wild-type and ECE-2 null mice. A significant elevation in both Aβ40 and Aβ42 was observed in the ECE-2 null animals compared to their wild-type littermates. These data provide direct evidence of a physiological role for this enzyme in limiting Aβ accumulation in the brain.
Acknowledgements: Supported by Smith Fellowships to C.E. and E.E., a Bursak Fellowship to E.E., and by the Mayo Foundation for Medical Education and Research.     

Rhamnose moiety of phosphoramidon is not required for in vivo inhibition of endothelin converting enzyme.

Experiments were conducted to determine the importance of the carbohydrate moiety of phosphoramidon in the inhibition of the pressor response to big endothelin-1 in anesthetized rats. Big endothelin-1 produced a 42% increase in mean arterial pressure which was nearly abolished by co-infusion of phosphoramidon. Similarly, when an analog of phosphoramidon lacking the rhamnose group (phosphoryl-leu-trp-OH) was co-infused, a significant attenuation of the pressor response was observed. These findings indicate that the rhamnose moiety of phosphoramidon is not responsible for the distinguishing this compound as an inhibitor of the response to big endothelin-1 in the rat.     

Inhibition by angiotensin converting enzyme inhibitors of endothelin secretion from cultured human endothelial cells.

We conducted a study to determine whether angiotensin converting enzyme inhibitors (ACEIs) inhibit endothelin secretion from cultured human endothelial cells. Confluent umbilical vein endothelial cells were incubated in multi-well plates with culture medium containing either captopril (10(-6), 10(-5), 10(-4) M) or enalaprilat (10(-7), 10(-6), 10(-5) M) for 6 hours. Immunoreactive endothelin in the medium was measured by radioimmunoassay. Calf serum (CS) stimulated endothelin release in a concentration-dependent manner, and both ACEIs inhibited 5% CS-stimulated endothelin release in a concentration-dependent manner. To explore the mechanisms of ACEI-induced suppression of endothelin release, the effects of angiotensin II (10(-8), 10(-7), 10(-6) M), angiotensin converting enzyme (0.1, 1, 10 mU/ml), bradykinin (10(-8), 10(-7), 10(-6) M), and sodium nitroprusside (10(-6), 10(-5), 10(-4) M) on endothelin release were also examined. Although angiotensin II and angiotensin converting enzyme had no significant effect on endothelin release, concentration-dependent suppression occurred with bradykinin and sodium nitroprusside. These results indicate that ACEIs inhibit the stimulated release of endothelin from human endothelial cells, and provide indirect evidence that ACEI-induced ET suppression may be mediated via potentiation of autacoid formation from the cells.     

Comparative properties of untreated andN-nitrosobenzylmethyl-amine-transformed rat esophageal epithelial cell lines

Summary A culture system utilizing rat esophageal epithelial cells has been developed. Four normal and eightN-nitrosobenzylmethylamine-treated lines were compared with respect to chromosome number, anchorage-independent growth in agarose, and tumorigenic potential in syngenic rats. All cell lines were aneuploid with nine in the near-tetraploid range and three in the near-diploid range. No relation between tumorigenic potential and chromosome number or structure was apparent. Similarly, anchorage-independent growth in agarose did not correlate with tumorigenic potential. Three of the 12 immortalized lines (two carcinogen-treated and 1 untreated) induced well-differentiated squamous cell carcinomas in syngeneic rats. These tumors had weak metastatic potentials suggesting that tumorigenic potential and metastatic ability are separately controlled. These cell lines will be useful for the investigation of factors involved in the conversion of immortalized rat esophageal epithelial cell lines to lines of high metastatic potential.     

Characterization of endothelin converting enzyme activities in soluble fraction of bovine cultured endothelial cells

Endothelin converting enzyme activities in the soluble fraction of cultured bovine aortic endothelial cells were characterized. The two major endothelin converting enzyme activities were eluted from a hydrophobic chromatography column and the elution profile of the endothelin converting enzyme activities was the same as that of cathepsin D activities. These activities had a same pH optimum at pH 3.5 and were effectively inhibited by pepstatin A. Furthermore, anti-cathepsin D antiserum absorbed these activities as well as cathepsin D activity. Immunoblotting analysis using the antiserum showed the major active fractions have immunostainable components of identical molecular weights with cathepsin D. From these results, we concluded that the major endothelin converting activities in the soluble fraction of endothelial cells are due to cathepsin D. In addition to these cathepsin D activities, a minor endothelin converting enzyme activity with an optimum pH at 3.5 was found, which does not have angiotensin I generating (cathepsin D) activity from renin substrate and needs much higher concentrations of pepstatin A to inhibit the activity than cathepsin D.     

Cardiorenal actions of endothelin, Part I: Effects of converting enzyme inhibition

We have evaluated the effects of endothelin-1, with and without captopril administration, on the circulating concentration of aldosterone in pentobarbital (60 mg/kg) anesthetized rats. Following surgery, rats (N = 5/group) were infused with saline intravenously, at a rate of 0.024 ml/min, with or without captopril (5 mg kg-1 hr-1) administration throughout the entire experiment. All rats were allowed 60 min to stabilize and 3 X 20 min control clearances collected. Endothelin-1 (100 eta kg-1 min-1) was then added to the infusate for 30 min. Plasma aldosterone concentration increased from 60 +/- 7 eta/dl to 171 +/- 14 eta/dl (p less than 0.01) with endothelin alone and from 101 +/- 6 eta/dl to 210 +/- 54 eta/dl (p less than 0.01) in rats treated with endothelin plus captopril. The endothelin-induced increment in blood pressure was not altered by captopril treatment. However, the endothelin-induced decrement in renal function was markedly attenuated in rats treated with captopril. These data demonstrate that endothelin stimulates the release of aldosterone from the rat adrenal and that the angiotensin II is not involved in this response. These data also demonstrate that the endothelin-induced systemic vasoconstriction is not affected by captopril whereas the endothelin-induced changes in renal function are abolished by captopril.     

Establishment of epithelial and fibroblast cell cultures and cell lines from primary renal cancer nephrectomies

Renal cell carcinoma (RCC) is one of the most lethal urogenital cancers and effective treatment of metastatic RCC remains an elusive target. Cell lines enable the in vitro investigation of molecular and genetic changes leading to renal carcinogenesis and are important for evaluating cellular drug response or toxicity. This study details a fast and easy protocol of establishing epithelial and fibroblast cell cultures or cell lines concurrently from renal cancer nephrectomy tissue. The protocol involves mechanical disaggregation, collagenase digestion and cell sieving for establishing epithelial cells while fibroblast cells were grown from explants. This protocol has been modified from previous published reports with additional antibiotics and washing steps added to eliminate microbial contamination from the surgical source. Cell characterisation was carried out using immunofluorescence and quantitative polymerase chain reaction. Eleven stable epithelial renal tumour cell lines of various subtypes, including rare subtypes, were established with a spontaneous immortalisation rate of 21.6% using this protocol. Eight fibroblast cell cultures grew successfully but did not achieve spontaneous immortalisation. Cells of epithelial origin expressed higher expressions of epithelial markers such as pan‐cytokeratin, cytokeratin 8 and E‐cadherin whereas fibroblast cells expressed high α‐smooth muscle actin. Further mutational analysis is needed to evaluate the genetic or molecular characteristics of the cell lines.