Conversion of porcine big endothelin to endothelin by an extract from the porcine aortic endothelial cells

Conversion of porcine big endothelin (big ET) to endothelin (ET) by an extract from cultured porcine aortic endothelial cells was investigated using a radioimmunoassay (RIA) specific for ET and reverse-phase high performance liquid chromatography (RP-HPLC). When big ET was incubated with the extract at an acid pH in the presence of E-64, a cysteine protease inhibitor, the amount of immunoreactive-ET (IR-ET) in the incubation mixture was greatly increased and the optimum pH for this increased reaction was 4.0. The extract-induced increase in IR-ET was completely inhibited by pepstatin-A. These immunoreactive alterations correlated with those in the vasoconstrictor activity. When the incubation mixture of big ET with the cell extract was applied to the RP-HPLC, the IR-ET eluted at the same retention time as seen with synthetic porcine ET. We suggest that a pepstatin-sensitive aspartic protease is involved in the conversion of big ET to ET in vascular endothelial cells.     

Determination of immunoreactive endothelin in medium from cultured endothelial cells and human plasma

We have developed a sensitive and selective radioimmunoassay for porcine/human endothelin (ET1). The assay has a detection limit of 0.62 pg/tube and exhibits no cross-reactivity to atrial natriuretic peptide, arginine vasopressin, or angiotensin II. Procedures were developed for extraction of endothelin from human plasma samples and samples of buffer from endothelial cell incubations using C18 Sep-Pak extraction cartridges. The mean recovery following extraction was approximately 80%. Both bovine and porcine aortic endothelial cells were found to produce immunoreactive endothelin (IR-ET) with porcine cells producing 4.7 +/- 1.1 ng of IR-ET/mg cell protein after 6 hours. Human plasma samples were extracted, assayed and found to contain a mean concentration of 2.0 +/- 0.4 pg/ml of IR-ET.     

Phosphoramidon inhibits the generation of endothelin-1 from exogenously applied big endothelin-1 in cultured vascular endothelial cells and smooth muscle cells

When cultured porcine aortic endothelial cells (ECs) were incubated with porcine big endothelin-1 (bit ET-1(1-39)), there was a time-dependent increase in immunoreactive (IR)-ET in the culture supernatant, in addition to an endogenous IR-ET release fron the cells. Reverse-phase HPLC of the culture supernatant revealed one major IR-ET component corresponding to the elution position of synthetic ET-1, thereby indicating that the additional increase in IR-ET was due to the conversion of big ET-1 to mature ET-1(1-21). Phosphoramidon, a metalloproteinase inhibitor, strongly suppressed this increase in IR-ET as well as the endogenous IR-ET release. Cultured vascular smooth muscle cells (VSMCs) also released IR-ET. The apparent conversion of exogenously applied big ET-1 to ET-1 and its inhibition by phosphoramidon were observed using cultured VSMCs, although the enzyme inhibitor did not influence the basal secretion of IR-ET from VSMCs. These results suggest that both cultured ECs and VSMCs can generate ET-1 from exogenously applied big ET-1 via action of the same type of phosphoramidon-sensitive metalloproteinase, which is also involved in the endogenous ET-1 generation in ECs.     

Phosphoramidon, a metalloproteinase inhibitor, suppresses the secretion of endothelin-1 from cultured endothelial cells by inhibiting a big endothelin-1 converting enzyme

Time-dependent secretion of immunoreactive-endothelin (IR-ET) from cultured porcine aortic endothelial cells was markedly suppressed by phosphoramidon is due to proteinase inhibitor. Analysis of the culture supernatant with or without phosphoramidon by reverse-phase high performance liquid chromatography confirmed that the suppression of IR-ET secretion by phosphoramidon is due to a decreae in secretion of endothelin-1-like materials. The secretion of the C-terminal fragment (CTF, 22-39)-like materials of big ET-1 was also decreased by phosphoramidon, whereas there was an increased secretion of big ET-1-like materials. These data strongly suggest that phosphoramidon suppresses the secretion of ET-1 from cultured endothelial cells by inhibiting the conversion of big ET-1 to ET-1. It is most likely that phosphoramidon-sensitive metalloproteinase is responsible for the processing of big ET-1 in vascular endothelial cells.     

Endothelin in human plasma and culture medium of aortic endothelial cells--detection and characterization with radioimmunoassay using monoclonal antibody

We have developed monoclonal (KY-ET-1-I) and polyclonal (ET-F5) antibodies against endothelin-1 (ET-1) and established sensitive radioimmunoassays (RIAs) with different specificities. The RIA with KY-ET-1-I detected ET-1, ET-2 and ET-3, while the RIA with ET-F5 recognized ET-3 very weakly. Using these RIAs, we have investigated the concentration and molecular forms of ET-1-like immunoreactivity (-LI) in culture medium of bovine aortic endothelial cells and human plasma. Culture medium of endothelial cells contained two major components compatible with big ET and ET-1. ET-1-LI was also detected in human plasma. ET-1-LI in human plasma consisted of apparent two components, the small molecular form emerging at the position of ET-1 and the large form with the peak eluting at the preceding fraction of the elution position of big ET. The concentration of the small form of ET in human plasma was about 5 pg/ml.     

Concomitant secretion of big endothelin and its C-terminal fragment from human and bovine endothelial cells

A specific radioimmunoassay (RIA) for the carboxyl-terminal fragment (CTF) of big porcine endothelin (pET), an intermediate form of pET, was established to characterize big ET-like and its CTF-like immunoreactivity (LI) secreted from cultured bovine and human endothelial cells (EC). The antibody used crossreacted equally with big pET(1-39) and its CTF(22-39), but not with pET(1-21). Serial dilution curves of the culture media from bovine and human EC were parallel to that of standard CTF. Reverse-phase HPLC coupled with RIAs for big ET and ET of the culture media from bovine and human EC revealed essentially the same elution profiles: two major CTF-LI components, one corresponding to big pET(1-39) and the other to its CTF(22-39), in addition to one major ET-LI component corresponding to pET(1-21). The amounts of CTF-LI were almost equal to that of ET-LI on a molar basis. These data suggest that big ET is processed by a putative ET converting enzyme to yield its CTF and the mature ET(1-21) in EC.     

Detection of immunoreactive endothelin in plasma of hemodialysis patients

Two types of radioimmunoassay (RIA) methods for measuring endothelin (ET) in human plasma were developed. One was an extraction procedure using a Sep-Pak C18 cartridge, the other being a direct method. By the extraction method, plasma ET levels were lower than the detectable limit (7 pg/ml) in normal subjects and elevated in hemodialysis patients. The absolute values obtained via the direct method were 20-times higher than those from extraction. Gel-filtration experiments revealed that this discrepancy was mainly due to immunoreactive (IR-) endothelin-like substances of high molecular mass near 11.6 kDa (large IR-ET). Extraction of the peptide by the C18 cartridge could eliminate interference by large IR-ET and is important in the accurate measurement of ET concentrations in plasma.     

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.     

Stimulation of bovine endothelial cell angiotensin-I-converting enzyme activity by cyclic AMP-related agents

Bovine pulmonary artery endothelial cells in culture were used to assess the influence of cyclic nucleotides, isoproterenol (beta adrenergic agonist), and theophylline (phosphodiesterase inhibitor) on angiotensin-I-converting enzyme (ACE) activity of the cells and culture medium. Dibutyryl cAMP (10(-3) M) but not cAMP or dibutyryl cGMP stimulated angiotensin-I-converting enzyme (ACE) activity of cells in culture approximately 50-100% but had little influence on ACE activity of the medium. Theophylline at 10(-3) M concentration produced a three- to fourfold stimulation of both cellular and medium ACE activity. Isoproterenol by itself had no effect on cellular ACE activity but produced a stimulatory effect at 10(-7)-10(-5) M concentration after pretreatment of cells for 24 hr with 10(-4) M theophylline. The results support the concept that ACE activity of endothelial cells is influenced by the cyclic AMP system. ACE activity in cells and that released into medium may be under different regulatory controls.     

Metabolism of endothelial cell-bound lipoprotein lipase. Evidence for heparan sulfate proteoglycan-mediated internalization and recycling

Lipoprotein lipase (LPL) hydrolyzes triglyceride in plasma lipoprotein primarily while bound to vascular endothelial cells. LPL metabolism by cultured endothelial cells was studied. Purified radioiodinated bovine LPL bound to porcine aortic endothelial cells at 4 degrees C with an association constant of 0.18 x 10(7) m-1. Analysis of the time course of LPL dissociation from endothelial cells at 4 degrees C yielded a dissociation rate constant of 3.9 x 10(-6)s-1. After 1 h at 37 degrees C, 28% of the LPL initially bound to the cell surface was no longer releasable by heparin or trypsin treatments, suggesting that LPL was internalized by the cells. Addition of heparin to the medium or pretreatment of the cells with heparinase markedly reduced the amount of LPL internalized, establishing a requirement for cell surface heparan sulfate proteoglycans in the process. When cells containing internalized LPL were incubated at 37 degrees C, a time-dependent increase in the amount of LPL in the medium and a corresponding decrease in LPL associated with the cells was found. This suggested that internalized LPL was released back into the medium. The catalytic activity, molecular size, and heparin-binding characteristics of the released LPL was similar to native LPL. Addition of either heparin, heparinase, or excess unlabeled LPL to prevent the rebinding of released 125I-LPL to the cell surface increased the amount of 125I-LPL present in the medium, suggesting that there is a process of recycling of 125I-LPL bound to the cell surface. Studies examining the effect of pH on dissociation of LPL from its binding site showed less dissociation of cell surface bound LPL at pH 5.5 compared with pH 7.4 and 8.5. These results suggest that even at acidic pH as in endocytotic vesicles, LPL remains bound to proteoglycans and this may facilitate the recycling of internalized LPL molecules.     

Release of an endothelial cell growth factor from cultured porcine thyroid follicles

It has been proposed from in vivo studies that thyroid angiogenesis during thyroid enlargement may be due to paracrine mitogenic factors released by epithelial thyroid cells. To study this paracrine growth regulating communication between thyroid cells and endothelial cells in vitro, culture medium from isolated porcine thyroid follicles was investigated for a growth promoting effect on porcine aortal endothelial cells. Serum-free conditioned medium (CM) from thyroid follicles in suspension culture contains a dose-related mitogenic activity which stimulates endothelial cell growth up to 197%. Stimulation of the thyroid follicles with TSH (1 mU/ml) significantly reduced the mitogenic activity for endothelial cells in CM to 131%. Thyroid hormones had no influence on mitogenic activity in CM. When follicles were treated with iodide (20 microM) during CM production, no proliferation of endothelial cells was observed by this CM. In contrast, CM from epidermal growth factor-treated thyroid follicles significantly enhanced the mitogenic activity for endothelial cells up to 235%. The mitogenic activity was precipitable by saturated ammonium sulfate, showed high affinity to heparin by chromatography on heparin-sepharose, and was abolished after treatment of CM with trypsin. On gel electrophoresis the heparin-binding fraction showed a double band with a mol wt of 15 and 15.5 k. These data show a paracrine mitogenic activity on endothelial cells released by thyroid follicles which is regulated by TSH, epidermal growth factor, and iodide in parallel with the direct effect of these substances on thyroid cell growth. The data suggest that the mitogenic factor is a polypeptide, which belongs to the heparin-binding growth factors.     

Conversion of big endothelin-1 to endothelin-1 by two types of metalloproteinases derived from porcine aortic endothelial cells

Incubation of big endothelin-1 (big ET-1(1-39] with either the cytosolic or membrane fraction obtained from cultured endothelial cells, resulted in an increase in immunoreactive-endothelin (IR-ET), which was markedly inhibited by metal chelators. Phosphoramidon, a metalloproteinase inhibitor, specifically suppressed the membrane fraction-induced increase in IR-ET, whereas the increase in IR-ET observed with the cytosolic fraction was not influenced by phosphoramidon. Reverse-phase (RP)-HPLC of the incubation mixture of big ET-1 with the cytosolic or membrane fraction revealed one major IR-ET component corresponding to the elution position of synthetic ET-1(1-21). Simultaneously, immunoreactivities like the C-terminal fragment (CTF22-39) of big ET-1 were present, as deduced from the RP-HPLC coupled with the radioimmunoassay for CTF. Our results indicate the presence of two types of metalloproteinases, which convert big ET-1 to ET-1 via a single cleavage between Trp21 and Val22, in vascular endothelial cells.     

Radioimmunoassay for endothelin and immunoreactive endothelin in culture medium of bovine endothelial cells

Using a synthetic 21-residue endothelin as antigen, we have produced an antiserum for endothelin and developed a specific and sensitive radioimmunoassay (RIA) for endothelin. The minimum detection limit of the RIA was 1 pg/tube. Immunoreactive (ir-) endothelin was extracted from the culture medium by Bondelute C8 column. The ir-endothelin in the culture medium of endothelial cells (EC) from bovine pulmonary artery and carotid artery was 1.48 ng/ml and 3.31 ng/ml, respectively. Reverse-phase high performance liquid chromatography coupled with the RIA revealed that ir-endothelin in the culture medium comprised one major component corresponding to synthetic endothelin. In addition, the cultured EC of bovine pulmonary artery were specifically stained by immunohistochemical technique. These results suggest that endothelin could be produced in the EC of the pulmonary and carotid arteries besides the aorta. The RIA presented in this study could be an useful tool to investigate the pathophysiologic significance of endothelin.     

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.     

Presence of immunoreactive endothelin in human saliva and rat parotid gland.

The presence of immunoreactive endothelin (IR-ET) in human saliva and rat parotid gland was investigated by radioimmunoassay. The IR-ET concentration (mean +/- SEM) in saliva taken from normal volunteers was 2.0 +/- 0.2 pmol/l (n = 15). The IR-ET concentration in rat parotid gland was 19.2 +/- 2.2 fmol/g wet weight (n = 10). Fast protein liquid chromatography (FPLC) of human saliva extract revealed 6 peaks; one peak eluting in the void volume, one in a position between ET-1 and -3, and the other four in the positions of synthetic ET-1, -2, -3 and big ET(1-38), respectively. A similar pattern of rat parotid gland extract was noted with FPLC, except that there was no peak after the void volume. Presence of endothelin, a potent growth factor, in saliva and salivary gland points to a role in maintaining the integrity of the oral and gastrointestinal tract mucosa.     

Androgens and FSH affect androgen receptor and aromatase distribution in the porcine ovary

In the present study the authors investigated whether androgens could interact with FSH to induce aromatase and androgen receptor expression in porcine granulosa cells. Dissected whole porcine follicles (small, medium, and large) were incubated for 8 hours in M199 medium supplemented with testosterone (10(-7) M), FSH (100 ng/ml) or both those hormones. After incubation, the follicles were fixed and immunostained to visualise androgen receptor and aromatase. In cultures of granulosa cells isolated from small and large follicles, oestrogen secretion was measured by appropriate RIA. Incubation of follicles with testosterone and FSH increased aromatase immunoreactivity in preantral and early antral (i.e. small) follicles. The immunostaining for androgen receptor was slightly higher in medium follicles, while such hormonal stimulation had no effect on small and large follicles. Moreover, granulosa cells isolated from small follicles cultured with both testosterone and FSH produced more estradiol than control cultures (40 pg vs. 100 pg/10(5) cells). The level was relatively close to that obtained in the culture of control granulosa cells isolated from large preovulatory follicles (105 pg/10(5) cells). These results indicate that testosterone acts synergistically with FSH to increase aromatase expression in the small porcine follicles.