endothelin-1 and pepstatin

This study investigated the pruritogenic potency of cathepsin E, an aspartic protease, and its mechanisms in mice. An intradermal injection of cathepsin E to the rostral back elicited scratching, an itch-associated response, of the injection site. This action was inhibited by the aspartic protease inhibitor pepstatin A, the endothelin ET(A) receptor antagonist BQ-123, and the opioid receptor antagonists naltrexone and naloxone, but not by the H(1) histamine receptor antagonist terfenadine, the proteinase-activated receptor-2 antagonist FSLLRY-NH(2), or mast cell deficiency. Pepstatin A inhibited scratching induced by intradermal injection of the mast-cell degranulator compound 48/80, but not by tryptase, a mast-cell mediator. An intradermal injection of cathepsin E increased endothelin-1 levels in the skin at the injection site. Preproendothelin-1 mRNA was present in primary cultures of keratinocytes, and immunohistochemistry using an antibody recognizing endothelin-1 and big-endothelin-1 revealed immunoreactivity in the epidermis, especially in the prickle and granular cell layers, but not in the basal cell layer. These results suggest that cathepsin E is an endogenous itch inducer, and that its action is mediated at least in part by the production of endothelin-1 in the epidermis.

Topics: Animals; Behavior, Animal; Cathepsin E; Cells, Cultured; Endothelin Receptor Antagonists; Endothelin-1; Histamine Release; Keratinocytes; Male; Mice; Mice, Inbred ICR; Naloxone; Naltrexone; Narcotic Antagonists; Pepstatins; Peptides, Cyclic; Protease Inhibitors; Pruritus; Receptor, PAR-2; RNA, Messenger; Skin

To examine whether angiotensin II and endothelins produced in vascular smooth muscle cells can play roles in the regulation of mitogen-activated protein (MAP) kinase activity in vascular smooth muscle cells, we measured the activity of MAP kinases in cultured vascular smooth muscle cells, and determined effects of renin-angiotensin and endothelin systems activators and inhibitors. Angiotensin II and endothelin-1 produced an activation of MAP kinase activity in vascular smooth muscle cells, whereas the angiotensin receptor antagonist, losartan and the endothelin receptor antagonist, cyclo (D-alpha-aspartyl-L-prolyl-D-valyl-L-leucyl-D-tryptophyl, BQ123) inhibited the enzyme activity. MAP kinase activity in vascular smooth muscle cells was also inhibited either by the renin inhibitor pepstatin A or by the angiotensin-converting enzyme inhibitor captopril. The degree of the inhibition of MAP kinase activity by pepstatin A, captopril and losartan was almost the same. Renin produced a considerable increase in MAP kinase activity and the renin-induced MAP kinase activation was inhibited by pepstatin A. The endothelin precursor big endothelin-1 produced an increase of MAP kinase activity in vascular smooth muscle cells, whereas the endothelin-converting enzyme inhibitor phosphoramidon inhibited the enzyme activity. These findings suggest that functional renin-angiotensin system and endothelin system are present in vascular smooth muscle cells and these systems tonically serve to increase MAP kinase activity. It appears that renin or renin-like substances play the determining role in the regulation of renin-angiotensin system in vascular smooth muscle cells.

Topics: Angiotensin I; Angiotensin II; Angiotensin-Converting Enzyme Inhibitors; Animals; Captopril; Cells, Cultured; Dose-Response Relationship, Drug; Endothelin Receptor Antagonists; Endothelin-1; Endothelins; Glycopeptides; Losartan; Male; Mitogen-Activated Protein Kinases; Muscle, Smooth, Vascular; Pepstatins; Peptides, Cyclic; Protease Inhibitors; Protein Precursors; Rats; Rats, Wistar; Renin

1. It is generally accepted that endothelial cells secrete endothelin (ET) to the underlying media which mediates the contractile effects of ET. However, there is some evidence that animal vascular smooth muscle cells (VSMCs) also secrete ET. We cultured VSMCs from human vessels representative of a number of different vascular beds to determine whether human VSMCs endogenously secrete ET. 2. VSMCs explanted from adult arterial vessels secrete picomolar quantities of immunoreactive mature ET: coronary artery 226.6 +/- 58.8 pM/10(6) cells (n = 7), thoracic aorta 169.5 +/- 105.4 pM/10(6) cells (n = 3), left internal mammary artery 102.4 +/- 23.1 pM/10(6) cells (n = 3) and saphenous vein 69.4 +/- 19.9 pM/10(6) cells (n = 3), as well as from umbilical vein (HUVSMCs) 38.3 +/- 4.3 pM/10(6) cells (n = 3). Secretion of immunoreactive big ET-1 was also detected: coronary artery 249.1 +/- 59.4 pM/10(6) cells (n = 7), thoracic aorta 120.0 +/- 13.4 pM/10(6) cells (n = 3), left internal mammary artery 170.0 +/- 68.2 pM/10(6) cells (n = 3), saphenous vein 105.1 +/- 30.7 pM/10(6) cells (n = 3) and from umbilical vein 146.3 +/- 7.4 pM/10(6) cells (n = 3). Comparable, intracellular levels of immunoreactive big ET-1 and mature ET were also detected in cultured VSMCs. 3. Since enzyme-dispersed VSMCs are thought to be more differentiated and more closely resemble their in vivo counterparts, and these enzyme-dispersed VSMCs from human umbilical vein (HUVSMCs) also secreted the greatest levels of immunoreactive peptides, they were characterized further. Reverse transcription-polymerase chain reaction assay demonstrated that HUVSMCs express ET-1 mRNA. High performance liquid chromatography coupled to radioimmunoassay revealed that HUVSMCs secrete ET-1 and ET-3, in addition to big ET-1. However, levels of ET are not altered by 100 AM phosphoramidon,an inhibitor of metalloproteases or by 100 microM pepstatin A, an aspartyl protease inhibitor.4. In concordance, KD and Bmax values for [125I]-ET-l saturation binding are not altered in HUVSMC cultures incubated for 24 h with 100 microM phosphoramidon (431 +/- 218 PM and 31.1 +/- 12.7 fmol mg-1;mean =/- s.e.mean, n = 3) or 100 microM pepstatin A (381 +/- 169PM and 19.9 +/- 7.8 fmol mg-1, n = 3) as compared to controls (355 +/- 99 pM and 33.3 +/- 9.3 fmol mg-1; n = 3). This observation indicates the absence of an autocrine 'unmasking' effect for ET receptors.5. HUVSMCs synthesize and secrete immunoreactive ET-1, ET-3 and big ET-1, and p

Topics: Antimalarials; Base Sequence; Cells, Cultured; Chromatography, High Pressure Liquid; Endothelin-1; Endothelins; Female; Glycopeptides; Humans; In Vitro Techniques; Kinetics; Molecular Sequence Data; Muscle, Smooth, Vascular; Pepstatins; Protease Inhibitors; Protein Precursors; Proteins; Radioimmunoassay; RNA, Messenger; Umbilical Veins

1. Two-site enzyme-linked immunosorbent assays have been developed for the rapid, sensitive and non-isotopic measurement of endothelin-1 and big endothelin-1. The sensitivities of detection were 0.5 and 0.3 fmol/well, with ED50 values of 13 and 12 fmol/well for the endothelin-1 and big endothelin-1 assays, respectively. Each assay is highly selective for its corresponding antigen. The ET-1 assay showed no detectable cross-reactivity with ET-1-(1-20), indicating that the assay only recognizes the 21-amino acid biologically active peptide. 2. The two assays were used to measure the effects of two classes of protease inhibitor on the basal release of enothelin-1 and big endothelin-1 from cultured first-passage human umbilical vein endothelial cells. 3. The secretion of both peptides was time-dependent over 12 h. The metalloprotease inhibitor phosphoramidon (1 x 10(-4) mol/l) significantly reduced the amount of endothelin-1 secreted into the medium (P < 0.05), with a concomitant increase in the secreted levels of big endothelin-1 (P < 0.01). The aspartyl protease inhibitor, pepstatin A, also caused a significant decrease in the secretion of endothelin (P < 0.05). However, unlike phosphoramidon, there was no increase in the levels of big ET-1 compared with the controls. At these concentrations, neither inhibitor affected the viability of the cells as indicated by Trypan Blue exclusion. 4. The two assays permit the direct measurement of endothelin-1 and its precursor, and will be of use in the elucidation of the putative human endothelin-converting enzyme(s).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Antimalarials; Cells, Cultured; Endothelin-1; Endothelins; Endothelium; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Glycopeptides; Humans; Metalloendopeptidases; Pepstatins; Protein Precursors; Umbilical Veins

We examined four commercially available human cell lines for endothelin-converting-enzyme-(ECE) like activity and compared the results with primary porcine aortic endothelial cell enzymes. The cells that were investigated were 293 (transformed primary human embryonal kidney), Hep G2 (human hepatocellular carcinoma), HUVECs (human umbilical vein endothelial cells), and U937 (human histiocytic lymphoma). The relative ECE-like activities were determined in cytosolic and particulate extracts of each cell type. Enzyme activity against pro-ET-1 was measured at pH 4 and 7, using a C-terminal Trp-specific antibody to ET-1 radioimmunoassay and by high-performance liquid chromatography analysis of the enzyme hydrolysis products of pro-ET-1. Inhibition by EDTA at pH 7 or pepstatin at pH 4 was used to classify the pro-ET-1 processing enzymes from the human cell lines as either metallo- or aspartylproteinases. The particulate extract of the primary porcine aortic endothelial cells contained both aspartyl and metallo ECE-like enzymes. No ECE-like activity was present in either the cytosolic or particulate extracts of the U937 nor 293 cells. Neither the particulate extract of the Hep G2 cells nor the cytosolic extract of the HUVECs had any ECE-like activity. The cytosolic extract of the Hep G2 cells and the particulate extract of the HUVECs had an ECE-like activity at pH 7 that was inhibited by 10 mM EDTA, qualifying these enzymes as members of the metalloproteinase family.

Topics: Animals; Aorta; Aspartic Acid Endopeptidases; Cell Line; Chromatography, High Pressure Liquid; Edetic Acid; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Endothelium, Vascular; Humans; Hydrogen-Ion Concentration; Metalloendopeptidases; Pepstatins; Peptide Fragments; Protein Precursors; Radioimmunoassay; Swine; Tumor Cells, Cultured

CPAE endothelial cells were cultured in the presence of pepstatin, NH4Cl, or chloroquine in order to assess their effects on the secretion of endothelin-1 (ET-1). The first of these is an inhibitor of aspartyl proteases and the last two are known to neutralize acidic intracellular compartments. The pepstatin was encapsulated into liposomes to aid in its uptake, and uptake was confirmed by measuring the residual aspartyl protease activity in washed, lysed cells. Pepstatin had no effect (less than 5%) on the secretion of ET-1, 25 mM NH4Cl decreased secretion by 30-47%, and 25 microM chloroquine increased secretion by 37-79%. In contrast, each of these reagents is known to inhibit lysosomal degradation of intracellular proteins by 75-90%. Additionally, big ET was shown to be a very poor substrate, in terms of kcat/Km values, for aspartyl proteases. The rate constants were less than 10(4) M-1 s-1, which is approximately 1% of the value for the best substrates. The data, therefore, do not support a role for aspartyl proteases in the formation of ET-1. Similar to chloroquine, 0.5 microM monensin increased the secretion of ET-1 by 40-60%. Both of these reagents have previously been shown to increase the rate of constitutive secretion of peptides by affecting their partitioning between packaging into storage granules and constitutive secretion. The results would therefore provide supportive evidence for the existence of a storage form of ET-1 in endothelial cells.

Topics: Ammonium Chloride; Aspartic Acid; Aspartic Acid Endopeptidases; Cells, Cultured; Chloroquine; Chromatography, High Pressure Liquid; Endothelin-1; Endothelins; Endothelium; Hydrogen-Ion Concentration; Indicators and Reagents; Kinetics; Monensin; Pepstatins; Protein Precursors

A combination of HPLC elution patterns and peptide sequencing has been used to characterize two distinct activities present in subcellular fractions of bovine aortic endothelial cells (BAECs) capable of converting human big endothelin-1 (big ET-1) to mature (ET-1). A pepstatin-inhibitable activity with an acidic pH optimum present in a lysosome-enriched fraction cleaved big ET-1 at positions 18 and 21 at similar rates. A neutral pH activity present in a postlysosomal organelles subfraction was also able to convert big ET-1, and was inhibited by EDTA, but not by 1-chloro-3-tosylamido-4-phenyl-2-butanone (TPCK), an inhibitor of chymotrypsin-like serine proteases.

Topics: Animals; Aspartic Acid Endopeptidases; Cattle; Chromatography, High Pressure Liquid; Edetic Acid; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Endothelium, Vascular; Freezing; Hydrogen-Ion Concentration; Metalloendopeptidases; Pepstatins; Protein Precursors; Subcellular Fractions; Tosylphenylalanyl Chloromethyl Ketone

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.

Topics: Animals; Aorta; Chromatography, High Pressure Liquid; Cross Reactions; Endothelin-1; Endothelins; Endothelium, Vascular; Hydrogen-Ion Concentration; Muscle Contraction; Muscle, Smooth, Vascular; Pepstatins; Peptides; Protein Precursors; Radioimmunoassay; Swine