phosphoramidon and kelatorphan

Using cultured human aortic endothelial cells, we examined the effects of phosphoramidon, an endothelin converting enzyme (ECE) inhibitor, on the release of endogenous endothelin-1 (ET-1) and big endothelin-1 (big ET-1), and on the generation of ET-1 from exogenously applied big ET-1. Phosphoramidon, at concentrations of 10(-6) to 2 x 10(-4) M, caused a biphasic alteration of the ET-1 release, i.e., at lower concentrations of the drug, there were slight but unexpected increases of the release, whereas higher concentrations led to a decrease which is due to the drug-induced inhibition of ECE. The former effect appears to be based on the inhibition of ET-1 degradation by neutral endopeptidase 24.11 (NEP), since kelatorphan, a specific NEP inhibitor, produced a similar increasing effect on ET-1 release. Phosphoramidon enhanced the big ET-1 release from the cells in a concentration-dependent manner. When high concentrations of phosphoramidon were added, there was a dramatic increase in the release of big ET-1, which cannot be explained only by the drug-induced inhibition of ECE. This increase in big ET-1 release appeared to be partly due to a transient stimulation of the expression of prepro ET-1 mRNA. The amount of ET-1 generated from exogenously applied big ET-1 was markedly decreased by phosphoramidon in a concentration-dependent manner. In a similar fashion, phosphoramidon markedly inhibited ECE activity of the membrane fraction of cultured cells. Thus, ET-1 generation from exogenously applied big ET-1 reflects the functional phosphoramidon-sensitive ECE activities in human aortic endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Analgesics; Aorta; Aspartic Acid Endopeptidases; Cell Membrane; Cells, Cultured; Dipeptides; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Endothelium, Vascular; Glycopeptides; Humans; Metalloendopeptidases; Protein Precursors; RNA, Messenger; Sensitivity and Specificity; Stimulation, Chemical

1. NK1 and NK2 tachykinin receptors were characterized in guinea-pig and rat bronchopulmonary systems and in the vasculature of the rat by use of radioligand binding and/or functional studies. 2. The radioligands for NK1 and NK2 receptors ([3H]-SP and [3H]-pNKA, respectively) did not label tachykinin receptors in homogenates of rat lungs or bronchi. In contrast, in the guinea-pig, [3H]-SP bound with high affinity to these tissues (KD = 0.23 +/- 0.08 nM and 0.34 +/- 0.05 nM, for lungs and bronchi, respectively). The total number of binding sites was 4.6 fold greater in bronchus (Bmax = 135 +/- 27 fmol mg-1 protein) than in lung homogenates (Bmax = 29.3 +/- 0.1 fmol mg-1 protein). Furthermore, this binding was markedly displaced by CP-96,345 (pKi = 9.5 +/- 0.1) and RP 67580 (pKi = 7.6 +/- 0.1), antagonists of NK1 receptors, slightly displaced by SR 48968 (pKi = 6.6 +/- 0.1), but not affected by actinomycin D or L-659,877, antagonists of NK2 receptors. Specific binding of [3H]-pNKA, detected in guinea-pig bronchi (KD = 5.2 +/- 0.1 nM, and Bmax = 203 +/- 19 fmol mg-1 protein) but not in lungs, was similarly (40 to 53%) displaced by RP 67580 (1 microM), CP-96,345 (10 and 100 nM) or SR 48968 (10 and 100 nM). The displacement approximately doubled (87 to 91%) when SR 48968 (10 nM) was combined with either RP 67580 (1 microM) or CP-96,345 (10 nM), but not when RP 67580 was combined with CP-96,345. 3. In urethane-anaesthetized guinea-pigs, i.v. injections of the NK1 receptor agonists SP, [Pro9]-SP, [Sar9,Met(O2)11]-SP and septide, as well as the NK2 receptor agonists NKA and [Lys5,MeLeu9,NLeu10]-NKA(4-10) (0.1-10 micrograms kg-1, i.v.), dose-dependently increased lung inflation pressure. The most potent of these peptides were septide and [Lys5, MeLeu9,NLeu10]-NKA(4-10) (EC50 = 0.38 +/- 0.07 and 0.07 +/- 0.02 microgram kg-1, respectively). Interestingly, septide was 130 fold less potent than SP in displacing [3H]-SP from its binding sites in the guinea-pig lung, whereas it was 14 fold more potent than SP as a bronchoconstrictor. RP 67580 (0.3-5 mg kg-1, i.v.) and CP-96,345 (0.01-3 mg kg-1, i.v.) dose-dependently reduced the bronchoconstriction produced by the NK1 receptor agonists. Conversely, the NK2 receptor antagonists actinomycin D (1-10 mg kg-1, i.v.) and SR 48968 (0.03-0.3 mg kg-1, i.v.) inhibited specifically the responses induced by NK2 receptor agonists.4. In pentobarbitone-anaesthetized rats, the NK1 and NK2 receptor agonists (0.01-4 microg kg-1, i.v.)pro

Topics: Animals; Binding Sites; Blood Vessels; Bronchi; Dipeptides; Glycopeptides; Guinea Pigs; Lung; Male; Neprilysin; Neurokinin A; Pulmonary Circulation; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Tachykinin; Substance P; Tachykinins; Tritium

The deduced amino acid sequences of CALLA, a cell surface marker of human acute lymphocytic leukemia, and human enkephalinase (neutral endopeptidase, EC 3.4.24.11) were recently reported to be almost identical. We show that membranes of CALLA+ cells of the REH lymphoblastic cell line as well as blast cells derived from the blood or bone marrow of patients with acute lymphocytic leukemia display high enkephalinase activity. This activity was abrogated by several enkephalinase inhibitors at concentrations closely similar to those required to inhibit pure human enkephalinase. However, these compounds did not significantly modify the rate of REH cell proliferation in vitro. Hence, the functional role, if any, of the high peptidase activity in lymphoblastic cells remains to be established.

Topics: Antigens, Differentiation; Antigens, Neoplasm; Bone Marrow; Cell Division; Cell Line; Cell Membrane; Dipeptides; Glycopeptides; Humans; In Vitro Techniques; Neprilysin; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Thiorphan; Tumor Cells, Cultured