The 21-amino acid vasoconstrictor peptide endothelin (Et) contains two disulfide bonds. We investigated the importance of the outer disulfide bond in Et-1 by replacing it with an amide linkage. Bioactivity was assessed in an isolated guinea pig lung preparation (perfused at constant flow with Ringer's solution/0.5% albumin) in which pulmonary artery pressure was monitored. Et-1 produced concentrationdependent pulmonary vasoconstriction at concentrations of 1 x 10-10 M and higher. [Dprl Asp15Et-1 (where Dpr is diamino propionic acid), in which the outer disulfide was replaced by an amide bond and the inner disuffide was left intact, showed no agonist activity at 1 x 10-6 M but 1 x 10-7 M [Dpr ispls]Et-1 inhibited Et-1-induced pulmonary vasoconstriction: effects of 1 x 10-10 M, 2 x 10-1' M, and 1 x 10-9 M Et-1 were inhibited by 98%, 75%, and 65%, respectively. Furthermore, this analog did not alter pulmonary vasoconstriction induced by thrombin, norepinephrine, or, most signiflcantly, Et-3. A monocyclic Et-1 analog with the same sequence but in which the amide bond was not formed showed weak pulmonary vasoconstrictor activity (300-500 times less potent than Et-1) but had no antagonist acty In addition, both the monocyclic control peptide and [Dpr',Aspls]Et-1 competed effectively with 12SI-labeled Et-1 for binding to cultured rat pulmonary artery smooth muscle cells. Thus, an Et-1 structural analog produced by replacement of the outer disulfide bond with an amide linkage displayed potent and specific Et-1 antagonism.Endothelin (Et) is a 21-amino acid potent vasoconstrictor peptide that is secreted by endothelial cells (1) and appears to be released in response to thrombin (1, 2) and other stimuli. Three distinct endothelin isopeptides exist (Et-1, Et-2, and Et-3) (3), all of which cause vasoconstriction in a number of vascular beds with an apparent potency order of Et-2 > Et-1 > Et-3 (4). Two distinct Et receptors have been cloned; one appears to be specific for Et-1 (5) whereas the other interacts with all three Et isopeptides (6). Competitive binding studies suggest that there are multiple classes of receptors with different affinities for Et isopeptides and that the distribution of the receptor subtypes is tissue specific (7-9). A recent report describes a substance P-based antagonist that blocks a spectrum of neuropeptide responses, including certain Et responses (10). To our knowledge, this is the only report of an Et antagonist, but there have been no reports of an antagonist specific to Et.To understand Et physiology definitively, antagonists specific for receptor subtypes would be helpful. In this report we demonstrate a structural analog of Et-1 that potently inhibits Et-1-induced pulmonary vasoconstriction but does not interfere with vasoconstriction secondary to norepinephrine, thrombin, or (most importantly) Et-3. MATERIALS AND METHODSSynthesis of Peptides. [Dpr1,Asp15IEt-1 § (where Dpr is diaminopropionic acid) was synthesized by solid-phase procedures (11) using a Biosearch model 9500 peptide ...
We examined whether the generation of tumor necrosis factor (TNF-alpha) after lipopolysaccharide (LPS) challenge contributes to increases in lung vascular permeability and water content. Guinea pig lungs perfused at constant flow with Ringer-albumin solution (0.5 g/100 ml) were challenged for 120 min with LPS (Escherichia coli; final concentration 33 ng/ml; n = 5). Lung effluent samples were assayed for TNF-alpha activity using the modified L-929 fibroblast cytolytic assay. TNF-alpha concentrations increased in a time-dependent manner with a peak value of 100 +/- 20 pg/ml noted 90-120 min after LPS. Human neutrophils [polymorphonuclear leukocytes (PMN; 2 x 10(7)] added to the perfusion solution after endotoxin challenge (n = 5) produced a threefold increase in lung tissue myeloperoxidase (MPO) activity over control values. PMN, added after LPS and activated using phorbol 12-myristate 13-acetate (PMA; 5 x 10(-9) M; n = 6), produced three- to sixfold increases in mean pulmonary arterial pressure (Ppa) and pulmonary capillary pressure (Pcap), wet weight-to-dry weight ratio (W/D), and the pulmonary capillary filtration coefficient (Kf,c) over control values (P < 0.05). Activation of PMN with PMA in non-LPS-challenged lungs produced only threefold increases in Ppa and Pcap and did not change W/D and Kf,c. Infusion of an anti-TNF-alpha antibody before the LPS challenge reduced by approximately 50% the increases in Ppa, Pcap, MPO content, Kf,c, and lung wet weight gain (P < 0.05). Therefore, endotoxin-induced TNF-alpha generation in lungs significantly contributes to pulmonary sequestration of PMN. Activation of the sequestered PMN increases pulmonary vascular permeability and tissue water content.
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