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The rabbit isolated superior mesenteric artery, when precontracted with phenylephrine, relaxes in a sustained manner in less than 30 min when exposed to recombinant interleukin-1 (IL-1), and this is a prostaglandin (PG)-dependent, endothelium- and 5-lipoxygenase-independent process. We have studied the dependency of IL-1-induced relaxation on protein synthesis and trafficking using isolated rings of rabbit mesenteric arteries. Three chemically unrelated protein synthesis inhibitors (PSIs) (cycloheximide, anisomycin and puromycin) completely prevented the response to IL-1. Moreover, the PSIs reversed IL-1-induced relaxations within 15 to 30 min of application. The amplitude and/or the duration of the relaxation induced by arachidonic acid and iloprost (a PGI2 mimetic) were partially inhibited by cycloheximide treatment, but not those induced by nitroglycerin or by cromakalim. However, tissues initially relaxed by IL-1 and then recontracted by a PSI are still able to relax when challenged with either arachidonic acid or iloprost, suggesting that cyclooxygenase is not depleted and that the responsiveness to PGs is intact under these conditions. Manoalide, thioether amide glycerophosphocholine (type II phospholipase A2 inhibitors) or brefeldin A (an inhibitor of intracellular protein trafficking) did not inhibit IL-1-induced relaxation. The data suggest the involvement of newly synthesized protein(s) with a very rapid turnover in the vascular response to IL-1. PSIs probably act at several levels to inhibit the relaxant response to IL-1, but depletion of tissue cyclooxygenase does not appear to be the limiting mechanism for the PSI effect on IL-1-induced relaxations.(ABSTRACT TRUNCATED AT 250 WORDS)

The isolated rabbit jugular and human umbilical veins respond to bradykinin (BK) by contractions that are mediated by the BK B2 type receptors. In this report, the pharmacology of recently developed BK B2 receptor antagonists is assessed by using these preparations. The nonpeptide kinin antagonist WIN 64338 (phosphonium, [[4-[[2-[[bis(cyclohexylamino)methylene]amino]- 3-(2-naphthalenyl)-1-oxopropyl]amino]phenyl]methyl]tributyl chloride monohydrochloride) demonstrates competitive and surmountable antagonism of BK in both the jugular and the umbilical veins (pA2 values of 6.14 and 5.99, respectively). WIN 64338 shows selectivity in its antagonist action as it does not inhibit the effect of various other contractile agents in either of the preparations. HOE-140 (D-Arg[hydroxyproline3,beta-thienylalanine5, D-Tic7, octahydroindol-2-yl-carbonyl residue8]-BK), a "second generation" peptide antagonist of BK, behaves as an insurmountable and irreversible antagonist in the rabbit jugular vein, but appears to be competitive in the umbilical vein (pA2 = 8.2). In the jugular vein, [L-Tic7]HOE-140 is an insurmountable antagonist about 2000-fold less potent than HOE-140; the L-Tic7 isomer demonstrates no significant antagonist activity on the umbilical vein at 30 microM. This study confirms that WIN 64338 behaves as a competitive and selective kinin antagonist of the BK B2 type receptors. The pharmacological profile of the L-Tic7 analog of HOE-140 may provide useful information in discerning the molecular interaction of noncompetitive BK antagonists with their receptors.

Four major guinea-pig blood vessels were isolated and studied in vitro for their contractile responses to anaphylatoxins C3a and C5a. Both peptides were assayed in the 10(-9) to 10(-7) M range and their effects were parallel, although C3a was less potent and more tachyphylactic than C5a. The most responsive blood vessel, relative to a histamine maximal response, was the portal vein, followed by the pulmonary artery and the thoracic aorta. The abdominal vena cava did not respond to either C3a or C5a. Participation of secondary mediators in the vasoconstrictor effect of C3a and C5a was assessed by exposing portal veins and pulmonary arteries to specific inhibitors. The four drugs were: indomethacin, a cyclooxygenase inhibitor; pyrilamine, an antagonist of H1 receptors for histamine; FPL55712, an antagonist of the leukotriene component of slow-reacting substance of anaphylaxis; and phentolamine, an alpha adrenergic blocker. This pharmacological analysis suggests that prostaglandin-like substances mediate most of the anaphylatoxin vasoconstrictor effects, with a biologically significant participation of leukotrienes in the case of C3a and histamine in the case of C5a. High variability between individual tissue responses to the anaphylatoxins was investigated in terms of previous exposure to activated complement. Cobra venom factor (200 micrograms) was injected i.v. in five guinea pigs 2 hr before sacrifice. This treatment reduced the serum concentration of C3 and C5 to 9 and 32%, respectively, as compared with saline-treated animals. The portal vein excised from the cobra venom factor-treated animals exhibited reduced sensitivities to C3a, but showed normal response to C5a. The selective desensitization to C3a after complement activation in vivo is presumably related to the highly tachyphylactic effect of C3a on blood vessels.

A rapid and sensitive method for measuring carboxypeptidase N (CPN) activity in human plasma is described. The procedure is based on the hydrolysis of a high-specificity/low-affinity substrate, hippuryl-L-lysine, to its products hippuric acid and lysine. The substrate and product are separated quantitatively by high-performance liquid chromatography in less than 10 min following a minimal sample preparation time. The advantages of this method over previous ones are discussed and data are presented demonstrating the reliability of this method for the routine clinical determination of CPN activity.