Phtsmin mai,dy cle;tved the ArlI~,$eP bond o1' ArlpVaI.Leu.Pro.Arll.inletleukin.8 (AVLPR-IL-a} produced by human derm.I Itbroblast=t, which r¢=ulted in the conversion orAVLPR.IL.8 to IL.~t and the in,clive pentapeptide, though, minor cleavall~ of AVLPR-IL.It by phtsmin at Ly~':,Ght'* bond occurred. MATERIALS AND METHODS Purtfication of FDNCFHuman dermal fibroblasts (SF-TY cell line) were maintained in Dulbecco's modified Eagle's medium supplemented with 5°7o fetal calf sert~,m, 25 mM Hepes, penicillin (0, I mg/ml) and streptomycin (O. 1 mg/ml) until they reached confluence. The cells were cultured in serum-free culture medium supplemented with tO "m M recombinant human IL-I# and 0.1o70 bovine serum albumin. After culture for 2 days, cell.free conditioned medium was obtained by centrifugation at 1600× g for 20 rain. The chemoattractant in the conditioned medium Abbreviations; AVLPR-IL-8, Ala-Val-Leu-Pro-Arg-interleukin-8; FDNCF fibroblast-derived neutrophil chemot~ctic factor; RP. HPLC, reverse-phase high performance liquid chromatography; SDS-PAGE, sodium dodccyl sulfate polyacrylamide gel electrophoresJs.Correspondence address: H. Nakagawa, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Sugitani, Toyarna 930-01, Japan, was purified essentially ns described previously [11]; the sample was chromatoliraphedsequential/y on CM-Seph~dex, Sephadex O-?$ and RP-HPLC, Tree/men/of FDNCF =+'ith plasminFDNCF (40 j+g) in a final volume of 0,2 ml ¢0f 50 mM Tris.HCI buffer (pH 8.0) was incubated with 0,gpg of plasmin (purified from human plasma; Sigma, MO. USA) at 37*C for 30 rain, and the rent. lion was stopped by the addition of leupeptin (final 1 raM), The reae. lion mixture was concentrated by a Speed-Vac centrifuge, dissolved in 6 M 8uanidine solution and loaded onto a C.18 reverse-phase col. umn (0.45 x l$cm; ODS.120T, Tosoh Co, Tokyo, Japan).Chemotaetic factors were ehtted with a linear co~centration gradient of aeetonitrile from 0% to 50.4% in 0.05% trifluoroacetic acid at z~ flow rate of 0.8 ml/min, Neutrophil chemotaxls of plasmin.treated FDNCF was performed in vitro by using rat and human neutrophils as described previously [121, As an index of ehemotaxis, the number of neutrophils migrated into the lower chamber was expressed as percentage (migration rate) of that of neutrophils applied in the upper chamber, $DS-PAGE was carried out on a slab gel of a discontinuous 9,6%/16,507o acrylamide in tricine buffer system [13]. NH2.terminal atnino acid sequencing analysisNH,-terminal amino acid sequence determination was performed by automated Edman degradation on a gas-plmse protein sequencer (model 470A, Applied Biosystems, CA, USA) e¢~uipped with a PTH analyzer (model 120A HPLC system). RESULTS AND DISCUSSIONHuman dermal fibroblasts stimulated with 10 -t° M IL-1 mainly produced FDNCF, which is the NH2-termina] extended 77-residue variant of IL-8 (AVLPR-IL-8). The homogeneity of the purified FDNCF was confirmed by SDS-PAGE, its NH2-terminal amino acid sequence and its COOHterminal amino...
The antianginal effects of JTV-506, a newly synthesized 2,2-bis-methoxymethyl benzopyran-derivative potassium channel opener, were evaluated in experimental angina models in rats and compared with those of levcromakalim, nicorandil, and nifedipine. JTV-506 (0.01-0.1 mg/kg, i.d.) dose-dependently inhibited S-wave elevation induced by injection of methacholine but caused almost no changes in blood pressure or heart rate. Other vasodilators including levcromakalim, nicorandil, and nifedipine, when administered intraduodenally, also inhibited the S-wave elevation and elicited a decrease in blood pressure. Oral administration of JTV-506 (1 and 3 mg/kg), levcromakalim (1 and 3 mg/kg), and nicorandil (30 mg/kg) significantly inhibited the S-wave depression induced by intravenous administration of arginine vasopressin (AVP). Thus JTV-506 exerts a potent protective effect on angina pectoris models to an extent similar to those of levcromakalim, nicorandil, and nifedipine, whereas its action on systemic blood pressure is different from that of other vasodilators, including reference potassium channel openers. These results suggest that JTV-506 may clinically be useful as an antianginal agent.
The vascular effects of JTV-506 ((-)-(3S,4R)-2.2-bis(methoxymethyl)- 4-[(1,6-dihydro-l-methyl-6-oxo-3-pyridazinyl)amino]-3-hydroxychroman+ ++-6- carbonitrile hemihydrate, CAS 170148-29-5), a new potassium channel opener, was evaluated in isolated coronary arteries and anesthetized dogs. JTV-506 (1 nmol/l-3 mumol/l) produced a concentration-dependent relaxation in porcine isolated epicardial large coronary arteries precontracted with KCl (30 mmol/l), phenylephrine (3 mumol/l), histamine (3 mumol/l), serotonin (5-HT; 300 nmol/l), prostaglandin F2 alpha (PGF2 alpha; 10 mumol/l), U-46619 (100 nmol/l), endothelin-1 (ET-1; 30 nmol/l) and Bay K-8644 (100 nmol/l). JTV-506 was 2.5-8.5 and 13.3-81.5 times more potent than levcromakalim (CAS 94535-50-9) and nicorandil (CAS 65141-46-0), respectively, but was less potent than nifedipine (CAS 21829-25-4). JTV-506 and levcromakalim produced almost a complete relaxation in arteries precontracted with various kinds of vasoconstrictor, except for KCl. In contrast, nifedipine produced about 80-90% relaxation in arteries, precontracted with PGF2 alpha, U-46619 and ET-1. Thus, this potassium channel opener can be characterized as an agonist-nonselective vasorelaxant. The relaxing effects of JTV-506 and levcromakalim on coronary arteries precontracted with 30 mmol/l KCl was competitively antagonized by 3 mumol/l glibenclamide, an ATP-sensitive potassium (KATP) channel blocker. In canine isolated epicardial large coronary arteries, 10 mumol/l JTV-506, 10 mumol/l levcromakalim, 100 mumol/l nicorandil and 0.1 mumol/l nifedipine eliminated 10 mmol/l 3,4-diaminopyridine-induced rhythmic contractions. In anesthetized dogs, when administered directly into the coronary artery, JTV-506 induced dose-dependent increases in coronary arterial diameter and coronary blood flow. These results suggest that JTV-506 elicits coronary vasorelaxation through activation of the KATP channel. It is expected that JTV-506 might be useful in the treatment of coronary vasospasm in angina pectoris.
Recombinant human interleukin-1 beta (IL-1) and tumor necrosis factor-alpha (TNF) synergistically stimulated BALB/c 3T3 cells to produce a chemotactic factor for rat polymorphonuclear leukocytes (PMNs), whereas an addition of 10(-11)-10(-8) M IL-1 or TNF alone to the cell culture resulted in a slight increase in the production of chemotactic factor. The partially purified factor was not a chemokinetic but chemotactic factor for PMNs when analyzed by checkerboard analysis. The partially purified factor was trypsin sensitive and heat stable; its isoelectric point was 8.5-10, and its molecular weight was about 10 kDa as estimated by gel filtration. These results suggest that fibroblasts may participate in PMN migration to the inflammatory site where both IL-1 and TNF are released by activated inflammatory cells, including macrophages.
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