Arao Ujiie

Inhibition of PDGF- and TGF-β1-induced collagen synthesis, migration and proliferation by tranilast in vascular smooth muscle cells from spontaneously hypertensive rats

Vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) proliferate faster and are more sensitive to transforming growth factor-beta 1 (TGF-beta 1) than those of normotensive Wistar-Kyoto rats. We studied the in vitro effects of tranilast, an anti-allergic drug, on the proliferation, migration and extracellular matrix synthesis in the SHR-VSMC. There were many inhibitory effects of tranilast (30-300 microM) on SHR-VSMC. One is the effect on the proliferation stimulated with fetal bovine serum (FBS), TGF-beta 1 and platelet-derived growth factor-BB (PDGF-BB). Another is the effect on the PDGF-BB-induced migration. Lastly, tranilast exhibited inhibitory effects on spontaneous collagen synthesis and TGF-beta 1-induced collagen and glycosaminoglycan synthesis. On the other hand, collagen induced the VSMC migration concentration-dependently. These results suggest that tranilast may prevent restenosis after percutaneous transluminal coronary angioplasty.

Effects of thromboxane synthetase inhibitors on aggregation of rabbit platelets

Thromboxane (TX) synthetase activity was selectively inhibited by (E)-3-[4-(1-imidazolylmethyl)phenyl]-2-propenoic acid hydrochloride monohydrate (OKY-046) and sodium (E)-3-[4-(3-pyridylmethyl)phenyl]-2-methyl-propenoate (OKY-1581) (OKYs). Their IC50 for the rabbit platelet enzyme were found to be 11nM and 3nM respectively. Arachidonic acid (AA) or collagen induced platelet aggregation, and generated TXA 2 and prostaglandins (PGs) in rabbit platelets. OKYs inhibited platelet aggregation and TXA2 generation without affecting PGs generation, while aspirin inhibited platelet aggregation, and TXA2 and PGs generation. There was a parallel relation between the degree of inhibition of platelet aggregation and TXA2 generation by OKYs, but the inhibitory effects of aspirin on platelet aggregation was related to that on both TXA2 and PGs generation. However, OKYs and aspirin did not inhibit ADP-induced platelet aggregation which did not involve the generation of TXA2 and PGs. These results suggested that TXA2 generation is related to platelet aggregation induced by AA or collagen, and that the inhibitory effect of OKYs on platelet aggregation is due to the inhibition of TX synthetase.

Effect of KMD-3213, an α1a-adrenoceptor-selective antagonist, on the contractions of rabbit prostate and rabbit and rat aorta

KMD-3213, (-)-(R)-1-(3-hydroxypropyl)-5-[2-[[2-[2-(2,2,2-trifluoroethoxy) phenoxy]ethyl]amino]propyl]indoline-7-carboxamide, a newly synthesized alpha 1-adrenoceptor antagonist, has been shown to have potent action toward, and to be selective for human cloned and native alpha 1-adrenoceptors. In the present study, we characterized the inhibitory effect of KMD-3213 on the phenylephrine (alpha 1-adrenoceptor-selective agonist)-induced contraction of rabbit prostate, rabbit thoracic aorta and rat thoracic aorta to functionally confirm the tissue selectivity of KMD-3213. The mean pA2 value for KMD-3213 for the inhibition of the rabbit prostatic contraction was 10.05, whereas the values for the rabbit and rat aortic contractions were 9.36 and 8.13, respectively. The order of mean pA2 values for the inhibition of the rabbit prostatic contraction was KMD-3213 > or = tamsulosin >> prazosin, whereas that for the rabbit and rat aortic contractions was tamsulosin > KMD-3213 > prazosin and tamsulosin > or = prazosin >> KMD-3213, respectively. KMD-3213 produced a sigmoidal inhibition curve for single-dose phenylephrine-induced contractions of rabbit prostate, whereas it produced a non-sigmoidal curve for that of rabbit aorta. KMD-3213 had no effect on isoproterenol-induced chronotropic action in guinea-pig atria, and 5-hydroxytryptamine-, histamine- and acetylcholine-mediated contractions of rabbit aorta. These results indicate that the potency of the inhibitory activity of KMD-3213 depends on the tissue subtype expression and that KMD-3213 preferentially antagonizes prostatic contraction.

Antiproliferative and c‐myc mRNA suppressive effects of tranilast on newborn human vascular smooth muscle cells in culture

1 Newborn human vascular smooth muscle cells (VSMCs) proliferated faster and were more sensitive to platelet‐derived growth factor‐BB (PDGF‐BB) than those from adults. In this study, we investigated mechanism of the inhibitory effect of tranilast on PDGF‐BB‐induced proliferation of VSMCs from newborns. 2 Tranilast (30–300 μm) concentration‐dependently inhibited the VSMC proliferation in randomly growing cultures stimulated with PDGF‐BB. 3 Tranilast (30–300 μm) concentration‐dependently inhibited the [3H]‐thymidine incorporation into DNA in VSMCs that had been synchronized by 48 h serum depletion and then stimulated by addition of PDGF‐BB. However, tranilast had little influence on unscheduled DNA synthesis in quiescent cells or on RNA and protein synthesis, unlike aphidicolin, actimomycin D, and cycloheximide. 4 In synchronized VSMC cultures, tranilast still inhibited the PDGF‐BB‐induced DNA synthesis even when added 18 h after stimulation of the quiescent cells. The mode of the antiproliferative action of tranilast was different from that of NiCl2, genistein, or staurosporin. In addition, flow cytometry of synchronized VSMCs treated with tranilast revealed a blockade of PDGF‐inducible cell‐cycle progression at the G1/S checkpoint. 5 Northern blotting showed that tranilast (30–300 μm) concentration‐dependently suppressed constitutive c‐myc mRNA expression even when added 18 h after PDGF‐BB‐stimulation of quiescent VSMCs. Tranilast still had an inhibitory effect on the induction of c‐myc mRNA when de novo protein synthesis was inhibited by cycloheximide and did not shorten the degradation of c‐myc mRNA at the post‐transcriptional level, demonstrating that tranilast directly inhibited c‐myc mRNA expression at the transcriptional level. 6 These results suggest that the inhibitory effect of tranilast on PDGF‐BB‐induced proliferation is due to S‐phase blockade and may be, at least in part, involved in the direct suppression of c‐myc gene expression. Tranilast did not cause cell toxicity and may therefore hold promising potential for the prevention of vascular proliferative diseases.

In vitro effect of KCA-098, a derivative of coumestrol, on bone resorption of fetal rat femurs

The effects of 3,9-bis(N,N-dimethylcarbamoyloxy)-5H-benzofuro[3,2-c]quinoli ne-6-one (KCA-098), a derivative of coumestrol, on bone resorption was studied in organ cultures of 20-day fetal rat femora. KCA-098 increased the length, dry weight, and calcium and phosphorus contents of parathyroid hormone (PTH)-treated fetal rat femur. As PTH significantly reduced the calcium and phosphorus contents of the femora, probably by stimulating bone resorption, KCA-098 seems to inhibit bone resorption. In fact, KCA-098 inhibited the PTH-induced release of 45Ca from pre-labeled fetal rat femora into the medium in organ culture. Coumestrol also inhibited the release of 45Ca from bone into the medium. However, KCA-098 did not increase the uterine weight of ovariectomized rats, whereas coumestrol did so. Thus KCA-098 is a unique, new inhibitor of bone resorption that has no estrogenic activity.

Augmentation of prostacyclin and depression of PGE2, PGF2α and thromboxane A2 by TSH in cultured porcine thyroid cells: An important role of prostacyclin in maintaining thyroid cell function

The importance of prostaglandins (PCs) in thyroid physiology has long been a controversial subject. In [l-3] 6-ketoprostaglandin Fr, was isolated and found to be an end-metabolite of the unstable compound, prostacyclin [4,.5]. Prostacyclin producing activity has been reported in many tissues and cells [6-81, and biological significance of prostacyclin in maintaining homeostasis has become apparent. Thromboxane Ba, an end-metabolite of thromboxane Aa, has been isolated [9] and this thromboxane A* seems to play some role to maintain homeostasis. However, prostacyclin and thromboxane Aa producing activities in the thyroid have not been reported and their biological significance needs to be studied. To evaluate the role of endogenous PGs in the regulation of thyroid function, we have estimated prostaglandin Ea (PGE,), prostaglandin Fzor (PGFa,), 6-ketoprostaglandin Fr, and thromboxane Bz (TXBa) and correlations of these PGs and thyroid functions were studied using cultured porcine thyroid cells. Here, we show that in the absence of TSH, the cells are unable to take up iodide or organify it but product; PGE2, PGFzo, and TXBz and that in the presence of TSH, the cells are able to take up iodide and organify it but preferentially produce 6-ketoprostaglandin Fr,, an end-metabolite of prostacyclin.

Simultaneous determination of glycyl-L-histidyl-L-lysine and its metabolite, L-histidyl-L-lysine, in rat plasma by high-performance liquid chromatography with post-column derivatization.

A selective and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of glycyl-L-histidyl-L-lysine (GHK), a liver-cell growth factor isolated from human plasma, and its metabolite, L-histidyl-L-lysine (HK), in rat plasma. Both high selectivity and sensitivity were achieved by the use of solid-phase extraction with a Bond-Elut Certify cartridge, ion-pair chromatography with 1-pentanesulfonate on a 5-microm Capcell Pak C18 UG120 column (250x4.6 mm I.D.) with a guard column, and by post-column derivatization with o-phthalaldehyde (OPA). GHK and HK were extracted from 0.1 ml of rat plasma after addition of o-phenanthroline to protect against degradation. The limit of detection for GHK and HK were 50 and 15 ng/ml, respectively, and the calibration curves were linear in the range 0.1-5.0 microg/ml. The developed method was applied to the pharmacokinetic study of GHK after a single dose was administered intravenously to rats. GHK was rapidly degraded to HK, which was eliminated rapidly.

Role of thromboxane (Tx) A2 in guinea pig forssman shock and the effect of OKY-046, TX A2 synthetase inhibitor

To study the role of thromboxane (Tx) A2 in Forssman systemic shock (FSS) in guinea pig, the effect of (E)-3-[p-(1H-Imidazol-1-ylmethyl)phenyl]-2-propenoic acid hydrochloride (OKY-046), a specific Tx A2 synthetase inhibitor, was studied. OKY-046 administered intravenously clearly prolonged survival time and protected against fatal shock. In shocked animals, definite decreases in serum complement hemolytic activity (CH50), leucocyte counts and platelet counts and an increase in lactate dehydrogenase (LDH) activity were observed. In addition, a significant increase of Tx B2 and incoagulability of blood were observed after shock. Whereas OKY-046 had no effect on the decreases in CH50, platelet counts and leucocyte counts, it inhibited the increase of Tx B2 and increased the amount of 6-keto PG F1 alpha. When Forssman antibody (half a lethal dose) was injected, a diphasic increase in airway resistance was observed. OKY-046 inhibited this diphasic increase in airway resistance. These data suggest a pathophysiological role for Tx A2 in FSS. OKY-046 inhibited the Forssman antibody induced respiratory disorders probably due to the inhibition of Tx A2 synthesis after shock.

Influence of epiphyseal cartilages on bone resorption of fetal rat femora

We developed a facile method for studying bone resorption using fetal rat femur by labelling the bone with 45Ca in vitro. We found that cartilages stimulated the bone resorption of a shaft which was obtained by cutting off both distal and proximal cartilages from the femur. When the shaft was co-cultured with the cartilages isolated by a 0.4-microns microporous membrane in the same Transwell, the bone resorption of the shaft was increased. This finding suggests that the stimulation of bone resorption by the cartilages is not a result of recruitment of osteoclasts or the precursor cells from the cartilages. Indomethacin (10(-6) M) failed to influence the bone resorbing activity of the cartilages. The bone resorbing activity in the supernatant obtained from the cartilage-culture was decreased by heating. The bone resorbing activity of the supernatant did not remain in the lipid-extract or the pronase-digested supernatant, but was present in a fraction whose molecular weight was greater than 50,000. These results collectively suggest that the cartilages produce a bone resorption-stimulating factor(s) which is water-soluble, is a non-prostanoid material, contains protein and has a molecular weight greater than 50,000.