Kazuaki Naito

Identification of 1,4-dihydropyridine binding domains within the primary structure of the α1 subunit of the skeletal muscle L-type calcium channel

Calcium channel blockers are drugs that bind to the α1 subunit of L‐type calcium channels and selectively inhibit ion movements through these channels. Determination of the mechanism of channel blockade requires localization of drug‐binding sites within the primary structure of the receptor. In this study the 1,4‐dihydropyridine‐binding site of the membrane bound receptor has been identified. The covalently labeled receptor was purified and digested with trypsin. The labeled peptide fragments were immunoprecipitated with sequence‐directed antibodies. The data indicate the existence of at least three distinct dihydropyridine‐binding domains within the primary structure of the α1 subunit.

Inhibition of Pulmonary Eosinophilia Does Not Necessarily Prevent the Airway Hyperresponsiveness Induced by Sephadex Beads

Background: The Lewis rat among highly inbred strains exhibits significant airway hyperresponsiveness (AHR) following intravenous administration of Sephadex G-200 (Sephadex). The aim of this study was to investigate the association of Sephadex-induced AHR with changes in airway inflammation in Lewis rats. Methods: A suspension (0.5 mg/ml/rat) of Sephadex was intravenously administered to male Lewis rats on days 0, 2 and 5. Measurement of airway responsiveness to serotonin, bronchoalveolar lavage (BAL) and histological study were performed on day 2–11. Results: Significant AHR induced by Sephadex was recognized on day 2 (p < 0.05), and AHR reached a maximum on day 7 (p < 0.001). In the BAL study, eosinophils increased on day 2 (p < 0.01) with a peak on day 5 (p < 0.05). In the histological study, we found Sephadex beads trapped in small arteries of the lung and granulomatous arteritis on day 2 or later. Pulmonary granulomas, horseshoe-shaped multinuclear giant cells, eosinophils and goblet cell hyperplasia were observed on day 2, and the degree became intense on day 5–7. GCC-AP0341 (10 mg/kg, i.p. × 3) inhibited the recruitment of eosinophils in BAL fluid and in lung tissue, but it did not inhibit AHR. The compound also inhibited pulmonary granulomas and goblet cell hyperplasia. Conclusion: The mechanism of Sephadex-induced AHR may not be directly associated with inflammatory changes such as recruitment of eosinophils, pulmonary granulomas and hyperplasia of goblet cells in rats.

Evidence that cyclosporin A and dexamethasone inhibit allergic airway eosinophilic inflammation via suppression of interleukin-5 synthesis by T cells

1 We have recently demonstrated that airway eosinophilic inflammation can be transferred to unprimed mice by infusing interleukin (IL)‐5‐producing T cell clones. Using that murine model, we performed this study to delineate the mechanism of cyclosporin A and dexamethasone to inhibit allergic airway eosinophilic inflammation. 2 The ovalbumin‐reactive murine T cell clones, FJ17, produced IL‐2, IL‐4 and IL‐5 upon stimulation with relevant antigen. In FJ17‐transferred mice, messenger RNA (mRNA) of IL‐2 and IL‐5 expressed in the lungs, the number of eosinophils in bronchoalveolar lavage fluid (BALF) was increased and the bronchial responsiveness to acetylcholine was enhanced after antigen provocation. 3 Cyclosporin A (10, 100 ng ml−1) and dexamethasone (10, 100 ng ml−1 suppressed the production of IL‐5 as well as IL‐2 and IL‐4 by FJ17 in vitro. 4 Subcutaneously administered cyclosporin A (30 mg kg−1) and dexamethasone (10 mg kg−1) inhibited antigen‐induced mRNA expression of IL‐2 and IL‐5, increase of BALF eosinophils and bronchial hyperresponsiveness of FJ17‐transferred mice in vivo. The number of BALF eosinophils was correlated with the bronchial responsiveness to acetylcholine (r = 0.672). 5 The results clearly indicated that the suppression of IL‐5 synthesis by T cells is involved in the effects of cyclosporin A and dexamethasone to inhibit allergic airway eosinophilic inflammation.

Binding characteristics of a new 1,5-benzothiazepine, clentiazem, to rat cerebral cortex and skeletal muscle membranes

The binding properties of a new 1,5-benzothiazepine, clentiazem (TA-3090), were investigated in rat cerebral cortex and skeletal muscle membranes with [3H]diltiazem and [3H]nitrendipine as radioligands. Clentiazem inhibited [3H]diltiazem binding to cerebral cortex membranes at the same concentrations as diltiazem at 2 degrees C. However, at 37 degrees C clentiazem was 3 times more potent to inhibit binding than diltiazem. [3H]Nitrendipine binding was modulated by clentiazem in a temperature-dependent manner. At 37 degrees C clentiazem significantly enhanced [3H]nitrendipine binding to rat cerebral cortex membranes, whereas it has an inhibitory effect on [3H]nitrendipine binding at 0 degree C and no effect at 25 degrees C. Of two optical isomers of clentiazem and four of diltiazem, only d-cis isomers (clentiazem and diltiazem) increased [3H]nitrendipine binding, indicating that both compounds have the same stereoselectivity for increasing [3H]nitrendipine binding. These results suggest that clentiazem binds to the same 1,5-benzothiazepine binding sites as diltiazem but with greater affinity.

Prevention of ozone-induced airway hyperresponsiveness and epithelial injury by phosphodiesterase inhibitors in guinea pigs.

We investigated the effects of phosphodiesterase (PDE) inhibitors on ozone-induced airway hyperresponsiveness (AHR) in guinea pigs. Theophylline (10-100 mg/kg), rolipram (1-100 mg/kg) and T-440 (1-100 mg/kg) were orally administered 30 min before ozone exposure (3 ppm, for 30 min). Ozone exposure caused an increase in airway responsiveness to methacholine aerosol, and log PC(10) (log-transformed methacholine concentration causing a 10 cm H(2)O increase in pulmonary inflation pressure) in the control and ozone-exposed group was 4.43±0.05 (n=6) and 3.26±0.15 (n=12), respectively. Theophylline at 100 mg/kg, 10 mg/kg rolipram and 10 mg/kg T-440 significantly inhibited AHR with log PC(10) value of 4.73±0.16 (n=7), 3.74±0.11 (n=7), 3.82±0.15 (n=6), respectively. On histological examination, epithelial damage in the trachea and peripheral airways was recognized after ozone exposure. At 100 mg/kg, rolipram, T-440 and theophylline caused complete inhibition of AHR, and prevented epithelial damage of the trachea and peripheral airways. These results indicate that PDE inhibitors prevent not only ozone-induced AHR but also airway epithelial injury by ozone.

Synthesis and biological activities of 1-pyridylisoquinoline and 1-pyridyldihydroisoquinoline derivatives as PDE4 inhibitors.

A novel series of 1-pyridylisoquinoline and 1-pyridyldihydroisoquinoline derivatives has been prepared. These compounds showed potent PDE4 inhibitory activities and a broad margin between the K(i) value of the rolipram binding affinity and the IC(50) value of PDE4 inhibition. They also exhibited potent inhibitory activities toward LPS-induced TNF-alpha production in mice.

Augmentation of tumor immunity by 6-MPG, a water-soluble derivative of 6-mercaptopurine, in mice

Inhibitory effects on some immunological responses and MethA fibrosarcoma in the double grafted tumor system in mice were compared between 6-mercaptopurine (6-MP) and its novel water-soluble derivative, gamma-(9H-purine-6-yl)thiomethyl L-glutamate (6-MPG). The dose-dependent inhibitory effects by 6-MPG on the hemagglutinin response to SRBC, DTH reaction to MBSA, contact sensitivity to oxazolone, GVH response and growth of the primary tumor were 3-10 times weaker than those by 6-MP, probably reflecting the difference in their cytotoxicities antimetabolites. However, the two drugs were nearly equipotent in reproducing inhibition of the secondary tumor growth, which is a host-mediated immunological response to tumor antigen as shown by its dependency on the primary inoculation with 1 x 10(4) or more MethA cells and by the production of anti-tumor splenocytes in tumor-bearing animals (the Winn assay). Thus, 6-MPG may point to the direction of derivatization towards anti-tumor immunopotentiators with an improved therapeutic index.

Study on the mechanism of immunopotentiating antitumor effect of 6-MPG, a water-soluble derivative of 6-mercaptopurine

We investigated possible mechanisms of the antitumor action of gamma-(9H-purine-6-yl) thiomethyl L-glutamate (6-MPG), a water-soluble derivative of 6-MP. In the double grafted tumor system, BALB/c mice were inoculated intradermally with 10(6) cells of MethA fibrosarcoma at the right inguinal region on day 0 (the primary tumor) and later with 3 x 10(6) cells at the left on day 10 (the secondary tumor). Intraperitoneal administration of 6-MPG at a dose of 100 mg/kg/day from day 3 through 7 completely prevented growth of the secondary tumor. 6-MPG showed no effect on growth of colon 26 adenocarcinoma cells inoculated in place of the secondary MethA cells (antigen specificity). 6-MPG did not inhibit the secondary MethA growth in the BALB/c (nu/nu) mouse. The inhibitory effect of 6-MPG on the secondary tumor growth was diminished by prior treatment of the primed animals with cyclosporin A and anti-Thy antibody. Spleen cells from the tumor-bearing mice treated with 6-MPG showed a tumor-neutralizing activity (Winn assay). Treatment of the spleen cells with anti-CD8 antibody plus complement diminished the tumor-neutralizing effect but that with anti-CD4 antibody plus complement did not, indicating that CD8-positive cells are responsible for potentiation of the tumor immunity. These results suggest that the antitumor effect of 6-MPG against the secondary tumor is elicited by augmenting tumor specific T-cell production.