Mutumi Muramatu

Inhibitory effect of anti-allergic agent NCO-650 on histamine release induced by various secretagogues

Histamine release from rat peritoneal mast cells induced by antigen and anti-IgE was essentially complete within 2 min and 3 min, respectively, but that due to Concanavalin A (Con A) was complete only within 9 min. An anti-allergic agent NCO-650 [trans-4-Guanidinomethylcyclohexanecarboxylic acid p-tert-butylphenyl ester hydrochloride], which is a strong inhibitor of trypsin, dose-dependently inhibited anti-IgE-induced histamine release from rat peritoneal mast cells. Moreover, the rate and extent of histamine release from rat peritoneal mast cells induced by various histamine liberators such as antigen, concanavalin A, ionophore A 23187 and compound 48/80 are significantly diminished in samples incubated with NCO-650. The IC50 values of NCO-650 on histamine release induced by antigen, anti-IgE, Concanavalin A, A23187 and compound 48/80 were in the order of micromolar range, i.e. 1.9, 3.6, 4.6, 2.9 and 6.1 μM, respectively. On a molecular basis, NCO-650 is 1000-fold more potent than DSCG, an anti-allergic drug, in inhibiting the antigen-induced histamine release. The present results suggest that the effect of NCO-650 might be due to the inhibition of a common process underlying the release of histamine by various histamine liberators.

Antibacterial Effects of Esters of Guanidino-and Amidino-Acids Trypsin Inhibitors

Inhibitory effects of various esters of trans-4-guanidinomethylcyclohexanecarboxylic acid and the 4-tert- butylphenyl esters of amidinopiperidine-4-alkanoic, trans-4- amidinocyclohexanealkanoic, trans-4-guanidinoethylcyclohexanecarboxylic and trans-4-guanidinocyclohexanealkanoic acids, all trypsin inhibitors, on the growth of E. coli, B. subtilis, S. aureus and S. epidermidis were examined. 4-tert-Butylphenyl esters strongly inhibited the growth of E. coli and the order of the inhibitory effects correlated with that for the inhibitory effects on proteinase In, which appears immediately before initiation of DNA synthesis in E. coli and closely correlates with the onset of DNA synthesis. No correlation was observed between the inhibitory effects and Ki values for trypsin. The 4-tert-butylphenyl esters also strongly inhibited B. subtilis, S. aureus and S. epidermidis, and the order, of the inhibitory effects on these bacteria roughly coincided with that on E. coli. The order of the inhibitory effects of each ester, on these bacteria was S. epidermidis > S. aureus > B. subtilis > E. coli. Among the esters examined, the biphenyl ester of trans-4-guanidinoethylcyclohexanecarboxylic acid was the most inhibitory on these four bacteria and proteinase In. Hydrolysis of tert-butyloxycarbonyl-L-valyl-L-prolyl-L-arginine 4-methylcoumarin-7-amide, which is a substrate for proteinase In, in crude extracts of E. coli, B. subtilis and S. epidermidis was examined. The order of this activity in these bacteria was E. coli > B. subtilis > S. epidermidis.

INHIBITION OF PHOSPHOLIPID METHYLATION BY AN ANTI-ALLERGIC AGENT, NCO-650, DURING HISTAMINE RELEASE

Abstract Antigen, anti-IgE and concanavalin A (Con A) induced an increase in both the incorporation of the 3 H-methyl moiety into phospholipids and histamine release. Maximal incorporation of the 3 H-methyl moiety into the lipid fraction of the cells was observed within 15 sec and 1 min after being challenged with antigen (100 μg/mL) and anti-IgE (200 μg/mL) respectively. However, the methylated phospholipid decreased rapidly. The addition of Con A (10 μg/mL) also increased phospholipid methylation, which reached a maximum at 5 min after challenge. Trans -4-guanidinomethylcyclohex-anecarboxylic acid p - tert -butylphenyl ester hydrochloride (NCO-650; 27 μM) strongly inhibited the incorporation of the 3 H-methyl moiety into phospholipid by antigen, anti-IgE and Con A. The ic 50 values of NCO-650 for phospholipid methylation in response to antigen, anti-IgE and Con A were 1.5, 4.7 and 1.1 μM respectively. Although the Ca 2+ -ionophore A23187 did not induce phospholipid methylation, it caused histamine release.

A trypsin-like proteinase appearing at 17 h and 17 min in the cell cycle time of HeLa cells correlates with the onset of DNA synthesis

A trypsin-like proteinase appearing sharply at 17 h 17 min (17:17) in the cell cycle time of the synchronized and growing HeLa cells correlates with the onset of DNA synthesis, and the inhibition of the proteinase activity by trans-4-guanidinomethylcyclohexanecarboxylic acid 4-tert-butylphenyl ester (GMCHA-OPhBut) results in a 3 h retardation of the onset of the DNA synthesis. The proteinase activity is cell density dependent and completely inhibited by 100 microM GMCHA-OPhBut. The proteinase was named HeLa tryptase 17:17. The fact that the DNA synthesis occurred after the 3 h retardation in the presence of GMCHA-OPhBut strongly suggests the involvement of the alternative trigger for DNA synthesis in HeLa cells.

Fluctuation of trypsin-like proteinase activity in the cell cycle of synchronized and growing HeLa cells, and the effect of GMCHA-OPhBut

HeLa cells synchronized by double-thymidine block were grown in Eagles minimum essential medium supplemented with 10% calf serum, and the fluctuation of trypsin-like protease activity in the cell cycle was examined. Seven distinct activity peaks were observed in one cell cycle at a cell density of 2%: two peaks in S phase, one peak at the S/G2 boundary, one peak in early M phase and one at the M/G1 boundary, and two peaks in G1 phase. HeLa cells synchronized by a mitotic detachment technique also showed similar results at cell density of 4.8%. The appearance of trypsin-like proteinase activity in the cell cycle was markedly affected by cell density, and no definite peak was observed above 8%. trans-Guanidinomethylcyclohexanecarboxylic and 4-tert-butylphenyl ester (GMCHA-OPhBut), a specific inhibitor for trypsin and a strong inhibitor of HeLa cell growth, had no effect on the various events in the first S, G2 and M phases, such as the incorporation of [methyl-3H]thymidine into DNA, the increase in the cell concentration, and the appearance of trypsin-like proteinase activity, whereas it retarded the onset of the second S phase and the various events in the second S, G2 and M phases for 3 h. In particular, it induced the appearance of a new proteinase peak at the G1/S boundary.

Inhibition of cAMP Increase by an Anti-Allergic Agent, NCO-650, during Histamine Release

Antigen and concanavalin A (Con A) induced an increase in cAMP and histamine release from rat peritoneal mast cells. In a dose-dependent manner, the compound, NCO-650, significantly inhibited both the initial and secondary increases in cAMP stimulated by antigen, anti-IgE and Con A in rat peritoneal mast cells. IC50 values of NCO-650 for cAMP increase stimulated by antigen, anti-IgE and Con A were 3.8, 3.4 and 2.8 microM, respectively.