Tryptase Inhibitors Research Articles

Synergistic combinations and peptides in the inhibition of human immunodeficiency virus

Various combinations of inhibitors of HIV reverse transcriptase were tested for inhibition of HIV replication in order to reveal any potential synergism or antagonism. PFA, a pyrophosphate analogue, gave synergistic inhibition of HIV replication in combination with both of the thymidine analogues AZT and FLT. The combination of PFA and AZT-TP gave only additive or weakly synergistic inhibition in a reverse transcriptase enzyme assay. The combination of AZT and FLT also gave synergistic inhibition of HIV replication, whilst the combination of AZT-TP and FLT-TP gave only additive or weakly synergistic inhibition of reverse transcriptase. Thus, the synergy does not arise from effects on reverse transcriptase alone but must be owing to other, cellular factors, such as effects on nucleoside metabolism or metabolism of the analogues. The results are consistent with the hypothesis that AZT may have an alternative mechanism of inhibition other than inhibition of reverse transcriptase. The diminished cytotoxicity observed in addition to the synergistic inhibition makes these combinations attractive from the point of view of combination chemotherapy. The inhibition of HIV replication by peptides from various parts of the V3 region of gp120 whose sequences were homologous with the tryptase inhibitor trypstatin was tested. Inhibitory activity was displayed by two peptides containing cysteine in their sequence. Antibodies to two peptides containing the two conserved cysteine residues from opposite sides of the neutralizing loop of gp120 were previously associated with protection from vertical transmission of HIV. The V3 region thus seems to be important for the function of gp120 and the transmission of HIV.

The mast cell as an effector of connective tissue degradation: A study of matrix susceptibility to human mast cells

The susceptibility of connective tissue elements to degradation by human mast cells was explored using purified mast cell tryptase and sonicated mast cell preparations. The R-22 strain of smooth muscle cells from rat heart was used for preparation in vitro of a labelled anchored matrix. Digestion of 11.9 ± 1.2% (n=5) of this matrix was observed after overnight incubation with the mast cell sonicates. Pretreatment of the sonicate with a tryptase inhibitor TLCK reduced the digestion by 42%. Digestion of 12 ± 1% (n=4) of the matrix was observed with purified tryptase. The susceptible substrate within this anchored insoluble matrix resided in the glycoprotein compartment as defined by enzymatic characterization of the residual matrix. Mast cells may play a role in mediating connective tissue degradation through the release of proteases specifically synthesized by this cell.

Mast cell tryptase causes airway smooth muscle hyperresponsiveness in dogs.

Supernatants obtained by degranulation of dog mastocytoma cells greatly increased the sensitivity and the magnitude of the contractile response of isolated dog bronchial smooth muscle to histamine. The enhanced contractile response was reversed completely by H1-receptor antagonists and was prevented by an inhibitor of tryptase (a major protease released with histamine from secretory granules of mast cells). The potentiation of histamine-induced contractions was reproduced by active tryptase in pure form. The contractions due to the combination of histamine and purified tryptase were abolished by the Ca2+ channel blockers nifedipine and verapamil. The bronchoconstricting effects of KCl and serotonin, which, like histamine, contract airway smooth muscle by a mechanism predominantly involving membrane potential-dependent Ca2+ transport, were also potentiated by tryptase. However, the contractile effects of acetylcholine, which contracts dog airway smooth muscle by a mechanism independent of Ca2+ channels, were unaffected by tryptase. These findings show a striking promotion of agonist-induced bronchial smooth muscle contraction by mast cell tryptase, via direct or indirect effects on Ca2+ channels, and the findings therefore suggest a novel potential mechanism of hyperresponsiveness in dog bronchi.

Role of Calcium in Histamine Release from Rat Mast Cells Activated by Various Secretagogues; Intracellular Calcium Mobilization Correlates with Histamine Release

Anti-IgE, con A or antigen caused an increase in the intracellular calcium concentration, [Ca2+]i, of mast cells. The increase occurred in two stages: a rapid initial rise caused by Ca-mobilization from intracellular Ca-stores and a second sustained rise caused by an influx of extracellular calcium (White, J.R., Pluznik, D.V., Ishizaka, K. & Ishizaka, T. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 8193-8197). The rapid initial rise was followed by a release of histamine, which seemed to coincide with the second rise. A23187 and compound 48/80 induced a rapid initial rise in [Ca2+]i, followed by a gradual decrease in [Ca2+]i, GMCHA-OPhBut, a specific pH 7 tryptase inhibitor (Muramatu, M., Ito, T., Takei, M. & Endo, K. (1988) Biol. Chem. Hoppe-Seyler 369, 617-625), strongly inhibited both the initial and second rises of [Ca2+]i, as well as histamine release by these secretagogues, and its effects on the initial rise were closely correlated with those on histamine release. Addition of GMCHA-OPhBut immediately after the initial rise strongly inhibited the second rise, thereby decreasing the final [Ca2+]i. These results strongly suggested a possible involvement of pH 7 tryptase, not only in Ca-mobilization leading to the initial rise in [Ca2+]i, but also in the second rise. Trapping of extracellular calcium by 3mM EGTA decreased both the initial rise in [Ca2+]i and histamine secretion induced by anti-IgE or con A; the magnitude of this effect depended on the time between induction and EGTA addition. Histamine release was closely correlated with the initial rise in [Ca2+]i. Similar results were obtained with A23187, but even 5 min after the addition of EGTA an initial rise of [Ca2+]i could still be induced, and histamine (30% of total histamine) was still released. However, A23187 did not induce a rise in [Ca2+]i in mast cells which had been exhaustively washed with Tyrode/Hepes solution containing 3mM EGTA, followed by suspension in the same solution. Even at 20 min after depletion of the extracellular calcium, compound 48/80 still caused an initial rise in [Ca2+]i to above half the maximal value, and histamine secretion was even less affected. The above results indicated that the initial rise in [Ca2+]i, due to Ca-mobilization, correlates with the histamine release promoted by the secretagogues described. On the other hand, isoproterenol strongly induced histamine secretion with no change of [Ca2+]i, while EGTA treatment prior to isoproterenol stimulation had no effect on histamine release, indicating a different secretion mechanism.

Biological functions of serine proteases in mast cells in allergic inflammation

Serine proteases in mast cell granules, such as chymase, atypical chymase, and tryptase, which are major proteins in the granules, may play important roles in the process of immunoglobulin E (IgE)-mediated degranulation and in pathobiological alterations in tissues. Indeed, inhibitors of chymase, substrate analogs, and antichymase F(ab')2, but not inhibitors of tryptase, markedly inhibited histamine release induced by IgE-receptor bridging but not that induced by Ca ionophore. In contrast, inhibitors of metalloprotease inhibited histamine release induced not only by IgE-receptor bridging but also by Ca ionophore. These results suggest that chymase and metalloprotease are involved at different steps in the process of degranulation. The extents of inhibition of histamine release were closely correlated with the amounts of the inhibitors of chymase accumulated in the granules. After degranulation, the released proteases may in part contribute to pathobiological alterations in allergic disorders through generations of C3a anaphylatoxin and thrombin by human and rat tryptase, respectively, and those of angiotensin II and a chemotactic factor of neutrophils by human and rat chymase, respectively. Moreover, chymase and atypical chymase from rat were shown to destroy type IV collagen, and human tryptase was found to hydrolyze various plasma proteins, such as fibrinogen and high-molecular-weight kininogen. The biological activities of tryptase and chymase from rat may be regulated by their dissociation from and association with trypstatin, an endogenous inhibitor of these proteases.

Biological functions of serine proteases in the granules of rat mast cells

The effects of specific low- and high-molecular weight inhibitors of chymase and tryptase and F(ab′) 2 of antichymase on histamine release from activated mast cells were examined. The release of histamine induced by anti-rat immunoglobulin E was markedly inhibited by F(ab′) 2 fragments of antichymase and the low-molecular weight inhibitor of chymase chymostatin, whereas release of histamine induced by calcium ionophore A23187 was inhibited only by chymostatin. Neither the inhibitor nor the antibody affected histamine release induced by compound 48 80 . These results suggest that two main chymotrypsin-type proteases are involved in process of degranulation: one is chymase, which acts at a step before calcium entry, and the other is an unidentified protease, which acts at a step after calcium entry. These results are summarized in Figure 8. After degranulation, released chymase remains associated with the cell surface while released tryptase was found in the extracellular milieu. Tryptase converted bovine prothrombin to thrombin, as shown by increase in thrombin activity with a synthetic substrate, t-butyloxy-carbonyl-Val-Pro-Arg-4-methyl-coumaryl-7-amide. The apparent K m value toward prothrombin was relatively low (2.3 μ m), suggesting that tryptase contributes to blood coagulation or the process of fibrosis in tissues. The proteolytic products of IgG 1 produced by chymase had chemotactic activity for neutrophil leukocytes in vitro and in vivo. These findings indicate the possible functions of these proteases after degranulation.