Involvement Of Proteolytic Activity Research Articles

Antiproteases attenuate the release of neutrophil chemotactic activity from bronchial epithelial cells induced by smoke.

The released neutrophil chemotactic activity (NCA) from bronchial epithelial cells (BECs) in response to smoke extract was evaluated by reverse-phase, high-performance liquid chromatography (RP-HPLC) and the involvement of proteolytic activity was assessed for the release of NCA from BECs. Smoke extract stimulated the release of NCA (55.3 +/- 5.2 vs. 17.3 +/- 4.1 cells per high-power field [HPF], p < .001). The released activity determined by RP-HPLC analysis was 15-hydroxyeicosatetraenoic acid and leukotriene B4. Several structurally and functionally different serine protease inhibitors, including alpha-1-protease inhibitor (alpha-1-PI), chloromethyl ketone (CK) derivatives, N-tosyl-L-lysine CK (TLCK), methoxysuccinyl-Ala-Ala-Pro-Val CK (SPCK), N-alpha-tosyl-L-phenylalanine CK (TPCK), and N-alpha-p-tosyl-L-arginine methyl ester hydrochloride (TAME), attenuated the release of NCA (P < .01) in a dose-dependent fashion. Leupeptin, a cysteine protease inhibitor, has only a small effect on the release of NCA (p < .05), and phosphoramidon, a neutral endopeptidase inhibitor, had no effect. The measurement of proteolytic enzyme activity using synthetic substrate S-2288 revealed that smoke extract significantly (p < .05) augmented the serine protease activity in BEC layers. Culture supernatant fluids and cell lysates of BECs in response to smoke extract solubilized 14C-labeled casein. These results suggest that BECs may release lipoxygenase-derived NCA in response to smoke extract and that the release of NCA may involve the activation of proteolytic activity of BECs which was inhibited by serine protease inhibitors.

Thrombin-induced expression of the KC gene in cultured aortic endothelial cells. Involvement of proteolytic activity and protein kinase C

The KC gene, first identified in platelet-derived growth factor-stimulated BALB/c 3T3 cells, shares structural similarities with a new family of genes that code for secreted cytokines which appear to be involved in wound healing and inflammation. Thrombin is a coagulation system proteinase likely to be present in vivo at sites of tissue injury. This enzyme is known to stimulate multiple responses in cultured endothelial cells (EC), including the production of eicosanoids, the expression of growth factor genes and the adhesion of leukocytes. The present experiments were designed to examine the effect of thrombin on KC mRNA expression in EC and to explore the molecular mechanisms involved. Thrombin caused a marked concentration-dependent increase in the steady state level of KC mRNA in confluent porcine aortic EC. The level of KC mRNA reached a peak 2 h after thrombin treatment and returned to near control levels by 8 h. Thrombin that was pretreated with phenylmethylsulfonyl fluoride (PMSF) to block proteolytic activity did not stimulate KC gene expression. Trypsin (2 μg/ml) but not PSMF-trypsin also caused a substantial increase in the level of KC mRNA. We postulated a role for protein kinase C in thrombin-induced KC gene expression since previous work had demonstrated a similar EC response to phorbol esters. This hypothesis was further supported by the finding that thrombin-induced KC expression was suppressed by the C kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, but not by its structural analogue. The results of the present study demonstrate that thrombin augments KC mRNA expression by vascular EC in a process that requires intact proteinase activity. The activation of protein kinase C may be a necessary component of the intracellular signalling pathway involved in this response.

Trypanosoma cruzi: involvement of proteolytic activity during cell fusion induced by epimastigote form.

Human erythrocytes were fused by Trypanosoma cruzi from 7 and 14 day old culture (stationary and declination phases, respectively) while only lysis was induced by 4 day old culture parasite (exponential phase). Lysis and erythrocyte fusion were studied by phase contrast microscopy, measuring of hemolysis and gel electrophoresis. The fusogenicity is Ca2+-dependent while lysis is delayed in the absence of exogenous Ca2+. The proteolysis of erythrocyte protein bands 1, 2, 2.1, 2.3 and 3 are common features of both fusion and lysis processes. Nevertheless the breakdown rate of ankyrin (band 2.1) and band 3 are different in fused or in lysed cells. The lysis process is associated with a faster degradation of band 2.1 and increase of band 2.3 than in the case of the fusion process. By contrast, degradation of band 3 occurs faster in the fusion than in the lytic event. Treatment of fusogenic parasites but not erythrocytes with TPCK, soybean trypsin inhibitor or FCS inhibited to some extent the fusion process and the decrease of bands 1, 2, 2.1, 2.3 and 3. The results suggest that proteases from fusogenic parasites may be directly or indirectly involved in the proteolysis of band 2.1 in a way related to induction of fusion.

Involvement of proteolytic activity in early events in lymphocyte transformation by phytohemagglutinin

The stimulation by phytohemagglutinin (PHA) of DNA synthesis in cultured blood lymphocytes of guinea pig was markedly inhibited by addition of leupeptin, a well-characterized, powerful protease inhibitor of tripeptide nature. About 30 to 40 per cent inhibition was observed at 40 μg/ml of leupeptin when leupeptin was added 30 min prior to or together with PHA. Per cent inhibition by the appropriate amount of leupeptin was proportional to the amount of PHA added in the range of 0.6 to 3.0 μg of PHA at which the per cent inhibition reached maximum. This inhibitory effect of leupeptin on PHA stimulation was abolished when the lymphocytes were preincubated with PHA for more than 10 min before addition of leupeptin or preincubated with leupeptin for more than 60 min prior to PHA addition.