Bronchial tissue kallikrein activity is regulated by hyaluronic acid binding.
Tissue kallikrein (TK) is secreted by serous cells of tracheobronchial submucosal glands and plays a role in allergic airway responses. To better understand the regulation of TK, we used primary cultures of submucosal gland cells that release TK upon stimulation. Media from cultures stimulated with chymase (10(-7) M) showed increased TK activity (0.50 +/- 0.22 mU/ml mean +/- standard error) in comparison with the control group (0.08 +/- 0.02 mU/ml). The increased TK activity was significantly correlated with increases in the levels of the serous cell marker, secretory leukoprotease inhibitor. Anion exchange chromatography of the conditioned culture media showed that TK activity eluted as a broad peak between 1.6 and 1.8 M NaCl, unlike the reported elution (0.3 to 0.6 M NaCl) of kallikreins from other tissues, suggesting that secreted bronchial TK was bound to a negatively charged molecule. Hyaluronidase digestion increased TK activity in both pre- and post-chymase-stimulated culture media, whereas no such change was seen after samples were digested with heparinase or chondroitinase ABC. Further, after hyaluronidase digestion of media, TK eluted from an anion exchange column between 0.3 and 0.6 M NaCl. Enzymatic detection of TK after nondenaturing gel electrophoresis showed that hyaluronidase digestion also reduced the electrophoretic heterogeneity of TK to a single band, whereas adding back hyaluronic acid (HA) to hyaluronidase-digested samples restored the original heterogeneity. Finally, TK activity bound to HA-Sepharose and could be eluted with HA. These studies show that primary cultures of ovine submucosal gland cells secrete TK in a regulated fashion, and that secreted TK binds to HA. This binding reduces TK enzymatic activity; therefore, factors that affect HA turnover could modify the TK activity in the airway lumen. These events could be important in the regulation of kinin-mediated airway inflammation.
Forteza
R
R
Division of Pulmonary Disease, University of Miami at Mt. Sinai Medical Center, Miami Beach, Miami, Florida. rforteza@msmc.com
Lauredo
I
I
Abraham
W M
WM
Conner
G E
GE
D000818:Animals; D001980:Bronchi / Q000201:enzymology*; D002478:Cells, Cultured; D019765:Chondroitin ABC Lyase / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D053818:Chymases; D017077:Culture Media, Conditioned / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D019763:Heparin Lyase / Q000378:metabolism; D006820:Hyaluronic Acid / Q000494:pharmacology*; D006821:Hyaluronoglucosaminidase / Q000378:metabolism; D011485:Protein Binding; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D012756:Sheep; D020840:Tissue Kallikreins / Q000378:metabolism*; D014132:Trachea / Q000201:enzymology
D017077:Culture Media, Conditioned; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins; D006820:Hyaluronic Acid; D006821:Hyaluronoglucosaminidase; D012697:Serine Endopeptidases; D020840:Tissue Kallikreins; D053818:Chymases; D019765:Chondroitin ABC Lyase; D019763:Heparin Lyase