The influence of lidocaine and verapamil on the proliferation, CD44 expression and apoptosis behavior of human chondrocytes

Abstract

Ion channels, which are responsible for the controlled functioning of many cell biological processes, are present on the cell membrane of all living human cell systems under physiological conditions. A relationship between ion channel activity and proliferation behavior has been demonstrated in various cell systems. We showed in earlier studies that there is a resting membrane potential in the cell membranes of human chondrocytes, which can be influenced by various ion channel modulators. The question is to what extent can specific modulation of the ion channel activity regulate proliferation, CD44 expression and the apoptosis behavior of human chondrocytes. Human chondrocytes were isolated from osteoarthritic knee joint cartilage. The culture was made as a monolayer in RPMI medium with the addition of 10% fetal calf serum, 50 microg/ml gentamycinsulfate and 2 microg/ml amphotericin B at 37 degrees C and 5% carbon dioxide. The voltage dependent Na+ channel blocker, lidocaine, and the calcium antagonist, verapamil, were used as ion channel modulators. Proliferation was determined using 3H-thymidine incorporation as the measure. Proof of the CD44 membrane protein was performed by flow cytometry with an FITC-conjugated CD44 antibody (anti-CD44H-FITC). For apoptosis detection, the translocation of phosphatidylserine (Annexin V-FITC assay), Apo2.7 and the Caspase activity on cytokeratin 18 were determined by flow cytometry. The results show that proliferation behavior can be regulated by lidocaine and verapamil, whereby lidocaine results in a transient increase in 3H-thymidine incorporation. Both substances resulted in marked suppression of proliferation after longer incubation times. At the same time, no influence of lidocaine could be determined on the apoptosis of human chondrocytes, whereas marked cytotoxic effects occurred under verapamil. With respect to CD44 receptor expression, incubation with lidocaine resulted in an increase of up to 43%, while suppression of up to 56% was observed with verapamil. The possibility of specific modulation of ion channel activity on the cell membrane of human chondrocytes may serve as the basis for development of new therapeutic options for the treatment of arthrosis.