Influence of ion channels on the proliferation of human ... - CiteSeerX

Biorheology 39 (2002) 55–61 IOS Press

55

Influence of ion channels on the proliferation of human chondrocytes David Wohlrab ∗ , Susanne Lebek, Thomas Krüger and Heiko Reichel Department of Orthopaedics, Martin Luther University, Halle-Wittenberg, Germany Abstract. The goal of the study was to examine connections between ion channel activity and the proliferation of human chondrocytes. Chondrocytes were isolated form human osteoarthritic knee joint cartilage. In this study the concentration-dependent influence of the ion channel modulators tetraethylammonium (TEA), 4-aminopyridine (4-AP), 4 ,4 diisothiocyanato-stilbene2,2 -disulfonic acid (DIDS), 4-acetamido-4 -isothiocyano-2,2 -disulfonic acid stilbene (SITS), verapamil (vp) and lidocaine (lido) on the membrane potential and the proliferation of human chondrocytes was investigated using flow cytometry and the measurement of 3 H-thymidine incorporation as measure for the cell proliferation. The results show an effect of the used ion channel modulators causing a change of the membrane potential of human chondrocytes. The maximal measurable effects of the membrane potential were listed with 0.25 mmol/l verapamil (−18%) and 0.1 mmol/l lidocaine (+20%). When measuring DNA distribution, it became apparent that the human chondrocytes are diploid cells with a very low proliferation tendency. After 12 days culture duration, lidocaine and 4-AP cause an increase of the DNA synthesis rate being a limited effect. These results allow the conclusion of an influence of ion channel modulators on chondrocyte proliferation. To gain knowledge of the regulation of chondrocyte proliferation via ion channel modulators could serve the research of new osteoarthritis treatment concepts. Keywords: Chondrocytes, ion channel modulators, membrane potential, proliferation

1. Introduction As all living cell systems, human chondrocytes are provided with a membrane potential. For its origin the existence of ion channels at the cell membrane is an essential prerequisite. For this development, different active and passive transportation systems are responsible, especially ion channels in the cell membrane [6]. In non-human chondrocytes, different ion channels could already be identified [9,11,15]. A connection between the potassium channel activity and the proliferation has already been detected in different human cell systems [12]. Whereas, the proof of a connection between ion channel activity of human chondrocytes and the proliferation has yet to be established. The objective of the performed experiments was to gain the membrane potential of human chondrocytes by flow cytometry. Furthermore, it should be determined the concentration dependent influence of the ion channel modulators tetraethylammonium (TEA), 4-aminopyridine (4-AP), 4 ,4 diisothiocyanato-stilbene-2,2 disulfonic acid (DIDS), 4-acetamido-4 -isothiocyano-2,2 -disulfonic acid (SITS), verapamil (vp) and lidocaine (lido) on the DNA distribution of human chondrocytes and the 3 H-thymidine incorporation as measure for the DNA synthesis capacity. * Address for correspondence: David Wohlrab, MD, Department of Orthopaedics, Martin Luther University, HalleWittenberg, 06097 Halle, Germany. Tel.: +49 345 557 4802/05; Fax: +49 345 557 4809; E-mail: david.wohlrab@medizin. uni-halle.de.

0006-355X/02/$8.00  2002 – IOS Press. All rights reserved

56

D. Wohlrab et al. / Influence of ion channels on the proliferation of human chondrocytes

2. Material and methods Electrophysiological investigations were performed on human chondrocytes isolated from the osteoarthitic knee joint cartilage of 11 patients (5 female, 6 male) suffering from gonarthritis between the ages of 40 to 79 years (mean 63.5 ± 12.8 years). 2.1. Chondrocyte preparation The cartilage was isolated from cartilage- bone- fragments resected during the insertion of knee prostheses. Immediately after the resection, the cartilage- bone- fragments were potted in sterile L15 medium (Seromed, Berlin, Germany). Thereafter, the cartilage was separated from the bone, reduced to 1 mm3 pieces and handled as described previously [17]. 2.2. Electrophysiological investigations To determine and characterize the membrane potential of the chondrocytes, these were incubated in solutions with known ionic concentrations (10 mmol/l hepes, 10 mmol/l glucose, 0.5 mmol/l MgCl2 , 0.3 mmol/l CaCl2 , pH = 7.4), distinguished only by the concentration of potassium. Additionally, these solutions contained the ionophor valinomycin (Molecular Probes, Leiden, Holland) and the fluorescent pigment oxonol VI (Molecular Probes). By valinomycin, the cell membrane becomes permeable for potassium and the fluorescent intensity of oxonol VI is altered depending on the membrane potential. First, the chondrocytes were incubated in a 150 mmol/l potassium solution and then, dissolved in a solution with less potassium concentration. Immediately after, a flow cytometric analysis of approximately 70,000 cells was performed by the determination of the fluorescence intensity of oxonol VI. Using the Nernst equation and knowing the exact ionic concentration of the used solutions it is possible to calculate the according membrane potentials. With the obtained curve and its mathematical function all measured fluorescence intensities of oxonol VI could be calculated. The influence of ion channel modulators on the membrane potential has been measured in human chondrocytes after incubation in different solutions. These solutions (10 mmol/l Hepes, 10 mmol/l glucose, 0.5 mmol/l MgCl2 , 0.3 mmol/l CaCl2 ) contained an ion channel modulator of different concentration each. For the measurement of the influence of different ion channel modulators on the membrane potential and the proliferation of human chondrocytes we used TEA, 4-AP, DIDS, SITS, verapamil and lidocaine. TEA (Fluka GmbH, Buchs, Switzerland) and 4-AP (Sigma GmbH, Steinheim, Germany) are known unspecific potassium channel blockers [4,7,14]. DIDS (Sigma GmbH) is an anion channel blocker with an inhibiting effect of chloride exchange simultaneously; ion channels with conducting capacity for Cl− and some other anions (e.g., aspartate) are mainly blocked [14]. SITS (Sigma GmbH) is known as a strong inhibitor of the anion transport. Verapamil (ICN-Biomedicals Inc., Aurora/Ohio, USA) blocks Ca2+ -channels in smooth and heart muscle cells. Additionally, it has vasodilatative and antiarrhythmic properties. Lidocaine (Sigma) is known as a Na+ -channel blocker. Different concentrations of the ion channel modulations were used (20 and 40 mmol/l TEA, 1 and 2 mmol/l 4-AP, 0,1 and 0.3 mmol/l DIDS, 0.25 and 0.5 mmol/l SITS, 0.25 and 0.5 mmol/l verapamil, lidocaine 0.1 and 0.2 mmol/l).


57

2.3. Determination of the DNA distribution The determination of the DNA distribution of human chondrocytes denoting the proliferation were performed using the Cycle TestTM PLUS DNA Reagent Kit (Becton Dickinson, San Jose, USA) with the fluorescent pigment propidium iodide. The fluorescence intensity being proportional of the DNA distribution has been measured by flow cytometry. For data evaluation the average as well as the standard deviation were calculated. The testing of the values for statistical significance (p < 0.05) was carried out with the aid of the t test and the one-way variance analysis, respectively. 2.4. 3 H-thymidine incorporation For proliferation measurement, chondrocytes were cultivated from 6, 12 or 18 days at 37.0◦ C with 5% CO2 . Thereafter measuring of the 3 H-thymidine incorporation as measure for the cell proliferation was carried out. Subsequently, this was followed by the addition of 20 µl 3 H-methyl-thymidine (specific activity 60.3 Ci/mmol; American Radio Labeled Chemicals Inc., St. Louis, USA). Two hours after administering 3 H-thymidine, the medium was siphoned off from the cell chamber with the aid of a cell harvester (Berthold Inc., Bad Wildbad, Germany). Every culture chamber was fed with 200 µl trypsin and after 20 minutes, the cell suspension was siphoned off through a filter. Subsequently, this was followed by measuring the radioactivity of the cells in the filter paper with the aid of a Tricarb (Berthold Inc., Bad Wildbad, Germany). The ion channel modulators dissolved in PBS were added on the second day of cell culture. The control investigations done at the same time were mixed with the same amount of PBS. To gain a start value, uncultivated cells were exposed to the influence of 3 H-thymidine.

3. Results 3.1. Membrane potential calibration curve The chondrocytes incubated in different extracellular potassium concentrations [K+ ]e were measured by flow cytometry to gain a calibration curve of the membrane potential. According to the measured fluorescence values the actual membrane potentials were calculated with the Nernst equation. For the calibration curve a mathematical function allowing the calculation of the membrane potential for each fluorescence value was determined [6]. The results as well as their mathematical function are shown in Fig. 1. 3.2. The influence of ion channel modulators on the membrane potential A considerable decrease of the membrane potential of the chondrocytes has been registered with 4-AP, verapamil and SITS. There were no differences in the different concentrations, however. The influence of TEA leads to a more positive membrane potential compared with a control group. With a concentration of 0.1 mmol/l, lidocaine causes an increase of the membrane potential of 20% compared to the control. This is a significant increase compared to TEA (Fig. 2). An influence of DIDS on the membrane potential of the chondrocytes could not be found (Fig. 2).

58


Fig. 1. Results of the flow cytometric characterization of the membrane potential of human chondrocytes using the fluorescent pigment oxonol VI and the ionophor valinomycin (n = 10).

Fig. 2. Behaviour of the membrane potential of human chondrocytes under the concentration dependent influence of different ion channel modulators (n = 6) (mmol).

3.3. DNA distribution The analysis of the results of the DNA distribution of human chondrocytes denoting the proliferation showed the following pattern: only approximately 95% of the cells are in G0/G1 phase of the cell cycle, 1% in the S phase and 4% in the G2/M phase. The subsumption of the results of the evaluation (Fig. 3) shows even after the analysis of the distribution in the several cell cycle phases a small drift of the populations in different donators. In Fig. 4 the results of all investigations are represented in a diagram.


59

Fig. 3. Cell cycle distribution of human chondrocytes (n = 11).

Fig. 4. Example of the DNA synthesis rate of human chondrocytes depending on the cultivation period. 3 H-thymidine was measured as a unit of proliferation. Each assessment consisting of eight single measurements provided the mean value.

These results show that the investigated human chondrocytes are diploid cells with an extremely low proliferative tendency. 3.4. 3 H-thymidine incorporation The different ion channel modulators were added to the human chondrocytes on the 2nd culture day. incorporation was measured on the 6th, 12th and 18th culture day. By the 6th culture day (4 days after adding ion channel modulators) there were no changes detected in the 3 H-thymidine incorporation rate compared to the control group. On the 12th culture day, an increase of the 3 H-thymidine incorporation was verified with 0.1 mmol/l lidocaine as well as with 1.0 mmol/l 4-AP. However, these ef3 H-thymidine

60


fects are limited. After 18 culture days, with all used ion channel blockers a decrease of the 3 H-thymidine incorporation rate was observed compared with the control group (Fig. 4).

4. Discussion Chondrocytes are considered non-excitable cell. However, like all living cell systems, they have a rest membrane potential. For this development, there are several mechanisms responsible, in particular, different active and passive transportation systems [6]. These mechanisms result in the development of an equilibrium potential called rest membrane potential. The existence of ion channels is a prerequisite for ion transport through the cell membrane and, following, the development of a rest membrane potential. In non-human chondrocytes there has already been proven several ion channels regulated by different ion channel modulators. Long et al. [9] proved the existence of Ca2+ activated K+ channels being blocked by TEA or Ba2+ and activated by a catalytic subunit of protein kinase A. Furthermore, Sugimoto et al. [16] described specific ion channels for Na+ as well as K+ and Cl− in rabbit’s chondrocytes. These ion channels could be inhibited by ion channel specific blockers tetrodotoxine (TTX), TEA, 4-AP and SITS. Martina et al. [11] successfully identified a K+ channel in pig’s chondrocytes activated by stretching of the channel. The findings presented show that a rest membrane potential on the cell membrane of chondrocytes exists, too. This rest membrane potential can be influenced by certain ion channel modulators. By adding potassium channel blockers, sodium channel blockers, anion channel blockers and calcium channel blockers change the membrane potential of the chondrocytes. Adding the ion channel blockers 4-AP, verapamil and SITS result in a more negative membrane potential by 18% compared to a control group. The unspecific K+ channel blocker TEA and the Na+ channel blocker lidocaine lead to a more positive membrane potential by 8% and 20%, respectively. These changes of the membrane potential caused by specific ion channel modulators are first evidence of the existence of specific ion channels for K+ , Ca2+ , Na+ , Cl− and anions, respectively. Further investigations will specify the ion channels more precisely. To gain knowledge of the connection between ion channel activity and cell proliferation, already proven in some and presumed in other cell systems, is essential. In 1988, Deutsch et al. [2] proved such a connection between K+ channel activity and the proliferation of Schwann cells and B lymphocytes. Thereafter, similar connections were identified in many other cell systems, such as tumor cell populations, human [12] and mouse keratinocytes [5], melanoma cells [13], neuroblastoma and astrocytoma cells [8]. A connection between free intracellular Ca2+ concentration and cell proliferation has already been known [1–3]. Whether the proliferation of chondrocytes can be influenced by ion channel activity is not known yet. First results show changed proliferation behaviour by modifying the ion channel activity. Up to now, it has been established that an increased proliferation by modification of ion channel activity can be traced to a conversion of cells from the G0 phase into the G1 phase of the cell cycle [10, 16]. Furthermore, it appears likely that the retention of cells in the G1 phase is partly abrogated [10,16]. These mechanisms lead to an increase of cells into the cell cycle resulting in a numerical shifting of cells within the phases by changing the regulation mechanisms in the course of cell proliferation. Accordingly, as approximately 90% of human osteoarthritic chondrocytes are in the G0/G1 phase, many cells can be stimulated to proliferate by ion channel modulation. So far connections of this kind are unknown in human as well as in non-human chondrocytes. Our results show, that by inhibition of different ion channels on the cell membrane of human chondrocytes


61

the proliferation behaviour can be influenced. The inhibition of Na+ and K+ channels, respectively, lead temporarily to a distinct increase of the proliferation. The inhibition of ion channels over a longer period lead presumably to a decrease of proliferation. As 95% of human chondrocytes exist in G0/G1 phase (Fig. 3) an increased proliferation by increased conversion of cells from G0 into G1 or G1 into S is suppositive. Certainly, further investigations are necessary to characterize possible connections between ion channel activity and proliferation behaviour as well as cell cycle distribution of human chondrocytes. References [1] A.M. Al Ani, A.G. Messenger, J. Lawry, S.S. Bleehen and S. MacNeil, Calcium/calmodulin regulation of the proliferation of human epidermal keratinocytes, dermal fibroblasts and mouse B16 melanoma cells in culture, Br. J. Dermatol. 119 (1988), 295–306. [2] S. Amigorena, D. Choquet, J.L. Teillaud, H. Korn and W.H. Fridman, Ion channels and B cell mitogenesis, Mol. Immunol. 27 (1990), 1259–1268. [3] D. Breitkreutz, H.J. Stark, P. Plein, M. Baur and N.E. Fusenig, Differential modulation of epidermal keratinization in immortalized (HaCaT) and tumorigenic human skin keratinocytes (HaCaT-ras) by retinoic acid and extracellular Ca2+ , Differentiation 54 (1993), 201–217. [4] C. Deutsch, K+ channels and mitogenesis, Prog. Clin. Biol. Res. 334 (1990), 2–21. [5] S.C. Harmon, D. Lutz and J. Ducote, Potassium channel openers stimulate DNA synthesis in mouse epidermal keratinocyte and whole hair follicle cultures, Pharmacol. Rev. 6 (1993), 170–178. [6] B. Hille, Ionic Channels of Excitable Membranes, Sinauer Associates Inc., Sunderland, 1992, pp. 504–524. [7] S. Inohara, Studies and perspectives of signal transduction in the skin, Exp. Dermatol. 1 (1992), 207–220. [8] Y.S. Lee, M.M. Sayeed and R.D. Wurster, Inhibition of cell growth by K+ channel modulators is due to interference with agonist-induced Ca2+ release, Cellular Signaling 5 (1993), 803–809. [9] K.J. Long and K.B. Walsh, A calcium-activated potassium channel in growth plate chondrocytes: regulation by protein kinase A, Biochem. Biophys. Res. Commun. 201 (1994), 776–781. [10] J. Lübbe, P. Kleihues and G. Burg, Das Tumorsupressor-Gen p53 und seine Bedeutung für die Dermatologie, Hautarzt 45 (1994), 741–745. [11] M. Martina, J.W. Mozrzymas and F. Vittur, Membrane stretch activates a potassium channel in pig articular chondrocytes, Biochim. Biophys. Acta 1329 (1997), 205–210. [12] M.T. Mauro, R.R. Isseroff, R. Lasarow and A.P. Pappone, Ion channels are linked to differentiation in keratinocytes, J. Membrane Biol. 132 (1993), 201–209. [13] B. Nilius and G. Droogmans, A role for potassium channels in cell proliferation?, News in Physiological Sciences 16 (1994), 1–12. [14] B. Nilius, G. Schwarz and G. Droogmans, Control of intracellular calcium by membrane potential in human melanoma cells, Am. J. Physiol. 265 (1993), 1501–1510. [15] T. Sugimoto, M. Yoshino, M. Nagao, S. Ishii and H. Yabu, Voltage-gated ionic channels in cultured rabbit articular chondrocytes, Comp. Biochem. Physiol. 115C (1996), 223–232. [16] D. Wohlrab, Der Einfluß elektrophysiologischer Membraneigenschaften auf die Proliferation humaner Keratinozyten, Tectum, Marburg, 1998, pp. 1–89. [17] D. Wohlrab, J. Wohlrab, H. Reichel and W. Hein, Is the proliferation of human chondrocytes regulated by ionic channels?, J. Orthop. Sci. 6 (2001), 155–159.