Cartilage degradation by matrix metalloproteinases (MMPs) is a major factor in the progression of ostearthritis. We previously found that synovial cells of patients with osteoarthritis contained an increased concentration of gap-junction channels made of connexin 43, and that the channels were needed to maintain the interleukin-1β (IL-1β) signaling cascade that leads to increased MMP production. In the present study we sought evidence that the amplification mechanism by which pg/ml levels of IL-1β controlled MMP secretion involved propagation of a depolarization wave through the gap junctions in aggregates of synovial cells, resulting in the opening of membrane calcium channels, a known critical process in IL-1β signal transduction by synovial cells. Our specific aims were to experimentally detect the depolarization wave, and to provide a theoretical explanation of the wave propagation.
The nystatin double-patch method was used to study depolarization wave propagation in aggregates of ∼20 HIG-82 cells (nontransformed rabbit synovial fibroblasts) grown in petri-dishes (Fig. 1). Two electrodes were connected to different cells in the aggregate, and cell resting potentials at zero current and ionic currents through the cell membranes at clamped voltages were measured using Axopatch 200B amplifiers. The nystatin permitted use of the whole-cell configuration while preventing diffusion of small signaling molecules from the cell into the electrode, thereby preserving intracellular regulation. Each measurement was done 5 times and the results were presented as mean ± SE.
|Fig. 1. Nystatin double-patch of an aggregate of HIG-82 cells. Electrode 1 (left) was used to clamp membrane voltage of cell 1 at −30 mV for 5 minutes while adding IL-1β. Electrode 2 (right) measured resting potential of cell 2.|
We proved the existence of gap-junction channels between neighbor HIG-82 cells by direct measurements of the channel (Fig. 2); the channel current was blocked by the gap-junction inhibitors 18alpha-glycyrrhetinic acid and octanol.
|Fig. 2. Single gap junction channel recording between HIG-82 cells. Two electrodes were connected to two aggregated cells by the nystatin perforated-patch method. The current was measured at clamped 100 mV. Conductance of a single gap junction channel was 70±10 pS.|
The voltage dependence of membrane L-type calcium channels was studied by measuring integral current with 10 mM Ba2+ in the bath solution as current carrier. Ca2+ channels were opened at the membrane voltage greater than about −30 mV, and closed at more negative voltages.
Experiments using one electrode showed that the cells had a resting potential of −67±6 mV. Addition of 100 pg/ml IL-1β alone did not change the resting potentials. However, addition of the cytokine in the presence of a voltage clamp at −30 mV induced a permanent decrease in the resting potential of the patched cell to −33±10 mV. Experiments with Ca2+ channel blockers showed that pre-exposure of the cell to −30 mV was needed to open calcium channels, thereby increasing Ca2+ concentration in the cell.
Experiments with two electrodes attached to different cells in an aggregate (Fig. 1) revealed the following phenomenon. Pre-exposure to −30 mV of only one cell in an aggregate and subsequent application of IL-1β to the bath solution induced permanent depolarization of other cell in the aggregate, even though none of the other cells were pre-exposed directly to −30 mV (Fig. 3), indicating that the low voltage applied to one cell spread to other cells in the aggregate through gap-junction channels.
|Fig. 3. Resting potential of cell #2 in the aggregate shown in Fig. 1. (1) Cell #1 was temporally pre-exposed to −30 mV and 100 pg/ml IL-1β was added to the bath solution. In 7±3 minutes, resting potential of cell #2 dropped to −33±10 mV. (2) IL-1β was added without pre-exposition to low voltage.|
Control experiments showed that the clamped voltage (−30 mV) could propagate through gap junction channels without IL-1β only for one–two cells, which was significantly shorter in comparison with the distance between cells in the aggregate (Fig. 1).
We developed a mathematical model of the excitation wave propagation in monolayer of synovial cells taking into account paracrine activation of the cells by IL-1β and positive feedback of its production. The equations predicted the observed rate of spread of the depolarization wave.
The experimental evidence indicated that a −30 mV voltage clamp of a cell on one edge of the aggregate spread passively to adjacent cells through gap junction channels, thereby opening Ca2+channels and resulting in increased Ca2+ concentration in the adjacent cells. Following addition of IL-1β, the signaling cascade worked synergistically with the elevated Ca2+ levels causing a decrease of resting potential in these cells which spread through gap junctions to previously unaffected cells (Fig. 4).
|Fig. 4. Excitation wave propagation through gap-junction channels between synovial cells. Cells with high (−67 mV) and low (−33 mV) resting potential are shown by white and dark colors respectively. Opened and closed calcium and gap junction channels are shown.|
The obtained results demonstrated important principle that diffusion of signaling molecules like cytokines is not a simple passive process involving delivering the molecules to their receptors. In conjunction with nonlinear systems like gap junctions and other ion channels, diffusion can result in active signal propagation over relatively long distances. Such effects can play an important role in regulation in the organism, and in the development of disease such as osteoarthritis.