Signal transduction in electrically stimulated articular chondrocytes involves translocation of extracellular calcium through voltage-gated channels

Objective: To ascertain, using specific inhibitors, the potential role of calcium-related signal transduction pathways in the mechanism of cartilage matrix protein gene induction and metalloproteinase gene suppression by capacitively coupled electric fields. Methods: Articular chondrocytes were isolated from adult bovine patellae and cultured in high density for 7 days. To study matrix protein expression, cells cultured in the absence or presence of specific calcium pathway inhibitors were exposed to a capacitively coupled electrical field (60 kHz, 20 mV/cm): for aggrecan 1 h at 50% duty cycle and for type II collagen 6 h at 8.3% duty cycle. To study metalloproteinase expression in the presence of interleukin 1 beta (IL-1 beta), cells were cultured as above but exposed for only 30 min to a 100% duty cycle signal. At harvest, total mRNA was isolated and aggrecan, type II collagen, matrix metalloproteinase (MMP-1, -3 and -13) and aggrecanase [a disintegrin and metalloproteinase with thrombospondin repeats (ADAMTS-4 and -5)] mRNA expression were measured by quantitative real-time polymerase chain reaction (qPCR). Results: (1) In the absence of inhibitors, appropriate electrical stimulation induces a 3-4-fold up-regulation of both aggrecan and type II collagen mRNA and a 3.7-9.6-fold down-regulation of IL-1 beta-induced metalloproteinases; (2) the presence of inhibitors alone does not affect any target mRNA levels; (3) inhibitors of intracellular calcium regulation and inositol 1,4,5-triphosphate (IP3) formation [8-(diethylamino)octyl-3,4,5,trimethoxybenzoate hydrochloride (TMB-8) and neomycin, respectively] have no effect on regulation of target mRNA levels by electrical stimulation; and (4) inhibitors of voltage-gated calcium channels (verapamil), calmodulin activation (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride, W-7), calcineurin activity (cyclosporin A), phospholipase C activity (bromophenacyl bromide, BPB) and prostaglandin E-2 (PGE(2)) synthesis (indomethacin) completely inhibit the effects of electrical stimulation. Conclusions: The results are consistent with the effects of electrical stimulation involving a pathway of extracellular Ca2+ influx via voltagegated calcium channels rather than from intracellular Ca2+ repositories; and with downstream roles for calmodulin, calcineurin and nuclear factor of activated T-cells (NF-AT) rather than for phospholipase C and IP3. (c) 2008 Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International.