Signal transduction in electrically stimulated articular chondrocytes involves translocation of extracellular calcium through voltage-gated channels 1

SummaryObjectiveTo 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.MethodsArticular 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-1β), 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-1β-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 E2 (PGE2) synthesis (indomethacin) completely inhibit the effects of electrical stimulation.ConclusionsThe results are consistent with the effects of electrical stimulation involving a pathway of extracellular Ca2+ influx via voltage-gated 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.