Aims: 

Cytosolic free Ca²⁺ concentration exhibits a characteristic pattern during differentiation of chondrifying primary high density cell cultures (HDC) established from distal limb buds of 4-day-old chicken embryos. During our experiments, we undertook to characterise short-term, high frequency Ca²⁺ oscillations that characterise the differentiation of cells in HDC. We also aimed to identify ion channels that may regulate these oscillatory Ca²⁺ transients.

Methods: 

In the experimental model applied, spontaneous cartilage matrix formation occurs within a 6-day-long culturing period; cells of HDC differentiate into chondroblasts on day 3 of culturing. Spontaneous Ca²⁺ oscillations were detected in Fluo-4-loaded cells by confocal microscopy on various days of culturing.

Results: 

Oscillations with the highest amplitude and frequency were identified in cells of 1-day-old cultures. As Ca²⁺ oscillations were completely abolished when free Ca²⁺ ions were eliminated from the extracellular space, we concluded that influx of extracellular ions was required for this phenomenon. Voltage gated Ca²⁺ ion channels were also involved in the regulation of Ca²⁺ oscillations, since administration of the aspecific Ca²⁺ channel blocker CdCl₂, as well as the more specific nifedipine considerably interfered with both parameters of these oscillatory Ca²⁺ transients.

Conclusion: 

We hypothesise that the observed unique Ca²⁺ oscillations in differentiating cells of HDC might play a role in the activation of Ca²⁺ sensitive transcription factors required for the onset of their differentiation programme.

Support: 

Hungarian Science Research Fund (OTKA CNK 80709), Hungarian Ministry of Health (ETT 022/09)