Background: 

1-Ethyl-2-benzimidazolinone (1-EBIO) is a direct activator of SKCa channels and induces cellular hyperpolarization. This has been demonstrated in mouse embryonic stem cells (CGR8) by using the membrane potential sensitive dye Di-8-ANEPPS. Following prolonged treatment with EBIO stem cells are forced to differentiate preferentially into pacemaker-like cells. The detailed signalling pathway which contributes to this effect is still vague.

Methodology and Results: 

We identified an increase in nitric oxide (NO) (DAF-fluorescence microfluorometry) in ES-cells at day 5 following EBIO treatment. In 70% of the EBIO treated cells Ca²⁺ signals were observed. In comparison only 20% of the cells responded with Ca²⁺ transients to ATP at this time of cell culture. The Ca²⁺ response was still present in Ca²⁺ free external medium for EBIO as well as ATP. In contrast EBIO- and ATP-induced calcium signals were abolished upon preincubation with the calcium chelator BAPTA-AM. Furthermore the calcium store depleting agent thapsigargin completely abrogated the ATP and EBIO induced Ca²⁺-signal. When cells were incubated with the PLC inhibitor U73122 a complete inhibition of ATP-induced Ca²⁺-responses was observed, whereas upon EBIO treatment still 50 % of cells displayed Ca²⁺-signals. Dantrolene (ryanodine receptor inhibitor) inhibited the EBIO induced Ca²⁺-signal in 70% of the cells. Upon treatment with the NO-synthase inhibitor L-NAME the number of cells displaying Ca²⁺-responses towards EBIO were decreased by 30%.

Conclusion: 

We conclude that NO and Ca²⁺-signals contribute to cardiomyogenesis of murine ES-cells. The EBIO induced Ca²⁺-signal is generated partially from intracellular Ca²⁺ stores via IP3 receptors and ryanodine receptors.