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. 1998 Dec 28;143(7):2045–2056. doi: 10.1083/jcb.143.7.2045

Figure 8.

Figure 8

Functional expression of the sarcoplasmic Ca2+-ATPase and IP3-sensitive Ca2+ stores in ES cells and ES cell–derived cardiac precursor cells. The expression of the sarcoplasmic Ca2+-ATPase was investigated by addition of the selective antagonist Tg (1 μM) (left panels). (A) Tg evoked a sustained increase of [Ca2+]i in ES cells, indicating activation of a capacitative Ca2+ influx pathway. This was confirmed by application of Gd3+ (1 μM), which resulted in a fast decline of elevated [Ca2+]i to control levels. (B) In contrast, application of Tg in a cardiac precursor cell (7 d) resulted in a transient rise of [Ca2+]i. The expression of IP3-sensitive Ca2+ stores was tested by intracellular dialysis of ES cell–derived cells during different stages of development with IP3 (100 μM) and the Ca2+ indicator fura-2 (125 M) through patch-pipette (right panels). Changes in [Ca2+]i were monitored using measurements of the 360/390 ratio. Establishment of the classic whole-cell configuration (arrow) evoked in ES cells (A) and 7-d-old cardiomyocytes (B) a fast increase of the 360/390 ratio because of an increase of [Ca2+]i, indicating the functional expression of IP3-sensitive stores. In control experiments, omission of IP3 from the pipette solution evoked a decrease of the 360/390 ratio (insets, identical calibration as for IP3 experiments).