Figure 7. A caged IRL2500 analog inhibits rhodamine-ET-3 binding in isolated nuclei and the increase in [Ca²⁺]_n induced by caged ET-1.

A) Nuclei freshly isolated from rat ventricular myocytes were plated onto a laminin-coated Fluorodish and preincubated for 30 min with photolysed (uIRL2500) or not photolysed (cIRL2500) caged IRL2500 (10⁻⁶ M). Rhodamine ET-3 (7.5 × 10⁻⁹ M) was then added for an additional 30 min incubation. Following three washes, nuclei were stained with fluorescent DNA dye, DRAQ5. Images were acquired using a Zeiss LSM 710 microscope. Scale Bar = 4.8 μm. B) Changes in nucleoplasmic [Ca²⁺] were recorded, as described in Materials and Methods, before and after photolysis in cells preincubated with or without caged ET-1 (cET-1) and in presence or absence of caged IRL2500 (cIRL2500). DRAQ5 was used to target a region corresponding to the nucleoplasm. Signals are presented as background-subtracted normalized fluorescence (%F/F₀), where F is the fluorescence intensity, and F₀ is the resting fluorescence recorded in the same cell under steady-state conditions prior to photolysis. For each condition, the mean ± s.e.m. of nucleoplasmic Fluo-4 fluorescence at 50 s is presented.