Cell membrane permeable esters of d-myo-inositol 1,4,5-trisphosphate

d-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃, or IP₃) is a ubiquitous second messenger that regulates cytosolic Ca²⁺ activities ([Ca²⁺]_i). To study this signaling branch in intact cells, we have synthesized a caged and cell permeable derivative of IP₃, ci-IP₃/PM, from myo-inositol in 9 steps. Ci-IP₃/PM is a homologue of cm-IP₃/PM, a caged and cell permeable IP₃ ester developed earlier. In ci-IP₃/PM, 2- and 3-hydroxyl groups of myo-inositiol are protected by an isopropylidene group; whereas in cm-IP₃/PM, a methoxymethylene is used. Ci-IP₃/PM can be loaded into cells non-invasively to high concentrations without activating IP₃ receptors (IP₃Rs). UV uncaging of loaded ci-IP₃ released i-IP₃, a potent agonist of IP₃Rs, and evoked Ca²⁺ release from internal stores. Interestingly, elevations of [Ca²⁺]_i by i-IP₃ lasted longer than [Ca²⁺]_i transients by m-IP₃, the uncaging product of cm-IP₃. To understand this difference, we measured the metabolic stability of i-IP₃ and m-IP₃. Like natural IP₃ which is known to be rapidly metabolized in cells, m-IP₃ could only be detected within several seconds after uncaging cm-IP₃. In contrast, i-IP₃ was metabolized at a much slower rate. By exploiting different metabolic rates of m-IP₃ and i-IP₃, we developed two procedures for activating IP₃Rs in cells without UV uncaging. The first method involves photolyzing ci-IP₃/PM in vitro to generate i-IP₃/PM. Successive additions of low micromolar i-IP₃/PM to NIH 3T3 cells caused graded Ca²⁺ releases, confirming that "quantal Ca²⁺ release" occurs in fully intact cells with normal ATP supplies and undisrupted endoplasmic reticulum. The second technique utilizes two photon uncaging. After locally illuminating cells loaded with cm-IP₃ with femtosecond-pulsed near-infrared light (730 nm), we observed a burst of Ca²⁺ activity in the uncaging area. This local Ca²⁺ rise rapidly propagated across cells and could be repeated many times in different sub-cellular locations to produce artificial Ca²⁺ oscillations of defined amplitudes and frequencies. The complementary advantages of these IP₃ prodrugs should provide new approaches for studying IP₃-Ca²⁺ signaling in intact cell populations with high spatiotemporal resolutions.