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

D-myo-inositol 1.4,5-trisphosphate (Ins(1,4,5)P-3, or IN 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 (IP(3)Rs). UV uncaging of loaded Ci-IP3 released i-IP3, a potent agonist Of IP3Rs. and evoked Ca2+ 2,] 2,], release from internal stores. Interestingly, elevations of [Ca i by i-IP3 lasted longer than [Ca 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 fermosecond-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. (c) 2007 Elsevier Ltd. All rights reserved.