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

Abstract

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