TY - JOUR
T1 - Cell membrane permeable esters of d-myo-inositol 1,4,5-trisphosphate
AU - Dakin, Kenneth
AU - Li, Wen Hong
N1 - Funding Information:
We thank the financial support from the American Diabetes Association (7-01-CD-13) and the Welch Foundation (I-1510).
PY - 2007/9
Y1 - 2007/9
N2 - 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.
AB - 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.
KW - Calcium signaling
KW - Cell permeable ester of IP3
KW - IP3
KW - IP3/PM
KW - Prodrug design
KW - Quantal calcium release
KW - Second messenger
KW - Two photon uncaging
KW - ci-IP3/PM
KW - cm-IP3/PM
KW - i-IP3/PM
KW - m-IP3/PM
UR - http://www.scopus.com/inward/record.url?scp=34447560168&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=34447560168&partnerID=8YFLogxK
U2 - 10.1016/j.ceca.2006.12.003
DO - 10.1016/j.ceca.2006.12.003
M3 - Article
C2 - 17307252
AN - SCOPUS:34447560168
SN - 0143-4160
VL - 42
SP - 291
EP - 301
JO - Cell Calcium
JF - Cell Calcium
IS - 3
ER -