CELL-PERMEANT CAGED INSP(3) ESTER SHOWS THAT CA2-EXPRESSION( SPIKE FREQUENCY CAN OPTIMIZE GENE)

Inositol 1,4,5-trisphosphate (InsP(3)) releases calcium from intracell ular stores and triggers complex waves and oscillations in levels of c ytosolic free calcium(1-5). To determine which longer-term responses a re controlled by oscillations in InsP(3) and cytosolic free calcium, i t would be useful to deliver exogenous InsP(3), under spatial and temp oral control, into populations of unpermeabilized cells. Here we repor t the 15-step synthesis of a membrane-permeant, caged InsP(3) derivati ve from myo-inositol. This derivative diffused into intact cells and w as hydrolysed to produce a caged, metabolically stable InsP(3) derivat ive. This latter derivative accumulated in the cytosol at concentratio ns of hundreds of micromolar, without activating the InsP(3) receptor. Ultraviolet illumination uncaged an InsP(3) analogue nearly as potent as real InsP(3), and generated spikes of cytosolic free calcium, and stimulated gene expression via the nuclear factor of activated T cells (6,7). The same total amount of InsP(3) analogue elicited much more ge ne expression when released by repetitive flashes at 1-minute interval s than when released at 0.5- or greater than or equal to 2-minute inte rvals, as a single pulse, or as a slow sustained plateau, Thus, oscill ations in cytosolic free calcium levels at roughly physiological rates maximize gene expression for a given amount of InsP(3).