| Abstrakt: |
Nicotinic acid adenine dinucleotide phosphate (NAADP) is a metabolite of NADP with Ca2+mobilizing activity. The Ca2+release mechanism activated by NAADP as well as the Ca2+stores that it acts on are different from those activated by either cyclic ADP-ribose or inositol 1,4,5-trisphosphate (IP3) (Lee, H. C., and Aarhus, R. (1995) J. Biol. Chem.270, 2152-2157). In order to demonstrate unambiguously that NAADP can mobilize Ca2+stores in live cells, a caged analog was synthesized by reacting NAADP with 1-(2-nitrophenyl)diazoethane. Anion exchange high pressure liquid chromatography (HPLC) was used to purify one particular caged form from the mixture of products. Phosphate analyses following specific enzymatic cleavage indicate that the caging group is on the 2′-phosphate. This is confirmed by 31P NMR spectroscopy, showing that the 2′-phosphate of the caged compound exhibits an altered chemical shift of −2.6 ppm as compared with 2.3 ppm determined for the 2′-phosphate of NAADP. Caged NAADP had no Ca2+releasing activity at a concentration as high as 1 μMwhen tested on sea urchin egg microsomes. After photolysis, it released Ca2+, was effective in nanomolar range, and was indistinguishable from authentic NAADP. The regeneration of NAADP after photolysis was also confirmed by HPLC analyses. The analog is particularly susceptible to UV and can be efficiently photolyzed using a spectrofluorimeter. To demonstrate its utility in live cells, caged NAADP was microinjected into sea urchin eggs. Photolysis effectively regenerated NAADP and activated Ca2+oscillations in the eggs. Removal of external Ca2+did not prevent the Ca2+oscillations but only delayed the second Ca2+peak by about 45 s, indicating that the oscillations are due to release from internal stores and not caused by Ca2+influx. A mechanism based on sensitization of the Ca2+release by Ca2+loading is proposed to account for the Ca2+oscillation observed. |
