Caged Nicotinic Acid Adenine Dinucleotide Phosphate

doi:10.1074/jbc.272.7.4172

Caged Nicotinic Acid Adenine Dinucleotide Phosphate

SYNTHESIS AND USE*

From the Department of Physiology, University of Minnesota, Minneapolis, Minnesota 55455,
^§ Molecular Probes, Inc., Eugene, Oregon 97402, and
^¶ 3M Company, St. Paul, Minnesota 55144-1000

^‡ To whom correspondence should be addressed:
6-182 Lyon Laboratory, Dept. of Physiology, University of Minnesota, Minneapolis, MN 55455.
Tel.: 612-625-7120, 612-625-4641; Fax: 612-625-0991, 612-625-5941; E-mail: leehc{at}maroon.tc.umn.edu

Abstract

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 μM when 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.

Footnotes

↵* This work was supported by National Institutes of Health Grants HD17484 and HD32040 (to H. C. L.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

cADPR

cyclic ADP-ribose

NAAD

nicotinic acid adenine dinucleotide

NAADP

nicotinic acid adenine dinucleotide phosphate

IP₃

inositol 1,4,5-trisphosphate

NPE

1-(2-nitrophenyl)diazoethane

HPLC

high pressure liquid chromatography

ASW

artificial seawater

0CaSW

Ca²⁺-free seawater.
- Received July 23, 1996.
- Revision received November 1, 1996.