title: Fluorescent analogs of NAADP with calcium mobilizing activity.
abstract: Nicotinic acid adenine dinucleotide phosphate (NAADP) mobilizes Ca2+ through a mechanism totally independent of cyclic ADP-ribose or inositol trisphosphate. Fluorescent analogs of NAADP were synthesized in this study to facilitate further characterization of this novel Ca2+ release mechanism. The base-exchange reaction catalyzed by ADP-ribosyl cyclase was utilized to convert nicotinamide 1,N6-ethenoadenine dinucleotide phosphate to a fluorescent product, nicotinic acid 1,N6-ethenoadenine dinucleotide phosphate (etheno-NAADP). The excitation spectrum of the product showed two maxima at 275 nm and 300 nm and an emission maximum at 410 nm. An aza derivative of etheno-NAADP was also synthesized by sequential treatments with NaOH and nitrite. The product, nicotinic acid 1,N6-etheno-2-aza-adenine dinucleotide phosphate (etheno-aza-NAADP) had excitation maxima at 280 nm and 360 nm and an emission maximum at 470 nm. The fluorescence of both analogs was sensitive to polarity and exhibited a 3-4-fold enhancement going from an aqueous buffer to an organic solvent. Proton-NMR measurements confirmed the presence of the etheno ring in both analogs. In the aza derivative the proton at the 2-position of the adenine ring was absent, consistent with the conversion of the 2-carbon to a nitrogen. Both analogs could activate Ca2+ release from sea urchin egg homogenates and the half-maximal concentrations for etheno-aza-NAADP and etheno-NAADP were at about 2.5 microM and 5 microM, respectively. At sub-threshold concentrations, both analogs could also function as antagonists, inactivating the NAADP-sensitive Ca2+ release with a half-maximal concentration of 60-80 nM. Microinjection of etheno-aza-NAADP into live eggs activated Ca2+ increase and triggered a cortical exocytotic reaction confirming its effectiveness in vivo. These fluorescent analogs are potentially useful for visualizing the novel Ca2+ stores that are sensitive to NAADP in live cells.
authors.affiliation: Department of Physiology, University of Minnesota, Minneapolis 55455, USA. leehc@maroon.tc.umn.edu
authors.forename: H C
authors.initials: HC
authors.lastname: Lee
authors.forename: R
authors.initials: R
authors.lastname: Aarhus
mesh_terms: D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D000818:Animals; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D005260:Female; D005456:Fluorescent Dyes / Q000737:chemistry; D066298:In Vitro Techniques; D017136:Ion Transport / Q000187:drug effects; D009682:Magnetic Resonance Spectroscopy; D009244:NAD+ Nucleosidase / Q000378:metabolism; D009249:NADP / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D010063:Ovum / Q000187:drug effects / Q000378:metabolism; D012617:Sea Urchins; D013050:Spectrometry, Fluorescence
chemical_list: D015703:Antigens, CD; D000943:Antigens, Differentiation; D005456:Fluorescent Dyes; C115597:nicotinic acid 1,N(6)-etheno-2-azaadenine dinucleotide phosphate; C115596:nicotinic acid 1,N(6)-ethenoadenine dinucleotide phosphate; D009249:NADP; C024376:NAADP; D036541:ADP-ribosyl Cyclase; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1; D002118:Calcium