Thio-NADP is not an antagonist of NAADP.
Nicotinic acid adenine dinucleotide phosphate (NAADP) is a metabolite of NADP, which can release Ca2+ from stores that are distinct from those activated by either cyclic ADP-ribose or inositol 1,4,5-trisphosphate (IP3). It has previously been suggested that thio-NADP is a specific antagonist of NAADP (Chini et al. [1995] J. Biol. Chem. 270, 3216-3223). Its effects in sea-urchin egg homogenates were investigated. At 50 microM, thio-NADP activates partial Ca2+ release and totally inhibits subsequent challenge with a saturating concentration of NAADP. Purification by HPLC eliminates the Ca2+ releasing activity of 50 microM thio-NADP and reduces the subsequent inhibition by 73.7 +/- 1.3%. The residual inhibitory effect is no more than that exerted by 50 microM of either NADP itself or nicotinic acid adenine dinucleotide (NAAD). These results are confirmed by 32P-NAADP binding studies. Unpurified thio-NADP inhibits the specific 32P-NAADP binding to egg microsomes with an IC50 of 40 microM. After HPLC purification, only 20% inhibition is seen at a concentration as high as 50 microM, similar to the extent of inhibition effected by 40 microM NADP. These results indicate the inhibitory substance in thio-NADP is a contaminant. The partial Ca2+ release activity of unpurified thio-NADP suggests the contaminant is NAADP itself. This is supported by the fact that pretreatment with a subthreshold concentration of only 2 nM NAADP totally desensitizes the egg homogenates such that no Ca2+ response is seen with saturating NAADP. Estimation from the binding studies shows that a contamination of 0.012% of NAADP in the unpurified thio-NADP samples is sufficient to account for the inhibitory effects. These results indicate thio-NADP is not an antagonist of NAADP.
Dickey
D M
DM
Department of Pharmacology, University of Minnesota, Minneapolis 55455, USA.
Aarhus
R
R
Walseth
T F
TF
Lee
H C
HC
D000246:Adenosine Diphosphate Ribose / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D000837:Anion Exchange Resins; D002118:Calcium / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D036563:Cyclic ADP-Ribose; D004340:Drug Contamination; D009243:NAD / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors; D009249:NADP / Q000031:analogs & derivatives* / Q000494:pharmacology; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D012117:Resins, Synthetic; D012617:Sea Urchins
D000837:Anion Exchange Resins; D012117:Resins, Synthetic; D009243:NAD; D036563:Cyclic ADP-Ribose; C015541:thionicotinamide adenine dinucleotide phosphate; D000246:Adenosine Diphosphate Ribose; D009249:NADP; C018348:nicotinic acid adenine dinucleotide; C042855:Mono Q; D002118:Calcium