Thio-NADP is not an antagonist of NAADP |
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Authors: | Deborah M Dickey Robert Aarhus Timothy F Walseth Hon Cheung Lee |
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Institution: | (1) Department of Pharmacology, University of Minnesota, 55455 Minneapolis, MN;(2) Department of Physiology, University of Minnesota, 55455 Minneapolis, MN |
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Abstract: | 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 μM, 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 μM thio-NADP and reduces the subsequent inhibition by 73.7±1.3%. The residual inhibitory effect is no more than that exerted
by 50 μM of either NADP itself or nicotinic acid adenine dinucleotide (NAAD). These results are confirmed by32P-NAADP binding studies. Unpurified thio-NADP inhibits the specific32P-NAADP binding to egg microsomes with an IC50 of 40 μM. After HPLC purification, only 20% inhibition is seen at a concentration as high as 50 μM, similar to the extent of inhibition effected by 40 μM 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. |
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Keywords: | NAADP Ca2+ release Thio-NADP |
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