Functions of NAADP as a Second Messenger

cl+ is an important intracellular messenger with a role in a wide range of processes. Ca2 + is normally maintained at a very low concentration on the cytosol, but in order to generate a signal, its concentration may be increased by influx through channels in the plasma membrane, or by means of release from intracellular stores. Release of Ca2 + from intracellular stores may be initiated by ci+ itself, or by a Ca:!T-releasing second messenger such as inositol 1,4,5-trisphosphate. Another such Ca2+-releasing second messenger is the recently discovered nicotinic acid adenine dinucleotide phosphate (NAADP). The second messenger functions of NAADP have thus far been defined onlv in a limited munber of model tissues. This thesis aims to develop a versatile and sensitive method for measuring changes in NAADP levels in a \vide variety of cell and tissue types. This method is then used to address a munber of outstanding questions \\1th regard to the physiological functions ofNAADP. The sea urchin sperm model system is used to investigate possible steps in the transduction cascade and the synthesis pathway for NAADP. The first data regarding a possible function for NAADP within the speml is also presented. The possible fimction ofNAADP as a second messenger bas then been investigated in two mammalian tissue types. Changes in NAADP levels in response to extracellular agol1ists are measured in cardiac ventricular and neuronal tissues, providing important insight into exactly which receptor types use NAADP as a part of their intracellular transduction cascade. For example, stimulation of dopamine D2-like, but not DJ-like receptors produces an increase in NAADP. The development of two new chemical tools, NAADP-AM and Ned-14 is also detailed. These are the first cell-permeant compolUlds to he identified for NAADP. Ned-14 is an antagonist of the NAADP receptor with an IC50 of 2 J.!M. As such, it is hoped that their use will allow much more rapid and definitive elucidation of the pathways of NAADP-mediated Ca2+signalling than has previously been possible.