Fertilization and Nicotinic Acid Adenine Dinucleotide Phosphate Induce pH Changes in Acidic Ca2+ Stores in Sea Urchin Eggs

Abstract

The second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) releases Ca 2؉ from the acidic Ca 2؉ stores of many organisms, including those of the sea urchin egg. We investigated whether the pH within the lumen of these acidic organelles changes in response to stimuli. Fertilization activates the egg by Ca 2؉ release dependent upon NAADP, and accordingly, we report that fertilization also alters organellar pH in a spatio-temporally complex manner. Upon sperm fusion, vesicles deep in the egg center slowly acidify, whereas cortical vesicles undergo a rapid alkalinization. The cortical vesicle alkalinization is independent of exocytosis and cytosolic pH but coincides with the NAADP-dependent fertilization Ca 2؉ wave. Microinjection of NAADP mimicked the fertilization cortical response, suggesting that it occurred within NAADP-sensitive acidic Ca 2؉ stores. Our data show that NAADP and physiological stimuli alter the pH within intracellular organelles and suggest that NAADP signals through pH as well as Ca 2؉ .The sea urchin egg is an invaluable cell model for studying Ca 2ϩ signaling since the discovery of new second messengers and new Ca 2ϩ stores in this system has subsequently proven to have a major impact on mammalian biology (1). The mechanism of activation of the egg upon fertilization encompasses the rapid increases in the cytosolic pH and intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) important for fertilization envelope formation, DNA and protein synthesis, and embryological development (2). The pattern of [Ca 2ϩ ] i changes within the egg are highly organized spatio-temporally and reflect the precisely timed and placed recruitment of different families of Ca 2ϩ channels resident upon either the plasma membrane or intracellular organelles (2, 3). After the initial transient "cortical flash" caused by Ca 2ϩ influx across the plasma membrane (2-5), Ca 2ϩ release from intracellular stores is manifest as a Ca 2ϩ wave that propagates across the entire egg initiating from the point of sperm entry (2). These phasic elevations in [Ca 2ϩ ] i are driven by a complex interplay between the second messengers inositol 1,4,5-trisphosphate, cyclic adenosine diphosphoribose, and nicotinic acid adenine dinucleotide phosphate (NAADP) 2 (1). Importantly, the relative contribution of each messenger to each phase of the Ca 2ϩ signal is different, e.g. NAADP is the only messenger implicated in the cortical flash (4, 5), whereas cyclic adenosine diphosphoribose probably plays a later role in prolonging the main Ca 2ϩ spike (6).NAADP not only evokes a cortical flash but, as first revealed in sea urchin egg, also releases Ca 2ϩ from acidic Ca 2ϩ stores that are lysosome-like organelles (possibly yolk platelets in eggs), whereas inositol 1,4,5-trisphosphate and cyclic adenosine diphosphoribose release Ca 2ϩ from the neutral endoplasmic reticulum (1, 7). Recently in sea urchin egg homogenate, we revealed that the luminal pH (pH L ) of these acidic stores is dynamic and increases upon NAADP-induced Ca 2ϩ release, whic...

Calcium signals regulated by NAADP and two-pore channels - their role in development, differentiation and cancer

“…Subsequently, NAADP-sensitive Ca 2+ stores, but not IP 3 -or cADPR-sensitive stores, were shown to be sensitive to disruption of the lysosomal proton gradient via blockers of the vacuolar H + ATPase such as bafilomycin A1, and to osmotic lysis of lysosomal stores by glycyl-L-phenylalanine-beta-naphthylamide (GPN), indicating that NAADP releases Ca 2+ from lysosome-related acidic organelles (Churchill et al, 2002). NAADP-induced Ca 2+ release was also found to be accompanied by an increase in lysosome luminal pH (Morgan and Galione 2007). Lysosome-related stores were subsequently confirmed as the target of NAADP in mammalian cells (Kinnear et al, 2004, Yamasaki et al, 2004.…”

NAADP Receptors

“…Thus, fluctuations in luminal Ca 2þ because of cycles of release and uptake of Ca 2þ could be important determinants of the effects of NAADP on Ca 2þ release, and offers one explanation for how constant NAADP levels may elicit trains of Ca 2þ spikes, as widely observed in various cell types (Cancela et al 1999). Another variable is luminal pH of acidic stores, since NAADP has also been found to alkalinize acidic stores in sea urchin eggs and homogenates (Morgan and Galione 2007a;Morgan and Galione 2007b), and it is possible that luminal pH has significant effects on TPC2 channel properties. Importantly, the NAADP antagonist was also found to block single channel TPC2 currents (Pitt et al 2010).…”

The Ecto-enzyme CD38 Is a Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Synthase That Couples Receptor Activation to Ca2+ Mobilization from Lysosomes in Pancreatic Acinar Cells

“…The Ca 2ϩ -mobilizing messenger nicotinic acid adenine dinucleotide phosphate (NAADP) 5 is the most potent of the three established Ca 2ϩ -mobilizing messengers. NAADP was first shown to evoke Ca 2ϩ release from intracellular stores in sea urchin eggs (1).…”