Properties of the nicotinic acid adenine dinucleotide phosphate-binding protein in sea urchin eggs.

Nicotinic acid adenine dinucleotide phosphate (NAADP) has recently emerged as a novel intracellular calcium mobilising messenger in a variety of cells. Whilst increasing evidence suggests that NAADP acts on a distinct binding protein, little is known regarding the biochemical properties of the putative NAADP "receptor". My thesis investigates properties of the NAADP-binding protein in sea urchin eggs. Firstly, I show that NAADP binding to its target protein is inhibited by altering the protein:lipid ratio of soluble sea urchin egg homogenates - an effect prevented and reversed specifically by addition of exogenous phospholipids. These data highlight the importance of the lipid environment in maintenance of NAADP binding to its target protein. In addition, I show that upon binding its ligand, the NAADP-binding protein undergoes an unusual stabilization process that is dependent upon the time the receptor is exposed to its ligand. This property endows the NAADP-binding protein with the extraordinary ability to detect the duration of its activation. Finally I describe the development of a highly sensitive radioreceptor assay (based upon the sea urchin egg NAADP-binding protein) that is capable of detecting low levels of NAADP from cellular extracts. I apply this technique to determine NAADP levels in a variety of extracts prepared from cells under resting and stimulated conditions

The two-pore channel TPCN2 mediates NAADP-dependent Ca2+-release from lysosomal stores

Second messenger-induced Ca2+-release from intracellular stores plays a key role in a multitude of physiological processes. In addition to 1,4,5-inositol trisphosphate (IP3), Ca2+, and cyclic ADP ribose (cADPR) that trigger Ca2+-release from the endoplasmatic reticulum (ER), nicotinic acid adenine dinucleotide phosphate (NAADP) has been identified as a cellular metabolite that mediates Ca2+-release from lysosomal stores. While NAADP-induced Ca2+-release has been found in many tissues and cell types, the molecular identity of the channel(s) conferring this release remained elusive so far. Here, we show that TPCN2, a novel member of the two-pore cation channel family, displays the basic properties of native NAADP-dependent Ca2+-release channels. TPCN2 transcripts are widely expressed in the body and encode a lysosomal protein forming homomers. TPCN2 mediates intracellular Ca2+-release after activation with low-nanomolar concentrations of NAADP while it is desensitized by micromolar concentrations of this second messenger and is insensitive to the NAADP analog nicotinamide adenine dinucleotide phosphate (NADP). Furthermore, TPCN2-mediated Ca2+-release is almost completely abolished when the capacity of lysosomes for storing Ca2+ is pharmacologically blocked. By contrast, TPCN2-specific Ca2+-release is unaffected by emptying ER-based Ca2+ stores. In conclusion, these findings indicate that TPCN2 is a major component of the long-sought lysosomal NAADP-dependent Ca2+-release channel

Molecular Characterization of a Novel Intracellular ADP-Ribosyl Cyclase

Background. ADP-ribosyl cyclases are remarkable enzymes capable of catalyzing multiple reactions including the synthesis of the novel and potent intracellular calcium mobilizing messengers, cyclic ADP-ribose and NAADP. Not all ADP-ribosyl cyclases however have been characterized at the molecular level. Moreover, those that have are located predominately at the outer cell surface and thus away from their cytosolic substrates. Methodology/Principal Findings. Here we report the molecular cloning of a novel expanded family of ADP-ribosyl cyclases from the sea urchin, an extensively used model organism for the study of inositol trisphosphate-independent calcium mobilization. We provide evidence that one of the isoforms (SpARC1) is a soluble protein that is targeted exclusively to the endoplasmic reticulum lumen when heterologously expressed. Catalytic activity of the recombinant protein was readily demonstrable in crude cell homogenates, even under conditions where luminal continuity was maintained. Conclusions/Significance. Our data reveal a new intracellular location for ADP-ribosyl cyclases and suggest that production of calcium mobilizing messengers may be compartmentalized

NAADP-evoked Ca2+ signals through two-pore channel-1 require arginine residues in the first S4-S5 linker

Two-pore channels (TPCs) are two-domain members of the voltage-gated ion channel superfamily that localize to acidic organelles. Their mechanism of activation (ligands such as NAADP/PI(3,5)P2 versus voltage) and ion selectivity (Ca(2+) versus Na(+)) is debated. Here we report that a cluster of arginine residues in the first domain required for selective voltage-gating of TPC1 map not to the voltage-sensing fourth transmembrane region (S4) but to a cytosolic downstream region (S4-S5 linker). These residues are conserved between TPC isoforms suggesting a generic role in TPC activation. Accordingly, mutation of residues in TPC1 but not the analogous region in the second domain prevents Ca(2+) release by NAADP in intact cells. Our data affirm the role of TPCs in NAADP-mediated Ca(2+) signalling and unite differing models of channel activation through identification of common domain-specific residues

Native bamboo Arundinaria gigantea restoration for climate smart land management: A review

Bamboo, a versatile woody grass with over 1200 species, plays a critical role in the livelihoods of approximately 2.5 billion people globally, particularly in Asia, due to its diverse applications ranging from bioenergy production to construction and furniture manufacturing. Bamboo's significant potential in carbon farming and trading has garnered global attention. In the United States of America (U.S.A.), native bamboo species, collectively known as ''cane'', are represented by four species, including the historically abundant species giant river cane (Arundinaria gigantea). Historically forming extensive canebrakes in the southeastern U.S.A. and across 22 states, giant river cane is a resilient, perennial monocot capable of thriving across varied environments, from floodplains to mountain slopes. However, habitat loss due to agricultural expansion, urban development, overgrazing and fire suppression has reduced canebrake coverage to less than 2 % of its historical range. Giant river cane demonstrates exceptional environmental benefits, including sediment filtration, nutrient attenuation and habitat provision for diverse wildlife. Studies highlight its effectiveness in riparian buffers, significantly reducing sediment, nitrate and phosphorus levels in agricultural runoff, while its dense rhizome system aids in soil stabilization and water infiltration. Advanced propagation techniques, including rhizome planting and novel container-based methods, offer promising solutions for large-scale restoration of canebrakes, particularly in bottomland forest and riparian zones. Restoring giant river cane in degraded habitats not only supports biodiversity but also enhances ecosystem services, making it a critical component of riparian and agricultural land management. Giant cane’s contribution to ecological restoration is further supported by its impressive biomass production and carbon sequestration capabilities. In a 1720 m² plot, giant cane sequestered an estimated 5.8 metric tonnes of carbon, with a significant portion allocated to its below-ground system, emphasizing its role in soil stabilization and long-term carbon storage. Soil properties, such as calcium and phosphorus levels, also influence its growth dynamics, with soil calcium correlating with culm counts and cation exchange capacity affecting rhizome internodes. GIS-based site suitability analyses further enable targeted restoration efforts by identifying ideal conditions for giant cane growth. Future research should focus on optimizing propagation methods, evaluating its ecological impacts at watershed scales, and expanding its use in carbon trading frameworks to maximize its economic and environmental potential

Determination of cellular nicotinic acid-adenine dinucleotide phosphate (NAADP) levels.

Nicotinic acid-adenine dinucleotide phosphate (NAADP) is fast emerging as a new intracellular Ca2+-mobilizing messenger. In sea urchin egg homogenates, binding of NAADP to its receptor is not readily reversible; hence, prior incubation with low concentrations of NAADP is more effective in inhibiting subsequent binding of radiolabelled NAADP than incubating the preparation with the two ligands simultaneously [Patel, Churchill and Galione (2000) Biochem. J. 352, 725-729]. We extend this finding to show that NAADP is more effective still in inhibiting the subsequent radioligand binding at lower homogenate concentrations, an effect again quite probably due to the non-reversible nature of the receptor-ligand interaction. Enhanced sensitivity of the preparation to NAADP afforded by simple manipulation of the experimental conditions has been applied to determine low levels of NAADP in acid extracts from human red blood cells, rat hepatocytes and Escherichia coli without interference from NADP breakdown. Our improved method for the quantification of NAADP should prove useful in the further assessment of its signalling role within cells

Similarities of KATP channel expression and Ca2+ changes in pancreatic beta cells and hypothalamic neurons

The mechanisms through which pancreatic β cells recognize and respond to changes in circulating glucose are well understood. Evidence is accumulating that a subpopulation of neurons in the ventromedial hypothalamus (VMH) use similar cellular mechanisms to sense changes in extracellular glucose. In the present study, we used PCR and single-cell calcium imaging techniques to investigate whether glucose-sensing cells in the pancreas and hypothalamus employ a similar set of stimulus-response elements. Dispersed cells from mouse pancreata and hypothalamus were used in conjunction with the insulin-secreting cell line MIN6. We present functional data suggesting that both pancreatic and a subpopulation of hypothalamic cells exhibit glucose- and tolbutamide-evoked changes in cytosolic calcium and consider some clinical implications of different glucose sensors using the same mechanisms. Copyright © 2005 by Lippincott Williams & Wilkins