Recent evidence has shown that NAD(P) plays a variety of roles in cell-signaling processes. Surprisingly, the presence of NAD(P) utilizing ectoenzymes suggests that NAD(P) is present extracellularly. Indeed, nanomolar concentrations of NAD have been found in plasma and other body fluids. Although very high concentrations of NAD have been shown to enter cells, it is not known whether lower, more physiological concentrations are able to be taken up. Here we show that two mammalian cell types are able to transport low NAD concentrations effectively. Furthermore, extracellular application of NAD was able to counteract FK866-induced cell death and restore intracellular NAD(P) levels. We propose that NAD uptake may play a role in physiological NAD homeostasis.Pyridine nucleotides, although often considered as simple redox cofactors in the cell, are in fact multifunctional molecules involved in a wide range of cellular processes (1). It is becoming clear that NAD(P) is involved in more pharmacologically attractive cellular processes such as cell signaling, transcriptional regulation, and post-translational protein modification (2, 3). For example, NAD(P) has been shown to be the precursor of molecules involved in calcium signaling (e.g. cADPR, 2 NAADP, and ADPR), to be involved in the regulation of epigenetic changes via sirtuins and to be a substrate for both monoand poly-ADP-ribosylation (3-5).The general perception is that cellular NAD is synthesized either de novo from tryptophan or via one of two possible recycling pathways: from nicotinic acid or nicotinamide (together referred to as vitamin PP, or niacin) (6). Recently, a third biosynthesis pathway has been described, which uses nicotinamide riboside as a precursor (7).The presence of membrane-bound ectoenzymes that use NAD(P) (8) has led to the investigation and description of several mechanisms for the export of NAD across the plasma membrane, including transport through connexins and stimulus-induced exocytotic release (9 -11). Surprisingly, though, the possibility that low NAD concentrations can be imported across the membrane to directly replenish the cellular NAD(P) pools bypassing biosynthetic pathways has not been conclusively addressed. However, circumstantial evidence suggests that this may occur: (i) High concentrations of extracellularly applied NAD(H) have been shown to increase intracellular NAD levels (10, 12-14): (ii) extracellular NAD counteracts PARP-induced intracellular NAD depletion (12, 15); (iii) CD38 knock-out mice, which are impaired in their ability to degrade extracellular NAD, display higher endogenous SIRT1 activity (16); (iv) uptake processes have been shown for the Ca 2ϩ -mobilizing NAD(P) metabolites cADPR and NAADP in a variety of diverse mammalian cell types (17-21); (v) NAD and at least one of the enzymes involved in biosynthesis are present extracellularly (13,22,23); and (vi) Wallerian degeneration can be slowed by the addition of extracellular NAD (24).Here we show that various mammalian cell types are able to transport pico...

Neurofibromatosis type 2 (NF2) is an inherited predisposition cancer syndrome characterized by the development of multiple benign tumors in the nervous system including schwannomas, meningiomas, and ependymomas. Using a disease model comprising primary human schwannoma cells, we previously demonstrated that adherens junctions (AJs) are impaired in schwannoma cells because of a ubiquitous, upregulated Rac activity. However, the mechanism by which loss of contact inhibition leads to proliferation remains obscure in merlin-deficient tumors. In this study, we show that proliferative Wnt/β-catenin signaling is elevated as active β-catenin (dephosphorylated at serine 37 and threoine 41) localizes to the nucleus and the Wnt targets genes c-myc and cyclin D1 are upregulated in confluent human schwannoma cells. We demonstrate that Rac effector p21-activated kinase 2 (PAK2) is essential for the activation of Wnt/β-catenin signaling because depletion of PAK2 suppressed active β-catenin, c-myc, and cyclin D1. Most importantly, the link between the loss of the AJ complex and the increased proliferation in human schwannoma cells is connected by Src and platelet-derived growth factor receptor-induced tyrosine 654 phosphorylation on β-catenin and associated with degradation of N-cadherin. We also demonstrate that active merlin maintains β-catenin and N-cadherin complex at the plasma membrane through direct regulation. Finally, we demonstrate that phosphorylation of tyrosine 654 is critical for the increased proliferation in human schwannoma cells because overexpression of a Y654F mutant β-catenin reduces hyperproliferation of schwannoma cells. We suggest a model that these pathways are coordinated and relevant for proliferation in merlin-deficient tumors.

The resumption of the meiotic cycle (maturation) induced by 1-methyladenine in prophase-arrested starfish oocytes is indicated by the breakdown of the germinal vesicle and is characterized by the increased sensitivity of the Ca 2؉ stores to inositol 1,4,5-trisphosphate (InsP 3 ) to InsP 3 starting at the animal hemisphere (where the germinal vesicle was originally located) and propagating along the animal/vegetal axis of the oocyte. This initiates Ca 2؉ signals around the germinal vesicle before nuclear envelope breakdown. Previous studies have suggested that the final activation of the maturation-promoting factor (MPF), a cyclin-dependent kinase, which is the major element controlling the entry of eukaryotic cells into the M phase, occurs in the nucleus. MPF is then exported to the cytoplasm where its activity is autocatalytically amplified following a similar animal/vegetal spatial pattern. We have investigated whether activated MPF was involved in the increased sensitivity of the Ca 2؉ response to InsP 3 . We have found that the development of increased sensitivity of the Ca 2؉ stores to InsP 3 receptors together with the Ca 2؉ signals in the perinuclear region was blocked in oocytes treated with the specific MPF inhibitor roscovitine. That the nuclear MPF activation is indeed required for changes of the InsP 3 receptors sensitivity was shown by enucleating or by dissecting oocytes into vegetal and animal hemispheres prior to the addition of 1-MA. MPF activity 50 min after 1-methyladenine addition was much lower in the enucleated oocytes and in the vegetal hemisphere, which did not contain the germinal vesicle, as compared with the animal hemisphere, which did contain it. The Ca 2؉ increase induced by InsP 3 under these experimental conditions correlated with the changes in actin cytoskeleton induced by MPF.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is involved in the Ca2+ response observed at fertilization in several species, including starfish. In this study, we have employed Ca2+ imaging and the single-electrode voltage-clamp technique to investigate whether the NAADP-mediated Ca2+ entry discovered in our laboratory in starfish oocytes was underlain by a membrane current and whether the response to NAADP required an intact cytoskeleton. Uncaging of preinjected NAADP evoked a cortical Ca2+ flash that was followed by the spreading of the wave to the remainder of the cell. No Ca2+ increase was detected in Ca2+-free sea water. Under voltage-clamp conditions, the photoliberation of NAADP activated an inward rectifying membrane current, which reversed at potentials more positive than +50 mV and was abolished by removal of Ca2+ but not of Na+. The current was affected by preincubation with verapamil, SKF 96356, and thapsigargin but not by preinjection of heparin, 8-NH2- cyclic ADP-ribose, or both antagonists. The membrane current and the Ca2+ wave were inhibited by latrunculin-A and jasplakinolide, which depolymerize and stabilize actin cytoskeleton, respectively. These data offer the first demonstration that NAADP initiates a Ca2+ sweep by activating a Ca2+-permeable membrane current that requires an intact F-actin cytoskeleton as other Ca2+-permeable currents, such as ICRAC and IARC.