Muscarinic Receptor-mediated Dual Regulation of ADP-ribosyl Cyclase in NG108-15 Neuronal Cell Membranes

Abstract

Cyclic ADP-ribose (cADP-ribose) 1 is synthesized from ␤-NAD ϩ , an abundant intracellular substrate, by ADP-ribosyl cyclase in sea urchin eggs and in mammalian cells (1, 2). Pharmacological studies suggest that cADP-ribose is an endogenous modulator of ryanodine-sensitive Ca 2ϩ release channels (3-10). If cADP-ribose acts as an intracellular second messenger, ADPribosyl cyclase, as an effector enzyme, should be activated or inhibited in response to stimulation by hormones or neurotransmitters, which should simultaneously be associated with a transient decrease in the intracellular NAD ϩ concentration ([NAD ϩ ] i ) and an increase in cADP-ribose concentration (11).ADP-ribosyl cyclase seems to be present in both cytosolic and membrane-bound forms (1, 2, 12). The mammalian membranebound form of ADP-ribosyl cyclase has been identified as a cellsurface antigen, CD38 (13-19) and .Recently, it has been shown that the formation of cADPribose is regulated by nitric oxide or cGMP (21-23) and that nitric oxide or cGMP is increased by stimulation with agonists (24, 25). These findings suggest the hypothesis that the regulation of the cADP-ribose level is located far downstream in the signal transduction cascade from receptors (11). An alternative hypothesis is that the cADP-ribose formation is regulated by ADP-ribosyl cyclase through the direct action of G proteins activated by receptors within the surface membrane, as already shown for the formation of cyclic AMP, inositol 1,4,5-trisphosphate, and diacylglycerol (26 -28). To test this hypothesis, we used NG108-15 neuroblastoma ϫ glioma hybrid cells (29), in which signal transduction from receptors to effectors has been extensively characterized (29,30). In particular, in NGPM1-27 cells (31), which overexpress muscarinic acetylcholine receptors (mAChRs), it has been shown that intracellular NAD ϩ or NAD ϩ metabolites are involved in signal transduction from m1 mAChRs to K ϩ channels (32,33). In this context, such neuronal cell lines have advantages for analyzing receptor-ADP-ribosyl cyclase coupling in detail.For measurement of ADP-ribosyl cyclase, high pressure liquid chromatography (HPLC) is commonly used to separate cADP-ribose-related compounds (1,2,8,14,15,17,19,34,35). However, since it takes 30 -60 min to process one sample, it is essential to develop a much more rapid method that can allow processing of multiple samples at once. There are two papers that describe ADP-ribosyl cyclase assay by TLC (21,36), in which NAD ϩ migrates faster than cADP-ribose. The methods used in those reports seem to be affected by large amounts of radiolabeled substrates. We here developed a TLC method that overcomes this problem and allows separation of cADP-ribose in up to 19 samples within 40 -50 min. Our TLC method was first tested on COS-7 cells overexpressing human CD38 and was shown to be applicable for measuring ADP-ribosyl cyclase activity. We demonstrate that crude cell membranes of NG108-15 cells possess ADP-ribosyl cyclase activity and that such activity is activated or inhibi...

Ryanodine receptors are involved in muscarinic antinociception in mice

“…Carbachol-evoked increase of intracellular calcium concentration in cerebellar granule cells was inhibited by ryanodine [30]. Activation of muscarinic acetylcholine receptors in NG108-15 neuroblastoma × glioma cells produces cyclic ADP-ribose, a known endogenous modulator of RyR, which may upregulate the release of calcium from the ryanodine receptors [18,19]. Carbachol-induced contractions of rat stomach fundus strips were reduced by ryanodine and thapsigargin [31].…”

Oxytocin-induced elevation of ADP-ribosyl cyclase activity, cyclic ADP-riboseor Ca2+ concentrations is involved in autoregulation of oxytocin secretionin the hypothalamus and posterior pituitary in male mice

“…ADP-ribosyl cyclase activity in the hypothalamus and pituitary were determined fluorometrically using nicotinamide guanine dinucleotide (NGD + ) as a substrate, with a slight modification (Higashida et al, 1997 and1999) of the method described previously (Graeff et al, 1994). The tissues were homogenized in 10 mM Tris-HCl buffer, pH 7.4.…”

“…Intracellular cADPR concentrations are known to be regulated in many different ways: in one such mechanism, ADP-ribosyl cyclase or CD38 seems to be coupled directly with neurotransmitter or hormone receptors such as muscarinic acetylcholine or metabotropic glutamate receptors via different G proteins on the membrane surface (Higashida et al, 1997(Higashida et al, , 1999(Higashida et al, , 2007; or phosphorylation downstream of the G-protein-coupled receptor signaling pathway (Boittin et al, 2003;Sternfeld et al, 2003). Specifically, the activation of ADP-ribosyl cyclase or CD38 by cyclic GMP-or cyclic AMP-dependent protein kinases has been reported in Aplysia californica (Graeff et al, 1998), LAK cells (Rah et al, 2005) and artery smooth muscle cells (Boittin et al, 2003).…”

cADP-ribose potentiates cytosolic Ca2+ elevation and Ca2+ entry via L-type voltage-activated Ca2+ channels in NG108-15 neuronal cells

“…Since ADPR has no facilitating effect, the action of β-NAD + seems to be mediated by cADPR [23,24], after conversion of β-NAD + to cADPR by ADP-ribosyl cyclase in NG108-15 cells. Recently, we have reported membrane-associated intrinsic ADP-ribosyl cyclase modulated by muscarinic receptor stimulation [18].…”

“…To pursue these issues, we used differentiated NG108-15 cells which possess low-and highthreshold VACCs [15] and RyRs that are sensitive to caffeine [14,16]. In this preparation, we have found that β-NAD + and cADPR function as modulators of the M-type K + channel [17] and have characterized ADP-ribosyl cyclase activity, which converts cADPR from β-NAD + [18]. medium supplemented with 5 % fetal calf serum, 100 µM hypoxanthine, 0.1 µM aminopterin and 16 µM thymidine, as described previously [17].…”