2'-deoxy cyclic adenosine 5'-diphosphate ribose derivatives: importance of the 2'-hydroxyl motif for the antagonistic activity of 8-substituted cADPR derivatives.

The structural features needed for antagonism at the cyclic ADP-ribose (cADPR) receptor are unclear. Chemoenzymatic syntheses of novel 8-substituted 2'-deoxy-cADPR analogues, including 8-bromo-2'-deoxy-cADPR 7, 8-amino-2'-deoxy-cADPR 8, 8- O-methyl-2'-deoxy-cADPR 9, 8-phenyl-2'-deoxy-cADPR 10 and its ribose counterpart 8-phenyl-cADPR 5 are reported, including improved syntheses of established antagonists 8-amino-cADPR 2 and 8-bromo-cADPR 3. Aplysia californica ADP-ribosyl cyclase tolerates even the bulky 8-phenyl-nicotinamide adenine 5'-dinucleotide as a substrate. Structure-activity relationships of 8-substituted cADPR analogues in both Jurkat T-lymphocytes and sea urchin egg homogenate (SUH) were investigated. 2'-OH Deletion decreased antagonistic activity (at least for the 8-amino series), showing it to be an important motif. Some 8-substituted 2'-deoxy analogues showed agonist activity at higher concentrations, among which 8-bromo-2'-deoxy-cADPR 7 was, unexpectedly, a weak but almost full agonist in SUH and was membrane-permeant in whole eggs. Classical antagonists 2 and 3 also showed previously unobserved agonist activity at higher concentrations in both systems. The 2'-OH group, without effect on the Ca (2+)-mobilizing ability of cADPR itself, is an important motif for the antagonistic activities of 8-substituted cADPR analogues.

Retinoic acid receptor antagonist inhibits CD38 antigen expression on human hematopoietic cells in vitro.

The CD34+ CD38- subset of human hematopoietic stem cells are crucial for long-term ex-vivo expansion; conditions that decreased this specific sub-population reduced the self-renewal capacity and shortened the duration of the proliferative phase of the culture. Retinoids, such as all-trans retinoic acid (ATRA), have been shown to induce CD38 expression. ATRA present in serum may be responsible for the high CD38 of cells grown in serum-containing medium. In the present study we analyzed the effects of AGN 194310, a retinoic acid receptor pan-antagonist, on CD38 expression of human hematopoietic cells. Normal cells (cord blood derived CD34+ cells) and abnormal cells (myeloid leukemic lines) were studied when grown in either serum-containing or serum-free media. The results showed that both serum and ATRA enhanced differentiation and, thereby, reduced the proportion of CD34+ CD38- cells and total CD34+ cell expansion. AGN reversed these effects of serum and ATRA: it delayed differentiation and increased CD34+ CD38- cells. These results suggest that physiological ATRA levels in serum may prevent efficient cell expansion. AGN, by neutralizing ATRA, improves cell expansion in serum-containing cultures, thus making AGN a useful agent for ex vivo expansion of stem cells and other specific sub-populations for research and clinical use.

Molecular mechanism of ADP-ribosyl cyclase activation in angiotensin II signaling in murine mesangial cells.

ADP-ribosyl cyclase (ADPR-cyclase) produces a Ca(2+)-mobilizing second messenger cyclic ADP-ribose (cADPR) from NAD(+). In this study, we investigated the molecular basis of ADPR-cyclase activation and the following cellular events in angiotensin II (ANG II) signaling in mouse mesangial cells (MMCs). Treatment of MMCs with ANG II induced an increase in intracellular Ca(2+) concentrations through a transient Ca(2+) release via an inositol 1,4,5-trisphosphate receptor and a sustained Ca(2+) influx via L-type Ca(2+) channels. The sustained Ca(2+) signal, but not the transient Ca(2+) signal, was blocked by a cADPR antagonistic analog, 8-bromo-cADPR (8-Br-cADPR), and an ADPR-cyclase inhibitor, 4,4'-dihydroxyazobenzene (DHAB). In support of the results, ANG II stimulated cADPR production in a time-dependent manner, and DHAB inhibited ANG II-induced cADPR production. Application of pharmacological inhibitors revealed that activation of ADPR-cyclase by ANG II involved ANG II type 1 receptor, phosphoinositide 3-kinase, protein tyrosine kinase, and phospolipase C-gamma1. Moreover, DHAB as well as 8-Br-cADPR abrogated ANG II-mediated Akt phosphorylation, nuclear translocation of nuclear factor of activated T cell, and uptake of [(3)H]thymidine and [(3)H]leucine in MMCs. These results demonstrate that ADPR-cyclase in MMCs plays a pivotal role in ANG II signaling for cell proliferation and protein synthesis.

Nicotinic acid-adenine dinucleotide phosphate activates the skeletal muscle ryanodine receptor.

Calcium is a universal second messenger. The temporal and spatial information that is encoded in Ca(2+)-transients drives processes as diverse as neurotransmitter secretion, axonal outgrowth, immune responses and muscle contraction. Ca(2+)-release from intracellular Ca(2+) stores can be triggered by diffusible second messengers like Ins P (3), cyclic ADP-ribose or nicotinic acid-adenine dinucleotide phosphate (NAADP). A target has not yet been identified for the latter messenger. In the present study we show that nanomolar concentrations of NAADP trigger Ca(2+)-release from skeletal muscle sarcoplasmic reticulum. This was due to a direct action on the Ca(2+)-release channel/ryanodine receptor type-1, since in single channel recordings, NAADP increased the open probability of the purified channel protein. The effects of NAADP on Ca(2+)-release and open probability of the ryanodine receptor occurred over a similar concentration range (EC(50) approximately 30 nM) and were specific because (i) they were blocked by Ruthenium Red and ryanodine, (ii) the precursor of NAADP, NADP, was ineffective at equimolar concentrations, (iii) NAADP did not affect the conductance and reversal potential of the ryanodine receptor. Finally, we also detected an ADP-ribosyl cyclase activity in the sarcoplasmic reticulum fraction of skeletal muscle. This enzyme was not only capable of synthesizing cyclic GDP-ribose but also NAADP, with an activity of 0.25 nmol/mg/min. Thus, we conclude that NAADP is generated in the vicinity of type 1 ryanodine receptor and leads to activation of this ion channel.

Subtype-specific coupling with ADP-ribosyl cyclase of metabotropic glutamate receptors in retina, cervical superior ganglion and NG108-15 cells.

Cyclic ADP-ribose (cADP-ribose) is a putative second messenger or modulator. However, the role of cADP-ribose in the downstream signals of the metabotropic glutamate receptors (mGluRs) is unclear. Here, we show that glutamate stimulates ADP-ribosyl cyclase activity in rat or mouse crude membranes of retina via group III mGluRs or in superior cervical ganglion via group I mGluRs. The retina of mGluR6-deficient mice showed no increase in the ADP-ribosyl cyclase level in response to glutamate. GTP enhanced the initial rate of basal and glutamate-stimulated cyclase activity. GTP-gamma-S also stimulated basal activity. To determine whether the coupling mode of mGluRs to ADP-ribosyl cyclase is a feature common to individual cloned mGluRs, we expressed each mGluR subtype in NG108-15 neuroblastoma x glioma hybrid cells. The glutamate-induced stimulation of the cyclase occurs preferentially in NG108-15 cells over-expressing mGluRs1, 3, 5, and 6. Cells expressing mGluR2 or mGluRs4 and 7 exhibit inhibition or no coupling, respectively. Glutamate-induced activation or inhibition of the cyclase activity was eliminated after pre-treatment with cholera or pertussis toxin, respectively. Thus, the subtype-specific coupling of mGluRs to ADP-ribosyl cyclase via G proteins suggests that some glutamate-evoked neuronal functions are mediated by cADP-ribose.