On a Magical Mystery Tour with 8-Bromo-Cyclic ADP-Ribose: From All-or-None Block to Nanojunctions and the Cell-Wide Web.

A plethora of cellular functions are controlled by calcium signals, that are greatly coordinated by calcium release from intracellular stores, the principal component of which is the sarco/endooplasmic reticulum (S/ER). In 1997 it was generally accepted that activation of various G protein-coupled receptors facilitated inositol-1,4,5-trisphosphate (IP3) production, activation of IP3 receptors and thus calcium release from S/ER. Adding to this, it was evident that S/ER resident ryanodine receptors (RyRs) could support two opposing cellular functions by delivering either highly localised calcium signals, such as calcium sparks, or by carrying propagating, global calcium waves. Coincidentally, it was reported that RyRs in mammalian cardiac myocytes might be regulated by a novel calcium mobilising messenger, cyclic adenosine diphosphate-ribose (cADPR), that had recently been discovered by HC Lee in sea urchin eggs. A reputedly selective and competitive cADPR antagonist, 8-bromo-cADPR, had been developed and was made available to us. We used 8-bromo-cADPR to further explore our observation that S/ER calcium release via RyRs could mediate two opposing functions, namely pulmonary artery dilation and constriction, in a manner seemingly independent of IP3Rs or calcium influx pathways. Importantly, the work of others had shown that, unlike skeletal and cardiac muscles, smooth muscles might express all three RyR subtypes. If this were the case in our experimental system and cADPR played a role, then 8-bromo-cADPR would surely block one of the opposing RyR-dependent functions identified, or the other, but certainly not both. The latter seemingly implausible scenario was confirmed. How could this be, do cells hold multiple, segregated SR stores that incorporate different RyR subtypes in receipt of spatially segregated signals carried by cADPR? The pharmacological profile of 8-bromo-cADPR action supported not only this, but also indicated that intracellular calcium signals were delivered across intracellular junctions formed by the S/ER. Not just one, at least two. This article retraces the steps along this journey, from the curious pharmacological profile of 8-bromo-cADPR to the discovery of the cell-wide web, a diverse network of cytoplasmic nanocourses demarcated by S/ER nanojunctions, which direct site-specific calcium flux and may thus coordinate the full panoply of cellular processes.

Inhibition of CD38/Cyclic ADP-ribose Pathway Protects Rats against Ropivacaine-induced Convulsion.

BACKGROUND: The CD38/cyclic ADP-ribose (cADPR) pathway plays a role in various central nervous system diseases and in morphine tolerance, but its role in local anesthetic intoxication is unknown. The aim of this study was to determine the role of the CD38/cADPR pathway in ropivacaine-induced convulsion. METHODS: Forty male Sprague-Dawley rats were randomly divided into five groups (n = 8 per group): sham group, ropivacaine group, ropivacaine+8-Br-cADPR (5 nmol) group, ropivacaine+8-Br-cADPR (10 nmol) group, and ropivacaine+8-Br-cADPR (20 nmol) group (no rats died). Rats were intracerebroventricularly injected with normal saline or 8-Br-cADPR 30 min before receiving an intraperitoneal injection of ropivacaine. Electroencephalography and convulsion behavior scores were recorded. The hippocampus was harvested from each group and subjected to nicotinamide adenine dinucleotide and cADPR assays, Western blotting analysis, and malondialdehyde (MDA) and superoxide dismutase (SOD) assays. RESULTS: Intraperitoneal injection of ropivacaine (33.8 mg/kg) induced convulsions in rats. CD38 and cADPR levels increased significantly following ropivacaine-induced convulsion (P = 0.031 and 0.020, respectively, compared with the sham group). Intraventricular injection of 8-Br-cADPR (5, 10, and 20 nmol) significantly prolonged convulsion latency (P = 0.037, 0.034, and 0.000, respectively), reduced convulsion duration (P = 0.005, 0.005, and 0.005, respectively), and reduced convulsion behavior scores (P = 0.015, 0.015, and 0.000, respectively). Intraventricular injection of 8-Br-cADPR (10 nmol) also increased the B-cell lymphoma-2 (Bcl-2)/Bcl-2-associated X protein ratio (P = 0.044) and reduced cleaved Caspase 3/Caspase 3 ratio, inducible nitric oxide synthase, MDA and SOD levels (P = 0.014, 0.044, 0.001, and 0.010, respectively) compared with the ropivacaine group. CONCLUSIONS: The CD38/cADPR pathway is activated in ropivacaine-induced convulsion. Inhibiting this pathway alleviates ropivacaine-induced convulsion and protects the brain from apoptosis and oxidative stress.

Blocking Cyclic Adenosine Diphosphate Ribose-mediated Calcium Overload Attenuates Sepsis-induced Acute Lung Injury in Rats.

BACKGROUND: Acute lung injury (ALI) is a common complication of sepsis that is associated with high mortality. Intracellular Ca2+ overload plays an important role in the pathophysiology of sepsis-induced ALI, and cyclic adenosine diphosphate ribose (cADPR) is an important regulator of intracellular Ca2+ mobilization. The cluster of differentiation 38 (CD38)/cADPR pathway has been found to play roles in multiple inflammatory processes but its role in sepsis-induced ALI is still unknown. This study aimed to investigate whether the CD38/cADPR signaling pathway is activated in sepsis-induced ALI and whether blocking cADPR-mediated calcium overload attenuates ALI. METHODS: Septic rat models were established by cecal ligation and puncture (CLP). Rats were divided into the sham group, the CLP group, and the CLP+ 8-bromo-cyclic adenosine diphosphate ribose (8-Br-cADPR) group. Nicotinamide adenine dinucleotide (NAD+), cADPR, CD38, and intracellular Ca2+ levels in the lung tissues were measured at 6, 12, 24, and 48 h after CLP surgery. Lung histologic injury, tumor necrosis factor (TNF)-µ, malondialdehyde (MDA) levels, and superoxide dismutase (SOD) activities were measured. RESULTS: NAD+, cADPR, CD38, and intracellular Ca2+ levels in the lungs of septic rats increased significantly at 24 h after CLP surgery. Treatment with 8-Br-cADPR, a specific inhibitor of cADPR, significantly reduced intracellular Ca2+ levels (P = 0.007), attenuated lung histological injury (P = 0.023), reduced TNF-µ and MDA levels (P < 0.001 and P = 0.002, respectively) and recovered SOD activity (P = 0.031) in the lungs of septic rats. CONCLUSIONS: The CD38/cADPR pathway is activated in the lungs of septic rats, and blocking cADPR-mediated calcium overload with 8-Br-cADPR protects against sepsis-induced ALI.

Blocking NAD(+)/CD38/cADPR/Ca(2+) pathway in sepsis prevents organ damage.

BACKGROUND: Although the nicotinamide adenine dinucleotide (NAD(+))/CD38/cyclic ADP ribose (cADPR)/Ca(2+) signaling pathway has been shown to regulate intracellular calcium homeostasis and functions in multiple inflammatory processes, its role in sepsis remains unknown. The aim of this study was to determine whether the NAD(+)/CD38/cADPR/Ca(2+) signaling pathway is activated during sepsis and whether an inhibitor of this pathway, 8-Br-cADPR, protects the organs from sepsis-induced damage. MATERIALS AND METHODS: Male Sprague-Dawley rats were subjected to cecal ligation and puncture (CLP) or sham laparotomies. NAD(+), cADPR, CD38, and intracellular Ca(2+) levels were measured in the hearts, livers, and kidneys of septic rats at 0, 6, 12, 24, and 48 h after CLP surgery. Rats were also divided into sham, CLP, and CLP+8-Br-cADPR groups, and the hearts, livers, and kidneys were hematoxylin-eosin-stained and assayed for malondialdehyde and superoxide dismutase activities. RESULTS: NAD(+), cADPR, CD38, and intracellular Ca(2+) levels increased in the hearts, livers, and kidneys of septic rats as early as 6-24 h after CLP surgery. Treatment with 8-Br-cADPR inhibited sepsis-induced intracellular Ca(2+) mobilization, attenuated tissue injury, reduced malondialdehyde levels, and increased superoxide dismutase activity in septic rats. CONCLUSIONS: The NAD(+)/CD38/cADPR/Ca(2+) signaling pathway was activated during sepsis in the CLP rat model. Blocking this pathway with 8-Br-cADPR protected hearts, livers, and kidneys from sepsis-induced damage.