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Cyclic ADP-ribose requires CD38 to regulate the release of ATP in visceral smooth muscle Leonie Durnin and Violeta N. Mutafova-Yambolieva Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV, USA Keywords ATP; bladder; cADP-ribose; CD38; NAD; purinergic neurotransmission Correspondence V. N. Mutafova-Yambolieva, Department of Physiology and Cell Biology, University of Nevada School of Medicine, Center for Molecular Medicine ⁄ MS 575, Reno, NV 89557-0575, USA Fax: +1 775 784 6903 Tel: +1 775 784 6274 E-mail: vmutafova@medicine.nevada.edu (Received 30 April 2011, revised 24 June 2011, accepted 30 June 2011) doi:10.1111/j.1742-4658.2011.08233.x It is well established that the intracellular second messenger cADP-ribose (cADPR) activates Ca 2+ release from the sarcoplasmic reticulum through ryanodine receptors. CD38 is a multifunctional enzyme involved in the for- mation of cADPR in mammals. CD38 has also been reported to transport cADPR in several cell lines. Here, we demonstrate a role for extracellular cADPR and CD38 in modulating the spontaneous, but not the electrical field stimulation-evoked, release of ATP in visceral smooth muscle. Using a small-volume superfusion assay and an HPLC technique with fluorescence detection, we measured the spontaneous and evoked release of ATP in bladder detrusor smooth muscles isolated from CD38 + ⁄ + and CD38 ) ⁄ ) mice. cADPR (1 nM) enhanced the spontaneous overflow of ATP in blad- ders isolated from CD38 + ⁄ + mice. This effect was abolished by the inhibi- tor of cADPR receptors on sarcoplasmic reticulum 8-bromo-cADPR (80 l M) and by ryanodine (50 lM), but not by the nonselective P2 puriner- gic receptor antagonist pyridoxal phosphate 6-azophenyl-2¢ ,4¢-disulfonate (30 l M). cADPR failed to facilitate the spontaneous ATP overflow in bladders isolated from CD38 ) ⁄ ) mice, indicating that CD38 is crucial for the enhancing effects of extracellular cADPR on spontaneous ATP release. Contractile responses to ATP were potentiated by cADPR, suggesting that the two adenine nucleotides may work in synergy to maintain the resting tone of the bladder. In conclusion, extracellular cADPR enhances the spontaneous release of ATP in the bladder by influx via CD38 and subse- quent activation of intracellular cADPR receptors, probably causing an increase in intracellular Ca 2+ in neuronal cells. Introduction Cyclic ADP-ribose (cADPR) is an intracellular second messenger that can release Ca 2+ from ryanodine-sensi- tive stores [1] in a wide variety of cells [2], including cells in the nervous system [3]. In mammals, cADPR is generated from NAD by ADP-ribosyl cyclase associ- ated with CD38, a multifunctional type II integral membrane glycoprotein with ADP-ribosyl cyclase and NAD-glycohydrolase activities [2,4,5]. The catalytic site of CD38 faces the ectocellular space [6,7], making this enzyme suitable as a regulator of extracellular b- NAD + and cADPR levels [8]. Therefore, cADPR could be produced extracellularly in each system that releases b-NAD + and expresses membrane-bound CD38. In 3T3 murine fibroblasts and HeLa cells, CD38 also mediates intracellular influx of cADPR [9,10]. Furthermore, extracellular cADPR can stimu- late NG108-15 cells, a neurally derived clonal cell line, and elevate intracellular Ca 2+ levels [11]. It is presently Abbreviations ADPR, ADP-ribose; BoNTA, botulinum neurotoxin A; cADPR, cADP-ribose; CBX, carbenoxolone; cGDPR, cGDP-ribose; eADPR, 1,N 6 -etheno- ADPR; EFS, electrical field stimulation; FFA, flufenamic acid; NGD, nicotinamide guanine dinucleotide; PPADS, pyridoxal phosphate 6-azophenyl-2¢,4¢-disulfonate; PS, prestimuation; SE, standard error; TTX, tetrodotoxin. FEBS Journal 278 (2011) 3095–3108 ª 2011 The Authors Journal compilation ª 2011 FEBS 3095 unknown whether such mechanisms play a role in complex systems such as visceral smooth muscle. Likewise, the role of extracellular cADPR in modulat- ing neurotransmission at the nerve–smooth muscle junction remains to be determined. In a number of smooth muscle tissues, the precursor of cADPR b-NAD + is released at rest and upon firing of action potentials, and serves as a neurotransmitter and a neuromodulator [12–16]. CD38 is expressed exclusively on nerve terminals in some smooth muscle preparations [14], and hence cADPR is present extracellularly, probably because of degradation of b-NAD + by CD38. Exogenous cADPR modifies the release of neurotransmitter in blood vessels [12], but it is unclear whether neuromodulation by cADPR is mediated by receptors on the presynaptic membrane or by receptors on intracellular Ca 2+ stores and subse- quent changes in intracellular Ca 2+ . It is also unknown whether cADPR can modulate equally the spontaneous and evoked release of neurotransmitters. ATP is believed to be a cotransmitter with acetyl- choline in the urinary bladder [17,18]. To address some of the aforementioned unresolved issues, we examined how exogenous cADPR modulates the amounts of ATP released in the bladder. In particular, we studied the effects of exogenous cADPR on spontaneous and electrical field stimulation (EFS)-evoked overflow of ATP in bladder detrusor smooth muscle isolated from CD38-deficient (CD38 ) ⁄ ) ) mice and from control C57 ⁄ BL6 mice, referred to as CD38 + ⁄ + mice through- out this article. We report here that exogenous cADPR facilitates the spontaneous release of ATP, probably because of influx of cADPR through CD38 and subse- quent activation of intracellular ryanodine-sensitive cADPR receptors. The EFS-evoked release of ATP, however, appears to be unaffected by extracellular cADPR, suggesting that the spontaneous and EFS- evoked release of ATP in the bladder are mediated differentially by CD38. Results Mechanisms of spontaneous and EFS-evoked release of ATP in bladder detrusor muscles from CD38 + ⁄ + and CD38 ) ⁄ ) mice We first determined the spontaneous and EFS-evoked release of ATP in bladder detrusor smooth muscles isolated from CD38 + ⁄ + and CD38 ) ⁄ ) mice. As shown in Fig. 1, superfusate samples collected before stimula- tion [prestimulation (PS)] or during EFS [16 Hz, 0.1 ms for 60 s; stimulation (ST)] of bladder detrusor muscles from CD38 + ⁄ + and CD38 ) ⁄ ) mice contained ATP along with other adenine compounds, including ADP, AMP, b-NAD + , ADP-ribose (ADPR), cADPR and Ado, suggesting that there is spontaneous and evoked release of ATP in the murine bladder. As demonstrated previously [12], b-NAD + , ADPR and cADPR eluted as one peak, owing to conversion to 1,N 6 -etheno-ADPR (eADPR) during etheno-derivatiza- tion of tissue superfusate samples (see Experimental procedures). There were no significant differences between the spontaneous and EFS-evoked overflow of ATP in CD38 + ⁄ + and CD38 ) ⁄ ) mice. The EFS- evoked release of ATP, determined by the difference ST ) PS, was 3.18 ± 0.52 fmolÆmg )1 tissue in bladders from CD38 + ⁄ + mice (n = 55) and 2.48 ± 0.41 fmo- lÆmg )1 tissue in bladders from CD38 ) ⁄ ) mice (n = 40) (P > 0.05). Tetrodotoxin (TTX) (0.30.5 lm, for 30 min) had no effect on the spontaneous release of ATP in bladders isolated from CD38 + ⁄ + mice or CD38 ) ⁄ ) mice (P > 0.05 versus controls; Fig. 1). The EFS-evoked overflow of ATP was reduced by TTX in bladders isolated from CD38 + ⁄ + mice (ST ) PS was 0.18 ± 0.65 fmolÆmg )1 tissue, n = 12, P < 0.05 versus control), but not in bladders isolated from CD38 ) ⁄ ) mice (ST ) PS was 2.05 ± 0.46 fmolÆmg )1 tissue, n = 22, P > 0.05 versus controls; Fig. 1). Incubation of bladders isolated from CD38 + ⁄ + mice with botulinum neurotoxin A (BoNTA) (100–300 nm for 2.5 h) led to cleavage of SNAP25 (Fig. 2, inset). The spontaneous overflow of ATP in BoNTA-treated tissues remained unchanged in bladders from CD38 + ⁄ + and CD38 ) ⁄ ) mice (Fig. 2) (P > 0.05 versus PS values in nontreated tissues). As expected, no additional overflow was observed upon EFS. As ATP release from cells can also occur via hemichannels [19–22], we next examined whether the spontaneous or evoked overflow of ATP is affected by two widely used hemichannel blockers, namely carbe- noxolone (CBX) and flufenamic acid (FFA) [19,22,23]. In bladders isolated from CD38 + ⁄ + mice, the sponta- neous overflow of ATP was as follows (fmolÆmg )1 tis- sue): 0.34 ± 0.08 (n = 4), 0.28 ± 0.04 (n = 4) and 0.58 ± 0.04 (n = 3) in the presence of vehicle, CBX (100 lm) and FFA (100 lm), respectively (P > 0.05 versus vehicle controls). The evoked overflow of ATP, determined from the ST ) PS values, was as follows (fmolÆmg )1 tissue): 0.82 ± 0.21 (n = 4), 1.15 ± 0.27 (n = 4) and 0.36 ± 0.20 fmolÆ mg )1 tissue in the pres- ence of vehicle, CBX and FFA, respectively (P > 0.05 versus controls). Therefore, neither the spontaneous nor the evoked release of ATP appeared to be affected by CBX or FFA in bladders isolated from CD38 + ⁄ + mice. Likewise, in bladders isolated from CD38 ) ⁄ ) mice, the spontaneous release of ATP was as follows cADPR and CD38 modulate ATP release in the bladder L. Durnin and V. N. Mutafova-Yambolieva 3096 FEBS Journal 278 (2011) 3095–3108 ª 2011 The Authors Journal compilation ª 2011 FEBS (fmolÆmg )1 tissue): 0.25 ± 0.018 (n = 5), 0.30 ± 0.06 (n = 3) and 0.58 ± 0.19 (n = 4) in the presence of vehicle, CBX and FFA, respectively (P > 0.05). The EFS-evoked overflow of ATP (ST ) PS values, fmo- lÆmg )1 tissue) was as follows: 1.16 ± 0.14 (n = 5), 1.32 ± 0.18 (n = 3) and 0.69 ± 0.44 (n = 4) in the presence of vehicle, CBX and FFA, respectively (P > 0.05). As shown in Fig. 1, tissue superfusates contained not only ATP, but also b-NAD + , as well as other adenine compounds, including ADP, AMP, Ado, ADPR, and cADPR. These adenine compounds are metabolites of either ATP, b-NAD + , or both: ADP is a direct metab- olite of ATP, whereas AMP and Ado can be formed by both ATP and b-NAD + [2,4,24]. Table 1 shows the values of ADP, AMP, b-NAD + + ADPR + cADPR (eluted as eADPR) and Ado accumulated in tissue superfusates before (spontaneous overflow) and during (evoked overflow) nerve stimulation in control experi- ments in bladder detrusor muscles isolated from CD38 + ⁄ + and CD38 ) ⁄ ) mice. In control CD38 + ⁄ + mice, the overflow of adenine purines was increased during nerve stimulation. No significant differences were observed in the spontaneous overflow of all ade- nine purines in CD38 + ⁄ + and CD38 ) ⁄ ) preparations. The amounts of b-NAD + + ADPR + cADPR, adeno- sine and total purines were reduced in the samples col- lected during nerve stimulation of bladders isolated from CD38 ) ⁄ ) mice. CD38 carries the ADP-ribosyl cyclase activity in the murine bladder detrusor muscle Next, we tested whether ADP-ribosyl cyclase activity in the bladder is associated with CD38. We first examined whether there is a difference between the degradation of nicotinamide guanine dinucleotide (NGD) to cGDP- ribose (cGDPR) in bladders isolated from CD38 + ⁄ + and CD38 ) ⁄ ) mice as a measure of GDP-ribosyl (and possibly ADP-ribosyl) cyclase activity [4]. As shown in Fig. 3, production of cGDPR from NGD was increased during incubation of NGD with bladders CD38 +/+ PS ST ATP ADP β-NAD + ADPR + cADPR AMP Ado ATP ADP β-NAD + ADPR + cADPR AMP Ado CD38 +/+ ATP overflow (fmol·mg –1 tissue) 0 2 6 4 CD38 –/– PS ST ATP ADP β-NAD + ADPR + cADPR AMP Ado ATP ADP β-NAD + ADPR + cADPR AMP Ado B A C ATP ADP β-NAD + ADPR + cADPR AMP Ado ST, TTX 10 12 14 16818 Min 100 LU 10 12 14 16818 Min *** (55) (55) (12) ATP ADP β-NAD + ADPR + cADPR AMP Ado ST, TTX TTX PS ST PS ST Controls (12) (fmol·mg –1 tissue) CD38 –/– ATP overflow 0 2 6 4 D TTX PS ST PS ST Controls *** ** (22) (22) (40) (40) Fig. 1. ATP is released at rest and during EFS in murine bladder detrusor muscle. (A, B) Original chromatograms of tissue superfusate samples collected before EFS (PS) and during EFS (16 Hz, 0.1 ms for 60 s; ST) in CD38 + ⁄ + mice and CD38 ) ⁄ ) mice, respectively. Chromatograms from ST samples collected during superfusion with TTX (0.5 l M, 30 min) are also shown. Spontaneous overflow of ATP and the metabolites ADP, AMP and Ado, and b-NAD + + ADPR + cADPR, occurred in PS samples. EFS (ST) resulted in increased overflow of all nucleotides and nucleosides. LU, luminescence units: scale applies to all chromatograms. (C, D) ATP overflow in CD38 + ⁄ + mice and CD38 ) ⁄ ) mice, respec- tively, before EFS (PS) and during EFS (ST) in the absence and presence of TTX (0.3– 0.5 l M) (averaged data in fmolÆmg )1 tissue, presented as means ± SE; ***P < 0.001, **P < 0.05). Numbers of observations are in parentheses. Enhanced overflow of all purines was observed during EFS. TTX had no effect on the spontaneous overflow of ATP. TTX significantly reduced the evoked overflow of ATP during EFS of bladders isolated from CD38 + ⁄ + mice, but not in bladders isolated from CD38 ) ⁄ ) mice. L. Durnin and V. N. Mutafova-Yambolieva cADPR and CD38 modulate ATP release in the bladder FEBS Journal 278 (2011) 3095–3108 ª 2011 The Authors Journal compilation ª 2011 FEBS 3097 isolated from CD38 + ⁄ + mice. In contrast, bladders iso- lated from CD38 ) ⁄ ) mice failed to degrade NGD. Thus, the entire GDP-ribosyl cyclase activity in the murine bladder detrusor muscle appears to be associ- ated with CD38. We next carried out an HPLC fraction analysis [12] to determine whether cADPR and ADPR are present in tissue superfusates from bladders isolated from CD38 ) ⁄ ) mice along with their precursor b-NAD + . The amounts of ADPR and cADPR were negligible: samples collected before EFS contained 94.71% ± 1.93% b-NAD + , 2.9% ± 0.69% ADPR, and 2.38% ± 1.24% cADPR, whereas samples collected during EFS contained 98.42% ± 0.35% b-NAD + , 0.66% ± 0.31% ADPR, and 0.91% ± 0.42% cADPR (n = 3, 12–16 chambers in each experiment). There- fore, the ADP-ribosyl cyclase activity in the murine bladder detrusor appears to be attributable exclusively to CD38. Effects of exogenous cADPR on spontaneous and evoked overflow of ATP To determine whether extracellular cADPR is a neuro- modulator and can modify the release of ATP, we next examined the effects of exogenous cADPR (1 nm)on the spontaneous and EFS-evoked overflow of ATP. cADPR caused a significant increase in the spontane- ous overflow of ATP in bladders isolated from CD38 + ⁄ + mice, but not in bladders isolated from CD38 ) ⁄ ) mice (Fig. 4), suggesting that CD38 is impor- tant for the enhancing effect of exogenous cADPR in the bladder. However, cADPR (1 nm) did not enhance the EFS-evoked release of ATP in bladders isolated from either CD38 + ⁄ + mice or CD38 ) ⁄ ) mice (Fig. 5): The evoked release, determined by the difference in ATP amounts between ST and PS samples (ST ) PS), was 3.97 ± 1.88 fmolÆmg )1 tissue in bladders from CD38 + ⁄ + mice (n = 16) and 2.077 ± 0.87 fmolÆmg )1 CD38 +/+ PS ST ATP ADP β-NAD + ADPR + cADPR AMP Ado ATP ADP β-NAD + ADPR + cADPR AMP Ado CD38 –/– PS ST ATP β-NAD + ADPR + cADPR AMP Ado ATP ADP β-NAD + ADPR + cADPR AMP Ado B A ATP ADP β-NAD + ADPR + cADPR AMP Ado ST, BoNTA 10 12 14 16818 Min 100 LU 10 12 14 16818 Min ATP ADP β-NAD + ADPR + cADPR AMP Ado ST, BoNTA ADP SNAP-25 25 kDa Control Bo NTA CD38 +/+ ATP overflow (fmol·mg –1 tissue) (fmol·mg –1 tissue) 0 2 4 CD38 –/– ATP overflow DC * (4) (4) (4) BoNTA PS ST PS ST ControlsBoNTA PS ST PS ST Controls (4) * (3) (3) (3) (3) 0 2 4 Fig. 2. Differential effects of BoNTA on the spontaneous and EFS-evoked release of ATP. (A, B) Original chromatograms of tissue superfusate samples collected before EFS (PS) and during EFS (16 Hz, 0.1 ms for 60 s; ST) in CD38 + ⁄ + mice and CD38 ) ⁄ ) mice, respectively. Chromatograms from ST samples collected during superfusion of BoNTA-treated (100 n M for 2.5 h) tissues are also shown. EFS (ST) resulted in increased overflow of all nucleotides and nucleosides, and this was reduced by BoNTA. LU, luminescence units: scale applies to all chromatograms. (C, D) ATP overflow in CD38 + ⁄ + mice and CD38 ) ⁄ ) mice, respectively, before EFS (PS) and dur- ing EFS (ST) in controls and BoNTA-treated tissues (averaged data in fmolÆmg )1 , presented as means ± SE; *P < 0.05). Numbers of observations are in parenthe- ses. Enhanced overflow of all purines was observed during EFS. BoNTA significantly reduced the EFS-evoked, but not the spontaneous, overflow of ATP in bladders isolated from CD38 + ⁄ + and CD38 ) ⁄ ) mice. (C) Inset: western immunoblot analysis of SNAP-25 shows a single band at 25 kDa in homogenates from control (vehicle-treated) tissues. An additional 24-kDa band appears in BoNTA-treated tissues, indicating cleav- age of SNAP-25 induced by BoNTA. cADPR and CD38 modulate ATP release in the bladder L. Durnin and V. N. Mutafova-Yambolieva 3098 FEBS Journal 278 (2011) 3095–3108 ª 2011 The Authors Journal compilation ª 2011 FEBS tissue in bladders from CD38 ) ⁄ ) mice (n = 11) (P > 0.05). These values were not significantly differ- ent from the ST ) PS amounts of ATP in the absence of cADPR. Note that the peak of eADPR (standing for b-NAD + + ADPR + cADPR) was increased in the samples collected during superfusion with cADPR (Figs 4 and 5), because the exogenous cADPR was also derivatized to eADPR during the precolumn derivatization [12]. Thus, the peaks of b-NAD + + ADPR + cADPR, AMP and Ado represented the amounts of endogenously formed nucleotides and nucleosides plus products of the degradation of the exogenous cADPR, and therefore were not analyzed in detail. The enhancing effect of cADPR on ATP overflow was not reduced by the nonselective P2 receptor antag- onist pyridoxal phosphate 6-azophenyl-2¢,4¢-disulfonate (PPADS) (30 lm) (Fig. 6), suggesting that prejunction- al P2 receptors were not involved in the facilitating effects of cADPR. In contrast, the inhibitors of intra- cellular cADPR receptors 8-Br-cADPR (80 lm) and ryanodine (50 lm for 45 min) abolished the enhancing effect of cADPR (Fig. 6). Therefore, the responses to exogenous cADPR are probably mediated by intracel- lular ryanodine-sensitive cADPR receptors. cADPR is hydrolyzed to ADPR [4], which is degraded to AMP by nucleotide pyrophosphatases [25]. AMP, in turn, is degraded to Ado by ecto-5¢- nucleotidase [26], but AMP can also synthesize ADP and ATP via backward ecto-phosphotransfer reactions, provided that enzymes such as adenylate kinase, nucleoside diphosphate kinase and ATP syn- thase [27] are present on the cell surface. Therefore, we next examined whether the increase in ATP during su- perfusion with cADPR is, rather, attributable to regen- eration of ATP from AMP or ADP, distant products of cADPR. The commercially available ADP sub- stance used in these experiments at a concentration of 10 nm contained a small amount of ATP, which, nor- malized to tissue weight, is about 0.78 ± 0.09 fmo- lÆmg )1 tissue (n = 4). Perfusion with ADP did not result in additional formation of ATP: thus, the level of ATP was 0.85 ± 0.06 fmolÆmg )1 tissue in the sam- ples collected during perfusion with ADP (n =4, P > 0.05 versus nontissue controls). Likewise, perfu- sion of tissue with AMP (10 nm) caused no additional formation of ATP: 0.514 ± 0.081 fmolÆmg )1 in nontis- sue controls (n = 4), and 0.466 ± 0.023 fmolÆmg )1 tis- sue in bladders perfused with 10 nm AMP (n =4, P > 0.05). Therefore, superfusion of tissues with either ADP or AMP caused no additional formation of ATP in tissue superfusates, suggesting that kinase activities mediating production of ATP from ADP or AMP Table 1. Spontaneous and EFS-evoked (16 Hz, 0.1 ms for 60 s) overflow of ADP, AMP, Ado, b-NAD + ADPR + cADPR and total purines (ATP + ADP + AMP + Ado + b-NAD + ADPR + cADPR) in CD38 + ⁄ + (n = 55) and CD38 ) ⁄ ) (n = 40) bladder detrusor muscle in fmolÆmg )1 tissue ± SE. Significant differences between PS and ST: ***P < 0.001, **P < 0.01, and *P < 0.05. Significant differences between CD38 + ⁄ + and CD38 ) ⁄ ) preparations: P < 0.001, P < 0.01, and P < 0.05) (one-way ANOVA followed by post hoc Bonfer- roni multiple comparison tests). ADP AMP Ado eADPR for b-NAD + ADPR + cADPR Total purines CD38 + ⁄ + CD38 ) ⁄ ) CD38 + ⁄ + CD38 ) ⁄ ) CD38 + ⁄ + CD38 ) ⁄ ) CD38 + ⁄ + CD38 ) ⁄ ) CD38 + ⁄ + CD38 ) ⁄ ) Spontaneous overflow (PS) 2.29 ± 0.28 2.6 ± 0.37 2.96 + 0.47 2.3 ± 0.32 11.86 ± 1.18 11.08 ± 1.7 17.55 ± 7.53 6.88 ± 1.28 36.17 ± 8.17 24.07 ± 3.1 Evoked overflow (ST) 5.30 ± 0.64*** 5.63 ± 0.69*** 6.46 ± 0.79*** 4.52 ± 0.54 94.83 ± 14.66*** 49.4 ± 6.7* ,  42.12 ± 7.52* 20.82 ± 4.9 153.3 ± 22.0*** 84.0 ± 11.2* ,  L. Durnin and V. N. Mutafova-Yambolieva cADPR and CD38 modulate ATP release in the bladder FEBS Journal 278 (2011) 3095–3108 ª 2011 The Authors Journal compilation ª 2011 FEBS 3099 (and ultimately from cADPR) were undetectable under our experimental conditions. To determine whether b-NAD + , a precursor of cADPR, affects the spontaneous or EFS-evoked over- flow in a manner similar to cADPR, we superfused bladder detrusor muscles isolated from CD38 + ⁄ + mice with b-NAD + (1 nm). The resting overflow of ATP was 1.81 ± 0.22 fmolÆmg )1 tissue (n = 12) and 3.72 ± 0.85 fmolÆmg )1 tissue (n = 12) in the absence and presence of b-NAD + (P > 0.05). The EFS- evoked overflow of ATP was 5.91 ± 0.91 fmolÆmg )1 tissue (n = 12) in the presence of b-NAD + (P > 0.05 versus PS in b-NAD + -treated tissues; P > 0.05 versus ST in controls). To determine whether ADPR, a product of cADPR, has an effect on the ATP release, we superfused blad- ders isolated from CD38 + ⁄ + mice with 1 nm ADPR. The overflow of ATP was 3.56 ± 0.51 fmolÆmg )1 tissue (n =6,P > 0.05 versus controls) in samples collected before EFS and 10.07 ± 0.94 fmolÆmg )1 tissue (n =6, P < 0.05 versus controls) in superfusate samples col- lected during EFS. It has been proposed that, in PC12, cells acetylcho- line induces the production of cADPR via CD38-medi- ated mechanisms [28]. To determine whether acetylcholine that might have been released during EFS of murine bladder detrusor smooth muscles caused increased formation of ATP, we examined the effect of carbachol, a stable analog of acetylcholine, on the spontaneous overflow of ATP. Carbachol (1 lm) caused no additional formation of ATP in bladder detrusor muscles isolated from CD38 + ⁄ + and CD38 ) ⁄ ) mice: the amounts of ATP were 0.86 ± 0.14 and 0.70 ± 0.13 fmolÆmg )1 tissue in the absence and presence of carbachol, respectively (n =4,P > 0.05). Therefore, stimulation of acetylcholine receptors or smooth muscle contraction per se did not induce addi- tional release of ATP. 35791 11 Min CD38 +/+ cGDPR NGD (–) Tissue (+) Tissue CD38 –/– cGDPR NGD 200 LU 0 2 3 1 cGDPR formation (nmol·mg –1 tissue) (nmol·mg –1 tissue) (–) Tissue (+) Tissue 200 LU BA DC CD38 +/+ CD38 –/– (+) Tissue cGDPR formation 0 3 1 2 (–) Tissue ** (–) Tissue (+) Tissue 35791 11 Min (9) (9) (6) (6) Fig. 3. CD38 carries the GDP-ribosyl cyclase activity in bladder detrusor muscle. (A) Original chromatograms showing the formation of cGDPR from NGD (0.2 m M)in the absence of tissue [()) tissue)] and in the presence of tissue for 2 min [(+) tissue)] in CD38 + ⁄ + mice. A significant increase in cGDPR production occurred within 2 min of tissue contact. LU, luminescence units. (B) Averaged data (in nmolÆmg )1 tissue) presented as means ± SE; **P < 0.01. (C) Original chromatograms showing the forma- tion of cGDPR from NGD (0.2 m M) in the absence of tissue [()) tissue)] and in the presence of tissue for 2 min [(+) tissue)] in CD38 ) ⁄ ) mice. Increased production of cGDPR from NGD did not occur within 2 min of tissue contact when CD38 was absent (P > 0.05). (D) Averaged data (nmolÆmg )1 tissue) presented as means ± SE. Numbers of observations are in parentheses. cADPR and CD38 modulate ATP release in the bladder L. Durnin and V. N. Mutafova-Yambolieva 3100 FEBS Journal 278 (2011) 3095–3108 ª 2011 The Authors Journal compilation ª 2011 FEBS cADPR facilitates the contractile responses to ATP ATP at 1–10 lm for 1 min caused transient contractile responses in bladder detrusor strips. cADPR (1 nm) did not cause measurable changes in the resting smooth muscle tone, but the responses to ATP were enhanced in the presence of cADPR (Fig. 7). Discussion This study demonstrates several new features of presynaptic neuromodulation in a visceral smooth muscle. Stimulation of intrinsic neurons in murine bladder detrusor muscle caused release of ATP and b-NAD + . b-NAD + was degraded by CD38 to cADPR and ADPR. cADPR enhanced the spontane- ous release of ATP but not the release of ATP evoked by action potential firings. The enhancing effect of cADPR on spontaneous release of ATP was: (a) unaf- fected by inhibition of P2 purinoreceptors; (b) abol- ished by inhibition of intracellular cADPR receptors; (c) eliminated by prolonged treatment with ryanodine; and (d) absent in bladders isolated from mice lacking the CD38 gene. These data suggest that, in the bladder detrusor muscle, extracellular cADPR can be trans- ported by CD38 to the cytosol, activate cADPR recep- tors on ryanodine-sensitive Ca 2+ stores, and facilitate spontaneous ATP release. ATP is a proposed neurotransmitter at the nerve– smooth muscle junction in the urinary bladder [17,29], enteric nervous system [30–32], and blood vessels [33]. b-NAD + is another adenine-based nucleotide that is released upon stimulation of neurosecretory cells [34] and nerves in the bladder [12,13], mesenteric blood vessels [12,14], and large intestine [15,16]. In all of these tissues, ATP and b-NAD + coexist in tissue superfusates, and, in some cases, b-NAD + mimics the effects of the endogenous neurotransmitter better than ATP [15,16]. b-NAD + is degraded to ADPR and cADPR by NAD-glycohydrolase and ADP-ribosyl cyclase, respectively [2,4]. In mammals, both enzymatic activities are associated with CD38 [2,10]. The cyclase activity of CD38 is relatively weak [2], but even small CD38 +/+ ATP ADP eADPR for β-NAD + ADPR + cADPR AMP Ado ATP ADP eADPR for cADPR (1 nM) AMP Ado ATP ADP eADPR for β-NAD + ADPR + cADPR AMP Ado ATP ADP AMP Ado CD38 –/– Control, no EFS Control, no EFS cADPR, no EFS cADPR, no EFS BA 10 12 14 16818 Min 25 LU 10 12 14 16818 Min eADPR for cADPR (1 nM) 0 6 2 4 Control cADPR (1 nM) CD38 +/+ DC 0 6 2 Spontaneous (PS) ATP overflow (fmol·mg –1 tissue) (fmol·mg –1 tissue) 4 Control cADPR (1 nM) *** (40) (11) Spontaneous (PS) ATP overflow (55) (12) CD38 –/– Fig. 4. cADPR enhances the spontaneous overflow of ATP. (A, B) Original chromato- grams showing spontaneous overflow of ATP in the absence (upper panels) and presence of cADPR (1 n M) (lower panels) in CD38 + ⁄ + mice and CD38 ) ⁄ ) mice, respec- tively. cADPR caused a significant increase in the spontaneous overflow of ATP in CD38 + ⁄ + mice. In CD38 ) ⁄ ) mice, spontane- ous overflow of ATP was not increased in the presence of cADPR (P > 0.05). LU, luminescence units: scale applies to all chromatograms. (C, D) Averaged data (fmolÆmg )1 tissue) presented as means ± SE; ***P < 0.001. Numbers of observations are in parentheses. L. Durnin and V. N. Mutafova-Yambolieva cADPR and CD38 modulate ATP release in the bladder FEBS Journal 278 (2011) 3095–3108 ª 2011 The Authors Journal compilation ª 2011 FEBS 3101 amounts of the second messenger cADPR [1,2] might have an effect on the release of cotransmitters in the smooth muscle. CD38, in addition to producing cAD- PR from extracellular b-NAD + , can also transport cADPR in the intracellular compartment [9–11]. This might not be a universal mechanism, however, as some cells, such as T-lymphocytes [35], do not express CD38-mediated transport of cADPR. If this mecha- nism were present in ATP-releasing nerve terminals, then cADPR, formed extracellularly, would affect the release of neurotransmitters, a process that depends heavily on elevated Ca 2+ in the cytosol [36,37]. To test this hypothesis, we used murine bladder detrusor mus- cle as a smooth muscle organ with established puriner- gic cotransmission in the parasympathetic nervous system [17,18,29]. In agreement with previous studies in the bladder [12,13], we found that both ATP and b-NAD + are released spontaneously and upon action potential firing. As expected, the evoked release of ATP in bladders isolated from CD38 + ⁄ + mice was inhibited by TTX, and ATP during EFS therefore appeared to originate from excitable cells containing fast Na + channels, such as neurons. Interestingly, the evoked release of ATP in bladders isolated from CD38 ) ⁄ ) mice demonstrated lack of sensitivity to TTX, despite the large number of observations. Fur- ther studies are warranted to examine the mechanisms underlying the switch to TTX-resistant release of ATP during EFS in bladders from CD38 ) ⁄ ) mice. As expected, the EFS-evoked release in bladders isolated from both CD38 + ⁄ + and CD38 ) ⁄ ) mice was abolished by BoNTA, suggesting that this release was mediated by SNAP-25-dependent vesicle exocytosis. Multiple mechanisms may be involved in the basal release of ATP from cells [38], including numerous types of membrane channel, such as connexin and pannexin hemichannels [39,40], maxi-ion channels [41], volume-regulated anion channels [42], the P2X7 receptor [43], ATP-binding cassette transporters [44], or vesicle exocytosis [45]. The mechanisms responsible for this release may differ among different types of cell. In the present study, the spontaneous release of cADPR, 16 Hz ATP ADP eADPR for cADPR (1 nM) AMP Ado Control, 16 Hz ATP ADP eADPR for β-NAD + ADPR + cADPR AMP Ado cADPR, 16 Hz ATP ADP eADPR for cADPR (1 nM) AMP Ado ATP ADP eADPR for β-NAD + ADPR + cADPR AMP Ado Control, 16 Hz BA DC 0 6 8 2 4 Control cADPR (1 nM) 0 6 8 2 EFS-evoked (ST – PS) ATP overflow (fmol·mg –1 tissue) (fmol·mg –1 tissue) 4 Control cADPR (1 nM) CD38 +/+ CD38 –/– CD38 +/+ CD38 –/– 25 LU 10 12 14 16818 Min 10 12 14 16818 Min (40) (55) (12) (11) EFS-evoked (ST – PS) ATP overflow Fig. 5. cADPR does not change the EFS- evoked overflow of ATP. (A, B) Original chromatograms showing EFS-evoked (16 Hz, 0.1 ms for 60 s) overflow of ATP in the absence (upper panels) and presence of cADPR (1 n M) (lower panels) in CD38 + ⁄ + mice and CD38 ) ⁄ ) mice, respectively. cADPR did not affect the EFS-evoked over- flow of ATP in CD38 + ⁄ + mice or CD38 ) ⁄ ) mice (P > 0.05). LU, luminescence units: scale applies to all chromatograms. (C, D) Averaged data (fmolÆmg )1 tissue) presented as means ± SE. Numbers of observations are in parentheses. cADPR and CD38 modulate ATP release in the bladder L. Durnin and V. N. Mutafova-Yambolieva 3102 FEBS Journal 278 (2011) 3095–3108 ª 2011 The Authors Journal compilation ª 2011 FEBS ATP in bladders from both CD38 + ⁄ + and CD38 ) ⁄ ) mice was insensitive to inhibition of fast Na + channels with TTX, inhibition of connexin and pannexin hemi- channels with CBX and FFA, and cleavage of SNAP- 25 with BoNTA. Importantly, the spontaneous release of ATP in the bladder was activated by stimulation of intracellular cADPR receptors with cADPR (discussed below). The spontaneous release of ATP also tended to be reduced by inhibition of ryanodine recep- tor ⁄ channels, although statistical significance was not reached. The precise mechanisms of spontaneous release of ATP in the bladder remain to be determined, but the present study suggests that this release is not induced by action potential firing in peripheral nerves, by opening of hemichannels, or by vesicle exocytosis, and requires intact ryanodine-sensitive and cADPR- sensitive intracellular Ca 2+ stores. cADPR is formed in the murine bladder, as it does express ADP-ribosyl cyclase activity measured as GDP-ribosyl cyclase activity. Although the ADP-ribo- syl cyclase and GDP-ribosyl cyclase activities are not always equivalent [46], in the mouse bladder the cyclase activities appear to be carried entirely by CD38: bladders isolated from CD38 ) ⁄ ) mice failed to form cGDPR from NGD, which is in contrast to the findings in bladders isolated from CD38 + ⁄ + mice. Furthermore, tissue superfusates from bladders iso- lated from CD38 ) ⁄ ) mice contained b-NAD + , but almost no cADPR and ADPR (the present study), whereas bladders isolated from CD38 + ⁄ + mice also contained the b-NAD + metabolites cADPR and ADPR [12]. cADPR, in particular, constituted  12% of the b-NAD + + ADPR + cADPR cocktail in the PS samples in bladders isolated from CD38 + ⁄ + mice [12], whereas the PS samples from CD38 ) ⁄ ) bladders contained < 2% cADPR in the b-NAD + + ADPR + cADPR mixture. Furthermore, the overflow of Ado and total purines was reduced in the bladders isolated from CD38 ) ⁄ ) mice, suggesting that, in control tissues, a significant proportion of Ado is formed by the degradation of b-NAD + via CD38. The data from the overflow experiments and HPLC fraction analysis demonstrate that ATP and cADPR can simultaneously exist in the vicinity of the neuro- muscular junction at rest and during action potential firing. Spontaneous ATP overflow (fmol·mg –1 tissue) 0 4 8 12 *** *** (55) (12) (9) (6) (4) (4) (40) (11) (7) (3) CD38 –/– CD38 +/+ Fig. 6. Effects of cADPR on spontaneous overflow of ATP in blad- der detrusor smooth muscle isolated from CD38 + ⁄ + mice or CD38 ) ⁄ ) mice. Averaged data (in fmolÆmg )1 tissue) presented as means ± SE. Numbers of observations are in parenthesis. cADPR (1 n M) significantly increased the spontaneous overflow of ATP in CD38 + ⁄ + mice (***P < 0.001). The enhancing effect was also observed in the presence of PPADS (30 l M), a nonselective P2 pur- ine receptor antagonist (***P < 0.001). The inhibitor of intracellular cADPR receptors, 8-Br-cADPR (80 l M), and ryanodine (50 lM) abol- ished the enhancing effect on spontaneous ATP overflow (P > 0.05). cADPR did not affect spontaneous ATP overflow when CD38 was absent (CD38 ) ⁄ ) , P > 0.05). 1 mN ATP cADPR, 1 nM 0 2 1 Force (mN) ATP cADPR + ATP ** ATP 30 s (11) (11) A B Fig. 7. Exogenous cADPR facilitates the contractile responses to ATP in bladder smooth muscle strips. (A) ATP (1 l M) caused tran- sient contractile responses, which were enhanced in the presence of cADPR (1 n M). (B) Averaged data (mN force) presented as means ± SE. Numbers of observations are in parentheses. L. Durnin and V. N. Mutafova-Yambolieva cADPR and CD38 modulate ATP release in the bladder FEBS Journal 278 (2011) 3095–3108 ª 2011 The Authors Journal compilation ª 2011 FEBS 3103 The amounts of cADPR produced by released b-NAD + may be relatively low, given that the mamma- lian ADP-ribosyl cyclase associated with CD38 converts only 2% of b-NAD + to cADPR [2,10]. We therefore sought to determine whether low concentrations of cADPR can affect the amounts of released ATP in the bladder. We found that a low nanomolar concentration of cADPR enhances the spontaneous overflow of ATP, but does not change the release of ATP evoked by action potential firing. These differential effects of cAD- PR can be explained by differences in the dependence of ‘spontaneous’ and ‘evoked’ release of neurotransmit- ters on extracellular and intracellular Ca 2+ . For example, it is well accepted that physiological neurotransmitter release is largely triggered by action potential-evoked Ca 2+ influx through voltage-gated Ca 2+ channels localized on presynaptic nerve terminals [36]. Unlike this ‘evoked’ release, the ‘spontaneous’ release of neurotransmitters is not triggered by action potential firing. Spontaneous vesicle fusion is thought to be a Ca 2+ -independent process, because it occurs both in the absence of action potentials and without any apparent stimulus. However, increasing evidence shows that this form of neurotransmitter release can be modulated by changes in intracellular Ca 2+ concentra- tion [37,47]. Modulation of spontaneous discharge at the level of the release machinery is not always accompanied by corresponding modulation of action potential-evoked release, suggesting that two indepen- dent processes underlie spontaneous and action potential-evoked exocytosis [47]. In agreement with this notion, the present study demonstrates that exogenous cADPR modulates the spontaneous but not the action potential-evoked release of ATP. Therefore, the neuro- modulator effects of cADPR are not mediated by influx of extracellular Ca 2+ , but are probably caused by Ca 2+ release from intracellular stores. Similar to cADPR, its precursor b-NAD + did not affect the evoked release of ATP, but tended to increase the spontaneous release of ATP, suggesting that the effects of b-NAD + might be mediated by its metabolite cADPR. ADPR, a product of both b-NAD + and cADPR [2,10], did not enhance the spontaneous overflow of ATP, suggesting that the effect of cADPR was not caused by its breakdown product ADPR. Unlike cADPR and b-NAD + , however, ADPR facilitated the EFS-evoked release of ATP. Further studies are needed to determine the mechanisms of purine-mediated presynaptic neuromod- ulation in the bladder. The enhancing effect of cADPR on the spontaneous release of ATP is not caused by activation of membrane-bound P2 purinoceptors, backward ecto- phosphotransfer reactions and formation of ATP from either ADP or AMP [27] potentially produced by the exogenous cADPR, or acetylcholine-induced produc- tion of cADPR [28]. Instead, the enhancing effect of cADPR on the spontaneous release of ATP is inhibited by 8-Br-cADPR, a specific antagonist of cADPR receptors in intracellular Ca 2+ stores [48], and by ryanodine, which, at higher concentrations and with prolonged application, also inhibits Ca 2+ release chan- nels (receptors) in intracellular Ca 2+ stores [49]. These findings suggest that the effect of exogenous cADPR on the spontaneous release of ATP is mediated by receptors localized in the intracellular compartment. Mechanisms for cADPR influx must, then, be present in this preparation. Of particular importance is the finding that exogenous cADPR failed to increase the spontaneous release of ATP in the absence of CD38. In other words, the presence of CD38 is mandatory for the occurrence of intracellular actions of extracellu- lar cADPR. Low concentrations of cADPR, which do not produce measurable changes in mechanical force in bladder preparations, potentiated the contractile responses to ATP, suggesting that our observations that cADPR enhances the spontaneous release of ATP may imply novel mechanisms of cotransmission that might be important for the fine tuning of bladder functions. In conclusion, the present study suggests that the enhancing effects of extracellular cADPR on ATP release are mediated by the triggering of intracellular signal transduction pathways in response to cADPR transported into the cytosol via membrane-bound CD38. Thus, similar to studies in some cell lines [9,10], the present study suggests that extracellular cADPR can be transported into the cytosol by CD38 on nerve cell membranes in a smooth muscle organ. The extracellular b-NAD + –cADPR system, together with CD38, may thus participate in the complex mech- anisms of synaptic regulation of smooth muscle functions. Experimental procedures Animals used C57BL ⁄ 6 mice (45–60 days of age; Charles River Laborato- ries, Wilmington, MA, USA) and CD38 knockout mice (CD38 ) ⁄ ) ; The Jackson Laboratory, Bar Harbor, ME, USA) were anesthetized with isoflurane and decapitated after cervical dislocation. This method is approved by the Institutional Animal Care and Use Committee at the University of Nevada. Urinary bladders were dissected out and placed in oxygenated cold (10 °C) Krebs solution with the following composition: 118.5 mm NaCl, 4.2 mm KCl, cADPR and CD38 modulate ATP release in the bladder L. Durnin and V. N. Mutafova-Yambolieva 3104 FEBS Journal 278 (2011) 3095–3108 ª 2011 The Authors Journal compilation ª 2011 FEBS [...]... techniques in conjunction with fluorescence detection [51] The increase in the amount of the product cGDPR in the presence of tissue was used as a measure for ecto-ADPribosyl cyclase activity cADPR and CD38 modulate ATP release in the bladder HPLC assay of etheno-purines, NGD, and cGDPR To prepare the samples for HPLC analysis, chloroacetaldehyde was added, and the samples were heated to 80 °C for 40 min to. .. (2006) Tachykinins and tachykinin receptors in the gut, with special reference to NK2 receptors in human Auton Neurosci 126–127, 232–249 32 Burnstock G (2008) The journey to establish purinergic signalling in the gut Neurogastroenterol Motil 20(Suppl 1), 8–19 33 Starke K, von Kugelgen I & Bultmann R (1992) Noradrenaline ATP cotransmission: operation in blood vessels and cotransmitter release ratios... to near the optimum length for tension development In all experiments, tissues were initially equilibrated for 45 min, and this was followed by three 2-min exposures to KCl (60 mm) every 20 min in order to establish viability and equilibrate the tissue Contractile responses to ATP (1–100 lm) in the absence or presence of cADPR (1 nm) were recorded ATP was applied at 45-min intervals to avoid receptor... Durnin and V N Mutafova-Yambolieva 1.2 mm MgCl2, 23.8 mm NaHCO3, 1.2 mm KH2PO4, 11.0 mm dextrose, and 1.8 mm CaCl2 (pH 7.4) The bladders were opened along the longitudinal axis After removal of urothelium, the detrusor smooth muscles were used for experiments All experiments were carried out in pure detrusor smooth muscles, to avoid the in uence of the urothelium, which is a significant source of ATP in. .. signaling in astrocytes via ATP release through connexin hemichannels J Biol Chem 277, 10482–10488 21 Cotrina ML, Lin JH, Alves-Rodrigues A, Liu S, Li J, Azmi-Ghadimi H, Kang J, Naus CC & Nedergaard M (1998) Connexins regulate calcium signaling by controlling ATP release Proc Natl Acad Sci USA 95, 15735–15740 cADPR and CD38 modulate ATP release in the bladder 22 Dahl G & Locovei S (2006) Pannexin: to. .. Bouron A (2001) Modulation of spontaneous quantal release of neurotransmitters in the hippocampus Prog Neurobiol 63, 613–635 FEBS Journal 278 (2011) 3095–3108 ª 2011 The Authors Journal compilation ª 2011 FEBS 3107 cADPR and CD38 modulate ATP release in the bladder L Durnin and V N Mutafova-Yambolieva 38 Corriden R & Insel PA (2010) Basal release of ATP: an autocrine–paracrine mechanism for cell regulation... L, Franco L & Bruzzone S (2004) Autocrine and paracrine calcium signaling by the CD38 ⁄ NAD+ ⁄ cyclic ADP-ribose system Ann NY Acad Sci 1028, 176–191 11 Amina S, Hashii M, Ma WJ, Yokoyama S, Lopatina O, Liu HX, Islam MS & Higashida H (2010) Intracellular calcium elevation induced by extracellular application of cyclic- ADP-ribose or oxytocin is temperaturesensitive in rodent NG108-15 neuronal cells... beta-Nicotinamide adenine dinucleotide is an inhibitory neurotransmitter in visceral smooth muscle Proc Natl Acad Sci USA 104, 16359–16364 16 Hwang SJ, Durnin L, Dwyer L, Rhee PL, Ward SM, Koh SD, Sanders KM & Mutafova-Yambolieva VN (2011) beta-Nicotinamide adenine dinucleotide is an enteric inhibitory neurotransmitter in human and nonhuman primate colons Gastroenterology 140, 608–617 17 Andersson KE & Wein... Pharmacology of the lower urinary tract: basis for current and future treatments of urinary incontinence Pharmacol Rev 56, 581–631 18 Burnstock G (2009) Purinergic cotransmission Exp Physiol 94, 20–24 19 Evans WH, De VE & Leybaert L (2006) The gap junction cellular internet: connexin hemichannels enter the signalling limelight Biochem J 397, 1–14 20 Stout CE, Costantin JL, Naus CC & Charles AC (2002) Intercellular... previously [53] NGD and cGDPR were detected at an excitation wavelength of 270 nm and an emission wavelength of 400 nm, according to previous optimization of the HPLC application for non-etheno-derivatized nucleotides [12] The degradation of NGD was determined by the increase in the amount of the product cGDPR Each compound was quantified against known standards Results were normalized for sample volume and . Cyclic ADP-ribose requires CD38 to regulate the release of ATP in visceral smooth muscle Leonie Durnin and Violeta N. Mutafova-Yambolieva Department of. the spontaneous release of ATP in the absence of CD38. In other words, the presence of CD38 is mandatory for the occurrence of intracellular actions of extracellu- lar