Báo cáo khoa học: Cyclic ADP-ribose requires CD38 to regulate the release of ATP in visceral smooth muscle ppt

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:
(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
1.2 mm MgCl
2
, 23.8 mm NaHCO
3
, 1.2 mm KH
2
PO
4
,
11.0 mm dextrose, and 1.8 mm CaCl
2
(pH 7.4). The blad-
ders 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 influence of the
urothelium, which is a significant source of ATP in the
bladder [50].
Overflow experiments
Detrusor muscles (one to three bladders per chamber) were
placed in 200-lL water-jacketed superfusion chambers
equipped with platinum electrodes, as described previously
[12,13,15], and superfused with oxygenated Krebs solution

at 37 °C. Nerves were stimulated with EFS at 16 Hz and
0.1 ms for 60 s. Samples of the superfusion solution were
collected before and during EFS. In some experiments,
tissues were superfused with cADPR (1 nm for 5 min)
and samples were collected: (a) before suferfusion with
cADPR; (b) during superfusion with cADPR, but in the
absence of EFS; and (c) during superfusion with EFS in
the presence of cADPR. In some experiments, tissues were
superfused with PPADS (30 lm) or 8-Br-cADPR (80 lm)
for 30 min, or with ryanodine (50 lm) for 45 min, before
cADPR. In other experiments, tissues were superfused with
TTX (0.3–0.5 lm) for 30 min. In some experiments, tissues
were superfused with carbachol (1 lm for 5 min), ADP or
AMP (each 10 nm). In a separate set of experiments
tissues, were incubated with BoNTA (100–300 nm) for
2.5–4 h prior to being loaded into the superfusion system.
In some experiments, tissues were superfused with CBX or
FFA (each 100 lm) for 60 min before overflow experi-
ments were performed. Finally, in some experiments,
tissues were superfused with either b-NAD
+
or ADPR
(1 nm for 5 min), and samples were collected before and
during EFS.
Degradation of NGD
ADP-ribosyl cyclase converts both b-NAD
+
into cADPR
and NGD into cGDPR [4]. However, unlike cADPR,
cGDPR is not hydrolyzed by tissue cADPR hydrolase.

Therefore, we used the conversion of NGD into cGDPR to
determine the GDP-ribosyl cyclase activity ⁄ ADP-ribosyl
cyclase activity, as described previously [51]; this avoided
the influence of cADPR hydrolysis to ADPR. Tissues were
loaded into small-volume superfusion chambers as
described above, and superfused with NGD (0.2 mm) for
2 min. NGD and cGDPR in substrate solution in the
absence and presence of tissue were detected by RP-HPLC
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-ADP-
ribosyl cyclase activity.
HPLC assay of etheno-purines, NGD, and cGDPR
To prepare the samples for HPLC analysis, chloroacetalde-
hyde was added, and the samples were heated to 80 °C for
40 min to form 1,N
6
-etheno-derivatives of endogenous ade-
nine nucleotides and nucleosides present in the superfusates
[52,53]. Thus, ATP, ADP, AMP and Ado were converted
to their 1,N
6
-etheno-derivatives: 1,N
6
-etheno-ATP, 1,N
6
-
etheno-ADP, 1,N
6
-etheno-AMP, and 1,N

6
-etheno-Ado,
respectively. The endogenous b-NAD
+
and its metabolites
cADPR and ADPR formed eADPR as described previously
[12,13]. Likewise, exogenous cADPR was also converted to
eADPR during the etheno-derivatization of superfusates.
Samples were processed through gradient reverse-phase
Agilent Technologies 1100 or 1200 liquid chromatography
module systems equipped with fluorescence detectors, as
described previously [53]. The fluorescence detector detected
etheno-purines at an excitation wavelength of 230 nm and
an emission wavelength of 420 nm, as determined previ-
ously [53]. NGD and cGDPR were detected at an excita-
tion 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 quan-
tified against known standards. Results were normalized for
sample volume and tissue wet weight.
HPLC fraction analysis
HPLC fraction analysis [12,13,15] was performed to iden-
tify the compounds forming eADPR. Tissue superfusates
from 16 chambers (one to three bladders per chamber) were
concentrated, and the combined samples were injected into
the HPLC apparatus. Fractions were collected according to
retention times for cADPR (7.0–7.4 min), ADPR (8.3–
8.7 min), and b-NAD

+
(10.3–10.7 min), etheno-derivatized,
and analyzed by HPLC for eADPR content. The HPLC
fraction analysis was performed in three sets of combined
overflow experiments.
Western immunoblot analysis of SNAP-25
Bladders from control and BoNTA-treated groups were
frozen by immersion in liquid nitrogen. Frozen tissues were
pulverized, and total protein was extracted by glass–glass
homogenization with a RIPA buffer composed of 20 mm
Tris, 150 mm NaCl, 10% glycerol, 1% NP40, 2.5 mm NaF,
0.1 mm sodium orthovanadate, 1 mm benzamidine, 2.5 mm
b-glycerophosphate, 100 lm 4-(2-Aminoethyl) benzenesulfo-
nyl fluoride hydrochloride, and 1 lm leupeptin. Insoluble
material was pelleted by centrifugation at 15 000 g for
20 min at 4 °C. The total protein concentration of the
supernatant was determined by the bicinchoninic acid
assay, with BSA as the standard. Tissue homogenates were
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 3105
reduced with Laemmli reagent, and equal amounts of total
protein (60 lg) were resolved by SDS ⁄ PAGE (12% acryl-
amide) and transferred onto nitrocellulose membranes for
1.5 h at 100 V and 4 °C (Bio-Rad, Hercules, CA, USA).
Membranes were blocked for 1 h with 2% nonfat dry milk
plus 0.2% Tween, and probed for 18 h at 4 °C with a pri-
mary rabbit mAb against SNAP-25 (Epitomics, Burlin-
game, CA, USA), diluted 1000-fold in the blocking
solution. After removal of excess primary antibody, mem-
branes were incubated for 1.5 h at room temperature with

a secondary horseradish peroxidase-conjugated rabbit anti-
body. Immunostained protein bands were detected with
ECL Advantage (GE HealthCare Biosciences, Piscataway,
NJ, USA), and visualized with a CCD camera-based detec-
tion system (Epi Chem II; UVP Laboratory Products,
Upland, CA, USA).
Mechanical force measurements
Detrusor smooth muscle strips (5 mm in length) were
mounted in 10-mL organ baths by means of loop sutures,
and force displacements were monitored with Fort 10
isometric force transducers in a Myobath 4 system (World
Precision Instruments, Sarasota, FL, USA). A resting force
of 1 g was applied to each muscle segment. This was found
to stretch tissue segments to near the optimum length for
tension development. In all experiments, tissues were ini-
tially 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 desensitization.
Statistics
Data are presented as means ± standard errors (SEs).
One-way ANOVA with a post hoc Bonferroni multiple com-
parison test was performed with graphpadprism v. 3 for
Windows (GraphPad Software, San Diego, CA, USA) when
three or more groups of data were compared. Paired or
unpaired two-tailed Student’s t-test was performed with the
same software when two groups of data were compared.
A P-value of < 0.05 was considered to be significant.

Chemicals
ATP, ADP, AMP, Ado, ADPR, 8-Br-cADPR, carbachol,
CBX, FFA, NGD, PPADS and Aplysia cyclase were pur-
chased from Sigma-Aldrich (St Louis, MO, USA). cADPR
was purchased from Biolog (Bremen, Germany). Ryanodine
was purchased from Axxora, LLC (San Diego, CA, USA),
and BoNTA was purchased from List Biological Laborato-
ries (Campbell, CA, USA). FFA and ryanodine were
dissolved in 70% ethanol and then diluted in Krebs solu-
tion. BoNTA was dissolved in 1 mgÆmL
)1
BSA according
to the manufacturer’s instructions, and further diluted in
Krebs solution. All other chemicals were initially dissolved
in double-distilled water and further diluted in the superfu-
sion solution.
Acknowledgements
This work was supported by R01 HL60031 and
P01 DK41315 grants. We are grateful to M. Mendoza
and D. Russell for technical assistance.
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