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

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

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

+ ⁄ +
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

)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

+ ⁄ +
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

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

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

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
) ⁄ )

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

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

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

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
) ⁄ )

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.

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

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).

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

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
) ⁄ )

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

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

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

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

+ ⁄ +
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)

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-

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

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

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

+
(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

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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