�-Nicotinamide adenine dinucleotide is an inhibitoryneurotransmitter in visceral smooth muscleVioleta N. Mutafova-Yambolieva*, Sung Jin Hwang*, Xuemei Hao†, Hui Chen*, Michael X. Zhu†, Jackie D. Wood‡,Sean M. Ward*, and Kenton M. Sanders*§

*Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557; and †Department of Neuroscience, Center forMolecular Neurobiology, and ‡Department of Physiology and Cell Biology, Ohio State University, Columbus, OH 43210

Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved August 28, 2007 (received for reviewJune 13, 2007)

Peripheral inhibitory nerves are physiological regulators of thecontractile behavior of visceral smooth muscles. One of the trans-mitters responsible for inhibitory neurotransmission has beenreputed to be a purine, possibly ATP. However, the exact identityof this substance has never been verified. Here we show that�-nicotinamide adenine dinucleotide (�-NAD), an inhibitory neu-rotransmitter candidate, is released by stimulation of entericnerves in gastrointestinal muscles, and the pharmacological profileof �-NAD mimics the endogenous neurotransmitter better thanATP. Levels of �-NAD in superfusates of muscles after nervestimulation exceed ATP by at least 30-fold; unlike ATP, the releaseof �-NAD depends on the frequency of nerve stimulation. �-NADis released from enteric neurons, and release was blocked bytetrodotoxin or �-conotoxin GVIA. �-NAD is an agonist for P2Y1receptors, as demonstrated by receptor-mediated responses inHEK293 cells expressing P2Y1 receptors. Exogenous �-NAD mimicsthe effects of the enteric inhibitory neurotransmitter. Responses to�-NAD and inhibitory junction potentials are blocked by theP2Y1-selective antagonist, MRS2179, and the nonselective P2receptor antagonists, pyridoxal phosphate 6-azophenyl-2�,4�-disulfonic acid and suramin. Responses to ATP are not blocked bythese P2Y receptor inhibitors. The expression of CD38 in gastro-intestinal muscles, and specifically in interstitial cells of Cajal,provides a means of transmitter disposal after stimulation. �-NADmeets the traditional criteria for a neurotransmitter that contrib-utes to enteric inhibitory regulation of visceral smooth muscles.

enteric nervous system � gastrointestinal motility � P2Yreceptor � purinergic neurotransmission � interstitial cells of Cajal

Inhibitory neurons participate in the control of the involuntarymovements of visceral smooth muscles and are essential for

complex motor patterns such as peristalsis, receptive relaxation,and sphincter opening (1). The transmitters responsible forinhibitory neurotransmission have been the subject of studies forseveral decades, and several substances have been proposed ascotransmitters mediating the postjunctional effects of inhibitoryneurotransmission. ATP has been a candidate inhibitory (alsocalled a nonadrenergic, noncholinergic) neurotransmitter invisceral muscles for many years (2, 3). However, the identity ofATP as an inhibitory transmitter remains controversial (4, 5),and many investigators continue to refer to the actual transmittersubstance as a purine or purine-like substance. Nitric oxide (NO)also has been considered a primary neurotransmitter, and theevidence supporting a role for NO in inhibitory neural responsesis convincing (6–8). Neuropeptides, such as vasoactive intestinalpeptide and pituitary adenylate cyclase-activating polypeptide,also are expressed in inhibitory neurons and may contribute tohigher threshold responses (9, 10).

In the present study, we investigated the hypothesis that apurine not previously considered a neurotransmitter mediatesenteric inhibitory neurotransmission in visceral smooth muscles.�-nicotinamide adenine dinucleotide (�-NAD) is released in avariety of smooth muscle tissues during stimulation of nerves (11,

12). This substance has been referred to as a neuromodulator,but here we show that �-NAD meets the classical criteria for aneurotransmitter (13) in murine colonic muscles, where entericinhibitory responses are not well mimicked by the release oractions of ATP.

ResultsElectrical field stimulation (EFS) (0.1- to 0.5-ms pulses) ofintrinsic nerves evoked biphasic inhibitory junction potentials(IJPs) in murine colonic muscles. IJPs were composed of aninitial fast hyperpolarization, followed by a slower secondaryhyperpolarization, and they were blocked by 1 �M tetrodotoxin(TTX). The secondary hyperpolarization was nitrergic andcompletely blocked by pretreatment with N(�)-nitro-L-arginine(L-NNA) (14, 15).

After blocking the nitrergic component, the initial componentof the IJP was reduced by 1 �M apamin by 64 � 2.0% (n � 6;P � 0.0001) (Fig. 1a) (15, 16). The apamin-sensitive neuralresponses inhibited spontaneous action potentials and relaxedcolonic muscle strips. This response has been described aspurinergic and is possibly due to the release of ATP or a relatedpurine-like substance (3–5, 15, 17–19).

A recent report showed that P2Y1 receptors are the dominantreceptors mediating postjunctional enteric inhibitory responsesin human colonic muscles (19). We confirmed the expression ofP2Y1 receptors in murine colonic muscles and also found P2Y4receptors by RT-PCR and Western blot analysis (Fig. 1 k and l).Further, 10 �M pyridoxal phosphate 6-azophenyl-2�,4�-disulfonic acid and suramin (PPADS), a nonselective inhibitor ofP2 receptors, reduced IJP amplitudes by 52 � 2.2% (P � 0.0001;n � 6) (Fig. 1b). 2�-Deoxy-N6-methyladenosine-3�,5�-biphos-phate (MRS2179), a selective, competitive P2Y1 antagonist (20,21), inhibited IJPs in a concentration-dependent manner (e.g., 1�M reduced IJPs by 41 � 6.3%) (P � 0.0005; n � 8) (Fig. 1c).Also, 100 �M suramin reduced IJP amplitude by 55 � 5.6% (P �0.0001; n � 5) (Fig. 1d). IC50 for the effects of MRS2179 variedfrom 1.1 � 0.2 �M with 0.1-ms pulses to 2.9 � 0.3 �M with0.5-ms pulses, confirming that IJPs are mediated primarily byP2Y1 receptors (19) and demonstrating similarities in inhibitoryneurotransmission in mice and humans.

Author contributions: V.N.M.-Y. and S.J.H. contributed equally to this work; V.N.M.-Y.,M.X.Z., S.M.W., and K.M.S. designed research; V.N.M.-Y, S.J.H., X.H., H.C., and J.D.W.performed research; and V.N.M.-Y., M.X.Z., S.M.W., and K.M.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Abbreviations: ADPR, adenosine 5�-diphosphate-ribose; �-NAD, �-nicotinamide adeninedinucleotide; c-ADPR, cyclic adenosine 5�-diphosphate-ribose; EFS, electrical field stimula-tion; IJP, inhibitory junction potential; L-NNA, N(�)-nitro-L-arginine; PPADS, pyridoxalphosphate 6-azophenyl-2�, 4�-disulfonic acid and suramin; TTX, textrodotoxin.

§To whom correspondence should be addressed. E-mail: kent@unr.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0705510104/DC1.

© 2007 by The National Academy of Sciences of the USA

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We next sought to determine whether the drugs that inhibitedneural responses also inhibit responses to exogenous ATP. ATPwas applied close to the recording site by a picospritzer pipette(50-ms pulses at 10 psi) to better mimic the rapid and localizeddelivery of a neurotransmitter. Spritzes of ATP caused 13 � 1mV hyperpolarizations (n � 20) in the presence of 1 �M atropineand 100 �M L-NNA. These responses were not affected by 0.3�M TTX, but were reduced by 70 � 4% by 0.3 �M apamin (n �5; P � 0.006).

In contrast to neutrally evoked responses, the hyperpolariza-tion responses to exogenous ATP were not significantly inhibitedby 30 �M PPADS (ATP hyperpolarizations were 86 � 4% ofcontrol) (P � 0.48; n � 5) (Fig. 1e), 10 �M MRS2179 (ATPresponses were 90 � 12% of control) (n � 5; P � 0.7) (Fig. 1f ),or 100 �M suramin (ATP responses were 92 � 5% of control)(P � 0.78; n � 5) (Fig. 1g). Thus, responses to the transmitter

released from inhibitory nerves and responses to exogenousATP displayed different pharmacological profiles, suggestingthat these responses were mediated by different receptors.Spritzing UTP hyperpolarized muscles by 15 � 2 mV (n � 5), andthis response also was not blocked by PPADS (n � 5) (data notshown). Then 10–100 �M adenosine failed to induce hyperpo-larization of muscles (n � 5), and 10–30 �M adenosine A1receptor blocker, 8-(p-sulfophenyl)theophylline, had no effecton IJPs (data not shown), suggesting that the breakdown of ATPis not responsible for postjunctional responses.

HPLC, coupled with fluorescence detection of ethenoderi-vatized purines, was used to measure the release of �-NAD,ATP, and their metabolites from colonic muscles in response tostimulation of intrinsic nerves. ATP, ADP, AMP, adenosine, aswell as �-NAD and its metabolites, ADP-ribose (ADPR) andcyclic ADP-ribose (cADPR), accumulated in superfusates dur-ing EFS (Fig. 2). ATP quickly degrades in tissues to ADP, AMP,and adenosine by ectonucleoside-triphosphate diphosphohydro-lases and 5�-nucleotidase (22). Likewise, �-NAD is degraded tocADPR and ADPR by CD38-associated glycohydrolase andADP-ribosyl cyclase (23), and ADPR is degraded to AMP andadenosine by ectonucleotide pyrophosphatase (24) and 5�-nucleotidase. Therefore, the amounts of ATP and �-NADdetected in tissue superfusates are the remnants of the releasedsubstances less their metabolic products. ADP is a product ofATP, AMP and adenosine are products of ATP and �-NAD, andcADPR and ADP-ribose are products of �-NAD.

�-NAD, ADPR, and cADPR were coeluted as 1,N6-etheno-ADPR at �11.2 min. HPLC fraction analysis was performed todetermine the major compound in the 11.2-min peak (11, 12). Theratio of �-NAD to ADPR and cADPR in superfusates was 93:6:1.The concentration of �-NAD exceeded the concentration of ATPby �30-fold (Fig. 2 k, m, and o). Thus, during stimulation ofinhibitory nerves, the concentration of �-NAD near postjunctionalreceptors is likely to be much greater than the concentration ofATP.

EFS-evoked release of �-NAD was neural in origin because itwas nearly abolished by 0.3 �M TTX (Fig. 2 c and i), a blockerof neural Na� channels, and 50 nM �-conotoxin GVIA (Fig. 2d and i), an inhibitor of N-type Ca2� channels in nerve terminals.�-conotoxin GVIA also blocks purinergic IJPs (25). EFS-evokedrelease of �-NAD increased as a function of stimulation fre-quency. In contrast, release of ATP, ADP, and AMP did notincrease with stimulation frequency, and levels of these com-pounds were less sensitive to TTX and �-conotoxin GVIA than�-NAD, ADPR, and cADPR (Fig. 2 e–g). Thus, a significantfraction of the ATP released during EFS may not be released atnerve terminals. The overflow of total purines followed the samefrequency dependence and pharmacology as �-NAD (and itsmetabolites), whereas the overflow of ATP and ADP did notfollow this pattern (Fig. 2). These data show that both ATP and�-NAD are released upon EFS. However, the release of �-NADis more compatible with mechanisms known to control neuro-transmitter release (i.e., frequency dependence, TTX sensitivity,and �-conotoxin GVIA sensitivity).

CD38, the enzyme responsible for metabolizing �-NAD toADPR and cADPR, was found to be expressed in colonicmuscles, and transcripts of CD38 were up-regulated in interstitialcells of Cajal (ICCs), the cells innervated by inhibitory neuronsin gastrointestinal muscles (Fig. 2 o and p) (26). A mechanism forthe local degradation of �-NAD may explain the presence ofADPR and cADPR in superfusates.

We next examined whether �-NAD is an agonist for P2Y1receptors because these receptors mediate nonnitrergic entericinhibitory responses (ref. 19 and this study). Receptor-mediatedresponses were assayed by changes in intracellular Ca2� ([Ca2�]i)in HEK293 cells stably transfected with guinea pig P2Y1 recep-tor cDNA (27). �-NAD (�100 �M) and ATP (�0.1 �M) had

Fig. 1. Membrane responses to enteric inhibitory neurotransmitter andexogenous purines. (a–d) IJPs evoked by 0.3-ms single pulses of EFS (black dots)of colonic muscles strips (in the presence of 1 �M atropine to block the majorexcitatory input and 100 �M L-NNA to block the nitrergic component of theIJP). The nonnitrergic IJPs were blocked by 0.3 and 1 �M apamin (a), 10 and 30�M PPADS (b), 1 and 10 �M MRS2179 (c), and 100 �M suramin (d). (e–g)Hyperpolarization responses to picospritzing of ATP onto muscles near the siteof recording (10 mM ATP in spritz pipet; 50-ms pulses at 10 psi). Unlike theresponses to the endogenous neurotransmitter, the hyperpolarization re-sponses to ATP were not blocked by 30 �M PPADS (e), 10 �M MRS2179 ( f), or100 �M suramin (g). (h–j) Hyperpolarization responses to picospritzing of�-NAD onto the muscles near the site of recording (50 mM �-NAD in spritzpipet; 50-ms pulses at 10 psi). Similar to responses to the endogenous neuro-transmitter, the hyperpolarization responses to �-NAD were reduced by 30�M PPADS (h), 10 �M MRS2179 (i), and 100 �M suramin (j). (Scale bars in j applyto all traces in e–j.) (k) RT-PCR detected expression of P2Y1 and P2Y4 receptortranscripts in the muscularis externae of the proximal colon. P2Y1 and P2Y4receptor transcripts were observed at 1,204 and 1,228 bp, respectively. Tran-scripts also were detected in mouse brain used as a positive control. (l) Westernblots were performed on membrane fractions of muscle homogenates. Spe-cific labeling of P2Y1 and P2Y4 receptors was localized at �42 kDa fromjejunum, ileum, and colon muscles.

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little effect on [Ca2�]i in nontransfected cells (Fig. 3a). Cellstransfected with P2Y1 receptors displayed increased sensitivityto a variety of agonists, including ATP, 2-methylthio-ADP, and2-methylthio-ATP, and MRS2179 antagonized these responses(27). Further, 0.1–100 �M �-NAD increased [Ca2�]i in cells

The 16-Hz EFS-evoked overflow of purine nucleotides and ADO was re-duced by 50 nM �-conotoxin GVIA (�-Ctx; 30-min perfusion). (e–j) Aver-aged data. Overflow (femtomoles per milligram of tissue) is the overflowduring EFS less overflow before EFS (ST-PS) (n � 4 – 6 per group). Data arepresented as means � SEM. (e–g) The evoked overflow of ATP, ADP, andAMP did not increase with stimulation frequency (4 and 16 Hz; P � 0.05).The evoked overflow of ATP was not affected by TTX (P � 0.2594) or �-Ctx(P � 0.2247). Likewise, the overflow of ADP was not reduced by TTX (P �0.1358) or �-Ctx (P � 0.5618). The overflow of AMP also was not changedby either TTX (P � 0.2928) or �-Ctx (P � 0.3820). (h–j) The evoked overflowof �-NAD/cADPR/ADPR, and ADO, as well as of total purines, increased withstimulation frequency (P � 0.0437, 0.0429, and �0.0001 for �-NAD/cADPR/ADPR, ADO, and total purines, respectively) and was reduced by both TTX(P � 0.0372, 0.0001, and �0.0001 for �-NAD/cADPR/ADPR, ADO, and totalpurines, respectively) and �-CtxGVIA (P � 0.0492, 0.0027, and �0.0001 for�-NAD/cADPR/ADPR, ADO, and total purines, respectively). Asterisks de-note significant differences from 16-Hz controls (P � 0.05). Open circlesdenote significant differences from 4-Hz controls (P � 0.05). (k–m) Originalchromatograms of the 7.2-min (cADPR-containing) fraction (k), the 8.5-min(ADPR-containing) fraction (l), and the 10.5-min (�-NAD-containing) frac-tion (m) of tissue superfusate samples (30 chambers combined) collectedduring EFS at 16 Hz, 0.5 ms, for 30 s and ethenoderivatized with 2-chloro-acetaldehyde at 80°C (pH 4.0) for 40 min. Note that the 7.2-min fraction alsocontained ATP. (n) Highest amounts of 1,N6-etheno-ADPR (eADPR) wereobserved in the 10.5-min (�-NAD-containing) fraction. The amount of�-NAD exceeded the amount of ATP by at least 30-fold. (o) QuantitativePCR data for expression of Kit in whole-muscle homogenates (filled bar)and sorted ICCs (open bar). The relative amount of Kit expression isenriched in ICCs (P � 0.001). (p) Quantitative PCR data for expression ofCD38 in whole-muscle homogenates (filled bar) and ICCs (open bar). Therelative amount of CD38 expression is enriched in ICCs (P � 0.001). Data ino and p are means � SEM from three experiments.

Fig. 2. Release and metabolism of ATP and �-NAD. (a–d) Original chromato-grams for tissue superfusate samples collected before [prestimulation (PS)] orduring EFS (16 Hz, 0.5 ms, 15 V, 480 pulses) in the absence or presence of neuralblockers. Scale applies to all chromatograms. LU, luminescence units. (a) Asample collected PS contained small amounts of ATP, ADP, AMP, and adeno-sine (ADO). (b) EFS (16 Hz) evoked overflow of ATP, ADP, AMP, ADO, and amixture of �-NAD, cADPR, and ADPR. (c) TTX (0.3 �M; 30-min perfu-sion) reduced the overflow of all purines evoked by EFS at 16 Hz. (d)

Fig. 3. Initiation of Ca2� transients in HEK293 cells transfected with guineapig P2Y1 receptor cDNA. (a) Responses to 100 �M �-NAD, 0.1 �M ATP, and 100�M charbachol (CCh) in a nontransfected control cell. (b) Concentration-dependent increases in [Ca2�]i in response to 0.1–100 �M �-NAD in cellstransfected with P2Y1 receptor cDNA. These responses are compared withresponses to 0.1 �M ATP and 100 �M CCh. (c) Responses to �-NAD in trans-fected cells were completely blocked by MRS2179. The response to �-NAD wasrestored after washout of MRS2179. (d) Concentration-response curves for theP2Y1-mediated effects of �-NAD on [Ca2�]i normalized to either ATP (Upper)or CCh (Lower) responses.

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expressing P2Y1 receptors in a concentration-dependent man-ner (Fig. 3b). The response to 10 �M �-NAD was completelyblocked by 10 �M MRS2179 (n � 3) (Fig. 3c). Responses to�-NAD, normalized against 0.1 �M ATP or 100 �M carbacholresponses, gave EC50 values of 6.1 � 0.6 and 6.2 � 0.6 �M,respectively (n � 8 and 9, respectively) (Fig. 3d). These datademonstrate that �-NAD is an agonist for P2Y1 receptors. ATPis more potent than �-NAD for P2Y1 receptors expressed inHEK293 cells, but the relative insensitivity of the release of ATPand metabolites to �-conotoxin GVIA in overflow experimentssuggests that a significant fraction of the ATP released inmuscles is not in close proximity to the P2Y1 receptors mediatinginhibitory neurotransmission.

Colonic muscles spontaneously generated action potentialsand phasic contractions (4.25 � 0.2/min) (n � 20) that wereinhibited by nerve stimulation (Fig. 4a). Then 0.5–10 mM�-NAD caused concentration-dependent inhibition of contrac-tions (Fig. 4 b and d). Inhibition of contractions by nervestimulation was blocked by MRS2179 (Fig. 4e). Picospritzing�-NAD (50 ms at 10 psi) caused repeatable hyperpolarizationsaveraging 16.8 � 1.2 mV (n � 20). These responses were reduced79 � 4% by 0.3 �M apamin (n � 5; P � 0.002), 81 � 2.5% by30 �M PPADS (n � 5; P � 0.012) (Fig. 1h), 91 � 3% by 10 �MMRS2179 (n � 5; P � 0.007) (Fig. 1i), and 73 � 6% by 100 �Msuramin (n � 7; P � 0.016) (Fig. 1j). The responses wereunaffected by 0.3 �M TTX (1 � 6%) (n � 5; P � 0.9). Thus,exogenous �-NAD mimicked the responses and pharmacologyof the neurotransmitter released during the stimulation ofenteric inhibitory neurons.

�-NAD also hyperpolarized membrane potential and inhib-ited action potentials and contractions throughout the murinegastrointestinal tract (i.e., gastric fundus and antrum and smallintestine); in Cymologous macaque stomach, small intestine, andcolon; and in human jejunum (Fig. 4 c and d and data not shown).

DiscussionEccles (13) listed several criteria that need to be satisfied for asubstance to be considered a neurotransmitter. �-NAD metthese criteria in our experiments investigating the substance usedfor nonnitrergic inhibitory neural responses in gastrointestinalmuscles: (i) �-NAD, and enzymatic processes to synthesize it, areubiquitously present in cells (much like ATP); (ii) �-NAD isreleased from neurons in a frequency-dependent manner, and itsrelease depends on stimulation frequency and the availability ofneuronal Na� and Ca2� channels; (iii) exogenous �-NAD is anagonist for P2Y1 receptors and mimics the nonnitrergic hyper-polarization and relaxation responses caused by the stimulationof enteric inhibitory neurons; (iv) CD38, an extracellular met-abolic enzyme for �-NAD, is expressed in murine colonicmuscles and is up-regulated in ICCs that are innervated byinhibitory neurons (26, 28); and (v) receptor antagonists(PPADS, suramin, and MRS2179) block postjunctional inhibi-tory neural responses and responses to �-NAD, but these drugsdid not block responses to ATP. Taken together, these obser-vations suggest that �-NAD serves as an inhibitory (nonadren-ergic, noncholinergic) neurotransmitter in gastrointestinalmuscles.

�-NAD is best known for its important intracellular functions.�-NAD serves as a coenzyme for cellular oxidation-reductionreactions, a donor of ADPR in the posttranslational modifica-tion of proteins, and a precursor to cADPR and other intracel-lular second messengers with Ca2�-releasing activity (23). Inrecent years, �-NAD also has been shown to have extracellularfunctions (29, 30). For example, extracellular �-NAD inducesCa2� signaling and apoptosis in human osteoclastic cells (31),activates P2Y11 receptors in human granulocytes (32), and,together with ADPR, activates Ca2� influx in human monocytesthrough plasma membrane Ca2� uptake (33). �-NAD and its

metabolites, ADPR and cADPR, are released during EFS ofcanine mesenteric blood vessels and bladder detrusor smoothmuscles from several species, including mouse, rat, guinea pig,rabbit, dog, monkey, and human (11, 12). The release of �-NAD

Fig. 4. Inhibitory neural regulation of colonic muscles. (a) Simultaneous intra-cellular electrical (Upper) and contractile (Lower) activity from a colonic muscle.Under control conditions, spontaneous action potentials were organized intoperiodic clusters, causing phasic contractile behavior. Stimulation of inhibitoryneurons (at arrowhead) caused immediate cessation of action potentials, repo-larization, and relaxation from contraction. After addition of L-NNA to block thenitrergic component of inhibitory neurotransmission, cells depolarized and ac-tionpotentialsfiredcontinuously. Stimulationofenteric (nonnitrergic) inhibitoryneurons caused a transient hyperpolarization, brief cessation of action poten-tials, and transient relaxation. (b and c) Concentration-dependent inhibition ofspontaneous colonic and ileal contractions by 0.1–10 mM �-NAD, respectively.Drugs were added (black bars) and washed out, and control contractile activitywere recovered between drug doses. (d) Summary of eight experiments (colon)and six experiments (ileum) like the ones in b and c. The IC50 for the effects of�-NAD on contractions was 2.5 � 0.5 and 0.23 � 0.08 mM for colonic and ilealmuscles, respectively. (e) (Left) Inhibition of spontaneous contractile activity bynonnitrergic inhibitory neural inputs (experiment performed in the presence of200 �M L-NNA and 1 �M atropine). Nerves were stimulated at 5 Hz for 30 s (0.3-spulses) (n � 6). (Right) The inhibitory response to nerve stimulation was blockedby 10 �M MRS2179.

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is not induced by the classic neurotransmitter substances orcontraction of smooth muscle per se (11). Rather, �-NAD releasedepends on the availability of neuronal Ca2� channels andSNARE proteins (i.e., SNAP-25), and hence �-NAD appears tobe released by vesicular exocytosis (12, 34). Exogenous �-NADreduces the release of norepinephrine in blood vessels (11) andinhibits spontaneous contractions and smooth muscle tone in thehuman bladder (12). �-NAD has been suggested to be a neu-romodulator released from neural cells in vascular and visceralsmooth muscles. Previous studies have not tested whether�-NAD meets the criteria for a neurotransmitter.

The characteristics of �-NAD and ATP release demonstrateimportant differences in the mechanisms responsible for therelease of these substances. In the present study, both com-pounds were released in response to nerve action potentialsbecause TTX reduced the release of both substances. However,a significant fraction of the ATP (and its metabolites) releasedwas not blocked by �-conotoxin. Thus, a substantial portion ofthe ATP released could have been extrajunctional or could haveresulted from stimulation of another population of neurons with�-conotoxin-insensitive Ca2� entry pathways at nerve terminals(i.e., intrinsic nerves other than enteric inhibitory neurons orextrinsic nerves). Fast IJPs in gastrointestinal muscles areblocked by �-conotoxin (25), and �-conotoxin strongly inhibitedthe release of �-NAD (and its metabolites). Thus, it appears thata substantial fraction of the �-NAD released came from nerveterminals of enteric inhibitory nerves.

Spritzing ATP on muscles resulted in hyperpolarization re-sponses that were blocked by apamin, but not by MRS2179,PPADS, or suramin. Thus, a second population of P2Y receptorsnot available to the transmitter released by enteric inhibitoryneurons must be present and be the dominant receptors boundby exogenous ATP. P2Y2 and P2Y4 receptors also are expressedin colonic tunica muscularis (35). Hyperpolarization also wascaused by UTP, and these responses were not blocked byPPADS. Thus, a combination of P2Y2 and P2Y4 receptors mightmediate responses to exogenous ATP and UTP. Exogenous�-NAD, which should have access to the same populations ofreceptors as exogenous ATP, preferentially activated the samepopulation of receptors used in neurotransmission because�-NAD displayed the same pharmacological profile as theinhibitory neurotransmitter. Thus, �-NAD does not appear to bea good agonist for the extrajunctional receptors that dominate inresponses to exogenous ATP.

Our data cannot strictly eliminate the possibility that ATP servesas a cotransmitter in enteric inhibitory neurotransmission. How-ever, the fact that the release of ATP was not frequency-dependentand not significantly affected by �-conotoxin suggests that much ofthe ATP released does not occur at the same sites or by the samemechanisms controlling the release of �-NAD. If the release ofATP is extrajunctional, then the amount of ATP does not appearto be sufficient to stimulate the receptors affected by exogenousATP. Responses of P2Y1 receptors expressed in HEK cells showedthat ATP was more potent than �-NAD. However, the mass of�-conotoxin-sensitive �-NAD released from neurons far exceededthat of ATP. The greater mass of �-NAD, coupled with thepossibility that ATP is not released at nerve terminals, may meanthat the levels and sites of release do not favor ATP as a factor ininhibitory neurotransmission.

An important question is how different populations of receptorscan be stimulated by exogenous ATP and purines released fromintrinsic sources such as neurons. One explanation for this obser-vation might be compartmentalization of P2Y receptors withclustering of specific types of receptors at postjunctional effector(synaptic) sites. In fact, synaptic specializations are present ingastrointestinal muscles, and several studies have shown closesynaptic-like contacts between enteric motor nerves and ICCs (26,28, 36–39). When these structures are absent, postjunctional re-

sponses to highly diffusible neurotransmitters such as NO andacetylcholine are greatly reduced (26). Both pre- and postjunctionalsynaptic proteins also have been observed in varicosities of entericmotor neurons and ICCs, respectively (39). P2Y1 receptors may beclustered in postjunctional membranes within synaptic regions.Outside synaptic clefts, P2Y4 and P2Y2 receptors might dominatein responses to ATP. We tested several antibodies against P2Yreceptors in immunohistochemical studies and verified the speci-ficity of these antibodies with Western blots (data not shown).However, none of the antibodies tested was suitable for thedetermination of subcellular distributions of these receptors.

After the release of a neurotransmitter, mechanisms forrecovering or metabolizing the neurotransmitter substance areneeded to terminate neural signals. We found that CD38 isexpressed in colonic muscles, and transcripts of CD38 areup-regulated in ICCs, which are innervated by inhibitory neu-rons in gastrointestinal muscles (26, 39). CD38 is a multifunc-tional enzyme that degrades �-NAD to ADPR by NAD glyco-hydrolase, �-NAD to cADPR by ADP-ribosyl cyclase, andcADPR to ADPR by cADPR hydrolase activities (23). The NADglycohydrolase activity of CD38 appears to be most efficient(30). The catalytic site of CD38 faces the extracellular space (40,41), making this enzyme suitable as a regulator of extracellular�-NAD levels (30). Thus, a mechanism for local degradation of�-NAD and terminating its neurotransmitter action is availablein colonic muscles.

MethodsElectrophysiological and Isometric Force Experiments. Thirty- to50-day-old C57BL/6 mice (Charles River Laboratories, Wilming-ton, MA) were anesthetized and decapitated after cervicaldislocation. Proximal colon and ileum segments were removedand opened along the mesenteric border. After removing mu-cosa, 10 � 6-mm sheets of muscularis were placed in a recordingchamber with the circular muscle facing upward. Muscles weremaintained in 37.5 � 0.5°C flowing Krebs-Ringer bicarbonatesolution containing 120.4 mM NaCl, 5.9 mM KCl, 15.5 mMNaHCO3, 11.5 mM glucose, 1.2 mM MgCl2, 1.2 mM NaH2PO4,and 2.5 mM CaCl2. The solution was bubbled with 97% O2–3%CO2 (pH 7.3–7.4). The circular muscle cells were impaled with50–80 M microelectrodes, and transmembrane potentials weremeasured and analyzed as previously described (26). Alterna-tively, 6-mm-wide circular muscle strips were fixed at one endand attached to a Fort 10 isometric force transducer (WPI, Fl).Isometric force was measured as described previously (26). Datain the text are expressed as means � SEM. Differences wereevaluated by Student’s t test by using SigmaPlot (Systat SoftwareInc., Richmond, CA). P values �0.05 were taken as statisticallysignificant.

Electrical Stimulation and Pressure Ejection of Drugs. EFS wasapplied (0.1- to 0.5-ms pulses; 150 V) by platinum electrodesby using a square wave stimulator (Grass 588; Grass Instru-ments, Quincy, MA). Receptor antagonists and neural toxinswere added to the bath solution. Purine agonists were loadedinto micropipettes positioned close to the electrical recordingsite, and drugs were applied by pressurized ejection (Pico-spritzer; General Valve, East Hanover, NJ). Responses todrugs applied by picospritzer were calculated as the area underthe curve by using Clampfit (Axon Instruments, Sunnyvale,CA). ATP, �-NAD, adenosine, UTP, TTX, PPADS, suramin,apamin, L-NNA, atropine, and �-conotoxin GVIA were pur-chased from Sigma–Aldrich (St. Louis, MO). MRS2179 waspurchased from Tocris Bioscience (Ellisville, MO).

Purine Overflow Experiments. Muscles were placed in 200 �l ofwater-jacketed superfusion chambers equipped with platinumelectrodes (42–44). The muscles were superfused with oxygen-

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ated Krebs solution (37°C), and nerves were stimulated with EFS(480 pulses at 4 or 16 Hz, 0.5 ms, supramaximal voltage).Superfusion solution was collected before and during EFS inice-cold tubes and acidified to pH 4.0 with citrate phosphatebuffer. Chloroacetaldehyde was added, and the samples wereheated for 45 min at 80°C to form f luorescent 1,N6-ethenoderivatives of endogenous ATP, ADP, AMP, and aden-osine. 1,N6-etheno-ADPR was formed by endogenous �-NAD,cADPR, and ADPR as described (11).

HPLC Assay of 1,N6-Ethenopurines. Derivatized samples were pro-cessed through a gradient HP1100 liquid chromatography mod-ule system equipped with a fluorescence detector as described(44). Each 1,N6-ethenopurine was quantified against knownstandards. Results (femtomoles per milligram) were normalizedfor volume and tissue weight.

HPLC Fraction Analysis of �-NAD, ADPR, and ADPR. HPLC Fractionanalysis (11, 12) was performed to identify the compoundsforming 1,N6-etheno-ADPR. Tissue superfusates from 30chambers were concentrated, and samples were injected intothe HPLC. Then 400-�l fractions corresponding to retentiontimes of cADPR and ATP (7.0–7.4 min, 7.2-min fraction),ADPR (8.3–8.7 min, 8.5-min fraction), and �-NAD (10.3–10.7min, 10.5-min fraction) were collected and derivatized. Thederivatized samples were analyzed for 1,N6-etheno-ADPR and1,N6-etheno-ATP content. Means � SEM were compared bya two-tailed, unpaired t test (GraphPadPrism, Version 3.02;GraphPad Software, Inc., San Diego, CA).

�-NAD-Induced Intracellular Ca2� Concentration ([Ca2�]i) Changes inCells Expressing P2Y1 Receptors. HEK293 cells expressing guineapig P2Y1 receptors (27) were grown on 20 �g/ml poly(L-ornithine)-coated glass coverslips for �16 h and loaded with 2

�M fura2-AM. Changes in [Ca2�]i in 10–15 cells were monitoredby using a photometry system (PTI, Birmingham, NJ) (27). Fordose-response curves to �-NAD, peak [Ca2�]i increases werenormalized to responses to 100 nM ATP or 100 �M carbachol.

Measurement of CD38, P2Y Receptor Transcripts, and Western Blots ofP2Y Receptors. Primers specific for P2Y1 and P2Y4 receptorswere designed to amplify full-length transcripts of these recep-tors. Murine brain was used as a positive control. Expression ofP2Y1 and P2Y4 receptor mRNAs in colon and brain weredetermined by RT-PCR. P2Y1 and P2Y4 protein expression wastested by Western blot analysis. Crude homogenates of jejunum,ileum, and colon muscles were separated into cytosolic andmembrane-bound fractions. Membrane fraction proteins wereseparated on SDS/10% PAGE and probed with antibodiesagainst P2Y1 and P2Y4 receptors. Specific labeling of P2Y1 andP2Y4 receptors was localized to bands at �42 kDa.

ICCs from C57BL/6 mice were sorted by fluorescence-activated cell sorting as described previously (45). The transcrip-tional expression of CD38 in cells sorted in three separate sortswas measured by quantitative RT-PCR and expressed in �-actin(a housekeeping gene) units. An RT-PCR experiment also wasperformed by using GAPDH as a housekeeping gene to checkthe results with �-actin. The transcriptional expression of CD38in colonic tunica muscularis also was measured and used as areference for ICC levels. The CD38 expression in sorted cells andtissues was compared by a Student’s t test (n � 3). Detailedmethods for these experiments are given in supporting informa-tion (SI) Text.

We thank David Westfall for comments on the paper. This work wassupported by National Institutes of Health Grant P01 DK41315 (toK.M.S. and S.M.W.), R01 HL 60031 (to V.N.M.-Y.), and R21 NS056942(to M.X.Z.).

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16364 � www.pnas.org�cgi�doi�10.1073�pnas.0705510104 Mutafova-Yambolieva et al.