Membrane responses to enteric inhibitory neurotransmitter and exogenous purines. (a–d) IJPs evoked by 0.3-ms single pulses of EFS (black dots) of colonic muscles strips (in the presence of 1 μM atropine to block the major excitatory input and 100 μM l-NNA to block the nitrergic component of the IJP). The nonnitrergic IJPs were blocked by 0.3 and 1 μM apamin (a), 10 and 30 μM PPADS (b), 1 and 10 μM MRS2179 (c), and 100 μM suramin (d). (e–g) Hyperpolarization responses to picospritzing of ATP onto muscles near the site of recording (10 mM ATP in spritz pipet; 50-ms pulses at 10 psi). Unlike the responses to the endogenous neurotransmitter, the hyperpolarization responses to ATP were not blocked by 30 μM PPADS (e), 10 μM MRS2179 (f), or 100 μM suramin (g). (h–j) Hyperpolarization responses to picospritzing of β-NAD onto the muscles near the site of recording (50 mM β-NAD in spritz pipet; 50-ms pulses at 10 psi). Similar to responses to the endogenous neurotransmitter, the hyperpolarization responses to β-NAD were reduced by 30 μM PPADS (h), 10 μM MRS2179 (i), and 100 μM suramin (j). (Scale bars in j apply to all traces in e–j.) (k) RT-PCR detected expression of P2Y1 and P2Y4 receptor transcripts in the muscularis externae of the proximal colon. P2Y1 and P2Y4 receptor transcripts were observed at 1,204 and 1,228 bp, respectively. Transcripts also were detected in mouse brain used as a positive control. (l) Western blots were performed on membrane fractions of muscle homogenates. Specific labeling of P2Y1 and P2Y4 receptors was localized at ≈42 kDa from jejunum, ileum, and colon muscles.

Initiation of Ca²⁺ transients in HEK293 cells transfected with guinea pig P2Y1 receptor cDNA. (a) Responses to 100 μM β-NAD, 0.1 μM ATP, and 100 μM charbachol (CCh) in a nontransfected control cell. (b) Concentration-dependent increases in [Ca²⁺]_i in response to 0.1–100 μM β-NAD in cells transfected with P2Y1 receptor cDNA. These responses are compared with responses to 0.1 μM ATP and 100 μM CCh. (c) Responses to β-NAD in transfected cells were completely blocked by MRS2179. The response to β-NAD was restored after washout of MRS2179. (d) Concentration-response curves for the P2Y1-mediated effects of β-NAD on [Ca²⁺]_i normalized to either ATP (Upper) or CCh (Lower) responses.

Release and metabolism of ATP and β-NAD. (a–d) Original chromatograms for tissue superfusate samples collected before [prestimulation (PS)] or during EFS (16 Hz, 0.5 ms, 15 V, 480 pulses) in the absence or presence of neural blockers. Scale applies to all chromatograms. LU, luminescence units. (a) A sample collected PS contained small amounts of ATP, ADP, AMP, and adenosine (ADO). (b) EFS (16 Hz) evoked overflow of ATP, ADP, AMP, ADO, and a mixture of β-NAD, cADPR, and ADPR. (c) TTX (0.3 μM; 30-min perfusion) reduced the overflow of all purines evoked by EFS at 16 Hz. (d) The 16-Hz EFS-evoked overflow of purine nucleotides and ADO was reduced by 50 nM ω-conotoxin GVIA (ω-Ctx; 30-min perfusion). (e–j) Averaged data. Overflow (femtomoles per milligram of tissue) is the overflow during EFS less overflow before EFS (ST-PS) (n = 4–6 per group). Data are presented as means ± SEM. (e–g) The evoked overflow of ATP, ADP, and AMP 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 ω-Ctx (P = 0.2247). Likewise, the overflow of ADP was not reduced by TTX (P = 0.1358) or ω-Ctx (P = 0.5618). The overflow of AMP also was not changed by either TTX (P = 0.2928) or ω-Ctx (P = 0.3820). (h–j) The evoked overflow of β-NAD/cADPR/ADPR, and ADO, as well as of total purines, increased with stimulation frequency (P = 0.0437, 0.0429, and <0.0001 for β-NAD/cADPR/ADPR, ADO, and total purines, respectively) and was reduced by both TTX (P = 0.0372, 0.0001, and <0.0001 for β-NAD/cADPR/ADPR, ADO, and total purines, respectively) and ω-CtxGVIA (P = 0.0492, 0.0027, and <0.0001 for β-NAD/cADPR/ADPR, ADO, and total purines, respectively). Asterisks denote significant differences from 16-Hz controls (P < 0.05). Open circles denote significant differences from 4-Hz controls (P < 0.05). (k–m) Original chromatograms of the 7.2-min (cADPR-containing) fraction (k), the 8.5-min (ADPR-containing) fraction (l), and the 10.5-min (β-NAD-containing) fraction (m) of tissue superfusate samples (30 chambers combined) collected during EFS at 16 Hz, 0.5 ms, for 30 s and ethenoderivatized with 2-chloroacetaldehyde at 80°C (pH 4.0) for 40 min. Note that the 7.2-min fraction also contained ATP. (n) Highest amounts of 1,N⁶-etheno-ADPR (eADPR) were observed in the 10.5-min (β-NAD-containing) fraction. The amount of β-NAD exceeded the amount of ATP by at least 30-fold. (o) Quantitative PCR data for expression of Kit in whole-muscle homogenates (filled bar) and sorted ICCs (open bar). The relative amount of Kit expression is enriched in ICCs (P < 0.001). (p) Quantitative PCR data for expression of CD38 in whole-muscle homogenates (filled bar) and ICCs (open bar). The relative amount of CD38 expression is enriched in ICCs (P < 0.001). Data in o and p are means ± SEM from three experiments.

Inhibitory neural regulation of colonic muscles. (a) Simultaneous intracellular electrical (Upper) and contractile (Lower) activity from a colonic muscle. Under control conditions, spontaneous action potentials were organized into periodic clusters, causing phasic contractile behavior. Stimulation of inhibitory neurons (at arrowhead) caused immediate cessation of action potentials, repolarization, and relaxation from contraction. After addition of l-NNA to block the nitrergic component of inhibitory neurotransmission, cells depolarized and action potentials fired continuously. Stimulation of enteric (nonnitrergic) inhibitory neurons caused a transient hyperpolarization, brief cessation of action potentials, and transient relaxation. (b and c) Concentration-dependent inhibition of spontaneous colonic and ileal contractions by 0.1–10 mM β-NAD, respectively. Drugs were added (black bars) and washed out, and control contractile activity were 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 β-NAD on contractions was 2.5 ± 0.5 and 0.23 ± 0.08 mM for colonic and ileal muscles, respectively. (e) (Left) Inhibition of spontaneous contractile activity by nonnitrergic inhibitory neural inputs (experiment performed in the presence of 200 μM l-NNA and 1 μM atropine). Nerves were stimulated at 5 Hz for 30 s (0.3-s pulses) (n = 6). (Right) The inhibitory response to nerve stimulation was blocked by 10 μM MRS2179.