β-nicotinamide adenine dinucleotide is an enteric inhibitory neurotransmitter in human and nonhuman primate colons

Abstract

Background & aims: An important component of enteric inhibitory neurotransmission is mediated by a purine neurotransmitter, such as adenosine 5'-triphosphate (ATP), binding to P2Y1 receptors and activating small conductance K(+) channels. In murine colon β-nicotinamide adenine dinucleotide (β-NAD) is released with ATP and mimics the pharmacology of inhibitory neurotransmission better than ATP. Here β-NAD and ATP were compared as possible inhibitory neurotransmitters in human and monkey colons.

Methods: A small-volume superfusion assay and high-pressure liquid chromatography with fluorescence detection were used to evaluate spontaneous and nerve-evoked overflow of β-NAD, ATP, and metabolites. Postjunctional responses to nerve stimulation, β-NAD and ATP were compared using intracellular membrane potential and force measurements. Effects of β-NAD on smooth muscle cells (SMCs) were recorded by patch clamp. P2Y receptor transcripts were assayed by reverse transcription polymerase chain reaction.

Results: In contrast to ATP, overflow of β-NAD evoked by electrical field stimulation correlated with stimulation frequency and was diminished by the neurotoxins, tetrodotoxin, and ω-conotoxin GVIA. Inhibitory junction potentials and responses to exogenous β-NAD, but not ATP, were blocked by P2Y receptor antagonists suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS), 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate (MRS 2179), and (1R,2S,4S,5S)-4-[2-Iodo-6-(methylamino)-9H-purin-9-yl]-2-(phosphonooxy)bicyclo[3.1.0]hexane-1-methanol dihydrogen phosphate ester tetraammonium salt (MRS 2500). β-NAD activated nonselective cation currents in SMCs, but failed to activate outward currents.

Conclusions: β-NAD meets the criteria for a neurotransmitter better than ATP in human and monkey colons and therefore may contribute to neural regulation of colonic motility. SMCs are unlikely targets for inhibitory purine neurotransmitters because dominant responses of SMCs were activation of net inward, rather than outward, current.

Figure 1. Overflow of ATP and ß-NAD in human colonic muscle

(A) Chromatograms of tissue superfusates collected during EFS (4 and 16 Hz) and with neural blockers at 16 Hz. EFS-evoked overflow of ß-NAD, but not ATP, increased with stimulation frequency and decreased with TTX (0.5μmol/L) or ω-conotoxin GVIA (50nmol/L); LU, luminescence units. Note that each chromatogram shows data from a different experimental tissue. (B and C) Averaged data are means±SEM; (^o) denote significant differences from 4 Hz controls (P<.05); (*) denote significant differences from 16 Hz controls (*P<.05, **P<.01). Experiment number shown in parentheses. (D and E) HPLC fraction analysis demonstrated that ß-NAD is the primary purine nucleotide in ß-NAD+ADPR+cADPR peak. (E) Fraction analysis showed that the amount of ß-NAD exceeded ATP; (*) denote significant differences from ATP (**P<.01).

Figure 2. ATP and ß-NAD released in monkey whole and circular muscle

(A) NADPH-diaphorase staining of LM and CM of monkey colon. Left panel shows NADPH-diaphorase⁺ neurons (arrows) in myenteric plexus LM preparation. Inset is magnification showing neurons within MG (*). Right panel shows that CM is free of ganglia. Nerve fibers parallel to CM remain (arrow heads). Inset is magnification showing nerve fibers. Scale bars are 500μm in both panels and 50μm in insets. (B and C) ATP and ß-NAD released from monkey WM and CM (4 and 16 Hz) and with TTX (0.5 μmol/L) and ω-conotoxin-GVIA (50 nmol/L). Averaged data (fmol/mg tissue) are means±SEM; (^o) denote significant differences from 4 Hz controls (P<.05); (*) denote significant differences from 16 Hz controls (*P<.05, **P<0.01). (D and E) HPLC fraction analysis demonstrates that ß-NAD is the primary nucleotide in ß-NAD+ADPR+cADPR peak in WM and CM. ß-NAD was greater in WM than CM. (^o) denote significant difference from WM (P<.05). ß-NAD exceeded ATP in WM and CM (**P<0.01).

Figure 3. Purinergic component of IJPs

IJPs generated by EFS (0.5 ms; 1 pulse; solid circles) in monkey (A) and human colonic muscles (B) in the presence of atropine (1 μmol/L), L-NNA (100 μmol/L) and nifedipine (1 μmol/L). Purinergic component of IJP in monkey colon was reduced by apamin, suramin, PPADS, and MRS2179. In human colon apamin only partially reduced IJPs, but surmain, PPADS, MRS2179 and MRS2500 significantly reduced IJPs.

Figure 4. Membrane responses to exogenous purines

Picospritzed ATP (10mmol/L, left panels) and ß-NAD (50mmol/L, right panels) hyperpolarized monkey (A-D) and human muscles (E, F). Responses to ß-NAD, but not to ATP, were inhibited by purine receptor antagonists (as labeled). In monkey muscles, apamin inhibited both ATP and ß-NAD induced hyperpolarization (A, left and right panels). In some muscles ß-NAD caused transient depolarization after MRS2179 (D, right). In human muscles MRS2179 and MRS2500 did not affect ATP response (E,F left), but significantly reduced ß-NAD response (E, F right)). Scale bars in D&F apply to traces in A-D and E-F, respectively.

Figure 5. Expression of P2Y receptors in monkey and human tissues

(A) Expression of P2Y1, P2Y2, P2Y4, P2Y6 and P2Y11 receptor transcripts in the tunica muscularis of the monkey fundus, antrum, jejunum and proximal colon. (B) Expression of P2Y1, P2Y2, P2Y4, P2Y6 and P2Y11 receptor transcripts in human brain, fundus, gastric body (G. Body), antrum, ascending colon (A. Colon), distal colon (D. Colon) and sigmoid colon (S. Colon). C ytoglobin was used as a house keeping gene and M represents base pair marker.

Figure 6. ß-NAD and ATP activate inward currents in SMCs of monkey (A-C) and human (D-F)

(A) In cell-attached patches (pipette CaPSS; bath HK), channel openings were negligible. ß-NAD (1mmol/L) increased openings at -80 mV. (B) Stepping from -80 to +80 mV showed ß-NAD-activated currents reversed at 0 mV in monkey SMC. a (control) and b (ß-NAD) show expanded traces from panel A during ramp depolarization (-80 mV to +80 mV). (C) ATP (1mmol/L) increased channel activity at -80 mV in monkey SMC. (D) Perforated whole-cell conditions (pipette Cs-TEA, bath CaPSS), ß-NAD (5mmol/L) activated inward current at -80 mV. ß-NAD activated-currents reversed upon washout. (E) a, b, and c show expanded traces from panel D during ramp depolarization. ß-NAD activated-currents reversed at 0 mV, demonstrating non-selective cation conductance was activated by ß-NAD. Dotted lines in B and E denote 0 mV and 0 pA. (F) ATP (1 mmol/L) activated inward currents at -80 mV.