Violeta N. Mutafova-Yambolieva

β-Nicotinamide adenine dinucleotide is an inhibitory neurotransmitter in visceral smooth muscle

Peripheral inhibitory nerves are physiological regulators of the contractile behavior of visceral smooth muscles. One of the transmitters responsible for inhibitory neurotransmission has been reputed to be a purine, possibly ATP. However, the exact identity of this substance has never been verified. Here we show that β-nicotinamide adenine dinucleotide (β-NAD), an inhibitory neurotransmitter candidate, is released by stimulation of enteric nerves in gastrointestinal muscles, and the pharmacological profile of β-NAD mimics the endogenous neurotransmitter better than ATP. Levels of β-NAD in superfusates of muscles after nerve stimulation exceed ATP by at least 30-fold; unlike ATP, the release of β-NAD depends on the frequency of nerve stimulation. β-NAD is released from enteric neurons, and release was blocked by tetrodotoxin or ω-conotoxin GVIA. β-NAD is an agonist for P2Y1 receptors, as demonstrated by receptor-mediated responses in HEK293 cells expressing P2Y1 receptors. Exogenous β-NAD mimics the effects of the enteric inhibitory neurotransmitter. Responses to β-NAD and inhibitory junction potentials are blocked by the P2Y1-selective antagonist, MRS2179, and the nonselective P2 receptor antagonists, pyridoxal phosphate 6-azophenyl-2′,4′-disulfonic acid and suramin. Responses to ATP are not blocked by these P2Y receptor inhibitors. The expression of CD38 in gastrointestinal muscles, and specifically in interstitial cells of Cajal, provides a means of transmitter disposal after stimulation. β-NAD meets the traditional criteria for a neurotransmitter that contributes to enteric inhibitory regulation of visceral smooth muscles.

P2Y1 purinoreceptors are fundamental to inhibitory motor control of murine colonic excitability and transit

Key points  •  Normal colonic motility is regulated by excitatory and inhibitory motor neurons, and previous studies have shown that both components of neural regulation are important for normal propulsion of colonic contents. •  Inhibitory neural control consists of two main components, and the major neurotransmitters have been identified as nitric oxide and purines; we investigated the nature of the receptors responsible for purine inhibitory motor control of the colon using mice with P2Y1 receptors deactivated. •  Inhibitory control of the colon by purine neurotransmitters was dramatically decreased in these animals and transit of fecal pellets was delayed. •  Inhibitory responses to purine neurotransmission and exogenous β‐NAD, a neurotransmitter candidate, were completely abolished in P2Y1 receptor knockouts. •  These studies demonstrate the importance of purinergic neural regulation of colonic motility and suggest this form of neural regulation depends upon P2Y1 receptors to receive and transduce inhibitory neural signals.

Inhibitory and facilitatory presynaptic effects of endothelin on sympathetic cotransmission in the rat isolated tail artery.

The present study was undertaken to determine the modulatory effects of the endothelin peptides on the neurogenically‐induced release of endogenous noradrenaline (NA) and the cotransmitter adenosine 5′‐triphosphate (ATP) from the sympathetic nerves of endothelium‐free segments of the rat isolated tail artery. The electrical field stimulation (EFS, 8 Hz, 0.5 ms, 3 min) evoked overflow of NA and ATP, in the absence of endothelins, was 0.035±0.002 pmol mg−1 tissue and 0.026±0.002 pmol mg−1 tissue, respectively. Endothelin‐1 (ET‐1; 1–30 nM) significantly reduced the EFS evoked overflow of both NA and ATP. The maximum inhibitory effect was produced by a peptide concentration of 10 nM, the amount of NA overflow being 0.020±0.002 pmol mg−1 and that of ATP overflow 0.015±0.001 pmol mg−1. Higher peptide concentrations (100 and 300 nM) reversed the EFS‐evoked overflow of NA to control levels and that of ATP to above control levels. The inhibitory effect of ET‐1 (10 nM) was resistant to the selective ETA receptor antagonist cyclo‐D‐Trp‐D‐Asp(ONa)‐Pro‐D‐Val‐Leu (BQ‐123) but was prevented by ETB receptor desensitization with sarafotoxin S6c (StxS6c) or by ETB receptor blockade with N, cis‐2,6‐dimethylpiperidinocarbonyl‐L‐gmethylleucyl‐D‐1‐methoxycarbonyltryptophanyl‐D‐norleucine (BQ‐788). StxS6c, upon acute application, exerted a dual effect on transmitter release. At concentrations of 0.001–0.3 nM the peptide significantly reduced the EFS‐evoked NA overflow, whereas at concentrations of 1–10 nM it caused a significant increase in the evoked overflow of both ATP and NA. Both the maximum inhibitory effect of StxS6c at a concentration of 0.003 nM (approximately 85% reduction of NA overflow and 40% of ATP overflow) and the maximum facilitatory effect of the peptide at a concentration of 3 nM (approximately 400% increase of ATP overflow and 200% of NA overflow) were completely antagonized by either BQ‐788 or by StxS6c‐induced ETB receptor desensitization. ET‐3 (10–100 nM) did not affect the EFS evoked overflow of either ATP or NA, but at a concentration of 300 nM significantly potentiated the release of both transmitters (0.118± 0.02 pmol mg−1 tissue ATP overflow and 0.077±0.004 pmol mg−1 NA overflow). This effect was prevented either by BQ‐123 or by BQ‐788. In summary, the endothelin peptides exerted both facilitatory and inhibitory effects on the neurogenically‐induced release of the sympathetic cotransmitters ATP and NA in the rat tail artery. Both transmitters were modulated in parallel indicating that the endothelins do not differentially modulate the release of NA and ATP in this tissue.

Adenosine 5′-diphosphate-ribose is a neural regulator in primate and murine large intestine along with β-NAD+

Key points  •  Normal gastrointestinal activity depends upon orderly movement of nutrients and wastes through the alimentary canal. These movements require coordinated contractions of the muscular wall and regulation by excitatory and inhibitory motor neurons of the enteric nervous system. •  We examined the nature of candidate purine neurotransmitters (ATP and β‐NAD) and their metabolites (ADP and ADP‐ribose) and the effects of these compounds on electrical and mechanical responses of colonic muscles. •  After release, ATP and β‐NAD were rapidly degraded to ADP and ADP‐ribose, suggesting that inhibitory neural responses may include actions of primary transmitters and metabolites. •  Metabolites of both neurotransmitter candidates elicited responses similar to responses to inhibitory nerve stimulation. However, only ADP‐ribose had pharmacology that mimicked the effects of the endogenous inhibitory neurotransmitter. •  These results help us better understand neural regulation of colonic motility and provide new insights about how defects in neural responses might lead to motility disorders such as constipation.

Platelet-derived growth factor receptor-α-positive cells and not smooth muscle cells mediate purinergic hyperpolarization in murine colonic muscles.

Enteric inhibitory neurotransmission is an important feature of the neural regulation of gastrointestinal motility. Purinergic neurotransmission, via P2Y1 receptors, mediates one phase of inhibitory neural control. For decades, ATP has been assumed to be the purinergic neurotransmitter and smooth muscle cells (SMCs) have been considered the primary targets for inhibitory neurotransmission. Recent experiments have cast doubt on both of these assumptions and suggested that another cell type, platelet-derived growth factor receptor-α-positive (PDGFRα(+)) cells, is the target for purinergic neurotransmission. We compared responses of PDGFRα(+) cells and SMCs to several purine compounds to determine if these cells responded in a manner consistent with enteric inhibitory neurotransmission. ATP hyperpolarized PDGFRα(+) cells but depolarized SMCs. Only part of the ATP response in PDGFRα(+) cells was blocked by MRS 2500, a P2Y1 antagonist. ADP, MRS 2365, β-NAD, and adenosine 5-diphosphate-ribose, P2Y1 agonists, hyperpolarized PDGFRα(+) cells, and these responses were blocked by MRS 2500. Adenosine 5-diphosphate-ribose was more potent in eliciting hyperpolarization responses than β-NAD. P2Y1 agonists failed to elicit responses in SMCs. Small hyperpolarization responses were elicited in SMCs by a small-conductance Ca(2+)-activated K(+) channel agonist, cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine, consistent with the low expression and current density of small-conductance Ca(2+)-activated K(+) channels in these cells. Large-amplitude hyperpolarization responses, elicited in PDGFRα(+) cells, but not SMCs, by P2Y1 agonists are consistent with the generation of inhibitory junction potentials in intact muscles in response to purinergic neurotransmission. The responses of PDGFRα(+) cells and SMCs to purines suggest that SMCs are unlikely targets for purinergic neurotransmission in colonic muscles.

Co‐Release Of Endogenous ATP And Noradrenaline From Guinea‐Pig Mesenteric Veins Exceeds Co‐Release From Mesenteric Arteries

1. The present study was designed to compare the overflow of sympathetic neurotransmitters of guinea‐pig inferior mesenteric artery and mesenteric vein evoked by electrical field stimulation (EFS) with special emphasis on the simultaneous release of ATP and noradrenaline (NA). The stimulation‐evoked overflow of ADP, AMP and adenosine was also evaluated.

Differential expression of genes related to purinergic signaling in smooth muscle cells, PDGFRα-positive cells, and interstitial cells of Cajal in the murine colon.

Purinergic signaling provides regulation of colonic motility. Smooth muscle cells (SMC), interstitial cells of Cajal (ICC), and platelet‐derived growth factor receptor α‐positive (PDGFRα+) cells are electrically coupled and form a functional (SIP) syncytium that constitutes the receptive field for motor neurotransmitters in the tunica muscularis. Each cell type in the SIP syncytium has specialized functions in mediating motor neurotransmission. We compared gene transcripts for purinergic receptors and membrane‐bound enzymes for purine degradation expressed by each cell type of the SIP syncytium.

Oxidative stress disrupts purinergic neuromuscular transmission in the inflamed colon

•  Colitis is associated with an attenuation of purinergic inhibitory neuromuscular transmission. •  In this study we tested the hypothesis that purine release is disrupted due to an effect of oxidative stress on mitochondrial purine synthesis. •  Stimulus‐induced release of purines was decreased in inflamed colons. •  Disruption of mitochondrial purine synthesis, or induction of oxidative stress, mimicked the effects of inflammation on purinergic neuromuscular transmission. •  Treatment of animals with a free radical scavenger resulted in a protection of the purinergic neuromuscular transmission. •  Treatment with a free radical scavenger also resulted in an improvement of propulsive motility in inflamed colons.

Uridine adenosine tetraphosphate is a novel neurogenic P2Y1 receptor activator in the gut.

Significance Millions of people suffer of gastrointestinal (GI) motility disorders. P2Y1 purine receptors and Ca2+-activated small-conductance K+ (SK) channels are established as key mediators of enteric inhibitory neurotransmission in the distal GI tract. However, the identity of the purinergic neurotransmitter in the bowel is controversial. We describe uridine adenosine tetraphosphate (Up4A) as a highly potent native activator of purinergic P2Y1 receptors and SK channels that is released spontaneously and during nerve stimulation in the human and mouse colons. We characterized potential sites of release, mimicry of the endogenous neurotransmitter, action on postjunctional targets, and metabolic pathways for Up4A. Our data identify Up4A as a novel factor in the purinergic signaling in the gut, including enteric inhibitory motor neurotransmission. Enteric purinergic motor neurotransmission, acting through P2Y1 receptors (P2Y1R), mediates inhibitory neural control of the intestines. Recent studies have shown that NAD+ and ADP ribose better meet criteria for enteric inhibitory neurotransmitters in colon than ATP or ADP. Here we report that human and murine colon muscles also release uridine adenosine tetraphosphate (Up4A) spontaneously and upon stimulation of enteric neurons. Release of Up4A was reduced by tetrodotoxin, suggesting that at least a portion of Up4A is of neural origin. Up4A caused relaxation (human and murine colons) and hyperpolarization (murine colon) that was blocked by the P2Y1R antagonist, MRS 2500, and by apamin, an inhibitor of Ca2+-activated small-conductance K+ (SK) channels. Up4A responses were greatly reduced or absent in colons of P2ry1−/− mice. Up4A induced P2Y1R–SK-channel–mediated hyperpolarization in isolated PDGFRα+ cells, which are postjunctional targets for purinergic neurotransmission. Up4A caused MRS 2500-sensitive Ca2+ transients in human 1321N1 astrocytoma cells expressing human P2Y1R. Up4A was more potent than ATP, ADP, NAD+, or ADP ribose in colonic muscles. In murine distal colon Up4A elicited transient P2Y1R-mediated relaxation followed by a suramin-sensitive contraction. HPLC analysis of Up4A degradation suggests that exogenous Up4A first forms UMP and ATP in the human colon and UDP and ADP in the murine colon. Adenosine then is generated by extracellular catabolism of ATP and ADP. However, the relaxation and hyperpolarization responses to Up4A are not mediated by its metabolites. This study shows that Up4A is a potent native agonist for P2Y1R and SK-channel activation in human and mouse colon.

Differential release of β-NAD(+) and ATP upon activation of enteric motor neurons in primate and murine colons.

Background  The purinergic component of enteric inhibitory neurotransmission is important for normal motility in the gastrointestinal (GI) tract. Controversies exist about the purine(s) responsible for inhibitory responses in GI muscles: ATP has been assumed to be the purinergic neurotransmitter released from enteric inhibitory motor neurons; however, recent studies demonstrate that β‐nicotinamide adenine dinucleotide (β‐NAD+) and ADP‐ribose mimic the inhibitory neurotransmitter better than ATP in primate and murine colons. The study was designed to clarify the sources of purines in colons of Cynomolgus monkeys and C57BL/6 mice.