Leonie Durnin

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.

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.

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.

Storage and secretion of β-NAD, ATP and dopamine in NGF-differentiated rat pheochromocytoma PC12 cells

In nerve–smooth muscle preparations β‐nicotinamide adenine dinucleotide (β‐NAD) has emerged as a novel extracellular substance with putative neurotransmitter and neuromodulator functions. β‐NAD is released, along with noradrenaline and adenosine 5′‐triphosphate (ATP), upon firing of action potentials in blood vessels, urinary bladder and large intestine. At present it is unclear whether noradrenaline, ATP and β‐NAD are stored in and released from common populations of synaptic vesicles. The answer is unattainable in complex systems such as nerve–smooth muscle preparations. Adrenal chromaffin cells are thus used here as a single‐cell model to examine mechanisms of concomitant neurosecretion. Using high‐performance liquid chromatography techniques with electrochemical and fluorescence detection we simultaneously evaluated secretion of dopamine (DA), ATP, adenosine 5′‐diphosphate, adenosine 5′‐monophosphate, adenosine, β‐NAD and its immediate metabolites ADP‐ribose and cyclic ADP‐ribose in superfused nerve growth factor‐differentiated rat pheochromocytoma PC12 cells. β‐NAD, DA and ATP were released constitutively and upon stimulation with high‐K+ solution or nicotine. Botulinum neurotoxin A tended to increase the spontaneous secretion of all substances and abolished the high‐K+‐evoked release of β‐NAD and DA but not of ATP. Subcellular fractionation by continuous glycerol and sucrose gradients along with immunoblot analysis of the vesicular marker proteins synaptophysin and secretogranin II revealed that β‐NAD, ATP and DA are stored in both small synaptic‐like vesicles and large dense‐core‐like vesicles. However, the three substances appear to have different preferential sites of release upon membrane depolarization including sites associated with SNAP‐25 and sites not associated with SNAP‐25.

Release, neuronal effects and removal of extracellular β-nicotinamide adenine dinucleotide (β-NAD+) in the rat brain

Recent evidence supports an emerging role of β‐nicotinamide adenine dinucleotide (β‐NAD+) as a novel neurotransmitter and neuromodulator in the peripheral nervous system –β‐NAD+ is released in nerve‐smooth muscle preparations and adrenal chromaffin cells in a manner characteristic of a neurotransmitter. It is currently unclear whether this holds true for the CNS. Using a small‐chamber superfusion assay and high‐sensitivity high‐pressure liquid chromatography techniques, we demonstrate that high‐K+ stimulation of rat forebrain synaptosomes evokes overflow of β‐NAD+, adenosine 5′‐triphosphate, and their metabolites adenosine 5′‐diphosphate (ADP), adenosine 5′‐monophosphate, adenosine, ADP‐ribose (ADPR) and cyclic ADPR. The high‐K+‐evoked overflow of β‐NAD+ is attenuated by cleavage of SNAP‐25 with botulinum neurotoxin A, by inhibition of N‐type voltage‐dependent Ca2+ channels with ω‐conotoxin GVIA, and by inhibition of the proton gradient of synaptic vesicles with bafilomycin A1, suggesting that β‐NAD+ is likely released via vesicle exocytosis. Western analysis demonstrates that CD38, a multifunctional protein that metabolizes β‐NAD+, is present on synaptosomal membranes and in the cytosol. Intact synaptosomes degrade β‐NAD+. 1,N 6‐etheno‐NAD, a fluorescent analog of β‐NAD+, is taken by synaptosomes and this uptake is attenuated by authentic β‐NAD+, but not by the connexin 43 inhibitor Gap 27. In cortical neurons local applications of β‐NAD+ cause rapid Ca2+ transients, likely due to influx of extracellular Ca2+. Therefore, rat brain synaptosomes can actively release, degrade and uptake β‐NAD+, and β‐NAD+ can stimulate postsynaptic neurons, all criteria needed for a substance to be considered a candidate neurotransmitter in the brain.

A commonly used ecto-ATPase inhibitor, ARL-67156, blocks degradation of ADP more than the degradation of ATP in murine colon

Adenosine 5′‐triphosphate (ATP) is released extracellularly as a neurotransmitter and an autocrine or paracrine mediator in numerous systems, including the gastrointestinal tract. It is rapidly degraded to active and inactive metabolites by membrane‐bound enzymes. Investigators frequently use inhibitors of ATP hydrolysis such as ARL‐67156 and POM‐1 to suppress the catabolism of ATP and prolong its effects in pharmacological studies. Our aim was to investigate directly the effects of ARL‐67156 and POM‐1 on the degradation of ATP and adenosine 5′‐diphosphate (ADP) in mouse colonic muscles.

Urothelial purine release during filling of murine and primate bladders.

During urinary bladder filling the bladder urothelium releases chemical mediators that in turn transmit information to the nervous and muscular systems to regulate sensory sensation and detrusor muscle activity. Defects in release of urothelial mediators may cause bladder dysfunctions that are characterized with aberrant bladder sensation during bladder filling. Previous studies have demonstrated release of ATP from the bladder urothelium during bladder filling, and ATP remains the most studied purine mediator that is released from the urothelium. However, the micturition cycle is likely regulated by multiple purine mediators, since various purine receptors are found present in many cell types in the bladder wall, including urothelial cells, afferent nerves, interstitial cells in lamina propria, and detrusor smooth muscle cells. Information about the release of other biologically active purines during bladder filling is still lacking. Decentralized bladders from C57BL/6 mice and Cynomolgus monkeys (Macaca fascicularis) were filled with physiological solution at different rates. Intraluminal fluid was analyzed by high-performance liquid chromatography with fluorescence detection for simultaneous evaluation of ATP, ADP, AMP, adenosine, nicotinamide adenine dinucleotide (NAD+), ADP-ribose, and cADP-ribose content. We also measured ex vivo bladder filling pressures and performed cystometry in conscious unrestrained mice at different filling rates. ATP, ADP, AMP, NAD+, ADPR, cADPR, and adenosine were detected released intravesically at different ratios during bladder filling. Purine release increased with increased volumes and rates of filling. Our results support the concept that multiple urothelium-derived purines likely contribute to the complex regulation of bladder sensation during bladder filling.

Cyclic ADP-ribose requires CD38 to regulate the release of ATP in visceral smooth muscle

It is well established that the intracellular second messenger cADP‐ribose (cADPR) activates Ca2+ release from the sarcoplasmic reticulum through ryanodine receptors. CD38 is a multifunctional enzyme involved in the formation 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+/+ and CD38−/− mice. cADPR (1 nm) enhanced the spontaneous overflow of ATP in bladders isolated from CD38+/+ mice. This effect was abolished by the inhibitor of cADPR receptors on sarcoplasmic reticulum 8‐bromo‐cADPR (80 μm) and by ryanodine (50 μm), but not by the nonselective P2 purinergic receptor antagonist pyridoxal phosphate 6‐azophenyl‐2′,4′‐disulfonate (30 μm). cADPR failed to facilitate the spontaneous ATP overflow in bladders isolated from CD38−/− mice, indicating that CD38 is crucial for the enhancing effects of extracellular cADPR on spontaneous ATP release. Contractile responses to ATP were potentiated by cADPR, suggesting that the two adenine nucleotides may work in synergy to maintain the resting tone of the bladder. In conclusion, extracellular cADPR enhances the spontaneous release of ATP in the bladder by influx via CD38 and subsequent activation of intracellular cADPR receptors, probably causing an increase in intracellular Ca2+ in neuronal cells.