Spatial mapping of ectonucleotidase gene expression in the murine urinary bladder.

Purinergic signaling is important for normal bladder function, as it is thought to initiate the voiding reflex and modulate smooth muscle tone. The availability of adenine nucleotides and nucleosides (aka purines) at receptor sites of various cell types in the bladder wall is regulated by ectonucleotidases (ENTDs). ENTDs hydrolyze purines such as adenosine 5'-triphosphate (ATP) and adenosine 5'-diphosphate (ADP) with varying preference for the individual substrate. Therefore, the end effect of extracellular purines may depend significantly on the type of ENTD that is expressed in close proximity to the target cells. ENTDs likely have distinct cellular associations, but the specific locations of individual enzymes in the bladder wall are poorly understood. We used RNAscope™, an RNA in situ hybridization (ISH) technology, to visualize the distribution and measure the levels of gene expression of the main recognized ectonucleotidases in large high-resolution images of murine bladder sections. The relative gene expression of ENTDs was Entpd3 > Alpl >> Enpp1 = Entpd2 >> Enpp3 > Entpd1 (very low to no signal) in the urothelium, Entpd1 ≥ Entpd2 >> Enpp3 > Enpp1 = Alpl ≥ Nt5e (very low to no signal) in the lamina propria, and Entpd1 >> Nt5e = Entpd2 >> Enpp1 > Alpl = Enpp3 in the detrusor. These layer-specific differences might be important in compartmentalized regulation of purine availability and subsequent functions in the bladder wall and may explain reported asymmetries in purine availability in the bladder lumen and suburothelium/lamina propria spaces.

Differential Influences of Endogenous and Exogenous Sensory Neuropeptides on the ATP Metabolism by Soluble Ectonucleotidases in the Murine Bladder Lamina Propria.

Bladder urothelium and suburothelium/lamina propria (LP) have prominent sensory and transducer functions with the active participation of afferent neurons and urothelium-derived purine mediators such as adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine (ADO). Effective concentrations of purines at receptor targets depend significantly on the extracellular degradation of ATP by ectonucleotidases (ENTDs). We recently reported the regulated release of soluble ENTDs (s-ENTDs) in the LP and the consequent degradation of ATP to ADP, AMP, and ADO. Afferent neurons in the LP can be activated by urothelial ATP and release peptides and other transmitters that can alter the activity of cells in their vicinity. Using a murine decentralized ex vivo detrusor-free bladder model, 1,N6-etheno-ATP (eATP) as substrate, and sensitive HPLC-FLD methodologies, we found that exogenous neuropeptides calcitonin gene-related peptide (CGRP), substance P (Sub P), neurokinin A (NKA), and pituitary adenylate cyclase-activating polypeptide [PACAP (1-38)] all increased the degradation of eATP by s-ENTDs that were released in the LP spontaneously and/or during bladder filling. Using antagonists of neuropeptide receptors, we observed that endogenous NKA did not modify the ATP hydrolysis by s-ENTDs, whereas endogenous Sub P increased both the constitutive and distention-induced release of s-ENTDs. In contrast, endogenous CGRP and PACAP (1-38) increased the distention-induced, but not the spontaneous, release of s-ENTDs. The present study puts forward the novel idea that interactions between peptidergic and purinergic signaling mechanisms in the LP have an impact on bladder excitability and functions by regulating the effective concentrations of adenine purines at effector cells in the LP.

The Pannexin 1 Channel and the P2X7 Receptor Are in Complex Interplay to Regulate the Release of Soluble Ectonucleotidases in the Murine Bladder Lamina Propria.

The bladder urothelium releases ATP into the lamina propria (LP) during filling, which can activate P2X receptors on afferent neurons and trigger the micturition reflex. Effective ATP concentrations are largely dependent on metabolism by membrane-bound and soluble ectonucleotidases (s-ENTDs), and the latter are released in the LP in a mechanosensitive manner. Pannexin 1 (PANX1) channel and P2X7 receptor (P2X7R) participate in urothelial ATP release and are physically and functionally coupled, hence we investigated whether they modulate s-ENTDs release. Using ultrasensitive HPLC-FLD, we evaluated the degradation of 1,N6-etheno-ATP (eATP, substrate) to eADP, eAMP, and e-adenosine (e-ADO) in extraluminal solutions that were in contact with the LP of mouse detrusor-free bladders during filling prior to substrate addition, as an indirect measure of s-ENDTS release. Deletion of Panx1 increased the distention-induced, but not the spontaneous, release of s-ENTDs, whereas activation of P2X7R by BzATP or high concentration of ATP in WT bladders increased both. In Panx1-/- bladders or WT bladders treated with the PANX1 inhibitory peptide 10Panx, however, BzATP had no effect on s-ENTDS release, suggesting that P2X7R activity depends on PANX1 channel opening. We concluded, therefore, that P2X7R and PANX1 are in complex interaction to regulate s-ENTDs release and maintain suitable ATP concentrations in the LP. Thus, while stretch-activated PANX1 hinders s-ENTDS release possibly to preserve effective ATP concentration at the end of bladder filling, P2X7R activation, presumably in cystitis, would facilitate s-ENTDs-mediated ATP degradation to counteract excessive bladder excitability.

Sensory Neurons, PIEZO Channels and PAC1 Receptors Regulate the Mechanosensitive Release of Soluble Ectonucleotidases in the Murine Urinary Bladder Lamina Propria.

The urinary bladder requires adequate concentrations of extracellular adenosine 5'-triphosphate (ATP) and other purines at receptor sites to function properly. Sequential dephosphorylation of ATP to ADP, AMP and adenosine (ADO) by membrane-bound and soluble ectonucleotidases (s-ENTDs) is essential for achieving suitable extracellular levels of purine mediators. S-ENTDs, in particular, are released in the bladder suburothelium/lamina propria (LP) in a mechanosensitive manner. Using 1,N6-etheno-ATP (eATP) as substrate and sensitive HPLC-FLD methodology, we evaluated the degradation of eATP to eADP, eAMP and eADO in solutions that were in contact with the LP of ex vivo mouse detrusor-free bladders during filling prior to substrate addition. The inhibition of neural activity with tetrodotoxin and ω-conotoxin GVIA, of PIEZO channels with GsMTx4 and D-GsMTx4 and of the pituitary adenylate cyclase-activating polypeptide type I receptor (PAC1) with PACAP6-38 all increased the distention-induced but not spontaneous release of s-ENTDs in LP. It is conceivable, therefore, that the activation of these mechanisms in response to distention restricts the further release of s-ENTDs and prevents excessive hydrolysis of ATP. Together, these data suggest that afferent neurons, PIEZO channels, PAC1 receptors and s-ENTDs form a system that operates a highly regulated homeostatic mechanism to maintain proper extracellular purine concentrations in the LP and ensure normal bladder excitability during bladder filling.

Mechanosensitive Hydrolysis of ATP and ADP in Lamina Propria of the Murine Bladder by Membrane-Bound and Soluble Nucleotidases.

Prior studies suggest that urothelium-released adenosine 5'-triphosphate (ATP) has a prominent role in bladder mechanotransduction. Urothelial ATP regulates the micturition cycle through activation of purinergic receptors that are expressed in many cell types in the lamina propria (LP), including afferent neurons, and might also be important for direct mechanosensitive signaling between urothelium and detrusor. The excitatory action of ATP is terminated by enzymatic hydrolysis, which subsequently produces bioactive metabolites. We examined possible mechanosensitive mechanisms of ATP hydrolysis in the LP by determining the degradation of 1,N 6 -etheno-ATP (eATP) at the anti-luminal side of nondistended (empty) or distended (full) murine (C57BL/6J) detrusor-free bladder model, using HPLC. The hydrolysis of eATP and eADP was greater in contact with LP of distended than of nondistended bladders whereas the hydrolysis of eAMP remained unchanged during filling, suggesting that some steps of eATP hydrolysis in the LP are mechanosensitive. eATP and eADP were also catabolized in extraluminal solutions (ELS) that were in contact with the LP of detrusor-free bladders, but removed from the organ chambers prior to addition of substrate. The degradation of both purines was greater in ELS from distended than from nondistended preparations, suggesting the presence of mechanosensitive release of soluble nucleotidases in the LP. The released enzyme activities were affected differently by Ca2+ and Mg2+. The common nucleotidase inhibitors ARL67156, POM-1, PSB06126, and ENPP1 Inhibitor C, but not the alkaline phosphatase inhibitor (-)-p-bromotetramisole oxalate, inhibited the enzymes released during bladder distention. Membrane-bound nucleotidases were identified in tissue homogenates and in concentrated ELS from distended preparations by Wes immunodetection. The relative distribution of nucleotidases was ENTPD1 >> ENPP1 > ENTPD2 = ENTPD3 > ENPP3 = NT5E >> ENTPD8 = TNAP in urothelium and ENTPD1 >> ENTPD3 >> ENPP3 > ENPP1 = ENTPD2 = NT5E >> ENTPD8 = TNAP in concentrated ELS, suggesting that regulated ectodomain shedding of membrane-bound nucleotidases possibly occurs in the LP during bladder filling. Mechanosensitive degradation of ATP and ADP by membrane-bound and soluble nucleotidases in the LP diminishes the availability of excitatory purines in the LP at the end of bladder filling. This might be a safeguard mechanism to prevent over-excitability of the bladder. Proper proportions of excitatory and inhibitory purines in the bladder wall are determined by distention-associated purine release and purine metabolism.

Urinary ATP Levels Are Controlled by Nucleotidases Released from the Urothelium in a Regulated Manner.

Adenosine 5'-triphosphate (ATP) is released in the bladder lumen during filling. Urothelial ATP is presumed to regulate bladder excitability. Urinary ATP is suggested as a urinary biomarker of bladder dysfunctions since ATP is increased in the urine of patients with overactive bladder, interstitial cystitis or bladder pain syndrome. Altered urinary ATP might also be associated with voiding dysfunctions linked to disease states associated with metabolic syndrome. Extracellular ATP levels are determined by ATP release and ATP hydrolysis by membrane-bound and soluble nucleotidases (s-NTDs). It is currently unknown whether s-NTDs regulate urinary ATP. Using etheno-ATP substrate and HPLC-FLD detection techniques, we found that s-NTDs are released in the lumen of ex vivo mouse detrusor-free bladders. Capillary immunoelectrophoresis by ProteinSimple Wes determined that intraluminal solutions (ILS) collected at the end of filling contain ENTPD3 > ENPP1 > ENPP3 ≥ ENTPD2 = NT5E = ALPL/TNAP. Activation of adenylyl cyclase with forskolin increased luminal s-NTDs release whereas the AC inhibitor SQ22536 had no effect. In contrast, forskolin reduced and SQ22536 increased s-NTDs release in the lamina propria. Adenosine enhanced s-NTDs release and accelerated ATP hydrolysis in ILS and lamina propria. Therefore, there is a regulated release of s-NTDs in the bladder lumen during filling. Aberrant release or functions of urothelial s-NTDs might cause elevated urinary ATP in conditions with abnormal bladder excitability.

Neurotransmitters responsible for purinergic motor neurotransmission and regulation of GI motility.

Classical concepts of peripheral neurotransmission were insufficient to explain enteric inhibitory neurotransmission. Geoffrey Burnstock and colleagues developed the idea that ATP or a related purine satisfies the criteria for a neurotransmitter and serves as an enteric inhibitory neurotransmitter in GI muscles. Cloning of purinergic receptors and development of specific drugs and transgenic mice have shown that enteric inhibitory responses depend upon P2Y1 receptors in post-junctional cells. The post-junctional cells that transduce purinergic neurotransmitters in the GI tract are PDGFRα+ cells and not smooth muscle cells (SMCs). PDGFRα+ cells express P2Y1 receptors, are activated by enteric inhibitory nerve stimulation and generate Ca2+ oscillations, express small-conductance Ca2+-activated K+ channels (SK3), and generate outward currents when exposed to P2Y1 agonists. These properties are consistent with post-junctional purinergic responses, and similar responses and effectors are not functional in SMCs. Refinements in methodologies to measure purines in tissue superfusates, such as high-performance liquid chromatography (HPLC) coupled with etheno-derivatization of purines and fluorescence detection, revealed that multiple purines are released during stimulation of intrinsic nerves. ß-NAD+ and other purines, better satisfy criteria for the purinergic neurotransmitter than ATP. HPLC has also allowed better detection of purine metabolites, and coupled with isolation of specific types of post-junctional cells, has provided new concepts about deactivation of purine neurotransmitters. In spite of steady progress, many unknowns about purinergic neurotransmission remain and require additional investigation to understand this important regulatory mechanism in GI motility.

Extracellular metabolism of the enteric inhibitory neurotransmitter ß-nicotinamide adenine dinucleotide (ß-NAD) in the murine colon.

KEY POINTS: ß-Nicotinamide adenine dinucleotide (ß-NAD) is a key inhibitory neurotransmitter in the colon. The neuroeffector junction in the gut consists of enteric motor neurons and SIP syncytium, including smooth muscle cells (SMCs), interstitial cells of Cajal (ICC), and cells expressing platelet-derived growth factor receptor α (PDGFRα+ cells). Measuring metabolism of 1,N6 -etheno-NAD (eNAD) in colonic tunica muscularis and in SMCs, ICC and PDGFRα+ cells with HPLC-FLD, we report that (1) in tissues, eNAD is degraded to eADP-ribose, eAMP and e-adenosine (eADO) by CD38, ENPP1 and NT5E, (2) with SMCs and PDGFRα+ cells, eNAD is metabolized to eADO by ENPP1 and NT5E, (3) eNAD is not metabolized by ICC, (4) NT5E is expressed chiefly by SMCs and moderately by PDGFRα+ cells, (5) SIP cells are not the primary location of CD38. These data argue that the duration and strength of purinergic neurotransmission can be modulated by targeting multiple enzymes with specialized cellular distribution in the colon. ABSTRACT: Prior studies suggest that ß-nicotinamide adenine dinucleotide (ß-NAD) is an important inhibitory motor neurotransmitter in the enteric nervous system. Metabolism of ß-NAD at the neuroeffector junction (NEJ) is likely to be necessary for terminating inhibitory neurotransmission and may also produce bioactive metabolites. The enteric NEJ consists of enteric neurons and postjunctional cells of the SIP syncytium, including smooth muscle cells (SMCs), interstitial cells of Cajal (ICC), and cells expressing platelet-derived growth factor receptor α (PDGFRα+ cells). We examined possible specialized functions of the NEJ in ß-NAD metabolism by determining the degradation of 1,N6 -etheno-NAD (eNAD) in colonic tunica muscularis of wild-type, Cd38-/- , Nt5e-/- , Enpp1-/- and Cd38-/- /Nt5e-/- mice and in SIP cells from mice expressing cell-specific fluorescent reporters purified by fluorescence activated cell sorting (FACS). We measured eNAD and its metabolites eADP-ribose (eADPR), eAMP and e-adenosine (eADO) from tissues and sorted SIP cells using liquid chromatography. eNAD exposed to colonic muscularis of wild-type mice produced eADPR, eAMP and eADO. CD38 mediated the conversion of eNAD to eADPR, whereas ENPP1 mediated degradation of eNAD and eADPR to eAMP. NT5E (aka CD73) was the primary enzyme forming eADO from eAMP. PDGFRα+ cells and SMCs were involved in production of eADO from eNAD, and ICC were not involved in extracellular metabolism of eNAD. CD38 mediated the eNAD metabolism in whole tissues, but CD38 did not appear to be functionally expressed by SMCs or ICC. NT5E was expressed in SMCs > PDGFRα+ cells. Our data show that extracellular metabolism of ß-NAD in the colon is mediated by multiple enzymes with cell-specific expression.

A Decentralized (Ex Vivo) Murine Bladder Model with the Detrusor Muscle Removed for Direct Access to the Suburothelium during Bladder Filling.

Previous studies have established the release of chemical substances from flat bladder mucosa sheets affixed in Ussing chambers and exposed to changes in hydrostatic pressure or mechanical stretch and from cultured urothelial cells upon hydrostatic pressure changes, stretch, cell swelling, or drag forces, and in bladder lumen at end of filling. Such findings led to the assumption that these mediators are also released in suburothelium (SubU)/lamina propria (LP) during bladder filling, where they affect cells deep in the bladder wall to ultimately regulate bladder excitability. There are at least two obvious limitations in such studies: 1) none of these approaches provide direct information about the presence of mediators in SubU/LP, and 2) the stimuli used are not physiological and do not recapitulate authentic filling of the bladder. Here, we discuss a procedure that enables direct access to the suburothelial surface of the bladder mucosa in the course of bladder filling. The murine detrusor-free preparation we created closely resembles filling of the intact bladder and allows pressure-volume studies to be performed on the bladder in the absence of confounding signaling from spinal reflexes and detrusor smooth muscle. Using the novel detrusor-free bladder model, we recently demonstrated that intravesical measurements of mediators cannot be used as a proxy to what has been released or present in the SubU/LP during bladder filling. The model enables examination of urothelium-derived signaling molecules that are released, generated by metabolism and/or transported into the SubU/LP during the course of bladder filling to transmit information to neurons and smooth muscle of the bladder and regulate its excitability during continence and micturition.

An ex vivo bladder model with detrusor smooth muscle removed to analyse biologically active mediators released from the suburothelium.

KEY POINTS: Studies of urothelial cells, bladder sheets or lumens of filled bladders have suggested that mediators released from urothelium into suburothelium (SubU)/lamina propria (LP) activate mechanisms controlling detrusor excitability. None of these approaches, however, has enabled direct assessment of availability of mediators at SubU/LP during filling. We developed an ex vivo mouse bladder preparation with intact urothelium and SubU/LP but no detrusor, which allows direct access to the SubU/LP surface of urothelium during filling. Pressure-volume measurements during filling demonstrated that bladder compliance is governed primarily by the urothelium. Measurements of purine mediators in this preparation demonstrated asymmetrical availability of purines in lumen and SubU/LP, suggesting that interpretations based solely on intraluminal measurements of mediators may be inaccurate. The preparations are suitable for assessments of release, degradation and transport of mediators in SubU/LP during bladder filling, and are superior to experimental approaches previously used for urothelium research. ABSTRACT: The purpose of this study was to develop a decentralized (ex vivo) detrusor smooth muscle (DSM)-denuded mouse bladder preparation, a novel model that enables studies on availability of urothelium-derived mediators at the luminal and anti-luminal aspects of the urothelium during filling. Urinary bladders were excised from C57BL6/J mice and the DSM was removed by fine-scissor dissection without touching the mucosa. Morphology and cell composition of the preparation wall, pressure-volume relationships during filling, and fluorescent dye permeability of control, protamine sulfate- and lipopolysaccharide-treated denuded bladders were characterized. The preparation wall contained intact urothelium and suburothelium (SubU)/lamina propria (LP) and lacked the DSM and the serosa. The utility of the model for physiological research was validated by measuring release, metabolism and transport of purine mediators at SubU/LP and in bladder lumen during filling. We determined asymmetrical availability of purines (e.g. ATP, ADP, AMP and adenosine) in lumen and at SubU/LP during filling, suggesting differential mechanisms of release, degradation and bilateral transurothelial transport of purines during filling. Some observations were validated in DSM-denuded bladder of the cynomolgus monkey (Macaca fascicularis). The novel model was superior to current models utilized to study properties of the urothelium (e.g. cultured urothelial cells, bladder mucosa sheets mounted in Ussing chambers or isolated bladder strips in organ baths) in that it enabled direct access to the vicinity of SubU/LP during authentic bladder filling. The model is particularly suitable for understanding local mechanisms of urothelium-DSM connectivity and for broad understanding of the role of urothelium in regulating continence and voiding.

Loss of nitric oxide-mediated inhibition of purine neurotransmitter release in the colon in the absence of interstitial cells of Cajal.

Regulation of colonic motility depends on the integrity of enteric inhibitory neurotransmission mediated by nitric oxide (NO), purine neurotransmitters, and neuropeptides. Intramuscular interstitial cells of Cajal (ICC-IM) and platelet-derived growth factor receptor-α-positive (PDGFRα+) cells are involved in generating responses to NO and purine neurotransmitters, respectively. Previous studies have suggested a decreased nitrergic and increased purinergic neurotransmission in KitW/KitW-v (W/Wv ) mice that display lesions in ICC-IM along the gastrointestinal tract. However, contributions of NO to these phenotypes have not been evaluated. We used small-chamber superfusion assays and HPLC to measure the spontaneous and electrical field stimulation (EFS)-evoked release of nicotinamide adenine dinucleotide (NAD+)/ADP-ribose, uridine adenosine tetraphosphate (Up4A), adenosine 5'-triphosphate (ATP), and metabolites from the tunica muscularis of human, monkey, and murine colons and circular muscle of monkey colon, and we tested drugs that modulate NO levels or blocked NO receptors. NO inhibited EFS-evoked release of purines in the colon via presynaptic neuromodulation. Colons from W/Wv, Nos1-/- , and Prkg1-/- mice displayed augmented neural release of purines that was likely due to altered nitrergic neuromodulation. Colons from W/Wv mice demonstrated decreased nitrergic and increased purinergic relaxations in response to nerve stimulation. W/Wv mouse colons demonstrated reduced Nos1 expression and reduced NO release. Our results suggest that enhanced purinergic neurotransmission may compensate for the loss of nitrergic neurotransmission in muscles with partial loss of ICC. The interactions between nitrergic and purinergic neurotransmission in the colon provide novel insight into the role of neurotransmitters and effector cells in the neural regulation of gastrointestinal motility.NEW & NOTEWORTHY This is the first study investigating the role of nitric oxide (NO) and intramuscular interstitial cells of Cajal (ICC-IM) in modulating neural release of purines in colon. We found that NO inhibited release of purines in human, monkey, and murine colons and that colons from KitW/KitW-v (W/Wv ) mice, which present with partial loss of ICC-IM, demonstrated augmented neural release of purines. Interactions between nitrergic and purinergic neurotransmission may affect motility in disease conditions with ICC-IM deficiencies.

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.

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

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 Ca(2+)-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 Ca(2+) 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.

The purinergic neurotransmitter revisited: a single substance or multiple players?

The past half century has witnessed tremendous advances in our understanding of extracellular purinergic signaling pathways. Purinergic neurotransmission, in particular, has emerged as a key contributor in the efficient control mechanisms in the nervous system. The identity of the purine neurotransmitter, however, remains controversial. Identifying it is difficult because purines are present in all cell types, have a large variety of cell sources, and are released via numerous pathways. Moreover, studies on purinergic neurotransmission have relied heavily on indirect measurements of integrated postjunctional responses that do not provide direct information for neurotransmitter identity. This paper discusses experimental support for adenosine 5'-triphosphate (ATP) as a neurotransmitter and recent evidence for possible contribution of other purines, in addition to or instead of ATP, in chemical neurotransmission in the peripheral, enteric and central nervous systems. Sites of release and action of purines in model systems such as vas deferens, blood vessels, urinary bladder and chromaffin cells are discussed. This is preceded by a brief discussion of studies demonstrating storage of purines in synaptic vesicles. We examine recent evidence for cell type targets (e.g., smooth muscle cells, interstitial cells, neurons and glia) for purine neurotransmitters in different systems. This is followed by brief discussion of mechanisms of terminating the action of purine neurotransmitters, including extracellular nucleotide hydrolysis and possible salvage and reuptake in the cell. The significance of direct neurotransmitter release measurements is highlighted. Possibilities for involvement of multiple purines (e.g., ATP, ADP, NAD(+), ADP-ribose, adenosine, and diadenosine polyphosphates) in neurotransmission are considered throughout.

Oxidative stress disrupts purinergic neuromuscular transmission in the inflamed colon.

Colitis, induced by trinitrobenzene sulfonic acid (TNBS) in guinea pig, leads to decreased purinergic neuromuscular transmission resulting in a reduction in inhibitory junction potentials (IJPs) in colonic circular muscle. We explored possible mechanisms responsible for this inflammation-induced neurotransmitter plasticity. Previous studies have suggested that the deficit in inflamed tissue involves decreased ATP release. We therefore hypothesized that decreased purinergic transmission results from inflammation-induced free radical damage to mitochondria, leading to decreased purine synthesis and release. Stimulus-induced release of purines was measured using high-performance liquid chromatography, and quantities of all purines measured were significantly reduced in the inflamed colons as compared to controls. To test whether decreased mitochondrial function affects the IJP, colonic muscularis preparations were treated with the mitochondrial ATP synthase inhibitors oligomycin or dicyclohexylcarbodiimide, which resulted in a significant reduction of IJP amplitude. Induction of oxidative stress in vitro, by addition of H2O2 to the preparation, also significantly reduced IJP amplitude. Purinergic neuromuscular transmission was significantly restored in TNBS-inflamed guinea pigs, and in dextran sodium sulfate-inflamed mice, treated with a free radical scavenger. Furthermore, propulsive motility in the distal colons of guinea pigs with TNBS colitis was improved by in vivo treatment with the free radical scavenger. We conclude that oxidative stress contributes to the reduction in purinergic neuromuscular transmission measured in animal models of colitis, and that these changes can be prevented by treatment with a free radical scavenger, resulting in improved motility.

Neuronal and extraneuronal release of ATP and NAD(+) in smooth muscle.

Adenosine 5'-triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD(+) ) are key intracellular constituents involved in energy transfer and redox homeostasis in the cell. ATP is also released in the extracellular space and in the past half century it has been assumed to be the purinergic neurotransmitter in many systems including smooth muscle. In some smooth muscles (i.e., the human urinary bladder detrusor muscle), ATP does appear to be primarily released from nerves upon action potential firings, but in other smooth muscles (i.e., the human large intestine), ATP does not mimic the endogenous purine neurotransmitter. It was recently found that NAD(+) , another ubiquitous intracellular adenine nucleotide, also follows a regulated release in neurosecretory cells, vascular and visceral smooth muscles, and the brain. In some cases, NAD(+) fulfills presynaptic and postsynaptic criteria for a neurotransmitter better than ATP. Therefore, the purine hypothesis of neural regulation in smooth muscle is in need of reevaluation. This article will briefly review the current understanding of neuronal and extraneuronal release of purines in smooth muscle with emphasis on the roles of extracellular ATP and NAD(+) and, further, will discuss more recent information about the likely involvement of multiple purines in smooth muscle neurotransmission.

Adenosine 5-diphosphate-ribose is a neural regulator in primate and murine large intestine along with ß-NAD(+).

Adenosine 5'-triphosphate (ATP) has long been considered to be the purine inhibitory neurotransmitter in gastrointestinal (GI) muscles, but recent studies indicate that another purine nucleotide, ß-nicotinamide adenine dinucleotide (ß-NAD(+)), meets pre- and postsynaptic criteria for a neurotransmitter better than ATP in primate and murine colons. Using a small-volume superfusion assay and HPLC with fluorescence detection and intracellular microelectrode techniques we compared ß-NAD(+) and ATP metabolism and postjunctional effects of the primary extracellular metabolites of ß-NAD(+) and ATP, namely ADP-ribose (ADPR) and ADP in colonic muscles from cynomolgus monkeys and wild-type (CD38(+/+)) and CD38(−/−) mice. ADPR and ADP caused membrane hyperpolarization that, like nerve-evoked inhibitory junctional potentials (IJPs), were inhibited by apamin. IJPs and hyperpolarization responses to ADPR, but not ADP, were inhibited by the P2Y1 receptor antagonist (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 (MRS2500). Degradation of ß-NAD(+) and ADPR was greater per unit mass in muscles containing only nerve processes than in muscles also containing myenteric ganglia. Thus, mechanisms for generation of ADPR from ß-NAD(+) and for termination of the action of ADPR are likely to be present near sites of neurotransmitter release. Degradation of ß-NAD(+) to ADPR and other metabolites appears to be mediated by pathways besides CD38, the main NAD-glycohydrolase in mammals. Degradation of ß-NAD(+) and ATP were equal in colon. ADPR like its precursor, ß-NAD(+), mimicked the effects of the endogenous purine neurotransmitter in primate and murine colons. Taken together, our observations support a novel hypothesis in which multiple purines contribute to enteric inhibitory regulation of gastrointestinal motility.

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

Recent evidence supports an emerging role of ß-nicotinamide adenine dinucleotide (ß-NAD(+) ) as a novel neurotransmitter and neuromodulator in the peripheral nervous system -ß-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 ß-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 ß-NAD(+) is attenuated by cleavage of SNAP-25 with botulinum neurotoxin A, by inhibition of N-type voltage-dependent Ca(2+) channels with ω-conotoxin GVIA, and by inhibition of the proton gradient of synaptic vesicles with bafilomycin A1, suggesting that ß-NAD(+) is likely released via vesicle exocytosis. Western analysis demonstrates that CD38, a multifunctional protein that metabolizes ß-NAD(+) , is present on synaptosomal membranes and in the cytosol. Intact synaptosomes degrade ß-NAD(+) . 1,N (6) -etheno-NAD, a fluorescent analog of ß-NAD(+) , is taken by synaptosomes and this uptake is attenuated by authentic ß-NAD(+) , but not by the connexin 43 inhibitor Gap 27. In cortical neurons local applications of ß-NAD(+) cause rapid Ca(2+) transients, likely due to influx of extracellular Ca(2+) . Therefore, rat brain synaptosomes can actively release, degrade and uptake ß-NAD(+) , and ß-NAD(+) can stimulate postsynaptic neurons, all criteria needed for a substance to be considered a candidate neurotransmitter in the brain.

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 Ca(2+) 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 Ca(2+) in neuronal cells.