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 Kit^W/Kit^W-v ( W/W^v) 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/W^v, Nos1^−/−, and Prkg1^−/− mice displayed augmented neural release of purines that was likely due to altered nitrergic neuromodulation. Colons from W/W^v mice demonstrated decreased nitrergic and increased purinergic relaxations in response to nerve stimulation. W/W^v 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 Kit^W/Kit^W-v ( W/W^v) 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.

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,N⁶-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.

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.

The urinary bladder mucosa (urothelium and suburothelium/lamina propria) functions as a barrier between the content of the urine and the underlying bladder tissue. The bladder mucosa is also a mechanosensitive tissue that releases signaling molecules that affect functions of cells in the bladder wall interconnecting the mucosa with the detrusor muscle and the CNS. Adenosine 5ʹ-triphosphate (ATP) is a primary mechanotransduction signal that is released from cells in the bladder mucosa in response to bladder wall distention and activates cell membrane-localized P2X and P2Y purine receptors on urothelial cells, sensory and efferent neurons, interstitial cells, and detrusor smooth muscle cells. The amounts of ATP at active receptor sites depend significantly on the amounts of extracellularly released ATP. Spontaneous and distention-induced release of ATP appear to be under differential control. This review is focused on mechanisms underlying urothelial release of ATP in response to mechanical stimulation. First, we present a brief overview of studies that report mechanosensitive ATP release in bladder cells or tissues. Then, we discuss experimental evidence for mechanosensitive release of urothelial ATP by vesicular and non-vesicular mechanisms and roles of the stretch-activated channels PIEZO channels, transient receptor potential vanilloid type 4, and pannexin 1. This is followed by brief discussion of possible involvement of calcium homeostasis modulator 1, acid-sensing channels, and connexins in the release of urothelial ATP. We conclude with brief discussion of limitations of current research and of needs for further studies to increase our understanding of mechanotransduction in the bladder wall and of purinergic regulation of bladder function.