Increased Graft Survival through Ectonucleotidases Modulation in Platelets and Lymphocytes of Kidney Transplanted Patients

Background: despite limited long-term survival, kidney transplantation is the best form of renal replacement therapy for terminal disease patients. Components of extracellular purinergic signaling plays a fundamental role on inflammation and immune response related to organ transplantation. They could be alternative targets to avoid graft rejection. Materials and Methods: The hydrolysis of ATP, ADP and AMP nucleotides was analyzed in both lymphocytes and platelets, as well as the quantification of ATP and ADA activity. A sample of 30 patients who underwent kidney transplants was obtained, of which 15 had a transplant time of less than one year (acute response) and 15 had a transplant time between one and three years (chronic response). Results: In the group with transplantation time between one and three years, it was possible to identify a significant decrease in the amount of ATP, increase in ATP hydrolysis in platelets, decrease in AMP hydrolysis and increase in ADA activity, also in platelets. In the lymphocyte sample, there was a significant reduction in ADA activity as well as a decrease in the amount of ATP. Conclusions: From the data obtained in the study, it can be inferred that adenosine can reduce pro-inflammatory cytokines, providing greater graft survival and reducing the intensity of graft-versus-host disease. ATP signaling exerts inflammatory effects and modulates the purinergic signaling cascade, offering new avenues for drug therapies to combat chronic graft rejection.

The ATP-hydrolyzing ectoenzyme E-NTPD8 attenuates colitis through modulation of P2X4 receptor–dependent metabolism in myeloid cells

Extracellular adenosine triphosphate (ATP) released by mucosal immune cells and by microbiota in the intestinal lumen elicits diverse immune responses that mediate the intestinal homeostasis via P2 purinergic receptors, while overactivation of ATP signaling leads to mucosal immune system disruption, which leads to pathogenesis of intestinal inflammation. In the small intestine, hydrolysis of luminal ATP by ectonucleoside triphosphate diphosphohydrolase (E-NTPD)7 in epithelial cells is essential for control of the number of T helper 17 (Th17) cells. However, the molecular mechanism by which microbiota-derived ATP in the colon is regulated remains poorly understood. Here, we show that E-NTPD8 is highly expressed in large-intestinal epithelial cells and hydrolyzes microbiota-derived luminal ATP. Compared with wild-type mice, Entpd8−/− mice develop more severe dextran sodium sulfate–induced colitis, which can be ameliorated by either the depletion of neutrophils and monocytes by injecting with anti–Gr-1 antibody or the introduction of P2rx4 deficiency into hematopoietic cells. An increased level of luminal ATP in the colon of Entpd8−/− mice promotes glycolysis in neutrophils through P2x4 receptor–dependent Ca2+ influx, which is linked to prolonged survival and elevated reactive oxygen species production in these cells. Thus, E-NTPD8 limits intestinal inflammation by controlling metabolic alteration toward glycolysis via the P2X4 receptor in myeloid cells.

Panx1 channels promote both anti- and pro-seizure-like activities in the zebrafish via p2rx7 receptors and ATP signaling

The molecular mechanisms of excitation-inhibition imbalances promoting seizure generation in epilepsy patients are not fully understood. Experimental evidence suggests that Pannexin1 (Panx1), an ATP release channel, modulates excitability of the brain. In this report, we have performed behavioral and molecular phenotyping experiments on zebrafish larvae bearing genetic or pharmacological knockouts of panx1a or panx1b channels, each highly homologous to human PANX1. When Panx1a function is lost or both channels are under pharmacological blockage, treatment with pentylenetetrazol to induce seizures causes reduced ictal-like events and seizure-like locomotion. These observations were extended by transcriptome profiling, where a spectrum of distinct metabolic and cell signaling states correlates with the loss of panx1a. The pro- and anticonvulsant activities of both Panx1 channels affects ATP release and involves the purinergic receptor p2rx7. We propose that Panx1 zebrafish models offer opportunities to explore the molecular and physiological basis of seizures and assist anticonvulsant drug discovery.

ATP signaling in the integrative neural center of Aplysia californica

ATP and its ionotropic P2X receptors are components of the most ancient signaling system. However, little is known about the distribution and function of purinergic transmission in invertebrates. Here, we cloned, expressed, and pharmacologically characterized the P2X receptors in the sea slug Aplysia californica—a prominent neuroscience model. AcP2X receptors were successfully expressed in Xenopus oocytes and displayed activation by ATP with two-phased kinetics and Na+-dependence. Pharmacologically, they were different from other P2X receptors. The ATP analog, Bz-ATP, was a less effective agonist than ATP, and PPADS was a more potent inhibitor of the AcP2X receptors than the suramin. AcP2X were uniquely expressed within the cerebral F-cluster, the multifunctional integrative neurosecretory center. AcP2X receptors were also detected in the chemosensory structures and the early cleavage stages. Therefore, in molluscs, rapid ATP-dependent signaling can be implicated both in development and diverse homeostatic functions. Furthermore, this study illuminates novel cellular and systemic features of P2X-type ligand-gated ion channels for deciphering the evolution of neurotransmitters.

Urothelium-Specific Deletion of Connexin43 in the Mouse Urinary Bladder Alters Distension-Induced ATP Release and Voiding Behavior

Connexin43 (Cx43), the main gap junction and hemichannel forming protein in the urinary bladder, participates in the regulation of bladder motor and sensory functions and has been reported as an important modulator of day–night variations in functional bladder capacity. However, because Cx43 is expressed throughout the bladder, the actual role played by the detrusor and the urothelial Cx43 is still unknown. For this purpose, we generated urothelium-specific Cx43 knockout (uCx43KO) mice using Cre-LoxP system. We evaluated the day–night micturition pattern and the urothelial Cx43 hemichannel function of the uCx43KO mice by measuring luminal ATP release after bladder distention. In wild-type (WT) mice, distention-induced ATP release was elevated, and functional bladder capacity was decreased in the animals’ active phase (nighttime) when Cx43 expression was also high compared to levels measured in the sleep phase (daytime). These day–night differences in urothelial ATP release and functional bladder capacity were attenuated in uCx43KO mice that, in the active phase, displayed lower ATP release and higher functional bladder capacity than WT mice. These findings indicate that urothelial Cx43 mediated ATP signaling and coordination of urothelial activity are essential for proper perception and regulation of responses to bladder distension in the animals’ awake, active phase.

ATP Signaling in the Integrative Neural Center of Aplysia Californica

ATP and its ionotropic P2X receptors are components of the most ancient signaling systems. However, little is known about the distribution and function of purinergic transmission in invertebrates. Here, we cloned, expressed, and pharmacologically characterized P2X receptors in the sea slug Aplysia californica – the prominent neuroscience model. acP2X receptors were successfully expressed in Xenopus oocytes and were displayed activation by ATP with two-phased kinetics and Na+-dependence. Pharmacologically, they were quite different from other P2X receptors. The ATP analog, Bz-ATP, was a less effective agonist than ATP, and PPADS was a more potent inhibitor of the acP2X receptors than the suramin. acP2X were uniquely expressed within the cerebral F-cluster, which contains multiple secretory peptides (e.g., insulins, interleukins, and potential toxins), ecdysone-type receptors, and a district subset of ion channels. We view F-cluster as the multifunctional integrative center, remarkably different from other neurosecretory cells. acP2X receptors were also found in the chemosensory structures and the early cleavage stages. Therefore, in molluscs, rapid ATP-dependent signaling can be implicated both in development and diverse homeostatic functions. Furthermore, this study illuminates novel cellular and systemic features of P2X-type ligand-gated ion channels for deciphering evolution of neurotransmitters.