Extracellular adenosine 5’-triphosphate concentrations changes in rat spinal cord associated with the activation of urinary bladder afferents. A microdialysis study

Jeová Nina Rocha,Universidade De São Paulo, Brazil

doi:10.1590/s1679-45082016ao3794

Abstract

ABSTRACTObjective To determine adenosine 5’-triphosphate levels in the interstice of spinal cord L6-S1 segment, under basal conditions or during mechanical and chemical activation of urinary bladder afferents.Methods A microdialysis probe was transversally implanted in the dorsal half of spinal cord L6-S1 segment in female rats. Microdialysate was collected at 15 minutes intervals during 135 minutes, in anesthetized animals. Adenosine 5’-triphosphate concentrations were determined with a bioluminescent assay. In one group of animals (n=7) microdialysate samples were obtained with an empty bladder during a 10-minutes bladder distension to 20 or 40cmH2O with either saline, saline with acetic acid or saline with capsaicin. In another group of animals (n=6) bladder distention was performed and the microdialysis solution contained the ectonucleotidase inhibitor ARL 67156.Results Basal extracellular adenosine triphosphate levels were 110.9±35.34fmol/15 minutes, (mean±SEM, n=13), and bladder distention was associated with a significant increase in adenosine 5’-triphosphate levels which was not observed after bladder distention with saline solution containing capsaicin (10µM). Microdialysis with solution containing ARL 67156 (1mM) was associated with significantly higher extracellular adenosine 5’-triphosphate levels and no further increase in adenosine 5’-triphosphate was observed during bladder distension.Conclusion Adenosine 5’-triphosphate was present in the interstice of L6-S1 spinal cord segments, was degraded by ectonucleotidase, and its concentration increased following the activation of bladder mechanosensitive but not of the chemosensitive afferents fibers. Adenosine 5’-triphosphate may originate either from the central endings of bladder mechanosensitive primary afferent neurons, or most likely from intrinsic spinal neurons, or glial cells and its release appears to be modulated by capsaicin activated bladder primary afferent or by adenosine 5’-triphosphate itself.

Highlights

Adenosine 5’-triphosphate (ATP) was detected in dorsal ganglia extracts more than 50 years ago.[1]. A few years later, it was observed that antidromic stimulation of primary afferent fibers caused an increase in extracellular ATP levels in tissues innervated by the stimulated afferent fibers.[2]. Since accumulated evidence have indicated that ATP, besides its established role as an intracellular energy source, can act as an extra- and intercellular messenger released by a variety of cells, including neurons in the central and peripheral nervous system.[3,4]
Electrophysiological studies on spinal cord slices showed that two purported P2 receptor selective antagonists [pyridoxalphosphate-6-azophenyl2 ́,4 ́-disulphonic acid (PPADS) and suramin] decreased, whereas the ectonucleotidase inhibitor (ARL 67156) increased the amplitude of lamina V neurons excitatory postsynaptic currents (EPSC), evoked by dorsal root stimulation.[6]. In addition, studies in vitro, with primary cultures of neonate rat dorsal horn neurons, showed that postsynaptic currents were reduced by purinergic receptor antagonists.[7]. Synaptosomes prepared from spinal dorsal horns release ATP when depolarized with K.(8) Capsaicin pre-treatment of the animals did not alter the amount of ATP released by synaptosomes
Experiments in mice lacking the expression of P2X3 receptors, as well as the use of selective P2X antagonists, suggested that peripheral P2X1 and P2X3 receptors, at the bladder level, are involved in the micturition reflex.[11,12,13,14] More recently, it has been shown that ATP is released by the urothelium to activate purinergic receptors present in the peripheral endings of bladder primary afferents.[15] recent experiments conducted in genetically engineered mice that do not express P2X2 or P2X3 receptors showed that these receptors are not essential for the normal micturition reflex.[16]

Summary

Introduction

Adenosine 5’-triphosphate (ATP) was detected in dorsal ganglia extracts more than 50 years ago.[1]. Adenosine 5’-triphosphate has been proposed to be a neurotransmitter released by the central endings of some primary afferent neurons,(5,6) most evidence that support this proposal is indirect. Since the quantity of ATP released by spinal cord dorsal horn synaptosomes was only partially reduced by dorsal rhizotomy, it was proposed that ATP originated mainly from capsaicin-insensitive primary afferents fibers (Ab e Ad), as well as from spinal cord intrinsic neurons.[8,9] In addition, by using intrathecal administration of novel and selective P2X antagonists, Kaan et al, suggested that spinal purinergic receptors may regulate afferent signals coming from the bladder.[10]. Recent experiments conducted in genetically engineered mice that do not express P2X2 or P2X3 receptors showed that these receptors are not essential for the normal micturition reflex.[16]

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Discussion

Conclusion