Botulinum toxin A inhibits ATP release from bladder urothelium after chronic spinal cord injury

Abstract

The effects of mechanoreceptor stimulation and subsequent ATP release in spinal cord injured and normal bladders was examined to demonstrate if spinal cord injury (SCI) modulates the basal or evoked release of ATP from bladder urothelium and whether intravesical botulinum toxin A (BTX-A) administration inhibits urothelial ATP release, a measure of sensory nerve activation. A Ussing chamber was used to isolate and separately measure resting and mechanoreceptor evoked (e.g. hypoosmotic stimulation) ATP release from urothelial and serosal sides of the bladder. Following spinal cord injury, resting urothelial release of ATP was ninefold higher than that of normal rats. Botulinum toxin A instillation did not significantly affect the resting release of ATP after spinal cord injury. Evoked ATP release following hypoosmotic stimulation was significantly higher in chronic spinal cord injured compared to normal rat bladders. However, botulinum toxin A treatment markedly reduced ATP release in spinal cord injured bladders by 53% suggesting that ATP release by mechanoreceptor stimulation, as opposed to basal release, occurs by exocytotic mechanisms. In contrast, there was no significant difference in basal or evoked ATP release from bladder serosa following spinal cord injury. Moreover, intravesical instillation of botulinum toxin A did not affect ATP release from the serosal side after spinal cord injury, suggesting that its effects were confined to the urothelial side of the bladder preparation. In summary: (1) increased release of ATP from the urothelium of spinal cord injured bladders may contribute to the development of bladder hyperactivity and, (2) mechanoreceptor stimulated vesicular ATP release, as opposed to basal non-vesicular release of ATP, is significantly inhibited in spinal cord injured bladders by intravesical instillation of botulinum toxin A. These results may have important relevance in our understanding of the mechanisms underlying plasticity of bladder afferent pathways following SCI.

Introduction

Spinal cord injury (SCI) induces significant plasticity within neural pathways innervating the lower urinary tract that leads to functional changes such as increased bladder activity (e.g. detrusor hyperreflexia) and bladder outlet obstruction (e.g. detrusor sphincter dyssynergia). Underlying these changes in efferent outflow are significant increases in bladder afferent activity that are secondary, in large part, to the unmasking of normally quiescent C-fiber nociceptors (De Groat et al., 1990). Following pathologic injury such as SCI or bladder inflammation, C-fiber afferent nerves respond to physiologic (pressure and volume) changes in addition to noxious stimuli. However, increased afferent nerve activity results not only from the recruitment of normally silent C-fiber nerves, but also because responding nerves become more excitable (Yoshimura and de Groat, 1997). Maggi has presented evidence that bladder sensory nerves have dual afferent and efferent nerve functions (Maggi, 1993). Sensory efferent functions include the release of transmitters such as ATP, substance P and CGRP that can act on nearby tissues as well as on afferent nerve terminals in an autocrine fashion to increase afferent nerve activity.

In addition to the role that afferent nerve transmitter release plays in sensory function, significant recent interest has been directed to the existence and function of non-neuronal sources of transmitters. For example, it has been shown that acetylcholine (Reinheimer et al., 1996, Klapproth et al., 1997) as well as ATP (Ferguson et al., 1997, van der Wijk et al., 2003) can be released from the epithelium of bronchi, intestine and urinary bladder. Investigators postulate that these non-neuronal sources of neurotransmitters have important paracrine functions on surrounding tissues to modulate transmitter release and afferent nerve activity. Neuro-epithelial interactions between epithelium and afferent nerves has gained widespread interest among investigators focused on the role that sensory input plays in the development of various models of bladder dysfunction (Wessler et al., 1998).

In this regard, urothelium is thought to play an important role in sensory transduction mechanisms that modulate micturition, particularly in conditions of increased sensory nerve transmission such as following chronic inflammation and SCI. Studies have demonstrated that urothelial cells can release the neurotransmitter ATP when stimulated by stretch (Sun et al., 2001). Released ATP can activate suburothelially located P2X₃ receptors to increase sensory nerve transmission, possibly in a non-synaptic manner, a phenomenon that has been previously described regarding the interaction between efferent nerve terminals (Vizi, 1979). The importance of P2X₃ receptors in sensory nerve transduction mechanisms is underscored by the significant decrease in bladder activity found in P2X₃ knockout mice (Vlaskovska et al., 2001).

Botulinum toxin A (BTX-A) is well established as an inhibitor of vesicular acetylcholine release from motor nerve terminals that acts by cleaving the SNARE protein SNAP-25 (Schiavo et al., 1993). Our lab has demonstrated that ACh and norepinephrine (NE) release from the rat bladder and urethra, respectively, is inhibited by BTX-A (Smith et al., 2003b). Furthermore, contractile data suggests that BTX-A may impair ATP release in addition to ACh release from isolated bladder tissue (Smith et al., 2003a). Yet, while traditionally used to treat disorders of muscle spasticity, recent basic and clinical evidence suggests that BTX-A may have antinociceptive effects unrelated to its actions on efferent nerve terminals (Cui and Aoki, 2000; Vemulakonda et al., 2004a, Vemulakonda et al., 2004b; Smith and Chancellor, 2004). By impairing urothelial or afferent nerve transmitter release, particularly under conditions of chronic inflammation or SCI, BTX-A could reduce peripheral sensitization mechanisms that are thought to play an important role in increasing afferent nerve activity. In fact, experiments have shown that hypoosmotic stimulus evoked release of ATP from intestinal epithelium is inhibited by botulinum toxin (van der Wijk et al., 2003). These results suggest not only that evoked ATP release from epithelium is via vesicular mechanisms, but also demonstrate that BTX-A can inhibit transmitter release from non-neuronal tissue.

In the present experiments, we studied the effects of mechanoreceptor stimulation and subsequent ATP release in spinal cord injured and normal bladders to examine whether: (1) SCI modulates the basal or evoked release of ATP from bladder urothelium and, (2) BTX-A administration inhibits urothelial ATP release, a measure of sensory nerve activation.