Spinal ERK activation contributes to the regulation of bladder function in spinal cord injured rats

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Abstract

The extracellular signal-regulated kinase 1 and 2 (ERK) pathway, regulated by phosphorylation on specific amino acids, is emerging as an important signaling cascade in neurones, transducing sensory input into cellular responses. In the mammalian nervous system, the ERK pathway has been found to mediate plasticity events. Particularly, in the spinal cord, ERK play an important role in allodynia and hyperalgesia. Recently, it was demonstrated that ERK activation is upregulated in the spinal cord of rats with chronic bladder inflammation and contributes to bladder overactivity. Thus, in this study we sought to assess the involvement of ERK in micturition reflexes associated to spinal cord injury (SCI) in the rat.

Bladder function in chronic SCI rats was altered compared to spinal intact rats. PhosphoERK levels were upregulated in the L6 spinal cord segment, particularly after saline infusion for 2 h. The increase in spinal ERK phosphorylation was specifically restricted to L6 spinal segment. No variation in the levels of total ERK protein was observed. Intrathecal administration of PD98059, a specific inhibitor of ERK phosphorylation, reduced the frequency and amplitude of bladder contractions in SCI animals but not in spinal intact ones.

Overall, our results demonstrate increased activation of the ERK pathway in the spinal cord from SCI rats, restricted to spinal segments that receive sensory input arising from the bladder. Since the use of PD98059 reduced the frequency and amplitude of bladder contractions, ERK inhibitors may provide a new therapeutic approach to the treatment of bladder overactivity after spinal injuries.

Introduction

The activation of extracellular signal-regulated kinases 1 and 2 (ERK) is known to lead to a variety of functional changes in both neuronal and non-neuronal cells. These plastic changes include alterations in gene expression and post-translational modulation of membrane receptors and channels (Ji, 2004). In the spinal cord, ERK phosphorylation is regulated by the synaptic actions of neurotrophins and glutamate on neurones. Upon binding to specific Trk receptors, neurotrophins promote the sequential activation of the Ras/Raf/MEK pathway which, ultimately, will cause the phosphorylation of ERK on specific threonine and tyrosine residues (Grewal et al., 1999). On the other hand, glutamate, upon binding to ionotropic and metabotropic receptors, activates ERK by increasing intracellular calcium levels (via ionotropic receptors) or by activating the small GTPase Ras (via metabotropic receptors) (Kawasaki et al., 2004, Lever et al., 2003).

Recently, we showed that, at the lumbosacral spinal cord level, ERK phosphorylation in neurones receiving sensory input from the urinary bladder occurs swiftly upon bladder distension. In addition, ERK phosphorylation was also shown to contribute to inflammatory allodynia and hyperalgesia and plays a role in the regulation of bladder reflex activity in animals with chronic cystitis (Cruz et al., 2005). In that study, we observed that PD98059, a specific inhibitor of ERK phosphorylation, decreased the inflammation-induced bladder overactivity.

Another important cause of bladder overactivity is spinal cord injury (SCI) above lumbosacral segments which induces important changes in micturition control (de Groat, 1997). In this case, bladder contractions necessary for bladder emptying are no longer dependent on suprasacral regulation but are instead controlled by an involuntary sacral micturition reflex usually inactive in the adult life. The enhancement of the sacral reflex involves complex and poorly understood reorganization of the neuronal circuitry at the lumbosacral spinal cord level. However, it is accepted that neurotrophins might play a role in such reorganization. In fact, immunoneutralization of spinal nerve growth factor (NGF) with specific antibodies reduces bladder overactivity (Seki et al., 2002, Seki et al., 2004) in chronic SCI animals. Furthermore, glutamate also contributes to bladder dysfunction caused by SCI as the use of specific antagonists of AMPA receptors reduced bladder overactivity (Nishizawa et al., 1999, Yoshiyama et al., 1999). However, it is unknown if neurotrophin- and glutamate-dependent increased bladder reflex activity after SCI involves ERK activation in spinal cord neurones.

In this study, we evaluated the levels of phosphorylated and total ERK proteins in the spinal cord of animals submitted to chronic SCI. Furthermore, we also tested the effect of the intrathecal administration of a specific inhibitor of ERK phosphorylation to assess the involvement of this signaling cascade in bladder overactivity that follows SCI.

Section snippets

Experimental animals

Adult female Wistar rats (220–250 g) from the IBMC colony (Porto, Portugal) were used. All procedures were performed according to the European Communities Council Directive (86/609/EEC) and to the ethical guidelines for investigation of experimental pain in animals (Zimmermann, 1983).

Antibodies and reagents

The antibodies against the phosphorylated isoforms of ERK 1 and 2 (phosphoERK) and total ERK, made in rabbit, came from Cell Signalling, UK. Biotin-conjugated swine anti-rabbit was bought from Dakopatts A/5,

Bladder reflex activity after SCI

Voiding was totally abolished in SCI animals for 8–10 days after surgery, making the manual expression of the bladder necessary until reflex voidings occurred. At 7 weeks after SCI, cystometries showed that the number of bladder contractions per min was 1.01 ± 0.27, a value significantly higher than that of control animals (0.52 ± 0.84; P < 0.01). The mean peak pressure reached by bladder contractions was significantly higher in SCI animals (44.33 ± 4.04 cm H2O) than in intact animals

Discussion

In this study, we found that spinal cord transection at the thoracic level enhanced ERK phosphorylation in L6 spinal cord segment. Such enhancement was particularly intense following bladder stimulation, but it was also evident in sham manipulated and in non-stimulated SCI animals. IR-cells were found bilaterally in laminae I–II, DCM and in ILG. The presence of strong immunoreactivity in L6 spinal cord segment, but not in L4, suggested that bladder sensory input was the driving stimulus for ERK

Acknowledgments

Support by FCT project POCI/SAU-NEU/55983/2004, Portugal. C.D.C. is a student of the Gulbenkian PhD Program in Biomedicine, Portugal and received a FCT scholarship (SFRH/BD/5826/2001). The authors wish to acknowledge the helpful support of Dr. Jorge Ferreira.

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