Colon mustard oil instillation induced cross-organ reflex sensitization on the pelvic–urethra reflex activity in rats

Abstract

We investigated the participation of cyclin-dependent kinase-5 (Cdk5)-mediated N-methyl-D-aspartate receptor (NMDAR) NR2B subunit phosphorylation in cross-organ reflex sensitization caused by colon irritation. The external urethral sphincter electromyogram (EUSE) reflex activity evoked by the pelvic afferent nerve test stimulation (TS, 1 stimulation/30s) and protein expression in the spinal cord and dorsal root ganglion tissue (T13-L2 and L6-S2 ipsilateral to the stimulation) in response to colon mustard oil (MO) instillation were tested in anesthetized rats. When compared with a baseline reflex activity with a single action potential evoked by the TS before the administration of test agents, MO instillation into the descending colon sensitized the evoked activity characterized by elongated firing in the reflex activity in association with increased protein levels of Cdk5, PSD95, and phosphorylated NR2B (pNR2B) but not of total NR2B (tNR2B) in the spinal cord tissue. Both cross-organ reflex sensitization and increments in protein expression were reversed by intra-colonic pretreatments with ruthenium red (a non-selective transient receptor potential vanilloid, TRPV, antagonist), capsaizepine (a TRPV1-selective antagonist), lidocaine (a nerve conduction blocker) as well as by the intra-thecal pretreatment with APV (a NRMDR antagonist) Co-101244 (a NR2B-selective antagonist) and roscovitine (a Cdk5 antagonist). Moreover, compared with the control group, both the increase in pNR2B and the cross-organ reflex sensitization were attenuated in the si-RNA of NR2B rats. All these results suggested that Cdk-dependent NMDAR NR2B subunit phosphorylation mediates the development of cross-organ pelvic-urethra reflex sensitization caused by acute colon irritation which could possibly underlie the high concurrence of pelvic pain syndrome with irritable bowel syndrome.

Endogenous ephrinB2 mediates colon-urethra cross-organ sensitization via Src kinase-dependent tyrosine phosphorylation of NR2B

“…The right lumbosacral (L6 -S2) dorsal horns were dissected out and were immediately snap-frozen on liquid nitrogen. The protocol for Western blotting has been described elsewhere (22,23); in brief, the sample was homogenized and the extracts were centrifuged to retain supernatants. The supernatant was separated on an acrylamide gel and transferred to a polyvinylidene difluoride membrane and then were incubated for 1 h at room temperature in either rabbit antiephrinB2 (1:2,000, Santa Cruz Biotechnology), anti-phosphorylated EphB1/2 (pEphB1/2l 1:2,000, Millipore), rabbit anti-phospho-Src antibody (1:1,000, Millipore), or rabbit anti-phosphotyr1336 or anti-phosphotyr1472-NR2B (1:1,000 Millipore).…”

“…At the lumbosacral spinal cord, our laboratory has recently demonstrated a novel form of pain-related central sensitization, cross-organ sensitization, in which instillation of mustard oil (MO) into the descending colon could sensitize the reflex activity of the urethra (21,22). Pharmacological investigations indicated spinal NR2B subunit phosphorylation is crucial in the induction of cross-organ sensitization (19,20,23).…”

EphrinB2 induces pelvic-urethra reflex potentiation via Src kinase-dependent tyrosine phosphorylation of NR2B

“…In the last decade, studies have demonstrated that the interactions between EphBRs and their ephrinB ligands modulate neural plasticity induction in the mammalian central nervous system, mainly, but not exclusively, via exhibiting effects on NMDAR (8,12). A recent study also demonstrated that, in association with thermal hyperalgesia, EphBR activation caused by immunoglobulin fusion protein of ephrinB2 (ephrinB2-Fc)-induced, Src kinase-dependent NR2B phosphorylation in the spinal cord (2), suggesting that ephrinB2-EphBR tyrosine kinase interactions could probably modulate pain signaling via spinal Src-dependent NMDAR phosphorylation (2).Although further proof is still needed for the physiological and/or pathophysiological relevance, the induction of pelvicurethra reflex potentiation (15, 16), a form of NR2B phosphorylation-dependent neural plasticity (5,25,31,41), has been linked to the development of visceral pain from pelvic organs (26,27,30,32,33). Studies have shown that the activation of nociceptive afferent fibers expressing transient receptor potential vanilloid/transient receptor potential ankyrin by the instillation of irritants into the uterus (32, 33) and the descending colon (29) facilitated pelvic-urethra reflex activity in a crossorgan manner.…”

“…Although further proof is still needed for the physiological and/or pathophysiological relevance, the induction of pelvicurethra reflex potentiation (15, 16), a form of NR2B phosphorylation-dependent neural plasticity (5,25,31,41), has been linked to the development of visceral pain from pelvic organs (26,27,30,32,33). Studies have shown that the activation of nociceptive afferent fibers expressing transient receptor potential vanilloid/transient receptor potential ankyrin by the instillation of irritants into the uterus (32, 33) and the descending colon (29) facilitated pelvic-urethra reflex activity in a crossorgan manner.…”

The effect of spinal cord injury on vagal afferents.

“…Cross-sensitization between the bladder and other pelvic viscera is a clinical concern that makes diagnosing and treating the etiology difficult (Berkley, 2005, Baranowski et al, 2008, Theoharides et al, 2008, van de Merwe et al, 2008. In humans and in animals, the organs that seem to be most often involved in pelvic cross-sensitization besides bladder and colon, with respect to spinal afferents, appear to be the pelvic urethra and the uterus (Dmitrieva et al, 2001, Giamberardino et al, 2001, Dmitrieva and Berkley, 2002, Morrison et al, 2006, Peng et al, 2008a, Peng et al, 2008b, Peng et al, 2009). …”