Hsien Yu Peng

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/30 s) 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.

Neuroactive steroids inhibit spinal reflex potentiation by selectively enhancing specific spinal GABAA receptor subtypes

ABSTRACT Recently, we demonstrated a spinal GABAA receptor (GABAAR)‐dependent inhibition on the induction of repetitive stimulation‐induced spinal reflex potentiation. However, it remains unclear whether steroid hormones modulate such an inhibition. Here, we show that progesterone is capable of producing GABAARs‐dependent inhibition of the induction of spinal reflex potentiation by actions through neurosteroid metabolites. Progesterone (5 mg/kg, twice daily for 4 days) up‐regulates the expression of GABAAR α2, α3, α4 and δ subunits, and is associated with attenuated repetitive stimulation‐induced spinal reflex activity in ovariectomized rats. These changes were blocked by finasteride (50 mg/kg, twice daily), an antagonist of neurosteroid synthesis from progesterone, but not by the progesterone receptor antagonist, RU486 (100 mg/kg, twice daily). The induction of spinal reflex potentiation was attenuated after a short (30 min) intrathecal treatment with the neurosteroids, allopregnanolone (ALLOP, 10 μM, 10 μL) and 3α,5α‐tetrahydrodeoxycorticosterone (THDOC, 10 μM, 10 μL). Acute intrathecal administration of the GABAAR antagonist, bicuculline (10 μM, 10 μL) reversed the inhibition produced by progesterone, THDOC and allopregnanolone. These results imply that progesterone‐mediated effects on GABAAR expression and neural inhibition are regulated by neurosteroids synthesis rather than progesterone receptor activation.

TRPV1 mediates the uterine capsaicin-induced NMDA NR2B-dependent cross-organ reflex sensitization in anesthetized rats

Spinal cord-mediated cross-organ sensitization between the uterus and the lower urinary tract may underlie the high concurrence of obstetrical/gynecological inflammation and chronic pelvic pain syndrome characterized by urogenital pain. However, the neural pathway and the neurotransmitters involved are still unknown. We tested the hypothesis that the excitation of capsaicin-sensitive primary afferent fibers arising from the uterus through the stimulation of transient receptor potential vanilloid 1 (TRPV1) induces cross-organ sensitization on the pelvic-urethra reflex activity. Capsaicin (1-1,000 microM, 0.05 ml) was instilled into the uterus to induce cross-organ reflex sensitization. Activation of capsaicin-sensitive primary afferent fibers by capsaicin instillation into the uterine horn sensitized the pelvic-urethra reflex activity that was reversed by an intrauterine pretreatment with capsaizepine, a TRPV1-selective antagonist. Intrathecal injection of AP5, a glutamatergic N-methyl-D-aspartate (NMDA) antagonist, and Co-101244, an NMDA NR2B-selective antagonist, both abolished the cross-organ reflex sensitization caused by capsaicin instillation. These results demonstrated that TRPV1 plays a crucial role in contributing to the capsaicin-sensitive primary afferent fibers mediating the glutamatergic NMDA-dependent cross-organ sensitization between the uterus and the lower urinary tract when there is a tissue injury.

Estrogen-dependent facilitation on spinal reflex potentiation involves the Cdk5/ERK1/2/NR2B cascade in anesthetized rats

Cyclin-dependent kinase-5 (Cdk5), a proline-directed serine/threonine kinase, may alter pain-related neuronal plasticity by regulating extracellular signal-related kinase-1/2 (ERK1/2) activation. This study investigated whether Cdk5-dependent ERK activation underlies the estrogen-elicited facilitation on the repetitive stimulation-induced spinal reflex potentiaton (SRP) that is presumed to be involved in postinflammatory/neuropathic hyperalgesia and allodynia. Reflex activity of the external urethra sphincter electromyogram evoked by pelvic afferent nerve test stimulation (TS; 1 stimulation/30 s for 10 min) and repetitive stimulation (RS; 1 stimulation/1 s for 10 min) was recorded in anesthetized rats. TS evoked a baseline reflex activity, whereas RS produced SRP. Intrathecal (it) beta-estradiol facilitated the repetitive stimulation-induced SRP that was reversed by pretreatment with the estrogen receptor anatogonist ICI 182,780 (10 nM, 10 microl it), Cdk5 inhibitor roscovitine (100 nM, 10 microl it), ERK inhibitor (U-0126; 100 microM, 10 microl it) and N-methyl-D-aspartate (NMDA) NR2B subunit antagonist (Co-101244; 100 nM, 10 microl it). Moreover, ERalpha (propylpyrazoletriol; 100 nM, 10 microl it) and ERbeta (diarylpropionitrile; 100 microM, 10 microl it) agonists both facilitated the SRP, similar to results with a beta-estradiol injection. In association with the facilitated RS-induced SRP, an intrathecal beta-estradiol injection elicited ERK1/2 and NR2B subunit phosphorylation that were both reversed by intrathecal roscovitine and U-0126. These results indicated that the Cdk/ERK cascade, which is activated by ERalpha and ERbeta, may subsequently phosphorylate the NR2B subunit to develop NMDA-dependent postinflammatory hyperalgesia and allodynia to maintain the protective mechanisms of the body.

Orexin-A modulates glutamatergic NMDA-dependent spinal reflex potentiation via inhibition of NR2B subunit

Glucose-sensitive neurons in the lateral hypothalamic area produce orexin-A (OxA) as well as orexin-B (OxB) and send their axons to the spinal dorsal horn, which predominantly expresses orexin receptor-1 (OX-1), showing a higher sensitivity to OxA. The purpose of the present study was to assess the effects of OxA on the induction of a novel form of activity-dependent reflex potentiation, spinal reflex potentiation (SRP), in the pelvic-urethral reflex activity. External urethra sphincter electromyogram in response to pelvic afferent nerve test stimulation (TS; 1/30 Hz) or repetitive stimulation (RS; 1 Hz) was recorded in anesthetized rats. TS evoked a baseline reflex activity, whereas RS produced SRP, which was abolished by intrathecal OxA (30 nM, 10 mul). Intrathecal SB-408124 (10 muM, 10 mul), an OX-1 antagonist, reversed the abolition on SRP caused by OxA. Although there is, so far, no NR2A- and NR2B-specific agonist available, N-methyl-d-aspartate (NMDA) reversed the abolition on the RS-induced SRP caused by the co-administration of OxA and Co-101244 (30 nM, 10 mul; an NMDA NR2B subunit antagonist), but it did not reverse the abolition by the co-administration of OxA and PPPA (300 nM, 10 mul; an NMDA NR2A subunit antagonist). In conclusion, the activation of descending orexinergic fibers may inhibit the repetitive afferent input-induced central sensitization of pelvic-urethral reflex activity and urethra hyperactivity, indicating that spinal orexinergic neural transmission may be a novel target for the treatment of patients with neuropathetic or postinflammatory pain of pelvic origin.

Spinal SIRPα1-SHP2 interaction regulates spinal nerve ligation-induced neuropathic pain via PSD-95-dependent NR2B activation in rats

Summary Spinal SIRPα1‐SHP2 interaction, which subsequently triggers SHP2/PSD‐95/NR2B cascade, plays a pivotal role in neuropathic pain development caused by L5 spinal nerve ligation in rats. ABSTRACT The fact that neuropathic pain mechanisms are not well understood is a major impediment in the development of effective clinical treatments. We examined whether the interaction between signal regulatory protein alpha 1 (SIRPα1) and Src homology‐2 domain‐containing protein tyrosine phosphatase 2 (SHP2), and the downstream spinal SHP2/postsynaptic density 95 (PSD‐95)/N‐methyl‐D‐aspartate receptor NR2B subunit signaling cascade play a role in neuropathic pain. Following spinal nerve ligation (L5), we assessed tactile allodynia using the von Frey filament test and analyzed dorsal horn samples (L4‐5) by Western blotting, reverse transcription polymerase chain reaction, coimmunoprecipitation, and immunofluorescence. Nerve ligation induced allodynia, SIRPα1, SHP2, phosphorylated SHP2 (pSHP2), and phosphorylated NR2B (pNR2B) expression, and SHP2‐PSD‐95, pSHP2‐PSD‐95, PSD‐95‐NR2B, and PSD‐95‐pNR2B coimmunoprecipitation in the ipsilateral dorsal horn. In allodynic rats, injury‐induced SHP2 immunoreactivity was localized in the ipsilateral dorsal horn neurons and coincident with PSD‐95 and NR2B immunoreactivity. SIRPα1 silencing using small interfering RNA (siRNA; 1, 3, or 5 μg/rat for 7 days) prevented injury‐induced allodynia and the associated changes in protein expression, phosphorylation, and coimmunoprecipitation. Intrathecal administration of NSC‐87877 (an SHP2 antagonist; 1, 10, or 100 μM/rat) and SIRPα1‐neutralizing antibodies (1, 10, or 30 μg/rat) suppressed spinal nerve ligation‐induced allodynia, spinal SHP2 and NR2B phosphorylation, and SHP2/phosphorylated SHP2‐PSD‐95 and PSD‐95‐NR2B/phosphorylated NR2B coprecipitation. SHP2 siRNA led to similar effects as the NSC‐87877 and SIRPα1 antibody treatments, except it prevented the allodynia‐associated spinal SHP2 expression. In conclusion, our results suggest that a spinal SIRPα1‐SHP2 interaction exists that subsequently triggers SHP2/PSD‐95/NR2B signaling, thereby playing a role in neuropathic pain development.

Spinal SGK1/GRASP-1/Rab4 is involved in complete Freund’s adjuvant-induced inflammatory pain via regulating dorsal horn GluR1-containing AMPA receptor trafficking in rats

TOC summary SGK1/GRASP‐1/Rab4 participated in complete Freund’s adjuvant‐induced inflammatory pain via regulating GluR1‐containing AMPAR recycling at the dorsal horn neurons. ABSTRACT The elusiveness of the mechanism underlying pain is a major impediment in developing effective clinical treatments. We examined whether the phosphorylation of spinal serum‐ and glucocorticoid‐inducible kinase 1 (SGK1) and downstream glutamate receptor interacting protein (GRIP)‐associated protein‐1 (GRASP‐1)/Rab4‐dependent GluR1‐containing α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor (AMPAR) recycling play a role in inflammatory pain. After intraplantar injection of complete Freund’s adjuvant (CFA), we assessed thermal hyperalgesia using the Hargreaves test and analyzed dorsal horn samples (L4‐5) using Western blotting, coprecipitation, and immunofluorescence. CFA administration provoked behavioral hyperalgesia along with SGK1 phosphorylation, GluR1 trafficking from the cytosol to the membrane, and phosphorylated SGK1 (pSGK1)‐GRASP‐1, GRASP‐1‐Rab4, and Rab4‐GluR1 coprecipitation in the ipsilateral dorsal horn. In the dorsal horns of hyperalgesic rats, CFA‐enhanced pSGK1 was demonstrated to be colocalized with NeuN, GRASP‐1, Rab4, and GluR1 by immunofluorescence. GSK‐650394 (an SGK1 activation antagonist, 1, 10, and 30 μM, 10 μL/rat, intrathecally) dose‐dependently prevented CFA‐induced pain behavior and the associated SGK1 phosphorylation, GluR1 trafficking, and protein‐protein interactions at 1 day after CFA administration. Intrathecal 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX, an AMPAR antagonist, 1, 3, and 10 μM, 10 μL/rat) attenuated the hyperalgesia and GluR1 trafficking caused by CFA; however, it had no effect on SGK1 phosphorylation. Small interfering RNA targeting Rab4 hindered the CFA‐induced hyperalgesia and the associated GluR1 trafficking and Rab4‐GluR1 coprecipitation. Our results suggest that spinal SGK1 phosphorylation, which subsequently triggers the GRASP‐1/Rab4 cascade, plays a pivotal role in CFA‐induced inflammatory pain by regulating GluR1‐containing AMPAR recycling in the dorsal horn.

PI3K modulates estrogen-dependent facilitation of colon-to-urethra cross-organ reflex sensitization in ovariectomized female rats

J. Neurochem. (2010) 10.1111/j.1471‐4159.2010.06577.x

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

Recently, the role of EphB receptor (EphBR) tyrosine kinase and their ephrinB ligands in spinal pain-related neural plasticity has been identified. To test whether Src-family non-receptor tyrosine kinase-dependent glutamatergic N-methyl-d-aspartate receptor (NMDAR) NR2B subunit phosphorylation underlies lumbosacral spinal EphBR activation to mediate cross-organ sensitization between the colon and the urethra, external urethra sphincter electromyogram activity evoked by pelvic nerve stimulation and protein expression in the lumbosacral (L6-S2) dorsal horn were studied before and after intracolonic mustard oil (MO) instillation. We found MO instillation produced colon-urethra reflex sensitization along with an upregulation of endogenous ephrinB2 expression as well as phosphorylation of EphB 1/2, Src-family kinase, and NR2B tyrosine residues. Intrathecal immunoglobulin fusion protein of EphB1 and EphB2 as well as PP2 reversed the reflex sensitization and NR2B phosphorylation caused by MO. All these results suggest that EphBR-ephrinB interactions, which provoke Src-family kinase-dependent NMDAR NR2B phosphorylation at the lumbosacral spinal cord level, are involved in cross-organ sensitization, contributing to the development of viscero-visceral referred pain between the bowel and the urethra.

Glutamate-mediated spinal reflex potentiation involves ERK 1/2 phosphorylation in anesthetized rats

The extracellular signal-regulated kinase (ERK) cascades are suggested to contribute to excitatory plasticity in the CNS, including the spinal cord. This study investigated whether the ERK involves in the repetitive stimulation-induced spinal reflex potentiation (SRP) in the pelvic nerve-to-external urethra sphincter reflex activities. External urethra sphincter electromyogram in response to pelvic afferent nerve test stimulation (TS, 1/30 Hz) or repetitive stimulation (RS, 1 Hz) was recorded in anesthetized rats. TS evoked a baseline reflex activity, whereas RS produced SRP in associated with significant ERK 1/2 phosphorylation. RS-induced SRP and ERK 1/2 phosphorylation were both abolished by pretreatment of U0126 (MEK inhibitor). Intrathecal CNQX (AMPA receptor antagonist) attenuated, while AP5 (NMDA receptor antagonist) abolished the RS-induced SRP and ERK 1/2 phosphorylation. Pretreated U0126 abolished the SRP elicited by glutamatergic agonists including glutamate, NMDA and AMPA. Intrathecal H89 and BIS7 (PKA and PKC inhibitors, respectively) both abolished the RS- and glutamate agonist-induced SRP as well as ERK 1/2 phosphorylation. In addition, forskolin and PMA (PKA and PKC activator, respectively) induced SRP, which were both abolished by pretreated U0126. Saline distension, mimicking the storage phase of the urinary bladder, induced SRP and ERK 1/2 phosphorylation. In conclusion, activated ERK 1/2 may produce SRP in the pelvic nerve-to-external urethra sphincter reflex activity, which is essential for urine continence. In addition, blockage of spinal ERK 1/2 activation decreases the physiological function of the urethra, indicating that phosphorylation of the ERK 1/2 cascade may represent a novel target for the treatment of patients with neurological incontinence of spinal origin.