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To clarify the role of descending dopaminergic innervation in reflexive urethral closure, the impacts of dopaminergic D2 receptor (DR2)-selective agonists and antagonists on repetitive stimulation-induced pelvic-to-urethra spinal reflex potentiation (SRP) were tested using in vivo rat preparations. Pelvic afferent nerve test stimulation (TS; 1 pulse/30 s for 30 rain) evoked baseline reflex activity with single spikes in the external urethral sphincter electromyogram (EUSE), whereas, repetitive stimulation (RS; 1 pulse/s for 30 min) induced SRP. Intrathecal application of quinelorane dihydrochloride (Q110; 10, 30, and 100 nM, l0 [micro]l, a selective DR2 agonist) dose dependently inhibited the RS-induced SRP. Pretreatment with L135 (100 nM, 10 [micro]L it, a selective DR2 antagonist) antagonized the Q110-dependent inhibition (100 nM, 10 [micro]l it). Intrathecal AMPA (10 [micro]M, 10 [micro]l, a selective glutamatergic AMPA receptor agonist), and NMDA (10 [micro]M, 10 [micro]l, a selective glutamatergic NMDA receptor agonist) reversed the Q110-dependent inhibition. Intrathecal forskolin (100 nM, l0 [micro]l, a PKA activator) prevented the Q110-dependent inhibition that was reversed by CNQX (10 [micro]M, 10 [micro]l it, a selective glutamate AMPA receptor antagonist) and APV (10 [micro]M, 10 [micro]l it, a selective glutamate NMDA receptor antagonist). Our results suggest that DR2 activation, which inactivates intracellular PKA, may be involved in descending dopaminergic inhibition of NMDA/AMPA receptor-dependent SRP at the lumbosacral spinal cord, which is thought to be involved in reflexive urethral closure. D2 receptor; cAMP; urethra closure; Q110; L135; rat doi: 10.1152/ajprenal.00090.2010.

Am J Physiol Renal Physiol 298: F109-F117, 2010. First published October 28, 2009; doi: 10.1152/ajprenal.00287.2009.--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 crossorgan 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 EphB1/2, 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. pelvic pain syndrome; urethra; irritable bowel syndrome; colon; NMDA

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 [micro]l it), Cdk5 inhibitor roscovitine (100 nM, 10 [micro]l it), ERK inhibitor (U-0126; 100 [micro]M, 10 [micro]l it) and N-methyl-D-aspartate (NMDA) NR2B subunit antagonist (Co-101244; 100 nM, 10 [micro]l it). Moreover, ER[alpha] (propylpyrazoletriol; 100 nM, 10 [micro]l it) and ER[beta] (diarylpropionitrile; 100 [micro]M, 10 [micro]l 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 ER[alpha] and ER[beta], may subsequently phosphorylate the NR2B subunit to develop NMDA-dependent postinflammatory hyperalgesia and allodynia to maintain the protective mechanisms of the body. estradiol; cyclin-dependent kinase-5; NR2B; extracellular signal-related kinase; pelvic pain; spinal reflex potentiaton; hyperalgesia

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 (TRPVI) induces cross-organ sensitization on the pelvic-urethra reflex activity. Capsaicin (1-1,000 [micro]M, 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 [TRPV.sup.1]-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 [TRPV.sub.1] 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. spinal cord; C-fiber; pain; desensitization

The impact of acute anal stretch on the pelvic-urethra reflex potentiation was examined in urethane-anesthetized rats by recording the external urethra sphincter electromyogram activity evoked by the pelvic afferent stimulation. Test stimulation (1 stimulation/30 s) evoked a baseline reflex activity with a single action potential that was abolished by gallamine (5 mg/kg iv). On the other hand, the repetitive stimulation (1 stimulation/1 s) induced spinal reflex potentiation (SRP) that was attenuated by intrathecal 6-cyano-7-nitroquinoxaline-2,4-dione (a glutamatergic [alpha]-amino-3-hydroxy-5methyl-4-isoxazoleproprionat receptor antagonist, 100 [micro]M, 10 [micro]l). and D-2-amino-5-phosphonovalerate [a glutamatergic N-methyl-D-aspartate (NMDA) antagonist, 100 [micro]M, 10 [micro]l]. Acute anal stretch using a mosquito clamp with a distance of 4 mm exhibited no effect, whereas distances of 8 mm attenuated and 12 mm abolished the repetitive stimulation-induced SRP. Intrathecal NMDA (100 [micro]M, 10 [micro]l) reversed the abolition on SRP caused by anal stretch. On the other hand, pretreated bicuculline [gamma]-aminobutyric acid (GABA) A receptor antagonist, 100 [micro]M, 10 [micro]l] but not hydroxysaclofen ([GABA.sub.B] receptor antagonist) counteracted the abolition on the repetitive stimulation-induced SRP caused by the anal stretch. All of the results suggested that anal stretch may be used as an adjunct to assist voiding dysfunction in patients with overactive urethra sphincter and that GABAergic neurotransmission is important in the neural mechanisms underlying external urethra sphincter activity inhibited by anal stretch. spinal cord: voiding dysfunction; glutamate; bicuculline; hydroxysaclofen

Cross-organ sensitization between the uterus and the lower urinary tract (LUT) underlies the high concurrence of pelvic pain syndrome and LUT dysfunctions, and yet the role of gonadal steroids is still unknown. We tested the hypothesis that cross-organ sensitization on pelvic-urethra reflex activity caused by uterine capsaicin instillation is estrous cycle dependent. When compared with the baseline reflex activity (1.00 [+ or -] 0.00 spikes/stimulation), uterine capsaicin instillation significantly increased reflex activity (45.42 [+ or -] 9.13 spikes/stimulation, P < 0.01, n = 7) that was corroborated by an increase in phosphorylated NMDA NR2B (P < 0.05, n = 4) but not NR2A subunit (P > 0.05, n = 4) expression. Both intrauterine pretreatment with capsazepine (5.02 [+ or -] 2.11 spikes/stimulation, P < 0.01, n = 7) and an intrathecal injection of AP5 (3.21 [+ or -] 0.83 spikes/stimulation, P < 0.01, n = 7) abolished the capsaicin-induced cross-organ sensitization and the increment in the phosphorylated NR2B level (P < 0.05, n = 4). The degrees of the cross-organ sensitization increased in a dose-dependent manner with the concentration of instilled capsaicin from 100 to 300 [micro]M in both the proestrus and metestrus stages, whereas they weakened when the concentrations were higher than 1,000 [micro]M. Moreover, the cross-organ sensitization caused by the uterine capsaicin instillation increased significantly in the rats during the proestrus stage when compared with the metestrus stage (P < 0.01, n = 7). These results suggest that estrogen levels might modulate the cross-organ sensitization between the uterus and the urethra and underlie the high concurrence of pelvic pain syndrome and LUT dysfunctions. transient receptor potential vanilloid subfamily member 1; N-methyl-D-aspartate; pelvic pain syndrome; central sensitization; spinal reflex potentiation; capsaicin; spinal cord

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 pelvicurethral 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 [micro]1). Intrathecal SB408124 (10 [micro]M, 10 [micro]1), 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 coadministration of OxA and Co-101244 (30 nM, 10 [micro]1; an NMDA NR2B subunit antagonist), but it did not reverse the abolition by the co-administration of OxA and PPPA (300 nM, 10 [micro]1; 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 cord; pelvic nerve; urethra; rats

This study was conducted to investigate the possible neurotransmitter that activates the descending pathways coming from the dorsolateral pontine tegmentum (DPT) to modulate spinal pelvic-urethra reflex potentiation. External urethra sphincter electromyogram (EUSE) activity in response to test stimulation (TS, 1/30 Hz) and repetitive stimulation (RS, 1 Hz) on the pelvic afferent nerve of 63 anesthetized rats were recorded with or without microinjection of nicotinic cholinergic receptor (nAChR) agonists, ACh and nicotine, to the DPT. TS evoked a baseline reflex activity with a single action potential (1.00 [+ or -] 0.00 spikes/stimulation, n = 40), whereas RS produced a long-lasting reflex potentiation (16.14 [+ or -] 0.96 spikes/ stimulation, n = 40) that was abolished by D-2-amino-5-phosphonovaleric acid (1.60 -+ 0.89 spikes/ stimulation, n = 40) and was attenuated by 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo (F) quinoxaline (7.10 [+ or -] 0.84 spikes/stimulation, n = 40). ACh and nicotine microinjections to DPT both produced facilitation on the RS-induced reflex potentiation (23.57 [+ or -] 2.23 and 28.29 [+ or -] 2.36 spikes/stimulation, P < 0.01, n = 10 and 20, respectively). Pretreatment of selective nicotinic receptor antagonist, chlorisondamine, reversed the facilitation on RS-induced reflex potentiation caused by nicotine (19.41 [+ or -] 1.21 spikes/ stimulation, P < 0.01, n = 10) Intrathecal WAY-100635 and spinal transection at the T1 level both abolished the facilitation on reflex potentiation resulting from the DPT nicotine injection (12.86 [+ or -] 3.13 and 15.57 [+ or -] 1.72 spikes/stimulation, P < 0.01, n = 10 each). Our findings suggest that activation of nAChR at DPT may modulate N-methyl-D-aspartic acid-dependent reflex potentiation via descending serotonergic neurotransmission. This descending modulation may have physiological/pathological relevance in the neural controls of urethral closure. acetylcholine; serotonin; WAY-100635; intrathecal; rats; N-methyl-D-aspartic acid; spinal reflex potentiation