Activation of extracellular signal-regulated protein kinase 5 is essential for cystitis- and nerve growth factor-induced calcitonin gene-related peptide expression in sensory neurons.

BACKGROUND: Cystitis causes considerable neuronal plasticity in the primary afferent pathways. The molecular mechanism and signal transduction underlying cross talk between the inflamed urinary bladder and sensory sensitization has not been investigated. RESULTS: In a rat cystitis model induced by cyclophosphamide (CYP) for 48 h, the mRNA and protein levels of the excitatory neurotransmitter calcitonin gene-related peptide (CGRP) are increased in the L6 dorsal root ganglia (DRG) in response to bladder inflammation. Cystitis-induced CGRP expression in L6 DRG is triggered by endogenous nerve growth factor (NGF) because neutralization of NGF with a specific NGF antibody reverses CGRP up-regulation during cystitis. CGRP expression in the L6 DRG neurons is also enhanced by retrograde NGF signaling when NGF is applied to the nerve terminals of the ganglion-nerve two-compartmented preparation. Characterization of the signaling pathways in cystitis- or NGF-induced CGRP expression reveals that the activation (phosphorylation) of extracellular signal-regulated protein kinase (ERK)5 but not Akt is involved. In L6 DRG during cystitis, CGRP is co-localized with phospho-ERK5 but not phospho-Akt. NGF-evoked CGRP up-regulation is also blocked by inhibition of the MEK/ERK pathway with specific MEK inhibitors U0126 and PD98059, but not by inhibition of the PI3K/Akt pathway with inhibitor LY294002. Further examination shows that cystitis-induced cAMP-responsive element binding protein (CREB) activity is expressed in CGRP bladder afferent neurons and is co-localized with phospho-ERK5 but not phospho-Akt. Blockade of NGF action in vivo reduces the number of DRG neurons co-expressing CGRP and phospho-CREB, and reverses cystitis-induced increases in micturition frequency. CONCLUSIONS: A specific pathway involving NGF-ERK5-CREB axis plays an essential role in cystitis-induced sensory activation.

Up-regulation of brain-derived neurotrophic factor in primary afferent pathway regulates colon-to-bladder cross-sensitization in rat.

BACKGROUND: In humans, inflammation of either the urinary bladder or the distal colon often results in sensory cross-sensitization between these organs. Limited information is known about the mechanisms underlying this clinical syndrome. Studies with animal models have demonstrated that activation of primary afferent pathways may have a role in mediating viscero-visceral cross-organ sensitization. METHODS: Colonic inflammation was induced by a single dose of tri-nitrobenzene sulfonic acid (TNBS) instilled intracolonically. The histology of the colon and the urinary bladder was examined by hematoxylin and eosin (H&E) stain. The protein expression of transient receptor potential (TRP) ion channel of the vanilloid type 1 (TRPV1) and brain-derived neurotrophic factor (BDNF) were examined by immunohistochemistry and/or western blot. The inter-micturition intervals and the quantity of urine voided were obtained from analysis of cystometrograms. RESULTS: At 3 days post TNBS treatment, the protein level of TRPV1 was increased by 2-fold (p < 0.05) in the inflamed distal colon when examined with western blot. TRPV1 was mainly expressed in the axonal terminals in submucosal area of the distal colon, and was co-localized with the neural marker PGP9.5. In sensory neurons in the dorsal root ganglia (DRG), BDNF expression was augmented by colonic inflammation examined in the L1 DRG, and was expressed in TRPV1 positive neurons. The elevated level of BDNF in L1 DRG by colonic inflammation was blunted by prolonged pre-treatment of the animals with the neurotoxin resiniferatoxin (RTX). Colonic inflammation did not alter either the morphology of the urinary bladder or the expression level of TRPV1 in this viscus. However, colonic inflammation decreased the inter-micturition intervals and decreased the quantities of urine voided. The increased bladder activity by colonic inflammation was attenuated by prolonged intraluminal treatment with RTX or treatment with intrathecal BDNF neutralizing antibody. CONCLUSION: Acute colonic inflammation increases bladder activity without affecting bladder morphology. Primary afferent-mediated BDNF up-regulation in the sensory neurons regulates, at least in part, the bladder activity during colonic inflammation.

Uroguanylin, an intestinal natriuretic peptide, is delivered to the kidney as an unprocessed propeptide.

Orally delivered salt stimulates renal salt excretion more effectively than does iv delivered salt. Although the mechanisms that underlie this "postprandial natriuresis" are poorly understood, the peptide uroguanylin (UGn) is thought to be a key mediator. However, the lack of selective assays for UGn gene products has hindered rigorous testing of this hypothesis. Using peptide-specific assays, we now report surprisingly little UGn in rat intestine or plasma. In contrast, prouroguanylin (proUGn), the presumed-inactive precursor of UGn, is plentiful (at least 40 times more abundant than UGn) in both intestine and plasma. The intestine is the likely source of the circulating proUGn because: 1) the proUGn portal to systemic ratio is approximately two under normal conditions, and 2) systemic proUGn levels decrease rapidly after intestinal resection. Together, these data suggest that proUGn itself is actively involved in enterorenal signaling. This is strongly supported by our observation that iv infusion of proUGn at a physiological concentration produces a long-lasting renal natriuresis, whereas previously reported natriuretic effects of UGn have required supraphysiological concentrations. Thus, our data point to proUGn as an endocrine (i.e. circulating) mediator of postprandial natriuresis, and suggest that the propeptide is secreted intact from the intestine into the circulation and processed to an active form at an extravascular site.

In vivo regulation of brain-derived neurotrophic factor in dorsal root ganglia is mediated by nerve growth factor-triggered Akt activation during cystitis.

The role of brain-derived neurotrophic factor (BDNF) in sensory hypersensitivity has been suggested; however the molecular mechanisms and signal transduction that regulate BDNF expression in primary afferent neurons during visceral inflammation are not clear. Here we used a rat model of cystitis and found that the mRNA and protein levels of BDNF were increased in the L6 dorsal root ganglia (DRG) in response to bladder inflammation. BDNF up-regulation in the L6 DRG was triggered by endogenous nerve growth factor (NGF) because neutralization of NGF with a specific NGF antibody reduced BDNF levels during cystitis. The neutralizing NGF antibody also subsequently reduced cystitis-induced up-regulation of the serine/threonine kinase Akt activity in L6 DRG. To examine whether the NGF-induced Akt activation led to BDNF up-regulation in DRG in cystitis, we found that in cystitis the phospho-Akt immunoreactivity was co-localized with BDNF in L6 DRG, and prevention of the endogenous Akt activity in the L6 DRG by inhibition of phosphoinositide 3-kinase (PI3K) with a potent inhibitor LY294002 reversed cystitis-induced BDNF up-regulation. Further study showed that application of NGF to the nerve terminals of the ganglion-nerve two-compartmented preparation enhanced BDNF expression in the DRG neuronal soma; which was reduced by pre-treatment of the ganglia with the PI3K inhibitor LY294002 and wortmannin. These in vivo and in vitro experiments indicated that NGF played an important role in the activation of Akt and subsequent up-regulation of BDNF in the sensory neurons in visceral inflammation such as cystitis.

Endogenous PI3K/Akt and NMDAR act independently in the regulation of CREB activity in lumbosacral spinal cord in cystitis.

The integral interaction of signaling components in the regulation of visceral inflammation-induced central sensitization in the spinal cord has not been well studied. Here we report that phosphoinositide 3-kinase (PI3K)-dependent Akt activation and N-methyl-d-aspartic acid receptor (NMDAR) in lumbosacral spinal cord independently regulate the activation of cAMP response element-binding protein (CREB) in vivo in a rat visceral pain model of cystitis induced by intraperitoneal injection of cyclophosphamide (CYP). We demonstrate that suppression of endogenous PI3K/Akt activity with a potent PI3K inhibitor LY294002 reverses CYP-induced phosphorylation of CREB, however, it has no effect on CYP-induced phosphorylation of NR1 at Ser(897) and Ser(896); conversely, inhibition of NMDAR in vivo with MK801 fails to block CYP-induced Akt activation but significantly attenuates CYP-induced CREB phosphorylation in lumbosacral spinal cord. This novel interrelationship of PI3K/Akt, NMDAR, and CREB activation in lumbosacral spinal cord is further confirmed in an ex vivo spinal slice culture system exposed to an excitatory neurotransmitter calcitonin gene-related peptide (CGRP). Consistently we found that CGRP-triggered CREB activation can be blocked by both PI3K inhibitor LY294002 and NMDAR antagonists MK801 and D-AP5. However, CGRP-triggered Akt activation cannot be blocked by MK801 or D-AP5; vice versa, LY294002 pretreatment that suppresses the Akt activity fails to reverse CGRP-elicited NR1 phosphorylation. These results suggest that PI3K/Akt and NMDAR independently regulate spinal plasticity in visceral pain model, and target of a single pathway is necessary but not sufficient in treatment of visceral hypersensitivity.

Up-regulation of brain-derived neurotrophic factor is regulated by extracellular signal-regulated protein kinase 5 and by nerve growth factor retrograde signaling in colonic afferent neurons in colitis.

Brain-derived neurotrophic factor (BDNF) plays an essential role in sensory neuronal activation in response to visceral inflammation. Here we report that BDNF up-regulation in the primary afferent neurons in the dorsal root ganglia (DRG) in a rat model of colitis is mediated by the activation of endogenous extracellular signal-regulated protein kinase (ERK) 5 and by nerve growth factor (NGF) retrograde signaling. At 7 days of colitis, the expression level of BDNF is increased in conventional neuronal tracing dye Fast Blue labeled primary afferent neurons that project to the distal colon. In these neurons, the phosphorylation (activation) level of ERK5 is also increased. In contrast, the level of phospho-ERK1/2 is not changed in the DRG during colitis. Prevention of the ERK5 activation in vivo with an intrathecal application of the MEK inhibitor PD98059 significantly attenuates the colitis-induced increases in BDNF expression in the DRG. Further studies show that BDNF up-regulation in the DRG is triggered by NGF retrograde signaling which also involves activation of the MEK/ERK pathways. Application of exogenous NGF exclusively to the compartment containing DRG nerve terminals in an ex vivo ganglia-nerve preparation markedly increases the BDNF expression level in the DRG neuronal cell body that is placed in a different compartment; this BDNF elevation is attenuated by U0126, PD98059 and a specific ERK5 inhibitor BIX02188. These results demonstrate the mechanisms and pathways by which BDNF expression is elevated in primary sensory neurons following visceral inflammation that is mediated by increased activity of ERK5 and is likely to be triggered by the elevated NGF level in the inflamed viscera.