CYP-induced bladder overactivity is decreased in Trpc1/c4^−/− mice.

<p><b>A,</b> Typical urodynamic traces of WT and Trpc1/c4^−/− mice in vehicle or following CYP treatment. <b>B,C,</b> Analysis of intercontractile interval in vehicle (<b>B</b>) and CYP-treatment mice (<b>C</b>) in WT and Trpc1/c4^−/− mice shows that double knock-out mice are functionally less affected by CYP treatment. <b>D,</b> Typical muscle strip contractility traces of WT mice in control and in CYP_c rats activated by 10 nM of carbachol. The statistical analysis of carbachol-induced contraction of muscle strips did not show difference between the two genotypes in CYP_c mice. <b>E,</b> Typical traces of muscle strip contractility of WT mice stimulated with high KCl concentration (122 mM). There is no difference between WT and Trpc1/c4^−/− in control and CYP_c conditions.</p

Sensory neurites sprouting in bladder wall in CYPc rats.

<p><b><b><i>A</i></b><b>,</b></b> Nerve fibers stained for PGP9.5 and Tuj1 in whole mount urothelium - scale bar : 50 µm. <b>B,</b> Fibers are stained by CGRP antibody but not by VAChT antibody in control condition. <b><i>C,</i></b> PGP9.5 staining of whole mount bladder mucosa in control and CYP-treated rats. <b>D,</b> Line representation showing neurites based on the images in panel <b>C</b>, illustrating increased outgrowth in CYP_c. <b>E-G,</b> Statistical comparison of neurite segments (<b>E</b>), neurite length (<b>F</b>) and neurite density (<b>G</b>) between control and CYP_c rats. <b>H</b>, Neurite fibers observed in CYP_c are stained by CGRP but by VAChT antibody.</p

TRP expression screening in DRG of CYPc rats.

<p><b>A–D,</b> qRT-PCR analysis of Trpc (<b>A</b>), Trpm (<b>B</b>), Trpv (<b>C</b>), and Trpa1 and Trpml (<b>D</b>) mRNA shows an up-regulation of Trpc1 and Trpc4 transcripts and a decreased expression of Trpc5 and Trpc6 transcripts in L6-S1 DRG of CYP-treated rats.</p

CYP-induced peripheral sprouting is abolished in Trpc1/c4^−/− mice.

<p><b>A,</b> PGP9.5 staining in whole mount bladder mucosa in control and CYP-treated wild type, TRPC1, TRPC4 and TRPC1/C4 deficient mice - scale bar : 50 µm. <b>B–D,</b> Quantitative analysis of neurite segments (<b>B</b>), neurite length (<b>C</b>) and neurite density (<b>D</b>) shows a normal innervation in treated Trpc1/c4^−/− mice.</p

Structure of the SthK Carboxy-Terminal Region Reveals a Gating Mechanism for Cyclic Nucleotide-Modulated Ion Channels

<div><p>Cyclic nucleotide-sensitive ion channels are molecular pores that open in response to cAMP or cGMP, which are universal second messengers. Binding of a cyclic nucleotide to the carboxyterminal cyclic nucleotide binding domain (CNBD) of these channels is thought to cause a conformational change that promotes channel opening. The C-linker domain, which connects the channel pore to this CNBD, plays an important role in coupling ligand binding to channel opening. Current structural insight into this mechanism mainly derives from X-ray crystal structures of the C-linker/CNBD from hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels. However, these structures reveal little to no conformational changes upon comparison of the ligand-bound and unbound form. In this study, we take advantage of a recently identified prokaryote ion channel, SthK, which has functional properties that strongly resemble cyclic nucleotide-gated (CNG) channels and is activated by cAMP, but not by cGMP. We determined X-ray crystal structures of the C-linker/CNBD of SthK in the presence of cAMP or cGMP. We observe that the structure in complex with cGMP, which is an antagonist, is similar to previously determined HCN channel structures. In contrast, the structure in complex with cAMP, which is an agonist, is in a more open conformation. We observe that the CNBD makes an outward swinging movement, which is accompanied by an opening of the C-linker. This conformation mirrors the open gate structures of the K_v1.2 channel or MthK channel, which suggests that the cAMP-bound C-linker/CNBD from SthK represents an activated conformation. These results provide a structural framework for better understanding cyclic nucleotide modulation of ion channels, including HCN and CNG channels.</p></div

CYP_c bladders are not innervated by more sensory neurons.

<p><b>A,</b> Imaging of retrolabelled bladder neurons with DiI dye - scale bar: 50 µm. <b>B,</b> The quantitative analysis shows that bladder is not innervated by more afferent neurons.</p

Cyclophosphamide treatment induces overexpression of GAP-43 in DRG.

<p><b>A,</b> qRT-PCR analysis of GAP-43 transcripts in L6-S1 DRG shows a significant increase of GAP-43 expression after CYP treatment. <b>B,</b> Neurite outgrowth marker GAP-43 staining in DRG of control and CYP_c rats. <b>C,</b> Quantification of number of GAP-43 positive cells in bladder-innervating DRG - scale bar: 50 µm.</p

TRPC4 is upregulated in bladder-innervating neurons in CYP_c rats.

<p><b>A, B</b> Immunocytochemistry of TRPC4 in DRG of control and CYP_c rats, retrolabelled with DiI - scales bar: 50 µm. <b>C,</b> Quantitative analysis of colocalization of DiI and TRPC4.</p

TRPC1 is upregulated in bladder-innervating neurons in CYP_c rats.

<p><b>A, B</b> Immunocytochemistry of TRPC1 in DRG of control and CYP_c rats, retrolabelled with DiI - scales bar: 50 µm. <b>C,</b> Quantitative analysis of colocalization of DiI and TRPC1. <b>D,</b> Immunohistochemistry showing that a majority of GAP-43 positive neurons are TRPC1 positive in CYP_c rats – scale bar: 20 µm.</p

Crucial Role of TRPC1 and TRPC4 in Cystitis-Induced Neuronal Sprouting and Bladder Overactivity

<div><p>Purpose</p><p>During cystitis, increased innervation of the bladder by sensory nerves may contribute to bladder overactivity and pain. The mechanisms whereby cystitis leads to hyperinnervation of the bladder are, however, poorly understood. Since TRP channels have been implicated in the guidance of growth cones and survival of neurons, we investigated their involvement in the increases in bladder innervation and bladder activity in rodent models of cystitis.</p><p>Materials and Methods</p><p>To induce bladder hyperactivity, we chronically injected cyclophosphamide in rats and mice. All experiments were performed a week later. We used quantitative transcriptional analysis and immunohistochemistry to determine TRP channel expression on retrolabelled bladder sensory neurons. To assess bladder function and referred hyperalgesia, urodynamic analysis, detrusor strip contractility and Von Frey filament experiments were done in wild type and knock-out mice.</p><p>Results</p><p>Repeated cyclophosphamide injections induce a specific increase in the expression of TRPC1 and TRPC4 in bladder-innervating sensory neurons and the sprouting of sensory fibers in the bladder mucosa. Interestingly, cyclophosphamide-treated Trpc1/c4^−/− mice no longer exhibited increased bladder innervations, and, concomitantly, the development of bladder overactivity was diminished in these mice. We did not observe a difference neither in bladder contraction features of double knock-out animals nor in cyclophosphamide-induced referred pain behavior.</p><p>Conclusions</p><p>Collectively, our data suggest that TRPC1 and TRPC4 are involved in the sprouting of sensory neurons following bladder cystitis, which leads to overactive bladder disease.</p></div