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. 2013 Oct;51(2):602-14.
doi: 10.1007/s12031-013-0033-5. Epub 2013 May 21.

Increased TRPV4 expression in urinary bladder and lumbosacral dorsal root ganglia in mice with chronic overexpression of NGF in urothelium

Beatrice M Girard  1 , Liana Merrill, Susan Malley, Margaret A Vizzard
Affiliations

Increased TRPV4 expression in urinary bladder and lumbosacral dorsal root ganglia in mice with chronic overexpression of NGF in urothelium

Beatrice M Girard et al. J Mol Neurosci. 2013 Oct.

Abstract

Transient receptor potential vanilloid (TRPV) family member 4 (TRPV4) expression has been demonstrated in urothelial cells and dorsal root ganglion (DRG) neurons, and roles in normal micturition reflexes as well as micturition dysfunction have been suggested. TRP channel expression and function is dependent upon target tissue expression of growth factors. These studies expand upon the target tissue dependence of TRPV4 expression in the urinary bladder and lumbosacral DRG using a recently characterized transgenic mouse model with chronic overexpression of nerve growth factor (NGF-OE) in the urothelium. Immunohistochemistry with image analyses, real-time quantitative polymerase chain reaction, and Western blotting were used to determine TRPV4 protein and transcript expression in the urinary bladder (urothelium + suburothelium, detrusor) and lumbosacral DRG from littermate wild-type (WT) and NGF-OE mice. Antibody specificity controls were performed in TRPV4(-/-) mice. TRPV4 transcript and protein expression was significantly (p ≤ 0.001) increased in the urothelium + suburothelium and suburothelial nerve plexus of the urinary bladder and in small- and medium-sized lumbosacral (L1, L2, L6-S1) DRG cells from NGF-OE mice compared to littermate WT mice. NGF-OE mice exhibit significant (p ≤ 0.001) increases in NGF transcript and protein in the urothelium + suburothelium and lumbosacral DRG. These studies demonstrate regulation of TRPV4 expression by NGF in lower urinary tract tissues. Ongoing studies are characterizing the functional roles of TRPV4 expression in the sensory limb (DRG, urothelium) of the micturition reflex.

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Figures

Figure 1
Figure 1
Increased protein expression of nerve growth factor (NGF) in urinary bladder (A) and lumbosacral dorsal root ganglia (DRG) (B) in mice with chronic urothelial overexpression of NGF (NGF-OE). NGF protein content was significantly (p ≤ 0.001) increased in urinary bladder of NGF-OE compared to littermate wildtype (WT) mice (A). NGF protein content was significantly (p ≤ 0.001) increased in lumbosacral (L1, L2, L5-S1) DRG of NGF-OE compared to WT mice (B). No change in NGF protein content was observed in L5 DRG of NGF-OE mice compared to WT mice. Increased NGF expression was similar in L1, L2, L6 and S1 DRG from NGF-OE mice. Samples size are n of 7 – 9; *, p ≤ 0.001 versus WT.
Figure 2
Figure 2
Regulation of NGF (A) and TRPV4 (B, C) transcript levels in lumbosacral (L6-S1) dorsal root ganglia (DRG; A, C) and urothelium (Uro) + suburothelium (SU) (B) or detrusor smooth muscle (B) from wildtype (WT) and nerve growth factor-overexpressing (NGF-OE) mice. NGF transcript expression is significantly (p ≤ 0.001) increased in L6 and S1 DRG (A) of NGF-OE mice. TRPV4 transcript expression is significantly (p ≤ 0.001) increased in Uro + SU (B) and L6-S1 DRG (C) of NGF-OE mice compared to littermate WT mice. No change in NGF transcript expression was demonstrated in the detrusor of NGF-OE mice (B). Relative expression of NGF (A) and TRPV4 (B, C) transcript expression is normalized to the relative expression of the reference gene, L32. Samples size are n of 7 – 9; *, p ≤ 0.001.
Figure 3
Figure 3
Western blot analyses of TRPV4 expression in lumbar (L) 6 dorsal root ganglia (DRG) (A) and urothelium (Uro) + suburothelium (SU) from wildtype (WT), NGF-overexpressing (OE) and TRPV4-/- mice. Representative western blot of TRPV4 protein expression in L6 DRG from WT, NGF-OE and TRPV4-/- mice (A). Summary histogram of the relative expression of TRPV4 protein normalized to actin expression in L6 DRG from WT, NGF-OE and TRPV4-/- mice (A). Representative western blot of TRPV4 protein expression in Uro + SU from WT, NGF-OE and TRPV4-/- mice (B). Summary histogram of the relative expression of TRPV4 protein normalized to actin expression in Uro + SU from WT, NGF-OE and TRPV4-/- mice (B). Samples size are n of 7 – 9; *, p ≤ 0.001 versus WT.
Figure 4
Figure 4
TRPV4-immunoreactivity (IR) in cryostat sections of urinary bladder from WT and NGF-OE mice. In WT mice, faint TRPV4-IR was present in urothelium (U) and detrusor smooth muscle (sm) (A, C). Chronic overexpression of NGF in the U increased TRPV4-IR in U (B, D, F) and detrusor sm (B). Higher power fluorescence image of TRPV4-IR in U (F). TRPV4-IR was exhibited in all cellular layers of the U (A-D, F) in WT and NGF-OE mice. TRPV4-IR was present in the U throughout the bladder dome, body and neck regions. For all images, exposure times were held constant, and all tissues were processed simultaneously. E: Histogram of TRPV4 expression above threshold in the U of NGF-OE and WT mice expressed as a percentage of control. E. TRPV4-IR above threshold in U was significantly (p ≤ 0.001) increased in NGF-OE mice. Calibration bar represents 50 μm in A-D and 25 μm in F. Data are a summary of n = 7-9 for each group. L, lumen.
Figure 5
Figure 5
Fluorescence photographs of TRPV4-immunoreactivity (IR) in the suburothelial plexus in the bladder neck region in whole mount preparations of the urinary bladder from WT and NGF-OE mice. Dense TRPV4-IR was present in the suburothelial plexus in the bladder neck region of NGF-OE mice (A) compared to WT mice with less dense TRPV-IR in the suburothelial plexus (B). In NGF-OE mice, small clusters of TRPV4-IR cells were located within the suburothelial plexus of NGF-OE mice (C, arrows). Higher power immunofluorescence images of TRPV4-IR in the suburothelial plexus and in clusters of cells (D, arrows) within the suburothelial plexus in NGF-OE mice. WT mice did not exhibit clusters of TRPV4-IR cells in the suburothelial plexus. E. TRPV4-IR above threshold in the suburothelial plexus was significantly (p ≤ 0.001) increased in NGF-OE mice. Calibration bar represents 50 μm in A, B and 25 μm in C, D. Data are a summary of n = 7-9 for each group.
Figure 6
Figure 6
TRPV4-immunoreactivity (IR) in wholemount preparations of the urinary bladder from NGF-OE mice. In wholemount preparations, robust TRPV4-IR was present throughout the urothelium in the bladder dome, body and neck regions (A, B). With the urothelium dissected, TRPV4-IR is observed in the suburothelial nerve plexus (B, bracketed region). The bracketed region (B) is an area with the urothelium removed showing the suburothelial nerve plexus as in C. TRPV4-IR was present in the suburothelial plexus (C) in the bladder dome, body and neck and with greatest density of TRPV4-immunoreactive nerve fibers being present in the bladder neck. The specificity of the TRPV4 antibody used was verified in urinary bladder harvested from TRPV4-/- mice that did not exhibit TRPV4-IR (D). Calibration bar represents 50 μm in A - D.
Figure 7
Figure 7
TRPV4-IR in lumbosacral dorsal root ganglia in WT and NGF-OE mice. Lower power immunofluorescence images of TRPV4-IR in L1 DRG from WT (A) and NGF-OE mice (B). Higher power images of TRPV4-IR in L1 (C) and L6 (D) DRG from NGF-OE mice. TRPV4-IR nerve fibers were present throughout DRG in WT and NGF-OE mice (A-D, white arrows). TRPV4-IR was present in both small (C, D, arrowheads) and larger (C, D, yellow arrows) DRG cells. E. Summary histogram of the number of TRPV4-IR cells per section in L1, L2, L5-S1 DRG in WT and NGF-OE mice. Numbers of TRPV4-IR DRG cells were significantly (*, p ≤ 0.001) increased in L1, L2, L6 and S1 DRG from NGF-OE mice compared to WT. No changes in TRPV4-IR were observed in L5 DRG between NGF-OE and WT mice. Numbers of TRPV4-IR DRG cells were significantly (#, p ≤ 0.01) greater in L1 and L2 DRG compared to L6 and S1 DRG in NGF-OE mice. Calibration bar represents 80 μm in A, B; 25 μm in C, D. Data are a summary of n = 7-9 for each group.
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