PACAP/VIP and receptor characterization in micturition pathways in mice with overexpression of NGF in urothelium

Abstract

Urothelium-specific overexpression of nerve growth factor (NGF) in the urinary bladder of transgenic mice stimulates neuronal sprouting or proliferation in the urinary bladder, produces urinary bladder hyperreflexia, and results in increased referred somatic hypersensitivity. Additional NGF-mediated changes might contribute to the urinary bladder hyperreflexia and pelvic hypersensitivity observed in these transgenic mice such as upregulation of neuropeptide/receptor systems. Chronic overexpression of NGF in the urothelium was achieved through the use of a highly urothelium-specific, uroplakin II promoter. In the present study, we examined pituitary adenylate cyclase activating polypeptide (PACAP), vasoactive intestinal polypeptide (VIP), and associated receptor (PAC1, VPAC1, VPAC2) transcripts or protein expression in urothelium and detrusor smooth muscle and lumbosacral dorsal root ganglia in NGF-overexpressing and littermate wildtype mice using real-time quantitative reverse transcription-polymerase chain reaction and immunohistochemical approaches. Results demonstrate upregulation of PAC1 receptor transcript and PAC1-immunoreactivity in urothelium of NGF-OE mice whereas PACAP transcript and PACAP-immunoreactivity were decreased in urothelium of NGF-OE mice. In contrast, VPAC1 receptor transcript was decreased in both urothelium and detrusor smooth muscle of NGF-OE mice. VIP transcript expression and immunostaining was not altered in urinary bladder of NGF-OE mice. Changes in PACAP, VIP, and associated receptor transcripts and protein expression in micturition pathways resemble some, but not all, changes observed after induction of urinary bladder inflammation known to involve NGF production.

Figure 1

Regulation of PAC1, VPAC1 and VPAC2 receptor transcript levels in littermate wildtype (WT) and NGF overexpressing (NGF-OE) mice in urothelium and detrusor smooth muscle. Relative expression of the urothelium and detrusor receptor transcripts are expressed as a percentage of WT urothelium and normalized to the relative expression of the housekeeping gene, 18S. A: PAC1 mRNA expression. B: VPAC1 mRNA expression. C: VPAC2 mRNA expression. Samples size are n of 5 – 7; *, p ≤ 0.01 versus control.

Figure 2

PAC1-immunoreactivity in urothelium (U) (A–C) and detrusor smooth muscle (sm) (D–E) of littermate wildtype (WT) and NGF overexpressing (NGF-OE) mice. Faint PAC1-IR was present in apical urothelial cells of WT urothelium (A, arrows). In NGF-OE mice, PAC1-IR was increased in apical urothelial cells (B–C, arrows) but was also expressed in all urothelial cell layers (B, bracket). PAC1-IR was expressed in detrusor smooth muscle of WT and NGF-OE mice (D–E). F. Summary histogram of PAC1 expression in the U and detrusor sm in WT and NGF-OE mice. Values are mean ± SEM; (n = 5). *, p ≤ 0.01. L, lumen. Calibration bar equals 50 μm.

Figure 3

Regulation of PACAP and VIP transcript levels in littermate wildtype (WT) and NGF overexpressing (NGF-OE) mice in urothelium and detrusor smooth muscle. Relative expression of the urothelium and detrusor receptor transcripts are expressed as a percentage of WT urothelium and normalized to the relative expression of the housekeeping gene, 18S. A: PACAP mRNA expression. B: VIP mRNA expression. Samples size are n of 5 – 7; *, p ≤ 0.01 versus control.

Figure 4

PACAP-immunoreactivity in urothelium (U) (A–C) and detrusor smooth muscle (sm) (C) of littermate wildtype (WT) and NGF overexpressing (NGF-OE) mice. Robust PACAP-IR was present throughout the WT urothelium (A). Some evidence of PACAP-IR in presumptive suburothelial nerve fibers was observed (A, arrows). In NGF-OE mice, PACAP-IR was decreased in all urothelial cell layers (B). C. Summary histogram of PACAP expression in the U and detrusor sm in WT and NGF-OE mice. No changes in PACAP-IR were observed in WT or NGF-OE sm. Values are mean ± SEM; (n = 5). *, p ≤ 0.01. L, lumen; lp, lamina propria. Calibration bar equals 50 μm.

Figure 5

VIP-immunoreactivity in urothelium (U) (A–C) and detrusor smooth muscle (sm) (C) of littermate wildtype (WT) and NGF overexpressing (NGF-OE) mice. VIP-IR was present throughout the WT urothelium (A, C) and NGF-OE (B) urothelium. VIP-IR in presumptive suburothelial nerve fibers was observed in WT and NGF-OE urinary bladder (C, arrows). C. Summary histogram of VIP expression in the U and detrusor sm in WT and NGF-OE mice. No changes in VIP-IR were observed in WT or NGF-OE U or sm. Values are mean ± SEM; (n = 5). *, p ≤ 0.01. L, lumen; lp, lamina propria. Calibration bar equals 100 μm in A, B and 30 μm in C.

Figure 6

Regulation of PACAP and VIP mRNA expression in lumbosacral (L6-S1) dorsal root ganglia (DRG) in littermate wildtype (WT) and NGF overexpressing (NGF-OE) mice. Relative expression of the L6 and S1 receptor transcripts are expressed as a percentage of WT L6 DRG and normalized to the relative expression of the housekeeping gene, 18S. A: PACAP mRNA expression. B: VIP mRNA expression. Samples size are no 5 to 7; *, p ≤ 0.01 versus control.