Postnatal expression of corticotropin releasing factor (CRF) in rat urinary bladder

Abstract

Corticotropin releasing factor (CRF) is a neuropeptide expressed in micturition reflex circuitry and different roles in these reflexes have been suggested. These studies examined the expression of CRF/CRF receptors in the urinary bladder during postnatal development in the rat. Urinary bladder was harvested from rats (postnatal (P) day 0-adult) euthanized by isoflurane (4%) and thoracotomy. CRF protein expression significantly (p<or=0.01) decreased in the urothelium with increasing postnatal age. In contrast, CRF-immunoreactivity (IR) was increased in nerve fibers in the suburothelial plexus during the second-third postnatal week. Total CRF protein from urinary bladder significantly increased during the second-third postnatal weeks as determined with ELISAs. CRF receptor 2 (CRFR(2)) transcript was expressed in urinary bladder of all postnatal ages examined whereas no CRFR(1) transcript was expressed at any postnatal age examined. We also demonstrated changes in urinary bladder mRNA expression for the neuropeptides, galanin, substance P, vasoactive intestinal polypeptide and pituitary adenylate cyclase activating polypeptide during postnatal development. These studies demonstrate changes in the CRF expression in urinary bladder, specifically in the urothelium and nerve fibers of the suburothelial plexus during postnatal development. Changes in CRF expression and neuropeptide expression in general in the urinary bladder may contribute to the emergence of mature voiding reflexes.

Figure 1

Semi-quantitative analysis of CRF-immunoreactivity (IR) in the urothelium during postnatal development. The urothelium was outlined (green) and measured in total pixels area (A–F). A threshold encompassing an intensity range of 100–250 grayscale values was applied to the region of interest (A–F). The same threshold was subsequently used for all images. Percent CRF expression above threshold in the total area selected was then calculated. Grayscale versions of CRF-IR in urinary bladder at birth, postnatal (P) day 0 (A), P7 (B), P14 (C), P21 (D), P28 (E) and adult (F) with the urothelium (U) outlined in green are demonstrated. Images are thresholded and little CRF (absence of yellow within the outlined region) is above threshold in adult bladder compared to significant CRF-IR that is above threshold at younger ages (A–E, presence of yellow within U). Calibration bar represents 50 µm.

Figure 2

CRF expression in urothelium in postnatal rat bladder. Fluorescence images of CRF expression in urinary bladder sections of P0 (A), P7 (B), P14 (C), P21 (D), P28 (E) and adult (F) rats. For all images, exposure times were held constant and all tissues were processed simultaneously. In adult and P28 rats, little CRF expression was visible in the urothelium (U; E, F). In younger rats (P0–P21; A–D) greater expression of CRF in the urothelium in all layers (apical, intermediate and basal) of the U was observed. In the youngest rats examined (P0–P7; A, B), some diffuse CRF expression was present in the suburothelium (SU) region and in some regions of smooth muscle (sm) but this staining was non-homogeneous. Calibration bar represents 50 µm.

Figure 3

CRF-immunoreactivity (IR) in urothelial cells from urinary bladder with less (A–E) or more distention (F–H). A. CRF-IR is present in urothelial cells (arrows) throughout the thickness of the urothelium in a cryostat section of urinary bladder from postnatal day (P)14. B. Same section of urinary bladder stained with YOYO-1 to visualize cell nuclei. C. Merged image of A and B. The area enclosed by the rectangle (white lines) is magnified in panels D and E. D, E. Higher power magnification of urothelial cells exhibiting CRF-IR in the cytoplasm with visible nuclei (YOYO-1; arrows). F. CRF-IR in urothelial cells (arrows) from a urinary bladder with greater distention and flatter urothelial cells as a result of the distention. G. Same section of urinary bladder stained with YOYO-1 to visualize cell nuclei. H. Merged image of F and G with CRF-IR urothelial cells indicated (arrows). I. Absorption of CRF antiserum with CRF peptide (10 µg/ml) significantly reduced staining to background levels. U, urothelium; L, lumen. Calibration bar represents 80 µm in A–H, I 90 µm in D and 100 µm in E.

Figure 4

Age-dependent reduction in urothelial CRF expression. Summary histogram of CRF expression in the urothelium during postnatal development expressed as a percentage of adult CRF expression. Histogram represents the average of n = 6 samples per postnatal age. An age-dependent reduction in urothelial CRF expression was evident (R²= 0.978; p ≤ 0.001). *, p ≤ 0.01 compared to P14–P36; **, p ≤ 0.01 compared to P14–P36; #, p ≤ 0.01 compared to P36.

Figure 5

Postnatal expression of corticotropin releasing factor (CRF) in urinary bladder. Changes in total urinary bladder CRF as detected with immunoassays during postnatal (P) development (P0-Adult). Significant increases in total urinary bladder CRF were detected at P0–P4 (##, p ≤ 0.01) compared to P36 and adult as well as at P7 (#, p ≤ 0.01) compared to P28-Adult. At P14, total urinary bladder CRF significantly (*, p ≤ 0.01) increased compared to P0–P7 and P28-Adult. At P21, total urinary bladder CRF significantly (**, p ≤ 0.01) increased compared to P0–P4 and P28-Adult. Histogram represents the average for n = 6 samples per group.

Figure 6

CRF-immunoreactive nerve fibers in suburothelial plexus of urinary bladder. Fluorescence photographs of CRF-immunoreactive nerve fibers in nerve fibers in the suburothelial plexus in whole mount preparations of the urothelium/suburothelium from postnatal rats (P0, A; P7, B; P14, C; P21, D; P36, E; Adult, F). CRF-immunoreactive nerve fibers in the suburothelial plexus in the urothelium/suburothelium whole mount preparation were abundant during the second-third postnatal weeks (C, D; arrows) compared to an absence of CRF expression in nerve fibers from P0–P7. Although CRF expression in nerve fibers was not evident at P0–P7, CRF expression in urothelial cells was present (A, B; arrows). With increasing age (P36-Adult; arrows) CRF expression in nerve fibers appeared to decrease in density. G. As a comparison, the suburothelial plexus was stained with the pan neuronal antibody, protein gene product 9.5, that labels all nerve fibers. All images (A–G) are from the bladder neck region that together with the trigone region exhibited the greatest density of CRF staining from P14-adult. Calibration bar represents 80 µm.

Figure 7

CRF receptor (CRFR₂) expression in postnatal urinary bladder. Urinary bladder from all postnatal ages examined express CRFR₂ transcript (B). In contrast, CRFR₁ is not detected in urinary bladder under the specified conditions despite the robust expression of CRFR₁ in lumbar (L) 6 spinal cord (A).

Figure 8

Postnatal plasticity in neuropeptide mRNA expression in urinary bladder. For each postnatal age, total RNA from individual urinary bladder was reverse transcribed, and the cDNA templates synthesized from urinary bladder were analyzed by real-time quantitative PCR using primers specific for rat pituitary adenylate cyclase activating polypeptide (PACAP), vasoactive intestinal polypeptide (VIP), galanin and substance P (sub P). Transcript levels are normalized to the housekeeping gene, 18S, and expressed as relative change compared to P7 (100%). For all neuropeptides examined, adult transcript expression in urinary bladder was less than that observed in younger urinary bladder. n = 4 for Adult; n = 8 each for P7, P14. *, p ≤ 0.01 versus adult; #, p ≤ 0.001 versus P7.