P2X2 and P2X3 receptor expression in postnatal and adult rat urinary bladder and lumbosacral spinal cord

Abstract

P2X receptors mediate the effects of ATP in micturition and nociception. During postnatal maturation, a spinobulbospinal reflex and voluntary voiding replace primitive voiding reflexes. This may involve changes in neuroactive compounds and receptors in bladder reflex pathways. We examined P2X2 and P2X3 receptors in bladder and spinal cord from postnatal (P0-P36, indicating number of days) and adult Wistar rats. Western blot of whole bladders for P2X2 and P2X3 expression was performed. Immunostaining for P2X2 and P2X3 receptors in urothelium and detrusor smooth muscle whole mounts and spinal cord sections was examined. Western blot demonstrated an age-dependent decrease (R(2) = 0.96, P </= 0.005) in P2X2 receptor expression in bladder, whereas P2X3 receptor expression in bladder peaked (P </= 0.005) during P14-P21. P2X2-immunoreactivity (IR) was present in urothelial cells, suburothelial plexus, detrusor smooth muscle, and serosa at birth, with staining in urothelial cells and serosa being most predominant. With increasing postnatal age, the intensity of P2X2-IR decreased in urothelial cells but increased in suburothelial plexus. P2X3-IR increased in urothelial cells and suburothelial plexus with postnatal age, whereas staining in detrusor and serosa remained relatively constant. At birth, P2X3-IR was present in the dorsal horn, lateral collateral pathway, and dorsal commissure. With increasing age, P2X3-IR was restricted to superficial dorsal horn and lateral collateral pathway. P2X2-IR was present in ependyme cells (S-100-IR) of the central canal as early as P2. These studies demonstrate plastic expression of P2X2 and P2X3 receptors in bladder and spinal cord during early postnatal development at times coincident with appearance of mature voiding patterns.

Figure 1

A. Western blot of whole urinary bladder (40 mg) for P2X2 receptor expression in postnatal (P0-P36) and adult (A) rats. Erk staining was used as a loading control. B. Preabsorption of P2X2 antibody with immunogen (1 mg ml⁻¹) eliminated the band at 90kD. C. Histogram of relative P2X2 band density in all groups examined normalized to erk-1 staining. P2X2 receptor expression in urinary bladder demonstrated an age-dependent (R² = 0.96, p ≤ 0.005) decrease in expression. P2X2 receptor expression in urinary bladder from P0-P14 is significantly greater compared to adult expression. *, p ≤ 0.005.

Figure 2

A. Western blot of whole urinary bladder (40 mg) for P2X3 receptor expression in postnatal (P0-P36) and adult (A) rats. Erk staining was used as a loading control. B. Preabsorption of P2X3 antibody with immunogen (1 mg ml⁻¹) eliminated the band at 75kD. C. Histogram of relative P2X3 band density in all groups examined normalized to erk-1 staining. P2X3 receptor expression in urinary bladder is significantly increased from P14-P21 compared to adult rat bladder. *, p ≤ 0.005, #, p ≤ 0.01.

Figure 3

Fluorescence images of P2X2 receptor expression in urothelium (A, E), suburothelial plexus in bladder neck or trigone (B, F), detrusor (C, G) and serosa (D, H) from postnatal rats, P0-P14. For all images, exposure times were held constant. Expression of P2X2 receptor in the detrusor and serosa was relatively constant. On occasion, P2X2-IR cells were observed in close proximity or adjacent to detrusor smooth muscle (G, arrow). P2X2 receptor expression was intense in urothelial cells but relatively weak in the suburothelial plexus. Calibration bar represents 50 μm.

Figure 4

Fluorescence images of P2X2 receptor expression in urothelium (A, E), suburothelial plexus in bladder neck or trigone (B, F), detrusor (C, G) and serosa (D, H) from postnatal rats, P21-adult (A). For all images, exposure times were held constant. From P21-A, P2X2 receptor expression in detrusor and serosa was relatively constant and P2X2-IR cells were still occasionally observed adjacent to detrusor smooth muscle (G, arrow). P2X2 receptor expression in urothelial cells from P21-A was dramatically reduced compared to that observed from P0-P14. The suburothelial plexus staining for P2X2 receptor in bladder neck or trigone increased from P21-P28 and then staining decreased to that observed from P0-P14. Calibration bar represents 50 μm.

Figure 5

Fluorescence images of P2X2 (A-F) or P2X3 (G-L) receptor expression in the suburothelial plexus (A-C; G-I) and serosa (D-F; J-L) of adult animals. Whole mounts were also stained for protein gene product (PGP9.5) (B,E,H,K) to confirm that P2X2 and P2X3-immunoreactive structures were nerve fibers. In all tissues examined, the P2X2 and P2X3-IR in the suburothelial plexus or serosa was colabeled with PGP9.5. Merged images (C,F,I,L) demonstrating overlap between P2X2 and PGP9.5 (C,F) or P2X3 and PGP9.5 (I,L). Some isolated nerve fibers demonstrating P2X2- and PGP9.5-IR (C, arrow) or P2X3-and PGP9.5-IR (I, arrow) are shown in the suburothelial plexus. Calibration bar represents 50 μm.

Figure 6

Fluorescence images of P2X3 receptor expression in urothelium (A, E), suburothelial plexus in bladder trigone or neck (B, F), detrusor (C, G) and serosa (D, H) from postnatal rats, P2-P7. For all images, exposure times were held constant. Expression of P2X3 receptor in the detrusor and serosa was relatively constant. On occasion, isolated or small groups of P2X3-IR cells were observed in close proximity or adjacent to detrusor smooth muscle (C, arrows). P2X3 receptor expression was weak in urothelial cells and sparse P2X3-IR was observed in the suburothelial plexus from P0-P14. Calibration bar represents 50 μm.

Figure 7

Fluorescence images of P2X3 receptor expression in urothelium (A, E) suburothelial plexus in bladder trigone or neck (B, F), detrusor (C, G) and serosa (D, H) from postnatal rats, P21-adult (A). For all images, exposure times were held constant. Expression of P2X3 receptor in the detrusor and serosa was relatively constant. P2X3 receptor expression increased in urothelial cells from P21 through adulthood and a more extensive P2X3-IR suburothelial nerve plexus was observed in the bladder trigone and neck from P21 through adult. Calibration bar represents 50 μm.

Figure 8

P2X2 receptor expression in cells lining the central canal (CC) of the lumbosacral spinal cord from P2-adult (A). As early as P2 (A, B), P2X2-IR cells (B, arrows) were observed surrounding the CC but no staining was present at P0. Similar P2X2-immunoreactive cells surrounding the CC were observed at P14 (D, white arrows) and A (F, white arrows). In some instances, long processes could be observed emerging from these P2X2-immunoreactive cells and projecting from the CC laterally and ventrally (D, F, yellow arrows). P2X2-IR was also expressed in the anterior spinal artery (A, C, E, white arrow) and in the anterior corticospinal tract (C, E, yellow arrow). Calibration bar represents 120 μm in A, C, E and 60 μm in B, D.

Figure 9

P2X2-immunoreactive cells lining the central canal (CC) are presumptive ependyme cells, modified glial cells. P2X2-immunoreactive cells lining the CC did not exhibit immunoreactivity to the pan neuronal marker, HuC/D (A-D) and were therefore not neuronal. Numerous HuC/D-immunoreactive cells were present throughout the spinal cord parenchyma (C, arrows) but P2X2-immunoreactive cells did not exhibit HuC/D-IR (C, D). C, D are merged images demonstrating P2X2- (red) and HuC/D-IR (green). In addition, P2X2-IR cells surrounding the CC did not exhibit immunoreactivity to glial fibrillary acidic protein (GFAP) (E, G, H) and were therefore not astrocytes (E-H). G, H are merged images demonstrating P2X2- (red) and GFAP-IR (green) surrounding the CC. Some ependyme cells express immunoreactivity to the protein S-100 (19). S-100-IR cells (I) were observed surrounding the CC and many of these cells expressed a similar morphology to the P2X2-IR cells (D, H versus I) surrounding the CC. Calibration bar represents 60 μm.

Figure 10

P2X3-IR in lumbosacral spinal cord from young postnatal (P0-P14) rats. Low power images of P2X3-IR in lumbosacral spinal cord from P0 (A), P7 (C) and P14 (E). To demonstrate the location of the sacral parasympathetic nucleus (SPN) in these young rat pups, tissues were stained for neuronal nitric oxide synthase (nNOS, green; A-F). In animals from P0-P7, P2X3-IR was diffusely distributed in the dorsal horn being present in lamina II but in deeper laminae as well (A, C). In addition, dense P2X3-immunoreactive fiber bundles were present in the dorsal commissure (DCM; A, C). Higher power images (B, D, F) of the boxed regions in A, C, E that demonstrate P2X3-IR (red) in close association with nNOS-IR cells (green). Prominent P2X3-IR (red) was present in the lateral collateral pathway (LCP) of Lissauer extending from lamina I to the region of the SPN (B, D, F). Reduction in P2X3-IR in the DCM and LCP was apparent by P14 (E). By P14, P2X3-IR was more restricted to lamina II in the DH (E). Calibration bar represents 80 μm in A, C, E and 50 μm in B, D, F.

Figure 11

P2X3-IR in lumbosacral spinal cord from older postnatal (P21-P28) and adult (A) rats. Low power fluorescence images of the L6 spinal cord showing restricted P2X3-IR in the superficial dorsal horn (DH; A, C, E). Higher power fluorescence images of the dorsal, lateral quadrant of the L6 spinal cord showing restricted P2X3-IR in lamina II (B, D, F). Weak P2X3-IR was present in the lateral collateral pathway (LCP) of Lissauer in the lumbosacral spinal cord (F, arrows) of older postnatal and adult rats. P2X3-IR was observed in the anterior spinal artery (A, C, E, arrow). Calibration bar represents 100 μm in A, C, E and 60 μm in B, D, F.

Figure 12

A. The area occupied by P2X3-immunoreactive nerve fibers in three representative regions of the superficial dorsal horn (DH) in lamina II (lateral, medial and intermediate) of the L6 spinal cord was determined and averaged across spinal cord sections and ages analyzed. Only P2X3-immunoreactive nerve fibers (green) that exceeded the threshold level were quantified. B. Summary histogram of the percentage (%) of the DH area containing P2X3-immunoreactive nerve fibers during postnatal (P0-P36) development and adulthood of the rat. The percentage of the DH occupied by P2X3-immunoreactive nerve fibers significantly (*, p ≤ 0.001) increased from P7-P28 compared to adult. In addition, the percentage of the DH occupied by P2X3-immunoreactive nerve fibers was significantly (#, p ≤ 0.001) increased from P14-P21 compared to P0. Calibration bar represents 40 μm.