Role of gap junctions in spontaneous activity of the rat bladder

Abstract

Increased gap junction expression in lamina propria myofibroblasts and urothelial cells may be involved in detrusor overactivity, leading to incontinence. Immunohistochemistry was used to compare connexin (Cx) 26, 43, and 45 expression in the bladders of neonatal, adult, and spinal cord-transected rats, while optical imaging was used to map the spread of spontaneous activity and the effects of gap junction blockade. Female adult Sprague-Dawley rats were deeply anesthetized, a laminectomy was performed, and the spinal cord was transected (T8/T9). After 14 days, their bladders and those of age-matched adults (4 mo old) and neonates (7-21 day old) were excised and studied immunohistochemically using frozen sections or optically using whole bladders stained with voltage- and Ca(2+)-sensitive dyes. The expression of Cx26 was localized to the urothelium, Cx43 to the lamina propria myofibroblasts, and Cx45 to the detrusor smooth muscle. While the expression of Cx45 was comparable in all bladders, the expression of Cx43 and Cx26 was increased in neonate and transected animals. In the bladders of adults, spontaneous activity was initiated at multiple sites, resulting in a lack of coordination. Alternatively, in neonate and transected animals spontaneous activity was initiated at a focal site near the dome and spread in a coordinated fashion throughout the bladder. Gap junction blockade (18beta-glycyrrhetinic acid, 1 microM) abolished this coordinated activity but had no effect on the uncoordinated activity in adult bladders. These data suggest that coordinated spontaneous activity requires gap junction upregulation in urothelial cells and lamina propria myofibroblasts.

Figures

Fig. 1

Effect of 18β-glycyrrhetinic acid on intravesical bladder pressure. Intravesical pressure recordings from normal adult (A), spinal cord-injured adult (SCI; B) and normal neonatal (C) rat bladders. At the first break, 10 μM 18β-glycyrrhetinic acid was added to the bath fluid and recordings were made ~15 min later. The agent was washed out at the second break, and a similar interval was left before recording was resumed.

Fig. 2

Intracellular [Ca2+] and membrane potential, Em, recordings from the ventral surface of rat bladders. A:16 × 16 array of Ca2+ transients from a normal adult bladder. Bottom: isochronal map from the transient array. Isochronal intervals, 92 ms. B: corresponding Em transients from the same recording, with the derived isochronal map below. Isochronal intervals, 8 ms. C: isochronal map of Ca2+ transients from a SCI adult bladder. Isochronal intervals, 42 ms. D: corresponding Em transients from the same recording, with the derived isochronal map. Isochronal intervals, 4 ms.

Fig. 3

Effect of 18β-glycyrrhetinic acid on Ca2+ transient isochronal maps. A: control isochronal map of a normal adult bladder and a comparison isochronal map of the effect of 100 μM 18β-glycyrrhetinic acid. In normal adult bladders, there was a disorganized spread of activity that was not affected by 18β-glycyrrhetinic acid. B: control isochronal map of a spinal cord-transected rat bladder and the effect of 1 μM and 10 mM 18β-glycyrrhetinic acid on the same bladder. The activity originated from a single focus that spread across the bladder surface. In the presence of 18β-glycyrrhetinic acid, activity was severely curtailed at 1 mM, and abolished completely at 10 μM, as seen by an absence of isochrones. The outline demarcates the visible boundary of the bladder.

Fig. 4

Connexin 43 (Cx43) labeling in the urothelial/suburothelial layer of the bladder wall: green, Cx43; blue, nuclei; red, smooth muscle. A: labeling in the normal adult bladder; the urothelium (uro), and suburothelium (suburo) layer are demarcated. An invagination of the lumen with no nuclear stain can be seen in the top of the section. The white box indicates an area used for analysis. See text for details. B: labeling in the SCI adult bladder. C: plot of normalized Cx43 labeling in sections from normal and SCI adult rats. Median data (25%, 75% interquartiles). *P < 0.05 vs. normal adult bladder. D: labeling in the normal neonatal bladder. Magnification ×400 for all sections.

Fig. 5

Cx labeling in the detrusor layer of the bladder wall: green, Cx; blue, nuclei; red, smooth muscle. A: Cx43 labeling in a section from a SCI animal. B: Cx45 labeling in a section from a normal adult rat bladder. Magnification ×400 for all sections.

Fig. 6

Cx26 labeling in the bladder wall: green, Cx26; blue, nuclei; red, smooth muscle. A: normal adult bladder. B: neonatal bladder. C: SCI adult bladder. D: normalized Cx26 labeling in samples from the 3 bladder groups. Median data (25%, 75% interquar-tiles). *P < 0.05 vs. normal adult bladder. Magnification ×400 for all sections.