Origin of spontaneous activity in neonatal and adult rat bladders and its enhancement by stretch and muscarinic agonists

Abstract

This study examined the origin of spontaneous activity in neonatal and adult rat bladders and the effect of stretch and muscarinic agonists and antagonists on spontaneous activity. Rats were anesthetized and their bladders were excised, cannulated, and loaded with voltage- and Ca(2+)-sensitive dyes. Intracellular Ca(2+) and membrane potential transients were mapped using photodiode arrays in whole bladders, bladder sheets, or cross-section preparations at 37 degrees C. Intravesical pressure was recorded from whole bladders. In neonatal bladders and sheets, spontaneous Ca(2+) and electrical signals arose at a site near the dome and spread in a coordinated manner throughout the bladder with different dome-to-neck conduction velocities (Ca(2+): 3.7 +/- 0.4 mm/s; membrane potential: 46.2 +/- 3.1 mm/s). In whole bladders, optical signals were associated with spontaneous contractions (10-20 cmH(2)O). By contrast, in adult bladders spontaneous Ca(2+) and electrical activity was uncoordinated, originating at multiple sites and was associated with smaller (2-5 cmH(2)O) contractions. Spontaneous contractions and optical signals were insensitive to tetrodotoxin (2 muM) but were blocked by nifedipine (10 muM). Stretch or low carbachol concentrations (50 nM) applied to neonatal whole bladders enhanced the amplitude (to 20-35 cmH(2)O) of spontaneous activity, which was blocked by atropine. Bladder cross sections revealed that Ca(2+) and membrane potential transients produced by stretch or carbachol began near the urothelial-suburothelial interface and then spread to the detrusor. In conclusion, spontaneous activity in neonatal bladders, unlike activity in adult bladders, is highly organized, originating in the urothelium-suburothelium near the dome. Activity is enhanced by stretch or carbachol and this enhancement is blocked by atropine. It is hypothesized that acetylcholine is released from the urothelium during bladder filling to enhance spontaneous activity.

Fig. 1

Optical system to record intracellular Ca²⁺ and membrane potential signals from bladder preparations. A: optical system showing the arrangement of the light source, filters, and photodiode arrays. B: neonatal bladder observed in situ with a 16 × 16 array grid superimposed to show the individual photodiode recording fields. C: Ca²⁺ signals recorded from the photodiode array, the observed transient signals correspond to the field occupied by the bladder surface in B. The recording duration was 60 s. D: isochronal map derived from the data in C. The initiation site was identified as the individual signal in C with the earliest Ca²⁺ transient (“focal point” in D). The neck-to-dome axis is shown from top to bottom. Isochrones represent successive conduction delays of 44 ms (D).

Fig. 2

Intravesical pressure transients recorded from a whole adult bladder before and after addition of 2 μM tetrodotoxin (TTX). The period of electrical stimulation (EFS; 20-s trains, 50 Hz, 0.25 ms, 140-V pulses) is shown.

Fig. 3

Intravesical pressure transients recorded from a neonatal bladder before and after addition of 2 μM TTX. The layout and period of electrical stimulation are similar to that of Fig. 2.

Fig. 4

Ca²⁺ and membrane potential transients in the neonatal rat bladder. Top traces: successive Ca²⁺ transients above the corresponding isochronal maps. Middle trace: membrane potential transient and the associated isochronal map corresponding to middle Ca²⁺ transient above. Bottom traces: spontaneous intravesical pressure changes, with the middle 3 corresponding to the Ca²⁺ and the middle one corresponding to the membrane potential transients shown above. Isochrones represent successive conductions delays of 44 ms for the Ca²⁺ array and 4 ms for the membrane potential array.

Fig. 5

Ca²⁺ and membrane potential transients in the adult rat bladder. The layout is similar to that of Fig. 4. Isochrones represent successive conductions delays of 89 ms for the Ca²⁺ array and 6 ms for the membrane potential array.

Fig. 6

Ca²⁺ maps in the presence of 2,3-butanedione monoxamine (BDM). Bottom: effect of 5 mM BDM on the pressure recording from an intact neonatal rat bladder. Top: spontaneous Ca²⁺ trace data superimposed over isochronal maps generated from these intracellular Ca²⁺ transients. While BDM abolished smooth muscle contractile activity, and therefore bladder pressure generation, spontaneous Ca²⁺ transients persisted.

Fig. 7

Ca²⁺ maps in sheets of bladder from neonatal (A–D) and adult (E–G) rats. A: bladder sheet from a neonatal rat with the mucosal face uppermost and a superimposed photodiode array grid. The positions of the dome and neck of the bladder are shown. B and E: isochronal Ca²⁺ maps after stretch (0.8 mm or 10%) in the neck-dome axis, indicated by the double headed arrows. C and F: isochronal Ca²⁺ maps after the topical application of carbachol (50 nM), indicated by *. D and G: spontaneous isochronal Ca²⁺ maps. Isochrones represent successive conduction delays of 44 ms (B–D) and 89 ms (E–G).

Fig. 8

Ca²⁺ maps in bladder transverse sections from neonatal (A–C) and adult (E–F) rats. A: transverse section of a neonatal rat bladder, the mucosal and serosal surfaces are shown. B and E: isochronal Ca²⁺ maps after point mechanical stimulation at the sites marked by *. C and F: isochronal Ca²⁺ maps after topical application of carbachol (50 nM). D: diagram of the bladder wall on the same transverse scale as in A–C and E–F. Isochrones represent successive conduction delays of 7 ms (B–C) and 15 ms (E–F).