Recent studies have shown that specialised pacemaking cells, resembling the interstitial cells of Cajal (ICC) in the gastrointestinal tract, are present in the walls of the lower urinary tract (Sergeant et al. 2000). While ICC have not yet been identified in the bladder wall, an investigation on the origin of spontaneous activity in the guinea-pig bladder reported that it originated in cells located on the boundary of smooth muscle bundles (Hashitani et al. 2001). The purpose of the present investigation was to establish whether interstitial-like cells exist in the bladder using specific ICC antibodies and confocal and multiphoton fluorescence imaging.

Guinea-pigs were killed by cervical dislocation, and bladders were removed and pinned out, mucosal surface uppermost, on sylgard-filled dishes. The mucosa was removed by sharp dissection, tissues were fixed in 4 % formaldehyde followed by incubation in an antibody to the ICC-specific marker, ckit, and then incubated in fluorescent secondary antibodies. Examination of whole mount preparations revealed that ckit-positive cells were arranged in diagonal orientations throughout the bladder wall (n = 10). Double labelling with anti-smooth muscle myosin (n = 4) revealed that the ckit-positive cells were located on the boundary of smooth muscle bundles, running in parallel with the smooth muscle cells.

Enzymatic dissociation of fresh, bladder tissues yielded a heterogeneous population of cells, comprising spindle-shaped smooth muscle cells and cells having a stellate or branched morphology reminiscent of ICC in other preparations. After loading with fluo-4 AM (1 µM), the ICC-like cells were studied using a confocal microscope acquiring images at a rate of 6 frames per second. Application of 10 µM carbachol or 10 mM caffeine evoked Ca²⁺ waves, typically starting from a focal point and spreading axially or bi-axially along the length of the cell. Such responses were either biphasic or consisted of a complex of transients having mean durations of 17.1 ± 3.15 s (S.E.M.) in carbachol and 26.8 ± 1.53 s (S.E.M.) when evoked by caffeine. In addition, cells were observed to fire Ca²⁺ waves spontaneously in the absence of stimulation by drugs (13.5 ± 0.63 waves min^-1, n = 5).

In conclusion, we have identified a subpopulation of ckit-positive cells in the guinea-pig bladder, which are immunohistochemically distinct from smooth muscle cells and found in a location indicative of a physiological function either in pacemaking or in the mediation of neurotransmission.Financial support from The Wellcome Trust is gratefully acknowledged.

Hashitani, H., Fukuta, H., Takano, H., Klemm, M.F. & Suzuki, H. (2001). J. Physiol. 530, 273-286. abstract

Sergeant, G.P., Hollywood, M.A., McCloskey, K.D., Thornbury, K.D. & McHale, N.G. (2000). J Physiol. 526, 359-366.