Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, PC355

Poster Communications

Responses of ICC populations in the bladder to electrical field stimulation suggests functional innervation

S. M. Gray1, G. J. McGeown2, G. McMurray3, K. D. McCloskey1

1. Centre for Cancer Research and Cell Biology, Queen's University, Belfast, Belfast, Northern Ireland, United Kingdom. 2. Centre for Vision and Vascular Sciences, Queen's University Belfast, Belfast, United Kingdom. 3. Pfizer UK, Sandwich, United Kingdom.

Populations of interstitial cells of Cajal (ICC) in the bladder comprise interconnected networks of stellate shaped ICC-LP in the lamina propria and elongated, branched intramuscular ICC-IM in the detrusor. ICC-LP and ICC-IM lie in close proximity to nerves; moreover they respond to exogenous agonists by firing Ca2+-transients (1, 2). While this implies neuronal regulation of ICC activity, there is currently little evidence of functional innervation. This study aimed to investigate whether ICC are directly innervated in bladder sheet preparations containing urothelium, smooth muscle, nerves and ICC. Bladders from male guinea-pigs were opened longitudinally and the mucosa removed from the underlying detrusor by sharp dissection. Mucosal preparations were pinned over 2 silver electrodes in a recording chamber and loaded with the calcium indicator fluo-4 AM. Electrical field stimulation (EFS) delivered trains (10.5s) of 0.2ms pulses of 40V at frequencies 0.5, 2, 5, 10Hz. Calcium activity (dF/F0) was recorded with an electron multiplying CCD camera imaging system. Data is expressed as mean±SEM; n refers to number of preparations. Fluo-4AM loaded preparations contained ICC-LP, ICC-IM and detrusor smooth muscle (DSM) readily identified by their distinctive morphologies. Prior to stimulation, in the majority of preparations, the three cell types exhibited characteristic patterns of spontaneous Ca2+-signalling (3). Responses to EFS were abolished by 1µM TTX. In DSM, EFS at 0.5Hz, 2Hz or 5Hz evoked a prolonged rise in baseline dF/F0 of 0.89±0.23 (n=7), 0.94±0.18 (n=8) and 1.18±0.44 (n=3) respectively, and temporarily prevented spontaneous activity which typically returned after several minutes. ICC-IM responded to EFS (0.5Hz or 2Hz) with an increase in baseline Ca2+ or an increase in amplitude and duration of Ca2+ transients compared with pre-stimulation events. Similar results were obtained in 4 representative experiments. Spontaneous Ca2+-transients in cells from a group of ICC-LP (within a field of view) appeared to be synchronized by 0.5Hz and 2Hz EFS with several cells firing within 2.35±1.13s (n=3) and 1.78±0.66s (n=5) of each other respectively. Prior to stimulation, there was little apparent synchronization of events. ICC-LP also responded to 10Hz with a large, prolonged rise in intracellular Ca2+, which occurred after a delay of 3.16 ± 0.47s (n=15) from the onset of EFS. These results demonstrate that bladder ICC subpopulations respond to EFS and are thus likely to be functionally innervated. The synchronicity of ICC-LP following EFS suggests that they could act as a functional network, and serve as targets for parasympathetic neurotransmission, which may be integral in regulating activity within the bladder.

Where applicable, experiments conform with Society ethical requirements