title: cGMP-generating cells in the bladder wall: identification of distinct networks of interstitial cells.
abstract: OBJECTIVE
  To identify cells which might contribute to the complex physiological responses of the guinea-pig bladder, and specifically to describe the distribution and types of cell in the bladder wall of the guinea pig which respond to nitric oxide (NO) with an increase in intracellular cGMP, i.e. putative interstitial cells (ICs).
  
  
  METHODS
  The whole bladder was removed from 11 male guinea pigs killed by cervical dislocation. Sections of the bladder wall, from the dome lateral wall and base, were isolated and incubated separately in Krebs' solution at 36 degrees C, gassed with 95% O(2) and 5% CO(2), and containing 1 mmol/L of the nonspecific phosphodiesterase inhibitor isobutyl-methyl-xanthene. Individual pieces of tissue were then exposed to 100 micromol/L of the NO donor NONOate for 10 min; control tissues remained in Krebs' solution. Tissues were then fixed in 4% paraformaldehyde and processed for immunohistochemistry. cGMP and neuronal NO synthase (nNOS) were subsequently visualized using appropriate primary and secondary antibodies.
  
  
  RESULTS
  Cells responding to NO with an increase in cGMP were detected in the dome, lateral wall and base, with positive cells in the thin outer surface of the wall (muscle coat), associated with muscle bundles in an outer layer of muscle, and in a region immediately beneath the urothelium. These cells (not urothelium, smooth muscle or vascular) are described as interstitial cells. Superficial urothelial umbrella cells were apparent and were strongly cGMP-positive. A high density of interstitial cells was associated with muscle bundles on the outer aspects of the wall, while few cells were detected on inner bundles. Thus there appeared to be two distinct types of muscle, inner and outer, with no obvious orientation of the fibres in each layer. Both muscle groups contained fibres expressing nNOS. In the outer muscle layer most of these fibres co-localized with cGMP, suggesting that different populations of nerves innervate each layer. There were more nNOS-positive fibres in the base of the bladder than in the dome. Three populations of cGMP-positive interstitial cells were associated with the outer muscle layer; cells in the outer surface (muscle coat interstitial cells, MC-ICs), cells on the surface of the bundles (superficial, SM-ICs) and cells within the muscle bundles (intramuscular, IM-ICs). The IM-ICs form a network in close apposition to the smooth muscle cells while the SM-ICs may connect adjacent muscle bundles and connect to the MC-ICs. Thus, there is a network linking potentially the muscle cells in the outer muscle bundles. cGMP-positive cells were also detected in the suburothelial layer (suburothelial, SU-ICs) which had a different structure to the cells associated with muscle, had a oval cell bodies with bifurcating processes and appeared to form a complex network; they were prevalent in the base and virtually absent in the dome.
  
  
  CONCLUSIONS
  There are structures within the bladder wall that can be identified and categorized by the ability of the constituent cells to increase intracellular cGMP in response to NO; these cells have been defined as ICs. Two distinct networks were identified, one associated with the outer muscle layers and another lying immediately beneath the urothelium, predominantly in the base of the bladder. The functions of these cells and networks are unknown; their possible roles in complex motor activity, urothelial signalling and bladder pathophysiology are discussed.
authors.affiliation: The Urophysiology Research Group, School of Surgical and Reproductive Sciences, The Medical School, The University, Newcastle upon Tyne, UK. j.i.gillespie@ncl.ac.uk
authors.forename: James I
authors.initials: JI
authors.lastname: Gillespie
authors.forename: Marjanne
authors.initials: M
authors.lastname: Markerink-van Ittersum
authors.forename: Jan
authors.initials: J
authors.lastname: de Vente
mesh_terms: D000818:Animals; D006152:Cyclic GMP / Q000378:metabolism*; D005453:Fluorescence; D006168:Guinea Pigs; D007150:Immunohistochemistry / Q000379:methods; D008297:Male; D009132:Muscles / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D001743:Urinary Bladder / Q000166:cytology / Q000378:metabolism*
chemical_list: D009569:Nitric Oxide; D006152:Cyclic GMP