PurposeWe investigated the 3-dimensional morphological arrangement of KIT positive interstitial cells of Cajal in the human bladder and explored their structural interactions with neighboring cells.Materials and MethodsHuman bladder biopsy samples were prepared for immunohistochemistry/confocal or transmission electron microscopy.ResultsWhole mount, flat sheet preparations labeled with anti-KIT (Merck, Darmstadt, Germany) contained several immunopositive interstitial cell of Cajal populations. A network of stellate interstitial cells of Cajal in the lamina propria made structural connections with a cholinergic nerve plexus. Vimentin positive cells of several morphologies were present in the lamina propria, presumably including fibroblasts, interstitial cells of Cajal and other cells of mesenchymal origin. Microvessels were abundant in this region and branched, elongated KIT positive interstitial cells of Cajal were found discretely along the vessel axis with each perivascular interstitial cell of Cajal associated with at least 6 vascular smooth muscle cells. Detrusor interstitial cells of Cajal were spindle-shaped, branched cells tracking the smooth muscle bundles, closely associated with smooth muscle cells and vesicular acetylcholine transferase nerves. Rounded, nonbranched KIT positive cells were more numerous in the lamina propria than in the detrusor and were immunopositive for anti-mast cell tryptase. Transmission electron microscopy revealed cells with the ultrastructural characteristics of interstitial cells of Cajal throughout the human bladder wall.ConclusionsThe human bladder contains a network of KIT positive interstitial cells of Cajal in the lamina propria, which make frequent connections with a cholinergic nerve plexus. Novel perivascular interstitial cells of Cajal were discovered close to vascular smooth muscle cells, suggesting interstitial cells of Cajal-vascular coupling in the bladder. KIT positive detrusor interstitial cells of Cajal tracked smooth muscle bundles and were associated with nerves, perhaps showing a functional tri-unit controlling bladder contractility.

Rabbit urethral smooth muscle cells were studied at 37 degrees C by using the amphotericin B perforated-patch configuration of the patch-clamp technique, using Cs(+)-rich pipette solutions. Two components of current, with electrophysiological and pharmacological properties typical of T- and L-type Ca(2+) currents, were recorded. Fitting steady-state inactivation curves for the L current with a Boltzmann equation yielded a V(1/2) of -41 +/- 3 mV. In contrast, the T current inactivated with a V(1/2) of -76 +/- 2 mV. The L currents were reduced by nifedipine (IC(50) = 225 +/- 84 nM), Ni(2+) (IC(50) = 324 +/- 74 microM), and mibefradil (IC(50) = 2.6 +/- 1.1 microM) but were enhanced when external Ca(2+) was substituted with Ba(2+). The T current was little affected by nifedipine at concentrations <300 nM but was increased in amplitude when external Ca(2+) was substituted with Ba(2+). Both Ni(2+) and mibefradil reduced the T current with an IC(50) = 7 +/- 1 microM and approximately 40 nM, respectively. Spontaneous electrical activity recorded with intracellular electrodes from strips of rabbit urethra consisted of complexes comprising a series of spikes superimposed on a slow spontaneous depolarization (SD). Inhibition of T current reduced the frequency of these SDs but had no effect on either the number of spikes per complex or the amplitude of the spikes. In contrast, application of nifedipine failed to significantly alter the frequency of the SD but reduced the number and amplitude of the spikes in each complex.

The urethra generates sufficient tone to prevent leakage from the bladder and thus plays an important role in maintaining urinary continence. Despite this central role, relatively little is known about the mechanisms that underlie the generation and modulation of urethral tone, although it can be influenced by a number of factors including blood flow through the lamina propria . There is little doubt that a myogenic mechanism also contributes significantly to urethral tone, since it is unaffected in vitro by nerve blockade in a variety of species including pigs (Bridgewater et al. 1993), sheep (Thornbury et al. 1992, rats (McKeag et al. 2001) and humans .A number of studies have demonstrated that urethral myogenic tone is critically dependent on the influx of Ca 2+ across the cell membrane, since removal of external Ca 2+ or inhibition of L-type Ca 2+ channels reduces tone significantly in rats, humans and pigs in vitro (Bridgewater et al. 1993;Brading, 1999;Shafei et al. 2003). Shafei et al. (2003) have demonstrated that application of nifedipine or Ni 2+ significantly reduces tone in an isolated rat whole urethra preparation, suggesting that Ca 2+ influx through both T and L channels contributes to urethral tone. Recent studies by Bradley et al. (2003) have characterised the Ca 2 currents in isolated rabbit urethral myocytes and demonstrated the existence of currents with biophysical and pharmacological properties typical of L-and T-type Ca 2+ currents in arterial (Benham et al. 1987), venous (Yatani et al. 1987) and bladder myocytes (Sui et al. 2001).To date, no study has examined successfully the electrophysiology of human urethral myocytes, presumably because of the poor availability of suitable tissue and the difficulty of obtaining viable cells from small biopsy samples. In this study we provide the first electrophysiological data from freshly dispersed human myocytes obtained from adults undergoing treatment for bladder or prostate cancer. Our results suggest that human urethral myocytes possess Ca 2+ currents with electrophysiological and pharmacological properties typical of T and L channels (for reviews see Kotlikoff et al. 1999;Perez-Reyes, 2003 The purpose of the present study was to characterise Ca 2+ currents in smooth muscle cells isolated from biopsy samples taken from the proximal urethra of patients undergoing surgery for bladder or prostate cancer. Cells were studied at 37°C using the amphotericin B perforated-patch configuration of the patch-clamp technique. Currents were recorded using Cs

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