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1 Smooth Muscle Research Centre, Dundalk Institute of Technology, Dublin Road, Dundalk, Co. Louth, Ireland
In the present study we used a combination of patch clamping and fast confocal Ca2+ imaging to examine the effects of activators of the nitric oxide (NO)/cGMP pathway on pacemaker activity in freshly dispersed ICC from the rabbit urethra, using the amphotericin B perforated patch configuration of the patch-clamp technique. The nitric oxide donor, DEA-NO, the soluble guanylyl cyclase activator YC-1 and the membrane-permeant analogue of cGMP, 8-Br-cGMP inhibited spontaneous transient depolarizations (STDs) and spontaneous transient inward currents (STICs) recorded under current-clamp and voltage-clamp conditions, respectively. Caffeine-evoked Cl– currents were unaltered in the presence of SP-8-Br-PET-cGMPs, suggesting that activation of the cGMP/PKG pathway does not block Cl– channels directly or interfere with Ca2+ release via ryanodine receptors (RyR). However, noradrenaline-evoked Cl– currents were attenuated by SP-8-Br-PET-cGMPs, suggesting that activation of cGMP-dependent protein kinase (PKG) may modulate release of Ca2+ via IP3 receptors (IP3R).When urethral interstitial cells (ICC) were loaded with Fluo4-AM (2 µM), and viewed with a confocal microscope, they fired regular propagating Ca2+ waves, which originated in one or more regions of the cell. Application of DEA-NO or other activators of the cGMP/PKG pathway did not significantly affect the oscillation frequency of these cells, but did significantly reduce their spatial spread. These effects were mimicked by the IP3R blocker, 2-APB (100 µM). These data suggest that NO donors and activators of the cGMP pathway inhibit electrical activity of urethral ICC by reducing the spatial spread of Ca2+ waves, rather than decreasing wave frequency.
(Received 1 March 2006;
accepted after revision 14 April 2006;
first published online 27 April 2006)
Corresponding author M. A Hollywood: Smooth Muscle Research Centre, Dundalk Institute of Technology, Dublin Road, Dundalk, Co. Louth, Ireland. Email: mark.hollywood{at}dkit.ie
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