Activation of the cGMP/PKG pathway inhibits electrical activity in rabbit urethral interstitial cells of Cajal by reducing the spatial spread of Ca2+waves

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Abstract
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 IP3receptors (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.