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

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 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.