Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC360
Effect of modulators of the nitric oxide/cGMP pathway on L-type Ca2+ current in rabbit corpus cavernosum smooth muscle cells.
C. Doyle1, G. P. Sergeant1, M. A. Hollywood1, N. G. McHale1, K. D. Thornbury1
1. Smooth Muscle Research Centre, Dundalk Institute of Technology, Dundalk, Ireland.
Although it is well known that nitric oxide (NO) induces erection by inhibiting tone in corpus cavernosum smooth muscle cells (CCSMC), the mode of action is not yet fully elucidated. It has been suggested that an important contributory factor is inhibition of L-type Ca2+ current, although this has never been directly tested in CCSMC1. The purpose the present study was to examine the effect of an NO donor and several modulators of the NO/cGMP pathway on L-type Ca2+ current in rabbit CCSMC. Male rabbits were humanely killed and their penises removed. The corpus cavernosum was cut into 1 mm3 pieces and CCSMC isolated using a collagenase/protease mixture2. Cells were superfused with physiological saline at 37°C and studied using the perforated patch voltage clamp technique2. YC-1 (30 μM), an activator of soluble guanylate cyclase, reversibly reduced the L-type current evoked by stepping from -60 to 0 mV (from -129±40 pA to -79±23 pA, p<0.05, paired t test, n=8). This effect was completely blocked by ODQ (30 μM), an inhibitor of soluble guanylate cyclase (-132±31 in ODQ and -125±29 in ODQ + YC-1, n.s., n=8). Similar results were obtained with a NO donor, DEA-NO (30 μM; current reduced from -104±14 pA to -80±13 pA, p<0.05, n=9) and 8-bromo cGMP (1 mM; from -83±21 pA to -70±22 pA, p<0.05, n=6). When the effect of sildenafil (1 μM) was examined it was found to produce only a small reduction in the current (from -132±28 pA to -116±25 pA, p<0.05, n=7) and even at the supratherapeutic concentration of 10 μM the effect was modest (reduced from -160±32 pA to -123±24 pA, p<0.05, n=5). Indeed the effects of the all of the above compounds on L-type Ca2+ current were modest, compared to their more potent effects on blocking intracellular Ca2+ waves, in CCSMC3, suggesting that the latter mechanism may be more important. For comparison, we examined the effect of phenylephrine (PE, 10 μM), a potent tone-inducing agonist in the corpus cavernosum. Similar to the above agents, PE caused a small reduction in L-type Ca2+ current (from -197±35 pA to -123±21 pA, p<0.05, n=8). However, despite their similar actions on L-type Ca2+ current, PE and YC-1 had markedly contrasting effects on spontaneous depolarisations (SD) in CCSMC studied in current clamp recordings. SDs have been previously shown to be mediated by Ca2+ waves activating calcium-activated Cl- currents in these cells3. PE (10 μM) increased the frequency of SDs from 37±6 min-1 to 65±4 min-1 (p<0.05, n=5), while YC-1 (30 μM) reduced their frequency from 41±8 min-1 to 5±2 min-1 (p<0.05, n=4). In conclusions, these results suggest that inhibition of L-type Ca2+ current plays only a minor role, if any, in mediating the inhibitory effects of the NO/cGMP pathway on CCSMC.
Where applicable, experiments conform with Society ethical requirements