Inhibitory effect of vesamicol on vascular smooth muscle TRPC channels

Jian Shi1, Preet Chadha1, Rebecca Lever2, Anthony Albert1, James Moffatt1. 1St George's, University of London, London, UK, 2University College London, London, UK

In the course of unrelated experiments, we recently observed that the acetylcholine vesicular transport inhibitor vesamicol caused relaxation of preparations of vascular smooth muscle. In the present experiments we investigated the mechanism by which vesamicol exerted this effect.

Vesamicol caused maximal (98 ± 8%; n=5) relaxation of Wistar rat (male; 200-300g) mesenteric arteries maximally contracted with phenylephrine with a pEC50 of 5.53 ± 0.13; near maximal relaxation occurred at 10 µM. This effect could be washed out; preparations contracted equally well to phenylephrine and contractions were inhibited by vesamicol with the same pEC50 (5.52 ± 0.11; n=5). Vesamicol produced only minor relaxations (17.3 ± 9.5%; n=4) of preparations contracted by potassium-induced depolarisation, and only at very high concentrations (100 µM). When preparations were made permeable to Ca2+ ions with ionomycin, and after intracellular Ca2+ stores had been depleted, vesamicol had no effect on the remaining phenylephrine-induced contraction, although sodium nitroprusside (10 µM) elicited consistent and complete relaxation (n=5), albeit rather slowly. The phenylephrine-induced contraction that persisted in the presence of nifedipine (1 µM) was reversed by 88 ± 3% (n=4) by vesamicol (10 µM). These findings suggest that vesamicol blocks non-voltage-dependent calcium entry into smooth muscle cells. Vesamicol had no effect on contracted preparations of rat aorta, trachea, bladder or stomach, suggesting a degree of tissue selectivity.

In outside out patches of membranes from freshly isolated rat mesenteric arterial smooth muscle cells held at -70 mV, application of noradrenaline induced cation channel currents with a unitary amplitude of about -0.2 pA. The single channel properties of these 10 µM noradrenaline-evoked cation channel currents are similar to those features of TRPC1-containing channels previously described in different vascular preparations. Co-application of 10 µM vesamicol significantly inhibited the mean open probability (NPo) of 10 µM noradrenaline-evoked cation channel activity from 0.30 ± 0.03 to 0.02 ± 0.01 (n=7, p<0.05). TRPC1-containing channels have distinct activation mechanisms compared to non-TRPC1-containing channels; stimulation of protein kinase C (PKC) is obligatory and not inhibitory for channel gating. Bath application of the PKC activator, PDBu (10 µM), stimulated cation channel currents in outside-out patches held at -70 mV, which also had an unitary amplitude of about -0.2 pA. Moreover, co-application of 10 µM vesamicol significantly attenuated the mean NPo of PDBu-evoked cation channel activity from 0.25 ± 0.03 to 0.01 ± 0.01 (n=5, p<0.05)

These results suggest that vesamicol is a selective inhibitor of TRPC channels, and of TRPC1-containing channels in particular.