TRPC1 is involved in receptor-activated calcium entry in rat aortic smooth muscle cells

Khalid Tai, Marie-Christine Hamaide, Huguette Debaix, Maurice Wibo, Nicole Morel Laboratoire de pharmacologie expérimentale – UCL 5410 - avenue Hippocrate 54, B1200 Brussels, Belgium.

In vascular smooth muscle, activation of plasma membrane ion channels is an important mechanism allowing for the increase in cytosolic calcium concentration and the development of contraction. In rat aorta, agonist-stimulated calcium entry is only partly inhibited by blockade of voltage-operated calcium channels (Ghisdal et al., 2003), suggesting that another calcium entry pathway is activated. Canonical transient receptor potential (TRPC) proteins have been proposed as candidates for receptor- or store-operated calcium channels in smooth muscle cells (Beech et al., 2004). In order to determine the role of TRPC1 in the calcium signal evoked by agonists in rat aorta smooth muscle cells (VSMC), its expression was inhibited by transfecting the cells with small interfering RNA directed against TRPC1 (siRNA-TRPC1). Functional evaluation was performed by measuring the calcium signal by fluorescence microscopy in fura-2 loaded VSMC. Male Wistar rats (150 g) were anaesthetised with ether and killed by decapitation. Aorta was quickly removed, cleaned from adherent tissue and endothelium was gently rubbed off. Primary culture of VSMC was performed by the explant technique. Investigation of the gene expression of TRPC isoforms in VSMC by RT-PCR revealed that TRPC1 was the most abundant. In cells transfected with siRNA-TRPC1, TRPC1 mRNA expression was inhibited by 72 ± 3 % (n=4). In contrast, transfection with nonsilencing control siRNA did not affect the level of expression of TRPC1 mRNA. Immunocytochemistry revealed that TRPC1 protein expression was attenuated by 86 ± 8 % in siRNA-TRPC1 transfected VSMC (n=39 cells) but was not modified in cells transfected with nonsilencing siRNA (n=26 cells). Measurement of calcium signal evoked by endothelin-1 (10 nM) in VSMC showed that, in the absence of external calcium, endothelin-1 induced a rapid but transient increase in cytosolic calcium, reflecting the release of intracellular calcium. Addition of calcium into the external solution in the presence of endothelin-1 evoked a smaller but more sustained increase in cytosolic calcium, which was completely blocked by the ET-A receptor antagonist BQ-123 (1 μM) and by Gd3+.(1 μM). VSMC transfection with siRNA-TRPC1 did not modify endothelin-1 evoked calcium release but abolished the increase in cytosolic calcium observed after the addition of calcium into the external solution. Calcium entry evoked by endothelin-1 was estimated by the quenching of fura-2 fluorescence with Mn 2+. It was markedly and similarly depressed in cells transfected with siRNA-TRPC1 and in cells pretreated with BQ-123.

These results indicate that TRPC1 channel protein is involved in receptor-activated calcium entry in aortic VSMC.

Beech D.J. et al. (2004)J. Physiol. 559, 685-706.
Ghisdal P. et al. (2003). J Physiol, 551, 855-867.