An increase in endothelial cell calcium ([Ca²⁺]_i) is an obligatory precursor to endothelium-dependent relaxations (Nilius & Droogmans, 2001). The principal candidate channels for this influx are the mammalian transient receptor protein (TRP) homologues, the TRPC family. These channels may be stimulated by receptor and/or store-depletion-dependent mechanisms. The contribution of endothelial TRPC channels to relaxation of rat isolated mesenteric artery was investigated using flufenamic acid (FFA) and SKF96365. These compounds are reported to modulate TRPC function (Halaszovich et al. 2000; Inoue et al. 2001). The expression and localization of these TRPC channels was further investigated using RT-PCR and immunoconfocal microscopy.

Male Wistar rats (200-225 g) were stunned and killed by cervical dislocation. Second-order mesenteric arteries were dissected free, cleaned of connective tissue and mounted in a Mulvany-Halpern wire myograph under normalized tension for isometric recording of force. Arteries were preconstricted with phenylephrine (PE) to 70 % of maximum force (2-5 mM). The application of FFA and SKF96365 to these arteries abolished PE-induced tone in endothelium-intact and endothelium-denuded arteries in a concentration-dependent manner (n = 7 and n = 8, respectively).

Sequence-specific primers were designed to rat TRPC1-7. Using RNA isolated from whole arterial homogenates the expression of TRPC mRNA was investigated. RT-PCR demonstrated the expression of TRPCs 1, 3 and 6 (n = 3). Appropriate positive and negative controls were carried out for each experiment. Immunoconfocal microscopy was used to investigate the localization of TRPC 1, 3 and 6 in fixed arterial sections. TRPC1 and TRPC6 were shown to be expressed in both smooth muscle and endothelial cells. However, expression of TRP3 appeared to be restricted to endothelial cells.

The non-specific effects of FFA and SKF96365 on PE-induced tone demonstrate the unsuitability of wire myography for the study of arterial TRPC channels. Further experiments will investigate the effects of these compounds on Ca²⁺ handling in isolated endothelial cells. Both RT-PCR and immunoconfocal microscopy demonstrate the expression of arterial TRPCs 1, 3 and 6. In conclusion, our data suggest that TRPC1, 3 and 6 are the principal TRPC channels expressed in rat mesenteric arteries. We suggest these channels are candidates involved in [Ca²⁺]_i influx necessary for endothelium-dependent relaxations.

All procedures accord with current UK legislation.