In normoxic conditions, 5-HT induces contraction and proliferation in pulmonary artery (MacLean et al. 2000). Plasmatic concentration of 5-HT is increased in patients with primary pulmonary hypertension (Kéreveur et al. 2000). Since intracellular calcium is strongly involved in contraction, we investigated the effect of 5-HT on intracellular calcium concentration ([Ca²⁺] _i ) in primary cultured smooth muscle cells from rat small intrapulmonary artery (IPA) in normoxia and chronic hypoxia.

Wistar male rats were either housed in room air at a normal atmospheric pressure (101 kPa) or kept in a hypobaric chamber for 3 weeks at a pressure of 50.5 kPa. Rats were then humanely killed according to national guidelines. IPA were isolated from the left lung by dissection. Smooth muscle cells (SMC) were enzymatically isolated and primary cultured (48-72 hours) before they were loaded with the calcium-sensitive fluorescent dye fura-PE3 (2 µM). All results are expressed as the mean ± S.E.M.; significance was tested with unpaired Student’s t test with P < 0.05 and n represents the number of cells.

Calcium signal to 5-HT 10 µM was characterized by a transient followed by a sustained phase. The amplitude of these both phases were decreased in chronic hypoxia (change in 345/380 ratio, ΔR = 0.08 ± 0.01 vs. 0.2 ± 0.02 in control for the transient phase, ΔR = 0.02 ± 0.003 vs. 0.06 ± 0.01 in control for the sustained phase, n = 69 and 173 respectively). An antagonist of the 5-HT _2A receptor, ritanserin 0.1 µM blocked the calcium signal to 5-HT 10 µM in normoxia (n = 22) and R-DOI 10 µM, a 5-HT _2A agonist increased [Ca²⁺]_i in the same manner than 5-HT (n = 38). In chronic hypoxia, the calcium signal to 5-HT was also blocked by ritanserin. Calcium sources involved in the response to 5-HT were then investigated. In normoxia, the calcium response to 5-HT was not modified by thapsigargin 1 µM, which depletes intracellular calcium store (n = 28). The sustained phase was suppressed by bath perfusion with a calcium free solution (n = 9) or RHC80267 50 µM (n = 31), an inhibitor of the diacylglycerol lipase which thus inhibits the arachidonic acid production. In chronic hypoxia, both phases of the calcium signal to 5-HT were fully blocked by a calcium free solution (n = 42). Immunolabelling of the SMC with antibodies against proteins coding for non-voltage gated calcium permeable channels (TRPC or transient receptor potential channels) demonstrated the presence of TRPC1, 3, 4 and 6 and TRPC 3, 4, 5 and 6 in normoxic and hypoxic rats respectively. There was no labelling for TRPC1 in hypoxic rats.

In conclusion, 5-HT activates 5-HT _2A receptors and induces a calcium signal which is independent on intracellular calcium stores but strongly depends on extracellular calcium in normoxic and hypoxic rats. TRPCs appeared to be present and may be responsible for the calcium influxes.

This work was funded by INSERM and Conseil Regional d’Aquitaine. L.R. was supported by Fondation pour la Recherche Medicale (FRM).