Bioactive sphingolipids and their metabolites such as sphingosylphosphorylcholine (SPC) are believed to play an important role in regulating various cellular processes, primarily via receptors of the EDG family. Recent evidence has suggested a role for sphingolipids in the control of vascular tone, but the signalling pathways involved may differ significantly depending on the vascular bed, with calcium entry, release from stores, and activation of RhoA and Rho kinase having variable degrees of importance (Coussin et al. 2002; Shirao et al. 2002). The action of sphingolipids has not been investigated in the pulmonary circulation, where there is evidence that Rho kinase may be of particular importance. We therefore examined the effect of SPC on small (300-500 µm i.d.) intrapulmonary arteries (IPA) of the rat.

Rats were humanely killed by cervical dislocation. IPA were mounted on a small vessel myograph. In some cases intracellular calcium was estimated simultaneously using Fura-PE3. Data are given as means ± S.E.M. and were tested for significance using ANOVA.

SPC (3-100 µM) caused a relatively slowly developing, concentration-dependent vasoconstriction in IPA, which reached a plateau after ~30 min. The maximum tension was 111 ± 15 % of the response to 80 mM KCl (n = 7), with an EC₅₀ of 18 ± 6 µM. Removal of the endothelium or application of 100 µM L-NAME caused ~20 % increase in tension without altering the EC₅₀ (n = 4 and 7). Simultaneous recording of tension and intracellular calcium showed that the development of tension was associated with a rise in intracellular calcium, although tension continued to increase after calcium reached a steady state. Removal of calcium from the bathing solution shifted the concentration-response curve to the right (EC₅₀: 97 ± 43 µM, n = 6, P < 0.05), without having a significant effect on maximum tension (135 ± 39 %, n = 6). Once tension had reached steady state following application of 10 µM SPC, application of the L-type calcium channel blocker diltiazem (10 µM) had a relatively minor effect on tension and intracellular calcium. However, the Rho kinase inhibitor Y27632 (10 µM) caused partial relaxation of ~35 % (n = 5).

We conclude that SPC-induced constriction of rat IPA is mediated through both calcium entry, primarily via a voltage-independent pathway, and Rho kinase-dependent calcium sensitisation, but that different receptors may be responsible for these two actions.

This work was funded by the Wellcome Trust