C2-ceramide induces vasodilation in phenylephrine-induced pre-contracted rat thoracic aorta: role of RhoA/Rho-kinase and intracellular Ca2+ concentration.

It is known that ceramide may play an important regulatory role in vascular tone although its effect on vascular tone and the mechanisms involved are controversial. The present study was designed to investigate the effects of ceramide and its key initial regulators, TNF-alpha and neutral sphingomyelinase (SMase), on vascular tone of isolated rat thoracic aortic rings and elucidate the mechanisms involved in the changes in vascular tone induced by ceramide. Contractile responses and Fura-2 Ca2+ signals were measured in rat thoracic aortic rings or strips. 10(-5) M C2-ceramide, 0.1 U/ml neutral sphingomyelinase (SMase), and 5x10(-7) g/ml TNF-alpha had no effect on resting tone in rat thoracic aortic rings. However, in phenylephrine-induced pre-contracted rings, treatment with ceramide, SMase, and TNF-alpha evoked a gradual but sustained vasodilation. Vasodilation effect in response to 10(-5) M C2-ceramide was not significantly changed by the absence or presence of endothelium, a cyclooxygenase pathway inhibitor (10(-6) M indomethacin), or PKC inhibitors (10(-5) M H-7 & 5x10(-7) M calphostin-C). Pretreatment with 1 microM Y-27632, a RhoA/Rho-kinase inhibitor, significantly inhibited the phenylephrine-induced contraction itself as well as the C2-ceramide-induced vasodilation. Pre-treatment with 10(-5) M C2-ceramide had no effect on phasic rise in [Ca2+]i and tension evoked by stimulation with 10(-8) M phenylephrine, but post-treatment of C2-ceramide significantly reduced the phenylephrine-induced secondary tonic [Ca2+]i and tension plateau. Our results indicate that C2-ceramide induces vasodilation in phenylephrine-induced pre-contracted rat thoracic aorta. Furthermore, inhibition of phenylephrine-induced activation of RhoA/Rho-kinase pathway as well as phenylephrine-induced elevations in [Ca2+]i are clearly a key factors in C2-ceramide-induced vasodilation.