Cell-Culture–Dependent Change of Ca²⁺ Response of Rat Aortic Myocytes to Sphingosine-1-Phosphate

Abstract

We characterized the effects of sphingosine-1-phosphate (S1P) on rat aortic myocytes with or without culture. Application of S1P induced a small Ca²⁺ response in 40% freshly dispersed aortic myocytes, whereas S1P caused a larger Ca²⁺ response in 90% myocytes cultured for 72 h. Concentration–response relationships of S1P in cultured myocytes were significantly different from that in non-cultured myocytes. Analysis of the expression of S1P-receptor mRNA transcripts revealed that S1P-receptor type 3 (S1P₃) was significantly increased when myocytes were cultured for 24 h. Neither the removal of serum from culture medium nor pretreatment with pharmacological agents, such as ERK, Rho, and PI3 kinase inhibitors, affected the progression of the S1P-induced Ca²⁺ response during culture. The sustained component of the Ca²⁺ response to S1P was sensitive to the removal of external Ca²⁺ and was effectively inhibited by inorganic Ca²⁺-channel blockers such as Gd³⁺, Cd²⁺, and Ni²⁺. However, application of S1P did not induce any contraction in organ-cultured as well as the intact aorta muscle strip. Aortic myocytes freshly dispersed from the organ-cultured muscle were also ineffective against S1P. Taken together, cell-culture changes the S1P₃-mediated Ca²⁺ response to S1P in rat aortic myocytes.