Reduction in cardiolipin reduces expression of creatine transporter-1 and creatine transport in hBMEC/D3 human brain microvessel endothelial cells

Abstract

AbstractBackgroundCardiolipin (CL) is the signature phospholipid of mitochondria and regulates a plethora of cellular functions including mitochondrial energy production. There is little knowledge of how CL regulates uptake and membrane transport processes in mammalian cells. Endothelial cells of the blood brain barrier (BBB) play a vital role in uptake of metabolites into the brain and are enriched in mitochondria compared with peripheral endothelial cells. We examined how deficiency in BBB endothelial CL regulates the expression of selected drug and metabolite transporters and their function.MethodsCardiolipin synthase-1 (hCLS1) was knocked down in a human brain microvessel endothelial cell line, hBMEC/D3, by Lipofectamine® transfection with hCLS1 siRNA and CL levels, CL synthase activity and the mRNA expression of selected blood brain barrier drug and metabolite transporters examined. Mock transfected hBMEC/D3 cells served as controls. The incorporation of [14C]creatine and [14C]oleate into hBMEC/D3 cells was determined as a measure of solute metabolite transport and protein expression of the creatine transporter determined.ResultsKnockdown of the CL biosynthetic enzyme hCLS1 in hBMEC/D3 reduced CL and CL synthase activity and the mRNA expression of creatine transporter-1, p-glycoprotein and breast cancer resistance protein compared to controls. In contrast, mRNA expression of ATP binding cassette subfamily C members-1, -3, multidrug resistance-associated protein-4 variants 1, -2, and fatty acid transport protein-1 were unaltered. Although ATP production was unaltered by hCLS1 knockdown, incorporation of [14C]creatine into hBMEC/D3 cells was reduced compared to controls. The reduction in [14C]creatine incorporation was associated with a reduction in creatine transporter-1 protein expression. In contrast, incorporation of [14C]oleic acid into hBMEC/D3 cells and the mRNA expression of fatty acid transport protein-1 was unaltered by knockdown of hCLS1 compared to controls.ConclusionKnockdown of hCLS1 in hBMEC/D3, with a corresponding reduction in CL, results in alteration in expression of specific solute membrane transporters.