Stratification of Sphingosine Kinase 1 Expression and activity in rat kidney

MM FACCHINETTI; F LEOCATA NIETO; MG MARQUEZ; N STERIN-SPEZIALE

CELLS TISSUES ORGANS

Karger

Lugar: Basilea; Año: 2008 vol. 188 p. 384 - 392

1422-6405

Abstract Background It is now well known that several enzymes of lipid metabolism serve as signaling modules, and their  products serve as bioeffectors. Sphingosine1-phosphate (Sph1P), the product of Sph kinase activity, is an important bioactive sphingolipid metabolite that regulates diverse biological processes such as cell growth and survival, morphogenesis and migration. Sphingosine 1-phosphate also modulates renovascular contraction, diuresis and natriuresis in rats. Mammalian tissues contain a cytosolic and membrane-associated Sph kinase. This key enzyme for sphingolipid signaling has been first purified from cytosolic fractions of rat kidney  and then cloned. Methods Sphingosine kinase activity was measured in tissue homogenates by incubation of the lysates with 2 sphingosine, [32P]ATP. Lipids were extracted, and [32P]S1P was resolved by TLC  and quantified with a Phosphor Imager. To study the modulation of Sph kinase activity, tissue slices were preincubated in the presence of  activators and inhibitors of signal transduction pathways. Endogenous production of Sphingosine-1-phosphate was extracted, isolated by TLC and the inorganic phosphorous was quantified. The expression pattern of the enzyme in the various zones of the kidney  was assayed by immunoblotting by using the SK1 antibody and by immunohystochemistry of the paraffin-embedded tissue sections.