Ceramide Is a Potent Activator of Plasma Membrane Ca2+-ATPase from Kidney Proximal Tubule Cells with Protein Kinase A as an Intermediate

The kidney-proximal tubules are involved in reabsorbing two-thirds of the glomerular ultrafiltrate, a key Ca(2+)-modulated process that is essential for maintaining homeostasis in body fluid compartments. The basolateral membranes of these cells have a Ca(2+)-ATPase, which is thought to be responsible for the fine regulation of intracellular Ca(2+) levels. In this paper we show that nanomolar concentrations of ceramide (Cer(50) = 3.5 nm), a natural product derived from sphingomyelinase activity in biological membranes, promotes a 50% increase of Ca(2+)-ATPase activity in purified basolateral membranes. The stimulatory effect of ceramide occurs through specific and direct (cAMP-independent) activation of a protein kinase A (blocked by 10 nm of the specific inhibitor of protein kinase A (PKA), the 5-22 peptide). The activation of PKA by ceramide results in phosphorylation of the Ca(2+)-ATPase, as detected by an anti-Ser/Thr specific PKA substrate antibody. It is observed a straight correlation between increase of Ca(2+)-ATPase activity and PKA-mediated phosphorylation of the Ca(2+) pump molecule. Ceramide also stimulates phosphorylation of renal Ca(2+)-ATPase via protein kinase C, but stimulation of this pathway, which inhibits the Ca(2+) pump in kidney cells, is counteracted by the ceramide-triggered PKA-mediated phosphorylation. The potent effect of ceramide reveals a new physiological activator of the plasma membrane Ca(2+)-ATPase, which integrates the regulatory network of glycerolipids and sphingolipids present in the basolateral membranes of kidney cells.