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

Abstract

The kidney proximal tubules are involved in reabsorbing twothirds 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 Important molecular transport processes take place across the epithelium of kidney proximal tubules. The basolateral membranes (BLM) 4 of kidney proximal tubules cells contain different active transporters, or ion pumps, such as the very abundant Na ϩ K ϩ -ATPase, which is considered to be the molecular machinery responsible for Na ϩ reabsorption (1). Other ion pumps are not so numerous, but some, such as the plasma membrane calcium pump, play important roles in the fine regulation of intracellular ion concentrations. Our group has recently shown that Ca 2ϩ -ATPase is exclusively located and active in caveolin-cholesterol-rich membrane microdomains or lipid rafts in the kidney BLM (2). These membranes have also been shown to house different cell signaling systems that are initiated by the activation of either different lipid kinases, with further generation of bioactive molecules (3, 4), or protein kinases associated with the BLM (5-8).The location of the BLM Ca 2ϩ -ATPase in caveolin-cholesterol-rich domains adds ceramides to the emerging potential regulatory network in these membranes, because those microdomains are also rich in sphingolipids (9). Rafts are thought to be present in the outer leaflet of the cell membrane, where sphingomyelin, the precursor of ceramide (Cer) in a pathway catalyzed by sphingomyelinases, appears to be predominantly located. An important concept is that the assembly of the outer leaflet lipid rafts would alter the inner leaflet, thus enabling the different steps required for signal transduction to be coordinated (for review, see Ref. 10).Many studies during the past decade have shown that smaller lipid rafts are merged into large membrane domains when sphingomyelin is hydrolyzed and Cer is generated (Refs. 11 and 12; see also Ref. 10 for review). The generation of Cer molecules and their self-association leads to dramatic changes in plasma membranes with further formation of small Cer-rich microdomains, which are able to fuse spontaneously to others resulting in large domains called platforms (12)(13)(14). Therefore, Cer-enriched microdomains seem to play an important role in facilitating and amplifying signaling processes, via different types of cell surface receptors, resulting in clusters of receptors and other cell signaling machinery that facilitate the effective transduction of different signals (10).The importance of Cer in different cell processes is not only related to its physicochemical properties. It can be also considered a c...

Undernutrition Affects Cell Survival, Oxidative Stress, Ca2+ Handling and Signaling Pathways in Vas Deferens, Crippling Reproductive Capacity

“…In this regard, the huge increase in PKA-mediated phosphorylation of PMCA in the CM group compared to the lesser increase in PKC-mediated phosphorylation (Figure 6B and 6D) could account for this imbalance. We demonstrated that PKA is a key activator of PMCA in the innervated faces of electrocytes from Electrophorus electricus L. [31] and in renal cells [28,29], whereas PKC acts as an inhibitor [41], and the undernutrition-induced imbalance between these two kinases could account for an altered Ca 2+ handling. To date, it has not been demonstrated that SERCA is a substrate for PKA.…”

“…The protein kinase activities were analyzed in homogenized vas deferens tissue by measuring the incorporation of the γ-phosphoryl group of [γ- 32 P] ATP into histone in the absence or presence of the specific cyclic AMP-dependent protein kinase (PKA) and protein kinase C (PKC) inhibitors, 10 nM PKAi 5-24 and 10 nM calphostin C, respectively, as previously described [15,28]. The reaction was started by adding [γ- 32 P] ATP (10 µM; specific activity ~1.5 × 10 11 Bq/mmol) to a medium (0.1 ml) containing 20 mM HEPES-Tris (pH 7.0), 4 mM MgCl 2 , 1.5 mg/mL histone and 0.7 mg/ml protein.…”

“…Kinase-mediated phosphorylation of Ca 2+ -ATPase in homogenized vas deferens tissue was determined as previously described [28,29]. The membranes (1 mg/ml) were preincubated for 10 min at 37 o C in a reaction medium containing 50 mM Hepes-Tris (pH 7.4), 0.5 mM ouabain, 5 mM MgCl 2 , 10 mM NaN 3 , 10 mM NaF, 0.3 mM EGTA, 0.34 mM CaCl 2 (10 µM free Ca 2+ ) and 1.1 M hydroxylamine in the absence of protein kinase inhibitors, or in the presence of either 10 nM PKAi 5-24 (to block PKA activity) or 10 nM calphostin C (to ensure inhibition of the diacylglycerol-dependent PKC isoforms).…”

Exposure of luminal membranes of LLC-PK1cells to ANG II induces dimerization of AT1/AT2receptors to activate SERCA and to promote Ca2+mobilization

“…32 P]ATP into histone H8, which was used as substrate as described elsewhere (11). About 5 ϫ 10 5 cells were incubated with different ANG II concentrations for 30 min, in the absence or presence of 5 ϫ 10 Ϫ8 M calphostin C (PKC inhibitor) that had been added to the culture 10 min before ANG II.…”

Diacylglycerol regulates the plasma membrane calcium pump from human erythrocytes by direct interaction

“…This is the case of the some sphingolipids (ceramide and sphingosine), which act in many systems in combination with Ca 2+ and even regulating the [Ca 2+ ] i [15,16]. Ceramide, which is a signal sphingolipid that induces apoptosis in many cancer cell lines [17] and regulates other enzyme processes [18], stimulates the PMCA from human erythrocytes [19] and from renal cells [20]. Ceramide affects positively both the affinity for Ca 2+ and the V max of the ATPase activity of PMCA.…”