Biphasic regulation of type II phosphatidylinositol-4 kinase by sphingosine: cross talk between glycero- and sphingolipids in the kidney.

Phosphatidylinositol-4 kinase (PI-4K) is responsible for the generation of phosphatidylinositol-4 phosphate (PtdIns(4)P), a bioactive signaling molecule involved in several biological functions. In this study, we show that sphingosine modulates the activity of the PI-4K isoform associated with the basolateral membranes (BLM) from kidney proximal tubules. Immunoblotting with an anti-α subunit PI-4K polyclonal antibody revealed the presence of two bands of 57 and 62kDa in the BLM. BLM-PI-4K activity retains noteworthy biochemical properties; it is adenosine-sensitive, not altered by wortmanin, and significantly inhibited by Ca(2+) at the µM range. Together, these observations indicate the presence of a type II PI-4K. Endogenous phosphatidylinositol (PI) alone reaches PI-4K half-maximal activity, revealing that even slight modifications in PI levels at the membrane environment promote significant variations in BLM-associated-PI-4K activity. ATP-dependence assays suggested that the Mg.ATP(2-) complex is the true substrate of the enzyme and that free Mg(2+) is an essential cofactor. Another observation indicated that higher concentrations of free ATP are inhibitory. BLM-associated-PI-4K activity was ~3-fold stimulated in the presence of increasing concentration of sphingosine, while in concentrations higher than 0.4mM, in which S1P is pronouncedly formed, there was an inhibitory effect on PtdIns(4)P formation. We propose that a tightly coupled regulatory network involving phosphoinositides and sphingolipids participate in the regulation of key physiological processes in renal BLM carried out by PI-4K.

Spectral-domain optical coherence tomography for early glaucoma assessment: analysis of macular ganglion cell complex versus peripapillary retinal nerve fiber layer.

OBJECTIVE: We sought to compare the glaucoma discrimination ability of macular inner retinal layer (MIRL) thickness with that of conventional peripapillary retinal nerve fiber layer (pRNFL) thickness as measured by spectral-domain optical coherence tomography (SD-OCT) in patients with early glaucoma. DESIGN: Cross-sectional study. PARTICIPANTS: We studied 67 patients with early glaucoma (visual field mean deviation index ≥-6 dB), and 56 healthy subjects were prospectively enrolled. METHODS: All patients underwent MIRL thickness measurement (ganglion cell complex [GCC] scan) and pRNFL thickness measurement (3.45 mm scan) by SD-OCT. Whenever both eyes were eligible, one was randomly selected. Receiver operating characteristic curves and sensitivities at fixed specificities were generated for different parameters. The areas under the receiver operating characteristic curves (AUCs) of each parameter were compared. RESULTS: The average mean deviation for the glaucomatous eyes was -2.5 ± 1.6 dB. The AUCs for average (0.815); superior (0.807); and inferior (0.788) MIRL thicknesses were not significantly different (p ≥ 0.18). The AUCs for average (0.735); superior (0.728); and inferior (0.697) pRNFL thicknesses were also similar (p ≥ 0.15). Average MIRL thickness had a significantly larger AUC compared to average pRNFL thickness analysis (0.815 vs 0.735; p = 0.03). Sensitivities at 80% specificity for average MIRL and pRNFL thicknesses were 66.7% (cutoff, 89.9 µm) and 62.9% (cutoff, 111.8 µm), respectively. CONCLUSIONS: The GCC scan showed a similar or even a slightly better ability to discriminate between healthy and early glaucomatous eyes compared to the pRNFL scan. Different from previous analyses considering total macular thickness, the GCC macular scan seems to be a useful tool for identification of early structural damage in patients with glaucoma.

Diacylglycerol kinase activity in purified basolateral membranes of kidney tubules. I. Evidence for coupling with phospholipase C.

The diacylglycerol kinase (DGK) catalyzes the phosphorylation of diacylglycerol (DAG) yielding phosphatidic acid (PA) signaling molecules which are involved in the modulation of different cell responses. The aim of this work was to characterize the DGK activity associated to the basolateral membranes (BLM) of kidney proximal tubules, in a native preparation that preserves the membrane microenvironment. The Arrhenius plot of DGK activity was non-linear, indicating a complex influence of the lipid environment of the native membrane. The formation of PA was strongly impaired by U73122, an inhibitor of PLC, whereas remained unmodified when exogenous DAG or PLC were added. The Mg.ATP2- complex is the true phosphoryl-donor substrate, and the very narrow peak of activation at pH 7.0 suggests that amino acids that dissociate at this pH, i.e. hystidine residues, play a role by acting in the coordination of the Mg2+ atoms. The renal DGK is almost completely blocked by 0.1 mM sphingosine, but it is insensitive to micromolar free Ca2+ concentrations and to R59499, the most potent inhibitor of the classical DGKs. Taken as a whole, these data suggest that the DGK isoform present in BLM of proximal tubules is different from those included in the type I family, and that membranous PLC could be the main source of DAG for DGK catalysis.