Glymphatic fluid transport is suppressed by the aquaporin-4 inhibitor AER-271.

The glymphatic system transports cerebrospinal fluid (CSF) into the brain via arterial perivascular spaces and removes interstitial fluid from the brain along perivenous spaces and white matter tracts. This directional fluid flow supports the clearance of metabolic wastes produced by the brain. Glymphatic fluid transport is facilitated by aquaporin-4 (AQP4) water channels, which are enriched in the astrocytic vascular endfeet comprising the outer boundary of the perivascular space. Yet, prior studies of AQP4 function have relied on genetic models, or correlated altered AQP4 expression with glymphatic flow in disease states. Herein, we sought to pharmacologically manipulate AQP4 function with the inhibitor AER-271 to assess the contribution of AQP4 to glymphatic fluid transport in mouse brain. Administration of AER-271 inhibited glymphatic influx as measured by CSF tracer infused into the cisterna magna and inhibited increases in the interstitial fluid volume as measured by diffusion-weighted MRI. Furthermore, AER-271 inhibited glymphatic efflux as assessed by an in vivo clearance assay. Importantly, AER-271 did not affect AQP4 localization to the astrocytic endfeet, nor have any effect in AQP4 deficient mice. Since acute pharmacological inhibition of AQP4 directly decreased glymphatic flow in wild-type but not in AQP4 deficient mice, we foresee AER-271 as a new tool for manipulation of the glymphatic system in rodent brain.

Novel caged fluorescein diphosphates as photoactivatable substrates for protein tyrosine phosphatases.

We have characterized some novel caged fluorescein diphosphates as photoactivatable, cell-permeable substrates for protein tyrosine phosphatases and explored their usefulness in identifying inhibitors of tyrosine phosphatases. 1-(2-Nitrophenyl)ethyl protected fluorescein diphosphate (NPE-FDP) undergoes rapid photolysis to release FDP upon irradiation with a 450-W UV immersion lamp and its by-product does not inactivate protein tyrosine phosphatase 1B (PTP1B) or alters the viability of cells. The generated FDP from photolysis of NPE-FDP was shown to have exactly the same properties as FDP, which can be used as a PTP substrate in pure enzyme assays. We have also demonstrated that the PTP activity can be measured using NPE-FDP in small droplets. Its advantage as an inert substrate before photolysis allows the possibility of applying nanospray technology in screening and optimizing PTP inhibitors through a large chemical library. Like other caged bioeffectors such as nucleotide and inositol trisphosphate, NPE-FDP is cell-permeable. The NPE-FDP can be photolyzed to generate FDP inside cells, and then can be hydrolyzed by phosphatases to produce fluorescein monophosphate and subsequently to fluorescein. Although Jurkat cells contain high concentrations of CD45, it has not been possible to use FDP as a substrate to measure CD45 activity in the intact cell. This is due to the hydrolysis of FDP by several other cellular phosphatases. However, NPE-FDP can be useful as a cell-permeable substrate for overexpressed phosphatases such as alkaline phosphatase.

In vitro PKA phosphorylation-mediated human PDE4A4 activation.

The PDE4 catalytic machinery comprises, in part, two divalent cations in a binuclear motif. Here we report that PDE4A4 expressed in Sf9 cells exhibits a biphasic Mg(2+) dose-response (EC(50) of 0.15 and >10 mM) in catalyzing cAMP hydrolysis. In vitro phosphorylation of PDE4A4 by the PKA-catalytic subunit increases the enzyme's sensitivity to Mg(2+), leading to 4-fold increased cAMP hydrolysis without affecting its K(m). The phosphorylation also increases the potencies of (R)- and (S)-rolipram without affecting CDP-840 and SB-207499. The results support that modulating the cofactor binding affinity of PDE4 represents a mechanism for regulating its activity.

Monotonicity of interleukin-1 receptor-ligand binding with respect to antagonist in the presence of decoy receptor.

We consider the interaction between interleukin-1 IL-1, its receptor IL-1RI, the receptor antagonist IL-1Ra and a decoy receptor (or trap) that binds both with the ligand and the antagonist. We study how the interaction between IL-1Ra and the decoy receptor influences the effect of either reagent on reducing the equilibrium concentration of the receptor-ligand complex. We obtain that, given a certain relationship among the equilibrium constants and the total concentrations of solutes, IL-1Ra can reverse the effect of the decoy receptor of decreasing the equilibrium concentration of the receptor-ligand complex. This finding derives from a mathematical result applicable to any reversible chemical reaction system comprising four species arranged in a square such that each species binds its two immediate neighbors. The result gives the monotonicity of the equilibrium concentrations of the complex species as functions of the total concentrations of the simple species.

Catalytic inactivation of protein tyrosine phosphatase CD45 and protein tyrosine phosphatase 1B by polyaromatic quinones.

Polyaromatic quinones, such as the environmental pollutants 9,10-phenanthrenediones, elicit a wide range of responses including growth inhibition, immune suppression, and glucose normalization in diabetic models. Yet the molecular mechanisms behind these effects remain controversial. Here we report that many of them are oxygen-dependent and catalytic inactivators of protein tyrosine phosphatases (PTP). Under aerobic conditions, the PTP inactivation by 2-nitro-9,10-phenanthrenedione followed a pseudo-first-order process, with the rate of inactivation increasing nearly linearly with increasing inhibitor concentration, yielding apparent inactivation rate constants of 4300, 387, and 5200 M(-1) s(-1) at pH 7.2 against CD45, PTP1B, and LAR, respectively. The rate of CD45 inactivation increased approximately 25-fold from pH 6.0 to 7.5, with complete inactivation achieved using a catalytic amount (0.05 molar equiv) of the inhibitor. The quinone-catalyzed CD45 inactivation was prevented by catalase or superoxide dismutase. Inactivated CD45 after (125)I-9,10-phenanthrenedione treatment carried no radioactivity, indicating the absence of a stable inhibitor/enzyme complex. The activity of inactivated CD45 was partially restored ( approximately 10%) by hydroxylamine or dithiothreitol, supporting the presence of a small population of sulfenic acid or sulfenyl-amide species. Treatment of PTP1B with 2-nitro-9,10-phenanthrenedione resulted in the specific and sequential oxidation of the catalytic cysteine to the sulfinic and sulfonic acid. These results suggest that reactive oxygen species and the semiquinone radical, continuously generated during quinone-catalyzed redox cycling, mediate the specific catalytic cysteine oxidation. Naturally occurring quinones may act as efficient regulators of protein tyrosine phosphorylation in biological systems. Aberrant phosphotyrosine homeostasis resulting from continued polyaromatic hydrocarbon quinone exposure may play a significant role in their disease etiology.