Molecular mechanisms underlying the heterogeneous barrier responses of two primary endothelial cell types to sphingosine-1-phosphate.

Sphingosine-1-phosphate (S1P) signals to enhance or destabilize the vascular endothelial barrier depending on the receptor engaged. Here, we investigated the differential barrier effects of S1P on two influential primary endothelial cell (EC) types, human umbilical vein endothelial cells (HUVECs) and human pulmonary microvascular endothelial cells (HPMECs). S1PR1 (barrier protective) and S1PR3 (barrier disruptive) surface and gene expression were quantified by flow cytometry and immunofluorescence, and RT-qPCR, respectively. Functional evaluation of EC monolayer permeability in response to S1P was quantified with transendothelial electrical resistance (TEER) and small molecule permeability. S1P significantly enhanced HUVEC barrier function, while promoting HPMEC barrier breakdown. Immunofluorescence and flow cytometry analysis showed select, S1PR3-high HPMECs, suggesting susceptibility to barrier destabilization following S1P exposure. Reevaluation of HPMEC barrier following S1P exposure under inflamed conditions demonstrated synergistic barrier disruptive effects of pro-inflammatory cytokine and S1P. The role of the Rho-ROCK signaling pathway under these conditions was confirmed through ROCK1/2 inhibition (Y-27632). Thus, the heterogeneous responses of ECs to S1P signaling are mediated through Rho-ROCK signaling, and potentially driven by differences in the surface expression of S1PR3.

γ-Secretase Partitioning into Lipid Bilayers Remodels Membrane Microdomains after Direct Insertion.

γ-Secretase is a multisubunit complex that catalyzes intramembranous cleavage of transmembrane proteins. The lipid environment forms membrane microdomains that serve as spatio-temporal platforms for proteins to function properly. Despite substantial advances in the regulation of γ-secretase, the effect of the local membrane lipid microenvironment on the regulation of γ-secretase is poorly understood. Here, we characterized and quantified the partitioning of γ-secretase and its substrates, the amyloid precursor protein (APP) and Notch, into lipid bilayers using solid-supported model membranes. Notch substrate is preferentially localized in the liquid-disordered (Ld) lipid domains, whereas APP and γ-secretase partition as single or higher complex in both phases but highly favor the ordered phase, especially after recruiting lipids from the ordered phase, indicating that the activity and specificity of γ-secretase against these two substrates are modulated by membrane lateral organization. Moreover, time-elapse measurements reveal that γ-secretase can recruit specific membrane components from the cholesterol-rich Lo phase and thus creates a favorable lipid environment for substrate recognition and therefore activity. This work offers insight into how γ-secretase and lipid modulate each other and control its activity and specificity.

Assessment of mazindane, a pro-drug form of mazindol, in assays used to define cocaine treatment agents.

The current studies compared mazindane (5-(4-chlorophenyl)-2,3-dihydro-5H-imidazo [2,1a] isoindole) hydrogen sulfate, a water soluble pro-drug of mazindol (5-(4-chlorophenyl-2,3-dihydro-5H-imidazo [2,1-a] isoindol-5-ol), with mazindol in assays used to define cocaine treatment agents. Both compounds enhanced motor activity (LMA) in Swiss Webster mice with ED(50) values of 2.5 mg/kg i.p. for mazindane and 3.9 mg/kg i.p. for mazindol. At 25 mg/kg mazindane displayed toxic effects and death while mazindol was effect/death free at 50 mg/kg. In Sprague-Dawley rats trained to discriminate cocaine from saline both compounds fully substituted for cocaine with mazindane being fourfold more potent in the total session (0.33 vs. 1.3 mg/kg i.p.) and first reinforcer (0.29 vs. 1.2 mg/kg i.p). Complete substitution was observed in rhesus monkeys trained to discriminate cocaine from saline with ED(50) values for mazindane (0.134 mg/kg i.m.) and mazindol (0.119 mg/kg i.m.). Mazindol exhibited little or no activity at 10(-5) M in inhibiting radioligand binding at 14 neurotransmitter sites while mazindane gave weak activity at the histamine H(1) and 5-hydroxytryptamine 5-HT(3) sites. These results demonstrate that mazindane could be a useful alternative to mazindol as a pharmacological tool because of its similar profile of activity and enhanced water solubility.

Mazindol analogues as potential inhibitors of the cocaine binding site at the dopamine transporter.

A series of mazindol (2) and homomazindol (3) analogues with a variety of electron-donating and electron-withdrawing groups in the pendant aryl group and the benzo ring C, as well as H, methoxy, and alkyl groups replacing the hydroxyl group were synthesized, and their binding affinities at the dopamine transporter (DAT) on rat or guinea pig striatal membranes were determined. Several active analogues were also evaluated for their ability to block uptake of DA, 5-HT, and NE and inhibit binding of [(125)I] RTI-55 at HEK-hDAT, HEK-hSERT, and HEK-hNET cells. Mazindane (26) was found to be a pro-drug, oxidizing (5-H --> 5-OH) to mazindol on rat striatal membranes and HEK-hDAT cells. The 4',7,8-trichloro analogue (38) of mazindol was the most potent and selective ligand for HEK-hDAT cells (DAT K(i) = 1.1 nM; SERT/DAT = 1283 and NET/DAT = 38). Experimental results strongly favor the cyclic or ol tautomers of 2 and 3 to bind more tightly at the DAT than the corresponding keto tautomers.

Three-dimensional quantitative structure-activity relationships of mazindol analogues at the dopamine transporter.

A three-dimensional quantitative structure-activity relationship (3D-QSAR) study was performed on a series of mazindol analogues using the comparative molecular field analysis (CoMFA) method with their corresponding binding affinities for the displacement of [(3)H]WIN 35 428 from rat caudate putamen tissue. The cross-validated CoMFA models were derived from a training set of 50 compounds, and the predictive ability of the resulting CoMFA models was evaluated against a test set of 21 compounds. A set of alignment rules was derived to superimpose these compounds onto a template structure, mazindol (1). These CoMFA models yielded significant cross-validated r(2)(cv) values. Inclusion of additional descriptors did not improve the significance of the CoMFA models; thus, steric and electrostatic fields are the relevant descriptors for these compounds. The best QSAR model was selected on the basis of the predictive ability of the activity on the external test set of compounds. The analysis of coefficient contour maps provided further insight into the binding interactions of mazindol analogues with the DAT. The aromatic rings C and D are involved in hydrophobic interactions in which ring D may bind in a large hydrophobic groove. The relative orientation of these two rings is also important for high binding affinity to the DAT.

Benzo- and cyclohexanomazindol analogues as potential inhibitors of the cocaine binding site at the dopamine transporter.

A series of mazindol (1), homomazindol (2), and bishomomazindol (3) derivatives with a benzo or cyclohexano ring fused at various sites were prepared as part of an SAR study to determine the effect of increased aliphatic and aromatic lipophilicity on selected in vitro assays used to identify potential cocaine-like and cocaine antagonism activity. Very good (IC(50) = 2-3 nM) inhibition of [(3)H] WIN 35,428 and [(125)I] RTI-55 binding on rat or guinea pig striatal membranes and HEK cells expressing cDNA for the human dopamine transporter (HEK-hDAT) was shown by the 8,9-benzomazindol 25 and 9,10-benzohomomazindol 28. All new compounds were weaker inhibitors of [(3)H] DA uptake in HEK-hDAT cells than 1 and 2. No improvement in the binding selectivity ratio (SERT/DAT and NET/DAT) was found when compared to 2. Compounds 25and 28 showed a considerable increase versus 1 in uptake/binding discrimination ratios at the DAT (311.0 and 182.1 vs 0.9), SERT (33.6 and 127.3 vs 1.9), and NET (7.3 and 10.0 vs 0.3).