Distribution of the enantiomers of hydroxychloroquine and its metabolites in ocular tissues of the rabbit after oral administration of racemic-hydroxychloroquine

The disposition of the enantiomers of hydroxychloroquine (HCQ) and its major metabolites in ocular tissues of rabbits has been studied. Both albino, New Zealand White (NZW), and pigmented animals were administered daily oral doses of rac-HCQ, (S)-HCQ or (R)-HCQ (20 mg/kg) over 1, 6, or 8 day periods or for 8 days followed by a 7-day washout period. At the end of the study periods, plasma and whole blood samples were collected and the rabbits were sacrificed. The eyes were collected, the aqueous humor removed with a syringe, and the eyes separated into the cornea, lens, vitreous body, iris, choroid-retina, sclera, and conjunctiva. The concentrations of (R)-HCQ, (S)-HCQ, and their respective metabolites were determined using a validated enantioselective liquid chromatographic assay. The data from these studies indicate that HCQ accumulated in both pigmented and nonpigmented ocular tissues. In the pigmented tissues, HCQ and its metabolites were bound to melanin and the binding was not enantiospecific. In the nonpigmented tissues and in the iris and retina-choroid of the NZW rabbits, the accumulation appeared to be the result of a reversible and enantioselective binding of HCQ and its metabolites to an unidentified biopolymer present in these ocular tissues. © 1994 Wiley-liss, Inc.     

Low force decelerates L-selectin dissociation from P-selectin glycoprotein ligand-1 and endoglycan

Selectin-ligand interactions mediate the tethering and rolling of circulating leukocytes on vascular surfaces during inflammation and immune surveillance. To support rolling, these interactions are thought to have rapid off-rates that increase slowly as wall shear stress increases. However, the increase of off-rate with force, an intuitive characteristic named slip bonds, is at odds with a shear threshold requirement for selectin-mediated cell rolling. As shear drops below the threshold, fewer cells roll and those that do roll less stably and with higher velocity. We recently demonstrated a low force regime where the off-rate of P-selectin interacting with P-selectin glycoprotein ligand-1 (PSGL-1) decreased with increasing force. This counter-intuitive characteristic, named catch bonds, might partially explain the shear threshold phenomenon. Because L-selectin-mediated cell rolling exhibits a much more pronounced shear threshold, we used atomic force microscopy and flow chamber experiments to determine off-rates of L-selectin interacting with their physiological ligands and with an antibody. Catch bonds were observed at low forces for L-selectin-PSGL-1 interactions coinciding with the shear threshold range, whereas slip bonds were observed at higher forces. These catch-slip transitional bonds were also observed for L-selectin interacting with endoglycan, a newly identified PSGL-1-like ligand. By contrast, only slip bonds were observed for L-selectin-antibody interactions. These findings suggest that catch bonds contribute to the shear threshold for rolling and are a common characteristic of selectin-ligand interactions.     

Depletion of mammalian target of rapamycin (mTOR) via siRNA mediated knockdown leads to stabilization of β-catenin and elicits distinct features of cardiomyocyte hypertrophy

Cardiac myocyte growth is under differential control of mammalian target of rapamycin (mTOR) and glycogen-synthase-kinase-3β (GSK3β). Whereas active GSK3β negatively regulates growth and down-regulates cellular protein synthesis, activation of the mTOR pathway promotes protein expression and cell growth. Here we report that depletion of mTOR via siRNA mediated knockdown causes marked down-regulation of GSK3β protein in cardiac myocytes. As a result, GSK3β target protein β-catenin becomes stabilized and translocates into the nucleus. Moreover, mTOR knockdown leads to increase in cardiac myocyte surface area and produces an up-regulation of the fetal gene program. Our findings suggest a new type of convergence of mTOR and GSK3β activities, indicating that GSK3β-dependent stabilization of β-catenin in cardiac myocytes is influenced by mTOR.     

Endoglycan,a member of the CD34 family,functions as an L-selectin ligand through modification with tyrosine sulfation and sialyl Lewis x

During lymphocyte homing to secondary lymphoid organs and instances of inflammatory trafficking, the rolling of leukocytes on vascular endothelium is mediated by transient interactions between L-selectin on leukocytes and several carbohydrate-modified ligands on the endothelium. Most L-selectin ligands such as CD34 and podocalyxin present sulfated carbohydrate structures (6-sulfated sialyl Lewis x or 6-sulfo-sLex) as a recognition determinant within their heavily glycosylated mucin domains. We recently identified endoglycan as a new member of the CD34 family. We report here that endoglycan, like the two other members of this family (CD34 and podocalyxin) can function as a L-selectin ligand. However, endoglycan employs a different binding mechanism, interacting with L-selectin through sulfation on two tyrosine residues and O-linked sLex structures that are presented within its highly acidic amino-terminal region. Our analysis establishes striking parallels with PSGL-1, a leukocyte ligand that interacts with all three selectins, mediating leukocyte-endothelial, leukocyte-leukocyte, and platelet-leukocyte interactions. Since the distribution of endoglycan includes hematopoietic precursors and leukocyte subpopulations, in addition to endothelial cells, our findings suggest several potential settings for endoglycan-mediated adhesion events.     

Endoglycan, a member of the CD34 family of sialomucins, is a ligand for the vascular selectins

The interactions of the selectin family of adhesion molecules with their ligands are essential for the initial rolling stage of leukocyte trafficking. Under inflammatory conditions, the vascular selectins, E- and P-selectin, are expressed on activated vessels and interact with carbohydrate-based ligands on the leukocyte surface. While several ligands have been characterized on human T cells, monocytes and neutrophils, there is limited information concerning ligands on B cells. Endoglycan (EG) together with CD34 and podocalyxin comprise the CD34 family of sialomucins. We found that EG, previously implicated as an L-selectin ligand on endothelial cells, was present on human B cells, T cells and peripheral blood monocytes. Upon activation of B cells, EG increased with a concurrent decrease in PSGL-1. Expression of EG on T cells remained constant under the same conditions. We further found that native EG from several sources (a B cell line, a monocyte line and human tonsils) was reactive with HECA-452, a mAb that recognizes sialyl Lewis X and related structures. Moreover, immunopurified EG from these sources was able to bind to P-selectin and where tested E-selectin. This interaction was divalent cation-dependent and required sialylation of EG. Finally, an EG construct supported slow rolling of E- and P-selectin bearing cells in a sialic acid and fucose dependent manner, and the introduction of intact EG into a B cell line facilitated rolling interactions on a P-selectin substratum. These in vitro findings indicate that EG can function as a ligand for the vascular selectins.     

Transient receptor potential vanilloid type 1 channels contribute to reflex cutaneous vasodilation in humans

Mechanisms underlying the cutaneous vasodilation in response to an increase in core temperature remain unresolved. The purpose of this study was to determine a potential contribution of transient receptor potential vanilloid type 1 (TRPV-1) channels to reflex cutaneous vasodilation. Twelve subjects were equipped with four microdialysis fibers on the ventral forearm, and each site randomly received 1) 90% propylene glycol + 10% lactated Ringer (vehicle control); 2) 10 mM l-NAME; 3) 20 mM capsazepine to inhibit TRPV-1 channels; 4) combined 10 mM l-NAME + 20 mM capsazepine. Whole body heating was achieved via water-perfused suits sufficient to raise oral temperature at least 0.8°C above baseline. Maximal skin blood flow was achieved by local heating to 43°C and infusion of 28 mM nitroprusside. Systemic arterial pressure (SAP) was measured, and skin blood flow was monitored via laser-Doppler flowmetry (LDF). Cutaneous vascular conductance (CVC) was calculated as LDF/SAP and normalized to maximal vasodilation (%CVC(max)). Capsazepine sites were significantly reduced compared with control (50 ± 4%CVC(max) vs. 67 ± 5%CVC(max), respectively; P < 0.05). l-NAME (33 ± 3%CVC(max)) and l-NAME + capsazepine (30 ± 4%CVC(max)) sites were attenuated compared with control (P < 0.01) and capsazepine (P < 0.05); however, there was no difference between l-NAME and combined l-NAME + capsazepine. These data suggest TRPV-1 channels participate in reflex cutaneous vasodilation and TRPV-1 channels may account for a portion of the NO component. TRPV-1 channels may have a direct neural contribution or have an indirect effect via increased arterial blood temperature. Whether the TRPV-1 channels directly or indirectly contribute to reflex cutaneous vasodilation remains uncertain.     

Enantioselective determination of hydroxychloroquine and its major metabolites in urine and the observation of a reversal in the (+)/(−)-hydroxychloroquine ratio

A sequential achiral-chiral high-performance liquid chromatographic system has been developed for the quantitation in urine of the enantiomers of hydroxychloroquine (HCQ), and of its 3 major metabolites, desethylhydroxychloroquine (DHCQ), desethylchloroquine (DCQ), and bisdesethylchloroquine (BDCQ). HCQ and its metabolites were separated and quantified on a cyano-bonded phase, and the enantiomeric ratios were determined using a Chiral-AGP chiral stationary phase. The assay validation and application of this method to a preliminary study in a human volunteer are presented. In this subject, the initial 0-4 h urine contained the 2 HCQ enantiomers in a ratio of (+)-HCQ:(?)-HCQ of 3:2; by the 2,064 h of the study, this ratio had reversed to (+)-HCQ:(?)-HCQ of 3:7. © 1993 Wiley-Liss, Inc.     

H (0) blood group determinant is present on soluble human L-selectin expressed in BHK-cells.

In the present study we show that the H (0) blood group determinant Fuc alpha1-2Gal beta1-4GlcNAc beta1-R is present on N-linked glycans of soluble human L-selectin recombinantly expressed in baby hamster kidney (BHK) cells. The glycans were isolated using complementary HPLC techniques and characterized by a combination of exoglycosidase digestion and mass spectrometry. The linkage of the fucose residues was determined by incubation of the glycans with specific fucosidases. The H blood determinant Fuc alpha1-2Gal beta1-4GlcNAc beta1 was detected for bi-, 2,4 branched tri- and tetraantennary structures. To our knowledge, the proposed oligosaccharide structures represent a new glycosylation motif for recombinant glycoproteins expressed on BHK cells.