ABSTRACT
AIM: Platelet-activating factor acetyl hydrolase 1B1 (PAFAH1B1, also known as Lis1) is a protein essentially involved in neurogenesis and mostly studied in the nervous system. As we observed a significant expression of PAFAH1B1 in the vascular system, we hypothesized that PAFAH1B1 is important during angiogenesis of endothelial cells as well as in human vascular diseases. METHOD: The functional relevance of the protein in endothelial cell angiogenic function, its downstream targets and the influence of NONHSAT073641, a long non-coding RNA (lncRNA) with 92% similarity to PAFAH1B1, were studied by knockdown and overexpression in human umbilical vein endothelial cells (HUVEC). RESULTS: Knockdown of PAFAH1B1 led to impaired tube formation of HUVEC and decreased sprouting in the spheroid assay. Accordingly, the overexpression of PAFAH1B1 increased tube number, sprout length and sprout number. LncRNA NONHSAT073641 behaved similarly. Microarray analysis after PAFAH1B1 knockdown and its overexpression indicated that the protein maintains Matrix Gla Protein (MGP) expression. Chromatin immunoprecipitation experiments revealed that PAFAH1B1 is required for active histone marks and proper binding of RNA Polymerase II to the transcriptional start site of MGP. MGP itself was required for endothelial angiogenic capacity and knockdown of both, PAFAH1B1 and MGP, reduced migration. In vascular samples of patients with chronic thromboembolic pulmonary hypertension (CTEPH), PAFAH1B1 and MGP were upregulated. The function of PAFAH1B1 required the presence of the intact protein as overexpression of NONHSAT073641, which was highly upregulated during CTEPH, did not affect PAFAH1B1 target genes. CONCLUSION: PAFAH1B1 and NONHSAT073641 are important for endothelial angiogenic function.
Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/physiology , Microtubule-Associated Proteins/physiology , 1-Alkyl-2-acetylglycerophosphocholine Esterase/genetics , Calcium-Binding Proteins/biosynthesis , Calcium-Binding Proteins/genetics , Cells, Cultured , Extracellular Matrix Proteins/biosynthesis , Extracellular Matrix Proteins/genetics , Female , Gene Knockdown Techniques , Histones/metabolism , Human Umbilical Vein Endothelial Cells , Humans , Hydroxyeicosatetraenoic Acids/metabolism , Hypertension, Pulmonary/etiology , Hypertension, Pulmonary/metabolism , Microtubule-Associated Proteins/genetics , Neovascularization, Physiologic/genetics , Neovascularization, Physiologic/physiology , RNA Polymerase II/metabolism , RNA, Long Noncoding/physiology , Thromboembolism/complications , Thromboembolism/metabolism , Wound Healing , Matrix Gla ProteinABSTRACT
LC-MS/MS has been applied for the rapid determination of the nucleoside analogue ribavirin in human plasma and red blood cells. The incorporation of ribavirin to the erythrocytes has been assayed after in vitro incubation of the cells at different concentrations of the antiviral drug. After protein precipitation, samples were injected into a C8 column, achieving a complete separation of ribavirin from the endogenous isobaric compound uridine. Calibration ranges varied from 10 to 10,000 ng/mL in plasma and from 0.2 to 200 ng/cell pellet in red blood cells. Precision and accuracy values were always below 10 and 13%, respectively, in all assayed matrices. Ribavirin was demonstrated to remain unchanged after short and long time storage. No matrix effects could be assessed for the analyzed matrices. The developed method has been fully validated. Monitoring of ribavirin concentration in red blood cells in addition to the classic plasma monitoring of the drug could help to explain its efficacy and safety profiles in patients.
Subject(s)
Chromatography, High Pressure Liquid/methods , Erythrocytes/chemistry , Ribavirin/blood , Tandem Mass Spectrometry/methods , Humans , Plasma/chemistryABSTRACT
BACKGROUND AND PURPOSE: Selective nociceptor fibre block is achieved by introducing the cell membrane impermeant sodium channel blocker lidocaine N-ethyl bromide (QX-314) through transient receptor potential V1 (TRPV1) channels into nociceptors. We screened local anaesthetics for their capacity to activate TRP channels, and characterized the nerve block obtained by combination with QX-314. EXPERIMENTAL APPROACH: We investigated TRP channel activation in dorsal root ganglion (DRG) neurons by calcium imaging and patch-clamp recordings, and cellular QX-314 uptake by MS. To characterize nerve block, compound action potential (CAP) recordings from isolated nerves and behavioural responses were analysed. KEY RESULTS: Of the 12 compounds tested, bupivacaine was the most potent activator of ruthenium red-sensitive calcium entry in DRG neurons and activated heterologously expressed TRPA1 channels. QX-314 permeated through TRPA1 channels and accumulated intracellularly after activation of these channels. Upon sciatic injections, QX-314 markedly prolonged bupivacaine's nociceptive block and also extended (to a lesser degree) its motor block. Bupivacaine's blockade of C-, but not A-fibre, CAPs in sciatic nerves was extended by co-application of QX-314. Surprisingly, however, this action was the same in wild-type, TRPA1-knockout and TRPV1/TRPA1-double knockout mice, suggesting a TRP-channel independent entry pathway. Consistent with this, high doses of bupivacaine promoted a non-selective, cellular uptake of QX-314. CONCLUSIONS AND IMPLICATIONS: Bupivacaine, combined with QX-314, produced a long-lasting sensory nerve block. This did not require QX-314 permeation through TRPA1, although bupivacaine activated these channels. Regardless of entry pathway, the greatly extended duration of block produced by QX-314 and bupivacaine may be clinically useful.
Subject(s)
Anesthetics, Local/pharmacology , Bupivacaine/pharmacology , Lidocaine/analogs & derivatives , Nerve Block , Sodium Channel Blockers/metabolism , Anesthetics, Local/administration & dosage , Animals , Behavior, Animal/drug effects , Bupivacaine/administration & dosage , Calcium/metabolism , Cell Line , Foot Injuries , Ganglia, Spinal/drug effects , Ganglia, Spinal/metabolism , Injections , Lidocaine/metabolism , Male , Mice, Knockout , Patch-Clamp Techniques , Peripheral Nerves/drug effects , Primary Cell Culture , Rats , Rats, Sprague-Dawley , Sciatic Nerve/drug effects , TRPA1 Cation Channel , Transient Receptor Potential Channels/genetics , Transient Receptor Potential Channels/metabolismABSTRACT
Cannabis is not only a widely used illicit drug but also a substance which can be used in pharmacological therapy because of its analgesic, antiemetic, and antispasmodic properties. A very rapid and sensitive method for determination of ∆(9)-tetrahydrocannabinol (THC), the principal active component of cannabis, and two of its phase I metabolites in plasma has been developed and validated. After solid-phase extraction of plasma (0.2 mL), the clean extracts were analyzed by tandem mass spectrometry after a 5-min liquid chromatographic separation. The linear calibration ranges were from 0.05 to 30 ng mL(-1) for THC and 11-nor-∆(9)-carboxy-tetrahydrocannabinol (THC-COOH) and from 0.2 to 30 ng mL(-1) for ∆(9)-(11-OH)-tetrahydrocannabinol (11-OH-THC). Imprecision and inaccuracy were always below 7 and 12 % (expressed as relative standard deviation and relative error), respectively. The method has been successfully applied to determination of the three analytes in plasma obtained from healthy volunteers after oral administration of 20 mg dronabinol.
Subject(s)
Chromatography, High Pressure Liquid/methods , Dronabinol/blood , Illicit Drugs/blood , Tandem Mass Spectrometry/methods , Dronabinol/metabolism , Humans , Illicit Drugs/metabolism , Sensitivity and SpecificityABSTRACT
A new analytical method for the quantitation of the orally active immunomodulatory drug FTY720 and its phosphate derivative in human plasma and murine subcellular compartments has been developed. The samples undergo a liquid-liquid extraction process before they are injected into a liquid chromatographic system coupled to a tandem mass spectrometer operating in positive ion mode. The quantitation is based on the analysis of two multiple reaction monitoring transitions per drug. The recovery of the analytical process is around 80% for all analytes. Intra- and interday precision and accuracy, as relative standard deviation and relative error, respectively, are lower than 12.5% in all cases. No important matrix effects were observed. The lower limits of quantitation for the analysed substances were 0.875 ng/mL and 2 ng/mL for FTY720 and FTY720-phosphate, respectively. Since no deuterated derivatives of the analytes were commercially available, the developed method was applied for quantifying the studied compounds using C17-sphingosine and C-17-sphingosine-1-phosphate as internal standards, in subcellular compartments of murine splenocytes. This method could be applied in the future for monitoring purposes in multiple sclerosis patients, since FTY720 has been approved by the American Food and Drug Administration and the European Medicines Agency for the pharmacological treatment of this disease.