Optimal Use of the Fluorescent PicoGreen Dye for Quantitative Analysis of Amplified Polymerase Chain Reaction Products on Microplate

Proc. Natl. Acad. Sci. U.S.A.

et al. 2006

Leukotriene (LT) B4 is a powerful chemotactic and immune modulating agent that signals via two receptors denoted BLT1 and BLT2.Here we report that BLT1 and BLT2 are expressed at low levels in an apparently silent state in human umbilical vein endothelial cells (HUVEC). However, treatment with LPS leads to a >10 fold increase in the levels of BLT1 mRNA without any significant effects on BLT2 mRNA. In parallel, LPS also increases the amounts of BLT1 protein.Tumor necrosis factor-␣ (TNF-␣) increases the expression of BLT2 mRNA Ϸ6 times above basal levels with only a modest increase in BLT1 mRNA. Interleukin-1␤ causes variable and parallel increases of both BLT1 and BLT2 mRNA. The natural ligand LTB4 also increases BLT1, but not BLT2, mRNA and protein expression. Along with the induction of BLT1 and͞or BLT2, HUVEC acquire the capacity to respond to LTB4 with increased levels of intracellular calcium and these signals can be blocked by isotype selective BLT antagonists, CP-105696 and LY-255283. In addition, treatment of HUVEC with LTB4 causes increased release of both nitrite, presumably reflecting nitric oxide (NO), and monocyte chemoattractant protein-1. Our data indicate that expression of functional BLT receptors may occur at the surface of endothelial cells in response to LPS, cytokines, and ligand, which in turn may have functional consequences during the early vascular responses to inflammation. Moreover, the results point to BLT receptors as potential targets for pharmacological intervention in LT-dependent inflammatory diseases such as asthma, rheumatoid arthritis, and arteriosclerosis.inflammation ͉ arteriosclerosis ͉ rheumatoid arthritis ͉ asthma ͉ nitric oxide T he leukotrienes (LTs) are a family of lipid mediators that play important roles in a variety of allergic and inflammatory reactions (1, 2). These molecules are divided into two classes, the spasmogenic cysteinyl-LTs (cys-LT) and LTB 4 , which is a classical nM chemotactic agent produced by neutrophils, macrophages, and mast cells. Thus, LTB 4 is a potent chemoattractant for polymorphonuclear leukocytes, comparable to complement peptide C5a and fMet-Leu-Phe (3, 4). Recent data also indicate that LTB 4 is a strong chemoattractant for T cells, creating a functional link between early innate and late adaptive immune responses (5-7). Because of these biological effects, LTB 4 is regarded as an important chemical mediator in a variety of acute and chronic inflammatory diseases and only recently, genetic and biochemical evidence strongly implicate LTB 4 as a mediator of vascular inflammation and arteriosclerosis (8-10). LTB 4 , is synthesized from arachidonic acid via the concerted action of 5-lipoxygenase, assisted by 5-lipoxygenase-activating protein and the terminal LTA 4 hydrolase (11), usually in a single cell or via transcellular routes; this mechanism has been shown to occur in vivo (12,13). LTB 4 signals primarily via a specific, high-affinity, G proteincoupled seven-transmembrane receptor, termed BLT 1 (14). The BLT 1 gene is located on the huma...

Section: Methodsmentioning

confidence: 99%

Differential induction of BLT receptor expression on human endothelial cells by lipopolysacharide, cytokines, and leukotriene B₄

Qiu

Johansson

Proc. Natl. Acad. Sci. U.S.A.

et al. 2006

“…Aliquots of the PCR mixtures (10 ml) were analyzed by electrophoresis using a 1 % agarose gel containing 0.5 mg/ml ethidium bromide. For semi-quantitative analysis of amplified PCR products the fluorescent dye PicoGreen (Molecular Probes, Eugene, OR) was used according to the manufacturer's instructions [26,27]. Two microliters of amplified DNA in 100 ml TE buffer were mixed with an equal volume of diluted PicoGreen reagent (1:200, v/v in TE buffer).…”

Section: Lps Challenge Of Rats and Organ Preparationsmentioning

confidence: 99%

Microsomal glutathione S-transferases: selective up-regulation of leukotriene C4 synthase during lipopolysaccharide-induced pyresis

Schröder

CMLS, Cell. Mol. Life Sci.

Qiu

et al. 2005

Cysteinyl-leukotrienes (cys-LTs) are potent smooth muscle contracting agents, which play key roles in inflammatory and allergic diseases. The committed step in cys-LT biosynthesis is catalyzed by leukotriene C(4) synthase (LTC4S) as well as microsomal glutathione S-transferase type 2 (MGST2) and type 3 (MGST3). Here we report that intraperitoneal injections of lipopolysaccharide in rats lead to a strong increase of LTC4S messenger RNA (mRNA) levels after approximately 1 h, particularly in the heart, brain, adrenal glands and liver, without any significant effect on MGST2 and MGST3 mRNA levels. After 6 h, LTC4S mRNA returns to basal levels, concomitant with a 4.9-, 4.0-, 2.9- and 2.3-fold induction of LTC4S protein in brain, heart, liver and adrenal gland, respectively. Hence, challenge with lipopolysaccharide in vivo causes an organ-selective, local priming for leukotriene C(4) synthesis. Moreover, these data suggest that LTC4S and cys-LTs may be involved in acute systemic inflammatory responses such as fever and tachycardia.

“…For semiquantitative analysis of PCR products, a fluorescent dye (PicoGreen, Molecular Probes) was used according to the manufacturer's instructions. 15 The fluorescence was measured (excitation wavelengthϭ485 nm; emission wavelengthϭ538 nm) in a fluorometer (SpectraMax GeminiXS). For comparative RT-PCR, total RNA was diluted until the PCR signal for CysLT 2 R was comparable to that of CysLT 1 R in undiluted RNA, with the use of 40 ϩ40 cycles.…”

Section: Analysis Of Mrna By Rt-pcr and Real-time Rt-pcrmentioning

confidence: 99%

Dominant Expression of the CysLT₂Receptor Accounts for Calcium Signaling by Cysteinyl Leukotrienes in Human Umbilical Vein Endothelial Cells

Johansson

Schröder

et al. 2003

ATVB

HUVECs almost exclusively express the CysLT2R. Furthermore, Ca2+ fluxes elicited by CysLT in these cells emanate from perturbation of the CysLT2R, rather than the expected CysLT1R. Hence, signaling events involving CysLT2R might trigger functional responses involved in the critical components of LT-dependent vascular reactions, which in turn have implications for ischemic heart disease and myocardial infarction.