Binding characteristics of the doxepin E/Z-isomers to the histamine H receptor revealed by receptor-bound ligand analysis and molecular dynamics study.

Doxepin is an antihistamine and tricyclic antidepressant that binds to the histamine H receptor (HR) with high affinity. Doxepin is an 85:15 mixture of the E- and Z-isomers. The Z-isomer is well known to be more effective than the E-isomer, whereas based on the crystal structure of the HR/doxepin complex, the hydroxyl group of Thr112 is close enough to form a hydrogen bond with the oxygen atom of the E-isomer.

Sp1-mediated downregulation of P2X4 receptor gene transcription in endothelial cells exposed to shear stress.

Endothelial purinoceptors play an important role in vascular responses to extracellular adenine nucleotides and hemodynamic forces. Here we report that P2X4 purinoceptor expression in human umbilical vein endothelial cells is transcriptionally downregulated by fluid shear stress. When human umbilical vein endothelial cells were subjected to a laminar shear stress of 15 dyn/cm(2), P2X4 mRNA levels began to decrease within 1 h and further decreased with time, reaching 60% at 24 h.

Fluid shear stress activates Ca(2+) influx into human endothelial cells via P2X4 purinoceptors.

Ca(2+) signaling plays an important role in endothelial cell (EC) responses to shear stress generated by blood flow. Our previous studies demonstrated that bovine fetal aortic ECs showed a shear stress-dependent Ca(2+) influx when exposed to flow in the presence of extracellular ATP. However, the molecular mechanisms of this process, including the ion channels responsible for the Ca(2+) response, have not been clarified.

P2X(4) receptors mediate ATP-induced calcium influx in human vascular endothelial cells.

ATP induces Ca(2+) influx across the cell membrane and activates release from intracellular Ca(2+) pools in vascular endothelial cells (ECs). Ca(2+) signaling leads to the modification of a variety of EC functions, including the production of vasoactive substances such as nitric oxide and prostacyclin. However, the molecular mechanisms for ATP-induced Ca(2+) influx in ECs have not been thoroughly clarified.

The effect of flow on the neutrophil-mediated Ca2+ responses in human vascular endothelial cells stimulated by endotoxin.

Leukocyte-vascular endothelial cell (EC) interactions which promote inflammatory and immune reactions involve bidirectional signaling between two cell types. We investigated the effects of flow on neutrophil-mediated changes in endothelial intracellular Ca2+ levels ([Ca2+]i). Cultured human umbilical vein ECs stimulated by endotoxin were labeled with Fura-2 and exposed to fluid flow with neutrophils.

Fluid shear stress transcriptionally induces lectin-like oxidized LDL receptor-1 in vascular endothelial cells.

Fluid shear stress has been shown to modulate various endothelial functions, including gene expression. In this study, we examined the effect of fluid shear stress on the expression of lectin-like oxidized LDL receptor-1 (LOX-1), a novel receptor for atherogenic oxidized LDL in cultured bovine aortic endothelial cells (BAECs). Exposure of BAECs to the physiological range of shear stress (1 to 15 dyne/cm2) upregulated LOX-1 protein and mRNA in a time-dependent fashion.

Endothelial Ca2+ waves preferentially originate at specific loci in caveolin-rich cell edges.

Stimulation of endothelial cells (ECs) with ATP evoked an increase in intracellular Ca2+ concentration ([Ca2+]i). In a single bovine aortic EC, the [Ca2+]i rise started at a specific peripheral locus and propagated throughout the entire cell as a Ca2+ wave. The initiation locus was constant upon repeated stimulation with ATP or other agonists (bradykinin and thrombin).

Physiologic shear stress suppresses endothelin-converting enzyme-1 expression in vascular endothelial cells.

Shear stress dilates blood vessels and exerts an antiproliferative effect on vascular walls. These effects are ascribed to shear stress-induced, endothelium-derived vasoactive substances. Endothelin-converting enzymes (ECEs), the enzymes that convert big endothelin-1 (ET-1) to ET-1, have recently been isolated and the corresponding proteins have been termed ECE-1 and ECE-2.

Fluid shear stress increases the production of granulocyte-macrophage colony-stimulating factor by endothelial cells via mRNA stabilization.

To investigate whether the production of colony-stimulating factors (CSFs) by vascular endothelial cells is regulated by hemodynamic force, we exposed cultured human umbilical vein endothelial cells (HUVECs) to controlled levels of shear stress in a flow-loading apparatus and examined changes in the production of CSFs at both the protein and mRNA level. Exposure of HUVECs to a shear stress of 15 and 25 dyne/cm2 markedly increased the release of granulocyte-macrophage CSF (GM-CSF) detected by ELISA to 5.0 and 9.

Negative transcriptional regulation of the VCAM-1 gene by fluid shear stress in murine endothelial cells.

To explore the mechanism of shear stress-induced downregulation of vascular cell adhesion molecule 1 (VCAM-1) expression in murine endothelial cells (ECs), we examined the effect of shear stress on VCAM-1 gene transcription and assessed the cis-acting elements involved in this phenomenon. VCAM-1 mRNA expression was downregulated at the transcriptional level as defined by nuclear run-on assay and transient transfection of VCAM-1 promoter-luciferase gene constructs. The luciferase assay on the VCAM-1 deletion mutants revealed that the cis-acting element is contained between -694 and -329 bp upstream from the transcription initiation site.

Cloning of cDNAs encoding G protein-coupled receptor expressed in human endothelial cells exposed to fluid shear stress.

A cDNA library of human umbilical vein endothelial cells exposed to fluid shear stress was constructed to search for functional endothelial genes expressed under flow conditions, and cDNAs encoding members of the G protein-coupled receptor (GPCR) family were cloned by a polymerase chain reaction (PCR) method using degenerate oligonucleotide primers. One of the two GPCR clones obtained was edg-1, and the other clone is a novel gene named FEG-1 that encodes a 375-amino acid protein similar to the receptors for both angiotensin II and chemokines. Reverse transcriptase-PCR showed that the FEG-1 and edg-1 mRNA levels in endothelial cells increased markedly in response to fluid flow.

Shear stress augments expression of C-type natriuretic peptide and adrenomedullin.

Shear stress is known to dilate blood vessels and exert antiproliferative effects on vascular walls: these effects have been ascribed to shear stress-induced upregulation of endothelium-derived vasoactive substances, mainly nitric oxide and prostacyclin. We have demonstrated the significance of C-type natriuretic peptide (CNP) as a novel endothelium-derived relaxing peptide (EDRP) that shares a cGMP pathway with nitric oxide. Adrenomedullin is a recently isolated EDRP that elevates intracellular cAMP as prostacyclin does.

Differential display and cloning of shear stress-responsive messenger RNAs in human endothelial cells.

To investigate the effect of shear stress on endothelial gene expression, we performed differential display of mRNAs from cultured human umbilical vein endothelial cells either incubated under static conditions or exposed to shear stress (15 dynes/cm2) for 6 h in a flow-chamber. Around 4% of the total number of mRNAs detected were either up- or down-regulated by shear stress. DNA sequencing of some of these shear stress-responsive mRNAs revealed homology of several clones to known gene sequences and many other clones for unknown genes.

Contribution of sustained Ca2+ elevation for nitric oxide production in endothelial cells and subsequent modulation of Ca2+ transient in vascular smooth muscle cells in coculture.

To elucidate the intracellular Ca2+ (Ca2+i ) transient responsible for nitric oxide (NO) production in endothelial cells (ECs) and the subsequent Ca2+i reduction in vascular smooth muscle cells (VSMCs), we administrated four agonists with different Ca2+i-mobilizing mechanisms for both cells in iso- or coculture. We monitored the Ca2+i of both cells by two-dimensional fura-2 imaging, simultaneously measuring NO production as NO2-. The order of potency of the agonists in terms of the peak Ca2+i in ECs was bradykinin (100 nM) > ATP (10 microM) > ionomycin (50 nM) > thapsigargin (1 microM).

Flow stimulates ICAM-1 expression time and shear stress dependently in cultured human endothelial cells.

Human umbilical vein endothelial cells were subjected to controlled levels of shear stress in a flow-loading apparatus, and changes in the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) were measured by flow cytometry. Application of shear stress (15 dynes/cm2) increased the cell surface expression of ICAM-1 2.7 times the control level 4 hr after the onset of flow, while it caused no change in VCAM-1 expression.

Fluid shear stress increases the expression of thrombomodulin by cultured human endothelial cells.

Endothelial cells (ECs) cultured from human umbilical vein were exposed to medium flow in a flow-loading chamber, and changes in thrombomodulin (TM) expression were examined by flow cytometry and enzyme linked immunosorbent assay with monoclonal antibody. The expression of TM antigen was increased time- and shear stress-dependently by flow, and when exposed to a shear stress of 15 dynes/cm2 for 24 hr, it increased to approximately 200% of the stationary control level. Reverse transcriptase-polymerase chain reaction showed that TM mRNA levels in ECs also increased in response to flow.

Exogenous nitric oxide inhibits proliferation of cultured vascular endothelial cells.

Cultured bovine fatal aortic endothelial cells (BAECs) were stimulated with nitric oxide (NO)-releasing vasodilators and NO gas-saturated solution, and changes in the cell proliferation were examined. Sodium nitroprusside (SNP) and nitroglycerin (NTG) shifted the growth curve downward, and inhibited 3H-thymidine incorporation by the ECs in a dose-dependent manner. Application of NO solution also reduced 3H-thymidine incorporation.

Shear stress inhibits adhesion of cultured mouse endothelial cells to lymphocytes by downregulating VCAM-1 expression.

Monolayers of endothelial cells (EC) cultured from mouse lymph nodes were exposed to controlled levels of shear stress (0-7.1 dyn/cm2) in a parallel plate flow chamber, and binding between the flow-loaded EC and mouse lymph node-derived lymphocytes was assayed. A large number of lymphocytes adhered to the stationary control EC, but in EC exposed to a shear stress of 1.

Laminar flow stimulates ATP- and shear stress-dependent nitric oxide production in cultured bovine endothelial cells.

Based on the fact that nitric oxide (NO) production is associated with changes in intracellular cGMP levels and is selectively inhibited by N omega-methyl L-arginine (L-NME), we investigated the shear stress dependency of NO production in endothelial cells (ECs) from its cGMP responses to various shear stress loads. Cultured fetal bovine aortic ECs treated with a phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX; 1 mM), were exposed to a laminar flow of Krebs buffer solution for 5 minutes in a parallel-plate flow chamber and examined for changes in intracellular cGMP levels by radioimmunoassay using an [125I] cGMP kit. Application of flow increased the cGMP levels.

Intracellular calcium response to directly applied mechanical shearing force in cultured vascular endothelial cells.

We studied the responses of cultured endothelial cells to mechanical shearing force directly applied to those cells in vitro to determine changes in the concentration of intracellular calcium ion (Ca++), one of the factors that transfers information within the cell. Cultured bovine fetal aortic endothelial cells containing the Ca++ fluorescence indicator, Fura-2, were rubbed with a latex balloon in a specially designed system, and changes in the fluorescence of Fura-2 caused by this shear stimulation were determined by photometric fluorescence microscopy. Immediately after shear stimulation, the concentration of Ca++ in the cells was increased and reached a peak (511 +/- 165 nM, n = 12) within 15 seconds after stimulation.

[Release of endothelium-derived relaxing factor (EDRF) from cultured vascular endothelial cells depends on extra- and intracellular Ca2+ concentrations].

The objective of this study was to elucidate the role of extracellular and intracellular Ca2+ in EDRF release from vascular endothelial cells. Cultured fetal bovine aortic endothelial cells were stimulated by adenosine triphosphate (ATP), calcium ionophore (A23187), bradykinin (BKN) and acetylcholine (ACh). The amount of EDRF released was determined on the basis of the relaxation of rabbit aortic ring and the cGMP content of cultured smooth muscle cells derived from fetal bovine aorta.

[Role of hemodynamic factors in atherogenesis].

Since atherosclerotic lesions are apt to occur at specified regions in the blood vessels, hemodynamic factors, such as shear stress generated by blood flow, have been considered to play a role in atherogenesis. Atheroma, which is characterized by the localized accumulation of lipid and the proliferation of smooth muscle cells in the intima, appears at branching or curving sites of blood vessels, where both geometrical shape and blood flow change suddenly. In such sites, both stagnant and turbulent blood flow can occur and the direction and intensity of shear stress, acting on the vascular wall, changes transiently.