Estrogen Metabolite 2-Methoxyestradiol Attenuates Blood Pressure in Hypertensive Rats by Downregulating Angiotensin Type 1 Receptor.

The therapeutic potential of 2-Methoxyestradiol (2ME2) is evident in cardiovascular disease. Our laboratory has previously demonstrated the mechanism involved in the 2ME2 regulation of angiotensin type 1 receptor (AT1R) . However, 2ME2 regulation of angiotensin receptors and its effects on blood pressure (BP) and resting heart rate (RHR) are uncertain.

2-Methoxyestradiol causes matrix metalloproteinase 9-mediated transactivation of epidermal growth factor receptor and angiotensin type 1 receptor downregulation in rat aortic smooth muscle cells.

Studies have demonstrated the therapeutic potential of estrogen metabolite 2-methoxyestradiol (2ME2) in several cardiovascular disorders, including hypertension. However, the exact mechanism(s) remains unknown. In this study, primary rat aortic smooth muscle cells (RASMCs) were exposed to 2ME2, and angiotensin type 1 receptor (ATR) expression, function, and associated signaling pathways were evaluated.

Competitive Binding Assay for the G-Protein-Coupled Receptor 30 (GPR30) or G-Protein-Coupled Estrogen Receptor (GPER).

The role of 2-methoxyestradiol is becoming a major area of investigation because of its therapeutic utility, though its mechanism is not fully explored. Recent studies have identified the G-protein-coupled receptor 30 (GPR30, GPER) as a high-affinity membrane receptor for 2-methoxyestradiol. However, studies aimed at establishing the binding affinities of steroid compounds for specific targets are difficult, as the tracers are highly lipophilic and often result in nonspecific binding in lipid-rich membrane preparations with low-level target receptor expression.

2-methoxyestradiol binding of GPR30 down-regulates angiotensin AT(1) receptor.

Controlling angiotensin AT1 receptor function has been shown to be protective for many pathophysiological disorders. Although estrogen metabolite, 2-methoxyestradiol (2ME2) can down-regulate angiotensin AT1 receptor expression independently of nuclear receptors, no specific cellular targets have been identified. This study was focused on identification and validation of a cellular target responsible for 2ME2-mediated angiotensin AT1 receptor down-regulation in a continuously passaged rat liver epithelial cell line.

Interplay between EGR1 and SP1 is critical for 13-cis retinoic acid-mediated transcriptional repression of angiotensin type 1A receptor.

Recently, we have demonstrated that 13-cis retinoic acid (13cRA) downregulates rat angiotensin type 1A receptor (Agtr1a) gene transcription through a MAP kinase (ERK1/2)-dependent mechanism in rat liver epithelial and aortic smooth muscle cells. However, the exact mechanism remained unknown. In this study, we determined the signaling intermediates activated by ERK1/2 involved in 13cRA-mediated Agtr1a downregulation.

Angiotensin type 1 receptor resistance to blockade in the opossum proximal tubule cell due to variations in the binding pocket.

Blockade of the angiotensin (ANG) II receptor type 1 (AT(1)R) with angiotensin receptor blockers (ARBs) is widely used in the treatment of hypertension. However, ARBs are variably effective in reducing blood pressure, likely due, in part, to polymorphisms in the ARB binding pocket of the AT(1)R. Therefore, we need a better understanding of variations/polymorphisms that alter binding of ARBs in heterogeneous patient populations.

Functional role of sodium glucose transporter in high glucose-mediated angiotensin type 1 receptor downregulation in human proximal tubule cells.

Previously, we have demonstrated human angiotensin type 1 receptor (hAT(1)R) promoter architecture with regard to the effect of high glucose (25 mM)-mediated transcriptional repression in human proximal tubule epithelial cells (hPTEC; Thomas BE, Thekkumkara TJ. Mol Biol Cell 15: 4347-4355, 2004). In the present study, we investigated the role of glucose transporters in high glucose-mediated hAT(1)R repression in primary hPTEC.

Pharmacologic effects of 2-methoxyestradiol on angiotensin type 1 receptor down-regulation in rat liver epithelial and aortic smooth muscle cells.

Background: Delayed onset of cardiovascular disease (CVD) in female patients is not well understood, but could be due in part to the protective effect of estrogen before menopause. Experimental studies have identified the angiotensin type 1 receptor (AT1R) as a key factor in the progression of CVD.

Objective: We examined the effects of the estrogen metabolite 2-methoxyestradiol (2ME2) on AT1R expression.

Tannic acid down-regulates the angiotensin type 1 receptor through a MAPK-dependent mechanism.

In the present study, we investigated the effects of tannic acid (TA), a hydrolysable polyphenol, on angiotensin type 1 receptor (AT1R) expression in continuously passaged rat liver epithelial cells. Under normal conditions, exposure of cells to TA resulted in the down-regulation of AT1R-specific binding in concentrations ranging from 12.5-100 μg/ml (7.

13-cis-Retinoic acid specific down-regulation of angiotensin type 1 receptor in rat liver epithelial and aortic smooth muscle cells.

Transcriptional repression through cis- and trans-acting factors enabling an alternate approach to control angiotensin type 1 receptor (AT1 or AGTR1 as listed in the MGI database) expression has not been studied. In previous investigations, treatment with retinoic acid was found to be associated with enhanced insulin sensitivity. In our previous study, expression of AT1 was found to be inversely correlated with intracellular glucose concentrations.

Effects of transient overexpression or knockdown of cytochrome P450 reductase on reactive oxygen species generation and hypoxia reoxygenation injury in liver cells.

1. Literature data suggest that the electron-donating enzyme, cytochrome P450 reductase (CPR), might act as a source of reactive oxygen species (ROS). However, the role of CPR in pathophysiological conditions associated with oxidative stress is unknown.

Delivery of NADPH-cytochrome P450 reductase antisense oligos using avidin-biotin approach.

Although avidin-mediated intracellular delivery of oligonucleotides or proteins has been shown before, the efficacy studies are lacking. Here, we tested the effectiveness of avidin for delivery of a cytochrome P450 reductase (CPR) antisense oligo in rat liver epithelial cells. A phosphorodiamidate morpholino oligo (PMO) against CPR was biotinylated using four reagents with short, cleavable, or long linkers, followed by conjugation with avidin.

A functional role for sodium-dependent glucose transport across the blood-brain barrier during oxygen glucose deprivation.

In the current study, we determined the functional significance of sodium-dependent/-independent glucose transporters at the neurovasculature during oxygen glucose deprivation (OGD). Confluent brain endothelial cells cocultured with astrocytes were exposed to varying degrees of in vitro stroke conditions. Glucose transporter (GLUT) 1 and sodium glucose cotransporter (SGLT) activity were investigated by luminal membrane uptake and transport studies using [(3)H]D-glucose and also by [(14)C]alpha-methyl D-glucopyranoside (AMG), a specific, nonmetabolized substrate of SGLT.

Regulation of megalin expression in cultured proximal tubule cells by angiotensin II type 1A receptor- and insulin-mediated signaling cross talk.

Impairment of proximal tubular endocytosis of glomerular-filtered proteins including albumin results in the development of proteinuria/albuminuria in patients with chronic kidney disease. However, the mechanisms regulating the proximal tubular function are largely unknown. This study aimed to investigate the role of angiotensin II type 1A receptor (AT(1A)R)- and insulin-mediated signaling pathways in regulating the expression of megalin, a multiligand endocytic receptor in proximal tubule cells (PTCs).

Protective effects of diallyl sulfide, a garlic constituent, on the warm hepatic ischemia-reperfusion injury in a rat model.

Purpose: The aim of this study is to evaluate the effects of diallyl sulfide (DAS) on the warm hepatic ischemia-reperfusion (IR) injury in a rat model.

Methods: Rats (n = 8-10/group) were subjected to sham operation or warm ischemia (1 h)-reperfusion (3 h) preceded by a single intraperitoneal dose (1.75 mmol/kg) of DAS or vehicle, and relevant biochemical parameters were monitored.

Angiotensin II induces phosphorylation of glucose-regulated protein-75 in WB rat liver cells.

Studies in vascular smooth muscle cells suggest that, angiotensin II (Ang II)-mediated cellular response requires transactivation of epidermal growth factor receptor (EGF-R), and involves tyrosine phosphorylation of caveolin-1. Here we demonstrate that, exposure of WB rat liver cells to Ang II does not cause transactivation of EGF-R, but did rapidly activate p42/p44 mitogen-activated protein (MAP) kinases suggesting that it activates MAP kinases independent of EGF-R transactivation. We observed that the phospho-specific anti-caveolin-1 antibody detected a tyrosine phosphorylated, 75kDa protein in Ang II-treated cells which we identified as glucose regulated protein-75 (GRP-75).

NGP1-01 is a brain-permeable dual blocker of neuronal voltage- and ligand-operated calcium channels.

Calcium overload of neurons leads to cell death and is a key feature in neurodegenerative diseases. The polycyclic amine NGP1-01 blocks L-type voltage operated calcium channels in cardiomyocytes. Here, we tested whether NGP1-01 blocks neuronal calcium channels.

Acute effect of high glucose on long-term cell growth: a role for transient glucose increase in proximal tubule cell injury.

Although chronic exposure of renal cells to high glucose has been shown to cause cell injury, the effect of acute exposure has not been elucidated. In this study, we demonstrate that acute (10 min) exposure of human proximal tubule epithelial cells (hPTEC) to high glucose (25 mM) induces a time-dependent dual effect consisting of an early proliferation and a late apoptosis. Acute exposure of hPTEC to high glucose induced a twofold increase in DNA synthesis and cell number at 12 h.

Protein kinase C family members as a target for regulation of blood-brain barrier Na,K,2Cl-cotransporter during in vitro stroke conditions and nicotine exposure.

Purpose: The aim of the study is to identify specific protein kinase C (PKC) isoforms involvement in K(+) transport mediated at altered blood-brain barrier (BBB) response to stroke conditions with prior nicotine exposure, which provides ways to intervene pharmacologically in PKC-mediated molecular pathways that could lead to effective treatment for smoking stroke patients.

Methods: Changes in PKC isoform levels were studied in the cytosolic and membrane fractions of bovine brain microvessel endothelial cells subjected to stroke conditions as well as nicotine/cotinine exposure. Furthermore, abluminal Na,K,2Cl-cotransporter (NKCC) activity regulated by specific conventional PKC isoform activators and inhibitors was investigated using rubidium ((86)Rb) uptake studies.

ATR blockade reduces IFN-gamma production in lymphocytes in vivo and in vitro.

Background: Type 1 angiotensin II (Ang II) receptor (AT(1)R) signaling induces proinflammatory responses. Recent studies suggest that T lymphocytes express AT(1)R; yet the effects of Ang II binding to AT(1)R on T cells are poorly understood. We examined the effect of AT(1)R blockade on release of the proinflammatory cytokine, interferon-gamma (IFN-gamma) by human lymphocytes in vivo and in vitro.

alpha-thrombin rapidly induces tyrosine phosphorylation of a novel, 74-78-kDa stress response protein(s) in lung fibroblast cells.

We demonstrated previously that exposure of CCL39 lung fibroblasts to alpha-thrombin rapidly inhibits interleukin 6-induced tyrosine phosphorylation of signal transducers and activators of transcription 3 (Stat3). While studying the cross-talk between alpha-thrombin and interleukin 6, we observed that the phospho-specific (tyrosine) anti-Stat3 antibody specifically cross-reacted with a 74-78-kDa protein(s) in alpha-thrombin-treated cells. In this study, we demonstrate that in alpha-thrombin-treated CCL39 cells, the 74-78-kDa protein(s) rapidly undergoes tyrosine phosphorylation.

Glucose mediates transcriptional repression of the human angiotensin type-1 receptor gene: role for a novel cis-acting element.

Human angiotensin type 1 receptor (hAT1R) gene is regulated by hormones, second messengers, and both pathophysiological and developmental states. The focus of the present study was to determine the role of glucose in the trans-repression of hAT1R gene transcription and to identify the functional cis-acting response element(s). Serial deletions of the hAT1R promoter region indicated that an area between -1717 and -1543 base pairs upstream of the 5' end of the cDNA sequence has a glucose responsive regulatory element (GluRE) to down-regulate the gene expression.

Evidence for involvement of 3'-untranslated region in determining angiotensin II receptor coupling specificity to G-protein.

The mRNA 3'-untranslated region (3'-UTR) of many genes has been identified as an important regulator of the mRNA transcript itself as well as the translated product. Previously, we demonstrated that Chinese-hamster ovary-K1 cells stably expressing angiotensin receptor subtypes (AT(1A)) with and without 3'-UTR differed in AT(1A) mRNA content and its coupling with intracellular signalling pathways. Moreover, RNA mobility-shift assay and UV cross-linking studies using the AT(1A) 3'-UTR probe identified a major mRNA-binding protein complex of 55 kDa in Chinese-hamster ovary-K1 cells.

Role of internalization in AT(1A) receptor function in proximal tubule epithelium.

Angiotensin II (ANG II), acting through angiotensin type I (AT(1)) receptors on apical and basolateral surfaces of proximal tubule epithelial cells, increases sodium reabsorption in proximal tubules. Apical and basolateral receptors internalize after exposure to ANG II, but the role of internalization in receptor signaling and transport is not well defined. To determine the role of receptor internalization in ANG II-mediated receptor signaling and sodium transport, we stably expressed full-length and truncated AT(1A) receptors in opossum kidney cells.