We tested the hypothesis that exogenous administration of the ET-1 precursor, bigET-1, would regulate adult rat ventricular myocyte (ARVM) contractility in a p38-mitogen activated protein kinase (p38-MAPK)-dependent mechanism during sepsis. Ventricular myocytes from adult rat hearts (both sham and septic) were stimulated to contract at 0.5 Hz and mechanical properties were evaluated using an IonOptix Myocam system. Immunoblot analysis was used to determine the phosphorylation of p38-MAPK and extracellular signal-regulated kinase 1/2 (ERK1/2). ARVMs were treated with vehicle, bigET-1 and inhibitors for 24 h and then subjected to functional and biochemical estimations. Septic ARVM displayed a distorted cell membrane and irregular network within the cells along with increased cell contractility as evidenced by elevated peak shortening (PS), maximal velocity of shortening (+dL/dt) and relengthening (-dL/dt) in comparison to sham ARVM. BigET-1 treatment caused ARVM enlargement in both sham and sepsis groups. BigET-1 (100 nM) produced an increase in ARVM contractility in sham group as compared to vehicle treatment. However, septic ARVM treated with bigET-1 exhibited unaltered ARVM contractility, and upregulated ET(B) receptors as compared to respective sham group. BigET-1 increased the concentration of ET-1 and upregulated phosphorylation of p38-MAPK but not of ERK1/2 in sham and septic ARVM. Furthermore, inhibition of p38-MAPK by SB203580 (10 microM) increased ARVM contractility in sham but not in sepsis group. BigET-1 reversed SB203580-induced increase in PS in sham group but accentuated it in sepsis group. BigET-1 also reversed SB203580-induced inhibition of p38-MAPK phosphorylation in sham but not in septic ARVM. SB203580 pretreatment followed by bigET-1 administration significantly decreased p38-MAPK phosphorylation and downregulated ET(B) receptor expression as compared to bigET-1 treatment per se in sepsis group but not in sham. We concluded that a bigET-1-induced non-responsive effect on septic ARVM contractile function could be due to upregulation of p38-MAPK phosphorylation and ET(B) receptor expression.
Despite advances in the understanding of pathophysiological mechanisms, there are limited pharmacotherapeutic options for sepsis, septic shock, and related pathologies. It is surprising that although sepsis-induced myocardial depression is documented in clinics, the cellular mechanisms are from clear. Alterations in molecular signaling mechanisms activated by cytokines and potent mediators such as ET-1 could pose the risk for myocardial dysfunction in sepsis. Our laboratory data suggest that the septic heart, in vivo, exhibits an increased time constant of left ventricular relaxation, tau, along with changes in LVEDP. We also observed that bigET-1-induced elevation of ET-1 correlates with cardiodynamic alterations, induction of apoptosis, and activation of p38-MAPK phosphorylation during sepsis. In light of these evidences, we emphasize that these molecular alterations in heart, both at organ and cellular level during early sepsis, need to be elucidated thoroughly.
ABSTRACT:Human cytosolic sulfotransferase SULT1E1 catalyzes the sulfation of endogenous estrogens as well as xenobiotic estrogen-like chemicals. This reaction increases the water solubility of the molecule, which may affect its cellular distribution and biological activity. This could alter estrogen signaling to the estrogen receptor in human estrogen receptor-positive cells. The current work characterized the cellular distribution of SULT1E1 in the human embryonic kidney 293 (HEK293) cell line using green fluorescent protein (GFP) tagging and immunochemistry methods. The GFP-tagged recombinant SULT1E1 protein was expressed and localized in the cytoplasm of HEK293 cells. By using a commercial anti-SULT1E1 peptide antibody, a 35.7-kDa protein was detected in HEK293 cells via Western blot. The molecular mass of the protein detected suggested that it may be related to native SULT1E1 protein. However, reverse transcription-polymerase chain reaction (RT-PCR) with gene-specific primers could not confirm the presence of the SULT1E1 transcript in the total RNA sample of HEK293 cells. The discrepancy between protein and transcript data could be due to the instability of SULT1E1 mRNA or the specificity of the anti-SULT1E1 antibody used. In the present work, RT-PCR analysis with gene-specific primers also identified a transcript fragment of human estrogen-related receptor ␥. Future studies on the functional relationship between estrogen-related receptors and sulfotransferases are expected to provide additional insights into the physiological and toxicological roles of human estrogen sulfotransferases.
ABSTRACT:Human cytosolic sulfotransferase SULT1E1 catalyzes the sulfation of estrogens and estrogenic drugs in human reproductive tissues. Logically, this estrogen-preferring sulfotransferase isoform could play a regulatory role in estrogen signaling activities in human reproductive cells, including the prostate cells. This hypothesis was tested using DNA microarray and real-time reverse transcription-polymerase chain reaction methods in the present work. Potential changes in the transcriptional expression of selected signal transduction-related genes in human prostate cancer CA-HPV-10 cell line after SULT1E1 transfection were examined by DNA microarray methods. Notable changes were observed in the mRNA expression levels of TFRC, a cell membrane transferrin receptor gene, and TMEPAI, a gene encoding a steroid-dependent mRNA product. Expression of TFRC was down-regulated, whereas expression of TMEPAI was up-regulated by SULT1E1 transfection in CA-HPV-10 cells. Data from the current studies also showed that the estrogen-induced estrogen response element activation in CA-HPV-10 cells was repressed after the cells were transfected with SULT1E1. These results indicate that SULT1E1 may function as a transcriptional mediator in human prostate cancer CA-HPV-10 cells.
Human cytosolic sulfotransferase SULT1E1 catalyzes the sulfation of estrogens and estrogenic drugs in human hepatic and non‐hepatic tissues. This estrogen‐preferring sulfotransferase isoform could play a regulatory role in intracellular estrogen signaling activities. Previous studies have suggested that estrogens induce the expression of TFRC, a cell membrane transferrin receptor gene. Studies using DNA microarray and quantitative real‐time PCR methods by our group also showed that TFRC expression was down‐regulated by SULT1E1 transfection in human prostate cancer CA‐HPV‐10 cell line. Because human CA‐HPV‐10 cell line is an estrogen receptor positive cell line, the estrogen receptor‐coupled signaling pathways could relate to the molecular mechanism underlying the observed effect of SULT1E1 on TFRC expression. To test this hypothesis, the effect of SULT1E1 transfection on the estrogen receptor‐coupled signaling pathways was examined using an estrogen response element reporter gene assay in the present studies. The results showed that β‐estradiol was able to induce the estrogen response element controlled reporter gene expression in CA‐HPV‐10 cells. This β‐estradiol‐induced signal transduction process was repressed after the cells were transfected with SULT1E1. These results suggest that SULT1E1 may function as an estrogen signaling mediator in human prostate cancer CA‐HPV‐10 cells.
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