omology-based cloning approaches and genome-sequencing efforts have revealed the existence of a large number of human genes encoding 'orphan' G-protein-coupled receptors (GPCRs), receptors that bind unidentified natural ligands. Discovery of these natural ligands is the first necessary step in understanding the biological significance of the orphan GPCRs. We 1 and others 2 have developed an approach by which to successfully isolate endogenous ligands from complex tissue libraries. This approach, referred to as the orphan-receptor strategy 3 , uses orphan receptors as baits to isolate their native ligand from tissue extracts and has been successfully applied to identify new neuropeptides 4-6 . Here we apply this strategy to the orphan receptor GPR14 and show that it binds the bioactive peptide known as urotensin II.The complementary DNA encoding the orphan receptor GPR14, or SENR, was cloned using degenerate oligonucleotides directed at conserved regions of known GPCRs 7,8 . Phylogenetic analysis positioned the GPR14 sequence closely to somatostatin, opioid and galanin receptors 8 , indicating that the endogenous ligand of GPR14 may also be peptidergic. We set out to identify the natural ligand of GPR14 from peptide extracts prepared from a variety of different mammalian tissues. Extracts were fractionated by preparative reverse-phase high-performance liquid chromatography (rpHPLC) into 72 individual fractions, and aliquots were tested for induction of changes in intracellular Ca 2+ ([Ca 2+ ] i ) in Chinese hamster ovary (CHO) cells transiently transfected with GPR14 cDNA. Intracellular Ca 2+ changes were monitored using a fluorescence imaging plate reader (FLIPR) system 9 . A reproducible and robust change in Ca 2+ concentration was observed in two adjacent fractions (Fig. 1a). This change could not be detected either in nontransfected cell lines or in cells transfected with other orphan receptors. Highest levels of activity were detected in bovine hypothalamic tissue, which was consequently used for large-scale purification. The active component was purified over a seven-step purification strategy combining reverse-phase and cationic-exchange HPLCs. One single activity peak was detected, indicating that the activity can be attributed to a unique molecular entity. The bioactive compound was extremely scarce, preventing us from carrying out a total structural analysis. However, the Ca 2+ response to the active material showed a distinctive time course (Fig. 1a, inset). Furthermore, the active material was sensitive to trypsin (Fig. 1a, inset) and alkylating agents, leading us to conclude that the biological activity could be attributed to a peptide containing basic amino acid(s) and alkylation-sensitive amino acids.As GPR14 is similar to the somatostatin receptors we decided to screen somatostatin-like, cysteine-bridge-containing peptides on GPR14 and to compare their biological activities and physicochemical properties with that of the endogenous compound identified in our purification scheme. Peptides tested wer...
Chronic estrogen treatment increases levels of eNOS protein in cerebral microvessels of male and female rats. This increase in eNOS protein correlates with our previous functional findings indicating that estrogen exposure increases NO modulation of cerebrovascular reactivity in both male and female animals. Upregulation of eNOS expression may contribute to the neuroprotective effect of estrogen.
A cute heart failure (AHF) is common and is associated with adverse outcomes among chronic HF patients. According to the US National Discharge Survey, it is estimated that more than 1.1 million hospitalizations with a principal diagnosis of AHF occurred in 2004. 1 Data from Europe have also reported high AHF rates. [2][3][4][5] In patients with chronic HF, hospitalization for AHF is associated with increased mortality. 6 AHF patients are also particularly prone to readmission, with reported readmission rates between 44% to 60% within 6 to 9 months of discharge. 7,8 AHF hospitalizations account for more than half of the costs of HF treatment, 2,9 which in the United States are projected to reach $37 billion in 2009. 10 Renal impairment (RI) is common in HF and is associated with adverse outcomes in AHF patients. [11][12][13][14] In addition, approximately 20% to 30% of patients admitted for AHF have worsening renal function (WRF), which further worsens prognosis. [15][16][17] Consistent with these findings, guidelines for HF treatment acknowledge that RI or WRF compromise prognosis in AHF and recommend regular monitoring of renal function in these patients. 18 -21 However, despite its importance, there is no clear consensus on how RI and WRF should be defined and to what extent these conditions affect outcomes in AHF specifically. 22 Two recent systematic reviews and meta-analyses have evaluated the association of RI and long-term mortality in HF 23 as well as the association of WRF and mortality and readmission risk. 17,23 Smith et al, 23 in a meta-analysis of 7 chronic HF (nϭ16 106) and 2 AHF (nϭ54 305) studies found a 56% increase in long-term mortality risk with severe renal impairment; moreover, WRF was associated with a 47% increase in the risk of 6-month mortality, but there was only marginal association with readmissions in AHF patients. 23 Damman et al 17 reported a 61% increase in risk of mortality and 30% risk of all-cause readmission associated with WRF after 2 to 6 months of follow-up. Although these studies provide valuable insights, they were not designed to assess outcomes specifically in AHF and did not address short-term health and health care resource outcomes such as in-hospital mortality, risk of complications, and length of stay (LOS), which are uniquely relevant to AHF patients. 17,23 The objective of this systematic review was to enumerate the range of studies that have assessed the impact of RI and WRF on short-and long-term health outcomes and health care resource use in adult AHF patients. We also describe the range of definitions for RI and WRF as well as compare the associations of these definitions with relevant outcomes in AHF. Literature SearchA MEDLINE (PubMed) literature search was performed to identify peer-reviewed studies that assessed the association between RI or WRF and health outcomes and health care resource use in adult AHF patients (Figure). Because WRF epidemiology has been mainly defined after the Evaluation of Losartan in the Elderly (ELITE) trial was published in 1...
Background and Purpose-In vivo and in vitro rat models of hormone therapy were used to test the following hypotheses:(1) estrogen acts directly on cerebrovascular estrogen receptors to increase endothelial nitric oxide synthase (eNOS); (2) increased protein correlates with higher NOS activity; and (3) effects of estrogen on eNOS are altered by concurrent treatment with either medroxyprogesterone acetate (MPA) or progesterone. Methods-Blood vessels were isolated from brains of ovariectomized female rats; some were treated for 1 month with estrogen, estrogen and progesterone, or estrogen and MPA. The latter effect persists in the presence of either progesterone or MPA. Thus, increased NO production by eNOS may contribute to the neuroprotective effects of estrogen.
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