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The cerebral vasculature is a target tissue for sex steroid hormones. Estrogens, androgens, and progestins all influence the function and pathophysiology of the cerebral circulation. Estrogen decreases cerebral vascular tone and increases cerebral blood flow by enhancing endothelial-derived nitric oxide and prostacyclin pathways. Testosterone has opposite effects, increasing cerebral artery tone. Cerebrovascular inflammation is suppressed by estrogen but increased by testosterone and progesterone. Evidence suggests that sex steroids also modulate blood-brain barrier permeability. Estrogen has important protective effects on cerebral endothelial cells by increasing mitochondrial efficiency, decreasing free radical production, promoting cell survival, and stimulating angiogenesis. Although much has been learned regarding hormonal effects on brain blood vessels, most studies involve young, healthy animals. It is becoming apparent that hormonal effects may be modified by aging or disease states such as diabetes. Furthermore, effects of testosterone are complicated because this steroid is also converted to estrogen, systemically and possibly within the vessels themselves. Elucidating the impact of sex steroids on the cerebral vasculature is important for understanding male-female differences in stroke and conditions such as menstrual migraine and preeclampsia-related cerebral edema in pregnancy. Cerebrovascular effects of sex steroids also need to be considered in untangling current controversies regarding consequences of hormone replacement therapies and steroid abuse.

We report here that estrogen (E(2)) modulates mitochondrial function in the vasculature. Mitochondrial dysfunction is implicated in the etiology of vascular disease; thus, vasoprotection by estrogen may involve hormonal effects on the mitochondria. To test this hypothesis, mitochondria were isolated from cerebral blood vessels obtained from ovariectomized female rats, with or without E(2) replacement. Estrogen receptor-alpha (ER-alpha) was detected in mitochondria by immunoblot and confocal imaging of intact vessels. E(2) treatment in vivo increased the levels of specific proteins in cerebrovascular mitochondria, such as ER-alpha, cytochrome c, subunit IV of complex IV, and manganese superoxide dismutase, all encoded in the nuclear genome, and subunit I of complex IV, encoded in the mitochondrial genome. Levels of glutathione peroxidase-1 and catalase, however, were not affected. Functional assays of mitochondrial citrate synthase and complex IV, key rate-limiting steps in energy production, showed that E(2) treatment increased enzyme activity. In contrast, mitochondrial production of hydrogen peroxide was decreased in vessels from E(2)-treated animals. In vitro incubation of cerebral vessels with 10 nM 17beta-estradiol for 18 h also elevated levels of mitochondrial cytochrome c. This effect was blocked by the estrogen receptor antagonist fulvestrant (ICI-182,780, Faslodex) but was unaffected by inhibitors of nitric-oxide synthase or phosphoinositide-3-kinase. Nuclear respiratory factor-1 protein, a primary regulator of nuclear gene-encoded mitochondrial genes, was significantly increased by long-term estrogen treatment in vivo. In summary, these novel findings suggest that vascular protection by E(2) is mediated, in part, by modulation of mitochondrial function, resulting in greater energy-producing capacity and decreased reactive oxygen species production.

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...

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