Effects of urotensin II on functional activity of late endothelial progenitor cells

Urotensin II (UII) is a potent vasoactive cyclic peptide which has multiple effects on the cardiovascular system. However, the effects of UII on late endothelial progenitor cells (EPCs) are still unclear. The aim of the present study is to investigate whether UII influences the functional activity of late EPCs. Late EPCs were isolated from human umbilical cord blood by Ficoll density gradient centrifugation and treated with UII (10(-10), 10(-9), 10(-8), 10(-7) and 10(-6)M), or vehicle control. Expression of urotensin II receptor (UT) in late EPCs was confirmed by indirect immunofluorescence staining. Late EPCs proliferation, migration and in vitro vasculogenesis activity were assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, transwell chamber assay, and matrigel tube formation assay. Late EPCs adhesive assay was performed by replating cells on fibronectin-coated dishes, and then adherent cells were counted. Incubation with UII increased the migratory, adhesive and in vitro vasculogenesis capacity and inhibited the proliferative activity of late EPCs. Furthermore, these UII-mediated effects on late EPCs were attenuated by pretreatment with the UT antagonist urantide. These findings indicate that UII may exert multiple effects on functional activity of late EPCs through UT.     

Cardiovascular effects of native and non-native urotensin II and urotensin II-related peptide on rat and salmon hearts

Urotensin II (UII) was first discovered in the urophyses of goby fish and later identified in mammals, while urotensin II-related peptide (URP) was recently isolated from rat brain. We studied the effects of UII on isolated heart preparations of Chinook salmon and Sprague–Dawley rats. Native rat UII caused potent and sustained, dose-dependent dilation of the coronary arteries in the rat, whereas non-native UII (human and trout UII) showed attenuated vasodilation. Rat URP dilated rat coronary arteries, with 10-fold less potency compared with rUII. In salmon, native trout UII caused sustained dilation of the coronary arteries, while rat UII and URP caused significant constriction. Nω-nitro-_l-arginine methyl (l-NAME) and indomethacin significantly attenuated the URP and rat UII-induced vasodilation in the rat heart. We conclude that UII is a coronary vasodilator, an action that is species form specific. We also provide the first evidence for cardiac actions of URP, possibly via mechanisms common with UII.     

Identification of urotensin II-related peptide as the urotensin II-immunoreactive molecule in the rat brain

Urotensin II (UII) has been reported as the most potent known vasoconstrictor. While rat and mouse orthologs of UII precursor protein have been reported, only the tentative structures of UII peptides of these animals have been demonstrated, since prepro-UII proteins lack typical processing sites for their mature peptides. In the present study, we isolated a novel peptide, UII-related peptide (URP), from the extract of the rat brain as the sole immunoreactive substance to anti-UII antibody; the amino acid sequence of the peptide was determined as ACFWKYCV. cDNAs encoding rat, mouse, and human precursor proteins for URP were cloned and revealed that the sequences of mouse and human URP peptides are the same as that for rat URP. Prepro-URP gene is expressed in several rat tissues such as those of the thymus, spleen, testis, and spinal cord, although with lower levels than the prepro-UII gene. In the human, the prepro-URP gene is expressed comparably to prepro-UII in several tissues except the spinal cord. URP was found to bind and activate the human or rat UII receptors (GPR14) and showed a hypotensive effect when administered to anesthetized rats. These results suggest that URP is the endogenous and functional ligand for UII receptor in the rat and mouse, and possibly in the human. We also describe the preparation of specific monoclonal antibodies raised against UII peptide and the establishment of a highly sensitive enzyme immunoassay system for UII peptides.     

Biochemical and functional characterization of high-affinity urotensin II receptors in rat cortical astrocytes

The urotensin II (UII) gene is primarily expressed in the central nervous system, but the functions of UII in the brain remain elusive. Here, we show that cultured rat astrocytes constitutively express the UII receptor (UT). Saturation and competition experiments performed with iodinated rat UII ([(125)I]rUII) revealed the presence of high- and low-affinity binding sites on astrocytes. Human UII (hUII) and the two highly active agonists hUII(4-11) and [3-iodo-Tyr9]hUII(4-11) were also very potent in displacing [(125)I]rUII from its binding sites, whereas the non-cyclic analogue [Ser5,10]hUII(4-11) and somatostatin-14 could only displace [(125)I]rUII binding at micromolar concentrations. Reciprocally, rUII failed to compete with [(125)I-Tyr0,D-Trp8]somatostatin-14 binding on astrocytes. Exposure of cultured astrocytes to rUII stimulated [(3)H]inositol incorporation and increased intracellular Ca(2+) concentration in a dose-dependent manner. The stimulatory effect of rUII on polyphosphoinositide turnover was abolished by the phospholipase C inhibitor U73122, but only reduced by 56% by pertussis toxin. The GTP analogue Gpp(NH)p caused its own biphasic displacement of [(125)I]rUII binding and provoked an affinity shift of the competition curve of rUII. Pertussis toxin shifted the competition curve towards a single lower affinity state. Taken together, these data demonstrate that rat astrocytes express high- and low-affinity UII binding sites coupled to G proteins, the high-affinity receptor exhibiting the same pharmacological and functional characteristics as UT.     

Structural requirements at the N-terminus of urotensin II octapeptides

Urotensin II is the latest of a growing list of peptides exhibiting potent cardiovascular effects. It is an extremely potent vasoconstrictor in primates; its excretion is elevated in hypertensive patients thus suggesting therapeutic potential for urotensin II analogues, particularly receptor antagonists. In the present study, a number of interesting structural features pertaining to the N-terminus of urotensin II have been evaluated for binding to cloned human and rat urotensin II receptors and functional effects on rat upper thoracic aorta smooth muscle preparations. Shortened octapeptides retained full binding affinities and functional activities, did not require a free N-terminal amino group, and could tolerate an amidated C-terminus. The N-terminal Asp residue present in the octapeptides did not require a negatively charged side chain, merely one which contained a hydrogen bond acceptor CO group which could be present at a variety of positions on the side chain. The side chain could be constrained into a trans-olefinic configuration with full retention of potency, but potency was lost in the cis configuration. N-terminal aromatic amino substituted with polar groups such as OH and NO₂ also resulted in high affinity analogues. Overall, the correlation between binding affinities for the human and rat receptors was quite good. These findings could be of value in the development of more potent urotensin II receptor antagonists based on the previously identified somatostatin antagonist octapeptides which we have recently found, function as relatively weak urotensin II antagonists.     

Isolation and amino acid sequence of two urotensin II peptides from Catostomus commersoni urophyses

Two peptides with urotensin II (UII = smooth muscle stimulating) activity have been isolated from urophyses of the sucker, Catostomus commersoni. The amino acid sequences of the two peptides, designated UIIA and UIIB, are as follows: UIIA, H-Gly-Ser-Gly-Ala-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Val-OH; UIIB, H-Gly-Ser-Asn-Thr-Glu-Cys-Phe-Trp-Lys-Tyr-Cys-Val-OH. These peptides have the part sequence Cys-Phe-Trp-Lys-Tyr-Cys-Val in common with the UII peptide from Gillichthys mirabilis and the part-sequence Phe-Trp-Lys in common with somatostatin.     

Diabetes alters LDL receptor and PCSK9 expression in rat liver

Since the hepatic LDL receptor is regarded as a major determinant of plasma LDL levels, the effect of diabetes on the expression of this receptor was examined in rat liver. Inducing diabetes with streptozotocin caused a significant reduction in hepatic LDL receptor mRNA levels in concert with an increase in serum cholesterol levels. However, LDL receptor protein levels were unaffected by the diabetic state. Further investigation revealed that protein levels of PCSK9, which has been shown to enhance the degradation of the LDL receptor protein, were significantly decreased in the diabetic rats explaining the lack of reduction in LDL receptor protein levels. These observations indicate that the rate of LDL receptor cycling (function) in diabetic rats is decreased resulting in higher serum LDL levels.     

H_2O_2诱导小鼠肌原细胞系C2C12 UII/UT系统的高表达

目的:观察外源给予H2O2对C2C12细胞中UII/UT系统表达的影响。方法:培养小鼠肌原细胞系C2C12细胞株,应用胎盘蓝染色和测定细胞培养液中LDH的含量检测H2O2对细胞损伤的影响,应用放射免疫的方法检测细胞培养液和裂解液中UII的含量,应用RT-PCR法测定C2C12中UT mRNA的表达,应用Western Blot检测不同浓度H2O2对肌原细胞系C2C12中UT蛋白表达的影响。结果:外源给予不同浓度的H2O2,C2C12细胞裂解液中UII的含量各组间无明显差异,但增加了细胞孵育液中UII的含量,10-4 M和10-3 M时分别增加了83.1%(p0.01)和94.5%(P0.01)。低浓度H2O2(0.5×10-6 M,10-6 M,10-5 M,10-4 M)刺激明显增加了UT m RNA的表达,而高浓度的H2O2(10-3M)刺激反而使UT mRNA的表达降低。高浓度的H2O2(10-6 M,10-5M,10-4 M,10-3M)刺激使UT蛋白表达分别增加了200.1%、255.6%、111.1%和100.1%(均p0.05)。结论:H2O2作为T2DM发病因素之一的活性氧族,可能是T2DM时骨骼肌组织中UII/UT系统表达增加的一个因素。     

Interaction with dopamine D2 receptor enhances expression of transient receptor potential channel 1 at the cell surface

Receptor signaling is mediated by direct protein interaction with various types of cytoskeletal, adapter, effector, and additional receptor molecules. In brain tissue and in cultured neurons, activation of dopamine D2 receptors (D2Rs) has been found to impact cellular calcium signaling. Using a yeast two-hybrid approach, we have uncovered a direct physical interaction between the D2R and the transient receptor potential channel (TRPC) subtypes 1, 4 and 5. The TRPC/D2R interaction was further validated by GST-pulldown assays and coimmunoprecipitation from mammalian brain. Ultrastructural analysis of TRPC1 and D2R expression indicates colocalization of the two proteins within the cell body and dendrites of cortical neurons. In cultured cells, expression of D2Rs was found to increase expression of TRPC1 at the cell surface by 50%. These findings shed new light on the constituents of the D2R signalplex, and support the involvement of D2Rs in cellular calcium signaling pathways via a novel link to TRPC channels.     

Heterodimerization of the alpha and beta isoforms of the human thromboxane receptor enhances isoprostane signaling

Isoprostanes are free radical catalyzed products of arachidonic acid that are elevated in pro-oxidant disease states. Two isoprostanes, 8-isoprostaglandin F(2alpha) (iPF(2alpha)III) and 8-isoprostaglandin E2 (iPE2III), act at the receptor for thromboxane A2 (the TP) to mediate pro-atherogenic effects in vivo. We confirmed dimerization of the human TP isoforms, TPalpha and TPbeta, and determined the impact on isoprostane signaling. No overt changes in ligand binding at the TP were observed as a result of TPalpha/TPbeta coexpression. The response to iPF(2alpha)III or iPE2III was enhanced in HEK293 cells stably coexpressing TPalpha and TPbeta, as measured by inositol phosphate generation or intracellular calcium mobilization, relative to cells expressing TPalpha or TPbeta individually. In contrast, the response to traditional thromboxane analogs was unaltered. Augmented isoprostane signaling was similarly observed in HEK 293 cell transiently transfected with TPalpha and TPbeta. These results indicate that TPalpha/TPbeta dimerization enhances isoprostane-mediated signal transduction.     

Photoaffinity scanning in the mapping of the peptide receptor interface of class II G protein-coupled receptors.

The family of G protein-coupled receptors constitutes about 50% of the therapeutic drug targets used in clinical medicine today, although the mechanisms of ligand binding, activation and signal transduction for G protein-coupled receptors are not yet well defined. This review discusses ongoing research using the photoaffinity scanning method to map the bimolecular interface between class II G protein-coupled receptors and their ligands. Furthermore the available computer model of class II peptide ligand docking into the receptor, based on the positional constraints imposed by the photoaffinity scanning analyses, will be discussed briefly. The ultimate goal of these efforts is to understand the molecular basis of receptor binding and therefore to generate a template for rational drug design.     

Characterization of a tyramine receptor from Caenorhabditis elegans

Octopamine (OA) plays an important role in the regulation of a number of key processes in nematodes, including pharyngeal pumping, locomotion and egg-laying. However, while putative OA receptors can be tentatively identified in the Caenorhabditis elegans database, no OA receptors have been functionally characterized from any nematode. We have isolated two cDNAs, ser-2 and ser-2a, encoding putative C.elegans serotonin/OA receptors (C02D4.2, ser-2). The sequences of these cDNAs differ from that predicted by GeneFinder and lack 42 bp of exon 2. In addition, ser-2a appears to be alternatively spliced and lacks a predicted 23 amino acids in the third intracellular loop. COS-7 cells expressing SER-2 bind [3H]LSD in the low nM range and exhibit Kis for tyramine, octopamine and serotonin of 0.07, 2, and 13.7 micro m, respectively. Significantly, tyramine reduces forskolin-stimulated cAMP levels in HEK293 cells stably expressing SER-2 with an IC50 of about 360 nm, suggesting that SER-2 is a tyramine receptor.     

Identification and characterization of the diuretic hormone 31 receptor in the silkworm Bombyx mori

The receptor for diuretic hormone 31 (DH31R) was identified in the silkworm Bombyx mori. A heterologous expression system revealed that an orphan G-protein coupled receptor, BNGR-B1, responded to DH31 and upregulated the intracellular cAMP level. DH31R (BNGR-B1) was predominantly expressed in the anterior silk gland, midgut, and ovary, whereas DH31 was predominantly expressed in the central nervous system and midgut.     

Decreased expression of the pancreatic secretory trypsin inhibitor II gene during aging of the rat liver

The genetic constitution and differential gene expression of an organism play important roles in controlling the species-specific rate of aging and the maximum life span potential. We utilized a differential-display polymerase chain reaction technique to identify the age-dependent expression of genes in the rat liver. We demonstrate in this report, for the first time, that expression of the pancreatic secretory trypsin inhibitor II (PSTI-II) gene declines drastically during aging. We confirmed this decrease by Northern blot analysis. Low PSTI-II levels in aged animals might result in a lack of protection from prematurely activated trypsin-like proteases, which would thus enhance inflammation.Supported by funds from the Department of Biotechnology and by Research Fellowships (to H.P. and G.Z.) from the Council of Scientific and Industrial Research (Government of India).     

Lipidated peptides derived from intracellular loops 2 and 3 of the urotensin II receptor act as biased allosteric ligands

Over the last decade, the urotensinergic system, composed of one G protein-coupled receptor and two endogenous ligands, has garnered significant attention as a promising new target for the treatment of various cardiovascular diseases. Indeed, this system is associated with various biomarkers of cardiovascular dysfunctions and is involved in changes in cardiac contractility, fibrosis, and hypertrophy contributing, like the angiotensinergic system, to the pathogenesis and progression of heart failure. Significant investment has been made toward the development of clinically relevant UT ligands for therapeutic intervention, but with little or no success to date. This system therefore remains to be therapeutically exploited. Pepducins and other lipidated peptides have been used as both mechanistic probes and potential therapeutics; therefore, pepducins derived from the human urotensin II receptor might represent unique tools to generate signaling bias and study hUT signaling networks. Two hUT-derived pepducins, derived from the second and the third intracellular loop of the receptor (hUT-Pep2 and [Trp¹, Leu²]hUT-Pep3, respectively), were synthesized and pharmacologically characterized. Our results demonstrated that hUT-Pep2 and [Trp¹, Leu²]hUT-Pep3 acted as biased ago-allosteric modulators, triggered ERK_1/2 phosphorylation and, to a lesser extent, IP₁ production, and stimulated cell proliferation yet were devoid of contractile activity. Interestingly, both hUT-derived pepducins were able to modulate human urotensin II (hUII)- and urotensin II-related peptide (URP)-mediated contraction albeit to different extents. These new derivatives represent unique tools to reveal the intricacies of hUT signaling and also a novel avenue for the design of allosteric ligands selectively targeting hUT signaling potentially.     

Inhibition of squalene synthase upregulates PCSK9 expression in rat liver

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a critical role in cholesterol metabolism by enhancing the degradation of the LDL receptor protein in the liver. It has previously been shown that administration of zaragozic acid A (ZA), a potent inhibitor of squalene synthase, also significantly increases the rate of degradation of hepatic LDL receptor protein. Thus, we decided to determine whether ZA administration might act to up regulate hepatic expression of the rat PCSK9 gene. Administration of ZA resulted in increased PCSK9 mRNA and protein levels in rat liver surprisingly in concert with an increase in hepatic LDL receptor mRNA levels, LDL receptor protein turnover, and decreased serum cholesterol levels. These observations suggest an involvement of PCSK9 in hepatic LDL receptor protein degradation and perhaps, in increasing the rate of LDL receptor cycling resulting in lower serum cholesterol levels in response to cholesterol biosynthesis inhibitors.     

G-protein coupled receptor solubilization and purification for biophysical analysis and functional studies,in the total absence of detergent

G-protein coupled receptors (GPCRs) constitute the largest class of membrane proteins and are a major drug target. A serious obstacle to studying GPCR structure/function characteristics is the requirement to extract the receptors from their native environment in the plasma membrane, coupled with the inherent instability of GPCRs in the detergents required for their solubilization. In the present study, we report the first solubilization and purification of a functional GPCR [human adenosine A_2A receptor (A_2AR)], in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid (SMA) co-polymer direct from the membrane into a nanoscale SMA lipid particle (SMALP). Furthermore, the A_2AR–SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (∼5°C) compared with detergent [DDM (n-dodecyl-β-D-maltopyranoside)]-solubilized A_2AR controls. The A_2AR–SMALP was also stable when stored for prolonged periods at 4°C and was resistant to multiple freeze-thaw cycles, in marked contrast with the detergent-solubilized receptor. These properties establish the potential for using GPCR–SMALP in receptor-based drug discovery assays. Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([³H]ZM241385) capability. SMALP-solubilization of GPCRs, retaining the annular lipid environment, will enable a wide range of therapeutic targets to be prepared in native-like state to aid drug discovery and understanding of GPCR molecular mechanisms.